Characterisation of the yeast Pichia membranifaciens and its possible use in the biological control of Botrytis cinerea, causing the grey mould disease of grapevine

Pichia membranifaciens strain FY-101, isolated from grape skins, was found to be antagonistic to Botrytis cinerea, the causal organism of the grey mould disease of the grapevine. When grown together on solid as well as liquid media, the yeast brings about the inhibition of this parasitic fungus, coagulation and leakage of its cytoplasm, and suppression of its ability to produce the characteristic grey mould symptoms on the grapevine plantlets. In vitro experiments confirm that this yeast can be used as a biological control organism against B. cinerea. An account of the molecular characterisation of P. membranifaciens (complete sequence of the ITS region of its ribosomal DNA, GenBank accession No. AF 270935), as well as the interaction between B. cinerea and the yeast, are given here.     

A STUDY OF ABANDONED ASH PONDS RECLAIMED THROUGH GREEN COVER DEVELOPMENT

Green capping is one of the popular methods to re-vegetate abandoned ash ponds of coal based thermal power plants thereby lowering the risk of contamination to the surrounding environment. It has innumerable advantages such as prevention of dust emission, checking soil erosion, stabilizing the surface areas of ash, preventing potential ground water contamination, and finally, adding native vegetation cover, which is very vital in the long term. During the early nineties and later, various reclamation projects were carried out on fly ash dumps, but until date, there have not been any initiatives to assess the alterations in physicochemical and biological properties of fly ash resulting from implementation of these reclamation projects. In the present study, three abandoned ash ponds, located in India, that were reclaimed during 1998–2003 are investigated. Marked alterations in nutritional status, microbial population, and microbial activities have been observed in reclaimed ash ponds.     

Freeze-drying vegetative mycelium of Laccaria fraterna and its subsequent regeneration

Laccaria fraterna, a basidiomycetous, filamentous, non-sporulating (in vitro) fungus, was subjected to freeze-drying and tested for viability after rehydration. The optimization of the freeze-drying protocol included selection of the candidate fungus; optimizing physiological growth conditions and age; standardizing the protectant type and concentration; optimizing the pre-freezing method, freeze-drying run and extent of drying; choice of the rehydrant and the extent of rehydration. The culture retained its viability after lyophilization and when subjected to quality assurance tests gave consistent results similar to the non-lyophilized culture, indicating the stability of the product and the applicability of the developed process to freeze-dry vegetative mycelium of filamentous non-sporulating fungi.     

Enhanced production of ligninolytic enzymes and decolorization of molasses distillery wastewater by fungi under solid state fermentation

Selected isolates of fungi were grown on wheat straw and corncob in the presence of different moistening agents such as water, molasses, potato dextrose broth and distillery effluent. All the fungal isolates responded differently with respect to growth and ligninolytic enzyme production. Fungal growth on different substrates was checked by calculating ergosterol content, which varied widely within a single species when grown on different substrates. The maximum laccase production was obtained for Aspergillus flavus TERI DB9 grown on wheat straw with molasses. For manganese peroxidase, highest production was in Aspergillus niger TERI DB20 grown on corncob with effluent. Among the two isolates positive for lignin peroxidase, the highest production was in Fusarium verticillioides ITCC 6140. This immobilized fungal biomass was then used for decolorization of effluent from a cane molasses based distillery. Maximum decolorization (86.33%) was achieved in Pleurotus ostreatus (Florida) Eger EM 1303 immobilized on corncob with molasses in a period of 28 days.     

Growth responses and dependence of Acacia nilotica var. cupriciformis on the indigenous arbuscular mycorrhizal consortium of a marginal wasteland soil

 The responses of Acacia nilotica L. var. cupriciformis to phosphorus application and inoculation with the indigenous consortium of arbuscular mycorrhizal (AM) fungi were evaluated in a nursery experiment using soil from a marginal wasteland. A positive growth response to mycorrhizal inoculation was observed at an Olsen-P level of 20 ppm in the presence of the natural population of AM fungi. There was growth stimulation by either inoculation or additional P at the highest soil P of 40 ppm. Colonization was negatively correlated to soil P but P content of both shoot and root were positively correlated. Inoculation with the indigenous AM consortium significantly increased the uptake of P at all levels of applied P. Acacia is moderately dependent upon the AM symbiosis and exhibited a maximal mycorrhizal dependence (MD) of 18.25% at 20 ppm Olsen-P level under the conditions studied. A sharp and considerable reduction in MD and dry matter yield observed at 40 ppm P suggests that the external P requirement for maximal production of biomass was met at approximately 20 ppm Olsen-P. Accepted: 25 June 1996     

Concentration of fungal ligninolytic enzymes by ultrafiltration and their use in distillery effluent decolorization

Extracellular ligninolytic enzymes secreted by seven different fungi grown under solid-state fermentation using agro-residue as substrate were extracted through successive extractions. In general, most of the enzymes were recovered during first and second extractions. These extractants were then subjected to ultrafiltration using a 10 kDa membrane for further concentration. The permeates collected after every filtration and the final retentate were analyzed for protein and enzymes. In all the isolates, enzyme concentration was maximum in first retentate, which reduced significantly in second, third, and fourth retentate. Maximum per unit laccase (14.44 U g⁻¹) and MnP production (142.2 U g⁻¹) was observed in Fusarium verticillioides TERIDB16 while maximum LiP production (137.42 U g⁻¹) was in Alternaria gaisen TERIDB6. The retentate was further used for checking its decolorization efficiency of undiluted distillery effluent. The maximum decolorization (37%) was obtained using the enzyme extract of Pleurotus florida EM1303.     

