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

Incidence of potato virus diseases and their significance for a seed certification program in Lebanon

Potato fields in the two main production areas of Lebanon, the Bekaa and Akkar plains, were surveyed in two growing seasons for viruses and other pathogens of significance for a potato seed certification program. ELISA tests showed that Potato virus Y (PVY) was the predominant virus, followed by Potato virus A (PVA), Potato virus X (PVX), Potato virus M (PVM), Potato virus S (PVS) and Potato leaf roll virus (PLRV). Of 789 samples tested by ELISA during the two growing seasons, 372 samples were infected by one or more viruses. Single, double and multiple infections represented 75.3, 21.2 and 3.5% of all infected samples, respectively. Incidence of viruses was higher on crops from locally produced uncertified seed-potatoes than on crops from imported certified seed-potatoes. In nucleic acid spot hybridization assays, all 109 samples tested for potato spindle tuber viroid were negative. Other important pathogens of quarantine interest, including Clavibacter michiganensis, Ralstonia solanacearum and Synchytrium endobioticum, were not detected

Virus Diseases Infecting Almond Germplasm in Lebanon

Cultivated and wild almond species were surveyed for virus diseases. Four viruses infected cultivated almonds (Prunus dulcis): Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV), Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV). Only ACLSV and ApMV were detected on wild almonds, (Prunus orientalis and P. korschinskii). The occurence of PNRSV or PDV on seeds used for the production of rootstocks, on seedlings in nurseries, and on mother plants revealed the need for a certification program. There was no evidence for the occurrence of Plum pox virus (PPV) or Tomato ringspot virus (ToRS)

Antifungal Activities of Extracts from Selected Lebanese Wild Plants against Plant Pathogenic Fungi

Extracts of nine plant species growing wild in Lebanon were tested for their efficacy against seven plant pathogenic fungi: Botrytis cinerea, Alternaria solani, Penicillium sp., Cladosporium sp., Fusarium oxysporum f. sp. melonis, Rhizoctonia solani and Sphaerotheca cucurbitae. Extracts of three of the plants, Origanum syriacum, Micromeria nervosa and Plumbago maritima, showed the highest levels of in vitro activity against spore germination and mycelial growth of the fungi tested. Inula viscosa showed high activity against spore germination but only moderate activity against mycelial growth. The other five plant species tested Calamintha origanifolia, Micromeria juliana, Ruta sp., Sideritis pullulans and Urginea maritima showed only moderate to low activity against these fungi. Preventive sprays with extracts of O. syriacum, M. nervosa, P. maritima and I. viscosa, applied at concentrations ranging between 4 and 8% to squash and cucumber seedlings, gave efficient protection against gray mold caused by B. cinerea and powdery mildew caused by S. cucurbitae. However, these extracts did not control green mold of citrus fruits caused by Penicillium sp. Thin layer chromatography revealed three inhibitory bands in extracts of O. syriacum, two in I. viscosa and only one in each of the other plants tested: M. nervosa, P. maritima, C. origanifolia and Ruta sp

The potential of grafting with selected stone fruit varieties for management of almond witches’ broom

A lethal disease of stone fruit trees, characterized by proliferation of axillary shoots and witches’ broom symptoms, has caused severe problems for more than 200,000 almond, peach and nectarine plants in Lebanon since the 1990s. The agent associated with almond witches’ broom (AlmWB) was identified as ‘Candidatus Phytoplasma phoenicium’, belonging to subgroup 16SrIX-B. Management of the disease has relied on uprooting of affected trees. Since no disease-resistant cultivars have been identified, grafting experiments in the field and in the greenhouse we performed to develop a new option for the integrated management of the disease. AlmWB-affected almond trees were grafted with apricot or plum scions, and their growth was symptomless for over 2 years in the field. Similarly, in greenhouse trials, grafting AlmWB-affected almond scions onto seedlings of plum and apricot resulted in growth of symptomless shoots. One year post-grafting in the greenhouse the phytoplasma was not detected by PCR in almond grafted on Angeleno and Red plum and Early Blush apricot used as rootstocks. The phytoplasma was, however, detected in almond scions grafted on Farclo apricot and Jawhara plum, although their growth was symptomless. Shoots developing from Farclo apricot grafted on AlmWB-affected trees in the field showed severe symptoms 2 months after grafting but recovered 3 months later, and remained symptomless for about 2.5 years. Similarly, in the greenhouse trial, the growth of phytoplasma-infected scions grafted on Early blush apricot developed symptoms 2 months after grafting, did not show symptoms 2 months later, and remained symptomless 1 year later. Quantitative PCR analysis of almond scions grafted on Early blush apricot seedlings confirmed reduction of phytoplasma titre from 44 GU/ng DNA to below detection level

Biological Control of Three Major Cucumber and Pepper Pests: Whiteflies, Thrips, and Spider Mites, in High Plastic Tunnels Using Two Local Phytoseiid Mites

To enhance food security, food safety, and environmental health, a bio-based integrated pest management (BIPM) strategy was evaluated at two coastal locations in Lebanon as an alternative to toxic pesticide sprays in commercial high-arched plastic tunnels common in many countries. The evaluation occurred during two cucumber and pepper cropping seasons: spring and fall. At each site, two commercial tunnels were used; farmers’ conventional practices were applied in one tunnel, while the BIPM approach was followed in the second tunnel. In the farmers’ practices, a total of 14 sprays of insecticide/acaricide mixtures were applied during the spring growing season, and 6 sprays were applied during the fall. In the BIPM tunnels, hotspot releases of local strains of Amblyseius swirskii and Phytoseiulus persimilis were applied. By the end of the spring season, the number of whitefly nymphs (WFNs)/leaf and thrips/leaf in the pesticide treatment were 4.8 and 0.06, respectively, compared to 0.1 and 0.33, respectively, in the BIPM treatment. Similarly, at the end of the fall season, the WFNs reached 19.7/leaf in the pesticide control as compared to 1.2/leaf in the BIPM treatment, proving the efficacy of A. swirskii. Farmers using conventional acaricides during both cropping seasons failed to control Tetranychus urticae, the two-spotted spider mite (TSSM). However, hotspot releases of P. persimilis were successful in controlling TSSM. By the end of June, the number of TSSMs reached 7.8/leaf in the BIPM treatment compared to 53/leaf in the pesticide treatment. Likewise, in December, TSSM numbers reached 9/leaf in the BIPM treatment compared to 40/leaf in the pesticide treatment. Preliminary observations of pepper showed that both predatory mites (A. swirskii and P. persimilis) gave similar or better efficacy against the three pests. The two local predatory phytoseiid mites seem to be effective in controlling these three major pests and to be adapted to local environmental conditions. A rate of increase of 0.86 was observed for P. persimilis and 0.22 for A. swirskii, in June, when maximum temperatures were close to 40 °C. This also shows a compatibility between the two predators. In conclusion, our BIPM approach was efficient under a Mediterranean climate in arched plastic tunnels with relatively poor aeration

Survey of Leafhopper Species in Almond Orchards Infected with Almond Witches'-Broom Phytoplasma in Lebanon

Leafhoppers (Hemiptera: Auchenorrhyncha: Cicadellidae) account for more than 80% of all “Auchenorrhynchous” vectors that transmit phytoplasmas. The leafhopper populations in two almond witches'-broom phytoplasma (AlmWB) infected sites: Tanboureet (south of Lebanon) and Bourj El Yahoudieh (north of Lebanon) were surveyed using yellow sticky traps. The survey revealed that the most abundant species was Asymmetrasca decedens, which represented 82.4% of all the leafhoppers sampled. Potential phytoplasma vectors in members of the subfamilies Aphrodinae, Deltocephalinae, and Megophthalminae were present in very low numbers including: Aphrodes makarovi, Cicadulina bipunctella, Euscelidius mundus, Fieberiella macchiae, Allygus theryi, Circulifer haematoceps, Neoaliturus transversalis, and Megophthalmus scabripennis. Allygus theryi (Horváth) (Deltocephalinae) was reported for the first time in Lebanon. Nested PCR analysis and sequencing showed that Asymmetrasca decedens, Empoasca decipiens, Fieberiella macchiae, Euscelidius mundus, Thamnottetix seclusis, Balclutha sp., Lylatina inexpectata, Allygus sp., and Annoplotettix danutae were nine potential carriers of AlmWB phytoplasma. Although the detection of phytoplasmas in an insect does not prove a definite vector relationship, the technique is useful in narrowing the search for potential vectors. The importance of this information for management of AlmWB is discussed

'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

Inhibition of Citrinin Production in Penicillium citrinum Cultures by Neem [Azadirachta indica A. Juss (Meliaceae)]

The efficacy of different concentrations of aqueous neem leaf extract (3.12 to 50 mg/mL) on growth and citrinin production in three isolates of Penicillium citrinum was investigated under laboratory conditions. Mycotoxin production by the isolates was suppressed, depending on the concentration of the plant extract added to culture media at the time of spore inoculation. Citrinin production in fungal mycelia grown for 21 days in culture media containing 3.12 mg/mL of the aqueous extract of neem leaf was inhibited by approximately 80% in three isolates of P. citrinum. High-performance liquid chromatography was performed to confirm the spectrophotometric results. Vegetative growth was assessed, but neem extract failed to inhibit it. Neem leaf extract showed inhibition of toxin production without retardation in fungal mycelia growth

Identification of new 16SrIX subgroups, -F and -G, among ‘Candidatus Phytoplasma phoenicium’ strains infecting almond, peach and nectarine in Lebanon

Almond witches’-broom is a lethal almond disease associated with ‘Candidatus Phytoplasma phoenicium’, subgroups 16SrIX-B and -D. In Lebanon, where the disease was first reported, analogous diseases affecting peach and nectarine were recently associated with presence of ‘Ca. P. phoenicium’. In the present study, genetic diversity among 24 ‘Ca. P. phoenicium’ strains infecting almond, peach and nectarine plants in diverse geographic regions of Lebanon was investigated by virtual restriction fragment length polymorphism (RFLP) analysis of 16S rDNA nucleotide sequences. Calculation of virtual restriction similarity coefficients revealed the presence of two new subgroups, -F and -G, in group 16SrIX that were confirmed by real RFLP analyses. Obtained results open new opportunities for in-depth studies of the distribution of ‘Ca. P. phoenicium’ strains in plant hosts and insect vector populations from different geographic areas of Lebanon