'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.

Specific PCR and real-time PCR assays for detection and quantitation of 'Candidatus Phytoplasma phoenicium'.

Almond witches' broom (AlmWB) is a fast-spreading lethal disease of almond, peach and nectarine associated with 'Candidatus Phytoplasma phoenicium'. The development of PCR and quantitative real-time PCR (qPCR) assays for the sensitive and specific detection of the phytoplasma is of prime importance for early detection of 'Ca. P. phoenicium' and for epidemiological studies. The developed qPCR assay herein uses a TaqMan(®) probe labeled with Black Hole Quencher Plus. The specificity of the PCR and that of the qPCR detection protocols were tested on 17 phytoplasma isolates belonging to 11 phytoplasma 16S rRNA groups, on samples of almond, peach, nectarine, native plants and insects infected or uninfected with the phytoplasma. The developed assays showed high specificity against 'Ca. P. phoenicium' and no cross-reactivity against any other phytoplasma, plant or insect tested. The sensitivity of the developed PCR and qPCR assays was similar to the conventional nested PCR protocol using universal primers. The qPCR assay was further validated by quantitating AlmWB phytoplasma in different hosts, plant parts and potential insect vectors. The highest titers of 'Ca. P. phoenicium' were detected in the phloem tissues of stems and roots of almond and nectarine trees, where they averaged from 10(5) to 10(6) genomic units per nanogram of host DNA (GU/ng of DNA). The newly developed PCR and qPCR protocols are reliable, specific and sensitive methods that are easily applicable to high-throughput diagnosis of AlmWB in plants and insects and can be used for surveys of potential vectors and alternative hosts.

Frequent migration of introduced cucurbit-infecting begomoviruses among Middle Eastern countries.

BACKGROUND: In the early 2000s, two cucurbit-infecting begomoviruses were introduced into the eastern Mediterranean basin: the Old World Squash leaf curl virus (SLCV) and the New World Watermelon chlorotic stunt virus (WmCSV). These viruses have been emerging in parallel over the last decade in Egypt, Israel, Jordan, Lebanon and Palestine. METHODS: We explored this unique situation by assessing the diversity and biogeography of the DNA-A component of SLCV and WmCSV in these five countries. RESULTS: There was fairly low sequence variation in both begomovirus species (SLCV π = 0.0077; WmCSV π = 0.0066). Both viruses may have been introduced only once into the eastern Mediterranean basin, but once established, these viruses readily moved across country boundaries. SLCV has been introduced at least twice into each of all five countries based on the absence of monophyletic clades. Similarly, WmCSV has been introduced multiple times into Jordan, Israel and Palestine. CONCLUSIONS: We predict that uncontrolled movement of whiteflies among countries in this region will continue to cause SLCV and WmCSV migration, preventing strong genetic differentiation of these viruses among these countries.

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.

Tolerance in Cucumber to Cucurbit yellow stunting disorder virus.

Cucurbit yellow stunting disorder virus (CYSDV), genus Crinivirus and family Closteroviridae, has emerged as a serious whitefly-transmitted virus of cucurbit crops, causing between 30 and 50% yield losses. Development of resistant cultivars represents an economically and environmentally sound approach to management of this disease. In all, 124 cucumber accessions were evaluated for reaction to CYSDV under high inoculum pressure over three growing seasons. Seven accessions showed delayed expression of symptoms, milder final symptoms, and lower percentages of infected plants compared with susceptible cucumbers. Although none of these accessions were immune to CYSDV, virus concentrations in the middle leaves of the tolerant accessions were significantly lower than those of susceptible accessions.

Immunodiagnosis of Prune dwarf virus using antiserum produced to its recombinant coat protein.

Certification represents the first line of defense against fruit tree viruses. For certification or surveys dealing with large number of samples, ELISA is still considered the technique of choice and requires a continuous supply of good quality antibodies. Prune dwarf virus (PDV) is among the major viruses affecting stone fruits; it belongs to the genus Ilarvirus named so for its isometric labile particles. Recombinant DNA technology was investigated for production of PDV antiserum to avoid labile virus purification and virus maintenance problems. The PDV coat protein gene (CP) was cloned into a protein expression bacterial plasmid vector which allowed a good level of expression of up to 2mg native protein/L culture. The recombinant PDV CP was injected into rabbits and the crude antiserum was successfully used in indirect ELISA at dilutions of up to 1:5000 to detect PDV in infected leaf samples. Similar results were obtained in dot blot immunoassays (DBIA). The antibodies were used in double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and results were comparable to a reference commercial kit. The crude antiserum was efficiently used for coating ELISA plates, thereby reducing test costs.

Antimycotic activities of selected plant flora, growing wild in Lebanon, against phytopathogenic fungi.

Petroleum ether (PE) and methanolic extracts of nine wild plant species were tested in vitro for their antimycotic activity against eight phytopathogenic fungi. The efficacy of PE extracts against all pathogens tested was higher than that of methanolic extracts. Wild marjoram (Origanum syriacum) PE extract showed the highest and widest range of activity. It resulted in complete inhibition of mycelial growth of six of eight fungi tested and also gave nearly complete inhibition of spore germination of the six fungi included in the assay, namely, Botrytis cinerea, Alternaria solani, Penicillium sp., Cladosporium sp., Fusarium oxysporum f. sp. melonis, and Verticillium dahlia. The other plant extracts showed differential activities in the spore germination test, but none was highly active against mycelial growth. Inula viscosa and Mentha longifolia were highly effective (>88%) in spore germination tests against five of six fungi tested, whereas Centaurea pallescens, Cichorium intybus, Eryngium creticum, Salvia fruticosa, and Melia azedarach showed >95% inhibition of spore germination in at least two fungi. Foeniculum vulgare showed the least antimycotic activity. Fractionation followed by autobiography on TLC plates using Cladosporium sp. as a test organism showed that O. syriacum PE extracts contained three inhibition zones, and those of Inula viscosa and Cichorium intybus, two, whereas the PE extracts of the remaining plants showed each one inhibition zone. Some of the major compounds present in these inhibition zones were identified by GC-MS. The possibility for using these extracts, or their mixtures, to control plant diseases is discussed.

An Epidemic of Almond Witches'-broom in Lebanon: Classification and Phylogenetic Relationships of the Associated Phytoplasma.

An epidemic of almond witches'-broom has devastated almond production in Lebanon. Thousands of almond trees have died over the past 10 years due to the rapid spread of the disease. The symptoms, which include early flowering, stunted growth, leaf rosetting, dieback, off-season growth, proliferation of slender shoots, and witches'-brooms arising mainly from the main trunk and roots, resemble those caused by phytoplasmal infections. For the detection of the putative causal agent, nested polymerase chain reaction (PCR) was performed using universal primers (P1/P7, R16mF2/R16mR1, and R16F2n/R16R2) commonly used for the specific diagnosis of plant pathogenic phytoplasmas. Phytoplasmas were readily detected from infected trees with witches'-broom symptoms collected from three major almond growing regions in Lebanon. Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified by the primer pair R16F2n/R16R2 revealed that the phytoplasma associated with infected almonds is similar to, but distinct from, members of the pigeon pea witches'-broom phytoplasma group (16SrIX). A new subgroup, 16SrIX-B, was designated. Sequencing of the amplified products of the phytoplasma 16S rRNA gene indicated that almond witches'-broom (AlmWB) phytoplasma is most closely related to members of the pigeon pea witches'-broom phytoplasma group (with sequence homology ranging from 98.4 to 99.0%). Phylogenetic analysis of 16S rDNA sequences from AlmWB phytoplasma and from representative phytoplasmas from GenBank showed that the AlmWB phytoplasma represents a distinct lineage within the pigeon pea witches'-broom subclade. The same phytoplasma appears also to infect peach and nectarine seedlings.

Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR.

Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100-1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.