百香果内生细菌多样性及促生长特性

doi:10.13560/j.cnki.biotech.bull.1985.2021-0144

摘要/Abstract

摘要：

内生菌可为植物提供营养成分,也可以通过代谢产物促进植物生长,目前很少出现关于百香果（Passiflora edulia Sims）内生菌的研究。百香果是西番莲科西番莲属的一种草质藤本植物,主要生长于亚热带与热带地区,对其内生菌进行分离纯化,依据插入序列指纹图谱（IS-PCR）结果对所得菌株聚类,经16S rRNA基因测序并进行系统发育分析,之后对聚类得到的各类群代表菌株进行固氮酶活性、产生长素能力、溶磷能力、解钾能力、产蛋白酶能力与生理生化特性试验。共分离纯化得到51株百香果内生细菌,经聚类分为12个类群,分别属于Bacillus altitudinis、Bacillus circulans、Lysinibacillus macroides、Brevibacillus antibioticus、Paenibacillus illinoisensis、Microbacterium zeae、Rhizobium pusense、Beijerinckia fluminensis、Achromobacter mucicolens、Stenotrophomonas maltophilia、Klebsiella michiganensis和Klebsiella pneumoniae,对分离得到的内生菌进行促生特性试验,结果表明分离所得的百香果内生菌各类群代表菌株具有丰富的功能：其中10株具有分泌生长素的能力,5株具有固氮功能,6株具溶磷功能,7株具有解钾能力,3株具有分泌铁载体功能,5株具有产蛋白酶能力。本研究表明,从百香果中分离得到的内生菌不但具有种群多样性,而且具有功能多样性,这些具有不同特性的菌株有望应用在农业生产中以发挥其价值。

关键词: 百香果, 内生菌, 固氮能力, 产生长素, 促生特性

Abstract:

Endophytes can provide nutrients for plants and also promote plant growth through metabolites. Currently,there are few studies on endophytes in Passiflora edulia Sims. P. edulia Sims is a herbaceous vine of the Passiflora genus of the Passiflora family and mainly distributed in tropical and subtropical regions. By isolating and purifying the endophytes of P. edulia Sims,the obtained strains were clustered according to the IS-PCR results,the 16S rRNA genes of the representative strains were sequenced and phylogenetic analysis was performed,and their nitrogenase activity,auxin-producing capacity,phosphorus-dissolving ability,potassium-dissolving ability,protease production ability and physiological and biochemical characteristics were tested. The isolated and purified 51 strains of P. edulia Sims endophytic bacteria,clustered into 12 groups,belonging to Bacillus altitudinis,Bacillus circulans,Lysinibacillus macrolides,Brevibacillus antibioticus,Paenibacillus illinoisensis,Microbacterium zeae,Rhizobium pusense,Beijerinckia fluminensis,Achromobacter mucicolens,Stenotrophomonas maltophilia,Klebsiella michiganensis and Klebsiella pneumonia,the growth-promoting characteristics of the isolated endophytes were determined,and the results showed that the isolated representative endophytes had abundant functions,10 of them had the ability of secreting auxin,5 of them had nitrogen-fixing function,6 of them had phosphorus-dissolving function,7 of them had potassium-dissolving ability,3 of them had the ability of secreting siderophores,and 5 of them had the ability of producing protease. This experiment shows that the endophytes isolated from P. edulia Sims have both population diversity and functional diversity. These strains with different characteristics are expected to be utilized in agricultural production to exert their values.

Key words: Passiflora edulia Sims, endophytic bacteria, nitrogen fixation, auxin production, growth-promoting effect

唐嘉城, 梁毅珉, 马葭思, 彭桂香, 谭志远. 百香果内生细菌多样性及促生长特性[J]. 生物技术通报, 2022, 38(1): 86-97.

TANG Jia-cheng, LIANG Yi-min, MA Jia-si, PENG Gui-xiang, TAN Zhi-yuan. Diversity and Growth Promotion of Endophytic Bacteria Isolated from Passiflora edulia Sims[J]. Biotechnology Bulletin, 2022, 38(1): 86-97.

图/表 9

参考文献 37

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类群 Group	代表菌株（GenBank登录号） Representative strain（GenBank accession number）	最相似菌株名称（GenBank登录号） Closely related strain（GenBank accession number）	相似性 Similarity/%
Ⅰ	BXG95（MW714902）	Bacillus altitudinis 41KF2b^T（NR_042337）	98.20
Ⅱ	BXG129（MW714912）	Bacillus circulans strain NBRC 13626^T（NR_112632）	97.99
Ⅲ	BXG111（MW714903）	Lysinibacillus macroides strain LMG 18474^T（NR_114920）	98.42
Ⅳ	BXG92（MW714901）	Brevibacillus antibioticus strain TGS2-1^T（NR_165725）	98.70
Ⅴ	BXY92（MW714907）	Paenibacillus illinoisensis strain NBRC 15959^T（NR_113828）	97.49
Ⅵ	BXJ201（MW714905）	Microbacterium zeae strain 1204^T（NR_149816）	97.61
Ⅶ	BXG81（MW714904）	Rhizobium pusense strain NRCPB10^T（NR_116874）	99.00
Ⅷ	BXG212（MW714911）	Beijerinckia fluminensis strain UQM 1685^T（NR_116306）	98.57
Ⅸ	BXG101（MW714910）	Achromobacter mucicolens strain R-46658^T（NR_117613）	98.13
Ⅹ	BXG201（MW714909）	Stenotrophomonas maltophilia strain ATCC 19861^T（NR_040804）	98.29
Ⅺ	BXJ71（MW714906）	Klebsiella michiganensis strain W14^T（NR_118335）	97.13
Ⅻ	BXG53（MW714908）	Klebsiella pneumoniae strain DSM 30104^T（NR_117683）	98.62

