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Detection and Molecular Characterization of ‘Candidatus Liberibacter asiaticus’ and Citrus Tristeza Virus Associated with Citrus Decline in Bhutan

    Affiliations
    Authors and Affiliations
    • Dilip Kumar Ghosh1 †
    • Amol D. Kokane1
    • Sunil B. Kokane1
    • Jigme Tenzin2
    • Mrugendra G. Gubyad1
    • Phuntsho Wangdi3
    • Ashutosh A. Murkute1
    • Ashwani Kumar Sharma4
    • Siddarame Gowda5
    1. 1Indian Council of Agricultural Research–Central Citrus Research Institute, Nagpur-440 033, Maharashtra, India
    2. 2National Citrus Program, Department of Agriculture, Royal Government of Bhutan, Thimphu 11001, Bhutan
    3. 3National Citrus Repository, Department of Agriculture, Royal Government of Bhutan, Tsirang, Bhutan
    4. 4Department of Biotechnology, Indian Institute of Technology, Roorkee – 247 667, India
    5. 5University of Florida, Citrus Research and Education Centre, Lake Alfred, FL 33850, U.S.A.
    Published Online:14 Apr 2021https://doi.org/10.1094/PHYTO-07-20-0266-R

    Abstract

    Citrus, mainly mandarin (Citrus reticulata Blanco), is an economically important fruit crop in Bhutan. Despite having favorable agroclimatic conditions for citrus cultivation, the early decline of fruit-bearing orchards coupled with low crop productivity is a major concern among citrus growers. During a recent survey, an association of ‘Candidatus Liberibacter asiaticus’ (citrus greening) and citrus tristeza virus (CTV), either singly or as mixed infections in declined citrus trees, was recorded in all four major citrus-growing districts (Tsirang, Dagana, Zhemgang, and Sarpang). Using PCR-based diagnosis, a higher incidence of citrus greening (27.45%) and tristeza (70.58%) was observed in symptomatic field samples. Detection and characterization of ‘Ca. L. asiaticus’ was performed based on the 16S ribosomal DNA, prophage gene, 50S ribosomal rplA-rplJ gene, and tandem repeats of the CLIBASIA_01645 locus. Similarly, the coat protein, p23, and p18 genes were used as genetic markers for the detection and characterization of Bhutanese CTV. The ‘Ca. L. asiaticus’ isolates from Bhutan segregated into classes II and III based on the CLIBASIA_01645 locus, analogous to Indian isolates from the northeast region and Term-A based on the CLIBASIA_05610 locus. CTV isolates of Bhutan were observed as closely related to the VT strain, which is considered to be the most devastating. To the best of our knowledge, this is the first study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan.

    Bhutan is a small landlocked country in the Himalayan region, between India and China, and its economy is traditionally based on agriculture, horticulture, and forestry that provide the main livelihood for more than 60% of its population. Diverse agroclimatic conditions prevalent in the country favor the production of a wide range of horticultural crops, with citrus being the most important fruit crop (Joshi and Gurung 2009). Citrus, mainly mandarin (Citrus reticulata Blanco), is considered not just a commodity but also a source of foreign exchange, livelihood, and employment opportunities and stands first in export earnings compared with other horticultural commodities (Joshi and Gurung 2009, Trienekens et al. 2008). Citrus is cultivated in 17 of Bhutan’s 20 districts and amounts to more than 95% of total citrus production in the country (Dorji et al. 2016). The predominant mandarin-growing districts of Bhutan are Tsirang, Dagana, Zhemgang, and Sarpang (Tipu and Fantazy 2014). Decline of citrus trees within a few years of orchard establishment is a major concern faced by citrus growers. Among different biotic factors, severe infections of citrus greening (huanglongbing) and tristeza have been major impediments for higher fruit yield and orchard longevity (Dorji et al. 2016).

