J Gen Plant Pathol (2007) 73:152–155 DOI 10.1007/s10327-006-0333-5

VIRAL AND VIROID DISEASES

© The Phytopathological Society of Japan and Springer 2007

Short communication

Toru Kondo · Kyushiro Kasai · Kazuo Yamashita Masahiro Ishitani

Selection and discrimination of an attenuated strain of Chinese yam necrotic mosaic virus for cross-protection

Received: May 24, 2006 / Accepted: September 22, 2006

Abstract An attenuated strain of Chinese yam necrotic mosaic virus (CYNMV), designated KM3, was selected from among CYNMV field isolates. KM3 causes a mild mosaic on leaves of Chinese yam (Dioscorea opposita) cv. Nagaimo during the early stages of growth, and the severity of the symptoms is reduced with growth of the plants. In crossprotection tests using aphids to transmit the virus, Nagaimo plants that had been infected with KM3 were protected from infection with CYNMV severe strains YS117 and IW5. A reverse transcription-polymerase chain reactionrestriction fragment length polymorphism (RT-PCR-RFLP) method was developed to discriminate KM3 from other isolates in the field. Key words Attenuated strain · Chinese yam necrotic mosaic virus (CYNMV) · Cross-protection · Dioscorea opposita · RT-PCR-RFLP

The Chinese yam (Dioscorea opposita Thunb.) cultivar Nagaimo is the most popular and widely cultivated yam in Japan. Two species of viruses have been reported to infect Nagaimo plants: Chinese yam necrotic mosaic virus (CYN-

T. Kondo (*) Aomori Green BioCenter, Aomori Prefectural Agriculture and Forestry Research Center, 221-10 Yamaguchi, Nogi, Aomori 030-0142, Japan Tel. +81-17-728-1015; Fax +81-17-728-1017 e-mail: [email protected] K. Kasai Aomori Prefectural Agriculture and Forestry Research Center, Aomori, Japan K. Yamashita · M. Ishitani Field Crops and Horticultural Experiment Station, Aomori Prefectural Agriculture and Forestry Research Center, Aomori, Japan The nucleotide sequence reported in this article is available in the DDBJ/EMBL/GenBank databases under accession number AB255747.

MV) (Fukumoto and Tochihara 1978) and Broad bean wilt virus 2 (BBWV-2) (Kondo et al. 2005). Infection of seed tubers with CYNMV, the most abundant virus in Nagaimo plants, causes a loss of yield of as much as 30%–45% (Tochihara 1993). The host range of CYNMV is restricted to Dioscorea spp. (Fukumoto and Tochihara 1978). CYNMV is a flexuous, filamentous virus 660 nm long that is transmitted by aphids in a nonpersistent manner (Fukumoto and Tochihara 1978). Based on these characteristics, CYNMV was first presumed to be a member of the genus Carlavirus, but the nucleotide sequence of the 3′-terminal portion of the genome revealed that it is a member of the genus Macluravirus in the family Potyviridae (Kondo 2001). To avoid necrotic mosaic disease in Nagaimo plants caused by infection with CYNMV, growers start with virusfree nursery plants. However, after the virus-free plants are transplanted in the field, they are easily infected with CYNMV through an aphid vector. Purchasing virus-free Nagaimo plants, spraying with an insecticide, and removing infected plants from the field all require a great deal of expense and labor on the part of farmers. Therefore, crossprotection by an attenuated strain may offer an alternative strategy for the control of severe strains of CYNMV that are responsible for yield losses. The extent of cross-protection is correlated with the degree of genetic relatedness between the protecting and challenging virus strains (Hall et al. 2001). There has been some research into the use of attenuated strains to protect perennial plants from viruses that damage economically important crops (Costa and Müller 1980; Fuji et al. 2000; Kajihara et al. 2000; Uga et al. 2004). Kondo et al. (2003) reported that cross-protection using an attenuated strain may be useful for controlling CYNMV in the field because the genetic diversity of CYNMV in the main Nagaimo-producing areas of Japan and Korea is very low. In this study, an attenuated strain of CYNMV, designated KM3, was selected from CYNMV isolates obtained in the field. A reverse transcription-polymerase chain reaction-restriction fragment length polymorphism (RT-PCRRFLP) method was then developed to discriminate KM3 from other isolates in the field.

