Disease Of Watermelon Research Articles

Editing eIF4E in the Watermelon Genome Using CRISPR/Cas9 Technology Confers Resistance to ZYMV

Watermelon is one of the most important cucurbit crops, but its production is seriously affected by viral infections. Although eIF4E proteins have emerged as the major mediators of the resistance to viral infections, the mechanism underlying the contributions of eIF4E to watermelon disease resistance remains unclear. In this study, three CleIF4E genes and one CleIF(iso)4E gene were identified in the watermelon genome. Among these genes, CleIF4E1 was most similar to other known eIF4E genes. To investigate the role of CleIF4E1, CRISPR/Cas9 technology was used to knock out CleIF4E1 in watermelon. One selected mutant line had an 86 bp deletion that resulted in a frame-shift and the expression of a truncated protein. The homozygous mutant exhibits developmental defects in plant growth, leaf morphology and reduced yield. Furthermore, the mutant was protected against the zucchini yellow mosaic virus, but not the cucumber green mottled mosaic virus. In summary, this study preliminarily clarified the functions of eIF4E proteins in watermelon. The generated data will be useful for elucidating eIF4E-related disease resistance mechanisms in watermelon. The tissue-specific editing of CleIF4E1 in future studies may help to prevent adverse changes to watermelon fertility.

First Report of Rhizoctonia solani AG 4 HG-III Causing Stem Lesion of Watermelon in Idaho.

In July 2022, stem lesions, approximately 4 to 5 cm in length as well as leaf wilt and dark brown necrosis on stems and roots were observed in two fields in Southwest Idaho on 20 to 30% of watermelons (Citrullus lanatus). To determine the causal agent, isolations were attempted from symptomatic tissue. The surface of the affected material was disinfected with 0.6% sodium hypochlorite for 1 min and rinsed three times with sterile water. Approximately 2 mm3 sections of tissue were plated on water agar amended with 0.02% penicillin and 0.08% streptomycin and incubated at room temperature for 7 days. Fungal colonies were tentatively identified as Rhizoctonia from right-angle branching and septate hyphal structures, slight constriction and septum near the branch base, and the production of 1 to 2 mm white to light brown irregularly shaped sclerotia. Single hyphal tips were transferred to potato dextrose agar (PDA) and grown at room temperature. Approximately ten isolates from each field with a consistent macromorphology were observed. These isolates had light brown mycelia, produced sclerotia at ambient temperature with no exposure to continuous light, and a representative isolate, designated D22-110 was selected for sequencing and pathogenicity testing. For isolate D22-110, mycelia were removed with a scalpel after 7 days of growth, for DNA extraction and sequencing of the rDNA internal transcribed spacer (ITS) region as previously described (White et al., 1990). A 726 bp product was generated and the sequence was submitted to GenBank (Accession No. OQ794049). NCBI-BLAST indicated this sequence was 99% identical (631 of 634 bp and 632 of 634 bp identical) with known reference isolates previously identified as R. solani AG 4 HG-III (Accession No. AF354075 and AF354076, respectively) from a phylogenetic study (Gonzalez et al., 2001). Pathogenicity testing was performed twice on two-week-old seedlings of watermelon cultivars Endless Summer and Wingman in greenhouse conditions (29oC, 12 h daylight). Two disks (3 mm diam) from 7-day-old plates of PDA were placed around each seedling at the root and stem convergence point. Ten seedlings were mock-inoculated with sterile PDA plugs as a control. Approximately 35% damping-off incidence was observed on inoculated seedlings six days post-inoculation, while control seedlings remained healthy. At 20 days post-inoculation, 20 (first trial) and 34 seedlings (second trial) were assessed for visible stem and root lesions. Incidence of stem lesions occurred on 90% of seedlings, with 80% of seedlings possessing lesions greater than 10 mm in diameter. Seedlings without R. solani inoculation were free of stem and root lesions. R. solani was re-isolated from symptomatic tissue, with 40% frequency of isolation, identified by right-angle branching of the hyphae thus confirming Koch's postulates. R. solani AG 4 has been reported in watermelon in the US since 1994 (Hall and Summer, 1994) but the AG 4 subgroup was not reported. AG 4 HG-III was reported in melon seedlings causing damping-off in Kyrgyzstan (Erper et al. 2016). In other hosts, AG 4 HG-III was found in potatoes in South Africa (Muzhinji et al., 2014), buckwheat and foxtail millet in China (Zhou et al., 2015; Hao et al., 2023), broccoli and spinach (Kuramae et al., 2003) and turnip green (Sekiguchi et al., 2015). To the best of our knowledge, this is the first report of R. solani AG 4 HG-III causing disease in watermelon in Idaho. Given the the rate of disease incidence observed in the field, growers should consider avoiding planting alternative host crops to minimize inoculum buildup.

