1 PROFORMA PSF-I ® SUBMISSION OF RESEARCH PROPOSAL FOR FINANCIAL ASSISTANCE TO PAKISTAN SCIENCE FOUNDATION Name & Address of the Institution CABI South Asia Opposite 1-A Data Gunj Baksh Road, Satellite Town Rawalpindi- Pakistan Title of Research Proposal: Exploration of bacloviruses prevalent in different agroecological zones of Pakistan and their evaluation against target lepidoptrans pests Main Field of Study: Agriculture Sciences/ Crop protection Nature of Research: (a) Basic * Principal Investigator: Aamir Hummayun Malik ** Co-Principal Investigator: Shakeel Ahmad (Attach Biodata) Proposed Duration: Two Years May-2009-April 2001 (24 months) Total Funds Requested: 1,840,740 ENDORSEMENT: Signature Principal Investigator Institutional Head*** Official Stamp Date: Address: Pakistan Science Foundation, 1-Constitution Avenue, G-5/2, Islamabad, P.O. Box 1121, Telex 54080 PSFIDPK Fax 9202468
Transcript
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PROFORMA PSF-I ®
SUBMISSION OF RESEARCH PROPOSAL FOR FINANCIAL
ASSISTANCE
TO PAKISTAN SCIENCE FOUNDATION Name & Address of the Institution
CABI South Asia Opposite 1-A Data Gunj Baksh Road, Satellite Town Rawalpindi-Pakistan
Title of Research Proposal:
Exploration of bacloviruses prevalent in different agroecological zones of Pakistan and
their evaluation against target lepidoptrans pests
Main Field of Study: Agriculture Sciences/ Crop protection
Exploration of bacloviruses prevalent in different agroecologiical zones of
Pakistan and their evaluation against target lepidoptrans pests 2. Project Abstract (Summary):
There has been a continuous demand for agents that are not harmful to the environment, human health and can control pest insect populations. Family Baculoviridae contain the viruses (Nucleopolyhydrosis virus (NPV)/Granulovirus (GV)) that are pathogens only for arthropods, mainly insects, most to a narrow range of Lepidoptera. As these viruses are environment friendly and do not replicate in vertebrates, these have been the subject of detailed study in many countries of the world as a alternative to chemical insecticides. Now a days different formulation of indigenous NPV and GV isolates in different countries are commercially available and being successfully utilized for insect control. In contrast to other countries no research has been conducted in Pakistan on exploration and application of bacloviruses as biocontrol agent. The main objective of the project is to recover NPV/GV from different insect hosts throughout Pakistan, isolation of most virulent strains of the virus and test against insects of economic importance (Spodoptra
litura, Helicoverpa armigera, Agrotis epsilon/segetum and Cydia pomonella (codyling moth)) by making different formulation. This initial selection from the natural pool of isolates would be important for their success because the indigenous viruses are well adapted to the climatic conditions and agro-ecosystem of the country and can be stable in the environment for longer period of time as compared to the imported ones. The proposed research work will lead to successfull bio-control of insect population (Lepidoptrous pests) in all vegetables, cotton, sorghum, chickpeas, maize and forest trees. As it is quite inexpensive to formulate and easy to apply, it will also benefit the poor farming community of the country by decreasing extra expenses on pesticides and health of agriculture workers by toxicity of pesticides.
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3. Project Narrative:
i) Significance of the proposed research duly supported with review of literature and bibliography to indicate current trends in the proposed field of study
Lepidopterous insects complex is a major pest problem in food, fibre and horticultural crops in Pakistan.
Farmers have been using huge amounts of pesticides to control these pests but the prevailing strains have developed resistance against many groups of insecticides (Ahmed et al., 2001; Kranthi et al., 2001). Similarly consumer concern about chemical pesticide residues on food is driving the search for alternatives and excessive use of pesticides is badly affecting the health of agricultural workers, wildlife, domestic animals, biological diversity and environment. The result is chronic pesticide poisoning, with its symptoms ranging from mild headache to skin allergies to cancer of internal organs. In Pakistan evaluation of toxicity due to commercial pesticides in female workers based on reproductive hormonal assay ranged between 9.9-22.22% in different age groups. They reported that agricultural pesticides are the endocrine disrupting chemicals which poses a health threat, particularly to the sensitive gender, frequent farm workers and onward into their children. (Ahmed et al., 2004). Similarly DNA damage in Pakistani pesticide manufacturing workers assayed using comet assay was significant compared to healthy controls (Bhalli et al., 2006). New research suggests that bio-control, using virus, bacteria or fungi to control plant disease and pests, could be developed as an effective alternative. Bio-control is an environment friendly method of controlling pests and bio-pesticides are much less toxic than chemical pesticides. There in no danger of insect resistance development as it is in case of synthetic chemicals.
A large number of viruses offer potential as microbial control agents of insects. Those with the greatest microbial control potential are in the Baculoviridae. The family is taxonomically subdivided into two genera, Nucleopolyhedrovirus (NPV) and Granulovirus (GV), distinguished by occlusion bodies (OB) morphology (Volkman et al., 1995). NPV is an enveloped double-stranded DNA (80 to 180 kbp) viruses that almost exclusively infect insects (Adams and McClintock, 1991; Ukuda et al. 2007). Studies on baculovirus pathology concluded that after infection of midgut cells, tracheal epithelial cells become infected, and then baculovirus appears in the hemolymph followed by the infection of other tissues (Keddie et al., 1989; Trudeau et al., 2001). The hemolymph may be the main route of virus distribution in some infected insects (Adams et al., 1977; Granados and Lawler, 1981).
The first objective of the project is to recover bacloviruses isolates from different insect hosts and to test them against lepidoptrous insects of key importance. It is of prime importance to evaluate and develop the indigenous strains of viruses to adopt them as bio-control agent because they show more efficiency, stability and host range under unfavorable condition during the action phase and in hibernation phase also. It has also been recorded that some viral show more virulence even being a part of single population. So the survey and bioassay for most virulent strains will lead us to effective bio-control strategy. Lavina et al. (2001) reported that a Philippine Spodoptera litura
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nucleopolyhedrovirus (SltMNPV) isolated from an uncloned NPV population exhibited high insecticidal activity against S. litura as compared to commercially available formulations of different viruses. Results showed that SltMNPV was a unique isolate which could be developed into a more effective microbial insecticide. Smits et al. (1988) reported the virulence of five NPV infectious for larvae of Spodoptera exigua and their potential as biological control agents. Comparison of the biological activity of these 5 isolates showed that the Spodoptera exigua MNPV (SeMNPV) was more virulent against S. exigua than the other isolates. SeMNPVs had broader host range and potential production in alternate hosts or cell-lines, and is considered to be the most suitable candidate as biological control agent. Possee et al. (1997) reported that the use of baculoviruses for insect control had high degree of host specificity. They described various approaches to the genetic modification of baculoviruses to decrease the infection time and increase infectivity. These modifications resulted in improvement in insecticidal activity during laboratory trials which was also confirmed in field experiments.
