Genome sequencing and functional genomics approaches in tomato

Tomato genome sequencing has been taking place through an international, 10-year initiative entitled the International Solanaceae Genome Project (SOL). The strategy proposed by the SOL consortium is to sequence the approximately 220Mb of euchromatin that contains the majority of genes, rather than the entire tomato genome. Tomato and other Solanaceae plants have unique developmental aspects, such as the formation of fleshy fruit, not afforded by Arabidopsis or rice. Divergent phenotypes and habitats of the Solanaceae also make the family an ideal model to explore the bases of diversification and adaptation. Current progress in genome sequencing, genetic and genomic resources, and functional genomics approaches for tomato is summarized. Given the foreseen wealth of information in the upcoming genome sequence, the role of the laboratory-grown miniature tomato cultivar Micro-Tom as a valuable functional genomics tool for plant pathology and emerging areas of biology, such as omics biology, is emphasized.     

Phylogenetic analysis of elongation factor Tu gene of phytoplasmas from Japan

The elongation factor Tu (tuf) gene from nine Japan phytoplasma isolates was amplified with the polymerase chain reaction, and the DNA sequences of the tuf gene were determined. The tuf gene from 14 phytoplasma isolates, including reference isolates and other bacteria, were phylogenetically analyzed. A nucleotide sequence of the tuf gene among seven aster yellows group (16Sr I-B and I-D) phytoplasmas had 97%–100% similarity, and the tuf gene of two phytoplasmas of the X-disease group (16Sr III-B) had 99% similarity. The tuf genes had lower homology than did the 16S rRNA gene in the phytoplasma groups. A phylogenetic tree of amino acid sequences of the tuf gene was nearly equal to that of the 16S rRNA gene but differed somewhat from the tree based on the 16S rRNA gene in that paulownia witches broom (PaW: 16Sr I-D) and American aster yellows (AAY: 16Sr I-B) were in a subclade.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB095495, AB095667, AB095668, AB095669, AB095670, AB095671, AB095672, AB095673 and AB095674     

Characterization and classification of phytoplasmas from wild and cultivated plants by RFLP and sequence analysis of ribosomal DNA

Restriction fragment length polymorphism and sequence analysis of PCR-amplified ribosomal DNA were used to identify and classify phytoplasmas associated with diseases of various wild and cultivated plants. The diseases examined were either not known before or the presumable causal agents were not yet identified and characterized or were only known from other geographic areas. New diseases examined were those causing virescence and phyllody of Bunias orientalis and Cardaria draba. Both were associated with strains of the aster yellows phytoplasma. The same type of aster yellows phytoplasma was also found to be associated with yellows and phyllody diseases of Portulaca oleracea, Stellaria media, Daucus carota ssp. sativus, and Cyclamen persicum. In German and French DNA samples from diseased Trifolium repens, the clover phyllody phytoplasma was identified, which could clearly be distinguished from other phytoplasmas of the aster yellows group. Strains of the stolbur phytoplasma were detected in big bud-affected tomatoes and almost exclusively in Convolvulus arvensis. In Cirsium arvense and Picris echioides two distinct phytoplasmas were identified which showed relationship to the sugarcane white leaf phytoplasma group but may represent a new group or subgroup. In Conyza (syn.: Erigeron) canadensis a phytoplasma of the X-disease group was detected. A strain from Gossypium hirsutum showed the same restriction profiles as the faba bean phyllody phytoplasma.     

宁夏马铃薯僵顶植原体的分子鉴定

通过透射电子显微镜,在从宁夏回族自治区固原市彭阳县红河镇采集的表现叶片上卷、红叶、气生薯症状的马铃薯样品叶脉韧皮部筛管细胞内观察到大量直径为500~700 nm的球形植原体粒子。以提取的感病和健康马铃薯叶片总DNA为模板,应用植原体16S rRNA基因和rp基因通用引物进行PCR扩增,从感病样品中扩增得到了长度均约为1.2 kb的片段。对获得基因核酸一致性比较分析表明,马铃薯僵顶植原体宁夏株系16S rRNA基因与‘Candidatus Phytoplasma fragariae’槭树株系(MK501642)16S rRNA基因核酸一致性最高,为99.7%,rp基因与‘Ca.P.fragariae’云南马铃薯YN-2G株系(KJ144889)rp基因核酸一致性最高,为100%;基于16S rRNA基因和rp基因构建系统进化树发现,马铃薯僵顶植原体宁夏株系与16SrⅫ-E亚组成员聚在一起。基于透射电镜观察和基因序列比较分析,证明宁夏发生的马铃薯僵顶病与植原体侵染相关,该植原体在分类地位上属于植原体16SrⅫ-E亚组。     

