Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.     

Multilocus genotyping of a ‘Candidatus Phytoplasma aurantifolia’‐related strain associated with cauliflower phyllody disease in China

A new cauliflower disease characterised by the formation of leaf‐like inflorescences and malformed flowers occurred in a seed production field located in Yunnan, a southwest province of China. Detection of phytoplasma‐characteristic 16S rRNA gene sequences in DNA samples from diseased plants linked the cauliflower disease to phytoplasmal infection. Results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence indicated that the cauliflower‐infecting agent is a ‘Candidatus Phytoplasma aurantifolia’‐related strain and is a new member of the peanut witches'‐broom phytoplasma group, subgroup A (16SrII‐A). Multilocus genotyping showed close genetic relationship between this cauliflower phytoplasma and a broad host range phytoplasma lineage found only in East Asia thus far. Molecular markers present in the secY and rp loci distinguished this phytoplasma from other members of the subgroup 16SrII‐A.     

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.     

Molecular Identification of a ‘Candidatus Phytoplasma ziziphi’‐related Strain Infecting Amaranth (Amaranthus retroflexus L.) in China

Amaranth (Amaranthus retroflexus L.) is a common weed that grows vigorously in orchards, roadside verges, fields, woods and scrubland in China. In 2009, phytoplasma disease surveys were made in orchards in Beijing, China, and stem/leaf tissues were collected from asymptomatic amaranths. Direct PCR using universal phytoplasma primers P1/P7 detected 16S rRNA gene sequences in every DNA sample extracted from the symptomless amaranths. Sequence alignment and phylogenetic analyses of the 16S rRNA gene determined that the amaranth phytoplasma strain was related to ‘Candidatus Phytoplasma ziziphi’. Furthermore, virtual RFLP pattern analysis showed that the amaranth phytoplasma belonged to the 16SrV‐B subgroup. This is the first report of symptomless plants containing a ‘Candidatus Phytoplasma ziziphi’‐related strain.     

Surveys reveal the occurrence of phytoplasmas in plants at different geographical locations in Peru

Two independent surveys were performed in Peru during February and November 2007 to detect the presence of phytoplasmas within any crops showing symptoms resembling those caused by phytoplasmas. Molecular identifications and characterisations were based on phytoplasma 16S and 23S rRNA genes using nested PCR and terminal restriction fragment length polymorphism (T‐RFLP). The surveys indicated that phytoplasmas were present in most of the locations sampled in Peru in both cultivated crops, including carrots, maize, native potatoes, improved potato, tomato, oats, papaya and coconut, and in other plants such as dandelion and the ornamental Madagascar periwinkle (Catharanthus roseus). Phylogenetic analysis of the sequences confirmed that while most of the isolates belong to the 16SrI aster yellows group, which is ubiquitous throughout other parts of South America, one isolate from potato belongs to the 16SrII peanut witches’ broom group, and one isolate from tomato and one from dandelion belong to the 16SrIII X‐disease group. The use of T‐RFLP was validated for the evaluation of phytoplasma‐affected field samples and provided no evidence for mixed infection of individual plants with more than one phytoplasma isolate. These data represent the first molecular confirmation of the presence of phytoplasmas in a broad range of crops in Peru.     

Molecular Detection and Identification of a 16SrVI Group Phytoplasma Associated with Tomato Big Bud Disease in Xinjiang,China

In 2010, tomato plants with big bud symptoms were observed in Xinjiang, China. PCR products of approximately 1.2 and 2.8 kb were amplified from infected tomato tissues but not from asymptomatic plants. A comparison of 16S rDNA sequences showed that the casual tomato big bud (TBB) phytoplasma was closely (99%) related to the ‘Candidatus Phytoplasma trifolii’ (16SrVI group). The TBB phytoplasma clustered into one branch with the Loofah witches'‐broom phytoplasma according to the 23S rDNA analysis but with no other member of the 16SrVI group. The cause of TBB symptoms was identified as ‘Ca. Phytoplasma trifolii' (16SrVI group) by PCR, virtual RFLP and sequencing analyses. This is the first report of a phytoplasma related to ‘Ca. Phytoplasma trifolii' causing TBB disease in China.     

Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, Iran

In 2002, garden beet witches’ broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR‐amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII‐E, in the peanut witches’ broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722 ) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII‐E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma.     

Molecular Identification of a 16SrII‐A Group‐Related Phytoplasma Associated with Cinnamon Yellow Leaf Disease in China

Chinese cinnamon (Cinnamomum cassia Presl), an evergreen tree native to China, is a multifaceted medicinal plant. The stem bark of cinnamon is used worldwide in traditional and modern medicines and is one of the most popular cooking spices. In recent years, cinnamon with pronounced yellow leaf symptoms has been observed in their natural habitat in Hainan, China. Phytoplasmas were detected from symptomatic cinnamon trees via polymerase chain reaction using phytoplasma universal primers P1/P7 followed by R16F2n/R16R2. No amplification products were obtained from templates of asymptomatic cinnamon trees. These results indicated a direct association between phytoplasma infection and the cinnamon yellow leaf (CYL) disease. Sequence analysis of the CYL phytoplasma 16S rRNA gene determined that CYL phytoplasma is a ‘Candidatus Phytoplasma australasiae’‐related strain. Furthermore, virtual restriction fragment length polymorphism pattern analysis and phylogenetic studies showed that CYL phytoplasma belongs to the peanut witches’‐broom (16SrII) group, subgroup A. This is the first report of a 16SrII group phytoplasma infecting cinnamon under natural conditions.     

Identification of Elm Yellows Phytoplasma in Plum Trees in China

Plum plants (Prunus cerasifera Ehrh) with small and rolled leaves resembling symptoms of phytoplasma infection were observed during 2008 and 2009 in the ornamental garden of Northwest A&F University (Republic of China). Nested polymerase chain reaction (PCR) using a combination of phytoplasma‐specific universal primer pairs (R16F2m/R16R1m‐R16F2n/R16R2) amplified 16S rDNA with the expected size (1.2 kb) from all samples of symptomatic plum plants. Sequencing results and restriction fragment length polymorphism (RFLP) analysis of the 1248 bp R16F2n/R16R2 products showed that the phytoplasma belongs to group 16SrV. Phylogenetic analysis showed that the phytoplasma had a close relation to JWB phytoplasma. This is, we believe, the first report of elm yellows phytoplasma infecting plum plants in China.     

Detection and Identification of Group 16SrVI Phytoplasma in Willows in China

In 2010 and 2011, willow proliferation disease was observed in Erdos, Inner Mongolia, China. The phytoplasma‐specific 16S rRNA gene fragment of 1.2 kb was amplified by a nested PCR with universal primer pair P1/P7 followed by R16F2n/R2. Phylogenetic and virtual RFLP analyses revealed that the phytoplasma associated with willow proliferation was a member of subgroup 16SrVI‐A. The field survey indicated that the incidence of willow proliferation in Erdos was approximately 36.84%. To our knowledge, this is the first record of group 16SrVI phytoplasma infecting willow in China.     

Molecular Identification of a Candidatus Phytoplasma asteris Associated with Cabbage Witches’‐Broom in China

In 2010, cabbages (Brassica oleracea L.) showing symptoms of proliferated axillary buds, crinkled leaves and plant stunting with shortened internodes typical to phytoplasma infection were found in a breeding facility in Beijing, China. Three symptomatic plants and one symptomless plant were collected, and total DNA was extracted from the midrib tissue and the flowers. With phytoplasma universal primers R16F2n/R16R2, a special fragment of 1247 bp (16S rDNA) was obtained from all three symptomatic cabbage plants, but not from the one symptomless cabbage plant. The 16S rDNA sequence showed 99% similarity with the homologous genes of the aster yellows group phytoplasma (16SrI group), and the phytoplasma was designed as CWBp‐BJ. Phylogenetic and computer‐simulated restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA gene revealed that CWBp‐BJ belongs to subgroup 16SrI‐B. This is the first report of a phytoplasma associated with cabbage witches’‐broom in China.     

