1) Utilize both genetic and biological approaches to identify genes involved in vector-borne microbial cotton boll infections. Sub-objective 1.A: Confirm the ability of the verde plant bug to transmit cotton seed and boll rot pathogens. Sub-objective 1.B: Sequence the complete genome of a representative opportunistic bacterial strain that is vectored by the verde plant bug into bolls causing seed and boll rot. Sub-objective 1.C: Conduct bioinformatics analyses of the generated sequence data to putatively identify virulence and pathogenicity genes. 2) Determine the transport efficiency of each of the Fov4 fusaric acid transporter isoforms and their stoichiometric combinations, as well as their membrane localization or co-localization, to identify an optimal transport system that can be used to increase resistance to Fov4 in cotton. Sub-objective 2.A: Tag one fusaric acid transporter isoform with GFP and the other with RFP and overexpress them in Fov individually or in combination, determine their membrane localizations, and test their role in fusaric acid resistance. Sub-objective 2.B: Overexpress the two forms of fusaric acid transporter individually or in combination in a biocontrol agent, Trichoderma virens, to test whether fusaric acid resistance can be transferred to another organism. Sub-objective 2.C: Test the transporter isoform(s) overexpressing in Fov for pathogenicity on cotton. Sub-objective 2.D: Test the transporter isoform(s) overexpressing in T. virens for enhanced biocontrol activity against root rotting Fov. 3) Identify genetic sources and molecular DNA markers for resistance to Fov4 in cotton by screening germplasm. Sub-objective 3.A: Determine the population structure of F. oxysporum isolates from cotton and identify those isolates best suited to a resistance breeding program directed at all F. oxysporum populations. Sub-objective 3.B: Develop reliable inoculation protocols and identify different sources of resistance to Fusarium wilt with and without root-knot nematode. Sub-objective 3.C: Determine the inheritance of resistance and association with specific DNA markers.
Two emerging diseases, internal boll rots and Fusarium wilt caused by race 4, confront U.S. cotton producers. To address boll rots, we will identify genes for pathogenicity that are common to boll rotting pathogens with the long-term goal to develop a test kit to be used in the field that can determine if field insects harbor pathogens. To this end, we previously identified and sequenced the genome of bacterial pathogens transmitted by stink bugs and fleahoppers. In this study, we will confirm that the verde plant bug is a vector of boll rot pathogens, and identify and sequence the genome of a representative pathogen transmitted by this insect. In addition, a new strain of Fusarium oxysporum f. sp. vasinfectum (CA-Fov4) has been found for the first time in the western hemisphere in California fields from which cottonseed for planting originates. Spread of race 4 in the U.S. could be catastrophic. Race 4 is unique in that it produces prodigious quantities of fusaric acid, a plant toxin to which cotton is particularly sensitive. To address this problem, localization and efficiencies of transporter isoforms involved in secretion and detoxification of fusaric acid will be elucidated and their role in pathogenicity will be determined. The isoforms will be incorporated into a biocontrol agent to improve its biocontrol activity against Fov, because fusaric acid may enhance Fov’s ability to compete with other microbes in the soil. Sources of resistance to race 4 in cotton germplasm will be identified and incorporated into nematode-resistant germplasm; DNA markers will be identified to facilitate use of this germplasm.
Significant progress was made in FY 2019 on understanding the dynamics of major disease issues that affect cotton production in the U.S. Work under Objective 1 expanded the knowledge base on plant bugs that are important in transmitting boll rot pathogens to cotton bolls, which then seriously diminish lint production and quality. A boll rot pathogen transmitted by the verde plant bug was studied intensively using modern molecular techniques; the pathogen was identified as most likely to be Serratia marcescens. Accurate taxonomical categorization of boll rot pathogens is not only critical in determining the virulence and pathogenicity of boll rot pathogens, but is also essential for understanding the mechanisms involved in the infection and disease processes. Under Objective 2, genetic variants of Fusarium oxysporum (Fov), a soil fungus that attacks cotton, were shown to produce different levels of fusaric acid; this acidic compound is known to be involved in the toxicity of Fov in cotton. Manipulation of fusaric acid expression, or introducing fusaric acid resistance into cotton through traditional breeding efforts, offer promise as Fov management approaches. Work under Objective 3 on a particularly virulent Fov strain known as Fov4 identified several different genetic sub-types of the fungus; a modern genetic technique known as Polymerase Chain Reaction or PCR was used to develop diagnostic methods that accurately detect Fov4 and its variants. This PCR-based detection method will be of great value in detecting and monitoring the Fov4 pathogen which has been a serious problem in California since 2001 and was most recently detected in West Texas cotton production regions in 2017. Work on developing cotton types that will be resistant or even immune to the pathogenic effects of Fov, particularly Fov4 (Objective 3), made significant progress in FY 2019. New cotton germplasm was developed by appropriate breeding techniques that shows significantly increased resistance in successive generations. Overall, work under this project during FY 2019 was closely focused on major disease issues affecting cotton production efficiency and profitability in the U.S. The work resulted in significant advances in understanding disease dynamics and, with Fov4, a much clearer definition of the genetic make-up and variability; and in developing effective strategies for disease management with major focus on the development of new, Fov-resistant cotton types.
1. Genetic characterization of Fusarium oxysporum f. sp. vasinfectum race 4(Fov4). A highly virulent cotton wilt fungal pathogen known as Fov4 was found in cotton growing in West Texas in 2017; the disease had previously been confined to California since 2001. Although most of the genetic types of FOV that are found in U.S. agricultural fields require the presence of nematodes to cause disease, Fov4 causes severe disease in the absence of nematodes and represents a major threat to cotton production. ARS scientists at College Station, Texas, working on Fov4 collections from diverse geographic origins, identified four distinct genetic groups within Fov4 and, using modern molecular techniques, clearly defined their relationships with each other. Specific detection methods incorporating a molecular technique known as Polymerase Chain Reaction were developed for identifying Fov4 and distinguishing the four genetic sub-types. These methods will be extremely valuable in precisely identifying the specific Fov4 sub-type in a given field, or in harvested cotton seed. This accomplishment represents a significant advancement in Fov4 research, will help to minimize the further spread of this devastating disease, and will facilitate the work of cotton breeders in developing Fov4 resistant varieties.
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Mukherjee, P.K., Hurley, J.F., Taylor, J.T., Puckhaber, L.S., Lehner, S., Druzhinina, I., Schumacher, R., Kenerley, C.M. 2018. Ferricrocin, the intracellular siderophore of Trichoderma virens, is involved in growth, conidiation, gliotoxin biosynthesis and induction of systemic resistance in maize. Biochemical and Biophysical Research Communications. 505:606-611. https://doi.org/10.1016/j.bbrc.2018.09.170.
Janga, M.R., Pandeya, D., Campbell, L.M., Konganti, K., Toinga, S., Puckhaber, L.S., Pepper, A., Stipanovic, R.D., Scheffler, J.A., Rathore, K. 2018. Genes regulating gland development in the cotton plant. Plant Biotechnology Journal. 16:1-12. https://doi.org/10.1111/pbi.13044.