Molecular characterization of recombinant citrus yellow mosaic badnavirus infecting Coorg mandarin exhibiting yellow mosaic disease symptoms in high humid tropic region of Western Ghats

Abstract

The citrus yellow mosaic badnavirus (CMBV) is one of the most important viruses causing yellowing and declining in different Citrus species. The Coorg mandarin, pomelo and grapefruit showing the yellow mosaic disease symptoms were collected from different famers field during the survey. Further viral pathogenicity was confirmed through grafting on Rangpur lime as root stock. To confirm the identity of the pathogen, total genomic DNA was extracted from Coorg mandarin, Pomelo and grapefruit were subjected to PCR amplification using ORF III specific primers. Further the complete genome of CMBV amplified using different sets of specific primers were cloned and sequenced. The sequence analysis showed that CMBV from the Coorg mandarin showed maximum nt identity of 94.5% with CMBV-AL infecting acid lime. Recombination and GC plot analysis showed that the recombination occurred at in low GC content regions of genome of the CMBV and are derived from the previously reported Badnaviruses infecting different Citrus species.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13337-024-00864-z.

Introduction

Citrus is considered as one of the most important tropical fruit crops in the world and the total world production of citrus is 131.41 million metric tons (MMT) [1]. India ranked third among the world’s top citrus producing countries after China and Brazil. It also plays a vital role in the fruit economy of the country, next to banana and mango, with 13.98 MMT production and huge export potential [2]. Citrus is largely cultivated in Andhra Pradesh, Maharashtra, Telangana, Punjab, Madhya Pradesh, Gujarat, Karnataka and Assam states of India. Andhra Pradesh is top in citrus output, generating 1805.64 tonnes, accounting for 24.19% of overall production in India [1].

Citrus farming in India faces multiple challenges such as the unavailability of disease-free planting materials, the spread of infections through bud wood, and the prevalence of major pests and diseases. Over 150 diseases and disorders caused by various pathogens affect Citrus spp. from nursery to harvesting stages, resulting in significant losses. Coorg mandarin, a well-known ecotype cultivated in Karnataka, Tamil Nadu, and Kerala for over 150 years, has dwindled to less than 2,000 hectares due to factors such as Phytophthora rot and citrus greening disease. Inadequate crop management techniques and excessive shade have also affected plant development and fruiting in the region. Efforts are needed to revive citrus farming in India, particularly for valuable varieties like Coorg mandarin [3].

Citrus yellow mosaic badnavirus (CMBV) causes yellow mosaic disease in Citrus species. It belongs to the genus Badnavirus of the family Caulimoviridae. CMBV is well documented in different citrus species in India [4, 5]. The CMBV was first reported in sweet oranges (Citrus sinensis) [6] and it has since spread to other Citrus species, causing significant yield losses in citrus growing regions throughout the country. Some citrus groves have suffered losses of up to 70% due to this disease [7, 8]. Infection with CMBV has not been reported in any other citrus-growing region around the world, and currently appears to be restricted to India. This disease has been ranked as one of the 30 most significant pest hazards to plant health by the European Food Safety Authority (EFSA) [9]. This virus is spread by mechanical inoculation, grafting, dodders to a lesser extent to other Citrus species [7, 10, 11]. Despite reports of viral transmission by aphids (Myzus persicae and Aphis craccivora) and mealybugs (Planococcus citri) [12] further research is required to determine the precise function of these insect vectors in disease transmission in nature.

The citrus yellow mosaic badnavirus is characterized by a size of 130 × 30 nm and a bacilliform shape [7, 11]. The viral genome comprises a single circular double-stranded DNA molecule, measuring 7.5 kb in size, and consists of six open reading frames (ORFs). These ORFs encode proteins on the plus strand, exhibiting sizes that span from 10 to 224 kDa. [10–12]. The RNA binding region with a high cysteine content and a zinc finger-like structure (CXCX2CX4HX4C), aspartic protease, reverse transcriptase, and RNase H domains are conserved domains found in the large polyprotein encoded by CMBV ORF III, similar to other Badnaviruses [13–15]. In addition to the first cysteine-rich domain, the ORF III encoded protein in CMBV also contains a second cysteine-rich conserved domain (CX2CX11CX2CX4CX2C), which is only shared by Badnaviruses [4]. An estimated 700 nucleotides of the intergenic region comprise a potential promoter and transcription start signal [4].

