Isolation and identification of pathogen causing damping off at seedling stage of Trollius chinensis / 中国中药杂志

Trollius chinensis is a traditional Chinese medicinal material in China, the wild resource of T. chinensis are now exhausted, and commercial medicinal T. chinensis mainly depends on artificial cultivation. As one of the most severely happened diseases at the seedling period, damping off has been a serious threaten to the breeding of T. chinensis seedlings. However, no related research have been reported so far. So, the authors collected damping-off samples of T. chinensis in 2018 from seedling breeding nursery in Guyuan, Hebei province, and carried out study on taxonomic identification of the pathogen. Damping off occurs in the T. chinensis production area from mid-May to late June every year. At the beginning, brown lesions were observed on the basal stem, then the lesions circumferential expanded and constricted, and finally resulted in the fall and death of T. chinensis seedlings. Pathogenic isolate was growing rapidly on the PDA medium, well developed aerial mycelia were grey white at first, then turned brown gradually, and a great number of small dark brown sclerotia were developed in the middle and periphery of the colony. Mycelial diameter of the pathogen was about 7 to 10 μm, near right angle or acute angle branches, near branches with septa, branches and septa with constriction. After the healthy T. chinensis seedlings were inoculated by pathogenic isolate, damping-off was observed soon, and the symptom was as same as those observed in the field. Through homogenous blast, the rDNA-ITS sequence of the pathogenic isolate shown 99.49% to 99.84% homology with Rhizoctonia solani, R. solani AG-1 IC mycelium anastomosis group and Thanatephorus cucumeris, the sexual type of Rhizoctonia. Furthermore, obvious mycelial anastomosis phenomena were observed when the pathogenic isolate and R. solani AG-1 IC strain were confronting cultured. Based on the results above, the pathogenic isolate causing damping off of T. chinensis was identified as R. solani AG-1 IC mycelial anastomosis group. RESULTS:: in the present work have important significance for further research on basic biology of the pathogen and integrated control of damping off causing by it on T. chinensis.

Setting a rating scale for assess Rhizoctonia solani lesions on cotton, soybean and common bean seedlings / Ajuste de uma escala de notas para avaliar lesões de Rhizoctonia solani em plântulas de algodão, soja e feijão

Diseases incited by soilborne fungi are responsible for reducing the yield and cause significant impact to almost all crops. Among then, Rhizoctonia solani AG-4 is considered the most important in the cotton, common bean and soybean crops in Brazil. The use of diagrammatic scale or rating scales, as a tool to help in the quantification of the severity of a particular disease, is more common for foliar diseases. Considering the lack of a standardized, illustrated and easy-to-apply methodology for assessing the severity of R. solani lesions in cotton, soybean and common bean seedlings, a simple and precise rating scale was developed with the objective to fill this gap. The proposed scale shows four levels of disease severity, with the descriptions and illustrations for each type of lesion observed in the cotton, soybean and common bean seedlings. This developed scale was validated in many experiments and proved to be adequate for severity assessments of R. solani lesions on cotton, soybean and common bean seedlings.

Doenças causadas por fungos de solo reduzem a produtividade e impactam de forma significativa quase todas as culturas. Dentre elas, Rhizoctonia solani AG-4 é considerada a mais importante nas culturas de algodão, feijão e soja no Brasil. A utilização de escalas diagramáticas ou escalas de notas, como ferramenta para auxiliar na quantificação da severidade de uma determinada doença, é mais comum quando se trata de doenças foliares. Considerando a inexistência, até então, de metodologia padronizada, ilustrada e de fácil aplicação para a avaliação da severidade de lesões de R. solani em plântulas de algodão, soja e feijão, desenvolveu-se uma escala de notas, simples e precisa, com o objetivo de suprir essa lacuna. A escala proposta apresenta quatro níveis de severidade de doença, com descrições e ilustrações para cada tipo de lesão observada nas plântulas de algodão, soja e feijão. Essa escala foi validade em inúmeros experimentos, provando ser adequada para a avaliação da severidade de lesões de R. solani nas plântulas de algodão, soja e feijão

Control Efficacy of Streptomyces sp. A501 against Ginseng Damping-off and Its Antifungal Substance

