(This chapter has been published in Taiwania 68: 7-15, 2018. DOI information:
10.6165/tai.2018.63.7)
Abstract
Legume-rhizobia symbioses of seven leguminous species growing along Xindian riverbank of Northern Taiwan were investigated in this study. These legumes form either determinate or indeterminate types of root nodules. The determinate nodules of Alysicarpus vaginalis, Desmodium. triflorum, D. heterophyllum, Sesbania cannabina
and the indeterminate nodules of Mimosa pudica harbored bacteroids of morphological uniformity (length of 1-3 μm), while the indeterminate nodules of Crotalaria zanzibarica and Trifolium repens contained bacteroids of highly pleomorphism (size
varying from 1 to 5 μm). The enclosed bacteria were isolated from respective nodules,
and twenty slow-growing and nine fast-growing rhizobial isolates were recovered. The slow-growing isolates were classified to the genus Bradyrhizobium based on the 16S rRNA sequences, whereas the fast-growing rhizobia comprise four genera, Neorhizobium, Rhizobium, Cupriavidus and Paraburkholderia. Results of stable isotope
analyses revealed that the seven leguminous species had similar and consistently negative δ15N values in leaves (mean of -1.2 ‰), whereas the values were positive (varying from 3.7 to 7.3 ‰) in the nodules. These values were significantly higher in the
indeterminate nodules than those in the determinate ones. In addition, variations in the values of leaf δ13C (varying from -29 to -34‰) among the seven legumes were
measured, indicating their photosynthetic water use efficiencies were different. This is the first field survey to report the rhizobial diversity and the nutrient relationships of sympatric legume in Taiwan.
Introduction
Nitrogen, one of the most important nutrients for plant growth and reproduction, is often limited in the ecosystems (Vitousek and Howarth, 1991). Some plants evolved symbiosis with bacteria capable of nitrogen fixation and overcame the limitation.
Consequently, the nitrogen availability and the primary productivity of the ecosystems are improved by the symbiotic activity (Vitousek et al., 2002). Because the interaction plays an important role in affecting primary productivity and is of great application in agriculture, the symbiotic relationship has received much attention from the researchers worldwide.
Most leguminous plants are capable of fixing atmospheric N2 via symbiosis with rhizobia (Sprent, 2001, 2009). There were 217 legume species, including 148 native species and 59 exotic species, recorded in Taiwan (Huang, 1993; Wu et al., 2003).
Despite the fact that legume is one of the largest plant families in Taiwan (Huang, 1993) and the importance of the symbiotic relationship in contribution to the nitrogen availability and primary productivity of ecosystems, the bacteria symbionts have been investigated only in few species of leguminous plants in Taiwan (Chen et al., 2000, 2003, 2005; Chen and Lee, 2001; Hung et al., 2005; Huang et al., 2016). Field survey of root-nodulating rhizobia and concomitantly measurements of nutrient of sympatric
leguminous species are lacking in Taiwan.
Xindian River is located in Northern Taiwan. The soil along riverbank is mainly sandy and frequently subjected to disturbances, such as flooding and human activity.
Despite this, leguminous plants are prevalent in this habitat. The nitrogen-fixing symbiosis between the leguminous plants and the soil rhizobia might provide these plants competitive advantages in the nitrogen-poor and arid habitats. Seven leguminous species were commonly observed in this area, including three native species (Alysicarpus vaginalis, Desmodium triflorum and D. heterophyllum) and four exotic species (Crotalaria zanzibarica, Mimosa pudica, Sesbania cannabina and Trifolium repens) (Table 1). These species belong to distantly related legume groups, including
Genistoids (C. zanzibarica), Milletioids (A. vaginalis, D. triflorum, and D.
heterophyllum), Robinioids (S. cannabina), Inverted Repeat-lacking clade (T. repens)
and Mimosoids (M. pudica) (Lewis et al., 2005; Sprent, 2007). In addition, the geographic origins of these legumes are also disparate. A. vaginalis, D. triflorum, and D.
heterophyllum are native species in Taiwan, while C. zanzibarica, S. cannabina, T.
