2018 CucCAP Watermelon Team Annual Report

cuccap

Progress Reports and Work Plans

Watermelon Team members: Amnon Levi (USDA, ARS), Shaker Kousik (USDA, ARS), Kai-shu Ling (USDA, ARS), Cecilia McGregor (Univ. Georgia), Pat Wechter (USDA, ARS), and Todd Wehner (North Carolina St. Univ.) reported on team progress and work plans.

Overall objectives: Identifying quantitative trait loci (QTL) associated with resistance to major and emerging diseases, developing useful molecular markers and utilizing the genomic tools to incorporate resistance into watermelon cultivars. 

Major diseases: Gummy stem blight, Fusarium wilt, Powdery mildew, Phytophthora fruit rot, Papaya ringspot virus (PRSV) and Cucumber green motile mosaic virus (CGMMV).

Work in progress and plans

1.2. Perform GBS analysis of PI collections, establish core populations of the four species, and provide community resource for genome wide association studies (GWAS)

(Takshay Patel and Todd C. Wehner)

1.2.1. GBS of cucurbit species, establish molecular-informed core populations

Objective: Develop molecular markers for high resistance to gummy stem blight (GSB) using genome-wide association studies (GWAS) in the USDA watermelon germplasm collection, and introgress GSB resistance into watermelon cultivars.

We are collecting and increasing Citrullus PI accessions, heirloom cultivars, and gene mutant type-lines. Seed increase of the 2000 PI accessions is being accomplished by seed companies, USDA scientists, and university researchers. Each is increasing 1 to 10 accessions per year using controlled pollination in greenhouse or field.

Association analysis: Collected phenotypic and genotypic data will be analyzed using R packages: SNPassoc, snpMatrix, GenABEL and pbatR. The result of the analysis will allow us to locate and identify SNP markers associated with GSB resistance.

Gene type lines                                                                                                                 

Sixty-two available accessions.

Collection and seed increase of the watermelon gene type-lines will include all cultivars, breeding lines, and PI accessions in the gene mutant list at Cucurbit Genetics Cooperative. Examples include:
PI 189225 (db, Ar-2-1), NC-517 (C), PI 482261 (Ctr), Bush Charleston Gray (dw-1), PI 595203 (zym-CH, zym-FL).

Watermelon gummy stem blight resistance

(Luis Rivera and Todd C. Wehner)

Objective: a) Evaluate a RIL population of watermelon (Citrullus lanatus × C. amarus) for resistance to gummy stem blight and fruit quality traits and b) Map GSB resistant genes through genome-wide association studies (GWAS).

Phenotyping: A watermelon GSB population was developed by intercrossing the most resistant accessions of Citrullus four times (I4), followed by crossing with elite cultivars of watermelon (I4F1), followed by intercrossing without selection, while maintaining wild and elite types in the populations (I4F1I4), followed by self-pollinations of plants at random (I4F1I4S1)

Figure 1. Greenhouse test for resistance to gummy stem blight

Figure 2. Gummy stem blight symptoms during the greenhouse test

The 300 RILs and 20 controls (10 PIs and 10 commercial cultivars) were evaluated for resistance to gummy stem blight in greenhouses at North Carolina State University in Raleigh, North Carolina (Figure 1 and 2), and in the field at the Horticultural Crops Research Station at Clinton, North Carolina (Figure 3). We inoculated plants with S. cucurbitacearum at a concentration of 5×105 spores/ml (Figure 4). To evaluate disease severity, we adopted an ordinal disease assessment scale (Gusmini et al. 2002). Plants were rated four times, in an experiment with, 2 locations, and 10 replications (at greenhouse and field). We also evaluated fruit quality in the gummy stem blight field trial. We also collected data of fruit shape, rind pattern and toughness, seed size and color, flesh color and intensity and hollow heart. We will identify RILs with high yield of excellent fruit quality.

Additionally, genomic DNA of gummy stem blight isolates collected from field outbreaks was extracted, and a PCR-based marker test for distinguishing the three morphologically identical, but genetically distinct species causing gummy stem blight was performed (Figure 4). We used three sets of primers, including Db05 that produces a 216 to 224-bp fragment in all three species, Db06 that produces a 283- to 289-bp in S. citrulli and a 268-bp and slightly fainter fragment in S. cucurbitacearum, and Db01 that produces a 256- to 364-bp fragment in S. citrulli (Brewer et al. 2015). Two of the isolates were S. cucurbitacearum (syn. Dydymella bryoniae) and one isolate was S. caricae.

Genotyping: The 300 RILs will be planted in spring 2018, at greenhouses of NC State, to sample leaf tissue for DNA extraction. The DNA will be send for SNPs discovery through genotyping by sequencing (GBS) method at Cornell University. We expect to get several thousand of SNPs for the association analysis (GWAS).

Association analysis: Collected phenotypic and genotypic data will be analyzed using R packages: GWASTools, GWASdata, SNPassoc, snpMatrix, GenABEL and pbatR. The result of the analysis will allow us to locate and identify SNP markers associated with GSB resistance.

Figure 3. Field test for resistance to gummy stem blight

Figure 4. Gummy stem blight spore mass production and identification through PCR and electrophoresis

QTL Mapping, Marker Validation and Trait introgression of Gummy Stem Blight resistance in Watermelon

(Cecilia McGregor and Winnie Gimode, University of Georgia, Athens, Georgia).

1.1 Population Development:

  • WPop GSB1: PI 482276 x Crimson Sweet population of 225 F2:3 Complete
  • Backcross population: WPop GSB 1BC: BCF2 for PI 482276 x Crimson Sweet (recurrent) for trait introgression and marker validation. In progress
  • WPop GSB2: PI 526233 x Sugar Baby population for 96 F2:3 Complete – This population will however not be phenotyped because the ‘resistant’ parent (PI 526233) has proven to be rather susceptible from the screens. This population has therefore been replaced with another population: WPop GSB2B: PI 189225 x Sugar Baby. In progress
  • WPop GSB3: Backup population of resistant amarus (PI 482276) x susceptible C. amarus. In progress

1.2 Phenotyping

  • GSB1 F2:3 Population (6 plants x 225 lines = 1,350 plants) was phenotyped in walk-in growth chamber. Disease symptoms for each seedling were scored on a 0 – 5 scale. Parents and F1 and 4 other control genotypes were also included.
  • All the parental lines and a subset (14 plants x 95 lines = 1330 plants) of the GSB1 population were also phenotyped in the field in Attapulgus (GA) in summer 2017. Natural infection was allowed to take place. The purpose of this was to compare field level resistance to what we observe in the growth chamber screens. A subset of the population was used due to space limitations at the field site.
  • Generally, symptoms in the growth chamber screens were more severe than in the field. Correlation between the field and growth chamber screens was high for controls, but low for the population. We are screening more plants per line in the growth chamber for the population.

1.3 Genotyping

At the 2016 meeting in East Lansing it was proposed that QTL-Seq could be used. Since we use QTL-Seq for other traits in our lab and had an extra spot for sequencing, we decided to include resistant and susceptible bulks (selected using growth chamber phenotypic data from 2 screens)

  • of WPop GSB 1 (PI 482276 x Crimson Sweet population). However, we did not detect any significant QTL.
  • Genotyping-by-sequencing will be used for this population (WPop GSB1) and 2 plates of the WPop GSB1 have been sent to MSU for DNA extraction and quality checks and GBS at Cornell.

Goals for 2017-2018

  • Complete phenotyping for GSB1, GSB2 in the growth chamber
  • Field phenotyping for subset of GSB1 and parental lines
  • Mapping of resistance to GSB for GSB1 population, KASP marker development.

