ANALYSIS OF SPECIALIZED PESTICIDE PROBLEMS
INVERTEBRATE CONTROL AGENTS - EFFICACY TEST METHODS
VOLUME IX
BACULOVIRUSES AND ENTOMOGENOUS BACTERIA
JANUARY
1978
EPA-540/10-784W1
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REPORT To THE
ENVIRONMENTAL PROTECTION AGENCY
ANALYSIS OF SPECIALIZED PESTICIDE PROBLEMS
INVERTEBRATE CONTROL AGENTS - EFFICACY TEST METHODS
VOLUME IX
BACULOVIRUSES AND ENTQMOGENOUS BACTERIA
The work upon which this publication is based was performed in whole or
in part under Contract No. 68-01-2457 with the Office of Pesticide Programs,
Environmental Protection Agency.
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Report To The
Environmental Protection Agency
By The
American Institute of Biological Sciences
Arlington, Virginia 22209
EPA REVIEW NOTICE
This Report has been reviewed by the Office of Pesticide Programs,
Criteria and Evaluation Division, and approved for publication.
Approval does not signify that the contents necessarily reflect
the views and policies of the Environmental Protection Agency, nor
does mention of trade names or commercial products constitute
endorsement of recommendation for use.
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BACULOVIRUSES AND ENTOMOGENOUS BACTERIA TASK GROUP
Chairman:
DR. WILLIAM G. YENDOL
Penn State University
DR. TERRY L. COUCH
Abbott Laboratories
DR. JA1-IES D. HARPER
Auburn University
DR. LOUIS A. FALCON
University of California, Berkeley
DR. 'JILLIA1-! H. McGAUGHEY
USDA/ARS Grain Marketing Research
Center
DR. WILLIAM YEARIAN
University of Arkansas
EPA Observer
AIBS Coordinators:
MR. ROGER PIERPONT
Criteria and Evaluation Division
MS. PATRICIA RUSSELL
MR. DONALD R. 3EEX
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BACULOVIRUSES AND ENTOMOGENOUS BACTERIA
Table of Contents
Page
Introduction . 1.
General Methods 4
Laboratory Studies 4
Small-Scale Field Tests 5
Large-Scale Field Tests 7
Reporting Microbial Agent Test Results 8
Annual Row Crops 12
Fiber Crops 12
Cotton 12
Boll and Square Feeders : Bollworm and Tobacco Budworm 13
Pink Bollworm 14
Foliage Feeders: Armyworms, Cabbage Looper, Cotton Leaf Worm 16
Oil Crops 16
Corn 16
Peanuts 17
Foliage Feeders: Corn Earwonn, Fall Armyworm, Velvetbean
Caterpillar, Rednecked Peanutworm 17
Peg and Pod Feeders, Lesser Cornstalk Borer . . . . 18
Soybean 18
Pod Feeders , Corn Earworm 19
Foliage Feeders: Soybean Looper, Green Cloverworm, Velvetbean
Caterpillar, Beet Armyworm, Cabbage Looper, Fall Armyworm 20
Sunflower 21
Seed Head Feeders: Sunflower Moth, Corn Earworm, Tobacco Budworm..21
Sugar Crops 22
Sugarbeets 22
Foliage Feeders : Beet Armyworm, Fall Armyworm 22
Sugarcane 23
Stalk Borers : Sugarcane Borer 23
Tobacco 23
Bud and Seed Pod Feeders: Tobacco Budworm, Corn Earworm, 24
Foliage Feeders: Tobacco Hornworm, Tomato Hornworm, Cabbage Looper..25
Stalk and Foliage Feeders : Dark-side Cutworm 26
Vegetable Crops 26
Crucif ers 27
Cabbage Looper 29
Curcurbits 29
Pickleworm and Melonworm 30
Cabbage Looper 31
Tomatoes 31
Tomato Fruitwonn, Tomato Hornworm, Western Yellow-striped Armyworm,
Beet Armyworm, Southern Armyworm, Cabbage Looper, Tomato Pinworm...31
Tomato Fruitworm and Tomato Pinworm 32
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Sweet Corn . 34
Corn Earworm » 34
Fall Armyworm 35
European Corn Borer 35
Irish Potatoes 37
Potato Tuberworm - 37
European Corn Borer 38
Peppers 39
European Corn Borer. . « 39
Snap Bean, Lima Bean, Southern Pea, Celery 39
Cabbage Looper 39
Pome and Stone Fruits and Tree Nuts 41
Deciduous Pome and Stone Fruits 41
Chewing Insect Pests .42
Twig Borers . . , * 45
Trunk Borers 46
Tree Nuts 48
Chewing Insect Pests - Codling Moth and Filbertworm - Walnut.. 48
Navel Orangeworm - Almond. 49
Hickory Shuckworm - Pecan 49
Twig Borers - Peach Twig Borer - Almond 50
Tree Borers - Peachtree Borer - Walnut 50
Defoliators - Redhumped Caterpillar - Walnut 50
Greenhouse Plants 52
General Methods 52
Application Techniques and Equipment 52
Efficacy Testing 53
Specific Insects and Crops 54
Tomato Pinworm. 55
Cutworm 57
Forage Crops 58
Alfalfa Caterpillar 59
Green Cloverworm 60
Alfalfa Webworm and Garden Webworm 60
Introduced European Skipper k 60
Rangeland 62
Grasshoppers 63
Range Caterpillar 63
Lawns , Turf grasses , Pastures 65
Small-Scale Field Tests 65
Large-Scale Field Tests 67
Webworms 67
White Grubs ... 69
Fores t, Shade, and Ornamental Trees 72
A looper 75
Bagworm. 76
Western Tent Caterpillar, Black-Headed Budworm 77
California Oakworm - 77
Douglas Fir Tussock Moth 78
European Pine Sawfly
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Fall Cankerworm 80
Mimosa Webworm, Fall Webworm, Walnut Caterpillar 81
Forest Tent Caterpillar 81
Great Basin Tent Caterpillar 82
Gypsy Moth 83
Hemlock Looper 84
Lodgepole Needle Miner 85
Omnivorous Leaf Roller 85
Orangestriped Oakworm 86
Pine Butterfly 86
Pitch Pine Looper 87
Redheaded Pine Sawfly 88
Spring Cankerworm and Saddled Prominent 88
Spruce Budworm 89
Western Hemlock Looper 91
Stored Products 93
Insect Rearing 94
Bioassay 95
Laboratory Testing 95
Pilot-Scale Testing 97
Commercial-Scale Testing 100
Lepidoptera in Stored Grain 101
Laboratory Testing 101
Pilot-Scale Testing 102
Commercial-Scale Testing 103
Lepidoptera in Stored Tobacco 104
Laboratory Testing 104
Pilot-Scale Testing 105
Comercial-Scale Testing 105
Lepidoptera in Dried Fruits and Nuts 106
Storage Infestation 106
Laboratory Testing 106
Pilot-Scale Testing 107
Comercial-Scale Testing 107
Field Investigation 108
Lepidoptera in Processed Cereal Products 108
Other Stored Commodities 109
Beeswax and Beecomb 109
Exhibits:
1 Assessment of Plant Coverage and Field Persistance of Entomogenous
Bacteria and Baculoviruses Applied for Pest Control Ill
2 Suggested Protocol for Evaluation of Microbial Pathogens for
Control of Selected Insects on Soybeans 114
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INTRODUCTION
Social and political concerns for the quality of man's environment
as well as increasing problems of pest resistance to chemical pesticides
have stimulated the research and development of biological agents as al-
ternatives to chemicals for pest control. Insect pathogens, especially
the entomogenous bacteria and baculoviruses, Ideally fit into this approach.
The purpose of this report is to provide guidance for conducting
tests to determine the efficacy of entomogenous bacteria and baculoviruses.
The methods described herein are not to be considered exclusive of
other methods. Methodologies and recommended practices for evaluating
microbial agents for pest control are few in number compared to those
available for testing chemical pesticides. Different situations may re-
quire special methods, and new approaches will have to be developed.
Every effort was made to keep the suggested test methods broad to cover a
wide range of conditions. More detailed information may be obtained by
referring to the literature citations in the guidelines.
The entomogenous bacteria developed for pest control are the spore-
formers. Non-sporeformers may offer potential but have not been adequately
studied as pest control agents. The baculoviruses are the nuclear poly-
hedrosis and granulosis viruses. They are the most studied group of in-
sect viruses and several have been used for pest control in recent years.
For additional information see publications listed in the General Reference
section.
Entomogenous bacteria and baculoviruses possess many unique features.
Special considerations and, in some cases, modifications of the methods
used to test chemical insecticides for efficacy, may be required. Factors
to be considered are many:
1. The pathogens must be ingested to be effective. Therefore, con-
sideration must be given to the reproductive activity and feeding
behavior of the insect. Method and timing of application can be
very critical.
2. All insect pathogens have an incubation period, and observable
effects may be delayed for several days. Evaluation intervals
should be adjusted accordingly.
3. Insect pathogens may be adversely affected by ultraviolet radiation,
and protective measures are often necessary.
4. Biological activity of stored microbial agents can be reduced by
exposure to high temperatures, and cool storage conditions should
be used.
5. Microbial agents are pH sensitive and the pH of formulations,
tank mixes, as well as the substrate to be treated should be
monitored. Buffering agents can be used to control problem
situations.
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6. Treated plants can grow rapidly and dilute field deposits of
microbial agents. Repeated applications at close intervals may
be necessary to maintain an effective level of control.
7. Anti-bacterial or anti-viral substances produced by target plants
may interfere with the effectiveness of a microbial agent.
8. Formulation, method of application and application equipment may
influence coverage, persistence and overall performance of a
microbial insecticide.
9. Some pathogens applied for insect control may persist in an area
for several generations of the target species, or several seasons,
and may contribute to long-term suppression of a pest.
Much of the interest in utilizing baculoviruses and entomogenous
bacteria for pest control has been stimulated by research results which have
shown that (1) they are host-selective, environmentally nondisruptive con-
trol agents well-suited for use in integrated control and other pest manage-
ment programs; and (2) they have not been shown to be hazardous to man,
other mammals and plant life. They are very adaptable, and may be employed:
1. As one would use a chemical insecticide;
2. As replacement for a chemical insecticide where the latter is no
longer effective because of insect resistance;
3. To provide relief in situations where the use of an otherwise
effective pesticide is restricted;
4. As a substitute for parasites and predators where they have been
reduced by chemical pesticide;
5. To complement naturally-occurring parasites, predators, and
pathogens for pest control; and
6. As an aid in controlling several pest species simultaneously when
used in a mixture of pesticides.
The most common approach to the dissemination of microbial agents has
been to employ the same equipment used for the application of chemical in-
secticides. Other ways demonstrated for the application of microbial agents
include (1) seeding a microbial pathogen into the target population; (2) the
use of traps to capture, externally contaminate with a pathogen and then re-
lease wild insects; (3) rear and release parasites which have been externally
contaminated with a pathogen.
In the United States, all pesticides are registered under FIFRA. The
requirements for the registration of pesticides of biological origin are
essentially the same as those prescribed for chemical products. Thus, for
registration purposes, there must be clear and convincing evidence that the
product to be marketed is effective as stated on the label, and safe when
so used. The registration of microbial agents must follow a carefully
planned approach which can be summarized as follows: (1) identification of
the insect pathogen by various criteria including morphology, growth re-
quirements, stability, bioassay and infectious process; (2) assessment of
effects on vertebrate and invertebrate non-target organisms, including acute,
subacute, and long-term toxicological studies; (3) small-scale field tests
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to gather data on efficacy, as well as to monitor effects on the environ-
ment; and (4) large-scale field tests to determine efficacy and usefulness
under commercial conditions.
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GENERAL METHODS
The early stages of development of a microbial agent include the
isolation, characterization and identification of the pathogen and the
application of the Koch's postulates. The safety of the pathogen for non-
target organisms, especially humans, is determined (see "Guidance for
Safety Testing of Baculoviruses", published by the U.S.E.P.A., Summers
et al, 1975). The requirements for safety testing are influenced by the
proposed uses of the pathogens. In some situations a temporary exemption
from the requirement of a tolerance must be obtained with an experimental
use permit for field testing. In other situations where it has been demon-
strated that the use of an insect pathogen does not alter the diversity
of insect pathogens naturally present in the environment, and does not
increase the residue above that which occurs naturally, the negligible
residue concept may be applied.
Laboratory Studies
Bioassay: Bioassays are necessary in the initial stages of microbi-
al insecticide development for studying the comparative pathbgenicity of
various pathogen isolates either in crude or formulated form, and for study-
ing the susceptibility of various target and non-target species or popula-
tions. Also, formulated products may be standardized using bioassays.
Bioassays in insects are used to establish the virulence, pathogenic!ty,
and infectivity of a candidate pathogen. This will aid in selecting initial
trial dosages for field tests. Bioassay can be employed also to determine
compatibility of a candidate pathogen and other agent, chemical, etc.; the
effect of application methods and equipment on the activity of a pathogen;
and plant coverage, specifically if deposition of the pathogen was in the
target areas of the plant. It can also be utilized to study residual persist-
ence of the applied pathogen.
Dosage Selection: The selection of an appropriate dosage of a patho-
gen for field studies depends on laboratory data accumulated against the
specific pest. Results obtained from bioassay followed by greenhouse trials
are useful for rate determinations. It is important to establish the lowest
level at which initial control is first detected and maximum level where
additional quantities will not result in substantial increase in control.
A standard insecticide treatment commonly used in actual control, untreated
checks, and where practical, a diluent control should be included for com-
parison with experimental materials.
Insect-Rearing and Propagation of Pathogens: The establishment and
maintenance of laboratory insect cultures is useful and necessary to provide
the test animals for bioassay studies, and as a means of propagating the
pathogens to be tested. Methods for establishing and maintaining insect
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cultures are many and varied, and are described in the literature for
many pest species (see General Reference section).
In addition to using the living whole organism several other methods
have been suggested for propagation of insect pathogens. These include
(1) embryonated eggs or cultures of organs, tissue, dispersed cells, or
established cell lines; (2) fermentation media; (3) completely defined
chemical substrates; and (4) the use of nonhomologous hosts, produced by
fermentation. The available systems for mass production of microbial in-
secticides are categorized and described in the literature (see General
Reference section).
Small-Scale Field Tests
Small plot field testing to support registration is usually begun
after investigation of the mode of action, growth characteristics in vivo
and in vitro, and formulation development in the laboratory. Testing
during these early stages is adaptable for early screening and subsequent
performance evaluations. Small plots facilitate thorough and uniform
coverage of the host substrate and permit a maximum number of observations.
Such plots facilitate control over variables which may influence efficacy
under large scale tests or actual use conditions. Also, they minimize the
quantity of experimental material required and the crop acreage necessary
for testing. Data on optimal rates, formulations, damage prevention, phyto-
toxicity, compatibility with adjuvant and behavior of infected insects can
be obtained in the small-scale field tests.
Site Selection; The general area selected should have a previous
history of infestations by the target pests. Uniform population densities
are highly desirable. Pest populations should be increasing at the time the
tests are initiated, or expected to appear in sufficient numbers to provide
measurable differences between treatments. Under certain conditions it may
be advantageous to artificially infest a selected number of plants within
each plot to ensure uniform distribution of the pest. The test site should
be sufficiently isolated (by noncrop areas or untreated crop areas) to
reduce the hazard of pesticide drift from treatment of other crops.
Test sites selected should have an even uniform stand of the host plant
or a uniform mixture of host plants. For agronomic crops, all tests should
be established on commercially grown varieties within the selected test site.
The variety selected should be susceptible to feeding by the target pest
species. The soil type should be uniform, and prepared with methods consist-
ent with growing the crop commercially. The crop should be planted, grown,
and maintained in accordance with accepted local agronomical practices.
Climatic conditions, cropping practices, composition of the ecosystem
and other factors often result in a wide variation of target insect popula-
tion levels. Thus, tests should be conducted in as many locations as possi-
ble to provide reliable and applicable information. When possible, test
locations should cover the target pest-crop range.
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Plot Size; Plot size will differ with specific target pests and
crops, and will be discussed under the individual pests. Plots should be
sufficiently large, or protected by buffer space, to prevent drift of materi-
als that are applied to adjacent plots. They should also be large enough
that removal of pest species or plant parts during data collection will not
interfere with the overall pest populatiori density or normal development and
maturation of the crops. Minimum plot length and width will in part be
dictated by the application equipment used. Small plots (0.025 ha) may be
used with hand-operated applicators whereas larger plots are required with
larger equipment.
Experimental Design and Data Analysis: Selection of the experimental
design will vary somewhat with pest species, crop and individual preference.
Randomized complete block is the most commonly used experimental design for
small plot efficacy evaluations. Other designs, i.e., Latin Square, split
plot and split block are also applicable and in certain instances it may be pre-
ferred. A minimum of 3 replicas is suggested. When target pest density,
plant age, plant density, plant vigor and soil type are not uniform, more
than 3 replications may be needed. A chemical insecticide may be used as a stand-
ard serves as a reference point only. Efficacy less than that of the in-
secticide standard may not preclude the usefulness of a microbial agent as
a pest management tool. Treatment effects should be compared to the untreated
checks and where possible, diluent controls.
Initially candidate microbial agents should be applied as a single com-
ponent rather than in combination with insecticides and fungicides. If
combination treatments are used the biological compatibility of the additive
with the microbial agent must be determined previously. Compatibility of
other additives, adjuvants, carriers, mixes, etc., should also be determined.
The activity of the mixture should be checked by bioassay.
The number of field trials conducted with each microbial agent must be
sufficient to allow accumulation of data on: (1) optimum dosage, (2) proper
timing, (3) treatment intervals, (4) performance at different target pest
densities and stages, (5) effects on the various cultivars of the host plant,
(6) effects on nontarget species, (7) effects complimentary oi: antagonistic
to naturally occurring biological control agents, (8) influence of applica-
tion on existing titer of the microbial agent in the environment, (9) per-
formance under different climatic conditions, (10) persistence in the test
area, and(11) compatibility with all application systems which may be used
to apply the microbial agent.
Due to the relative host specificity of microbial agents, it may be
necessary to implement control measures for nontarget pest species during
efficacy evaluations. Whenever possible the additional control measures
should be nonchemical in nature. When a chemical pesticide is the only
alternative, it should be one with a minimum of effect against the target
pest species and its naturally occurring biological control complex.
An analysis of variance and multiple range test or other appropriate
statistical aaalyses are employed where necessary to determine the statistical
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reliability of differences between treatments. Treatment means presented
alone should be accompanied by the standard deviation or standard error.
Application and Equipment: Candidate microbial agents are applied
with equipment and methods known to provide adequate coverage of the plant
parts requiring protection. In small plots, knapsack, high clearance,
tractor-mounted sprayers, mist blowers and other suitable equipment may
be used for liquid applications. The addition of wetting and sticking
agents to a candidate microbial agent preparation may be desirable. The
volume, pressure and flow rate of the application equipment as well as the
number and arrangement of the spray nozzles will vary with the insect
and crop under test. Dusts and granular preparations are usually applied
with hand dusters and granular applicators. Application equipment should
always be accurately calibrated before applying the materials to be tested.
It is desirable to express the dosage in terms of potency or activity
e.g., International Units (IU) for Bacillus thuringiensis. Potency of
each candidate microbial agent formulation should be monitored throughout
the test period using appropriate bioassay methods.
Timing of applications and their number will vary depending upon the
properties of the candidate microbial agent, crop and target insect.
Meteorological conditions should be recorded during application periods,
Information relative to plant coverage and persistence of candidate microbi-
al agent at the target site should be determined (see Exhibit 1).
Equipment should be thoroughly cleaned before and after use. When 2
or more rates of the same microbial agent are to be applied, begin applica-
tions with the lowest rate in order to maintain the integrity of the desired
dosage.
Sampling Techniques: Scientifically valid standardized procedures
should be employed for assessing the efficacy of a candidate microbial agent.
Methods will vary with crop management procedures, candidate microbial agent
and the target species, and will be discussed under each pest or pest com-
modity in the sections following. In general, criteria to be used include
pest population densities and damage estimates before and after,treatments
as well as yield and/or marketability of the crop at harvest.
Large-Scale Field Tests
Field tests should be conducted using application techniques commonly
employed for control of the particular target pest on the crop. A sufficient
number of trials should be conducted to cover the host range and geographical
distribution of the pest. Testing at this level provides data to indicate
efficacy of a candidate microbial agent under operational conditions.
Site Selection: Large field tests are conducted at sites in which
host-pest conditions are representative of the areas for which the product
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registration is desired. Where applicable, the tests include the host
varieties, host plant ages, cultural practices, pest populations and
weather conditions likely to be encountered in actual field operation.
Plot Size and Design. Plots must be large enough to permit utiliza-
tion of commercial equipment and practices. Optimal plot size may vary
with candidate microbial agent and commodity being tested. Plots should
be replicated; however, if this is not possible, a sufficient number of
subsamples must be taken within the treated and untreated areas to provide
a reliable measure of effectiveness.
Dosage Selection: Rates utilized in large-scale field testing may
consist of a range of dosages including the minimum effective rate deter-
mined in small plot experiments. Comparison with standard control practices
is useful.
Application and Equipment: The method of application used must
provide adequate coverage of the plant surfaces. High or low-volume (9.38 -
93.81/ha) ground or aerial applications with conventional low-volume sys-
tems may be employed. Test materials may be combined with other components
of the typical spray program if they are known to be compatible. (See
Small-Scale Field Tests Application and Equipment, and Exhibit 1.)
Sampling Techniques: Sampling techniques may differ with the pest
and crop under study and are discussed in the specific commodity or pest
sections that follow. Comparisons of yield data including quality and
marketability of the crop treated with a candidate microbial agent, standard
treatment materials and untreated controls are made. Supportive state-
ments from the investigator in testimony of the degree of control of the
candidate materials may be useful.
Reporting Microbial Agent Test Results
Information on the following should be provided as completely as
possible in reporting the results of efficacy tests. However, it is re-
cognized that all information listed below may not be available or needed
for every situation.
Name and address of investigator
Objectives and purpose of study (crop and target species)
Product used
formulation
lot number
storage conditions (temperature)
potency
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Cropping practices
variety and planting date
plant density and spacing
other agronomic practices (irrigation, cultivation,
fertilization, other pesticides applied)
Location of study (longitude and latitude, elevation,
exposure, orientation, soil type and analysis)
Experimental design
plot size
number of replicates
sampling procedure
statistical methods followed
Application
methods
type of equipment (nozzles, type, number, arrangement,
direction, pressure)
materials applied
- treatment dates
- dosage per hectare
- volume of application (ground or air speed)
- tank mix (pH, water quality)
- coverage (actual particles/unit area, droplet
size and density, volume emitted vs. volume
deposited)
Timing of application
stage of crops
density and stage of target species
time of application (day, hour)
climatic conditions (temperature, relative humidity,
cloud cover, wind speed and direction, precipitation,
crop wet or dry)
Assessment pre- and post-treatment
sample dates
crop data
- stage of growth and development
- parts examined
- damage
- phytotoxicity
- yields
- quality
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target species data
- population densities
- stages present
data on associated species (pests, parasites, predators, other)
- identification
- where sampled
- population densities
- stages present
- distribution
data on microbial insecticide
- residue on crop
- incidence/persistence (in host, in other species)
Determining effectiveness and usefulness
degree of protection provided
cost of applications
- material
- equipment
- time
return
- value of crop (e.g., recreation value, aesthetic value,
non-timber value)
- production
practicality of methods
t Other comments
General References
Bulla, L. A., edr 1973. Regulation of insect populations by microorganisms.
Ann. N.I. Aoad. Sci. 217: 243.
Surges, H. D., and N. W. Hussey, eds. 1971. Microbial Control of Insects
and Mites. Academic Press, New York. 861 pp.
Cantwell, G. E., ed. 1974. Insect Diseases, vols. 1 and 2. Marcel Dekker,
New York.
DeBach, P., and E. I. Schlinger, eds. 1964. Biological Control of Insect
Pests and Weeds. Reinhold Book Division, New York, chapters 18, 19,
20, 21.
Falcon, L. A. 1971. Microbial control as a tool in integrated control programs.
C. B. Huffaker, ed. Chapter 15, Biological Control. Plenum Press,
New York. 511 pp.
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Ignoffo, C. G. 1975. Entomopathogens as Insecticides. Env. Lett.
8(1): 23-40. "
Maddox, J. V. 1975. Use of diseases in pest management. Chapter 6 in
Introduction to Insect Pest Management. R. L. Metcalf and W. H.
Luckman, eds. John Wiley and Sons, New York. 587 pp.
Maxwell, F. G., and F. A. Harris, eds. 1974. Proceedings of the Summer
Institute of Biological Control of Plant Insects and Diseases.
University Press of Mississippi, Jackson. 647 pp.
National Academy of Sciences. 1969. Microbial control of insects. Chapter
8 in Insect Pest Management and Control Publication 1659 . NAS, Washington,
D.C. 508 pp.
Steinhaus, E. A., ed. 1963. Insect Pathology, An Advanced Treatise,
Vol. 1, 661 pp.; Vol. 2, 689 pp. Academic Press, New York.
Summers, M., R. Engler, L. A. Falcon, and P. V. Vail, eds. 1975. Baculo-
viruses for insect pest control: safety considerations. American
Society for Microbiology, Washington, D.C. 186 pp.
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ANNUAL ROW CROPS
Methods of efficacy evaluation of baculoviruses and bacteria on annual row
crops may not differ appreciably from that for chemical insecticides. The
relatively short time that the host plant is present, and the relative high
crop value per production unit, particularly for such crops as vegetables and
tobacco, may necessitate that a strict regimen be followed to provide eco-
nomic suppression of the pest population. To gain producer acceptance and
use, results with microbial agents alone or as part of the overall pest
management program must provide good crop protection.
In general, the lepidopterous pests of annual row crops are multi-
voltine and feed on a variety of hosts. The crop to be protected may be
only one in a succession of hosts that is attacked during the seasonal
activity of the pest. As a result, protection against a specific pest may
be required for only 1 or 2 generations. With the short-term characteristics
of the crop and pest problem, multi-cropping and crops rotation, long-term
evaluation of microbial agents on most annual crops may be difficult. In
this section emphasis is placed on short-term effects of microti^l agents on
pest population levels and crop protection. The long-term effects of microbi-
al agents should not be neglected. Data should be collected on the total
seasonal effects of the microbial agent on the target pest and non-target species,
The following test methods for annual row crops apply to dilute and
concentrate sprays, dusts, granules and baits. Only exceptions to and
variations from the procedures described in the General Methods are presented.
In addition, an attempt was made to standardize methods with those described
in Analysis of Pesticide Problems, Invertebrate Control Agents Effioaey Test
Methods: Volume II - Foliar Treatments II. (AIBS Report to EPA. EPA-
540110-77-001. 1977.)
FIBER CROPS
Cotton., Gossypiion hirsutwn
Insect pests are generally present on cotton throughout the producing
areas of the United States in sufficient numbers to affect yields seriously
unless control measures are applied. More chemical pesticides are used on
cotton than on any other crop. Thus the development and eventual widespread
use of microbial agents on cotton could contribute considerably to reduction
of the chemical pesticide load in the cotton agroecosystem. Cotton insect
pests currently amenable to suppression by baculoviruses and bacteria in-
clude the boll and square feeders bollworm, Heliothis zea (Boddie),
tobacco budworm, Heliothis viresoens (Fabricius), and pink bollworm,
Peatinophora gossypiella (Saunders); and the foliage feeders armyworms,
Spodoptera spp.,cabbage looper, Triohoplusia ni (Hubner), and cotton leaf
worm, Alabama agr-illaaea (Hubner).
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Boll and Square Feeders: Bollworm, Helioth-is zea and Tobacco Budworm,
Eeli-oth-is virescens
Plot Size: Plot size may vary considerably depending upon uniformity
of infestation, population density, application equipment used and personal
preference. In small plot tests 0.04 ha is generally accepted as standard.
Smaller plots, 8-12 rows by 15-30 m, have been used successfully by a number
of investigators. Large plots are usually 24-96 rows by 150 m for ground
equipment and 3 or more 12 m swaths by 300 m for aerial application. In
the latter stages of product development large plots may cover entire fields.
Application Equipment; A variety of equipment has been used for small
plot applications, ranging from individually constructed sprayers to commerci-
ally produced apparatus. Knapsack or high clearance sprays with a minimum
of 2 hollow cone nozzles per row operating at appropriate pressures to deliver
36-124 1 per hectare are commonly used. Dusts or granules are usually applied
by hand or with small commercial applicators.
Large plot applications may be made with high clearance sprayers (36-
62 1/ha) or by air (24-62 1/ha). Most late season applications are by air.
Timing and Frequency of Application: The initial application may be
made when 6,200-16,400 eggs or 1st stage larvae are present per hectare.
Repeated applications at 4-6 day intervals may be required through the
fruiting cycle of the plant.
Sampling and Evaluation; Weekly egg, larval, damaged square and
damaged boll counts made weekly through the treatment period from the center
rows of each plot may be helpful in evaluating efficacy. It is suggested
that the data be reported in numbers per hectare. Methods of sampling have
varied with individual researchers, with whole plant counts on 2-4 m of
successive row per plot most commonly used. In addition to damage data,
reporting of number of undamaged squares and bolls per hectare may also be
useful in evaluating effectiveness.
Yield samples from small plots have been most commonly obtained from
the center two or more rows of each plot and reported in kilograms seed
cotton per hectare. In large plots, the entire plot or sample area may be
mechanically harvested.
