​Morphological and Molecular Diversity of Botrytis cinerea Infecting Chickpea in India

Author(s):  
L. Manjunatha ◽  
Upasana Rani ◽  
T. Basavaraja ◽  
R. Jagadeeswaran ◽  
Yogesh Kumar

Background: The Botrytis gray mold (BGM) caused by Botrytis cinerea Pers. ex Fr., has become a major threat for chickpea cultivation in India. In view of monetary significance of this disease and growing its spread into new regions through seed and soil (sclerotia) encouraged us to study variability of this pathogen from diverse regions. Methods: Botrytis cinerea infected chickpea samples were collected from Punjab and Uttarakhand states. Pathogen was isolated using chickpea dextrose agar media. Pathogenicity was proved by inoculation of spore suspension (3x105 spores’mL-1) of the fungus on susceptible cultivar. In vitro studies were conducted for observations of morphological and cultural variability of the fungus. The pathogenic fungus was also identified through molecular characterization using ITS primers. Result: The isolates were categorized into different groups based on growth rate of the pathogen on PDA media. Pathogenicity test proved that B. cinerea isolates from Punjab were showed less disease severity scale compared to Uttarakhand state isolates. The B. cinerea fungus prefers PDA for efficient growth and multiplication than Chickpea Dextrose Agar (CDA) media. There was no conspicuous difference in colour of the colony on PDA and CDA media. The colour of the mycelium were initially white or dirty white or greyish and sporulated culture shown to have as grey with profuse mass on the surface of the mycelium. The fungus B. fabae, a different species was also responsible for causing BGM disease which was confirmed through pathogenicity and ITS sequencing. Twenty-eight isolates were studied for diversity in cultural, morphological and molecular level. The diversity in colony colour, growth type and diameter of mycelial growth, sclerotia initiation, number and pattern of sclerotia formation were observed amongst the isolates. This study would help in designing breeding strategy for development of disease resistant cultivars.

2017 ◽  
Vol 107 (3) ◽  
pp. 362-368 ◽  
Author(s):  
Wayne M. Jurick ◽  
Otilia Macarisin ◽  
Verneta L. Gaskins ◽  
Eunhee Park ◽  
Jiujiang Yu ◽  
...  

Botrytis cinerea causes gray mold and is an economically important postharvest pathogen of fruit, vegetables, and ornamentals. Fludioxonil-sensitive B. cinerea isolates were collected in 2011 and 2013 from commercial storage in Pennsylvania. Eight isolates had values for effective concentrations for inhibiting 50% of mycelial growth of 0.0004 to 0.0038 μg/ml for fludioxonil and were dual resistant to pyrimethanil and thiabendazole. Resistance was generated in vitro, following exposure to a sublethal dose of fludioxonil, in seven of eight dual-resistant B. cinerea isolates. Three vigorously growing B. cinerea isolates with multiresistance to postharvest fungicides were further characterized and found to be osmosensitive and retained resistance in the absence of selection pressure. A representative multiresistant B. cinerea strain caused decay on apple fruit treated with postharvest fungicides, which confirmed the in vitro results. The R632I mutation in the Mrr1 gene, associated with fludioxonil resistance in B. cinerea, was not detected in multipostharvest fungicide-resistant B. cinerea isolates, suggesting that the fungus may be using additional mechanisms to mediate resistance. Results from this study show for the first time that B. cinerea with dual resistance to pyrimethanil and thiabendazole can also rapidly develop resistance to fludioxonil, which may pose control challenges in the packinghouse environment and during long-term storage.


FLORESTA ◽  
2013 ◽  
Vol 43 (2) ◽  
pp. 225
Author(s):  
Miriam Machado Cunico ◽  
Celso Garcia Auer ◽  
Marlon Wesley Machado Cunico ◽  
Obdulio Gomes Miguel ◽  
Patricio Peralta Zamora ◽  
...  

