李想，劉艷霞，夏范講，蔡劉體，張恒，石俊雄

1 貴州省煙草科學(xué)研究院，貴陽(yáng) 550000；2 中國(guó)煙草總公司貴州省公司，貴州貴陽(yáng) 550000

煙草根際促生菌（PGPR ）的篩選、鑒定及促生機(jī)理研究

李想1，劉艷霞1，夏范講2，蔡劉體1，張恒1，石俊雄1

1 貴州省煙草科學(xué)研究院，貴陽(yáng) 550000；2 中國(guó)煙草總公司貴州省公司，貴州貴陽(yáng) 550000

篩選并鑒定了貴州煙區(qū)煙草根際促生菌（PGPR）菌株。測(cè)定PGPR產(chǎn)激素能力、定殖能力以及對(duì)煙草苗期促生作用，以具備多項(xiàng)促生能力為篩選標(biāo)準(zhǔn)，確定目標(biāo)PGPR，再通過(guò)16S rDNA、平板對(duì)峙以及PCR技術(shù)對(duì)目標(biāo)PGPR進(jìn)行屬鑒定、抗病能力測(cè)定。通過(guò)篩選獲得的7株目標(biāo)PGPR均能產(chǎn)生脫落酸、細(xì)胞分裂素、赤霉素和生長(zhǎng)素。煙苗盆栽試驗(yàn)表明7株P(guān)GPR均具有促生效果，其中LX-7處理的煙苗鮮重和根系活力最大；大田移栽30d后，LX-7根際定殖數(shù)量可維持107cfu/g根，顯著高于其它菌株；抑菌試驗(yàn)證實(shí)LX-4、LX-5和LX-7具有廣譜抗菌作用。綜合考量，LX-4、LX-5和LX-7具有顯著的促生效果，經(jīng)16S rDNA鑒定分別為枯草芽孢桿菌、地衣芽孢桿菌和解淀粉芽孢桿菌。

煙草根際促生菌；篩選鑒定；促生能力；16SrDNA；促生基因

根際促生菌（PGPR）的種類繁多, 主要集中于細(xì)菌的20 多個(gè)屬，部分放線菌以及真菌也具有促生作用[1]。PGPR對(duì)植物的生長(zhǎng)促進(jìn)可分為直接和間接作用[2]，直接促生的機(jī)理包括提高作物氮素吸收能力[3]、改善根系溶磷能力[4]、產(chǎn)鐵載體[5]以及產(chǎn)生植物激素類物質(zhì)[6]等，間接促生機(jī)理主要體現(xiàn)在對(duì)病原菌拮抗能力[7]。PGPR不僅能促進(jìn)植物生長(zhǎng)降低化肥的使用量，還可一定程度減少農(nóng)藥的施用，起到“減肥、減藥”的作用[8]，植物根際促生菌的研究越來(lái)越受到關(guān)注[9]。目前有關(guān)PGPR的研究與應(yīng)用主要集中在水稻[10]、小麥[11]、高粱[12]、玉米[13]等重要糧食作物上，煙草根際促生菌研究主要集中在病害生防方面[14]，對(duì)促生減肥方面研究甚少[15]，煙草PGPR能否有效地發(fā)揮促生抗病功能，很大程度上取決于菌群在植物根際的存活與增殖能力。從貴州不同植煙區(qū)域土壤樣品中篩選鑒定了具有多種促生機(jī)制的PGPR 菌株，并進(jìn)行了抗病能力測(cè)定，以期為構(gòu)建高效廣適促生菌群提供菌株資源。

1 材料與方法

1.1 植物根際土壤樣品處理及PGPR菌株初篩

從貴州省典型植煙生態(tài)區(qū)域（道真、天柱、威寧、興義和貴定5縣）采集根際土壤共26份[16]。采用稀釋平板涂布法從土壤樣品中分離、純化PGPR菌株。所用培養(yǎng)基為牛肉膏蛋白胨培養(yǎng)基（牛肉膏 5.0 g，氯化鈉5.0 g，蛋白胨10 g，瓊脂20 g，蒸餾水1000 mL，pH 7.0～7.2，121 ℃滅菌20 min）。平板共分離PGPR菌株99株，綜合其菌落形態(tài)和生長(zhǎng)速度差異初步篩選出20株菌株，并對(duì)其進(jìn)行純化[17]。

