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United States Department of Agriculture

Agricultural Research Service

Research Project: DEVELOPMENT AND APPLICATION OF MOLECULAR PHYLOGENETICS OF FUNGI TO ENHANCE FOOD SAFETY AND FOOD SECURITY
2011 Annual Report


1a.Objectives (from AD-416)
Establish a molecular evolutionary framework for understanding the genetic diversity, pathogenicity and mycotoxin potential of fusarial pathogens that cause Fusarium head blight (FHB) of wheat and barley and sudden death syndrome (SDS) of soybeans. Determine from multilocus sequence analysis the genetic diversity of Aspergillus and Pencillium species that produce ochratoxin-A, and develop rapid detection methods for these fungi using oligonucleotide probes. Develop molecular genetic methods, based on analysis of multilocus gene sequences, for rapid detection and identification of yeasts responsible for food and beverage spoilage as well as for biocontrol yeasts that are applied to fruit, vegetables and grains to ensure species safety and to determine the fate of these species in the environment.


1b.Approach (from AD-416)
Multiple diagnostic gene sequences will be developed for phytopathogenic and toxigenic species for molds in the genera Aspergillus, Fusarium, Penicillium, and their relatives, and for food spoilage and biocontrol yeasts. The gene sequences will be used to develop population- and species- specific molecular probes for rapid detection and for understanding the genetic diversity and relationships of each group of pathogens, food spoilage, and biocontrol yeasts.


3.Progress Report
The primary goal of this research was to develop rapid, accurate methods utilizing gene sequences for detection and identification of yeasts that cause food spoilage or are used for biocontrol of fruit rotting molds, and for filamentous fungi that produce toxic compounds and/or cause plant diseases. This research had two approaches. First, genetically define the lineages (species or subspecies) of the spoilage organisms and pathogens. Following this, gene-based systems were used to place the species in genetically defined groups such as genera. Using these data, rapid DNA-based systems were developed that identify species and groups of species. Correct identification is essential for solving problems in food spoilage, plant disease, and formation of toxins by fungi. Progress toward accomplishing these objectives included the following: .
1)Multigene analysis was completed for the ascomycetous yeasts resulting in redefinition of present genera and description of nearly 20 new genera. Included in the 100 genera now recognized are many groups that cause food spoilage. Multigene analysis was applied to 400 strains assigned to the genus Metschnikowia to recognize species in this genus that are used for biocontrol of fruit rotting molds and to correlate biocontrol activity with genetically defined species; .
2)A multigene-based phylogeny of the genus Aspergillus, which includes Ochratoxin-A (OTA) producing species, has been completed and published. Species of Aspergillus and Penicillium produce toxins in grains and other food products, which can severely affect human and animal health. The databases generated in the phylogenetic study have been used to develop DNA-based probes that accurately identify the species that produce OTA contamination in foods and commodities. OTA is a regulated toxin in the European Union and some other parts of the world and its presence in U.S. produced commodities can affect exports; .
3)We constructed a gene sequence database for 850 isolates spanning the phylogenetic breadth of the Fusarium oxysporum species complex (FOSC). Members of the FOSC are ubiquitous soil borne pathogens responsible for vascular wilts, rots, and damping-off diseases of over 80 agronomically and horticulturally important crops. In addition, some species of Fusarium produce potent toxins. This database represents a unique resource for typing plant host-specific pathogens and opportunistic pathogens of humans. Of the 850 isolates compared, 562 strains were genetically differentiated using two genes. This analysis also revealed that Fusarium isolates associated with human mycoses are genetically diverse. Experiments were also conducted to assess the potential of genetically diverse members of the FOSC to produce the mycotoxins moniliformin, fumonisin, and enniatin. The purpose of this work was to develop gene probes for rapid detection of Fusarium plant pathogens, but because some Fusarium species are human pathogens, the gene sequence database developed has immediate use in the diagnosis of human disease.


