Protoplast generation in the nematode trapping fungi, Arthrobotrys oviformis and Dactylaria parvispora (original) (raw)
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Protoplast generation in nematode trapping fungi
2008
A protocol for generating viable protoplasts from isolate of the nematophagous fungus Arthobotrys oviformis is developed. The lytic enzyme Novozyme 234 is used in the study. Maximum protoplasts are released with 24 hours old A. oviformis mycelia after 35 minutes of treatment with the lytic enzyme Novozyme 234 at a concentration of 10 mg ml. The nematophagous fungus Dactylaria parvispora however is found to be refractive to the process and protoplasts could not be generated under the range of conditions used in this study. The yield of protoplasts and the frequency of regeneration of A. oviformis is found to be highest when 0.6 molar Sodium Chloride is used as the osmotic stabilizer. This is the first report of an optimized method for generating viable protoplast from young mycelia of the nematophagous fungus A. oviformis.
Transformation of the nematode-trapping fungus Arthrobotrys oligospora
FEMS Microbiology Letters, 1999
The nematode-trapping fungus Arthrobotrys oligospora was transformed to hygromycin resistance using the hygromycin-B phosphotransferase gene from Escherichia coli under the control of various heterologous fungal promoters. Plasmid DNA was introduced into fungal protoplasts by polyethylene glycol/CaCl P treatment. Transformation frequencies varied between 1^6 transformants per Wg DNA. Seven out of 13 integration events analyzed from transformants were single copy integrations, whereas the remaining were multiple and more complex integrations. The addition of restriction enzymes during transformations increased the frequency of single copy integrations. Co-transformation, using the E. coli uidA gene encoding the L-glucuronidase reporter gene under the control of an Aspergillus nidulans promoter, occurred at frequencies of up to 63%.
Journal of Invertebrate Pathology, 1988
Nomuraea rileyi (UF178-6) conidia isolated from larval cadavers of Anticarsia gemmatalis and from an attenuated culture (UF178-6a) were used to establish separate hyphai body and mycelial cultures in liquid media, respectively. Studies on the growth of each strain were performed under submerged liquid fermentation conditions. Analysis of the respective utilization of carbohydrates and amino acids from the media, production of extracellular polysaccharide, and alterations in pH of the media demonstrated differences between the mycelial and hyphal body cultures. Using an enzyme mixture of j3-glucuronidase and chitinase, protoplasts were produced from hyphal bodies and mycelia throughout each fermentation. Maximum yields of protoplasts were isolated from each strain midway through their vegetative growth phases. o 1988 Academic PESS. IK.
Annals of Microbiology, 2009
Optimization of different factors for efficient protoplast release fromMetarhizium anisopliae was investigated. Factors like culture media, age of the mycelium, incubation time, different enzymatic combinations and osmotic stabilizer were studied. Mycelium harvested at 40th h in Sabouraud Dextrose broth showed the best protoplast yield over the other media and age of mycelium tested. An incubation time of 3 h and Lysing enzyme at a concentration of 10 mg/ml was the best among the different enzymatic concentrations and combinations tested and yielded a protoplast release of 7.3×108 protoplasts/ml. The most suitable osmotic stabilizer for efficient protoplast release was 0.7M KCl.
Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae
Journal of Phytopathology, 1997
Preparation and regeneration of mycelial protoplasts from Aliernaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 X 10*/gfresh weight mycelia) was obtained from 24h-old mycelia digested with Novozym 234 (20 mg/m!) in a stabilized buffer of pH 6.4 and incubated in the dark at 30"C on a rotary shaker (90 r.p.m.) for 6h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration.
Isoenzyme analysis of Arthrobotrys, a nematode-trapping fungus
Brazilian Journal of Medical and Biological Research, 1997
Extraction and isoenzyme analysis of four isolates of Arthrobotrys including A. musiformis, A. robusta and A. conoides were conducted. Among the 14 enzymes studied by starch gel electrophoresis, using morpholine-citrate as gel/electrode buffer, the following nine enzymes showed interpretable banding patterns: α-esterase, fumarase, hexokinase, isocitrate dehydrogenase, leucine aminopeptidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase and phosphoglucoisomerase. All isolates studied displayed typical isoenzyme phenotypes for each species. Two isolates of A. conoides differed in their α-isoesterase banding patterns, but no differences were observed for the other enzymes. The assay was satisfactory for enzyme extraction and resolution of Arthrobotrys and could be used in future taxonomic and genetic studies of this organism.
Low temperature induced pseudo-mycostasis in nematode-trapping fungus Arthrobotrys oviformis
Artificial mycostasis was induced in conidial spores of the nematode trapping fungus Arthrobotrys oviformis through a method of low temperature preservation. Spore viability and predatory property was confirmed after 10 months of storage at -200C through specific regeneration studies. Constitutive dormancy appeared to be absent in the organism. A minimum of 1 day spore maturity was associated with 42.9% larval predation where as germination for 4 days was associated with cent percent trapping. Data indicate feasibility to undertake delivery related application study with these conidia forming genera.