Didymella Bryoniae Research Articles

Determination of fungal root and stem rot agents of melons grown in Kumluca/Antalya

Kumluca has an important place in terms of greenhouse vegetable cultivation. Melon is among one of the main vegetables grown in the district on about 3200 decare of land. Various diseases cause decrease in the yield and quality of melons grown undercover in Türkiye. Among them, Fusarium wilt and gummy stem blight diseases especially have caused significant losses in recent years. In this research, the incidence and severity of the root and stem rot disease in Kumluca were determined by surveys made in 72 melon greenhouses in this area. Plant and soil samples were taken to the laboratory and isolations were made. As a result, Fusarium oxysporum and Didymella bryoniae were the most frequently isolated pathogens from the plant samples, followed by other Fusarium species. Fungi with the highest isolation frequency from the soil samples were Fusarium spp., Rhizoctonia solani and Macrophomina phaseolina. In the pathogenicity test, F. oxysporum, F. solani, F. verticillioides, M. phaseolina and R. solani isolates caused severe symptoms on melon seedlings. Virulence of the F. oxysporum isolates on different cucurbit species was also investigated and it was determined that they caused severe wilting on melon and watermelon seedlings, while symptoms on squash and cucumber were moderate or slight. Additionally, reactions of five melon cultivars (Yusufbey, Çıtırex, Niovi, Ferdevs and Memory) commonly grown in the region against D. bryoniae were investigated using randomly selected four pathogen isolates. All the cultivars were susceptible to the disease.

Biological Control of Gummy Stem Blight Caused by Didymella bryoniae (Auersw.) on Watermelon by Bacillus sp. Strains

Background: The gummy stem blight caused by the fungal pathogen Didymella bryoniae (Auersw.) is a common and serious disease of watermelon plants in Vietnam. Bacillus species have been used to control plant diseases as biological control agents. The objectives of this studies were to determine the fungal inhibition of D. bryoniae and disease suppression of gummy stem blight under greenhouse by Bacillus sp. strains. Methods: The study was carried out at University of Agriculture and Forestry, Hue University during 2022-2023. Six potential Bacillus strains namely S1A1, S1F3, S13E2, S13E3, S18F11 and S20D12 isolated from stem-base of groundnut in central Vietnam were tested its fungal inhibition of D. bryoniae and disease suppression of gummy stem blight. Result: All tested strains of Bacillus sp. showed inhibition of the hyphal growth of D. bryoniae strain DB-01 in potato dextrose broth. Among the strains, Bacillus sp. S20F12 had highest inhibition of the growth of mycelia of D. bryoniae strain DB-01 with an antagonistic efficiency of 65.7% at 10 days after fungal inoculation. By 21 days after fungal inoculation, disease incidences were low in the plants treated with Bacillus sp. S20D12 and S1F3 by 46.6 and 50.0% and disease severity by 15,3% and 20.6%, respectively. As results, watermelon plants were prevented from damage of gummy stem blight caused by D. bryoniae strain BD-01 by Bacillus sp. S20D12 and S1F13. The results would contribute to the knowledge of antagonistic activities of the Bacillus to optimize the biological control against D. bryoniae.

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.

Infection Biology of Stagonosporopsis cucurbitacearum in Watermelon and Defence Responses in the Host

Infection biology and defence responses of watermelon attacked by Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae) were studied in two genotypes, accessions PI189225 (moderately resistant) and 232-0125/B (susceptible). On intact leaf surfaces, spores started to germinate 14 h after inoculation (hai) with one to three germ tubes, which subsequently developed and formed appressoria. Invasion of the host tissue started at 20 hai by direct penetration from appressoria or occasionally indirectly through stomata. In the susceptible accession, a significantly higher number of direct penetrations were observed than in the moderately resistant. After invasion, hyphal colonisation was restricted in the intercellular spaces in the moderately resistant accession, whereas they developed extensively, causing tissue decay, in the susceptible accession. Macroscopic symptoms were seen in leaves of the moderately resistant accession as small and dry lesions, whereas big, water-soaked lesions developed on the susceptible accession within 48 hai. Investigations of the defence responses of the two accessions showed accumulation of H2O2 at penetration sites beneath appressoria in the moderately resistant, but to a lesser extent in the susceptible accession. Such H2O2 accumulation correlated with a reduction in penetration frequency and a lower level of hyphal growth after infection in the moderately resistant accession. There was a rapid and early increase in total peroxidase as well as β-1,3-glucanase activity in the moderately resistant compared to the susceptible accession. These results indicate that fungal penetration and development in watermelon are inhibited by a consorted action of different responses including accumulation of H2O2, peroxidase and β-1,3-glucanase.

