四川攀枝花地区印度块菌菌根根际土壤内可培养放线菌功能多样性
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摘要
以四川攀枝花地区印度块菌菌根根际土壤为对象,利用4种放线菌分离培养基筛选其中的放线菌,采用基于16S rRNA基因的PCR-RFLP方法结合传统的形态学观察进行鉴定。通过测定各分离菌株摇瓶培养发酵液中酶活性或代谢产物获取各菌株降解碳水化合物以及产促生剂的能力,采用平板对峙法测定各菌株抗病原真菌活性。结果共分离得到125株放线菌,经形态学观察去重复得到57株(W1-W57),经多样性分析将其归于3个属的23个种,其中以链霉菌属最多(91.3%),功能分析表明:有23株、17株、15株和18株放线菌能分别降解1,3-β-葡聚糖、几丁质、纤维素和果胶,其中有47.8%、26.1%和26.1%的菌株能同时降解4、3和2类碳水化合物;有19株、3株和2株放线菌分别能产含铁载体、IAA和ACC脱氨酶,其中有21.7%、56.6%和21.7%的菌株能分别产生3、2和1种根际促生剂;有10株放线菌表现出对不止一种病源真菌的拮抗能力,其中菌株W2和W19对4种病源真菌同时具有拮抗活性,且这2株菌对4种病源真菌抑菌能力比其他菌强;同时获得了包括W2、W3、W17、W19、W20、W34、W36和W54等在内的多功能高活性菌株,该研究表明四川攀枝花地区印度块菌菌根土壤内含丰富的多功能放线菌。所获结果可为我国印度块菌人工种植新技术的开发提供新思路。
In this study, we isolated actinomyces from Tuber inducum mycorrhizal soils in Panzhihua, Sichuan by four types of medium, and all isolates were identified by PCR-RFLP based on 16 S rRNA gene in combination with morphological observation.The abilities of each strain to degrade carbohydrates and produce growth promoters were obtained by measuring the enzyme activity or metabolites in shaking flask fermentation broth of each isolate. Then, dual-culture inhibition was adopted for screening strains antifungal activity. A total of 125 actinomyces were isolated, of which 57 strains(W1-W57) were selected after morphological character observation and assigned to 3 genera. Actinomyces could be divided into 23 species, and among all isolates the Streptomyces were the most(91.3%). Result of functional analysis was as follows: 23, 17, 15, and 18 strains of all isolates degraded 1,3-β-glucan, chitin and cellulose and pectin, respectively, of which 47.8%, 26.1% and 26.1% were able to degrade simultaneously 4, 3 and 2 carbohydrates, respectively. 19 isolates produced the siderophore, 3 isolates secreted indoleacetic acid, and 2 strains produced ACC deaminase, of which 21.7%, 56.6% and 21.7% respectively had simultaneously the above bioactivities of 3, 2 and 1, respectively. 10 isolates had more than one ability to resist pathogenic fungi; W2 and W19 showed better inhibitory ability to four pathogenic fungi than other isolates. At the same time, the multifunctional functions and high bioactive strains including W2, W3, W17, W19, W20, W34, W36 and W54 were obtained. This study shows that there are lots of multifunctional actinomycetes in T. inducum mycorrhizal of Panzhihua, Sichuan, and the results can also provide new ideas for the development of new planting techniques in artificial cultivation of T. inducums.
In this study, we isolated actinomyces from Tuber inducum mycorrhizal soils in Panzhihua, Sichuan by four types of medium, and all isolates were identified by PCR-RFLP based on 16 S rRNA gene in combination with morphological observation.The abilities of each strain to degrade carbohydrates and produce growth promoters were obtained by measuring the enzyme activity or metabolites in shaking flask fermentation broth of each isolate. Then, dual-culture inhibition was adopted for screening strains antifungal activity. A total of 125 actinomyces were isolated, of which 57 strains(W1-W57) were selected after morphological character observation and assigned to 3 genera. Actinomyces could be divided into 23 species, and among all isolates the Streptomyces were the most(91.3%). Result of functional analysis was as follows: 23, 17, 15, and 18 strains of all isolates degraded 1,3-β-glucan, chitin and cellulose and pectin, respectively, of which 47.8%, 26.1% and 26.1% were able to degrade simultaneously 4, 3 and 2 carbohydrates, respectively. 19 isolates produced the siderophore, 3 isolates secreted indoleacetic acid, and 2 strains produced ACC deaminase, of which 21.7%, 56.6% and 21.7% respectively had simultaneously the above bioactivities of 3, 2 and 1, respectively. 10 isolates had more than one ability to resist pathogenic fungi; W2 and W19 showed better inhibitory ability to four pathogenic fungi than other isolates. At the same time, the multifunctional functions and high bioactive strains including W2, W3, W17, W19, W20, W34, W36 and W54 were obtained. This study shows that there are lots of multifunctional actinomycetes in T. inducum mycorrhizal of Panzhihua, Sichuan, and the results can also provide new ideas for the development of new planting techniques in artificial cultivation of T. inducums.
