Simultaneous editing of two DMR6 genes in grapevine results in reduced susceptibility to downy mildew.

The reduction of pesticide treatments is of paramount importance for the sustainability of viticulture, and it can be achieved through a combination of strategies, including the cultivation of vines (Vitis vinifera) that are resistant or tolerant to diseases such as downy mildew (DM). In many crops, the knock-out of Downy Mildew Resistant 6 (DMR6) proved successful in controlling DM-resistance, but the effect of mutations in DMR6 genes is not yet known in grapevine. Today, gene editing serves crop improvement with small and specific mutations while maintaining the genetic background of commercially important clones. Moreover, recent technological advances allowed to produce non-transgenic grapevine clones by regeneration of protoplasts edited with the CRISPR/Cas9 ribonucleoprotein. This approach may revolutionize the production of new grapevine varieties and clones, but it requires knowledge about the targets and the impact of editing on plant phenotype and fitness in different cultivars. In this work we generated single and double knock-out mutants by editing DMR6 susceptibility (S) genes using CRISPR/Cas9, and showed that only the combined mutations in VviDMR6-1 and VviDMR6-2 are effective in reducing susceptibility to DM in two table-grape cultivars by increasing the levels of endogenous salicylic acid. Therefore, editing both genes may be necessary for effective DM control in real-world agricultural settings, which could potentially lead to unwanted phenotypes. Additional research, including trials conducted in experimental vineyards, is required to gain a deeper understanding of DMR6-based resistance.

Plasmopara viticola infection affects mineral elements allocation and distribution in Vitis vinifera leaves.

Plasmopara viticola is one of the most important pathogens infecting Vitis vinifera plants. The interactions among P. viticola and both susceptible and resistant grapevine plants have been extensively characterised, at transcriptomic, proteomic and metabolomic levels. However, the involvement of plants ionome in the response against the pathogen has been completely neglected so far. Therefore, this study was aimed at investigating the possible role of leaf ionomic modulation during compatible and incompatible interactions between P. viticola and grapevine plants. In susceptible cultivars, a dramatic redistribution of mineral elements has been observed, thus uncovering a possible role for mineral nutrients in the response against pathogens. On the contrary, the resistant cultivars did not present substantial rearrangement of mineral elements at leaf level, except for manganese (Mn) and iron (Fe). This might demonstrate that, resistant cultivars, albeit expressing the resistance gene, still exploit a pathogen response mechanism based on the local increase in the concentration of microelements, which are involved in the synthesis of secondary metabolites and reactive oxygen species. Moreover, these data also highlight the link between the mineral nutrition and plants' response to pathogens, further stressing that appropriate fertilization strategies can be fundamental for the expression of response mechanisms against pathogens.

Ecological impact of a rare sugar on grapevine phyllosphere microbial communities.

Plants host a complex microbiota inside or outside their tissues, and phyllosphere microorganisms can be influenced by environmental, nutritional and agronomic factors. Rare sugars are defined as monosaccharides with limited availability in nature and they are metabolised by only few certain microbial taxa. Among rare sugars, tagatose (TAG) is a low-calories sweetener that stimulates and inhibits beneficial and pathogenic bacteria in the human gut microbiota, respectively. Based on this differential effect on human-associated microorganisms, we investigated the effect of TAG treatments on the grapevine phyllosphere microorganisms to evaluate whether it can engineer the microbiota and modify the ratio between beneficial and pathogenic plant-associated microorganisms. TAG treatments changed the structure of the leaf microbiota and they successfully reduced leaf infections of downy mildew (caused by Plasmopara viticola) and powdery mildew (caused by Erysiphe necator) under field conditions. TAG increased the relative abundance of indigenous beneficial microorganisms, such as some potential biocontrol agents, which could partially contribute to disease control. The taxonomic composition of fungal and bacterial leaf populations differed according to grapevine locations, therefore TAG effects on the microbial structure were influenced by the composition of the originally residing microbiota. TAG is a promising biopesticide that could shift the balance of pathogenic and beneficial plant-associated microorganisms, suggesting selective nutritional/anti-nutritional properties for some specific taxa. More specifically, TAG displayed possible plant prebiotic effects on the phyllosphere microbiota and this mechanism of action could represent a novel strategy that can be further developed for sustainable plant protection.

Reducing copper use in the environment: the use of larixol and larixyl acetate to treat downy mildew caused by Plasmopara viticola in viticulture.

