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Group Title: Annual report, Bean/Cowpea Collaborative Research Support Program
Title: Annual report
ALL VOLUMES CITATION THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
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Permanent Link: http://ufdc.ufl.edu/UF00055292/00004
 Material Information
Title: Annual report
Physical Description: v. : ill. ; 23-28 cm.
Language: English
Creator: Bean/Cowpea Collaborative Research Support Program
Publisher: Michigan State University
Place of Publication: East Lansing Mich
Publication Date: 1982
Frequency: annual
regular
 Subjects
Subject: Beans -- Periodicals   ( lcsh )
Genre: periodical   ( marcgt )
 Notes
Numbering Peculiarities: Issued in parts: Part one. Technical summary.--Part two. External review panel.
General Note: Description based on: 1983.
 Record Information
Bibliographic ID: UF00055292
Volume ID: VID00004
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: oclc - 19930082
lccn - sn 89013327

Table of Contents
    Front Cover
        Page 1
    Introduction
        Page 2
    Main
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Full Text































































Staff
I fliversit
48824 .S.A


V







INTRODUCTION


The Bean/Cowpea CRSP is a coordinated effort established on September 30,
1980 to address hunger and malnutrition in Africa and Latin America through
research on the production and utilization of beans (Phaseolus vulgaris) and
cowpeas (Vigna unguiculata).

Beans and cowpeas are dietary staples in the countries associated with
this CRSP. Among the poor, these legumes provide the major source of high
quality, affordable protein, as well as an important source of B vitamins.
The CRSP focus is on beans and cowpeas grown as food for household consumption,
rather than as export crops.

Based on a global plan developed in concert with Host Country colleagues,
the CRSP is made up of a series of discrete butintegrated international
reasarh projects involving teams of scientists collaborating in a study of
individual facets of the overall plan. Eighteen research projects, each led
by a Principal Investigator from one of the U.S. research institutions, were
developed from over 80 proposals initially received.

These vigorous international research partnerships directly involve
research institutions in 13 Host Countries, 2 International Centers and 14
U.S. agricultural research institutions, including the nine having lead roles
in the CRSP. One of the ten institutions identified by the CRSP Grant has
withdrawn, reducing the total number of lead institutions.

Special emphasis is placed on the needs and resources of the subsistence
farm family. These family units are major producers and consumers of beans
ana cowpeas. As a group they are highly susceptible to problems of hunger,
malnutrition, and poverty. Resources for food production are very limited,
including productive soils, water, improved seed, fertilizer, pesticides, and
machinery. Climates are often hostile, and farming operations are usually
conducted by hand labor, although animal power is used when available.

Storage, preparation, and human utilization of beans and cowpeas present
additional problems. Storage loss to insects is high, and represents
debilitating loss of labor inputs as well as other resources. Traditional
bean and cowpea preparation methods require high investments of not only water
and fuel, but time and labor as well.

In much of the world, women are uniquely involved in production, storage,
and utilization of beans and cowpeas. Therefore, it is reasonable that the
Bean/Cowpea CRSP should maintain a special purview of the role of women and of
the implication of CRSP research on women in its activities and objectives.
The objective that application of CRSP findings must maintain or improve the
quality of family ecology follows easily from the focus on women in
international development.







Goal

Reflecting the mission of the "Famine Prevention and Freedom from Hunger
Act" (Title XII) under which the program is funded, the goal of the
Bean/Cowpea CRSP is to make a substantive contribution to the eradication of
hunger and malnutrition in identified developing countries where beans and
cowpeas are a major source of calories and protein.


Objectives Distributed Among Projects

Variety improvement ut } C &7t,< ).
Insect and disease control
Productive and stable farming systems, within which beans and cowpeas
are grown
Efficient nitrogen fixation and soil phosphorus utilization
Drought and heat tolerance
Improved nutrition and digestibility'
Improved seed and seed availability
Reduced cooking requirements
Improved storage and methods of preparation
Understanding of the socioeconomic implications of agronomic intervention


Objectives Shared by All Projects

Training Host Country professional and technical personnel
Development of research capability in collaborating institutions
Participation of senior U.S. researchers with their counterparts in
research institutions in Host Countries
Appropriate attention to the involvement of women

Bean/Cowpea CRSP Strategy

The Bean/Cowpea CRSP strategy is to identify universal constraints to
production, availability and consumption of beans and cowpeas, and to address
them through research in settings where they have unique local importance.

The CRSP avoids duplicating existing research. It participates with
national programs and regional and international centers in identifying -
constraints, and in planning and executing research. It will utilize the same
linkages to disseminate its research findings.

Unique Features of the Bean/Cowpea CRSP .

While the Bean/Cowpea CRSP shares common features with other CRSPs it has
organizational or administrative characteristics which tend to give it. a
"separate identity. These include:








1. A manageable number (9) of Title XII Lead Institutions and a
straight-forward organizational structure.

2. Collaboration with a number of Host Countries including: Botswana,
Brazil, Cameroon, Dominican Republic, Ecuador, Guatemala, Honduras,
Kenya, Malawi, Mexico, Nigeria, Senegal, Tanzania.

3. Diffusion of 18 projects in East and West Africa, Central and South
America and the Caribbean, averaging $123,000 USAID annual
contribution per project (FY83).

One country hosts 3 projects
Three countries host 2 projects
Nine countries host single projects

4. Identification of the entire array of projects, U.S. and Host Country
institutions, and investigators in the planning process, prior to
operational establishment of the CRSP.

5. A Women in International Development Specialist on the CRSP
Ma4ngement Office staff.

6. U.S. Lead Institutions prepared to provide programmatic and fiscal
management of research on either beans or cowpeas, supplemented by
researchers from collaborating U.S. institutions to create teams with
broad technical expertise and unique resources.

One project includes 5 collaborating U.S. institutions
One project includes 3 collaborating U.S. institutions
Five projects include 2 collaborating U.S. institutions
The remaining 10 projects involve a single U.S. institution

In addition, Boyce Thompson Institute for Plant Research will
manage one project.

Features of CRSPs in General

The Bean/Cowpea CRSP is one of seven current CRSP programs. Unique
advantages of CRSPs include:

a. Involving leading scientists from U.S. institutions, many of whom
would not otherwise be engaged in international work.

b. Obtaining major resource contributions from U.S. and Host Country
institutions, which combine to nearly equal the USAID contributions.

c. Creating scientist-to-scientist and institution-to-institution
linkages, with major emphasis on program activities in Host Countries.

d. Accruing dual nfi to both U.S. and Host Country agriculture
which offer an incentive to state legislatures and universities to
participate.








ORGANIZATION OF THE BEAN/COWPEA CRSP


The Bean/Cowpea CRSP Grant was awarded by USAID to Michigan State
University on September 30, 1980. Michigan State University was designated as
the Management Entity (ME) of the CRSP. The University created a Management
Office (MO) for CRSP activities. On January 2, 1981 the CRSP Management
Office staff was expanded to its present size, and moved into new offices.
Three groups--a Board of Directors, a Technical Committee, and an External
Review Panel--work closely with the University and MO to guide the CRSP in
areas of policy, budget management, technology, and review.

The Board of Directors

The Board of Directors, herein referred to as the Board, is the executive
committee for CRSP policy and budget. It consists of 5 institutional
representatives (IR's), elected for 2-year terms by the entire group of 9
IR's. IR's are designated by the presidents of their institutions to
represent them for CRSP policy and administrative matters. They are typically
administrators of international programs, deans, or experiment station
directors. The Board elects a chairman and secretary.

The members of the Board of Directors for FY-82 were:

Dr. J.F. Metz, Jr. Dr. Donal Johnson
(Chairperson of the Board) (Secretary of the Board)
Director, International Agriculture Dean, College of Agricultural
Cornell University Sciences
Colorado State University

Dr. Dale Harpstead, Chairperson Dr. Robert Hougas, Director
Department Crop and Soil Sciences Agricultural Experiment Station
Michigan State University University of Wisconsin

Dr. Calvin Qualset, Associate Dean
College of Agriculture and Environmental Sciences
University of California-Davis

The Board held two meetings during the year. Action taken at those
meetings included:

1. Approval of a procedure for effecting changes in projects. , *

2. Recommendation of External Review Panel members forwarded to JRC for
approval...

3. Review and approval of project proposals, following recommendation to
the Board by the Technical Committee and independent review by the
Board.

4. Review and approval of budgets for research projects and the'i-
Management Office.







5. Affirmed, by motion, the requirement that not less than 50% of USAID
funds for support of projects be spent in or directly on behalf of
Host Country CRSP activities.

6. Met with Dr. Jack Robins, USAID, who has responsibility for CRSPs,
and other AID and BIFAD staff.

The Technical Committee

The Technical Committee, herein referred to as TC, advises the Board, ME,
and MO in areas of research technology, project management, and technical
review. It has specific responsibility for review of candidate projects for
inclusion in the CRSP. Having completed intensive review of the 18 CRSP
projects, the TC has identified an array of new initiatives including project
review, review/revision of the CRSP global plan, evaluation of proposals for
new CRSP research, establishment of priorities for new and existing research,
development of criteria for project evaluation, development of plans for
regional meetings of TC with PIs from East Africa, West Africa/Botswana, South
America, and Central America/Carribean/Mexico. The TC consists of five
investigators engaged in CRSP projects from U.S. institutions, plus two
international members one from an international research center, and one
from a participating Host Country institution. TC members are appointed to
2-year terms by the Board.

The members of the Technical Committee for FY-82 were:


Dr. Barbara Webster (Chairperson)
Department Agronomy & Range Science
University of California-Davis

Dr. Fred Bliss
Department of Horticulture
University of Wisconsin


Dr. Dermot
Department
University


Coyne
of Horticulture
of Nebraska


Dr. Aart van Schoonhoven,
Coordinator
Bean Program CIAT
Cali, Colombia


Dr. Larry R. Beuchat (Secretary)
Department of Food Science
University of Georgia

Dr. Azuka Dike
Department of Sociology/Anthropology
University of Nigeria

Dr. Julio Lopez-Rosa
Coordinator of International
Programs
College of Agricultural Sciences
University of Puerto Rico


The Technical Committee held 4 meetings during the year. Actions taken
included:

1. Substantive review of each project proposal on the basis of initial
review by committee members individually, the TC agreed as a group to
forward proposals to the Board for approval, or return to the
Principal Investigator for adjustments.








2. Participation on the Bean Workshop held at CIAT, Colombia.

3. Endorsed the nomination of a replacement (Dr. P. Markakis) for P.I.
Dr. A. Akpom, who retired from MSU.

4. Supported choice of Mexico as the site for Adams' stress physiology
project which was originally slotted for CIAT.

5. Supported the goal of baseline data collection for each project.

6. Responded, positively, to proposal for Nitrogen Fixation Workshop for
CRSP trainees in 1983 at the University of Wisconsin.

7. Developed procedures for pre-publication exchange of technical
information.

8. Reaffirmed the policy of priority for training Host Country
scientists.

9. Developed an array of proposed TC activities for future years.

The External Review Panel

The External Review Panel, herein referred to as ERP, is advisory to
USAID, the ME, and the CRSP, and will be responsible for review and evaluation
of CRSP management and the progress of research activities. The Panel
members, nominated by the Board, and approved by JRC are:

Clarence C. Gray,III (Chairman)
Associate Director for Agricultural Sciences
The Rockefeller Foundation

Mel Blase, Professor Hugh Bunting, Professor
Agricultural Economics Agricultural Development Overseas
The University of Missouri University of Reading

Luis Camacho Peter E. Hildebrand, Professor
INSTOY Plant Breeder Food & Resource Economics Department
University of Puerto Rico University of Florida

Antonio M. Pinchinat, Coordinator Charlotte E. Roderuck, Director
Agricultural Production Committee World Food Institute
IICA, Costa Rica Iowa State University

The ERP will convene for the first time October 31 November 5, 1982.








The Management Office


The Management Office, herein referred to as MO, is charged with
facilitating activities of the CRSP on behalf of the ME, providing staff
resources to the Board and TC, and supporting activities of the ERP. MO
consists of a Director, Deputy Director, Women in n-ternational development
(WID) Specialist, and Administrative Officer, plus office staff, totalling
5 1/4 FTEs.

The Management Office staff and facilities were established within FY-81.
In January, 1981, the staffing was complete, and presently includes:

Dr. Donald R. Isleib, Director
Dr. Pat Barnes-McConnell, Deputy Director
Mrs. Nancy W. Axinn, WID Specialist
Mr. George A. Davies, Administrative Officer
Mrs. Darlene Romsos, Administrative Assistant
Mrs. Mary K. Carter, Secretary

The offices, in 200 Center for International Programs, provide adequate
work space and facilities for the on-going activities of the CRSP. Word
processing equipment, capable of handling documentation and the accounting and
bookkeeping for the multiple budgets of the CRSP, was purchased and installed.

Staff activities in the Management Office in FY-82 focused on support to
the research projects. This included completion of documentation for those
projects not initiated in 1981; initiation of budget mechanisms for each
project; negotiation, as needed, with the grants and contracts offices of the
lead institutions to establish appropriate vouchering procedures; interaction
with appropriate USAID officials to obtain equipment purchase and travel
clearances; and mediation between research scientists to implement the
research process. Telephone conferences with PIs occur daily.

Management Office staff have participated in collaborative planning
meetings with project personnel in the following projects: the team from
Senegal with their University of California-Riverside collaborators;
representatives from the 5 universities collaborating with INCAP with Dr.
Bressani; Dr. Ndunguru from Morogoro, Tanzania and the scientists from the
Universities of Illinois and Washington State; the Experiment, Georgia
scientists with Dr. Ngoddy from the University of Nigeria; the University of
Nebraska team with Dr. Paniagua from the Dominican Republic; and Dr. Drew from
the Nigeria-Jos project with MSU researchers. The CRSP Director travelled to
Botswana with the PI from Colorado State to develop the research project there
in collaboration with the Ministry of Agriculture in Botswana, and
USAID/Botswana.

Visitors to the Management Office from the Host Countries have included
scientists from Brazil, Nigeria and FAO.








The Management Office staff participated in Regional meetings with all the
PIs, to begin preparation for the External Review (FY83), and to develop work
plans and budgets for the next years of the projects.

Members of the MO participated in the Bean Scientists Workshop in CIAT and
the Cowpea Review at IITA.

Interface with other organizations included presentations to Association
of U.S. University Directors of International Agricultural Programs
(AUSUDIAP), and initial discussions with the Latin American FAO Office.

The MO maintains an ongoing interaction with the Management Offices of the
other CRSPs, and with USAID and BIFAD staff members concerned with CRSPs.
This included a two day presentation by the Bean/Cowpea and other CRSPs to
USAID and BIFAD in January, and a two-day Bean/Cowpea meeting of the Board and
Technical Committee in Washington in May with representatvies of these same
organizations.

Considerable effort this year went into preparations for the first annual
review of the Bean/Cowpea CRSP. Candidates were nominated by the TC for
consideration by the Board. The Board developed a roster which was
recommended to JRC. Director Isleib presented this roster to JRC, which
requested changes. A revised roster was developed after consultation with AID
staff, chairpersons of the TC and Board, and an ad hoc committee of JRC. The
revised roster was approved by JRC.

A planning meeting of the ERP Chairman, AID staff, and CRSP Director and
Deputy Director was held in Washington for the purpose of developing
pre-review and review week procedures and schedules. These plans include
pre-review visits by review panelists to U.S. principal investigators at the
U.S. lead institutions.

Preparations were made for 4 meetings of the Board of Directors, although
only 2 were held. The MO staff also prepared agenda and background papers for
the 4 Technical Committee meetings.

Publications from the MO during FY82 included the first Annual Report, and
two issues of the newsletter.

The Women in Development Specialist focused on increasing the awareness of
the scientists on women as cultivators, processors and consumers of beans and
cowpeas. News items and publications to strengthen the WID component of the
CRSP were shared with the scientists. Many research teams reflected this
awareness by including women as trainees and as collaborating scientists.
Several Host Country teams include women scientists. Additionally, the male
scientists are expressing a sensitivity to WID issues in developing their
research plans, and in data analysis.








Part of the WID work plan was to share insights as they evolve from the
CRSP, and to document the research process as it related to WID.
Contributions to this goal include a presentation on "Women in Farming
Systems" made to AUSUDIAP and a chapter, co-authored with other social
scientists "International Interdisciplinary Research Management: A Case Study"
for the University of Minnesota publication: Enabling Interdisciplinary
Research in Agriculture. Continual interaction with the WID group at MSU and
WID groups and individuals on other campuses in the United States also
supports this goal.

PROJECT ANNUAL REPORTS: FY-82

Introduction

Initiation of the eighteen research projects identified in the CRSP Grant
was among the first objectives adopted by the MO. The CRSP Board of Directors
and MO staff agreed on the concept that no project activity should be
undertaken with CRSP funds in the U.S. until such time as the project was
ready for invitation in an identified Host Country. Furthermore, all
participants, including Host Country governments and USAID Missions, would be
involved in project development and committed to project proposals and budgets
before any Subgrant to a U.S. Lead Institution would be established.

Unlike the project reports for FY-81 which emphasized project
administrative arrangements for initiation of research, these annual reports
focus on the substance of the research accomplished. Both in Host Countries,
and in the United States, considerable research data have been reported. In
some projects unusual amounts of research have been forthcoming in the first
full year of activity. Delays in providing approval for equipment essential
to some research activities has slowed progress in some projects.

A number of the research scientists have been able to present their data
to professional meetings during this year, and several articles in
professional journals have been published about CRSP related research. These
are identified in the individual project reports which follow.

Review of Principal Investigator's annual reports reveals that in addition
to the planned collaborative research, there is a spin-off of interaction
among concerned scientists, some who are project participants, and some who
are not, culminating in a greater focus on beans and cowpeas than was
envisioned originally. A network of research practitioners is developing
which will strengthen the scientific field in the United States and throughout
the developing world.

Women scientists are involved in many of the Bean/Cowpea research
projects. In the U.S. they are Principal Investigators in two projects. They
are co-investigators in five projects. Women technicians and graduate
assistants are involved in the research in many of the projects.








In the Host Countries, a few women scientists have been involved as
members of the research teams. In Brazil, women scientists have been
identified and will be used as consultants. Women have been nominated to come
to the United States for training in several of the projects. In Malawi,
women from Bunda College participated in the field survey conducted during the
summer months of 1982. Most of the women scientists from the United States
spent extended periods of time this year in field work at Host Country sites.

Training of Host Country scientists is an important component of each CRSP
research project. Candidates for advanced graduate training, or specific
short term training appropriate for the research project have been
identified. Nearly every project has people from Host Countries already in
graduate school at the collaborating U.S. institutions in Masters and PhD
degree programs. U.S. research scientists are also receiving training in
language and socio-cultural aspects of the areas in which they are working.
Short-term training in the U.S. for Host Country scientists in specific
scientific techniques needed for research activities is in process. Details
of these training arrangements can be found in the full text of the Principal
Investigators' Annual Reports.

MO summaries from Principal Investigators' annual reports follow. The
complete FY-82 annual reports as submitted by the Principal Investigators are
available as appendices on request.








BRAZIL/BOYCE THOMPSON INSTITUTE/ROBERTS

"INSECT PATHOGENS IN COWPEA PEST MANAGEMENT SYSTEMS FOR DEVELOPING NATIONS"


Cowpeas are a principal staple in the diet of subsistence farmers in
sub-saharan Africa, parts of Central America, and northern South America.
Insect pests are among the most serious constraints to adequate cowpea
production in these truly poor areas of the world. The areas involved all
suffer from imbalance of payments, and one of the objectives of the CRSP
project will be to develop insect control measures based on insect disease
agents which can be produced locally with a minimum of capital outlay.
Another objective will be to train Latin American scientists in the use of
this relatively new methodology so they can expand the work into larger
areas. Surveys conducted to date by combined CRSP and Brazilian research
teams have found that insect diseases are active in cowpea fields, sometimes
at high levels. This work has been conducted from the newly established
CRSP-supported insect pathology laboratory at the Brazilian National Rice and
Bean Research Station (CNPAF) in Goiania. College students are currently
studying with the full-time CRSP-supported insect pathologist assigned by the
project to CNPAF. A short course on insect pathology and microbial control
will soon be conducted at this station for 15 Latin American students. The
outcome of this project will be insect control measures of low cost and high
efficacy available to subsistence farmers for cowpea production and possibly
for other crops. In addition, a cadre of newly trained insect pathologists
will be available to a number of developing nations to assist them with their
* insect-control problems.


'1









BRAZIL/BOYCE THOMPSON INSTITUTE/ROBERTS


"Insect Pathogens in Cowpea Pest Management Systems for Developing Nations"

Dr. Donald W. Roberts, Insect Pathology Resource Center, Boyce Thompson
Institute (PI); Dr. Richard Soper, USDA Insect Pathology Research
Unit, Boyce Thompson Institute, Co-PI; Dr. Richard A. Daoust,
Research Associate in Brazil, Insect Pathologist, Boyce Thompson
Institute; Mr. Michael Ward, Boyce Thompson Institute; and
Dr. Almiro Blumenschein, Centro Nacional de Pesquisa de Arroz e Feijao
(CNPAF), EMBRAPA (PI); Dr. Evane Ferreira, Entomological Research,
CNPAF (Co-PI); Dr. Gerson Pereira Rios, Cowpea Plant Pathologist,
CNPAF, CO-PI; Mr. Belmiro Periera das Neves, Cowpea Entomologist,
CNPAF: Co-PI; Mr. Joao Prategil Pereira de Araujo, Cowpea Research,
CNPAF

Introduction

Insect pests and plant diseases are the most important constraints to
cowpea production and storage in Brazil and other countries where cowpea is
grown as a staple food. Chemical insecticides are effective in the control of
some insect pests, but since cowpea is frequently produced by poor farmers as
a subsistence crop, the cost of importation and utilization of chemicals is
prohibitive. The development of varietal resistance against some of the major
pests has been unsuccessful; and some cowpea breeders have expressed the
* opinion that the development of varietal resistance against many different
pests simultaneously will be difficult to achieve.

Therefore, an inte rated approach to pest control must be sought that can
incorporate information concerning the socioeconomic structure of rural
subsistence farmers#. "-crobial agents pathogenic to the principal cowpea
pests are being considered. Since virtually no research information of either
an applied or basic nature is available, and virtually no experienced insect
pathologists have worked on this crop, alleviation of these limitations will
be sought by (a) conducting basic and applied research in the cowpea-producing
nation of Brazil and in the U.S. (b) stationing an experienced insect
pathologist in Brazil to conduct research with Brazilian scientists, and (c)
training scientists and aspiring scientists.

