Molecular Characteristics of Bean Common Mosaic Virus Occurring in Inner Mongolia, China

Bean common mosaic virus (BCMV) was detected on common bean (Phaseolus vulgaris) plants showing wrinkled and/or narrow leaves, curling, shrinking and chlorosis of leaves, dwarfing of plants, and mottled pods in Inner Mongolia and named BCMV-22Huhe. Its genome has a size of 10,062 bp and was deposited in GenBank under the accession number OR778613. It is closely related to BCMV-Az (GenBank accession no. KP903372, in China) in the lineage of AzBMV. A recombination event was detected for BCMV-22Huhe among the 99 BCMV isolates published in the NCBI GenBank database, showing that BCMV-CJ25 (MK069986, found in Mexico) was a potential major parent, and the minor parent is unknown. This work is the first description of the occurrence of BCMV in Inner Mongolia, China.


Introduction
Bean common mosaic virus (BCMV) is a member of the genus Potyvirus [1], it has a genome of a single-stranded RNA molecule of about 10,000 nucleotides.BCMV is widely distributed worldwide, and yield loss due to BCMV can be as high as 100% [2][3][4].It is a potential threat to the bean industry in China [5].BCMV can be transmitted through various ways, including mechanical inoculation, seeds, pollen grains, and aphids [6,7].
Common bean (P.vulgaris) is herbaceous annual plant grown worldwide for its edible dry seeds or green, unripe pods, and it is known by many different names, including French beans, string beans, and snap beans.The world production of string beans was about 1.3 million tons from about 0.14 million ha in 2021 (https://www.fao.org/home/en,accessed on 21 December 2023).Inner Mongolia is one of the main production areas for common beans in China.With an increasing cultivation area and continuous cropping for years, disease and pest problems have become increasingly prominent, especially the occurrence of viral diseases [5,8].In recent years, mosaic, wrinkling and other viral diseases of beans have been observed on land and in greenhouses in Inner Mongolia, affecting the yield and quality of beans and limiting the sustainable development of the bean industry.Here, we described the molecular characteristics of BCMV occurring on common bean in Inner Mongolia, China.

Virus Source
In 2022, common bean (P.vulgaris) plants showing wrinkled leaves, chlorosis of leaves, and mottled or deformed pods (Figure 1a) were observed in Hohhot, Inner Mongolia, China, suggesting viral infection.A visual incidence rate of about 50% occurred for the disease.Samples of fresh leaves of the diseased common bean plants were collected for pathogen diagnosis.
In 2022, common bean (P.vulgaris) plants showing wrinkled leaves, chlorosis of leaves, and mottled or deformed pods (Figure 1a) were observed in Hohhot, Inner Mongolia, China, suggesting viral infection.A visual incidence rate of about 50% occurred for the disease.Samples of fresh leaves of the diseased common bean plants were collected for pathogen diagnosis.

Amplification of the Genomic Fragments of BCMV
The total RNA of each sample was extracted using a Spectrum TM plant total RNA kit (Sigma-Aldric, St. Louis, MO, USA), and its concentration was determined using a microspectrophotometer, a Nano-300 (Allsheng, Hangzhou, China).The 5′ and 3′ termini of the BCMV isolate were amplified using a SMARTer RACE kit (Takara, Dalian, China), following the manufacturer's instructions.The middle part of the BCMV isolate was cloned in 3 fragments (Table 1).Briefly, using the RNA samples as templates, cDNAs were synthesized using a PrimeScript II 1st strand cDNA synthesis kit (Promega Biotechnology, Beijing, China) following the manufacturer's instructions.The cDNAs served as templates in PCR assays for amplifying the genomic cDNA fragments of BCMV.The PCR reaction mixture (20 μL) was prepared as follows: cDNA, 2.0 μL; M5 superlight mix (Mei5 Biotechnology, Beijing, China), 10 μL; nuclease-free H2O, 6.0 μL; forward and reverse primers, 1.0 μL each.The primers (Table 1) were designed based on the conserved parts of the complete genome sequences of 96 isolates of BCMV deposited in the NCBI GenBank database.The PCR program was set as follows: pre-denaturation at 95 °C for 3 min, 35 cycles of 30 s at 94 °C, 30 s at Tm (Table 1), and 1 kb/min at 72 °C, and a final extension of 10 min at 72 °C.

