A multiplex nested PCR assay for simultaneous detection of Corchorus golden mosaic virus and a phytoplasma in white jute (Corchorus capsularis L.)

A multiplex nested PCR assay was developed by optimizing reaction components and reaction cycling parameters for simultaneous detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma (Group 16Sr V‐C) causing little leaf and bunchy top in white jute (Corchorus capsularis). Three sets of specific primers viz. a CoGMV specific (DNA‐A region) primer, a 16S rDNA universal primer pair P1/P7 and nested primer pair R16F2n/R2 for phytoplasmas were used. The concentrations of the PCR components such as primers, MgCl₂, Taq DNA polymerase, dNTPs and PCR conditions including annealing temperature and amplification cycles were examined and optimized. Expected fragments of 1 kb (CoGMV), 674 bp (phytoplasma) and 370 bp (nested R16F2n/R2) were successfully amplified by this multiplex nested PCR system ensuring simultaneous, sensitive and specific detection of the phytoplasma and the virus. The multiplex nested PCR provides a sensitive, rapid and low‐cost method for simultaneous detection of jute little leaf phytoplasma and CoGMV. Based on BLASTn analyses, the phytoplasma was found to belong to the Group 16Sr V‐C.

Significance and Impact of the Study

Incidence of phytoplasma diseases is increasing worldwide and particularly in the tropical and subtropical world. Co‐infection of phytoplasma and virus(s) is also common. Therefore, use of single primer PCR in detecting these pathogens would require more time and effort, whereas multiplex PCR involving several pairs of primers saves time and reduces cost. In this study, we have developed a multiplex nested PCR assay that provides more sensitive and specific detection of Corchorus golden mosaic virus (CoGMV) and a phytoplasma in white jute simultaneously. It is the first report of simultaneous detection of CoGMV and a phytoplasma in Corchorus capsularis by multiplex nested PCR.     

Molecular biology and pathogenicity of phytoplasmas

Phytoplasmas 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.     

Classification of a new phytoplasmas subgroup 16SrII-W associated with <Emphasis Type="Italic">Crotalaria</Emphasis> witches’ broom diseases in Oman based on multigene sequence analysis

Background

Crotalaria aegyptiaca, a low shrub is commonly observed in the sandy soils of wadis desert and is found throughout all regions in Oman. A survey for phytoplasma diseases was conducted. During a survey in a wild area in the northern regions of Oman in 2015, typical symptoms of phytoplasma infection were observed on C. aegyptiaca plants. The infected plants showed an excessive proliferation of their shoots and small leaves.

Results

The presence of phytoplasma in the phloem tissue of symptomatic C. aegyptiaca leaf samples was confirmed by using Transmission Electron Microscopy (TEM). In addition the extracted DNA from symptomatic C. aegyptiaca leaf samples and Orosius sp. leafhoppers were tested by PCR using phytoplasma specific primers for the 16S rDNA, secA, tuf and imp, and SAP11 genes. The PCR amplifications from all samples yielded the expected products, but not from asymptomatic plant samples. Sequence similarity and phylogenetic tree analyses of four genes (16S rDNA, secA, tuf and imp) showed that Crotalaria witches’ broom phytoplasmas from Oman is placed with the clade of Peanut WB (16SrII) close to Fava bean phyllody (16SrII-C), Cotton phyllody and phytoplasmas (16SrII-F), and Candidatus Phytoplasma aurantifolia’ (16SrII-B). However, the Crotalaria’s phytoplasma was in a separate sub-clade from all the other phytoplasmas belonging to Peanut WB group. The combination of specific primers for the SAP11 gene of 16SrII-A, ?B, and -D subgroup pytoplasmas were tested against Crotalaria witches’ broom phytoplasmas and no PCR product was amplified, which suggests that the SAP11 of Crotalaria phytoplasma is different from the SAP11 of the other phytoplasmas.

