Biotic Stress (BS)
View articles in the conference PDF booklet.
Aphid-Triggered Immunity in Melon / Key Determinants for Durable Resistance to Virus and Aphids
Nathalie Boissot
INRA-GAFL, Montfavet, 84143, France
The Vat gene in melon is unique in conferring resistance to both A. gossypii and the viruses it transmits. This double phenotype is controlled by a cluster of genes including a CC-NLR which has been characterized in detail. Copy-number polymorphisms (for the whole gene and for a domain that stands out in the LLR region) and single-nucleotide polymorphisms have been identified in the Vat cluster. The Vat gene structure suggests a functioning so called effector- triggered immunity (ETI), with separate recognition and response phases. During the recognition phase, the VAT protein is thought to interact (likely indirectly) with an aphid effector introduced by aphid salivation within the plant cells. A few hours later, several miRNAs are upregulated in Vat plants. Peroxidase activity increases, and callose and lignin are deposited in the walls of the cells adjacent to the stylet path, disturbing aphid behavior. In aphids feeding on Vat plants, the levels of miRNAs are modified. At the plant level, resistance to aphids is quantitative (aphids escape the plant and display low rates of reproduction). ‘Aphid-ETI’ has qualitative and local effect against non-circulative viruses Cucumber mosaic virus ( CMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV) and but quantitative effect against circulative virus such as Cucurbit aphid-borne yellows virus (CABYV. Durability of ETI is highly variable. At the population level, ‘Aphid-ETI’ reduces aphid density and genetic diversity, and durability of the ‘Aphid-ETI’ strongly depends on the agro-ecosystem. Some clones are adapted to Vat resistance, putatively either by introducing a polymorphic effector not triggering ETI, or by adapting to the defenses they triggered. Several ways to enhance ‘Aphid-ETI’ durability will be proposed. ‘Aphid-ETI’ against viruses decreases the intensity of CMV and CABYV epidemics. Laboratory experiments strongly suggested that non circulative viruses cannot adapt to ‘Aphid-ETI’ and therefore durability of ‘Aphid-ETI’ against CMV is predicted long. Field experiments combined to modelling suggested that highly durable resistance against CABYV could be obtained by combining within a same melon genotype the Vat gene with recessive genes conferring resistance to CABYV. Extension of those results to any other circulative viruses will be discussed.
Genomic Characterization of Xanthomonas cucurbitae, the Causal Agent of Bacterial Spot Disease of Cucurbits
Sarah Hind, Rikky Rai
University of Illinois at Urbana-Champaign, Urbana, IL, USA
Plants in the Cucurbitaceae family comprise several economically-important vegetable and fruit crops including pumpkin, squash, zucchini, cucumber, melon, and watermelon. In 2017, world production of pumpkins, squash, and gourds exceeded 24 million tons worth more than $4 billion, and U.S. production of pumpkins, squash, and gourds was worth $370 million. Illinois farmers are responsible for 40% of total pumpkin production and 85% of the processing pumpkins grown in the U.S., making it a top vegetable commodity in Illinois. Economically important diseases, including bacterial spot on cucurbits, are a problem globally and particularly in Illinois where pumpkin fields can have both high disease incidence and extensive (up to 90%) yield losses. Bacterial species in the genus Xanthomonas can cause bacterial spot disease on a variety of plants. In recent years, bacterial spot disease caused by X. cucurbitae has spread throughout the U.S. and globally, and has become an important bacterial diseases of cucurbit plants. Large-scale genomic studies of other Xanthomonas species has increased our general understanding of virulence mechanisms, evolution, and host specificity factors of these phytopathogens. However, as bacterial species in this genus have many host-specific factors essential to their virulence functions, and as our current genetic and genomic knowledge of X. cucurbitae is limited, a high-quality reference genome would significantly aid in characterizing this particular bacterial species. Since the X. cucurbitae strain ATCC 23378 has been utilized in many previous studies, we used this strain to create a X. cucurbitae reference genome. We employed Oxford Nanopore long-read sequencing technology along with Illumina paired-end short-read sequences to generate a high-quality hybrid genome assembly. The assembly consists of a single, circular, 4.6 Mbp chromosome and one 14 kb plasmid. Initial gene prediction using Prokka estimated approximately 4,000 genes, and characterization of several genes of interest, include one TAL (transcription activator-like) effector, are currently in progress. Additionally, we are using RAD-seq to evaluate population genomics of field isolates from the Midwest region, and we plan to expand our analysis to include strains from different regions of the U.S. isolated from different cucurbit host plants.
Cucurbit Rootstocks Resistant to Fusarium Wilt of Watermelon Retain Resistance When Co- Infected by Southern Root-Knot Nematode
Anthony Keinath, Paula Agudelo
Clemson University, Charleston, SC, USA.
William Rutter, W. Patrick Wechter
U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA
The interspecific hybrid squash (Cucurbita maxima × C. moschata) rootstock ‘Carnivor’ is resistant to Fusarium oxysporum f. sp. niveum (FON) but susceptible to Meloidogyne incognita, the southern root-knot nematode. A new citron (Citrullus amarus) rootstock ‘Carolina Strong Back’ is resistant to FON and M. incognita. The objectives of this study were to determine if an interaction between M. incognita and FON race 2 occurred on ‘Carnivor,’ ‘Carolina Strong Back,’ or watermelon ‘Fascination,’ which is susceptible to FON race 2. In 2016 and 2018 field experiments, plants of non-grafted ‘Fascination’ and ‘Fascination’ grafted onto ‘Carnivor’ and ‘Carolina Strong Back’ were inoculated with one of four pathogen treatments: no pathogens, M. incognita alone, FON alone, or both pathogens. In both years, 20 g wheat grain colonized by FON was added to the transplanting holes, while in 2018, an additional 10 g was applied per 0.3 m of row in infested plots. M. incognita was applied as 2000 eggs (2016) or eggs plus juvenile nematodes (2018) to seedlings before transplanting. After 9 weeks, incidence of Fusarium wilt and area under the disease progress curve did not differ when hosts were inoculated with FON alone or with FON and M. incognita together. Plants not inoculated with FON did not wilt. Fusarium wilt was greater on nongrafted watermelon (78% incidence) than on both grafted rootstocks and lower on ‘Carnivor’ (1%) than on ‘Carolina Strong Back’ (12%) (P≤0.05). In 2016 after 16 weeks, ‘Carnivor’ had a greater percentage of the root system galled than the other two hosts, and watermelon had more galling than ‘Carolina Strong Back.’ In conclusion, cucurbit rootstocks that are susceptible and resistant to M. incognita retain resistance to FON when they are co-infected with M. incognita. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Cucurbit Powdery Mildew Population Virulence Variation – a Complex View from a Global Perspective
Ales Lebeda, Bozena Sedlakova, Eva Kristkova
Palacký University in Olomouc, Faculty of Science, Department of Botany, Olomouc-Holice, 78371, Czech Republic.
James D. McCreight
U.S. Department of Agriculture, Agricultural Research Service, Crop Improvement and Protection Research Unit, Salinas, California, CA, USA.
Evsey Kosman
Institute for Cereal Crops Improvement, The George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv, Israel
Cucurbit powdery mildew (CPM) is mainly caused by two obligate ectoparasites, Golovinomyces orontii s.l. (Go) and Podosphaera xanthii (Px), that are highly variable in virulence. Various independent systems of CPM race determination and denomination have been used in recent decades. We recently developed new tools to enhance research of CPM virulence variation. Diversity models were applied to analyses of virulence variation of Go and Px populations (115 Czech isolates) from 2010 through 2012. Diversity within and distances between Go and Px populations and each other in spatio-temporal context and with regard to original host plant species were analyzed, based on virulence patterns of individual isolates (races) on a set of 21 melon (Cucumis melo L.) race differentials. Significant differentiation among the Go and Px pathogen populations was revealed. The results clearly demonstrate that the set of differential melo genotypes is well composed because of significant differentiation capacity for both species. There were no significant differences between Go isolates from different host plant species due to high variability, but there was significant host-specific differentiation among Px isolates. This approach was used to evaluate virulence variation of CPM populations in other European countries, South Africa and Asia (Thailand). Preliminary results obtained for Px showed enormous virulence variation in other European countries outside of Czech Republic, as well as in South Africa. Px isolates from Netherlands that originated from wild Cucumis species, grown in greenhouse however expressed lower virulent variation than the isolates from other countries. A Px isolate from Momordica charantia in Thailand differed in virulence compared with isolates from other countries. The approach used in this study provides revealed complex virulence structures of CPM populations of diverse origins, and when completed by race determination and denomination on melon, it may serve as a base to understand virulence variation of both CPM species on a global perspective.
