Characterization of a novel strain of Candidatus Phytoplasma aurantifolia infecting cowpea (Vigna unguiculata) based on 16S rDNA sequence analysis

Jameel Akhtar (  jameelnbpgr@gmail.com ) National Bureau of Plant Genetic Resources https://orcid.org/0000-0002-3587-3851 Kuldeep Tripathi National Bureau of Plant Genetic Resources Mohammad Akram Indian Institute of Pulses Research https://orcid.org/0000-0002-2707-4355 Naimuddin Kamaal Indian Institute of Pulses Research Utkarsh Singh Rathore Indian Institute of Pulses Research Ashok Kumar National Bureau of Fish Genetic Resources Vasimalla Celia Chalam National Bureau of Plant Genetic Resources


Introduction
Cowpea (Vigna unguiculata L.) is one of the most important legumes across the semi-arid tropics valued for its pods and dried seeds (Kumar et al. 2012). Pests and diseases are the major biotic stresses that decrease yield, raise production costs, and limit the storability and marketability of food and feed. Among biotic stresses, phytoplasma diseases have been reported to infect a large number of plants that includes ornamentals, fruits trees, vegetables, cereals, legumes and grapevines worldwide. Phytoplasma is prokaryote without cell-wall restricted to sieve tubes in plants and transmitted by phloem-feeding insects.
Association of phytoplasma caused by Candidatus Phytoplasma asteris, a 16SrI-B subgroup in cowpea showing bud proliferation on the main shoot and stunting was rst reported from India by Kumar et al (2012). Subsequently, Thorat et al (2016) and Rao et al (2017b) reported the association of respectively, Ca. P. aurantifolia, 16SrII-D subgruop and Ca. P. cynodontis, 16SrXIV-A subgroup from cowpea plants showing little leaf, witches' brooms and at stem symptoms. Phytoplasma 16SrVI-A subgroups have also been reported infecting cowpea from Iran (Kardani and Jamshidi 2018) and 16SrXIV-A subgroup from Iraq (Al-Kuwaiti et al. 2019). Besides these reports, some sequences of phytoplasma obtained from cowpea have also been submitted at NCBI database indicating the presence of phytoplasma16SrIIpeanut WB group from China (KC953009 to KC953019) and Taiwan (KU170534 and KU170535).
Symptoms of bud proliferation and stunting suspected to be caused by phytoplasma were noticed in cowpea (accession number IC16966) growing at Experimental Farm, New Area, ICAR-National Bureau of Plant Genetic Resources (ICAR-NBPGR), New Delhi. Since there is meagre information available on the association of phytoplasma in cowpea from India, the present investigation was carried out to identify the phytoplasma in symptomatic cowpea and to study diversity in cowpea infecting phytoplasma, if any in India.