Conclusion

We propose to assign the Crotalaria witches’ broom from Oman in a new lineage 16SrII-W subgroup depending on the sequences analysis of 16S rRNA, secA, imp, tuf, and SAP11 genes. To our knowledge, this is the first report of phytoplasmas of the 16SrII group infecting C. aegyptiaca worldwide.

     

Detection and Characterization of Phytoplasmas Infecting Ornamental and Weed Plants in Iran

During field surveys in 2004, ornamental and weed plants showing symptoms resembling those caused by phytoplasmas were observed in Mahallat (central Iran). These plants were examined for phytoplasma infections by polymerase chain reaction (PCR) assays using universal phytoplasma primers directed to ribosomal DNA (rDNA). All affected plants gave positive results. The detected phytoplasmas were characterized and differentiated through restriction fragment length polymorphism (RFLP) and sequence analysis of PCR‐amplified rDNA. The phytoplasmas detected in diseased Asclepias curassavica and Celosia argentea were identified as members of clover proliferation phytoplasma group (16SrVI group) whereas those from the remaining plants examined proved to be members of aster yellow phytoplasma group (16SrI group) (‘Candidatus Phytoplasma asteris’). In particular, following digestion with AluI, HaeIII and HhaI endonucleases, the phytoplasma detected in Limonium sinuatum showed restriction profiles identical to subgroup 16SrI‐C; phytoplasmas from Gomphocarpus physocarpus, Tanatacetum partenium, Lactuca serriola, Tagetes patula and Coreopsis lanceolata had the same restriction profiles as subgroup 16SrI‐B whereas Catharanthus roseus‐ and Rudbeckia hirta‐infecting phytoplasmas showed restriction patterns of subgroup 16SrI‐A. This is the first report on the occurrence of phytoplasma diseases of ornamental plants in Iran.     

Biological and molecular evidence for the transmission of aster yellows phytoplasma to French marigold (Tagetes patula) by the flatid planthopper Metcalfa pruinosa

Aster yellows (AY) phytoplasmas (Candidatus Phytoplasma asteris) are associated with a number of plant diseases throughout the world. Several insect vectors are responsible for spreading AY diseases resulting in wide distribution and low host specificity. Because the role of sucking insects as vectors of phytoplasmas is widely documented, and the citrus flatid planthopper Metcalfa pruinosa is a phloem feeder, it has been incriminated as a possible vector of phytoplasmas. However, its ability to transfer phytoplasma has not been confirmed. The present work shows that M. pruinosa (Hemiptera: Flatidae), a polyphagous planthopper, is able to vector Ca. P. asteris to French marigold (Tagetes patula). Transmission experiments were conducted in 2017 and 2018 in central Hungary by two approaches: (a) AY-infected M. pruinosa were collected from an area with severe incidence of the disease on T. patula and caged on test plants for an inoculation-access period of 2 weeks, and (b) presumably phytoplasma-free insects were collected from apparently healthy grapevines (Vitis vinifera L.) and fed on AY-infected T. patula plants for 2 weeks prior to being caged on test plants. AY disease symptoms developed on 4 out of 10 and 10 out of 15 test plants, respectively. All phytoplasma-positive marigold and M. pruinosa samples showed identical RFLP patterns and shared 100% 16S rDNA sequence identity with each other and with the aster yellows phytoplasma strain AJ33 (GenBank accession number MK992774). These results indicated that the phytoplasma belonged to the phytoplasma subgroup 16SrI-B Ca. P. asteris. Therefore, the work presented here provides experimental evidence that M. pruinosa is a vector of a 16SrI-B subgroup phytoplasma to T. patula.     