Pathotypes and Races of Pseudoperonospora cubensis – Different Concepts of Virulence Differentiation
Ales Lebeda, Eva Kristkova, Bozena Sedlakova
Palacký University in Olomouc, Faculty of Science, Department of Botany, Olomouc-Holice, 78371, Czech Republic
Host-parasite interactions between Cucurbitaceae and P. cubensis exhibit significant variation. This contribution reviews the current state of knowledge regarding characterization of P. cubensis virulence variation on the level of pathotypes (variation in host genera and species host-range) and races (variation in intraspecific level). However, our knowledge of the interactions between P. cubensis isolates and the most important genera and species of cultivated cucurbits is limited. The concept of pathotypes identification was introduced in 1980s with some modifications later. The former concept was missing some crucial requirements to be applied and comparable internationally. It was the reason why an improved differential set of six cucurbit genera and 12 genotypes (Benincasa, Citrullus, Cucumis, Cucurbita, Lagenaria, and Luffa) was developed to characterize pathotypes among P. cubensis isolates, and is broadly used. This concept allows application of various mathematical approaches for virulence comparison. Long-lasting research of interactions between cucurbits (Cucumis spp., Cucurbita spp.) and Pseudoperonospora cubensis demonstrated frequent expression of race-specific reaction patterns. The differential set of 21 genotypes of Cucumis melo was developed for determination of cucurbit powdery mildew races. Most recent research of virulence variation of cubensis population in the Czech Republic showed very broad spectrum of virulence patterns on Cucumis melo demonstrating existence of huge number of races by this pathogen. The approach of how to determine races of P. cubensis is discussed and demonstrated. Utilization and combination of both approaches (establishment of pathotypes and races) are important for science as well as for practical application in cucurbit resistance breeding.
Resistance to Fungicides in Cucurbit Powdery Mildew in Europe
Bozena Sedlakova, Martin Srajbr, Ales Lebeda
Palacký University in Olomouc, Faculty of Science, Department of Botany, Olomouc-Holice, 78371, Czech Republic
One hundred cucurbit powdery mildew (CPM) isolates (41 Golovinomyces orontii s.l. [Go], 59 Podosphaera xanthii [Px]) from the Czech Republic (2012 to 2015), were screened for fungicide efficacy to the six frequently used fungicides (quinoxyfen /Atlas 500 SC/, propiconazole/Bumper 25 EC/, fenpropimorph /Corbel/, dinocap /Karathane LC/ azoxystrobin /Ortiva/) and penconazole /Topas 100 EC/). Fungicide efficacy was determined by a modified leaf-disc bioassay with three concentrations. Highly susceptible Cucumis sativus ‘Stela F1’ was used for preparation of leaf discs. Efficacy of fungicides towards screened CPM isolates varied significantly. There were observed also differences in efficacy of some fungicides between both CPM species, and as well as, among years. In the case of quinoxyfen, propiconazole, fenpropimorph and penconazole, there have been no reports from Czech Republic since the year 2012. Fenpropimorph was 100% effective and showed some phytotoxicity to C. sativus ‘Stela F1’ leaf discs. Propiconazole was also highly effective; the same phenomenon was recorded for penconazole, however only for Go during the studied period. Nevertheless, efficacy of penconazole has decreased since 2014 when frequency of Px strains with moderately resistant reactions to the lower and recommended concentrations increased. The highest number of various reaction patterns of CPM populations was observed in response to quinoxyfen. There was recorded decreased efficacy, except the year 2015, when a majority of CPM isolates were controlled by recommended concentration. Dinocap showed high efficacy and the majority of screened CPM isolates expressed sensitive reaction to recommended concentration. However, there were also observed occurrence of strains (Go, Px) with moderately resistant or resistant responses to screened concentrations. This phenomenon reflects the situation in Czech CPM populations from 2001 to 2011. Azoxystrobin showed low efficacy, this situation started in 2007 (1st year of study), when a continual shift towards prevalence of azoxystrobin-resistant strains in Czech CPM populations was observed (Lebeda et al. 2010a,b; Sedláková et al. 2017).
