1 Rhizobia with Broad Host Ranges

Every rhizobium has its own host range. Some rhizobia like Sinorhizobium meliloti can nodulate with only specific host legume, Melilotus and Medicago, and these rhizobia are classified as narrow host-range rhizobia. In contrast, some rhizobia can infect and nodulate many different kinds of legumes, and they are described as broad or wide host-range rhizobia.

Strain NGR234 (NGR is the abbreviation for New Guinea Rhizobium) was the only fast-growing strain among 30 isolates prepared from Lablab purpureus nodules grown in Papua New Guinea (soil pH 8.5) by Trinick in 1965 (Pueppke and Broughton 1999). The bacterium NGR234 nodulates a very wide range of hosts, with more than 112 genera of legumes (Pueppke and Broughton 1999) as well as the nonlegumes Parasponia andersonii (Op den Camp et al. 2012), Parasponia rugosa and Parasponia parviflora in the family Cannabaceae (Behm et al. 2014). The hosts infected by strain NGR234 are evolutionarily divergent legumes with fundamental differences in nodule morphology and physiology. Strain NGR234-infected nodules of different legumes include both determinate and indeterminate nodules. The common legumes forming determinate nodules infected by NGR234 are represented by Lablab purpureus (Li et al. 2013), Vigna unguiculata (Li et al. 2013; Pueppke and Broughton 1999) and Lotus japonicus (Pueppke and Broughton 1999), while indeterminate nodules formed by NGR234 are exemplified by its well-studied interactions with Leucaena leucocephala (Li et al. 2013) and Sophora flavescens (Jiao et al. 2015b).

However, the broad host-range strain NGR234 cannot nodulate some widely used and studied legumes such as Medicago sativa, Medicago truncatula, Pisum sativum, Vicia faba, Cicer arietinum, Astragalus sinicus, Arachis hypogaea, Glycine max (only some varieties could be nodulated by NGR234 and the nodules formed are inefficient), Trifolium repens and Phaseolus vulgaris. The inability to nodulate these important model legumes restrict its use in some extensive and deep studies, but NGR234 has nevertheless been used in some important studies of the symbiotic relationship between rhizobia and legumes (Li et al. 2013; Müller et al. 2001; Xin et al. 2012; Xu et al. 2018; Zamani et al. 2017).

NGR234 is a Sinorhizobium, closely related to S. fredii but perhaps representing a distinct species (Tian et al. 2012). Besides strain NGR234, other rhizobial strains have also been found to have wide host ranges. Examples are various strains of Sinorhizobium fredii: HH103, USDA257 and CCBAU 45436. Strain HH103 can nodulate Lotus burttii, Glycine max (Acosta-Jurado et al. 2016), Glycyrrhiza uralensis (Crespo-Rivas et al. 2016) and P. vulgaris (Sadowsky et al. 1988). Strain USDA257 can form nodules with 79 legume plant genera (Pueppke and Broughton 1999; Schuldes et al. 2012). Strain CCBAU 45436 can establish effective symbiosis with G. max (determinate nodules) and Cajanus cajan (indeterminate nodules) (Li et al. 2016).

2 Rhizobia Associated with Symbiotically Specific Plants

2.1 Mesorhizobia and Chickpea

Belonging to the tribe Cicereae, chickpea (Cicer arietinum) is, after soybean, the second most widely grown legume crop. It is grown in nearly 50 countries around the world (Varshney et al. 2013), particularly in India, Australia and arid and semi-arid countries around the Mediterranean Basin. In relation to its symbiotic nitrogen fixation (SNF) with rhizobia and its ability to grow on depleted soils with low rainfalls, chickpea cultivation presents both huge agricultural significance and environmental security, decreasing fertiliser input, promoting the growth of chickpea itself and the crops rotated with it and providing human dietary protein, especially in developing counties. Through SNF, chickpea can supply nitrogen to the soil ranging from 80 to 141 kg ha−1 when inoculated with an efficient rhizobial strain (Herridge et al. 1995). Rhizobial population richness, genetic diversity, geographic distribution, symbiotic matching, soil properties and inoculation with an effective strain can influence the effectiveness of SNF between the two symbiotic partners.