repens and M. pudica, are exotic and originated from Africa, Asia, Europe and America,
respectively (Wu et al., 2003). Among the seven species, the bacterial symbionts of A.
vaginalis, C. zanzibarica, M. pudica and S. cannabina in Taiwan have been reported
(Chen and Lee, 2001; Chen et al., 2003; Hung et al., 2005; Huang et al., 2016) but not the other three species. However, the reported symbionts were mostly isolated from central and southern Taiwan, except that of C zanzibarica was recently isolated from a greenhouse in northern Taiwan (Huang et al., 2016). These phylogenetically distant legume species originated from disparate geographic sources in combination with the heterogeneous soil conditions of the riverbank might result in novel symbiotic properties.
Two major types of nodules are classified by their growth. Determinate nodules usually have a round shape and are short-lived (lasting for days to weeks), while indeterminate nodules may have few or many branches and last for several months (Sprent, 2001, 2007). Within the nodules, the rhizobia differentiate into nitrogen-fixing bacteroids. The morphology of bacteroids is either similar to or different from that of free-living bacteria (short rod, about 1μm long). For example, bacteroids in nodules of pea display pleomorphism, such as swollen, elongated, or branched (Mergaert et al., 2006), while those in nodules of soybean uniformly rod-shaped (Oono et al., 2009). The swollen bacteroids might optimize nitrogen-fixing efficiency (Oono and Denison, 2010).
Stable isotopes techniques have been widely used in ecological studies (Peterson
and Fry, 1987). The nitrogen isotope ratio (δ15N) of individual plants is often used to
assess the forms of nitrogen source, i.e. NO3-, NH4+ or N2 (Robinson, 2001). In general, foliar δ15N values of non-N2-fixing plants vary widely (could be positive or negative
values), while N2-fixing legumes often display consistently negative foliar δ15N values
(Virginia and Delwiche, 1982; Sprent et al., 1996). In contrast to the leaves, nodules of legume plants commonly have positive and variable δ15N values which might depend on
their nodule symbionts and reflect the nitrogen fixing activities (Shearer et al., 1982;
Steele et al., 1983; Wanek and Arndt, 2002). The carbon isotope ratio, δ13C, can be used to identify photosynthetic pathway. In addition, δ13C of C3 plants is a proxy of water
use efficiency (WUE) (Farquhar et al., 1982). Studies have shown that increases in N supply improve WUE hence enhance plant productivity (Brueck, 2008). It is also found that water use efficiency (WUE) was positively related to leaf nitrogen content for woody nitrogen fixing plants (Adams et al., 2016). The analyses of nitrogen and carbon isotopes could provide information of nitrogen sources and water use efficiency of the sympatric plants.
In this study, we analyzed 16S rRNA genes to classify the genus of rhizobial symbionts, examined nodule and bacteroid morphologies, and analyzed nitrogen and carbon contents and δ15N and δ13C values, of seven leguminous plants growing
sympatrically along the bank of Xindian River in northern Taiwan. The objective of the study is to understand the diversity of rhizobial symbionts associated with the seven leguminous plant and the nutrient and water relationships of the host plants.
Materials and methods
Sampling site and plant materials
Xiandan (XD) River is located in northern Taiwan. The dominant leguminous species, A. vaginalis, C. zanzibarica, D. triflorum, D. heterophyllum, M. pudica, S.
cannabina and T. repen (Figure 2-1 and Table 2-1) co-existing in an area about 10 ×
150 m along the XD riverbank (24°98′ N, 121°52′ E), was investigated in May 2013,
March and June 2014. Six individuals of each legume species were surveyed, and 1-2 nodules per individual were collected for rhizobial isolation. Mature leaves and all
available nodules were collected from additional individuals of the seven legumes species (4 to 9 individuals per species) for nitrogen and carbon contents, and δ15N and δ13C analyses. For comparison purpose, leaves of a non-legume species, Bidens pilosa
var. radiata, growing about 100 m away from the legume community were also analyzed.
