Bacillus velezensis BE2 controls wheat and barley diseases by direct antagonism and induced systemic resistance.

Wheat and barley rank among the main crops cultivated on a global scale, providing the essential nutritional foundation for both humans and animals. Nevertheless, these crops are vulnerable to several fungal diseases, such as Septoria tritici blotch and net blotch, which significantly reduce yields by adversely affecting leaves and grain quality. To mitigate the effect of these diseases, chemical fungicides have proven to be genuinely effective; however, they impose a serious environmental burden. Currently, biocontrol agents have attracted attention as a sustainable alternative to fungicides, offering an eco-friendly option. The study aimed to assess the efficacy of Bacillus velezensis BE2 in reducing disease symptoms caused by Zymoseptoria tritici and Pyrenophora teres. This bacterium exhibited significant antagonistic effects in vitro by suppressing fungal development when pathogens and the beneficial strain were in direct confrontation. These findings were subsequently confirmed through microscopic analysis, which illustrated the strain's capacity to inhibit spore germination and mycelial growth in both pathogens. Additionally, the study analysed the cell-free supernatant of the bacterium using UPLC-MS (ultra-performance liquid chromatography-mass spectrometry). The results revealed that strain BE2 produces, among other metabolites, different families of cyclic lipopeptides that may be involved in biocontrol. Furthermore, the beneficial effects of strain BE2 in planta were assessed by quantifying the fungal DNA content directly at the leaf level after bacterization, using two different application methods (foliar and drenching). The results indicated that applying the beneficial bacterium at the root level significantly reduced pathogens pressure. Finally, gene expression analysis of different markers showed that BE2 application induced a priming effect within the first hours after infection. KEY POINTS: • BE2 managed Z. tritici and P. teres by direct antagonism and induced systemic resistance. • Strain BE2 produced seven metabolite families, including three cyclic lipopeptides. • Application of strain BE2 at the root level triggered plant defense mechanisms.

Deep Learning Based Barley Disease Quantification for Sustainable Crop Production.

Net blotch disease caused by Drechslera teres is a major fungal disease that affects barley (Hordeum vulgare) plants and can result in significant crop losses. In this study, we developed a deep-learning model to quantify net blotch disease symptoms on different days post-infection on seedling leaves using Cascade R-CNN (Region-Based Convolutional Neural Networks) and U-Net (a convolutional neural network) architectures. We used a dataset of barley leaf images with annotations of net blotch disease to train and evaluate the model. The model achieved an accuracy of 95% for cascade R-CNN in net blotch disease detection and a Jaccard index score of 0.99, indicating high accuracy in disease quantification and location. The combination of Cascade R-CNN and U-Net architectures improved the detection of small and irregularly shaped lesions in the images at 4-days post infection, leading to better disease quantification. To validate the model developed, we compared the results obtained by automated measurement with a classical method (necrosis diameter measurement) and a pathogen detection by real-time PCR. The proposed deep learning model could be used in automated systems for disease quantification and to screen the efficacy of potential biocontrol agents to protect against disease.

Exploration of the Diversity of Vicine and Convicine Derivatives in Faba Bean (Vicia faba L.) Cultivars: Insights from LC-MS/MS Spectra.

While numerous Fabaceae seeds are a good nutritional source of high-quality protein, the use of some species is hampered by toxic effects caused by exposure to metabolites that accumulate in the seeds. One such species is the faba or broad bean (Vicia faba L.), which accumulates vicine and convicine. These two glycoalkaloids cause favism, the breakdown of red blood cells in persons with a glucose-6-phosphate dehydrogenase deficiency. Because this is the most common enzyme deficiency worldwide, faba bean breeding efforts have focused on developing cultivars with low levels of these alkaloids. Consequently, quantification methods have been developed; however, they quantify vicine and convicine only and not the derivatives of these compounds that potentially generate the same bio-active molecules. Based on the recognition of previously unknown (con)vicine-containing compounds, we screened the fragmentation spectra of LC-MS/MS data from five faba bean cultivars using the characteristic fragments generated by (con)vicine. This resulted in the recognition of more than a hundred derivatives, of which 89 were tentatively identified. (Con)vicine was mainly derivatized through the addition of sugars, hydroxycinnamic acids, and dicarboxylic acids, with a group of compounds composed of two (con)vicine residues linked by dicarboxyl fatty acids. In general, the abundance profiles of the different derivatives in the five cultivars mimicked that of vicine and convicine, but some showed a derivative-specific profile. The description of the (con)vicine diversity will impact the interpretation of future studies on the biosynthesis of (con)vicine, and the content in potentially bio-active alkaloids in faba beans may be higher than that represented by the quantification of vicine and convicine alone.

