三株硫氧化细菌的筛选鉴定及其生物活性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0731

摘要/Abstract

摘要：

目的针对农业土壤硫有效性低、作物缺硫的问题，从云南油菜根际土壤中筛选兼具高效硫氧化能力与多种生物活性的硫氧化细菌（sulfur-oxidizing bacteria, SOB），明确其农业应用潜力。方法采用定向富集分离法，通过pH降低和硫酸盐生成量评估硫氧化活性，结合固氮、溶锌、磷酸盐溶解等功能测定筛选多功能菌株；进一步测定活性菌株分泌铁载体、吲哚乙酸和拮抗病原菌等生物活性，并验证促种子发芽和番茄植株生长效果。结果分离获得313株SOB，其中16株具有较强的硫氧化能力（硫酸盐产量38.53-66.54 mg/L）；结合形态学、生理生化特性和16S rDNA序列测序，3株高活性菌株YNK-FB0053、YNK-FB0056和YNK-FB0057分别鉴定为唐菖蒲伯克霍尔德氏菌（Burkholderia gladioli）、泛菌（Pantoea endophytica）和阿氏芽胞杆菌（Priestia aryabhattai）；3个菌株兼具固氮、溶锌、磷酸盐溶解和分泌铁载体活性并携带srfAB、yndJ、ituC、sboA、bioA、NRPS和PKSI等抗生素基因；种子萌发试验显示，3株菌对油菜、番茄、黑麦萌发率提升16.67%-44.33%，整株长增加17.95%-33.70%；番茄盆栽试验中，3株菌均显著（P<0.05）促进生长，其中叶绿素含量、株高和茎粗提高约15%-30%，根长增加超过55%，地下部生物量增幅最为显著。养分积累方面，全氮、全磷、全钾和全硫均有所提升，其中铁元素含量增加最为明显，提升了112.50%。结论云南油菜根际土壤富含兼具高效硫氧化与多种生物活性的SOB，YNK-FB0053、YNK-FB0056和YNK-FB0057可显著促进种子萌发、番茄生长及植株元素吸收，具有良好的农业应用潜力。

关键词: 土壤硫循环, 硫氧化细菌, 唐菖蒲伯克霍尔德氏菌, 泛菌, 阿氏芽胞杆菌, 微生物资源

Abstract:

Objective To address the issues of low sulfur availability in agricultural soils and sulfur deficiency in crops, this study is aimed to screen sulfur-oxidizing bacteria (SOB) with both high sulfur oxidation efficiency and multiple bioactivities from the rhizosphere soil of oilseed rape in Yunnan, China, and to evaluate their potential for agricultural application. Method SOB were isolated using targeted enrichment and isolation techniques. Sulfur oxidation activity was evaluated based on pH reduction and sulfate production. Isolates were screened for multifunctional PGP traits, including nitrogen fixation, zinc solubilization, and phosphate solubilization. Promising strains were further assessed for biological activities such as siderophore production, heteroauxin (IAA) secretion, and antagonism against plant pathogens. Their efficacy in promoting seed germination and Solanum lycopersicum plant growth was validated. Result A total of 313 SOB strains were isolated. Among them, 16 Strains presented strong sulfur-oxidizing capacity (sulfate production: 38.53-66.54 mg/L). Based on morphological, physiological, biochemical characteristics, and 16S rDNA sequence analysis, three high-activity strains, YNK-FB0053, YNK-FB0056, and YNK-FB0057, were identified as Burkholderia gladioli, Pantoea endophytica, and Priestia aryabhattai, respectively. All three strains possessed the activities of nitrogen fixation, zinc solubilization, phosphate solubilization, and siderophore secretion. They also harbored genes associated with antibiotic synthesis (srfAB, yndJ, ituC, sboA, bioA, NRPS, and PKSI). Seed germination tests demonstrated that these strains increased germination rates of Brassica napus L, Solanum lycopersicum, and Secale cereale by 16.67%–44.33% and total seedling length by 17.95%–33.70%. In pot experiments with Solanum lycopersicum, all three strains significantly (P<0.05) promoted plant growth, with chlorophyll content, plant height, and stem diameter increased by approximately 15%–30%, root length extended by more than 55%, and belowground biomass showing the most pronounced improvement. In terms of nutrient accumulation, total nitrogen, phosphorus, potassium, and sulfur contents were all enhanced, while iron content had the greatest increase, reaching 112.50%. Conclusion The rhizosphere soil of Yunnan oilseed rape harbors SOB strains with both high-efficiency sulfur oxidation and multiple plant growth-promoting functions. Strain YNK-FB0053 (B. gladioli), YNK-FB0056 (P. endophytica), and YNK-FB0057 (P. aryabhattai) significantly enhance seed germination, Solanum lycopersicum plant growth, and mineral element accumulation, demonstrating substantial potential for agricultural application as novel multifunctional bioinoculants.

