Utilization of fungal biocontrol agents against rice sheath blight disease provides insight into their role in plant defense responses

Biotic and abiotic factors have an effect on rice production all around the world. Diseases are regarded as major restrictions among the biotic stressors, and rice sheath blight (Rhizoctonia solani Kühn) is one of the most calamitous diseases that significantly damage the crop. Lately, biocontrol of fungal plant pathogens has appeared as an appealing approach. The present investigation was undertaken to evaluate different biocontrol agents like Talaromyces flavus, Chaetomium globosum, Pseudomonas fluorescens and Aspergillus niger against sheath blight disease. Prior to sowing, seeds were bioprimed with each isolate and sown in the nursery. After 21 days, seedlings were transplanted in-vivo and were inoculated with a virulent isolate of Rhizoctonia solani at maximum tillering stage. Observations on biochemical parameters and gene expression studies were carried out at 24, 48, 72, and 96 hpi. Enzymatic activity viz., chitinase, ?-1,3-glucanase, catalase, and PAL was observed maximum in Chaetomium globosum. PR-genes viz., IPT, BrD, HmPr, AMP, AldD, NIC and LisH showed up-regulation at 96 hpi. Chaetomium globosum had the highest yield, maximum number of tillers with least RLH% as compared to other treatments. However, results indicated biocontrol agents are helpful and they induce multitude of defence responses against R. solani in rice.

Hongos formadores de micorrizas aislados a partir de raíces de la orquídea Rodriguezia granadensis (LINDL.) RCHB. F / Mycorrhizal forming fungi isolated from roots of the orchid Rodriguezia granadensis (LINDL.) RCHB. F

RESUMEN Rodriguezia granadensis (Lindl.) Rchb. f. es una orquídea epífita que crece sobre árboles, en zonas poco intervenidas. Para la germinación de sus semillas y en los estadios iniciales de su desarrollo forma relaciones simbióticas con hongos micorrícicos, los cuales, brindan los nutrientes que necesita. En el municipio de Fusagasugá esta especie crece en condiciones naturales y debido a la belleza de sus flores es promisoria para su producción comercial, pero hay poca información sobre los microorganismos asociados a esta planta; por lo tanto, el presente trabajo tuvo como propósito identificar, a nivel de género, los hongos asociados a las raíces de R. granadensis, en el agroecosistema Hacienda Betania. Se tomaron muestras de raíz, anotando el forofito donde se encontraban las plantas. En laboratorio, se dividieron en tres extremos: proximal, medio y distal y se realizaron cortes transversales, para identificar enrollamientos hifales. Adicionalmente, se sembraron explantes en agar papa dextrosa, agar Sabouraud y medio de Ko y Hora. Se identificó al género Rhizoctonia, con una frecuencia del 95 %. Se encontró que el 70 % de las plantas muestreadas crecían sobre árboles de guayabo y el 30 % sobre cítricos. Los resultados indican que R. granadensis pueden tener interacciones con hongos del género Rhizoctonia.

ABSTRACT Rodriguezia granadensis (Lindl.) Rchb. f. is an epiphytic orchid that grows on trees in areas with little intervention. For seed germination and the initial stages of development, it forms symbiotic relationships with mycorrhizal fungi, which provide the necessary nutrients. In the municipality of Fusagasugá, this species grows in natural conditions and, due to the beauty of its flowers is a promissory specie to commercial production, but there is little information about the microorganisms associated with this plant; therefore, the purpose of this work was to identify, at the genus level, the fungi associated with the roots of R. granadensis in Betania farm. Root samples were taken, noting the phorophyte where the plants grew. In the laboratory, they were divided into three sections: proximal, middle, and distal, and made cross-sections to identify hyphal curls. Additionally, explants were put in potato dextrose agar, Sabouraud agar, and Ko and Hora media. The genus Rhizoctonia was identified, with a frequency of 95 %. It was found that 70 % of the sampled plants grew on guava trees and 30 % on citrus. The results indicate that R. granadensis may have interactions with fungi of Rhizoctonia genus.

