Stefano Panno

Advanced methods of plant disease detection. A review

Plant diseases are responsible for major economic losses in the agricultural industry worldwide. Monitoring plant health and detecting pathogen early are essential to reduce disease spread and facilitate effective management practices. DNA-based and serological methods now provide essential tools for accurate plant disease diagnosis, in addition to the traditional visual scouting for symptoms. Although DNA-based and serological methods have revolutionized plant disease detection, they are not very reliable at asymptomatic stage, especially in case of pathogen with systemic diffusion. They need at least 1–2 days for sample harvest, processing, and analysis. Here, we describe modern methods based on nucleic acid and protein analysis. Then, we review innovative approaches currently under development. Our main findings are the following: (1) novel sensors based on the analysis of host responses, e.g., differential mobility spectrometer and lateral flow devices, deliver instantaneous results and can effectively detect early infections directly in the field; (2) biosensors based on phage display and biophotonics can also detect instantaneously infections although they can be integrated with other systems; and (3) remote sensing techniques coupled with spectroscopy-based methods allow high spatialization of results, these techniques may be very useful as a rapid preliminary identification of primary infections. We explain how these tools will help plant disease management and complement serological and DNA-based methods. While serological and PCR-based methods are the most available and effective to confirm disease diagnosis, volatile and biophotonic sensors provide instantaneous results and may be used to identify infections at asymptomatic stages. Remote sensing technologies will be extremely helpful to greatly spatialize diagnostic results. These innovative techniques represent unprecedented tools to render agriculture more sustainable and safe, avoiding expensive use of pesticides in crop protection.

Emergence and Phylodynamics of Citrus tristeza virus in Sicily, Italy

Citrus tristeza virus (CTV) outbreaks were detected in Sicily island, Italy for the first time in 2002. To gain insight into the evolutionary forces driving the emergence and phylogeography of these CTV populations, we determined and analyzed the nucleotide sequences of the p20 gene from 108 CTV isolates collected from 2002 to 2009. Bayesian phylogenetic analysis revealed that mild and severe CTV isolates belonging to five different clades (lineages) were introduced in Sicily in 2002. Phylogeographic analysis showed that four lineages co-circulated in the main citrus growing area located in Eastern Sicily. However, only one lineage (composed of mild isolates) spread to distant areas of Sicily and was detected after 2007. No correlation was found between genetic variation and citrus host, indicating that citrus cultivars did not exert differential selective pressures on the virus. The genetic variation of CTV was not structured according to geographical location or sampling time, likely due to the multiple introduction events and a complex migration pattern with intense co- and re-circulation of different lineages in the same area. The phylogenetic structure, statistical tests of neutrality and comparison of synonymous and nonsynonymous substitution rates suggest that weak negative selection and genetic drift following a rapid expansion may be the main causes of the CTV variability observed today in Sicily. Nonetheless, three adjacent amino acids at the p20 N-terminal region were found to be under positive selection, likely resulting from adaptation events.

Detection and identification of Fabavirus species by one-step RT-PCR and multiplex RT-PCR

The genus Fabavirus of the family Secoviridae comprises a group of poorly characterized viruses. To date, only five species have been described: Broad bean wilt virus 1 (BBWV-1), Broad bean wilt virus 2 (BBWV-2), Lamium mild mosaic virus (LMMV), Gentian mosaic virus (GeMV) and Cucurbit mild mosaic virus (CuMMV). The development is described of two RT-PCR procedures for the detection and identification of Fabavirus species: a one-step RT-PCR using a single pair of conserved primers for the detection of all fabaviruses, and a one-step multiplex RT-PCR using species-specific primers for the simultaneous detection and identification of the above-mentioned species of the genus Fabavirus. These methods were applied successfully to field samples and the results were compared with those obtained by molecular hybridization and ELISA. The combination of the two techniques enables rapid, sensitive and reliable identification of the five known fabavirus species, as well as the possibility of discovering new species of this genus.

Population genetics of cucumber mosaic virus infecting medicinal, aromatic and ornamental plants from northern Italy

The genetic variation and evolution of cucumber mosaic virus (CMV) from aromatic, medicinal and ornamental plants in northern Italy was studied by sequence analysis of the movement protein gene and comparison with equivalent sequences of isolates from other countries. Comparison of nonsynonymous and synonymous substitutions suggested that 30% of amino acid sites were under negative selection and only one was under positive selection. Phylogenetic, nucleotide diversity and genetic differentiation analyses suggested that long-distance migration plays a role in the evolution and determination of the genetic structure and diversity of CMV in northern Italy and other areas.

The complete genome sequence of Lamium mild mosaic virus , a member of the genus Fabavirus

Lamium mild mosaic virus (LMMV) is the only one of the five members of the genus Fabavirus for which there are no nucleotide sequence data. In this study, the complete genome sequence of LMMV was determined and compared with the available complete genome sequences of other members of the genus Fabavirus. The genome was the largest of the genus but maintained the typical organization, with RNA 1 of 6080 nucleotides (nt), RNA 2 of 4065 nt, and an unusually long 3′ untranslated region in RNA 2 of 603 nt. Phylogenetic analysis of the amino acid sequences of the protease-polymerase (Pro-Pol) region and the two coat proteins confirmed that LMMV belongs to a distinct species within the genus Fabavirus.

