Research Project: Characterization of Host Resistance and Biology of Diseases and Nematodes in Vegetable Crops

Location: Vegetable Research

2023 Annual Report

Objectives
1. Identify and characterize disease and nematode resistance in vegetable crops, and develop molecular tools to enhance breeding efforts for resistance. 1.A. Determine genetic basis of resistance to powdery mildew (PM) and Phytophthora fruit rot in watermelon and develop molecular markers linked to resistance genes. 1.B. Fine map quantitative trait loci (QTL) conferring resistance to watermelon vine decline (WVD) caused by Squash vein yellowing virus (SqVYV) and develop molecular markers. 1.C. Develop markers for and characterize the molecular mechanisms of resistance to root-knot nematode in cucumber. 2. Identify and develop vegetable germplasm and breeding lines with enhanced resistance to diseases and nematodes. 2.A. Identify sources of resistance to the whitefly-transmitted Cucurbit leaf crumple virus (CuLCrV) in watermelon. 2.B. Develop pepper breeding lines with pyramided nematode resistance genes. 3. Characterize population dynamics to improve biological understanding of existing and emerging fungal and nematode pathogens in the southeastern United States. 3.A. Characterize powdery mildew (Podosphaera xanthii) isolates collected from cucurbits in southeastern U.S. 3.B. Characterize the influence of temperature on the development of the guava root-knot nematode (Meloidogyne enterolobii).

Approach
This approach will identify and develop resistant germplasm and breeding lines for managing Phytophthora fruit rot(PFR),powdery mildew(PM),whitefly-transmitted Squash vien yellowing virus(SqVYV), Cucurbit leaf crumple virus(CuLCrV), and root-knot nematodes(RKN). Modern reisstance phenotyping and breeding techniques will be utilized to accomplished our objectives. A recombinant inbred line(RIL) population will be developed by crossing a multiple disease resistant watermelon line (PFR and PM) and a susceptible line. The RIL population will be phenotyped for resistance to PFR and PM to determine genetics of resistance. Analysis of differentially expressed genes by RNA-Seq resulting from Phytophthora and watermelon fruit interactions will be used to further elucidate genetics of fruit rot resistance. Red fleshed RILs with resistance to PFR and/or PM will be evaluated for fruit quality and selected for release. Similarly, a RIL population developed from a cross of SqVYV resistant watermelon line and a susceptible cultivar will be phenotyped to detremine genetics of resistance and develop molecular markers for use in breeding programs. We will phenotype cucumber lines from a mapping population to fine map the mj resistance gene. The resulting candidate resistance genes will be cloned and characterized to provide pathologists with a better understanding of the biological underpinnings of RKN resistance and provide molecular tools to assist breeders in developing resistant cucumbers. Agrobacterium infectious clones of CuLCrV have been developed for phenotyping watermelon and other susceptible vegetable crops for resistance. The clones will be used to phenotype the watermelon core collection available with USDA and sources of resistance will be identified for use in breeding programs. We will cross the M. enterolobii resistant pepper line PMER-2 with the M. incognita resistant 'Charleston Belle' and develop populations to assess the inheritance pattern of M. enterolobii resistance. If resistance from PMER-2 is simply inherited, wewill be able to select M. enterolobii resistant breeding lines. Development of new RKN resistant pepper lines with resistance against both the invasive M. enterolobii and the endemic RKN species will provide valuable tools to help pepper growers in southern U.S. to manage these pests in the field. We will conduct side-by-side developmental embryogenesis assays with M. enterolobii and M. incognita over a range of temperatures to compare the thermal developmental profiles of these two species and for the first time calculate an estimate of the thermal base temperature and optimal thermal temperature for M. enterolobii. A greater understanding of the thermal factors that underlie the development in M. enterolobii will provide insight into the growing regions in the U.S. that may be threatened by this nematode as it continues to spread. We have a large collection of isolates of cucurbit powdery mildrew pathogen collected from accross the U.S. This collection will be genotyped using simple sequence repeat(SSR) markers developed based on an available sequence and used to characterize diversity among the pathogen population.

