| Panax ginseng, one of the most well-known Chinese herbal medicines, is mainly dependent on artificial cultivation in China. However, ginseng cannot be cultivated on the same plot of land consecutively for several years or even decades due to high requirements for soil quality and strongly discontinuous cultivation. In the past several years, though some researchers have carried out the investigations on mechanisms of discontinuous cultivated, the underlying mechanisms responsible for relationship between microbial diversity and discontinuous cultivation are still poorly understood. Recently, agricultural and chemical methods were used to control the soilborne diseases of ginseng; however, chemical fungicide, using for a long time, not only pollute the environment, but also affect the quality and yield of ginseng because of excessive pesticide residues. In order to reveal the status of soil micro ecological imbalance on the occurrence of ginseng continuous cropping obstacles, the microbial genetic diversity, metabolic function diversity, microbial activities and microbial biomass accumulation of ginseng rhizosphere soil were determined by PCR-DGGE, BIOLOG and culture-depended method, respectively. Meanwhile, a screening program for biocontrol fungi based on the rhizosphere microflora of healthy ginseng plants was evaluated to control ginseng fungal diseases. The stability of the fermented liquid and antagonistic mechanism of biocontrol fungi were investigated, respectively. Meanwhile, experiments under pot conditions or in field were conducted to elucidate the functional mechanisms of biocontrol fungi from view point of colonization, diseases prevention and growth-promoting affects as well as induced resistance, which not only enriched the biocontrol fungal resources of ginseng, but also provided theoretical basis for prevention and cure of ginseng continuous cropping obstacle utilizing bio-control agents. Main results and conclusions are listed as follows:1. The analysis of microbial genetic diversity by PCR-DGGE showed that microbial communities were significantly variable in composition compared with the forest soil uncultured ginseng, of which 6 bacterial phyla and 7 fungal classes were detected in ginseng soil. Among them, Proteobacteria and Hypocreales dominated in ginseng rhizosphere soil. With the increase of cultivation ages, some groups such as actinobacterium, Alcaligenaceae and Comamonadaceae gradually decreased and disappeared, while Acidobacteria and Verrucobacteria were dominant groups in higher cultivation soil and fallow soil of ginseng. Some fungal groups also disappeared because of ginseng cultivation, such as Creolimax fragrantissima from Ichthyophonida, Gliocladium roseum from Bionectriaceae and some protists, whereas the amount of some other species, such as Cryptococcus humicolus and Fusarium oxysporum were increased due to ginseng cultivation. Out-of-balance of micro-ecology in ginseng rhizosphere soil was associated with the changes of microbial community composition.2. Metabolic function diversity of microbe in ginseng rhizosphere soil was characterized by community level physiological profiles(CLPP) using Biolog EcoPlate. It was showed that ginseng cultivation had a remarkable influence on the carbon substrates metabolic activities of soil microbes. The microbial activity in direct-seeding soil was enhanced with the increase of cultivation ages. The increase of cultivation ages resulted in disequilibrium in microbial diversity, while microbial diversity indices increased strikingly in direct-seeding soil but decreased in transplanting soil with the increase of cultivation ages. And ginseng fallow soil abandoned for ten years is helpful to improve the soil microbial metabolic diversity.3. The results of soil microbial activity and biomass showed that ginseng rhizosphere soil microbial activity and biomass accumulation were inhibited observably while the increase of cultivation ages. The soil respiration, soil cellulose decomposition and soil nitrification of ginseng rhizosphere soil microorganism were inhibited significantly, in contrast to the control soil uncultivated ginseng(R0). And the inhibition was gradually increased with the number of growing years or ages of ginseng. The SMB-C and SMB-N in ginseng rhizosphere soil had a decreased tendency with the number of growing years and ages of ginseng.4. 400 kinds of soil fungi were isolated from rhizosphere soil of healthy ginseng by traditional separation named dilution plate method. Among of them, 15 biocontrol fungi, which had broad spectrum antimicrobial effect on 7 kinds of ginseng pathogens, were screened using face to face culture on the plates of PDA. The results of morphological and ITS-rDNA sequence analysis showed that 15 biocontrol fungi were divided into 6 kinds of genus. Among of them, FSR-10, FSR-43 and FSR-106 were identified as Trichoderma atroviride, FSR-38 as Epicoccum nigrum, FSR-46 as T. longibrachiatum, FSR-55 as T. hamatum, FSR-72 as T. citrinoviride, FSR-74 as Chaetomium globosum, FSR-97 as T. polysporum, FSR-91 as Daldinia childiae, as well as FSR-25, FSR-67 and FSR-121 as Penicillium sp..5. The pot experiment results showed that FSR-74 and FSR-97 were successful colonization for 35 d in the soil and ginseng root. FSR-97 was also colonized in ginseng stems with the largest content in 21 d. FSR-74 and FSR-97, which was endogenous, had better potential application.6. By means of mycelial growth cultivating in poisonous PDA medium, the stabilities of fermentation broths from FSR-74 and FSR-97 in different conditions were studied. The results showed that antimicrobial activity of FSR-74 and FSR-97 fermentation broths were stable lower than 40 ℃. Fermentation broth of FSR-74 had a stable activity to UV but it was unstable for fermentation broth of FSR-97. The fermentation broths of FSR-74 and FSR-97 were stable when storing in lower temperature(4 ℃). Meanwhile, the fermentation broths of FSR-74 and FSR-97 were unstable to trypsin K, while the antibacterial activity of fermentation broths had no significant changes when the two strains were transferred for 10 generations. The temperature, illumination, as well as pH must be controlled in a proper range in the prevention and control of plant diseases.7. The objective of this study was to verify the antimicrobial mechanism of both FSR-74 and FSR-97 through determining of mycelial growth rate in PDA amended with the culture filtrate of FSR-74 and FSR-97. The results were showed that competition and mycoparasitism were the important antagonistic mechanism. FSR-74 and FSR-97 were able to inhibit mycelial growth and spores germination through the secretion of antibiotic substance. It was observed that mycelia, with increased of abnormal branching, were twisted and inflated in apex or local, and mycelial protoplasm was condensed. The spores were deformed and germinations rates were decreased.8. The induced ginseng resistance was detected using root-irrigating in pot experiment. The activities of defense enzyme(POD, PAL, PPO, SOD and CAT) were analyzed as the indicators of ginseng resistance to diseases, after inoculated with Cylindrocarpon destructans or/and biocontrol fungi(FSR-74 and FSR-97). The results showed that the individual inoculation of FSR-74 and FSR-97 could increase the POD, PAL, PPO, SOD and CAT enzymes activities of ginseng root, which could improve root physiological function enhancement and increase the induced host plant resistance.9. A pot experiment was conducted to evaluate the control efficiency of FSR-74 and FSR-97 against ginseng fungal diseases caused by Alternaria panax and C.destructans. The results showed that the control efficiency against A. panax and C. destructans of FSR-74 were 62.29% and 57.99% separately, which was not significant compared with pesticides(carbendazim). The control efficiency of FSR-97 against Alternaria panax was 73.06%, which was higher than carbendazim treatment. A field experiment was used to assess the control efficiency of FSR-74 and FSR-97 against Phytophthora cactorum. The results showed that the control efficiency against P. cactorum of FSR-74 and FSR-97 were 55.63% and 74.43%, respectively. |
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