Genetic variation and evolutionary analysis of Pepino mosaic virus in Sicily: insights into the dispersion and epidemiology

Pepino mosaic virus (PepMV) is a highly infectious potexvirus that causes a severe disease in tomato (Solanum lycopersicum) crops worldwide. In Sicily, the first outbreak was detected in a single greenhouse in 2005 and it was promptly eradicated. However, in 2008, a large number of greenhouses were simultaneously affected, and it was impossible to eradicate or control the virus. This study addressed the dispersion and the genetic diversity of PepMV isolates obtained from the outbreak in Sicily, in comparison with worldwide PepMV isolates, to gain insight into the factors determining the evolution and epidemiology of the virus. A total of 1800 samples from plants with and without symptoms were collected in the Sicilian provinces of Agrigento, Caltanissetta, Palermo, Ragusa, Siracusa and Trapani. Three isolates collected at different times were biologically characterized. The incidence of the virus increased rapidly from 13% in 2011 to 63% in 2013, and phylogenetic analysis showed that all Sicilian isolates of PepMV belonged to the CH2 strain, one of the six strains previously described. Nucleotide diversity of the Sicilian isolates was low, thus suggesting rapid spread and genetic stability.

Citrus rootstock breeding: response of four allotetraploid somatic hybrids to Citrus tristeza virus induced infections

Four allotetraploid somatic hybrids of citrus, with potential for rootstock improvement, have been evaluated for their response to Citrus tristeza virus (CTV) infection. CTV is the most important viral pathogen affecting citrus production worldwide. Somatic combinations of ‘Milam’ lemon (Citrus jambhiri Lush.) + Sour orange (C. aurantium L Osb.), Calamondin (C. madurensis Lour.) + ‘Keen’ sour orange (C. aurantium L.), Calamondin + ‘Femminello‘ lemon (C. limon L. Burm. F.) and Cleopatra mandarin (C. reshni Hort. ex Tan.) + ‘Femminello’ lemon, were studied. Plants were grafted with CTV-infected “Valencia” sweet orange budwood. Two different CTV strains collected in Sicily, considered as “mild” and “severe”, were used to inoculate candidate rootstocks. The goal of this work is to select a rootstock alternative to CTV susceptible Sour orange, still the prevalent rootstock in the Mediterranean basin. DAS-ELISA and real-time PCR assays confirmed a decreased level of viral replication in tested somatic hybrids, as compared to the susceptible genotypes sour orange and Citrus alemow. The Calamondin+‘Keen’ sour orange genotype did not support any replication in either CTV strain. Somatic hybridization is confirmed to be an effective tool to obtain functionally new rootstocks. Our results can be considered a starting point to open new approaches for the Mediterranean citrus industry. Indeed, the four somatic hybrids have been propagated for agronomical multisite evaluation trials to further assess if their horticultural performance, fruit holding capacity, and soil adaptation are adequate to replace sour orange.

The nucleotide sequence of a recombinant tomato yellow leaf curl virus strain frequently detected in Sicily isolated from tomato plants carrying the Ty-1 resistance gene

In July 2016, an aggressive syndrome of tomato yellow leaf curl disease was reported in Sicily in tomato plants carrying the Ty-1 resistance gene. A total of 34 samples were collected and analyzed. Twenty-seven out of the 34 samples analyzed appeared to contain only recombinant molecules. One full sequence was obtained after cloning. Alignments and plot similarity analysis showed that the genome of the recombinant, named TYLCV-IL[IT:Sic23:16], was mostly derived from tomato yellow leaf curl virus (TYLCV), with a small region of 132 nucleotides in the non-coding region between the stem-loop and the start of the V2 ORF replaced by 124 nucleotides derived from a virus of a different species, tomato yellow leaf curl Sardinia virus. All plants in which the new recombinant was detected belonged to resistant tomato cultivars.

Antimicrobial Activity of the Extracts of Terfezia Claveryi and Tirmania Pinoyi Against Gram-positive and Gram-negative Bacteria Causal Agent of Diseases in Tomato

Antimicrobial Activity of the Extracts of Terfezia claveryi and Tirmania pinoyi Against Gram-positive and Gram-negative Bacteria Causal Agent of Diseases in Tomato Maria Letizia Gargano , Patrizia Bella , Stefano Panno, Vincenzo Arizza, Luigi Inguglia, Vittoria Catara, Giuseppe Venturella, Salvatore Davino* Department of Agricultural and Forest Science (SAF), University of Palermo, Viale delle Scienze Bld. 5, 90123 Palermo (Italy) Department of Biological, Biochemistry and Pharmacological Science and Technology (STEBICEF), Via Archirafi 18-28, 90122 Palermo, (Italy) Department of Agricultural, Food and Environment (Di3A), Via Santa Sofia 100, 91123 Catania, (Italy) salvatore.davino@unipa.it # These authors contributed equally to the study