 Extratos etanólicos de anestesia, Ottonia martiana Miq., foram reavaliados quanto à inibição do crescimento micelial dos fungos Cylindrocladium spathulatum (pinta-preta da erva-mate) e Botrytis cinerea (mofo-cinzento do eucalipto), por meio do planejamento fatorial. A ocorrência de decomposição de bioativos no processo de autoclavagem também foi investigada, por meio de teste de eficiência de extratos filtrados (filtro Millipore) e esterilizados (autoclave) no controle dos fitopatógenos, nas concentrações de 1, 10, 100 e 1000 ppm. Os extratos etanólicos filtrado e esterilizado inibiram o crescimento micelial dos fungos e foram mais ativos frente a B. cinerea.O extrato filtrado exibiu maior potencial antifúngico que o extrato esterilizado. O processo de esterilização por autoclavagem causou pequena decomposição dos bioativos presentes no extrato de anestesia.Palavras-chave: Anestesia; mofo-cinzento; pinta-preta. Abstract Fungitoxic potential of ethanolic extracts of anestesia in the control of phytopathogenic diseases. The antifungal potential of anestesia, Ottonia martiana Miq. was reassessed by factorial design, in vitro testing of fungal mycelial growth compared to the pathogenic isolates Cylindrocladium spathulatum, causal agent of black spot onyerba mate, and Botrytis cinerea causal agent of gray-mold on eucalypts. Occurrence of decomposition of bioactive of the autoclaving process was investigated using foliar detached test compared to the pathogens (1000 ppm). Ethanolic extracts - EBEtOH (filtered and autoclaved) inhibited the mycelial growth of C. spathulatum and B. cinerea (1000 ppm) and were more pronounced against B. cinerea (43.6 % and 68.9 %). EBEtOH filtered (0.22 µm) presented higher activity than EBEtOH autoclaved (C. spathulatum: 52.8 % and 43.6 %, B. cinerea: 68.9 % and 43.6 %), suggesting little decomposition ofbioactive after autoclaving. EBEtOH filtrate presented potential inhibition of 28 % in eucalypt leaves against B. cinerea.  Keywords: Ottonia martiana; black spot; gray-mold.


FLORESTA ◽  
2013 ◽  
Vol 43 (1) ◽  
pp. 145 ◽  
Author(s):  
José Antonio Sbravatti Junior ◽  
Celso Garcia Auer ◽  
Ida Chapaval Pimentel ◽  
Álvaro Figueredo dos Santos ◽  
Bruno Schultz

   O Eucalyptus benthamii é uma das principais espécies de eucalipto plantadas na região Sul do Brasil, por sua resistência a geadas e por seu uso na produção florestal de madeira para fins energéticos. Na produção de mudas, uma das principais doenças ocorrentes em viveiros é o mofo-cinzento, causado pelo fungo Botrytis cinerea. Uma das alternativas para o controle dessa doença é o controle biológico com fungos endofíticos, os quais podem competir com os patógenos foliares de mudas de eucalipto. O objetivo deste trabalho foi isolar os fungos endofíticos provenientes de mudas de E. benthamii, identificá-los e selecioná-los para o controle de B. cinerea. Eles foram isolados do interior de tecidos vegetais desinfectados, identificados de acordo com critérios macro e micromorfológicos e classificados a partir de testes de controle biológico in vitro. Os resultados evidenciaram o potencial antagonista dos fungos Aspergillus sp., Penicillium sp. e Trichoderma sp. Nenhum desses fungos causou lesões em mudas de E. benthamii.Palavras-chave: Mofo-cinzento; eucalipto; viveiro.AbstractIn vitro selection of endophytes for biological control of Botrytis cinerea in Eucalyptus benthamii. Eucalyptus benthamii is one of the main eucalypt species planted in Southern Brazil, due to its resistance to frost and its use in the production of forest wood for energy purposes. During the production of seedlings, the main disease occurring in forest nurseries is gray-mold caused by the fungus Botrytis cinerea. One alternative for control this disease is biological control with fungal endophytes, which can compete with the foliar pathogens of eucalypt seedlings. The objective of this study was to isolate endophytic fungi from seedlings of Eucalyptus benthamii, identify and select them for B. cinerea control. These were isolated from the interior of disinfected plant tissues, identified according to macro and micromorphological criteria, and based on tests of biological control in vitro. The results revealed the potential antagonist of Aspergillus sp., Penicillium sp. and Trichoderma sp. No fungi caused lesions in E. benthamii seedlings.Keywords: Gray-mold; eucalypt; nursery.    