Fusarium wilt races 1 and 2 resistance in watermelon (Citrullus amarus)

(Patrick Wechter, Sandra Branham, Melanie Katawczik, and Amnon Levi)

Fusarium oxysporum f. sp. niveum (Fon), which causes Fusarium wilt of watermelon, is considered one of the most important diseases of watermelon production in the United States.  There are currently no economical or even viable chemical control strategies or methods that can control this soil-borne pathogen.  To date, only a few watermelon lines have been identified and reported as resistant or tolerant to this pathogen.  Unfortunately, although some of these lines were reported more than twenty-five years ago, no commercial cultivar is available with resistance to race 2 of Fon.

Year 3 progress: 2.1.1; 2.2.1; 2.3.1

Development of Germplasm lines and Genetic Populations
  • Seeds of C. amarus USVL246-FR2 and USVL252-FR2, both developed in our work, have been requested and disseminated to ten seed companies and numerous researchers for use in breeding programs and Fusarium studies.
  • A recombinant inbred line (RIL) population has been generated from 225, single seed descent lines at the F7-F8 stage from a cross of Fon race 1 and 2 resistant C. amarus USVL246 by a susceptible C. amarus PI582114.
  • Two reciprocal F2:3 genetic populations USVL252-FR2 x PI 244019-S3; with resistance to both races of Fon and papaya ringspot virus (PRSV) have been developed. The first population includes 178 F2:3 families, while the population derived from the reciprocal cross includes 220 F2:3 These populations have been developed with the generous support of Sakata Seeds (Dr. Nihat Guner).
Genetic mapping of QTL associated with resistance to Fusarium wilt race 2
  • DNA was isolated from 203 RIL families (USVL246 x PI582114) and 203 F2:F3 (USVL252 x PI 244019) and GBS sequencing was performed at Cornell University.
  • Three rounds of Fon race 1 greenhouse inoculation / resistance assays were performed using the F2:3 (USVL246 x PI582114) families.
  •  Two rounds of Fon race 2 greenhouse inoculation / resistance assays were performed using the F2:3 (USVL252 x PI 244019), in separate experiments at the U.S. Vegetable Laboratory.
  • Genotype and phenotype data have been analyzed and we have identified one major QTL for race 1 resistance on Chromosome 9, making this resistance unique from the currently available Fon race 1 resistance in C. lanatus and PI296341-FR.
  • Genetic mapping with this population resulted in a saturated map with 2495 SNP markers.
  • KASP analysis has begun on Fon race 2 and race 1 resistance QTL for better marker development.
  • USVL252-FR2 and USVL246-FR2 have been crossed into Sugar Baby, Charleston Gray and Calhoun Gray.
  • Backcrossing and selfing of the above into the recurrent parent have been performed.

Figure 1. Distribution pattern of 220 F2:3 families (USVL-252FR x PI 244019-PRSV-R) for resistance to Fusarium wilt race 2 (left), following their evaluation in a greenhouse at the U.S. Vegetable Laboratory (right).

Converting QTL to SNP markers tightly linked to Fusarium wilt race 1 resistance in C. lanatus

(Sandra Branham, Patrick Wechter, Laura Massey, and Amnon Levi)

DNA of the resistant and susceptible parents and the F2 plants of most resistant versus most susceptible F2:3 families (Lambel et al. 2014) were submitted for whole genome resequencing. QTL-seq analysis of the bulks narrowed the known Fon race 1 resistance QTL interval on chromosome 1 of watermelon (Lambel et al. 2014). SNPs from the QTL were converted to Kompetitive Allele Specific PCR (KASP) primers and used to genotype the original F2:3 population. A genetic map of these SNPs yielded several KASP markers tightly linked to Fon race 1 resistance (Figure 2). In collaboration with the HM.Clause team in Davis, California we will validate the KASP markers using advanced populations segregating for FW race 1 resistance. Also, we are testing 40 cultivars and PIs for Fon race 1 resistance and will use them to validate the KASP markers.

