高寒草地耐低温植物根际促生菌的筛选鉴定及特性研究

doi:10.11686/cyxb2021311

摘要/Abstract

摘要：

为获得优良植物根际促生菌（PGPR）并明确其促生特性，以红原地区广泛分布的3种高寒草地优势植物为材料，采用选择性培养基从3种高寒植物根际分离、筛选具有固氮、溶磷、分泌植物激素能力的耐低温植物根际促生菌，并进行16S rRNA分子生物学鉴定。结果表明：毛稃羊茅、洽草、紫穗鹅观草植物根际分布着大量根际细菌，且PGPR在根际不同部位的数量呈现出根系表面（RP）>根表土壤（RS）>根内（HP）的分布规律。经筛选共获得76株PGPR菌株，其中固氮菌30株，其固氮酶活性为124.61~311.04 nmol C₂H₄·h^-1·mL^-1；溶解无机磷菌株24株，溶解有机磷菌株22株，溶磷量分别为249.85~558.07 μg·mL^-1和52.25~158.77 μg·mL^-1。挑选固氮酶活性较高、溶磷能力较强的27株PGPR菌株进行分泌植物激素能力测定，26株菌株具有分泌赤霉素（GA₃）的能力，分泌量为0.60~52.91 μg·mL^-1，分泌吲哚-3-乙酸（IAA）菌株18株，分泌量为0.11~1.53 μg·mL^-1，分泌玉米素（t-Z）含量均小于0.31 μg·mL^-1。筛选出综合性能优良的菌株14株，经鉴定分别属于假单胞菌属、不动杆菌属、假节杆菌属、贪噬菌属。研究结果可为研制适合高寒地区植被恢复的微生物肥料提供宝贵的菌种资源及理论基础。

关键词: 植物根际促生菌, 固氮菌, 溶磷菌, 植物激素, 耐低温菌

Abstract:

The study aimed to isolate and screen low-temperature-tolerant plant growth promoting rhizobacteria （PGPR） from the rhizosphere of the dominant plant species in alpine meadows， with a view to using the PGPRs in restoration of grassland degradation. Nitrogen-fixing， phosphorus-dissolving and phytohormone secreting bacterial species were screened by selective media and subsequently the superior strains were identified by 16S rRNA molecular biology techniques. The results indicated large numbers of bacteria in the rhizosphere of Festuca kirilowii， Koeleria cristata and Roegneria purpurascens. The quantitative distribution of bacteria exhibited a strong rhizosphere effect， with bacteria numbers on the rhizoplane or surface of roots （RP）>on soil adhering to roots （RS）>histoplane or interior of roots （HP）. In total， we obtained 76 strains of PGPR， of which 30 were nitrogen-fixing bacteria with their nitrogenase activity ranging between 124.61 and 311.04 nmol C₂H₄·h^-1·mL^-1. A further 24 strains were active in dissolution of inorganic phosphorus （249.85-558.07 μg·mL^-1in test solution）， while 22 strains could dissolve organic phosphorus 52.25-158.77 μg·mL^-1 in test solution）. Of the strains of PGPR with high nitrogenase activity or phosphate-solubilizing ability 27 strains were further selected for determination of plant hormone secretion. Results indicated that 26 strains of PGPR secreted GA₃， with the secretion amounts ranging from 0.60 to 52.91 μg·mL^-1 of test solution. Meanwhile， 18 strains of PGPR secreted IAA with the levels in test solution ranging from 0.11-1.53 μg·mL^-1， and all strains secreted t-Z at less than 0.31 μg·mL^-1. Preliminary identification of 14 superior PGPR strains using phylogenetic analysis determined that these belong to the bacterial genera Pseudomonas， Acinetobacter， Pseudarthrobacter and Variovorax. The superior PGPR strains detected in this study are potentially valuable bacterial resources that could be used for the development of microbial fertilizers suitable for vegetation restoration in alpine regions.

Key words: plant growth promoting rhizobacteria （PGPR）, nitrogen-fixing rhizobacteria, phosphorus-solubilizing rhizobacteria, plant hormones, low-temperature-tolerant rhizobacteria

刘晓婷, 姚拓. 高寒草地耐低温植物根际促生菌的筛选鉴定及特性研究[J]. 草业学报, 2022, 31(8): 178-187.

