Bertaccini, Assunta

From 2016, witches’ broom and stunting symptoms were observed in Lolium rigidum grown in some fruit tree nurseries in Faragheh (Abarkouh, Yazd province, Iran). Total DNAs were extracted from symptomatic and asymptomatic plants and assessed for phytoplasma presence using direct and nested PCR to detect the 16S ribosomal RNA gene. From all symptomatic L. rigidum plant samples, expected length PCR amplicons were obtained. RFLP analysis with informative restriction enzymes showed identical profiles in all the samples resulted positive, that were also consistent with those of one of the subgroups of the aster yellows phytoplasmas (16SrI). The 16S rRNA gene sequence of Faragheh L. rigidum bushy stunt strain was 100% identical to some ‘Candidatus Phytoplasma asteris’ related strains, and 99.12% similar to the reference ‘Ca. P. asteris’ strain. The virtual RFLP pattern was identical (similarity coefficient 1.00) to the pattern of phytoplasmas in subgroup 16SrI-F. This is the first report of occurrence and molecular identification of this phytoplasma strain in L. rigidum and indicates a potential phytoplasma reservoir for trees in fruit tree nurseries where insect vectors may be present. This phytoplasma strain has been reported in symptomatic stone fruits in Spain and in potato in Ecuador. Further research on the epidemiology of witches’ broom and stunting in L. rigidum is required to develop elimination the phytoplasma from areas surrounding agricultural crops and avoid the risks of epidemics.

Cultivation of prickly pear (Opuntia ficus-indica L., family Cactaceae) is of high value in dry-land agriculture in Jordan. In May 2021, symptoms including thickening and severe stunting of the cladodes and deformation of fruits were observed on prickly pear plants cultivated in southern Jordan, Madaba region (31.593565 N, 35.850111 E), with a 15% incidence across three cactus fields. To verify the occurrence of a graft-transmissible disease, wedge grafting was performed on asymptomatic opuntia rootstocks, resulting in thickened cladodes and deformed fruits within five weeks. Samples of cladodes from naturally infected plants were collected from fourteen symptomatic and one asymptomatic plant. A nested polymerase chain reaction (PCR) performed by using primer pair P1/P7 (Deng and Hiruki 1991; Schneider et al. 1995), followed by R16F2n/R16R2 (Gundersen and Lee 1996), amplified a fragment of the expected size only from the symptomatic samples. Direct amplicon sequencing followed by blast comparison to both, GenBank and EPPO-QBank databases (https://qbank.eppo.int/phytoplasmas/) allowed the identification of a ’Candidatus Phytoplasma australasiae=australasiaticum’ strain (GenBank accession no. PQ319761, designated strain ´Cact1´), with a 100% sequence identity to the reference strain (GenBank accession no. Y10097, ribosomal subgroup 16SrII-D) (White et al., 1998; Rodrigues et al., 2023). The phylogenetic analysis of strain ´Cact1´ with phytoplasma strains of ribosomal subgroups in group 16SrII and detected in cactus in China, Italy and Turkey showed that strain ´Cact1´ is not clustering with any of them (Figure 1). Indeed, comparison with all sequences in GenBank, show that the phytoplasma from Jordan clusters with those enclosed in the 16SrII group under the species ‘Ca. P. australasiae=australasiaticum’, showing about 99.00% identity to this phytoplasma and having an identity to ‘Ca. P. aurantifolia=citri’ of about 98.50%, that is below the accepted threshold for ‘Ca. Phytoplasma’ strains differentiation (Bertaccini et al. 2022). This identification was confirmed by amplifying and sequencing the leucyl transfer RNA synthetase (leuS) gene (Abeysinghe et al. 2016) (GenBank accession no. PQ349195), that showed 100% sequence identity with 100% coverage to the Parthenium hysterophorus phyllody phytoplasma strain ´PR08´ from India (GenBank accession no. CP097207), identified as a strain of ‘Ca. P. australasiae=australasiaticum’ (White et al., 1998; Rodrigues et al., 2023). Other phytoplasma strains having 100% identity on the leuS gene but with 95-96% coverage were reported in pearl millet, soybean and alfalfa from India (GenBank accession no. MW020555, MW020562 and MW020559 respectively). The leuS gene sequence has been relevant to confirm the identification of phytoplasmas infecting various agricultural important crops especially in Asian countries (Tiwari et al., 2023). It is thus necessary to investigate insect vector(s) presence and search for other economically important hosts and for alternative host weeds for this phytoplasma considering that the disease and the associated phytoplasmas are present and spreading in other regions of Jordan.