引文
[1]BARBIERI E,CECCAROLI P,SALTARELLI R,et al.New evidence for nitrogen fixation within the Italian white truffle Tuber magnatum[J].Fungal Biology,2010,114(11/12):936-942.
[2]MARJANOVIC?,GLISIC A,MUTAVDZIC D,et al.Ecosystems supporting Tuber magnatum Pico production in Serbia experience specific soil environment seasonality that may facilitate truffle lifecycle completion[J].Applied Soil Ecology,2015,95(9):179-190.
[3]NAVARRORODENAS A,BERNA LM,LOZANO-CARRILLO C,et al.Beneficial native bacteria improve survival and mycorrhization of desert truffle mycorrhizal plants in nursery conditions[J].Mycorrhiza,2016,26(7):769-779.
[4]ANTONY-BABU S,DEVEAU A,VAN J D,et al.Black truffle-associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles[J].Environmental Microbiology,2014,16(9):2831-2847.
[5]PICCERI G G,LEONARDI P,IOTTI M,Gallo M,et al.Acteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles(Tuber borchii)[J].Applied Microbiology and Biotechnology,2018,102(3):1429-1441.
[6]GRYNDLER M,SOUKUPOVA L,HRSELOVA H,et al.Aquest for indigenous truffle helper prokaryotes[J].Environmental Microbiology Reports,2013,5(3),346-352.
[7]SORRENTINO E,SUCCI M,TIPALDI L,et al.Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool toimprove the shelf life of fresh black truffles[J].International Journal of Food Microbiology,2018,266,183-189.
[8]王冉,刘培贵,万山平,等.印度块菌(Tuber indicum)菌根促生细菌的研究[J].微生物学通报,2015,42(12):2366-2376.
[9]S BRANA C,AGNOLUCCI M,BEDINI S.Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T.borchii mycelial growth[J].Fems Microbiology Letters,2002,211(2),195-201.
[10]ALFONSO N R,LUIS M B.Beneficial native bacteria improve survival and mycorrhization desert truffle mycorrhizal plants in nursery conditions[J].Mycorrhiza,2016,26(7):769-779.
[11]宋漳.不同林分土壤放线菌的分布及其与土壤因子的关系[J].浙江林学院学报,1997,14(1):41-44.
[12]马腾,王雪薇,阮继生,等.三江源地区不同退化程度草地的土壤放线菌区系[J].微生物学通报,2008,35(12):1879-1883.
[13]LIU Xiao,CONG Jing,LU Hui,et al.Community structure and elevational distribution pattern of soil Actinobacteria in alpine grasslands[J].Acta Ecologica Sinica,2017,37(4):213-218.
[14]PIAO Zhe,YANG Lingzhang,ZHAO Liping,et al.Actinobacterial community structure in soils receiving long-term organic and inorganic amendments[J].Applied and Environmental Microbiology,2008,74(2):526-530.
[15]BARBIERI E,GUIDI C,BERTAUX J,et al.Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation[J].Environmental Microbiology,2007,9(9):2234-2246.
[16]PAVIC A,STANKOVIC S,SALJNIKOV E,et al.Actinobacteria may influence white truffle(Tuber magnatum Pico)nutrition,ascocarp degradation and interactions with other soil fungi[J].Fungal Ecology,2013,6(6):527-538.
[17]GOUDJIA Y,ZAMOUML M,MEKLAT A,et al.Plantgrowth-promoting potential of endosymbiotic actinobacteria isolated from sand truffles(Terfezia leonis Tul.)of the Algerian Sahara[J].Annals of microbiology,2016,66:91-100.
[18]孙亚明.攀枝花块菌资源可持续经营研究[J].中国农业资源与区划,2016.37(4):198-202.
[19]万山平,郑毅,汤利,等.攀枝花块菌-华山松菌根土壤可培养细菌的多样性研究[J].植物分类与资源学报,2015,37(6):861-870.
[20]王冉,刘培贵,万山平,等.印度块菌(Tuber indicum)菌根促生细菌的研究[J].微生物学通报,2015,42(12):2366-2376.
[21]TOKALA R K,STRAP J L,JUNG C M.Novel plantmicrobe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant(Pisum sativum)[J].Applied and Environmental Microbiology,2002,68(5):2161-2171.