Ginseng damping-off, caused by the fungal pathogens Rhizoctonia solani and Pythium sp., is a critical disease in ginseng seedling. In a continuing effort to find microorganisms with the potential of acting as a biocontrol agent against Rhizoctonia damping-off, we found that a Streptomyces sp. A501 showed significant antifungal activity against Rhizoctonia solani. In field experiment to test the efficacy of Streptomyces sp. A501 in controlling ginseng damping-off, the incidence of damping-off disease was meaningfully reduced when ginseng seeds were soaked in the culture broth of Streptomyces sp. A501 before sowing. To perform characterization of the antifungal compound, we isolated it from the culture broth of strain A501 through Diaion HP-20 and silica gel column chromatographies and preparative high-performance liquid chromatography. The structure of the antifungal compound was assigned as fungichromin by spectroscopic methods, mainly nuclear magnetic resonance and electrospray ionization-mass analysis.

Antifungal Substances from Streptomyces sp. A3265 Antagonistic to Plant Pathogenic Fungi

In a previous study, we identified a Streptomyces sp., A3265, as exhibiting potent antifungal activity against various plant pathogenic fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Rhizoctonia solani. This strain also exhibited a biocontrolling effect against ginseng root rot and damping-off disease, common diseases of ginseng and other crops. In this study, we isolated two antifungal substances responsible for this biocontrolling effect via Diaion HP-20 and Sephadex LH-20 column chromatography, medium pressure liquid chromatography, and high-performance liquid chromatography. These compounds were identified as guanidylfungin A and methyl guanidylfungin A by spectroscopic methods. These compounds exhibited potent antimicrobial activity against various plant pathogenic fungi as well as against bacteria.

A quitosana como fungistático no crescimento micelial de Rhizoctonia solani Kuhn / Chitosan as fungistatic mycelial growth of Rhizoctonia solani Kuhn

Rhizoctonia solani é um fungo causador de tombamento de plântulas em várias espécies vegetais. A quitosana é um polímero derivado do processo de desacetilação da quitina, a qual é encontrada em grande quantidade na carapaça de crustáceos, insetos e parede celular de fungos. A quitosana tem sido testada para diversos usos, inclusive no controle de fitopatógenos em agricultura, já que apresenta atividade antimicrobiana, para controle de patógenos. O presente trabalho teve como objetivo avaliar o efeito fungistático de diferentes concentrações de quitosana (0; 0,25; 0,5; 1 e 2%) no crescimento micelial do fungo R. solani in vitro. Os resultados obtidos demonstraram efeito significativo de quitosana nas diferentes concentrações utilizadas, na redução do crescimento micelial de R. solani. Observou-se também aumento do efeito fungistático da quitosana conforme o aumento da dose.

Rhizoctonia solani is a fungus that causes damping-off of seedlings in various plant species. Chitosan is a polymer derived from the process of desacetylation of chitin, which is found in large quantities in the exoskeleton of crustaceans, insects and fungal cell wall. Chitosan has been tested for various uses, including the control of plant pathogens in agriculture, since it presents antimicrobial activity to control pathogens. This study aimed to evaluate the fungistatic effect of different chitosan concentrations (0; 0.25; 0.5; 1 and 2%) in mycelial growth in vitro of the fungus R. solani. The results showed a significant effect of different concentrations of chitosan, in reduccing the mycelial growth of R. solani. It was also observed increased fungistatic effect with increasing of the concentration.

Influência da temperatura sobre isolados de Rhizoctonia solani (Kühn) obtidos em áreas produtoras de algodão nos estados brasileiros / Influence of temperature on the Rhizoctonia solani (Kühn) strains obtained from cotton fields on the brazilian states