Harnessing the antagonistic potential and unraveling the bioactive compounds of Streptomyces sp. isolate WAB2 to protect watermelon crop from Colletotrichum orbiculare

ABSTRACT Watermelon (Citrullus lanatus Thumb) is a significant vegetable crop globally and is prone to anthracnose disease caused by Colletotrichum spp. The use of chemical fungicides for managing crop diseases has adverse impacts on the environment and human health. Therefore, it is important to use biocontrol agents for disease management. In this study, streptomycetes were assessed as a potential biocontrol agent against anthracnose pathogen, Colletotrichum orbiculare WEC2. The potent Streptomyces sp. isolate WAB2 was isolated from healthy watermelon plants and showed remarkable antifungal efficacy against C. orbiculare WEC2. Rigorous assays confirmed its performance against C. orbiculare WEC2. Bioactive compounds produced by Streptomyces sp. isolate WAB2 were identified using Gas Chromatography–Mass Spectrometry (GC–MS). An SEM study indicated the parasitic action of Streptomyces sp. isolate WAB2 against the pathogen. The formulated talc-based product of Streptomyces sp. isolate WAB2 was effective when applied as a seed treatment + foliar spray against C. orbiculare WEC2 under pot culture conditions. These treatments reduced the disease incidence (31.81%) and exhibited a higher disease reduction (52.56%), compared to the untreated control. Therefore, treatment with talc-based Streptomyces sp. isolate WAB2 is a sustainable solution for anthracnose disease management, and it facilitates eco-friendly crop protection practices against the disease in watermelon. RESEARCH HIGHLIGHTS Isolated streptomycete strains from healthy watermelon plants and screened for its efficacy against the pathogen, Colletotrichum orbiculare WEC2. Streptomyces sp. isolate WAB2 showed significant antifungal activity against the pathogen. Bio-active compounds produced by Streptomyces sp. isolate WAB2 was identified by Gas Chromatography–Mass Spectrometry (GC–MS). Using scanning electron microscopy (SEM), the parasitic activity of Streptomyces sp. isolate WAB2 against the pathogen was detected. Developed a talc-based formulation of Streptomyces sp. isolate WAB2 and tested it as a seed treatment plus foliar spray, effectively reducing the disease incidence.

Bioactivity and Resistance Risk of Fluxapyroxad, a Novel SDHI Fungicide, in Didymella bryoniae.

Gummy stem blight, caused by Didymella bryoniae, is an important disease in watermelon in China. Fluxapyroxad, a new succinate dehydrogenase inhibitor fungicide, shows strong inhibition of the mycelia growth of D. bryoniae. However, its resistance risk in D. bryoniae is unclear. In this research, the sensitivities of 60 D. bryoniae strains to fluxapyroxad were investigated. The average EC50 value and MIC values of 60 D. bryoniae strains against fluxapyroxad were 0.022 ± 0.003 μg/ml and ≤0.1 μg/ml for mycelial growth, respectively. Eight fluxapyroxad-resistant mutants with medium resistance levels were acquired from three wild-type parental strains. The mycelial growth and dry weight of mycelia of most mutants were significantly lower than those of their parental strains. However, four resistant mutants showed a similar phenotype in pathogenicity compared with their parental strains. The above results demonstrated that there was a medium resistance risk for fluxapyroxad in D. bryoniae. The cross-resistance assay showed that there was positive cross-resistance between fluxapyroxad and pydiflumetofen, thifluzamide, and boscalid, but there was no cross-resistance between fluxapyroxad and tebuconazole and mepronil. These results will contribute to evaluating the resistance risk of fluxapyroxad for managing diseases caused by D. bryoniae and further increase our understanding about the mode of action of fluxapyroxad.

