John D. Vandenberg

Agrobacterium-Mediated Disruption of a Nonribosomal Peptide Synthetase Gene in the Invertebrate Pathogen Metarhizium anisopliae Reveals a Peptide Spore Factor

ABSTRACT Numerous secondary metabolites have been isolated from the insect pathogenic fungus Metarhizium anisopliae, but the roles of these compounds as virulence factors in disease development are poorly understood. We targeted for disruption by Agrobacterium tumefaciens-mediated transformation a putative nonribosomal peptide synthetase (NPS) gene, MaNPS1. Four of six gene disruption mutants identified were examined further. Chemical analyses showed the presence of serinocyclins, cyclic heptapeptides, in the extracts of conidia of control strains, whereas the compounds were undetectable in ΔManps1 mutants treated identically or in other developmental stages, suggesting that MaNPS1 encodes a serinocyclin synthetase. Production of the cyclic depsipeptide destruxins, M. anisopliae metabolites also predicted to be synthesized by an NPS, was similar in ΔManps1 mutant and control strains, indicating that MaNPS1 does not contribute to destruxin biosynthesis. Surprisingly, a MaNPS1 fragment detected DNA polymorphisms that correlated with relative destruxin levels produced in vitro, and MaNPS1 was expressed concurrently with in vitro destruxin production. ΔManps1 mutants exhibited in vitro development and responses to external stresses comparable to control strains. No detectable differences in pathogenicity of the ΔManps1 mutants were observed in bioassays against beet armyworm and Colorado potato beetle in comparison to control strains. This is the first report of targeted disruption of a secondary metabolite gene in M. anisopliae, which revealed a novel cyclic peptide spore factor.

Exchange of Inoculum of Beauveria bassiana (Bals.) Vuill. (Hyphomycetes) Between Adult Flies of the Cabbage Maggot Delia radicum L. (Diptera: Anthomyiidae)

Adult cabbage maggots ( Delia radicum L.) were exposed to dry conidia of isolates of several hyphomycetous fungi by placing them in a centrifuge tube containing conidia, then releasing them into small screened plastic cages. Mortality was assessed after 48, 120 and 160 h. A Beauveria bassiana isolate (P89 from Musca domestica ) caused the highest mortality after 48 h, resulting in 100% mortality and 100% infection. Isolate L90 ( B. bassiana ) and one Metarhizium anisopliae isolate (ARSEF 2521) also caused fatal infection in more than 50% of the flies. To investigate exchange of inoculum, flies were placed in a small container with a dry powder formulation containing B. bassiana (Mycotrol ® ) on the bottom. The flies were removed to small screened cages containing untreated flies. This experiment confirmed the ability of flies to pass inoculum to other flies. In a similar experiment, one treated fly was placed in each cage with one untreated fly. When each fly died, one untreated fly was added to each cage after the dead fly was removed. This study showed that fly to fly transfer of fatal doses of inoculum was possible for a series of at least six flies. When female flies were exposed to the inoculum, then transferred to small cages containing males and an oviposition substrate, no eggs were laid. Further studies are being conducted to develop a system where flies attracted to a trap will be inoculated with the fungus and spread it to a field population.

Use of RAPD analysis to trace the origin of the weevil pathogen Zoophthora phytonomi in North America.

In North America, the fungal pathogen Zoophthora phytonomi has been known to cause significant levels of infection in introduced clover leaf weevil populations, Hypera punctata , since 1885. This pathogen was never noted in introduced populations of alfalfa weevil, H. postica , sympatric with clover leaf weevil until 1973 when it was found in alfalfa weevil in Ontario. From 1973 through 1981, Z. phytonomi was progressively found further south from Ontario. Whether these reports of Z. phytonomi infecting H. postica actually demonstrate spread by a novel genotype has previously been proposed and disputed. PCR-RAPD analysis was used to compare isolates of Z. phytonomi from both hosts in North America, and from H. postica in Israel with Z. radicans and Conidiobolus osmodes as outgroups. Both phenetic and cladistic analyses demonstrate that two main genotypes of Z. phytonomi occur in North America; one genotype including only H. punctata isolates with a second more homogeneous and principally including isolates from H. postica . The genotype principally including isolates from H. postica was more closely related to isolates from H. postica in Israel than to the other North American group, but also included one isolate from H. punctata . Based on affinity with Israeli genotypes, this latter strain may have originated in the Eurasian areas where H. postica is endemic. The degree of host specificity of these two North American genotypes of Z. phytonomi will require further investigation.

Biology of two members of the Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae), recently invasive in the U.S.A., reared on an ambrosia beetle artificial diet

Recent molecular studies have found that the ambrosia beetle Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is a complex of cryptic species, each carrying a different species of symbiotic fungus, in the genus Fusarium, which they farm within galleries inside woody hosts. Several of these beetle species have become invasive pests around the world for attacking and infecting healthy trees with their phytopathogenic fungal symbionts. Diet and rearing protocols were developed for two members of the E. fornicatus species complex, polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB), using sawdust from host trees, allowing collection of data on beetle biology, phenology and sex ratios. Adults developed within 22 days at 24 °C. Single PSHB or TSHB foundresses averaged 32.4 and 24.7 adult female offspring, respectively, and up to 57 and 68 female adults within 6–7 weeks. A strong predictor of the number of offspring in a colony was the number of entry holes. Average sex ratios (% male) for PSHB and TSHB, respectively, were 7.4% and 7.2%. Being haplodiploid, virgin PSHB foundresses were able to produce and mate with male offspring, then subsequently produce female offspring, confirming that they have arrhenotokous reproduction. A cold tolerance study found significant mortality rates among PSHB colonies exposed to −5° or −1 °C but not colonies exposed to 0°, 1° or 5 °C. Given Hamiltons local mate competition (LMC) theory, a number of LMC predictions were violated. PSHB sex ratios were not affected by the number of foundresses; approximately 14% of broods did not contain males; males did not usually eclose before females but eclosed around the same time (22–23 days); and PSHB males were found walking outside of their natal galleries on the trunk of a heavily infested tree in the field. Alternatives to LMC are considered, such as early forms of sociality (maternal care, cooperative brood care), local resource enhancement and kin selection.

