Reactive Oxygen Species Play a Role in the Infection of the Necrotrophic Fungi, Rhizoctonia solani in Wheat

Rhizoctonia solani is a nectrotrophic fungal pathogen that causes billions of dollars of damage to agriculture worldwide and infects a broad host range including wheat, rice, potato and legumes. In this study we identify wheat genes that are differentially expressed in response to the R. solani isolate, AG8, using microarray technology. A significant number of wheat genes identified in this screen were involved in reactive oxygen species (ROS) production and redox regulation. Levels of ROS species were increased in wheat root tissue following R. solani infection as determined by Nitro Blue Tetrazolium (NBT), 3,3'-diaminobenzidine (DAB) and titanium sulphate measurements. Pathogen/ROS related genes from R. solani were also tested for expression patterns upon wheat infection. TmpL, a R. solani gene homologous to a gene associated with ROS regulation in Alternaria brassicicola, and OAH, a R. solani gene homologous to oxaloacetate acetylhydrolase which has been shown to produce oxalic acid in Sclerotinia sclerotiorum, were highly induced in R. solani when infecting wheat. We speculate that the interplay between the wheat and R. solani ROS generating proteins may be important for determining the outcome of the wheat/R. solani interaction

Biological detoxification of the mycotoxin deoxynivalenol and its use in genetically engineered crops and feed additives

Deoxynivalenol (DON) is the major mycotoxin produced by Fusarium fungi in grains. Food and feed contaminated with DON pose a health risk to humans and livestock. The risk can be reduced by enzymatic detoxification. Complete mineralization of DON by microbial cultures has rarely been observed and the activities turned out to be unstable. The detoxification of DON by reactions targeting its epoxide group or hydroxyl on carbon 3 is more feasible. Microbial strains that de-epoxidize DON under anaerobic conditions have been isolated from animal digestive system. Feed additives claimed to de-epoxidize trichothecenes enzymatically are on the market but their efficacy has been disputed. A new detoxification pathway leading to 3-oxo-DON and 3-epi-DON was discovered in taxonomically unrelated soil bacteria from three continents; the enzymes involved remain to be identified. Arabidopsis, tobacco, wheat, barley, and rice were engineered to acetylate DON on carbon 3. In wheat expressing DON acetylation activity, the increase in resistance against Fusarium head blight was only moderate. The Tri101 gene from Fusarium sporotrichioides was used; Fusarium graminearum enzyme which possesses higher activity towards DON would presumably be a better choice. Glycosylation of trichothecenes occurs in plants, contributing to the resistance of wheat to F. graminearum infection. Marker-assisted selection based on the trichothecene-3-O-glucosyltransferase gene can be used in breeding for resistance. Fungal acetyltransferases and plant glucosyltransferases targeting carbon 3 of trichothecenes remain promising candidates for engineering resistance against Fusarium head blight. Bacterial enzymes catalyzing oxidation, epimerization, and less likely de-epoxidation of DON may extend this list in future

Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction

Rhizoctonia solani and R. oryzae are the principal causal agents of Rhizoctonia root rot in dryland cereal production systems of the Pacific Northwest. To facilitate the identification and quantification of these pathogens in agricultural samples, we developed SYBR Green I-based real-time quantitative-polymerase chain reaction (Q-PCR) assays specific to internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA of R. solani and R. oryzae. The assays were diagnostic for R. solani AG-2-1, AG-8, and AG-10, three genotypes of R. oryzae, and an AG-I-like binucleate Rhizoctonia species. Quantification was reproducible at or below a cycle threshold (Ct) of 33, or 2 to 10 fg of mycelial DNA from cultured fungi, 200 to 500 fg of pathogen DNA from root extracts, and 20 to 50 fg of pathogen DNA from soil extracts. However, pathogen DNA could be specifically detected in all types of extracts at about 100-fold below the quantification levels. Soils from Ritzville, WA, showing acute Rhizoctonia bare patch harbored 9.4 to 780 pg of R. solani AG-8 DNA per gram of soil.. Blastn, primer-template duplex stability, and phylogenetic analyses predicted that the Q-PCR assays will be diagnostic for isolates from Australia, Israel, Japan, and other countries

First Report of Damping-Off of Canola Caused by Rhizoctonia solani AG 2-1 in Washington State

