Location: Vegetable Research
2018 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
Powdery mildew of watermelon is a major factor limiting production throughout the United States where the crop is grown. 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 to the public in 2018.
The hypocotyls, cotyledons and true leaves of these four PMR lines were highly resistant to powdery mildew compared to the susceptible watermelon line USVL677-PMS or cultivar ‘Mickey Lee’ on which severe powdery mildew was observed. These four powdery mildew resistant lines have red-pink flesh with brix content ranging from 6 to 8. Currently commercial watermelon cultivars with powdery mildew resistance are rare and these lines will be useful sources for incorporating resistance into commercially acceptable cultivars. These lines readily hybridize with commercial cultivars and inbred lines to develop breeding populations. Three seed companies were provided with seeds of these four lines upon request for use in their breeding programs.
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 and powdery mildew was crossed with a susceptible line to develop breeding populations. Several studies to determine inheritance of resistance to Phytophthora fruit rot using these populations were completed. 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 F6 stage and will be developed till the F8 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. Further selections will be made from these lines including additional back crosses to cultivated type watermelon varieties. USVL-531 was provided to two seed companies and a university upon request through outgoing material transfer agreements (MTA).
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. Seeds of 392291-VDR were provided to numerous seed companies upon request. Crosses to develop populations of 392291-VDR x Crimson sweet for genetic studies (F1, F2, BCF1R and BCF1S) have been made. Generation of an F3 population is in progress. We identified five new accessions with resistance to SqVYV in screens conducted in Immokalee, Florida in fall 2017 and spring 2018. Two of these accessions continue to show high levels of resistance to WVD and Cucurbit leaf crumple virus (CuLCrV). Resistant germplasm lines will be developed from these sources for use in breeding programs.
There is currently no known source of pepper resistance to the northern root-knot nematode (RKN). To try and identify new sources of resistance, three purportedly resistant PI lines were evaluated for susceptibility to the northern RKN in the USVL greenhouses. One of the three lines (P1479) displayed a relatively high level of resistance. Seed from P1479 has been shared with a researcher in Connecticut who will test this line for resistance in the field at the Connecticut Agricultural Experiment Station. P1479 is currently being advanced to the next generation to fix this resistance trait and prepare for future genetic analysis studies.
As a part of Objective 1, four powdery mildew resistant watermelon germplasm lines were advanced and released for public use. 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 (USVL527-PFR, USVL531-MDR) and ‘Dixie Lee’ were made and breeding populations were developed.
F1 hybrid and subsequent first backcross populations were developed by crossing both commercial sweet-banana type and cubanelle-type peppers with nematode resistant pepper lines Carolina Cayenne and Charleston Hot. These first backcross plants will be screened and selected for resistance to the southern RKN using a KASP marker recently developed at the U.S. Vegetable Laboratory (USVL) which is closely linked to the ‘N’ resistance gene. Use of this marker will reduce the time it takes to develop advanced breeding lines by removing the need to perform time and space consuming nematode resistance screens on every successive generation. Surveys of South Carolina grower fields for the presence of the hypervirulent and invasive RKN species Meloidogyne (M.) enterolobii, has led to the first discovery of this nematode in South Carolina. The discovery of this new RKN species within the state allows us to begin evaluating USVL germplasm for resistance to this pest. Cultures of M. enterolobii collected within the state are now being maintained at the USVL and all advanced pepper breeding lines will now be tested for resistance against this hypervirulent nematode.
There is currently no known source of resistance to southern RKN in cucumber. Two lines from a wild relative of cucumber (Cucumis hystrix), which had previously been reported as being resistant to southern RKN, were tested for resistance in greenhouse screens at the USVL. Unfortunately, both of these lines proved to be highly susceptible to our southern RKN isolates. In an effort to search for other sources of resistance to the southern RKN we are collaborating with USDA, ARS, in Madison, Wisconsin and this has led to the development of a diversity panel consisting of fifty wild cucumber accessions that will be screened for resistance to southern RKN at the USVL. Lines are currently in the process of being bulked in Wisconsin to be sent to the USVL to prepare for these screens.
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 2018 and were rated for disease development. As during the past 5 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, New York and South Carolina.
Experiments to determine the effect of biological treatments for managing cucurbit powdery mildew were conducted during fall 2017. Squash and watermelon plants were treated with various formulations of a biocontrol bacteria. At higher concentration one of the biocontrol formulations was effective in reducing disease severity compared to the check and looks promising. Further experiments are in progress (summer 2018) to determine if integrating less susceptible cultivars with the biocontrol agent will be effective.
Experiments to determine if biological mulch combined with fungicide sprays can be effective for Managing Fruit Rot of Watermelon is in progress. Similarly experiments to determine if Phytophthora capsici can infect watermelon seeds and subsequently infest soil is also ongoing. These trials will be completed by December 2018.
Studies on combining Insecticides and Systemic Acquired Resistance Induced by Actigard to Manage Watermelon Vine Decline was conducted during spring 2018 in Immokalee, Florida and the samples are being processed and the data is being analyzed.
Watermelon grafting is slowly gaining popularity in the United States. A hybrid of Cucurbita (C.) 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 have identified sixteen C. moschata accessions with high levels of resistance to Phytophthora crown rot. Of these, 13 accessions were reevaluated and advanced to S2 generation and are currently being screened for resistance to crown rot (S3) for development of resistant germplasm lines.