Chickpea rhizobia were first assigned to the genus Rhizobium by Nour et al. with two species described, Rhizobium ciceri in 1994 (Nour et al. 1994) and Rhizobium mediterraneum in 1995 (Nour et al. 1995). However, it became apparent that these species were sufficiently diverged to require the creation of a new genus, and because the intermediate growth speed of chickpea rhizobia is slower than that of fast growers like Rhizobium species but faster than the slow growers like Bradyrhizobium species, a novel genus Mesorhizobium (meso-, middle, intermediate) was proposed, and these two species were subsequently transferred to Mesorhizobium gen. nov. (Jarvis et al. 1997), with Mesorhizobium ciceri and Mesorhizobium mediterraneum as their new combined names in 1997. In 2012, a third novel species Mesorhizobium muleiense was proposed by Zhang et al. (2012a) when they studied the distinct chickpea rhizobia in Xinjiang China (Zhang et al. 2012b). An additional species was isolated from root nodules of chickpea grown in Gansu Province of Northwest China (Zhang et al. 2017) and has now been named Mesorhizobium wenxiniae (Zhang et al. 2018). There are reports of other potential novel Mesorhizobium species nodulating chickpea in southern and central Ethiopia (Tena et al. 2017) and in Indian soils (Nandwani and Dudeja 2009).

Besides the above four species, chickpea can be nodulated by many other Mesorhizobium that were originally described from hosts other than chickpea, including M. amorphae, M. tianshanense and M. opportunistum isolated from chickpea nodules in Spain and Portugal (Laranjo et al. 2012; Rivas et al. 2007) and M. tarimense, M. gobiense, M. tamadayense, M. opportunistum, M. loti and M. amorphae isolated from chickpea nodules in eastern Algeria (Dekkiche et al. 2017).

Endophytic bacteria, including Sinorhizobium (Ensifer) spp., were also isolated from chickpea nodules in some arid regions. In a survey of chickpea rhizobia in Algeria, 41 isolates were assigned to Ensifer meliloti, while 19 isolates could firmly be considered as Mesorhizobium strains (Dekkiche et al. 2017). Of the 15 randomly chosen Ensifer isolates, only 2 could form root nodules and were verified to be identical to the original isolated strain (Dekkiche et al. 2017). However, nodC gene could not be amplified from any of the 41 E. meliloti isolates, though 9 isolates had a nifH gene closely related to the nifH gene in E. meliloti and E. medicae (Dekkiche et al. 2017). Other studies also reported the existence of Ensifer spp. in chickpea nodules (Aouani et al. 2001; Ben Romdhane et al. 2007). Some other bacteria, including Agrobacterium sp., Ochrobactrum sp. (Naseem et al. 2005) and Rhizobium (Ogutcu et al. 2009), have also been isolated from chickpea nodules. The efficiency of nitrogen fixation of these endophytic non-Mesorhizobium isolates on chickpea needs to be checked. The role of these apparently non-symbiotic endophytic bacteria is unclear, but they might be helping in the process of nodulation (Hameed et al. 2004) or have the function of increasing phosphate solubilisation (Hameed et al. 2005).

From studies on the phylogeny of symbiosis genes (nodC and nifH) of chickpea rhizobia, it is clear that all these different chickpea-nodulating Mesorhizobium spp. have very similar symbiosis genes and these differ from the genes of rhizobia associated with non-chickpea legumes (Fig. 7.1). This could explain the highly host specificity of chickpea (Dadarwal 1980), and all these chickpea-nodulating rhizobia should be assigned to the symbiovar ciceri, i.e. they are Mesorhizobium spp. sv. ciceri. The distinct symbiosis genes are found in different Mesorhizobium spp. in different countries, indicating strong positive selection by chickpea. However, specific chickpea Mesorhizobium species were found only in specific regions. Retention and horizontal transfer of the chickpea-specific symbiosis genes among different mesorhizobia may be common to keep the specific symbiosis with chickpea.

Fig. 7.1
figure 1

Phylogenetic tree based on nodC genes of different sv. ciceri strains nodulating Cicer arietinum and Cicer canariense. Sinorhizobium medicae A-321 was used as outgroup. Strains from different continents (Europe, Asia, Africa) were highlighted with different colours. Bar, 5% nucleotide substitution

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So far, only M. muleiense and M. wenxiniae have been found in nodules of chickpea in China. M. ciceri is, however, found to be widely distributed in the Mediterranean, North Africa, North America, Indian subcontinent and Russia (Nour et al. 1995). The wide distribution of M. ciceri may be due to its adaption to soils with a wide range of pH from 5.0 to 10.0 and its temperature resistance up to 40 °C (Jarvis et al. 1997). M. muleiense from chickpea nodules has only been found in China, especially in alkaline soils (pH 8.46–9.16) in Xinjiang Province, where chickpea has been cultivated for over 2500 years (Zhang et al. 2012a, b, 2014). Ten strains isolated from nodules of Astragalus in Ningxia and Shanxi Provinces were classified as M. muleiense, but their symbiosis genes (nodC and nifH) were different from those of sv. ciceri (Yan et al. 2016a). The complete genome sequence of M. ciceri sv. biserrulae strain WSM1271 (isolated from root nodules of the pasture legume Biserrula pelecinus introduced into Australian soil in 1994) (Nandasena et al. 2014) and the ongoing genome sequencing (unpublished) of four sv. ciceri strains belonging to different species (M. ciceri, M. mediterraneum, M. muleiense, M. wenxiniae) will help scientists to elucidate the distinct origin of their symbiosis genes and their transfer among different Mesorhizobium species.

