Pathogenic and coat protein characterization confirming the occurrence of Bean common mosaic virus on common bean (Phaseolus vulgaris) in Kashmir, India Aflaq Hamid, Mushtaq Ahmad, B. A. Padder, M. D. Shah, Sehar Saleem, T. A. Sofi & A. A. Mir Phytoparasitica ISSN 0334-2123 Phytoparasitica DOI 10.1007/s12600-013-0362-5
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Author's personal copy Phytoparasitica DOI 10.1007/s12600-013-0362-5
Pathogenic and coat protein characterization confirming the occurrence of Bean common mosaic virus on common bean (Phaseolus vulgaris) in Kashmir, India Aflaq Hamid & Mushtaq Ahmad & B. A. Padder & M. D. Shah & Sehar Saleem & T. A. Sofi & A. A. Mir
Received: 29 June 2013 / Accepted: 25 October 2013 # Springer Science+Business Media Dordrecht 2013
Abstract Bean common mosaic virus (BCMV), belonging to the family Potyviridae, is a serious pathogen of common bean (Phaseolus vulgaris L.) causing considerable economic losses owing to seed, sap and aphid transmissibility. The viral nature of the test isolates and identity of the virus as BCMV were confirmed by mechanical transmission and DAS-ELISA using BCMV antiserum. Pathogenic variability studies in BCMV infecting common bean (Phaseolus vulgaris L.) in Jammu and Kashmir (a northwestern Himalayan state of India), revealed the existence of three pathogroups – PG-I, PG-II and PG-VII, accommodating five strains (NL-1, NL-1n, NL-4, NL-7 and NL-7n). Comparative sequence analysis of coat protein gene revealed that the strains NL-1, NL-4 and NL-7 shared more than 90% amino acid sequence homology with other BCMV isolates from other countries. DAG motif as well as BCMV specific conserved motif MVWCIDN were present in all the three strains. Phylogenetic analysis of coat protein also clustered them in the BCMV group. This study confirmed the occurrence of BCMV and its strains on common bean in Kashmir.
Electronic supplementary material The online version of this article (doi:10.1007/s12600-013-0362-5) contains supplementary material, which is available to authorized users. A. Hamid (*) : M. Ahmad : B. A. Padder : M. D. Shah : S. Saleem : T. A. Sofi : A. A. Mir Division of Plant Pathology, SKUAST- Kashmir, Shalimar, Srinagar 191121 Jammu & Kashmir, India e-mail:
[email protected]
Keywords BCMV . Molecular characterization . Variability
Introduction Common bean (Phaseolus vulgaris L.), a premier grain legume crop in the northwestern Himalayan states of India, is cultivated both as snap and dry bean over an area of approximately 26.75 thousand hectares with an annual production of approx. 14.2 thousand metric tonnes (Anon. 2010). Like other crops, common bean is infected by numerous viruses worldwide, mostly transmitted by aphids and some carried by whiteflies. However, Bean common mosaic virus (BCMV) and closely related Bean common mosaic necrosis virus (BCMNV) are seed-transmitted and widely distributed in bean crops throughout the world. Pathogenic strains of the virus first identified by Drijfhout et al. (1978) on an international differential set of bean varieties were categorized into two serotypes, A and B, causing temperature-insensitive necrosis and mosaic symptoms on differential cultivars carrying I and ii resistance genes. Both produce similar type of symptoms in bean plants, except “black root” or “top necrosis” caused by BCMNV at all temperatures or necrosis caused by strains of BCMV at high temperatures. Hence, the similarity of symptoms produced by some strains of both viruses makes it very difficult to distinguish between them in the field. The knowledge of strain spectrum of a particular pathogen is the first prerequisite to exploit their
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management through host resistance, which is the only durable and economic method of managing the viral pathogens. BCMV has been reported to possess a high degree of pathogenic variability in Europe, Africa and the USA (Drijfhout 1978; Omunyin et al. 1995; Sengooba et al. 1997). The serological and biological data have suggested that complex interrelationship exists among BCMV strains and other potyviruses that infect legumes. As a result, different methods of strain identification, viz., serology through polyclonal and monoclonal antibodies, n-terminal serology, etc., have proved ineffective in differentiation of virus strains (Mink & Silbernagel 1992; Shukla et al. 1989). Hence, the reliable criterion for differentiation of BCMV is molecular characterization of viral genome sequences. Molecular data on BCMV have shown that 3′ terminal region is highly variable and can be used as a reliable criterion to distinguish the strains (Sharma et al. 2011). Most of the work in India on BCMV has been carried out in Himachal Pradesh (Kapil et al. 2011; Sharma et al. 2011), where four BCMV strains were identified, including two new novel strains not known elsewhere in the world, viz., NL-1n and NL-7n. Since nothing was known about BCMV status in Kashmir valley, the objective of the present study was to elucidate the virulence behavior of the BCMV population in Kashmir valley vis-à-vis their relatedness with Himachal Pradesh strains.
