Unearthing the power of microbes as plant microbiome for sustainable agriculture.

In recent years, research into the complex interactions and crosstalk between plants and their associated microbiota, collectively known as the plant microbiome has revealed the pivotal role of microbial communities for promoting plant growth and health. Plants have evolved intricate relationships with a diverse array of microorganisms inhabiting their roots, leaves, and other plant tissues. This microbiota mainly includes bacteria, archaea, fungi, protozoans, and viruses, forming a dynamic and interconnected network within and around the plant. Through mutualistic or cooperative interactions, these microbes contribute to various aspects of plant health and development. The direct mechanisms of the plant microbiome include the enhancement of plant growth and development through nutrient acquisition. Microbes have the ability to solubilize essential minerals, fix atmospheric nitrogen, and convert organic matter into accessible forms, thereby augmenting the nutrient pool available to the plant. Additionally, the microbiome helps plants to withstand biotic and abiotic stresses, such as pathogen attacks and adverse environmental conditions, by priming the plant's immune responses, antagonizing phytopathogens, and improving stress tolerance. Furthermore, the plant microbiome plays a vital role in phytohormone regulation, facilitating hormonal balance within the plant. This regulation influences various growth processes, including root development, flowering, and fruiting. Microbial communities can also produce secondary metabolites, which directly or indirectly promote plant growth, development, and health. Understanding the functional potential of the plant microbiome has led to innovative agricultural practices, such as microbiome-based biofertilizers and biopesticides, which harness the power of beneficial microorganisms to enhance crop yields while reducing the dependency on chemical inputs. In the present review, we discuss and highlight research gaps regarding the plant microbiome and how the plant microbiome can be used as a source of single and synthetic bioinoculants for plant growth and health.

Agroecological Management of the Grey Mould Fungus Botrytis cinerea by Plant Growth-Promoting Bacteria.

Botrytis cinerea is the causal agent of grey mould and one of the most important plant pathogens in the world because of the damage it causes to fruits and vegetables. Although the application of botrycides is one of the most common plant protection strategies used in the world, the application of plant-beneficial bacteria might replace botrycides facilitating agroecological production practices. Based on this, we reviewed the different stages of B. cinerea infection in plants and the biocontrol mechanisms exerted by plant-beneficial bacteria, including the well-known plant growth-promoting bacteria (PGPB). Some PGPB mechanisms to control grey mould disease include antibiosis, space occupation, nutrient uptake, ethylene modulation, and the induction of plant defence mechanisms. In addition, recent studies on the action of anti-Botrytis compounds produced by PGPB and how they damage the conidial and mycelial structures of the pathogen are reviewed. Likewise, the advantages of individual inoculations of PGPB versus those that require the joint action of antagonist agents (microbial consortia) are discussed. Finally, it should be emphasised that PGPB are an excellent option to prevent grey mould in different crops and their use should be expanded for environmentally friendly agricultural practices.

Deciphering the role of endophytic microbiome in postharvest diseases management of fruits: Opportunity areas in commercial up-scale production.

As endophytes are widely distributed in the plant's internal compartments and despite having enormous potential as a biocontrol agent against postharvest diseases of fruits, the fruit-endophyte-pathogen interactions have not been studied detail. Therefore, this review aims to briefly discuss the colonization patterns of endophytes and pathogens in the host tissue, the diversity and distribution patterns of endophytes in the carposphere of fruits, and host-endophyte-pathogen interactions and the molecular mechanism of the endophytic microbiome in postharvest disease management in fruits. Postharvest loss management is one of the major concerns of the current century. It is considered a critical challenge to food security for the rising global population. However, to manage the postharvest loss, still, a large population relies on chemical fungicides, which affect food quality and are hazardous to health and the surrounding environment. However, the scientific community has searched for alternatives for the last two decades. In this context, endophytic microorganisms have emerged as an economical, sustainable, and viable option to manage postharvest pathogens with integral colonization properties and eliciting a defense response against pathogens. This review extensively summarizes recent developments in endophytic interactions with harvested fruits and pathogens-the multiple biocontrol traits of endophytes and colonization and diversity patterns of endophytes. In addition, the upscale commercial production of endophytes for postharvest disease treatment is discussed.

Can Bacterial Endophytes Be Used as a Promising Bio-Inoculant for the Mitigation of Salinity Stress in Crop Plants?-A Global Meta-Analysis of the Last Decade (2011-2020).

Soil salinity is a major problem affecting crop production worldwide. Lately, there have been great research efforts in increasing the salt tolerance of plants through the inoculation of plant growth-promoting endophytic bacteria. However, their ability to promote plant growth under no-stress and salinity-stress conditions remains largely uncertain. Here, we carried out a global meta-analysis to quantify the plant growth-promoting effects (improvement of morphological attributes, photosynthetic capacity, antioxidative ability, and ion homeostasis) of endophytic bacteria in plants under no-stress and salinity-stress conditions. In addition, we elucidated the underlying mechanisms of growth promotion in salt-sensitive (SS) and salt-tolerant (ST) plants derived from the interaction with endophytic bacteria under no-stress and salinity-stress conditions. Specifically, this work encompassed 42 peer-reviewed articles, a total of 77 experiments, and 24 different bacterial genera. On average, endophytic bacterial inoculation increased morphological parameters. Moreover, the effect of endophytic bacteria on the total dry biomass, number of leaves, root length, shoot length, and germination rate was generally greater under salinity-stress conditions than no-stress conditions. On a physiological level, the relative better performance of the bacterial inoculants under the salinity-stress condition was associated with the increase in total chlorophyll and chlorophyll-b, as well as with the decrease of 1-aminocylopropane-1-carboxylate concentration. Moreover, under the salinity-stress condition, bacterial inoculation conferred a significantly higher increase in root K+ concentration and decrease in leaf Na+ concentration than under the no-stress condition. In SS plants, bacterial inoculation induced a higher increase in chlorophyll-b and superoxide dismutase activity, as well as a higher decrease in abscisic acid content, than in ST plants. Under salinity-stress, endophytic bacterial inoculation increased root K+ concentration in both SS and ST plants but decreased root Na+ concentration only in ST plants. Overall, this meta-analysis suggests that endophytic bacterial inoculation is beneficial under both no salinity-stress and salinity-stress conditions, but the magnitude of benefit is definitely higher under salinity-stress conditions and varies with the salt tolerance level of plants.

The Perception of Rhizosphere Bacterial Communication Signals Leads to Transcriptome Reprogramming in Lysobacter capsici AZ78, a Plant Beneficial Bacterium.

The rhizosphere is a dynamic region governed by complex microbial interactions where diffusible communication signals produced by bacteria continuously shape the gene expression patterns of individual species and regulate fundamental traits for adaptation to the rhizosphere environment. Lysobacter spp. are common bacterial inhabitants of the rhizosphere and have been frequently associated with soil disease suppressiveness. However, little is known about their ecology and how diffusible communication signals might affect their behavior in the rhizosphere. To shed light on the aspects determining rhizosphere competence and functioning of Lysobacter spp., we carried out a functional and transcriptome analysis on the plant beneficial bacterium Lysobacter capsici AZ78 (AZ78) grown in the presence of the most common diffusible communication signals released by rhizosphere bacteria. Mining the genome of AZ78 and other Lysobacter spp. showed that Lysobacter spp. share genes involved in the production and perception of diffusible signal factors, indole, diffusible factors, and N-acyl-homoserine lactones. Most of the tested diffusible communication signals (i.e., indole and glyoxylic acid) influenced the ability of AZ78 to inhibit the growth of the phytopathogenic oomycete Pythium ultimum and the Gram-positive bacterium Rhodococcus fascians. Moreover, RNA-Seq analysis revealed that nearly 21% of all genes in AZ78 genome were modulated by diffusible communication signals. 13-Methyltetradecanoic acid, glyoxylic acid, and 2,3-butanedione positively influenced the expression of genes related to type IV pilus, which might enable AZ78 to rapidly colonize the rhizosphere. Moreover, glyoxylic acid and 2,3-butanedione downregulated tRNA genes, possibly as a result of the elicitation of biological stress responses. On its behalf, indole downregulated genes related to type IV pilus and the heat-stable antifungal factor, which might result in impairment of twitching motility and antibiotic production in AZ78. These results show that diffusible communication signals may affect the ecology of Lysobacter spp. in the rhizosphere and suggest that diffusible communication signals might be used to foster rhizosphere colonization and functioning of plant beneficial bacteria belonging to the genus Lysobacter.

The Differential Growth Inhibition of Phytophthora spp. Caused by the Rare Sugar Tagatose Is Associated With Species-Specific Metabolic and Transcriptional Changes.

Tagatose is a rare sugar with no negative impacts on human health and selective inhibitory effects on plant-associated microorganisms. Tagatose inhibited mycelial growth and negatively affected mitochondrial processes in Phytophthora infestans, but not in Phytophthora cinnamomi. The aim of this study was to elucidate metabolic changes and transcriptional reprogramming activated by P. infestans and P. cinnamomi in response to tagatose, in order to clarify the differential inhibitory mechanisms of tagatose and the species-specific reactions to this rare sugar. Phytophthora infestans and P. cinnamomi activated distinct metabolic and transcriptional changes in response to the rare sugar. Tagatose negatively affected mycelial growth, sugar content and amino acid content in P. infestans with a severe transcriptional reprogramming that included the downregulation of genes involved in transport, sugar metabolism, signal transduction, and growth-related process. Conversely, tagatose incubation upregulated genes related to transport, energy metabolism, sugar metabolism and oxidative stress in P. cinnamomi with no negative effects on mycelial growth, sugar content and amino acid content. Differential inhibitory effects of tagatose on Phytophthora spp. were associated with an attempted reaction of P. infestans, which was not sufficient to attenuate the negative impacts of the rare sugar and with an efficient response of P. cinnamomi with the reprogramming of multiple metabolic processes, such as genes related to glucose transport, pentose metabolism, tricarboxylic acid cycle, reactive oxygen species detoxification, mitochondrial and alternative respiration processes. Knowledge on the differential response of Phytophthora spp. to tagatose represent a step forward in the understanding functional roles of rare sugars.

Lysobacter enzymogenes antagonizes soilborne bacteria using the type IV secretion system.

Soil microbiome comprises numerous microbial species that continuously interact with each other. Among the modes of diverse interactions, cell-cell killing may play a key role in shaping the microbiome composition. Bacteria deploy various secretion systems to fend off other microorganisms and Type IV Secretion System (T4SS) in pathogenic bacteria was shown to function as a contact-dependent, inter-bacterial killing system only recently. The present study investigated the role played by T4SS in the killing behaviour of the soilborne biocontrol bacterium Lysobacter enzymogenes OH11. Results showed that L. enzymogenes OH11 genome encompasses genes encoding all the components of T4SS and effectors potentially involved in inter-bacterial killing system. Generation of knock-out mutants revealed that L. enzymogenes OH11 uses T4SS as the main contact-dependent weapon against other soilborne bacteria. The T4SS-mediated killing behaviour of L. enzymogenes OH11 decreased the antibacterial and antifungal activity of two Pseudomonas spp. but at the same time, protected carrot from infection by Pectobacterium carotovorum. Overall, this study showed for the first time the involvement of T4SS in the killing behaviour of L. enzymogenes and its impact on the multiple interactions occurring in the soil microbiome.

Characterisation of the Antibiotic Profile of Lysobacter capsici AZ78, an Effective Biological Control Agent of Plant Pathogenic Microorganisms.

Determining the mode of action of microbial biocontrol agents plays a key role in their development and registration as commercial biopesticides. The biocontrol rhizobacterium Lysobacter capsici AZ78 (AZ78) is able to inhibit a vast array of plant pathogenic oomycetes and Gram-positive bacteria due to the release of antimicrobial secondary metabolites. A combination of MALDI-qTOF-MSI and UHPLC-HRMS/M was applied to finely dissect the AZ78 metabolome and identify the main secondary metabolites involved in the inhibition of plant pathogenic microorganisms. Under nutritionally limited conditions, MALDI-qTOF-MSI revealed that AZ78 is able to release a relevant number of antimicrobial secondary metabolites belonging to the families of 2,5-diketopiperazines, cyclic lipodepsipeptides, macrolactones and macrolides. In vitro tests confirmed the presence of secondary metabolites toxic against Pythium ultimum and Rhodococcus fascians in AZ78 cell-free extracts. Subsequently, UHPLC-HRMS/MS was used to confirm the results achieved with MALDI-qTOF-MSI and investigate for further putative antimicrobial secondary metabolites known to be produced by Lysobacter spp. This technique confirmed the presence of several 2,5-diketopiperazines in AZ78 cell-free extracts and provided the first evidence of the production of the cyclic depsipeptide WAP-8294A2 in a member of L. capsici species. Moreover, UHPLC-HRMS/MS confirmed the presence of dihydromaltophilin/Heat Stable Antifungal Factor (HSAF) in AZ78 cell-free extracts. Due to the production of HSAF by AZ78, cell-free supernatants were effective in controlling Plasmopara viticola on grapevine leaf disks after exposure to high temperatures. Overall, our work determined the main secondary metabolites involved in the biocontrol activity of AZ78 against plant pathogenic oomycetes and Gram-positive bacteria. These results might be useful for the future development of this bacterial strain as the active ingredient of a microbial biopesticide that might contribute to a reduction in the chemical input in agriculture.

Ecological Role of Volatile Organic Compounds Emitted by Pantoea agglomerans as Interspecies and Interkingdom Signals.

Volatile organic compounds (VOCs) play an essential role in microbe-microbe and plant-microbe interactions. We investigated the interaction between two plant growth-promoting rhizobacteria, and their interaction with tomato plants. VOCs produced by Pantoea agglomerans MVC 21 modulates the release of siderophores, the solubilisation of phosphate and potassium by Pseudomonas (Ps.) putida MVC 17. Moreover, VOCs produced by P. agglomerans MVC 21 increased lateral root density (LRD), root and shoot dry weight of tomato seedlings. Among the VOCs released by P. agglomerans MVC 21, only dimethyl disulfide (DMDS) showed effects similar to P. agglomerans MVC 21 VOCs. Because of the effects on plants and bacterial cells, we investigated how P. agglomerans MVC 21 VOCs might influence bacteria-plant interaction. Noteworthy, VOCs produced by P. agglomerans MVC 21 boosted the ability of Ps. putida MVC 17 to increase LRD and root dry weight of tomato seedlings. These results could be explained by the positive effect of DMDS and P. agglomerans MVC 21 VOCs on acid 3-indoleacetic production in Ps. putida MVC 17. Overall, our results clearly indicated that P. agglomerans MVC 21 is able to establish a beneficial interaction with Ps. putida MVC 17 and tomato plants through the emission of DMDS.

Pseudomonas chlororaphis metabolites as biocontrol promoters of plant health and improved crop yield.

The Pseudomonas fluorescens complex contains at least eight phylogenetic groups and each of these includes several bacterial species sharing ecological and physiological traits. Pseudomonas chlororaphis classified in a separate group is represented by three different subspecies that show distinctive traits exploitable for phytostimulation and biocontrol of phytopathogens. The high level of microbial competitiveness in soil as well as the effectiveness in controlling several plant pathogens and pests can be related to the P. chlororaphis ability to implement different stimulating and toxic mechanisms in its interaction with plants and the other micro- and macroorganisms. Pseudomonas chlororaphis strains produce antibiotics, such as phenazines, pyrrolnitrine, 2-hexyl, 5-propyl resorcinol and hydrogen cyanide, siderophores such as pyoverdine and achromobactine and a complex blend of volatile organic compounds (VOCs) that effectively contribute to the control of several plant pathogens, nematodes and insects. Phenazines and some VOCs are also involved in the induction of systemic resistance in plants. This complex set of beneficial strategies explains the high increasing interest in P. chlororaphis for commercial and biotechnological applications. The aim of this review is to highlight the role of the different mechanisms involved in the biocontrol activity of P. chlororaphis strains.

Selection of plant growth promoting rhizobacteria sharing suitable features to be commercially developed as biostimulant products.

Plant biostimulants (PBs) are an eco-friendly alternative to chemical fertilisers because of their minimal or null impact on human health and environment, while ensuring optimal nutrient uptake and increase of crop yield, quality and tolerance to abiotic stress. Although there is an increasing interest on microbial biostimulants, the optimal procedure to select and develop them as commercial products is still not well defined. This work proposes and validates a procedure to select the best plant growth promoting rhizobacteria (PGPR) as potential active ingredients of commercial PBs. The stepwise screening strategy was designed based on literature analysis and consists of six steps: (i) determination of the target crop and commercial strategy, (ii) selection of growth media for the isolation of microbial candidates, (iii) screening for traits giving major agronomical advantages, (iv) screening for traits related to product development, (v) characterisation of the mode of action of PGPR and (vi) assessment of plant growth efficacy. The strategy was validated using a case study: PGPR combined with humic acids to be applied on tomato plants. Among 200 bacterial strains isolated from tomato rhizosphere, 39 % were able to grow in presence of humic acids and shared the ability to solubilise phosphate. After the screening for traits related to product development, only 6 % of initial bacterial strains were sharing traits suitable for the further development as potential PBs. In fact, the selected bacterial strains were able to produce high cell mass and tolerated drought, aspects important for the mass production and formulation. These bacterial strains were not able to produce antibiotics, establish pathogenic interaction with plants and did not belong to bacterial species associated to human, animal and plant diseases. Most importantly, five of the selected bacterial strains were able to promote tomato seedling vigour in experiments carried out in vitro. These bacterial strains were furtherly characterised for their ability to colonize effectively tomato plant roots, produce phytohormones and solubilise soil minerals. This characterisation led to the selection of two candidates that showed the ability to promote tomato plant growth in experiments carried out in greenhouse conditions. Overall, this work provides a flow diagram for the selection of PGPR candidates to be successfully developed and commercialized as PBs. The validation of the flow diagram led to the selection of two bacterial strains belonging to Pantoea and Pseudomonas genera, potential active ingredients of new commercial PBs.

Isolation of 2,5-diketopiperazines from Lysobacter capsici AZ78 with activity against Rhodococcus fascians.

Inhibitory activity of the biocontrol bacterial strain Lysobacter capsici AZ78 is related to the production of cyclo(l-Pro-l-Tyr), a 2,5-diketopiperazine with in vitro and in vivo toxic activity against Phytophthora infestans and Plasmopara viticola. Further investigation of culture filtrate organic extracts showed its ability to produce other 2,5-diketopiperazines. They were isolated and identified by spectroscopic (1H NMR and ESIMS) and physic (specific optical rotation) methods as cyclo(l-Pro-l-Val), cyclo(d-Pro-d-Phe), cyclo(l-Pro-l-Leu), and cyclo(d-Pro-l-Tyr). When tested against the phytopathogenic Gram-positive bacterium Rhodococcus fascians LMG 3605, cyclo(l-Pro-l-Val) showed a toxic activity similar to chloramphenicol at a comparable concentration. Overall, these data suggest that 2,5-diketopiperazines represent a class of metabolites characterizing the metabolome of L. capsici AZ78. Furthermore, the toxic activity showed by cyclo(l-Pro-l-Val) against R. fascians LMG 3605 broaden the spectrum activity of 2,5-diketopiperazines against phytopathogenic microorganisms enforcing their potential development as biopesticides.[Figure: see text].

Functional divergence of flagellar type III secretion system: A case study in a non-flagellated, predatory bacterium.

The lack of functional flagella and the ability to prey upon other microorganisms are well-known traits of Lysobacter enzymogenes, a plant beneficial bacterial species. Here, we report a possible link between these two traits in the model strain L. enzymogenes OH11 (OH11). The genome of OH11 encompasses several homologous genes involved in the flagellum formation but it lacks a functional fliC, encoding the flagellin. Despite the lack of the main component of the flagellum, OH11 genome includes genes involved in the flagellar type III secretion system (FT3SS), which is commonly deployed by flagellated bacteria to transport flagellar subunit proteins. To understand the role played by FT3SS in OH11, we showed that the remaining FT3SS genes were expressed under laboratory conditions. Subsequently, we showed that the identified FT3SS genes involved in the secretion of the hook-capping protein FlgD, suggesting OH11 likely possessed a functional FT3SS system. Blocking FT3SS in OH11 via inactivation of the ATPase FliI impaired the secretion of the proteins Le3970 (protease), Le4493 (ß-1,3-glucanase A) and Le1659 (halo acid dehalogenase family), that showed a toxic activity against the yeast Saccharomyces cerevisiae. The possible link between FT3SS and OH11 antagonism towards S. cerevisiae was also confirmed by loss of toxicity in both mutants of ΔfliI and ΔflhB that lacks the FT3SS structural gene flhB when co-cultured with the yeast strain. The design of synthetic proteins toxic against the Gram-negative bacterium Ralstonia solanacearum further supported the involvement of FT3SS in the ability of OH11 to parasitize other microorganisms. Overall, these results revealed a possible cooption of components of FT3SS system in the competition with other microorganisms in the plant beneficial bacterium OH11 and highlighted a functional divergence of FT3SS between flagellated and non-flagellated bacteria.

Volatile-Mediated Inhibitory Activity of Rhizobacteria as a Result of Multiple Factors Interaction: The Case of Lysobacter capsici AZ78.

Plant beneficial rhizobacteria may antagonize soilborne plant pathogens by producing a vast array of volatile organic compounds (VOCs). The production of these compounds depends on the medium composition used for bacterial cell growth. Accordingly, Lysobacter capsici AZ78 (AZ78) grown on a protein-rich medium was previously found to emit volatile pyrazines with toxic activity against soilborne phypathogenic fungi and oomycetes. However, the discrepancy between the quantity of pyrazines in the gaseous phase and the minimum quantity needed to achieve inhibition of plant pathogens observed, lead us to further investigate the volatile-mediated inhibitory activity of AZ78. Here, we show that, besides VOCs, AZ78 cells produce ammonia in increased amounts when a protein-rich medium is used for bacterial growth. The production of this volatile compound caused the alkalinization of the physically separated culture medium where Rhizoctonia solani was inoculated subsequently. Results achieved in this work clearly demonstrate that VOC, ammonia and the growth medium alkalinization contribute to the overall inhibitory activity of AZ78 against R. solani. Thus, our findings suggest that the volatile-mediated inhibitory activity of rhizobacteria in protein-rich substrates can be regarded as a result of multiple factors interaction, rather than exclusively VOCs production.

Humic Acid Enhances the Growth of Tomato Promoted by Endophytic Bacterial Strains Through the Activation of Hormone-, Growth-, and Transcription-Related Processes.

Plant growth-promoting bacteria (PGPB) are promising alternatives in the reduction of the use of chemical fertilizers. Likewise, humic acid (HA) can improve plant growth and/or the establishment of endophytic PGPB. Although the effects of PGPB colonization or HA treatment have been studied separately, little information is available on plant response to the combined applications of PGPB and HA. Thus, the aim of this work was to understand the physiological effects, bacterial colonization and transcriptional responses activated by endophytic bacterial strains in tomato roots and shoots in the absence (control condition) and presence of HA (HA condition). Tomato shoot length was promoted by seed inoculation with Paraburkholderia phytofirmans PsJN, Pantoea agglomerans D7G, or Enterobacter sp. 32A in the presence of HA, indicating a possible complementation of PGPB and HA effects. Tomato colonization by endophytic bacterial strains was comparable in the control and HA condition. The main transcriptional regulations occurred in tomato roots and the majority of differentially expressed genes (DEGs) was upregulated by endophytic bacterial strains in the HA condition. Half of the DEGs was modulated by two or three strains as possible common reactions to endophytic bacterial strains, involving protein metabolism, transcription, transport, signal transduction, and defense. Moreover, strain-specific tomato responses included the upregulation of signal transduction, transcription, hormone metabolism, protein metabolism, secondary metabolism, and defense processes, highlighting specific traits of the endophyte-tomato interaction. The presence of HA enhanced the upregulation of genes related to signal transduction, hormone metabolism, transcription, protein metabolism, transport, defense, and growth-related processes in terms of number of involved genes and fold change values. This study provides detailed information on HA-dependent enhancement of growth-related processes stimulated by endophytic bacterial strains in tomato plants and reports the optimized dosages, complementation properties and gene markers for the further development of efficient PGPB- and HA-based biostimulants.

Volatile Organic Compounds From Lysobacter capsici AZ78 as Potential Candidates for Biological Control of Soilborne Plant Pathogens.

The genus Lysobacter includes several bacterial species which show potential for being used in biological control of plant diseases. It was shown recently that several Lysobacter type strains produce volatile organic compounds (VOCs) which controlled the growth of Phytophthora infestans in vitro when the bacteria were grown on a protein rich medium. In the present study, Lysobacter capsici AZ78 (AZ78) has been tested for its potential to produce VOCs that may contribute to the bioactivity against soilborne plant pathogens. To this end, split Petri dish assays of bacterial cultures have been combined with GC-MS measurements with the aim to reveal the identity of the VOCs which inhibit the growth of Pythium ultimum Rhizoctonia solani, and Sclerotinia minor. While AZ78 completely suppressed the growth of P. ultimum and S. minor, the growth of R. solani was still reduced significantly. The GC-MS analysis revealed 22 VOCs to be produced by AZ78, the majority of which were (putatively) identified as mono- and dialkylated methoxypyrazines. Based on additional cultivation and GC-MS experiments, 2,5-dimethylpyrazine, 2-ethyl-3-methoxypyrazine and 2-isopropyl-3-methoxypyrazine were selected as presumable bioactive compounds. Further bioassays employing indirect exposure to standard solutions (1-10 mg per Petri dish) of the synthetic compounds via the gas phase, revealed that each of these pyrazines was able to suppress the growth of the pathogens under investigation. The results of this study highlight the possible future implementation of pyrazine derivatives in the control of soilborne plant diseases and further support the biocontrol potential of L. capsici AZ78.

Impact of spontaneous mutations on physiological traits and biocontrol activity of Pseudomonas chlororaphis M71.

Three morphological mutants (M71a, M71b, M71c) of the antagonist Pseudomonas chlororaphis M71, naturally arose during a biocontrol trial against the phytopathogenic fungus Fusarium oxysporum f.sp. radicis-lycopersisci. In this study, the three mutants were investigated to elucidate their role in the biocontrol of plant pathogens. M71a and M71b phenotypes were generated by a mutation in the two-component system GacS/GacA. The mutation determined an increase in siderophore production and an impaired ability to release proteases, to swarm, to produce phenazine and AHLs and to colonize tomato roots. In vitro antagonistic activity against different plant pathogens was partially reduced in M71a, while M71b resulted effective only against Pythium ultimum. Biocontrol efficacy against Fusarium oxysporum f.sp. radicis-lycopersisci, was partially reduced in M71a and completely lost in M71b. M71c phenotype was impaired in swarming motility, did not produce biofilms and its antagonistic activity was similar to the parental M71 strain. M71c showed an enhanced ability to colonize tomato roots, on which its progeny in part reverted to the M71 parental phenotype. Volatile organic compounds (VOCs) emitted by all four strains, inhibited the growth of Clavibacter michiganensis subsp. michiganensis and Seiridium cardinale in vitro. Real-time screening of VOCs by PTR-MS combined with GC-MS analysis, showed that methanethiol was the main component of the blend produced by all four M71 strains. However, the emissions of hydrogen cyanide, dimethyl disulfide, 1,3-butadiene and acetone were significantly affected by the three different mutations. These findings highlight that the simultaneous presence of different M71 phenotypes may improve, through the integration of different mechanisms, the ecological fitness and biocontrol efficacy of P. chlororaphis M71.

The Rare Sugar Tagatose Differentially Inhibits the Growth of Phytophthora infestans and Phytophthora cinnamomi by Interfering With Mitochondrial Processes.

Rare sugars are monosaccharides with limited availability in nature and their biological functions are largely unknown. Among them, tagatose was developed as a low-calorie sweetener and showed beneficial effects on human health. Tagatose is metabolized by only certain microbial taxa and inhibits the growth of important crop pathogens (e.g., Phytophthora infestans), but its mode of action and the microbial responses are unknown. The aim of this study was to understand the tagatose mode of action against Phytophthora spp., with the final aim of developing new plant protection products. Tagatose inhibited P. infestans growth in vitro and caused severe ultrastructural alterations, with the formation of circular and concentric mitochondrial cristae. Decreased ATP content and reduced oxygen consumption rate (OCR) were found in tagatose-incubated P. infestans as compared to the control, with the consequent accumulation of reactive oxygen species (ROS) and induction of genes related to apoptosis and oxidative stress response. On the other hand, tagatose did not, or only slightly, affect the growth, cellular ultrastructure and mitochondrial processes in Phytophthora cinnamomi, indicating a species-specific response to this rare sugar. The mode of action of tagatose against P. infestans was mainly based on the inhibition of mitochondrial processes and this rare sugar seems to be a promising active substance for the further development of eco-friendly fungicides, thanks to its anti-nutritional properties on some phytopathogens and low risk for human health.

The rhizosphere signature on the cell motility, biofilm formation and secondary metabolite production of a plant-associated Lysobacter strain.

Lysobacter spp. are common bacterial inhabitants of the rhizosphere of diverse plant species. However, the impact of the rhizosphere conditions on their physiology is still relatively understudied. To provide clues on the behaviour of Lysobacter spp. in this ecological niche, we investigated the physiology of L. capsici AZ78 (AZ78), a biocontrol strain isolated from tobacco rhizosphere, on a common synthetic growth medium (LBA) and on a growth medium containing components of the plant rhizosphere (RMA). The presence of a halo surrounding the AZ78 colony on RMA was a first visible effect related to differences in growth medium composition and it corresponded to the formation of a large outer ring. The lower quantity of nutrients available in RMA as compared with LBA was associated to a higher expression of a gene encoding cAMP-receptor-like protein (Clp), responsible for cell motility and biofilm formation regulation. AZ78 cells on RMA were motile, equipped with cell surface appendages and organised in small groups embedded in a dense layer of fibrils. Metabolic profiling by mass spectrometry imaging revealed increased diversity of analytes produced by AZ78 on RMA as compared with LBA. In particular, putative cyclic lipodepsipeptides, polycyclic tetramate macrolactams, cyclic macrolactams and other putative secondary metabolites with antibiotic activity were identified. Overall, the results obtained in this study shed a light on AZ78 potential to thrive in the rhizosphere by its ability to move, form biofilm and release secondary metabolites.

Ecological impact of a rare sugar on grapevine phyllosphere microbial communities.

Plants host a complex microbiota inside or outside their tissues, and phyllosphere microorganisms can be influenced by environmental, nutritional and agronomic factors. Rare sugars are defined as monosaccharides with limited availability in nature and they are metabolised by only few certain microbial taxa. Among rare sugars, tagatose (TAG) is a low-calories sweetener that stimulates and inhibits beneficial and pathogenic bacteria in the human gut microbiota, respectively. Based on this differential effect on human-associated microorganisms, we investigated the effect of TAG treatments on the grapevine phyllosphere microorganisms to evaluate whether it can engineer the microbiota and modify the ratio between beneficial and pathogenic plant-associated microorganisms. TAG treatments changed the structure of the leaf microbiota and they successfully reduced leaf infections of downy mildew (caused by Plasmopara viticola) and powdery mildew (caused by Erysiphe necator) under field conditions. TAG increased the relative abundance of indigenous beneficial microorganisms, such as some potential biocontrol agents, which could partially contribute to disease control. The taxonomic composition of fungal and bacterial leaf populations differed according to grapevine locations, therefore TAG effects on the microbial structure were influenced by the composition of the originally residing microbiota. TAG is a promising biopesticide that could shift the balance of pathogenic and beneficial plant-associated microorganisms, suggesting selective nutritional/anti-nutritional properties for some specific taxa. More specifically, TAG displayed possible plant prebiotic effects on the phyllosphere microbiota and this mechanism of action could represent a novel strategy that can be further developed for sustainable plant protection.