Osmotic Stress and ABA Affect Immune Response and Susceptibility of Grapevine Berries to Gray Mold by Priming Polyamine Accumulation

Abiotic factors inducing osmotic stress can affect plant immunity and resistance against pathogen attack. Although a number of studies have characterized grapevine responses to various forms of biotic and abiotic stresses, the relationships between osmotic stress response and susceptibility of mature berries to Botrytis cinerea still remain unknown. In this study, we investigated the effects of osmotic stress and abscisic acid (ABA) on defense responses of mature grapevine berries before and after B. cinerea infection. We focused on the possible involvement of polyamines in the interaction between osmotic stress response and susceptibility to B. cinerea. We showed that osmotic stress induced by PEG or sucrose, and exogenous ABA induce transient but low defense responses, including weak expression of PR genes and phytoalexin synthesis in mature berries. This was accompanied by an upregulation of NCED2 involved in ABA biosynthesis and a large production of free polyamines. However, osmotic stress followed by B. cinerea infection primed berries for enhanced accumulation of polyamines, but slowed down the defense responses and increased susceptibility to the pathogen. A weak increase of diamine- and polyamine-oxidase activities was also recorded in stressed berries, but declined after pathogen infection. The pretreatment of stressed berries with appropriate inhibitors of diamine- and polyamine-oxidases further increased polyamine level and greatly lowered defense responses, leading to higher susceptibility to B. cinerea. These results suggest that increased polyamine titer through low activation of their oxidative degradation in grape berries may contribute at least in part to the weakening of defense responses and subsequent disease susceptibility

Bacillus subtilis PTA-271 Counteracts Botryosphaeria Dieback in Grapevine, Triggering Immune Responses and Detoxification of Fungal Phytotoxins

Plant pathogens have evolved various strategies to enter hosts and cause diseases. Particularly Neofusicoccum parvum, a member of Botryosphaeria dieback consortium, can secrete the phytotoxins (-)-terremutin and (R)-mellein during grapevine colonization. The contribution of phytotoxins to Botryosphaeria dieback symptoms still remains unknown. Moreover, there are currently no efficient control strategies of this disease, and agro-environmental concerns have raised increasing interest in biocontrol strategies to limit disease spread in vineyards, especially by using some promising beneficial bacteria. Here, we first examined in planta the biocontrol capacity of Bacillus subtilis PTA-271 against N. parvum Np-Bt67 strain producing both (-)-terremutin and (R)-mellein. We then focused on the direct effects of PTA-271 on pathogen growth and the fate of pure phytotoxins, and explored the capacity of PTA-271 to induce or prime grapevine immunity upon pathogen infection or phytotoxin exposure. Results provided evidence that PTA-271 significantly protects grapevine cuttings against N. parvum and significantly primes the expression of PR2 (encoding a β-1,3-glucanase) and NCED2 (9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis) genes upon pathogen challenge. Using in vitro plantlets, we also showed that PTA-271 triggers the expression of salicylic acid- and jasmonic acid-responsive genes, including GST1 (encoding a glutathione-S-transferase) involved in detoxification process. However, in PTA-271-pretreated plantlets, exogenous (-)-terremutin strongly lowered the expression of most of upregulated genes, except GST1. Data also indicated that PTA-271 can detoxify both (-)-terremutin and (R)-mellein and antagonize N. parvum under in vitro conditions. Our findings highlight (-)-terremutin and (R)-mellein as key aggressive molecules produced by N. parvum that may weaken grapevine immunity to promote Botryosphaeria dieback symptoms. However, PTA-271 can efficiently attenuate Botryosphaeria dieback by enhancing some host immune responses and detoxifying both phytotoxins produced by N. parvum

Bacillus subtilis PTA-271 counteracts Botryosphaeria dieback in grapevine, triggering immune responses and detoxification of fungal phytotoxins