Figure 2. QTL-seq based on resequencing of resistant versus susceptible bulks identified SNPs tightly linked to Fusarium wilt race 1 and cover a small genomic region of 500 kb within a major QTL identified on Chromosome 1 (left) in C. lanatus. KASP markers tightly linked to Fusarium wilt race 1 resistance (qFon1-1) on chromosome 1 of watermelon (right).  (Branham et al. )

References:

Lambel, S., B. Lanini, E. Vivoda, J.Fauve, W.P. Wechter, K.R. Harris-Shultz, L. Massey, and A. Levi. 2014.  A major QTL associated with Fusarium oxysporum race 1 resistance identified in genetic populations derived from closely related watermelon lines using selective genotyping and genotyping-by-sequencing for SNP discovery. Theor. Appl. Genet. 127: 2105-15.

Wechter, W.P., M. McMillan, M. Farnham, and A. Levi. 2016. Watermelon germplasm lines USVL246-FR2 and USVL252-FR2 tolerant to Fusarium oxysporum f. sp. niveum race 2. HortScience 51:1065-1067.

Branham, S.E., A. Levi, M.W. Farnham, W.P. Wechter. 2016. A GBS-SNP based linkage map and quantitative trait loci (QTL) associated with resistance to Fusarium oxysporum f. sp. niveum race 2 identified in Citrullus lanatus var. citroides. Theor. Appl. Genet. 130:319-330.

Identifying QTL associated with Papaya ringspot virus (PRSV) resistance

(Amnon Levi and Kai-shu Ling, USDA, ARS, U.S. vegetable Laboratory (USVL), Charleston, SC)

Several F2 and BC1 populations derived from the cross USVL-252FR x PI 244019-PRSV-R(S3) were constructed with the help of Dr. Nihat Guner and team at Sakata Seeds. Two populations were evaluated for PRSV-resistance and soon be analyzed using GBS procedure for identification of QTL associated with the resistance. 

What do you plan to do during the next reporting period to accomplish the goals?

  • Complete GBS and QTL analysis of population segregating for FW race 2 resistance (Levi and Wechter).
  • Complete validation of KASP markers tightly linked to FW race 1 resistance (Levi and Wechter).
  • Complete analysis of PRSV-resistance F2 and BC1 populations and identify QTL associated with resistance (Levi and Ling).

Powdery mildew of watermelon

(Shaker Kousik, Mihir Mandel and Jennifer Ikerd)

Powdery mildew (PM, Podosphaera xanthii) of watermelon (Citrullus lanatus) continues to be a constant problem throughout the southeast.  Our recent survey of watermelon researchers also indicated that powdery mildew was considered an important priority for research across the U.S.A.  We have developed USVL531-MDR which is resistant to powdery mildew and Phytophthora fruit rot and have provided the seeds of this germplasm lines recently to two seed companies (Voloagri and Syngenta) through a Materials transfer agreement (MTA). Resistance to powdery mildew in cotyledons and true leaves appears to be a dominant trait in USVL531-MDR. We have completed extracting DNA from parents, and 180 F2 plants for analysis. We will send out the 20 most susceptible and 20 most resistant F2 lines and the parents for QTL-seq analysis.

Segregating populations (F1, F2, BCF1R, BCF1S) from cross of USVL003-MDR and USVL677-PMS were developed in 2016-2017. USVL003-MDR is resistant to powdery mildew and Phytophthora fruit rot whereas USVL677 is susceptible to both these diseases. USVL003 is an egusi type watermelon with white flesh and low brix and was derived from PI 560003 after five cycles of screening and selections.  Studies on inheritance of resistance to PM will be conducted in 2018. Since resistance to Phytophthora fruit rot is complex we are developing recombinant inbred lines (RIL) of the cross between USVL531-MDR and USVL677-PMS. 

Advancing Powdery mildew resistant inbred lines.

