Location:2008 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 2008 resulted in significant progress in developing new cotton lines resistant to the reniform and root knot nematodes, in generating mutants of bacteria that contribute to seed and boll rots, and in identifying and cloning a gene involved in the biosynthesis of the phytotoxin fusaric acid. Thirty-nine lines carrying resistance to the reniform nematode were advanced to the BC7S1 generation, and seven new lines with resistance to both the reniform and root knot nematodes were developed using molecular markers located on the long arm of chromosome 11. Potential DNA markers were identified that are linked to reniform resistance in Gossypium barbadense Accession No. 713. In research focused on seed and boll rots, more than 1,000 mutants of bacteria that cause boll/seed rot were developed and tested for their ability to cause the disease. Two of these mutants were identified as potentially attenuated in their ability to produce the disease in cotton bolls. This completed the first step in identifying genes that are involved in seed and boll rot caused by these pathogens. If these genes are confirmed to be involved in disease development, they will be used as targets to rapidly detect seed/boll rot pathogens in samples collected from the field. This work will assist producers in timing pesticide applications to control various insects that have been identified as vectors for these pathogens. In work on fusaric acid, significant progress was made in identifying and cloning genes from Fusarium oxysporum f. sp. vasinfectum (Fov) that are involved in the biosynthesis of this phytotoxin. Utilizing a sequence homology-based strategy, a candidate gene, along with flanking sequences, was identified and cloned. This gene is strongly expressed when the isolates produce fusaric acid and is turned off when fusaric acid production is suppressed. In preliminary experiments, when this gene is knocked out in Fusarium verticillioides, fusaric acid production is completely suppressed, data that further confirms the identity of the cloned gene as the target gene. Identification of the fusaric acid synthesis gene will provide the basis for establishing the role of fusaric acid in the virulence of the newly emerging Fov isolates and for developing new control strategies for these highly pathogenic isolates. (NP 303, Component 3, Problem Statement 3B)
1. Cotton germplasm with stacked resistance to the root knot and reniform nematodes: Root knot and reniform nematodes cause losses of almost 5% of the U.S. cotton crop. Commercial cottons have limited resistance to the root knot nematode, and none are resistant to the reniform nematode. Scientists in the Cotton Pathology Research Unit at the Southern Plains Agricultural Research Center, College Station, Texas, in cooperation with scientists at Texas A&M University, used marker-directed breeding to combine and link the rkn-1 gene from Acala Nemex cotton (which confers resistance to the root knot nematode) with the Ren gene from wild Gossypium longicalyx (which confers resistance to the reniform nematode). The resulting lines are resistant to both nematodes. This accomplishment demonstrates that the molecular markers utilized in this study can be readily used by seed companies to introduce these combined resistance traits into commercial cottons, which will accelerate the release of new resistant cotton germplasm that will be highly resistant, if not immune, to both the reniform and root knot nematodes. (NP 303, Component 3, Problem Statement 3B)
5. Significant Activities that Support Special Target Populations
Taylor, R.E., French, A.D., Gamble, G.R., Himmelsbach, D.S., Stipanovic, R.D., Thibodeaux, D.P., Wakelyn, P.J., Dybowski, C. 2008. 1H and 13C Solid-state NMR of Gossypium barbadense (Pima) Cotton. Journal of Molecular Structure. 878(1-3):177-184.