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Selection of an attenuated strain of CYNMV In 1997, 390 aerial tubers were collected from Nagaimo plants in fields in which CYNMV was spreading in Aomori Prefecture, Japan. The aerial tubers were grown in an insect-proof greenhouse for 2 years. Plants with either mild mosaic or no symptoms were assayed by RT-PCR for the presence of CYNMV. Total RNA was extracted from fresh leaf samples using SepaGene RV-R (Sanko Junyaku, Tokyo, Japan). RT-PCR was performed using Ready-To-Go RT-PCR beads (GE Healthcare, Buckinghamshire, UK). Two oligonucleotide primers (CYV1655P and CYV1906 M, Fig. 1) were designed to amplify 252 nt of the CYNMV RNA, including the 3′-untranslated region. RT-PCR was carried out as follows: 1 cycle at 42°C for 15 min for reverse transcription, and then 95°C for 5 min and 35 cycles of 95°C for 1 min, 57°C for 1 min, and 72°C for 1 min for PCR. The RT-PCR assay detected no CYNMV from symptomless plants at any time during the growing period. Thirteen of the plants that were positive for CYNMV that also had mild mosaic symptoms were grown in the field according to the custom in Aomori Prefecture: seed tubers were planted in late May and new tubers were harvested in November. After 3 years of symptom observations in the field, one plant with mild symptoms was selected as a virus source. An attenuated strain of CYNMV, designated KM3, was isolated from this plant and transmitted to a healthy Nagaimo plant by Aulacorthum solani, a major aphid vector of CYNMV to Nagaimo plants in Aomori Prefecture (Ishitani and Niwata 1993). A 3′-terminal region of about 1.1 kb of the KM3 genomic RNA was isolated and sequenced as 195(A→G)

NIb

CP CYV1655P

Bbs I 1028(U→C) poly(A) CYV1906M

CYV1655P: 5'-CAGCGCCCAGCTTTATATTAGT-3' CYV1906M: 5'-CCTTCTATGGAACAGGATAAGT-3'

Fig. 1. Nucleotide sequences of reverse transcription-polymerase chain reaction (RT-PCR) primers and their location in the Chinese yam necrotic mosaic virus (CYNMV) genome. The positions of two point substitutions specific to KM3 are indicated at the top of the figure. NIb, large nuclear inclusion protein; CP, coat protein Fig. 2A–C. Leaf symptoms of Chinese yam cv. Nagaimo plants infected with CYNMV strains. A Plant infected with severe strain YS117. B Plant infected with attenuated strain KM3. C Healthy plant

A

described previously (Kondo et al. 2003). The nucleotide sequence (1098 nucleotides) will appear in the DDBJ/ EMBL/GenBank databases under accession number AB255747. In field conditions, leaves of plants infected with the severe strain YS117 (Kondo et al. 2003) rapidly developed necrotic mosaic symptoms after they expanded. Most leaves developed severe necrotic mosaic symptoms throughout the growing period (Fig. 2A) and turned yellow in mid-October. In contrast, plants infected with attenuated strain KM3 developed mild mosaic symptoms on leaves only during the early growing stage. The mosaic symptoms of the leaves lessened in August as the plants aged, with most of the leaves having no symptoms and only a small number of leaves expressing any necrotic mosaic symptoms (Fig. 2B). In October, some of the younger leaves developed a mild mosaic, but most of the leaves remained green and looked similar to leaves of virus-free plants. In field cultivation, the CYNMV infection rate per year for plants grown from virus-free seed tubers was 1.1%–9.1%.

Cross-protection test via aphid transmission Tubers infected with KM3 were grown in pots, and shoots approximately 10 cm long were inoculated with one of two severe CYNMV strains using aphids. Severe strains YS117 and IW5 (Kondo et al. 2003) were selected for crossprotection tests because these strains were distinguishable from KM3 in preliminary tests using single-strand conformation polymorphism (SSCP) analysis of RT-PCR products. The aphids were starved for 2 h and then allowed a 5-min acquisition feeding period on a detached leaf infected with the severe strain. After the acquisition feeding, 15 or 30 aphids were transferred to individual plants for a 1-day inoculation feeding period. At 3 months after inoculation, total RNA was extracted from the plants and subjected to RT-PCR as described earlier. SSCP analysis of the RTPCR products was performed using a GeneGel Excel 12.5/24 kit (GE Healthcare). Samples were resolved by electrophoresis for 4 h at 600 V and 5°C using a GenePhor electrophoresis unit (GE Healthcare). The SSCP pattern was visualized using silver staining. As shown in Table 1, plants infected with KM3 were completely protected from infection with either YS117 or B

C

154

1

2 3

4

5

6

7

8

9

10

Fig. 3. RT-PCR-single-strand conformation polymorphism analysis of Chinese yam cv. Nagaimo plants. Lanes 1–5, virus-free plants inoculated with IW5; lanes 6–8, KM3-infected plants inoculated with IW5; lane 9, IW5-infected plant; lane 10, KM3-infected plant

Table 1. Cross-protection of plants of Chinese yam cv. Nagaimo infected with an attenuated strain of Chinese yam necrotic mosaic virus (KM3) against challenge inoculation with severe strains via an aphid vector Plants

