Phytoplasma Universal Primer Pairs Research Articles

First report of 16SrII-V phytoplasma associated with cowpea (Vigna unguiculata) phyllody in China

Cowpea plants exhibiting virescence, phyllody and little leaf symptoms were observed in the fields of Zhanjiang City, Guangdong province of China in October 2020. Total DNA was extracted from symptomatic cowpea leaves. Using phytoplasma universal primer pairs (R16mF2/R16mR1, P1/P7) and a secA primer pair (secAfor1/secArev3), expected PCR fragments of approximately 1400, 1800 and 800 bp were amplified from all symptomatic cowpea leaves. MUSCLE analysis revealed that the representative 16S rRNA gene sequence of cowpea phyllody phytoplasma (CPP) shared 98.67% nucleotide identity with the reference strain WBDL of ‘Ca. Phytoplasma aurantifolia’ (GenBank accession number U15442); and 100% nucleotide identity with 16SrII-V phytoplasmas associated with ‘Praxelis clematidea’ phyllody (GenBank accession number KY568717); ‘Desmodium triflorum’ little leaf (GenBank accession number MT452308); and ‘Ixeris chinensis’, ‘Emilia sonchifolia’, and ‘Desmodium ovalifolium’ witches’ broom (GenBank accession number MT416114, MT420682 and MK956144, respectively). Phylogenetic analyses based on the 16S rRNA and secA gene sequences revealed that the CPP strain clustered with the 16SrII-V subgroup and 16SrII group, respectively. Virtual restriction fragment length polymorphism analysis of the 16S rRNA sequence revealed that CPP was associated with the 16SrII-V subgroup. To our knowledge, this is the first report of the16SrII-V subgroup of phytoplasmas associated with cowpea phyllody disease.

Detection, Identification, and Molecular Characterization of the 16SrII-V Subgroup Phytoplasma Strain Associated with Digera muricata in Taiwan.

Digera muricata (L.) Mart. is a pantropical annual herb belonging to the Amaranthaceae family. In August 2021, D. muricata with indicative phytoplasma symptoms of phyllody, witches'-broom, and virescence was discovered adjacent to a peanut field in Mailiao, Yunlin, Taiwan. The causal agent of the observed symptoms was detected and identified by a series of molecular characterizations. Sieve elements of the phloem tissue were perused under the transmission electron microscope and revealed the presence of pleomorphic phytoplasma-like organisms. Nested PCR using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 was able to amplify a 1.2-kb DNA fragment for the 16S rRNA gene only from the symptomatic D. muricata. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP further affirmed that the phytoplasma associated with the diseased D. muricata can be classified into the 16SrII-V subgroup. Moreover, displayed evident symptoms were explained by the concomitant detection of PHYL1 and SAP11, the virulence genes responsible for the development of leaf-like flowers and shoot proliferation, respectively. Although phytoplasma infection on the noncrop species does not have a direct economic impact, its role in disease spread and perpetuation is indubitable.

First Report of 16SrII-V Peanut Witches' Broom Phytoplasma in Snake Gourd (Trichosanthes cucumerina L.) in Taiwan.

Snake gourd (Trichosanthes cucumerina L.), an annual climbing plant belonging to the family of Cucurbitaceae, is native to Southeast Asia countries, e.g., India, Pakistan, Malaysia, China, and Indonesia. It is commonly consumed as a vegetable and also used as a traditional herbal medicine due to the antidiabetic, anti-inflammatory, antibacterial, hepatoprotective, and cytotoxic activities (Devi 2017). In September 2020, phytoplasma-induced disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom were observed on snake gourd in Yunlin County, Taiwan. The cross-sectional examination of the symptomatic plant by transmission electron microscopy showed typical phytoplasma-like pleomorphic bodies with spherical, oval and tubular shapes in sieve elements. Further examination by nested PCR revealed that a 1.2 kb DNA fragment for 16S rRNA gene was only amplified from symptomatic leaf of snake gourd using the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2. BLAST and iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi) analyses on the amplified DNA fragment (accession no. MW309142) revealed that it shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches' broom (PnWB) phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain (Firrao et al. 2004), and could be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further characterized by western blotting using the polyclonal antibody raised against the Imp of PnWB phytoplasma (Chien et al. 2020a, b). An expected signal of 19 kDa specific for Imp was only detected in the symptomatic snake gourd, but not in healthy snake gourd. Since the disease symptoms caused by phytoplasma infection are highly dependent on the secreted effectors (Namba 2019), phyllogen gene that is responsible for phyllody and virescence symptoms was amplified from symptomatic snake gourd by PCR. BLAST analysis revealed that phyllogen identified in snake gourd is identical with that of PnWB phytoplasma. In Taiwan, species of family Cucurbitaceae such as loofah, bitter gourd, and pumpkin are commonly infected by 16SrVIII phytoplasma (Davis 2017). In this study, we report for the first time that snake gourd, a species of family Cucurbitaceae, was infected by 16SrII-V PnWB phytoplasma in Taiwan.

