Didymella Bryoniae Research Articles

Manejo da adubação potássica e da irrigação no progresso de doenças fúngicas e produtividade da melancia

Entre as principais doenças da melancia, o crestamento gomoso do caule (Didymella bryoniae) e o míldio (Pseudoperonospora cubensis) se destacam devido às grandes perdas que causam na produtividade e qualidade de frutos. Para investigar a influência da adubação potássica no progresso do crestamento gomoso e na produtividade da melancia e a influência da lâmina de água e do intervalo de irrigação no progresso do míldio, foram conduzidos dois ensaios em condições de campo em Gurupi-TO, nos anos de 2009 e 2010. O ensaio I foi conduzido em blocos ao acaso e os tratamentos se constituíram em três doses de potássio aplicadas em cobertura (0, 50 e 100 kg ha-1 de KCl) e 12 repetições, sendo avaliada a severidade do crestamento gomoso, por meio de uma escala de notas e os dados de produção e qualidade dos frutos. No ensaio II, utilizou-se o delineamento em blocos ao acaso com quatro repetições, em esquema fatorial de 3 x 3, três lâminas de irrigação (100, 300 e 500 mm de água durante o ciclo da cultura) e três intervalos de irrigação. Avaliou-se além da severidade do míldio, também a produção e a qualidade dos frutos. Constatou-se que as doses de potássio não influenciaram no progresso do crestamento gomoso e no número, peso médio e qualidade dos frutos. As lâminas de irrigação aplicadas influenciaram no progresso do míldio. Maiores níveis de severidade do míldio foram observados nas plantas mantidas sob irrigação com lâmina de 500 mm de água. As lâminas de água aplicadas também influenciaram significativamente na produção e qualidade dos frutos. Observou-se a presença de maior número de frutos deformados ou tortos nas plantas mantidas sob déficit hídrico, na lâmina de 100 mm. As maiores produtividades comerciais e totais foram obtidas nas plantas irrigadas com a lâmina de 500 mm de água.

Genetic mapping of gummy stem blight (Didymella bryoniae) resistance genes in Cucumis sativus-hystrix introgression lines

Gummy stem blight (GSB, Didymella bryoniae (Auersw.) Rehm) is a devastating disease occurring worldwide in cucumber (Cucumis sativus L.) production and causing considerable yield loss. No commercially available cultivars are resistant to GSB. By screening 52 introgression lines (ILs) derived from the cross of C. hystrix × C. sativus and eight cucumber cultivar/lines through a whole plant assay, three ILs (HH1-8-1-2, HH1-8-5, HH1-8-1-16) were identified as GSB resistant lines. Six common introgression regions in these three ILs were on Chromosomes 1, 4, and 6. To further map the resistance in the ILs, three mapping populations (2009F2, 2009F2′ and 2010F2) from a cross between resistant IL HH1-8-1-2 and susceptible 8419 were constructed and used for QTL mapping with SSR markers. Two quantitative trait loci (QTLs) were identified; one on Chromosome 4 and the other on Chromosome 6. The interval for Chromosome 4 QTL is 12 cM spanning 3.569 Mbp, and the interval for Chromosome 6 QTL is 11 cM covering 1.299 Mbp. The mapped QTLs provide a foundation for map-based cloning of the genes and establishing an understanding of the associated mechanisms underlying GSB resistance in cucumber.

Diagnostic Guide for Gummy Stem Blight and Black Rot on Cucurbits

Gummy stem blight and black rot affect all commonly cultivated cucurbits, including melon, watermelon, cucumber, pumpkin, and squash. Symptoms may be found on all vegetative and reproductive parts of plants, although there is variation in susceptibility among species and horticultural types. Characteristic symptoms include foliar blight, crown and stem cankers, and fruit rot. The pathogen, Didymella bryoniae, can be identified from its fruiting bodies of pycnidia and pseudothecia on diseased tissue and in culture. Accepted for publication 27 August 2013. Published 24 October 2013.