Screening potential insect vectors in a museum biorepository reveals undiscovered diversity of plant pathogens in natural areas

Phytoplasmas (Mollicutes, Acholeplasmataceae), vector‐borne obligate bacterial plant parasites, infect nearly 1,000 plant species and unknown numbers of insects, mainly leafhoppers (Hemiptera, Deltocephalinae), which play a key role in transmission and epidemiology. Although the plant–phytoplasma–insect association has been evolving for >300 million years, nearly all known phytoplasmas have been discovered as a result of the damage inflicted by phytoplasma diseases on crops. Few efforts have been made to study phytoplasmas occurring in noneconomically important plants in natural habitats. In this study, a subsample of leafhopper specimens preserved in a large museum biorepository was analyzed to unveil potential new associations. PCR screening for phytoplasmas performed on 227 phloem‐feeding leafhoppers collected worldwide from natural habitats revealed the presence of 6 different previously unknown phytoplasma strains. This indicates that museum collections of herbivorous insects represent a rich and largely untapped resource for discovery of new plant pathogens, that natural areas worldwide harbor a diverse but largely undiscovered diversity of phytoplasmas and potential insect vectors, and that independent epidemiological cycles occur in such habitats, posing a potential threat of disease spillover into agricultural systems. Larger‐scale future investigations will contribute to a better understanding of phytoplasma genetic diversity, insect host range, and insect‐borne phytoplasma transmission and provide an early warning for the emergence of new phytoplasma diseases across global agroecosystems.     

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.     

First Report of a 16SrI‐C Phytoplasma Infecting Celery (Apium graveolens) with Stunting,Bushy Top and Phyllody in the Czech Republic

Apium graveolens L. plants showing stunting, purplish/whitening of new leaves, flower abnormalities and bushy tops were observed in South Bohemia (Czech Republic) during 2011 and 2012. Transmission electron microscopy observations showed phytoplasmas in phloem sieve tube elements of symptomatic but not healthy plants. Polymerase chain reactions with universal and group‐specific phytoplasma primers followed by restriction fragment length polymorphism analyses and sequencing of 16S rDNA enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐C. Identical analyses of the ribosomal protein genes rpl22 and rps3 were used for further classification and revealed affiliation of the phytoplasmas with the rpIC subgroups. This is the first report of naturally occurring clover phyllody phytoplasma in A. graveolens in both the Czech Republic and worldwide.     

Transmission of “Candidatus Phytoplasma pruni”‐related strain associated with broccoli stunt by four species of leafhoppers

A disease known as broccoli stunt, associated with “Candidatus Phytoplasma pruni”‐related strain, has been responsible by significant economic losses in crops grown in the State of São Paulo, Brazil. Previous investigations evidenced some species of leafhoppers observed in broccoli fields as potential vectors of the phytoplasma. In this study, the six species more frequently found in broccoli crops were collected to confirm that evidence. Group of five insects of each species were confined per broccoli seedling for an inoculation access period (IAP) of 48 hr. After the IAP, each group was tested for detection of phytoplasma. Evaluation of plants was performed 60 days after inoculation based on the presence of phytoplasma in their tissues. When transmission was positive, genomic fragments corresponding to 16S rDNA were sequenced both for the infected plants and its respective group of insects. The results revealed that the species Agallia albidula, Agalliana sticticollis, Atanus nitidus and Balcluta hebe were able to transmit phytoplasma to broccoli seedlings. Based on the estimates of transmission probability by single insects (P), the highest transmission rate was observed for A. nitidus (24.2%) and the lowest for B. hebe (1.9%). The sequencing of 16S rDNA revealed complete similarity between the sequences of the phytoplasma transmitted to broccoli test plants and the sequences of the phytoplasma found in the field‐collected leafhoppers. These findings support the inclusion of those species as vectors of phytoplasmas belonging to 16SrIII group in broccolis, providing additional information to improve management of this important disease of endemic occurrence.     

DNA Barcoding for Identification of ‘Candidatus Phytoplasmas’ Using a Fragment of the Elongation Factor Tu Gene

Background

Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported.

Methodology/Principal Findings

We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases.