A temperatura é importante para estudos com Rhizoctonia solani por ser um patógeno cosmopolita e polífago. Nas temperaturas adequadas o patógeno pode ser favorecido, o qual obtém sucesso no processo doença. Já em temperaturas inadequadas, o seu crescimento e desenvolvimento pode ser retardado. O objetivo foi avaliar a influência da temperatura no crescimento micelial, na produção de escleródios e na patogenicidade de isolados de R. solani. Obtiveramse 18 isolados de plântulas de algodão, oriundos dos estados de Minas Gerais (8), Bahia (3), Goiás(2), Mato Grosso (4) e Mato Grosso do Sul (1), que foram testados nas temperaturas de 15°C, 18°C, 21°C, 24°C, 27°C e 30°C. Para o crescimento micelial, os isolados foram dispostos em placas de Petri (9 cm de diâmetro), contendo meio batata-dextroseágar. As placas foram acondicionadas em câmaras de germinação com fotoperíodo de 12 horas. Realizaram-se medições ortogonais do diâmetro da colônia, diariamente, por 8 dias e quantificou-se o índice de crescimento micelial (ICM). As placas foram mantidas por três meses nas respectivas câmaras de crescimento para análise da produção de escleródios. Para a determinação de patogenicidade e a avaliação da severidade da doença, seguiu-se o método descrito por Oliveira et al. (2008). Os dados obtidos foram submetidos à analise de variância. Houve interação significativa entre isolados e temperaturas. Quanto aos oito isolados de Minas Gerais, um apresentou maior ICM a 24ºC e três a 27°C, observando-se relação com o modelo quadrático. Três isolados apresentaram melhor ajuste ao modelo linear e um não diferiu estatisticamente para as temperaturas avaliadas. Os isolados de GO apresentaram maior ICM nas temperaturas de 24°C e 27°C. Para os isolados do MT, dois tiveram ajuste ao modelo linear, enquanto os outros dois tiveram ao modelo quadrático, nas temperaturas de 21°C e 24°C. Já o isolado do MS foi ajustado ao modelo quadrático a 27°C, enquanto todos os três isolados da BA foram ajustados ao modelo linear. O maior número de escleródios foi observado nas temperaturas de 15°C e 18ºC com exceção do isolado do MS, o qual obteve o maior número a 27ºC. Verificou-se que 14 isolados (6 de MG, 2 da BA, 2 de GO, 3 de MT e 1 de MS) apresentaram maior severidade entre 24°C e 27°C, ajustandose ao modelo quadrático, enquanto três isolados (2 de MG e 1 de MT) não diferiram significativamente para as temperaturas avaliadas e apenas um isolado (BA 2 ­ I01) ajustou-se ao modelo linear.

The temperature is important for studies of Rhizoctonia solani (Kühn), since it is a cosmopolitan and polyphagous pathogen. Appropriate temperatures can favor the pathogen, which starts the infection process. On the other hand inappropriate temperatures can impose a delay to its growth and development. The objective were evaluate the influence of temperature on mycelial growth, sclerotia production and pathogenicity of R. solani strains. In the fields were obtained 18 strains from cotton seedlings on the States of Minas Gerais-MG (8), Bahia-BA (3), Goias-GO (2), Mato Grosso-MT (4) and Mato Grosso do Sul-MS (1), which were tested at temperatures of 15°C, 18°C, 21°C, 24°C, 27°C and 30°C. For mycelial growth, strains were placed in Petri dishes (9 cm diameter) containing potato-dextrose-agar (PDA). The dishes were placed into a germination chamber with a photoperiod of 12 hours. There were orthogonal daily measurements of the diameter of the colony during 8 days and the rate of mycelial growth was quantified afterwards. The dishes were kept for three months in the respective growth chambers for the sclerotia production analysis. For the pathogenicity determination and evaluation of disease severity the method described by Oliveira et al. (2008) was followed. The data were subjected to analysis of variance. There was significant interaction between isolates and temperatures. Among the eight strains of Minas Gerais, one had a higher rate of mycelial growth at 24 ° C and three at 27 ° C, adjusting to the quadratic model. Three strains showed better fit to a linear model and did not differ statistically for the temperatures. Strains from GO had a higher rate of mycelial growth temperatures of 24°C and 27°C. Concerning about the strains from MT, two were fit to a linear model, while the other two had the quadratic model at temperatures of 21°C and 24ºC. Strains from MS was adjusted to quadratic model at 27°C, while all three strains from BA were fitted to the linear model. The largest number of sclerotia was observed at 15°C and 18°C except for MS strain, which obtained the highest number at 27ºC. It was found that 14 strains (six from MG, two from BA, two from GO, three from MT and one from MS) showed a higher severity between 24°C and 27°C, adjusting to the quadratic model, while three isolates (two from MG and one from MT) did not differ significantly for the temperatures evaluated and only one isolate (BA 2 - I01) had set better to the linear model.

