First Report of the Association of the Psyllid Vector Bactericera trigonica (Hemiptera: Triozidae) with 'Candidatus Liberibacter Solanacearum' in Italy.

Psyllids, members of the family Triozidae, represent a potential threat to the cultivation of solanaceous and apiaceous crops worldwide, mainly as vectors of the phloem-restricted bacterium 'Candidatus Liberibacter solanacearum' (Lso). The Lso haplotypes C, D and E are known to affect apiaceous crops, such as carrot and celery, in several European countries. In Italy, data on the incidence and natural spread of both Lso and psyllids have not been reported so far. In this study, the presence of the vectors was investigated in a main Italian district for carrot production, the "Altopiano del Fucino" area (Central Italy). Both occasional and regular surveys were carried out on a total of five carrot fields and one potato field in 2021 and 2022. Bactericera trigonica (Hodkinson), which is known to efficiently transmit Lso to carrots, was found to be well-established in the area. High levels of population density were recorded in the summer period (more than 100 adult specimens per trap caught every two weeks) and then sharply decreased after the carrot harvest, confirming the strict association of this psyllid species with crop availability. In 2022, 27.5% of the total tested psyllid samples resulted in being positive for Lso haplotypes D and E, the latter being prevalent. This survey revealed, for the first time in Italy, the presence of B. trigonica adults associated with Lso in carrot crops. Although this study was limited to a few fields located in one area, it provided important evidence of the risks for Lso outbreaks and prompted further research to assess the spread and incidence of the disease in apiaceous cultivations in Italy.

Trans-grafting plum pox virus resistance from transgenic plum rootstocks to apricot scions.

Introduction: Trans-grafting could be a strategy to transfer virus resistance from a transgenic rootstock to a wild type scion. However contradictory results have been obtained in herbaceous and woody plants. This work was intended to determine if the resistance to sharka could be transferred from transgenic plum rootstocks to wild-type apricot scions grafted onto them. Methods: To this end, we conducted grafting experiments of wild- type apricots onto plum plants transformed with a construction codifying a hairpin RNA designed to silence the PPV virus and studied if the resistance was transmitted from the rootstock to the scion. Results: Our data support that the RNA-silencing-based PPV resistance can be transmitted from PPV-resistant plum rootstocks to non-transgenic apricot scions and that its efficiency is augmented after successive growth cycles. PPV resistance conferred by the rootstocks was robust, already occurring within the same growing cycle and maintained in successive evaluation cycles. The RNA silencing mechanism reduces the relative accumulation of the virus progressively eliminating the virus from the wild type scions grafted on the transgenic resistant PPV plants. There was a preferential accumulation of the 24nt siRNAs in the scions grafted onto resistant rootstocks that was not found in the scions grafted on the susceptible rootstock. This matched with a significantly lower relative accumulation of hpRNA in the resistant rootstocks compared with the susceptible or the tolerant ones. Discussion: Using transgenic rootstocks should mitigate public concerns about transgenes dispersion and eating transgenic food and allow conferring virus resistance to recalcitrant to transformation cultivars or species.

Fighting Sharka in Peach: Current Limitations and Future Perspectives.

Sharka, caused by Plum Pox Virus (PPV), is by far the most important infectious disease of peach [P. persica (L.) Batsch] and other Prunus species. The progressive spread of the virus in many important growing areas throughout Europe poses serious issues to the economic sustainability of stone fruit crops, peach in particular. The adoption of internationally agreed-upon rules for diagnostic tests, strain-specific monitoring schemes and spatial-temporal modeling of virus spread, are all essential for a more effective sharka containment. The EU regulations on nursery activity should be modified based on the zone delimitation of PPV presence, limiting open-field production of propagation materials only to virus-free areas. Increasing the efficiency of preventive measures should be augmented by the short-term development of resistant cultivars. Putative sources of resistance/tolerance have been recently identified in peach germplasm, although the majority of novel resistant sources to PPV-M have been found in almond. However, the complexity of introgression from related-species imposes the search for alternative strategies. The use of genetic engineering, particularly RNA interference (RNAi)-based approaches, appears as one of the most promising perspectives to introduce a durable resistance to PPV in peach germplasm, notwithstanding the well-known difficulties of in vitro plant regeneration in this species. In this regard, rootstock transformation to induce RNAi-mediated systemic resistance would avoid the transformation of numerous commercial cultivars, and may alleviate consumer resistance to the use of GM plants.

