Characterization of phytoplasmas detected in Chinaberry trees with symptoms of leaf yellowing and decline in Bolivia

Polymerase chain reaction (PCR) assays were used to detect phytoplasmas in foliage samples from Chinaberry ( Melia azedarach ) trees displaying symptoms of yellowing, little leaf and dieback in Bolivia. A ribosomal coding nuclear DNA (rDNA) product (1·8 kb) was amplified from one or more samples from seven of 17 affected trees by PCR employing phytoplasma-universal rRNA primer pair P1/P7. When P1/P7 products were reamplified using nested rRNA primer pair R16F2n/R16R2, phytoplasmas were detected in at least one sample from 13 of 17 trees with symptoms. Restriction fragment length polymorphism (RFLP) analysis of P1/P7 products indicated that trees CbY1 and CbY17 harboured Mexican periwinkle virescence (16SrXIII)-group and X-disease (16SrIII)-group phytoplasmas, respectively. Identification of two different phytoplasma types was supported by reamplification of P1/P7 products by nested PCR employing X-disease-group-specific rRNA primer pair R16mF2/WXint or stolbur-group-related primer pair fSTOL/rSTOL. These assays selectively amplified rDNA products of 1656 and 579 bp from nine and five trees with symptoms, respectively, of which two trees were coinfected with both phytoplasma types. Phylogenetic analysis of 16S rDNA sequences revealed Chinaberry yellows phytoplasma strain CbY17 to be most similar to the chayote witches'-broom (ChWBIII-Ch10) agent, a previously classified 16SrIII-J subgroup phytoplasma. Strain CbY1 resembled the Mexican periwinkle virescence phytoplasma, a 16SrXIII-group member. The latter strain varied from all known phytoplasmas composing group 16SrXIII. On this basis, strain CbY1 was assigned to a new subgroup, 16SrXIII-C.     

Genetic diversity in the coconut lethal yellowing disease phytoplasmas of East Africa

DNA primers, based on the ribosomal sequences of lethal yellowing-type disease (LYD) phytoplasmas, were used to analyse genetic variation within the lethal yellowing-type diseases of coconut in East Africa. Samples were collected from palms in Kenya, Mozambique and high, medium and low disease incidence areas of Tanzania. The mollicute-specific primer pair P1 and P6 amplified a 1.5 kbp product from all diseased palms and no product from symptomless palms, indicating that phytoplasmas were associated with all of these diseases. However, the Rohde forward and Rohde reverse primers (a second rRNA primer pair designed to detect East African LYD-associated phytoplasmas) only amplified products from Tanzanian and Kenyan diseased palms and not from those of Mozambique. Conversely, primers Ghana 813 and AK-SR, designed for specific detection of coconut-associated phytoplasmas in West Africa, amplified products only from the Mozambique palms, indicating that the phytoplasma associated with LYD in Mozambique is more closely related to those from West Africa. This was supported by restriction enzyme digestion of PCR products. DNA sequencing of PCR products from phytoplasmas within Tanzania revealed no detectable differences in the rDNA sequences of isolates from high, medium and low incidence areas.     

Partial characterization of phytoplasmas associated with lettuce and wild lettuce phyllodies in Iran

Phytoplasmas associated with lettuce phyllody (LP) and wild lettuce phyllody (WLP) in southern Iran were partially characterized by molecular analyses and host-range studies. Agents of both diseases were transmitted by Neoaliturus fenestratus , a leafhopper colonizing lettuce and wild lettuce, to lettuce, wild lettuce, sowthistle and periwinkle, but not to safflower, sunflower, calendula and sesame. Both phytoplasmas induced bud proliferation, virescence, phyllody and witches' broom in infected plants. Total DNA extracted from infected lettuce and wild lettuce or from vector tissues was subjected to PCR using phytoplasma-specific primer pair P1/P7 or nested PCR using P1/P7 followed by R16F2n/R16R2. PCR product of nested PCR (1·2 kbp) was subjected to restriction fragment length polymorphism (RFLP). RFLP analysis of nested PCR product identified the LP, WLP and N. fenestratus -associated phytoplasmas as members of the pigeon pea witches' broom group, 16SrIX. Phylogenetic analysis of the 16S rRNA gene sequence also clustered LP and WLP phytoplasmas with other known members of the 16SrIX group. While no significant differences could be detected between LP and WLP phytoplasmas, both isolates differed from Lebanese wild lettuce phyllody in molecular properties.     