Key words: soil sulfur cycle, sulfur-oxidizing bacteria, Burkholderia gladioli, Pantoea endophytica, Priestia aryabhattai, microbial resources

陈艳, 刘鑫, 施竹凤, 普特, 刘恩德, 张义杰, 曹艳茹, 杨佩文. 三株硫氧化细菌的筛选鉴定及其生物活性[J]. 生物技术通报, 2026, 42(1): 315-328.

CHEN Yan, LIU Xin, SHI Zhu-feng, PU Te, LIU En-de, ZHANG Yi-jie, CAO Yan-ru, YANG Pei-wen. Screening， Identification and Biological Activity of Three Sulfur-oxidizing Bacteria[J]. Biotechnology Bulletin, 2026, 42(1): 315-328.

图/表 16

表1 土壤采样点

Table 1 Soil sampling location

序号 No.	州（市） Prefectures （Cities）	海拔 Height （m）	经度（E） Longitude	纬度（N） Latitude
1	丽江	1 832	105°17′09″	27°51′27″
2	大理	1 969	100°08′28.77″	25°37′1.82″
3	曲靖	1 590	104°23′38.07″	25°21′39.99″
4	保山	1 650	98°32′31″	24°56′48.93″
5	楚雄	1 816	101°37′01.35″	25°18′33.10″
6	昭通	2 141	105°17′09″	27°51′27″
7	迪庆	3 122	98°39′11.83″	27°58′6.77″
8	怒江	2 963	99°51′35.64″	27°10′58.80″

表2 抗生素基因检测引物

Table 2 Primers for antibiotic gene detection

引物名称Primer name	引物序列Primer sequence （5′-3′）	产物长度Product length （bp）	参考文献Reference
SrfAB	GTTCTCGCAGTCCAGCAGAAG GCCGAGCGTATCCGTACCGAG	308	［25］
yndJ	CAGAGCGACAGCAATCACAT TGAATTTCGGTCCGCTTATC	212	［25］
fenD	CCTGCAGAAGGAGAAGTGAAG TGCTCATCGTCTTCCGTTTC	293	［25］
ituC	TTCACTTTTGATCTGGCGAT CGTCCGGTACATTTTCAC	575	［25］
bamC	CTGGAAGAGATGCCGCTTAC AAGAGTGCGTTTTCTTCGGA	957	［26］
dhbC	ATGTTGGATCAAAACGTTAT TCATATGTGATCCACGCCCA	1 197	［25］
sboA	TCGGTTTGTAAACTTCAACTGC GTCCACTAGACAAGCGGCTC	334	［26］
ysnE	GGCTGTGAACCTTTGCTATG GCTGTTCGGGTCCTCTTTAT	462	［25］
bioA	TTCCACGGCCATTCCTATAC TTTGTCCCCTTATCCTGCAC	210	［26］
PKSI	TSAAGTCSAACATCGGB CGCAGGTTSCSGTACCAG	1 200—1 400	［27］
PKSII	ACCGGCT GCACSTCSGGSCT ACGTAGTCSAGGTCGCASAC	700	［28］
NRPS	GCSTACSYSATSTACACSTCSGG SASGTCVCCSGTSCGGTAS	800	［27］

图1 菌株的营养转化效果图菌株YNK-FB0053的固氮（A）、溶锌（B）、解磷（C）、溶磷（D）效果；YNK-FB0056的固氮（E）、溶锌（F）、解磷（G）、溶磷（H）效果；YNK-FB0057的固氮（I）、溶锌（J）、解磷（K）、溶磷（R）效果

Fig. 1 Nutrient transformation effect diagrams of strainsEffect diagram of nitrogen fixation(A), zinc dissolution(B), phosphorus solution(C), dissolved phosphorus(D) of YNK-FB0053; nitrogen fixation(E), zinc dissolution(F), phosphorus solution(G), dissolved phosphorus(H) of YNK-FB0056; nitrogen fixation(I), zinc dissolution(J), phosphorus solution(K), dissolved phosphorus(R) of YNK-FB0057

表3 菌株的营养转化指数

Table 3 Nutrient transformation indexes of the strain

D/d	固氮指数 Nitrogen fixation	溶锌指数 Zinc dissolution	解磷指数 Phosphorus solution	溶磷指数 Phosphorus dissolution
YNK-FB0053	4.106±0.694a	5.129± 0.459a	3.453±0.320a	1.506±0.425b
YNK-FB0056	4.476±0.240a	5.087± 0.302a	3.687±0.251a	3.460±0.441a
YNK-FB0057	2.060±0.183b	2.660±0.613b	1.157±0.562b	2.910±0.571ab

图2 菌株YNK-FB0053（A）、YNK-FB0056（B）和YNK-FB0057（C）的分泌铁载体效果图

Fig. 2 Secretory siderophores of strain YNK-FB0053(A), YNK-FB0056(B), and YNK-FB0057(C)