BACKGROUND: Plant extracts might provide sustainable alternatives to copper fungicides, which are still widely used despite their unfavourable ecotoxicological profile. Larch bark extract and its constituents, larixyl acetate and larixol, have been shown to be effective against grapevine downy mildew (Plasmopara viticola) under semi-controlled conditions. The aim of this study was to reduce the gap between innovation and the registration of a marketable product, namely to develop scalable extraction processes and to evaluate and optimise the performance of larch extracts under different conditions. RESULTS: Toxicologically and technically acceptable solvents like ethanol were used to extract the active compounds larixyl acetate and larixol from bark in sufficient amounts and their combined concentration could be increased by up to 39% by purification steps. The combined concentration of larixyl acetate and larixol from larch turpentine could be increased by up to 66%. The Minimal Inhibitory Concentration (MIC100 ) against P. viticola in vitro (6-23 µg mL-1 ) and the Effective Concentration (EC50 ) in planta under semi-controlled conditions (0.2-0.4 mg mL-1 ) were promising compared with other plant extracts. In vineyards, efficacies of larch extracts reached up to 68% in a stand-alone strategy and 84% in low-copper strategies. CONCLUSION: Larch extracts represent valid candidates for copper reduction in organic vineyards, and their development into a sustainable plant protection product might be feasible. © 2017 Society of Chemical Industry.

Monitoring Lysobacter capsici AZ78 using strain specific qPCR reveals the importance of the formulation for its survival in vineyards.

Survival in the phyllosphere is a critical feature for biofungicides based on non-spore forming bacteria. Moreover, knowledge of their persistence on plants is important to design effective formulations and application techniques. With this scope, the aim of this work was to develop a specific method to monitor the fate in the environment of Lysobacter capsici AZ78, a biocontrol agent of Plasmopara viticola, and to evaluate the contribution of formulation in its persistence on grapevine leaves. A strain-specific primer pair derived from REP-PCR fingerprinting was used in quantitative PCR experiments to track the evolution of L. capsici AZ78 population in vineyards. The population reached between 5 and 6 log10 cells gram of leaf(-1) after application and decreased by more than 100 times in one week. Multiple regression analysis showed that unfavourable temperature was the main environmental factor correlating with the decrease of L. capsici AZ78 persistence on grapevine leaves. Importantly, the use of formulation additives protected L. capsici AZ78 against environmental factors and improved its persistence on the leaves by more than 10 times compared to nude cells. Formulation and the knowledge about the persistence of L. capsici AZ78 in vineyards will be useful to develop commercial biofungicides for foliar application.

Stepwise flow diagram for the development of formulations of non spore-forming bacteria against foliar pathogens: The case of Lysobacter capsici AZ78.

The formulation is a significant step in biopesticide development and is an efficient way to obtain consistency in terms of biological control under field conditions. Nonetheless, there is still a lack of information regarding the processes needed to achieve efficient formulation of non spore-forming bacterial biological control agents. In response to this, we propose a flow diagram made up of six steps including selection of growth parameters, checking of minimum shelf life, selection of protective additives, checking that the additives have no adverse effects, validation of the additive mix under field conditions and choosing whether to use additives as co-formulants or tank mix additives. This diagram is intended to provide guidance and decision-making criteria for the formulation of non spore-forming bacterial biological control agents against foliar pathogens. The diagram was then validated by designing an efficient formulation for a Gram-negative bacterium, Lysobacter capsici AZ78, to control grapevine downy mildew caused by Plasmopara viticola. A harvest of 10(10)L. capsici AZ78cellsml(-1) was obtained in a bench top fermenter. The viability of cells decreased by only one order of magnitude after one year of storage at 4°C. The use of a combination of corn steep liquor, lignosulfonate, and polyethyleneglycol in the formulation improved the survival of L. capsici AZ78 cells living on grapevine leaves under field conditions by one order of magnitude. Furthermore, the use of these additives also guaranteed a reduction of 71% in P. viticola attacks. In conclusion, this work presents a straightforward stepwise flow diagram to help researchers develop formulations for biological control agents that are easy to prepare, stable, not phytotoxic and able to protect the microorganims under field conditions.

A complex protein derivative acts as biogenic elicitor of grapevine resistance against powdery mildew under field conditions.