Differential detection and genetic relatedness of phytoplasmas in papaya

The genetic relatedness of phytoplasmas associated with dieback (PDB), yellow crinkle (PYC) and mosaic (PM) diseases in papaya was studied by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and 16S rRNA/23S rRNA spacer region (SR). RFLP and SR sequence comparisons indicated that PYC and PM phytoplasmas were identical and most closely related to members of the faba bean phyllody strain cluster. By comparison the PDB phytoplasma was most closely related to Phormium yellow leaf (PYL) phytoplasma from New Zealand and the Australian grapevine yellows (AGY) phytoplasma from Australia. These three phytoplasmas cluster with the stolbur and German grapevine yellows (VK) phytoplasmas within the aster yellows strain cluster. Primers based on the phytoplasma tuf gene, which amplify gene products from members of the AY strain cluster, also amplified a DNA product from the PDB phytoplasma but not from either the PYC or PM phytoplasmas. Primers deduced from the 16S rRNA/SR selectively amplified rDNA sequences from the PDB and AGY phytoplasmas but not from other members of the stolbur strain cluster. Similarly, primers designed from 16S rRNA/SR amplified rDNA from the PYC and PM phytoplasmas but not from the PDB phytoplasma. These primers may provide for more specific detection of these pathogens in epidemiological studies.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Detection and identification of phytoplasmas in yellows-diseased weeds in Italy

Yellows-diseased plants of Crepis setosa (hawksbeard), Knautia arvensis (field scabious), Convolvulus arvensis (field bindweed), Picris echioides (bristly oxtongue), Echium vulgare (blueweed) and Calendula officinalis (pot marigold) collected in central and southern Italy were examined for phytoplasma infection by means of polymerase chain reaction (PCR) technology using universal phytoplasma primers directed to ribosomal sequences. The detected phytoplasmas were characterized and differentiated using restriction fragment length polymorphism analysis of PCR-amplified DNA. The phytoplasma detected in diseased pot marigold plants was identified as a member of the aster yellows group and proved indistinguishable from a strain of the American aster yellows phytoplasma. The phytoplasma identified in diseased field bindweed plants is a putative new type of the stolbur group that differed from the typical stolbur phytoplasma. Phytoplasmas detected in diseased hawksbeard, blueweed and field scabious plants are all putative new members of the sugarcane white leaf group while the phytoplasma detected in diseased bristly oxtongue plants represents a new member of the faba bean phyllody group. For hawksbeard and field scabious this is the first report on the occurrence of phytoplasma diseases, whereas phytoplasmas infecting bristly oxtongue and blueweed have never been characterized before.     

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.     

Identification of fruit tree phytoplasmas and their vectors in Bosnia and Herzegovina

Surveys were carried out in autumn 2004 and spring 2005 in the traditional areas dedicated to pome and stone fruit cultivation in Bosnia and Herzegovina to assess the presence, distribution and incidence of phytoplasma diseases in fruit trees. The occurrence of psyllid vectors was also considered. The detection of phytoplasmas in plant and insect samples and their identification were carried out by symptom observations in the field, double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA), nested polymerase chain reaction (nested-PCR) and restriction fragment length polymorphism (RFLP) analyses. Laboratory analyses showed the presence of phytoplasmas belonging to: (i) 16SrX group, subgroup A (' Candidatus Phytoplasma mali') in 23 out of 25 apple samples, in 4 groups out of 18 of Cacopsylla picta (synonym Cacopsylla costalis ) and in 2 groups out of 9 of Cacopsylla melanoneura ; (ii) 16SrX group, subgroup C (' Candidatus Phytoplasma pyri') in 11 out of 30 pears samples and in 2 groups out of 9 of Cacopsylla pyri ; (iii) 16SrX group, subgroup B (' Candidatus Phytoplasma prunorum') in 4 apricots, 2 peaches out of 42 stone fruit samples and in 1 group out of 14 of Cacopsylla pruni . The presence of different subtypes of Candidatus Phytoplasma mali, both in apple trees and in insects, was proven.     

