萎缩芽孢杆菌CNY01的生防特性及其对玉米的抗盐促生作用

doi:10.13560/j.cnki.biotech.bull.1985.2023-1174

摘要/Abstract

摘要：

【目的】从黄河三角洲盐碱地中筛选到具有抗盐促生作用的一株根际菌，制成菌剂用于缓解玉米植株的高盐胁迫。【方法】通过功能培养基、16S rDNA序列和全基因组分析，确定菌株种属以及对玉米植株的抗盐促生效果。【结果】从黄河三角洲盐碱地中筛选到一株可在0-16%的NaCl和pH 5-8的条件下正常生长的萎缩芽孢杆菌CNY01，该菌株可促进玉米在高盐条件下生长，具有降蛋白和解钾的能力，还可抑制青枯雷尔氏菌、串珠镰刀菌等病原菌的生长。玉米植株在含100 mmol/L NaCl的营养液中生长8 d时，施加菌株CNY01可使植株的生理株高、根长和鲜重分别提高38.80%（P<0.01）、23.73%和28.19%（P<0.01）。玉米植株在加100 mmol/L NaCl的高盐盆栽条件下生长28 d时，菌株CNY01可使植株的株高、地上鲜重和地下鲜重分别提高10.84%、41.87%（P<0.01）和23.29%。全基因组序列分析也预测出该菌株基因组中含有维持细胞渗透压、合成应激蛋白等缓解高盐胁迫的基因。【结论】从黄河三角洲盐碱地中筛选到一株具有防病促生功能的萎缩芽孢杆菌CNY01，对玉米植株有显著的抗盐促生作用，结合基因组结果预测到该菌株含有与抗盐促生相关基因，是重要的菌种资源。

关键词: 玉米, 植物根际促生细菌, 萎缩芽孢杆菌, 抗盐促生, 全基因组测序

Abstract:

【Objective】A rhizobacterium with salt-resistant and growth-promoting effects was screened from the saline-alkali soil in Yellow River Delta to prepare an agent for alleviating high-salt stress of maize seedlings. 【Method】The salt-resistant and growth-promoting functions of the strain were identified on functional mediums, and the species of the strain was determined by 16S rDNA sequence and whole genome analysis. The strain was applied to maize rhizosphere to verify the salt-resistant and growth-promoting effects of the strain on maize seedlings.【Result】A strain Bacillus atrophaeus CNY01 was screened from the saline-alkali soil of Yellow River Delta. Strain CNY01 grew normally under the condition of 0-16% NaCl and pH 5-8. Strain CNY01 had the ability of degrading protein and dissolving potassium, and inhibited the growth of plant pathogens such as Ralstonia solanacearum and Fusarium moniliforme. The application of strain CNY01 promoted the growth of maize seedlings under high-salt condition and alleviated the stress of high salt on maize seedlings. When strain CNY01 was applied to maize seedlings in nutrient solution containing 100 mmol/L NaCl for 8 d, the physiological plant height, root length, and fresh weight of maize seedlings increased by 38.80%（P<0.01）, 23.73%, and 28.19%（P<0.01）, respectively. When strain CNY01 was applied to maize seedlings in pot experiments adding 100 mmol/L NaCl for 28 d, the physiological plant height, the aboveground fresh weight, and the underground fresh weight of maize seedlings increased by 10.84%, 41.87%（P<0.01）, and 23.29%, respectively. Genes that maintain cell osmotic pressure and synthesize stress proteins to alleviate high salt stress were also predicted in the genome sequence of this strain.【Conclusion】A strain of B. atrophaeus CNY01 is screened from saline-alkali soil of Yellow River Delta, which has the function of preventing disease and promoting growth, and has significant salt-resistant and promoting growth effect on maize plants. Meanwhile, strain CNY01 contains genes related to salt-resistant and growth-promoting. B. atrophaeus CNY01 is an important strain resource.

Key words: maize, plant growth-promoting rhizobacteria, Bacillus atrophaeus, salt-resistant and growth-promoting, whole-genome sequencing

孙亚楠, 王春雪, 王欣, 杜秉海, 刘凯, 汪城墙. 萎缩芽孢杆菌CNY01的生防特性及其对玉米的抗盐促生作用[J]. 生物技术通报, 2024, 40(5): 248-260.

