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.

The community structure of macroscopic basidiomycetes (Fungi) in Brazilian mangroves influenced by temporal and spatial variations / La estructura de la comunidad de basidiomicetos macroscópicos (Fungi) en los manglares brasileños influenciada por las variaciones temporales y espaciales

Mangroves are transitional ecosystems between terrestrial and marine environments, and are distinguished by a high abundance of animals, plants, and fungi. Although macrofungi occur in different types of habitat, including mangroves, little is known about their community structure and dynamic. Therefore the aim of this study was to analyze the diversity of macrofungi in a number of Brazilian mangroves, and the relationship between such diversity, precipitation and area of collection. A total of 32 field trips were undertaken from 2009 to 2010, and macrofungi were studied in four 250×40m transects: Timbó and Santa Cruz Channel on the Northern coast, and Maracaípe and Ariquindá on the Southern coast. All basidiomata found along the transects were placed in paper bags, air-dried and identified using existing literature. It was found that Northern areas predominantly featured Avicennia schaueriana mangroves, while Rhizophora mangle dominated in Southern transects. A total of 275 specimens were collected, and 33 species, 28 genera, 14 families and six orders were represented. Overall abundance and species richness did not vary significantly among areas, but varied according to time, being higher during the rainy season. Subtle differences in composition were observed over time and between areas, probably due to variations in plant species occurrence. Further studies with collections during months of greater precipitation in transects dominated by different mangrove species of the same ecosystem are suggested to assess the overall diversity of mycobiota in these ecosystems.

Los manglares son ecosistemas de transición entre los ambientes terrestres y marinos, y se distinguen por la gran abundancia de animales, plantas y hongos. Aunque los macrohongos se encuentran en diferentes tipos de hábitat, incluidos los manglares, poco se sabe acerca de la estructura de su comunidad y dinámica. Por lo tanto, el objetivo de este estudio fue analizar la diversidad de macrohongos en los manglares de Brasil y su relación con la precipitación y área de recolección. Se realizaron un total de 32 salidas de campo entre 2009 y 2010, y los macrohongos fueron estudiados en cuatro transectos de 250×40m: Timbó y Canal de Santa Cruz en la costa norte y Maracaípe y Ariquindá en la costa sur. Todos los basidiomas encontrados a lo largo de los transectos se colocaron deshidratados en bolsas de papel, y se identificaron con ayuda de la literatura preexistente. Se encontró que las zonas del norte predominantemente presentaron Avicennia schaueriana, mientras Rhizophora mangle domina en transectos del sur. Se recolectaron un total de 275 especímenes y 33 especies, 28 géneros, 14 familias y seis órdenes estuvieron representados. Abundancia y riqueza de especies en general no varió significativamente entre las áreas, pero si varió en el tiempo, siendo mayor durante la estación lluviosa. Se observaron diferencias sutiles en la composición a través del tiempo y entre áreas, probablemente debido a las variaciones en la presencia de las especies de plantas. Otros estudios con recolectas durante los meses de mayor precipitación en transectos dominados por diferentes plan- tas de manglar en el mismo ecosistema son deseables para acceder a la diversidad de la micobiota.

Isolation of Polysaccharides and Terpenoids from Some Basidiomycota and Their Antibacterial Activities.

Aims: Antibacterial chemicals were isolated from fruit bodies of three basidiomycota [Coltricia perennis (L) Murrill, Onnia tomentosa (Fr.) P. Karst., and Polyporus mori (Pollini) Fr. ] fungi and their antibacterial potential were screened against five bacteria. Study Design: All experiments were performed thrice in completely randomized design (CRD) each, with five replications per treatment (antibacterial activity). The data was subjected to ANOVA. Means of three observations were compared with Duncan’s Multiple Range Test (DMRT). Place and Duration of Study: Molecular Mycopathology Laboratory, Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, between January 2012 and February 2013. Methodology: During the rainy season in the year of 2012, a survey for mushroom collection in the forest beds, infected logs in the plain of west Bengal was conducted .The fruit bodies of some basidiomycota were collected in sterile biodegradable polythene begs and brought to laboratory. The morphology, anatomy of fruit bodies and measurement of reproductive organs were recorded. The spore prints of all collected basidiocarps were taken.The collected basidiomycota were identified. The polysaccharides from the basidiocarps of the test fungi were isolated employing the methods of Mizuno et al. [17] and Wang et al.[12].Terpeniods were isolated according to the method followed by Anke and Werte [24] and Chairul et al. [25]. Their antibacteral activities were assayed against five bacteria [three Gram positive bacteria (Staphylococcus aureus, Micrococcus roseus and Bacillus brevis ) and two Gram negative bacteria (Ralstonia solanacearum and Escherichia coli )] following the agar plates cup diffusion techniques. Results: Terpenoid isolated from Coltricia perennis was most active in inhibiting the growth of all five bacteria. This terpenoid inhibited maximum (25 ±2.4mm) growth against Staphylococcus aureus and minimum against Micrococcus roseus (17±1.1mm). The polysaccharides isolated from these three mushrooms were less active against the test five bacteria. The terpenoids isolated from Onnia tomentosa and Polyporus mori also inhibited the growth of the test bacteria. Conclusion: These three basidiomycetous mushrooms have antibacterial activity. After further research, their activity can be employed in medical science.

