The rhizosphere microbiome: Plant-microbial interactions for resource acquisition.

While horticulture tools and methods have been extensively developed to improve the management of crops, systems to harness the rhizosphere microbiome to benefit plant crops are still in development. Plants and microbes have been coevolving for several millennia, conferring fitness advantages that expand the plant's own genetic potential. These beneficial associations allow the plants to cope with abiotic stresses such as nutrient deficiency across a wide range of soils and growing conditions. Plants achieve these benefits by selectively recruiting microbes using root exudates, positively impacting their nutrition, health and overall productivity. Advanced knowledge of the interplay between root exudates and microbiome alteration in response to plant nutrient status, and the underlying mechanisms there of, will allow the development of technologies to increase crop yield. This review summarizes current knowledge and perspectives on plant-microbial interactions for resource acquisition and discusses promising advances for manipulating rhizosphere microbiomes and root exudation.

Root exudates drive soil-microbe-nutrient feedbacks in response to plant growth.

Although interactions between plants and microbes at the plant-soil interface are known to be important for plant nutrient acquisition, relatively little is known about how root exudates contribute to nutrient exchange over the course of plant development. In this study, root exudates from slow- and fast-growing stages of Arabidopsis thaliana plants were collected, chemically analysed and then applied to a sandy nutrient-depleted soil. We then tracked the impacts of these exudates on soil bacterial communities, soil nutrients (ammonium, nitrate, available phosphorus and potassium) and plant growth. Both pools of exudates shifted bacterial community structure. GeoChip analyses revealed increases in the functional gene potential of both exudate-treated soils, with similar responses observed for slow-growing and fast-growing plant exudate treatments. The fast-growing stage root exudates induced higher nutrient mineralization and enhanced plant growth as compared to treatments with slow-growing stage exudates and the control. These results suggest that plants may adjust their exudation patterns over the course of their different growth phases to help tailor microbial recruitment to meet increased nutrient demands during periods demanding faster growth.

Differential Effects of Phosphorus Fertilization on Plant Uptake and Rhizosphere Microbiome of Cultivated and Non-cultivated Potatoes.

There is evidence that shows that phosphorus (P) fertilization has a moderate effect on the rhizosphere microbial composition of cultivated crops. But how this effect is manifested on wild species of the same crop is not clear. This study compares the impact of phosphorus fertilization with rhizosphere bacterial community composition and its predicted functions, related to P-cycling genes, in both cultivated and non-cultivated potato (Solanum sp.) plants. It was found that the biomass of non-cultivated potatoes was more responsive to P fertilization as compared with cultivated plants. Differences in general bacterial community composition patterns under increasing P amendments were subtle for both potato groups. However, potato genotype significantly influenced community composition with several bacterial families being more abundant in the cultivated plants. In addition, the predicted phosphatases had lower abundances in modern cultivars compared with non-cultivated potatoes. In summary, despite higher accumulation of differentially abundant bacteria in the rhizosphere of cultivated plants, the responsiveness of these plants to increase P levels was lower than in non-cultivated plants.

Isolation of Klebsiella pneumoniae and Pseudomonas aeruginosa from entomopathogenic nematode-insect host relationship to examine bacterial pathogenicity on Trichoplusia ni.

Klebsiella pneumoniae was isolated from infected pupae of Galleria mellonella and Pseudomonas aeruginosa was isolated from the entomopathogenic nematode Heterorhabditis bacteriophora hosted within the pupae of G. mellonella. Insect consumption and surface application of P. aeruginosa resulted in 83.33% and 81.66% mortality of Trichoplusia ni larvae, respectively. In contrast, 50% mortality was shown when T. ni larvae were fed with K. pneumoniae, and no larvae were killed when applying the bacterium to the larval cuticle. This report shows that two opportunistic human pathogens found in the insect-nematode ecosystem could kill insect pests.

Bacterial Microbiome and Nematode Occurrence in Different Potato Agricultural Soils.