Performance of a selected Trichoderma strain as plant pathogen inhibitor and biofertilizer

Aims@#The application of beneficial microbes is a suitable alternative to synthetic pesticides and fertilizers for agriculture. This study was aimed to evaluate the potential of a selected Trichoderma strain as a biocontrol agent against Rhizoctonia sp. and as a biofertilizer to improve paddy growth.@*Methodology and results@#Four Bipolaris strains were identified via DNA barcoding as the cause of brown spot disease, whereas two Rhizoctonia strains were similarly identified as the cause of sheath blight disease in Brunei Darussalam. Eight Trichoderma strains were initially screened using confrontation assay and were found to substantially inhibit the growth of Rhizoctonia sp. Hybrid rice named BDR5 was treated with Trichoderma sp. UBDFM01 and/or Rhizoctonia sp. It was found that the selected strain showed the potential as a biofertilizer by significantly increasing the vigour index I, chlorophyll a, chlorophyll b, total chlorophyll and dry shoot weight of the rice plants. The pathogen negatively affected the plants by significantly reducing the vigour index II, chlorophyll a, chlorophyll a/b ratio, total chlorophyll, and total weight of grains. Trichoderma strain showed the potential as a biocontrol agent by significantly diminishing the negative effects of the pathogen on the chlorophyll a, chlorophyll a/b ratio and total chlorophyll.@*Conclusion, significance and impact of study@#This study highlights the potential of Trichoderma sp. UBDFM01 as a biocontrol agent against Rhizoctonia sp. and also as a biofertilizer for rice plants. In addition, this study is the first to provide DNA-based evidence of Bipolaris sp. and Rhizoctonia sp. as the fungi that caused rice diseases in Brunei Darussalam.

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.

Effectiveness of indigenous fluorescent pseudomonads in suppressing Rhizoctonia solani root rot disease and promoting plant growth in chilli seedlings

Aims@#This study aimed to isolate and evaluate the indigenous fluorescent Pseudomonas spp. with bio-control potential against Rhizoctonia solani and promoting growth in chilli seedlings. @*Methodology@#A total of 120 fluorescent bacterial were isolated from the healthy chilli rhizosphere soil from the seven major chilli cultivation localities in Terengganu, Malaysia. Only 115 Gram negative fluorescent isolates were further invitro screened for antagonistic activities against R. solani and plant growth-promoting properties. The 50 most effective fluorescent Pseudomonads antagonist against R. solani with minimum percentage inhibition of radial growth (PIRG) of 65% were selected. Hierarchical cluster analysis was further conducted with two dendrograms derived from SPSS Statistic 20 to facilitate the comparison between these 50 isolates for antagonistic and growth-promoting properties. A total of 40 fluorescent isolates within the most potential cluster were further selected and identified using 16S rRNA sequencing. Thirty four fluorescent isolates were identified as Pseudomonas spp. and six isolates as Burkholderia spp. The top 13 ranked fluorescent Pseudomonas spp. from the scoring index were evaluated for seed germination and vigor index in chilli seedlings. There was no significant difference in germination rate between fluorescent Pseudomonas inoculated with control. However, vigor index of chilli seeds pre-inoculated with fluorescent P. putida (B5C1), P. aeruginosa (B3C56) and P. putida (B5C7) were significantly increased with 4684.9, 4657.3 and 4401.0 over control (P ≤ 0.05).@*Conclusion, significance and impact of study@#These selected fluorescent isolates: P. putida (B5C1), P. aeruginosa (B3C56) and P. putida (B5C7) have the potential to be developed as biofungicide against R. solani and as growthpromoter in chilli production system.

Fructosan form Paenibacillus kribbensis PS04 enhance disease resistance against Rhizoctonia solani and tobacco mosaic virus

BACKGROUND: Rice sheath blight (caused by Rhizoctonia solani) and tobacco mosaic virus are very important plant diseases, causing a huge loss in global crop production. Paenibacillus kribbensis PS04 is a broad-spectrum biocontrol agent, used for controlling these diseases. Previously, extracellular polysaccharides (EPS) from P. kribbensis PS04 had been purified and their structure was inferred to be fructosan. This study aimed to evaluate the effects of exogenous EPS treatment on plant­pathogen interactions. RESULTS: Plant defense genes such as phenylalanine ammonia-lyase, catalase, chitinase, allene oxide synthase, and PR1a proteins were significantly induced by exogenous EPS treatment. Moreover, subsequent challenge of EPSpretreated plants with the pathogens (R. solani or tobacco mosaic virus) resulted in higher expression of defenseassociated genes. Increased activities of defense-associated enzymes, total phenols, and flavonoids were also observed in EPS pretreated plants. The contents of malondialdehyde in plants, which act as indicator of lipid peroxidation, were reduced by EPS treatment. CONCLUSIONS: This study comprehensively showed that EPS produced from P. kribbensis PS04 enhances disease resistance in plants by the activation of defense-associated genes as well as through the enhancement of activities of defense-related enzymes.

