Evaluación del cultivo in vitro de una cepa mexicana de Ganoderma lucidum / Evaluation of in vitro cultivation of a Mexican strain of Ganoderma lucidum

RESUMEN La cepa mexicana CP-145 de Ganoderma lucidum debido a la importancia medicinal que ha presentado últimamente, la presente investigación tuvo como objetivo evaluar el efecto de la temperatura y medio de cultivo sobre el crecimiento micelial óptimo en diferentes rangos de pH. Los tratamientos correspondieron en la utilización del medio de cultivo papa dextrosa agar (PDA) y extracto de malta agar (EMA), con dos niveles de temperatura (25 y 28 °C) y seis rangos de pH (4.0, 4.5, 5.0, 5.5, 6.0 y 6.5). El diseño experimental fue completamente al azar con medidas repetidas a través del tiempo, analizados con el paquete REPEATED MEASURE y el efecto tiempo con PROC MIXED de SAS. Como resultado se obtuvieron que el efecto de la temperatura y medios de cultivo en los diferentes rangos de pH, presentaron diferencias significativas (P ≤ 0.05). El crecimiento micelial óptimo de la cepa mexicana de G. lucidum fue en el medio de cultivo EMA en los rangos de pH de 4.0 y 4.5 con 8.3 y 8.2 cm respectivamente. De igual forma, en los rangos de pH 4.0 y 4.5 se obtuvieron los crecimientos miceliales óptimos a temperatura de 25 °C con 8.1 y 8.0 cm respectivamente. El cual concluyó esta investigación que el crecimiento micelial óptimo de la cepa mexicana fueron a pH 4.0 y 4.5, temperatura de 25 °C y medio de cultivo EMA.

ABSTRACT The Mexican strain CP-145 of Ganoderma lucidum due to the medicinal importance it has presented lately, the present investigation had as objective to evaluate the effect of temperature and culture medium on the optimal mycelial growth in different pH ranges. The treatments corresponded to the use of potato dextrose agar (PDA) and malt extract agar (EMA), with two temperature levels (25 and 28 °C) and six pH ranges (4.0, 4.5, 5.0, 5.5, 5.5, 6.0 and 6.5). The experimental design was completely randomised with repeated measures over time, analysed with the REPEATED MEASURE package and the time effect with PROC MIXED of SAS. As a result, the effect of temperature and culture media in the different pH ranges showed significant differences (P ≤ 0.05). The optimal mycelial growth of the Mexican strain of G. lucidum was in the EMA culture medium in the pH ranges of 4.0 and 4.5 with 8.3 and 8.2 cm respectively. Similarly, in the pH ranges 4.0 and 4.5 the optimum mycelial growth was obtained at 25 °C with 8.1 and 8.0 cm respectively. This research concluded that the optimal mycelial growth of the Mexican strain was at pH 4.0 and 4.5, temperature of 25 °C and EMA culture medium.

Sewage Sludge Biochar Effects on Phytopathogenic Fungi and Beneficial Microorganisms

Abstract Biochar (BCH) is a solid product, rich in carbon, obtained by heating biomass under controlled conditions of oxygenation, in a process known as pyrolysis. Its benefits are associated with improvements to the physical, chemical and biological properties of soil. Furthermore, BCH can affect the growth of phytopathogenic microorganisms. Despite research advances in this area, there is still a lack of information on the effect of BCH concentration on different soilborne microorganisms. This work evaluated the direct effect of sewage sludge biochar (SSB) on the in vitro growth of different soilborne fungi. Eight phytopathogens [Fusarium oxysporum, F. oxysporum f. sp. lycopersici (CEN 1456), Macrophomina phaseolina (429), Sclerotinia sclerotiorum, S. sclerotiorum (CEN 1147), Sclerotium rolfsii (CEN 216), Sclerotium cepivorum (CEN 1357), Rhizoctonia solani] were evaluated. Additionally, a biological control agent [Trichoderma afroharzianum (T-22)] was also studied. Microorganisms were subjected to growth in PDA (Potato-Dextrose-Agar) culture medium enriched with SSB. Biochars, produced at 300 and 500 ºC, were applied at different doses: 0.0 (control), 0.2, 0.4, 0.6, 0.8 and 1.0 (g of SSB/100 ml of PDA). Biochars showed inhibitory effects on the mycelial growth of the different fungi evaluated. It was observed that there is a certain specificity of biochar concentration that must be evaluated for the control of phytopathogens. In most cases, the 0.4 and 0.6% concentrations had a greater inhibitory effect on phytopathogens and did not affect the biological control agent. Therefore, SSB proved to be a promising product for the control of different soilborne phytopathogens.

