Isolation and Identification of a Cellulase-producing Endophytic Fungus in Paris polyphylla var. yunnanensis

doi:10.13560/j.cnki.biotech.bull.1985.2021-0266

Abstract

Abstract:

This paper aims to isolate and identify an endophytic fungus with cellulase activity in Paris polyphylla var. yunnanensis. Surface disinfection method was applied to isolate the endophytic fungi from P. polyphylla var. yunnanensis rhizome. CMC plate assay was used to detect the cellulase activity of the isolated strains. Morphological features and sequence characters based techniques were applied to identify the strains producing high-yield cellulase. The factors affecting the cellulase activity were also explored. Finally,plate activity screen method was used to test the activity of other extracellular hydrolases production. A total of 41 fungal endophytes were isolated from 3 origins of P. polyphylla var. yunnanensis. Among the 41 isolates,strains named as AS-5,AS-7,AS-9 and AS-18 were found to be able to produce cellulase,and strain AS-9 showed the strongest cellulase production activity. AS-9 was identified as Setophoma terrestris based on the morphological observations and ITS,LSU sequences analysis. AS-9 presented maximal cellulase activity at pH7.0 and 28℃. UV exposure did not have a significant effect on cellulase production of AS9. Meanwhile,S. terrestris AS-9 also demonstrated the activities of producing caseinase,lipase,asparaginase,glutaminase and urease production. To the best of our knowledge,S. terrestris AS-9 is first detected as an endophytic fungus in P. polyphylla var. yunnanensis and shows a better cellulase production capacity. It is worth for further study.

Key words: cellulase, Paris polyphylla var. yunnanensis, isolation and identification, Setophoma terrestris, extracellular hydrolytic enzyme

ZHANG Gong-you, WANG Yi-han, GUO Min, ZHANG Ting-ting, WANG Bing, LIU Hong-mei. Isolation and Identification of a Cellulase-producing Endophytic Fungus in Paris polyphylla var. yunnanensis[J]. Biotechnology Bulletin, 2022, 38(2): 95-104.

Figures/Tables 10

Table 1 Isolation of fungal endophytes from P. polyphylla var. yunnanensis

位置Location	菌株编号Strain No.	来源Source	数量Number
105°58'13.22″E 26°02'22.85″N	AS-1,AS-2,AS-3,AS-4,AS-5,AS-7,AS-8,AS-9,AS-12,AS-13,AS-14,AS-15,AS-16,AS-17,AS-18,AS-19,AS-20	块茎Tuber	17
107°01'10.49″E 28°14'28.36″N	ZY-1,ZY-2,ZY-5,ZY-7,ZY-16	块茎Tuber	5
105°05'37.79″E 24°29'27.58″N	GX-E,GX-F,GX-G,GX-H,GX-I,GX-J,GX-K,GX-L,GX-M,GX-N,GX-O,GX-P,GX-Q,GX-R,GX-S,GX-T,GX-U,GX-V,GX-W	块茎Tuber	19

Fig. 1 Screening of cellulase-producing strains

Table 2 Size of transparent circle produced by cellulase-producing endophytic fungus

菌株编号 Strain No.	透明圈直径Transparent circle diameter D/cm	菌落直径 Colony diameter d/cm	D/d
Penicillium	1.8	1.2	1.64
AS-5	1.5	0.7	2.14
AS-7	0.8	0.55	1.45
AS-9	1.7	0.75	2.27
AS-18	1.5	1.0	1.5

Fig. 2 Morphology of AS-9 strain A：The colony surface. B：The colony dorsal surface. C-F：Fungal hyphae were observed using the optical microscope. G：Conidia. H,I：Fungal hyphae were observed using scanning electron microscopy

Fig. 3 Results of ITS and LSU PCR amplification

Fig. 4 Phylogenetic trees based on the ITS and LSU gene sequences A：Phylogenetic tree based on the ITS sequence. B：Phylogenetic tree based on the LSU sequence

Fig. 5 Effects of different pH,temperature and UV on cellulase activity A：pH. B：Temperature. C：UV irradiation

Table 3 Size of transparent circle produced by AS-9 strain under different culture conditions

培养条件Culture condition	透明圈直径 Transparent circle diameter D/cm	菌落直径 Colony diameter d/cm	D/d
pH4.0	1.5	0.95	1.58
pH5.0	1.5	1.05	1.43
pH6.0	1.4	0.85	1.65
pH7.0	1.9	0.75	2.53
pH8.0	1.3	1.05	1.24
25℃	1.7	0.75	2.27
28℃	2.0	0.8	2.5
32℃	1.3	0.55	2.36
28℃（无紫外）	1.7	0.65	2.61
28℃（紫外）	1.25	0.45	2.78

Fig. 6 Standard curve of cellulase activity

Fig. 7 Detection of extracellular hydrolytic enzyme activity A：Caseinase. B：Lipase. C：L-asparaginase. D：Glutaminase. E：Urease. F：Gelatinase. G：Phytase. H：Pectinase. I：Amylase. J：Ligninase

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