Rhizobial isolation and bacteroid morphology
Fresh nodules were surface sterilized by immersion in 0.5% SDS for 1 min, then 70% ethanol for 5 min, and washed three times by sterile deionized-distilled water (DDW). Nodule suspension was obtained by crushing the nodule in DDW and spread onto yeast extract-mannitol (YEM) plate (Vincent, 1970). A single of putative rhizobium was isolated from each nodule sample and checked for the unity by repeated streaking on YEM plate. For observation of bacteroid morphology, the nodule
suspension were stained with DAPI (4,6-diamidino-2-phenylindole; Sigma-Aldrich, St.
Louis, MO, USA) at 50 μg/ml for 10 min at 25℃ and examined by a fluorescent
microscopy (BX51, Olympus, Tokyo, Japan).
Phylogenetic analysis of nodule symbionts by using 16S rRNA genes
Total genomic DNA was extracted from the pure cultures of each rhizobial isolates grown in YEM broth until the late exponential phase of growth. Extraction of the DNA was performed by using Geneaid DNA Mini kit (Geneaid Biotech, New Taipei, Taiwan).
PCR amplification of 16S rRNA genes were performed by using bacterial universal primer pairs, 27F-1492R (Marchesi et al., 1998) and Taq DNA Polymerase 2x Master Mix RED (Ampliqon, Copenhagen, Denmark). PCR products were first checked on
1.5% agarose gel and purified with Gel/PCR DNA fragments extraction kit (Geneaid Biotech, New Taipei, Taiwan). Sequencing reactions were performed by using the ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). The sequences assembled and quality checked were conducted by using BioEdit 7.2.5 (Hall, 1999).
To analyze the phylogenetic relationships between the isolates and defined rhizobial species, the 16S rRNA sequences of reference strains which are highly similar with the isolates were download from the NCBI GenBank database (https://www.ncbi.nlm.nih.gov/) based upon BLAST results. The 16S rRNA gene sequences of isolates and reference strains were then aligned by MUSCLE program as implemented in MEGA version 6 (Tamura et al., 2013). The Kimura’s 2-parameter distance correction model was used to reconstruct neighbor-joining (NJ) phylogenetic trees by software MEGA6. The topology of the tree was evaluated by bootstrapping with 1,000 replications. The GenBank accession numbers of the 16S rRNA gene sequences generated in this study are shown in Figure 2-3.
Nitrogen and carbon contents, and δ15N and δ13C values analyses
Leaf and nodule samples were washed with distilled water then dried at 60 °C in an oven for three days. Dried samples were ground to a homogenized powder with a
mortar and pestle. A 2 mg of ground material was loaded into a tin capsule for further analysis (Kao, 2010). Nitrogen content (Nmass, mg g-1) and carbon content (Cmass, mg g-1)
was determined with an elementary analyzer (FlashEA 1112 series, Thermo Fisher Scientific, Italy). Stable nitrogen isotope ratio (δ15N) was determined by an isotope ratio mass spectrometer (DeltaV Advantage, Finnigan Mat, Germany) and calculated as: δ15N (‰) = [(R sample / R standard) - 1]*1000, where R is the ratio of 15N to 14N. Stable carbon isotope ratio (δ13C) was calculated as: δ13C (‰) = [(R sample / R standard) - 1]*1000, where
R is the ratio of 13Cto 12C (Ehleringer and Osmond, 1989).The standards for δ15N and δ13C are atmospheric N2 and Pee Dee Belemnite, respectively.
Statistical analysis
To determine whether variables (leaf/nodule N, C contents, δ15N and δ13C values) were significantly different among seven legumes and B. pilosa, one way analysis of variance (ANOVA) was conducted by using the software SAS 9.4 (SAS inst. Inc. USA).
If the null hypothesis was rejected after the analysis of ANOVA, then SNK (Student-Newman-Keuls) test was used for multiple comparisons.
Results
Nodule types and morphology of bacteroids
Fig. 1 shows the morphology of nodules collected from field-growing legumes. A.
vaginalis, D. triflorum, D. heterophyllum and S. cannabina formed determinate nodules
(Figure 2-2A-D), while T. repens, M. pudica and C. zanzibarica produced indeterminate nodules with no branch, few branches and many branches, respectively (Figure 2-2E-G). In addition, the lenticels (as white stripes) were observed on the nodules of A. vaginalis, D. triflorum, D. heterophyllum (Figure 2-2A-C).
