丛枝菌根真菌和有机土基质栽培对甜椒根际微生物分子多态性的影响

Effects of Arbuscular Mycorrhizal Fungus andOrganic Substrate on the Molecular Diversity ofMicrobes in Sweet Pepper Rhizosphere underProtected CultivationChaoxing HE*, Linchuang WANG, Zhibin ZHANGInstitute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, ChinaSupported by the National Key Technology RTechnically Assisted by the Key Laboratory of Horticultural Genetic Improvement of theMinistry of Agriculture.*Corresponding author. E-mail: hechaoxing126.comReceived: December 12, 2011 Accepted: April 8, 2012AAgricultural Science Organic soil substrate; Denaturing gradientgel electrophoresis; Microbial diversity; Sweet pepperSoil microorganisms are the keycomponent of soil. Act as thepromoters in transformationand recycling of soil organic mattersand energy, soil microorganisms areinvolved in the decomposition of soilorganic matters, the formation of soilhumus and recycling soil nutrients.The structure and activities of soil mi-crobiota not only determine the fertilityand quality of soil, but also link to theproductivities of plants growing onthem1. Polymerase chain reaction-denaturing gradient gel electrophore-sis (PCR-DGGE) is a novel molecularmethod provides faster and more ac-curate analysis and comparison of mi-crobiota variation than the convention-al plate culture method2. DGGE tech-nique can effectively separate DNAsequences with the same molecularweight but different base sequences.Researchers have investigated thestructure of soil microbiota using PCR-DGGE, for example, some studieshave been taken to understand theenvironmental, spatial and temporalvariation of bacterial populations3.Protected vegetable cultivationand production has recently gainedrapid progress in China which stronglyguarantees the year-round vegetableprovidence. However, the protectedvegetable cultivation in China is facingsome constraints which affect the sus-tainable development of the industry,such as the low productivity, con-straints caused by the continuouscropping, imbalance in soil microbiota,soil secondary salinization, etc. Duringthe previous years, the application ofsome organic cultivation techniques inprotected vegetable production inte-grated with the straw and manure re-turning into the field. With these tech-niques, problems in protected cultiva-tion, e.g. soil crust, frequent epidemicof soil-borne diseases, continuouscropping constraints and undergroundwater pollution, are partly solved. Fur-thermore, these techniques can alsoimprove the quality of vegetables andsave the cost of production4. Othertechniques, such as inoculating veg-etable seedlings with some beneficialsymbiotic microorganism, integratedwith the organic protected cultivation,can increase the vegetable productivi-ty,improve the quality of the agro-products, and decrease the input offertilizers and chemicals. Consequent-ly, they show their significance in envi-ronmental protection and improvementof economic value5. On this basis, thisstudy was designed to elucidate theeffects of these new techniques on thediversity of soil microbiota. The currentstudy was to investigate the moleculardiversity of soil microbiota in the sweetpepper following different soil treat-ments of arbuscular mycorrhizal fun-gus inoculation (untreated soil as thecontrol). The results were used to un-derstand the variation of microbialstructure after application of organicmatters and arbuscular mycorrhizafungus.DOI:10.16175/j.cnki.1009-4229.2012.05.042AgriculturalScience however the number of clonesobtained was notably changed. Thetreatments also changed the structureof bacterial population with somespecies absent or present in compari-son with the control. In