Powdery mildew caused by Erysiphe necator is one of the most important grapevine diseases in several viticulture areas, and high fungicide input is required to control it. However, numerous synthetic chemical pesticides are under scrutiny due to concerns about their impact on human health and the environment. Biopesticides, such as biogenic elicitors, are a promising alternative to chemical fungicides. Although several studies have reported on effective elicitors against grapevine diseases, their efficacy under field conditions has not been investigated extensively or has occurred at rather limited levels. Our goal was to examine the efficacy of a protein-based composition, namely nutrient broth (NB), against powdery mildew under field conditions and to characterize its mechanism of action. Weekly treatments with NB was highly effective in controlling powdery mildew on grapevine across seasons with different disease pressures. The level of disease control achieved with NB was comparable to standard fungicide treatments both on leaves and bunches across three different years. NB has no direct toxic effect on the germination of E. necator conidia, and it activates plant resistance with both systemic and translaminar effect in experiments with artificial inoculation under controlled conditions. NB induced the expression of defense-related genes in grapevine, demonstrating stimulation of plant defense mechanisms, prior to and in the early stages of pathogen infection. NB is a natural derivative from meat and yeast, substances that tend not to raise concerns about toxicological and ecotoxicological properties. NB represents a valid control tool for integrated plant protection programs against powdery mildew, to reduce the use of synthetic pesticides on grapevine.

Resilience of the natural phyllosphere microbiota of the grapevine to chemical and biological pesticides.

The phyllosphere is colonized by complex microbial communities, which are adapted to the harsh habitat. Although the role and ecology of nonpathogenic microorganisms of the phyllosphere are only partially understood, leaf microbiota could have a beneficial role in plant growth and health. Pesticides and biocontrol agents are frequently applied to grapevines, but the impact on nontarget microorganisms of the phyllosphere has been marginally considered. In this study, we investigated the effect of a chemical fungicide (penconazole) and a biological control agent (Lysobacter capsici AZ78) on the leaf microbiota of the grapevine at three locations. Amplicons of the 16S rRNA gene and of the internal transcribed spacer were sequenced for bacterial and fungal identification, respectively. Pyrosequencing analysis revealed that the richness and diversity of bacterial and fungal populations were only minimally affected by the chemical and biological treatments tested, and they mainly differed according to grapevine locations. Indigenous microbial communities of the phyllosphere are adapted to environmental and biotic factors in the areas where the grapevines are grown, and they are resilient to the treatments tested. The biocontrol properties of phyllosphere communities against downy mildew differed among grapevine locations and were not affected by treatments, suggesting that biocontrol communities could be improved with agronomic practices to enrich beneficial populations in vineyards.

Lysobacter capsici AZ78 can be combined with copper to effectively control Plasmopara viticola on grapevine.

The bacterial genus Lysobacter represents a still underdeveloped source of biocontrol agents able to protect plants against pathogenic oomycetes. In this work the L. capsici strain AZ78 was evaluated with regard to the biological control of Plasmopara viticola, the causal agent of grapevine downy mildew. L. capsici AZ78 is able to resist copper ions and its resistance to this metal is probably due to the presence of genes coding for copper oxidase (copA) and copper exporting PIB-type ATPases (ctpA). The presence of both genes was also detected in other members of the Lysobacter genus. Resistance to copper allowed L. capsici AZ78 to be combined with a low-dose of a copper-based fungicide, leading to more effective control of grapevine downy mildew. Notably, prophylactic application of L. capsici AZ78 alone to grapevine leaves reduced downy mildew disease to the same degree as a copper-based fungicide. Furthermore, L. capsici AZ78 persists in the phyllosphere of grapevine plants and tolerates environmental stresses such as starvation, freezing, mild heat shock and UV light irradiation. These traits suggest that L. capsici AZ78 could be a suitable candidate for developing a new biofungicide to be used in combination with copper to control grapevine downy mildew.

Salvia officinalis Extract Can Protect Grapevine Against Plasmopara viticola.

The ability of sage (Salvia officinalis) extract to control grapevine downy mildew under greenhouse and field conditions was tested. The persistence and rainfastness of sage extract were also investigated. Sage extract provided a high level of sustained disease control in artificially inoculated, potted grapevine under greenhouse conditions. However, even small amounts of simulated rainfall (10 mm) significantly reduced efficacy of sage extract. In a field experiment in 2006, sage extract provided 94% reduction in disease incidence and 63% reduction in area under the disease progress curve for disease severity on berries and leaves, respectively, reaching a level of disease control not significantly different from that provided by copper hydroxide. In 2007, the sage extract provided only a partial reduction (less than 30%) of downy mildew on leaves, probably as a result of a long rainy period between two of the consecutive treatments. Overall, sage extract effectively controlled grapevine downy mildew and could be a promising alternative to copper in organic viticulture. However, the low rainfastness of this treatment adversely affected its efficacy.