Grass White Leaf Research Articles

Detection and Practical Differentiation of Phytoplasmas from Several Host Plants Using PCR-RFLP

Phytoplasma as a phytopathogenic prokaryote with a wide host range is a pathogen that needs more attention in Indonesia. This pathogen is relatively difficult to detect and identify due to its complicated biological properties. This study involved detection of phytoplasmas by polymerase chain reaction (PCR) technique with P1/P7 primers from seven symptomatic plants, i.e. Bermuda grass white leaf, bamboo yellows, witches’ broom of peanut, soybean, yard long bean, and cactus, and sweet potato little leaf. The phytoplasma DNA of the 16S rRNA gene resulting from PCR amplification was examined by digestion reaction using three endonuclease enzymes AluI, RSaI, and MSeI to generate restriction fragment length polymorphism (RFLP) profile. The seven diseased plants were confirmed positive to be associated with phytoplasma as indicated by the PCR product of 1800 bp. Based on the RFLP profiles of the three enzymes, the phytoplasmas were divided into two groups, namely group I (Bermuda grass and bamboo) and group II (peanuts, soybeans, yard long beans, cactus, and sweet potatoes). Cactus phytoplasma is a sub-group (strain) because it has a slightly different fragment of MSeI RFLP profile.

Complete genome sequence of "Candidatus Phytoplasma cynodontis" GY2015, a plant pathogen associated with Bermuda grass white leaf disease in Taiwan.

The complete genome sequence of "Candidatus Phytoplasma cynodontis" strain GY2015, which consists of one 498,922-bp circular chromosome, is presented in this work. This uncultivated plant-pathogenic bacterium is associated with Bermuda grass white leaf disease in Taoyuan, Taiwan.

Geographical Distribution and Identification of Phytoplasma Strain Associated with Sugarcane White Leaf Disease in Sri Lanka

Sugarcane white leaf (SCWL) is one of the major sugarcane diseases in the Asian continent associated with phytoplasmas. It occurs in all sugarcane-growing areas of Sri Lanka, causing substantial economic losses. However, subgroup level identification has been not reported so far for the phytoplasma strains associated with this disease in Sri Lanka. In the present study, the geographical distribution of phytoplasma strains associated with SCWL in Sri Lanka, identification of their weed reservoirs and ribosomal subgroup analysis based on phytoplasma 16S rRNA gene were carried out. A total of 27 SCWL samples representing six main sugarcane-growing areas of Sri Lanka and six samples of two grass species (Cynodon dactylon and Brachiaria distachya) showing putative symptoms of phytoplasma were analyzed. A nested PCR product of 1.2 kb size was consistently amplified in all the symptomatic samples with primers amplifying the 16S rRNA gene. A higher level of sequence identity from 99.03 to 100% was shared among the 16S rRNA gene sequences of all tested SCWL phytoplasma strains. Both Bermudagrass white leaf (BGWL) and Brachiaria grass white leaf (BraWL) phytoplasma strains shared 97.5–98.1% identity of their 16S rRNA gene with SCWL strains. The 16S rRNA gene sequence comparisons and virtual RFLP analysis of SCWL, BGWL, and BraWL phytoplasma isolates allowed their affiliations with ‘Candidatus Phytoplasma sacchari’ (16SrXI-B subgroup) with SCWL and ‘Ca. P. cynodontis’ (16SrXIV-A subgroup) with the grasses samples. The phytoplasma subgroup 16SrXI-B was identified as the most widespread SCWL phytoplasma subgroup in commercial sugarcane varieties in Sri Lanka.

'Candidatus Phytoplasma sacchari', a novel taxon - associated with Sugarcane Grassy Shoot (SCGS) disease.

