森林根际土壤细菌的分离、鉴定及生物活性筛选

doi:10.13560/j.cnki.biotech.bull.1985.2023-0586

生物技术通报 ›› 2024, Vol. 40 ›› Issue (1): 294-307.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0586

森林根际土壤细菌的分离、鉴定及生物活性筛选

冯路遥¹^,²(), 赵江源³, 施竹凤², 莫艳芳²^,⁴, 杨童雨²^,⁴, 申云鑫²^,⁴, 何飞飞¹, 李铭刚³, 杨佩文²()

1.云南大学资源植物研究院，昆明 650504
2.云南省农业科学院农业环境资源研究所，昆明 650205
3.云南大学云南省微生物研究所，昆明 650504
4.云南农业大学植物保护学院，昆明 650201

收稿日期:2023-06-20 出版日期:2024-01-26 发布日期:2024-02-06
通讯作者: 杨佩文，男，博士，研究员，研究方向：植物病害生物防治；E-mail: peiwenyang@yaas.org.cn
作者简介:冯路遥，女，硕士研究生，研究方向：微生物资源的开发与利用；E-mail: huan2004212@163.com
基金资助:
云南省科技计划重大科技专项(202202AE090015);云南省科技计划重大科技专项(202202AE090010);云南省专家基层科研工作站-杨佩文专家工作站（嵩明县）(云人社通「2022」17 号);云南省专家基层科研工作站-杨佩文专家工作站（陆良县）(云人社通「2022」17 号)

Isolation and Identification of Bacteria in Forest Rhizosphere Soil and Their Biological Activity Screening

FENG Lu-yao¹^,²(), ZHAO Jiang-yuan³, SHI Zhu-feng², MO Yan-fang²^,⁴, YANG Tong-yu²^,⁴, SHEN Yun-xin²^,⁴, HE Fei-fei¹, LI Ming-gang³, YANG Pei-wen²()

1. Resource Plant Research Institute, Yunnan University, Kunming 650504
2. Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences, Kunming 650205
3. Yunnan Institute of Microbiology, Yunnan University, Kunming 650504
4. College of Plant Protection, Yunnan Agricultural University, Kunming 650201

Received:2023-06-20 Published:2024-01-26 Online:2024-02-06

摘要/Abstract

摘要：

【目的】从无量山国家级自然保护区森林根际土壤发掘具有多种生物活性的功能菌株，探究其开发应用潜力。【方法】采集无量山地区25个区域植物的根际土壤，采用选择培养基，分离鉴定磷酸盐溶解、固氮、溶锌和拮抗等活性菌株，进一步测定菌株分泌铁载体、ACC脱氨酶和吲哚乙酸等生物活性，并验证促番茄种子发芽和生长效果。【结果】分离鉴定得到解磷菌70株，固氮菌27株，解钾菌8株，拮抗镰刀菌的菌株51株。其中，YIM B08401和YIM B08402形态学结合生理生化特性和16S rDNA序列测序，鉴定为白色伯克霍尔德氏菌（Burkholderia alba）和青岛假单胞菌（Pseudomonas qingdaonensis），两个菌株均具有磷酸盐溶解、固氮、溶锌和分泌铁载体的活性，最大可溶性磷含量为（455.63±59.65）mg/L和（878.95±64.78）mg/L；两株菌的促种子发芽试验结果接近，施加稀释10倍、10²倍和10³倍的发酵上清液后，发芽率都维持在82%-93%，明显高于对照组的56%和49%，施加菌株发酵液的处理组相较于空白对照组的长度都有显著增加。盆栽实验证明，两株菌株促生效果最明显的处理组在地上部长度、鲜重、干重、茎粗、根长、根鲜重、根干重方面的数据都显著优于对照组，YIM B08401的上述指标相对于对照组分别显著增加了89%、495%、268%、62%、53%、385%和469%，YIM B08402的上述指标相对于对照组分别显著增加了118%、528%、477%、55%、37%、413%和747%。此外，菌株YIM B08401还具有拮抗病原菌和分泌ACC脱氨酶活性，YIM B08402则还具有分泌吲哚乙酸的活性。【结论】无量山森林根际土壤蕴含具有多种生物活性的功能菌株，白色伯克霍尔德氏菌（B. alba, YIM B08401）和青岛假单胞菌（P. qingdaonensis, YIM B08402）具备开发为新型微生物肥料的应用潜力。

