The rhizosphere of a drought-tolerant plant species in Morocco: A refuge of high microbial diversity with no taxon preference.

Arid and semi-arid areas are facing increasingly severe water deficits that are being intensified by global climate changes. Microbes associated with plants native to arid regions provide valuable benefits to plants, especially in water-stressed environments. In this study, we used 16S rDNA metabarcoding analysis to examine the bacterial communities in the bulk soil, rhizosphere and root endosphere of the plant Malva sylvestris L. in Morocco, along a gradient of precipitation. We found that the rhizosphere of M. sylvestris did not show significant differences in beta-diversity compared to bulk soil, although, it did display an increased degree of alpha-diversity. The endosphere was largely dominated by the genus Rhizobium and displayed remarkable variation between plants, which could not be attributed to any of the variables observed in this study. Overall, the effects of precipitation level were relatively weak, which may be related to the intense drought in Morocco at the time of sampling. The dominance of Rhizobium in a non-leguminous plant is particularly noteworthy and may permit the utilization of this bacterial taxon to augment drought tolerance; additionally, the absence of any notable selection of the rhizosphere of M. sylvestris suggests that it is not significatively affecting its soil environment.

Evaluating Rhizobacterial Antagonists for Controlling Cercospora beticola and Promoting Growth in Beta vulgaris.

Cercospora beticola Sacc. is an ascomycete pathogen that causes Cercospora leaf spot in sugar beets (Beta vulgaris L.) and other related crops. It can lead to significant yield losses if not effectively managed. This study aimed to assess rhizosphere bacteria from sugar beet soil as a biological control agent against C. beticola and evaluate their effect on B. vulgaris. Following a dual-culture screening, 18 bacteria exhibiting over 50% inhibition were selected, with 6 of them demonstrating more than 80% control. The bacteria were identified by sequencing the 16S rRNA gene, revealing 12 potential species belonging to 6 genera, including Bacillus, which was represented by 4 species. Additionally, the biochemical and molecular properties of the bacteria were characterized in depth, as well as plant growth promotion. PCR analysis of the genes responsible for producing antifungal metabolites revealed that 83%, 78%, 89%, and 56% of the selected bacteria possessed bacillomycin-, iturin-, fengycin-, and surfactin-encoding genes, respectively. Infrared spectroscopy analysis confirmed the presence of a lipopeptide structure in the bacterial supernatant filtrate. Subsequently, the bacteria were assessed for their effect on sugar beet plants in controlled conditions. The bacteria exhibited notable capabilities, promoting growth in both roots and shoots, resulting in significant increases in root length and weight and shoot length. A field experiment with four bacterial candidates demonstrated good performance against C. beticola compared to the difenoconazole fungicide. These bacteria played a significant role in disease control, achieving a maximum efficacy of 77.42%, slightly below the 88.51% efficacy attained with difenoconazole. Additional field trials are necessary to verify the protective and growth-promoting effects of these candidates, whether applied individually, combined in consortia, or integrated with chemical inputs in sugar beet crop production.

Does soil history decline in influencing the structure of bacterial communities of Brassica napus host plants across different growth stages?

Soil history has been shown to condition future rhizosphere microbial communities. However, previous experiments have also illustrated that mature, adult plants can "re-write," or mask, different soil histories through host plant-soil community feedbacks. This leaves a knowledge gap concerning how soil history influences bacterial community structure across different growth stages. Thus, here we tested the hypothesis that previously established soil histories will decrease in influencing the structure of Brassica napus bacterial communities over the growing season. We used an on-going agricultural field experiment to establish three different soil histories, plots of monocrop canola (B. napus), or rotations of wheat-canola, or pea-barley-canola. During the following season, we repeatedly sampled the surrounding bulk soil, rhizosphere, and roots of the B. napus hosts at different growth stages-the initial seeding conditions, seedling, rosette, bolting, and flower-from all three soil history plots. We compared composition and diversity of the B. napus soil bacterial communities, as estimated using 16S rRNA gene metabarcoding, to identify any changes associated with soil history and growth stages. We found that soil history remained significant across each growth stage in structuring the bacterial bulk soil and rhizosphere communities, but not the bacterial root communities. This suggests that the host plant's capacity to "re-write" different soil histories may be quite limited as key components that constitute the soil history's identity remain present, such that the previously established soil history continues to impact the bacterial rhizosphere communities, but not the root communities. For agriculture, this highlights how previously established soil histories persist and may have important long-term consequences on future plant-microbe communities, including bacteria.

Cultural techniques capture diverse phosphate-solubilizing bacteria in rock phosphate-enriched habitats.

Phosphorus (P) deficiency is a common problem in croplands where phosphate-based fertilizers are regularly used to maintain bioavailable P for plants. However, due to their limited mobility in the soil, there has been an increased interest in microorganisms that can convert insoluble P into a bioavailable form, and their use to develop phosphate-solubilizing bioinoculants as an alternative to the conventional use of P fertilizers. In this study, we proposed two independent experiments and explored two entirely different habitats to trap phosphate-solubilizing bacteria (PSBs). In the first experiment, PSBs were isolated from the rhizoplane of native plant species grown in a rock-phosphate (RP) mining area. A subset of 24 bacterial isolates from 210 rhizoplane morphotypes was selected for the inorganic phosphate solubilizing activities using tricalcium phosphate (TCP) as the sole P source. In the second experiment, we proposed an innovative experimental setup to select mycohyphospheric bacteria associated to arbuscular mycorrhizal fungal hyphae, indigenous of soils where agronomic plant have been grown and trapped in membrane bag filled with RP. A subset of 25 bacterial isolates from 44 mycohyphospheric morphotypes was tested for P solubilizing activities. These two bacterial subsets were then screened for additional plant growth-promoting (PGP) traits, and 16S rDNA sequencing was performed for their identification. Overall, the two isolation experiments resulted in diverse phylogenetic affiliations of the PSB collection, showing only 4 genera (24%) and 5 species (17%) shared between the two communities, thus underlining the value of the two protocols, including the innovative mycohyphospheric isolate selection method, for selecting a greater biodiversity of cultivable PSB. All the rhizoplane and mycohyphospheric PSB were positive for ammonia production. Indol-3-acetic acid (IAA) production was observed for 13 and 20 isolates, respectively among rhizoplane and mycohyphospheric PSB, ranging, respectively, from 32.52 to 330.27 µg mL-1 and from 41.4 to 963.9 µg mL-1. Only five rhizoplane and 12 mycohyphospheric isolates were positively screened for N2 fixation. Four rhizoplane PSB were identified as siderophore producers, while none of the mycohyphospheric isolates were. The phenotype of one PSB rhizoplane isolate, assigned to Pseudomonas, showed four additive PGP activities. Some bacterial strains belonging to the dominant genera Bacillus and Pseudomonas could be considered potential candidates for further formulation of biofertilizer in order to develop bioinoculant consortia that promote plant P nutrition and growth in RP-enriched soils.

Draft Genome of Nocardia canadensis sp. nov. Isolated from Petroleum-Hydrocarbon-Contaminated Soil.

The bacterial strain WB46 was isolated from the rhizosphere of willow plants (Salix purpurea L.) growing in soil contaminated with petroleum hydrocarbons. The strain was subjected to whole-genome shotgun sequencing using Illumina HiSeq. Its draft genome is 7.15 Mb, with a 69.55% GC content, containing 6387 protein-coding genes and 51 tRNA and 15 rRNA sequences. The quality and reliability of the genome were assessed using CheckM, attaining an estimated genome completeness of 98.75% and an estimated contamination of 1.68%. These results indicate a high-quality genome (>95%) and low contamination (<5%). Many of these genes are responsible for petroleum hydrocarbon degradation, such as alkane 1-monooxygenase (alkB) and naphthalene dioxygenase (ndo). 16S rRNA gene analysis, including in silico DNA-DNA hybridization (DDH) and average nucleotide identity (ANI), showed that strain WB46 belongs to the genus Nocardia, and the most closely related species is Nocardia asteroides. The strain WB46 showed a distance of 63.4% and sequence identity of 88.63%, respectively. These values fall below the threshold levels of 70% and 95%, respectively, suggesting that the strain WB46 is a new species. We propose the name of Nocardia canadensis sp. nov. for this new species. Interestingly, the sequence divergence of the 16S rRNA gene showed that the divergence only occurred in the V2 region. Therefore, the conventional V3-V4, V5-V7, or V8-V9 targeting metabarcoding, among others, would not be able to assess the diversity related to this new species.

Enhanced production of select phytocannabinoids in medical Cannabis cultivars using microbial consortia.

The root microbiome of medical cannabis plants has been largely unexplored due to past legal restrictions in many countries. Microbes that live on and within the tissue of Cannabis sativa L. similar to other plants, provide advantages such as stimulating plant growth, helping it absorb minerals, providing protection against pathogen attacks, and influencing the production of secondary metabolites. To gain insight into the microbial communities of C. sativa cultivars with different tetrahydrocannabinol (THC) and cannabidiol (CBD) profiles, a greenhouse trial was carried out with and without inoculants added to the growth substrate. Illumina MiSeq metabarcoding was used to analyze the root and rhizosphere microbiomes of the five cultivars. Plant biomass production showed higher levels in three of five cultivars inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis and microbial suspension. The blossom dry weight of the cultivar THE was greater when inoculated with R. irregularis and microbial suspension than with no inoculation. Increasing plant biomass and blossom dry weight are two important parameters for producing cannabis for medical applications. In mature Cannabis, 12 phytocannabinoid compounds varied among cultivars and were affected by inoculants. Significant differences (p ≤ 0.01) in concentrations of cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), and cannabigerolic acid (CBGA) were observed in all Cannabis cultivars when amended with F, K1, and K2 inoculants. We found microbes that were shared among cultivars. For example, Terrimicrobium sp., Actinoplanes sp., and Trichoderma reesei were shared by the cultivars ECC-EUS-THE, CCL-ECC, and EUS-THE, respectively. Actinoplanes sp. is a known species that produces phosphatase enzymes, while Trichoderma reesei is a fungal train that produces cellulase and contributes to organic matter mineralization. However, the role of Terrimicrobium sp. as an anaerobic bacterium remains unknown. This study demonstrated that the use of inoculants had an impact on the production of phytocannabinoids in five Cannabis cultivars. These inoculants could have useful applications for optimizing cannabis cultivation practices and increasing the production of phytocannabinoids.

Microbial-Based Plant Biostimulants.

Beneficial microorganisms offer essential ecological services to both natural and agricultural ecosystems [...].

Disentangling arbuscular mycorrhizal fungi and bacteria at the soil-root interface.

Arbuscular mycorrhizal fungi (AMF) are essential components of the plant root mycobiome and are found in approximately 80% of land plants. As obligate plant symbionts, AMF harbor their own microbiota, both inside and outside the plant root system. AMF-associated bacteria (AAB) possess various functional traits, including nitrogen fixation, organic and inorganic phosphate mobilization, growth hormone production, biofilm production, enzymatic capabilities, and biocontrol against pathogen attacks, which not only contribute to the health of the arbuscular mycorrhizal symbiosis but also promote plant growth. Because of this, there is increasing interest in the diversity, functioning, and mechanisms that underlie the complex interactions between AMF, AAB, and plant hosts. This review critically examines AMF-associated bacteria, focusing on AAB diversity, the factors driving richness and community composition of these bacteria across various ecosystems, along with the physical, chemical, and biological connections that enable AMF to select and recruit beneficial bacterial symbionts on and within their structures and hyphospheres. Additionally, potential applications of these bacteria in agriculture are discussed, emphasizing the potential importance of AMF fungal highways in engineering plant rhizosphere and endophyte bacteria communities, and the importance of a functional core of AAB taxa as a promising tool to improve plant and soil productivity. Thus, AMF and their highly diverse bacterial taxa represent important tools that could be efficiently explored in sustainable agriculture, carbon sequestration, and reduction of greenhouse gas emissions related to nitrogen fertilizer applications. Nevertheless, future studies adopting integrated multidisciplinary approaches are crucial to better understand AAB functional diversity and the mechanisms that govern these tripartite relationships.

Soil Chemistry and Soil History Significantly Structure Oomycete Communities in Brassicaceae Crop Rotations.

Oomycetes are critically important in soil microbial communities, especially for agriculture, where they are responsible for major declines in yields. Unfortunately, oomycetes are vastly understudied compared to bacteria and fungi. As such, our understanding of how oomycete biodiversity and community structure vary through time in the soil remains poor. Soil history established by previous crops is one factor known to structure other soil microbes, but this has not been investigated for its influence on oomycetes. In this study, we established three different soil histories in field trials; the following year, these plots were planted with five different Brassicaceae crops. We hypothesized that the previously established soil histories would structure different oomycete communities, regardless of their current Brassicaceae crop host, in both the roots and rhizosphere. We used a nested internal transcribed spacer amplicon strategy incorporated with MiSeq metabarcoding, where the sequencing data was used to infer amplicon sequence variants of the oomycetes present in each sample. This allowed us to determine the impact of different soil histories on the structure and biodiversity of the oomycete root and rhizosphere communities from the five different Brassicaceae crops. We found that each soil history structured distinct oomycete rhizosphere communities, regardless of different Brassicaceae crop hosts, while soil chemistry structured the oomycete communities more during a dry year. Interestingly, soil history appeared specific to oomycetes but was less influential for bacterial communities previously identified from the same samples. These results advance our understanding of how different agricultural practices and inputs can alter edaphic factors to impact future oomycete communities. Examining how different soil histories endure and impact oomycete biodiversity will help clarify how these important communities may be assembled in agricultural soils. IMPORTANCE Oomycetes cause global plant diseases that result in substantial losses, yet they are highly understudied compared to other microbes, like fungi and bacteria. We wanted to investigate how past soil events, like changing crops in rotation, would impact subsequent oomycete communities. We planted different oilseed crops in three different soil histories and found that each soil history structured a distinct oomycete community regardless of which new oilseed crop was planted, e.g., oomycete communities from last year's lentil plots were still detected the following year regardless of which new oilseed crops we planted. This study demonstrated how different agricultural practices can impact future microbial communities differently. Our results also highlight the need for continued monitoring of oomycete biodiversity and quantification.

A Comprehensive Insight of Current and Future Challenges in Large-Scale Soil Microbiome Analyses.

In the last decade, various large-scale projects describing soil microbial diversity across large geographical gradients have been undertaken. However, many questions remain unanswered about the best ways to conduct these studies. In this review, we present an overview of the experience gathered during these projects, and of the challenges that future projects will face, such as standardization of protocols and results, considering the temporal variation of microbiomes, and the legal constraints limiting such studies. We also present the arguments for and against the exhaustive description of soil microbiomes. Finally, we look at future developments of soil microbiome studies, notably emphasizing the important role of cultivation techniques.

Glomerales Dominate Arbuscular Mycorrhizal Fungal Communities Associated with Spontaneous Plants in Phosphate-Rich Soils of Former Rock Phosphate Mining Sites.

Arbuscular mycorrhizal fungi (AMF) are key drivers of soil functioning. They interact with multiple soil parameters, notably, phosphorus (P). In this work, AMF communities of native plants grown spontaneously on former mining sites either enriched (P sites) or not enriched with P (nP sites) by mining cuttings of rock phosphate (RP) were studied. No significant differences were observed in the root mycorrhizal rates of the plants when comparing P and nP sites. The assessment of AMF diversity and community structure using Illumina MiSeq metabarcoding and targeting 18S rDNA in roots and rhizospheric soils showed a total of 318 Amplicon Sequence Variants (ASVs) of Glomeromycota phylum. No significant difference in the diversity was found between P and nP sites. Glomeraceae species were largely dominant, formed a fungal core of 26 ASVs, and were persistent and abundant in all sites. In the P soils, eight ASVs were identified by indicator species analysis. A trend towards an increase in Diversisporaceae and Claroideoglomeraceae and a reduction in Paraglomeraceae and Glomeraceae were noticed. These results provide new insights into AMF ecology in former RP mining sites; they document that P concentration is a driver of AMF community structures in soils enriched in RP long term but also suggest an influence of land disturbance, ecosystem self-restoration, and AMF life history strategies as drivers of AMF community profiles.

Antibiotic resistance in plant growth promoting bacteria: A comprehensive review and future perspectives to mitigate potential gene invasion risks.

Plant growth-promoting bacteria (PGPB) are endowed with several attributes that can be beneficial for host plants. They opened myriad doors toward green technology approach to reduce the use of chemical inputs, improve soil fertility, and promote plants' health. However, many of these PGPB harbor antibiotic resistance genes (ARGs). Less attention has been given to multi-resistant bacterial bioinoculants which may transfer their ARGs to native soil microbial communities and other environmental reservoirs including animals, waters, and humans. Therefore, large-scale inoculation of crops by ARGs-harboring bacteria could worsen the evolution and dissemination of antibiotic resistance and aggravate the negative impacts on such ecosystem and ultimately public health. Their introduction into the soil could serve as ARGs invasion which may inter into the food chain. In this review, we underscore the antibiotic resistance of plant-associated bacteria, criticize the lack of consideration for this phenomenon in the screening and application processes, and provide some recommendations as well as a regulation framework relating to the development of bacteria-based biofertilizers to aid maximizing their value and applications in crop improvement while reducing the risks of ARGs invasion.

The Potential Applications of Commercial Arbuscular Mycorrhizal Fungal Inoculants and Their Ecological Consequences.

Arbuscular mycorrhizal fungal (AMF) inoculants are sustainable biological materials that can provide several benefits to plants, especially in disturbed agroecosystems and in the context of phytomanagement interventions. However, it is difficult to predict the effectiveness of AMF inoculants and their impacts on indigenous AMF communities under field conditions. In this review, we examined the literature on the possible outcomes following the introduction of AMF-based inoculants in the field, including their establishment in soil and plant roots, persistence, and effects on the indigenous AMF community. Most studies indicate that introduced AMF can persist in the target field from a few months to several years but with declining abundance (60%) or complete exclusion (30%). Further analysis shows that AMF inoculation exerts both positive and negative impacts on native AMF species, including suppression (33%), stimulation (38%), exclusion (19%), and neutral impacts (10% of examined cases). The factors influencing the ecological fates of AMF inoculants, such as the inherent properties of the inoculum, dosage and frequency of inoculation, and soil physical and biological factors, are further discussed. While it is important to monitor the success and downstream impacts of commercial inoculants in the field, the sampling method and the molecular tools employed to resolve and quantify AMF taxa need to be improved and standardized to eliminate bias towards certain AMF strains and reduce discrepancies among studies. Lastly, inoculant producers must focus on selecting strains with a higher chance of success in the field, and having little or negligible downstream impacts.

Limited effect of thermal pruning on wild blueberry crop and its root-associated microbiota.

Thermal pruning was a common pruning method in the past but has progressively been replaced by mechanical pruning for economic reasons. Both practices are known to enhance and maintain high yields; however, thermal pruning was documented to have an additional sanitation effect by reducing weeds and fungal diseases outbreaks. Nevertheless, there is no clear consensus on the optimal fire intensity required to observe these outcomes. Furthermore, fire is known to alter the soil microbiome as it impacts the soil organic layer and chemistry. Thus far, no study has investigated into the effect of thermal pruning intensity on the wild blueberry microbiome in agricultural settings. This project aimed to document the effects of four gradual thermal pruning intensities on the wild blueberry performance, weeds, diseases, as well as the rhizosphere fungal and bacterial communities. A field trial was conducted using a block design where agronomic variables were documented throughout the 2-year growing period. MiSeq amplicon sequencing was used to determine the diversity as well as the structure of the bacterial and fungal communities. Overall, yield, fruit ripeness, and several other agronomical variables were not significantly impacted by the burning treatments. Soil phosphorus was the only parameter with a significant albeit temporary change (1 month after thermal pruning) for soil chemistry. Our results also showed that bacterial and fungal communities did not significantly change between burning treatments. The fungal community was dominated by ericoid mycorrhizal fungi, while the bacterial community was mainly composed of Acidobacteriales, Isosphaerales, Frankiales, and Rhizobiales. However, burning at high intensities temporarily reduced Septoria leaf spot disease in the season following thermal pruning. According to our study, thermal pruning has a limited short-term influence on the wild blueberry ecosystem but may have a potential impact on pests (notably Septoria infection), which should be explored in future studies to determine the burning frequency necessary to control this disease.

The Effects of an Arbuscular Mycorrhizal Fungus and Rhizobium Symbioses on Soybean Aphid Mostly Fail to Propagate to the Third Trophic Level.

The cascading effects of microbe-plant symbioses on the second trophic level, such as phytophagous insects, have been most studied. However, few studies have examined the higher third trophic level, i.e., their natural enemies. We investigated the effects of the symbiotic associations between an arbuscular mycorrhizal (AM) fungus, Rhizophagus irregularis (Glomerales: Glomeraceae), a nitrogen-fixing bacterium, Bradyrhizobium japonicum (Rhizobiales: Bradyrhizobiaceae), and soybean, Glycine max (L.) Merr. (Fabaceae) on two natural enemies of the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), the ladybird beetle Coleomegilla maculata (De Geer) (Coleoptera: Coccinellidae), and the parasitoid Aphelinus certus Yasnosh (Hymenoptera: Aphelinidae). We measured the growth and survival in the predator and parasitoid reared on aphids feeding on soybean inoculated seedlings. The rhizobium symbiosis alone was affected with a decreased rate of parasitoid emergence, presumably due to decreased host quality. However, number of mummies, sex-ratio, development time, and parasitoid size were all unaffected by inoculation. AM fungus alone or co-inoculated with the rhizobium was unaffected with any of the parameters of the parasitoid. For the predator, none of the measured parameters was affected with any inoculant. Here, it appears that whatever benefits the microbe-plant symbioses confer on the second trophic level are little transferred up to the third.

Influence on Soybean Aphid by the Tripartite Interaction between Soybean, a Rhizobium Bacterium, and an Arbuscular Mycorrhizal Fungus.

The inoculation of arbuscular mycorrhizal (AM) fungi and rhizobia in legumes has been proven to increase plant growth and yield. To date, studies of the effects of these interactions on phytophagous insects have shown them to be context-dependent depending on the inoculant strain, the plant, and the insect species. Here, we document how a symbiosis involving an AM fungus, Rhizophagus irregularis; a rhizobium, Bradyrhizobium japonicum; and soybean, Glycine max, influences the soybean aphid, Aphis glycines. Soybean co-inoculated with the AM fungus-rhizobium pair increased the plant's biomass, nodulation, mycorrhizal colonization, nitrogen, and carbon concentrations, but decreased phosphorus concentration. Similar effects were observed with rhizobium alone, with the exception that root biomass was unaffected. With AM fungus alone, we only observed an increase in mycorrhizal colonization and phosphorus concentration. The aphids experienced an increased reproductive rate with the double inoculation, followed by rhizobium alone, whereas no effect was observed with the AM fungus. The size of individual aphids was not affected. Furthermore, we found positive correlation between nitrogen concentration and aphid population density. Our results confirm that co-inoculation of two symbionts can enhance both plant and phytophagous insect performance beyond what either symbiont can contribute alone.