溶磷内生菌的筛选鉴定及其对薏苡生长发育的影响

普凤雅; 谷书杰; 何永宏; 陈崧林; 杨志清

doi:10.19303/j.issn.1008-0384.2022.007.016

溶磷内生菌的筛选鉴定及其对薏苡生长发育的影响

doi: 10.19303/j.issn.1008-0384.2022.007.016

普凤雅^1,,
谷书杰¹,
何永宏²,
陈崧林¹,
杨志清^{1, 3, 4, ,}

1.
云南农业大学农学与生物技术学院，云南　昆明　650201
2.
云南农业大学植物保护学院，云南　昆明　650201
3.
云南农业大学云南省药用植物生物学重点实验室，云南　昆明 650201
4.
云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心，云南　昆明 650201

基金项目: 云南省科技计划项目（2014RE002）

详细信息

作者简介:
普凤雅（1996−），女，硕士研究生，研究方向：药用植物栽培与生理状态（E-mail：pfy996@163.com）

通讯作者:
杨志清（1968−），女，硕士，教授，研究方向：药食同源作物栽培与资源评价研究（E-mail: yzq1468@126.com）

中图分类号: S 182
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- 被引次数: 0
出版历程
- 收稿日期: 2022-03-01
- 修回日期: 2022-05-09
- 网络出版日期: 2022-08-07
- 刊出日期: 2022-07-28

Identification and Characterization of Phosphate-solubilizing Endophytes in Coix lacryma-jobi L.

PU Fengya^1
,,
GU Shujie¹,
HE Yonghong²,
CHEN Songlin¹,
YANG Zhiqing^{1, 3, 4
, ,}

1.
College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan　650201, China
2.
College of Plant Protection, Yunnan Agricultural University, Kunming, Yunnan　650201, China
3.
Key Laboratory of Medicinal Plant Biology of Yunnan Province, Yunnan Agricultural University, Kunming, Yunnan　650201, China
4.
National & Local Joint Engineering Research Center on Germplasm Innovation & Utilization of Chinese Medicinal Materials in Southwestern China, Yunnan Agricultural University, Kunming, Yunnan　650201, China

摘要

摘要: 目的筛选具有溶磷能力的薏苡内生细菌并明确其类型，为后续研制生物菌肥提供优良菌株资源。方法以播种25 d的薏苡幼苗为试验材料，采用研磨稀释涂板法从薏苡的根、茎、叶中分离筛选具有溶磷能力的薏苡内生菌，探索该类菌溶解有机磷、无机磷、分泌磷酸酶的能力；通过盆栽试验探究溶磷内生菌对薏苡幼苗的促生长作用。结果筛选到溶解有机磷细菌8株，溶解无机磷细菌5株。其中，编号为R24的菌株溶解有机磷能力最强，摇瓶5 d后，上清液可溶性磷含量为37.20 mg·L⁻¹；编号为L21的菌株溶解无机磷能力最强，摇瓶5 d后，上清液可溶性磷含量为62.93 mg·L⁻¹。2株溶磷菌及等比例混合菌液能够促进盆栽薏苡的株高、茎粗、分蘖数，促生长能力从大到小为：L21＞R24＞混合菌液[V（R24）∶V（L21）=1∶1]。通过16S rDNA序列鉴定，R24为短小芽孢杆菌，L21为贝莱斯芽孢杆菌。结论溶磷菌L21和R24对薏苡生长有显著促进作用。
- 薏苡 /
- 内生菌 /
- 溶磷细菌 /
- 溶磷能力 /
- 鉴定
Abstract: Objective Screening bacteria with phosphate-solubilizing ability in Coix were identified and provide excellent strain resource for bio-fertilization agent. Methods Endophytic bacteria were isolated and purified from specimens the roots, stems, and leaves of 25-d-old seedlings of Coix lacryma-jobi L. Abilities of the isolates in dissolving organophosphate and inorganic phosphate as well as in secreting phosphatase were determined. In a pot experiment, effects of two selected isolates on the growth of Coix seedlings were examined. Results Eight strains of bacteria capable of dissolving organophosphate and 5 of degrading inorganic phosphate were isolated. In the 5-d culture supernatants, the highest concentration of phosphorous converted from organophosphate at 37.20 mg·L⁻¹ was reached by R24, and that from inorganic phosphate at 62.93 mg·L⁻¹ by L21. The enhancing effects on plant height, stem girth, and tillering number of the potted Coix seedlings by each and mixture in equal proportion of the two supernatants ranked in the order of L21＞R24＞mixed bacterial liquid[ V （R24）∶ V （L21）=1∶1]. The 16S rDNA sequence phylogenetic analysis identified R24 as Bacillus pumilus and L21 as B. velezensis. Conclusion The phosphobacteria L21 and R24 displayed a significant effect in promoting the growth of Coix seedlings.
- Coix /
- endophytes /
- phosphobacteria /
- phosphate-solubilizing ability /
- identification