Morphological, Pathogenic and Molecular Characterisation of Rhizoctonia solani Strains Isolated from Potato

Rhizoctonia solani Kühn [teleomorph: Thanatephorus cucumeris (Frank) Donk.] is an important fungal pathogen widespread in all potato growing areas of the world that causes stem canker and black scurf of potato (Solanum tuberosum L.). The aim of this study was to find a simple and reliable technique for determining the pathogenicity of Rhizoctonia solani isolates. Sixty (60) isolates of R. solani obtained from sclerotia on potato tubers, collected from different market of Agadir and Casablanca regions (Morocco), were studied for their morphology, pathogenicity and molecular characteristics. They were morphologically characterised by the production of sclerotia and moniloïd cells, and by the mycelium growth capacity at 15, 20, 25, 30 and 35°C. This morphological characterisation leads to three groups of isolates. The first group contained P01 and P03 isolates, which were able to develop under 35°C. However, under 25°C, they did not develop sclerotia. The second group, only formed by L17.1 isolate, did not form sclerotia under 25°C and was not able to develop under 35°C. The third group, formed by several isolates, developed sclerotia under 25°C conditions and were not able to grow under 35°C. Also, a positive correlation was consistent between the production of sclerotia and moniloïd cell formation. The anastomosis reaction revealed that P01, P03, L17.1, and L4.1 isolates were identified as AG-4 and for the other isolates as AG-3. The pathogenic characterisation has shown that P01, P03, L4.1, and L17.1 isolates caused important damping off of radish, tomato, beans, zucchini, and melon. However, the other isolates showed only a minor damping off rate. Molecular characterisation confirmed the classical anastomosis grouping of the isolates into AG-3 and AG-4 Anastomosis Groups. The molecular characterisation is the most rapid and reliable technique to determine the anastomosis group of unknown isolates. The three tests including the pathogenicity, the cultural anastomosis grouping, and the molecular method helped to separate the studied isolates to two groups AG-3 and AG-4.

Isolation and Biochemical Characterisation of Endophytic Bacillus spp from Urtica dioica and Study of Their Antagonistic Effect against Phytopathogens

Endophytic bacteria have been isolated from the roots of Urtica dioica. A total of 54 endophytic bacteria were isolated from the underground parts using suitable surface sterilisation protocol. Three isolates R45a; R45b; R21a were tested for antagonism effect against Fusarium oxysporum, Colletotrichum gloeosporioides, Rhizoctonia solani, Phytophthora parasitica in dual culture method. Significant inhibitory effects on mecylial radial growth have been revealed with a percentage superior or equal to 75%. These strains were Gram-positive rods. Cultures on nutrient agar showed irregular, entirely cream coloured colonies that are strictly aerobic and capable of forming endospore. They belong probably to the genus of Bacillus spp.

In silico analysis of disease resistance and defence-related genes for a major sheath blight qShb 9-2 QTL in rice

Aims@#Sheath blight disease (Rhizoctonia solani) is an important rice disease that causes heavy yield losses in rice annually. To date, no rice variety has been found to be completely resistant to this disease. The most desirable approach for the management of sheath blight disease is to introgress genes with major and durable resistance into the rice genome. Therefore, this study aims to identify disease resistance and defence genes within qShb 9-2, a major QTL found within moderately resistant rice population via in silico analysis. @*Methodology and results@#The sequences of tightly linked markers of qShb 9-2 from GRAMENE database was used to derive the 10.24 Mbp QTL region that contains 1581 genes according to MSU Rice Genome Annotation Project database. BLAST results showed that 11.4% of these genes were transposable elements which may be involved in gene duplication. Through Blast2GO, fifty-four (2.9%) defence-related genes were annotated within this QTL and can be classified into 5 major defence mechanisms. Further, fifty (2.7%) disease resistance genes were identified in qShb 9-2 based on the presence of NB-ARC, LRR-receptor kinase, Ser/Thr protein kinase and protein kinase domains. Lastly, directed acyclic graphs showing the interaction between all the disease resistance and defence-related genes were generated. @*Conclusion, significance and impact of study@#The presence of these genes indicates that qShb 9-2 region may contribute towards the defence against sheath blight disease. By deciphering the gene landscape within the QTL, it may be possible to further fine map the QTL into a smaller region for QTL pyramiding in breeding programmes. The resistance and defence genes are also a source for genetic engineering studies and a good source for marker development.