A CRISPR/LbCas12a-based method for detection of bacterial fruit blotch pathogens in watermelon.

Acidovorax citrulli is the main pathogen causing bacterial fruit blotch, which seriously threatens the global watermelon industry. At present, rapid, sensitive, and low-cost detection methods are urgently needed. The established CRISPR/LbCas12a visual detection method can specifically detect A. citrulli and does not cross-react with other pathogenic bacteria such as Erwinia tracheiphila, Pseudomonas syringae, and Xanthomonas campestris. The sensitivity of this method for genomic DNA detection is as low as 0.7 copies/μL, which is higher than conventional PCR and real-time PCR. In addition, this method only takes 2.5 h from DNA extraction to quantitative detection and does not require complex operation and sample treatment. Additionally, the technique was applied to test real watermelon seed samples for A. citrulli, and the results were contrasted with those of real-time fluorescence quantitative PCR and conventional PCR. The high sensitivity and specificity have broad application prospects in the rapid detection of bacterial fruit blotch bacterial pathogens of watermelon.IMPORTANCEBacterial fruit blotch, Acidovorax citrulli, is an important seed-borne bacterial disease of watermelon, melon, and other cucurbits. The lack of rapid, sensitive, and reliable pathogen detection methods has hampered research on fruit spot disease prevention and control. Here, we demonstrate the CRISPR/Cas12a system to analyze aspects of the specificity and sensitivity of A. citrulli and to test actual watermelon seed samples. The results showed that the CRISPR/Cas12a-based free-amplification method for detecting bacterial fruit blotch pathogens of watermelons was specific for A. citrulli target genes and 100-fold more sensitive than conventional PCR with quantitative real-time PCR. This method provides a new technical tool for the detection of A. citrulli.

Genome-Wide Association Mapping and Genomic Prediction of Fusarium Wilt Race 2 Resistance in the USDA Citrullus amarus Collection.

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum (Fon) race 2 is a serious disease in watermelon and can reduce yields by 80%. Genome-wide association studies (GWAS) are a valuable tool in dissecting the genetic basis of traits. Citrullus amarus accessions (n = 120) from the USDA germplasm collection were genotyped with whole-genome resequencing, resulting in 2,126,759 single nucleotide polymorphic (SNP) markers that were utilized for GWAS. Three models were used for GWAS with the R package GAPIT. Mixed linear model (MLM) analysis did not identify any significant marker associations. FarmCPU identified four quantitative trait nucleotides (QTN) on three different chromosomes (i.e., chromosomes 1, 5, and 9), and Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) identified one QTN on chromosome 10 as significantly associated with Fon race 2 resistance. FarmCPU identified four QTN that explained 60% of Fon race 2 resistance, and the single QTN from BLINK explained 27%. Relevant candidate genes were found within the linkage disequilibrium (LD) blocks of these significant SNPs, including genes encoding aquaporins, expansins, 2S albumins, and glutathione S-transferases which have been shown to be involved in imparting resistance to Fusarium spp. Genomic predictions (GP) for Fon race 2 resistance using all 2,126,759 SNPs resulted in a mean prediction accuracy of 0.08 with five-fold cross-validation employing genomic best linear unbiased prediction (gBLUP) or ridge-regression best linear unbiased prediction (rrBLUP). Mean prediction accuracy with gBLUP leave-one-out cross-validation was 0.48. Thus, along with identifying genomic regions associated with Fon race 2 resistance among the accessions, this study observed prediction accuracies that were strongly influenced by population size.

