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Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) is a major fungal disease of watermelon (Citrullus lanatus) and other cucurbits. Thirty-five isolates of Didymella and Phoma spp. associated with symptoms of gummy stem blight on watermelon, Canary melon (Cucumis melo), muskmelon (C. melo), and winter squash (Cucurbita maxima) from Florida and Georgia were characterized based on morphology on agar media, pathogenicity on 'Melody' watermelon, the internal transcribed spacer (ITS) sequence of ribosomal DNA (rDNA), random amplified polymorphic DNA (RAPD) analysis, and polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. All of the isolates were pathogenic on watermelon but differed in virulence. RAPD and ITS sequence analysis indicated genetic variability among the isolates but PCR-RFLP analysis did not show any variability. ITS sequence phylogenetic analysis identified two isolates, DB-05 and DB-33, which had a greater identity to that of D. bryoniae isolates from China (98 to 100% sequence homology) than other isolates from Florida and Georgia (95 to 98%). These two isolates possessed a single nucleotide substitution of A to G at position 131 of the ITS1 region. The study characterized the genetic profile of a collection of D. bryoniae isolates from Florida and Georgia in relation to isolates from other U.S. states and countries. [ABSTRACT FROM AUTHOR]

Bacterial spot of tomato (BST), predominantly caused by Xanthomonas perforans ( Xp ) in Florida, is one of the most devastating diseases in hot, humid environments. Bacterial resistance to copper-based bactericides and antibiotics makes disease management extremely challenging. This necessitates alternative new solutions to manage the disease. In this study, we used two novel hybrid copper and magnesium nanomaterials, noted as magnesium double-coated (Mg-Db) and magnesium-copper (Mg-Cu), to manage BST. In in vitro experiments, no viable cells were recovered following 4 h of exposure to 500 μg/ml of both Mg-Db and Mg-Cu, while 100 and 200 μg/ml required 24 h of exposure for complete inhibition. In a viability assay using the live/dead cell straining method and epifluorescence microscopy, copper-tolerant Xp cells were killed within 4 h by both Mg-Cu and Mg-Db nanomaterials at 500 μg/ml but not by copper hydroxide (Kocide 3000). In the greenhouse, Mg-Db and Mg-Cu at 100 to 500 μg/ml significantly reduced BST severity compared with micron-sized commercial copper bactericide Kocide 3000 and the growers' standard (copper hydroxide + mancozeb) ( P < 0.05). In field studies, Mg-Db and Mg-Cu nanomaterials significantly reduced disease severity in two out four field trials. Mg-Db at 500 μg/ml reduced BST severity by 34% compared with the nontreated control without affecting yield in fall, 2020. The use of hybrid nanomaterials at the highest concentrations (500 μg/ml) evaluated in the field experiments can reduce copper use by 90% compared with the growers' standard. In addition, there was no phytotoxicity observed with the use of hybrid nanomaterials in the field. These results suggest the potential of novel magnesium-copper-based hybrid nanomaterials to manage copper-tolerant bacterial pathogens., Competing Interests: The author(s) declare no conflict of interest.

Whiteflies (Bemisia tabaci) are a significant pest of cucurbits and vectors many viruses leading to substantial economic losses. Modern diagnostic tools offer the potential for early detection of viruses in the whiteflies before crop production. One such tool is the multiplex reverse transcriptase quantitative PCR (RT-qPCR) probe-based technique, which can detect multiple targets in a single reaction and simultaneously quantify the levels of each target, with a detection limit of 100 copies per target. In this study, a multiplex RT-qPCR-based detection system capable of identifying one DNA virus and three RNA viruses in whiteflies: cucurbit leaf crumple virus (CuLCrV), cucurbit chlorotic yellows virus (CCYV), cucurbit yellow stunting disorder virus (CYSDV), and squash vein yellowing virus (SqVYV) was developed. To ensure the reliability of the assay, an internal gene control as the fifth target to monitor false-negative results was incorporated. This newly developed molecular diagnostic tool possesses several advantages. It can detect up to five desired targets from a single whitefly RNA sample, even at concentrations as low as 1 ng/µl. To evaluate its sensitivity, we conducted experiments using serially diluted cloned plasmids and in vitro transcribed RNA transcripts of the target viruses. We also assessed the specificity of the assay by including aphid-transmitted viruses and other viruses known to infect cucurbits. The diagnostic method successfully detected all five targets simultaneously and allowed for the quantification of up to 100 copies using a mixture of healthy? RNA and in vitro transcribed RNA. Our aim with this study was to develop a highly specific and sensitive one-step multiplex RT-qPCR system for the simultaneous detection of viruses transmitted by whiteflies in cucurbits. This system offers significant advantages for early detection, enabling prompt control measures to mitigate the further spread of viral infections and reduce yield losses. Additionally, we demonstrated the ability to simultaneously detect mixed viruses (CCYV, CYSDV, CuLCrV, and SqVYV) in individual whiteflies and quantify the number of viral copies carried by each whitefly. The multiplex RT-qPCR assay outperforms currently available techniques for detecting many samples at a given time and can be effectively utilized for early monitoring of plant viruses in individual whiteflies and symptomless plants.