Biotechnological strategies and tools for Plum pox virus resistance: trans-, intra-, cis-genesis, and beyond.

Plum pox virus (PPV) is the etiological agent of sharka, the most devastating and economically important viral disease affecting Prunus species. It is widespread in most stone fruits producing countries even though eradication and quarantine programs are in place. The development of resistant cultivars and rootstocks remains the most ecologically and economically suitable approach to achieve long-term control of sharka disease. However, the few PPV resistance genetic resources found in Prunus germplasm along with some intrinsic biological features of stone fruit trees pose limits for efficient and fast breeding programs. This review focuses on an array of biotechnological strategies and tools, which have been used, or may be exploited to confer PPV resistance. A considerable number of scientific studies clearly indicate that robust and predictable resistance can be achieved by transforming plant species with constructs encoding intron-spliced hairpin RNAs homologous to conserved regions of the PPV genome. In addition, we discuss how recent advances in our understanding of PPV biology can be profitably exploited to develop viral interference strategies. In particular, genetic manipulation of host genes by which PPV accomplishes its infection cycle already permits the creation of intragenic resistant plants. Finally, we review the emerging genome editing technologies based on ZFN, TALEN and CRISPR/Cas9 engineered nucleases and how the knockout of host susceptibility genes will open up next generation of PPV resistant plants.

Control of pome and stone fruit virus diseases.

Many different systemic pathogens, including viruses, affect pome and stone fruits causing diseases with adverse effects in orchards worldwide. The significance of diseases caused by these pathogens on tree health and fruit shape and quality has resulted in the imposition of control measures both nationally and internationally. Control measures depend on the identification of diseases and their etiological agents. Diagnosis is the most important aspect of controlling fruit plant viruses. Early detection of viruses in fruit trees or in the propagative material is a prerequisite for their control and to guarantee a sustainable agriculture. Many quarantine programs are in place to reduce spread of viruses among countries during international exchange of germplasm. All these phytosanitary measures are overseen by governments based on agreements produced by international organizations. Also certification schemes applied to fruit trees allow the production of planting material of known variety and plant health status for local growers by controlling the propagation of pathogen-tested mother plants. They ensure to obtain propagative material not only free of "quarantine" organisms under the national legislation but also of important "nonquarantine" pathogens. The control of insect vectors plays an important role in the systemic diseases management, but it must be used together with other control measures as eradication of infected plants and use of certified propagation material. Apart from the control of the virus vector and the use of virus-free material, the development of virus-resistant cultivars appears to be the most effective approach to achieve control of plant viruses, especially for perennial crops that are more exposed to infection during their long life span. The use of resistant or tolerant cultivars and/or rootstocks could be potentially the most important aspect of virus disease management, especially in areas in which virus infections are endemic. The conventional breeding for virus-tolerant or resistant fruit tree cultivars using available germplasm is a long-term strategy, and the development and production of these cultivars may take decades, if successful. Genetic engineering allows the introduction of specific DNA sequences offering the opportunity to obtain existing fruit tree cultivars improved for the desired resistance trait. Unfortunately, genetic transformation of pome and stone fruits is still limited to few commercial genotypes. Research carried out and the new emerging biotechnological approaches to obtain fruit tree plants resistant or tolerant to viruses are discussed.

Robust RNA silencing-mediated resistance to Plum pox virus under variable abiotic and biotic conditions.

