Location: Foreign Disease-weed Science Research2009 Annual Report
1a. Objectives (from AD-416)
Identify genomic and phenotypic elements to characterize emerg9ing and foreign fungal plant pathogens. Develop an understanding of the biology, genetics and epidemiology of emerging and foreign fungal plant pathogens. Screen germplasm for resistant sources to emerging and foreign fungal plant pathogens. Per PDRAM for Research on Emerging Fungal Plant Pathogens at Frederick, MD #R07 Objective 4: Develop rapid and reliable diagnostics for new and emerging foreign fungal plant pathogens that can be transferred to regulatory agencies and the private sector.
1b. Approach (from AD-416)
Utilize the Bio-Safety Level 3 Plant Pathogen Containment facilities to investigate and characterize virulence, genetic variability, epidemiology, host range, and survival of foreign and emerging fungal plant pathogens considered to be a threat to the U.S. Establish pathogen collections, compare exotic and endemic isolates for morphology, virulence, and genomics using a variety of scientific methods. Develop detection techniques using PCR and immunological methodologies. Investigate pathogen genomics and host plant resistance using classical and molecular approaches.
3. Progress Report
Chrysanthemum white rust: Conventional and real-time PCR protocols developed in the previous year for detection of Puccinia horiana in chrysanthemums prior to the onset of symptoms were optimized. Gladiolus rust: Research was completed on the effects of temperature and humidity on spore germination. Sorghum ergot: Real-time PCR protocols for the detection of Claviceps africana, C. sorghi, and C. sorghicola were optimized, and multiplex PCR experiments were initiated using several primer and probe sets. Wheat blast: Sixty-seven isolates of Magnaporthe grisea were acquired: 58 of the wheat pathotype, 5 of the rice pathotype, and 4 of the Kentucky bluegrass pathotype. Two hundred U.S. wheat cultivars were inoculated with M. grisea and rated for foliar disease resistance, while 100 U.S. winter wheat cultivars and 20 spring barley cultivars were tested for resistance to spike infection. Soybean rust: Eighteen simple sequence repeat (SSR) markers were used to generate populations lineages of 28 Phakopsora pachyrhizi isolates in the ARS collection at Ft. Detrick, MD, and each isolate was inoculated and evaluated on soybean accessions containing the 4 single rust resistance genes, Rpp1-4. A suppressive subtraction hybridization cDNA library was constructed from mRNA extracted from P. pachyrhizi appressoria formed on the surface of polysytrene plates. Approximately 1150 EST clones were sequenced, and compared to EST clones previously generated from P. pachyrhizi germinating spores. A total of 44 unique sequences were found and comparisons were made with all available nucleic acid and protein sequences in GenBank to determine potential function. We are developing diagnostic immunological reagents against two high titer proteins, PHEP107 and PHEP369 (for PHakopsora Extracellular Protein) found in the spore wall of P. pachyrhizi. Transmission electron microscopy immunolocalization studies confirmed that PHEP107 and PHEP369 are located in the spore and appressorial cell wall, and Western blots confirmed that they are present spore germination media as well. Six hybridoma cell lines were commercially produced that express monoclonal antibodies against recombinant forms of PHEP107 and PHEP369. One each of the monoclonal antibodies were found to be specific for PHEP 369 or PHEP 107, while two of the monoclonal antibodies were non-specific and reacted with both proteins Red leaf blotch: Replicated experiments were performed with soybean cultivar Williams 82 to investigate environmental conditions allowing infection by Phoma glycinicola. Approximately 2 week-old soybeans were inoculated with a suspension of ground mycelium of P. glycinicola and incubated in dew chambers for 4 days. Significantly greater numbers of lesions were obtained in a dew chamber at 20 degrees C rather than 24 degrees C; however, the percent lesion area did not differ significantly. The effects of incubation time on infection were evaluated, and the highest levels of disease both in terms of lesion numbers and lesion areas were obtained after 5 days incubation at 20 degrees C.
1. Temperature affects viability of gladiolus rust spores: Gladiolus rust is caused by the fungal pathogen, Uromyces transversalis, and is of plant quarantine importance in Europe and the U.S. In 2006, the pathogen was found in the U.S. for the first time in commercial nurseries in Florida and California. In response to requests from stakeholders, experiments were conducted to determine the effects of temperature and humidity on spore germination using controlled environmental conditions. While changes in relative humidity did not have any significant effect on spore germination, temperature did have a significant effect on spore germination. No spores germinated after 79 days at any of the temperature treatments, indicating the spores are no longer viable after this time. This information will be valuable to government, academic and private researchers in developing accurate predictive gladiolus rust disease models.
