Climate change is intensifying the simultaneous occurrence of biotic and abiotic stresses in fruit crops, but yet the molecular mechanisms underlying plant responses remain poorly understood. The physiological and transcriptomic responses of two sweet cherry (Prunus avium L.) cultivars, Santina and Bing, grafted onto Gisela 12, were investigated under single and combined stresses imposed by Pseudomonas syringae pv. syringae and water deficit. Although biomass, gas exchange, and hormone accumulation showed only minor changes, combined stress triggered distinct cultivar-dependent transcriptional reprogramming. The cultivar Bing exhibited a pronounced response with 4261 differentially expressed genes (DEGs), characterized by strong repression of photosynthetic processes and activation of defense- and hormone-related pathways. In contrast, the cultivar Santina showed a moderate response with 674 DEGs, primarily reinforcing structural and secondary metabolism. Cultivar-specific modulation of abscisic acid sensitivity was associated with the contrasting regulation of WRKY40 and Sin3-like repressors, despite comparable ABA levels. Strikingly, both cultivars upregulated the GIGANTEA gene, underscoring its role as a central regulatory hub linking circadian rhythm, stomatal function, and hormonal crosstalk under dual stress. Collectively, these results reveal non-additive, genotype-specific transcriptional strategies in sweet cherry trees, providing insights into stress integration in fruit trees and identifying regulatory genes that may inform breeding and management strategies for resilience under climate change.

Sesame is an oilseed crop threatened by a phyllody disease associated with the presence of phytoplasmas, which can reduce yields by up to 80%. The molecular identification of these bacteria in crops located in Western Iran was achieved from samples showing symptoms of diverse intensity and types. For biological characterization, the pathogen was also dodder-transmitted to periwinkle plants. After nucleic acid extraction and nested PCR using phytoplasma-specific primer pairs amplifying part of the 16S rRNA gene, it was possible to amplify DNA fragments from both symptomatic sesame samples and dodder-inoculated periwinkle plants. The virtual RFLP pattern from the 16S rRNA gene sequences using iPhyClassifier indicated the presence of phytoplasma strains in 16SrII-D and 16SrIX-C subgroups. The identity percentage values of the obtained amplified sequences corroborated by the phylogenetic analysis identified them as ‘Candidatus Phytoplasma australasiaticum’ and ‘Ca. P. phoenicium’, respectively. The two phytoplasma strains were detected in different sesame samples collected in the same field. The coexistence of two phytoplasmas may influence the observed differences in disease severity and suggests appropriate management strategies, since diverse insect vectors were reported alongside diverse phytoplasmas associated with this disease. Moreover, the widespread disease presence strongly suggests the breeding of resistant varieties.

Pseudomonas syringae pv. syringae and Xanthomonas arboricola pv. juglandis are the causal agents of bacterial canker in cherry and walnut blight, respectively, which cause significant production losses worldwide. These diseases have traditionally been controlled by copper-based agrochemicals and, more recently, antibiotics. However, the prolonged use of these compounds has led to the emergence of resistant bacterial strains. The search for new, efficient, and environmentally friendly biocontrol alternatives has intensified. Phages are promising candidates due to their ability to specifically infect and lyse bacterial pathogens. Endolysin enzymes are responsible for bacterial cell wall degradation, and although they have been extensively studied in medical and veterinary contexts, their application in agriculture remains limited. In this study, 17 putative endolysins were identified from bacteriophages infecting X. arboricola pv. juglandis and P. syringae pv. syringae. Based on conserved domain analyses, 12 were classified as glycosidases, four as amidases, and one as an endopeptidase. From these, a recombinant amidase (LysP) and a recombinant glycosidase (LysX) were expressed in E. coli, purified, and evaluated as pure enzymes. Both endolysins exhibited significant antimicrobial activity, reducing P. syringae pv. syringae viability by 62-78.3% and X. arboricola pv. juglandis viability by 51.5-53.1%, respectively. These findings highlight these recombinant endolysins as promising candidates for the development of biocontrol strategies against bacterial plant pathogens.

Over the past decade, in Ecuador it was reported the presence of severe epidemic outbreaks of a disease called purple top, which affects potatoes and other solanaceous plants such as Solanum betaceum (“tamarillo”). Molecular analyses were therefore performed on symptomatic plants of this species to verify the presence of 'Candidatus Phytoplasma' and 'Ca. Liberibacter solanacearum'. The detected phytoplasmas were identified on the 16S rRNA gene analyses by PCR, RFLP and sequencing. The haplotype of 'Ca. L. solanacearum' was identified by amplification and sequencing of 16S rRNA, 50S rplJ and rplL ribosomal protein genes, and the 16S/23S intergenic space region. A 'Ca. P. hispanicum' related strain (16SrXIII group) was detected in the symptomatic leaf samples, while the haplotype A of 'Ca. L. solanacearum' was detected in “tamarillo” seeds. This is the first identification of a phytoplasma never reported in Ecuador, moreover the presence of ‘Ca. L. solanacearum’ in “tamarillo” seeds opens new questions about the purple top disease epidemiology in solanaceous crops. Research on insect transmission, reservoir host species and seed transmission will further clarify the disease epidemiology, while appropriate management practices to reduce the further spread of these bacteria in the Ecuadorian agro-ecosystems should be applied such as insect control and certification of propagation materials.