Pratylenchus neglectus and Meloidogyne chitwoodi are the main plant-parasitic nematodes in potato crops of the San Luis Valley, Colorado. Bacterial microbiome (16S rRNA copies per gram of soil) and nematode communities (nematodes per 200 g of soil) from five different potato farms were analyzed to determine negative and positive correlations between any bacterial genus and P. neglectus and M. chitwoodi. Farms showed differences in bacterial communities, percentage of bacterivorous and fungivorous nematodes, and numbers of P. neglectus and M. chitwoodi. The farm with the lowest population of P. neglectus and M. chitwoodi had higher abundances of the bacterial genera Bacillus spp., Arthrobacter spp., and Lysobacter spp., and the soil nematode community was composed of more than 30% of fungivorous nematodes. In contrast, the farm with higher numbers of P. neglectus and M. chitwoodi had a lower abundance of the abovementioned bacterial genera, higher abundance of Burkholderia spp., and less than 25% of fungivorous nematodes. The α-Proteobacteria Rhodoplanes, Phenylobacterium, and Kaistobacter positively correlated with M. chitwoodi, and the Bacteroidia and γ-Proteobacteria positively correlated with P. neglectus. Our results, based largely on co-occurrence analyses, suggest that the abundance of Bacillus spp., Arthrobacter spp., and Lysobacter spp. in Colorado potato soils is negatively correlated with P. neglectus and M. chitwoodi abundance. Further studies will isolate and identify bacterial strains of these genera, and evaluate their nematode-antagonistic activity.

Supplementing Blends of Sugars, Amino Acids, and Secondary Metabolites to the Diet of Termites (Reticulitermes flavipes) Drive Distinct Gut Bacterial Communities.

Although it is well known that diet is one of the major modulators of the gut microbiome, how the major components of diet shape the gut microbial community is not well understood. Here, we developed a simple system that allows the investigation of the impact of given compounds as supplements of the diet on the termite gut microbiome. The 16S rRNA pyrosequencing analysis revealed that feeding termites different blends of sugars and amino acids did not majorly impact gut community composition; however, ingestion of blends of secondary metabolites caused shifts in gut bacterial community composition. The supplementation of sugars and amino acids reduced the richness significantly, and sugars alone increased the evenness of the gut bacterial community significantly. Secondary metabolites created the most dramatic effects on the microbial community, potentially overriding the effect of other types of compounds. Furthermore, some microbial groups were stimulated specifically by particular groups of compounds. For instance, termites fed with secondary metabolites contained more Firmicutes and Spirochaetes compared to the other treatments. In conclusion, our results suggest that the termite (Reticulitermes flavipes) can be used as a simple and effective system to test the effects of particular chemical compounds in modulating the gut microbiome.

Linking Jasmonic Acid Signaling, Root Exudates, and Rhizosphere Microbiomes.

Jasmonic acid (JA) is an essential hormone in plant development and defense responses in Arabidopsis thaliana. Exogenous treatment with JA has recently been shown to alter root exudate profiles and the composition of root-associated bacterial communities. However, it is currently unknown whether disruptions of the JA in the rhizosphere affect root exudation profiles and the relative abundance of bacteria and archaea in the rhizosphere. In the present study, two Arabidopsis mutants that are disrupted in different branches of the jasmonate pathway, namely myc2 and med25, were cultivated in nutrient solution and soil to profile root exudates and bacterial and archaeal communities, respectively. Compared with the wild type, both mutants showed distinct exudation patterns, including lower amounts of asparagine, ornithine, and tryptophan, as well as distinct bacterial and archaeal community composition, as illustrated by an increased abundance of Streptomyces, Bacillus, and Lysinibacillus taxa in the med25 rhizosphere and an Enterobacteriaceae population in myc2. Alternatively, the Clostridiales population was less abundant in the rhizosphere of both mutants. Similarities between plant genotypes were highly correlated, as determined by operational taxonomic units in the rhizosphere and metabolites in root exudates. This strongly suggests that root exudates play a major role in modulating changes in microbial community composition upon plant defense responses.