Cicer canariense is a perennial wild chickpea endemic to the Canary Islands, and nine Mesorhizobium genospecies, including M. ciceri, were found to nodulate this wild plant (Armas-Capote et al. 2014). The other eight species included M. caraganae, M. opportunistum, M. tamadayense, M. australicum, M. loti and three (M. tianshanense/M. gobiense/M. metallidurans) that could not be unambiguously assigned based on 16S rRNA gene phylogeny (Armas-Capote et al. 2014). Only a small group of isolates had nodC sequences identical to those of sv. ciceri, and these were all M. ciceri (Armas-Capote et al. 2014). The other isolates have two distinct nodC lineages, one close to Lotus symbionts such as M. loti NZP2213T and the other differing from all known mesorhizobia (Armas-Capote et al. 2014). Besides these Mesorhizobium spp., some fast-growing strains belonging to Rhizobium leguminosarum whose symbiotic nodC gene was not close to bv. ciceri but close to R. leguminosarum bv. trifolii ATCC 14480 phylogenetically have been reported to nodulate C. canariense effectively (Martinez-Hidalgo et al. 2015). A LacZ-labelled derivative of strain R. leguminosarum bv. trifolii RCCHU01 was used to confirm the effective nodulation on C. canariense and Trifolium repens (Martinez-Hidalgo et al. 2015). These results indicate that the domesticated chickpea (C. arietinum) is more selective in its choice of rhizobia than the wild chickpea (C. canariense).

2.2 Sinorhizobium/Ensifer and Alfalfa

It is considered that the Sinorhizobium (Ensifer) meliloti is the most effective and widely distributed rhizobial species in interacting with alfalfa (Medicago sativa L.) in symbiotic nitrogen-fixing (Wang et al. 2018), though the other minor species of S. medicae (Rome et al. 1996), Rhizobium tibeticum (Hou et al. 2009) and R. favelukesii (Torres Tejerizo et al. 2016) were reported to nodulate alfalfa.

Results from the analyses of 581 nodule isolates of alfalfa grown in Tibetan Plateau have been shown, 579 of them were classified as S. meliloti, only 1 isolate was identified as S. medicae, and 1 isolate was identified as Rhizobium sp. (close to R. mongolense and R. yanglingense based on nodC phylogeny) (Wang et al. 2018), supporting the extremely wide distribution and preponderance of S. meliloti in Tibetan Plateau. Similarly, the dominant species from alfalfa nodules in Serbian soil was also S. meliloti, and only one strain was identified as S. medicae, two strains as R. tibeticum and one strain as Rhizobium sp. (Stajković-Srbinović et al. 2012). This is also true in Argentina and Uruguay (Segundo et al. 1999).

In summary, alfalfa is a highly selective and specific legume that prefers S. meliloti as its effective symbiont around the world.

2.3 Mesorhizobium amorphae and Amorpha fruticosa

Amorpha fruticosa is a shrub legume indigenous to North America (Allen and Allen 1981) that was introduced to China as a windbreak and as soil cover for erosion control. The majority of rhizobia forming an effective symbiosis with A. fruticosa growing in different regions of China were characterised as a sole species of Mesorhizobium, M. amorphae (Wang et al. 1999). The type strain of M. amorphae, ACCC 19665T, only nodulates its host plant A. fruticosa in cross-nodulation tests (Wang et al. 1999). In 2002, Wang et al. (2002) reported that A. fruticosa in its native North America was also nodulated by M. amorphae, which was probably transmitted from the USA to China.

In 2007, Gu et al. identified several rhizobial strains isolated from Lespedeza species as M. amorphae (Gu et al. 2007), and two strains (CCBAU 45224 and CCBAU 25056) had nodC and nifH genes completely identical those of type strain ACCC 19665T. Cross-nodulation tests among the M. amorphae populations isolated, respectively, from A. fruticosa and Lespedeza spp. would be helpful to reveal if they can nodulate both the host plants.