Evaluation of Bacillus sp. strains for biological control of gummy stem blight, Didymella bryoniae (Auersw.) in watermelon (Citrullus lanatus)

The gummy stem blight caused by Didymella bryoniae (Auersw.) is a major and destructive disease of watermelon in Vietnam. Bacillus species have been used to control plant diseases as biological control agents. This study was carried out at the Department of Crop Production and Plant Protection, Phu Yen province, Vietnam during 2020 – 2021 to determine fungal inhibition of D. bryoniae DB-03 in in vitro and disease suppression of gummy stem blight under greenhouse conditions by Bacillus sp. strains namely S1A1, S1F3, S13E3, S18F11, and S20D12, which were isolated from stem-base of groundnut in central Vietnam. Results revealed that, all the tested strains of Bacillus sp. showed good inhibitory effect on the hyphal growth of D. bryoniae strain DB-03 in potato dextrose broth. Among the strains, Bacillus sp. S20F12 had highest inhibition of the mycelia growth of D. bryoniae strain DB-03 with an antagonistic efficiency of 60.7% at 10 days after fungal inoculation. Bacillus sp. reduced gummy stem blight under greenhouse conditions. At 21 days after fungal inoculation, disease incidences were low in the plants treated with Bacillus sp. S18F11 and S20D12 by 60.0% and 63.3%, respectively. The results would contribute to the knowledge of antagonistic activities of the Bacillus to optimize the biological control programme against D. bryoniae.

Identification of the Causal Agent of Bacterial Leaf Spot on Watermelon in China.

Watermelon diseases caused by pathogenic bacteria were endemic in Liaoning and Jilin Provinces from 2019 to 2020 in China, resulting in serious economic losses to the watermelon industry. This study characterized 56 strains isolated from symptomatic watermelon leaves collected from Liaoning and Jilin Provinces. Through morphological observation, 16S rRNA and gyrB sequence analysis, and BIOLOG profiles, the pathogen was identified as Pseudomonas syringae. In China, the watermelon disease caused by P. syringae was reported for the first time. The multilocus sequence analysis showed that the isolated strains belonged to three different clades within P. syringae phylogroup 2. Interestingly, most of them (79%) belonged to clade 2a, 14% were clade 2b, and 7% were clade 2d. This indicates that bacterial leaf spot outbreaks of watermelon in China were caused by multiple sources and mainly by P. syringae clade 2a.

Genome-Wide Association Study of Resistance to Pseudomonas syringae in the USDA Collection of Citrullus amarus.

Pseudomonas leaf spot (PLS), caused by Pseudomonas syringae pv. syringae, is an emerging disease of watermelon in the United States with the potential to severely reduce yield under humid conditions. The genetic basis of resistance to this disease is not known and no resistant germplasm is available. Because Citrullus amarus is an important reservoir of resistance genes for the cultivated watermelon, C. lanatus, we screened the United States Department of Agriculture plant introduction collection of C. amarus for resistance to PLS. Accessions (n = 117) were phenotyped for their level of resistance to PLS in two separate tests. Accession means of percent leaf area affected ranged from 1.5 to 99.4%. The broad-sense heritability for the trait was 0.51. Whole-genome resequencing generated 2,126,759 single-nucleotide polymorphisms (SNPs) which were used to perform a genome-wide association study (GWAS) aimed at discovering molecular markers for resistance. Three different models-BLINK, FarmCPU, and MLM-were included in the GWAS analyses. BLINK and FarmCPU, which are multilocus models, found eight SNPs, located on chromosomes Ca01, Ca05, Ca06, Ca08, and Ca10, that were significantly associated with resistance to PLS. Two of these SNPs were found by both BLINK and FarmCPU. The MLM model did not detect any significant associations. BLINK and FarmCPU estimated an explained phenotypic variance of 43.6 and 28.5%, respectively, for SNP S6_19327000 and 25.0 and 26.0%, respectively, for SNP S1_33362258, the two most significant SNPs found. In total, 43 candidate genes with known involvement in disease resistance were discovered within the genomic intervals of seven of the eight peak SNPs. Eleven of the candidate genes that were found have been reported to be involved in resistance to P. syringae in other plant species. Two significant SNPs were within resistance genes previously documented to play important roles of plant resistance specific to P. syringae in other pathosystems. The SNPs identified in this study will be instrumental in finding causal genes involved in PLS resistance in watermelon and developing resistant germplasm through breeding.

