Alternata Japanese Pear Pathotype Research Articles

Regiospecific and Enantiospecific Effects of the β-Benzyl-α-benzylidene-γ-butyrolactone Structure on Phytotoxic Fungi

Novel derivatives of E-β-benzyl-α-benzylidene-γ-butyrolactone (3-benzyl-2-benzylidene-4-butanolide) with lignano-9,9'-lactone structures were developed as anti-phytopathogenic fungal compounds. Their regiospecific and enantiospecific characteristics were determined, with the E-form and 3R-configuration showing higher activities against the Alternaria alternata Japanese pear pathotype. By the syntheses of benzyl compounds instead of benzylidene and aromatic derivatives, followed by an bioassay experiment, the importance of the benzylidene structure and effects of the substituents of the aromatic ring were clarified. The (2-OCH3, 4'-CH3/4'-CF3)-derivatives, 19 and 25, and (2-OCH3, 6-CH3/6-F/6-Br, 4'-OCH3)-derivatives, 34, 38, and 42, were more effective with EC50 values of 0.1-0.3 μM. It was assumed that the 2-OCH3 group, a hydrophobic group at the 6-position, and some size of the hydrophobic group at the 4'-position were necessary for the increased activity.

Searching for the Stereoisomer of 7,7′-Epoxylignan Showing the Most Potent Antifungal Activity and Finding the 3-(Trifluoromethyl)-4-hydroxy-3′-fluoro Derivative to Have the Highest Activity

The effect of the stereochemistry of 9,9′-epoxylignan, 7,9′-epoxylignan, and 7,7′-epoxylignan bearing the 4-hydroxy-3-methoxyphenyl group on their antifungal activity was estimated by employing stereoisomers of these compounds. The results suggest that the (7S,7′R,8R,8′R)-stereoisomers of 7,7′-epoxylignan are the most potent. (−)-Verrucosin (16) had an EC50 value of 89 μM against the Alternaria alternata Japanese pear pathotype, whereas the most effective derivative against the A. alternata Japanese pear pathotype was (7S,7′R,8R,8′R)-3-(trifluoromethyl)-4-hydroxy-3′-fluoro-7,7′-epoxylignan derivative 38, which was found to be ∼124 times more potent (EC50 = 0.72 μM) than natural (−)-verrucosin (16). The presence of both hydroxy and electron-withdrawing groups on the 7-aromatic ring enhanced the activity. Compared with that of butane type structure 42, the activity of 7,7′-epoxylignan structure 38, which is fixed between positions 7 and 7′, was 38 times higher.

A new interspecific pear cultivar Yutaka: highly resistant to the two major diseases scab and black spot on Asian pears

Scab, caused by Venturia nashicola, is the most important disease of Asian pears. In Japan, the most popular and widely grown pear cultivar (cv.) Kousui is resistant to black spot, caused by the Japanese pear pathotype of Alternaria alternata, but highly susceptible to scab. Recently, a new interspecific pear cv. Yutaka carrying high fruit quality was registered officially after a cross between the Chinese pear cv. Hongli and the Japanese pear cv. Housui. Accordingly, tests were conducted to assess resistance of cv. Yutaka to scab, black spot and anthracnose. When conidial suspensions of V. nashicola were spray inoculated onto potted trees, leaves of cv. Yutaka showed no disease symptoms although severe scab development was recorded on the susceptible reference cv. Kousui. Furthermore, neither black spot symptoms nor necrosis formation were observed when leaves of cv. Yutaka were inoculated with conidial suspensions of the A. alternata Japanese pear pathotype or treated with a culture filtrate containing the host-specific AK-toxin of this pathogen, respectively. These results strongly indicate that cv. Yutaka is highly resistant to scab and black spot. Inoculation tests also suggested the potential high levels of resistance of cv. Yutaka to anthracnose caused by Colletotrichum gloeosporioides sensu lato. The introduction of this new pear cultivar will reduce fungicide applications in pear orchards and thus lower the risk of fungicide resistance development in these pathogens.

