Microsatellite Markers Diagnostic for the
Bacterial Blight Resistance Gene, xa-5

M. Blair
Department of Plant Breeding
Cornell University
Ithaca, NY 14850

Abstract: Microsatellite markers tightly linked to the bacterial leaf blight (BLB) resistance gene, xa-5, were identified in this study. PCR-based markers are economical and easy to use, and would have immediate applicability in efforts to pyramid the recessive xa-5 gene with other BLB resistance genes. A molecular genetic survey was conducted to find microsatellite markers that detected polymorphisms between a nearly isogenic line (IRBB5) carrying xa-5, the donor parent and the susceptible recurrent parent (IR24). Microsatellite markers were used to show that the isoline contained one large introgression around xa-5, and three spurious introgressions on other chromosomes. Two F2 populations were evaluated for disease reaction phenotype and for marker genotype, and used to estimate linkage. Disease reaction was scored by clip-inoculating leaves at maximum tillering stage with two Philippine races of Xanthomonas oryzae pv. oryzae. Both RFLP and microsatellite markers were used to select individual F2 plants from the IR24 x IRBB5 population that were more nearly-isogenic to the recurrent parent. Two microsatellite markers were located at approximately 2 cM and 18 cM from the xa-5 locus. A microsatellite marker was developed based on the sequence of a cDNA probe encoding cytochrome b5 and containing a simple sequence repeat in its 5' untranslated region. The new microsatellite marker mapped to the same location as the original RFLP and was tightly linked to the targeted xa-5 gene. A germplasm survey of diverse lines containing BLB resistance genes, indicated the range of allelic diversity for each of the linked microsatellite loci. Microsatellite amplification was tested in a panel of genetically distant grass taxa, including wild rice species, bamboo, wheat, barley, oats, maize, millet, sugarcane and sorghum. A relationship between the number of alleles detected in cultivated rice and the ability to amplify in genetically distant species was observed and may be related to the location of the microsatellite locus in gene-coding versus non-coding regions of the genome.

Transformation of Tobacco and Tomato
with a Fungal Endochitinase Gene

A. Brants and E.D. Earle
Department of Plant Breeding and Biometry
Cornell University
Ithaca, New York

Abstract: The biocontrol fungus Trichoderma harzianum can substantially decrease damage caused by pathogenic fungi by excreting the hydrolytic enzyme endochitinase, which randomly cleaves chitin of fungal cell walls. The goal of this study was to transform tobacco and tomato with constructs encoding the T. harizanum endochitinase in order to obtain plants with enhanced resistance to fungal pathogens. The four constructs used differed in promoters, presence of enhancers, and presence of extracellular targeting sequences. Many tobacco and tomato plants (15-40) were recovered after transformation with each construct. Transformants were screened using PCR and NPTII protein-specific ELISA. Endochitinase activity was measured using a fluorimetric assay. Tobacco plants transformed with pStudI (35S-35S promoter + AMV enhancer; no intron; fungal targeting sequence) averaged 12-21 times higher endochitinase activity than control plants. pStudI tomato plants averaged 6 times higher enzyme activity than controls. Considerable variation in enzyme activity was observed among the transgenic plants. In contrast, endochitinase activity in p35ThEn42 tobacco plants (35S promoter, intron) was comparable to that of controls. Callus was initiated from high-expressing pStudI tobacco plants. Endochitinase activity was detected both in the medium under the calli (50-70 times higher than controls) and in the calli. Medium from cell suspensions initiated from the calli had 116 times higher endochitinase activity than controls. This suggests that the endochitinase is secreted from the transformed cells. Further characterization of the various transgenic materials is under way.

