2. 浙江大学生物技术研究所，杭州 310058
2. Institute of Biotechnology, Zhejiang University, Hangzhou 310058
实现非豆科主粮作物自主利用生物固氮，满足作物生长发育的氮素营养需求，是农业生物技术的重要目标。实现这一目标主要有3条途径：改造非豆科作物和根瘤菌而形成有效根瘤共生固氮；将原核生物的固氮酶系统导入植物细胞的叶绿体或线粒体，使非豆科作物自主固氮；利用或优化自然界存在的高效非豆科植物联合固氮系统[1]。联合固氮（Associative nitrogen fixation）是指自生固氮菌定殖于植物体表或体内，从植物体表分泌物和植物体内获得能源，在氧浓度适合的微环境中固氮，为植物提供氮素，与植物互惠共生，但没有形成像根瘤这样特异分化的共生固氮器官。本文描述的联合固氮包括了Johanna Döbereiner定义的联合固氮和内生固氮（Endophytic nitrogen fixation）[2]。相应地，联合固氮菌包括定殖于植物体表的附生固氮菌（Epiphytic diazotrophs）和植物体内的内生固氮菌（Endophytic diazotrophs）。固氮菌与非豆科植物联合固氮为植物提供氮素的水平一般较低。那些植株高大、生物量大，能在贫瘠土壤中常年茂盛生长的禾本科C4植物，如象草、芒草、柳枝稷（Panicum virgatum）和Kallar草（Leptochloa fusca），甚至作物（如甘蔗、玉米和高粱），可能通过高效的生物固氮系统获得生长所需氮素，因而较早受到研究者的关注[3-4]。
甘蔗是重要的糖料作物和能源作物，生长周期长，需氮量高。在巴西，很多地区种植甘蔗通常每年施氮60-70 kg/hm2，而甘蔗积累氮100 kg以上，却能保持连年高产且土壤肥力不减[5-6]。从20世纪50年代开始，研究者从巴西甘蔗根际土壤和体内分离到多种固氮细菌，通过乙炔还原乙烯法[7]、15N2-掺入法[8-9]、氮平衡法[6, 10-11]、15N自然丰度法[12-13]和15N稀释法[6, 11]证实了巴西某些甘蔗品种中存在高水平的生物固氮，从生物固氮获得的氮素最高可达甘蔗积累氮素的70%[11-12]。
中国是世界第三大产糖国，糖产量的90%来源于甘蔗。中国蔗田一般施氮量是每年400-800 kg/hm2，不仅远高于巴西蔗田的施氮量，也高于印度、澳大利亚和美国蔗田的施氮量（150-200 kg/hm2）。过度施肥不仅增加生产成本，而且导致更多氮肥流失，生态环境恶化，已成为中国甘蔗产业可持续发展的重要限制因素[14]。
对巴西甘蔗联合固氮菌的研究曾引领了非豆科植物联合固氮和植物内生细菌的研究。Baldani等[15-16]回顾了前期关于巴西甘蔗联合固氮的研究。而本文基于巴西甘蔗联合固氮研究的经验和近年来甘蔗微生物组学研究进展，探讨利用和优化甘蔗联合固氮的策略，以发挥生物固氮在中国甘蔗产业可持续发展中的作用。
1 奇特的Gluconacetobacter diazotrophicusAcetobacter diazotrophicus（后分类为Gluconacet-obacter diazotrophicus）被Döbereiner[2]称为“最奇特的固氮菌”（Most extraordinary diazotroph）。G. diazotrophicus在含10%（W/V）蔗糖、0.5%（V/V）甘蔗汁、pH 5.5（接近甘蔗茎中蔗糖的浓度和pH）的无氮培养基[17-18]中生长良好，在含30%蔗糖的培养基中还能生长[17]。在培养基中含10%蔗糖时，G. diazotrophicus的固氮酶活性对氧、铵、氨基酸及盐胁迫有较强的耐受能力[19]。G. diazotrophicus不能直接转运和同化蔗糖，通过分泌果聚糖蔗糖酶（Levansucrase，一种果糖基转移酶Fructosyltransfer-ase）水解蔗糖产生葡萄糖和果糖；而且，果聚糖蔗糖酶能催化合成果聚糖，形成胞外多糖限制氧扩散，保护固氮酶在较高氧浓度下固氮[20-21]。