Location: Insect Control and Cotton Disease Research
2020 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 2020 on understanding the dynamics of major disease issues that affect cotton production in the U.S. Work under Objective 1 to sequence the genome of a bacterial boll rot pathogen transmitted by the verde plant bug resulted in a complete draft assembly. This genome assembly is essential for identifying the gene(s) responsible for virulence and pathogenicity of bacterial boll rot pathogens and, subsequently, gaining a better understanding of the mechanisms involved in the infection and disease processes. Under Objective 2, isolates of Trichoderma virens were genetically modified to overexpress the fusaric acid transporter gene. These transformed isolates were shown to be more resistant than 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 in cotton. Manipulation of fusaric acid resistance traits in biocontrol agents such as Trichoderma virens or in cotton through traditional breeding techniques may offer novel approaches for managing Fov diseases in cotton. Work under this objective also confirmed that the presence of root-knot nematodes was not needed for Fov race 4 to cause severe disease in cotton, but the presence of these nematodes appeared to enhance disease severity. Work under Objective 3 led to the development of three germplasm lines having a combination of four genes that reduce reproduction of root-knot and reniform nematodes by 95% and 90%, respectively. Under controlled environmental conditions, the lines showed resistance to 20 genotypes of Fov race 1 that are dependent on these nematodes to cause severe disease in cotton. Substantial progress under Objective 3 was also made in breeding these nematode-resistant lines with wild cotton species that produce unique caryophyllene derivatives that appear to deter insects or reduce insect feeding. Overall, work under this project during fiscal year 2020 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. Manipulating cotton to enhance resistance to Fusarium wilt. Terpenoid compounds derived from the gossypol and lacinilene pathways play an important role in defending cotton plants against herbaceous insects, animals, and pathogens. Both pathways share a common initial biosynthetic step that can be manipulated to affect each pathway. Using a molecular technique known as RNAi suppression, ARS researchers at College Station, Texas, blocked the gossypol pathway and observed increased resistance to Fusarium wilt pathogens. Comparison of terpenoid contents in the roots of RNAi suppressed and non-suppressed plants revealed increased production of lacinilene compounds in the RNAi plants. The concentration of lacinilenes found in the roots of RNAi suppressed plants were also shown to inhibit the growth of the Fov fungal pathogen by more than 93%. Thus, the increased induction of the lacinilene pathway likely enhanced resistance to Fov in the RNAi suppressed plants. Genetic manipulation of these two major cotton chemical defense pathways may provide a new approach to increase host resistance to Fov wilt pathogens in cotton.
Review Publications
Wagner, T.A., Cai, Y., Bell, A.A., Puckhaber, L.S., Magill, C., Duke, S.E., Liu, J. 2020. RNAi suppression of a gossypol biosynthetic CYP82D P450 hydroxylase enhances Fusarium wilt disease resistance in cotton. Journal of Phytopathology. 168:103-112. https://doi.org/10.1111/jph.12873.
Esquivel, J.F., Medrano, E.G. 2019. Transmission of Eremothecium coryli (syn. Nematospora coryli) to consecutive cotton bolls by individual stink bugs. Southwestern Entomologist. 44(4):853-860. https://doi.org/10.3958/059.044.0414.
Glover, J.P., Medrano, E.G., Isakeit, T., Brewer, M.J. 2019. Transmission of cotton seed and boll rotting bacteria by the verde plant bug. Journal of Economic Entomology. 113(2):793-799. https://doi.org/10.1093/jee/toz334.
