Peanut Witches' Broom Phytoplasma Research Articles

First Report of 'Candidatus Phytoplasma aurantifolia' Associated with the Invasive Weed Eclipta prostrata (L.) in Taiwan.

Eclipta prostrata (L.), commonly known as false daisy of the family Asteraceae, is an erect or prostrate annual herb that grows 5 to 45 cm tall. It is widespread mainly in tropical and subtropical regions like India, China, Taiwan, Thailand, and Brazil (Chung et al., 2017). E. prostrata has very wide medicinal properties accounted by several phytochemicals like thiophene derivatives, steroids, flavonoids, and polypeptides (Feng et. al., 2019). It is also used as a traditional herbal medicine for the treatment of bleeding, hemoptysis and itching, hepatitis diarrhea, and even hair loss (Timalsina et al., 2021). In September 2021, E. prostrata displaying branch proliferation and phyllody symptoms with about 30% (6 were symptomatic and 14 were healthy) incidence rate was observed in Mailiao, Yunlin, Taiwan where phytoplasma disease is permeating and has affected many crops and non-crop species including peanut, mungbean, curl-leaved tobacco, false amaranth, etc. Compared to healthy E. prostrata bearing white ray florets and cream or dull white disk florets, symptomatic ones developed phyllody which is more pronounced on the severely infected ones. Further examination by transmission electron microscope revealed a pleomorphic (circular, elliptical, and bell-shaped) phytoplasma-like organisms accumulated in the sieve elements of the symptomatic leaves. Phytoplasma infection was further confirmed by nested polymerase chain reaction using universal primers P1/P7 (carried out for 12 cycles), followed by R16F2n/R16R2 (carried out for 35 cycles) on the genomic DNA extracted by Plant Genomic DNA Purification Kit (DP022-150, GeneMark) (Lee et al. 1993). Results revealed that the conserved 16S rRNA gene with a 1.2 kb fragment size was amplified only by the symptomatic samples. Furthermore, western blotting was done using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of peanut witches'-broom (PnWB) phytoplasma, a 'Candidatus Phytoplasma aurantifolia' in Taiwan that belongs group to 16SrII (Chen et al. 2021). Consistent with the nested PCR, only the symptomatic samples revealed a specific Imp signal with a size of 19 kDa. To classify the phytoplasma associated with the symptomatic E. prostrata, the DNA sequence (No. OM397418) of the P1/P7 primer pair-amplified DNA fragment was obtained using P1 and a nested primer (5'-GGGTCTTTACTGACGCTGAGG-3'), which shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of PnWB phytoplasma. Further analysis of the virtual RFLP pattern of OM397418 by iPhyClassifier confirmed that the phytoplasma identified in the symptomatic E. prostrata belongs 16SrII-V subgroup. To the best of our knowledge, this is the first report of phytoplasma disease in E. prostrata associated with the 'Ca. P. aurantifolia' in Taiwan.

First report of 16SrII group (Peanut witches' Broom) Phytoplasmas associated with the Leucas aspera Phyllody in India.