    Citrus greening disease was first reported in Bhutan in 2003 (Ahlawat et al. 2003; Doe et al. 2003). It is associated with a phloem-limited yet uncultured, Gram-negative α-proteobacterium, ‘Candidatus Liberibacter’ (Jagoueix et al. 1994). Presently, three different species—‘ Candidatus Liberibacter asiaticus’, ‘Ca. L. americanus’, and ‘Ca. L. africanus’—are known to infect citrus plants (Bove 2006; Lopes et al. 2009). ‘Ca. L. asiaticus’ is widely prevalent and responsible for maximum citrus destruction worldwide (Hung et al. 2004; Miyakawa 1980). ‘Ca. L. asiaticus’ and ‘Ca. L. americanus’ are transmitted by Asian citrus psyllid (Diaphorina citri Kuwayama) whereas ‘Ca. L. africanus’ is vectored by Trioza erytreae Del Guercio (Bhose et al. 2015; Halbert and Manjunath 2004; Lopes et al. 2009).

    The typical symptoms of the greening disease include asymmetrical blotchy mottling of leaves which often resembles zinc deficiency and ultimately leads to the development of yellow shoots (da Graça and Korsten 2004). Field-level identification of greening is confused at times with other plant diseases or nutrient deficiency (Lama and Amatya 1993; Wang et al. 2016). PCR-based diagnostic tools have been routinely used for reliable ‘Ca. L. asiaticus’ detection (Ghosh et al. 2018c). Various molecular markers have been developed to study the genetic variation within ‘Ca. L. asiaticus’ populations. Single-nucleotide polymorphism (SNP)-based genetic diversity targeting ribosomal protein genes and 16S ribosomal DNA (rDNA) (Adkar-Purushothama et al. 2009; Kokane et al. 2020a), prophage terminase gene-based genetic variability (Liu et al. 2011), and analysis of tandem repeat numbers (TRNs) in a hypervariable genomic locus, CLIBASIA_01645 (Chen et al. 2010; Ghosh et al. 2015), have been some of the reported methods of determining genetic variability among ‘Ca. L. asiaticus’ isolates.

    Citrus tristeza virus (CTV), is another graft-transmissible and economically important pathogen that has affected the citrus industry worldwide by destroying millions of productive trees (Dawson et al. 2013; Moreno et al. 2008). CTV belongs to the genus Closterovirus, family Closteroviridae, and has long flexuous filamentous virions of 2,000 by 11 nm. Different aphid species—Aphis gossypii Glover, A. (Toxoptera) citricidus Kirkaldy, and A. spiraecola Patch—act as vectors and transmit CTV in a semipersistent manner (Marroquín et al. 2004). CTV infections result in reduced production and fruit quality, and increased disease severity leads to the decline of citrus trees, particularly on sour orange (C. aurantium) rootstock. Numerous biological strains of CTV present worldwide infect almost all citrus species and produce a variety of symptoms; namely, stem pitting, vein clearing, general stunting, and slow or quick decline (Brlansky et al. 2003; Warghane et al. 2020). The single-stranded positive-sense RNA genome of CTV, approximately 19.3 kb, potentially encodes at least 19 proteins. Open reading frame (ORF)1a and ORF1b, which encompass half the genome toward the 5′ end, encode the replication-related proteins translated directly from the genomic RNA (Karasev et al. 1995). ORF2 to ORF11, expressed via 3′ coterminal subgenomic RNAs that comprise the 3′ half of the genome, encode for p33, p6, p65, p61, p27 (minor coat protein), p25 (major coat protein), p18, p13, p20, and p23 (RNA binding protein) (Hilf et al. 1995; Pappu et al. 1994). Genetic diversity of the CTV populations has been determined based on sequence evaluations of different genomic regions of the virus (Biswas et al. 2012; Martín et al. 2009; Rubio et al. 2001). Although preliminary reports on the incidence of citrus greening and CTV in Bhutan are available in the literature (Ahlawat et al. 2003; Doe et al. 2003; Dorji et al. 2016), no detailed work has been reported to date on molecular characterization of these two important systemic pathogens associated with citrus decline.

