根际溶磷伯克霍尔德菌Paraburkholderia spp.对马尾松苗的促生作用

doi:10.13560/j.cnki.biotech.bull.1985.2022-1226

摘要/Abstract

摘要：

为筛选溶磷效果较好的根际细菌（phosphate-solubilizing rhizobacteria, PSB），并明确其对马尾松苗的促生效果和作用机制。利用土壤稀释平板法进行PSB菌株的分离和纯化，通过形态学和16S rDNA分子测序等方法进行PSB菌株的鉴定，最后将PSB菌株接种至马尾松苗，培养60 d后测定马尾松苗生长、生理、苗根际土壤理化性质和根际细菌群落结构和组成。结果表明：由马尾松根际土中分离获得溶磷能力较强的3个PSB菌株WJ10、WJ25和WJ41均为伯克霍尔德菌Paraburkholderia spp.；3个PSB菌株对磷酸铝的增溶能力最强，其次是磷酸三钙、磷酸氢钙和磷酸铁；盆栽试验表明，3个PSB菌株均可促进幼苗的生长，其中WJ25对苗高、根长的促进效果最明显，WJ41和WJ10次之。3个PSB菌株对苗促生的主要机制包括，PSB提高了马尾松苗的根系活力、叶绿素b、可溶性蛋白等生长指标及氮、磷和钾等养分含量；同时，提升了根际土有效磷、速效钾、活性氮、土壤养分含量、土壤酶活性；此外，3个PSB菌株的添加还影响了马尾松苗根际细菌群落的组成和多样性，促进了Bacillus、Nitrosospira、Gemmata和Cytophaga等有益菌在根际土壤中的显著富集。综上，本研究筛选获得的3个溶磷伯克霍尔德菌，它们能够通过调控植物生理及改变根际微环境从而促进马尾松苗的生长。通过本研究，为马尾松根际溶磷细菌菌肥的开发和应用提供了理论依据。

关键词: 根际溶磷细菌, 促生, 伯克霍尔德菌, 马尾松

Abstract:

It is aimed to screen phosphate-solubilizing rhizobacteria（PSB）and to clarify its growth-promoting effect on Pinus massoniana seedlings and the mechanism. The soil-dilution plate method was used to isolate and purify the PSB strains. The PSB strains were identified by morphology and 16s rDNA sequencing methods. Finally, the masson pine seedlings were inoculated with PSB strains for 60 d, then the seedling growth and physiological indexes, soil physical and chemical properties, and rhizosphere bacterial community were determined. The results showed that three PSB strains（WJ10, WJ25 and WJ41）with strong phosphate-solubilizing abilities were isolated from the P. massoniana rhizosphere soil and belonged to Paraburkholderia spp. The three PSB strains had the strongest phosphate-solubilizing abilities to aluminum phosphate, followed by tricalcium phosphate, calcium monophosphate and ferric phosphate. Based on the pot experiment, the three PSB strains effectively promoted seedlings growth, with the best effects on the seedling height and root length promotion for WJ25, followed by WJ41 and WJ10. The main mechanisms of seedling promotion of the three PSB strains include: 1）The PSB strains increased the root activity, chlorophyll b, soluble protein, nitrogen, phosphorus and potassium contents in masson pine seedlings; 2）meanwhile, PSBs enhanced the contents of available phosphorus, available potassium, active nitrogen, soil urease and phosphatase in rhizosphere soil; 3）in addition, the addition of three PSB strains also affected the composition and diversity of the rhizosphere bacterial community, and promoted the significant enrichment of beneficial bacteria such as Bacillus, Nitrosospira, Gemmata and Cytophaga in the rhizosphere soil. In conclusion, the three Paraburkholderia spp. strains could promote masson pine seedlings growth by regulating plant physiology and changing rhizosphere micro-environment. This study provides a theoretical basis for the development and application of rhizosphere PSB fertilizer of P. massoniana.

Key words: phosphate-solubilizing bacteria, growth promotion, Paraburkholderia spp., Pinus massoniana

徐红云, 吕俊, 于存. 根际溶磷伯克霍尔德菌Paraburkholderia spp.对马尾松苗的促生作用[J]. 生物技术通报, 2023, 39(6): 274-285.

