Summary Plant Growth Promoting Microorganisms or Plant Probiotics (PGPMs) constitute a promising solution for agricultural sustainability. The concept that inoculation of PGPM mixtures may perform better in enhancing agricultural production than single strain application dates back to the discovery of plant growth rhizobacteria (PGPR) and is gaining ground in our days. This shift is highlighted by the increasing number of research publications dealing with the positive impact of microbial mixtures in promoting plant growth, controlling plant pathogens, as well as providing abiotic stress tolerance. The continuous deposition of patents as well as commercially available formulations concerning bioprotective and/or biostimulant multistrain mixtures also underlines this shift. A major issue in engineering an effective and consistent synthetic multistrain mixture appears to be the compatibility of its components. The present review provides a thorough literature survey supporting the view that treatment of plants with compatible multistrain mixtures generally exerts a better effect in plant growth and health than single-strain inoculation. Our study focuses on multistrain mixtures based on Pseudomonas, Bacillus and beneficial fungal strains, while commercial products are also being referred.

Botrytis bunch rot caused by Botrytis cinerea is one of the most economically significant post-harvest diseases of grapes. In the present study, we showed that the bacterial strain Bvel1 is phylogenetically affiliated to Bacillus velezensis species. The strain Bvel1 and its secreted metabolites exerted an antifungal activity, under in vitro conditions, against B. cinerea. UHPLC–HRMS chemical analysis revealed that iturin A2, surfactin-C13 and -C15, oxydifficidin, bacillibactin, L-dihydroanticapsin, and azelaic acid were among the metabolites secreted by Bvel1. Treatment of wounded grape berries with Bacillus sp. Bvel1 cell culture was effective for controlling grey mold ingress and expansion in vivo. The effectiveness of this biological control agent was a function of the cell culture concentration of the antagonist applied, while preventive treatment proved to be more effective compared to curative. The strain Bvel1 exhibited an adequate colonization efficiency in wounded grapes. The whole-genome phylogeny, combined with ANI and dDDH analyses, provided compelling evidence that the strain Bvel1 should be taxonomically classified as Bacillus velezensis. Genome mining approaches showed that the strain Bvel1 harbors 13 antimicrobial biosynthetic gene clusters, including iturin A, fengycin, surfactin, bacilysin, difficidin, bacillaene, and bacillibactin. The results provide new insights into the understanding of the endophytic Bacillus velezensis Bvel1 biocontrol mechanism against post-harvest fungal pathogens, including bunch rot disease in grape berries.

Summary Plant-associated environments harbor a huge number of diverse bacteria that compete and/or cooperate for the occupation of the most nutrient-rich ecological niches. Motility, a common trait among bacteria, has long been assumed to provide a survival advantage to skilful bacteria in invading these environments. Bacterial surface motility, such as swarming, a flagella-driven type of surface movement, although mostly observed and studied on agar substrates, is emerging as a major trait involved in many functions of plant-associated bacteria in regard to their ability to colonize and spread on their host. In this review, we address some novel swarming motility strategies, which enable bacteria to colonize, disperse and compete in plant surfaces.

The endophytic bacterial strain Hil4 was isolated from leaves of the medicinal plant Hypericum hircinum. It exhibited antifungal activity against Botrytis cinerea and a plethora of plant growth promoting traits in vitro. Whole genome sequencing revealed that it belongs to Bacillus halotolerans and possesses numerous secondary metabolite biosynthetic gene clusters and genes involved in plant growth promotion, colonization, and plant defense elicitation. The Mojavensin cluster was present in the genome, making this strain novel among plant-associated B. halotolerans strains. Extracts of secreted agar-diffusible compounds from single culture secretome extracts and dual cultures with B. cinerea were bioactive and had the same antifungal pattern on TLC plates after bioautography. UHPLC-HRMS analysis of the single culture secretome extract putatively annotated the consecutively produced antimicrobial substances and ISR elicitors. The isolate also proved efficient in minimizing the severity of gray mold post-harvest disease on table grape berries, as well as cherry tomatoes. Finally, it positively influenced the growth of Arabidopsis thaliana Col-0 and Solanum lycopersicum var. Chondrokatsari Messinias after seed biopriming in vitro. Overall, these results indicate that the B. halotolerans strain Hil4 is a promising novel plant growth promoting and biocontrol agent, and can be used in future research for the development of biostimulants and/or biological control agents.

The endophytic strain Cal.l.30, isolated from the medicinal plant Calendula officinalis, was selected among seven Bacillus strains with plant growth promoting activity and strong biological potential against the postharvest fungal pathogen Botrytis cinerea. Treatment by inoculating Cal.l.30 bacterial cell culture or cell free supernatant on harvested grapes and cherry tomato fruits, significantly reduced gray mold disease severity index and disease incidence. Based on 16S rRNA sequence analysis and whole genome phylogeny, Cal.l.30 was identified as Bacillus halotolerans. Genome mining revealed that B. halotolerans Cal.l.30 is endowed with a diverse arsenal of secondary metabolite biosynthetic gene clusters (SM-BGCs) responsible for metabolite production with antimicrobial properties. A sub-set of the identified SM-BGCs (mojavensin A, ‘bacillunoic acid’) appears to be the result of recent horizontal gene transfer events. Its genome was also mined for CAZymes associated with antifungal activity. Further UHPLC-HRMS analysis indicated that Cal.l.30 synthesizes and secretes secondary metabolites with antimicrobial activity, including the lipopeptides, fengycin, surfactin and mojavensin A, bacillaene isoforms, L-dihydroanticapsin and bacillibactin. Other compounds with known antimicrobial activity were also detected, such as azelaic acid, 15- hydroxypentadecanoid acid and 2-hydroxyphenylacetic acid. The genomic and metabolomic features of the B. halotolerans Cal.l.30 provided new perspectives on the exploitation of novel Bacillus sp. as a biocontrol agent.

The application of beneficial bacteria may present an alternative approach to chemical plant protection and fertilization products as they enhance growth and resistance to biotic and abiotic stresses. Plant growth-promoting bacteria are found in the rhizosphere, epiphytically or endophytically (Plant Growth Promoting Endophytic Bacteria, PGPEB). In the present study, 36 out of 119 isolated endophytic bacterial strains from roots, leaves and flowers of the pharmaceutical plant Calendula officinalis were further identified and classified into Bacillus, Pseudomonas, Pantoea, Stenotrophomonas and Rhizobium genera. Selected endophytes were evaluated depending on positive reaction to different plant growth promoting (PGP) traits, motility, survival rate and inhibition of phytopathogenic fungi in vitro and ex vivo (tomato fruit). Bacteria were further assessed for their plant growth effect on Arabidopsis thaliana seedlings and on seed bio-primed tomato plantlets, in vitro. Our results indicated that many bacterial endophytes increased seed germination, promoted plant growth and changed root structure by increasing lateral root density and length and root hair formation. The most promising antagonistic PGPEB strains (Cal.r.29, Cal.l.30, Cal.f.4, Cal.l.11, Cal.f.2.1, Cal.r.19 and Cal.r.11) are indicated as effective biological control agents (BCA) against Botrytis cinerea on detached tomato fruits. Results underlie the utility of beneficial endophytic bacteria for sustainable and efficient crop production and disease control.

Evaluating microbial-based alternatives to conventional fungicides and biofertilizers enables us to gain a deeper understanding of the biocontrol and plant growth-promoting activities. Two genetically distinct Bacillus halotolerans strains (Cal.l.30, Cal.f.4) were evaluated for the levels of their compatibility. They were applied individually or in combination under in vitro and greenhouse conditions, using seed bio-priming and soil drenching as inoculum delivery systems, for their plant growth-promoting effect. Our data indicate that application of Cal.l.30 and Cal.f.4 as single strains and as a mixture significantly enhanced growth parameters of Arabidopsis and tomato plants. We investigated whether seed and an additional soil treatment with these strains could induce the expression of defense-related genes in leaves of young tomato seedling plants. These treatments mediated a long lasting, bacterial-mediated, systemic-induced resistance as evidenced by the high levels of expression of RP3, ACO1 and ERF1 genes in the leaves of young tomato seedlings. Furthermore, we presented data showing that seed and soil treatment with B. halotolerans strains resulted in an effective inhibition of Botrytis cinerea attack and development on tomato leaves. Our findings highlighted the potential of B. halotolerans strains as they combine both direct antifungal activity against plant pathogens and the ability to prime plant innate immunity and enhance plant growth.

Deltamethrin and imidacloprid are commonly used insecticides for controlling sub-sucking insects in greenhouses. However, their application may cause sublethal effects on the aphid coccinellid predator Coccinella septempunctata (Coleoptera: Coccinellidae). Here, we study (i) the toxicity and the effect of two sublethal doses (LD10 and LD30) of deltamethrin and imidacloprid on C. septempunctata in a laboratory microcosm and (ii) the residual toxicity of the two insecticides in a greenhouse. The results showed that both insecticides reduced fecundity, longevity, the intrinsic rate of increase, the finite rate of increase and the net reproductive rate. However, the developmental time of the fourth instar larvae was prolonged by both insecticides at LD10 and LD30. Deltamethrin residues were toxic 21 DAT (days after treatment) to C. septempunctata fourth instar larvae. In contrast, imidacloprid began in the slightly harmful category (75%) 1 DAT and declined to the harmless category (18.33%) 21 DAT. These results indicate that deltamethrin and imidacloprid have potential risks to C. septempunctata. This study provides information to guide the development of integrated pest management (IPM) strategies in greenhouses.