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.
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.
Race 4 of Fusarium oxysporum f. sp. vasinfectum (Fov-4) has caused serious losses to cotton production in California since 2001. Project work has previously: 1) shown that Fov-4 produces copious amounts of the plant toxin, fusaric acid; 2) identified fusaric acid biosynthetic genes and generated gene knock-out mutants; and 3) demonstrated that fusaric acid plays an important role in the virulence of this pathogen. In FY 2015, project work identified and characterized a fusaric acid transporter in Fov by creation of mutants with the transporter deleted (Objective 2). The mutants were less viable than the wild type when exposed to high concentrations of fusaric acid concentration, demonstrating that the transporter plays an important role in protecting Fov cells from the toxic effect of fusaric acid. Such a transporter gene may provide a source of host resistance to Fusarium wilt in cotton which would represent a major step forward in developing effective approaches for disease management. Cotton boll rot bacterial pathogens have been previously shown by project work to significantly reduce yield; these bacteria are transmitted by insect pests harboring the pathogen. Project work in FY 2015 on boll rot (Objective 1) sequenced and annotated the entire genome of the opportunistic boll rot pathogen Klebsiella pneumoniae. This strain was isolated from the southern green stink bug which is a known pathogen vector and an important pest of cotton. The putative virulence genes identified can be used as molecular targets to directly monitor the presence of boll rot pathogens in insect vectors. This work will guide cotton producers to combat production losses due to the presence of insect vectors harboring the pathogens.
1. Deficiencies of a Fusarium oxysporum f. sp. vasinfectum race 4 (Fov-4) detection kit. Fov-4, first identified in California in 2001, has spread to new fields planted to cotton in the San Joaquin Valley. Fov-4 is a serious seed borne pathogen that can cause disease without the root knot nematode, and is thus a potential threat to all U.S. cotton production. ARS researchers at College Station, Texas, evaluated an existing race 4 specific Polymerase Chain Reaction test kit for ability to discriminate between Fov races and genotypes. The test kit is currently the only available method to rapidly identify Fov-4 in the field. Inclusion of more representative races and genotypes in the evaluation revealed that these diagnostic tests are able to distinguish race 4 from races 1, 2, 6, and 8 but not from race 3 and race 7 isolates. Knowing the limitations of diagnostic tests is important in correctly identifying diseased fields and seed lots.
Medrano, E.G., Forray, M.M., Bell, A.A. 2014. Complete genome sequence of a Klebsiella pneumoniae strain isolated from a known cotton insect boll vector. Genome Announcements. 2(4):e00850-14.
Puckhaber, L.S., Frelichowski, J.E., Bell, A.A., Stipanovic, R.D. 2014. New HPLC methods to quantitate terpenoid aldehydes in foliage of cotton (Gossypium). Journal of Natural Products. 4(3):188-195.
Gu, A., Liu, J., Li, D., Chen, Q., Wang, L., Qu, Y. 2015. Screening and evaluation of molecular markers linked with the factors affected Verticillium wilt resistance in cotton. Xinjiang Agricultural Sciences. 52:493-502.
Crutcher, F.K., Moran-Diez, M.E., Liu, J., Horowitz, B.A., Mukherjee, P.K., Kenerley, C.M. 2015. A paralog of the proteinaceous elicitor sm1 affects colonization of maize roots by Trichoderma virens. Fungal Biology. 119(6):476-486.
Suh, C.P., Medrano, E.G., Lan, Y. 2015. Detecting cotton boll rot with an electronic nose. Journal of Cotton Science. 18:435-443.
Crutcher, F.K., Liu, J., Puckhaber, L.S., Stipanovic, R.D., Bell, A.A., Nichols, R.L. 2015. FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum. Journal of Microbiology. 161(Pt4):875-883.
Wagner, T.A., Liu, J., Williams, H., Puckhaber, L.S., Bell, A.A., Stipanovic, R.D. 2015. RNAi construct of a P450 gene CYP82D109 blocks an early step in the biosynthesis of hemigossypolone and gossypol in transgenic cotton plants. Phytochemistry. 115:59-69.
Bell, A.A., Robinson, A.F., Quintana, J., Duke, S.E., Starr, J.L., Stelly, D.M., Zheng, Z., Prom, S., Saladino, V., Gutierrez, O.A., Stetina, S.R., Nichols, R.L. 2015. Registration of BARBREN-713 germplasm line of Upland cotton resistant to reniform and root-knot nematodes. Journal of Plant Registrations. 9:89-93.
Medrano, E.G., Bell, A.A., Greene, J.K., Roberts, P.M., Bacheler, J.S., Marios, J.J., Wright, D.L., Esquivel, J.F., Nichols, R.L., Duke, S.E. 2015. Relationship between piercing-sucking insect control and internal lint and seed rot in Southeastern cotton (Gossypium hirsutum). Journal of Economic Entomology. 108:1540-1544.