Comparison of phytoplasmas infecting winter oilseed rape in the Czech Republic with Italian Brassica phytoplasmas and their relationship to the aster yellows group

Winter oilseed rape grown in several areas in South Bohemia showed symptoms of stunting, leaf reddening and extensive malformation of floral parts. Phytoplasmas were consistently observed by using electron microscopy only in phloem tissue of symptomatic plants. DNA isolated from infected and healthy control plants was used in PCR experiments. Primer pairs R16F2/R2, P1/P7 and rpF2/R2, amplifying, respectively, 16S rDNA, 16S rDNA plus spacer region and the beginning of the 23S and ribosomal protein gene L22 specific for phytoplasmas, were used. According to RFLP and sequence analyses of PCR products, the phytoplasma from rape was classified in the aster yellows phytoplasma group, subgroup 16SrI-B. The PCR products from the Czech phytoplasma-infected rape also had RFLP profiles identical to those of phytoplasma strains from Italian Brassica . This first molecular characterization of phytoplasmas infecting rape compared with strains from Brassica does not, however, clearly indicate differences among isolates of the same 16SrI-B subgroup. Further studies on other chromosomal DNA portions could help the research on host specificity or on geographical distribution of these phytoplasmas.     

Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.     

Evidence for the physical integrity of flavescence dorée phytoplasmas purified by immunoaffinity from infected plants or leafhoppers and the plant pathogenicity of phytoplasmas from leafhoppers

Phytoplasmas were extracted from flavescence dorée-infected broadbean ( Vicia faba ) using a vacuole isolation medium, and were immunoaffinity purified from infected leafhoppers. Purified phytoplasmas from both host sources were immunolabelled and observed under the electron microscope. The infectivity of the purified phytoplasmas from leafhoppers was checked by injecting healthy leafhoppers which were then allowed to feed on healthy V. faba seedlings. The appearance of typical symptoms in these plants, and the positive results obtained in ELISA with extracts of some of the injected leafhoppers and with symptomatic V. faba , indicated that the purified phytoplasmas had retained their infectivity and had multiplied in the injected leafhoppers which had become infective. These results support a previous report that phytoplasmas purified by immunoaffinity chromatography are well preserved.     

Comparison of the ribosomal RNA operon from Texas Phoenix decline and lethal yellowing phytoplasmas

A comparison was made of the two palm yellows phytoplasmas affecting palms to determine if the entire ribosomal RNA operon portion of the phytoplasma genome, or portions thereof, could account for the observed palm host differences. Polymerase chain reaction (PCR) was used to amplify a 5.0?kb DNA fragment consisting of the entire ribosomal RNA operon from a subgroup 16SrIV-D phytoplasma that causes Texas Phoenix palm decline (TPD) in cabbage (Sabal palmetto) palm in west central Florida and from a subgroup 16SrIV-A phytoplasma that causes lethal yellowing (LY) in coconut (Cocos nucifera) palm in Jamaica. Before the PCR reaction, we sequenced by 454 sequencing a draft genome of the coconut LY phytoplasma, strain LYFL, that infects C. nucifera in Florida, and obtained from this draft sequence both copies of the entire ribosomal operon. Sequence analysis of the ribosomal RNA operons from both the LY and TPD phytoplasmas revealed the gene composition and orientation for the operons to be 5′16S rRNA-tRNA^Ile-23S rRNA-5S rRNA3′ and a tRNA^Val3′ downstream of the 5S rRNA gene. Based on molecular comparisons using the sequences of the ribosomal RNA operon, the TPD (16SrIV-D) strain was 98?% similar to the LY (16SrIV-A) strains.     

Molecular identification,characterization and transmission of phytoplasmas associated with sesame phyllody in Turkey

Phyllody is a destructive disease of sesame in Turkey. The disease has been causing significant economic losses by stunting the plants and altering their floral parts into leafy structures with no capsule and hence no seeds in sesame fields of the country. This research was undertaken to examine symptomatology, etiology, taxonomy and transmission of two recently discovered phyllody phytoplasmas infecting sesame in Turkey. Direct and nested PCR amplifications of 16S rRNA gene with the phytoplasma-specific universal primers P1/P7 and R16F2n/R2, respectively were employed for identification of the phytoplasmas associated with sesame phyllody. Phytoplasma-specific PCR amplicons of 1.8 kb and 1.2 kb were amplified only from symptomatic sesame plants and insect vector samples. Sequencing of the PCR amplicons and computer simulated restriction fragment length polymorphism analysis allowed classification of the phytoplasmas with pigeon pea witches’-broom (16SrIX-C) and peanut witches’-broom (16SrII-D) groups. The sequence homology and phylogenetic analyses further confirmed this classification. Among the insects collected from the sesame fields, the leafhopper Orosius orientalis Matsumara (Syn: O. albicinctus Distant) was the only vector proven to transmit the sesame phyllody phytoplasmas from diseased to healthy sesame plants in transmission assays. The results demonstrated that the 16SrIX-C and 16SrII-D group phytoplasmas were the agent of sesame phyllody and O. orientalis was the vector insect of the disease in Turkey.     

