成都平原蚕豆高效根瘤菌的筛选及其促生功能初步评价
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摘要
【目的】筛选适用于成都平原的高效广谱蚕豆根瘤菌,并对其相关促生功能进行初步评价,为成都平原高效蚕豆根瘤菌剂的研制与应用提供科学依据。【方法】供试6株根瘤菌由课题组前期分离自成都平原,其与四川主栽蚕豆‘大白蚕豆’匹配良好,采用常规方法测定了这6个菌株分泌生长素及溶磷能力。菌株与蚕豆品种匹配试验采用低氮砂培法,供试蚕豆品种为成都平原主栽品种‘成胡14’、‘成胡15’;两个品种的蚕豆种子播种后,分别接种6个菌株,以不接种为对照(CK),光照(控温22～25℃、光照强度2800 lx左右、日照时间14 h)下培养41天后收获,测定植株生物量和根瘤数。然后,对匹配性试验筛到的两株高效广谱根瘤菌进行田间验证,供试蚕豆品种为成胡15,将2个根瘤菌制备的菌剂(活菌数5.0×108 CFU/g以上,载体为泥炭)进行拌种,以不接菌处理的灭菌泥炭为对照。在盛花期(生育期105 d)采样测定株高、根瘤数、地上部分植株干重;收获期(生育期200 d)采样测产;测定两个时期植株样品氮、磷、钾含量。盛花期采用BOX-PCR分子标记法测定接种根瘤菌占瘤率,同时提取接种菌株SCAUf73、SCAUf76的总DNA,比较接种菌株及相应根瘤类菌体根瘤菌DNA的BOX-PCR分子指纹图谱。用多位点基因序列分析法对田间验证的优良菌株SCAU73进行分类地位研究。【结果】1)通过匹配性砂培试验,筛选到2株与2个成都平原主栽蚕豆品种均高效匹配的根瘤菌SCAUf73、SCAUf76。SCAUf76、SCAUf73能使‘成胡14’、‘成胡15’植株干重较CK显著增加40.5%～61.6%。2)通过两株菌田间接种试验发现,接种SCAUf76处理的蚕豆产量与CK差异不显著;接种SCAUf73处理蚕豆植株干重、全氮含量等指标均高于CK,籽粒鲜产比CK显著增加25.0%,并显著高于SCAUf76,其占瘤率达到33%。3)多位点基因序列分析表明,SCAUf73可能是Rhizobium的一个新类群。4)促生性试验表明,6株菌都能分泌生长素(IAA),最大分泌量为21.0 mg/L(SCAUf76);供试菌株的溶磷能力不明显。【结论】从成都平原上筛选的6个菌株中,SCAUf73具有分泌IAA能力,与蚕豆接种后,占瘤率达33%,可显著促进蚕豆氮素吸收积累,提高蚕豆籽粒产量。与成都平原的主栽蚕豆品种匹配的高效广谱根瘤菌SCAUf73,适用于成都平原的蚕豆生产。
【Objectives】High efficient and widely adaptive faba bean rhizobia from Chengdu Plain were screened and preliminary evaluated for their growth promotion ability, to provide a reference for the development and application of the effective faba bean rhizobia inoculant in this region.【Methods】A sand culture experiment was carried out using double-layer pots and low-N nutrition solution. The six tested rhizobia strains were isolated and screened from previous experiment, and matched well with the predominant faba bean cultivar Dabaidou in Chengdu Plain and Sichuan. In this experiment, their matching efficiency with faba bean cultivars of Chenghu-14 and Chenghu-15 were compared. Two cultivars of faba bean were sown in vermiculite of double-layer pots. Six strains were inoculated into each faba bean cultivar, and the plant biomass and nodule number were measured after 41 days of cultivation under light(22–25 ℃, 2800 lx, 14 h) and without inoculation as control(CK). The phosphate solubilization ability and IAA excretion ability of the 6 strains were determined by the conventional method. Two strains screened by matching test were tested in the field, the tested faba bean cultivar was Chenghu-15. The two microbial agents(the number of viable count was more than 5.0 × 108 CFU/g and the carrier was peat) were mixed with faba bean, and without inoculation and sterilization of peat as control(CK). Plant height, nodule number and dry weight were measured in blooming stage(105 days of growing period). Yield was measured in harvest stage(200 days of growing period). N, P and K contents of plant samples were measured at two stages, and competitive nodulation ability of two strains was compared in field test using BOX Molecular Marker Method in blooming stage. Multilocus sequence analyses were applied to identify the phylogenetic position of this strain which was verified suitable for field application in Chengdu Plain by field experiments.【Results】1) The matching experiment indicated that 2 strains, SCAUf73 and SCAUf76,matched efficiently with 2 cultivars of faba bean in Chengdu Plain area were screened out. When inoculated with SCAUf76 and SCAUf73, the plant dry weight of faba bean were higher than those of CK. The plant dry weight increased significantly by 40.5%–61.6% compared with CK. 2) Field experiments showed that faba bean yield difference between SCAUf76 inoculated and CK was not significant, when inoculated with SCAUf73, the plant dry weight and total nitrogen content of faba bean were higher than those of CK. The fresh grain yield was increased significantly by 25.0% compared with CK, and significantly higher than those of SCAUf76 infected. Its nodule occupancy reached 33.3%. 3) Sequence analysis of multilocus genes showed that strain SCAUf73 might be a novel strain of Rhizobium. 4) Six strains could secrete IAA, and the maximum secretion amount was 21.0 mg/L happened in SCAUf76, indicated by promotion ability test. All strains did not show significant P solubilization ability.【Conclusions】This study indicated that the strain SCAUf73 was the best one matched with main planting cultivars of faba bean in Chengdu Plain area. It had the ability of secreting IAA, and was highly efficient. Field experiments showed that faba bean yield and total nitrogen content were higher than those of CK, the tumour rate reached 33%.
