【作者】 成艳红；

【导师】 李辉信；

【作者基本信息】 南京农业大学 ， 生态学， 2010， 博士

【摘要】 食细菌线虫作为土壤中重要的食细菌微型动物,与微生物相互作用对植物生长的影响已有大量研究报导。但关于食细菌线虫对植物根系生长影响的机理尚不明确。有些研究认为是其取食微生物所产生的直接或间接的养分作用(主要是氮素矿化)为植物生长提供更多有效氮,进而促进植物的生长。最近有研究表明：原生动物促进植物生长存在激素效应,而食细菌线虫在生理及生态上与原生动物很相似,因此有研究者推测食细菌线虫对植物生长的促进作用机理也可能存在激素效应。本文从土壤微生物及植物两条途径研究食细菌线虫对植物根系生长影响的机理。首先采用简化的悉生培养系统,研究食细菌线虫与不同产IAA细菌的相互作用及其对土壤IAA含量及氮素矿化的影响；并在同样模式下研究不同食细菌线虫与不同细菌的相互作用对水稻根系生长的影响,并采用实时荧光定量PCR(Quantitative Real-time qRT-PCR)检测激素IAA早期响应基因GH3-2在水稻根系内的表达,旨在揭示土壤食细菌线虫与细菌的相互作用对植物生长的激素作用机理。另一方面,在自然土培条件下,研究供试土壤土著食细菌线虫对水稻根系生长的影响,及根系内部响应基因对土壤IAA含量变化的响应,同时采用BIOLOG和PCR-DGGE两种方法研究了食细菌线虫对根际土壤微生物群落结构的影响,旨在进一步阐明土壤食细菌线虫与细菌的相互作用及其对植物生长的促进作用机理。所获主要研究结果如下：1.悉生培养(接种单一线虫和单一细菌)条件下,食细菌线虫(Cephalobus sp.)对不同产IAA细菌Pseudomonas sp.(P)和Burkholderia sp.(B)数量、活性及分泌IAA能力的影响在整个培养期,食细菌线虫的取食对土壤中细菌的数量虽无增殖作用,却促进了细菌的活性及分泌IAA的能力,且这种影响受采样时间和接种细菌种类的影响。接种线虫在培养第8天时显著促进了细菌P的活性,在20天时显著促进了细菌B的活性；接种线虫在培养14天显著增加了接种细菌P处理土壤IAA的含量,在20天时显著增加了接种细菌B处理土壤中IAA的含量。然而,不同的细菌种类也影响土壤中线虫的数量,除培养4天外,Cephalobus sp线虫的数量在接种P细菌处理中均高于接种B细菌处理,但两处理培养结束时线虫的数量都是起始数量的2倍以上。2.悉生培养条件下,不同食细菌线虫(Cephalobus sp.和C. elegans)与产IAA细菌(Pseudomonas sp和Burkholderia sp.)(?)司相互作用对水稻氮含量及根系生长的影响接种食细菌线虫[Cephalobus sp. (N1)和C. elegans(N2)]对产IAA细菌的数量均无明显促进作用,但是对产IAA细菌的活性都有显著影响,且因线虫种类、细菌种类和采样时间的不同而不同。接种线虫N1、N2对细菌P活性的促进作用在14天才表现出来；而接种线虫N2对细菌B活性的促进作用表现的更早,这种差异可能是由不同细菌特性的差异导致的。然而,不同产IAA细菌对接种线虫数量的影响也因线虫种类、细菌种类的不同有一定差异。接种线虫数量在接种细菌P处理中高于接种细菌B处理,且C. elegans线虫的数量高于Cephalobus sp线虫。食细菌线虫对水稻根系的总长度、根表面积及根尖数都有显著影响,且因接种细菌的种类及采样时间的不同而不同。接种线虫处理的水稻氮含量均高于单独接种细菌处理,且在接种细菌B处理的含量显著高于接种细菌P处理,而整个培养期内接种线虫种类对水稻氮含量无显著影响。3.悉生培养条件下,食细菌线虫对土壤IAA含量及水稻根系内GH3-2表达的影响接种食细菌线虫增加了土壤中IAA的含量,但是不同线虫和细菌的组合效应有所不同。与土壤中IAA含量的变化一致,水稻根系中IAA早期响应基因GH3-2的表达量也与土壤中接种的线虫与否及种类有明显的关系。土壤中IAA的含量与GH3-2基因的表达量之间存在显著的正相关关系(相关系数为0.663,P=0.019)。结合食细菌线虫对水稻根系形态的影响进一步从植物内在根系发育角度证明了食细菌线虫对根系生长的促进作用存在明显的激素作用。4.原位富化条件下,食细菌线虫对水稻根系生长的影响原位富化试验条件下,食细菌线虫使水稻的根系形态(根总长、根表面积、根平均直径和根尖数等)发生明显改变,使植物的根系都发育成了更长、有更多分支的根系系统,且这种影响受采样时间的影响很大。5.原位富化条件下,土壤食细菌线虫对土壤中氮素矿化的影响种植水稻系统中,有大量线虫富集处理(SM1)和有少量线虫富集处理(SM5)土壤中矿质氮(Mineral Nitrogen) 6勺含量均表现出先升高后降低的变化趋势。在培养10天时,SM1处理土壤中矿质氮的含量显著高于SM5处理土壤；随着培养时间的延长,两处理土壤中矿质氮含量均显著降低,且SM1处理MN的含量下降幅度为82.6%,显著高于SM5处理的50.5%。这一结果表明：食细菌线虫促进了土壤中氮的矿化,提高了其可利用性,而植物生长的吸收和固定氮素量的增加降低了土壤中矿质氮的含量,同时水稻对NH4+-N的偏好性使得土壤中NH4+-N和N03--N出现差异。6.食细菌线虫对根际土壤(种植水稻)微生物(细菌)群落结构的影响(Biolog和PCR-DGGE方法)Biolog结果表明：食细菌线虫影响了土壤微生物群落的总体活性(AWCD值表示),并显著地改变了土壤微生物群落结构(微生物对底物碳源的利用表征),但是这种影响随采样时间的不同而有所变化。在种植水稻20天后,食细菌线虫的存在增加了微生物碳源利用丰富度。种植水稻的土壤微生物群落结构DGGE分析结果也表明：培养20天时,SMl处理土壤微生物多样性指数和丰富度指数都显著高于SM5处理,这与Biolog方法得出的结果相同。培养30天时,两种方法得出的两个处理在多样性指数和丰富度指数的不一致性可能是因为BIOLOG和DGGE对微生物群落结构组成表征的方法不同,但是却从不同角度说明食细菌线虫改变了根际土壤微生物的群落结构组成。7.原位富化条件下,食细菌线虫对土壤IAA含量和植物体内GH3-2基因表达的影响与SM5处理相比,种植水稻的SM1处理土壤中IAA的含量及GH3-2基因在水稻根系内的表达量均显著提高。相关性分析得出二者的相关系数达0.865(p=0.0261),说明外源IAA含量的增加上调了GH3-2基因在水稻根系内的表达。这一结果从自然的土培试验,利用线虫原位富化的培养体系,进一步证实了悉生培养体系中食细菌线虫促进植物根系生长所存在的养分和激素作用。本研究从土壤微生物群落结构及植物内部相关基因的调控探讨了食细菌线虫对植物根系生长影响的养分效应和激素效应机理。本研究可为今后有效开发利用土壤有益线虫资源、改善土壤养分管理、提高作物生产提供理论依据和潜在的技术储备,有重要的理论意义和广阔的应用前景。更多还原

【Abstract】 Bacterial-feeding nematodes, one of the primary grazers of soil bacteria, have been shown to affect root growth of different plant species. Most researches realized that the effects of bacterial-feeding nematodes on plant root growth resulted from direct or indirect nutrient effects (mainly nitrogen mineralization). Bacterial-feeding nematodes grazing on bacteria accelerate bacterial turnover and increase the turnover of soil organic matter, increased the nitrogen mineralization and improved the supply of inorganic nitrogen, and subsequently the plant growth was stimulated. Several researchers, however, have claimed that this process may not fully explain the mechanism of bacterial-feeding nematode stimulating plant growth. Considering the similarly of the physiology and zoology between bacterial-feeding nematodes and protozoa, and that the bacterial-feeding nematodes occur at equal or greater biomass in the rhizosphere than protozoa, it is believed that the activity of bacterial-feeding nematodes in the rhizosphere will also stimulate root proliferation by hormonal