Amplification of the Genomic Fragments of BCMV
The total RNA of each sample was extracted using a Spectrum TM plant total RNA kit (Sigma-Aldric, St. Louis, MO, USA), and its concentration was determined using a micro-spectrophotometer, a Nano-300 (Allsheng, Hangzhou, China).The 5 ′ and 3 ′ termini of the BCMV isolate were amplified using a SMARTer RACE kit (Takara, Dalian, China), following the manufacturer's instructions.The middle part of the BCMV isolate was cloned in 3 fragments (Table 1).Briefly, using the RNA samples as templates, cDNAs were synthesized using a PrimeScript II 1st strand cDNA synthesis kit (Promega Biotechnology, Beijing, China) following the manufacturer's instructions.The cDNAs served as templates in PCR assays for amplifying the genomic cDNA fragments of BCMV.The PCR reaction mixture (20 µL) was prepared as follows: cDNA, 2.0 µL; M5 superlight mix (Mei5 Biotechnology, Beijing, China), 10 µL; nuclease-free H 2 O, 6.0 µL; forward and reverse primers, 1.0 µL each.The primers (Table 1) were designed based on the conserved parts of the complete genome sequences of 96 isolates of BCMV deposited in the NCBI GenBank database.The PCR program was set as follows: pre-denaturation at 95 • C for 3 min, 35 cycles of 30 s at 94 • C, 30 s at Tm (Table 1), and 1 kb/min at 72 • C, and a final extension of 10 min at 72 • C.

Cloning and Sequencing of the BCMV Fragments
The PCR products were separated on 1% agarose gel, and the fragments of expected sizes were purified using a Tiangen Midi purification kit (Tiangen, Beijing).The purified fragments were inserted into a pTOPO-TA cloning vector (Aidlab Biotechnologies, Beijing).The ligation mixture included 5 µL of the purified fragments, 1 µL of pTOPO-TA Vector, 1 µL of 10× Enhancer, and 3 µL of nuclease-free H 2 O, and the mixture was incubated 2 h at 37 • C. The ligation product was used for the transformation of Escherichia coli JM109 competent cells.Positive colonies were screened using a colony PCR, and the positive ones were sent to Sangon Biotech (Beijing) for Sanger sequencing.

BCMV Genome Assembly and Sequence Analysis
The obtained query sequences were submitted to the BLASTn online tool to retrieve homologous sequences from the NCBI GenBank database, and the sequences, verified BCMV genome fragments, were assembled using Vector NTI 11.5 (Invitrogen, Carlsbad, CA, USA), generating the full-length genome of the BCMV isolated in Hohhot, named BCMV-22Huhe.
A recombination analysis was conducted for BCMV-22Huhe and the 99 BCMV isolates (including the 15 representative strains) and published in the NCBI GenBank database using the Recombination Detection Program 4 (RDP4) software, in which seven different methods were used for the recombination analysis, namely RDP, GENECO NV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN, and 3SEQ.With p ≤ 0.05 as a standard [11], if more than three methods were detected at the same time, it was judged to be a meaningful recombination event.

BCMV Host Range
To identity potential host plants for BCMV, healthy plants of seven different species were sap-inoculated using the juice of the BCMV 22Huhe-infected common beans.The tested plants included N. benthamiana, N. occidentalis, N. tabacum, N. glutinosa, Chenopodium quinoa, C. amaranticolor, and Datura stramonium.The inoculated plants were maintained in a glasshouse under 16/8 h of light and darkness at 20-25 • C and 60% humidity.The inoculated plants were observed daily for possible symptom development.Eight days post inoculation, an RT-PCR was performed to detect BCMV in the inoculated plants.

