A landscape of transcriptome analysis of three sclerotia growth stages in Polyporus umbellatus / 中国中药杂志

Polyporus umbellatus,a traditional Chinese precious medicine as long been used for eliminating dampness,diuresis and have effect on cancer,getting more and more popularly in China recently. And the developmental metabolic process of the medicinal fungus,P. umbellatus,has been gotten more attention. This study is for the first time to explore the three sclerotial growth stages in P. umbellatus,named " white Polyporus"( initial phase), " grey Polyporus"( developmental phase) and " black Polyporus"( mature phase),by utilizing the de novo transcriptome assembly analysis technology. Finally,we obtained 88. 12 Gb sequence containing85 235 unigenes( ≥200 bp) assembled and 100% were annotated. We identified genes differentially expressed among the three stages of the sclerotia and screened out MFSgst,ERG4/ERG24,WD40,Rho A,CYP450,PKS,GSase and CHS1,which may contribute to the production of medicinal secondary metabolites and the defense mechanism against the environmental stress and biological invasion. We did the qRT-PCR trial to verify our results,which is in line with expectations. Our results are purposed to unearth the molecular mechanism of the accumulation of active constituents in different stages of Polyporus sclerotia which can be applied in the production and protection of Polyporus effectively.

Correlative analysis advance of chemical constituents of Polyporus umbellatus and Armillaria mellea / 中国中药杂志

Medicinal Polyporus umbellatus is the dry sclerotia of P. umbellatus, with the effect of diuresis; Armillaria mellea is a parasitic fungus which can infect plants up to 300 genera, with sedative, anticonvulsant and some other biological activities. As the medicinal value of P. umbellatus and A. mellea is increasingly wide concerned, the market quantity demanded of them is gradually increased and the demand outstrips the supply. The symbiotic A. mellea and P. umbellatus are both the medicinal and edible fungi with diverse activities, including hypoglycemic action, improve immunity and antitumor and so on. The growth of the sclerotia forming from the mycelium of P. umbellatus is related to the infection of the symbiotic A. mellea and their secondary products. In this study, by comparing the chemical constituents of the mycelium and sclerotia of P. umbellatus and A. mellea, we found that they all produced steroids and nitrogen-containing heterocycles. The sclerotia of P. umbellatus and A. mellea also produced triterpenes secondary metabolites. In addition, the mycelium and infected sclerotia of P. umbellatus mainly produced different steroids, and the sclerotia produced some other special secondary metabolites, such as long-chain fatty acids, ceramides, phenol and so on. By analyzing above all kinds of differences, speculated that these may be caused by the infection of the symbiotic A. mellea which mainly produced sesquiterpenes, diterpenes and other secondary metabolites. The contents and types of compounds of P. umbellatus and A. mellea are closely related to their symbiosis and reproduction, therefore, many symbiosis mechanisms should be found by utilizing more molecular biology technology to elucidate this complex symbiotic infection and provide scientific basis for improving the yield and quality of P. umbellatus and A. mellea.

Reduction of white mold level on soybean by fungicide management strategies / Redução do nível de mofo-branco na soja pelo manejo de fungicidas

The objective of this study was to verify the efficiency of the control of white mold on soybean with the use of fungicides applied alone and in rotation, at different growth stages and in a mixture of two active ingredients at three locations: Arapoti, PR, Mauá-da-Serra, PR and Pinhão, PR, Brazil. The fungicides used were carbendazim (Ca), thiophanate methyl (Tm), procymidone (Pr) and fluazinam (Fl). The experiments consisted on 17 treatments and 4 replications in a randomized block design. The analyzed variables were severity, incidence, number of sclerotia and yield. Mauá-da-Serra and Pinhão presented the highest incidences (31% and 29.8% in the control, respectively). At these two locations most of treatments with fungicides decreased the incidence and production of sclerotia, when compared to control; however, no differences in terms of yield were observed. Arapoti presented the lowest incidence (15.8% in the control) where most of treatments with fungicides did not present differences for the variables incidence, production of sclerotia and yield, when compared to the control. No differences were also observed for severity in any of three locations. In conclusion, fungicides applied in soybean areas with historically white mold incidence up to 31% can reduce the disease incidence and sclerotia production levels.

O objetivo deste estudo foi o de verificar a eficiência de controle do mofo-branco na soja com o uso de diferentes fungicidas aplicados isoladamente, alternados, em diferentes estádios fenológicos e em mistura de dois princípios ativos, em três locais: Arapoti/PR, Mauá-da-Serra/PR e Pinhão/PR, Brasil. Os fungicidas utilizados foram carbendazim (Ca), tiofanato metílico (Tm), fluazinam (Fl) e procimidona (Pr). Os experimentos foram compostos por 17 tratamentos e 4 repetições distribuídas em blocos aleatorizados. As variáveis analisadas foram incidência, severidade, produção de escleródios e o rendimento da cultura. Mauá-da-Serra e Pinhão apresentaram as maiores incidências, 31% e 29,8% no tratamento controle, respectivamente, onde a maioria dos tratamentos com fungicidas proporcionou menor incidência e produção de escleródios, quando comparados ao controle, porém sem diferenças quanto ao rendimento. Em Arapoti foi observada a menor incidência, 15,8% no controle, onde a maioria dos tratamentos com fungicidas não apresentou diferenças para incidência, produção de escleródios e rendimento, quando comparados ao controle. Em nenhum dos três locais foram verificadas diferenças entre os tratamentos e o controle para a variável severidade. Como conclusão, fungicidas aplicados em áreas de soja com histórico de incidência de mofo-branco de até 31% podem reduzir a incidência da doença e o nível de produção de escleródios.