2011 ◽  
Vol 101 (12) ◽  
pp. 1433-1445 ◽  
Author(s):  
Anne-Sophie Walker ◽  
Angélique Gautier ◽  
Johann Confais ◽  
Daniel Martinho ◽  
Muriel Viaud ◽  
...  

Botrytis cinerea is a major crop pathogen infesting >220 hosts worldwide. A cryptic species has been identified in some French populations but the new species, B. pseudocinerea, has not been fully delimited and established. The aim of this study was to distinguish between the two species, using phylogenetic, biological, morphological, and ecological criteria. Multiple gene genealogies confirmed that the two species belonged to different, well-supported phylogenetic clades. None of the morphological criteria tested (spore size, germination rate, or mycelial growth) was able to discriminate between these two species. Sexual crosses between individuals from the same species and different species were carried out. Only crosses between individuals from the same species were successful. Moreover, population genetics analysis revealed a high level of diversity within each species and a lack of gene flow between them. Finally, a population survey over time showed that B. cinerea was the predominant species but that B. pseudocinerea was more abundant in spring, on floral debris. This observation could not be explained by temperature adaptation in tests carried out in vitro or by aggressiveness on tomato or bean leaves. This study clearly establishes that B. cinerea and B. pseudocinerea constitute a complex of two cryptic species living in sympatry on several hosts, including grapevine and blackberry. We propose several biological or molecular tools for unambiguous differentiation between the two species. B. pseudocinerea probably makes a negligible contribution to gray mold epidemics on grapevine. This new species has been deposited in the MycoBank international database.


PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9626
Author(s):  
Huiyu Hou ◽  
Xueying Zhang ◽  
Te Zhao ◽  
Lin Zhou

Background Botrytis cinerea causes serious gray mold disease in many plants. This pathogen has developed resistance to many fungicides. Thus, it has become necessary to look for new safe yet effective compounds against B. cinerea. Methods Essential oils (EOs) from 17 plant species were assayed against B. cinerea, of which Origanum vulgare essential oil (OVEO) showed strong antifungal activity, and accordingly its main components were detected by GC/MS. Further study was conducted on the effects of OVEO, carvacrol and thymol in vitro on mycelium growth and spore germination, mycelium morphology, leakages of cytoplasmic contents, mitochondrial injury and accumulation of reactive oxygen species (ROS) of B. cinerea. The control efficacies of OVEO, carvacrol and thymol on tomato gray mold were evaluated in vivo. Results Of all the 17 plant EOs tested, Cinnamomum cassia, Litsea cubeba var. formosana and O. vulgare EOs had the best inhibitory effect on B. cinerea, with 0.5 mg/mL completely inhibiting the mycelium growth of B. cinerea. Twenty-one different compounds of OVEO were identified by gas chromatography–mass spectrometry, and the main chemical components were carvacrol (89.98%), β-caryophyllene (3.34%), thymol (2.39%), α-humulene (1.38%) and 1-methyl-2-propan-2-ylbenzene isopropyl benzene (1.36%). In vitro experiment showed EC50 values of OVEO, carvacrol and thymol were 140.04, 9.09 and 21.32 μg/mL, respectively. Carvacrol and thymol completely inhibited the spore germination of B. cinerea at the concentration of 300 μg/mL while the inhibition rate of OVEO was 80.03%. EC50 of carvacrol and thymol have significantly (P < 0.05) reduced the fresh and dry weight of mycelia. The collapse and damage on B. cinerea mycelia treated with 40 μg/mL of carvacrol and thymol was examined by scanning electron microscope (SEM). Through extracellular conductivity test and fluorescence microscope observation, it was found that carvacrol and thymol led to increase the permeability of target cells, the destruction of mitochondrial membrane and ROS accumulation. In vivo conditions, 1000 μg/mL carvacrol had the best protective and therapeutic effects on tomato gray mold (77.98% and 28.04%, respectively), and the protective effect was significantly higher than that of 400 μg/mL pyrimethanil (43.15%). While the therapeutic and protective effects of 1,000 μg/mL OVEO and thymol were comparable to chemical control. Conclusions OVEO showed moderate antifungal activity, whereas its main components carvacrol and thymol have great application potential as natural fungicides or lead compounds for commercial fungicides in preventing and controlling plant diseases caused by B. cinerea.