Within the NCBI repository, only eight full-length sequences of CMBV are available, showcasing isolates from various Citrus species, including sweet orange, pomelo, acid lime, Rangpur lime, and rough lemon [16]. Despite numerous reports documenting CMBV infections in different Citrus species across India, it is noteworthy that no recorded instances have been found on Coorg mandarin (Citrus reticulata) to date, even though the virus has affected other citrus varieties such as sweet orange. Considering this knowledge gap, our study is strategically designed to characterize the complete genome of CMBV specifically from instances of yellow mosaic disease observed in Coorg mandarin.

Materials and methods

Survey and collection of CMBV infected samples from different Citrus species

A survey was undertaken across various locations in three southern states, namely Karnataka, Tamil Nadu, and Kerala, with the aim of assessing the prevalence of citrus yellow mosaic disease in citrus species such as Coorg mandarin, pomelo, and grapefruit. The surveyed areas predominantly featured Coorg mandarin, followed by pomelo and grapefruit, cultivated within a multi-tier cropping system alongside coffee and pepper plantations. The incidence of yellow mosaic disease on Coorg mandarin, pomelo, and grapefruit was determined by observing 100 plants, and the percentage incidence was calculated by dividing the number of infected plants by the total observed plants and multiplying by 100. The percentage of disease incidence was estimated using formula.

$$\text{PDI}\left(\%\right)=\frac{\text{Total infected citrus trees}}{\text{Total number of trees observed}}\times 100$$

The surveyed region, situated in the Western Ghats, experiences an annual rainfall exceeding 1500 mm over a span of 100 days (from July to September). A total of 152 plants infected with citrus mosaic yellow virus (CMBV), displaying symptoms of yellow mosaic disease in Coorg Mandarin, Pomelo, and grapefruits, were gathered from various farmers' fields in three Southern states. Among these, there were 70 Coorg Mandarin samples, 37 Pomelo samples, and 20 grapefruit samples. Additionally, 25 samples were collected from CHES-Chetalli, including 15 Coorg Mandarin clones maintained as mother plants in a polyhouse, and 5 each of Pomelo and grapefruit maintained in an experimental field (Table 2). Along with the infected samples, healthy leaf samples were also collected as control and all these samples were transported to the Plant Pathology laboratory at CHES, Chettalli, Madikeri, Karnataka, India, where they underwent molecular characterization.

Table 2.

Citrus yellow mosaic infecting citrus samples collected from three Southern States and confirmation through PCR using Citrus yellow mosaic virus specific primers

Place	No. of orchards	Age group (years)	No. of samples collected			Type of symptoms	Av, PDI of CMBV	No. of samples PCR positive for CBMV
			CM	PM	GF			CM	PM	GF
Karnataka	25	5–15	60	25	15	Yellow mosaic and complete yellowing	10 to 35%	50	20	10
Tamil Nadu	15	5–15	15	10	5	Yellow mosaic and complete yellowing	10 to 20%	10	5	2
Kerala	10	5–10	10	7	5	Yellow mosaic and complete yellowing	15 to 25%	8	6	2
			85	42	25			68	31	14

CM Coorg mandarin, PM Pumelo, GF Grape fruit

Pathogenicity test using graft transmission

Side veneer grafting was performed using scions obtained from infected Coorg mandarin, pomelo, and grapefruit grafted onto non-symptomatic Rangpur lime rootstocks. In each instance, twenty Rangpur lime seedlings were individually utilized for grafting with each scion. The grafted section was securely fastened with a polythene strip, and a polythene bag was used to cover the scion. These plants were placed in a cool area within the glasshouse and monitored on a weekly basis for the emergence of mosaic symptoms.

