A quantitative PCR assay for the detection and quantification of Septoria pistaciarum, the causal agent of pistachio leaf spot in Italy.

Septoria leaf spot is one of the most widespread diseases affecting pistachio (Pistacia vera) in countries of the Mediterranean region. Septoria pistaciarum was recently confirmed as the causal agent of this disease in Italy. Currently, the detection of S. pistaciarum relies on isolation techniques. These require significant amounts of labor, and time for completion. Also, a reliable identification requires the sequencing of at least two housekeeping genes, in addition to the morphological observations. To accurately detect the presence and quantify S. pistaciarum in pistachio tissues, a molecular tool was necessary. We designed applicable primers that allow reliable amplification of the ß-tubulin gene. The amplification of target DNA was highly efficient, with a 100% success rate, and the assay was able to detect as little as 100 fg/rxn of pure fungal DNA. When tested in artificial mixtures of plant and pathogen DNAs, the assay was able to detect the pathogen consistently at a limit of detection of 1 pg/rxn. The assay was also effective in identifying the pathogen in naturally infected samples, providing rapid detection in all symptomatic specimens. The resulting qPCR assay is an improved detection tool for accurate diagnosis of S. pistaciarum that can also contribute to better understand the population dynamics of the pathogen in the orchard.

Hybrid de novo genome assembly and comparative genomics of three different isolates of Gnomoniopsis castaneae.

The first genome assemblies of Gnomoniopsis castaneae (syn. G. smithogilvyi), the causal agent of chestnut brown rot of kernels, shoot blight and cankers, are provided here. Specifically, the complete genome of the Italian ex-type MUT401 isolate was compared to the draft genome of a second Italian isolate (GN01) and to the ICMP 14040 isolate from New Zealand. The three genome sequences were obtained through a hybrid assembly using both short Illumina reads and long Nanopore reads, their coding sequences were annotated and compared with each other and with other Diaporthales. The information offered by the genome assembly of the three isolates represents the base of data for further application related to -omics strategies of the fungus and to develop markers for population studies at a local and global scale.

Complete genome assembly of the levan-positive strain PVFi1 of Pseudomonas savastanoi pv. savastanoi isolated from olive knots in Central Italy.

Pseudomonas savastanoi pv. savastanoi, the causal agent of olive knot disease, is a fluorescent Gram-negative bacterium classified, according to the specific LOPAT profile, as Ib. However, during the 90s, a number of atypical non-fluorescent levan-positive strains of Pseudomonas savastanoi pv. savastanoi have been unexpectedly isolated from olive knots in Central Italy. Since its first report, several studies were conducted on this species variant, but its genome sequence has never been reported. The complete genome sequence and two additional plasmids of PVFi1, a representative strain, were here obtained using a hybrid sequencing approach with both Oxford Nanopore Technology and Illumina sequencing. A thorough genomic analysis unravelled several genetic features of this peculiar strain, showing a transposase insertion downstream a fragmented copy of the levansucrase gene. The same features were previously reported on levan-negative Pseudomonas savastanoi pv. savastanoi strains. In addition, a second copy of the levansucrase gene fully equipped for a gene expression and comparable to the levan-positive Pseudomonas savastanoi pv. glycinea, may explain the levan-positive test. This result provides a solid genetic demonstration that the bacterial species Pseudomonas savastanoi contains either levan-positive or levan-negative strains, providing insights for an update of the related LOPAT classification.

Draft Genome Sequence of a New Fusarium Isolate Belonging to Fusarium tricinctum Species Complex Collected From Hazelnut in Central Italy.

In summer 2019, during a survey on the health status of a hazelnut orchard located in the Tuscia area (the province of Viterbo, Latium, Italy), nuts showing symptoms, such as brown-grayish spots at the bottom of the nuts progressing upward to the apex, and necrotic patches on the bracts and, sometimes, on the petioles, were found and collected for further studies. This syndrome is associated with the nut gray necrosis (NGN), whose main causal agent is Fusarium lateritium. Aiming to increase knowledge about this fungal pathogen, the whole-genome sequencing of a strain isolated from symptomatic hazelnut was performed using long Nanopore reads technology in combination with the higher precision of the Illumina reads, generating a high-quality genome assembly. The following phylogenetic and comparative genomics analysis suggested that this isolate is caused by the F. tricinctum species complex rather than F. lateritium one, as initially hypothesized. Thus, this study demonstrates that different Fusarium species can infect Corylus avellana producing the same symptomatology. In addition, it sheds light onto the genetic features of the pathogen in subject, clarifying facets about its biology, epidemiology, infection mechanisms, and host spectrum, with the future objective to develop specific and efficient control strategies.