Antifungal effects and phytochemical screening of Andrographis paniculata extracts on dermatomycoses

Aims@#Andrographis paniculata (AP), a medicinal herb was selected to investigate the antifungal activity on selected dermatophyte fungi. The phytochemical screening was also carried out to evaluate its chemical constituents.@*Methodology and results@#The potato dextrose agar (PDA) incorporated with aqueous, ethanol and methanol AP extracts at concentrations 0.99% (v/v), 1.96% (v/v) and 7.41% (v/v) were used for selected fungi culturing; Trichophyton mentagrophytes, T. rubrum, T. interdigitale, Microsporum fulvum, M. nanum, M. gypseum, M. canis, Fusarium solani and Aspergillus fumigatus. Phytochemical screening showed the presence of flavonoids, saponins and tannins in the ethanol extract and flavonoids alone in both aqueous and methanol extracts. Studies on antifungal effects indicated that the ethanol extract significantly increased the mycelial inhibition percentage of all tested fungi, especially at a concentration of 7.41% (v/v). All ethanol AP extract concentrations inhibited M. gypseum and M. canis (p<0.05) with at least 36.00% mycelial inhibition. In aqueous AP extract, it significantly increased the mycelial inhibition of T. mentagrophytes, T. interdigitale and M. gypseum (p<0.05), while the methanol AP extract significantly inhibited all fungi at a concentration of 7.41% (v/v) except for T. rubrum, M. gypseum and F. solani (p<0.05). No spore sedimentation was recorded for the fungal spores of T. rubrum, M. nanum, T. mentagrophytes, M. gypseum and T. interdigitale at 7.41% (v/v) ethanol AP. @*Conclusion, significance and impact of study@#It is concluded that the ethanol AP extract contained phytochemical constituents and showed the highest antifungal activity. In addition, this extract has a great potential to treat dermatophytes effectively.

Mycelial biomass cultivation of Lentinus crinitus / Crescimento micelial de Lentinus crinitus

Lentinus crinitus is a medicinal basidiomycete, little studied regarding the basic cultivation conditions, which is used in bioremediation and consumed by native Indians from the Brazilian Amazon. Also, it produces a fungal secondary metabolite panepoxydone that has been described as an essential regulator of the inflammatory and immune response. This study aimed to evaluate basic conditions of temperature, pH, and nitrogen concentration and source in the cultivation of L. crinitus mycelial biomass. In order to evaluate fungal growth temperature, 2% malt extract agar (MEA) medium, pH 5.5, was utilized from 19 to 40 °C. For pH, MEA had pH adjusted from 2 to 11 and cultivated at 28 °C. Urea or soybean meal was added to MEA to obtain final concentration from 0.5 and 16 g/L of nitrogen, pH of 5.5, cultivated at 28 °C. The best temperature growth varies from 31 to 34 ºC and the optimal one is 32.7º C, and the best pH ranges from 4.5 to 6.5 and the optimal one is 6.1. Protein or non-protein nitrogen concentration is inversely proportional to the mycelial biomass growth. Nitrogen concentrations of 2.0 g/L soybean meal and urea inhibit mycelial biomass growth in 11% and 12%, respectively, but high concentrations of 16.0 g/L nitrogen inhibit the growth in 46% and 95%, respectively. The fungus is robust and grows under extreme conditions of temperature and pH, but smaller adaptation with increasing nitrogen concentrations in the cultivation medium, mainly non-protein nitrogen.

Lentinus crinitus é um basidiomiceto medicinal consumido por índios nativos da Amazônia brasileira. Este fungo tem sido estudado quanto ao potencial de biorremediação de metais, mas ainda carece de estudos sobre às condições básicas de crescimento. L. crinitus produz panepoxidona - um metabólito secundário fúngico - descrito como regulador da resposta inflamatória e imune em células animais. Este trabalho teve como objetivo avaliar as condições básicas de temperatura, pH e concentração e fonte de nitrogênio para o crescimento micelial de L. crinitus. O fungo foi crescido em meio agar extrato de malte a 2% (MEA), pH 5,5 e mantido entre 19 e 40 °C. Para a avaliação de pH o MEA teve o pH ajustado de 2 a 11 e o crescimento foi realizado a 28 °C. As fontes de nitrogênio estudadas foram a uréia e o farelo de soja adicionado ao MEA para obter entre 0,5 a 16 g/L de nitrogênio, pH de 5,5, cultivado a 28 ° C. A melhor faixa temperatura para o crescimento micelial foi de 31 a 34 ºC com ótimo a 32,7 º C; a melhor faixa de pH de 4,5 a 6,5 e com ótimo de 6,1. A concentração de nitrogênio proteico ou não proteico é inversamente proporcional ao crescimento do fungo. Concentrações de nitrogênio de 2,0 g/L reduzem o crescimento da biomassa micelial em 11% e 12%, respectivamente e meios com nitrogênio de 16,0 g/L reduzem o crescimento em 46% e 95%, respectivamente. O fungo é robusto e cresce sob condições extremas de temperatura e pH, mas menor adaptação em meios com alta concentração de nitrogênio, principalmente não proteico.