Sugarcane Grassy Shoot (SCGS) disease is known to be related to Rice Yellow Dwarf (RYD) phytoplasmas (16SrXI-B group) which are found predominantly in sugarcane growing areas of the Indian subcontinent and South-East Asia. The 16S rRNA gene sequences of SCGS phytoplasma strains belonging to the 16SrXI-B group share 98.07 % similarity with 'Ca. Phytoplasma cynodontis' strain BGWL-C1 followed by 97.65 % similarity with 'Ca. P. oryzae' strain RYD-J. Being placed distinctly away from both the phylogenetically related species, the taxonomic identity of SCGS phytoplasma is unclear and confusing. We attempted to resolve the phylogenetic positions of SCGS phytoplasma based on the phylogenetic analysis of 16S rRNA gene (>1500 bp), nine housekeeping genes (>3500 aa), core genome phylogeny (>10 000 aa) and OGRI values. The draft genome sequences of SCGS phytoplasma (strain SCGS) and Bermuda Grass White leaf (BGWL) phytoplasma (strain LW01), closely related to 'Ca. P. cynodontis', were obtained. The SCGS genome was comprised of 29 scaffolds corresponding to 505 173 bp while LW01 assembly contained 21 scaffolds corresponding to 483 935 bp with the fold coverages over 330× and completeness over 90 % for both the genomes. The G+C content of SCGS was 19.86 % while that of LW01 was 20.46 %. The orthoANI values for the strain SCGS against strains LW01 was 79.42 %, and dDDH values were 22. Overall analysis reveals that SCGS phytoplasma forms a distant clade in RYD group of phytoplasmas. Based on phylogenetic analyses and OGRI values obtained from the genome sequences, a novel taxon 'Candidatus Phytoplasma sacchari' is proposed.

Genetic diversity of phytoplasma strains inducing phyllody, flat stem and witches' broom symptoms in Manilkara zapota in India.

During a survey performed in sapota orchards of India, from 2015 to 2018, symptoms of phyllody, little leaf, flat stem and witches' broom were observed in three states: Karnataka, Kerala and Tripura. The association of phytoplasmas was confirmed in all the symptomatic sapota samples by using nested PCR specific primers (P1/P7, R16F2n/R16R2 and 3Far/3Rev) with amplification of fragments of ~ 1.25kb and ~ 1.3kb. Association of three phytoplasma groups, aster yellows with flat stem from Tripura (Lembucherra), clover proliferation with phyllody symptoms at Karnataka (Bengaluru) and bermuda grass white leaf with flat stem and little leaf from Kerala (Thiruvananthapuram) and Tripura (Cocotilla) were confirmed by 16S rRNA gene sequence comparison analysis. Virtual RFLP analysis of 16S rRNA gene sequences using pDRAW32 further classified the sapota phytoplasma isolates into 16SrI-B, 16SrVI-D and 16SrXIV-A subgroups. This is the first report on identification of three phytoplasma groups in sapota in world.

Draft Genome Sequences of Two Phytoplasma Strains Associated with Sugarcane Grassy Shoot (SCGS) and Bermuda Grass White Leaf (BGWL) Diseases

We performed whole-genome sequencing of two phytoplasmas associated with sugarcane grassy shoot (SCGS) and Bermuda grass white leaf diseases. These are the first draft genomes of SCGS phytoplasma (strain SCGS) and 'Candidatus Phytoplasma cynodontis' (strain LW01) and may help to delineate these phytoplasmas at a finer taxonomic level.

AFM VISUALIZATION OF PHYTOPLASMA DNA IMMOBILIZATION AND HYBRIDIZATION EVENT ON CHITOSAN MODIFIED SPCE SURFACE

The evidence of DNA immobilization and hybridization on a functional surface is demonstrated in this presentation following the high-resolution atomic force microscopic (AFM) direct observation. A planar functional surface was fabricated by self-assembly of a biopolymer chitosan onto screen-printed carbon electrode (SPCE) concerning to its excellent covalent bonding binding of biomolecules. The target DNA molecules were extracted from important phytoplasma diseased plants such as sugarcane white leaf (SCWL), burmuda grass white leaf (BGWL) and sesame phyllody (SP). The real image in actual posture of long chain natural DNA and the well-known structure of the DNA double helix were obviously visualized following the DNA immobilization and subsequent transitional hybridization with their complementary DNA. The coordinate quantitative parameters of AFM images such as line profile analysis, surface roughness, power spectrum density (PSD) algorithm and fractal dimension were also determined. The width size of double stranded DNA and the specific hybrid DNA were apparently almost 2 folds bigger than their single stranded DNA. Whereas an aggregation and growth mechanism of those DNAs on chitosan modified SPCE surfaces were mainly stacked in vertical direction and grown beyond a diffusion limited model as indicated by their fractal dimension values around 2 to 3. This phenomenon is hence valuable for the verification and the efficient development of diagnostic DNA-based devices.

