Location: Vegetable Research
2022 Annual Report
Objectives
1. Determine genetics of resistance to diseases and nematodes and develop molecular markers linked to resistance genes in vegetable crops with emphasis on cucurbit and solanaceous crops.
Sub-objective 1.A. Determine inheritance of resistance to powdery mildew (Podosphaera xanthii) in watermelon and identify molecular markers closely linked to resistance genes.
Sub-objective 1.B. Determine the genetic basis of resistance to Phytophthora fruit rot in watermelon.
Sub-objective 1.C. Determine inheritance of resistance to watermelon vine decline caused by Squash vein yellowing virus (SqVYV) and identify molecular markers closely linked to resistance genes.
Sub-objective 1.D. Determine genetic basis of resistance to northern root-knot nematodes (Meloidogyne hapla) in pepper.
2. Develop and release cucurbit and solanaceous germplasm with resistance to diseases and nematodes.
Sub-objective 2.A. Develop and release disease resistant (Phytophthora fruit rot and Powdery mildew) watermelon breeding lines.
Sub-objective 2.B. Develop sweet peppers (sweet banana and Cubanelle-types) with resistance to southern root-knot nematode (Meloidogyne incognita).
Sub-objective 2.C. Develop germplasm resources for cucumber with improved resistance to southern root-knot nematode (M. incognita).
3. Monitor, collect and characterize emerging cucurbit fungal pathogens to aid in improving management practices for growers and processors.
Approach
This project will identify and develop cucurbit and solanaceous germplasm and breeding lines with enhanced resistance to diseases caused by fungal, viral and other plant pathogens. Specifically, resistant germplasm and breeding lines will be developed for managing major limiting diseases, including Phytophthora fruit rot, powdery mildew, watermelon vine decline, and root-knot nematodes (RKN). We will utilize conventional and contemporary resistance phenotyping and crop improvement techniques to accomplish our objectives.
Populations of watermelon segregating for resistance to powdery mildew will be generated by crossing a highly resistant selection developed from a Citrullus lanatus var. lanatus accession with susceptible cultivars. Resulting populations will be phenotyped for reaction to powdery mildew and analyzed to determine inheritance of resistance. Molecular markers linked to resistance will be identified and used in marker assisted selection to develop resistant breeding lines.
A recombinant inbred line (RIL) population from a cross between Phytophthora fruit rot resistant and susceptible lines will be developed and phenotyped for resistance, and the information will be used to determine genetics of resistance. Analysis of differentially expressed transcriptomes by RNA-Seq resulting from Phytophthora-watermelon fruit interactions will also be used to further elucidate the genetics of fruit rot resistance. Red fleshed RILs with resistance will be used to develop Phytophthora-resistant lines.
Advanced watermelon lines resistant to vine decline caused by the whitefly-transmitted squash vein yellowing virus, (SqVYV) will be developed using known sources of resistance in wild watermelon accessions and by employing pure line selection. A watermelon vine decline (WVD) resistant line developed previously (392291-VDR) will be crossed with a susceptible commercial cultivar to develop segregating populations that can be assessed for disease response to determine inheritance of SqVYV resistance.
Sweet banana and Cubanelle pepper types resistant to southern RKN will be developed by using conventional recurrent backcross breeding procedures to transfer the dominant ‘N’ gene, which confers resistance, from a bell pepper to the different sweet pepper types. Populations of pepper segregating for resistance to northern RKN will be developed by crossing a highly resistant pepper with a susceptible pepper cultivar. These populations will be phenotyped for resistance to northern RKN, and the data will be used to determine the mode of inheritance.
Select cucumber accessions will be screened for resistance to southern RKN. Resistant selections will be advanced by multiple cycles of selfing and resistance screening to develop southern RKN resistant cucumber lines.
Isolates of cucurbit powdery mildew will be collected from across the U.S. and used to infect cucurbit differentials to determine the prevalence of particular powdery mildew races.
Progress Report
This is the final report for this project. Refer to project 6080-22000-031-000D, "Characterization of Host Resistance and Biology of Diseases and Nematodes in Vegetable Crops" for additional information.