2021 ◽  
Vol 48 (3) ◽  
Author(s):  
Hind Lahmyed ◽  
◽  
Rachid Bouharroud ◽  
Redouan Qessaoui ◽  
Abdelhadi Ajerrar ◽  
...  

The present work aims to isolate actinomycete bacteria with antagonistic abilities towards Botrytis cinerea, the causal agent of gray mold, from a soil sample collected from the rhizosphere of a healthy tomato grove. In vitro confrontation led to the isolation of 104 actinomycete isolates; fifteen isolates have shown the most significant mortality rate of the mycelial growth of B. cinerea (>50%). Based on the results of this screening, representative strains were selected to verify their in vivo antagonistic activity on tomato fruits; the reduction of B. cinerea has a percentage ranging from 52.38% to 96.19%. Furthermore, the actinomycete isolates were evaluated for their plant growth-promoting (PGP) properties and their ability to produce biocontrol-related extracellular enzymes viz., amylase, protease, cellulase, chitinase, esterases, and lecithinase. Indeed, Ac70 showed high β-1,3-glucanase activity and siderophore production (17U/ml and 43% respectively), and the highest chitinase activity (39μmol/ml) was observed for Ac24. These results indicated that these actinomycetes might potentially control gray mold caused by B. cinerea on tomato fruits. Investigations on enhancing the efficacy and survival of the biocontrol agent in planta and finding out the best formulation are recommended for future research.


2020 ◽  
Vol 11 ◽  
Author(s):  
Carole Balthazar ◽  
Gabrielle Cantin ◽  
Amy Novinscak ◽  
David L. Joly ◽  
Martin Filion

Cannabis (Cannabis sativa L.) offers many industrial, agricultural, and medicinal applications, but is commonly threatened by the gray mold disease caused by the fungus Botrytis cinerea. With few effective control measures currently available, the use of beneficial rhizobacteria represents a promising biocontrol avenue for cannabis. To counter disease development, plants rely on a complex network of inducible defense pathways, allowing them to respond locally and systemically to pathogens attacks. In this study, we present the first attempt to control gray mold in cannabis using beneficial rhizobacteria, and the first investigation of cannabis defense responses at the molecular level. Four promising Pseudomonas (LBUM223 and WCS417r) and Bacillus strains (LBUM279 and LBUM979) were applied as single or combined root treatments to cannabis seedlings, which were subsequently infected by B. cinerea. Symptoms were recorded and the expression of eight putative defense genes was monitored in leaves by reverse transcription quantitative polymerase chain reaction. The rhizobacteria did not significantly control gray mold and all infected leaves were necrotic after a week, regardless of the treatment. Similarly, no systemic activation of putative cannabis defense genes was reported, neither triggered by the pathogen nor by the rhizobacteria. However, this work identified five putative defense genes (ERF1, HEL, PAL, PR1, and PR2) that were strongly and sustainably induced locally at B. cinerea’s infection sites, as well as two stably expressed reference genes (TIP41 and APT1) in cannabis. These markers will be useful in future researches exploring cannabis defense pathways.


Plant Disease ◽  
2020 ◽  
Vol 104 (5) ◽  
pp. 1298-1304 ◽  
Author(s):  
Ting-ting Li ◽  
Jing-di Zhang ◽  
Jia-quan Tang ◽  
Zhi-cheng Liu ◽  
Ya-qian Li ◽  
...  