Evaluation of hybrids and single spore isolates of paddy straw mushroom Volvariella volvacea (Bull.) Singer for fruiting body yield and nutritional quality

Aim: Developing high yielding single spore isolates and hybrid strains of paddy straw mushroom (Volvariella volvacea) with superior nutritional composition of the fruiting bodies.Methodology: Two contrasting high yielding strains (DMRO-463 and DMRO-484) of V. volvacea previously released for higher fruiting body yield were used for isolation of single spore isolates (SSIs). The slow growing SSIs were used for developing the hybrid strains by mating on Malt Extract Agar Medium Petri dishes. Conversely, the fast growing SSIs and the developed hybrid strains were screened for downward mycelial growth on paddy straw filled in wide mouth test tubes. Based upon downward mycelial growth, only eight SSIs and ten hybrid strains with numerically higher downward mycelial growth compared to two parents' checks were further evaluated for fruiting body yield on composted cotton ginning mill waste substrate under indoor conditions. Results: In successive yield evaluation trials (one preliminary and three full scale), hybrid strains VvH-11, VvH-13 and VvH-18 gave fruiting body yield higher than the two parents. Out of these hybrids, the yield distribution at different height (selves) in growing room was consistent in hybrid VvH-13 in trial-2, while rest two hybrids and parents showed significantly lower yield in lowest self (30 cm above floor level). The fruiting body weight also showed similar trend. The fruiting bodies from the highest yielding hybrid VvH-11 exhibited highest level of crude fiber (2.07%) and ash (10.95%) contents, while the hybrid VvH-13 giving consistent yield across growing room exhibited higher level of crude fiber (2.00 %), vitamin C (52.35 mg 100 g-1) and vitamin D (1434.7 µg 100 g-1). The third hybrid VvH-18 was superior in crude fiber (1.74%), manganese (22.46 ppm), selenium (0.26 ppm) and vitamin C (43.13 mg 100 g-1). All three hybrids exhibited lower fat (1.54 to 1.86 %) content compared to parents (2.54 to 2.59 %). Interpretation: High yielding hybrid strains can be developed in paddy straw mushroom (V. volvacea) through mating of slow growing SSIs, and their screening involving downward mycelial growth on paddy straw and repetitive grow out trials.

Obtaining monokaryotic and dikaryotic mycelial cultures of two Amazonian strains of Geastrum (Geastraceae, Basidiomycota)

The high diversity of the genus Geastrum and the difficulty of obtaining mycelial cultures impairs the study of the ecophysiology and the exploration of the biotechnological potential of the taxon. In this study, different culture media were tested to obtain mycelial cultures for G. lloydianum and G. subiculosum collected in the Brazilian Amazon. Data on spore germination, and isolation of monokaryotic cultures and in vitro sexual reproduction are presented, as well as a brief morphological description of the cultures obtained. For both species, Potato Dextrose Agar (PDA) was the most promising of the tested culture media. The highest growth in agar culture ever recorded for this genus is reported (4.9 mm per week for G. lloydianum and 7.5 mm for G. subiculosum). In the PDA culture medium, spores germinated after 35-40 days of incubation and the isolation of monokaryotic cultures of the two species, as well as in vitro sexual crosses, were successfully performed. (AU)

New Rabbit Erythrocyte Specific Mycelial Lectins from Fusarium sp. with Complex Saccharide Specificity

Eight Fusarium sp. namely, F. acutatum, F. globosum, F. graminearum, F. lactis, F. nivale, F. proliferatum, F.pseudoanthophilum and F. robustum were screened for the presence of lectins by hemagglutination activityusing human ABO, porcine, ovine, goat and rabbit erythrocytes. Mycelial extracts of all the fungal culturesexcept F. graminearum displayed unique lectin activity with only rabbit erythrocytes. Enzymatic treatment ofrabbit erythrocytes with neuraminidase has significantly enhanced the titre of all the lectin-positive extractsof fungal cultures. In contrast, most of the lectins showed a decline in lectin activity with protease treatedrabbit erythrocytes. Saccharide specificity studies have shown that majority of the lectins are inhibitory towardsO-acetyl sialic acids. None of the lectins from Fusarium sp. were inhibited by dextran, meso-inositol, andN-acetyl-D-glucosamine. Most of the fungal cultures displayed highest hemagglutination activity during the10th day of growth in broth cultures. The unique saccharide specificity of Fusarium sp. lectins can be used forelucidating their clinical role in glycobiology research.