Plant pathogens have evolved various strategies to enter hosts and cause diseases. Particularly Neofusicoccum parvum, a member of Botryosphaeria dieback consortium, can secrete the phytotoxins (-)-terremutin and (R)-mellein during grapevine colonization. The contribution of phytotoxins to Botryosphaeria dieback symptoms still remains unknown. Moreover, there are currently no efficient control strategies of this disease, and agro-environmental concerns have raised increasing interest in biocontrol strategies to limit disease spread in vineyards, especially by using some promising beneficial bacteria. Here, we first examined in planta the biocontrol capacity of Bacillus subtilis PTA-271 against N. parvum Np-Bt67 strain producing both (-)- terremutin and (R)-mellein. We then focused on the direct effects of PTA-271 on pathogen growth and the fate of pure phytotoxins, and explored the capacity of PTA- 271 to induce or prime grapevine immunity upon pathogen infection or phytotoxin exposure. Results provided evidence that PTA-271 significantly protects grapevine cuttings against N. parvum and significantly primes the expression of PR2 (encoding a β-1,3-glucanase) and NCED2 (9-cis-epoxycarotenoid dioxygenase involved in abscisic acid biosynthesis) genes upon pathogen challenge. Using in vitro plantlets, we also showed that PTA-271 triggers the expression of salicylic acid- and jasmonic acid-responsive genes, including GST1 (encoding a glutathione-S-transferase) involved in detoxification process. However, in PTA-271-pretreated plantlets, exogenous (-)-terremutin strongly lowered the expression of most of upregulated genes, except GST1. Data also indicated that PTA-271 can detoxify both (-)- terremutin and (R)-mellein and antagonize N. parvum under in vitro conditions. Our findings highlight (-)-terremutin and (R)-mellein as key aggressive molecules produced by N. parvum that may weaken grapevine immunity to promote Botryosphaeria dieback symptoms. However, PTA-271 can efficiently attenuate Botryosphaeria dieback by enhancing some host immune responses and detoxifying both phytotoxins produced by N. parvum

Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum

[EN] Grapevine trunk diseases (GTDs) are a big threat for global viticulture. Without effective chemicals, biocontrol strategies are developed as alternatives to better cope with environmental concerns. A combination of biological control agents (BCAs) could even improve sustainable disease management through complementary ways of protection. In this study, we evaluated the combination of Bacillus subtilis (Bs) PTA-271 and Trichoderma atroviride (Ta) SC1 for the protection of Chardonnay and Tempranillo rootlings against Neofusicoccum parvum Bt67, an aggressive pathogen associated to Botryosphaeria dieback (BD). Indirect benefits offered by each BCA and their combination were then characterized in planta, as well as their direct benefits in vitro. Results provide evidence that (1) the cultivar contributes to the beneficial effects of Bs PTA-271 and Ta SC1 against N. parvum, and that (2) the in vitro BCA mutual antagonism switches to the strongest fungistatic effect toward Np-Bt67 in a three-way confrontation test. We also report for the first time the beneficial potential of a combination of BCA against Np-Bt67 especially in Tempranillo. Our findings highlight a common feature for both cultivars: salicylic acid (SA)-dependent defenses were strongly decreased in plants protected by the BCA, in contrast with symptomatic ones. We thus suggest that (1) the high basal expression of SA-dependent defenses in Tempranillo explains its highest susceptibility to N. parvum, and that (2) the cultivar-specific responses to the beneficial Bs PTA-271 and Ta SC1 remain to be further investigated.Funding This work was supported by a French Grant from the Region GRAND-EST France and the City of GRAND-REIMS France through the BIOVIGNE Ph.D. program, whose functioning is supported by BELCHIM Crop Protection France. DG was supported by the Ramon y Cajal program, Spanish Government (RyC-2017-23098).Leal, C.; Richet, N.; Guise, J.; Gramaje, D.; Armengol Fortí, J.; Fontaine, F.; Trotel-Aziz, P. (2021). Cultivar Contributes to the Beneficial Effects of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to Protect Grapevine Against Neofusicoccum parvum. Frontiers in Microbiology. 12:1-17. https://doi.org/10.3389/fmicb.2021.726132S1171

Pseudomonas spp.-induced systemic resistance to Botrytis cinerea is associated with induction and priming of defence responses in grapevine