the organic soil,there appeared to be more not closelyrelated bacterial populations than thecontrol soil. It also favored the growthand reproduction of some dominantspecies. Such results might be due tothe fact that organic soil had improvedsoil quality, and high organic mattercontent and humus, which provided afavorable condition for the growth ofmicroorganisms. Treatments with fun-gal inoculation caused the increase ofbacterial species; among them weresome new species. Some specieswere disappeared in those treatmentsand the abundance of some universalbacteria was increased. Suggestedfrom the previous reports, such varia-tion might be related to the interactionsof arhusclar mycorrhiza on host plantsfor plant growth and disease resis-tance, e.g. the increase and abun-dance of some effective microorgan-isms11,13,24,25,27,28, the decrease and ab-senceofsomepathogenicbacteria15-20.Judged from the similarity analysis, itwas concluded that the soil type hadmore influence on the diversity of rhi-zosphere microbiota than G.M inocu-lation, and the control soil with G.M in-oculation had stronger effect on the di-versity variation than the organic soil.Some consistent changes in microbialpopulations occurred for both types ofsoil after G.M fungi inoculation and thesimilarity of soil microbiota of two soiltypes was increased.Comprehensively, from theDGGE analysis on the rhizospheremicrobiota diversity in soils from differ-ent treatments, the bacterial diversityand population structure was affectedby organic soil cultivation andarhusclar mycorrhiza fungi inoculationwith some bacterial species were in-creased or decreased or their abun-dance changed. Further studies werestill needed to clarify the specific bac-terial species significantly influencedand their effects on crop yield. Suchstudies would be helpful in the de-velopment and utilization of some1025AgriculturalScience&TechnologyAgricultural Science & Technology Vol.13, No.5, 20122012Responsible editor: Ze LIU Responsible proofreader: Xiaoyan WUnovel microbial agents and microbialfertilizers.References1 ZELLES L. Fatty acid patterns of phos-pholipids and lipopolysaccharides in thecharacterisation of microbial communi-ties in soil: a review J. Biology andFertility of Soils, 1999, 29: 111-129.2 TORSVIK V. Microbial diversity and fun-ctioninsoil:fromgenestoecosystemsJ.Curr Opin Microbiol, 2002, 5: 240-245.3 NAKATSU CH. Soil microbial communi-ty analysis using denaturing gradientgel electrophoresisJ. Soil Science So-ciety of America, 2007, 71: 562-571.4 HE SX (贺超兴 ), ZHANG ZB (张志斌 ).New techniques in organic cultivation ofenvironmental friendly protected veg-etable production(设施蔬菜环境友好型有机栽培新技术 )J. Journal of North-west Horticulture (西北园艺 ), 2007, 3:4-6.5 SHI ZY(石兆勇 ), LIU RJ(刘润进 ), et al.Effects of culture substrates and AMfungus on horticultural crops (栽培基质与 AM 真菌对园艺作物的影响 )J. Chi-nese Journa1 of Eco-agriculture(中国生态农业学报 ), 2002, 10(2): 50-52.6 MUYZER, ELLEN CW, ANDRE GU.Profiling of complex microbial popula-tions by denaturing gradient gel elec-trophoresis analysis of polymerasechain reaction genes coding for 16SrRNA J. Appl Environ Microbiol, 1993,59: 695-700.7 ZHOU J, BRUNS MA, TIEDJE JM. DNArecovery from soils of diverse composi-tion J. Applied and Environmental Mi-crobiology, 1996, 62: 316-322.8 SMITH SE, READ DJ. Mycorrhizal sym-biosis M. 2nded. London: AcademicPress, 1997: 1-10.9 KOIDE RG, MOSSE B. A history of re-search on arbuscular mycorrhiza J.Mycorrhiza, 2004, 14:145-163.10 CRUZ AF, ISHII T, MATSUMOTOI, et al. Evaluation of the mycelialnetwork formed by arbuscular mycor-rhizal hyphae in the rhizosphere of pa-paya and other plants under intercrop-ping systemJ. Braz