Detection of a pigeon pea witches'-broom-related phytoplasma in trees of Gliricidia sepium affected by little-leaf disease in Central America

The polymerase chain reaction (PCR) incorporating phytoplasma-specific ribosomal RNA primer pair P1 and P7 consistently amplified a product of expected size (1.8 kb) when template DNA for PCR was extracted from leaf and shoot samples of Gliricidia sepium from Honduras exhibiting symptoms of little-leaf disease (LLD). Restriction fragment length polymorphism analysis (RFLP) of amplified rDNA products indicated that phytoplasmas detected in LLD-affected G. sepium were very similar although not identical to phytoplasmas associated with pigeon pea witches'-broom (PPWB) disease in the western Caribbean region. Phytoplasma detection in trees was enhanced by reamplification of the P1/P7-primed PCR products with nested primers PPf1 and Tint. Nested reactions enabled additional positive detections in foliage of several G. sepium trees showing only mild or no apparent LLD symptoms. Neither rDNA RFLP nor sequence analyses of 16–23 S rRNA spacer regions revealed differences among Honduran LLD phytoplasma or between these strains and phytoplasmas detected in G. sepium with LLD-like symptoms in El Salvador, Guatemala, and Nicaragua, indicating that closely related, possibly identical, pathogens presently affect this tree species in all four countries.     

Disease progression of a lethal decline caused by the 16SrIV‐D phytoplasma in Florida palms

Recently, a new phytoplasma was discovered in Hillsborough County in the state of Florida, USA. This phytoplasma belongs to the 16SrIV taxonomic group and is classified as subgroup D. It is the causal agent of lethal bronzing disease (LBD) of palm. Since the discovery of LBD in 2006, the disease has spread throughout much of the state. In 2014 and 2015, stands of cabbage palm and queen palms that had been present at the University of Florida's Fort Lauderdale Research and Education Center in Davie, FL began showing symptoms of LBD. After confirming the presence of the LBD phytoplasma in initially infected palms by nested PCR and RFLP analysis, all palms were systematically sampled over the period of 1 year to monitor and quantify disease spread. A total of 30 cabbage palms were tested monthly by qPCR, with five testing positive on the first sample date. By the end of the study period, 16 cabbage palms had died from the infection. A total of 16 queen palms were surveyed, with three palms initially testing positive. By the end of the study, four queen palms had tested positive and died from the infection. To the authors’ knowledge, this study is the first to document and quantify spread of palm‐infecting phytoplasmas. This data provides important insights into the ecology of palm‐infecting phytoplasmas and highlights the impact that the movement of infective insects can pose to established stands of palms.     

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.     

Sensitive Detection of a Phytoplasma Associated with Little Leaf Symptoms in Withania somnifera

Withania somnifera is an important medicinal plant native to the Indian-sub continent. Owing to the presence of a number of precious alkaloids, flavonoids and withanolides, it is widely used in the Indian and African systems of medicines. It is severely affected by phytoplasma present in the sieve tubes of phloem. With a view to micropropagate phytoplasma-free W. somnifera plants, an efficient and effective nested PCR-based system was developed for detection of associated phytoplasmas. Universal primers, designed from the 16S rDNA sequences of phytoplasmas, were applied in direct/nested-PCR. Total DNA extracts from leaf tissues of 33 suspected symptomatic and 11 non-symptomatic plants were subjected to direct PCR. The direct PCR products were subsequently employed as templates in nested PCR. The nested PCR could reamplify direct PCR products yielding a DNA fragment of 1.4 kb. A phytoplasma was detected in all the diseased plants and not from the healthy looking plants. Further, it was sensitive enough to amplify phytoplasma DNA obtained from crude DNA diluted up to 2500 times from naturally infected plants and also from various stages of in vitro-propagated diseased plants. Identical restriction fragment polymorphism enzyme profiles were obtained following restriction enzyme digestion of nested PCR products, obtained from five different plants, by EcoRI, AluI and RsaI restriction endonucleases. The developed nested PCR based system should facilitate indexing of the phytoplasma in different stages of in vitro-generated plants and probably identification of, as yet unknown, hosts and vectors of phytoplasma associated with phytoplasma disease of W. somnifera.     