SUN Ya-nan, WANG Chun-xue, WANG Xin, DU Bing-hai, LIU Kai, WANG Cheng-qiang. Biocontrol Characteristics of Bacillus atrophaeus CNY01 and Its Salt-resistant and Growth-promoting Effect on Maize Seedling[J]. Biotechnology Bulletin, 2024, 40(5): 248-260.

图/表 10

图1 菌株CNY01的菌落和细胞形态特征 A：菌株CNY01菌落图片；B：菌株CNY01的革兰氏染色图片

Fig. 1 Colony and cell morphological characteristics of strain CNY01 A: Colony picture of strain CNY01; B: Gram stain picture of strain CNY01

图2 菌株CNY01的系统发育进化树

Fig. 2 Phylogenetic tree based on strain CNY01

图3 菌株CNY01的耐盐和耐酸碱能力测定 A：菌株CNY01在不同盐浓度固体培养基中的长势；B：菌株CNY01的液体培养耐盐测定；C：CNY01的液体培养耐酸碱测定

Fig. 3 Determination of salt and acid-alkali tolerance of strain CNY01 A: Growth of CNY01 on solid medium with different salt concentrations; B: salt tolerance test of CNY01 in liquid medium; C: determination of acid and alkali resistance of CNY01 in liquid medium

图4 菌株CNY01的拮抗促生能力测定 A：干酪素培养基培养CNY01；B：硅酸盐培养基培养CNY01；C：CNY01与枯草芽孢杆菌拮抗培养；D：CNY01与青枯雷尔氏菌拮抗培养；E：CNY01与串珠镰刀菌拮抗培养

Fig. 4 Determination of antagonistic and growth-promoting ability of strain CNY01 A: CNY01 cultured in casein medium; B: CNY01 cultured in silicate medium; C: CNY01 cultured in antagonism with Bacillus subtilis; D: CNY01 cultured in antagonism with Ralstonia solanacearum; E: CNY01 cultured in antagonism with Fusarium moniliforme

图5 菌株CNY01对玉米植株的促生作用（第8天） A：玉米生理株高；B：玉米根长；C：植株鲜重。CK：无菌无盐组；BK：有菌无盐组；CN：无菌有盐组；BN：有菌有盐组。*P<0.05，**P<0.01，下同

Fig. 5 Growth-promoting effect of strain CNY01 on maize seedlings（Day 8） A: Maize physiological plant height; B: maize root length; C: plant fresh weight. CK: Sterile and salt-free group; BK: the salt-free group with strain CNY01; CN: sterile salt group; BN: the salt group with strain CNY01. *P< 0.05 and **P < 0.01, the same below

图6 盆栽条件下不同时期玉米植株的农艺性状分析 A：株高；B：生理株高；C：茎粗；D：地上鲜重（28 d）；E：地下鲜重（28 d）；F：地上干重（28 d）；G：地下干重（28 d）

Fig. 6 Analysis of agronomic traits of maize seedlings in different periods under potted conditions A: Plant height; B: physiological plant height; C: stem thick; D: aboveground fresh weight（28 d）; E: underground fresh weight（28 d）; F: aboveground dry weight（28 d）;G: underground dry weight（28 d）

表1 萎缩芽孢杆菌CNY01、GQJK17、BA59、PENSV20、1942、SRCM101359、NS2、NX-12和MBLB1156基因组信息比对

Table 1 General genome features of B. atrophaeus CNY01, GQJK17, BA59, PENSV20, 1942, SRCM101359, NS2, NX-12, and MBLB1156

