Location: Vegetable Research
2024 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. A recombinant inbred line (RIL) population developed from the cross of USVL531-MDR X UVL677-PMS was screened for resistance to PM in the field. Similarly populations (F1, F2, BCF1R and BCF1S) developed by crossing PM resistant USVL608-PMR crossed with susceptible lines USVL677-PMS, Sugar Baby and Dixie Lee were screened for resistance to PM. A major QTL in chromosome 2 was identified, and Kompetitive allele specific polymorphisms (KASP) markers were developed. KASP markers identified the appropriate phenotype with 99% accuracy in the F2 progeny of all the populations. KASP markers to identify specific resistance gene loci in different susceptible back grounds (USVL677-PMS, Sugar Baby & Dixie Lee) have been developed. In addition, KASP markers were used for marker assisted selection and progenies with good quality flesh were advanced to F4 generation.
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 and 2024. The RIL population described above was screened for Phytophthora fruit rot resistance in 2022 and twice in 2023 (Summer and Early Fall). Quantitative Trait Loci (QTL) analysis by combining the DNA bulks of the most resistant and susceptible RIL lines identified significant QTLs in Chromosome 2 and 6. In addition data on fruit qualities including, rind color and pattern, rind thickness, flesh color, flesh firmness, bitterness, brix, seed size, and seed type were also collected. Total DNA from 187 RIL lines used in these studies were extracted and whole genome resequencing (25X) has been completed. Phytophthora fruit rot resistant selections with red flesh and decent brix from the RIL population have been identified and are being evaluated in the field in 2024 for potential release. Results from these studies will provide markers and germplasm lines 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 and continues to be a problem in Southwest Florida. A RIL population from the cross of 392291-VDR (resistant) x Crimson sweet (susceptible) was advanced to the next generation. Experiments to identify potential genes involved in resistance using a transcriptomic approach were completed. The plasmodesmata callose binding protein (PDCB) gene, which is responsible callose deposition in the cells, was more highly expressed in the resistant line 392291-VDR than in Crimson Sweet before and after inoculation, suggesting inhibition of cell-to-cell movement of the virus. Several other genes that were being upregulated and play a role in plant defense were identified. Results from these studies will help fine map chromosomal regions associated with resistance and will provide markers that will enable breeders to develop new cultivars that are resistant to SqVYV.
We confirmed that a highly conserved gene in cucumber is responsible for the mj resistance trait, which confers resistance to specific species of root-knot nematode. This resistance gene, known as Systemic Acquired Resistance Deficient 1 (SARD1), is a transcription factor that is known in other plants to positively regulate plant defenses against a wide range of pathogens. Using a comparative transcript expression approach, we found for the first time that SARD1 is constitutively higher expressed in mj resistant cucumbers compared to susceptible cucumbers. In addition, we observed that the virulent southern root-knot nematode reduces the expression of SARD1 during infection, while the avirulent javanese root-knot nematode and the peanut root-knot nematode were unable to reduce the expression of SARD1 in resistant plants. These results show that the mj resistance trait has a unique mechanism of action that relies on the expression of SARD1. Because of the highly conserved nature of SARD1 and its responsiveness to nematode infection similar resistance mechanisms may be used to develop resistance in other plants and against other root-knot nematode species. This work contributes to Objective 1C.
The following progress is relative to objective 2. We are currently increasing the seeds of the S3 generation of the watermelon core collection. Screening watermelon plant introductions (PI) for resistance to Cucurbit leaf crumple virus (CuLCrV) using Agrobacterium-Infectious clones of) is being continued. In repeated tests, resistance to CuLCrV was identified in several wild watermelon germplasm (e.g. PI 560012, PI 560002, PI326516, and PI 560016). All Sweet and USVL677-PMS were highly susceptible to CuLCrV. ARS scientists in Charleston developed a system to quantify virus particles in host tissue using the QIAcuity digital polyemerase chain reaction system (dPCR) that was very effective in repeated tests. Screening of additional PI in the core collection is currently being conducted. Field studies were conducted to determine if watermelon PI identified as resistant in growth chamber studies were also resistant in the field at Fort Valley State University in Georgia. However, because of low populations of whiteflies CuLCrV was not observed in the susceptible varieties. Similarly, we conducted field studies 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). Additional screening Seeded and Seedless Watermelon Varieties for Resistance to the Whitefly-transmitted Cucurbit Leaf Crumple Virus is in progress (6080-22000-031-007S).
ARS researchers at Charleston, South Carolina, completed phenotyping screens of a biparental genetic mapping population generated from a cross between the guava root-knot nematode tolerant pepper line PMER-2 and the southern root-knot nematode resistant pepper line ‘Charleston Belle’ to produce mapping populations.
Individual F2 plants were sequenced in order to perform a Bulk Segregant Analysis that could identify genomic regions associated with guava root-knot nematode tolerance. At the same time, we are also using our new controlled environment pods to implement a new speed breeding approach to more quickly develop a RIL population of F6-F8 individuals that will facilitate additional mapping experiments and utilization of this population. Once we have identified this region, we will use marker assisted selection to transfer it into a horticulturally favorable pepper lines. This work directly contributes to Objective 2B.
