枫香根际解有机磷细菌筛选及其促生效应

doi:10.3969/j.issn.1000-2006.201908022

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南京林业大学学报（自然科学版） ›› 2020, Vol. 44 ›› Issue (3): 95-104.doi: 10.3969/j.issn.1000-2006.201908022

枫香根际解有机磷细菌筛选及其促生效应

宋娟¹，徐国芳²，赵邢³，姚尧³，杨学祥³，唐荣林³，崔家旺¹，陈凤毛^1*，任嘉红²

^1.南京林业大学林学院, 南方现代林业协同创新中心, 江苏南京 210037
^2.长治学院生物科学与技术系，山西长治 046011
^3.句容市句容林场，江苏句容 212424

收稿日期:2019-08-15 修回日期:2020-01-18 出版日期:2020-05-30 发布日期:2020-06-11
作者简介:宋娟（Sjj818388@outlook.com），ORCID（0000-0002-6612-428X）。
基金资助:
国家自然科学基金项目(31100471);国家重点研发计划(2017YFD0600104);江苏高校优势学科建设工程资助项目;江苏省林业科技创新与推广项目（LYKJ[2017]20）

Screening of indigenous phosphate⁃solubilizing bacteria from Liquidambar formosana Hance rhizosphere and its potential applications for improving plant growth

SONG Juan¹(), XU Guofang², ZHAO Xing³, YAO Yao³, YANG Xuexiang³, TANG Ronglin³, CUI Jiawang¹, CHEN Fengmao¹(), REN Jiahong²

^1.Co -Innovation Center of the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China
^2.The Department of Biological Science and Technology, Changzhi University, Changzhi 046011, China
^3.Forest Farm of Jurong City, Jurong 212424, China

Received:2019-08-15 Revised:2020-01-18 Online:2020-05-30 Published:2020-06-11
Contact: CHEN Fengmao

摘要/Abstract

摘要： 目的

从枫香（Liquidambar formosana Hance）根际土壤中分离和鉴定有效解磷菌（PSB），并研究其在溶磷方面的应用。

方法

采用形态学及生理生化特征、微生物鉴定系统（MIDI）、BIOLOG细菌鉴定和16SrDNA序列分析等方法鉴定解磷菌。应用离子发射光谱法测定各解磷菌菌株（JX7、JX18、JX21、WHP）处理下枫香叶片大量及微量元素的含量，鉴定PSB菌株对植物生长的影响。

结果

解磷细菌菌株JX7鉴定为争论产碱菌（Variovorax paradoxus），JX18鉴定为蜡状芽孢杆菌（Bacillus cereus），WH10鉴定为蕈状芽孢杆菌（Bacillus mycoides），JX21鉴定为蜡状芽孢杆菌（Bacillus cereus）。菌株JX21的可溶性磷酸盐含量最高，为（9.35±0.89） mg/L，其次是JX7[（6.63±0.09) mg/L]，WH10[（6.16±0.12) mg/L]和JX18[（5.32±0.07) mg/L]。以上解磷菌在培养72 h后，其溶磷量均达到最大值。筛选自枫香根际的解磷菌菌株（JX7、WH10、JX18、JX21）接种枫香幼苗比对照显著提高了枫香的苗高和地径（P < 0.05)。在室温条件下接种解磷菌显著提高枫香叶片内的P、Ca、Mg、Zn的含量（P < 0.05)。

结论

筛选解磷菌均显示出良好的溶磷特性，并具有作为生物肥料的潜力。可进一步评估所筛选菌株的特性，开发其商业应用。

关键词: 枫香, 植物促生菌, 植物营养素, 解磷菌, 生物肥料

Abstract: Objective

Isolate and identify efficient phosphate solubilizing bacteria(PSB) from Liquidambar formosana Hance rhizosphere soil and investigate the application in phosphorus release.

Method

the PSB strains were graded based on morphological and physiological-biochemical characteristics, microbial identification system (MIDI), BIOLOG analysis and 16S rRNA identification methods. The effect of these PSB strains (JX7，JX18，JX21 and WH10）on plant growth was evaluated by determining the minor and major mineral contents of L. formosana leaves using inductively-coupled plasma atomic emission spectrometry.

