Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines, bindweeds and vegetables.

AIM: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. MATERIALS AND RESULTS: Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf-a type and 68% by tuf-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf-b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences' analysis and virtual digestions. CONCLUSIONS: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. SIGNIFICANCE AND IMPACT OF THE STUDY: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

Verticillium Wilt of Xanthium italicum Caused by Verticillium dahliae in Italy.

During the late summer of 2003, a wilt disease of the weed Italian cockleburr (Xanthium italicum Mor.) was observed in the Basilicata Region of southern Italy. Diseased plants were growing near an apricot orchard in which some trees were severely affected by Verticillium wilt. The most characteristic symptoms of the wilt disease affecting Italian cockleburr were yellowing, stunting, and gradual wilting. Also, diagonal and cross sections of stems revealed brown discoloration of their vascular tissues. To elucidate the etiology of the disease, we attempted detection and identification of the causal agents using traditional and polymerase chain reaction (PCR)-based methods. Small pieces of petiole and stem tissues from diseased and asymptomatic plants were surface disinfested in NaOCl solution, rinsed in sterile distilled water, blotted dry, and plated onto water agar (WA) medium. Following incubation at 22°C, the emerging colonies were transferred to potato dextrose agar (PDA). Verticillium dahliae (one isolate) was consistently identified on the basis of its morphological features according to the description of Smith (2). Using PCR assays with the primer pair ITS5/ITS4 (3), which are directed to fungal nuclear ribosomal DNA (rDNA) repeat sequences, an amplification product of approximately 560 bp was obtained by using total DNA extracted from wilt-affected Italian cockleburr plant tissues (five plants examined) as well as fresh mycelium from the V. dahliae-infected Italian cockleburr pure culture-maintained isolate mentioned above. No visible PCR products were obtained with total DNA from asymptomatic Italian cockleburr plants. Sequence analysis of the ITS5/ITS4 amplicons revealed no differences in their nucleotide positions. The obtained sequence of the V. dahliae-infected Italian cockleburr isolate (GenBank Accession No. AJ865691) was then used as query sequences in a BLAST 2.0 search (1). Sequence of the southern Italian isolate proved to be identical to that of the Greek strain "76 Greece" of V. dahliae (GenBank Accession no. AF104926). To prove Koch's postulates, 10 healthy Italian cockleburr seedlings were experimentally inoculated by dipping trimmed roots in a single-conidial suspension (1.5 × 106 CFU/ml) obtained from 10-day-old colonies of the V. dahliae-infected Italian cockleburr pure culture-maintained isolate. After 4 weeks, all inoculated Italian cockleburr plants showed symptoms identical to those of naturally infected field-grown plants. V. dahliae was consistently reisolated from inoculated plants. Additional inoculation experiments revealed that pepper and eggplant were also susceptible to the V. dahliae-infected Italian cockleburr isolate showing typical Verticillium wilt symptoms. To our knowledge, this is the first report of the occurrence of Verticillium wilt of X. italicum. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) H. C. Smith. N.Z. J. Agric. Res. 8:450, 1965. (3) T. J. White et al. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

'Candidatus Phytoplasma spartii', 'Candidatus Phytoplasma rhamni' and 'Candidatus Phytoplasma allocasuarinae', respectively associated with spartium witches'-broom, buckthorn witches'-broom and allocasuarina yellows diseases.

Spartium witches'-broom (SpaWB), buckthorn witches'-broom (BWB) and allocasuarina yellows (AlloY) are witches'-broom and yellows diseases of Spartium junceum (Spanish broom), Rhamnus catharticus (buckthorn) and Allocasuarina muelleriana (Slaty she-oak), respectively. These diseases are associated with distinct phytoplasmas. The SpaWB, BWB and AlloY phytoplasmas share <97.5 % 16S rDNA sequence similarity with each other and with other known phytoplasmas, including the closely related phytoplasmas of the apple proliferation group. Also, the SpaWB, BWB and AlloY phytoplasmas each have a different natural plant host. Based on their unique properties, it is proposed to designate the mentioned phytoplasmas as novel 'Candidatus' species under the names 'Candidatus Phytoplasma spartii', 'Candidatus Phytoplasma rhamni' and 'Candidatus Phytoplasma allocasuarinae', respectively.

'Candidatus Phytoplasma asteris', a novel phytoplasma taxon associated with aster yellows and related diseases.

Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri) virescence in Michigan, USA, was selected as the reference strain for the novel taxon 'Candidatus Phytoplasma asteris'. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Teste de Vôo como Critério de Avaliação da Qualidade de Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae): Adaptação de Metodologia / Flight Test as Evaluation Criterion for the Quality of Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae): Adaptation of the Methodology

The goal of this research was to evaluate the quality of populations of Trichogramma pretiosum Riley, in the laboratory, by using the flight test developed by the International Organization of Biological Control (IOBC). The parasitoids were obtained from eggs of Helicoverpa zea (Bod.) collected in corn fields, and kept in the laboratory on eggs of the factitious host Anagasta kuehniella (Zeller). First, we made a comparison between the standard model developed by the IOBC (with some modifications), and a customized model named ESALQ. For each model, we evaluated the quality of three populations of T. pretiosum, kept in the laboratory for 3, 35 and 72 generations. Both models showed no difference in the quality of the studied populations, based on the percentage of parasitoids that showed initial flight activity after emergence. The ESALQ model allowed better discrimination between "non-flyers" and "flyers". Second, we monitored the quality of three sexual populations of T. pretiosum, started with one, five and ten couples, during 21 generations, using the ESALQ model. The population started with a single couple showed an inferior quality when compared to the populations started with five and ten couples. The flight test was highly efficient to determine the quality of T. pretiosum populations, under laboratory conditions, and the modifications made in the standard model provided a better discrimination of flyers and non-flyers of the parasitoid. Thus, we indicate the ESALQ model as a substitute of the standard model, developed by the IOBC.

O objetivo do trabalho foi avaliar a qualidade de populações sexuadas de Trichogramma pretiosum Riley, em laboratório, utilizando-se o teste de vôo desenvolvido pela Organização Internacional de Controle Biológico (IOBC). Os parasitóides foram obtidos de ovos de Helicoverpa zea (Bod.), coletados na cultura de milho, e mantidos em laboratório em ovos do hospedeiro alternativo Anagasta kuehniella (Zeller). Numa primeira etapa, realizou-se a comparação entre o modelo padrão, desenvolvido pela IOBC (com algumas modificações) e um modelo adaptado e denominado ESALQ. Para cada modelo, avaliou-se a qualidade de três populações de T.pretiosum, mantidas em laboratório por 3, 35 e 72 gerações. Independente da geração, ambos os modelos não registraram diferença na qualidade das populações estudadas, tomando-se por base a porcentagem de parasitóides que apresentaram aptidão para vôo, após a emergência. O modelo ESALQ permitiu melhor discriminação entre "voadores" e "não voadores". Numa segunda etapa, utilizou-se o modelo ESALQ para monitorar, durante 21 gerações, a qualidade de três populações sexuadas de T. pretiosum, iniciadas respectivamente com um, cinco e dez casais. A população iniciada com um casal apresentou qualidade inferior em comparação com as populações iniciadas com cinco e dez casais. O teste de vôo mostrou-se eficiente na determinação da qualidade de populações de T. pretiosum, em condições de laboratório, e o modelo ESALQ favoreceu a melhor separação de voadores e não voadores do parasitóide, podendo o mesmo ser indicado como substituto do modelo padrão, desenvolvido pela IOBC.

Genetic variability among flavescence dorée phytoplasmas from different origins in Italy and France.

Flavescence dorée is a devastating disease of grapevine widespread in several countries in EU such as France, Italy and Spain. Genetic variability among 17 Italian and 3 French FD strains was investigated by RFLP analyses based on a fragment of the ribosomal protein operon and on the non-ribosomal DNA fragment FD9. RFLP analysis of the PCR amplified ribosomal protein fragment, coding for the 3' end of rpl22 and the entire rps3 genes, differentiated 4 rp-subgroups among the FD strains and 4 subgroups among the reference strains belonging to elm yellows group (16SrV). Sequencing and phylogenetic analysis of the same ribosomal protein DNA fragment validated the delineation of 4 distinct FD strain types derived by RFLP analyses. The results supported the differentiation based on analysis of the non-ribosomal DNA fragment FD9. The phylogenetic analysis further revealed relationships and a probable evolutionary trend among the FD strains and the other representatives of elm yellows group. All the FD strains together with the reference strains ALY, RuS and JWB formed a cluster very well distinct from the EY/ULW cluster. Moreover, ALY was shown to be more closely related to three FD strain types: the Lombardia/Piemonte, the French FD70, and the French FD88/Italian FD-D strain clusters.

Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) and its relationship with the citrus canker bacterium Xanthomonas axonopodis pv citri in Brazil / Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae) e sua relação com a bactéria do cancro cítrico Xanthomonas axonopodis pv citri no Brasil