Fruit from F2 plants from a cross of USVL531-MDR and USVL677-PMS with powdery mildew resistance, uniform red flesh and decent brix (>7) were collected and have been advanced till F5 and further advancement to F6 is in progress. We conducted a progeny test on 23 red fleshed F4 lines using 16 plants per line and identified several lines that are homozygous for resistance.  We completed assessment of fruit quality from F4 and F5 progenies that were homozygous for resistance to PM. We grew 6 lines from F5 to obtain fruit and move to F6 for further screening and advancement.

Release of pink to red fleshed powdery mildew resistant lines with broad resistance to isolates from across U.S.A.

We have developed and released four PM resistant lines with broad resistance to P. xanthii isolates from across the U.S.A. These lines: USVL608-PMR (powdery mildew resistant), USVL255-PMR, USVL313-PMR, and USVL585-PMR are watermelon (Citrullus lanatus var. lanatus (Thunb.) Matsum. & Nakai) germplasm lines that exhibit high levels of resistance to powdery mildew (PM) caused by Podosphaera xanthii (Castagne) U. Braun & Shishkoff (syn.  Sphaerotheca fuliginea). Specifically, the hypocotyls, cotyledons and true leaves of these four PMR lines are highly resistant to powdery mildew compared to the susceptible watermelon line USVL677-PMS or cultivar ‘Mickey Lee’ on which severe powdery mildew and abundant development of conidia can be observed (Figure 1). The true leaves of these four PMR lines were also resistant to P. xanthii isolates from other states including; CA, FL, GA, NY, and SC. Each of these four PMR lines are uniform for various growth characteristics including, fruit size, shape and color with red to pink flesh (Figure 2) and brix content ranging from 6 to 8. Currently, commercial watermelon cultivars with powdery mildew resistance are rare. Hence, USVL608-PMR, USVL255-PMR, USVL313-PMR, and USVL585-PMR will be useful sources for incorporating resistance in commercially acceptable cultivars. These lines are fertile and can easily be crossed with commercial watermelon cultivars to develop new breeding populations.

We have also completed making crosses with these powdery mildew resistant lines (USVL608-PMR, USVL313-PMR, USVL585-PMR and USVL225-PMR) with USVL677-PMS and ‘Dixie Lee’ to develop populations for conducting inheritance studies in 2018 and developing resistant inbred lines with high fruit quality. We have also completed collecting fruit quality on the four PM resistant lines at two locations (Charleston, SC and Fort Pierce, FL). We have also completed collecting PM resistance data in summer and fall of 2017 on these four PM resistant lines. A paper documenting the release of these four PM resistant watermelon lines was submitted for publication to HortScience in February 2018.

Figure 1. Reaction of powdery mildew resistant (PMR) lines (USVL608-PMR, USVL585-PMR, USVL313-PMR, USVL255-PMR) to Podosphaera xanthii isolate from Florida compared to susceptible line USVL677-PMS.

Figure 2. Fruit characteristics of USVL developed powdery mildew resistant (PMR) watermelon (Citrullus lanatus var. lanatus) germplasm lines USVL608-PMR, USVL255-PMR, USVL313-PMR and USVL585-PMR. USVL608-PMR has deep red flesh.

Whole genome resequencing of USVL531-MDR (PM resistant) and USVL677-PMS (PM susceptible) watermelon lines.

To identify SNPs between PM resistant and susceptible watermelon lines with either resistant (red flesh / white flesh) or susceptible (red fleshed) to powdery mildew (PM) pathogen Podosphaera xanthii, we resequenced three watermelon PI lines: USVL531-PMR (derived from PI 494531, white flesh), USVL608-PMR (PI 307608, red flesh) and USVL677-PMS (PI 269677, red flesh). Based on preliminary analysis we have identified 1176825 SNPs among the resistant and susceptible watermelon lines. Most of the sequenced reads mapped to the draft watermelon (Citrullus lanatus, 97103) genome sequence. Currently we are pursuing comparative analysis of our SNPs data with SNPs obtained from twenty already resequenced watermelon cultivar/PIs that have differential response to PM infection to identify candidate markers for PM resistance to be utilized for molecular breeding. In addition, the whole genome resequencing of the PM resistant and susceptible lines will help in analysis of transcriptome profiling data that we have generated using RNAseq during PM and resistant or susceptible host interactions.