Structure-Antifungal Activity Relationship of Fluorinated Dihydroguaiaretic Acid Derivatives and Preventive Activity against Alternaria alternata Japanese Pear Pathotype.

The structure-activity relationship of the antifungal fluorinated dihydroguaiaretic acid derivatives was evaluated. Some of the newly synthesized lignan compounds were found to show higher antifungal activity against phytopathogenic fungi such as Alternaria alternata (Japanese pear and apple pathotypes) and A. citri than the lead compound, 3-fluoro-3'-methoxylignan-4'-ol (3). The broad antifungal spectrum of 3'-hydroxyphenyl derivative 16 was observed, and the 3'-fluoro-4'-hydroxyphenyl derivative 38 was found to show the highest activity against the A. alternata Japanese pear pathotype, with an EC50 value of 11 μM. The preventive effect of the potent lignan on the infection of A. alternata in the Japanese pear's leaves was also shown.

Physical mapping of black spot disease resistance/susceptibility-related genome regions in Japanese pear (Pyrus pyrifolia) by BAC-FISH.

Black spot disease, caused by Alternaria alternata Japanese pear pathotype, is one of the most harmful diseases in Japanese pear cultivation. In the present study, the locations of black spot disease resistance/susceptibility-related genome regions were studied by fluorescence in situ hybridization using BAC clone (BAC-FISH) on Japanese pear (Pyrus pyrifolia (Burm. f.) Nakai) chromosomes. Root tips of self-pollinated seedlings of ‘Osa Gold’ were used as materials. Chromosome samples were prepared by the enzymatic maceration and air-drying method. The BAC clone adjacent to the black spot disease-related gene was labeled as a probe for FISH analysis. Black spot disease-related genome regions were detected in telomeric positions of two medium size chromosomes. These two sites and six telomeric 18S-5.8S-25S rDNA sites were located on different chromosomes as determined from the results of multi-color FISH. The effectiveness of the physical mapping of useful genes on pear chromosomes achieved by the BAC-FISH method was unequivocally demonstrated.

Syntheses of natural 1,3-polyol/α-pyrone and its all stereoisomers to estimate antifungal activities against plant pathogenic fungi.

All stereoisomers of 1,3-polyol/α-pyrone 1-8 with more than 99% ee were synthesized to estimate the effect of stereochemistry on the antifungal activity. The absolute configuration of natural compound was determined as (6R,2'S,4'R)-2. The eight stereoisomers showed the antifungal activity against plant pathogenic Alternaria alternata Japanese pear pathotype and Colletotrichum lagenarium. The large difference of activity level was not observed between stereoisomers, showing 43-72% of growth ratio against control at 0.5mM. The most potent stereoisomer was (6S,2'S,4'S)-8 and the activity of (6R,2'S,4'S)-1 was weakest against both fungi.

Durable and broad-spectrum disease protection measure against airborne phytopathogenic fungi by using the detachment action of gelatinolytic bacteria

We had previously obtained collagenolytic/gelatinolytic bacteria, which degrade the fungal extracellular matrix, to establish a novel biological control measure that inhibits germling adhesion of airborne phytopathogenic fungi on the host plant surface. By using barley-Magnaporthe oryzae pathosystem, Chryseobacterium sp. was most effective biocontrol agents as tested. The selected bacteria were evaluated for durable disease protection against M. oryzae on barley leaves by using chloramphenicol-resistant mutants. Chryseobacterium sp. from the soil was less likely to settle on leaf surfaces. Therefore, we tried to manipulate Chryseobacterium sp. to inhabit the leaf’s surface. The gelatin supplementation dramatically improved the settlement of gelatinolytic bacteria Chryseobacterium sp. from the soil, and the disease protection effect lasted for more than 2weeks on barley. Moreover, exploitation of Chryseobacterium sp. for disease protection was extended against other airborne pathogens, Alternaria alternata Japanese pear pathotype on Japanese pear and Colletotrichum orbiculare on cucumber.