关键词: 无量山森林根际土壤, 可培养微生物, 白色伯克霍尔德氏菌, 青岛假单胞菌, 养分转化, 拮抗活性, 生物肥料

Abstract:

【Objective】Functional strains with multiple biological activities were excavated from the forest rhizosphere soil of Wuliangshan National Nature Reserve, and their potential for development and application was explored. 【Method】The 25 rhizosphere soil samples from different regions and plants in Wuliangshan National Nature Reserve were collected. The active strains with functions such as phosphate dissolution, nitrogen fixation, zinc dissolution, and antibacterial activity were isolated and identified using selective culture medium. The biological activities of these strains, such as secreted siderophore, ACC deaminase, and indoleacetic acid, were tested while verifying their effectiveness in promoting tomato seed germination and plant growth. 【Result】70 phosphorus-dissolving bacteria, 27 nitrogen-fixing bacteria, 8 potassium-dissolving bacteria, and 51 antibacterial bacteria were isolated and identified. Among them, YIM B08401 and YIM B08402 were identified as Burkholderia alba and Pseudomonas qingdaonensis based on morphological, physiological, biochemical characteristics, and 16S rDNA sequencing. The two strains both had the activities of phosphate lysis, nitrogen fixation, zinc lysis and siderophore secretion. Their maximum soluble phosphorus content was（455.63±59.65）mg/L and（878.95±64.78）mg/L. The results of seed germination promotion tests of the two strains were similar, and the germination rates of the two strains were maintained at 82%-93% after applying the fermentation supernatant diluted 10 times, 10² times and 10³ times, which was significantly higher than 56% and 49% in the control group. The length of the treatment group with the fermentation liquid of the strain significantly increased compared with the blank control group. Pot experiment showed that the above ground length, fresh weight, dry weight, stem diameter, root length, fresh weight and dry weight of the two strains in the treatment group with the most obvious growth promotion effect were significantly better than that in the control group. Compared with the control group, the above indexes of YIM B08401 significantly increased by 89%, 495%, 268%, 62%, 53%, 385% and 469%, and the above indexes of YIM B08402 significantly increased by 118%, 528% and 477%, 55%, 37%, 413% and 747%, respectively. In addition, strain YIM B08401 also had activities of antagonizing pathogenic bacteria and secreting ACC deaminase, and YIM B08402 also had the activity of secreting indoleacetic acid. 【Conclusion】The rhizosphere soil in the forest of Wuliang National Nature Reserves contained a variety of strains with biological activity. B. alba YIM B08401 and P. qingdaonensis YIM B08402 demonstrates the potential for the development and application of new microbial fertilizers.

Key words: Wuliangshan forest rhizosphere soil, culturable microorganism, Burkholderia alba, Pseudomonas qingdaonensis, nutrient transformation, antibacterial activity, biological fertilizer

冯路遥, 赵江源, 施竹凤, 莫艳芳, 杨童雨, 申云鑫, 何飞飞, 李铭刚, 杨佩文. 森林根际土壤细菌的分离、鉴定及生物活性筛选[J]. 生物技术通报, 2024, 40(1): 294-307.

FENG Lu-yao, ZHAO Jiang-yuan, SHI Zhu-feng, MO Yan-fang, YANG Tong-yu, SHEN Yun-xin, HE Fei-fei, LI Ming-gang, YANG Pei-wen. Isolation and Identification of Bacteria in Forest Rhizosphere Soil and Their Biological Activity Screening[J]. Biotechnology Bulletin, 2024, 40(1): 294-307.

