Ultrastructural study on acibenzolar-S-methyl-induced scab resistance in epidermal pectin layers of Japanese pear leaves

The infection behavior of Japanese pear scab pathogen Venturia nashicola race 1 was studied ultrastructurally in acibenzolar-S-methyl (ASM)-pretreated susceptible Japanese pear (cv. Kousui) leaves to determine the mechanism of ASM-induced scab resistance. On ASM-pretreated leaf surfaces, the infection behavior (conidial germination and appressorial formation) was similar to that on distilled water (DW)-pretreated leaves prior to cuticle penetration by the pathogen. However, after penetration, differentiated behavior was found in epidermal pectin layers and middle lamellae of the ASM-pretreated leaves. Subcuticular hyphae in epidermal pectin layers and middle lamellae of ASM-pretreated pear leaves were observed at lower frequency than in DW-treated leaves. The results indicated that fungal growth was suppressed in ASM-pretreated pear leaves. In the pectin layers of ASM- and DW-pretreated leaves, some hyphae showed morphological modifications, which were used as criteria to judge collapse of hyphal cells, including plasmolysis, necrotic cytoplasm, and cell wall destruction. More hyphae had collapsed in ASM-pretreated leaves than in DW-treated ones. In addition, the cell walls of collapsed hyphae broke into numerous fibrous and amorphous pieces, suggesting that ASM-induced scab resistance might be associated with cell-wall-degrading enzymes from pear plants. In addition, results from morphometrical analysis suggested that the activity or production of pectin-degrading enzyme from hyphae were inhibited by ASM application when compared with DW treatment.     

Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab

Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars.     

Molecular cloning and expression analysis of genes related to phosphatidic acid synthesis in Japanese pear leaves inoculated with <Emphasis Type="Italic">Venturia nashicola</Emphasis>

Phosphatidic acid, which can be generated by the action of phospholipase D (PLD) and by the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK), is a plant defense signal. To identify the role of the PLD, PLC, and DGK genes encoding these enzymes in pear resistance to the scab fungus Venturia nashicola, we report the cloning of these genes and analysis of their expression in inoculated pear leaves. The results showed that PLD mRNA increased in the leaves of the susceptible pear cv. Kousui 1 day after inoculation (dai), then decreased to the basal level. In resistant pear cv. Kinchaku, PLD mRNA did not change significantly except for a small increase at 2 and 3 dai. However, the level of PLD mRNA in Kinchaku was higher than in Kousui 2 dai. The level of PLC mRNA significantly increased in cvs. Kousui and Kinchaku by 1 dai, peaked at day 2, then decreased to the basal level. Thus, the gene expression pattern did not differ between the two cultivars. PLC gene expression was also induced by environmental stress. The DGK gene seemed to be constitutively expressed and was not induced by inoculation in either the susceptible or resistant cultivars. Together, this data showed that both PLD and PLC are possibly involved in a defense response to V. nashicola in Japanese pear leaves, and the PLD gene seems to play a more important role than does the PLC gene.     

Infection Behavior of Venturia nashicola, the Cause of Scab on Asian Pears

ABSTRACT The infection of Japanese pear by Venturia nashicola, the cause of scab on Asian pears (Japanese pear, Pyrus pylifolia var. culta; Chinese pear, P. ussuriensis), was examined using light and electron microscopy to determine the mechanism of resistance in pears. Early stages of infection were similar on the susceptible cv. Kosui, the resistant cv. Kinchaku, and the nonhost European pear (P. communis) cv. Flemish Beauty. V. nashicola penetrated only the cuticle layer on pear leaves and formed subcuticular hyphae on all three cultivars. Hyphae were localized in the pectin layer of pear leaves and never penetrated into the cytoplasm of epidermal cells. This restriction of fungal growth suggested that pectinases released by infection hyphae or subcuticular hyphae may be important in infection. Subcuticular hyphae were modified ultrastructurally in the pectin layer of resistant pear cultivars accompanied by fungal cell death. In contrast, fungal cells appeared intact in susceptible pear cultivars, suggesting the existence of resistance mechanisms.     