Design, Synthesis, Antifungal Evaluation, Structure-Activity Relationship (SAR) Study, and Molecular Docking of Novel Spirotryprostatin A Derivatives.

Phytopathogenic fungi cause plant diseases and economic losses in agriculture. To efficiently control plant pathogen infections, a total of 19 spirotryprostatin A derivatives and 26 spirooxindole derivatives were designed, synthesized, and tested for their antifungal activity against ten plant pathogens. Additionally, the intermediates of spirooxindole derivatives were investigated, including proposing a mechanism for diastereoselectivity and performing amplification experiments. The bioassay results demonstrated that spirotryprostatin A derivatives possess good and broad-spectrum antifungal activities. Compound 4d exhibited excellent antifungal activity in vitro, equal to or higher than the positive control ketoconazole, against Helminthosporium maydis, Trichothecium roseum, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, Alternaria brassicae, Alternaria alternate, and Fusarium solan (MICs: 8-32 µg/mL). Compound 4k also displayed remarkable antifungal activity against eight other phytopathogenic fungi, including Fusarium oxysporium f. sp. niveum and Mycosphaerella melonis (MICs: 8-32 µg/mL). The preliminary structure-activity relationships (SARs) were further discussed. Moreover, molecular docking studies revealed that spirotryprostatin A derivatives anchored in the binding site of succinate dehydrogenase (SDH). Therefore, these compounds showed potential as natural compound-based chiral fungicides and hold promise as candidates for further enhancements in terms of structure and properties.

Natural field diagnosis and molecular confirmation of fungal and bacterial watermelon pathogens in Bangladesh: A case study from the Natore and Sylhet districts.

The study investigated watermelon diseases characterized by contrasting climatic conditions in the Sylhet and Natore Districts of Bangladesh. Sylhet experiences lower temperatures and high rainfall, while Natore has higher temperatures and low rainfall. In these survey regions, 40 watermelon fields were selected, and 10 diseases were observed, including 4 fungal, 3 bacterial, 2 water mold, and 1 viral disease. The observed diseases were Anthracnose, Cercospora leaf spot, Fusarium wilt, Gummy stem blight, Downy mildew, Phytophthora fruit rot, Bacterial fruit blotch, Angular leaf spot, Yellow vine, and Watermelon mosaic disease. Molecular analysis was done in the Plant Pathology Lab at Sher-e-Bangla Agricultural University using the specific primers for fungal (ITS1/ITS4) and bacterial (27F/1492R) DNA regions and identified nine pathogen species, excluding the causal organism of the viral disease. The identified pathogens included Colletrotrichum orbiculare, Cercospora citrullina, Fusarium oxysporum, Stagonosporopsis cucurbitacearum, Pseudoperonospora cubensis, Phytophthora capsici, Acidovorax citrulli, Pseudomonas syringae, and Serratia marcescens. The sequencing of the identified pathogens revealed high homology (98.91-99.71%) with known sequences in the GenBank database. Phylogenetic analysis showed six clusters for fungal and water mold pathogen isolates and three for bacterial isolates where the percentages of replicate trees were 100% in all the cases. Among the identified diseases, the highest disease occurrence was caused by Fusarium wilt (47.5%) followed by Gummy stem blight (41.5%) in the Sylhet region and Angular leaf spot (37.5%) followed by Yellow vine (33%) in the Natore area. Fusarium wilt also has a high disease intensity, demonstrating its devastating impact on yield. This study highlights the influence of environmental conditions on disease incidence and underscores the need for tailored management strategies. These findings provide a foundation for developing targeted disease management practices for sustainable watermelon cultivation in Bangladesh.

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.