G. diazotrophicus能通过增强呼吸来降低固氮酶周围的氧浓度[22]，通过FeSII蛋白与固氮酶形成复合体改变固氮酶构象，使暂时失活的固氮酶不受胞内高浓度氧的伤害[23]。在10%蔗糖下，G. diazotrophicus的铵同化率降低2/3，能在10 mmol/L铵下维持固氮酶活性[19]，而25 mmol/L铵不仅抑制G. diazotrophicus的固氮酶活性，而且诱导部分细胞变形为长细胞，转变为不可培养状态[24]。G. diazotrophicus没有硝酸还原酶，不能还原硝酸盐，固氮酶活性不受硝酸盐抑制。G. diazotrophicus在含葡萄糖和蔗糖的培养基中产酸，在pH 2.5还能生长和固氮[17, 25]。G. diazotrophicus与裂解淀粉酵母在含10%蔗糖或2%淀粉的无氮培养基中共培养时，固氮酶活性是单独培养时的2倍，而且将固定氮素的一半转移到了酵母中[26]。这些特性显示G. diazotrophicus适应甘蔗体内环境，能在高糖有氮有氧的条件下固氮，并可能将固定的氮素转给甘蔗细胞。
G. diazotrophicus的数量在不同品种甘蔗中有量级上的差异，在同一品种甘蔗的不同生长期也可发生量级上的变化[24, 27-29]。土壤中高水平的化合态氮抑制G. diazotrophicus在甘蔗中定殖[29-31]。在低氮土壤，甘蔗根、茎和叶中G. diazotrophicus的数量最高分别可达每克鲜重107、106和107个[5, 24, 27-28]，在菌量上有可能实现高水平的联合固氮。
用G. diazotrophicus野生型菌株和nifD-突变株接种适应低氮甘蔗品种SP70-1143的无菌组培苗，野生型和突变株能等量定殖于甘蔗苗和成熟甘蔗，15N2掺入法检测到接种野生型的甘蔗苗中掺入15N的量显著高于接种nifD-突变株和不接种的甘蔗苗，而后两者间没有显著差异，表明G. diazotrophicus在甘蔗苗体内固氮。在缺氮条件下，接种野生型甘蔗苗的长势（高度、分蘖数和叶数）强于接种nifD-突变株和不接种的甘蔗苗，而且苗中氮总量显著高于接种nifD-突变株和不接种的甘蔗苗，G. diazotrophicus可能为甘蔗苗生长提供了氮素。在不缺氮条件下，接种野生型和nifD-突变株都能显著促进甘蔗苗生长和积累氮素，其间没有显著差异，氮肥可能抑制野生型菌株与甘蔗联合固氮，nifD-突变株可能通过固氮外的其他因子促进甘蔗生长[32]。用G. diazotrophicus PAL5接种SP70-1143甘蔗组培苗后盆栽12个月，用15N稀释法测得G. diazotrophicus的固氮率（Ndfa%，nitrogen derived from air）是9.2%[27]；接种另一个适应低氮甘蔗品种RB 867515，从甘蔗根和叶鞘中提取RNA，用RT-PCR扩增nifH mRNA并测序cDNA发现G. diazotrophicus能在叶鞘中表达nifH[33]。这些证据表明G. diazotrophicus能与巴西适应低氮的甘蔗品种联合固氮，但测得联合固氮的水平比相应甘蔗品种在田间所显示的生物固氮水平低得多，而且G. diazotrophicus对甘蔗生长的促进作用不仅是固氮的贡献。G. diazotrophicus PAL5能合成吲哚乙酸[34]和赤霉素[35]，可能由此促进甘蔗根的伸长和植株的发育，扩展的甘蔗根系能更有效地吸收水分和氮素等营养。