Detection of cucumber and watermelon diseases based on image processing techniques using K-means algorithm

Agriculture in Nigeria is a branch of its economy providing employment for over 70% of its population and contributing about 41% to it gross domestic production (GDP). Nigeria’s wide range of climate variations allows it to produce a variety of food and cash crops. Cucumber/watermelon is among the few varieties of fruits and vegetables produced in the country. The need for steady monitoring of the plants is necessary to detect and control the spread of its diseases. Common practices for detecting these plants diseases are mostly based on direct observation of the affected plant, which are sometimes erroneous. Laboratory analysis can also be used for plant diseases detection but mostly costly and time consuming. Digital image processing can identify and grade the diseases in cucumber/watermelon. This will aid in describing and predicting the performance of the said cultivated crops, hence increasing the production yield. Although, official disease recognition is a responsibility of professional agriculturists, low-cost observation and computational assisted diagnosis can effectively help in the recognition of a plant disease in its early stages. The application software was designed based on Object Oriented System Analysis and Design Methodology while UML was used to model the system; MATLAB was used in designing the front-end and the database is MYSQL Server. The developed system works efficiently and can successfully detect and classify the specified diseases in cucumber/watermelon with a precision between 92% and 98%.

Reactive oxygen species-related genes participate in resistance to cucumber green mottle mosaic virus infection regulated by boron in Nicotiana benthamiana and watermelon.

Cucumber green mottle mosaic virus (CGMMV) infection causes acidification and rot of watermelon flesh, resulting in serious economic losses. It is widely reported the interaction relationship between boron and reactive oxygen species (ROS) in regulating normal growth and disease resistance in plants. Our previous results demonstrated that exogenous boron could improve watermelon resistance to CGMMV infection. However, the roles of ROS-related genes regulated by boron in resistance to CGMMV infection are unclear. Here, we demonstrated that CGMMV symptoms were alleviated, and viral accumulations were decreased by boron application in Nicotiana benthamiana, indicating that boron contributed to inhibiting CGMMV infection. Meanwhile, we found that a number of differentially expressed genes (DEGs) associated with inositol biosynthesis, ethylene synthesis, Ca2+ signaling transduction and ROS scavenging system were up-regulated, while many DEGs involved in ABA catabolism, GA signal transduction and ascorbic acid metabolism were down-regulated by boron application under CGMMV infection. Additionally, we individually silenced nine ROS-related genes to explore their anti-CGMMV roles using a tobacco rattle virus (TRV) vector. The results showed that NbCat1, NbGME1, NbGGP and NbPrx Q were required for CGMMV infection, while NbGST and NbIPS played roles in resistance to CGMMV infection. The similar results were obtained in watermelon by silencing of ClCat, ClPrx or ClGST expression using a pV190 vector. This study proposed a new strategy for improving plant resistance to CGMMV infection by boron-regulated ROS pathway and provided several target genes for watermelon disease resistance breeding.

Dissection of race 1 anthracnose resistance in a watermelon (Citrullus lanatus var. lanatus) biparental mapping population