Production of high molecular-ordered stilbene oligomers for the study of their biological activity: total synthesis, bio-catalyzed synthesis and production by plant systems.

Though the iconic stilbene resveratrol and its related dimers constitute a top storyline in the field of natural product research, resveratrol oligomers (condensation >2) have been left aside despite their higher biological activity compared to that of the monomers. This situation largely results from the difficulty of getting them in sufficient quantities to enable evaluation of their biological properties in vivo. We present here a synthetic and critical analysis of the methods used for the production of high molecular-ordered stilbene oligomers of potential biomedical interest, gathering the most salient data regarding the approaches employed to prepare them by total synthesis, use of biomimetic approaches or through plant systems.

Pseudomonas Lipopeptide-Mediated Biocontrol: Chemotaxonomy and Biological Activity.

Pseudomonas lipopeptides (Ps-LPs) play crucial roles in bacterial physiology, host-microbe interactions and plant disease control. Beneficial LP producers have mainly been isolated from the rhizosphere, phyllosphere and from bulk soils. Despite their wide geographic distribution and host range, emerging evidence suggests that LP-producing pseudomonads and their corresponding molecules display tight specificity and follow a phylogenetic distribution. About a decade ago, biocontrol LPs were mainly reported from the P. fluorescens group, but this has drastically advanced due to increased LP diversity research. On the one hand, the presence of a close-knit relationship between Pseudomonas taxonomy and the molecule produced may provide a startup toolbox for the delineation of unknown LPs into existing (or novel) LP groups. Furthermore, a taxonomy-molecule match may facilitate decisions regarding antimicrobial activity profiling and subsequent agricultural relevance of such LPs. In this review, we highlight and discuss the production of beneficial Ps-LPs by strains situated within unique taxonomic groups and the lineage-specificity and coevolution of this relationship. We also chronicle the antimicrobial activity demonstrated by these biomolecules in limited plant systems compared with multiple in vitro assays. Our review further stresses the need to systematically elucidate the roles of diverse Ps-LP groups in direct plant-pathogen interactions and in the enhancement of plant innate immunity.

Synthetic Mono-Rhamnolipids Display Direct Antifungal Effects and Trigger an Innate Immune Response in Tomato against Botrytis Cinerea.

Natural rhamnolipids are potential biocontrol agents for plant protection against bacterial and fungal diseases. In this work, we synthetized new synthetic mono-rhamnolipids (smRLs) consisting in a rhamnose connected to a simple acyl chain and differing by the nature of the link and the length of the lipid tail. We then investigated the effects of these ether, ester, carbamate or succinate smRL derivatives on Botrytis cinerea development, symptoms spreading on tomato leaves and immune responses in tomato plants. Our results demonstrate that synthetic smRLs are able to trigger early and late immunity-related plant defense responses in tomato and increase plant resistance against B. cinerea in controlled conditions. Structure-function analysis showed that chain length of the lipidic part and type of acyl chain were critical to smRLs immune activity and to the extent of symptoms caused by the fungus on tomato leaves.

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.

Impact of Paraburkholderia phytofirmans PsJN on Grapevine Phenolic Metabolism.