Evaluation of the fungitoxic activity of vegetal extracts on the mycelial growth of phytopathogens / Avaliação da atividade fungitóxica de extratos vegetais sobre o crescimento micelial de fitopatógenos

Although the productivity of common bean in Tocantins is economically favorable, it has been infected by various pathogens found in soil. Among the major diseases is the web blight and root rot caused by Rhizoctonia solani and collar rot caused by the fungus Sclerotium rolfsii. This study aimed to evaluate the fungitoxic activity of methanol extracts of eight plant species on the inhibition of mycelial growth of S. rolfsii and R. solani. The fungitoxic activities were carried out over the inhibition of mycelial growth by means in vitro assays. The extracts were applied in concentrations of 250, 500, 1000, 2500 e 5000 µg ml-1 in PDA culture medium. In bioassays, it was found the significant effect of plant, concentration and also their interaction on the antifungal activity of the extracts. However, some extracts showed no inhibition of mycelial growth of the pathogens studied. Among those who had higher inhibitions is the extract of Lantana trifolia, which inhibited the mycelial growth of S. rolfsii in all concentrations, being the same as 97% for the highest concentration. When the methanol extract of Piper amplum Kunth, inhibition of the highest concentration was 83% for S. rolfsii and 74% for R. solani. These results show the potential of methanolic extract of Lantana trifolia and Piper amplum Kunth in the control set of plant pathogens studied.

Apesar da produtividade do feijão comum no Tocantins ser economicamente favorável, o mesmo pode ser infectado por vários patógenos habitantes do solo, dentre as principais doenças encontra-se a mela e a podridão radicular causadas pelo fungo Rhizoctonia solani e a podridão do colo causada pelo fungo Sclerotium rolfsii. Este trabalho teve como objetivo avaliar a atividade fungitóxica dos extratos metanólicos de oito espécies vegetais sobre a inibição do crescimento micelial de Sclerotium rolfsii e Rhizoctonia solani. As atividades fungitóxicas foram realizadas perante a inibição do crescimento micelial por meio de ensaios in vitro, sendo os extratos aplicados nas concentrações de 250, 500, 1000, 2500 e 5000 µg ml-1 em meio de cultura BDA. Observou-se o efeito significativo dos fatores planta, concentração e também da interação destes sobre as atividades fungitóxicas. No entanto, alguns extratos não apresentaram inibição do crescimento micelial dos fitopatógenos estudados. Entre os que apresentaram maiores inibições encontra-se o extrato de Lantana trifolia, que inibiu o crescimento micelial do S. rolfsii em todas as concentrações, sendo o mesmo de 97% para a maior concentração. Já a concentração mais elevada do extrato metanólico de Piper amplum apresentou inibição de 83% sobre o crescimento micelial de S. rolfsii e 74% sobre o crescimento micelial de R. solani. Tais resultados evidenciam a potencialidade dos extratos metanólicos das folhas de Lantana trifolia e de Piper amplum no controle dos fitopatógenos estudados.

Characterization and pathogenicity of Rhizoctonia SPP. isolated from vegetable crops grown in greenhouses in Samsun province, Turkey / Caracterização e patogenicidade de Rhizoctonia SPP. isolada a partir de culturas vegetais crescidas em estufas na província de Samsun, Turquia

A total of one hundred and five isolates of Rhizoctonia belonging to 7 anastomosis groups (AGs) were obtained from the diseased roots and rhizosphere soils of bean, cucumber, eggplant, pepper and tomato plants grown in greenhouses in Samsun province (Black Sea region, Turkey) during the period 2011­2012. The isolates of Rhizoctonia spp. were examined for their cultural characteristics, anastomosis groups and pathogenicity. Of these, 83.8% were multinucleate Rhizoctonia solani (AG-2, AG-4, AG-5 and AG-6) and 16.2% were binucleate Rhizoctonia (AG-A, AG-E and AG-F). Sixty five of the isolates belonged to AG-4 which was the most frequent group (61.9%) in all greenhouses surveyed. Numbers of the isolates belonging to AG-2 (7.6%), AG-5 (6.7%) and AG-6 (7.6%) were 8, 7 and 8, respectively. Seventeen isolates recovered from greenhouses surveyed were identified as binucleate Rhizoctonia AG-A (1.9%), AG-E (6.7%) and AG-F (7.6%). All isolates of Rhizoctonia spp. tested for growth rates grew at temperatures of 10, 15, 20, 25 and 30°C, whereas they were completely inhibited at 5°C. The results of pathogenicity tests showed that the differences in virulence among isolates of Rhizoctonia spp. were statistically significant (P < 0.001). The tests on bean seedlings showed that the highest disease severity was caused by AG-4 isolates. The disease severity index (DSI) of the R. solani AG-4 isolates ranged from 3.2 to 3.8. In addition, the isolates of three AGs belonging to binucleate Rhizoctonia spp. were generally found to be moderately virulent (DSI 2.0­2.4).

