**3. Plants shape the microbial community**

Microbial interactions play a crucial role in plant community ecology and performance. How do plants harbor unique microbial communities? How do plants shape a unique rhizosphere microbial community? These are the questions that must be addressed. Modern genomic technologies (e.g., high throughput sequencing) can provide clues to the answer. Lundberg et al. attempted to define the core *Arabidopsis thaliana* root microbiome [64]. They collected more than 600 *A. thaliana* plants and compared the bacterial communities using 16S rRNA gene sequencing. They observed that the root microbial communities of plants are sufficiently dependent on the host genotype to vary between inbred *A.* accessions. However, the mechanisms were not clear but included differences in the host physiology and immune responses.

### **3.1. Plant genes responsible for defense affect the variation of the microbial community**

Several studies showed that plant genotype has a small but significant effect on the composition of the endophytic, rhizosphere, or phyllosphere microbial communities [17, 64–67]. A quantitative trait locus (QTL) analysis and a genome-wide association study (GWAS) were used to identify taxa linked to host genes in humans, mice, plants, and flies [17, 68–70]. A GWAS of the *A. thaliana* leaf microbial community suggested that the *A. thaliana* loci are responsible for defense and that cell wall integrity affected the composition of this community [17]. Furthermore, host genetic variation shaped species richness in the bacterial community. Horton et al. showed that defense response associated genes against pathogens shaped this microbial ecosystem. In Matthew's study, 196 accessions of *A. thaliana* were sown in a greenhouse and transferred to a field site. The field experiment data suggested that the plant tissue structure (i.e., the cell wall integrity) might affect the leaf microbial community. To understand the plant host genetic factors that affect the associated microbial population, Bodenhausen et al. used a candidate gene approach to investigate the host effects on the composition and abundance of the *A. thaliana* phyllosphere community [67]. A panel of 55 *A. thaliana* mutants with alterations in the cell wall, surface structure, defense signaling, secondary metabolism, and pathogen recognition was constructed to reveal the effect on the microbiota composition and/or abundance in a small number of single host mutations. The results showed that *lacs* and *pec1* mutants affected cuticle formation, which led to an increased bacterial abundance and community composition. Moreover, the ethylene signaling gene *ein2* was observed to be a host factor that modulated the community composition. Peiffer et al. also noted that approximately 19% of the interline variation in species richness could be attributed to the host genotype in different maize lines grown in the same geographical regions [65].

### **3.2. Role of root exudates in shaping rhizosphere microbial community**

More than a century ago, Lorenz Hiltner defined the term "rhizosphere" as the soil compartment affected by plant roots [71]. Soil microbes are chemotactically attracted to plant root exudates, volatile organic carbon, and rhizodeposition, and then proliferate in this carbon-rich environment [72]. Plant root exudates differ between plant species, so differences in rhizosphere microbiomes of different plant species are expected [73]. More recent studies have provided strong evidence for plant species-specific microbiomes [74, 75]. Plants can also shape the microbial community via root exudates. Root exudates can be categorized as sugars, amino acids, organic acids, nucleotides, flavonoids, antimicrobial compounds, and enzymes [4, 73].

### *3.2.1. The types of root exudate*

their symbionts inhibit the ability of the native symbiotic community to acquire resources, indirectly reducing the performance of native plants [60, 61] (**Figure 1**). Positive feedback might be exemplified by the enhanced growth and survival of exotic seedlings near native established symbionts [62, 63]. Plant monodominance, coexistence, and invasion ecology

Microbial interactions play a crucial role in plant community ecology and performance. How do plants harbor unique microbial communities? How do plants shape a unique rhizosphere microbial community? These are the questions that must be addressed. Modern genomic technologies (e.g., high throughput sequencing) can provide clues to the answer. Lundberg et al. attempted to define the core *Arabidopsis thaliana* root microbiome [64]. They collected more than 600 *A. thaliana* plants and compared the bacterial communities using 16S rRNA gene sequencing. They observed that the root microbial communities of plants are sufficiently dependent on the host genotype to vary between inbred *A.* accessions. However, the mechanisms were not clear but included differences in the host physiology and immune responses.

**Figure 1.** Plant soil community feedback in low diversity communities (modified after Bever et al. [16]).

**3.1. Plant genes responsible for defense affect the variation of the microbial community**

Several studies showed that plant genotype has a small but significant effect on the composition of the endophytic, rhizosphere, or phyllosphere microbial communities [17, 64–67]. A quantitative trait locus (QTL) analysis and a genome-wide association study (GWAS) were

have high relation to symbiotic microbial interactions.