Detection, identification and significance of phytoplasmas in grasses in northern Australia

Sugarcane yields have been severely reduced by white leaf and grassy shoot phytoplasma diseases in many parts of Asia. Australian sugarcane crops are not known to be affected by these diseases, but plant pathogenic phytoplasmas found in other introduced and native grasses in northern Australia could pose a serious threat to the Australian sugarcane industry. To further evaluate this threat, leaves from plants of 20 grass species, with and without symptoms, were collected during field surveys in northern Australia and tested to determine whether phytoplasmas were present and whether symptoms were reliable indicators of phytoplasma presence. Molecular tools were used to detect and characterize phytoplasmas. Four different phytoplasmas were found in seven grass species known to grow near healthy sugarcane crops. All the phytoplasmas were closely related to sugarcane white leaf phytoplasma (SCWL), one of the phytoplasmas that causes disease in sugarcane in Asia. Four of the host plant species and two of the phytoplasmas were new records. The relationship between symptoms and phytoplasma presence was poor. Because some plants with symptoms tested negative for phytoplasmas, a series of surveys was carried out in which flowers, leaves, roots and stems of two known host plant species, Whiteochloa cymbiformis and Sorghum stipoideum, were tested separately on nine occasions during two wet seasons. This was done to investigate the distribution of phytoplasmas within plants over time. Results showed that spatial and temporal variation of phytoplasmas occurred in these two host plant species. Hence, evaluation of disease distribution within a region requires repeated testing of all plant parts from plants without symptoms, as well as those with symptoms. To date, there is no report of a vector capable of transmitting to Australian sugarcane the phytoplasmas found in grasses in this study. If one is present, or occurs in the future, then native and introduced grasses could constitute a large reservoir of phytoplasma for vectors to draw on. This work provides an early warning for the sugarcane industry that the potential for infection exists.     

Survey and molecular detection of phytoplasmas associated with potato in Romania and southern Russia

In recent years, emerging phytoplasma diseases of potato (Solanum tuberosum L.) have increasingly become important in central and eastern Europe. Accurate identification of phytoplasmas and their insect vectors is essential to developing effective management strategies for diseases caused by these plant pathogens. Potato phytoplasma diseases in Europe were for a long time diagnosed only on the basis of visual symptoms. However, this approach is not very reliable and the use of modern molecular techniques such as polymerase chain reaction (PCR) is required in order to accurately determine the etiology of these phytoplasma diseases. A survey and identification of phytoplasmas associated with potato crops in Romania and southern Russia were conducted based on modern molecular techniques. Symptomatic potato plants were collected from several fields and tested for phytoplasmas by PCR. Also, selected crops and weeds in the vicinity of these potato fields were sampled and tested for phytoplasmas. Stolbur (“Candidatus Phytoplasma solani”; 16SrXII-A) was the only phytoplasma detected in potato and adjacent crops, including tomato (Solanum lycopersicum), pepper (Capsicum annuum), eggplant (Solanum melongena), and beet (Beta vulgaris). This phytoplasma was also detected in weeds, particularly Convolvulus arvensis, Cuscuta sp., and Euphorbia falcata. Genotyping of obtained stolbur isolates on tuf genes revealed that they all had the same RFLP profile corresponding to the tuf-type ‘b’ (VK Type II). Stolbur-affected potato plants produced a large number of spongy tubers that resulted in commercially unacceptable potato chips upon processing.     

Molecular identification of 16SrII-D subgroup phytoplasmas associated with chickpea and faba bean in Sudan

In January 2011, symptomatic chickpea and faba bean plants were observed in fields located in the Gezira state (Sudan). Faba bean plants showed yellowing and stunting, whereas chickpea plants presented yellowing, reddening and little leaves. The disease etiology was investigated using nested polymerase chain reaction (PCR) with phytoplasma-specific primers which amplify a fragment of the 16S rRNA gene. Sequencing and restriction fragment length polymorphism (RFLP) analyses revealed that the tested phytoplasmas belonged to the group 16SrII. Phylogenetic analyses of the 16S rRNA gene of the obtained sequences indicated that the chickpea and faba bean phytoplasmas from Sudan were more closely related to the phytoplasmas subgroup 16SrII-D. To our knowledge, this is the first report of phytoplasmas from the group 16SrII-D infecting chickpea in Sudan, and faba bean worldwide.