Total nucleic acid isolation, polymerase chain reaction and sequencing

Total nucleic acid was isolated from 152 CMBV infected Coorg mandarin (85 samples), pomelo (42 samples) and grape fruit (25 samples) and each one healthy leaf samples by using CTAB method [17]. The presence of CMBV in the 152 infected Coorg mandarin, pomelo, and grapefruit samples was evaluated through PCR amplification utilizing ORF III specific primers (forward primer 5’-GTGGCTTTCATCAGGTAGC 3´ and reverse primer 5´ CATGCATCCATCCGTTTCG-3´). Since the full genome sequences for pomelo and grapefruit infecting CMBV were already available in GenBank, a representative Coorg mandarin sample was selected for complete genome amplification through PCR, employing five sets of overlapping primers specific to the CMBV genome (18) (Table 1). PCR amplifications were carried out in a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, CA) thermocycler. Each reaction had a volume of 25 μL, comprising 100 ng of DNA template, 0.5U Taq DNA polymerase, 25 mM MgCl₂, 2 mM dNTPs (Fermentas, Germany), and 25 pmol of each primer. The thermocycler settings involved 35 cycles, with denaturation at 94 °C for 45 s, annealing at 52 °C for 30 s, and elongation at 72 oC for 1 min for all primers. The runs included an initial denaturation at 94 °C for 3 min and a final extension at 72 °C for 15 min for all primers. PCR products were separated on 0.8% agarose gels stained with ethidium bromide (10 mg/mL) and visualized using a gel documentation system (Alpha Innotech, USA).

Table 1.

Primers used to amplify and sequence full genome of CMBV

Primer name	Sequence	Nucleotide position	Apprx.PRODUCT size in kbp
CMBVOL 2F	5’ GCATAGTTCAGGTTCGGATCCN 3’	1493–1513	1.7
CMBVOL 2R	5’ ACATGTTTTCAATGAACTCGGCN 3’	3229–3208
CMBVOL 3F	5’ GCCGAGTTCATTGAAAACATGTN 3’	3208–3229	1.5
CMBVOL 3R	5’ ACGCTCCTGTGTCAAGAATCGN 3’	4706–4688
CMBVOL 4F	5’ CGATTCTTGACACAGGAGCGN 3’	4688–4706	1.0
CMBVOL 4R	5’ TGAGAATACTAGGATGTCGTCGATN 3’	5766–5743
CMBVOL 5F	5’ ATCGACGACATCCTAGTATTCTCAN 3’	5743–5766	1.6
CMBVOL 5R	5’ CAAGGCTATGGCATATCCTTATATAN 3’	7384–7360
CMBVOL 6F	5’ TACGGTTCTCTAAATTGCCTATATAAN 3’	7343–7366	1.5
CMBVOL 1R	5’ GGATCCGAACCTGAACTATGCN 3’	1513–1493

The PCR-amplified products were cloned into the pTZ57R/T vector (Fermentas, Germany) and subsequently transformed into DH5α competent Escherichia coli cells (Invitrogen, Carlsbad, CA, USA) following the manufacturers' provided protocols. The sequencing of three transformed clones thrice (repeated 3 times) using the ABI PRISM 3730 automated sequencer (Applied Biosystems) at the Medauxin DNA sequencing facility in Bengaluru, India. The repeated sequences obtained in each PCR fragments were 100% similar to the CBMV viral genome indicates absence mixed infection in the plants. The obtained sequences overlapping region of each PCR fragment were curated and realign using different bioinformatics programs (BioEdit, Clustal X2, and Sea View).