Detection and characterization of phytoplasmas in diseased stone fruits and pear by PCR-RFLP analysis in Turkey

During the late summer-early autumn of 2002, surveys were carried out in Turkey to determine the presence of phytoplasma diseases in fruit trees. Phytoplasmas were detected and characterized by PCR-RFLP analysis and TEM technique in stone fruit and pear trees in the eastern Mediterranean region of the country. Six out of 24 samples, including almond, apricot, peach, pear and plum, gave positive results in PCR assays. RFLP analysis usingSspI andBsaAI enzymes of PCR products obtained with primer pair f01/r01 enabled identification of the phytoplasmas involved in the diseases. Stone fruit trees, including a local apricot variety (‘Sakıt’) and a pear sample, were found to be infected with European stone fruit yellows (ESFY, 16SrX-B) and pear decline (PD, 16SrX-C) phytoplasmas, respectively. This is the first report in Turkey of PD phytoplasma infecting pear and of ESFY phytoplasma infecting almond, apricot, myrobalan plum and peach; ESFY phytoplasma infecting Japanese plum was previously reported. http://www.phytoparasitica.org posting July 21, 2005.     

Phytoplasmas associated with disease of coconut in Malaysia: phylogenetic groups and host plant species

Coconut palm ( Cocos nucifera ), oil palm ( Elaeis guineensis ), Bermudagrass ( Cynodon dactylon ) and Madagascar periwinkle ( Catharanthus roseus ) with symptoms indicative of phytoplasma disease were collected from different locations in Malaysia. PCR assays employing phytoplasma universal rRNA gene primers P1/P7 alone or P1/P7 followed by R16F2n/R16R2 detected phytoplasmas in eight out of 20 Malayan Red Dwarf (MRD), nine out of 12 Malayan Yellow Dwarf (MYD) and 12 out of 12 Malayan Tall (MT) coconut palms displaying coconut yellow decline symptoms. Positive detections were also obtained from six out of six oil palm seedlings showing symptoms of yellowing and necrosis, from 10 out of 10 Bermudagrass samples with white leaf symptoms, and from eight out of eight periwinkle plants showing phyllody, virescence, little leaf, proliferation and foliar yellowing. Phytoplasmas were not detected in any of the symptomless plants tested. Sequencing and phylogenetic analysis of PCR products determined that phytoplasmas infecting both MRD and MT coconuts and Bermudagrass in Serdang, Selangor State, were all members of the 16SrXIV ' Candidatus Phytoplasma cynodontis' group, whereas isolates in periwinkle in Serdang were all members of the 16SrI ' Ca. Phytoplasma asteris' group. However, the phytoplasmas detected in MYD coconuts and oil palms from Banting, Selangor State, and in periwinkle from Putrajaya were collectively very similar (99%), but shared <97·5% similarity with 16S rDNA sequences of all other known phytoplasmas, indicating that they represent a novel taxonomic group. Thus, at least two phylogenetically distinct phytoplasmas are associated with the coconut yellow decline syndrome in Malaysia, both of which were also detected in other plant species.     

Detection and differentiation of grapevine yellows phytoplasmas belonging to the elm yellows group and to the stolbur subgroup by PCR amplification of non-ribosomal DNA

Primer pairs were designed from a cloned DNA probe of a strain of flavescence dorée (FD) phytoplasma and from a cloned DNA probe of a strain of stolbur phytoplasma. Among an array of reference phytoplasma strains maintained in periwinkle, pair FD9f/r amplified a 1.3 kb DNA fragment only with phytoplasma strains of elm yellows (EY) group, i.e. two strains of FD and two strains of EY. Tru9I restriction analysis of the fragment amplified by FD9f/r revealed a diversity among EY-group phytoplasmas. The FD strains differed from the strains isolated from elm. The profile of the phytoplasmas infecting the grapevine samples from Catalonia and most of the samples from Northern Italy were identical to that of a FD strain. Three other profiles were detected in grapevine from Palatinate, in Germany.The two primer pairs derived from a stolbur strain, STOL4f/r and STOL11f2/r1, specifically amplified a 1.7 kb and a 0.9 kb DNA fragment, respectively, with all strains in the stolbur subgroup. However, the pair STOL4f/r did not recognise strain MOL. Both pairs allowed to detect phytoplasmas in diseased grapevines from France, Italy, Spain and Israel. Attempts to differentiate between phytoplasmas in the stolbur subgroup by restriction analyses failed. The pairs FD9f/r and STOL11f2/r1 could be used in the same reaction (multiplex PCR) to detect EY-group phytoplasmas, stolbur-subgroup phytoplasmas or both phytoplasmas simultaneously when template DNAs were mixed.     

