青海一株蚕豆根瘤菌的鉴定及抗旱性评价

Identification of Vicia faba Rhizobium and drought resistance verification in arid region of Qinghai

摘要:

【目的】研究青海干旱地区蚕豆根瘤菌的遗传多样性，获得与蚕豆品种共生匹配且具有耐旱性的根瘤菌株，促进蚕豆耐旱根瘤菌在青海干旱地区生产中的应用。【方法】以分离自青海干旱地区一株菌株QHCD22为材料，利用细菌形态学、生理生化指标鉴定、Biolog细菌鉴定系统、16S rRNA基因序列分析、全基因组分析等进行菌种鉴定和系统发育分析，进一步通过PEG6000模拟干旱胁迫、盆栽回接干旱胁迫处理及旱作田间接种验证试验对该菌株的耐旱性进行综合评价。【结果】QHCD22菌株属快生型根瘤菌属(Rhizobium)，Rhizobium indicum种。随着PEG6000模拟干旱胁迫程度的加剧，在−0.6 mPa这一更低渗透势时菌株存活数量增高，浊度由61.48%上升到69.42%，表现出较强的耐旱性。盆栽试验表明，接种根瘤菌处理(NA)的株高、植株鲜干比、根瘤数、根瘤鲜重、叶绿素含量(SPAD)、叶片相对含水量(RWC)、脯氨酸含量(PRO)、超氧化物歧化酶活性(SOD)、根系活力(TCC)均高于不接种根瘤菌处理(NN)，并且在正常供水条件下，NA处理的各指标也均高于NN处理。旱作田间验证试验表明接种该菌株显著提高固氮酶活性，青海13号蚕豆根瘤固氮酶活性由不接种的42.07 C₂H₄ nmol/(g·h)显著增加到221.78 C₂H₄ nmol/(g·h)，青蚕14号蚕豆由40.60 C₂H₄ nmol/(g·h)显著增加到109.78 C₂H₄ nmol/(g·h)，马牙蚕豆由33.41 C₂H₄ nmol/(g·h)显著增加到643.15 C₂H₄ nmol/(g·h)。接种根瘤菌对于增加产量具有促进作用，其中青蚕14号的增产效果显著，增产幅度达32.3%。【结论】QHCD22菌株可能为快生型根瘤菌属的一个种Rhizobium indicum，具有一定的耐旱性，研究表明接种根瘤菌可以提高蚕豆的耐旱性，尤其对干旱敏感型蚕豆品种增产效果显著，具有潜在的应用前景。

Abstract:

[Objective] To analyze the genetic diversity of Vicia faba Rhizobium in arid region of Qinghai,so as to obtain the Rhizobium strain with drought tolerance and matching V.faba varieties and thus promote the application of drought-tolerant Rhizobium of V.faba in arid region of Qinghai.[Methods] With the strain QHCD22 isolated from the arid region of Qinghai as the research subject,species identification and phylogenetic analysis were performed by morphology,physiological and biochemical characteristics,Biolog microbial identification system,16S rRNA gene sequencing and whole genome analysis.Further,polyethylene glycol (PEG)6 000 simulated drought stress,pot experiment and field plot trial were used to evaluate the drought resistance of the strain.[Results] Strain QHCD22 belonged to Rhizobium indicum.With the increased addition of PEG 6 000 to yeast mannitol agar (YMA) broth medium,the strain had an intermediate lethal osmotic potential under medium PEG concentration.But the number of viable strains increased under higher PEG concentration (osmotic potential of−0.6 mPa),and the turbidity of the strain rose from 61.48% to 69.42%,showing a strong drought tolerance.Pot experiment revealed that V.faba inoculated with the strain had higher plant height,GW/DW ratio,nodule number,nodule fresh weight,SPAD values,leaf relative water content (RWC),proline (PRO) content,superoxide dismutase (SOD) activity and root vatality (triphenyltetrazolium chloride,TTC) than that non-inoculated.In addition,under the condition of normal water,the indexes of the inoculated V.faba were higher than those of non-inoculated.The field plot trial indicated that the nitrogenase activity of strain QHCD22 was significantly elevated.Specifically,the nitrogenase activities of Qinghai 13 variety,Qingcan 14 variety and Maya variety ranged from 42.07 to 221.78 C₂H₄ nmol/(g·h),40.60 to 109.78 nmol C₂H₄ nmol/(g·h),and 33.41 to 643.15 C₂H₄ nmol/(g·h),respectively.Rhizobium inoculation promoted the increase of V.faba yield,with Qingcan 14 variety having a significant increase of 32.3%.[Conclusion] Strain QHCD22 belonged to Rhizobium indicum,which had certain drought-tolerant characteristics.The study confirmed that Rhizobium inoculation could improve the drought tolerance of V.faba,especially the drought-sensitive V.faba variety.Therefore the strain had potential application prospects.