Detection of a 16SrXII phytoplasma associated with sugarcane yellow leaf syndrome in India

Phytoplasma strain was detected in leaves of sugarcane in India exhibiting symptoms of yellowing of midribs. A phytoplasma characteristic 1.2 kb rDNA PCR product was amplified from DNAs of all diseased samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs P1/P7 and f5U/r3U. Restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA indicated that diseased sugarcane was infected by phytoplasma. The 16S rDNA sequence of the Indian sugarcane yellow leaf phytoplasma (SCYLP) showed the closest identity (99%) to that of SCYLP in Cuba identified as Macroptilium lathyroides (AY725233), which belongs to 16SrXII (Stolbur group). This is the first record of the detection of SCYLP, and identification of the 16SrXII group of phytoplasma associated with yellow leaf syndrome (YLS) in India.     

Association of a Phytoplasma with Pistachio Witches’ Broom Disease in Iran

Pistachio is an important crop in Iran, which is a major producer and exporter of pistachio nuts. The occurrence of a new disease of pistachio trees, characterized by the development of severe witches’ broom, stunted growth and leaf rosetting, was observed in Ghazvin Province. A phytoplasma was detected in infected trees by polymerase chain reaction (PCR) amplification of rRNA operon sequences. Nested PCR with primer pairs P1/P7 and R16F2n/R16R2 was used for specific detection of the phytoplasma in infected trees. To determine its taxonomy, the random fragment length polymorphism (RFLP) pattern and sequence analysis of the amplified rRNA gene were studied. Sequencing of the amplified products of the phytoplasma 16S rRNA gene indicated that pistachio witches’ broom (PWB) phytoplasma is in a separate 16S rRNA group of phytoplasmas (with sequence homology 97% in Blast search). The unique properties of the DNA of the PWB phytoplasma indicate that it is a representative of a new taxon.     

Molecular Identification and Phylogenetic Analysis of Sugarcane Yellow Leaf Phytoplasma (SCYLP) in Egypt

Samples of sugarcane leaves were collected from different commercial fields and breeding stations in Egypt. Aetiology of sugarcane phytoplasma disease was investigated using nested PCR. Phytoplasma‐specific primers (P1/P7 and R16F2n/R16R2) were used to amplify a fragment of the 16S rRNA gene. Sequencing and restriction fragment length polymorphism analyses revealed that the tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group. Phylogenetic analyses of 60 screened accessions of 16S ribosomal RNA gene sequences of Candidatus phytoplasmas comprising those collected from Egypt (this study) and those extracted from GenBank showed that they split into two distinct clusters. All the phytoplasmas form a stable phylogenetic subcluster, as judged by branch length and bootstrap values of 100% in the 16S group cluster. Results of phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. Conversely, based on the analysis of the 16S‐23S region, examined isolates segregated into four different clusters suggesting a notable heterogeneity between them. These results are the first record of the presence of phytoplasma in association with sugarcane yellow leaf in Egypt.     

Transmission of sugarcane white leaf phytoplasma by Yamatotettix flavovittatus, a new leafhopper vector

Sugarcane white leaf disease is caused by plant pathogenic phytoplasmas that are transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). To determine whether there are other insect vectors that transmit this disease pathogen, leafhopper species in sugarcane, Saccharum officinarum L., fields in northeastern Thailand were monitored by using light traps. Sixty-nine leafhopper species from family Cicadellidae were found. Using nested polymerase chain reaction (PCR) with specific primers, a 210-bp amplified DNA fragment corresponding to phytoplasma associated with sugarcane white leaf disease was detected from 12 species of leafhoppers [Balclutha rubrostriata (Melichar), Balclutha sp., Bhatia olivacea (Melichar), Exitianus indicus Distant, Macrosteles striifrons Anufriew, Matsumuratettix hiroglyphicus (Matsumura), Recilia distincta (Motschulsky), Recilia dorsalis (Motschulsky), Recilia sp., Thaia oryzivora Ghauri, Yamatotettix flavovittatus Matsumura, and Xestocephalus sp.]. The percentage of individual infection with phytoplasma varied from 5% in B. olivacea to 35% in Xestocephalus sp. The most abundant leafhopper species, i.e., E. indicus, Y. flavovittatus, and M. hiroglyphicus were used in transmission tests to determine their vector status for the sugarcane white leaf phytoplasma transmission. Infected insects were reared on healthy plants and specific PCR followed by sequencing of the amplicons was used to determine whether the phytoplasma was transmitted to the plants. The results showed that both Y. flavovittatus and M. hiroglyphicus, but not E. indicus, can transmit sugarcane white leaf phytoplasma to healthy sugarcane plants. The transmission efficiency of M. hiroglyphicus (55%) was higher than that of Y. flavovittatus (45%). We conclude that Y. flavovittatus is a newly discovered vector for sugarcane white leaf disease, in addition to M. hiroglyphicus. These two species peak at different times of the year and therefore complement each other in the transmission of the phytoplasma. Because there are no known alternative host plants for the sugarcane white leaf, management of the disease will necessarily require the control of both Y. flavovittatus and M. hiroglyphicus.     

Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.     

Artemisia vulgaris,a new host of 16SrV-C phytoplasma related strains infecting black alder in Poland

Yellowing of leaf tissue and strongly deformed shoots were observed in common mugwort (Artemisia vulgaris L.) growing in a nature reserve in Southern Poland. Similar foliage chlorosis together with abnormal shoot proliferation was noticed on alder tree (Alnus glutinosa Gaertn.) growing next to the common mugwort. DNA specific fragments coding 16S rRNA and ribosomal proteins (rp) were amplified from mugwort and alder samples using direct and nested PCR (Polymerase Chain Reaction) assays. Phylogenetic relationships inferred from 16S and rps3 genes indicated that strains infecting mugwort and alder were most closely related to phytoplasmas of subgroups 16SrV-C and 16SrV-D. Based on the restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA, the investigated phytoplasma strains were classified to subgroup 16SrV-C. Two sequence variants of the rps3 gene which differed by a single nucleotide were detected in all analysed samples by pairwise analysis of the aligned reads. Taking into account that this single-nucleotide polymorphism (SNP) occurs among 16SrV-C and 16SrV-D related phytoplasmas and that the phytoplasmas have a single copy of rp operon, we concluded that each plant species was infected by two distinct, closely related phytoplasma strains. To the best of our knowledge, this is the first report of group 16SrV-C related phytoplasmas infecting common mugwort worldwide, adding a new host species that is possibly linked to the spread of the alder pathogen in Eastern Europe. Although alder yellows phytoplasma has been frequently found in Europe, this is the first detection of phytoplasmas associated with alder in Poland.     

Cloning and Sequencing of Phytoplasma Ribosomal DNA (rONA) Associated with Kerala Wilt Disease of Coconut Palms

Using the polymerase chain reaction (PCR) the 165 rRNA gene of phytoplasma associated with Kerala wilt disease of coconut palm (Cocos nucifera L) was amplified from infected leaf samples. Within the three universal primer pairs P1/P6, P1/P7and P41 P7, the primer pair P4/P7 only showed an amplification of 650 bp DNA fragment. 5ince P4/P7 amplifies the 16S-23S intergenic spacer region of 165 rRNA gene, the PCR product 650 bp of Kerala wilt disease palm indicates the phytoplasma DNA. The amplified fragment was sequenced and deposited in Genbank data library (Accession No. AY158660). The absence of restriction sites for Bcll and Rsa/l in 650 bp indicates phytoplasmic nature of DNA and its strain difference. A comparison of the 650 bp sequence with other phytoplasmas and its restriction profile indicates Kerala wilt disease phytoplasma as a separate 165 rRNA group in the classification of phytoplasmas. To our knowledge, this report records the first finding of the phytoplasma DNA using universal primers and its sequence analysis in coconut palms of Kerala, south India.     