Xiao-ting LIU, Tuo YAO. Screening， identification and characteristics of low-temperature-tolerant plant growth promoting rhizobacteria in alpine meadow[J]. Acta Prataculturae Sinica, 2022, 31(8): 178-187.

图/表 6

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菌株 Strain	部位 Part	pH	固氮酶活性NA （nmol·h^-1·mL^-1）	菌株 Strain	部位 Part	pH	固氮酶活性NA （nmol·h^-1·mL^-1）
NCM3	HP	产碱Alkali-producing	124.61±2.79h	NBQ6	RP	中性Neutral	169.22±3.11bcdefg
NCM4	HP	产酸Acid-producing	133.62±10.09fgh	NBQ7	RP	产酸Acid-producing	150.69±13.77bcdefgh
NCZ1	HP	产碱Alkali-producing	311.04±2.82a	NBQ9	RP	产酸Acid-producing	146.73±18.59cdefgh
NCQ2	HP	产酸Acid-producing	176.81±1.91bcde	NAM6	RS	产碱Alkali-producing	180.82±4.14bcde
NCQ3	HP	中性Neutral	168.94±1.35bcdefg	NAM7	RS	产酸Acid-producing	187.22±8.46bc
NCQ4	HP	产碱Alkali-producing	132.09±1.11gh	NAZ1	RS	产碱Alkali-producing	143.39±6.06defgh
NCQ6	HP	中性Neutral	162.57±5.15bcdefgh	NAZ4	RS	产酸Acid-producing	159.55±13.20bcdefgh
NCQ7	HP	产碱Alkali-producing	154.59±6.16bcdefgh	NAZ6	RS	产碱Alkali-producing	151.59±2.90bcdefgh
NCQ9	HP	产碱Alkali-producing	140.92±6.32efgh	NAZ9	RS	产酸Acid-producing	169.49±8.19bcdefg
NAQ6	RS	产碱Alkali-producing	159.82±3.07bcdefgh	NAZ11	RS	产酸Acid-producing	188.74±6.27b
NAQ8	RS	产碱Alkali-producing	146.53±3.86cdefgh	NAZ12	RS	产碱Alkali-producing	155.83±5.65bcdefgh
NAQ9	RS	产酸Acid-producing	159.78±6.30bcdefgh	NAZ16	RS	产碱Alkali-producing	179.56±0.79bcde
NAQ11	RS	产碱Alkali-producing	150.14±6.72bcdefgh	NAZ17	RS	产碱Alkali-producing	150.03±10.05bcdefgh
NAQ15	RS	产酸Acid-producing	187.31±7.58bc	NAZ19	RS	中性Neutral	183.73±4.37bcd
NBZ7	RP	产碱Alkali-producing	173.12±1.39bcdef	NBM5	RP	产酸Acid-producing	165.47±6.77bcdefg

菌株 Strain	部位 Part	pH	固氮酶活性NA （nmol·h^-1·mL^-1）	菌株 Strain	部位 Part	pH	固氮酶活性NA （nmol·h^-1·mL^-1）
NCM3	HP	产碱Alkali-producing	124.61±2.79h	NBQ6	RP	中性Neutral	169.22±3.11bcdefg
NCM4	HP	产酸Acid-producing	133.62±10.09fgh	NBQ7	RP	产酸Acid-producing	150.69±13.77bcdefgh
NCZ1	HP	产碱Alkali-producing	311.04±2.82a	NBQ9	RP	产酸Acid-producing	146.73±18.59cdefgh
NCQ2	HP	产酸Acid-producing	176.81±1.91bcde	NAM6	RS	产碱Alkali-producing	180.82±4.14bcde
NCQ3	HP	中性Neutral	168.94±1.35bcdefg	NAM7	RS	产酸Acid-producing	187.22±8.46bc
NCQ4	HP	产碱Alkali-producing	132.09±1.11gh	NAZ1	RS	产碱Alkali-producing	143.39±6.06defgh
NCQ6	HP	中性Neutral	162.57±5.15bcdefgh	NAZ4	RS	产酸Acid-producing	159.55±13.20bcdefgh
NCQ7	HP	产碱Alkali-producing	154.59±6.16bcdefgh	NAZ6	RS	产碱Alkali-producing	151.59±2.90bcdefgh
NCQ9	HP	产碱Alkali-producing	140.92±6.32efgh	NAZ9	RS	产酸Acid-producing	169.49±8.19bcdefg
NAQ6	RS	产碱Alkali-producing	159.82±3.07bcdefgh	NAZ11	RS	产酸Acid-producing	188.74±6.27b
NAQ8	RS	产碱Alkali-producing	146.53±3.86cdefgh	NAZ12	RS	产碱Alkali-producing	155.83±5.65bcdefgh
NAQ9	RS	产酸Acid-producing	159.78±6.30bcdefgh	NAZ16	RS	产碱Alkali-producing	179.56±0.79bcde
NAQ11	RS	产碱Alkali-producing	150.14±6.72bcdefgh	NAZ17	RS	产碱Alkali-producing	150.03±10.05bcdefgh
NAQ15	RS	产酸Acid-producing	187.31±7.58bc	NAZ19	RS	中性Neutral	183.73±4.37bcd
NBZ7	RP	产碱Alkali-producing	173.12±1.39bcdef	NBM5	RP	产酸Acid-producing	165.47±6.77bcdefg