References
Allen, G. E. , B. G. Gregory, and J. R. Brazzel. 1966. Integration of the
Heliothis nuclear polyhedrosis virus into a biological control program
on cotton. J. Eoon. Entomol. 59: 1333-1336.
Allen, G. E. , B. G. Gregory, and T. L. Pate. 1967. Field evaluation of a
nuclear polyhedrosis virus in control of Hel-ioth-is zea and Eeliothis
virescens on cotton. J. Invevtebv. Pat-hoi. 9: 40-42.
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Andrews, G. L., F. A. Harris, P. P. Sikorowski, and R. E. Mclaughlin.
1975. Evaluation of Eeliothis nuclear polyhedrosis virus in a cotton-
seed oil bait for control of Eeliothis viresoens and H. zea on cotton.
J. Eoon. Entomol. 68: 87-90.
Bull, D. L., R. L. Ridgway, V. S. House, and N. W. Pryor. 1976. Improved
formulations of the Heliothis nuclear polyhedrosis virus. J. Eoon.
Entomol. 69: 731-736.
Chapman, A. J., and C. M. Ignoffo. 1972. Influence of rate and spray volume
of a nucleopolyhedrosis virus on control of Eeliothis on cotton.
J. Invertebr. Pathol. 20: 183-186.
Ignoffo, C. M. , A. J. Chapman, and D. F. Martin. 1965. The nuclear poly-
hedrosis virus of Heliothis zea (Boddie) and Eeliothis vivesoens
(Fabricius). III. Effectiveness of the virus against field populations
of Eeliothis on cotton, corn, and grain sorghum. J. Eoon. Entomol.
7: 227-235.
McGarr, R. L. 1968. Field tests with nuclear polyhedrosis virus against
the bollworm and tobacco budworm. J. Eaon. Entomol. 61:342.
McGarr, R. L. , H. T. Dulmage, and D. A. Wolfenbarger. 1970. The delta
endotoxin of Baoillus thwcingiensis, HD-1, and chemical insecticides
for control of the tobacco budworm and the bollworm. J. Eoon. Entomol,
63: 1357-1358.
McGarr, R. L., H. T. Dulmage, and D. A. Wolfenbarger. 1972. Field tests with
HD-1, delta endotoxin of Baoillus thuringiensis, and with chemical in-
secticides for control of tobacco budworm and bollworm in 1970. J. Eoon.
Entomol. 65: 897-899.
McGarr, R. L. and C. M. Ignoffo. 1966. Control of Eeliothis spp. with a
nuclear polyhedrosis virus, EPN and two newer insecticides. J. Eoon.
Entomol. 59.< 1284-1285.
!
Montoya, E. L., C. M. Ignoffo, and R. L. McGarr. 1966. A feeding stimulant
to increase effectiveness of and a field test with a nuclear polyhedrosis
virus of Heliothis. J. Invertebr. Pathol. 8: 320-324.
Pink Bollworm, PeotinophoTa gossypiella
Plot Design: Field cages, 1.8 x 1.8 x 10.9 m, have been used for
efficacy evaluation of microbials against Peotinophora gossypiella. It is sug-
gested that the plants in the cages be sprayed with a non-residual insecticide to
remove other pest species. 10 pairs of 2-day old P. gossypiella adults
released in each cage 1 day prior to the 1st application with additional
releases at 10 and 15 days provide infestation pressure. Adults from over-
wintering larvae have provided more reliable infestation than laboratory
reared adults.
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For open field tests, plot sizes may be comparable to those given for H. sea
and H. viresoens.
Application Equipment; See Heliothis zea and Heliothis virescens.
Timing and Frequency of Application: In field cage studies the first
application may be made 1 day following release of adults with subsequent
applications at 5-day intervals until the test is terminated.
In open field tests, applications may be initiated when susceptible
squares are present on the plants and adult pink bollworms are detected
in the field. This early application would be directed toward the larval
generation that develop in squares. Others have initiated application
when 850 or more larvae per hectare were present in blooms. Applications
may be continued at 4-6 day intervals as long as infestation pressure per-
sists.
Sampling and Evaluation; Bloom counts, i.e., normal vs. resetted,
may be useful in evaluating the effect of the treatment, especially on that
generation developing during the 1st 6 weeks of the season. An estimation
of the total number of blooms and total resetted blooms in each plot or cage
may be determined. When bolls are present, the larval population may be
estimated by determining the number of bolls per plot and collecting a
minimum of 50 bolls per plot. These bolls may be incubated in suitable
containers with paper for pupation sites and the number of larvae exiting
the bolls determined. Others have obtained estimates of the number of larvae
entering bolls by opening samples of bolls and examining the inside of the
carpel for larval mines.
Yield data may be taken as outlined for Heliothis zea and Heliothis virescens.
References
Bell, M. R., and R. F. Kanavel. 1977. Field tests of a nuclear poly-
hedrosis virus in a bait formulation for control of pink bollworms
and Heliothis spp. on cotton in Arizona. J. Econ. Entomol. (in press).
Bullock, H. R., and H. T. Dulmage. 1969. Bacillus thuringiensis against
pink bollworms on cotton in field cages. J. Econ. Entomol. 62: 994-995.
Ignoffo, C. M., and H. M. Graham. 1967. Laboratory and field cage tests
with Bacillus thwcingiensis against pink bollworm larvae. J. Irwertebr.
Pathol. 9: 390-394.
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Foliage Feeders: Armyworms, Spodopt'era spp.; Cabbage Looper, Tvichoplusia
ni\ Cotton Leaf Worm, Alabama argillacea
Plot Design: See also Boll and Sqaure Feeders: Heliothis sea and Helio-
this viresaens. With heavy, uniform infestations small plots, 4 rows by 23 m,
may be adequate. When small plots are used, a minimum buffer of 4.5 m between
blocks and 2.0 m between plots within blocks is recommended.
Application Equipment; See Boll and Square Feeders: Heliothis sea
and Heliothis virescens.
Timing and Frequency of Application; Multiple applications may not
be required with discrete populations. Treatment is recommended when a
majority of the larvae are in the 1st or 2nd instars. If multiple applica-
tions are necessary, they may be made at 4-6 day intervals.
Sampling and Evaluations; Larval counts, defoliation estimates,
and yield may prove useful in evaluating treatments. For other foliage
feeders, larval counts may be made with sweep net, D-vac, or shake cloth
methods. 4 random samples are normally taken from the center of each
plot pretreatment, and 5, 10, and 14 days posttreatment with counts con-
verted to numbers per hectare.
Yield data may be taken as outlined for Heliothis sea and Heliothis vireseens.
References
Ignoffo,^C. M., A. J. Chapman, and D. F. Martin. 1964. Control of Alabama
argillaoea (Hubner) with Bacillus ' thuringiensis Berliner. J. Insect
Pathol. 6: 411-416.
OIL CROPS
Corn, Zea mays
Insect pests of corn that are promising candidates for efficacy
evaluations with bacteria and baculoviruses include ear feeders corn
earworm, Heliothis sea (Boddie); whorl and ear feeders corn earworm,
H. sea, and fall armyworm, Spodoptera frugiperda (J. E. Smith); and stalk
borers European corn borer, Ostrinia nubilalis (Hubner) and Southwestern
corn borer, Diatraea grandiosella (Dyar). Procedures for evaluating efficacy
of microbial agents on corn were developed on sweet corn. These procedures
are also applicable to field corn and are presented in the Vegetable Crops:
Sweet Corn section.
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Peanuts, fwadkis hypsgaea
Insect pests of peanuts that may be amenable to control with
bacteria and baculoviruses include the foliage feeders corn earworm,
Heliothis zea (Boddie), fall armyworm, Spodopteva frugiperda (J. E. Smith),
velvet-bean caterpillar, Ant'iaca'S'ia gemmatal'is (Hubner) , rednecked pea-
nutworm, Stegasta bosquella (Chambers); and the pod and peg feeder
lesser corn stalk borer, Elasmopalpus lignosella Zeller. Literature on
efficacy evaluations of microbials for control of these pests is wanting.
The following procedures are based upon those used for these pests on other
crops and with chemical insecticides on peanuts.
Foliage Feeders; Corn Earworm, Heliothis sea; Fall Armyworm, Spodoptera
frugiperda; Velvetbean Caterpillar, Anticarsia gemmatal'is; Rednecked
Peanutworm, Stegasta bosquelia
Plot Design; Small plot size averages 4-8 rows by 15-30 m with
large plots 24-96 rows by 150 m for L. V. ground application and 45-60 m by
150-300 m for aerial or mistblower application.
Application Equipment; The first application should be made when
the number of 1st or 2nd stage larvae per row meter approaches 3-4. Larval
number may be determined by use of sweep net, shake cloth or D-vac. One
application is usually adequate, but should oviposition occur over an extend-
ed period, repeated applications at 4-6 day intervals may be required.
Sampling and Evaluation; Efficacy evaluations are usually based upon
larval numbers, defoliation, or yield. Larval numbers may be determined by
sweep net, 20 sweeps with a 0.4 m net across 2 adjacent rows in the center
of each plot; by shake cloth, shake the plants at 4 locations per plot over
aim cloth; or D-Vacing 12 m of row per plot. Larval counts are usually
made 5 and 10 days following the last application.
Defoliation may be determined visually and rated as follows: none,
light 1-20%, moderate 21-40%, heavy greater than 41%.
Yield samples should be taken from the center two rows of each plot
either manually or with a commercial thresher.
References
Yearian, W. C. 1977. Unpublished data.
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Peg and Pod Feeders, Lesser Cornstalk Borer, Elasmopalpus lignosellus
Plot Design; See Foliage Feeders: Heliothis zea, Spodoptera fmgi-
perda, Ant^oarsia gemmatalis3 Stegasta bosquella.
Application Equipment; See Foliage Feeders: Heliothis zea, Spodoptera
frugiperda} A. gemmatalis} S. 'bosquella. Granular applications may be made
by hand or with commercial applicators.
For basal directed sprays, a flat fan 80° nozzle should be directed
on each side of the row so as to cover the soil and lower leaves. A mini-
mum of 180 1 total volume per hectare is recommended.
Granular or bait applications should be made when the foliage is dry
in a 0.45-0.51 m band over the row.
Timing and Frequency of Application; Foliar applications should be
timed at peak moth flight with granular applications made 20-30 days after
planting.
Sampling and Evaluation: At 7, 14, 21 and 28 days posttreatment 10-20
plants at 4 locations in the center of each plot are examined for live borers
and damage. Number of larvae, plants infested, pegs damaged and nuts damaged
are recorded.
The center two rows of each plot should be harvested with yield and grade
of cured peanuts recorded.
References
Cunningham, W. H. Jr., D. R. King,.and B. C. Langley. 1959. Insecticidal
control of lesser cornstalk borer on peanuts. J. Eoon. Entomol. 52:
329-330.
Harding, J. A. 1960. Control of the lesser cornstalk borer attacking
peanuts. J. Eoon. Entomol. 53: 664-667
Leuck, D. B. 1966. Biology of the lesser cornstalk borer in south
Georgia. J. Boon. Entomol. 59: 797-801.
Leuck, D. B. 1967. Lesser cornstalk borer damage to peanut plants. J.
Eoon. Entomol. 60: 1549-1551.
Soybean, Glyoine max
Insect pests may be present in soybean in sufficient numbers throughout
the United States to seriously affect yield unless control measures are
applied. Of these the pod feeder, Heliothis sea (Boddie) and the foliage
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feeders soybean looper, Psuedoplusia inaludens (Clemens); green clover-
worm, Plathypena scabra'(Fabriclus); velvetbean caterpillar, Antiearsia
gemmatalis (Hubner); beet armywonn, Spodovtera exigua (Hubner) ; cabbage
looper, Tri,jlioplt>sia ni. (Hubner); and fall armyworm, Spodoptera frugiperda
(J. E. Smith) are the most likely candidates for control with" bacteria and
baculoviruses.
Pod Feeders, Corn Earworm, Heliothis sea
Plot Design: Plots 4-8 rows by 15-30 m are adequate for small scale
evaluations. Suggested plot size for large scale evaluations is 24-96 rows
by 150 m for ground equipment and 36 by 300 m for aerial application.
Application Equipment: Knapsack, high clearance or other L. V. small
plot equipment may be used. Sprayers should be equipped with 2 hollow cone
nozzles per row operating at appropriate pressures and ground speed to de-
liver 36-60 1 per hectare. Dusts may be applied by hand or with commercial
dusters.
Timing and Frequency of Application: Treatments are usually made when
4 or more 1st or 2nd stage larvae are present per row meter. Additional
applications may not be required.
Sampling and Evaluation: At 5 and 10 days posttreatment larval counts
are made by shaking or beating the plants over a ground cloth, 3 m in length.
A minimum of 4 points should be sampled per plot. The number of live larvae
are recorded and converted to numbers per hectare.
Yield samples may be taken from the center rows of each plot,
Peferences
Anonymous. 1974. Suggested protocol for evaluation of microbial pathogens
for control of selected insects on soybeans. NSF-IPM Mimeo. Rpt.
(Exhibit 2).
Shepard, M., G. R. Garner, and S. G. Turnipseed. 1974. A comparison of
3 sampling methods for arthropods in soybeans. Environ. Entomol.
3: 227-232.
Turnipseed, S. G., J. W. Todd, and G. L. Greene. 1974. Minimum rates of
insecticides on soybeans, Mexican bean beetle, green cloverworm,
corn earworm, velvetbean caterpillar. J. Eoon. Entomol-. 67: 287-291.
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Foliage Feeders: Soybean Looper, Pseudoplusia includens; Green Cloverworm,
Plathypena scabra; Velvetbean Caterpillar, Anticarsia gemmatalis; Beet Army-
worm, Spodoptera exigua; Cabbage Looper, Trichoplusia ni; Fall Armyworm,
Spodoptera frugiperda
Plot Design: See Pod Feeders: Heliothis zea.
Application Equipment: See Pod Feeders: Heliothis zea.
Timing and Frequency of Application; The first treatment is recom-
mended when 50-65,000 1st or 2nd stage larvae are present per hectare. Ap-
plications are repeated at 4-6 day intervals on an as-needed basis.
Sampling and Evaluation: See Pod Feeders: Heliothis zea.
References
Anonymous. 1974. Suggested protocol for evaluation of microbial pathogens
for control of selected insects on soybeans. NSF-IPM Mimeo. Rpt.
(Exhibit 2).
Beegle, C. C., L. P. Pedigo, F. L. Poston, and J. D. Stone. 1973. Field
effectiveness of the granulosis virus of the green cloverworm as com-
pared with Bacillus thuringiensis and selected chemical insecticides
on soybean. J. Econ. Entomol. 66: 1137-1138
Begum, A., and W. G. Eden. 1965. Influence of defoliation on yield and
quality of soybeans. J. Econ. Entomol. 58: 591-592.
Moscandi, Flavio. 1977. Control of Anticarsia gemmatalis (Hubner) on
soybean with a baculovirus and selected insecticides and their effect
on natural epizootics of the entomogenous fungus Nomuraea riley (Farlow)
Samson. M.S. Thesis. Univ. of Florida. 67 pp.
Shepard, M. G. R. Garner, and S. G. Turnipseed. 1974. A comparison of
three sampling methods for arthropods in soybeans. Environ. Entomol.
3: 227-232.
Turnipseed, S. G., J. W, Todd, G. L. Greene, and M. H. Bass. 1974.
Minimum rate of insecticides on soybeans: Mexican beetle,
green cloverworm,, corn earworm, and velvetbean caterpillar. J.
Econ. Entomol. 67: 287-291.
Yearian, W. C., J. M. Livingston, and S. Y. Young. 1973. Field evaluation
of Bacillus thuringiensis for control of selected lepidopterous
foliage feeders on soybeans. Ark. Agric. Exp. Sta. Rpt. Ser. 212.
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Sunflower, Eel'Lanthus annus
The sunflower moth, Homoeosoma electellwn (Hulst) is considered the
major insect pest of sunflower. The larvae of this insect feed on the
developing seeds in the seed head. The corn earworm, Heliothis zea
(Boddie), and tobacco budworm, Hel-iothis wiresoens (Fabricius) , may also
attack seed heads of sunflower.
Seed Head Feeders; Sunflower Moth, Homoeosoma eleetelluryi Corn Earworm,
Heliothis zea; Tobacco Budworm, Heliot'h^s virescens
Plot Design; Small scale evaluations may be conducted with plots
4-8 rows by 15-30 m. Large plots range from 0.4-4.0 hectares with a 7.5
m buffer area between plots.
Application and Equipment: Most applications have been made with a
high clearance sprayer or by air. When a high clearance sprayer is used 2
flat fan nozzles per row are directed on the face of the seed head. Volumes
from 120-240 1 per hectare are suggested.
Timing and Frequency of Application: Treatments should be begun
when 20% of the heads are in the flower stage. Repeated applications at
4-6 day intervals may be required as dictated by infestation counts.
Sampling and Evaluation: At weekly intervals during the treatment
period and through 21 days following the last application, 10-25 seed heads
should be selected from each plot and examined. The number of heads in-
fested, damaged seed per head and seed yields per head are recorded.
References
Adams, A. L., and J. C. Gaines. 1950. Sunflower insect control. J.
Eeon. Entomol. 43: 181-184
Beckham, C. M., and H. H. Tippins. 1972. Observations of sunflower
insects. J. Econ. Entomol. 65: 865-866.
Carlson, E. C. 1967. Control of sunflower moth larvae and their damage
to sunflower seeds. J. Eoon. Entomol. 60: 1068-1071.
Carlson, E. C. 1971. New insecticides to control sunflower moth. J.
Eoon. Entomol. 64: 208-210.
Teetes, G. L., N. M. Randolph, and M. L. Kinman. 1970. Notes on noctuid
larvae attacking cultivated sunflower. J. Eoon. Entomol. 63: 1031-
1032.
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Sugar Crops
Sugarbeets, Beta vulgaris and sugarcane, Saccharum spp. are the major
sugar producing crops in the United States. Lepidopterous pests of these
crops amenable to control with bacteria and baculoviruses include the
beet armyworm, Spodoptera exigua (Hubner), and fall armyworm, Spodoptera
frugiperda (J. E. Smith), on sugarbeets and the sugarcane borer, Diatraea
saocharalis (Fabricius) on sugarcane,
Sugarbeets: Beta vulgaris
Foliage Feeders: Beet Armyworm, Spodoptera exigua; Fall Armyworm,
Spodoptera frugiperda
Plot design: Minimum plot size is 2 rows by 15 m. An untreated
buffer row between plots and 1.5-1.8 m buffer area between blocks is re-
commended.
Large-scale plots with ground equipment may be 4-6 rows by 30 m and 36
m by 180 m for aerial application.
Application Equipment: Small plot applications may be made with
knapsack, tractor-mounted, or other similar types of equipment. Fan-type
nozzles are usually selected and operated at appropriate pressures to pro-
vide 95-151 1 per hectare.
Large-scale applications are made with commercial equipment. For
ground equipment, the above volume per hectare is suggested. Volume for
aerial application usually ranges from 19-38 1 per hectare.
Timing and Frequency of Application: Treatment is suggested when
50-65,000 1st or 2nd stage larvae are present per acre. Repeated applica-
tions should be at 5-7 day intervals on an as-needed basis.
Sampling and Evaluation: Efficacy evaluations may be based on larval
counts. At 5, 10 and 14 days posttreatiaent, 5-10 plants are selected at
random from 2 or more rows per plot with the number of larvae counted. A
visual estimate of damage may be made.
References
Harris, C. R., H. J. Svec, S. A. Turnbill, and W. W. Sans. 1975. Laboratory
and field studies on the effectiveness of some insecticides in control-
ling the armyworm. J. Econ. Entomol. 68: 513-516.
Weinman, C. J.s and G. C. Decker. 1951. The toxicity of eight organic
insecticides to the armyworm. J. Econ. Entomol. 44: 547-552.
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Sugarcane: Saccharum spp.
Stalk Borers: Sugarcane Borer, Diatraea saccharal-ls
Plot Design; Small-scale evaluations may be conducted in plots 3 rows
by 7 m. Large-scale plots for ground equipment average 4-6 rows by 30 m long.
Aerial applications should be on plots a minimum of 36 m wide by 180 m in
length.
Application; Small plot spray applications may be made with knapsack,
high clearance or other adaptable sprayers. Fan-type nozzles are suggested
and should be operated at appropriate pressures to provide 95-151 1 per
hectare.
Granules may be applied by hand or with commercial applicators.
Timing and Frequency of Application; Treatments should begin after
2nd generation larvae hatch, but before the young larvae bore into the stalk.
A minimum of 5% of the stalks should be infested.
Repeated applications are made at bi-weekly intervals.
Sampling and Evaluation: At weekly intervals during, and up to two
weeks following the treatment period, 25-50 stalks are selected at random
in each plot. The number of stalks with leaf sheath feeding and number of
live and dead larvae are recorded.
At harvest, 25-50 stalks are selected from each plot and the number of
joints tunneled determined.
References
Charpentier, L. J., R. D. Jackson, and W. J. McCormick. 1973. Sugarcane
borer. Control by delta-endotoxin of Bacillus thuringiensis
HD-1 in field tests. J. Econ. Entomol. 66: 249-251.
Hensley, S. D., W. J. McCormick, W. H. Long, and E. J. Concienne. 1961.
Field tests with new insecticides for control of the sugarcane borer
in Louisiana in 1959. J. Econ. Entomol. 54: 1153-1154.
Long, W. H., S. E. Hensley, E. J. Concienne, and W. J. McCormick. 1961.
Field tests with new insecticides for control of sugarcane borer in
Louisiana in 1969. J. Econ. Entomol. 54: 1155-1156.
Tobacco
Tobacco, Nicotiana tobacum, is a relatively localized, restricted acreage
crop with a high cash value per production unit. The crop is susceptible to
damage by a number of lepidopterous pests. These include bud and seed
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-24-
f eeders tobacco budworm, Heliothis virescens (Fabricius) , and corn
earworm, Heliothis zea (Boddie); and foliage feeders tobacco horn-
worm, Manduoa sexta (Linneaus), tomato hornworm, Manduca quinquemaaulata
(Haworth), cabbage looper, Triohoplusia ni., and a variety of cutworms.
Bud and Seed Pod Feeders: Tobacco Budworm, Heliothis vireseens; Corn
Earworm, Heliothis zea
Plot Design: For small scale field testing, plots may range from 2-4
rows by 10-15 m with a two-row buffer between plots and 3-6 m buffer between
blocks. Large plots average 2-4 times the size of small plots.
Application Equipment: Knapsack, high clearance, and other small plot
sprayers may be used in small-scale testing. The sprayers are usually equipped
with 1 full or disc cone nozzle per row positioned 0.3-0.45 m above the plant.
Recommended volume ranges from 370-560 1 per hectare.
Dust or granules may be applied by hand or with commercial applicators.
Timing and Frequency of Application: Treatments may be begun when
5-10% of the plants are infested with 1st and 2nd stage larvae. Repeated
applications may be made at 4-6 day intervals as long as the infestation per-
sists or until flower buds appear. Where infestations are irregular or non-
uniform artificial infestation of selected plants in each plot with newly
hatched larvae from laboratory colonies has proven useful.
Sampling and Evaluation; Larval counts and damage estimates are helpful
in evaluating efficacy. At 3, 6 and 10 days posttreatment, 25-30 plants are
examined in the center of each plot. With more than one application, larval
counts may be made at 3-day intervals and 3, 6 and 10 days following the
last application. The number of larvae and percent of plants infested are
recorded. At the final examination the number of leaves consumed per larva
is visually estimated to the nearest 0.1 leaf. When damage ratings are used
the parameters and limit of each rating category should be defined.
References
Chamberlain, F. S., and S. R. Dutky. 1958. Tests of pathogens for the
control of tobacco insects. J. Eoon. Entomol. 51: 560.
Gentry, G. R., W. W. Thomas, and J. M. Stanley. 1969. Integrated control
as an improved means of reducing populations of tobacco pests. J. Eoon.
Entomol. 62: 1274-1277.
Mistric, W. J., Jr., and F. D. Smith. 1971. Control of tobacco budworm on
flue cured tobacco with insecticides applied mechanically. J. Eoon.
Entomol. 64: 126-132.
Mistric, J. W., Jr., and F. D. Smith. 1973. Tobacco budworm control on flue-
cured tobacco with certain microbial pesticides. J. Eoon. Entomol. 66: 979-982,
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Reagan, T. E., R. L. Rabb, and W. K. Collins. 1974. Tobacco budworm:
topping and sucker control practices on infestations in flue cured
tobacco. J. Eoon. Entomol, 67: 551-552.
Foliage Feeders; Tobacco Hornworm, Manduoa sexto.', Tomato Hornworm, Manduoa
quinquemaoulata; Cabbage Looper, Triohoplusia ni
Plot Design; See Bud and Seed Pod Feeders: Eeliothis vivescens and
Eeliothis zea.
Application Equipment: See Bud and Seed Pod Feeders: Eeliothis
vivesoens and Eeliothis zea.
Timing and Frequency of Application; Applications may begin when
5-10% of the plants are infested with 3rd and 4th stage larvae. One appli-
cation is usually sufficient for hornworm control. Multiple applications
may be required for cabbage looperand made at 5-7 day intervals until larval
density drops below 5% infested plants.
Sampling and Evaluation; See Bud and Seed Pod Feeders: Eeliothis
viresoens and Eeliothis zea.
References
Begg, J. A. 1974. Microbial and chemical control of hornworms attacking
tobacco in Ontario. J. Eoon. Entomol. 57: 646-649.
Chamberlain, F. S., and S. R. Dutky. 1958. Tests of pathogens for the
control of tobacco insects. J. Eoon. Entomol. 51: 560.
Gentry, C. R., W. W. Thomas, and J. M. Stanley. 1969. Integrated control
as an improved means of reducing populations of tobacco pests. J. Eoon.
Entomol. 62: 1274-1277.
Guthrie, F. E., R. L. Rabb, and T. G. Bowery. 1959. Evaluation of
candidate insecticides and insect pathogens for tobacco hornworm
control, 1956-1958. J. Eoon. Entomol. 52: 798-804.
Mistric, W. J.. Jr., and F. D. Smith. 1973. Tobacco hornworm: methomyl,
monocroptophos, and other insecticides for control on flue-cured
tobacco. J. Eoon. Entomol. 66: 581-583.
Rabb, R. L., E. A. Steinhaus, and F. E. Guthrie. 1957. Preliminary tests
using Baoillus thuringiensis Berliner against hornworms. J. Eoon.
Entomol. 50: 259-262.
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Stalk and Foliage Feeders: Dark-side Cutworm, Euxoa messoria (Harris)
A number of cutworm species are reported to attack transplanted tobacco.
Although the procedures outlined below are for Euxoa messoria, they are
applicable to other species. Further, these procedures may also be appli-
cable for efficacy evaluations of bacteria and baculoviruses against cut-
worms on other annual row crops.
Plot Design: See Bud and Seed Pod Feeders : Heliothis vLrescens and Heliothis sea.
Application Equipment: Due to the mode of action of bacteria and
baculoviruses and the habits of cutworms, only granular baits are suggested.
These may be applied by hand or with commercial applicators.
Timing and Frequency of Application: Treatments are made preplant
over the soil surface of the bed or postplant over the plants. Repeated
applications are not advised.
Sampling and Evaluation: All plants are examined in the center 2
rows of each plot for cutworm damage and larval populations. Counts should
be made at weekly intervals until larval development is completed. Damage
is usually recorded as percent damaged plants per plot.
Other; To prevent interplot cutworm migration, aluminum barrier
strips 20.3 cm may be embedded in the soil to a depth of 7.6 m encircling
each plot.
References
Cheng, H. H. 1971. Field studies on the chemical control of the dark-
sided cutworm (Lepidoptera: Noctuidae) on tobacco in Ontario, with
particular reference to Dursban. Can. Entomol. 103: 649-653.
Cheng, H. H. 1973. Laboratory and field tests with Bae-illus thuringiensis
against dark-sided cutworm, Euxoa messeria (Lepidoptera: Noctuidae)
on tobacco. Can. Entomol. 105: 941-145.
Cheng, H. H. 1973. Further field evaluation of insecticides for control
of the dark-sided cutworm (Lepidoptera: Noctuidae) on tobacco in
Ontario. Can. Entomol. 105: 1351-1357.
Vegetable Crops
Crops which will be examined in this section include crucifers, lettuce,
cucurbits, potatoes, beans, peas, peppers, snap beans, lima beans, southern
peas, sweet corn, and tomatoes. Insect pests are confined to the order
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Lepidoptera. Often test methods described are not specific for microbial
insecticides but are indicative of adequate test procedures for the in-
sect on the specific crop reviewed.
Crucifers
Crops in this group include cabbage, Brassieae oleraaea var. eapitata;
broccoli, Brassiea oleraeea var. italica cauliflower, Brass-tea oleracea
var. capitata.', Brussels sprouts, Brassica olepacea var. germi-fera; kale,
Bpassiaa oleraeea var. oeephala; collards, Brassica oleraoea var. vird-is;
turnip, Brassioa Gconpestris var. rapa; mustard, Brassioa junoea var. crispifolia;
spinach, Spinacia oleTaoea^ and kohlrabi, Brassiea oaulorapa.
Pests in this group include cabbage looper, Tvichoplusia ni (Hubner);
imported cabbage worm, P-ler^Ls rapae (Linnaeus); diamond back moth, Plutella
xylostella (Linnaeus); fall armyworm, Spodoptera fTugiperda (J. E. Smith);
beet armyworm, Spodoptera exigua (Hubner); garden webworm, Loxostege rantalis
(Guenee); Hawaiian beet webworm, Eymenia TeQWCval'is (Fabricius) ; and corn
earworm, Hel'io't'h'Ls zea (Boddie).