Since the project commenced almost one year later than anticipated, what
had been planned for 1981 was done in 1982. This one year offset will, of
course, continue throughout the life of the project. With this adjustment
taken into consideration, the work is fully on schedule.

First Year (1982) Project Objectives

a. Establish the Insect Pathology Resource Center (Brazil) in
CNPAF/EMBRAPA, Goiania, including: 1) research laboratory with specialized
insect pathology supplies; 2) training facility; 3) pathogen culture
collection, primarily of bacterial and fungal isolates, with an indexing
system, and which utilizes several storage methodologies for long-term
maintenance of strains collected locally and obtained from culture
collections; and 4) literature and information center on insect pathology
(Including books, reprints, and a computer-generated literature survey).








S b. Condtct surveys in cowpea producing areas of Brazil to 1) collect
(isolate in culture) some of the principal pathogens associated with the major
insect-pest species; 2) provide information on the natural incidence of
diseases (i.e., provide epizootiological data); and 3) identify researchers
and field stations which will collaborate with the CRSP project for further
survey work and for preliminary field trials.

c. Establish lines of communication.

d. Identify and characterize newly discovered pathogens.

e. Establish insect colonies, develop bioassays, and initiate screening
tests to identify microbial isolates with promise for pest control.

f. Seek Latin American students for training in insect pathology and
microbial control.

g. Provide basic training in insect pathology techniques to the
entomologists, plant pathologists, and technicians at CNPAF.

Summary of Achievements

Our CRSP grant started in mid-September with a trip by Dr. D. Roberts,
Principal Investigator, and Dr. R. Daoust, U.S., host-country investigator, to
CNPAF, Goiania, Goias, Brazil to discuss our research and training program
With host country scientists and to finalize details of the execution of our
program. In late October, Dr. R. Daoust and family was moved to Brazil to
begin the program. Three major survey trips were made by the host country
research associate (Dr. Daoust) and the Brazilian counterpart entomologist
(Dr. Belmiro das Neves) to the North and Northeast regions of Brazil. The PI
(Dr. Roberts) and Co-PI (Dr. Soper) as well as other staff scientists from
CNPAF accompanied Dr. Daoust and Mr. das Neves'on two of these trips. In
addition, surveys were conducted continually at CNPAF during the initial 9
months of the program. This was possible at the National Bean and Rice Center
since irrigation systems now make it possible to grow cowpea throughout the
year. Entomopathogenic fungi were the most common pathogen group recovered
although some virus-diseased insects were also found. Bacterial diseases
appeared to be considerably less common among pest species on cowpeas. Six
entomopathogenic fungi were recovered from 6 field sites in the Amazonas
region and 12 were recovered from 50 small subsistence farms and 14
experimental sites in the Northeast. Several viruses have also been recovered
from the Northeast and CNPAF, but since these are difficult to identify they
have not been classified but will be stored for future reference. The
predominant pathogens recovered from the principal cowpea pests have been
Beauveria bassiana and Metarhizium anisopliae. Eleven fungal isolates have
been recovered from the cowpea curculio, considered the principal pest in
Northeast Brazil. Similarly, 11 isolates were obtained from Chrysomelid,
leaf-feeding beetles including Ceratoma arcuata and Diabrotica paranoense.
Numerous isolates have been recovered from other principal pest species
including the leafhopper, Empoasca kraemeri, various pod-sucking bugs such as
Piezodorus guildini, and the pod borer Maruca testulalis. In the case of E.
kraemeri, several extensive epizootics were located. The entomophthorales
isolate was cultured and returned to CNPAF and BTI for preservation,
identification, and possible development for field control while the H. guyana
strain was highly fastidious, and attempts to culture it were unsuccessful.








In addition to surveys made to discover pathogens of pest species,
I attempts were made to determine which insect species were most important in
cowpea-producing regions of Northeast Brazil. Pest species varied
significantly in different geographical locations with both climatic and
agronomic differences. In general, the cowpea curculio, Chalcodermus aeneus,
the leafhopper, E. kraemeri, the lesser cornstalk borer, E. lignosellus, the
lepidopteran pod borer Etiella sp., and pod-sucking pentatomids were most
important.

In addition to pathogen surveys, the gathering of epizootiological data,
and estimations of pest populations, other progress made on survey trips
included the establishment of contacts with scientists, extension agents, and
administrators in various research centers and universities throughout the
North and Northeast. By visiting more than 50 small-scale and subsistence
farmers in the North and Northeast, and through lengthy discussions involving
sociological, economic, political, cultural, and agricultural aspects of
cowpea production and utilization, both CRSP and cooperating Brazilian
scientists became more aware of factors associated with cowpea production and
use in Brazil.

Several insect colonies were established, and screening tests with
entomopathogenic fungi were initiated to identify microbial isolates with high
potential for pest control. Since cowpea-pest species were not being
maintained at CNPAF, Brazil prior to the arrival of Dr. Daoust, a major effort
wqs devoted during the first year to the establishment of laboratory
colonies. At present, colonies of Chalcodermus aeneus, Callososobruchus
maculatus, Ceratoma arcuata, and Diabrotica paranoense are being established
in a newly constructed insectary at CNPAF, Brazil. This work has been
hampered by delays in construction of the rearing facility and delays in
hiring technical help. The above mentioned species have all been successfully
reared on natural foods for at least 2 generations; however, a production
schedule for supplying large numbers of insects for bioassays has still not
been developed. As a result, preliminary screenings of fungal isolates
against Chalcodermos aeneus and Ceratoma arcuata were conducted using
field-collected insects, while those against Collosobruchus maculatus used
laboratory-reared insects. The use of field-collected Chalcodermus aeneus
presented no problems except that adult ages were unknown, and specimens were
only intermittently available.

Various screening bioassays were performed to assess infection
methodologies applicable to Chalcodermus aeneus. Spores of Metarhizium
anisopliae and Beauveria bassiana were applied both to soil and foliage, the
foliage applications employing known and unknown spore concentrations. Fungal
strains isolated from naturally infected cowpea curculios in Brazil and
others obtained from the BTI culture collection were tested. In general, it
was shown that the B. bassiana isolates were considerably more virulent to C.
aeneus than were those of M. anisopliae.

In tests conducted with entomopathogenic fungi against Ceratoma arcuata,
several application methodologies were used, and 17 M. anisopliae, 16 B.
bassiana, and 1 Nomuraea rileyi isolates were tested. Control mortalities
* were high in many experiments. Nevertheless, one isolate (CP5) of B.
bassiana, obtained from a naturally infected C. arcuata adult found in a
cowpea field in the Amazonas region of Brazil, proved highly virulent to C.








arcuata adults, producing 100% mortality with LT50 values of 4.57 and 7.09
days in two laboratory tests. In addition, the isolate grew and sporulated
well on artificial media. In screening tests with strains for M. anisopliae
and B. bassiana isolated from a variety of insects from world-wide
geographical regions, it was found that while both fungal species were
pathogenic, virulence was highly dependent upon the isolate tested. More
quantitative and extensive screening tests to be conducted during the second
year should elucidate the potential of these and other isolates against
coleopterous pests. The Nomuraea rileyi isolate proved to be relatively
avirulent even though spore concentrations were high and spore viability was
91%.

Although work in the production of microbial control agents area was not
planned to commence until the second year, studies were initiated on the
production and formulation of Beauveria bassiana, the fungal pathogen we have
isolated most frequently from cowpea pests in Brazil.

The entomopathogenic fungus, B. bassiana, is produced at the farm-commune
level in the P.R.C. for local control of insect pests such as the European
corn borer, Ostrinia nubilalis. The field efficacy of a granular formulation
of Beauveria against the corn borer is reported to be 80 to 90%. Beauveria
bassiana produced at county agricultural stations in the P.R.C. has proved to
be an effective method of control for the pine caterpillar, Dendrolimus
punctatus.

The culture media for the fungus consist of raw materials produced on the
farms: wheat and ricebran, cornstalk powder, grasses, or compost humus.
Inoculum buildup is accomplished in 3 stages on 1) sterile slices of potato in
test tubes, 2) sterile bran and water in jars, and 3) nonsterile media in
wooden trays in incubation rooms or spread on outdoor soil beds. Production
on soil bveds is utilized in P.R.C. to afford a heat-sink for the excessively
high level of heat of metabolism associated with fungus growth.

The production of B. bassiana using this low technology method was
undertaken at Boyce Thompson Institute in order to assess the potential of the
procedure as a means of large-scale production of inoculum by local control
programs in cowpea and bean producing areas of Brazil and elsewhere. In
particular, the methods investigated in this study may have applications in
subsistence-level agricultural systems where cost is a severely limiting
factor.

The results successfully demonstrated the capacity of this production
system to produce large quantities of infectious material at extremely low
cost.

1983 Objectives

a. Continue development of the Insect Pathology Resource Center (Brazil).

b. Continue dissemination and exchange of insect pathology and microbial
control related information through the various lines of communication
*established in Brazil in 1982 (including contacts with Brazilian scientists,
regional grower associations, and individual subsistence farm families).








c. Continue and extend the survey for pathogens of cowpea pests in
S Brazil. New sites will be surveyed, and productive sites found in 1982 will
be revisited in 1983.

d. Continue work on the development and improvement of insect rearing
and bioassay methods. Pathogens isolated from the field and obtained from
culture collections such as that maintained by IPRC (BTI) will be screened
against the appropriate pest insects.

e. Continue research on methods of formulation and low-technology
production of fungal pathogens. Initiate small-scale production of selected
pathogens to provide material for preliminary field trials.

f. Initiate studies on strain improvement.

g. Conduct preliminary field trials with pathogens demonstrating promise
for control of selected insect pests.

h. Commence training of Brazilian students and scientists in insect
pathology and microbial control. An insect pathology workshop will be
conducted for cooperators at IPRC (Brazil) in fiscal 1983.


2531B













TECHNICAL ANNUAL REPORT
for FISCAL YEARS 1981 and 1982


on research and training sponsored by



BEAN/COWPEA COLLABORATIVE RESEARCH
SUPPORT PROGRAM (BC/CRSP)
MICHIGAN STATE UNIVERSITY
EAST LANSING, MICHIGAN 48824 USA


and


BOYCE THOMPSON INSTITUTE
TOWER ROAD, CORNELL UNIVERSITY
ITHACA, NEW YORK 14853 USA


and


CENTRO NATIONAL DE PESQUISA DE ARROZ (Rice) E
FEIJAO (Beans) (CNPAF)
EMPRESA BRASILEIRA DE PESQUISA AGROPECUARIA (EMBRAPA)
GOIANIA, GOIAS 74.000 BRAZIL


entitled


INSECT PATHOGENS IN COWPEA PEST MANAGEMENT SYSTEMS

FOR DEVELOPING NATIONS


prepared in English by


Donald W. Roberts, PI
Richard S. Soper, CoPI
Richard A. Daoust, Research Associate seconded to Brazil
Stephen P. Wraight, Research Assistant






Brazil/Roberts


TABLE OF CONTENTS


I. Introduction

A. Description of Project

B. United States and Host Country Personnel

1. U.S.
a. D.W. Roberts
b. R.S. Soper
c. R.A. Daoust
d. M. Ward
e. S.P. Wraight
2. H.C.
a. A. Blumenschein
b. E. Ferreira
c. G.P. Rios
d. B.P. Neves
e. J.P.P. Araujo
f. M. Yokoyama

II. Original Approved Plan

A. Goals

B. Methods

III. Adjustments and Justification

A. Goals

B. Methodologies

IV. Summary of Achievements

A. Planned H.C. Activities

1. Establish Insect Pathology Resource Center in Goiania,
Goias
2. Training Program
3. Surveys for Pathogens
4. Screening of Pathogens for Virulence

B. Planned U.S. Activities

1. Develop Bioassay Techniques
2. Fungus Mass Production and Formulation

C. Unplanned Activities






Brazil/Roberts


V. Budget Expenditures to Date

VI. Plans

A. 1983 Program

B. 1983 Budget

VII. References

APPENDIX

1. Culture Collection (Brazil)

2. Undergraduate Student Project Report (Brazil)

3. Training Course on Microbial Control of Cowpea Pests (Brazil)

4. Training Course on Cowpea Production (Brazil)

5. Research Data (Tables 1-10)






Brazil/Roberts -1


I. Introduction

A. Brief Description of Project

Insect pests and plant diseases are the most important constraints
to cowpea production and storage in Brazil and other countries where
cowpea is grown as a staple food. Chemical insecticides are effective
in the control of some insect pests, but since cowpea is frequently
produced by poor farmers as a subsistence crop, the cost of importation
and utilization of chemicals is prohibitive. Varietal resistance has
been considered as an alternative to the widespread application of
chemical pesticides. However, the development of resistance against
some of the major pests has been unsuccessful; and some cowpea breeders
have expressed the opinion that the development of varietal resistance
against many different pests simultaneously will be difficult to
achieve. This is particularly true in Brazil where cowpea consumers
tend to be very fixed as to size, color, and plant type acceptable to
them, which means that many breeding programs will be needed to cover
the whole of the country.

Therefore, an integrated approach to pest control must be sought
that can incorporate information concerning the socioeconomic structure
of the poor, rural subsistence farmers. As part of an integrated
system, microbial agents pathogenic to the principal cowpea pests are
being considered. These agents have feasibility for implementation even
in poor areas where the cash value of cowpea crops is low, since they
could potentially be produced locally, and since the possibility exists
for colonization of pathogenic agents that could operate in pest
population regulation by the establishment of enzootic or epizootic
infection foci. Since virtually no research information of either an
applied or basic nature is available on microbiol control of cowpea
pests, and since virtually no experienced insect pathologists have
worked on this crop, alleviation of these limitations will be sought by
(a) conducting basic and applied research in the cowpea-producing nation
of Brazil and in the U.S. (b) stationing an experienced insect
pathologist in Brazil to conduct research with Brazilian scientists, and
(c) training scientists and aspiring scientists, including women, from
Latin America in insect pathology and microbial control. Training will
include short-term courses for cowpea researchers and students as well
as training of individuals at the graduate and undergraduate levels.

Although portions of Brazil are economically advanced, the very
large northern half of the nation where cowpea is the staple food of
subsistence farmers is one of the poverty areas of the world. It is
anticipated that the findings of this CRSP project will be applicable to
cowpea culture in other nations as well as Brazil.

Progress to date, assuming because of a one-year delay in start-up
that the 1981 proposed research should be done in 1982, has progressed
as planned.






Brazil/Roberts-2


B. Name and Background of Major U.S. and H.C. Personnel and Their
Role in the Project

1. Major U.S. Personnel

a. Dr. Donald W. Roberts, P.I.

Background: Dr. Roberts is an insect pathologist with broad
experience in terms of pathogens, insects, and nations. He is Insect
Pathologist in the Boyce Thompson Institute Biological Control Program
and Coordinator of the Insect Pathology Resource Center. The latter
(IPRC) consists of scientists from BTI, USDA, and Cornell University
working with insect pathogens. The functions of the Center are
training, respository, consultation, and research (both basic and
applied) with emphasis on microbial control of pests.

Dr. Roberts was in Brazil six times prior to initiation of the
BC/CRSP project, and on four of these occasions discussed the
desirability and feasibility of conducting the project with the
principal collaborators, the CNPAF staff in Goiania, and several EMBRAPA
administrators. They strongly encouraged his proceeding with the
project and provided material to help prepare the grant application. He
has a beginning knowledge of Portugese which he intends to improve. He
lived in Switzerland one year (1964) as a NSF Postdoctoral Fellow, and
since 1974 he has conducted short-term work in Nigeria, India,
Philippines, Malaysia, Thailand, and, as mentioned above, Brazil. He is
interested in both agricultural and medically important pests. He has
studied viral, protozoan, bacterial, and nematode diseases of insects;
but his principal research activity at present is with entomopathogenic
fungi. Although he was engaged in only basic studies for several years,
he became convinced about ten years ago that entomopathogenic
microorganisms were underutilized in insect control programs, including
the majority of "integrated pest management" programs. He has since
been actively engaged in discovery, production, formulation, and
application of pathogens against a variety of pests in the nations
listed above and the USA. The principal targets have been mosquito
larvae, bollworm (Heliothis armigera), rice brown planthopper,
spittlebugs, and Colorado potato beetle.

Role in project: Dr. Roberts, as Principal Investigator,
coordinates the project in the U.S. and acts as the direct liaison in
all communications with the host country. He directs the research in
the U.S. and participates in the research, particularly survey and field
trials, in Brazil. In addition, he is involved in the training portion
of the project in both Brazil and U.S.

b. Dr. Richard S. Soper, CoPI

Background: Dr. Soper is in charge of the USDA, SEA-AR Insect
Pathology Research Unit consisting of 3 research scientists and 3
support staff. The mission of IPRU is broadly defined "... to develop
entomopathogenic fungi as alternatives to chemical insecticides".
Personal research efforts are generally orientated towards
epizootiology. However he has conducted production and formulation





Brazil/Roberts -3


research and holds patents in this area. He has also published on
strain selection bioassayy) and fungal systematics.

Dr. Soper has conducted research outside the US including
pathogen surveys in Brazil, Argentina, Venezuela, Chile, Peru, Central
America, France, Yugoslavia, Sri Lanka, and Indonesia; epizootiological
studies in Australia and the Philippines; and laboratory studies in
Kenya and England.

Role in project: Dr. Soper participated in the 1982 survey
work in Brazil and will participate in field trials in Brazil in 1983.
In addition, he functions in administrative roles such as assisting in
budget preparation.

c. Dr. Richard A. Daoust, Research Associate

Background: The insect pathologist stationed in Brazil, Dr.
Richard A. Daoust, has had extensive academic and professional
experience in insect pathology and entomology. He has worked with
viral, bacterial and fungal pathogens of a wide variety of pest insects.
In addition he has had extensive experience in the isolation,
cultivation, identification, and characterization of bacterial and
fungal pathogens of insects. This includes thesis work for the Ph.D. on
an investigation into the microbiological control of the gypsy moth.
These studies emphasized the use of chitinolytic bacteria and Bacillus
thuringiensis for control of this important forest defoliator. Later
work emphasized the development of the entomopathogenic fungus
Metarhizium anisopliae for mosquito control. This has resulted in
strain improvement, and the development of effective storage,
formulation, and production techniques.

Dr. Daoust has a particular interest and strong background in
applied solution-oriented research which is demonstrated by extensive
involvement in field experiments using microbial control agents both in
the U.S. and abroad. Overseas experience involved a two-year residence
in Africa where bacterial and viral pathogens were developed for control
of the major pest (Heliothis armigera) of the most important subsistence
crop (sorghum) in Botswana. Other overseas experience included field
studies for two months (1979-1980) in Brazil on the control of the
pasture spittlebug with fungal pathogens as part of a national effort by
the Brazilian government research organization, EMBRAPA, to increase
beef production. In recent years he has assumed major roles in
laboratory management and direction of technical assistants and
students, first in Ithaca and later in Brazil.

Role in project: As the insect pathologist stationed in
Brazil, Dr. Daoust coordinates and participates in the research and
training efforts in Brazil and serves as the principal link with host
country investigators.

d. Michael Ward

Background: Mr. Ward is an agricultural industry-trained
formulation chemist and applications specialist with experience in





Brazil/Roberts -4


dealing with both microbial and chemical pesticides.

Role in project: As a full-time consultant at Boyce Thompson
Institute, Mr. Ward's role in this project is to develop low-cost,
low-technology microbial formulation and application techniques which
could be employed by farmers or state agricultural agencies in
developing nations.

e. Stephen Wraight

Background: Mr. Wraight is a doctoral candidate with
experience in the fields of insect pathology and microbial control. His
dissertation research dealt with factors affecting the laboratory and
field efficacy of entomopathogenic bacteria. Previous experience also
includes in vivo production of insect viruses and nematodes. In
conducting these investigations he acquired a.working knowledge of
bioassay design and data analysis.

Role in project: Mr. Wraight's responsibilities include the
conduct of the screening, bioassay, and field trial research in the
U.S., production of pathogens for laboratory and field tests,
acquisition and shipment of essential items to Brazil, maintenance of
insect colonies, and coordination and supervision of technical staff.

2. Major H.C. personnel

a. Dr. Almiro Blumenschein, H.C.-P.I.

Background: Dr. Blumenschein is one of Brazil's most
distinguished agricultural scientists. In addition, he is active in
international agriculture, most recently as chairman of the committee
which reviewed the International Rice Research Institute.

Role in project: As chief of CNPAF, Dr. Blumenschein's
enthusiastic support of the BTI BC/CRSP insect pathology project has
been crucial to our success. He is involved in our decisions on
training of scientists and students and collaboration with CNPAF staff,
and he provides administrative support in areas such as research space
allotment and communications.

b. Dr. Evane Ferreira, H.C.-Co P.I.

Background: Dr. Ferreira is the administrative leader of
entomological research at CNPAF. His personal research emphases are
biological control and rice pests.

Role in project: Dr. Ferreira assists development of our
insect pathology/microbial control project through discussion of
research programs and through administrative assistance.

c. Dr. Gerson Pereira Rios, H.C.-Co P.I.

Background: Dr. Rios is the CNPAF cowpea plant pathologist.
As such, he is familiar with research on fungi in Brazil and with the
diseases of cowpea.






Brazil/Roberts-5


Role in project: Dr. Rios has been personally involved in the
surveys for pathogens of cowpea-associated insects. Also, he has served
as our advisor on cowpea diseases and on insect vectors of these
diseases.

d. Mr. Belmiro Periera das Neves, H.C. Co P.I.

Background: Mr. das Neves is the CNPAF cowpea entomologist.
As such, he is the person at CNPAF most familiar with the insects
associated with cowpea and with handling these species in the
laboratory.

Role in project: Mr. das Neves frequently is involved in our
research in Goiania and he has travelled with us in all of our survey
trips outside of Goiania. His advice is constantly sought on insect-
related problems such as life cycles and identification. Through such
interactions his awareness of insect diseases, which initially was very
low, has been significantly increased and he has expressed some interest
in receiving advanced training in this area.

e. Joao Pratagil Pereira de Araujo

Background: Mr. de Araujo is the project leader for cowpea
research at CNPAF and the cowpea national program for Brazil. He is a
plant breeder by training.

Role in project: Mr. de Argujo is personally involved-in our
cowpea research at CNPAF and has been very helpful in organizing our
survey trips to North and Northeast Brazil. He has also travelled with
us on these trips and assisted with the searches in the field for
diseased insects.

f. Mr. Massaru Yokoyama

Background: Mr. Yokoyama is the bean entomologist for CNPAF.