Occurrence of QoI-resistant Strains of Didymella bryoniae, Causal Fungus of Gummy Stem Blight of Cucurbits in Kanagawa Prefecture

Occurrence of QoI-resistant Strains of <i>Didymella bryoniae</i>, Causal Fungus of Gummy Stem Blight of Cucurbits in Kanagawa Prefecture

Isolates of Didymella bryoniae from South Carolina Remain Sensitive to DMI Fungicides Despite Multiyear Exposure

AbstractTriazole fungicides, which are sterol demethylation inhibitors, have become the primary systemic fungicides applied to cucurbits to control gummy stem blight caused by Didymella bryoniae. Isolates of D. bryoniae from South Carolina that were never exposed to tebuconazole or exposed for several years were tested for sensitivity to tebuconazole and difenoconazole. Colony diameters, percentage germination of ascospores and conidia, and germ tube lengths were measured when isolates were grown on agar amended with 0.10–10.0 mg/l tebuconazole and 0.01–1.0 mg/l difenoconazole. All 147 isolates tested were sensitive to tebuconazole and difenoconazole with mean EC50 values of 0.41 and 0.054 mg/l, respectively. Ascospore germination was greater than conidia germination on fungicide‐amended agar. Although the length of germ tubes arising from both spore types was reduced by both fungicides, the reduction was greater for ascospore germ tubes than for conidia germ tubes. Because many watermelon growers rotate crops among fields every two years, local populations of D. bryoniae have not been exposed repeatedly to tebuconazole. In addition, growers often apply a rotation of systemic and contact fungicides. Thus, despite exposure to tebuconazole for up to nine years, isolates of D. bryoniae from South Carolina remain sensitive to triazole fungicides.

Composição e fungitoxicidade do óleo essencial de capim citronela em função da adubação orgânica

O objetivo deste trabalho foi avaliar a influência da adubação orgânica sobre o teor e a composição do óleo essencial de capim citronela (Cymbopogon nardus), bem como a fungitoxicidade desse óleo ao crescimento micelial dos fungos Didymella bryoniae, Colletotrichum gloeosporioides e Amphobotrys ricini. A extração do óleo essencial foi realizada por hidrodestilação, a partir de plantas cultivadas em quatro doses de adubação orgânica (0, 3, 6 e 9 kg de esterco bovino curtido por cova), e a identificação dos constituintes químicos foi conduzida por cromatografia gasosa/espectrometria de massas. Para avaliar a inibição do crescimento micelial, o experimento foi instalado no delineamento inteiramente casualizado, em arranjo fatorial. Foram testadas sete alíquotas do óleo essencial (0, 5, 10, 15, 20, 25 e 30 µL), em cinco épocas de avaliação, com quatro repetições. O teor de óleo essencial do capim citronela não foi afetado pela adubação orgânica, e o óleo essencial do capim citronela apresentou 24 compostos químicos, entre estes monoterpenos e sesquiterpenos, com maior concentração dos compostos citronelal, β-citronelol, geraniol e elemol. O óleo essencial do capim citronela apresenta maior efeito de inibição a Amphobotrys ricini, em comparação aos fungos Didymella bryoniae e Colletotrichum gloeosporioides.

Relationship Between Fungicide Sensitivity and Control of Gummy Stem Blight of Watermelon Under Field Conditions.

Gummy stem blight (GSB), caused by the fungus Didymella bryoniae, is the most destructive disease of watermelon and is managed primarily with fungicides. D. bryoniae has developed resistance to many fungicides that were once very effective, including azoxystrobin, boscalid, and thiophanate-methyl. Field experiments were conducted in Tifton (TN) and Reidsville (RV), GA in 2009 and 2010 to establish a relationship between frequency of resistance to a fungicide based on in vitro assays and its efficacy in the management of GSB. Frequency of resistance to boscalid, thiophanate-methyl, and azoxystrobin was >0.80 in isolates collected from nontreated plots in both locations and years. All isolates collected after six applications of boscalid, thiophanate-methyl, or azoxystrobin were resistant to the respective fungicide. All isolates collected from treated and nontreated plots were sensitive to tebuconazole and difenoconazole. GSB severity was assessed on a weekly basis from 63 days after planting. GSB severity in plots treated with boscalid, thiophanate-methyl, or azoxystrobin was not significantly different from that in the nontreated plots (39%, TN-2009; 45%, TN-2010; and 16%, RV-2010). GSB severity in tebuconazole-treated plots (27%, TN-2009; 14%, TN-2010; and 4%, RV-2010) was significantly lower than all other treatments and the nontreated control. There was a consistent negative association between frequency of fungicide resistance and disease control in the field. Thus, knowledge of the frequency of fungicide resistance in the pathogen population will be helpful in selecting the most effective fungicides for the management of GSB in watermelon fields.

