产生促生挥发性物质的潜在PGPR菌株筛选及其促生特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0619

摘要/Abstract

摘要：

旨在筛选出能产生促进植物生长的挥发性有机化合物（volatile organic compounds,VOCs）的潜在植物根际促生菌（plant growth promoting rhizobacteria,PGPR）,考察其VOCs对植物的促生作用及其它促生功能,为研发微生物肥料提供新的思路及可靠的材料。以本实验室从海洋样品中分离保存的48株功能菌为供试菌株,通过二分隔平板实验、VOCs盆栽实验进行筛选,结合菌株的16S rRNA进行鉴定,通过气相色谱-质谱联用的方法（Gas Chromatography-Mass,GC-MS）对菌株所产VOCs成分进行分析。通过平板活性实验对细菌的固氮,溶磷及产IAA进行活性检测。研究结果显示：筛选得到1株潜在PGPR - GX14001,其所产生的VOCs对本氏烟草（Nicotiana benthamiana）和上海青（Brassica chinensis L.）均表现出明显的促生作用,菌株经16S rRNA鉴定为橙色微杆菌（Microbacterium aurantiacum）。经GC-MS对其VOCs成分分析,共得到7种特异性化合物。经平板活性检测,GX14001具有较强的溶有机/无机磷活性和一般固氮活性,而其产IAA能力较弱,为1.737 μg/mL。PGPR可通过不同的促生机制对植物表现出促生作用,研究结果表明GX14001所产VOCs对植物有明显促生作用,其他方面的促生活性并不高,两者之间不存在较高的正相关性,说明其最主要的促生作用是通过所产生的VOCs获得的。

关键词: 植物促生菌, 挥发性有机物, 橙色微杆菌, 溶磷

Abstract:

The aim of this work is to screen the potential plant growth-promoting rhizobacteria（PGPR）whose volatile organic compounds（VOCs）can promote plant growth,and to investigate the growth-promoting effect of the VOCs on plants and other growth-promoting functions,which provides new ideas and reliable materials for the research and development of microbial fertilizers. Using 48 functional bacteria isolated from marine samples and preserved in our laboratory as test strains,two-compartment plate test and VOCs pot experiment were used to screen them,16S rRNA to identify them,and GC-MS to analyze the VOCs components produced by the strains. Finally plate activity test was applied to detect the activities of the bacteria for nitrogen fixation,phosphorus solubilization and IAA production. The results showed that a strain of potential PGPR GX14001 was obtained. The VOCs produced by PGPR-GX14001 showed obvious growth-promoting effects on both Nicotiana benthamiana and Brassica chinensis L. seedlings. The strain GX14001 was identified as Microbacterium aurantiacum by 16S rRNA. Seven specific compounds were obtained by GC-MS analysis of VOCs. The GX14001 had strong dissolved organic/inorganic phosphorus activity and general nitrogen fixation activity,but weak IAA production capacity,which was 1.737 μg/mL. PGPR promoted the growth of plants through different growth-promoting mechanisms. The results showed that the VOCs produced by GX14001 had obvious growth-promoting effect on plants,but not high in the other aspects of growth-promoting activity;and there was no high positive correlation between them,indicating that the most important growth-promoting effect was via the produced VOCs.

Key words: plant growth-promoting rhizobacteria（PGPR）, volatile organic compounds（VOCs）, Microbacterium aurantiacum, phosphorus solubilization

高亚慧, 姜明国, 丰景, 周桂. 产生促生挥发性物质的潜在PGPR菌株筛选及其促生特性研究[J]. 生物技术通报, 2022, 38(3): 103-112.

GAO Ya-hui, JIANG Ming-guo, FENG Jing, ZHOU Gui. Screening of Potential PGPR Strains Producting Growth-promoting Volatile Compounds and Study on Their Growth-promoting Characteristics[J]. Biotechnology Bulletin, 2022, 38(3): 103-112.

