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United States Department of Agriculture

Agricultural Research Service



2009 Annual Report

1a. Objectives (from AD-416)
Develop control strategies that will minimize yield and fiber quality losses caused by nematodes and microbial pathogens that are emerging as significant impediments to sustained profitability by cotton producers. Of particular concern is the spread of the reniform nematode in the southern U.S. and south Texas, the emergence of a new disease called South Carolina seed rot in the southeast, the introduction of a particularly virulent isolate of Fusarium oxysporum f. sp. vasinfectum that was inadvertently imported from Australia on cottonseed and that could adversely affect 50% of U.S. cotton production, and the identification of race 4 of F.o.v. for the first time in the U.S. To address these emerging diseases we will: 1) Complete the introgression of reniform nematode (Rotylenchulus reniformis) resistance from Gossypium longicalyx and G. barbadense into Upland cotton (G. hirsutum) and develop markers for the trait suitable for marker-assisted selection; 2) Describe mechanisms of pathogenesis and identify virulence genes in the bacterial pathogen(s) causing the disease known as South Carolina seed and boll rot; and develop detection methods for the pathogen(s); and 3) Determine whether fusaric acid contributes to differences in virulence among races and biotypes of Fusarium oxysporum f. sp. vasinfectum to provide a basis for developing strategies for controlling Fusarium wilt of cotton.

1b. Approach (from AD-416)
1) Introgression of Reniform Nematode Resistance: Standard procedures for cotton flower emasculation and pollen transfer will be followed for backcross breeding. After each cross, progeny will be bioassayed for resistance to the reniform nematode, and the most highly resistant progeny will be retained for subsequent crossing, self seed generation, and DNA extraction for marker development. Plants will be grown under field conditions and evaluated for agronomic performance. Standard techniques and commercially available technology will be used for cotton DNA extraction. PCR amplification, electrophoresis, and fragment size detection will be utilized to identify molecular markers. 2) South Carolina Seed and Boll Rot: A mutagenesis system will be used to identify genes involved in production, regulation, and/or secretion of factors that cause boll rot. Based on these results, a set of predicted gene sequences associated with pathogenicity will be used to develop a PCR based method for detecting seed and boll rotting bacteria in field samples. Bolls from greenhouse grown plants will be used in initial testing to determine the efficacy of the developed amplification system. 3) Relation of Fusaric Acid to Virulence of Fusarium oxysporum f. sp. vasinfectum (F.o.v.): Biotypes of F.o.v. will be monitored for their ability to produce high levels of phytotoxins, and virulence of biotypes will be determined. The biosynthesis of those phytotoxins that correlate with virulence will be determined by feeding labeled substrates to the pathogens. Genes involved in the biosynthesis of these phytotoxins will be identified. Knock-out mutants will be generated to assess the role of specific phytotoxins in virulence and pathogenicity.

3. Progress Report
Work under this project during FY 2009 resulted in significant progress in advancing by one or two generations more than 400 cotton lines carrying new sources of resistance to the reniform nematode. These lines were evaluated for nematode resistance and agronomic performance in the field. All lines showed excellent resistance to the nematode in several fields. In the absence of Photosystem II herbicides and seedling diseases, many lines yielded better than susceptible siblings or commercial cultivars. Most lines with nematode resistance from Gossypium longicalyx were sensitive to the herbicides Caporal and Cotoran, and showed severe stunting and yield losses. Many lines also were very susceptible to seedling diseases and showed severe stunting. One of the pathogens responsible was identified as Thielaviopsis basicola. Selections for resistance to herbicides and seedling disease were made and are being evaluated. In our seed and boll rot project, significant progress was made in culturing over 2200 mutants of bacteria that potentially can cause boll/seed rot, and in testing their ability to cause the disease. Studies of two mutants, initially identified in FY 2007 as potentially non-pathogenic, showed that neither transconjugant's infectivity was totally lost. A new mutant with an apparent loss in pathogenicity was identified in FY 2008. If we can confirm that the mutated gene is involved in disease development, it will be used as the target to develop technology for rapid detection of boll-rotting bacteria in samples collected from the field. This will assist producers in timing pesticide applications to control various insects that have been identified as vectors for these pathogens. In work on Fusarium oxysporum f. sp. vasinfectum (Fov), significant progress was made in the identification of genes from Fov that are involved in the biosynthesis of the phytotoxin fusaric acid, and in targeted disruption of these genes. Two of the pathway genes were identified and cloned through a homology based strategy, and confirmed by expression analysis. Disruption of either one of the genes resulted in complete loss of fusaric acid production. The pathogenicity of these mutants was significantly reduced. Fusaric acid has been implicated as a virulence factor of wilt in Fov isolates, but its role has not been established unequivocally. Our work provides an essential tool to assess the role of fusaric acid in the virulence of the newly emerging and highly pathogenic Fov isolates and for development of new and effective control strategies.