Role in project: Some of the pests affecting cowpeas also
occur on beans. Mr. Yokoyama has been very helpful in bringing diseases
of these pests in beans to our attention.

g. Dr. Earl Watt

Background: Dr. Watt is a U.S. citizen in the employ of IITA
(Nigeria) and seconded to CNPAF as a cowpea breeder.

Role in project: Dr. Watt is recognized by the Brazilians as
an important catalyst in the organization of the national cowpea
program. Although he is not currently personally involved in our
research, he has provided useful information on such topics as where to
conduct surveys and.promising scientific contacts in the cowpea national
program.





Brazil/Roberts-6


II. Original approved plan

A. Goals and objectives

1. Five-year goals

The project is designed to develop insect pathogens as effective,
economical, and safe cowpea pest management tools fully compatible
(integrated) with other insect control practices used by LDC farmers,
and train LDC scientists in insect pathology so they can function
independently in microbial control projects in cowpeas and other crops.

Insect pests are a major constraint to production and storage of
cowpeas in Brazil and other LDCs. Ten-fold increases in cowpea yield
have been realized in some areas by extensive use of insecticides.
Since they can be produced in LDCs, microorganisms pathogenic to the
major pests of cowpeas will be developed by integration into insect
control programs. The current limitations to microbial control on this
crop are (a) virtually no research data, either basic or applied,
available on the topic, and (b) virtually no experienced insect
pathologists working on this crop worldwide. Alleviation of these
limitations will be sought by (a) conducting basic and applied research
in a cowpea-producing nation (Brazil) and in the US to increase the data
base; (b) sending experienced insect pathologists to Brazil to conduct
experiments with Brazilian scientists, and (c) training scientists and
aspiring scientists from LDCs in insect pathology and microbial control.
Training will include basic as well as applied concepts to provide the
trainee with adequate knowledge to function independently in insect
pathology in his or her home LDC.

2. First year (1982) project objectives

a. Establish the Insect Pathology Resource Center (Brazil) in
CNPAF/EMBRAPA, Goiania, including: 1) research laboratory with
specialized insect pathology supplies; 2) training facility; 3)
pathogen culture collection, primarily of bacterial and fungal
isolates, with an indexing system, and which utilizes several
storage methodologies for long-term maintenance of strains
collected locally and obtained from culture collections; and
4) literature and information center on insect pathology (including
books, reprints, and a computer-generated literature survey).

b. Conduct surveys in cowpea producing areas of Brazil to
1) collect (isolate in culture) some of the principal pathogens
associated with the major insect-pest species; 2) provide
information on the natural incidence of diseases (i.e., provide
epizootiological data); and 3) identify researchers and field
stations which will collaborate with the CRSP project for further
survey work and for preliminary field trials.

c. Establish lines of communication. A BTI staff member will be
stationed full-time in Brazil at the Brazilian national rice and bean
station (CNPAF) and will make brief excursions to the Northeast.
Accordingly, we will have daily interactions with Brazilian scientists






Brazil/Roberts -7


and/or farmers. In addition, the PI and CoPI will spend substantial
periods of time in Brazil. Every opportunity will be taken to exchange
information with Brazilian entomologists, plant pathologists, plant
breeders, and farmers to assist us in integrating insect pathogens into
insect pest management systems. Women as well as men will be consulted,
since women work in cowpea fields, assist in harvest and storage, and
prepare seeds for meals. We will also maintain close contact with a
CRSP group at BTI (PI = A. Renwick) working on resistance of cowpeas to
insects in the Cameroons. This work is in collaboration with the
University of Georgia, so we expect opportunities to interact with them
as well. There will be two CRSP projects on beans from the University
of Wisconsin affiliated with CNPAF also. We will discuss common
problems during their visits to Brazil.

d. Identify and characterize newly discovered pathogens.

e. Establish insect colonies, develop bioassays, and initiate
screening tests to identify microbial isolates with promise for pest
control.

f. Seek Latin American students for training in insect pathology
and microbial control.

g. Provide basic training in insect pathology techniques to the
entomologists, plant pathologists, and technicians at CNPAF.

3. Second year (1983) project objectives.

a. Continue development of IPRC (Brazil).

b. Extend survey for pathogens of cowpea pests.

c. Continue information exchange through communication.

d. Continue bioassay development and screening for promising
microbial isolates. Bioassays will be developed for each of the major
pests. Pathogens collected from the field and obtained from culture
collections will be screened against the appropriate pest insect. It is
expected that both time-mortality (LT50) and dosage-mortality (LD50)
data will be generated for at least some of the pathogens showing
promise for control of the major pests. Several pathogens will be
identified which can be produced for small scale field tests.

e. Initiate studies on fungal strain improvement.

f. Initiate small-scale production and formulation of selected
pathogens (for use in field trials).

g. Conduct preliminary field trials with pathogens demonstrating
promise-for control.

h. Commence training of Brazilian students and scientists in
insect pathology and microbial control. Graduate students will be
selected and will complete several courses in Brazil or in the






Brazil/Roberts-8


U.S., depending on students' programs. One to several Brazilian
scientists selected as project cooperators will be invited to spend
several months at IPRC (Brazil). An insect pathology workshop will
be conducted for cooperators at IPRC (Brazil) in fiscal year 1983.

B. Methodology

1. Target insects.

This CRSP project will be concerned with the control of several
different pests and will utilize several pathogen groups (see Table 1).
Chalcodermus sp. (probably aeneus or bimaculatus) (a coleopterous
podfeeder) is considered to be a very important pest and in fact
probably is the most important pest in the Northeast north of Petrolina,
but it occurs as far south as Goiania (Watt et al., 1979). The insect
was first noted in the NE in 1971, and it has aow spread to most of the
cowpea-growing areas of Brazil. The insect pest with the widest
distribution is Empoasca kraemeri, a leafhopper (Watt et al., 1979).
The most severe damage occurs on late plantings, especially as
dry-season river-bottom plantings. The CNPAF cowpea breeders and
entomolgists have had some success in identifying varieties with reduced
susceptibility to Empoasca, but they have advised us that studies on
other approaches to control should be conducted as well. The weevil,
Callosobruchus maculatus causes severe damage to stored cowpeas and has
a universal distribution including many parts of Africa. Other cowpea
pests which are generally restricted to isolated locations or sporadic
infestations include Elasmopalpus lignosellus the lesser cornstalk
borer; other Lepidoptera- Spodoptera sp., Maruca testulalis; chrysomelid
leaf-feeding beetles Diabrotica spp. and Ceratoma arcuata; pentatomid
stink bugs Acrosthermum sp., Nezara sp., and Crinocerus gildini; the
coreid bug Crinocerus sanctus; several mirid bugs; and occasionally
aphids (CNPAF Technical Report, 1980). Since data and specimen
collection on all pest species is beyond our limited resources, the
emphasis during the first two years will be on the three major pests,
with some attention to lepidopterous and hemipterous pests. Research
priorities will be modified in accordance with changes in pest
populations resulting from farming systems or agronomic practices. For
example, Taylor (1978) indicated that the introduction of high yielding
varieties in Sierra Leone have caused increased problems with
Callosobruchus sp.

2. Organize insect pathology laboratory.

The initial priority will be to establish an Insect Pathology
Resource Center (Brazil)(IPRCBR) with an experienced insect pathologist
(RA) at the National Center for Rice and Bean Research (CNPAF, Goiania,
Brazil). This is to be closely associated physically and intellectually
with the overall operation of the host institution. There will be
extensive sharing of facilities, equipment, and knowledge and most of
the work will be of a collaborative nature. The CRSP-supported US
scientist is to be treated as much as practicable as a member of the
0






Brazil/Roberts-9



Table 1. List of Potential Pathogens for the Control of Cowpea Insects


Insect Pest


Candidate Pathogen


Coleopterous Pests


Chalcodermus aeneus or
bimaculatus (Adults)
Note: There is disagreement in
Brazil as to the species of
Chalcodermus in that nation.
Expert opinion is being sought.

Callosobruchus maculatus

Chrysomelid beetles (Ceratoma and
Diabrotica)


Homopterous Pests

Empoasca kraemeri


Aphids (various species)


Beauveria bassiana
Metarhizium anisopliae
Cordyceps sp.
Paecilomyces sp.
Bacillus thuringiensis


same as for Chalcodermus

Beauveria bassiana
Entomophthora spp.


Zoophthora radicans
Metarhizium anisopliae

Erynia neoaphidis
Conidiobolus obscurus
Zoophthora radicans


Hemipterous Pests


Pentatomid stink bugs (Acrosthermum
sp., Nezara, and Piezodorus
gildini), coreid bugs (Crinocerus
sanctus), and several species of
mirid bugs


Beauveria bassiana
Metarhizium anisopliae


Lepidopterous Pests


Elasmopalpus, Spodoptera, Maruca, etc.


Entomophthora gammae
Zoophthora radicans
Beauveria bassiana
Nomuraea rileyi
Bacillus thuringiensis
Nuclear polyhedrosis virus






Brazil/Roberts-lO


CNPAF senior staff. The IPRC(BR) laboratory will have the specialized
supplies and equipment necessary to (a) identify and characterize
pathogens, (b) transfer and handle diseased specimens and pathogen
isolates, (c) test the pathogenicity of agents against pest species,
(d) propogate and culture pathogens, and (e) formulate and apply
pathogens in the field. The establishment of the laboratory will also
stress training of technicians, graduate students, and counterpart
Brazilian scientists in insect pathology techniques. IPRC(BR) will also
serve as a location from which frequent contact can be made with
subsistence producers. This will aid in determining the most important
needs of subsistence farmers and will facilitate the development of
methods which can be easily transferred into existing agricultural
practices. Surveys conducted by the IPRC(BR) will include consulting
with women in fields as to current agricultural practices, since they
are involved in growing cowpeas. This will aid in understanding the
role of women in cowpea production and allow greater involvement of
women in the pest management programs of the CRSP. Finally, the
maintenance of an active research program in Brazil will facilitate
increased communication and coordination between our CRSP project and
other cowpea research programs in Brazil. Since the CNPAF has
researchers developing higher yielding, more resistant cowpea varieties
both for Brazil and other countries, the close association will allow
more extensive communication to occur. Researchers at CNPAF have
identified the Northeast as the principal area of production and highest
per capital consumption of cowpea in Brazil (15.9 Kg/year as compared to
only 1.2 Kg/year for the next highest region). Watt et al. (1979)
stated,

"This high level of bean consumption coupled with low total per
family income means the Northeasterners spend the highest percent
of their income (39 versus 20-30%) for food and in particular beans
(8 versus 4.0-5.5%) of any other region in Brazil. Beans, and thus
cowpea, are very important in the economics of consumption of the
Northeast, more important than for any other region."

3. Survey for pathogens of cowpea pests.

The experimental plan will involve surveys in the North and
Northeast of Brazil during the cropping season (April through July).
During this period both survey work and farmer contact will be made.
During the remainder of the year, except during years where later second
plantings occur, work will continue at CNPAF and BTI to develop
pathogens. However, trips necessary to increase communication between
researchers and subsistence farmers will be considered high priority.

The PI (or CoPI), the RA and, when possible, a member of the CNPAF
staff will travel to the Northeast to conduct surveys and establish
epizootiological data. The Northeastern region will include field sites
in the states of Piaui, Ceara, Rio Grande do Norte, Pernambuco, Paraiba
and Bahia. As indicated in Table 2, promising field sites and
collaborating entomologists have been identified in many of these
states. Survey in northern Brazil will probably be centered around
Belem, Para. Substantial cowpea production also occurs in the east and






Table 2. Field Sites for Surveys and Field Trials in the North and Northeast of Brazil.


Location and Description of
State Field Station Specialist Activities


Piaui


Ceara


Pernambuco


Bahia


Maranhao


Rio Grande
do Norte


Alagoas


Teresina, EMBRAPA
field station (UEPAE)



Picos (EMATER)
Barbalha (EPACE)


Univ. Fortaleza of
Ceara (UFC):
Quixada
Pentecostas
Fortaleza
Petrolina
EMBRAPA field
station (CPATSA)
Serra Talhada
Recife (IPA)
Univ. of Pernambuco
Irece (EPABA)



Bacabal (Exp. St.)
S. Luiz (EMAPA)
Natal (EMPARN)


Maceio (EPEAL)


Paulo H.S. da Silva Entomologist
Antonio Apoliano don Santos -
Plant Path.
Francisco F. filho Crop
Improvement Specialist
Davi Josue da Costa Agronomist
Mary Ann Quindere Plant Protection
Paulo Diogenes Barreto Plant
Technology Agronomist
Jose Higimo Santos Entomologist
Jose Julio da Ponte Plant Path.
Jose A.M. Bastos Entomologist
Fernando J. Montenegro Sales -
Biol. Control
Gilberto Joao de Moraes -
Entomologist

Paulo Roberto Fernandes Agronomist
Paulo Miranda Crop Improvement
Jose Vargas de Oliveira Entomologist
Cuaracy Campelo Melo Plant Pathology
Regina C=Guilherme da Silva Plant
Technology Agronomist
Ubiracy Mendes Soares Agronomist
Evandro Ferreira Chagas Entom.
Duri Alaeacio Simplicio Plant Path.


Joao Batista Fernandes Entomol.
Jose William Veras Lemos Plant Path.
Cleonor Cavalcanti Entomol.


Field trials, survey, farm
visits



Survey
Field trials, survey, farm
visits

Field trials, survey, farm
visits


Survey, farm visits, field
trials


Survey,
Survey

Survey



Survey

Survey


farm visits


Survey. Possibly field trials





Brazil/Roberts-12



south of Brazil. Since geographic locations with both climatic and
agronomic differences may substantially change the host-pathogen
complex, survey work may also be conducted in these regions. However,
since cowpea is frequently grown as a cash crop in the south, survey
work there may not be initiated until after the second year. CNPAF has
committed one staff member, probably the leader of their cowpea team, to
accompany us on our first survey trip in 1981 at their expense. Belmiro
Neves, cowpea entomologist, and Gerson P. Rios, cowpea pathologist, will
similarly accompany us on later trips.

The normal method of transportation for CNPAF staff in the NE is to
fly to a major city and be carried by a local scientist interested in
their program to the sites he considers most promising to the needs of
the trip. This can be a state or federal scientist. If the vehicle is
state owned, the scientist cannot cross state lines, and a scientist
from the next state meets the CNPAF staff member near the border.

a. Sampling methodologies will be crucial to the success of
identifying candidate control agents and establishing epizootiological
data. The latter will elucidate natural disease incidence and indicate
how pathogens and pests can be manipulated. Sampling strategies will
depend on a number of factors which can be summarized as follows:

aa. The sampling methods will be specific for each pest species.
The discovery of pathogens and predicting disease incidence will be
largely dependent on sampling methodologies (Newman and Carner, 1975).
Both adults and nymphs of Empoasca can be sampled by relative sampling
methods already available for soybeans, including direct observations
and sweep net collections for adults, and examinations and plant-shake
collections for nymphs (Helm et al., 1980). (Absolute sampling methods
will be used during field trials where population estimates are
critical.) Sampling methods for Chalcodermus and Callosobruchus will
involve direct examination of infested pods and may include soil samples
taken with specialized core samples. Collection and extraction of
coleopterous pests in all life stages (primarily adults of Chalcodermus)
from soil samples will utilize techniques similar to those of Kogan and
Herzog (1980) and Eastman (1980) for soybean pests. Specimen collection
will include both live and dead insects. Disease incidence will be
predicted by allowing live field specimens to develop infections in the
laboratory, but dead specimens probably will serve as the major source
of new pathogens.

bb. Sampling initially will be confined to collecting specimens of
the pest species on cowpea plants. However, later surveys may include
samples from soil (especially for Chalcodermus and other soil inhabiting
species), foliage, or cowpea pests on other crops.

cc. Sample locations will initially be confined to the regions
discussed above, but within sampling regions different climatic and
geographical locations will be selected. For example, Watt (1978)
indicates that the Northeast has four major ecological regions which
includes the "transicao ao Amazonas", the Zonaa da mata", "literal" and
the "agreste". These regions differ widely in their elevation,





Brazil/Roberts-13


rainfall, humidity, and soil fertility and also in the pest complex on
cowpea. In addition, pathogens may vary from one region to another.

dd. Sampling time is critical to determine disease incidence and
to identify the pathogen complex associated with insects. Many
pathogens demonstrate a seasonal periodicity not necessarily correlated
with pest densities (Carner, 1980; Ignoffo et al., 1975; Harper and
Carner, 1973). Therefore, surveys and epizootiological studies need to
be extensive and conducted over the entire cropping season to insure the
best selection of pathogen candidates.

b. Culture of the pathogens from diseased specimens will be
attempted during the survey trips using media brought from the US (1st
survey trip) or from Goiania (subsequent survey trips). The PI and
Co-PI have recently conducted surveys in SE Asia and therefore are
familiar with details of the proper media, containers, equipment, etc.
necessary for successful initiation of cultures while travelling. As in
our previous studies, specimens also will be stored over 80% glycerol
for culture attempts on return to Goiania.

c. Identification of the organisms found on or in insects will be
attempted at IPRC(BR). Dr. Gerson P. Rios will assist with the fungi.
He has many years' experience with fungi, and he will be invaluable in
helping us recognize the numerous fungi we will find, most of which will
be saprophytes. Mr. Aloisio Sartorato, also of the CNPAF plant
pathology group, will assist with identification of bacteria. These
< persons also will assist in attempts to culture fastidious
microorganisms. Assistance from taxonomic specialists will be sought
where identification help is needed. IPRC(BTI) also will be involved in
identification of difficult cases and will be responsible for strain
characterization (primarily by isozyme analysis) and storage in liquid
nitrogen of isolates which can be cultured. Attempts will be made to
multiply insect-pathogenic viruses at IPRC(BR) to afford sufficient
material for characterization at IPRC(BTI) or other interested research
groups.

4. Screening for pathogenicity

Insect colonies for screening tests will be maintained in
conjunction with Brazilian entomologists at CNPAF (primarily Belmiro
Neves) and at BTI. It is anticipated that colonies of 3 pests
(Callosobruchus, Chalcodermus, and Empoasca) will be maintained at CNPAF
the first year. Chalcodermus and Empoasca are already being grown and
Callosobruchus is an easily cultured storage pest. Colonies of
Callosobruchus maculatus currently exist at BTI in Ithaca (Dr. A.
Renwick). Empoasca kraemeri is not present in N.A., so Empoasca fabae
will be utilized at IPRC/BTI. A strong colony of E. fabae is maintained
in the Cornell Department of Entomology, and a colony of Chalcodermus
aeneus can be initiated in 1981 from material generously promised by
colleagues in the southern U.S. Chalcodermus will be maintained on
cowpeas grown in the greenhouse. Our greenhouse staff has worked with
cowpeas since 1978 in conjunction with our N2-fixation group and produce
excellent plants. During the latter part of the first year, screening





Brazil/Roberts -14


tests will be initiated to identify microbial isolates with promise for
pest control. These studies will continue during the second year.
Fungal pathogens will be emphasized since this pathogen group is known
to be most effective against beetles and leafhoppers (Roberts and
Humber, 1981; Yevlakhova, 1978). However, other pathogens will be
obtained and preliminary pathogenicity studies conducted. For example,
several isolates of Bacillus thuringiensis have recently been shown to
be pathogenic to the Colorado potato beetle, Leptinotarsa decemlineata
(Cantwell, personal communications). An attempt will be made to procure
these isolates and other promising species from culture collections.
Since researchers at BTI have a large pathogen collection obtained from
numerous insect species worldwide, it will serve as a major source of
pathogens for screening against cowpea pests. Pathogen groups expected
to demonstrate the greatest success against cowpea pests in Brazil are
listed in Table 1. Efforts will also be made in the second year (and
later years) to work on strain improvement. This will involve the use
of mutagenic agents (e.g. EMS or UV irradiation) and cultural techniques
(e.g. passage of strains through alternate hosts or pest species) to
enhance virulence of promising isolates. Such techniques have been
useful in producing increased pathogenicity in other studies (Daoust and
Roberts, 1979; Al-Aidroos and Roberts, 1978; Aizawa, 1971). This work
will be conducted both in Brazil at CNPAF and primarily at BTI.

Initial pathogen screening will be conducted in the laboratory at
CNPAF using methods to establish time-mortality data (LT50) as a
relative estimate of infectivity and virulence. Bioassay techniques
will vary with each pest species but will emphasize systems that rely on
natural food and produce pathogenesis by the same route of infection as
would occur under field conditions. For example, fungal pathogens
against leaf feeding pests (Chrysomelid beetles, or lepidopterous leaf-
feeders) will be screened using test procedures which employ foliage
(Ignoffo et al. 1976). Bioassays involving Chalcodermus will probably
involve application of fungal conidia to soil infested with adults.
Dr. Evane Ferreira and Belmiro Neves will participate fully in the
design and execution of the screening tests.

5. Field tests

Wojciechowska et al. (1977) showed that Beauveria bassiana conidia
applied to soil gave good activity against the Colorado potato beetle
for up to two years. Since Callosobruchus spends the majority of its
life cycle within the bean, attempts to control this pest will primarily
include two approaches. Early season treatment of fields with fungal
conidia might reduce egg laying on cowpea since infestations usually
originate from scattered cowpea remaining in the field from the previous
year (Metcalf et al., 1962). Alternatively, pathogens could be used in
storage bins. Beetles would be considerably more susceptible during
storage since breeding and egg laying by adult beetles occurs constantly
in storage until the cowpea is depleted (Metcalf et al., 1962).

To prevent overlooking slower acting pathogens, which nevertheless
may ultimately produce effective control, dosage studies (LD50) may be
included. As candidate pathogens are identified, comparative
pathogenicity of strains will be determined using dosage-mortality





Brazil/Roberts -15


methods. Pathogens selected for further testing will be studied to
develop appropriate technologies for existing traditional farming
practices of subsistence level farmers or farm cooperatives. Production
methodologies will emphasize techniques for small farmers. This will be
determined by visits to subsistence farms and farm cooperatives in the
Northeast. Local pathogen production will be designed to be
economically feasible for small farmers and probably will involve women
in "cottage-type production facilities". The role of women may be
crucial to success since they will be involved in pathogen surveys,
dissemination of pathogens, and production of pathogens. Such a system
already exists to some extent in Brazil in which farmer cooperatives are
producing the fungus Metarhizium anisopliae for use against froghoppers
(Cercopidae) in sugarcane.