Cabbage looper is the major pest of the crucifers. The other pests may
be sampled and recorded using the same basic techniques used for cabbage
loopers.
Plot Design: Plot size will vary depending on population uniformity,
distribution and density. Randomized complete blocks, with three or more
replicates are recommended. Plots may consist of either single or multiple
rows, 8-15 m in length. Buffer rows are essential to l and 2 row plots
and desirable in larger plots.
Aerial applications require larger plots. These should consist of a
minimum of 2, and preferably 3, swaths at least 12 m wide. This aids
in minimizing the effects of drift and ensures a sufficiently sized central
area for collection of representative samples.
Application Equipment; Knapsack sprayers, if calibrated carefully,
will give satisfactory results. When single nozzles are used the top and
sides of each row should be covered.
A wide variety of pressures and rates are used in commercial application.
Generally, the type of application equipment is of secondary importance to good
coverage. To optimize coverage spraybooms should be equipped with at least 3
nozzles per row and volumes of tank mix ranging from 187-935 1/ha should be
applied.
Equipment adaptable and consistent with accepted practices in the given
locality should be used for tests in large plots. Tank, boom and nozzles
should be thoroughly cleaned before use and when changing treatments. If
more than one rate is tested, start test sequence with the lowest rate to lessen
chances of cross-contamination from other treatment material.
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Timing and Frequency of Application; Spraying should begin when an
economic population level is present in the plots. Efforts should be made
to initiate treatment when insects are in the first instar. Spray inter-
vals should be 5-7 days during the active cycle of the target insect.
Observations should be made at 5-7 day intervals to determine degree of
initial kill and relative residual activity of the microbial insecticide.
Sampling and Evaluation: Larvae should be counted on at least 10-25
plants per plot. Surviving loopers should also be grouped according to size.
Larval counts should be supplemented with ratings of the crop injury.
Various ratings can be employed (1-10 scale, 1-6 scale, gradation of damage
on edible parts of the plant). Terminology such as medium, severe, etc.,
should not be used unless clearly explained.
Yield records detailing head weight, marketability, etc., should be
taken.
References
Chalfant, R. B. , and C. H. Brett. 1965. Cabbage looper and imported
cabbageworms: Feeding damage and control on cabbage in western
North Carolina. J. Eoon. Entomol. 58(1): 28-33.
Chalfant, R. B., W. G. Genung, and R. B. Workman. 1973. Control of the
cabbage looper in Florida and Georgia. J. Econ. Entomol. 66(1): 276-277.
Creighton, C. S., and T. L. McFadden. 1974. Complementary actions of low
rates of Bacillus thuringi-ensis and chlordimeform hydrochloride for
control of caterpillars on cole crops. J. Econ. Entomol. 67(1): 102-104.
Creighton, C. S., and T. L. McFadden. 1975. Cabbage caterpillars: Effects
of chlordimeform and Bacillus thuringiensis in spray mixtures and the
comparative efficacy of several chemical and B. thuTi-ngiensis formulations.
J. Econ. Entomol. 68(1): 57-60.
Creighton, C. S., T. L. McFadden, and R. B. Cuthbert. 1971. Control of
cabbage looper, Trichoplusia ni , and two associated species on cabbage
with Bacillus thuringiensis and chemical insecticides. J. Entomol. Soc.
8(2): 132-136.
Greene, G. L., and R. B. Workman. 1971. Cabbage looper control on collards
in Florida. J. Econ. Entomol. 64(5): 1331-1332.
Hale, R. L., and H. H. Shorey. 1972. Cabbage looper control on cole crops
in southern California: Granular insecticides in the soil indicate
lack of promise. J. Econ. Entomol. 65(6): 1658-1661.
Jacques, R. P. 1973. Tests on microbial and chemical insecticides for
control of Trichoplusia ni (Lepidoptera: Noctuidae) and Pieris rapae
(Lepidoptera: Pieridae) on cabbage. Can. Entomol. 105: 21-22.
Kouskolekas, C. A., and J. D. Harper. 1973. Control of insect defoliants
of collards in Alabama. J. Econ. Entomol. 66(5): 1159-1161.
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Cabbage Looper, Triehoplusia ni (Hubner)
The following methods will also apply to other pests such as imported
cabbageworm, armyworm, diamond back moth, and beet armyworm.
Plot Design: Small-scale ground applications may consist of single
plots one row wide by 8-15 m in length. Buffer rows (1-2) should be in-
cluded between plots to prevent drift and movement of insects. Commercial
ground applications should be sized to handle the common spray applicators
normally used by growers. These should be 6-8 beds wide by 15-20 meters
in length. Aerial applications require large plots consisting of 2 or pre-
ferably 3 swaths at least 12 m wide. This aids in preventing drift and
ensures a sufficiently sized central area for collection of representative
samples.
Application Equipment: See section under crucifers.
Timing and Frequency of Application: Spraying should begin when an
economic population level is present in the plots. Efforts should be made
to initiate treatment when larvae are in the first instar. Spray intervals
should be 5-7 days during the active cycle of the target insect. Observa-
tions should be made at 5-7 day intervals to determine degree of initial
kill and relative residual activity of the microbial insecticide.
Sampling and Evaluation: A larval count on at least 10 plants per
plot should be made. Care should be taken when examining single row hand
plots to keep at least 1.5 m away from the ends of each plot. For large
test plots, where application has been made by ground or air, evaluations
should be restricted to a well-defined buffer zone within the test area.
To determine effects on yield all mature lettuce heads in the central
part of each plot should be graded and marketable heads put in cartons.
Convert data to cartons per hectare.
References
McCalley, N. F., and Der-I-Wang. 1972. Field evaluation of insecticides
for control of green peach aphid and alfalfa looper on head lettuce.
J. Eeon. Entomol. 65(3): 794-796.
Vail, P. V., C. F. Soo Hoo, R. S. Seary, R. G. Killinen, and W. W. Wolf.
1972. Microbial control of lepidopterous pests of fall lettuce in
Arizona and effects of chemical and microbial pesticides on parasitoids.
Env. Entomol. 1(6): 780-785.
Curcurbits
Crops in this group include cantaloupe, Cucumis melo var. oantalupensis;
cucumber, Cueumis sativus; pumpkin, Cueurbita pepo; squash, Cuouvbita maxima;
and watermelon, Citrullus vulgaris.
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Pickleworm, Diaphania nitidalis (Stoll) and melonworm, Diaphania hyalinata
(Linnaeus)
The insects are similar and can be evaluated by essentially the same
techniques. However, to date insecticides manufactured from bacteria or
baculoviruses have been unsuccessful in controlling this pest complex.
Methods described will therefore be general.
The stems, terminal buds and blossoms of muskmelon, cucumber and
squash are severely attacked by the pickleworm, Diaphania nitidalis (Stoll).
The melonworm, Diaphania hyalinata (Linnaeus) feeds extensively on the leaves.
Unless foliage injury is severe, treatments should be begun one week
before fruit set and weekly applications should begin thereafter.
Plot Design: A randomized complete block design and 3 or more
replicates per treatment are suggested.
Plot size will vary depending on density and uniformity of the insect
population. Plot sizes which have been used have ranged from approximately
8 meters in length by 1-8 rows wide to commercial size plots 4-16 rows in
width extending to field length.
Aerial Application: 2 and preferably 3 swaths covering an area
approximately 182 m long by 36 m wide is suggested,
Application Equipment: Ground applications may be made using knap-
sack sprayers delivering 200-1,000 1 per hectare. Sprayers should be cali-
brated and spray applied along the top and each side of the row. This can
be accomplished with a boom having 3 nozzles suitably positioned or a
single nozzle. With single nozzle equipment, 3 separate trips down
each row will be required to provide thorough coverage of the plants.
Commercial-scale applications should be made according to the accepted
spray practices normally used on the crop and area in which the trial is
being conducted. Aerial application should use 19-94 1/ha using commercially
acceptable nozzle and boom arrangements.
Timing and Frequency of Application: Application should begin before
fruit set and at weekly intervals thereafter.
Sampling and Evaluation; Because microbial agents are slower-acting,
evaluations should not be made sooner than 5 days following the final spray.
This will ensure that the pathogen has sufficient time to be ingested and
kill the insect pest.
Counts should be made of 10-25 fruit or more per plot. The number in-
fested should be recorded and, if possible, the actual worm counts. Fre-
quent checks are necessary since damaged fruit rot and disappear rapidly.
A thorough record should be made of infested stems, terminal buds or
blossoms per 10-25 plants, or per plot in the case of the pickleworm. Check
foliage of 10-25 plants for melonworm.
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-31-
Take yields. Count damaged and undamaged fruit; record weight of
marketable fruit and average weight.
References
Canerday, T. D. 1967. Control of pickleworm on cucurbits. J. Eaon. Entomol.
60(6): 1705-1708.
Cabbage Looper, Tviohoplusia ni (Hubner)
In recent years cabbage looper has become a major pest of cucurbits
in Virginia and bordering states. Published references concerning looper
control in cucumbers and other cucurbits are unavailable. The experimental
techniques and reporting procedures described in the General Methods section
and under Crucifers can be used.
Plot Design: See section under melonworm and pickleworm.
Application Equipment: See section under melonworm and pickleworm.
Timing and Frequency of Application: Begin application when loopers
first appear on the crop. Apply microbial agent at 5-7 day intervals as re-
quired during the active cycle of the pest.
Sampling and Evaluation: Efficacy should be evaluated by enumerating
looper larvae on 10-25 plants or leaves per plot after each application.
Loopers should be divided into size classifications.
References
Hale, R. L., and H. H. Shorey. 1972. Cabbage looper control on crops in
southern California: Granular insecticides in the soil indicate lack
of promise. J. Eoon. Entomol. 65(6): 1658-1661.
Tomatoes, Lycopersioon esculentum
Tomato fruitworm, Eeli-ofhis z&a (Boddie); tomato hornworm, Manduoa
qui-nquemacu'iata (Haworth); western yellow-striped armyworm, Spodoptera
praefica (Grote);beet armyworm, Spodoptera exi-gua (Hubner); southern army-
worm, Spodoptera eridania (Cramer); cabbage looper, Trichoplusia ni (Hubner);
Tomato pinworm, Kei-feria lyoopsersi-cella(Busck)
The tomato fruitworm, Eel'Lotlrls sea, also known as the corn earworm
and cotton bollworm, is the major pest of tomatoes in the United States.
When testing bacteria or baculoviruses against this pest, foliar sprays
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applied during a specific spray schedule at a prescribed interval of 5-7
days should be followed.
Plot Design: A randomized complete block design and 3 or more
replicates per treatment is suggested.
Single row plots 1.8m wide and 12 m long have been used, but should
be considered minimum. More typical plot sizes are 5.4 m wide (1.8 m
rows by 3.6 m long), 4 rows wide by 7.5 m long, and 3 rows wide by 3.6 m
long.
Application Equipment: Knapsack sprayers and compressed air sprayers
(7.6 1) are satisfactory for applying test material to small plots. Volumes
per hectare of experimental materials should be in the range of 465-830 1.
Uniform coverage should be stressed and the addition of a suitable wetter-
sticker is recommended.
Best results in large plots will probably be obtained by using commer-
cial spray practices and equipment.
Timing and Frequency of Application: Applications begin at fruit set
and at 5-7 day intervals thereafter during the active cycle of the insect.
Sampling and Evaluation: Efficacy ratings of the treatments should
be based on the percentage of harvested fruit with fruitworm injury. Per-
centage control is estimated from comparison of the percent injured fruit
from treated and untreated test plots.
Because fruitworm injury is often light, 100-200 fruit per plot should
be examined. Fruitworm may destroy young tomatoes. If this situation exists,
count the total number of infested fruit in 10-25 or more plants per plot and
remove the small, infested tomatoes as they are observed. Armyworm damage
can be evaluated in a similar manner. Pinworm control is evaluated by re-
cording numbers of marketable and cull fruit lost, yield, mines per leaflet
and pupae on mulch beneath plants and at harvest.
Tomato hornworms and cabbage looper control is determined by direct
counts of surviving larvae on at least 10 plants in the treated plots. These
are compared to the untreated control and percent population reduction deter-
mined .
References
See citations following tomatoes: poled (Fresh Market).
Tomato fruitworm, Heliofnis zea (Boddie) and tomato pinworm, Keifevia
lyoopersicella (Busck)
Plot Design; A randomized complete block design and 3 or more
replicates per treatment is suggested.
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Plots for initial screening can be 3 rows wide by 7.5 m long. For
secondary screening larger plots 4-8 rows wide by 15 m long should be used.
This will help prevent migration of ovipositing adults into the untreated
plots.
Since most commercial ground applicators treat 4 rows per swath,
plots under commercial evaluation should be 8 rows wide by 30 m long. The
length should be sufficient to harvest a representative sample of fruit.
Application Equipment: Hy-Boy type spray equipment with vertical
booms on each side of each plant row using 3-5 nozzles (depending on the
height of the tomato plant) on each boom would provide good coverage with
ground equipment. Properly calibrated aerial equipment may also be used.
For ground equipment the microbial insecticide should be applied in a
volume of 930-1860 1/h. Aerial application should be made in the range
of 140-184 1/h.
Application should begin when small tomatoes first appear. Preventive
treatment schedules should be used and the pathogen applied at 7-day inter-
vals until harvest. When feasible, depending on geographical area and popu-
lation pressure, more than one time interval should be evaluated.
Sampling and Evaluation; Since poled tomatoes are harvested over a
period of several weeks, the sampling of fruit for examination should be
done when there are enough mature fruit for a representative sample. To-
matoes should be harvested at random over a wide area but at all times with-
in a well-defined buffer zone.
Small screening plots that are 3 rows wide by 7.5 m long should have a
fruit selection area one bed wide and 5 ft. from the end of each plot. This
method provides a 2-row buffer zone on each side of each plot.
Aerial plots 36 m by 180 m should have a fruit selection area in the
middle third of the plot.
A total number of 350-400 marketable tomatoes should be selected at
random per treatment regardless of the number of plots.
Each tomato should be examined and the number, species of larvae and
damage recorded. Extreme care should be taken to determine if damage is the
result of the fruitworm or pinworm.
The total number of tomatoes infested as well as percent tomatoes in-
fested is recorded.
References
Creighton, C. S. 1976. Field tests of insecticidal sprays and baits for
control of tomato fruitworm in tomatoes. J. Ga. Entomol. Soo. 11(2):
101-105.
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Creighton, C. S. , T. L. McFadden, and R. B. Cuthbert. 1971. Control of
caterpillars on tomatoes with chemicals and pathogens. J. Eoon.
Entomol. 64(3): 737-739.
Creighton, C. S. , T. L. McFadden, and R. B. Cuthbert. 1973. Tomato fruit-
worm: Control in South Carolina with chemical and microbial insecti-
cides 1970-1971. J. Eoon. Entomol. 66(2): 473-475.
Harding, J. A. 1971. Field comparisons of insecticidal sprays for control
of four tomato insects in South Texas. J, Eoon. Entomol. 64(5):
1302-1304.
Poe, S. L., and P. H. Everett. 1974. Comparison of single and combined
insecticides for control of tomato pinworm in Florida. J. Eoon.
Entomol. 67(5): 671-674.
Sweet Corn
Corn Earworm, Heliothis zea (Boddie)
Plot Design; Randomized complete block design with 3 or more
replicates per treatment is suggested.
In the early stages, small-scale applications are useful for demon-
strating efficacy. Dusts, sprays, granules and baits may be evaluated. Plots
may be 3-5 rows wide by 7.5-15 m long.
]
Commercial ground applications and larger plots should be done in the
final stages of development. Plots should be a minimum of 4 rows wide by 60
m long.
Application Equipment; Hand applications of treatment material may
be used in preliminary tests. A compressed air or knapsack sprayer is satis-
factory. Large-scale field tests should be made with a high-clearance sprayer
with 2-4 nozzles per row adjusted to cover only the ear area, particularly the
silks.
Spray volumes from 465-930 1/ha should give satisfactory results. Full
and uniform coverage should be stressed.
Timing and Frequency of Application; Application should coincide with
the first appearance of silk on the corn ears. Young larvae hatch from eggs
laid in the silk and migrate down into the silk channel between husks. 3-6
applications are required at 2-3 day intervals for adequate control.
Sampling and Evaluation; 25-50 ears should be selected at random
from the center of each plot. Each ear should be examined and rated as
damaged or clean. Data are recorded as "percent ears damaged."
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-35-
Referenoes
See citations following the Sweet Corn section.
Fall Armyworm, Spodoptera frugiperda (Smith)
The fall armyworm is often found associated with corn earworm. How-
ever, when the fall armyworm infests young sweet corn prior to silk forma-
tion, severe damage often occurs. Once the silk stage is reached, plot
size and application techniques described for corn earworm apply.
Plot Design: See section under corn earworm.
Application Equipment: A compressed air or knapsack sprayer is
satisfactory for small plots but the test material must be applied from
overhead into the whorl. This is necessitated because the armyworm feeds
deeply into the whorl of the plant. For larger plots, commercial equipment
may be used.
Volumes of spray per hectare are similar to that listed for corn earworm.
Timing and Frequency of Application: A 2-3 day application schedule
prior to silking or a shorter application period after silking will be re-
quired.
Sampling and Evaluation; Direct counts of armyworms or grading of
armyworm injury in 10-50 corn plants should be done. Leaves should be unrolled
and whorls thoroughly examined. Records should be based on fresh damage.
Foliar injury ratings may also be useful.
Armyworm infestation in samples of at least 25 ears per plot should be
counted. Carefully observe and record the degree of injury into the same
classes as used for earworms.
References
See citations following the Sweet Corn section.
European Corn Borer, Ostpina nubilalis (Hubner)
The European corn borer is often associated with corn earworm, and general-
ly will be controlled using the same sprays and schedules used for earworms.
However, the corn borer deposits its eggs in masses on the undersides of the
leaves in clusters up to 50. Young worms bore into various parts of the plant,
ears included, and often cause stalks to break and the ear section to come
into contact with the ground. Damage to the ears is often extensive rendering
it entirely unfit for marketing and processing.
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Plot Design: See section under corn earworm.
Application Equipment: See section under fall armyworm and corn ear-
worm. Basically, when the silk stage is reached application techniques used
for earworm apply. The technique recommended for fall armyworm should be used
for first generation corn borers, and insecticide sprays, baits, or granules
should be applied from an overhead boom directly into the whorl of the plant.
Timing and Frequency of Application: A 2-3 day application schedule
will be required after silking begins.
Sampling and Evaluation: Direct counts of corn borers and borer injury
in 25-100 sweet corn plants should be made.
Count the corn borer infestation in 25-100 ears per plot. Observe and
record the degree of injury into the same classes used for earworms.
References
Greene, G. L., and J. J. Jones. 1970. Control of budworms on sweet corn
in central and south Florida. J. Eoon. Entomol. 62(2):579-582.
Harrison, F. P., and J. W. Press. 1971. Tuning of insecticide applications
for European corn borer control in sweet corn. J. Eoon, Entomol. 64(6):
1496-1499.
Henderson, C. F., H. G. Kinzer, and J. H. Hatchett. 1962. Insecticidal field
screening tests against the fall armywonn in sorghum and corn. J. Eoon.
Entomol. 55(6): 1005-1006.
Hudson, M. 1962. Field experiments with Bacillus thuringiensis and chemical
insecticides for the control of European corn borer, Ostrinia nubilalis,
on sweet corn in southwestern Quebec. J. Eoon. Entomol. 55(1): 115-117.
Hudson, M. 1963. Further field experiments on the use of Baoillus thuringiensis
and chemical insecticides for the control of European corn borer, Ostrinia
nubilalis, on sweet corn in southwestern Quebec. J. Eoon. Entomol.
56(6): 804-808.
Janes, M. J. 1973. Corn earworm and. fall armywonn occurrence and control in
sweet corn ears in Florida. J. Eoon. Entomol. 66(4): 973-974.
Janes, M. J. 1974. Foam application of methomyl to sweet corn and leafy
vegetables. J. Eoon. Entomol. 67(2): 249-250.
Janes, M. J. 1975. Corn earworm and fall armyworm: Comparative larval popu-
lations and insecticidal control in sweet corn in Florida. J. Eoon.
Entomol. 68(2): 657-658.
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McWhorter, G. M., E. C. Berry, and L. C. Lewis. 1972. Control of European
corn borer with two varieties of Baoillus thwri-ngiensis. J. Econ. Entomol.
65(5): 1414-1417.
Oatman, E. R. , I. M. Hall, K. Y. Arakawa, G. R. Platner, L. A. Bascam, and
C. C. Beegle. 1970. Control of corn earworm of sweet corn in southern
California with a nuclear polyhedrosis virus and Bacillus thuringiensis.
J. Boon. Entomol. 63(2): 415-521.
Irish Potatoes
Irish potatoes, Solanwn tuberoswn, are attacked by two species of
Lepidoptera which could possibly be controlled with entomogenous bacteria or
baculoviruses. These are the potato tuberworm, Gnori-mosohema o'peTculella
(Zeller) and European corn borer, Ostrinia nubilalis (Hubner). Test methods
for these insects will be described below.
Potato tuberworm, Gnorimosohema, operculella (Zeller)
Plot Design; For early tests, plots may be 3 beds wide by 2.5 m long.
Plots this size will help to prevent normal movement of adults from adversely
affecting results under normal population pressures.
For commercial trials, size of the plot is dictated by available ground
applications which treat 6-8 beds per swath. Therefore, plots should be 18-24
beds wide by 60 m long. This will also provide enough area in the middle of
the plot to take representative samples.
Plots for aerial application should be 36 m wide or 3 swaths of 12 m in
width to minimize drift and allow sufficient area to harvest a representative
sample of tubers.
Application Equipment; A broadcast boom with flat fan tips (8004 is
suggested) arranged equidistant should provide optimum coverage when using
ground equipment. Knapsack sprayers will give satisfactory results in small
plot tests. Full coverage should be stressed with this method and the use of
flat fan nozzles i§ preferred.
Finished spray volume is dependent on plant size but satisfactory results
can be expected with ground applications of from 561-935 1/ha. Aerial spray
rates of from 93-140 1/ha are recommended.
Timing and Frequency of Application: Control of tuberworms requires
preventative-type treatments and sprays should be applied on 5-7 day and 10-14
day schedules. This will permit observations on residual activity of the pathogen.
Sampling and Evaluation; Normal harvest procedures for the area in which
the test is being conducted should be followed.
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-38-
Harvested tubers should be selected from the center of the experimental
plots. Total numbers of tubers selected should be in the range of 500-600
regardless of the total number of plots.
Tubers should be examined and recorded as damaged if a tuberworm mine is
found. The number of mines per tuber is not important since one is sufficient
to necessitate culling.
The total number of tubers infested is recorded as well as the percent
of tubers infested. Care should also be taken to separate "green tubers"
(those that are exposed on the surface) from the "marketable" tubers when
recording those that are infested.
References
See citations following Irish Potato section.
European corn borer, Ostrinia nubitalis (Hubner)
European corn borer attacks both spring and fall crops of Irish potatoes
in Virginia and the Atlantic seaboard states. Although pathogens are not
particularly effective against this insect because of its feeding behavior,
the method described below may be acceptable for test purposes.
Plot Design: See section under potato tuberworm.
Application Equipment; See section under potato tuberworm.
Timing and Frequency of Application; Treatments must be preventative in
nature. Criteria which may be used for initiating the spray program are:
consistent moth flights as determined by light trap collections, and/or ap-
pearance of egg masses in the field. Application should be made on a 5-7 day
schedule and must be started before the borers have entered the plant.
Sampling and Evaluation; Corn borer infestations are self-evident since
the plants either break over or wilt and die. Only one field count will be
necessary but at least 5 plants per plot and preferably 10 or more will have
to be dissected.
Insect data may be taken as number of plants injured by corn borer or
number of borers per plant or plants.
Supporting data should consist of yields or specific gravities. Evalu-
ate as U.S. No. 1 or U.S. No. 2 grade. Yields converted to kg/ha or cwt/acre
should be taken from the whole plot or a representative portion therefrom.
Secondary effects on tubers such as stem and discoloration, reduced size,
etc., should also be recorded.
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References
Hofmaster, R. N., R. L. Waterfield, and J. C. Boyd. 1967. Insecticides ap-
plied to the soil for control of eight species of insects on Irish po-
tatoes in Virginia. J. Econ. Entomol. 60(5): 1311-1318.
Reed, E. M., and B. P. Springett. 1971. Largescale field testing of a
granulosis virus for the control of the potato moth Phthorimaea
operculella (Zell). (Lep., Gelechidae). Bull. Entomol. Res.
61: 223-233.
Shorey, H. H., A. S. Deal, R. L. Hale, and M. J. Snyder. 1967. Control of
potato toberwonns with phosphamidon in southern California. J. Econ.
Entomol. 60(3): 892-893.
Peppers, Capsicum annum
European corn borer, Ostrinia nubilalis
Corn borer is the primary lepidopterous pest of peppers. Because of
its biology on this vegetable it is extremely difficult to control with
chemicals and entomopathogens. Results with microbial agents under optimum
conditions have been favorable. Because of the mode of action of
microbials there is little point in testing against this pest and no specific
method will be reviewed. The reader is referred to the General Methods section
and Crucifers section for test design information. The following publications
are recommended.
References
Burbutis, P. P., D. J. Fieldhouse, D. F. Crossanan, R. S. VanDenburgh, and
L. P. Ditman. 1962. European corn borer, green peach aphid, and cabbage
looper control in peppers. J. Econ. Entomol. 55(3): 285-288.
Burbutis, P. 0., R. S. VanDenburgh, D. F. Bray, and L. P. Ditman. 1960.
European corn borer control in peppers. J. Econ. Entomol. 53(4):
590-592.
Hofmaster, R. N., D. F. Bray, and L. P. Ditman. 1960. Effectiveness of in-
secticides against the European corn borer and green peach aphid on
peppers. J. Econ. Entomol. 53(4): 624-626.
Snap Bean, Phaseolus vulgai"Ls; Lima Bean, Phaseolus lunatus;
Southern Pea, Vigna sinensis; Celery, Ap-ium graveolens dulce
Cabbage Looper, Tvichoplusia ni
The primary lepidopterous pest of this group of vegetables is the
cabbage looper, Trichoplusia ni. (Hubner).
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Method description and design to control this pest on these crops can
be found in the General Methods section and Crucifers section.
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Pome and Stone Fruits and Tree Nuts
The evaluation of baculoviruses and entomogenous bacteria for efficacy
and usefulness on fruit and nut crops has been rather limited. The basic
approach for conducting required field testing is described in the General
Methods section. Additional information is provided below and selected ex-
amples of methods employed by researchers for specific pests are included with
appropriate referencing.
DECIDUOUS POME AND STONE FRUITS
The most significant pest groups in deciduous tree fruits which are known
to be susceptible to control with bacterial and baculoviral insecticides are
listed below.
Chewing Insect Pests
Twig Borers
Codling Moth
Laspeyresia pomonella (Linnaeus)
Oriental Fruit Moth
Grapholifha molesta (Busck)
Fruittree Leafroller
Archips argyrosp-ilus (Walker)
Redbanded Leafroller
Argyrotaenia Delutinana (Walker)
Tufted Applfe Budmoth
Platynota idaeusal'is (Walker)
Variegated leafroller
Platynota flavedana (Clemens)
Obliquebanded leafroller
Ckoristoneura rosaceana (Harris)
Peach Twig Borer
Ansaria li-neatella (Zeller)
Oriental fruit moth
frapholitha molesta (Busck)
Wood Borers
Peach Tree Borer
Sanninoidea eccitiosa (Say)
Lesser Peach Tree Borer
Sananthedon piotipes (Grote and Robinson)
American Plum Borer
Euzophera semi-funeralis (Walker)
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Chewing Insect Pests
Plot Design: Small-scale testing of microbial agents should employ
a minimum of 4 single-tree replicates per treatment in a randomized block
design. In some situations, buffer trees to prevent drift of spray from
one treatment to another may be desirable. Tree size and planting distance
should allow each tree to be treated as a unit. Varieties chosen should
be typical of those common to the area and susceptible to injury. A stand-
ard, treatment (one which has a background of information on its performance)
and an untreated check plot should be included. The check plot is needed
to determine the magnitude of the insect infestations on which the microbial
agent is being tested.
Orchards used in large-scale tests must be representative of the area
in such matters as varieties, ages of trees,, cultural practices, and insect
populations. Plots may be replicated in one orchard or in different orchards.
The effectiveness of experimental agents can be compared to a standard in-
secticide product applied in an adjacent area of the orchard. Untreated
check plots are required for a valid comparison of treatment results.
Application Equipment: A portable, high-pressure sprayer equipped
with a pump capable of delivering 10-35 gpm (38-132 1) at 30-60 psi, a single
or multi-compartmented tank, high-pressure sprayhose and adjustable individual
spray guns are commonly used for small-scale orchard tests. Where test trees
are uniform in size, a spray-mast fitted with spray guns may be substituted
for the spray-hose and individual spray guns. The tank should be designed
for easy rinsing and if divided into compartments, pipes and valves must be
arranged to limit delivery and throw-back of spray mixture to and from the
pump from only one compartment at a time. Upon changing spray output from
one compartment to another, the new spray mixture should be directed to the
ground for 15 seconds to clear the pump, hose and gun(s) of previously used
spray mixture.