First Report of Didymella bryoniae Causing Gummy Stem Blight of Chayote in Taiwan

Chayote (Sechium edule (Jacq.) Swartz, Cucurbitaceae), originally native to Mexico, is an important vegetable known as "dragon-whisker vegetable" and is cultivated for its shoots in Ji-an, Hualien County in eastern Taiwan. In June 2010, 70 to 80% of the chayote plants grown in Ji-an developed necrotic spots on stems, leaves, and fruits. The disease was severe during the warm and rainy season from June to August. The symptoms on stems, leaves, and fruits were water-soaked lesions that eventually dried up, cracked, and produced perithecia on necrotic tissues. A single ascospore was isolated from perithecia harvested from diseased stems and cultured on potato dextrose agar (PDA) at 25°C for 1 month. Colonies of three isolates (SE5, SE6, and SE7) were white to olivaceous green bearing unicellular conidia measuring 2 to 5 × 3 to 10 μm, which is consistent with the morphological characteristics of Didymella bryoniae (Auersw.) Rehm (anamorph Phoma cucurbitacearum (Fr.:Fr.) Sacc.) (1,2,3). DNA of SE5, SE6, and SE7 isolates were obtained using microwave-based method (4). The internal transcribed spacer (ITS) rDNA (GenBank accessions AB714984, AB714985, and AB714986), PCR-amplified using primers ITS1 and ITS4, had 98 to 99% nucleotide sequence identity with D. bryoniae (GenBank Accession Nos. GU045304 and GU592001). A pathogenicity test was conducted in a greenhouse with temperature ranging from 20 to 30°C. Three-day-old mycelial plugs (5 × 5 mm) of the three isolates were placed on the needle-pricked wounds of stems and leaves of 36 4-month-old potted chayote plants wrapped in plastic bags to maintain 100% relative humidity for 2 days. Six days after inoculation, water-soaked lesions formed on the stems and leaves. Controls inoculated with sterile water had no symptoms. The fungus reisolated from the lesions of diseased stems and leaves had morphological characteristics of D. bryoniae. Based on the results of morphology, molecular data, and pathogenicity tests, we reported for the first time to our knowledge that gummy stem blight of chayote is caused by D. bryoniae in Taiwan.

Promotion of growth, health and stress tolerance of Styrian oil pumpkins by bacterial endophytes

Substantial yield losses of Styrian oil pumpkin caused by the fungus Didymella bryoniae and bacterial pathogens were recently reported. Here we applied bacterial endophytes with a broad antagonistic activity to pumpkin plants by seed priming. Effects of the bacterial inoculants with and without chemical seed treatments on plant growth and health were evaluated during three different field trials in two consecutive years (2010 and 2011). Biological seed treatments strongly supported the germination of pumpkin seeds. In 2010, the germination of the biologically treated seeds was comparable to the rate following a chemical treatment; whilst in 2011 effects of biological seed treatments were more obvious, including an increased emergence rate up to 109 % by Serratia plymuthica S13. Furthermore, tolerance against desiccation stress was observed for Serratia as well as for Lysobacter gummosus L101 treatment. The biological treatment showed different effects against fungal diseases: no effect on fruit rot was observed, whereas powdery mildew could be significantly suppressed by Paenibacillus polymyxa PB71 and L. gummosus L101 in 2010. In addition, both strains led to reproducible increases in harvest yields. In this study, we found bacterial endophytes suitable as inoculants for plant growth promotion, biocontrol, as well as for enhancing stress tolerance in Styrian oil pumpkins.

Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase-Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae.