Viral genome analysis

Genome was assembled and sequences were analyzed from the contigs using the CAP Assembly program of BioEdit Package V 5.0.9. The sequence similarity search for viral genome performed by BLASTn Program (http://www.ncbi.nlm.nih.gov/BLAST) and the selected Badnavirus sequence (Table 2) accessions displaying the highest percentage of nucleotide (nt) identity were retrieved from the NCBI database for analysis. The per cent pairwise nt identities of CMBV and selected badnavirus were calculated using SDT version 1.2 [19]. Neighbor-joining method analysis was carried out to determine the evolutionary relationship between CMBV associated with Coorg mandarin and GenBank Badnavirus isolates using MEGA X software by 1,000 bootstrapped replications [20]. Recombination break point analysis was carried out between CMBV and other selected Badnavirus using RDP5 with defaults settings (P value of 0.05) [21]. The GC content in the viral genome was analyzed and per cent GC-plot graph generated through Artemis DNA plotter analysis tool v18.1.0 [22].

Results

Symptomatology of citrus yellow mosaic virus on different citrus species

Various citrus species cultivated in orchards across three states displayed various intriguing symptoms. These include light mosaic patterns, yellow mosaic patches spreading between veins, and complete yellowing, as observed in Coorg mandarin plants (Fig. 1A, B, C). Similarly, Pomelo plants exhibited mosaic patches and overall yellowing of leaves (Fig. 1D). Meanwhile, grapefruit plants in all surveyed locations across three southern states displayed uniform yellow leaves (Fig. 1E). The survey also identified a significant infestation of mealy bugs on the infected plants in their natural environment. On each leaf and twig, a substantial cluster of more than 10 to 20 mealy bugs was observed (Fig. 1F). The presence of mealy bugs, combined with mosaic and vein clearing symptoms, raised suspicions of the citrus yellow mosaic virus association with the affected plants.

Fig. 1.

Sequential viral symptoms development in Coorg Madarin plant a light mosaic b, yewllo mosaic spreads between the veins, c complete yellow and Vein clearing, d Pemelo plant showing complete yellowing e Grape fruit plant exhibiting complete yellowing leaves under natural conditions, f Mealy bugs feeding on citrus yellow mosaic infected coorg mandarin plant

Survey and incidence of CMBV on different Citrus species

A roving survey conducted across 25 orchards in Karnataka, 15 in Tamil Nadu, and 10 in Kerala, revealed that the incidence of CMBV disease varied across different citrus species. In Karnataka, the disease affected Coorg mandarin, Pomelo, and grapefruit plants raised from both true seeds and grafted plants in commercial groves, with an incidence ranging from 10 to 35%. In Tamil Nadu, the incidence was recorded at 10% to 20%, while in Kerala, it ranged from 15 to 25% on grafted plants in commercial groves. Additionally, it was noted that the yellowing of leaves was more pronounced in budded plants six years after planting compared to plants raised from true seeds. The infected plants displayed yellow mosaic symptoms, which were consistently observed across all surveyed areas (Table 2, Table S1).

Graft transmission of CMBV on Coorg mandarin

Citrus yellow mosaic disease was successfully graft transmitted from field-infected Coorg mandarin, Pemelo and Grape fruit plants (CMBV, CHES isolate) to healthy seedlings of Rangpur lime in the greenhouse. All graft-inoculated plants displayed yellow mosaic symptoms—within 3 months after grafting in each experiment (Fig. 2a, b, c).

Fig. 2.

Graft transmission of citrus yellow mosaic virus on different citrus species (H) Healthy plant coorg mandarin plant a Pemelo plant showing mosaic, b Coor Mandarin plant exhibiting mosaic, c Grapefruit plant expressing mosaic symptoms

Complete genome characterization

Initially, 152 CMBV-infected citrus samples were gathered from various farmers’ fields in three Southern states. These included 70 Coorg mandarin samples, 37 Pomelo samples, and 20 grapefruit samples. Additionally, 25 samples from CHES-Chetalli comprised 15 Coorg mandarin clones maintained as mother plants in a polyhouse, along with five pomelo and grapefruit samples maintained in an experimental field. PCR amplification using CMBV ORF III specific primers was performed on these samples. Out of the 152 CMBV-infected samples collected during the survey, 68 Coorg mandarin samples, 31 Pomelo samples, and 14 grapefruit samples exhibited positive amplification, producing approximately a 650 bp DNA fragment representing the ORF III region from the virus-infected plants but not from healthy ones (Table 2). The PCR-amplified fragments from only 100 samples were directly sequenced to confirm the virus identity in different citrus species. Sequence analysis revealed that the ORF III region of all 100 CMBV-infected samples (60 Coorg mandarin, 20 pomelos, and 20 grapefruit) exhibited more than 98% nucleotide identity with CMBV infecting acid lime (Data not shown).