1.2 PGPR促生能力、產(chǎn)生植物激素以及定殖能力的測(cè)定

PGPR的產(chǎn)NH3[18]、產(chǎn)HCN[19]、產(chǎn)鐵載體[20]以及溶磷解鉀能力[4]采用相關(guān)文獻(xiàn)方法測(cè)定。采用Elisa定量檢測(cè)試劑盒（GENMED SCIENTIFICS INC.U.S.A）[21]測(cè)定根際促生菌的牛肉膏蛋白胨液體培養(yǎng)基培養(yǎng)液中生長(zhǎng)素、脫落酸、分裂素、赤霉素含量。煙苗根際PGPR數(shù)量采用牛肉膏蛋白胨培養(yǎng)基平板計(jì)數(shù)法測(cè)定，方法是稱取育苗缽中的煙苗根系1 g加9 mL無(wú)菌水，28 ℃恒溫?fù)u床振蕩20 min，采用系列稀釋法涂布平板，置25℃～30 ℃培養(yǎng)箱中暗培養(yǎng)24h～72 h計(jì)數(shù)[22]。

1.3 PGPR對(duì)煙苗的促生作用

將PGPR進(jìn)行牛肉膏蛋白胨液體發(fā)酵擴(kuò)繁，當(dāng)培養(yǎng)液中菌株數(shù)量達(dá)到108cfu/mL時(shí)，在3000 rpm、4℃下離心3 min，用滅菌去離子水重懸微生物。以2 %接種量接種育苗缽（體積為250mL）基質(zhì)（滅過(guò)菌）中，拌勻后將煙苗（K326）移栽于育苗缽中，以接滅菌去離子水為對(duì)照，每處理重復(fù)5缽。定期觀察煙苗生長(zhǎng)情況，并測(cè)定煙苗的鮮重、根系活力和葉綠素含量。采用WinRhizo根系分析系統(tǒng)測(cè)定根系形態(tài)指標(biāo)，使用根系掃描儀(型號(hào)為EPSON1680)及其配套的WinRhizo Pro 5.0 根系分析軟件測(cè)定根系指標(biāo)[23]。

1.4 PGPR的16S rDNA的鑒定

將篩選得到的 LX4、LX5和LX7在液體培養(yǎng)基中培養(yǎng)至對(duì)數(shù)期，按凍融法提取細(xì)菌基因組DNA，進(jìn)行16S r DNA鑒定[24]。

1.5 PGPR對(duì)其它病原菌的抑菌圖譜

對(duì)真菌性病害采用在土豆葡萄糖瓊脂培養(yǎng)基（PDA：1000 mL含200 g土豆浸提物，50 g葡萄糖，pH 自然，瓊脂粉20 g）平板中心位置接種直徑5 mm7d齡的菌塊，在距離菌塊2 cm處分別用滅菌牙簽點(diǎn)接目標(biāo)PGPR，28 ℃恒溫培養(yǎng)培養(yǎng)7d，以不接PGPR只接病原菌的平板作為對(duì)照，觀察病原菌的生長(zhǎng)是否受到抑制[25]。對(duì)細(xì)菌性病害將目標(biāo)PGPR點(diǎn)接在改良后的牛肉膏蛋白胨平板上，病原細(xì)菌搖成菌懸液，然后均勻噴在平板上。在30 ℃恒溫培養(yǎng)48～72 h后，觀察PGPR周圍是否有抑菌圈。

1.6 數(shù)據(jù)處理

試驗(yàn)數(shù)據(jù)采用Microsoft Excel 2003處理，顯著性分析采用SPSS Base Ver.13.0統(tǒng)計(jì)軟件 (SPSS, IL,Chicago, USA)進(jìn)行, LSD、Duncan 新復(fù)極差進(jìn)行多重比較（P≤0.05）。

