慢生根瘤菌及其與花生共生機(jī)制研究進(jìn)展

吳月，隋新華，戴良香，鄭永美，張智猛，田云云，于天一，孫學(xué)武，孫棋棋，馬登超，吳正鋒*

慢生根瘤菌及其與花生共生機(jī)制研究進(jìn)展

吳月1，隋新華2，戴良香1，鄭永美1，張智猛1，田云云1，于天一1，孫學(xué)武1，孫棋棋1，馬登超3，吳正鋒1*

1山東省花生研究所，山東青島 266100；2中國(guó)農(nóng)業(yè)大學(xué)生命科學(xué)學(xué)院，北京 100193；3濟(jì)寧市農(nóng)業(yè)科學(xué)研究院，山東濟(jì)寧 272009

氮是植物生長(zhǎng)發(fā)育所必需的大量元素之一，豆科植物通過(guò)與根瘤菌的共生固氮獲得氮素。這種共生關(guān)系的建立包括結(jié)瘤和固氮兩個(gè)過(guò)程，涉及復(fù)雜的互作調(diào)控機(jī)理，并受環(huán)境因素的顯著影響?；ㄉc慢生根瘤菌的共生對(duì)花生生產(chǎn)尤為重要，具有較多特異和未知的共生機(jī)制。本文綜述了慢生根瘤菌及其與花生共生的相關(guān)內(nèi)容，具體包括：（1）花生的慢生根瘤菌多樣性和基因組功能；（2）花生與慢生根瘤菌的共生機(jī)制，包括慢生根瘤菌的裂隙侵染及與花生的共生信號(hào)交流，花生的結(jié)瘤固氮和根瘤數(shù)調(diào)控機(jī)制；（3）田間環(huán)境因素（土壤氮素、pH、溫度、水分）對(duì)花生結(jié)瘤固氮及產(chǎn)量的影響。本文從慢生根瘤菌、慢生根瘤菌與花生的共生以及在花生田間的應(yīng)用三方面指出目前研究中存在的問(wèn)題主要為：針對(duì)花生的慢生根瘤菌基因組功能研究較少、慢生根瘤菌與花生互作調(diào)節(jié)機(jī)理細(xì)節(jié)未知、慢生根瘤菌菌劑田間應(yīng)用利用率差等?；诖?，未來(lái)研究重點(diǎn)應(yīng)該集中在花生慢生根瘤菌基因組及基因功能分析；慢生根瘤菌與花生的信號(hào)交流、根瘤數(shù)調(diào)節(jié)和營(yíng)養(yǎng)交換機(jī)制；與根瘤固氮規(guī)律相配合的化學(xué)氮肥合理施用技術(shù)、通過(guò)合成生物學(xué)手段獲得適用于花生種植的新型根瘤菌劑等方面。本文為深入了解豆科植物與根瘤菌的共生機(jī)制、提高豆科作物結(jié)瘤固氮效率和產(chǎn)量、減少化學(xué)氮肥施用和改善農(nóng)業(yè)生態(tài)環(huán)境等提供理論基礎(chǔ)。

慢生根瘤菌；花生；共生固氮；結(jié)瘤固氮機(jī)制；多樣性；環(huán)境因素

0 引言

大氣中存在78%的游離氮?dú)?，但植物卻只能吸收利用化合態(tài)氮。在這些化合態(tài)氮中，生物固定的氮占據(jù)主導(dǎo)地位，約為70%[1]。固氮生物主要是原核生物中細(xì)菌和古菌的某些屬種，按照與植物的關(guān)系這些屬種可以分為共生固氮、自生固氮、聯(lián)合固氮和內(nèi)生固氮等幾種類型[2]。根瘤菌與豆科植物的共生固氮是能力最強(qiáng)的共生體系，為植物提供生長(zhǎng)所需60%—65%氮素，在可持續(xù)農(nóng)業(yè)生產(chǎn)和生態(tài)環(huán)境保護(hù)中意義重大[3-4]。固氮根瘤菌屬于原核生物細(xì)菌域（）、變形桿菌門（）、阿拉法-變形桿菌綱（α-）和貝塔-變形桿菌綱（β-）、根瘤菌目（）和伯克霍爾德氏菌目（），至2020年7月已有360多個(gè)種[5]。根瘤菌是一類廣泛分布于土壤中的革蘭氏陰性細(xì)菌，具有可運(yùn)動(dòng)、無(wú)芽孢、好氧等特性[6]。比較常見(jiàn)且占比例最大的根瘤菌主要屬于根瘤菌屬（）、慢生根瘤菌屬（）、中慢生根瘤菌屬（）和中華根瘤菌屬（）等[4]。

花生（L.）是世界上種植最廣的豆科作物之一[7]。能夠與花生建立共生關(guān)系的根瘤菌主要分布在慢生根瘤菌屬，研究證明花生-慢生根瘤菌共生體系的固氮量相當(dāng)于純氮100—152 kg·hm-2，可滿足花生生長(zhǎng)需要氮量的30%—80%，并提高后茬作物產(chǎn)量[8]。因此，慢生根瘤菌在減少化學(xué)氮肥的前提下，能夠解決土壤生態(tài)問(wèn)題，維持土壤環(huán)境可持續(xù)發(fā)展，在花生栽培上有著較高的應(yīng)用潛力和價(jià)值。

1 花生的慢生根瘤菌

1.1 花生的慢生根瘤菌多樣性

近幾年，關(guān)于花生根瘤菌多樣性的研究較多，發(fā)現(xiàn)這些根瘤菌主要屬于慢生根瘤菌屬（）。國(guó)外對(duì)花生慢生根瘤菌多樣性的研究主要集中在阿根廷和非洲等花生種植國(guó)家。阿根廷花生慢生根瘤菌的系統(tǒng)發(fā)育地位與.、相近[9]。摩洛哥花生慢生根瘤菌的系統(tǒng)發(fā)育地位與、、、相近[10]。同時(shí)也發(fā)現(xiàn)了少量新種慢生根瘤菌，例如和等[11-12]。我國(guó)花生根瘤菌也屬于慢生根瘤菌屬，優(yōu)勢(shì)種群為、和。山東省和河北省花生慢生根瘤菌優(yōu)勢(shì)種群為，河南省的優(yōu)勢(shì)種群為[6]；江蘇、廣東和廣西三省花生慢生根瘤菌優(yōu)勢(shì)種群為[13]；四川省花生慢生根瘤菌在系統(tǒng)發(fā)育上與關(guān)系最近[14]。此外，在我國(guó)也發(fā)現(xiàn)了大量慢生根瘤菌新種，例如、、、和等，這說(shuō)明我國(guó)花生的慢生根瘤菌具有較大遺傳多樣性，潛在慢生根瘤菌屬的新種較多[15-18]。

1.2 花生的慢生根瘤菌基因組功能

目前，關(guān)于花生的慢生根瘤菌基因組功能研究較少。李永華[19]根據(jù)結(jié)瘤基因和固氮基因?qū)⒒ㄉ穆鼍譃镮型和II型兩種類型，共生表型檢測(cè)結(jié)果顯示II型慢生根瘤菌接種花生形成的根瘤數(shù)為I型根瘤菌接種處理的1.5—2倍。比較基因組學(xué)分析發(fā)現(xiàn)I型和II型慢生根瘤菌有338個(gè)共生相關(guān)基因與上述分型結(jié)果一致；所有II型慢生根瘤菌具有共生質(zhì)粒；I型和II型慢生根瘤菌在影響表達(dá)的重要區(qū)域上游的UAS位點(diǎn)及啟動(dòng)子區(qū)域差異較大。推測(cè)這些基因組特征可能與兩種類型慢生根瘤菌的根瘤數(shù)差異相關(guān)。此外，筆者通過(guò)遺傳操作方法發(fā)現(xiàn)II型慢生根瘤菌CCBAU 53363T（）多個(gè)染色體基因正調(diào)控花生結(jié)瘤，qRT-PCR分析結(jié)果表明CCBAU 53363T的這些染色體基因通過(guò)延長(zhǎng)花生共生信號(hào)通路中AhSYMRK蛋白（symbiosis receptor- like kinase）表達(dá)的時(shí)間、提高花生耐受根瘤數(shù)自調(diào)控系統(tǒng)AON（autoregulation of nodulation）和乙烯調(diào)控系統(tǒng)的反饋調(diào)節(jié)而誘導(dǎo)花生大量結(jié)瘤[20]。