Despite setbacks that arose from COVID 19 pandemic during this five-year cycle, significant progress was made toward developing new resources of resistance to help stakeholders manage diseases and root-knot nematodes in vegetable crops. The project with 2 researchers, successfully published 31 peer reviewed journal manuscripts and released 9 disease resistant vegetable germplasm lines, which included four powdery mildew resistant watermelon lines, a phytophthora fruit rot and powdery mildew resistant watermelon line, 2 powdery mildew resistant bottle gourd lines, and root knot nematode resistant pepper and sweetpotato lines. In addition disease resistant pepper and watermelon germplasm was provided to several national and international seed companies and universities through 15 outgoing material transfer agreements (MTA). The project also developed molecular markers linked to resistance genes for powdery mildew of watermelon. We completed quantitative trait loci (QTL) analysis to determine the chromosomal regions where potential resistance gene are located for PM, Phytophthora fruit rot (PFR) and watermelon vine decline (WVD). We mapped resistance to the Javanese root-knot nematode in cucumber. We developed new methods for detecting root-knot nematodes in sweetpotato roots. And we identified new sources of resistance to the guava root-knot nematode in both sweetpotato and pepper. All of this work as well as new avenues of research will be continued into the next five-year project plan cycle to continue to help stakeholders manage these pests within their fields.
The following progress is relative to Objective 1. Powdery mildew (PM) of watermelon is a major factor limiting production throughout the United States. Resistant line USVL531-MDR was crossed with susceptible line USVL677-PMS to develop breeding populations (F1, F2, BCF1R and BCF1S). Kompetitive allele specific polymorphisms (KASP) markers were developed using a single nucleotide polymorphism (SNP) in the resistance gene ClaPMR2 and used to assay segregating F2 populations. KASP markers identified the appropriate phenotype with 99% accuracy. KASP markers also helped identify powdery mildew resistant phenotype in another population developed from a cross of USVL608-PMR X USVL677-PMS.
Phytophthora fruit rot is a serious disease that has plagued watermelon growers in the eastern U.S. and the National Watermelon Association (NWA) has considered it their top research priority. Evaluation of F2 breeding population of USVL531-MDR X USVL677-PMS indicated that inheritance of resistance is complex and hence, we developed a recombinant inbred line (RIL) population (F10). This RIL population was phenotyped for resistance in 2021 and 2022. We are currently also increasing the seeds of RIL lines for use in genetic studies next year. Screening populations developed using USVL003-MDR X ‘Dixie Lee’ for fruit rot indicated that at least four QTLs are significantly associated with resistance, with a major QTL in Chromosome 4 and numerous minor QTL. These results will provide markers that will enable breeders to develop new cultivars that are resistant to fruit rot.
Watermelon vine decline (WVD) caused by the whitefly transmitted Squash Vein Yellowing Virus (SqVYV) is a serious disease that has plagued watermelon growers for the past several years. Populations (F1, F2, BCF1R and BCF1S) developed from a cross of 392291-VDR x Crimson sweet were evaluated for resistance to SqVYV in a growth chamber. Preliminary analysis indicated that resistance is governed by recessive genes. QTLseq using extreme phenotypes of the F2 population identified 5 major QTL’s significantly associated with resistance. Generation of an RIL population from this cross for fine mapping is in progress. Genome wide association mapping (GWAS) analysis based on screening of 1200 plant introductions (PI) with SqVYV indicated two major QTLs associated with resistance in chromosome 5. Resistance was confirmed in three new accessions to SqVYV in screens conducted in a growth chamber. These results will provide markers that will enable breeders to develop new cultivars that are resistant to SqVYV.
Advanced BC3F3 pepper plants have been selected in the field for agronomically favorable traits and genotyped using the KASP-N337 marker which has now been shown to be closely linked to the ‘N’ resistance gene for resistance to the southern root-knot nematode (SRKN). Greenhouse tests were conducted using selected lines and all three selections were confirmed to be resistant to SRKN. Fall field trials in root-knot nematode infected and uninfected fields are underway to perform field evaluations for these advanced lines and make a final selection before release. The recessive resistance locus (mj) confers resistance to the Javanese root-knot nematode (JRKN) in cucumber. Having previously mapped the interval where this gene resides within the cucumber genome, we developed new markers that were used to screen 1400 back cross individuals and successfully identify 18 recombination events that are being used for fine mapping of the mj resistance trait. These new markers were also used to select advanced recombinant inbred lines that retain the mj resistance trait and have more favorable agronomic traits. Additional genomic and transcriptomic experiments will be used to identify the gene responsible for mj resistance, so that it can be easily moved into cultivar quality lines.