Association of a phytoplasma with a yellow leaf syndrome of sugarcane in Africa

Evidence is presented for the association of a phytoplasma, provisionally named sugarcane yellows phytoplasma (ScYP), in sugarcane affected by a yellow leaf syndrome. The phytoplasma was consistently detected in leaves of more than 40 varieties from eight African countries. It was present in all symptomatic as well as some asymptomatic field grown cane samples but not in plants grown from true seed, and it was also observed in phloem sieve tubes by transmission electron microscopy. Phytoplasma 16S rDNA was confirmed by PCR, and restriction fragment analysis using Rsal and Haelll confirmed that PCR-amplified products were of phytoplasma rather than of plant or of other pathogen origin. Sequences obtained from the intergenic spacer region, between the 16S and 23S rDNA genes, confirmed the identity of the phytoplasma as belonging to the western X group of phytoplasmas.     

狗牙根白化病植原体16S rDNA序列

[目的]狗牙根(草坪草的一种)白化病是狗牙根的一种重要病害,在全世界均有分布.为了确定中国大陆狗牙根白化病病原,进一步与世界其他地区的病原相比有无特异性.[方法]采用植原体16S rDNA PCR扩增、序列分析、Southern分子杂交等技术对狗牙根健康植株及白化病患病植株进行研究.[结果]仅白化病植株扩增获得1.3 kb的特异片段,序列分析表明特异片段与CandidatusPhytoplasm Cynodontis有99%同源性.Southern杂交有特异条带,大小在预期范围之内.证实了在中国大陆发生的狗牙根健株型白化病病原含有植原体.[结论]大陆狗牙根白化病病原中含有植原体,为该病病原的进一步鉴定与防治奠定了基础.     

Detection and Identification of a 16SrII Group Phytoplasma Causing Clover Little Leaf Disease in Iran

White clover plants showing little leaf and leaf reddening symptoms were observed in Isfahan Province in central Iran. Restriction fragment length polymorphism analyses of nested PCR‐amplified fragments from Iranian clover little leaf phytoplasma isolates and representative phytoplasmas from other phytoplasma groups using AluI, CfoI, KpnI and RsaI restriction enzymes indicated that the clover phytoplasma isolates are related to the peanut WB group. Sequence analyses of partial 16S rRNA fragments showed that Iranian clover little leaf phytoplasma has 99% similarity with soybean witches'‐broom phytoplasma, a member of the peanut WB (16SrII) phytoplasma group. This is the first report of clover infection with a phytoplasma related to the 16SrII group.     

16S rRNA interoperon sequence heterogeneity distinguishes strain populations of palm lethal yellowing phytoplasma in the Caribbean region

DNA of phytoplasmas in lethal yellowing (LY)‐diseased palms was detected by a nested polymerase chain reaction (PCR) assay employing rRNA primer pair P1/P7 followed by primer pair LY16Sf/ LY16‐23Sr. Polymorphisms revealed by Hinfl endonuclease digestion of rDNA products differentiated coconut‐infecting phytoplasmas in Jamaica from those detected in palms in Florida, Honduras and Mexico. A three fragment profile was generated for rDNA from phytoplasmas infecting all 21 Jamaican palms whereas a five fragment profile was evident for phytoplasmas infecting the majority of Florida (20 of 21), Honduran (13 of 14) and Mexican (5 of 5) palms. The RFLP profile indicative of Florida LY phytoplasma was resolved by cloning into two patterns, one of three bands and the other of four bands, that together constituted the five fragment profile. The two patterns were attributed to presence of two sequence heterogeneous rRNA operons, rrnA and rrnB, in most phytoplasmas composing Florida, Honduran and Mexican LY strain populations. Unique three and four fragment RFLP profiles indicative of LY phytoplasmas infecting Howea forsteriana and coconut palm in Florida and Honduras, respectively, were also observed. By comparison, the Jamaican LY phytoplasma population uniformly contained one or possibly two identical rRNA operons. No correlation between rRNA interoperon heterogeneity and strain variation in virulence of the LY agent was evident from this study.     