The Use of Pseudomonas syringae  508 for
Biocontrol of Apple Scab (Venturia inaequalis) 

M.R. Corral-Garcia, T.J. Burr
M.C. Matteson, and C.A. Smith*, J.Y. Takemoto**
*Department of Plant Pathology
Cornell University, NYSAES
Geneva, New York 14456
**Department of Biology
Utah State University
Logan, UT 84322-5305

Abstract: Apple scab, caused by the fungus Venturia inequalis,  is one of the most important apple diseases worldwide. Control of scab has relied almost exclusively on the use of fungicides. The future of fungicide use may be limited by concerns about pesticide damage to health and the environment, and by the development of pathogen resistance. As an alternative to chemicals, the use of microorganisms that colonize apple leaves and fruits to reduce scab incidence during the growing season is being investigated. A total of 931 bacteria and yeasts were isolated from apple orchards in New York and tested against V. inaequalis  in vitro and on apple seedlings. One isolate, 508, provided the greatest reduction of scab. It provides control on apple seedlings comparable to the fungicide captan. It also has fungicidal activity against conidia of V. inaequalis.  508 was identified as a non pathogenic Pseudomonas syringae  strain. Following introduction on trees, 508 survived well in the orchard, remaining around 105 cfu/gr of leaf tissue over a five-day period. It was discovered that 508 produces antibiotics syringomycin E and G. Both antibiotics kill conidia of V. inaequalis  as do cultures of 508. Syringomycin may not be the only factor involved in the mechanism of control since 508 suppressed scab to a greater extent than several other P. syringae  strains that also produce syringomycin. The importance of syringomycin production by 508 on biocontrol of apple scab is currently being evaluated. A syringomycin deficient P. s. 508 mutant has been developed for this purpose.

Unique Applied and Basic Biology
of a Trichoplusia ni  SNPV

T.R. Davis and H.A. Wood
Boyce Thompson Institute
Cornell University
Ithaca, NY 14853

Abstract: The unique properties of a cloned isolate of the Trichoplusia ni single-nucleocapsid, nuclear polyhedrosis virus (TnSNPV) were studied in vitro and in vivo. Unlike previous in vitro studies with other SNPVs, the BTI-Tn-5B1-4 cells are highly susceptible to TnSNPV infection, producing an average of 500 polyhedra per cell. Experiments examining viral-induced protein synthesis, genomic organization, and occluded and budded virions were conducted. Other experiments comparing TnSNPV and Autographa californica MNPV replication in BTI-Tn-5B1-4 cells were performed. Although onset of viral replication and polyhedrin production were similar for both virus types, there are several features of the TnSNPV:host cell interaction which are unique. For example, at 3 days post inoculation the mean yield of polyhedra per cell for TnSNPV was significantly higher than AcMNPV. Also, TnSNPV-infected host cell protein synthesis continued much later into the virus replication cycle than AcMNPV-infected cells. These novel characteristics suggest that a TnSNPV expression vector system could be superior to the AcMNPV system in the production and processing of foreign proteins. Finally, in bioassays performed with neonate T. ni larvae, the mean LD50 for TnSNPV polyhedra was significantly lower than AcMNPV polyhedra. Based on this result, and the high number of polyhedra produced per cell, TnSNPV appears to possess excellent biopesticidal properties.

Biocontrol of Grape Powdery Mildew and
Grape Downy Mildew with Mycoparasitic Fungi

S.P. Falk, D.M. Gadoury, A. Sztejnberg*,
P. Cortesi**, W.F. Wilcox, and R.C. Seem
Department of Plant Pathology
NYSAES, Cornell University
Geneva, NY 14456-0462
*Department of Plant Pathology and Microbiology
The Hebrew University of Jerusalem
Rehovot 76100, Israel
**Istituto di Patologia Vegetale
Università degli Studi di Milano
20133 Milano, Italy