    During a survey conducted in 2018–19, the incidence of citrus greening and tristeza disease, as single or mixed infections, was recorded in different citrus orchards of Bhutan. PCR-based diagnostic tools were employed for the reliable detection of ‘Ca. L. asiaticus’ and CTV in field-collected samples. In the present study, detailed molecular characterization of ‘Ca. L. asiaticus’ was performed based on sequence variations of four different genomic regions; namely, the 16S rDNA, 50S ribosomal rplA-rplJ gene, prophage gene, and TRNs of hypervariable genomic locus CLIBASIA_01645. Similarly, three genomic regions—namely, the coat protein (CP) gene (p25), RNA binding protein gene (p23), and p18 gene—were analyzed for the molecular characterization of CTV populations.

    MATERIALS AND METHODS

    Sample collection and processing.

    Leaf samples showing citrus greening-like and tristeza-like symptoms from declined citrus trees along with samples from apparently healthy trees were collected from different orchards of four major citrus-growing districts of Bhutan (i.e., Tsirang, Dagana, Zhemgang, and Sarpang) (Table 1; Fig. 1). The symptomatic and nonsymptomatic leaves (Fig. 2) were washed and wiped with 70% ethanol to avoid any surface contamination and blot dried. Midrib tissues were excised and ground in liquid nitrogen, and approximately 100 mg of the powdered sample was used for total genomic DNA and RNA extractions.

    TABLE 1. Details of samples collected from four districts of Bhutan and presence (+) or absence (−) of ‘Candidatus Liberibacter asiaticus’ (huanglongbing [HLB]) and citrus tristeza virus (CTV) as tested by PCR and reverse-transcription PCR, respectivelya

    Fig. 1.

    Fig. 1. Map of Bhutan, showing details of incidence and distribution of ‘Candidatus Liberibacter asiaticus’ (huanglongbing [HLB]) and citrus tristeza virus (CTV) in Bhutan. Districts showing the sampling of citrus are depicted in the bottom panel. All of the ‘Ca. L. asiaticus’ isolates segregated under two groups (class II and class III) based on different tandem repeat numbers (TRNs) at the CLIBASIA_01645 locus have been depicted in the map with different shapes.

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    Fig. 2.

    Fig. 2. Citrus orchards in Bhutan. A, View of an apparently healthy orchard in Tsirang district, Bhutan. B, Symptomatic and healthy trees in the field. C, Citrus decline in a mandarin tree surrounded by seemingly healthy trees. D, Yellow leaf symptoms on citrus. E, Blotchy mottling symptoms of citrus greening (huanglongbing).

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    Primer design.

    Different genomic regions—namely, the 16S rDNA, 50S ribosomal rplA-rplJ gene, prophage gene, and CLIBASIA_01645 locus for ‘Ca. L. asiaticus’ and CP, p23, and p18 genes for CTV—were selected to design primer sets based on the primer 3v.0.4.0 tool (https://bioinfo.ut.ee/primer3-0.4.0/). The primers were synthesized through Integrated DNA Technologies (Coralville, IA, U.S.A.).

    Nucleic acid extraction.

    Total DNA was extracted from leaf midrib tissue using the cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1990). Briefly, 100 mg of tissue of the fine powder from each sample was homogenized in 1 ml of preheated extraction buffer (2% CTAB, 1.4 M NaCl, 100 mM Tris-Cl [pH 8.0], and 20 mM EDTA [pH 8.0]) and incubated in the dry bath at 65°C for 30 min. The mixture was extracted with an equal volume of chloroform/isoamyl alcohol (24:1) and mixed well, and the DNA in the aqueous phase was precipitated with ethanol. The DNA pellet was washed with 70% ethanol, air dried, and resuspended in 50 µl of nuclease-free water. Total RNA isolation was performed with 100 mg of liquid nitrogen ground powder using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), based on the manufacturer’s guidelines.

