Hyperparasitism on Plasmopara viticola by Simplicillium lanosoniveum

A previously unknown association between Plasmopara viticola, the causal pathogen of grape downy mildew, and the mycoparasite strain F2 was described in this paper. F2 could be consistently isolated from the mildew of diseased spots on grape leaves of Kyoho (Vitis labrusca × V. vinifera cv. Kyoho) infected with P. viticola, but neither from the surfaces of healthy grape leaves nor within lesions of other diseases. It could be observed that strain F2 was capable of coiling around sporangiophores and sporangia of P. viticola under microscope, causing hyphae deformation, decreasing the pathogen growth, and reducing the number of sporangia. This fungal strain was identified as Simplicillium lanosoniveum on the basis of morphological characterizations and 28S rDNA sequencing. To our knowledge, this is the first report of S. lanosoniveum with hyperparasitic characteristics on P. viticola, and the hyperparasite has potential application in the control of grape downy mildew.

Comprehensive Genomic Analysis of the Endophytic Bacillus altitudinis Strain GLB197, a Potential Biocontrol Agent of Grape Downy Mildew

Bacillus has been extensively studied for agricultural application as a biocontrol agent. B. altitudinis GLB197, an endophytic bacterium isolated from grape leaves, exhibits distinctive inhibition to grape downy mildew based on unknown mechanisms. To determine the genetic traits involved in the mechanism of biocontrol and host-interaction traits, the genome sequence of GLB197 was obtained and further analyzed. The genome of B. altitudinis GLB197 consisted of one plasmid and a 3,733,835-bp circular chromosome with 41.56% G + C content, containing 3,770 protein-coding genes. Phylogenetic analysis of 17 Bacillus strains using the concatenated 1,226 single-copy core genes divided into different clusters was conducted. In addition, average nucleotide identity (ANI) values indicate that the current taxonomy of some B. pumilus group strains is incorrect. Comparative analysis of B. altitudinis GLB197 proteins with other B. altitudinis strains identified 3,157 core genes. Furthermore, we found that the pan-genome of B. altitudinis is open. The genome of B. altitudinis GLB197 contains one nonribosomal peptide synthetase (NRPS) gene cluster which was annotated as lichenysin. Interestingly, the cluster in B. altitudinis has two more genes than other Bacillus strains (lgrD and lgrB). The two genes were probably obtained via horizontal gene transfer (HGT) during the evolutionary process from Brevibacillus. Taken together, these observations enable the future application of B. altitudinis GLB197 as a biocontrol agent for control of grape downy mildew and promote our understanding of the beneficial interactions between B. altitudinis GLB197 and plants.

Semi-Targeted Metabolomics to Validate Biomarkers of Grape Downy Mildew Infection Under Field Conditions

Grape downy mildew is a devastating disease worldwide and new molecular phenotyping tools are required to detect metabolic changes associated to plant disease symptoms. In this purpose, we used UPLC-DAD-MS-based semi-targeted metabolomics to screen downy mildew symptomatic leaves that expressed oil spots (6 dpi, days post-infection) and necrotic lesions (15 dpi) under natural infections in the field. Leaf extract analyses enabled the identification of 47 metabolites belonging to the primary metabolism including 6 amino acids and 1 organic acid, as well as an important diversity of specialized metabolites including 9 flavonols, 11 flavan-3-ols, 3 phenolic acids, and stilbenoids with various degree of polymerization (DP) including 4 stilbenoids DP1, 8 stilbenoids DP2, and 4 stilbenoids DP3. Principal component analysis (PCA) was applied as unsupervised multivariate statistical analysis method to reveal metabolic variables that were affected by the infection status. Univariate and multivariate statistics revealed 33 and 27 metabolites as relevant infection biomarkers at 6 and 15 dpi, respectively. Correlation-based networks highlighted a general decrease of flavonoid-related metabolites, whereas stilbenoid DP1 and DP2 concentrations increased upon downy mildew infection. Stilbenoids DP3 were identified only in necrotic lesions representing late biomarkers of downy mildew infection.