Abstract: The non grata status and phytotoxicity of some fungicides has led us to investigate alternative controls for grape diseases. Ampelomyces quisqualis was found to naturally parasitize grape powdery mildew, caused by Uncinula necator, in the fall in vineyards in New York. To establish a level of parasitism sufficient to reduce disease, we introduced inoculum of the mycoparasite into the vineyard in the spring by suspending cotton wicks colonized by the mycoparasite over grape vines. Rain dispersed conidia of the mycoparasite and facilitated colonization of the developing powdery mildew colonies. In wet years, parasitism reduced the severity of powdery mildew on berries by 56-59%, compared to untreated vines. In addition, parasitism reduced the number of cleistothecia overwintering on grape vine bark by 50-60%. This is significant since cleistothecia provide the only source of primary inoculum of the pathogen in New York. A commercial formulation of A. quisqualis provided moderate control when sprayed on vines in 1995 field trials, but will be further evaluated in 1996. Fusarium proliferatum isolated from atypical colonies of grape downy mildew, caused by Plasmopara viticola, was found to parasitize hyphae and sporangiophores and reduce production of sporangia in vitro. Weekly sprays using microconidia of the mycoparasite reduced severity of berry infections from 53-99% under field conditions, depending on the year and grape cultivar. Use of these mycoparasites might be practical in combination with some fungicides, on moderately resistant cultivars, or in areas where disease pressure is not severe.

Use of Genetically Modified Plants
to Control Virus Diseases, and Assessment
of Environmental Risks

M. Fuchs and D. Gonsalves
Department of Plant Pathology
NYSAES, Cornell University
Geneva, NY 14456

Abstract: Genetic engineering has been a major breakthrough in the development of virus resistant crops. For example, transgenic plants that express viral coat protein (CP) genes are effective in controlling aphid-borne viruses. Our field evaluations show that transgenic tomato plants expressing the CP gene of cucumber mosaic cucumovirus (CMV) are resistant to infection by CMV, transgenic squash plants containing the CP genes of zucchini yellow mosaic potyvirus (ZYMV) and watermelon mosaic potyvirus 2 (WMV 2) exhibit resistance to mixed infections by both viruses, and transgenic muskmelons expressing the CP genes of CMV, ZYMV, and WMV 2 are resistant to mixed infections by these three viruses. Virus-resistant transgenic crops offer significant benefits to agriculture including: increased yield, reduced pesticide use to control insect vectors, multiple virus resistance, reduced incidence of mixed infections, and development of resistant plants when none are available to growers. Despite these benefits, concerns have been raised regarding the commercial release of virus-resistant transgenic crops. We are addressing two environmental risk issues: the development of new viruses through transencapsidation and recombination, and transgene escape into free-living crop relatives through pollen flow. We are analyzing under field conditions the potential of transgenic plants expressing CP genes derived from aphid transmissible viruses to mediate the spread of viruses that are aphid non transmissible. Also, we are evaluating the potential of CP genes to introgress from transgenic Cucurbita pepo squash into wild C. texana squash. This work will allow us to assess if risks outweigh benefits.

BTI-Tn5B1-4 cells, Derived from the Cabbage Looper,
Trichoplusia ni,  are High Producers of
Baculoviruses and Recombinant Proteins

R. R. Granados and K. A. McKenna
Boyce Thompson Institute
Cornell University
Ithaca, NY 14853

Abstract: The embryonic cell line, BTI-Tn5B1-4, is a clonal isolate of the parental line BTI 5B1, which was established in 1986 (Granados et al., Virology, 1986). The Tn5B1-4 cells grow as an attached monolayer, have a doubling time of approximately 21 hrs, and reach densities of 2-3 x 106 cells/ml in both serum-containing and serum-free media. Studies showed that this cell line was highly susceptible to the Autographa californica MNPV and the Trichoplusia ni SNPV. Yields of AcMNPVocclusion bodies (OBs) under stationary conditions ranged from 70-80 and 20-30 per cell for Tn5B1-4 and Spodoptera frugiperda (Sf-21) cells, respectively. The Tn5B1-4 cells produced high levels of TnSNPV OBs (500-800 OBs/cell) (Granados et al., 1994), whereas the Sf-21 cells are not susceptible to this virus. In addition, expression of many recombinant proteins have been found to be expressed to much higher levels in Tn5B1-4 cells compared to Sf-21 cells or its clonal isolate, Sf-9. For example, §-galactosidase expression was found to be twice as much in Tn5B1-4 cells when compared to Sf-9 and Sf-21 cells. Likewise, a glycosylated protein, secreted alkaline phosphatase (SEAP), was found to be expressed approximately 20 times greater per 106 cells when compared to Sf-9 and Sf-21 cells. High levels of protein expression can be achieved in cells grown as attached monolayers or in suspension.