Accomplishments
1. Melatonin plays a role in plant defense against powdery mildew in cucurbits. Melatonin is a naturally occurring hormone present in animals and humans and plays an important role in patients suffering from sleep disorders and dementia, and for treatment of delirium and Alzheimer’s disease. Recently, melatonin has been discovered in plants and is now known to help plant growth and development. ARS researchers at Charleston, South Carolina, detected melatonin in watermelon plants and showed that melatonin plays a role in disease resistance against two destructive diseases; powdery mildew and Phytophthora fruit rot. Application of melatonin to watermelon reduced the severity of these two diseases. Moreover, increased melatonin levels in watermelon plants transformed with the melatonin biosynthetic gene SNAT were found to exhibit greater resistance against powdery mildew. Further, results indicated that melatonin alters the expression of genes involved in plant defenses. This work indicates that developing strategies to increase melatonin levels in specialty crops such as watermelon may lead to enhanced resistance against diverse plant pathogens. The genetic information developed by this study will also help plant pathologists and physiologists to further elucidate the role of melatonin in plants and help scientists develop potential alternative approaches for managing watermelon diseases.
2. Powdery mildew resistant rootstocks impart tolerance to grafted susceptible watermelon scion seedlings. Powdery mildew is an important fungal disease that can infest watermelon seedlings and can cause reduced vigor or death of seedlings. ARS scientists in Charleston, South Carolina, developed watermelon rootstocks that are resistant to powdery mildew. Watermelon seedlings that were not resistant to powdery mildew showed less severe powdery mildew when grafted on resistant rootstocks. The resistant rootstocks can help reduce powdery mildew severity on commercial watermelon cultivars thus potentially reducing pesticide use. 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 where watermelon seedlings are generally grown.
3. Fruit of watermelon from resistant germplasm lines are resistant to fruit rot at all stages of development. Watermelon is an important crop grown throughout the USA. Many different pests and diseases attack watermelon plants causing extensive damage. In recent years, an old, but re-emerging disease called Phytophthora fruit rot has been causing serious problems in watermelon production in many states including Florida, Georgia, South Carolina, North Carolina, Delaware, Michigan and Maryland. The National Watermelon Association, a group made up of watermelon growers and shippers from across the United States, considers Phytophthora fruit rot an important problem for which management solutions are needed. Weather conditions for fruit rot development are always prevalent in the southeastern United States and hence the pathogen can infect the fruits at any stage. ARS researchers at Charleston, South Carolina, identified, developed and released Phytophthora fruit rot resistant germplasm lines for use by seed companies and University plant breeders. They also undertook a study to determine if the developed germplasm lines produced resistant fruit at all growth stages. Results showed that watermelon fruit from susceptible commercial varieties were highly susceptible at all fruit ages while USDA developed resistant germplasm lines were resistant at all fruit ages. These findings suggest that disease management actions may need to begin as early as fruit set for commercial cultivars that are susceptible and currently being cultivated. The fruit rot resistant watermelon germplasm and the information gained from these age studies will be useful for public and private plant breeders for incorporating fruit rot resistance in watermelon cultivars.
Review Publications
Kousik, C.S., Ikerd, J.L., Turechek, W. 2017. Development of Phytophthora fruit rot caused by Phytophthora capsici on resistant and susceptible watermelon fruit of different ages. Plant Disease. 102(2)370-374. https://doi.org/10.1094/PDIS-06-17-0898-RE.
Adkins, S.T., Kousik, C.S. 2017. Cucumber vein yellowing virus. American Phytopathological Society. 2017:143-144.
Rennberger, G., Kousik, C.S., Keinath, A.P. 2017. First report of powdery mildew on cucumis zambianus, cucurbita digitata and zehneria scabraCaused by podosphaera xanthii. Plant Disease. 102(1):246. https://doi.org/10.1094/PDIS-06-17-0916-PDN.
Levi, A., Jarret, R.L., Kousik, C.S., Wechter, W.P., Nimmakayala, P., Reddy, U. 2017. Genetic Resources of Watermelon. In: Grumet R., Garcia-Mas J., and Katzir N., editors. Genetics and Genomics of Cucurbitaceae. Springer International Publishing AG 2016. p. 87-110. https://doi:10.1007/7397_2016_34.
Cutulle, M.A., Harrison Jr, H.F., Kousik, C.S., Wadl, P.A., Levi, A. 2017. Bottle gourd genotypes vary in clomazone tolerance. HortScience. 52:1687-1691.
Rutter, W.B., Salcedo, A., Akhunova, A., Wang, S., Bolus, S., Chao, S., Rouse, M.N., Szabo, L.J., Bowden, R.L., Akhunov, E., Dubcovsky, J. 2017. Variation in the AvrSr35 effector determines Sr35 resistance against wheat stem rust race Ug99. Science. 358(6370):1604-1606. https://doi:10.1126/science.aao7294.
Mandal, M., Suren, H., Ward, B., Boroujerdi, A., Kousik, C.S. 2018. Differential roles of melatonin in plant-host resistance and pathogen suppression in cucurbits. Journal of Pineal Research. e12505. https://doi.org/10.1111/jpi.12505.
Rutter, W.B., Salcedo, A., Akhunova, A., Wang, S., Hanquan, L., Bowden, R.L., Akhunova, E. 2017. Divergent and convergent modes of interaction between wheat and Puccinia graminis f. sp. tritici isolates revealed by the comparative gene co-expression network and genome analyses. Biomed Central (BMC) Genomics. 18:291. doi:10.1186/s12864-017-3678-6.
Kousik, C.S., Mandal, M., Hassell, R. 2018. Powdery mildew resistant rootstocks impart tolerance to grafted susceptible watermelon scion seedlings. Plant Disease. 102(7):1290-1298. https://doi.org/10.1094/PDIS-09-17-1384-RE.