As mentioned previously, the most promising isolates will be
characterized for isozymes, structural peptides, and serologically
detectable differences. This will be used to provide positive
identifications of materials used in safety and field trials. This work
will be conducted at the BTI but it is expected that it will not be a
major activity until after the second year. Pathogens may be produced
on a limited scale by commercial companies and/or at the BTI for field
testing in cowpea fields in Brazil. In addition, production using
simple technologies may be conducted in Goiania.

Several conceptual approaches for the use of microorganisms to
control insects exist. These include: (a) colonization of pathogens
which once established will perpetuate themselves and naturally control
a pest species, (b) microbial insecticides and (c) integration of
insect pathogens with other control strategies. Epizootiological data
gathered during survey trips and by counterpart field entomologist in
the Northeast, will be important in determining control strategies.
Since control through natural epizootics often occurs after significant
crop damage, it may be necessary to manipulate these agents by early
seasonal introduction using a modification of the colonization approach.
This approach has been shown to be important with other pathogens, most
notably with Hirsutella thompsonii, Nomuraea rileyi (Roberts and Humber,
1981), and Zoophthora radicans (Milner et al., 1981).

The development of pathogens as microbial insecticides will
constitute the most important approach to field control of cowpea
insects. Therefore, every effort will be made to identify candidate
pathogens during the first two years for field testing. During the
second year, field tests will be initiated on those major pest species
for which candidate pathogens have been developed. Field trials will be
conducted where possible, both on Northeast small farms (where 80% of
the cowpeas are produced in intercropping systems) and in Goiania. The
success of field experiments in the Northeast will depend on both the
identification of cowpea growing areas in which farmers are receptive to
such tests and on the availability of field entomologists in nearby
locations. However, every effort will be made to involve small farmers
and women when possible. The exact experimental design of field trials
will depend on numerous factors but generally small plots will be used.
Cowpea plots used for preliminary screening of germplasm under both mono
and intercropped conditions consist of 2 rows spaced 0.5 by 5 M and 1.5





Brazil/Roberts -16


M between plots (Watt et al., 1979). A similar design with 4-5
replicates per treatment will be used for pathogen field trials although
larger plots may be used. Generally, experiments with cowpea
intercropped with tree cotton, corn, cassava or sweet potato will be
preferred. Multiple intercropping may also be included since it is
frequently practiced. Some trials may also be performed with cowpea as
the sole crop. Field applications can either be applied to leaf foliage
and pods or to soil for control of Chalcodermus. Field experiments will
be extended with larger plots and additional pathogens in later years.
Pathogen combinations alone or with other approaches (e.g. chemical
insecticides) may also be considered.

Field efficacy of microbial insecticides may be substantially
increased through improved formulation and application procedures.
Therefore, as candidate pathogens are identified studies will be
initiated to examine these aspects. Formulation experiments will be
performed at BTI. -

6. Methods of Data Analysis

Statistical advice, when needed, will be sought for experimental
design and data analysis from statisticians located both at CNPAF and
the BTI. Computer facilities will be employed at both locations when
necessary. It is anticipated that laboratory bioassay will be analyzed
using probit analysis methods (Finney, 1971) using computer program
POLO. Field experiments will be analyzed to determine efficacy and
compare treatments using an analysis of variance coupled with Duncan's
multiple range test analysis. Other statistical methods will be used as
needed.

7. Training

We consider the proposed project as primarily a training project.
In addition to conducting research we intend to train at various, well
defined levels, several Latin American scientists, both men and women,
so they can improve on our microbial control systems after we leave
Brazil. We feel the trainees should spend a considerable portion of
their time on problems which relate to developing-nation food
production. In Brazil, good basic research as well as field studies can
be carried out at CNPAF. Our basic philosophy on training appears to be
similar to others in the CRSP, but our emphasis is slightly shifted
toward activity in the host country. This was done because of the
strengths of our host country and because it would permit our training
larger numbers of people, including scientists who wish to use insect
pathology as a supplement to their existing skills. Training at BTI
(Cornell) also is desirable. The available funds, however, will not
permit extensive training in both the US and Brazil. Our compromise
will be to do the majority of training in Brazil, with course work and
short-term research at Cornell University for key trainees.

a. U.S. Students. During the first several years of this
project, all available training funds will be concentrated on scientists





Brazil/Roberts -17


from cowpea-producing less-developed nations. Therefore no US students
will be supported by title XII funds during this period.

b. H.C. Students. We plan to train Brazilian students (or
students from other Latin American countries) through a structured
program which will provide a comprehensive theoretical as well as a
practical background. We believe this can best be accomplished by
identifying highly qualified students who have a strong interest in
cowpeas and related crops who are committed to solving malnutrition and
food scarcity problems. Female and male students will be sought in the
recruitment process. Students ideally will be trained intensively on a
short-term basis at Cornell University (for approximately 1 year). This
will entail coursework which will provide basic theoretical knowledge,
and research skills will be gained primarily by working with BTI
scientists. The students will then return to Brazil and continue
coursework in a Brazilian University. The order may be reversed (Brazil
first, Cornell-BTI second) if this is more appropriate to the needs of
the student or the program. EMBRAPA maintains an intensive language
school for English which, through our affiliation with EMBRAPA/CNPAF,
will be available to our students. This is a comprehensive program
which takes four months to complete, but prepares the student to
function reasonably well on entrance into the USA. Their dissertations
will be on cowpea pests under Brazilian conditions at CNPAF.

It should be emphasized that the student will have a structured
education through a Brazilian University (with coursework taken at
Cornell applicable toward his or her degree) with a specific faculty
advisor. However, extensive training should also be gained by working
directly with the experienced BTI Insect Pathologist at CNPAF who will
provide structured guidance for the Ph.D. or M.S. dissertation. CNPAF
has advised us that acceptance of US scientists on dissertation
committees is common in Brazil.

Other training will include conducting workshops and bringing
scientists and technicians from other portions of the nation for
short-term research projects at CNPAF.

c. US Principal Researchers Training. Dr. Daoust (RA in Brazil)
completed a comprehensive Portuguese language course (4 credit hours) at
Cornell. He will engage a tutor to continue these studies following
assignment to Brazil. The PI, Dr. Roberts, will likewise engage a tutor
to improve his Portuguese language skills. Working with CNPAF
scientists and travelling in Brazil on cowpea surveys will afford
opportunities for the US principal researchers to become knowledgeable
on all aspects of cowpeas and several other crops.

d. H.C. Principal Researchers Training. Depending on adequate
English skills, the Brazilian scientists associated with the cowpea
program will be encouraged to spend time at IPRC (BTI). This would
allow overall program orientation. They would also be exposed to
techniques used in a wide range of insect pathology investigations. The
principal training for most CNPAF principals will be at IPRC (Brazil)
through workshops as mentioned above and through direct participation in





Brazil/Roberts -18


the research program. It is likely, however, that one of the first
Ph.D. candidates trained under the CRSP project will be a member of the
CNPAF staff, for example, Belmiro Neves.


III. Adjustments and Justification

A. Major Adjustments

There were no major adjustments needed except that, since the
project commenced almost one year later than anticipated, what had been
planned for 1981 was done in 1982. This one year offset will, of
course, continue throughout the life of the project. With this
adjustment taken into consideration, the work if fully on schedule.

B. Minor Adjustments

1. Insect colonies

We were informed that several colonies were being reared at CNPAF,
but upon arrival there in October, Dr. Daoust found that no colonies
were being maintained. In part, this was due to a recent move to new
research facilities. However, in some cases pests were not maintained
due to the extensive labor required and also because of incomplete
knowledge of the life cycles of the pests. As a result, a laboratory
assistant was hired (only in June, 1982) to rear at least 3 important
cowpea pests, including 1) cowpea curculio (Chalcodermus aeneus),
2) cowpea storage weevil (Callosobruchus maculatus), and 3) chrysomelid
leaf-feeding beetles (Ceratoma arcuata and Diabrot6ca paranoense). The
first several months will involve considerable training, after which
sufficient numbers of stock should be available for bioassays and
small-scale screen-house or outdoor infectivity tests. A fourth pest of
cowpeas, Empoasca kraemeri, was initially programmed to be maintained at
CNPAF. However, since a very closely related species, E. fabae, can be
reared in the U.S., and since technology is already operating for this
species, research on Empoasca in the beginning will be restricted to the
U.S. until promising candidate pathogens are found. Surveys for
pathogens of Empoasca kraemeri will continue in Brazil, however, and
pathogens found will be sent to the U.S.

2. Purchase of vehicle in Brazil

The CRSP technical committee had initially recommended that a
vehicle be leased in Brazil. After completion of extensive paperwork
including approval of a bank-financial arrangement, EMBRAPA informed us
that the the car could not be leased through a Brazilian bank in
EMBRAPA's or Boyce Thompson's names. Applications and justifications
were then submitted to CRSP, MO, to receive A.I.D. approval to purchase
a vehicle. Approval was granted, and a vehicle was purchased and
registered in Boyce Thompson Institute's name. As.stipulated by CNPAF,
this vehicle utilizes alcohol fuel.





Brazil/Roberts -19


3. Formulation and production of microbial agents

Although work in these areas was not planned to commence until the
second year, studies were initiated on the production and formulation of
Beauveria bassiana, the most frequently isolated fungal pathogen of
cowpea pests (see culture collection list in appendix). For details,
see section IV-B-2.



IV. Summary of Achievements

A. Planned H.C. Activities

Our CRSP grant started in mid-September with a trip by Dr. D.
Roberts, Principal Investigator, and Dr. R. Daoust, U. S., host-country
investigator, to CNPAF, Goiania, Goias, Brazil to discuss our research
and training program with host country scientists and to finalize
details of the execution of our program. In late October, Dr. R. Daoust
and family was moved to Brazil to begin the program. From September to
August the following progress was made in the program:

1. Administrative Progress and the Establishment of an Insect
Pathology Resource Center in Brazil

a) Establishment of U.S., host-country investigator in Brazil.
Housing wag located and furniture was purchased in Goiania. In
addition, language training in Portuguese began for Dr. and Mrs. Daoust.

b) Importation of specialized supplies and small equipment into
Brazil to establish the insect pathology laboratory. An importation
license was obtained, after a considerable amount of Brazilian
bureaucracy, to import approximately $5,000 worth of American supplies.
This was an important achievement since many questions were resolved
concerning equipment and small supplies importation. In conjunction
with EMBRAPA personnel in the International Importation Division in
Brazilia, we worked out the details of procedures to be followed for all
future importations. The precedence used in this case will facilitate
all future importations for this and other CRSP programs located in
Brazil.

c) Importation of large equipment into Brazil. Import licenses and
full translations of all equipment specifications for 2 wide-range
incubators, 1 laminar flow vertical containment hood, and
stereomicroscope with full photographic equipment have been requested.
A license has been obtained for the incubators, and they are being
shipped. The laminar flow hood was delayed due to extensive
justifications needed. The justifications were submitted and we were
recently granted an import license. The stereoscope license has still
not been received but is expected shortly. Once again the procedures
worked out for the importation of this equipment'should facilitate
equipment importation by other CRSP researchers, e.g., University of
Wisconsin.





Brazil/Roberts -20


d) Purchase of vehicle in Brazil. An automobile (a 1982 Fiat
Panorama alcohol-fueled station wagon) was purchased for CRSP related
work (see Adjustments section). Unfortunately, due to numerous
bureaucratic difficulties, the car was not obtained until July. This
caused a considerable delay in our field work and precluded more
extensive surveys for insect pathogens in the cowpea-producing areas
near Goiania.

e) Establishment of the Insect Pathology Resource Center in Brazil.
A laboratory in the new research facility at CNPAF was devoted to insect
pathology research. This laboratory is situated between the plant
pathology and soil microbiology laboratories on one side and the
entomology laboratory and insect rearing room complex on the other. Dr.
Blumenschien was instrumental in designing this space arrangement so
that we may serve as a research bridge between the microbiology, plant
pathology, and entomology disciplines. The laboratory is now equipped
to perform insect pathology research, except for the equipment still
being awaited for importation and the construction of permanent microscope
tables. Research on our title XII program was delayed considerably due
to the fact that running water, distilled water, electricity, gas lines
and autoclave were not functional in most laboratories until March 1982.
The research staff moved into the new facility in October 1982, but it
was difficult to perform laboratory research during the first 3 months.
The insect pathology laboratory now has the capacity and specialized
supplies and equipment to a) identify and characterize pathogens, b)
transfer and handle diseased specimens and pathogen isolates, c) test
the pathogenicity of microbial agents against pest species, d) propagate
and culture pathogens, and e) formulate and apply pathogens in the
field.

f) Establishment of insect pathogen culture collection at CNPAF.
A collection of approximately 20 isolates of Bacillus thuringiensis, 3
insect viruses and an extensive collection of entomopathogenic fungi
including 61 isolates of Metarhizium anisopliae, 36 isolates of
Beauveria bassiana, 4 isolates of Paecilomyces spp., 1 isolate of
Nomuraea rileyi and 1 of Verticillium lecanii, 2 isolates of Hirsutella
thompsonii and 7 Entomophthorales has been established. In addition, a
collection of 63 isolates of entomogenous fungi and several viruses
collected in Brazil as part of the Title XII survey work is being
maintained at CNPAF and duplicate isolates have been sent to the Boyce
Thompson Institute culture collection in Ithaca, NY for preservation in
liquid nitrogen. Isolates will be distributed upon request to any
interested cowpea scientist or other researchers in or outside Brazil.
A list of all newly discovered isolates can be found in the appendix
(Appendix 1).

g) Development of an information and literature repository at CNPAF
on insect pathology and microbial control. Literature including
approximately 2,000 scientific reprints and technical references and
more than 10 books on insect pathology and microbial control have been
obtained for inclusion in a special insect pathology section to be
established in the library at CNPAF. A special indexing system is being
established for the holdings on insect pathology. This literature and






Brazil/Roberts -21


information repository will be increased substantially during the next
year, and a considerable amount of general information on insect
pathology will be translated into Portuguese and distributed to
libraries, research centers and Universities to increase knowledge on
insect pathology and microbial control in Brazil. A computer profile to
survey the current literature on pests and diseases of pests of cowpea
has been established. Computer printouts are regularly forwarded from
IPRC (BTI) to IPRC (CNPAF, Brazil).

2. Progress in the Training Program in Insect Pathology and the
Microbial Control of Cowpea Pests

As specified in our grant proposal our program is primarily a
training program, and it will be oriented to training Latin American and
Brazilian scientists and students, both men and women, at various well
defined levels in the areas of insect pathology and microbial control of
cowpea pests.

a. On-site training of cowpea research scientists in Northeast (NE)
Brazil. Three survey trips were made to the NE by the Host-country
Title XII Research Associate, Dr. R. A. Daoust, and the PI, Dr. D. W.
Roberts or the CoPI, Dr. R. S. Soper. Cowpea researchers, including
cowpea entomologists and cowpea plant protection specialists,
accompanied us on field trips and participated in informal training in
insect pathology and microbial control of cowpea pests. Among the
techniques demonstrated were 1) Recognition of diseased insects in the
field and the recognition and estimation of enzootic and epizootic
levels of infection in the pest population, 2) Collection, diagnosis,
and handling of diseased insect specimens, and 3) Isolation of
insect pathogens from pests of cowpeas and other crops and methods used
to ship pathogens and pure cultures.

A list of cowpea scientists who participated in this program is
presented below.



Area of
Participant Specialization Location


Joao Pratagil Pereira de Cowpea Breeder CNPAF/Goiania, Goias
Araujo

Belmiro Pereira das Neves Cowpea Entomologist CNPAF/Goiania, Goias

Jocicler da Silva Carneiro Entomologist UEPAE/Manaus,Amazonas

Maria de Fatima Batista Plant Pathologist UEPAE/Manaus,Amazonas

Gerson Pereira Rios Plant Pathologist CNPAF/Goiania, Goias


EPABA/Itaberaba, Bahia


Guaracy de Campelo


PI. Protect. Spec.






Brazil/Roberts-22


Area of
Participant Specialization Location


Eduardo Assis Menezes Plant Breeder EPABA/Itaberaba, Bahia

Paulo Diogenes Barreto Pl.&Cowpea Breeder EPACE/Barbalha,Ceara

Francisco Edson de Araujo P1. Protect. Spec. EPACE/Pacajus, Ceara

Reginaldo Barros Entomologist Student at UFC/Fortaleza,
Ceara

Auri Alaecio Simplicio Plant Pathologist EMPARN/Caico,Rio Grande
do Norte

Gilson Soares de Melo Entomologist UEP/Itapirema,Pernambuco



In addition to field and laboratory instruction on methods used in
insect pathology, discussions were held with various cowpea researchers
on the microbial control of insects, and literature was provided on the
techniques and theories used in microbial control. An extension-type
reference was prepared and translated into Portuguese to aid cowpea
researchers in the collection, diagnosis, handling and shipping of
diseased insects, and this was distributed to various researchers in NE
Brazil. As a result of this training effort we have established strong
contacts with various cowpea researchers in Brazil and have already
begun to receive diseased insect specimens by mail at CNPAF. Pathogen
isolations from these specimens will be incorporated into the culture
collection.

b. Short-term training of cowpea researchers at CNPAF. CNPAF has
made arrangements for cowpea researchers from NE Brazil to visit CNPAF
for periods of 2 4 weeks for intensive short-term specialized training
in cowpea research. Two scientists from the research organization in
the state of Maranhao of EMAPA have already entered into this training
program. These workers spent several days in the microbial control of
cowpea pest research program to learn field and laboratory methods and
to discuss the theories involved in microbial control. This included
more than 2 hours of formal presentations with slides, 4 6 hours of
discussions, and field and laboratory demonstrations of techniques used
in insect pathology and microbial control. Epizootics of an as yet
unidentified entomophthorales infection on Empoasca kraemeri and
Paecilomyces sp. on Lagria vilosa, were occurring during this time, and
the trainees were shown methods of collection, diagnosis,
identification, and isolation, and bioassay techniques were demonstrated
in the field and laboratory. The participants for this training were:






Brazil/Roberts-23


1. Ubiracy Mendes Soares He is an agronomist stationed at
Bacaba, Maranhao through EMAPA.

2. Iranilde Silva de Sousa She is a plant protection
specialist stationed at Brejo, Maranhao through EMAPA.

This training was held from 10 June to 30 June, 1982. Many more
short-term trainees from field stations in NE Brazil are expected to
participate in our training program during the coming year.

c. Training of students at CNPAF. EMBRAPA/CNPAF has a strong
interest in training students from Brazilian Universities at all levels.
For example, CNPAF invited 10 undergraduate students from the University
of Sao Paulo in Piracicaba, S.P. to visit the research center for the
month of July, 1982. Our program was included in the discussions with
these students. One student, Marina Yukie Murayama, had a particularly
strong interest in our program since she had had former experience in
insect pathology. She spent a considerable amount of time discussing
microbial control with Dr. Daoust and she was given many references on
insect pathology and microbial control of cowpea pests.

Two students, both seniors at the Federal University of Goias,
Goiania, Goias, have started a research training program under the
direction of Dr. Daoust. This project involves experimentation on a
part-time student basis with the cowpea storage weevil, Callosobruchus
maculatus. A complete description of this project with the results of
experiments can be found in the appendix.

Students at the graduate level have not been finally selected for
participation in our training program, but the following progress has
been made. One graduate student who has just started her training at
the Federal University of Ceara in Fortaleza, CE, Ms. Mary Ann Weyne
Quindere (an M.S. candidate), has been contacted. Mary Ann has
expressed a strong interest in participating in our training program and
in doing her thesis on some aspect of microbial control of cowpea pests.
The University of Fortaleza has a history of cooperation in such
programs and the faculty coordinator of graduate students in the
Department of Plant Technology (Fitotecnia, Dr. Jose'Albersio Araujo
Lima, and the entomologist, Dr. Jose Higino Ribeiro dos Santos, have
indicated that arrangements can be made for Dr. Daoust to serve on a
graduate student committee and to be given status as an adjunct
professor in the Department. Dr. Lima also expressed a strong interest
in cooperating with us on short-term training of University students or
other personnel in microbial control of cowpea pests. In order for Ms.
Quindere to participate in our graduate training program, however, it
will be necessary for her to gain permission to stay in Goiania at CNPAF
for at least 1 year through her sponsoring agency, EPACE, CE. She has
worked on cowpea research for several years through this state research
organization and they are paying for her education at the M.S. level.
As of August, 1982 they had still not agreed to allow Mary. Ann to do her
thesis work in Goiania.

Other potential trainees for our graduate training program include
the following candidates:





Brazil/Roberts -24



1. Maria de Lourdes Barbosa dos Santos She is a cowpea
scientist working for the Departamento Nacional Obras Contra Seca
(Irrigation systems) in Teresina, Piaui. She will be beginning her
Ph.D. program, probably in a university in southern Brazil in the near
future. She has written Dr. Daoust a letter expressing strong interest
in participating in our microbial control program.

2. Marcia Regina Albertoni She is a senior B.S. candidate
at UFG in Goiania, Boias and she is presently doing a project under the
direction of Dr. Daoust at CNPAF. She is expecting to enter a graduate
program between March and September next year and she has expressed a
strong interest in doing her thesis in our program.

3. Paulo Marcal Fernandes He is also a senior B.S.
candidate at UFG and similarly is doing a project at CNPAF with Dr.
Daoust. He will also enter a graduate program.next year and has a
strong interest in our program.