Bacteroids isolated from the nodules of A. vaginalis, D. triflorum, D.
heterophyllum, S. cannabina and M. pudica displayed morphological uniformity with
1-3 μm in length (Fig. 1H-K and M). In contrast, those of C. zanzibarica, and T. repens were highly pleomorphic (included rod-shaped, branched and club-like cells) and varied in size from 1 to 5 μm (Figure 2-2L and N).
16S rRNA gene phylogeny of rhizobial isolates
A total of 29 putative rhizobial isolates, 3 from A. vaginalis, 6 from C. zanzibarica, 6 from D. triflorum, 5 from D. heterophyllum, 4 from M. pudica, 2 from S. cannabina and 3 from T. repens, were recovered from the nodules of the seven legume species. The isolates from A. vaginalis, C. zanzibarica, D. triflorum, and D. heterophyllum displayed
slow-growing phenotype, forming visible colonies on YEM plates after 5-7 days at 30℃.
In contrast, the isolates from M. pudica, S. cannabina and T. repens formed detectable colonies after 1-2 days, indicating that they were fast-growing strains.
The 1,300 bp of 16S rRNA gene sequences were used to analyze the relationship among the 29 isolates and defined genus of the strains (Figure 2-3). These isolates were separated into two distinct groups, belonging to alpha- and beta-rhizobia. In alpha-rhizobia group, a total of 20 isolates from A. vaginalis, C. zanzibarica, D.
triflorum and D. heterophyllum were situated within genus Bradyrhizobium. One isolate,
ScHDB, from S. cannabina belongs to Neorhizobium, a novel genus recently been separated from Rhizobium (Mousavi et al. 2014). Additionally, one isolate from S.
cannabina (ScHDA) and three isolates from T. repens (TrHDA, TrHD1 and TrHD2)
were grouped together with Rhizobium strains. In beta-rhizobia group, four isolates from M. pudica are closely related to Cupriavidus taiwanensis (MpHDA), Paraburkholderia phymatum (MpHD2) and P. mimosarum (MpHD and MpHD3),
respectively (Figure 2-3). The latest two species have been recently separated from genus Burkholderia and reclassified as members of the Paraburkholderia (Sawana et al.
2014).
Nitrogen and carbon contents, δ13C and δ15N analyses
Table 2-2 shows the nitrogen content and δ
15N values of nodules and leaves.Variations in nodule and leaf N contents and δ15N of nodules were found among the seven leguminous plants, but similar δ15N values were found in their leaves. In
comparison of nodules and leaves of the same plants, in general, nodules had higher N contents and more positive δ15N values than leaves. In comparison to legume plants, B.
pilosa had significantly more positive and variable leaf δ15N values, ranging from
-0.3‰ to +2.1‰.
The carbon and δ13C contents of nodules and leaves are shown in Table 3. All
legume plants had similar C content in the nodules (about 43%), while their leaf C content varied from 42% to 47%. In each of the legume species, nodules had
significantly more positive δ13C values than leaves, the enrichment was approximate 1.5‰. The relationship between leaf and nodule δ13C values among seven legume
species was significantly positive (Figure 2-4).
Discussion
Few rhizobial species have been isolated from Taiwan soils in previous studies (Chen et al., 2000, 2003, 2005; Chen and Lee, 2001; Hung et al., 2005; Huang et al.
2016). However, the natural diversity of nodulating bacteria in field growing, sympatric leguminous species has not been reported in Taiwan. This is the first study conducted to explore the diversity of nodulating rhizobia associated with sympatric leguminous plants in the field of Taiwan and the results provide rhizobial diversity at genus level.