First report of an association of ‘Candidatus Phytoplasma cynodontis’ with Napier grass white leaf disease in India

Napier grass is an important forage crop grown in India for livestock feed. White leaf symptoms with multiple shoot proliferation (NGWL) were observed in Napier grass fields at Baramati, Pune and Devanahalli, Bengaluru. The symptomatic samples were collected and analysed for the presence of phytoplasma by PCR assays. Amplicons of the expected sizes (1800 and 1250 bp) were amplified in all the symptomatic plants using the universal primer pair P1/P7 and R16F2n/R16R2, respectively, but not from any non-symptomatic plants. The ~ 1.2 kb product of 16S rDNA sequence of NGWL showed that they had 99% nucleotide identity with ‘Candidatus Phytoplasma cynodontis’-related strains. The phylogeny and virtual RFLP analysis revealed that NGWL phytoplasma strain belonged to the 16SrXIV-A subgroup. This is the first report of association of Napier grass by a strain of 16SrXIV-A phytoplasma worldwide.

Immunological detection of the Weligama coconut leaf wilt disease associated phytoplasma: Development and validation of a polyclonal antibody based indirect ELISA.

Weligama coconut leaf wilt disease (WCLWD) causes heavy losses in the coconut cultivations of southern Sri Lanka. The in-house developed and validated indirect ELISA was based on specific polyclonal antibodies raised in female New Zealand White rabbits, against partially purified WCLWD associated phytoplasma. This ELISA has the potential to distinguish secA PCR confirmed, WCLWD associated phytoplasma positive palms from phytoplasma free palms at high accuracy (93%) and sensitivity (92.7%), but with marginal specificity (79%). The calculated ELISA cross reactivity index (CRI) values were low for sugarcane white leaf (7%) and sugarcane grassy shoot (8%) infected leaves, but with marked highCRIfor both Bermuda grass white leaf (69%) and areca nut yellow leaf (70%) infected leaves. SecA gene based phylogenetic relationships of the WCLWD associated phytoplasma with these other locally prevalent phytoplasma strains elucidated this immunological cross reactivity, which was further reiterated by virtual restriction fragment length polymorphism analysis. Based on scanning electron microscopy, this study provides additional visual evidence, for the presence of phytoplasmas in WCLWD infected tissues.

Identification of grass white leaf disease associated with a ‘Candidatus Phytoplasma asteris’-related phytoplasma strain (16SrI-B and cpn60 I-IIIB) in Mexico

Samples of a wild grass showing white leaf symptoms were collected in 2015 in Veracruz, Mexico around sugarcane plantations. DNA amplification, sequencing and phylogenetic analysis revealed the presence of a ‘Candidatus Phytoplasma asteris’-related strain in the plants that were positive using 16S-based and cpn60 PCR assays. The strain determined to be associated with the grass white leaf disease was identified as a member of the 16SrI-B subgroup and a member of the cpn60 I-IIIB subgroup through in silico RFLP analysis. This is the first report of phytoplasma-infected grass plants associated with white leaf disease in Mexico. The implications of these findings are vital for the management of other plant hosts of the family Poaceae such as sugarcane and corn, and point to weedy grasses as a potential source of phytoplasma inoculum for nearby crop plants.

