ETIOLOGY AND EPIDEMIOLOGY OF PHYTOPLASMA-ASSOCIATED DISEASES OF STONE FRUITS AND GRAPEVINE IN JORDAN

Abstract In this study, a national survey on phytoplasma-associated diseases was conducted in Jordan from 2019 to 2021 targeting almond, pomegranate, and grapevine, three of the main fruit crops cultivated in all country as commercials and family farming. The activities included: (i) monitoring and sampling symptomatic and symptomless plants from early summer to autumn; (ii) total nucleic acids extraction and phytoplama detection by 16S rDNA amplification in nested PCRs using the primer pairs P1/P7 followed by F1/R0; (iii) sequencing and bioinformatic analyses (BlastN, iPhyClassifier) of F1/R0 amplicons. During field surveys, almond yellows and witches’-broom (incidence ranging from 20- 85%), pomegranate exhibiting leaf chromatic alteration and rolling, little leaf and witches’-broom (incidence ranging from 30- 65%), and grapevine yellows (incidence ranging from 10-55%) were observed. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 23, 17, 22 and 15.7% of collected symptomatic almond, pomegranate trees and symptomatic wine and table grape cultivar plants, respectively. Molecular detection and 16S rDNA nucleotide sequence analyses revealed the presence of different ‘Candidatus Phytoplasma’ species within samples from symptomatic plants, while no amplification was obtained from symptomless plant samples. Five categories of phytoplasma-like symptoms, including early flowering along with evergreen pattern; witches’-broom, yellowing, and dieback; slim leaf and leaf rolling; stem fasciation, were observed in almond trees. Disease incidence in the investigated orchards ranged from 20 to 85%. Nested PCR-based amplification of 16S rRNA gene detected phytoplasmas in 23% of collected symptomatic almond trees. Amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma asteris’ (taxonomic subgroups 16SrI-B and -R), ‘Ca. P. aurantifolia’ (16SrII-B and -C), ‘Ca. P. omanense’ (16SrXXIX-A and -B) (16SrXXIX-B described for the first time), ‘Ca. P. phoenicium’(16SrIX-B), ‘Ca. P. pyri’ (16SrX-C), ‘Ca. P. solani’ (16SrXII-A), and ‘Ca. P. ulmi’(16SrV-A). Moreover, further investigation identified ‘Ca. P. asteris’ (subgroup 16SrI-R) in putative insect vectors Agalmatium sp., Empoasca sp., Reptalus quinquecostatus, and Hyalesthes obsoletus, ‘Ca. P. pyri’ in Cacopsylla bidens, Cicadulina bipunctata, Laodelphax striatellus, and Tettigometra sp., and ‘Ca. P. omanense’ (subgroup 16SrXXIX-B) in the non-crop plant Amaranthus sp. In pomegranate symptomatic plants, four genetically distinct phytoplasmas were identified and attributed to ‘Candidatus Phytoplasma solani’ (16SrXII-A), ‘Ca. Phytoplasma aurantifolia’ (16SrII-B), ‘Ca. Phytoplasma asteris’ (16SrI-B, -R), and ‘Ca. Phytoplasma ulmi’ (16SrV-A). Additionally, the presence of three cicadellids (Macrosteles sexnotatus, Cicadulina bipunctata, Psammotettix striatus) and two non-crop plants (Plantago major, Capsicum annuum) hosting the same pomegranate-infecting ‘Ca. Phytoplasma asteris’ strains, and one cicadellid (Balclutha incisa) carrying the same pomegranate-infecting ‘Ca. Phytoplasma solani’ strain was found. In conclusion, this study described a new pomegranate disease, called pomegranate witches’-broom and leaf alteration, associated with multiple phytoplasmas. Interestingly, ‘Ca. P. ulmi’, Ca. P. pyri’, and ‘Ca. P. omanense’ in association with almond, and ‘Ca. P. ulmi’ in association with pomegranate are reported for the first time in this study. The other phytoplasma species identified in almond and pomegranate were previously reported in the Middle East. In grapevine yellows (GY) affected plants, amplicon nucleotide sequence analyses allowed attributing the detected phytoplasmas to ‘Candidatus Phytoplasma solani’ (taxonomic subgroup 16SrXII-A), ‘Ca. P. omanense’ (16SrXXIX-A and -B), ‘Ca. P. aurantifolia’ (16SrII-C), and ‘Ca. P. asteris’ (16SrI-R) in 72.4%, 17.2%, 6.9%, 3.4% of infected plants, respectively. Further investigation allowed identifying ‘Ca. P. solani’ in the putative insect vectors Orosius cellulosus (firstly reported in Jordan), Euscelidius mundus, Laodelphax striatellus, and Circulifer sp., and in Convolvulus arvensis; ‘Ca. P. aurantifolia’ in the insect O. cellulosus and in bindweed; ‘Ca. P. omanense’ in the insect Psammotettix striatus; ‘Ca. P. asteris’ in the insects Arboridia adanae, Cicaduliana bipunctata, Circulifer sp., L. striatellus, Hyalesthes obsoletus, and P. striatus. Based on this preliminary data, ecological cycles of such phytoplasmas are discussed. Obtained results suggest that GY phytoplasma diversity and ecology in Jordan are more complex than previously known, leading to a potential risk of disease outbreaks. Data obtained in this study revealed a great genetic diversity of phytoplasmas infecting important crops in Jordan. Further studies concerning the epidemiology of these phytoplasma-associated diseases, including the identification of putative insect vectors and reservoir plants, are in progress. Overall results will allow developing integrated strategies for the management of such diseases.