Development of a Novel Spawn (Block Spawn) of an Edible Mushroom, Pleurotus ostreatus, in Liquid Culture and its Cultivation Evaluation

Mushroom cultivation has gained increased attention in recent years. Currently, only four types of spawn, including sawdust spawn, grain spawn, liquid spawn, and stick spawn, are commonly available for mushroom cultivation. This limited spawn diversity has led to difficulty in selecting suitable inoculum materials in some cultivation. In this study, three small blocks of lignocellulosic agro-wastes and one block of a synthetic matrix were prepared as support for growing Pleurotus ostreatus in liquid medium. Mycelium-adsorbed blocks were then evaluated for their potential as block spawn for fructification. Our results indicated that the edible fungus was adsorbed and abundantly grew internally and externally on loofah sponge and synthetic polyurethane foam (PUF) supports and also has the ability to attach and grow on the surface of sugarcane bagasse and corncob supports. The mycelia of P. ostreatus adhered on corncob exhibited the highest metabolic activity, while those on the PUF showed the least activity. Mycelial extension rates of block spawns made of agro-waste materials were comparable to that of sawdust spawn, but the block spawn of PUF showed a significantly lower rate. No significant differences in cropping time and yield were observed among cultivations between experimental block spawns and sawdust spawns. Moreover, the corncob block spawn maintained its fruiting potential during an examined period of 6-month storage. The developed block spawn could be practically applied in mushroom cultivation.

Long-term cryopreservation of basidiomycetes

Abstract Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to climatic features, the preservation of several basidiomycetes is threatened, and to guarantee the preservation of this genetic resource, the development of long-term preservation techniques is necessary once there is no universal protocol for the cryopreservation of basidiomycetes. Cryopreservation is a technique in which microorganisms are submitted to ultralow temperatures. Therefore, this study aimed to collect information on the main conditions for long-term cryopreservation of basidiomycetes in the last 20 years. Scientific articles on cryopreservation of basidiomycetes published from 1997 to 2016, were researched, and only the studies on two intervals of cryopreservation were considered: from 1 to 2 years and for longer than 2 years. The analyzed conditions of basidiomycete cryopreservation were: most studied genera, cryopreservation temperature, substrate, cryoprotectant (and preservation substrate), cryopreservation period, thawing temperature and cultivation medium after thawing, physiological and genetic stability of basidiomycetes after thawing in cryopreservation. In this review, the viability of the main cryopreservation conditions of basidiomycetes studied in the last 20 years are presented and discussed.

Antifungal potential of crude extracts of Trichoderma spp / Potencial antifúngico de extratos brutos de Trichoderma spp

Abstract Antibiosis is the mechanism by which certain microorganisms respond to the presence of others, secreting compounds or metabolites capable of inhibiting or impeding their development. The crude extract of Trichoderma contains a mixture of secondary compounds, which may show antibiotic effect, and has been used for the prospect of this fungus for biological control and other industrial purposes. Faced with the increasing demand of agriculture for ecologically compatible alternatives for the management of diseases, this work aimed to investigate the spectrum of action of Non-Volatile Metabolites (NVMs) of Trichoderma isolates against different plant pathogenic fungi. The antagonistic potential of NVMs was evaluated through the incorporation method of the filtered liquid extract in PDA medium. The assays showed that all the NVMs produced inhibited the fungus Sclerotinia sclerotiorum similarly. On the other hand, strains CEN1245 and CEN1274, both belonging to the species Trichoderma brevicompactum, showed broad spectrum against Sclerotium rolfsii, Colletotrichum gloesporioides, Verticillium dahliae, Fusarium oxysporum and Cylindrocladium sp. The present study describes isolates producing non-volatile metabolites with broad spectrum of antifungal action, as well as pathogen-specific. The Trichoderma spp. NVMs obtained from different soil samples cultivated with vegetables, cassava and maize were efficient in inhibiting plant pathogenic fungi belonging to other patossystems, such as forest or fruit, which could increase their potential application in biological control of plant diseases. In addition, these antagonistic fungi should be studied in greater depth for the identification of bioactive molecules of industrial interest or in commercial formulations of products for biological control of plant pathogens.

Resumo Antibiose é um mecanismo pelo qual certos microrganismos respondem à presença de outros, secretando compostos ou metabólitos capazes de inibir ou impedir o seu desenvolvimento. O extrato bruto de Trichoderma contém uma mistura de compostos secundários e tem sido utilizado na prospecção deste fungo para o controle biológico e demais fins industriais. Diante da crescente demanda da agricultura por alternativas ecologicamente compatíveis para o manejo de doenças, este trabalho teve como objetivo investigar o espectro de ação de Metabólitos Não Voláteis (MNVs), produzidos por isolados de Trichoderma, contra diferentes fungos fitopatogênicos. O potencial antagônico dos MNVs foi avaliado através do método de incorporação do extrato líquido filtrado em meio BDA. Os ensaios mostraram que todos os MNVs produzidos inibiram de forma semelhante o fungo Sclerotinia sclerotiorum. Por outro lado, os isolados CEN1245 e CEN1274, ambos Trichoderma brevicompactum, mostraram um amplo espectro de ação, atuando contra Sclerotium rolfsii, Colletotrichum gloesporioides, Verticillium dahliae, Fusarium oxysporum e Cylindrocladium sp. O presente estudo descreve isolados que produziram metabólitos não voláteis com amplo espectro de ação antifúngico, assim como patógeno-específico. Os MNVs de Trichoderma spp. obtidos de diferentes amostras de solo cultivadas com vegetais, mandioca e milho, foram eficientes na inibição de fungos fitopatogênicos pertencentes a outros patossistemas, como os de espécies florestais e frutíferas, o que poderia aumentar sua potencial aplicação no controle de doenças de plantas. Adicionalmente, estes fungos antagonistas devem ser mais bem estudados para identificação de moléculas bioativas de interesse industrial ou formulação de produtos para o controle biológico de patógenos de plantas.