Um total de cento e cinco isolados de Rhizoctonia pertencentes a 7 grupos de anastomose (AGs) foram obtidos a partir de raízes doentes e solos rizosféricos de plantas de feijão, pepino, berinjela, pimenta e tomate cultivados em estufas na província de Samsun (região do Mar Negro, Turquia) durante o período 2011-2012. Os isolados de Rhizoctonia spp. foram examinados por suas características culturais, grupos de anastomose e patogenicidade. Destes, 83,8% eram Rhizoctonia solani multinucleadas (AG-2, AG-4, AG-5 and AG-6) e 16,2% era Rhizoctonia binucleadas (AGA, AG-E and AG-F). Sessenta e cinco dos isolados pertenciam ao AG-4, que foi o grupo mais freqüente (61,9%) em todas as estufas pesquisadas. O número de isolados pertencentes a AG-2 (7,6%), AG-5 (6,7%) e AG-6 (7,6%) foi de 8, 7 e 8, respectivamente. Dezessete isolados recuperados de estufas pesquisadas foram identificados como Rhizoctonia binucleada AG-A (1,9%), AG-E (6,7%) e AG-F (7,6%). Todos os isolados de Rhizoctonia spp. testados para taxas de crescimento cresceram a temperaturas de 10, 15, 20, 25 e 30ºC, enquanto que foram completamente inibidos a 5ºC. Os resultados dos testes de patogenicidade mostraram que as diferenças de virulência entre os isolados de Rhizoctonia spp. foram estatisticamente significativas (P <0,001). Os testes em mudas de feijão mostraram que a maior severidade da doença foi causada por isolados AG-4. O índice de gravidade da doença (do inglês, disease severity index - DSI) dos isolados de R. solani AG-4 variou de 3,2 a 3,8. Além disso, os isolados de três AGs pertencentes à Rhizoctonia spp. binucleadas foram geralmente encontrados como moderadamente virulentos (DSI 2,0-2,4).

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.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Efecto antifúngico de cuatro mezclas de monoterpenos como alternativa para el control de Rhizoctonia solani Kün / Antifungal effect of four mixtures from monoterpenes like alternative by control of Rhizoctonia solani Kün

Rhizoctonia solani Kühn, es un patógeno de suelo, que causa enfermedades en un amplio rango de hospedantes de cultivos agrícolas. Las necesidades de reducir el uso indiscriminado de fungicidas químicos conllevan al estudio de otras alternativas menos agresivas al medio ambiente. Los monoterpenos presentes en los aceites esenciales de plantas, presentan una marcada actividad biocida y son compuestos que resultan menos perjudiciales desde el punto de vista ambiental. Se prepararon cuatro mezclas de monoterpenos (timol-entol, timolalcanfor, timol-citronelal y timol-1,8 cineol) con el objetivo de evaluar la actividad antifúngica in vitro sobre R. solani, mediante su efecto sinérgico. Se empleó el método de adición al medio de cultivo de cada una de las mezclas a concentraciones de: 0,5; 0,1; 0,05; 0,03 y 0,01 por ciento, partiendo de una solución madre al 10 por ciento en dimetilsufóxido (DMSO) al 5 por ciento. Se calcularon los porcentajes de inhibición y los resultados se analizaron estadísticamente. Se determinaron las dosis inhibitorias medias y se clasificaron la toxicidad de cada una de las mezclas en: inocua, ligera, moderada y tóxica a cada concentración. Las cuatro mezclas mostraron 100 por ciento de inhibición a las concentraciones de: 0, 5; 0, 1; 0,05 y 0,03 por ciento. A la menor concentración las mezclas de timol-mentol y timol-cineol fueron las de mayor efecto inhibitorio del crecimiento micelial de R. solani.