    Detection of ‘Ca. L. asiaticus’ using PCR.

    Primer pair OI1/OI2c specific to ‘Ca. L. asiaticus’ was used for partial amplification of 16S rDNA (approximately 1,160 bp) (Bove 2006; Garnier et al. 2000; Ghosh et al. 2018a,c, 2019; Jagoueix et al. 1996; Warghane et al. 2017). The PCR was performed as described by Ghosh et al. (2018b). The amplified PCR products were separated on a 1% agarose gel, stained with ethidium bromide at 0.5 μg/ml, and visualized in the UV GelDoc system (G:Box; Syngene). The rplA-rplJ region corresponding to the conserved β operon of the 50S subunit ribosomal protein gene was amplified using primer pair A2/J5 (Bhose et al. 2015; Ghosh et al. 2018b; Hocquellet et al. 1999). The amplification was performed as described by Ghosh et al. (2018b) and separated on a 1% agarose gel.

    Detection of CTV using RT-PCR.

    Total RNA extracted from the leaf midrib of suspected CTV samples were used for reverse-transcription (RT)-PCR using CTV CP-specific primer set CN150/CN151 (Karasev et al. 1995; Pappu et al. 1993). The RT-PCR was performed in two-steps, as described by Warghane et al. (2017). The other two genomic regions of CTV (i.e., p23 and p18) were also used for detection. The primer pair RBP-23F (5′-ATGAACGATACTAGCGGAC-3′) and RBP-23R (5′GATGAAGTGGTGTTCACGG3′) specific to the p23 gene and AR18F/R (Roy et al. 2005) specific to the p18 gene were used to perform the RT-PCR. The first-strand cDNA synthesis and PCR amplification were carried out as previously described (Kokane et al. 2020b; Warghane et al. 2017). The annealing time and temperatures in the PCR program were 45 s at 52°C and 40 s at 62°C for the p23 and p18 genes, respectively. The resulting RT-PCR products were separated on a 1% agarose gel and visualized in the UV GelDoc system.

    Nucleotide sequencing and sequence analysis.

    Amplified PCR products were excised and eluted from the gel using the GenElute Gel Extraction Kit (Sigma-Aldrich, Bengaluru, India) and sequenced by the Sanger dideoxy method at an automated DNA sequencing facility (Eurofins Genomics, Bengaluru, India), and the results were analyzed using Bioedit software version 7.2. The chromatograms of both forward and reverse sequences were checked with the Bioedit Sequence Alignment Editor and sequence similarity searches were performed using nucleotide BLAST. The assembled sequences have been deposited in GenBank using BankIt software (https://www.ncbi.nlm.nih.gov/WebSub/).

    Diversity analysis of ‘Ca. L. asiaticus’ based on tandem repeats in the CLIBASIA_01645 locus.

    The CLIBASIA_01645 locus-specific primer pair LapGP-1f/LapGP-1r (Chen et al. 2010; Ghosh et al. 2015) was used to investigate the ‘Ca. L. asiaticus’ diversity. PCR assays were performed using genomic DNA as a template for each ‘Ca. L. asiaticus’ isolate, as described previously (Ghosh et al. 2015). The amplified products were purified using the GenElute Gel Extraction Kit and sequenced by Sanger dideoxy method. Sequences were aligned in Bioedit software as well as ClustalW. The tandem repeats (AGACACA) present in the CLIBASIA_01645 genomic locus were identified as described earlier (Ghosh et al. 2015; Kokane et al. 2020a).

    Analysis of prophage gene of ‘Ca. L. asiaticus’.

    The primer set 766F/766R (Liu et al. 2011) was used to amplify the prophage terminase gene (CLIBASIA_05610) of ‘Ca. L. asiaticus’, and two different sets of primers, CT3f/CT3r specific for Term-A and FC3f/FC3r specific for Term-G, were used for PCR amplification using ‘Ca. L. asiaticus’ genomic DNA as a template (Deng et al. 2014).