The following progress is relative to objective 3. Seed increase of various powdery mildew differential lines is a continuing process. Melon and watermelon powdery mildew race differentials were planted in April 2023 and May 2024 and rated for disease development. As during the past 10 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. Isolates of the PM pathogen are maintained on squash cotyledons in the lab by transferring once every three weeks, as these need a live host to survive. 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. The cucurbit powdery mildew pathogen genus was confirmed using molecular markers and microscopic studies.
A second round of embryogenesis assays comparing the development of the guava root-knot nematode with that of the southern root-knot nematode at high temperatures was completed. Our results confirm that guava root-knot nematode develops much faster and is much more viable at higher temperatures then the southern root-knot nematode. Ongoing experiments at lower temperatures will allow us to calculate an accurate baseline temperature for the guava root knot nematode. By comparing the relative baseline temperature of this nematode to that of the endemic southern root-knot nematode we should get a better understanding of the potential climatic regions where this guava root-knot nematode is likely to pose a threat. This work directly contributes to objective 3B.
ARS researchers are Charleston, South Carolina, collaborated with Clemson University to screen pepper lines with known sources of nematode resistance against the peach root-knot nematode. We confirmed that the peach root-knot nematode breaks all the previously known root-knot nematode resistance genes in pepper. Interestingly, we also found that one of previously released USDA ARS pepper lines PA136, that was previously thought to be universally susceptible, was highly resistant to this nematode species, indicating that it contains a yet unknown resistance gene that could be useful in managing the peach root-knot nematode in pepper. We have begun generating a biparental mapping population by crossing PA136 with ‘Charleston Belle’ in hopes of mapping this resistance gene in the future. This work contributes to both objective 1 and objective 2 of the project plan.
Accomplishments
1. Development of a High-throughput Method for Detecting Guava Root-knot Nematode in Sweetpotato. Guava Root-knot nematode (GRKN) is an invasive and quarantined pest that is spreading across the US sweetpotato industry. The most popular sweetpotato varieties in the United States are highly susceptible to this nematode, and the transport and planting of nematode infected sweetpotato ‘seed’ roots is a major avenue by which GRKN is spread to new fields. To help prevent the further spread of this nematode, ARS researchers in Charleston, South Carolina, developed and tested a high-throughput method for detecting GRKN in commercial sweetpotato operations and provided training and assistance to state extension services to help them implement this new detection method. Compared to traditional nematode survey methods, this new method allows for quicker sampling and more sensitive detection of GRKN in batches of sweetpotato roots. This will significantly reduce the labor and technical expertise needed for GRKN sampling and identification and could help slow the spread of GRKN to new sweetpotato fields.
Review Publications
Culbreath, J.R., Wram, C.L., Bechtel, T., Wadl, P.A., Muller, J., Khanal, C., Rutter, W.B. 2023. A high-throughput sampling method for detection of Meloidogyne enterolobii and other root-knot nematodes in sweetpotato storage roots. Crop Protection. 174. Article 106401. https://doi.org/10.1016/j.cropro.2023.106401.
Wu, S., Sun, H., Gao, L., Branham, S., Mcgregor, C., Xu, Y., Kousik, C.S., Wechter, W., Levi, A., Fei, Z. 2023. A Citrullus genus super-pangenome reveals extensive variations in wild and cultivated watermelons and sheds light on watermelon evolution and domestication. Plant Biotechnology Journal. 21(10):1926-1928. https://doi.org/10.1111/pbi.14120.
Zheng, Y., Wu, S., Bai, Y., Sun, H., Jiao, C., Guo, S., Zhao, K., Blanca, J., Zhang, Z., Huang, S., Xu, Y., Weng, Y., Mazourek, M., Reddy, U.K., Ando, K., Mccreight, J.D., Schaffer, A., Burger, J., Tadmor, Y., Katzir, N., Tang, Z., Liu, Y., Giovannoni, J.J., Ling, K., Wechter, P.W., Levi, A., Garcia-Mas, J., Grumet, R., Fei, Z. 2018. Cucurbit Genomics Database (CuGenDB): a central portal for comparative and functional genomics of cucurbit crops. Nucleic Acids Research. (2019): Vol. 4747(D1):D1128-D1136.. https://doi.org/10.1093/nar/gky944.
Delorean, E.E., Youngblood, R.C., Simpson, S.A., Schoonmaker, A.N., Scheffler, B.E., Rutter, W.B., Hulse-Kemp, A.M. 2023. Representing true plant genomes: haplotype-resolved hybrid pepper genome through trio binning. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2023.1184112.
Alam, M.S., Khanal, C., Roberts, J., Rutter, W.B., Wadl, P.A. 2024. Enhancing reniform nematode management in sweet potato by complementing host-plant resistance with non-fumigant. Plant Disease. https://doi.org/10.1094/PDIS-07-23-1412-RE.
Quesada-Ocampo, L.M., Parada-Rojas, C.H., Hansen, Z., Vogel, G., Smart, C., Hausbeck, M.K., Carmo, R.M., Huitema, E., Naegele, R.P., Kousik, C.S., Tandy, P., Lamour, K. 2023. Phytophthora capsici: recent progress on fundamental biology and disease manangement 100 years after its description. Annual Review of Phytopathology. 61:185-208. https://doi.org/10.1146/annurev-phyto-021622-103801.