Submission of

TNSCST-STUDENTS PROJECT PROPOSAL

Microbial Secondary Metabolite Mediated Predator Attraction

in arresting Aphids (Acyrthosiphon pisum) infesting
on Cowpea (Vigna unguiculata L.)

Submitted to

Tamilnadu State Council for Science and Technology

DOTE campus, Chennai - 600 025

Under the Supervision of Submitted by

Dr. K. Kannan
Sabareesh.M
Assistant Professor
II-M.Sc. Microbiology
Department of Microbiology
Department of Microbiology
Madura College
Madurai - 625011 Madura College
Madurai - 625011
 TNSCST-STUDENT PROJECT PROPOSAL

1 Name of the Student : A.Uttam Singh

.
e-mail ID : uttam67.us@gmail.com
2 Name of the Guide : Dr.P.N.Rajarajan
.
Designation : Assistant Professor
Institutional Address : Department of Microbiology
Madura Collegee
Madurai – 625 011
Phone No. : -
Mobile No. : 9047740928,
r.raja56@gmail.com
3 Project title
.
:Enumeration of microorganisms from sewage
water sample at Madurai city.
4 Sector in which your Project
. proposal is to be Considered : Environment
5 Project Details : Annexure –I
.
6 Has similar project carried out in
. your college/elsewhere. If so : No
furnish details of the previous
project and highlight the
improvements suggested in the
present one

CERTIFICATE

This is to certify that Mr.A.Uttam Singh is a bonafide final year student of P.G. Science courses
of our college and it is also certified that two copies of utilization certificate and final report along
with seminar paper will be sent to the Council after completion of the project by the end of April
2022.

Signature of the Guide Signature of the HOD Signature of the Principal/

Head of the Institution
INTRODUCTION

Cowpea, Vigna unguiculata L. is one of the most important legume crop grown in the tropical
belt (Singh et al., 1997). This crop provides food, animal feed and cash for the rural populace in
addition to benefits to farmlands via in situ decay of root residues and ground cover due to the
spreading habit of the plant. In addition, cowpea grain provides a cheap and nutritious food for
relatively poor urban communities. Cowpea is cultivated for its grains, leaves, and green pods.
The seeds are important in diets for the high protein content providing protein to rural as well as
the urban dwellers as a substitute for the animal protein. Cowpea's high protein content, its
adaptability to different types of soil and intercropping systems, its resistance to drought, and its
ability to improve soil fertility and prevent erosion makes it an important economic crop in many
developing countries.

The major constraint for cowpea grain production is insect damage. Aphids, jassids,
sucking bugs, pod borers were considered as key pests of cowpea affecting 90% of plants
according to the field study. Aphis craccivora (Koch) is polyphagous by nature affecting more
than 15 different crops, mainly pertaining to the family Leguminosae. It is considered as major
threat to the agricultural and horticultural crops particularly in the drier regions of the tropics
attacking 50 host plants species belonging to 19 different families throughout the world. Due to a
large number of host range, aphids seem to be present throughout the year (Soratur et al., 2017).
In order to control the aphid’s damage in cowpea we plan to introduce the “Microbial
Pheromones” to attract the insects resulting in reduce the yield loss (10-90%) to the economically
important crops depending upon severity of damage by sucking pests. This has been known that
the Pheromones are using in agriculture field for controlling the sucking insects but here we
going to use “Microbial Pheromones and secondary metabolites” for the control of aphids in
cowpea.
Microbial secondary metabolites have the ability to control the insect pests. Pheromones
are one of the class of semiochemicals that insects and other animals release to communicate
with other individuals of the same species. The key to all of these behavioral chemicals is that
they leave the body of the first organism, pass through the air (or water) and reach the second
organism, where they are detected by the receiver.
Our core aim is to use “microbial pheromones” extracted from the gut flora of aphids
leads to attract the population of predators in the meantime controls the proliferation of aphids
population. The population of predators increased with the increase in aphid population and also
the ovi positions. This becomes the evidence of reducing number of aphids in the cowpea field
for the better crop yield.
OBJECTIVES

 To isolate and identify gut microorganisms from cowpea aphids;

 To extract the microbial secondary metabolites compounds from isolated
microorganisms;
 To characterize the microbial compounds involved in attractions and oviposition
improvement of predators;
 To identify the microbial secondary metabolite mediated biocontrol of aphids in
cowpea – in vitro and in vivo study.

METHODOLOGY
Sample Collection
Aphids will be collected from seriously affected seedling of cowpea plant (Vigna unguiculata L.)
cultivated in and around Madurai district.

Isolation and Identification of Gut Flora from Aphids

Each surface sterilized aphid from cowpea was crushed with a sterile glass rod in a test tube
containing 1 ml distilled water with 0.5 % NaCl. One loopful of the gut suspension was streaked
on Petri plates containing Nutrient agar to obtain a pure isolated colony and incubated at 37 ºC
for 24 hours and make assured observations for the appearance of typical colonies of gut micro
flora.