[22]CHERNIN L,ISMAILOV Z,HARAN S,et al.Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens[J].Applied and Environmental Microbiology,1995,61(5):1720-1726.
[23]MAHASNEH A M,STEWART D J.A medium for detecting beta-(1-3)glucanase activity in bacteria[J].Journal of Applied Bacteriology,2010,48(3):457-458.
[24]HENDRICKS C W,DOYLE J D,HUGLEY B.A new solid medium for enumerating cellulose-utilizing bacteria in soil[J].Applied and Environmental Microbiology,1995,61(5):2016-2019.
[25]HANKIN L,ZUCKER M,SANDS D C.Improved solid medium for the detection and enumeration of pectolytic bacteria[J].Applied and Environmental Microbiology,1971,22(2):205-209.
[26]RUANPANUM P,TANGCHITSOMKID N,HYDE K D,et al.Actinomycetes and fungi isolated from plant-parasitic nematode infested soils:screening of the effective biocontrol potential,indole-3-acetic acid and siderophore production[J].World journal of microbiology and biotechnology,2010,26(9):1569-1578.
[27]SELEH S S,GLICK B R.Involvement of gacS and rpoS in enhancement of the plant growth-promoting capabilities Enterobacter cloacae CAL2 and UW4[J].Canadian journal of microbiology,2001,47(8):698-705.
[28]SADEGHI A,KARIMI J,ABASZADEH D,et al.Plant growth promoting activity of an auxin and siderophore producing isolate of Streptomyces under saline soil conditions[J].World journal of microbiology and biotechnology,2012,28(4):1503-1509.
[29]BREDHOLDT H,GALATENKO O A,ENGELHARDT K,et al.Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord,Norway:isolation,diversity and biological activity[J].Environmental Microbiolog,2007,9(11):2756-2764.
[30]TOKALA R K,STAP J L,JUNG C M,et al.Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant(Pisum sativum)[J].Applied and Environmental Microbiology,2002,68(5):2161-2171.
[31]PANDEY P,BISH S,SOOD A,et al.Consortium of Plant-Growth-Promoting Bacteria:Future Perspective in Agriculture[M]//SHIMIZU M.Bacteria in Agrobiology:Plant Probiotics.Springer Berlin Heidelberg.Germany:Springer-Verlag Berlin Heidelberg,2012,201-220.
[32]BHATTACHARYYA P,JHA D.Plant growth-promoting rhizobacteria(PGPR):emergence in agriculture[J].World journal of microbiology and biotechnology,2012,28(4):1327-1350.
[33]张盼盼,秦盛,袁博,等.南方红豆杉内生及根际放线菌多样性及其生物活性[J].微生物学报,2016,56(2):241-252.
[34]DIB-BELLAHOUEL S,FORTAS Z.Activity of the desert truffle Terfezia boudieri Chatin,against associated soil microflora[J].African Journal Microbiology Research,2014,8(32):3008-3016.
[35]SBRANA C,AGNOLUCCI M,BEDINI S,et al.Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T.borchii mycelial growth[J].Fems Microbiology Letters,2002,211(2):195-201.
[36]FREY-KLETT P,GARBAYE J,TARKKA M.The mycorrhiza helper bacteria revisited[J].New Phytologist,2007,176(1):22-36.
[37]BRAGATO G,VIGNOZZI,N,PELLEGRINI S,et al.Physical characteristics of the soil environment suitable for Tuber magnatum production in fluvial landscapes[J].Plant Soil,2010,329(1):51-63.
[38]BRADAI L,BISSATI S,CHENCHOUNI H,et al.Effects of climate on the productivity of desert truffles beneath hyper-arid conditions[J].International journal of biometeorology,2015,59(7):907-915.
[39]IOTTI M,LEONARDI P,VITALI G,et al.Effect of summer soil moisture and temperature on the vertical distribution of Tuber magnatum mycelium in soil[J].Biology and Fertility of Soils,2018,54(6):1-10.
[40]QIAO Peng,TIAN Wei,LIU Peigui,et al.Phylogeography and population genetic analyses reveal the speciation of the Tuber indicum complex[J].Fungal Genetics and Biology,2018,113:14-23.
[41]YE Lei,LI Qiang,FU Yu,et al.Host species effects on bacterial communities associated with the fruiting bodies of Tuber species from the Sichuan Province in Southwest China[J].Mycological Progress,2018,17(7):833-840.
[42]SCHNEIDER-MAUNOURY L,SECLERCQ S,CLEMENTC,et al.Is Tuber melanosporum colonizing the roots of herbaceous,non-ectomycorrhizal plants[J].Fungal Ecology,2018,31,59-68.