图/表 16

图1 无量山根际土壤可培养微生物门水平（A）和属水平（B）分布特征

Fig. 1 Distribution characteristics of cultivable microbial phylum level（A）and genus level（B）of Wuliangshan mountain rhizosphere soil

图2 无量山根际土壤中32属代表菌株基于16S rDNA基因构建的邻接法系统发育树

Fig. 2 Phylogenetic tree of 32 genera representative strains in the Wuliangshan rhizosphere soil, constructed by neighbor-joining tree based on 16S rDNA gene

表1 部分活性菌株促生特性

Table 1 Growth-promoting and disease-resistant properties of some active strains

菌株 Strain	种属名 Top-hit taxon	溶磷 Phosphorus solution	固氮 Nitrogen fixation	解钾 Potassium solution	拮抗镰刀菌 Antagonistic fusarium
YIM B08401	B. alba	+	+	-	+
YIM B08402	P. qingdaonensis	+	+	-	-
82-a15	P. costantinii	+	+	-	-
61-b2	P. juntendi	+	+	-	-
66-3	P. jessenii	+	+	-	-
60-a4	B. altitudinis	+	-	-	-
62-a15	P. peoriae	+	-	-	-
66-a4	P. costantinii	+	+	-	-
82-a5	P. granadensis	+	+	-	-
66-a3	P. costantini	+	+	-	-
60-a14	B. cereus	+	-	-	-
60-a6	B. cereus	+	+	-	-
84-a4	P. monteilii	+	+	-	-
60-a1	P. aryabhattai	+	+	+	-
66-a9	B. amyloliquefaciens	+	-	-	+
74-4	P. borealis	+	-	-	+
82-a14	B. siamensis	+	-	-	+
82-a16	B. siamensis	+	-	-	+
83-a6	P. umsongensis	-	+	-	-
84-a16	B. subtilis	-	-	-	+

图3 菌株YIM B08401（A）和菌株YIM B08402（B）基于16S rDNA基因构建的邻接法系统发育树

Fig. 3 Neighbor-joining trees of the strain YIM B08401（A）and YIM B08402（B）based on 16S rDNA gene

图4 菌株YIM B08401的溶磷（A）、解磷（B）、固氮（C）、溶锌（D）和YIM B08402的溶磷（E）、解磷（F）、固氮（G）、溶锌（H）效果图

Fig. 4 Effect diagram of dissolved phosphorus（A）, phosphorus solution（B）, nitrogen fixation（C）, zinc dissolution（D）of YIM B08401 and phosphorus dissolution（E）, phosphorus solution（F）, nitrogen fixation（G）, and zinc dissolution（H）of YIM B08402

表2 菌株的营养转化指数

Table 2 Nutrient transformation indexes of the strain

D/d	溶磷指数Phosphorus dissolution	解磷指数Phosphorus solution	固氮指数Nitrogen fixation	溶锌指数Zinc dissolution
YIM B08401	1.207±0.01b	2.731±0.442a	4.519±0.403a	2.41±0.216a
YIM B08402	2.316±0.159a	2.730±0.423a	2.595±0.884b	2.71±0.375a

图5 菌株YIM B08401（A）和YIM B08402（B）发酵液可溶性磷含量及pH变化

Fig. 5 Soluble phosphorus content and pH in the fermentation broth of the strain YIM B08401（A）and YIM B08402（B）

图6 菌株YIM B08401（A）和YIM B08402（B）的分泌铁载体效果图

Fig. 6 Secretory siderophores of strain YIM B08401（A）and YIM B08402（B）

图7 菌株YIM B08402的分泌生长素活性测定左侧为空白对照，右侧为菌株YIM B08402的显色结果

Fig. 7 Determination of IAA activity of strain YIM B08402 On the left it is a blank control, and on the right it is the color rendering result of strain YIM B08402

图8 菌株YIM B08401与具有ACC脱氨酶的菌株蛋白质序列的多序列比对

Fig. 8 Multiple sequence alignment of proteins in strain YIM B08401 and strain with ACC deaminase

