Severe decline symptoms (reaching 60–80

Phytoplasmas are known to infect different kind of fruits and forest trees worldwide including apples, plum, pears, almond, apricot, peach, cherry, mulberry, citrus and paulownia. The most devastating diseases on trees are Apple’s proliferation (AP) disease, Elm yellows, Pear decline, Citrus witches’ broom and mulberry dwarf that causes upto 50 to 80% yield losses in trees globally. This review paper depicts that the Candidatus Phytoplasma asteris, and Candidatus Phytoplasma prunorum, are the most diverse phytoplasma groups affecting tree populations. This review article explores various phytoplasma diseases affecting trees worldwide, addressing their distribution, symptomatology, effects and approach towards management. Phytoplasmainfected forest and fruit trees not only lead to economic losses but also disrupts the natural aesthetics of the environment.

Chestnut phytosanitary emergencies are rising in recent years also due to climate changes, which allow new treats to find favourable environmental conditions in previously unsuitable places and the rapidity of movements which aids their survival. Among the emerging fungal pathogens, Sirococcus smithogilvyi the causal agent of chestnut brown rot (CBR), a very damaging chestnut disease, stands out. Between 2023-2024, S. smithogilvyi was frequently isolated from 1200 nuts collected randomly in three chestnut locations of Basilicata region (Southern Italy) and initially identified based on morphology features. Other fungal taxa were less frequently isolated: Alternaria alternata, Botrytis cinerea, Penicillium sp., Neofusicoccum parvum, Mucor sp., Cladosporium sp. and Trichoderma sp. Sequencing of two common fungal barcodes, the Internal Transcribed Spacer (ITS) region of ribosomal DNA and the beta-tubulin (tub2) gene confirmed the morphological identification. Furthermore, phylogenetic analyses of S. smithogilvyi isolates, based on tub2 nucleotide sequences, showed the occurrence of two separate evolutionary lineages in Basilicata, previously recognized as haplotypes A and B. The haplotype A, was predominant registering 86% frequency while the haplotype B registered only a 14% frequency. The prevalent presence of S. smithogilvyi haplotype A (reported to be more aggressive) along with other additional factors could explain the serious damage and the chestnut production losses recorded for the past years in Basilicata, which urges to find and implement suitable measures to control this emerging and very damaging chestnut pathogen.

Pear decline (PD), associated with ‘Candidatus Phytoplasma pyri’, is one of the most severe diseases affecting pear cultivation in Europe and the United States. Several psyllid species act as vectors of phytoplasmas belonging to the 16SrX group and play a key role in the epidemiology of the disease. This study aimed to characterize the epidemiology of pear decline in Sicily using integrated field, molecular, vector, and remote sensing approaches, four years after the first detection of PD in the region. Visual surveys and molecular analyses were conducted over two years in eight pear orchards. A total of 115 plant samples and 101 Cacopsylla spp. specimens, selected from 1435 collected individuals, were analysed, confirming the presence of ‘Ca. P. pyri’ in 69% of symptomatic plants and in 4.6% of C. pyri individuals. Genetic characterization revealed a high degree of similarity among the phytoplasma isolates analysed. Remote sensing analyses conducted since 2018, combined with vector population monitoring, confirmed the epidemic nature of PD and indicated the persistence of a risk of further pathogen spread within the region, supporting the use of remote sensing as a complementary tool for large-scale disease monitoring.

Phytoplasmas of the X-disease group (16SrIII) are economically significant pathogens in South America, causing severe crop losses. Traditional classification based on the 16S rRNA gene has limitations in resolving closely related strains, prompting the exploration of alternative markers. This study focuses on the immunodominant membrane proteins imp and idpA, which exhibit high variability and play crucial roles in host–pathogen interactions. Through molecular characterization of imp and idpA genes in 16SrIII subgroups, we identified significant genetic diversity and distinct evolutionary pressures. The imp gene, under positive selection, showed high variability in its hydrophilic extracellular domain, suggesting adaptation to host immune responses. In contrast, idpA exhibited strong negative selection, indicating functional conservation. Phylogenetic analyses revealed that imp and idpA provide higher resolution than the 16S rRNA gene, enabling finer differentiation within subgroups. These findings highlight the potential of imp and idpA as complementary markers for phytoplasma classification and diagnostics.