A summary of the situation for detecting and naming phytoplasmas associated with different diseases in the last 30 years is presented in this chapter. In the last decades, a great amount of research has been devoted to classifying and differentiating these prokaryotes by molecular and bioinformatic tools applied to the analysis of specific phytoplasma genes. The availability of a robust classification system has greatly facilitated phytoplasma identification, leading to increased knowledge of their associated plant diseases worldwide. Scientific knowledge on phytoplasma biology still needs to be improved to allow better and more focused management solutions with minimal impact on the environment to mitigate the severity of these diseases in both agricultural and natural environments, also contributing to reduce the loss of biodiversity.

Phytoplasmas (‘Candidatus Phytoplasma’) are obligate, phloem-limited, cell wall-lacking bacterial pathogens with highly reduced genomes that infect diverse plant and insect vector species. They are associated with economically important diseases worldwide, including grapevine yellows, coconut lethal yellowing, sesame phyllody, apple proliferation, potato purple top, European stone fruit yellows, sugarcane white leaf, tomato big bud, and witches’ broom of several crops, posing significant threats to global agriculture, trade, and biosecurity. Phytoplasmas induce characteristic symptoms such as yellows, witches’ broom, phyllody, virescence, little leaf, and abnormal plant development, and are primarily transmitted by phloem-feeding insects, as well as through grafting, vegetative propagation, and occasionally seeds. Advances in molecular diagnostics, including PCR, qPCR, digital PCR, LAMP, RPA, WGS and CRISPR/Cas-based assays, have greatly enabled rapid, sensitive, and quantitative detection of phytoplasmas in both plants and insects. Comparative analysis of 16S rRNA gene and whole-genome sequencing has enabled the identification and classification of more than 52 ‘Ca. Phytoplasma’ species belonging to 40 ribosomal groups and numerous subgroups. Phytoplasma effectors mainly delivered through the Sec pathway manipulate host hormonal regulation, immunity, development, and insect vector interactions to promote their own colonization and transmission. Sustainable management remains challenging and requires integrated approaches involving resistant cultivars, insect vector control, quarantine restrictions, advanced diagnostics, surveillance, and emerging biotechnological and AI based tools for effective disease detection and mitigation.

Potato purple top is a complex phytoplasma disease that poses a serious threat to potato cultivation worldwide. To verify the presence of different phytoplasma strains in potato disease outbreaks in Iran, six major potato-growing provinces in the central and western regions of the country were surveyed, and a total of 270 potato plants, 230 symptomatic and 40 asymptomatic, was sampled. Nested PCR analysis revealed the phytoplasma presence in 45% of symptomatic and 7% of asymptomatic plants. Molecular analysis was performed, analyzing the sequences of the 16S rRNA gene and the ribosomal protein rp, secY, and tufB genes. Four ‘Candidatus Phytoplasma’ species were identified in the tested potato samples: ‘Ca. P. asteris’ (16SrI-B), ‘Ca. P. tritici’ (16SrI-R), ‘Ca. P. trifolii’ (16SrVI-A), and ‘Ca. P. solani’ (16SrXII-A). The ‘Ca. P. solani’ strains were prevalent, occurring in all the surveyed provinces, whereas the ‘Ca. P. tritici’ strains were restricted to the Chaharmahal and Bakhtiari province. Phylogenetic and multilocus sequence analyses provided a finer resolution, distinguishing among some of the closely related phytoplasma strains. This study represents the first comprehensive molecular survey of potato-infecting phytoplasmas across a wide geographical region of Iran. The findings will aid studies regarding insect vector(s), pathogen biology, host range and disease management strategies.

Periploca aphylla witches' broom (PapWB) is a phytoplasma-associated disease first reported in south-western Iran in 2010. Recent observations showed that the disease is spreading to new areas. PapWB phytoplasmas, detected in symptomatic P. aphylla plants, exhibited identical nucleotide sequences in the 1.8 kb PCR amplicon encompassing the 16S rRNA gene, the 16S-23S spacer region and the 5'-end of the 23S rRNA gene. When compared with the reference strains of previously described 'Candidatus Phytoplasma' species, the selected representative strain PapWB-S exhibited the highest 16S rRNA gene sequence identity (98.15%) with salt cedar witches' broom (SCWB1) phytoplasma, the reference strain of 'Ca. P. tamaricis'. Moreover, the sequence identity values for the tufB, rplV-rpsC and secY genes of PapWB-S strain, when compared with those of reference strains of previously described 'Ca. Phytoplasma' species, were below the thresholds adopted for describing new species. Phylogenetic analyses confirmed the close relationship of the strain PapWB-S with SCWB1 followed by other 'Ca. Phytoplasma' species belonging to the ribosomal groups 16SrX, 16SrXX and 16SrXXXIII. Following the revised guidelines for describing 'Ca. Phytoplasma' species, 'Ca. P. periplocae' sp. nov., associated with PapWB disease, is proposed, and the 16S rRNA gene sequence (accessible at GenBank/NCBI accession number OQ819043) is its nomenclatural type.