Materials and methods Collection and maintenance of isolates Twenty-one leaf samples exhibiting characteristic symptoms of BCMV, viz., mosaic, leaf distortion and stunting, were collected from diverse commercial bean growing areas of the Kashmir valley. The virus inoculum of each isolate was prepared in 0.01M phosphate buffer (pH 7.0) and mechanically inoculated using the leaf rub method for production of local lesions on Chenopodium amaranticolor. Pure culture of the virus was established by inoculation of the sap obtained from a single local lesion. Similar kinds of symptoms (as observed under field conditions) were produced on common bean upon back inoculation from C. amaranticolor, thus confirming pathogenicity of the virus. The presence of BCMV in each isolate was confirmed through the DAS-ELISA test using polyclonal antiserum.
Pathogenic variability A standard set of differential cultivars (Drijfhout 1978) was used to identify the virus strains. Five plants from each differential cultivar raised in an insect-proof glasshouse, were mechanically inoculated with each isolate and one plant was left uninoculated to serve as healthy control. The plants of host group 6-9 were transferred to a greenhouse, and maintained at >30°C for observing systemic necrosis if any. Each isolate was tested twice and the strain(s) were identified by following the procedure of Drijfhout (1978). Molecular variability Total RNA of most prevalent strains, viz., NL-1, NL-4 and NL-7, was extracted using Nucleo-pore RNASure® Plant Kit (Genetix Biotech Asia Pvt Ltd) following the manufacturer’s instructions. For RT specific primers 5′-GGATCCATGTCAGG AC/TCTGGACAGCC-3′ (forward) and 5′CTAGACTGCGAAGGACCCATGCC-3′ (reverse) with restriction sites XbaI site: CTAGA; BamHI site: GGATCC were designed and the reaction was carried out in thin-walled tubes as per Padder et al. (2011). The PCR products were eluted from agarose gel using the gel extraction kit (Promega Corporation) as described by the manufacturer’s instructions and ligated into the Ttailed cloning vector (pGEM-T Easy, Promega). Ligation reactions were transformed into E. coli strain DH5α using standard transformation techniques and recombinant colonies were identified by colony PCR. The freeze-dried cloned product was directly submitted for custom sequencing through custom sequence services (Life Technologies India Pvt. Ltd). The sequences were retrieved from the chromatograms using BioEdit version 7.0 and aligned. All the three sequences were submitted to GenBank (NCBI) and accession numbers of sequences were obtained (JN692256, JN692257, and JN692258). For sequencing analysis, the CP sequences of the three BCMV strains were compared with 23 sequences of BCMV including one out-group (Beet mosaic virus) obtained from NCBI gene bank http:// www.ncbi.gov. Sequence polymorphism was analyzed and a neighbor-joining tree was constructed according to Kimura 2-parameter model using MEGA, in a bootstrap test with 1000 replicates. The coat protein nucleotide sequences from NL-1, NL-4 and NL-7 strains of BCMV from Kashmir were translated using Expasy translation tool (www.expasy.org/tools/dna.html). The deduced amino-acid sequences were compared with amino-acid sequences of BCMV from NCBI gene bank as described above.
BCMV-10
BCMV-17
BCMV-12
+ -
+ -
+ -
+
-
-
-
-
-
-
-
2
3
4
5
6
7
8
9
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
-
+
+
-
-
-
N
-
-
-
+
+
-
-
-
N
-
-
-
+
+
BCMV-21
-
-
-
-
-
+
+
+
+
BCMV-2
-
-
-
-
-
+
+
+
+
BCMV-15
+ N -
-
-
-
-
-
+
+
+
+
BCMV-18
+ N -
BCMV-9
*Differential cultivars (Drijfhout 1978); +, Host group susceptible to systemic infection; –, Resistant, no systemic infection; N, Systemic necrosis >30°C (susceptible)
+
1
BCMV-13
BCMV-4
+ -
BCMV-1
+ -
Strain group VII (NL-4)
BCMV-20
+ -
Strain group IIb (NL-7n)