Conclusions/Significance

This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.     

First detection and molecular characterization of grapevine phytoplasmas in Kosovo

A search for phytoplasma-associated diseases was conducted for the first time in the main grapevine-growing localities of the Dukagjini plain in Kosovo. A total of 144 samples were collected from grapevine cultivars displaying leaf yellowing, reddening, discolouration and irregular wood ripening, and analysed using nested and quantitative PCR assays. These assays showed that 35.4% of samples belonging to eight cultivars were positive to the presence of phytoplasmas in the 16SrXII group. The 16S rDNA phytoplasma sequences obtained from 15 samples shared identity greater than 99.5% with ‘Candidatus Phytoplasma solani’. Sequence analysis of the tuf gene showed that the strains found in Kosovar grapevines are in the tuf-type b1 group, sharing 99.6% to 99.8% identity with ‘Ca. P. solani'-related strains associated with the “bois noir” grapevine disease in many European countries; the secY gene sequences, on the other hand, shared 100% identity with ‘Ca. P. solani' strains from Bosnia and Herzegovina, Serbia, Croatia and Turkey. This study constitutes the first report on the presence and molecular characterization of phytoplasmas in Kosovar vineyards. Based on these results, it is recommended that testing for phytoplasma be included in the certification program for grapevine in Kosovo.     

Comparative Molecular Analyses of Phytoplasmas infecting Sophora japonica cv. golden and Robinia pseudoacacia

Phytoplasmas were detected in Sophora japonica cv. golden and Robinia pseudoacacia with diseased branches of witches'‐broom collected in Haidian district, Beijing, China. Phytoplasma cells were observed in phloem sieve elements of symptomatic S. japonica cv. golden by transmission electron microscopy. The presence of phytoplasmas was further confirmed by sequence determination of partial gene sequences of 16S rDNA, rp (ribosomal protein) and secY. Phylogenetic trees and virtual restriction fragment length polymorphism (RFLP) analyses indicated that the phytoplasmas causing S. japonica cv. golden witches'‐broom (SJGWB) and R. pseudoacacia witches'‐broom (RPWB) belong to the 16SrV (elm yellows) group, and they are most closely related to subgroup 16SrV‐B, rpV‐C and secYV‐C jujube witches'‐broom (JWB) phytoplasma. Comparative analyses indicated that the phytoplasma of RPWB was closer to the JWB and that R. pseudoacacia might serve as an alternative host plant of JWB phytoplasma.     

Detection and Molecular Characterization of a 16SrII‐A* Phytoplasma in Grapefruit (Citrus paradisi) with Huanglongbing‐like Symptoms in China

In the year 2010, in a survey in Guangxi Province, China, to detect and characterize phytoplasmas in a huanglongbing (HLB)‐infected grapefruit (Citrus paradisi) orchard, 87 leaf samples with symptoms of blotchy mottle were collected from symptomatic grapefruit trees, and 320 leaf samples from symptomless trees adjacent to the symptomatic trees. Nested polymerase chain reaction (PCR) using universal phytoplasma primer set P1/P7 followed by primer set fU5/rU3 identified 7 (8.0%) positive samples from symptomatic samples but none from symptomless samples. Of the 87 symptomatic samples, 77 (88.5%) were positive for ‘Candidatus Liberibacter asiaticus’ and 5 for both phytoplasma and ‘Ca. L. asiaticus’. Sequence analysis indicated that seven 881‐bp amplicons, amplified by nested phytoplasma primer sets P1/P7 and fU5/rU3, shared 100.0% sequence identity with each other. Genome walking was then performed based on the 881 bp known sequences, and 5111 bp of upstream and downstream sequences were obtained. The total 5992 bp sequences contained a complete rRNA operon, composed of a 16S rRNA gene, a tRNA^Ile gene, a 23S rRNA gene and a 5S rRNA gene followed by eight tRNA genes. Phylogenetic analysis and virtual restriction fragment length polymorphism analysis confirmed the phytoplasma was a variant (16SrII‐A*) of phytoplasma subgroup 16SrII‐A. As phytoplasmas were only detected in blotchy‐mottle leaves, the 16SrII‐A* phytoplasma identified was related to HLB‐like symptoms.     