Recent research has demonstrated a presence inside the seeds of several plant species of endophytic bacteria that can directly or indirectly interact with germination and seedling growth. Phytoplasmas are plant-pathogenic bacteria that severely impact the agricultural productivity of several crops, including alfalfa, a crucial forage crop in which seed transmission was reported. Therefore, understanding the transmission pathways of phytoplasmas is essential for developing effective control strategies. This study investigates the seed transmission of phytoplasmas in alfalfa using seeds collected in Oman in 2002 and kept in a dry environment in a laboratory for 20 years. The sterilized seeds were germinated and grown in agar medium under sterile conditions and transplanted in soil under greenhouse-controlled insect-proof conditions. Utilizing polymerase chain reaction (PCR) and nested PCR followed by RFLP and sequencing analyses, the alfalfa seedlings were screened for the phytoplasma presence. The detection of phytoplasmas in 16SrIII, 16SrV, 16SrX, and 16SrXII groups was achieved, confirming the preliminary results obtained in the 2002 testing of the same seed batches. This finding indicates that seed transmission could be a critical pathway for the spread of these pathogens in alfalfa, considering their survival in seeds for more than 20 years. Further investigations into the mechanisms of seed transmission and the development of resistant alfalfa varieties are essential to enhance the sustainability and productivity of alfalfa cultivation, thereby supporting the agricultural sector’s efforts to meet the growing demand for high-quality forages.

Persimmon (Diospyros kaki) plants showing yellowing, reddening, die-back, and decline symptoms were observed in Mehriz (Yazd province), Iran. Total DNAs, extracted from samples collected from symptomatic and symptomless plants, were subjected to direct and nested PCR, amplifying the 16S rRNA gene of phytoplasmas using specific primer pairs. PCR amplicons of expected lengths were obtained, mainly from nested PCR, and only from samples collected from symptomatic plants. Real and virtual RFLP, phylogenetic, and DNA identity analyses of the partial 16S rRNA gene sequences suggested the presence of diverse phytoplasmas in the analyzed samples. The identified phytoplasmas were referable to ‘Candidatus Phytoplasma omanense’ (16SrXXIX group) and ‘Ca. P. australasiae = australasiaticum’ (16SrII-D subgroup). The results of the sampling and testing highlight the urgent need for an accurate survey to verify the presence and identity of phytoplasmas in symptomatic fruit trees in Iran, in order to be able to plan appropriate management strategies. Further investigations of the possible role of ‘Ca. P. omanense’ strains as an emerging threat to fruit orchards in Iran should also be performed.

In recent years, the cultivated area of hazelnuts in Chile has increased significantly. Along with this rapid expansion, biotic constraints that affect the optimal development of the crop have been identified. Among these, bacterial blight disease caused by Xanthomonas arboricola pv. corylina has been particularly relevant. This pathogen has a global distribution and is present in all hazelnut-producing countries. In the spring of 2023, hazelnut orchards were sampled from the Maule to Biobío Regions of Chile. The Chilean isolates recovered from hazelnut tissues showing symptoms of bacterial blight were characterized by their ability to grow on different semi-selective media, their carbohydrate utilization profiles, hypersensitivity response in tobacco plants, and biochemical tests. Additionally, the isolates were identified based on the 16S rRNA gene and multilocus sequence analysis (MLSA) on the rpoD, gyrB, and atpD genes. The results showed that the X. arboricola pv. corylina Chilean isolates differed from previously reported isolates in other geographic areas as they are capable of metabolizing sorbitol and mannitol. Using MLSA and average nucleotide identity (ANI) comparison, these isolates were grouped into four and five phylogenetic clades, respectively, representing a significant difference from what has been reported in similar international studies.

Evidence from an increasing number of studies indicates that plant endophytic microorganisms play a significant role during biotic and abiotic stress resistance. To date, however, only a handful of studies on endophytes in response to the presence of phytoplasmas have been conducted. The production of jujube (Ziziphus jujuba) is threatened by jujube witches’ broom (JWB) disease, which is associated with the presence of the JWB phytoplasma ‘Candidatus Phytoplasma ziziphi’. To investigate the impact of jujube witches’ broom phytoplasma on the endophyte populations in jujube, high-throughput sequencing was performed in healthy and JWB-infected orchard jujube trees and in vitro jujube shoots. The results showed that the presence of JWB phytoplasma in jujube altered the abundance, diversity, and community structure of endophytic bacteria and fungi. In the branches and the roots, the presence of JWB phytoplasma was associated with an increase in the richness of the endophytic communities and a decrease in their diversity, with the phyla Proteobacteria, Firmicutes, and Bacteroidota and the genus ‘Ca. Phytoplasma’ becoming the most abundant. The presence of phytoplasmas was also associated with the remodeling of the endophytic microorganisms’ interaction network, shifting to a simpler biodiversity state. These results demonstrate the response of the jujube endophytic community to the presence of JWB phytoplasmas and shed light on the possible antagonistic agents that could be further evaluated for JWB disease biocontrol.

'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'.

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.