Location: Insect Control and Cotton Disease Research
2018 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
In FY 2018, verde bugs were caged with bolls from greenhouse grown cotton plants of known age (Objective 1). Following the incubation period of two weeks, disease was observed and vectored pathogens were recovered from both the insects and diseased bolls. A small percentage of bolls (<5%) were fed upon, but disease symptoms were absent in seed and lint tissues. The putative pathogenic microbes isolated are both being re-tested for infectivity, and characterized using both classical and molecular taxonomical techniques. In fusarium research in FY 2018, both Fov and Trichoderma virens transformants overexpressing isoforms of fusaric acid transporter were generated and confirmed (Objective 2). This will allow functional characterization of the transporter genes and testing their potential to control Fusarium wilt of cotton cause by Fov4. Work characterized Fov4 isolates from India, China, California, and West Texas with respect to virulence and genetic variation (Objective 3), and confirmed the Fov isolates causing Pima cotton wilting as Fov4. This work established the first occurrence of Fov4 outside California. PCR primers were developed to assess the genetic variations, as well as distinguish them from non-pathogenic MDS-12 mid-South isolates that have DNA sequences identical with those of Fov4. Resistance to Fusarium wilt caused by race 4 (Fov4) was transferred from six genetic sources for two addition generations into Upland cotton. BC5 plants from six successive crosses of resistant progeny into Upland cottons were self-pollinated to give BC5S1 plants. Test crosses were made using susceptible Pima S-7, and test-cross progeny were evaluated for resistance to Fov4. This allowed the identification of BC5S1 plants that were homozygous for resistance to Fov 4. BC5S2 seed from the homozygous plants are being grown in the greenhouse to produce seed for field evaluation and release as germplasm lines (Objective 3). New sources of resistance to Fov4 and nematodes were developed by creating 7 different triple-species plants and backcrossing these to Upland cotton cultivars and lines (Objective 3). All 7 sources have yielded some plants that are highly resistant to Fov4. The BC1 plants from the triple-species hybrids (170 total) were backcrossed to Fibermax 966’ and Barbren 713-32-X germplasm lines, and progeny will be analyzed for resistance to root-knot and reniform nematodes with and without Fusarium wilt race 1 isolates.
Accomplishments
1. Verde bug transmits cotton boll rot pathogens. As of 2009, an upward trajectory of periodic occurrences in cotton seed and boll damage associated with the verde plant bug (Creontiades signatus) and stink bugs have been reported in south Texas. Previously, it has been shown that in the southeastern Cotton Belt, the piercing-sucking southern green stink bug (Nezara viridula) is a vector of boll rot pathogens commonly called cotton boll rot. ARS scientists at College Station, Texas, collected verde plant bugs along the Texas Gulf Coast, and caged them with field grown cotton bolls previously protected with insecticides. The bolls were harvested two weeks following infestation; boll rot occurred. Microorganisms were recovered and retested for pathogenicity. Infective bacteria were recovered, thus demonstrating conclusively that the verde bug transmits boll rot pathogens.
2. Detection of Fov4 in Texas. Fusarium wilt of cotton is a widespread disease; the fungal causal agent, Fov, is genetically diverse. Most of the genotypes of U.S. Fov require the presence of nematodes to cause disease. Fov4, however, causes severe disease in the absence of nematodes, and can represent a major constraint to cotton production. In the U.S., Fov4 was first reported in California in 2001 and was confined to that state for many years. ARS scientists at College Station, Texas, working with a number of collaborators, demonstrated that the wilting of Pima cotton in fields of west Texas observed in 2017 was caused by Fov4. Using newly developed specific PCR tests, two subpopulations of Fov4 were identified in Texas based on the number of transposon insertions in the PHO gene. These discoveries will be very useful in monitoring spread of Fov4 in the U.S. and in development of new techniques and protocols for disease control.
3. Development of Fov4-resistant cotton lines. Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum race 4 (Fov4) has become a major problem for cotton production in parts of California and west Texas and poses a threat to the entire U.S. cotton belt. Many Gossypium arboreum cultivars and accessions are immune to Fov4. ARS scientists at College Station, Texas, successfully transferred resistance from G. arboreum Acc. No. 190 into agronomically desirable lines of Upland cotton. These lines will be released as new germplasm lines that can be used by breeders to develop cultivars resistant to Fov4.
Review Publications
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.
Crutcher, F.K., Puckhaber, L.S., Stipanovic, R.D., Bell, A.A., Nichols, R.L., Lawrence, K.S., Liu, J. 2017. Microbial resistance mechanisms to the antibiotic and phytotoxic fusaric acid. Journal of Chemical Ecology. 43(10):996-1006.
Halpern, H.C., Bell, A.A., Wagner, T.A., Liu, J., Nichols, R.L., Olvey, J., Woodward, J.E., Sonogo, S., Jones, C.A., Chan, C.T., Brewer, M.T. 2018. First report of Fusarium wilt of cotton caused by Fusarium oxysporum f. sp. vasinfectum race 4 in Texas, USA. Plant Disease. 102(2):446.
Mitchell, P.L., Zeilinger, A., Medrano, E.G., Esquivel, J.F. 2018. Pentatomoids as vectors of plant pathogens. In: J.E. McPherson, editor. Invasive Stink Bugs and Related Species (Pentatomoidea): Biology, Higher Systematics, Semiochemistry, and Management. Boca Raton, FL: CRC Press. p. 611-640.