Growth profile of ectomycorrhizal fungal mycelium: emphasis on substrate pH influence

A knowledge of behaviour of ectomycorrhizal fungal isolates to substrate pH would help in identifying candidate fungi for plantation programs. Ectomycorrhizal fungus isolates were studied to determine the pH optima for growth, substrate acidification by the culture and the effect of substrate acidification on culture growth. Of the isolates tested, the members of Agaricales (except Laccaria laccata) and Aphyllophorales favored neutral to near neutral pH, while members of the order Sclerodermatales strictly favored acidic pH. The change in substrate pH (ΔpH) was maximum at the optimum growth pH for acidophilic isolates but minimum for neutro/basophilic isolates. The experiments indicate that the substrate pH would not only determine the growth rate of the fungus but also limits further proliferation of the fungus in medium. This gives vital information for determining the subculture frequency and for designing substrate parameters for nursery/plantation programs. This revised version was published online in June 2006 with corrections to the Cover Date.     

Response of Eucalyptus tereticornis to inoculation with indigenous AM fungi in a semiarid alfisol achieved with different concentrations of available soil P

Eucalyptus tereticornis was grown in a green house in a low phosphorus (0.67 ppm Olsen's P) soil (Typic Haplustalf) inoculated with mixed indigenous arbuscular mycorrhizal (AM) fungi. Soil was amended to achieve P levels of 10, 20, 25, 30 and 40 ppm to evaluate the growth response and dependence of E. tereticornis to inoculation with AM fungi. A positive response to mycorrhizal inoculation was evident at the first two levels of soil P, i.e., at 0.67 and 10 ppm but not at the higher levels of soil P. Dry matter yield of inoculated plants beyond 20 ppm soil P was similar or even less compared to their uninoculated counterparts. Inoculated plants produced maximum dry matter (root and shoot) at 10 ppm soil P, whereas uninoculated plants did not produce until the level reached 20 ppm. The percentage root length colonized by AM fungi decreased from 31% to 3% as the concentration of P increased beyond 10 ppm soil P. Higher levels of soil P depressed the AM colonization significantly. Inoculated plants had higher shoot P and N contents compared to their uninoculated counterparts at all levels of soil P. However, at the first two lower levels of soil P, inoculated plants showed significantly higher shoot P and N contents over their respective uninoculated counterparts. The increasing shoot P accumulation beyond 10 ppm did not enhance dry matter yields. Inoculated plants had lower values of phosphorus utilization efficiency (PUE) and nitrogen utilization efficiency (NUE) at all levels of soil P except at the unamended level (0.67 ppm) where the inoculated plants showed higher values of NUE compared to uninoculated control plants. Taking dry matter yield into consideration, Eucalyptus plants were found to be highly dependent on 10 ppm of soil P for maximum dry matter production. Any further amendment of P to soil was not beneficial neither for AM symbiosis nor plant growth.     

Congruence of ribosomal DNA sequencing, fatty acid methyl ester profiles and morphology for characterization of the genus Rhizophagus (arbuscular mycorrhiza fungus)

Difficulties in obtaining sterile axenic cultures and heterogeneity in nuclear-encoded ribosomal DNA (n-rDNA) sequences within a single arbuscular mycorrhizal spore make genetic analysis of arbuscular mycorrhizal fungi (AMF) a complicated task, and currently available methods of genotyping are inadequate for identification to the species level. Therefore, we applied a multipronged approach on different isolates grown in root organ culture (ROC) belonging to the genus Rhizophagus which were not characterized at species level. Each strain was characterized using the fatty acid methyl ester profile (FAME), partial sequencing of a small subunit-internal transcribed spacer (SSU-ITS) and a large subunit (LSU) region of n-rDNA, and morphological examination of spores. Neighbor-joining trees obtained from the SSU-ITS rDNA sequences were broadly similar to those obtained from the LSU rDNA sequences. FAME profiles of the same isolates used for molecular characterization were obtained using fatty acid datasets, and results were compared to a neighbor-joining tree of n-rDNA sequence. Based on the results of these studues, a combination of morphology, biomarkers (FAME), and molecular sequencing (of highly variable D1-D2 of LSU and ITS) is recommended for phylogenetic analysis and characterization of species/strain of Glomeromycota.