Location: Vegetable Research
2020 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 of watermelon is a major factor limiting production throughout the United States where the crop is grown. Five powdery mildew resistant (PMR) watermelon germplasm lines (USVL531-MDR, USVL608-PMR, USVL255-PMR, USVL313-PMR, and USVL585-PMR) were crossed with a powdery mildew susceptible line USVL677-PMS and a susceptible commercial cultivar Dixie Lee to develop populations (F1, F2, BCF1R and BCF1S) to determine genetics of resistance and develop molecular markers. Chi-square analyses of segregating F2 plants of cross of USVL608-PMR X USVL677-PMS indicated that two genes control PM resistance with a good fit for a 7:9 resistance to susceptibility ratio. The proposed model for this ratio is two genes with one recessive for high resistance and one dominant for high resistance. Quantitative trait loci (QTL)-seq analysis of the extremes from the F2 populations indicated a major QTL on Chromosome 2. RNA-seq identified significantly upregulated resistance genes in the major QTL region in USVL608-PMR compared to USVL677-PMS. Similarly, a gene associated with PM resistance was identified in USVL531-MDR using RNAseq and whole genome sequencing and was named ClaPMR2. Molecular marker was developed using a single nucleotide polymorphism (SNP) in ClaPMR2 and used to assay segregating F2 populations. USVL608-PMR and USVL531-MDR will serve as a useful source to incorporate PM resistance into commercial cultivars. The molecular marker will be useful to University and commercial breeders.
Phytophthora fruit rot is a serious disease that has plagued watermelon growers in the eastern U.S. for the past several years, and the National Watermelon Association (NWA) has considered it their top research priority. USVL531-MDR, which is resistant to Phytophthora fruit rot was crossed with a susceptible line to develop breeding populations. Studies indicated that inheritance of resistance is complex and hence a recombinant inbred line (RIL) population is being developed. At present the RIL lines are at the F9 stage and will used in genetic studies next year as field trials could not be done due to the pandemic. Several Phytophthora fruit rot resistant watermelon breeding lines with uniform red flesh and satisfactory sugar content (brix of 8) have been evaluated and advanced. RNAseq experiments to determine genes expressed during resistance vs susceptible Phytophthora and fruit interactions have been completed. Data analysis is in progress.
Watermelon vine decline (WVD) is a serious disease that has plagued watermelon growers for the past several years and has resulted in losses of over $60 million in southwest and west central Florida. Watermelon vine decline is caused by a virus called Squash Vein Yellowing Virus (SqVYV) which is transmitted by whiteflies. 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 tolerance to SqVYV in a growth chamber by mechanical inoculations. Preliminary analysis indicated that resistance may be governed by recessive genes. Generation of an RIL (F4) population is in progress for use in fine mapping. We confirmed resistance in three new accessions to SqVYV in screens conducted in Immokalee, Florida, in spring 2019 and in a growth chamber in Charleston, South Carolina. Two of these accessions continued to show high levels of resistance to WVD and Cucurbit leaf crumple virus (CuLCrV).
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 and are being re-evaluated using another isolate of the NRKN from North Carolina is being used confirm that the resistance trait the CoVID19 shutdown has interrupted this screen which will have to be repeated at a later date when the team can enter the building full time.
The following progress is relative to Objective 2. As a part of Objective 1, four powdery mildew resistant watermelon germplasm lines were advanced and released in 2018. Crosses were made between USVL608-PMR and ‘Dixie Lee’ to develop breeding populations (F1, backcrosses). Backcross population has been screened and the most resistant selections advanced to the next generation. PM resistant selections with red flesh and decent brix from a cross of USVL531-MDRX USVL677-PMS have been advanced to F6. These will be evaluated in the field 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). BC3F3 seed has been developed, and depending on the status of the CoVID19 shutdown will be advanced and screened for nematode resistance in the greenhouse before field evaluations planned for spring 2021.
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 BC3 lines that retain the mj resistance trait and have more favorable agronomic traits. Depending on the status of the CoVID19 shutdown, recombination event lines will be screened against JRKN in order to further fine map the mj trait, and advanced BC4F2 seed will be developed for evaluation and future release.
