EPIDEMIOLOGICAL AND MOLECULAR STUDY ON `CANDIDATUS PHYTOPLASMA PHOENICIUM¿ IN LEBANON

‘Candidatus Phytoplasma phoenicium’, a member of the 16SrDNA phytoplasma group IX, is considered the presumptive aetiological agent of Almond witches'-broom (AlmWB) disease, which caused in Lebanon the death of more than 100,000 almond trees in the last decade. In the last few years, severe infections, frequently associated with a noticeable yield reduction, have also been observed on peach and nectarine trees. The aim of this work is to improve the knowledge of AlmWB epidemiology through (i) the symptoms description in almond, peach and nectarine trees in order to select the most suitable period for observing the typical alterations induced by the disease and for collecting samples for a fast and effective diagnosis, (ii) the update of the data concerning the AlmWB spread in Lebanon, (iii) the molecular characterization of AlmWB phytoplasma strains isolated from different host plants and from different Lebanese regions, and (iv) a preliminary screening of the insect(s) that could be candidate vector(s) responsible for the disease transmission. First of all, the symptom evolution was described through one-year-Iong observations of infected almond, peach and nectarine trees in three key-orchards located in three different Lebanese regions: Jbeil in the North, Hasbaya and Marjayoun in the South. Leaf and flower samples were collected from symptomatic and asymptomatic plants and analysed by direct and nested polymerase chain reaction (PCR) assays in order to detect AlmWB phytoplasma. Due to the importance of stone fruit in Lebanon and to the serious impact of the disease on these cultures, a national survey on AlmWB, based on the criteria derived from the symptom observation in the examined key-orchards, was carried out in 24 Lebanese districts. Leaf and flower samples were collected from 368 plants in order to detect the phytoplasma and characterize the infected regions. Moreover, molecular characterization of 24 representative 'Ca. Phytoplasma phoenicium' strains was carried out through virtual and actual RFLP analysis of the 16S rRNA gene, in order to study the genetic variability of the pathogen and to find out possible relationships with the different hosts and the various cultivation regions. Furthermore, since the AlmWB phytoplasma insect vector(s) is(are) still unknown, a wide insect collection was carried out in two infected almond and nectarine orchards during two consecutive years in order to identify and analyze candidate phytoplasma vector(s). The observations carried out on infected peach and nectarine trees were used to describe the symptom evolution on these two new AlmWB hosts. Even if the presence of witches’-broom is more common in almond trees than in peach/nectarine, the most important difference between peach/nectarine and almond symptoms is the development, in peach/nectarine trees, of phyllodies, never recorded on almond. They appear usually in April/May and are easy to recognize on field. By using the specific primer pair AlWF2/AlWR2, AlmWB phytoplasma was identified in 95% of symptomatic almonds and in 100% of symptomatic peaches and nectarines selected during the national survey on AlmWB. The disease was found to be present in 16 out of 24 Lebanese districts, where it affects almond, nectarine and peach trees at different rates. A national map indicating the location of all the affected and healthy monitored villages was developed using the GIS software. Numerous meetings were held in these regions, in order to describe the disease and its possible management to the farmers. Molecular characterization of 24 representative 'Ca. P. phoenicium' strains by virtual RFLP assays lead to the identification of two new 16SrIX subgroups, indicated as 16SrIX-F and IX-G, distinguished by the use of BstUI and TaqI restriction enzymes. The geographical distribution of the phytoplasma subgroups here identified (IX-D, IX-F, IX-G) were also showed in the GIS map elaboration. During a wide survey on putative AlmWB phytoplasma insect vectors, 45 species of leafhoppers, 4 genera of cixiids, and 9 species of psyllids were collected and identified. Since leafhoppers were previously investigated as AlmWB phytoplasma vectors in Lebanon, the research focused on Cixiidae and Psyllidae taxa. In detail, 64 Cixiidae and 53 Psyllidae specimens were analyzed by direct and nested PCR, using respectively the specific primers AlWF2/AlWR2 and the universal primers P1/P7 followed by F2n/R2. Whereas all the psyllids tested negatives, 11 PCR reactions on Cixiidae specimens have shown positive results using the universal primers, whereas 16 reactions gave positive results using the specific primer pair, opening new possibilities about the research of the 'Ca. Phytoplasma phoenicium' vector(s). Results obtained in the present study evidenced the wide diffusion of 'Ca. P. phoenicium'-related strains in Lebanon. The pathogen affects different hosts and can be spread in territories characterised by very different climate and environmental conditions, representing a risk because of its adaptability to the neighbouring regions/Countries. The preliminary results obtained on Cixiidae analysis highlighted the presence of several phytoplasma-infected insects; their vectoring activity must be confirmed through green-house transmission assays, in order to demonstrate their role on 'Ca. P. phoenicium' transmission. In-depth investigating on Cixiidae biology, ecology and host range will allow planning a possible management of the disease. The results obtained during the present research work suggest that regulation and control measures are urgently necessaries to limit the diffusion of Almond Witches’-broom in Lebanon but also to avoid its spread in the Middle East and in Europe.