Genomic information	CNY01	GQJK17	BA59	PENSV20	1942	SRCM101359	NS2	NX-12	MBLB1156
Genome Size/Mb	4.14	4.33	4.26	4.15	4.17	4.18	4.26	4.16	4.15
G+C content/%	43.50	43.30	43.10	43.50	43.20	43.30	43.30	43.30	43.40
CDSs（with protein）	3950	4045	3889	3932	3995	3923	4018	3946	3870
Pseudo genes number	146	161	383	157	152	157	159	158	232
rRNA genes number	24	24	26	24	21	24	24	24	24
tRNA genes number	82	84	84	83	75	82	82	82	82
The source of isolation	Soil （China）	Rhizophere （China）	Cotton root （China）	Soil （Canada）	-	Food （South Korea）	-	Cotton rhizosphere soil	Fermented to fu

表2 菌株CNY01的ANI比对结果

Table 2 ANI contrast test of strain CNY01

Name of strain	Bacillus haynesii	Bacillus sonorensis	Bacillus siamensis	Bacillus licheniformis	Bacillus atrophaeus	Bacillus paralicheniformis	CNY01
Bacillus haynesii	*	81.18	71.86	95.14	72.29	95.14	72.23
Bacillus sonorensis	80.85	*	71.84	80.97	72.47	80.72	72.38
Bacillus siamensis	72.04	71.92	*	71.93	76.64	71.84	76.73
Bacillus licheniformis	95.40	81.42	71.94	*	72.45	94.38	72.43
Bacillus atrophaeus	72.28	72.57	76.66	72.30	*	72.32	98.06
Bacillus paralicheniformis	95.03	81.00	71.98	94.02	72.25	*	72.13
CNY01	72.32	72.63	76.89	72.33	98.36	72.34	*

图7 菌株CNY01基因的COG和KEGG功能聚类分类 A：COG聚类分析；B：KEGG分类

Fig. 7 COG and KEGG functional classification analysis of genes in strain CNY01 A: COG functional classification; B: KEGG classification

表3 菌株CNY01的部分耐盐促生相关基因分析

Table 3 Analysis of some genes related to salt tolerance and growth promotion in strain CNY01

位置Location	长度Length/bp	基因Gene	产物Product
KCX77_05795	453	nhaC	Na⁺/H⁺ antiporter NhaC
KCX77_09890	429	/	Na⁺/H⁺ antiporter NhaC family protein
KCX77_12210	468	nhaC	Na⁺/H⁺ antiporter NhaC
KCX77_15895	806	/	Na⁺/H⁺ antiporter subunit A
KCX77_15910	493	/	Na⁺/H⁺ antiporter subunit D
KCX77_15915	158	/	Na⁺/H⁺ antiporter subunit E
KCX77_15925	124	/	Na⁺/H⁺ antiporter subunit G
KCX77_16910	671	/	Na⁺/H⁺ antiporter
KCX77_01700	418	opuAA	Glycine/proline betaine ABC transporter
KCX77_01705	282	opuAB	Glycine/proline betaine ABC transporter permease subunit OpuAB
KCX77_01805	303	/	Proline dehydrogenase
KCX77_01815	504	putP	Sodium/proline symporter PutP
KCX77_04225	492	putP	Sodium/proline symporter PutP
KCX77_09365	564	proS	Proline--tRNA ligase
KCX77_16595	302	/	Proline dehydrogenase
KCX77_17080	381	/	Betaine/proline/choline family ABC transporter ATP-binding protein
KCX77_00320	205	/	General stress protein Ctc
KCX77_04635	115	/	General stress protein
KCX77_15790	130	yugI	General stress protein 13
KCX77_19470	547	katX	Catalase KatX
KCX77_02490	273	/	Manganese catalase family protein
KCX77_05335	483	katA	Catalase KatA
KCX77_12565	285	/	Manganese catalase family protein
KCX77_08110	302	/	Manganese catalase family protein
KCX77_19655	685	/	Catalase
KCX77_07800	449	/	TrkH family potassium uptake protein
KCX77_08320	221	ktrC	Ktr system potassium transporter KtrC
KCX77_15595	222	/	TrkA family potassium uptake protein
KCX77_15600	445	/	TrkH family potassium uptake protein
KCX77_15755	328	/	Potassium channel family protein
KCX77_19400	221	/	TrkA family potassium uptake protein
KCX77_17200	394	/	Phosphoglycerate kinase
KCX77_14540	585	pyk	Pyruvate kinase

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