Antimicrobial activity of Agaricus brasiliensis on Plasmopara viticola and its effect on the induction of resistance to the control of downy mildew on Isabel Precoce / Atividade antimicrobiana de Agaricus brasiliensis sobre Plasmopara viticola e seu efeito na indução de resistência para o controle de míldio em videiras Isabel Precoce

Agaricus brasiliensis include bioactive compounds that can act as antibiotics, bacteriostatic, fungistatic and nematostatic substances. In this sense, this study aimed to evaluate the effect of a single application of aqueous mycelial suspension (AMS) of A. brasiliensis in control of downy mildew (Plasmopara viticola) and resistance induction in 'Isabel Precoce' grapevines under greenhouse conditions. Treatments consisted of three doses of 1%, 5%, 10%, 15% and 20% AMS A. brasiliensis, as well as treatment with acibenzolar-S-methyl (ASM). The variables analyzed were: sporangiospore germination, disease severity, represented by the area under the disease progress curve (AUDPC), catalase enzyme activity, peroxidase and polyphenol. The 10%, 15% and 20% doses of AMS caused approximately 80% reduction in germination of P. viticola sporangiospores. The treatments did not show significant effects in reducing both the AUDPC of mildew and polyphenol oxidase enzyme activity. The A. brasiliensis aqueous mycelial suspension showed a fungitoxic effect on the germination of sporangiopores; however, it was not enough to reduce the severity of mildew in the 'Isabel Precoce' grapevines, even when acting on the catalase and peroxidase enzymes. Thus, experiments should be performed to verify the viability of the reproductive structures of the pathogen externalized in the vines when treated with A. brasiliensis AMS.(AU)

Agaricus brasiliensis possui compostos bioativos que apresentam atividade antimicrobiana e induz mecanismos de defesa em plantas contra patógenos. O objetivo deste trabalho foi avaliar a aplicação da suspensão miceliada aquosa de A. brasiliensis no controle do míldio (Plasmopara viticola) e indução de resistência em videiras Isabel Precoce. Os tratamentos foram: 0, 1, 5, 10, 15 e 20% da suspensão miceliada aquosa de A. brasiliensis, além do tratamento com acibenzolar-S-metil. As variáveis analisadas foram: germinação de esporangiósporos; severidade da doença, representada pela área abaixo da curva de progresso da doença; atividade da enzima catalase; peroxidase e polifenoloxidase. As doses 10, 15 e 20% de suspensão miceliada aquosa de A. brasiliensis proporcionaram redução de aproximadamente 80% na germinação dos esporangiósporos de P. viticola. Os tratamentos não apresentaram efeitos significativos na redução da área abaixo da curva de progresso da doença do míldio e na atividade da enzima polifenoloxidase. A dose de 10% da suspensão miceliada aquosa de A. brasiliensis reduziu a atividade de catalase e induziu a atividade da peroxidase. A suspensão miceliada aquosa de A. brasiliensis apresentou efeito fungitóxico na germinação de esporangióporos, entretanto não foi suficiente para reduzir a severidade do míldio da videira Isabel Precoce, mesmo atuando na atividade das enzimas catalase e peroxidase. Assim, experimentos deverão ser realizados para verificar a viabilidade das estruturas reprodutivas do patógeno exteriorizadas nas videiras quando tratadas com suspensão miceliada aquosa de A. brasiliensis.(AU)

European apple canker: morphophysiological variability and pathogenicity in isolates of Neonectria ditissima in southern Brazil / Cancro europeu da macieira: variabilidade morfofisiológicas e patogenicidade de isolados de Neonectria ditissima no sul do Brasil

ABSTRACT: European apple canker (EC) is caused by Neonectria ditissima, a pathogen officially registered as a quarantine pest in 2012. Thirty-five isolates of N. ditissima of different geographical regions of southern Brazil from apple branches showing symptoms of EC were identified by the specific pair primers Ch1 and Ch2 and analyzed concerning the virulence on Gala apple cultivar and morphophysiological characteristics. The disease symptoms were characterized and the isolates compared based on average mycelium growth (AMG), mycelium growth index (MGI), colony color, conidia type, dimensions and growth on potato dextrose agar (PDA), malt agar (AM), and synthetic SNAY (SN) culture media. Nineteen isolates showed the greatest AMG on PDA, forming three growth groups of 35.56 (GI), 52.71 (GII), and 62.67mm (GIII). Seven isolates showed MGI greater than 4.0mm diameter on PDA compared with that on AM and SN. The highest conidia production was on SN, and the predominant colony color in all media was white to beige with central pigmentation of brown and borders colored in shades of beige. There were significant differences among the average dimensions of micro- and macroconidia on PDA, AM, and SN. The pathogenicity was confirmed for all isolates despite of different morphophysiological characteristics. There was no correlation among isolates morphophysiological variability, virulence, and geographical origin.

