First report of phytoplasma presence in a phyllody disease of buckwheat

Abstract

Buckwheat is an ancient herbaceous pseudocereal crop belonging to Polygonaceae family which occupy a crucial part of the human diet and is consumed globally. Typical symptoms of a phytoplasma disease were collected from buckwheat grown during October 2019 with a disease incidence of 10 to 15% in an experimental field at University of Agricultural Sciences, GKVK, Bengaluru, India. Symptomatic and asymptomatic plant samples were collected, and the DNA was extracted using a CTAB method. A nested-PCR assay was used to amplify the 16S rRNA region of phytoplasmas with the primer pairs P1/P7 and R16F2n/R16R2. All the symptomatic plants and the positive control produced an expected amplicon size of about 1.2-kb, but no products were obtained from the asymptomatic plants. PCR products were cloned and sequenced from both ends. BLASTn analysis of 16S rDNA sequence showed 99.49% nucleotide identity with ‘Candidatus Phytoplasma australasiae = australasiaticum’. This is the first report of phytoplasma presence in buckwheat.

 Keywords: nested PCR, phylogenetic analysis, 16S rRNA gene, molecular identification

Introduction

Buckwheat (Fagopryum esculentum Monch) is a gluten-free dicotyledonous crop that belongs to the family Polygonaceae. It is referred to as a pseudocereal due to its use and chemical composition, which resembles that of conventional wheat (Campbell, 1997). It is believed to have originated in the mountainous provinces of southern China and is now widely cultivated and distributed globally, including in Asia, Europe, and America (Ji et al., 2019; Sinkovic et al. 2020). It has many ecological adaptations, so it can be grown in high-altitude regions with low rainfall and temperature (Ge and Wang, 2020; Liu, 2015). It is a good source of various nutrient-rich and bioactive components with different health and pharmaceutical effects (Christa and Soral-Smietana, 2008; Sinkovic et al., 2020), so it attract more attention as a potentially valuable food source with curative effects on chronic diseases with antioxidant, anticancer, cardioprotective, antihypertensive, hepatoprotective, antitumor, anti-inflammatory, antidiabetic, neuroprotective, and cholesterol-lowering properties (Ge and Wang, 2020; Kwon et al., 2018; Lv et al., 2017). Although the scope and demand for buckwheat is increasing due to its medicinal and nutritional value, its yield potential is decreasing due to various biotic and abiotic constraints. The reason for the low productivity of most crops is the presence of numerous plant diseases at various stages of crop growth caused by fungi, bacteria, and viruses.

 Phytoplasmas are bacteria without cell walls, associated with more than 700 plant diseases worldwide, affecting a wide range of crops and plants. These can have significant economic implications due to reduced crop production and quality. Phytoplasmas are transmitted by phloem-feeding insects, with high economic impact on crop production and quality (Lee et al., 2000; Rao et al., 2018). Plants infected with phytoplasmas exhibit various symptoms following a significant disruption in their normal behaviour and physiology (Bertaccini et al., 2014). These symptoms can include yellowing, stunted growth, and abnormal development of flower parts (phyllody). A survey was conducted in October 2019 at the Zonal Agricultural Research Station, University of Agricultural Sciences, in Bengaluru, India to verify the presence of phytoplasmas in buckwheat plants showing symptoms of phytoplasma disease

 Materials and Methods

 A field survey was performed in experimental fields to collect the leaves from buckwheat plants showing chlorosis, stunting, shoot proliferation with small and crinkled leaves, and the transformation of flower organs into leaf-like structures. Ten symptomatic and 10 asymptomatic plants were sampled randomly and used for phytoplasma detection through molecular methods. The genomic DNA was extracted using a CTAB method in 100 µl of elution buffer and stored at -20oC. The extracted nucleic acids were used as templates in PCRs using the universal primer pair P1/P7 (Deng and Hiruki, 1991; Schneider et al., 1995) followed by R16F2n/ R16R2 (Lee et al., 1993; Gundersen and Lee, 1996) in nested-PCR assays. The amplification cycling consisted of initial denaturation at 95oC for 2 minutes followed by 35 cycles of denaturation at 94oC for 30 seconds, primer annealing at 55oC for 1 minute, primer extension at 72oC for 2 minute and at 72oC for 10 minutes for final extension. Fifteen microliters of PCR products were analysed by electrophoresis through a 1.5% agarose gel, stained with ethidium bromide, visualized using a UV transilluminator and photografed in a gel documentation unit.