Some abiotic and biotic conditions are known to have a negative impact on post-transcriptional gene silencing (PTGS), thus representing a potential concern for the production of stable engineered virus resistance traits. However, depending on the strategy followed to achieve PTGS of the transgene, different responses to external conditions can be expected. In the present study, we utilized the Nicotiana benthamiana­Plum pox virus (PPV) pathosystem to evaluate in detail the stability of intron-hairpin(ihp)-mediated virus resistance under conditions known to adversely affect PTGS. The ihp plants grown at low or high temperatures were fully resistant to multiple PPV challenges, different PPV inoculum concentrations and even to a PPV isolate differing from the ihp construct by more than 28% at the nucleotide level. In addition, infections of ihp plants with viruses belonging to Cucumovirus, Potyvirus or Tombusvirus, all known to affect PTGS at different steps, were not able to defeat PPV resistance. Low temperatures did not affect the accumulation of transgenic small interfering RNAs (siRNAs), whereas a clear increase in the amount of siRNAs was observed during infections sustained by Cucumber mosaic virus and Potato virus Y. Our results show that the above stress factors do not represent an important concern for the production,through ihp-PTGS technology, of transgenic plants having robust virus resistance traits.

Genetically engineered resistance to Plum pox virus infection in herbaceous and stone fruit hosts.

Plum pox virus (PPV), a Potyvirus, is the causal agent of sharka, the most detrimental viral disease affecting stone fruit trees. This review focuses on research carried out to obtain PPV- resistant transgenic plants and on how biotechnological strategies evolved in light of the scientific advances made during the last several years. Successful RNA silencing strategies that confer high level of resistance to strains of PPV have been developed and tested under laboratory and greenhouse conditions. Moreover, field tests showed that transgene-mediated RNA silencing was effective in protecting plum plants against aphid-mediated PPV infection. The new emerging biotechnological approaches for conferring PPV resistance are discussed.

Silencing of Plum pox virus 5'UTR/P1 sequence confers resistance to a wide range of PPV strains.

An effective disease-control strategy should protect the host from the major economically important and geographically widespread variants of a pathogen. Plum pox virus (PPV) is the causal agent of sharka, the most devastating viral disease of Prunus species. We have shown previously that the hairpin RNA expression driven by h-UTR/P1, h-P1/HCPro, h-HCPro and h-HCPro/P3 constructs, derived from the PPV-M ISPaVe44 isolate, confers resistance to the homologous virus in Nicotiana benthamiana plants. Since the production of transgenic stone fruits and their evaluation for PPV resistance would take several years, the ISPaVe44-resistant plant lines were used to evaluate which construct would be the best candidate to be transferred to Prunus elite cultivars. To do that, nine PPV isolates of the D, M, Rec, EA and C strains originally collected from five Prunus species in different geographical areas, were typed by sequencing and used to challenge the transgenic N. benthamiana lines; 464 out of 464 virus-inoculated plants of lines h-UTR/P1, h-HCPro and h-HCPro/P3 showed complete and long-lasting resistance to the seven PPV isolates of D, M and Rec strains. Moreover, the h-UTR/P1 plants were also fully resistant to PPV-C and -EA isolates. Our data suggest that the h-UTR/P1 construct is of particular practical interest to obtain stone fruit plants resistant to the sharka disease.

Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus.

We report the application of the hairpin-mediated RNA silencing technology for obtaining resistance to Plum pox virus (PPV) infection in Nicotiana benthamiana plants. Four sequences, covering the P1 and silencing suppressor HC-Pro genes of an Italian PPV M isolate, were introduced into N. benthamiana plants as two inverted repeats separated by an intron sequence under the transcriptional control of the Cauliflower Mosaic Virus 35S promoter. In a leaf disk infection assay, 38 out of 40 T0 transgenic plants were resistant to PPV infection. Eight lines, 2 for each construct, randomly selected among the 38 resistant plants were further analysed. Two hundred forty eight out of 253 T1 transgenic plants were resistant to local and systemic PPV infection. All transgenic single locus lines were completely resistant. These data indicate that the RNA silencing of PPV P1/HCPro sequences results in an efficient and predictable PPV resistance, which may be utilized in obtaining stone fruit plants resistant to the devastating Sharka disease.