Low volume sprays may be applied using a portable airblast sprayer equipped
with a 100-gallon (378.5 liters) tank or larger, a pump capable of operating
at 200 psi or higher (lower on highly specialized equipment) at a capacity of
20 gpm (75.7 liters) or more and an air delivery equal to or greater than
20,000 cfm at a velocity of 80 mph (128.7 km) can be used for large-scale
orchard tests. (Sprayers with 2.5 times or more air delivery produce more
repeatable results. Smaller equipment may be used in some plantings.)
When changing the delivery of spray from one compartment to another in
a multi-compartmented sprayer, the sprayer should be operated in an area
away from test plots to clear previously used pesticides from pump and lines.
Timing and Frequency of Application: Timing of application can be
determined by monitoring insect activity. The use of traps (bait pan,
light traps and pheromone traps) has been successfully employed to provide
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information on the activity and abundance of some moth species including
codling moth and oriental fruit moth. Techniques for monitoring oviposition
and egg hatch have been developed for the codling moth. Computerized pro-
grams which forecast the occurrence of life stage events for codling moth
are useful for determining when applications can be made for maximum ef-
fectiveness (Pickel 1976).
Sampling and Evaluation: A primary method for determining the ef-
fectiveness of a candidate microbial agent against a chewing insect pest is
to determine fruit injury. The status of control may be estimated any time
during the season and at harvest by scoring the injury on a sample of fruit
from each replicate. Both fruit in the tree and on the ground should be
sampled. In some cases estimates of fruit load per tree or other unit area
provide useful information in determining impact of the pest and usefulness
of the microbial agent. Samples taken too long after an application may
permit other factors besides the effectiveness of a pathogen to interfere
with the results. As infestations may differ according to variety, separate
records should be maintained.
Each fruit should be examined individually for evidence of insect in-
jury. For codling moth or oriental fruit moth each fruit should be cut open
and the extent of damage recorded (deep entry - worm, or a shallow entry -
sting).
To test microbial agents against fruittree leafroller, employ a mimi-
mum of 6-tree sub-plots replicated 3 times. The sample unit should consist
of 100 fruit spurs per sub-plot (post-bloom) or 300 fruit spurs per plot.
The number of live larvae found by examining the fruit spurs should be re-
corded and the results reported as the number of larvae per 300 clusters.
At harvest the sample unit should consist of all the fruit from the 2 center
trees in each sub-plot. The number of fruit per tree and the number of fruit
per tree damaged by the fruittree leafroller should be recorded. Results
should be reported as the percent of injured fruit.
Additional information may be obtained in the case of the redbanded
leafroller, the obliquebanded leafroller, the tufted apple budmoth, and the
variegated leafroller by counting the egg masses on a predetermined number
of oviposition sites per treatment. With some of these pests, timed counts
of larval feeding sites or examinations of a predetermined number of fruit
clusters for larvae may also be of value.
A more complete evaluation of the effect of a microbial agent on insect
species infesting deciduous fruit trees can best be made by following the pest
situation during an entire season. In this way the effect of a candidate ma-
terial can be tested for effectiveness on the complex of pests in the test
area.
Sampling and counting methods in the large plots are similar to those
used for small plots. In those situations where replication is not possible,
collect data from at least 10 trees or from several bulk bins throughout the
treated area. Compare means and standard deviations to determine efficacy
of candidate materials.
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References
Asquith, D., and E. R. Krestensen. 1977. Test method for control of major
insect pests on apple and other deciduous fruit trees. Pages 62-68 in
American Institute of Biological Sciences. Analysis of Specialized
Pestieide Problems, Invertebrate Control Agents3 Efficacy Test Methods:
Vol. I Foliar Treatments I. Report to the Environmental Protection
Agency. EPA-540/10-77-001.
Bode, W. M. 1971. The codling moth, Laspeyresia pomonella (Lepidoptera:
Olethreutidae): effects of an introduced granulosis virus on a field
population and laboratory rearing on artificial diets. Ph.D. Thesis,
Ohio State Univ. 127 pp. Univ. Microfilms. Ann Arbor, MI (Intern.
Diss. Abstr. 32: 348).
Dolphin, R. E., M. L. Cleveland, and T. E. Mouzin. 1966. Field tests with
Baaillus thuringiensis (Berliner) in an apple orchard. Proa. Indiana
Aoad. Sai. : 265-269.
Falcon, L. A., W. R. Kane, and R. S. Bethell. 1968. Preliminary Evaluation
of a granulosis virus for control of the codling moth. J* Econ. Entomol.
61(5): 1208-1213.
Falcon, L. A. 1971. Microbial control as a tool in integr, ' od control pro-
grams. C. B. Huffaker, ed. Pages 346-364 in Biological Control.
Plenum Press. New York/London.
Huber, J., and E. Dickler. 1975. Codling moth granulosis virus: its effi-
ciency in the field in comparison with organophosphorus insecticides.
Z. Pflanzenkr. Pflanzenschutz. 82: 540-546.
Jacques, R. P. 1961. Control of some lepidopterous pests of apple with
commercial preparations of Bacillus thuringiensis Berliner. J. Invertebr.
Pathol. 3: 167-182.
Jacques, R. P. 1963. The influence of some fungicides on the effectiveness
of sprays of Bacillus thuringiensis Berliner . Can. J. Plant Sci.
43: 301-306.
Jacques, R. P. 1965. The effect of Bacillus thuringiensis Berliner on
the fauna of an apple orchard. Can. Entomol. 97: 795-802.
Keller, S. 1973. Microbiological control of the codling moth Laspeyresia
pomonella (Carpocapsa pomonella) with a specific granulosis virus,
Z. Agnew. Entomol. 73: 137-181.
Legner, E. F., and E. R. Oatman. 1962. Effects of Thuricide on the eye-
spotted bud moth, Spilonota ocellana. J. Econ. Entomol. 55: 677-678.
Madsen, H. F. 1969. Integrated control of the fruittree leafroller and
the white apple leafhopper in British Columbia. J. Econ. Entomol.
62(6) 1351-1353.
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-45-
Morris, W. 1972. A cooperative programme of research into the management
of pome-fruit pests in Southeastern Australia. III. Evaluation of a
nuclear granulosis virus for control of codling moth. Page 238 in
Abstracts of the 14th International Congress of Entomology. Canberra,
Australia, 22-30 August.
Oatman, E. R. 1965. The effect of Bacillus thuringiensis Berliner on some
lepidopterous larval pests, apple aphid and predators and on phytophagous
and predaceous mites on young apple trees. J. Boon, Entomol. 58(6):
1144-1147.
Oatman, E. R. 1966. Studies on integrated control of apple pests. J. Eoon.
Entomol. 59: 368-373.
Oatman, E. R., and E. F. Legner. 1964. Additional studies on the effect of
Bacillus thuringiensis on the eye-spotted bud moth, Spilonota ocellana.
J. Econ. Entomol. 57: 294.
Pickel. 1976. Computerized forecasting as an aid for timing codling moth
control. Unpublished Master of Science Thesis. University of California,
Berkeley. 37 pp.
Sheppard, R. F., and G. F. Stairs. 1976. Effects of dissemination of low
dosage levels of a granulosis virus in populations of the codling moth.
J. Econ. Entomol. 69: 583-586.
Twig Borers
The larvae of the peach twig borer burrow into tender new shoot growth
about the time the first peach leaves appear. The injury results in the
death of the terminal and is accompanied by an exudate of gum from the site
of injury. Larvae also attack the fruit, usually at the stem end where the
feeding excavations become filled with gum mixed with frass.
Oriental fruit moth eggs are laid on the underside of leaves at or near
the time peaches are in bloom. After the larvae hatch they burrow into
tender new twig growth near the base of the terminal bud. There may be
several generations a year, and when succulent twig growth is not available
the larvae may attack the fruit.
Plot Design: The plot design for twig borers is the same as that
used for chewing insects.
Sampling and Evaluation: Injury by these pests should be recorded
as the number of damaged terminals per tree (peach twig borer dormant treat-
ments or foliar sprays) or as percent injured fruit (foliar sprays only).
A minimum of 100 fruits per replicate should be selected at random for each
examination. Where evaluation is based on damaged terminals, determine
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which species caused the damage. The same techniques are used for large-
scale field tests, but a larger fruit sample should be taken.
Eeferenaes
Bobb, M. L. 1973. Insect and mite pests of apple and peach in Virginia.
Va. Polyteoh. Inst. State Univ. Ext. D-iv. Pub. 566. Blacksbiirg,
Virginia 24061.
Trunk Borers
The larvae of the peach tree borer and the American plum borer feed at
or below ground level and may girdle the trunk. This may result in the death
of young peach, nectarine or apricot trees in a single season if several
borers are feeding. The first evidence of injury is frass on the trunk
of the tree in the early fall. The following spring, an exudate of frass
mixed with gum will be evident at the base of the tree.
The lesser peach tree borer restricts its feeding to the larger scaf-
fold limbs of the tree and is inclined to inhabit large pruning wounds or
other similar suitable points of entry. Several larvae may develop at a
single site and limbs or whole trees may be killed by their feeding. Se-
cretions of gum mixed with frass at the site of injury clearly indicate the
presence of these borers.
Plot Design: Usually 6-10 trees per treatment in small-scale tests,
randomly selected, should be included in each treatment and the check.
The possibility of tree mortality due to injury by the peach tree borer may
make it impractical to establish large-scale plots for testing candidate
pathogens in orchards other than those that have been abandoned for com-
mercial ventures. The same consideration may preclude using large untreated
check plots in the experimental design. Depending on the microbial agent
used, it may be advisable to make repetitive tests for at least 2 seasons
in order to get meaningful results with peach tree borers, particularly
with small-scale tests where total numbers of insects will be very small.
Application Equipment: An adjustable handgun attached to a high-
pressure hydraulic sprayer that can deliver up to 35 gpm (132.5 1) at from
200-600 psi can be used for application. The candidate microbial agent
should be applied uniformly over the target area until it has been thoroughly
wetted. For the peach tree borer, the spray material should be directed to
the trunk of the tree. In the case of the lesser peach tree borer, it is
important that the trunk and the larger limbs be thoroughly sprayed. Accur-
ate measurement of quantity of pathogen applied per tree or per hectare
related to the diameter of the tree trunk at a predetermined height is re-
ported.
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Timing of Application: Timing of applications should be correlated
with the seasonal development of the target pest, so that the susceptible
life stage is present when treatments are applied. Male moth emergence
can be monitored with pheromone traps and other developmental information
may be obtained form close observation of caged or field populations.
Sampling and Evaluation: The appropriate pheromone may be used to
determine commencement, duration, intensity and termination of male moth
activity. Moth catches should be recorded in such a manner that the number
of trapping days included in each recording is clearly indicated. Weather
information is included whenever possible.
Evaluation of candidate microbial agents for control of boring insects
requires that a detailed examination of the trunk and larger limbs be made
in the late fall following the application of spray treatments. Data for
the treatments and control should be recorded as live larvae per tree.
Preliminary population level estimates of peach tree borer and
lesser peach tree borer on each tree may be obtained by counting fresh
frass poles in the fall (peach tree borer, American plum borer) or in the
summer (lesser peach tree borer), excavating larvae from infested trees,
and also counting (weekly) the number of cast pupal cases extending from
the bark once the moths begin to emerge. Each pupal case should be
destroyed once it has been recorded to avoid overestimating the population.
Data should be presented as cast pupal cases per tree.
If soil applications of pesticides as surface sprays are made, the
components of the vegetative cover should be noted and the pH of the soil
determined and recorded.
References
Bobb, M. L. 1943. Ethylene dichloride emulsion and paradichlorobenzene
crystals in peach tree borer control. Virginia Polytechnic Institute
Bulletin 347.
Bobb, M. L. 1973. Insects and mite pests of apple and peach in^Virginia.
Ext. Div.,, Virginia Polytechnic Institute and State University Pub. 566.
King, J. L. 1917. The lesser Peachtree borer. Ohio Agric. Exp. Sta.
Bull. 307: 399-435.
Madsen, H. F., and J. B. Bailey. 1959. Control of Sanninoides exitiosa
graefi (Hy-Edw) on apricots. J. Econ. Entomol. 52: 804-806.
Tumlinson, J. H., C. E. Yonce, R. E. Doolittle, R. R. Heath, C. R. Gentry,
and E. R. Mitchell. 1974. Sex pheromones and reproduction isolation
of the lesser peachtree borer and the peachtree borer. Sci. 185: 614-616,
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TREE NUTS
The following list presents the most significant pest groups which are
known to be susceptible to bacterial and baculoviral agents. Test methods
and supporting information for the evaluation of pathogens are described
only when they differ from the General Methods section.
Chewing Insect Pests
Codling Moth (Walnut)
Laspeyresi-a pomonella
Filbertworm (Walnut)
Melissopus lat'iferreanus
Navel Qrangeworm (Almond)
Paramyelo'is trans'itella
Hickory shuckworm (Pecan)
Laspeyresia oaryana
Twig Borers
Peach Twig Borer (Almond)
Anarsia li-neatella
Tree Borers
Peachtree Borer (Walnut)
Sann-ino'idea exitiosa
Defoliators
Redhumped caterpillar (Walnut)
Schizura conc'inna
Chewing Insect Pests Codling Moth and Filbertworm Walnut
Plot Design: A minimum of 5 single tree replicates in random dis-
tribution is recommended.
Sampling and Evaluation: The sample unit should consist of 100
nuts per replicate randomly collected from the ground at strategic times
during the season and from the tree at harvest. The nuts are cracked to
determine the number damaged by codling moth larvae and the results reported
as the percent of nuts infested by codling moth.
References
Falcon, L. A. 1970. Field studies with Baoillus thuringiensi-s for the
control of codling moth on walnuts in northern California. Progress
Report. University of California, Berkeley. 18 pp.
Madsen, H. F., L. A. Falcon, and T. T. Y. Wong. 1964. Control of the walnut
aphid and codling moth on walnuts in northern California. J. Boon.
Entomol. 57(6): 950-952.
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Michelbacher, A. E., and W. W. Middlekauff. 1949. Codling moth investi-
gations on the Payne variety of English walnut in northern California.
J. Econ. Entomol. 42(5): 736-746.
Navel Orangeworm Almond
Plot Design; A minimum plot size of 1 acre should be used.
Sampling and Evaluation; 10 trees should be selected from the center
of the plot. The sample unit for the treatment should consist of 100 nuts
taken from each of the 10 trees. The nuts in the composite sample should
be hulled and shelled and a determination be made as to whether the navel
orangeworm has attacked the nut meat. Results should be expressed as the
percent of kernels damaged by the larvae. If an untreated plot is included
in the test, the results should further be expressed in terms of the percent
reduction of damaged kernels.
References
Pinnock, D. E., and J. E. Milstead. 1972. Evaluation of Bacillus thur-
ingiensis for suppression of navel orangeworm infestation of almonds.
J. Econ. Entomol. 65: 1747-1749.
Summers, F. M., and D. W. Price. 1964. Control of navel orangeworm.
Calif. Agvic. 18(2): 14-16.
Hickory Shuckworm Pecan
Plot Design; A minimum of 8 single tree replicates in random dis-
tribution should be used.
Sampling and Evaluation: The sample unit should consist of 50 shucks
per tree sampled. A determination should be made as to whether the shuck is
infested and the results expressed as the percent of shucks infested. Ancil-
lary data should be obtained with regard to the number of nuts per pound,
based on a random sampling from the harvest of each count tree. The results
should be expressed as the average number of nuts per pound for the treatment
References
Osburn, N. R. 1954. EPN for control of the hickory shuckworm on pecan.
J. Econ. Entomol. 47(5): 931.
Payne, J. A., W. L. Tedders, and C. R. Gentry. 1971. Biology and control
of a pecan serpentine leafminer, Nept-icula juglandifoliella. J. Econ.
Entomol. 64(1): 92-93.
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Twig Borers Peach Twig Borer Almond
Plot Design; A minimum of 10 single tree replicates in random dis-
tribution should be used. The trees should be 2-5 years of age.
Sampling and Evaluation: The number of worm-damaged terminals
("strikes") found on each tree counted is recorded. If the treatments are
applied before bloom or during the petal-fall period, the counts should
not be made until the surviving overwintering generation larvae have matured.
If the treatments are directed against the next generation, counts should be
delayed until early summer. The results should be reported as the number of
"strikes" per plot of 10 trees.
On the older bearing trees the extent of damage to nuts should be de-
termined. At harvest, a 30-pound (13.5 kg) sample of nuts should be random-
ly selected from each treatment plot. The nuts should be hulled and cracked
and the number of wormy meats and the total number of nuts examined recorded.
Results should be reported as the percent of injury to nuts.
References
Bailey, S. F. 1948. The peach twig borer. Calif. Agric. Exp. Sta. Bull.
76: 56 pp.
Summers, F. M. 1951. Tests of new materials to control peach twig borer
on almonds and peaches. J. Econ. Entomol. 44(6): 935-939.
Tree Borers Peachtree Borer Walnut
Plot Design: A minimum of 5 trees per sub-plot, replicated 5 times
in random distribution, should be used.
Sampling and Evaluation: Each tree of the treatment group should be
examined during the spring period following applications made the previous
year to determine the number of live borers present. Results should be ex-
pressed as the number of live borers per tree. See also Feachtree Borer in
Deciduous Pome and Stone Fruit section.
References
Snapp, 0. I. 1962. Peach tree borer experiments in peach orchards.
J. Econ. Entomol. 55(3): 418-419.
Defoliators Redhumped Caterpillar Walnut
Plot Design; Single tree plots with up to 6 trees may be used.
However, plot design may vary according to the host tree and larval density.
Application Equipment: All formulations are applied to the point of
runoff; the volume applied per tree depends on the surface area of foliage
treated and may exceed 60 1 per tree.
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Timing of Application: The pathogen to be tested is applied when
the majority of the larval population is in the 1st to 3rd instar. In these
stages the larvae are gregarious and easily detectable, and the defoliation
sustained by the host tree is kept to a minimum.
Sampling and Evaluation: Trees are individually coded so that
populations of larvae may be monitored on a tree-by-tree basis. At each
observation time, total larval counts classified by instar are recorded
for each tree by pooling results for a],l broods found on that tree.
References
Pinnock, D. E. , J. E. Milstead, N. F. Coe, and R. J. Brand. 1974. The
effectiveness of Baci-'Llus thuri,ngiensi,s formulations for the control
of larvae of Schizwca conoi-nna on Cerc'is occidental-is trees in
California. Entomophaga 19: 221-227.
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GREENHOUSE PLANTS
Crops grown in the greenhouse are either ornamentals grown in pots
or beds, or vegetables grown hydroponically or in soil. In either case,
the crops and the greenhouse have several unique characteristics which
can greatly influence the determination of levels of efficacy and testing
procedures. Perhaps the most important of these is the extreme suscepti-
bility of greenhouse plants to phytotoxic reactions. This factor is im-
portant enough that separate tests may be necessary in which all stages
of plant growth are exposed to each formulation of microbial insecticides.
Another important characteristic of greenhouse crops which has many rami-
fications is that they generally have a much higher value than field crops,
which causes a lowering of economic injury levels. In the case of ornament-
als, the market dictates that this level is zero, that is, both a flower
and its attached foliage roust be free from damage.
High value of crop and space under glass also affects application
and testing. In maximizing the utilization of space, plants are crowded
together and aisles between beds are kept so narrow that the choice of
spray equipment is limited. Testing is affected by the low tolerance for
damage. A pathogen must be able to prevent damage, but in order to ob-
tain significant efficacy data, a high pest population must be allowed
to develop.
A third characteristic of the greenhouse is that crops are usually
in all stages of growth. This requires that workers handle the crops
continuously and thus their exposure time to the candidate material will
be very high. This may be an advantage for microbial agents.
There are other factors which can affect application and testing.
High humidity may cause a fungus problem which can require frequent appli-
cation of fungicides. Fungicides may interfere with the microbial agents
applied for insect control. Frequent overhead watering can wash off the
microbial agent. Temperatures are moderate and probably optimum for the
growth of most insect pests and microbial agents. Ultraviolet light radia-
tion may be decreased, which is beneficial to the survival of the microbial
agents.
Microbial pest control efforts in the greenhouse have been extremely
limited; Lepidoptera have been the primary targets for control using ento-
mogenous bacteria.
General Methods
Application Techniques and Equipment
Pathogens may be applied with any of the following techniques:
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High or low-volume pressure sprayer and handgun. The spray should
be put on to the point of runoff. High volume sprays may be simu-
lated by dipping plants into spray mixtures.
Aerosols. The material cannot be viscous or particulate.
Ultra low volume microdroplets. Cold fogging applicators must
be used. If highly viscous or impure preparations are used, an
air-shear, venturi nozzle would be most appropriate to avoid clogging.
Tests should be conducted to determine coverage in dense foliage (see
General Methods section).
Dusts.
Contaminated mobile pests.
Efficacy Testing
Plot Design: Individual plot size will vary depending on the type
of application, type of pest and pest population. Plots can consist of
single potted plants, an entire bed or section of the greenhouse, or an
entire greenhouse. In general, the smaller the plot size, the larger the
number of replications that will be needed. Single plants should be repli-
cated at least 10 times. When using foggers or aerosols, each greenhouse
will constitute one plot. However, it is sometimes possible to cover beds
or plots with plastic to provide an untreated check. In all tests, the
smallest practical plot size should be used. To avoid cross-contamination,
polyethylene plastic tarps should be placed between plots. Replicated un-
treated controls must be included and contaminated insects should be prevent-
ed from wandering into adjacent plots.
Sampling: Damage to the crop must be assessed, as well as pest
mortality. Pretreatment counts should be made to establish level of in-
festation. Lepidopterous pests can be sampled by removing foliage or
flowers and examining them under a dissecting microscope. This method has
the drawback of removing pests from the population before they succumb to
the pathogen. An alternative method is the time-count method. In this
method, the searcher examines as much foliage as can be sampled in a given
amount of time. 10 minutes for 10 feet of plants is the minimum amount.
Sampling Interval: Because pathogens are slow-acting, counts for
mortality should be done no sooner than 7-14 days after treatment. Patho-
gens, however, may act as repellents or feeding suppressors, and to detect
these types of activity, a count should be made within 24 hours.
Small-Scale Tests: Small-scale tests should be used wherever possi-
ble to determine the efficacy of the pathogens and any possible phytotoxic
reactions.
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Large-Scale Tests: Large-scale plots are for demonstrating the
ability of pathogens to prevent economic damage.
Specific Insects and Crops
Armyworms of several species including the fall armyworm, Pseudaleti-a
unipunctata (Haw.) and the yellow striped armywomij Prodenia ornithogalli
Guen, often infest greenhouse vegetables in the fall season. Night flying
moths of the corn earworm or tomato fruit worm enter the greenhouses in
late summer or early fall and lay their eggs on tomato foliage. The young
larvae feed at first on the foliage; later, they cut small entrance holes
in the fruits and devour the interior. A single larva may damage several
fruits. The cabbage looper, Trichoplusia ni (Hbn.) moths enter greenhouses
in the fall. This insect is probably more widespread and the most general-
ly serious caterpillar pest of greenhouse vegetables and ornamentals. If
uncontrolled, cabbage loopers continue to breed on greenhouse crops through-
out the winter. Cabbage loopers feed on foliage of tomato, cucumber, cress
and radishes and are especially damaging to lettuce where they are also
most difficult to control. Prompt and regular applications of an effective
insecticide are essential.
The early larval instars of armyworms, cabbage loopers, corn earworms
and other lepidopterous pests are more susceptible to microbial agents than
are older larvae. Early detection of infestations and prompt treatment in
greenhouse vegetable crops are important for efficient control.
As the pest species discussed above are also important pests of out-
door commercial vegetable crops in many parts of the country, data on
promising new microbial agents that results from field experiments should
give leads to materials that may be adaptable to greenhouse crops.
Plot Design: Grow lettuce, tomato, cucumber or other plants in 10-
15 cm (4-6") pots in isolation to prevent infestation by unwanted pests.
To obtain caterpillars of each species, infest groups of plants with eggs
from moths reared from local infestations or captured in black lights, or
hold eggs for hatching and transfer known numbers of larvae to plants one
day before applying the test insecticide. Use larvae of same age, stage
of development and weight.
For the initial tests, groups of 4 or more pots of plants containing
a total of 50 or more larvae in each age group are treated as a unit for
each dosage level and within the range of greenhouse temperatures required
for production of the crop involved. 3 or more replications per treat-
ment are required for analysis of results. Include untreated controls,
and if possible, treatment with a product of known performance.
Application and Equipment: Sprays should be utilized for control
of localized infestations of these pests. Knapsack sprayers operating at
2.1-4.2 kg per cm2 (30-60 psi) and delivering 187.1-935.4 1/ha (20-100 gal/
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acre) are satisfactory for preliminary tests on infested plants. For appli
cation of aerosols, individual compartments with volumes of 28.3 m3 (1000
or more may be required. For these tests, which would precede treatments
in larger greenhouses, groups of plants infested as for preliminary spray
tests could be placed throughout the compartment to provide infestations
for mortality counts. All tests should be conducted within the temperature
range required for proper growth of the plant and at a time of day or night
when host plant injury might be critical due to closing of ventilators. As
sick caterpillars usually drop from treated plants, sheets of polyethylene
or other material may be placed on the soil or mulched surface around the
plants before applying the test chemical.
Make direct counts on a representative sample of plants from each
replicate. Make insect injury ratings of 1-5 or 1-10 on host plants and
carefully describe leaf damage such as percentage of leaf area destroyed
or market acceptability for each category. Record number of injured and
uninjured fruits in treated and check plots. Record yield of treated and
untreated plots and make note of the extent of feedirig injury to market-
able parts of the plants. Record host plant injury following application
of test material, including foliage injury as chlorosis, marginal burn,
and also flower bud abscission on tomatoes and cucumbers.
References
Harris, C. R., H. J. Svec, S. A. Turnbull, and W. W. Sans. 1975.
Laboratory and field studies on the effectiveness of some insecti-
cides in controlling the armyworm. J. Eoon. Entomol. 68: 513-516.
Lindquist, R. K. 1972. Bacillus thuringiensis formulations for cabbage
looper control on greenhouse lettuce. Greenhouse Veg. Res.: Res.
Swm. 58. OARDC. Wooster, Ohio.
Smith, F. F. 1959. Control of insects of greenhouse vegetables. USDA
Agric. Handbook 142. 25 pp.
Tomato Pinworm
The tomato pinworm, Keiferia lycopevsioe'lla (Busck) , has a long history
of sporadically infesting greenhouse tomatoes in northern states and is
a regular pest on greenhouse tomatoes as well as field-grown tomatoes in
warmer parts of the United States. The tomato pinworm does not survive
out of doors in northern states but infests field-grown tomatoes near green-
houses where infestations persist from year to year. It is transported
to new areas on infested tomato plants or in fruits or in used containers.
Larvae make blotch mines in leaves, feed in growing tips and flower
buds, and enter the fruit through pinholes under the calyx of ripening
fruit. Effective control efforts should be directed toward the insect
before it invades the fruit where it is difficult to control with a microbial
agent but causes the greatest damage to the tomato crop.
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Crop and Location; Select a variety of tomato commonly grown in
commercial greenhouses. Plants should be grown in containers and under
isolation to prevent unwanted infestation with pinworms and other insects.
Less desirable for preliminary tests is the selection of plots in infested
greenhouses where active flying adults can reinfest treated as well as
untreated plants.
Plot Design: Groups of plants infested with 50-100 or more insects
for each treatment should be replicated 3 or more times. Each series of
tests should include comparable groups of plants that receive (a) a known
effective treatment as standard, and (b) no treatment. Treated and un-
treated groups of plants should be isolated to prevent posttreatment re-
infestation.
Application and Equipment: Expose plants for 1-2 days to egg-laying
adults in a cage or greenhouse compartment that contains infested plants.
Application of the test materials should be made to groups of plants prior
to egg hatching to determine their potential for destroying larvae before
they penetrate the leaves or fruit.
To test the effect of materials against larvae within leaf mines, make
application to plants when the larvae are in their early instars and the
mines are small, and against more mature larvae when mines are larger.
After the effectiveness of the candidate microbial agent has been
determined as above, a series of treatments at timed intervals can be
made to plots or preferably to entire sections of commercial greenhouses.
Sampling: For determining effectiveness against hatching larvae,
count and examine the leaf mines, as they will indicate larvae that sur-
vived the treatment. Make direct counts of older larvae in mines or adults
in cages at 7 and 14 days after treatment.
In tests conducted on a growing crop in a commercial situation where
2-8 or more applications per week were made, record the number of dead
larvae in mines, the number of mines per leaf on treated plants, and the
number of fruits with pinworm injuries.
References
Anderson, L. D., and H. G. Walker. 1944. Tomato pinworm control in the
greenhouse. J. Eaon. Entomol. 37: 264-268.
Lindquist, R. K. 1975. Insecticides and insecticide combinations for
control of tomato pinworm larvae on greenhouse tomatoes: A progress
report. Greenhouse Veg, Res.: Res. Summ. 82. OARDC. Wooster, Ohio.
Neiswander, R. B. 1950. The tomato pinworm. Ohio Agrio. Exp. Sta. Res. Bull.
702.
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Thomas, C. A. 1932. The tomato pinworm Cnorimoschema ly coper si, oella
(Busck), a new pest in Pennsylvania. J. Eoon. Entomol. 25: 137-138.
Thomas, C. A. 1936. Status of the tomato pinworm Cnorimoschema
lycopersicella (Busck) in Pennsylvania. J. Econ. Entomol. 29: 313-317.