2 慢生根瘤菌與花生的共生機(jī)制

2.1 慢生根瘤菌對(duì)花生的侵染

植物根部在生長(zhǎng)時(shí)會(huì)向土壤中釋放氨基酸、類黃酮等有機(jī)分泌物，根瘤菌對(duì)這些分泌物做出反應(yīng)并通過(guò)趨化作用運(yùn)動(dòng)到植物根系，在根瘤菌多糖（環(huán)葡聚糖，類似于凝膠和黏附素）與植物凝集素（根瘤菌多糖特異性受體）匹配性識(shí)別之后，植物凝集素幫助根瘤菌定殖在根表及根毛上，為侵染根系做準(zhǔn)備[21-23]。對(duì)于慢生根瘤菌來(lái)說(shuō)，鈣結(jié)合黏附素細(xì)胞表面蛋白幫助慢生根瘤菌吸附在花生根表[24]。此外，菌體的生長(zhǎng)狀態(tài)也對(duì)附著效果存在較大影響，即處于生長(zhǎng)對(duì)數(shù)期末、穩(wěn)定期初的慢生根瘤菌具有最強(qiáng)的花生根表定殖能力[25]。

比較常見(jiàn)的根瘤菌對(duì)豆科植物的侵染方式為根毛侵染，根毛侵染是指根瘤菌經(jīng)由豆科植物根毛進(jìn)入根部并形成根瘤的過(guò)程。這種侵染模式主要發(fā)生在含羞草亞科和蝶形花亞科的植物上，如豌豆（）、大豆（）、截型苜蓿（）和日本百脈根（）等[25]。在侵染過(guò)程中，根瘤菌首先誘導(dǎo)植物根毛發(fā)生卷曲，并通過(guò)釋放植物細(xì)胞壁降解酶侵入到根毛細(xì)胞中[26]；隨后，根瘤菌以侵染線（infection thread，IT）的形式經(jīng)由根毛細(xì)胞和皮層細(xì)胞，進(jìn)入根瘤原基細(xì)胞[27-29]。另一種侵染方式為裂隙侵染，裂隙侵染是指根瘤菌通過(guò)植物根部表皮裂開(kāi)的傷口或側(cè)根與主根之間的裂隙進(jìn)入到植物根部并形成根瘤的過(guò)程。該侵染類型主要屬于亞熱帶豆科植物，包括花生屬（）、田菁屬（）和合萌屬（）等。目前研究較多的是合萌共生體系，在該體系中光合慢生根瘤菌通過(guò)合萌腋生根毛與主根之間的裂隙進(jìn)入根表皮細(xì)胞間隙并大量繁殖形成侵染袋，侵染袋中的光合慢生根瘤菌通過(guò)皮層細(xì)胞間隙進(jìn)入位于皮層的根瘤原基細(xì)胞中[30]。慢生根瘤菌與花生的共生也屬于裂隙侵染類型，但關(guān)于該共生體系的侵染路徑研究較少，僅了解到慢生根瘤菌經(jīng)由花生側(cè)根與主根之間的裂隙進(jìn)入到花生根部細(xì)胞間隙，并通過(guò)細(xì)胞間隙向根瘤原基運(yùn)動(dòng)[31]。

2.2 慢生根瘤菌與花生的信號(hào)交流

對(duì)于根毛侵染體系來(lái)說(shuō)，豆科植物首先向土壤中釋放類黃酮，類黃酮與根瘤菌的NodD蛋白識(shí)別并結(jié)合，誘導(dǎo)根瘤菌結(jié)瘤基因（）表達(dá)合成結(jié)瘤因子（nod factors，NF）[32-33]。結(jié)瘤因子與植物根表的結(jié)瘤因子受體蛋白（nod factor receptor，NFR）正確識(shí)別后激活植物共生信號(hào)通路，在該信號(hào)通路中SymRK（leucine-rich repeat receptor kinase）信號(hào)轉(zhuǎn)導(dǎo)蛋白首先被誘導(dǎo)表達(dá)，進(jìn)而將信號(hào)傳入根毛的細(xì)胞核中并經(jīng)由Ca2+振蕩激活Ca2+依賴蛋白激酶CCaMK（Calcium calmodulin-dependent protein kinase）[34]。CCaMK一方面通過(guò)調(diào)節(jié)與肌動(dòng)蛋白重排相關(guān)的Nap1蛋白及轉(zhuǎn)錄調(diào)節(jié)因子NSP1、NSP2和NIN（nodule inception）的表達(dá)而調(diào)控侵染線在根毛中的形成及延伸，另一方面通過(guò)細(xì)胞分裂素等信號(hào)分子調(diào)節(jié)轉(zhuǎn)錄調(diào)節(jié)因子NSP1、NSP2和NIN的表達(dá)而調(diào)控根部皮層細(xì)胞分裂促進(jìn)根瘤原基形成，為根瘤菌侵入根瘤原基細(xì)胞做準(zhǔn)備[35]。此外，根瘤菌胞外多糖EPS（exopolysaccharides）作為侵染線基質(zhì)的重要組成成分參與到了侵染線的延伸過(guò)程中，同時(shí)作為植物防御體系抑制劑提高了根瘤菌在侵染線和根瘤細(xì)胞中的存活率和共生效率[36-37]。

對(duì)于慢生根瘤菌與花生的裂隙侵染體系來(lái)說(shuō)，研究發(fā)現(xiàn)結(jié)瘤因子缺陷型慢生根瘤菌sp.SEMIA 6144 V2突變體和野生型菌株sp.BTAI 1在與花生共生時(shí)分別表現(xiàn)出了不結(jié)瘤和結(jié)瘤的性質(zhì)，說(shuō)明花生的裂隙侵染過(guò)程是否需要結(jié)瘤因子還未完全確定[38-39]；此外，也有研究發(fā)現(xiàn)結(jié)瘤因子受體蛋白AhLYR3和AhEPR3（EPS receptor 3）廣泛存在于花生植株中并在共生時(shí)顯著表達(dá)，推測(cè)該蛋白在慢生根瘤菌-花生共生時(shí)發(fā)揮著重要作用[40-41]；對(duì)于花生共生信號(hào)通路來(lái)說(shuō)，磷酸化的AhSYMRK蛋白能夠幫助慢生根瘤菌侵染花生根部和根瘤原基細(xì)胞[42]；AhCCaMK蛋白通過(guò)調(diào)控轉(zhuǎn)錄調(diào)節(jié)因子AhCYCLOPS的磷酸化而控制下游蛋白AhHK1（Histidine Kinase1）、AhNIN及AhENOD40表達(dá)，最終調(diào)節(jié)根瘤的形成和固氮以及類菌體的分布和分化[43-45]；、、等基因也參與到了花生與慢生根瘤菌共生關(guān)系的建立中[46]。此外，有研究證明慢生根瘤菌EPS缺陷型突變體接種花生的根瘤數(shù)、地上干重、組織中氮含量及根瘤細(xì)胞中類菌體密度均顯著低于野生型菌株處理，說(shuō)明EPS也影響了慢生根瘤菌與花生的共生效率[47-48]。

2.3 花生根瘤的形成

對(duì)于根毛侵染體系來(lái)說(shuō)，當(dāng)侵染線延伸至根瘤原基處時(shí)會(huì)將根瘤菌釋放入根瘤原基細(xì)胞中，隨后，根瘤菌隨著根瘤原基細(xì)胞的不斷分裂而大量繁殖，最終充滿整個(gè)根瘤細(xì)胞。在此過(guò)程中，根瘤菌脫去細(xì)胞壁并被源于植物的共生體膜包裹，分化為具有固氮能力的共生體[49]。對(duì)于裂隙侵染體系的花生-慢生根瘤菌共生來(lái)說(shuō)，慢生根瘤菌經(jīng)由花生細(xì)胞間隙運(yùn)動(dòng)到位于皮層的根瘤原基處，通過(guò)改變花生細(xì)胞壁結(jié)構(gòu)進(jìn)入到根瘤原基細(xì)胞中[31,50]。慢生根瘤菌原有的細(xì)胞壁被花生分泌的細(xì)胞壁降解酶水解，隨著水解小孔的逐步變大，根瘤菌原生質(zhì)體被源于花生的共生體膜包裹，最終膨脹、發(fā)育為球狀成熟共生體。當(dāng)所有根瘤菌發(fā)育為共生體后，根瘤完全成熟，固氮能力達(dá)到頂峰[50]。