In the proposed study, characterization of bacloviruses at gene level will aid to accurate identification of different strains and will provide the basis for understanding of molecular alterations involved in the change of virulence. The sequence data generated from the different strains will be compared with the results of invitro infectivity of the viruses on target insects which will help us to understand the genetic difference among more and less virulent strains. Hughes et al. (1983) were able to show a correlation between virulence and a DNA restriction enzyme pattern. Jakubowska et al. (2005) characterized the SeMNPV. Phylogenetic analysis based on three conserved baculovirus genes, polh, lef-8 and pif-2, showed the highest homology of SeMNPV to Mamestra
brassicae MNPV and M. configurata MNPV, and lesser to SeMNPV. Bioassays revealed that SeMNPV isolated from the commercial bioinsecticide Spod-XR was the most infectious for S. exigua, while the infectivity of SeMNPV (P) and MbMNPV was significantly lower.
Valicente et al. (2007) reported that Spodoptera fruigiperda nucleopolyhedrovirus (SfMNPV) could be very effective in controlling fall armyworm (S. frugiperda). Widespread application of this baculovirus had been limited mainly due its low production on commercial scale. Liquefaction generally causes significant losses of virus. A SfMNPV isolate that doesn’t disrupt the integument was recently found. Their results showed that between 80 to 120 larvae (from 11 to 13 grams of body weight) are sufficient to produce a dose for one hectare. Seufi (2008) reported an Egyptian isolate of S.
littoralis NPV (SpliNPV) for its potential as biocontrol agent in comparison with AcMNPV. Comparative assays of SpliNPV and AcMNPV against 2nd instar larvae of Spodoptera littoralis revealed 4-fold greater susceptibility to AcMNPV than to SpliNPV based on LC50 values for the two viruses. DNA segment was cloned and sequenced. Sequence alignment results revealed that Polh-cr showed significant similarities with 91 different baculovirus isolates.
GVs are more specific than NPVs, as they have been reported only from Lepidoptera (Bilimoria 1991; Granados and Federici, 986; Miller, 1997). OBs are formed in the nuclei of cells (NPVs) or in both nuclei and cytoplasm for some GVs (Granados and Federici, 1986). Infected defoliating larvae usually climb to the upper parts of the plants, dying in 5 days, although cessation of feeding may occur in 2–4 days (Evans, 1986). The
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Granulovirus of codling moth (CpGV) was first isolated from infected larvae in Mexico and described by Tanada in 1964. CpGV is one of the most virulent baculoviruses (insect-specific viruses). Following ingestion by neonate larvae, the proteinaceous coat or granule is dissolved in the alkaline pH of the gut.
The efficacy, specificity, and production of secondary inoculum make baculoviruses attractive alternatives to broad-spectrum insecticides and ideal components of IPM systems due to their lack of untoward effects on beneficial insects including other biological control organisms. Despite their potential, viral insecticides are employed much less than they could be in crops and forests (Cunningham and Howse, 1984). Few examples of bacloviruses used as biocontrol agents in different countries are given below. Many countries and companies of the world have registered their indigenous NPV isolates for insect control (Table.1). A NPV of Pseudoplusia (Chrysodeixis) includens is possibly the best example of a baculovirus implemented as a classical biocontrol agent in a row crop. This NPV was released on 200–250 ha of soybean in Louisiana and provided control 12–15 years later (Fuxa et al.,1992). Its success seems to be related to those viruses with efficient horizontal (insect to insect) and vertical (from generation to generation and year to year) transmission as well as to a diversified complex of natural enemies that interact with introduced viruses to maintain host populations below damaging levels. One of the key advantages of baculoviruses is that they replicate and persist in the environment and may be able to maintain host populations below damaging levels with fewer applications compared with chemicals (Cunningham and Howse 1984; Moscardi and Sosa-G´omez 1992; Moscardi and Sosa-G´omez 1996). In United States in 1975 of the first viral insecticide (Elcar™, by Sandoz Inc.), was used against the cotton bollworm, Helicoverpa zea (Huber 1990; Ignoffo and Couch, 1981). It infects all the major Helicoverpa/Heliothis species and provides efficient control in soybean, sorghum, maize, and tomato, as well as in chickpea and navy beans. In 1996, Biosys introduced GemStar™ LC, a liquid concentrated formulation of H. zea NPV, for control of H. zea
and H. virescens in US cotton and Australia. In China H. zea NPV isolate has beenproduced on H. armigera larvae reared on artificial diet and formulated as a wettable powder or as an emulsion and applied to aproximately 100,000 ha annually (Vlak and Hu, 1997) mainly to control H. armigera and H. assulta on cotton, but also on tobacco, cayenne pepper, and tomato. Isolates of H. armigera NPV have also been produced and used in Thailand and Vietnam (Deseo-Kovaks and Rovesti 1992). In Brazil, an indigenous isolate of S. frugiperda NPV (SfNPV) has been used to control the insect on maize (Valicente and Cruz 1991). A S. exigua NPV was developed in the United States through collaboration between Crop Genetics International (CGI) and DuPont from 1992 to 1994. In 1994, CGI began selling SPOD-X™ for control of the insect on greenhousegrown cut flowers and ornamental plants in Netherlands and USA. In China, a NPV of S. litura was successfully applied to control this insect in vegetables, cotton, rice, and peanuts (Yi and Li 1989). Also a GV of the cabbage worm, Pieris rapae, has been mass produced in China as PrGV since 1978 and applied in many regions, totaling 100,000 ha (Yi and Li 1989). Similarly a GV of the cassava hornworm, Erinnyis ello, has been used in Brazil (Schmitt 1985). It has been used in crude preparations (10 larval equivalents/ha), providing good control of the pest with only one application during the season.