图9 菌株YIM B08401对烟草疫霉（A）、茎点霉（B）、木贼镰刀菌（C）的拮抗效果

Fig. 9 Antagonistic effects of strain YIM B08401 against Phytophthora parasitica var. nicotianae（A）, Phoma matteuciicola（B）and Fusarium equiseti（C）

图10 菌株YIM B08401（A）和YIM B08402（B）的各处理番茄苗

Fig. 10 Tomato seedlings under each treatment by strain YIM B08401（A）and YIM B08402（B）

图11 菌株YIM B08401（A、B）和YIM B08402（C、D）的各处理番茄种子发芽长度和发芽率

Fig. 11 Germination lengths and germination rates of tomato seeds of strain YIM B08401（A, B）and YIM B08402（C, D）

图12 菌株YIM B08401（A）和YIM B08402（B）的各处理番茄植株

Fig. 12 Tomato plants treated with strain YIM B08401（A）and YIM B08402（B）

图13 菌株YIM B08401和YIM B08402各处理对番茄植株地上部鲜重（A）、地上部干重（B）、地上部长度（C）、根鲜重（D）、根干重（E）、根长（F）、茎粗（G）的影响

Fig. 13 Effects on the fresh weight of above ground part（A）, dry weight of above ground part（B）, length of above ground part（C）, fresh weight of root（D）, dry weight of root（E）, root length（F）, stem diameter（G）of tomato plants under each treatment of strain YIM B08401 and YIM B08402

表3 番茄根际土壤理化指标

Table 3 Physicochemical indexes in the rhizosphere soil of tomato plants

处理 Treatment	CK1	CK2	YIM B08401			YIM B08402
处理 Treatment	CK1	CK2	T1	T2	T3	T1	T2	T3
pH	7.22±0.03 c	7.1±0.01 e	7.16±0.02 d	7.42±0.02 b	7.49±0.02 a	7.38±0.04 b	7.16±0.05 d	7.43±0.03 b
有机质Organic matter/（g·kg）	169.22±0.09 b	183.51±1.14 a	93.54±0.92 f	99.01±0.15 e	100.9±0.33 d	90.92±0.15 g	121.02±0.07 c	120.94±0.14 c
全氮 Total nitrogen/g·kg）	4.19±0.01 b	4.28±0.01 a	3.37±0.01 f	3.34±0.01 g	3.42±0.02 e	3.37±0.01 f	3.77±0 d	3.89±0.01 c
全磷Total phosphorus/（g·kg）	2.19±0.02 e	2.29±0.03 cd	2.34±0.04 c	2.41±0.02 b	2.26±0.02 d	2.54±0.05 a	2.45±0.04 b	2.27±0 d
全钾Total potassium/（g·kg）	6.22±0.08 f	6.99±0.11 d	7.01±0.08 d	7.83±0.13 a	7.28±0.08 c	7.32±0.11 c	7.58±0.23 b	6.74±0.03 e
水解性氮Hydrolyzed nitrogen/（mg·kg）	276.53±2.21 b	296.33±1.11 a	245.24±3.32 d	241.4±0 e	258.65±0 c	241.4±3.32 e	175.62±1.11 f	238.85±1.11 e
有效磷Available phosphorus/（mg·kg）	102.17±2.78 a	97.1±0.61 b	67.75±1.72 e	81.86±1 c	67.75±0.24 e	71.89±1.87 d	72.51±1.65 d	71.37±1.15 d
速效钾Fast-acting potassium/（mg·kg）	293.41±0.86 g	400.87±3.19 f	517.33±9.44 e	603.08±7.04 b	619.83±6.68 a	543.47±12.12 d	588.95±9.14 c	612.87±1.83 ab

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森林根际土壤细菌的分离、鉴定及生物活性筛选

Isolation and Identification of Bacteria in Forest Rhizosphere Soil and Their Biological Activity Screening

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