Cutworm
Several species of cutworms including the black cutworm, Agrotis
ipsilon (Hufnagel) ; varietaged cutworm, Peridroma sauc-La (Hubner) ; and
dingy cutworm, Felt-la subgoth-ioa (Haw.) attack lettuce, cucumbers, tomatoes
and cress, especially in the seedling stage. The variegated cutworm and
others known as climbing cutworms also climb older plants and feed on
leaves, buds and fruit. All hide in the soil or mulch during the day and
feed at night. In the greenhouse, adults and young larvae have been
controlled by aerosols containing parathion or malathion. Baits containing
bran, molasses, sometimes other ingredients, and a toxicant are effective
against older cutworm larvae.
Plot Design: For tests in greenhouses with cutworm infestations
on lettuce, tomato, cucumber or cress, select plots of adequate size to
reduce influence of larval dispersal from contiguous plots. If facilities
are available, cutworm larvae may be reared and released in plots 1 or 2
days before treatment.
Application and Equipment: Bran baits prepared with or without
molasses and with the test compound as toxicant may be used. These are
broadcast late in the afternoon at a rate of 11.2-22.4 kg per ha (10-20
Ib/acre). It should not be scattered on the plants, but directed to the
ground.
Sampling: Make direct counts of dead larvae found in soil depressions
around the base of injured plants. Make counts of cutworm-injured plants
at 1 and 7 days posttreatment.
References
Metcalf, C. L., W. P. Flint, and R. L. Metcalf. 1962. Destructive and
useful insects. McGraw-Hill Book Co., New York. 1083 pp.
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FORAGE CROPS
Forage crops and principally alfalfa and clovers are attacked by
a wide variety of insects, but only a few are known to be susceptible
to infection by entomogenous bacteria or baculoviruses. These are all
foliage feeders and are similar in enough respects that general method-
ologies for determination of microbial agent efficacy may be applied to
all with specific details pertinent to each being covered under a dis-
cussion of the individual pests. In addition to the specific test methods
listed below, the reader is referred to the introductory section of this
document for general considerations applicable to the efficacy testing
of all baculoviruses and entomogenous bacteria.
Crop Variety and Location of Tests; The variety tested should be
one recommended for cultivation in the test region. It should be suscept-
ible to damage by the insects to be controlled. If mixtures of legumes
are planted, a common agronomic practice, the percent stand composition
of each species should be determined. Plots should be located in areas
which have a history of past infestations of the desired pest.
Experimental Design: A randomized complete block design is appro-
priate for determination of efficacy of microbials on lepidopterous defolia-
tors of alfalfa, clovers, and other forage crops. Plot size for most work
with viruses and Eaoi-ltus thuring'L&ns'is has been relatively large, i.e.,
6-9 m wide by 45-120 m long for ground application plots and three swath
widths by 300-450 m long for aerially applied formulations.
Most caterpillars do not move laterally to significant extents unless popu-
lations are so high that complete defoliation occurs. Thus the plot sizes
above should be adequate from this standpoint if sampling is confined to
their central portions.
Application and Equipment; Entomogenous bacterial formulations and
baculoviruses can be applied in initial, small plot tests by compressed air
sprayers or low pressure ground apparatus. Aerial applications can be ap-
plied with standard fixed wing aircraft or by helicopter. Volumes of final
formulations applied have been in the range of 15-95 1/ha for nuclear poly-
hedrosis virus and Bao-illus thwc'ing'iens-Ls applied by ground equipment and
48-95 1/ha when applied aerially.
Sampling: Populations of Lepidoptera on alfalfa and clover are normal-
ly sampled with the standard 15-inch sweep net. One sweep is considered to
be a 180° arc made horizontally over the crop. The number of sweeps should
be adjusted to the population levels so numbers of insects obtained are valid
for statistical analysis.
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Samples should be taken immediately prior to spray applications and
again at 1, 3 and 7-day intervals post-application. The residual effects
of viruses may require that additional samples be made on the same plots
for several host generations. Such samples should be compared to adjacent
untreated plots and larvae collected should be examined to determine the
presence of virus in the tissues.
For certain insect-microbial interactions, mortality may not provide
an accurate assessment of efficacy. In such cases, yield of hay per unit
area may be a more valid measure of efficacy.
Analysis and Reporting of Data: Both insect counts and yield data
should be subjected to analysis of variance tests and treatment means com-
pared by multiple range tests. In addition, the results of laboratory tests
on percent incidence of the pathogenic agents in field-collected larvae,
changes in insect counts over time, plant height and amount of defoliation
at time of treatment, and standard weather parameters should be reported.
Alfalfa Caterpillar
The alfalfa caterpillar, Colias eurytheme Boisduval, is principally
a pest of alfalfa in southwestern United States and the alfalfa growing
regions along the Pacific slope. It only occasionally damages clover and
other legumes. In the southwest, it may have from 5-7 generations per
year. It is highly susceptible to Baoillus thuvingiensis infection and
is also susceptible to a nuclear polyhedrosis virus. Methods for determin-
ing efficacy of these agents conform to those described above.
References
Hall, I. M., and V. M. Stern. 1962. Comparison of Bacillus thuvingiensis
Berliner var. thuringiensis and chemical insecticides for control
of the alfalfa caterpillar. J. Econ. Entomol. 55(6): 862-865.
Steinhaus, E. A., and C. G. Thompson. 1949. Preliminary field tests
using a polyhedrosis virus to control the alfalfa caterpillar.
J. Econ. Entomol. 42(2): 301-305.
Stern, V. M., I. M. Hall, and G. D. Peterson. 1959. The utilization of
Baoillus thuringiensis Berliner as a biotic insecticide to suppress
the alfalfa caterpillar. J. Invertebr. Pathol. 1: 142-151.
Stern, V. M., V. Sevacherian, A. Mueller, and J. Ryan. 1968. Effect
of naled, trichlorfon, and Bacillus thuringiensis on three species
of lepidopterous larvae attacking alfalfa in California. J. Econ.
Entomol. 61(5): 1324-1327-
Thompson, C. G. 1951. Field tests during 1950 using a polyhedrosis virus
to control the alfalfa caterpillar. J. Econ. Entomol. 44(2): 255-256.
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Thompson, C. G., and E. A. Steinhaus. 1950. Further tests using a poly-
hedrosis virus to control the alfalfa caterpillar. Hilgavdia 19(14):
411-445.
Green Cloverworm
The green cloverworm, Plafhypena scdbva (Fabricius) is an occasional
pest of alfalfa and clover although it is almost always present on these
crops. It frequently causes economic damage to other crops such as soy-
beans and other legumes and is most serious in the southeastern states.
It is highly susceptible to Bacillus thuringiensis and is also infected by
a granulosis virus. Methods for determining efficacy for these agents of
forage crops conform to those described for forage defoliators above.
Alfalfa Webworm and Garden Webworm
The alfalfa webworm, Loxostege commixtalis (Walker), and the garden
webworm, L. similalis (Guenee), can severely damage alfalfa, and they
occasionally reach pest status on clover. Both form webs over leaves and
consume these leaves under cover of the webs. Found throughout the United
States, they may have from 2-5 generations per year depending on the latitude.
Both insects are susceptible to Bacillus thuringiensis and the methods for
determining efficacy as described for lepidopterous forage pests in general
are also applicable here.
References
Stern, V. M., V. Sevacherian, A. Mueller, and J. Ryan. 1968. Effect
of naled, trichlorfon, and Bacillus thuringiensis on three species
of lepidopterous larvae attacking alfalfa in California. J. Econ.
Entomol. 61(5): 1324-1327.
Introduced European Skipper
A pest of southern Canada as well as northcentral and northeastern
United States, the introduced European skipper, Thymelicus lineola
(Lepidoptera:Hesperiidae) damages many hay and pasture crops, principally
grasses. Larvae feed on leaves of these crops, severely reducing yields.
The methods utilized for determination of efficacy are essentially the same
as described for forage crops in general. It has been shown to be highly
susceptible to Bacillus thwcingiensis under field conditions.
References
Arthur, A. P. 1968. Further information on the control of the introduced
European skipper, Thymelicus lineola, with Bacillus thuringiensis.
J. Invertebr. Partial. 10: 146-150.
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Arthur, A. P., and T. A. Angus. 1965. Control of a field population of
the introduced European skipper, Thymeli-ous li-neola (Ochsenheimer)
(Lepidoptera:Hesperiidae) with ~BaQ-i11us fhu^Lngi, ens-is Berliner.
J. Invertebr. Pathol. 7(2): 180-183.
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P.ANGELAND
A discussion of the problems associated with control of pests on
rangeland has been prepared and published in a previous report (AIBS. 1977.
Analysis of Specialized Pesticide Problems, Invertebrate Control Agents
Efficacy Test Methods: Volume II Foliar Treatments II. Report to the
Environmental Protection Agency EPA 540/10-77-009.) An excellent description
of the rangeland caterpillar, a pest of rangeland grasses which is suscepti-
ble to both bacterial and baculovirus infections, is also presented in that
document. Portions of the discussion presented there are repeated here where
appropriate. Additional discussion is included only where necessary to
modify that text for the demonstration of efficacy of Bacillus thuringiensis
or baculoviruses. The reader is referred to the introductory section of
this document for general considerations applicable to the efficacy testing of
all baculoviruses and entomogenous bacteria.
Rangeland pest control presents unique problems because of the vast
acreages usually involved and the relatively unfavorable cost-benefit ratios
involved in making insect control applications to these crops. Nevertheless,
defoliation can at times be so severe that control measures are a necessity
if economically feasible methods are available.
The above-mentioned factors, in addition to the rough terrain, usually
necessitate aerial application of insecticides in almost all instances.
Many of the species of insects which damage rangeland forage are quite mobile
and this factor, coupled with the variability in population densities and
the necessity of evaluating aerial application, has led to the use of large
(20-259 ha) plots for efficacy testing. Plot size often must be reduced in
mountain or forest rangeland.
Careful attention should be given to the overall efficacy evaluation
program to ensure that the tests in different areas are a part of the same
experimental design so that the number of replicates at a given site can be
reduced. Sub-plots are often selected at random within the large plots to
improve efficiency. Care should be given to selecting plot locations which
will not involve spray applications over ponds or water courses unless it is
the intention of the researcher to monitor for pesticides in those areas.
Minimum plot size for aerial application will usually be three swaths
wide by long enough to assume sustained level flight of the aircraft. Plots
must be long enough to allow for variation in initiation of spray at each
end. Plot size may be reduced by the use of smaller, slower aircraft.
Width of plots or separation between plots must be adequate to prevent drift
onto adjacent plots. Preliminary "screening" can be accomplished by low
pressure - low volume sprayers. Minimum plot size is dictated by the mobility
of the insect species involved. Border treatment with pesticides labeled
for the particular use can be used to further reduce plot size.
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Microbial agents should be applied with carefully calibrated equip-
ment under acceptable weather conditions. To make certain of this, more
than one day may be required for aerial application of several treatments.
Grasshoppers
Grasshoppers are not known to be susceptible to baculoviruses and
show little response to Bacillus fhuyingi-ensis. Therefore, no detailed
descriptions of techniques for testing these agents against grasshoppers
will be presented here. Information on field testing of chemical insecti-
cides is contained in the previously mentioned Report to the Environmental
Protection Agency 540/10-77-009. Many considerations presented in that source
would be applicable to field testing of baculoviruses and entomogenous bac-
teria if and when candidate pathogens are discovered.
Range Caterpillar
The range caterpillar, HemLleuea oli-viae Cockerell, feeds primarily
on range grasses in areas in northeastern and south central New Mexico
at elevations between 1734-2438 meters (4,700-8000 feet). The infestation
has extended into southeastern Colorado and the western edge of the Texas
Panhandle. Range caterpillars consume large amounts of foliage, waste
additional unconsumed parts of leaves and cause other foliage to be ungrazed
because of the presence of irritating spines on the active larvae and cast
skins (Hewitt et al. 1974). Heavy populations may destroy all grass down
to the crown, producing conditions conducive to wind and water erosion.
Crop and Location of Tests: Species composition of the vegetation
and the terrain are usually dictated by the location of economic infestations
of the pest. Care should be given to uniformity of vegetation and terrain
among treatments. Proximity to watering sites will affect forage utilization
and distribution of livestock.
Experimental Design; Since migration is limited in the early instars,
plot size can be considerably smaller than is required when the caterpillars
are large. Migration by large caterpillars is increased as the density of the
population and percentage of standing crop foliage consumed increases.
For small worms, plots should be at least three swaths wide by 402.2 m
(h mile) in length. For larger worms, the plot width should be at least
doubled. To reduce drift, plots should be separated by an adequate distance
which will depend on wind velocity and direction.
Plots as small as 2.43 hectares (6 acres) have been used for efficacy
tests by airplane against first and second instars.
Watts, et al. (unpublished data) has experimented with circular arenas
encompassing approximately 4.18 m^ (5 yard^) to confine known numbers of
small caterpillars within the test plots on an experimental basis to reduce
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variation in density. These arenas were made of 6-inch strips of tin
forced ca 2.54 cm (1 inch) into the soil. Larger plots, from 20.25-259.2
ha (50-640 acres) have been used on other studies in New Mexico. Coppeck
(unpublished data) conducted preliminary screening test with a compressed
air hand sprayer on small plots.
Application and Equipment: Aerial application equipment that is de-
signed for the aircraft being used should be properly calibrated. Aircraft
designation; boom size and length; number, size of nozzle, or atomizers and
position on aircraft; pressure; aircraft speed; altitude and swath width;
type of solvent; concentration of solution and quantity of solution used
should be reported. Dye cards for evaluation of deposit uniformity are re-
commended. Flight runs made crosswind usually increase the uniformity of
deposits. Applications should be made under conditions that avoid exces-
sive convection currents.
Sampling: Population densities are usually evaluated by counting the
number of caterpillars in square yard sampling areas located well within the
plot. Various sampling schemes, designed to remove bias and assure coverage
of an adequate area, have been used. The sampling scheme used should assure
that an entire swath width and preferably more be included in the area to
be sampled. A minimum of 10.84 m^ (1 yard^) samples or more, until at least
50 worms are counted, is needed per plot. Additional samples will increase
precision. When 5, 4.18 m2 (5 yard^) arenas are used, 100 caterpillars per
arena should be used.
Because of the habits of the range caterpillar, the accuracy of visual
counts is increased by delaying initiation of counting until mid-morning
when ground temperatures have increased enough to initiate activity in the
caterpillars.
Counts should be taken immediately prior to treatment and at intervals
estimated to embrace partial and maximum kill.
Analysis and Reporting of Data; Where possible, treatment means
should be compared using a valid statistical test for significance. The
standard material used in private-state-federal control programs in the geo-
graphic area should be included. Replicated untreated plots are also recom-
mended .
References
Hewitt, B. G., E. W. Huddleston, R. J. Lavigne, D. N. Veckert, and J. G.
Watts. 1974. Rangeland entomology. Range Soi. Ser. 2, 127 pp.
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LAWNS3 TURFGRASSES, PASTURES
Lawns and turfgrasses are generally considered high value crops or
commodities, principally because of their aesthetic values. Determination
of efficacy of microbial agents for control of insect pests of these com-
modities may,, therefore, be conducted on a labor and cost intensive basis
because while control costs are usually high on a per acre basis, they
are borne by the owners because of the aesthetic values and because total
land areas treated are normally small. These commodities may often sustain
considerable levels of many pest species before noticeable damage or damage
requiring treatment occurs.
Pastures, on the other hand, have a much lower monetary value as a
commodity and correspondingly lower amounts of money may be spent for con-
trol of pests on them. Economic threshold levels on pasture grasses, how-
ever, may be higher than on lawns because yield losses may occur even
though unsightly or aesthetically unpleasing damage is not occurring.
While the above differences may dictate different management decisions
for pest control, methodologies for determining efficacy of microbials on
small plots will be essentially the same. Insect control on lawns, turf
and pastures will normally be accomplished with ground equipment. In ad-
dition to specific test methods listed below, the reader is referred to the
introductory section of this document for general considerations applicable
to the efficacy testing of baculoviruses and entomogenous bacteria.
Small-Scale Field Tests
Site Selection: The site or sites selected for tests should be of a
uniform host plant or of a uniform mixture of host plants. If damage has
already occurred, it should be of uniform severity and the pest population
should be of uniform density over the entire field plot layout. If such
conditions cannot be met, treatments should be limited so that any compari-
sons are valid and meaningful.
Normally, established lawns, golf fairways and greens, pastures and
any other turf will be infested and will provide conditions wherein typical
grass varieties for a given locality will be planted. Pest populations
should be increasing at the time the tests are conducted. If this is not
the case, a measure of population quality should be given. Pretreatment
population counts taken just prior to treatment should be made.
If standard management practices are to be employed on the treatment
area (e.g., application of fertilizers, growth regulators, fungicides, etc.)
they should be applied uniformly over the entire test site. In the case
of pastures, some provision may be needed to exclude livestock from grazing
on treated plots following the tests if test materials do not have an exemp-
tion from tolerance or if season-long or multiseasonal effects are to be
monitored.
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Plot Size and Design: Most turf grass, lawn and pasture pests
which are susceptible to Bacillus sp. and baculoviruses are not highly
motile in their damaging stages. Thus relatively small plots (e.g., 1.5 x
3 to 3 x 3 m) will normally provide satisfactory data. Unusually heavy
populations of certain pests may necessitate larger plots to overcome im-
migration effects whereby the edges of the treated plots would serve as'
buffer zones with data being taken from the central portions of the plot.
Normally a minimum of 4 replicates should be included for each treatment.
This number might need to be increased for pests which show discontinuous
or contagious distribution patterns.
Application and Equipment: Formulations should be applied when the
pest is present in sufficient numbers to provide meaningful population re-
duction and damage estimates when the treatments are evaluated at a later
date or dates. Applications should be made with equipment that distributes
the formulations in the same manner as would be accomplished using standard
application equipment for that area. Every care should be taken to accu-
rately calibrate the equipment and distribution procedure. The latter may
simply involve uniform application of pre-measured amounts of material to
each plot as an alternative to calibration. Materials should be applied
in their commercial formulations where possible.
Dosage Selection, Treated Check, Untreated Check; Dosage is normally
dictated by the manufacturer, but where possible, it is desirable to include
treatments of 3gx and 2x the recommended rates. Where no previous data is
available, these intervals should be increased or a wider range of treat-
ments should be tested, especially in the first year of tests against a
given host.
If the goal of treatment with the microbial insecticide is rapid con-
trol, efficacy should be compared to a standard insecticide recommended for
use in the test area. Similarly, untreated check plots are an absolute
necessity for evaluating the influence of natural infections on the pest
population during the relatively long period of time required for evalua-
tion of efficacy of these products. If season-long, multiseasonal or
permanent establishment (colonization) of the pathogen is sought, standard
insecticidal check plots and untreated check plots should also be included
as a basis for comparison of costs of treatments as well as providing an
estimate of the pest potential in the absence of treatment.
Number of Trials: The number of trials cannot be rigidly established.
Sufficient numbers should be included to permit accumulation of data on:
1. Efficacy over the range of the pest for which registration is sought
2. Persistence or self-perpetuation in the environment.
3. Compatibility with all application systems with which it would
be applied.
4. Effects of weather factors on efficacy.
5. Proper timing with respect to:
stage of insect host
stage of host plant
need (under the concept of pest management programs).
6. Effects on non-target insects.
7- Activity in the target insect.
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Statistical Analysis; The experimental design utilized should be
such that a measure in variation of response by the host insect is obtained.
At the very least this should be a measure of the standard deviation in the
host-pathogen treatment which can be compared to the effects in the untreated
host population. When several rates, formulations and products are tested,
an analysis of variance with a-multiple range test applied to treatment means
should be performed.
Sampling Methods: Sampling methods vary from crop to crop and in-
sect to insect and will be described for each host plant-pest insect inter-
action discussed.
Large-Scale Field Tests
For the commodities listed in this section, large-scale tests may be of
little value. Most ownerships will involve relatively small acreages with
area-limited infestation. Therefore, ground applications using techniques
applied to small plots should be applicable to most pest problems.
Webworms
Sod webworms (LepidopterarPyralidae) are pests of lawns and grass in
most areas of the United States. Several genera and species are included
under the general term sod webworm. The most common pest species are members
of the genera Herpetogpcorma and Crambus. Sod webworms nip and clip grass
blades and pull them into silken tunnels which they construct near the
ground line. Under certain weather conditions this damage is noticed as small,
circular brown areas over a lawn or in turf. If the infestation is severe
enough, such areas may be so numerous as to run together causing large
patches of dead grass (Scheibner 1972). Many natural control agents, cli-
matic factors, and cultural methods reduce severity of sod webworm damage.
Insecticidal control programs should be implemented to complement action
by these other factors in cases where their activity is insufficient to re-
duce damage below acceptable levels.
Crop Variety and Location of Tests: The crop variety will vary con-
siderably in tests on sod webworm since different grasses are utilized as
lawns throughout the United States and many are attacked. Thus the variety
of crop will normally be that commonly grown in a particular region. In
any event, the species of grass or grasses being treated should be clearly
identified. Plots are normally located in areas where infestations occur
naturally because of the difficulty of establishing and maintaining turf for
this specific purpose. Infestation areas should be large enough to allow
sufficient replications of all treatments to be established over uniformly
infested turf. Populations have been encouraged in some tests by application
of fertilizer to maintain a vigorous stand of grass (Reinert 1976).
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Experimental Design: Sod webworm control tests normally should be
established in randomized complete block designs with three or four blocks.
Within block variation was reduced in one test (Reinert 1973) by establishing
each block in areas of uniform infestation where between block infestation
differences were present. This would be acceptable if no uniform test areas
are available.
Relatively small plots of 4-5 m^ may be utilized effectively
for determination of efficacy of Baoi-llus thwc'ing'iensis because of the low
mobility of these insects under normal infestation levels and because treat-
ment of such plots can closely simulate techniques utilized on a commercial
or homeowner scale. These plots can be relatively close together with only
a small (0.5 m) buffer strip between plots to reduce possible overlapping
of spray applications.
Application and Equipment: Bacillus tkuvingi ens-is can be applied
uniformly to small plots with a compressed air sprayer. Large volumes of
water 1077 1/ha (436 gal/acre) have been used (Reinert 1976) on such small
plots. The use of sticking agents may be desirable. Granular formulations
can be dispersed by hand shakers and watered into the turf.
Sampling: Sod webworm sampling is effected by use of pyrethrins
applied to uniform areas within the plots. Normally 0.02-0.13% solutions
are applied with a sprinkling can at a rate of approximately 0.95 1/0.36 m2.
Larvae are counted as they come to the surface following treatment and counts
are made over a standard time period, usually 10 minutes. Counts should be
continued at weekly intervals, and care should be taken to sample different
areas within each plot at each sampling date.
Larval counts, if used as the only measure of effectiveness of Baci-llus
thuringiensis, may give misleading estimates of product efficacy. Many sod
webworms may ingest B. thuringiensis and not be killed immediately if at
all, yet feeding activity may be stopped or severely reduced. It is thus
important to monitor stand condjt^on, monitoring the rate of continued brown-
ing or of "greening up" in both-S. thur-ingiensis-treated plots, untreated
control plots and plots treated with standard chemical insecticides. Nu-
merical ratings which can be analyzed statistically should be taken for all
tests of B. thuringiensis efficacy. Because of this mode of action, evalua-
tion of B. thuringiensis should be continued at least as long as the genera-
tion treated is still present in the field and new damage from progeny of
immigrating moths does not interfere with assessment of residual effects on
the population originally present.
Analysis and Reporting of Data: In addition to data listed in Re--
porting Microbial Agent Test Report, the following should be reported:
» Larval counts per unit made immediately prior to spraying and
at weekly intervals following treatment,
Stand condition or damage rating at each sampling date.
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o Temperature, weather conditions and rainfall throughout the test.
o Larval counts and mean damage ratings for each treatment should
be compared by use of a multiple range test.
References
Heinrichs, E. A. 1973. Bionomics and control of sod webworms. Bull.
Entomol. Soo. Amer. 19(2): 94-95.
Jefferson, R. N., I. M. Hall, and F. S. Morishita. 1964. Control of
lawn moths in southern California. J. Eoon. Entomol. 57(1): 150-152.
Reinert, J. A. 1973. Sod webwonn control in Florida turfgrass. Fla.
Entomol. 56(4): 333-337.
Reinert, J. A. 1974. Tropical sod webwonn and southern chinch bug control
in Florida. Fla. Entomol. 57(3): 275-279.
Reinert, J. A. 1976. Control of sod webworms, (Herpetogrcmma spp. and
Crambus spp.) on Bermudagrass. J. Econ. Entomol. 69(5): 669-672.
Scheibner, R. A. 1972. Controlling sod webworms. Ky. Agric. Exp. Sta. ,
Dept. of Entomology, ENT-7, 4 pp.
White Grubs
Certain white grubs, the larval stages of scarabaeid beetles, feed on
the roots of turfgrasses, often causing severe damage to them. White grubs
are found as turf pests throughout the entire United States and include
several different genera and many different species. Suppression of many
of these pests may be Drought about through the use of Baci-llus popill-lae,
B. lentimorbus or various strains of each (Tashiro 1973). Use of this
method has the advantage of permanence in that once introduced into the soil,
the bacterial spores remain inactive but viable for years until ingested by
grubs. These become infected and produce more spores to add to the soil
inoculum. Under ideal conditions, an equilibrium between pest and pathogen
is established in which the numbers of grubs present is maintained near the
economic threshold which Tashiro (1973) states as being between 1-5 grubs
per square foot, depending on the locality in the United States.
Crop and Location of Tests; Most, if not all grasses are subject to
damage by white grubs. The species of grasses tested should be dictated
by the grass species being damaged by grubs in a given locality.
Location of tests is most important, since evaluation may be desirable
over a period of years. Plots should thus be placed in areas where land
uses are to remain constant for at least 3-5 years. It is also desirable
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to establish plots in a variety of localities so that as many environmental
factors as possible, i.e., soil type, soil pH, soil temperatures, etc., may
be examined as to their influence on the host-pathogen interaction. The
sites selected should be protected from other insecticide applications
throughout the test period.
Plot Size and Design: Plots of ca. 4 x 4 to 6 x 6 m have been utilized
successfully for determining efficacy of various strains of Bacillus popilliae
and B. lentimorbus. 3 or 4 replications of each treatment should be established
and standard recommended chemical control plots as well as untreated check
plots should be included. Plots should be well-separated if possible since
infected beetles are known to move and spread the inoculum laterally at rates
of over 1.5 m in 3 years and have in fact been shown to move at rates of
1 foot/day under extreme experimental conditions. If the demonstration of
efficacy for one season only is desired, this factor is of less importance
and plots may be placed closer together.
Application and Equipment: Application is achieved simply by placing
measured quantities of spore-carrier mixtures at uniformly spaced points over
the surface of the plots. One manufacturer recommends that these points be
on 4-foot centers. It is important to thus provide centers of heavy concen-
trations of spores so that grubs feeding in those areas will consume lethal
quantities of spores and later die. Their cadavers then release spores and
form new epicenters away from the original release site. Natural soil mix-
ing by both grubs and other soil invertebrates tends to distribute spores
even further until they are distributed throughout the entire plot at infec-
tious levels.
The rate of spread and thus the rate of reduction of grubs is dependent
upon the initial dosages applied and the grub population density. Since
complete coverage of a plot with high rates would be economically unfeasible,
although rapid results might be obtained, it is suggested that application
using field control methods be followed in field test plots for determining
efficacy.
Sampling: White grub counts are made from 0.1 m2 soil
samples randomly located within plots and excavated to a depth appropriate
for beetle activity under the conditions at the time of sampling. Grubs
move vertically within the soil as moisture and temperature conditions change.
Notes on stand condition should accompany larval counts. Counts should be
made every 2 weeks with sufficient samples being taken to provide statisti-
cally analyzable data. Larvae collected should be returned to the laboratory
for microscopic examination of hemolymph to confirm infection. Grubs
should be counted by developmental stages as well as in total because of
the correlation between species, time of year, susceptibility and lethality
as discussed by Tashiro (1969).
Analysis and Reporting of Data: The data should be analyzed through
multiple range tests for comparisons of means if possible. All environmental
conditions should be accurately and faithfully recorded as stated above.
Weather conditions during the entire test period should be recorded and
summarized for possible correlation with efficacy data.
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RefeTenoes
Beard, R. L. 1945. Studies of the milky disease of Japanese beetle latvae.
Conn. Agr"ic. Exp. Sta. Bull. 491: 505-582.
Dutky; S. R. 1941. Testing the possible value of milky diseases for con-
trol of soil-inhabiting larvae. J. Econ. Entomol. 34: 217-218.
Fleming, W. E. 1968. Biological control of the Japanese beetle. USDA
Tech. Bull. 1383, 78 pp.
Fleming, W. E. 1976. Integrating control of the Japanese beetle a
historical review. USDA Tech. Bull. 1545, 64 pp.
Hurpin, B., and P. H. Robert. 1972. Comparison of the activity of certain
pathogens of the cockchafer Melolontha melolontha in plots of natural
meadowland. J. Invertebr. Pathol. 19: 291-298.
Tashiro, J. 1957. Susceptibility of European chafer and Japanese beetle
larvae to different strains of milky disease organisms. J. Econ.
Entomol. 50(3): 350-352.
Tashiro, H. 1973. Bionomics and control of root feeding insect pests:
grubs and billbugs. Bull. Entomol. Soo. Amer. 19(2): 92-94.
Tashiro, H., G. G. Gyrisco, F. L. Gambrell, B. J. Fiori, and H. Breitfield.
