2009 Annual Report
1a.Objectives (from AD-416)
The long-term objective of this project is to characterize and develop rapid molecular-based protocols for detecting high-risk foreign plant pathogenic bacteria. During the next 5 years we will focus on collecting germplasm, characterize, fingerprint, and determine the taxonomy of high-risk foreign and domestic bacteria which may threaten U.S. agriculture through natural or deliberate introduction. We will collect cultures of Burkolderia andropogonis, B. glumae, B. caryophylli and B. gladioli and conduct sequencing, fingerprinting, and determine taxonomy. Citrus seedlings (infected with the non-cultureable Candidatus Liberibacter asiaticus, L. africanus, and L. americanus) will be collected, and we will develop a means to grow these fastidious organisms. Same-day on-site molecular techniques will be developed for rapid identification and detection of B. andropogonis, glumae, B. caryophylli, B. gladioli, L. asiaticus, L. africanus, and L. americanus.
1b.Approach (from AD-416)
Collect and add new germplasm of domestic and foreign sources to the International Collection of Phytopathogenic Bacteria maintained at ARS/Ft. Detrick. Develop improved biosensors, DNA-based techniques including real-time PCR, and other novel techniques for rapid, sensitive detection and identification of bacteria. Determine the genetic profiles of foreign bacteria using molecular techniques, including DNA/DNA hybridization, AFLP, pulse gel electrophoresis, and sequencing. Various media designed for growing insect bacteria and phytoplasma will be tested for culturing Liberibacter.
We collected citrus seedlings infected with the HLB bacterium (Liberibacter) from Thailand for our studies on cultivation of the HLB bacterium. All three species of Liberibacter have been successfully cultivated for the first time. Several DNA samples of Liberibacter asiaticus were collected from Thailand and other Southeast Asian countries for developing a sequence typing scheme to track the pathogen. Although DNA from the cultured organism was submitted to Los Alamos National Laboratory for sequencing the entire genome, the sequencing was not successful due to lack of enough DNA. We therefore submitted DNA to The National Genome Resource Center where they have equipment to sequence small amounts of DNA. Libraries have been made and the whole genome sequence will be available by September 15, 2009. An improved multiplex real-time PCR assay has been developed for rapid field diagnosis of HLB and detection of infested vectors. We collected strains of Bulkholderia from Thailand but his project has been put on hold due to lack of the assigned technician (sick leave) and priority of the HLB project. We have identified for the first time the presence of the wheat gumming bacterium Rathayibacter iranicus in Turkey. An environmentally friendly seed treatment has been developed for eradicating Xanthomoans campestris pv. campestris from crucifer seeds.
Identified Rathayibacter iranicus in Turkey: Rathayibacter iranicus (Ri), originally reported in Iran in 1961 (Sharif, 1961), has not been reported outside Iran and only one strain is known to exist. Like R. tritici (Rt), Ri causes a gumming disease of wheat in association with the nematode Anguina tritici. Asymptomatic wheat seeds from six central provinces in Turkey were assayed for the presence of the Rathayibacter species. Of the 25 isolated strains presumptively identified as Rathayibacter species based on biochemical properties, three new strains were found by additional characterization to be most similar to R. tritici and R. iranicus. Analysis of cell wall composition, DNA fingerprinting, and sequencing identified and confirmed the new strains to be R. iranicus. This is the first report of this species causal agent of wheat gumming disease outside of Iran.
Developed a seed treatment for eradicating Acidovorax avenae subsp. citrulli (Aac) from watermelon and melon seeds: Acidovorax avenae subsp. citrulli (Aac), the causal agent of a watermelon seedling blight and fruit blotch (WFB), has emerged as a serious seedborne pathogen of watermelon, melons, pumpkin, and citron world-wide. Although attempts have been made to eradicate the pathogen from seeds with chemical seed treatments, none has worked well enough to be routinely used by the seed industry. Seeds contaminated with Aac remain a problem. We describe an environmental friendly seed treatment using acidified water for eradicating Aac from watermelon and melon seeds. This inexpensive seed treatment should prove useful as a routine seed treatment for eradication of Aac from watermelon and melon seeds.
Cultured and sequenced of the causal agent of the citrus Huanglongbing (HLB) disease: Huanglongbing (HLB) disease of citrus is one of over 20 known plant diseases where the suspected phloem-limited causal bacterium has yet to be cultured. The disease causes a rapid tree decline. A new medium designated Liber A was designed and used to cultivate all three ‘Candidatus Liberibacter species,’ the suspect causative agents of HLB for 60 to 90 days before viability declined. Suspect‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ cells grown on Liber A medium were pathogenic on citrus and could be isolated from noninoculated tissues of inoculated trees and seedlings 9 and 2 months later, respectively. This is the first report of the cultivation and pathogenicity of ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ associated with symptoms of HLB. The ability to culture this important fastidious bacterium will allow researchers to conduct studies and elucidate the conditions required for infection and transmission of the causal agent of Huanglongbing disease.
Zhao, T., Feng, J., Sechler, A.J., Randhawa, P., Schaad, N.W. 2009. An improved assay for detection of Acidovorax avenae subsp. citrulli in watermelon and melon seed. Seed Science and Technology. 37:337-347.
Postnikova, E.N., Agarkova, I., Altundag, S., Eskandari, F., Sechler, A.J., Karahan, A., Vidaver, A.K., Schneider, W.L., Ozakman, M., Schaad, N.W. 2009. Rathayibacter iranicus isolated from asymptomatic wheat seeds in Turkey. Plant Pathology. 58:796.
Feng, J., Schuenzel, E., Li, J., Schaad, N.W. 2009. Multilocus sequence typing reveals two evolutionary lineages of the watermelon pathogen, Acidovorax avenae subsp. citrulli. Phytopathology. 99:913-920.
Sechler, A.J., Schuenzel, E., Cooke, P.H., Donnua, S., Thaveechai, N., Postnikova, E.N., Stone, A.L., Schneider, W.L., Damsteegt, V.D., Schaad, N.W. 2009. Cultivation of Candidatus Liberibacter asiaticus and Ca. L. americanus associated with Huanglongbing. Phytopathology. 99:480-486.