First Report of 16SrII-V Phytoplasma Associated with Green Manure Soybean (Glycine max L.) in Taiwan.

QING PI DOU, a local variety of soybean (Glycine max (L.) Merrill) with small seed size, is primarily cultivated in the southern region of Taiwan. Due to the advantage of high germination rate, fast growth and high nitrogen fixation capacity, QING PI DOU has widely used as green manure in rotation with rice to increase soil fertility in Taiwan. In the summer of 2020, phytoplasma-induced disease symptoms were observed in QING PI DOU with 23% (18/78) disease incidence in Yunlin County, Taiwan. These plants exhibited severe disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom compared to healthy plants. Leaf samples of the symptomatic plants were subsequently collected and examined through transmission electron microscopy (TEM), PCR, and western blotting analyses. The ultrathin sections of the diseased QING PI DOU were double-stained with uranyl acetate and lead citrate. The typical phytoplasma-like pleomorphic bodies were observed in sieve elements of leaf veins by TEM. To investigate the association of phytoplasma with the diseased QING PI DOU, total DNA extracted by the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) was examined by nested PCR using the phytoplasma universal primer pair P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). The 1.2 kb PCR product specific for 16S ribosomal RNA (16S rRNA) gene was only amplified from symptomatic plants but not from healthy plants. BLAST analysis demonstrated that the sequence (accession no. MW393690) of amplified DNA fragment of 16S rRNA is identical to that of GenBank accession no. NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches' broom (PnWB) phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain (Firrao et al. 2004). Further analysis on the virtual RFLP pattern of MW393690 generated by iPhyClassifier confirmed that the phytoplasma identified in the diseased QING PI DOU can be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further selected for total cell extracts preparation and characterized by western blotting using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of PnWB phytoplasma (Chien et al. 2020). An expected signal of 19 kDa specific for Imp was only detected in symptomatic plants but not in healthy plants. Moreover, the PCR products encoding SAP11 and phyllogen, the virulence factors responsible for phytoplasma-induced witches' broom and phyllody symptoms (Namba 2019), were also amplified from symptomatic QING PI DOU by PCR using the primer pairs 5'-ATGGCTCCCGAAAAAAATGATAAAGG-3'/5'-TTTTTTAGAATCATCAGGCTTTTTAG-3' (0.28 kb) and 5'-ATGGATCCAAAACTTCCAGAAACT-3'/5'-GTTTTTTTCATCATTTAAATCAT-3' (0.27 kb), respectively. Further analysis by BLAST revealed that SAP11 and phyllogen identified in symptomatic QING PI DOU are identical with those of PnWB phytoplasma. To the best of our knowledge, this report is the first to describe phytoplasma-associated soybean (Glycine max L.) witches' broom disease in green manure soybean in Taiwan.

Ixeris Chinensis is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan.

Ixeris chinensis (Thunb. ex Thunb.) Nakai, a perennial herbaceous plant that belongs to the family of Asteraceae, is widely distributed at mid-low altitude regions in Taiwan. I. chinensis is commonly used as traditional herbal medicine for the treatment of inflammation, bronchitis, pneumonia, and diarrhea. In March 2020, disease symptoms such as shoot proliferation, phyllody, virescence, purple top, and witches' broom were observed on I. chinensis at the sansheng community park in Mailiao, Yunlin County, Taiwan. Totally, eight I. chinensis plants were checked and half of them were symptomatic. These disease symptoms are similar to those associated with peanut witches' broom (PnWB) disease identified in the same area (Liu et al. 2015). Three samples mixed with leaf, stem, and flower were tested including one healthy and two symptomatic I. chinensis. The total DNA of each sample was extracted and examined by nested PCR for the amplification of 16S rDNA with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). A specific signal of expected size (1.2 kb) for 16S rDNA was only detected in the symptomatic I. chinensis, but not in healthy I. chinensis. The nucleotide sequence (accession no. MT416114) of the amplified DNA fragment using primer pairs P1/P7 from symptomatic I. chinensis is identical to that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Analysis of the virtual RFLP pattern of MT416114 generated by iPhyClassifier revealed that the phytoplasma detected in symptomatic I. chinensis belongs to a 16SrII-V subgroup. The total protein of each sample was also extracted and examined by western blotting using the polyclonal antibody raised against Imp protein of purple coneflower witches' broom phytoplasma (Chien et al. 2020), which is identical with that (accession no. ADD59806) of PnWB phytoplasma. An expected signal of 19 kDa specific for Imp was detected in symptomatic I. chinensis, but not in healthy I. chinensis. Subsequent PCR, DNA sequencing and western blotting assays further confirmed that the gene encoding a SAP11-like protein was only detected in symptomatic I. chinensis, and shares 100% identity with that (accession no. EMR14684) of PnWB phytoplasma. Our results indicate that PnWB phytoplasma causes disease in I. chinensis, a common weed, which may act as an alternative natural host and facilitate the spreading of phytoplasma disease in Taiwan.

