In this study we investigated the secA gene as possible marker to supplement the classification s... more In this study we investigated the secA gene as possible marker to supplement the classification scheme based on 16S rRNA sequences. We cloned and sequenced secA gene and its flanking region of ‘Ca. P. asteris’ CPh strain phytoplasma, as well as 1997 bp length secA gene fragment of Canada peach X-disease CX and 1327 bp fragment of ‘Ca. P. ziziphi’ JWB. This helped us to select a primer pair for a single step PCR that can amplify ~1200 kb length fragment of secA gene from different phytoplasma groups. We propose that this fragment could be used in RFLP analysis to quickly identify and distinguish 16SrI-A, I-B, I-C, I-D, III-A, III-B, III-E, III-F, III-H, V-B, XII-B and XXI-A subgroup phytoplasmas using just two restriction endonucleases.
Previously undescribed phytoplasmas were detected in diseased plants of dandelion (Taraxacum offi... more Previously undescribed phytoplasmas were detected in diseased plants of dandelion (Taraxacum officinale) exhibiting virescence of flowers, thistle (Cirsium arvense) exhibiting symptoms of white leaf, and a Gaillardia sp. exhibiting symptoms of stunting and phyllody in Lithuania. On the basis of restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in PCR, the dandelion virescence (DanVir), cirsium whiteleaf (CirWL), and
During recent years, increasing attention has been devoted to the development of field floricultu... more During recent years, increasing attention has been devoted to the development of field floriculture in Lithuania. Since the quality of ornamental plants is adversely affected by diseases attributed to phytoplasmas, we surveyed plant collections in botanical gardens and floriculture farms in Lithuania for phytoplasmal diseases. 37 ornamental species belonging to 18 plant families exhibiting disease symptoms including general yellowing and stunting, proliferation of shoots, phyllody, virescence and reduced size of flowers, and reddening of leaves were collected in commercial floriculture farms and botanical gardens. Analysis of phytoplasmal 16S rRNA gene sequences amplified in the polymerase chain reaction revealed that the plants were infected by phytoplasmas belonging to five distinct subgroups (16SrI-A, 16SrI-B, 16SrI-C, 16SrI-L, and 16SrI-M) of group 16SrI (aster yellows phytoplasma group) and two subgroups (16SrIII-B, 16SrIII-F) of group 16SrIII (X-disease phytoplasma group).
... 6. Clark MF, Adams AN. ... IEDIKOSIOS DĖMĖTLIGĖS VIRUSO IDENTIFIKAVIMAS RT-PCR METODU Santr... more ... 6. Clark MF, Adams AN. ... IEDIKOSIOS DĖMĖTLIGĖS VIRUSO IDENTIFIKAVIMAS RT-PCR METODU Santrauka Pomidorų iedikosios dėmėtligės virusas (Tomato ringspot nepovirus, ToRSV), plačiai paplitęs visame pasaulyje, pa-eidia daugelį darovių, dekoratyvinių ...
Our previous studies reported that phytoplasma was the causative agent of the pine disease in the... more Our previous studies reported that phytoplasma was the causative agent of the pine disease in the Curonian Spit, Lithuania. In this study, insects from diseased pine trees and their adjacent areas were collected from 2016 to 2019 to further identify potential insect vectors that spread phytoplasmas. A total of 1018 phloem-feeding insects (order Hemiptera) were identified, 98.62% of which were aphids (Aphididae), and no known phytoplasma vectors were found. The results from semi-nested polymerase chain reaction (PCR) using phytoplasma-specific universal primers revealed that phytoplasmas were detected in Scots pine aphids (Cinara pini (Linnaeus, 1758)), waxy grey pine needle aphids (Cinara pineti (Fabricius, 1781)), and species-unknown aphids. Further sequence analysis and virtual RFLP (restriction fragment length polymorphism) analysis of aphid-harbored phytoplasma strains indicated that they were closely related to ‘Candidatus Phytoplasma pini’ (16SrXXI-A), but mainly 16SrXXI-A var...
Mountain pine (Pinus mugo Turra) is a coniferous native to the highlands of central Europe. Our p... more Mountain pine (Pinus mugo Turra) is a coniferous native to the highlands of central Europe. Our previous study revealed that mountain pine proliferation decline (MPPD) disease in the Curonian Spit of Lithuania is caused by a ‘Candidatus Phytoplasma pini’-related strain (16SrXXI-A). However, the insect vector of MPPD has not been identified. In this study, we conducted a survey to determine potential insect vectors of MPPD phytoplasma for three consecutive years (2016–2019). More than 1000 insect samples were collected from four locations in the Curonian Spit. These insects were identified as belonging to six families and ten genera. The presence of phytoplasma in insect samples was examined by nested polymerase chain reaction (PCR) using phytoplasma-specific primers (P1A/16S-SR and R16F2n/R16R2n). Phytoplasmas were detected in Cinara (Cinara) pini (Scots pine aphid), Cinara (Cinara) piniphila and Cinara (Schizolachnus) pineti (waxy grey pine needle aphid) insect samples. Subsequent ...
This paper provides an updating of information of a selected number of major phytoplasma diseases... more This paper provides an updating of information of a selected number of major phytoplasma diseases of forest trees, with a focus on the associated phytoplasma taxa. Phytoplasma diseases of forest trees have been less extensively studied than those affecting fruit trees. Research on the role of phytoplasmas as the cause of diseases of forest trees has only in the last few years been intensified, after sensitive and specific detection methods greatly based on PCR technology became available. Various phytoplasma taxa have been identified in naturally infected elm, ash, conifer, sandal, and eucalyptus trees, whereas only one phytoplasma taxon has been recorded in naturally infected alder trees. However, for almost all of the reviewed diseases, there is still sparse information about insect vectors, plant host range, strain virulence, pathogenicity, and host tolerance and resistance. Knowledge of these aspects is the basis for appropriate disease management. In particular, further researc...
MATERIALS AND METHODS Phenotypic characterization of strains. Size and morphology of vegetative c... more MATERIALS AND METHODS Phenotypic characterization of strains. Size and morphology of vegetative cells and endospores were studied in fresh cultures (grown for 17-24 h at 60 ° C on nutrient agar) under an Olympus AX70 microscope. Gram staining was performed using the Merck kit; the Gram reaction was additionally checked by the express method with 3% KOH. Minimum, optimum and maximum growth temperatures were established after cultivation at 40, 45, 50, 55, 60, 65, 70, 75, and 80 ° C. The medium used for this purpose was nutrient agar (Difco). NaCl requirements were tested in nutrient broth (Difco) at the following NaCl concentrations: 0, 5, 10, 15, and 20%. The strains isolated from the geothermal site were routinely stored at 4 ° C after growth on nutrient agar for 20-24 h at 60 ° C. Casein digestion and catalase production were determined according to Bergey's Manual of Systematic Bacteriology [9]. Collagen digestion was determined in water agar (20 g/l) supplemented with 2% collagen. DNA extraction. Bacterial genomic DNA was extracted from fresh cultures after cultivation on nutrient agar plates for 14 h at 60 ° C. The Genomic DNA Purification Kit (MBI Fermentas) was used for this purpose. Genomic DNA was extracted according to the manufacturer's instructions. Amplification of 16S rDNA. Synthetic oligonucleotides were purchased from MBI Fermentas. Primers
Symptoms of general stunting and yellowing of leaves were observed in diseased cultivated strawbe... more Symptoms of general stunting and yellowing of leaves were observed in diseased cultivated strawberry (Fragaria x ananassa Duchesne) in Lithuania. Analysis of 16S rRNA gene sequences amplified by PCR indicated that the symptoms were associated with infection by a phytoplasma, designated strawberry yellows (StrawY) phytoplasma. Phylogenetic analysis of 16S rRNA gene sequences indicated that StrawY phytoplasma, 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma asteris', stolbur phytoplasma and 'Candidatus Phytoplasma…
The Genus Phytophthora with over 64 spp. includes many of the most important plant-pathogens. Spe... more The Genus Phytophthora with over 64 spp. includes many of the most important plant-pathogens. Species classification has relied on morphological characterization. Frequent overlapping of species justifies molecular characterization. After evaluating morphological characters and RFLP analysis, sequences of the ITS (ITS1-5.8S-ITS2) rDNA from isolates of P. glovera, and P. bisheria were compared by phylogenetic analysis with those of 53 Phytophthora spp. (GenBank). The resulting trees showed two major groups for papillated and non-papillated forms. Papillated species grouped in two clusters: semi and conspicuously papillated. Integrating morpho/molecular characterization of P. glovera and P. bisheria consistently showed that these species are in the semipapillated group but in different clusters. An innovative key to Phytophthora spp. has been developed which integrates a pictorial morphological key with known molecular characteristics for each species and enhances reliable identification.