图/表 9

参考文献 32

[1]	Adesemoye AO, Kloepper JW. Plant-microbes interactions in enhanced fertilizer-use efficiency[J]. Appl Microbiol Biotechnol, 2009, 85(1):1-12. doi: 10.1007/s00253-009-2196-0 pmid: 19707753
[2]	李俊, 姜昕, 马鸣超, 等. 我国微生物肥料产业需求与技术创新[J]. 中国土壤与肥料, 2019(2):1-5.
	Li J, Jiang X, Ma MC, et al. Development demand and technical innovation for bio-fertilizer industry in China[J]. Soil Fertil Sci China, 2019(2):1-5.
[3]	Ryu CM, Farag MA, Hu CH, et al. Bacterial volatiles promote growth in Arabidopsis[J]. PNAS, 2003, 100(8):4927-4932. doi: 10.1073/pnas.0730845100 URL
[4]	Zhang H, Kim MS, Krishnamachari V, et al. Rhizobacterial volatile emissions regulate auxin homeostasis and cell expansion in Arabidopsis[J]. Planta, 2007, 226(4):839-851. doi: 10.1007/s00425-007-0530-2 URL
[5]	Rudrappa T, Biedrzycki ML, Kunjeti SG, et al. The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana[J]. Commun Integr Biol, 2010, 3(2):130-138. doi: 10.4161/cib.3.2.10584 URL
[6]	Zhang H, Sun Y, Xie X, et al. A soil bacterium regulates plant acquisition of iron via deficiency-inducible mechanisms[J]. Plant J, 2009, 58(4):568-577. doi: 10.1111/tpj.2009.58.issue-4 URL
[7]	Cappellari LDR, Banchio E. Microbial volatile organic compounds produced by Bacillus amyloliquefaciens GB03 ameliorate the effects of salt stress in Mentha piperita principally through acetoin emission[J]. J Plant Growth Regul, 2020, 39(2):764-775. doi: 10.1007/s00344-019-10020-3 URL
[8]	Yasmin H, Rashid U, Hassan MN, et al. Volatile organic compounds produced by Pseudomonas pseudoalcaligenes alleviated drought stress by modulating defense system in maize(Zea mays L.)[J]. Physiol Plant, 2021, 172(2):896-911. doi: 10.1111/ppl.v172.2 URL
[9]	Choudoir M, Rossabi S, Gebert M, et al. A phylogenetic and functional perspective on volatile organic compound production by actinobacteria[J]. mSystems, 2019, 4(2):e00295-18.
[10]	Lee S, Behringer G, Hung R, et al. Effects of fungal volatile organic compounds on Arabidopsis thaliana growth and gene expression[J]. Fungal Ecol, 2019, 37:1-9. doi: 10.1016/j.funeco.2018.08.004 URL
[11]	Paul D, Park KS. Identification of volatiles produced by Cladosporium cladosporioides CL-1, a fungal biocontrol agent that promotes plant growth[J]. Sensors:Basel, 2013, 13(10):13969-13977. doi: 10.3390/s131013969 URL
[12]	Tahir HA, Gu Q, Wu H, et al. Plant growth promotion by volatile organic compounds produced by Bacillus subtilis SYST2[J]. Front Microbiol, 2017, 8:171.
[13]	Zhang HM, Murzello C, Sun Y, et al. Choline and osmotic-stress tolerance induced in Arabidopsis by the soil microbe Bacillus subtilis(GB03)[J]. Mol Plant Microbe Interact, 2010, 23(8):1097-1104. doi: 10.1094/MPMI-23-8-1097 URL
[14]	Lee B, Farag MA, Park HB, et al. Induced resistance by a long-chain bacterial volatile:elicitation of plant systemic defense by a C13 volatile produced by Paenibacillus polymyxa[J]. PLoS One, 2012, 7(11):e48744. doi: 10.1371/journal.pone.0048744 URL
[15]	白变霞, 陈艳彬, 任嘉红. 蜡状芽孢杆菌CLY07菌株的解有机磷特性研究[J]. 西南林业大学学报, 2016, 36(4):75-81.
	Bai BX, Chen YB, Ren JH. Study on Organophosphate-solubilizing characteristics of Bacillus cereus CLY07[J]. J Southwest For Univ, 2016, 36(4):75-81.
[16]	Nautiyal CS. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms[J]. FEMS Microbiol Lett, 1999, 170(1):265-270. pmid: 9919677
[17]	徐晓芬. 类芽孢杆菌BD3526在无氮固体培养基上产胞外多糖的研究[J]. 食品工业科技, 2017, 38(16):95-100.
	Xu XF. Study on the exopolysaccharides produced by Paenibacillus bovis sp. nov BD3526 in nitrogen-free solid culture medium[J]. Sci Technol Food Ind, 2017, 38(16):95-100.