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.     

Occurrence and Characterization of a 16SrII‐D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran

During 2010–2013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches’ broom, stunting, flower virescence and phyllody. The disease causative agent was dodder transmitted from symptomatic parsley to periwinkle and from periwinkle to periwinkle by grafting inducing phytoplasma‐type symptoms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally inoculated periwinkle plants in direct polymerase chain reaction (PCR) using phytoplasma primer pair P1/P7 or nested PCR using the same primer pair followed by R16F2n/R16R2 primers. Restriction fragment length polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd province belong to 16SrII‐D phytoplasma subgroup. This is the first report of association of a 16SrII‐related phytoplasma with PrWB disease in Iran.     

First Report of Phytoplasma (16SrI) Associated with Yellow Decline Disease of Royal Palms [Roystonea regia (Kunth) O. F. Cook] in Malaysia

Royal Palms (Roystonea regia) with symptoms such as severe chlorosis, stunting, collapse of older fronds and general decline were observed in the state of Selangor, Malaysia. Using polymerase chain reaction (PCR) amplification with phytoplasma universal primer pair P1/P7 followed by R16F2N/R16R2 and fU5/rU3 as nested PCR primer pairs, all symptomatic plants tested positively for phytoplasma. Results of phylogenetic and virtual RFLP analysis of the 16S rRNA gene sequences revealed that the phytoplasma associated with Royal Palm yellow decline (RYD) was an isolate of ‘Candidatus Phytoplasma asteris’ belonging to a new 16SrI‐subgroup. These results show that Roystonea regia is a new host for the aster yellows phytoplasma (16SrI). This is the first report on the presence of 16SrI phytoplasma on Royal Palm trees in Malaysia.     

Detection and molecular characterization of phytoplasmas associated with vegetable and alfalfa crops in Qassim region

During a survey conducted in Qassim province, Saudi Arabia, in the year in 2015, 120 samples of carrot (Daucus carota subsp. sativus), onion (Allium cepa), faba bean (Vicia faba), green mustard (Brassica juncea) and alfalfa (Medicago sativa) plants displaying symptoms reminiscent of phytoplasma diseases were collected and tested for phytoplasma infection. Phytoplasma-specific PCR products were only amplified from symptomatic plants by nested-PCR. Disease incidence ranged from 3.14% in alfalfa crop fields 1 year after cultivation to 77.48% in 3-year-old fields. In the five carrot fields sampled in this study, the incidence changed from 3.2% to 100% after 7 months of cultivation. Phylogenetic analysis revealed that all Qassim phytoplasma isolates belong to the 16SrII group. Most of them shared 100% identity with papaya yellow crinkle (16SrII-D Y10097). The results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence confirmed that the phytoplasma of Qassim isolates under study is a member of 16SrII-D subgroup. To the best of my knowledge, the onion and green mustard are considered new hosts for the 16SrII group; therefore, this is the first report on the association of phytoplasma with diseases of faba bean, onion, carrot, mustard and alfalfa in Qassim province, Saudi Arabia.     

The Distribution of Phytoplasmas in Myanmar

Phytoplasma‐infected plants with symptoms of general yellowing, stunting, little leaves, white leaves, virescence, phyllody and witches’ broom growth of axillary shoots were collected from various plant species in Myanmar during 2010 and 2011. Restriction fragment length polymorphism (RFLP), sequence analysis of the PCR‐amplified 16S ribosomal RNA gene and phylogenetic analyses were used to identify and classify the phytoplasmas. Based on RFLP and sequence analyses, 13 isolates were identified and classified into one subgroup of 16SrI‐B, two subgroups of 16SrII‐A and 16SrII‐C, and one of 16SrXI group phytoplasmas. Phylogenetic analyses also supported the relationship of Myanmar isolates with the three 16Sr groups. This study showed that at least three 16Sr groups exist and 16SrII group phytoplasmas are widely distributed in Myanmar.     