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The potato tuber moth (PTM), Phthorimaea operculella, is an important pest of potatoes worldwide. A GV isolated from PTM has been developed as a microbial insecticide in Peru by the International Potato Center (CIP) which has high efficacy in protecting the potato crop in the field or potato tubers under storage (Raman et al., 1992). The formulated product is widely used in Colombia, Ecuador, Peru, and Bolivia. The codling moth, C. pomonella, is a worldwide key pest of apples, pears, and walnuts. A GV isolated from C. pomonella (CpGV) was highly virulent to the insect and killed it rapidly, protecting fruit from economic damage, in numerous field trials in several countries (Falcon 1985; Falcon and Berlowitz 1986; Huber 1990; Jaques 1990). Currently, commercial formulations of CpGV are available in France (Carpovirusine®, by Natural Plant Protection), in Switzerland (Madex®, by Andermatt Biocontrol AG), in Germany (Granusal®, by Behringwerke AG) and in Russia (Virin-CyAP) (Filippov 1990). A number of forests Lepidoptera have been subjected to applications of baculoviruses (Filippov 1990) these include include the gypsy moth, L. dispar; the Douglas-fir tussock moth, Orgyia pseudotsugata; the spruce budworm, Choristoneura fumiferana; the western spruce budworm, Choristoneura occidentalis; the jackpine budworm, Choristoneura pinus and the pine beauty moth, P. flammea (Cunningham 1995; Huber 1990). The AgNPV occurs naturally in Brazil in A. gemmatalis populations with pathogenesis similar to other NPVs (Carner and Turnipseed 1977). A pilot program for AgNPV use was conducted during the 1980/1981 (Moscardi 1989). In all AgNPV plots, reductions of over 80% of A. gemmatalis larval populations were attained and yields were not significantly different from insecticide-treated plots. Approximately 20,000 ha were treated in the 1983/1984 season. Use increased substantially in the 1984/1985 season (approximately 200,000 ha) after the implementation of regional NPV production units (Moscardi 1989). The treated area increased to 500,000 ha in the 1986/1987 season, to 700,000 ha in 1988/1989 and to 1 million ha in 1989/1990. Currently, all commercial production of AgNPV in Brazil is made in farmers' fields. Virus-killed larvae are collected and stored in large rooms at -5°C until the virus is processed into a wettable-powder formulation.
Human Health and Ecological Risk Assessment
According to USDA, Forest services report (2004) on “Human Health and Ecological Risk Assessment for Gypchek (NPV)” there is no indication that LdNPV is pathogenic in any mammalian species, even when the animal’s immune function is compromised. There is no indication that LdNPV is likely to cause dermal or respiratory irritation (Becker and Park, 1997). One specific study conducted on wildlife mammals that may consume contaminated gypsy moth larvae indicates no adverse effects in mice, shrews, and opossums (Lautenschlager, 1980). Similar there is no indication that LdNPV or the Gypchek formulation of LdNPV has the potential to cause any adverse effects in any nontarget insect species including honey bees (Cantwell et al 1990). Based on bioassays of LdNPV on the large number of nontarget insect species and supported by the general high species specificity of related baculoviruses, the hazard identification for NPV in nontarget insects is essentially identical to mammals (Doller G. 1985). Few studies are available in birds which indicate that exposures to NPV at levels that are substantially higher than those likely to occur in the environment can not be associated with any adverse effects (Lautenschlager and Podgwaite 1979). Relatively few studies have been
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conducted in fish and aquatic invertebrates but these studies are consistent with studies in terrestrial species and indicate that effects on fish or aquatic invertebrates are unlikely (Streams 1976).
Keeping in view all studies described a separate survey will be conducted for Lepidoptera occurring in non crop plants. Bioassays for NPV will be conducted with special reference to waxmoth (Gallaria mellonella), Honey bee (Apis domestica) and other lepidoptrous pests controlling weed after survey.
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Table 1 Baculoviruses developed as microbial insecticides to control Lepidoptera
Host insect Baculovirus Crops Commercial name
Country References
Adoxophyes orana GV Apple Capex 2 Switzerland Cunningham 1995.
Adoxophyes sp. GV Tea Japan Nishi and Nonaka ,1996
*These NPVs have been developed primarily for use against lepidopteran species other than original hosts because of their wide host range
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ii) A brief account of work done in Pakistan/papers published thereof. and relationship of the proposed research to the socio-economic development of the country.
Lepidopteran insects (bollworm/budworm) complex is a primary insect pest problem in Pakistan. The most important species are Helicoverpa armigera, S. litura, S. exigua, Autographa nigrisigna, Pieris rapae, Pieris brassiceae and Agrotis spinifera. The genera Helicoverpa, and spodoptra are important on a global basis attacking over 60 crops. Ghouri (1960) listed S. litura as pest of sugarcane, cotton, castor, groundnut, linseed, rice, wheat, cabbage, brinjal, tomato, clover, lucerne, tobacco and maize. Alam et al. (1969) reported it from mint, willow and spinach. In addition to these CABI SA in different surveys recorded it from potato, sugar beat, cauliflower, turnip, yellow sarson (Brassica
camppestris), bittergourd, radish, okra, pea, kale (Brassica oleracea), dock and pig weed (Chenopodium album). Similarly Heliothis armigera has been reported from gram, potato, tobacco, horse purslane (Trianthema portulacastrum), sugarbeet, clover (Trifolium alexandrianum), wheat, rose (Rosa indica), cabbage and cauliflower (CABI SA project report on cutworms: 1978-1983). A lot of studies have been conducted on the natural enemies (predators and parastoids) of Lepidopteran insects of these crops and a number of projects were started. Unfortunately in contrast to other countries of the world no dedicated work has been carried out in Pakistan to explore and utilize bacloviruses as natural source for insect control. In Pakistan no published literature exists and very little knowledge exists about the application of bacloviruses as bio-control agent. Working on a project on natural enemies of cutworms (1978-83) a multiembedded NPV from S.litura larvae on cauliflower was recorded and identified by National History Museum U.K. Researchers at some institutes record their observations for presence of NPV infected larvae in field conditions and forests (personal communication) but it never has been isolated and augmentated.
The high prices of insecticides and their application frequency has created economic burden for the formers which can be decreased to certain level by using NPV as very heap source of insect control. Effective control strategies can be developed by the information generated by the proposed project which will enhance the agriculture productivity by minimizing pest losses. Presently, suggested project “Exploration of bacloviruses prevalent in different agroecologiical zones of Pakistan and their evaluation against target lepidoptrans pests”will ultimately help to increase income of farming community and reduce poverty in rural areas. This will be greatly helpful in decreasing pesticide use and their ill effects on environment and consequently strengthen renewable sources and minimize health hazard.
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iii) Specific Objectives and expected results.
Objectives
1. To explore the occurrence and diversity of bacloviruses in Pakistan
2. Gene sequencing of viral isolates from different insect sources and different
agroecological zones will be done to aid accurate identification
3. To invitro assess the bio-control potential of bacloviruses isolates against S. litura,
H. armigera, A. epsilon/ segetum and Cydia pomonella (codyling moth)
4. To Identify most virulent bacloviruses strains
5. Testing of different formulations of viruses against target insects
6. To provide alternative to chemical pesticides to safe human health and
environment
7. To save foreign exchange spent on import of chemical pesticides
Expected results
1. Availability of basic data on occurrence and diversity of bacloviruses in
Pakistan
2. Availability of most stable and virulent strains of virus to test against S. litura, H.
armigera, A. epsilon/ segetum and Cydia pomonella (codyling moth) at pilot scale
3. Recommendations of formulation for insect control of different crops in
different seasons
4. Availability of safe, environment friendly and non-toxic bio-pesticides
5. Three research publications
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iv) Description of research methodology/techniques to be used including critical or difficult phases or factors and how these will be investigated.