Preparation of Emulsions of some Vegetable Oils and Evaluation of Their Effectiveness in Inhibiting Pathogenic Fungus Didymella bryoniae

Five pathogenic fungi were isolated from the infected melon/Cucumis melo, Cucumber/Cucumis sativus, Watermelon/Citrullus lanatus and Squash/Cucurbita pepo with gum stem blight disease. The fungi identified to the species level by the nucleotide sequence analysis method for the ITS region within the 5.8SrRNA gene as Stagonosporopsis cucurbitacearum strain Has. AA-16, S. cucurbitacearum strain Has. AA-17, S. cucurbitacearum strain Has. AA-18, S. cucurbitacearum strain Has. AA-19 and S. cucurbitacearum strain Has. AA-19 and S. cucurbitacearum strain Has. AA-20 then registered in the World Genetic Bank at the NCBI website under the accession numbers OP247683.1, OP247685.1, OP247686.1, OP247709.1 and OP247707.1, respectively. The physical and antagonistic properties of five types of emulsions produced from lavender, thyme, cinnamon and cloves oils with surfactants Inex A and Spam 80 were studied. The results showed a decrease in the surface tension of all emulsions produced from these oils with both surfactants. Increasing the temperature to 40°C led to a more decrease in the surface tension compared to 20°C. The lowest surface tension values for emulsions produced from clove, thyme and cinnamon oils with the Index A (1:6;oil:Inex A) at 40°C, the surface tension reached to 11.98, 13.22 and 14.17 Dyne/cm, respectively. The stability of all emulsions resulting from the studied oils increased with an increase in the ratios of the Inex A and Span 80, the highest stability was reached by decreasing the separation layer when emulsions of thyme and lavender oils with Inex A at a ratio of 1:6 at 20°C, which reached 0.61 and 0.66 cm, respectively. All emulsions consisting of oils and surfactants inhibited the growth of pathogenic fungus D. bryoniae, the highest inhibition zones were 1.9, 1.8, 1.7 and 1.6 cm for emulsions resulting from clove, cinnamon, thyme and lavender oils with Inex A at a ratio of 1:4 at a 20°C, respectively.

The Extracellular Lipopeptides and Volatile Organic Compounds of Bacillus subtilis DHA41 Display Broad-Spectrum Antifungal Activity against Soil-Borne Phytopathogenic Fungi.

Fusarium oxysporum f. sp. niveum (Fon) is a devastating soil-borne fungus causing Fusarium wilt in watermelon. The present study investigated the biochemical mechanism underlying the antifungal activity exhibited by the antagonistic bacterial strain DHA41, particularly against Fon. Molecular characterization based on the 16S rRNA gene confirmed that DHA41 is a strain of Bacillus subtilis, capable of synthesizing antifungal lipopeptides, such as iturins and fengycins, which was further confirmed by detecting corresponding lipopeptide biosynthesis genes, namely ItuB, ItuD, and FenD. The cell-free culture filtrate and extracellular lipopeptide extract of B. subtilis DHA41 demonstrated significant inhibitory effects on the mycelial growth of Fon, Didymella bryoniae, Sclerotinia sclerotiorum, Fusarium graminearum, and Rhizoctonia solani. The lipopeptide extract showed emulsification activity and inhibited Fon mycelial growth by 86.4% at 100 µg/mL. Transmission electron microscope observations confirmed that the lipopeptide extract disrupted Fon cellular integrity. Furthermore, B. subtilis DHA41 emitted volatile organic compounds (VOCs) that exhibited antifungal activity against Fon, D. bryoniae, S. sclerotiorum, and F. graminearum. These findings provide evidence that B. subtilis DHA41 possesses broad-spectrum antifungal activity against different fungi pathogens, including Fon, through the production of extracellular lipopeptides and VOCs.

Identification and fine mapping of gummy stem blight resistance gene Gsb-7(t) in Melon.

Gummy stem blight (GSB), caused by the Didymella bryoniae (Auersw.) Rehm, is a devastating fungal disease of melon worldwide. Breeding GSB-resistant cultivars with host resistance genes is considered to be the most economic and effective strategy to control this disease. In this study, 260 melon germplasm resources were screened for resistance to GSB, and an inbred line H55R exhibited immunity to GSB was identified. To further understand the resistance mechanism of H55R against GSB, an F2 population was obtained from a cross between the GSB-susceptible line A15 and H55R, and genetic analysis indicated that the GSB resistance in H55R was controlled by a single dominant gene, tentatively named Gsb-7(t). The Gsb-7(t) gene was finally delimited to a 140 kb interval on chromosome 7 using bulked-segregant analysis and chromosome walking strategies. Ten putative genes were annotated in this region that contains a wall-associated receptor kinase (WAK) gene MELO3C010403. The MELO3C010403 gene contains two alternative transcripts, MELO3C010403-T1 and MELO3C010403-T2, with five and seven non-synonymous mutation sites, respectively. Gene expression analysis showed that expression of MELO3C010403-T2 but not the MELO3C010403-T1 was significantly induced by the D. bryoniae at 24 hours post-inoculation (hpi), indicating that the MELO3C010403-T2 transcript of MELO3C010403 was the most likely candidate gene of Gsb-7(t). Our results offer new genetic resources and will be helpful for the development of GSB-resistant melon cultivars in the future.