Threeflower Tickclover (Desmodium triflorum) is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan.

Three-flower Tick-clover (Desmodium triflorum) is a perennial herbaceous plant that belongs to the family of Leguminosae. Threeflower tickclover widely grows at mid-low altitude regions in Taiwan and is commonly used as a traditional herbal medicine for the treatment of dysmenorrheal, muscle spasm, cough, pain and poisoning. In March 2020, disease symptoms such as little leaf, phyllody, virescence, and witches' broom were observed on threeflower tickclover at the sansheng community park in Mailiao, Yunlin County, Taiwan. Similar disease symptoms were observed on peanut infected with peanut witches' broom (PnWB) phytoplasma grown in the same area (Liu et al. 2015). Leaf samples collected from the healthy and symptomatic threeflower tickclover were used to extract total DNA and protein for PCR and western blotting assays, respectively. Nested PCR was performed with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 for the amplification of 16S ribosomal RNA (rRNA) gene (Lee et al. 1993). A specific DNA fragment of expected size (1.2 kb) for 16S rRNA was only amplified from leaf samples of symptomatic threeflower tickclover. The nucleotide sequence of the amplified DNA fragment using primer pairs P1/P7 was deposited into the GenBank (accession no. MT452308). Blast analysis revealed that MT452308 shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Based on the virtual RFLP pattern of MT452308 generated by iPhyClassifier, the phytoplasma detected in symptomatic threeflower tickclover could be classified into the 16SrII-V subgroup. For western blotting, the polyclonal antibody raised against Imp protein of purple coneflower witches' broom phytoplasma (Chien et al. 2020), which is identical with that (accession no. ADD59806) of PnWB phytoplasma, was used. An expected signal of 19 kDa specific for Imp was only detected in threeflower tickclover exhibiting disease symptoms. Subsequent assays including PCR, DNA sequencing and western blotting further confirmed that the gene encoding a SAP11-like protein (accession no. EMR14684) identified in PnWB phytoplasma was also found in samples of symptomatic threeflower tickclover, and shares 100% identity with each other. Our results indicate that threeflower tickclover, a common weed in Taiwan, may act as an alternative natural host for PnWB phytoplasma, and contributes to the spreading of phytoplasma disease.

Fringed Spiderflower (Cleome rutidosperma) Is a New Host for Purple Coneflower Witches’ Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan

Fringed spiderflower (Cleome rutidosperma) is an annual herbaceous plant that grows as a weed. Originally from tropical Africa, fringed spiderflower has been introduced and is becoming widely distributed at low altitudes in Taiwan. In June 2019, symptoms such as small leaves, witches’ broom, floral virescence, and phyllody were observed on fringed spiderflowers grown at the agricultural experiment station in Wufeng, Taichung City, Taiwan. These disease symptoms are similar to those associated with purple coneflower (Echinacea purpurea) witches’ broom disease caused by a 16SrII-A subgroup phytoplasma identified in the same area (Chang et al. 2015; Tseng et al. 2012). Leaf samples of spiderflowers exhibiting disease symptoms were collected for western blotting and PCR assays. Total proteins were extracted from plant samples and examined by western blotting using the polyclonal antibody raised against immunodominant membrane protein (Imp, accession no. KPN61379) of purple coneflower witches’ broom phytoplasma. A specific signal of expected size (19 kDa) for Imp was detected in the symptomatic leaf samples but not in healthy leaf samples. For PCR analysis, the total DNA of each sample was extracted using the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) and used as a template in nested PCR with the phytoplasma universal primer pairs P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). DNA extracted from leaves sampled on healthy plants were used as a negative control. An amplified DNA fragment of 1.2 kb specific for 16S ribosomal RNA (16S rRNA) obtained from symptomatic leaf samples was gel purified and sequenced. The nucleotide sequence was deposited into GenBank (accession no. MN213634). BLAST analysis revealed that MN213634 is identical to the nucleotide sequence of GenBank accession LKAC01000001.1 (complement [31093 to 32611]) of phytoplasma associated with purple coneflower witches’ broom disease. Using iPhyClassifier, the virtual restriction fragment length polymorphism pattern of the 16S rDNA fragment (accession no. MN213634) confirmed that the phytoplasma detected in symptomatic fringed spiderflower belongs to the 16SrII-V subgroup. Subsequent PCR and DNA sequencing assays further confirmed that the genes encoding the putative secreted proteins (accession nos. WP_054598519 and KPN61138) identified in purple coneflower witches’ broom phytoplasma were also found in samples of symptomatic fringed spiderflowers. Our findings revealed that the fringed spiderflower is a new weed plant host for purple coneflower witches’ broom phytoplasma. This result expands our knowledge of the natural host range of 16SrII-V subgroup strains.