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.     

Phytoplasma from little leaf disease affected sweetpotato in Western Australia: detection and phylogeny

Symptoms of leaf and stem chlorosis and plant stunting were common in sweetpotato plants (Ipomoea batatas) in farmers’ fields in two widely separated locations, Kununurra and Broome, in the tropical Kimberley region in the state of Western Australia in 2003 and 2004. In the glasshouse, progeny plants developed similar symptoms characteristic of phytoplasma infection, consisting of chlorosis and a stunted, bushy appearance as a result of proliferation of axillary shoots. The same symptoms were reproduced in the African sweetpotato cv. Tanzania grafted with scions from the plant Aus1 with symptoms and in which no viruses were detected. PCR amplification with phytoplasma‐specific primers and sequencing of the 16S‐23S rRNA gene region from two plants with symptoms, Aus1 (Broome) and Aus142A (Kununurra), revealed highly identical sequences. Phylogenetic analysis of the 16S rRNA gene sequences obtained from previously described sweetpotato phytoplasma and inclusion of other selected phytoplasma for comparison indicated that Aus1 and Aus142A belonged to the Candidatus Phytoplasma aurantifolia species (16SrII). The 16S genes of Aus1 and Aus142A were almost identical to those of sweet potato little leaf (SPLL‐V4) phytoplasma from Australia (99.3%–99.4%) but different from those of the sweetpotato phytoplasma from Taiwan (95.5%–95.6%) and Uganda (SPLL‐UG, 90.0%–90.1%). Phylogenetically, Aus1, Aus142A and a phytoplasma previously described from sweetpotato in the Northern Territory of Australia formed a group distinctly different from other isolates within Ca. Phytoplasma aurantifolia species. These findings indicate that novel isolates of the 16SrII‐type phytoplasma pose a potential threat to sustainable sweetpotato production in northern Australia.     

Occurrence,Molecular Characterization and Vector Transmission of a Phytoplasma Associated with Rapeseed Phyllody in Iran

Symptoms of rapeseed phyllody were observed in rapeseed fields of Fars, Ghazvin, Isfahan, Kerman and Yazd provinces in Iran. Circulifer haematoceps leafhoppers testing positive for phytoplasma in polymerase chain reaction (PCR) successfully transmitted a rapeseed phyllody phytoplasma isolate from Zarghan (Fars province) to healthy rapeseed plants directly after collection in the field or after acquisition feeding on infected rapeseed in the greenhouse. The disease agent was transmitted by the same leafhopper from rape to periwinkle, sesame, stock, mustard, radish and rocket plants causing phytoplasma‐type symptoms in these plants. PCR assays using phytoplasma‐specific primer pair P1/P7 or nested PCR using primers P1/P7 followed by R16F2n/R2, amplified products of expected size (1.8 and 1.2 kbp, respectively) from symptomatic rapeseed plants and C. haematoceps specimens. Restriction fragment length polymorphism analysis of amplification products of nested PCR and putative restriction site analysis of 16S rRNA gene indicated the presence of aster yellows‐related phytoplasmas (16SrI‐B) in naturally and experimentally infected rapeseed plants and in samples of C. haematoceps collected in affected rapeseed fields. Sequence homology and phylogenetic analysis of 16S rRNA gene confirmed that the associated phytoplasma detected in Zarghan rapeseed plant is closer to the members of the subgroup 16SrI‐B than to other members of the AY group. This is the first report of natural occurrence and characterization of rapeseed phyllody phytoplasma, including its vector identification, in Iran.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.