J Microbiol, 2003,34(1): 72-76.11 TSIMILLI-MICHAEL M, EGGENBERGP, BIRO B, et al. Synergistic and an-tagonistic effects of arbuscular mycor-rhizal fungi and Azospirillum and Rhi-zobium nitrogen-fixers on the photo-synthetic activity of alfalfa, probed bythe polyphasic chlorophyll a fluores-cence transient O-J-I-P J. AppliedSoil Ecology, 2000, 15(2): 169-182.12 HIJRI M, REDECKER D, PETETOTJAMC, et al. Identification and isolationof two ascomycete fungi from sporesof the arbuscular mycorrhizal fungusScutellospora castaneaJ. Appl Env-iron Microbiol, 2002, 68 (9): 4567 -4573.13 MAMATHA G, BAGYARAJ DJ, JA-GANATH S. Inoculation of field-es-tablished mulberry and papaya witharbuscular mycorrhizal fungi and amycorrhiza helper bacterium J. Myc-orrhiza, 2002, 12(6): 313-316.14 LIU RJ (刘润进 ), SHEN CY (沈崇尧 ).Inductive effects of VA fungus andVerticillium dahliae on cotton patho-genesis proteins(菌根对大丽轮枝菌于棉花体内 PR 蛋白的诱导作用 )J. ActaPhytopathologica Sinica (植物病理学报 ), 1993, 23(2): 162-167.15 HU ZJ (胡正嘉 ), WANG P (王平 ). Ef-fects of VA fungus on cotton wilt dis-ease(菌根真菌对棉花枯萎病的影响 )J. Acta Pedologica Sinica(土壤学报 ),1994, 31(supplement): 212-217.16 FILION M, ST-ARNAUD M, FORTINJA. Interactive effects of arbuscularmycorrhizal fungus Glomus in-traradices and different rhizospheremicroorganisms J. New Phytologist,1999, 141: 525-533.17 ZHU HH(朱红惠 ), LONG LK(龙良坤 ),YANG SZ (羊宋贞 ), et al. Influence ofAM fungus on Ralstonia solanacearumpopulation and bacterial communitystructure in rhizosphere (AM 真菌对青枯菌和根际细菌群落结构的影响 )J.Mycosystema(菌物学报 ), 2005, 24(1):137-142.18 ABDEL-FATTAH GM, SHABANA YM.Efficacy of the arbuscular mycorrhizalfungus Glomus clarum in protection ofcowpea plants against root rotpathogen Rhizoctonia solani J. Jour-nal of Plant Disease and Protection,2002, 109: 207-215.19 ABDALLA ME, ABDEL-FATTAH GM.Influence of the endomycorrhizal fun-gus Glomus mosseae on the develop-ment of peanut pod rot disease inEgyptJ.Mycorrhiza,2000,10:29-35.20 VIGO C, NORMAN JR, HOOKER JE.Biocontrol of the pathogen Phytoph-thora parasitica by arbuscular mycor-rhizal fungi is a consequence of effectson infection loci J. Plant Pathology,2000, 49: 509-514.21 ARTURSSON V, FINLAY RD, JANS-SON JK. Combined bromodeoxyuri-dine immunocapture and terminal re-striction fragment length polymorphismanalysis highlights differences in theactive soil bacterial metagenome dueto glomus mosseae inoculation orplant speciesJ. Environmental Micro-biology, 2005, 7(12): 1952-1966.22 BHATIA NP, ADHOLEYA A, SHARMAA. Biomass production and changes insoil productivity during long term culti-vation of Prosopis juliflora (Swartz) DCinoculated with VA mycorrhiza andRhizobium spp.in a semiarid waste-landJ. Biol Fertil Soils, 1998, 26:208-214.23 BAREA JM, AZCóN R, AZCóN-AGU-LAR C. Mycorrhizosphere interactionsto improve plant fitness and soil qualityJ. Antonie Van Leeuwenhoek, 2002,81(1/4): 343-351.24 GAMALERO E, TROTTA A, MASSAN, et al. Impact of two fluorescentpseudomonad sand an arbuscular my-corrhizal fungus on tomato plantgrowth, root architecture and P acquisi-tionJ.Mycorrhiza,2004,14:185-192.25 QIN FL (秦芳玲 ), WANG JG (王敬国 ).Effects of VA fungus and Bacillusmegaterium on the growth and nitro-gen utilization in red clover (Trifoliumpratense)(菌根真菌和解磷细菌对红三叶草生长和氮磷营养的影响 )J. ActaPratacultuae Sinica (草业学报 ), 2000,9(1): 9-14.26 CAI XM(蔡宣梅 ), ZHANG QF(张秋芳 ),ZHENG WW (郑 伟 文 ). Effect of VAmycorrhizal fungi and Acetobacter di-azotrophicus on the growth of supersweet corn(VA 菌根菌与重氮营养醋杆菌双接种对超甜玉米生长的影响 )J.Fujian Journal of Agricultural Sciences(福建农 业 学 报 ), 2004, 19 (3): 125-159.27 LI SL (李树林 ), ZHAO SJ (赵士杰 ),ZHENG HL (郑 红 丽 ). Effect of VAMfungi and plastic-cover on Verticilliumwilt of eggplant and