Didymella bryoniae, which causes gummy stem blight (GSB) of watermelon, has a history of developing resistance to fungicides, most recently the succinate-dehydrogenase-inhibiting (SDHI) fungicide boscalid. To facilitate fungicide resistance monitoring, baseline sensitivity distributions were established for demethylation-inhibiting (DMI) fungicides tebuconazole and difenoconazole and the SDHI fungicide penthiopyrad, and reestablished for the SDHI fungicide boscalid. In all, 71 isolates with no known prior exposure to SDHIs or DMIs were used to determine the effective concentration at which mycelial growth was inhibited by 50% (EC50). EC50 values for boscalid, penthiopyrad, tebuconazole, and difenoconazole were 0.018 to 0.064, 0.015 to 0.057, 0.062 to 0.385, and 0.018 to 0.048 μg/ml, with median values of 0.032, 0.026, 0.118, and 0.031 μg/ml, respectively. Significant positive correlations between the sensitivity to penthiopyrad and boscalid (P < 0.0001, r = 0.75) and between tebuconazole and difenoconazole (P < 0.0001, r = 0.59) indicate a potential for cross-resistance between chemically related fungicides. In 2009, 103 isolates from fungicidetreated watermelon fields were tested for sensitivity to boscalid and penthiopyrad using a discriminatory concentration of 3.0 μg/ml. Of the isolates tested, 82 were insensitive and 14 were sensitive to both fungicides. Because of the significant potential for cross-resistance between closely related fungicides, growers will be advised not to use both SDHIs or both DMIs successively in the same fungicide spray program.

Net melon resistance to Didymella bryoniae according to grafting and potassium levels

Chemical control of the fungus Didymella bryoniae, the causal agent of the disease gummy stem blight in melon, is frequently inefficient; thus, alternatives such as grafting and nutrition must be studied. Rootstocks and potassium levels were tested aimed at controlling this disease in net melon under protected environment. The melon hybrid 'Bônus II', ungrafted and grafted onto 'Dinero' melon and 'Strong Tosa' pumpkin rootstocks, was cultivated and inoculated by using the toothpick insertion method with 7-mm mycelial disks from the isolate D. bryoniae Dbr 37; for control, only toothpick insertion was used. The plants were subjected to the following potassium levels: 0, 62.5, 125, 187.5, 250 mg L-1. Grafted 'Bônus II' melon plants were resistant to the fungus, whereas ungrafted ones were susceptible. The adopted potassium levels did not influence the stem lesion size or the survival of plants.

Anthracnose and Gummy Stem Blight Are Reduced on Watermelon Grown on a No-Till Hairy Vetch Cover Crop.

Multiple applications of fungicides are used to manage anthracnose caused by Colletotrichum orbiculare and gummy stem blight caused by Didymella bryoniae, the two most common and destructive diseases on watermelon (Citrullus lanatus) in the mid-Atlantic region of the United States. To develop a sustainable, nonchemical management option, a split-plot experiment was conducted over 3 years to evaluate the effects of a no-till hairy vetch (Vicia villosa) cover crop on disease severity, plant growth, and fruit yield compared with two conventional bedding systems and fungicide application. The main plots were bedding strategies consisting of bare ground, polyethylene covering, or a hairy vetch cover crop that was planted in the fall, killed the following spring, and left on the soil surface as an organic mulch. The subplots were a nonfungicide control or a weekly application of a standard fungicide program. Hairy vetch mulch provided greater than a 65% reduction in the area under the disease progress curves of anthracnose and gummy stem blight and greater than an 88% decrease in diseased fruit compared with bare ground or polyethylene mulch. The reductions were comparable with those achieved by fungicide applications. Watermelon vine lengths in plots with hairy vetch were similar to or greater than those in plots with polyethylene or bare ground that were treated with fungicides. Marketable fruit in plots with hairy vetch was higher compared with bare ground in 2 of 3 years and was similar to that in plots treated with fungicides in all 3 years. Addition of fungicide application to hairy vetch treatment further reduced anthracnose in 1 year and gummy stem blight in 2 years but did not significantly increase fruit yield in all 3 years. This is the first demonstration that a no-till hairy vetch production system can reduce anthracnose and gummy stem blight on watermelon and that the production system has the potential to mitigate damage caused by these diseases.

Differential Sensitivity to Boscalid in Conidia and Ascospores of Didymella bryoniae and Frequency of Boscalid-Insensitive Isolates in South Carolina.