Surveys reveal a complex association of phytoplasmas and viruses with the blueberry stunt disease on Canadian blueberry farms

Surveys for phytoplasmas and viruses were conducted during September 2014 and 2015 on highbush blueberry farms in the Région Montérégie, Quebec. Total DNA and RNA were extracted from blueberry bushes showing blueberry stunt (BBS) symptoms and from symptomless blueberry bushes, and utilised as templates for PCR and RT‐PCR assays, respectively. Phytoplasma DNA was amplified with universal phytoplasma primers that target the 16S rRNA, secA and secY genes from 12 out of 40 (30%) plants tested. Based on 16S rRNA, secA and secY gene sequence identity, phylogenetic clustering, actual and in silico RFLP analyses, phytoplasma strains associated with BBS disease in Quebec were identified as ‘Candidatus Phytoplasma asteris’‐related strains, closely related to the BBS Michigan phytoplasma strain (16SrI‐E). The secY gene sequence‐based single nucleotide polymorphism analysis revealed that one of the BBS phytoplasma strains associated with a leaf marginal yellowing is a secY‐I RFLP variant of the subgroup 16SrI‐E. Two viruses were detected in blueberry bushes. The Blueberry Red Ringspot Virus (BRRV) was found in a single infection in the cultivar Bluecrop with no apparent typical BRRV symptoms. The Tobacco Ringspot Virus (TRSV) was found singly infecting blueberry plants and co‐infecting a BBS phytoplasma‐infected blueberry cv. Bluecrop plant. This is the first report of TRSV in the cv. Bluecrop in Quebec. The Quebec BBS phytoplasma strain was identified in the leafhopper Graphocephala fennahi, which suggests that G. fennahi may be a potential vector for the BBS phytoplasma. The BBS disease shows a complex aetiology and epidemiology; therefore, prompt actions must be developed to support focused BBS integrated management strategies.     

Detection and Identification of an Elm Yellows Group Phytoplasma Associated with Camellia in China

In 2012, yellowing of camellias was observed in Tai'an in Shandong province, China. Transmission electron microscopy (TEM) revealed phytoplasma in the phloem sieve tube elements of symptomatic plants. A specific fragment of phytoplasma 16S rRNA gene was amplified by polymerase chain reaction (PCR) using the universal phytoplasma primers P1/P7 followed by R16F2n/R16R2. Sequence and restriction fragment length polymorphism (RFLP) analyses allowed us to classify the detected phytoplasma into the elm yellows (EY) group (16SrV), subgroup 16SrV‐B. Sequence analyses of the ribosomal protein (rp) gene confirmed a close relationship with phytoplasmas belonging to the rpV‐C subgroup. Thus, the phytoplasma associated with yellows disease in camellia, designated as ‘CY’, is a member of the 16SrV‐B subgroup. This is the first report of phytoplasma associated with camellia.     

Detection of Putative Recombination Events in the 16S Ribosomal RNA Gene of Some Members of Candidatus phytoplasma

Recombination plays a major evolutionary role by creating genetic diversity and provides the potential to find rapid adaptation to new environmental conditions. We sought the occurrence of possible recombination events in the 16S ribosomal RNA gene of 60 accessions belonging to the group 16SrI of Candidatus phytoplasma (aster yellows phytoplasma). Three bioinformatic programs were used (TOPALI v2.5, RECCO and RDP package). All the three programs indicated the presence of putative recombination signals in aligned sequences. Recombination events located in the 16S ribosomal RNA gene revealed the presence of four recombining accessions gathering sugarcane grassy shoot phytoplasmas (JF928001, DQ459439, EF614269 and JN223446).     

Effect of Antibiotics on the Symptoms of Stunting Disease of Magnolia liliiflora Plants

Treatment of diseased magnolia plants with Oxytetracycline, Baytril or Tylan did not reduce the number of symptomatic plants, but promoted shoot growth, development of symptomless leaves and flower buds. The most efficient were 500 ppm Baytril, 200 ppm Tylan and 500 or 1000 ppm Oxytetracycline. Lower concentrations of Baytril and Oxytetracycline were less effective and higher concentrations of Tylan decreased the growth of magnolia shoots. All the tested antibiotic treated and untreated magnolias were shown by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) to contain the AY (16SrI) phytoplasma and two also to contain a phytoplasma related to apple proliferation phytoplasma group (16SrX). The results indicate that Magnolia is a natural host of phytoplasmas belonging to the aster yellows and apple proliferation phytoplasma groups, and support the suggestion that phytoplasmas are the cause of magnolia stunting disease.     