Cucumber Green Mottle Mosaic Virus: Seed Transmissibility, Seed Health Assays and Screening Watermelon Germplasm for Disease Resistance
Kai-Shu Ling, Rugang Li, Andrea Gilliard, Amnon Levi
U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA.
Xuelian Sui, Zujian Wu
Fujian Agriculture and Forestry University, Institute of Plant Virology, Fuzhou, China
Cucumber green mottle mosaic virus (CGMMV), a tobamovirus in the family Tobamoviridae, seriously affects cucurbit crop productions around the world, with its epidemics in Asia and Europe, and as an emerging disease in North America and Australia. This seed-borne virus is very contagious and poses serious threat to all types of cucurbit crops, including cucumber, melon, watermelon, and squash. Planting clean and certified CGMMV-free seeds is an important measure in disease prevention. Currently, there is no standard method of seed health assay and no known resistant genetic materials that are available in watermelon. In the present study, we investigated the nature of seed transmissibility and compared various molecular and serological methods for CGMMV detection. In an effort to breed watermelon for resistance, we evaluated the USDA watermelon germplasm (1,486) for resistance to CGMMV. Using a Canadian isolate of CGMMV (in Asian genotype), through mechanical inoculation on seedlings and symptom observation, plants from seven accessions with putative resistance (tolerance) to CGMMV were selected. A repeat screening was conducted using seedlings generated through self-pollination of selected plants, resistance (tolerance) to CGMMV was confirmed on those Citrullus colocynthis lines, but not C. lanatus lines. Those CGMMV-tolerant colocynthis plants had no apparent visible symptom, but virus-titers could be detectable using lab tests. The genetic materials from those advance-selected C. colocynthis lines could be useful for breeding watermelon cultivar or rootstock with resistance to CGMMV. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Molecular Diagnosis and Characterization of Cucumber green mottle mosaic virus (G: Tobamovirus, F: Virgaviridae) Infections on Bottlegourd (Lagenaria siceraria L.)
B. Mahesha, Samuel Duleep, P. Shivarama, M. Krishna
Indian Institute of Horticultural Research, Bangalore, Karnataka, India
During the year 2017-18 the symptoms of green mottling on leaves, vein banding, leaf distortion and green patches were notified on bottlegourd fruits with 60-70% incidence. Further, the infected leaf samples of bottlegourd were collected; diagnosed and characterized through Transmission Electron Microscopy (TEM), Atomic Force Microscopy and RT-PCR analysis. The TEM results confirmed the presence of rigid rod virus particles. It was suspected that the pathogen might be Tobamovirus. RT-PCR analysis was conducted with Tobamovirus specific primer and results confirmed the infections by Cucumber green mottle mosaic virus (CGMMV) on bottlegourd. The sap transmission (Mechanical transmission) method was followed to screen the bottlegourd genotypes and to identify the host ranges of CGMMV. Under glass house conditions 27 genotypes of bottlegourd were screened for resistance against CGMMV; none of them found resistant. Host range studies revealed that CGMMV was easily transmitted to squash, pumpkin and cucumber. Currently, CGMMV is designated as global pathogen under quarantine perspective due to its seed borne and highly contagious nature. Hence needs immediate attention to nab the pathogen at national level through quarantine measures and integrated disease management approaches.
Role of Antioxidant Molecule Melatonin in Plant-Host Resistance and Pathogen Suppression in Cucurbits
Mihir Mandal (ORISE Participant), Chandrasekar Kousik
U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, USA.
Haktan Suren
Department of Forest Resources and Environmental Conservation, Virginia Tech, Blacksburg, VA, USA.
Brian Ward
Clemson University, CREC, Charleston, SC, USA.