effects as seen for protozoan grazing.Two ways, which are soil microbilal and plant, were constructed to study the mechanisms of the effect of bacterial-feeding nematode on plant root growth. First, a gnotobiotic microcosm experiments were conducted to study the interactions between different bacterial-feeding nematodes and different IAA-producing bacteria and their effects on soil IAA content and nitrogen mineralization; Then, with the same system to study the effects of interactions between different bacterial-feeding nematodes and different IAA-producing bacteria on rice root growth and rice root intrinsic gene responding to alteration of IAA content in soil, which is then illustrate the mechanisms of the effect of bacterial-feeding nematodes on plant root growth. Then, experiments with natural soil (soil didn’t sterile but directly incubated for enriched in soil native nematodes) were manipulated to determine the effects of bacterial-feeding nematodes on the root development of rice seedlings. Nematode grazing changing the microbial community was also discussed by physiology method Biolog and molecule method PCR-DGGE; Moreover, in order to illustrate the auxin effect of interactions between bacterial-feeding nematodes and bacteria on plant growth, the relative expression of the early auxin response genes GH3-2 in rice root was also measured by quantitative Real-time PCR. The main results were as follows:1. The effect of bacterial-feeding nematodes (Cephalobus sp.) on the number、activity and IAA production of IAA-producing bacteria (P and B) in gnotobiotic microcosm experimentThe results showed that:Grazing on bacteria did not stimulate the growth of the bacteria; however, Grazing on bacteria stimulated bacteria activity and the ability of producing IAA significantly. They were influenced by bacteria species and sampling time; the presence of nematodes increased the activity of P bacteria after 8 days, while after 20 days for B bacteria; the nematodes increased soil IAA cntent after 14 days for P bacteria and 20 days after incubation for B bacteria. Meanwhile, the number of nematodes was influenced by bacteria species. The number of Cephalobus sp. in P bacteria was higher in B bacteria, but the number of the nematode was 2 times more than initial for both treatments.2. The effect of different bacterial-feeding nematodes (Cephalobus sp. and C. elegans) on IAA-producing bacteria (P and B) and the consequence of these interactions on rice plant growth in gnotobiotic microcosm experimentThe results showed that the presence of nematodes affected the activity of IAA-producing acteria significantly, and the effects were varied with nematodes species, bacteria species and sampling time. C. elegans (N2) increased bacterial activity and soil IAA content much earlier than Cephalobus sp. (N1), which may be explained by different characteristic of nematode from different family. The effect of bacteria on the number of nematodes was different between different treatments. The number of nematodes in P treatment was higher than that in B treatment, and C. elegans was more than Cephalobus sp.. The presence of nematodes influences the root growth of rice including the total length, surface area and average diameter; the effects were varied with bacteria species and sampling time. The higher nitrogen uptake by the plant in the presence of nematodes than absence of nematodes, and the nitrogen content was higher in B treatment than that in P treatment. However, there was no significantly effect of nematode species on nitrogen uptake by the plant during the whole incubation.3. The effect of bacterial-feeding nematodes on soil IAA content and GH3-2 expression in rice root in gnotobiotic microcosm experiment The presence of nematodes increased soil IAA content, but the effect varied with different nematode and bacteria. Consistent with soil IAA content, the nematodes increased the GH3-2 expression in rice root. The correlations analysis showed that IAA