The following progress is relative to Objective 2. PM resistant selections with red flesh and decent brix from a cross of USVL531-MDR X USVL677-PMS have been advanced to F10 and will be evaluated in the field next year. Phytophthora fruit rot resistant watermelon germplasm lines with red flesh have been identified in the RIL population (F10) and will be phenotyped for horticultural traits prior to release.
The guava root-knot nematode (GRKN, Meloidogyne enterolobii) is a hyper-virulent and invasive species in the southeastern U.S. and is causing serious damage on normally RKN resistant vegetable cultivars. We previously identified twenty sweetpotato Plant Introductions (PIs) for resistance against GRKN isolates from both South Carolina and North Carolina. This resistant germplasm has been incorporated to the sweetpotato breeding program at the USVL to begin developing GRKN resistant sweetpotatoes, and we have already selected and confirmed advanced lines with resistance to this nematode that could help farmers manage this destructive pest. After screening 192 pepper PIs for resistance to GRKN we have successfully selected line PMER-2 with enhanced resistance to GRKN, which was confirmed in replicated Greenhouse screens. We crossed this GRKN resistant line with the RKN resistant pepper cultivar ‘Charleston Belle’ to develop a mapping population that will be phenotyped and genotyped in order to map the genomic regions that are responsible for GRKN resistance in pepper. We also conducted a Genome Wide Association Scan for GRKN resistance in a population of 109 wild watermelon lines from the USVL collection and successfully identified QTL associated with resistance that will be used to develop new watermelon lines with enhanced resistance.
The following progress is relative to Objective 3. Seed increase of the various powdery mildew differential lines is a continuing process. Melon and watermelon powdery mildew race differentials were planted in April 2021 and April 2022 and rated for disease development. As during the past 6 years powdery mildew melon race 1 was the most prevalent in the area based on melon differentials. Isolates of powdery mildew on cucurbits including watermelon and other cucurbits were collected from California and South Carolina. Individual isolates in our collection were evaluated for their reaction on four-week-old watermelon seedlings in reach in growth chambers. The study further confirmed the presence of at least two races based on watermelon differentials USVL677-PMS and Mickey Lee.
Agrobacterium-Infectious clones of Cucurbit leaf crumple virus (CuLCrV) were used to phenotype sixteen watermelon lines for resistance. PI 386015, SP-6 and USVL531-MDR were resistant compared to All Sweet which was highly susceptible to CuLCrV. Trials to determine effect of silver plastic mulch on whiteflies and CuLCrV are in progress (#6080-22000-029-20I). Project to evaluate lines for resistance to PM has been on hold as we are waiting to receive a new set of seeds for screening (0000065259).
This project contributes to the NP 303 (Plant Diseases), Component 3A, Development and Deployment of Host Resistance. It also contributes to NP301 Components 1 and 2C.
Accomplishments
1. Resistance to Phytophthora crown rot in butternut squash. Butternut squash is an important vegetable crop grown and consumed in most states in the USA. Squash plants are also used as rootstocks for grafting watermelon and melon plants to help manage soil-borne diseases. In the southeastern USA it is difficult to grow squash and other vegetable crops as they are constantly infected by various diseases. One important disease-causing serious yield loss in squash is Phytophthora crown rot. Currently commercial varieties resistant to Phytophthora crown rot are not available and hence growers use pesticides to manage this problem. ARS researchers in Charleston, South Carolina, identified and developed several squash lines with resistance to Phytophthora crown rot. These new sources of resistance can be utilized for developing new crown and root rot resistant rootstocks for watermelon grafting and for developing resistant varieties for human consumption which will ultimately result in reduced pesticide use. The information will be useful for seed company breeders, private plant breeders, USDA, and university researchers for developing squash varieties with disease resistance.