Phytoplasma diseases of ornamental plants: An Indian overview

The economic importance of ornamentals has been progressing significantly in many countries with international demand expanding continuously. Cut flowers represent the largest segment of the industry followed by flowering potted plants, trees, shrubs, annuals, flower bulbs and other propagation materials. Like other crops, ornamental plants are also affected by a number of pathogens such as fungi, bacteria, viruses, phytoplasmas etc. Among them, the phytoplasmas are equally important group of pathogens which drastically damage growth and marketing parameters of ornamental plants and thereby affecting their commercial value. Phytoplasmas cause diseases in several commercial cut flowers and ornamental plants causing serious economic losses all over the world. Phytoplasma diseases are a major constraint in commercial production of ornamental plants by lowering the quantum and quality gaining international importance. So far more than 26 ornamental plant species have been reported to be associated with phytoplasma infection from all over India belonging to six different phytoplasma groups, viz. aster yellows (16SrI), peanut witches ’broom (16SrII), clover proliferation(16SrVI), pigeon pea witches ’broom(16SrIX), rice yellow dwarf(16SrXI) and bermuda grass white leaf (16SrXIV) phytoplasma. Limited studies are made towards epidemiological aspects of these diseases in India. Major management practices adapted for phytoplasma disease management are using tetracycline treatment, insecticides for vector control and micropropagation for emission and elimination of phytoplasmas. In this review, an update status of progress on research work done on phytoplasma diseases of ornamentals in India is discussed.

Refinement of the Taxonomic Structure of 16SrXI and 16SrXIV Phytoplasmas of Gramineous Plants using Multilocus Sequence Typing.

Phytoplasmas that infect gramineous plants, including Napier grass stunt, sugarcane whiteleaf, sugarcane grassy shoot, and Bermuda grass whiteleaf, have been classified into two closely related groups, 16SrXI and 16SrXIV, based on the 16S ribosomal RNA (rRNA) gene. Subsequently, phytoplasmas associated with coconut and Areca palm in southern India and Sri Lanka have been added into the 16SrXI group. However, the 16S rRNA gene gives relatively poor resolution between these phytoplasmas. In this study, a new set of universal phytoplasma primers that amplify approximately 1 kb of the leucyl transfer RNA synthetase (leuS) gene have been validated on a broad range of phytoplasma taxonomic groups. These have been used along with partial sequences of the secA gene to clarify the taxonomic classification of 16SrXI and 16SrXIV phytoplasmas. Based on this data, the sugarcane whiteleaf and grassy shoot phytoplasmas appear to be the same phytoplasma. The Napier grass stunt phytoplasma forms a distinct group from the Bermuda grass whiteleaf and sugarcane phytoplasmas, suggesting that Napier grass stunt should be in its own 'Candidatus Phytoplasma sp.'. The phytoplasmas associated with coconut and arecanut in southern India and Sri Lanka, which are in the same 16SrXI group, appear in different groups based on secA analysis.

Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province, Saudi Arabia

ABSTRACTTypical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400 bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.

Molecular biology and pathogenicity of phytoplasmas

AbstractPhytoplasmas are a large group of plant‐pathogenic wall‐less, non‐helical, bacteria associated with diseases, collectively referred to as yellows diseases, in more than a thousand plant species worldwide. Many of these diseases are of great economic importance. Phytoplasmas are difficult to study, in particular because all attempts at culturing these plant pathogens under axenic conditions have failed. With the introduction of molecular methods into phytoplasmology about two decades ago, the genetic diversity of phytoplasmas could be elucidated and a system for their taxonomic classification based on phylogenetic traits established. In addition, a wealth of information was generated on phytoplasma ecology and genomics, phytoplasma–plant host interactions and phytoplasma–insect vector relationships. Taxonomically, phytoplasmas are placed in the class Mollicutes, closely related to acholeplasmas, and are currently classified within the provisional genus ‘Candidatus Phytoplasma’ based primarily on 16S rDNA sequence analysis. Phytoplasmas are characterised by a small genome. The sizes vary considerably, ranging from 530 to 1350 kilobases (kb), with overlapping values between the various taxonomic groups and subgroups, resembling in this respect the culturable mollicutes. The smallest chromosome, about 530 kb, is known to occur in the Bermuda grass white leaf agent ‘Ca. Phytoplasma cynodontis’. This value represents the smallest mollicute chromosome reported to date. In diseased plants, phytoplasmas reside almost exclusively in the phloem sieve tube elements and are transmitted from plant to plant by phloem‐feeding homopteran insects, mainly leafhoppers and planthoppers, and less frequently psyllids. Most of the phytoplasma host plants are angiosperms in which a wide range of specific and non‐specific symptoms are induced. Phytoplasmas have a unique and complex life cycle that involves colonisation of different environments, the plant phloem and various organs of the insect vectors. Furthermore, many phytoplasmas have an extremely wide plant host range. The dynamic architecture of phytoplasma genomes, due to the occurrence of repetitive elements of various types, may account for variation in their genome size and adaptation of phytoplasmas to the diverse environments of their plant and insect hosts. The availability of five complete phytoplasma genome sequences has made it possible to identify a considerable number of genes that are likely to play major roles in phytoplasma–host interactions. Among these, there are genes encoding surface membrane proteins and effector proteins. Also, it has been shown that phytoplasmas dramatically alter their gene expression upon switching between plant and insect hosts.