Anthracnose, caused by the fungal pathogen Colletotrichum orbiculare (Berk. & Mont.) Arx syn. lagenaria, is one of the most important diseases of watermelon in the United States and worldwide. The study was conducted to identify C. orbiculare race 1 resistance quantitative trait loci (QTL) in a ‘Charleston Gray’, resistant parent, and ‘New Hampshire Midget’, susceptible parent, biparental mapping population. The mapping population consisted of 228 F2 and the validation population consisted of 60 individuals each in BC1P1 and BC1P2. The disease severity was rated using a disease index comprising a rating scale of 0–100%. IciMapping was used to draw the linkage map and R/qtl non-parametric method (‘model = np’) was used to identity QTL. We identified a major disease resistance QTL, Qar1-8, on chromosome 8. The significant SNP marker S8_5149002, part of a putative coiled-coil (CC)–nucleotide-binding site (NBS)–leucine-rich repeat (LRR) (CC-NBS-LRR or CNL; ClCG08G002410), had a LOD of 14.06. The significant marker was validated on mapping populations using R package functions ‘chisq.test’, ‘wilcox.test’, ‘kruskal.test’, and ‘dunn.test’. The significant marker S8_5149002 was also tested for its ability to differentiate race 1 anthracnose resistance on 61 watermelon germplasm including 41 plant introduction (PI) lines. Hence, the diagnostic SNP marker S8_5149002 could be used for marker assisted selection (MAS) for race 1 anthracnose resistance in watermelon breeding programs.

Fusarium falciforme and F. oxysporum causing postharvest fruit rot of watermelon (Citrullus lanatus) in Malaysia: A first report

Watermelon is one of the fruit crops being produced in large scale (9000 ha in the year of 2021) in Malaysia. However, fungal diseases are a major factor in suppressing yield. In November 2019, field surveys in the states of Kelantan, Terengganu, and Penang found harvested watermelon showing fruit rot disease symptoms. In the present study, we delineated the pathogens causing these symptoms using morphological observations, molecular methods, and pathogenicity tests. Morphological characterization, DNA sequencing and phylogenetic analysis using ITS and TEF loci revealed that two isolates, S9 and S11, isolated from symptomatic fruits were Fusarium falciforme and F. oxysporum, respectively. Pathogenicity tests confirmed the both Fusarium species caused the observed symptoms, fulfilling Koch's postulates. This is the first report of these Fusarium species as the pathogens of watermelon fruit rot in Malaysia. Findings from the present study contribute to the knowledge of watermelon diseases in Malaysia, provide an essential foundation for development of effective pre-harvest and postharvest disease management strategies to curb disease spread.

SISTEM PAKAR UNTUK MENDIAGNOSIS HAMA PENYAKIT TANAMAN SEMANGKA MENGGUNAKAN METODE CERTAINTY FACTOR DAN METODE BAYES

Watermelon is one of the most popular fruits by the general public. Apart from the benefits of watermelon, watermelon also has several diseases that are difficult for farmers to know, such as fruit rot (anthracnose), fruit scabies (bacterial fruit blotch), and others. Although watermelon disease is common, there are still many farmers who do not know about this problem. Therefore, an expert system for watermelon disease detection can be used using the certainty factor algorithm and the Bayes method. Based on the problems that occurred, this study raised the title "Expert System for Diagnosing Pests and Diseases of Watermelon Plants using the Certainty Factor Method and the Bayes Method" to overcome the ignorance of farmers in detecting watermelon disease by entering various characteristics listed in the system based on plant events, the system will provide insight based on existing characteristics using a certainty factor algorithm which will then compare the results with the Bayes method

QTL associated with Gummy Stem Blight (GSB) resistance in watermelon

BackgroundGummy stem blight (GSB), caused by Didymella bryoniae (syn. Stagonosporopsis cucurbitacearum), produces devastating symptoms on whole plants of watermelon (Citrullus lanatus) and other cucurbits, significantly reducing yield and quality. Identification of genetic determinants and sources of resistance to this devastating GSB disease in watermelon is essential for developing resistant varieties.ResultsIn this study, we aimed at identifying quantitative trait loci (QTLs) linked to GSB resistance in melon. We identified the genome-wide single nucleotide polymorphisms (SNPs) by genotyping by sequencing (GBS) of an F2 population developed from C. lanatus lines, ‘PI 279461’ (resistant) ✕ ‘PI 223764’ (susceptible). Inheritance analysis indicated that resistance to GSB is a multi-genic trait in this population. Three QTLs namely, ClGSB1.1, ClGSB10.1, and ClGSB11.1 associated with GSB resistance, explaining approximately 10% of the phenotypic variation, were identified. Among these, the QTL ClGSB1.1 on chromosome 1 is identified as a major QTL harboring five candidate genes associated with GSB resistance including two RLKs (ClC01G014900 and ClC01G015010), two WRKY transcription factors (ClC01G014910 and ClC01G014990), and one AvrRpt-cleavage domain protein (ClC01G015130).ConclusionTwo high resolution melting (HRM) markers, WmGSB1.1–2 and WmGSB1.1–7 having a high positive correlation with the phenotypic variations, were developed. Five potential candidate genes were predicted to be associated with GSB resistance. These findings will help breeders to develop watermelon cultivars resistant to GSB.