    Phylogenetic study and comparative sequence analysis.

    Maximum-likelihood trees were deduced using MEGA X, assuming the Tamura three-parameter model (Kumar et al. 2018), with 1,000 bootstrap replicates. The phylogenetic tree was constructed based on the CP gene of 12 Bhutanese CTV isolates along with 31 reported CP gene sequences in GenBank (Harper 2013; Tarafdar et al. 2013). In total, 25 sequences of the 50S ribosomal subunit gene were used for the construction of the phylogenetic tree for ‘Ca. L. asiaticus’.

    The pairwise nucleotide sequences of the CP gene of Bhutanese isolates were aligned and compared among themselves and also with corresponding sequences of seven major CTV strains—namely, T36 (U16304), T30 (AF260651), VT (EU937519), T3 (KC525952), T68 (JQ454870), HA16-5 (GQ454870), and NZRB-M17 (FJ525435)—that represent major CTV genotypes (Dawson et al. 2015; Harper 2013; Hilf et al. 2005; Yokomi et al. 2018).

    RESULTS

    PCR detection of ‘Ca. L. asiaticus’.

    In total, 51 symptomatic samples collected from different citrus-growing regions of Bhutan (Table 1) were tested by PCR with the ‘Ca. L. asiaticus’-specific OI1/OI2c primer set, among which 14 samples were found positive because they amplified ‘Ca. L. asiaticus’-specific approximately 1,160-bp amplicons. The remaining 37 samples were observed to be negative for ‘Ca. L. asiaticus’. Amplification was not observed in a healthy citrus plant (negative control) and nontemplate control (Fig. 3A). The amplicons of six representative isolates were excised from the gel, purified, sequenced, and deposited in GenBank (Table 2).

    Fig. 3.

    Fig. 3. Agarose gel electrophoresis of PCR-amplified product of Bhutanese ‘Candidatus Liberibacter asiaticus’ isolates. A, 16S ribosomal DNA (16S rDNA) gene with primer set OI1/OI2c. B, 50S ribosomal subunit rplA-rplJ gene with primer set A2/J5. C, CLIBASIA_01645 region with primer set LapGP-1f/LapGP-1r. D, Prophage terminase gene (CLIBASIA_05610) with primer set 766F/766R. E, Prophage terminase gene (CLIBASIA_05610) with primer set CT3f/CT3r. Lane M, 100-bp DNA Ladder; lanes 1 to 6, amplified product of representative Bhutanese isolates; lane -Ve, nontemplate control; lane C, healthy plant control; and lane +Ve, positive control.

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    TABLE 2. GenBank accession numbers obtained for sequences of ‘Candidatus Liberibacter asiaticus’ and citrus tristeza virus (CTV) isolates of Bhutana

    Molecular characterization of ‘Ca. L. asiaticus’.

    The citrus-greening-positive samples were used for molecular characterization by targeting different genomic regions of ‘Ca. L. asiaticus’. The rplA-rplJ region, corresponding to the conserved β operon subunit of the 50S ribosomal protein gene, was amplified using primer set A2/J5, and a ‘Ca. L. asiaticus’-specific approximately 700-bp amplicon was observed (Fig. 3B). The amplified PCR products of 10 representative isolates were gel purified, sequenced, and deposited in GenBank (Table 2). These sequences showed a maximum 99% similarity with the sequences of other ‘Ca. L. asiaticus’ isolates (GenBank accession numbers FJ827780, DQ303211, and GQ890155). It was also observed that the sequences were 81% similar to a ‘Ca. L. africanus’ isolate (GenBank accession number GU120038). A phylogenetic relationship study based on the 50S ribosomal rplA-rplJ gene suggested that all ‘Ca. L. asiaticus’ isolates from Bhutan could be grouped into a single taxon (Fig. 4).

    Fig. 4.