Arezue Boroujerdi
Claflin University, Dept of Chemistry, Orangeburg, SC, USA
Melatonin (N-acetyl-5-methoxytryptamine) is a naturally occurring low molecular weight indole-based metabolite and serves as an antioxidant molecule in various plants and animals. Melatonin, as an animal neurohormone has multi-regulatory effect on patients suffering from insomnia, cancer, Alzheimer’s and other neurobiological disorders. In plants, melatonin plays a wide and diverse range of cellular and physiological functions including plant growth and development, and is inducible in response to diverse biotic and abiotic stresses. However, studies on the direct role of melatonin in disease suppression and as a signaling molecule in host-pathogen defense mechanism are lacking. Our study provides insight into the conserved nature of the biosynthetic pathway of melatonin in watermelon (Citrullus lanatus) and how exogenous application of melatonin, an environmental-friendly immune inducer, can boost plant immunity and suppress pathogen growth. Melatonin (1mM) applied as a spray suppressed powdery mildew development on various cucurbit leaves and Phytophthora fruit rot development on cucumber. Watermelon plants transformed with the melatonin biosynthetic gene SNAT (serotonin N-acetyl transferase) from a powdery mildew resistant plant also helped reduce PM development compared to non-transformed controls. Increased melatonin levels in plants were found to boost resistance against the foliar pathogen Podosphaera xanthii (powdery mildew). Our data also suggests there is subcellular exchange/flow of melatonin intermediates between cytoplasm and chloroplast during melatonin biosynthesis in watermelon. Further, transcriptomic data on melatonin sprayed (1mM) watermelon leaves, suggests that melatonin alters the expression of genes (Pathogenesis related protein PR1a, receptor kinases, NAC domain TFs, Elicitor-responsive protein 3) involved in both PAMP (pathogen-associated molecular pattern) and ETI (effector- triggered immunity) mediated defenses in a salicylic acid (SA) dependent pathway. Developing strategies by using CRISPR/Cas9 genome-editing method to increase melatonin levels in specialty crops such as watermelon and other cucurbit crops can have dual effect: (a) enhanced disease resistance against diverse plant pathogens, (b) serve as a source of natural antioxidant molecules for human nutrition. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Inhibitory Effects of Cucumber Age-Related Resistance to Phytophthora capsici Manifest Within 24 Hours of Inoculation
Ben Mansfeld, Rebecca Grumet
Michigan State University, East Lansing, MI, USA
Cucumber (Cucumis sativus) fruit are largely susceptible to infection by Phytophthora capsici. However, some cucumber cultivars develop a fruit surface-associated age-related resistance (ARR) to P. capsici. Young, rapidly growing fruit are highly susceptible, but become resistant as they complete exponential growth [~16 days post-pollination (dpp); 2-3 weeks prior to ripening]. Prior transcriptomic and metabolomic comparisons of peel of ARR expressing and non-expressing uninoculated fruit identified changes associated with resistance possibly functioning as preformed defenses. Here we performed scanning electron microscopy and transcriptomic analyses of inoculated fruit at resistant (16 dpp) and susceptible (8 dpp) ages, providing a unique opportunity to examine compatible and incompatible interactions in the same genotype. Strong transcriptional changes were observed at 4 hours post inoculation (hpi), with approximately 2000 genes differentially expressed in either age. Gene Ontology term enrichment analysis of upregulated genes at 4 hpi revealed both common (including response to wounding, response to oxidative stress, defense response) and unique responses. In addition to the mutually upregulated defense genes at 4hpi, several other resistance-associated genes were uniquely upregulated in resistant fruit. At later time points the transcriptional responses markedly diverged. At 24 and 48 hpi, susceptible 8 dpp fruit continued to mount defense along with strong downregulation of genes involved in photosynthesis, cell wall synthesis and modification, lipid and cuticle biosynthesis, cell division and growth. In contrast, resistant 16 dpp samples largely downregulated defense responses while upregulating photosynthesis and other biological processes. Further investigation of the transcriptome network dynamics and pathogen development during the first 24 hours of infection (0, 2, 4, 8, 12, 18, 24 hpi) is underway. Scanning electron microscopy of resistant peels showed evidence for infection failure as early as 4 hpi, including aberrant long germ tubes, and ungerminated, deflated and/or disintegrated spores and hyphae, that were not observed on susceptible fruit. Together these results suggest that in ARR-expressing fruit, a successful defense is mounted within the first 24 hours.