A representative sample from Coorg mandarin was selected for further analysis, as no previous reports of CMBV infecting Coorg mandarin existed. Full-length genome amplification was conducted using various sets of primers. PCR-amplified genomic regions were cloned and sequenced, and the raw sequences underwent curation and alignment using various bioinformatics programs, including Clustal X, Sea View, and BioEdit. The compiled consensus full viral genome of CMBV infecting Coorg mandarin (CMBV-CM) was then submitted to the NCBI GenBank under the accession number OR765856.

The complete genome of CMBV-CM spans 7467 nucleotides, which is somewhat smaller than the earlier reported CMBV-AL isolate (13). Table 3 presents the pairwise percent identities of nt and derived amino acid (aa) sequences for the six open reading frames (ORFs), whole genomes, and the intergenic region (IR) sequence of CMBV-CM. In comparison, the complete genome of CMBV-CM was analyzed against nine CMBV isolates and 18 other Badnavirus sequences retrieved from the NCBI Database. The results indicate that the CMBV-CM isolate exhibits maximum nucleotide identity, with more than 94.5%, compared to CMBV-AL infecting acid lime (Table 3). Among the CMBV isolates infecting various citrus species, the nucleotide identity ranges from 88.3 to 94.5%. Conversely, the CMBV-CM isolate displays a very low nucleotide identity, less than 50%, compared to the other 18 Badnavirus isolates infecting different crops. These findings were further corroborated by the sequence demarcation tool, affirming that CMBV-CM shares greater homology with CMBV isolates infecting different citrus species in India (Supplementary Fig. 1a).

Table 3.

Pair wise identities between Citrus yellow mosaic virus infecting coorg mandarin with other Badnaviruses available in GenBank

Badnaviruses#	Crops	Acc. No	Genome	IR	ORF1	ORF2	ORF3	ORF4	ORF5	ORF6
CMBV-SO	Sweet orange	AF347695	88.3	79.8	95.1	91.2	94.5	75.1	72.6	96.1
CMBV-P	Pumello	EU489745	94.0	95.3	95.8	94.1	96.5	85.6	70.1	98.7
CMBV-AL	Acid Lime	EU489744	94.5	97.4	95.8	94.8	97.2	86.1	70.5	100
CMBV- SOP	Sweet orange	EU708316	89.9	79.8	97.2	92.7	95.0	36.4	69.9	95.4
CMBV-SO Nagri	Sweet orange	FJ617224	88.4	79.8	95.8	90.5	–	68.5	68.9	93.5
CMBV-AL AP	Acid lime	EU708317	88.7	77.7	97.2	91.2	95.0	35.9	73.6	97.4
CMBV-RL	Rangpur lime	DQ875213	88.8	76.6	97.9	94.2	–	58.0	74.7	96.1
CMBV-RoL	Rough lemon	JN006806	90.7	80.9	95.8	94.8	94.0	86.7	65.0	94.1
CMBV-RoL	Rough lemon	JN006805	89.2	82.5	94.4	94.8	94.4	74.0	73.6	97.4
BsCVBV	Bougainvillea	EU034539	42.3	32.5	20.9	20.6	32.9	10.6	–	–
BSVGF	Banana	AY493509	43.2	34.0	22.4	20.0	37.1	–	–	–
BSVMYS	Banana	AY805074	43.3	27.6	30.1	23.4	37.6	–	–	–
CSSV	Cacao	NC_001574	50.2	31.9	53.8	26.2	49.7	96.0	89.0	25.9
CSSV Peki	Cacao	AJ609019	51.7	36.7	55.2	23.4	50.0	74.0	12.0	27.2
CoYMV	Commelina	X52938	42.4	36.1	19.0	18.0	36.1	–	–	–
CLNV	Cycad	EU853709	39.0	27.1	25.7	20.0	39.0	–	–	–
DBV	Dioscorea	DQ822073	49.5	31.1	34.4	–	–	–	–	–
DrMV	Dracaena	DQ473478	45.5	29.8	36.6	28.3	39.4	63.0	12.2	19.2
GVBaV	Gooseberry	HQ852251	42.0	33.9	10.7	28.2	35.7	–	–	–
KTSV	Kalanchoe	AY180137	41.6	28.5	17.8	22.8	34.5	–	–	–
PBCoV	Pineapple	GU121676	43.2	31.3	25.7	22.6	37.9	–	–	–
PVBV	Pelargonium	GQ428155	43.0	31.8	42.6	28.1	37.0	–	–	–
SPBVA	Sweet potato	FJ560943	43.5	33.5	46.1	19.3	39.6	85.0	–	–
SPBVB	Sweet potato	FJ560944	43.5	27.7	46.1	19.3	39.7	85.0	–	–
ScBIMV	Sugarcane	AJ277091	41.4	27.8	45.4	18.1	40.4	11.6	–	–
ScBMorV	Sugarcane	M89923	40.5	29.6	23.4	23.4	32.3	–	–	–
TaBV	Taro	AF357836	42.8	27.8	36.6	23.7	35.1	–	–	–

