Candidatus Phytoplasma Oryzae Research Articles

Virulence effectors encoded in the rice yellow dwarf phytoplasma genome participate in pathogenesis.

Bacteria-like phytoplasmas alternate between plant and insect hosts, secreting proteins that disrupt host development. In this study, we sequenced the complete genome of 'Candidatus Phytoplasma oryzae' strain HN2022, associated with rice yellow dwarf (RYD) disease, using PacBio HiFi technology. The strain was classified within the 16Sr XI-B subgroup. Through SignalP v5.0 for prediction and subsequent expression analysis of secreted proteins in Nicotiana benthamiana and rice (Oryza sativa L.), we identified the key virulence effector proteins RY348 and RY378. RY348, a homologue of Secreted Aster Yellows Phytoplasma Effector 54 (SAP54), targets and degrades the MADS-box transcription factors MADS1 and MADS15, causing pollen sterility. Meanwhile, RY378 impacts the strigolactone and auxin signaling pathways, substantially increasing tillering. These findings offer insights into the interactions between plants and phytoplasmas.

First report of “Candidatus Phytoplasma oryzae” associated with lemongrass in India

First report of “Candidatus Phytoplasma oryzae” associated with lemongrass in India

Characterization and genetic diversity of phytoplasmas associated with elephant foot yam (Amorphophallus paeoniifolius) in India.

During the growing season of 2015 and 2016, leaf yellowing, stunting, and declining symptoms were observed on elephant foot yam in three states of India.The 1.3kb 16S rDNA fragments were amplified from genomic DNA extracted from all the symptomatic elephant foot yam samples in nested PCR assays using primer pairs, P1/P7 followed by 3F/3R. Pair wise sequence comparison, virtual RFLP and phylogenetic analysis of 16S rRNA gene sequences confirmed association of 'Candidatus Phytoplasma trifolii' (16SrVI-D) and 'Candidatus Phytoplasma oryzae' (16SrXI-B) related strains in Uttar Pradesh, Uttarakhand and Tripura states, respectively. To our knowledge, this is the first report of association of 'Ca. P. trifolii' and 'Ca. P. oryzae' related strains in elephant foot yam in world. In the present study, we also reported Datura stramonium showing witches' broom as a natural weed host for 'Ca. P. trifolii' phytoplasma in Gorakhpur and Kushinagar districts of Uttar Pradesh state, India.

A new phytoplasma strain associated with the sugarcane white leafdisease in Vietnam

Sugarcane white leaf (SCWL) disease is one of the most important diseases of sugarcane in Vietnam. The presence of phytoplasmas in phloem tubes of infected plants was confirmed by DAPI staining. A nested PCR assay using R16mF2/R16mR1 and R16F2n/R16R2 primer pairs amplified products with approximate length of 1,250 bp from the symptomatic sugarcane samples. The sequence analysis indicated that the phytoplasma strains from Khanh Hoa province shared 100% sequence identity with previously sequenced SCWL phytoplasma strains that were related to ‘Candidatus Phytoplasma oryzae’ (16SrXI). The virtual RFLP patterns of a 1,127 bp sequence obtained with the restriction enzymes BfaI, BstUI, EcoRI, HaeIII, HinfI, HpaI, RsaI and TaqI indicate that this SCWL-associated phytoplasma from Vietnam is a different strain from those in the 16SrXI described ribosomal subgroups. The two insect species, Matsumuratettix hiroglyphicus and Yamatotettix flavovittatus, identified as the vectors of SCWL in Thailand, were identified and found to be infected also in the SCWL symptomatic fields in Vietnam. The transmission experiments from SCWL-infected leafhoppers to healthy sugarcane and to insect feeding medium failed, suggesting that the infected planting material is the major transmission manner of SCWL disease in Vietnam.

