Common bean, Phaseolus vulgaris, is commercially important in Okinawa Prefecture, Japan. It can be cultivated in unheated greenhouses during winter in subtropical Okinawa. In November 2016, leaf curling and yellowing were observed in commercial beans (cv. Bikkuri Jumbo) on Ishigaki Island, Okinawa, Japan. Symptoms were first observed on a few plants near the entrance of the greenhouse before they were found on other plants during the cultivation period. This pattern suggested that the causative agent was spread by an insect-transmitted virus. The aim of the present study was to identify the pathogen.

Three common bean plants with typical leaf curling and yellowing (Fig. 1a) were collected from the field. All samples were stored at − 80 °C until use in the analysis described below. The symptoms suggested that the disease was caused by infection with a virus such as begomovirus. Since ageratum yellow vein virus (AYVV) and tomato yellow leaf curl virus (TYLCV) are major begomoviruses infecting crops and weeds in Okinawa Prefecture (Andou et al. 2010; Ueda et al. 2009), we conducted PCR with specific primers for AYVV and TYLCV (AYmulti-V and AYmulti-C for AYVV, TYIL-V3 and TYCON-C for TYLCV-Israel [IL] strain, TYMld-V3 and TYCON-C for TYLCV-Mild [Mld] strain) (Table 1). Total DNA was extracted from the leaves showing yellow leaf curl symptoms using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The concentration of the extracts was measured using a NanoDrop 2000 (Thermo Fisher Scientific, Witham, MA, USA). PCR was performed with Ex Taq DNA polymerase (TaKaRa Bio, Kusatsu, Japan) using the extracted DNA (100 ng) as a template. Total DNA extracted from AYVV-Ishigaki isolate (GenBank accession AB306314), TYLCV-IL isolate, or TYLCV-Mld isolate infected tomato plants were used for this experiment as positive control. The following conditions were used for PCR: 98 °C for 2 min followed by 35 cycles of 10 s at 98 °C, 30 s at 53°C, and 2 min at 72 °C. PCR products were then separated by 2% agarose gel electrophoresis. Amplicons of the expected size (approximately 200 bp) from all affected common bean samples and AYVV positive control were observed in the AYVV lanes, whereas there were no amplicons of DNA-A in the TYLCV-IL or TYLCV-Mld lanes in any common bean sample (Fig. 2).

Fig. 1
figure 1

Leaf curling and yellowing on Phaseolus vulgaris cv. Bikkuri Jumbo infected with ageratum yellow vein virus in the field (a) and in a transmission test (b)

Full size image
Table 1 Primers used to detect and sequence genomes of ageratum yellow vein virus (AYVV) and tomato yellow leaf curl virus (TYLCV)
Full size table
Fig. 2
figure 2

PCR detection of ageratum yellow vein virus (AYVV) and tomato yellow leaf curl virus (TYLCV) from diseased common beans using specific primers. Tomato plants infected with TYLCV-Israel (IL), TYLCV-Mild (Mld), or AYVV-Ishigaki isolates were used as positive control. Primers are presented in Table 1. PCR products were separated by 2% agarose electrophoresis. Expected amplified products are marked by arrowheads. Lane M, 100 bp DNA ladder (Promega, Madison, WI, USA)

Full size image

To detect DNA-A, DNA-β, and DNA-α, we also conducted PCR with universal primers (V/C for DNA-A, Beta01/Beta02 for DNA-β, DNA101/DNA102 for DNA-α) of begomoviruses, previously reported (Briddon et al. 2002; Briddon and Markham 1994; Bull et al. 2003) (Table 1). Total DNAs of AYVV-Ishigaki isolate and common bean samples as described above were used in the PCR analyses. In addition, the complete nucleotide sequence of DNA-α of AYVV-Pakistan isolate (GenBank accession LT840069), synthesized by FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), was used as a positive control for PCR detection of DNA-α. PCR was performed with Ex Taq DNA polymerase (Takara Bio). The PCR conditions to detect DNA-A were 98 °C for 2 min, followed by 35 cycles of 10 s at 98 °C, 30 s at 45 °C, and 4 min at 72 °C. The PCR conditions for DNA-β were 98 °C for 2 min, followed by 35 cycles of 10 s at 98 °C, 30 s at 45 °C, and 2 min at 72 °C, and conditions for DNA-α were 98 °C for 2 min, followed by 35 cycles of 10 s at 98 °C, 30 s at 50 °C, and 2 min at 72 °C.

