Cryphonectria hypovirus 1 (CHV-1) has been widely studied and used as a biocontrol agent because of its ability to infect the chestnut blight fungus, Cryphonectria parasitica, and to reduce its virulence. Knowledge about the hypovirus, its presence, and diversity is completely lacking in South Tyrol (northern Italy), which may obstruct biocontrol measures for chestnut blight based on CHV-1. This work aimed to study the occurrence of CHV-1 infecting C. parasitica in South Tyrol and to perform a genetic characterization of the hypovirus. In South Tyrol, CHV-1 was found to occur in 29.2% of the fungal isolates investigated, varying in frequency between different regions and chestnut stands. Twenty-three haplotypes based on partial cDNA (complementary DNA) sequences of open reading frame (ORF)-A and 30 haplotypes based on partial cDNA sequences of ORF-B were identified among 47 and 56 hypovirulent fungal isolates, respectively. Phylogenetic analysis showed that all the haplotypes belonged to the Italian subtype of CHV-1 and that they were closely related to the populations of Italy, Switzerland, Croatia and Slovenia. Evidence of recombination was not found in the sequences and point mutations were the main source of diversity. Overall, this study indicated that the prevalence of CHV-1 in South Tyrol is low compared to many other central and western European populations and determined a need to actively impose biocontrol measures. Using sequence analysis, we identified some variants of interest of CHV-1 that should be studied in detail for their potential use in biocontrol.

Downy mildew, caused by Plasmopara viticola, is a highly significant grapevine disease causing severe yield losses and affecting wine quality. Disease control mainly relies on use of copper and synthetic fungicides. Today there is a growing interest in developing microbial biopesticides for downy mildew control in integrated and organic viticulture. Here, the bacterial biocontrol agent Lysobacter capsici AZ78 (AZ78) was tested following a holistic approach including in planta efficacy test (large-scale trial) combined with bioassays aiming to assess potential adverse effects on non-target organisms: beneficial arthropods (Aphidius rhopalosiphi and Typhlodromus pyri), the crustacean Daphnia magna, the algae Selenastrum capricornutum and soil organisms [earthworm Eisenia (Ei.) fetida and worm Enchytraeid (En.) albidus]. Moreover, side effects of AZ78 treatments on grape quality and wine-making process were assessed. The results showed that AZ78 was equally effective to reference fungicide (Kocide (R) 2000) in controlling downy mildew on leaves and bunches. AZ78 had no significant effect on the mortality of the indicator arthropod species at a Limit test Rate, which was equal to four times the recommended field rate. Additionally, no significant effects (mortality, growth rate or reproduction) were observed on D. magna, Ei. albidus, En. fetida and S. capricornutum, indicating AZ78 low toxicity on non-target organisms. Likewise, the yeast population dynamics in fermenting musts was not affected, without compromising the fermentation processes and wine quality. Overall, this study provides new insights into the characterisation of the biocontrol agent AZ78 showing that this bacterium has the potential to be further developed as a commercial biopesticide.

In an era of growing international competition in modern viticulture, the study and implementation of innovative technologies to increase the production of high-quality grapes and wines are of critical importance. In this study, the non-destructive portable sensor Multiplex, based on fluorescence sensing technique, was applied to evaluate grape maturity parameters and flavonol content of the understudied Pinot blanc variety. The effects of environmental and agronomical factors on flavonol content of Pinot blanc grapes were investigated in eight vineyards characterised by different microclimatic and agronomic conditions. Furthermore, the direct impact of canopy management treatment on the flavonol dynamics of the grapes oriented in the four cardinal directions was assessed. Results highlight the positive role of moderate temperatures and direct sunlight exposure on Pinot blanc flavonol content; however, no direct vineyard-elevation effect was observed. The ability to modulate and evaluate the flavonol content in field represent crucial factors because of their potential effect on flavonoids-dependent wine characteristics, such as stability and ageing. In the present study, for the first time, two calibration curves were reported for pre- and post-veraison periods between flavonol indices and the berry skin flavonol content and a good correlation was observed between Multiplex measurement and the total polyphenolic content of grape juice. Moreover, the strong correlation between the chlorophyll index with grape juice sugar content and titratable acidity revealed the practical application of non-destructive sensors to predict the optimal harvest time for Pinot blanc grapes. In conclusion, the non-destructive fluorescence sensor Multiplex is a high-potential tool for innovative viticulture, for evaluating grape skin composition variables in white grape varieties.