2 結(jié)果與分析

2.1 促生菌株的篩選

分別測(cè)定20株細(xì)菌的5項(xiàng)促生指標(biāo)（產(chǎn)NH3、產(chǎn)HCN、產(chǎn)鐵載體、溶磷能力、解鉀能力），結(jié)果見(jiàn)表1, 由表1可以看出：T1-2、A1-5、B4-5和B5-8不具備產(chǎn)NH3能力，占測(cè)定菌株的20%；A1-5、LX-2、B3-1、LX6、B5-8不具備產(chǎn)HCN能力，占測(cè)定菌株的25%；LX1、B2-4、B5-5、B5-8、LX4、LX5和LX7共7株菌株可以產(chǎn)鐵載體，占測(cè)定菌株的35%；在溶磷能力方面B2-4、B3-1、LX4、LX5、LX7和LY-1表現(xiàn)好于其它菌株；在解鉀能力上 B2-4、B3-4、LX6、LX4、LX5、LX7和 LY-1表現(xiàn)好于其它菌株。綜合各項(xiàng)指標(biāo)，初步篩選B2-4、B3-1、LX4、LX5、B3-6、LX7、LY-1作為促生目標(biāo)菌株。

表1 細(xì)菌體外促生能力Tab. 1 Plant promotion ability of rhizobacteria

續(xù)表1

2.2 促生菌株產(chǎn)生植物激素

從圖1可以看出，篩選到的7株促生菌均能產(chǎn)生脫落酸、細(xì)胞分裂素、赤霉素和生長(zhǎng)素，產(chǎn)生的赤霉素總量顯著高于細(xì)胞分裂素；其中LX4的脫落酸含量與LY-1無(wú)顯著差異，但顯著高于其它菌株，是LX5產(chǎn)生的脫落酸的7.38倍；LX4菌株產(chǎn)生的細(xì)胞分裂素最多，顯著高于其它菌株， B3-1、B3-6和LX7之間無(wú)顯著差異；LX4分泌的赤霉素顯著高于其它菌株，其次為L(zhǎng)X7，再次為L(zhǎng)X5，分別比LX7和LX5多13.67%和31.18%；LX5分泌的生長(zhǎng)素顯著高于其它菌株，其次為L(zhǎng)X7，再次為L(zhǎng)X4，分別比LX7和LX4多31.08%和45.04%。

圖1 促生菌產(chǎn)生植物激素的含量Fig. 1 Content of phytohormone produced by plant-promoting bacteria

2.3 促生菌株的煙草根際定殖

起始接種量為1×108cfu/mL，移栽10d檢測(cè)PGPR菌株在煙草根表的定殖數(shù)量，B3-1菌體數(shù)量下降最為明顯，降幅顯著高于其它菌株（表2），其次為B2-4，其余5株促生菌均可維持在107cfu/g根；移栽20d后，LX4和LX7的數(shù)量在107cfu/g根以上，而其它菌株的數(shù)量有所下降，LX5、LX6和LY-1下降到106cfu/g根。移栽30d后，LX7的數(shù)量顯著高于其它菌株，仍然維持在107cfu/g根，而LX4和LX5的數(shù)量在106cfu/g根，其它菌株的定殖數(shù)量下降明顯，可見(jiàn)LX7、LX4和LX5在煙草根系具有較高的定殖能力。

表2 目標(biāo)PGPR在煙草根系的定殖能力Tab. 2 Colonized ability of target PGPR in rhizosphere soils

2.4 促生菌株對(duì)煙苗的促生作用

如圖2所示，篩選的7個(gè)促生菌株對(duì)煙苗的促生效果各有不同。

圖2 7種促生菌株對(duì)烤煙煙苗的促生效果Fig. 2 Promotion effect of 7 plant-promoting bacteria on seedlings of fl ue-cured toabcco

2.4.1 對(duì)烤煙苗期的影響

如表3所示，所有PGPR處理的煙苗鮮重均比對(duì)照顯著增加，分別增加13.78%（B2-4）、27.89%（B3-1）、52.04%（LX4）、57.99%（LX5）、41.68%（B3-6）、69.35%（LX7）、39.58%（LY-1）；LX7處理的根系活力最高，與LX4和LX5處理之間無(wú)顯著差異，顯著高于其它高處理；與對(duì)照相比，LX4、LX5和LX-7處理的根系活力顯著增加，分別增加84.8%、83.1%和91.0%。同時(shí)，LX5處理葉綠素含量最高，與LX4、LX7處理的之間無(wú)差異，顯著高于對(duì)照，是對(duì)照的1.74倍。

2.4.2 對(duì)煙株苗期根系發(fā)育的影響

LX5、LX-7和LX4促生菌株處理煙苗的根總長(zhǎng)之間無(wú)顯著差異（圖3），但顯著高于其它處理，分別比對(duì)照處理增加33.56%、29.21%和24.87%；LX5和LX7處理的煙苗根直徑無(wú)顯著差異，但顯著高于其它處理，比對(duì)照處理分別高出12.22%和8.89%；LX5、LX7和LX4處理的煙苗根面積無(wú)顯著差異，但顯著高于其它處理，分別比對(duì)照處理增加66.49%、60.31%和54.12%；LX5、LX7和LX4處理的煙苗根體積無(wú)顯著差異，但顯著高于其它處理，分別是對(duì)照處理的1.99、1.97和1.95倍；LX4、LX5和LX7處理的平均促生效果分別在根總長(zhǎng)、根直徑、根面積和根體積上比對(duì)照處理增加29.21%、8.49%、60.3%和96.94%（圖3）。

表3 不同PGPR對(duì)苗期烤煙的影響Tab. 3 Effect of different PGPR on fl ue-cured tobacco seedlings

圖3 7種促生菌株對(duì)煙苗根系發(fā)育的影響Fig. 3 Effect of 7 plant-promoting bacteria on root growth

2.5 促生菌株LX4、LX5和LX7的16S rDNA鑒定

16S rDNA基因序列測(cè)序結(jié)果表明，LX4、LX5、LX7分別為：枯草芽孢桿菌（Bacillussubtilis）、地衣芽孢桿菌（Bacilluslicheniformis）和解淀粉芽孢桿菌（Bacillusamyloliquefaciens）（圖4）。

圖4 促生菌株LX4、LX5和LX7的系統(tǒng)發(fā)育樹(shù)Fig. 4 Diagram of LX4, LX5 and LX7

2.6 LX4、LX5和LX7對(duì)病原菌的拮抗作用

LX4、LX5和LX7對(duì)試驗(yàn)用病原菌都有抑制作用（表4），具有防控多種土傳病害的潛力。

表4 LX4、LX5和LX7對(duì)土傳病原菌的拮抗作用Tab. 4 Antagonistic effect of LX4, LX5 and LX7

3 討論

在5項(xiàng)促生指標(biāo)（產(chǎn)NH3、產(chǎn)HCN、產(chǎn)鐵載體、溶磷能力、解鉀能力）中，經(jīng)過(guò)初篩的20株P(guān)GPR均具有多項(xiàng)促生指標(biāo)，同時(shí)采用原位篩選保證了20株P(guān)GPR具備田間應(yīng)用條件。但是否可以用于實(shí)際生產(chǎn)還需要進(jìn)一步通過(guò)模擬試驗(yàn)驗(yàn)證。LX4、LX5和LX7的各項(xiàng)能力在20株P(guān)GPR中促生能力表現(xiàn)突出（表1），LX7產(chǎn)HCN的能力最強(qiáng)，可能在抗病能力上具有顯著的效果，同時(shí)也有研究表明細(xì)菌產(chǎn)生鐵載體對(duì)病原真菌具有一定的拮抗作用[26]，本研究中的20株P(guān)GPR具有產(chǎn)鐵載體能力的比較少，結(jié)合產(chǎn)HCN和產(chǎn)鐵載體能力，LX7可作為貴州生態(tài)條件下煙草病害生防菌來(lái)進(jìn)行探究和利用。