Biocontrol of Dendrobium candidum damping-off disease with endophytic fungi / 中国药学杂志

OBJECTIVE: To screen out endophytic fungi isolates with good biological control against damping-off in Dendrohium candidum seedlings among the isolates owned by our laboratory, thus to lay a foundation on developing biological control technique and field application. METHODS: Eighteen isolates of endophytic fungi identified by the PDA Petri dish confrontation experiment and athogen of damping-off were used in the biological control experiment. Co-incubation in bottles was conducted to screen out isolates with weak pathogenic and growth promoting effect on Dendrobium candidum seedlings. The chosen isolates were then enrolled in the potted seedlings experiment to determine their biological control effect. RESULTS: Isolates 4829 and 3952 were confirmed to have good biological control effect by statistical analysis. The survival rates of Dendrobium candidum seedlings reached 66.7% and 60.5% respectively. CONCLUSION: Endophytic fungi isolated from Dendrobium sp. shows good biological control against Dendrobium candidum damping-off.

Potential strain of trichoderma spp. to control damping off of disease in chilli.

The present investigation deals with the population of soil fungi in chilli field of Thiruvarur District. The antibiotic ability and potential strain of Trichoderma spp.isolated from chilli field soil were used against damping off disease and its tolerance to antibiotics. Among the soil fungi, tested Trichoderma spp. showed maximum activity in controlling the disease with influence of soil physico-chemical parameters. The antibiotic interactions of some soil fungi against Pythium debaryanum were studied in vitro dual culture experiments. All the Trichoderma spp. Showed the ability to inhibit the pathogen, some of the species showed variability in the percentage of inhibition were studied.

Phenology, seed dispersal and difficulties in natural recruitment of the canopy tree Pachira quinata (Malvaceae)

Life history and recruitment information of tropical trees in natural populations is scarce even for important commercial species. This study focused on a widely exploited Neotropical canopy species, Pachiraquinata (Malvaceae), at the southernmost, wettest limit of its natural distribution, in the Colombian Amazonia. We studied phenological patterns, seed production and natural densities; assessed the importance of seed dispersal and density-dependent effects on recruitment, using field experiments. At this seasonal forest P. quinata was overrepresented by large adult trees and had very low recruitment caused by the combination of low fruit production, high seed predation and very high seedling mortality under continuous canopies mostly due to damping off pathogens. There was no evidence of negative distance or density effects on recruitment, but a clear requirement of canopy gaps for seedling survival and growth, where pathogen incidence was drastically reduced. In spite of the strong dependence on light for survival of seedlings, seeds germinated readily in the dark. At the study site, the population of P. quinata appeared to be declining, likely because recruitment depended on the rare combination of large gap formation with the presence of reproductive trees nearby. The recruitment biology of this species makes it very vulnerable to any type of logging in natural populations. Rev. Biol. Trop. 59 (2): 921-933. Epub 2011 June 01.

Información acerca de la historia de vida y reclutamiento de poblaciones naturales de árboles tropicales es escasa, incluso para especies de alto interés comercial. Este estudio se centró en una especie de dosel altamente explotada, Pachira quinata (Malvaceae), en la zona más húmeda de su distribución geográfica, en la Amazoní a colombiana. Estudiamos sus patrones fenológicos, producción de semillas y densidades naturales, además de la importancia de la dispersión de semillas y efectos denso-dependientes en el reclutamiento natural, todo lo anterior se logró con el uso de experimentos y observaciones en el campo. En este bosque estacional, la población de P. quinata estaba dominada por grandes árboles adultos y el reclutamiento era extremadamente bajo, como consecuencia de la baja producción de frutos, alta depredación de semillas y muy alta mortalidad de plántulas bajo el bosque por infección de patógenos. No encontramos evidencia de efectos de distancia al árbol parental o densidad de semillas en el reclutamiento, pero si hallamos un evidente requerimiento de claros de luz para la supervivencia y el crecimiento de plántulas, donde la mortalidad por patógenos se reduce drásticamente. Sin embargo, a pesar de la dependencia de la luz para la supervivencia, las semillas germinan en la oscuridad. En la zona de estudio, la población de P. quinata parece estar en claro declive, probablemente porque el reclutamiento depende de la coincidencia de la formación de grandes claros con la presencia cercana de árboles reproductivos. Los requerimientos de reclutamiento de esta especie la hacen muy vulnerable a cualquier actividad extractiva de árboles adultos en poblaciones naturales.