    Fig. 4. Phylogenetic tree showing the 50S ribosomal subunit rplA-rplJ gene-based genetic relationship among Bhutanese ‘Candidatus Liberibacter asiaticus’ isolates with other reported isolate sequences using a maximum-likelihood method in MEGA X and the significance of the node was estimated with 1,000 bootstrap replicates.

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    The CLIBASIA_01645 region amplified approximately 600-bp amplicons with the LapGP-1f/LapGP-1r primer set (Fig. 3C). Amplified products of 10 representative isolates were sequenced and the sequences were deposited in GenBank (Table 2). The sequences were analyzed to determine the variable tandem repeats, as reported by Ghosh et al. (2015). It was observed that isolates Bhu-Ts-1, Bhu-Ts-3, Bhu-Ts-9, Bhu-Ts-20, Bhu-Da-76, Bhu-Sa-82, Bhu-Da-36, and Bhu-Sa-78 belong to class II because they have seven and eight TRN copy numbers. Isolates Bhu-Zh-68 and Bhu-Zh-71 were classified under class III, with 12 and 13 TRN copy numbers, respectively (Fig. 5).

    Fig. 5.

    Fig. 5. Nucleotide sequence alignment of the CLIBASIA_01645 loci of Bhutanese ‘Candidatus Liberibacter asiaticus’ isolates with variable tandem repeats. Individual variable tandem repeats are indicated with different shading.

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    The amplification of the prophage terminase gene (CLIBASIA_05610) using primer pair 766F/766R produced the expected approximately 766-bp amplicons (Fig. 3D), and sequences of 10 isolates were deposited in GenBank (Table 2). All isolates showed 100% sequence similarity with the ‘Ca. L. asiaticus’ prophage terminase large-subunit gene (GenBank accession number KX348043) and the phage complete genome (GenBank accession number CP019958). Moreover, sequences showed 99% similarity with other sequences, with accession numbers HM105498, CP001677, and HQ377374. The results also suggested that all Bhutanese isolates belonged to Term-A because specific, approximately 370-bp amplifications were observed with primer set CT3f/CT3r (Fig. 3E) and no amplification was observed with primer set FC3f/FC3r, specific for Term-G.

    RT-PCR detection of CTV.

    In all, 51 samples collected from different regions of Bhutan were also tested for CTV by RT-PCR using different primer sets—namely, CN150/CN151 (CP gene), RBP-23F/RBP-23R (p23 gene), and AR18F/AR18R (p18 gene). In total, 36 samples were found positive for CTV, because all of these Bhutanese isolates amplified products of expected sizes of approximately 672-bp (Fig. 6A), approximately 630-bp (Fig. 6B), and approximately 511-bp (Fig. 6C) amplicons with CP, p23, and p18 gene-specific primers, respectively. Representative amplified products of the CP gene of CTV isolates were excised from the gel, purified, sequenced, and deposited in GenBank (Table 2).

    Fig. 6.

    Fig. 6. Agarose gel electrophoresis of PCR amplified product of Bhutanese citrus tristeza virus isolates. A, Coat protein (CP) gene with primer set CN150/CN151; B, p23 gene with primer set RBP23F/R; and C, p18 gene with primer set AR18F/R. Lane M, 100-bp DNA Ladder; lanes 1 to 8, representative Bhutanese isolates; lane -Ve, nontemplate control; lane C, healthy plant control; and lane +Ve, positive control.

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    Molecular characterization of CTV.

    The partial sequences of the CP gene of Bhutanese CTV isolates were compared with published sequences of seven CTV strains. Phylogenetic analysis of the CP sequences revealed that the majority of the Bhutanese CTV isolates clustered with the VT strain. The nine Bhutanese isolates clustered to genogroup III, similar to isolates from Meghalaya and Andhra Pradesh States of India (GenBank accession numbers KC590492, KC590495, and KC590501) and the VT genotype (GenBank accession number EU937519). A single Bhutanese isolate (Bhu-Da-76) segregated into genogroup I, similar to Indian isolates (GenBank accession numbers GQ475547, HQ912023, and GQ475549), and two isolates (Bhu-Ts-18 and Bhu-Ts-20) belonged to genogroup IV, similar to isolates from India (GenBank accession numbers KC590508, KY012241, and KC590499) and Jamaica (GenBank accession numbers HM160503 and HM160511) (Fig. 7).