1. Survey for collection of infected larvae
The diseased larvae of Lepidopteran insects will be collected from grasses, vegetables, gardens and field crops grown in fallowing agro ecological zones of Pakistan. The most critical point in sampling is to differentiate between the viral infected larvae and the larvae found dead with any other reason. So one should be very careful and clear about the symptoms exhibited by the infected viruses
No Areas to be visited No. of trips Date Purpose
1 Western Hills (Quetta, Ziarat, Naseerabad, and Mastung)
1 June (2009)
To search and collect the virus infected larvae of codling moth (C.
pomonella) from apple trees, and S. litura and H. armigera from vegetables and forest trees
2 Foot Hills (Rawalpindi, Attock and Haripur) (Mardan, Kohat and Banu)
1 1
Start May (2009)
August (2010)
To collect virus infected S. litura, Pieris rapae and H. armigera
larvae from vegetables and forest trees
3 Peshawar Valley (Peshawar, Kohat) 1 August (2009) To collect virus infected S. litura, S. exigua and H. armigera larvae from vegetables and forest trees
4 Northern Plains (Faisalabad, Lahore, Gujranwala, Okara and Sahiwal)
(Sialkot, Gujrat)
1
1
April (2010)
September (2010)
To collect virus infected S. litura and H. armigera larvae from
vegetables and other crops plants
5 Semi Deserts (Multan, Rahim Yar Khan, Bahwalpur) (Layyah, Jhang, Sargodha, Toba Tek Singh)
1 1
September (2009)
March (2010)
To collect virus infected S. litura, S. exigua and H. armigera larvae from cotton,
Tomato, okra and other host crops
To collect virus infected H. armigera larvae from chickpea
6 Sub-Costal areas (Hyderabad, Tandojam, Badin)
(Turbat, Lasbella, Panjgur, Khuzdar)
1
1
June (2009)
Start May (2010)
To collect virus infected S. litura, S. exigua and H. armigera larvae
from cotton, Tomato, okra and other host crops To collect virus infected S. litura and H. armigera larvae from
vegetables and cotton and other crops plants
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2. Viruses isolate, virus production and isolation of OBs
The larvae thought to show baculovirus infection symptoms will be brought to laboratory and keep at 20 °C until the isolation of Obs (polyhedra). NPV will be purified using a modification of the method described by (Hanzlik et al., 1993). Virus will be multiplied in healthy S. litura larvae reared at 27–28°C on an artificial diet (Li et al., 1988).
3. Bioassay procedure
Lab cultures for Helicoverpa, Spodoptra, Autographa, Pieris and Agrotis will be maintained on artificial diet described by Shorey and Hale, 1965. The test larvae will be infected by feeding with baclovirus infected leaf discs. A single larva will be placed in each rearing tube containing a treated leaf disc. After consumption of food larvae will be transferred to a virus free diet for the rest of the experimental period. A minimum of 30 larvae will be used per replicate and three replicates will be maintained for each dose. Different dose concentrations will be tested. In the control set, distilled water will be used instead of the virus. The test larvae will be maintained in an incubator at different temperatures and relative humidity and will be examined and recorded daily for symptoms and virus death.
4. Long term preservation of viral isolates
Infectivity may be destroyed by degradative enzymes that destroy nucleic acids, high temperature, detergents that solubilize the lipid-containing envelopes and UV radiations. The viruses will be preserved at low or ultra-low temperatures, and/or in the absence of water, that will slows down these destructive processes sufficiently to increase significantly the length of time that the virus can be stored as infectious material.
5. Molecular Characterization
5.1 Isolation of genomic DNA:
DNA will be extracted by the method described by (Verhagen et al., 1999). The method is based on extraction of DNA in a buffer and chloroform, precipitation by ethanol and collection of precipitate.
5.2 Primer designing and PCR amplification:
PCR of isolated genomic DNA will be carried out by the procedure described by (Michot et
al., 1984). Primers will be designed on conserved genes (lef-8, lef-9 and polh) reported for molecular characterization of different NPV and GV isolates. To avoid non specific amplifications primer designing is quite technical and need expertise. Target gene sequences will be aligned first and then primers will be designed in highly conserved regions. PCR product will be run on 1% agarose gel with ethidium bromide (100µg/ml)
7.3 Cloning and sequencing of the PCR product
Amplified product will be ligated into cloning vector Ptz57/RT and will be transformed into 10b electrocompetent cells by heat shock method. Clone will be confirmed by restricting plasmids with endonuleases and sequencing
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v) Year-wise plan of work.
Plan of work (2009)
Activity Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Remarks
Survey of the targeted areas for infected larvae sampling
Isolation of NPV isolates from infected samples
Host insects collection and maintaining their lab culture
Conducting different bioassays for screening the most suitable strains for different hosts in hot and tropic regions of the country
Primer designing for molecular identification and PCR amplifications
Data analysis and report writing
Plan of work (2010)
Activity Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Remarks
Survey of the targeted areas for infected larvae sampling
Isolation of NPV isolates from infected samples
Conducting different bioassays for screening the most suitable strains for different hosts in hot and tropic regions of the country
Bioassays for non target lepidoptrans
Continued PCR amplifications and cloning of candidate genes
Sequencing of cloned genes
Data analysis, report and paper writing
Plan of work (2011)
Activity Jan Feb Mar Apr
Conducting different bioassays for screening the most suitable strains for hot regions of the country
Continued PCR amplifications and cloning of candidate genes
Sequencing of cloned genes
Data analysis, report and paper writting
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vi) Expected benefits of the proposed study.
� Availability of basic data on occurrence, diversity and host range of bacloviruses in Pakistan
� Availability of alternate and safe source of insect control.
� Microbial solutions to agricultural, environmental, forestry and public health problems
� Utilization of microbial diversity to contribute towards the national development strategy of poverty eradication
� Trained scientific staff
4. Scientific personnel required for the project.
Principal Investigator: Aamir Humayum Malik
Designation: Biotechnology Specialist
Qualifications Doctor of Philosophy in Biotechnology (Ph.D)-2004-2008- School of Biotechnology,
National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad Campus. Quaid-i-Azam University Islamabad. Pakistan. Thesis title “Molecular characterization of melon leaf curl disease and
development of control strategies” Master of Science in Plant Pathology (M.Sc Hons.)-2004- University of Agriculture
Faisalabad, Department of Plant Pathology. Thesis title “Studies on survey
of Quick Decline of Mango disease in Punjab and isolation of pathogenic
fungi and control measures of disease”.