根據(jù)形態(tài)結(jié)構(gòu)可將成熟根瘤分為無(wú)限型根瘤（不定型根瘤）和有限型根瘤（定型根瘤）。無(wú)限型根瘤存在明顯的結(jié)構(gòu)分層，包括具有持續(xù)分裂分化能力的頂端分生區(qū)（I）、被根瘤菌侵染的侵染區(qū)（II）、同時(shí)含有未分化和分化類菌體的過(guò)渡區(qū)（Ⅱ—Ⅲ）、充滿成熟類菌體且具有高效固氮能力的固氮區(qū)（Ⅲ）以及類菌體大量解體并喪失固氮能力的衰老區(qū)（Ⅳ）[51]。有限型根瘤為球形，無(wú)明顯結(jié)構(gòu)差異，主要由表皮層、分生組織、輸導(dǎo)組織和含菌組織（中央固氮區(qū)，III）構(gòu)成。其中，皮層起到儲(chǔ)存養(yǎng)分、保護(hù)根瘤的作用；分生組織僅存在于根瘤形成早期，隨后分化為其他組織；疏導(dǎo)組織負(fù)責(zé)在植物和根瘤間傳輸水分和養(yǎng)分；中央固氮區(qū)包括少量不含類菌體的根瘤細(xì)胞和大量充滿類菌體的根瘤細(xì)胞[52-53]?；ㄉ鰹榈湫偷挠邢扌透?，直徑1—5 mm，中央固氮區(qū)細(xì)胞較大、含有細(xì)胞核、充滿膨脹的球狀共生體[20,54-55]。此外，花生根瘤細(xì)胞中含有與類菌體膜緊密接觸的脂質(zhì)體，這些脂質(zhì)體通過(guò)β-過(guò)氧化和乙醛酸降解途徑為類菌體供給碳源[56-57]。

2.4 花生根瘤固氮機(jī)理

成熟根瘤的共生體中含有固氮酶，固氮酶能夠在微氧環(huán)境中將N2轉(zhuǎn)化為NH3[58-59]。固氮酶由鉬鐵蛋白和鐵蛋白兩部分構(gòu)成，鐵蛋白是由2個(gè)相同亞基構(gòu)成的同源二聚體；鉬鐵蛋白由4個(gè)亞基構(gòu)成，并含有2個(gè)鉬原子和不同數(shù)量的Fe-S簇。在固氮過(guò)程中，鐵氧還蛋白將電子傳遞給固氮酶的鐵蛋白組分，鐵蛋白在水解ATP的同時(shí)將固氮酶的鉬鐵蛋白還原，鉬鐵蛋白將電子傳遞至分子態(tài)氮并將其還原為NH3[60]。NH3與共生體膜內(nèi)的H+結(jié)合形成NH4+，隨后NH4+與植物細(xì)胞中的谷氨酸結(jié)合形成谷氨酰胺為植物提供氮源，或者谷氨酰胺將氨基傳遞給天冬氨酸以天冬酰胺的形式供給氮源[61]。根瘤菌的固氮基因（和）直接參與上述固氮過(guò)程，這些基因呈簇狀排列，一般為，其中和參與合成鉬鐵固氮酶的Fe-S聚簇，基因調(diào)控固氮酶成熟，操縱子調(diào)節(jié)固氮基因的表達(dá)[62]。由于固氮酶固定1分子N2需要消耗16分子ATP，因此類菌體的固氮反應(yīng)需要宿主植物持續(xù)提供碳源[63]。植物光合作用固定的碳以四碳二羧酸的形式提供給類菌體，如蘋果酸在蘋果酸酶和丙酮酸脫氫酶催化下生成乙酰輔酶A，用于類菌體三羧酸循環(huán)（tricarboxylic acid cycle，TCA）形成ATP[64]。此外，植物細(xì)胞也為類菌體提供大量的鐵、硫、鉬、磷、鎂、錳、鋅等多種礦質(zhì)元素以及各種糖類、氨基酸和精胺等物質(zhì)[65-68]。

目前，關(guān)于花生根瘤固氮機(jī)理的研究較少，研究發(fā)現(xiàn)花生根瘤鮮重、豆血紅蛋白含量和固氮酶活性均與根瘤固氮積累量和供氮比例以及莢果產(chǎn)量正相關(guān)[69]?；ㄉ蛞簿哂杏绊懜龉痰富钚缘哪芰43]。此外，不同花生慢生根瘤菌對(duì)花生根瘤固氮酶活性及動(dòng)態(tài)變化影響顯著[20]。

2.5 花生對(duì)根瘤數(shù)的調(diào)控

雖然根瘤菌為豆科植物提供大量氮素，但由于根瘤固氮消耗過(guò)高的能源成本，因此植物進(jìn)化出了多種機(jī)制嚴(yán)格控制根瘤數(shù)量。其中，最為重要的是植物根瘤數(shù)自調(diào)控系統(tǒng)AON，在AON調(diào)控系統(tǒng)中共生信號(hào)通路的下游蛋白NIN調(diào)節(jié)植物合成結(jié)瘤抑制蛋白CLE （CLAVATA3/endosperm-surrounding region-related），該蛋白將信號(hào)傳遞到植株地上部位并激活葉片中的NARK受體激酶（nodule autoregulation receptor kinase），NARK誘導(dǎo)KAPP蛋白（kinase-associated protein phosphatase）合成結(jié)瘤抑制因子SDI（shoot-derived inhibitor）或細(xì)胞分裂素等信號(hào)分子，這些信號(hào)分子再次將信號(hào)傳遞回根部并通過(guò)抑制NIN等共生信號(hào)通路中下游蛋白的表達(dá)而阻斷結(jié)瘤[70-72]。第二種根瘤數(shù)調(diào)節(jié)系統(tǒng)是植物激素信號(hào)調(diào)節(jié)網(wǎng)絡(luò)，在該調(diào)控網(wǎng)絡(luò)中細(xì)胞分裂素及局部積累的生長(zhǎng)素具有促進(jìn)根瘤發(fā)育的能力；乙烯、茉莉酸、脫落酸和赤霉素抑制根毛侵染體系中侵染線的形成和根瘤發(fā)育[73]。此外，植物激素間也存在相互調(diào)節(jié)作用，例如過(guò)量分泌的細(xì)胞分裂素和生長(zhǎng)素促進(jìn)乙烯大量合成，進(jìn)而通過(guò)乙烯信號(hào)調(diào)節(jié)通路激活植物免疫反應(yīng)而反饋抑制根瘤形成和發(fā)育[74]。

對(duì)于花生根瘤數(shù)調(diào)控系統(tǒng)來(lái)說(shuō)，在結(jié)瘤前期，AON調(diào)控系統(tǒng)中的AhCLE13、AhSUNN、AhKLAVIER蛋白表達(dá)量上調(diào)，說(shuō)明AON系統(tǒng)在花生中發(fā)揮了調(diào)控作用[20, 41]。在植物激素信號(hào)網(wǎng)絡(luò)中，細(xì)胞分裂素通過(guò)調(diào)節(jié)花生細(xì)胞分裂素受體AhHK1的表達(dá)參與根瘤原基形成[44]；此外，與乙烯相關(guān)的乙烯響應(yīng)因子AhERF（ethylene response factor）、乙烯信號(hào)調(diào)節(jié)因子AhEIN2（ethylene signaling protein）等蛋白在花生結(jié)瘤時(shí)大量表達(dá)，參與調(diào)控根瘤原基細(xì)胞分裂、分化及根瘤發(fā)育[20, 45]。

3 影響慢生根瘤菌與花生結(jié)瘤固氮的環(huán)境因素

3.1 氮

化學(xué)氮肥的施用對(duì)豆科作物產(chǎn)量的提升尤為重要，然而過(guò)量施氮卻影響作物產(chǎn)量和品質(zhì)的提高，引起土壤酸化板結(jié)和地下水污染[75]，并抑制作物結(jié)瘤和根瘤固氮。研究表明，土壤中高濃度的氮（尤其是硝態(tài)氮）通過(guò)抑制根瘤菌侵染、根瘤原基形成和生長(zhǎng)而降低植物結(jié)瘤數(shù)量，通過(guò)降低豆血紅蛋白的合成而降低根瘤固氮酶活性并加速根瘤衰老和崩解[76-78]。進(jìn)一步研究證明這種抑制過(guò)程涉及植物氮反饋調(diào)控系統(tǒng)，該系統(tǒng)能夠保證植株在土壤氮素充足的情況下免于浪費(fèi)能量供給根瘤菌進(jìn)行結(jié)瘤和固氮。在氮反饋調(diào)控系統(tǒng)中，過(guò)量的氮素首先誘導(dǎo)植物根部大量表達(dá)CLE蛋白，該蛋白通過(guò)AON調(diào)控系統(tǒng)中的HAR1、NARK和SUNN蛋白促進(jìn)細(xì)胞分裂素或生長(zhǎng)素等信號(hào)激素的合成，最終反饋抑制根部NIN蛋白表達(dá)以及根瘤形成[77, 79-83]。