Phenotypic plasticity of the basidiomata of Thelephora sp. (Thelephoraceae) in tropical forest habitats

Phenotypic plasticity in macroscopic fungi has been poorly studied in comparison to plants or animals and only general aspects of these changes have been described. In this work, the phenotypic variation in the basidiomata of Thelephora sp. (Thelephoraceae) was examined, as well as some aspects of its ecology and habitat, using 24 specimens collected in the tropical forests of the Chamela Biological Station, Jalisco, Mexico. Our observations showed that this taxon has clavarioid basidiomata that can become resupinate during development and growth if they are in contact with rocks, litter or live plants, establishing in the latter only an epiphytic relationship. This tropical species may form groups of up to 139 basidiomata over an area of 32.2m2, and in both types of vegetation (tropical sub-evergreen and deciduous forest) were primarily located on steep (>20°) South-facing slopes. It is found under closed canopy in both tropical forests, but its presence in sub-evergreen forests is greater than expected.

La plasticidad fenotípica en hongos macroscópicos ha sido poco estudiada en comparación con la de plantas o animales y solo se conocen aspectos generales de estos cambios. En este trabajo se examinó la variación fenotípica en los basidiomas de una especie de Thelephora sp. (Thelephoraceae), así como algunos aspectos de su ecología y hábitat a partir del estudio de 24 ejemplares recolectados en bosques tropicales de la Estación de Biología de Chamela, Jalisco, México. Nuestras observaciones mostraron que este taxon presenta basidiomas en forma clavarioide, los cuales pueden modificarse a resupinados si en su proceso de desarrollo se interponen obstrucciones físicas como rocas, restos vegetales o plantas vivas, estableciendo en estas últimas solo una relación epifítica. Esta especie llega a formar conjuntos de hasta 139 basidiomas en un área de 32.2m2; con localización predominante en laderas orientadas hacia el sur, de pendientes mayores a 20°, bajo doseles cerrados y con presencia mucho más significativa de lo esperado en el bosque tropical subperennifolio.

Identification of leaf rust resistance genes in selected Argentinean bread wheat cultivars by gene postulation and molecular markers

Leaf rust, caused by Puccinia triticina Eriks. is a common and widespread disease of bread wheat (Triticum aestivum L.), in Argentina. Host resistance is the most economical, effective and ecologically sustainable method of controlling the disease. Gene postulation helps to determine leaf rust resistance genes (Lr genes) that may be present in a large group of wheat germplasm. Additionally presence of Lr genes can be determined using associated molecular markers. The objective of this study was to identify Lr genes that condition leaf rust resistance in 66 wheat cultivars from Argentina. Twenty four differential lines with individual known leaf rust resistance genes were tested with 17 different pathotypes of leaf rust collected from Argentina. Leaf rust infection types produced on seedling plants of the 66 local cultivars were compared with the infection types produced by the same pathotypes on Lr differentials to postulate which seedling leaf rust genes were present. Presence of Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr25, Lr26, Lr29, Lr34, Lr35, Lr37, Lr47 and Lr51 was also determined using molecular markers. Eleven different Lr genes were postulated in the material: Lr1, Lr3a, Lr3ka, Lr9, Lr10, Lr16, Lr17, Lr19, Lr24, Lr26, Lr41. Presence of Lr21, Lr25, Lr29, and Lr47 could not be determined with the seventeen pathotypes used in the study because all were avirulent to these genes. Eleven cultivars (16.7 percent) were resistant to all pathotypes used in the study and the remaining 55 (83.3 percent) showed virulent reaction against one or more local pathotypes. Cultivars with seedling resistance gene combinations including Lr16 or single genes Lr47 (detected with molecular marker), Lr19 and Lr41, showed high levels of resistance against all pathotypes or most of them. On the opposite side, cultivars with seedling resistance genes Lr1, Lr3a, Lr3a + Lr24, Lr10, Lr3a + Lr10, Lr3a + Lr10 + Lr24 showed the highest number of virulent reactions against local...