Döbereiner和同事最初仅从甘蔗和含糖量高且行营养繁殖的象草和甘薯体内分离到G. diazotrophicus，没有从甘蔗行间土壤和田间多种杂草的根中分离到，但从侵染甘薯的VA菌根真菌的孢子中[36]和吸食甘蔗汁的甘蔗粉红粉蚧中分离到G. diazotrophicus[37]，因此认为G. diazotrophicus是专性内生菌（Obligate endophyte）[38]，在植物体的传播主要通过植物营养繁殖、VA菌根真菌和粉蚧。后来，从咖啡树[39-40]、穇子（Eleusine coracana）[41]、菠萝[42]、胡萝卜、萝卜、甜菜[40]和水稻[43]等植物体内，从咖啡树、穇子、水稻和甘蔗的根际土壤中也分离到了G. diazotrophicus[24, 39, 41, 43]。而用G. diazotrophicus接种甘蔗组培苗后，G. diazotrophicus在根际的数量甚至高于在根内的数量[29]。提高土壤湿度能延长G. diazotrophicus在土壤中的存活时间[44]。总之，G. diazotrophicus适应甘蔗体内高糖和偏酸环境，主要通过甘蔗营养繁殖而传播，但它的生境并不局限在高糖和行营养繁殖植物的体内。相应地，人们用G. diazotrophicus接种甘蔗以外的作物，如高粱[45]、玉米[46]、菜豆[47]和模式植物拟南芥[48]，以促进植物生长并研究其机理。
Cocking等[49]在含3%蔗糖的MS培养基上培养拟南芥、玉米、水稻、小麦、油菜、番茄和白花三叶草的无菌幼苗，接种约5个细胞的G. diazotrophicus，让G. diazotrophicus进入了这些植物根分生组织的细胞，存在于由膜包裹的囊泡内，进而由根分生组织进入根伸长区细胞和叶细胞内。G. diazotrophicus可能通过细胞壁降解酶作用穿过根分生组织细胞的细胞壁，在蔗糖诱导的植物细胞内吞作用[50]下进入根细胞。G. diazotrophicus如何从根分生组织实现系统侵染目前还不清楚。用nifH启动子融合GUS报告基因（nifH-GUS）标记的G. diazotrophicus接种植物显示G. diazotrophicus在植物根细胞质中表达nifH-GUS，有可能形成不结瘤的细胞内共生固氮系统[51]。
自然界中有固氮细菌与非豆科植物形成了不结瘤的细胞内共生固氮系统，如Gunnera属被子植物与念珠藻（Nostoc）属的固氮蓝细菌形成了独特的细胞内共生固氮系统。Nostoc细菌进入Gunnera植物茎上分泌粘液的腺体，在腺体细胞细胞质中由膜包裹的囊泡中分裂并分化成异形胞。异形胞的由多糖和糖脂形成的双层被膜能限制氧的扩散。异形胞固定的氮素能转给植物，满足Gunnera植物对氮的需求[51-52]。目前已发现多种内生细菌能定殖在植物细胞内，如Thomas等发现在香蕉和木瓜的细胞内有活的内生细菌[52-53]。固氮菌与植物形成高效的共生固氮系统或许需要细菌内生于植物细胞，但结瘤不是共生固氮必须的。Cocking等[49]的操作可能让G. diazotrophicus与植物的联合固氮系统转变为细胞内共生固氮系统，植物细胞质中包裹着固氮菌的囊泡或可演化成固氮细胞器[51]。G. diazotrophicus与拟南芥或水稻形成的内共生系统可作为模式系统用于研究细菌内生于植物细胞的机制和建立高效的细胞内共生固氮系统。
2 固氮菌联盟（Diazotroph consortium）巴西甘蔗高水平的联合固氮可能是多种固氮菌共同实现的。除了G. diazotrophicus，前期从巴西高固氮品种甘蔗中分离培养的固氮菌主要还有Herbaspirillum seropedicae、H. rubrisubalbicans、Burkholderia tropica（后分类为Paraburkholderia tropica）、Azospirillum lipoferum、A. brasilense和A. amazonense（后分类为Nitrospirillum amazonense）等[16]。5个固氮菌株G. diazotrophicus PAL5、H. seropedicae HRC54、H. rubrisubalbicans HCC103、N. amazonense CBAmC和P. tropica Ppe8经混合组成“固氮菌联盟”接种甘蔗。它们都能合成吲哚乙酸。