All PCR products were separated by 1% agarose gel electrophoresis. Amplicons of the expected size for DNA-A (approximately 2,700 bp) and DNA-β (approximately 1,300 bp) from all AYVV samples were observed (Fig. 3a and b), whereas there were no amplicons of the expected size for DNA-α in any common bean sample (Fig. 3c) or in a tomato sample infected with the AYVV-Ishigaki isolate (Fig. 3c).

Fig. 3
figure 3

PCR detection of DNA-A (a), DNA-β (b), and DNA-α (c) of ageratum yellow vein virus (AYVV) from diseased common beans. Tomato plants infected with AYVV-Ishigaki isolate were used as a positive control. In PCR detection of DNA-α of AYVV, synthesized DNA-α of AYVV-Pakistan isolate (GenBank Accession LT840069) was also used as a positive control. Primer sets are in Table 1. PCR products were separated by 1% agarose gel electrophoresis. Expected amplified products are marked by arrowheads. Lane M, 1 kbp DNA ladder (Promega)

Full size image

Expected DNA fragments were purified using a QIAquick PCR Purification Kit (Qiagen) or a QIAquick gel extraction kit (Qiagen). The nucleotide sequences of extracted DNA fragments were determined using a BigDye Terminator v1.1 Cycle Sequencing Ready Reaction Kit and a Genetic Analyzer DNA Model 3100 (Thermo Fisher Scientific). The nucleotide sequences were collected using Geneious version R10 software (http://www.geneious.com). Primers used for sequencing are listed in Table 1. According to a BLASTn analysis, the partial and complete nucleotide sequences shared 100% and 99.9%, respectively, with the DNA-A sequence of the AYVV isolate previously reported in Ishigaki Island (GenBank accession AB306314). The partial and complete nucleotide sequences were deposited in DDBJ/EMBL/NCBI (GenBank Accessions LC475505 and LC487406).

According to a BLASTn analysis of the complete nucleotide sequence of DNA-β, the nucleotide sequence of the fragments shared 97.9% identity with the complete sequence of DNA-β of the TYLCV isolate reported in Taiwan (GenBank Accession AJ542495). The nucleotide sequence was deposited in DDBJ/EMBL/NCBI (GenBank accession LC48883).

Healthy P. vulgaris cv. Bikkuri Jumbo seedlings were used in a transmission test with Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) and Mediterranean (MED). Ageratum conyzoides plants infected with AYVV were collected from around a common bean field for source of acquisition by whitefly. Single infection of the AYVV was confirmed by PCR as described above (data not shown). Fifty adult whiteflies were given an acquisition access period of 4 days on A. conyzoides leaves infected with AYVV. They were then collected and transferred to healthy common bean seedlings (6 viruliferous whiteflies per plant) for an inoculation access period of 2 weeks, after which whiteflies were killed with the insecticide Dinotefuran (Mitsui Chemicals, Tokyo, Japan). Plants were then grown for 2 months in the greenhouse. Healthy plants and nonviruliferous whiteflies were used as controls. The results of this transmission test showed that the virus was transmitted by both B. tabaci MEAM1 and MED, and leaf curling and yellowing similar to those observed in the original plants were seen on all test plants (Fig. 1b and Table 2). Additionally, AYVV was detected by PCR in diseased plants but not in asymptomatic healthy plants. We thereby confirmed AYVV transmission from diseased plants to healthy plants via the insect vector B. tabaci MEAM1 and MED.

Table 2 Test for transmission of ageratum yellow vein virus by genetic groups of Bemisia tabaci
Full size table

The results of the present study indicate that the causative agent of leaf curling and yellowing disease is AYVV. The first report of AYVV was from A. conyzoides in Singapore (Swanson et al. 1993). Since then, the virus has been reported in several dicotyledonous plants (Liu et al. 2008; Tsai et al. 2011). In Japan, AYVV was reported in tomato plants in Okinawa and Tokyo Prefectures (Andou et al. 2010; Shahid et al. 2014). The present study is the first observation of AYVV in a common bean plant. Andou et al. (2010) reported that A. conyzoides was an important reservoir of AYVV. Notably, we found A. conyzoides infected with AYVV around the greenhouse in which the original infected bean plants were growing; thus, AYVV likely transmitted by whiteflies from A. conyzoides to the bean plants. Therefore, management of weeds and whitefly are likely to be the most important control measures of this disease. Because AYVV causes serious damage to tomato and common bean plants, it is essential to carefully monitor the spread of AYVV in other areas, including the main island of Okinawa. To the best of our knowledge, this is the first report of leaf curling and yellowing caused by AYVV in P. vulgaris in Japan. We therefore propose adding yellow leaf curl disease as a new disease of common bean.