 The amplicons generated from buckwheat plant samples were directly sequenced in both directions by the primer pairs R16F2n/R16R2. The 16S rDNA nucleotide sequence similarity was assessed, and multiple alignments were done using ClustalW. A phylogenetic tree was constructed using the 16S rDNA sequence from the buckwheat phyllody sample along with 20 sequences of ‘Candidatus Phytoplasma’ strains from GenBank database (Table 1). The phylogenetic tree was constructed using the neighbour joining method (Saitou and Nei, 1987) in MEGA7 program (Kumar et al., 2016) with 1,000 bootstrap replications (Dopazo, 1994; Rzhetsky and Nei, 1992). The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980). Acholeplasma laidlawii was used as outroot for the phylogenetic tree.

Results and Discussion

The disease incidence was recorded 10-15% in the sampled region. Diseased plants exhibited typical symptoms of phytoplasmas including transformation of flower organs into leaf like structures, chlorosis, stunting, shoot proliferation and small, crinkled leaves (Figure 1). A DNA fragment of approximately 1.2 kb was amplified by nested PCR using the primer pair P1/ P7 and R16F2n/R2 for the 16S rDNA gene, indicating the presence of phytoplasmas in symptomatic buckwheat plants but not in asymptomatic plants (data not shown).

 The nucleotide sequence of buckwheat phyllody phytoplasma resulted 100% identical to the previously deposited sequence of Crotalaria juncea phyllody phytoplasma strain GKVK from India (GenBank accession number MW559981) therefore if was not deposited in the GenBank. Comparing with the reference strains ‘Candidatus Phytoplasma australasiae = australasiaticum’ (Rodrigues Jardim et al., 2023) (GenBank accession number Y10097) it showed 99.49% identity while its identity to ‘Ca. P. aurantifolia = citri’  (Rodrigues Jardim et al., 2023) (GenBank accession number U15442) was 97.96%. Following the updated guidelines for ‘Ca. Phytoplasma’ designation (Bertaccini et al., 2022) the buckwheat phyllody phytoplasma is therefore ‘Ca. P. australasiae = australasiaticum’. Comparing with other sequences from the NCBI

Figure 1. Symptoms in phytoplasma infected buckwheat plants: (a) asymptomatic, (b) phyllody infected plant.

Figure 2. Phylogenetic tree by neighbour joining method generated by using 16S rRNA gene sequences from the phytoplasma detected in buckwheat plants and other genetically related ‘Candidatus Phytoplasma’ strains. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary analyses were conducted in MEGA7.

database the phytoplasma was found to have 99.25% nucleotide identity with the phytoplasmas from eggplant strain Al-Zubair3, in Iran (GenBank accession number MN121115), ‘Ca. P. australasiae = australasiaticum’ from bamboo in India (GenBank accession number MH547068), Chrysanthemum morifolium phyllody phytoplasma NDL (GenBank accession number KY612250), Albizia lebbeck witches’ broom phytoplasma strain AWB (GenBank accession number KX008309), Allium cepa phytoplasma On Gharb 2 (GenBank accession number LT715991.1) and Tylophora indica little leaf phytoplasma Gurgaon-1 (GenBank accession number KF773149) (Table 1). Similar results were obtained in earlier work of Naik et al. (2015) on a phytoplasma that was very similar on the 16S ribosomal gene to the one detected in buckwheat. This finding is the first identification of a phytoplasma in this species and show a further new host species for ‘Ca. P. australasiae = australasiaticum’ suggesting its large spreading in the Indian subcontinent.

Acknowledgements

The authors expresses sincere gratitude to Department of Plant Pathology, University of Agricultural Sciences, GKVK, Bengaluru for providing access to the necessary facilities and materials essential for this study. The first author acknowledge the financial support provided by the Rajiv Gandhi National Fellowship from the University Grants Commission, Government of India

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