2. Broad resistance to Phytophthora fruit rot in USDA developed watermelon germplasm. Watermelon is an important crop grown in forty-four states in the USA. Many different pests and diseases attack watermelon plants causing extensive damage. In recent years, an old, but re-emerging disease called Phytophthora fruit rot has been causing serious problems in watermelon production in many states in the U.S. including FL, GA, SC, NC, DE, MI, MD, etc. Weather conditions for fruit rot development is always prevalent in southeastern United states and hence the pathogen can infect the fruits at any stage. ARS researchers in in Charleston, South Carolina, developed and released Phytophthora fruit rot resistant germplasm lines for use by plant breeders. The present study demonstrated that the USDA ARS developed germplasm were resistant to 20 isolates of the plant pathogen collected from nine different states and different crops from across the United States. These resistant germplasm lines can be used to develop resistant cultivated type watermelon for managing the disease, thus helping reduce pesticide use. These fruit rot resistant watermelons accessions, and the information will be useful for public and private plant breeders for incorporating fruit rot resistance in watermelon cultivars.
3. Managing stubborn oomycete plant pathogens. Many kinds of plant Pathogens cause devastating losses to agriculture and native forests. Once such group of plant pathogens are known as oomycetes. One of the well-known oomycete called Phytophthora infestans was the cause of the great Irish famine in the mid-19th century. Significant research efforts have been conducted on these plant pathogens and helped advanced our understanding of these organisms. Nonetheless, managing them continues to be challenging. ARS researchers in Charleston, South Carolina, in collaboration with researchers from University and the private industry, brought together their expertise and have written papers for the first focus issue of Plant Health Progress published by the American Phytopathological Society (APS). This focus issue contains 28 peer-reviewed papers including three diagnostic guides, three mini reviews, three briefs, two surveys, and 17 research papers. All these peer-reviewed papers help advance our understanding of these stubborn oomycete pathogens. The information presented will be useful to plant pathologists, extension agents, for seed company breeders, private plant breeders, ARS and university researchers for developing strategies to manage oomycete plant pathogens.
4. Molecular markers to help breed powdery mildew resistant melons. Melon is a nutritious and delicious fruit crop grown across the United States. Powdery mildew is one of the most important diseases of melon that is known to reduce fruit quality and yield. Powdery mildew can infect the hypocotyls, cotyledons, true leaves, and stems of the melon plant and reduce its vigor. ARS researchers in Charleston, South Carolina, developed two molecular markers closely linked to powdery mildew resistance genes for use in breeding programs. The molecular markers will enable efficient marker-assisted introgression of powdery mildew resistance into improved melon cultivars. The information on easy to use molecular markers will be useful to vegetable seed company and University Plant breeders. Information on powdery mildew resistance will be useful to seed company and University researchers and extension agents.
Review Publications
Kousik, C.S., Ikerd, J.L., Wechter, W.P., Barnham, S., Turechek, W. 2022. Broad resistance to fruit rot in USVL watermelon germplasm lines to isolates of Phytophthora capsici from across USA. Plant Disease. 106:711-719. https://doi.org/10.1094/PDIS-11-20-2480-RE.
Branham, S.E., Kousik, C.S., Mandal, M., Wechter, W.P. 2021. QTL mapping of resistance to powdery mildew race 1 in a recombinant inbred line population of melon. Plant Disease. https://doi.org/10.1094/PDIS-12-20-2643-RE.
Kousik, C.S., Quesada-Ocampo, L.M., Keinath, A.P., Hausbeck, M., Granke, L., Naegele, R.P., Ji, P. 2021. Managing stubborn Oomycete plant pathogens. Plant Health Progress. 22:215-218. https://doi.org/10.1094/PHP-02-21-0047-FI.
Kousik, C.S., Vogel, G.M., Ikerd, J.L., Mandal, M.K., Mazourek, M., Smart, C.D., Turechek, W. 2021. New sources of resistance in winter squash (Cucurbita moschata) to phytophthora crown rot and their relationship to cultivated squash. Plant Health Progress. 22:323-331. https://doi.org/10.1094/PHP-02-21-0047-FI.
Rutter, W.B., Franco, J., Gleason, C. 2022. Rooting out the mechanisms of root-knot nematode-plant interactions. Annual Review of Phytopathology. https://doi.org/10.1146/annurev-phyto-021621-120943.
He, F., Wang, W., Rutter, W.B., Jordan, K., Ren, J., Akhunova, A., Szabo, L.J., Rouse, M.N., Akhunov, E. 2022. Genomic variants affecting homoeologous gene expression dosage contribute to agronomic trait variation in allopolyploid wheat. Nature Genetics. 13(1):1-15. https://doi.org/10.1038/s41467-022-28453-y.