Bacterial leaf spot of cucurbits (BLS) is an emerging disease in the southeastern United States that is capable of causing widespread outbreaks under conducive conditions. Historically attributed solely to the bacterium Pseudomonas syringae pv. lachrymans , recent studies have identified additional P. syringae pathovars as causal agents of the disease. To further investigate the identity and diversity of P. syringae strains associated with BLS in the southeastern United States, 47 bacterial isolates were recovered from symptomatic cucurbits from Florida, Alabama, and Georgia. Strains were characterized using the LOPAT testing scheme, fluorescence, and pathogenicity to watermelon and squash seedlings. Thirty-eight fluorescent isolates underwent whole-genome sequencing and were further characterized with 16S rRNA, four gene multilocus sequence analysis (MLSA) phylogeny, and average nucleotide identity analysis. Thirty-four isolates were identified as members of the P. syringae species complex, including P. syringae sensu stricto (12), P. alliivorans (12), P. capsici (nine), and P. viridiflava (one). An additional four isolates were found to belong to the Pseudomonas genus outside of the syringae species complex, though they did not share 95% or greater average nucleotide identity to any validly published species and are believed to belong to three novel Pseudomonas species. These results reveal an unpredicted level of diversity of Pseudomonas strains associated with BLS in the region and show the benefits of whole-genome sequencing for strain identification. Identification of P. capsici , which is capable of causing disease at higher temperatures than P. syringae , as a causal agent of BLS may also affect management strategies in the future., Competing Interests: The author(s) declare no conflict of interest.

Watermelon (Citrullus lanatus) is a high nutrient crop, high in vitamins and very popular in the U.S and globally. The crop was harvested from 101,800 acres with a value of $560 million in the U.S (USDA-NASS, 2020). California, Florida, Georgia and Texas are the four-leading watermelon-producing states in the U.S. During the fall season of 2020, plants in two North Florida watermelon fields, one in Levy County (~20 acres) and one in Suwannee County (~80 acres) with varieties Talca and Troubadour, respectively, exhibited viral-like symptoms. The fields had 100% disease incidence that led to fruit quality issues and yield losses of 80% and above. Symptoms observed in the watermelon samples included leaf crumpling, yellowing and curling, and vein yellowing similar to that of single/and or mixed infection of cucurbit leaf crumple virus (CuLCrV; genus: Begomovirus, family: Geminiviridae), cucurbit yellow stunting disorder virus (CYSDV; genus: Crinivirus, family: Closteroviridae) and squash vein yellowing virus (SqVYV; genus: Ipomovirus, family: Potyviridae), although the vine decline symptoms often associated with SqVYV infection of watermelon were not observed. All three viruses are vectored by whiteflies and previously described in Florida (Akad et al., 2008; Polston et al., 2008; Adkins et al., 2009). To confirm the presence of these viruses, RNA was isolated from 20 symptomatic samples using the RNeasy Plant Mini Kit (Qiagen, USA) as per protocol. This was followed by RT-PCR (NEB, USA) using gene-specific primers described for CuLCrV, CYSDV and SqVYV (Adkins et al., 2009). Amplicons of expected sizes were obtained for all the viruses with the infection of CuLCrV in 17/20, CYSDV in 16/20, and SqVYV in 8/20 samples. In addition, the presence of cucurbit chlorotic yellows virus (CCYV; genus: Crinivirus, family: Closteroviridae) in mixed infection was confirmed in 4/20 samples (3 leaves and 1 fruit) by RT-PCR with primers specific to the CCYV coat protein (CP), heat shock protein 70 homolog (HSP70h) and RNA dependent RNA polymerase (RdRp) designed based on the available CCYV sequences (Sup Table. 1). The RT-PCR amplification was performed using a symptomatic watermelon sample and the amplicons of RdRp, HSP70h and CP were directly sequenced by Sanger method, and the sequences of the amplicons were deposited in GenBank under the accession number: MW527462 (RdRp, 952 bp), MW527461 (HSP70h, 583 bp) and MW527460 (CP, 852 bp). BLASTn analysis demonstrated that the sequences exhibited an identity of 99% to 100% (RdRp and HSP70h, 100%; and CP, 99%) with the corresponding regions of the CCYV isolate Shanghai from China (accession number: KY400636 and KY400633). The presence of CCYV was further confirmed in the watermelon samples by ELISA (Loewe, Germany) using crude sap extracted from the RT-PCR-positive, symptomatic watermelon samples. CCYV was first identified in Kumamoto, Japan in 2004 on melon plants (Gyoutoku et al. 2009). The CCYV was previously reported on melon from Imperial Valley, California (Wintermantel et al., 2019), and more recently on squash in Tifton, Georgia (Kavalappara et al., 2021) and cantaloupe in Cameron, Texas (Hernandez et al., 2021). To our knowledge, this is the first report of CCYV on field watermelon production in the U.S. Continued monitoring of the CCYV in spring and fall watermelon crop, and cucurbit volunteers and weeds will be critical toward understanding the spread of this virus and its potential risk to watermelon in Florida and other regions of the U.S.