Mixed infection of phytoplasma and begomovirus associated with leaf curling and witches’ broom disease of Zinnia elegans in India

Zinnia elegans belonging to the asteraceae family is a flowering annual summer plant and is native to Mexico. Symptoms of excessive leaf curling, little leaf, stunting and witches’ broom were observed on Z. elegans in Tripura state of India during 2016–2017. PCR assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 and tomato leaf curl virus coat protein specific primers yielded specific amplifications of ~ 1.25 kbp and ~ 800 bp, respectively from the symptomatic zinnia leaf samples. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene sequences of zinnia isolate confirmed the presence of ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup). Further coat protein sequence comparison and phylogenetic tree analysis of coat protein gene confirmed association of begomovirus with zinnia symptomatic samples and identified it as a strain of tomato leaf curl virus. Our results confirmed a mixed infection of ‘Ca. P. asteris’ and begomovirus with zinnia leaf curl and witches’ broom symptoms.

First Report of ‘Candidatus Phytoplasma solani’ Infecting Plum (Prunus domestica) in Jordan

Plum (Prunus domestica L., Rosaceae) is among the most important stone fruit species grown in Jordan, especially in the northeastern part of the country. In September 2017, 30 trees from a 2-ha orchard of cultivar ‘Early Queen’ showing possible phytoplasma symptoms of leaf yellowing and reddening, stunted growth, and witches’ broom were observed. Additionally, there were around 20 bindweed plants (Convolvulus arvensis L.) among the plum trees showing stunting and leaf reddening in the Mafraq region in northeast Jordan. Leaf samples were collected from 15 symptomatic and five asymptomatic plum trees and three symptomatic bindweed plants. Total DNA was extracted from all samples and analyzed by PCR using the phytoplasma universal primer pair P1/P7 (16S/23S RNA, Deng and Hiruki 1991; Schneider et al. 1995), followed by nested PCR with primer pair R16F2n and R16R2 (16S rDNA, Gundersen and Lee 1996). The same samples were also tested by PCR using the 16SrXII-specific primers SecY1a and SecYR1 (secY, Lee et al. 2010) and by “stolbur” phytoplasma specific quantitative PCR (16S rDNA, Hren et al. 2007). DNA samples of five out of 15 P. domestica and all three bindweed samples tested positive for the presence of a phytoplasma by qPCR (Ct values of 19.5 to 26.2 and 33 to 34 for plum and bindweed, respectively) and nested PCR, yielding the expected PCR amplicons. Interestingly, no phytoplasma was detected in 10 samples from symptomatic plum trees as well as from all the asymptomatic trees. Bidirectional sequencing (Macrogen, Amsterdam, The Netherlands) was done for each PCR amplicon after gel purification. A BLASTn similarity analysis of the assembled consensus sequences derived from the plum and the bindweed host plants revealed that the sequences of phytoplasmas infecting P. domestica (accession nos. MH085227, MH085228, and MH085229) and C. arvensis (MH085225 and MH085226) in Jordan had identity of 99% to the deposited sequence of ‘Candidatus Phytoplasma solani’ from of Vitis vinifera L. in Jordan (KC835139) and the ‘Ca. P. solani’ strain sequence from Capsicum annuum L. from Serbia (AF248959). The same analysis based on the secY gene confirmed the highest identity (99%) of analyzed sequences of ‘Ca. P. solani’ (MK928324 to MK928326) with strains from Russia and Germany (GU004346 and FO393427, respectively). The identity of ‘Ca. P. solani’ was confirmed in plum and bindweed by virtual RFLP (iPhyClassifier, Zhao et al. 2009) and phylogenetic analyses (BioNumerics, Applied Math, Belgium). These results supported the first occurrence of ‘Ca. P. solani’ in plum in Jordan, where it may represent a potential threat to other already reported susceptible stone fruit hosts, such as peach, almond, and cherry. The reported presence of the bindweed in Jordan (Salem et al. 2013), a well-known phytoplasma natural reservoir (Mehle et al. 2011), and potential insect vectors (e.g., Hyalesthes obsoletus) (Quaglino et al. 2013) along with vegetatively propagated plant material (Bertaccini et al. 2014) may have contributed to the plum infection in Jordan.

