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:
Project work in FY 2016 sequenced and annotated the entire genome of the opportunistic boll rot pathogen, Pantoea ananatis (Objective 1). This strain was isolated from a cotton fleahopper, which is an important pest of cotton. The putative virulence genes identified can be used as molecular targets to directly monitor the presence of cotton pathogens in insect vectors. This work will guide cotton producers to combat production losses due to the presence of insect vectors harboring the pathogens. Race 4 of Fusarium oxysporum f. sp. vasinfectum (Fov-4) has caused serious losses to cotton production in California since 2001. Project work (Objective 2) 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; 3) demonstrated that fusaric acid plays an important role in the virulence of this pathogen, 4) identified and characterized a fusaric acid transporter in Fov and demonstrated that the transporter plays an important role in protecting cells from the toxic effect of fusaric acid. In FY 2016, project work further characterized the transporter function and discovered that the transporter in Fov is composed of two isoforms due to alternative splicing (Objective 2). Determining the effective transporter form(s) and the cellular mechanisms of the transport may provide a source of host resistance to Fusarium wilt in cotton or provide an additional mechanism to resist Fov in biocontrol agents. Root-knot and reniform nematodes, among all pests, cause the greatest yield loss on cotton (3-5% of the potential crop). Project work previously resulted in release of the LONREN-1 and LONREN-2 germplasm lines for resistance to reniform nematode and BARBREN 713 for resistance to both nematodes. In FY 2016, we characterized the nature of a stunt disease associated with the Renlon gene and generated ca. 40,000 backcross progeny to identify 7 recombinants near the gene (Objective 3). However, stunting still was associated with the Renlon gene in the recombinants. Eight new germplasm lines which have both nematode resistance and elite agronomic performances were released, and in FY 2016 we increased seed for the registration, publication, and deposit of seed to the National Seed Depository in Colorado.
1. Release of nematode-resistant cotton lines. Among pests, root-knot and reniform nematodes cause the greatest yield losses in cotton (ca. 4% of potential yield). The nematodes also act synergistically to increase the severity of seedling diseases and Fusarium wilt. ARS researchers at College Station, Texas, jointly with workers at Texas A&M AgriLife Research and Cotton Incorporated released eight new nematode-resistant germplasm lines. All eight lines are resistant to the reniform nematode, and five are also resistant to the root-knot nematode. The lines also have elite agronomic performance; they will be valuable to plant breeders in their work to reduce pest losses and cost of pest control in cotton.
5. Significant Activities that Support Special Target Populations:
Zheng, X., Hoegenauer, K.A., Quintana, J., Bell, A.A., Hulse-Kemp, A.M., Nichols, R.L., Stelly, D.M. 2016. SNP-based MAS in cotton under depressed-recombination for Renlon-flanking recombinants: Results and inferences on wide-cross breeding strategies. Crop Science. 56:1-14.
Stipanovic, R.D., Puckhaber, L.S., Frelichowski, J.E., Esquivel, J.F., Westbrook, J.K., O Neil, T.M., Bell, A.A., Dowd, M.K., Hake, K., Duke, S.E. 2015. Gossypolhemiquinone, a dimeric sesquiterpenoid identified in cotton (Gossypium). Phytochemistry. 122:165-171.
Crutcher, F.K., Doan, H.K., Bell, A.A., Davis, R.M., Stipanovic, R.D., Nichols, R.L., Liu, J. 2015. Evaluation of methods to detect the cotton wilt pathogen Fusarium oxysporum f. sp. vasinfectum race 4. European Journal of Plant Pathology. 144(1):225-230.
Medrano, E.G., Bell, A.A. 2015. Genome sequence of Pantoea annatis strain CFH 7-1, which is associated with a vector-borne cotton fruit disease. Genome Announcements. 3(5):e01029-15.
Stipanovic, R.D., Esquivel, J.F., Westbrook, J.K., Puckhaber, L.S., O Neil, M., Bell, A.A., Duke, S.E., Dowd, M.K., Lopez, J.D., Hake, K. 2014. The effect of gossypolone on the growth and development of Helicoverpa zea (Boddie). Southwestern Entomologist. 39(1):1-7.
Ortiz, C.S., Bell, A.A., Magill, C.W., Liu, J. 2016. Specific PCR detection of Fusarium oxysporum f. sp. vasinfectum California Race 4 based on a unique Tfo1 insertion event in the PHO gene. Plant Disease. 101(1):34-44.