Source: PARATECHS CORP. submitted to
DEVELOPMENT OF A NEW PRODUCTION SYSTEM FOR BACULOVIRUS PESTICIDES
 
PROJECT DIRECTOR: Fath-Goodin, A.
 
PERFORMING ORGANIZATION
(N/A)
PARATECHS CORP.
LEXINGTON,KY 40546
 
NON TECHNICAL SUMMARY: Insects are major pests of economically important plant-based food and fiber crops and also vector diseases causing millions of human deaths and untold suffering annually. Moreover, our efforts to control insect pests have caused massive environmental contamination and continue to cause human and animal fatalities annually. Biotechnology offers alternatives to conventional pest control that have great promise although, like other technological innovations, there is resistance to its use and it carries some risks with which we have little prior experience. Baculoviruses, which are common pathogens of many important pest insect species causing frequent and dramatic epizootics that produce major declines in pest populations in cropping and landscape systems have been intensively investigated as biological pesticides. However, baculovirus biopesticides, with a few notable exceptions, have not been commercially successful due to high production costs. One increasingly important aspect of biotechnology is its potential to replace conventional products with process and materials that are more environmentally sound. Paratechs biology-based baculovirus production (B3P) technology is an example of such a green process in that it has the potential to produce cost-competitive biopesticides that can be used to replace chemical pesticides and relies upon natural biological processes rather than synthetic chemicals to do so. Such a technology would have important commercial impact and also have beneficial environmental and health effects.
 
OBJECTIVES: Insects are major pests of plants and animal food and fiber crops and also vector diseases causing millions of human deaths and untold suffering annually. Efforts to control insect pests have caused massive environmental contamination and continue to cause human and animal fatalities annually. Biotechnology offers alternatives to conventional pest control that have great promise although there is resistance to its use and some risks with which we have little prior experience. One increasingly important aspect of biotechnology is its potential to replace conventional products with processes and materials that are more environmentally sound. Paratechs biology-based baculovirus production (B3P) technology is an example of such a green process in that it has the potential to produce biopesticides capable of replacing some chemical pesticides and relies upon natural biological processes rather than synthetic chemicals to do so. Such a technology would have important commercial impact and also have beneficial environmental and health impacts. Baculoviruses are common pathogens of many important pest insect species causing frequent and dramatic epizootics that produce major declines in pest populations in cropping and landscape systems. Baculoviruses have also been intensively investigated in university, commercial and government laboratories as biological pesticides, but, with a few exceptions, have not been commercially successful. In developing this proposal, we found it instructive to consider the apparent reasons for commercial success and failure of baculovirus pesticides and believe that cost of production has not received sufficient emphasis. However, we do believe that there is significant opportunity for innovative approaches to reduce the cost of producing baculovirus pesticides so as to make them much more cost-competitive with chemical pesticides. Here, we address the fundamental issue of cost of production of baculovirus pesticides. The B3P system described in this proposal has novel features that derive from the biology of the insect used for virus production that should enable production of baculovirus pesticides in quantities and at costs that can compete with any existing insect virus production system in the world. During Phase I, ParaTechs demonstrated the utility of the B3P technology as part of an integrated plan for cost-effective commercial production of baculovirus biopesticides. In this proposal we seek support to quantify yield and cost of the production of one metric ton of baculovirus biopesticide on a pilot scale, convert the crude material into a marketable biopesticide, expand the host range of the B3P technology for other economically important baculoviruses and evaluate another Lepidopteran species as a B3P system. We consider it likely that ParaTechs B3P technology may make baculoviruses production cost-competitive with chemical pesticides. We also believe that the favorable regulatory and environmental traits of baculovirus biopesticides will become an increasingly important advantage relative to competitive products. This proposal will implement, test and quantify the potential of the B3P system.
 
APPROACH: The overall technical objective of this proposal is to test the hypothesis that ParaTechs proprietary in vivo baculovirus production system offers a superior system for inexpensive production of baculovirus pesticides. We describe specific technical objectives that will quantify yield and cost of the production of one metric ton of baculovirus biopesticide on a pilot scale, convert the crude material into a marketable biopesticide, expand the host range of the B3P technology for other economically important baculoviruses and evaluate another lepidopteran species as a B3P system. With our proposed production of a viral biopesticide in the environment in tremendous quantities, the presence of pathogens and microbial contaminants of concern to regulators is to be expected. Perhaps the biggest concern of regulators may be the difficulty in knowing what is in the crude feedstock material. Therefore, we will deploy the 454 state-of-the-art DNA sequencing technology, to define the complete array of organisms of potential concern. Once the array of commensals and pathogens are known it becomes relatively easy to establish purification procedures or treatments to inactivate the targeted viral, bacterial and fungal pathogens in the baculovirus preparation procedures and then establish quality control methods, such as PCR and diet feeding assays, to demonstrate the efficacy of these procedures.
 
