Salvatore Davino

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.

Spread of Tomato yellow leaf curl virus in Sicily : partial displacement of another geminivirus originally present

The geminivirus Tomato yellow leaf curl virus (TYLCV) was reported for the first time in Italy in 2002. We have followed its spread in Sicily, where Tomato yellow leaf curl Sardinia virus (TYLCSV), another tomato-infecting geminivirus, is endemic and has been causing severe crop losses since 1989. The presence of the two viruses was monitored in the main tomato growing area, the Ragusa province, analyzing samples with yellow leaf curling symptoms. At first (spring–summer 2002) both viruses were always found in mixed infections, but in 2003 and 2004 18–35% of plants were found infected by TYLCV alone and 8–28% by TYLCSV alone, with 41–69% carrying both viruses. TYLCV has spread quickly in the area, demonstrating, as in other parts of the world, its high virulence and invasiveness; however it has not, so far, completely displaced TYLCSV. An infectious clone of TYLCV from Sicily (TYLCV-IT) was sequenced. The nucleotide sequence was 97% identical to other TYLCV strains of the ‘severe’ type, found in many countries worldwide.

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.

Molecular analysis suggests that recent Citrus tristeza virus outbreaks in italy were originated by at least two independent introductions

AbstractCitrus tristeza virus (CTV) is the causal agent of the most important virus disease of citrus. Numerous CTV isolates differing in biological and molecular characteristics have been reported worldwide. Recently, CTV was detected in Italy in several citrus crops from three separate areas: (1) Cassibile, province of Syracuse; (2) Massafra, province of Taranto; and (3) Belpasso, province of Catania. CTV isolates from Massafra and Cassibile were mild, whereas isolates from Belpasso induced severe symptoms. To study the genetic variation of CTV populations of these areas, 150 samples per area were examined by single strand conformation polymorphism (SSCP) and nucleotide sequence analysis of CTV gene p20. All isolates from the same area showed the same SSCP pattern whereas for each area a different SSCP pattern was obtained. The Massafra and the Cassibile isolates had a nucleotide identity higher than 99% with a mild isolate from Spain and about 92% with the Belpasso isolates, which were similar (identity higher than 99%) to severe isolates from California and Japan. These results suggest at least two independent introductions of CTV in Italy, probably by import of CTV-infected budwoods. Within each area, the virus population was homogeneous suggesting diffusion of CTV by aphid transmission.

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.

OCCURRENCE OF TOMATO LEAF CURL NEW DELHI VIRUS INFECTING ZUCCHINI IN SARDINIA (ITALY)

Tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) is a bipartite, circular, ssDNA virus, able to infect species within the Cucurbitaceae and Solanaceae. In August 2016, field observations carried out in Sardinia (Italy) highlighted in one location (Decimoputzu, CA) some plants of zucchini squash (Cucurbita pepo L.) showing a systemic disease never observed before, even in a previous survey (end-June 2016) on cucurbit viruses. The symptoms consisting of leaf curling and yellowing, swelling of veins and plant stunting resembled those caused ToLCNDV, recently reported in Sicily (Panno et al., 2016). Symptomatic leaves from three plants were collected and used for total DNA extraction (Qiagen, Hilden, Germany). Extracts were first tested using degenerate primers for begomovirus detection (Lecoq and Desbiez, 2012) obtaining positive results. Subsequently, the same extracts were amplified by PCR using ToLCNDV specific primers for both DNA A and DNA B (Mizutani et al., 2011). For a partial characterization, PCR amplicons of DNA A were directly sequenced in both directions. The assembled sequences of the three samples, each including part of AV1, complete AC2 and AC3, and part of AC1 (1869 bp), were identical and one was deposited in GenBank (KX826050). When compared to other ToLCNDV isolates, the Sardinia isolate showed the highest nucleotide identity (99%) with isolates from Murcia and Almeria, Spain (KF749223-5; KF891468) and the Sicilian isolate (KU145141). When comparing the amino acid sequences of all above isolates, the Sardinian isolate showed two conservative and one non-conservative substitutions in the AV1 and AC1 regions, respectively. This August outbreak of ToLCNDV is most likely the first one occurred in the region. Transfer of infected plants or viruliferous whiteflies via plant trade with Spain, and to a lesser extent with Sicily, could have been the pathway of introduction of this emergent pathogen in Sardinia. This new finding stresses the possibility of a rapid spreading of ToLCNDV in Italy.

Evolutionary analysis of Citrus tristeza virus outbreaks in Calabria, Italy: two rapidly spreading and independent introductions of mild and severe isolates

The evolution of citrus tristeza virus (CTV) from outbreaks occurred in Calabria, Italy, was compared with that of CTV outbreaks reported previously in another two proximal Italian regions, Sicily and Apulia. Examination of four genomic regions (genes p20, p25 and p23, and one fragment of open reading frame 1) showed two recombination events, and phylogenetic analysis disclosed two divergent CTV groups in Calabria: one formed by severe and the other by mild isolates. This analysis, together with others involving population genetic parameters, revealed a low migration rate of CTV between the three Italian regions, as well as significant differences in selective pressures, epidemiology and demography, all affecting the genetic structure of CTV populations.

Survey of the distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) in Lazio region (Central Italy): a threat for the northward expansion of Tomato leaf curl New Delhi virus ( Begomovirus : Geminiviridae) infection

Bemisia tabaci (Hemiptera: Aleyrodidae) is responsible for severe damage to horticultural and ornamental crops worldwide, mainly for its role as virus vector. In Italy, the B. tabaci Mediterranean (MED) and Middle East–Asia Minor 1 (MEAM1) cryptic species are widespread in the Southern regions as well as in Sicily and Sardinia. During the last two decades, MED populations progressively increased, in those areas where intensive farming is applied. The recent introduction of the begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) prompted extensive surveys of both vector and symptomatic plants. In 2016 and 2017, monitoring activities were carried out in Lazio region (Central Italy) where begomovirus epidemics had never occurred before and the presence of B. tabaci was thought to be only occasional. ToLCNDV-infected zucchini plants were found in Southern Lazio together with whitefly populations belonging only to the MED cryptic species. The MED-Q2 haplotype was the most abundant, likely favored by high temperatures and intensive agricultural practices. Single and mixed populations of MED and MEAM1 were found in Central and Northern Lazio, suggesting that agro-ecological factors still limit MED outbreaks in these areas. This preliminary survey indicates that B. tabaci is well established in Lazio, making ToLCNDV potentially able to spread to the rest of the region as well as to the nearby regions of Central Italy that have similar climatic and cultural conditions. The northward spread of B. tabaci is a critical issue for viral disease epidemiology and the management of whitefly-transmitted viruses in Central Italy, and must be kept under strict surveillance.