4. Numerous other potential M.S. or Ph.D. candidates are
expected to be identified at CNPAF during the months of September and
October, 1982 since we will be receiving many applications from
interested candidates for participation in our course in microbial
control (see next paragraph for explanation).

d. First training course entitled, 1o Curso de Controle
Microbiologico de Pragas do Caupi (First Course on the Microbiological
Control of Cowpea Pests). We will be sponsoring a course for between 10
and 15 cowpea researchers and students, principally from NE Brazil, from
11 October to 15 October at CNPAF. A brochure has been prepared and
sent to more than 200 locations in Brazil which describes the course
(see Appendix 3 for copy of the brochure and English translation of the
subjects to be covered). Thirty-five potential candidates applied for
participation in the course. These scientists represent 11 NE cowpea-
producing states and other areas of Brazil. Candidates were selected in
September, 1982. The majority are women. Expenses for the course will
be provided by the Title XII CRSP program on microbial control. We will
provide air passage for approximately 10 researchers, one Brazilian
scientist to provide lectures on diseases of soybean pests, and one
scientist (D.W. Roberts) from the U.S.A. to assist in teaching the
course. In addition, the employers of 5 more invitees will be requested
to support their employees. Dr. Blumenschein has agreed to contribute
to the course also by providing housing at CNPAF, meals at CNPAF at a
nominal cost, some transportation, classroom and laboratory space, and
other associated costs in conducting the course.

e. First course on the production of cowpeas entitled, 1 Curso de
Producao de Caupi. From 25 October to 13 November, 1982 a course
sponsored by CNPAF will be held at Goiania for approximately 30 cowpea
researchers from Brazil, Africa and other Latin American countries on
the production of cowpeas. Dr. Daoust has been asked to contribute to
the training of all participants by giving ca. 4 hours of formal
lectures and 2 hours of practical field and/or laboratory experience on
the microbial control of cowpea pests. The training course description





Brazil/Roberts-25



can be found in the brochure attached to Appendix 4. A list of the
talks to be given by Dr. Daoust is also included.

f. Training of laboratory personnel in microbial control and
insect pathology. Mr. Sebastiao Martins dos Santos was employed March
1, 1982 to perform technical assistance in our Title XII CRSP Program.
He has had previous experience in microbiology laboratory work and in
lab analyses. During the last 6 months he has received intensive
training in insect pathology and microbial control. He will work
primarily in a technical capacity including responsibilities for
conducting bioassays with insect pathogens, preparing various
specialized bacterial and mycological media, producing microbial control
agents, conducting field surveys for diseased insects, transferring and
preserving all isolates in the CNPAF culture collection (including more
than 200 insect pathogenic isolates) and aiding in field trials. He
will receive more specialized training during the next year in insect
pathology also. Sebastiao is currently taking an English course, paid
for by the Title XII CRSP Program, at the Fisk Language School in
Goiania, Goias.

Miss Heloisa da Silva Coelho was employed June 17 by our Title XII
Program. She has no previous experience in related fields, but she will
receive training in insect rearing and microbial control. She will
assist in many insect pathology techniques and will have complete
responsibility for the insectary. She has already received training in
insect rearing and in maintaining colonies of Chalcodermus aeneus,
Diabrotica paranoense, Ceratoma arcuata and Callosobruchus maculatus.
Soon we will be adding a colony of Elasmopalpus sp., the lesser stalk
borer, to this list.

g. Language training for Title XII CRSP scientists. Dr. Daoust
and his wife have received Portuguese instruction in privately tutored
classes for approximately 3 months (3 nights a week for 2 hours/class).
Dr. Roberts has received 6 weeks training of 1 hour per day.

3. Progress in the survey for pathogens of cowpea pests and
Related Activities

In our experimental plan, extensive surveys for pathogens of cowpea
pests were proposed for the first year. Three major survey trips were
made by the host country research associate (Dr. Daoust) and the
Brazilian counterpart entomologist (Dr. Belmiro das Neves) to the North
and Northeast regions of Brazil. The PI (Dr. Roberts) and Co-PI
(Dr. Soper) as well as other staff scientists from CNPAF accompanied Dr.
Daoust and Mr. das Neves on two of these trips. In addition, surveys
were conducted continually at CNPAF during the initial 9 months of the
program. This was possible at the National Bean and Rice Center since
irrigation systems now make it possible to grow cowpea throughout the
year. Locations visited in North and Northeast Brazil and the number
and types of pathogen isolates obtained in pure culture are shown in
Appendix 5, Table 1. Entomopathogenic fungi were the most common
pathogen group recovered although some virus-diseased insects were also
found. Bacterial diseases appeared to be considerably less common among
pest species on cowpeas. Six entomopathogenic fungi were recovered from





Brazil/Roberts-26


6 field sites in the Amazonas region and 12 were recovered from 50 small
subsistence farms and 14 experimental sites in the Northeast. However,
most isolates were obtained from CNPAF including 43 fungal isolates from
the following genera: Beauveria, Metarhizium, Nomuraea, Paecilomyces,
Erynia, Entomophaga, unidentified entomophthorales, and several as yet
unidentified fungal isolates. Several viruses have also been recovered
from the Northeast and CNPAP, but since these are difficult to identify
they have not been classified but will be stored for future reference.
The predominant pathogens recovered from the principal cowpea pests have
been Beauveria bassiana and Metarhizium anisopliae as shown in Appendix
5, Table 2. Eleven fungal isolates have been recovered from the cowpea
curculio, which is considered to be the principal pest in Northeast
Brazil. Similarly, 11 isolates were obtained from Chrysomelid,
leaf-feeding beetles including Ceratoma arcuata and Diabrotica
paranoense. These pests are particularly important on cowpea since they
have been shown to be involved in the transmission of the principal
disease of cowpeas -in Brazil, the severe mosaic virus. In addition,
numerous isolates have been recovered from other principal pest species
including the leafhopper, Empoasca kraemeri, various pod-sucking bugs
such as Piezodorus guildini, and the pod borer Maruca testulalis. In
the case of E. kraemeri, which causes severe damage and loss of yield to
cowpeas during the dry period of the cropping season, especially during
pod maturity, several extensive epizootics were located. In the state
of Rio Grande da Norte, cowpea fields in some areas with extremely high
Empoasca populations had epizootics with mixed infections of Hirsutella
guyana and an entomophthorales (possibly Entomophaga australiensis).
The entomophthorales isolate was cultured and returned to CNPAF and BTI
for preservation, identification, and possible development for field
control while the H. guyana strain was highly fastidious, and attempts
to culture it were unsuccessful. Fungal epizootics have also been found
on E. kraemeri at CNPAF. Several fungal pathogens have been isolated
but not yet identified.

In addition to surveys made to discover pathogens of pest species,
attempts were made, with the help of Brazilian entomologists, to
determine which insect species were most important in cowpea-producing
regions of Northeast Brazil. The information collected by CRSP
scientists and cooperating Brazilian scientists was compiled by Mr.
Belmiro das Neves and is presented in Appendix 5, Table 3. Pest species
varied significantly in different geographical locations with both
climatic and agronomic differences. For example, in wetter areas in the
litorall" regions of cowpea-producing areas, curculionid weevils and
pod-sucking bugs were more serious constraints on production, while
drier areas in the "sertao", especially late in the season during pod
maturation, had higher populations of E. kraemeri and Elasmopalpus
lignosellus, the lesser cornstalk borer. In general, the cowpea
curculio, Chalcodermus aeneus, the leafhopper, E. kraemeri, the lesser
cornstalk borer, E. lignosellus, the lepidopteran pod borer Etiella sp.,
and pod-sucking pentatomids were most important. However, considerable
variability occurred between states. For example, E. kraemeri
populations were extremely high and highly damaging to cowpea in Rio
Grade da Norte while they were only of minor importance in Ceara and
Bahia. Alternatively, E. lignosellus was very important in Bahia, but
was not found in Pernambuco. In other states not presented in Table 3,






Brazil/Roberts-27


such as Piaui and Alagoas, the most important pest species were the
cowpea curculio, C. aeneus, Chrysomelid leaf-feeding beetles (C. arcuata
and D. paranoense), pod-sucking pentatomids, and E. kraemeri. In
contrast, C. aeneus and E. kraemeri were not found in the state of
Amazonas; but Maruca testulalis, pod-sucking bugs, and chrysomelid
leaf-feeding beetles were highly damaging to cowpea.

In addition to pathogen surveys, the gathering of epizootiological
data, and estimations of pest populations, other progress made on survey
trips included the establishment of contacts with scientists, extension
agents, and administrators in various research centers and universities
throughout the North and Northeast. Also, potential trainees working
with insect pests of cowpeas were identified for participation in our
first training course on microbiological control of cowpea pests (see
above for description). Many of these individuals are also expected to
be future collaborators in our research program in the Northeast.
Private farmers and their families were consulted regarding the
production of cowpeas in the Northeast. By visiting more than 50
small-scale and subsistence farmers in the North and Northeast, and
through lengthy discussions involving sociological, economic, political,
cultural, and agricultural aspects of cowpea production and utilization,
both CRSP and cooperating Brazilian scientists became more aware of
factors associated with cowpea production and use in Brazil. Women
played a significant role in these discussions in aspects of cowpea
production, harvesting, storage and utilization.

4. Progress in the screening of entomogenous fungi for
pathogenicity against cowpea pests

Several insect colonies were established, and screening tests with
entomopathogenic fungi were initiated to identify microbial isolates
with high potential for pest control. Since cowpea-pest species were
not being maintained at CNPAF, Brazil prior to the arrival of Dr. Daoust
(see adjustments section), a major effort was devoted during the first
year to the establishment of laboratory colonies. At present, colonies
of Chalcodermus aeneus, Callososobruchus maculatus, Ceratoma arcuata,
and Diabrotica paranoense are being established in a newly constructed
insectary at CNPAF, Brazil. This work has been hampered by delays in
construction of the rearing facility (still not fully completed), delays
in hiring technical help to maintain colonies (a full-time, high-
school-level laboratory assistant was hired only in June, 1982),
training of technical personnel in rearing techniques, and the
incompleteness of present knowledge regarding life cycles and rearing
methods. Nevertheless, the above mentioned species have all been
successfully reared on natural foods for at least 2 generations;
however, a production schedule for supplying large numbers of insects
for bioassays has still not been developed. As a result, preliminary
screenings of fungal isolates against Chalcodermus aeneus and Ceratoma
arcuata were conducted using field-collected insects, while those
against Callosobruchus maculatus used laboratory-reared insects. The
use of field-collected Chalcodermus aeneus presented no problems except
that adult ages were unknown, and specimens were only intermittently
available. In contrast, bioassays with field-collected Ceratoma arcuata






Brazil/Roberts -28


and Diabrotica paranoense were difficult due to high control mortalities
resulting from naturally acquired diseases and parasites.

Various screening bioassays were performed to assess infection
methodologies applicable to Chalcodermus aeneus. Spores of Metarhizium
anisopliae and Beauveria bassiana were applied both to soil and foliage,
the foliage applications employing known and unknown spore
concentrations (Appendix 5, Tables 4 and 5, respectively). Fungal
strains isolated from naturally infected cowpea curculios in Brazil and
others obtained from the BTI culture collection were tested. In
general, it was shown that the B. bassiana isolates were considerably
more virulent to C. aeneus than were those of M. anisopliae.

In tests conducted with entomopathogenic fungi against Ceratoma
arcuata, several application methodologies were used, and 17 M.
anisopliae, 16 B. bassiana, and I Nomuraea rileyi isolates were tested
(Appendix 5, Tables 6-9). Control mortalities were high in many
experiments. Nevertheless, one isolate (CP5) of B. bassiana, obtained
from a naturally infected C. arcuata adult found in a cowpea field in
the Amazonas region of Brazil, proved highly virulent to C. arcuata
adults, producing 100% mortality with LT50 values of 4.57 and 7.09 days
in two laboratory tests. In addition, the isolate grew and sporulated
well on artificial media. In screening tests with strains for M.
anisopliae and B. bassiana isolated from a variety of insects from
world-wide geographical regions, it was found that while both fungal
species were pathogenic, virulence was highly dependent upon the isolate
tested (Tables 8 and 9). More quantitative and extensive screening
tests to be conducted during the second year should elucidate the
potential of these and other isolates against coleopterous pests. The
Nomuraea rileyi isolate proved to be relatively avirulent even though
spore concentrations were high and spore viability was 91%.

B. Planned U.S. Activities

1. Progress in bioassay development and pathogen screening.
(Study Conducted by Research Assistants Stephen Wraight and Susan
Raba in Collaboration with Dr. D. W. Roberts, P.I.).

It was decided to conduct the pathogen screening and bioassay
studies against leafhoppers of the genus Empoasca which are considered
major pests of cowpea and other bean crops in Latin America. The
decision to concentrate on this pest group was made for several reasons.

a. Empoasca is a common pest of legumes in the Cornell University
area, providing opportunity for field research.

b. Empoasca fabae, the most important species of Empoasca in the
U.S. (including the Cornell area) is very closely related to E.
kraemeri, its counterpart in Brazil (E. karemeri is a member of the
fabae complex). Pathogenicity data collected on E. fabae will therefore
likely be applicable to E. kraemeri.

c. Boyce Thompson Institute maintains a laboratory colony of E.
fabae. The original colony, held on fava beans, has been successfully





Brazil/Roberts -29


transferred to cowpeas and is currently supplying specimens for the
screening of pathogens.

d. The reviewers of the grant application proposed that this
insect be emphasized in the US portion of the research.

Much of the first year was spent in the development of a bioassay
procedure. Initial attempts to hold insects on excised leaf disks
proved unsatisfactory due to high rates of control mortality. A system
was therefore developed in which the insects are held on whole leaves,
the stems of which are embedded in water-saturated cotton. Control
mortality now is usually less than 10%.

Also sought was an effective and repeatable method of treatment
(exposure of the insects to the infectious units of the pathogen).
Initial treatments were- made by treating leaves with surface-wetting
agents followed by direct application of a desired volume of pathogen
suspension onto the leaf surface. This method was time consuming and
did not produce a uniform distribution of infectious material on the
leaf surface. To overcome these problems, a spray apparatus originally
designed for fungus treatment of spruce budworm was constructed and
modified. A comparison was then made of mortality attained by exposing
leafhoppers to treated leaves to that obtained by spraying the pathogen
directly onto the insects. Mortality rates resulting from the two
treatment methods were about equal or somewhat higher when the insects
were sprayed directly, and since this method has proven faster and
easier to carry out than spraying of the leaves, it has been adopted.

The bioassay procedure resulting from these investigations is
outlined below.

a) Mid-late-instar nymphs are anesthetized with CO2 and placed in
the center of a small (35 mm diam.) petri dish (6 nymphs/dish).

b) The dish is then centered beneath an atomizing spray nozzle and
either distilled water (control) or a pathogen (fungal spore) suspension
is applied at the rate of approximately 0.04 ml/cm' (exact amounts are
determined by replicate weighing of spray deposits);

c) The sprayed insects are then transferred (allowed to walk) into
a larger (90 mm diam.) petri dish containing fresh cowpea leaves
supplied with water as described above.

d) The petri dishes are closed and incubated at 27 + 1 C under
16:8 photoperiod.

e) Dead insects are counted, and the survivors are transferred to
fresh leaves every 48 hr. Assays are ended after 4-6 days depending on
the pathogen being tested.

This bioassay procedure is being developed using the well known
entomopathogenic deuteromycete, Metarhizium anisopliae, as a model.





Brazil/Roberts -30


The bioassay, as described, represents a maximum challenge
situation. The humidity in the closed dishes is at or near 100%, which
coupled with the warm temperature, provides optimal conditions for
fungal sp2re germination and penetration. High doses (10 and 10
spores/cm ) are applied during the initial screening. The test system
is now operational and screening of fungal pathogens is underway.

2. Progress in formulation and large-scale production of microbial
control agents

Although work in this area was not planned to commence until the
second year, studies were initiated on the production and formulation of
Beauveria bassiana, the fungal pathogen we have isolated most frequently
from cowpea pests in Brazil (see culture collection list).

a) Large-scale production of B. bassiana-using low-technology
methods developed in the People's Republic of China. (Study conducted
by Robert C. Everich, a Cornell University undergraduate student in
collaboration with Dr. D. W. Roberts, P.I., and Mr. Zengzhi Li, a
visiting scientist from the P.R.C.)

The entomopathogenic fungus, B. bassiana, is produced at the
farm-commune level in the P.R.C. for local control of insect pests such
as the European corn borer, Ostrinia nubilalis. The field efficacy of a
granular formulation of Beauveria against the corn borer is reported to
be 80 to 90%. Beauveria bassiana produced at county agricultural
stations in the P.R. C. has proved to be an effective method of control
for the pine caterpillar, Dendrolimus punctatus.

The culture media for the fungus consist of raw materials produced
on the farms: wheat and ricebran, cornstalk powder, grasses, or compost
humus. Inoculum buildup is accomplished in 3 stages on 1) sterile
slices of potato in test tubes, 2) sterile bran and water in jars, and
3) nonsterile media in wooden trays in incubation rooms or spread on
outdoor soil beds. Production on soil beds is utilized in P.R.C. to
afford a heat-sink for the excessively high level of heat of metabolism
associated with fungus growth.

The production of B. bassiana using this low technology method was
undertaken at Boyce Thompson Institute in order to assess the potential
of the procedure as a means of large-scale production of inoculum by
local control programs in cowpea and bean producing areas of Brazil and
elsewhere. In particular, the methods investigated in this study may
have applications in subsistence-level agricultural systems where cost
is a severely limiting factor.

The production procedure, results, and observations are presented
in Appendix 5, Table 10. The results successfully demonstrated the
capacity of this production system to produce large quantities
(approximately 10 conidia per gram of bran) of infectious material at
extremely low cost.







Brazil/Roberts-31


b. Formulation of B. basiana spores with ultraviolet light
protectants. (Study conducted by BTI formulations specialist Michael
Ward in collaboration with Drs. D. W. Roberts, P.I., and R. S. Soper,
Co.-P.I.).

The degradation of microbial pathogens by ultraviolet (UV) light is
a primary problem in their development as commercial insecticides. For
this reason, numerous photoactive compounds are being investigated for
their ultraviolet protection capabilities. These UV screening compounds
were selected from five chemical groups: hydroxybenzophenones,
benzotriazoles, salicylates, aryl-substituted acrylates, and amino
benzoates. Several commercially available representatives from each
chemical group were tested for compatibility by plating out a simulated
tank mix of B. bassiana spores and the protectant and quantifying the
spore viability. Compounds that were found compatible (i.e., did not
significantly reduce spore viability) were mixed with B. bassiana spores
and exposed to natural sunlight. Artificial light was not used since
the only UV lamps available to us provided wave lengths much shorter
than the 290 millimicron and longer present in sunlight.

The protectanis tested thus far include generic brands of p-amino
benzoate, Giv-TanF (Givaudan Corp., Clifton, NJ), and UVF (Agra-K
corp., Minneapolis, MN). Sunscreen-coated spores were sprayed onto
potato leaves and exposed to four hours of direct sunlight. Leaf washes
were then used to recover the spores. The recovered, irradiated spores
were subsequently bioassayed using first-instar Colorado potato beetle
(Leptinotarsa decemlineata) larvae. The resulting LT50 of Giv-Tan F
(2-ethoxyethyl p-methoxycinnamate)-protected spores was 4.96 days. This
was lower than that of unprotected, irradiated spores (5.40 days), but
higher than nonirradiated (control) spores (4.53 days), indicating that
partial protection was afforded by this compound. None of the other
compounds tested provided a measurable amount of UV protection.

C. Unplanned U.S./H.C. Activities

All activities, except mass production and formulation of fungi,
were planned for the first year of the project. The two exceptions were
originally intended for second-year attention, but were moved to year
one to assist in preparing for anticipated field trials in year two.





Brazil/Roberts -32


VI. Plans

A. 1983 program

1. Objectives

a) Continue development of the Insect Pathology Resource
Center (Brazil).

b) Continue dissemination and exchange of insect
pathology and microbial control related information through the various
lines of communication established in Brazil in 1982 (including contacts
with Brazilian scientists, regional grower associations, and individual
subsistence farm families).

c) Continue and extend the survey for pathogens of
cowpea pests in Brazil. New sites will be surveyed, and productive
sites found in 1982 will be revisited in 1983.



d) Continue work on the development and improvement of
insect rearing and bioassay methods. Pathogens isolated from the field
and obtained from culture collections such as that maintained by IPRC
(BTI) will be screened against the appropriate pest insects. It is
expected that both time-mortality (LT50) and dose-mortality (LC50) data
will be generated for at least some of the pathogens showing promise for
control. Tests will be conducted against a number of important cowpea
pests, including Chalcodermus bimaculatus, Callosobruchus maculata,
Ceratoma arcuata, Diabrotica paranoense, and Empoasca spp. Work on a
lepidopteran pest, Maruca or Elasmopalpus lignosellus, will be added as
well in 1983.

e) Continue research on methods of formulation and
low-technology production of fungal pathogens. Initiate small-scale
production of selected pathogens to provide material for preliminary
field trials.

f) Initiate studies on strain improvement. This will
involve use of mutagenic agents (e.g. EMS or UV irradiation) and
cultural techniques (e.g. passage of strains through alternate hosts or
pest species) to enhance virulence of promising isolates.

g) Conduct preliminary field trials with pathogens
demonstrating promise for control of selected insect pests.

h) Commence training of Brazilian students and
scientists in insect pathology and microbial control. Graduate students
will be selected and will complete several courses in Brazil or in the
U.S., depending on the students' programs. One to several Brazilian
scientists selected as project cooperators will be invited to spend
several months at IPRC (Brazil). An insect pathology workshop will be
conducted for cooperators at IPRC (Brazil) in fiscal 1983.






Brazil/Roberts-33




2. Methodologies

The original approved plan included methods for accomplishing
the objectives of the first two years of the project, and these are
presented in section II of this report. Since we have adhered closely
to the original plan, there is little to add here. Detailed
descriptions of methods developed for specific problems such as the
establishment of lines of communication, production and formulation of
Beauveria bassiana, bioassay of selected insect pest species, and
training of Brazilidn scientists and students can be found in section IV
(Summary of Achievements).






Brazil/Roberts -36


VII. References

Aizawa, K. 1971. Strain improvement and preservation of virulence of
pathogens, pp.655-672. In Microbial Control of Insects and Mites.
(H.D. Burges and N.W. Hussey, eds.) Academic Press, NY.

Al-Aidroos, K., and D.W. Roberts. 1978. Mutants of Metarhizium
anisopliae with increased virulence toward mosquito larvae. Can.
J. Genet. Cytol. 20, 211-219.

Carner, G.R. 1980. Sampling pathogens of soybean insect pests.
pp.559-574. In Sampling Methods in Soybean Entomology. (M. Kogan
and D.C. Herzog, eds.) Springer-Verlag, NY.

CNPAF. 1980. Subsidies para elaboracao do program national de
pesquisas com caupi. Internal report., maio, 1980.

Daoust, R.A. and D.W. Roberts. 1979. Virulence of natural and insect-
passaged strains of Metarhizium anisopliae to larvae representing
three mosquito genera. Abstr. Ann. Meeting of the Society of
Invert. Pathol., Gainesville, FL.

Eastman, C.E. 1980. Sampling phytophagous underground soybean
arthropods. pp.327-354. In Sampling Methods in Soybean Entomology.
(M. Kogan and D.C. Herzog, eds.) Springer-Verlag, NY.

Finney, D.J. 1971. Probit Analysis. 3rd Edition, Cambridge University
Press, London.

Harper, J.D. and G.R. Carner. 1973. Incidence of Entomophthora sp. and
other natural control agents in populations of Pseudoplusia
includes and Trichoplusia ni. J. Invert. Pathol. 22, 80-85.

Helm, C.G., M. Kogan and B.G. Hill. 1980. Sampling leafhoppers on
soybean. pp.260-282. In Sampling Methods in Soybean Entomology.
(M. Kogan and D.C. Herzog, eds.) Springer-Verlag, NY.