4.Accomplishments
1. Phylogenetic recognition of Fusarium plant pathogens by gene typing. Novel toxigenic Fusarium pathogens have been discovered on cereal crops in Russia. Fusarium pathogens and their toxins pose a significant threat to the production of cereals worldwide. Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens & Mycology Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, collaborated with scientists in Russia, Finland, and Japan, and discovered and characterized a novel Fusarium head blight pathogen on wheat, barley, and oats in Russia. The pathogen contaminates these cereals with a highly toxic compound called T-2 toxin. A molecular diagnostic assay was developed that distinguishes the novel pathogen named Fusarium sibericum so that its movement can be monitored. Because T-2 toxin is highly toxic to humans, active surveillance of this pathogen is essential to minimize human exposure.

2. Identification of Ochratoxin A producing fungi from gene sequences. The toxin Ochratoxin A (OTA) is often detected in foods and beverages and results from growth in these products of species of the fungi Aspergillus and Penicillium. Proper identification of the fungi is critical to preventing this toxin from entering the food supply. Because these fungi are so similar in appearance, accurate identification is only possible from examination of the gene sequences. Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens & Mycology Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, investigated the DNA sequences from numerous fungal isolates and developed a database that can now be used to accurately identify toxin producing species as well as look-alike non-toxin producing species. This work will allow food producers to identify fungal contamination and modify processing to prevent toxin production.

3. Molecular genetic diversity of biocontrol yeasts. Species of the yeast genus Metschnikowia are used internationally to control mold-induced rots of fruits, however these species are difficult to differentiate. To provide accurate identification, Agricultural Research Service (ARS) scientists in the Bacterial Foodborne Pathogens & Mycology Research Unit at the National Center for Agricultural Utilization Research in Peoria, IL, developed a diagnostic database of DNA sequences from four genes and tested the database on 400 strains that include all known yeast species. From this database, diagnostic probes can be developed to identify the species used by producers for biocontrol of fruit rots and to follow survival and dispersal of the biocontrol species in the field. This work will facilitate application of biocontrol technology to prevent fruit storage rots.


Review Publications
Kurtzman, C.P. 2011. Kregervanrija Kurtzman (2006). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 497-501.

Kurtzman, C.P. 2011. Citeromyces Santa Maria (1957). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 345-348.

Kurtzman, C.P. 2011. Pachysolen Boidin & Adzet (1957). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 673-675.

Kurtzman, C.P. 2011. Pichia E.C. Hansen (1904). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 685-707.

Kurtzman, C.P. 2011. Barnettozyma Kurtzman, Robnett & Basehoar-Powers (2008). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 333-339.

Kurtzman, C.P. 2011. Lindnera Kurtzman, Robnett & Basehoar-Powers (2008). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 521-543.

Kurtzman, C.P. 2011. Wickerhamomyces Kurtzman, Robnett & Basehoar-Powers (2008). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 899-917.

Kurtzman, C.P. 2011. Yarrowia van der Walt & von Arx (1980). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 927-929.

Kurtzman, C.P. 2011. Protomyces Unger (1833). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 725-731.

Kurtzman, C.P. 2011. Meyerozyma Kurtzman & M. Suzuki (2010). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 621-624.

Kurtzman, C.P. 2011. Nakazawaea Y. Yamada, Maeda & Mikata (1994). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 637-639.

Kurtzman, C.P. 2011. Komagataella Y. Yamada, Matsuda, Maeda & Mikata (1995). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 491-495.

Vaughan-Martini, A., Lachance, M.-A., Kurtzman, C.P. 2011. Kazachstania Zubkova (1971). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 439-470.

Kurtzman, C.P. 2011. Torulaspora Lindner (1904). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 867-874.

Kurtzman, C.P. 2011. Ogataea Y. Yamada, K. Maeda & Mikata (1994). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 645-671.

Lachance, M.-A., Kurtzman, C.P. 2011. Lachancea Kurtzman (2003). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 511-519.

Kurtzman, C.P., Smith, M.Th. 2011. Saccharomycopsis Schionning (1903). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 751-763.

Kurtzman, C.P., Fell, J.W., Boekhout, T. 2011. Gene sequence analyses and other DNA-based methods for yeast species recognition. In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 1, 5th edition. New York, NY: Elsevier. p. 137-144.