研究證明2.5 mmol/棵KNO3能夠顯著降低花生根瘤數(shù)量；5 mmol/棵KNO3幾乎完全抑制了花生結(jié)瘤，僅有的幾個(gè)根瘤缺少豆血紅蛋白、無(wú)固氮能力[20]。這說(shuō)明由硝酸鹽誘導(dǎo)的氮反饋調(diào)節(jié)機(jī)制在花生-慢生根瘤菌共生體系中依舊發(fā)揮著降低根瘤數(shù)和根瘤固氮酶活性的作用，但具體調(diào)節(jié)機(jī)制還需進(jìn)行深入研究。此外，也有研究發(fā)現(xiàn)，結(jié)莢期花生根瘤的固氮速率及根瘤供氮量達(dá)到最高，此時(shí)過(guò)高的氮肥供應(yīng)會(huì)通過(guò)氮反饋調(diào)控系統(tǒng)抑制根瘤固氮，促進(jìn)根瘤早衰[84-85]。因此，根據(jù)花生氮反饋調(diào)節(jié)機(jī)制和根瘤固氮?jiǎng)討B(tài)，在花生的不同生育時(shí)期合理施用氮肥是一項(xiàng)符合花生生產(chǎn)實(shí)際的舉措。

3.2 pH

土壤酸化是農(nóng)業(yè)種植中面臨的一個(gè)尤為嚴(yán)重的問(wèn)題，而連作的豆科作物更能加速這種酸化進(jìn)程，因此土壤pH對(duì)豆科作物與根瘤菌共生固氮的影響值得關(guān)注。研究表明，當(dāng)pH低于4.5時(shí)，根瘤菌的生長(zhǎng)和存活量相對(duì)較少[86-87]；當(dāng)pH為5.0時(shí)，根瘤菌與豆科植物的共生效率受到顯著抑制[88]；此外，酸化土壤通過(guò)降低植物類黃酮等物質(zhì)誘導(dǎo)根瘤菌結(jié)瘤因子合成的效率而阻斷二者共生關(guān)系建立[89]。耐酸根瘤菌是一類可以在酸化土壤中與花生正常結(jié)瘤和固氮的慢生根瘤菌，這類根瘤菌通過(guò)增強(qiáng)質(zhì)子排斥及細(xì)胞質(zhì)緩沖能力、合成酸休克蛋白、提高膜滲透能力、調(diào)控鈣離子代謝及谷氨酰胺合成酶/谷氨酸合成酶途徑等方式將細(xì)胞質(zhì)的pH維持在中性水平（pH 7.2—7.5），進(jìn)而維持根瘤菌正常定殖和共生能力[87, 90-96]。因此，耐酸慢生根瘤菌可以作為潛在根瘤菌劑菌種應(yīng)用于酸化土壤的花生種植中，為提高酸化土壤中豆科作物固氮效率、減少氮肥施用、延緩?fù)寥浪峄峁┛赡堋?/p>

3.3 溫度

土壤溫度對(duì)根瘤菌的存活、結(jié)瘤和固氮能力存在較大影響，大部分根瘤菌最適生長(zhǎng)溫度為28—31℃[87]。同時(shí)溫度也影響了根瘤菌對(duì)植物根毛的侵染、類菌體分化、根瘤結(jié)構(gòu)和功能[97-98]?；ㄉ?慢生根瘤菌的共生對(duì)根溫較其他豆科植物更為敏感。研究發(fā)現(xiàn)，37℃時(shí)花生慢生根瘤菌sp.ATCC 10317、SEMIA 6144和TAL 1371生物量輕微下降，細(xì)胞內(nèi)低分子量的低聚糖含量顯著增加，中性葡聚糖的合成被完全抑制。40℃處理4 h時(shí)，根瘤菌細(xì)胞會(huì)合成兩個(gè)分子質(zhì)量分別為17 kD和18 kD的熱休克蛋白[99]。對(duì)于花生來(lái)說(shuō)，當(dāng)根溫為40℃時(shí)花生結(jié)瘤和固氮能力被完全抑制[100]。因此，施加根瘤菌劑時(shí)需要考慮田間溫度，以保證根瘤菌在土壤中的存活率以及與花生的共生效率。

3.4 水分

干旱脅迫是限制作物生長(zhǎng)發(fā)育和生產(chǎn)的重要環(huán)境因素之一，同時(shí)也顯著降低豆科作物的結(jié)瘤和固氮效率[101]。研究證明干旱脅迫通過(guò)韌皮部水體積流量影響豆科作物體內(nèi)的碳代謝、根瘤透氧性和氮反饋體系等3個(gè)方面，進(jìn)而限制根瘤的固氮能力[102]。對(duì)于花生來(lái)說(shuō)，干旱脅迫影響花生生長(zhǎng)，降低植株地上干重、根瘤數(shù)和含氮量；誘導(dǎo)植株大量合成H2O2，損害脂質(zhì)和蛋白質(zhì)；抑制根瘤的發(fā)育程度和固氮能力[101, 103]。因此，在花生結(jié)瘤和固氮過(guò)程中應(yīng)保證充足的水分供應(yīng)，為花生結(jié)莢準(zhǔn)備足夠的營(yíng)養(yǎng)物質(zhì)。

4 存在問(wèn)題及展望

慢生根瘤菌與花生共生機(jī)制的研究及田間生產(chǎn)存在如下問(wèn)題：（1）花生慢生根瘤菌基因組遺傳進(jìn)化及功能方面，隨著基因組測(cè)序技術(shù)及分析手段的發(fā)展，目前初步了解了花生慢生根瘤菌基因組功能及進(jìn)化機(jī)制[19]，但缺乏試驗(yàn)驗(yàn)證和系統(tǒng)的深入研究；（2）共生信號(hào)交流方面，部分研究證明花生與慢生根瘤菌的共生需要結(jié)瘤因子，但另一部分研究卻表明不分泌結(jié)瘤因子的根瘤菌也能與花生結(jié)瘤共生[38-39]，因此結(jié)瘤因子是否發(fā)揮功能還需進(jìn)一步證明。此外，雖然在花生共生信號(hào)通路中發(fā)現(xiàn)了一些調(diào)控結(jié)瘤的蛋白，但這些蛋白具體調(diào)控機(jī)制依舊未知；（3）侵染路徑方面，對(duì)于裂隙侵染來(lái)說(shuō)，研究中關(guān)注更多的是光合慢生根瘤菌在合萌上的侵染路徑，發(fā)現(xiàn)根瘤菌的熒光標(biāo)記配合植物組織的激光共聚焦顯微觀察是最簡(jiǎn)便的檢測(cè)方法[30]。但由于熒光基因及其他標(biāo)記基因在慢生根瘤菌中遺傳穩(wěn)定性差，難以標(biāo)記成功。因此，為了了解慢生根瘤菌在花生上的侵染路徑，首先需要克服慢生根瘤菌遺傳標(biāo)記這一問(wèn)題；（4）根瘤數(shù)量調(diào)節(jié)方面，幾乎所有研究均認(rèn)為只有豆科植物調(diào)控了根瘤數(shù)，筆者發(fā)現(xiàn)花生慢生根瘤菌具有影響花生根瘤數(shù)的能力[20]，說(shuō)明慢生根瘤菌-花生共生體系可能存在特殊的共生機(jī)制，但具體機(jī)制未知；（5）養(yǎng)分交流方面，根瘤菌與豆科植物養(yǎng)分交流的研究主要集中在根毛侵染體系上，完全忽略了裂隙侵染體系。而早期研究發(fā)現(xiàn)花生根瘤細(xì)胞中含有一種特異的參與碳源供應(yīng)的脂質(zhì)體[56]，說(shuō)明花生-慢生根瘤菌的共生可能存在某種較為特殊的碳-氮源交換路徑，值得進(jìn)行深入研究。（6）根瘤菌菌劑方面，市面上已經(jīng)出現(xiàn)了一些用于花生栽培的慢生根瘤菌菌劑，但這些菌劑存在普適性低、花生品種匹配性差、結(jié)瘤固氮效率低等問(wèn)題；（7）化學(xué)肥料施用方面，化學(xué)氮肥的大量施用，導(dǎo)致慢生根瘤菌與花生的共生固氮作用被顯著抑制，造成了資源浪費(fèi)和生態(tài)環(huán)境污染；（8）遺傳育種方面，目前幾乎所有育種研究均集中在花生的高產(chǎn)、高油、高油酸、抗病及抗逆等方面，完全忽略了花生的高效結(jié)瘤和固氮等特性以及慢生根瘤菌與花生品種的匹配性。

