Grass White Leaf Research Articles

Mobile units of DNA in phytoplasma genomes

Phytoplasmas are obligate symbionts of plants and insects that are responsible for significant yield losses in diverse crops. Genome sequencing has revealed that many phytoplasma genomes appear to contain repeated genes organized in units of approximately 20 kb. These 'potential mobile units' (PMUs) resemble composite replicative transposons. PMUs contain several genes for recombination and some also contain putative 'virulence genes'. Genome alignments suggest that PMUs are involved in phytoplasma genome instability and recombination. In this edition of Molecular Microbiology, Hogenhout and colleagues report that one PMU from the aster yellows phytoplasma strain Witches' Broom (AY-WB) can exist as both a linear PMU within the chromosome and as an extrachromosomal circular form. The copy number of the circular form is much higher in the insect vector compared with the plant, and expression levels of genes present on the PMU are also higher in the insect. These observations suggest not only that this PMU could be a mobile element, but that it could also be involved in a phase-variation mechanism that allows the phytoplasma to adapt to its different hosts.

First report of a phytoplasma associated with Bermuda grass white leaf disease in Kenya

Bermuda grass (=Star grass), Cynodon dactylon , is a perennial plant of the family Poaceae that is native to the African Savannah but is now widely distributed over many warm countries of the world (Marcone et al ., 1997). C. dactylon plants…

'CandidatusPhytoplasma cynodontis' (16SrXIV group) affectingOplismenus burmannii(Retz.) P. Beauv. andDigitaria sanguinalis(L.) Scop. in India

A phytoplasma has been detected in Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. plants with symptoms of chlorotic streaks and white leaf disease in India. By sequence and phylogenetic analyses of polymerase chain reaction-amplified rDNA sequences, the detected phytoplasma was identified as ‘Candidatus Phytoplasma cynodontis’, a member of the 16SrXIV group, and the causal agent of Bermuda grass white leaf disease. This is the first report on the occurrence of ‘Ca. Phytoplasma cynodontis’ in O. burmannii and D. sanguinalis grasses.

MOLECULAR CHARACTERIZATION AND TRANSMISSION OF BERMUDA GRASS WHITE LEAF PHYTOPLASMA IN IRAN

SUMMARY Symptoms of Bermuda grass white leaf (BGWL) were observed in several provinces of Iran. Among leafhoppers collected in BGWL-affected areas, Exitianus capicola tested positive for phytoplasma in PCR assays and transmitted the BGWL agent to healthy Bermuda grass plants. Using P1/P7 primer pair, target DNA fragments of approximately 1.8 kbp were amplified from diseased samples of Bermuda grass plants from 10 widely separated areas in six provinces. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 PCR products identified two BGWL isolates from Firouzabad and Juyom (Fars province) denoted FBGWL and JBGWL as members of the Bermuda grass white leaf group, 16SrXIV. Percent homology and phylogenetic analysis of 16S rDNA and 16S-23S spacer region (SR) sequences showed also that FBGWL and JBGWL are related to the 16SrXIV group. Based on RFLP and phylogenetic analyses, FBGWL and JBGWL isolates were distinguishable from each other and were classified in subgroups 16SrXIV-A and 16SrXIV-B, respectively. This is the first report of BGWL disease and leafhopper transmission of the associated phytoplasma in Iran.

First report of a ‘Candidatus Phytoplasma cynodontis’-related strain (group 16SrXIV) associated with white leaf disease of Dichanthium annulatum in India

A phytoplasma has been detected in a formerly undescribed white leaf disease of Dichanthium annulatum (Kleberg’s bluestem) in India. By sequence and phylogenetic analyses of polymerase chain reaction-amplified rDNA sequences, the detected phytoplasma proved to be closely related to Bermuda grass white leaf agent (‘Candidatus Phytoplasma cynodontis’) which is a member of the 16SrXIV group.

First report of a 16SrXIV, ‘Candidatus Phytoplasma cynodontis’ group phytoplasma associated with coconut yellow decline in Malaysia

The coconut palm (Cocos nucifera) is an important perennial oil crop cultivated throughout Malaysia and is used for its aesthetic appeal in landscapes. Symptoms of yellow decline have been observed in a plantation in Selangor, similar to those caused by phytoplasma groups of the lethal yellowing (16SrIV) type recorded from the Americas, Caribbean and Africa, and similar also to Stolbur (16SrXII) associated with Kalimantan wilt in Indonesia. Although not widespread, this is the first report on such symptoms in coconut in Malaysia. The lower canopy foliage initially turns light yellow and eventually light-brown, spreading rapidly to the younger leaves. Also severe chlorosis of the emerging spear leaf, inflorescence necrosis, premature nut fall, and gradual collapse of fronds occur, and immature palms generally die within 5 months of initial symptom appearance. Samples were collected from 20 affected and five symptomless Malayan red dwarf coconut palms. Malayan yellow dwarf and some Malayan tall coconuts exhibited similar symptoms, but were not tested in this study. Total DNA was extracted and assayed in a nested PCR with universal phytoplasma primer pairs P1/P7 followed by either R16F2/R16R2 or fU5/ rU3. Products of the expected size (approximately 1200 bp and 880 bp, respectively) were amplified from 8/20 plants with symptoms but not from any symptomless coconut palms. Amplification also failed when the LY (16SrIV) specific primers LY16Sf/LY16Sr were used for nested PCR on P1/P7 first round PCR samples (Harrison et al., 2002), as well as when LY non-ribosomal specific primers LYC24F/LYC24R (Harrison et al ., 1994) were used, indicating that the phytoplasma was not from the 16SrIV group. The phytoplasma 16SrDNA was cloned and sequenced (GenBank Acc. No. EU328159) and showed the highest homology (99%) with those of Bermuda grass white leaf phytoplasma from Thailand (AF248961) and Malaysia (EU294011), members of the 16SrXIV ‘ Candidatus Phytoplasma cynodontis’ group. This group has previously been found associated with a decline disease in date palm in Sudan (Cronje et al ., 2000). However, this is the first report of a 16SrXIV phytoplasma associated with yellow decline in Malaysia.

Molecular identification of ‘Candidatus Phytoplasma cynodontis’ associated with Bermuda grass white leaf disease in India

Molecular identification of ‘<i>Candidatus</i> Phytoplasma cynodontis’ associated with Bermuda grass white leaf disease in India

Detection of sugarcane grassy shoot phytoplasma infecting sugarcane in India and its phylogenetic relationships to closely related phytoplasmas

In several sugarcane-growing areas of India, sugarcane plants showing typical symptoms of sugarcane grassy shoot (SCGS) disease as well as non-symptomatic sugarcane plants and sugarcane with non-specific yellowing and stunting symptoms were examined for phytoplasmal infections by means of polymerase chain reaction (PCR) technology, using primers directed to phytoplasma ribosomal DNA (rDNA) sequences. From all field-collected, symptomatic sugarcane plants and those with non-specific symptoms as well as from most the non-symptomatic plants, detectable PCR products were obtained through the highly sensitive nested-PCR assays. Also, the phylogenetic relationships of SCGS phytoplasma strains including those whose rDNA sequences were determined in this study, to each other and to other closely related phytoplasmas, at both 16S rRNA gene and 16S/23S rDNA spacer region sequence levels were examined. The SCGS phytoplasma is a member of the rice yellow dwarf (RYD) phytoplasma group or 16SrXI group, and proved very closely related to the sugarcane with leaf (SCWL) agent. More distantly related to the SCGS agent, are sorghum grassy shoot (SGS), Bermuda grass white leaf (BGWL) (=‘Candidatus Phytoplasma cynodontis’) and RYD (= ‘Candidatus Phytoplasma oryzae’) phytoplasmas. The extensive survey of phytoplasmal infections in sugarcane plants in India as well as analysis of phylogenetic relationships of SCGS phytoplasma strains to each other and to other closely related phytoplasmas, at both 16S rRNA gene and 16S/23S rDNA spacer region sequence levels, had previously not been known.

'Candidatus Phytoplasma cynodontis', the phytoplasma associated with Bermuda grass white leaf disease.

Bermuda grass white leaf (BGWL) is a destructive, phytoplasmal disease of Bermuda grass (Cynodon dactylon). The causal pathogen, the BGWL agent, differs from other phytoplasmas that cluster in the same major branch of the phytoplasma phylogenetic clade in <2.5% of 16S rDNA nucleotide positions, the threshold for assigning species rank to phytoplasmas under the provisional status 'Candidatus'. Thus, the objective of this work was to examine homogeneity of BGWL isolates and to determine whether there are, in addition to 16S rDNA, other markers that support delineation of the BGWL agent at the putative species level. Phylogenetic analyses revealed that the 16S rDNA sequences of BGWL strains were identical or nearly identical. Clear differences that support separation of the BGWL agent from related phytoplasmas were observed within the 16S-23S rDNA spacer sequence, by serological comparisons, in vector transmission and in host-range specificity. From these results, it can be concluded that the BGWL phytoplasma is a discrete taxon at the putative species level, for which the name 'Candidatus Phytoplasma cynodontis' is proposed. Strain BGWL-C1 was selected as the reference strain. Phytoplasmas that are associated with brachiaria white leaf, carpet grass white leaf and diseases of date palms showed 16S rDNA and/or 16S-23S rDNA spacer sequences that were identical or nearly identical to those of the BGWL phytoplasmas. However, the data available do not seem to be sufficient for a proper taxonomic assignment of these phytoplasmas.