Rice yellow dwarf phytoplasma (16Sr XI-B subgroup) infecting Jasminum sambac in India

Leaf yellowing and witches’ broom symptoms were recorded on Jasminum sambac (L.) plants in August 2012 at Jaipur, Rajasthan, India. Total DNA was isolated from symptomatic jasmine plants and nested PCR was performed using primers pairs P1/P7 and 3Far/3Rev. Expected size amplicons of ~1.3 kb were obtained in nested PCR from symptomatic jasmine plants which were cloned and sequenced. BLASTn analysis of partial 16S rRNA gene sequence of jasmine leaf yellows and witches’ broom (Jas LY and WB) isolate revealed highest sequence identity (99%) with phytoplasma associated with sugarcane white leaf, sugarcane grassy shoot, coconut root wilt, sorghum grassy shoot and goose grass white leaf, all members of the rice yellow dwarf phytoplasma (16Sr XI) group. Phylogenetic analysis of the Jas LY and WB phytoplasma sequence revealed further its closest relationship with members of the 16Sr XI group. This confirms the association of 16Sr XI group phytoplasma with Jas LY and WB disease. Real and virtual RFLP analysis of 16S rRNA gene of jasmine phytoplasma further confirmed that it belonged to the 16Sr XI-B subgroup. This is the first report of 16SXI-B subgroup phytoplasma infection on Jasminum sambac in the world.

First Report of Two Distinct Phytoplasma Species, 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma asteris,' Simultaneously Associated with Yellow Decline of Wodyetia bifurcata (Foxtail Palm) in Malaysia.