Since 2003, a 2:1 mixture of the fungicides boscalid and pyraclostrobin (Pristine) has been used widely on watermelon and other cucurbits, primarily to control gummy stem blight caused by Didymella bryoniae. Several isolates of D. bryoniae that were insensitive to boscalid at 10 mg/liter were found in a watermelon research plot in South Carolina in 2008. In total, 201 isolates collected between 1998 and 2009 were tested for sensitivity to boscalid by determining percentage germination of ascospores and conidia on media amended with boscalid at 0.01 to 10.0 mg/liter. All 31 isolates collected in 1998, 2002, or 2005 were sensitive to boscalid. Of the 170 isolates collected in or after 2006, 84.7% were insensitive to boscalid, including 19 of 30 isolates recovered from greenhouse-grown seedlings. The oldest insensitive isolates were obtained in 2006 from a greenhouse and in 2008 from a commercial field. Ascospores were less sensitive to boscalid than conidia. With boscalid at 1.0 mg/liter, 22.4% of ascospores but only 4.1% of conidia of 31 sensitive isolates germinated. Similarly, a mean of 68.6% of the ascospores and 54.1% of the conidia of 120 insensitive isolates germinated at 1.0 and 10.0 mg/liter. The 50% effective concentration (EC50) values based on ascospore germination were two to three times higher than values based on conidia germination. The significance of miscalculating EC50 values by considering only conidia was demonstrated in a greenhouse experiment. Twelve isolates that were sensitive, moderately insensitive, or highly insensitive based on conidia germination did not differ in relative virulence on boscalid-treated muskmelon seedlings when inoculum suspensions comprised ascospores alone or ascospores and conidia. This is the first report of differential sensitivity to a fungicide between conidia and ascospores in D. bryoniae. Because D. bryoniae produces conidia and ascospores on diseased hosts, both spore types should be used when calculating EC50 values for boscalid.

Evaluation of Streptomyces spp. for biocontrol of gummy stem blight (Didymella bryoniae) and growth promotion of Cucumis melo L.

Gummy stem blight of Cucumis melo L. (melon) caused by Didymella bryoniae is a serious disease in the major production area of northwest China. Two Streptomyces isolates (Streptomyces pactum A12 and S. globisporus subsp. globisporus C28) previously isolated from the Qinghai-Tibet Plateau were investigated regarding their biocontrol of gummy stem blight and growth promotion of melon under controlled conditions. Streptomyces A12 and C28 indicated obvious antagonistic activity against D. bryoniae in vitro. Both A12 and C28 significantly decreased disease severity and AUDPC (area under the disease progress curve) of melon gummy stem blight in vivo (P < 0.05). Ten-fold dilution of C28 culture filtrate was more effective in controlling the disease compared with other treatments, the disease reduction effects were 41.0–64.2%. The mean fresh weights were increased by 40.4% for plants, 44.2% for roots, and 40.3% for aerial parts, when A12 was applied in both nursery soil and transplanted soil. Streptomyces C28 also increased the mean fresh weights of melon plants by 18.4–49.0% compared with the control in pot trial. Streptomyces A12 and C28 showed substantial colonization abilities in the rhizosphere and on the rhizoplane of melon plants. Results demonstrated that Streptomyces A12 and C28 were of positive effect on the biocontrol of gummy stem blight and growth promotion of Cucumis melo L.

Boscalid Insensitivity Documented in Didymella bryoniae Isolated from Watermelon in Florida and North Carolina

The fungicide Pristine, a commercial mixture of pyraclostrobin and boscalid, has been used widely on watermelon and other cucurbits to control gummy stem blight, caused by the fungus Didymella bryoniae. Since 2007, isolates of D. bryoniae insensitive to boscalid have been found in Georgia, Indiana, and South Carolina. Most isolates of D. bryoniae obtained in 2009 and 2010 from diseased watermelon leaves collected in several counties in Florida and North Carolina were found to be insensitive to boscalid using in vitro assays. Gummy stem blight would not be effectively managed with Pristine in any of the counties where samples were collected due to the high frequency of insensitivity to boscalid. Fortunately growers can instead use several triazole fungicides registered for use on cucurbits since 2009. Accepted for publication 30 March 2012. Published 18 May 2012.