KM3-infected Virus-free

Challenge inoculuma YS117

IW5

0/9b 5/8

0/3 5/5

a

Each plant was inoculated with 15 (YS117) or 30 (IW5) aphids Number of plants infected with the severe strain/number of inoculated plants

b

IW5, whereas virus-free plants were easily infected with both severe strains. No SSCP pattern corresponding to the severe strains was detected in plants infected with KM3 after inoculation with either of the severe strains (Fig. 3). Plants infected with a severe strain by aphid transmission developed severe necrotic mosaic symptoms on leaves by 1–2 months after inoculation. In contrast, KM3-infected plants inoculated with either of the severe strains developed only a mild mosaic early in their growth, and the symptoms decreased with further growth of the plant. Symptoms on plants inoculated with a severe strain and on uninoculated plants infected with KM3 did not differ.

Discrimination of attenuated strain KM3 from other CYNMV isolates by RT-PCR-RFLP To evaluate the cross-protective ability of KM3 in the field, we must be able to discriminate KM3 from other CYNMV isolates. In a preliminary test, the RT-PCR-SSCP analysis described earlier was used for this purpose. KM3 and 6 other isolates collected from the same field as KM3 (Misawa, Aomori Prefecture, Japan) were examined. However, the SSCP patterns of 4 of the 6 isolates were similar to that of KM3, which was thus difficult to differentiate (data not shown). RT-PCR-RFLP was reported to be a reliable method for evaluating the efficacy of attenuated strains of several plant viruses (Fuji et al. 2000; Nakazono-Nagaoka et al. 2005; Uga et al. 2004). A comparison of the nucleotide sequences of KM3 and 16 other CYNMV isolates (Kondo et al. 2003) revealed that two base substitutions at positions 195 and 1028 are specific to KM3 (Fig. 1). Because the base substitution at position 1028 (uracil to cytosine) generates a BbsI site specific to KM3, RFLP analysis was applied. KM3 and

bp 500 400 300 200 100

1

2

3

4

5

6

7

8

Fig. 4. RT-PCR-restriction fragment length polymorphism analysis of Chinese yam cv. Nagaimo plants infected with attenuated strain KM3 (lane 8) or one of the six other isolates collected from the same field as KM3 (lanes 2–7). Lane 1, 1 kb Plus DNA Ladder (Invitrogen)

6 other isolates collected from the same field as KM3 (described earlier) were examined for RT-PCR-RFLP. RTPCR was performed as described. The PCR products were purified using a QIAquick PCR Purification kit (Qiagen, Hilden, Germany) and then digested with BbsI (New England Biolabs, Ipswich, MA, USA). The digested products were electrophoresed in 2.0% agarose gels. As expected, the KM3 PCR product was split into two fragments of 173 bp and 79 bp, whereas those of the other 6 isolates remained intact (Fig. 4). No PCR product was obtained from leaf samples of virus-free plants (data not shown). Therefore, RT-PCR-RFLP allowed clear discrimination of KM3 from other CYNMV isolates in the field. As described, the attenuated strain KM3, which was able to induce cross-protection against severe strains, was selected from among several CYNMV field isolates. There has been some research on the use of attenuated strains [e.g., Citrus tristeza virus (Costa and Müller 1980), Japanese yam mosaic virus (Fuji et al. 2000; Kajihara et al. 2000), and Bean yellow mosaic virus (Uga et al. 2004)] to protect perennial plants. Each of these attenuated strains was selected from native isolates occurring naturally in the field. An attenuated virus strain selected from native isolates is probably effective for cross-protection because the attenuated strain already demonstrates a cross-protective ability to survive in the field. This study has demonstrated that Nagaimo plants infected with KM3 were protected from subsequent infection with the severe CYNMV strains YS117 and IW5. This result supports the idea that cross-protection using attenuated virus strains is useful for controlling CYNMV in the field (Kondo et al. 2003). Further field experiments are in progress to confirm the cross-protective ability of KM3 against severe strains. In our preliminary tests, the decrease in tuber yield for plants infected with KM3 was only 2.0%–6.0% less than that for plants grown from virus-free seed tuber, while that for plants infected with CYNMV occurring in the field was 18.5%–18.7%. The RT-PCR-RFLP method developed in this study is a powerful tool for evaluating the utility of KM3 in the control of CYNMV severe strains. To make cross-protection practical, the stability of symptom expression and tuber yield during long-term field cultivation, the effects of super-infection caused by BBWV-2 and/or other viruses, and the viscosity and other characteristics of the plants infected with KM3 still need to be studied. Acknowledgments We thank Dr. T. Sano for critically reading the manuscript and Ms. N. Sato for technical assistance.

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