First Report of ‘Candidatus Phytoplasma ulmi’ on Elm in Belgium

In April 2017, the project ‘Belgian Plant Sentinel Network’ started as part of the Euphresco-IPSN2 network. The botanical gardens and arboreta of the network contain diverse plant collections with both exotic and native taxa that can be used as sentinels by recording their sensitivity to either native or to (newly) introduced pests and diseases. One of the case studies focused on monitoring phytoplasma diseases in trees, especially on Ulmus spp. To monitor these species, a total of 117 root samples were taken from collection trees in five botanical gardens and arboreta in Flanders, Belgium. Total genomic DNA was extracted by a CTAB protocol (Doyle and Doyle 1990). The 16S rRNA gene was partially amplified using the phytoplasma universal primer pairs P1/P7 (Deng and Hiruki 1991) followed by nested PCR with primer pairs R16F2/R2 (Lee et al. 1995) as diagnostic test, and P1/Tint (Smart et al. 1996) on positive samples for sequencing reasons. Additionally, specific group V nPCR targeting the secY (primer sets FD9f2L/FD9r followed by FD9f3L/FD9r2L) gene, according to Arnaud et al. (2007), was done to yield a nested PCR product of 1,174 bp, and finally, a short fragment from the tuf gene was generated following the Q-Bank methodology for Q-phytoplasma (www.Q-bank.eu). In one location in Vlaams Brabant, one sample, roots from an Ulmus minor subsp. canescens tree (∼8 years old) tested positive for the presence of a phytoplasma. The sample was taken in March 2018 (early spring) and only consisted of roots. An amplified fragment of ∼1,600 bp (P1/Tint) was gel purified (SmartPure, Eurogentec) and directly sequenced (Macrogen, Amsterdam, the Netherlands). The obtained partial sequence of the 16S ribosomal RNA gene (1,516 bp) was submitted to GenBank (accession MH583020.2). This sequence revealed the closest homology (99%) with ‘Candidatus Phytoplasma ulmi’ strains from the U.S.A. (AF189214.1) and Czech Republic (EU184021). In addition, the secY and tuf fragment were directly bidirectionally sequenced and the sequences of the resulting contigs, 1,027 bp (MH727566) and 367 bp (MK491655), respectively, were also submitted to GenBank. Phylogenetic analysis was undertaken (MEGA version 7.0.26) on the 16S, secY, and tuf fragments, indicating the phytoplasma to be a member of ‘Ca. P. ulmi’, 16SrV subgroup A, and showing the genetic relationship with other ‘Ca. P. ulmi’ isolates previously reported in Europe. Virtual RFLP on the 16S fragment using iPhyClassifier (Zhao et al. 2009) confirmed this result. All other Ulmus trees from the five botanical gardens and arboreta, as well as related Zelkova trees (Z. carpinifolia and Z. serrata) and a Celtis aetnensis tree, tested negative. In addition, the phytoplasma survey on elm has expanded to the complete affected botanical garden (28 Ulmus or related trees) and to lane trees from the surrounding area. A second sampling of the same tree in May 2018 confirmed the presence of ‘Ca. P. ulmi’ on new root and leaf samples. From July onward, yellowing and partial branch decay could be observed on the affected tree. All additional samples from the botanical garden and surrounding area tested negative. To our knowledge, this is the first report of ‘Ca. P. ulmi’ on Ulmus in Belgium. Considering the other recent findings in Europe, we suspect that ‘Ca. P. ulmi’ is likely to be distributed throughout the region, but with few previous reports, rather than being a new emerging risk.

Molecular Identification of Sugarcane White Leaf in Puer, Yunnan Province, China

To confirm the phytoplasma association in 13 sugarcane white leaf (SCWL) symptomatic samples collected from Puer, China, nested PCR assays with phytoplasma universal primer pairs was performed. A ~ 1240 bp product was obtained from 10 out of 13 symptomatic samples, and 16S rRNA gene sequencing results showed that all sequences of SCWL isolates were 100% identical to each other. BLASTn analysis revealed 100% sequence similarity with the 16S rRNA genes from SCWL phytoplasma strains LC7 and LC9 from Lincang, Yunnan province, China belonging to 16SrXI-B subgroup. Virtual restriction fragment length polymorphism also confirmed that Puer SCWL strains were member of the 16SrXI-B subgroup.

