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United States Department of Agriculture

Agricultural Research Service

Research Project: Cool Season Grain Legume Genetic Enhancement and Pathology

Location: Grain Legume Genetics Physiology Research

Title: Oxalate-minus mutants of Sclerotinia sclerotiorum via T-DNA insertion accumulate fumarate in culture and retain pathogenicity on plants

Authors
item Xu, Liangsheng -
item Xiang, M -
item White, D -
item Chen, Weidong

Submitted to: Fungal Genetics Reports
Publication Type: Abstract Only
Publication Acceptance Date: February 25, 2013
Publication Date: March 11, 2013
Citation: Xu, L., Xiang, M., White, D., Chen, W. 2013. Oxalate-minus mutants of Sclerotinia sclerotiorum via T-DNA insertion accumulate fumarate in culture and retain pathogenicity on plants. Fungal Genetics Reports. 60 (Suppl): 598.

Technical Abstract: Sclerotinia sclerotiorum is a ubiquitous necrotrophic pathogen capable of infecting over 400 plant species including many economically important crops. Oxalic acid production has been shown in numerous studies to be a pathogenicity factor for S. sclerotiorum through several mechanisms. During our random mutagenesis study of S. sclerotiorum using Agrobacterium-mediated transformation, we identified three mutants that had lost oxalate production. Southern hybridization blots showed the mutation was due to a single T-DNA insertion, and plasmid rescue and DNA sequencing confirmed that the T-DNA insertion site was located in the ORF of oxaloacetate acetylhydrolase (Ssoah, SS1G_08218) of S. sclerotiorum. The mutants did not change the color of a pH-indicating medium (PDA amended with 50 mg/L bromophenol blue). The pH values of 6-day PDB culture filtrates were 1.8-2.0 for the wild type and 2.8-3.1 for the mutants. No oxalic acid was detected using HPLC in culture filtrates or in the mycelium of the mutants, but another acid compound was accumulated in culture filtrates of the mutants and detected by HPLC, and the compound was identified as fumaric acid using LC-MS. The mutants showed reduced vegetative growth on PDA and produced sclerotia that are beige in color and soft in texture. Artificial acidic conditions (pH 3.4 and 4.2) enhanced vegetative growth and promoted normal (black and hard) sclerotial formation of the mutants. Furthermore, the oxalate-minus mutants retained pathogenicity on pea, green bean and faba bean in detached leaf assays and on intact plants of Arabidopsis thaliana, and their virulence levels were similar to that of the wild type strain on certain host plants, but varied depending on the plant species tested. The mutant had increased expression levels of cell wall-degrading enzymes such as polygalacturonases compared to the wild type strain during the process of infecting pea leaves. The results showed that a low pH condition is very important for growth and virulence of S. sclerotiorum on its wide range of host.

Last Modified: 8/29/2014
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