3 Rhizobia Associated with Promiscuous Plants

3.1 Various Rhizobia and Soybean (Glycine max)

Two genera, the Bradyrhizobium and Sinorhizobium (syn. Ensifer), are the publicly recognised rhizobia that can establish effective symbiosis with soybean (Glycine max). Two isolates from nodules of soybean grown in Xinjiang were previously classified as Rhizobium (now Mesorhizobium) tianshanense (Chen et al. 1995), but their authentication could not be verified later (our unpublished observations). The current subsection only focuses on the two well-known genera of soybean rhizobia.

In the genus Bradyrhizobium, seven rhizobial species including B. elkanii (Kuykendall et al. 1992), B. japonicum (Jordan 1982), B. diazoefficiens (Delamuta et al. 2013), B. daqingense (Wang et al. 2013a), B. liaoningense (Xu et al. 1995), B. huanghuaihaiense (Zhang et al. 2012c) and B. ottawaense (Yu et al. 2014) have been described until now to nodulate soybean. In the phylogenetic tree based on six concatenated housekeeping gene sequences (Fig. 7.2), these seven soybean bradyrhizobial species occupied their distinct positions. However, only two nodC gene types were found among these seven species, with B. elkanii as a sole type; the other six species had identical nodC gene sequences (see Chap. 5).

Fig. 7.2
figure 2

Bayesian phylogenetic tree of atpD-glnII-recA-gyrB-rpoB-dnaK concatenated gene sequences for species in genus Bradyrhizobium (Yu et al. 2014)

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In the genus Sinorhizobium, four fast-growing species of soybean symbionts have been established: S. (E.) fredii (de Lajudie et al. 1994; Jarvis et al. 1992), E. sojae (Li et al. 2011), E. glycinis (Yan et al. 2016b) and E. shofinae (Chen et al. 2017). Based on the maximum likelihood phylogenetic tree of three housekeeping genes (Chen et al. 2017), the four fast-growing soybean rhizobia occupy distinct positions among the other known species in Ensifer/Sinorhizobium (Fig. 7.3).

Fig. 7.3
figure 3

Maximum likelihood (ML) phylogenetic tree based on concatenated sequences of recA, atpD and glnII showing the genetic relationship among type strains of species in genus Ensifer (Sinorhizobium) (Reprint from Chen et al. (2017))

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As with the nodC gene in Bradyrhizobium species, all the four Ensifer species had identical nodC genes, too (see Chap. 5). In addition, the common components and structure of Nod factors (LCOs) secreted by these soybean-associating Bradyrhizobium and Sinorhizobium/Ensifer species may endow them with same host range (see Chap. 5).

3.2 Various Rhizobia and Sophora

Sophora is a leguminous genus in the tribe Sophoreae, the subfamily Faboideae. This genus contains about 50 species (Sprent 2009) of shrubs and small trees and is distributed worldwide. About 19 species in the genus Sophora have been documented to have nitrogen-fixing nodules (Sprent 2009), and for some species, their rhizobial diversity and taxonomy have been studied in detail since 1995 when Chen et al. described a novel species, Rhizobium tianshanense (Chen et al. 1995) (now renamed Mesorhizobium tianshanense (Jarvis et al. 1997)), from Sophora alopecuroides growing in Xinjiang, China. Another two rhizobial species, Bradyrhizobium elkanii (Han et al. 2008a) and Rhizobium multihospitium (Han et al. 2008b) were isolated from S. alopecuroides by Han et al. Five rhizobial strains isolated from other Sophora species growing in New Zealand were defined as groups related to two Mesorhizobium species and one Rhizobium species (Weir et al. 2004). Seventy-five nodule isolates of S. alopecuroides grown in China’s Loess Plateau were clustered into nine genospecies in five genera, Mesorhizobium, Agrobacterium, Phyllobacterium, Rhizobium and Sinorhizobium (Zhao et al. 2010), revealing the promiscuous symbiosis of this legume. Cross-nodulation tests further confirmed that not only alpha-rhizobia (α-rhizobia) but also beta-rhizobia (β-rhizobia) could nodulate Sophora flavescens (Jiao et al. 2015a), another extremely promiscuous host plant, sister of S. alopecuroides. The extreme diversity of nodC types of various rhizobia that nodulate S. flavescens has been confirmed (Jiao et al. 2015a; Liu et al. 2018a).

