Development and Application of Attenuated Plant Viruses as Biological Control Agents in Japan.

In 1929, it was reported that yellowing symptoms caused by a tobacco mosaic virus (TMV) yellow mosaic isolate were suppressed in tobacco plants that were systemically infected with a TMV light green isolate. Similar to vaccination, the phenomenon of cross-protection involves a whole plant being infected with an attenuated virus and involves the same or a closely related virus species. Therefore, attenuated viruses function as biological control agents. In Japan, many studies have been performed on cross-protection. For example, the tomato mosaic virus (ToMV)-L11A strain is an attenuated isolate developed by researchers and shows high control efficiency against wild-type ToMV in commercial tomato crops. Recently, an attenuated isolate of zucchini yellow mosaic virus (ZYMV)-2002 was developed and registered as a biological pesticide to control cucumber mosaic disease. In addition, attenuated isolates of pepper mild mottle virus (PMMoV), cucumber mosaic virus (CMV), tobacco mild green mosaic virus (TMGMV), melon yellow spot virus (MYSV), and watermelon mosaic virus (WMV) have been developed in Japan. These attenuated viruses, sometimes called plant vaccines, can be used not only as single vaccines but also as multiple vaccines. In this review, we provide an overview of studies on attenuated plant viruses developed in Japan. We also discuss the application of the attenuated strains, including the production of vaccinated seedlings.

The entry of cucumber mosaic virus into cucumber xylem is facilitated by co-infection with zucchini yellow mosaic virus.

We investigated the synergistic effects of co-infection by zucchini yellow mosaic virus (ZYMV) and cucumber mosaic virus (CMV) on viral distribution in the vascular tissues of cucumber. Immunohistochemical observations indicated that ZYMV was present in both the phloem and xylem tissues. ZYMV-RNA was detected in both the xylem wash and guttation fluid of ZYMV-inoculated cucumber. Steam treatment at a stem internode indicated that ZYMV enters the xylem vessels and moves through them but does not cause systemic infection in the plant. CMV distribution in singly infected cucumbers was restricted to phloem tissue. By contrast, CMV was detected in the xylem tissue of cotyledons in plants co-infected with CMV and ZYMV. Although both ZYMV-RNA and CMV-RNA were detected in the xylem wash and upper internodes of steam-treated, co-infected cucumbers grown at 24 °C, neither virus was detected in the upper leaves using an ELISA assay. Genetically modified CMV harboring the ZYMV HC-Pro gene was distributed in the xylem and phloem tissues of singly inoculated cucumber cotyledons. These results indicate that the ZYMV HC-Pro gene facilitates CMV entry into the xylem vessels of co-infected cucumbers.

Effectiveness of an Attenuated Zucchini yellow mosaic virus Isolate for Cross-Protecting Cucumber.

To cross-protect cucumber plants from Zucchini yellow mosaic virus (ZYMV), we used cold treatment to obtain an attenuated isolate of ZYMV, designated ZYMV-2002. ZYMV-2002 was obtained from a virulent ZYMV isolate after repeated low temperature treatment at 12.5 to 15°C followed by five cycles of single-plant transfer. The isolate produced very mild or no symptoms on cucurbit plants. In addition, inoculated cucumber plants had very similar fruit productivity to healthy control plants under field conditions. During field experiments in 2002 and 2003, when other viruses were also present, protected plants significantly suppressed infection with ZYMV, progression of disease severity, and reduction of fruit yield and quality. These results demonstrate that ZYMV-2002 is a potentially useful attenuated ZYMV isolate for reducing the impact of ZYMV.

A New Technique to Select Mild Strains of Cucumber mosaic virus.

An efficient technique to select a good attenuated virus to control Cucumber mosaic virus (CMV) disease was developed. Preliminary screenings were conducted to assess the virulence of virus recovered from dark-green islands and yellow tissues of mosaic leaves of Nicotiana rustica after co-inoculation with an attenuated mutant P2bR46C of CMV and its original severe isolate Pepo. All single-lesion isolates (SLIs) obtained from dark-green islands had the attenuated P2bR46C phenotype, but the SLIs from yellow tissue had either the virulent Pepo or the P2bR46C phenotype. When Pepo-infected N. rustica and tomato plants were grown at 15 or 36°C for 30 days, 17 of 288 SLIs obtained from the treated leaves elicited mosaic and dark-green spots without malformation. Dark-green tissue from each plant infected with 1 of these 17 SLIs then was used to inoculate one plant of N. rustica. All 17 plants had either very mild mosaic or no visible symptoms. One of these potential mild strains, 36R37, had an amino acid substitution on the 2b gene encoding the 2b protein. Isolate 36R37 also was highly cross-protective, and its symptom attenuation was stable for three serial host passages. After cold or heat treatment, the dark-green tissue proved to be a good source for isolating mild strains of the virus.

Cucumber mosaic virus 2b protein compensates for restricted systemic spread of Potato virus Y in doubly infected tobacco.

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) inoculated with a necrotic strain of Potato virus Y (PVY, T01 isolate) developed necrotic symptoms in some systemically infected leaves, but not in younger leaves. However, PVY expressed distinct symptoms not only in the older leaves, but also in the younger leaves, of plants that had been doubly inoculated with PVY and with Cucumber mosaic virus (CMV, strain Pepo). A tissue blot immunoassay of tissues from various positions of the stem detected PVY weakly in each stem, but not in the shoot apex, of singly infected plants, whereas PVY was detected at high levels in almost all sections of doubly infected plants. CMV was also detected at high levels in sections of singly and doubly infected plants. Immunohistochemistry of stem tissues showed that in singly infected plants, PVY was confined to external phloem cells and was not detected in internal phloem cells. However, in doubly infected plants, PVY was distributed uniformly throughout whole tissues, including the external phloem, xylem parenchyma and internal phloem cells. In plants that were doubly infected with PVY and Pepo Delta 2b, a modified CMV that cannot translate the 2b protein, the spread of PVY was restricted as in singly infected plants. These results suggested that the plant host has a counterdefence mechanism that restricts systemic spread of PVY T01, and that the 2b protein of CMV strain Pepo negates this restriction.