Elevated yield of Monacolin K in Monascus purpureus by fungal elicitor and mutagenesis of UV and LiCl

In China, Monascus spp., a traditional fungus used in fermentation, is used as a natural food additive. Monascus spp. can produce a secondary metabolite, monacolin K namely, which is proven to be a cholesterol-lowering and hypotensive agent. Hence, recently, many researchers have begun focusing on how to increase the production of monacolin K by Monascus purpureus. In the present study, we investigated the effect of the fungal elicitor and the mutagenesis of UV & LiCl on the amount of monacolin K produced by Monascus purpureus. The fugal elicitor, Sporobolomyces huaxiensis, was isolated from tea leaves and its filtrate was added into the culture filtrate of Monascus purpureus during growth to induct the production of monacolin K. The results showed that the highest amount of monacolin K produced by the liquid fermentation was 446.92 mg/mL, which was produced after the fungal elicitor was added to the culture filtrate of Monascus purpureus on the day 4; this amount was approximately 6 times greater than that of the control culture filtrate, whereas the highest amount of monacolin K produced by the mutated strain was 3 times greater than the control culture after the irradiation of UV light in the presence of 1.0 % LiCl in the medium.

In vitro synthesis of Frankia and mycorrhiza with Casuarina equisetifolia and ultrastructure of root system.

Casuarina equisetifolia is one of the ecologically and economically important tropical coastal trees nodulated by nitrogen-fixing actinomycete Frankia and forming symbiotic associations with both ecto- and endomycorrhizal fungi. The present study aims at the ultrastructural study of interactions between C. equisetifolia, Frankia, and mycorrhiza. C. equisetifolia seeds were sterilised and germinated under in vitro condition. The seedlings were transferred to conical flasks containing vermiculite and saw dust with Hoagland's solution. After 30 days, the inoculum of AM fungus--Glomusfasciculatum (A), ectomycorrhizal fungus-Pisolithus tinctorius (E) and actinorhizal Frankia (F) were inoculated individually and in various combinations, (A+E), (A+F), E+F) and (A+E+F). After 90 days, the experimental plant roots and nodules were harvested for assessment of growth characters of mycorrhizal and actinorhizal association by light and scanning electron microscope methods. C. equisetifolia roots were infected with arbuscles and vesicles of G. fasciculatum; P. tinctorius formed fungal sheath but no Hartig net. Large number of cortical cells were seen infected with Frankia, hyphae of Frankia were frequently seen penetrating from cell to cell directly through cell walls and Frankia occupied majority of the cell volume.

Arbuscular mycorrhiza-like biotechnological potential of Piriformospora indica, which promotes the growth of Adhatoda vasica Nees

We report the growth promoting potential of Piriformospora indica, which is a newly discovered arbuscular mycorrhiza-like fungus. It is a facultative symbiont and unlike arbuscular mycorrhizal fungi, it can be cultured in vitro. Adhatoda vasica is a medicinal plant. Rapid proliferation of roots was recorded in A. vasica withan important root colonization estimated to 95 percent after 6 months. P. indica improved growth of A. vasica. This association forms a new host-symbiont combination.

Antagonistic effect of fluorescent pseudomonads against Macrophomina phaseolina that causes charcoal rot of groundnut.