【Objectives】High efficient and widely adaptive faba bean rhizobia from Chengdu Plain were screened and preliminary evaluated for their growth promotion ability, to provide a reference for the development and application of the effective faba bean rhizobia inoculant in this region.【Methods】A sand culture experiment was carried out using double-layer pots and low-N nutrition solution. The six tested rhizobia strains were isolated and screened from previous experiment, and matched well with the predominant faba bean cultivar Dabaidou in Chengdu Plain and Sichuan. In this experiment, their matching efficiency with faba bean cultivars of Chenghu-14 and Chenghu-15 were compared. Two cultivars of faba bean were sown in vermiculite of double-layer pots. Six strains were inoculated into each faba bean cultivar, and the plant biomass and nodule number were measured after 41 days of cultivation under light(22–25 ℃, 2800 lx, 14 h) and without inoculation as control(CK). The phosphate solubilization ability and IAA excretion ability of the 6 strains were determined by the conventional method. Two strains screened by matching test were tested in the field, the tested faba bean cultivar was Chenghu-15. The two microbial agents(the number of viable count was more than 5.0 × 108 CFU/g and the carrier was peat) were mixed with faba bean, and without inoculation and sterilization of peat as control(CK). Plant height, nodule number and dry weight were measured in blooming stage(105 days of growing period). Yield was measured in harvest stage(200 days of growing period). N, P and K contents of plant samples were measured at two stages, and competitive nodulation ability of two strains was compared in field test using BOX Molecular Marker Method in blooming stage. Multilocus sequence analyses were applied to identify the phylogenetic position of this strain which was verified suitable for field application in Chengdu Plain by field experiments.【Results】1) The matching experiment indicated that 2 strains, SCAUf73 and SCAUf76,matched efficiently with 2 cultivars of faba bean in Chengdu Plain area were screened out. When inoculated with SCAUf76 and SCAUf73, the plant dry weight of faba bean were higher than those of CK. The plant dry weight increased significantly by 40.5%–61.6% compared with CK. 2) Field experiments showed that faba bean yield difference between SCAUf76 inoculated and CK was not significant, when inoculated with SCAUf73, the plant dry weight and total nitrogen content of faba bean were higher than those of CK. The fresh grain yield was increased significantly by 25.0% compared with CK, and significantly higher than those of SCAUf76 infected. Its nodule occupancy reached 33.3%. 3) Sequence analysis of multilocus genes showed that strain SCAUf73 might be a novel strain of Rhizobium. 4) Six strains could secrete IAA, and the maximum secretion amount was 21.0 mg/L happened in SCAUf76, indicated by promotion ability test. All strains did not show significant P solubilization ability.【Conclusions】This study indicated that the strain SCAUf73 was the best one matched with main planting cultivars of faba bean in Chengdu Plain area. It had the ability of secreting IAA, and was highly efficient. Field experiments showed that faba bean yield and total nitrogen content were higher than those of CK, the tumour rate reached 33%.