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     

亚洲梨黑星病菌致病性及其寄主抗病机制的研究进展

梨黑星病是亚洲梨的主要病害之一。该病是由纳雪黑星病菌(Venturia nashicola)感染所致。V.nashicola主要寄生在亚洲梨叶片表皮细胞壁的果胶质层中。该菌的感染可能主要与分泌的细胞外分泌物质、角质分解酶、过氧化氢和果胶质分解酶有关。而亚洲梨对V.nashicola的抗性可能主要与多聚半乳糖醛酸酶抑制蛋白、多种病程相关蛋白、富亮氨酸重复类受体蛋白激酶等有关。另外,不具直接杀菌能力的系统抗性诱导剂acibenzolar-S-methyl(ASM)在大田试验中对梨黑星病菌有较好控制效果。这与ASM诱导的植物防御反应,包括多聚半乳糖醛酸酶抑制蛋白和几丁质酶等有关。     

多堆柄锈菌侵染玉米的细胞学及超微结构特征

为明确玉米对多堆柄锈菌Puccinia polysora侵染后病理反应的细胞学特征,利用扫描和透射电镜技术分析了玉米自交系与多堆柄锈菌互作中二者的细胞变化过程。多堆柄锈菌对玉米的侵染主要以直接穿透叶片表皮侵入为主,少量可从气孔和细胞间隙侵入。接种后,病菌夏孢子在感病自交系叶片上快速并大量萌发,在叶表生长蔓延并侵入表皮组织细胞,7 d后形成夏孢子堆;在抗病自交系上,病菌萌发、菌丝生长均受到明显抑制,少量入侵的病菌也由于寄主细胞死亡而导致菌丝和夏孢子干瘪死亡。侵染早期在感病寄主细胞间隙出现菌丝并穿透细胞壁,在胞内产生分枝菌丝,此时寄主细胞结构正常;随着菌丝进一步扩展,叶绿体等结构发生紊乱,被侵染细胞逐渐死亡。在抗病自交系上,接菌24 h后寄主即出现过敏性坏死反应,侵入位点与周围细胞快速坏死,抑制菌丝生长蔓延;叶绿体中清晰可见深色颗粒状物质;72 h后细胞壁外侧产生大量致密的深色结晶体,应为与抗病反应相关的酚类物质。表明抗多堆柄锈菌的玉米材料可能存在2种抗病途径,即寄主与病菌互作中由分子识别引起的免疫反应和病菌侵入后的系统防卫反应。     

Responses of cucumber cultivars to induction of systemic resistance against anthracnose by plant growth promoting fungi

Initial experiment on the reactions of five Japanese cultivars of cucumber toColletotrichum orbiculare infection in the greenhouse revealed that cv Suyo and Gibai were susceptible and moderately susceptible, respectively, while cv Shogoin fushinari and Sagami hanjiro were resistant to infection byC. orbiculare; cv Ochiai fushinari was moderately resistant. The ability of 16 plant growth promoting fungi (some isolates belonged to species ofPhoma and some non-sporulating isolates) isolated from zoysiagrass rhizospheres to induce systemic resistance in the above five cucumber cultivars was tested by growing plants in potting medium infested with barley grain inocula of PGPF in the greenhouse. The second true leaves of 21-day-old plants were challenge inoculated withC. orbiculare and disease assessed. Nine, out of 16 isolates, caused significant reduction of disease caused byC. orbiculare in at least two cultivars.Phoma isolates (GS8-1 and GS8-2) and non-sporulating isolates (GU21-2, GU23-3, and GU24-3) significantly reduced the disease in all the five cultivars. The disease suppression in cucumber was due to the induction of systemic resistance, since the inducer(s) and the pathogen were separated spatially and that the inducer did not colonize aerial portions. The resistance induced by certain isolates in a susceptible cultivar was less than that in a resistant cultivar. Disease suppression caused by isolate GU21-2 was similar to theC. orbiculare induced control in certain cultivars. The average rate of expansion of lesion diameter on leaves due toC. orbiculare was slower due to induction with the selected plant growth promoting fungi compared to the uninduced control plants. Roots of four cultivars were colonized by only three isolates, however, roots of one cultivar (Suyo) was colonized by five isolates suggesting the cultivar-specific root colonization ability.Abbreviations cv cultivar(s) - PGPF plant growth promoting fungal isolates - PGPR plant growth promoting rhizobacteria     

Silicon-induced cell wall fortification of rice leaves: a possible cellular mechanism of enhanced host resistance to blast