Rhizoctonia solani Kühn is a soil pathogen that causes diseases in a wide host range from agricultural crops. The need to reduce the indiscriminate use of chemical fungicide it has led to the study of other less aggressive environment alternatives. Monoterpenes, common components in plants essential oils, shows a maked biocide activity and are compounds less harmful from an environmental point of view. With the objective of evaluate the in vitro antifungal activity against R. solani, four binary monoterpenes mixtures (thymol-menthol, thymol-camphor, thymol-citronellal and thymol-1,8 cineol) with the objective of evaluate the in vitro antifungal activity against R. solani, four monoterpenes mixtures were prepared. The method of addition to culture media of each of the mixtures in concentrations of: 0, 5; 0, 1; 0, 05; 0, 03 and 0, 01 percent, from a stock solution to 10 percent in dimethylsulfoxide (DMSO) to 5 percent were used. The inhibition percentages were calculated and the results were statistically analyzed. Median inhibitory dose was determined and the toxicity of each mixture were classified in: harmless, light, moderate and toxic in each concentration. The four mixtures showed 100 percent of micelial growth inhibition in concentrations of 0, 5; 0, 1; 0,05 and 0,03 percent. The thymol-cineol and thymol-menthol mixtures had the higher inhibitory effect on micelial growth of R. solani at the lower concentration.

Mycorrhizal fungi isolated from native terrestrial orchids of pristine regions in Córdoba (Argentina) / Hongos micorrízicos aislados de orquídeas terrestres nativas de regiones pristinas en Córdoba (Argentina)

Orchidaceae is a highly dependent group on the Rhizoctonia complex that includes Ceratorhiza, Moniliopsis, Epulorhiza and Rhizoctonia, for seed germination and the development of new orchid plants. Thus, the isolation and identification of orchid mycorrhizal fungi are important to understand the orchid-fungus relationship, which can lead to the development of efficient conservation strategies by in vivo germination of seeds from endangered orchid plants. The aim of our work was to isolate and characterize the different mycorrhizal fungi found in roots of terrestrial orchids from Córdoba (Argentina), and, to learn about the natural habit and fungal associations in the Chaco Serrano woodland pristine region. In this study, bloomed orchid root and rhizosphere soil samples were obtained in two times from Valle de Punilla during spring of 2007; samples were kept in plastic bags until processed within 48 hours, and mycorrhizal condition confirmed assessing peloton presence. A total of 23 isolates of the orchideous mycorrhizal Rhizoctonia complex were obtained. The isolates were studied based on morphological characters and ITS-rDNA sequences. Morphological characteristics as color of colonies, texture, growth rate, hyphal diameter and length and presence of sclerotia were observed on culture media. To define the number of nuclei per cell, the isolates were grown in Petri dishes containing water-agar (WA) for three days at 25°C and stained with Safranine-O solution. The mycorrhizal fungi were grouped into binucleate (MSGib, 10 isolates) and multinucleate (MSGim, 13 isolates) based on morphological characteristics of the colonies. We obtained the ITS1-5.8s-ITS4 region that was amplified using primers ITS1 and ITS4. Based on DNA sequencing, isolates Q23 and Q29 were found to be related to species of Ceratobasidium. Isolates Q24 and Q4 were related to the binucleated anastomosis group AG-C of Rhizoctonia sp. The rest of the isolates grouped in the Ceratobasidium clade without grouping. From our knowledge this is the first report of the association of the AG-C testers with terrestrial orchids. A high specificity was observed in the symbiotic relationship. As the mycorrhizal fungal isolates were obtained from native orchids, they could be incorporated in conservation programes of endangered orchids in Argentina.