Duties:
� Survey for collection of bacloviruses infected larvae from different agroecological zones if Pakistan
� Isolation and purification of viruses from infected samples � Identification of virus polyhedral occlusion bodies under light microscope � Molecular identification of viral isolates � Long term preservation of viral isolates � Preparation of different formulations to test against target insects � Data analysis, report writing and research paper writing
Training and research experience:
� Molecular identification of plant infecting viruses � Investigation of synergistic interaction between viruses of unrelated species in
muskmelon � Isolation and Identification of pathogenic fungi associated with decline disease of
mango. � PCR based identification of mealybug complex found in Pakistan
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� International Certificate in “Project Management in controlled environment” (PRINCE2)
Co-Principal Investigator: Shakeel Ahmad
Designation: Bio-pesticide Specialist
Qualifications:
Master of Science in Agricultural Entomology M.Sc (Hons)-1996-99: University of Arid Agriculture Rawalpindi Department of Entomology. Thesis Title “Survey of
entomopathogenic nematodes in Potohar region and their virulence against H. armigera
(Lepidoptera: Noctuidae)”
Master of Science in nematology 2006-07: University of Ghent, Belgium Department of Science. Thesis Title “Persistence and activation of entomopathogenic nematodes in a
granular formulation”
Duties:
� To guide in establishing insect lab cultures � To conduct bioassays for testing virulence of bacloviruses against lab reared insects � Helping in data analysis and technical report and paper writing
Training and research experience:
� Training on “Nematode isolation, culturing and application” in National Nematological Research Centre (NNRC) Karachi University, Pakistan.
� Training on rearing of different lepidopterous insect pests on artificial diet � Training on “Integrated Pest Management” organized by UNDP, Pakistan. � Training on Bioassay techniques used for testing biological control agents against
different pests in Germany
5. Existing institutional facilities.
Established molecular biology lab equipped with the basic laboratory and library facilities required for the proposed study. Institute also has an insect culture lab well equipped with necessary items and trained staff
Molecular biology lab includes items
No. Item Name Condition
1 PCR machine Working
2 Gel electrophoresis Working
3 Gel documentation apparatus Working
4 Controlled temp. Incubator Working
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6 Refrigerators Working
7 Laminar Flow hood Working
8 Sensitive electric balance Working
9 Desktop centrifuge (In purchase pipeline)
10 All sorts of glassware Available
11 Microwave oven Working
12 Micro-centrifuge Working
13 Hot plate with stirrer Working
14 Compact thermostat Working
15 Ovens Working
16 CCTV Camera fitted Stereomicroscopes
Working
17 Binocular microscopes Working
18 Water distillation app. With deionizer
(In purchase pipeline)
19 Digital Autoclave (In purchase pipeline)
20 Sensitive electric balance Working
21 Pipetman sets Out of two sets available one is in working condition
State if the scheme has been submitted to some other aid giving agency for financial support.
This project has not been submitted to any other funding agency
6. Patentability of the project results. Not applied
7. End user (s) of the project results in case of applied research project (attach letter (s) of intent from the end user (s) Not applied.
8. Indicate, as per details given below, other research projects being conducted or previously guided by the Principal and Co-Principal Investigator, if any.
This will be the first project as PI. The PI has the responsibilities as “Biotechnology specialist” in project entitled “Biological control of major cotton pests including mealybug in Pakistan” funded by MinFA. He has good experience of working with both DNA and RNA viruses in doctoral studies (detail in CV).
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10. Research Proposal Budget:
TABLE-I
Estimated Cost of the Project in Rupees (Table-II to V)
(III) Travel within country: (Details (p.12) and justification (p19)
provided)
220,000
220,000
440,000
Total:- 435,000 435,000 870,000
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Justification for the Technical person Research associate will be working on isolation, infectivity and bioassays of NPV on lapedoptrous pests larvae and will be assisting PI in molecular identification and microscopy. He will also help in field surveys for collection of diseased samples. Proposed minimum qualification for technical person is B.Sc (Hons) Plant pathology but preferably M.Sc (Hons) Plant pathology having work experience in virology. The salary cost has been calculated @ 10,000/month which we believe is minimum to attract a suitable candidate for the described post
Justification for Field Surveys In Pakistan information is scanty on the use of bacloviruses for control of insect pests. No comprehensive information is available on diversity and distribution of these viruses in different ecological zones of Pakistan. As these viruses are being used as one of the alternates of synthetic chemicals for insect pest management in the world and it has also been recorded that we can find some strains of viruses which are more virulent even being a part of single population. Under this research project, visits to different area of Pakistan will be paid to explore the diversity, natural distribution of bacloviruses. For this very purpose sampling of infected larvae of the lepidopterous pests from the above mentioned areas will be conducted. This extensive survey will give us the information about the virus strains in different agroecological zones. Based on this knowledge, we can proceed forward to identify the most virulent starins which later on can be used as one of the biological control agents against economically important pests.
TABLE-III
Expenditure on Equipment & Supplies
Sr.No. Item Ist 2nd Total
1. Shaking water bath (Justification provided)
Pipetman set (Justification provided)
200,000 ----
56,666
256,666
2. Chemicals (List provided on page 20)
100,000 120,000 220,000
3. Expandable equipment (Justification provided
on page 20)
125,000 125,000 250,000
4. Glass-ware
10,000 10,000 20,000
5. Stationery
5,000 5,000 10,000
6. Literature
5,000 5,000 10,000
7. Contingencies, Postage etc.
10,000 10,000 10,000
Total: 455,000 331,666 786,666
20
Justification for shaking water bath
It is necessary item for the proposed study. Control temperature shaker will be used for growth of bacterial cultures at 37°C. It is one of the main steps in gene cloning. Transformed E. coli cells are kept in shaking at 120-150 rpm over night for optimum growth. It will be also used in making of competent E. coli cells. Note: Price of the shaker in proposal is added with 10% margin as compared to the quotation
Specifications of equipment.
Temperature range ambient to +50°C Timer min. 50hr Material used Steel Capacity 12 lit min Speed 30-130 /min
Justification for Pipetman set
As described in list of available equipment only one set of pipetman is in working condition which is quite insufficient when the equipment is in need of more than one researcher at the same time. During the quantification and cloning of viral genes the work load will increase and then it will be necessary to have at least one more set (three micropipets measuring 20µ, 200µ and 1000µ) of pipetmans. Gilson micropipets have been coated in the proposed study which are preferably sued in microbiology labs in Pakistan as well as in other countries
Note: Price of the shaker in proposal is added with 10% margin as compared to the quotation
Specifications of equipment.