Progress Report
The following progress is relative to Objective 1. Powdery mildew (PM) of watermelon is a major factor limiting production throughout the United States. Resistant lines USVL531-MDR and USVL608-PMR were 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 in both populations. KASP markers also helped identify powdery mildew resistant phenotype using other population developed from cross of USVL608-PMR X Sugar Baby. 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 in 2023. A recombinant inbred line (RIL) population developed from the cross of USVL531-MDR X UVL677-PMS was pheotyped for Phytophthora fruit rot resistance in 2022 and 2023. The data from these trials is being analyzed. Total DNA from 192 RIL lines used in these studies has been extracted and will be used for whole genome resequencing (20X). Seeds of RIL lines are also being increased for use in genetic studies next year. Results from these studies will provide markers that will enable breeders to develop new cultivars that are resistant to fruit rot. 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 and decent brix have been identified in the RIL population (F10) and were phenotyped for horticultural traits in Fort Pierce and similar experiments are in progress in Charleston. 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 and has been advanced to F7. Results from these studies will help fine map chromosomal regions significantly associated with resistance and will provide markers that will enable breeders to develop new cultivars that are resistant to SqVYV. Studies to develop markers and characterize the molecular mechanisms of resistance to root-knot nematode in cucumber have been progressing well. A candidate resistance gene that underlies the mj nematode resistance trait in cucumber was identified. Marker assisted selection (MAS) was utilized to screen over 1400 backcross lines and identify 18 cucumber lines with recombination events within the mj genomic region. Each recombination line was phenotyped for the mj resistance trait, and based on the results from these experiments we conducted whole genome resequencing on select lines and were able to use these data to identify the specific gene responsible for the mj resistance trait. New experiments have been initiated with the mj resistance gene to try and understand how this gene functions in root-knot nematode parasitism, and how we can potentially use it to transfer this resistance trait to other crops and other root-knot nematodes. This work directly contributes to Objective 1C. The following progress is relative to Objective 2. Agrobacterium-Infectious clones of Cucurbit leaf crumple virus (CuLCrV) were used to phenotype watermelon cultivars and plant introductions (PI) for resistance. PI 386015, SP6 and USVL531-MDR were resistant compared to All Sweet which was highly susceptible to CuLCrV. Resistance to CuLCrV was identified in several wild watermelon germplasm (e.g. PI 560012, PI 560002, PI326516, and PI 560016). Several susceptible wild watermelon PI were also identified (PI 247398 and PI 254740) that were as susceptible as some of the commercialy cultivated watermelon type such as All sweet and sugar Baby. A system to quantify virus particles in host tissue using the QIAcuity digital polyemerase chain reaction system (dPCR) was also developed and used to determine actual copy numbers of CuLCrV virus particles in in growing tips of inoculated plants. Significantly greater copy numbers of CuLCrV were detected in All Sweet (>4000 copies / 0.1 ng total DNA) compared to very low numbers in SP6 and USVL531-MDR (<200 copies) and PI 386015 (<10 copies). Significant (=0.0001) correlations between disease ratings and CuLCrV copy numbers was also observed. Field studies were conducted to determine the combined effect of silver plastic mulch, chemical treatments to manage whiteflies and resistance on CuLCrV development. Study indicated that Chemical treatments helped manage whiteflies. However, we did not observe any CuLCrV infection in the field (#6080-22000-029-20I). Screening Seeded and Seedless Watermelon Varieties for Resistance to the Whitefly-transmitted Cucurbit Leaf Crumple Virus is in progress (6080-22000-031-007S) 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. The guava root-knot nematode tolerant pepper line PMER-2 was successfully crossed with the southern root-knot nematode resistant pepper line ‘Charleston Belle’ to produce mapping populations. The genomes of both of these parent plants have been sequenced to facilitate genetic mapping. Greenhouse trials are underway to simultaneously phenotype and genotype the mapping populations to try and identify the genomic regions responsible for the root-knot nematode tolerance in line PMER-2. Once this region is identified, we will use marker assisted selection to transfer it into a horticulturally acceptable line. This work directly contributes to Objective 2B. 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 2022 and April 2023 and rated for disease development. As during the past 8 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 various states in the USA. 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. Using a transcriptomics based approach molecular markers for the cucurbit powdery mildew pathogen have been identified and used on quantifying PM development of resistant and susceptible genotypes. A temperature sensor was successfully designed to accurately monitor and log the temperatures experienced by developing nematodes on a microscope slide. This sensor will allow us to accurately model and characterize the development of guava root-knot nematode eggs during embryogenesis assays at different temperatures and there by better understand the potential climatic regions where this nematode is likely to pose a threat. This work directly contributes to Objective 3B. A machine learning approach was utilized to train a computer algorithm to count nematode eggs in microscope images. Researchers at U.S. Vegetable Laboratory, USDA ARS in Charleston, SC worked with the USDA ARS Breeding Insight group to develop an image analysis pipeline to automate the process of counting root-knot nematode eggs under a microscope. Once the system is validated and published this pipeline will significantly increase our capacity to test plants for root knot nematode resistance and speed the process of developing new root-knot nematode resistant vegetable lines and cultivars. This work contributes to both objective 1 and objective 2 of the project plan and other related projects (6080-22000-031-004R). Over 100 additional sweetpotato genotypes were phenotyped for resistance to the guava root-knot nematode which has been causing significant damage and regulatory concerns in the sweetpotato industry. These screens were part of a genome wide association study designed to identify the genes in sweetpotato responsible for resistance to this nematode. By identifying these genes we will be providing sweetpotato breeders with valuable tools to speed the process of developing new guava root-knot nematode resistant sweetpotato varieties. This work contributes to both Objective 1 and Objective 2 of the project plan and other related projects (6080-22000-031-003R). 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. Identification of guava root-knot nematode resistance in wild watermelon. The guava root-knot nematode is an invasive plant parasite that poses a significant threat to many crops in the southeastern United States, including watermelon. ARS researchers in Charleston, South Carolina, are working to develop new watermelon varieties that are resistant to this pest and will protect stakeholder yields while also reducing the need to spray costly and environmentally damaging nematicides in their fields. To help develop guava root-knot nematode resistant watermelon varieties we screened 109 wild watermelon lines for resistance to this nematode. We successfully identified 28 resistant wild watermelon lines that will provide useful germplasm for breeding new watermelon varieties. We also conducted a genome wide association scan to identify regions of the wild watermelon genome that are responsible for this resistance. We published the results of this study in the journal Plant Disease. The data from this study will provide watermelon breeders with valuable germplasm and genetic markers to help select new guava root-knot nematode resistant watermelon varieties.