Alternaria属菌の宿主特異的毒素：植物病害の決定因子

Seven diseases caused by pathotypes of Alternaria alternata, which produced host-selective toxins (HSTs), a diverse group of low-molecular-weight secondary metabolites, are known, and each HST has an essential role as a determinant of pathogenicity in all interactions between the plant host and A. alternata. Although these HST-producing pathotypes are morphologically indistinguishable, each has a distinct host range and can be distinguished by its specificity on the respective host plant, hence their designation as pathotypes of A. alternata. In 1933, Tanaka made the first discovery of a HST; fungus-free culture filtrates of A. kikuchiana (now called A. alternata Japanese pear pathotype) were toxic to susceptible cultivar Nijisseiki, but not to resistant cultivars. Over the 80 years since then, the structure of HST molecules, target sites and mode of actions of HSTs, and the molecular genetics of HST production regulating by supernumerary chromosomes encoding HST gene clusters have been studied extensively. We focus this review on studies of low-molecular-weight HSTs produced by A. alternata and give an overview of various types of HST studies.

Alternaria host-selective toxins: determinant factors of plant disease

Seven diseases caused by pathotypes of Alternaria alternata, which produced host-selective toxins (HSTs), a diverse group of low-molecular-weight secondary metabolites, are known, and each HST has an essential role as a determinant of pathogenicity in all interactions between the plant host and A. alternata. Although these HST-producing pathotypes are morphologically indistinguishable, each has a distinct host range and can be distinguished by its specificity on the respective host plant, hence their designation as pathotypes of A. alternata. In 1933, Tanaka made the first discovery of a HST; fungus-free culture filtrates of A. kikuchiana (now called A. alternata Japanese pear pathotype) were toxic to susceptible cultivar Nijisseiki, but not to resistant cultivars. Over the 80 years since then, the structure of HST molecules, target sites and mode of actions of HSTs, and the molecular genetics of HST production regulating by supernumerary chromosomes encoding HST gene clusters have been studied extensively. We focus this review on studies of low-molecular-weight HSTs produced by A. alternata and give an overview of various types of HST studies.

Quantitative Structure–Activity Relationship Analysis of Antifungal (+)-Dihydroguaiaretic Acid Using 7-Phenyl Derivatives

The relationship between antifungal activity against Alternaria alternata and structure on the 7-phenyl group of (+)-dihydroguaiaretic acid ((+)-DGA) was clarified by employing 38 synthesized (+)-DGA derivatives. The results were identified by quantitative structure-activity relationship (QSAR) analysis employing the Hansch-Fujita method. Some compounds showed higher activity than (+)-DGA. The compound showing highest activity was 3,5-difluorophenyl derivative 37. It was suggested that the small electron-withdrawing group at the meta-position of the 7-phenyl group is important for the higher activity by antifungal test and Hansch-Fujita analysis. The whitening activity of 3-hydroxy-4-methoxyphenyl derivative 28, 3-hydroxy-4-ethoxyphenyl derivative 29, and 3-hydroxy-4-isopropoxyphenyl derivative 30 against A. alternata Japanese pear pathotype was also discovered.

Appressorium‐localized NADPH oxidase B is essential for aggressiveness and pathogenicity in the host‐specific, toxin‐producing fungus Alternaria alternata Japanese pear pathotype

Black spot disease, Alternaria alternata Japanese pear pathotype, produces the host-specific toxin AK-toxin, an important pathogenicity factor. Previously, we have found that hydrogen peroxide is produced in the hyphal cell wall at the plant-pathogen interaction site, suggesting that the fungal reactive oxygen species (ROS) generation machinery is important for pathogenicity. In this study, we identified two NADPH oxidase (NoxA and NoxB) genes and produced nox disruption mutants. ΔnoxA and ΔnoxB disruption mutants showed increased hyphal branching and spore production per unit area. Surprisingly, only the ΔnoxB disruption mutant compromised disease symptoms. A fluorescent protein reporter assay revealed that only NoxB localized at the appressoria during pear leaf infection. In contrast, both NoxA and NoxB were highly expressed on the cellulose membrane, and these Nox proteins were also localized at the appressoria. In the ΔnoxB disruption mutant, we could not detect any necrotic lesions caused by AK-toxin. Moreover, the ΔnoxB disruption mutant did not induce papilla formation on pear leaves. Ultrastructural analysis revealed that the ΔnoxB disruption mutant also did not penetrate the cuticle layer. Moreover, ROS generation was not essential for penetration, suggesting that NoxB may have an unknown function in penetration. Taken together, our results suggest that NoxB is essential for aggressiveness and basal pathogenicity in A. alternata.

