A ready-to-use single- and Duplex-TaqMan-qPCR assay to detect and quantify the biocontrol agents Trichoderma asperellum and Trichoderma gamsii

Trichoderma asperellum strain icc012 and Trichoderma gamsii strain icc080, the microbial active ingredients of Remedier™ (ISAGRO, Novara, Italy), are biocontrol agents (BCAs) employable for crop protection against a wide range of fungal pathogens, including soil-borne pathogens and fungi involved in grapevine trunk disease. In this study, single and duplex real-time quantitative PCR (qPCR) methods to detect and quantify T. asperellum and T. gamsii were developed. Primers/probe sets were designed on the T. asperellum and T. gamsii rpb2 genes and tested for specificity on a panel of microorganisms commonly associated with grape wood and soil. No differences were observed comparing single- and duplex-qPCR assays on different BCAs, 1 pg of target DNA was detected approximately at Cq= 34. R2-values and the efficiency were always equal to 0.99 and > 80%, respectively. The detection limit of the duplex-qPCR assay on artificially inoculated samples was 2 × 103and 4 × 104conidia g-1of grape wood tissue and soil, respectively. The methods will be useful to better schedule BCA application in the field and in grapevine nurseries, as well as for investigating the dynamic of BCA populations

First Report of Pseudomonas Grapevine Bunch Rot Caused by Pseudomonas syringae pv. syringae .

Pseudomonas syringae pv. syringae, a Gammaproteobacterium belonging to genomospecies 2 within the P. syringae complex, is distributed worldwide, and it is responsible for bacterial canker on >100 different hosts, including the grapevine. P. syringae pv. syringae induces necrotic lesions in the leaf blades, veins, petioles, shoots, rachis, and tendrils on grapevine cultivars in different areas. P. syringae pv. syringae has been associated with severe economic losses in different grape cultivars in Australia, where it causes inflorescence rot. In midsummer to late summer 2017, symptoms of berry rots differing from those caused by the common berry rots agents were observed in different cultivar Red Globe vineyards of Apulia (southern Italy). As proven by fulfillment of Koch's postulates, these symptoms were caused by a bacterium that, according to the results of biochemical, physiological, nutritional, antimicrobial activity, and pathogenicity tests and sequencing of 16S ribosomal DNA, gyrB, rpoB, and rpoD genes, was identified as P. syringae pv. syringae. This is the first report of Pseudomonas grapevine bunch rot

Molecular Characterization of a Recombinant Isolate of Tomato Leaf Curl New Delhi Virus Associated with Severe Outbreaks in Zucchini Squash in Southern Italy

The molecular characterization of a tomato leaf curl New Delhi virus (ToLCNDV) isolate, denoted ToLCNDV-Le, is reported. The virus was associated with severe and recurrent outbreaks in protected crops of zucchini squash grown in the Province of Lecce (Apulia, southern Italy). The fully sequenced genome of ToLCNDV-Le consists of two genomic components named DNA-A and DNA-B of 2738 and 2683 nt in size, respectively. Like other ToLCNDV isolates, ToLCNDV-Le DNA-A contains the AV2 and AV1 open reading frames (ORFs) in the virion-sense orientation and five additional ORFs named AC1, AC2, AC3, AC4 and AC5 in the complementary-sense orientation. The DNA-B contains BV1 ORF in the virion-sense orientation and BC1 ORF in the complementary-sense orientation. No DNA betasatellites were found associated with ToLCNDV-Le in naturally infected samples. Phylogenetic analysis clustered ToLCNDV-Le with the ToLCNDV-ES strain of western Mediterranean Basin isolates. Consequently, the ToLCNDV-ES-[IT-Zu-Le18] name is proposed as the descriptor for ToLCNDV-Le. Using recombination detection program RDP4, one putative recombination breakpoint (Rbp) was identified close to nucleotide positions 2197–2727, covering approximately half of the AC1 region, including the AC4 ORF and the 3′ UTR. RDP4 indicated the event represents an Rbp of an isolate similar to ToLCNDV [Pk-06] (Acc. No. EF620534) found in Luffa acutangula in Pakistan and identified as putative minor parent into the background of ToLCNDV [BG-Jes-Svr-05] (Acc. No. AJ875157), found in tomato in Bangladesh, and identified as putative major parent. To the best of our knowledge, this is the first report of a ToLCNDV-ES recombinant isolate in the AC1-AC4 region in Italy

Phenotypic and Molecular Investigations on Hypovirulent Cryphonectria parasitica in Italy

Chestnut blight is caused by the fungus Cryphonectria parasitica. As one of the most ecologically important diseases of Castanea spp., C. parasitica can rapidly kill trees. In Europe, mitigation of disease severity took place spontaneously through colonization of C. parasitica by mycoviruses, which reduced the virulence of the fungus. In the framework of a survey, 138 C. parasitica isolates were identified, and virulent/hypovirulent phenotypes were determined through morphological properties and pathogenicity tests. For a pool of four hypovirulent isolates, dsRNA was extracted, cDNA synthesized, and a library subjected to next-generation sequencing. The bioinformatics analysis allowed detecting and reconstructing the complete genome of Cryphonectria hypovirus 1 (CHV-1), denoted as CHV-1 Marche. When compared with the available genomes of other hypoviruses that affected the virulence of C. parasitica, available in databases, CHV-1 Marche showed some nucleotide diversity. The approach used in this study was effective to explore the virome inside a pool of hypovirulent C. parasitica isolates. Next-generation sequencing allowed us to exclude the presence of any other ssRNA and dsRNA viruses infecting the fungus and determine CHV-1 as the only responsible of hypovirulence of C. parasitica in the analyzed samples

Genetic diversity assessments of brown rot pathogen Monilinia fructicola based on the six simple sequence repeat loci

Monilinia fructicola, causal agent of brown rot of stone fruits, is an economically important problem worldwide. Six of the sequence tagged microsatellite sites developed for M. fructicola were used to genotype 68 M. fructicola isolates, which included isolates from three cities in Turkey (n = 42) that were compared to isolates from the USA (n = 15) and Italy (n = 11). AMOVA indicated a significant differentiation among samples from the three different countries. Samples from Turkey represented with nine haplotypes indicating a low diversity according to these markers. Samples from the USA with 12 haplotypes showed the highest genetic diversity values among the sample group from the three countries. However, all the samples from Italy were found as a single haplotype with those markers. Based on Nei’s genetic distance measurements, the single genotype from Italy was distinct from the others and samples from Turkey and the USA were genetically closer to each other, which also presented on the principal coordinate plot. While informative, these results suggest that ascertainment bias in marker development may limit the power of these markers when applied to populations of M. fructicola from other locations in the world. Knowledge on genetic diversities and comparative analysis provides valuable insight for recent changes and movements in pathogen populations which is important for the disease management

La muffa grigia.

Si descrive Botrytis cinerea, agente causale della muffa grigia e gli interventi di protezione della vite dalla malattia