Extraction of Microbial secondary metabolites:

Mass culturing of the microbes by using nutrient medium for 24 – 48 hrs. After proper incubation
at 37°C and sufficient growth the culture centrifuged at 10000 rpm. Supernatant collected and
extracted by using the polar solvents (Kannan et al., 2018).
Identification of compounds through Analytical Techniques
Frequent analytical techniques such as FTIR, GC-MS to analyse the presence of microbial
pheromone compounds present in the aphid honey dew and microbial extracts (Lee et al., 2017).
Field Study - Attractant-baited Traps:
Several unique types of traps are used for the control of aphids and other flies. The microbial
pheromone coated traps used as an attractant baited traps which cannot escape the aphids. And
also the microbial pheromones spray also going to use to trap the flies. Funnel traps were hung
between trees by a rope, and the height of the collection cup was about 30 cm above ground
level. Traps were deployed in randomized complete blocks with traps 15–20 m apart,
approximately 50 m between blocks and 200 m between experiments. (Campion, Lester and
Nesbitt, 1978; Campion, 1983).

HYPOTHESIS OF THE PROJECT

Pictorial representation of proposed hypothesis of the project

WORK PLAN

Cowpea Plant

Aphids Sample collection

Isolation and Identification of Gut micro flora and surface

Mass culture and Extraction of Microbial

Secondary Metabolite compounds

Analysis of Microbial compounds

FTIR, GC-MS

In vitro and In vivo study using extracted compound

to control aphids

Increase cowpea yield by controlling

aphids using Microbial secondary
metabolites
 Proposed Time
Work Duration
Collection of aphids from cowpea January 1st to January
10th
Isolation and identification of microbes January 11th to January
25th
Mass culture of isolates January 26th to February
14th
Extraction and identification of secondary February 15th to March
25th
metabolites from the isolated microbes

In vitro and in vivo study March 26th to April 15th

Data interpretation and report preparation April 16th to April

30th

BUDGET

REQUIREMENTS AMOUNT (Rs.)

500
Sample collection(Travel)
Analytical charges 4500
Report preparation 500

TOTAL 5500

OUTCOME OF THE PRODUCED PROJECT

Widespread use of chemicals to control plant diseases has extremely disturbed soil environment,
soil flora and other biodiversity. An alternate to these synthetic chemicals is the use of certain
biocontrol agents which are inexpensive and eco-friendly, and have no harmful effects on human
population. Microbial secondary metabolites are stepping up into the pest control management
leads to better alternative to the synthetic chemicals for the higher yield of the crops.
REFERENCE
 Bakke, A and Kvamme, T. (1981). Kairomone response in Thanasimus predators in
pheromone components of Ips typographus. J. Chem. Ecol., 7, 303–312.
 Campion, DG and Nesbitt, F. (1981). Recent advances in the use of pheromones in
developing countries with particular reference to mass-trapping for the control of the
Egyptian cotton leaf worm Spodoptera littoralis and mating disruption for the control of
pink bollworm Pectinophora gossypiella. In Les Mediateurs Chimiques Agissant sur le
Comportement des Insectes, Institut National de la Recherche Agronomique, Paris, pp.
335–342.
 El-Sayed, AM., Suckling, DM and Wearing, CH. (2006). Potential of mass trapping for
long term pest management and eradication of invasive species. Journal of Economic
Entomology, 99: 1550–1564.
 Kannan K. (2018). Analysis of PGPR Secondary Metabolites Efficiency against Bacterial
Blight Disease in Vigna unguiculata (L.) Walp. through In vitro and In vivo Conditions.
Ph. D. thesis.
 Kuwahara, Y and Casida, JE. (1973). Quantitative analysis of the sex pheromone of
several phycitid moths by electron capture gas chromatography. Agric. Biol. Chem., 37,
681–684.
 Lee, HR., Lee, SC., Lee, DH., Choi, WS., Jung, CS., Jeon, JH., Kim, JE and Park.
(2017). Identification of the Aggregation-Sex Pheromone Produced by Male
Monochamus saltuarius, a Major Insect Vector of the Pine Wood Nematode. J Chem
Ecol. 43:670–678.
 Mazomenos, BE and Haniotakis, GE. (1985). Male olive fruit fly attraction to synthetic
sex pheromone components in laboratory and field tests. J. Chem. Ecol., 11, 397–405.

 Soratur, M., Rani, D and Naik, SM. (2017). Population dynamics of major insect pests of
cowpea [Vigna unguiculata L. Walp] and their natural enemies. Journal of Entomology
and Zoology Studies. 5(5): 1196-1200.
 Tarawali, SA., Singh, BB., Peters, M and Blade, SF. (1997). Cowpea haulms as
fodder. Advances in Cowpea Research, 313 - 325.