^#Definition for acronyms as per the Bhat et al. (2016)

The length of the intergenic region (IR) in CMBV-CM measured 656 nucleotides, in contrast to the 731 nucleotides observed in the CMBV-SO isolate infecting sweet orange. This discrepancy primarily results from deletions in the IR region of CMBV-CM compared to CMBV-SO. The distinct regulatory sequence found in the IR region of CMBV-CM closely resembles that of CMBV-AL. Within the IR region is an 18-nucleotide consensus sequence (5'TGGTATCAGAGCTTGGTT3'), representing the plant tRNAmet, which serves as the start site of replication. This consensus sequence is conserved across all members of the Family Caulimoviridae and in all reported CMBV isolates to date. Additionally, the IR includes a TATAA consensus sequence, potentially serving as a TATA box, which remains conserved in all CMBV isolates. The IR region of CMBV-CM exhibits a maximum nucleotide identity of 97.4% with the CMBV-AL isolate. Alongside the TATA box, several other consensus sequences (TGACG and CACAAT) in CMBV-CM are similar to those observed in CMBV-AL. Notably, a potential polyadenylation consensus of AATAAA is also identified in CMBV-CM.

The CMBV-CM genome includes highly conserved regions in its ORF I, encoding a 143-amino acid predicted protein. Alignment CMBV-CM with other CMBV isolates, especially CMBV-AP infecting acid lime, reveals five conserved 'pockets' (SHTANLEYLDLAS, HNLAV, PLSK, QPKLVE, and LTE). Similarly, ORF II of CMBV-CM, which encodes a 137-amino acid protein exhibits conserved 'pockets' (KDPY, KQNN, and ALPDDLIDKL), with maximum identity to CMBV-AL infecting acid lime.

ORF III, a polyprotein post-translationally modified into movement protein, coat protein, protease, and reverse transcriptase/RNAse H domains, is highly conserved in CMBV-CM, resembling other reported CMBV isolates. The amino acid sequence of ORF III of CMBV-CM showed maximum identity to CMBV-AL infecting acid lime.

ORF IV encodes a 95-amino acid protein with 96.7% identity to CMBV-RoL infecting rough lemon. Additionally, ORF V and VI display 73.6% and 100% maximum amino acid identity with CMBV-RoL and CMBV-AL infecting rough lemon and acid lime, respectively.