Phenolic compounds are implied in plant-microorganisms interaction and may be induced in response to plant growth-promoting rhizobacteria (PGPRs). Among PGPR, the beneficial bacterium Paraburkholderia phytofirmans PsJN was previously described to stimulate the growth of plants and to induce a better adaptation to both abiotic and biotic stresses. This study aimed to investigate the impact of PsJN on grapevine secondary metabolism. For this purpose, gene expression (qRT-PCR) and profiling of plant secondary metabolites (UHPLC-UV/DAD-MS QTOF) from both grapevine root and leaves were compared between non-bacterized and PsJN-bacterized grapevine plantlets. Our results showed that PsJN induced locally (roots) and systemically (leaves) an overexpression of PAL and STS and specifically in leaves the overexpression of all the genes implied in phenylpropanoid and flavonoid pathways. Moreover, the metabolomic approach revealed that relative amounts of 32 and 17 compounds in roots and leaves, respectively, were significantly modified by PsJN. Once identified to be accumulated in response to PsJN by the metabolomic approach, antifungal properties of purified molecules were validated in vitro for their antifungal effect on Botrytis cinerea spore germination. Taking together, our findings on the impact of PsJN on phenolic metabolism allowed us to identify a supplementary biocontrol mechanism developed by this PGPR to induce plant resistance against pathogens.

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.

Genome sequencing and traits analysis of Burkholderia strains reveal a promising biocontrol effect against grey mould disease in grapevine (Vitis vinifera L.).

Grey mould caused by Botrytis cinerea is among the most important disease affecting the production of grapevine worldwide. The high economical loss each year has led producers to become more dependent on chemical pesticides for protection. However, environmental impacts of the pesticides overuse have sparked crescent interest in developing alternative biocontrol methods. The use of plant-associated bacteria has, thus, received many attentions as a promising strategy for sustainable agriculture. Three strains, isolated from the rhizosphere of crops cultivated in the northeast of France, were evaluated for their antagonistic effect. They were found to exhibit an antagonistic effect against a set of phytopathogenic fungi. Phenotypic and molecular characterization showed that isolates belong to the genus Burkholderia. The genome sequencing and analysis of isolated strains revealed the presence of gene clusters coding for secondary metabolites potentially involved in the biocontrol. When the grapevine plantlets were infected with B. cinerea, all plants associated with isolated strains showed a significant protection against B. cinerea compared to non-inoculated plants. To understand the mechanisms contributing to the biocontrol effect of selected isolates, the production of reactive oxygen species (ROS) and the expression of several defense genes were investigated. The maximum accumulation of H2O2 was detected in the inoculated cell suspension medium 30 min after the challenge with B. cinerea. After pathogen challenge, results showed that grapevine cell culture inoculated with isolated strains exhibited significant over expression of defense markers genes PR5, PR10, and chit4c, in response to B. cinerea, confirming their priming effect.

Pseudomonas knackmussii MLR6, a rhizospheric strain isolated from halophyte, enhances salt tolerance in Arabidopsis thaliana.

AIMS: The study aimed for evaluate the efficacy of Pseudomonas knackmussii MLR6 on growth promotion, photosynthetic responses, pigment contents and gene expression of the plant model Arabidopsis thaliana under NaCl stress. METHODS AND RESULTS: The strain MLR6 was isolated from the rhizopshere of the halophyte Salsola tetrandra collected from a natural saline Algerian soil. Results showed the ability of MLR6 to induce plant growth promoting traits even under NaCl stress. The inoculation with MLR6 improved the stomatal conductance, the transpiration rate, the total chlorophyll and carotenoids contents under salt stress. It conferred also an increase of fresh/dry weight as well as plant height. MLR6 inoculation further provided a positive effect on cell membrane stability by reducing the electrolyte leakage and priming the ROS accumulation after the salt exposition. Additionally, the expression of NHX1, HKT1, SOS2, and SOS3 as well as SAG13 and PR1 was maintained in MLR6-bacterized plant at a similar level of controls. CONCLUSIONS: The inoculation of Arabidopsis thaliana with MLR6 improves plant growth and reduces damages caused by salt stress. SIGNIFICANCE AND IMPACT OF STUDY: The use of Pseudomonas knackmussii MLR6 appears as a promising strategy to improve the sustainable agriculture under saline conditions. This article is protected by copyright. All rights reserved.

Kluyveromyces marxianus, an Attractive Yeast for Ethanolic Fermentation in the Presence of Imidazolium Ionic Liquids.