Bioactivity of essential oils on the fungus Didymella bryoniae of the cucumber culture

The objective of this study was to evaluate the in vitro antifungal activity of the essential oil of five plant species in the inhibition of mycelial growth of the fungus Didymella bryoniae causer of the gummy stem blight of cucumber ( Cucumis sativa ). In the inhibition of the mycelial growth of the fungus D. bryoniae was used five concentrations (250, 500, 750, 1000 and 1250 ppm) of essential oil of citronella grass (Cymbopogon nardus) , lemon grass (Cymbopogon citratus) , peppermint (Mentha piperita) , lemongrass (Lippia alba) and eucalipto citriodora (Eucalyptus citriodora) , in five times of assessment (2, 4, 6, 8 and 10 days after transplanting). Another experiment was performed with lower concentrations of essential oil of citronella grass (150, 300, 450 and 600 ppm) in four times of assessment (3, 5, 7 and 9 days after transplanting). The essential oils were distributed on the surface of the PDA meium crop (potato-dextrose-agar) for assessment of mycelial growth in different concentrations. The experiments were conducted in a completely randomized design with four replications. The essential oil of lemongrass provided the highest effect of inhibition of mycelial growth of D. bryoniae, 100% of inhibition at all tested concentrations. The essential oil of eucalipto citriodora ( Eucalyptus citriodora) provided the smaller effect of inhibition.

Molecular characterization of boscalid‐ and penthiopyrad‐resistant isolates of Didymella bryoniae and assessment of their sensitivity to fluopyram

Didymella bryoniae has a history of developing resistance to single-site fungicides. A recent example is with the succinate-dehydrogenase-inhibiting fungicide (SDHI) boscalid. In laboratory assays, out of 103 isolates of this fungus, 82 and seven were found to be very highly resistant (B(VHR) ) and highly resistant (B(HR) ) to boscalid respectively. Cross-resistance studies with the new SDHI penthiopyrad showed that the B(VHR) isolates were only highly resistant to penthiopyrad (B(VHR) -P(HR) ), while the B(HR) isolates appeared sensitive to penthiopyrad (B(HR) -P(S) ). In this study, the molecular mechanism of resistance in these two phenotypes (B(VHR) -P(HR) and B(HR) -P(S) ) was elucidated, and their sensitivity to the new SDHI fluopyram was assessed. A 456 bp cDNA amplified fragment of the succinate dehydrogenase iron sulfur gene (DbSDHB) was initially cloned and sequenced from two sensitive (B(S) -P(S) ), two B(VHR) -P(HR) and one B(HR) -P(S) isolate of D. bryoniae. Comparative analysis of the DbSDHB protein revealed that a highly conserved histidine residue involved in the binding of SDHIs and present in wild-type isolates was replaced by tyrosine (H277Y) or arginine (H277R) in the B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Further examination of the role and extent of these alterations showed that the H/Y and H/R substitutions were present in the remaining B(VHR) -P(HR) and B(HR) -P(S) variants respectively. Analysis of the sensitivity to fluopyram of representative isolates showed that both SDHB mutants were sensitive to this fungicide as the wild-type isolates. The genotype-specific cross-resistance relationships between the SDHIs boscalid and penthiopyrad and the lack of cross-resistance between these fungicides and fluopyram should be taken into account when selecting SDHIs for gummy stem blight management.

English

To search for the dematiaceous fungi in nature, clarify their habitat and the environmental circumstances in which they may infect man, we studied the phylogenetic relationships of these isolates. 60 dematiaceous fungal strains out of 367 soil samples were isolated. They were further identified by molecular biological method. The phylogenetic relationships were demonstrated by using internal transcribed spacer (ITS) region of ribosomal DNA sequences. In the neighbor-joining (NJ) tree,&nbsp;Phialophora&nbsp;sp.,Cladophialophora chaetospira,&nbsp;Exophiala spinifera,&nbsp;Phaeococcomyces&nbsp;sp. and&nbsp;Exophiala eucalyptorum&nbsp;formed cluster A,&nbsp;Didymella bryoniae,&nbsp;Leptosphaeriaceae&nbsp;sp.,&nbsp;Ascomycetesp.,&nbsp;Microdiplodia hawaiiensis&nbsp;and&nbsp;Cochliobolus lunatus&nbsp;formed cluster B,&nbsp;Staninwardia suttonii,&nbsp;Cladosporium oxysporum,&nbsp;Cladosporium cladosporioides, Cladosporium sp.&nbsp;andMelanized limestone ascomycete&nbsp;formed cluster C, and&nbsp;Scolecobasidium tereum,Scolecobasidium humicola&nbsp;formed cluster D. The phylogenetic relationships between cluster B, cluster C and cluster D were closer than that of cluster A.&nbsp;Dematiaceous species were found&nbsp;widely&nbsp;in Guangdong soil, and the distribution amounts were not in a specific pattern, the phylogenetic method based on the rDNA ITS sequence&nbsp;was proven to be a&nbsp;quick and accurate fungi identify method. There is a relationship between genetic distances and some biological habits of some strains, but lacking of connection was found between the genetic distances and the geographical factors. &nbsp; Key words:&nbsp;Dematiaceous fungi, isolation,&nbsp;phylogenetic tree,&nbsp;internal transcribed spacer (ITS) region,&nbsp;ribosomal DNA&nbsp;(rDNA).