H. seropedicae和H. rubrisubalbicans还有1-氨基环丙烷-1-羧酸（ACC）脱氨酶基因[55]，ACC脱氨酶分解植物产生的乙烯合成前体ACC，降低植物在逆境下产生乙烯的水平，从而缓解逆境下植物生长所受的抑制。G. diazotrophicus在蔗糖中产酸，而Herbaspirillum嗜用有机酸，它们在甘蔗体内共处可能稳定共栖微环境的pH。5个菌株都有合成细菌素（Bacteriocin）的基因，G. diazotrophicus PAL5合成的细菌素gluconacin在体外能不同程度地抑制多种甘蔗病原细菌，也能抑制其他G. diazotrophicus菌株、N. amazonense CBAmC和P. tropica Ppe8[56]，但大体上，用5个菌株共同接种甘蔗产生了协同增效的作用。
用N. amazonense CBAmC和P. tropica Ppe8共同接种SP70-1143甘蔗组培苗后5 d，P. tropica Ppe8的定殖量是N. amazonense CBAmC的近1 000倍，而用5个菌株共同接种，N. amazonense CBAmC的定殖量比与P. tropica Ppe8共同接种时提高了100倍[28]。用5个菌株共同接种后7 d，细菌总数大于每克鲜重107个，对甘蔗苗的生长产生负荷，让甘蔗苗在45 d驯化期的存活率降低；接种后200 d，5个菌株在甘蔗根和地上部的数量都接近每克鲜重106个，在接种后400 d的甘蔗根中的数量也接近每克鲜重106个。接种后12个月，5个菌株的联合固氮率（29.2%）比单独接种G. diazotrophicus PAL5（9.2%）、共同接种H. seropedicae HRC54和H. rubrisubalbicans HCC103（14.6%）、共同接种N. amazonense CBAmC和P. tropica Ppe8（8.1%）的固氮率高[27]。而且，在不施氮肥的贫瘠土壤中，用“固氮菌联盟”接种新植蔗和宿根蔗，都发生了较高水平的联合固氮并增产[57]。
用“固氮菌联盟”接种RB 867515和RB 72454甘蔗的三芽种茎，在不施氮、施磷钾和钼的土壤中生长，能显著促进甘蔗增产，增产幅度接近施120 kg/hm2 N的作用，但无论接种与否，甘蔗都表现出高水平的生物固氮，固氮水平不受接种影响[58]。另一研究用“固氮菌联盟”接种RB 867515新苗和每季收获后的宿根蔗，发现仅G. diazotrophicus PAL5在接种甘蔗的叶鞘中表达nifH，P. tropica Ppe8在接种甘蔗的根中表达nifH；而在不接种不施氮的甘蔗根和叶鞘中，有P. tropica表达了nifH[33]。可能这5个固氮菌中仅G. diazotrophicus和P. tropica与甘蔗联合固氮，或5个固氮菌在不同时间和甘蔗体内不同空间联合固氮，而不接种的甘蔗中同样有P. tropica或其他固氮菌在不同时空发生联合固氮。
用“固氮菌联盟”接种RB 867515和适应中高氮甘蔗品种IACSP95-5000的由种茎催芽获得的幼苗，能促进幼苗生根、增加细根数、增加根长、根体积和表面积，增加根干重近50%；但在不施氮、施磷钾和微量元素的土壤中，无论接种与否，接种单个菌株或组合菌株，两个品种都表现出高水平的生物固氮，不受接种固氮菌影响[59]。
总之，“固氮菌联盟”能协同促进甘蔗的生长，通过产生植物生长调节物质，促进根的伸长和发育，提高根吸收水分和氮素等营养，进而促进甘蔗的生长发育和抗逆；在低氮土壤中，“固氮菌联盟”与某些甘蔗品种联合固氮，实现减氮不减产。