The following progress is relative to Objective 3. Seed increase of the various powdery mildew differential lines were done and is being continued. Melon and watermelon powdery mildew race differentials were planted in April 2019 and July 2020 and were rated for disease development. As during the past 6 years powdery mildew melon race 1 was the only race prevalent in the area based on disease reaction on the melon differentials in the field. Isolates of powdery mildew on cucurbits including watermelon and other cucurbits were collected from California, Florida, Texas, and South Carolina. Individual isolates were evaluated for their reaction on true leaves of watermelon. This study indicated the presence of at least two races based on watermelon differentials.
The following progress is relative to incoming agreements. Progress on project 6080-22000-029-19-S. 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 cultivars of sweetpotato. We previously identified twenty 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. We have also screened 191 pepper PIs for resistance to GRKN, and identified seven individual plants with potential resistance to this nematode. These plants have been propagated and are being grown for seed in the greenhouse. Depending on the status of the CoVID19 shutdown these lines will be evaluated further for GRKN resistance.
Experiments to determine the effect of biological treatments for managing cucurbit powdery mildew were conducted during fall 2018 and summer 2019. Squash and watermelon plants were treated with various formulations of a biocontrol bacteria. At higher concentrations, one of the biocontrol formulations was effective in reducing disease severity compared to the check and looks promising. (Project# 6080-22000-029-18H). These studies have been completed.
Sequencing of local isolate of CuLCrV was completed. Clones are being developed for use in evaluation of watermelon PI for resistance. Two new sources of resistance to CuLCrV were identified in evaluations conducted in Immokalee, Florida, in 2019. Cucurbit Yellow Stunt Disorder Virus (CYSDV) another whitefly transmitted was detected for the first time in South Carolina. Now all three whitefly transmitted viruses (SqVYV, CuLCrV and CYSDV) on cucurbits are present in the state (#6080-22000-029-20I).
The project to evaluate lines for resistance to PM has been on hold due to the maximum telework during the pandemic (65259). Three projects (62394, 62395, 62397) were completed in 2018-2019 and reports submitted to National Watermelon Association and presented in last years annual report (AD421).
Watermelon grafting is slowly gaining popularity in the United States. A hybrid of Cucurbita moschata X Cucurbita maxima is generally used in many regions of the world as rootstock for watermelon grafting because of its natural resistance to Fusarium wilt of watermelon. However, these rootstocks are highly susceptible to Phytophthora crown rot. We identified sixteen C. moschata accessions with high levels of resistance to Phytophthora crown rot. Of these, 13 accessions were reevaluated and advanced to S3 generation for development of resistant germplasm lines. Similarly, two C. maxima lines with resistance to crown rot have been advanced to S3 generation. Crosses between resistant C. moschata and C. maxima have been made. Two pepper lines with resistance to crown rot have been developed. Large scale grower field trials using these lines as rootstocks is currently being conducted in Ohio with promising results (Project# 432455).
Accomplishments
1. Relative susceptibility of watermelon varieties to powdery mildew. 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, evaluated many available commercial varieties for their reaction to powdery mildew and identified that a few pollenizers and one variety has some level of resistance. The results indicate a need to develop newer varieties with resistance to powdery mildew. Varieties with resistance to powdery mildew will help reduce the amount of pesticides applied on watermelon. The results of this study will be useful to watermelon growers, extension workers, seed industry and University researchers to help manage powdery mildew in the greenhouse and the field.
Review Publications
Kousik, C.S., Ikerd, J.L., Mandal, M. 2019. Relative susceptibility of commercial watermelon varieties to powdery mildew. Crop Protection. https://doi.org/10.1016/j.cropro.2019.104910
Anco, D., Rouse, L., Lucas, L., Parks, F., Mellinger, C., Adkins, S.T., Kousik, C.S., Roberts, P., Stansly, P., Ha, M., Turechek, W. 2019. Spatial and temporal physiognomies of whitefly and tomato yellow leaf curl virus epidemics in southwestern Florida tomato fields. Phytopathology. 110(1):130-145. https://doi.org/10.1094/PHYTO-05-19-0183-FI.
Hajihassani, A., Rutter, W.B., Schwarz, T., Woldemariam, M.W., Ali, E., Hamidi, N. 2020. Characterization of resistance to major tropical root-knot nematodes (Meloidogyne spp.) in solanum sisymbriifolium. Phytopathology. 110:666-673. https://doi.org/10.1094/PHYTO-10-19-0393-R.
Kousik, C.S., Adkins, S.T. 2020. Detection of cucurbit yellow stunting disorder virus (CYSDV) infecting watermelon in South Carolina. Plant Health Progress. https://doi.org/10.1094/php-03-20-0016-br.