Kurtzman, C.P., Fell, J.W., Boekhout, T., Robert, V. 2011. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 1, 5th edition. New York, NY: Elsevier. p. 87-110.

Kurtzman, C.P. 2011. Discussion of teleomorphic and anamorphic Ascomycetous yeasts and yeast-like taxa. In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 293-307.

Otero-Colina, G., Rodriguez-Alvarado, G., Fernandez-Pavia, S., Maymon, M., Ploetz, R.C., Aoki, T., O Donnell, K., Freeman, S. 2010. Identification and characterization of a novel etilogical agent of mango malformation disease in Mexico, Fusarium mexicanum sp. nov. Phytopathology. 100(11):1176-1184.

O Donnell, K., Sutton, D.A., Rinaldi, M.G., Sarver, B.J., Balajee, S., Schroers, H., Summerbell, R.C., Robert, V.G., Crous, P.W., Zhang, N., Aoki, T., Jung, K., Park, J., Lee, Y., Kang, S., Park, B., Geiser, D.M. 2010. An internet-accessible DNA sequence database for identifying Fusaria from human and animal infections. Journal of Clinical Microbiology. 48(10):3708-3718.

Yli-Mattila, T., Ward, T.J., O Donnell, K., Proctor, R., Burkin, A., Kononenko, G., Gavrilova, O., Aoki, T., McCormick, S.P., Gagkaeva, T. 2011. Fusarium sibiricum sp. nov, a novel type A trichothecene-producing Fusarium from northern Asia closely related to F. sporotrichioides and F. langsethiae. International Journal of Food Microbiology. 147(1):58-68.

Edupuganti, S., Rouphael, N., Mehta, A., Eaton, M., Heller, J., Bressler, A., Brandt, M., O Donnell, K. 2011. Fusarium falciforme vertebral abscess and osteomyelitis: case report and molecular classification. Journal of Clinical Microbiology. 49(6):2350-2353.

Scandiani, M.M., Aoki, T., Luque, A.G., Carmona, M.A., O Donnell, K. 2010. First report of sexual reproduction by the soybean SDS pathogen Fusarium tucumaniae in nature. Plant Disease. 94(12):1411-1416.

Hemashettar, B.M., Patil, R.N., O Donnell, K., Padhye, A.A. 2011. Chronic rhino-orbital mucormycosis caused by Mucor irregularis (Rhizomucor variabilis) in India. Journal of Clinical Microbiology. 49(6):2372-2375.

Kurtzman, C.P. 2011. Recognition of yeast species from gene sequence comparisons. The Open Applied Informatics Journal. 5:20-29.

Kurtzman, C.P. 2011. Eremothecium Borzi emend. Kurtzman. De Hoog, G.S., Miller, M.W. In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 405-412.

Kurtzman, C.P. 2011. Phylogeny of the ascomycetous yeasts and the renaming of Pichia anomala to Wickerhamomyces anomalus. Antonie Van Leeuwenhoek. 99(1):13-23.

Smith, J.A., O Donnell, K., Mount, L.L., Shin, K., Peacock, K., Trulock, A., Spector, T., Determann, R. 2011. A novel Fusarium species causes a canker disease of the critically endangered conifer, Torreya taxifolia. Plant Disease. 95(6):633-639.

Vaughan-Martini, A., Lachance, M., Kurtzman, C.P. 2011. Naumovozyma Kurtzman (2008). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 641-643.

Lachance, M., Kurtzman, C.P. 2011. Wickerhamiella van der Walt (1973). In: Kurtzman, C.P., Fell, J.W., Boekhout, T., editors. The Yeasts, a Taxonomic Study. Volume 2, 5th edition. New York, NY: Elsevier. p. 891-897.

Rich, J.O., Manitchotpisit, P., Peterson, S.W., Leathers, T.D. 2011. Laccase production by diverse phylogenetic clades of Aureobasidium pullulans. Rangsit Journal of Arts and Sciences. 1(1):41-47.

Last Modified: 12/18/2014
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