Tomato gray mold caused by Botrytis cinerea is one of the main diseases of tomato and significantly impacts the yield and quality of tomato fruit. The overuse of chemical fungicides has resulted in the development of fungicide-resistant strains. Biological control is becoming an alternative method for the control of plant diseases to replace or decrease the application of traditional synthetic chemical fungicides and genus Trichoderma is widely used as a biological agent for controlling tomato gray mold. Brassinolide (BR) is a plant-growth-promoting steroid. To enhance the efficiency and stability of Trichoderma activity against B. cinerea, an optimal combination of Trichoderma atroviride CCTCCSBW0199 and BR that controls B. cinerea infection in tomato was identified. Strain CCTCCSBW0199 was found to have antagonistic activity against B. cinerea both in vitro and in vivo. In addition, a fermented culture of chlamydospores and metabolites, or metabolites only of strain CCTCCSBW0199 also reduced growth of B. cinerea. BR reduced growth of B. cinerea and had no effect on the sporulation and growth of Trichoderma spp. An application of metabolites of a Trichoderma sp. + BR reduced gray mold on tomato leaves by approximately 70.0%. Furthermore, the activities of induced defense response-related enzyme, such as peroxidase, superoxide dismutase, catalase, and phenylalanine ammonia-lyase were increased in tomato plants treated with a Trichoderma sp. + BR. Our data suggested that applying a mix of metabolites of T. atroviride CCTCCSBW0199 + BR was effective at reducing gray mold of tomato and may lay a theoretical foundation for the development of novel biofungicides.


2020 ◽  
Author(s):  
Veronica Ambrosini ◽  
Mohammad Issawi ◽  
Vincent Sol ◽  
Catherine Riou

Abstract Botrytis cinerea is a necrotic fungal plant pathogen responsible for the gray mold disease on more than 200 crops, including grapevine. Due to its genetic plasticity, this fungus presents a strong resistance to a large spectrum of fungicides. Thus, new fighting strategies against B. cinerea are urgently needed. In this context, antimicrobial photodynamic treatment (APDT) was considered. APDT involves the use of a photosensitizer that generates reactive oxygen species upon illumination. Tetra-4-sulfonatophenyl porphyrin tetra-ammonium (TPPS) was tested on B. cinerea; upon light exposure. 1.5 µM TPPS was shown to completely inhibit mycelial growth. A concentration of 12.5 µM TPPS was tested on three genetic background clones from Chardonnay, Merlot and Sauvignon, grown in vitro for 2 months. Treated root apparatus of the three backgrounds was able to increase thiol production as a molecular protection against photoactivated TPPS, leading to a normal phenotype as compared with control plantlets. Finally, two-month-old grapevine leaves were infected with four-day-old mycelium of B. cinerea pre-incubated or not with TPPS. The pre-treated mycelium was unable to infect the isolated leaves of any of the three grapevine varieties after 72 h growth when subjected to a 16 h photoperiod, contrary to untreated mycelium. These results on fungus, plantlets and Botrytis-grapevine leaves, in contact with a very low concentration of TPPS, suggest a strong potential of photo-treatment against Botrytis mycelium for future agricultural practices in vineyard or other cultures.


Processes ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 2231
Author(s):  
Cátia Santos ◽  
Joana Monte ◽  
Natália Vilaça ◽  
João Fonseca ◽  
Henrique Trindade ◽  
...  

Composts are widely used in horticulture as organic amendments to improve the properties of soils. Composts have also been reported to enhance the disease suppressive potential of soils and, therefore, could be used as a strategy for managing plant diseases. The aim of this study was to test the ability of soils amended with four different agro-industrial waste-based composts (chestnut peels and shells, spent coffee grounds, grape marc, and olive leaves) to inhibit the growth and activity of Botrytis cinerea and several soilborne pathogens. First, the capacity of aqueous compost extracts to inhibit the growth of Botrytis cinerea and five soilborne fungi was evaluated in vitro using a broth macrodilution method. Second, lettuce plants were grown on soils amended with composts and inoculated either with B. cinerea or the soilborne fungus Fusarium oxysporum Schlechtendahl isolated from lamb’s lettuce. The determination of minimal inhibitory concentrations indicated that none of the composts inhibited the mycelium growth of the selected fungal pathogens. However, the pathogens did not cause any damage on plants grown on the chestnut- and olive-based composts. Lettuce yields were also highest for plants grown with composts made from chestnut and olive, irrespective of the amount of compost incorporated into soils (5% or 10%, weight basis). The grape-based compost also exhibited a fertilization effect, although the effect was associated with increased Fusarium wilt severity. Both N immobilization and symbiosis with the compost’s microflora were used to explain the pathogenicity of F. oxysporum Schlechtendahl in response to amendment with composts made from grape and coffee wastes. The beneficial effects of the chestnut- and olive-based composts reported in this study could be exploited in strategies aimed at reducing reliance on synthetic pesticides for the control of fungi in lettuce cultivation.


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