菌株Strain	部位 Part	D/d	溶磷量PSC （μg·mL^-1）	pH	菌株Strain	部位 Part	D/d	溶磷量PSC （μg·mL^-1）	pH
PAM2	RS	2.59±0.04d	322.59±3.44k	4.76±0.02cdef	PBQ6	RP	3.32±0.01ab	272.14±3.20l	4.27±0.05k
PAM5	RS	2.34±0.05e	321.08±4.90k	4.47±0.02ghijk	PAQ1	RS	1.69±0.02j	399.87±8.72fg	4.66±0.02defgh
PAM9	RS	3.36±0.02a	387.60±0.21gh	4.93±0.03abc	PAQ2	RS	1.51±0.01l	361.13±7.23j	4.93±0.07abc
PAM11	RS	3.25±0.04b	249.85±4.38m	4.84±0.03bcde	PAQ3	RS	2.22±0.03f	433.47±3.95e	4.40±0.01ijk
PAM12	RS	2.02±0.01h	361.12±2.34j	4.90±0.01abcd	PAQ4	RS	1.70±0.01j	509.77±5.06bc	4.34±0.02jk
PAM15	RS	2.24±0.01f	330.77±0.65k	4.43±0.30hijk	PAQ7	RS	1.98±0.01h	527.19±7.97b	4.70±0.02cdefg
PBM3	RP	2.12±0.01g	363.56±2.90ij	4.77±0.02cde	PAQ8	RS	2.22±0.02f	491.68±8.40cd	4.75±0.01cdef
PBM5	RP	1.59±0.02k	339.34±1.91k	5.03±0.00ab	PAQ14	RS	2.75±0.03c	319.89±4.78k	4.71±0.02cdefg
PBM6	RP	2.74±0.02c	378.89±8.67ghi	5.10±0.02a	PAZ2	RS	1.83±0.00i	498.46±8.47cd	4.82±0.01bcde
PBQ1	RP	2.17±0.03fg	414.58±2.35ef	4.52±0.02fghij	PAZ10	RS	2.15±0.01fg	480.82±10.92d	4.90±0.05abcd
PBQ3	RP	2.39±0.01e	381.58±2.66ghi	4.62±0.01efghi	PAZ16	RS	1.51±0.03l	424.40±1.91e	4.31±0.08jk
PBQ4	RP	2.74±0.01c	399.25±4.85fg	4.06±0.01l	PAZ17	RS	1.85±0.02i	558.07±6.97a	4.59±0.01efghi