ESTUDO DO CRESCIMENTO MICELIAL DE MACROPHOMINA SPP. NO BRASIL, SUA PATOGENICIDADE E SENSIBILIDADE A FUNGICIDA

ABSTRACT Macrophomina (Botryosphaeriaceae) is one of the main genera of soilborne phytopathogenic fungi, which causes root and seed rot in more than 800 host plants worldwide. Recent phylogenetic studies identified the species M. phaseolina and M. pseudophaseolina in Trianthema portulacastrum and Boerhavia diffusa in melon and watermelon production areas in northeastern Brazil. Therefore, the objective of this study was: i) to verify the effect of temperature and salinity on the mycelial growth of M. phaseolina, M. pseudophaseolina and M. euphorbiicola, ii) to assess their pathogenicity on melon and watermelon seedlings, and iii) to determine their sensitivity to the fungicide carbendazim. The optimal temperature for mycelial growth rate (MGR) for Macrophomina spp. ranged from 27.18 ºC (CMM4771 – M. pseudophaseolina) to 31.80 ºC (CMM4763 – M. phaseolina). For the effect of salinity on mycelial growth of Macrophomina isolates, the EC50 ranged from 103.76 (CMM4868 – M. euphorbiicola) to 315.25 mM (CMM4801 – M. pseudophaseolina). The pathogenicity test demonstrated that M. phaseolina, M. pseudophaseolina and M. euphorbiicola are pathogenic on melon with M. phaseolina exhibiting a higher level of virulence. Macrophomina euphorbiicola isolates did not cause disease in watermelon. The most sensitive isolates to the fungicide carbendazim were CMM4868, CMM4867 (M. euphorbiicola) and CMM1531 (M. phaseolina) with EC50 of 0.003, 0.012 and 0.012 mg.L-1 a.i., respectively. All Macrophomina spp. used in these experiments were pathogenic to the tested melon and watermelon cultivars with the exception of the M. euphorbiicola isolate that did not cause damage to watermelon.

Essential Acidovorax citrulli Virulence Gene hrpE Activates Host Immune Response against Pathogen.

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli (Ac) is a devastating watermelon disease that severely impacts the global watermelon industry. Like other Gram-negative bacteria, the type three secretion system (T3SS) is the main pathogenicity factor of A. citrulli. The T3SS apparatus gene hrpE codes for the Hrp pilus and serves as a conduit to secret effector proteins into host cells. In this study, we found that the deletion of hrpE in A. citrulli results in the loss of pathogenicity on hosts and the hypersensitive response on non-hosts. In addition, the A. citrulli hrpE mutant showed a reduction in in vitro growth, in planta colonization, swimming and twitching motility, and displayed increases in biofilm formation ability compared to the wild type. However, when HrpE was transiently expressed in hosts, the defense responses, including reactive oxygen species bursts, callose deposition, and expression of defense-related genes, were activated. Thus, the A. Citrulli growth in HrpE-pretreated hosts was suppressed. These results indicated that HrpE is essential for A. citrulli virulence but can also be used by hosts to help resist A. citrulli. Our findings provide a better understanding of the T3SS pathogenesis in A. citrulli, thus providing a molecular basis for biopesticide development, and facilitating the effective control of BFB.

Colletotrichum Species Associated with Anthracnose Disease of Watermelon (Citrullus lanatus) in China.