    Fig. 7.

    Fig. 7. Phylogenetic tree constructed based on the coat protein (CP) (p25) gene of Bhutanese citrus tristeza virus isolates with other reported CP gene sequences using a maximum-likelihood method in MEGA X. The significance of the node was estimated with 1,000 bootstrap replicates.

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    The pairwise sequence analysis revealed that the nucleotide identity of CP sequences ranged from 90.05 to 100% among the Bhutanese CTV isolates (Table 3). The highest pairwise sequence identity (100%) was observed between Bhu-Ts-4 and Bhu-Ts-9, while the lowest identity (90.05%) was recorded between isolates Bhu-Ts-20 and Bhu-Ts-9. The sequence identity of studied isolates with reported major CTV genotypes varied from 88.99 to 98.47% (Table 3). The CP genes revealed that most of the Bhutanese CTV isolates were highly similar to the VT strain of CTV, with 93.11 to 98.47% identities (Table 3).

    TABLE 3. Pairwise nucleotide sequence identities (%) of the coat protein genea

    DISCUSSION

    A survey was conducted to examine the present status of citrus greening and tristeza diseases in Bhutan and to characterize these two pathogens (‘Ca. L. asiaticus’ and CTV) based on the sequences of different genes. Suspected citrus samples were collected primarily from the orchards of four major citrus-growing districts of Bhutan (namely, Tsirang, Dagana, Zhemgang, and Sarpang). A higher incidence of citrus greening (27.45%) and tristeza disease (70.58%) was recorded in collected symptomatic leaf samples in the present investigation. Incidentally, single and mixed infections of citrus greening and CTV were found in declined citrus trees, which suggested that either one of these two systemic and vector-transmitted pathogens could cause the citrus decline disease in Bhutan (Fig. 2). All four surveyed districts were in the southern part of Bhutan, geographically closer to the northeast region of India (Borah et al. 2014; Ghosh et al. 2015).

    Most citrus orchards in the country were in a neglected condition and infestations by aphids and psyllids were abundantly observed in the surveyed areas. The application of insecticides by the growers to control insect pests was not prioritized. Heavy infestation of aphids was earlier recorded in this area, which act as an efficient vector for CTV (Kashyap et al. 2015; Singh et al. 2017). Similarly, the presence of citrus psyllid (D. communis Mathur) was also recorded in some of the districts of Bhutan (Donovan et al. 2012). These insects, in the absence of any chemical control measures, possibly play an important role as vectors for the potential spread of tristeza and greening within the citrus orchards. Thus, many citrus orchards have been destroyed due to these two major pathogens.