Chestnut is a valuable species that grows widely throughout the Italian peninsula. It is susceptible to different diseases and among them chestnut blight caused by Cryphonectria parasitica is the most devastating one. This study aimed to molecularly characterize and distinguish virulent and hypovirulent C. parasitica isolates from the Basilicata region. Pure fungal cultures were obtained from symptomatic plant material, their genomic DNA was extracted and amplified using rDNA Internal Transcribed Spacer (ITS), M13 minisatellite and (GACA)4 microsatellite primers. ITS sequencing allowed the identification of the fungal species while minisatellite and microsatellite Polymerase Chain Reactions (PCRs) differentiate between virulent and hypovirulent isolates placing them in distinct clusters. Additionally, sequence analysis of hypovirus partial genome showed that all identified hypoviruses, belonged to Cryphonectria hypovirus 1 (CHV-1) subtype, sharing a 99% sequence identity with the Italian isolate Marche of CHV-1. To our knowledge, this is the first study to differentiate virulent and hypovirulent isolates of C. parasitica at molecular level and identify the hypovirus subtype associated with the hypovirulent isolates in the Basilicata region.

Pear decline (PD), associated with ‘Candidatus Phytoplasma pyri’, is a major disease of pear in Europe and the United States. Several psyllid species are involved in the tritrophic system of PD as vectors of phytoplasmas belonging to the 16SrX group. Four years after the first detection of PD in Sicily, an integrated approach was applied to investigate the epidemic in a major pear-growing area. Visual surveys and molecular analyses were conducted over two years in eight orchards. A total of 115 plant samples and 101 Cacopsylla spp. specimens selected from a total of 1,435 collected individuals were analysed, confirming ‘Ca. P. pyri’ in 69% of symptomatic plants and in 4.6% of C. pyri individuals. Multilocus sequence typing (MLST) revealed high genetic similarity among 16SrX isolates. Remote sensing analyses since 2018, combined with vector monitoring, confirmed the epidemic nature of PD and the persistence of a risk of further pathogen spread within the region, proving, inter alia, to be a valid method for identifying the syndrome even on a large scale.

In the spring of 2023 in Eboli and Caserta (Campania, southern Italy), strawberry plants (var. Marimbella) grown in organic open fields showed an outbreak of a severe and unprecedented decline (disease incidence reaching > 80%) associated with root rot, crown rot, and leaf spot and closely resembling symptoms reported previously in other countries for Neopestalotiopsis spp. infection. Therefore, the present study was undertaken with the aim of determining the aetiology of this serious disease. Fungal isolates were obtained from symptomatic strawberry plants and investigated in detail for molecular identification. Phylogenetic analysis was conducted by amplifying and sequencing three DNA barcodes: the internal transcribed spacer (ITS) region of rDNA, the beta-tubulin (tub2) partial gene, and the translation elongation factor 1 alpha (tef1) partial gene. Symptoms observed in the field were replicated in pathogenicity tests, conducted by inoculating strawberry (var. Marimbella) leaves, fruits and plants, thus satisfying Koch's postulates. Phylogenetic analyses identified the causal agent as Neopestalotiopsis rosae. To our knowledge, this is the first report of the emerging and serious fungal pathogen N. rosae infecting strawberry in Italy.

