Studies On Coprinus Spawn Quality Control Technique

Spawn is a source of factor for edible fungi cultivation, and the quality of spawn directly influences the courses of the cultivation and management of edible fungi, and also influences the quality and production of edible fungi, and finally influnces the mushroom industry can have a good development environment or not. In this study, three major aspects including spawn activity,spawn purity and spawn fingerprinting were investigated on four Coprinus comatus strains, the final aim is to establish an relitavely intergrated set of spawn control technique. The main contents and results of this research are as follows:1.Spawn FingerprintingIn this experiment,total DNA of four Coprinus comatus, three Flammulina velutipes and two Volvariellas volvacea strains were isolated and used to the PCR analysis of ITS. Coprinus comatus and Flammulina velutipes can be recognized according to the length of ITS.However, Coprinus comatus and Volvariellas volvacea can not be recognized. In this case, Coprinus comatus and Volvariellas volvacea were analized with ITS-RFLP. The results indicated that the conversation of the sequence of ITS among Coprinus comatus specie was significant, ITS-RFLP can not get to the goal of recognization among Coprinus comatus specie, but it can recognize the spawn between species. The DNAMAN analysis of the sequence of Coprinus comatus in NCBI Combined with the features of restriction enzyme of another 29 spawns, Coprinus comatus can be distinguished from another 29 spawns. Therefore, the identification of the Coprinus comatus in the level of specie with ITS-RFLP can be realized.The individual in the specie should be identified after the Coprinus comatus spawn was made sure of. In this experiment, the analysis of IGS was made of four individuals in the Coprinus comatus specie, the results showed that the features of four related restriction enzyme were completely same, the individual in the specie could not be identified by IGS1-RFLP. The length of IGS2 were different between four Coprinus comatus. IGS2-PCR could identify Co.0001 from another three Coprinus comatus. In this experiment , Co.0058 strain was taken as an example ,the same strain of Co.0058 was copied to two different strains, one was made as standard strain, the other made as tested strain, and made Co.0001 as control strain. Then IGS2-PCR,RAPD-PCR,SRAP-PCR was carried on on the above three strains at the same time. 9 and 8 were selected respectively from 60 RAPD random primers and 80 pairs of SRAP random primers. Within these 18 kinds of primers combinations, the standard strain and tested strain have identical fingerprint,compared with the control strain, 25 bands with good stability and high reproducibility could be amplified.Therefore, when identifying the tested spawn , whichever techniques are carried on , so long as the amplication features are completely same between tested strain and standard strain , and there are enough unique bands between tested strain and control strain , the individual can be made sure of in the corprinus comatus.2.Spawn purityThe normal Coprinus comatus strains without contamination were inoculated with different concentrations of E.coli in order to acquire Coprinus comatus strains with invisible pollution.Foru kinds of methods were carried on the invisible contamination-strains to detect E.coli.The results were as follows:Four kinds of methods all could detect the presence of bacteria.Among them,liquid shake flask method had the highest sensitivity,it can detect the limit of 1000 bacterias per gram mycelium;coated plate method and the boiling PCR method could detect 10000 bacterias per gram mycelium;flat dash method detection limited minimum of 100000 bacterias per gram mycelia.Artifically mixed the normal Coprinus comatus strains with different concentrations of mold spores,the PDA culture method exploited in this experiment could detect the minimum level of 67 spores per gram mycelium.Isolating dsRNA from three Coprinus comatus comatus and digesting the extracts with DNaseâ… and RNaseA could detect the presence of dsRNA virouses,this experiment found that three kinds of Coprinus comatus did not exist dsRNA virous. 3.Spawn activityThe physiological and biochemical factors were studied on three Coprinus comatus strains with different ages.The results indicated that the mycelium growth capacity of all the three Coprinus comatus strains had different degrees of weakening with the aging of strains. Among the activity of six enzymes,the activity of polyphenol oxidase,cellulase and protease had significant correlation with ages.In which, polyphenol oxidase and strains age had significant positive correlation; The cellulase,protease and strain age had negative correlation.The amylase, laccase and TTC-dehydrogenase and strain age had no significant internal relations. Therefore, from mycelium growth capacity, heat capacity, polyphenol oxidase and protease activity could determine the degree of aging in the Coprinus comatus specie.