Location: Insect Control and Cotton Disease Research
2021 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
Significant progress was made in Fiscal Year (FY) 2021 on understanding the dynamics of major disease issues that affect cotton production in the U.S. Work under Objective 1 identified a bacterial boll rot pathogen transmitted by the verde plant bug. The genome of the pathogen was sequenced and assembled to identify genes responsible for virulence and pathogenicity. Under Objective 2, isolates of Trichoderma virens were genetically modified to overexpress the fusaric acid transporter gene. The transformed isolates showed slightly reduced virulence compared with non-transformed isolates against Fusarium oxysporum (Fov) strains that produce high levels of fusaric acid; this acidic compound is known to be involved in the toxicity of Fov race 4 (Fov4) in cotton. However, the virulence of the transformed or non-transformed isolates was not significantly different from that of the wild-type isolate, suggesting that other factors are also involved in Fov4 virulence. Work under this objective also confirmed that the presence of root-knot nematodes was not needed for Fov4 to cause severe disease in cotton, but the presence of these nematodes enhanced disease severity. Understanding and identifying fungal toxin resistance mechanisms will be critical for creating new sources of Fusarium wilt resistance. Work under Objective 3 elucidated the variation in virulence among Fov isolates. A classification scheme was developed for Fov isolates based on vegetative compatibility testing, DNA sequences of key genes, and virulence to cotton species. Significant progress under this objective was also made in 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. Some of these lines have been bred with wild cotton species that produce unique caryophyllene derivatives that deter insect pests or reduce insect feeding. Efforts are underway to increase seed production of these caryophyllene derivative and nematode-resistant/Fov lines for large-scale field trials; release of one or more germplasm lines is expected in FY 2022. Overall, work under this project during FY 2021 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 of boll rot pathogens and, with Fov, identification of new strategies or novel approaches for managing this disease in cotton.
Accomplishments
1. Identification of a boll rot pathogen vectored by the verde plant bug. Accurate taxonomical categorization of boll rot pathogens is not only critical in determining the virulence and pathogenicity of these pathogens, but is also essential for understanding the mechanisms involved in the infection and disease processes. ARS researchers at College Station, Texas, identified a bacterial boll rot pathogen (Serratia marescens) that is vectored by the verde plant bug. The genome of the bacterium was sequenced and assembled, and the genes responsible for virulence and pathogenicity were identified. This accomplishment provides the necessary foundation for the development of a molecular-based diagnostic tool that can be used in the field to rapidly detect insects harboring bacterial boll rot pathogens.
2. Root-knot nematodes enhance Fusarium wilt severity. Unlike other strains of Fusarium oxysporum f. sp. vasinfectum (Fov), Fov race 4 (Fov4) does not require the presence of nematodes to cause severe disease of cotton plants. However, the interaction between root-knot nematodes and Fov4 pathogenicity remains unclear. ARS researchers at College Station, Texas, developed an assay procedure for assessing the interaction between root-knot nematodes and Fov4, and for assessing the subsequent impact of nematodes on disease severity. Fov4 disease assays using this protocol confirmed that nematodes were not required for Fov4 infection, but also established that the presence of root-knot nematodes significantly enhanced Fov4 disease severity on many cotton cultivars, including those that were previously shown to be moderately resistant to Fov4. This work is important because it shows that if Fov4 spreads to nematode-infested fields, some cotton cultivars that are moderately resistant to Fov4 may not retain that resistance.
Review Publications
Wagner, T., Gu, A., Duke, S.E., Bell, A.A., Magill, C., Liu, J. 2021. Genetic diversity and pathogenicity of Verticillium dahliae isolates and their co-occurrence with Fusarium oxysporum f. sp. vasinfectum causing cotton wilt in Xinjiang, China. Plant Disease. 105:978-985. https://doi.org/10.1094/PDIS-09-20-2038-RE.
Esquivel, J.F., Medrano, E.G. 2020. Retention of Pantoea agglomerans Sc1R across stadia of the southern green stink bug, Nezara viridula (L.) (Hemiptera: Pentatomidae). PLoS ONE. 15(12). https://doi.org/10.1371/journal.pone.0242988.
Medrano, E.G., Smith, T.P., Bell, A.A., Brewer, M.J., Glover, J.P. 2020. Complete genome sequence of Serratia sp. strain CC119 associated with inner cotton boll rot via insect vector transmission. Microbiology Resource Announcements. 9(50). https://doi.org/10.1128/MRA.01077-20.