3 联合固氮根瘤菌从发现甘蔗内生固氮菌并组合“固氮菌联盟”进行田间试验的近20年里，受技术限制，人们并不清楚哪些固氮菌是巴西甘蔗高水平生物固氮的主要贡献者。2000年以来应用RT-PCR等技术的研究揭示了固氮菌在不同生境下nifH的表达水平与固氮水平相符[60-61]。Fischer等[33]发现“固氮菌联盟”接种RB 867515甘蔗后，仅G. diazotrophicus和P. tropica在甘蔗体内表达nifH；发现Bradyrhizobium在接种和不接种甘蔗的根中、Rhizobium在接种和不接种甘蔗的叶鞘中、Ideonella/Herbaspirillum近缘固氮菌在接种和不接种甘蔗的根和叶鞘中，Methylocapsa在不接种甘蔗根中表达nifH；而且检测到非接种固氮菌的nifH cDNA克隆数量比接种菌株的高。这些非接种固氮菌有可能是巴西甘蔗高水平生物固氮的主要贡献者。Burbano等[62]发现在巴西、墨西哥和纳米比亚田间甘蔗根部活跃表达nifH的是Rhizobium rosettiformans的近缘菌，在巴西和墨西哥甘蔗茎中也发现有Bradyrhizobium属的细菌表达nifH，但没有发现“固氮菌联盟”中的菌种表达nifH。在日本Miyako岛种植的甘蔗品种NiF8有显著的生物固氮[12, 63-64]。Thaweenut等[64]发现NiF8甘蔗仅在高温季节有显著的生物固氮，用RT-PCR检测到甘蔗茎和根中有近似Bradyrhizobium属、Azorhizobium caulinodans、Sinorhizobium fredii、Derxia gummosa和Nostoc commune等细菌表达nifH。这些研究揭示在甘蔗体内活跃固氮的主力可能是根瘤菌。
在澳大利亚昆士兰种植的甘蔗（澳大利亚品种Q186和Q208、巴西品种SP 79-2313）[65]和在云南开远种植的甘蔗野生种Saccharum officinarum、S. spontaneum、S. barberi和栽培品种ROC22[66]的根内，丰度最高的固氮菌属是Bradyrhizobium属，Rhizobium属固氮菌的丰度也较高。
Rouws等[67]用豇豆结瘤诱捕或YMA培养基直接分离，从RB 867515甘蔗的根和茎基部获得遗传多样的Bradyrhizobium属菌株，其中2个能结瘤和2个不结瘤的Bradyrhizobium菌株能在自由培养下表现固氮酶活性，有可能在甘蔗体内以不结瘤的状态固氮。
另有研究发现Bradyrhizobium可能与水稻、甘薯和高粱联合固氮。非洲野生稻与合萌属豆科植物（Aeschynomene sensitiva）一起生长，非洲野生稻中有能与合萌结瘤的光合Bradyrhizobium细菌，用野生稻的内生Bradyrhizobium菌株和从合萌分离的Bradyrhizobium菌株ORS278接种水稻后能测到固氮酶活性并促进水稻生长和结实[68]。与豆科作物轮作的水稻中有能分别与不同豆科植物结瘤的光合和非光合Bradyrhizobium细菌[69]，非光合Bradyrhizobium菌株SUTN9-2在缺氮下与水稻联合能表达nifH[70]。在与花生间作的水稻中发现了没有nodABC基因的Bradyrhizobium属细菌[71]。甘薯适合在贫瘠土壤中生长并形成块根。在日本种植的甘薯品种Beniazuma可能从生物固氮获得约44%的氮素[72]，Beniazuma甘薯的茎和块根中丰度最高且表达nifH的固氮菌属于Bradyrhizobium属[73]，从表面灭菌的甘薯块根中分离到的Bradyrhizobium菌株AT1[74]有nif基因簇但没有nodABC基因[75]，能在自由培养下表现固氮酶活性[74]，用AT1接种无菌的甘薯幼苗增加了甘薯苗的鲜重和氮含量，甘薯叶和茎中的固氮率为10%-14%[74]。在日本福岛田间生长的高粱品系KM1和KM2的根在生长后期表现出较高的固氮活性，KM1和KM2根中有较高丰度的与不结瘤Bradyrhizobium菌株S23321和光合B. oligotrophicum菌株S58T近缘的固氮菌；从高粱根中分离到的Bradyrhizobium中，菌株TM122和TM124没有nod基因，能在自由培养下表现固氮酶活性；TM122还能在接种高粱种子后，在根中表现固氮酶活性，有可能以不结瘤方式与高粱联合固氮[76]。