Application of natural blends of phytochemicals derived from the root exudates of Arabidopsis to the soil reveal that phenolic-related compounds predominantly modulate the soil microbiome.

The roots of plants have the ability to influence its surrounding microbiology, the so-called rhizosphere microbiome, through the creation of specific chemical niches in the soil mediated by the release of phytochemicals. Here we report how these phytochemicals could modulate the microbial composition of a soil in the absence of the plant. For this purpose, root exudates of Arabidopsis were collected and fractionated to obtain natural blends of phytochemicals at various relative concentrations that were characterized by GC-MS and applied repeatedly to a soil. Soil bacterial changes were monitored by amplifying and pyrosequencing the 16 S ribosomal small subunit region. Our analyses reveal that one phytochemical can culture different operational taxonomic units (OTUs), mixtures of phytochemicals synergistically culture groups of OTUs, and the same phytochemical can act as a stimulator or deterrent to different groups of OTUs. Furthermore, phenolic-related compounds showed positive correlation with a higher number of unique OTUs compared with other groups of compounds (i.e. sugars, sugar alcohols, and amino acids). For instance, salicylic acid showed positive correlations with species of Corynebacterineae, Pseudonocardineae and Streptomycineae, and GABA correlated with species of Sphingomonas, Methylobacterium, Frankineae, Variovorax, Micromonosporineae, and Skermanella. These results imply that phenolic compounds act as specific substrates or signaling molecules for a large group of microbial species in the soil.

Rhizosphere Microbiome Co-Occurrence Network Analysis across a Tomato Domestication Gradient.

When plant-available phosphorus (P) is lost from a soil solution, it often accumulates in the soil as a pool of unavailable legacy P. To acquire legacy P, plants employ recovery strategies, such as forming associations with soil microbes. However, the degree to which plants rely on microbial associations for this purpose varies with crop domestication and subsequent breeding. Here, by generating microbial co-occurrence networks, we sought to explore rhizosphere bacterial interactions in low-P conditions and how they change with tomato domestication and breeding. We grew wild tomato, traditional tomato (developed circa 1900), and modern tomato (developed circa 2020) in high-P and low-P soil throughout their vegetative developmental stage. Co-occurrence network analysis revealed that as the tomatoes progressed along the stages of domestication, the rhizosphere microbiome increased in complexity in a P deficit. However, with the addition of P fertilizer, the wild tomato group became more complex, surpassing the complexity of traditional and modern tomato, suggesting a high degree of responsiveness in the rhizosphere microbiome to P fertilizer by wild tomato relatives. By illustrating these changing patterns of network complexity in the tomato rhizosphere microbiome, we can further understand how plant domestication and breeding have shaped plant-microbe interactions.

Tomato domestication rather than subsequent breeding events reduces microbial associations related to phosphorus recovery.

Legacy phosphorus (P) is a reservoir of sparingly available P, and its recovery could enhance sustainable use of nonrenewable mineral fertilizers. Domestication has affected P acquisition, but it is unknown if subsequent breeding efforts, like the Green Revolution (GR), had a similar effect. We examined how domestication and breeding events altered P acquisition by growing wild, traditional (pre-GR), and modern (post-GR) tomato in soil with legacy P but low bioavailable P. Wild tomatoes, particularly accession LA0716 (Solanum pennellii), heavily cultured rhizosphere P solubilizers, suggesting reliance on microbial associations to acquire P. Wild tomato also had a greater abundance of other putatively beneficial bacteria, including those that produce chelating agents and antibiotic compounds. Although wild tomatoes had a high abundance of these P solubilizers, they had lower relative biomass and greater P stress factor than traditional or modern tomato. Compared to wild tomato, domesticated tomato was more tolerant to P deficiency, and both cultivated groups had a similar rhizosphere bacterial community composition. Ultimately, this study suggests that while domestication changed tomato P recovery by reducing microbial associations, subsequent breeding processes have not further impacted microbial P acquisition mechanisms. Selecting microbial P-related traits that diminished with domestication may therefore increase legacy P solubilization.

Harnessing Phosphorous (P) Fertilizer-Insensitive Bacteria to Enhance Rhizosphere P Bioavailability in Legumes.

Phosphorous (P) is widely used in agriculture; yet, P fertilizers are a nonrenewable resource. Thus, mechanisms to improve soil P bioavailability need to be found. Legumes are efficient in P acquisition and, therefore, could be used to develop new technologies to improve soil P bioavailability. Here, we studied different species and varieties of legumes and their rhizosphere microbiome responses to low-P stress. Some varieties of common beans, cowpeas, and peas displayed a similar biomass with and without P fertilization. The rhizosphere microbiome of those varieties grown without P was composed of unique microbes displaying different levels of P solubilization and mineralization. When those varieties were amended with P, some of the microbes involved in P solubilization and mineralization decreased in abundance, but other microbes were insensitive to P fertilization. The microbes that decreased in abundance upon P fertilization belonged to groups that are commonly used as biofertilizers such as Pseudomonas and Azospirillum. The microbes that were not affected by P fertilization constitute unique species involved in P mineralization such as Arenimonas daejeonensis, Hyphomicrobium hollandicum, Paenibacillus oenotherae, and Microlunatus speluncae. These P-insensitive microbes could be used to optimize P utilization and drive future sustainable agricultural practices to reduce human dependency on a nonrenewable resource.

Potential impact of soil microbiomes on the leaf metabolome and on herbivore feeding behavior.

It is known that environmental factors can affect the biosynthesis of leaf metabolites. Similarly, specific pairwise plant-microbe interactions modulate the plant's metabolome by stimulating production of phytoalexins and other defense-related compounds. However, there is no information about how different soil microbiomes could affect the plant growth and the leaf metabolome. We analyzed experimentally how diverse soil microbiomes applied to the roots of Arabidopsis thaliana were able to modulate plant growth and the leaf metabolome, as assessed by GC-MS analyses. Further, we determined the effects of soil microbiome-driven changes in leaf metabolomics on the feeding behavior of Trichopulsia ni larvae. Soil microbiomes differentially impacted plant growth patterns as well as leaf metabolome composition. Similarly, most microbiome-treated plants showed inhibition to larvae feeding, compared with unamended control plants. Pyrosequencing analysis was conducted to determine the soil microbial composition and diversity of the soils used in this study. Correlation analyses were performed to determine relationships between various factors (soil microbial taxa, leaf chemical components, plant growth patterns and insect feeding behavior) and revealed that leaf amino acid content was positively correlated with both microbiome composition and insect feeding behavior.

Isolation and characterization of lignin-degrading bacteria from rainforest soils.

The deconstruction of lignin to enhance the release of fermentable sugars from plant cell walls presents a challenge for biofuels production from lignocellulosic biomass. The discovery of novel lignin-degrading enzymes from bacteria could provide advantages over fungal enzymes in terms of their production and relative ease of protein engineering. In this study, 140 bacterial strains isolated from soils of a biodiversity-rich rainforest in Peru were screened based on their oxidative activity on ABTS, a laccase substrate. Strain C6 (Bacillus pumilus) and strain B7 (Bacillus atrophaeus) were selected for their high laccase activity and identified by 16S rDNA analysis. Strains B7 and C6 degraded fragments of Kraft lignin and the lignin model dimer guaiacylglycerol-ß-guaiacyl ether, the most abundant linkage in lignin. Finally, LC-MS analysis of incubations of strains B7 and C6 with poplar biomass in rich and minimal media revealed that a higher number of compounds were released in the minimal medium than in the rich one. These findings provide important evidence that bacterial enzymes can degrade and/or modify lignin and contribute to the release of fermentable sugars from lignocellulose.

Variations in diversity and richness of gut bacterial communities of termites (Reticulitermes flavipes) fed with grassy and woody plant substrates.

Diets shape the animal gut microbiota, although the relationships between diets and the structure of the gut microbial community are not yet well understood. The gut bacterial communities of Reticulitermes flavipes termites fed on four individual plant biomasses with different degrees of recalcitrance to biodegradation were investigated by 16S rRNA pyrosequencing analysis. The termite gut bacterial communities could be differentiated between grassy and woody diets, and among grassy diets (corn stover vs. sorghum). The majority of bacterial taxa were shared across all diets, but each diet significantly enriched some taxa. Interestingly, the diet of corn stover reduced gut bacterial richness and diversity compared to other diets, and this may be related to the lower recalcitrance of this biomass to degradation.

Effect of plant sterols and tannins on Phytophthora ramorum growth and sporulation.

Elicitin-mediated acquisition of plant sterols is required for growth and sporulation of Phytophthora spp. This study examined the interactions between elicitins, sterols, and tannins. Ground leaf tissue, sterols, and tannin-enriched extracts were obtained from three different plant species (California bay laurel, California black oak, and Oregon white oak) in order to evaluate the effect of differing sterol/tannin contents on Phytophthora ramorum growth. For all three species, high levels of foliage inhibited P. ramorum growth and sporulation, with a steeper concentration dependence for the two oak samples. Phytophthora ramorum growth and sporulation were inhibited by either phytosterols or tannin-enriched extracts. High levels of sterols diminished elicitin gene expression in P. ramorum; whereas the tannin-enriched extract decreased the amount of 'functional' or ELISA-detectable elicitin, but not gene expression. Across all treatment combinations, P. ramorum growth and sporulation correlated strongly with the amount of ELISA-detectable elicitin (R (2) = 0.791 and 0.961, respectively).

Shifts of the soil microbiome composition induced by plant-plant interactions under increasing cover crop densities and diversities.

Interspecific and intraspecific competition and facilitation have been a focus of study in plant-plant interactions, but their influence on plant recruitment of soil microbes is unknown. In this greenhouse microcosm experiment, three cover crops (alfalfa, brassica, and fescue) were grown alone, in paired mixtures, and all together under different densities. For all monoculture trials, total pot biomass increased as density increased. Monoculture plantings of brassica were associated with the bacteria Azospirillum spp., fescue with Ensifer adhaerens, and alfalfa with both bacterial taxa. In the polycultures of cover crops, for all plant mixtures, total above-ground alfalfa biomass increased with density, and total above ground brassica biomass remained unchanged. For each plant mixture, differential abundances highlighted bacterial taxa which had not been previously identified in monocultures. For instance, mixtures of all three plants showed an increase in abundance of Planctomyces sp. SH-PL14 and Sandaracinus amylolyticus which were not represented in the monocultures. Facilitation was best supported for the alfalfa-fescue interaction as the total above ground biomass was the highest of any mixture. Additionally, the bulk soil microbiome that correlated with increasing plant densities showed increases in plant growth-promoting rhizobacteria such as Achromobacter xylosoxidans, Stentotrophomonas spp., and Azospirillum sp. In contrast, Agrobacterium tumefaciens, a previously known generalist phytopathogen, also increased with alfalfa-fescue plant densities. This could suggest a strategy by which, after facilitation, a plant neighbor could culture a pathogen that could be more detrimental to the other.

A Microbiological Approach to Alleviate Soil Replant Syndrome in Peaches.