[18]	胡江春, 薛德林, 马成新, 等. 植物根际促生菌(PGPR)的研究与应用前景[J]. 应用生态学报, 2004, 15(10):1963-1966.
	Hu JC, Xue DL, Ma CX, et al. Research advances in plant growth-promoting rhizobacteria and its application prospects[J]. Chin J Appl Ecol, 2004, 15(10):1963-1966.
[19]	Ahemad M, Kibret M. Mechanisms and applications of plant growth promoting rhizobacteria:Current perspective[J]. J King Saud Univ Sci, 2014, 26(1):1-20. doi: 10.1016/j.jksus.2013.05.001 URL
[20]	Xie SS, Wu HJ, Zang HY, et al. Plant growth promotion by spermidine-producing Bacillus subtilis OKB105[J]. Mol Plant Microbe Interact, 2014, 27(7):655-663. doi: 10.1094/MPMI-01-14-0010-R URL
[21]	Cordovez V, Schop S, Hordijk K, et al. Priming of plant growth promotion by volatiles of root-associated Microbacterium spp[J]. Appl Environ Microbiol, 2018, 84(22):e01865-18.
[22]	Mills GA, Walker V. Headspace solid-phase microextraction procedures for gas chromatographic analysis of biological fluids and materials[J]. J Chromatogr A, 2000, 902(1):267-287. pmid: 11192159
[23]	Blom D, Fabbri C, Connor EC, et al. Production of plant growth modulating volatiles is widespread among rhizosphere bacteria and strongly depends on culture conditions[J]. Environ Microbiol, 2011, 13(11):3047-3058. doi: 10.1111/j.1462-2920.2011.02582.x pmid: 21933319
[24]	Velázquez-Becerra C, Macías-Rodríguez LI, López-Bucio J, et al. A volatile organic compound analysis from Arthrobacter agilis identifies dimethylhexadecylamine, an amino-containing lipid modulating bacterial growth and Medicago sativa morphogenesis in vitro[J]. Plant Soil, 2011, 339(1/2):329-340. doi: 10.1007/s11104-010-0583-z URL
[25]	Yamagiwa Y, Inagaki Y, Ichinose Y, et al. Talaromyces wortmannii FS₂ emits β-caryphyllene, which promotes plant growth and induces resistance[J]. J Gen Plant Pathol, 2011, 77(6):336-341. doi: 10.1007/s10327-011-0340-z URL
[26]	Jishma P, Hussain N, Chellappan R, et al. Strain-specific variation in plant growth promoting volatile organic compounds production by five different Pseudomonas spp. as confirmed by response of Vigna radiata seedlings[J]. J Appl Microbiol, 2017, 123(1):204-216. doi: 10.1111/jam.13474 pmid: 28423218
[27]	Xu Z, Mulchandani A, Chen W. Detection of benzene, toluene, ethyl benzene, and xylenes(BTEX)using toluene dioxygenase-peroxidase coupling reactions[J]. Biotechnol Prog, 2003, 19(6):1812-1815. doi: 10.1021/bp0341794 URL
[28]	Bhattacharyya PN, Jha DK. Plant growth-promoting rhizobacteria(PGPR):emergence in agriculture[J]. World J Microbiol Biotechnol, 2012, 28(4):1327-1350. doi: 10.1007/s11274-011-0979-9 URL
[29]	Dastager SG, Damare S. Marine actinobacteria showing phosphate-solubilizing efficiency in chorao island, Goa, India[J]. Curr Microbiol, 2013, 66(5):421-427. doi: 10.1007/s00284-012-0288-z pmid: 23288302
[30]	Kishore N, Pindi PK, Ram Reddy S. Phosphate-solubilizing microorganisms:A critical review[M]//Plant Biology and Biotechnology. New Delhi:Springer India, 2015:307-333.
[31]	Karlidag H, Esitken A, Turan M, et al. Effects of root inoculation of plant growth promoting rhizobacteria(PGPR)on yield, growth and nutrient element contents of leaves of apple[J]. Sci Hortic, 2007, 114(1):16-20. doi: 10.1016/j.scienta.2007.04.013 URL
[32]	Sheng XF, Xia JJ, Jiang CY, et al. Characterization of heavy metal-resistant endophytic bacteria from rape(Brassica napus)roots and their potential in promoting the growth and lead accumulation of rape[J]. Environ Pollut, 2008, 156(3):1164-1170. doi: 10.1016/j.envpol.2008.04.007 pmid: 18490091