Leucas aspera (Wild.) Linn. (Family: Lamiaceae) is a commonly found weed throughout India, known for its pharmacological properties. Its white flowers and leaves are used in many Ayurvedic formulations for the treatment of chronic rheumatism, psoriasis, snake bites and skin eruptions (Prajapathi et al., 2010). During a survey of commercial flower crop fields in May 2018, a few L. aspera plants, growing as unwanted weeds in the fields and surrounding agricultural wastelands with the symptoms of phyllody, virescence and little leaves were observed in Emmekoppalu (12.2106, 76.2511; n= 1/26 plants) and Beerihundi (12.1630, 76.3225; n= 2/59 plants) localities of Mysuru district, and Srirangapatna in Mandya district (12.2541, 76.411; 1/67 plants), Karnataka- India(Figure 1). 'n' denotes the symptomatic/ asymptomatic samples observed. The disease incidence in the surveyed localities ranged less than four per cent. The total genomic DNA was extracted from the leaf midrib tissues of three representative symptomatic and two asymptomatic samples using the CTAB method. The phytoplasma 16S rRNA gene was amplified in nested PCR assay by P1/P7 followed by R16F2n/R16R2 primers using Long Amplification (LA) Taq polymerase (Takara, Japan). Additionally, the PCR assays were performed for the amplification of phytoplasma secA gene using the primers SecAfor1/SecArev3 and SecAfor2/SecArev3 (Hodgetts et al., 2008). The DNA templates from all the symptomatic samples generated amplicons of approximately 1.25kb (16S rRNA gene) and 480 bp (secA gene) revealing the association of phytoplasma strains. No amplifications were observed for the asymptomatic L. aspera samples. The obtained 16S rRNA gene sequences (MN223676, MT807111 and MZ093053) showed 97.96, 98.37 and 98.18 % sequence identity, respectively; with the 'Candidatus Phytoplasma aurantifolia', strain 'WBDL (U15442) using EzBiocloud database. The NCBI-BLAST analysis revealed maximum identity to various Peanut witches' Broom (PWB) phytoplasma strains. The virtual RFLP tool, iPhyClassifier delineated the Leucas phyllody phytoplasma strains (MN223676, MT807111 and MZ093053) to group 16SrII (PWB, Peanut Witches' broom group) subgroup D with the similarity coefficient 1.0 (Zhao et al. 2009). The obtained secA gene sequences (MZ151944, MZ151945 and MZ151946) were 98.15 to 100 % similar to the strain sequences of PWB phytoplasma strains. Further, the clustering pattern in the phylogenetic trees (16S rRNA and secA genes) constructed using MEGA 7 confirmed that the Leucas phyllody phytoplasma sequences were closely related to PWB strains. To the best of our knowledge, this is the first report on the association of 16SrII-D subgroup phytoplasma with the phyllody disease of L. aspera. In India, many weeds and wild plants serve as alternative hosts of PWB phytoplasmas and aid in the emergence of related diseases in economically important crops (Thorat et al., 2016; Thorat et al., 2017). The close genetic association of phytoplasma strains found in L. aspera and many other crops indicates the presence of common insect vector(s) transmitting these phytoplasmas (Yadav et al. 2015). This report is an addition to the catalogue of the weed species harboring phytoplasma strains associated with economically important crop plants (Rao et al., 2017). The screening of phytoplasma strains in weeds, alternate hosts and known/ unknown insect vectors is therefore essential to develop management strategies and effective management of phytophagous insect vectors feeding on both weeds and crop plants.

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.

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.

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.

Lilac Tasselflower (Emilia sonchifolia) is a New Host for Peanut Witches' Broom Phytoplasma, a 16SrII-V Subgroup Strain in Taiwan.

Lilac tasselflower (Emilia sonchifolia) is an annual herbaceous plant that belongs to the family of Asteraceae. Lilac tasselflower is widely distributed at mid-low altitude regions in Taiwan, and is commonly used as traditional herbal medicine for the treatment of inflammation, rheumatism, dysentery, and analgesic. In March 2020, disease symptoms such as shoot proliferation, phyllody, and witches' broom were observed on lilac tasselflower at the sansheng community park in Mailiao, Yunlin County, Taiwan. Totally, four lilac tasselflower plants were checked and half of them were symptomatic. At the same area, similar symptoms associated with peanut witches' broom (PnWB) disease were observed (Liu et al. 2015). Samples including one healthy and two symptomatic lilac tasselflower were collected for total DNA and protein extraction used for PCR and western blotting assays, respectively. First, two sets of phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2 were used to perform nested PCR for detection of 16S ribosomal RNA (rRNA) gene (Lee et al. 1993). A specific signal of expected size (1.2 kb) for 16S rRNA was only detected in samples of lilac tasselflower exhibiting disease symptoms. The amplified DNA fragment using primer pairs P1/P7 was partially sequenced (accession no. MT420682) with P1 and a nested primer (5'-GGGTCTTTACTGACGCTGAGG-3'). The 1.4 kb nucleotide sequence shares 100% identity with that of GenBank accession NZ_AMWZ01000008 (complement [31109 to 32640]) of phytoplasma associated with PnWB disease (Chung et al. 2013). Further analysis by iPhyClassifier, the virtual RFLP pattern of MT420682 confirmed that the phytoplasma detected in symptomatic lilac tasselflower could be classified into the 16SrII-V subgroup. For western blotting, total protein of each sample was examined using the polyclonal antibody raised against Imp protein of purple coneflower witches' broom phytoplasma (Chien et al. 2020), which shares 100% identity with that (accession no. ADD59806) of PnWB phytoplasma. A specific signal of expected size (19 kDa) for Imp was detected in symptomatic lilac tasselflower, but not in healthy lilac tasselflower. Subsequent PCR, DNA sequencing and western blotting assays further confirmed that the gene encoding a SAP11-like protein detected in samples of lilac tasselflower exhibiting disease symptoms is identical to that (accession no. EMR14684) of PnWB phytoplasma. Our results indicated that lilac tasselflower, which is recognized as a common weed in Taiwan, may facilitate the spreading of phytoplasma disease by acting as an alternative natural host for PnWB phytoplasma.

