Agent Of Gummy Stem Blight Research Articles

Widespread Resistance to Tebuconazole and Cross-Resistance to Other DMI Fungicides in Stagonosporopsis citrulli Isolated from Watermelon in South Carolina.

Tebuconazole, a demethylation-inhibitor (DMI) fungicide, is widely used on watermelon and muskmelon because it is inexpensive and has been effective against Stagonosporopsis citrulli, the primary causal agent of gummy stem blight in the southeastern United States. Most isolates (94% of 251) collected from watermelon in South Carolina in 2019 and 2021 were moderately resistant to tebuconazole at 3.0 mg/liter in vitro. Ninety isolates were identified as S. citrulli, and no isolates of S. caricae were found in this study. On watermelon and muskmelon seedlings treated with the field rate of tebuconazole, sensitive, moderately resistant, and highly resistant isolates were controlled 99, 74, and 45%, respectively. In vitro, tebuconazole-sensitive isolates were moderately resistant to tetraconazole and flutriafol but sensitive to difenoconazole and prothioconazole, while highly resistant isolates were highly resistant to tetraconazole and flutriafol and moderately resistant to difenoconazole and prothioconazole. On watermelon seedlings treated with field rates of five DMI fungicides in the greenhouse, severity of gummy stem blight did not differ significantly from the nontreated control when seedlings were inoculated with a highly resistant isolate, while severity was lower with all DMIs on seedlings inoculated with a sensitive isolate, although severity was greater with tetraconazole than with the other four DMIs. In the field, tetraconazole rotated with mancozeb did not reduce severity of gummy stem blight caused by a tebuconazole-sensitive isolate when compared to the nontreated control, while the other four DMIs did. With a highly resistant isolate, all DMIs rotated with mancozeb reduced severity of gummy stem blight compared to the nontreated control, but severity with tetraconazole and tebuconazole was greater than with mancozeb alone, and severity with flutriafol, difenoconazole, prothioconazole, and difenoconazole plus cyprodinil did not differ from mancozeb applied alone. Results from in vitro, greenhouse, and field experiments with the five DMI fungicides were highly correlated with each other. Thus, determining relative colony diameters with a discriminatory dose of 3 mg/liter of tebuconazole is an effective way to identify isolates of S. citrulli highly resistant to tebuconazole.

Direct antifungal activity of tiadinil, a systemic acquired resistance inducer, and thymol formulations on Stagonosporopsis citrulli and control of watermelon gummy stem blight

This study evaluated the direct antifungal activity of tiadinil [N-(3-chloro-4-methylphenyl)-4-methyl-1,2,3-thiadiazole-5-carboxamide], a systemic acquired resistance (SAR) inducer and two formulations of thymol (thymol I and thymol II) against Stagonosporopsis citrulli, the causal agent of gummy stem blight (GSB) disease of watermelon. Tiadinil, thymol I and thymol II completely inhibited the mycelial growth of S. citrulli in vitro at ≥ 100 ppm. Conidial germination and germ tube elongation were completely inhibited by tiadinil at ≥ 2000 ppm and by thymol-based formulations at ≥ 100 ppm. A single foliar application of tiadinil at ≥ 10 ppm or a single application of thymol I and II at ≥ 1 ppm, 48 h before or after pathogen inoculation, significantly reduced disease severity of watermelon seedlings inoculated with 105/ml conidial suspension of S. citrulli, compared to respective nontreated controls. Plants treated with foliar application of tiadinil at ≥ 1000 ppm before pathogen inoculation had significantly lower disease severity than plants that received an equivalent drench application. The efficacy of foliar application of tiadinil was affected by concentration and frequency of application. The study suggests direct antifungal activity of tiadinil, indicating a new mode of action of tiadinil against GSB disease of watermelon. The study also demonstrated direct antifungal action of thymol, a formulated active compound of essential oils, against S. citrulli and GSB disease of watermelon.

Identification and characterization of Didymella bryoniae causing gummy stem blight disease of watermelon (Citrullus lanatus) in Turkey

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is an important plant in the family Cucurbitaceae. Didymella bryoniae (Auersw.) Rehm [anamorph Phoma cucurbitacearum (Fr.) Sacc.] is the causative agent of gummy stem blight (GSB), a disease affecting members of the family Cucurbitaceae. Watermelon seedlings showing varied degree (20–90%) of suspected GSBs were collected from seedling companies in Antalya province, Turkey. The symptoms included angular water-soaked lesions, which turned tan over time; defoliation; dry, pale green stem; stem necrosis; gummy exudates; wilt; and the eventual death of the upper parts of the seedling plants. The growth of the fungal isolate in vitro showed white aerial and olivaceous mycelium, and olive to dark green or black substrate mycelium at the latter period of fungal growth. The colony surface was rough and undulated. The conidia were round-ended, cylindrical, monoseptate, and hyaline. The conidia ranged from 6.4 to 13.6 μm in length and 3.69–4.68 μm in diameter. The isolates were subjected to PCR using three primers, namely, DB-F3/DB-R3, GSBF1/GSBR1, and ITS1/ITS4, which produced fragment sizes of approximately 120, 780, and 560 bp, respectively. The real-time PCR using DB-F3/DB-R3 primer and SYBR Green fluorescence dye produced positive result for the samples tested. The D. bryoniae isolate was pathogenic to all the four different cucurbits tested and were consistently re-isolated from the diseased seedlings confirming their pathogenic status and thereby satisfying Koch's postulates. This is the second report confirming the presence of D. bryoniae in Turkey after its first detection in cucumber in 2015.