In early September 2003, winter canola (Brassica napus L) cv. Inca was direct seeded into plots previously cropped with spring barley at the Washington State University Dryland Research Station at Lind, WA. Before planting, the plots received 80 mm of water by sprinkler irrigation, and 2 weeks later, volunteer barley was killed with Paraquat contact herbicide. In late September, 3 weeks after planting, canola seedlings exhibited postemergence damping-off and lesions on the hypocotyls, resulting in significant stand reductions. Rhizoctonia solani was isolated from infected hypocotyls using water agar amended with chloramphenicol (100 μg/ml). Cultures on potato dextrose agar produced dark brown colonies with dark brown microsclerotia. Three isolates were grown on autoclaved oat seed for 3 weeks in 1-liter Erlenmeyer flasks at 22°C, and colonized seed was air dried in a laminar flow hood, ground in a coffee grinder, and added to a Thatuna silt loam at 1% (w/w). The infested soil was placed into 4- × 20.5-cm plastic tubes and planted with five canola seeds per tube, five tubes per isolate. In the control treatment, soil was not infested. Plants were grown in a temperature-controlled room in a greenhouse at 16°C, 12-h light/dark. Isolates caused pre- and postemergence damping-off after 1 week, and the surviving seedlings had significantly less plant height and dry weight. Isolates were identified as AG 2-1 by pairing cultures with AG 8, 2-1, and 10 on agar-coated slides (1). Selected isolates were also identified as AG 2-1 by sequencing of the ITS 1 and 2 regions of the rDNA and matching them to sequences in GenBank. On a farm north of Pullman, WA in 2004, R. solani was isolated from soil in spring and winter wheat fields using a toothpick baiting method (2). R. solani was found primarily from sites previously cropped with winter and spring canola. These isolates were identified as AG 2-1 and five isolates were tested in the greenhouse, as described above, on canola (cv. Inca), lentil (Lens culinaris Medik. cv. Merrit), wheat (Triticum aestivum L. cv. Madsen), barley (Hordeum vulgare L. cv. Baronesse), pea (Pisum sativum L. cv. Stirling), and chickpea (Cicer arietinum L. cv. Sierra). Three of five isolates significantly reduced emergence of canola, and all isolates significantly reduced dry weight of canola seedlings and caused lesions on hypocotyls. None of the isolates reduced emergence of the other crops. All isolates reduced the dry weight of pea and three isolates reduced plant height. None of the isolates reduced the dry weight of lentil, chickpea, wheat, or barley. One of the isolates was also tested on Arabidopsis thaliana and found to be pathogenic. R. solani AG 2-1 has been reported as an important pathogen on canola in Canada and Australia, but has not been reported from the Pacific Northwest of the United States. R. solani AG 2-1 is also pathogenic on rapeseed, mustard, and subterranean clover and has been isolated from wheat, sugar beets, and potato (3). Canola is a minor rotation crop in cereal-based cropping systems in eastern Washington (1,600 ha in 2005), but there is increasing interest in this oilseed crop for biodiesel production. However, R. solani AG 2-1 may reduce stands and yield of canola. References: (1) W. C. Kronland and M. E. Stanghellini. Phytopathology 78:820, 1988. (2) T. C. Paulitz and K. L. Schroeder. Plant Dis.89:767, 2005. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society. St. Paul, MN, 1991

Symbiotic root nodules of the actinorhizal plant Datisca glomerata express Rubisco activase mRNA

N2-fixing symbiotic root nodules of the actinorhizal host Datisca glomerata express Dgrca (D. glomerata Rubisco activase) mRNA, a transcript usually associated with photosynthetic organs or tissues. In northern blots a mature, 1700-nucleotide Dgrca mRNA was detected in green plant organs (leaves, flowers, and developing fruits) and in nodules but was not detected in roots. A second message of 3000 nucleotides was observed only in nodules. Both size classes of transcripts were polyadenylated. The larger transcript was 2- to 5-fold more abundant than the mature mRNA; it was hybridized to an intronic probe, indicating that a stable, incompletely spliced transcript was accumulating. Treatment with light on excised nodules did not alter the relative abundance of the two species. In in situ hybridizations the Dgrca message was expressed intensely in the nuclei of infected cells. The Dgrca transcripts also accumulated at lower levels in uninfected cortical cells adjacent to the periderm and the vascular cylinder. mRNA encoding the large subunit of Rubisco (DgrbcL) was abundant in mature infected cells and in the amyloplast-rich sheath of uninfected cortical cells lying between the infected cells and nodule periderm. The proteins Rubisco activase, Rubisco, and the 33-kD O2-evolving complex subunit did not accumulate to detectable levels, indicating that a functional photosynthetic apparatus was not prevalent in nodule tissue. Signals or factors required for the transcription of Dgrca appeared to be present in nodules, but efficient splicing and translation of the message were not observed in Frankia-infected tissue where transcript accumulation was highest

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction

Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp