Paraburkholderia phytofirmans PsJN delays Botrytis cinerea development on grapevine inflorescences.

Grapevine flowering is an important stage in the epidemiology of Botrytis cinerea, the causal agent of gray mold disease. To prevent infection and to minimize postharvest losses, the control of this necrotrophic fungus is mainly based on chemical fungicides application. However, there is a growing interest in other control alternatives. Among them, the use of beneficial microorganisms appears as an eco-friendly strategy. This study aims to investigate the effect of Paraburkholderia phytofirmans PsJN, root-inoculated or directly sprayed on fruiting cuttings inflorescences to control B. cinerea growth. For this purpose, quantification by real time PCR of Botrytis development, direct effect of PsJN on fungal spore germination and chemotaxis were assayed. Our results showed a significant protective effect of PsJN only by direct spraying on inflorescences. Moreover, we demonstrated an inhibition exerted by PsJN on Botrytis spore germination, effective when there was a direct contact between the two microorganisms. This study showed that PsJN is positively attracted by the pathogenic fungus B. cinerea and forms a biofilm around the fungal hyphae in liquid co-culture. Finally, microscopic observations on fruit cuttings revealed a co-localization of both beneficial and pathogenic microorganisms on grapevine receptacle and stigma that might be correlated with the protective effect induced by PsJN against B. cinerea via a direct antimicrobial effect. Taking together, our findings allowed us to propose PsJN as a biofungicide to control grapevine gray mold disease.

Biofilm-Constructing Variants of Paraburkholderia phytofirmans PsJN Outcompete the Wild-Type Form in Free-Living and Static Conditions but Not In Planta.

Members of the genus Burkholderia colonize diverse ecological niches. Among the plant-associated strains, Paraburkholderia phytofirmans PsJN is an endophyte with a broad host range. In a spatially structured environment (unshaken broth cultures), biofilm-constructing specialists of P. phytofirmans PsJN colonizing the air-liquid interface arose at high frequency. In addition to forming a robust biofilm in vitro and in planta on Arabidopsis roots, those mucoid phenotypic variants display a reduced swimming ability and modulate the expression of several microbe-associated molecular patterns (MAMPs), including exopolysaccharides (EPS), flagellin, and GroEL. Interestingly, the variants induce low PR1 and PDF1.2 expression compared to that of the parental strain, suggesting a possible evasion of plant host immunity. We further demonstrated that switching from the planktonic to the sessile form did not involve quorum-sensing genes but arose from spontaneous mutations in two genes belonging to an iron-sulfur cluster: hscA (encoding a cochaperone protein) and iscS (encoding a cysteine desulfurase). A mutational approach validated the implication of these two genes in the appearance of variants. We showed for the first time that in a heterogeneous environment, P. phytofirmans strain PsJN is able to rapidly diversify and coexpress a variant that outcompete the wild-type form in free-living and static conditions but not in plantaIMPORTANCEParaburkholderia phytofirmans strain PsJN is a well-studied plant-associated bacterium known to induce resistance against biotic and abiotic stresses. In this work, we described the spontaneous appearance of mucoid variants in PsJN from static cultures. We showed that the conversion from the wild-type (WT) form to variants (V) correlates with an overproduction of EPS, an enhanced ability to form biofilm in vitro and in planta, and a reduced swimming motility. Our results revealed also that these phenotypes are in part associated with spontaneous mutations in an iron-sulfur cluster. Overall, the data provided here allow a better understanding of the adaptive mechanisms likely developed by P. phytofirmans PsJN in a heterogeneous environment.

On a Cold Night: Transcriptomics of Grapevine Flower Unveils Signal Transduction and Impacted Metabolism.

Low temperature is a critical environmental factor limiting plant productivity, especially in northern vineyards. To clarify the impact of this stress on grapevine flower, we used the Vitis array based on Roche-NimbleGen technology to investigate the gene expression of flowers submitted to a cold night. Our objectives were to identify modifications in the transcript levels after stress and during recovery. Consequently, our results confirmed some mechanisms known in grapes or other plants in response to cold stress, notably, (1) the pivotal role of calcium/calmodulin-mediated signaling; (2) the over-expression of sugar transporters and some genes involved in plant defense (especially in carbon metabolism), and (3) the down-regulation of genes encoding galactinol synthase (GOLS), pectate lyases, or polygalacturonases. We also identified some mechanisms not yet known to be involved in the response to cold stress, i.e., (1) the up-regulation of genes encoding G-type lectin S-receptor-like serine threonine-protein kinase, pathogen recognition receptor (PRR5), or heat-shock factors among others; (2) the down-regulation of Myeloblastosis (MYB)-related transcription factors and the Constans-like zinc finger family; and (3) the down-regulation of some genes encoding Pathogen-Related (PR)-proteins. Taken together, our results revealed interesting features and potentially valuable traits associated with stress responses in the grapevine flower. From a long-term perspective, our study provides useful starting points for future investigation.

Paraburkholderia phytofirmans PsJN-Plants Interaction: From Perception to the Induced Mechanisms.

The use of plant-associated bacteria has received many scientific and economic attention as an effective and alternative method to reduce the chemical pesticides use in agriculture. The genus Burkholderia includes at least 90 species including pathogenic strains, plant pathogens, as well as plant beneficial species as those related to Paraburkholderia, which has been reported to be associated with plants and exerts a positive effect on plant growth and fitness. Paraburkholderia phytofirmans PsJN, a beneficial endophyte able to colonize a wide range of plants, is an established model for plant-associated endophytic bacteria. Indeed, in addition to its plant growth promoting ability, it can also induce plant resistance against biotic as well as abiotic stresses. Here, we summarized an inventory of knowledge on PsJN-plant interaction, from the perception to the resistance mechanisms induced in the plant by a way of the atypical colonization mode of this endophyte. We also have carried out an extensive genome analysis to identify all gene clusters which contribute to the adaptive mechanisms under different environments and partly explaining the high ecological competence of P. phytofirmans PsJN.

How Streptomyces anulatus Primes Grapevine Defenses to Cope with Gray Mold: A Study of the Early Responses of Cell Suspensions.

Gray mold, caused by Botrytis cinerea, is one of the most destructive diseases of grapevine and is controlled with an intense application of fungicides. As alternatives to chemicals, beneficial microbes may promote plant health by stimulating the plant's immune system. An actinomycete, Streptomyces anulatus S37, has been screened from the rhizosphere microbiome of healthy Vitis vinifera on the basis of its ability to promote grapevine growth and to induce resistance against various phytopathogens, including B. cinerea. However, molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain unknown. This study focuses on local defense events induced in grapevine cells during interactions with S. anulatus S37 before and after pathogen challenge. We demonstrated that S. anulatus S37 induced early responses including oxidative burst, extracellular alkalinization, activation of protein kinases, induction of defense gene expression and phytoalexin accumulation, but not the programmed cell death. Interestingly, upon challenge with the B. cinerea, the S. anulatus S37 primed grapevine cells for enhanced defense reactions with a decline in cell death. In the presence of the EGTA, a calcium channel inhibitor, the induced oxidative burst, and the protein kinase activity were inhibited, but not the extracellular alkalinization, suggesting that Ca2+ may also contribute upstream to the induced defenses. Moreover, desensitization assays using extracellular pH showed that once increased by S. anulatus S37, cells became refractory to further stimulation by B. cinerea, suggesting that grapevine cells perceive distinctly beneficial and pathogenic microbes.