+ -
Strain group IIa (NL-7 ) BCMV-16
BCMV-8
BCMV-3
BCMV-7
BCMV-5
BCMV-6
Strain group Ib (NL-1n)
Strain group Ia (NL-1)
Pathogroup - I
Reaction of BCMV isolates
Pathogroup -VII
BCMV-14
I+ I+ bc-u bc-1 I+ bc-u bc-12 I+ bc-u bc-2 I+ bc-u bc-12 bc-22 I bc-1 1 bc-1 I I, bc-12
Allele of the inhibitor gene
Pathogroup - II
Reaction of BCMV isolates
Stringless Green Refugee (SGR)\Double Witte Redland’s Greenleaf ‘C’ Redland’s Greenleaf ‘B’ Michelite ‘62’ Monroe Jubila Top crop Black turtle soup Amanda
1 2 3 4 5 6 7 8 9
Host group
Differential bean variety
Host group
Table 1 Reaction pattern of BCMV isolates on differential bean varieties*
-
-
-
-
-
+
+
+
+
BCMV-19
+ N -
BCMV-11
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Results Pathogenic variability Twenty-one isolates when inoculated on differential cultivars of host group 1 through 5 were categorized into three pathogroups (PG) I, II and VII, accommodating 10, 7 and 4 isolates, respectively (Table 1). Isolates placed in PGI caused only mosaic symptoms on differential cultivar ‘Double Witte’, whereas isolates grouped in PGII showed symptoms on two differential cultivars, viz., ‘Double Witte’ and ‘Redlands Greenleaf C’. Isolates placed in PGVII showed symptoms on four of the differential cultivars, viz., ‘Double Witte’, ‘Redlands Greenleaf C’, ‘Redlands Greenleaf B’ and Michelite ‘62’. Isolates of PG I and PGII were divided into two strain groups, viz., Ia, Ib, IIa and IIb as isolates of Ib and IIb induced veinal necrosis on cultivar ‘Jubila’ (supplementary Fig. 1) at high temperature (>30°C). Fig. 1 Phylogenetic tree showing nucleotide relationship of Kashmir strains with other BCMV strains at coat protein
Results of pathogenic variability studies revealed the existence of five strains (Ia, Ib, IIa, IIb and VII) of BCMV in Kashmir valley. Strain group Ia was classified as NL-1 strain and Ib as NL-1n (NL-1necrotic) based on the necrotic reaction of Ib strain on differential cv. Jubila; and in a similar fashion, strain groups IIa and IIb were classified as NL-7 and NL-7n (NL-7 necrotic), respectively. Molecular variability of most prevalent strains Of the five BCMV strains identified in the present study, three strains, viz., NL-1, NL-4 and NL-7, found to be most prevalent in Kashmir valley, were used to characterize their coat protein gene for comparison. The pair-wise nucleotide homology between various sequences varied from 74% to 99%. The pair-wise homology between the nucleotide sequence of NL-1 and NL-4 was 95%, whereas the homology between NL-1 and NL-7 and between NL-4 and NL-7 was 93%, respectively. The
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nucleotide sequence of NL-4 of Kashmir valley showed maximum homology of 96% with EU492546 and AF328753 BCMV strains from India and Mexico, respectively. The nucleotide sequence of NL-7 showed maximum homology of 97% with BCMV strain EU492546 of India. Phylogenetic analysis of 25 BCMV strains and one out-group (Beet mosaic virus) revealed that the strains were grouped into two major clusters, viz., A and B, accommodating 1 and 25 sequences, respectively (Fig. 1). The CP sequences of NL-1, NL-4 and NL-7 strains of BCMV from Kashmir valley were most closely related to BCMV strains from Himachal Pradesh (India), USA, and other countries. The comparison of coat protein amino-acid sequences (supplementary Fig. 2) showed 17-97% amino acid homology, maximum being with BCMV strain of India followed by that with BCMV strain of USA and BCMV strain of England, respectively. Phylogenetic analysis revealed that BCMV strains from Kashmir valley and other translated sequences of selected viruses were clustered into two groups. In all the three Kashmir valley strains, DAG motif at amino-acid 13 of CP and BCMV specific conserved motif MVWCIDN was present.