The relationship of the citrus canker bacterium Xanthomonas axonopodis pv citri with the citrus leafminer (CLM), Phyllocnistis citrella Stainton was investigated. The experiment was conducted under laboratory conditions at 28±2ºC, 70±10 percent RH and 14h photophase and in a greenhouse. Sweet orange (Citrus sinensis L. Osbeck) "Caipira"cv was used to rear CLM. Plants inoculated with 2nd and 3rd instar larvae or pupae showed high percentages (94.3, 98.3 and 100 percent, respectively) of bacterium-infected leaves. The damage caused by this insect was responsible for the increase in citrus canker infestation. The leaf infection rate by X. axonopodis pv citri on pre-injured leaves was similar to that observed on mechanically damaged leaves inoculated with the bacterium, with 94.1 percent to 97.0 percent of the leaves presenting bacterial pustules. The bacterium can also penetrate through the stomata. An 11-fold lower infection rate was observed as compared to the leaves injured by the insect seven days after inoculation. Under such conditions the percentage of cankered leaves increased to 41.2 percent at 14 days, a value corresponding to about 50 percent of the leaves attacked by the insect. In this paper it is also pointed out the significance of the damages caused by CLM in terms of the increase of citrus canker, since the favorable microclimatic conditions of temperature and relative humidity inside the mines built by the larvae account for an improved development of the bacterium.

Estudou-se em laboratório (28±2ºC, 70±10 por cento UR e fotofase de 14h) e em casa-de-vegetação a relação entre as lesões provocadas pelo ataque do minador-dos-citros, Phyllocnistis citrella Stainton, e a infecção causada pela bactéria do cancro cítrico Xanthomonas axonopodis pv citri. Utilizaram-se, como hospedeiro do minador, plantas de laranja caipira cultivadas em tubetes. Folhas inoculadas com lagartas de segundo e terceiro ínstares ou pupas apresentaram índices de infecção bacteriana de 94,3, 98,3 e 100 por cento, respectivamente. A taxa de infecção foliar por X. axonopodis pv citri em folhas lesionadas pelo inseto foi semelhante àquela observada em folhas danificadas mecanicamente e inoculadas posteriormente com bactéria (94,1 a 97 por cento de pústulas bacterianas). A bactéria penetra também através dos estômatos. Aos sete dias após a inoculação, constatou-se uma taxa de infecção 11 vezes menor nas folhas que não foram lesionadas pelo inseto. O percentual de folhas com cancro aumentou aos 14 dias para 41,2 por cento; esse valor é cerca de 50 por cento menor se comparado às folhas que foram atacadas pelo inseto. Ficou demonstrada a importância dos danos provocados pelo minador-dos-citros no aumento do cancro cítrico, uma vez que as condições favoráveis de temperatura e umidade relativa no interior das minas construídas pelas lagartas, contribuem para um melhor desenvolvimento da bactéria.

Classification of aster yellows-group phytoplasmas based on combined analyses of rRNA and tuf gene sequences.

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

Chromosome sizes of phytoplasmas composing major phylogenetic groups and subgroups.

ABSTRACT Chromosome sizes of 71 phytoplasmas belonging to 12 major phylogenetic groups including several of the aster yellows subgroups were estimated from electrophoretic mobilities of full-length chromosomes in pulsed-field gels. Considerable variation in genome size, from 660 to 1,130 kilobases (kb), was observed among aster yellows phytoplasmas. Chromosome size heterogeneity was also observed in the stolbur phytoplasma group (range 860 to 1,350 kb); in this group, isolate STOLF contains the largest chromosome found in a phytoplasma to date. A wide range of chromosome sizes, from 670 to 1,075 kb, was also identified in the X-disease group. The other phytoplasmas examined, which included members of the apple proliferation, Italian alfalfa witches' broom, faba bean phyllody, pigeon pea witches' broom, sugarcane white leaf, Bermuda grass white leaf, ash yellows, clover proliferation, and elm yellows groups, all have chromosomes smaller than 1 megabase, and the size ranges within each of these groups is narrower than in the aster yellows, stolbur, and X-disease groups. The smallest chromosome, approximately 530 kb, was found in two Bermuda grass white leaf phytoplasma isolates. This not only is the smallest mollicute chromosome found to date, but also is the smallest chromosome known for any cell. More than one large DNA band was observed in several phytoplasma preparations. Possible explanations for the occurrence of more than one band may be infection of the host plant by different phytoplasmas, the presence of more than one chromosome in the same organism, or the presence of large extrachromosomal DNA elements.

Detection of Bermuda Grass White Leaf Disease in Italy and Characterization of the Associated Phytoplasma by RFLP Analysis.

Bermuda grass showing symptoms of a white leaf disease has been observed in fruit orchards, vegetable fields, and uncultivated areas in the Latium and Campania regions of central and southern Italy. Using polymerase chain reaction (PCR) amplification with phytoplasma-specific primers, all symptomatic plants tested positively; whereas no amplification product was obtained from nonsymptomatic plants. Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified ribosomal DNA revealed a uniform pattern that was similar to that of the Bermuda grass white leaf phytoplasma collected in Thailand, which is known to be a member of the sugarcane white leaf phytoplasma group. By RFLP analysis, the phytoplasma infecting Bermuda grass could be distinguished from other group members, including the phytoplasmas associated with sugarcane white leaf and Brachiaria white leaf. This is the first report on the presence of the Bermuda grass white leaf phytoplasma in Europe.