Cyclamen (Cyclamen persicum) is a small perennial flowering plant with fragrant, showy flowers on long stems rising above the foliage. Between 2018 and 2022, about 6% of C. persicum plants belonging to diverse varieties showed stunting, leaf yellowing, virescence and phyllody in commercial nurseries at three locations (Tiszabög, Szombathely and Kecskemét) in Hungary. These symptoms are similar to those associated with the phytoplasma disease described in Italy known as cyclamen little leaf (Bertaccini, 1990) were observed in plants of six cyclamen cultivars: in 21 out of 352 plants of Super Serie Mini Winter 'Mix', 19 out of 286 plants of Super Serie Micro 'Mix', 12 out of 199 plants of Halios 'Mix', 3 out of 17 plants of Fantasia 'Purple', 1 out of 7 plants of Curly 'Early Mix Evolution' and 4 out of 66 plants of Halios Curly 'Rose' plants. Total DNA was extracted from petioles collected when possible from 10 symptomatic and 5 symptomless plants from each cultivar by a CTAB method (Ahrens and Seemüller 1992) and used as templates for PCR. Phytoplasma 16S rDNA was amplified using universal primers P1/P7 and R16F2n/R16R2 (Lee et al. 1998 and references therein). Translocase protein (secY) gene was amplified with AYsecY_F-46 (5'-AAGCAGCCATTTTAGCAGTTG-3') and AYsecY_R1450 (5'-AAGTAATCAGCTATCATTTGGTTAGT-3') primer pair, which was designed on the basis of aster yellows (AY) phytoplasma secY sequences available in Genbank. Elongation factor Tu (tuf) was amplified with fTuf1/rTuf1 (Schneider et al. 1997a) primer pairs. Thermocycler conditions consisted of 98°C for 2 min, 32 cycles at 98°C for 30 s, 60°C or 55°C (in case of tuf) for 30 s and 72°C for 1 min, followed by a final extension of 72°C for 10 min with Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA). Amplicons of the expected sizes (P1/P7: 1.8 kb, R16F2n/R16R2: 1.1 kb, AYsecY_F-46/AYsecY_R1450: 1.5 kb, fTuf1/rTuf1: 1.1 kb) were produced from all symptomatic plants but not from the asymptomatic ones. Amplified PCR products were gel purified and ligated into the pJET1.2/blunt cloning vector using a CloneJET PCR cloning kit (Thermo Fisher Scientific, Waltham, MA). The cloned PCR fragments (at least three from each PCR reaction) were sequenced from both directions by LGC Genomics (Berlin, Germany) using pJET1.2 forward and reverse primers, and the obtained sequence was deposited in GenBank. The 16S rRNA gene sequences (GenBank Accession Nos. ON594635 and ON594636) showed 100% and 99.95% identity, respectively, with Onion yellows phytoplasma strain OY-M (GenBank AP006628) from the ‘Candidatus Phytoplasma asteris’ 16SrI-B subgroup. . In iPhyClassifier analysis, the virtual RFLP pattern of 16S rDNA was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank AP006628). This is in agreement with the results of Schneider et al. (1997b) and Seemüller et al. (1998) in Germany, where phytoplasmas associated with a cyclamen disease were enclosed in the 16SrI-B subgroup. Other researches in Italy (Alma et al., 2000) and Israel (Weintraub et al., 2007) revealed that phytoplasmas belonging to the 16SrI-C and 16SrXII-A groups have been associated with cyclamen diseases. The obtained secY and tuf gene fragments (GenBank ON564432 and ON515746) shared 99.3% and 99.9% sequence identity, respectively, with Onion yellows phytoplasma strain OY-M. To our knowledge this is the first identification of 'Candidatus Phytoplasma asteris' in cyclamen in Hungary.
“Candidatus Liberibacter solanacearum” (Lso) causes disease symptoms and economic losses in potato, tomato, and other solanaceous crops in North America. Lso is transmitted to plants by potato psyllid, Bactericera cockerelli, which occurs as distinct haplotypes named western, central, and northwestern that differ in presence or absence of the bacterial endosymbiont, Wolbachia. Previous work showed that all three vector haplotypes can transmit Lso, but it was not clear whether acquisition and transmission rates of Lso were equal among the haplotypes. The goal of our study was to compare Lso infection rates among psyllids of the western, central, and northwestern haplotypes. Using data collected from several years of periodic testing of Lso infection of laboratory-reared potato psyllid colonies, we showed that psyllids of the western and central haplotypes are more likely to harbor Lso than are psyllids of the northwestern haplotype. We then used greenhouse assays to demonstrate that psyllids of the northwestern haplotype are less likely to acquire and transmit Lso compared with those of the western haplotype. Lso infection rates corresponded with Wolbachia infection among the three psyllid haplotypes. The Wolbachia-infected central and western haplotypes were more likely to harbor and transmit Lso compared with the Wolbachia-free northwestern haplotype. Results demonstrate that potato psyllids of the western and central haplotypes pose a greater risk for spread of Lso in crops and suggest a pattern between infection with Lso and Wolbachia in potato psyllid.
Candidatus Liberibacter solanacearum (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signal from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 non-target plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA, for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks, for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.
