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United States Department of Agriculture

Agricultural Research Service

Research Project: SYSTEMATICS OF BIOLOGICAL CONTROL MICROFUNGI FOR MANAGEMENT OF PLANT DISEASES AND INSECT PESTS

Location: Systematic Mycology and Microbiology

2013 Annual Report


1a.Objectives (from AD-416):
1. Develop and validate molecular identification and genotyping resources for insect biocontrol fungi Beauveria spp. and Metarhizium. 2. Distinguish fungal pathogens from species with potential for the biological protection of plants through molecular systematics investigations of complex coelomycete genera, including Colletotrichum and Pestalotiopsis.


1b.Approach (from AD-416):
Genomic sequences will be mined with specialized software to develop novel molecular identification and genotyping methods for the insect pathogens Beauveria and Metarhizium. Marker data obtained from strains collected by the scientists, collaborators or acquired from culture collections will form the basis for systematic and taxonomic revisions and the creation of on-line molecular and morphological identification resources. A molecular epidemiological analysis of Beauveria pathogens of coffee berry borer, an important insect pest of coffee, will be conducted in coffee farms in Puerto Rico. Molecular methods for species identification will be critically tested. Population genetic analyses combining microsatellite, mating type and SNP data will be used to analyze the genetic structure of epizootics against the environmental diversity of indigenous Beauveria. In addition, the efficacy and persistence and geographic spread of GHA, the Beauveria mycoinsecticide Mycotrol® strain that has been released for control of coffee berry borer, will be assessed. For the project on pathogenic and endophytic strains of Colletotrichum and Pestalotiopsis, strains will be obtained from a network of cooperators in the Neotropics. Initial diversity screens of Colletotrichum strains will be conducted using markers APN2 and APN2/MATigs; diversity screens for Pestalotiopsis will utilize variable intron regions of elongation factor-1 alpha (EF-1a) and beta tubulin (B-tub). Strains selected from the diversity screens will be used for multi-locus phylogenetic analyses (B-tub, EF-1a, RPB1, RPB2 and other loci) to delineate species boundaries, infer species relationships, and determine whether endophytic and pathogenic ecologies are phylogenetically associated.


3.Progress Report:
Progress was made on both objectives, which fall under Problem Statement 1, Diagnostics, Etiology and Systematics of Plant Disease and use molecular and morphological approaches to characterize fungi with potential for biological control using both molecular sequence data and microscopy.

Under Objective 1, progress was made in developing improved methods for identifying species of insect pathogenic fungi. These methods are needed to conduct epidemiological studies of these organisms and to track the field behavior of strains released for insect control applications. A pipeline has been developed for the comparative analysis of genome sequences of the insect control fungi Beauveria and Metarhizium.

Under Objective 2, progress was made developing eight nuclear DNA markers for use in species discovery and diagnostics. These markers will be used to resolve the phylogeny of endophytic and pathogenic fungi in the complex genera Colletotrichum and Pestalotiopsis, the most frequently isolated pathogens and endophytes of cacao and other tropical tree crops. Due to sequestration, the scientist vacancy to be hired to conduct this research was cancelled.


4.Accomplishments
1. An inexpensive technique developed for finding insect-killer fungi. Fungi can be used to control insects, however, it is difficult to determine precisely which fungal isolate will be the most effective at destroying insects. Also, it is necessary to determine if the fungus is really working or if some other factor has killed the insects. The most accurate method of identifying a fungal isolate is using DNA sequencing, however, this is expensive and time-consuming especially when screening hundreds or thousands of isolates. In this study, we used a low-cost, rapid method to separate 42 different groups of an bug-killing fungus. Some groups of fungi differed only by a single base pair. This method is inexpensive yet does a good job of sorting the isolates into groups. Using this approach scientists can find the very best single isolate of a fungus to use for killing insects.

2. Anthracnose fungi cause fruit and stem diseases of cranberry in North America but until now it was not known which fungi occur on cranberry and whether or not they occur on nearby plants. To understand the kinds of disease-causing fungi and the plants they infect, anthracnose fungi were studied from cranberry plants and associated plants. Seven different kinds of anthracnose fungi were found to be associated with cranberry, three of which were previously unknown to science. Three of these new species cause fruit and stem disease in cranberry of which two also infect different kinds of plants growing near the cranberry bogs. Knowing about these disease-causing fungi is essential for plant breeders and plant pathologists who are working to grow healthy cranberry, one of North America’s few native crops.


Review Publications
Cai, Y., Chang, P., Rehner, S.A., Huang, B. 2013. Discrimination of Chinese Beauveria strains by DGGE genotyping and taxonomic identification by sequence analysis of the Bloc nuclear intergenic region. Applied Entomology and Zoology. DOI: 10.1007/s13355-013-0179-1.

Doyle, V.P., Oudemans, P.V., Rehner, S.A., Litt, A. 2013. Habitat and host indicate lineage identity in Colletotrichum gloeosporioides s.l. from wild and agricultural landscapes in North America. PLoS Pathogens. DOI: 10.1371/journal.pone.0062394.

Du, X., Zhao, Q., Yang, Z.L., Hansen, K., Taskin, H., Buyukalaca, S., Dewsbury, D., Moncalvo, J., Douhan, G.Q., Robert, V.A., Crous, P.W., Rehner, S.A., Rooney, A.P., Sink, S.L., O'Donnell, K. 2012. How well do ITS rDNA sequences differentiate species of true morels (Morchella)? Mycologia. 104(6):1351-1368.

O'Donnell, K., Rooney, A.P., Proctor, R., Brown, D.W., McCormick, S.P., Ward, T.J., Frandsen, R.N., Lysoe, E., Rehner, S.A., Aoki, T., et al. 2013. Phylogenetic analyses of RPB1 and RPB2 support a middle Cretaceous origin for a clade comprising all agriculturally and medically important fusaria. Fungal Genetics and Biology. 52:20-31.

Kepler, R.M., Rehner, S.A. 2013. Genome-assisted development of nuclear intergenic sequence markers for entomopathogenic fungi of the Metarhizium anisopliae species complex. Molecular Ecology Resources. 13:210-217.

Toth, K., Van Gool, M., Schols, H., Samuels, G., Gruppen, H., Szakacs, G. 2013. Diversity in production of xyaln-degrading enzymes among species belonging to the Trichoderma section Longibrachiatum. BioEnergy Research. 6(2):631-643.

Last Modified: 9/22/2014
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