RESUMO: O cancro europeu (CE) da macieira é causado pelo fungo Neonectria ditissima e foi oficialmente registrado como praga quarentenária em 2012. Foram avaliados 35 isolados de N. ditissima provenientes de ramos de macieira com sintomas típicos de CE de diferentes regiões do sul do Brasil. Os isolados foram identificados pelos primers específicos Ch1 e Ch2 e analisados quanto as características morfofisiológicas e virulência na cultivar de macieira Gala. Os sintomas foram caracterizados e os isolados comparados com base no crescimento micelial médio (CMM), índice de crescimento micelial (ICM), coloração das colônias e tipos, dimensões e produção de conídios em meios de cultura batata-dextrose-ágar (BDA), malte-ágar (MA) e meio sintético SNAY (SS). Dezenove isolados tiveram os maiores CMM sobre BDA, formando três grupos de crescimento de 35.56 (GI), 52.71 (GII) e 62.67mm (GIII). Sete isolados tiveram ICM maior de 4mm de diâmetro sobre BDA quando comparados com os meios MA e SS. A maior produção de conídios ocorreu sobre o meio SS e a coloração predominante foi de branco-à-bege com pigmentação central marrom e bordas de colônia em tons de bege. Foram observadas diferenças significativas nas dimensões médias dos micro- e macroconídios sobre os meios de culturas BDA, MA e SS. Todos os isolados foram patogênicos na cultivar de macieira gala, independentemente das diferenças morfofisiológicas. Não foram observadas correlações entre a variabilidade morfofisiológicas, virulência e origem geográfica dos isolados.

Characterization of Alternaria isolates and reaction of potato genotypes to early blight / Caracterização de isolados de Alternaria e reação de genótipos de batata à pinta preta

ABSTRACT: Early blight is one of the most important fungal diseases of potato. The objective of this study was to evaluate the in vitro reaction of potato genotypes to the severity of early blight and compare the species Alternaria solani and Alternaria grandis , etiologic agents of disease, through the evaluation of physiological characteristics. Twenty-two genotypes of the Potato Breeding Program at UFLA, two cultivars, Aracy and Bintje, patterns of resistance and susceptibility, respectively were evaluated. For inoculation, three isolates of A. grandis and two isolates of A. solani were used. Plantlets obtained in vitro were inoculated with a 5mm diameter mycelial disk of isolates Alternaria spp. of. These were incubated in severity and classified according to the rating scale. Isolates of Alternaria spp. were evaluated for mycelial growth index (MGI) and for formation of mycelial compatibility groups (MCG). The methodology for evaluating the in vitro reaction of genotypes to severity of early blight was efficient for both isolates. The isolates of A. solani and A. grandis were similar in their aggressiveness. The isolates of A. grandis MGI's had higher, and therefore mycelium grew faster when compared to those isolated from A. solani . The occurrence of mycelial compatibility among isolates of A. solani and A. grandis indicated the possible occurrence of parasexual cycle.

RESUMO: A pinta preta é uma das doenças fúngicas mais importantes da cultura da batata. O objetivo deste trabalho foi avaliar, in vitro, a reação de genótipos de batata à pinta-preta e comparar as espécies Alternaria grandis e Alternaria solani , por meio da avaliação de características fisiológicas. Foram avaliados 22 genótipos do Programa de Melhoramento Genético de Batata da UFLA, sendo duas cultivares, padrões de resistência e suscetibilidade. Foram inoculados três isolados de A. grandis e dois de A. solani . As plântulas, obtidas in vitro, foram inoculadas com os isolados de Alternaria spp., incubadas e classificadas quanto à severidade. Os isolados de Alternaria spp. foram avaliados quanto ao índice de velocidade de crescimento micelial (IVCM) e a formação de grupos de compatibilidade micelial (GCM). A metodologia de avaliação precoce in vitro da reação dos genótipos à severidade da pinta-preta foi eficiente para os isolados de ambas as espécies de Alternaria . Os isolados de A. grandis e A. solani apresentaram comportamento semelhante quanto à agressividade. Os isolados de A. grandis apresentaram IVCMs superiores e, portanto, crescimento micelial mais rápido. A ocorrência de compatibilidade micelial entre isolados de A. grandis e A. solani indica a possibilidade de ocorrência do ciclo parassexual.