Materials And Methods
This study was undertaken jointly at ICAR-NBPGR, New Delhi and ICAR-IIPR, Kanpur, India during the year 2020-21.
Collection of samples and DNA isolation: Five samples (Cow-1, Cow-2, Cow-3, Cow-4 and Cow-5) of cowpea genotype (accession no. IC016966) showing bud proliferation and stunting along with two healthy samples (Cow-6 and Cow-7) collected from Experimental Farm, New Area, ICAR-NBPGR, New Delhi ( Fig. 1a & b) were brought to the laboratory. Total DNA was extracted from 100 mg of symptomatic as well as healthy plant parts using DNeasy Plant Mini Kit (QIAGEN GmbH, Hilton) following the manufacturer's instructions. The DNA extracted from the phytoplasma affected chickpea sample (Akram et al. 2016) was used as a positive control.
Another primer pair (R16F2n-5'GAAACGACTGCTAAGACTGG3'/ R16R2-5'TGACGGGCGGTGTGTACAAAACCCC3' ) which amplify a DNA fragment of ~1,250 bp from a portion of the 16S rDNA was used in nested-PCR (Lee et al. 1998). The rst round-PCR and nested-PCR were conducted simultaneously in a total volume of 50 μl PCR reaction mix prepared using Dream Taq Green Master Mix 2X (Fermentas) containing 25 μl 2x master mix, 25 pmol each primer, 2 μl template DNA and nuclease-free H 2 O. The PCR conditions involved an initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 2 min, annealing at 55°C for 2 min for P1/P7 and 56°C for 1 min for R16F2n/R16R2, primer extension at 72°C for 2 min and nal extension at 72°C for 10 min. PCR products were separated by electrophoresis in 1% (w/v) agarose gel prepared in a 1xTAE buffer. The DNA was stained with ethidium bromide added to the gel. The DNA bands were visualized on a UV trans-illuminator and photographed using a mobile digital camera. The DNA extracted from two healthy plants and doubledistilled water (negative control) was used as an experimental control. The DNA extracted from the phytoplasma affected chickpea plant (Akram et al. 2016) was used as a positive control.
The amplicons of the expected size ~1250bp were excised from the gel and puri ed using Nucleopore Gel/ PCR clean up kit. The concentration of puri ed fragments was measured using Nanodrop 2000 spectrophotometer (Thermo Scienti c) and the 50 ng DNA was ligated into CloneJET/1.2 blunt vector and transformed in E. coli (DH5á) cells using CloneJET and Bacterial Transformed Aid Kits (Fermentas) following the manufacturer's instructions. Initially, the transformed bacterial cells were con rmed by colony-PCR using primers (pJETF/pJETR) and nally by using restriction digestion of the plasmids. The cloned 16S rDNA fragments were sequenced from both sides through the sequence service provider (Genotypic Technology). For each sample, two clones were sequenced. Sequence analysis: The sequence data obtained were blasted and trimmed using Bioedit (v.7.2) to remove the vector sequences. Since the sequences obtained from ve samples were identical, only two representative 16S rDNA sequences were submitted to GenBank under accession no. MW827058 and MW827059. These sequences were subjected to the program iPhyclassi er online tool (http:// plantpathology.ba.ars.usda.gov/ cgi-bin/ resource/ iphyclassi er. cgi) for further analysis. The computersimulated restriction analysis of the subjected sequences (MW827058 and MW827059) generated the restriction pro le with 17 restriction enzymes (AluI, BamHI, BfaI, BstUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI, MseI, RsaI, SspI and TaqI) which gave output in the form of a virtual gel. The 16Sr DNA sequences obtained from the present study and phytoplasma sequences consisting of one representative sequence of each known phytoplasma group, subgroups of 16SrII and those infecting cowpea (Table 1) retrieved from NCBI database were used to generate phylogeny using MEGA X (Kumar et al. 2018).