Transcriptomic profiling of watermelon-powdery mildew (Podosphaera xanthii) resistant and susceptible interactions.

To gain a better understanding of the innate and activated molecular defense mechanisms involved during compatible (susceptible) and incompatible (resistant) powdery mildew-watermelon interactions, we conducted RNA-seq analysis.

The PM susceptible (USVL677-PMS) and resistant (USVL531-PMR) watermelon plants were inoculated with 105 conidia-ml of P.xanthii.  Symptom development was observed every day. In addition, leaf samples were collected for microscopy and for RNA extraction. RNA-seq profiling was done on leaf samples collected at 0, 1, 3, and 8 days post inoculation (DPI). The compatible interactions resulted in distinct plant gene activation (>2 fold unique transcripts, 335:191:1762 :: 1:3:8 DPI) as compared to incompatible interaction (>2 fold unique transcripts, 314:681:487 :: 1:3:8 DPI). Compatible interactions mostly involved pathogenesis events including carbohydrate metabolism, ethylene signaling and activation of stress responsive transcripts. Incompatible interaction results in activation of both TIR and CC-NBS-LRR mediated signaling events including induced transcripts of shikimate kinase, receptor kinase, ankyrin repeat containing protein and RIN4 like protein within 24 hr of PM infection. A detailed study on NBS-LRR genes activated during watermelon-PM interaction is under progress. A manuscript is in preparation and a poster of the study was presented at the American Phytopathological Society meeting in August 2017.

Figure 1. Time course of powdery mildew (PM) infection process in watermelon resistant (USVL531-MDR) and susceptible (USVL677-PMS) true leaves. PM conidia do germinate on leaves of the resistant line USVL531-MDR, however they do not develop rapidly or cause significant infection compared to the susceptible line USVL677-PMS where development of conidia can be observed by 8 days after inoculation.

Figure 4. Venn diagram representing unique and overlapping differentially expressed genes (DEGs) in resistant (USVL531-PMR) and susceptible (USVL677-PMS) watermelon lines at 24hr, 72hr and 8 days post inoculation with P. xanthii conidia.

Additional research on powdery mildew as an offshoot of the CuCAP research

We hired a post-doctoral associate, Dr. Mihir Mandal through ORISE to help us with the molecular work on CuCAP project. Dr. Mandal has conducted the research on transcriptomic profiling and whole genome resequencing of the resistant and susceptible lines. During the process of our research on the CuCAP objectives on PM susceptible (USVL677-PMS) and resistant (USVL531-MDR) lines, we identified that melatonin could play a role in plant defense. To further study this we conducted additional research and the abstract of a manuscript that was recently submitted and is acceptable for publication (upon revisions) in Journal of Pineal Research and some additional details are presented below.

Since the 1950s, research on the animal neurohormone melatonin, has focused on its multi-regulatory effect on patients suffering from insomnia, cancer, and Alzheimer’s.  Previous studies on melatonin in plants have focused primarily on plant growth and development. However, studies on the physiological function of melatonin in host-pathogen defense mechanism are lacking. This study provides insight on the predicted biosynthetic pathway of melatonin in watermelon (Citrullus lanatus) and how application of melatonin, an environmental-friendly immune inducer, can boost plant immunity and suppress pathogen growth where fungicide resistance and lack of genetic resistance are major problems. We evaluated the effect of spray-applied melatonin and also transformed watermelon plants with the melatonin biosynthetic gene SNAT to determine the role of melatonin in plant defense. Increased melatonin levels in plants were found to boost resistance against the foliar pathogen Podosphaera xanthii (powdery mildew), and the soilborne oomycete Phythophthora capsici in watermelon and other cucurbits. Further, transcriptomic data on melatonin sprayed (1mM) watermelon leaves, suggests that melatonin alters the expression of genes involved in both PAMP and ETI mediated defenses. Twenty seven upregulated genes were associated with constitutive defense as well as initial priming of the melatonin induced plant resistance response. Our results indicate that developing strategies to increase melatonin levels in specialty crops such as watermelon can lead to resistance against diverse filamentous pathogens.