Universal and group-specific real-time PCR diagnosis of flavescence dorée (16Sr-V), bois noir (16Sr-XII) and apple proliferation (16Sr-X) phytoplasmas from field-collected plant hosts and insect vectors

Three real‐time PCR–based assays for the specific diagnosis of flavescence dorée (FD), bois noir (BN) and apple proliferation (AP) phytoplasmas and a universal one for the detection of phytoplasmas belonging to groups 16Sr‐V, 16Sr‐X and 16Sr‐XII have been developed. Ribosomal‐based primers CYS2Fw/Rv and TaqMan probe CYS2 were used for universal diagnosis in real‐time PCR. For group‐specific detection of FD phytoplasma, ribosomal‐based primers fAY/rEY, specific for 16Sr‐V phytoplasmas, were chosen. For diagnosis of BN and AP phytoplasmas, specific primers were designed on non‐ribosomal and nitroreductase DNA sequences, respectively. SYBR^® Green I detection coupled with melting curve analysis was used in each group‐specific protocol. Field‐collected grapevines infected with FD and BN phytoplasmas and apple trees infected with AP phytoplasma, together with Scaphoideus titanus, Hyalesthes obsoletus and Cacopsylla melanoneura adults, captured in the same vineyards and orchards, were used as templates in real‐time PCR assays. The diagnostic efficiency of each group‐specific protocol was compared with well‐established detection procedures, based on conventional nested PCR. Universal amplification was obtained in real‐time PCR from DNAs of European aster yellows (16Sr‐I), elm yellows (16Sr‐V), stolbur (16Sr‐XII) and AP phytoplasma reference isolates maintained in periwinkles. The same assay detected phytoplasma DNA in all test plants and test insect vectors infected with FD, BN and AP phytoplasmas. Our group‐specific assays detected FD, BN, and AP phytoplasmas with high efficiencies, similar to those obtained with nested PCR and did not amplify phytoplasma DNA of other taxonomic groups. Melting curve analysis was necessary for the correct identification of the specific amplicons generated in the presence of very low target concentrations. Our work shows that real‐time PCR methods can sensitively and rapidly detect phytoplasmas at the universal or group‐specific level. This should be useful in developing defence strategies and for quantitative studies of phytoplasma–plant–vector interactions.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

Molecular Characterization of Phytoplasmas Associated with Peach Diseases in Iran

Recently, peach trees showing leaf rolling, little leaf, rosetting, yellowing, bronzing of foliage and tattered and shot‐holed leaves symptoms were observed in peach growing areas in the central and north‐western regions of Iran. Polymerase chain reaction (PCR) and nested PCR using phytoplasma universal primer pairs P1/Tint, R16F2/R2, PA2F/R and NPA2F/R were employed to detect phytoplasmas. The nested PCR assays detected phytoplasma infections in 51% of symptomatic peach trees in the major peach production areas in East Azerbaijan, Isfahan, ChaharMahal‐O‐Bakhtiari and Tehran provinces. Restriction fragment length polymorphism (RFLP) analyses of 485 bp fragments amplified using primer pair NPA2F/R in nested PCR revealed that the phytoplasmas associated with infected peaches were genetically different and they were distinct from phytoplasmas that have been associated with peach and almond witches’‐broom diseases in the south of Iran. Sequence analyses of partial 16S rDNA and 16S–23S rDNA intergenic spacer regions demonstrated that ‘Candidatus Phytoplasma aurantifolia’, ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma trifolii’ are prevalent in peach growing areas in the central and north‐western regions of Iran.     

