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United States Department of Agriculture

Agricultural Research Service

Research Project: ENHANCING PATHOGEN DETECTION AND CROP PROTECTION IN SUGARBEET USING MOLECULAR TECHNOLOGIES
2011 Annual Report


1a.Objectives (from AD-416)
Three of the most significant field diseases of sugarbeet in the U.S. are root rot, caused by Aphanomyces cochlioides; Rhizomania, caused by a fungal/viral complex; and wilt, caused by the sugarbeet cyst nematode, Heterodera schachtii, and concomitant infection by Fusarium fungi. The objectives of this project are to investigate methods to reproduce the field diseases of these pathogens in controlled environments, to develop qualitative and quantitative detection reagents and protocols for these organisms, and to determine genetic changes in viruses of the Rhizomania complex that condition heightened virulence to sugarbeet. Since the incorporation of natural genetic resistance into crops remains the most cost-effective strategy for disease control, an additional objective of the project is to obtain molecular genetic tags for disease resistance genes in sugarbeet in collaborative studies with ARS sugarbeet geneticists and pathologists.


1b.Approach (from AD-416)
Gradients of saturation across seedbeds will be tested as a means to evaluate sugarbeet varieties with known resistance to Aphanomyces cochlioides using stand loss as a measure of disease severity. Protocols for the inoculation of sugarbeet with Polymyxa betae will be modified to select for clonal isolates of the organism, an aspect lacking in past studies on this pathogen. Probe primers will be designed to perform in conjunction with specific primer sets in the development of real-time PCR (qPCR) methods for quantifying these pathogens in soil and plant samples. Disruptions (insertions) in the chromosomes of beet black scorch virus and beet necrotic yellow vein virus will be engineered in efforts to determine the role of virus genes in pathogen virulence. Plants typed for either resistance or susceptibility to the sugarbeet cyst nematode and Fusarium stalk blight will be subjected to DNA fingerprinting for generation of molecular markers linked to resistance genes.


3.Progress Report
Progress was made in all three Objectives and their Sub-objectives. The ability to reproduce sugarbeet diseases in controlled settings has important implications for understanding pathogen biology and disease resistance screening. In Sub-objective 1a, protocols were developed that allowed easy and reproducible disease development to occur with two important sugarbeet pathogens. Parameters were optimized for zoospore production in Aphanomyces cochlioides, an organism typically recalcitrant for production of high numbers of zoospores in vitro. Since A. cochlioides zoospores typically initiate disease in nature, the ability to mass-produce zoospores in vitro is critical for disease screening and other greenhouse or growth chamber experiments. In addition, procedures were optimized Rhizomania disease reproduction in the greenhouse. This relied on diluting field soils containing the disease complex with potting soil at certain ratios. Rhizomania is caused by beet necrotic yellow vein virus and molecular techniques to distinguish this virus from others are important for the specific detection of Rhizomania in diseased plants. In Sub-objective 1b, primers were developed on single nucleotide polymorphisms in the RNA3 region of the BNYVV genome that distinguish it from other viruses causing disease in sugarbeet. In order to gain an understanding of effectors used to cause Aphanomyces root rot disease, experiments were initiated to study secreted proteins from A. cochlioides. In subobjective 2a, proteins were harvested from A. cochlioides growing in vitro utilizing media that has been shown to initiate secretion of effectors from other sugarbeet pathogens rather than protein samples harvested from infected sugarbeet that contain complex protein samples from both organisms. In Sub-objective 2b, Cercospora beticola isolates harvested from more than 10 fields were assessed for mating type and fungicide sensitivity. It was found that fields with reduced sensitivity to fungicides were predominated by one mating type or the other, likely a result of fungicide selection pressure and subsequent asexual reproduction by the fungus. In Sub-objective 3a, apoplastic fluid was harvested from sugarbeet infected with C. beticola. Proteins were harvested that contain sugarbeet defense-related and pathogen virulence-related proteins.


4.Accomplishments
1. Disease management in sugarbeets. Evaluating resistance to fungicides is major focus for leaf spot disease management in sugarbeets. Utilizing fungicide sensitivity values obtained from over 3,500 isolates collected over four years, ARS scientists in Fargo, ND have shown that fungicide resistant isolates are becoming more common in commercial fields. In addition, no relationship between fungicide sensitivity and aggressiveness exists, increasing the chances that fungicide resistance will become even more common in the future if fungicides usage is not managed properly. The results of this study show that monitoring fungicide resistance is critical in order to prevent outbreaks of disease caused by fungicide-resistant isolates. This information will assist growers in selecting the optimum fungicide for efficient disease control thereby reducing production costs while also increasing yields.

2. Molecular characterization of CbCyp51, a gene encoding a fungicide target in Cercospora beticola. Characterizing genes encoding proteins that are targeted by fungicides gives critical information on how fungicide resistance develops in plant pathogens. ARS scientists in Fargo, ND cloned CbCyp51 from C. beticola and sequenced the gene from 20 isolates with varying levels of resistance to fungicides to show that no mutations in the gene relate to fungicide sensitivity. However, the gene was over-expression in resistant isolates. This information is useful for plant pathologists that wish to utilize molecular tools to monitor fungicide resistance in C. beticola.

3. Identification of Fusarium sp. novum, a new pathogen of sugarbeet. Identification of new and emerging diseases of sugarbeet is critical in order to develop resistant varieties before epidemics occur. ARS scientists in Fargo, ND used molecular tools to show that the new Fusarium species causing the novel disease termed Fusarium yellowing decline is distinct from all other Fusarium species. This information is useful for plant breeders for developing germplasm resistant to Fusarium yellowing decline and for plant pathologists to develop molecular probes for specific detection of this pathogen.


Review Publications
Gonzalez, M., Pujol, M., Metraux, J.P., Gonzalez-Garcia, V., Bolton, M.D., Borras-Hidalgo, O. 2011. Tobacco leaf spot and root rot caused by Rhizoctonia solani Kuhn. Molecular Plant Pathology. 12(3):209-216.

Dejonge, R., Bolton, M.D., Thomma, B.P.H.J. 2011. How filamentous pathogens co-opt plants; the ins and outs of fungal effectors. Current Opinion in Plant Biology. 14:400-406.

Last Modified: 11/27/2014
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