The rhizobial species isolated from nodules of Sophora species are listed in Table 7.1, and they are classified into five genera as mentioned above. Altogether, 15 rhizobial (geno)species were clustered into the genus Mesorhizobium, isolated from 6 Sophora species. Fourteen rhizobial (geno)species were classified into the genus Rhizobium; five species were found in Sinorhizobium/Ensifer; two species in each of Phyllobacterium and Bradyrhizobium; and only one species was in the genus Agrobacterium (Table 7.1). Clearly, the predominant rhizobia of Sophora belong to the genera Mesorhizobium and Rhizobium.

Table 7.1 Various rhizobia isolated from root nodule of Sophora species
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The phylogeny of nodC and nodA symbiosis genes of the Sophora-associated rhizobia revealed diverse branches (Jiao et al. 2015a; Zhao et al. 2010). Five clades (13 types) of nodA genes were found in different Sophora rhizobia by Zhao et al. (Jiao et al. 2015a). Seven types of nodC genes were detected among different Sophora rhizobia in the study of Jiao et al. (Jiao et al. 2015a). Clearly, Sophora can be nodulated by rhizobia with phylogenetically different symbiosis genes. Interestingly, all the 51 Mesorhizobium strains belonging to 7 different species collected from New Zealand had almost identical nodC genes, and they clustered closely with the mesorhizobia collected from China (Jiao et al. 2015a; Nguyen et al. 2017), indicating their common origin. Furthermore, identical nodC genes were found in isolates of two different genera, R. mongolense CCBAU 11559 and M. septentrionale CCBAU 11523, supporting the lateral gene transfer of nodulation genes among different rhizobial genera (Jiao et al. 2015a).

To test the promiscuous nodulation of Sophora, more rhizobial species belonging to different genera with different nodC phylogeny in the classes Alphaproteobacteria and Betaproteobacteria were chosen and cross-inoculated on Sophora. This identified another 26 rhizobial species that could nodulate S. flavescens (Table 7.2) (Jiao et al. 2015a). Consistent with the observed diversity of rhizobial species and symbiosis genes among isolates from S. flavescens, the cross-nodulation tests further confirmed the promiscuity of this plant, in that not only does it not select strictly the rhizobial species, but it does not select strictly the symbiosis gene types and accepts rhizobia originally isolated from different cross-nodulation groups. For example, S. meliloti and M. huakuii are two very host-specific rhizobial species, only selecting Medicago and Astragalus sinicus, respectively, as their host plant, but both of them can nodulate S. flavescens (Jiao et al. 2015a).

Table 7.2 Cross-nodulation on Sophora flavescens by different rhizobia
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Although S. flavescens is nodulated promiscuously by many different rhizobia, there are still some rhizobial strains that cannot nodulate it, including the following: Bradyrhizobium japonicum USDA 6, B. diazoefficiens USDA 110, B. yuanmingense CCBAU 10071, Bradyrhizobium sp. strain ORS278, B. oligotrophicum LMG 10732, Ochrobactrum lupini LMG 22726, M. muleiense CCBAU 83963, M. amorphae ATCC 19665, R. galegae HAMBI 1174, Burkholderia nodosa LMG 23741 and Burkholderia phymatum STM 815 (Jiao et al. 2015a). One of these specific rhizobia, USDA 110, a strain isolated from soybean (Glycine max), was studied further to find the molecular mechanism determining its host range (Liu et al. 2018a). Tn5 transposon mutants of USDA 110 were constructed, and 14 mutants were able to form normal nodules on S. flavescens, while wild-type USDA 110 only induced small pseudonodules with no bacteroids on this plant (Liu et al. 2018a). Analyses of the mutated genes found that they were involved in metabolism, transportation and chemotaxis and did not participate in the biosynthesis of Nod factors directly (Liu et al. 2018a). These results indicate that mutation of some nonessential genes could expand rhizobial host ranges (Brewin et al. 1980) and that promiscuity or specificity is dependent both on host and rhizobia (Liu et al. 2018a).

Ensifer adhaerens, a bacterial predator attacking and lysing Micrococcus luteus cells, was originally isolated from Hagerstown silty clay loam soil (Casida 1980, 1982). Controversy over the nomenclature of E. adhaerens and Sinorhizobium adhaerens is not our concern here, but S. adhaerens was previously isolated from root nodules of Sophora tomentosa grown in Brazil and Taiwan and China (Hung et al. 2005; Toma et al. 2017). Although S. (E.) adhaerens was reported to be a predominant occupant in nodules of S. tomentosa (Toma et al. 2017), the capability of nodulation and nitrogen fixation of this species need to be further checked carefully. Other endophytic bacterial species belonging to the genera Bacillus, Brevibacillus and Paenibacillus were also found within S. tomentosa nodules (Toma et al. 2017), further supporting the importance of nodulation tests with S. (E.) adhaerens.