基于上述問(wèn)題，未來(lái)的研究重點(diǎn)及方向應(yīng)該集中在以下幾個(gè)方面：（1）花生的慢生根瘤菌基因功能，找到適合編輯慢生根瘤菌基因組的遺傳操作手段，檢測(cè)這些基因與花生共生表型的關(guān)系，通過(guò)測(cè)定突變體的轉(zhuǎn)錄組來(lái)分析被突變基因的功能及可能存在的調(diào)控網(wǎng)絡(luò)；（2）慢生根瘤菌對(duì)花生的侵染，篩選能夠穩(wěn)定標(biāo)記慢生根瘤菌的質(zhì)粒，結(jié)合激光共聚焦顯微鏡觀察技術(shù)檢測(cè)慢生根瘤菌在花生根部的侵染路徑。此外，根據(jù)已知豆科植物共生信號(hào)通路蛋白的編碼基因在花生基因組中篩選出相應(yīng)的同源基因，通過(guò)基因編輯方法敲除花生植株中的這些基因并驗(yàn)證功能，找到共生過(guò)程所涉及的花生共生信號(hào)通路；（3）花生根瘤數(shù)調(diào)節(jié)，為了深入了解慢生根瘤菌調(diào)控花生根瘤數(shù)的機(jī)制，首先需要分析慢生根瘤菌結(jié)瘤和固氮基因與花生根瘤數(shù)的表型關(guān)系，隨后通過(guò)轉(zhuǎn)錄組測(cè)定方法確定這些基因或蛋白與花生根瘤數(shù)調(diào)控蛋白的互作機(jī)制，找到慢生根瘤菌與花生根瘤數(shù)間的調(diào)節(jié)通路；（4）合成根際微生物菌劑代替單一根瘤菌菌劑，由于單一種屬的根瘤菌在施入土壤后可能被土著微生物同化或抑制，造成菌劑的田間效果不佳。因此，通過(guò)合成微生物群落的方法，將根瘤菌及花生根際主要促生微生物按照一定比例混合制成菌劑，能夠提高有益微生物在土壤中的存活率及根際定殖率，達(dá)到促進(jìn)花生結(jié)瘤固氮和生長(zhǎng)的目的；（5）了解田間花生根瘤的固氮?jiǎng)討B(tài)，合理配施化學(xué)氮肥，在充分利用生物固氮的基礎(chǔ)上減少化學(xué)氮肥施用量；（6）基于花生基因組學(xué)信息，利用遺傳育種方法，對(duì)花生的結(jié)瘤相關(guān)基因進(jìn)行遺傳操作，提高主流花生品種的結(jié)瘤固氮能力。

5 結(jié)語(yǔ)

花生是重要的油料、蛋白質(zhì)和經(jīng)濟(jì)來(lái)源。深入了解花生的慢生根瘤菌結(jié)瘤和固氮基因功能以及慢生根瘤菌和花生共生機(jī)制是改善二者間結(jié)瘤和固氮效率、提高花生產(chǎn)量和品質(zhì)的關(guān)鍵步驟，關(guān)系到化學(xué)氮肥充分利用、農(nóng)業(yè)生態(tài)可持續(xù)發(fā)展和食品安全，為誘導(dǎo)非豆科植物結(jié)瘤固氮提供可能。此外，由于慢生根瘤菌在進(jìn)化上的特異性以及與花生在共生模式上的特殊性，通過(guò)探索二者間的共生機(jī)制可以擴(kuò)展我們?cè)诙箍浦参锷飳W(xué)和根際生物學(xué)領(lǐng)域的知識(shí)。

[1] 張秋磊, 林敏, 平淑珍.生物固氮及在可持續(xù)農(nóng)業(yè)中的應(yīng)用.生物技術(shù)通報(bào), 2008, 2: 1-4.

ZHANG Q L, LIN M, PING S Z.Biological nitrogen fixation and its application in sustainable agriculture.Biotechnology Bulletin, 2008, 2: 1-4.(in Chinese)

[2] 陳文新, 汪恩濤, 陳文峰.根瘤菌-豆科植物共生多樣性與地理環(huán)境的關(guān)系.中國(guó)農(nóng)業(yè)科學(xué), 2004, 37(1): 81-86.

CHEN W X, WANG E T, CHEN W F.The relationship between the symbiotic promiscuity of rhizobia and legumes and their geographical environments.Scientia Agricultura Sinica, 2004, 37(1): 81-86.(in Chinese)

[3] 常月立.中國(guó)南方地區(qū)花生、扁豆根瘤菌的多相分類[D].北京: 中國(guó)農(nóng)業(yè)大學(xué), 2010.

CHANG Y L.Polyphasic systematics of rhizobia isolated fromandgrown in southern China[D].Beijing: China Agricultural University, 2010.(in Chinese)

[4] 陳文新, 汪恩濤.中國(guó)根瘤菌.北京: 科學(xué)出版社, 2011.

CHEN W X, WANG E T.Rhizobia in China.Beijing: Science Press, 2011.(in Chinese)

[5] de Lajudie P, Mousavi S A, Young J P W.International committee on systematics of prokaryotes subcommittee on the taxonomy of rhizobia and agrobacteria minutes of the closed meeting by videoconference, 6 July 2020.International Journal of Systematic and Evolutionary Microbiology, 2021, 71: 4784.

[6] 張丹.中國(guó)北方花生主產(chǎn)區(qū)花生根瘤菌多樣性及其與土壤生態(tài)因子之間關(guān)系的研究[D].北京: 中國(guó)農(nóng)業(yè)大學(xué), 2010.

ZHANG D.Diversity of rhizobia isolated from peanut nodules in main peanut producing region of northern China and relationship between the diversity and soil factors[D]. Beijing: China Agricultural University, 2010.(in Chinese)

[7] CHEN J Y, GU J, WANG E T, MA X X, KANG S T, HUANG L Z, CAO X P, LI L B, WU Y L.Wild peanutare nodulated by diverse and novelspecies in acid soils.Systematic and Applied Microbiology, 2014, 37: 525-532.

[8] 劉保平.根瘤菌菌劑研究[D].武漢: 華中農(nóng)業(yè)大學(xué), 2005.

LIU B P.Study on rhizobium inoculants[D].Wuhan: Huazhong Agricultural University, 2005.(in Chinese)

[9] BOGINO P, BANCHIO E, GIORDANO W.Molecular diversity of peanut-nodulating rhizobia in soils of Argentina.Journal of Basic Microbiology, 2010, 50: 274-279.

[10] El-AKHAL M R, RINCON A, El-MOURABIT N, PUEYO J J, BARRIJAL S.Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of peanut (L.) grown in Moroccan soils.Journal of Basic Microbiology, 2009, 49: 415-425.

[11] GRONEMEYER J L, CHIMWAMUROMBE P, REINHOLD-HUREKB.sp nov., a symbiotic nitrogen-fixing bacterium from root nodules of groundnuts.International Journal of Systematic and Evolutionary Microbiology, 2015, 65: 3241-3247.

[12] GRONEMEYER J L, HUREK T, BUNGER W, REINHOLD-HUREK B.sp.nov., a nitrogen-fixing symbiont isolated from effective nodules ofand.International Journal of Systematic and Evolutionary Microbiology, 2016, 66: 62-69.

[13] 王蕊.中國(guó)南方花生根瘤菌多樣性及其與土壤因子相關(guān)性研究[D].北京: 中國(guó)農(nóng)業(yè)大學(xué), 2013.

WANG R.Biodiversity of peanut rhizobia collected from southern China and its correlation with soil factors[D].Beijing: China Agricultural University, 2013.(in Chinese)

[14] 張小平.四川花生根瘤菌的遺傳多樣性和系統(tǒng)發(fā)育研究[D].武漢: 華中農(nóng)業(yè)大學(xué), 2001.

ZHANG X P.Diversity and phylogeny ofstrains isolated from the root nodules of peanut () in Sichuan[D].Wuhan: Huazhong Agricultural University.(in Chinese)

[15] Chang Y L, Wang J Y, Wang E T, Liu H C, Sui X H, Chen W X.sp.nov., isolated from effective nodules ofandgrown.International Journal of Systematic and Evolutionary Microbiology, 2011, 61: 2496-2502.