    In the present investigation, all ‘Ca. L. asiaticus’-infected samples were used for PCR amplification of the 50S ribosomal rplA-rplJ gene of ‘Ca. L. asiaticus’, and the nucleotide sequences of the amplified products were compared with other submitted sequences of ‘Ca. L. asiaticus’, ‘Ca. L. africanus’, and ‘Ca. L. americanus’ isolates at NCBI. Phylogenetic analysis showed that all ‘Ca. L. asiaticus’ isolates grouped into a single taxon separated from ‘Ca. L. africanus’ and ‘Ca. L. americanus’. The variability in TRN in the CLIBASIA_01645 region was also examined using primer set LapGP-1f/LapGP-1r (Chen et al. 2010; Ghosh et al. 2015). In an earlier study, Indian ‘Ca. L. asiaticus’ isolates were grouped into four classes (class I to class IV), with most ‘Ca. L. asiaticus’ isolates of the northeast region of India belonging to TRN class II (Ghosh et al. 2015). In the present study, it was observed that the Bhutanese ‘Ca. L. asiaticus’ isolates contained 7, 8, 12, or 13 TRNs and, thus, were closely related to the ‘Ca. L. asiaticus’ isolates of the northeastern part of India. Based on previously reported prophage terminase gene analysis, ‘Ca. L. asiaticus’ isolates were divided into two groups, where isolates from the Asian continent and Brazil were classified under Term-A (for adenine), while Term-G (for guanine) included isolates from Florida (Deng et al. 2014). Based on results of the present study, Bhutanese ‘Ca. L. asiaticus’ isolates were placed into Term-A along with Asian ‘Ca. L. asiaticus’ isolates, consistent with previous reports (Deng et al. 2014). Liu et al. (2011) also reported the two-strain classification system based on an SNP study.

    Molecular characterization of CTV has been studied by either full genome sequencing (Moreno et al. 2008) or characterization based on genetic diversity of different genomic regions (Biswas et al. 2012), multiple molecular marker assays (Hilf et al. 2005), and average nucleotide identity assays for different genomic regions (Harper 2013). Based on the complete genome analysis, three biologically distinct CTV strains (namely, T30, T36, and VT) were categorized initially (Moreno et al. 2008). Subsequent studies revealed the existence of seven genotypes (namely, T36, VT, T30, T68, T3, HA16-5, and RB) based on the complete genome analysis and biological indexing (Dawson et al. 2015; Harper 2013; Hilf and Garnsey 2000; Melzer et al. 2010). Recently, a new CTV genotype (S1) has been observed based on the full-length genome sequence (Yokomi et al. 2018). Based on the CP gene analysis, Biswas et al. (2012) classified Indian CTV isolates into seven genogroups with the subsequent addition of more genogroup (Tarafdar et al. 2013; Warghane et al. 2020).

    In the present study, a CP gene-based phylogenetic tree of Bhutanese isolates along with other reported CTV isolates have identified eight genogroups (Biswas et al. 2012; Tarafdar et al. 2013; Warghane et al. 2020), in which the Bhutanese isolates clustered into genogroups I, III, and IV. However, a majority of the isolates revealed a close phylogenetic association with severe isolate VT (EU937519). Furthermore, CP gene-based pairwise nucleotide identity of Bhutanese isolates, compared among themselves and with reported major CTV genotypes, revealed 96.71, 93.35, 92.74, 95.71, 93.77, 93.34, and 91.68% similarity with VT, T36, T30, T68, T3, NZRB, and HA16-5, respectively. Based on the CP gene, Harper (2013) reported that the pairwise nucleotide identity ranged from 91.1 to 96.4%. The CP gene is a historical legacy, because it is the most highly conserved, and shared 90.05 to 100% nucleotide identity between isolates examined in this study. In brief, the majority of the Bhutanese isolates were closely related to the VT genotype, showed minimal nucleotide sequence variability for the CP gene analyzed, and displayed the greatest variability with other major CTV genotypes.

    To the best of our knowledge, this is the first detailed study on molecular characterization of ‘Ca. L. asiaticus’ and CTV isolates and their association with citrus decline in Bhutan. Integrated management strategies to avoid citrus decline include the use of disease-free planting material, early pathogen detection, and control of insect vectors. The information generated in the present investigation would act as a documentary platform for policy frameworks and to enable more timely and effective management decisions toward a healthy and productive citrus industry in Bhutan.

    ACKNOWLEDGMENTS

    We thank J. Freitas-Astúa, EMBRAPA, Brazil for editing and commenting on the manuscript.

    The author(s) declare no conflict of interest.

    LITERATURE CITED

    The author(s) declare no conflict of interest.

    Funding: Support was provided by the Indian Council of Agricultural Research grant number F. No.16-11/PP/ICAR-CRP/17-18/06.