3.3 Rhizobia and Common Bean (Phaseolus vulgaris)

As mentioned in Chap. 5, the promiscuous legume common bean (Phaseolus vulgaris) can be nodulated by various species mainly in the genus Rhizobium, including Rhizobium etli (Aguilar et al. 2004), R. leguminosarum (García-Fraile et al. 2010; Mulas et al. 2011), R. lusitanum (Valverde et al. 2006), R. gallicum (Amarger et al. 1997), R. phaseoli (Ramírez-Bahena et al. 2008), R. tropici (Amarger et al. 1994; Martinez-Romero et al. 1991), R. leucaenae (Ribeiro et al. 2012), R. paranaense (Dall’Agnol et al. 2014), R. vallis (Wang et al. 2011) and R. sophoriradicis (Jiao et al. 2015c; Ormeño-Orrillo et al. 2018), as well as R. giardinii (Amarger et al. 1997), which is now Pararhizobium giardinii (Mousavi et al. 2015). In addition, some isolates containing symbiosis genes (nod and nif) were found in the genera Agrobacterium (Wang et al. 2016), Bradyrhizobium (Cao et al. 2014) and Ensifer (Wang et al. 2016), and one non-nodulating species Phyllobacterium endophyticum (Flores-Félix et al. 2012) was reported to be isolated from root nodules of P. vulgaris grown in different soils of China, Mexico and Spain.

The phylogeny of nodC symbiosis genes of the rhizobia associated with common bean is discussed in Chap. 5 of this book.

3.4 Bradyrhizobia and Peanut (Arachis hypogaea)

Most of the effective rhizobia associated with peanut (Arachis hypogaea) belong to the genus Bradyrhizobium (Chen et al. 2003, 2016; El-Akhal et al. 2009; Santos et al. 2017; Taurian et al. 2006), though some effective fast-growing rhizobia have also been described (El-Akhal et al. 2009; Jaiswal et al. 2017; Osei et al. 2018; Taurian et al. 2006). Bradyrhizobium arachidis (Wang et al. 2013b), B. guangdongense and B. guangxiense (Li et al. 2015) have been described as novel species, and the type strains of these species were isolated originally from nodules of peanut. Strain CCBAU 23160, isolated from peanut grown in Anhui province, China, was identified as B. lablabi, though the type strain of B. lablabi, CCBAU 23086T, was isolated from nodules of Lablab purpureus (Chang et al. 2011).

Bacteria from Ghana, closely related to B. yuanmingense, were reported to be effective peanut microsymbionts (Osei et al. 2018). In the same study, fast-growing isolates KNUST 1003 and 1007 were isolated that were highly similar to Rhizobium tropici (Osei et al. 2018). In another study, Bradyrhizobium isolates obtained from native peanut grown in Córdoba, Argentina, were classified as B. iriomotense and B. yuanmingense based on phenotypic and phylogenetic comparisons (Muñoz et al. 2011). Other isolates, also from Córdoba, were identified as B. japonicum, Bradyrhizobium sp., R. gardinii and R. tropici (Taurian et al. 2006).

Other potentially novel species of peanut symbionts are being described by researchers at China Agricultural University (personal communication). The symbiosis genes of peanut bradyrhizobia are described in Chap. 5 of this book.

3.5 Bradyrhizobia and Erythrophleum fordii

Erythrophleum fordii, commonly known as ironwood tree, is an evergreen legume in the subfamily Caesalpinioideae indigenous to the south of China and the north of Vietnam (Sein and Mitlöner 2011; Yao et al. 2014). Previously, only one rhizobial strain closely related to B. elkanii was reported to be a symbiotic bacterium of E. fordii (Lu et al. 2011). Later, systematic studies on the rhizobia of this ironwood tree were carried out by Yao et al. (2014, 2015).

A total of 166 bacterial isolates were obtained from the nodules of E. fordii growing in Guangdong and Guangxi provinces in southern China. All these isolates were found to be in the genus Bradyrhizobium, with 22 genotypes. Based on multilocus sequence analyses, five genospecies were identified: B. elkanii and B. pachyrhizi comprising the dominant symbionts and B. yuanmingense, B. erythrophlei and B. ferriligni comprising the minor symbionts (Yao et al. 2014, 2015).