Production of mycelial biomass by the Amazonian edible mushroom Pleurotus albidus

ABSTRACT Edible mushroom species are considered as an adequate source of food in a healthy diet due to high content of protein, fiber, vitamins, and a variety of minerals. The representatives of Pleurotus genus are characterized by distinct gastronomic, nutritional, and medicinal properties among the edible mushrooms commercialized worldwide. In the present study, the growth of mycelial biomass of Pleurotus albidus cultivated in submerged fermentation was evaluated. Saccharose, fructose, and maltose were the three main carbon sources for mycelial biomass formation with corresponding yields of 7.28 g L−1, 7.07 g L−1, and 6.99 g L−1. Inorganic nitrogen sources did not stimulate growth and the optimal yield was significantly higher with yeast extract (7.98 g L−1). The factorial design used to evaluate the influence of saccharose and yeast extract concentration, agitation speed, and initial pH indicated that all variables significantly influenced the production of biomass, especially the concentration of saccharose. The greater amount of saccharose resulted in the production of significantly more biomass. The highest mycelial biomass production (9.81 g L−1) was reached in the medium formulated with 30.0 g L−1 saccharose, 2.5 g L−1 yeast extract, pH 7.0, and a speed of agitation at 180 rpm. Furthermore, P. albidus manifested different aspects of morphology and physiology under the growth conditions employed. Media composition affected mycelial biomass production indicating that the diversification of carbon sources promoted its improvement and can be used as food or supplement.

In vitro Stimulation of NK Cells and Lymphocytes Using an Extract Prepared from Mycelial Culture of Ophiocordyceps sinensis

Ophiocordyceps sinensis is a natural fungus that has been valued as a health food and used in traditional Chinese medicine for centuries. The fungus is parasitic and colonizes insect larva. Naturally occurring O. sinensis thrives at high altitude in cold and grassy alpine meadows on the Himalayan mountain ranges. Wild Ophiocordyceps is becoming increasingly rare in its natural habitat, and its price limits its use in clinical practice. Therefore, the development of a standardized alternative is a great focus of research to allow the use of Ophiocordyceps as a medicine. To develop an alternative for wild Ophiocordyceps, a refined standardized extract, CBG-CS-2, was produced by artificial fermentation and extraction of the mycelial strain Paecilomyces hepiali CBG-CS-1, which originated from wild O. sinensis. In this study, we analyzed the in vitro immune-modulating effect of CBG-CS-2 on natural killer cells and B and T lymphocytes. CBG-CS-2 stimulated splenocyte proliferation and enhanced Th1-type cytokine expression in the mouse splenocytes. Importantly, in vitro CBG-CS-2 treatment enhanced the killing activity of the NK-92MI natural killer cell line. These results indicate that the mycelial culture extract prepared from Ophiocordyceps exhibits immune-modulating activity, as was observed in vivo and this suggests its possible use in the treatment of diseases caused by abnormal immune function.

Effect of surfactants and temperature on germination and vegetative growth of Beauveria bassiana

Three non-ionic surfactants: Tween20, Tween80 and Breakthru® were screened for their effects on spore germination and mycelial growth rates and for their influence on three isolates of Beauveria bassiana spore germination at various temperatures. Tween20 and Tween80 were compatible with all the B. bassiana isolates in the germination studies, but inhibited germination at higher surfactant concentrations, irrespective of the conidial concentrations. Breakthru® had an inhibitory effect on germination even at the lowest concentration of 0.1% on all the B. bassiana isolates. The effects of the surfactants on spore germination did not correspond with their effects on colony growth. Conidial viability within the same formulation declined significantly with increases in temperature, irrespective of the surfactant. The optimal temperature for conidial germination of B. bassiana isolates was approximately 25 °C with an upper limit at 30 °C. Isolate 7320 was identified as the least affected by the different surfactants. This isolate was able to germinate rapidly in a broad temperature range of 25–30 °C after 24 h, this characteristic being an essential factor in controlling house fly populations in poultry houses.

Evaluation of Culture Media for Mycelial and Sporangial Production of Phytophtora Colocasiae.

Eight different culture media were used to determine where Phytophthora colocasiae would bet grow and reproduce. Mycelia growth of 86 mm, 79.6 mm, 80.6 mm, 72 mm and 50 mm growth of P. colocasiae were obtained in Carrot Agar, Carrot Potato Agar (CPA) medium, Papaya Sucrose Agar, Host leaf extract agar, Oat meal agar respectively. P. colocasiae grown on Carrot agar for 4-7 days gave Carrot agar was supplement the nutrients to Phytophthora spp. to enhanced production of sporangial growth.

