β-1,3-D-Glucan Transported from Golgi Apparatus of Japanese Pear Leaves is a Component of Extracellular Polysaccharides Accumulated after AK-toxin I Treatment

This fluorescence and immunoelectron microscopic study showed that β-1,3-D-glucan accumulated only in leaves of a susceptible cultivar of Japanese pear after treatment with a host-specific toxin, AK-toxin I, from Alternate, alternata Japanese pear pathotype. The positive fluorescent reaction of callose was detected only in aniline blue fluorochrome-stained sections from toxin-treated leaves of the susceptible cultivar: positive sites were observed on cell walls of leaf cells. The sites of callose deposition were probably consistent spatially with modified sites on the plasma membrane that were observed only in the toxin-treated leaves of the susceptible cultivar. The toxin-induced modifications, identified as damage to the plasma membrane, were characterized by invagination of the plasmalemma specifically at plasmodesmata and as the concomitant accumulation of extracellular polysaccharides at the invaginated sites. A positive reaction to anti-β-1,3-D-glucan antibody was detected at the polysaccharides, Golgi vesicles, and trans-Golgi network (TGN) of toxin-treated leaves of the susceptible cultivar, but not at Golgi vesicles and TGN of water-treated ones. The cis-, medial and trans-Golgi stacks of toxin-treated leaves of the susceptible cultivar were negative for the antibody. The results showed that the polysaccharides, Golgi vesicles and TGN contained abundant β-1,3-D-glucan and that the glucan was transported from the Golgi apparatus via Golgi vesicles to the modified sites in cells of toxin-treated leaves of the susceptible cultivar. Received 7 March 2002/ Accepted in revised form 10 June 2002     

Reactive oxygen species (ROS) generation and ROS-induced lipid peroxidation are associated with plasma membrane modifications in host cells in response to AK-toxin I from Alternaria alternata Japanese pear pathotype

AK-toxin I caused plasma membrane modifications with plasma membrane-derived membrane fragments only in sensitive Japanese pear tissues. H₂O₂ generation was abundant in both the membrane fragments and the plasma membranes of the toxin-treated sensitive tissues. Whether lipid peroxidation was induced in plasma membranes of the toxin-treated sensitive tissues was examined biochemically and histochemically. Lipid peroxidation was caused only in the toxin-treated sensitive tissues or the toxin-treated plasma membrane-enriched fractions from sensitive young pear fruits. The results indicated that the peroxidation was probably induced by reactive oxygen species in the modified plasma membranes by action of toxin, suggesting that peroxidation is closely associated with plasma membrane modifications.     

Genetic Analysis of Pathogenicity and Host-specific Toxin Production of Alternaria alternata Tomato Pathotype by Protoplast Fusion

Several pathotypes of Alternaria alternata are known to produce host-specific toxins (HSTs) as agents of pathogenicity or virulence. However, investigations into the genetic controls of HST biosynthesis and pathogenicity of Alternaria pathogens have been limited by the lack of a sexual stage in the life cycle of these pathogens. We report here the development of a protoplast fusion system and its use for genetic analysis of HST production and specific pathogenicity of the tomato pathotype of A. alternata that produces AAL-toxin as a HST. Drug-resistant transformants have been isolated by genetic transformation of nonpathogenic A. alternata (strain O-94) and A. alternata tomato pathotype (strain As-27) with vectors conferring resistance to hygromycin B and geneticin, respectively. Protoplasts of the respective transformants were fused by polyethylene glycol treatment or electrofusion. Fusion products were selected by culturing in the presence of both hygromycin B and geneticin, then confirmed by amplification using a polymerase chain reaction with specific primers to the transforming drug-resistance genes. Stable fusants were purified by successive subcultures on selective medium and single-spore isolation. The resultant stable fusants, probably inter-strain hybrids, had the same pathogenicity and toxin production as the wild-type strain As-27. These results suggest that protoplast fusion has potential applications for genetic analysis of A. alternata pathogens. Received 8 September 2000/ Accepted in revised form 27 October 2000     

Alternaria Leaf Spot on Three Species of Pelargonium Caused by Alternaria alternata in Japan

A new disease of pelargonium (Pelargonium domesticum Bailey), ivy geranium (P. peltatum (L.) L'Hér. ex Ait.) and scented geranium (P. graveolens L'Hér.), primarily causing brown spots on leaves, was found in Kawasaki-shi in Kanagawa Prefecture and Tachikawa-shi in Tokyo. An Alternaria sp. was consistently isolated from these diseased leaves, and the isolates were pathogenic to their host leaves. Based on morphological characteristics, the causal fungus in all three cases was identified as Alternaria alternata (Fr.) Keissler. Because Alternaria leaf spot of geranium by A. alternata has already been reported, the pathogenicity of isolates from four groups of genus Pelargonium was investigated. The isolates from scented geranium were pathogenic only to their original host, but the isolates from pelargonium, ivy geranium and geranium were pathogenic to all groups of pelargonium. This is the first report of this disease on pelargonium, ivy geranium and scented geranium caused by A. alternata in Japan. We propose the names for these diseases as Alternaria leaf spot of pelargonium (kappan-byo), Alternaria leaf spot of ivy geranium (kappan-byo) and Alternaria leaf spot of scented geranium (kappan-byo). Received 11 December 2000/ Accepted in revised form 19 July 2001     

Microscopic detection of reactive oxygen species generation in the compatible and incompatible interactions of Alternaria alternata Japanese pear pathotype and host plants

 Reactive oxygen species (ROS) generation was examined in the interaction of Alternaria alternata Japanese pear pathotype and host plants using three methods: nitro blue tetrazolium (NBT) method for microscopic detection of O₂ ⁻, diaminobenzidine (DAB) methods for microscopic detection of H₂O₂, and cerium chloride methods for ultrastructural detection of H₂O₂. ROS generation was detected by NBT and DAB methods at appressoria on leaves of susceptible cultivars and heat-shocked leaves of resistant cultivars but not in leaves of resistant cultivars. Ultrastructural detection by the cerium chloride method identified ROS generation at cell walls of appressoria and penetration pegs in susceptible, resistant leaves and heat-shocked leaves. These differences in the ultrastructural and microscopic data in resistant areas were due to the restriction of ROS generation in limited areas, the side facing the plant surface, of appressoria and penetration pegs. Therefore, ROS generation was apparently induced regardless of the resistance or susceptibility of the cultivar with the difference being in the volumes generated. After evaluating the pathological role of ROS generation in fungal structures, such generation was found to be associated with early penetration of cell walls in pear plants. Additionally, ROS generation in plants was also found in degrading pectin layers near infected hyphae and in plasma membrane modification sites in susceptible leaves but not in resistant leaves. ROS generation in susceptible leaves might be accompanied with plasma membrane damage, although the role of ROS generation in the pectin layers is not clear. ROS generation in both fungal and plant cells during their interaction was likely associated with the expression of susceptibility. Received: June 3, 2002 / Accepted: July 31, 2002