4 Rhizobial Infection Through Root Hair Entry or Crack Entry

The establishment of symbiosis between legumes and rhizobia starts from the molecular recognition and interaction between them, followed by infection and entry of rhizobia into the leguminous cell. To form an efficient nitrogen-fixing nodule, live rhizobia must enter the root cell of legume through a specific pathway. Two major infection patterns can be found commonly: root hair infection thread (Fig. 7.4a) and crack entry (Fig. 7.4b). In addition, intercellular infection (Fig. 7.4c) was observed in some other legumes. Most legumes have only one of the infection patterns, while certain legumes can have two kinds of infection patterns.

Fig. 7.4
figure 4

Infection patterns. (a) Root hair infection thread. (b) Crack entry with or without infection threads. (c) Intercellular infection. The lower part of the figure shows endocytosis into cortical cells and the host-derived peribacteroid membrane surrounding the rhizobial bacteroids. (Figure redrawn from Madsen et al. (2010))

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The root hair infection thread pattern (Fig. 7.4a) is found in most well-studied legumes, such as alfalfa (Medicago sativa) (Gage 2004), Medicago truncatula (Fournier et al. 2008), soybean (Glycine max) (Rao and Kerster 1978), pea (Pisum sativum), Lotus japonicus (Markmann et al. 2012; Xie et al. 2012) and Sophora flavescens (Jiao et al. 2015a). Rhizobial entry by the crack pattern (Fig. 7.4b) is found in certain legumes including peanut (Arachis hypogaea) (Boogerd and vanRossum 1997), joinvetch (Aeschynomene americana) (Grant and Trese 1996) and Adesmia bicolor (Bianco 2014), as well as the nonlegume, Parasponia andersonii (Bender et al. 1987). Besides these two major patterns of infection, other distinct patterns are observed in some legumes. A woody legume, tagasaste (Chamaecytisus proliferus ssp. proliferus var. palmensis), forms N2-fixing indeterminate nodules in response to infection by strains of Bradyrhizobium sp. (Chamaecytisus) (Vega-Hernandez et al. 2001). The infection process in tagasaste starts with deformed root hairs, but the infection aborts early, and, instead, the rhizobia use the crack entry mode to move along the intercellular route, eventually entering the altered cell walls directly (Vega-Hernandez et al. 2001). In Lotus japonicus, two alternative intercellular infection modes, crack entry and infection thread independent single cell infection, were discovered using symbiotic mutants (Markmann et al. 2012). Similar to tagasaste, the semiaquatic legume Sesbania rostrata is also infected via an intercellular invasion; the bacteria colonise epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium (Capoen et al. 2010).

In a study of the infection pattern of Sophora flavescens by different rhizobial strains (Fig. 7.5), the authors found the distinguished deformation of the root hairs infected by different rhizobia, leading to different morphologies (Fig. 7.5b–h) (Jiao et al. 2015a). Though different deformed root hairs, the rhizobia invade the root through infection thread clearly (Fig. 7.5h).

Fig. 7.5
figure 5

Light micrograph of root hair, infection thread and nodule primordium of Sophora flavescens inoculated by different rhizobia (Jiao et al. 2015a). (a) Uninoculated root hairs at 6 days. (b and c) Root hairs, 6 days after inoculation (DAI) with Sinorhizobium fredii CCBAU 45436. (d and e) Root hairs, 6 DAI with Rhizobium yanglingense CCBAU 01603. (f) Aggregating cells of the lacZ-labelled Sinorhizobium fredii CCBAU 45436 surrounding the tip of a biforked root hair, 10 DAI. (g) Infection pocket formed by the lacZ-labelled R. yanglingense CCBAU 01603, 10 DAI. (h) Infection thread induced by lacZ-labelled Sinorhizobium fredii CCBAU 45436, 11 DAI. Bars: 40 μm in (a), (b), (d), (e) and (h); 20 μm in (c), (f) and (g)

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5 Determinate or Indeterminate Root Nodules

Two major types of nodules, indeterminate (Fig. 7.6a, b) and determinate (Fig. 7.6c, d), are found on the roots of legumes. Types of nodules are classified according to their mode of development. The indeterminate type is characterised by a persistent nodule meristem (Fig. 7.6b), while the determinate nodule type lacks such a meristem (Fig. 7.6d). Therefore, indeterminate nodules are often clavate and elongated, while determinate nodules are usually spherical and have limited size.