'Candidatus Phytoplasma', a taxon for the wall-less, non-helical prokaryotes that colonize plant phloem and insects.

The trivial name 'phytoplasma' has been adopted to collectively name wall-less, non-helical prokaryotes that colonize plant phloem and insects, which were formerly known as mycoplasma-like organisms. Although phytoplasmas have not yet been cultivated in vitro, phylogenetic analyses based on various conserved genes have shown that they represent a distinct, monophyletic clade within the class Mollicutes. It is proposed here to accommodate phytoplasmas within the novel genus 'Candidatus (Ca.) Phytoplasma'. Given the diversity within 'Ca. Phytoplasma', several subtaxa are needed to accommodate organisms that share <97.5% similarity among their 16S rRNA gene sequences. This report describes the properties of 'Ca. Phytoplasma', a taxon that includes the species 'Ca. Phytoplasma aurantifolia' (the prokaryote associated with witches'-broom disease of small-fruited acid lime), 'Ca. Phytoplasma australiense' (associated with Australian grapevine yellows), 'Ca. Phytoplasma fraxini' (associated with ash yellows), 'Ca. Phytoplasma japonicum' (associated with Japanese hydrangea phyllody), 'Ca. Phytoplasma brasiliense' (associated with hibiscus witches'-broom in Brazil), 'Ca. Phytoplasma castaneae' (associated with chestnut witches'-broom in Korea), 'Ca. Phytoplasma asteris' (associated with aster yellows), 'Ca. Phytoplasma mali' (associated with apple proliferation), 'Ca. Phytoplasma phoenicium' (associated with almond lethal disease), 'Ca. Phytoplasma trifolii' (associated with clover proliferation), 'Ca. Phytoplasma cynodontis' (associated with Bermuda grass white leaf), 'Ca. Phytoplasma ziziphi' (associated with jujube witches'-broom), 'Ca. Phytoplasma oryzae' (associated with rice yellow dwarf) and six species-level taxa for which the Candidatus species designation has not yet been formally proposed (for the phytoplasmas associated with X-disease of peach, grapevine flavescence dorée, Central American coconut lethal yellows, Tanzanian lethal decline of coconut, Nigerian lethal decline of coconut and loofah witches'-broom, respectively). Additional species are needed to accommodate organisms that, despite their 16S rRNA gene sequence being >97.5% similar to those of other 'Ca. Phytoplasma' species, are characterized by distinctive biological, phytopathological and genetic properties. These include 'Ca. Phytoplasma pyri' (associated with pear decline), 'Ca. Phytoplasma prunorum' (associated with European stone fruit yellows), 'Ca. Phytoplasma spartii' (associated with spartium witches'-broom), 'Ca. Phytoplasma rhamni' (associated with buckthorn witches'-broom), 'Ca. Phytoplasma allocasuarinae' (associated with allocasuarina yellows), 'Ca. Phytoplasma ulmi' (associated with elm yellows) and an additional taxon for the stolbur phytoplasma. Conversely, some organisms, despite their 16S rRNA gene sequence being <97.5% similar to that of any other 'Ca. Phytoplasma' species, are not presently described as Candidatus species, due to their poor overall characterization.

Detection of phytoplasmas associated with grassy shoot and white leaf diseases of sugarcane in sri lanka using fta™ papers

The most reliable and rapid detection method has been the use of nested PCR directed to conserved sequences in the 16S rRNA gene. DNA isolation follows a phytoplasmal enrichment procedure. This process is both expensive and time-consuming. The present study was conducted at the Department of Pathology, South African Sugar Association Experiment Station, using sugarcane leaves and grass samples with GS, WL and Bermuda Grass White Leaf (BGWL) symptoms collected in Sri Lanka to investigate the use of FTA™ paper (Whatman BioScience) for DNA extraction and the use of a radioactive probe in conjunction with a tissue blot procedure to detect phytoplasmas in sugarcane leaf samples exhibiting symptoms of GS and SCWL. Bermuda grass leaf samples showing symptoms of Bermuda Grass White Leaf (BGWL) were also included in the study. It was found that the FTA™ paper method was a rapid and cost effective in detecting sugarcane phytoplasma compared to the enrichment method.

"Candidatus Phytoplasma oryzae", a novel phytoplasma taxon associated with rice yellow dwarf disease.

In addition to rice yellow dwarf (RYD) phytoplasma, several phytoplasmas infect gramineous plants, including rice orange leaf, bermuda grass white leaf, brachiaria grass white leaf and sugarcane white leaf phytoplasmas. To investigate whether the RYD phytoplasma is a discrete, species-level taxon, several isolates of the aforementioned phytoplasmas were analysed using PCR-amplified 16S rDNA sequences. Two RYD isolates, RYD-J(T) and RYD-Th, were almost identical (99.2 %), but were distinct (similarities of 96.3-97.9 %) from other phytoplasma isolates of the RYD 16S-group. The notion that the RYD phytoplasma constitutes a unique taxon is also supported by its unique insect vector (Nephotettix sp.), its unique host plant in nature (rice) and its limited geographical distribution (Asia). In Southern blot analysis, chromosomal and extrachromosomal DNA probes of the RYD phytoplasma reportedly did not hybridize with those of closely related phytoplasmas. These properties of the RYD phytoplasma clearly indicate that it represents a novel taxon, 'Candidatus Phytoplasma oryzae'.