Genome Structure and Sequence Identity Analysis of BCMV Isolates
The complete genome of BCMV-22Huhe consists of 10,062 nucleotides.Through alignment mainly with the representative strain of BCMV-R in Zhejiang, China (GenBank accession no.NC_003397), it was annotated and encodes a large open reading frame (ORF), expressing a polymeric protein of about 358 kDa which can be spliced into 10 functional proteins, i.e., P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb, CP, and a small frameshift ORF from the P3 gene, producing PIPO.The genome of BCMV-22Huhe had nucleotide sequence identities of 81.7-93.8% to the genomes of the 99 BCMV isolates published in NCBI GenBank database, while their amino acid sequence identities ranged from 87.0% to 96.3%.Moreover, BCMV-22Huhe has the highest nucleotide sequence identity with BCMV-C54 and -313615 (GenBank accession no.OP828732 in China and MH024840 in the USA), and the lowest nucleotide sequence identity with BCMV-RU1P (KF919300 in the USA); the same is true for its amino acid sequence identity.

Phylogeny and Recombination Analyses of BCMV Isolates
To determine the classification of BCMV-22Huhe, a phylogenetic tree including 17 representative strains of BCMV was constructed based on the complete genome sequences of the BCMV isolates (Figure 2).On the tree, BCMV 22Huhe was closely related to BCMV-Az (KP903372), suggesting the AzBMV lineage of BCMV-22Huhe.
(ORF), expressing a polymeric protein of about 358 kDa which can be spliced into 10 functional proteins, i.e., P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb, CP, and a small frameshift ORF from the P3 gene, producing PIPO.The genome of BCMV-22Huhe had nucleotide sequence identities of 81.7-93.8% to the genomes of the 99 BCMV isolates published in NCBI GenBank database, while their amino acid sequence identities ranged from 87.0% to 96.3%.Moreover, BCMV-22Huhe has the highest nucleotide sequence identity with BCMV-C54 and -313615 (GenBank accession no.OP828732 in China and MH024840 in the USA), and the lowest nucleotide sequence identity with BCMV-RU1P (KF919300 in the USA); the same is true for its amino acid sequence identity.

Phylogeny and Recombination Analyses of BCMV Isolates
To determine the classification of BCMV-22Huhe, a phylogenetic tree including 17 representative strains of BCMV was constructed based on the complete genome sequences of the BCMV isolates (Figure 2).On the tree, BCMV 22Huhe was closely related to BCMV-Az (KP903372), suggesting the AzBMV lineage of BCMV-22Huhe.The phylogenetic tree based on the whole-genome sequences of BCMV.The tree was constructed using the maximum likelihood method (general time-reversible model + GI).GenBank accession numbers, strains, and countries are labeled for each node; BCMIV-22Huhe in this work is in red, its potential major parent of CJ25 is in blue, and a possibly potential minor parent of Ir_GoB is in brown.Closing braces show the lineage of related clades.
Among the 99 BCMV isolates published in the NCBI GenBank database, a recombination event was detected for BCMV-22Huhe which was supported by all seven algorithms (Table 2).The recombination site was found from 2121 nt (99% confidence interval 1903 nt-3443 nt) to 6626 nt (99% confidence interval 5717-6824 nt); the major parent was BCMV-CJ25 (MK069986 found in Mexico), while the minor parent was unknown.BCMV-IrGoB (MF498886 found in Iran) was suggested as a possibly potential minor parent (Figure 3a).Using the recombinant fragment of the representative strains, a phylogenetic tree (Figure 3b) was rebuilt on which BCMV-22HuHe was closely related to the potential major parent, BCMV-CJ25 (MK069986), in the US1 lineage.This supported the result of the recombination analysis.While removing the recombinant sequences from the complete genome and assembling the non-recombinant sequences for constructing another phylogenetic tree, on the tree, BCMV-22HuHe was found to be closely related to BCMV-Az (KP903372) in the AzBMV lineage (Figure 3c), in agreement with the result of the phylogenetic analysis using the completed genome sequences above.Among the 99 BCMV isolates published in the NCBI GenBank database, a recombination event was detected for BCMV-22Huhe which was supported by all seven algorithms (Table 2).The recombination site was found from 2121 nt (99% confidence interval 1903 nt-3443 nt) to 6626 nt (99% confidence interval 5717-6824 nt); the major parent was BCMV-CJ25 (MK069986 found in Mexico), while the minor parent was unknown.BCMV-IrGoB (MF498886 found in Iran) was suggested as a possibly potential minor parent (Figure 3a).Using the recombinant fragment of the representative strains, a phylogenetic tree (Figure 3b) was rebuilt on which BCMV-22HuHe was closely related to the potential major parent, BCMV-CJ25 (MK069986), in the US1 lineage.This supported the result of the recombination analysis.While removing the recombinant sequences from the complete genome and assembling the non-recombinant sequences for constructing another phylogenetic tree, on the tree, BCMV-22HuHe was found to be closely related to BCMV-Az (KP903372) in the AzBMV lineage (Figure 3c), in agreement with the result of the phylogenetic analysis using the completed genome sequences above.