A phylogenetic analysis was conducted to ascertain the evolutionary relationship of the CMBV-CM isolate with nine selected CMBV isolates and 18 other Badnavirus sequences obtained from the database. The Neighbor-Joining (NJ) method implemented in MEGA X software, with 1000 bootstrap replications and Cacao swollen shoot virus (CSSV) as an outgroup, was employed. The phylogenetic tree revealed that the CMBV-CM isolate forms a close cluster with nine CMBV isolates infecting various citrus species in India (Fig. 3a). This clustering was further supported by the Sequence Demarcation Tool (SDT) (Supplementary Fig. 1a).

Fig. 3.

Phylogenetic tree showing the relationship of citrus yellow mosaic virus infecting Coorg Mandarin and other badnaviruses. The trees were drawn using MEGA 11 tool, using the Neighbor-Joining method

GC plot analysis

The proportion of guanine (G) and cytosine (C) in a given stretch fragment of the genome is referred to as guanine-cytosine (GC) content. The GC content of the CMBV-CM genome from Coorg mandarin was determined using Artemis DNA plotter version 18.1.0. (Supplementary Fig. 2). The GC analysis revealed variation in GC content along different stretches of the CMBV-CM genome. With a window size of 100, the innermost circle and bar represent above-average (orange) and below-average (black) GC content in the genome of CMBV-CM associated yellow mosaic disease of Coorg mandarin. Except in stretches of the IR region, there is variation in GC content across the genome (ORFI, ORFII, ORFIIII, ORFIV, ORFV, and ORFVI). All the genes had stretches of GC rich and GC low regions in the viral genome, except the IR region, which had completely below average GC, content (Supplementary Fig. 2).

Neighbor-net and recombination analysis

Neighbor-net analysis of complete genome of CMBV-CM along with nine CMBV isolates and 18 other Badnavirus retrieved from the NCBI Database using SplitTree program revealed extensive networked evolution in CMBV isolates in the present study with other 18 other Badnavirus indicating recombination. Split tree analysis revealed a "rectangular" network structure in which CMBV-CM isolate is clearly differentiated according to their respective species CMBV isolates (Supplementary Fig. 3). Further recombination analysis was carried out between CMBV-CM DNA sequence from Coorg mandarin and other Badanaviruses using RDP5, which indicated intra-specific recombination in the genome of CMBV-CM associated with Coorg mandarin. The genome of CMBV-CM associated with Coorg mandarin was shown to be derived from citrus yellow mosaic badnavirus (CMBV-SO, AF347695) and (CMBV-RL, DQ875213) as major and minor parents infecting Sweet orange and Rangpur lime. Recombination was found at nucleotide positions 2005 and 4055, with a probability of 5.071X10⁻¹². Another 1413 nt recombination breakpoint was identified in the genome of CMBV-CM and may be derived from CMBV-RL (DQ875213) and CMBV-SO (AF347695) as major and minor parents infecting Rangpur lime and sweet orange, respectively. Recombination break point was predicted at nucleotide, 4927 and 6340 with the P-value of 5.8277 × 10^–4. In the genome of CMBV-CM, a recombination fragment of 2836 nts was identified, with the minor and major parents resembling CMBV-AL (EU489744) and CMBV-RoL (JN006806) infecting acid lime and rough lemon, respectively. Recombination was found at nucleotide positions 7454 and 4618 with a probability value of > 1 (Supplementary Fig. 1b).

Discussion

Coorg mandarin, a well-known mandarin ecotype, has been grown for over 150 years in Southern India alongside Pomelo and grapefruit, usually in a multi-tier cropping system with coffee and pepper plantations. Coorg mandarins, recognized as man-made hybrids, are prized for their tight skin, sweet–sour taste, and extended shelf life. Unfortunately, Coorg mandarin, Pomelo, and grapefruit cultivation have sharply declined due to challenges such as the scarcity of virus-free planting materials and the impact of diseases like Phytophthora rot and citrus greening. This has resulted in a significant reduction in yield, with a single plant now producing around 10 kg, compared to the once abundant yield of over 50 kg per plant [23].