Diversity and Pathogenicity on Watermelon of Six New Fungal Species in the Family Stachybotriaceae
Gabriel Rennberger
Anthony Keinath Clemson University, Charleston, USA
The fungal family Stachybotriaceae includes 210 species in 33 genera with high genetic diversity. Although most species from this family are saprophytic, some are plant pathogens with relatively wide host ranges. One member of this family, Myrothecium roridum, has been reported to cause leaf lesions on watermelon, but due to a lack of molecular identification, the species may have been misidentified. A total of 92 isolates from lesions on watermelon leaves was collected in five fields in South Carolina throughout three seasons from 2015 to 2017. In fall 2016 symptomatic leaves were found in 5 of 11 fields, and leaf lesions caused by Stachybotriaceae were the most prevalent disease in two fields (present on 68 and 43% of leaves). For all isolates, partial gene sequences were determined for cmdA, ITS and tub2. Based on these sequences, six species, Albifimbria verrucaria, Gregatothecium humicola, Paramyrothecium foliicola, Paramyrothecium humicola, Xenomyrothecium tongaense and Xepicula leucotricha, were identified. G. humicola was most common, followed by P. foliicola, whereas the other species occurred infrequently. In phylogenetic trees based on Bayesian inference, the South Carolina isolates grouped with the corresponding species reported previously (Lombard et al., 2016, Persoonia 36:156). In a greenhouse pathogenicity test, plants of watermelon ‘Tri X-313’ were inoculated with suspensions of 1×106 conidia/ml prepared for one isolate of each species. Water-sprayed plants were used as controls. Plants were held at approx. 95% RH and 25°C for four days and then transferred to greenhouse benches. Disease incidence and severity were rated after seven days. The test was repeated once with twice the inoculum concentration. All six species caused leaf lesions on watermelon and were recovered successfully. There were few significant differences in disease severity among the six species, with the exception that G. humicola and X. tongaense were significantly more virulent than X. leucotricha (P = 0.05). A. verrucaria and X. leucotricha were only slightly pathogenic. This study is the first report of more than one species in Stachybotriaceae causing disease on watermelon, which might be an emerging disease under humid fall environments in the southeastern United States.
Detection of the Gummy Stem Blight-Causing Pathogens (Stagonosporopsis spp.) in Watermelon, Using Three Species-Specific LAMP Assays
Jorge Reyes, Cecilia McGregor
Department of Horticulture, University of Georgia, Athens, Georgia, USA. 2
Caterina Villari
Warnell School of Forestry & Natural Resources, University of Georgia, Athens, Georgia, USA.
Marin Brewer
Department of Plant Pathology, University of Georgia, Athens, Georgia, USA
Gummy stem blight (GSB) is one of the most important diseases of watermelon in the southeastern U.S. GSB is caused by three cryptic Stagonosporopsis species; S. citrulli, S. cucurbitacearum and S. caricae, which can infect most of the above ground parts of the watermelon plant. Since there is not currently resistance to GSB in commercial watermelon cultivars, its management relies on cultural practices and costly preventative fungicide applications. Furthermore, a difference in sensitivity/resistance for the most common fungicide chemistries among the three species imposes a serious challenge for its management. Efficient methods for pathogen detection and diagnosis are therefore required for appropriate management decisions. In this study, three species-specific loop-mediated isothermal amplification (LAMP) assays were developed for the GSB-causing pathogens. Species-specific LAMP primer sets were designed in the mating type (MAT1-1-1) gene of the different Stagonosporopsis spp. to target single nucleotide polymorphisms (SNPs) different among species. LAMP assays show specificity for the intended targets and sensitivity tests are currently underway to determine their detection thresholds using gDNA and GSB pathogen spore suspensions. The LAMP assays have potential application for point-of-care diagnosis, allowing growers and county agents to make informed decisions depending on the presence and type of GSB species at any given time. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project)