Colletotrichum species are important plant pathogens, causing anthracnose in virtually every crop grown throughout the world. However, little is known about the species that infect watermelon. A total of 526 strains were isolated from diseased watermelon samples of eight major watermelon growing provinces in China. Phylogenetic analyses using seven loci (ITS, gadph, chs-1, his3, act, tub2, and gs) coupled with morphology of 146 representative isolates showed that they belonged to 12 known species of Colletotrichum, including C. aenigma, C. chlorophyti, C. fructicola, C. jiangxiense, C. karstii, C. magnum, C. nymphaeae, C. nigrum, C. orbiculare, C. plurivorum, C. sojae, and C. truncatum and three new species, here described as C. citrulli, C. kaifengense, and C. qilinense. Colletotrichum orbiculare was the dominant species. Pathogenicity tests revealed that all isolates of the species described above were pathogenic, with C. magnum and C. kaifengense being the most aggressive to leaves and fruits, respectively. This is the first report of C. aenigma, C. chlorophyti, C. fructicola, C. jiangxiense, C. nymphaeae, C. nigrum, C. plurivorum, and C. sojae on watermelon. These findings shed light on the Colletotrichum spp. involved in watermelon anthracnose and provide useful information for implementing effective control of watermelon anthracnose in China.

Dissipation Residue Behaviors and Dietary Risk Assessment of Boscalid and Pyraclostrobin in Watermelon by HPLC-MS/MS.

Fungicides containing active ingredients of boscalid and pyraclostrobin have been widely applied in watermelon disease control. To provide data for avoiding health hazards caused by fungicides, we investigated its terminal residues and evaluated the dietary risk. In this work, watermelon samples were collected from field sites in six provinces and analyzed with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The average recoveries of boscalid and pyraclostrobin in the watermelon matrix were 97–108% and 93–103%, respectively, with the relative standard deviations (RSDs) ≤ 9.1%. The limits of quantifications (LOQs) were 0.01 and 0.005 mg/kg for boscalid and pyraclostrobin. Twenty-one days after applying the test pesticide with 270 g a.i./ha, the terminal residues of boscalid and pyraclostrobin were all below 0.05 mg/kg and below the maximum residue limits (MRLs) recommended by European Food Safety Authority (EFSA). According to the national estimated daily intake (NEDI), the risk quotients (RQs) of boscalid and pyraclostrobin were 48.4% and 62.6%, respectively. That indicated the pesticide evaluated in watermelon exhibited a low dietary risk to consumers. All data provide a reference for the MRL establishment of boscalid in watermelon for China.

Identification and Classification of Downy Mildew Severity Stages in Watermelon Utilizing Aerial and Ground Remote Sensing and Machine Learning.

Remote sensing and machine learning (ML) could assist and support growers, stakeholders, and plant pathologists determine plant diseases resulting from viral, bacterial, and fungal infections. Spectral vegetation indices (VIs) have shown to be helpful for the indirect detection of plant diseases. The purpose of this study was to utilize ML models and identify VIs for the detection of downy mildew (DM) disease in watermelon in several disease severity (DS) stages, including low, medium (levels 1 and 2), high, and very high. Hyperspectral images of leaves were collected in the laboratory by a benchtop system (380–1,000 nm) and in the field by a UAV-based imaging system (380–1,000 nm). Two classification methods, multilayer perceptron (MLP) and decision tree (DT), were implemented to distinguish between healthy and DM-affected plants. The best classification rates were recorded by the MLP method; however, only 62.3% accuracy was observed at low disease severity. The classification accuracy increased when the disease severity increased (e.g., 86–90% for the laboratory analysis and 69–91% for the field analysis). The best wavelengths to differentiate between the DS stages were selected in the band of 531 nm, and 700–900 nm. The most significant VIs for DS detection were the chlorophyll green (Cl green), photochemical reflectance index (PRI), normalized phaeophytinization index (NPQI) for laboratory analysis, and the ratio analysis of reflectance spectral chlorophyll-a, b, and c (RARSa, RASRb, and RARSc) and the Cl green in the field analysis. Spectral VIs and ML could enhance disease detection and monitoring for precision agriculture applications.