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Scientia Silvae Sinicae ›› 2015, Vol. 51 ›› Issue (4): 116-125.doi: 10.11707/j.1001-7488.20150415

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Isolation and Identification of Bio-control Microorganisms in Soil to Poplar Canker

Yang Lei2,3, Liang Jun1,3, Zhou Guoying2, Ni Yang3, Lü Quan3, Zhang Xingyao1,3   

  1. 1. Southern modern forestry cooperative innovation center, Nanjing Forestry University Nanjing 210037;
    2. College of Forestry, Central South University of Forestry & Technology Changsha 410004;
    3. The Key Laboratory of Forest Protection of China State Forestry Administration, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry Beijing 100091, China
  • Received:2013-04-09 Revised:2015-01-12 Online:2015-04-25 Published:2015-05-20

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Abstract:

【Objective】 Poplar canker is one of the major biological disasters of poplar plantations in China. The biological control method for regulating poplar canker is sustained and effective. The aim of this study is to find and isolate antagonistic microorganisms which have good bio-control effect on poplar canker from the poplar forest soil. 【Method】 In this study, poplar canker pathogen Botryosphaeria dothidia was used as the target strain, and the bio-control microorganisms in the soil were isolated, screened and identified. The dilution plate coating method was used to separate soil microorganisms. Three procedures were applied to screen the antagonistic microbes, including primary screening, fermentation liquor secondary screening and the inhibitory action in vitro. The dual culture method was used in primary screening, the "sandwich method" and the "improved agar diffusion method" were combined in fermentation liquor secondary screening, and the objective bio-control strain was used ultimately to determine the control effect on poplar canker in vitro tissue. The bio-control strain was identified by combining morphological observation and the the molecular biology method. 【Result】 Totally 48 soil samples were collected in upper, middle and deeper soil layers of poplar forest in Langfang Hebei and Yuanyang Henan. A total of 259 strains were isolated in the 48 soil samples, including 122 bacteria strains, 106 actinomycetes strains and 31 fungi strains. The microbial distribution pattern overall followed as: top layer soil> middle layer soil > lower layer soil. There were 8 bacteria and actinomycetes strains, screened in primary screening, which had antibiological average diameters > 4 mm, and there were 8 fungi strains, selected by dual culture, which had colony diameter > 40 mm. Eight antagonistic microorganisms strains were selected by fermentation medium secondary screening. They were bacteria strains of TYZ1B3 and YX5B1, and fungi strains of LS10F1, LX5F1, LZ10F1, LS6F1, LX6F2 and TLZ2F2, respectively. Through determination of the control effect in vitro tissue, a bio-control fungus strain, LX6F2, was obtained, and the control effect of the strain could be up to 76.04%. The LX6F2 feature, including the colonies, hyphae and spores, was matched with Fusarium sp. by morphological observation. Through rDNA ITS sequence and phylogenetic analysis, the LX6F2 sequence length was 563 bp, and the sequence registration number was FR872729.1. There was 100% similarity between LX6F2 rDNA ITS sequence and the Fusarium sp strain's sequence which number is JN038467 in GenBank. Based on these results, the strain LX6F2 was identified as Fusarium equiseti. 【Conclusion】 The discovery of LX6F2 provides a new raw material for biological control of poplar canker. It's important for sustainable control of poplar canker. In a follow-up study its antibacterial mechanism, effective antagonist compositions, bacterium agent and so on will be investigated.

Key words: soil antagonistic microorganisms, poplar canker, Fusarium equiseti, antagonistic activity, bio-control strain

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