Patricia Trotel-Aziz

Chitosan oligomers and copper sulfate induce grapevine defense reactions and resistance to gray mold and downy mildew.

ABSTRACT Chitosan (CHN), a deacetylated derivative of chitin, was shown to be efficient in promoting plant defense reactions. CHN oligomers of different molecular weight (MW) and degree of acetylation (DA) triggered an accumulation of phytoalexins, trans- and cis-resveratrol and their derivatives epsilon-viniferin and piceid, in grapevine leaves. Highest phytoalexin production was achieved within 48 h of incubation with CHN at 200 mug/ml with an MW of 1,500 and a DA of 20% (CHN1.5/20), while oligomers with greater MW were less efficient, indicating that a specific MW threshold could be required for phytoalexin response. Treatment of grapevine leaves by highly active CHN1.5/20 also led to marked induction of chitinase and beta-1,3-glucanase activities. CHN1.5/20 applied together with copper sulfate (CuSO(4)) strongly induced phytoalexin accumulation. CuSO(4) alone, especially at low concentrations also elicited a substantial production of phytoalexins in grapevine leaves. Evidence is also provided that CHN1.5/20 significantly reduced the infection of grapevine leaves by Botrytis cinerea and Plasmopara viticola, and in combination with CuSO(4) conferred protection against both pathogens.

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.

Biological control of Botrytis cinerea by selected grapevine-associated bacteria and stimulation of chitinase and β-1,3 glucanase activities under field conditions

In this study, the biocontrol ability of seven grapevine-associated bacteria, previously reported as efficient against Botrytis cinerea under in vitro conditions, was evaluated in two vineyard orchards with the susceptible cv. Chardonnay during four consecutive years (2002–2005). It was shown that the severity of disease on grapevine leaves and berries was reduced to different levels, depending on the bacterial strain and inoculation method. Drenching the plant soil with these bacteria revealed a systemic resistance to B. cinerea, even without renewal of treatment. Accordingly, this resistance was associated with a stimulation of some plant defense responses such as chitinase and β-1,3-glucanase activities in both leaves and berries. In leaves, chitinase activity increased before veraison (end-July) while β-1,3-glucanase reached its maximum activity at ripening (September). Reverse patterns were observed in berries, with β-1,3-glucanase peaking at full veraison (end-August) and chitinase at a later development stage. Highest activities were observed with Acinetobacter lwoffii PTA-113 and Pseudomonas fluorescens PTA-CT2 in leaves, and with A. lwoffii PTA-113 and Pantoea agglomerans PTA-AF1 in berries. These results have demonstrated an induced protection of grapevine against B. cinerea by selected bacteria under field conditions, and suggest that induced resistance could be related to a stimulation of plant defense reactions in a successive manner.

Drought stress tolerance in grapevine involves activation of polyamine oxidation contributing to improved immune response and low susceptibility to Botrytis cinerea

Environmental factors including drought stress may modulate plant immune responses and resistance to pathogens. However, the relationship between mechanisms of drought tolerance and resistance to pathogens remained unknown. In this study, the effects of drought stress on polyamine (PA) homeostasis and immune responses were investigated in two grapevine genotypes differing in their drought tolerance; Chardonnay (CHR), as sensitive and Meski (MSK), as tolerant. Under drought conditions, MSK plants showed the lowest leaf water loss and reduction of photosynthetic efficiency, and expressed a lower level of NCED2, a gene involved in abscisic acid biosynthesis, compared with CHR plants. The improved drought tolerance in MSK was also coincident with the highest change in free PAs and up-regulation of the genes encoding arginine decarboxylase (ADC), copper amine-oxidase (CuAO), and PA-oxidases (PAO) and their corresponding enzyme activities. MSK plants also accumulated the highest level of amino acids, including Arg, Glu, Gln, Pro, and GABA, emphasizing the participation of PA-related amino acid homeostasis in drought tolerance. Importantly, drought-tolerant plants also exhibited enhanced phytoalexin accumulation and up-regulation of PR genes, especially PR-2 and Chit4c, compared with the sensitive plants. This is consistent with a lower susceptibility of MSK than CHR to Botrytis cinerea. Data suggest a possible connection between water stress tolerance and immune response in grapevine. Pharmacological experiments revealed that under drought conditions CuAO and PAO pathways were involved in the regulation of photosynthetic efficiency, and also of immune response and resistance of grapevine to a subsequent pathogen attack. These results open new views to improve our understanding of crosstalk between drought tolerance mechanisms and immune response.

