间作豆科作物对甘蔗联合固氮的影响

Some sugarcane germplasms are able to obtain high levels of nitrogen via N2-fixation by associated N2-fixing bacteria. Recent studies showed that the transcripts of the nifH gene encoding the iron protein of nitrogenase were predominantly expressed by rhizobia in the N2-fixing sugarcane plants in Japan, Brazil, Mexico, and Namibia. Rhizobia likely fix N2 in sugarcane in a non-nodulation state as endophytes based on the correlation between bacterial N2-fixation and the levels of nifH expression in diverse environments. We have obtained rhizobia, which is able to fix N2 in soybean nodules and in association with sugarcane, from roots of sugarcane crops intercropping with soybean crops. We think that legume intercropping may work as inoculation of rhizobia to sugarcane crops and may increase legume symbiotic N2-fixation and sugarcane-associated N2-fixation and thus the overall nitrogen utilization in sugarcane fields under right N-fertilization. We plan to use sugarcane germplasms shown different levels of adaption to low-nitrogen stresses to experiment sugarcane monoculture and intercropping with soybean and peanut crops under a moderately low N-fertilization. We will use 15N natural abundance technique to compare the N2-fixing levels of the monoculture and intercropping sugarcane crops. We will also amplify nifH transcripts and analyze the nifH expression frequencies from the sugarcane crops, and thus evaluate if sugarcane/legume intercropping supports high levels of N2-fixation associated with sugarcane. Moreover, we will analyze the nifH transcriptoms to know the active N2-fixing bacterial communities in the crops, isolate the functional N2-fixing bacteria especially rhizobia, and investigate their N2-fixation associated with monoculture and intercropping sugarcane crops. Together, this study aims to find the right sugarcane cultivars, functional N2-fixing bacteria, and planting ways to achieve a highly efficient N2-fixing system for sugarcane crops.

某些甘蔗种质能进行高水平联合固氮。最新研究发现多地甘蔗体内活跃表达nifH的固氮菌主要是根瘤菌，根瘤菌能以不结瘤的方式作为内生固氮菌与甘蔗联合固氮。我们已从甘蔗/大豆间作的甘蔗根中分离筛选到既能与大豆结瘤共生固氮又能与甘蔗联合固氮的根瘤菌，认为间作豆科作物可能为甘蔗接种联合固氮根瘤菌，通过蔗豆间作和适度施氮可能提高豆科作物共生固氮和甘蔗联合固氮的效率，提高蔗田氮肥利用率。本项目拟在适度低氮条件下，单作和用大豆、花生间作对低氮胁迫适应能力不同的甘蔗品种，用15N自然丰度法比较不同甘蔗种质在单作和间作下的联合固氮水平，用RT-PCR扩增甘蔗体内功能固氮菌的nifH转录组来检测甘蔗联合固氮的频率，了解间作豆科作物能否提高甘蔗联合固氮效率；而且，解析间作下甘蔗体内的主要功能固氮菌，分离筛选高效的联合固氮根瘤菌，为找到切实提高甘蔗联合固氮效率的甘蔗种质、固氮菌和栽培方式提供依据。

甘蔗是世界上最重要的糖料和能源作物。有些甘蔗种质能进行高水平联合固氮，最新研究发现根瘤菌能以不结瘤的方式作为内生固氮菌与甘蔗联合固氮。本项目在适当低氮（150 kg尿素/hm2）条件开展甘蔗单作及与大豆、花生间作试验，在甘蔗分蘖期和伸长期从不同甘蔗品种的根和茎中共分离获得224个菌株，分属4个门6个纲的42个属，以变形菌门和放线菌门的细菌在数量和种类上都占优势，其中从与大豆和花生间作的甘蔗组织中分离到的不同类型的根瘤菌明显比较单作的多。甘蔗根系的优势固氮细菌类群分别为慢生根瘤菌属、克雷博氏菌属、伯克霍尔德菌、草螺菌属的细菌，以及未鉴定的变形杆菌细菌，根瘤菌细菌，变形菌细菌。对分离到的根瘤菌株Rhizobium sp. BIG27在甘蔗根部定殖的特性以及根瘤菌株GL003和放线菌株Streptomyces chartreusis WZS021的固氮和促生特性进行了分析，初步确定了其促生特性及对不同甘蔗品种接种后的固氮效率差异，证明B9是一个固氮效率高的甘蔗品种，放线菌株WZS021接种不仅对甘蔗具有显著的促生作用，而且显著提高甘蔗的耐旱能力。间作花生对甘蔗的增产效果比大豆好。从根际土壤中分离得到255株固氮菌株，均具有固氮酶活性，其中菌株MYSP104的活性最高，MISP58、MYSP90、CTSS241和MISP39次之。通过16S rRNA测序比对，聚类结果显示，经筛选后的21株菌株共归为厚壁菌门、变形菌门（α, β, γ-变形菌）、放线菌门和拟杆菌门四个门6个，其中Bacillus tequilensis、Variovorax paradoxus、Acidovorax facilis、Leucobacter aridicollis、Streptomyces fimicariusand、Pseudomonas nitroreducens在广西首次作为固氮菌被报道。结果表明，间作花生和大豆有利于改善土壤理化性质并增加土壤的微生物总量，而以间作大豆效果较好。本研究理清了肠杆菌目中固氮菌的分布和系统分类；理清了Klebsiella属和Raoultella属细菌的系统分类；理清了Kosakonia属和Phytobacter属细菌的系统分类。研究结果为分离筛选高效的联合固氮根瘤菌、筛选高固氮效率的甘蔗种质以及合理的甘蔗与豆科作物间作方式提供了依据。