[2]MARJANOVIC?,GLISIC A,MUTAVDZIC D,et al.Ecosystems supporting Tuber magnatum Pico production in Serbia experience specific soil environment seasonality that may facilitate truffle lifecycle completion[J].Applied Soil Ecology,2015,95(9):179-190.
[3]NAVARRORODENAS A,BERNA LM,LOZANO-CARRILLO C,et al.Beneficial native bacteria improve survival and mycorrhization of desert truffle mycorrhizal plants in nursery conditions[J].Mycorrhiza,2016,26(7):769-779.
[4]ANTONY-BABU S,DEVEAU A,VAN J D,et al.Black truffle-associated bacterial communities during the development and maturation of Tuber melanosporum ascocarps and putative functional roles[J].Environmental Microbiology,2014,16(9):2831-2847.
[5]PICCERI G G,LEONARDI P,IOTTI M,Gallo M,et al.Acteria-produced ferric exopolysaccharide nanoparticles as iron delivery system for truffles(Tuber borchii)[J].Applied Microbiology and Biotechnology,2018,102(3):1429-1441.
[6]GRYNDLER M,SOUKUPOVA L,HRSELOVA H,et al.Aquest for indigenous truffle helper prokaryotes[J].Environmental Microbiology Reports,2013,5(3),346-352.
[7]SORRENTINO E,SUCCI M,TIPALDI L,et al.Antimicrobial activity of gallic acid against food-related Pseudomonas strains and its use as biocontrol tool toimprove the shelf life of fresh black truffles[J].International Journal of Food Microbiology,2018,266,183-189.
[8]王冉,刘培贵,万山平,等.印度块菌(Tuber indicum)菌根促生细菌的研究[J].微生物学通报,2015,42(12):2366-2376.
[9]S BRANA C,AGNOLUCCI M,BEDINI S.Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T.borchii mycelial growth[J].Fems Microbiology Letters,2002,211(2),195-201.
[10]ALFONSO N R,LUIS M B.Beneficial native bacteria improve survival and mycorrhization desert truffle mycorrhizal plants in nursery conditions[J].Mycorrhiza,2016,26(7):769-779.
[11]宋漳.不同林分土壤放线菌的分布及其与土壤因子的关系[J].浙江林学院学报,1997,14(1):41-44.
[12]马腾,王雪薇,阮继生,等.三江源地区不同退化程度草地的土壤放线菌区系[J].微生物学通报,2008,35(12):1879-1883.
[13]LIU Xiao,CONG Jing,LU Hui,et al.Community structure and elevational distribution pattern of soil Actinobacteria in alpine grasslands[J].Acta Ecologica Sinica,2017,37(4):213-218.
[14]PIAO Zhe,YANG Lingzhang,ZHAO Liping,et al.Actinobacterial community structure in soils receiving long-term organic and inorganic amendments[J].Applied and Environmental Microbiology,2008,74(2):526-530.
[15]BARBIERI E,GUIDI C,BERTAUX J,et al.Occurrence and diversity of bacterial communities in Tuber magnatum during truffle maturation[J].Environmental Microbiology,2007,9(9):2234-2246.
[16]PAVIC A,STANKOVIC S,SALJNIKOV E,et al.Actinobacteria may influence white truffle(Tuber magnatum Pico)nutrition,ascocarp degradation and interactions with other soil fungi[J].Fungal Ecology,2013,6(6):527-538.
[17]GOUDJIA Y,ZAMOUML M,MEKLAT A,et al.Plantgrowth-promoting potential of endosymbiotic actinobacteria isolated from sand truffles(Terfezia leonis Tul.)of the Algerian Sahara[J].Annals of microbiology,2016,66:91-100.
[18]孙亚明.攀枝花块菌资源可持续经营研究[J].中国农业资源与区划,2016.37(4):198-202.
[19]万山平,郑毅,汤利,等.攀枝花块菌-华山松菌根土壤可培养细菌的多样性研究[J].植物分类与资源学报,2015,37(6):861-870.
[20]王冉,刘培贵,万山平,等.印度块菌(Tuber indicum)菌根促生细菌的研究[J].微生物学通报,2015,42(12):2366-2376.
[21]TOKALA R K,STRAP J L,JUNG C M.Novel plantmicrobe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant(Pisum sativum)[J].Applied and Environmental Microbiology,2002,68(5):2161-2171.
[22]CHERNIN L,ISMAILOV Z,HARAN S,et al.Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens[J].Applied and Environmental Microbiology,1995,61(5):1720-1726.