This study reports the draft genome of 'Candidatus Phytoplasma pyri' strain P1, isolated from Argentina. The genome assembly consisted of 17 contigs, with a total length of 575,431 bp, a GC content of 20.35%, and 125x coverage. A total of 537 genes were annotated, including those related to metabolism, genetic information processing, and signaling. Phylogenetic analysis placed 'Ca. Phytoplasma pyri' within the 16SrX group, supporting its classification as a distinct species from 'Ca. Phytoplasma mali' and 'Ca. Phytoplasma prunorum', with average nucleotide identity values ranging from 90.3 to 90.5%. In contrast, strain P1 shares 98.1% average nucleotide identity with the Chilean strain 'Ca. Phytoplasma pyri' UCh_1, supporting their classification as members of the same species. The investigation also identified 12 secreted proteins, including homologs of known effectors, as well as conserved proteins associated with virulence mechanisms. A homolog of the immunodominant membrane protein was also characterized, revealing conserved gene organization across related strains. These findings contribute to the understanding of 'Ca. Phytoplasma pyri' pathogenicity and provide valuable genomic data for future research.Copyright (c) 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In the Campania region of southern Italy, a formerly undescribed witches’-broom disease of cultivated strawberry characterized by symptoms similar to those of strawberry witches’-broom and multiplier diseases occurring in North America, has been observed. Strawberry witches’-broom and multiplier diseases are not known to occur in Europe. To elucidate the etiology of the new strawberry disease occurring in southern Italy and to determine the taxonomic position of the presumable causal agent, field observations and PCR assays using universal and group-specific phytoplasma primers followed by multigene sequence analysis were carried out. All of the symptomatic strawberry plants examined tested phytoplasma positive with universal primers and primers specific to the elm yellows (EY) phytoplasma group or 16SrV group. The percentage of diseased plants in the fields was about 30%. Data obtained from sequence and phylogenetic and virtual RFLP analyses of PCR-amplified rDNA (16S rDNA and 16S/23S rDNA spacer region), rpsV (rpl22) and rpsC (rps3), map, imp and groEL gene sequences, showed that the diseased strawberry plants harbored phytoplasma strains which were identical or nearly identical to each other and to strains of the rubus stunt (RuS) agent ‘Ca. Phytoplasma rubi’, a member of the 16SrV group, subgroup 16SrV-E. The 16S rDNA sequence similarity among the strawberry-infecting phytoplasma strains ranged from 99.1 to 99.9%. These strains shared the same range of 16S rDNA sequence similarity with RuS phytoplasma strains including the reference strain RUS of ‘Ca. Phytoplasma rubi’. This is the first report on the occurrence of RuS phytoplasma in naturally affected strawberry plants.

‘Candidatus Phytoplasma ulmi’ (16SrV-A) is the causal agent of elm yellows (EY), a lethal and/or decline disease of several Ulmus (elm) species and hybrids in North America and Europe. In this study, field observations and PCR assays were used to detect phytoplasma infections in diseased U. minor, U. pumila and U. glabra trees in southern Italy. Also, a multigene sequence analysis employing various less conserved genes was carried out to explore the genetic variation in detected strains. All the symptomatic elm trees tested were infected with ‘Ca. Phytoplasma ulmi’. No other phytoplasmas or variants could be detected. Although ‘Ca. Phytoplasma ulmi’ was already known to occur in southern Italy on European field elm, the current work expands the information on the presence, disease incidence and severity, plant host range and molecular aspects of EY phytoplasma strains occurring in southern Italy. In addition, this is the first report from Italy on the molecular characterization of EY phytoplasma strains through map, imp and groEL gene sequence and phylogenetic analyses. Among the newly detected EY phytoplasma strains, some proved distantly related to each other and to other previously characterized EY phytoplasma strains within the genes examined. This implies the presence of distinct taxonomic entities within the material examined. The occurrence of different strains was not linked to the biological traits and geographical distribution. However, the data obtained may provide a basis for further studies aimed at elucidating several other unknown aspects of the EY agent, knowledge of which is essential for effective disease management and control strategies. The results of the current work also show that the EY phytoplasma is particularly widespread in southern Italy and is of considerable economic and ecological relevance.

Among grapevine yellows, Bois noir (BN), associated with 'Candidatus Phytoplasma solani', represents the biggest threat in the main wine-growing areas worldwide, causing significant losses in berry quality and yields. BN epidemiology involves multiple plant hosts and several insect vectors, making considerably complex the development of effective management strategies. Since application of insecticides on the grapevine canopy is not effective to manage vectors, BN management includes an integrated approach based on treatments to the canopy to make the plant more resistant to the pathogen and/or inhibit the vector feeding, and actions on reservoir plants to reduce possibilities that the vector reaches the grapevine and transmit the phytoplasma. Innovative sustainable strategies developed in the last twenty years to improve the BN management are reviewed and discussed.

This collaborative work by over 180 researchers from 40+ countries addresses the challenges posed by “phantom agents”—putative pathogenic agents named in literature without supporting data on their existence. Those agents remain on regulatory lists, creating barriers in trade and plant certification. Historically identified based solely on symptoms, these agents lack isolates or sequence data, making reliable detection or risk assessment impossible. After reviewing over 120 such agents across 10 key plant genera, we recommend their removal from regulatory lists and call for revised standards aligned with modern diagnostics. This effort seeks to streamline germplasm exchange, benefiting global agriculture by removing the constraints imposed by phantoms.

