Location: Vegetable Research
2021 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
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). Screening the population indicated that resistance was conditioned by a dominant trait. RNA-seq identified significantly upregulated resistance genes in a major QTL region in chromosome 2 in USVL531-MDR. Using RNAseq and whole genome re-sequencing we identified a gene encoding an NBS-LRR resistance protein in chromosome 2 which was named ClaPMR2. Kompetitive allele specific polymorphisms (KASP) markers were developed using a single nucleotide polymorphism (SNP) in ClaPMR2 and used to assay segregating F2 populations. KASP markers identified the appropriate phenotype with 99% accuracy.
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 breeding population of USVL531-MDR X USVL677-PMS indicated that inheritance of resistance is complex and hence, a recombinant inbred line (RIL) population is being developed. We are currently increasing the seeds of RIL lines (F9) for use in genetic studies next year. A partial trail is being conducted this year using the RIL lines. 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.
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 and has resulted in losses of over $60 million in Florida. ARS scientists developed and released a source of resistance to SqVYV called 392291-VDR. 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 (F5) population from this cross for fine mapping is in progress. We completed RNAseq on 392291-VDR and Crimson Sweet to identify potentially upregulated genes involved in resistance. 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.
There is currently no known source of pepper resistance to the northern root-knot nematode (NRKN). Pepper line P1046 displayed a relatively high level of resistance in previous greenhouse screens at the USVL. Self-fertilized populations from all P1046 parents have also been generated from each parent plant. NRKN cultures were lost during the CoVID19 shutdown and will need to be required and bulked before the resistance in line P1046 can be confirmed. This will have to be performed at a later date when the team can enter the building full time.
The following progress is relative to Objective 2. Four powdery mildew resistant and one multiple disease resistant watermelon germplasm lines were released in 2018. PM resistant selections with red flesh and decent brix from a cross of USVL531-MDR X USVL677-PMS have been advanced to F7. These lines will be evaluated in the field next year. Backcross population of USVL608-PMR and ‘Dixie Lee has been screened and the most resistant selections advanced to the next generation. Several Phytophthora fruit rot resistant watermelon breeding lines with uniform red flesh and satisfactory sugar content (brix of 8) have been evaluated and advanced. Fruit rot resistant lines identified from a cross of USVL003 X Dixie lee were also advanced and screened. However, none of these progenies were resistant. We will screen additional lines of this cross next year.
Advanced BC3F2 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 BC3F3 lines and all three selections were confirmed to be resistant to SRKN. Root-knot nematode fields are being prepared to perform field evaluations for these advanced lines in Spring 2022 to 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 200 BC1 individuals and successfully identify 14 recombination events that will be used to develop lines for fine mapping of the mj resistance trait. These new markers were also used to select advanced BC4 lines that retain the mj resistance trait and have more favorable agronomic traits. Greenhouse screens were conducted to further fine map the mj resistance trait in cucumber.
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 July 2020 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 desktop growth chambers. The study 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 developed using the A and B particles. Appropriate concentrations of CuLCrV infectious clones for evaluation was determined and used for evaluating commercial watermelon varieties and select germplasm lines. Several commercial watermelon varieties and some germplasm lines displayed tolerance to CuLCrV.
Project on evaluation of biological treatments for controlling cucurbit powdery mildew was completed and submitted to AGBIOME. Project to evaluate lines for resistance to PM has been on hold due to maximum telework requirements during the pandemic. Project on environmental bacteria as potent biocontrol agents for controlling Phytophthora on Watermelon was completed in July 2021.
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 that will help farmers manage this destructive pest. After screening 191 pepper PIs for resistance to GRKN we have successfully selected line PMER-2 with enhanced resistance to GRKN. Greenhouse screens are underway to further confirm and quantify the level of resistance within this pepper line. We also conducted an initial screen of 126 watermelon lines from the USVL collection and have identified promising lines with potential resistance to GRKN. Additional screens are underway to confirm these results and potentially map QTL for this resistance. Depending on the status of the COVID 19 work restrictions we plan to initiate a similar set of screens for cucumber. We are conducting a second year of field tests to assess the economic viability and efficacy of using anaerobic soil disinfestation (ASD) and RKN resistant root stocks to manage RKN in tomato fields. Our early numbers show promising results with certain ASD-root stock combinations showing a significant reduction in RKN soil numbers. Due to the COVID-19 restrictions we were unable to hire any temporary workers for some projects, and very little progress was made on our portion of these grants.
Accomplishments
1. Resistance to guava root knot nematode in sweetpotato. The guava root-knot nematode (GRKN, Meloidogyne enterolobii) is a hyper-virulent and invasive species in the southeastern U.S. where it causes serious damage on a wide range of historically root-knot nematode resistant crops, including sweetpotato. ARS researchers in Charleston, South Carolina, screened sweetpotato from the USDA germplasm collections and identified twenty lines with strong resistance against GRKN isolates from both South Carolina and North Carolina. The information on these lines has been published and shared with sweetpotato breeding programs across the U.S. who are using this information to develop new GRKN resistant varieties. Once developed, these GRKN resistant sweetpotato varieties will help stakeholders manage and slow the spread of this emerging pest in the field. The information ARS has provided will be useful to both breeders and university researchers.
2. Powdery mildew resistant watermelon and markers for use in breeding cultivars. Watermelon is an important vegetable crop grown in 44 states in the U.S.A. Many diseases and pests attack watermelon seedlings and plants and reduce their yield resulting in monetary loss for growers. One such disease, known as powdery mildew, can infest watermelon seedlings and can cause reduced vigor or death of seedlings. Growers routinely spray pesticides to manage powdery mildew of watermelon. ARS researchers in Charleston South Carolina, developed an excellent source of resistance to this disease and characterized the mechanisms of resistance in this line called USVL531-MDR. They developed a molecular marker to be used in breeding programs to incorporate this resistance into commercial cultivars. The results of this study will be useful to seed industry and University vegetable breeders to develop new watermelon varieties with resistance to powdery mildew. The manuscript has been accessed over 2000 times since it was published. The results will also be useful to watermelon growers, extension workers, seed industry and University researchers to help manage powdery mildew in the greenhouse where watermelon seedlings are generally grown.
Review Publications
Mihir, M., Haktan, S., Kousik, C.S. 2020. Elucidation of resistance signaling and identification of powdery mildew resistant mapping loci (ClaPMR2) during watermelon-podosphaera xanthii interaction using RNA-seq and whole-genome resequencing approach. Scientific Reports. https://doi.org/10.1038/s41598-020-70932-z.
Kasmita, K., Coolong, T., Mandal, M., Kousik, C.S., Gitaitis, R., Hajihassani, A., Dutta, B. 2022. Micronutrients affect induced resistance in watermelon against Fusarium oxysporum f. sp. niveum and Meloidogyne incognita infection. Plant Disease. 10:796. https://doi.org/10.3390/pathogens10070796.
Rutter, W., Wadl, P.A., Mueller, J.D., Agudelo, P. 2021. Identification of sweetpotato germplasm resistant to pathotypically distinct isolates of Meloidogyne enterolobii from the Carolinas. Plant Disease. https://doi.org/10.1094/PDIS-02-20-0379-RE