Fig. 7.6
figure 6

Determinate and indeterminate nodules. (a and b) Indeterminate nodules of Leucaena leucocephala; (c and d) determinate nodules of cowpea (Vigna unguiculata). (b) and (d) are nodule sections showing the dark red N2-fixing zone containing leghaemoglobin

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Determinate nodules are found on root of cowpea (Vigna unguiculata) (Fig. 7.6c, d), soybean (Glycine max), peanut (Arachis hypogaea), mungbean (Vigna radiata), common bean (Phaseolus vulgaris), Lotus japonicus, etc. Legumes having indeterminate nodules include alfalfa (Medicago sativa), Medicago truncatula, white clover (Trifolium repens), red clover (Trifolium pratense), common vetch (Vicia sativa), broad bean (Vicia faba) and Leucaena leucocephala (Fig. 7.6a, b), pea (Pisum sativum) and Sophora flavescens (Jiao et al. 2015a, d). The type of nodule is determined by the host plant, not by the rhizobial strain (itself). The phenomenon is well demonstrated by the observation of root nodules of S. flavescens that only indeterminate nodules are formed regardless of the different rhizobial strains originally isolated from determinate nodule-forming legumes or the indeterminate nodule-forming legumes (Jiao et al. 2015a, d). Club-shaped pseudonodule, that no rhizobial bacteroid inside the nodule, could produce by infection of wild-type Bradyrhizobium diazoefficiens (formerly Bradyrhizobium japonicum) USDA 110 on S. flavescens (Liu et al. 2018b). Also nodule could initialise and develop induced only by the existence of Nod factor, lipooligosaccharide (LCO) (Stokkermans and Peters 1994). Therefore, the initiation of nodule primordium could be independent on the alive rhizobia but is dependent on LCO of rhizobia (D’Haeze and Holsters 2002). Another study showed that even without the LCO as well as rhizobia, the mutants of Lotus japonicus could form spontaneous nodules (Tirichine et al. 2006).

The biology of these two types of nodules has been fully described in a previous Tansley review (Hirsch 1992). Readers could refer this review for the comparison and differences of these two types of nodules.

6 Swollen or Non-swollen Bacteroids

Inside the legume nodule cells, rhizobia differentiate into N2-fixing bacteroids which are physiologically and morphologically different from the free-living bacteria. Two kinds of bacteroids can be recognised: terminal or swollen bacteroids and nonterminal or non-swollen bacteroids. The size and shape of non-swollen bacteroids are similar to those of free-living bacteria, and these bacteroids can reproduce when they are re-isolated from the nodule cells. On the other hand, the swollen or terminal bacteroids have lost their reproductive capacity and no longer divide normally when they are re-isolated from the nodules (Zhou et al. 1985).

Typically, the differentiation of bacteroids to swollen or non-swollen types is determined by the host plant. Swollen/terminal bacteroids are observed usually in nodules of pea (Pisum sativum) (Oono and Denison 2010), alfalfa (Medicago sativa), peanut (Arachis hypogaea) (Fig. 7.7a, c) and clover (Trifolium repens) (Zhou et al. 1985). Branched, “Y”-shaped terminal bacteroids are found in pea, and spherical bacteroids (Fig. 7.7c) are found in peanut. Non-swollen/nonterminal bacteroids are observed in nodules of soybean (Glycine max) (Zhou et al. 1985), bean (Phaseolus vulgaris), cowpea (Vigna unguiculata) (Oono and Denison 2010) and Sophora flavescens (Fig. 7.7b, d).

Fig. 7.7
figure 7

Nodule section and transmission electron microscope of ultrathin nodule sections photographed by Xiang-Fei Meng. General light microscope of nodule sections of Arachis hypogaea (a) and Sophora flavescens (b). Transmission election microscope of ultrathin nodule sections of Arachis hypogaea (c) and Sophora flavescens (d). Bac bacteroid, PBS peribacteroid space, PBM peribacteroid membrane, PP electron-opaque polyphosphate inclusions, MZ meristem zone, NF nitrogen fixation zone, PHB poly-β-hydroxybutyrate

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Swollen bacteroids have higher fixation efficiency as well as greater benefit to the legume host than non-swollen ones (Florian et al. 2018; Oono and Denison 2010).

7 Concluding Remarks and Perspectives

In this chapter, we have reviewed the diversity of interaction between rhizobia and legumes. Some rhizobia, like NGR234, can nodulate more than a hundred genera of legumes. However, this strain cannot nodulate some common leguminous crops, such as soybean, peanut, alfalfa and chickpea. Chickpea selects only Mesorhizobium with a specific nodC gene as its effective microsymbionts. In contrast, Sophora flavescens is an extremely promiscuous legume and can nodulate with different genera of rhizobia in the Alphaproteobacteria and Betaproteobacteria. The infection pattern, nodule and bacteroid development are also diverse in different symbioses.

With further investigation of the relationship between rhizobia and legumes, more novel rhizobia will be discovered. The range of symbiotic relationships among them may become more complex, but it will be illuminated more clearly.