ABSTRACT Locations of silicon accumulation in rice leaves and its possible association with resistance to rice blast were investigated by electron microscopy and X-ray microanalysis. A blast-susceptible cultivar, Jinmi, and a partially resistant cultivar, Hwaseong, were grown under a hydroponic culture system with modified Yoshida's nutrient solution containing 0, 50, 100, and 200 ppm of silicon. Electron-dense silicon layers were frequently found beneath the cuticle in epidermal cell walls of silicon-treated plants. Increasing levels of silicon were detected in the outer regions of epidermal cell walls. Silicon was present mainly in epidermal cell walls, middle lamellae, and intercellular spaces within subepidermal tissues. Furthermore, silicon was prevalent throughout the leaf surface, with relatively small deposition on stomatal guard cells in silicon-treated plants. Silicon accumulation and epidermal cell wall thickness in leaves were greater in cv. Jinmi than in cv. Hwaseong. However, the thickness ratios of the silicon layers to epidermal cell walls were greater in cv. Hwaseong (53.25 to 93.28%) than in cv. Jinmi (36.58 to 66.54%). Leaf blast severity was lower in cv. Hwaseong than in cv. Jinmi and was significantly reduced in silicon-treated plants of both cultivars. These results suggest that silicon-induced cell wall fortification of rice leaves may be closely associated with enhanced host resistance to blast.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

Latency- and Defense-Related Ultrastructural Characteristics of Apple Fruit Tissues Infected with Botryosphaeria dothidea

ABSTRACT Apple fruit tissues infected with Botryosphaeria dothidea were examined by transmission electron microscopy using susceptible cv. Fuji and resistant cv. Jonathan. Immature (green) and mature (red) fruits of cv. Fuji with restricted or expanding lesions were also examined to reveal subcellular characteristics related with latent and restricted disease development. In infected susceptible mature fruits, cytoplasmic degeneration and organelle disruption commonly occurred, accompanying cell wall dissolution around invading hyphae. Cell wall dissolution around invading hyphae in subepidermis was rare in immature, red halo-symptomed cv. Fuji and resistant cv. Jonathan fruits. In infected immature fruits of cv. Fuji, presumably at the latent state of disease development, cellular degeneration was less severe, and invading hyphae contained prominent microbody-lipid globule complexes or the deposition of thin electron-dense outer layer around cell wall of intercellular hyphae. Both mature fruits with red halos and resistant apple fruits formed cell wall protuberances at the outside of cell walls. In addition, electron-dense extramural layers were formed in the resistant apple fruits. Aberrant hyphal structures such as intrahyphal hyphae were found only in resistant fruit tissues, indicating the physiologically altered fungal growth. These ultrastructural changes of host tissues and fungal hyphae may reflect the pathogenesis of apple white rot under varying conditions of apple fruits.     

Impact of carrot resistance on development of the <Emphasis Type="Italic">Alternaria</Emphasis> leaf blight pathogen (<Emphasis Type="Italic">Alternaria dauci</Emphasis>)

The interaction between Alternaria dauci and two carrot cultivars differing in their resistance to leaf blight was investigated by microscopy. The fungal development between 1 and 15 days post-inoculation was quite similar in the susceptible cv. Presto and the partially resistant cv. Texto: After conidial germination, leaf adhesion of the pathogen was achieved with mucilaginous filaments; hyphae penetrated the leaves directly with/without the formation of appressoria-like structures or via stomata; the fungus spread by epiphytic hyphae with hyphopodia and subcuticular mycelia. Intense necrotic plant cell reactions occurred under the fungal structures. At 21 days post-inoculation, typical features of fungal development were noted for each cultivar: growing hyphae emerged from stomata in cv. Presto, whereas conidiophores without conidia were observed in cv. Texto. Leaf tissues of both cultivars were strongly damaged and vesicle-like structures (assumed to be plant phenolics) were abundantly present between mesophyll cells. A real-time PCR method was developed for in planta quantification of A. dauci. Between 1 and 15 days post-inoculation, the fungal biomass was equivalent in the two cultivars and was about fourfold higher in cv. Presto than cv. Texto at 21 and 25 days post-inoculation. Taken together, our results indicated that A. dauci was able to colonize both cultivars in a similar manner during the first steps of the interaction, then fungal development in the partially resistant cultivar was restricted due to putative plant defence reactions. The results of this study enhance the overall understanding of infection processes in the A. dauci-carrot pathosystem.     