Atividade de óleos e extratos vegetais sobre germinação carpogênica e crescimento micelial de Sclerotinia sclerotiorum / Activity of plant extracts on the carpogenic germination and mycelial growth of Sclerotinia sclerotiorum

A germinação carpogênica e o crescimento micelial de Sclerotinia sclerotiorum foram avaliados sob extratos metanólicos de Annona cacans, A. coriacea, A. crassiflora, A. dioica, A. sylvatica, Geophila repens, Guettarda viburnoides, Palicourea crocea, Schinus terebinthifolius e Trichilia silvatica, e sob as frações hexânica, hidrometanólica, clorofórmica e acetato de etila de A. cacans e óleo essencial de S. terebinthifolius. A concentração utilizada foi de 1.000 ppm para os extratos e de 100 ppm para as frações. Os extratos vegetais e as frações foram incorporados em meio ágar-água, que foi vertido em caixas gerbox com 20 escleródios. O crescimento micelial foi avaliado em óleo essencial de S. terebinthifolius, nas concentrações de 0, 100 e 1.000 ppm, incorporado ao meio BDA (Batata-Dextrose-Ágar). A germinação carpogênica apresentou-se menor sob os extratos de G. repens, P. crocea e S. terebinthifolius e sob as frações acetato de etila e clorofórmica de A. cacans. O número de apotécios formados por gerbox foi menor com o extrato de A. cacans. O crescimento micelial apresentou 10% de inibição na maior concentração do óleo essencial de S. terebinthifolius.(AU)

The efect of methanolic extracts of Annona cacans, A. coriacea, A. crassiflora, A. dioica, A. sylvatica, Geophila repens, Guettarda viburnoides, Palicourea crocea, Schinus terebinthifolius e Trichilia silvatica, and A. cacans hexane, ethyl etila, aqueous and chloroform fractions and the essential oil of S. terebinthifolius on mycelial growth and carpogenic germination of Sclerotinia sclerotiorum was evaluated. Te concentrations are 1,000 ppm for the extracts and 100 ppm for the fractions. To evaluate the germination, carpogenic, extracts and fractions were incorporated in agar-water that was poured into gerboxes where 20 sclerotia were distributed. To evaluate the mycelial growth, essential oil of S. terebinthifolius in concentrations of 0, 100 and 1,000 ppm was incorporated into the PDA and then poured into Petri dishes, to where pathogen mycelial discs were transferred. Extracts of G. repens, P. crocea and S. terebinthifolius and fractions ethyl acetate and chloroform of Annona cacans reduced the carpogenic germination of sclerotia of S. sclerotiorum and the extract of A. cacans reduced the number of apothecia formed. Mycelial growth showed 10% inhibition at the highest concentration of essential oil of S. terebinthifolius.(AU)

Investigation of symbiotic Armillaria species with Chinese traditional medicinal fungus Polyporus umbellatus / 中国药学杂志

OBJECTIVE: To identify symbiotic Armillaria species with Polyporus umbellatus. METHODS: Armillaria was cultured on PDA paltes. To determine whether the strains were pure culture, the internal intergenic spacer (IGS) region were sequenced. Furthermore, the NCBI BLAST program was used to search similar sequences in the GenBanksequence database for the IGS sequence of the species on homology, and a phylogenetic tree was constructed based on neighbor-joining method. RESULTS: All isolates were similar in colony morphology and grew well in PDA medium with well-developed rhizomorphs. The isolated 22 strains were pure culture of the fungus. Phylogenetic analysis based on IGS sequence indicated that the symbiotic Armillaria species with Polyporus umbellatus belonged to A. cepistipes, A. gallica, A. ostoyae, and A. mellea. CONCLUSION: In the present study, phylogenetic analysis based on molecular method was carried out for the symbiotic Armillaria species with P. umbellatus for the first time. The results suggest that Armillaria species have no specificity with P. umbellatus, which is different from the previous reports. Further research will be done on the growth of P. umbellatus affected by different kinds of Armillaria species.

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.

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.