The survey recovered five genera of nodulating bacteria, Bradyrhizobium, Neorhiobium, Rhizobium, Cupriavidus and Paraburkholderia, associated with seven sympatric
leguminous species growing along the riverbank of northern Taiwan. Most of isolates (20 out of 29) in our survey belong to the genus Bradyrhizobium (Figure 2-3). The result confirms previous report that Bradyrhizobium is the most abundant and prevalent rhizobial genus contributing to the major symbiont of tropical and sub-tropical legume taxa (Sprent, 2007, 2009). Strains of Bradyrhizobium were isolated from the nodules of A. vaginalis, C. zanzibarica, D. triflorum and D. heterophyllum (Figure 2-3). A more
precise classification of these strains into discrete species is hampered by the exceptional conservation of the 16S rRNA gene sequence in the genus Bradyrhizobium (Rivas et al., 2009; Azevedo et al., 2015). Among the Bradyrhizobium spp. reported in Taiwan, B. arachidis was recently isolated from the nodules of C. zanibarica grown in a greenhouse (Huang et al., 2016) and B. japonicum from nodules of A. vaginalis growing in central Taiwan (Hung et al., 2005). Strains associated with D. heterophyllum and D.
triflorum in Taiwan have not been reported. To reveal the species identities of these
isolates, analyses combined multiple housekeeping genes are currently undertaken.
Rhizobium strains were isolated from S. cannabina and T. repens in this study
(Figure 2-3). Chen and Lee (2001) reported that S. cannabina growing in the southern part of Taiwan were nodulated by Rhizobium and Sinorhizobium (Ensifer) strains.
Although we did not identify any Sinorhizobium strain in the nodules of S. cannabina, we isolated a Neorhizobium strain (Figure 2-3). These results revealed that S.
cannabina can establish symbiosis with rhizobia of Rhizobium, Neorhizobium and
Sinorhizobium in Taiwan. In addition, there was no report with respect to rhizobia
establish symbiosis with T. repens in Taiwan, but Rhizobium is known to establish symbiosis with T. repens in China (Liu et al., 2007). Among the seven legume species investigated, M. pudica, an invasive plant in Taiwan, is the only species that establishes symbiosis with beta-rhizobia (Figure 2-3). This plant is known to establish symbiosis with both Cupriavidus and Paraburkholderia strains in Taiwan (Chen et al., 2003), while its nodule symbionts were restricted to Paraburkholderia in the native regions (Barrett and Parker, 2005). In consistent with the previous report by Chen et al. (2013), the isolates from M. pudica growing along Xindian riverbank had highly similar 16S rRNA gene sequences with several beta-rhizobia, including Cupriavidus taiwanensis,
Paraburkholderia phymatum and P. mimosarum (Figure 2-3). The results revealed that
these genera of beta-rhizobia co-existed in this habitat and only established symbiosis with M. pudica but not with other 6 species of legume.
Morphologies of nodule and bacteroid are two of the most noticeable traits in legume-rhizobia symbiosis. These traits are related to the evolution of the symbiosis.
For example, indeterminate nodules and non-swollen bacteroids are considered ancestral traits, while determinate nodules and swollen bacteroids are derived (Doyle, 2011; Oono et al., 2010). In this study, the seven legume species formed either indeterminate or determinate nodules. Within each group of the legume, formed either indeterminate or determinate nodules, plants were nodulated by phylogenetically distant rhizobia (Table 2-1). This result confirms that the formation of nodule types is dependent on the host plant not on the specific rhizobia (Oono et al., 2010). The relationship between the nodule types and the morphologies of the enclosed bacteroids are not discreet, the determinate nodules of A. vaginalis, D. triflorum, D. heterophyllum, S. cannabina harbored exclusively non-swollen bacteroids, while the indeterminate
nodules of C. zanzibarica and T. repens harbored swollen but that of M. pudica harbored non-swollen bacteroids (Table 2-1). It is reported that the morphology of the bacteroids was determined by host plants (Haag et al., 2013)
As shown in Table 2-2, the 7 leguminous species had similar and consistently negative foliar δ15N and their foliar δ15N differed significantly from that of the
non-N2-fixing B. pilosa var. radiata (with variable foliar δ15N), strongly suggested that these leguminous plants depend on the same nitrogen source (from atmospheric N2) differing from the sources (NH4 and/or NO3 in soil) utilized by the non-N2-fixing plant.