Molecular and biological characterization of lab-induced benzovindiflupyr-resistant mutants of Didymella bryoniae

Benzovindiflupyr is a novel succinate dehydrogenase inhibitor fungicide effective against many plant pathogens. However, the resistance risk of Didymella bryoniae to this fungicide is unknown. This study determined the baseline sensitivity of 69 wild D. bryoniae strains to benzovindiflupyr on mycelial growth with half-maximal effective concentrations (EC50) of 0.0021–0.0983 μg/mL with an average of 0.027 ± 0.0216 μg/mL. The minimum inhibitory concentration was 3 μg/mL. Four benzovindiflupyr-resistant mutants were obtained by fungicide taming and their resistance could stably inherited. Most mutants were not strikingly different from wild-type strains in mycelial growth, dry weight, pathogenicity, and sensitivity to various environmental stress factors. Most benzovindiflupyr-resistant mutants showed positive cross-resistance to pydiflumetofen, fluopyram, fluxapyroxad, fluindapyr, pyraziflumid, isopyrazam, and boscalid, but not tebuconazole and fluazinam. Consequently, the resistance risk of D. bryoniae to benzovindiflupyr would be medium to high. A point mutation at codon 277 in SdhB, changing CAC (histidine) to TAC (tyrosine), was identified in highly-resistant mutant XN51BR-1 but not in other mutants. However, a point mutation at codon 248 in SdhB of Fusarium graminearum (equivalent to the H277Y change in D. bryoniae) conferred medium resistance to benzovindiflupyr. These results provide further information about the resistance risk and resistance mechanism of D. bryoniae to benzovindiflupyr.

Suppression of Fusarium Wilt in Watermelon by Bacillus amyloliquefaciens DHA55 through Extracellular Production of Antifungal Lipopeptides

Fusarium wilt caused by Fusarium oxysporum f. sp. niveum is one of the most devastating fungal diseases affecting watermelon (Citrullus lanatus L.). The present study aimed to identify potent antagonistic bacterial strains with substantial antifungal activity against F. oxysporum f. sp. niveum and to explore their potential for biocontrol of Fusarium wilt in watermelon. Out of 77 isolates from watermelon rhizosphere, six bacterial strains-namely, DHA4, DHA6, DHA10, DHA12, DHA41, and DHA55-exhibited significant antifungal activity against F. oxysporum f. sp. niveum, as well as other phytopathogenic fungi, including Didymella bryoniae, Sclerotinia sclerotiorum, Fusarium graminearum, and Rhizoctonia solani. These Gram-positive, rod-shaped, antagonistic bacterial strains were able to produce exo-enzymes (e.g., catalase, protease, and cellulase), siderophore, and indole-3-acetic acid and had the ability to solubilize phosphate. In greenhouse experiments, these antagonistic bacterial strains not only promoted plant growth but also suppressed Fusarium wilt in watermelon. Among these strains, DHA55 was the most effective, achieving the highest disease suppression of 74.9%. Strain DHA55 was identified as Bacillus amyloliquefaciens based on physiological, biochemical, and molecular characterization. B. amyloliquefaciens DHA55 produced various antifungal lipopeptides, including iturin, surfactin, and fengycin, that showed significant antifungal activities against F. oxysporum f. sp. niveum. Microscopic observations revealed that B. amyloliquefaciens DHA55 exhibited an inhibitory effect against F. oxysporum f. sp. niveum on the root surface of watermelon plants. These results demonstrate that B. amyloliquefaciens DHA55 can effectively promote plant growth and suppress the development of watermelon Fusarium wilt, providing a promising agent for the biocontrol of Fusarium wilt in watermelon.