Note: A comparison of molecular diagnostic procedures for the detection of aster yellows phytoplasmas (16Sr-I) in leafhopper vectors

Different molecular procedures were compared for the detection of aster yellows phytoplasmas (AYP) in the leafhopper vectorsMacrosteles quadripunctulatus (Kirschbaum),Euscelidius variegatus (Kirschbaum) andEuscelis incisus (Kirschbaum). Polymerase chain reaction (PCR) with universal and group-specific primers designed on the 16S-rDNA sequence was most sensitive in nested assays. A dot-blot procedure with an oligoprobe designed on the 16S-rDNA was less sensitive and consistent to detect phytoplasmas in total insect DNA, but consistently detected amplicons from direct PCR. The dot-blot assay with a probe based on a phytoplasma plasmid sequence detected AYP in most vector specimens and did not react with DNAs from leafhoppers infected by flavescence dorée and psyllids infected by apple proliferation phytoplasmas. This last assay is almost devoid of contamination risks, faster and cheaper compared to PCR, therefore it has to be preferred for field-scale analysis of leafhopper populations. http://www.phytoparasitica.org posting Feb. 24, 2004.     

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.     

Range of phytoplasma concentrations in various plant hosts as determined by competitive polymerase chain reaction

ABSTRACT For competitive polymerase chain reaction (PCR), an internal standard DNA template was developed that consisted of a highly conserved, internally deleted 16S rDNA fragment of an aster yellows phytoplasma. The internal standard was calibrated using a quantified culture of Acholeplasma laidlawii. Serial dilutions of the internal standard and fixed amounts of target templates from infected plants were coamplified with the same primers, and the products obtained were quantified using an enzyme-linked immunosorbent assay procedure. Analysis of the data revealed that the phytoplasma concentration in the plants examined differed by a factor of about 4 x 10(6). Phytoplasma concentrations of 2.2 x 10(8) to 1.5 x 10(9) cells per g of tissue were identified in periwinkles infected with various phytoplasmas. High to moderate concentrations were detected in Malus domestica (apple) genotypes infected with the apple proliferation phytoplasma, Alnus glutinosa (alder) genotypes infected with the alder yellows phytoplasma, and most aster yellows-infected Populus (poplar) genotypes examined. Very low phytoplasma concentrations, ranging from 370 to 34,000 cells per g of tissue, were identified in proliferation-diseased apple trees on resistant rootstocks 4551 and 4608, yellows-diseased Quercus robur (oak) trees, and Carpinus betulus (hornbeam) trees. Such low concentrations, which corresponded to about 4 to 340 cells in the reaction mixture, could only be detected and quantified by nested PCR.     

Kerala wilt disease phytoplasma: Phylogenetic analysis and identification of a vector, Proutista moesta