These normally anchorage-dependent insect cells can be adapted to suspension growth by gradual selection of cells over a period of 15 to 20 passages under spinner flask condition. We have developed a method by which the Tn5B1-4 cells can be quickly put into suspension by the addition of heparin to the cell culture medium. Cells grown in the presence of heparin are less susceptible to AcMNPV but are still able to express acceptable levels of recombinant protein.

The Tn5B1-4 cells are widely used for expression of pharmaceutical proteins in many laboratories around the world. They also show excellent potential for production of baculovirus pesticides. Two commercially available cell culture media, ExCell 405® (JRH Biosciences) and Express Five® (Gibco/LTI) were specifically developed for the growth of these cells. A patent on the BTI-Tn5B1-4 cells has been issued and these cells are available for research purposes from Invitrogen, Inc., San Diego, CA., under the trade name "High Fives®".

Evidence for an Allee Effect due to
Mate Limitation in a Chrysomelid Beetle
Introduced Against Purple Loosestrife

Fritzi S. Grevstad
Section of Ecology and Systematics
Cornell University
Ithaca, NY 14850

Abstract: Most unsuccessful attempts in classical biological control are the result of the natural enemy species failing to establish a permanent population in the new location. The presence of an Allee effect (a reduction in population growth rate at low densities) in newly released biocontrol agent populations may be an important cause of failed establishement. Here I present evidence for an Allee effect due to mate limitation in one of two species of chrysomelid beetles (Galerucella calmariensis  and Galerucella pusilla ) currently being introduced into North America for the biological control of purple loosestrife (Lythrum salicaria ). I placed unmated beetles of both beetle species into large field cages at two densities. After ten days I removed and dissected the females to determine their mating status. The proportion of G. calmariensis  females that had mated was not influenced by density. However, for G. pusilla , mating frequency was much higher in the high density treatment (95%) than it was in the low density treatment (42%). The differential responses to density between the two species can be explained by differences in their local mobility. G. calmariensis  moves more frequently between host plants and therefore is more likely to encounter potential mates, even if beetle densities are low. G. pusilla  tends to move less frequently between plants and individuals are likely to remain isolated from mates when beetle densities are low.

Control of Silverleaf Whitefly
on Poinsettia by Natural Enemies

M. Hoddle*, J. Sanderson**, & R. VanDriesche*
*Department of Entomology
University of Massachusetts
Amherst, MA
**Department of Entomology
Cornell University
Ithaca, NY 14850

Abstract: In New York, poinsettias are sprayed for whiteflies an average of 14 times during the 3 1/2 month growing period. Because of increasing problems associated with chemical control in greenhouses, biological control offers a viable non-chemical whitefly control option, particularly for whitefly control on poinsettias. Chances for success are good because whiteflies are essentially the only key pests on poinsettias, compatible control strategies exist for the other pests, and parasitoids are or will be commercially available.

We are evaluating the use of weekly inundative releases of Encarsia formosa  (Beltsville strain) and Eretmocerus  sp. nr. californicus  for silverleaf whitefly control in small replicated greenhouses. The impact of the parasitoids on the whitefly population is measured by:

1. contrasting lifetable data from whitefly populations in the presence and absence of parasitoid releases, using twice-weekly photography of whitefly cohorts wherein the fate of each individual is observed; and
2. weekly population counts of whiteflies on foliage in the presence and absence of parasitoids.

A low release rate of 1 female wasp per plant per week was compared with a high rate of 3 females per plant per week. Control was better at the higher release rate for both parasitoid species. Based on data from the whitefly cohorts, there was no difference in overall whitefly mortality caused by the two parasitoid species at the high release rate (99%). For both species, parasitism was higher at the low release rate, while mortality caused by host-feeding or superparasitism was higher at the high release rate. Successful parasitism was higher for En. formosa  than for Er. californicus . For both species, successful parasitism increased as the crop canopy grew, suggesting that superparasitism decreased as the amount of leaf area to be searched increased. Overall control was unacceptable at the low release rate.