Microbial Diversity Inside Pumpkins: Microhabitat-Specific Communities Display a High Antagonistic Potential Against Phytopathogens

Recent and substantial yield losses of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) are primarily caused by the ascomycetous fungus Didymella bryoniae but bacterial pathogens are frequently involved as well. The diversity of endophytic microbial communities from seeds (spermosphere), roots (endorhiza), flowers (anthosphere), and fruits (carposphere) of three different pumpkin cultivars was studied to develop a biocontrol strategy. A multiphasic approach combining molecular, microscopic, and cultivation techniques was applied to select a consortium of endophytes for biocontrol. Specific community structures for Pseudomonas and Bacillus, two important plant-associated genera, were found for each microenvironment by fingerprinting of 16S ribosomal RNA genes. All microenvironments were dominated by bacteria; fungi were less abundant. Of the 2,320 microbial isolates analyzed in dual culture assays, 165 (7%) were tested positively for in vitro antagonism against D. bryoniae. Out of these, 43 isolates inhibited the growth of bacterial pumpkin pathogens (Pectobacterium carotovorum, Pseudomonas viridiflava, Xanthomonas cucurbitae); here only bacteria were selected. Microenvironment-specific antagonists were found, and the spermosphere and anthosphere were revealed as underexplored reservoirs for antagonists. In the latter, a potential role of pollen grains as bacterial vectors between flowers was recognized. Six broad spectrum antagonists selected according to their activity, genotypic diversity, and occurrence were evaluated under greenhouse conditions. Disease severity on pumpkins of D. bryoniae was significantly reduced by Pseudomonas chlororaphis treatment and by a combined treatment of strains (Lysobacter gummosus, P. chlororaphis, Paenibacillus polymyxa, and Serratia plymuthica). This result provides a promising prospect to biologically control pumpkin diseases.

Emerging multi-pathogen disease caused by Didymella bryoniae and pathogenic bacteria on Styrian oil pumpkin

The Styrian oil pumpkin, Cucurbita pepo L. subsp. pepo var. styriaca Greb. is a crop of cultural, commercial, and medical importance. In the last decade, yield losses of pumpkins increased dramatically. The ascomycetous fungus Didymella bryoniae (Fuckel) Rehm was identified as main causal agent provoking gummy stem blight as well as black rot of pumpkins. We observed a remarkable phenotypic diversity of the fungal pathogen, which contrasted with a high genotypic similarity. Evidence of pathogenictiy of D. bryoniae on Styrian oil pumpkin was demonstrated in a newly developed greenhouse assay. Isolates representing the five observed phenotypic groups fulfilled the Koch’s postulates. In the field, the fungal disease was often associated with bacterial colonization by Pectobacterium carotovorum, Pseudomonas viridiflava, Pseudomonas syringae and Xanthomonas cucurbitae. The pathogenic behaviour of bacterial isolates on pumpkin was confirmed in the greenhouse assay. The high co-incidence of fungal and bacterial disease suggests mutualistic effects in pathogenesis. With a new assay, we found that bacteria can use the mycelium of D. bryoniae for translocation. We argue that the rapid rise of the multi-pathogen disease of pumpkins results from combined action of versatile pathogenic bacteria and the rapid translocation on a structurally versatile mycelium of the fungal pathogen.