Result

Isolates JX7, JX18, WH10 and JX21 were closely related to Variovorax paradoxus, Bacillus cereus, B. mycoides and B.cereus, respectively. The JX21 showed the highest content of soluble phosphate was (9.35 ± 0.89) mg/L, followed by JX7 [（6.63±0.09) mg/L], WH10[ (6.16 ± 0.12 ) mg/L] and JX18 [(5.32 ± 0.07 ) mg/L], all the maximum phosphate was fermented after 72 h of incubation. Phosphate-solubilizing bacteria (JX7, WH10, JX18 and JX21) selected from L. formosana rhizosphere showed a significantly improvement in shoot height and ground diameter of L. formosana (P < 0.05). As well as, these selected strains promoted L. formosana in taking up mineral elements such as phosphorus, magnesium, calcium and zinc under greenhouse conditions.

Conclusion

The isolates showed good phosphate-solubilizing activity and had potential for use as biofertilizer agents. Further study is needed to evaluate beneficial traits to develop commercial applications.

Key words: Liquidambar formosana Hance, plant growth promoting bacteria, plant nutrients, phosphate-solubilizing bacteria, biofertilizer

中图分类号:

S763

宋娟,徐国芳,赵邢,等. 枫香根际解有机磷细菌筛选及其促生效应[J]. 南京林业大学学报（自然科学版）, 2020, 44(3): 95-104.

SONG Juan, XU Guofang, ZHAO Xing, YAO Yao, YANG Xuexiang, TANG Ronglin, CUI Jiawang, CHEN Fengmao, REN Jiahong. Screening of indigenous phosphate⁃solubilizing bacteria from Liquidambar formosana Hance rhizosphere and its potential applications for improving plant growth[J].Journal of Nanjing Forestry University (Natural Science Edition), 2020, 44(3): 95-104.DOI: 10.3969/j.issn.1000-2006.201908022.

图/表 10

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strain 菌株	D／mm (dissolved phosphorus circle diameter 溶磷圈直径)	d／mm (colony diameter 菌落直径)	D/d	ring type 圈类型
WH10	32.46 ± 0.60	14.34 ± 0.28	2.26 ± 0.003	grease ring溶脂圈
WH18	44.84 ± 0.40	36.34 ± 0.34	1.23 ± 0.002	grease ring溶脂圈
WH19	37.67 ± 0.80	18.62 ± 0.44	2.02 ± 0.005	grease ring溶脂圈
JX1	47.15 ± 0.30	34.70 ± 0.52	1.36 ± 0.012	grease ring溶脂圈
JX7	8.91 ± 0.50	3.50 ± 0.19	2.55 ± 0.005	hydrolysis ring水解圈
JX8	8.33 ± 0.70	4.02 ± 0.53	2.07 ± 0.101	hydrolysis ring水解圈
JX9	9.60 ± 0.34	4.22 ± 0.26	2.27 ± 0.060	hydrolysis ring水解圈
JX11	13.22 ± 0.26	4.66 ± 0.33	2.84 ± 0.146	hydrolysis ring水解圈
JX12	7.13 ± 0.43	5.10 ± 0.23	1.40 ± 0.021	hydrolysis ring水解圈
JX13	7.00 ± 0.54	3.38 ± 0.13	2.07 ± 0.080	hydrolysis ring水解圈
JX15	29.53 ± 1.38	15.10 ± 0.85	1.96 ± 0.019	grease ring溶脂圈
JX18	44.77 ± 1.64	29.08 ± 1.32	1.54 ± 0.014	grease ring溶脂圈
JX19	40.02 ± 1.14	29.97 ± 0.76	1.34 ± 0.005	grease ring溶脂圈
JX20	39.96 ± 0.83	27.04 ± 0.54	1.48 ± 0.002	grease ring溶脂圈
JX21	42.45 ± 0.53	25.48 ± 0.36	1.67 ± 0.004	grease ring溶脂圈