Studies of the genetic diversity of ‘Candidatus Liberibacter asiaticus’ strains based on housekeeping genes have been unsuccessful. The increasing availability of complete genome sequences of several strains from different countries has allowed the identification of regions having greater variability, which have been successfully implemented for the bacterium characterization, including microsatellites, genes of prophage origin, and miniature transposable elements with inverted-repeats (MITEs). In the present work, the genetic structure of 147 ‘Ca. L. asiaticus’ strains from nine provinces of Cuba were investigated using two polymorphic regions, consisting of typing for prophages and MITEs. The results showed an important level of coexistence of type 1 and 2 prophages in the Cuban strains, while the type 3 prophage was not detected. Likewise, a high rate of co-occurrence of both types of MITEs (MCLas-A and -B) was also observed. However, the MITE MCLas-A was detected only in its empty form. The double-locus analysis allowed the identification of eight genotypes. Out of these, seven genotypes were present in the Western region, which constitutes the region with the highest genetic variability. This is the first report of a genetic characterization of Cuban strains of ‘Ca. L. asiaticus’ with polymorphic markers in orchards growing in commercial citrus regions.

‘ Candidatus Phytoplasma prunorum’ is associated with European stone fruit yellows disease, affecting wild and cultivated species of Prunus at different degrees of susceptibility, and so far is being mainly restricted to Europe. Here, we report draft genome sequences for ‘ Ca. Phytoplasma prunorum’ strains ESFY1 and LNS1, which represent the first available for this species. Strain ESFY1 is the causal agent of the European stone fruit yellows disease of P. persica in Germany, and LNS1 is the causal agent of leptonecrosis disease of P. salicina in Italy. Their draft genomes consist of 480,123 and 490,930 bases assembled into 28 and 16 contigs, with an average GC content of 21.1 and 20.7%, respectively. Analysis of average nucleotide identity (ANI) values between genomes further supported the delineation of ‘ Ca. P. prunorum’ as a species distinct from ‘ Ca. P. mali’, in agreement with previous phylogenetic data. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

‘ Candidatus Phytoplasma pyri’, the pathogen associated with pear decline, affects pear trees across both the old and new worlds. However, research on this phytoplasma has been limited by the lack of genomic data. This study presents the first draft genome of ‘ Ca. P. pyri’ using a strain from Chile, with its genomic features analyzed in comparison with the closely related ‘ Ca. Phytoplasma’ species ‘ Ca. P. mali’ and ‘ Ca. P. prunorum’. The draft genome spans 456,478 bp with a GC content of 20.4%. Key genes possibly associated with pathogenicity and potential pathogenic effectors were identified, and they are notably lacking orthologs of known effectors. A single potential mobile unit similar to that of ‘ Ca. P. mali’ was identified. It is characterized by the absence of the transposase tra5 and the presence of the IS3 family transposase iSErh1. Multilocus sequence analysis of six genetic markers (16S rRNA gene, LSU36p, tuf, aceF, secA, and secY) from 10 Chilean and 10 Italian samples revealed high genetic uniformity among the Chilean strains, collected from five geographically distant orchards over a span of 13 months; by contrast, strains with greater diversity were detected among those from Italy, collected from a few localities over approximately 30 years. These findings suggest limited evolutionary divergence of this phytoplasma in Chile. This study provides a foundational framework for investigations into the pathogenic mechanisms and evolutionary dynamics of ‘ Ca. P. pyri’.

AbstractDuring the past two decades, a high mortality of coconut palms was observed in the coastal areas of Equatorial Guinea. Reportedly, the palm population has been reduced by 60%–70%, and coconut production has decreased accordingly. To identify the cause of the mortality, a survey was carried out in April 2021 in various localities of the coconut belt. Molecular analyses carried out on 16S rRNA and secA genes detected phytoplasma presence in the majority of the samples. Sequencing and BLAST search of the 16S rRNA gene sequences showed >99% identity of the detected phytoplasmas to ‘Candidatus Phytoplasma palmicola’. The RFLP analyses of 16S ribosomal gene using Tru1I and TaqI enzymes led to assign these phytoplasmas to subgroup 16SrXXII‐A. In all samples that tested positive, including one from a hybrid coconut palm and two from oil palm the same phytoplasma was identified. The phylogenetic analyses of 16S rRNA and secA genes confirmed respectively 99.98%–100% and 97.94%–100% identity to ‘Ca. P. palmicola’. RFLP analyses using MboII enzyme on the secA gene amplicon differentiated the phytoplasma found in Equatorial Guinea from those present in Ghana and Ivory Coast. The Equatorial Guinean phytoplasma strain resulted to be identical to the strains from Mozambique, confirming the presence of a geographic differentiation among phytoplasma strains in the coastal areas of Western and Central Africa. The identified phytoplasma is different from the ‘Ca. P. palmicola’ strains found in Ghana and Ivory Coast and represents the first identification a 16SrXXII‐A strain in Equatorial Guinea and in Central Africa. Strict monitoring and surveillance procedures for early detection of the pathogen are strongly recommended to reduce its impact and further spread in the country and permit the recovery of coconut plantations.