Design, Synthesis, and Anti-Fungal Evaluation of Heterocyclic Benzoxazole Derivatives

In order to discover more promising anti-fungal agents, a series of benzoxazole family was synthesized by PPA-catalyzed condensation and a Raney nickel/hydrazine reduction. Altogether 45 compounds were obtained in good to excellent yields and characterized by FT-IR, NMR, MS, and X-ray crystal diffraction. Moreover, the biological activity against eight phytopathogenic fungi was investigated. All in all, most of these compounds bear moderate antifungal activities. Among them, three candidates show the strongest activities, compound 4ac, 4bc provided over 50% inhibition rate against five fungi. Especially, the inhibitory rate of compound 4ah on Mycosphaerella melonis reached 76.4%.

Influence of the Diffusivity and Transmittance of a Plastic Greenhouse Cover on the Development of Fungal Diseases in a Cucumber Crop

Mediterranean greenhouses are usually covered by plastic materials (films); these films allow light to pass through them, modifying some of their characteristics. The properties of the plastic cover influence the development of greenhouse crops. In addition, it can influence the stresses that the plants endure and the development of fungal diseases in the crop. The aim of this study is to analyze the effect that an experimental film cover, with high transmittance and high light diffusivity, produces on the development of fungal diseases on a cucumber crop (Cucumis sativus L.). Two different film covers were compared: (i) commercial film (transmittance of 85%; diffusivity of 60%); and (ii) experimental film (transmittance of 90%; diffusivity of 55%). The study was carried out across two autumn–winter crop cycles in a multi-span greenhouse divided into two isolated sectors. Three fungal diseases caused the main damage to the cucumber crop: downy mildew (Pseudoperonospora cubensis), powdery mildew (Sphaerotheca fuliginia) and gummy stem blight (Didymella bryoniae). In the case of powdery mildew, a greater severity in the sector was observed with the commercial film in comparison with the sector with the experimental film, with significant statistical differences between the two sectors in both crop cycles. Downy mildew and gummy stem blight were fungal diseases with less presence than downy mildew, and a greater presence of these two fungal diseases in the sector with the commercial film was also observed in both crop cycles.

Population Structure of Stagonosporopsis Species Associated with Cucurbit Gummy Stem Blight in Korea.

Gummy stem blight (GSB), a common and serious disease in cucurbits worldwide, is caused by three genetically distinct species: Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), S. citrulli, and S. caricae. In Korea, however, the three species of Stagonosporopsis have been barely characterized. In this study, 21 Stagonosporopsis isolates were recovered from watermelon (Citrullus lanatus) and muskmelon (Cucumis melo) leaves and stem showing blight symptoms collected from 43 fields in Korea. Sequence analysis performed with an internal transcribed spacer region was not competent to differentiate the Stagonosporopsis isolates. On the contrary, analysis of β-tubulin (TUB) genes and three microsatellite markers, Db01, Db05, and Db06, successfully differentiated Stagonosporopsis isolates. Further sequence analysis identified two Stagonosporopsis species, S. citrulli and S. caricae, and one previously unknown species of Stagonosporopsis. Representative isolates from three species caused dark water-soaked lesions on the detached watermelon and muskmelon leaves with no significant differences in the aggressiveness. Our results indicate that the S. citrulli, S. caricae, and unknown Stagonosporopsis sp. are all causal agents of GSB for both watermelon and muskmelon. This is the first report of a new species and the population structure of Stagonosporopsis species causing GSB in Korea.

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.

Fine mapping a quantitative trait locus underlying seedling resistance to gummy stem blight using a residual heterozygous lines-derived strategy in cucumber.

Gummy stem blight (GSB), caused by Didymella bryoniae, is one of the most devastating diseases that severely reduces cucumber production. Developing resistant varieties would be an effective strategy to control GSB. Although several GSB-resistant QTLs have been reported, causal genes for GSB resistance have not yet been identified in cucumber. A novel loci gsb3.1 for seedling GSB resistance from the “PI 183967” genotype was previously identified in a 1.7-Mb interval on chromosome 3. In this study, we developed a residual heterozygous line-derived strategy from Recombinant Inbred Lines to perform fine mapping, and with this approach, the gsb3.1 locus was narrowed to a 38 kb interval. There were six predicted genes at the gsb3.1 locus, four of which differed in expression in the GSB-resistant compared to the susceptible lines after fungal inoculation. These candidate genes (Csa3G020050, Csa3G020060, Csa3G020090, and Csa3G020590) within the gsb3.1 locus could be helpful for the genetic study of GSB resistance and marker-assisted selection in cucumber. Phylogenetic analyses indicated that the resistant gsb3.1 allele may uniquely exist in the wild species present in the Indian group, and that nucleotide diversity was significantly reduced in cultivated accessions. Therefore, the gsb3.1 allele could be introgressed into existing commercial cultivars and combined with other resistance QTLs to provide broad-spectrum and robust GSB resistance in cucumber.