CRIS NUMBER: 0214884 SUBFILE: CRIS
PROJECT NUMBER: KYK-2008-02301 SPONSOR AGENCY: NIFA
PROJECT TYPE: SMALL BUSINESS GRANT PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Sep 1, 2008 TERMINATION DATE: Aug 31, 2011

GRANT PROGRAM: SMALL BUSINESS AND INDUSTRY GRANTS
GRANT PROGRAM AREA: Small Business

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
211311011304.250%
215403011014.250%

CLASSIFICATION HEADINGS
KA215 - Biological Control of Pests Affecting Plants
KA211 - Insects, Mites, and Other Arthropods Affecting Plants
S3110 - Insects
S4030 - Viruses
F1130 - Entomology and acarology
F1101 - Virology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks


RESEARCH EFFORT CATEGORIES
BASIC (N/A)%
APPLIED 100%
DEVELOPMENTAL (N/A)%

KEYWORDS: baculovirus~biopesticide~baculovirus biopesticide~in vivo production system~lepidoptera

PROGRESS: Sep 1, 2008 TO Aug 31, 2011
OUTPUTS: Baculoviruses are common pathogens of many important pest insect species causing frequent and dramatic epizootics that produce major declines in pest populations in many cropping and landscape systems. We do believe that there is significant opportunity for innovative approaches to reduce the cost of producing baculovirus pesticides so as to make them more cost-competitive with chemical pesticides. This SBIR Phase II award evaluates the use of the Eastern Tent Caterpillar (ETC) as a production system for baculovirus pesticides. In this award we address the fundamental issue of cost associated with the production of Anagrapha falcifera multinuclear polyhedrosis virus (AfMNPV) biopesticide by optimizing yield and cost of production on a pilot scale, and implementing quality control measures. To obtain the best possible virus yield, two different virus application methods were evaluated by spraying the virus either on ETC nests in the field or on collected ETC nests in buckets. Therefore, trees containing four ETC nests harboring 4th instar larvae were sprayed with four different baculovirus dilutions of polyhedra inclusion bodies (PIB) in the presence or absence of optical brightener. Alternatively, different concentrations of AfMNPV in the presence or absence of optical brightener were applied to ETC nests containing 3rd or 4th instar larvae while in containers. The larvae were either supplied with food daily or only once at the time of virus application. Replicates of the experiments were carried out to ensure statistical significance of the results. Samples were taken from each treated nest and screened for the presence of AfMNPV, the ETC baculovirus Malacosoma americanum nuclear polyhedrosis virus (MaNPV) and microsporidia by PCR. Hence, PCR primers and conditions were developed to distinguish between AfMNPV, MaNPV and microsporidia. In addition, larvae were weighed and the virus titer was determined using a hemocytometer and light microscopy. The potency of the baculovirus recovered from ETC was determined by conducting feeding assays and comparing the mortality rate to that of AfMNPV purchased from Certis. In addition to AfMNPV other economically important baculoviruses were tested for their ability to replicate in the ETC and establish their potency in their respective host. Since field-collected larvae are used as a biopesticide production system, we expect ETC larvae to harbor insect commensals and pathogens, such as MaNPV and microsporidia, known to exist in the ETC population. A 454 sequencing approach was conducted to identify additional pathogens. Quality control measures are necessary to eliminate these contaminants and we have developed a virus purification procedure that removes microsporidia from the virus preparation but still contains a mixture of the two different baculoviruses. Furthermore, we surveyed ETC egg masses and nests for the natural occurrence of MaNPV and microsporidia. To reduce the load of MaNPV in the ETC population, ETC nests were screened by PCR for the presence of MaNPV. Moths from virus-free nests were used for breeding and the obtained egg masses were used to establish a virus-free colony. PARTICIPANTS: The following participants worked on this project during the cause of this project: Dr. Angelika Fath-Goodin, Dr. Angela Martin, Dr. Roland Hilgarth, Dr. Rebecca Scotland, John Webb, Cameron Freshwater, Andrew Lavey, Esther Fleming and Hannah Arvin. Dr. Angelika Fath-Goodin was the PI on this award and is responsible for overseeing the project, planning of experiments, analyses of data and report writing. After Drs. Hilgarth and Scotland's departure, Dr. Angela Martin one of ParaTechs' research scientists took over the responsibility for evaluating the field samples, purifying baculovirus, conducting PCR assays and performing feeding assays. Cameron Freshwater, Andrew Lavey and Hannah Arvin are undergraduate student helpers and were trained in conducting field work, purification of baculovirus from virus-infected Eastern Tent Caterpillar larvae, establishing PCR protocols and performing PCR and diet feeding assays. The student helpers were instructed by Dr. Angela Martin. John Webb and Dr. Rebecca Scotland helped the other participants during the field season by collecting caterpillars and food, and treating of the ETC nests with virus. Esther Fleming, a technician, helped with sample preparation. The D. Potter lab in the Entomology Department at the University of Kentucky is the collaborating entity on this SBIR Phase II award. Several meetings have been held between Dr. Potter, his students, Cody Elder and Andrea Bixby and Dr. Fath-Goodin. Cody Elder and Andrea Bixby conducted the experiments with Agrotis ipsilon larvae and AfMNPV and AgipMNPV(part of the subcontract). Dr. Fath-Goodin participated in the USDA Commercialization Assistance Training Program in 2009 with Brian Taylor, President and Principal Consultant, Growing Company Solutions, as her advisor. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