Exsudatos radiculares de plantas de cobertura no desenvolvimento de Sclerotinia sclerotiorum / Exudates of cover crops in the development of Sclerotinia sclerotiorum

O objetivo do trabalho foi avaliar a influência dos exsudatos radiculares das plantas de cobertura: crotalária (Crotalaria juncea L.), braquiária (Urochloa ruziziensis R. Germ. & Evrard), capim-mombaça (Panicum maximum cv. Mombaça Jacq.), milheto (Pennisetum glaucum (L.) R. Brown), feijão-guandu-anão (Cajanus cajan (L.) Millsp.) e estilosantes (Stylosantes capitata Vog.; Stylosanthes macrocephala Ferr. Et Costa) no desenvolvimento de Sclerotinia sclerotiorum (Lib.) de Bary. Para a liberação dos exsudatos, as raízes de plantas cultivadas durante 24 dias foram submetidas à centrifugação. Obtido os exsudatos, estes foram esterilizados por processos de filtragem com a utilização de membrana com poro de 0,22µm armazenados em tubos e acondicionados a -20ºC. O efeito dos exsudatos foi estudado em relação ao crescimento micelial, germinação micelial, desenvolvimento carpogênico do escleródio e germinação dos ascósporos de S. sclerotiorum. As concentrações utilizadas foram 1%, 10% e 25%, obtidas por meio da diluição em água destilada esterilizada. Os resultados mostraram que o índice de velocidade de crescimento micelial foi menor quando utilizado o exsudato de U. ruziziensis nas concentrações de 1% e 10%. Os exsudatos não influenciaram a germinação micelial dos escleródios. Quanto aos ascósporos, os exsudatos radiculares de Cajanus cajan e Styloshantes sp. inibiram a germinação quando não foi adicionado sulfato de streptomicina.

The objective was to explore the influence of root exudates of some cover plants known as Crotalaria juncea, Urochloa ruziziensis, Panicum maximum cv. mombaça, Pennisetum glaucum, Cajanus cajan e Stylosantes sp. For the release of exudates roots plants were grown for 24 days, were subjected to centrifugation. Retrieved these exudates were sterilized by filtering processes using a membrane with pore size of 0.22 micrometers. The filtrates were stored in tubes and stored at -20°C. The effect of the exudates were studied in relation to mycelial growth, mycelial and carpogenic germination of sclerotia and germination of ascospores of S. sclerotiorum. The concentrations used were 1%, 10% and 25% obtained by dilution in sterile water. The results showed mycelial growth rate was lower when using the exudate of U. ruziziensis concentrations of 1% and 10%. The exudates did not affect mycelial germination of sclerotia. Concerning the ascospores of Cajanus cajan and Stylosanthes spp. root exudates inhibited germination when it was added streptomycin.

Influence of light and spawn quantity on the growth of Nigerian mushroom Pleurotus tuber-regium.

The effects of light and quantity of spawn on the sporophore and sclerotial yields of Pleurotus tuber-regium, cultivated on cotton wastes, rice straw, cocoyam peel, corncob, groundnut shell and sawdusts of Mansonia altissima, Khaya ivorensis and Boscia angustifolia were observed.The organism had sporophore and sclerotial yield values of 36.8 and 27.6 g kg-1 waste, respectively, in cotton waste, at light quantum of 695 lux. There was a highly significant increase in yield of sclerotia (188.0 g kg-1 waste), in total darkness , while malformed fruitbodies (sporophores) were produced in all the substrates under the same condition. Increasing the quantity of spawn from 5 to 30% reduced the period of spawn run from 13 to 6, 15 to 8 and 24 to 17 days, respectively, in P. tuber-regium fruitbodies grown in cotton waste, rice straw and sawdust of B. angustifolia, with yield values of 38.0 and 20.0 g kg-1 waste in cotton waste and rice straw. The optimal spawn levels for sclerotia formation in the two wastes were 10 and 5%, respectively. .The mushroom did not produce sclerotia in corn cob and groundnut shell when exposed to light. However, maximal yield values of 286.8 and 288.4 g kg-1 waste were obtained in both substrates in total darkness.

EFFECT OF VARIOUS INORGANIC SALTS, CARBON AND NITROGEN SOURCES ON CAROTENOID YIELD IN SURFACE CULTURES OF PENICILLIUM SP.PT95 / 微生物学通报

Among inorganic salts tested, K 2HPO 4 was more essential to the sclerotia for mation and carotenogenesis of strain PT95 than KCl, MgSO 4 or FeSO 4 It wa s also shown that the combination of K 2HPO 4, KCl and MgSO 4 could produce t he best positive cooperation and give the highest sclerotia biomass (782mg/plate ) and pigment yield (328 ?g/plate) Five carbon sources, i e glucose sucros e, lactose, maltose and soluble starch, all could be utilized by the strain PT95 , and maltose was the best Among 8 nitrogen sources, yeast extract favooured t he sclerotia formation, and peptone fovoured the pigment accumulation; amine sal ts and urea were unfavourable to form sclerotia The medium containing 0 24 ～ 0 48 g/L sodium nitrate nitrogen was effective to both the sclerotia formation and the carotenoid production of strain PT95 when available maltose carbon con centrations were at 5 26～21 05 g/L The optimal C/N ratio was found to be 25 ∶1