2007 Annual Report 1a.Objectives (from AD-416) The long-term objective of this project is to reduce the impact of diseases on the productivity of the domestic sugarcane (Saccharum hybrids) industry by providing assistance to sugarcane breeders in identifying parental clones with resistance, improving the efficiency of selection for disease resistance traits, and providing the industry the information needed to guard against more virulent strain shifts and/or new pathogens. Our primary objectives will be to identify and develop germplasm with resistance to the major diseases affecting sugarcane, to identify the genetic variability among endemic pathogen populations, and to monitor the Louisiana sugarcane industry for the emergence of new pathogens. Disease resistant germplasm will be sought from among different taxa of Saccharum and related genera. Molecular markers that are linked to genes for disease resistance will be identified. Molecular approaches will be used to enhance the studies of host and pathogen genetics. 1b.Approach (from AD-416) To identify and develop germplasm with resistance to the major diseases affecting sugarcane in the United States, highly domesticated and wild clones of sugarcane and near relatives will be evaluated for resistance to the major sugarcane diseases following either natural and infections or artificial inoculation. To identify molecular markers that are linked to genes for disease resistance, polymerase chain reaction (PCR) based methods such as AFLP, SSR, or TRAP will be used to identify genetic markers closely linked to the resistance genes. Priority will be given to finding markers for smut, then ratoon stunting disease (RSD) and mosaic. Genotypic and phenotypic expressions of variability within populations of pathogens will be used to identify the genetic variability among pathogen populations and determine the distribution of races, strains, or other biotypes. The domestic sugarcane industry will be monitored for the introduction of exotic pathogens. 3.Progress Report • Project receives support from the American Sugar Cane League (6435-21000-012-04T) through a Trust Funded Cooperative Agreement as well as the in-house project, "Improving Sugarcane Production Efficiency", part of the in-house project 6435-21000-012-00D, "Genetic Improvement of Sugarcane by Conventional and Molecular Approaches." Additional details of research can be found in the reports of the subordinate and parent projects. • Greenhouse trials were initiated to test a collection of 86 sugarcane varieties and progeny of crosses using the wild sugarcane varieties that are being used in the SRU breeding program for the major diseases affecting sugarcane [mosaic, smut, leaf scald, and ratoon stunting disease (RSD)]. Inoculations for RSD and mosaic will be evaluated in early FY 2008. Inoculations and evaluations for leaf scald and smut will be made in FY 2008 on plants established in FY 2007. • A total of 132 candidate varieties that are being tested for possible release as commercial varieties in the next five years were screened through artificial inoculation for smut, leaf scald, and RSD. In other ARS breeding trials and nurseries, candidate varieties were observed for natural infection by pathogens that cause mosaic, rust, smut and leaf scald diseases. Pathology recommendations were made at variety advancement and variety release meetings. • A population of progeny from self pollination of variety LCP 85-384 was genotyped using simple satellite repeat (SSR) DNA markers to verify that they indeed were the result of fertilization between the male and female organs of the same flower. The progeny were multiplied and screened in greenhouse and field tests for susceptibility to RSD, mosaic, smut, and rust. Only one clone out of 286 was susceptible for mosaic, and only two were susceptible to smut. Susceptibility to RSD ranged from resistance to highly susceptible. The 25 most susceptible and the 25 most resistant were identified for future genetic analysis. • Nucleic acid sequence was determined for three isolates of SrMV that did not match the description of known SrMV strains. Future studies of the genetics of the viruses and their host range will be used to determine if they should be designated as a new strain of SrMV. Total nucleic acid extractions from plants with mosaic symptoms, but were negative for SrMV and SCMV, were sent to Dr. Ruhui Li, Plant Pathologist (National Germplasm Resources, Beltsville, MD), to test for possible presence of Sugarcane streak mosaic virus (SCSMV). This virus has only been reported in the Eastern Hemisphere, but produces symptoms very similar to those produced by SCMV and SrMV. The test was negative for SCSMV. 4.Accomplishments RANKING SUGARCANE VARIETIES FOR RESISTANCE TO RATOON STUNTING DISEASE (RSD) WITH QUANTITATIVE REAL-TIME PCR. Practical methods of ranking sugarcane varieties for resistance to RSD, primarily those based on immunoassays, have required the use of stalk tissue from plants older than 6 months where concentrations of the disease-causing bacteria are high enough to be detected by these methods. Differences in bacterial concentrations among varieties are used to rank them for susceptibility to the disease. A quantitative real-time polymerase chain reaction (PCR) protocol, that has the increased sensitivity to detect and quantify the lower levels of bacteria found in leaf or young stalk tissues at any growth stage, was developed to rank varieties of sugarcane for susceptibility to RSD. The real-time PCR protocol offers the flexibility to perform evaluations throughout the growing season and to evaluate greenhouse grown plants where it is not practical to grow them to maturity. This accomplishment addresses National Program 303 Component III, Plant Disease Resistance; Problem Statement, Disease resistance in new germplasm and varieties. GENETIC VARIABILITY AMONG VIRUSES CAUSING SUGARCANE MOSAIC. Strains H, I, and M of sorghum mosaic virus (SrMV) have been responsible for causing sugarcane mosaic disease in Louisiana since the 1960s. In surveys of mosaic affected plants conducted in 2006 and 2007 using molecular techniques, strain I continues to be the most commonly recovered strain from diseased plants, with strain H the next most commonly recovered, and strain M recovered occasionally. Approximately 20% of the virus isolates do not match the description of any known strain. Differences in the nucleic acid sequence of these isolates show that they differ genetically and represent new strains of SrMV. The discovery of the presence of new strains of SrMV indicates the virus has the potential to adapt to resistant sugarcane varieties and germplasm. This accomplishment addresses National Program 303 Component I, Disease Diagnosis: Detection, Identification and Characterization of Plant Pathogens; Problem Statement, Detection, Identification, Characterization, and Classification of Pathogens. EFFECT OF SUGARCANE YELLOW LEAF VIRUS (SCYLV) ON SUGARCANE. SCYLV infects all currently recommended sugarcane varieties in Louisiana. Yield loss associated with infection by SCYLV, the cause of sugarcane yellow leaf disease (SCYLD), varied among four Louisiana sugarcane varieties and among the crops of the varieties; however, infection by the virus consistently resulted in changes in the quality of juice recovered from infected plants. Our research shows that SCYLD can cause yield loss and juice quality, even when symptoms are absent, prompted the industry to request that the Louisiana Department of Agriculture and Forestry include testing of commercial seed cane fields for the presence of SCYLD and to restrict the known movement of the virus into the state in imported plant material. This accomplishment addresses National Program 303 Component V, Host Plant Resistance to Disease; Problem Statement, Disease resistance in new germplasm and varieties. 6.Technology Transfer Number of non-peer reviewed presentations and proceedings 5 Review Publications Grisham, M.P., Pan, Y., Richard Jr, E.P. 2007. Early detection of Leifsonia xyli subsp. xyli in sugarcane leaves by real-time polymerase chain reaction. Plant Disease. 91:430-434. Grisham, M.P., Pan, Y.B. 2007. A genetic shift in the virus strains that cause mosaic in Louisiana sugarcane. Plant Disease. 91:453-458.