In this study we investigated the secA gene as possible marker to supplement the classification s... more In this study we investigated the secA gene as possible marker to supplement the classification scheme based on 16S rRNA sequences. We cloned and sequenced secA gene and its flanking region of ‘Ca. P. asteris’ CPh strain phytoplasma, as well as 1997 bp length secA gene fragment of Canada peach X-disease CX and 1327 bp fragment of ‘Ca. P. ziziphi’ JWB. This helped us to select a primer pair for a single step PCR that can amplify ~1200 kb length fragment of secA gene from different phytoplasma groups. We propose that this fragment could be used in RFLP analysis to quickly identify and distinguish 16SrI-A, I-B, I-C, I-D, III-A, III-B, III-E, III-F, III-H, V-B, XII-B and XXI-A subgroup phytoplasmas using just two restriction endonucleases.
Previously undescribed phytoplasmas were detected in diseased plants of dandelion (Taraxacum offi... more Previously undescribed phytoplasmas were detected in diseased plants of dandelion (Taraxacum officinale) exhibiting virescence of flowers, thistle (Cirsium arvense) exhibiting symptoms of white leaf, and a Gaillardia sp. exhibiting symptoms of stunting and phyllody in Lithuania. On the basis of restriction fragment length polymorphism (RFLP) analysis of 16S rDNA amplified in PCR, the dandelion virescence (DanVir), cirsium whiteleaf (CirWL), and
During recent years, increasing attention has been devoted to the development of field floricultu... more During recent years, increasing attention has been devoted to the development of field floriculture in Lithuania. Since the quality of ornamental plants is adversely affected by diseases attributed to phytoplasmas, we surveyed plant collections in botanical gardens and floriculture farms in Lithuania for phytoplasmal diseases. 37 ornamental species belonging to 18 plant families exhibiting disease symptoms including general yellowing and stunting, proliferation of shoots, phyllody, virescence and reduced size of flowers, and reddening of leaves were collected in commercial floriculture farms and botanical gardens. Analysis of phytoplasmal 16S rRNA gene sequences amplified in the polymerase chain reaction revealed that the plants were infected by phytoplasmas belonging to five distinct subgroups (16SrI-A, 16SrI-B, 16SrI-C, 16SrI-L, and 16SrI-M) of group 16SrI (aster yellows phytoplasma group) and two subgroups (16SrIII-B, 16SrIII-F) of group 16SrIII (X-disease phytoplasma group).
... 6. Clark MF, Adams AN. ... IEDIKOSIOS DĖMĖTLIGĖS VIRUSO IDENTIFIKAVIMAS RT-PCR METODU Santr... more ... 6. Clark MF, Adams AN. ... IEDIKOSIOS DĖMĖTLIGĖS VIRUSO IDENTIFIKAVIMAS RT-PCR METODU Santrauka Pomidorų iedikosios dėmėtligės virusas (Tomato ringspot nepovirus, ToRSV), plačiai paplitęs visame pasaulyje, pa-eidia daugelį darovių, dekoratyvinių ...
Our previous studies reported that phytoplasma was the causative agent of the pine disease in the... more Our previous studies reported that phytoplasma was the causative agent of the pine disease in the Curonian Spit, Lithuania. In this study, insects from diseased pine trees and their adjacent areas were collected from 2016 to 2019 to further identify potential insect vectors that spread phytoplasmas. A total of 1018 phloem-feeding insects (order Hemiptera) were identified, 98.62% of which were aphids (Aphididae), and no known phytoplasma vectors were found. The results from semi-nested polymerase chain reaction (PCR) using phytoplasma-specific universal primers revealed that phytoplasmas were detected in Scots pine aphids (Cinara pini (Linnaeus, 1758)), waxy grey pine needle aphids (Cinara pineti (Fabricius, 1781)), and species-unknown aphids. Further sequence analysis and virtual RFLP (restriction fragment length polymorphism) analysis of aphid-harbored phytoplasma strains indicated that they were closely related to ‘Candidatus Phytoplasma pini’ (16SrXXI-A), but mainly 16SrXXI-A var...