Improved resistance against Botrytis cinerea by grapevine-associated bacteria that induce a prime oxidative burst and phytoalexin production.

Bacteria such as Pantoea agglomerans (Pa-AF2), Bacillus subtilis (Bs-271), Acinetobacter lwoffii (Al-113), and Pseudomonas fluorescens (Pf-CT2), originating from the vineyard, can induce defense responses and enhance resistance of grapevine against the fungal pathogen Botrytis cinerea. The perception of these bacteria by plant cells or tissues in relation to their activities remains unknown. In this study, we examined the relationships between the activity of each bacterium to induce or prime some defense responses, and its effectiveness to induce resistance in grapevine against B. cinerea. We showed that all selected bacteria are capable of inducing early oxidative burst and phytoalexin (trans-resveratrol and trans-ε-viniferin) production in grapevine cells and leaves. Pf-CT2 and Al-113 induced higher H(2)O(2) and trans-resveratrol accumulations, and were able to further prime plants for accelerated phytoalexin production after B. cinerea challenge. These two bacteria were also the most effective in inducing local and systemic resistance. A similar level of induced resistance was observed with live Pa-AF2 which also induced but not primed a greater accumulation of trans-resveratrol. However, Bs-271, which was less effective in inducing resistance, induced a lower trans-resveratrol synthesis, without priming activity. Treatment of grapevine cells with growing medium or crude extract of the bacteria quickly and strongly enhanced oxidative burst compared with the live bacteria. However, both treatments resulted in comparable amounts of phytoalexins and induced local and systemic resistance to B. cinerea as compared with those induced by living bacteria, with extracts from Pf-CT2 and Al-113 being the most effective. Together, these results indicate that induced resistance can be improved by treatment with bacteria or derived compounds which induced or primed plants for enhanced phytoalexin accumulation.

An assessment of the physiological properties of the so-called compatible solutes using in vitro experiments with leaf discs

Despite the considerable efforts directed over the past four decades to investigate, via correlative physiology and genetic engineering, the contribution of osmolytes to osmotolerance of higher plants, their physiological function(s) still remain a matter of debate. Here, we summarize the functions of proline and glycine betaine, recently documented in our group by using canola (Brassica napusL. var. oleifera), leaf discs treated in vitro with medium of low or high osmolarities. These discs accumulate proline when subjected to osmotic upshifts, whereas proline is rapidly consumed when the discs are further treated under non-stressing conditions. The period of time needed to osmoregulate the amount of proline is assumed to be too long to assign to the proline response a relevant function in turgor regulation which can be achieved with carbohydrates. In addition, proline accumulation is suppressed by supplying the osmotically stressing medium with polyamines, phyto-oxylipins and glycine betaine with no detrimental effect on the capacity to cope with osmotic stress. In opposition to proline, glycine betaine does not occur in canola. We take advantage of this situation to test the compatibility of exogenously supplied betaine in this plant material. Comparisons are done with spinach (Spinacia oleraceaL.) leaf discs, which contain an endogenous amount of betaine and also exhibit the capacity to absorb a large amount of it. The betaine absorbed is not degraded and thus cannot be considered in this situation as a true osmoregulator. In turgid tissues of plants that do not produce betaine, this compound induces an elevated level of glutamine, a decreased level of protein, and photo-dependent glycine accumulation. This can rely on senescent-like effects and on a blockage in the glycolate cycle. Such damages to nitrogen metabolism are not observed in leaf explants treated simultaneously with betaine and the stressing agent. We also find that the treatments of turgid canola leaf discs with proline or betaine induce important changes in polyamine contents, mimicking, but with a higher order of magnitude, the effects of osmotic stress. Interestingly, this inducing activity is not observed when osmolytes are supplied under osmotic upshift conditions. Antagonistic effects between osmolytes and osmotic stress are described for the first time in higher plants. They suggest that proline and betaine can act as signal molecules in counteracting destabilizing effects to polyamine metabolism and lead us to decipher other counter-stress effects of nitrogenous osmolytes which, obviously, do not behave just as inert osmobalancers.