Figure 1 .
Figure 1.BCMV-infected common bean and Nicotiana benthamiana plants.(a) A common bean plant with wrinkled leaves, chlorosis of leaves, and mottled pods.(b,c) Symptoms caused by BCMV-22Huhe on N. benthamiana plants eight days post inoculation.

Figure 1 .
Figure 1.BCMV-infected common bean and Nicotiana benthamiana plants.(a) A common bean plant with wrinkled leaves, chlorosis of leaves, and mottled pods.(b,c) Symptoms caused by BCMV-22Huhe on N. benthamiana plants eight days post inoculation.

Figure 2 .
Figure 2.The phylogenetic tree based on the whole-genome sequences of BCMV.The tree was constructed using the maximum likelihood method (general time-reversible model + GI).GenBank accession numbers, strains, and countries are labeled for each node; BCMIV-22Huhe in this work is in red, its potential major parent of CJ25 is in blue, and a possibly potential minor parent of Ir_GoB is in brown.Closing braces show the lineage of related clades.

Figure 2 .
Figure 2. The phylogenetic tree based on the whole-genome sequences of BCMV.The tree was constructed using the maximum likelihood method (general time-reversible model + GI).GenBank accession numbers, strains, and countries are labeled for each node; BCMIV-22Huhe in this work is in red, its potential major parent of CJ25 is in blue, and a possibly potential minor parent of Ir_GoB is in brown.Closing braces show the lineage of related clades.

Figure 3 .
Figure 3. Recombination analysis for BCMV-22Huhe.(a) The result of an RDP analysis of a recombination event in 22Huhe using RDP4; (b) the maximum likelihood phylogenetic tree based on recombinant sequences; (c) the maximum likelihood phylogenetic tree based on the assembled sequence of non-recombinant sequences.The trees were constructed using a bootstrapping method with 1000 replications.BCMIV-22Huhe in this work is in red, its potential major parent of CJ25 is in blue, and a possibly potential minor parent of Ir_GoB is in brown.

Figure 3 .
Figure 3. Recombination analysis for BCMV-22Huhe.(a) The result of an RDP analysis of a recombination event in 22Huhe using RDP4; (b) the maximum likelihood phylogenetic tree based on recombinant sequences; (c) the maximum likelihood phylogenetic tree based on the assembled sequence of non-recombinant sequences.The trees were constructed using a bootstrapping method with 1000 replications.BCMIV-22Huhe in this work is in red, its potential major parent of CJ25 is in blue, and a possibly potential minor parent of Ir_GoB is in brown.

Table 1 .
Primers used for the detection and amplification of the full-length genome of the BCMV.

Table 1 .
Primers used for the detection and amplification of the full-length genome of the BCMV.
editing, L.Z.; visualization, J.L.; supervision, Z.W.; project administration, Z.L. and Z.L.; funding acquisition, Z.L.All authors have read and agreed to the published version of the manuscript.This research was funded by the Natural Science Foundation of Inner Mongolia, grant number 2023LHMS03020, and the Higher Education Reform and Development Project-Young Science and Technology Talents Programme, grant number NJYT23079, and the Research Startup Funds for High-level Researchers in Inner Mongolia Agricultural University, grant number NDYB2019-1.
Funding:Informed Consent Statement: Not applicable.