Plant environments provide a diversity of ecological niches for microorganisms including
rhizobia, plant growth-promoting microbes, and pathogens. Among them, rhizobia have
been extensively studied for their dynamically-changing genome structures, polyphasic
interactions with host plants, and biogeochemical functions as representative plant-associated
microbes. Here, rhizobia are collectively termed as nodule-forming N2-fixing bacteria,
including the genera Rhizobium, Bradyrhizobium, Mesorhizobium, Ensifer (Sinorhizobium),
and Azorhizobium.
Rhizobial genes for nodulation (nod) and nitrogen fixation (nif) appear to be acquired
by genomes using lateral gene transfer. Recent studies provided several lines of evidence
for the horizontal transfer of symbiosis islands, which is a type of adaptation process
to host legumes from soil bacteria (5, 21, 46). Kasai-Maita et al. (15) demonstrated
the dynamics of symbiosis islands in three strains of Mesorhizobium loti: the integration
of symbiosis islands into a phenylalanine-tRNA gene and subsequent genome rearrangement.
Evidence for the horizontal transfer of symbiotic genes was also found in the phylogenetic
relationships of the nodC and 16S rRNA genes of hairy vetch rhizobia (54) and by the
presence of identical nodD and nifD sequences in the Bradyrhizobium and Ensifer species
of Afghanistan isolates from soybean nodules (4). Thus, it is widely accepted that
the horizontal transfer of symbiosis islands and genes frequently occurred between
rhizobia and other soil bacteria.
Bradyrhizobium sp. DOA9, a non-photosynthetic bacterial strain originally isolated
from the root nodules of Aeschynomene americana, efficiently nodulates on the roots
of many leguminous plants. The genome is composed of a single chromosome and single
megaplasmid (pDOA9) with symbiotic genes (31, 49), which is less common than the genome
structures of many other symbiotic bradyrhizobia (13, 14). Okubo et al. (34) compared
the nifDK gene sequences of rhizobial and non-rhizobial Bradyrhizobium strains in
order to examine the evolutionary history of nif genes in the genus Bradyrhizobium,
and suggested that the nif genes on symbiosis islands were forced to reduce GC contents
with higher substitution rates than the ancestral sequences. On the other hand, the
nifDK genes on the megaplasmid pDOA9 were derived from the non-symbiotic loci of Bradyrhizobium
with similar evolutionary rates to the ancestral sequences. The low GC pressure in
nif genes on symbiosis islands may be related to the evolutionary processes of symbiotic
bradyrhizobia through associations with plants.
Whole-genome sequencing and post-genomic studies on rhizobia have facilitated our
understanding of their lifestyle and strategies to adapt to environmental conditions.
The symbiotic systems are regulated by many environmental cues, such as legume host
flavonoids (47), plant hormone regulators (50), temperature (43), CO2 concentrations
(45), and rhizobial systems, including sigma factor (27) and cell division and differentiation
(9, 29). The distribution patterns of bradyrhizobial species and genotypes appear
to be associated with geographic locations (43) and soil types (42) in Japan, which
are more likely explained by the capabilities of anaerobic nitrate respiration (38,
44) and uptake hydrogenase (24).
Recent investigations have focused on the interactions between non-rhizobial bacteria
and plants. For example, the inoculation of specific bacteria into plant seedlings
has been shown to promote the growth of a number of plants, such as potato, rice,
and cacao. Tchinda et al. (48) isolated many Actinobacteria stains from cacao pods,
and evaluated the promotion of plant growth with their siderophore production and
biosynthesis of 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indole-3-acetic
acid (IAA). These findings suggested that the Actinobacteria strains colonizing cacao
pods function as plant health agents. A similar approach was conducted for Novosphingobium
strains to optimize rice cultivation (36). N2-fixing Novosphingobium strains were
isolated from rice plant tissue and their effects on the promotion of plant growth
were tested under nitrogen-free conditions. The selected strains of Novosphingobium
effectively colonized within rice plant interiors and consequently promoted its growth.
Not only the function of a single bacterial strain, but also the synergetic functions
of different bacterial species for the promotion of plant growth have been studied
(22, 39). Bacterial strains from potato roots and tubers were initially tested in
order to establish whether they produced plant growth-promoting substances or had
positive or negative effects on plant growth (39). The co-inoculation of two different
bacterial species exerted stronger effects on plant growth than the inoculation of
any single species, suggesting that the synergetic functions of multiple strains were
more effective on plant-bacteria interactions than those of a single specific strain
(39).