菌株Strain	部位 Part	D/d	溶磷量PSC （μg·mL^-1）	pH	菌株Strain	部位 Part	D/d	溶磷量PSC （μg·mL^-1）	pH
PAM2	RS	2.59±0.04d	322.59±3.44k	4.76±0.02cdef	PBQ6	RP	3.32±0.01ab	272.14±3.20l	4.27±0.05k
PAM5	RS	2.34±0.05e	321.08±4.90k	4.47±0.02ghijk	PAQ1	RS	1.69±0.02j	399.87±8.72fg	4.66±0.02defgh
PAM9	RS	3.36±0.02a	387.60±0.21gh	4.93±0.03abc	PAQ2	RS	1.51±0.01l	361.13±7.23j	4.93±0.07abc
PAM11	RS	3.25±0.04b	249.85±4.38m	4.84±0.03bcde	PAQ3	RS	2.22±0.03f	433.47±3.95e	4.40±0.01ijk
PAM12	RS	2.02±0.01h	361.12±2.34j	4.90±0.01abcd	PAQ4	RS	1.70±0.01j	509.77±5.06bc	4.34±0.02jk
PAM15	RS	2.24±0.01f	330.77±0.65k	4.43±0.30hijk	PAQ7	RS	1.98±0.01h	527.19±7.97b	4.70±0.02cdefg
PBM3	RP	2.12±0.01g	363.56±2.90ij	4.77±0.02cde	PAQ8	RS	2.22±0.02f	491.68±8.40cd	4.75±0.01cdef
PBM5	RP	1.59±0.02k	339.34±1.91k	5.03±0.00ab	PAQ14	RS	2.75±0.03c	319.89±4.78k	4.71±0.02cdefg
PBM6	RP	2.74±0.02c	378.89±8.67ghi	5.10±0.02a	PAZ2	RS	1.83±0.00i	498.46±8.47cd	4.82±0.01bcde
PBQ1	RP	2.17±0.03fg	414.58±2.35ef	4.52±0.02fghij	PAZ10	RS	2.15±0.01fg	480.82±10.92d	4.90±0.05abcd
PBQ3	RP	2.39±0.01e	381.58±2.66ghi	4.62±0.01efghi	PAZ16	RS	1.51±0.03l	424.40±1.91e	4.31±0.08jk
PBQ4	RP	2.74±0.01c	399.25±4.85fg	4.06±0.01l	PAZ17	RS	1.85±0.02i	558.07±6.97a	4.59±0.01efghi

菌株Strain	赤霉素GA₃	吲哚-3-乙酸IAA	玉米素t-Z	菌株Strain	赤霉素GA₃	吲哚-3-乙酸IAA	玉米素t-Z
PAZ10	-	0.13±0.01fg	0.13±0.01bc	MAQ19	2.59±0.87gh	-	0.18±0.01b
PAZ17	26.07±1.92c	0.39±0.08c	0.19±0.00ab	MBM11	1.60±0.09h	0.11±0.01g	0.21±0.00ab
PAM9	19.62±1.27d	0.15±0.01fg	0.11±0.00c	MCZ1	3.04±0.18gh	0.77±0.05b	0.14±0.02bc
PAQ2	52.91±3.85a	0.34±0.03cd	-	NAQ6	4.40±0.94fgh	-	0.31±0.13a
PAQ3	5.68±0.28fgh	-	0.12±0.00bc	NAQ11	14.73±1.07e	0.18±0.04fg	-
PAQ7	3.37±0.42gh	-	0.22±0.01ab	NAQ15	1.82±0.62h	-	0.12±0.00bc
PBQ4	3.29±1.20gh	0.14±0.01fg	0.16±0.02bc	NAZ12	33.98±2.50b	-	0.12±0.01bc
PBM6	0.78±0.01h	0.15±0.01fg	0.26±0.07ab	NAZ17	1.10±0.00h	0.12±0.01fg	0.11±0.00c
MAQ10	3.76±0.24gh	0.12±0.01fg	0.16±0.05bc	NAM6	1.77±0.12h	0.12±0.01fg	0.16±0.03bc
MAZ14	0.60±0.04h	0.30±0.02cde	0.12±0.01bc	NBQ7	1.10±0.01h	0.13±0.01fg	0.14±0.00bc
MAZ15	8.61±0.60f	0.26±0.02efg	0.15±0.01bc	NBQ9	3.39±1.31gh	-	0.13±0.00bc
MAQ23 MBQ3 MBQ13	0.71±0.04h 2.80±0.00gh 20.56±1.50d	- - 0.17±0.01fg	0.12±0.00bc 0.11±0.01c -	NBM5 NCZ1	7.89±0.75fg 2.05±0.24h	0.21±0.02fg 1.53±0.11a	0.14±0.03bc -