1a. Objectives (from AD-416):
1) Identify virulence/pathogenicity genes in Pantoea agglomerans, a causal agent of South Carolina seed rot that affects cotton, and apply this knowledge to disease management strategies. Subobjective 1A: Sequence the complete genome of the opportunistic P. agglomerans strain that is vectored by the southern green stink bug (Nezara viridula L.) into bolls causing seed and boll rot. Subobjective 1B: Conduct a bioinformatics analysis (BA) of the generated sequence data to putatively identify virulence and pathogenicity genes. Subobjective 1C: Generate mutants of the sequenced strain that contain a disruption in a gene presumptively involved in the infection process and test for pathogenicity in developing green cotton bolls. 2) Determine if a fusaric acid transporter gene can be used to increase resistance to Fusarium oxysporum f. sp. vasinfectum (F.o.v.) in cotton and apply this knowledge to disease management. Subobjective 2A: Identify and clone transporter genes located near the fusaric acid biosynthetic gene cluster to identify the putative gene(s) responsible for the pathogen's resistance to fusaric acid. Subobjective 2B: Generate knock-out mutants of the cloned genes to confirm their role in resistance to fusaric acid toxicity. Subobjective 2C: Determine if the targeted gene for knock-out is involved in self-protection against fusaric acid and test the mutant for pathogenicity. Subobjective 2D: Identify and clone a microbial gene for an enzyme that converts fusaric acid in a single step to a non-toxic product. 3) Develop and deploy new sources of cotton resistance to both the reniform nematode and root rotting fungal pathogens. Subobjective 3A: Develop germplasm lines with high levels of resistance to the reniform nematode and desirable agronomic properties derived from G. barbadense (GB713) using DNA marker assisted selections (MAS). Subobjective 3B: Determine the role of fungal root pathogens in the stunting of LONREN germplasm lines in reniform-infested fields. Subobjective 3C: Develop LONREN germplasm lines that have high levels of resistance to reniform nematodes and to stunting associated with the Ren1 **lon gene under field conditions.
1b. Approach (from AD-416):
To develop technology to identify piercing-sucking pests that harbor pathogenetic bacteria that cause cotton boll rot, the complete genome of an opportunistic bacterium will be sequenced. Statistics and computer programs will be used to map and analyze potential pathogenicity genes from the genome sequence. Mutants of the sequenced strain that contain a disruption in the gene putatively involved in the infection process will be generated, and these mutants will be tested for pathogenicity. To combat new virulent strains of the fungal pathogen Fusarium oxysporum f. sp. vasinfectum, genes that protect the pathogen from its own phytotoxic metabolites will be identified and cloned. Gene knock out mutants will be generated and the mutants will be tested for resistance to the metabolites. New cotton germplasm exhibiting resistance to the reniform nematode will be developed from Gossypium barbadense accession GB713 using marker assisted selection. In addition, the role of fungal root pathogens in causing stunting of cotton plants expressing the Renlon gene from G. longicalyx will be determined using greenhouse assays. Based on these studies, plants resistant to the reniform nematode will be identified and released as germplasm.
3. Progress Report:
Previous work established that the southern green and green stink bugs could act as vectors of a bacterium that causes cotton boll rot. Work under this project in FY 2013 established that the brown stink bug can also vector this pathogen. Project scientists have completely sequenced the genome of a boll-rotting bacterial pathogen vectored by the stink bug. The single chromosome of this bacterium is 4.4 Mbp and consists of over 4,000 potential genes. Work in this area in FY 2013 has contributed significantly to our goal of identifying specific boll rot pathogenicity genes. Project work also showed that, contrary to current theory, first instar stink bugs can and do feed on very young bolls. Prior work on the cotton wilt pathogen Fusarium oxysporum f. sp. vasinfectum (Fov) established that fusaric acid is an important component in highly pathogenic isolates of Fov like California race 4. In FY 2013, we identified a fusaric acid transporter gene in Fov, and generated corresponding transporter knock-out mutants that block expression of the gene. The mutants do not secrete fusaric acid into media, and they grow more slowly than the wild type under fusaric-acid-inducing conditions. This establishes that the transporter provides a protective function against the toxin. Prior work on resistance to the reniform nematode had shown that progeny derived from the cotton type known as Barbren-713 were highly resistant to the reniform nematode and had good agronomic properties. In FY 2013, new germplasm was developed from Barbren-713 that exhibits exceptional yield and fiber quality, with resistance to both the reniform and root knot nematodes. Seed will be released to breeders and other interested parties in FY 2014; this will accelerate development of commercial cottons with resistance to both nematodes and that are not limited by low yield and inferior fiber quality.
1. Classification of fusarium wilt biotypes that attack cotton. An exceptionally virulent Fusarium oxysporum f. sp. vasinfectum (Fov) race 4 fungus was identified in cotton fields in California in 2002. Race 4 is a grave threat to U.S. cotton production because seed-cotton grown in California has been distributed across the U.S. Cotton Belt; Fov is seed transmitted and thus U.S. fields may be infested with this pathogen. ARS scientists at College Station, Texas, investigated in detail a number of isolates of Fov and assigned them to one of three groups: 1) those capable of invading host plant xylem (water transport) cells and then spreading throughout the vascular system of the plant; 2) root-rotting pathogens; and 3) non-pathogens. This work established that earlier methods of determining Fov races and types are not reliable, and that only carefully controlled disease assays are reliable in determining virulence and thus the potential threat to cotton production. The accomplishment is foundational to ongoing research focused on protecting U.S. cotton from the devastating effects of extraordinarily virulent Fov types.
Uzbekov, V.V., Veshkurova, O.N., Stipanovic, R.D., Namazov, S.E., Salikhov, S.I. 2013. Prediction of cotton resistance to Helicoverpa armigera based on the percent (+)-gossypol in mature seed. Asian and Australasian Journal of Plant Science and Biotechnology. 7(Special Issue 2):61-63.