FIRST REPORT OF A 16SRXI GROUP PHYTOPLASMA ('CANDIDATUS PHYTOPLASMA ORYZAE') ASSOCIATED WITH CYPERUS spp. WHITE LEAF DISEASE IN IRAN

In 2014, Cyperus spp. (Cyperaceae) white leaf (CWL) disease was observed in lime orchards in Minab (Hormozgan province, Iran). Main disease symptoms were whitening of aerial plant parts and stunting. To investigate the phytoplasma presence, total DNAs extracted from five symptomatic and five symptomless Cyperus spp. plants were tested by nested PCR using primers P1/P7 followed by R16F2n/R16R2 (Lee et al., 1998). Amplicons of ca. 1.8 and 1.2 kb, were obtained from samples of symptomatic but not symptomless plants. All amplicons were cloned and sequenced. The obtained 16S rDNA sequences showed 100% identity with each other and a representative of the same sequences deposited in GenBank (accession No. MF136620). BLAST search using the full length 16S rRNA gene sequence revealed that the CWL sequence showed 99% identity with sugarcane white leaf and sugarcane grassy shoot phytoplasmas (AB646271 and KF908792, respectively), representatives of 16SrXI phytoplasma group. Computer-simulated restriction analysis using iPhyClassifier, showed that the RFLP profile of CWL phytoplasma was very similar to the reference pattern of the 16SrXI-B subgroup (X76432). Phylogenetic analysis using Mega software version 7 confirmed that the CWL phytoplasma clustered with16SrXI group phytoplasmas closest to 16SrXI-B subgroup. To our knowledge this is the first report of a 16SrXI group phytoplasma in Iran. Rice and sugarcane plants are two economically important hosts of 16SrXI group-related strains (Arocha and Jones, 2010), thus the CWL-associated phytoplasma could act as a source of inoculum for infecting rice and sugarcane crops in Iran.

Development of field-applicable tests for rapid and sensitive detection of Candidatus Phytoplasma oryzae

Napier grass Stunt Disease (NSD) is a severe disease of Napier grass (Pennisetum purpureum) in Eastern Africa, caused by the leafhopper-transmitted bacterium Candidatus Phytoplasma oryzae. The pathogen severely impairs the growth of Napier grass, the major fodder for dairy cattle in Eastern Africa. NSD is associated with biomass losses of up to 70% of infected plants.Diagnosis of NSD is done by nested PCR targeting the phytoplasma DNA, which is difficult to perform in developing countries with little infrastructure. We report the development of an easy to use, rapid, sensitive and specific molecular assay for field diagnosis of NSD. The procedure is based on recombinase polymerase amplification and targets the imp gene encoding a pathogen-specific immunodominant membrane protein. Therefore we followed a two-step process. First we developed an isothermal DNA amplification method for real time fluorescence application and then transferred this assay to a lateral flow format. The limit of detection for both procedures was estimated to be 10 organisms. We simplified the template preparation procedure by using freshly squeezed phloem sap from Napier grass. Additionally, we developed a laboratory serological assay with the potential to be converted to a lateral flow assay. Two murine monoclonal antibodies with high affinity and specificity to the immunodominant membrane protein IMP of Candidatus Phytoplasma oryzae were generated. Both antibodies specifically reacted with the denatured or native 17 kDa IMP protein. In dot blot experiments of extracts from infected plant, phytoplasmas were detected in as little as 12,5 μg of fresh plant material.

New Efficient Natural Leafhopper Vectors of Sugarcane Grassy Shoot Phytoplasma in India

Sugarcane grassy shoot (SCGS) disease is associated with the presence of 16SrXI group phytoplasmas that are transmitted by leafhoppers; limited studies have been performed in India toward its natural transmission. To determine the insect vectors that transmit the disease in nature, leafhopper species from SCGS-infected fields at Shahjahanpur, Central Uttar Pradesh, India, were collected and analyzed for phytoplasma presence using nested polymerase chain reaction with phytoplasma-specific primers. An ~1.2-kb amplified DNA fragment was detected in nested PCR from the three major leafhopper species, viz. Maiestas portica (Melichar), Exitianus indicus (Ross) and Cofana unimaculata (Signoret), and the symptomatic sugarcane leaves of variety CoS 07250. BLASTn analysis of ~1.2-kb 16S rDNA partial sequences obtained from symptomatic sugarcane plants and these leafhoppers revealed 99–100 % sequence identities among themselves and 99 % identity with other reported strains of ‘Candidatus Phytoplasma oryzae’ (16SrXI group). Phylogenetic analysis of 16S rRNA sequences of SCGS, M. portica, C. unimaculata and E. indicus phytoplasma strains also indicated the closest phylogenetic relationship with those of ‘Ca. P. oryzae’ group. Transmission tests and population sampling study further confirmed that M. portica and C. unimaculata were vectors of the SCGS phytoplasma from diseased to healthy sugarcane plants. The identification of new vectors of SCGS phytoplasma suggested that these leafhopper species may be responsible for secondary spread of SCGS phytoplasma.