First Report on the Occurrence of Virescence of Chrysanthemum Associated with 16SrII-A Group Phytoplasma in India

Chrysanthemum (Chrysanthemum indicum L.) is an important floriculture crop grown in different parts of Karnataka, India for its beautiful cut flowers. Recently, there has been a decline in flower quality due to major pests and diseases. A field survey conducted during 2012-2013 in chrysanthemum-growing areas revealed the occurrence of virescence with an incidence of 5 to 10% in nearly 6 to 8 ha of chrysanthemum fields in Mandya and Mysore districts. Symptoms including malformed flowers with greenish florets and reduced flower size and floral virescence with sepaloidy were observed. Diseased leaves and flower parts were collected and used for identification of the causative agent. Ten samples (6 symptomatic, 2 healthy, and 2 phytoplasma-associated with phyllody of Catheranthus roseus as positive control) were used for total genomic DNA isolation using GenElute Plant Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) and the DNA was used as template to amplify the phytoplasma-specific 16SrDNA gene using P1/P7 primers followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected only with DNA from symptomatic and positive control samples. Nested PCR products of all six phytoplasma-associated samples were purified and sequenced from both the directions using a ABIx370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The 16SrDNA sequences were aligned using CodonCode Aligner (Centerville, MA) and analyzed by BLASTn. The analysis revealed that all six sequences shared 100% identity with Cassava witches broom (KM280680.1) and Peanut witches broom (JX403944.1) phytoplasma. The in-silico RFLP pattern of F2n/R2 primed region of 16SrDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 99.8% nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup A (Peanut witches broom phytoplasma; Accession No. L33765.1). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chrysanthemum virescence was identified as a member of 16SrII-A group. The representative sequence was deposited in GenBank with Accession Number KP027534.1. Phytoplasma associated with little leaf and witches broom of chrysanthemum have been reported earlier from India and were assigned to groups 16SrI-A and 16SrII, respectively (Kumar et al. 2012; Raj et al. 2007). Chrysanthemum virescence phytoplasma belonging to Candidatus phytoplasma aurantifolia has been reported from Japan and has not been assigned to any group (Naito et al. 2007). The occurrence of virescence is of great importance as it affects the overall productivity and quality of flowers, which reduces the market value of chrysanthemum. To our knowledge, this is the first report of a group 16SrII-A phytoplasma associated with chrysanthemum virescence in India.

Detection and Inoculation of Peanut Witches' Broom Phytoplasma (16SrII‐A) and Periwinkle Leaf Yellowing Phytoplasma (16SrI‐B) in Citrus Cultivars in Taiwan

AbstractTo clarify the phytoplasma associated with Huanglongbing (HLB), a detection survey of phytoplasma in field citrus trees was performed using the standardized nested PCR assay with primer set P1/16S‐Sr and R16F2n/R16R2. The HLB‐diseased citrus trees with typical HLB symptoms showed a high detection of 89.7% (322/359) of HLB‐Las, while a low detection of phytoplasma at 1.1% (4/359) was examined in an HLB‐affected Wentan pummelo (Citrus grandis) tree (1/63) and Tahiti lime (C. latifolia) trees (3/53) that were co‐infected with HLB‐Las. The phytoplasma alone was also detected in a healthy Wentan pummelo tree (1/60) at a low incidence total of 0.3% (1/347). Healthy citrus plants were inoculated with the citrus phytoplasma (WP‐DL) by graft inoculation with phytoplasma‐infected pummelo scions. Positive detections of phytoplasma were monitored only in the Wentan pummelo plant 4 months and 3.5 years after inoculation, and no symptoms developed. The citrus phytoplasma infected and persistently survived in a low titre and at a very uneven distribution in citrus plants. Peanut witches' broom (PnWB) phytoplasma (16SrII‐A) and periwinkle leaf yellowing (PLY) phytoplasma belonging to the aster yellows group (16SrI‐B) maintained in periwinkle plants were inoculated into healthy citrus plants by dodder transmission. The PnWB phytoplasma showed infection through positive detection of the nested PCR assay in citrus plants and persistently survived without symptom expression up to 4 years after inoculation. Positive detections of the phytoplasma were found in a low titre and several incidences in the other inoculated citrus plants including Ponkan mandarin, Liucheng sweet orange, Eureka lemon and Hirami lemon. None of the phytoplasma‐infected citrus plants developed symptoms. Furthermore, artificial inoculation of PLY phytoplasma (16SrI‐B) into the healthy citrus plants demonstrated no infection. The citrus symptomless phytoplasma was identified to belong to the PnWB phytoplasma group (16SrII‐A).