The presence of double-stranded RNAs in Alternaria alternata Japanese pear pathotype is associated with morphological changes

Strains of the Japanese pear pathotype of Alternaria alternata were screened for double-stranded RNAs (dsRNAs). Four strains had several dsRNAs; strain N18 was associated with several dsRNAs and had impaired growth phenotypes such as irregular mycelium and abnormal pigmentation. We isolated dsRNA-cured isolates from strain N18 by single-conidium isolation. The dsRNA-cured isolates had recovered normal growth and pigmentation. Enlarged vesicles were observed in mycelial cells of the original dsRNA-carrying N18 strain. DAPI nuclear staining revealed regression of the nuclei in dsRNA-carrying N18 cells. These results indicate that the dsRNAs might have negative effects, such as apoptosis-like cell death, on the host fungus.

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.

Cytological and molecular analyses of non‐host resistance of Arabidopsis thaliana to Alternaria alternata

SUMMARY When challenged with the necrotrophic fungal pathogen Alternaria alternata Japanese pear pathotype, all tested ecotypes of Arabidopsis plants failed to show hypersensitive cell death, accumulation of detectable levels of reactive oxygen species or accumulation of phytoalexin. We operationally define A. alternata as a non-host pathogen for Arabidopsis plants and show that the protection against A. alternata demonstrated in this study is a non-host penetration resistance. To characterize non-host penetration resistance, we examined the expression patterns of c. 7000 genes by cDNA microarray analysis in Arabidopsis Col-0 plants after inoculation with A. alternata. After inoculation with A. alternata, the transcript levels of 48 genes increased in Col-0 plants. The expression of genes associated with hypersensitive reaction was induced in the non-host penetration resistance to A. alternata, despite the fact that A. alternata had no visible effect on the plants. The non-host penetration resistance to A. alternata was clearly associated with activation of the jasmonate- and ethylene-signalling pathways. In addition, analysis using histochemical staining of GUS activity suggests that defence reactions in non-host penetration resistance are activated locally. The characterization of non-host pathosystem involving Arabidopsis and A. alternata offers an overview of non-host penetration resistance.

Detection of fungi producing infection-inhibiting metabolites against Alternaria alternata Japanese pear pathotype from fungi inhabiting internal tissues of Japanese pear shoots

Fungi inhabiting Japanese pear were isolated from internal tissues of cv. Nijisseiki, and culture filtrates (CFs) of 100 isolates were evaluated for their inhibitory activity against infection by Alternaria alternata Japanese pear pathotype. CFs of 11 isolates inhibited lesion formation on the pear by the pathogen. Among these isolates, CFs of five isolates inhibited spore germination. CFs of the six other isolates inhibited appressorial formation, infection hypha formation, AK-toxin production, or a combination of these actions. Analysis of sequence homology in the rDNA ITS1 regions of these isolates showed that most isolates had high homology with some fungal endophytes.