Ascospores of the Japanese pear scab fungus (<Emphasis Type="Italic">Venturia nashicola</Emphasis> Tanaka &; Yamamoto) are discharged during the day

We investigated the diurnal pattern of ascospore discharge of the Japanese pear scab fungus (Venturia nashicola Tanaka & Yamamoto) in an orchard. Ascospores of V. nashicola were mainly discharged during the day. Most ascospores were discharged from 7:00 to 19:00: 99.6% in 2001, 99.3% in 2002, and 93.8% in 2005. Because the ascospores were discharged only when the fallen diseased leaves were wet from precipitation, the wetness of these leaves is probably imperative for spore discharge. Ascospore discharge began immediately after precipitation in the daytime. When it rained at night, however, ascospore discharge did not begin until the following morning and never began immediately after precipitation. We also investigated other meteorological factors. When fallen diseased leaves were wet, the percentage of ascospore discharge was positively correlated with the amount of solar radiation and atmospheric temperature and negatively correlated with relative humidity. Ascospore discharge was interrupted by a decrease in solar radiation and atmospheric temperature and by increased relative humidity at night. This report is the first that V. nashicola discharges ascospores primarily during the day.     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

Histopathology of Infection and Colonization of Susceptible and Resistant Port-Orford-Cedar by Phytophthora lateralis

ABSTRACT Port-Orford-cedar (POC) root disease, caused by Phytophthora lateralis, continues to kill POC in landscape plantings and natural forests in western North America. POC trees resistant to P. lateralis have been identified and propagated. Cytological observations of P. lateralis in susceptible and resistant roots and stems were made with light and transmission electron microscopy to identify resistance mechanisms. No differences in infection pathway and initial colonization were observed between susceptible and resistant roots, although there were differences in the rate and extent of development. Germ tubes formed appressoria, and penetration hyphae grew either between or directly through epidermal cell walls; inter- and intracellular hyphae colonized the root cortex. In susceptible roots, hyphae penetrated into the vascular system within 48 h of inoculation. In contrast, hyphae in roots of resistant seedlings grew more slowly in cortical cells and were not observed to penetrate to the vascular tissues. In resistant roots, infection was marked by general thickening of cortical cell walls, wall appositions around penetrating hyphae, collapse of cortical cells, and accumulation of osmophillic granules around hyphae. In susceptible stems, hyphae grew inter- and intracellularly in all cells of the secondary phloem except fiber cells, but were concentrated in sieve and parenchyma cells in the functional phloem. The pattern of penetration and colonization of hyphae was similar in the resistant stems, except that hyphae were found in the fiber cells of the xylem. In resistant stems, there were fewer hyphae in the functional phloem, and cytological changes such as damaged nuclei and disintegrated cytoplasm were evident. Structural changes in resistant stems included collapsed cells, wall thickening, secretory bodies, apposition of electron dense materials, and crystals in cell walls.     

Changes in soluble and cell wall-bound hydroxycinnamic and hydroxybenzoic acids in sugarcane cultivars inoculated with <Emphasis Type="Italic">Sporisorium scitamineum</Emphasis> sporidia

The accumulation of soluble and cell wall-bound phenolics in the sugarcane stems of young plants from highly resistant cv. My 5514 and susceptible cv. B 42231, inoculated or not inoculated with smut sporidia, was studied. The ratio of inoculated to uninoculated plants of some cell wall-bound phenolics, such as ferulic, caffeic, and syringic acids increased for the resistant cv. My 5514, whereas it was maintained more or less constantly for the susceptible cv. B 42231. The highest increase of this ratio in the resistant cv. My 5514 corresponded to both caffeic and syringic acids. This could result in a better capacity to cv. My 5514 for an increase in the frequency of bridges between lignin fragments through ester-ether linkages for reinforcing the cell wall and major resistance to the disease. This reinforcement of the cell wall could provide an effective barrier to pathogen entry and spread. Soluble sub-fractions of all phenolics detected showed non-stable patterns. Caffeic acid, that regulates phenylalanine ammonia-lyase activity in sugarcane, showed a significant decrease in its titre at 24 h in the resistant cultivar, principally in the free soluble fraction, whilst the susceptible cultivar enhanced it. We hypothesise that the pathway of hydroxybenzoic acids is only activated once the level of p-coumaric acid justifies the accumulation of hydroxycinnamic acids required for reinforcing the cell wall after inoculation.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.