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图 1 有机磷培养基摇瓶培养5 d后可溶性磷含量

不同小写字母表示不同材料间差异显著(P＜0.05) ；下同。

Figure 1. Solubilized phosphorus in organophosphate-containing liquid medium after 5 d culture in shaking-flask

Data with different lowercase letters indicate significant differences at P＜0.05.The same as below.

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图 2 无机磷培养基摇瓶培养5 d后可溶性磷含量

Figure 2. Solubilized phosphorus in inorganic phosphate-containing liquid medium after 5 d culture in shaking-flask

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图 3 溶磷菌株在有机磷培养基中分泌磷酸酶活性动态变化

a：酸性磷酸酶活性；b：碱性磷酸酶活性；c：有机磷培养基pH动态变化。

Figure 3. Dynamic changes on phosphatase secretion of two strains in organophosphate medium

a: Acid phosphatase activity; b: Alkaline phosphatase activity; c: Dynamic pH changes in organophosphate culture medium.

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图 4 溶磷菌株在无机磷培养基中分泌磷酸酶活性动态变化

a：酸性磷酸酶活性；b：碱性磷酸酶活性c：无机磷培养基pH动态变化。

Figure 4. Dynamic changes on phosphatase secretion of two strains in inorganic phosphate medium

a: Acid phosphatase activity; b: Alkaline phosphatase activity; c: Dynamic pH changes in inorganic phosphate culture medium.

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图 5 不同处理下薏苡的生长情况

Figure 5. Growth phenotype of coix seedlings after treatments

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图 6 菌株R24、L21的菌落形态

Figure 6. Colony morphology of R24 and L21

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图 7 溶磷菌株16S rDNA序列系统发育树

Figure 7. Phylogenetic trees generated by neighbor-joining method based on 16S rDNA sequences of selected strains

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表 1 菌株降解有机磷能力的比较

Table 1. Organic phosphate degrading ability of strains

菌株 Strains	菌株直径 Strain diameter/ cm	透明圈直径 Diameter of transparent ring/cm	D/d
R5	0.35±0.05 b	0.55±0.05 cd	1.58±0.09 b
R24	0.29±0.06 b	0.63±0.09 bc	2.15±0.13 a
R29	0.33±0.05 b	0.53±0.05 d	1.62±0.11 b
R401	0.50±0.01 a	0.77±0.05 a	1.53±0.12 b
S1	0.29±0.05 b	0.49±0.05 d	1.71±0.16 b
S4	0.37±0.03 b	0.54±0.03 cd	1.46±0.19 b
S22	0.39±0.03 b	0.66±0.06 b	1.69±0.09 b
L21	0.49±0.11 a	0.79±0.02 a	1.66±0.32 b
不同小写字母表示不同材料间差异显著（P＜0.05）；下同。 Different small letters indicate significant 0.05 among different materials.The same as below.