Infection behavior of Alternaria alternata Japanese pear pathotype and localization of 1,3-β- d -glucan in compatible and incompatible interactions between the pathogen and host plants

The infection behavior of Alternaria alternata Japanese pear pathotype and host responses were examined cytologically and ultrastructurally in compatible and incompatible interactions between Japanese pear plants and the pathogen during the infection process. No differences in spore germination, appressorial formation, or cuticle penetration were detected between susceptible and resistant leaves inoculated with spores of the pathogen. On the other hand, infection hyphae emerged from penetration pegs and invaded the pectin layers of susceptible leaves but not those of resistant leaves. The results indicate that expression of resistance to the pathogen may be induced before the formation of infection hyphae in pear tissues. Plasma membrane modifications were observed ultrastructurally 3 h after inoculation only in susceptible leaf cells. Papillae were usually formed in epidermal cells underneath the appressoria in resistant leaves at the stage when the progress of the pegs was stopped 9 h after inoculation. Callose deposition was observed fluorescence-microscopically as papillae at the infection sites in abaxial epidermis of both cultivars. The immunoelectron microscopic results demonstrated that the components of the papillae and the extracellular polysaccharides that had accumulated in plasma membrane modification sites were compatible with 1,3-β-d-glucan. These results suggest that the glucan deposition results from host cuticle damage by fungal invasion and from plasma membrane damage by AK-toxin.

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.

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.

NEW JAPANESE PEAR CULTIVAR 'YOUSUI'

'Yousui' is a new late maturing, russet-skin type cultivar of Japanese pear, released by the Aichi-ken Agricultural Research Center. This cultivar originated from a cross between 'Niitaka' and 'Kousui' made in 1990. In 2001, 'Yousui' was registered on March 13 under the Seeds and Seedlings Law of Japan. 'Yousui' trees are as vigorous as 'Niitaka' and twigs are thick. The tree form looks like that of 'Niitaka'. The tree exhibits few axillary flower buds on shoot as 'Kousui', but it is easy to maintain flower buds on spurs. It is considered that 'Yousui' is fairly productive as 'Niitaka'. It blooms at the same time as 'Housui', 3 to 4 days later than 'Niitaka'. 'Yousui' is cross-compatible with 'Kousui', 'Housui' and 'Niitaka'. It has large quantities of pollen. 'Yousui' matures between ‘Housui’ and 'Niitaka', late September to early October in Aichi prefecture. Fruit is oblate in shape and the skin is yellowish brown at maturity. Fruit is very large like that of 'Niitaka', about 900g. The flesh is white, soft, juicy and crisp. It is considered superior to 'Niitaka' in these points. Brix values are 14 to 15% in a usual year, 0.5 to 1% higher than 'Niitaka'. pH values are 5.2 to 5.3, less acid than 'Niitaka'. The shelf life is shorter than those of other late maturing cultivars such as 'Niitaka' and 'Shinsei'. The fruit quality remains good for about 15 days when the fruit is kept at room temperature (about 15°C). ‘Yousui’ is resistant to black spot disease (Alternaria alternata Japanese pear pathotype) and pear necrotic spot virus. 'Yousui' may be tolerant to other diseases and insect pests under standard cultivation condition. Furthermore, as for ‘Yousui’, a physiological disorder is not being observed at present.

Ultrastructural Localization of Hydrogen Peroxide in Host Leaves Treated with AK-toxin I Produced by Alternaria alternata Japanese Pear Pathotype

The rapid generation of reactive oxygen species (ROS), called the oxidative burst, is one of the earliest host responses to pathogen infection or elicitor treatments. Therefore, we looked for the induction of ROS generation in Japanese pear leaves by the host-specific toxin, AK-toxin I using a cytochemical method for detecting H2O2. A small amount of non-specific generation of H2O2 was found in the cell walls in toxin- and water-treated susceptible and resistant leaves. Thus, the generation of H2O2 at cell walls appears to be caused by wounding stress during sampling. Specific generation of ROS, however, was found only in the membrane fragments and extended desmotubules characteristic of modified sites of the plasma membrane in the toxin-treated susceptible leaves. In addition, generation of H2O2 at plasma membranes was observed with higher frequency in toxin-treated susceptible leaves. This result indicates that the H2O2 generation was associated with damaged sites in the plasmalemma after toxin treatment and perhaps with the formation of membrane fragments from altered portions of the invaginated plasma membrane.