Simultaneous Detection of Both RNA and DNA Viruses Infecting Dry Bean and Occurrence of Mixed Infections by BGYMV, BCMV and BCMNV in the Central-West Region of Mexico

Abstract

A multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed to simultaneously detect bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV), and bean golden yellow mosaic virus (BGYMV) from common bean leaves dried with silica gel using a single total nucleic acid extraction cetyl trimethyl ammonium bromide (CTAB) method. A mixture of five specific primers was used to amplify three distinct fragments corresponding to 272 bp from the AC1 gene of BGYMV as well as 469 bp and 746 bp from the CP gene of BCMV and BCMNV, respectively. The three viruses were detected in a single plant or in a bulk of five plants. The multiplex RT-PCR was successfully applied to detect these three viruses from 187 field samples collected from 23 municipalities from the states of Guanajuato, Nayarit and Jalisco, Mexico. Rates of single infections were 14/187 (7.5%), 41/187 (21.9%), and 35/187 (18.7%), for BGYMV, BCMV, and BCMNV, respectively; 29/187 (15.5%) samples were co-infected with two of these viruses and 10/187 (5.3%) with the three viruses. This multiplex RT-PCR assay is a simple, rapid, sensitive, and cost-effective method for detecting these viruses in the common bean and can be used for routine molecular diagnosis and epidemiological studies.

Keywords: Phaseolus vulgaris; multiplex RT-PCR; virus detection.

Figure 1

Specificity of four primer pairs for the detection of bean golden yellow mosaic virus (BGYMV) using polymerase chain reaction (PCR). The primer set used in each case is indicated above the lanes (A) Primer pairs reported for detection of the genus Begomovirus and for specific detection of BGYMV; (B) Primer pairs designed in the present study specifically for BGYMV. Lanes 1 and 10: non template control; lanes 2 and 11: negative control (cDNA from healthy plant); lanes 3 and 12: positive control (cDNA from common bean infected with BGYMV-MX strain); lanes 4 and 13: plasmid DNA of BGYMV-MX DNA A component; lanes 5 and 14: plasmid DNA of BGYMV-MX DNA B component; lanes 6 and 15: plasmid DNA of pepper huasteco yellow vein virus (PHYVV) DNA A component; lanes 7 and 16: plasmid DNA of PHYVV DNA B component; lanes 8 and 17: plasmid DNA of pepper golden mosaic virus (PepGMV) DNA A component; lanes 9 and 18: plasmid DNA of PepGMV DNA B component. M: Molecular marker 1 kb plus DNA ladder.

Figure 2

Determination of specificity and compatibility of primer pairs for the detection of bean common mosaic virus (BCMV), bean common mosaic necrotic virus (BCMNV) and bean golden yellow mosaic virus (BGYMV) in (A,B,C) uniplex, (A,B,C) duplex and (D) multiplex PCR. The combinations of the viruses detected with specific primers are shown above in each photograph. Lanes 1 and 10: non-template control; lanes 2 and 11: negative control (cDNA from healthy plant); lanes 3 and 12: single BGYMV control; lanes 4 and 13: single BCMV control; lanes 5 and 14: single BCMNV control; lanes 6 and 15: double BCMV and BGYMV control; lanes 7 and 16: double BCMNV and BGYMV control; lanes 8 and 17: double BCMV and BCMNV control; lanes 9 and 18: triple BCMV, BCMNV and BGYMV control. M: Molecular marker 1 kb plus DNA ladder. The sizes of the amplified product for each virus are shown at the right of the figure.

Figure 3

Detection of BCMV, BCMNV and BGYMV in composite samples. M: Molecular marker 1 kb plus DNA ladder. Lane 1: non-template control; lane 2: negative control (cDNA from healthy plants); lane 3: positive control (cDNA from common bean infected with BCMV, BCMNV and BGYMV); lanes 4, 5, 6, 7, 8 and 9: dilution 1:1, 1:2, 1:3, 1:4, 1:5, and 1:6, respectively.