[16] WANG R, CHANG Y L, ZHRNG W T, ZHANG D, ZHANG X X, SUI X H, WANG E T, HU J Q, ZHANG L Y, CHEN W X.sp.nov., isolated from effective nodules ofgrown in China.Systematic and Applied Microbiology, 2013, 36: 101-105.

[17] LI Y H, WANG R, ZHANG X X, YOUNG J P W, WANG E T, SUI X H, CHEN W X.sp.nov.andsp.nov., isolated from effective nodules of peanut.International Journal of Systematic and Evolutionary Microbiology, 2015, 65: 4655-4661.

[18] LI Y H, WANG R, SUI X H, WANG E T, ZHAGN X X, TIAN C F, CHEN W F, CHEN W X.sp.nov.,sp.nov.andsp.nov., isolated from effective nodules of peanut in southeast China.Systematic and Applied Microbiology, 2019, 42: 126002.

[19] 李永華.比較基因組學(xué)闡釋根瘤菌在花生和綠豆上的共生差異及慢生根瘤菌的進(jìn)化[D].北京: 中國(guó)農(nóng)業(yè)大學(xué), 2019.

LI Y H.Comparative genomic analysis of peanut bradyrhizobia reveals the genetic differences underlying two symbiotic phenotypes in peanut and mung bean and the evolution ofspp[D].Beijing: China Agricultural University, 2019.(in Chinese)

[20] 吳月.不同花生慢生根瘤菌共生差異的表型和遺傳比較[D].北京: 中國(guó)農(nóng)業(yè)大學(xué), 2020.

WU Y.Comparison of symbiotic difference in phenotype and genotype of peanut bradyrhizobia[D].Beijing: China Agricultural University, 2020.(in Chinese)

[21] D‘Haeze W, Gao M S, Rycke R D, Montagu M v, Engler G, Holsters M.Roles for azorhizobial Nod factors and surface polysaccharides in intercellular invasion and nodule penetration, respectively.Molecular Plant-Microbe Interactions, 1998, 11(10): 999-1008.

[22] HIRSCH A M.Role of lectins (and rhizobial exopolysaccharides) in legume nodulation.Current Opinion in Plant Biology, 1999, 2: 320-326.

[23] VAN RHIJN P, FUJISHIGE N A, LIM P O, Hirsch A M.Sugar- binding activity of pea lectin enhances heterologous infection of transgenic alfalfa plants bybiovar.Plant Physiology, 2001, 126: 133-144.

[24] DARDANELLI M, ANGELINI J, FABRA A.A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots.Canadian Journal of Microbiology, 2003, 49: 399-405.

[25] FABRA A, CASTRO S, TAURIAN T, ANGELINI J, IBANEZ F, DARDANELLI M, TONELLI M, BIANUCCI E, VALETTI L.Interaction amongL.(peanut) and beneficial soil microorganisms: How much is it known? Critical Reviews in Microbiology, 2010, 36(3): 179-194.

[26] BREWIN N J.Plant cell wall remodeling in the rhizobium-legume symbiosis.Critical Reviews in Plant Sciences, 2004, 23: 293-316.

[27] ROTH L E, STACEY G.Bacterium release into host-cells of nitrogen-fixing soybean nodules-the symbiosome membrane comes from 3 sources.European Journal of Cell Biology, 1989, 49(1): 13-23.

[28] Murray J D.Invasion by invitation: Rhizobial infection in legumes.Molecular Plant-Microbe Interactions, 2011, 24(6): 631-639.

[29] GAGE D J.Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes.Microbiology and Molecular Biology Reviews, 2004, 68(2): 280-300.

[30] BONALDI K, GARGANI D, PRIN Y, FARDOUX J, GULLY D, NOUWEN N, GOORMACHTIG S, GIRAUD E.Nodulation ofandby photosyntheticsp.strain ORS285: The Nod-dependent versus the Nod-independent symbiotic interaction.Molecular Plant-Microbe Interactions, 2011, 24(11): 1359-1371.

[31] Boogerd F C, van Rossum D.Nodulation of groundnut by: A simple infection process by crack infection.FEMS Microbiology Reviews, 1997, 21(1): 5-27.

[32] FOURNIER J, TIMMERS A C J, SIEBERER B J, JAUNEAU A, CHABAUD M, BARKER VAN RHIJN P, VANDERLEYDEN J.The-plant symbiosis.Microbiological Reviews, 1995, 59(1): 124-142.

[33] SPAINK H P.Root nodulation and infection factors produced by rhizobial bacteria.Annual Review of Microbiology, 2000, 54: 257-288.

[34] EHRHARDT D W, WAIS R, LONG S R.Calcium spiking in plant root hairs responding to rhizobium nodulation signals.Cell, 1996, 85: 673-681.

[35] MADSEN L H, TIRICHINE L, JURKIEWICZ A, SULLIVAN J T, HECKMANN A B, BEK A S, RONSON C W, JAMES E K, STOUGAARD J.The molecular network governing nodule organogenesis and infection in the model legume.Nature Communications, 2010, 1: 1-10.

[36] STACEY G, SO J S, ROTH L E, LAKSHMI B S K, CARLSON R W.A lipopolysaccharide mutant ofthat uncouples plant from bacterial differentiation.Molecular Plant-Microbe Interactions, 1991, 4(4): 332-340.

[37] LEIGH J A, COPLIN D L.Exopolysaccharides in plant-bacteria interactions.Annual Review Microbiology, 1992, 46: 307-346.

[38] IBANEZ F, FABRA A.Rhizobial Nod factors are required for cortical cell division in the nodule morphogenetic programme of the Aeschynomeneae legume.Plant Biology, 2011: 13: 794-800.

[39] GUHA S, SARKAR M, GANGULY P, UDDIN M R, MANDAL S, DASGUPTA M.Segregation of nod-containing and nod-deficient bradyrhizobia as endosymbionts ofand as endophytes ofin intercropped fields of Bengal Basin, India.Environmental Microbiology, 2016, 18(8): 2575-2590.

[40] IBANEZ F, ANGELINI J, FIGUEREDO M S, MUNOZ V, TONELLI M L, FABRA A.Sequence and expression analysis of putative(peanut) Nod factor perception proteins.Journal of Plant Research, 2015, 128: 709-718.

[41] Karmakar K, Kundu A, Rizvi A Z, Dubois E, Severac D, Czernic P, Cartieaux F, DasGupta M.Transcriptomic analysis with the progress of symbiosis in ‘Crack-Entry’ legumehighlights its contrast with ‘Infection Thread’ adapted legumes.Molecular Plant-Microbe Interactions,2019, 32(3): 271-285.

[42] SAHA S, PAUL A, HERRING L, DUTTA A, BHATTACHARYA A, SAMADDAR S, GOSHE M B, DASGUPTA M.Gatekeeper tyrosine phosphorylation of SYMRK is essential for synchronizing the epidermal and cortical responses in root nodule symbiosis.Plant Physiology, 2016, 171: 71-81.

[43] Sinharoy S, Dasgupta M.RNA interference highlights the role of CCaMK in dissemination of endosymbionts in the aeschynomeneae legume.Molecular Plant-Microbe Interactions, 2009, 22(11): 1466-1475.

[44] Kundu A, DasGupta M.Silencing of putative cytokinin receptor histidine kinase1 inhibits both inception and differentiation of root nodules in.Molecular Plant-Microbe Interactions, 2018, 31(2): 187-199.

[45] SHARMA V, BHATTACHARYYA S, KUMAR R, KUMAR A, IBANEZ F, WANG J, GUO B, SUDINI H K, GOPALAKRISHNAN S, DASGUPTA M, VARSHNEY R K, PANDEY M K.Molecular basis of root nodule symbiosis betweenand 'crack-entry' legume Groundnut (L.).Plants, 2020, 9: 276.

[46] Peng Z, Liu F, Wang L, Zhou H, Paudel D, Tan L, Maku J, Gallo M, Wang J.Transcriptome profiles reveal gene regulation of peanut (L.) nodulation.Scientific Reports, 2017, 7: 40066.

[47] MORGANTE C, ANGELINI J, CASTRO S, FABRA A.Role of rhizobial exopolysaccharides in crack entry/intercellular infection of peanut.Soil Biology and Biochemistry, 2005, 37: 1436-1444.