Models: P20, P200, P1000
P 20
Vol. range 2-20µl
Tips 200 µl
Accuracy 0.05 µl
P 200
Vol. range 20-200µl
Tips 200 µl
Accuracy 0.05 µl
P 1000
Vol. range 200-1000µl
Tips 1000 µl
Accuracy 0.01 µl
21
Justification for expandable equipment
No. Item (Kits) Qty Justification Remarks
1 Taq DNA polymerase kit
5 Used in PCR reaction for amplification from viral DNA
Essential
2 PCR cloning Kit
4 Used in cloning of PCR product in sequencing vector
Essential
3 Plasmid isolation kits
4 Used in isolation of high quality DNA for sequencing of clones
Essential
4 DNA isolation kit
2 Used for isolation of high quality DNA Essential at critical stages
List of Chemicals
Chemicals for molecular studies
S.No Items Qty
1 Tris base (1Kg)
2 Tris HCL (1Kg)
3 NaOH (1Kg)
4 CaCl2 (500g)
5 Agar (2 Kg)
6 Sodium Acetate (500 g)
7 Isomylachol (2.5Lit)
8 EDTA (1Kg)
9 MgCl2 (500g)
10 Glycerol (500 ml)
11 Tryptone (500g)
12 Yeast Extract (500g)
13 NaCl (1Kg)
14 6x loading dye (1 set)
15 1 kb DNA ladder (1 set)
16 dNTPs (2 sets)
17 Protenase K (5 ml)
18 Phenol chloroform (500ml)
19 Endonucleases As required
20 Primers As required
Chemicals for insect rearing
S.No Items Qty
1 Agar (ordinary) As required
2 Yeast (ordinary) As required
3 Vitamin mixture As required
22
4 Methyl p-hydroxybenzoate 100 g
5 Streptomycin As required
6 Formaldehyde 40% As required
7 Sorbic Acid 500 g
8 Ascorbic Asid 500 g
List of Glassware/Plasticware
S.No Items Qty
1 Tips for pipetmans (1ml, 200µl) As required
2 Sterile Petri dishes (Plastic) As required
3 Gloves 4 pks
4 Beakers (500 ml) 4
5 Autoclavable bottles (500ml,1lit) 4 each
6 Glass Vials As required
23
References Adams, J. R. and McClintock, J. T. 1991. Nuclear polyhedrosis viruses of insects. Atlas of
Keddie, B. A., G. W. Aponte and L. E. Volkman. 1989. The pathway of infection of Autographa californica nuclear polyhedrosis virus in an insect host. Bio. Sci., 243:1728-1730. Kranthi, K. R., Jadhav, D. R., Kranthi, S., Wanjari, R. R., Ali, S. S. and Russell, D. A. 2002.
Insecticide resistance in five major insect pests of cotton in India. Crop
Protection. 449-460
Lautenschlager R.A; Podgwaite J.D. 1979. Response of birds to aerial application of the
nucleopolyhedrosis virus of the gypsy moth, Lymantria dispar. Environmental
Entomology 8:760-764.
Lautenschlager RA; Podgwaite JD; Watson DE. 1980. Natural occurrence of the
nucleopolyhedrosis virus of the gypsy moth Lymantria dispar Lepidoptera
Lymantriidae in wild birds and mammals. Entomophaga. 253:261-267.
Lavina B. A., L. E. Padua., F. Q. Wu., N. Shirata., M. Ikeda and M. Kobayashi. 2001.
Biological Characterization of a Nucleopolyhedrovirus of Spodoptera litura
(Lepidoptera: Noctuidae) isolated from the Philippines. Bio. Cont., 20: 39-47.
Miller L. K. 1997. The Baculoviruses. New York: Plenum
Moscardi F. and Sosa-G´omez D. R. 1992. Use of viruses against soybean caterpillars in
Brazil. Pest Management in Soybean, ed. LG Copping, MB Green, RT Rees, 98–
109. London: Elsevier
Moscardi F. and Sosa-G´omez D. R. 1996. Utilizaci ´on de virus a campo. Microrganismos
Pat´ogenos Empleados en el Control Microbiano de Insectos Plaga, 261–76.
Buenos Aires:Mass
Moscardi F. 1989. Use of viruses for pest control in Brazil: the case of the nuclear
polyhedrosis virus of the soybean caterpillar, Anticarsia gemmatalis. Mem. Inst.
Oswaldo Cruz 84:51–56
Nishi Y, Nonaka T. 1996. Biological control of the tea tortrix—using granulosis virus in the
tea field. Agrochem. Jpn. 69:7–10
Possee, R. D., A. L. Barnett., R. E. Hawtin and L. A. King. 1997. Engineered baculoviruses
26
for pest control. Pesticide Sci., 51: 460-470
Raman K V, Alcazar J, Valdez A. 1992. Biological control of potato tuber moth using
Phthorimaea baculovirus. Int.Potato Cent. Lima CIP Train. Bull. 2. 27
Schmitt AT. 1985. Eficiˆencia da aplica¸c˜ao de Baculovirus erinnyis no controle do
mandarov´a da mandioca. Empasc, Itaja´ı, Com. Tec. 88. 7
Seufi, A. M. 2008. Characterization of an Egyptian Spodoptera littoralis
nucleopolyhedrovirus and a possible use of a highly conserved region from
polyhedrin gene for nucleopolyhedrovirus detection. J. Virol., 5:13.
Shoery, H. H. and R. L. Hale. 1965. Mass-Rearing of the Larvae of Nine Noctuid Species on
a Simple Artificial Medium. J. Eco. Entomology. 58: 522-524.
Smith L. and Bellotti A. C. 1996. Successful biocontrol projects with emphasis on the
Doctor of Philosophy in Biotechnology (Ph.D)-2004-2009- School of Biotechnology,
National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad Campus. Quaid-i-Azam University Islamabad. Pakistan. Thesis title “Molecular characterization of melon leaf curl disease and
development of control strategies” Master of Science in Plant Pathology (M.Sc Hons.)-2004- University of Agriculture
Faisalabad, Department of Plant Pathology. Thesis title “Studies on survey of Quick Decline of Mango disease in Punjab and isolation of pathogenic fungi and control measures of disease”.
Bachelor of Science in Plant Pathology (B.Sc. Hon).-2001- University of Agriculture
Faisalabad, Department of Plant Pathology
Curriculum Vitae
Name: Aamir Humayun Malik
29
Ph.D Research Work: Thesis Title: Molecular characterization of melon leaf curl disease and development of control strategies Project summary: The project was focussed on isolation and characterization of viruses involved in a severe disease on muskmelon in commercial fields. Experiments were performed to confirm the association and pseudorecombination of isolated viruses with the disease by making infectious dimeric molecules in binary vectors and a novel synergistic interaction between a DNA and an RNA virus was found which was responsible for causing disease syndrome with the involvement of HC-Pro gene of Zucchini Yellow Mosaic Virus (ZYMV). HC-Pro gene was also used to develop resistance by using RNA interference (RNAi). Transgenic Nicotiana benthamiana gave desired results against viral infection. Additionally valuable data was generated by conducting comprehensive surveys for identification of cucurbit infecting viruses in Punjab province each year (2004-06) by using different nucleic acid amplification, radioactively labelled probe hybridization and serological techniques.