Phytoplasma-Induced Floral Abnormalities in Catharanthus roseus Are Associated with Phytoplasma Accumulation and Transcript Repression of Floral Organ Identity Genes

Floral symptoms caused by phytoplasma largely resemble floral reversion in other plants. Periwinkle leaf yellowing (PLY) phytoplasma and peanut witches'-broom (PnWB) phytoplasma caused different degrees of floral abnormalities on infected periwinkle plants. The PLY phytoplasma-infected plants exhibited floral discoloration, virescence, small flowers, and only occasionally full floral reversion. In contrast, PnWB phytoplasma frequently induced complete floral reversion and resulted in a witches'-broom symptom from the floral reversion. Although different degrees of floral symptoms were induced by these two phytoplasmas, the morphological disorders were similar to those of other plants carrying SEPALLATA mutations or gene silencing. Here, we compared expression levels of organ-identity-related genes and pigmentation genes during floral symptom development. Accumulation of phytoplasmas in malformed flowers and their closely surrounding leaves was also compared. In infected plants, transcript abundance of all examined organ identity genes and pigmentation genes was suppressed. Indeed, CrSEP3, a SEPALLALA3 ortholog, showed the greatest suppression among genes examined. Of the pigmentation genes, transcript reduction of chalcone synthase was most highly correlated with the loss in floral pigmentation. Floral symptom severities were associated with the accumulation of either phytoplasmas. Interestingly, both phytoplasmas accumulated to higher levels in malformed flowers than in their surrounding leaves. Many plant pathogens manipulate host plant development to their advantage. It is intriguing to see whether phytoplasmas alter floral development to increase their population.

Characterization of Immunodominant Membrane Protein Gene from Peanut Witches’ Broom Phytoplasma and Bait Plasmid Construction

The molecular mechanisms involving insect-vector-phytoplasma-host-plant relationships were poorly understood.Our study focused on a cell-surface membrane protein of the phytoplasma named immunodominant membrane protein(Imp).The objective of experiment was to get the basic information between the mechanism of phytoplasma-host-plant relationships.The target ORF had been amplified using primers designed based on the conserved regions of imp gene from phytoplasma.The amplified gene was analyzed for phylogenetic tree,and the encoding protein was analyzed for transmembrane region,hydrophilic and hydrophobic region in this study.The imp gene was inserted into pBT vector including λcI gene.Construction of the pBT-Imp plasmid can be used for experiment followed by conformed that imp-λcI fusing protein was expressed by IPTG induction,western blot and self-validation testing.The immunodominant membrane protein gene from peanut witches' broom(PnWB) phytoplasma was 519 bp in length,encoding a predicted protein of 172 amino acids.Homology analysis of sequence from PnWB and 17 phytoplasma showed that imp gene of PnWB phytoplasma closely related to SPWB and WBDL phytoplasma,with homology rate of nucleotide being 99.9% and 79.8%,amino acids sequence,being 100% and 70.2%.Analysis of protein structure showed that the protein from PnWB phytoplasma possesses a region with C-terminal hydrophobic transmembrane anchor and N-terminal hydrophilic characterization,possibly internal to the phytoplasma cell.

Characterization of Catharanthus roseus Genes Regulated Differentially by Peanut Witches’ Broom Phytoplasma Infection

AbstractThe differential display (DD) strategy was applied to isolate periwinkle (Catharanthus roseus) cDNAs that were differentially expressed following infection with peanut witches’ broom (PnWB) phytoplasma. Sixty‐four clones were selected from differentially expressed cDNA fragments. Following screening by reverse Northern hybridization, ten transcripts were selected and sequenced. The expression level of each transcript was quantified by real‐time PCR, and seven DD transcripts were identified as truly differentially expressed following PnWB phytoplasma infection. Among these, one that was homologous with phi‐1 gene was up‐regulated, while the others were down‐regulated. Except two genes, other four down‐regulated genes shared homology with the genes encoding psaDa gene, ML domain protein gene, eukaryotic translation initiation factor SUI1 gene and plastidic aldolase NPALDP1 gene, respectively. The identities of homologous genes were further confirmed for three DD transcripts by isolating long cDNA fragments from the cDNA library that was established in this investigation. Verified genes were ML domain protein gene, translation initiation factor SUI1 gene and plastidic aldolase gene and were primarily involved in the innate immune response, the stress response and photosynthesis. The possible role of these genes in the periwinkle that was infected by PnWB phytoplasma is discussed.

An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom.

A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches' broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma. The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78. The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes. The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus. This major antigenic protein was also present in phytoplasma associated with peanut witches' broom (PNWB) and the two showed homology based on the results of Western blot analysis, Southern hybridization, Northern hybridization, primer extension analysis and PCR. The homologous genes of the antigenic protein of SPWB phytoplasma and PNWB phytoplasma were not found in other phytoplasmas tested.