Non-pathogenic rhizobacteria Pseudomonas spp. can reduce disease in plant tissues through induction of a defence state known as induced systemic resistance (ISR). This resistance is based on multiple bacterial determinants, but nothing is known about the mechanisms underlying rhizobacteria-induced resistance in grapevine. In this study, the ability of Pseudomonas fluorescens CHA0 and Pseudomonas aeruginosa 7NSK2 to induce resistance in grapevine against Botrytis cinerea is demonstrated. Both strains also triggered an oxidative burst and phytoalexin (i.e. resveratrol and viniferin) accumulation in grape cells and primed leaves for accelerated phytoalexin production upon challenge with B. cinerea. Treatment of cell cultures with crude cell extracts of bacteria strongly enhanced oxidative burst, but resulted in comparable amounts of phytoalexins and resistance to B. cinerea to those induced by living bacteria. This suggests the production of bacterial compounds serving as inducers of disease resistance. Using other strains with different characteristics, it is shown that P. fluorescens WCS417 (Pch-deficient), P. putida WCS358 (Pch- and SA-deficient) and P. fluorescens Q2-87 (a DAPG producer) were all capable of inducing resistance to an extent similar to that induced by CHA0. However, in response to WCS417 (Pch-negative) the amount of H2O2 induced is less than for the CHA0. WCS417 induced low phytoalexin levels in cells and lost the capacity to prime for phytoalexins in the leaves. This suggests that, depending on the strain, SA, pyochelin, and DAPG are potentially effective in inducing or priming defence responses. The 7NSK2 mutants, KMPCH (Pch- and Pvd-negative) and KMPCH-567 (Pch-, Pvd-, and SA-negative) induced only partial resistance to B. cinerea. However, the amount of H2O2 triggered by KMPCH and KMPCH-567 was similar to that induced by 7NSK2. Both mutants also led to a low level of phytoalexins in grapevine cells, while KMPCH slightly primed grapevine leaves for enhanced phytoalexins. This highlights the importance of SA, pyochelin, and/or pyoverdin in priming phytoalexin responses and induced grapevine resistance by 7NSK2 against B. cinerea

Use of Elicitors and Beneficial Bacteria to Induce and Prime the Stilbene Phytoalexin Response: Applications to Grapevine Disease Resistance

Phytoalexins are naturally occurring molecules with antimicrobial activity deriving from the secondary metabolism of plants; they are synthesized in response to physical agents or stresses and phytopathogenic agents (fungi, bacteria and viruses), as well as numerous chemical compounds and biological control agents. Among these, grapevine phytoalexins, which belong to the chemical group of stilbenes, exhibit biocidal activity against a large number and variety of plant pathogens. It is important to investigate whether induction of stilbene phytoalexin production can serve to protect this plant against its pathogens. Quite a few chemical compounds, derivatives of phytohormones bio-elicitors as well as biocontrol agents, have been used to induce the synthesis of stilbene phytoalexins with the aim of conferring protection to grapevine against its main diseases (gray mold, downy mildew, powdery mildew and esca). This article reviews the experiments that have been carried out in this direction during the last 30 years and shows that the observed protective effects towards pathogens are generally linked to induction and priming of the grapevine phytoalexin response, confirming the interest in using, in a more general way, stimulation of the production of phytoalexins in plants as a basis for crop protection

Maladies du bois de la vigne : les avancées présentées à Reims

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Evaluation of Bacillus subtilis PTA-271 and Trichoderma atroviride SC1 to control Botryosphaeria dieback and black-foot pathogens in grapevine propagation material

Background: Grapevine trunk diseases (GTDs) are a complex group of diseases that lead to major economic losses in all wine-producing countries. The investigation of biocontrol agents (BCAs) capable of forestalling or at least minimizing the development of GTDs has, recently, become a priority. Nursery experiments were set up to (i) assess the biocontrol effect of Trichoderma atroviride (Ta) SC1 and Bacillus subtilis (Bs) PTA-271, alone and in simultaneous application, against Botryosphaeria dieback (BOT)- and black-foot (BF)- associated pathogens during the grapevine propagation process and (ii) evaluate the success of the BCA inoculation during the grapevine propagation process, using quantitative reverse-transcription polymerase chain reaction techniques. RESULTS: The results demonstrated a significant reduction in the percentage of potentially infected plants and the percentage of fungal isolation from wood fragments of BOT and BF pathogens in nursery material treated with Ta SC1 and Bs PTA-271, respectively. In one of the experiments, simultaneous treatments with Bs PTA-271 and Ta SC1 caused a reduction in percentages of potentially infected plants and fungal isolation, from wood fragments containing BOT and BF pathogens. CONCLUSION: These biological treatments may be relevant components of an integrated approach, using complementary management strategies to limit infection by GTD pathogens, but further research is still needed to elucidate the effectiveness of Bs PTA-271 and the benefits of simultaneous application with Ta SC1 for the control of GTD pathogens in nurseries.This work was supported by a French grant from the regionGRAND-EST France and the city of GRAND-REIMS France throughthe BIOVIGNE PhD program, whose functioning is supported byBELCHIM Crop Protection France, and by Chaire Maldive. DGwas supported by the Ramón y Cajal program, Spanish Govern-ment (RyC-2017-23098). Part of this study wasfinanciallysupported by BELCHIM Crop Protection France. We also acknowl-edge the availability of nursery staff and vineyard farmers to facil-itate nursery andfield work, respectivel