strain 菌株	cell shape 菌体形态	Gram reaction 革兰氏染色	3% KOH	spores 芽孢	flagellum 鞭毛	aerobic condition 需氧情况	colony morphology 菌落形状
JX7	+	G-	+	-	-	+	colonies smaller, yellowish green, circular, slimy, moist 菌落较小，黄绿色，边缘整齐，光滑湿润
WH10	+	G+	-	+	+	+	larger colonies, creamy, circular, slimy, dry 菌落较大，乳白色，圆形,边缘整齐，光滑干燥
JX18	+	G+	-	+	+	+	larger colonies,whitish, circular, slimy, dry 菌落较大，白色圆形，边缘整齐，光滑干燥
JX21	+	G+	-	+	+	+	larger colonies, circular,the middle wrinkles, slimy, dry 菌落较大,圆形,边缘整齐，中间有皱褶，光滑干燥

project measureed 测定项目	JX7	WH10	JX18	JX21
BIOLOG result BIOLOG结果	Variovorax paradoxus 争论产碱菌	Bacillus mycoides 蕈状芽孢杆菌	Bacillus cereus 蜡状芽孢杆菌	Bacillus cereus 蜡状芽孢杆菌
oxidase test氧化酶	-	+	+	+
contact enzyme接触酶	+	+	+	+
lecithinase卵磷脂酶	-	+	+	+
gelatin hydrolysis 明胶水解	-	+	+	+
starch hydrolysis 淀粉水解	-	+	+	+
H₂S test产生H₂S	-	+	-	-
citrate utilization 柠檬酸盐利用	-	+	+	+
indole test 吲哚试验	+	-	+	+
methyl red test 甲基红试验	-	-	-	-
oges praskaure’s 伏普试验	+	+	+	+
nitrate reduction 硝酸盐还原	+	-	-	-

fatty acid 脂肪酸	JX18	JX21	WH10
13∶0 iso	6.72	6.77	6.08
14∶0 iso	3.10	3.40	3.11
14∶0	3.03	2.65	3.53
15∶0 iso	31.45	20.12	26.00
15∶0 anteiso	4.45	3.49	3.57
summed in feature 2 of 14∶0 3OH/16∶1 iso I	2.74	3.58	4.06
15∶1 w5c	no	4.30	3.51
16∶0 iso	5.25	5.13	5.10
summed in feature 3 of 16∶1 w6c/16∶1 w7c	8.21	8.95	7.61
16∶0	3.84	7.65	7.77
17∶1 iso w10c	3.94	2.38	2.98
17∶1 iso w5c	6.76	8.99	7.52
17∶0 iso	8.54	6.55	6.33
18∶0	2.18	6.40	no
summed in feature 8 of 18∶1 w7c	0.27	0.60	1.10
summed feature 2 of unknown 10.9525	2.74	3.58	4.06
summed feature 3 of 16∶1 w6c/16∶1 w7c	8.21	8.95	9.28
sim index	0.715	0.167	0.357

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[3]	史久洲, 姬晓悦, 陈继超, 卢雯, 徐莉. SPME/GC-MS分析不同产地枫香树脂中挥发性成分[J]. 南京林业大学学报（自然科学版）, 2020, 44(5): 239-244.
[4]	张昌伟,彭胜,张琳杰,王志宏,周云雷,彭密军. 以杜仲叶渣为载体固态发酵制备解磷生物肥料的工艺优化[J]. 南京林业大学学报（自然科学版）, 2014, 38(05): 134-138.
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枫香根际解有机磷细菌筛选及其促生效应

Screening of indigenous phosphate⁃solubilizing bacteria from Liquidambar formosana Hance rhizosphere and its potential applications for improving plant growth

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strain 菌株	specific name 种名	BIOLOG similarity		16SrDNA homology /% 16SrDNA同源性
strain 菌株	specific name 种名	Probability (PROB)/% BIOLOG可能性	(SIM) index value BIOLOG 相似性指数	16SrDNA homology /% 16SrDNA同源性
JX7	Variovorax paradoxus	99	0.648	97
WH10	Bacillus mycoides	83	0.636	95
JX18	Bacillus cereus	89	0.697	96
JX21	Bacillus cereus	94	0.720	97