This study collected samples of Coorg mandarin, pomelo, and grapefruit from both natural and polyhouse conditions and confirmed the presence of CMBV infection through PCR-based diagnostics and genome sequencing. Sequence analysis indicated that the CMBV associated with Coorg mandarin, Pomelo, and grapefruit is closely related to citrus yellow mosaic badnavirus, which infects acid lime in India. CMBV was initially reported in 1975 by Murthi and Reddy, followed by Dakshinamurti et al. [24] in sweet orange (Citrus sinensis), and later in other citrus species, including Pomelo (C. grandis [L.] Osbeck), acid lime (C. aurantifolia [L.] Swingle), and Rangpur lime (C. limonia) [3]. Citrus yellow mosaic badnavirus (CMBV) is a plant pararetrovirus, emerging as a disease in Southern India, extensively documented on various citrus species by different researchers [15, 16, 25, 26]. The virus spreads through infected budwood during grafting at nurseries [27, 28] and via Planococcus citri (citrus mealybug) in field conditions [7]. Coorg mandarin, Pomelo, and grapefruit plants supplied by private nurseries carry virus infections through grafted plants, significantly impacting major mandarin-growing states like Karnataka, Tamil Nadu, and Kerala. This phenomenon could be attributed to Coorg mandarin, Pomelo, and grapefruit plants infected by CMBV, displaying symptoms resembling mineral deficiencies commonly observed in citrus species. The challenge arises when nursery workers struggle to differentiate between diseased and healthy budwood plants, inadvertently utilizing budwood from infected plants for grafting. This practice contributes to the proliferation of the disease in newly established orchards.

The high GC content observed in the regions of the CBMV-CM genome, including ORFI, ORFII, ORFIII, ORFIV, ORFV, and ORFVI, suggests a potential recombination site. This phenomenon is well-documented in various viruses affecting plants and animals [29–31]. The stability of sequences with high GC content is attributed to triple hydrogen bonds, stacking interactions between bases, and DNA strands' overall topology and orientation [30, 32]. The presence of more bonds between bases in a DNA strand requires greater energy to break the strand. A parallel observation of high GC content in the intergenic region of the herpes simplex virus (HSV) genome further underscores its potential role in viral evolution and pathogenesis [33].

Recombination plays a crucial role in the rapid evolution of viruses, leading to changes in virulence and host range [34]. This phenomenon has been extensively documented worldwide, emphasizing its role in the emergence of new viruses [35–37]. Recombination events between different viruses result in new strains or viruses with enhanced virulence, representing a significant form of molecular variation in viruses. In some cases, recombination can be beneficial; for instance, recombination between different maize streak virus isolates increases pathogenicity in maize, indicating improved adaptation through recombination [38].

However, in some instances, recombination can be detrimental. For example, recombination between tomato yellow leaf curl virus and tomato yellow leaf curl Sardinia virus reduced the viruses' replicative capacity [39]. The present study unveils the presence of intraspecific recombination in CBMV-CM infecting Coorg mandarin, suggesting that this process may be responsible for the evolution of a new recombinant virus with severe effects on Coorg mandarin in India. Recombination between and within species of viruses is a significant factor in the emergence of novel virus species and their adaptation to new hosts in agricultural systems [40, 41].

Coorg mandarin is primarily propagated by seeds, excluding Pomelo and grapefruit. Due to wider heterozygosity and a prolonged juvenile phase, seedling plants are less favoured. Consequently, many farmers in all three states prefer grafted or budded plants. These grafted scions originate from CMBV-infected mother plants, and once the virus is established, its elimination from the planting material is challenging. Additionally, re-establishing disease-free desirable clones and preventing the virus from spreading to new locations pose difficulties [42]. The sequence information obtained from this study will be valuable for early virus detection in nursery planting materials, facilitating the production of clean planting materials and preventing further spread to fields across different states.

Supplementary Information

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Data availability

All data analyzed during this study are included in this article and its supplementary information files.

Declarations

Ethical approval

This article does not contain any studies with human participants performed by any of the authors.

Human and animal participation

This article does not contain any studies with human or animal subjects performed by any of the authors.