Ignoffo, C.M., B. Puttler, N.L. Marston, D.L. Hostetter and W.A.
Dickerson. 1975. Seasonal incidence of the entomopathogenic
fungus Spicaria rileyi associated with noctuid pests of soybeans.
J. Invert. Pathol. 25, 135-137.

Ignoffo, C.M., N.L. Marston, B. Puttler, D.L. Hostetter, G.D. Thomas,
K.D. Biever and W.A. Dickerson. 1976. Natural biotic agents
controlling insect pests of Missouri soybeans. pp.561-578. In
Proc. World Soybean Res. Conf., Sept. 1976. 1073 pp.

Kogan, M. and D.C. Herzog, eds. Sampling Methods in Soybean Entomology.
Springer-Verlag, NY.

Milner, R.J., R.S. Soper, and G.G. Lutton. 1982. Field release of an
Israeli strain of the fungus Zoophthora radicans (Brefeld) Batko
for biological control of Therioaphis trifolii (Monell) f.
maculata. J. Aust. Entomol. Soc., 21:113-118.






Brazil/Roberts -37


Metcalf, C.L., W.P. Flint and R.L. Metcalf. 1962. Destructive and
Useful Insects. Their Habits and Control. McGraw-Hill Book
Company, NY.

Newman, G.G. and G.R. Carner. 1975. Disease incidence in soybean
loopers collected by two sampling methods. Envir. Entomol. 4,
231-232.

Roberts, D.W. and R.A. Humber. 1981. Entomogenous fungi. pp.201-229.
In Biology of Conidial Fungi. Vol. 2. (G.T. Cole and B. Kendrick,
eds.) Academic Press, NY.

Taylor, T.A. 1978. Maruca testulalis: an important pest of tropical
grain legumes. In Pests of Grain Legumes: Ecology and Control.
(S.R. Singh, H.F. van Emden, and T.A. Taylor, eds.) Academic
Press, NY. 454 pp.

Watt, E.E. 1978. First annual report on IITA/EMBRAPA/IICA Cowpea
Program in Brazil, Goiania, Brazil.

Watt, E.E., et al. 1979. Second annual report on the EMBRAPA/IITA
Cowpea Program in Brazil, Goiania, Brazil.

Wojciechowska, M., K. Kmitowa, A. Fedorko and C. Bajan. 1977. Duration
of activity of entomopathogenic microorganisms introduced into
soil. Polish Ecol. Studies. 3, 141-148.

Yevlakhova, A.A. 1978. Basic trends in the use of entomopathogenic
fungi in the Soviet Union. pp. 35-50. In Proceedings of the First
Joint US/USSR Conference on the Production, Selection and
Standardization of Entomopathogenic Fungi of the US/USSR Joint
Working Group on the Production of Substances by Microbiological
Means. (C.M. Ignoffo, ed.)





Brazil/Roberts -38




APPENDIX
APPENDIX




Brazil/Roberts
Appendix 1


IPRC/BTI-CNPAF/EMBRAPA ENTOMOPATHOGENIC FUNGUS CULTURE COLLECTION
CNPAF Caixa Postal 179
Goiinia, Goiis 74000 Brazil


REVISED 01 August 1982


Beauveria bassiana
81-11-25-01
25 November 1981
Costa do Arapapa, Amazonas, Brazil
Vespidae (Adult)
Richard Daoust



Unknown
81-11-25-02
25 November 1981
Costa do Arapapa, Amazonas, Brazil
Vespidae (Adult)
Richard Daoust


Beauveria bassiana
81 -11 -25-04
25 November 1981
Costa do Surubin, Comunidade do
Cristo Rei, Amazonas, Brazil
Ceratoma arcuata
Richard Daoust


Beauveria bassiana
81-11-25-05
25 November 1981
Costa do Surubin, Comunidade do
Cristo Rei, Amazonas, Brazil
Ceratoma arcuata
Richard Daoust


CP 1
Acc. N9:
Date:
Site:
Host:
Coll:


Beuaveria bassiana
81-11-25-07
25 November 1981
Costa do Surubin, Comunidade do
Cristo Rei, Amazonas, Brazil
Ceratoma arcuata
Richard Daoust


Beauveria bassiana
82-1-6-01
6 January 1982
CNPAF, Fazenda Capivara, Goiania
Goiis, Brazil
Chalcodermus aeneus
(Coleoptera:curcul onidae)
Richard Daoust


CP 6
Acc. N9:
Date:
Site:

Host:
Coll:


CP 7
Acc. N9:
Date:
Site:

Host:

Coll:


CP 8
Acc. N9:
Date:
Site:
Host:
Coll:



CP 9
Acc. N9:
Date:
Site:
Host:
Coil:


Metarhizium anisopliae
82-1-6-02
6 January 1982
CNPAF
Chalcodermus aeneus
Richard Daoust



Beauveria bassiana
82-1-6-03
6 January 1982
CNPAF
Chalcodermus aeneus
(erson Pereira lios


CP 2
Acc. N9:
Date:
Site:
Host:
Coil:


CP 4
Acc. NO:
Date:
Site:

Host:
Coll:


CP 5
Acc. N9:
Date:
Site:

Host:
Coll:







CP 10
Acc. N9:
Date:
Site:
Host:
Coil:


CP 11
Acc. N9:
Date:
Site:
Host:
Coll:


Beauveria bassiana
82-1-6-04
6 January 1982
CNPAF
Chalcodermus aeneus
Gerson Pereira Rios


Metarhizium anisopliae
82-1-6-05
6 January 1982
CNPAF
Bruchidae
Richard Daoust


Coil:


Nomuraea rileyi
82-1-6-18
6 January 1982
CNPAF
Spodoptera sp.
Richard Daoust


Unknown
82-1-15-01
15 January 1982
CNPAF
Chalcodermus aeneus
Richard Daoust


Beauveria bassiana
82-1-15-02
15 January 1982
CNPAF
Chal codermus aeneus
Richard Daoust


CP 17
Acc. N9:
Date:
Site:
Host:
Coll:


CP 18
Acc. N9:
Date:
Site:
Host:
Col l:


CP 19
Acc. N9:
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CP 15
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Date :
Site:
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Coll:


CP 16
Acc. N9:
Date :
Site':
Host:


Brazil/Roberts
Appendix 1
Unknown
82-1-25-02
25 January 1982
CNPAF
Spodoptera sp.
Richard Daoust


Unknown
82-1-6-06
6 January 1982
CNPAF
Lagria vilosa
(Coleoptera: Lagriidae)
Richard Daoust


Paecilomyces fumosoroseus
82-1-6-10
6 January 1982
CNPAF
Lagria vilosa
Richard Daoust


Paecilomyces fumosoroseus
82-1-15-05
15 January 1982
CNPAF
Lagria vilosa
Richard Daous t


Paecilomyces fumosoroseus
82-1-15-06
15 January 1982
CNPAF
Lagria vilosa
Richard Daoust


CP 12
Acc. N9:
Date:
Site:
Host:
Coll:


CP 13
Acc. N9:
Date:
Site:
Host:
Coll:


CP 14
Acc. N9.
Date:
Site:
Host:
Coll:






Brazil/Roberts
Appendix 1


P 20
Acc. N9:
Date:
Site:
Host:
Coll:


CP 21
Acc. N9:
Date:
Site:
Host:
Coll:


CP 22
Acc. n9:
Date:
Site:
Host:
Coil:

I
CP 23
Acc. N9:
Date:
Site:
Host:
Coil:



CP 24
Acc. N9:
Date:
Site:
Host:

Coil:


Beauveria bassiana
82-1-26-01
26 January 1982
CNPAF
Pentatomidae
Evane Ferreira


Metarhizium anisopliae
82-2-2-01
2 February 1982
CNPAF
Tetyganidae
Yoshitaka Tanaka


Metarhizium anisopliae
82-2-3-02
3 February 1982
CNPAF
Unidentified orthopteran
Richard Daoust


CP 25
Acc. N9:
Date:
Site:
Host:
Col l.


CP 26
Acc. N9:
Date:
Site:
Host:
Coll:


CP 27
Acc. N9:
Date:
Site:
Host:

Coll:


CP 28
Acc. N9:
Date:
Site:
Host:

Coll:


CP 29
Acc. N9:
Date:
Site:
Host:
Coll:


Beauveria bassiana
82-2-9-01
9 February 1982
CNPAF
Chalcodermus aeneus
Ri chard Daoust


Metarhizium anisopliae
82-2-15-02
15 February 1982
CNPAF
Deois flavopicta
(Homoptera: Cereopidae)
Richard Daoust


Metarhizium anisopliae
82-2-15-04
15 February 1982
CNPAF
Ceratoma arcuata
Richard Daoust


Unknown
82-2-15-05
15 February 1982
CNPAF
Lagria vilosa
Richard Daoust


Beauveria bassiana
82-2-3-01
3 February 1982
CNPAF
Diabrotica paranoense
(Coleoptera: Chrysomelidae)
Richard Daoust


Beauveria bassiana
82-3-1-01
1 March 1982
CNPAF
Diabrotica paranoense
(Coleoptera: Chrysomelidae)
Richard Daoust


Beauveria bassiana
82-3-1-03
1 March 1982
CNPAF
Diabrotica paranoense
Richard Daoust






Brazil/Roberts
Appendix 1


CP 30
Acc. N9:
Date:
Site:
Host:

Coil:


CP 31
Acc. N9:
Date:
Site:
Host:

Coil:


CP 32
Acc. N9:
Date:
Site:
Host:
Col l:



CP 33
Acc. N9:
Date:
Site:
Host:


Beauveria bassiana
82-2-15-01
15 February 1982
CNPAF
Diabrotica paranoense
Richard Daoust


Metarhizium anisopl iae
82-2-15-06
15 February 1982
CNPAF
Piezodorus guildini
(Hemiptera: Pentatomidae)
Richard Daoust


Metarhizium anisopliae
82-2-15-03
15 February 1982
CNPAF
Ceratoma arcuata
(Coleoptera: Chrysomelidae)
Richard Daoust


Erynia sp.
82-3-10-01
10 March 1982
CNPAF
Unknown dipetran
Richard Soper



Conidiobolus sp.
82-3-10-04
10 March 1982
CNPAF
Unknown leafhopper
(Homoptera: Cicadellidae)
Richard Soper


Host:
Coll:


CP 35
Acc. NO:
Date:
Site:
Host:
Coil:



CP 36
Acc. N9:
Date:
Site:
Host:
Coll :



CP 37
Acc. N9:
Date:
Site:
Host:

Col l:


CP 38
Acc. N9:
Date:
Site:

Host:
Coll:


CP 39
Acc. N9:
Date:
Site:


Beauveria bassiana
82-3-23-01
23 March 1982
Missao Velha, Ceari, Brazil
Tenebrionidae
Richard Daoust



Beauveria bassiana
82-3-24-01
24 March 1982
Brejo Santo, Ceari, Brazil
Arocanthus sp.
Richard Soper



Metarhizium anisopliae
82-4-1-01
1 April 1982
CNPAF
Deois flavopicta
(Homoptera: Cercopidae)
Richard Daoust


Unknown
82-4-4-01
4 April 1982
Goiania, Garden behind house of
Richard Daoust
Unknown, probably Coleopterous
Richard Daoust


Metarhizium anisopliae
82-4-5-01
5 April 1982
Manaus, Amazon (UEPAE/Manaus),
Brazil
Pentatomidae
Jocicler da Silva Carneiro


CP 34
Acc. N9:
Date:
Site:
Host:
Coll:





Brazil/Roberts
Appendix 1


Acc. N9:
Date:
Site:
Host:

Coll:


CP 41
Acc. N9:
Date:
Site:
Host:
Coll:


CP 42
Acc. N9:
Date:
Site:
Host:

. 11:



CP 43
Acc. N9:
Date:
Site:

Host:
Coll:


Unknown
82-4-13-01
13 April 1982
CNPAF, old site in Goiania
Bemisia sp.
(Homoptera: Aleyr6didae)
Massaru Yokoyama


Unknown
82-4-13-02
13 April 1982
CNPAF, old site in Goiinia
Bemisia sp.
Massaru Yokoyama


Unknown
82-4-13-03
13 April 1982
CNPAF, old site in Goiinia
Bemisia sp.
Massaru Yokoyama


Erynia sp.
82-5-1-01B
1 May 1982
Municipality of Crateus Ceara,
Brazil
Unknown dipteran
Richard Daoust


CP 45
Acc. N9:
Date:
Site:

Host:
Coll:


CP 46
Acc. N9:
Date:
Site:
Host:
Coll:


CP 47
Acc. N9.
Date:
Site:
Host:

Coll:


CP 48
Acc. N9:
Date:
Site:
Host:

Coll:


Hirsutella guyana
82-5-7-30-2
7 May 1982
4 km from Goianinha, Rio Grande
do Norte, Brazil
Empoasca kraemeri
Richard Daoust and Donald Roberts


Beauveria bassiana
82-4-29-01
29 April, 1982
Caucaia, Ceara Brazil
Cerambycidae (Adult)
Richard Daoust


Beauveria bassiana
82-4-30-01C
30 April, 1982
Novo Oriente, Ceari, Brazil
Elateridae (Adult)
(Genera unknown)
Joao Pratagil P. de Araujo and
Richard Daoust


Beauveria bassiana
82-4-30-010
30 April 1982
Novo Oriente, Ceari, Brazil
Elateridae )Adult),
(Genera unknown)
Joao Pratagil P. de Araujo and
Richard Daoust


Entomophaga australiensis
82-5-7-3D-1
7 May 1982
4 km from Goianinha, Rio Grande
do Norte, Brazil
Empoasca kraemeri
Richard Daoust and Donald
Roberts
(previously undescribed species)


CP 49
Acc. N9:
Date:
Site:
Host:
Coll:


Beauveria bassiana
82-5-3-01
3 May 1982
Pacajus, Ceari, Brazil
Crimissa sp.
Liana Saraiva Teixeira


CP 44
Acc. N9:
Date:
Site:

Host:
Coil:






Brazil/Roberts
Appendix 1


CP 50
Acc. N9:
Date:
Site:

Host:
Coil:



CP 51
Acc. N9:
Date:
Site:

Host:
Coll:


Beauveria bassiana
82-5-3-03A
3 May 1982
20 km North of Pacajus, Ceara,
Brazil
Crimissa sp.
Richard Daoust and Francisco
Edson de Araujo


Beauveria bassiana
82-5-3-03B
3 May 1982
20 km North of Pacajus, Ceara,
Brazil
Crimissa sp.
Richard Daoust and Francisco
Edson de Araujo


Metarhizium anisopliae
82-5-4-01
4 May 1982
CNPAF, Goiania, Goias
Deois flavopicta
Sebastiao Martins dos Santos


Beauveria bassiana
82-5-4-06
4 May 1982
CNPAF, Goiinia, Goias
Deois flavopicta
Sebastiao Martins dos Santos



Paecilomyces sp.
82-5-4-03
4 May 1982
CNPAF, Goiania, Goias, Brazil
Chalcodermus aeneus
Richard Daoust


CP 57
Acc. N9:
Date:
Site:
Host:
Coil:


CP 58
Acc. N9:
Date:
Site:
Host:

Col 1 :


CP 59
Acc. N9:
Date:
Site:
Host:


Coll :


CP 55
Acc. N9:
Date:
Site:
Host:
Col l:




CP 56
Acc. N9:
Date:
Site:
Host:
Coll:


Unknown
82-5-27-02
27 May 1982
CNPAF, Goiania, Goias
Ceratoma arcuata
Sebastiao Martins dos Santos


Beauveria bassiana
82-5-6-01
6 May 1982
Barbalha, Ceara, Brazil
Tenebrionidae (Adult)
.(Genera unknown)
Joao Pratagil P. de Araujo


Beauveria bassiana
82-5-6-02
6 May 1982
Barbalha, Ceara, Brazil
Tenebrionidae (Adult)
(Genera unknown)
Joao Pratagil P. de Araujo


Unknown
82-5-12-04A
12 May 1982
CNPAF, Goiania, Goiis
Chalcodermus aeneus
Sebastiao Martins dos Santos




Unknown
82-4-27-03A
27 April 1982
CNPAF, Goiinia, Goias
Chalcodermus aeneus
Richard Daoust


CP 52
Acc. NO:
Date:
Site:
Host:
Coll:


CP 53
Acc. N9:
Date:
Site:
Host:
Col :



CP 54
Acc. N9:
Date:
Site:
Host:
Coll :





Brazil/Roberts
Appendix 1


0
CP 60
Acc. N9:
Date:
Site:
Host:
Col l:

CP 61
Acc. N9:
Date:
Site:
Host:
Coll:

CP 62
Acc. NO:
Date:
Site:
Host:

. 11:


Unknown
82-4-27-01
27 April 1982
CNPAF, Goiania, Goils, Brazil
Chalcodermus aeneus
Richard DaousTf

Beauveria bassiana
82-6-28-01
28 June 1982
CNPAF, Goiania, Goiis, Brazil
Diabrotica paranoense
Sebastiao Martins dos Santos

Metarhizium anisopliae
82-7-23-01
23 July 1982
CNPAF, Goiania, Goias, Brazil
Coleomegilla maculata
(Coleoptera: Coccinelidae)
Sebastiao Martins dos Santos


RAD/MOB-08.82.





Brazil/Roberts
Appendix 2


TRAINING PROGRAM FOR UNDERGRADUATE (B.S.) STUDENTS IN
AGRICULTURE AT THE FEDERAL UNIVERSITY OF
GOIAS IN GOIANIA, GOGLS, BRAZIL


Information about the students:

I. M'rcia Regina Albertoni
Rua 19 No. 55, Ed. Dom Abel Apt. 1004
Setor Central
Goiania, Goias 74.000 Brasil
Telephone: 062 225-0968

Program: 3.S. degree at Escola de Agronomia, Univeridade Federal de Goias,
Goiania, Goigs. Senior year.

II. Paulo Marcal Fernandes
Rua 210 ao. 05
Setor Vila Nova
Goiania, Goias 74.000 Brasil
Telephone: 062 261-3075 (brother)

Program: B.S. degree at Escola de Agronomia, Universidade Federal de Goias,
Goiania, Goias. Senior year.

Title of Project of Study in Title XII Program:

Studies on the Biology and Pathogenicity of Entomopathogenic Fungi to the
Cowpea Storage Weevil, Callosobruchus maculatus

Objectives of Project of Study:

1. Determine general biology, life cycle and ecology of the cowpea storage
weevil, Callosobruchus maculatus.
2. Determine the pathogenicity of entomopathogenic fungi, including strains
of Metarhizium anisopliae, Beauveria bassiana, Nomuraea rileyi and others,
against the cowpea storage weevil and to determine the potential of
fungi for use in microbial control.
3. Develop methodologies to study the pathology of the cowpea storage weevil.
U. Study the pathogenicity of serotypes of Bacillus thuringiensis against
the cowpea storage weevil.
5. Learn techniques and theories used in insect pathology and microbial control.

Progress of Project to Date:

The project was started in July, 1982 and will continue over the next year
on a part-time basis ( approximately 8 15 hours/week for each student and longer
during vacations). Before July the students had numerous discussions with Dr. R. A.
Daoust concerning insect pathology and microbial control and they spent a con-
siderable amount of time reading technical literature and books on the subject.
Since one of the students speaks English fluently (M'rcia) she has also translated
some of the relevant literature into Portuguese. Four experiments with Metarhizium
anisopliae and Beauveria bassiana have been completed and numerous more have been
planned over the next 2 3 months. Work on B. thuringiensis will be delayed for
a short period due to the highly effective results achieved with the fungi.





Student Projects 2


Brazil/Roberts
Appendix 2


Methods Used and Results:


Experiment I A suspension of M. anisopliae strain E ESMC Parent, obtained
from an infected pasture spittlebug, Deois flavopicta in Brazil, was prepared by plac-
ing spores from a 10 15 day old culture of the fungus on Emersons YpSs medium (agar)
into sterile distilled water containing 0.1% Sween 80, a surfactant. The final spore
concentration of the suspension was 3.26 x 10 spores/ml and the viability of the
suspension was 98%. Other conditions of the test were as follows:
i treatment methodologies were used for the fungus applications (see Table 1).
5 control treatments were used ( some were included to learn more about the insect's
biology and adult longevity).
3 replicates of 5 insects were held in glass petri dishes (100 x 15) with 20 cow-
pea seeds each.
Dead insects were removed at the intervals reported in Table 1 and they were
surface-sterilized by standard methods (1 min. in 0.1% NaOC1 and several rinses
in sterile distilled water) and placed into a sterile humid chamber (a petri
dish with filter paper and sterile distilled water to create an Relative humidity
of approx. 100%) to confirm the percent infection.

TABLE 1
Effect of Various Treatment Methods with Metarhizium anisopliae Against
Callosobruchus maculatus.

) Treatments Percent Mortality Percent
Confirmed
Da 3 Da 5 Da 7 Da 12 Infections
1. Control 1 Plates empty
with only insects; no 0 15 40 93 0
filter paper, no water and
no seeds.


2. Control 2 Plates with
dry seeds and insects
only; no filter paper
or water.

3. Seeds presoaked in a Water
solution of water + +
Tween 80 with or Tween
without the fungus; 80
no further water used only
and no filter paper put Water
into dishes. +
Tween
80

ungus


4. Seeds presoaked in a
solution of water +
Tween 80 with or without
the fungus; filter paper
included held at 100%
r.h. throughout the test


Water
+
Tween
only
Water
+
Tween
Vlrlmall


73 100


16 100


4o 100





Student Projects 3


Brazil/Roberts
Appendix 2


Table 1 continued


Treatments Percent mortality Percent
Confirmed
Da 3 Da 5 Da 7 Da 12 Infections


5. No seeds put into plates Water
but filter paper with
water + Tween 80 with Tween 7 33 38 93 0
or without fungus and 80
r. h. held at 100%
Only
throughout the test. Water
+
Tween 100 66
80
+
FunguE
6. Dry seeds put into dishes
with filter paper soaked
with water + Tween 80 + 40 100 -93
Fungus and r. h. held at
100% throughout the test..