Kerala wilt disease of coconut palm is a major threat of coconut production in Kerala caused by phytoplasma. The genomic DNA purified from the insect tissues of Proutista moesta (PM) and Stephanitis typica (ST) was subjected to PCR assay using the primer combination P1/P6, P1/P7 and P4/P7. The amplified products resolved a prominent band of 650 bp for the universal primer P4/P7 and no bands were noticed for the primer pairs P1/P6 and P1/P7 combination. Since P4/P7 amplifies the 16S–23S intergenic spacer region of 16SrRNA gene, the PCR product 650 bp of the insect PM indicate the phytoplasma DNA. The presence of 650 bp for the primer P4/P7 in the genomic DNA isolated from P. moesta indicates the vectoral ability of the insect. No sign of amplification was noticed in the case of ST for the three sets of primers suggesting the inability of this insect as vector. The amplified product 650 bp from the genomic DNA of KWD palms as well as the insect tissues of P. moesta was gel purified and sequenced. The sequential similarity of 650 bp of both KWD phytoplasma and the insect phytoplasma supports the transmission of phytoplasma through the vector PM. Moreover, the sequence of 650 bp was compared with other sequences of 26 coconut phytoplasmas so far reported internationally and a cladogram was prepared for determining the phylogenetic status. It is obvious from the cladogram that the KWD disease phytoplasma is evolutionarily closest to coconut phytoplasma of coconut lethal yellowing of Mexican palms within the group 16SrIV. Phylogenetically, KWD phytoplasma is grouped in the new subgroup 16SrIV-C subsequent to the groups 16SrIV-A and 16SrIV-B for Mexican coconut lethal yellowing and Tanzanian coconut lethal decline, respectively. The restriction enzyme analysis of the PCR product 650 bp using the enzymes AluI, BclI, HindIII and RsaI further supports the phytoplasmic nature of DNA. This data records the first finding of the vector of Kerala wilt disease by detecting KWD phytoplasma in insect tissue of PM by PCR based methods. Moreover, the study reveals the phylogenetic status of KWD phytoplasma compared to other coconut phytoplasmas internationally.     

A phytoplasma closely related to the pigeon pea witches'-broom phytoplasma (16Sr IX) is associated with citrus huanglongbing symptoms in the state of São Paulo, Brazil

In February 2007, sweet orange trees with characteristic symptoms of huanglongbing (HLB) were encountered in a region of S?o Paulo state (SPs) hitherto free of HLB. These trees tested negative for the three liberibacter species associated with HLB. A polymerase chain reaction (PCR) product from symptomatic fruit columella DNA amplifications with universal primers fD1/rP1 was cloned and sequenced. The corresponding agent was found to have highest 16S rDNA sequence identity (99%) with the pigeon pea witches'-broom phytoplasma of group 16Sr IX. Sequences of PCR products obtained with phytoplasma 16S rDNA primer pairs fU5/rU3, fU5/P7 confirm these results. With two primers D7f2/D7r2 designed based on the 16S rDNA sequence of the cloned DNA fragment, positive amplifications were obtained from more than one hundred samples including symptomatic fruits and blotchy mottle leaves. Samples positive for phytoplasmas were negative for liberibacters, except for four samples, which were positive for both the phytoplasma and 'Candidatus Liberibacter asiaticus'. The phytoplasma was detected by electron microscopy in the sieve tubes of midribs from symptomatic leaves. These results show that a phytoplasma of group IX is associated with citrus HLB symptoms in northern, central, and southern SPs. This phytoplasma has very probably been transmitted to citrus from an external source of inoculum, but the putative insect vector is not yet known.     

Ecological implications from a molecular analysis of phytoplasmas involved in an aster yellows epidemic in various crops in Texas

ABSTRACT In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.     

Development and evaluation of a one‐hour DNA extraction and loop‐mediated isothermal amplification assay for rapid detection of phytoplasmas

A rapid DNA extraction and loop‐mediated isothermal amplification (LAMP) procedure was developed and evaluated for the detection of two specific groups of phytoplasmas from infected plant material. Primers based upon the 16–23S intergenic spacer (IGS) region were evaluated in LAMP assays for amplification of group 16SrI (aster yellows group) and group 16SrXXII (Cape St Paul wilt group) phytoplasma strains. DNA could be extracted from leaf material (16SrI phytoplasmas) or coconut trunk borings (16SrXXII phytoplasmas) onto the membranes of lateral flow devices, and small sections of these membranes were then added directly into the LAMP reaction mixture and incubated for 45 min at 65°C. Positive reactions were detected through the hydroxyl napthol blue colorimetric assay within 1 h of the start of DNA extraction, and were confirmed by subsequent agarose gel electrophoresis of the LAMP products. The level of detection was comparable to that obtained by nested PCR using conventional 16S rDNA phytoplasma‐specific primers. Furthermore, the assays were specific for the phytoplasmas they were designed to detect – the 16SrI assay only detected 16SrI phytoplasmas and not those from any other phylogenetic groups, whilst the 16SrXXII assay only detected 16SrXXII phytoplasmas. The DNA extractions and LAMP assay are easy to perform, requiring minimal equipment, and may therefore form the basis of a rapid and reliable field‐detection system for phytoplasmas.     