The foxtail palm (Wodyetia bifurcata), an Australian native species, is an adaptable and fast-growing landscape tree. The foxtail palm is most commonly used in landscaping in Malaysia. Coconut yellow decline (CYD) is the major disease of coconut associated with 16SrXIV phytoplasma group in Malaysia (1). Symptoms consistent with CYD, such as severe chlorosis, stunting, general decline, and death were observed in foxtail palms from the state of Selangor in Malaysia, indicating putative phytoplasma infection. Symptomatic trees loses their green and vivid appearance as a decorative and landscape ornament. To determine the presence of phytoplasma, samples were collected from the fronds of 12 symptomatic and four asymptomatic palms in September 2012, and total DNA was extracted using the CTAB method (3). Phytoplasma DNA was detected in eight symptomatic palms using nested PCR with universal phytoplasma 16S rDNA primer pairs, P1/P7 followed by R16F2n/R16R2 (2). Amplicons (1.2 kb in length) were generated from symptomatic foxtail palms but not from symptomless plants. Phytoplasma 16S rDNAs were cloned using a TOPO TA cloning kit (Invitrogen). Several white colonies from rDNA PCR products amplified from one sample with R16F2n/R16R2 were sequenced. Phytoplasma 16S rDNA gene sequences from single symptomatic foxtail palms showed 99% homology with a phytoplasma that causes Bermuda grass white leaf (AF248961) and coconut yellow decline (EU636906), which are both members of the 16SrXIV 'Candidatus Phytoplasma cynodontis' group. The sequences also showed 99% sequence identity with the onion yellows phytoplasma, OY-M strain, (NR074811), from the 'Candidatus Phytoplasma asteris' 16SrI-B subgroup. Sequences were deposited in the NCBI GenBank database (Accession Nos. KC751560 and KC751561). Restriction fragment length polymorphism (RFLP) analysis was done on nested PCR products produced with the primer pair R16F2n/R16R2. Amplified products were digested separately with AluI, HhaI, RsaI, and EcoRI restriction enzymes based on manufacturer's specifications. RFLP analysis of 16S rRNA gene sequences from symptomatic plants revealed two distinct profiles belonging to groups 16SrXIV and 16SrI with majority of the 16SrXIV group. RFLP results independently corroborated the findings from DNA sequencing. Additional virtual patterns were obtained by iPhyclassifier software (4). Actual and virtual patterns yielded identical profiles, similar to the reference patterns for the 16SrXIV-A and 16SrI-B subgroups. Both the sequence and RFLP results indicated that symptoms in infected foxtail palms were associated with two distinct phytoplasma species in Malaysia. These phytoplasmas, which are members of two different taxonomic groups, were found in symptomatic palms. Our results revealed that popular evergreen foxtail palms are susceptible to and severely affected by phytoplasma. To our knowledge, this is the first report of a mixed infection of a single host, Wodyetia bifurcata, by two different phytoplasma species, Candidatus Phytoplasma cynodontis and Candidatus Phytoplasma asteris, in Malaysia.

Phylogenetic analysis identifies a ‘Candidatus Phytoplasma oryzae ’-related strain associated with yellow leaf disease of areca palm (Areca catechu L.) in India

Yellow leaf disease (YLD) with phytoplasmal aetiology is a serious disease of arecanut palm in India. The present study was undertaken to characterize the 16S rRNA and secA gene sequences of the Indian arecanut YLD phytoplasma for 'Candidatus Phytoplasma' species assignment and 16Sr group/subgroup classification. Phytoplasma 16S rRNA genes were amplified using three sets of semi-nested/nested primers, 1F7/7R3-1F7/7R2, 4Fwd/3Rev-4Fwd/5Rev and P1/P7-R16F2n/R16R2, producing amplicons of 491, 1150 and 1250 bp, respectively, from diseased samples. The amplicons were cloned and sequenced. A blast search showed that the sequences had 99 % similarity with sugar cane white leaf phytoplasma (16SrXI) and Napier grass stunt phytoplasma (16SrXI). Phylogenetic analysis based on the 16S rRNA gene revealed the clustering of YLD phytoplasma with the rice yellow dwarf and Bermuda grass white leaf groups. The YLD phytoplasma F2nR2 sequence shared 97.5 % identity with that of 'Candidatus Phytoplasma oryzae' and 97.8 % identity with that of 'Candidatus Phytoplasma cynodontis'. Hence, for finer differentiation, we examined the secA gene-based phylogeny, where the YLD phytoplasma clustered with Napier grass stunt and sugar cane grassy shoot phytoplasmas, both belonging to the rice yellow dwarf group. Hence, we are assigning the Indian arecanut YLD phytoplasma as a 'Candidatus Phytoplasma oryzae'-related strain. Virtual RFLP analysis of a 1.2 kb fragment of the 16S rRNA gene (F2nR2 region) identified the Indian arecanut YLD phytoplasma as a member of 16SrXI-B subgroup. We name the phytoplasma Indian yellow leaf disease phytoplasma, to differentiate it from the Hainan YLD phytoplasma, which belongs to group 16SrI.

'Candidatus Phytoplasma cynodontis' associated with white leaf disease of golden beard grass (Chrysopogon acicalatus)

A phytoplasma was detected in golden beard grass (Chrysopogon acicalatus) displaying white leaf symptoms near vegetative fields at the Tatkone region in Myanmar, 2011. Based on restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes, including the 16S-23S spacer region and part of the 23S rRNA gene, the phytoplasma was identified as a member of the Bermuda grass white leaf phytoplasma (BGWL) group. The golden beard grass white leaf phytoplasma (GBGWL) 16S rRNA gene sequence exhibited over 98.7% similarity with all members of BGWL group phytoplasmas and 99.5% similarity with Thailand Bermuda grass white leaf phytoplasma (AF248961). In addition, the GBGWL phytoplasma was confirmed to be a member of 'Candidatus Phytoplasma cynodontis' by phylogenetic analyses.