Phytophthora fruit rot of watermelon

(Shaker Kousik; USDA, ARS, U.S. vegetable Laboratory)

Phytophthora fruit rot of watermelon has been a major problem in watermelon growing areas in the Southeastern U.S.  (FL, GA, SC, NC and VA).  In recent years it has also become a problem in watermelon growing areas in Maryland (MD), Delaware (DE) and Indiana (IN). The National Watermelon Association considered Phytophthora fruit rot its top research priority in 2017 as well. At the U.S. Vegetable Laboratory (USDA, ARS) in Charleston we have developed several germplasm lines with high levels of resistance to Phytophthora fruit rot. In these studies we used the germplasm line USVL531-MDR which was resistant to 20 different P. capsici isolates from across the U.S.A.  Studies to determine inheritance of resistance to Phytophthora fruit rot using the same population described for powdery mildew (USVL531-MDR X USVL677-PMS) were conducted as USVL531 is resistant to both these diseases. However, based on this study it was difficult to assess the number of genes controlling resistance and hence we are in the process of developing a recombinant inbred line (RIL) population and are currently at the F7 stage.

We completed growing out the F3 families in summer-fall of 2018 (total 40 families, about 600 plants) and screened them for Phytophthora fruit rot. The data is being analyzed.

We have extracted DNA from parents and F2 plants for GBS. However, since resistance to powdery mildew is a dominant trait we will pool the DNA from 20 most susceptible lines and 20 most resistant lines and send it out for sequencing. We will perform QTLseq analysis on the resulting data.

We will phenotype the populations from USVL003-MDR x USVL677-PMS for resistance to powdery mildew and Phytophthora fruit rot in 2018.

We completed experiments to determine the transcriptomic profile during P. capsici infection of resistant and susceptible genotypes. Advanced germplasm lines of USVL531-MDR, USVL0020-PFR, Charleston, Gray and Sugar Baby were grown in the field and fruit were harvested when mature. Fruit of each of these lines was inoculated with 104 zoospores/ml and maintained in a humid chamber (26 ± 1 °C >95%RH). Fruit rind samples were collected from individual fruit after 12h, 24h, 48h, 72h, and 96h after inoculation and immersed in liquid nitrogen to quench all the metabolomics processes.  Rind samples were then processed for extraction of RNA and sent to Duke University Genomic center for RNA sequencing. Sequencing has been completed and we are currently analyzing the RNA-seq data.

We have identified three red fleshed (plants) with tolerance to Phytophthora fruit rot and high level of resistance to Powdery mildew (at the F5 stage). These will be screened for resistance to both the diseases and advanced further to F6

Our studies with melatonin have also shown that it can suppress the growth of Phytophthora capsici in culture plates.  Our research has also indicated that 1000mM melatonin solution is capable of reducing development of Phytophthora fruit rot on cucumbers.

Project metrics (time line) for research on Phytophthora fruit rot and powdery mildew of watermelon

  • Develop germplasm lines with resistance to Phytophthora fruit rot and powdery mildew for watermelon:
  • Develop populations for phenotyping resistance to Phytophthora fruit rot and powdery mildew of watermelon: Completed
  • Sequence and map Phytophthora fruit rot and powdery mildew QTL in watermelon: In progress.
  • Introgress Phytophthora and powdery mildew resistance into cultivated type watermelon: In progress
  • Participation in outreach to stakeholder groups per year via industry events and field days. Completed

2.1.1.1 CGMMV: Watermelon with resistance to CGMMV: Year-3 progress

(Ling and Levi)