PGPR產(chǎn)生適量的植物激素可以促進(jìn)作物生長(zhǎng)，而過(guò)量的激素反而會(huì)抑制植物生長(zhǎng)[27]，比如PGPR產(chǎn)生的脫落酸可以誘導(dǎo)作物產(chǎn)生抗干旱脅迫能力，減輕干旱對(duì)作物的氧化傷害[28]，但同時(shí)由于脫落酸和分裂素都有共同的前體物質(zhì)[29]，因此脫落酸數(shù)量增多也會(huì)導(dǎo)致細(xì)胞分裂素減少，進(jìn)而影響作物的生長(zhǎng)。本研究發(fā)現(xiàn)LX4產(chǎn)生脫落酸顯著大于其它菌株（圖1），但同時(shí)產(chǎn)生的細(xì)胞分裂素、赤霉素和生長(zhǎng)素也是最高的，LX4分泌的激素功能到底是促生或是抑制，取決于產(chǎn)物的濃度以及煙草的忍受力，因此通過(guò)煙草苗期試驗(yàn)發(fā)現(xiàn)LX7、LX4和LX5處理的煙苗鮮重?zé)o顯著差異，比對(duì)照增加52.04%～69.35%，LX4的根系活力以及根系指標(biāo)的促生效果基本與LX7和LX5相當(dāng)，說(shuō)明LX4盡管產(chǎn)生脫落酸以及赤霉素很高，但對(duì)煙草促生效果顯著，這與Porcel等的研究結(jié)果相類似[30]。

LX4、LX5和LX7經(jīng)鑒定分別為枯草芽孢桿菌（Bacillus subtilis）、 地 衣 芽 孢 桿 菌（Bacillus licheniformis）和解淀粉芽孢桿菌（Bacillus amyloliquefaciens）。本研究篩選得到的3株促生菌屬均有促生和防病作用的報(bào)道，Bacillus subtilis BEB-13bs作為PGPR可以提高西紅柿的產(chǎn)量和品質(zhì)[31]，Ramos等研究發(fā)現(xiàn)接種Bacillus licheniformis后可以顯著改善歐洲赤楊的根系和地上部生物量[32]，同時(shí)Bacillus amyloliquefaciens可以很好的定殖在玉米根際，同時(shí)形成生物膜進(jìn)行生物防控[33]。

LX7、LX4和LX5對(duì)大多數(shù)的病原菌均具有一定的拮抗作用，這3株促生菌在根際定殖的數(shù)量在30d后仍然維持在106cfu/g根以上，說(shuō)明該3株P(guān)GPR具有生防菌的潛力[34]、。本文篩選到LX4可以分泌植物類激素，促進(jìn)植物生長(zhǎng)， LX7在煙苗根際定殖能力強(qiáng)，還可以產(chǎn)生HCN，可作為生防菌，綜上所述LX4、LX5和LX7可以作為貴州煙草促生有機(jī)肥的功能菌株，具有較好的應(yīng)用潛力。

[1]Zaidi A, Ahmad E, Khan M S, et al. Role of plant growth promoting rhizobacteria in sustainable production of vegetables: Current perspective [J]. Scientia Horticulturae,2015, 193: 231-239.

[2]Arruda L, Beneduzi A, Martins A, et al. Screening of rhizobacteria isolated from maize (zea mays l.) in rio grande do sul state (south brazil) and analysis of their potential to improve plant growth [J]. Applied Soil Ecology, 2013, 63:15-22.

[3]Taulé C, Mareque C, Barlocco C, et al. The contribution of nitrogen fixation to sugarcane (saccharum officinarum l.), and the identi fi cation and characterization of part of the associated diazotrophic bacterial community [J]. Plant and Soil, 2012, 356(1): 35-49.

[4]Wu F, Wan J H C, Wu S, et al. E ff ects of earthworms and plant growth-promoting rhizobacteria (pgpr) on availability of nitrogen, phosphorus, and potassium in soil [J]. J. Plant Nutr. Soil Sci, 2012, 175: 423-433.