[48] MORGANTE C, CASTRO S, FABRA A.Role of rhizobial EPS in the evasion of peanut defense response during the crack-entry infection process.Soil Biology and Biochemistry, 2007, 39: 1222-1225.

[49] Jones, K M, Kobayashi H, Davies B W, Taga M E, Walker G C.How rhizobial symbionts invade plants: The-model.Nature Reviews, 2007, 5: 619-633.

[50] Bal A K, Sen D, Weaver R W.Cell wall (outer membrane) of bacteroids in nitrogen-fixing peanut nodules.Current Microbiology, 1985, 12: 353-356.

[51] Wang Q, Liu J, Zhu H.Genetic and molecular mechanisms underlying symbiotic specificity in legume-rhizobium interactions.Frontiers in Plant Science, 2018, 9: 313.

[52] SEN D, WEAVER R W, BAL A K.Structure and organization of effective peanut and cowpea root nodules induced by rhizobial strain 32H1.Journal of Experimental Botany, 1986, 37(176): 356-363.

[53] Fernandez-Luqueno F, Dendooven L, Munive A, Corlay-Chee L, Serrano-Covarrubias L M, Espinosa- Victoria D.Micro-morphology of common bean (L.) nodules undergoing senescence.Acta Physiologiae Plantarum, 2008, 30: 545-552.

[54] CORBY H D L.Types of rhizobial nodules and their distribution among leguminosae.Kirkia, 1988, 13(1): 53-124.

[55] Fabre S, Gully D, Poitout A, Patrel D, Arrighi J F, Giraud E, Czernic P, Cartieaux F.Nod factor-independent nodulation inrequired the common plant- microbe symbiotic toolkit.Plant Physiology, 2015, 169: 2654-2664.

[56] BAL A K, HAMEED S, JAYARAM S.Ultrastructural characteristics of the host-symbiont interface in nitrogen-fixing peanut nodules.Protoplasma, 1989, 150: 19-26.

[57] SIDDIQUE A M, BAL A K.Nitrogen fixation in peanut nodules during dark periods and detopped conditions with special reference to lipid bodies.Plant Physiology, 1991, 95: 896-899.

[58] Hunt S, Layzell D B.Gas exchange of legume nodules and the regulation of nitrogenase activity.Annual Review of Plant Physiology, 1993, 44: 483-511.

[59] Fischer H M.Genetic regulation of nitrogen fixation in rhizobia.Microbiology Review, 1994, 58(3): 352-386.

[60] 武維華.植物生理學(xué).第二版.北京: 科學(xué)出版社, 2008: 121-122.

WU W H.Plant Physiology.2nd edition.Beijing: Science Press, 2008: 121-122.(in Chinese)

[61] Udvardi M, Poole P S.Transport and metabolism in legume- rhizobia symbioses.Annual Review of Plant Biology, 2013, 64: 781-805.

[62] Rubio L M, Ludden P W.Biosynthesis of the iron-molybdenum cofactor of nitrogenase.Annual Review of Microbiology, 2008, 62: 93-111.

[63] Hoffman B M, Lukoyanov D, Yang Z, Dean D R, Seefeldt L C.Mechanism of nitrogen fixation by nitrogenase: The next stage.Chemical Reviews, 2014, 114: 4041-4062.

[64] Poole P, Allaway D.Carbon and nitrogen metabolism in.Advances in Microbial Physiology, 2000, 43: 117-163.

[65] MAUNOURY N, REDONDO-NIETO M, BOURCY M, DE VELDE W V, ALUNNI B, LAPORTE P, DURAND P, AGIER N, MARISA M, VAUBERT D, DELACROIX H, DUC G, RATET P, AGGERBECK L, KONDOROSI E, MERGAERT P.Differentiation of symbiotic cells and endosymbionts innodulation are coupled to two transcriptome-switches.PLoS ONE, 2010, 5(3): e9519.

[66] Li Y, tian c f, chen w f, wang L, sui x h, chen w x.High-resolution transcriptomic analyses ofsp.NGR234 bacteroids in determinate nodules ofand indeterminate nodules of.PLoS ONE, 2013, 8(8): e70531.

[67] Jiao J, Wu L J, Zhang B, Hu Y, Li Y, Zhang X X, Guo H J, Liu L X, Chen W X, Zhang Z, Tian C FMucR is required for transcriptional activation of conserved ion transporters to support nitrogen fixation ofin soybean nodules.Molecular Plant-Microbe Interactions, 2016, 29(5): 352-361.

[68] Hood G, Ramachandran V, East A K, Downie J A, Poole P S.Manganese transport is essential for N2‐fixation bybacteroids from galegoid but not phaseoloid nodules.Environmental Microbiology, 2017, 19: 2715-2726.

[69] 鄭永美, 杜連濤, 王春曉, 吳正鋒, 孫學(xué)武, 于天一, 沈浦, 王才斌.不同花生品種根瘤固氮特點(diǎn)及其與產(chǎn)量的關(guān)系.應(yīng)用生態(tài)學(xué)報(bào), 2019, 30(3): 961-968.

ZHENG Y M, DU L T, WANG C X, WU Z F, SUN X W, YU T Y, SHEN P, WAGN C B.Nitrogen fixation characteristics of root nodules in different peanut varieties and their relationship with yield.Chinese Journal of Applied Ecology, 2019, 30(3): 961-968.(in Chinese)

[70] Kosslak R M, Bohlool B B.Suppression of nodule development of one side of a split-root system of soybeans caused by prior inoculation of the other side.Plant Physiology, 1984, 75: 125-130.

[71] Reid D E, Ferguson B J, Gresshoff P M.Inoculation- and nitrate-induced CLE peptides of soybean control NARK-dependent nodule formation.Molecular Plant-Microbe Interactions, 2011, 24(5): 606-618.

[72] Ferguson B J, Mens C, Hastwell A H, Zhang M B, Su H, Jones C H, Chu X T, Gresshoff P M.Legume nodulation: The host controls the party.Plant Cell and Environment, 2019, 42: 41-51.

[73] Liu H, Zhang C, Yang J, Yu N, Wang E.Hormone modulation of legume-rhizobial symbiosis.Journal of Integrative Plant Biology, 2018, 60(8): 632-648.

[74] Guinel F C.Ethylene, a hormone at the center-stage of nodulation.Frontiers in Plant Science, 2015, 6: 1121.

[75] 崔賢, 王洪丹, 邱洪湘, 張國(guó)英, 謝金玉, 魏梅花.花生配方施肥技術(shù)肥料效應(yīng)試驗(yàn)研究.花生學(xué)報(bào), 2008, 37(3): 33-36.

CUI X, WANG H D, QIU H X, ZHANG G Y, XIE J Y, WEI M H.Effects of compounding application of fertilizer on peanut.Journal of Peanut Science, 2008, 37(3): 33-36.(in Chinese)

[76] OHYAMA T, FUJIKAKE H, YASHIMA H, TANABATA S, ISHIKAWA S, SATO T, NISHIWAKI T, OHTAKE N, SUEYOSHI K, ISHII S.Effect of nitrate on nodulation and nitrogen fixation of soybean//EL-SHEMY H A.In Soybean Physiology and Biochemistry.Croatia, Rijeka: InTech, 2011: 333-364.

[77] Nishida H, Suzaki T.Nitrate-mediated control of root nodule symbiosis.Current Opinion in Plant Biology, 2018, 44: 129-136.

[78] Du M, Gao Z, Li X, Liao H.Excess nitrate induces nodule greening and reduces transcript and protein expression levels of soybean leghaemoglobins.Annals of Botany, 2020, 126: 61-72.

[79] Carroll B J, McNeil D L, Gresshoff P M.A supernodulation and nitrate-tolerant symbiotic () soybean mutant.Plant Physiology, 1985, 78: 34-40.

[80] Nishimura R, Hayashi M, Wu G, Kouchi H, Imaizumi- Anraku H, Murakami Y, Kawasaki S, Akao S, Ohmori M, Nagasawa M, HARADA K, KAWAGUCHI M.HAR1 mediates systemic regulation of symbiotic organ development.Nature, 2002, 420: 426-429.

[81] Searle I R, Men A E, Laniya T S, Buzas D M, Iturbe- Ormaetxe I, Carroll, B J, Gresshoff P M.Long-distance signaling in nodulation directed by a CLAVATA1-like receptor kinase.Science, 2003, 299: 109-112.

[82] Jin J, Watt M, Mathesius U.The autoregulation genemediates changes in root organ formation in response to nitrogen through alteration of shoot-to-root auxin transport.Plant Physiology, 2012, 159: 489-500.