Greenhouse evaluation of Bacillus subtilis AP-01 and Trichoderma harzianum AP-001 in controlling tobacco diseases / Avaliação em estufa de dois agentes biológicos de controle de doenças do tabaco

Two biological control agents, Bacillus subtilis AP-01 (LarminarTM) and Trichoderma harzianum AP-001 (TrisanTM) alone or/in combination were investigated in controlling three tobacco diseases, including bacterial wilt (Ralstonia solanacearum), damping-off (Pythium aphanidermatum), and frogeye leaf spot (Cercospora nicotiana). Tests were performed in greenhouse by soil sterilization prior to inoculation of the pathogens. Bacterial-wilt and damping off pathogens were drenched first and followed with the biological control agents and for comparison purposes, two chemical fungicides. But for frogeye leaf spot, which is an airborne fungus, a spraying procedure for every treatment including a chemical fungicide was applied instead of drenching. Results showed that neither B. subtilis AP-01 nor T. harzianum AP-001 alone could control the bacterial wilt, but when combined, their controlling capabilities were as effective as a chemical treatment. These results were also similar for damping-off disease when used in combination. In addition, the combined B. subtilis AP-01 and T. harzianum AP-001 resulted in a good frogeye leaf spot control, which was not significantly different from the chemical treatment.

Dois agentes de controle biológico, Bacillus subtilis AP-01 (Larminar®) e Trichoderma harzianum AP-001 (Trisan®) foram avaliados separadamente ou em combinação quanto à capacidade de controlar três doenças do tabaco: murcha bacteriana (bacterial wilt, Ralstonia solanacearum), tombamento de mudas (damping-off, Pythium aphanidermatum), e mancha olho-de-rã (frogeye leaf spot, Cercospora nicotiana). Os testes foram realizados em estufa, esterilizando-se o solo antes da inoculação dos patógenos. Os patógenos causadores da murcha bacteriana e tombamento de mudas foram inicialmente encharcados e acompanhados com os agentes de controle biológico e, para comparação, com um fungicida químico. Para a mancha olho-de-rã, causada por um fungo anemófilo, utilizou-se um processo de spray ao invés do encharcamento. Os resultados indicaram que nenhum dos dois agentes de controle biológico, aplicado isoladamente, foi capaz de controlar a murcha bacteriana, mas quando em combinação a capacidade de controle foi similar ao do tratamento químico. Resultados semelhantes foram obtidos para o tombamento de mudas. Além disso, a combinação de Bacillus subtilis AP-01 e Trichoderma harzianum AP-001 resultou em um controle muito eficiente da mancha olho-de-rã, que não diferiu significativamente daquele obtido com o tratamento químico.

Biological Control of Pythium Damping-off of Bush Okra Using Rhizosphere Strains of Pseudomonas fluorescens

A severe damping-off disease of bush okra caused by Pythium aphanidermatum, was diagnosed in plastic houses in Der Attia village, 15 km southwest of El-Minia city, Egypt, during the winter of 2001. Bush okra seedlings showed low emergence with bare patches inside the plastic houses. Seedlings that escaped pre-emergence damping-off showed poor growth, stunting and eventually collapsed. Examination of the infected tissues confirmed only Pythium aphanidermatum, showing its typical intercalary antheridia, and lobulate zoosporangia. P. aphanidermatum was shown to be pathogenic on bush okra under pot and field experiments. Bacteria making inhibition zones against the damping-off fungus P. aphanidermatum were selected. Agar discs from rhizosphere soil of bush okra containing colonies were transferred onto agar plate culture of P. aphanidermatum. After 2 days of incubation, colonies producing clear zones of non-Pythium growth were readily detected. The two bacteria with the largest inhibition zones were identified as Pseudomonas fluorescens. Bush okra emergence(%) in both pot and plastic houses experiments indicated that disease control could be obtained by applying P. fluorescens to the soil or coating the bacteria to the bush okra seeds before sowing. In the plastic houses, application of the bacteria onto Pythium-infested soil and sowing bush okra seeds dressed with bacteria gave 100% emergence. In addition, This was the first reported disease of bush okra by this oomycete in Egypt.