96 Plant Ecology - Traditional Approaches to Recent Trends

**3. Plants shape the microbial community**

### *3.2.1.1. Organic acids and amino acids*

The composition of root exudates from different cultivars affects the growth of soil-borne pathogens. The susceptible peanut cultivar Ganhua-5 (GH) and the mid-resistant cultivar Quanhua-7 (QH) were chosen for a root exudate analysis and evaluated for the responses of the soil-borne pathogens *Fusarium oxysporum* and *Fusarium solani* [76]. The contents of total amino acids, alanine, and sugars in the root exudate of susceptible cultivars were significantly higher than in the mid-resistant cultivar, whereas the contents of total phenolic acids, p-hydroxybenzoic acid, benzoic acid, and p-coumaric acid were significantly lower than in mid-resistant cultivars. These differences in the root exudate composition of susceptible and resistant cultivars might be assumed to regulate the resistance mechanism in the peanut rhizosphere. However, the spore germination and mycelial growth of the soil-borne pathogens *F. oxysporum* and *F. solani* were significantly enhanced by treatment with the root exudates from both the susceptible and mid-resistant cultivars compared with a control. If root exudates do not directly inhibit the growth of pathogens, the effects of other factors must be considered. A previous report showed that organic acids modulated the colonization and enhanced the biofilm formation of the root microbiome. Yuan et al. demonstrated that organic acids from banana root exudates facilitated the root colonization by *Bacillus amyloliquefaciens* [77]. Fumaric acid significantly induced biofilm formation, whereas malic acid evoked the greatest chemotactic response. The results showed that organic acids from banana root exudates played a crucial role in attracting and initiating PGPR colonization on the plant roots. Rice exudates that primarily contained the amino acid residues of histidine, proline, valine, alanine, and glycine, and the carbohydrates glucose, arabinose, mannose, galactose, and glucuronic acid may induce a higher chemotactic response by the endophytic bacteria *Corynebacterium flavescens* and *Bacillus pumilus* [78].

### *3.2.1.2. Sugars*

The amount of sugar secretion might affect infection by plant pathogens. Gou et al. showed that the *Arabidopsis* vacuolar sugar transporter SWEET2 limited the Glc-derived carbon efflux from roots and inhibited *Pythium* infection [79]. They proposed that the expression of SWEET2 modulated sugar secretion, limiting the carbon loss to the rhizosphere. The reduction of available substrates in the rhizosphere contributed to the resistance to *Pythium*.

### *3.2.1.3. Antimicrobial compounds*

Root exudates can also participate in belowground plant defense. Low-molecular-weight antimicrobial chemicals can be divided into "phytoanticipins" and "phytoalexins" [80]. Phytoanticipins are defensive compounds that are constitutively produced and released by the plant root prior to a biotic stress such as pathogen infection. In a recent study, *Arabidopsis* roots deficient in diterpene rhizathalene A production were found to be more susceptible to insect herbivory [81]. Therefore, rhizathalene A was considered as a part of a constitutive direct defense system of the roots. Phytoalexins were defined as inducible defensive compounds that are not detected in healthy plants [80]. Five phenylpropanoid root-derived aromatic root exudates were induced by the attack of the soil-borne pathogen *Fusarium graminearum* and exhibited antifungal activity [82]. In general, root-secreted terpenoid and phenolic defensive compounds have strong antibacterial and antifungal activity [83, 84]. The largest class of plant defensive chemicals above- and below ground is terpenoids. Nonvolatile terpenoids can be secreted into the rhizosphere [85], and volatile organic compounds (VOCs) can be emitted from the roots as plant defensive compounds. Phenylpropanoids are a group of plant defensive phenolic root exudates. After a Fusarium graminearum infection, barley rapidly accumulated and secreted phenylpropanoids, which are cinnamic acid derivatives to resist a fungal attack [82]. Phenolic root exudates not only have antimicrobial activity but also beneficially attract soil-borne microorganisms that affect the native soil microbial community [86]. We have found that the same chemical compound, for example, the amino acid canavanine, can stimulate a specific group of microbes but suppress many other soil microbes. Plants can shape the specific rhizosphere microbial community via root exudates.

### *3.2.2. Environmental factors effects on root exudates*

Plants with different genotypes produce root exudates with different compositions. Abiotic and biotic factors also affect root exudates. Physico-chemical soil properties such as nutrient availability, organic matter content, pH, structure, and texture can affect the availability of root exudates and microbial recruitment by the plant roots. Some biotic factors such as soil microbial secondary metabolism can also affect the exudates.

### *3.2.2.1. Temperature*

peanut rhizosphere. However, the spore germination and mycelial growth of the soil-borne pathogens *F. oxysporum* and *F. solani* were significantly enhanced by treatment with the root exudates from both the susceptible and mid-resistant cultivars compared with a control. If root exudates do not directly inhibit the growth of pathogens, the effects of other factors must be considered. A previous report showed that organic acids modulated the colonization and enhanced the biofilm formation of the root microbiome. Yuan et al. demonstrated that organic acids from banana root exudates facilitated the root colonization by *Bacillus amyloliquefaciens* [77]. Fumaric acid significantly induced biofilm formation, whereas malic acid evoked the greatest chemotactic response. The results showed that organic acids from banana root exudates played a crucial role in attracting and initiating PGPR colonization on the plant roots. Rice exudates that primarily contained the amino acid residues of histidine, proline, valine, alanine, and glycine, and the carbohydrates glucose, arabinose, mannose, galactose, and glucuronic acid may induce a higher chemotactic response by the endophytic bacteria