Discussion Pathological variability Strain differentiation of the 21 isolates of BCMV determined on a set of differential bean cultivars revealed the existence of three pathogroups and five strains. Faccioli et al. (1982) also identified seven isolates of PG II and two of PG I in central Italy based on Drijfhout classification. Two strains, viz., Ia and IIa, resembled the type strains NL1 and NL-7 reported from the Netherlands and Peru (Drijfhout 1978) and Himachal Pradesh (Sharma et al. 2011) however, some isolates under study, grouped in strains NL-1n and NL-7n differed from previously reported strains of Drijfhout (1978) The same strains were reported by (Sharma et al. 2011) in Himachal Pradesh, India, thus confirming the presence of these two BCMV strains in this part of India again, hitherto unreported from Africa, Europe or the USA. The presence of NL-1n and NL-7n as distinct strains in Himachal Pradesh and Kashmir valley in India can also be explained as per Vavilo (1951), who claimed China as the secondary origin of beans which is nearest to Himachal Pradesh
and Kashmir valley. Therefore, these two distinct strains may be present in China also. The strains could be considered as mutants that might have evolved during the process of co-evolution of both bean and BCMV in this part of the world or could have evolved due to the recombination of different type strains infecting the same cultivar, as revealed by Silbernagel et al. (2001) in other host–pathogen systems. However, to establish the identity of newly designated strains, NL-1n and NL7n, requires confirmation through complete genome characterization. The strain NL-4 (PG VII), as recorded during the present study, was reported from Kashmir valley but not from other parts of India and its distribution was confined mainly to the relatively coolest places of Kashmir valley. The strains NL-1, NL-1n, NL-7 and NL-7n thus established were reported for the first time in Kashmir valley, whereas NL-4 was reported for the first time in India. Molecular variability A comparison of coat protein nucleotide sequences of the strains of Kashmir valley showed more than 90% sequence identity with other BCMV strains, thereby confirming that they are all strains of the same potyvirus. Similarly, the CP region of the Kashmir strains supports Shukla & Ward (1989) that strains of the virus shared 90–99% amino acid similarities. The phylogenetic analysis led to the grouping of 26 BCMV isolates into two different clades when a consensus tree using bootstrap procedure was drawn. BCMV strains NL-1, NL-4 and NL-7 from Kashmir valley were most closely related to BCMV strains from Himachal Pradesh, USA and other countries. Similarly, the results have been reported in comparison of the deduced amino acid sequence of BCMV strains with other BCMV strains and other related potyviruses (Sharma et al. 2011). In all three strains, the DAG motif was present at amino acid position 13 of CP, which is important for aphid transmission in potyviruses. The BCMV-specific conserved motif MVWCIDN was also present in all three strains. The DAG motif has also been reported from the same position in Indian isolates (Sharma et al. 2011).
References Anon. (2010). Digest of statistics (pp. 96–98). Jammu and Kashmir, India: Directorate of Economics and Statistic Planning and Development Department.
Author's personal copy Phytoparasitica Drijfhout, E. (1978). Genetic interaction between Phaseolus vulgaris and bean common mosaic virus with implications for strain identification and breeding for resistance. Verslagen van Landbouwkundige Onderzoekingen, 872, 1–89. Drijfhout, E., Silbernagel, M. J., & Burke, D. W. (1978). Differentiation of strains of bean common mosaic virus. Netherlands Journal of Plant Pathology, 84, 13–26. Faccioli, G., Cerato, C., & Ranalli, F. (1982). Characterization of Bean common mosaic virus isolates and genetic improvement for resistance. Informatore Fitopatologica, 37, 55–59. Kapil, R., Sharma, P., Sharma, S. K., Sharma, O. P., Sharma, O. P., Dhar, J. B., et al. (2011). Pathogenic and molecular variability in Bean common mosaic virus infecting common bean in India. Archives of Phytopathology and Plant Protection, 44, 1081–1092. Mink, G. I., & Silbernagel, M. J. (1992). Serological and biological relationships among viruses in the bean common mosaic virus subgroup. In Potyvirus Taxonomy (pp. 397-406). Dordrecht, the Netherlands: Springer. Omunyin, M. E., Gathuru, E. M., & Mukunya, D. M. (1995). Pathogenicity groups of Bean common mosaic virus isolates in Kenya. Plant Disease, 79, 985–989. Padder, B. A., Shah, M. D., Ahmad, M., Hamid, A., Sofi, T. A., Ahanger, F. A., et al. (2011). Status of apple mosaic virus in Kashmir Valley. Applied Biological Research, 13, 117–120.
Sengooba, T. N., Spence, N. J., Walkey, D. G. A., Allen, D. J., & Femi Lana, A. (1997). The occurrence of bean common mosaic necrosis virus in wild and forage legumes in Uganda. Plant Pathology, 46, 95–103. Sharma, P., Sharma, P. N., Kapil, R., Sharma, S. K., & Sharma, O. P. (2011). Analysis of 3′-terminal region of Bean common mosaic virus strains infecting common bean in India. Indian Journal of Virology, 22, 37–43. Shukla, D. D., Jilka, J., Tosic, M., & Ford, R. E. (1989). A novel approach to the serology of potyviruses involving affinitypurified polyclonal antibodies directed towards virus-specific N termini of coat proteins. Journal of General Virology, 70, 13–23. Shukla, D. D., & Ward, C. W. (1989). Structure of potyvirus coat proteins and its application in the taxonomy of the potyvirus group. In F. A. M. Karl Maramorosch & J. S. Aaron (Eds.), Advances in virus research (Vol. 36, pp. 273–314). New York, NY: Academic Press. Silbernagel, M. J., Mink, G. I., Zhao, R. L., & Zheng, G. Y. (2001). Phenotypic recombination between bean common mosaic and bean common mosaic necrosis potyviruses in vivo. Archives of Virology, 146, 1007– 1020. Vavilo, N. I. (1951). The origin, variation, immunity and breeding of cultivated plants. Waltham, MA, USA: Chronica Botanica.