Mountain pine (Pinus mugo Turra) is a coniferous native to the highlands of central Europe. Our p... more Mountain pine (Pinus mugo Turra) is a coniferous native to the highlands of central Europe. Our previous study revealed that mountain pine proliferation decline (MPPD) disease in the Curonian Spit of Lithuania is caused by a ‘Candidatus Phytoplasma pini’-related strain (16SrXXI-A). However, the insect vector of MPPD has not been identified. In this study, we conducted a survey to determine potential insect vectors of MPPD phytoplasma for three consecutive years (2016–2019). More than 1000 insect samples were collected from four locations in the Curonian Spit. These insects were identified as belonging to six families and ten genera. The presence of phytoplasma in insect samples was examined by nested polymerase chain reaction (PCR) using phytoplasma-specific primers (P1A/16S-SR and R16F2n/R16R2n). Phytoplasmas were detected in Cinara (Cinara) pini (Scots pine aphid), Cinara (Cinara) piniphila and Cinara (Schizolachnus) pineti (waxy grey pine needle aphid) insect samples. Subsequent ...
This paper provides an updating of information of a selected number of major phytoplasma diseases... more This paper provides an updating of information of a selected number of major phytoplasma diseases of forest trees, with a focus on the associated phytoplasma taxa. Phytoplasma diseases of forest trees have been less extensively studied than those affecting fruit trees. Research on the role of phytoplasmas as the cause of diseases of forest trees has only in the last few years been intensified, after sensitive and specific detection methods greatly based on PCR technology became available. Various phytoplasma taxa have been identified in naturally infected elm, ash, conifer, sandal, and eucalyptus trees, whereas only one phytoplasma taxon has been recorded in naturally infected alder trees. However, for almost all of the reviewed diseases, there is still sparse information about insect vectors, plant host range, strain virulence, pathogenicity, and host tolerance and resistance. Knowledge of these aspects is the basis for appropriate disease management. In particular, further researc...
MATERIALS AND METHODS Phenotypic characterization of strains. Size and morphology of vegetative c... more MATERIALS AND METHODS Phenotypic characterization of strains. Size and morphology of vegetative cells and endospores were studied in fresh cultures (grown for 17-24 h at 60 ° C on nutrient agar) under an Olympus AX70 microscope. Gram staining was performed using the Merck kit; the Gram reaction was additionally checked by the express method with 3% KOH. Minimum, optimum and maximum growth temperatures were established after cultivation at 40, 45, 50, 55, 60, 65, 70, 75, and 80 ° C. The medium used for this purpose was nutrient agar (Difco). NaCl requirements were tested in nutrient broth (Difco) at the following NaCl concentrations: 0, 5, 10, 15, and 20%. The strains isolated from the geothermal site were routinely stored at 4 ° C after growth on nutrient agar for 20-24 h at 60 ° C. Casein digestion and catalase production were determined according to Bergey's Manual of Systematic Bacteriology [9]. Collagen digestion was determined in water agar (20 g/l) supplemented with 2% collagen. DNA extraction. Bacterial genomic DNA was extracted from fresh cultures after cultivation on nutrient agar plates for 14 h at 60 ° C. The Genomic DNA Purification Kit (MBI Fermentas) was used for this purpose. Genomic DNA was extracted according to the manufacturer's instructions. Amplification of 16S rDNA. Synthetic oligonucleotides were purchased from MBI Fermentas. Primers
Symptoms of general stunting and yellowing of leaves were observed in diseased cultivated strawbe... more Symptoms of general stunting and yellowing of leaves were observed in diseased cultivated strawberry (Fragaria x ananassa Duchesne) in Lithuania. Analysis of 16S rRNA gene sequences amplified by PCR indicated that the symptoms were associated with infection by a phytoplasma, designated strawberry yellows (StrawY) phytoplasma. Phylogenetic analysis of 16S rRNA gene sequences indicated that StrawY phytoplasma, 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma asteris', stolbur phytoplasma and 'Candidatus Phytoplasma…
The Genus Phytophthora with over 64 spp. includes many of the most important plant-pathogens. Spe... more The Genus Phytophthora with over 64 spp. includes many of the most important plant-pathogens. Species classification has relied on morphological characterization. Frequent overlapping of species justifies molecular characterization. After evaluating morphological characters and RFLP analysis, sequences of the ITS (ITS1-5.8S-ITS2) rDNA from isolates of P. glovera, and P. bisheria were compared by phylogenetic analysis with those of 53 Phytophthora spp. (GenBank). The resulting trees showed two major groups for papillated and non-papillated forms. Papillated species grouped in two clusters: semi and conspicuously papillated. Integrating morpho/molecular characterization of P. glovera and P. bisheria consistently showed that these species are in the semipapillated group but in different clusters. An innovative key to Phytophthora spp. has been developed which integrates a pictorial morphological key with known molecular characteristics for each species and enhances reliable identification.
Uploads
Papers by Deividas Valiunas