图3 菌株YNK-FB0056和YNK-FB0057的分泌吲哚乙酸活性测定

Fig. 3 Determination of lAA activities of strain YNK-FB0056 and YNK-FB0057

图4 菌株YNK-FB0056和YNK-FB0057的连续7 d分泌吲哚乙酸情况

Fig. 4 Secretion of indole acetic acid in strains YNK-FB0056 and YNK-FB0057 for 7 consecutive days

图5 菌株对病原菌的拮抗效果菌株YNK-FB0053对烟草疫霉菌（A）、轮纹镰刀菌（B）、腐皮镰刀菌（C）、木贼镰刀菌（D）、锐顶镰刀菌（E）、尖孢镰刀菌（F）和禾谷镰刀菌（G）的拮抗效果

Fig. 5 Antagonistic effects of strains on pathogenic bacteriaAntagonistic effect of strain YNK-FB0053 on Phytophthora nicotiana (A), F. concentricum (B),F. solani (C),F. equiseti (D),F. acuminatum (E), F. oxysporum (F), and F. graminearum (G)

图6 硫氧化细菌YNK-FB0053（A）、YNK-FB0056（B）和YNK-FB0057（C）抗生素合成基因PCR的检测结果

Fig. 6 PCR detection results of antibiotic synthesis genes in sulfur-oxidizing bacteria strains YNK-FB0053(A), YNK-FB0056(B), and YNK-FB0057(C)

图7 菌株处理后的种子萌发效果菌株YNK-FB0053、YNK-FB0056和YNK-FB0057对油菜（A）、番茄（B）和黑麦（C）处理的种子苗

Fig. 7 Germination effect of strain-treated seedsStrain YNK-FB0053, YNK-FB0056 and YNK-FB0057 treated seedlings of Brassica napus L (A), Solanum lycopersicum (B), and Secale cereale (C)

图8 菌株YNK-FB0053（A）、YNK-FB0056（B）和YNK-FB0057（C）处理下种子发芽长度和发芽率

Fig. 8 Germination lengths and germination rates of seeds of strain YNK-FB0053(A), YNK-FB0056(B) and YNK-FB0057(C)

图9 菌株对番茄植株的促生效果

Fig. 9 Growth-promoting effect of strains on Solanum lycopersicum plants

图10 菌株对番茄各生长指标的促进效果

Fig. 10 Promoting effect of the strain on various growth indicators of Solanum lycopersicum

表4 番茄植株元素含量

Table 4 Mineral element contents in Solanum lycopersicum plants

处理 Treatment		全氮 Total nitrogen （g/kg）	全磷 Total phosphorus （g/kg）	全钾 Total potassium （g/kg）	全硫 Total sulfur （g/kg）	全铁 Total iron （g/kg）
CK1		37.33±0.99b	8.20±0.28c	60.27±1.69de	11.26±0.22c	0.48±0.04e
CK2		35.90±1.47b	7.80±0.38c	58.47±3.27ef	10.71±0.31de	0.50±0.05e
YNK-FB0053	T1	35.90±0.62b	9.20±0.29b	63.50±1.90cd	12.26±0.21b	0.83±0.03b
	T2	33.07±1.72c	8.75±0.35b	67.37±1.81b	11.99±0.52b	0.88±0.04b
	T3	35.17±0.64b	9.79±0.09a	73.43±0.83a	13.34±0.20a	0.78 ±0.01c
YNK-FB0056	T1	41.37±0.91a	9.11±0.65b	63.27±2.45cd	11.32±0.16c	0.73±0.10cd
	T2	30.53±0.06d	9.05±0.14b	55.00±0.82f	11.12±0.13cd	0.77±0.02c
	T3	27.33±1.18e	8.82±0.15b	64.80±1.05bc	11.47±0.16c	0.70±0.03d
YNK-FB0057	T1	35.37±1.19b	8.83±0.17b	62.77±0.81cd	10.45±0.33e	0.84±0.03b
	T2	42.40±1.35a	8.96 ±0.21b	66.63±1.56bc	10.78±0.08de	1.02±0.04a
	T3	26.27±1.81e	9.83 ±0.16a	64.80±3.77bc	11.43±0.33c	0.75±0.02cd

图11 菌株YNK-FB0053（A）、YNK-FB0056（B）和YNK-FB0057（C）的菌落形态及扫描电镜图

Fig. 11 Colony morphology and scanning electron microscope of strain YNK-FB0053(A), YNK-FB0056(B) and YNK-FB0057(C)

图12 菌株YNK-FB0053、YNK-FB0056和YNK-FB0057基于16S rRNA基因序列构建的系统发育树

Fig. 12 Phylogenetic tree of strain YNK-FB0053, YNK-FB0056 and YNK-FB0057 based on 16S rRNA gene sequences

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