IMPACT: 2008-09-01 TO 2011-08-31 Based on the results obtained during our 2009 field season, we have established that applying 25 ml of 1x10^6 PIBs/ml AfMNPV to ETC larvae in buckets with no food supplied is the most efficient and cost-effective way in obtaining high viral yields. The potency of the baculovirus recovered from ETC was determined by conducting feeding. Only 40-60% of the larvae died when treated with AfMNPV purified from ETC whereas 100% mortality was achieved when larvae ingested AfMNPV manufactured by Certis at the same viral concentration. The reduced virulence of AfMNPV purified from the in vivo production system can most likely by attributed to contamination with MaNPV. Four additional commercially important baculoviruses were tested for their ability to replicate in ETC. As determined by PCR between 20% and 60% of infected ETC larvae were affirmative for that respective virus. The virulence of the recovered viruses were tested by feeding virus-treated diet to their respective hosts and determining mortality over time. As it is the case with AfMNPV, these economically important viruses show reduced virulence when amplified in the ETC. Since field-collected larvae are used as a biopesticide production system, we expect ETC larvae to harbor insect commensals. Therefore, we surveyed ETC egg masses and nests for the natural occurrence of MaNPV and microsporidia by PCR to estimate the percentage of diseased ETC. We identified that 64% of all egg masses tested were infected with either MaNPV or microsporidia or both pathogens, indicating that both pathogens can be transmitted to the next generation. Examination of ETC larvae over three years revealed that the appearance of the pathogens fluctuated over the duration of the study being between 28 and 54% for microsporidia and between 46 and 85% for MaNPV. To further identify additional pathogens, a 454 sequencing approach was conducted and 393 out of 85,118 contigs showed sequence homology to microbial proteins. Quality control measures are necessary to eliminate these contaminants and we have developed a virus purification procedure that removes microsporidia from the virus. To reduce the load of MaNPV in the ETC population, ETC nests were screened by PCR for the presence of MaNPV and moths from pathogen-free nests were used for breeding to establish a virus-free colony. However, in our first field season none of the eggs hatched under the conditions the egg masses were kept over winter. This experiment was repeated during the following field season but the presence of MaNPV in larvae was very high and only one egg mass hatched. We proposed to evaluate the Forest Tent Caterpillar as a production system for baculovirus biopesticides. Due to difficulties associated with collecting these caterpillars we decided to abandon this objective. In summary, the reduced virulence of AfMNPV purified from the in vivo production system, most likely to be attributed to contamination with MaNPV which is present in a vast majority of ETC larvae collected in the field, and our inability to rear a virus-free ETC colony let us to conclude that the ETC is not suitable as a marketable biological-based bio pesticide production system.

PUBLICATION INFORMATION: 2008-09-01 TO 2011-08-31
No publications reported this period

PROJECT CONTACT INFORMATION
NAME: Fath-Goodin, A.
PHONE: 859-433-5293
FAX: 859-257-2489