Experiment 2. This experiment was conducted in a similar manner to the
first experiment to confirm the infectivity of M. anisopliae strain E ESMC Parent
to adults of C. maculatus. The same fungus suspension as was used in the first
experiment was used in this experiment to treat seeds. The only differences
between this and the first experiment was that only presoaked seeds in either
the water and Tween 80 solution or in the solution containing spores were used
for the treatments. In addition, seeds were placed in small plastic disposable
cups (capacity about 50 ml) rather than petri dishes and no further water was added.
Since cups had cloth with a coarse mesh-size held tight with elastic bands as lids
the seeds dried quickly and after day no further humidity was noted ( seeds were
very dry). As in the other experiments all treatments were held at room temper-
ature in the Insect Pathology Laboratory (temp. 2 25 C). One other difference
was that all the insects used in this test were newly emerged adults (1 day old)
while those used in the first experiment were of unknown age. The viability and
spore count was the same as in experiment 1. Other conditions were as follows:
5 cups with 10 insects were used for each treatment (5 replicates of 10 insects).
40 seeds were put into each cup.






Student Projects 4


Brazil/Roberts
Appendix 2


TABLE 2


Infectivity of Metarhizium anisopliae to Callosobruchus maculatus under

Conditions of Low Relative Humidity After Day 1.



Treatment Percent Mortality


Day 7 Day 9


Control
Seeds presoaked in a suspension
of water(sterile distilled) +
Tween 80 (0.1 %) 26 81

Treatment
Seeds presoaked in a suspension
of water(sterile distilled) +
Tween 80 (0.1,) + fungus solu-
tion. 95 98



Experiment 3. This experiment was a repeat of experiment 2 except that a
second strain of M. anisopliae was added to the test and only 5 insects were
included in each cup instead of 10 as in expt. 2 (5 replicates of 5 insects /
treatment). All C. maculatus adults were 1 day old. Strain E ESMC Parent was
981 viable and had a spore count of 3.26 x 10 spgres/ml. Strain E9ESMC Parent
was 100% viable and had a spore count of U.7 x 10 spores/ml.

TABLE 3
Infectivity of 2 Strains of Metarhizium anisopliae to Callosobruchus maculatus
under Conditions of Low Relative Humidity After Day 1.

Treatment Percent Mortality

Day 6 Day 8 Day 9

Control Seeds presoaked in
water + Tween 80 only 24 48 63

E6ESMC Parent Seeds presoaked
in fungus solution with this
strain. 50 92 100

E ESMC Parent Seeds presoaked
in fungus solution with this
strain. 31 77 100





Student Projects 5
Brazil/Roberts
Appendix 2

Experiment 4. This experiment was conducted to determine pathogenicity of
4 isolates of B. bassiana against C. maculatus. Seeds were presoaked in water
and Tween 80 (0.1%) with or without fungal spores of B. bassiana (B. bassiana
was grown on SDAy Agar for approximately 2 weeks at room temp. to obtain spores).
Twenty presoaked seeds were put into each petri dish and 5 dishes with 10 newly
emerged adults/dish were used for each treatment (5 replicates of 10 insects each).
Spore counts and viabilityof strains are shown below:
Strain Viability Spore Concentration
RS 32 100% 5.56 x 10/ml
RS 252 70% 1.Lo x 10o/ml
RS 502 99; 1.88 x 10 /ml
RS 285 99% l.65 x 10 /ml


TABLE 4

Pathogenicity of 4 Strains of Beauveria bassiana to Callosobruchus maculatus


Treatment Percent Mortality Number in Total Number on
Original Day
Da 3 Da 5 plates(5) 3 5

Control Seeds pre-
soaked in water +Tween 2 30 50 55 75

Seeds presoaked in water
+ Tween 80 + Fungus
RS285 38 81 50 64 75

RS 502 12 67 50 64 81

RS 252 17 58 50 57 70

RS 32 49 72 50 59 72


Discussion:

In the first experiment 4 methods of treatment with the fungus Metarhizium
anisopliae were used and high mortality was achieved in most treatments as early
as 3 days after exposure to the fungus. By 5 days after exposure 100% mortality
occurred in all treatments with the fungus while control insects showed mortal-
ities of only between 0 and 33%. In addition to the high mortality found in the
treatments with M. anisopliae between 87 and 100% of the insects which died in
treatments with the fungus were confirmed as having M. anisopliae infections. In
contrast, no insects removed from the control dishes had confirmed infections.
In one case in which seeds were presoaked in water and tween (control) or
water, Tween and the fungus suspension (test) and held at 100% relative humidity
in a petri dish on filter paper 100% mortality was achieved for the treated
insects within 5 days, while all controls remained alive (0% mortality). In
this treatment 100% of the insects dying in the treated plate developed infections.
... as +r t.he biology of C. maculatus, experiment 1 showed that humidity






Student Projects 6 Brazil/Roberts
Appendix 2

Discussion continued,

did not greatly affect longevity of adults. It was expected that humid conditions
would be detrimental to survival of adults since this insect normally lives under
very dry conditions. In contrast, survival of control insects was higher under
conditions of higher humidity.

In experiment 2 the results further corroborated those in experiment 1 demon-
strating high infectivity of M. anisopliae to the cowpea weevil. In this case
the experiment was conducted using plastic cups with only cloth tops to allow
moisture to evaporate rapidly. Therefore, the exposure period to high humidity
was for less than 1 day. High mortality was shown (95%) in the treatment in
which fungus was applied to seeds by day 7 while only a low mortality (26%)
occurred in the control cups. The percent infection was not confirmed in this
experiment.

The results of experiment 3 were similar to those shown in experiment 2, except
that mortalities were somewhat lower after 1 week in the latter test. Nevertheless,
100% mortality was reached by 9 days after exposure of adults to the fungus
treatments.

In experiment L,four isolates of Beauveria bassiana were tested against -.
maculatus. The methods used were similar to those used in experiment 1 in that
seeds presoaked in fungus solution were placed in petri dishes with high humidity
and held with 10 insects per plate. Strains RS 32 and RS 285 showed the highest vir-
ulence with 72% and 81% mortality, respectively, after 5 days exposure to the
fungus-treated seeds as compared with only 30% mortality for the control. In
this test old seeds were used which had eggs of C. maculatus an them and after
several days new insects began to emerge. The total numbers which emerged for each
treatment are shown in Table 4. It was assumed that the insects surviving after
5 days were mostly newly emerged adults and, therefore, mortalities among the
original test insects may have been considerably higher than those shown in
Table U.



NOTE: Control mortality appears high in these experiments. This, however, is
simply a reflection of the fact that the adults of Callosobruchus
maculatus have a short natural life span(5-7 days), and experiments were
read until the death of many or most of the untreated animals. As the
data indicate, such deaths were very seldom fungus related.






Brazil/Roberts
Appendix 3




GENERAL THEMES OF IPRC-CNPAF COURSE ON
MICROBIAL CONTROL OF COWPEA INSECT PESTS
Goiania, Goias, Brazil
October 11-15, 1982

INTRODUCTORY TOPICS

Introduction to the Microbiological
Control of Cowpea Pests.
Theory and Current Status of Microbial
Control
Biological Aspects and Damage of the
Principal Pests of Cowpeas

PATHOLOGY OF INSECTS

Viral Diseases
Bacterial Diseases
Fungal Diseases
SProtozoan and Nematode Diseases

(Taxonomy; Mode of Action; Infection
Process; Standardization and Application
and other Topics)

MICROBIAL CONTROL PROGRAMS AND PRODUCTS

Microbial Control Programs Worldwide
Commercial Microbial Products
Registration
Safety

MICROBIAL CONTROL TECHNOLOGY
Production Methods
Formulation and Application

MICROBIAL CONTROL TECHNIQUES
Survey and Collection of Pathogens
Sampling Insect Pathogens
SEpizootiology
Diagnosis, Isolation, Preservation,
Handling and Shipping Pathogens

CONCLUSIONS

Potential for Microbial Control Against
Cowpea Pests
Strategies and Constraints on the
Microbial Control of Pests in the Future





Brazil/Roberts
Appendix 3




LIST OF TE TAT I'.' .-T I TS =, C .': E '*'IC I CO TROL


TT : :A-.:i P 7, :: .. LOCATION
S??ECIALIZAT10


PERNAfMBUCO


*Cleonor Cavalcante Alves da



'*ocicer da Silva Carneirc


Guaracv Campelo


1. *Francisco Edson de Araujo



2. Marv Ann %'evne Ouindere


Iranilda Silva de Sousa


Bunrifaic Peixotc -Mazalhaes


1. Elizabeth Araujo de Albuquerque
Maranhao

2. *Francisca Nemaura Pedrosa Haji


ernomlorist



M.S. entomologist.


E.S. Plant


M.S. Plant
Protection

B.S. Plant
Protection


Plant Protection


E.S. En:to=logist


entomologist


Ph.D entomologist


EPEAL/Maceio, AL



LEPAE/Manaus, A.


EPABA/Itaberaba,
BA


EPACE/Pacajus,CE



EPACE/Barbalha,
(Univ.Fed.Ceara)


EMAPA/Brejo, MA


CPATU/Selem, PA


IPA/Serra Talha-
da, PE

CPATSA/Petroline


Maria de Lourdes Barbosa dos


Santos


F0U G7ANDE DO


Alessandra Perazzo Barbosa Xota


M.S. Plant
Technology


entomologist


DNOCS /PI
Teresina, PI


EMPARN/Natal, RK


Candidates to which top priority should be given.
40


.ALAGOAS



A:>!AZONAS


3AH IA


CEARA


:ORTE


.AR-AN.rLO






Brazil/Roberts
Appendix 4


I CURSO DE PRODUCAO DE CAUPI

Program


No. TITULO DATA HORARIO


1. Manejo de Solo e Agua em 28/10/82 08:00 horas
areas com Recursos Hidricos
Limitados.

2. Sistema de Producao e 01/11/82 08:00 horas
Ervas Daninhas.

3. Melhoramento e Estatistica. 03/11/82 19:10 horas

4. Sementes. 05/11/82 10:00 horas

5. Fisiologia e Caracteristicas 08/11/82 08:00 horas
do Caupi.

6. Microbiologia, Solos e 10/11/82 13:30 horas
Nutricao de Plantas.

7. *Entomologia e Controle 11/11/82 08:00 horas
Microbiologico.

8. Fitopatologia. 12/11/82 08:00 horas


*Dr. R.A. Daoust will give five, one-hour presentations on various
aspects of insect pathology and microbial control.

1. Program de control microbiologico. Produtos comerciais e
internacionais.

2. Colecao, diagnose, isolamento, prevencao, transport e
embalagem dos agents de control microbiologico.

3. Amostragem e levantamento de insetos contaminados com
patogenos.

4. Pratica de laboratorio.

5. Estrategias de aplicacao e limitacao no uso de agents de
control microbiologico.












O
01
,-A


N )
CIS 91

PQI _4


Dates
States and On Which
Region Locations Surveys Were
Surveyed Visited Conducted

North Brazil Amazonas Manaus. INov. 22-27.
Survey Trip I Iracrntiara. 9.in:.c.ipuru, 1981
and Experlneinal
Station at Caldeir5o
Northeast Bahia Salvador. Cruz March 14-27.
Brazil das Almas, Feira de 1982
Survey Trip 2 Santana, Itaberaha. and
Senhor do Bon fim
Pernambuco Petrolina.
Ourlcuri. Trinidad.
Salguciro, nom None, and
S3o JosC do Belmonte
Cearh Barbalha. M:issio
Velha, and Brejo Santo
Piaul Picos and Teresina
Survey Trip 3 Cenrt Fortaleza, April 28 -
Caucaia. Sobral. May 11. 1982
Crateus. Nova Oriente,
Independence, Canlnd1, and
Paca Jua
Rio :r.inde do Norte -
Natal. Gol.lnlnfh.I. Sa.to
Antonio, Calc6. Martins
Port Alegre. Umarlzdl. and
Mossor6
Paralba Brejo da Cruz and
eastern Paraiba
Pernambuco Itapirema and
Recife
Central Brazil CoLSs CNPAF and Periodic sur
COtania from Nov. 191


Aug. 1982


Nirijher of
Pathoigen Isolates
Obta ir.'d in
Pure Culture

6 Me


veys
81 -


Path,, p n
Groups
Recovered

tarhizium .iantsopine.
* iaveriai h i.is lina., .and
ntdentil led fungi


2 Reauverla bassiana












10 E ynLia sp.. Entomo-
phaga australiersils.
Hiqrsutelia gsuyna
(isolate not cultured).
.and nBea.ver'fr ha.;s lana











43 Beauveria bassiana,
.etarhizium aniospliae.
Noimraea rtiley .
Paeclomyce_ fImosno-
roseis., Erynia 9p..
Entomophaga australien--
sis and unidentified
fungi


*major pest of cowpeas


Insect Groups
from which Pathogens
were Isolated

Ceratoma arcuata*
Unidentified Vespldae
Unidentified Pentatomidae*


Arocanthus sp.*
Unidentifidd
Tenebrionldae


Brazil/Roberts, Appendix

No. of sites visited
Small Cowpea
Experi- (Subsistence)
mental Farmers

2 4


Empoasca kraemert*, 8
Crimlssa sp.
Unidentified Dipteran
Unidentified Cerambycldae
Unidentified ELateridie
Unidentified Tenehrlonidae










Chalcodermus aeneus*, 1
Spodoptera sp.*, Lagria
vilosa, Deois flavopicta,
Ceratoms arcuata*, Diabrotica
paranoense*. Piezodorus
guildint Bemisia sp..
Empoasca kraemert*,
Coleomegilla maculata,
Unidentified Bruchidae.
Pentatomidne. Tetygonidae,
Orthopteran. Dipteran, Cicadellidae


TAPlI.E 1. Surveys for Insect Pathogens Conducted in Rrazil.








Brazil/Roberts

Appendix 5


Brazil/Roberts


TABLE 2. Pachogen Isolates Obtained from Major and Minor Cowpea Pests and from Nonpest
Species Occurring in or Near Cowpea Fields.


Total
Number of Number of Number of Other
Pest Species Pathogen Isolates B. bassiana M. anisopltae Pathogens
Obtained in Isolates Isolates Isolated
Pure Culture


MAJOR COWPEA PESTS


Chalcodermus
aeneus

Ceratoma
arcuata

Diabrucica
paranoense

Empoasca
kraemeri




Piezodorus
quildini

Maruca cestulalis




Spodoptera sp.



Lagria vilosa


Acocanchus sp.

UnLdentified
Pentacomidae

Unidentified
Bruchidae



Deois flavopicta

Bemisia sp.


Crimissa sp.

Coleomegilla maculaca

Unidentified
Diptera
Orchoptera
Coleoptera

Cicadellidae

Cerambycidae
Elateridae
Tenebrionidae
Vespidae


MINOR COWPEA PESTS


5 probablyy
P'acilumyces sp.)

I unidentifiedd
fungus)

0


4 (Unidentified
Encomophchorales
and Hirsutella
guyana [isolate
not recovered])

0


2 (probable viral
infected larvae)



2 (Nomuraea rileyi
and unidentified
fungus and virus)

5 (Paecilomyces
fumosoroseus)

0

0


NON-PESTS ASSOCIATED WITH COWPEA FIELDS


0

3 (unidentified
fungi)

0

0


2 (Erynia sp.)
0
1 (unidentified
fungus)
1 (Entomophaga
australiensis)
0
0
0
I (unidentified





















TABIE 3. Occurrence of Cowpea Pests In 4 Northeastern States in Brazil.


Numhbr of
Properties
Visited

23

11


Percent Fr,.-1--v nt ; ich P'st Among Pest Species
Total
Are.
(ha) Emp. Ett. Cha. Pet. "ar. Li. \r,. Pant. Eu. Crin.

329 5 9 17 22 7 I/ 5 2 1

212 26 20 22 0 15 5 0 0


BiHfA 5 14.5 8 10 33 0 5 26 10
t' RNA' IO 10 697.5 22 9 31 11 0 0 15


TOTAl. 49 1253 13 12 22 13 5 10 11

Pl'repared by Belmiro P. dan Nrves, CNPAF.
Fmp. Empeapca n p., Et. Ettella sp., Chal. Chatcoderms; .nlen.ii. ent. P Ientrtonlrd.,e
SAro"lthl s sp. Pant. Pantomorus glaucos. Eu. Crin. Crninre.rai
Aphids, Thr. Thrips. --.


Collected

Dia. Cer. Aph. Thr.

2 1 6 0

0 0 3 3


0 0 8 0 0 0 0

0 0 0 5 0 0 0


4 1 2


2 1 4 1


Mar. Maruca testulalts. Elas. Elasmopatpun sp., Aro.
sp., Dia. Diabrotica sp., Cer. Ceratona sp., Aph. -


0


0


CEARA

R. (RANIDE
DO NORTF









0

Q ..14
NW


alsolates obtained from other host species outside Brazil.
Isolates obtained from C. aeneus in Brazil.
CConfirmed as positively infected with M. anisopliae or B. bassiana, respectively.


0


TABLE 4. Effect of Exposure of Cowpea Curculio Adults, Chalcodermus aeneus, to Soil Treated With Either
a Locally Collected Strain Isolated From C. aeneus or a Foreign-Collected Isolate From Another
Host of Two Entomogenous Fungi.


Species and Spore
Strain Tested Concentration Cumulative % Mortality After Percent with
/ g soil Day 6 Day 8 Day 15 Confirmed Infectionc


Metarhizium anisopliae
Ma 43a 5.0 x 106 17.5 25.0 42.5 16.6
CP 8b 3.1 x 106 7.5 12.5 20.0 20.0

Beauveria bassiana
RS 285a 1.4 x 107 35.0 55.0 71.0 82.6
CP 9b 1.1 x 107 45.0 67.0 95.0 66.7

Control -0- 10.0 10.0 10.0 -0-






U,


0


PQ
I- i


Cowpea Foliage With


Species and Spore Concentration Cumulative % Mortality After Percent with
Strain Tested and Exposure Method Day 5 Day 9 Day 14 Confirmed Infection


FOLIAGE DIPPED INTO SUSPENSION WITH CONCENTRATION

Control -0- 20.0 20.0 20.0 -0-

Metarhizium anisopliae
Strain CP 8 1.53 x 107 spores/ml 7.9 18.4 23.7 42.9

Beauveria bassiana
Strain RS 285 7.22 x 107 spores/ml 12.5 50.0 55.0 75.0


LEAF DISK WITH KNOWN CONCENTRATION /mm2

Control -0- 10.0 10.0 10.0 -0-

Metarhizium anisopliae
Strain Ma 43 2.47 x 104 spores/mm2 8.0 12.0 20.0 -0-
Strain CP 8 1.51 x 104 spores/mm2 0.0 0.0 8.0

Beauveria bassiana
Strain RS 285 7.13 x 104 spores/mm2 16.0 64.0 80.0 75.0
Strain CP 9 5.51 x 104 spores/mm2 8.0 40.0 68.0 90.0


TABLE 5. Effect of Exposure of Cowpea Curculio Adults, Chalcodermus aeneus, to
Known and Unknown Concentrations of Two Entomogenous Fungi.







14
W
0)

-4
*r4 d
No)
I.cda


aCP isolates were isolated
the BTI collection.


from various cowpea pest species in Brazil.


Other strains were obtained from


bViability of all strains was above 95%.


corrected by Abbott's Formula.
Corrected by Abbott's Formula.


TABLE 6. Actual and Corrected Percent Mortalities of Adults of Ceratoma arcuata Exposed to Leaf
Disks Treated With Various Strains of Entomogenous Fungi Isolated From Pest Species in
Brazil and Elsewhere.



Strain Spore Concentration Actual % Mortality Corrected % Mortality LT50
Testeda / mm2 Surface Areab Day 7 Day 12 Day 7 Day 12 (Days)


Control 28 40 0 0

M. anisopliae strains

CP 11 5.4 x 106 12 24 0 0
RS 346 1.7 x 107 36 48 11 13
E9 PARENT 1.2 x 107 16 36 0 0

B. bassiana strains

CP 4 3.8 x 105 32 64 6 40 -
CP 5 7.0 x 106 48 100 28 100 7.09
CP 14 6.2 x 106 36 40 11 0 -
CP 7 1.5 x 107 32 48 6 13
RS 220 3.5 x 105 12 28 0 0










TABLE 7. Actual and Corrected Percent Mortalities of Adults of Ceratoma arcuata Exposed to Soil
Treated With Various Strains of Entomogenous Fungi Isolated From Brazilian amd Other
Insect Species.


Strain Spore Concentration Actual % Mortality Corrected % Mortality LT50
Tested / g soil Day 6 Day 13 Day 6 Day 13 (Days)


Control 20 35 0 0

M. anisopliae strains
Eg CS 1 9.92 x 108 38 76 23 63 7.89
CP 11 4.32 x 108 50 89 38 83 6.00

B. bassiana strains
CP 1 1.11 x 109 23 59 4 37 -
CP 5 1.02 x 109 75 100 69 100 4.57
CP 10 5.32 x 108 25 45 6 15
RS 501 4.00 x 108 44 50 30 23


s







U,
$4
0

NGJ
$4 CL
PQ -


Adults Exposed to Strains of Metarhizium anisopliae
Brazil.


M. anisopliae Spore Concentration Cumulative % Mortality Afterb
Strains Tested / mm2'Surface Areaa 4 Days 8 Days 11 Days 13 Days


Control -0- 12 32 32 44

V14 3.6 x 105 54 86 92 94

RS 346 1.3 x 106 58 88 92 94

Ma 43 1.3 x 106 52 84 90 92

EPABA A24 1.4 x 106 26 48 58 66

31 (M-100) 1.3 x 106 24 52 74 74

E9 PARENT 1.8 x 106 46 90 98 98

MA 02 1.9 x 106 32 72 86 94

Phil #7 2.0 x 106 48 82 94 96

1140 2.3 x 106 40 72 86 94

Eg PARENT 2.7 x 106 42 74 88 96


aSpore viability for all strains was above 96%.
Cumulative results from 2 separate tests.


TABLE 8. Percent Mortality of Ceratoma arcuata
in Preliminary Screening Bioassays in








0


r4 d
Ndw
P3 <


TABLE 9. Percent Mortality of Ceratoma arcuata Adults Exposed to Strains of Beauveria bassiana
in Preliminary Screening Bioassays in Brazil.



B. bassiana Spore Concentration % Viability Cumulative % Mortality After
Strains Tested / mm2 Surface Area of Spores 4 Days 8 Days 12 Days 17 Days

Control -0- 0 12 16 29

RS 32 1.1 x 105 100 8 36 80 84

RS 149 3.9 x 104 100 4 36 64 88

RS 160 3.9 x 104 88 4 28 56 76

RS 220 3.0 x 104 69 0 28 56 92

RS 252 2.8 x 104 70 8 20 64 68

RS 285 9.2 x 10 99 8 44 92 100

RS 501 8.6 x 104 100 8 24 64 76

RS 502 3.7 x 104 99 4 16 52 84

Bb Pl 9.4 x 104 100 8 33 80 100


0






Brazil/Roberts
Appendix 5



TABLE 10. Methodology for Mass Production of Beauveria bassiana with
Minimal Technology and Capital Outlay.