First report of a phytoplasma associated with a disease of date palms in North Africa

White tip die-back (WTD) is a newly recognized disease of young date palms (Phoenix dactylifera L.) in northern Sudan and occurs in scattered foci throughout the region. It affects immature palms, 5–8 years old, which die within 6–12 months of symptom appearance. Symptoms appear as a severe chlorosis of the emerging spear leaf and at the tips of the pinnae of older fronds. Chlorotic streaks, mainly white with some necrosis, extend longitudinally along the sides of the frond mid-rib. There is no yellowing of the crown, which changes quickly from green to dry white. No phytoplasmas could be unequivocally identified by electron microscopy in the WTD-affected date palm tissues. Using a nested polymerase chain reaction together with general phytoplasma primers which amplify the 16S/23S rDNA (Cronjéet al., 1998), phytoplasma-specific products, 1250 bp in size, were consistently amplified from WTD palms but not from healthy date palms. The phytoplasma 16S/23S rDNA intergenic spacer region was sequenced and this showed a very high (99%) homology with comparable sequences of Bermuda grass white leaf (BGWL) phytoplasma strains from South-east Asia and Sudan (GenBank accession numbers: Y14645, AF100412). Further studies will be needed to establish if there is a connection between this date palm disease and the presence of BGWL in plantations. The 16S/23S spacer sequence from WTD date palm has been submitted to GenBank (accession number: AF 100411). Date palms are known to be susceptible to the lethal yellowing phytoplasma in the USA (Harrison et al., 1995), but there are no previous records for their susceptibility to phytoplasma infection in countries outside the lethal yellowing zone.

Slow decline: a new disease of mature date palms in North Africa associated with a phytoplasma

Slow Decline (SD) or El Arkish is an established lethal disease of uncertain aetiology that affects mature date palms (Phoenix dactylifera L.) in Northern Sudan. It is common along the Nile between Dongola and Merowe–Karema, where it causes steady annual losses estimated at 6%. Palm mortality occurs between 12 and 24 months after the first appearance of symptoms. SD symptoms include a yellowing of the lower canopy starting with the outermost (oldest) fronds and progressing towards the young central fronds and emerging spear leaf. All fronds dry white to light-brown and are then shed, leaving an erect dry tuft of young leaves at the top of the trunk which may break off to leave the trunk standing alone. In some case, the white dry spear leaves on palms can be seen from the ground prior to the appearance of any foliar yellowing. Young offshoots of SD-affected palms also show frond yellowing and white spear necrosis as the crown dies. Occasionally, the spear can be pulled out to reveal rotten, foul-smelling basal tissues. Although mature palms were too tall to observe any inflorescence or fruit symptoms, there was no evidence of fruit drop. Using a nested polymerase chain reaction together with general phytoplasma primers which amplify the 16S/23S rDNA (Cronje et al., 1998), phytoplasma-specific products, 1250-bp in size, were amplified from four of six SD-affected palms (one trunk and three of five petiole samples) but not from symptomless palms from the same area. The sequence of the SD phytoplasma 16S/23S rDNA intergenic spacer (GenBank Accession number AF268000) showed a very high (99%) homology with comparable sequences of phytoplasmas associated with White tip die-back, a newly reported disease of young date palms (Cronjéet al. 2000), and Bermuda grass white leaf (BGWL) disease from Sudan (Genbank Accession numbers AF100411, AF100412).

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.

Detection and characterization of a phytoplasma associated with annual blue grass (Poa annua) white leaf disease in southern Italy

A phytoplasma was detected in annual blue grass (Poa annua L. Fienardo), exhibiting white leaf symptoms, that was grown in the fields near Caserta in southern Italy. Based on restriction fragment length polymorphism analysis of PCR-amplified 16S rDNA sequences, the phytoplasma associated with annual blue grass white leaf disease was identified as a new member of phytoplasma 16S rRNA group XI (16SrXI) (type strain, rice yellow dwarf phytoplasma). The annual blue grass white leaf phytoplasma is most closely related to Bermuda grass white leaf phytoplasma found in Asia. Annul blue grass white leaf and Bermuda grass white leaf phytoplasmas were designated as the third subgroup (16SrXI-C) of group XI. This is the first report that a plant pathogenic phytoplasma belonging to group 16SrXI is present on the European continent.