Maximum colony growth inhibition was observed due to Pseudomonas PS2 (74%) as compared to PS1 (71%) on trypticase soy agar (TSM) plates after 5 days of incubation. Light and scanning electron microscopic examination showed hyphal coiling, vacuolation, coagulation and granulation of cytoplasm resulting in lysis of hyphae of M. phaseolina by pseudomonads. Cell free culture filtrates of strains PS1 and PS2 restricted the growth of mycelium of M. phaseolina. PS1 and PS2 caused maximum colony growth inhibition by 57 and 61% respectively at 20% concentration of culture filtrate after 4 days of incubation. Volatile substances produced by PS1 and PS2 also inhibited the colony growth of M. phaseolina by 25 and 32%, respectively. Inhibitory effect of volatile substances, however, decreased with advancing in incubation period. Colony growth of M. phaseolina was significantly decreased by PS1 and PS2 as compared to control both in iron- sufficient and iron-deficient conditions. PS2 showed higher antagonistic activity than PS1, as evidenced by pronounced colony growth inhibition.

Genetic analysis of Karnal bunt (Neovossia indica) resistance in wheat.

Embryos excised from seeds of six generations (P1, P2, F1, BC1, BC2 and F2) of a cross WH 283 WH 533 were cultured on modified MS medium already inoculated with secondary sporidia of Neovossia indica. Significant variations for callusing response (CR) (54 55-75 55%) were observed among generations but the presence or absence of N. indicia did not affect callusing response. A clear inhibition zone (IZ) was formed around each embryo showing callusing. The diameter of IZ varied significantly among generations and was maximum in the resistant genotype, WH 283 (3 60 cm). Fresh weight and dry weight of calli, initiated from embryo cultured and inoculated with N. indica, varied significantly among generations. Coefficient of infection as well as percentage of infection reflected the overdominance of susceptibility. Generation mean analysis showed that the three parameter model was adequate for diameter of IZ only. Six-parameter model showed that additive (in presence of N. indica), additive and additive dominance (in absence of N. indica) effects were also significant. Complementary type of epistasis for fresh weight of calli and dominance, and dominance dominance effects for dry weight of calli were observed in the presence of N. indica. Magnitude of additive effects was higher for diameter of IZ in three parameter model. Therefore, selection might assist in improving this trait and thus indirectly help in attaining the resistance towards N. indica.

Formación y regeneración de protoplastos de phaffia rhodozyma / Formation and regeneration of phaffia rhodozyma protoplasts

En este trabajo se describe la preparación y regeneración de protoplastos a partir de cultivos frescos de la levadura carotenogénica phaffia rhodozyma, en forma eficiente y con un alto grado de rendimiento en la sobrevida de las células tratadas. Para ello se han realizado una serie de experimentos para formar protoplastos de p.rhodozyma, utilizando tres cepas silvestres y cinco cepas afectadas en carotenogénesis. Se probó las enzimas bioglucanasa, biocelulasa, bioxilanasa, glucoronidasa, zimoliasa 100T, lisozima y novozima 234, encontrándose que novozima 234, es la que tiene mayor eficiencia. En las cepas silvestres UCD 67-210 y UCD 67-385 y las cinco cepas de color, se logra un 100 porciento de protoplastos entre 60 a 90 minutos de tratamiento con novozima a 37ºC. Sin embargo, no fue posible formar protoplastos en ninguna de las condiciones estudiadas con las cepas silvestres UCD 67-383. Tales resultados pueden ser devidos a diferencias en la pared celular entre dichas cepas. Además, se ha observado que la incubación a 37ºC reduce notablemente la sobrevida de las células tratadas. Para la formación y regeneración de protoplastos se utilizó una serie de condiciones, encontrando que un sorbitol 0.2 M la destrucción de las células es menor que a concentraciones de sorbitol 0.8 y 1 M. Sin embargo, el medio más adecuado para la formación y regeneración de los protoplastos debe contener KC1 0.8 M como agente osmolar, manteniendo la isotonía del medio y logrando que la destrucción celular sea menor

Comportamiento de una cepa extranjera de Flammulina velutipes en tres medios de cultivo / Behavior of one foreign strain of Flammalins velutipes in three culture media

Se cultivo Flamulina velutipes (Curt. ex Fr.) Sing. de una cepa de E.U.A. en agar de dextrosa Sabouraud, agar dextrosa papa y agar con extracto de malta, en tres temperaturas: 9-11-C, 13-15-C y 22-24-C, para conocer las condiciones óptimas de desarrollo. El primer crecimiento se obtuvo en agar dextrosa Sabouraud a 22-24-C