1969. Biology of the European chafer Amphimallon majalls (Coleoptera
Scarabaeidae) in northeastern United States. N. Y. St. Agric. Exp.
Sta. Geneva Bull. 828, 71 pp.
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FOREST3 SHADE AND ORNAMENTAL TREES
The lepidopterous defoliators and sawflies are among the most serious
pests of forests and much of our knowledge of insect population dynamics
is based on investigations of these species. Quantitative studies in the
field and laboratory have shown that viruses do cause epizootic diseases
in a number of these species. Among those important forest pests known
to be infected by baculoviruses are the gypsy moth, Lymantria dispar;
spruce budworm, ChoY"istoneia>a fum-iferana; Douglas fir tussock moth,
Orgyia pseudotsugata; tent caterpillars, Malaaosoma spp.; and the sawflies
CStairs 1972).
The use of Bacillus thuringiensis against forest pests has been ade-
quately reviewed by Harper (1974). This review covers the above-mentioned
pest species in addition to the spring and fall cankerworms and others.
Efficacy in pesticide usage can be measured in two general categories,
crop protection and pest population reduction. These categories are gener-
ally used in reference to the same treatment year. Usually little or no
inference is made to longer-term effects. Crop protection concepts as re-
lated to forest insect control generally means foliage protection. In some
cases, there are several levels of foliage protection that could be accept-
able and these depend upon the management objectives. Thus, in determining
efficacy the amount of permissible defoliation should be given, and its
basis related to a specific management objective.
When pest reduction is used in the evaluation it is usually expressed
in percent mortality. In the case of forest pests the residual population
levels might be more meaningful and possibly the quality of the population.
The "desired effects" might depend on the users' or managers' preferred
needs. As an example, regulatory officials might desire virtual elimina-
tion of the pest, but those in recreational areas would not need the popu-
lation level reduced so severely. Consequently, there can be several
levels of efficacy depending upon particular user's needs.
The following discussion considers some general guidelines for determining
efficacy of a microbial material applied to a forest pest species.
Plot Size; Usually plot size is determined by whether the test is
classified as an experimental or a pilot study. With the experimental test
the microbial material may be applied by either ground or air equipment.
If applied by ground, the minimum size should be no less than 0.041 ha, and
when applied by helicopter or fixed-wing the minimum plot sizes 2.02 and
10.1 ha, respectively, are desirable. Under the operational conditions of
the pilot tests the objectives are to establish experimental use and to
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determine operational feasibility. Using ground and aerial equipment,
4.04 and 4.4 ha, respectively, are preferred for the pilot test. Usual-
ly a minimum of 3-4 replications are considered for each treatment in the
experimental test. However, in pilot testing a minimum number of repli-
cations for each treatment should be established to yield statistically
significant results, and if possible, such tests should generally be con-
ducted in more than one geographical area.
Sampling and Evaluation; In evaluating the efficacy of microbial
insecticides applied to forests, general foliage protection and popula-
tion reduction are usually considered. When foliage protection is con-
sidered in evaluating the efficacy of a microbial insecticde, the net
defoliation and total defoliation may be estimated and/or the relative
level of defoliation of a particular tree species may be used. Popula-
tion reduction, on the other hand, may be estimated by determining the
mortality due to each treatment, the residual population and its relation-
ship to levels requiring retreatment or an arbitrary residual number, the
effect of the treatment as related to the mortality and the residual level
of the pest population in the year following treatment, and the reduction
of the pest population to acceptable lower levels as related to biological,
economic and/or esthetic concepts.
References
Harper, J. D. 1974. Forest Insect Control with Bacillus thuringiensis,
Survey of Current Knowledge. Auburn Univ., Auburn, AL. 64 pp.
Stairs, G. R. 1972. Pathogenic microorganisms in the regulation of
forest insect populations. Ann. Rev. Entomol. 17: 355-372.
The following table lists some defoliating insect pests that attack
forests, shade trees and some ornamentals, and have been subjected to
field treatment with microbial insecticides.
Common Name Scientific Name Hosts
A looper Lambdina athascana Eastern hemlock
athasaria (Walker)
Bagworm Thyridopteryx ephemev- Maple, pines, wild
aefomris (Haw.) cherry, poplar, oaks,
junipers, arborvitae
Black-headed budworm Aeleris variana (Fern) Western Hemlock
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California oakworm
Douglas fir tussock
moth
European pine sawfly
Fall cankerworm
Fall webworm
Forest tent caterpillar
Great basin tent
caterpillar
Gypsy moth
Hemlock looper
Lodgepole needle miner
Mimosa webworm
Omnivorous leaf roller
Orangestriped oakworm
Pine butterfly
Pitch pine looper
Redheaded pine sawfly
Phryganidia oalifovnioa
Packard
Ovgyia pseudotsugata
(McDonnough)
Neodiprion sertifer
(Geoffr.)
Alsophila pometaria
(Harris)
Eyphantyia cunea
(Drury)
Malaaosoma disstria
(Hbn)
Malaaosoma fragile
incurva (Smith)
Lymantria dispar (L.)
Lambdina fisaellaria
(Guenee)
Recwpbavia millevi
(Busck)
Homadanla albizziae
Platynota stuatana
(Walsingham)
Anisota sevratovia
(Smith)
Neop'hasia menapla
F. and F.
Lambdina arthasaria
pellueidaria (G and B)
Neodipvion leoontei
(Fitch)
Coast live oaks
Douglas fir, grand fir
Scotch pine, pine spp.
Elm, hickory, linden,
maple, ash, beech, box-
elder, basswood, cherry,
oaks
Persimmon, pecan, sour-
wood, black walnut,
hickory, cherry, sycamore,
crab apple, sweet gum
Aspen, water tupelo, hard
maple, gums, oaks
Cottonwood, willow
Oaks, basswood, birch,
willow, hemlock, pine,
cedar, spruce
Arborvitae, hemlock,
junipers
Lodgepole pine
Honey locust
Many ornamentals, Euonymus
Red oak
Ponderosa pine
Pitch pine
Shortleaf, loblolly,
lingleaf , slash pines
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Saddled prominent
Spring cankerworm
Spruce budworm
Walnut caterpillar
Western hemlock looper
Western tent
caterpillar
Eet&Tooampa gutt-iwitta Beech, sugar maple
(Walker)
Paleaorita vernata
(Peck)
White oak, red oak,
black cherry
Chortstoneura fioniferana Balsam fir, red and
(Clemens) white spruce, larch,
pine, hemlocks
Datana i,
Lambd'ina f-i
lugubrosa (Hulst)
Walnut
Western hemlock
Malaoosoma oalifoimieum Hawthorn
A looper, Lambd'ina athasaria athasaria (Walker)
Plot Design: Usually hemlocks occur in greatest abundance in moist
areas in draws or along water courses; consequently, spray areas may be ir-
regular in size and slope. Treated plots should be at least 20.3 ha for aer-
ial application, and each treatment replicated 3 times. Control areas should
be in the same general area and separated by sufficient distance to avoid
drift.
Application Equipment: A variety of aerial type equipment may be
used. Often the Bell 205 helicopter is employed applying the material
through 8003 flat fan nozzles, at a swath width of 60 m.
Timing and Frequency of Application: Application should be made during
the early larval instars.
Sampling and Evaluation: Two methods may be used to obtain efficacy
data: direct counts of larvae on branch tips and direct counts of larvae
knocked down into screens set under trees.
Twenty separate trees can be selected in each of the treatment replicates.
The criterion for the selection of an individual sample tree is that there is
positive evidence of larval activity prior to treatment. A branch tip sample,
38-46 cm long can be removed from each tree. The branch sample can then be
shaken vigorously over a drop cloth and the number of larvae determined
with observations made concerning their condition. Larval counts should be made
1 day prior to treatment at intervals of 1, 2, 4, 7 and 14 days after treat-
ment.
Larvae numbers may also be obtained by the amount of droppings onto
muslin-covered frames 59 . 2x59.2 cm (3054.64 cm2). Larval counts again
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should be made 1 day prior to treatment, and at Intervals of 1, 2, 4, 7,
14 and 21 days postspray.
References
Cameron, E. A., and V. C. Mastro. 1975. Control of a looper, Lambdina
athasaria athasaria, on hemlock with three chemical insecticides.
J. Eoon. Entomol. 68(6): 800-802.
Bagworm, Thyridopteryx ephemeraeformis (Haworth)
Plot Design: When ground applications are to be made, 50 trees, 1.5 m
2.5 m tall should be selected in alternate rows to minimize spray contamination
from drift. Three - 5 replicates should be treated for each dose. In some cases
2 trees may serve for each treatment and each treatment should be replicated
5 or more times. They should be adequately spaced to prevent any unneces-
sary drift. Individual ornamental trees about 1.5 m high, have also been
treated. Each treatment of this type should be replicated a sufficient
number of times to obtain acceptable statistical evaluation. Control groups
should be treated in the same manner.
Application Equipment: A variety of equipment is used r~>r ground
application ranging from 10-12 liter hand-operated compression sprayers to
solo-mist blowers. Application is usually made to run-off.
Timing and Frequency of Application: Treatments should be made after
all the eggs have hatched in the bags. Applications made one week after egg
hatch have been proven to be satisfactory.
Sampling and Evaluation: The postspray larval determinations may be
made by counting the number of living larvae on 25 cm terminals of the pre-
vious year. Evaluation should be made following each spray date if multi-
applications are investigated. 7-day posttreatment samples of 100 bags each
may be taken from each treated tree and untreated tree, and the number of
dead larvae determined. Foliage may also be removed and weighed to evaluate
the extent of bagworm damage.
References
Bishop, E. J., T. J. Helms, and K.A. Ludwig. 1972. Control of bagworm
with Bacillus thuringiensis. J. Eoon, Entomol. 66: 675-676.
English, L. L. 1959. Control of the bagworm with Debrom. J, Eoon.
Entomol. 52: 353.
English, L, L., and W. Hartstirn. 1962. Systemic insecticide control of
some pests of trees and shrubs a preliminary report. Nat. Hist.
Survey Div. Biol. Notes No. 48. 1-12.
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Kearby, W. H., D. L. Hostetter, and C. M. Ignoffo. 1972. Laboratory
and field evaluations of Bad-l^us fhuri,ng-iens-is for control of
bagworms. J. Econ. Entomol. 65: 477-480.
Western Tent Caterpillar, Malaeosoma oaliforniaim; Black-Headed Budworm,
Aoleris variana (Fern)
Plot Design; When individual trees are treated a minimum of 5 should
be sprayed with each material and each treatment should be replicated 2-3
times; an untreated group should also contain the same number of trees.
Application Equipment: Applications are made with a variety of
sprayers and dusters and include back-pack mist blowers and pressurized
hand sprayers.
Timing and Frequency of Application: Microbial materials should be
applied during the early instars and when sufficient foliage is present.
Sampling and Evaluation: When evaluating treatments for tent cater-
pillars pre-and postspray population estimations should be undertaken.
Tents may be selected at random from each treated tree and the number of
dead larvae determined.
Pre-and postspray population densities should be determined for the
budworm. Samples of an 18" branch tip, taken from the top third of the
crown with a sectional pole pruner may be used to estimate the population.
California Oakworm, Phryganidia oal-Lfornica Packard
Plot Design; When ground application is used, individual trees or
groups of trees may be treated with the selected material. When the indi-
vidual tree method is used, an adequate number should be employed for each
treatment with an appropriate replication. Thus, plot selection method
could also apply to groups of trees.
Application Equipment: The various materials may be applied with
conventional high pressure tree sprayer, single gun line pressure equipment
and motorized knapsack dusters.
Timing and Frequency of Application; The applications should be made
during the early 3rd larval instar, at which stage insignificant damage is
done. A single application is usually made, and applied to the point of run-
off.
Sampling and Evaluation: 25 shoots on each tree should be sampled at
random, and the number of eggs and larvae noted. Each shoot will contain
10 or 11 leaves on an average, and thus, more than 250 leaves per tree will be
sampled. The average number of larvae per 25 shoots may be used for comparing
treatment trees with untreated trees.
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Reference
Pinnock, D. E., and J. E. Milstead. 1971. Control of the California
oakworm with Bao-lllus thuringiens'Ls preparations. J. Boon. Entomol.
64: 510-513.
Douglas Fir Tussock Moth, Orgyia pseudosugata (McDonough)
Plot Design: Plots maybe 8.1-60.7 ha in size for aerial application.
Thoughout each plot, 25 trees in 5-tree clusters are designated for popu-
lation density sampling. Sample trees may be open grown Douglas fir at
about 12.2-15.2 m in height. The experimental design should be replicated 3
times and plot assignments should be made on a random basis. When tree
host and insect populations are initially different, efforts should be
made to distribute the treatment plots so as to consider any variation.
Control plots should also be included in the evaluation.
Application Equipment: A Cessna AG as well as other aerial equipment
may be used to apply the formulations using a conventional spray boom equipped
with T8010 flat fan tips. Swath spacing of 30.5 m results. A Bell 476 heli-
copter has also been used to apply the various formulations at the rate of
7.57 1 per 0.405 ha using a boom-equipped spray system with T8002 flat fan
tips. The helicopter may spray at 72.6 kph at ca 15.2 m above the canopy.
Timing and Frequency: Treatments should be applied when the majority
of the larvae are in the 2nd and 3rd instar.
Sampling and Evaluation: Near the center of each plot some 15 trees
can be designated for population density sampling. Population density should
be sampled prespray and at 7, 14, 21 and 35 days after treatment.
Sampling may Consist of counting larvae and measuring the foliage area
on three 46-50 cm branches cut with a sectional pole pruner from the mid-
clrown level of the 15 sample trees in each plot. Larvae counts should be con-
verted to larvae per 6452 cm2.
Larvae may also be collected at the sampling time and maintained in
containers with food for observations. They may be held at 23-24°C and 40-
50% RH for 14 days.
Visual estimates should be taken late in the season (September) on the 15
sampling trees. A 15.2 cm ruler be used to divide the foliated bole of
the tree into 6 approximately equal length units. Defoliation in each unit
may be given a numerical value of 0 (no defoliation), 50 (<^50% defoliation)
or 100 (>50% defoliation). The total for the 6 units maybe divided bv 6
thus giving a percent defoliation per tree. Individual tree values may then be
averaged for the plot.
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The surviving population density, percent population reduction, deposit
and defoliation data can be subjected to analysis of variance; Tukey's w-
procedure at the 95% level can be used to compare the treatment means. An
analysis of covariance can be conducted in which time can be a split plot
factor, to determine the treatment effects over the duration of the field
experiment.
References
Neisess, J., G. P. Marken, and R. Schaefer. 1976. Field evaluations of
Aceplhate and Dimilin against the Douglas-fir tussock moth. J. Eoon.
Entomol. 69: 783-786.
Stelzer, M. J., J. Neisess, and C. G. Thompson. 1975. Aerial application
of a nucleopolyhedrosis virus and Bao-Lllus thiwingiensis against the
Douglas fir tussock moth. J. Eoon. Entomol. 68: 269-272.
European pine sawfly, Neodipvion sertifer (Geoffr.)
Plot Design: For aerial applications, treated blocks may be 2-25.7 ha
and separated by a buffer strip of at least 91.4 m wide. Ten trees separated
from one another by 27-28 m or more may be selected randomly throughout each
treatment block and tagged for sampling. Twenty-21 ha has also been satis-
factory for evaluating plantation-type treatments. Four subplots 31 x 92 m
may be selected in such plantation blocks. Treatment blocks should be
replicated 2-3 times. Control should also be included in the evaluation.
For ground application, plots should consist of at least 10 trees for
each treatment, and treatments should be replicated 2-3 times.
Application Equipment: A Piper Pawnee 235 aircraft equipped with 8 or
10 flat-fan nozzles with 80015 tips may be used. Each nozzle will deliver
4.73 deciliters per min at 40 Ib per 6.45 cm2 line pressure. Spray boom is
canted 30° forward into airstream. The air speed is 161.3 kph.
For ground applications, speed sprayers and 11-12 1 pressurized hand
sprayers may be used.
Timing and Frequency of Application: Material should be applied during
early instars.
Sampling and Evaluation: When the larger blocks are used, approximately
200 trees should be selected for sampling in each subplot. 1 or 2 sawfly
colonies should be examined on each tree, and classified into the following
categories: (1) colonies all living, (2) colonies with some dead larvae, and
(3) colonies with all dead larvae. Posttreatment determinations should be made
on a weekly basis for 3 weeks.
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Referenoes
Bird, F. T. 1953. The use of a virus disease in the control of the
European pine sawfly, Neodiprion sertifer- (Geoffr.). Can. Entomol.
135: 347-446.
Wallners W. E. 1968. European pine sawfly control with aircraft applica-
tion of concentrated insecticides. J. Eoon. Entomol. 61: 1666-1667.
Fall Cankerwonn, Alsoph-ila pometaria (Harris)
Plot Design: For aerial applications, each treatment should be applied
to an area of 16.2-20.2 ha and separated by an untreated buffer strip.
For treatment with ground equipment, individual trees that are infested
with the larval stage may be selected.
Application Equipment; Applications may be made with a Bell G-2 Model
47-G helicopter fitted with a 9.14 m boom and 59 no. D-3W/23 cores nozzle type.
A 3.785 1 knapsack sprayer may be used for ground applications. Each
treatment is applied to 3 branches selected at random from the covered
branches of each of the trees.
Timing and Frequency of Application: One or 2 applications may be made
depending on the material being tested. Applications should be made on early
instars.
Sampling and Evaluation; Evaluation may be based upon larval numbers
collected from 3 3.05 x 3.05 polyethylene tarps within each treatment block.
Tarp placement should be made at random, and larval collections can be made at
2, 4, 7 and 21 days posttreatment.
Where smaller plots are used, individual trees infested with the larval
stage may be selected. Seven to 10 branches of each tree to be treated and which
are heavily infested with the fall cankerworm larvae should be selected. Selec-
tion of nontreatment trees should be done in a similar manner. On each tree, ca.
1.82 m of the distal portion of the selected branches should be covered with a
nylon mesh bag (2.74 x 1.22 m). The open end of the bag is then closed and
tied to the branch. Just prior to treatment, each bag is removed, sprays
applied, and the bag replaced.
References
Appleby, J. E., P. Bristol, and W. E. Eickhorst. 19 . Control of the fall
cankerworm. J. Eoon. Entomol. 68(2): 233-234.
Wallner, W. E. 1971. Suppression of four hardwood defoliators by helicopter
application of concentrate and dilute chemical and biological sprays.
J. Econ. Entomol. 64: 1487-1490.
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Mimosa Webworm, Homadaula alb'izz'iae Clarke; Fall Webworm, Hyphantria aunea
(Drury); Walnut caterpillar, Danata intege'pvi.na Grote and Robinson
Plot Design: Biological materials may be applied on individual trees,
and each treatment replicated 3 or more times. Untreated trees should be
treated in a similar fashion.
Application Equipment: Ground application may be made with mist blow-
ers or back pack mist blowers. Materials are usually applied to run-off
conditions.
Timing and Frequency of Application; The first application should be
made to larvae during their early instars. A second application may be neces-
sary 7-10 days later.
Sampling and Evaluation: Webworms and the walnut caterpillars may be
confined in sleeve cages (40-mesh cooper wire screen (15 cm dia x 46 cm long))
placed on each of the trees. Cages are slipped over the end of a branch which
has 25-30 undamaged crotches. Each week following applications, 25 larvae
are placed in each cage.
English, L. L., and W. Hartstirn. 1962. Systemic insecticide control of some
pests of trees and shrubs a preliminary report. Nat. Hist. Survey
Div. Biol. Notes. No. 48: 1-12.
Forest tent caterpillar, Malaoosoma disstria (Hubner)
Plot Design: For aerial treatments, plots should be 4-20 ha and, if pos-
sible, separated by an untreated buffer strip. Control plots of equal size
should be located near treatment plots to minimize differences in responses
that might be due to biotic and climatic factors.
Application and Equipment: Applications may be made with a Bell G-2
Model 47-G helicopter fitted with a 9.14 m boom and 50 no. D-3 W/23 cores
nozzle-type.
Applications have been made with a Piper Pawnee aircraft fitted with 10
Roto-spin nozzles (5/wing), each of which contained a No. 6 orifice, hollow-
core spray nozzle and a plastic, wind-driven propeller which further broke up
the emitted spray. Materials are applied from a height of 15 m above the
canopy at an air speed of 100 knots. Some materials may be applied at 3785
cm3/0.405 ha in water-based formulations. Whenever possible, aerial applica-
tion should be monitored from a spotter plane.
Timing and Frequency of Application: Egg hatch and subsequent larval
development should be monitored by weekly inspection of egg mass samples.
Treatment applications should be timed whenever possible to reach the
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-R7-
caterpillars when the majority are in the 2nd and 3rd instar. In normal
years, the foliage is ca. 1/2 to 2/3 expanded at this time. Second and 3rd
instar larvae will usually consume sufficient foliage to have a high
probability of ingesting the stomach-type microbials, yet not so much
feeding that average populations will cause severe damage to the tree
crowns. In some cases, a second application will be needed.
Sampling and Evaluation: Evaluation should be based upon larval numbers
collected from 3 3.05 x 3.05 m polyethylene tarps within each treatment
block. Tarp placement should be at random, and larval collections should be
made at 2, 4, 7 and 21 days posttreatment.
Branches may also be removed from the uppermost crowns of dominant
trees. Egg masses may then be counted and recorded as the average number per
1.23 m branch tip. Usually with water tupelo an average of 2.5 egg masses per
sample produces a caterpillar population capable of completely defoliating
the canopy and understory foliage of a stand by the end of the defoliation
period. Samples averaging less than 2.5 egg masses per sample will yield
larval populations that can cause partial stripping of the canopy, while
numbers greater than 2.5 cause accelerated defoliation.
Efficacy of eath treatment may be monitored by ground observations in
each plot beginning within 24 hours posttreatment and continuing at 7-day
or greater intervals until all larval feeding activity has ceased. On each
observation date, percentage of canopy defoliation should be estimated for each
treatment and control plot. Larvae on branches that are removed from tree-
tops in each treatment area should be examined, and the numbers, size and
general condition of these larvae determined.
References
Harper, J. D., and L. P. Abrahamson. 1977. Forest tent caterpillar control
with aerially applied formulations of Bacillus thuringi ens-is and Dimilin.
(In Press).
Wallner, W. E. 1971. Suppression of four hardwood defoliators by helicopter
application of concentrate and dilute chemical and biological sprays.
J. Eoon. Entomol. 64: 1487-1490.
Great basin tent caterpillar^ Malacosoma fragile incurva (Henry Smith)
Plot Design: For aerial application the plot size for each treatment
can be a 10.1 ha block and should be replicated. The untreated blocks should
be separated from the treated blocks by a buffer strip at least 91.4 m wide.
Application Equipment: Applications may be made with a helicopter or
other conventional air spray equipment.
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Sampling and Evaluation: Two 0.81 m^ subplots should be set up in each
hectare of the treated and untreated plots. Measurement of treatment effec-
tiveness should be based upon the following: (1) the rate of disease mortality
of larvae reared in dacron sleeve cages, and (2) the percent of the colonies
containing disease-killed larvae. Groups of 25-50 larvae may be caged on
the terminal of a 92 cm branch, located on each of a given number of cotton-
wood and/or willows in the subplots.
Colonies of the tent caterpillar may be removed from the host trees in
each subplot with a pole pruner at 1-3 day intervals after being sprayed.
They may tnen be examined for the presence or absence of disease-killed lar-
vae and the percent of infected colonies. Examination may be continued
until 25 days after spraying or until pupation.
References
Stelzer, M. J. 1969. Control of a tent caterpillar, Valacoscma fragile ir.aurva,
with an aerial application of a nuclear-polyhedrosis virus and Baai-llus
thur-ingiensis.
Gypsy Moth, Lymmtria dispar (L.)
Plot Size: When microbial insecticides are applied by air, plots
should be a minimum of 10.12-20.24 ha and replicated 3 times. Plot size
for ground applications may vary; but usually 0.1 ha has been found to be
satisfactory. The treatment site should be selected so as to contain at
least 50% primary gypsy moth host species. Efforts should be made to obtain
reasonable uniformity in the plot stand structure.
Application Equipment: Ground application may be made using truck-
mounted mist blowers. Adequate spray coverage of 0.1 ha plots is usually
achieved using ca. 76.0 1. Aerial application may be made with either heli-
copter or fixed wing aircraft. As an example, applications to experimental
plots have been made with a 450 Grumman Ag Cat equipped with 6 Beecomist
nozzles. Application is usually made at a height of 9-18 m above the canopy
at a speed of 2.1-2.4 km/min.
Timing and Frequency of Application: Usually 1 or 2 applications of
the microbial insecticide are made. The initial spray application is made
when at least 50% of the egg masses have hatched and when at least 50% leaf
expansion has occurred. If a 2nd application is to be made, it is usually
applied 5-10 days after the 1st spray.
Sampling and Evaluation: Egg mass determinations Subplots of
101.2 m2 size can be selected at random using about 10 10.12 ha treatment
plots. Egg mass determination should be made in each subplot prior to spray-
ing. These determinations should be made as thoroughly as possible including
fallen limbs, rock piles, loose bark, etc. Postspray determinations should be
made after leaf drop. For subplot selection the "prism point" plot selection
method may also be used (Wilson and Fontaine 1977).
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Burlap bands Five or more trees in each subplot should be banded with
burlap (a 15 cm burlap strip wrapped twice around the trunk 1.5 m above
ground, flap may be made by cutting into the burlap strip every 8 cm).
Larval density is then determined by counting larvae under the burlap
on a biweekly basis until pupation.
Terminal branch determination Terminal counts may also be made.
Select 25 59 cm terminals in each subplot and determine the number of larvae
on each of the terminals.
Defoliation estimates - Defoliation estimates should be made in each
101,2 m2 subplot immediately prior to spraying, and again at the completion
of larval feeding. The estimates may either be made from aerial photography
or ground observations. Five primary host trees can be selected or the entire
subplot may be estimated for defoliation. The damage level by tree or sub-
plot may be rated by the following scheme: 0-19, 20-39, 40-59, 60-79,
80-100% defoliation.
References
Connola, D. P., F. B. Lewis, and J. L. McDonough. 1966. Experimental field
technique used to evaluate gypsy moth, Porthetria dispar, control in
New York. J. Econ. Entomol. 59: 284-287.
Lewis, F. B., and D. P. Connola. 1966. Field and laboratory investigations
of Bacillus thuringiensis as a control agent for gypsy moth, Porfhety*ia
dispar (L.). U.S. Forest Service Research Paper NE-50, 1-38 pp. No.
East Forest Exper. Sta., Upper Darby, PA.
Lewis, F. B., D. P. Connola, and J. L. McDonough. 1962. Aerial application
of a Bacillus thuringiensis spore-crystal concentrate in fuel oil and
water formulation for gypsy moth control New York State, 1962.
Special Report. (Processed). Forest Insect Lab. Northeast Forest
Exp. Sta. U.S. For. Serv. 54 pp.
Lewis, F. B., N. R. Dubors, D. Grumble, W. Metterhouse, and J. Quimby.
1974. Gypsy moth; Efficacy of aerially-applied Bacillus thwfingiensis
J. Econ. Entomol. 67: 351-354.
Wilson, R. W., and G. A. Fontaine. 1977. A method for surveys of gypsy
moth infestations in forested areas. (In Press).
Yendol, W. G., R. A. Hamlen, and F. B. Lewis. 1973. Evaluation of Bacillus
thuringiensis for gypsy moth suppression. J. Econ. Entomol. 66: 183-186.
Hemlock looper, Lambdina fiscellar'ia (Guenee)
Plot Design: Trees or ornamentals planted in rows may be treated
by ground application. As an example, 6 rows containing approximately 150
plants may serve for each treatment.
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-85-
Application Equipment: Conventional, high-pressure, high-volume
hydraulic sprayer equipped with a spray gun may be used.
Timing and Frequency of Application: Single applications are usually
made during the 5th instc.r
Sampling and Evaluation; The number of larvae per individual plant
is usually determined. An appropriate statistical number of plants should
be used for sampling each treatment. Evaluation should be made on the 10th-20th
posttreatment day.
Eeferences
Kerr, T. W. 1971. Control of the hemlock looper. J. Eeon. Entomol.
64: 1552.
Lodgepole Needle Miner, Reaurvia m-illeri (Busck)
Plot Design: Individual trees may be used for testing each material
and specific concentration. A plot may consist of 3 trees and/or tree
group. Plots should be replicated 2-3 times.
Application Equipment: Ground applications may be made with a truck-
mounted mist blower or back pack mist blowers. Attempts should be made to
obtain thorough coverage.
Timing and Frequency of Application: Application should be directed
against the 1st instar larvae migrating to their 1st needles after eclosion.
Sampling and Evaluation: 10 twigs with 5 whorls of needles should be
taken from each treated tree to ascertain the larval population. Larval
counts are then made and the treatments compared with the controls.
Prespray sampling should be undertaken to determine the population.
References
Struble, G. R. 1965. Field test of Bacillus thuringiensis (Berliner) to
control lodgepole needle miner. J. Eeon. Entomol. 58: 1005-1006.
Omnivorous Leaf Roller, Platynota stultana (Walsingham)
Plot Design; Individual trees or ornamental bushes may be treated
individually. Each treatment should be replicated with sufficient numbers
(3-4 each).
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Application Equipment: Full coverage sprays may be applied with
small aerosol units.
Timing and Frequency of Application: Usually single applications
have been done.