2. Multiple disease resistant watermelon germplasm line USVL531-MDR. Watermelon is an important vegetable crop grown in 44 states in the U.S.A. Many diseases and pests attack watermelon seedlings and plants including fruit and reduce their yield resulting in monetary loss for growers. Phytophthora fruit rot and powdery mildew are two such diseases that can significantly reduce watermelon yield and quality. Growers generally spray expensive pesticides to manage these two diseases. ARS scientists in Charleston, South Carolina, have developed a watermelon germplasm line (USVL531-MDR) with resistance to powdery mildew and Phytophthora fruit rot. This resistant germplasm lines can be used by private and public sector plant breeders to develop watermelon varieties and breeding lines with resistance to these two diseases. Development of varieties with resistance to powdery mildew and Phytophthora fruit rot will help reduce pesticide use and allow watermelon growers to produce a healthy crop. The information will be useful to growers, seed company plant breeders, University researchers, extension agents and USDA scientists. The article made the cover of the April issue of the peer reviewed journal HortScience.

Review Publications
Alam, S., Khanal, C., Rutter, W.B., Roberts, J. 2022. Non-fumigant nematicides are promising alternatives to fumigants for the management of Meloidogyne enterolobii in tobacco. Journal of Nematology. 54(1):3922. https://doi.org/10.2478/jofnem-2022-0045.
Waldo, B.D., Branham, S.E., Levi, A., Wechter, W.P., Rutter, W.B. 2022. Distinct genomic loci underlie quantitative resistance to Meloidogyne enterolobii galling and reproduction in Citrullus amarus. Plant Disease. 107:2126-2132. https://doi.org/10.1094/PDIS-09-22-2228-RE.
Karki, K., Cooling, T., Kousik, C.S., Myers, B., Hajihassani, A., Mandal, M., Dutta, B. 2021. Transcriptomic profile of watermelon affected by zinc in presence of Fusarium oxysporum f. sp. niveum and Meloidogyne incognita. Pathogens. https://doi.org/10.3390/pathogens10070796.
Parada-Rojas, C.H., Granke, L.L., Naegele, R.P., Hansen, Z., Hausbeck, M.K., Kousik, C.S., Mcgrath, M.T., Smart, C., Quesada-Ocampo, L.M. 2021. A diagnostic guide for Phytophthora capsici infecting vegetable crops. Plant Health Progress. 22:404-414. https://doi.org/10.1094/PHP-02-21-0027-FI.
Wadl, P.A., Campbell, H.T., Rutter, W.B., Williams III, L.H., Murphey, V., Culbreath, J., Cutulle, M. 2023. A sustainable approach for weed and insect management in sweetpotato: breeding for weed and insect tolerant/resistant clones. Weed Technology. 37(1):60-66. https://doi.org/10.1017/wet.2022.99.
Kousik, C.S., Ikerd, J.L., Mandal, M., Adkins, S., Turechek, W. 2023. USVL531-MDR: Watermelon germplasm line with broad resistance to powdery mildew and phytophthora fruit rot. HortScience. 58(4):475-479. https://doi.org/10.21273/HORTSCI16907-22.
Sanago, S., Lamour, K., Kousik, C.S., Parado-Rojas, C., Quesada-Ocampo, L., Wyenandt, A., Babadoost, M., Hausbeck, M., Hansen, Z., Ali, E., Mcgrath, M., Hu, J., Crosby, K., Lozada, D., Miller, S. 2022. Phytophthora capsici, 100 years later: Research mile markers from 1922 to 2022. Phytopathology. https://doi.org/10.1094/PHYTO-08-22-0297-RVW.
Slonecki, T.J., Rutter, W.B., Olukolu, B., Yencho, G.C., Jackson, D.M., Wadl, P.A. 2023. Genetic diversity, population structure, and selection of core germplasm sets from the USDA sweetpotatoe (lpomomea batatas) collection. Horticulture Research. https://doi.org/10.3389/fpls.2022.1022555.