microbial popula-tion in eggplant root zone(VA 菌根真菌和覆膜结茄子黄萎病及茄根区微生物量的影响 )J. Journal of Inner Mongo-lia Agricultural University: Natural Sci-ence Edition (内蒙古农业大学学报 : 自然科学版 ), 1982, 26(1): 319-324.28 BAGYARAJ DJ, MENGE JA. Interac-tion between a VA mycorrhiza andazotobaeter and their effects on rhi-zosphere microflora and plant growthJ. New Phyto, 1978, 80: 567-573.29 KOTHARI SK, MARSCHNER H, RO-MHELD V. Effect of a vesicular-ar-buscular mycorrhizal fungus and rhi-zosphere andmanganese content andconcentration in red clover (Trifoliumpretense L.) plants J. New Phyto,1989, 112(2): 215-219.30 GENGGD(耿广东 ), LI L(李莉 ), ZHANGSQ(张素勤 ). Effects of VA-mycorrhizason absorption of mineral elements ofcucumber and quantity of microorgan-isms in the root medium (VA 菌根对黄瓜矿质元素吸收和根际土壤微生物数量的影响 )J. Jiangsu Agricultural Sci-ences(江苏农业科学 ), 2009 (1): 284-285.1026AgriculturalScience&TechnologyVol.13, No.5, 2012 Agricultural Science & Technology2012华中神农箭竹更新幼龄地下茎伸长规律研究王 玲，李 昆，孟银萍，赵丽雅，李兆华*（湖北大学资源环境学院，湖北武汉 430062）摘 要 目的 探讨华中神农箭竹更新幼龄地下茎的伸长规律 。方法 在神农架国家自然保护区内凉风垭设置样地，选取六丛独立生长的神农箭竹作为研究样本，测量各龄级地下茎长度与直径，地下茎龄级采用倒逐龄法确定 。结果 通过样地调查研究，可以发现在神农箭竹的幼苗期，地下茎随时间序列快速延长，呈指数曲线增长 。研究结果表明，克隆更新的神农箭竹幼苗虽然无性繁殖生长了 15 年，但是此新世代的种群还未达到稳定状态 。结论 该研究通过对神农箭竹地下茎伸长规律的探索，以期为掌握此物种生长周期的基本特性提供理论支持 。关键词 神农箭竹；克隆生长；地下茎；曲线拟合；神农架基金项目 国家自然科学基金（ 31070370） 。作者简介 王玲（ 1987-），女，湖北襄阳人，硕士研究生 , 从事植物生态学研究， E-mail: wlk_211126.com。* 通讯作者 。收稿日期 2012-02-02 修回日期 2012-03-12!Responsible editor: Qingqing YIN Responsible proofreader: Xiaoyan WUlation (实心狭叶方竹种群的生物量结构与地下茎生长规律研究 )J. Journal ofForest Research(森林研究杂志 ), 2009,22: 662-666.8 LI ZH, DENICH M, BORSCH T. Effectsof bamboo Fargesia murielae on plantdiversity in fir forest on Mountain Shen-nongjia J. Forest Stud China, 2004, 6(4):17-22.9 LI ZH , DENG L, ZHAO BY, et al.Species and distribution of mountainbamboos in Shennongjia, Central ChinaJ. J For Res, 2003, 14(1): 35-38.10 TIAN XQ. Natural regeneration of mou-ntain bamboos on Mount QinglingJ.Journal of Bamboo Research, 1991,10(3): 23-31.11 TAYLOR AH, QIN ZS. Tree regenera-tion after bamboo die-back in ChineseAbies-Betula forests J. Journal ofVegetation Science, 1992, 3: 253-260.12 LI ZH, DENICH M. Elevational diversityof arrow bamboo (Fargesia spath-acea) communities on Mount Shen-nongjia in Central ChinaJ. J For Res,2002, 13(3):171-176.13 LI ZH, DENICH M. Post-fire regenera-tion of umbrella bamboo (Fargesiamurielae) on Mount Shennongjia inCentral China J. Ecol Env & Cons,2002, 8: 201- 205.14 LI ZH , DENG L, ZHAO BY, et al.Shoot sprouting of arrow bamboo Far-gesia spathacea along an altitudinalgradient in Central China J. B am J,2003, 20: 1-11.15 LI ZH, DENICH M, DENG L. Seedproduction and dispersal of umbrellabamboo(Fargesia murielae)after massflowering in central china J. B am J,2004, 21: 8-17.16 LIU P(刘平 ), WANG N(王宁 ), SUN QJ(孙清江 ), et al. Study on growth modeland amount maturity of high-yieldingpoplar in Xinjiang Ili(新疆伊犁地区速生杨树生长模型及数量成熟研究 )J.Journal of Xinjiang Agricultural Univer-sity(新疆农业大学学报 ), 2003, 26: 45-48.17 LUO YT (罗应婷 ), YANG YJ (杨钰娟 ).SPSS statistical analysis from basisto practice(SPSS 统计分析 从基础到实践 )M. Beijing: Publishing Houseof Electronics Industry(北京 : 电子工业出版社 ), 2007.18 DAI GJ (戴国俊 ), WANG JY (王金玉 ),YANG JZ(杨建生 ), et al. Fitting growthcurve equation by software SPSS (应用统计软件 SPSS 拟合生长曲线方程 )J. Animal Husbandry & VeterinaryMedicine (畜牧与兽医 ), 2006, 38: 28-30.19 DE KH, VAN GJ. The ecology andevolution of clonal plants M. Leiden:Backhuys Publishers, 1997.20 ZHOU BZ (周本智 ), FU MY (傅懋毅 ).Review on bamboos undergroundrhizome-root system research (竹林地下鞭根系统研究进展 )J. Forest Re-search(林业科学研究 ), 2004, 17: 533-540.21 WANG KH (汪奎宏 ), HE QJ (何奇江 ),WENG FJ(翁甫金 ), et al. Investigationand Analysis on Und