Current status of phytoplasma diseases of forest and landscape trees and shrubs

This paper summarizes the current knowledge of phytoplasma diseases of forest and landscape trees and shrubs, with special reference to molecular and taxonomic aspects of the associated phytoplasmas in the light of the plethora of data generated over the last years. Phytoplasma diseases of such kind of trees and shrubs are of considerable economic and ecological significance throughout the world, either because of the local impact or of their widespread distribution. However, information is still missing on many aspects of some of these diseases, including insect vectors, phytoplasma-vector relationships, phytoplasma-host plant interactions, strain virulence, strain interference, host tolerance, host range, and impact of the infections on the growth and yield of affected plants. In some instances, phytoplasma-infected forest and landscape trees and shrubs can serve as reservoirs of phytoplasmas affecting agricultural crops. Latent phytoplasma infections in some forest and landscape trees and shrubs are common

Pulsed-field gel elecrophoresis for isolation of full-length phytoplasma chromosomes from plants

Pulsed-field gel electrophoresis (PFGE) is a powerful technique for genomic studies of unculturable plant-pathogenic phytoplasmas, which enables separation of full-length phytoplasma chromosomes from contaminating host plant nucleic acids. The PFGE method described here involves isolation of phytoplasmal DNA from high-titer phytoplasma-infected herbaceous plants using a phytoplasma enrichment procedure, embedding of phytoplasma chromosomes in agarose blocks, and separation of entire phytoplasma chromosomes from contaminating host plant nucleic acids by electrophoresis. Full-length phytoplasma chromosomes are resolved as single, discrete bands in the gel. The identity of these bands can be confirmed by Southern blot hybridization using a ribosomal DNA fragment as a probe. The method does not utilize gamma-irradiation to linearize phytoplasma chromosomes prior to electrophoresis

Widespread occurrence of apple proliferation disease in low-intensity orchards of Basilicata

Visual symptom assessment and PCR amplification were used to survey the occurrence of apple proliferation (AP) disease in low-intensity orchards in the Agri valley, a major cultivation area of Basilicata (southern Italy). The apple trees examined, whose cultivars were not determined as they consisted mostly of local types, were more than 20-year-old. Therefore, these plants had been exposed to insect vectors for a long time. The survey revealed that a high percentage of trees were infected reaching more than 50% in some locations. The symptoms of diseased trees were generally mild and consisted of enlarged stipules, rosettes, witches’-brooms as well as subterraneous witches’-broom-like growth arising from large roots. However, the incidence and severity of symptoms in the aerial parts of affected trees were more pronounced in trees which had been heavily pruned in the previous dormant season. Specificity of the primers used and RFLP analysis of PCR-amplified 16S rDNA sequences employing SspI and BsaAI restriction endonucleases showed that the trees testing positive by PCR were infected by the AP agent ‘Candidatus Phytoplasma mali’. The high incidence of AP infections in low-intensity orchards of the Agri valley is likely due to inappropriate vector control. The trees examined were not or rarely treated with insecticides. Although a few AP-affected apple trees grown in a low-intensity orchard in the Agri valley had previously been observed, our survey shows that the distribution of AP disease in Europe extends further south than previously thought and that the climatic conditions of southern Italy are not unsuitable for this quarantine disease

'Candidatus Phytoplasma cynodontis', the phytoplasma associated with Bermuda grass white leaf disease

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2·5 % of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S–23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name 'Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S–23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas

‘Candidatus Phytoplasma cynodontis’ (16SrXIV group) affecting Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. in India.

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Investigations on Fungi Isolated from Apple Trees with Die-Back Symptoms from Basilicata Region (Southern Italy)

Val d'Agri is an important orchard area located in the Basilicata Region (Southern Italy). A phenomenon affecting cv. "Golden Delicious" apples which lead to tree death has been observed in the past several years in this area. This phenomenon has already been detected in about 20 hectares and is rapidly expanding. The symptoms observed were "scaly bark" and extensive cankers, mainly located in the lower part of the trunk, associated with wood decay. Dead plants ranged from 20% to 80% and, in many cases, trees were removed by farmers. In order to identify the causes of this phenomenon, investigations were started in autumn/winter 2019. In order to determine the possible causal agents, fungal and bacterial isolations, from symptomatic tissues, were performed in laboratory. Bacterial isolations gave negative results, whereas pure fungal cultures (PFCs) were obtained after 3-4 passages on potato dextrose agar (PDA) media. Genetic material was extracted from each PFC and amplified by PCR using three pairs of primers: ITS5/4, Bt2a/Bt2b and ACT-512F/ACT-783R. The amplicons were directly sequenced, and nucleotide sequences were compared with those already present in the NCBI GenBank nucleotide database. All isolated fungi were identified based on morphological features and multilocus molecular analyses. Neofusicoccum parvum, Diaporthe eres and Trametes versicolor were most frequently isolated, while Pestalotiopsis funerea, Phomopsis spp. and Diaporthe foeniculina were less frequently isolated. All nucleotide sequences obtained in this study have been deposited into the EMBL database. Pathogenicity tests showed that N. parvum was the most pathogenic and aggressive fungus, while Phomopsis sp. was demonstrated to be the less virulent one. All the investigated fungi were repeatedly reisolated from artificially inoculated twigs of 2-year-old apple trees, cv. "Golden Delicious", and subsequently morphologically and molecularly identified. The role played by the above-mentioned fungi in the alterations observed in field is also discussed