类群 Group	代表菌株（GenBank登录号） Representative strain（GenBank accession number）	最相似菌株名称（GenBank登录号） Closely related strain（GenBank accession number）	相似性 Similarity/%
Ⅰ	BXG95（MW714902）	Bacillus altitudinis 41KF2b^T（NR_042337）	98.20
Ⅱ	BXG129（MW714912）	Bacillus circulans strain NBRC 13626^T（NR_112632）	97.99
Ⅲ	BXG111（MW714903）	Lysinibacillus macroides strain LMG 18474^T（NR_114920）	98.42
Ⅳ	BXG92（MW714901）	Brevibacillus antibioticus strain TGS2-1^T（NR_165725）	98.70
Ⅴ	BXY92（MW714907）	Paenibacillus illinoisensis strain NBRC 15959^T（NR_113828）	97.49
Ⅵ	BXJ201（MW714905）	Microbacterium zeae strain 1204^T（NR_149816）	97.61
Ⅶ	BXG81（MW714904）	Rhizobium pusense strain NRCPB10^T（NR_116874）	99.00
Ⅷ	BXG212（MW714911）	Beijerinckia fluminensis strain UQM 1685^T（NR_116306）	98.57
Ⅸ	BXG101（MW714910）	Achromobacter mucicolens strain R-46658^T（NR_117613）	98.13
Ⅹ	BXG201（MW714909）	Stenotrophomonas maltophilia strain ATCC 19861^T（NR_040804）	98.29
Ⅺ	BXJ71（MW714906）	Klebsiella michiganensis strain W14^T（NR_118335）	97.13
Ⅻ	BXG53（MW714908）	Klebsiella pneumoniae strain DSM 30104^T（NR_117683）	98.62

特性 Property	BXG95	BXG129	BXG111	BXG92	BXY92	BXJ201	BXG81	BXG212	BXG101	BXG201	BXJ71	BXG53
革兰氏染色 Gram stain	+	+	-	+	+	+	-	-	-	-	-	-
过氧化氢酶 Catalase	+	+	+	+	+	+	+	+	+	+	+	+
甲基红 Methyl red	+	+	-	+	+	+	+	+	-	-	-	+
产氨 Ammonia production	-	+	-	+	-	+	+	+	+	+	+	+
脲酶 Urease	-	+	-	-	-	+	+	+	+	+	-	+
乙酰甲基甲醇实验 V.P. test	-	-	+	-	-	-	-	-	+	+	+	-
NO₃^-还原测定 Nitrate reduction	-	+	-	+	-	-	+	-	+	+	+	+
明胶液化 Gelatin hydrolysis	+	+	-	-	+	-	-	-	-	+	+	-
固氮酶活性 Nitrogenase activity /（nmol C₂H₄/（mL·h））	0	0	0	0	0	0	23.97±0.43	100.94±0.89	0	21.11±0.66	477.93±3.85	191.33±1.27

特性 Property	BXG95	BXG129	BXG111	BXG92	BXY92	BXJ201	BXG81	BXG212	BXG101	BXG201	BXJ71	BXG53
革兰氏染色 Gram stain	+	+	-	+	+	+	-	-	-	-	-	-
过氧化氢酶 Catalase	+	+	+	+	+	+	+	+	+	+	+	+
甲基红 Methyl red	+	+	-	+	+	+	+	+	-	-	-	+
产氨 Ammonia production	-	+	-	+	-	+	+	+	+	+	+	+
脲酶 Urease	-	+	-	-	-	+	+	+	+	+	-	+
乙酰甲基甲醇实验 V.P. test	-	-	+	-	-	-	-	-	+	+	+	-
NO₃^-还原测定 Nitrate reduction	-	+	-	+	-	-	+	-	+	+	+	+
明胶液化 Gelatin hydrolysis	+	+	-	-	+	-	-	-	-	+	+	-
固氮酶活性 Nitrogenase activity /（nmol C₂H₄/（mL·h））	0	0	0	0	0	0	23.97±0.43	100.94±0.89	0	21.11±0.66	477.93±3.85	191.33±1.27

特性Characteristics	BXG95	BXG129	BXG111	BXG92	BXY92	BXJ201	BXG81	BXG212	BXG101	BXG201	BXJ71	BXG53
可溶性磷含量 Soluble P content/（mg·L^-1）	-	-	9.75	-	12.46	-	-	12.93	13.64	-	12.38	23.47
可溶性钾含量 Soluble K content/（mg·L^-1）	-	22.4	-	-	45.2	42.7	29	23.6	-	-	41	65.6
生长素 IAA/（mg·L^-1）	24.32	28.71	-	3.36	12.38	-	7.55	8.91	20.97	11.46	14.85	17.13
产铁载体 Siderophore production	-	-	-	-	-	-	+	-	-	-	+	+
产蛋白酶 Proteinase production	3.61	1.97	-	-	2.68	-	-	-	-	3.60	2.36	-