摘要
植物内生固氮菌是近年新发现的不同于经典共生固氮和联合固氮的高效固氮体系。内生固氮菌生活在非豆科植物体内,避免了化合态氮的抑制及土著微生物的竞争,表现出更高的固氮效率。同时其还具有分泌生长素、溶磷,增强植株抗病性等多方面特殊的生物学及生态学作用,在提高宿主的营养及竞争力,增强宿主对生物和非生物胁迫的抗性,促进其环境适应能力方面具有重要的应用价值。本文通过对塔里木河下游几种典型荒漠植物的根际微生物数量、群落组成及其活性的研究,揭示了根际微生物与植物间的相互关系和作用,以及植物生活型对根际效应的影响。在此基础上,从多枝柽柳(Tamarix ramosissima Ledeb.)、黑果枸杞(Lycium ruthenicum Murr.)中分离筛选出了固氮活性高,并具有溶磷、分泌植物生长激素等多种促生性能的内生固氮菌株,为创制应用于荒漠植被保育与恢复的、具有固氮、促生、抗逆的多功能接种剂奠定了基础。主要研究结果如下:
荒漠植物根际微生物数量、组成与根外有较大差异,并具有不同的根际效应,灌木的根际效应优于草本植物。塔里木河下游典型荒漠植物黑果枸杞、刚毛柽柳(Tamarix hispida Willd.)、花花柴(Karelinia caspia(Pall.)Less.)、盐穗木(Halostachys caspica(Bieb.)C.A.Mey.)具有显著的根际正效应,其根际细菌的R/S为1.26～150.42,以黑果枸杞根际效应最为突出(R/S为150.42)。疏叶骆驼刺(Alhagi sparsifolia Shap.ex Kell.et Shap.)、大叶白麻(Poacynum hendersonii(Hook.f.)Woodson)和河西苣(Hexinia polydichotoma(Ostent.)H.L.Yang)则产生负根际效应。根际生理群微生物氨化细菌和纤维素分解菌均表现出强烈的正根际效应,而硝化细菌未检出。荒漠植物的生长对根际土壤脲酶、碱性磷酸酶具有较强的促进作用,但不同荒漠植物根际对土壤呼吸作用强度、过氧化氢酶活性的影响表现出较大差异。
从黑果枸杞、多枝柽柳中分离获得58株内生固氮菌株,其固氮酶活性在0.26～388.58 nmolh-1mL-1之间,表明从荒漠灌木植物中可分离到具有较高固氮酶活性的菌株。通过测定溶磷强度最终筛选出25株溶磷能力较强的菌株,溶磷强度为5.457～24.212ug/mL,25株分泌植物生长素能力较高的菌株,其IAA产生量为0.114～1.392ug/mL。13株同时具有固氮、溶磷以及分泌生长素IAA的多种促生性能。其中EB20-THQ菌株促生性能较为稳定、突出。
应用电镜免疫胶体金染色技术定位人工回接内生固氮菌EB20-THQ菌株在荒漠灌木植物体内的定殖和传导,定位观察表明EB20-THQ菌株能在宿主植物根部定殖。根据16SrDNA序列测定结果及系统进行分析,将5株具有较高促生性能的固氮菌株初步归属为克雷伯氏菌属(Klebsiella)。
Endophytic nitrogen-fixing bacteria was an efficient nitrogen-fixing system, it was different from classical symbiotic nitrogen fixation and joint nitrogen fixation which were newly discovered in recent years. Endophytic nitrogen-fixing bacteria was living in non-leguminous plants, avoiding the inhibition of nitrogen compound and the competition of indigenous microbion, showing a higher efficiency of nitrogen fixation. At the same time, it has a wide range of biological and ecological role of IAA-Producing activity, solubilizing phosphate activity and enhanceing plant disease resistance, and has important application value in improving host nutrition and competitiveness, enhanceing the host of biotic and abiotic stress resistance and promoting the ability to adapt their environment. In this study, the microbial population, community composition and biological activity in rhizospheric soil of several kinds of typical dominant desert plants at the lower reaches of Tarim River, Xinjiang were studied, revealing the interaction between plants and rhizosphere microorganisms, and the affection of plant genotype to the rhizosphere effect. On this basis, isolated the endophytic bacteria from Tamarix ramosissima and Lycium ruthenicum, selected the varieties of functions Plant Growth-Promoting endophytic bacteria strains, These strains had the quality of high nitrogenase activity, solubilizing phosphate activity and IAA-Producing activity, in order to lay the foundation for creating and appling the multifunctional inoculant with the conservation and restoration of desert vegetation. The main results showed:
The rhizospher of the desert plants were quite different from non-rhizosphere in microbial population and composition, and had various rhizosphere effects, shrubs was superior to the herbs in rhizopheric effects. Several desert plants at the lower reaches of Tarim River exerted more obvious positive rhizosphere effects , R/S values reached from1.26 to 150.42, such as Lycium ruthenicum、Tamarix hispida、Karelinia caspica and Halostashys caspica, Lycium ruthenicum was the best one, its R/S values reached to 150.42. On contrary , Alhagi sparsifolia, Apocynum venetum and Hexinia polydichotoma showed negative rhizosphere effects. The physiological groups of ammonifiers and cellulosed ecomposing bacteria also exhibited remakable positive rhizosphere effect, however the nitrobacteria could not be examined.The desert plants growing could improve activity of urease and alkaline phosphatase in the rhizospheric soil. However, the effects of different desert plants on soil respiration intensity and the activity of the hydrogen peroxide showed different.
Fifty-eight of endophytic nitrogen-fixing bacteria strains were obtained in Lycium ruthenicum and Tamarix ramosissima, and the nitrogenase activity is from 0.26 to 388.58 nmolh-1mL-1. It is proved that endophytic bacterial strains with high nitrogenase activity can be isolated from desert shrubs. Tested the all endophytic bacterial strains isolated indicates that 25 strains have solubilizing phosphate activity by measuring activity of phosphate solubilization, its magnitude is from 5.457 to 24.212μgmL-1, 25 strains have the higher capacity to secrete auxine IAA and the productivity quantum of IAA is from 0.114 to 1.392μgmL-1, and 13 strains have multi-promoting function including azotification, solubilizing phosphate and the capacity to secrete auxine IAA. And the promoted function of the EB20-THQ strain among is stability and predominance.
Using the immune electronic microscope dyeing to localization the field planting and transimission of the endophytic diazotrophic bacteria EB20-THQ in the desert shrubs and indicates that the EB20-THQ strain can invade into the host of root. Determinating the whole 16SrDNA sequence and analysising the phylogenesis, the 5 endophytic diazotrophic bacteria strains of high growth-promoting belong to Klebsiella.
引文
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