Efeitos da temperatura e regime de luz sobre Corynespora cassiicola e da temperatura e período de molhamento foliar no desenvolvimento da mancha-alvo em acerola / Effects of temperature and light regime on Corynespora cassiicola and of temperature and of leaf wetness period in development of target spot in barbados cherry

O presente estudo teve como objetivos: (i) determinar, in vitro, o efeito da temperatura sobre o crescimento micelial (10 a 35oC) e a germinação de conídios (10 a 45oC) de Corynespora cassiicola ; (ii) avaliar, in vitro , o efeito do regime de luz (luz contínua, fotoperíodo de 12 h e escuro) sobre o crescimento micelial, a esporulação e a viabilidade dos esporos de C. cassiicola ; e (iii) verificar, in vivo , a influência da temperatura (20 a 30oC) e da duração do período de molhamento foliar (4 a 48 h) no desenvolvimento da mancha-alvo em folhas de acerola. As temperaturas ótimas, estimadas a partir das equações, para o crescimento micelial e a germinação de esporos foram de 30 e 29oC, respectivamente. O regime de luz não afetou o crescimento micelial e a germinação dos esporos, entretanto, luz contínua favoreceu a esporulação, e a ausência de luz durante a produção dos esporos diminuiu sua viabilidade. Houve um incremento na área das lesões da mancha-alvo em folhas de acerola conforme a temperatura aumentou até 30oC. Foram necessárias 12 h de molhamento foliar para que a infecção e as lesões ocorressem.(AU)

The objective of this study were: (i) to assess, in vitro , the effect of temperature on mycelial growth (10-35oC) and spores germination (10-45oC); (ii) to evaluate, in vitro¸the effect of the light regime (continuous light, 12 h photoperiod and continuous darkness) on mycelial growth, sporulation and viability of the spores of C. cassiicola ;and (iii) to assess, in vivo,the effect of temperature (20-30oC) and the duration of leaf wetness (4-48 h) in the development of target spot in leaves of barbados cherry. The estimated maximum temperatures for mycelia growth and spore germination were 30 and 29oC, respectively. The light regime did not affect the mycelial growth and spores germination. However, continuous light regime favored the sporulation, and the absence of light during spore production decreased the spore viability. The target spot lesions area in barbados cherry leaves increased with the increment in the temperature up to 30oC. Twelve hours of leaf wetness were necessary for the infection and lesion development of target spot.(AU)

Morphology and mycelial growth rate of Pleurotus spp. strains from the Mexican mixtec region

Two native Pleurotus spp. strains (white LB-050 and pale pink LB-051) were isolated from rotten tree trunks of cazahuate (Ipomoea murucoides) from the Mexican Mixtec Region. Both strains were chemically dedikaryotized to obtain their symmetrical monokaryotic components (neohaplonts). This was achieved employing homogenization time periods from 60 to 65 s, and 3 day incubation at 28 °C in a peptone-glucose solution (PGS). Pairing of compatible neohaplonts resulted in 56 hybrid strains which were classified into the four following hybrid types: (R1-n xB1-n, R1-n xB2-1, R2-n xB1-n and R2-n xB2-1). The mycelial growth of Pleurotus spp. monokaryotic and dikaryotic strains showed differences in texture (cottony or floccose), growth (scarce, regular or abundant), density (high, regular or low), and pigmentation (off-white, white or pale pink). To determine the rate and the amount of mycelium growth in malt extract agar at 28 °C, the diameter of the colony was measured every 24 h until the Petri dish was completely colonized. A linear model had the best fit to the mycelial growth kinetics. A direct relationship between mycelial morphology and growth rate was observed. Cottony mycelium presented significantly higher growth rates (p < 0.01) in comparison with floccose mycelium. Thus, mycelial morphology can be used as criterion to select which pairs must be used for optimizing compatible-mating studies. Hybrids resulting from cottony neohaplonts maintained the characteristically high growth rates of their parental strains with the hybrid R1-n xB1-n being faster than the latter.

Antibiosis In vitro of Trichoderma Strains Metabolic Extract on Mycelial Growth and Reproductive Capacity of Fusarium oxysporum Isolated from Pepper Plants (Capsicum annuum L.).

Aims: To inhibit the mycelia growth inhibition and reproductive capacity of important phyto-pathogen fungus: Fusarium oxysporum by cell-extracts from submerged cultures of Trichoderma. Study Design: A complete randomized experimental design with factorial fix was used. Place and Duration of Study: Laboratory of plant-pathology, Department of Agricultural Parasitology, Universidad Autónoma Agraria Antonio Narro (UAAAN), Mexico, between August 2012 and March 2012. Methodology: Metabolic extract of Trichoderma asperellum produced in liquid medium and Trichoderma dual cultures on F. oxysporum isolated infected plant pepper was evaluated; both strains were subsequently activated in PDA and tested in dual culture and poisoned culture with Trichoderma strains and metabolic extracts against F. oxysporum to determine their growth inhibition potential. Results: Strains Trichoderma were able to inhibit the growth of the plant pathogen, demonstrating to be an attractive alternative for biological control assays. Similar results were obtained with metabolic extracts, where the inhibition was affected up to 29%, the conidiogenesis by 30% and spore viability by 60% at the highest concentration tested. Conclusions: Metabolites produced have the power to reduce the reproductive capacity of F. oxysporum, decreasing sporulation and inhibit the germination of conidia, and this is extremely important, as reducing the quantity and viability of conidia, it is reducing the secondary inoculum of the pathogen.