Hyparrhenia grass white leaf disease, associated with a 16SrXI phytoplasma, newly reported in Kenya

Hyparrhenia rufa is a thatching grass commonly found throughout the tropics where it also serves as valuable cattle fodder and border grass to prevent soil erosion (Skerman & Riveros, 1990). In June 2010, we observed some H. rufa …

Molecular Detection of Phytoplasma Associated with Yellow Leaf Disease in Areca Palms (Areca catechu) in India.

The arecanut palm (Areca catechu L.), Arecaceae family, is one of the most important commercial crops in the world, which yields fruits called arecanut that are used as a medicine and chewing substance (1). Yellow leaf disease (YLD) is one of the most serious diseases in areca palms in India. It reduces the yield as much as 50% over a period of 3 years immediately following disease incidence. Foliar yellowing, the most conspicuous symptom, begins from the inner whorl and spreads to the outer parts of the crown. Chlorosis is observed on almost all leaves in the whorl from edges of the leaflet to the midrib region. Stems become spongy and friable and the conducting strands are destroyed. Microscopic detection is evidence of the association of phytoplasma in YLD-affected areca palms (3). There is no evidence for molecular level detection of phytoplasma in YLD-affected palms. To prove the phytoplasma association in YLD-affected palms in India, samples (inflorescence, spindle leaf, mature leaf, and root) were collected from 15 (5 severe, 5 middle, and 5 early stage of the disease) YLD-affected areca palms and two symptomless palms at Sullia District, Karnataka. DNA was extracted from rachis of inflorescence, midrib of spindle leaf, and meristem of root samples as previously described (2). With universal primers there was no consistency in amplification. Then we used two sets of seminested primers, 1F7/7R3-1F7/7R2 and 4Fwd/3Rev-4Fwd/5Rev, which were designed to amplify the coconut root (wilt) disease (RWD) phytoplasma (2). With the seminested primers, 1F7/7R3-1F7/7R2, a 493-bp amplicon was obtained from 15 of 15 palms. With the seminested primers, 4Fwd/3Rev-4Fwd/5Rev, a 1.3-kb amplicon was seen in 11 samples and the positive control sample (sugarcane grassy shoot DNA). The amplicons were cloned and sequenced and two representative sequences were deposited in GenBank (GU552782 and HM215624). A BLAST search showed that the sequence has 99% nt identity with sugarcane white leaf phytoplasma (FM208260, 16sr XI), coconut RWD phytoplasma (GQ850122, 16sr XI), 98% nt identity with bermuda grass white leaf phytoplasma (AJ550986, 16sr XIV), and only 91% nt identity with YLD-affected areca phytoplasma reported from China (FJ998269 and FJ694685). The phylogenetic analysis revealed the clustering of YLD phytoplasma with 16s rRNA XI and 16s rRNA XIV groups. However, the YLD phytoplasma is closely related to the 16s rRNA XI group. PhytoDB-group identifier tool (http://220.227.88.253/phytodb) showed YLD phytoplasma from India belongs to the 16sr XI group. Earlier we reported the association of 16sr XI group phytoplasma with coconut RWD in India (2) and the YLD phytoplasma reported here has 99% nt identity with RWD phytoplasma. In southern India, coconut and arecanut are grown together in adjacent fields and there is a possible occurrence of the same phytoplasma in two different hosts. The current study proved the association of phytoplasma through nested PCR in YLD-affected areca palms in India and it is clustered with 16sr RNA XI group. Purushothama et al. (4) couldn't detect the phytoplasma with YLD-affected areca palms. To our knowledge, this is first report of the association of 16SrXI group phytoplasma with the arecanut YLD in India.