Although plant-pathogenic bacteria cause significant damage to agricultural production,
some endophytic bacteria protect against pathogenic infections and subsequent disease
expression. Hassan et al. (6) clearly showed that the endophytic colonization of Streptomyces
humidus MBCN152-1 in cabbage plug seedlings increased host plant weight and protected
against disease expression caused by Alternaria brassicicola, with the percentage
of diseased seedlings becoming less than 10% with, but approximately 40% without the
inoculation of strain MBCN152-1. Hieno et al. (7) investigated the molecular mechanisms
of action of endophytic bacteria against the possible pathogen Pseudomonas syringae
pv. tomato DC3000 (Pst) in Arabidopsis thaliana. They demonstrated that the MYB44
gene of Penicillium, a transcription factor and stomata-specific enhancer of the ABA
signal for the stomatal closure of Arabidopsis thaliana, appeared to function by preventing
the penetration of pathogens through stomata, which is one of the mechanisms protecting
against plant diseases.
Recent studies on plant-associated bacteria have been depending more significantly
on culture-independent omic analyses of microbial ecologies than on conventional cultivation-based
techniques (8, 12, 28). Consequently, plants and their microbiota may be regarded
as holobionts, which embrace multiple plant-microbe and microbe-microbe interactions
(3, 37, 53).
Rice is one of the most important cereal crops in the world and is grown mainly in
flooded paddy fields. Important biogeochemical processes including the emission of
methane, a greenhouse gas, occur actively in paddy rice environments, and the rhizosphere
in a paddy field is considered to be a hot spot for the various inorganic redox reactions
of carbon, sulfur, and nitrogen compounds (17). Thus, the microbiomes of paddy rice
play an important role in carbon, sulfur, and nitrogen biogeochemical cycles (17).
An early metagenome analysis indicated that rice shoot microbiomes were dominated
by members of Alphaproteobacteria (51–52%), Actinobacteria (11–15%), Gammaproteobacteria
(9–10%), and Betaproteobacteria (4–10%) (32). Members of the uncharacterized phylum
Planctomycetes were also abundant in leaf sheaths (11). Shoot microbiomes harbored
more abundant genes for C1 compound metabolism and ACC deaminase than the rhizosphere
microbiome (32). In contrast, the root microbiomes of paddy rice were significantly
influenced by different environmental conditions, such as nitrogen fertilizer amendments
(10, 41), atmospheric CO2 concentrations (33), rice growth stages (33), temperature
(33), and rice genotypes (26). New findings have been obtained from these metagenomic
studies. A rice symbiotic gene (OsCCaMK), relevant to rhizobial nodulation and mycorrhization
in plants, appeared to function in the accommodation of N2-fixing methanotrophs in
root tissues under low-N fertilizer management conditions, which may lead to nitrogen
utilization by host plants via bacterial N2 fixation (26). Thus, CH4 oxidation and
methanotrophs are considered to be a driving force for shaping bacterial communities
in rice roots in CH4-rich environments (26). Amplicon sequence analyses of the 16S
rRNA gene indicated that rice root microbiomes responded to Azospirillum sp. B501
inoculations (2) and sulfur amendments (23). The abundance of uncharacterized phylum
™7 members in rice roots was increased by sulfur amendments (23). In addition, an
inoculation experiment of non-photosynthetic Bradyrhizobium sp. strain SUTN9-2 indicated
that the type III Secretion System (T3SS) of the bacterium is one of the key mechanisms
for endophyte colonization in rice roots (35).
Root microbiomes have also been characterized by 16S rRNA gene sequencing in other
plants, including sugar beet (30, 52), Arabidopsis grown under different conditions
of nitrogen availability (18), potato genotypes resistant and susceptible to S. turgidiscabies-induced
disease (16), and the healthy garden plant, Anthurium andraeanum (40). In A. andraeanum,
the different tissues of the leaf, stem, root, spathe, and spadix had often specific
microbiomes (40). By amplicon sequencing of the 16S rRNA gene, Lee et al. (19, 20)
compared the microbial community composition in soil in which tomato plants were planted
with and without Ralstonia solanacearum wilt symptoms, and suggested that several
genera of components (e.g., Hephaestia, Azospirillum, Dyella, and Choloroflexi) may
contribute to suppressing the soil-borne pathogens of bacterial wilt. In a metagenome
analysis, Minami et al. (25) found that Methylobacterium species dominated in the
shoot microbiomes in soybean plants. A functional gene analysis also indicated the
abundant occurrence of genes for urea degradation, such as the urease of Methylobacterium
species (25). This study demonstrated that ureide may serve as an important nitrogen
source of shoot-associated microbes even though it is a key substance of fixed nitrogen
transportation from legume nodules to shoots.
We would like to introduce some of the future perspectives in plant microbiome research
to further address community-level functions. New experimental approaches have recently
been developed for plant microbiome research: synthetic engineering approaches to
plant microbial communities in gnotobiotic systems (1, 53) and informatics approaches
to the identification of “hub microbes” (51, 53). The integration of these new approaches
with conventional techniques and knowledge as described herein will open a new dimension
of plant microbiomes and their application to agriculture.