Microbial biocontrol agents (mBCAs) are an eco-friendly alternative to chemical fungicides, due to their minimal or null impact on the environment and human health. The selection and development of effective commercial mBCAs begins with the adoption of appropriate screening procedures, which should first consider the environmental niche and epidemiological parameters of the plant pathogen targeted to be controlled. Following that, a stepwise selection strategy should be implemented to screen mBCAs for traits that provide significant agronomic advantages and biocontrol efficacy and to characterize the mBCAs’ mode of action, applying high-throughput omics technologies and in vitro methodologies. Despite the growing interest in mBCAs, few commercial products are currently available on the market. Nonetheless, the scientific community has made significant efforts in recent decades to foster research on new microbial species, including promising mBCAs, which might be developed as new active ingredients for novel microbial biopesticides, such as Clonostachys rosea, Lysobacter capsici, Pichia anomala and Piriformospora indica. In addition to a few thoroughly characterized mBCAs, hundreds of under-studied microbial species (e.g., Bdellovibrio bacteriovorus, Paenibacillus spp., Pseudozyma flocculosa) exhibiting effective biocontrol activity against economically relevant plant pathogen groups (e.g., powdery mildews, soilborne diseases and phytopathogenic bacteria) have been reported and might be successfully investigated in the future as a reservoir of novel microbial active ingredients.

Comprehensive phenolic composition of dried grape skin from cv. Pinot Blanc and its development during ripening is reported, with particular emphasis on flavonol glycosides profile. Extraction procedure and reversed -phase ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-QqQ-MS/ MS) method were optimized and validated for the determination of 39 phenolic compounds belonging to different groups (flavonols, dihydroflavonols, benzoic and hydroxycinnamic acids, flavan-3-ols). Beside selected reaction monitoring (SRM) for analytes available as standards, flavonols and dihydroflavonols profile was fur-therly explored performing precursor ion scan (PIS) with neutral loss (NL) for unavailable compounds. Quer-cetin-3-O-rutinoside occurred as major flavonol component, and rutinosides for kaempferol-and isorhamnetin-structures were also quantitable. Presence of different myricetin derivatives was unveiled, with myricetin-3-O- glucoside being quantifiable at all ripening time points. Besides high levels of astilbin, dihydroquercetins profile resulted highly complex. Moreover, ratio between caftaric and coutaric acid and between isomers of coutaric acid at harvest was uncommon if compared to other white cultivars.

Pinot blanc is a leading grapevine variety in South Tyrol (Italy) for wine production. The high quality of its wines derives from a typical aroma of elegant apple notes and lively acidity. The typicity of the final wine depends on the origin of the vine, the soil, the oenological practices and time of harvest. The South Tyrolean mountainous areas meet the cold climatic requirements of Pinot blanc, which guarantee its sweet-acidic harmony obtained when organic acids are in balance with the other components of the wine. However, increasing temperatures in valley sites during the berry development period boost the activity of malic acid (MA) enzymes, which negatively affect the final sugar/acid ratio. Researchers are currently focused on understanding acid dynamics in wines, and there are no references for the best sugar/acid ratio for Pinot blanc. Moreover, the contribution of individual acids to the sensory profile of this wine has not yet been studied. In this study we address the effect of different climate conditions and site elevations on the sugar/acid ratio in developmental grapevine berries, and we evaluate the effect on wine bouquet. Even if different models and indices have been proposed for predicting sugar content, no predictive models exist for MA in white grapes. In a three-year study (2017, 2018 and 2019) that involved eight vineyards in four different location in South Tyrol at various elevations ranging from 223 to 730 m a.s.l., the relationships between bioclimatic indices, such as growing-degree day (GDD) and grapevine sugar ripeness (GSR) and grapevine berry content were investigated. The analysis reveals that GDD may potentially predict MA dynamics in Pinot blanc; hence, a GDD-based model was used to determine the GDD to reach target MA concentrations (3.5, 3.0, 2.5, 2.0 g/L). This simple model was improved with additional temperature-based parameters by feature selection, and the best three advanced models were selected and evaluated by 5-fold cross-validation. These models could be used to support location and harvest date choice to produce high-quality Pinot blanc wines.

Omics technologies have had a tremendous impact on underinvestigated genera of plant disease biocontrol agents such as Lysobacter. Strong evidence of the association between Lysobacter spp. and the rhizosphere has been obtained through culture-independent methods, which has also contributed towards highlighting the relationship between Lysobacter abundance and soil suppressiveness. It is conceivable that the role played by Lysobacter spp. in soil suppressiveness is related to their ability to produce an impressive array of lytic enzymes and antibiotics. Indeed, genomics has revealed that biocontrol Lysobacter strains share a vast number of genes involved in antagonism activities, and the molecular pathways underlying how Lysobacter spp. interact with the environment and other micro-organisms have been depicted through transcriptomic analysis. Furthermore, omics technologies shed light on the regulatory pathways governing cell motility and the biosynthesis of antibiotics. Overall, the results achieved so far through omics technologies confirm that the genus Lysobacter is a valuable source of novel biocontrol agents, paving the way for studies aimed at making their application in field conditions more reliable.