Tomato (Solanum lycopersicum), pepper (Capsicum annum), and gboma (Solanum macrocarpon) are major vegetables in Togo, with many people depending on these crops for their livelihood. In December 2018, during the dry season with temperatures between 21°C to 35°C, tomato ('Petomech'), pepper ('Gboyebesse') and gboma (local landrace) showing wilt symptoms without foliar yellowing were collected from two locations, Tchouloum and CECO-AGRO sites in the Sotouboua Prefecture of Togo, ~300 km from the capital city of Lome. Disease incidence ranged between 10% to 50% in multiple fields. Cut stems of most wilting tomato, pepper and gboma plants produced bacterial ooze in water and vascular discoloration was visible in longitudinal stem sections. Ground cut stem tissue tested positive with Rs ImmunoStrips specific to the Ralstonia solanacearum species complex (RSSC) (Agdia Inc., Elkhart, IN, USA). Collected samples were stored at ambient temperature and cultured within 36 hr. Culturing sap from cut stems plated on modified SMSA medium (Engelbrecht 1994) yielded colonies with typical RSSC morphology: slow-growing, irregular, mucoid, and white with red centers. Genomic DNA was extracted from thirteen isolates: two from gboma, five from tomato and six from pepper. The expected 280-bp band was amplified from all 13 genomic DNAs following polymerase chain reaction (PCR) using the 759/760 RSSC-specific primer pair (Opina et al. 1997). PCR with the 630/631 primers, which identify the Race 3 biovar 2 RSSC subgroup, did not yield a product from any Togo isolate (Opina et al. 1997). The phylotype multiplex PCR identified all Togo isolates as belonging to the phylotype I subgroup, also called R. pseudosolanacearum (Prior et al. 2016; Fegan and Prior 2005). Phylotype control DNAs were from strains GMI1000 (phylotype I, Asia), K60 (phylotype II, Americas), CMR15 (phylotype III, Africa), and PSI07 (phylotype IV, Indondesia). Comparative genomic analysis of the partial endoglucanase (egl) gene, amplified with the Endo primer pairs (Poussier et al. 2000), revealed all Togo strains belonged to sequevar 17, a group known to cause bacterial wilt of peanut in China. (Xu et al. 2009). The egl sequences are in NCBI GenBank accessions MT572393 to MT572405. Koch's postulates were completed by inoculating 28-day-old bacterial wilt-susceptible 'Bonny Best' tomato plants by soil soak (Khokhani et al. 2018). Briefly, soil around each unwounded plant was drenched with 50 ml of a 108 CFU/mL suspension of bacteria grown from a single colony. Five plants were inoculated with each of four randomly selected Togo strains. RSSC phylotype I strain GMI1000 served as a positive control and water treated plants as negative controls. Plants were kept in a 28°C growth chamber with a 12 hr photoperiod. All RSSC inoculated plants were fully wilted within a week; symptoms resembled to those observed in the field. Water treated control plants did not wilt. Culturing sap from all inoculated plants on SMSA medium yielded colonies with typical RSSC morphology that tested positive with the Rs ImmunoStrips. This is the first identification of RSSC in Togo. These results will guide development of disease management strategies and regionally appropriate breeding of vegetable lines with resistance to the phylotype I RSSC strains present in Togo.