4. Accomplishments
1. Generation of fusaric acid knock-out mutants: Recent outbreaks of Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (Fov) race 4 lineage isolates in California, and of Australian biotype isolates in Australia, pose a major threat to U.S. cotton production. Because only limited Fov resistance is found in available cotton germplasm resources, new control strategies for Fov are needed. We have already established that high levels of fusaric acid correlate with virulence and pathogenicity of these aggressive Fov isolates, indicating that manipulation of fusaric acid levels may provide a new and efficient target to control Fusarium wilt. Unit scientists have now identified and developed techniques for disruption of two Fov genes that are involved in the biosynthesis of fusaric acid. The work successfully created knock-out mutants that were successful in eliminating fusaric acid biosynthesis within Fov and their pathogenicity was significantly reduced. This accomplishment provides an essential tool to elucidate the role of fusaric acid in Fov virulence and pathogenicity, and to develop new control strategies for reducing the negative impact of Fov on cotton production.

Review Publications
Liu, J., Stipanovic, R.D., Bell, A.A., Puckhaber, L.S., Magill, C.W. 2008. Stereospecific coupling of hemigossypol to (+)-gossypol in MOCO cotton is mediated by a dirigent protein. Phytochemistry. 69:3038-3042.

Isakett, T., Prom, L.K., Wheeler, M.H., Puckhaber, L.S., Liu, J. 2008. Mycotoxigenic potential of ten Fusarium species grown on sorghum and in vitro. Plant Pathology Journal. 7:183-186.

Wallace, T.P., Bowman, D., Campbell, B.T., Chee, P., Gutierrez, O.A., Kohel, R.J., McCarty, J., Myers, G., Percy, R., Robinson, F., Smith, W., Stelly, D.M., Stewart, J.M., Thaxton, P., Ulloa, M., Weaver, D.B. 2009. Status of the USA cotton germplasm collection and crop vulnerability. Genetic Resources and Crop Evolution 56:507-532.

Stipanovic, R.D., Lopez, J., Dowd, M.K., Puckhaber, L.S., Duke, S.E. 2008. Effect of racemic, (+)- and (-)-gossypol on the survival and development of the specialist herbivore Heliothis virescens (Fabricius). Environmental Entomology. 37:1081-1085.

Stipanovic, R.D., Puckhaber, L.S., Liu, J., Bell, A.A. 2009. Total and percent atropisomers of gossypol and gossypol-6-methyl ether in seeds from Pima cottons and accessions of Gossypium barbadense L. Journal of Agricultural and Food Chemistry. 57:566-571.

Medrano, E.G., Esquivel, J.F., Nichols, R.L., Bell, A.A. 2009. Temporal analysis of cotton boll symptoms resulting from southern green stink bug (Nezara viridula L.) feeding and transmission of a bacterial pathogen. Journal of Economic Entomology. 102:36-42.

Edwards, W., Hall, J.A., Rowlan, A.R., Barfield-Schneider, T., Sun, T., Patil, M.A., Pierce, M.L., Fulcher, R., Bell, A.A., Essenberg, M. 2008. Light filtering by epidermal flavonoids during the resistant response of cotton to Xanthomonas protects leaf tissue from light-dependent phytoalexin toxicity. Phytochemistry. 69:2320-2328.

Last Modified: 2/23/2016
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