Stage 1. Primary inoculum production. Peeled potato slices were
autoclaved in test tubes, inoculated with B. bassiana spores
from an agar plate, and incubated at room temperature for 8
days. Fungal growth and sporulation were excellent.

Stage 2. Inoculum build up. Raw, livestock-feed grade wheat bran with
1% CaO limewater added at the rate of 1 part bran to 0.7 parts
limewater by volume was placed in wide-mouth, clear glass
jars, covered with a cheesecloth and cotton stopper, and
autoclaved for 30 minutes at 121 C. After cooling, four
potato pieces from stage 1 production were added to each jar,
and the jar was agitated to distribute the spores and then
incubated at room temperature in light for 7 days. Spore
production was excellent.

Stage 3. Final production.

a. Production in trays. Unsterile bran was mixed with an equal
volume of a NaOH (pH 11) solution (high pH inhibits the growth
of contaminant microorganisms). The alkaline bran was then
mixed with inoculum from stage 2 production at a rate of 0.85
parts bran to 0.15 parts inoculum by volume. Large, shallow
trays were lined with ground corncob saturated with the NaOH
solution, filled to a depth of 4-5 cm with the inoculated,
alkaline bran, and covered with a thin layer of the alkaline
corncob to prevent moisture loss. The trays were then
incubated at room temperature in light. Approximately 18-24
hr after inoculation, a rapid rise in temperature occurred,
and ventilation was required to prevent the temperature of the
medium from rising above 280 C. Plain water was misted over
the culture beds twice a day for the first 5 days, and the
corncob covering was removed after 3 days when sporulation was
evident. After completion of the incubation period (7-10
days), the fungal mat was air dried and milled.

b. Production in Outdoor Soil Beds. A raised, level bed of fine
soil (1.3 x 1.3 x 2.5 m) was prepared, and covered with a
canvas tarp supported by a wooden frame. The soil was covered
with two layers of sterile newspaper, and the inoculated,
alkaline medium (prepared as described above) was spread to a
depth of 5-10 cm. Two additional layers of newspapers covered
the medium to prevent moisture loss. Water was added daily
until the third day at which time the newspaper cover was
removed and the culture allowed to dry. The final product
harvested after 8 days was found to contain approximately 1 x
10 spores/g.









CAMEROON/UNIVERSITY OF GEORGIA/CHALFANT


"PEST MANAGEMENT STRATEGIES FOR OPTIMIZING COWPEA YIELDS IN CAMEROON"


In northern Cameroon the cowpea is the major indigenous pulse; however,
production is severely limited by pre- and post-harvest insects which are
generally beyond the means of control by small farmers with existing
technology and finances. Lacking are strategies for management of these pest-
and the trained personnel to research and deliver new methods to the farmers.
This project is planned to develop methods for optimizing yields and quality
of cowpeas through a collaborative research and training program in cowpea
pest management involving Cameroonian and U.S. scientists. In Cameroon a
scientist trained in entomological research on cowpeas has been stationed at
the Institute de la Recherche Agronomique (IRA), and is actively engaged in
research. In the U.S. at Boyce Thompson Institute, U.S. and African strains
of the cowpea weevil have been established and biological studies are in
progress. A repellent substance deposited by the egg-laying female has been
found. Vegetable oils which have been reported to protect peas from the
cowpea weevil in storage were found to be effective for only a short period of
time.


2384B







CAMEROON/UNIVERSITY OF GEORGIA/CHALFANT


S"PEST MANAGEMENT STRATEGIES FOR OPTIMIZING COWPEA YIELDS IN CAMEROON"

Dr. Richard B. Chalfant, Department of Entomology and Fisheries,
University of Georgia (PI); Drs. J. A. A. Renwick and P. R. Hughes,
Boyce Thompson Institute; and
Dr. J. P.Eckebil, Institute of Agricultural Research, Department of
Agronomy and Forestry Research, Yaounde (PI); Dr. Moffi Ta'ama,
Institute de la Recherche Agronomique, Maroua

Introduction

The major goal is to improve yield and quality of cowpeas on small farms
in northern Cameroon through a cowpea insect pest management program involving
Cameroonian and U.S. scientists. Information developed is expected to form a
basis for continued use by Cameroon farmers. Cameroon is represented by
Delegation General de la Scientifique (DGRST) and Institute de la Recherche
Agronomique (IRA). U.S. is represented by the University of Georgia (UGA).

Basic studies in the U.S. will be directed toward the development of new
principles which can be used in the field. Studies in Cameroon will
concentrate on the practical aspects of pest management.

Summary of Achievements

In Cameroon

Dr. Moffi E. Ta'ama was approved by DGRST and IRA as the CRSP researcher
in Maroua. He established residence in a CRSP leased house on May 15, 1982
and began research at the IRA field station there.

The normal rainy season was delayed and research plots could not be
established until July following a trip by Dr. Ta'ama to IITA to obtain an
adequate supply of viable seed.

As of August 22, the peas are doing well, blossoming, and sampling is in
progress. Aphids appear to be an unexpected problem.

In early 1982 Mr. George Ntoukam, IRA-designated counterpart, was sent to
IITA in Nigeria to undergo a training program in cowpea research. He is now
in Maroua working with Dr. Ta'ama.

In the U.S.

Work at the Boyce Thompson Institute was initiated in February, 1982, when
Dr. Frank Messina was appointed. The major goals for the first year were:
(a) to establish a cowpea weevil colony and to develop a bioassay for
determining host preference; (b) to investigate the possible existence of an
oviposition deterring pheromone; (c) to characterize the protection qualities
of natural oils against the cowpea weevil.

Weevil colonies of distinct strains have been successfully established.
The first was obtained from IITA in Nigeria. Another colony originated from
cowpeas purchased by Dr. Chalfant at a market in Cameroon. The colonies have







been strictly isolated to investigate possible differences in behavior and
biology. Differences in coloration, markings and general activity of beetles
k have already been noted.

Studies on the oviposition behavior of the weevil have confirmed the
existence of a spacing mechanism that ensures even distribution of eggs. The
presence of eggs apparently deters further oviposition until all other beans
have received eggs. The deterrent appears to be chemical, and can be
extracted into diethylether. Treated and control beans were placed in
opposite sections, and the number of eggs on each were counted. The deterrent
has been collected by allowing female weevils to oviposit on glass beads.
Results indicate that the deterrent is associated with the act of oviposition.

The protection qualities of oils was investigated by treatment of
cowpeas with a wide range of oils. Peanut oil had been used in the past and
represents a natural oil that has a high content of unsaturated fatty acids.
Coconut oil was chosen for its high saturated fatty acid content. Safflower
oil was selected as a more general unsaturated oil. Completely different
chemical properties were provided by mineral oil, which consists entirely of
hydrocarbons, and Carbowax 300 (polyethylene glycol).

All of the oils tested appeared to be equally effective in reducing the
number of progeny from weevils that were added to cowpeas immediately after
treatment. The protective quality of the oils seems physical rather than
chemical. The deterrent effect of peanut oil was considerably higher when 60
days elapsed before addition of weevils to the treated beans. However, when
eggs were laid on these beans, no significant effect on larval development was
obtained. Furthermore, when no choice was provided, weevils confined to
treated and aged seeds laid as many eggs as those confined to control
cowpeas. These results indicate that oils may only be effective in protecting
stored cowpeas for a limited period of time. Although ovipositing weevils
show a strong preference for untreated seeds, the effect of the oils is not
enough to suppress egg laying when no choice is available.

Preliminary studies on the "active" flight form of the cowpea weevil
indicate that all strains of the insect may not have the potential for
production of this morph. Crowding of populations has resulted in the
appearance of the active form in both the Cameroonian and Florida strains, but
not in the IITA colony. It appears likely that natural selection in the
laboratory colony favors production of the "normal" form, so that the ability
to induce production of the active form disappears.

Unplanned:

In Cameroon

The cowpea aphid emerged as a potential key insect pest of cowpeas. This
may require revision of sampling procedures.

In the U.S.

The much reported protective vegetable oils were ineffective in protecting
* pea seeds under a no choice situation. This may require a revision of some of
the tactics for pest management.







1983 Program

* The 1983 program will be a continuation of the 1982 program with no
anticipated changes in goals, objectives or methodology.

In Cameroon

Field plots will include representative cultivars in treated and
non-treated plots, construction of insect sampling plots (for thrips, aphids),
evaluation of new cultivars for resistance to insects (thrips, aphids and
cowpea weevil), evaluation of experimental insecticides and application
methods for control of insects. Of particular interest is a new electrostatic
sprayer developed by Imperial Chemical Industries and which is very adaptable
for use by small farmers.

It is anticipated that a Cameroonian student will be enrolled in the
Graduate School of the University of Georgia by the 1983 Spring quarter.
Classes will be taken in Athens while research on cowpea insects will be at
Tifton.

In the U.S.

Work on host preference of cowpea weevils will be continued according to
the original plan. The oviposition deterrent will be further investigated to
determine the duration of its effect and whether suppression of oviposition in
a no-choice situation is possible. The effect of different concentrations
will be tested and the stability of the active material evaluated. Chemical
studies will be started to isolate and characterize the deterrent. Silica gel
chromatography will be utilized to separate ether extracts into lipid classes,
and fractions will be subjected to HPLC and/or GLC as appropriate.

The selection of host plants by active females will be investigated in
cages and a small room that allow flight. Little is known about the general
biology of the active form, which is believed to be responsible for initial
infestation of cowpeas in the field. These studies are thus expected to
provide new information on conditions influencing the success of the weevils.
Different cowpea varieties are already being grown in the greenhouse for
investigation of host preferences of active females. Data will be collected
on the effect of flight exercise on behavior and fecundity of active weevils.


2416B



















Pest Management Strategies for Optimizing

Cowpea Yields in Cameroon


Dr. Richard B. Chalfant

Department of Entomology and Fisheries

Coastal Plain Experiment Station

Tifton, GA 31793



Drs. J. A. A. Renwick and P. R. Hughes

Boyce Thompson Institute

Ithaca, N.Y.
















ANNUAL REPORT

1982








I. Introduction


A. The Project: The major goal is to develop methods for

improving yield and quality of cowpeas on small farms in

northern Cameroon through a collaborative research and train-

ing program in cowpea insect pest management involving

Cameroonian and U.S. scientists. Information obtained about

appropriate procedures developed are expected to form a basis
for continued use by Cameroon farmers over the long term.

Cameroon is represented by Delegation General de la Scientifique

(DGRST) and Institute de la Recherche Agronomique (IRA). U.S.

is represented by the University of Georgia (UGA).

Basic studies in the U.S. will be directed toward the

development of new principles which can be used in the field.

Studies in Cameroon will concentrate on the practical aspects

of pest management.

B. Participating Personnel:

1. Dr. Richard B. Chalfant, Principal Investigator. He

has had 23 years of experience in laboratory and field research

on the biology and management of insect pests of vegetable

crops in N. Carolina (1959-1966) and the University of Georgia

(1966-Present).

2. Drs. J. A. Renwick and P. R. Hughes, Co-Investi-

gators, Boyce Thompson Institute. Drs. Renwick and Hughes

have worked as a team for 10 years on problems of plant-insect

interactions.

3. Dr. Moffi E. Ta'ama, researcher in Maroua Cameroon.

Dr. Ta'ama has received special training in pest management








at the ICIPE Research Center, University of Nairobi in 1978

and Imperial College in London in 1981. His Ph.D. disseration

and previous research on cowpea insects has been at the Univ-

ersity of Ibadan and at IITA, Ibadan, Nigeria, 1977-1982.

4. Dr. Frank Messina, Post-Doctorate researcher at

Boyce Thompson. Dr. Messina obtained his Ph.D. in the

Department of Ecology and Systematics at Cornell University

in 1982 and was immediately hired for the CRSP project.


II. Original Approved Plan


A. Goals and Objectives

In Cameroon

1. Identify key insect pests of cowpeas and their

biology within the principal cowpea producing areas of northern

Cameroon.

2. Evaluate cowpea cultivars for resistance to major

insect pests and characterize mechanisms for resistance to

facilitate breeding for resistance.

3. Identify factors within cropping systems which

affect insect-plant relationships.

4. Develop and evaluate pest-management methods suitable

for small farmers in northern Cameroon.

5. Train Cameroonian students and technicians for

entomological research.

In the U.S.

6. Identify behavior-modifying chemicals potential

for management of major cowpea insect pests.








7. Characterize the chemical and ecological nature of

plant resistance.

B. Methodology

1. Establish replicated field plots in representative

sites. Plots will consist of several cultivars treated and

untreated with insecticides to elucidate the effects of

insect infestations on the plant. Other plots will evaluate

insect sampling methods and insect population dynamics. All

plots will be sampled frequently using established and experi-

mental methods (e.g., sticky traps, vacuum samples, direct

counts, etc.).

2. Cowpea cultivars obtained from IITA and other

sources will be evaluated for insect resistance in replicated

and unreplicated (negative screening) plots.

3. Representative cultivars will be grown in poly-

culture andmonoculture to determine the effects of poly-

culture on insect-plant interactions.

To select polyculture systems, visits will be made to

small farms.

4. Control tactics will be evaluated in small plots,

either singly or in combinations. Particular attention will

be given to suitability of methods to small farmers. Later,

successful methods will be tested in larger demonstration

plots.

NOTE: Data for the above methods will consist of insect

counts, plant damage, yield and market quality. Statistical

analysis will be performed.









5. Training of prospective graduate students, selected

jointly by Cameroon and Georgia will begin with several

months on-site familiarization with cowpeas and entomological

problems. Then steps will be taken to enroll a student in a

M.S. program at Georgia. Upon completion of the M.S., the

student will return to Cameroon to work but may return later

to the U.S. to take the Ph.D. degree.

Prospective technicians will be trained on site at

Maroua by the CRSP researcher stationed there.

NOTE: Attempts will be made to train women when they are

available. In Maroua the selection of candidates is done by

IRA and we do the final screening.

6. (a) Colonies of U.S. and Africian strains of the cowpea

weevil are to be established by Boyce Thompson for behavioral

studies (selection and oviposition behavior).

(b) A possible oviposition deterrent will be initiated

by allowing gravid females to oviposit on plants from which

deposited chemicals will be analyzed.

(c) The reported properties of vegetables in protecting

cowpea seeds from weevils will be investigated by bioassays,

topical application to the weevils and addition to larval

diets.

7. Possible insect resistance mechanisms will be

investigated by appropriate boassays.

III. Adjustments and Justification


None to report.









IV. Summary of Achievements

A. Planned:

In Cameroon

The memorandum of understanding between the U.S.A.

(University of Georgia) and Cameroon were signed by DGRST

and IRA on November 3 and 9 respectively.

Dr. Moffi E. Ta'ama was approved by DGRST and IRA as

the CRSP researcher in Maroua. He established residence

in a CRSP leased house on May 15, 1982 and began research

at the IRA field station there.

The normal rainy season was delayed and research plots

could not be established until July following a trip by

Ta'ama to IITA to obtain an adequate supply of viable seed.

Research plots consist of (1) 3 local and 3 improved cultivars

of peas in 15 m x 15 m plots divided into insecticide

treated and untreated sub-plots. (2) Thrips sampling trials

in 7 x 7 m plots.

As of August 22, the peas in the research plots are

doing well, are blossoming, and sampling is in progress.

Aphids appear to be an unexpected problem.

Dr. J. P. Eckebil, the director of IRA designated Mr.

George Ntoukam as the IRA counterpart for the CRSP researcher

at IRA-Maroua. In early 1982 Mr. Ntoukam was sent to IITA in

Nigeria to undergo a training program in cowpea research

methodology. The CRSP assumed the training costs. He

completed his training and is now in Maroua working with

Dr. Ta'ama. His application for CRSP funded graduate training









in the U.S. will be considered by DGRST after such scholarship

is advertised.

In June Dr. Eckebil met with Dr. Ta'ama at Maroua and

indicated that IRA would designate funds to support Mr.

Ntoukam and for office and laboratory space for CRSP at

Maroua. He also designated a vehicle for Ntoukam and Ta'ama

to be shared with Tim Shilling (Seed Multiplication Project)

until a U.S. vehicle arrived.

A phone conversation with Ta'ama on August 24 indicated

that office and lab construction would soon be underway.

Most of the supplies and equipment designated in the

1981-82 CRSP budget has been shipped via air freight and

has been received.

In the U.S.A.

Summary of achievements

Work at the Boyce Thompson Institute was initiated in

February, 1982, when Dr. Frank Messina was appointed to the

postdoctoral position. The research has proceeded directly

according to the original plans. The major goals for the

first year were: (a) to establish a cowpea weevil colony

and to develop a bioassay for determining host preference;

(b) to investigate the possible existence of an oviposition

deterring pheromone; (c) to characterize the protection

qualities of natural oils against the cowpea weevil. Con-

siderable progress has been made towards all three objectives.

Weevil colonies of three distinct strains have been

successfully established. The first of these was obtained







from IITA in Nigeria. A second colony was started from weevils

supplied by Dr. David Hagstrum of the USDA laboratory in Gainesville,

Florida. These insects had been collected from storage facilities

and had gone through only one generation in the laboratory. The

third colony originated from cowpeas purchased by Dr. Chalfant

at a market in Cameroon. The three colonies have been strictly

isolated to investigate possible differences in behavior and

biology of the different strains. Differences in coloration,

markings and general activity of beetles from the separate strains

have already been noted.

Studies on the oviposition behavior of the cowpea weevil

have confirmed the existence of a spacing mechanism that

ensures even distribution of eggs on available beans. The

presence of eggs on a bean apparently deters further ovi-

position on that bean until all other beans have received

eggs. The deterrent appears to be chemical, and can be

extracted into diethylether. A bioassay has been designed

to measure the preference of gravid females for treated or

untreated beans. The weevils are confined in a Petri dish

which is divided into four sections. Treated and control

beans are placed in opposite sections, and the number of eggs

on each are counted after various time periods. Black beans

are used for assays of chemical extracts so that the white

eggs are readily visible. The deterrent has been collected

by allowing female weevils to oviposit on glass beads. The

beads are then washed with ether and the extract reduced to a

standard concentration by evaporation of the solvent. Beans

for bioassays are dipped in the extract for 1 sec, and control









beans are dipped in pure ether. Results of such assays

indicate that the deterrent is associated with the act of

oviposition. Little effect was obtained from glass beads

that were offered to males alone or to virgin females.

The protection qualities of oils were investigated1 by

treatment of cowpeas with a wide range of oils before the

addition of weevils. The oils were chosen to provide widely

different chemical properties. Peanut oil has been used in

the past and represents a natural oil that has a high content

of unsaturated fatty acids, particularly C18:1 and C18:2-

Coconut oil was chosen for its high saturated fatty acid

content. The major components of this oil are the C12 and

C14 acids. Safflower oil was selected as a more general
unsaturated oil. Completely different chemical properties

were provided by mineral oil, which consists entirely of

hydrocarbons, and Carbowax 300 (polyethylene glycol), which

represents a more polar liquid of similar viscosity. Obser-

vations were made on oviposition, egg hatching, larval

development and adult emergence.

All the oils tested appeared to be equally effective in

reducing the number of progeny from weevils that were added

to cowpeas immediately after treatment. These results suggest

that the protective quality of oils is physical rather

than chemical. In a choice situation, more eggs were laid

on control than on treated beans, but this could not account

for most of the observed reduction in progeny production.

Mortality occurred at the egg stage and to first-instar


See Appendix









larvae before they could enter the seed. Those larvae which

succeeded in entering the seed were unaffected by the oil

treatments. The deterrent effect of peanut oil was consider-

ably higher when 60 days elapsed before addition of weevils

to the treated beans. However, when eggs were laid on these

beans, no significant effect on larval development was

obtained. Furthermore, when no choice was provided, weevils

confined to treated and aged seeds laid as many eggs as those

confined to control cowpeas. These results indicate that oils

may only be effective in protecting stored cowpeas for a

limited period of time. Although ovipositing weevils show a

strong preference for untreated seeds, the effect of the oils

is not enough to suppress egg laying when no choice is

available.

Preliminary studies on the "active" flight form of the

cowpea weevil indicate that all strains of the insect may

not have the potential for production of this morph. Crowding

of populations has resulted in the appearance of the active

form in both the Cameroonian and Florida strains, but not

in the IITA colony. It appears likely that natural selection

in the laboratory colony favors production of the "normal"

form, so that the ability to induce production of the

active form disappears.


B. Unplanned:

In Cameroon

The cowpea aphid emerged as a potential key insect pest

of cowpeas. This may require revision of sampling procedures.






10


In the U.S.

The much reported protective vegetable oils were

ineffective in protecting pea seeds under a no choice

situation. This may require a revision of some of the

tactics for pest management.









VI. Plans

A. 1983 Program: The 1982 program will be a continuation

of the 1982 program with no anticipated changes in goals,

objectives or methodology.


In Cameroon

Field plots will include representative cultivars in

treated and non-treated plots, construction of insect sampling

plots (for thrips, aphids), evaluation of new cultivars for

resistance to insects (thrips, aphids and cowpea weevil),

evaluation of experimental insecticides and application

methods for control of insects. Of particular interest is a

new electrostatic sprayer developed by Imperial Chemical

Industries and which is very adaptable for use by small

farmers.

It is anticipated that a Cameroonian student will be

enrolled in the Graduate School of the University of Georgia

by the 1983 Spring quarter. Classes will be taken in Athens

while research on cowpea insects will be at Tifton.

In the U.S.

Work on host preference of cowpea weevils will be con-

tinued according to the original plan. The oviposition

deterrent will be further investigated to determine the

duration of its effect and whether suppression of oviposition

in a no-choice situation is possible. The effect of dif-

ferent concentrations will be tested and the stability of

the active material evaluated. Chemical studies will be

started to isolate and characterize the deterrent. Silica








gel chromatography will be utilized to separate ether extracts
into lipid classes, and fractions will be subjected to HPLC
and/or GLC as appropriate.
The selection of host plants by active females will be
investigated in cages and a small room that allow flight.
Little is known about the general biology of the active
form, which is believed to be responsible for initial infestation
of cowpeas in the field. These studies are thus expected to
provide new information on conditions influencing the success
of the weevils. Different cowpea varieties are already
being grown in the greenhouse for investigation of host
preferences of active females. Data will be collected on
the effect of flight exercise on behavior and fecundity of
active weevils.






















.4



























APPENDIX




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