La Familia Orchidaceae se encuentra estrechamente relacionada con hongos micorrízicos que pertenecen al complejo Rhizoctonia, e incluyen los géneros Ceratorhiza, Moniliopsis, Epulorhiza y Rhizoctonia. Esta asociación es esencial para el desarrollo de nuevas plantas ya que favorecen el proceso de germinación de las semillas. Por lo tanto, el conocimiento de la naturaleza de esta interacción es importante para que los resultados de los programas de conservación de orquídeas sean efectivos. La fragmentación del bosque Chaqueño Serrano en el centro de Argentina, ha alcanzado un punto crítico en los últimos años, afectando el funcionamiento del ecosistema. El objetivo de este trabajo fue: a) aislar y caracterizar hongos micorrízicos presentes en orquídeas terrestres de la provincia de Córdoba (Argentina) y b) conocer el hábitat natural y las asociaciones fúngicas que se establecen en esta región prístina. A partir de las raíces de orquídeas terrestres, se obtuvieron 23 aislamientos de hongos micorrízicos que pertenecen al complejo Rhizoctonia. Estos aislamientos fueron caracterizados con base en caracteres morfológicos y moleculares. Las características morfológicas (color y textura de las colonias, cinética de crecimiento, diámetro y largo de la hifa y presencia de esclerocios) fueron observados en PDA y MEA a 25ºC. El número de núcleos por célula se observó en cultivos crecidos en AA (agar-agua) y teñidos con una solución de Safranine-O. La región ITS se amplificó usando los primers ITS1 e ITS4. Con base en las características morfológicas de la colonia, los aislamientos fueron agrupados en binucleados (MSGib) y multinucleados (MSGim). De acuerdo al cladograma obtenido con las secuencias de ADN, los aislamientos Q23 y Q29 están relacionados a especies de Ceratobasidium, aisladas de raíces de orquídeas. Los aislamientos Q24 y Q4 se asocian con el grupo de anastomosis de Rhizoctonia AG-C. Finalmente, se observó una alta variabilidad en el grado de especificidad existente en la simbiosis que se establece entre las raíces de estas orquídeas terrestres y los aislamientos obtenidos a partir de ellas. Este es el primer reporte de la asociación entre el grupo de anastomosis AG-C y orquídeas terrestres. Dado que estos aislamientos se obtuvieron de orquídeas terrestres nativas, podrían ser incorporados como nuevos patrones para micorrizas de orquídeas terrestres en Argentina. Este trabajo contribuye al conocimiento de la relación simbiótica que se establece entre orquídeas y hongos micorrízicos, así como también al desarrollo de estrategias de conservación de orquídeas terrestres nativas del bosque Chaco Serrano.

First Report of Web Blight of Rosemary (Rosmarinus officinalis) Caused by Rhizoctonia solani AG-1-IB in Korea

Herein, we report the first occurrence of web blight of rosemary caused by Rhizoctonia solani AG-1-IB in Gangneung, Gangwon Province, Korea, in August 2014. The leaf tissues of infected rosemary plants were blighted and white mycelial growth was seen on the stems. The fungus was isolated from diseased leaf tissue and cultured on potato dextrose agar for identification. The young hyphae had acute angular branching near the distal septum of the multinucleate cells and mature hyphal branches formed at an approximately 90degrees angle. This is morphologically identical to R. solani AG-1-IB, as per previous reports. rDNA-ITS sequences of the fungus were homologous to those of R. solani AG-1-IB isolates in the GenBank database with a similarity percentage of 99%, thereby confirming the identity of the causative agent of the disease. Pathogenicity of the fungus in rosemary plants was also confirmed by Koch's postulates.

Evolution of disease and potential biocontrol activity of Trichoderma SP. against Rhizoctonia solani on potato / Potencial do biocontrole por Trichoderma em relação à evolução da doença causada por Rhizoctonia solani em batata

Black scurf and stem canker disease cause by the fungal pathogen of Rhizoctonia solani and it is an economical important disease of potatoes in Bangladesh and throughout the world. This study evaluated the black scurf and stem canker disease development in potato and antagonistic activity of Trichoderma spp. against R. solani. The artificial infections were carried out using the inoculums of R. solani. The treatments (%inoculum) were: T1 (0% inoculum), T2 (5% inoculum), T3 (10% inoculum), T4 (20% inoculum), T5 (50% inoculum), and T6 (100% inoculum). The infection of stem canker and black scurf on progeny tubers increased with increase in inoculum levels. The highest disease incidence and severity was found in T6 (100% inocula). T6 showed the maximum black scurf infected tubers (russet, deformed and sclerotia). The lowest germination percentage, plant height and tuber yield were also obtained in the same treatment (100% inocula). Trichoderma spp reduced the growth of R. solani and the highest growth suppression was noted in isolate TM12. According to antagonistic activity, Trichoderma spp. reduced the growth of R. solani but was not able to stop the pathogen development. This finding showed management of this disease or R. solani invasion requires an integrated approach compared to Trichoderma single approach.

