| Isaria cicadae (IC, hereafters), also named as vegetable cicada or cicada flower, is a valuable cicada-parasitic fungus. Numerous studies confirmed the natural synnemata and cultured mycelia and synnemata containing a variety of active substances, has significant biological activity and pharmacological effects. Compared with natural IC and Cordyceps sinensis, cultured products of IC is more secure than wild products, and from the perspective of wildlife resource preservation and environmental protection, cultured products of IC has great application prospects. However, like other filamentous fungus, colony morphology and growth rate of IC change when a isolate is successively subcultured on artificial media, while the ability to produce synnymata and target bioactive substances decreased, which affects the output and quality of IC products. In this study, the phenomenon of degeneration in IC was concerned. Degeneration mechanisms of the impact on IC will be discussed from variation process of and relationship between the phenotype, metabolism and biological activity in IC.The results of study of biological characteristics of IC in successively subculture on artificial media suggest that periodic variation occurred with the sporulation decreased, growth rate of colony increased at the beginning of subculture and then flowed down after the 8th generation. The 8th and 9th generations were the key stages. Generally there was no rule in varying frequency during successive subculturings, and the variation showed isolate specificity.Based on metabolomic analysis, subculturing process of IC on artificial media divided into four stages:lst-5th generation were Stage I,7th and the 9th generation stage Ⅱ and stage Ⅲ, respectively, and 1 lth-15th generation were stage Ⅳ. Correspondence was built between metabolite difference and phenotypic difference in subcultures to a certain extent. Production of substances associated with the mycelium growth and sporulation capacity of subcultures at stage Ⅱ was significantly higher than at Stage I, such as choline, betaine and phospholipid. Metabolite differences expressed before phenotypic difference. Subculturing of IC on artificial media was under oxidative stress, with the 9th generation as a critical stage, at which the substances associated with oxidization stress were significantly higher in stage I, such as oxylipin, betaine, trehalose, mannitol, citric acid and pyruvic acid et al. Integrated result of studies on phenotypic differences and on metabolomics, the degeneration began from the 5th generation under environmental stress such as oxidization, with the 9th generation as a turning point, from which the ability to produce synnmata decreased significantly.Mycelia produced on solid plate culture and in liquid shake culture, respectively and synnyma produced in wide moth jar culture were analyzed for metabolite difference by metabolomic methods. The results showed that culture condition has significant effects on metabolism of ICly. The relative content of ulvaline, pyruvic acid, N6-(2-hydroxy) adenosine and so on were low in mycelia derived from liquid culture, while betaine, succinic acid, V-PYRRO/NO, and ampullosine were high. In the mycelia derived from solid plate culture, the relative content of lentinamycin, armillarian, beauvericin, trehalose were high. In the synnema derived by wide moth jar culture, the relative cotent of choline, cycloserine, asporyergosterol et al. were at high level while mannitol,17-Hydroxylinolenic acid, and 8-Hydroxy-linoleic acid at low level.The relative cotent of N6-(2-hydroxyethyl) adenosine was high in plate culture and the derived synnemata. It suggests that liquid culture and solid culture are both suitable for producing mannitol; solid culture is more appropriate for trehalose; liquid fermentation is unsuitable for production of antagonism inhibitor N6-(2-hydroxyethyl) adenosine.By using ISSR molecular markers, genetic diversity of subcultures was analyzed. The results showed that the IC isolates from the 15 generations isolate clustered into three groups:the former 5 generations clustered into one group,7th to 9th generation into one group and the latter 5 generations into a group. It shows genomic DNA of IC also changed with the successive subculturing. Variation accumulated gradually with subculturings, to a certain generation qualitative variation occurred. However, there might be a positive restore variation in the process of negative variation. Therefore, the variation of IC is very complex. The finding agreed with that from the former metabolomic analysis to a certain degree:based on genetic similarity coefficient,15 generation isolates of IC clustered into three groups, Generations G1, G3, and G5-generation clustered together, G7 and G9 together as a group, G11, G13 and G15 into one group, which was accordant with the clustering results of metabolomic study. It indicated a possibility of using a simple ISSR analysis to analyze the complex metabolite changes of IC.The results of study on radical scavenging activity of IC indicated that G1 generation had strong free radical scavenging capacity, and free radical scavenging ability of the isolates of different generations also showed a periodic nature:G7 and G9 generations were the turning point. The findings agreed with the results of the studies on phenotype difference, metabolomics analysis and ISSR analysis, further confirmed that generation 7-9 were the key stage in the process of variation and degeneration. Free radical scavenging activity of IC cultures from three different culture methods was different. DPPH free radical scavenging activity of mycelium from solid plate cultur was poor,while · OH free radical activity of the liquid culture mycelium was low, and the two free radical scavenging activities of the cultivated synnemaya were high.Based on the above findings, the subculturings of IC is accompanied by a variety of obvious variation in both morphological and biological characteristics, and in the mean time, metabolites of IC also showed profound changes. Simple ISSR fingerprinting can reflects metabolic changes at a certain degree. Different active substances will produce by different culture methods. Generation 7-9 took an important turning point in the process of variation and degeneration of IC, needing more attention paid to it in artificial culture. |
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