菌株编号Strain No.	菌株名称Name of strain
GX13159	Bacillus cereus
GX13529	Staphylococcus edaphicus
GX13571	Asticcacaulis excentricus
GX13580	Micrococcus aloeverae
GX13585	Microbacterium hydrothermale
GX13594	Microbacterium laevaniformans
GX13656	Bacillus acidiceler
GX13667	Bacillus aryabhattai
GX13668	Micrococcus aloeverae
GX13676	Streptomyces diastaticus subsp. ardesiacus
GX13678	Streptomyces sanglieri
GX13679	Falsibacillus pallidus
GX13685	Sinomonas humi
GX13699	Cellulosimicrobium funkei
GX13717	Kocuria indica
GX13747	Paenibacillus silvae
GX13767	Bacillus mangrovi
GX13774	Bacillus marisflavi
GX13777	Halobacillus marinus
GX13785	Kosakonia oryziphila
GX13790	Serratia oryzae
GX14001	Microbacterium aurantiacum
GX14008	Microbacterium maritypicum
GX14011	Exiguobacterium aestuarii
GX14016	Staphylococcus haemolyticus
GX14017	Staphylococcus hominis subsp. novobiosepticus
GX14022	Staphylococcus nepalensis
GX14028	Kytococcus sedentarius
GX14031	Sporosarcina koreensis
GX14032	Macrococcus canis
GX14046	Glutamicibacter creatinolyticus
GX14059	Pontibacter chinhatensis
GX14066	Kocuria tytonicola
GX14070	Micrococcus luteus
GX14080	Macrococcus caseolyticus subsp. Caseolyticus
GX14105	Bacillus altitudinis
GX14108	Brachybacterium squillarum
GX14201	Staphylococcus sciuri
GX14203	Bacillus haikouensis
GX14062	Agromyces indicus
GX14082	Psychrobacter pulmonis
GX14096	Pseudomonas deceptionensis
GX13112	Klebsiella Trevisan
GX13213	Klebsiella variicola subsp. Variicola
GX14104	Streptomyces griseus subsp. griseus
GX14111	Streptomyces koyangensis
GX14119	Streptomyces gramineus
GX14125	Streptomyces flavovirens

菌株编号Strain No.	菌株名称Name of strain
GX13159	Bacillus cereus
GX13529	Staphylococcus edaphicus
GX13571	Asticcacaulis excentricus
GX13580	Micrococcus aloeverae
GX13585	Microbacterium hydrothermale
GX13594	Microbacterium laevaniformans
GX13656	Bacillus acidiceler
GX13667	Bacillus aryabhattai
GX13668	Micrococcus aloeverae
GX13676	Streptomyces diastaticus subsp. ardesiacus
GX13678	Streptomyces sanglieri
GX13679	Falsibacillus pallidus
GX13685	Sinomonas humi
GX13699	Cellulosimicrobium funkei
GX13717	Kocuria indica
GX13747	Paenibacillus silvae
GX13767	Bacillus mangrovi
GX13774	Bacillus marisflavi
GX13777	Halobacillus marinus
GX13785	Kosakonia oryziphila
GX13790	Serratia oryzae
GX14001	Microbacterium aurantiacum
GX14008	Microbacterium maritypicum
GX14011	Exiguobacterium aestuarii
GX14016	Staphylococcus haemolyticus
GX14017	Staphylococcus hominis subsp. novobiosepticus
GX14022	Staphylococcus nepalensis
GX14028	Kytococcus sedentarius
GX14031	Sporosarcina koreensis
GX14032	Macrococcus canis
GX14046	Glutamicibacter creatinolyticus
GX14059	Pontibacter chinhatensis
GX14066	Kocuria tytonicola
GX14070	Micrococcus luteus
GX14080	Macrococcus caseolyticus subsp. Caseolyticus
GX14105	Bacillus altitudinis
GX14108	Brachybacterium squillarum
GX14201	Staphylococcus sciuri
GX14203	Bacillus haikouensis
GX14062	Agromyces indicus
GX14082	Psychrobacter pulmonis
GX14096	Pseudomonas deceptionensis
GX13112	Klebsiella Trevisan
GX13213	Klebsiella variicola subsp. Variicola
GX14104	Streptomyces griseus subsp. griseus
GX14111	Streptomyces koyangensis
GX14119	Streptomyces gramineus
GX14125	Streptomyces flavovirens