M.Sc Research Work: Thesis Title: Studies on survey of “Quick Decline of Mango” disease in Punjab and isolation
of pathogenic fungi and control measures of disease Project summary: In survey study there was no statistical difference among four commercial mango verities in susceptibility to disease and non of the cultivars under observation was found to be resistant. Botryodiplodia theobromae was proved to be associated with the disease. In case of physiological studies conducted on three different growth media, B. theobromae was found fast growing on (Potato dextrose Agar) PDA at 25˚C. Histopathological studies of infected tissue showed maximum invasion of fungus in cortical area which attained dark brown colour due to toxin production or mycelia of fungus. Comparison of in vitro effect of four commercially available fungicides showed that Topsin-M inhibited the growth of fungus at all concentrations as compared to other tested fungicides.
Training and research experience:
� Molecular identification of plant infecting viruses
In Molecular Virology and Gene silencing Lab, I have been primarily involved in experiments designed for identification of plant infecting viruses especially, in cucurbits and cotton. These experiments include PCR based amplification of geminiviral genes and complete components from the DNA isolated from infected plants followed by sequencing for precise identification of viruses responsible for the disease. Identification experiments of DNA viruses also included southern blot hybridization using radioactively labeled probes. Have also identified plant infecting RNA viruses, synthesizing cDNA from total RNA isolated from the infected plants and ELISA.
� Investigation of synergistic interaction between viruses of unrelated species in
muskmelon
Worked on synergism between Tomato leaf curl new Delhi virus (ToLCNDV) and Zucchini
yellow mosaic virus (ZYMV) and was able to identify a novel synergism between DNA and an
30
RNA virus first time to best our of knowledge. For this purpose partial duplication clones of both viruses were produced in plant expression vectors and N.benthamiana was used as model host plant. Also in order to block synergism an RNAi construct was produced and assessed. This work was presented at International Congress of Virology. San Francisco and 6TH Hangzhou International Symposium on Plant Pathology and Biotechnology, Hangzhou, China
� Isolation and Identification of pathogenic fungi associated with decline disease of
mango. A number of phytopathogenic fungi are associated with quick decline of mango (mangrifera
indica) and shisham (dalbergia sisso) in Pakistan. Seventy samples were collected from the stem and roots of symptomatic and asymptomatic plants. Isolates were identified based on mycelial growth (on three different media) and morphological (under light microscopes) characteristics. Pathogenicity of Botrodiplodia theobromae was assessed on two year old seedlings of mango plants.
� PCR based identification of mealybugs found in Pakistan
Now a days I am working on identification and genetic diversity of mealybugs insects found in Pakistan by cloning 18S ribosomal RNA and internal transcribed spacer (ITS) genes. I am also supervising a group of graduate student in learning biotechnological techniques. Honours and awards
� First class position in M.Sc. Plant Pathology, University of Agriculture Faisalabad,
Pakistan � Higher education commission scholarship for Ph.D
Technical skills:
Molecular Biology: Isolation of total RNA/DNA from various plant specimens and detection and amplification of viruses involved through ELISA, PCR and its modifications (PCR, Nested-PCR, real time PCR); cloning; transformation; radioactive and non radioactive probes labelling; southern and northen blotting; reverse transcription; RFLP; transient assays for gene function analysis in model system, pathogen derived RNAi construct development for insect and disease resistance. Plant tissue culture: Stable transformation of constructs based on pathogen derived resistance into plant tissues and development and analysis of performance of transgenic plants. Certifications
� Certified Professional in data base development Oracle (OCP, Developer track) � Certified Practitioner in PRINCE2 (Projects Management in controlled environment)
Computer skills:
� Excellence in use of Microsoft Office. � DNAStar, ClustalX, WinGene, and NCBI data base for data analysis.
31
Other skills
� Excellent research, analytical and conceptual skills � Ability to solve problems and implement innovative solutions � Ability to work under pressure and meet deadlines � Proven ability to work independently or as a part of team
PUBLICATIONS
Iram, S., Amrao, L., Mansoor, S., Malik, A. H., Briddon, R. W., Y.Zafar, (2005) First report of begomovrius associated with leaf curl disease of Duranta erecta in Pakistan. Plant Pathology
54, 260. (I.F, 1.76) Malik, A. H., Khan, S. M., Iqbal, Z., Malik, M. T., Saleem, A and Inam-Ul-Haq. (2005). histological and control studies on Botryodiplodia theobromae, the cause of mango decline in the Punjab. Pakistan Journal of Phytopathology. 17(1):18-21. Malik A. H., Mansoor S, Iram, S. Briddon, R. W. and Zafar Y. (2006). Severe disease of melon in North West Frontier Province is associated with simultaneous infection of two RNA viruses. Pakistan Journal of Botany. (In Press). (I.F, 0.15) Malik A. H., Mansoor, S, Iram S, Briddon R. W, and Zafar Y. (2006). A severe outbreak of melon yellow mosaic disease caused by Zucchini yellow mosaic virus in the Punjab province of Pakistan. Plant Pathology 55, 285. (I.F, 1.76)
Malik A. H., Mansoor, S, Iram S, Briddon R. W, (2009) Characterization of a bipartite begomovirus with defective DNA B associated with severe leaf curl disease on muskmelon in Pakistan. Ready for submission in Virus Research (I.F 2.8) Conferences and Symposiums Attended
Malik, A. H., Mansoor S, Iram, S, Briddon, R. W. and Zafar Y.(2005) Dual infection with a begomovirus and a Potyvirus of Melon showing severe leaf curl symptoms in Pakistan. International Congress of Virology. San Francisco 23-29 july 2005. Malik A. H., Mansoor, S, Briddon, R. W. and Zafar, Y. (2005). Symnergism between a Potyvirus and a Begomovirus is meadiated by Helper Component Protein. 18th Federation of Asian and Oceanian Biochemists and molecular biologists Symposium. Lahore, Pakistan. November 20-23 2005. Malik A. H., Mansoor, S, Briddon, R. W. and Zafar, Y. (2005).Multiple infection of plant viruses on cucurbits and their interaction in enhancement of severity of disease symptoms:
32
International Symposium on Recent Trends in Plant Disease Management. 20-22 December 2005.