[23]MAHASNEH A M,STEWART D J.A medium for detecting beta-(1-3)glucanase activity in bacteria[J].Journal of Applied Bacteriology,2010,48(3):457-458.
[24]HENDRICKS C W,DOYLE J D,HUGLEY B.A new solid medium for enumerating cellulose-utilizing bacteria in soil[J].Applied and Environmental Microbiology,1995,61(5):2016-2019.
[25]HANKIN L,ZUCKER M,SANDS D C.Improved solid medium for the detection and enumeration of pectolytic bacteria[J].Applied and Environmental Microbiology,1971,22(2):205-209.
[26]RUANPANUM P,TANGCHITSOMKID N,HYDE K D,et al.Actinomycetes and fungi isolated from plant-parasitic nematode infested soils:screening of the effective biocontrol potential,indole-3-acetic acid and siderophore production[J].World journal of microbiology and biotechnology,2010,26(9):1569-1578.
[27]SELEH S S,GLICK B R.Involvement of gacS and rpoS in enhancement of the plant growth-promoting capabilities Enterobacter cloacae CAL2 and UW4[J].Canadian journal of microbiology,2001,47(8):698-705.
[28]SADEGHI A,KARIMI J,ABASZADEH D,et al.Plant growth promoting activity of an auxin and siderophore producing isolate of Streptomyces under saline soil conditions[J].World journal of microbiology and biotechnology,2012,28(4):1503-1509.
[29]BREDHOLDT H,GALATENKO O A,ENGELHARDT K,et al.Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord,Norway:isolation,diversity and biological activity[J].Environmental Microbiolog,2007,9(11):2756-2764.
[30]TOKALA R K,STAP J L,JUNG C M,et al.Novel plant-microbe rhizosphere interaction involving Streptomyces lydicus WYEC108 and the pea plant(Pisum sativum)[J].Applied and Environmental Microbiology,2002,68(5):2161-2171.
[31]PANDEY P,BISH S,SOOD A,et al.Consortium of Plant-Growth-Promoting Bacteria:Future Perspective in Agriculture[M]//SHIMIZU M.Bacteria in Agrobiology:Plant Probiotics.Springer Berlin Heidelberg.Germany:Springer-Verlag Berlin Heidelberg,2012,201-220.
[32]BHATTACHARYYA P,JHA D.Plant growth-promoting rhizobacteria(PGPR):emergence in agriculture[J].World journal of microbiology and biotechnology,2012,28(4):1327-1350.
[33]张盼盼,秦盛,袁博,等.南方红豆杉内生及根际放线菌多样性及其生物活性[J].微生物学报,2016,56(2):241-252.
[34]DIB-BELLAHOUEL S,FORTAS Z.Activity of the desert truffle Terfezia boudieri Chatin,against associated soil microflora[J].African Journal Microbiology Research,2014,8(32):3008-3016.
[35]SBRANA C,AGNOLUCCI M,BEDINI S,et al.Diversity of culturable bacterial populations associated to Tuber borchii ectomycorrhizas and their activity on T.borchii mycelial growth[J].Fems Microbiology Letters,2002,211(2):195-201.
[36]FREY-KLETT P,GARBAYE J,TARKKA M.The mycorrhiza helper bacteria revisited[J].New Phytologist,2007,176(1):22-36.
[37]BRAGATO G,VIGNOZZI,N,PELLEGRINI S,et al.Physical characteristics of the soil environment suitable for Tuber magnatum production in fluvial landscapes[J].Plant Soil,2010,329(1):51-63.
[38]BRADAI L,BISSATI S,CHENCHOUNI H,et al.Effects of climate on the productivity of desert truffles beneath hyper-arid conditions[J].International journal of biometeorology,2015,59(7):907-915.
[39]IOTTI M,LEONARDI P,VITALI G,et al.Effect of summer soil moisture and temperature on the vertical distribution of Tuber magnatum mycelium in soil[J].Biology and Fertility of Soils,2018,54(6):1-10.
[40]QIAO Peng,TIAN Wei,LIU Peigui,et al.Phylogeography and population genetic analyses reveal the speciation of the Tuber indicum complex[J].Fungal Genetics and Biology,2018,113:14-23.
[41]YE Lei,LI Qiang,FU Yu,et al.Host species effects on bacterial communities associated with the fruiting bodies of Tuber species from the Sichuan Province in Southwest China[J].Mycological Progress,2018,17(7):833-840.
[42]SCHNEIDER-MAUNOURY L,SECLERCQ S,CLEMENTC,et al.Is Tuber melanosporum colonizing the roots of herbaceous,non-ectomycorrhizal plants[J].Fungal Ecology,2018,31,59-68.
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