Draft Genome Sequence of "Candidatus Phytoplasma oryzae" Strain Mbita1, the Causative Agent of Napier Grass Stunt Disease in Kenya.

Phytoplasmas are bacterial plant pathogens with devastating impact on agricultural production worldwide. In eastern Africa, Napier grass stunt disease causes serious economic losses in the smallholder dairy industry. This draft genome sequence of “Candidatus Phytoplasma oryzae” strain Mbita1 provides insight into its genomic organization and the molecular basis of pathogenicity.

'Candidatus Phytoplasma cirsii', a novel taxon from creeping thistle [Cirsium arvense (L.) Scop

Creeping thistle [Cirsium arvense (L.) Scop.] and dahlia (Dahlia sp.) plants showing typical symptoms of phytoplasma infection including yellowing, stunting, inflorescence and proliferation, were sampled; the presence of phytoplasma was confirmed by standard PCR using universal primers. RFLP analysis allowed classification of the detected phytoplasma strains CirYS, CirYS1 and DahlP within the 16SrXI group, the unique restriction profile F2nR2 fragment obtained in silico by iPhyClassifier indicated that they belong to the new 16SrXI-E subgroup. Genetic analysis of the 16S rRNA gene revealed that the studied strains shared less than 97.5% similarity with all of the previously described 'Candidatus Phytoplasma' species. The closest relatives are 'Candidatus Phytoplasma cynodontis' and 'Candidatus Phytoplasma oryzae' with 96.8% and 96.6% similarity. All strains studied bear three specific regions in the 16S rRNA gene, discriminating them from the other phytoplasma species. Phylogenetic analysis of the 16S rRNA and secA genes confirmed this specificity, as the creeping thistle and dahlia phytoplasma strains clustered in a distinguishable lineage group. The uniqueness of the genetic analysis agrees with the biological characterization of the studied phytoplasma strains, their host range, and geographical distribution. The strains only infect dicotyledonous plants in Europe, contrary to their closest relatives. Based on their unique properties, it could be concluded that the studied phytoplasma strains represent a discrete group that is proposed as a novel taxon 'Candidatus Phytoplasma cirsii', with strain CirYS as a reference strain.

English

Napier grass (Pennisetum purpureum Schumach), the most preferred fodder species for dairy production in East and Central Africa, is under threat from stunt disease caused by Candidatus Phytoplasma oryzae (Ns-phytoplasma). The disease reduces forage yield by 40 to 90%. Two alternative fodder grasses, Guinea grass (Panicum maximum Jacq) and Guatemala grass (Tripsacum laxum Scrib and Merr) and a new stunt disease tolerant Napier cv Ouma 3 were studied to determine their biomass production potential when subjected to three intervals harvest (4, 8, 12 weeks) alongside three basal heights of defoliations (5, 10 and 15 cm). The study was conducted at KARI, Kakamega (high rainfall zone) and KARI, Alupe (low rainfall zone) in Kenya. A split-split plot design with 3×3×3 factorial treatment arrangements was used. A 4-weekly interval of harvesting alongside defoliation heights of 10 and 15 cm significantly increased biomass yield in Napier cv Ouma (38.47 and 33.90 T/ha/year respectively) compared to Guatemala grass and Panicum maximum which yielded 23.3 and 27.4 T/ha/year respectively. There is appositive correlation between yield and morphological parameters with canopy diameter, plant height and number of leaves highly correlating in biomass yield. Key words: Frequency of harvest, alternative species, defoliation height, biomass yield, fodder species.

Molecular characterization identifies 16SrXI-B group phytoplasma (‘Candidatus Phytoplasma oryzae’-related strain) associated with root wilt disease of coconut in India