Pseudomonas fluorescens PTA-CT2 Triggers Local and Systemic Immune Response Against Botrytis cinerea in Grapevine

Although induced systemic resistance (ISR) is well-documented in the context of plant-beneficial bacteria interactions, knowledge about the local and systemic molecular and biochemical defense responses before or upon pathogen infection in grapevine is very scarce. In this study, we first investigated the capacity of grapevine plants to express immune responses at both above- and below-ground levels upon interaction with a beneficial bacterium, Pseudomonas fluorescens PTA-CT2. We then explored whether the extent of priming state could contribute to the PTA-CT2-induced ISR in Botrytis cinerea-infected leaves. Our data provide evidence that this bacterium colonized grapevine roots but not the above-ground plant parts and altered the plant phenotype that displayed multiple defense responses both locally and systemically. The grapevine roots and leaves exhibited distinct patterns of defense-related gene expression during root colonization by PTA-CT2. Roots responded faster than leaves and some responses were more strongly upregulated in roots than in leaves and vice versa for other genes. These responses appear to be associated with some induction of cell death in roots and a transient expression of HSR, a hypersensitive response-related gene in both local (roots) and systemic (leaves) tissues. However, stilbenic phytoalexin patterns followed opposite trends in roots compared with leaves but no phytoalexin was exuded during plant-bacterium interaction, suggesting that roots could play an important role in the transfer of metabolites contributing to immune response at the systemic level. Unexpectedly, in B. cinerea-infected leaves PTA-CT2-mediated ISR was accompanied in large part by a downregulation of different defense-related genes, including HSR. Only phytoalexins and glutathion-S-transferase 1 transcripts were upregulated, while the expression of anthocyanin biosynthetic genes was maintained at a higher level than the control. This suggests that decreased expression of HSR, as a marker of cell death, and activation of secondary metabolism pathways could be responsible for a reduced B. cinerea colonization capacity in bacterized plants.

Effectiveness of beneficial bacteria to promote systemic resistance of grapevine to gray mold as related to phytoalexin production in vineyards

Background and aimsThe rhizospheric Bacillus subtilis PTA-271 (271) and endophytic Pseudomonas fluorescens PTA-CT2 (CT2) and Pantoea agglomerans PTA-AF2 (AF2) bacteria are able to induce systemic resistance (ISR) in grapevine against B. cinerea, but ISR markers and their costs remained unknown in vineyards. In this study, we investigated the relationship between the effectiveness of single and binary combinations of selected bacteria to induce ISR and their ability to trigger phytoalexin accumulation, as a potential marker for disease resistance, in leaves and berries, as well as their impact on grape yield in vineyards.MethodsGrapevine plants were treated during 2006 in two vineyards by drenching soil with single or binary mixtures of bacteria. Induced resistance against B. cinerea was evaluated and stilbenic phytoalexins were analyzed by HPLC in both leaves and berries. Grape yield was also assessed as number and weight of clusters at ripening.ResultsBoth single and mixtures of bacteria were effective in reducing gray mold severity in the leaves and berries in vineyards. Disease control was accompanied by a significant accumulation of stilbenic phytoalexins, trans-resveratrol and ε-viniferin, in both leaves and berries in the bacterized plants. δ-Viniferin also accumulated, but only in berries of the treated plants. Reduction of disease symptoms and accumulation of resveratrol and viniferins were higher in the plants treated with single CT2 compared to AF2 and 271. Treatment of grapevine plants with binary mixtures of these isolates resulted in a significant performance of CT2+AF2 in leaves and CT2+271 in berries. On the other hand, bacterial treatments did not show any negative effect on grape yield.ConclusionsThese results revealed the efficacy of CT2 alone or in combination with AF2 or with 271 in triggering grapevine resistance against B. cinerea and enhancing systemic accumulation of resveratrol and viniferins, without compromising grape yield.