Bradyrhizobium属细菌在属分类水平上是全球土壤中最普遍的细菌[77-78]，也是陆生植物根部相对丰度最高的细菌[79]，是随陆生植物共同进化的核心细菌群的重要成员[79]。Bradyrhizobium属细菌有较大的基因组，进化中基因的获得和丢失让Bradyrhizobium具有遗传多样性、适应多样的环境，如有固氮或光合的能力、依赖nod基因或不依赖nod基因与豆科植物结瘤形成共生固氮系统，也可能与非豆科植物不结瘤联合固氮并成为非豆科植物的核心固氮菌群。
4 甘蔗微生物组植物微生物组（Microbiome）是与植物共栖的所有微生物及其遗传信息的集合。植物微生物组中相对数量较高、能稳定持久地与健康植物共栖的微生物被称为核心微生物菌群（Core microbiota），在维持植物健康生长和生态功能上发挥必要作用[80]。核心微生物菌群的分类结构（Taxonomic structure）有一定保守性，但可塑性高。核心微生物菌群的功能结构（Functional structure）比分类结构更稳定和持久，功能结构可以不依赖于分类结构[80-81]。
基于高通量测序16S rRNA基因和nifH基因的不依赖培养的微生物多样性分析显示在巴西、澳大利亚和中国种植的不同甘蔗种和品种的联合细菌及固氮菌的多样性有显著差异，但共有某些核心菌群[65-66, 82-83]。如在澳大利亚的昆士兰[65]和我国云南[66]种植的不同甘蔗栽培品种和野生种的共有核心菌群中有Chitinophagaceae等8个科的细菌，核心固氮菌群中都有Bradyrhizobium、Rhizobium和Burkholderia属的细菌。曾经被重点研究的与巴西甘蔗联合的Azospirillum，Herbaspirillum和Gluconacetobacter属固氮菌仅是巴西种植甘蔗品种SP80-3280核心菌群的次要成员[82]，在昆士兰[65]和云南[66]种植甘蔗的根中甚至检测不到Gluconacetobacter属固氮菌。而在巴西甘蔗中丰度最高的多个核心微生物种类，如Chitinophagaceae科的细菌，与甘蔗的联合还从来没有被研究过[82]。
澳大利亚田间的甘蔗没有显著的生物固氮[84]，甘蔗根的核心细菌菌群和固氮菌群的分类结构和相对丰度在低氮（40 kg/hm2）和常量氮（160-180 kg/hm2）下没有显著差异[65]；增加氮肥对土壤的碳循环有负面影响，提高了病原真菌在土壤和根际的丰度[85]；而甘蔗根的核心细菌菌群可能通过维持分类结构而维持必要功能。
Dong等[66]发现在施氮350 kg/hm2下生长的ROC22甘蔗根中Klebsiella属固氮菌的丰度高于Bradyrhizobium属。与此相符，Klebsiella属固氮菌是我们从广西田间ROC22甘蔗中分离到的优势固氮菌[86]。通过减少氮肥，用大豆和花生与甘蔗间作，能显著提高甘蔗根际土壤固氮菌的多样性[87]，提高根际核心固氮菌群中Bradyrhizobium属的相对丰度[88]；多因子诱导甘蔗联合固氮菌群分类结构的改变可能增强甘蔗联合固氮。
5 展望非豆科作物实现联合固氮取决于植物和固氮菌双方的基因型和环境因素。植物基因型决定植物为固氮菌提供的碳源和其他营养、植物对固氮菌定殖入侵的响应和植物吸收氮素的效率。固氮菌基因型决定固氮菌联合固氮、促进植物生长、定殖于植物和与其他微生物协作的能力。环境因素有植物和固氮菌共栖地土壤的碳氮比和含水量、气候决定的区域温度和降水量等因素。
中国甘蔗产业的可持续发展可以借鉴巴西经验：减施化肥；筛选适应低氮、氮利用率高且能联合固氮的甘蔗品种，如RB 867515（引进中国后简称B8）[89]；为新植蔗和宿根蔗接种固氮菌或包括固氮菌的合成功能菌群（Synthetic community），让接种菌与甘蔗紧密联合成为核心功能菌群（Functional core microbiota）；在甘蔗苗期施适量氮肥以支持甘蔗苗的生长发育和光合作用，为功能菌群提供碳源，建立稳定联合；在甘蔗整个生长期保持充足灌溉，施适量磷钾肥和钼肥支持甘蔗联合固氮[90]。