correlate with the GH3-2 expression in rice root (r=0.663, P=0.019). Combined these results with the effects of bacterial-feeding nematodes on plant root proliferation and to some extent illustrated that bacterial-feeding nematodes affect plant root growth likely through the hormonal effects by intrinsic genetic program in plant root.4. The effect of bacterial-feeding nematodes on rice root growthRice seedlings grown in the soils containing more bacterial-feeding nematodes developed a highly branched root system with longer roots; and the effects varied with sampling days. That may due to the difference growth period and sensitivity ability to variable environment of different species plant.5. The effect of bacterial-feeding nematodes on soil nitrogen mineralizationIn the system with rice, soil minerial nitrogen contents for both treatments with more bacterial-dfeeding nematodes and few bacterial-dfeeding nematodes were all increased during 0～10th day, and then decreased during 10～30th day. Soil minerial nitrogen contents in SM1 treatment were significant higher than SM5 treatment at day 10, with the increasing days of incubation, soil minerial nitrogen contents in SM1 treatment decreased (82.6%) more than that in SM5(50.5%). The results indicated that the nematodes increased soil nitrogen mineralization, increased the inorganic N available for uptake by the plant, thus decreased the content in soil. The results also showed that rice may be fond of different form of nitrogen, which induced the difference between NH4+-N and NO3--N contents in soil.6. The effect of bacterial-feeding nematodes on rhizosphere microbial community structure (Biolog and PCR-DGGE)The results showed that:Bacterial-feeding nematodes affect microbial activity (indicated by AWCD) and significantly changed the soil microbial community structure that the soil microbe’s functions shifted evidently based on the sole carbon use pattern derived form Biolog method, and the effects varied with sampling time. The presence of nematodes significantly increased bacterial diversity in rice system. The results of DGGE showed that the richness index and diversity index, evenness index of soil microbe was increased during 10-20th day, decreased during 20th-30th day, and the diversity of soil microbe in SM1 were significantly higher than that in SM5 at day 20. The UPGMA analysis showed that the microbial community is similar between SM1 and SM5 at day 20 and 30, while different from day10. It is similar with the results got from Biolog. DGGE fingerprint showed that the nematode affect soil bacterial community structure via appearances or disappearance bands or alteration band intensity.7. The effect of bacterial-feeding nematodes on soil IAA content and GH3-2 expression in rice rootCompared to the control soils (SM5), the soils containing more bacterial-feeding nematodes (SM1) significantly enhanced contents of IAA in soil planted with rice. The correlations analysis between IAA content in soil and GH3-2 expression measured by PT-PCR in rice root showed that IAA significantly correlated with the GH3 expression (P=0.0261, r=0.865), indicating that the GH3-2 was up regulated expression in rice root by increased exogenesis IAA. Combined these results with the effects of bacterial-feeding nematodes on plant root proliferation and to some extent illustrated that bacterial-feeding nematodes affect plant root growth likely through the hormonal effects by intrinsic genetic program in plant root.To summarize, the research discussed two main mechanisms of the effect of bacterial-feeding nematodes on plant growth by two ways, one is rhizosphere soil bacterial community structure, and the other is isintrinsic genetic program in plant root system. These findings supported theoretic knowledge and technical to host soil nematodes as an important bio-resource in soil nutrient management and plant growth. The present study may be valid not only for theory, but also for future application in agriculture.更多还原