2. Soil solarization effective against karnal bunt spores: Tilletia indica is a soilborne fungal pathogen that can infect wheat at the time of flowering and cause the disease known as Karnal bunt. Unlike the other wheat bunt pathogens, which are generally considered only as grain-quality issues, T. indica-infested wheat is considered by many countries as a phytosanitary pest. We conducted a 3-year study in a wheat field in Maricopa, Arizona to evaluate the efficacy of soil solarization to eliminate viable karnal bunt teliospores in soil. Our results showed a rapid decline in teliospore viability occurred at all treatment depths over 38 days, with efficacy comparable with methyl bromide protocols using clear plastic sheeting. Under current United States Department of Agriculture disease management strategies, soil solarization may be useful for the rapid deregulation of Karnal bunt-affected fields.
3. U.S. and European winter wheat susceptible to Karnal bunt: Karnal bunt of wheat, caused by the fungus Tilletia indica, is disease of international regulatory concern. While the disease does not impact yield significantly, it can have a tremendous effect on exports due to phytosanitary regulations. Karnal bunt was first found in the U.S. in 1996 in Arizona, California and Texas. Historically, karnal bunt was reported only on autumn-sown, spring wheat; however, in 1997, karnal bunt was discovered on winter wheat grown under rain-fed field conditions in central Texas and 4 years later in northern Texas. To better understand host resistance and the potential for KB to spread further into winter wheat production areas, 50 commonly-grown U.S. and European winter wheat cultivars were inoculated with T. indica. Results showed that the majority of winter wheat cultivars tested were susceptible to Karnal bunt. This information will be valuable to government, academic and private researchers and breeders developing new winter wheat cultivars.
4. High temperatures reduce soybean rust spore production: Soybean rust caused by the fungal pathogen, Phakopsora pachyrhizi, is a serious foliar disease of soybeans and has been reported in most countries where soybeans are grown. Experiments were conducted in controlled growth chambers to determine effects of temperature on lesion formation and spore production after infection has taken place. The maximum number of lesions peaked at 25 degrees C and spore production per plant decreased as temperature increased. Analysis of daily temperatures in the U.S. revealed temperatures in the mid-western states were usually conducive for spore production during May through September, whereas temperatures in the southern states peaked above 33 degrees C in July and August, suggesting that high day temperature limits spore production in the southern U.S. This information will be valuable to government, academic and private researchers in developing accurate predictive soybean rust disease models.
5. Soybean rust mitochondrial genome sequenced: Soybean rust is an important foliar disease in most countries where soybeans are grown including the U.S. Two closely related species of fungi, Phakopsora pachyrhizi and P. meibomiae, can cause soybean rust. P. pachyrhizi is the more aggressive species, results in more yield loss, and has been referred to as Asian soybean rust. We have sequenced the mitochondrial genomes of both Phakopsora species and have identified genes that encode for 15 proteins, 24 transfer RNAs, and the small and large ribosomal subunits. Gene order comparisons and phylogenetic analysis were made with all available fungal mitochondrial genomes, and our analysis supports current fungal taxonomy. Sequence differences were observed between the two soybean rust pathogens that might be useful for diagnostics assays.
6. Soybean rust spore proteins identified: Phakopsora pachyrhizi, the causal agent of Asian soybean rust, has become established over an expanding range in the U.S., resulting in significant annual costs to soybean producers in the Southeast. It is important to identify and characterize proteins in P. pachyrhizi in order to understand the early events in the infection process and develop strategies to disrupt the pathogen life cycle. We have applied a proteomic approach to identify 117 proteins expressed in germinating soybean rust urediniospores from public and custom sequence databases. Proteins with roles in primary metabolism, energy transduction, stress, cellular regulation and signaling were identified in this study, representing future molecular targets for development of disease mitigation strategies.
7. Soybean rust resistance gene identified: Phakopsora pachyrhizi is an aggressive fungal pathogen of soybeans and is the causal agent of the disease known as Asian soybean rust. The pathogen occurs in all major soybean-producing countries including the U.S., and has caused major yield losses in severely affected areas, resulting in billions of dollars lost annually. To date, five single genes conferring resistance to Asian soybean rust have been identified through germplasm screening efforts. Through the combined use of gene mapping data and virus-induced gene silencing technology, we identified and cloned a candidate gene, Rpp4, which confers resistance against all known isolates of the pathogen. The cloning of Rpp4 and the development of markers linked to this gene will assist in breeding efforts for resistance to Asian soybean rust and other economically important soybean pathogens.
Anderson, S.J., Stone, C.L., Posada-Buitrago, M., Boore, J.L., Neelam, B.A., Stephens, R.M., Luster, D.G., Frederick, R.D., Pedley, K.F. 2008. Development of simple sequence repeat markers for the soybean rust fungus, Phakopsora pachyrhizi. Molecular Ecology Resources. 8:1310-1312.