First report of ‘Candidatus Phytoplasma rubi’ on blackberry in Belgium

In May 2018 stunted blackberry plants were observed on a small-scale organic farm producing several small fruit types in West Flanders, Belgium. Besides severe stunting, leaf yellowing and distortions could also be observed (Figs. 1-2). Only Rubus fruticosus plants of the cultivar Obsidian showed symptoms but disease incidence was estimated to be almost 100%, of which almost half the plants were stunted. Other cultivars did not seem affected. Seven symptom-bearing plants, of which four had severe stunting, and three only leaf symptoms, were sampled and tested. Additionally, three symptomless plants from neighbouring rows and belonging to different cultivars, were tested. All samples consisted of leaves and roots, and tests were conducted on a pooled leaf/root sample. Total genomic DNA was extracted by a CTAB protocol. The 16S rRNA gene was partially amplified using the phytoplasma universal primer pair P1/P7 (Deng & Hiruki, 2; Schneider et al., 5) followed by a nested PCR with R16F2/R2 primers (Lee et al., 3) as a diagnostic test, and P1/Tint primers (Smart et al., 6) on positive samples for sequencing. All of the diseased plants tested positive for phytoplasma in nested PCR tests. Additionally, specific phytoplasma group V primers targeting the tuf (FDTUF-F1/R1 & FDTUF-F2/R2; Malembic-Maher et al., 4), and secY (primer sets FD9f2L/FD9r & FD9f3L/FD9r2L; Arnaud et al., 1) genes were used in nested PCRs and yielded fragments of 998 and 1174 bp respectively. The obtained 16SrRNA (P1/Tint), tuf and secY fragments were gel purified (SmartPure, Eurogentec), and sequenced directly (Genewiz, Leipzig, Germany). BLAST analysis of the 16SrRNA sequence, revealed the highest identity (99.85%) with a ‘Rubus stunt phytoplasma’ strain from blackberry (R. fruticosus) in Italy (isolate RuS400; GenBank Accession No. AY197649). Partial sequence of the 16S ribosomal RNA (1607 bp), and tuf (835 bp) and secY (1053 bp) genes were submitted to GenBank (MH801133, MH809672 and MH809673, respectively). Phylogenetic analysis was undertaken on all three fragments, and the phytoplasma was identified as ‘Candidatus Phytoplasma rubi’, ‘elm yellows’ group 16SrV-E (Figs. 3-5). To our knowledge, this is the first report of ‘Ca. P. rubi’ on blackberry in Belgium. An additional nationwide survey is needed to assess the phytopathological impact of this outbreak. Although a growing number of reports indicate the increased importance of Rubus stunt, associated with ‘Ca. P. rubi’, unlike the related ‘Ca. P. ulmi’, associated with elm trees, no regulatory actions are currently taken against this pathogen.

First report of a ‘Candidatus Phytoplasma trifolii‘‐related strain associated with rapeseed witches' broom in Iran

Rapeseed (Brassica napus) is an economically important oilseed crop grown in many countries worldwide. During recent years rapeseed cultivation has developed in regions of Iran. In April 2018, symptoms similar to phytoplasma-associated diseases including witches' broom (Fig. 1), stunting and little leaves (Fig. 2) were observed in rapeseed in several areas of Hormozgan province, Iran. To identify the association of phytoplasma with the diseased plants, total genomic DNA was extracted from midribs of eight symptom-bearing and two symptomless plants using the CTAB method. The samples were analysed for phytoplasma DNA by direct PCR using the phytoplasma universal primer pair P1/P7 and nested PCR using primers P1/P7 (first round) followed by R16F2n/R16R2 (second round) (Gundersen & Lee, 2). Amplicons of the expected sizes (1800 and 1250 bp) were amplified with direct and nested PCR in all symptom-bearing plants but not from symptomless plants or sterile distilled water used as negative controls. A positive nested PCR product was directly sequenced and deposited in GenBank under accession no. MH430092. A BLAST search using the sequenced fragment showed that the phytoplasma associated with rapeseed witches' broom (RsWB) had 100% nucleotide identity with the corresponding sequence of several members (KX773529, MG788318 and AM260486) of the 16SrVI phytoplasma group and 99.8% identity with the type strain, ‘Candidatus Phytoplasma trifolii’ (AY390261). The virtual RFLP revealed that the RsWB phytoplasma belonged to the 16SrVI-A subgroup. There are several reports regarding the association of aster yellows group (16SrI) phytoplasmas with rapeseed (e.g. rapeseed phyllody phytoplasma) in Iran (Salehi et al., 6) and other countries (Maliogka et al., 4; Olivier et al., 5). To our knowledge, this is the first report of natural infection of rapeseed by a 16SrVI phytoplasma in Iran and most probably in the world. Phytoplasmas belonging to the 16SrVI-A have been previously reported on several economically vegetable crops such as tomato and cucumber in Iran (Esmailzadeh Hosseini et al., 1; Jamshidi et al., 3). Therefore, the infected volunteer rapeseed around tomato and cucumber fields may act as an alternative host for this phytoplasma. The authors would like to thank Dr. Majid Siampour for his excellent technical assistance.