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表 2 菌株降解无机磷的能力比较

Table 2. Inorganic phosphate degrading ability of strains

菌株编号 Strains	菌株直径 Strain diameter/cm	透明圈直径 Diameter transparent ring/cm	D/d
R5	0.70±0.10 a	1.32±0.18 a	1.89±0.15 bc
R24	0.35±0.06 b	0.65±0.12 b	1.88±0.39 bc
R29	0.29±0.04 b	0.52±0.09 b	1.82±0.16 bc
R401	0.79±0.07 a	1.19±0.06 a	1.52±0.06 c
L21	0.33±0.02 b	0.67±0.04 b	2.01±0.25 a

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表 3 不同处理下盆栽薏苡农艺性状

Table 3. Agronomic traits of potted plants after treatments

时间 Time/d	处理 Treatment	株高 Plant height/cm	茎粗 Stem diameter/mm	分蘖 Tillers number/个	叶片 Blades number/片	叶面积 Leaf area/m²	叶绿素相对含量 SPAD
15	T1	38.80±3.49 a	5.34±0.09 a	0.00±0.00	4.40±0.55 a	0.29±0.03 a	19.45±0.85 ab
	T2	37.00±2.12 a	4.72±0.23 bc	0.00±0.00	4.00±0.00 a	0.28±0.05 a	21.14±1.37 a
	T3	39.40±2.07 a	5.05±0.20 ab	0.00±0.00	4.40±0.55 a	0.25±0.04 a	20.17±1.64 ab
	CK	36.80±3.96 a	4.66±0.45 c	0.00±0.00	4.20±0.45 a	0.25±0.06 a	18.12±1.83 b
30	T1	52.00±7.18 a	6.94±0.27 a	1.60±0.89 a	5.20±0.45 a	0.56±0.06 a	23.11±1.72 a
	T2	42.00±3.54 b	7.07±0.36 a	1.40±0.89 a	5.40±0.55 a	0.47±0.09 ab	21.78±1.69 ab
	T3	46.60±6.31 ab	6.82±0.54 a	1.40±0.55 a	5.40±0.55 a	0.54±0.09 a	20.84±1.62 bc
	CK	40.80±7.05 b	5.87±0.19 b	0.60±0.55 a	5.20±0.45 a	0.40±0.06 b	19.30±0.49 c
45	T1	55.80±4.21 a	8.91±0.42 a	2.20±0.45 a	6.00±0.00 a	0.79±0.06 a	22.79±0.78 a
	T2	51.00±4.30 a	8.56±0.3 a	2.20±0.45 a	5.60±0.55 a	0.70±0.09 b	20.38±0.91 b
	T3	52.80±9.73 a	7.21±0.36 b	1.80±1.10 ab	5.60±0.55 a	0.74±0.04 ab	18.90±0.48 c
	CK	54.40±2.88 a	6.76±0.35 b	1.00±0.71 b	5.40±0.55 a	0.61±0.02 c	16.90±1.16 d
60	T1	70.80±4.32 a	9.04±0.34 a	2.20±0.45 ab	6.20±0.45 a	0.92±0.04 a	18.42±0.92 a
	T2	67.20±4.44 a	8.68±0.19 a	2.40±0.55 a	6.00±0.00 a	0.77±0.06 b	18.70±1.01 a
	T3	66.00±4.30 a	7.70±0.12 b	1.80±1.10 ab	5.80±0.45 a	0.89±0.07 a	15.67±1.21 b
	CK	58.00±2.55 b	7.24±0.62 b	1.20±0.84 b	6.00±0.00 a	0.60±0.08 c	16.28±2.45 b

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表 4 菌株的生理生化特性

Table 4. Physiological and biochemical characteristics of strains

试验指标 Test index	菌株 Strains
试验指标 Test index	R24	L21
革兰氏染色 Gram stain	+	+
V-P试验 V-P test	+	+
甲基红试验 Methyl red test	−	+
淀粉水解试验 Starch hydrolysis test	−	+
吲哚试验 Indole test	+	+
柠檬酸试验 Citrate test	+	−
明胶液化 Gelatin liquefaction	+	+
硝酸盐试验 Nitrate reduction test	−	+
+：阳性；−：阴性。 +: Positive; −: Negative.

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