Understanding the Diversity of Cucumber Infecting Begomoviruses in Pakistan
Muhammad Shafiq
University of the Punjab Institute of Agricultural Sciences, Lahore, 75500, Pakistan
The cucumber (Cucumis sativus), of the family Cucurbitaceae is commonly known as cuke. Cucumber leaf curl disease complex(CucLCD) is one of the major factor for threatening cucumber crops in the Pakistan. Symptoms of CucLCD include severe upward and downward leaf curl with cup-shape, yellowing and stunted plant growth. This disease is caused by begomoviruses (single-stranded DNA viruses (family Geminiviridae) that are transmitted by whiteflies). The begomoviruses are either bipartite (with two genomic components known as DNA A and DNA B), monopartite (with a genome homolog of DNA A component of bipartite begomoviruses) or monopartite associated with DNA satellites (mainly betasatelites). All three types of begomoviruses are main player in CucLCD complex. Begomoviruses associated with shortening of leaves, vein swelling and enations in Cucumber, was cloned and sequenced. The preliminary results showed that clone MU6 and MU7 have highest nucleotide sequence identity of 97% and 98% to Tomato leaf curl Palampur virus (ToLCPMV) DNA-A and DNA-B isolated from tomato in India. Maximum likelihood phylogenetic analysis grouped MU6 (DNA-A) and MU7 (DNA-B) into well-supported clades together with ToLCPMV. Thus, both clones: MU6 and MU7 are new variant of ToLCPMV from Pakistan. Agrobacterium-mediated inoculation of the partial repeat construct of ToLCPMV clone obtained in this study to Nicotiana benthamiana induced severe upward leaf curl symptoms and flow of viral DNA were detected in infected plant leaves To our knowledge this is the first report of ToLCPMV infecting Cucumber in Pakistan. . In another other sample we also cloned Tomato leaf curl new Delhi virus from cucumber. In This presentation I will discus about the geographical distribution and management of CucLCD infecting Begomoviruses in Pakistan
Evaluating Cucurbit Rootstocks for their Utility in Preventing Disease Caused by the Soilborne Pathogens Pythium aphanidermatum and Pythium myriotylum
Sean Toporek, Anthony Keinath
Clemson University, Coastal Research and Education Center, Charleston, SC, USA
Root rot and damping-off of watermelon caused by the soilborne pathogen Pythium is a problem in watermelon production around the world. Grafting to resistant cucurbit rootstocks has been used to control other soilborne pathogens affecting watermelon. The objectives of this study were to assess grafting as a non-chemical control method against Pythium. In 2017 and 2018, a survey was conducted to find the most abundant species of Pythium associated with root disease in cucurbits in South Carolina. The two most common species identified were aphanidermatum and P. myriotylum. First, inoculum produced from one isolate of both species was mixed together and used to inoculate 21 non-grafted cultivars of watermelon (Citrullus lanatus) , citron (Citrullus amarus), bottle gourd (Lagenaria siceraria), and interspecific hybrid squash (Cucurbita maxima × C. moschata) in the field. Second, five rootstocks representing the previous three rootstock species were grafted with a Tri-X 313 watermelon scion and inoculated with the same inoculum mixture in the field. Non-grafted Tri- X 313 was included as a control. Third, 17 of the 21 cultivars were further challenged in a growth chamber against each Pythium species at a constant temperature of 30°C. Field experiments were conducted for 33 days and growth chamber experiments for 7 days. Area under the disease progress curve (AUDPC) was calculated from disease incidence for all three experiments. Based on AUDPC data from the non-grafted field trials, watermelon and bottle gourd cultivars were significantly more susceptible to Pythium disease than squash. In the grafting experiment, the non-grafted Tri-X 313 control was significantly more susceptible than any of the grafted rootstocks. In the growth chamber, the genus Citrullus was more susceptible to both Pythium species at 30°C than Cucurbita and Lagenaria. There was no significant difference in AUDPC between the two Pythium species. In conclusion, the genus of cucurbit rootstock has a significant impact on Pythium disease incidence and grafting watermelon to resistant rootstocks is a viable strategy to control Pythium diseases.
