Citrus are affected by many viruses and viroids, some globally widespread and some restricted to particular countries or areas. In this study, we simulated the use of high throughput sequencing (HTS) and the bioinformatic analysis of small interfering RNAs (siRNA) as a pre-screening method to guide bioindexing and molecular detection to enhance the surveillance survey of some key or emerging citrus viruses, such as non-European citrus tristeza virus isolates (non-EU CTV), citrus tatter leaf virus, citrus leprosis virus, citrus yellow mosaic virus, and citrus bark cracking viroid, present in the EPPO lists, and the citrus yellow vein clearing virus. The HTS’s ability to detect other citrus viroids was also evaluated. The results demonstrate that HTS provides a comprehensive phytosanitary status of citrus samples either in single and multiple infections of viruses and viroids. It also provides effective information on citrus tristeza virus mixed infections despite not being able to identify the non-EU variants of the virus. Bioindexing checks each single virus infection but does not differentiate viroids on the Etrog citron indicator and is time-consuming. Molecular assays are valuable as confirmation tests of viruses and viroids but many pairs of primers are needed for a full screening and new or non-target pathogens remain undetected. In addition, the genomes of two isolates of the citrus yellow vein clearing virus and the citrus tatter leaf virus, detected in a sample from China, are described.

The control of tristeza quick decline (QD) of citrus is based on the use of rootstocks that are tolerant or resistant to the Citrus tristeza virus (CTV), but some of them show bio-agronomic limits. The application of cross-protection (CP) has been insufficiently explored. The present study examined the possibility of QD control by cross-protection (CP) following reports showing the dependence of the CP strategy on the close genetic relationships between the protective and challenging CTV isolates. Taking advantage of deep sequencing technologies, we located six naturally infected trees harboring no-seedling yellow (no-SY) and no QD decline (mild) VT isolates and used these for challenge inoculation with three QD VT isolates. Symptom monitoring showed that all six Sicilian mild no-SY isolates, based on their genomic relatedness and mild symptoms reactions, provide effective protection against the three severe local VT isolates. The differences between the six mild and three severe isolates were confined to just a few nucleotide variations conserved in eight positions of three CTV genes (p23, p33, and Orf1a). These results confirm that the superinfection exclusion (SIE mechanism) depends on close genetic relatedness between the protective and challenging severe VT strain isolates. Ten years of investigation suggest that CP could turn into an efficient strategy to contain CTV QD infections of sweet orange trees on SO rootstock.

Recent phylogenetic studies have shown that different lineages representing mild and virulent Citrus tristeza virus (CTV) isolates have been present in Italy for a long time (Davino et al. 2013). The genetic structure of CTV population, based on p23 and p27 genes sequences, highlighted two main clusters referred to T30 and VT-like (Licciardello et al. 2012), as confirmed by the full genomes sequences of two representative isolates SG29 (KC748392) and Bau282 (KC748391) (Licciardello et al. 2015). According to the risk analysis, regulatory measures have been outlined including the recommendation to replace sour orange (Citrus aurantium) with tolerant rootstocks and to monitor the spread and the presence (if any) of different isolates not controlled by the change of rootstock. In 2014, within the frame of a large survey covering more than 25 thousand hectares located in Eastern Sicily, a subset of 249 alemow (Citrus macrophylla) seedlings, which are the preferred host of many citrus aphid species, was tested...

Mal secco is a vascular disease of citrus caused by the mitosporic fungus Plenodomus tracheiphilus. Soil containing infected plant material constitutes an inoculum source for root infections. In this study, the soil bacterial and fungal communities of five lemon orchards located in Syracuse Province (Sicily, Italy) affected by mal secco were analyzed. Soil samples were collected under lemon tree canopies and subjected to total genomic DNA extraction. The fungal DNA was detected through qPCR in all orchards, with variable concentrations. Bacterial and fungal communities were profiled using 16S and ITS amplicon-based high-throughput sequencing, respectively. According to our results, the relative abundances of the most represented bacterial phyla (e.g., Proteobacteria, Actinobacteriota, Acidobacteriota) changed across the orchards, while in the fungal community, the phylum Ascomycota was dominant, with Basidiomycota and Mortierellomycota abundances fluctuating. On the whole, β diversity analysis showed significant variation in the composition of the soil microbial communities across the orchards. This result was confirmed by the analysis of the core community (taxa present at ≥ 75% of total samples), where putative beneficial bacteria resulted in significantly enriched fungus-infected soil samples, suggesting complex microbial interactions. Our findings shed light on the composition and diversity of the soil microbiome in lemon orchards with the occurrence of mal secco infections.

ISHS XII International Citrus Congress - International Society of Citriculture GENETIC STRUCTURE OF CITRUS TRISTEZA VIRUS IN HUNAN PROVINCE (P.R. CHINA)

The Citrus tristeza virus (CTV), a member of the Closterovirus genus, is considered a serious threat to citrus trees grafted onto sour orange (SO) rootstock. In the Mediterranean area, the most prevalent CTV strains are VT and T30. The VT strain includes both mild and severe isolates, some of them associated with seedling yellows (SY) syndrome. Mild CTV-VT isolates that do not induce SY symptoms (no-SY) show minor variations in their Orf1a, p23, and p33 genes, with a single nucleotide polymorphism at position 161 of the p23 gene. These isolates can repress superinfection with homologous severe isolates. The aim of this study was to investigate the mechanism of cross-protection by means of biological indexing, real-time RT-PCR high-resolution melting (HRM), and p23 gene amplicon sequencing. Four no-SY CTV-VT isolates were inoculated onto SO seedlings and Hamlin sweet orange trees grafted on SO. These plants were later challenged with two homologous CTV-VT SY isolates and remained asymptomatic. The biological evaluation of the infection process in superinfected plants was investigated via inoculation of the bark on SO seedlings that were also asymptomatic. A parallel HRM analysis of midvein RNA extracts revealed that the melting temperature (Tm) of the no-SY isolates was statistically lower than that of the SY isolates. The Tm values of RNAs extracts from superinfected plants were not statistically different from those of the no-SY isolates. This suggests that the SY isolates failed to establish infection or replicate in plants pre-inoculated with no-SY isolates. This blockage of replication resembles superinfection exclusion, with attractive perspectives to prevent SY damage in field applications.