[5]Kurabachew H, Wydra K. Characterization of plant growth promoting rhizobacteria and their potential as bioprotectant against tomato bacterial wilt caused by ralstonia solanacearum [J]. Biological Control, 2013, 67(1): 75-83.

[6]Piromyou P, Buranabanyat B, Tantasawat P, et al. E ff ect of plant growth promoting rhizobacteria (pgpr) inoculation on microbial community structure in rhizosphere of forage corn cultivated in thailand [J]. European Journal of Soil Biology, 2011, 47(1): 44-54.

[7]Kheirandish Z, Harighi B. Evaluation of bacterial antagonists of ralstonia solanacearum, causal agent of bacterial wilt of potato [J]. Biological Control, 2015, 86:14-19.

[8]Erturk Y, Ercisli S, Cakmakci R. Yield and growth response of strawberry to plant growth-promoting rhizobacteria inoculation [J]. Journal of Plant Nutrition, 2012, 35: 817-826.

[9]Bhattacharyya P N, Jha D K. Plant growth-promoting rhizobacteria (pgpr): Emergence in agriculture [J]. World Journal of Microbiology & Biotechnology, 2012, 28: 1327-1350.

[10]Habibi S, Djedidi S, Prongjunthuek K, et al. Physiological and genetic characterization of rice nitrogen fixer pgpr isolated from rhizosphere soils of di ff erent crops [J]. Plant and Soil, 2014, 379(1): 51-66.

[11]Zhang J, Liu J, Meng L, et al. Isolation and characterization of plant growth-promoting rhizobacteria from wheat roots by wheat germ agglutinin labeled with fluorescein isothiocyanate [J]. The Journal of Microbiology, 2012, 50:191-198.

[12]Praveen Kumar G, Kishore N, Leo Daniel Amalraj E, et al.Evaluation of fl uorescent pseudomonas spp. With single and multiple pgpr traits for plant growth promotion of sorghum in combination with am fungi [J]. Plant Gowth Regulation,2012, 67: 133-140.

[13]Gholami A, Biyari A, Gholipoor M, et al. Growth promotion of maize (zea mays l.) by plant-growth-promoting rhizobacteria under fi eld conditions [J]. Communications in Soil Science and Plant Analysis, 2012, 43: 1263-1272.

[14]Wang S, Wu H, Qiao J, et al. Molecular mechanism of plant growth promotion and induced systemic resistance to tobacco mosaic virus by bacillus spp [J]. Journal of Microbiology and Biotechnology, 2009, 19: 1250-1258.

[15] 王豹祥, 李富欣, 張朝輝, 等. 應(yīng)用pgpr 菌肥減少烤煙生產(chǎn)化肥的施用量 [J]. 土壤學(xué)報(bào), 2011, 48(7): 813-822.Wang Baoxiang, Li Fuxin, Zhang Chaohui, Wu Fengguang,Xi Shuya, Zhu Bao, Cao Yubo, Liu Tianxiang, Qiu Liyou.Effect of application of PGPR on chemical fertilizer application tate for flue-cued tobacco.Acta Pedologica Sinica.2011, 48 (7): 813-822.

[16]Carlsen S C K, Pedersen H A, Spliid N H, et al. Fate in soil of fl avonoids released from white clover (trifolium repens l.)[J]. Appl Environ Soil Sci, 2012, 2012: 1-10.

[17]Usha Rani M, Arundhathi., Reddy G. Screening of rhizobacteria containing plant growth promoting (pgpr)traits in rhizosphere soils and their role in enhancing growth of pigeon pea [J]. African Journal of Biotechnology, 2012,11: 8085-8091.

[18] 康貽軍, 程潔, 梅麗娟, 等. 植物根際促生菌的篩選及鑒定 [J]. 微生物學(xué)報(bào), 2010, 50(7): 853-861.KANG Yijun, CHENG Jie, MEI Lijuan, et al. Screening and identi fi cation of plant growth-promotingrhizobacteria.Acta Microbiologica Sinica.2010, 50 (7): 853-861.