引文
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[3]Hardarson G,Atkins C.Optimising biological N2 fixation by legumes in farming systems[J].Plant and Soil,2003,252(1):41-54.
[4]王文丽,李娟,卢秉林.蚕豆根瘤菌的分离、筛选及其肥效研究[J].干旱地区农业研究,2010,28(6):112-115.Wang W L,Li J,Lu B L.Research on isolation,selection and fertility of horsebean rhizobium[J].Agricultural Research in the Arid Areas,2010,28(6):112-115.
[5]房增国,赵秀芬,孙建好,等.接种根瘤菌对蚕豆/玉米间作系统氮营养的影响[J].华北农学报,2009,24(4):124-128.Fang Z G,Zhao X F,Sun J H,et al.Effects of rhizobium inoculation on nitrogen nutrition in faba bean/maize intercropping system[J].Acta Agriculturae Boreali-Sinica,2009,24(4):124-128.
[6]Rugheim A M,Abdelgani M E.Effects of microbial and chemical fertilization on yield and seed quality of faba bean(Vicia faba)[J].International Food Research Journal,2012,19(2):417-422.
[7]葛诚.微生物肥料生产及其产业化[M].北京:化学工业出版社,2007.Ge C.Production and industrialization of microbial fertilizer[M].Beijing:Chemical Industry Press,2007.
[8]李俊,沈德龙,林先贵.农业微生物研究与产业化进展[M].北京:科学出版社,2011.Li J,Shen D L,Lin X G.Agricultural microbiology research and industrialized progress[M].Beijing:Science Press,2011.
[9]Chen Y X,Zou L,Penttinen P,et al.Faba bean(Vicia faba L.)nodulating rhizobia in Panxi,China,are diverse at species,plant growth promoting ability and symbiosis related gene levels[J].Frontiers in Microbiology,2018,9:1338.
[10]Tian C F,Wang E T,Wu L J,et al.Rhizobium fabae sp.nov.,a bacterium that nodulates Vicia faba[J].International Joumal of Systematic and Evolutionary Microbiology,2008,58:2871-2875.
[11]梅沛沛,王平,李隆,等.新开垦土壤上构建玉米/蚕豆-根瘤菌高效固氮模式[J].中国生态农业学报,2018,26(1):62-74.Mei P P,Wang P,Li L,et al.Construction of efficient nitrogen-fixing cropping pattern:Maize/faba bean intercrop with rhizobium inoculation in reclaimed low-fertility soils[J].Chinese Journal of EcoAgriculture,2018,26(1):62-74.
[12]陈文新,汪恩涛.中国根瘤菌[M].北京:科学出版社,2011.Chen W X,Wang E T.Rhizobium in China[M].Beijing:Science Press,2011.
[13]陈文新,汪恩涛,陈文峰.根瘤菌-豆科植物共生多样性与地理环境的关系[J].中国农业科学,2004,37:81-86.Chen W X,Wang E T,Chen W F.The relationship between the symbiotic promiscuity of rhizobia and legumes and their geographical environments[J].Scientia Agricultura Sinica,2004,37:81-86.
[14]杨武云,余东梅,罗菊枝.四川蚕豆育种及生产现状[J].四川农业科技,2003,(10):14.Yang W Y,Yu D M,Luo J Z.Breeding and production status of fababean in Sichuan[J].Sichuan Province Agriculture Science,2003,(10):14.
[15]Zou L,Chen Y X,Penttinen P,et al.Genetic diversity and symbiotic efficiency of nodulating rhizobia isolated from root nodules of faba bean in one field[J].PLoS One,2016,11(12):e0167804.
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