Replant syndrome (RS) is a global problem characterized by reduced growth, production life, and yields of tree fruit/nut orchards. RS etiology is unclear, but repeated monoculture plantings are thought to develop a pathogenic soil microbiome. This study aimed to evaluate a biological approach that could reduce RS in peach (Prunus persica) orchards by developing a healthy soil bacteriome. Soil disinfection via autoclave followed by cover cropping and cover crop incorporation was found to distinctly alter the peach soil bacteriome but did not affect the RS etiology of RS-susceptible 'Lovell' peach seedlings. In contrast, non-autoclaved soil followed by cover cropping and incorporation altered the soil bacteriome to a lesser degree than autoclaving but induced significant peach growth. Non-autoclaved and autoclaved soil bacteriomes were compared to highlight bacterial taxa promoted by soil disinfection prior to growing peaches. Differential abundance shows a loss of potentially beneficial bacteria due to soil disinfection. The treatment with the highest peach biomass was non-autoclaved soil with a cover crop history of alfalfa, corn, and tomato. Beneficial bacterial species that were cultivated exclusively in the peach rhizosphere of non-autoclaved soils with a cover crop history were Paenibacillus castaneae and Bellilinea caldifistulae. In summary, the non-autoclaved soils show continuous enhancement of beneficial bacteria at each cropping phase, culminating in an enriched rhizosphere which may help alleviate RS in peaches.

Root exudate-derived compounds stimulate the phosphorus solubilizing ability of bacteria.

Low phosphorus (P) availability in soils is a major challenge for sustainable food production, as most soil P is often unavailable for plant uptake and effective strategies to access this P are limited. Certain soil occurring bacteria and root exudate-derived compounds that release P are in combination promising tools to develop applications that increase phosphorus use efficiency in crops. Here, we studied the ability of root exudate compounds (galactinol, threonine, and 4-hydroxybutyric acid) induced under low P conditions to stimulate the ability of bacteria to solubilize P. Galactinol, threonine, and 4-hydroxybutyric acid were incubated with the P solubilizing bacterial strains Enterobacter cloacae, Pseudomonas pseudoalcaligenes, and Bacillus thuringiensis under either inorganic (calcium phosphate) or organic (phytin) forms of plant-unavailable P. Overall, we found that the addition of individual root exudate compounds did not support bacterial growth rates. However, root exudates supplemented to the different bacterial appeared to enhance P solubilizing activity and overall P availability. Threonine and 4-hydroxybutyric acid induced P solubilization in all three bacterial strains. Subsequent exogenous application of threonine to soils improved the root growth of corn, enhanced nitrogen and P concentrations in roots and increased available levels of potassium, calcium and magnesium in soils. Thus, it appears that threonine might promote the bacterial solubilization and plant-uptake of a variety of nutrients. Altogether, these findings expand on the function of exuded specialized compounds and propose alternative approaches to unlock existing phosphorus reservoirs of P in crop lands.

Soil microbiome disruption reveals specific and general plant-bacterial relationships in three agroecosystem soils.

Soil microbiome disruption methods are regularly used to reduce populations of microbial pathogens, often resulting in increased crop growth. However, little is known about the effect of soil microbiome disruption on non-pathogenic members of the soil microbiome. Here, we applied soil microbiome disruption in the form of moist-heat sterilization (autoclaving) to reduce populations of naturally occurring soil microbiota. The disruption was applied to analyze bacterial community rearrangement mediated by four crops (corn, beet, lettuce, and tomato) grown in three historically distinct agroecosystem soils (conventional, organic, and diseased). Applying the soil disruption enhanced plant influence on rhizosphere bacterial colonization, and significantly different bacterial communities were detected between the tested crops. Furthermore, bacterial genera showed significant abundance increases in ways both unique-to and shared-by each tested crop. As an example, corn uniquely promoted abundances of Pseudomonas and Sporocytophaga, regardless of the disrupted soil in which it was grown. Whereas the promotion of Bosea, Dyadobacter and Luteoliobacter was shared by all four crops when grown in disrupted soils. In summary, soil disruption followed by crop introduction amplified the plant colonization of potential beneficial bacterial genera in the rhizosphere.

Root secretion of defense-related proteins is development-dependent and correlated with flowering time.

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.