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United States Department of Agriculture

Agricultural Research Service



2012 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:
This project expired in FY 2012 and was replaced with 6202-22000-031-00D (Cotton Disease Management Strategies for Sustainable Cotton Production) which continues and expands upon the expired project. During the life of this project, all objectives were fully or substantially achieved. Australia is currently struggling with a new pathotype of Fusarium oxysporum f. sp. vasinfectum (Fov) that is exceptionally virulent to cotton plants. Fov-contaminated seed from these plants was inadvertently imported into the U.S. and used as feed for dairy cows. Thirteen Fov isolates and four isolates of a Fusarium sp. that resembled F. oxysporum were identified in the imported contaminated seed. Project scientists conducted studies to determine if the Australian and U.S. Fov isolates could fuse to form a viable new cell with genetic information from both parents [i.e., are vegetatively compatible (VC)]. The isolates from the contaminated seed formed four VC groups; one of these VC groups contained a single isolate that exhibited virulence equivalent to the biotypes originally identified in Australia. All of the Australian VC groups were incompatible with the 18 VC groups found in the U.S. Unlike U.S. isolates, all 17 of the Australian seed isolates and the original Australian biotypes readily attacked the vascular system of the cotton plant when roots were dipped into a flask containing inoculum. These aggressive Australian isolates have not yet been found in the U.S., but would cause severe wilt if they become established. However, new virulent isolates of Fov have been identified in California. Project scientists showed that both Australian and California biotypes produced copious amounts of fusaric acid, a potent phytotoxin. A Fov isolate that normally produces large amounts of fusaric acid was genetically modified to inhibit fusaric acid production. In a root dip assay this mutant, unlike the parent isolate, did not significantly reduce shoot weight, and the disease index was not significantly different from non-treated plants. This is the first direct proof that fusaric acid is a virulence factor in fungal plant pathogens. Project scientists previously established that adult green stink bugs vector bacterial pathogens that lead to cotton boll rot; work under this project established that newly hatched larvae (1st instars) of these insects can feed on green beans while more mature larvae (4th and 5th instars) can act as pathogen vectors. It was previously thought that the first instars of the stink bug do not feed and that juveniles could not vector the pathogen. In addition, plant sources that could harbor the pathogen were identified. These include snap beans and perhaps peanuts; corn did not harbor the pathogen. Thus, under field conditions, adjacent crops could affect boll rot problems. The reniform and root knot nematodes are major obstacles to profitable cotton production in many areas of the U.S. Work by this project developed cotton germplasm that contains elements that convey resistance to both of these nematodes. This cotton has been released to interested parties for analysis and testing under field conditions.

4. Accomplishments
1. Release of reniform/root knot nematode-resistant cotton germplasm. U.S. cotton producers lost almost 4% of their crop to the reniform and root knot nematodes in 2011. ARS researchers at College Station, Texas, had previously developed plants with significant resistance to the reniform nematode; however, these plants were badly stunted when planted in fields with high inoculum levels of pathogens that cause seedling diseases. The College Station researchers have now developed cotton lines with superior resistance to both the reniform and root knot nematodes. These new resistant lines exhibit excellent yields and exceptional fiber quality in the field. This dual resistant germplasm (called Barbren-713) has been released to cotton breeders and other interested parties for use in developing new, productive, nematode-resistant cotton varieties.

Review Publications
Liu, J., Bell, A.A., Wheeler, M.H., Stipanovic, R.D., Puckhaber, L.S. 2011. Phylogeny and pathogenicity of Fusarium oxysporum isolates from cottonseed imported from Australia into California for dairy cattle feed. Canadian Journal of Microbiology. 57:874-886.

Rathore, K.S., Sundaram, S., Sunilkumari, G., Campbell, L.M., Puckhaber, L.S., Marcel, S., Palle, S., Stipanovic, R.D., Wedegaertner, T.C. 2012. Ultra-low gossypol cottonseed: Generational stability of the seed-specific, RNAi mediated phenotype and resumption of terpenoid profile following seed germination. Plant Biotechnology Journal. 10(2):174-183.

Medrano, E.G., Esquivel, J.F., Bell, A.A., Greene, J.K., Roberts, P.M., Bacheler, J.S., Marois, J.J., Wright, D.L., Nichols, R.L. 2011. Analysis of microscopic cotton boll feeding injuries caused by southern green stink bug (Hemiptera: Pentatomidae). Southwestern Entomologist. 36(3):233-245.

Wagner, T.A., Liu, J., Stipanovic, R.D., Puckhaber, L.S., Bell, A.A. 2012. Hemigossypol, a constituent in developing glanded cottonseed (Gossypium hirsutum). Journal of Agricultural and Food Chemistry. 60:2594-2598.

Medrano, E.G., Bell, A.A. 2012. Genome sequence of Pantoea sp. strain Sc 1, an opportunistic cotton pathogen. Journal of Bacteriology. 194(11):3019.

Esquivel, J.F., Medrano, E.G. 2012. Localization of selected pathogens of cotton within the southern green stink bug. Entomologia Experimentalis et Applicata. 142:114-120.

Parkhi, V., Kumar, V., Campbell, L.M., Bell, A.A., Shah, J., Rathore, K.S. 2010. Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis NPR1. Transgenic Research. 19:959-975.

Last Modified: 2/23/2016
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