First report of ‘Candidatus Phytopalsma asteris’ subgroup 16SrI-Bassociation with a witches’ broom disease of fennel

Fennel is an economically important spice crop grown in many countries worldwide. In April 2016 witches’ broom symptoms were observed in fennel plants at fields of IARI, New Delhi, India. The symptomatic samples were analysed for phytoplasma DNA presence by PCR using the phytoplasma universal primer pair P1/P7 and nested PCR assay using primers R16F2n/R16R2. Amplicons of the expected sizes (1, 800 and 1, 250 bp respectively) were amplified in all the symptom-bearing fennel plants, but not from the symptomless plants used as negative controls. A nested PCR product was directly sequenced and deposited in GenBank. The BLASTn search analysis using the 16S rDNA sequenced fragment showed that the phytoplasma associated with fennel witches’ broom had 99% nucleotide identity with the sequences of ‘Candidatus Phytoplasma asteris’-related strains. The phylogeny and virtual RFLP analysis revealed that the phytoplasma belonged to the 16SrI-B subgroup. To our knowledge, this is the first report of infection of fennel by a strain of 16SrI-B phytoplasmas.

First Report of ‘Candidatus Phytoplasma trifolii’-Related Strain of 16SrVI-A Phytoplasma Subgroup, Associated with Elm Yellows Disease in American Elm (Ulmus americana L.) in Ohio, U.S.A

During the investigation of the sudden and early onset of yellowing, followed by mortality of American elm (Ulmus americana L.) trees at the USDA Forest Service Northern Research Station in Delaware, Ohio, a phytoplasma of the clover proliferation group (16SrVI) was detected as the putative causal agent of the disease outbreak. Onset of symptoms was rapid and widespread, occurring in late July 2016 and affecting ∼60 trees across two elm research plantations. Symptoms included a general yellowing of individual tree canopies, epinasty of foliage throughout the canopy, phloem discoloration, and on a subset of trees, a strong odor of methyl salicylate (observed in phloem tissue extracted from the lower stem). Similar symptoms in elms have been attributed to the classic elm yellows ‘Candidatus Phytoplasma ulmi’ (16SrV-A) (Lee et al. 2004) and the Illinois elm yellows phytoplasma (16SrVI-C) (Jacobs et al. 2003). In July 2016, samples were collected from 12 symptomatic and 8 asymptomatic American elm trees. DNA from the leaf midrib and branch phloem was isolated and analyzed for phytoplasma via seminested polymerase chain reactions (PCR). PCRs were first primed by phytoplasma universal primer pair P5/P7 (Jomantiene et al. 1998), followed by P7 and the reverse complement of the universal phytoplasma primer R16R2 for amplification of the phytoplasma 16S-23 ribosomal (r) DNA (16S-23 rRNA gene) sequences as per Gundersen and Lee (1996). The predicted band size of the second PCR product is 487 base pair (bp). The product bands were isolated, purified, and sequenced using primer P7. Sequencing results of the PCR products indicated that nine of the symptomatic and one of the asymptomatic American elm trees tested were infected by a phytoplasma. A BLAST search of the DNA sequences indicated high similarities to members of the ‘Ca. P. trifolii’ group 16SrVI-A. The sequences of all 10 phytoplasma-infected trees were identical to each other. To further confirm that a strain of ‘Ca. P. trifolii’ was infecting the elms, a PCR product was cloned and sequenced. The 1,557 bp band was the product of primers P1a/P7, followed by PCR primers designed from the sequence of ‘Ca. P. trifolii’. This band was cloned into the pMiniT 2.0 vector. Plasmid sequencing used standard sequencing primers (SP6 and T7 promoters), the primers included with the vector, and finally, custom designed phytoplasma primers that eliminate other bacterial DNA from getting amplified. The entire plasmid clone was sequenced in both directions with four to six times of coverage per base. Determination of the phytoplasma classification group was based on the nucleotide sequence within the phytoplasma universal primers F2n/R2 PCR fragment within the 16Sr gene. Using iPhyClassifier, the online tool for phytoplasma classification and taxonomic assignment (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi), the sequence similarity between the Delaware elm phytoplasma and ‘Ca. P. trifolii’ (GenBank accession no. AB279597.1) is 99.9% in the 16Sr region, which places the Delaware elm phytoplasma in the 16SrVI-A group. The sequence of the plasmid was deposited in GenBank under accession number MF385584. Elm decline and yellows diseases in North America have been associated with the Illinois elm yellows phytoplasma (16SrVI-C, GenBank accession no. AF268893.1) (Jacobs et al. 2003), ‘Ca. Phytoplasma ulmi’ (16SrV-A) (Lee et al. 2004), and phytoplasma in the aster yellows group (16SrI-C) (Lee et al. 1995).

Molecular detection and characterization of phytoplasma associated with China aster (Callistephus chinensis) phyllody in India

China aster (Callistephus chinensis L.) is one of the most popular annual flowering plant grown through-out the world. Phyllody disease of China aster is a phytoplasma associated disease that induces severe economic losses. Phytoplasmal disease in China aster was assessed for phytoplasma by direct polymerase chain reaction primed by using phytoplasma universal primer pairs PI/P7. A 1.8 Kb DNA fragments encoding the portion of phyto-plasma 16SrDNA amplified by PCR was cloned and sequenced. Sequencing of the PCR product and BLAST analy-sis indicated that China aster phyllody phytoplasma strain shared maximum sequence identity (99%) with strains of Peanut Witches’ broom (16SrII) phytoplasma group. Phylogenetic relationship of 16SrDNA sequence of China aster phyllody phytoplasma strain in the present study confirmed association of Peanut Witches’ broom (16SrII) group of phytoplasmas with China aster phyllody disease in India.