Identification and Characterization of the Causal Agent of Gummy Stem Blight from Muskmelon and Watermelon in East China

AbstractGummy stem blight (GSB) is one of the most destructive foliar diseases of cucurbits, worldwide. To identify and characterize the pathogen which causes GSB on watermelon and muskmelon in East China, morphological characteristics, pathogenicity assays as well as sequence characterization of the rDNA internal transcribed spacer (ITS) were performed on 41 isolates collected from Jiangsu, Zhejiang, Anhui and Jiangxi provinces. The mycelia of all the isolates were white on top and olivaceous green to black with concentric circles at the bottom on potato‐dextrose agar medium. The isolates differed significantly on aggressiveness based on pathogenicity assays. rDNA‐ITS sequences and phylogenetic analysis confirmed the isolates as Didymella bryoniae. The isolates were found to be highly identical with the exception of 13 isolates, which had a guanine substitution instead of adenine at position 131 of the ITS.

Development of a Real-time RT-PCR Assay for Squash Mosaic Virus Useful for Broad Spectrum Detection of Various Serotypes and its Incorporation into a Multiplex Seed Health Assay

Seed-borne pathogens pose a serious threat to modern agricultural cropping systems, as they can be disseminated to many geographical regions around the world. With trends of increasing global seed production and trade, seed-health testing is an important quality control step to prevent the introduction of harmful pathogens into agricultural production systems. An effective seed-health assay depends on a test that can provide timely, sensitive and broad-spectrum detection of all genetic variants of a pathogen, or in some cases, of several different pathogens. Previously, we developed a real-time PCR (qPCR) assay that would permit the simultaneous detection of two major seed-borne pathogens of cucurbits, the bacterium Acidovorax avenae subsp. citrulli (AAC, the causal agent of bacterial fruit blotch) and a fungus Didymella bryoniae (DB, the causal agent of gummy stem blight). The objective of the present study was to develop a sensitive, reverse transcriptase (RT)-based, qRT-PCR for broad spectrum detection of both serotypes of Squash mosaic virus (SqMV), that could be incorporated into a simultaneous detection of three pathogen types in a single PCR reaction. Converting SqMV RNA to cDNA prior to multiplexing stabilized the viral template that was then mixed with two other DNA templates (AAC and DB). To facilitate seed health testing, a generic plant nucleic acid extraction method was developed for cucurbit seeds. Using this method, nucleic acids extracted from seeds yielded strong signals for each target pathogen in multiplex qPCR. The ability to use a general nucleic acid extraction technique with subsequent PCR to detect bacterial, fungal and viral plant pathogens lends itself to a universal system for cucurbit seed health testing.

Infecção latente de Didymella bryoniae em meloeiro nobre

Didymella bryoniae, agente causal da podridão gomosa, é um importante patógeno em meloeiro nobre, em cultivo protegido e recentemente, resultados de alguns trabalhos têm evidenciado a ocorrência de infecção latente de D. bryoniae em meloeiro, sem, no entanto, registrar a comprovação. Objetivou-se com este trabalho constatar a ocorrência de infecção latente de D. bryoniae, em plantas dos híbridos de meloeiro nobre Sunrise, Bônus II e Prince Hakucho assintomáticas. Empregou-se a técnica de reação da polimerase em cadeia (PCR), com o conjunto de 'primers' D7S, D6 e UNLO28822, que amplificam regiões do DNA e distinguem D. bryoniae de outras espécies similares e de outros microrganismos comuns. Em todos os híbridos testados, constatou-se a presença de D. bryoniae no caule de plantas assintomáticas, através da amplificação das regiões específicas delimitadas pelos 'primers' utilizados.