Loop-Mediated Isothermal Amplification for the Rapid Detection of the Mutation of Carbendazim-Resistant Isolates in Didymella bryoniae

Gummy stem blight (GSB) caused by Didymella bryoniae (D. bryoniae) is a worldwide fungal soil-borne disease that can cause severe yield reduction of watermelon. To shorten the monitoring time of carbendazim-resistant strains of D. bryoniae in the field, in this study, we developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of carbendazim-resistant strains of D. bryoniae. The β-tubulin gene of carbendazim-resistant strains was selected as the target for primer design. Based on the color change of hydroxy naphthol blue (HNB) and gel electrophoresis, the optimal reaction conditions for LAMP were determined at 65 °C for 50 min. In specificity tests, the LAMP assay was able to distinguish between carbendazim-resistant and sensitive strains of D. bryoniae. Moreover, in sensitivity tests, the detection limit was 1 ng/μL D. bryoniae DNA of the carbendazim-resistant strain. In addition, the LAMP method was successfully applied to detect carbendazim-resistant strains in D. bryoniae-infested samples. Therefore, the developed LAMP assay provides a new method for the rapid detection of carbendazim-resistant strains of D. bryoniae.

Survey of cucumber target spot, in vitro sporulation and aggressiveness of Corynespora cassiicola

The objective was to carry out a survey of the occurrence of the target spot (Corynespora cassiicola) in cucumber crops in São Paulo State; to evaluate culture media for sporulation of the pathogen and the aggressiveness of isolates of the pathogen in cucumber plants. The target spot was found in nine municipalities, being the main disease in seven of the ten municipalities sampled, with leaf incidence above 50%, showing that the target spot of cucumber is widely distributed in São Paulo State. Other diseases found less frequently were scabies (Cladosporium cucumerinum), alternaria spot (Alternaria cucumerina), cercospora spot (Cercospora citrullina) and downy mildew (Pseudoperonospora cubensis), predominant in samples from one or two municipalities, but also found in most municipalities. The zoned spot (Leandria momordicae) was found to have a low incidence, in samples from three municipalities and with an incidence lower than 20%. Greater sporulation of the pathogen occurred in tomato juice and oat flour media, without scraping the surface of the colony maintained for 16 days at 25ºC, under continuous fluorescent light. The germination of C. cassiicola isolates used in the aggressiveness test was between 82.8 and 95.5%, with the 50 isolates separated into two groups. The isolates were separated into four groups within the range of 3.1 to 22.3% in severity, after ten days of inoculation of the pathogen, showing the genetic variability within the species, which should be considered in management studies, such as genetic improvement.

Fine mapping and candidate gene analysis of gummy stem blight resistance in cucumber stem.

Two candidate genes (Csa6G046210 and Csa6G046240) were identified by fine-mapping gsb-s6.2 for gummy stem blight resistance in cucumber stem. Gummy stem blight (GSB) is a serious fungal disease caused by Didymella bryoniae, that affects cucumber yield and quality worldwide. However, no GSB-resistant genes have been identified in cucumber cultivars. In this study, the wild cucumber accession 'PI 183967' was used as a source of resistance to GSB in adult stems. An F2 population was mapped using resistant line 'LM189' and susceptible line 'LM6' derived from a cross between 'PI 183967' and '931'. By developing InDel and SNP markers, the gsb-s6.2 QTL on Chr. 6 was fine-mapped to a 34kb interval harboring six genes. Gene Expression analysis after inoculation showed that two candidate genes (Csa6G046210 and Csa6G046240) were induced and differentially expressed between the resistant and susceptible parents, and may be involved in disease defense. Sequence alignment showed that Csa6G046210 encodes a multiple myeloma tumor-associated protein, and it harbored two nonsynonymous SNPs and one InDel in the third and the fourth exons, and two InDels in the TATA-box of the basal promoter region. Csa6G046240 encodes a MYB transcription factor with six variants in the AP2/ERF and MYB motifs in the promoter. These two candidate genes lay the foundation for revealing the mechanism of GSB resistance and may be useful for marker-assisted selection in cucumber disease-resistant breeding.