Bioassay of the chalkbrood fungus Ascosphaera aggregata on larvae of the alfalfa leafcutting bee, Megachile rotundata

Abstract Laboratory-reared larvae of the alfalfa leafcutting bee, Megachile rotundata, were inoculated at various ages with a range of doses of the chalkbrood fungus Ascosphaera aggregata. The calculated LD50 values (range 2.08–3.23) increased and the slopes of the dose-response curves remained the same (range 1.17–1.25) with increasing age at inoculation. For all larvae the time to death decreased with increasing dose (range 4–16 days). Larvae inoculated at 5 days of age took longer to die (average 9.5 days) than larvae inoculated at 1 to 3 days of age (average 6.7 days). A higher percentage of cadavers sporulated (95%) at intermediate doses (approximately 2000 spores/bee) than at both lower and higher doses. For larvae inoculated at 1 to 3 days of age, the weight of sporulated cadavers (an index of the number of spores) decreased as dose increased. For larvae inoculated at 5 days of age, however, dose did not affect percent sporulation or the weight of sporulated cadavers. The size of adults that survived inoculation as larvae, at any dose and age, was not different from controls.

Susceptibility of two hymenopteran parasitoids of Agrilus planipennis (Coleoptera: Buprestidae) to the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales).

Emerald ash borer (EAB), Agrilus planipennis Fairmaire, native to Asia, is killing ash trees (Fraxinus spp.) across 15 states and southeastern Canada. Integrated pest management using biological control is the only viable long-term approach for controlling the spread of EAB outside of host resistance. Three hymenopteran parasitoids, Spathius agrili Yang, Tetrastichus planipennisi Yang, and Oobius agrili Zhang and Huang were discovered attacking EAB in China and were approved for release in the United States in 2007. The objective of this study was to assess susceptibility of the larval parasitoid species S. agrili and T. planipennisi, relative to that of EAB, to Beauveria bassiana, an entomopathogenic fungus that infects and kills EAB adults when sprayed on ash bark or foliage. Adult EAB and parasitoids were exposed to B. bassiana inoculated ash twigs for 2 h and then monitored daily for death and signs of infection for up to 10 days. All EAB adults exposed to B. bassiana were fatally infected while mean survival for control EAB was 77%. Average survival in the treatment groups for T. planipennisi and S. agrili were 99% and 83%, respectively, indicating these parasitoids are relatively unaffected by exposure to B. bassiana. This research elucidates interactions between a fungal pathogen and two parasitoids of EAB, and provides data necessary to developing a successful multi-stage integrated management approach to control of EAB.

Modeling Agrilus planipennis (Coleoptera: Buprestidae) Within-Tree Colonization Patterns and Development of a Subsampling Technique

ABSTRACT Emerald ash borer, Agrilus planipennis Fairmaire, an insect native to central Asia, was first detected in southeast Michigan in 2002, and has since killed millions of ash trees (Fraxinus spp.) in North America. The objectives of this study were to 1) determine an optimal sampling location on girdled ash trap trees for detection of A. planipennis larvae based on measurements of tree characteristics, and 2) develop a whole-tree estimation method for extrapolating larval densities from subsampled heights. We conducted sampling at 1-m increments, recording larval presence, height on tree bole, bolt diameter, and bark roughness for 58 infested ash trees. Analyzing height and diameter separately, generalized linear mixed models indicated the probability of A. planipennis detection was maximized at 17.2 cm for diameter and increased linearly as vertical height increased. There was also a positive relationship between intermediate bark roughness and A. planipennis presence. Stepwise regression indicated the optimal bolts for extrapolating whole tree larval densities were, in order of importance, at heights of 1–2 m, 4–5 m, 7–8 m, and 0–1 m. Subsampling with just one or two bolts explained 70% and 86%, respectively, of the variance in A. planipennis densities. Our results can be used by resource managers to improve efficiency of detection efforts and estimate infestations of A. planipennis.

Sexual dimorphism in Drosophila melanogaster survival of Beauveria bassiana infection depends on core immune signaling

In many animal species, females and males differ in physiology, lifespan, and immune function. The magnitude and direction of the sexual dimorphism in immune function varies greatly and the genetic and mechanistic bases for this dimorphism are often unknown. Here we show that Drosophila melanogaster females are more likely than males to die from infection with several strains of the fungal entomopathogen Beauveria bassiana. The sexual dimorphism is not exclusively due to barrier defenses and persists when flies are inoculated by injection as well as by surface exposure. Loss of function mutations of Toll pathway genes remove the dimorphism in survivorship. Surprisingly, loss of function mutation of relish, a gene in the Imd pathway, also removes the dimorphism, but the dimorphism persists in flies carrying other Imd pathway mutations. The robust sexual dimorphism in D. melanogaster survival to B. bassiana presents opportunities to further dissect its mechanistic details, with applications for biological control of insect vectors of human disease and insect crop pests.