Identification of Different Armillaria spp. Strains and Extracellular Enzyme Activity

doi:10.11924/j.issn.1000-6850.casb20191000716

Abstract

Abstract:

The aim is to identify different Armillaria spp. strains associated with cultivated Gastrodia elata, and to provide a basis for screening the excellent strains by comparing the changes of extracellular enzyme activity and polysaccharide content. In this study, M1, M2, F2, and F3 strains were used as test materials, and the phylogenetic tree was constructed by the combined sequences of ITS, β-tubulin and Tef1-α gene fragments. The extracellular enzyme activity was determined by the ABTS and DNS method. The mycelial polysaccharide content was determined by the phenol-sulfuric acid method. According to phylogenetic analysis, the M1 and M2 strains isolated from the Yunnan G. elata base were Armillaria gallica Marxm. & Romagn. The F2 and F3 strains isolated from the Jilin G. elata base were Armillaria cepistipes Velen. The growth rate comparison showed that the growth rates of M1, M2, and F2 were relatively fast and that of F3 was the slowest. The results showed that the extracellular enzyme activity and polysaccharides content of the four Armillaria spp. strains increased at the beginning and then decreased. The M1 and M2 strains of Armillaria gallica had relatively high enzyme activities and polysaccharides content. Based on the comprehensive analysis, the four strains were identified as Armillaria gallica and Armillaria cepistipes, respectively. Combining with the previous G. elata production results, it could be preliminarily confirmed that M1 and M2 strains are excellent strains for cultivating G. elata.

Key words: Armillaria spp., molecular identification, extracellular enzyme, polysaccharide

CLC Number:

Liu Xiaomin, Chen Xiangdong, Zhang Weiwei, Wang Zhongqiao, Song Minghai, Lan Jin, Ma Lin. Identification of Different Armillaria spp. Strains and Extracellular Enzyme Activity[J]. Chinese Agricultural Science Bulletin, 2020, 36(24): 99-106.

Figures/Tables 10

References 25

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扩增对象	引物名称	引物序列(5′-3′)
rDNA	ITS-1	5′-TCCGTAGGTGAACCTGCGG-3′
rDNA	ITS-4	5′-TCCTCCGCTTATTGATATGC-3′
β-tubulin	TUB F	5′-GGTGCGGG TAACTGGGC-3′
β-tubulin	TUB R	5′-GAGGCAGCCATCATGTTCTT-3′
Tef1-α	EF526F	5′GTCGTYGTYATYGGHCAYGT- 3′
	EF1567R	5′ACHGTRCCRATACCACCRATCTT- 3′
	EF595F	5′CGTGACTTCATCAAGAACATG- 3′
	EF1160R	5′CCGATCTTGTAGACGTCCTG- 3′
阳性克隆菌液	M13F	5′-TGTAAAACGACGGCCAGT-3＇
阳性克隆菌液	M13R	5′-CAGGAAACAGCTATGACC-3′

扩增对象	引物名称	引物序列(5′-3′)
rDNA	ITS-1	5′-TCCGTAGGTGAACCTGCGG-3′
rDNA	ITS-4	5′-TCCTCCGCTTATTGATATGC-3′
β-tubulin	TUB F	5′-GGTGCGGG TAACTGGGC-3′
β-tubulin	TUB R	5′-GAGGCAGCCATCATGTTCTT-3′
Tef1-α	EF526F	5′GTCGTYGTYATYGGHCAYGT- 3′
	EF1567R	5′ACHGTRCCRATACCACCRATCTT- 3′
	EF595F	5′CGTGACTTCATCAAGAACATG- 3′
	EF1160R	5′CCGATCTTGTAGACGTCCTG- 3′
阳性克隆菌液	M13F	5′-TGTAAAACGACGGCCAGT-3＇
阳性克隆菌液	M13R	5′-CAGGAAACAGCTATGACC-3′

时间	生长速度/(cm/d)
时间	M1	M2	F2	F3
4	0.202±0.061bc	0.349±0.021a	0.254±0.014b	0.169±0.009c
8	0.738±0.059b	0.821±0.070a	0.703±0.072b	0.499±0.062c
12	0.810±0.008a	0.792±0.006a	0.752±0.078a	0.591±0.035b
16				0.534±0.011
20				0.487±0.011

时间	生长速度/(cm/d)
时间	M1	M2	F2	F3
4	0.202±0.061bc	0.349±0.021a	0.254±0.014b	0.169±0.009c
8	0.738±0.059b	0.821±0.070a	0.703±0.072b	0.499±0.062c
12	0.810±0.008a	0.792±0.006a	0.752±0.078a	0.591±0.035b
16				0.534±0.011
20				0.487±0.011

胞外酶	生长速度			多糖
胞外酶	菌株	r值	P值	菌株	r值	P值
漆酶	M1、M2、F2、F3	0.854	<0.01	M1、M2	0.811	<0.01
纤维素酶	M1、M2、F2、F3	0.785	<0.01	M1、M2、F2、F3	0.758	<0.01
淀粉酶	M1	0.796	<0.01	M1	0.961	<0.01
木聚糖酶	M1、M2、F2、F3	0.890	<0.01	M1、M2、F3	0.874	<0.01
果胶酶	M1、M2、F2、F3	0.918	<0.01	M1	0.784	<0.01