Results
Field observations: During Kharif 2020, symptoms of bud proliferation and stunting were observed in cowpea genotype (IC016966) ( Fig. 1a & b) with 4% incidence. The symptoms like bud proliferation on the main shoot and stunting (Kumar et al. 2012) and little leaf, witches' broom, leaf yellowing and stem fasciations (Thorat et al. 2016;Rao et al. 2017b, Al-Kuwaiti et al. 2019 have been reported to be caused by phytoplasma in cowpea. Similarly, symptoms of little leaf and thickened leaves, phyllody, the proliferation of shoot, wrinkled and malformed leaves, stem fasciations and stunting associated with phytoplasma infection in cowpea have also been reported from Iran (Kardani and Jamshidi 2018).
Molecular characterization: In the present study, the nested-PCR using universal primer pairs, P1/P7 and R16F2n/R16R2 gave positive results ( Fig. 1d) with all the ve cowpea samples (Cow-1 to Cow-5) showing symptoms of bud proliferation and stunting symptoms typically associated with phytoplasma infection in plants, whereas negative results were obtained with the healthy samples (Cow-6 and Cow-7). The presence of the DNA fragments of expected size ~1247 bp in the PCR products of all the ve samples (ampli ed by R16F2n/R16R2 primers pair) in the gel con rmed the association of the phytoplasma in symptomatic cowpea plants. These amplicons were successfully puri ed from the gel and cloned into pJET/1.2 cloning vector. The positive clones identi ed by colony-PCR were used to isolate plasmids. The plasmid DNA was subjected to restriction digestion released desired DNA fragments con rming them to be the correct clones (Fig. 1e). Two such clones of each sample were sequenced. Both the sequences obtained were found 100% similar. Only two sequences were, however, submitted at NCBI database under the accession numbers MW827058 and MW827059 and analyzed by iPhyClassi er (Zhao et al. 2009).
Sequence analysis: The sequence analysis of the sample Cow-1 and Cow-2 con rmed the presence of phytoplasma infection in cowpea samples tested as these sequences have a phytoplasma-speci c partial rRNA operon and a partial 16S-23S rRNA intergenic spacer. Sequences of 16S rRNA gene of these isolates subjected to iPhyClassi er for species/subspecies identi cation. Both of the sequences obtained in this study showed 98.6% identity with that of the reference strain of Ca. P. aurantifolia (GenBank accession: U15442). This indicated that the phytoplasma understudy is closely related to Ca. Phytoplasma aurantifolia.
The virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment (MW827058) indicated most similarity with a reference pattern of the 16Sr group II, subgroup D (GenBank accession: Y10097) with a similarity coe cient of 0.97, a threshold to designate/consider a different strain (Zhao et al. 2009). Among 17 restriction enzymes used to generate virtual RFLP, HaeI restriction enzyme gave different RFLP pattern from reference strain (Y10097-16SrII-D-Australia-Papaya). The enzyme (HaeI) released four DNA fragments in reference strain (Y10097), whereas in understudy (MW827058) phytoplasma, it produced ve DNA fragments (Fig. 1c & f). Thus, the phytoplasma found associated with cowpea samples in the present study is a new subgroup of Ca. Phytoplasma aurantifolia under the 16SrII group.

Discussion
In this study while compiling this manuscript, 24 subgroups designated after English alphabets A to X (Yang et al. 2017;Al-Subhi et al. 2017;Omar et al. 2020) have been reported. The 16S rDNA sequence generated in the present study and 61 sequences of different phytoplasma sequences representative of each known groups and 16SrII subgroups retrieved from GenBank were used to construct phylogeny by neighbour-joining method with MEGA X (Kumar et al. 2018). Phylogenetic analysis revealed that all the strains of phytoplasma belong to 16SrII group formed a major cluster. Further this cluster (16SrII group) was subdivided into clad for each subgroup. The present strain of Ca. Phytoplasma aurantifolia formed separate clad (Fig. 2). It is therefore proposed to name the phytoplasma associated with cowpea disease with symptoms of bud proliferation and stunting at Delhi as Ca. Phytoplasma aurantifolia, 16SrII-Y subgroup.
Phytoplasmas have been found associated with several diseases in plant species including crops and causes signi cant economic losses. Cowpea is one of the most important legumes used as pods, dried seeds and fodder in many places of the world. Various phytoplasma groups and subgroups are reported infecting cowpea crops from Iraq, Iran, India, China, Taiwan and Australia. However, in the present study, we reported that the phytoplasma strain is different from all the previously established 16SrII subgroups, which appears to represent a new subgroup within the 16SrII from India. Reports of association of phytoplasma with different plant species including agri-horticultural crops including legume crops are increasing. It is therefore imperative to conduct a countrywide survey to understand the current status of phytoplasma diseases per se and to decipher the diversity of this pathogen in the changing scenario of climatic conditions. Declarations Funding: Not applicable Con icts of interest/Competing interests: The authors declare that they have no con ict of interests.
Availability of the data and material (data transparency): Not applicable    Phylogentic relationship between cowpea phytoplasma understudy (16SrII-Y) with other selected phytoplasma groups and subgroups constructed from 16S rDNA sequences. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. 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. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 63 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1877 positions in the nal dataset. Evolutionary analyses were conducted in MEGA X. WB= Witches broom group; BWL=Bermuda white leaf group; CP=Clover proliferation group; BP= Bud proliferation group; AY=Ash yellows group; EY=Elm yellows group; YD=Yellow dwarf group; LY= Lethal yellows group; XD= X disease group; YCD= Yellow crinkle disease; NA= Not available