In an effort to maximize cost-effectiveness of biological control with these parasitoids, evaluations of variable weekly release rates at different stages of the crop are planned.

Multicolored Asian Lady Beetle, Harmonia axyridis ,
in North America: Friend or Foe?

E.R. Hoebeke* and J.J. Knodel**
*Department of Entomology
Cornell University
Ithaca, NY14850
**IPM Support Group-Entomology
New York State Agricultural Experiment Station
Geneva, NY 14456

Abstract: This relatively new Asian import is now widely distributed in eastern North America from Louisiana and Georgia to the Canadian Maritime Provinces and also occurs on the west coast (California, Oregon, and Washington). Researchers have suggested that populations of this Old World aphid predator probably originated either from an accidental introduction(s) from Asian freighter activity at the port of New Orleans, or from intentional USDA releases in the West in 1916, 1964-65, and in the East from 1977-1985. For biological control purposes, Multicolored Asian Lady Beetle is considered a principal and effective predator of injurious aphids, scales, and other homopterous insect pests on a variety of hosts plants, ranging from ornamental trees and shrubs, fruit and nut trees, Christmas trees, and numerous crops (alfalfa, cotton, wheat, tobacco, and small grains). However, in the limited time it has set up residence in North America, including New York State, it has become a major nuisance to homeowners due to its habit of invading and congregating inside houses and buildings in large numbers during the fall (mid October to early November). This poster will provide information on this lady beetle's native and North American distribution, identification characteristics, life cycle and habits, economic impact, management recommendations, and future outlook.

Construction and Characterization of a
Recombiinant AcMNPV with a Fungal Gene
Encoding an Insecticidal Protease

X.P. Huang, R.J. St. Leger, H. A. Wood, T. R. Davis and P. R. Hughes
Boyce Thompson Institute
Cornell University
Ithaca, NY 14853

Abstract: The entomopathogenic fungus, Metarhizium anisopliae  produces a subtilisin-like protease (designated Pr1) which solubilizes the cuticle of insect larval hosts and is highly toxic when injected into host larvae. The insertion and expression of the Pr1 gene into the Autographa californica  nuclear polyhedrosis virus (AcMNPV) genome was investigated to enhance the pesticidal properties of the virus. The AcMNPV polyhedrin gene was replaced with the Pr1 gene under the transcriptional control of the polyhedrin gene promoter. The recombinant virus, designated AcMNPV/Pr1, expressed Pr1 which was antigenically and functionally similar to the Pr1 produced by M. anisopliae . The expression of Pr1 caused 4th instar Trichoplusia ni  larvae injected with AcMNPV/Pr1 to exhibit an approximate 50% reduction in time-to-death compared to infections with a control Ac-E10, a polyhedrin-negative mutant of AcMNPV. All larvae injected with AcMNPV/Pr1 at 105 pfu/larvae died within 48 hr pi. Most of these larvae showed cessation of feeding by 24 hr pi, and no significant weight gain was observed from the time of injection to the death of the larvae. However, the level of per os  infection with the pre-occluded form (POV) of AcMNPV/Pr1 against T. ni  neonates were much lower compared with POV of Ac-E10. The Pr1 protease activity also reduced the infectivity levels of both POV or budded virion preparations of Ac-E10 by altering viral functional protein(s) which were required for per os  infection.

Mass Rearing Methods for
Galerucella calmariensis  and Galerucella pusilla ,
Biological Control Agents of Lythrum salicaria 

T.R. Hunt and B. Blossey
Department of Natural Resources
Cornell University
Ithaca NY 14853

Abstract: Effective control of purple loosestrife is facilitated by mass rearing of biological control agents to allow their widespread distribution.