XU Hong-Yun, LV Jun, YU Cun. Growth Promoting of Pinus massoniana Seedlings Regulated by Rhizosphere Phosphate-solubilizing Paraburkholderia spp.[J]. Biotechnology Bulletin, 2023, 39(6): 274-285.

图/表 10

图1 溶磷菌株在PVK培养基上产生的溶磷圈

Fig. 1 Phosphate solubilization zones on PVK agar plates A: WJ10; B: WJ25; C: WJ41

图2 溶磷菌株在电子显微镜下的形态结构

Fig. 2 Morphological structure of PSB strain under electron microscope A: WJ10; B: WJ25; C: WJ41

图3 采用Neighbor Joining法构建基于溶磷菌株16S rDNA序列的系统进化树

Fig. 3 Phylogenetic tree based on 16S rDNA sequence in PSB strains using Neighbor Joining method

图4 三个溶磷菌株对不同磷酸盐的溶磷能力及pH变化 A和B：WJ10; C和D：WJ25; E和F：WJ41。图中的数据是均值，误差线是标准偏差，下同

Fig. 4 P solubilizing capacity of three PSB in four different phosphate medium and acidification of medium during the phosphate solubilization A and B: Strain WJ10; C and D: strain WJ25; E and F: strain WJ41. The data in the figure are means, and the error line is the SE. The same blow

图5 接种溶磷菌株60 d后马尾松苗的生长差异

Fig. 5 Effects of three PSB strain inoculations on the growths of P. massoniana seedlings for 60 d

图6 接种3株溶磷菌株对马尾松苗生长、养分和生理的影响 A：苗高；B：根长；C：侧根数；D：鲜重；E：干重；F：可溶性蛋白含量；G：根系活力；H：叶绿素含量；I：植株养分。不同字母代表差异显著（P<0.05），下同

Fig. 6 Effects of PSB strain inoculations on the growth, nutrient uptake and physiological indexes of P. massoniana seedlings A: Seedling height; B: root length; C: lateral root number; D: fresh weight; E: dry weight; F: soluble protein content; G: root activity; H: chlorophyll content; I: plant nutrient. Different letters indicate siginificant difference（P<0.05）. The same below

图7 接种溶磷菌株对马尾松根际土养分含量和酶活性影响 A:有效养分；B:总养分含量；C:脲酶；D:过氧化氢酶；E:磷酸酶

Fig. 7 Effects of PSB strains on soil nutrient A: Available nutrient content of soil. B: Total nutrient content of soil. C: Soil urease. D: Soil catalase. E: Soil phosphatase

表1 不同处理马尾松根际土壤细菌α多样性指数

Table 1 Alpha diversity index of bacteria in rhizosphere soil of P. massoniana with different treatments

Treatment	OTUs	Shannon	ACE	Chao1	Simpson
CK	2181±100.00ab	5.67±0.07b	3126.18±107.24a	2968.00±175.58a	0.0134±0.002a
WJ10	1967±75.48bc	5.82±0.14ab	2713.49±60.82b	2636.03±78.64b	0.0090±0.001a
WJ25	2244±179.85a	5.86±0.18a	3109.78±98.45a	2960.25±57.96a	0.0116±0.002a
WJ41	1877±73.75c	5.66±0.17b	2604.44±45.00b	2474.54±47.17b	0.0121±0.002a

图8 接种溶磷菌株对马尾松苗根际细菌群落门水平和属水平组成的影响 A:门水平；B:属水平

Fig. 8 Relative abundance of bacteria at phyla levels and genus levels in the rhizosphere soils under different treatments A: Phyla levels; B: genus levels

表2 马尾松根际土壤有效养分、酶活性与细菌α多样性指数的相关性

Table 2 Correlation between the rhizosphere soil available nutrients, enzyme activity and bacterial alpha diversity index of P. massoniana

α多样性指数 Alpha diversity index	水解N Available N	有效P Available P	速效K Available K	尿素酶 Urease	过氧化氢酶 Catalase	磷酸酶 Phosphatase
Shannon	0.883	0.250	0.426	0.548	0.903	0.426
ACE	-0.139	-0.884	-0.441	-0.519	-0.157	-0.512
Chao1	-0.091	-0.850	-0.452	-0.504	-0.191	-0.516
Simpson	-0.689	-0.773	-0.378	-0.605	-0.464	-0.449

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