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.
In FY 2017, the brown stink bug was established as a vector of boll rot disease in cotton (Objective 1). Analysis of the genomes of 3 boll rot pathogens revealed a set of common infection associated genes. In fusarium research in FY 2017, the testing and characterization of fusaric acid transporter mutants was completed and it was established that both the transporter and other oxidizing enzymes provided protection against fusaric acid (Objective 2). Studies on the soil microbial fusaric acid resistance revealed that bacteria utilized efflux pumps (pumping out of the cell) while fungi deployed both efflux pumps and detoxification reactions to mitigate the toxic effect of fusaric acid. Over the life of this project, major advances were made in defining the causes of boll rot in cotton and in establishing vector/pathogen relationships (Objective 1). Project work developed new cotton germplasm that is crucial in developing commercial cottons that will be resistant or even immune to root-knot and reniform nematodes (Objective 3). Fusarium work under this project implicated fusaric acid as a significant contributor to plant toxicity, and provided significant advances in understanding the genetic mechanisms involved in fusarium pathogenicity (Objective 2). This project expired in FY 2017 and was replaced by project 3091-22000-035-00D which is continuing and expanding upon this work.
1. Characterization of Fusarium oxysporum isolates causing cotton wilt in Georgia. Locally severe outbreaks of Fusarium wilt of cotton in South Georgia have raised concerns about the genetic types of the causal pathogen, Fusarium oxysporum f. sp. vasinfectum. ARS researchers at College Station, Texas, in collaboration with Cotton Incorporated and the University of Georgia, characterized the pathogen population in Georgia and identified eight genetic Fusarium types including one previously unknown to science. These pathogens required the presence of nematodes to cause disease, establishing that use of nematode-resistant cotton cultivars will almost surely be required to manage the Fusarium wilt issue in Georgia.
Liu, J., Bell, A.A., Wagner, T.A., Stipanovic, R.D., Gu, A., Crutcher, F.K., Puckhaber, L.S. 2016. Physiology of host-pathogen interaction in wilt diseases of cotton in relation to pathogen management. In: Snider, J.L., Oosterhuis, D. M., editors. Cordova, TN: The Cotton Foundation Reference Book Series. p.117-146.
Crutcher, F., Puckhaber, L.S., Bell, A.A., Liu, J., Duke, S.E., Stipanovic, R.D., Nichols, R. 2017. Detoxification of fusaric acid by the soil microbe Mucor rouxii. Journal of Agricultural and Food Chemistry. 65(24):4989-4992.
Xiang, L., Liu, J., Wu, C., Deng, Y., Cai, C., Zhang, X., Cai, Y. 2017. Genome-wide comparative analysis of NBS-encoding genes in four Gossypium species. BMC Genomics. 18:292.
Bell, A.A., Kemerait, R.C., Ortiz, C.S., Prom, S., Quintana, J., Nichols, R.L., Liu, J. 2017. Genetic diversity, virulence, and Meloidogyne incognita interactions of Fusarium oxysporum isolates causing cotton wilt in Georgia. Plant Disease. 101:948-956.
Medrano, E.G., Bell, A.A., Duke, S.E. 2016. Cotton (Gossypium hirsutum L.) boll rotting bacteria vectored by the brown stink bug, Euschistus servus (Say) (Hemiptera: Pentatomidea). Journal of Applied Microbiology. 121:757-766.
Wagner, T., Suh, C.P., Liu, J., Puckhaber, L.S. 2017. Increased Helicoverpa zea (Boddie) larval feeding on cotton plants with RNAi construct CYP82D109 that blocks gossypol-related terpenoid synthesis. Southwestern Entomologist. 42:287-290.
Joshi, S.G., Kumar, V., Janga, M.R., Bell, A.A., Rathore, K.S. 2017. Response of AtNPR1-expressing cotton plants to Fusarium oxysporum f. sp. vasinfectum isolates. Physiology and Molecular Biology of Plants. 23:135-142.
Medrano, E.G., Bell, A.A. 2017. An opportunistic Pantoea sp. isolated from a cotton fleahopper that is capable of causing cotton (Gossypium hirsutum L.) bud rot. Journal of Agricultural Sciences. 8:64-76.
Medrano, E.G., Bell, A.A. 2017. Demonstration that a Klebsiella pneumoniae subsp. pneumoniae isolated from an insect (Nezara viridula) harbors a functional plasmid-borne type IV secretion system. Current Microbiology. 74:1033–1042.