An improved real-time PCR system for broad-spectrum detection of Didymella bryoniae, the causal agent of gummy stem blight of cucurbits

An improved real-time PCR system for broad-spectrum detection of Didymella bryoniae, the causal agent of gummy stem blight of cucurbits

Sensitivity to azoxystrobin in Didymella bryoniae isolates collected before and after field use of strobilurin fungicides

Isolates of Didymella bryoniae (Auersw.) Rehm, causal agent of gummy stem blight on cucurbits, developed insensitivity to azoxystrobin in the eastern United States 2 years after first commercial use in 1998. Baseline sensitivity of this fungus to azoxystrobin has never been reported. The objectives were to compare baseline sensitivities of D. bryoniae from South Carolina and other locations to sensitivities of isolates exposed to azoxystrobin for one or more seasons, and to compare sensitivity in vitro and in vivo. Sixty-one isolates of D. bryoniae collected before 1998 were sensitive. Median EC50 was 0.055 mg L(-1) azoxystrobin (range 0.005 to 0.81). Forty isolates collected after exposure during 1998 also were sensitive. Fifty-three of 64 isolates collected in South and North Carolina between 2000 and 2006 were insensitive to 10 mg L(-1) azoxystrobin. Sensitive and insensitive isolates were distinguished by disease severity on Cucumis melo L. seedlings treated with azoxystrobin (20 or 200 mg L(-1)). An azoxystrobin baseline sensitivity distribution was established in vitro for isolates of D. bryoniae never exposed to strobilurins. Baseline values were comparable with those of other ascomycetes. Insensitive isolates were found in fields with a history of strobilurin applications. An in vivo method distinguished sensitive and insensitive isolates.

Simultaneous Detection of Acidovorax avenae subsp. citrulli and Didymella bryoniae in Cucurbit Seedlots Using Magnetic Capture Hybridization and Real-Time Polymerase Chain Reaction

To improve the simultaneous detection of two pathogens in cucurbit seed, a combination of magnetic capture hybridization (MCH) and multiplex real-time polymerase chain reaction (PCR) was developed. Single-stranded DNA hybridization capture probes targeting DNA of Acidovorax avenae subsp. citrulli, causal agent of bacterial fruit blotch, and Didymella bryoniae, causal agent of gummy stem blight, were covalently attached to magnetic particles and used to selectively concentrate template DNA from cucurbit seed samples. Sequestered template DNAs were subsequently amplified by multiplex real-time PCR using pathogen-specific TaqMan PCR assays. The MCH multiplex real-time PCR assay displayed a detection threshold of A. avenae subsp. citrulli at 10 CFU/ml and D. bryoniae at 10(5) conidia/ml in mixtures of pure cultures of the two pathogens, which was 10-fold more sensitive than the direct real-time PCR assays for the two pathogens separately. Although the direct real-time PCR assay displayed a detection threshold for A. avenae subsp. citrulli DNA of 100 fg/microl in 25% (1/4 samples) of the samples assayed, MCH real-time PCR demonstrated 100% detection frequency (4/4 samples) at the same DNA concentration. MCH did not improve detection sensitivity for D. bryoniae relative to direct real-time PCR using conidial suspensions or seed washes from D. bryoniae-infested cucurbit seed. However, MCH real-time PCR facilitated detection of both target pathogens in watermelon and melon seed samples (n = 5,000 seeds/sample) in which 0.02% of the seed were infested with A. avenae subsp. citrulli and 0.02% were infested with D. bryoniae.

Development of a multiplex classical polymerase chain reaction technique for detection of Didymella bryoniae in infected cucumber tissues and greenhouse air samples

Two sets of primer pairs were evaluated for their usefulness in detecting and identifying Didymella bryoniae (anamorph Phoma cucurbitacearum), causal agent of gummy stem blight, in infected cucumber plant tissues and air samples, using polymerase chain reaction. One primer in each pair was a universal primer that has been used for amplification of septate fungi. The other primer in each pair was an oligonucleotide designed from sequence information in the GenBank database to be specific to D. bryoniae. Each primer pair on its own was specific to genus level, but gave some nonspecific results for other Phoma or Didymella species. However, when these primer pairs were combined in a multiplex PCR, a unique result for D. bryoniae was obtained. The multiplex PCR gave positive results for cucumber tissue infected with D. bryoniae, and negative results for uninfected tissue and for tissue infected with Botrytis cinerea. Positive results were obtained from air samples collected with a rotation impaction sampler situated in a greenhouse containing cucumber plants infected with gummy stem blight. This method is rapid, sensitive, and accurate for detecting and identifying D. bryoniae in pure culture and plant tissue and could be applied to the detection of this organism in air samples. This method, therefore, could be a valuable technique for use in disease prediction.

Internal Transcribed Spacer Regions 1 and 2 and Random Amplified Polymorphic DNA Analysis ofDidymella bryoniaeand RelatedPhomaSpecies Isolated from Cucurbits

ABSTRACT Didymella bryoniae (anamorph Phoma cucurbitacearum) is the causal agent of gummy stem blight, although other Phoma species are often isolated from cucurbit plants exhibiting symptoms of the disease. The molecular and phylogenetic relationships between D. bryoniae and these Phoma species are unknown. Isolates of D. bryoniae and Phoma obtained from cucurbits grown at various geographical locations in the United States were subjected to random amplified polymorphic DNA (RAPD) analysis and internal transcribed spacer (ITS) sequence analysis (ITS-1 and ITS-2) to determine the molecular and phylogenetic relationships within and between these fungi. Using RAPD fingerprinting, 59 isolates were placed into four phylogenetic groups, designated RAPD group (RG) I, RG II, RG III, and RG IV. D. bryoniae isolates clustered in either RG I (33 isolates), RG II (12 isolates), or RG IV (one isolate), whereas all 13 Phoma isolates clustered to RG III. There was greater than 99% sequence identity in the ITS-1 and ITS-2 regions between isolates in RG I and RG II, whereas isolates in RG III, P. medicaginis ATCC 64481, and P. exigua ATCC 14728 clustered separately. On muskmelon seedlings, a subset of RG I isolates were highly virulent (mean disease severity was 71%), RG II and RG IV isolates were slightly virulent (mean disease severity was 4%), and RG III isolates were nonpathogenic (disease severity was 0% for all isolates). The ITS sequences indicate that RG I and RG II are both D. bryoniae, but RAPD fingerprints and pathogenicity indicate that they represent two different molecular and virulence subgroups.

Development of PCR-ELISA for Detection and Differentiation of Didymella bryoniae from Related Phoma species.

The causal agent of gummy stem blight, Didymella bryoniae, often is isolated from infected cucurbits together with other Phoma spp. Polymerase chain reaction (PCR) primers specific to D. bryoniae and Phoma were used to develop and evaluate a microtiter-based PCR-enzyme-linked immunosorbent assay (ELISA) technique. Primers were modified by addition of a fluorescein and a biotin label to the 5' ends of the forward and reverse primers, respectively. After amplification, PCR products were detected in an ELISA using horseradish peroxidase-conjugated antifluorescein antibody and three substrates that yielded three colored products, one for each fungal group. The most sensitive substrate (highest signal:noise ratio) was 2,2' -azino-bis[3-ethylbenz-thiazoline-6-sulfonic acid]. PCR-ELISA successfully detected 45 of 46 D. bryoniae and all 13 Phoma isolates that were used. Results were comparable to those obtained with gel electrophoresis. Only one D. bryoniae isolate could not be detected with PCR-ELISA; this isolate also produced a fragment larger than other D. bryoniae isolates on agarose gels. PCR-ELISA was used successfully on crude extracts of "blind" fungal samples and identified seven of seven isolates as D. bryoniae or Phoma. Although less sensitive than gel electrophoresis, PCR-ELISA was a highly specific, yet simple, rapid and convenient assay for detection of D. bryoniae and Phoma sp.

Diagnosis and identification of Didymella bryoniae, causal agent of gummy stem blight of greenhouse cucumbers, using a dot blot technique

SummaryThe purpose of this study was to develop a molecular method for rapid diagnosis and identification of Didymella bryoniae, causal agent of gummy stem blight of greenhouse cucumbers. DNA from fungal cultures and from diseased plant tissue was extracted, amplified by PCR using primers specific to septate fungi, and fixed to nylon membranes. Using sequence information obtained from the GenBank database, specific oligonucleotides were designed and after labelling with digoxigenin-d-UTP, were used as probes in a dot-blot hybridization procedure. After preliminary testing, two of these probes were selected for further study. For probe D6, a positive reaction was obtained with all isolates of D. bryoniae and Phoma cucurbitacearum tested, and with fresh or frozen gummy stem blight diseased plant tissue. All other fungi tested gave a negative result. Cucumber stem tissue infected with Botrytis cinerea also gave a negative result. Uninfected, healthy cucumber stem tissue did not yield a PCR product. The other probe (D7) reacted slightly with D. applanata but otherwise showed the same results as D6. This dot blot assay can reliably be used to identify infection by Didymella bryoniae.

CENTRUM DEVELOPMENT IN DIDYMELLA BRYONIAE

Early stages of pseudothecium development consist of small pseudoparenchymatous stromata in which ascogonia differentiate. Deeply staining cells in the apical region of the young pseudothecium elongate to form pseudoparaphyses, which grow down to fill the centrum. Ascogenous hyphae grow out from ascogenous cells, located in the basal plectenchyma, and croziers arise and proliferate from the ascogenous hyphae. Bitunicate asci grow up among the pseudoparaphyses and forcibly discharge two‐celled hyaline ascospores at maturity. Because centrum development in Didymella bryoniae (Auersw.) Rehm is pseudoparaphysate, the causal agent of gummy stem blight in watermelon is properly placed in the order Pleosporales. The placement of this species in Didymella on the basis of the Ascochyta cucumis Fautr. et Roum. anamorph is supported by centrum structure.