These legume with symbiotic bacteria in root nodules can fix atmospheric nitrogen (N2), and this would give them an advantage in low soil nitrogen (N) habitats. Since the soil of riverbank is commonly nutrient-poor, and this result might explain the prevalence of legume plants along the bank of Xindian River. In contrast to the slightly depletion of
15N in their leaves, the seven legumes surveyed in this study all showed 15N enrichment
in their nodules (Table 2-2), regardless induced by distinct rhizobial symbionts (fast- or slow-growing rhizobia). Though Bergersen et al. (1986) reported that slow-growing rhizobial strains and fast-growing strains induces Lupinus plants produced 15N enriched nodules and little or no 15N enriched nodules, respectively. Explanations for the enrichment of 15N in nodules have been suggested, including denitrification in nodules preferentially releasing 14N (Shearer et al. 1980), importation from phloem of 15N enriched amino acids into nodules (Bergersen et al. 1988), exported of 15N depleted ureide from nodules (Shearer et al. 1982), or diffusion of NH3 from bacteroids causing
discrimination (Yoneyama et al. 1991). However, mechanism(s) causing the phenomenon have not been revealed unequivocally. The symbiotically fixed nitrogen can be assimilated and exported via aminde or ureide pathway depending on host species (Sprent, 2001). Among the seven leguminous species C. zanzibarica, M. pudica, S. cannabina and T. repens are aminde exporters while A. vaginalis, D. triflorum and D.
heterophyllum are ureide exporters (Sprent, 2001). Even so, no significant difference
was found neither in the nodule δ15N values nor in the leaf δ15N values between the two groups. Interestingly, the mean δ15N value of the indeterminate nodules of C.
zanzibarica, M. pudica and T. repens was significantly higher than that of the
determinate nodules of A. vaginalis, D. triflorum, D. heterophyllum and S. cannabina (Table 2-2). It is possible that 15N of nodule is also affected by nodule age.
Accordingly, indeterminate nodules, with longer life span, might accumulate more 15N thus resulting in higher 15N values than determined nodules.
The δ13C values of leaves of the sympatric legume varied from -29‰ to -34‰
(Table 2-3), indicating the legume species sampled in this study belong to C3 plants (O’Leary, 1988). The leaf δ13C values in C3 plants is known to reflect the ratio of
intercellular to ambient concentration of CO2 (Ci/Ca), which is affected by both stomatal conductance (CO2 diffusion) and photosynthesis (CO2 consumption) (Farquhar
et al., 1982). Hence, leaf δ13C value in C3 plants is often used as a proxy of photosynthetic water use efficiency (Farquhar et al., 1982). The more positive δ13C value showed the higher WUE. Variation in δ13C values of the seven leguminous indicates that they had different WUE. In addition, five of the seven leguminous plants had significantly more positive 13C values than their neighbor, the non-legume B.
pilosa var. raidiata (Table 2-3). Water use efficiency was found positively related to
leaf N content (on leaf area basis, Narea) for woody nitrogen fixing plants (Adam et al., 2016). The seven legumes also had significant differences in leaf N content (on dry weight basis, Nmass). However, because we did not measure specific leaf area thus cannot convert the Nmass (measured in this study) into Narea. Therefore, we cannot tell whether the differences in δ13C values of the leguminous species were attributed by their differences in leaf Narea. The significantly positive linear relationship between leaf δ13C and nodule δ13C (Figure 2-4) provides the evidence that nodule derived C from
leaf N content (on leaf area basis, Narea) for woody nitrogen fixing plants (Adam et al., 2016). The seven legumes also had significant differences in leaf N content (on dry weight basis, Nmass). However, because we did not measure specific leaf area thus cannot convert the Nmass (measured in this study) into Narea. Therefore, we cannot tell whether the differences in δ13C values of the leguminous species were attributed by their differences in leaf Narea. The significantly positive linear relationship between leaf δ13C and nodule δ13C (Figure 2-4) provides the evidence that nodule derived C from