The root wilt disease of coconut is a major threat to coconut cultivation in southern India. Here we report the species assignment and 16Sr sub group classification of phytoplasma associated with coconut root wilt disease. Leaf samples were collected from root wilt symptomatic palms in Kayankulam district of Kerala. The phytoplasma 16S rRNA gene was amplified using three sets of primers namely, 1F7/7R3-1F7/7R2, 3Fwd/3Rev-3Fwd/5Rev, and P1/P7-R16F2n/R16R2 producing amplicons of 490, 1300, and 1250bp respectively. Partial secA gene sequence of 480bp from root wilt disease phytoplasma was amplified using primers cocsf/cocsr. Sequence characterization and phylogenetic analysis of 16S rRNA and secA genes of root wilt disease phytoplasma grouped it with the rice yellow dwarf group phytoplasmas and identified coconut RWD phytoplasma as ‘Candidatus Phytoplasma oryzae’ related strain. Further, in silico restriction digestion study of phytoplasmal 16S rRNA gene region between primers R16F2n/R16R2 was performed for 16Sr group/sub group classification. The root wilt disease phytoplasma grouped with the 16SrXI group members. In the sub group classification it produced identical restriction profile as the sugarcane white leaf phytoplasma and arecanut yellow leaf disease phytoplasma, and hence, it was placed in the 16SrXI-B sub group.

Diversity of Bacterial Endosymbionts Associated with Macrosteles Leafhoppers Vectoring Phytopathogenic Phytoplasmas

Here, we investigate the endosymbiotic microbiota of the Macrosteles leafhoppers M. striifrons and M. sexnotatus, known as vectors of phytopathogenic phytoplasmas. PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA genes identified two obligate endosymbionts, "Candidatus Sulcia muelleri" and "Candidatus Nasuia deltocephalinicola," and five facultative endosymbionts, Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and a novel bacterium belonging to the Rickettsiaceae, from the leafhoppers. "Ca. Sulcia muelleri" and "Ca. Nasuia deltocephalinicola" exhibited 100% infection frequencies in the host species and populations and were separately harbored within different bacteriocytes that constituted a pair of coherent bacteriomes in the abdomen of the host insects, as in other deltocephaline leafhoppers. Wolbachia, Rickettsia, Burkholderia, Diplorickettsia, and the novel Rickettsiaceae bacterium exhibited infection frequencies at 7%, 31%, 12%, 0%, and 24% in M. striifrons and at 20%, 0%, 0%, 20%, and 0% in M. sexnotatus, respectively. Although undetected in the above analyses, phytoplasma infections were detected in 16% of M. striifrons and 60% of M. sexnotatus insects by nested PCR of 16S rRNA genes. Two genetically distinct phytoplasmas, namely, "Candidatus Phytoplasma asteris," associated with aster yellows and related plant diseases, and "Candidatus Phytoplasma oryzae," associated with rice yellow dwarf disease, were identified from the leafhoppers. These results highlight strikingly complex endosymbiotic microbiota of the Macrosteles leafhoppers and suggest ecological interactions between the obligate endosymbionts, the facultative endosymbionts, and the phytopathogenic phytoplasmas within the same host insects, which may affect vector competence of the leafhoppers.

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.

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.

First report of a 16SrXI group phytoplasma (Candidatus Phytoplasma oryzae) associated with Napier grass stunt disease in Uganda

Napier grass (Pennisetum purpureum), also known as elephant grass, is an important fodder crop in Uganda. The forage has been increasingly used in intensive (zero grazing) and semi-intensive dairy cattle production systems, in order to meet the increasing demand for milk in Uganda. Napier grass stunt disease, causing short internodes, bushy appearance, yellow to purple streaking and a low biomass yield, has spread among smallholder dairy farmers in Uganda since it was first observed in 2001 (Alacai et al., 2004). The disease has also been reported from western Kenya and shown to be associated with a phytoplasma (Jones et al., 2004) and in Tanzania (B. Pallangyo, personal communication). Leaf samples of stunted Napier grass were collected in the three major Napier grass-growing areas of Uganda. Air-dried leaf samples were ground in liquid nitrogen and total DNA was extracted using a DNeasy kit according to manufacturer's instructions (Qiagen). Leaves without symptoms were used as negative control. The presence of phytoplasma in plants with symptoms was confirmed by a real-time PCR method according to Christensen et al. (2004). Seven of these positive samples were sequenced in the 16S rRNA gene (deposited in GenBank Acc. No. EF012650) and the 16S/23S intergenic spacer (Acc. No. EF012649). No sequence variation was found. The sequences were 99·3% identical to the 16S rRNA sequences from the phytoplasma associated with Napier grass stunt from Kenya, which belongs to the 16SrXI rice yellow dwarf group (Candidatus Phytoplasma oryzae; Jones et al., 2004). This is the first report of a 16SrXI Group phytoplasma associated with Napier grass stunt disease in Uganda.

"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'.