组别 Group	鲜重 Fresh weight/g	根长 Root lengt/cm	侧根数 Number of lateral root/roots	叶片长 Leaf length/cm	叶片宽 Leaf width/cm
对照组Control	0.081±0.015	2.855±0.656	1.887±0.726	0.551±0.093	0.443±0.089
GX13594	0.503±0.214^**	4.627±1.481^**	4.400±1.183^**	0.990±0.217^**	0.730±0.158^**
GX13747	0.387±0.078^**	5.187±1.351^**	4.267±0.799^**	1.023±0.145^**	0.772±0.157^**
GX14001	0.703±0.300^**	4.530±1.129^**	4.267±1.870^**	1.250±0.260^**	0.880±0.190^**
GX14016	0.487±0.106^**	4.853±1.255^**	5.333±1.589^**	1.133±0.247^**	0.775±0.191^**
GX14066	0.620±0.100^**	4.243±0.918^**	4.533±1.552^**	1.285±0.244^**	0.958±0.260^**
GX14070	0.300±0.183^**	4.600±0.858^**	3.867±1.125^**	0.950±0.163^**	0.742±0.129^**
GX14201	0.457±0.093^**	4.486±1.143^**	4.643±1.082^**	1.107±0.289^**	0.764±0.209^**

组别 Group	鲜重 Fresh weight/g	根长 Root lengt/cm	侧根数 Number of lateral root/roots	叶片长 Leaf length/cm	叶片宽 Leaf width/cm
对照组Control	0.081±0.015	2.855±0.656	1.887±0.726	0.551±0.093	0.443±0.089
GX13594	0.503±0.214^**	4.627±1.481^**	4.400±1.183^**	0.990±0.217^**	0.730±0.158^**
GX13747	0.387±0.078^**	5.187±1.351^**	4.267±0.799^**	1.023±0.145^**	0.772±0.157^**
GX14001	0.703±0.300^**	4.530±1.129^**	4.267±1.870^**	1.250±0.260^**	0.880±0.190^**
GX14016	0.487±0.106^**	4.853±1.255^**	5.333±1.589^**	1.133±0.247^**	0.775±0.191^**
GX14066	0.620±0.100^**	4.243±0.918^**	4.533±1.552^**	1.285±0.244^**	0.958±0.260^**
GX14070	0.300±0.183^**	4.600±0.858^**	3.867±1.125^**	0.950±0.163^**	0.742±0.129^**
GX14201	0.457±0.093^**	4.486±1.143^**	4.643±1.082^**	1.107±0.289^**	0.764±0.209^**

编号No.	保留时间Retaining time/ min	化学名称Chemical name	峰面积Peak area
1	9.3353	邻乙基甲苯（1-ethyl-2-methyl- Benzene）	1335701
2	9.9132	1,2,3-三甲苯（1,2,3-trimethyl- Benzene）	2357671
3	11.1091	1,2,4-三甲苯（1,2,4-trimethyl- Benzene）	294469
4	11.298	4-甲基-癸烷（4-methyl-Decane）	917363
5	12.8658	十二烷（Dodecane）	3497194
6	32.2232	二十五烷（Pentacosane）	671059
7	34.1057	9-辛基-十七烷（9-octyl-Heptadecane）	387135