[83] Okamoto S, Kawaguchi M.Shoot HAR1 mediates nitrate inhibition of nodulation in.Plant Signaling and Behavior, 2015, 10: 5.

[84] 吳正鋒, 陳殿緒, 鄭永美, 王才斌, 孫學(xué)武, 李向東, 王興祥, 石程仁, 馮昊, 于天一.花生不同氮源供氮特性及氮肥利用率研究.中國(guó)油料作物學(xué)報(bào), 2016, 38(2): 207-213.

WU Z F, CHEN D X, ZHENG Y M, WANG C B, SUN X W, LI X D, WANG X X, SHI C R, FENG H, YU T Y.Supply characteristics of different nitrogen sources and nitrogen use efficiency of peanut.Chinese Journal of Oil Crop Sciences, 2016, 38(2): 207-213.(in Chinese)

[85] 鄭永美, 王春曉, 劉岐茂, 吳正鋒, 王才斌, 孫秀山, 鄭亞萍.氮肥對(duì)花生根系生長(zhǎng)和結(jié)瘤能力的調(diào)控效應(yīng).核農(nóng)學(xué)報(bào), 2017, 31(12): 2418-2425.

ZHENG Y M, WANG C X, LIU Q M, WU Z F, WANG C B, SUN X S, ZHENG Y P.Regulatory effects of nitrogen fertilizer on peanut root growth and nodulation.Journal of Nuclear Agricultural Sciences, 2017, 31(12): 2418-2425.(in Chinese)

[86] Vargas A A T, Graham P H.cultivar andstrain variation in acid-pH tolerance and nodulation under acid conditions.Field Crops Research, 1988, 19(2): 91-101.

[87] Graham P H.Stress tolerance inand, and nodulation under adverse soil conditions.Canadian Journal of Microbiology, 1992, 38: 475-484.

[88] Macció D, Fabra A, Castro S.Acidity and calcium interaction affect the growth ofsp.and the attachment to peanut roots.Soil Biology and Biochemistry, 2002, 34: 201-208.

[89] Angelini J, Castro S, Fabra A.Alterations in root colonization andgene induction in the peanut-rhizobia interaction under acidic conditions.Plant Physiology and Biochemistry, 2003, 41: 289-294.

[90] Krulwich T A, Agus R, Schneir M, Guffanti A A.Buffering capacity of bacilli that grow at different pH ranges.Journal of Bacteriology, 1985, 162(2): 768-772.

[91] Bhagwat A A, Apte S K.Comparative analysis of proteins induced by heat shock, salinity, and osmotic stress in the nitrogen- fixing cyanobacteriumsp.Strain L-31.Journal of Bacteriology, 1989, 171(9): 5187-5189.

[92] Graham P H.Stress tolerance inand, and nodulation under adverse soil conditions.Canadian Journal of Microbiology, 1992, 38: 475-484.

[93] Howieson J G, Robson A D, Abbott L K.Calcium modifies pH effects on the growth of acid-tolerant and acid-sensitive.Australian Journal of Agricultural Research, 1992, 43(3): 765-772.

[94] Chen H, Richardson A E, Rolfe B G.Studies of the physiological and genetic basis of acid tolerance inbiovar.Applied and Environmental Microbiology, 1993, 59: 1798-1804.

[95] Angelini J, Taurian T, Morgante C, Ibanez F, Castro S, Fabra A.Peanut nodulation kinetics in response to low pH.Plant Physiology and Biochemistry, 2005, 43: 754-759.

[96] Natera V, Sobrevals L, Fabra A, Castro S.Glutamate is involved in acid stress response insp.SEMIA 6144 (L.) microsymbiont.Current Microbiology, 2006, 53: 479-482.

[97] Roughley R J.The influence of root temperature,strain and host selection on the structure and nitrogen-fixing efficiency of the root nodules of.Annals of Botany, 1970, 34: 631-646.

[98] Roughley R J, Dart P J.Root temperature and root–hair infection ofL.cv.Cranmore.Plant Soil, 1970, 32: 518-520.

[99] Dardanelli M S, Woelke M R, González P S, Bueno M A, Ghittoni N E.The effects of nonionic hyperosmolarity and of high temperature on cell-associated low molecular weight saccharides from two rhizobia strains.Symbiosis, 1997, 23(1): 73-84.

[100] Michiels J, Verreth C, Vanderleyden J.Effects of temperature stress on bean-nodulatingstrains.Applied and Environmental Microbiology, 1994, 60(4): 1206-1212.

[101] PIMRATCH S, JOGLOY S, VORASOOT N, TOOMSAN B, PATANOTHAI A, HOLBROOK C C.Relationship between biomass production and nitrogen fixation under drought-stress conditions in peanut genotypes with different levels of drought resistance.Journal of Agronomy and Crop Science, 2008, 194: 15-25.

[102] SERRAJ R, SINCLAIR T R, PURCELL L C.Symbiotic N-2 fixation response to drought.Journal of Experimental Botany, 1999, 50(331): 143-155.

[103] FURLAN A, LLANES A, LUNA V, CASTRO S.Physiological and biochemical responses to drought stress and subsequent rehydration in the symbiotic association peanut-sp..International Scholarly Research Network ISRN Agronomy, 2012, 2012: 1-8.

Research advances of bradyrhizobia and its symbiotic mechanisms with peanut

WU Yue1, SUI Xinhua2, DAI Liangxiang1, ZHENG Yongmei1, ZHANG Zhimeng1, TIAN Yunyun1, YU Tianyi1, SUN Xuewu1, SUN Qiqi1, MA Dengchao3, WU Zhengfeng1*

1Shandong Peanut Research Institute, Qingdao 266100, Shandong;2College of Biological Sciences, China Agricultural University, Beijing 100193;3Jining Academy of Agricultural Sciences, Jining 272009, Shandong

Nitrogen is one of the essential elements for plant growth, which is obtained by legumes through symbiotic nitrogen fixation with rhizobia.The establishment of symbiotic relationship includes nodulation and nitrogen fixation, involving complex regulatory mechanisms, which is also significantly affected by environmental factors.Symbiosis between peanut and bradyrhizobia is essential for peanut growth and production, but contains many specific and unknown symbiotic mechanisms.In this review, symbiosis between peanut bradyrhizobia and peanut was reviewed, including: (1) Diversity and genomic functions of peanut bradyrhizobia; (2) Symbiotic mechanisms between peanut and bradyrhizobia: rhizobial crack infection and symbiotic signal exchange with peanut, peanut nodulation, nitrogen fixation, and nodule number regulation mechanisms; (3) Effects of environmental factors (soil nitrogen, pH, temperature and water content) on peanut nodulation, nitrogen fixation and yield.This review pointed out current problems in peanut bradyrhizobia, symbiosis between peanut and bradyrhizobia, and peanut field application, including few studies on genome functions of peanut bradyrhizobia, unknown interaction mechanisms between bradyrhizobia and peanut in details, as well as, poor utilization rate of peanut bradyrhizobia in the field, etc.Based on this analysis, the future researches should focus on genome omics analysis and gene functional analysis of peanut bradyrhizobia; signal communication pathways, nodule number regulation mechanisms, nutrient exchange systems between bradyrhizobia and peanut; rational application systems of nitrogen fertilizer that match with nodule nitrogen fixation rules, and obtain new peanut bradyrhizobia agents for peanut planting through synthetic biology.This article provided the theoretical basis for further understanding the symbiotic mechanisms of legumes and rhizobia, improving nodulation and nitrogen fixation efficiency of legume crops, reducing chemical nitrogen application, and improving agricultural ecological environment.

bradyrhizobia; peanut; symbiotic nitrogen fixation; mechanism of nodulation and nitrogen fixation; diversity; environmental factor

2021-07-14；

2021-10-09

國(guó)家重點(diǎn)研發(fā)計(jì)劃（2018YFD1000906）、山東省農(nóng)業(yè)科學(xué)院農(nóng)業(yè)科技創(chuàng)新工程（CXGC2021B33）、山東省農(nóng)業(yè)科學(xué)院農(nóng)業(yè)科技創(chuàng)新工程（CXGC2021A05）、現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)（CARS-13）、山東省重大科技創(chuàng)新工程（2019JZZY010702）、山東省花生產(chǎn)業(yè)技術(shù)體系濟(jì)寧綜合試驗(yàn)站（SDAIT-04-12）

吳月，E-mail：wuyuesw@163.com。通信作者吳正鋒，E-mail：wzf326@126.com

（責(zé)任編輯 楊鑫浩）