The effectiveness of mass production methods to rear the leaf beetles Galerucella calmariensis  and Galerucella pusilla  in the field or in the greenhouse was compared. The "quality" of the mass produced offspring was monitored by studying the fecundity of females and pre-overwintering survival of adults. Beetles were overwintered either in the field or for variable times in the refrigerator. Overwintering survival was not influenced by the type of material used for storage, nor by the length of storage time. Increasing the availability of overwintering materials (dry purple loosestrife stems) increased the survival rate. Oviposition studies showed that fecundity of females was positively correlated with duration of overwintering. Different cage sizes did not significantly influence fecundity, however, oviposition was reduced as number of beetles/cage increased. Rearings in the field produced 2-5 times more offspring than those in the greenhouse. Mortality during the pre-overwintering feeding period was higher for greenhouse reared beetles compared to those reared in the field. Maintenance of fitness and survival in all life stages, as well as monitoring offspring "quality", are important to mass produced biological control agents which have a high potential for successful establishment once they are released in the field. Mass rearing methods for Galerucella calmariensis  and Galerucella pusilla , biological control agents of Lythrum salicaria .

Using Bees to Disseminate Trichoderma  to Strawberries
for the Control of Botrytis  Fruit Rot

J. Kovach
IPM Program
NYSAES, Cornell University
Geneva, NY 14456

Abstract: In 1994 and 1995, tests were conducted at the NYS Agricultural Experiment Station to determine if bumble bees and honey bees could successfully disseminate the spores of the fungal antagonist, Trichoderma harzianum, to strawberry flowers for Botrytis fruit rot control. Botrytis cinerea causes gray mold on a variety of fruits and infection usually begins in the flowers. Current IPM recommendations call for one to two fungicide applications at bloom to manage this pest.

T. harzianum strain 1295-22 is a Cornell patented fungal biological control agent that has efficacy against B. cinerea . This strain, and some formulations based on it, have been registered with the Environmental Protection Agency.

Results from this study showed that the bee/Trichoderma combinations and the Ronilan treatments significantly reduced the number of berries with gray mold. In 1995, check plots had 23% diseased berries while the Ronilan treatment had only 4%. The honey bee/Trichoderma treatment had 5% infected berries and the bumble bees/Trichoderma treatment had 7% diseased berries. Although a single bloom spray of Trichoderma alone reduced the number berries with Botrytis (12%) compared to the check, it was not nearly as good as the other treatments.

Not only were plant protection benefits achieved through the use of bees as disseminators, but yields were increased by the present of these pollinators. Fruit weight was 25-35% larger in bee visited treatments versus treatments where bees were excluded. This increase was primarily due to increased seed numbers per fruit because of better pollination.

New destruxins from a new source:
The entomopathogenic fungus Aschersonia  sp

S.B. Krasnoff*, D.M. Gibson*, G.N. Belofsky**, K.B. Gloer**, and J.B. Gloer**
*Plant Protection Research Unit
USDA,ARS
Ithaca, NY 14853
**Department of Chemistry
University of Iowa
Iowa City, IA 52245

Abstract: Two new insecticidal cyclic depsipeptides, destruxins A4 and A5, were isolated by bioassay-guided fractionation of organic extracts from an undescribed species of entomopathogenic fungus from the genus Aschersonia . Another previously identified but related compound, homodestruxin B, was also isolated as a product of this fungus. The chemical structures of 1-3 were determined based on analysis of NMR and mass spectral data. In common with homodestruxin B, the novel compounds destruxins A4 and A5 have a MeIle moiety in place of the MeVal residue more typically found in destruxins. In addition, destruxin A5 has a b-MePro unit in place of the Pro residue found in most destruxins. LC50 values for 1 and 2 in an insecticidal assay against Drosophila melanogaster  were estimated at 41 and 52 ppm, respectively. Homodestruxin B showed no activity at 400 ppm in the same assay. Destruxins A4 and A5 are the first biologically-active secondary metabolites reported from the fungal genus Aschersonia , which represents a new source of destruxins.