The phytoplasma associated with purple woodnettle witches'‐broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group

In October 2013, a new disease affecting purple woodnettle, Oreocnide pedunculata, plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches'‐broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of polymerase chain reaction with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettles. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, rplV‐rpsC region revealed that purple woodnettle witches'‐broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rpI group and was designated as 16SrI‐AH and rpI‐Q, respectively, herein. RFLP analysis based on tuf gene region revealed that the PWWB belongs to tufI‐B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the tufI group. Taken together, our analyses based on 16S rRNA and rplV‐rpsC region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a Candidatus Phytoplasma asteris‐related strain in 16SrI‐AH, rpI‐Q and tufI‐B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas.     

Multigene analysis for differentiation of aster yellows phytoplasmas infecting carrots in Serbia

During a survey of large carrot fields in Serbia, plants showing leaf reddening and/or yellowing, adventitious shoot production and reduction in taproot size and quality were observed in a low percentage of plants. To verify phytoplasma association with the described symptoms and to carry out pathogen differentiation, PCR assays followed by restriction fragment length polymorphism (RFLP) analyses and/or sequencing of phytoplasma 16Sr DNA and ribosomal protein genes l22 and s3 , tuf , putative aa kinase plus ribosomal recycling factor genes and DNA helicase gene were carried out. Phytoplasmas belonging to 16SrI-A and 16SrI-B ribosomal subgroups and to rpI-A and rpI-B ribosomal protein subgroups, respectively, were identified by RFLP analyses in 13 of 15 symptomatic plants tested. No amplification was obtained with non-symptomatic carrot samples. The identification was confirmed by sequence analyses of the phytoplasma genes studied. In two carrot samples, presence of interoperon sequence heterogeneity was detected and phytoplasma strains were identified as belonging to 16SrI group but were not assigned to any 16S rRNA or ribosomal protein subgroup. This research allowed the first molecular identification of phytoplasmas infecting carrot in Serbia using several molecular markers, and it indicates that under field conditions in non-epidemic outbreaks a certain amount of genetic mutation may occur in conserved genes of these prokaryotes.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Little Leaf Disease of Brinjal (Solanum melongena L.) and Periwinkle (Catharanthus roseus) in Bangladesh

In Bangladesh little leaf disease was observed in brinjal ( Solanum melongena L.) and in periwinkle ( Catharanthus roseus ). Phloem-inhabiting phytoplasmas were consistently detected in both species of diseased plants using transmission electron microscopy (TEM) and polymerase chain reaction (PCR) techniques. The shape, size and within-tissue distribution of phytoplasmas appears to be similar in both hosts. Furthermore, the molecular characterization and identifications of observed phytoplasmas were carried out based on restriction fragment length polymorphism (RFLP) patterns of PCR-amplified products (1200 bp) using phytoplasma-specific universal primers and sequencing analysis of both 16S ribosomal DNA (rDNA) and intergenic spacer region (ISR) of 16S-23S rDNA phytoplasma genes. The patterns of RFLP analysis with seven restriction enzymes exhibited a similar pattern for both phytoplasma strains. The sequence homology between these two strains showed 100% similarity based on 16S rDNA and 16S-23S ISR. Therefore, in Bangladesh the causal agents of brinjal little leaf (BLL-Bd) and periwinkle little leaf (PLL-Bd) are probably the same or closely related phytoplasma strains. These strains, are very close or identical to the strain of brinjal little leaf phytoplasma in India (BLL-In), belonging to the clover proliferation group (Lee et al., Int. J. Syst. Bacteriol. 48, 1153–1169, 1998; Seemuller et al., J. Plant Pathol. 80, 3–26, 1998).     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Elimination of sugarcane grassy shoot disease through apical meristem culture

Elimination of sugarcane grassy shoot disease (SGSD) through apical meristem culture technique for producing clean planting material of sugarcane has been attempted in the present study. The results showed that meristems length of 2 and 3 mm were free from the SGSD pathogen at higher frequency than larger meristem length of 4 mm. However, the frequency of survival of explants during initiation of shoot cultures was higher in larger meristems (60%) in comparison to smaller ones (40%). The micropropagated plantlets raised from meristem culture were confirmed for disease-free by nested polymerase chain reaction (PCR) analysis at monthly interval up to 6 months. This is the first report on the elimination of SGSD phytoplasma through meristem culture in India.