Sampling and Evaluation: The total number of live larvae on each
treated plant should be determined at 10 days, 10 and 20 weeks posttreatment.
References
Campbell, R. L., and B. G. Ward, Jr. 1971. Insecticides tested against
Platynota stultana on Euonymus. J. Eoon, Entomol. 64: 1556.
Orangestriped oakworm, Anisota senatoria (J. E. Smith)
Plot Design: With small-scale ground applications one tree may serve
to test each treatment and/or concentration, and should be replicated 3
or more times. Care should be taken to have buffer trees between treated and
untreated trees.
Application Equipment: Materials may be applied with a hydraulic
sprayer at 400 Ib/m2.
Timing and Frequency of Application: Attempts should be made to
apply microbial materials during the early instars.
Sampling and Evaluation: Since Anisota senatoria are gregarious,
leaf counts should be made on branches only where the insect occurs. These
branches may be tagged and pre- and postspray leaf counts made. Twenty leaves
per tree are sampled and the number of larvae occurring on each leaf can be re-
corded. Counts should be taken at 1, 2 and 4 days posttreatment.
Two 0.835 m2 cloth trays may also be placed under each tree. The dead and
moribund larvae should then be determined.
References
Kaya, H. K. 1973. Laboratory and field evaluation of Baoillus thwringiensis
var. alesti for control of the orangestriped oakworm. J. Eoon. Entomol.
67: 390-392.
Pine Butterfly, Neophasia menapia F. and F.
Plot Design: For aerial application experimental blocks should be 16.2 ha
(20 x 20 chains). Each dosage should be applied to 3 randomly selected blocks.
3 blocks should be left untreated to serve as control blocks.
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Application Equipment: Conventional aerial application equipment
may be used.
Timing and Frequency of Application; Applications should be timed
so as to treat early instar larvae.
Sampling and Evaluation: 5 sample plots of 10 trees each may be
selected in each block. Prespray and postspray larval counts can be made
by removing 6 branches ca. 12.7 cm from each sample tree during each sample
period. Prespray larval counts should be made 24 hours prior to spraying.
Postspray counts should then be made 4, 8 and 12 days later. Prespray and
postspray larval densities can be expressed as the number of larvae per
lineal 2.54 cm of foliated branch.
Each plot may be aerially photographed with color IR late July to August
to measure the degree of defoliation.
Calculate the number of larvae per 2.54 cm of foliated branch.
References
Cessla, W. M., and J. E. Dewey. 1973. Control of pine butterfly in western
Montana with aerial applications of mexacarbate and Ba.ci.llus thuring'iensis.
Ann. Meeting Western Forest Pest. Comm. 64th West. Forestry Conf.,
San Jose, CA, December 5, 1973.
Pitch Pine Looper, Lambdina athasaria pellucidaria (G & Rl
Plot Design; Plots should be a minimum 10.1 ha for aerial application
and should be replicated 2-3 times. Distances between plots should range
between 92-183 m.
Application Equipment: The microbial insecticide may be delivered
with a Bell G-3 helicopter which travels about 97 kph, 3.5 m above the
canopy, with a swath about 31.1 m. Eight D-4 tip nozzles operated at 40 Ib.
per 6.452 cm2 have been used.
Timing and Frequency of Application: A single application is usually
made during the early instars when sufficient foliage is present.
Sampling and Evaluation: Four transects per treatment plot should be
established perpendicular to the direction of the spray operation. Larvae
are then collected randomly from selected trees along each transect. Five trees
should be used for every 2.02 ha sampled in each treatment. Samples 46 cm long
should be cut from terminal and lateral branches at a height of 92-299 cm.
Samples should not be selected within 15.2 m of the edge of the treatment
block. The 46 cm samples are beaten over a 76 x 76 cm piece of plastic until
no further loopers fall off the sample. Prespray determinations should
be made and comparative evaluations should be made 3 or more days postspray.
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ANOVA, t-tests and proportional comparisons can be used to determine
significance.
References
Sorenson, A., and P- Barbosa. 1975. Effectiveness of carbaryl and thuricide
16-B on a population of larval Lambdina athasaria peilucidana. J. Econ.
Entomol. 68: 561-562.
Redheaded Pine Sawfly, Neodiprion leeontei (Fitch)
Plot Design: Aerial applications should be made on 10.12-20.24 ha
blocks. Ground applications (mist blowers) may be made on 2.02 ha blocks.
Application Equipment: Application may be made with fixed wing air-
craft at 145.2 kph, ca 15.2 m above tree tops and a 30.5 m swath wide. Such
a spray system produced 22.7 kg per 6.452 cm2 of pressure with 8 no. 80015
flat fan spray nozzles.
With ground applications, mist blowers or smaller back pack equipment
may be used.
Timing and Frequency of Application: The first application should be
made when larvae are in the 2nd to 3rd instar.
Sampling and Evaluation: Before treatment, 100 larval colonies can
be selected randomly on the trees to be treated. On the day before treatment
larvae be counted in each tagged colony or estimated by tens (to minimize
disturbance) if the colony is large. Living larvae should then be counted in
the same colonies at 7, 14 and 21 days posttreatment.
References
Fowler, R. F., I. Miller, and L. F. Wilson. 1973. Aerial and ground
applications of malathion for control of the redheaded pine sawfly.
J. Econ. Entomol. 66: 288.
Wallner, W. E. 1968. European pine sawfly control with aircraft appli-
cation of concentrate insecticidal sprays. J. Econ. Entomol.
61: 1666-1667.
Spring Cankerworm, Paleacrita vemata (Peck) and Saddled Prominent, Heterocampa
guttivitta (Walker)
Plot Design: Each treatment may be applied to an area of 16-20 ha
separated by an untreated buffer strip.
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-89-
Application Equipment; Applications may be made with a Bell G-2
Model 47-G helicopter fitted with a 9.14 m boom and 59 no. D-3 W/23 cores
nozzle-type.
Timing and Frequency of Application; Initial applications should be
made when sufficient foliage is present, and if possible, when majority of
larvae are in the 2nd and early 3rd instar.
Sampling and Evaluation: Evaluation may be based upon larval numbers
collected from 3 3.05 x 3.05 m polyethylene tarps within each treatment
block. Tarp placement should be at random, and larval collections should be
made at 2, 4, 7 and 21 days posttreatment. Comparison should be made with
untreated blocks.
References
Wallner, W. E. 1971. Suppression of four hardwood defoliators by helicopter
application of concentrate and dilute chemical and biological sprays.
J. Eaon. Entomol. 64: 1487-1490.
Spruce Budworm, Chovistoneupq. fum-ifevana (Chem.)
Plot Size; Usually when aerial applicatons are made, 10-20 ha are used
and each treatment is replicated at least 3 times. Treatment blocks should
contain at least 0.4 km between each to act as a buffer zone. In the case of
ground applications, 1 ha blocks be used. Care should be taken to
select uniformity in blocks with regard to forest composition. In some
situations hand spraying of individual trees may be desirable and at least
30 or more trees be considered for each treatment.
Application Equipment: Usually sprays are applied in the early morning
and/or late evening. In the case of Morris and Armstrong (1975), a Cessna
Agtruck aircraft equipped with 4 AU 3000 Micronair emission units calibrated
to deliver droplets in the 50-100y dia. range was used. Diamond (1972) used
a helicopter to apply the microbial insecticide.
Timing and Frequency of Application: Usually 1 or 2 applications are
made and this may depend on the type of material being tested. Spraying is
usually done in the morning or evening and when the budworm development
reaches 60% in the 4th instar, 30% in the 3rd instar and the remainder in
the 2nd and 5th instars.
Sampling and Evaluation: Within each plot, 20 codominant fir trees
should be selected for sampling. If the stand is mixed spruce and balsam,
25 spruce and 25 balsam should be used. These trees should be selected at
random across the plot, but no closer than 30 m to the plot boundaries.
Population levels of the spruce budworm can be determined by making
counts of larvae and/or pupae on 2 38 cm branch terminals for 25 trees of
each species per plot (Diamond 1972). Other density determinations have
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-90-
been made by using 46 cm (18.1") of the branch tip of white spruce and
balsam fir. Prespray and postspray collections should be made in both
treatment and control plots. Posttreatment samples should be made 6-10 days
after spraying and then again at 2-3 weeks postspray which can be timed
with the termination of larval feeding (Diamond 1972). Morris (1975) on
the other hand, has used the 30 postspray day period as a final and probably
the most important postspray sample, the peak of pupation. Approximately
200-400 pupae should be collected from each plot and reared for emergence.
The actual residual population should be based on the live pupae which is
based on emergence. The corrected population reduction (i.e., reduction
due to treatment) may then be calculated on this residual population.
Defoliation estimates should be made after cessation of feeding. The
following method is proposed by Diamond (1972): ten 38 cm branches are
pruned from each 25-sample trees per plot. The current year's shoots should
be examined and classified as either destroyed (the entire year's shoot is
missing), damaged (shoot with apex intact and a bud formed to a healthy
fully-needled shoot with portions of 2 or 3 needles missing), or undamaged
(no signs of feeding). This can be converted to a single value. Although
somewhat time-consuming, the "Fettes1 branch examination method" may be used.
Destroyed Damaged Undamaged
Sum of buds from 5 branches in
each category
Percent of buds in each
category
Sum: destroyed % x 2,
damaged % x 1, undamaged
% x 0
Damage rating =
76
25%
207
69%
18
6%
(25 x 2) + (69 x 1) + (6x0)
50 + 69+0
119
Obviously, a tree with extreme damage would have 100% of its shoots
in the destroyed category and a damage rating calculated at 200. Con-
versely, a completely undamaged tree would calculate to a 0 damage rating.
Other trees would fall somewhere between on this 200-point scale.
Unusual defoliation estimates may also be used in comparing treatments
with non treated plots. The 25 trees in each plot may be rated using the
scale 0-19, 20-39, 40-59, 60-79, 80-100% defoliation. Visual estimates
may be made of the upper and lower crown, and the mean defoliation calculated
for the plot and treatments.
In some cases it is advisable to reassess 1 year after treatment to
determine any long-term effects on defoliation. The amount of defoliation
that stands can tolerate is perhaps still in question. Morris and Armstrong
(1975) suggest that less than 50% defoliation of the current year's growth
is generally considered to be not seriously injurious to tree growth and
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-91-
health. Using the damage conversion method Diamond (1972) suggests that
defoliation ratings of 100 or less are tolerable. For other information
concerning evaluation of microbials against the spruce budworm, consult
Morris and Hildebrand (1974) and Morris (1977).
References
Diamond, J. B. 1972. A demonstration of Bacillus thv^ringiensis plus the
enzyme chitinase, against the spruce budworm in Maine. Part I: Efficacy.
Misc. Report 144, Maine Agric. Exp. Sta. November 1972.
Morris, 0. N. 1977. Long-term studies of the effectiveness of Bacillus
thuringiensis - acephate (Orthene) combination, against the spruce
budworm, Choristoneura fumiferana (Clem.) Can. Entomol. (In Press).
Morris, 0. N. and J. A. Armstrong. 1975. Preliminary field trials with
Bacillus thuringiensis - chemical insecticide combinations in the
integrated control of the spruce budworm, Choristoneura fumiferana
(Lepidoptera: Tortricidae). Can. Entomol. 107: 1281-1288.
Morris, 0. N., and M. J. Hildebrand. 1074. Evaluation of commercial
preparations of Bacillus thuringiensis with and without chitinase
against spruce budworm. E. Assessment of Effectiveness of Aerial
Application. Algonquin Park, Ontario. Chem. Control Res. Inst.
Report No. CC-X-59.
Morris, O.N., G. M. House, and J. C. Cunningham. 1974. A two-year study
of virus-chemical insecticide combination in the integrated control
of the spruce budworm. Can. Entomol. 106: 813-824.
Western hemlock lodper, Lambdina fiscellari,a lugubrosa (Hulst)
Plot Design: For aerial application the treatment blocks may be 10
or more ha of infested forest. Each treatment block should then be replicated
3 times. An untreated area of the same size should also be developed.
Treatment blocks should contain at least 6-7 sampling plots, which can be
located on cardinal directional lines constructed through the block. Sampling
plots should be located at 137-146 m intervals along lines. Each sampling plot
should consist of 3 codominant hemlocks.
Application Equipment: The material may be applied with a Bell G-2
helicopter equipped with a 9.1 m boom and 18 no. 4664 Tee Jet spray nozzles,
delivered at the rate of 7.57 1 per 0.405 ha. Mass median diameter (M.M.D.)
should be about 160-170 microns.
Timing and Frequency of Application: Spraying should be timed with
the peak number of 3rd instar larvae.
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-92-
Sampling and Evaluation: Mortality should be assessed by counting and
collecting dead larvae found at intervals in ground trays. Ground trays
with an inside area of 0.19 m^ have been found to be adequate. The ground
trays are positioned under the hemlocks in each sampling plot. Ground
trays should be emptied prior to spraying, and then examined every 2 days
until 20 days after spraying.
Quantitative estimates of larval mortality should be obtained from periodic
counts of larvae on foliage samples collected with a pole pruner equipped
with a basket. At each sampling, 5 46 cm branch tips can be clipped from
each plot tree; the total larvae on the 15 branch tips from the 3 trees
represent the population estimate for the plot. Trees may be sampled
3 to 4 days before spraying and at 3 day intervals postspray. Estimates of
the populations can then be made by comparing the number of larvae on 15
branch tips before test-area spraying, and 19 days after spraying. Using
data collected in the spray area, a regression analysis may be performed
to determine the significance of downward population trends.
The frass-drop method may also be used to assess mortality. Its use
for estimating populations is based on determining: (1) the amount of
frass falling on a tray during a known period of time, and (2) the amount
of frass produced by an average larva during the same period.
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STORED PRODUCTS
Bacteria and baculoviruses appear to have potential for controlling
certain species of stored-product insects. Although studies have been
reported of the pathogen susceptibility of numerous species that infest
stored products, serious attempts at pest control have been limited to
three species of Lepidoptera: the Indian meal moth, Plodia interpunctella
(Hubner); the almond moth, Cadra eautella (Walker); and the tobacco moth,
Ephestia elutella (Hubner). These studies have dealt with infestations
in dried fruits, nuts, cereal grains and tobacco in very restricted com-
modity storage situations. More limited studies have been made on the
control of these pests and the Mediterranean flour moth, Anagasta
kuehniella (Zeller) in flour, and the greater wax moth, Galleria
mellonella (L.) and the lesser wax moth, Aahvoi^a grtsella (F.) in
beeswax and beecomb. The existing methodology will require modification
and extension as pathogen uses are proposed in other commodities and
storage situations, and when promising pathogens of species of stored-
product infesting Coleoptera are proposed as control measures. The
literature on test methods for chemical insecticide evaluation may be use-
ful for that purpose.
The general considerations and steps in product development that
introduce this report are generally applicable to stored commodities.
Only specialized procedures that are unique to tests on stored commodities
are discussed in this section. The methods presented are not to be
considered exclusive of other valid methods that have been used or that
will be developed in the future.
Unlike control measures for most phytophagous pest species, residual
insecticides may be used as preventive treatments on stored commodities.
They are usually applied as the commodity is moved into storage or before
infestation is apparent. Applications of pathogens made after infestations
are apparently effective only against subsequent generations of the pest. In
such cases other means of immediate population reduction such as fumigation
may be used prior to application of the pathogen. Because residual insecti-
cides are used primarily as preventive treatments, the frequently used
parameters of efficacy such as population reduction are not always applicable.
Furthermore, because the insecticides are usually intended to provide pro-
tection for several months or years, efficacy must be evaluated over a long
period of time.
Most stored commodities are subject to infestation by several species
of Lepidoptera and Coleoptera. Insect pathogens, because of their relatively
narrow spectrum of activity, will individually be unsuitable for controlling
all of the pest species that may be present in the commodity at one time.
They will be useful, however, in pest control programs in conbination with
other insecticides and pest control practices, and for controlling certain
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pests (particularly species of Lepidoptera) whose unique patterns of in-
festation necessitate the use of additional protective measures. Compati-
bility with other insecticides and with fumigants must be assured if they
are used together.
Because insects are considered to be contaminants as well as destroy-
ers in stored foods, the objective of all control measures should be com-
plete control or prevention. Presence of a detectable infestation would
indicate unsatisfactory control. It should be recognized, however, that
the best available control measures may fall short of this objective.
Harvested agricultural commodities that are subject to insect infest-
ation are stored under conditions that can be readily simulated in the
laboratory. Therefore, in contrast to pest control on growing plants,
extensive testing of stored-product pest control materials can be done
in the laboratory on a relatively small scale, using laboratory-reared
insects. Pilot and commercial scale tests are used to confirm the results
of the laboratory studies and to make any necessary adjustments in dose
or application procedure that may be required as a result of the larger
storage units and the wild insect populations.
Certain commodity quality factors including variety, moisture con-
tent, broken particles, and foreign material are known to affect the in-
festability of commodities and the performance of insecticides. These
factors should be considered when selecting commodities for use in tests,
and their interaction with the treatment should be evaluated in the ear-
lier stages of testing.
Insect Rearing
The extensive reliance on laboratory testing makes the maintenance
of healthy, vigorous, genetically-stable colonies of insects mandatory.
Several diets have been successfully used for rearing species of stored-
product infesting Lepidoptera. Most make use of mixtures of ground
cereal grains fortified with yeast, glycerol and honey, and some require
the addition of water and fungistatic agents. Sterilization, usually
be autoclaving of the cereal grain components is necessary to exclude
storage fungi and any insect pathogens that may be present on these
commodities as a result of prior insect infestation during storage. The
insect eggs are usually surface-sterilized with dilute solutions of form-
aldehyde or other suitable materials to reduce the transfer of pathogenic
microorganisms from generation to generation. Other laboratory practices
designed to assure a vigorous and genetically stable colony should be em-
ployed in order to obtain laboratory results that will be reproducible
under commercial commodity storage conditions.
References
Boles, H. P., and F. 0. Marzke. 1966. Lepidoptera infesting stored products.
Chapter 17, pages 259-270, in Insect Colonization and Mass Production,
C. N. Smith (ed.). Academic Press, New York.
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Finney, G. L., and D. Brinkman. 1967. Rearing the navel orangeworm in
the laboratory. J. Econ. Entomol. 60: 1109-1111.
Spitler, G. H. 1970. Protection of Indian meal moth cultures from a
granulosis virus. J. Econ. 'Entomol. 63: 1024-1025.
Strong, R. G., G. J. Partida, and D. N. Warner. 1968. Rearing stored-
product insects for laboratory studies: Six species of moths.
J. Econ. Entomol. 61: 1237-1249.
Bioassay
The larvae of species of stored-product infesting Lepidoptera develop
faster and with less mortality in fortified diets than in natural food
products. Therefore, bioassays that make use of these fortified diets
appear to provide greater precision with fewer test insects in a shorter
period of time. Conventional analytical techniques such as probit analy-
sis are satisfactory for estimating LC5Q values and the slopes of dose-
mortality relationships. Low slopes and variation in slope and LC5Q
estimates between assays necessitate ample replication and vigilance to
assure parallelism, particularly in comparative studies and standardiza-
tion tests.
A bioassay that has been useful for evaluating the efficacy of
Bacillus thuTingiensis against Indian meal moth and almond moth larvae
is presented in Exhibit 3. This assay has also been used for standardizing
a formulation of Indian meal moth granulosis virus. It probably will be
useful for the tobacco moth and other related species.
References
Bucher, G. E., and P. M. Morse. 1963. Precision of estimates of the
median lethal dose of insect pathogens. J. Insect Pathol. 5: 289-308.
Dulmage, H. T., A. J. Martinez, and T. Pena. 1976. Bioassay of Bacillus
fhuringiensis (Berliner) endotoxin using the tobacco budworm.
USDA, ARS. Tech. Bull. No. 1528. 15 pp.
McGaughey, W. H., and E. B. Dicke. 1977. A bioassay for evaluating the
efficacy of Bacillus thuringiensis isolates against Indian meal
moths and almond moths. (Exhibit 3).
Laboratory Testing
Purpose and Scope: The initial stages of laboratory testing may in-
volve very small samples of insect diet or commodity (10-200 g) treated with
suspensions of crude preparations of pathogens. Eventually, however, tests
are usually made in 1-20 kg samples of commodity treated with formulated
products. These latter tests involving formulated pathogen products may
be used to obtain estimates of minimum effective doses, to determine the
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longevity of effective insect control, to determine the stability of the
treatments under simulated storage conditions, to determine compatibility
with other pest control treatments such as fumigants, to compare alternative
formulations such as wettable powders, dusts, baits, etc., to study inter-
actions with nonsusceptible pest species, and in some cases to obtain pre-
liminary data regarding the efficacy of various application techniques. The
small size of the test units facilitates testing numerous dose rates.
Design and Analysis: A minimum of 3 replications are typically
used in these tests. More replications are sometimes needed when very
small numbers (less than 50) are tested in each experimental unit. Standard
statistical procedures such as analysis of variance and multiple range tests
are useful for interpreting differences.
Application: Because these tests are conducted primarily to evaluate
the product rather than the application technology, application can be made
by any available means that will assure thorough coverage. Large volumes
of diluent or carrier material may be used if provisions are made for drying
the commodity to prevent spoilage. For example, in testing on small samples
of cereal grains the insecticides have been suspended in water for appli-
cation at the rate of 21 ml/kg. The suspension was poured into a jar con-
taining the grain sample and the jar was tumbled or shaken until all free
moisture was absorbed by the grain and coverage appeared uniform. Dried
fruits have been dipped in the suspension, and spraying has been used on
tobacco leaves and on nuts. For some commodities commercial treatment pro-
cedures may be used to apply the insecticide to larger quantities of the
commodity. Small samples of the treated commodity can be held in appropriate
containers in the laboratory for determining efficacy.
Evaluation: After treatment the commodity samples are held under
laboratory conditions in containers that will confine insects, and are
subjected to infestation with insect eggs or neonate larvae. Reduction
of larval survival of adult emergence serve as a measure of efficacy. The
treated samples may be infested immediately after application or they may
be subjected to other treatments such as long-term storage in simulated or
actual storage environments, or application of other pest control products
or processes prior to infestation. The samples may also be held in containers
which simulate actual storage units such as vertical columns for grain, com-
mercial packages for dried fruits, or small bundles for tobacco.
References
Afify, A. M., and M. M. Matter. 1969. Retarded effect of Bacillus thuringiensis
Berliner on the fecundity of Anagasta kuehniella (Zell). Entomophaga
14: 447-456.
Gibson, N. H. E., and J. Wolf. 1964. Environmental humidity and the
susceptibility of larvae of Anagasta kuehniella (Zell). to Bacillus
thuringiensis Berliner. Entomophaga Mem. hors. Ser. No. 2. pp. 329-
336.
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Shaikh, M. U., and F. 0. Morrison. 1966. Susceptibility of nine insect
species to infection by Bacillus thuri,ngiensi,s var. thw?ingiensis.
J. Invertebr. Pathol. 8: 347-350.
Steinhaus, E. A., and C. R. Bell. 1953. The effect of certain micro-
organisms and antibiotics on stored-grain insects. J. Econ. Entomol.
46: 582-598.
Pilot-Scale Testing
Purpose and Scope: Pilot-scale testing may be considered intermediate
level testing to be used between laboratory and field testing in instances
where (1) field uses are of such magnitude as to make extrapolation from
laboratory results questionable, (2) resources are inadequate to enable
immediate testing on a commercial scale, or (3) an experimental use permit
is not available. The use of pilot scale tests will generally reduce the
number of doses that will need to be tested on a commercial scale.
Pilot-scale tests are usually made under actual storage conditions
(ambient) using a limited number of standard commodity storage units, or
numerous miniature simulated storage units. The test period should
coincide with a typical commodity storage period. The temperature and
moisture content of the commodity should be monitored during the test.
Insects should be permitted free access to and from the test units and
the units should generally be of sufficient size to accommodate the
normal behavioral patterns of the test insects. However, pilot-scale
tests need not rely exclusively on natural insect infestation. Insect
infestation can be supplemented in all of the individual bins or test
units or in the warehouse in which the test is conducted to the extent
necessary to assure heavy infestation of the untreated controls. At least
3 doses should be tested (the expected minimum effective dose plus one
higher and one lower dose) to assure an accurate determination of the
minimum effective dose.
Design and Analysis: Tests should be adequately replicated (usually
3-5 replications are used) and should include untreated control units to
accurately evaluate the efficacy of the treatments. Randomized block de-
sign is satisfactory, although other designs may be preferred under some
circumstances. Standard statistical procedures such as analysis of vari-
ance and multiple range tests are useful for interpreting the data.
Application: When possible, the pathogen should be applied using
techniques that are the same as or closely simulate those used in commer-
cial practice. The methods differ for each commodity and will be discussed
in the appropriate commodity sections. Because the moisture content of
stored commodities usually must be maintained within critical limits to
prevent spoilage, the minimum spray volume consistent with thorough coverage
should be used in these larger tests.
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Hunter, D. K. 1970. Pathogenicity of a granulosis virus of the Indian
meal moth. J. Invertebr. Pathol, 16: 339-341.
Hunter, D. K., S. J. Collier, and D. F. Hoffman, 1975. Compatibility
of malathion and the granulosis virus of the Indian meal moth.
J. Invertebr. Pathol. 25: 389-390.
Hunter, D. K., and D. F. Hoffman. 1973. Susceptibility of two strains
of Indian meal moth to a granulosis virus. J. Invertebr. Pathol.
21: 114-115.
Hunter, D. K., D. F. Hoffman, and S. J. Collier. 1973. Pathogenicity
of a nuclear polyhedrosis virus of the almond moth, Cadra caut ella.
J. Invertebr. Pathol.. 21: 282-286.
Kantack, B. H. 1959. Laboratory studies with Bacillus thuringiensis.
Berliner and its possible use for control of Plodia interpunatella.
(Hbn.). J. Boon. Entomol. 52: 1226-1227.
Kinsinger, R. A., and W. H. McGaughey. 1976. Stability of Bacillus
thuringiensis and a granulosis virus of Plodia interpunctella on
stored wheat. J. Eoon. Entomol. 69: 149-154.
van der Laan, P. A., and J. H. M. Wassink. 1965. Influence of crowding
and temperature on susceptibility of stored-products infesting
moths against Bacillus thuringiensis. Proc. XII. International
Congr. Entomol. London, pp. 740-741.
McGaughey, W. H. 1975. Compatibility of Bacillus thuringiensis and
granulosis virus treatments of stored grain with four grain fumi-
gants. J. Invertebr. Pathol. 26: 247-250.
McGaughey, W. H. 1975. A granulosis virus for Indian meal moth control
in stored wheat and corn. J. Econ. Entomol. 68: 346-348.
McGaughey, W. H. 1976. Bacillus thuringiensis for controlling three
species of moths in stored grain. Can. Entomol. 108: 105-112.
McGaughey, W. H., R. A. Kinsinger, and E. B. Dicke. 1975. Dispersal of
Bacillus thuringiensis spores by nonsusceptible species of stored
grain beetles. Environ. Entomol. 4: 1007-1010.
Nwanze, K. F., G. J. Partida, and W. H. McGaughey. 1975. Susceptibility
of Cadra cautella and Plodia interpunctella to Bacillus thuringiensis
on wheat. J. Econ. Entomol. 68: 751-752.
Schesser, J. H. 1976. Commercial formulations of Bacillus thuringiensis
for control of Indian meal moth. Appl. & Environ. Microbiol. 32:
508-510.
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Evaluation: -«- Due to< the larger size of the test units, efficacy must
be evaluated by different means in pilot scale tests than in laboratory
tests. A useful technique, particularly for dried fruits, nuts and tobacco
leaves, is simply to evaluate damage at suitable intervals throughout the
storage period. Random samples or entire test units can be evaluated.
Generally, the examination of entire test units will preclude their reuse,
making necessary the treatment of sufficient numbers of units to provide
replicates for examination at each time interval. Sampling also poses a
problem in that the insect infestations and resulting damage probably will
be localized within the test commodity units, and care must be exercised
to assure that the samples drawn are representative of the entire test
unit and that the sampling does not affect subsequent infestation of the
test unit. The level of damage may be assessed by applying industry grading
standards or other quantitative measurements such as percentage of nut meats
or leaves damaged. In making these assessments, insect counts should also
be made.
An indirect method of evaluating efficacy involves removing a repre-
sentative sample or group of samples from each test unit at appropriate
intervals. The samples can then be examined for infestation and placed
in containers in the laboratory and assayed for toxicity by infesting with
eggs or neonate larvae. This method will not in all cases conclusively
demonstrate efficacy, and must be supplemented by examination of repre-
sentative samples of the commodity for damage and infestation at the end
of the storage period.
A particularly useful method for detecting infestations in commodities
stored in individual bins or other containers involves the placement of
spools (ca. 2 cm wide x 4 cm in diameter) or strips (ca. 2 cm wide) of
corrugated paper on the commodity surface. Mature larvae of most species
of Lepidoptera that infest stored products will migrate to the surface
for pupation and many individuals will seek out and pupate in this
corrugated paper. The corrugated paper should be replaced weekly and
returned to the laboratory for examination. The paper can be separated
for counting the pupae, or it can be held in jars until adults emerge.
The latter is preferred if data are desired for more than one species,
as the adults are more easily identified. Other evidence of pupation
at the commodity surface such as webbing of particles or kernels together
to form cocoons may also be noted and quantitated. This means of efficacy
evaluation should be supplemented by damage assessment and laboratory
assays of the toxicity of periodically removed samples, and by damage
assessment upon termination of the test.
