Isolation, Identification And Characteristics Research Of Actinomycete From Agaricus Bisporus(Lange)Sing Cultivated Media

In agricultural production, there is a great deal of crop straw produced every year, which are seldom utilized effectively due to stable molecular structure of their major composition-cellulose and their particular existant state. In recent years, the burgeoning mushroom industry played an important role in straw utilization, which can cultivate a variety of edible fungi: Agaricus bisporus, Coprinus, Stropharia and so on. A. bisporus is a kind of grass rot mushroom, which can form fruiting body on heap fermented culture medium, their yield and quality are related to fermentation degree. In this paper, three actinomycetes strains LLJ-03, LLJ-04, LLJ-05 were separated in first heap fermented culture medium. According to cultural characteristics, 16S rDNA sequences, physiological and biochemical characteristics, they were identified as Streptomyces griseorubens LLJ-03, Streptomyces fungicidicus LLJ-04, Nocardioides thermolilacinus LLJ-05.S. griseorubens LLJ-03 grows well on straw substrate such as wheat straw, which can accelerate biotransformation of straw. Changes of carboxymethyl cellulase activity during fermentation was determined using DNS method: (1) during liquid fermentation, CMCase activity changed undulatedly in 70U/ml ~ 100U/ml interval, indicating the enzyme is relatively stable. At 24h the activity was 83.9U/ml, rised to 85.3U/ml at 48h, at 72h it reduced to 73.0U/ml, then rised to 96.4U/ml at 120h, and then declined slightly, at 192h the activity reached to peak-96.8U/ml; (2) during solid fermentation process, the CMCase activity reached the peak value-484.7U/ml at 120h after inoculation. During liquid fermentation process, the reducing sugar content was 5.12mg/ml at 24h, at 48h was 5.17mg/ml, which lowered slightly to 4.71mg/ml at 72h, then increased continually. As time goes on, the increasing rate declined gradually, the total reducing sugar content reaching a plateau-7.00mg/ml overall.Both S.fungicidicus LLJ-04 and N.thermolilacinus LLJ-05 have varying effects on 7 tested pathogenic fungi: Fusarium oxysporum, Fusarium oxysporum f.sp.cucum erinum, Fusarium graminearum, Fusarium oxysporum f.sp.niveurn, Aspergillus flavus, Neurospora crassa, Aspergillus fumigatus. Through plate confrontation experiments, inoculation pieces inhibited mycelial growth tests, organic solvent extract of mycelial inhibited Neurospora crassa, organic solvent extract of mycelial inhibited spore germination tests, the results showed that: (1) Streptomyces mycelium produced some antifungal agents during growing process, which have variable inhibitory effects on different fungi growth; (2) the antifungal agents was mainly existed in mycelial, a higher extraction productivity can be reached using high polarity solvents such as methanol; (3) the antifungal agents can effectively inhibit Neurospora crassa growing, and inhibit spore germination of tested pathogenic fungi with different inhibitory effects.Fermentation kinetics experiments results showed that the inhibition rate of S.fungicidicusLLJ-04 and N.thermolilacinus LLJ-05 for pathogenic fungi reached peaks at 240h after inoculation, while the producing rate of antifungal agents was different. S.fungicidicus LLJ-04 did not secrete antifungal agents until the mycelia matured, whih reached the peak at 7 days after inoculation, however, N.thermolilacinus LLJ-05 required about 11 days. The antifungal agents produced by S.fungicidicus LLJ-04 and N.thermolilacinus LLJ-05 are relatively stable, because the inhibition rate seldom changes with time. Optimized the fermentation conditions (temperature, shaking speed, volume of medium) by orthogonal test: the optimal fermentation condition for S.fungicidicus LLJ-04 was temperature 30℃, shaking speed 160 rpm, volume of medium 100 ml; the optimal fermentation condition for N.thermolilacinus LLJ-05 was temperature 30℃, shaking speed 180 rpm, volume of medium 75 ml.The antifungal agents produced by S.fungicidicus LLJ-04 and N.thermolilacinus LLJ-05 was heat-stable at temperature below 40℃, but the time should not be too long; they were acid resistance, alkali not, stable in neutral environment. We carried out chromatography on filter paper treated by different pH buffer , then biography. Rf values suggested they might be neural antibiotic. Similarly, the Doskochilova solvent system chromatogram showed that S. fungicidicus LLJ-04 extract had higher Rf values in systemⅡ&Ⅴ, while N.thermolilacinus LLJ-05 extract had higher Rf values in systemⅡ,Ⅳ&Ⅴ. UV-spectrometer analysis showed: the extract of S.fungicidicus LLJ-04 had two absorption peaks at 253nm, 338nm, in ultraviolet spectrum; the N.thermolilacinus LLJ-05 extract had one absorption peak at 407nm, in visible spectra. Isolation and purification are required for further verification in the future.