Engineering root microbiomes for healthier crops and soils using beneficial, environmentally safe bacteria.

The Green Revolution developed new crop varieties, which greatly improved food security worldwide. However, the growth of these plants relied heavily on chemical fertilizers and pesticides, which have led to an overuse of synthetic fertilizers, insecticides, and herbicides with serious environmental consequences and negative effects on human health. Environmentally friendly plant-growth-promoting methods to replace our current reliance on synthetic chemicals and to develop more sustainable agricultural practices to offset the damage caused by many agrochemicals are proposed herein. The increased use of bioinoculants, which consist of microorganisms that establish synergies with target crops and influence production and yield by enhancing plant growth, controlling disease, and providing critical mineral nutrients, is a potential solution. The microorganisms found in bioinoculants are often bacteria or fungi that reside within either external or internal plant microbiomes. However, before they can be used routinely in agriculture, these microbes must be confirmed as nonpathogenic strains that promote plant growth and survival. In this article, besides describing approaches for discovering plant-growth-promoting bacteria in various environments, including phytomicrobiomes and soils, we also discuss methods to evaluate their safety for the environment and for human health.

Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.

Reclassification of strains MAFF 303099T and R7A into Mesorhizobiumjaponicum sp. nov.

In this work we revise the taxonomic status of the Lotus-nodulating strains MAFF 303099T and R7A isolated in Japan and New Zealand, respectively. Their 16S rRNA gene sequences are identical and show 98.0, 99.7, 99.8 and 99.9 % similarity values with respect to Mesorhizobium loti NZP 2213T, M. jarvisii ATCC 33669T, M. huakuii USDA 4779T (=CCBAU 2609T) and M. erdmanii USDA 3471T, respectively. The analysis of recA and glnII gene sequeces showed that M. jarvisii ATCC 33669T and M. huakuii USDA 4779T (=CCBAU 2609T) are the most closely related strains to MAFF 303099T and R7A, with similarity values suggesting that these two strains belong to a different species for which MAFF 303099T is selected as the type strain. The DNA-DNA relatedness values between strain MAFF 303099T and its closest phylogenetic relatives ranged from 53 to 60 % in average. Strains MAFF 303099T and R7A presented slight differences in the proportions of C18 : 1ω7c 11-methyl and C19 : 0 cyclo ω8c fatty acids with respect to M. jarvisii ATCC 33669T and M. huakuii USDA 4779T, and also in several phenotypic characteristics. Therefore, we propose the reclassification of these two strains into a novel species named Mesorhizobium japonicum sp. nov., with the type strain being MAFF 303099T (=LMG 29417T=CECT 9101T).

Revision of the taxonomic status of type strains of Mesorhizobium loti and reclassification of strain USDA 3471T as the type strain of Mesorhizobiumerdmanii sp. nov. and ATCC 33669T as the type strain of Mesorhizobiumjarvisii sp. nov.

The species Mesorhizobim loti was isolated from nodules of Lotus corniculatus and its type strain deposited in several collections. Some of these type strains, such as those deposited in the USDA and ATCC collections before 1990, are not coincident with the original strain, NZP 2213T, deposited in the NZP culture collection. The analysis of the 16S rRNA gene showed that strains USDA 3471T and ATCC 33669T formed independent branches from that occupied by Mesorhizobium loti NZP 2213T and related to those occupied by Mesorhizobium opportunistum WSM2075T and Mesorhizobium huakuii IFO 15243T, respectively, with 99.9 % similarity in both cases. However, the analysis of concatenated recA, atpD and glnII genes with similarities lower than 96, 98 and 94 %, respectively, between strains USDA 3471T and M. opportunistum WSM2075T and between strains ATCC 33669T and M. huakuii IFO 15243T, indicated that the strains USDA 3471T and ATCC 33669T represent different species of the genus Mesorhizobium. These results were confirmed by DNA-DNA hybridization experiments and phenotypic characterization. Therefore, the two strains were reclassified as representatives of the two species Mesorhizobium erdmanii sp. nov. (type strain USDA 3471T = CECT 8631T = LMG 17826t2T) and Mesorhizobium jarvisii sp. nov. (type strain ATCC 33669T = CECT 8632T = LMG 28313T).

Paenibacillus lupini sp. nov., isolated from nodules of Lupinus albus.

A bacterial strain designated RLAHU15(T) was isolated from root nodules of Lupinus albus in Spain. Phylogenetic analyses based on 16S rRNA gene sequences placed the isolate in the genus Paenibacillus, with its closest relatives being Paenibacillus catalpae D75(T), Paenibacillus glycanilyticus DS-1(T), Paenibacillus endophyticus PECAE04(T) and Paenibacillus xinjiangensis B538(T) with 98.8 %, 98.9 %, 97.4 % and 97.4 % similarity, respectively. DNA-DNA hybridization studies showed values lower than 45 % between the strain RLAHU15(T) and any of these species. The isolate was a Gram-stain positive, motile and sporulating rod. Catalase activity was weak and oxidase activity was positive. Casein and starch were hydrolysed but gelatin was not. Growth was supported by many carbohydrates and organic acids as carbon sources. MK-7 was the only menaquinone detected and anteiso-C15 : 0 and iso-C16 : 0 were the major fatty acids. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, three unidentified phospholipids and an unidentified lipid. meso-Diaminopimelic acid was detected in the peptidoglycan. The DNA G+C content was 54.4 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain RLAHU15(T) represents a novel species of the genus Paenibacillus, for which the name Paenibacillus lupini sp. nov. is proposed. The type strain is RLAHU15(T) ( = LMG 27296(T) = CECT 8235(T)).

Inoculation With a Microbe Isolated From the Negev Desert Enhances Corn Growth.

Corn (Zea mays L.) is not only an important food source, but also has numerous uses, including for biofuels, fillers for cosmetics, glues, and so on. The amount of corn grown in the U.S. has significantly increased since the 1960's and with it, the demand for synthetic fertilizers and pesticides/fungicides to enhance its production. However, the downside of the continuous use of these products, especially N and P fertilizers, has been an increase in N2O emissions and other greenhouse gases into the atmosphere as well as run-off into waterways that fuel pollution and algal blooms. These approaches to agriculture, especially if exacerbated by climate change, will result in decreased soil health as well as human health. We searched for microbes from arid, native environments that are not being used for agriculture because we reasoned that indigenous microbes from such soils could promote plant growth and help restore degraded soils. Employing cultivation-dependent methods to isolate bacteria from the Negev Desert in Israel, we tested the effects of several microbial isolates on corn in both greenhouse and small field studies. One strain, Dietzia cinnamea 55, originally identified as Planomicrobium chinense, significantly enhanced corn growth over the uninoculated control in both greenhouse and outside garden experiments. We sequenced and analyzed the genome of this bacterial species to elucidate some of the mechanisms whereby D. cinnamea 55 promoted plant growth. In addition, to ensure the biosafety of this previously unknown plant growth promoting bacterial (PGPB) strain as a potential bioinoculant, we tested the survival and growth of Caenorhabditis elegans and Galleria mellonella (two animal virulence tests) as well as plants in response to D. cinnamea 55 inoculation. We also looked for genes for potential virulence determinants as well as for growth promotion.

High taxonomic diversity of Micromonospora strains isolated from Medicago sativa nodules in Western Spain and Australia.

The genus Micromonospora has been found in nodules of several legumes and some new species of this genus were isolated from these plant organs. In this study we analysed the taxonomic diversity of Micromonospora strains isolated from alfalfa nodules in Spain and Australia on the basis of three phylogenetic markers, the rrs and gyrB genes and 16S-23S intergenic spacer (ITS). The genome analysis of selected strains representative of different clusters or lineages found after rrs, gyrB and ITS analyses confirmed the results obtained with these phylogenetic markers. They showed that the analysed strains belong to at least 18 Micromonospora species including previously described ones, such as Micromonospora noduli, Micromonospora ureilytica, Micromonospora taraxaci, Micromonospora zamorensis, Micromonospora aurantiaca and Micromonospora tulbaghiae. Most of these strains belong to undescribed species of Micromonospora showing the high taxonomic diversity of strains from this genus inhabiting alfalfa nodules. Although Micromonospora strains are not able to induce the formation of these nodules, and it seems that they do not contribute to fix atmospheric nitrogen, they could play a role related with the mechanisms of plant growth promotion and pathogen protection presented by Micromonospora strains isolated from legume nodules.

Antifungal Activity of Bacillus Species Against Fusarium and Analysis of the Potential Mechanisms Used in Biocontrol.

Fusarium is a complex genus of ascomycete fungi that consists of plant pathogens of agricultural relevance. Controlling Fusarium infection in crops that leads to substantial yield losses is challenging. These economic losses along with environmental and human health concerns over the usage of chemicals in attaining disease control are shifting focus toward the use of biocontrol agents for effective control of phytopathogenic Fusarium spp. In the present study, an analysis of the plant-growth promoting (PGP) and biocontrol attributes of four bacilli (Bacillus simplex 30N-5, B. simplex 11, B. simplex 237, and B. subtilis 30VD-1) has been conducted. The production of cellulase, xylanase, pectinase, and chitinase in functional assays was studied, followed by in silico gene analysis of the PGP-related and biocontrol-associated genes. Of all the bacilli included in this study, B. subtilis 30VD-1 (30VD-1) demonstrated the most effective antagonism against Fusarium spp. under in vitro conditions. Additionally, 100 µg/ml of the crude 1-butanol extract of 30VD-1's cell-free culture filtrate caused about 40% inhibition in radial growth of Fusarium spp. Pea seed bacterization with 30VD-1 led to considerable reduction in wilt severity in plants with about 35% increase in dry plant biomass over uninoculated plants growing in Fusarium-infested soil. Phase contrast microscopy demonstrated distortions and abnormal swellings in F. oxysporum hyphae on co-culturing with 30VD-1. The results suggest a multivariate mode of antagonism of 30VD-1 against phytopathogenic Fusarium spp., by producing chitinase, volatiles, and other antifungal molecules, the characterization of which is underway.

Chitinase-producing bacteria and their role in biocontrol.

Chitin is an important component of the exteriors of insects and fungi. Upon degradation of chitin by a number of organisms, severe damage and even death may occur in pathogens and pests whose external surfaces contain this polymer. Currently, chemical fungicides and insecticides are the major means of controlling these disease-causing agents. However, due to the potential harm that these chemicals cause to the environment and to human and animal health, new strategies are being developed to replace or reduce the use of fungal- and pest-killing compounds in agriculture. In this context, chitinolytic microorganisms are likely to play an important role as biocontrol agents and pathogen antagonists and may also function in the control of postharvest rot. In this review, we discuss the literature concerning chitin and the basic knowledge of chitin-degrading enzymes, and also describe the biocontrol effects of chitinolytic microorganisms and their potential use as more sustainable pesticides and fungicides in the field.

The rhizosphere microbiome of burned holm-oak: potential role of the genus Arthrobacter in the recovery of burned soils.

After a forest wildfire, the microbial communities have a transient alteration in their composition. The role of the soil microbial community in the recovery of an ecosystem following such an event remains poorly understood. Thus, it is necessary to understand the plant-microbe interactions that occur in burned soils. By high-throughput sequencing, we identified the main bacterial taxa of burnt holm-oak rhizosphere, then we obtained an isolate collection of the most abundant genus and its growth promoting activities were characterised. 16S rRNA amplicon sequencing showed that the genus Arthrobacter comprised more than 21% of the total community. 55 Arthrobacter strains were isolated and characterized using RAPDs and sequencing of the almost complete 16S rRNA gene. Our results indicate that isolated Arthrobacter strains present a very high genetic diversity, and they could play an important ecological role in interaction with the host plant by enhancing aerial growth. Most of the selected strains exhibited a great ability to degrade organic polymers in vitro as well as possibly presenting a direct mechanism for plant growth promotion. All the above data suggests that Arthrobacter can be considered as an excellent PGP rhizobacterium that may play an important role in the recovery of burned holm-oak forests.

Analysis of rhizobial endosymbionts of Vicia, Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain).

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.

Induced systemic resistance against Botrytis cinerea by Micromonospora strains isolated from root nodules.

Micromonospora is a Gram positive bacterium that can be isolated from nitrogen fixing nodules from healthy leguminous plants, where they could be beneficial to the plant. Their plant growth promoting activity in legume and non-legume plants has been previously demonstrated. The present study explores the ability of Micromonospora strains to control fungal pathogens and to stimulate plant immunity. Micromonospora strains isolated from surface sterilized nodules of alfalfa showed in vitro antifungal activity against several pathogenic fungi. Moreover, root inoculation of tomato plants with these Micromonospora strains effectively reduced leaf infection by the fungal pathogen Botrytis cinerea, despite spatial separation between both microorganisms. This induced systemic resistance, confirmed in different tomato cultivars, is long lasting. Gene expression analyses evidenced that Micromonospora stimulates the plant capacity to activate defense mechanisms upon pathogen attack. The defensive response of tomato plants inoculated with Micromonospora spp. differs from that of non-inoculated plants, showing a stronger induction of jasmonate-regulated defenses when the plant is challenged with a pathogen. The hypothesis of jasmonates playing a key role in this defense priming effect was confirmed using defense-impaired tomato mutants, since the JA-deficient line def1 was unable to display a long term induced resistance upon Micromonospora spp. inoculation. In conclusion, nodule isolated Micromonospora strains should be considered excellent candidates as biocontrol agents as they combine both direct antifungal activity against plant pathogens and the ability to prime plant immunity.

Cicer canariense, an endemic legume to the Canary Islands, is nodulated in mainland Spain by fast-growing strains from symbiovar trifolii phylogenetically related to Rhizobium leguminosarum.

Cicer canariense is a threatened endemic legume from the Canary Islands where it can be nodulated by mesorhizobial strains from the symbiovar ciceri, which is the common worldwide endosymbiont of Cicer arietinum linked to the genus Mesorhizobium. However, when C. canariense was cultivated in a soil from mainland Spain, where the symbiovar ciceri is present, only fast-growing rhizobial strains were unexpectedly isolated from its nodules. These strains were classified into the genus Rhizobium by analysis of the recA and atpD genes, and they were phylogenetically related to Rhizobium leguminosarum. The analysis of the nodC gene showed that the isolated strains belonged to the symbiovar trifolii that harbored a nodC allele (ß allele) different to that harbored by other strains from this symbiovar. Nodulation experiments carried out with the lacZ-labeled strain RCCHU01, representative of the ß nodC allele, showed that it induced curling of root hairs, infected them through infection threads, and formed typical indeterminate nodules where nitrogen fixation took place. This represents a case of exceptional performance between the symbiovar trifolii and a legume from the tribe Cicereae that opens up new possibilities and provides new insights into the study of rhizobia-legume symbiosis.

Mining the phytomicrobiome to understand how bacterial coinoculations enhance plant growth.

In previous work, we showed that coinoculating Rhizobium leguminosarum bv. viciae 128C53 and Bacillus simplex 30N-5 onto Pisum sativum L. roots resulted in better nodulation and increased plant growth. We now expand this research to include another alpha-rhizobial species as well as a beta-rhizobium, Burkholderia tuberum STM678. We first determined whether the rhizobia were compatible with B. simplex 30N-5 by cross-streaking experiments, and then Medicago truncatula and Melilotus alba were coinoculated with B. simplex 30N-5 and Sinorhizobium (Ensifer) meliloti to determine the effects on plant growth. Similarly, B. simplex 30N-5 and Bu. tuberum STM678 were coinoculated onto Macroptilium atropurpureum. The exact mechanisms whereby coinoculation results in increased plant growth are incompletely understood, but the synthesis of phytohormones and siderophores, the improved solubilization of inorganic nutrients, and the production of antimicrobial compounds are likely possibilities. Because B. simplex 30N-5 is not widely recognized as a Plant Growth Promoting Bacterial (PGPB) species, after sequencing its genome, we searched for genes proposed to promote plant growth, and then compared these sequences with those from several well studied PGPB species. In addition to genes involved in phytohormone synthesis, we detected genes important for the production of volatiles, polyamines, and antimicrobial peptides as well as genes for such plant growth-promoting traits as phosphate solubilization and siderophore production. Experimental evidence is presented to show that some of these traits, such as polyamine synthesis, are functional in B. simplex 30N-5, whereas others, e.g., auxin production, are not.

Micromonospora from nitrogen fixing nodules of alfalfa (Medicago sativa L.). A new promising Plant Probiotic Bacteria.

Biotic interactions can improve agricultural productivity without costly and environmentally challenging inputs. Micromonospora strains have recently been reported as natural endophytes of legume nodules but their significance for plant development and productivity has not yet been established. The aim of this study was to determine the diversity and function of Micromonospora isolated from Medicago sativa root nodules. Micromonospora-like strains from field alfalfa nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene sequencing. The ecological role of the interaction of the 15 selected representative Micromonospora strains was tested in M. sativa. Nodulation, plant growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain abundant and highly diverse populations of Micromonospora, both at the intra- and at interspecific level. Selected Micromonospora isolates significantly increase the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the plant for nitrogen nutrition. Moreover, they promote aerial growth, the shoot-to-root ratio, and raise the level of essential nutrients. Our results indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition efficiency. Its ecological role, biotechnological potential and advantages as a plant probiotic bacterium (PPB) are also discussed.

The endemic Genista versicolor from Sierra Nevada National Park in Spain is nodulated by putative new Bradyrhizobium species and a novel symbiovar (sierranevadense).

Genista versicolor is an endemic legume from Sierra Nevada National Park which constitutes one of the UNESCO-recognized Biosphere Reserves. In the present study, a collection of strains nodulating this legume was analysed in characteristic soils of this ecosystem. Most strains nodulating G. versicolor belonged to rrs group I within the genus Bradyrhizobium and only one strain, named GV137, belonged to rrs group II from which only a single species, B. retamae, has been described in Europe to date. Strain GV137, and some strains from rrs group I, belonged to putative new species of Bradyrhizobium, although most strains from group I belonged to B. canariense, according to the ITS fragment and atpD gene analysis. This result contrasted with those obtained in Genista tinctoria in Northeast Europe whose endosymbionts were identified as B. japonicum. The analysis of the symbiotic nodC and nifH genes carried by G. versicolor-nodulating strains showed that most of them belonged to symbiovar genistearum, as did those isolated from G. tinctoria. Nevertheless, strain GV137, belonging to rrs group II, formed a divergent lineage that constituted a novel symbiovar within the genus Bradyrhizobium for which the name sierranevadense is proposed. This finding showed that the Genisteae are not restrictive legumes only nodulated by symbiovar genistearum, since Genista is a promiscuous legume nodulated by at least two symbiovars of Bradyrhizobium, as occurs in Retama species.

Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma, Canary Islands).

Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3-5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis.