First Report of a 16SrII Group Phytoplasma Associated with Shoot Proliferation of a Cactus (Opuntia monacantha) in Lebanon.

In October 2003, during a survey to evaluate the incidence of phytoplasma diseases in Lebanon, symptoms suggestive of phytoplasma infection in Opuntia monacantha (Haworth) were observed in Saghbine, Bekaa Valley. Symptoms were excessive stem and shoot proliferation. Three symptomatic and as well as symptomless plants were collected and analyzed for the presence of phytoplasmas. Nucleic acids were extracted from 0.5 g of shoot tissue and tested using polymerase chain reaction (PCR) with universal phytoplasma primers (fU5rU3) for partial amplification of the ribosomal 16SrDNA (4). PCR resulted in amplification of an expected 881-bp rDNA fragment from the symptomatic but not from symptomless samples. For characterization, sequence of the amplified DNA was determined (Genbank Accession No. AY939815). The sequence showed a high similarity with several isolates of the 16srII group of phytoplasmas. The highest similarity has been oserved with 16S rDNA of two isolates of cactus witches'-broom phytoplasma found in China (1) and Mexico (3) (Genbank Accession Nos. AJ293216 and AF320575, respectively) (99.8%) as well as faba bean phyllody phytoplasma (Genbank Accession No. X83432) (99.7%) and "Candidatus Phytoplasma aurantifolia" (Genbank Accession No. U15442) (99.3%). The presence of phytoplasmas was confirmed using nested-PCR with primers R16mF2/R1 and R16F2n/R2 (2). The Tru9I digestion pattern of the amplified product R16F2n/F16R2 detected in O. monacantha was identical to the digestion pattern obtained from periwinkle infected by "Ca. P. aurantifolia" (subgroup 16SrII-B) and soybean phyllody phytoplasma (subgroup 16SrII-C), but different from the Tru9I digestion pattern observed for cleome phyllody phytoplasma (subgroup 16SrII-A) and tomato big bud phytoplasma (subgroup 16SrII-E). To our knowledge, this is the first report of an infection with a phytoplasma belonging to16SrII group in Lebanon. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) N. E. Leyva-Lopez et al. Phytopathology. (Abstr.) 89(suppl):S45, 1999. (4) B. Schneider et al. Pages 369-380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, NY, 1995.

Fructose utilization and phytopathogenicity of Spiroplasma citri.

Spiroplasma citri is a plant-pathogenic mollicute. Recently, the so-called nonphytopathogenic S. citri mutant GMT 553 was obtained by insertion of transposon Tn4001 into the first gene of the fructose operon. Additional fructose operon mutants were produced either by gene disruption or selection of spontaneous xylitol-resistant strains. The behavior of these spiroplasma mutants in the periwinkle plants has been studied. Plants infected via leafhoppers with the wild-type strain GII-3 began to show symptoms during the first week following the insect-transmission period, and the symptoms rapidly became severe. With the fructose operon mutants, symptoms appeared only during the fourth week and remained mild, except when reversion to a fructose+ phenotype occurred. In this case, the fructose+ revertants quickly overtook the fructose- mutants and the symptoms soon became severe. When mutant GMT 553 was complemented with the fructose operon genes that restore fructose utilization, severe pathogenicity, similar to that of the wild-type strain, was also restored. Finally, plants infected with the wild-type strain and grown at 23 degrees C instead of 30 degrees C showed late symptoms, but these rapidly became severe. These results are discussed in light of the role of fructose in plants. Fructose utilization by the spiroplasmas could impair sucrose loading into the sieve tubes by the companion cells and result in accumulation of carbohydrates in source leaves and depletion of carbon sources in sink tissues.

Catharanthus roseus genes regulated differentially by mollicute infections.

A differential display of mRNAs was used to isolate periwinkle cDNAs differentially expressed following infection with one of three mollicutes: Spiroplasma citri, Candidatus Phytoplasma aurantifolia, and stolbur phytoplasma. Twenty-four differentially expressed cDNAs were characterized by Northern blots and sequence analysis. Eight of them had homologies with genes in databanks coding for proteins involved in photosynthesis, sugar transport, response to stress, or pathways of phytosterol synthesis. The regulation of these genes in periwinkle plants infected by additional phloem-restricted bacteria showed that they were not specific to a given mollicute, but correlations with particular symptoms could be established. Expression of transketolase was down regulated following infection with a pathogenic strain of S. citri. No down regulation was observed for the nonphytopathogenic mutant GMT553, which is deficient for fructose utilization.

First Report of Grapevine "Bois Noir" Disease and a New Phytoplasma Infecting Solanaceous Plants in Lebanon.

During a 2001 survey to evaluate the incidence of phytoplasma diseases in Lebanon, samples were collected from plants showing symptoms suggestive of phytoplasmal infections. Samples were also collected from symptomless plants. Sampled hosts from the Bekaa Valley included: 3 samples of tomato (Lycopersicum esculentum), 4 samples of pepper (Capsicum annuum), 10 samples of grapevine (Vitis vinifera) cvs. Chardonnay and Alicante Bouschet; 7 samples of ornamental periwinkle (Catharantus roseus) from the Tyr area; and 4 samples of weeds (Lactucca serratia). DNA was extracted from leaf midveins of diseased and symptomless plants, and from healthy periwinkle, grapevine, tomato, and pepper plants grown in a greenhouse in France. Polymerase chain reaction (PCR) with universal primers for the amplification of phytoplasma ribosomal RNA genes (3) only produced a 1.8-kbp rDNA fragment from symptomatic samples. The amplified DNAs were analyzed by restriction fragment length polymorphism (RFLP) with several restriction enzymes and sequenced. The analysis showed extracts of diseased grapevines, and two periwinkle plants had identical rDNA sequences and restriction profiles of the stolbur cluster (4). The sequences had 98% identity with two European stolbur isolates from grapevine and periwinkle (GenBank Accession Nos. X76428 and AF248959, respectively). In grapevine, the disease induced by the stolbur phytoplasma is "bois noir." Bois noir is present in Europe where its incidence is predominant in northern vineyards and has been reported in Israel (2). To our knowledge, this is the first report of the stolbur/bois noir disease in Lebanon. In tomato and pepper, the restriction profiles and sequences of the phytoplasma rDNAs were identical. Sequencing and phylogenetic analysis indicated that the phytoplasma belonged to the clover proliferation (CP) cluster, as does the eggplant little leaf phytoplasma of solanaceous plants in Asia. They differed from the stolbur phytoplasma, known to infect solanaceaous plants in Europe. Lastly, a phytoplasma belonging to the pigeon pea witches' broom (PPWB) cluster was found in L. serratia and in some periwinkle plants. A phytoplasma of the PPWB cluster was recently shown to be responsible for an emerging lethal disease of almond trees in Lebanon (1). References: (1) E. Choueiri et al. Plant Dis. 85:802, 2001. (2) X. Daire et al. Vitis 36:53, 1997. (3) B. Schneider et al. Pages 369-380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, NY, 1995. (4) E. Seemüller et al. J. Plant Pathol. 80:3, 1998.

Comparing the spatial genetic structures of the Flavescence dorée phytoplasma and its leafhopper vector Scaphoideus titanus.

The Nearctic leafhopper Scaphoideus titanus Ball is the vector of "Flavescence dorée" phytoplasma (FDp) in European vineyards. We studied the genetic diversity and structure of S. titanus populations in France and of the FDp they carried. A total of 621 S. titanus individuals, sampled in 24 FDp-infected and uninfected vineyards, were genotyped using seven polymorphic microsatellite loci. The mean observed heterozygosity in S. titanus populations was between 0.364 and 0.548. There was evidence of only a low level of population genetic differentiation (mean F(ST)=0.027) suggesting that there is long-distance gene flow between S. titanus populations. This may be a consequence of the high migration capacity of the vector associated with large effective population size and, at least in part, of passive dispersion over long distances by the transport of grapevine-planting material carrying eggs. For each insect, FDp was detected and typed by nested-PCR followed by RFLP and sequencing of a 674 bp fragment of the FDp map gene. Twelve of the 24 populations were found to be infected by FDp, with the percentage of infected individuals varying from 3% to 29%. FDp isolates were classified into two FDp genetic clusters (FD1 and FD2), which differed by 12-13 SNPs. FD1 genotypes were detected in the insect populations at two sites and the FD2 genotypes in the other ten populations. Both FD1 and FD2 genotypes were found to be transmitted by the insect. No significant relationship was found between the genetic structure of these French S. titanus populations and the distribution of the various FDp strain types they carried. Nevertheless, overall genetic differentiation between FDp-infected and healthy S. titanus "subsamples" was found to be significantly higher than zero. These results suggest that FDp-infected S. titanus individuals are more philopatric (disperse less) than healthy S. titanus.

Disruption of a gene predicted to encode a solute binding protein of an ABC transporter reduces transmission of Spiroplasma citri by the leafhopper Circulifer haematoceps.

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.

Gene disruption through homologous recombination in Spiroplasma citri: an scm1-disrupted motility mutant is pathogenic.

To determine whether homologous recombination could be used to inactivate selected genes in Spiroplasma citri, plasmid constructs were designed to disrupt the motility gene scm1. An internal scm1 gene fragment was inserted into plasmid pKT1, which replicates in Escherichia coli but not in S. citri, and into the S. citri oriC plasmid pBOT1, which replicates in spiroplasma cells as well as in E. coli. Electrotransformation of S. citri with the nonreplicative, recombinant plasmid pKTM1 yielded no transformants. In contrast, spiroplasmal transformants were obtained with the replicative, pBOT1-derived plasmid pCJ32. During passaging of the transformants, the plasmid was found to integrate into the chromosome by homologous recombination either at the oriC region or at the scm1 gene. In the latter case, plasmid integration by a single crossover between the scm1 gene fragment carried by the plasmid and the full-length scm1 gene carried by the chromosome led to a nonmotile phenotype. Transmission of the scm1-disrupted mutant to periwinkle (Catharanthus roseus) plants through injection into the leafhopper vector (Circulifer haematoceps) showed that the motility mutant multiplied in the insects and was efficiently transmitted to plants, in which it induced symptoms similarly to the wild-type S. citri strain. These results suggest that the spiroplasmal motility may not be essential for pathogenicity and that, more broadly, the S. citri oriC plasmids can be considered promising tools for specific gene disruption by promoting homologous recombination in S. citri, a mollicute which probably lacks a functional RecA protein.

Cloning of the spoT gene of "Candidatus Phlomobacter fragariae" and development of a PCR-restriction fragment length polymorphism assay for detection of the bacterium in insects.

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium "Candidatus Phlomobacter fragariae" is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between "P. fragariae" and other insect-associated proteobacteria, isolation of "P. fragariae" genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from "P. fragariae"-infected strawberry plants. It encodes part of a "P. fragariae" open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish "P. fragariae" from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry "P. fragariae." Interestingly however, the "P. fragariae" spoT sequence could be easily detected in whiteflies proliferating on "P. fragariae"-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.