Biological interactions in the microbial communities of the rhizosphere continuously shape the gene expression patterns of each individual microorganism. A dual RNA-Seq approach was applied to obtain a comprehensive overview of the molecular mechanisms activated during the interaction between the biocontrol rhizobacterium Lysobacter capsici AZ78 and the soilborne phytopathogenic oomycete Phytophthora infestans. The RNA-Seq transcriptional profile of L. capsici AZ78 was characterized by up-regulation of genes concerned in the biogenesis of type 4 pilus and lytic enzymes, involved, respectively, in host colonization and subsequent attack of the P. infestans cell wall. The activation of detoxification processes allowed L. capsici AZ78 to overcome the attempted defense processes of P. infestans. Moreover, the genes involved in antibiotic biosynthesis were up-regulated in L. capsici AZ78 and caused cell death in P. infestans, with the activation of putative apoptotic processes. The consequences of P. infestans cell death resulted in the down-regulation of primary metabolic pathways, such as carbohydrates, nucleic acids and protein metabolisms. Overall, the mechanism of action of L. capsici AZ78 was related to parasitism and predatory activities that cause the death of P. infestans.

Lysobacter capsici AZ78 has considerable potential for biocontrol of phytopathogenic microorganisms. However, lack of information about genetic cues regarding its biological characteristics may slow down its exploitation as a biofungicide. In order to obtain a comprehensive overview of genetic features, the L. capsici AZ78 genome was sequenced, annotated and compared with the phylogenetically related pathogens Stenotrophomonas malthophilia K729a and Xanthomonas campestris pv. campestris ATCC 33913. Whole genome comparison, supported by functional analysis, indicated that L. capsici AZ78 has a larger number of genes responsible for interaction with phytopathogens and environmental stress than S. malthophilia K729a and X. c. pv. campestris ATCC 33913. Genes involved in the production of antibiotics, lytic enzymes and siderophores were specific for L. capsici AZ78, as well as genes involved in resistance to antibiotics, environmental stressors, fungicides and heavy metals. The L. capsici AZ78 genome did not encompass genes involved in infection of humans and plants included in the S. malthophilia K729a and X. c. pv. campestris ATCC 33913 genomes respectively. The L. capsici AZ78 genome provides a genetic framework for detailed analysis of other L. capsici members and the development of novel biofungicides based on this bacterial strain.

Bacterial cells can display different types of motility, due to the presence of external appendages such as flagella and type IV pili. To date, little information on the mechanisms involved in the motility of the Lysobacter species has been available. Recently, L. capsici AZ78, a biocontrol agent of phytopathogenic oomycetes, showed the ability to move on jellified pea broth. Pea broth medium improved also the biocontrol activity of L. capsici AZ78 against Plasmopara viticola under greenhouse conditions. Noteworthy, the quantity of pea residues remaining on grapevine leaves fostered cell motility in L. capsici AZ78. Based on these results, this unusual motility related to the composition of the growth medium was investigated in bacterial strains belonging to several Lysobacter species. The six L. capsici strains tested developed dendrite-like colonies when grown on jellified pea broth, while the development of dendrite-like colonies was not recorded in the media commonly used in motility assays. To determine the presence of genes responsible for biogenesis of the flagellum and type IV pili, the genome of L. capsici AZ78 was mined. Genes encoding structural components an d regulatory factors of type IV pili were upregulated in L. capsici AZ78 cells grown on the above-mentioned medium, as compared with the other tested media. These results provide new insight into the motility mechanism of L. capsici members and the role of type IV pili and pea compounds on the epiphytic fitness and biocontrol features of L. capsici AZ78.

Introduction: Lysobacter capsici AZ78 (AZ78) is a Gram-negative bacterium that effectively controls oomycetes, in particular Plasmopara viticola. Non-spore forming bacteria populations quickly decline when exposed to harsh enviroments as phyllosphere and formulation can play an important role to achieve consistent efficacy in field applications. Limited information is available in literature on formulation of Gram-negative bacteria. Survival and fate in the environment are also important traits to be assessed for the registration as a biopesticide. Objectives: The main aim of this work was to set up and apply a stepwise flow diagram to design performing formulations for AZ78 and follow its fate in the environment (Figure 1). Materials and methods: Cell mass production was maximized in a benchtop fermenter and shelf life of the harvested cells was assessed. We screened compounds capable to protect the bacterial cells against desiccation, UV irradiation and wash-off. We tested the ability of combinations of selected compounds to preserve the efficacy of AZ78 against P. viticola and to establish populations on grapevine. A specific primer pair was developed starting from REP-PCR fingerprinting and subsequently used in a qPCR procedure for monitoring the fate of AZ78 in vineyards. Results: The optimised fermentation protocol gave a harvest of at least 10 10 AZ78 cell/ml. Viability of cells decreased only one order of magnitude after one year of storage at 4°C. The use of a combination of polyethyleneglycol, corn steep liquor and lignosulfonate in the formulation improved the survival of AZ78 cells in response to environmental stresses and the efficacy against P. viticola on grapevine in field conditions. Moreover, the qPCR procedure showed that the AZ78 population reached 10 6