  • Developing segregating breeding populations of watermelon germplasm (Citrulus colocynthis) with CGMMV-resistance.
  • Cucumber green mottle mosaic virus (CGMMV) is an emerging disease on watermelon and other cucurbit crops in North America (Ling et al., 2014; Tian et al., 2014). This seed-borne virus is contagious and poses a serious threat to the entire cucurbit industries in the U.S. In the last two years, through screening of the entire USDA watermelon germplasm, several lines from Citrulus colocynthis were confirmed to have some level of resistance/tolerance to CGMMV. Our focus in the last year was to develop segregating population of breeding lines for the inheritance of resistance study.
  • Through biological screening of the entire USDA watermelon germplasm (~1,400 accessions) through mechanical inoculation for CGMMV resistance, those plants with potential resistance based on symptom observation and appropriate lab tests (ELISA, PCR) were selected for self-pollination though single plant selection.
  • A repeat test on plants from seven accessions (including three Citrulus lanatus and four colocynthis) with potential resistance (tolerance) to CGMMV were conducted and resistance (tolerance) was confirmed on those C. colocynthis lines. Through single plant selection, S2 seeds were generated. One line was chosen to advance the seeds to S3, which will soon be released by ARS to the industry.
  • To study genetic inheritance of resistance, cross pollinations were made between the most resistance and a susceptible colocynthis line, as well as between the CGMMV-resistance C. colocynthis line and C. lanatus (cv. Charleston gray or Crimson Sweet). Currently, F1 and F2 seeds for C. colocynthis have been generated.  Due to self-incomparability from hybridization between C. colocynthis and C. lanatus, only two families of F1 and BC1 seeds were generated.
  • Development of those seeds is an important milestone for the evaluation of inheritance of resistance in colocynthis to CGMMV.
  • Seed Transmissibility of Cucumber Green Mottle Mosaic Virus in Cucurbits and Seed Health Assays.
  • CGMMV is a seed-borne virus, but its mechanism of virus transmission through contaminated seeds have not be well characterized and the seed health test methods have not been optimized.
  • In this study, through seedling grow-out or by mechanical inoculation to melon seedlings with CGMMV-contaminated seed extract, we determined that although seed transmission to seedlings through natural seeding grow-out process was fairly low, the mean of virus transmission to seedlings were more prevalent through mechanical inoculation of contaminated seed extract.
  • Comparative evaluation of various seed health test methods showed that serological methods (ELISA, immunostrips) could produce reliable results for seed health if a good source of antibody was used.  LAMP (isothermal amplification) was also sensitive for virus detection on seeds, but qRT-PCR results were poor and additional improvement is needed.
  • Genome sequencing of bottle gourd (lagenaria siceraria) and identification of SNPs associated with PRSV resistance. 
  • In recent years, several cucurbit genomes (cucumber, melon, watermelon and squash) have been sequence. In corporation with Fei’s group at Boyce Thompson Institute, we were able to obtain the genome sequence of bottle gourd using an advance selected line of L. siceraria with multiple virus resistance (Wu et al., 2017).
  • Using genotyping-by-sequencing technology, we were able to identify SNPs associated with resistance to Papaya ringspot virus (PRSV)
  • The molecular markers (CAPS, cleaved amplified polymorphic sequence) developed were shown to be useful for marker-assisted selection.

References:

  1. Ling KS, Li R, Zhang W. 2014. First report of Cucumber green mottle mosaic virus infecting greenhouse cucumber in Canada. Plant Disease, 98:701.
  2. Tian T, Posis K, Maroon-Lango CJ, Mavrodieva V, Haymes S, Pitman TL, Falk BW. 2014.  First report of Cucumber green mottle mosaic virus on melon in the United States.  Plant Disease 98:1163.
  3. Wu, S., Shamimuzzaman, M., Sun, H., Salse, J., Sui, X., Wilder, A.J., Wu, Z., Levi, A., Xu, Y., Ling, K.-S., Fei, Z. 2017. The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ringspot virus resistance locus. Plant Journal 92:963-975.