Molecular detection and characterization of 16SrI phytoplasma associated with rice orange leaf symptom in Vietnam

Rice plants showing orange leaf disease symptoms were observed in Southern Vietnam. A nested polymerase chain reaction assay using phytoplasma-universal primer pairs P1/P7 and R16F2n/R16R2 amplified products of approximately 1.2 kb only from symptomatic rice plants. Sequence analysis of 16Sr DNA amplicons showed that the phytoplasma strain VNTG-4 shared 100% sequence homology with phytoplasma strains from rice in Thailand, members of the 16SrI group. This is the first detection and identification of a phytoplasma associated with the orange leaf disease in Vietnam.

Molecular detection and characterization of a phytoplasma associated with maize bushy stunt in Vietnam

Maize bushy stunt symptoms were observed in Quang Ngai province, Vietnam. A nested polymerase chain reaction assay using phytoplasma-universal primer pairs P1/P7 and R16F2n/R16R2 amplified products of about 1, 200 bp from samples collected from these maize bushy stunt showing samples, whereas no amplicon was detected from the symptomless samples collected from northern Vietnam. Nucleotide sequence analysis of the 16S rDNA products revealed that phytoplasma associated with maize bushy stunt symptom in Quang Ngai shared 99% sequence homology with a phytoplasma strain from maize bushy stunt in Colombia, member of the 16SrI, ‘Candidatus Phytoplasma asteris’ group. This is the first detection and identification of a phytoplasma associated with the maize bushy stunt disease in Vietnam.

Differentiation and Classification of Phytoplasmas Associated with Almond and GF‐677 Witches'‐Broom Diseases using rRNA and rp Genes

AbstractStone fruits are affected by several diseases associated with plant pathogenic phytoplasmas. Previous studies have been shown that phytoplasma agents of almond and GF‐677 witches'‐broom (AlmWB and GWB, respectively) diseases belong to pigeon pea witches'‐broom (16SrIX) phytoplasma group. In this study, partial biological and molecular characterization was used to compare and classify phytoplasma agents of Khafr AlmWB (KAlmWB) and Estahban GWB (EGWB) diseases. Production of different symptoms in periwinkle indicated that agents of KAlmWB and EGWB are differentiable. Expected fragments were amplified from diseased almond and GF‐677 trees in direct PCR using phytoplasma universal primer pairs P1/P7 and rpF1/rpR1 and nested PCR using P1/P7 followed by R16F2n/ R16R2 primer pair. 16S‐rDNA Restriction fragment length polymorphism (RFLP) as well as phylogenetic analysis of rplV‐rpsC and 16S–23S rRNA spacer region sequences classified KAlmWB and EGWB phytoplasmas within 16SrIX‐C (rpIX‐C) and 16SrIX‐B (rpIX‐B) subgroups, respectively.

Group 16SrXI phytoplasma strains, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D, are associated with sugar cane white leaf.

Sugar cane white leaf (SCWL) is a serious disease caused by phytoplasmas. In this study, we performed nested PCR with phytoplasma universal primer pairs (P1/P7 and R16F2n/R16R2) for the 16S rRNA gene to detect SCWL phytoplasmas in 31 SCWL samples collected from Baoshan and Lincang, Yunnan, China. We cloned and sequenced the nested PCR products, revealing that the 16S rRNA gene sequences from 31 SCWL samples were all 1247 bp in length and shared more than 99 % nucleotide sequence similarity with the 16S rRNA gene sequences of SCWL phytoplasmas from various countries. Based on the reported 16S rRNA gene sequence data from SCWL isolates of various countries, we conducted phylogenetic and virtual RFLP analysis. In the resulting phylogenetic tree, all SCWL isolates clustered into two branches, with the Lincang and Baoshan SCWL phytoplasma isolates belonging to different branches. The virtual RFLP patterns show that phytoplasmas of the Lincang branch belong to subgroup 16SrXI-B. However, the virtual RFLP patterns revealed by HaeIII digestion of phytoplasmas of the Baoshan branch differed from those of subgroup 16SrXI-B. According to the results of phylogenetic and virtual RFLP analysis, we propose that the phytoplasmas of the Baoshan branch represent a new subgroup, 16SrXI-D. These findings suggest that SCWL is caused by phytoplasmas from group 16SrXI, including subgroup 16SrXI-B and a new subgroup, 16SrXI-D.