The amount of sugar secretion might affect infection by plant pathogens. Gou et al. showed that the *Arabidopsis* vacuolar sugar transporter SWEET2 limited the Glc-derived carbon efflux from roots and inhibited *Pythium* infection [79]. They proposed that the expression of SWEET2 modulated sugar secretion, limiting the carbon loss to the rhizosphere. The reduction of avail-

Root exudates can also participate in belowground plant defense. Low-molecular-weight antimicrobial chemicals can be divided into "phytoanticipins" and "phytoalexins" [80]. Phytoanticipins are defensive compounds that are constitutively produced and released by the plant root prior to a biotic stress such as pathogen infection. In a recent study, *Arabidopsis* roots deficient in diterpene rhizathalene A production were found to be more susceptible to insect herbivory [81]. Therefore, rhizathalene A was considered as a part of a constitutive direct defense system of the roots. Phytoalexins were defined as inducible defensive compounds that are not detected in healthy plants [80]. Five phenylpropanoid root-derived aromatic root exudates were induced by the attack of the soil-borne pathogen *Fusarium graminearum* and exhibited antifungal activity [82]. In general, root-secreted terpenoid and phenolic defensive compounds have strong antibacterial and antifungal activity [83, 84]. The largest class of plant defensive chemicals above- and below ground is terpenoids. Nonvolatile terpenoids can be secreted into the rhizosphere [85], and volatile organic compounds (VOCs) can be emitted from the roots as plant defensive compounds. Phenylpropanoids are a group of plant defensive phenolic root exudates. After a Fusarium graminearum infection, barley rapidly accumulated and secreted phenylpropanoids, which are cinnamic acid derivatives to resist a fungal attack [82]. Phenolic root exudates not only have antimicrobial activity but also beneficially attract soil-borne microorganisms that affect the native soil microbial community [86]. We have found that the same chemical compound, for example, the amino acid canavanine,

able substrates in the rhizosphere contributed to the resistance to *Pythium*.

*Corynebacterium flavescens* and *Bacillus pumilus* [78].

98 Plant Ecology - Traditional Approaches to Recent Trends

*3.2.1.2. Sugars*

*3.2.1.3. Antimicrobial compounds*

Since the onset of climate change and global warming, the resultant extreme heat and cold have affected the harvest of several crops. To elucidate the effects of temperature on root exudates, Husain and McKeen grew strawberry plants at 5–10°C and compared them with plants grown at 20–30°C. They found more amino acids in exudates in plants grown at a low soil temperature that markedly affected the pathogenicity of *Rhizoctonia fragariae* [87]. Pramanik demonstrated that in Japanese cucumber grown hydroponically in a growth chamber at high and low temperatures, the organic acid content increased with the elevation of temperature, and some of the compounds identified significantly inhibited plant root growth and/or germination of cucumber.

### *3.2.2.2. Soil moisture*

Flood and drought have reduced global cereal harvests. Several reports have demonstrated that the soil moisture affects the release of root exudates. The temporarily wilting of plants increased the release of amino acids from the plant roots, which might be related to the incidence of pathogens in the rhizosphere [88]. Plants such as peas, soybeans, wheat, barley, and tomatoes were grown in normal moist sand and dried, remoistened sand for the liberation of amino acids. The total amount of amino nitrogen in the temporarily dried sand was many times higher than in the normal moist sand.

### *3.2.2.3. Soil pH and nutrition*

The soil pH status and the availability of nutrients such as carbon, nitrogen, and phosphate have been found to affect the release of plant root exudates and the creation of specific chemical niches in the soil, as well as the abundance of plant pathogens and beneficial microbes [89–91]. Bowen first demonstrated a marked effect of nutrient status on the exudation of amides and amino acids from roots of *Pinus radiate* seedlings [89]. The results indicated a doubling of amides/amino acids in exudates from phosphate deficient plants. Toljander et al. analyzed the community of arbuscular mycorrhizal (AM) fungi in maize in a long-term fertilization trial and indicated the composition of AM fungi and bacteria was significantly affected and correlated with changes in pH, phosphate, and the soil carbon content [90]. Dumbrell et al. surveyed the AM fungal community of 425 individual plants from 28 plant species. The results showed the strong support for the hypothesis that niche differentiation was based on the structuring of the AM fungal community by soil pH [91]. Root secretion of phenolics was induced in Fe-deficient soil and altered the microbial community in the rhizosphere [92].

### *3.2.2.4. Microorganisms*

Soil microorganisms play a crucial role in plant growth and plant exudates. Microorganisms can affect exudation by affecting the permeability of root cells and root metabolism. Microorganisms can also absorb certain compounds in root exudates and excrete other compounds. Soil microbes can produce secondary metabolites that affect plant signaling and metabolism and can be considered as a "plant secondary genome" that provides plant hosts with microbe-derived compounds [93]. Some microbes and also some antibiotics (e.g., penicillin and polymyxin) increased the exudation of organic materials, altered cell permeability, and increased leakage [94, 95]. Soil microbes can also induce the exudation of phenolic compounds for enhancing plant Fe absorption in low-Fe availability soil [96].