构建高效的合成功能菌群可改良已有的甘蔗“固氮菌联盟”和基于微生物组学从头合成甘蔗功能菌群。由5个固氮菌组成的“固氮菌联盟”可视为合成功能菌群的雏形。“固氮菌联盟”有联合甘蔗固氮和提高甘蔗氮素利用率的基本功能，虽可能没有田间甘蔗原生核心菌群的成员，但通过无菌组培苗与细菌共培养[91]可将“固氮菌联盟”导入甘蔗而建立不依赖甘蔗原生核心菌群的合成功能菌群，通过调整菌群成员和接种方式可能提高合成功能菌群的效率。调整“固氮菌联盟”成员可增加甘蔗联合固氮的原生核心菌群，如Bradyrhizobium属固氮菌。调整接种方式如接种很少量的G. diazotrophicus与甘蔗组培苗共培养，诱导G. diazotrophicus进入甘蔗细胞而内共生固氮[49]。
在中国田间甘蔗中可能没有G. diazotrophicus[66]，人工接种可扩展G. diazotrophicus的宿主。我们将G. diazotrophicus导入ROC22甘蔗的组培苗，使甘蔗苗从氮气获得6.8%的氮素[92]，与ROC22甘蔗的土著固氮菌Klebsiella variicola的联合固氮率相当[93]。Dent[94]报告用G. diazotrophicus制成的菌剂产品NFix®处理玉米和小麦种子，在田间小区让不施氮肥和施不同水平氮肥下种植的玉米总体增产8%（830 kg/hm2），让小麦增产7%（460 kg/hm2）；NFix®的作用相当于给玉米减施27%的氮肥而不减产，给小麦减施61%的氮肥而不减产。NFix®对作物的作用是固氮和其他促植物生长因子的协同作用。Cocking等[49]接种G. diazotrophicus的技术可能让G. diazotrophicus实现细胞内共生固氮，是实现非豆科作物高效固氮的新途径。目前，还没有研究揭示甘蔗、甘薯和水稻体内功能固氮菌Bradyrhizobium的固氮状态，或许Bradyrhizobium也能进入非豆科作物细胞实现内共生固氮。
目前已具备基于微生物组学从头合成甘蔗功能菌群的技术。从田间成熟甘蔗植株尤其是从茎中提取DNA和RNA的难度较高，要优化多个步骤才能建立不依赖培养的甘蔗微生物组学方法[33, 95]。不依赖培养的微生物多样性分析已发现甘蔗各器官的核心微生物菌群[65-66, 82]，发现核心微生物菌群共存网络中关键的枢纽（Hub）微生物[83]。基于菌群培养（Community-based culture collection）的方法[96]结合96孔板收集可培养微生物和高通量测序两步扩增得到的16S rRNA基因序列，已能获得和鉴定甘蔗根际核心菌群中15.9%的细菌和茎秆内核心菌群中61.6%-65.3%的细菌，从中选出了对应20个丰度最高可操作分类单元（OTU）的培养物，合成菌群，接种去胚乳的玉米种子，使接种后4周幼苗的干重是不接种对照幼苗的3倍，证实由16S rRNA基因序列高丰度指导合成的核心菌群包括了重要的植物益生菌，能促进植物生长[97]。RT-PCR扩增nifH mRNA[33, 64]结合高通量测序能确定甘蔗体内核心功能固氮菌群（Functional diazotroph community）的组成；用不同配方的半固体无氮培养基分离富集固氮菌[98]，结合基于菌群培养的方法[96]和定向诱捕的方法[67]可能获得可培养的核心功能固氮菌群成员，基于nifH表达量指导合成功能固氮菌群，通过无菌组培苗与细菌共培养[91]将功能固氮菌群导入甘蔗，用15N稀释等方法检验功能固氮菌群的联合固氮功能。用甘蔗组培苗与细菌共培养建立的实验体系能用于调试优化合成功能菌群和筛选甘蔗品种，最终建立高效甘蔗-微生物共生体，实现甘蔗高效固氮和利用氮素，促进甘蔗产业可持续发展。
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