Flavescence dorée (FD) is the most important grapevine yellows, constituting a great threat in all major viticultural areas in Europe. The FD causal agent is an incidentally cited phytoplasma species termed ‘Candidatus Phytoplasma vitis’ (FDp), listed as a quarantine organism in Europe and mainly transmitted from vine to vine by the monophagous leafhopper Scaphoideus titanus. In the present study, the genetic variability of FDp identified in symptomatic plants located in a vineyard cv. Biancolella on the Ischia Island (Campania, Italy) was investigated. Our results unveiled for the first time the presence of FDp strains belonging to the subgroup 16SrV–C with a low genetic variability within map and rp genetic markers, reflecting an homogeneous genetic lineage of phytoplasma population examined in the present study. Phylogeny and nucleotide sequence analyses can also suggest the possible involvement of other insect vectors and plant hosts in the FD epidemiology on the Island. Further investigation to ascertain the presence of putative vectors and plant hosts representing inoculum sources should be carried out to reinforce the preliminary results obtained in the present study.

In recent years, the Asian chestnut gall wasp (ACGW) Dryocosmus kuriphilus has been reported to have a high incidence in Italy and other Mediterranean basin countries. In 2021-2022, a study was undertaken in the Basilicata Region (Southern Italy) to investigate the relationship between the galls produced by ACGW on sweet chestnut (Castanea sativa Mill.) and fungal pathogens. In particular, the fungal diversity from green and necrotic galls collected from two important sweet chestnut sites (Melfi and Rionero in Vulture) was investigated. Nineteen fungal taxa were identified based on their morphological and molecular traits. In both localities, the most frequent species isolated from green and necrotic galls were Gnomoniopsis castaneae, Colletotrichum acutatum, and Pestalotiopsis sp. It is essential to understand the role played by the galls as an inoculum source for sweet chestnut fungal pathogens, particularly for G. castaneae, an emerging pathogen of which biology is still poorly understood. Findings from the present study stressed that the complex relationship between host-insect-microbial community needs to be elucidated to be able to control the pathogenic fungi and consequently maintain sweet chestnut trees' health as they play a key role in the local agriculture (horticulture, forestry) and subsidiary economy

Alder yellows (ALY) phytoplasma (16SrV-C) is associated with ALY, a disease of several Alnus (alder) species in Europe and A. rubra in North America. In all affected species, the symptoms are similar. However, latent infections are common. ALY phytoplasma includes different strains which may be occasionally transmitted to grapevines leading to some grapevine yellows diseases. In the current study, visual symptom assessment and PCR-based methods using universal and group-specific phytoplasma primers were used to update and extend knowledge on the occurrence, impact, and genetic diversity of ALY phytoplasma in declining and non-symptomatic A. glutinosa and A. cordata trees in the Basilicata and Campania regions of southern Italy. ALY phytoplasma was detected in 80% of alder trees examined. In symptomatic trees, no other cause of disease was observed. More than half of alder trees that tested phytoplasma-positive proved to be latently infected. A considerable genetic variability was observed among the newly recorded ALY phytoplasma strains in southern Italy in almost of the genes examined. These included 16S rRNA, 16S/23S rDNA spacer region, ribosomal protein rpsV (rpl22) and rpsC (rps3), map, imp, and groEL genes. Eleven new genotypes were identified at map gene sequence level. However, the genetic differences observed were not related to plant host species, geographical origin, and symptoms shown by infected alder trees. Also, this study indicates that ALY phytoplasma is more widespread than previously thought.

'Candidatus Phytoplasma meliae' is a pathogen associated with chinaberry yellowing disease, which has become a major phytosanitary problem for chinaberry forestry production in Argentina. Despite its economic impact, no genome information of this phytoplasma has been published, which has hindered its characterization at the genomic level. In this study, we used a metagenomics approach to analyze the draft genome of the 'Ca. P. meliae' strain ChTYXIII. The draft assembly consisted of twenty-one contigs with a total length of 751.949 bp, and annotation revealed 669 CDSs, 34 tRNAs, and 1 set of rRNA operons. The metabolic pathways analysis showed that ChTYXIII contains the complete core genes for glycolysis and a functional Sec system for protein translocation. Our phylogenomic analysis based on 133 single-copy genes and genome-to-genome metrics supports the classification as unique 'Ca. P. species' within the MPV clade. We also identified 31 putative effectors, including a homolog to SAP11 and others that have only been described in this pathogen. Our ortholog analysis revealed 37 PMU core genes in the genome of 'Ca. P. meliae' ChTYXIII, leading to the identification of 2 intact PMUs. Our work provides important genomic information for 'Ca. P. meliae' and others phytoplasmas for the 16SrXIII (MPV) group.