Screening of the Melon Germplasms Resistant to Phytotpthora Rot Caused by Phytophthora capsici
Pingyong Wang
Zhengzhou Fruit Research Institute, Zhengzhou, China
Phytophthora blight, caused by Phytophthora capsici is one of the most devastating disease affecting melon (Cucumis melo) production worldwide. P. capsici can infect the host plants at any growth stage, causes necrosis on root, stem, leaves, crown and fruits. The infected plants can hardly overcome. Combinations of cultural and chemical prevention measures are commonly used to reduce the damage of P. capsici on melon production. However, food safety problems caused by chemical pesticides are causing more and more social panic. The use of host resistance to manage P. capsici is a more economical and environmentally friendly strategy, but the majority of current melon cultivars are susceptible to P. capsici. Moreover, very limited numbers of resistant melons have been reported, and no genes for resistance to this diseases have been fine mapped or cloned. In this study, 166 melon accessions were evaluated for resistance to P. capsici using the root-irrigating method. The tested accessions included 41 C. melo subsp. agrestis, 104 C. melo subsp. melo, 15 were C. melo var. agrestis, and 6 were wild relatives. Melon PI 140637, PI 165514 and PI 381772 were highly resistant to P. capsici. Sixty-six percent of the tested melons were at least at a middle resistant level, while, 98% were susceptible or highly susceptible to P. capsici. Correlation analysis revealed that the resistance level of C. melo subsp. melo was significantly higher than that of C. melo subsp. agrestis. The survey date of melon fruit showed that the single-fruit weight of 14 accessions was more than 1 kg, and the central soluble solid content of 8 accessions was more than 10%. The resistant germplasms with high-yield or high soluble solid content could be used for genetic research like gene mapping or cloning, and could also be used as parent lines to transfer the resistance gene(s) into the lines which owned good agronomic characters but with poor resistance.
Cucurbit chlorotic yellows virus, a New Crinivirus Infecting Cucurbits in California
William M. Wintermantel, Laura L.J. Hladky, Patti Fashing, Kaori Ando, James D. McCreight
U.S. Department of Agriculture, Agricultural Research Service, Agricultural Research Station, Crop Improvement and Protection Research Unit, Salinas, CA, USA
During the summer of 2018, melon (Cucumis melo L.) plants from a germplasm diversity study in Imperial Valley, California were found infected with Cucurbit chlorotic yellows virus (CCYV; genus Crinivirus, family Closteroviridae). Nearly all melon accessions in the study exhibited interveinal yellowing and chlorotic spot symptoms similar to those caused by Cucurbit yellow stunting disorder virus (CYSDV; genus Crinivirus), which has been prevalent in the region since 2006. However, nucleic acid extracts of two strongly symptomatic plants tested negative for CYSDV by RT-PCR. Subsequent RT-PCR evaluation of extracts of these two plants with primers specific for RNA-dependent RNA polymerase (RdRp) and coat protein (CP) gene sequences from CCYV RNA1 and RNA2, respectively, resulted in amplification. The 753 nt full-length CP gene and 1515 nt full-length RdRp gene of these isolates were sequenced, and each shared 99% sequence identity with the same regions of CCYV isolates from China, Greece, and Taiwan. Archived RNA extracts from Imperial Valley melon plants stored at -80C, and collected over the course of 9 years (2010-2018), were assayed for CCYV and CYSDV in order to determine how long CCYV may have been present in the region. Nineteen of 23 samples collected from 2014 to 2018 tested positive for CCYV, and many contained mixed infections of CCYV with CYSDV and/or the ipomovirus, Squash vein yellowing virus (SqVYV). Eighteen archived samples collected from 2010 to 2013 tested negative for CCYV, but CYSDV, the virus originally identified in the samples was successfully amplified from these archived extracts. Therefore, CCYV most likely emerged in the Imperial Valley in 2014, about the same time that SqVYV was first observed in California, but remained undetected due to similarity in symptomology on cucurbits to CYSDV. CCYV is transmitted efficiently by the whitefly, Bemisia tabaci MEAM1, which is common throughout the region. Preliminary observations suggest CYSDV resistance may not be effective for control of CCYV. Further studies will be necessary to evaluate epidemiology of CCYV in the southwestern U.S. desert crop production region, and to determine its impact on melon production and development of crinivirus-resistant melon cultivars. Partial Funding provided by the Specialty Crop Block Grant Program at the U.S. Department of Agriculture (USDA) through Grant 14-SCBGP-CA-0006 (The CucCAP Project).