A rizoctoniose ou crosta negra causada por Rhizoctonia solani é a mais importante doença nos campos de batata em Bangladesh, bem como em várias regiões do mundo. Este trabalho avaliou o potencial do biocontrole com Trichoderma spp. e sua ação antagonista contra R. solani em batateira. Realizou-se as avaliações do potencial antagonosta usando inoculação artificial de R. solani. Os tratamentos (% de inóculo) foram: T1 (0% de inóculo), T2 (5% ), T3 (10%), T4 (20%), T5 (50%) , e T6 (100% de inóculo). A infecção de rizoctoniose na haste e crosta negra nos tubérculos aumentou proporcionalmente com o aumento do nível de inóculo. A maior incidência e severidade da doença ocorreu no tratamento 6 (100 % de inóculo), o qual apresentou maior quantidade de tubérculos infectados e deformados com escleródios em sua superfície. A menor porcentagem de germinação e produção de tubérculos também foi encontrada no tratamento 6, o qual também apresentou menor altura de planta. Trichoderma spp reduziu o crescimento de R. solani e a maior atividade de supressão do crescimento foi encontrada pelo isolado TM12. Foi detectada a atividade antagonista de Trichoderma spp. em reduzir o crescimento de R. solani, mas este não inibiu o crescimento total do patógeno. Conclui-se que o manejo da rizoctoniose da batateira por colonização de R. solani necessita táticas de manejo integrado em detrimento do uso isolado do manejo ou biocontrole com Trichoderma spp.

Role of Sclerotia, Plant Debris and Different Hosts on Survival of Rice Sheath Blight Pathogen, Rhizoctonia Solani.

The survival of sclerotia stored under different conditions revealed that when they were kept in laboratory survived fully up to 7 months. However in soil at 5 cm and 10 cm depth, it survived 100 percent up to 8 and 10 months. The pathogen was viable in the sclerotial form for 17 months in the lab conditions however; it survived for 19 months and 20 months when kept at 5 cm and 10 cm depth in soil respectively. The survival of pathogen along with plant debris stored under different conditions revealed that it survived fully up to 3 months under lab conditions. However in soil at 5 cm and 10 cm depth, it survived 100 percent up to 5 and 6 months respectively. The pathogen survived in diseased plant debris for 9 months in lab conditions. However, the pathogen survives in plant debris up to 11 months and 13 months when kept at 5 cm and 10 cm depth of soil respectively. The viability of pathogen in plant debris was lost gradually. This states sclerotia and plant debris served as source of primary inoculum. Out of fourteen plant species belonging to three families tested, the pathogen produced disease symptoms on all the tested plants and stating pathogen has wide host range.

Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani

Background Rhizoctonia solani (teleomorph: Thanatephorus cucumeris) is one of the most important pathogens of rice (Oryza sativa L.) that causes severe yield losses in all rice-growing regions. Sclerotia, formed from the aggregation of hyphae, are important structures in the life cycles of R. solani and contain a large quantity of polysaccharides, lipids, proteins and pigments. In order to extract high-quality total RNA from the sclerotia of R. solani, five methods, including E.Z.N.A.™ Fungal RNA Kit, sodium dodecyl sulfate (SDS)-sodium borate, SDS-polyvinylpyrrolidone (PVP), guanidinium thiocyanate (GTC) and modified Trizol, were compared in this study. Results The electrophoresis results showed that it failed to extract total RNA from the sclerotia using modified Trizol method, whereas it could extract total RNA from the sclerotia using other four methods. Further experiments confirmed that the total RNA extracted using SDS-sodium borate, SDS-PVP and E.Z.N.A.™ Fungal RNA Kit methods could be used for RT-PCR of the specific amplification of GAPDH gene fragments, and that extracted using GTC method did not fulfill the requirement for above-mentioned RT-PCR experiment. Conclusion It is concluded that SDS-sodium borate and SDS-PVP methods were the better ones for the extraction of high-quality total RNA that could be used for future gene cloning and expression studies, whereas E.Z.N.A.™ Fungal RNA Kit was not taken into consideration when deal with a large quantity of samples because it is expensive and relatively low yield.

Pathogenicity Tests and Evaluation of Efficacy of Fungicides Against Rhizoctonia Bataticola, the Causal Agent of Dry Root Rot of Chickpea.

The pathogen was identified based on its mycelial and sclerotial characters and pathogenicity test was proved by soil inoculation method. Efficacy of two non systemic fungicides (copper oxychloride and captan), two systemic fungicides (hexaconazole and tebuconazole) and one antifungal antibiotic validamycin each at different concentrations were tested against Rhizoctonia bataticola, incitant of dry root rot of chickpea under in vitro conditions. The fungicides copper oxychloride, captan, hexaconazole and tebuconazole were found to be highly effective (100%) in inhibiting the mycelial growth of the highly virulent pathogen at all the concentrations tested.