Malik A. H., Mansoor, S, Briddon, R. W. and Zafar, Y. (2006). Severe disease of muskmelon is due to synergistic interaction between a DNA virus and RNA virus and synergism is mediated by helper component protein. National Symposium on Biotechnology for “Economic Prosperity”, 24-26 july, Nathiagali
Malik A. H, Mansoor, S, Zafar, Y and Briddon R.W (2007) Analysis of leaf curl disease of muskmelon suggests a synergestic interaction between a DNA and an RNA virus. 6TH Hangzhou International Symposium on Plant Pathology and Biotechnology People’s Republic of China May 31-June 3, 2007, Hangzhou, Workshops Attended.
International Thematic workshop on “Use of RNAi in Therapeutics” 25Nov.-8 Dec. 2006, COMSTECH Secretariat 33-Constitution Avenue, G5/2 Islamabad, Pakistan. International Thematic workshop on “DNA/Emerging Vaccines and Production Challanges” 3.-10 September, 2007, COMSTECH Secretariat 33-Constitution Avenue, G5/2 Islamabad, Pakistan 3rd International Conference on Plant Pathology & 7th Biennial Meeting of Phytopathological Society. 19-21 November 2007 Department of Mycology and Plant Pathology, Punjab University Lahore Academic referees
House No. 32/1, Street No.6, Rajput colony, Jehangir road Rawalpindi
Languages
English, Urdu (Can speak and write) Punjabi (Local languages)
Fluency in English
Good
Field of Specialization
Use of biological control agents in insect pests management
Employer
CABI
Unit where Employed
CABI South Asia, Pakistan
Present Job
Biopesticide Specialist
34
EDUCATIONAL QUALIFICATION
Examination
Board/University
Year
Division/Percentage
B.Sc. (Hons) Agri.
University of Agriculture, Faisalabad, Pakistan
1995
1st / 74.49%
M.Sc (Hons) Agri. Entomology
University of Arid Agriculture, Rawalpindi, Pakistan
1999
1st / 77.71%
M.Sc (Nematology)
University of Ghent, Ghent Belgium
2007 Great Distinction 76.30%
Trainings Received: Trainings in Pakistan:
� Training on “Nematode isolation, culturing and application” in National Nematological Research Centre (NNRC) Karachi University, Pakistan.
� Training on rearing of different lepidopterous insect pests on artificial diet from NARC, Islamabad.
� Training on “Technical Report Writing” organized by UNDP in Pakistan. � Training on “Integrated Pest Management” organized by UNDP, Pakistan. � Workshop on Facilitation Skills Development, organized by National IPM
Program and FAO.
� International Training:
� Training on “development of EPNs granular formulation e~nema, Kiel Germany (2007).
� Training on Bioassay techniques used for testing biological control agents against different pests different pests in Germany.
35
� Training on EPNs identification and application techniques in Christian Albrecht University, Germany.
� Visited different Rothemsted Research, CABI, Natural History Musium and Reading University, United Kingdom and University of Wageningon, Netherlands and discussions made with scientists working on use of Entomopathogenic Nematodes in year 2007.
Professional Experience: Biopesticide Specialist
Activities in brief:
� Technology development on use of biological control agents in Integrated Crop management.
� Research on Integrated pest management (IPM), and biological control strategies.
� Augmentative biological control activities.
� Monitoring & evaluation of participatory training and research activities.
� Cop Management in temperate fruits and vegetables.
� Responsible for project development and implementation on Integrated
Pest Management through biological control measures.
� Implementation of Participatory Learning & Action (PLA) programs through Training of Facilitator/Trainer (ToF/T) and Farmer Field School (FFS) in high value cropping systems.
� Sustainable agricultural with special emphasis on designing and managing
Programs/projects on extension of complex issues in rural setting using participatory approaches.
� Facilitation and development of curricula for season long ToF/T and FFS
activities for high value crops.
� Technology development on organic farming through compost making.
Honours and Awards
� Won scholarship from VLIR, Belgium, and recommended for M.Sc with
specialization on entomopathogenic nematodes.
36
� While studying for M.Sc at Ghent University, Leonardo da Vinci of Belgium govt. recommended for another scholarship to complete training on techniques for using EPNs in different crops.
� Got great distinction certificate from Ghent University, Belgium.
� Got distinction in M.Sc (Hons) Argi. Entomology from University of Arid
Agriculture, Rawalpindi.
Research Interests
� Technology development to explore biological control strategies in agriculture system.
� To explore natural ecosystem for introducing organic farming in Pakistan.
� To explore potential of biological control agents in minimizing the
dependency of farming community on synthetic chemicals. � To research out best possible ways towards environmental safety.
Achievements
� Successful establishment of Integrated Crop Management system in temperate fruits and vegetables.
� Reported entomopathogenic nematodes Heterorhabditis indica and
Steinernema sp. occurring in Pothwar region of Pakistan.
� Technology development on compost making. Project Reports:
� Integrated Crop Management of fruits and Vegetables in North West Frontier Province in Pakistan in collaboration with Swiss development Corporation (1999).
� Production of fruits and Vegetables with minimal use of synthetic
chemicals, in collaboration with UNDP, Pakistan (2000).
� Integrated Pest Management of Fruits in Chitral, in collaboration with AKRSP, Pakistan (2001-2004).
� Implementation of Best Agricultural Practices through Training of Facilitators and Farmer Field School, a project Funded by Asian Development Bank (2005).
37
� Implementation of Best Agricultural Practices through Training of Facilitators and Farmer Field School, a project Funded by Govt of Pakistan (2006).
� Compost making event completion report in year 2007 funded by German Agro Action.
�
Research Paper
� S. Ahmad and Z. Hussain, 2002. Effect of soil type and vegetation cover on occurrence of Entomopathogenic nematodes in Pakistan. Pakistan Journal of Biological Sciences 5 (6): 640-642.
� S. Ahmad., R. Ehlers and A. Peters. 2009. Assessment of survival and
infectivity of EPNs inside the granular formulation at different temperatures. Pakistan Journal of Biological Sciences(In Press)
Master Thesis M.Sc (Hons) Agri. Entomology
� Survey of Entomopathogenic Nematodes in Pothwar region
and their virulence against Helicoverpa armigera Hubner (Lepidoptera: Noctuidae).
M.Sc (Nematology)
� Persistence and activation of entomopathogenic nematodes in a granular formulation.
Computer Skill MS Office (MS Word, Excel, Power Point), Access
Referees 1. Prof. Dr. Ralf-Udo Ehlers
Institute Phytopathology Christian Albrect University, Kiel, Germany Email: [email protected]
2. Nic Smol Coordinator Postgraduate International Nematology Course, University of Ghent, Ghent, Belgium Email: [email protected]
