Location: Insect Control and Cotton Disease Research
2022 Annual Report
Objectives
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.
Approach
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.
Progress Report
Research by this project, culminating fiscal year (FY) 2022, provided a clearer understanding of the dynamics of two major diseases affecting cotton production in the United States; boll rot and Fusarium wilt (FOV). Work under Objective 1 identified a Serratia sp. as a new bacterial boll rot pathogen; the isolate's complete genome was sequenced and assembled. This work will be essential for identifying genes involved in boll rot virulence and pathogenicity. Work under Objective 1 confirmed that the verde plant bug was a major vector of the Serratia boll rot pathogen and was largely responsible for the yield losses observed along the coastal production area of Texas. Work addressing Objective 2 showed that genetic variants of FOV produced different levels of fusaric acid; this acidic compound is known to be involved in the toxicity of FOV in cotton. Several enzymes involved in the detoxification of fusaric acid were identified. In work addressing Objective 3, FOV isolates were obtained from various geographical areas and genotypically characterized to identify those best suited for use in a breeding program. Work under this objective revealed substantial variation in virulence among FOV isolates which led to the development of a classification scheme for FOV isolates based on vegetative compatibility testing, DNA sequences of key genes, and virulence to cotton species. Work under Objective 3 was also focused on breeding nematode-resistant lines with wild cotton species to develop germplasm lines that are resistant to nematodes and FOV. Six lines were developed and tested under controlled environmental conditions, with one line showing considerable promise against FOV. Some of these lines were bred with tri-species cotton lines that produce unique caryophyllene derivatives that deter insect pests or reduce insect feeding. This project expired in FY 2022 and was replaced by 3091-22000-040-000D which is continuing and expanding upon the work.
Accomplishments
1. Genotypic and virulence characterization of FOV4 in West Texas. Fusarium wilt of cotton, caused by the soilborne fungus Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4), is a serious cotton disease that had been found in cotton fields in California since 2001. However, in 2017, FOV4 was detected in several cotton fields near El Paso, Texas. ARS researchers at College Station, Texas, determined the FOV4 genotype distribution in the El Paso area. The genotype of FOV4 found in the Lower Valley of El Paso was more virulent than the genotype found in the Upper Valley region. This new knowledge will be critical for monitoring and identifying the dynamic population structure of FOV4 genotypes in fields, and represents a foundational contribution to the ultimate goal of developing effective strategies to manage this very destructive cotton disease.
2. Genome assembly of nematode-resistant cotton lines. Plant parasitic nematodes, including root-knot and reniform nematodes, are responsible for the greatest yield losses in U.S. cotton. These nematodes can interact synergistically with root rot pathogens such as Fusarium wilt to exacerbate disease severity. Although repeated applications of pesticides, along with crop rotation, can reduce nematode damage, the development and use of nematode-resistant cultivars is the most effective and economical approach for managing nematodes in cotton. ARS researchers at College Station, Texas, in collaboration with Mississippi State University, sequenced, assembled, and annotated the genomes of two nematode-resistant lines previously developed by the ARS College Station project. These genomes will expedite development of genetic markers, which will greatly facilitate incorporation of genes conferring nematode resistance into commercial Upland cotton lines that will be productively used by U.S. farmers.
Review Publications
Zhang, L., Chen, J., Liu, J., Sun, Q., Liu, J., Li, H., Wang, P., Chu, Z., Zhang, X., Yuan, Y., Shi, Y., Cai, Y. 2022. lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt. Plant Physiology. Article kiac041. https://doi.org/10.1093/plphys/kiac041.
Wagner, T.A., Davie, S.S., Mcgill, C., Liu, J. 2022. Interaction of Fusarium wilt race 4 with root-knot nematode increases disease severity in cotton. Plant Disease. https://10.1094/pdis-12-21-2725-sc.
Wagner, T.A., Bell, A.A., Castles, Z.A., Ali, A., Flores, O., Liu, J. 2022. Detection, genotyping, and virulence characterization of Fusarium wilt race 4 (VCG0114) causing cotton wilt in three Texas fields. Journal of Phytopathology. 170(7-8):492-503. https://doi.org/10.1111/jph.13100.
Perkin, L.C., Bell, A.A., Hinze, L.L., Suh, C.P., Arick II, M.A., Peterson, D., Udall, J.A. 2021. Genome assembly of two nematode-resistant cotton lines (Gossypium hirsutum L.). G3, Genes/Genomes/Genetics. 11(11). Article jkab276. https://doi.org/10.1093/g3journal/jkab276.
Liu, J., Wagner, T. 2021. Detection and genotyping of Fov4 (race 4, VCG0114), the fusarium wilt pathogen of cotton. In: Coleman, J., editor. Fusarium Wilt: Methods and Protocols, Methods in Molecular Biology. Switzerland: Springer Nature. 2391:191-205. https://doi.org/10.1007/978-1-0716-1795-3_16.
Medrano, E.G., Haydel, S.E. 2021. Complete genome sequence of the methicillin-resistant Staphylococcus aureus strain SQL1/USA300, used for testing the antimicrobial properties of clay phyllosilicates and customized aluminosilicates. Microbiology Resource Announcements. 10(45). Article e00861-21. https://doi.org/10.1128/MRA.00861-21.
Kodali, S., Medrano, E.G., Shuster, G. 2021. Response of commercial cotton varieties to Xanthomonas citri pv. malvacearum at early developmental stages in south Texas. Research Journal of Plant Pathology. 4(4):01.
