Project : USDA ARS

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Research Project: Biology, Etiology and Host Resistance in Vegetable Crops to Diseases and Nematodes

2019 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. Four powdery mildew resistant (PMR) watermelon germplasm lines (USVL608-PMR, USVL255-PMR, USVL313-PMR, and USVL585-PMR) with broad resistance to isolates from South Carolina, Georgia, Florida, New York and California were released in 2018. All the major seed companies (e.g. Sakata Seeds, Syngenta, Enza Zaden, HM Clause) were provided with seeds of these four lines upon request for use in their breeding programs. These four powdery mildew resistant lines have been 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 the genetics of resistance and develop molecular markers. 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 F8 stage and will be developed to the F9 for use in genetic studies. 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. Crosses to develop populations of 392291-VDR x Crimson sweet for genetic studies (F1, F2, BCF1R and BCF1S) have been made. Evaluation of these crosses to determine inheritance of resistance is in progress. Generation of an F3 population has been completed. We confirmed resistance in three new accessions to SqVYV in screens conducted in Immokalee, FL in spring 2019. Two of these accessions continued to show high levels of resistance to WVD and Cucurbit leaf crumple virus (CuLCrV). Crosses will be made with multiple virus resistant germplasm and susceptible commercial cultivar to determine genetics of resistance. 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. F1 populations have been generated between P1046 and two commercial cultivars, CM334 and Charleston Belle which are susceptible to NRKN. F2 and Backcross populations are currently being generated in the greenhouse, to prepare for genetic analysis of this resistance trait. Self-fertilized populations from all P1046 parents have also been generated from each parent plant and are being re-evaluated for NRKN resistance to confirm that the resistance trait has been fixed in all subsequent populations. 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 have been made between USVL608-PMR and ‘Dixie Lee’ to develop breeding populations (F1, backcrosses) and F2 seeds have been generated. Similarly crosses between Phytophthora fruit rot resistant lines (USVL003-MDR, USVL531-MDR) and ‘Dixie Lee’ were made and breeding populations were developed. Screening of breeding populations is in progress. Progress on developing southern root-knot nematode resistant pepper. 1st and 2nd backcross plants have been generated and screened for resistance to the southern root-knot nematode (SRKN) and genotyped using the KASP-N337 marker which has now been shown to be closely linked to the ‘N’ resistance gene and was developed at the USVL. 3rd backcross seed has been developed, and will be used for future field screens to select advanced lines with agronomically favorable traits. A biparental cucumber mapping population was screened for resistance to the javanese root-knot nematode (JRKN), and we were able to both confirm and map the presence of a recessive resistance locus (mj) within the cucumber genome. An F1 population has been generated by crossing a resistant inbred line from the mapping population with the commercial parent cultivar GY14. These crosses will help to fine map the genes within the mj locus, and to develop new cucumber germplasm with resistance to JRKN. The following progress is relative to Objective 3. Seed increase of the various powdery mildew differential lines was done and is being continued. Melon and watermelon powdery mildew race differentials were planted in April 2019 and were rated for disease development. Over 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, cucumber and squash were collected from California, Florida, Texas, and South Carolina. Lines were also evaluated in Florida in spring 2019. 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. 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 screened ninety-six sweetpotato Plant Introductions (PIs) for resistance against a GRKN isolate from South Carolina. Twenty of these PIs have been identified as potentially resistant to GRKN and are being tested further using other isolates of GRKN originating from North Carolina. The resistant germplasm will be incorporated to the sweetpotato breeding program at the USVL to begin developing GRKN resistant sweetpotatos that will help farmers manage this destructive pest. We have also been developing a new method for quickly surveying sweetpotato storage roots for the presence of GRKN. We have begun testing this new method by performing surveys of fresh market sweetpotatos, and have since found three new isolates of GRKN originating from different producers in North Carolina. We are currently testing the detection limits and error rates for this new method. Once published this new method will help researchers and regulatory agencies study, detect, and contain GRKN in sweetpotato (#6080-22000-029-19S). Experiments to determine if biological mulch combined with fungicide sprays can be effective for Managing Fruit Rot of Watermelon was completed (Project# 0000062394). Rye hay mulch was not effective in managing fruit rot. Studies on combining Insecticides and Systemic Acquired Resistance Induced by Actigard to Manage Watermelon Vine Decline was conducted during spring 2018 in Immokalee, FL (Project# 0000062397) and the results were inconclusive. Experiments to determine if Phytophthora capsici can infect watermelon seeds and subsequently infest soil is in progress (Project# 0000062395). Results obtained so far indicate that P.capsici can be seed borne, but may not be seed transmitted. 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 formulation was effective in reducing disease severity compared to the check and looks promising. (Project# 6080-22000-029-18H). Sequencing of local isolate of CuLCrV is in progress to develop clones for use in evaluation of watermelon PI for resistance. Two new sources of resistance to CuLCrV were identified in evaluations conducted in Immokalee, FL in 2019 (Project#6080-22000-029-20I). 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 (Project# 0000432455). 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. Development and release of a root-knot nematode resistant cherry pepper line. Development and release of a root-knot nematodes (RKN) are serious yield limiting pests of pepper production in the U.S. as well as the rest of the world. Incorporating RKN resistance genes into agronomically and commercially favorable pepper varieties can increase yield and reduce the need for nematicide applications. ARS scientists at Charleston, South Carolina, have released a new sweet cherry-type pepper line ‘PA-593’containing the ‘N’ resistance gene for RKN resistance. This new RKN resistant line will provide a valuable resource for stakeholders looking for new RKN resistant varieties, and for breeders looking to quickly incorporate this resistance into their cherry-type pepper breeding programs.

2. Identification of guava root-knot nematode in South Carolina. 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 RKN resistant crops. Through survey efforts, ARS scientists at Charleston, South Carolina, found GRKN for the first time in the state, where it was infecting the normally RKN resistant sweetpotato cultivar Covington. The identification of this invasive nematode in South Carolina has helped to initiated a collaborative multistate effort between researchers in four southeastern states to find, study, and manage GRKN in the field. This effort will help stakeholders to understand the threat posed by GRKN, slow the spread of this nematode to new areas, and provide resources to help farmers manage GRKN in their operations.

3. Development and release of powdery mildew resistant cucurbit germplasm lines. Watermelon is an important vegetable crop grown in 44 states in the U.S. 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 and also result in yield reduction in the field. Growers generally spray expensive pesticides to manage this disease. ARS scientists in Charleston, South Carolina, have developed four watermelon germplasm lines (USVL608-PMR, USVL313-PMR, USVL255-PMR and USVL585-PMR) with resistance to powdery mildew. Similarly, two bottle gourd lines (USVL351-PMR and USVL482-PMR) with resistance to powdery mildew were released in 2018. These resistant germplasm lines can be used by private and public sector plant breeders to develop watermelon or gourd varieties and breeding lines with resistance to powdery mildew. Development of varieties with resistance to powdery mildew will help reduce pesticide use and allow growers to produce a healthy crop. Private plant breeders and plant pathologists at vegetable seed companies, university researchers and extension agents can use these lines in their programs. Seeds of these powdery mildew resistant lines were provided to various national and international seed companies upon request including Sakata Seeds, Bayer, VNR and Enza Zaden. These lines are being used in their breeding and powdery mildew evaluation programs.

U.S. DEPARTMENT OF AGRICULTURE

Vegetable Research: Charleston, SC