New natural host plants of ‘Candidatus Phytoplasma pini’ in Poland and the Czech Republic

The presence of phytoplasmas in seven coniferous plant species (Abies procera, Pinus banksiana, P. mugo, P. nigra, P. sylvestris, P. tabuliformis and Tsuga canadensis) was demonstrated using nested PCR with the primer pairs P1/P7 followed by R16F2n/R16R2. The phytoplasmas were detected in pine trees with witches’ broom symptoms growing in natural forest ecosystems and also in plants propagated from witches’ brooms. Identification of phytoplasmas was done using restriction fragment length polymorphism analysis (RFLP) of the 16S rDNA gene fragment with AluI, MseI and RsaI endonucleases. All samples showed RFLP patterns similar to the theoretical pattern of ‘Candidatus Phytoplasma pini’, based on the sequence of the reference isolate Pin127S. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Comparison of the 16S rDNAs obtained revealed high (99·8–100%) nucleotide sequence identity between the phytoplasma isolates. The isolates were also closely related to four other phytoplasma isolates found in pine trees previously. Based on the results of RFLP and sequence analyses, the phytoplasma isolates tested were classified as members of the ‘Candidatus Phytoplasma pini’, group 16SrXXI.     

Molecular identification of a Candidatus phytoplasma (Group16SrV-D) coding partial uvrB gene and degV gene on a new host – mesta (Hibiscus sabdariffa) – with phyllody and reddening of leaves in India

Mesta (Hibiscus sabdariffa) is an important bast fiber crop. In August 2011, there was an outbreak of a phytoplasma-like disease on H. sabdariffa in different villages of the northern coastal mesta-growing region of Andhra Pradesh, India, covering mainly two districts – Srikakulam and Vijayanagaram. The infected plants showed characteristic symptoms such as phyllody and reddening of leaves. PCR with P1/P7 universal primer pair of 16 S rDNA yielded amplicons of 1850 bp from all symptomatic mesta leaf samples similar to samples of brinjal little leaf (phytoplasma positive reference control). However, asymptomatic samples were not amplified. Multiplex nested-PCR showed simultaneous amplification of DNA fragments with phytoplasma specific primers, viz., P1/P7 universal primer pair of 16 S rDNA, nested primer pair R16F2n/R2, uvrB and DegV gene-specific uvrB-degVF/R primer generating amplicons of 1850 bp, 1200 bp and 1023bp, respectively. However, SecY-map gene specific primer SecY-mapF/R was not amplified. The 1023 bp nucleotide sequence of uvrB and DegV gene of the phytoplasma was deposited in the GenBank (NCBI) with the accession no. JX975061. NCBI BLASTn analysis of the 1023 bp products showed that the phytoplasma strain belonged to elm yellows group (16SrV-D). This is the first report that Hibiscus sabdariffa is infected by a phytoplasma and we named it mesta phyllody disease (MPD).     

Detection and identification of a phytoplasma from lucerne with Australian lucerne yellows disease

Foliar and root symptoms are described for Australian lucerne yellows (ALuY), a disease common in Australian lucerne seed crops. A phytoplasma was detected in plants exhibiting symptoms, but not in symptomless lucerne plants. Oligonucleotide primers specific to the phytoplasma 16S-23S rRNA intergenic spacer region (SR) were used in polymerase chain reaction (PCR) assays on DNA extracted from lucerne plants with and without symptoms. Identical restriction fragment length polymorphism (RFLP) enzyme profiles were obtained for PCR products amplified from 10 yellows-affected lucerne samples. RFLP profiles obtained for four restriction enzymes were different from those of the tomato big bud (TBB) phytoplasma. ALuY phytoplasma PCR products were sequenced to determine phylogeny and were found to fall within the faba bean phyllody phytoplasma group, or phytoplasma group 16srII. Transmission electron microscopy revealed phytoplasmas in the phloem of yellows-affected plant samples, but not in symptomless plant samples. Fungal, bacterial and viral agents in the aetiology of Australian lucerne yellows were ruled out.