Imidazolium ionic liquids (ILs) are promising solvents for lignocellulosic biomass (LCB) pretreatment and allow the achievement of higher ethanolic yields after enzymatic hydrolysis and ethanolic fermentation. However, residual ILs entrapped in pretreated biomass are often toxic for fermentative microorganisms, but interaction mechanisms between ILs and cells are still unknown. Here we studied the effects of 1-ethyl-3-methylimidazolium acetate [Emim][OAc] and 1-ethyl-3-methylimidazolium methylphosphonate [Emim][MeO(H)PO2] on Kluyveromyces marxianus, a thermotolerant ethanologenic yeast. Morphological impacts induced by ILs on K. marxianus were characterized by Scanning Electron Microscopy analysis and showed wrinkled, softened, and holed shapes. In Yeast-Malt-Dextrose (YMD) medium, K. marxianus tolerated IL additions up to 2% for [Emim][OAc] and 6% for [Emim][MeO(H)PO2]. Below these thresholds, some IL concentrations enhanced ethanolic yields up to +34% by switching the metabolic status from respiratory to fermentative. Finally, K. marxianus fermentation was applied on several substrates pretreated with [Emim][OAc] or [Emim][MeO(H)PO2] and enzymatically hydrolyzed: a model long fiber cellulose and two industrial LCBs, softwood (spruce) and hardwood (oak) sawdusts. The maximum ethanolic yields obtained were 1.8 to 3.9 times higher when substrates were pretreated with imidazolium ILs. Therefore K. marxianus is an interesting fermentative yeast in a second-generation bioethanol process implying IL pretreatment.

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus.

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.

Cross-talk between environmental stresses and plant metabolism during reproductive organ abscission.

In plants, flowering is a crucial process for reproductive success and continuity of the species through time. Fruit production requires the perfect development of reproductive structures. Abscission, a natural process, can occur to facilitate shedding of no longer needed, infected, or damaged organs. If stress occurs during flower development, abscission can intervene at flower level, leading to reduced yield. Flower abscission is a highly regulated developmental process simultaneously influenced and activated in response to exogenous (changing environmental conditions, interactions with microorganisms) and endogenous (physiological modifications) stimuli. During climate change, plant communities will be more susceptible to environmental stresses, leading to increased flower and fruit abscission, and consequently a decrease in fruit yield. Understanding the impacts of stress on the reproductive phase is therefore critical for managing future agricultural productivity. Here, current knowledge on flower/fruit abscission is summarized by focusing specifically on effects of environmental stresses leading to this process in woody plants. Many of these stresses impair hormonal balance and/or carbohydrate metabolism, but the exact mechanisms are far from completely known. Hormones are the abscission effectors and the auxin/ethylene balance is of particular importance. The carbohydrate pathway is the result of complex regulatory processes involving the balance between photosynthesis and mobilization of reserves. Hormones and carbohydrates together participate in complex signal transduction systems, especially in response to stress. The available data are discussed in relation to reproductive organ development and the process of abscission.

Distinct regulation in inflorescence carbohydrate metabolism according to grapevine cultivars during floral development.

Carbohydrate metabolism is important in plant sexual reproduction because sugar contents are determining factors for both flower initiation and floral organ development. In woody plants, flowering represents the most energy-consuming step crucial to reproductive success. Nevertheless, in these species, the photosynthesis performed by flowers supplies the carbon required for reproduction. In grapevine (Vitis vinifera), the inflorescence has a specific status because this organ imports carbohydrates at the same time as it exports photoassimilates. In this study, fluctuations in carbohydrate metabolism were monitored by analyzing gas exchanges, photosynthetic electron transport capacity, carbohydrate contents and some activities of carbohydrate metabolism enzymes, in the inflorescences of Pinot noir and Gewurztraminer, two cultivars with a different sensitivity to coulure phenomenon. Our results showed that photosynthetic activity and carbohydrate metabolism are clearly different and differently regulated during the floral development in the two cultivars. Indeed, the regulation of the linear electron flow and the cyclic electron flow is not similar. Moreover, the regulation of PSII activity, with a higher Y(NPQ)/Y(NO) ratio in Gewurztraminer, can be correlated with the higher protection of the photosynthetic chain and consequently with the higher yield under optimal conditions of this cultivar. At least, our results showed a higher photosynthetic activity and a better protection of PSI in Pinot noir during the floral development.