[19]Subramanian J, Satyan K. Isolation and selection of fl uorescent pseudomonads based on multiple plant growth promotion traits and siderotyping [J]. Chilean journal of agricultural research, 2014, 74: 319-325.

[20]Jasim B, Benny R, Sabu R, et al. Metabolite and mechanistic basis of antifungal property exhibited by endophytic bacillus amyloliquefaciens bmb 1 [J]. Applied Biochemistry and Biotechnology, 2016, 179(5): 830-845.

[21]Lequin R M. Enzyme immunoassay (eia)/enzyme-linked immunosorbent assay (elisa) [J]. Clinical Chemistry, 2005,51(12): 2415-2418.

[22]Liu Y, Li X, Cai K, et al. Identification of benzoic acid and 3-phenylpropanoic acid in tobacco root exudates and their role in the growth of rhizosphere microorganisms [J].Applied Soil Ecology, 2015, 93: 78-87.

[23]王亞男, 程立娟, 周啟星. 鳶尾對(duì)石油烴污染土壤的修復(fù)以及根系代謝分析[J]. 環(huán)境科學(xué), 2016, 37(4): 1531-1538.WANG Yanan, CHENG Lijuan, ZHOU Qixing.Phytoremediation of petroleum contaminated soils withIris pseudacorusL. andthe metabolic analysis in roots. Chinese Journal of Environmental Science. 2016, 37(4): 1531-1538.

[24]Zhang H, Yu Z, Huang Q, et al. Isolation, identi fi cation and characterization of phytoplankton-lytic bacterium ch-22 against microcystis aeruginosa [J]. Limnologica, 2011, 41:70-77.

[25]Dinesh R, Anandaraj M, Kumar A, et al. Isolation,characterization, and evaluation of multi-trait plant growth promoting rhizobacteria for their growth promoting and disease suppressing effects on ginger [J]. Microbiological Research, 2015, 173: 34-43.

[26] 榮良燕, 姚拓, 趙桂琴, 等. 產(chǎn)鐵載體pgpr菌篩選及其對(duì)病原菌的拮抗作用 [J]. 植物保護(hù), 2011, 37(1): 59-64.RONG Liangyan, YAO Tuo, ZHAO Guiqin, et al.Screening of siderophore-producing PGPR bacteria andtheir antagonism against the pathogens[J]. Plant Protection. 2011,37 (1): 59-64

[27]Persello-Cartieaux F, Nussaume L, Robaglia C. Tales from the underground: Molecular plant-rhizobacteria interactions[J]. Plant,Cell and Environment, 2003, 26: 186-199.

[28]Kohler J, Hernández J A. Pgpr and am fungi modify alleviation biochemical mechanisms in water stressed plants[J]. Functional Plant Biology, 2008, 35: 141-151.

[29] 康貽軍, 程潔, 梅麗娟, 等. 植物根際促生菌作用機(jī)制研究進(jìn)展 [J]. 應(yīng)用生態(tài)學(xué)報(bào), 2010, 21(1): 232-238.Kang Yijun, Cheng Jie, Mei Lijuan, Hu Jian, Piao Zhe, Yin Shixue. Action mechanisms of plant growth-promoting rhizobacter ia (PGPR) : A review. Chinese Journal of Applied Ecology. 2010, 21 (1): 232-238

[30]Porcel R, Zamarre?o á M, García-Mina J M, et al.Involvement of plant endogenous aba in bacillus megaterium pgpr activity in tomato plants [J]. BMC Plant Biology, 2014, 14(1): 1-12.

[31]Mena-Violante H G, Olalde-Portugal V. Alteration of tomato fruit quality by root inoculation with plant growthpromoting rhizobacteria (pgpr): Bacillus subtilis beb-13bs[J]. Scientia Horticulturae, 2007, 113(1): 103-106.

[32]Ramos B, Garc??a J A L, Probanza A n, et al. Alterations in the rhizobacterial community associated with european alder growth when inoculated with pgpr strain bacillus licheniformis [J]. Environmental and Experimental Botany,2003, 49(1): 61-68.

[33]Zhang N, Yang D, Wang D, et al. Whole transcriptomic analysis of the plant-beneficial rhizobacterium bacillus amyloliquefaciens sqr9 during enhanced bio fi lm formation regulated by maize root exudates [J]. BMC Genomics,2015, 16(1): 1-20.

[34]Ling N, Deng K, Song Y, et al. Variation of rhizosphere bacterial community in watermelon continuous monocropping soil by long-term application of a novel bioorganic fertilizer [J]. Microbiological Research, 2014, 169(7/8):570-578.

：LI Xiang, LIU Yanxia, XIA Fanjiang, et al.Screening, identi fi cation and plant growth-promotion mechanism of tobacco plants rhizobacteria [J]. Acta Tabacaria Sinica, 2017, 23(3)

*Corresponding author.Email：iversonlyx@163.com

Screening, identi fi cation and plant growth-promotion mechanism of tobacco plants rhizobacteria

LI Xiang1, LIU Yanxia1*, XIA Fanjiang2,CAI Liuti1, ZHANG Heng1, SHI Junxiong1
1 Guizhou Academy of Tobacco Science, Guiyang 550000, China;2 Guizhou Tobacco Company, China National Tobacco Corporation,550000

Plant growth-promoting rhizobacteria was screened and identi fi ed in order to build a foundation for tobacco PGPR-bioorganic fertilizer development. Plant growth-promoting rhizobacteria in rhizosphere soils were pre-isolated by considering ammonia, HCN, and siderophores production, phosphate-solubilizing and potassium dissolving capacity. Phytohormone production, colonization and plantgrowth-promotion in seedling period were explored to screen PGPR. Target PGPR were identi fi ed by 16S rDNA sequencing. Diseaseresistance capacity and plant growth-promoting were evaluated. Twenty strains primarily selected had potential plant-growth-promotion ability. All seven re-screened PGPR strains produced abscisic acid, phytokinin, gibberellin and auxin. The population of LX-7 strain in rhizosphere soil was signi fi cantly higher than other selected strains and kept 107cfu/g soil 30 days after transplanting. The counts of LX-4 and LX-5 strains were 106cfu/g while the populations of other selected strains signi fi cantly decreased. Tobacco seedling experiment showed that seven PGPR had the ability of plant-growth-promotion. Fresh weight and root system activity of LX7 treatment were the highest among all treatments. Chlorophyll content of tobacco seedling in LX5 treatment was significantly higher than that of other treatments. Compared with control, average root length, root diameter, root area and root volume in LX4, LX5 and LX7 treatments increased by 29.21%, 8.49%, 60.3% and 96.94%. LX4, LX5 and LX7 were identified asBacillus subtilis,Bacillus licheniformisandBacillus amyloliquefaciens, respectively. These three PGPR had broad spectrum antibiotics. Three PGPR strains, LX4, LX5 and LX7, had remarkable e ff ect on plant-growth-promotion and can be employed as key bacteria in tobacco growth-promotion .

plant growth promoting rhizobacteria(PGPR); screening and identi fi cation; plant growth promoting ability; 16SrDNA; plant growth promoting gene

李想，劉艷霞，夏范講，等. 煙草根際促生菌（PGPR）的篩選、鑒定及促生機(jī)理研究[J]. 中國(guó)煙草學(xué)報(bào)，2017, 23（3）

國(guó)家自然科學(xué)基金項(xiàng)目（41461068）；貴州省科學(xué)技術(shù)基金項(xiàng)目（[2013]2197、2198）；中國(guó)煙草總公司貴州省公司科技項(xiàng)目（201410,110201402009）

李 想（1982—），博士，副研究員，主要研究方向：煙草營(yíng)養(yǎng)與土壤養(yǎng)分循環(huán)管理，Email：newcool1361214@163.com

劉艷霞（1982—），Email：iversonlyx@163.com

2016-08-26；< class="emphasis_bold">網(wǎng)絡(luò)出版日期：

日期：2017-05-16