References
La Hue, D. W. 1969. Evaluation of several formulations of malathion
as a protectant of grain sorghum against insects in small bins.
USDA, ARS. Marketing Research Report No. 828. 19 pp.
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LaHue, D. W. 1971. Controlling the Indian meal moth in shelled corn
with dichlorvos PVC resin strips. USDA, ARS. Research Report No.
52-42. 9 pp.
LaHue, D. W., and E. B. Dicke. 1976. Evaluating selected protectants
for shelled corn against stored-grain insects. USDA, ARS. Marketing
Research Report No. 1058. 9 pp.
McGaughey, W. H. 1970. Evaluation of dichlorvos for insect control
in stored rough rice. J. Econ. Entomol. 63: 1867-1970.
McGaughey, W. H. 1971. Malathion on milling fractions of three vari-
eties of rough rice duration of protection and residue de-
gradation. J. Econ. Entomol.
Commercial-Scale Testing
Purpose and Scope: Commercial-scale tests are made only after the
application rate has been narrowed to a single dose, or at most, 2 doses.
These tests are generally considered demonstrations of efficacy, and be-
cause of the large quantities and high values of the commodities involved
may or may not include control bins or storage units. Such demonstrations
should be conducted in all geographical areas in which the commodities
are normally stored. Standard industry handling and storage practices
should be employed, and the tests should be conducted throughout an entire
storage season. Repetition of the tests in a second year is desirable.
Design and Analysis: Because pest infestation pressures may be
sporadic and artificial infestation undesirable, extensive replication
may be required to obtain valid data. Statistical analysis of the results
may be impractical as control units may not be possible. Evaluation of
efficacy may be possible only by comparisons with earlier years, with re-
motely located bins or warehouses under differing storage and pest manage-
ment conditions and practices, or with laboratory determinations of in-
festability made on samples of the commodity prior to treatment.
Application: The proposed commercial application technique should
be used. The methods differ for each commodity and methods consistent with
industry practice should be used. Because the moisture content of stored
commodities usually must be maintained within critical limits to prevent
spoilage, the minimum spray volume consistent with thorough coverage should
be used.
Evaluation: Efficacy can be quantitatively measured in the same
manner as outlined for pilot-scale tests.
References
Quinlan, J. K. 1972. Malathion surface sprays for controlling insect popu-
lations in stored shelled corn, Proc. N. Central Branch Entomol. Soc.
Amer. 27: 65-67.
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Quinlan, J. K. 1977. Surface and wall sprays of malathion for controlling
insect populations in stored shelled corn. J. Econ. Entomol. 70: 335-336,
Quinlan, J. K., and R. F. Miller. 1958. Evaluation of synergized pyrethrum
for the control of Indian meal moth in stored shelled corn. USDA, AMS.
Marketing Research Report No. 222. 13 pp.
Lepidoptera in Stored Grain
Bacillus thuringiensis and a granulosis virus have been evaluated
for .controlling infestations of the Indian meal moth and the almond moth
in stored wheat and corn. Both insect species are serious pests of most
raw and processed grains. Infestations are confined primarily to the ex-
posed surface layers of the stored commodities. The larvae feed extensively
and contaiminate the commodities with frass, insect parts, and silk. In
severe infestations in grain the webbing may restrict air movement through
the grain mass, causing moisture condensation and subsequent grain spoilage.
Economic loss results from decreased quality of the commodity brought about
by contamination, weight and nutrient loss, and grain spoilage, and from
increased processing costs.
The test methods and procedures presented here should be applicable
for all bulk stored grains, and may be applicable for other commodities
such as inshell peanuts which are stored and handled in the same general
manner as grain.
Laboratory Testing
Methods and procedures have recently been published for laboratory-
scale evaluations of insect susceptibility, application techniques, formu-
lation efficacy, compatibility with grain fumigants, stability in the grain
storage environment, and interaction with nonsusceptible insect species.
The methods used follow the general laboratory testing procedure discussed
above. The references cited should be consulted for more specific details
of the test methods.
Test Size: 10 g - 27 kg of grain held in Mason jars, cloth bags,
or metal columns may be used.
Design and Analysis; 3-5 replications are normally used. Analysis
of variance and multiple range tests are useful in interpreting the data.
Application: Aqueous suspensions or dusts can be poured onto the
grain and incorporated by tumbling, shaking or rolling the grain in jars.
Liquid volumes as high as 20 ml per kg of grain are acceptable in labora-
tory studies if the grain is held in open containers for several hours
immediately following application to permit drying.
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Evaluation: Hold the grain in filter paper or cloth-covered contain-
ers and infest with eggs or neonate larvae. Monitor adult emergence. Use 10-
25 insects in samples smaller than 200 g, 50 insects in samples of 200 to
500 g and 100 or more insects in 500 g or larger samples.
Pilot-Scale Testing
Tests beyond laboratory-scale have not been reported, however pilot-
scale tests to evaluate the efficacy of Bacillus thuringiensis for Indian
meal moth and almond moth control on stored wheat and corn are in progress.
The procedures outlined here are being used in those tests.
Test Size: The bin size to be used in pilot-scale testing on grain
depends upon the insecticide application procedure. Tests to evaluate treat-
ment of the entire grain bulk can be accomplished in 3-5 bushel lots held
in garbage pail-size containers. Tests to evaluate surface-layer treatments
for moth control should be made in considerably larger lots of grain to
accommodate the surface-layer infestation behavior of the moths. The
minimum bin size for the latter is unknown, but bins of ca. 50 bushels
should be appropriate.
Design and Analysis: The treated and untreated bins should be
replicated at least 3 times. More replications are highly desirable. A
randomized block bin arrangement may be used. Infestation and damage in
the treated and untreated bins should be compared using standard statisti-
cal procedures.
Application: The pathogen can be applied to the grain as it is being
mixed in a cement mixer or other type of rotating drum, as it is borne on
a belt-type conveyor, or as it is being moved by auger from one bin to
another. Thorough mixing with the grain may be necessary. In tests of
surface-layer treatments, the insecticide can be applied in the same manner
to the portion of grain to be treated prior to or as that portion is being
placed in the bins. Or the pathogen may be applied to the grain surface
after all the grain is in the bin. When this method is used the dose
should be divided among 2 or more applications and the grain should be
thoroughly mixed to the appropriate depth using a scoop or other implement
following all but the final application. Conventional spraying equipment
can-be used to apply liquids, and liquids or powders can be sprinkled onto
the grain if the subsequent mixing is sufficient to assure uniform coverage
of the grain.
Evaluation: The bins should be individually infested with eggs or
pupae of the insect species to be tested at intervals throughout that part
of the storage period when the temperature will permit insect development.
Efficacy of moth preventive treatments should be monitored using spools
of corrugated paper as discussed in the general methods. Other evidence
of infestation such as cocoons, live insects and webbing should be recorded
at suitable intervals, Additionally small (ca. 300 g) samples of grain
should be removed periodically from the treated zone of grain in each
bin and examined for infestation. The samples can then be frozen to
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eliminate any infestation, placed in Mason jars in the laboratory, and
infested with eggs of the insect species being tested. Reduction of
adult emergence will be a measure of efficacy.
If tests are being made against species that infest the entire grain
mass, samples should be removed at suitable intervals from all parts of
the mass and examined for infestation.
Commercial-Scale Testing
Full-scale tests should be made to confirm the results obtained in
pilot scale testing. None have been made in grain. However, they should
conform to the general test methods discussed above.
References
Kantack, B. H. 1959. Laboratory studies with Bacillus thuringiensis
Berliner and its possible use for control of Plodia interpunctella
(Hbn.). J. Econ. Entomol. 52: 1226-1227.
Kinsinger, R. A., and W. H. McGaughey. 1976. Stability of Bacillus
thuringiensis and a granulesis virus of Plodia interpunctella
on stored wheat. J. Soon. Entomol. 69: 149-154.
LaHue, D. W. 1969. Evaluation of several formulations of malathion
as a protectant of grain sorghum against insects in small bins.
USDA, ARS. Marketing Research Report No. 828. 19 pp.
LaHue, D. W. 1971. Controlling the Indian meal moth in shelled corn
with dichlorvos PVC resin strips. USDA, ARS. Research Report
No. 51-42. 9 pp.
LaHue, D. W., and E. B. Dicke. 1976. Evaluating selected protectants for
shelled corn against stored-grain insects. USDA, ARS. Marketing
Research Report No. 1058. 9 pp.
McGaughey, W. H. 1970. Evaluation of dichlorvos for insect control in
stored rough rice. J. Econ. Entomol. 63: 1867-1870.
McGaughey, W. H. 1971. Malathion on milling fractions of three varieties
of rough rice duration of protection and residue degradation.
J. Eoon. Entomol. 64: 1200-1205.
McGaughey, W. H. 1975. Compatibility of Bacillus thuringiensis and
granulosis virus treatments of stored grain with four grain fumigants
J. Invertebr. PathoZ. 26: 247-250.
McGaughey, W. H. 1975. A granulosis virus for Indian meal moth control
in stored wheat and corn. J. Econ. Entomol. 68: 346-348.
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McGaughey, W. H. Bacillus thuringiensis for controlling three species
of moths in stored grain. Can. Entomol. 108: 105-112.
McGaughey, W. H., R. A. Kinsinger, and E. B. Dicke. 1975. Dispersal of
Bacillus thuringiensis spores by nonsusceptible species of stored
grain beetles. Environ. Entomol. 4: 1007-1010.
Mwanze, K. F., G. J. Partida, and W. H. McGaughey. 1975. Susceptibility
of Cadra cautella and Plodia interpunctella to Bacillus thuringiensis
on wheat. J. Econ. Entomol. 68: 751-752.
Quinlan, J. K. 1972. Malathion surface sprays for controlling insect
populations in stored shelled corn. Proc. N. Central Br. Entomol.
Soc. Amer. 27: 65-67-
Quinlan, J. K., and R. F. Miller, 1958. Evaluation of synergized pyrethrum
for the control of Indian meal moth in stored shelled corn. USDA, ARS.
Marketing Research Report No. 222. 13 pp.
Quinlan, J. K. 1977. Surface and wall sprays of malathion for controlling
insect populations in stored shelled corn. J. Econ. Entomol. 70: 335-336.
Schesser, J. H. 1976. Commercial formulations of Bacillus thuringiensis
for control of Indian meal moth. Appl. & Environ. Microbiol. 32: 508-510.
Lepidoptera in Stored Tobacco
Bacillus thuringiensis has been evaluated for controlling the tobacco
moth in tobacco stored in farm warehouses. This insect is a pest of numer-
ous stored agricultural products, but in flue-cured tobacco it feeds and
overwinters in stored or waste tobacco in warehouses. Newly hatched larvae
feed on the lamina of the stored leaves near the stem end and feeding pro-
gresses toward the leaf tips as the larvae mature. The presence of small
larvae can be noted only upon extremely close examination. Economic damage
is caused by half-grown and larger larvae which may or may not be present
at the time of examination. Heavy larval feeding may result in the presence
of abundant silk and frass, and in 10-20% loss of leaf lamina. Such infesta-
tions have little effect on the weight of tobacco, however the effect on
tobacco grades is drastic. The presence of foreign matter (silk, frass or
insects) or insect damage reduces the assigned grade of the tobacco to "no
grade" and results in significant economic loss.
Laboratory Testing
The susceptibility of the tobacco moth to candidate insect pathogens
may be determined in small-scale laboratory tests by incorporating the
pathogen into corn meal, fortified larval diet or other cereal products
that may be suitable for larval development. Extrapolation of doses from
these tests may be useful in selecting doses for pilot testing on tobacco leaves.
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Pilot-Scale Testing
Test Size: Tobacco is normally stored in warehouses in bundles or
sheets of 68-91 kg of leaves. In the initial testing when several doses
are used, smaller bundles containing approximately 180 leaves and weighing
1.3-1.6 kg may be used. These smaller units, assembled in the same manner
as the larger commercial size bundles, permit more extensive replication
while conserving commodity.
Design and Analysis; Treated bundles should be stored in completely
randomized block arrangement. 3-5 replications should be used. Standard
statistical procedures are useful for comparing the levels of damage in the
treated and untreated bundles.
Application: The tobacco leaves are sprayed with water as they are
assembled in bundles to raise the moisture content and prevent loss in
handling. Candidate insect pathogens may be incorporated in this water and
applied directly to the individual leaves as the bundles are assembled.
Evaluation: After treatment the bundles (including replicated control
bundles) should be stored in a heavily infested warehouse or room. Insect
infestation in the warehouse should be supplemented if necessary to assure
heavy infestation pressures. Other means of pest control which might affect
the maintenance of a heavy infestation should be avoided. Exclusive use
of a warehouse would be required as marketable tobacco could not be stored
there. A sufficient number of bundles should be treated to permit removal
of replicated bundles of each dose and the controls at various time inter-
vals throughout the normal storage period.
Upon removal from storage the individual leaves in each bundle should
be examined for damage and the presence of insects, frass or silk. The
degree of damage may be assessed by applying industry grading standards or
any other quantitative means of assessment. Other factors that have been
used are percentage of leaves damaged to any degree, percentage of leaves
damaged 5% or more, percentage loss per damaged leaf, percentage of leaf
area loss per bundle, weight loss per bundle and market value loss. In
general, however, noting the presence or absence of damage or foreign matter
is sufficient, given current grading and marketing standards. If leaf po-
sition on the growing plant or other quality factors affect the damage that
may result in storage, these factors should be evaluated in these small
bundle tests.
Commercial-Scale Testing
Test Size: Data from the small bundle tests should be adequate to
permit selection of a single dose for testing on a commercial scale using
standard-size leaf bundles.
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Design and Analysis: If possible, untreated bundles should be
stored along with the treated bundles to serve as controls. However,
pest management practices which should be in use in commercial warehouses
and treatement of most of the bundles stored there may prevent all but
sporadic infestation of the control bundles. The high value of the
commodity may preclude testing in artificially infested warehouses.
Statistical analysis may be impractical.
Application: The pathogen should be applied using the proposed
commercial practice. Incorporation with water sprayed onto leaves as
they are assembled in bundles would be most desirable.
Evaluation: At intervals throughout the storage period random
bundles should be removed from storage and the leaves in approximately
1.3 kg samples from the top, middle and bottom of representative bundles
examined for damage or foreign matter in the same manner as described
for pilot-scale tests.
References
Mistric, W. J., Jr. 1977. Personal Communication. Raleigh, North
Carolina.
Mistric, W. J., Jr. 1977. Tobacco moth control on farm-stored tobacco.
Proc. Entomol. Soc. 3 27th Tobacco Workers Conf. January 1977.
Atlanta, Georgia, pp. 4-6.
Lepidoptera in Dried Fruits and Nuts
Storage Infestation
A granulosis virus of the Indian meal moth has been evaluated to a
limited extent for controlling Indian meal moth infestations in stored
inshell almonds, walnuts and peanuts, and in field run and processed
(packaged) raisins. The Indian meal moth is a severe storage pest of each
of these commodities. Economic damage results from larval feeding. While
weight loss of the commodities may be minimal, the most serious losses are
in appearance and quality, resulting from larval feeding and from con-
tamination by frass, silk and insects. Market value may be lowered or
destroyed, and processing costs may be increased as a result of the need
to clean and sort the infested commodities.
Laboratory Testing
The susceptibility of the Indian meal moth larvae and larvae of
certain other lepidopterous pests of dried fruits and nuts to candidate
insect pathogens may be determined in small-scale laboratory tests by
incorporating the pathogen into cereal product diets or other suitable
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larval foods. Extrapolation of doses from these tests may be useful in
selecting doses for testing on the commodity.
Test Size: Initial testing^ of multiple doses on a commodity can be
accomplished by treating 100-1000 g samples. Tests of this size should pro-
vide adequate data to narrow the dose range to 2 or 3 levels in subsequent
tests.
Application: Application may be made by spraying, dusting or dipping.
Evaluation; The treated commodity can be held in Mason jars or other
suitable containers and infested with eggs or neonate larvae. Larval sur-
vival or adult emergence can be used as measures of efficacy.
Pilot-Scale Testing
Test Size: 6-12 kg of nuts per test unit have been used. However,
larger quantities have been treated using standard industry equipment and
subdivided into smaller samples for evaluation of efficacy-
Application: The pathogen may be applied by spraying the commodity
as it is borne on a belt or other type of mechanical conveyor.
Evaluation; The commodities treated with each dose can be divided
into replicate samples and held in jars or cloth-covered containers such
as garbage pails or barrels. The containers can then be held in actual or
simulated storage environments and periodically infested with eggs or neo-
nate larvae. The emergence of moths from the containers should be monitored
to evaluate efficacy. If the containers are large enough, small samples
of the commodity can be withdrawn periodically throughout the storage period
and subjected to quality determination, including taste panel tests, either
through the application of industry grading procedures or other reliable
means.
Commercial-Scale Testing
While no commercial-scale tests have been reported, these tests should
conform to the general procedures discussed above.
References
Hunter, D. K. 1977. Personal Communication. Fresno, California.
Hunter, D. K., S. J. Collier, and D. F. Hoffman. 1973. Effectiveness of
a granulosis virus- of the Indian meal moth as a protectant for stored
inshall nuts: preliminary observations. J, Invertebr. Pathol, 22: 481,
Spitler, G. H., J. D. Clark, J. A. Coffelt, and P. L. Hartsell. 1974.
Malathion as a protectant for inshell almonds during storage.
J. Soon. Entomol. 67: 535-536.
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Spitler, G. H., and P- L. Hartsell. 1967. Laboratory evaluation of malathion
as a protectant for almonds during storage. J. Eoon. Entomol. 60:
1456-1458.
Spitler, G. H., P- L. Hartsell, and H. D. Nelson. 1974. Malathion pro-
tection of inshell almonds in bulk storage pilot scale study.
USDA, ARS. Research Reprot W-26. 8 pp.
Field Investigation
Large-scale tests have been made of the efficacy of Baoillus thuringiensis
for controlling the navel orangeworm in almonds. Infestation of almonds by
this pest occurs in the field at about the time of hull crack. Larvae are
transported into storage with the harvested nuts where they continue to feed
on the nut meats until mature. Reproduction may occur in stored nuts. The
methods used appear to be appropriate, but the effectiveness of the treat-
ments is too inconclusive to permit a valid assessment of those methods.
References
Kellen, W. R., D. K. Hunter, J. E. Lindegren, D. F. Hoffman, and S. J.
Collier. 1977. Field evaluation of Bacillus thuringiennis for
control of navel orangeworms on almonds. J". Econ. Entomol. 70: 332-334.
Pinnock, D. E., and J. E. Milstead. 1972. Evaluation of Bacillus
thuringiensis for suppression of navel orangeworm infestation
of almonds. J. Econ. Entomol. 65: 1747-1749.
Lepidoptera in Processed Cereal Products
Processed cereal products are subject to infestation by a number of
species of Lepidoptera and Coleoptera. The damage and economic conse-
quences of these infestations closely parallel those occurring in whole
grains. Species of both orders may cause contamination, and species of
Lepidoptera may cause webbing in the products. Either results in economic
loss through the costs of and losses through cleaning of the infested
commodity.
Studies with insect pathogens have been made only on a very limited
laboratory scale. The insects studied have included the Indian meal moth,
almond moth and others, but the Mediterranean flour moth has been the pre-
dominant species. Because only limited tests have been made, detailed
methodology is unavailable. The General Methods section and the references
cited should be useful in developing appropriate procedures.
Bacillus thuringiensis powders have been mixed with small samples
(generally less than 1000 g) of wheat flour by tumbling in jars. Eggs
or neonate larvae were introduced and larval survival or adult emergence
were monitored to determine efficacy. In one test, Bacillus fhuringiensis
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was dusted and sprayed into small sheds containing residues of flour.
These sheds were designed and specially constructed to simulate a flour
storage warehouse that would contain deposits of spilled flour in cracks
and corners and on structural components of the building. The sheds were
subsequently infested with larvae of the Mediterranean flour moth. Effi-
cacy was monitored through 2 generations of the insects by comparing in-
sect counts and the percentage by weight of the flour that was webbed in
the treated and untreated sheds.
References
Surges, H. D. 1964. Insect pathogens and microbial control of insects
in stored products. I. Test with Bacillus thuringiensis Berliner
against moths. Entomophaga Mem. HOTS. Ser. 'No. 2. pp. 323-327.
Godavaribai, S., K. Krishnamurthy, and S. K. Majumder. 1962. Bacterial
spores with malathion for controlling Ephestia eautella. Pest
Technol. 4: 155-158.
Jacobs, S. E. 1951. Bacteriological control of the flour moth,
Ephestia kuehniella Z. Proc. Soc. Appl. Bact. 13: 83-91.
van der Laan, P. A., and H. J. M. Wassink. 1964. Susceptibility of
different species of stored-products moth larvae to Bacillus
thuringiensis. Enotmophaga Mem. HOTS. Ser. No. 2. pp. 315-322.
Other Stored Commodities
Beeswax and Beecomb
Specialized techniques are necessary for evaluating microbial agents
for controlling pests of beeswax and beecomb in storage or in beehives.
References are cited for studies that have been reported for controlling
the greater wax moth and the lesser wax moth using Bacillus thuringiensis.
These studies cite methods for determining larval susceptibility using
fortified larval diets and for determining the efficacy of pathogens in-
corporated in foundation comb wax.
References
Burges, H. D. 1966. Control of wax moth by Bacillus thuringiensis. Am.
Bee J. 106(2): 48-50.
Burges, H. D. 1976. Persistence of Bacillus thuringiensis in foundation
beeswax and beecomb in beehives for the control of Galleria mellonella.
J. Invertebr. Pafhol. 28: 217-222.
Burges, H. D. 1976. Leaching of Bacillus thuringiensis spores from
foundation beeswax into honey and their subsequent survival. J.
Invertebr. Pathol. 28: 393-394.
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Burges, H. D., and L. Bailey. 1968. Control of the greater and lesser
wax moths (Galleria mellonella and Aohroia grisella) with Bacillus
thuringiensis. J. Invertebr. Pathol. 11: 184-195.
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EXHIBIT
ASSESSMENT OF PLANT COVERAGE AND FIELD PERSISTANCE
OF ENTOMOGENOUS BACTERIA AND BACULOVIRUSES
APPLIED FOR PEST CONTROL
Several approaches have been utilized to determine coverage and field
persistence of pathogens including spore assay, bioassay, tracers, scan-
ning electron microscope and collection of organisms to assess impact
of treatment.
Spore Assay (Entomogenous Bacteria)
Parts to be examined (leaves, fruit, bark, stems) are removed from
treated trees and placed in separate vials without preservative. In the
laboratory, a known area is removed from each sample (e.g., 8 mm diameter
disc from the center of each leaf with a sterile cork-borer). These are
transferred to stoppered test tubes containing a measured amount of ster-
ile water (sufficient to cover) and about ten 3 mm diameter glass beads.
The tubes are shaken vigorously (10 minutes for leaf discs), and samples
removed for serial dilution and counting. The method of Miles and Misra
(1938) using Difco^ brain-heart infusion agar plates is employed. General-
ly, seven replicate counts are made of each suspension (Pinnock, et al.
1971, 1975; Brand et al. 1975). This method is useful to assess the cover-
age of products containing Bacriltus thuringiensis. Furthermore, this
technique can be employed to assess coverage of non-bacterial control
agents (e.g., baculoviruses, chemicals) by adding a known amount of a
B. thwnngi-ens-is product and following the procedures for spore assay
(Sorensen 1977).
Bioassay
One way to conduct bioassays to determine coverage is to place all
leaves comprising one sample in an 8-oz Dixie^ cup, add ten neonate lar-
vae and cover with a tightly fitted plastic lid. The larvae are allowed
to feed on the leaves for 48 hours. They are then transferred to indi-
vidual 7-dram plastic vials containing formaldehyde-free rearing diet.
The larvae are observed at frequent intervals and mortality recorded
(Card 1975). Other bioassay methods are available (see General Reference
section).
Tracer
Coverage may also be determined by the fluorescent particle spray drop-
let tracer method (Himel 1969). Calcofluor or other fluorescent paints can
be added to the spray mixture (Davidson and Pinnock 1971). A simple method
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is to place white cards in the area to be treated and add a dye to the
spray mixture. The spray droplets will be visible on the card and pro-
vide a basis for determining spray droplet size, density and distribution.
Scanning Electron Microscope (SEM)
Many types of SEM and various procedures for using them are avail-
able. An example given here is for use with a Cambridge Mark II SEM.
Treated plant parts are affixed to aluminum foil with silver conducting
paint. Specimens are placed in a vacuum evaporator and coated with
50 A° thickness of aluminum. The aluminum foil is attached to a speci-
men holder designed for the SEM, using a silver conducting paint as glue.
The specimens are placed in the SEM and scanned. Number of particles
visible for a known area can be determined.
Collecting Organisms to Assess Impact of Treatments
Available stages of target and non-target organisms are collected and
held individually under conditions favorable for survival and development.
The organisms are observed frequently, and all dead individuals are diag-
nosed to determine cause of death.
For codling moth on apple, for example, this can be done by collect-
ing a specific quantity of visibly infested fruit at predetermined inter-
vals following applications (e.g., ten fruit per plot at 0, 1, 2, 4, 8, 16
days following treatment) (Falcon et al. 1968).
Refevenees
Brand, R. J., D. E. Pinnock, K. L. Jackson, and J. E. Milstead. 1975.
Methods for assessing field persistence of Bacillus fhuving'iens-Ls
spores.
Falcon, L. A., W. R. Kane, and R. S. Bethell. 1968. Preliminary evaluation
of a granulosis virus for control of the codling moth. J. Eoon.
Entomol. 61(5): 1208-1213.
Card, I. 1975. Utilization of light traps to disseminate insect viruses
for pest control. Ph.D. Dissertation. University of California,
Berkeley. 174 pp.
Himel, C. M. 1969. The fluorescent particle spray droplet tracer method.
J. Eoon. Entomol. 62: 912-916.
Legner, E. F., and E. R. Oatman. 1962. Effects of Thuricide on the
eye-spotted bud moth, Spilonata ooellcma. J. Eaon. Entomol. 55: 677-678.
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Miles, A. A., and S. S. Misra. 1938. The estimation of the bactericidal
power of the blood. J. Eyg. 38: 732-748.
Oatman, E. R., and E. F. Legner. 1964. Additional studies on the effect
of Bacillus thuringiensis on the eye-spotted bud moth, Spilonota
ocellana. J. Eoon. Entomol. 57: 294.
Pinnock, D. E. 1975. Pest populations and virus dosage in relation to
crop productivity. In Baculoviruses for Insect Pest Control: Safety
Considerations. Am. Soa. Micro. PP- 145-154.
Pinnock, D. E., R. J. Brand, and J. E. Milstead. 1971. The field per-
sistence of Bacillus thuringiensis spores. J. Invertebr.
Pathol. 18: 405-411.
Pinnock, D. E., R. J. Brand, J. E. Milstead, and K. L. Jackson. 1975.
Effect of tree species on the coverage and field persistence of
Bacillus thuringiensis spores. J. Invevtebv. Pathol. 25: 209-214.
Sorensen, A. A. 1977. Research on the microdroplet application of insect
pathogens. Ph.D. Dissertation. University of California, Berkeley.
158 pp.
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EXHIBIT 2
SUGGESTED PROTOCOL FOR EVALUATION OF MICROBIAL PATHOGENS
FOR CONTROL OF SELECTED INSECTS ON SOYBEANS
Target Insects:
Soybean Looper, Pseudoplusia includens
Cabbage Looper, Triahoplusia ni
Corn Earworm, Heliothis sea
Microbial Pathogens:
Pseudoplusi-a NPV
Trichoplusia NPV
Eeliothis NPV
Bacillus thuringiensie var. kurstaki
Plot Size:
0.1 Acre (minimum)
Replications:
4 (minimum)
Treatments and Rates:
9 Pseudoplusia NPV for soybean looper control
(1) 20, 40 and 8, L.E.*/A and untreated check
L.E. = Larval Equivalent = 6 x 10^ Polyhedral Inclusion Bodies
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Triehoplusia NPV for cabbage looper control
(1) 10, 20 and 40 L.E./A and untreated check
Heliothis NPV for corn earworm control
(1) 20, 40 and 80 L.E./A and untreated check
B. Thur-ingiens-is var. kurstdki for soybean looper control
(1) 2 x 109> 4 x 109 and 8 x 109 I.D./A and untreated check
Combinations of NPV's for control of mixed target insect populations
(1) When mixed populations of the target insects occur, the NPV
of each species should be tested independently and in combina-
tion at the rates above, e.g., with mixed soybean looper and
cabbage looper populations, treatments would be as follows:
Pseudoplusia NPV - 20, 40 and 80 L.E./A; Triohoplusia NPV -
10, 20 and 40 L.E./A; P. NPV - L.E./A + T. NPV - 10 L.E./A;
P. NPV - 40 L.E./A + T. NPV - 20 L.E./A; P. NPV - 80 L.E./A
+ T. NPV - 40 L.E./A and untreated check.
Time and labor permitting, all possible rate combinations would
be desirable.
Formulations:
PseUdoplus'ia. NPV may be provided by University of Arkansas. (See
attached memorandum for others).
Application Equipment:
Boom-type sprayer
Initial Application:
To be determined by populations in each area a minimum of 10-20,000
larvae/A suggested. Applications should be made before larvae reach the
third stadium.
Spray Interval
With uneven aged populations, applications should be continued at 4-6
day intervals until a majority of the population reaches the fourth
stadium.
"U.S. GOVERNMENT PRINTING OFFICE: 1978 260-880/Z3 1-3
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