Double-stranded (ds) RNAs and imperfect hairpin RNAs of endogenous genes trigger post-transcriptional gene silencing (PTGS) and are cleaved by a Dicer-like nuclease into small interfering RNAs (siRNAs) and microRNs (miRNAs), respectively. Such small RNAs (siRNAs and miRNAs) then guide an RNA-induced silencing complex (RISC) for sequence-specific RNA degradation. While PTGS serves as an antiviral defense in plants, many plant viruses encode suppressors as a counter defense. Here we demonstrate that the PTGS suppressor (2b) of a severe strain (CM95R) of cucumber mosaic virus (CMV) can bind to in vitro synthesized siRNAs and even to long dsRNAs to a lesser extent. However, the 2b suppressor weakly bound to a miRNA (miR171) duplex in contrast to another small RNA-binding suppressor, p19 of tombusvirus that can effectively bind miRNAs. Because the 2b suppressor of an attenuated strain of CMV (CM95), which differs in a single amino acid from the 2b of CM95R, could barely bind siRNAs, we hypothesized that the weak suppressor activity of the attenuated strain resulted from a loss of the siRNA-binding property of 2b via a single amino acid change. Here we consider that 2b interferes with the PTGS pathway by directly binding siRNAs (or long dsRNA).

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Multiple inoculation of cucumber seedlings with attenuated isolates of cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), and watermelon mosaic virus 2 (WMV-2) somewhat reduced yield loss due to mixed infection by virulent strains of these viruses in field experiments under severe epidemic conditions in 1994 and 1995. In addition, this protective inoculation largely protected grafted cucumber plants from viral wilt syndrome. In greenhouse experiments, no significant differences were observed between single and multiple inoculations in the cross-protection effects of the attenuated viruses against their respective virulent strains. Triply inoculated plants developed synergistic yellow leaf mosaic symptoms and 15% fewer marketable fruits compared with healthy plants. Thus, multiple inoculation is appropriate for the summer-early autumn production, when economic losses due to the concurrent incidence of CMV, WMV-2, and ZYMV are significantly greater than the loss in yield due to the inoculation.

Six hundred sixty-three isolates of microorganisms, including fungi and bacteria, were collected from surface-sterilized roots of spinach (Spinacia oleracea L.) growing in commercial greenhouses in Kyoto Prefecture. These isolates were screened for their ability to control Fusarium wilt of spinach caused by Fusarium oxysporum f. sp. spinaciae. In primary screening, spinach seeds were treated with the isolates, sown in pots containing sterilized soil, and then challenge-inoculated with the pathogen. Nine bacteria were effective in reducing disease incidence. Subsequently, spinach seeds were treated with the selected isolates, then sown in an infested field and grown from June to July 1998. Four bacteria reduced disease incidence. One of these four, designated as SM10, significantly suppressed the disease. Based on bacteriological properties, SMlO was identified as a strain of Enterobacter cloacae. SMlO was observed within xylem vessels of spinach roots using light and immunoelectron microscopy, indicating E. cloacae SMlO was an endophytic bacterium of spinach.

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.