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. Plants support a complex micro-ecosystem, and they host distinct bacterial communities on and inside various plant organs (1). The aerial part of plants (phyllosphere) is normally colonized by a variety of bacteria, filamentous fungi and yeasts (2). Microbial phyllosphere communities are complex and composed by many uncultured microorganisms (2, 3), which are adapted to the harsh environmental conditions (4, 5). In particular, microbial epiphytes of the phyllosphere are exposed to the atmosphere and must deal with direct UV radiation, wide fluctuations in temperature, low water availability, and limited access to nutrients (2,6). Therefore, the composition of phyllosphere communities could be affected by environmental factors, such as UV radiation, air pollution, and nitrogen fertilization, as well as by biotic factors, such as plant species and invading microorganisms (6-8). Moreover, the phyllosphere is an open system and microbes can invade plant leaves by migration from the atmosphere, soil, other plants, insects, and animals (9).The phyllosphere has been less intensively studied than the rhizosphere and has received considerable attention in recent years (1). The interest in phyllosphere microbiology was initially driven by investigations into plant pathogens, but most phyllosphere-colonizing microorganisms live as commensals and/or mutualistic symbionts on their host plants (2,4,6). Phyllosphere communities are involved in functional processes as large in scale as the carbon cycle, nitrogen fixation, and degradation of organic pollu...

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The application of L. capsici AZ78 cells or its own culture filtrates effectively controls both P. infestans and Pl. viticola. Cyclo(L-Pro-L-Tyr) produced by L. capsici AZ78 is toxic against sporangia of both these oomycetes. These data enforce the potential in the use of Lysobacter members for the control of plant pathogenic oomycetes and provide the basis for the development of new low-impact fungicides based on cyclo(L-Pro-L-Tyr).

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.

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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.

Downy mildew, caused by Plasmopara viticola, is one of the most destructive diseases of grapevine and is controlled with intense application of chemical fungicides. Treatment with Trichoderma harzianum T39 (T39) or benzothiadiazole-7-carbothioic acid S-methyl ester (BTH) has been previously shown to activate grapevine resistance to downy mildew and reduce disease symptoms in the Pinot noir cultivar. However, enhancement of plant resistance can be affected by several factors, including plant genotype. In order to further extend the use of resistance inducers against downy mildew, the physiological and molecular properties of T39-and BTH-activated resistance in different cultivars of table and wine grapes were characterized under greenhouse conditions. T39 treatment reduced downy mildew symptoms, but the degree of efficacy differed significantly among grapevine cultivars. However, efficacy of BTH-activated resistance was consistently high in the different cultivars. Expression profiles of defence-related genes differed among cultivars in response to resistance inducers and to pathogen inoculation. T39 treatment enhanced the expression of defence-related genes in the responsive cultivars, before and after P. viticola inoculation. A positive correlation between the efficacy of T39 and the expression level of defence-related genes was found in Primitivo and Pinot noir plants, while different genes or more complex processes were probably activated in Sugraone and Negroamaro. The data reported here suggest that the use of a responsive cultivar is particularly important to maximize the efficacy of resistance inducers and new natural inducers should be explored for the less responsive cultivars.

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.