EPIDEMIOLOGICAL AND MOLECULAR STUDY ON `CANDIDATUS PHYTOPLASMA PHOENICIUM\ubf IN LEBANON

\u2018Candidatus Phytoplasma phoenicium\u2019, 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\u2019-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\u2019-broom in Lebanon but also to avoid its spread in the Middle East and in Europe

Almond witches’-broom phytoplasma (Candidatus Phytoplasma phoenicium): a real threat to almond, peach and nectarine.

Within less than a decade, Almond witches’-broom (AlmWB) phytoplasma killed over a hundred thousand almond trees in Lebanon (Abou-Jawdah et al., 2002). AlmWB belongs to the pigeon pea witches’ broom group (16SrIX), and the scientific name (Candidatus Phytoplasma phoenicium) was suggested (Verdin et al., 2003). Grafting experiments revealed that AlmWB may also affect peaches and nectarines (Abou-Jawdah et al, 2003). Later on, a similar disease was reported in Iran (Verdin et al., 2003; Salehi et al., 2006). This disease is still spreading on almond trees to new areas in North Lebanon, but more recently shoot proliferation with succulent small light green leaves were observed on peach and nectarine in South Lebanon, where the disease seemed to be spreading relatively fast. DNA sequencing showed over 99% sequence homology with AlmWB (Abou-Jawdah et al. 2008). This report shows that epidemics of AlmWB may occur also on peach and nectarine under field conditions, and strongly suggests the presence of an efficient vector

'Candidatus Phytoplasma phoenicium’ associated with almond witches’-broom disease: from draft genome to genetic diversity among strain populations

BACKGROUND: Almond witches'-broom (AlmWB), a devastating disease of almond, peach and nectarine in Lebanon, is associated with 'Candidatus Phytoplasma phoenicium'. In the present study, we generated a draft genome sequence of 'Ca. P. phoenicium' strain SA213, representative of phytoplasma strain populations from different host plants, and determined the genetic diversity among phytoplasma strain populations by phylogenetic analyses of 16S rRNA, groEL, tufB and inmp gene sequences. RESULTS: Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB-associated phytoplasma strains originating from diverse host plants, whereas their 16S rRNA, tufB and groEL genes shared 100 % sequence identity. Moreover, dN/dS analysis indicated positive selection acting on inmp gene. Additionally, the analysis of 'Ca. P. phoenicium' draft genome revealed the presence of integral membrane proteins and effector-like proteins and potential candidates for interaction with hosts. One of the integral membrane proteins was predicted as BI-1, an inhibitor of apoptosis-promoting Bax factor. Bioinformatics analyses revealed the presence of putative BI-1 in draft and complete genomes of other 'Ca. Phytoplasma' species. CONCLUSION: The genetic diversity within 'Ca. P. phoenicium' strain populations in Lebanon suggested that AlmWB disease could be associated with phytoplasma strains derived from the adaptation of an original strain to diverse hosts. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor (BI-1) in 'Ca. P. phoenicium' draft genome and within genomes of other 'Ca. Phytoplasma' species suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response

Leukocyte-derived ratios are associated with late-life any type dementia: a cross-sectional analysis of the Mugello study

Immunosenescence, vascular aging, and brain aging, all characterized by elevated levels of inflammatory markers, are thought to share a common pathogenetic pathway: inflamm-aging. Retrospective cross-sectional analysis was conducted using data from the Mugello study (Tuscany, Italy), a representative Italian cohort of free-living nonagenarians. to assess the association between specific peripheral inflammation markers derived from white blood cell counts, and the diagnosis of dementia. All the variables of interest were reported for 411 subjects (110 males and 301 females) out of 475 enrolled in the study. Anamnestic dementia diagnosis was obtained from clinical certificate and confirmed by a General Practitioner, whereas leukocyte ratios were directly calculated from white blood cell counts. Body mass index and comorbidities were considered potential confounders. Diagnosis of any type dementia was certified in 73 cases (17.8%). Subjects affected by dementia were older, more frequently reported a previous stroke, had lower body mass index, and lower Mini-Mental-State-Examination score. Moreover, they had a higher lymphocyte count and lymphocyte-to-monocyte ratio compared to the non-demented nonagenarians. We found that higher levels of lymphocyte counts are cross-sectionally associated with a clinical diagnosis of dementia. Furthermore, lymphocyte-to-monocyte ratio is directly associated with any type of dementia, independently of age, sex, lymphocyte count, and comorbidities. Lymphocyte-to-monocyte ratio may be considered a marker of immunological changes in the brain of dementia patients; moreover, it is low-cost, and easily available, thus enabling comparisons among different studies and populations, although the timeline and the extent of lymphocyte-to-monocyte ratio role in dementia development must be further investigated

Physical activity in older age: perspectives for healthy ageing and frailty.

Regular physical activity helps to improve physical and mental functions as well as reverse some effects of chronic disease to keep older people mobile and independent. Despite the highly publicised benefits of physical activity, the overwhelming majority of older people in the United Kingdom do not meet the minimum physical activity levels needed to maintain health. The sedentary lifestyles that predominate in older age results in premature onset of ill health, disease and frailty. Local authorities have a responsibility to promote physical activity amongst older people, but knowing how to stimulate regular activity at the population-level is challenging. The physiological rationale for physical activity, risks of adverse events, societal and psychological factors are discussed with a view to inform public health initiatives for the relatively healthy older person as well as those with physical frailty. The evidence shows that regular physical activity is safe for healthy and for frail older people and the risks of developing major cardiovascular and metabolic diseases, obesity, falls, cognitive impairments, osteoporosis and muscular weakness are decreased by regularly completing activities ranging from low intensity walking through to more vigorous sports and resistance exercises. Yet, participation in physical activities remains low amongst older adults, particularly those living in less affluent areas. Older people may be encouraged to increase their activities if influenced by clinicians, family or friends, keeping costs low and enjoyment high, facilitating group-based activities and raising self-efficacy for exercise