Development of molecular markers and a diagnostic tool for investigation of coinfections by and interactions between potato purple top and potato witches'‐broom phytoplasmas in tomato

Columbia Basin potato purple top (PPT) phytoplasma and Alaska potato witches'‐broom (PWB) phytoplasma are two closely related but mutually distinct pathogenic bacteria that infect potato and other vegetable crops. Inhabiting phloem sieve elements and being transmitted by phloem‐feeding insect vectors, both pathogens are affiliated with ‘Candidatus Phytoplasma trifolii’ and are members of the clover proliferation phytoplasma group (16SrVI). The polyphagous nature and wide geographic distribution of their insect vectors make mixed infection inevitable. In this study, we experimentally constituted a simultaneous PPT and PWB phytoplasma infection in tomato (Solanum lycopersicum) and developed a sensitive diagnostic tool to investigate mixed infections by and in planta interactions of the two phytoplasmas. The distribution and relative abundance of the two co‐infecting phytoplasmas were monitored over a 45‐day post‐infection time course and for three serial passages in planta. Our results revealed that dual infections of the two phytoplasmas induce a new symptom unseen in infection by either phytoplasma alone. Our results also raised an interesting question as to whether the two phytoplasmas differ in ability of competitive dominance under co‐infection conditions. The molecular markers and the diagnostic tool devised in this study should be useful for further investigations of the interactions between the two closely related phytoplasmas in their hosts.     

Multilocus sequence analysis of phytoplasma strains of 16SrII group in Iran and their comparison with related strains

Phytoplasmas of the group 16SrII (peanut witches'‐broom group) are among the most important phytoplasmas identified in Iran. These phytoplasmas are so diverse that they have been classified within 23 subgroups, among which phytoplasmas of subgroups 16SrII‐B, ‐C and ‐D have been recognised in Iran. In this study, we used multilocus sequence analysis as a tool to find the extent of genetic diversity and phylogeny of representative phytoplasmas of 16SrII in Iran in comparison to reference phytoplasma strains characterised elsewhere. The genes used were 16S rRNA, secY, rplV‐rpsC, imp and a hypothetical protein (inmp). Analysis of this study showed that phytoplasmas of 16SrII could be resolved into at least three main phylogenetic lineages. One lineage comprised phytoplasmas of the subgroups 16SrII‐A and II‐D, another included strains of subgroups 16SrII‐B and II‐C and the third lineage comprised phytoplasmas belonging to 16SrII‐E. The significance of host adaptation and geographical distribution in relation to the genetic diversity of these phytoplasmas is discussed. Among five different genetic loci used in this study, imp gene displayed the highest genetic diversity, hence considered as the most powerful genetic tool for differentiation of closely related phytoplasmas.     

Detection and Identification of Aster Yellows Phytoplasma Associated with Lipstick Yellow Frond Disease in Malaysia

Yellowing symptoms similar to coconut yellow decline phytoplasma disease were observed on lipstick palms (Cyrtostachys renda) in Selangor state, Malaysia. Typical symptoms were yellowing, light green fronds, gradual collapse of older fronds and decline in growth. Polymerase chain reaction assay was employed to detect phytoplasma in symptomatic lipstick palms. Extracted DNA was amplified from symptomatic lipstick palms by PCR using phytoplasma‐universal primer pair P1/P7 followed by R16F2n/R16R2. Phytoplasma presence was confirmed, and the 1250 bp products were cloned and sequenced. Sequence analysis indicated that the phytoplasmas associated with lipstick yellow frond disease were isolates of ‘Candidatus Phytoplasma asteris’ belonging to the 16SrI group. Virtual RFLP analysis of the resulting profiles revealed that these palm‐infecting phytoplasmas belong to subgroup 16SrI‐B and a possibly new 16SrI‐subgroup. This is the first report of lipstick palm as a new host of aster yellows phytoplasma (16SrI) in Malaysia and worldwide.