Huanglongbing (HLB) is a destructive citrus disease that affects citrus production worldwide. ‘Candidatus Liberibacter asiaticus’ (CLas), a phloem-limited bacterium, is the associated causal agent of HLB. The current standard for detection of CLas is real-time quantitative polymerase chain reaction (qPCR) using either the CLas 16S rRNA gene or the ribonucleotide reductase (RNR) gene-specific primers/probe. qPCR requires well-equipped laboratories and trained personnel, which is not convenient for rapid field detection of CLas-infected trees. Recombinase polymerase amplification (RPA) assay is a fast, portable alternative to PCR-based diagnostic methods. In this study, an RPA assay was developed to detect CLas in crude citrus extracts utilizing isothermal amplification, without the need for DNA purification. Primers were designed to amplify a region of the CLas RNR gene, and a fluorescent labeled probe allowed for detection of the amplicon in real-time within 8 mins at 39°C. The assay was specific to CLas, and the sensitivity was comparable to qPCR, with a detection limit cycle threshold of 34. Additionally, the RPA assay was combined with a lateral flow device for a point-of-use assay that is field deployable. Both assays were 100% accurate in detecting CLas in fresh citrus crude extracts from leaf midribs and roots from five California strains of CLas tested in the Contained Research Facility in Davis, California. This assay will be important for distinguishing CLas-infected trees in California from those infected by other pathogens that cause similar disease symptoms and can help control HLB spread.
Pearl millet [Pennisetum glaucum (L).R.Br.] also known as bajra, is one of the oldest millets and is cultivated in dry regions of arid and semi-arid tropics where no other cereal can be successfully grown. Pearl millet cultivation in India accounts for about two-thirds of millet production and is the fourth most cultivated food crop after rice, wheat and maize in India (Reddy et al. 2021a). In February 2021, the typical symptoms of stunting, phyllody and little leaf were observed after 25-30 days after sowing pearl millet seeds at Agricultural Research Station in Perumallapalle, Tirupati, India (Fig.1 A-C). The disease incidence was recorded up to 20% in the sampling regions. Total DNA was extracted from two symptomatic and two asymptomatic plant samples using CTAB DNA extraction method (Murray and Thompson, 1980). The extracted DNA was amplified in direct PCR and nested PCR assay using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (Gundersen and Lee.1996) and secA gene with secAfor1/SecArev3 and SecAfor2/SecArev3 primer pairs (Hodgetts et al. 2008). 16SrRNA (1.25 kb) and secA (600 bp) gene amplicons were obtained from two symptomatic samples by nested PCR. No amplicons were produced with DNA from healthy leaf samples. Nested PCR amplified products (1.25 kb and 600 bp) from the symptomatic samples corresponding to the F2nR2 region of 16S rRNA and secA were directly sequenced at automated DNA sequencing facility (Eurofin Genomics India Pvt., Ltd Bangalore) and sequence data was deposited to NCBI GenBank with accession number ON005559 and ON067810. BLAST analysis revealed that pearl millet phytoplasma strain shared 100% sequence identity in 16Sr RNA and secA genes to ‘Canditatus Phytoplasma aurantifolia’ related strains (Acc. Nos. OM616883 and MT952965) from India. The subgroup was identified as 16SrII-D using the iPhyClassifier based on the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment (Zhao et al. 2009). The virtual RFLP pattern is similar to the reference pattern of 16SrII-D (Y10096) with similarity coefficient 1.00. Phylogenetic analysis of 16S rRNA and secA gene sequences using MEGA version 7.0 revealed that the pearl millet phytoplasma strain clustered with ‘Ca. P. aurantifolia’ isolates of 16SrII-D subgroup. (Fig.1D-E) Earlier, one of 16SrI-B-phytoplasma strain (HM 134245) associated with green ear disease of pearl millet was reported in North India (Kumar et al. 2010). In this study, we reported the association of 16SrII-D subgroup phytoplasma with little leaves and witches’-broom disease of pearl millet in South India. Phytoplasmas belonging to the 16SrII-D subgroup have a wide range of hosts, including the agricultural and horticultural crops (Reddy et al., 2021b). Hence, this is the first report of ‘Ca. P aurantifolia’ infection in bajra in South India. The increase in the spread of 16SrII-D sub group phytoplasma diseases and the expansion of the host range strongly suggest further studies on the epidemiology of the dynamic dissemination of this disease in India.
“Candidatus Liberibacter asiaticus” (CLas) is the bacterium associated with the citrus disease known as Huanglongbing (HLB). This study evaluated the influence of CLas infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers’ activity, and osmoregulation of two different species of citrus (the pomelo Citrus maxima (Burm.) Merr. and the mandarin C. reticulata cv. Tankan), relative to their measured CLas infection load. Results indicated all measured physiological variables excepting soluble sugar were affected by increased CLas infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. CLas infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with CLas scions revealed a significant decrease in the photosynthesis variables Fv/Fm, Y(II) and QL, whilst NPQ increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables SOD, CAT, POD, and soluble protein initially increased and later decreased. In addition, progression of CLas replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize the observed interspecific differences in physiological change are related to their relative resistance against CLas infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research: ultimately these detailed observations can facilitate the diagnosis of CLas infection.
Moringa oleifera (family Moringaceae) also known as the ‘drumstick tree’ is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture & Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain ‘Candidatus Phytoplasma asteris’ (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with ‘Ca. Phytoplasma asteris’ (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B (‘Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. ‘Candidatus Phytoplasma asteris’ (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.