Enhancement of Salinity Stress Tolerance in Lettuce (Lactuca sativa L.) via Foliar Application of Nitric Oxide.

Salt stress negatively affects the growth, development, and yield of horticultural crops. Nitric oxide (NO) is considered a signaling molecule that plays a key role in the plant defense system under salt stress. This study investigated the impact of exogenous application of 0.2 mM of sodium nitroprusside (SNP, an NO donor) on the salt tolerance and physiological and morphological characteristics of lettuce (Lactuca sativa L.) under salt stress (25, 50, 75, and 100 mM). Salt stress caused a marked decrease in growth, yield, carotenoids and photosynthetic pigments in stressed plants as compared to control ones. Results showed that salt stress significantly affected the oxidative compounds (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX)) and non-oxidative compounds (ascorbic acid, total phenols, malondialdehyde (MDA), proline, and H2O2) in lettuce. Moreover, salt stress decreased nitrogen (N), phosphorous (P), and potassium ions (K+) while increasing Na ions (Na+) in the leaves of lettuce under salt stress. The exogenous application of NO increased ascorbic acid, total phenols, antioxidant enzymes (SOD, POD, CAT, and APX) and MDA content in the leaves of lettuce under salt stress. In addition, the exogenous application of NO decreased H2O2 content in plants under salt stress. Moreover, the exogenous application of NO increased leaf N in control, and leaf P and leaf and root K+ content in all treatments while decreasing leaf Na+ in salt-stressed lettuce plants. These results provide evidence that the exogenous application of NO on lettuce helps mitigate salt stress effects.

Plasmopara viticola the Causal Agent of Downy Mildew of Grapevine: From Its Taxonomy to Disease Management.

Plasmopara viticola (P. viticola, Berk. & M. A. Curtis; Berl. & De Toni) causing grapevine downy mildew is one of the most damaging pathogens to viticulture worldwide. Since its recognition in the middle of nineteenth century, this disease has spread from America to Europe and then to all grapevine-growing countries, leading to significant economic losses due to the lack of efficient disease control. In 1885 copper was found to suppress many pathogens, and is still the most effective way to control downy mildews. During the twentieth century, contact and penetrating single-site fungicides have been developed for use against plant pathogens including downy mildews, but wide application has led to the appearance of pathogenic strains resistant to these treatments. Additionally, due to the negative environmental impact of chemical pesticides, the European Union restricted their use, triggering a rush to develop alternative tools such as resistant cultivars breeding, creation of new active ingredients, search for natural products and biocontrol agents that can be applied alone or in combination to kill the pathogen or mitigate its effect. This review summarizes data about the history, distribution, epidemiology, taxonomy, morphology, reproduction and infection mechanisms, symptoms, host-pathogen interactions, host resistance and control of the P. viticola, with a focus on sustainable methods, especially the use of biocontrol agents.

Corrigendum: Genotypic Variation of Nitrogen Use Efficiency and Amino Acid Metabolism in Barley.

[This corrects the article DOI: 10.3389/fpls.2021.807798.].

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.

Pollen vacuoles and their significance.

Vacuoles of several types can be observed in pollen throughout its development. Their physiological significance reflects the complexity of the biological process leading to functional pollen grains. Vacuolisation always occurs during pollen development but when ripe pollen is shed the extensive translucent vacuoles present in the vegetative parts in previous stages are absent. Vacuole functions vary according to developmental stage but in ripe pollen they are mainly storage sites for reserves. Vacuoles cause pollen to increase in size by water accumulation and therefore confer some degree of resistance to water stress. Modalities of vacuolisation occur in pollen in the same manner as in other tissues. In most cases, autophagic vacuoles degrade organelles, as in the microspore after meiosis, and can be regarded as cytoplasm clean-up following the transition from the diploid sporophytic to the haploid gametophytic state. This also occurs in the generative cell but not in sperm cells. Finally, vacuoles have a function when microspores are used for pollen embryogenesis in biotechnology being targets for stress induction and afterwards contributing to cytoplasmic rearrangement in competent microspores.