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.     

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.     

Purification of polygalacturonases produced by the pear scab pathogens, Venturia nashicola and Venturia pirina

An exopolygalacturonase and three endopolygalacturonases were purified from mycelia of pear scab pathogens, Venturia pirina and Venturia nashicola. The molecular weight of the isolated exoPG from V. pirina was 43 kDa, and the endoPGs from V. nashicola were 42 kDa as estimated by SDS–polyacrylamide gel electrophoresis. The pH optimum of the exoPG activity from V. pirina was 5.0. TheK_m and V_maxvalues of the exoPG were 0.08 mg ml⁻¹and 4.44 × 10⁻³ mmol reducing group min⁻¹ mg protein⁻¹. The N-terminal amino acid sequence of the exoPG from V. pirina was similar to that of the exoPG from Fusarium oxysporum f. sp. melonis, and the N-terminal amino acid sequences of the three endoPGs fromV. nashicola races 1, 2 and 3 were similar to other fungal endoPGs with a conserved motif of ASxxxTFTxAAAxxxG.     

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.     

Evidence for loss of ontogenetic resistance of apple leaves againstVenturia inaequalis

The susceptibility of apple leaves toVenturia inaequalis was investigated by assessing disease on individual leaves of seedlings and labelled shoots of orchard trees. Four sets (A-D) of potted seedlings of cv. Golden Delicious, which had been grown in a glasshouse, each with approximately 30 mature leaves, were exposed to a high-inoculum orchard. Sets A and B were exposed after each other for 47 and 42 days, respectively. As a result of the six and three infection periods during exposure, 94% and 81%, respectively of the seedling shoot tips in set A and set B became infected. However, due to ontogenetic resistance, disease incidence was low in both sets on leaves which were fully expanded at the beginning of exposure. Set C was exposed during both periods (89 days) in which sets A and B were exposed. Not only were all the seedling shoot tips in set C infected, but also — due to the loss of ontogenetic resistance — nearly all of the mature leaves. Ontogenetic resistance was also lost in set D, which was exposed for 57 days at the end of the growing season. The symptoms on fully expanded leaves on plants in sets C and D were typical and similar to those on young leaves. A time course symptom assessment was performed on leaves which had developed early in the season on labelled, field-grown shoots of cv. Golden Delicious trees. A steady increase of disease incidence was detected, which could not only have resulted from infections followed by extended incubation periods. In addition to the increase of typical lesions on both leaf surfaces, there was also an abundance of diffuse mycelial development on the lower surfaces of the leaves of the field-grown trees.     

Detection of fungi producing infection-inhibiting metabolites against <Emphasis Type="Italic">Alternaria alternata</Emphasis> 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.     

梨黑星病菌对氟硅唑的敏感性及与不同杀菌剂之间的交互抗性

采用孢子芽管长度法,测定了采自河北省不同地区的88个梨黑星病菌 Venturia nashicola 菌株对氟硅唑的敏感性,并测定了10个相对敏感菌株和5个相对抗性菌株对甲基硫菌灵、醚菌酯、福美双和苯醚甲环唑的敏感性。结果表明:不同地区黑星病菌对氟硅唑的敏感性存在一定差异,平均EC50值为(1.3821±0.9276)μg/mL;氟硅唑对未使用过麦角甾醇生物合成抑制剂(EBI)类杀菌剂的75个梨黑星病菌菌株的平均EC50值为(1.1676±0.6128)μg/mL,最大值是最小值的16.91倍,其敏感性分布频率呈连续性单峰曲线,且符合正态分布,故可作为敏感基线用于监测田间梨黑星病菌对氟硅唑的敏感性变化。不同菌株对5种药剂的敏感性相互关系分析表明,氟硅唑和苯醚甲环唑之间呈显著正相关,但与醚菌酯、甲基硫菌灵和福美双之间均不存在交互抗性关系。     

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.     

Collar rot caused byPhytophthora citrophthora on pear trees in Greece

A severe crown rot of pear trees of cultivar ‘Kondoula’ grafted on quince rootstock was observed in Greece. Isolations from the affected tissues repeatadly yielded aPhytophthora sp. that was determined by morphological and physiological characteristics to beP. citrophthora. The pathogenicity of two of theP. citrophthora isolates was tested by inoculating trunks of 2-year-old pear trees by mycelial agar disks. Thirty-two days after inoculation all inoculated trees were infected. Although the pear isolates could not be differentiated from isolates ofP. palmivora orP. nicotianae based on isozyme profiles of α-esterase or lactate dehydrogenase, RAPD profiles with one selected primer differentiated the pear isolates from the other species and revealed an electrophoretic banding pattern similar to that of aP. citrophthora standard. This is the first report ofP. citrophthora on pear trees in Greece.     

梨抗黑星病AFLP标记筛选

 Pear scab caused by Venturia nashicola is one of the most destructive diseases of pears. Molecular markers linked to scab resistance gene is expected to be useful for improving pear. In this study, the F₁ population derived from the cross of ‘Huangguan’ and ‘Yali’ was analyzed genetically. The resistance of pear to scab was proved to be controlled by a single gene in a dominant manner. Bulked segregant analysis (BSA) was conducted to screen 64 fluorescent AFLP primer pairs. A marker designated as D3-365 was found to be linked to the resistant locus. Selective genotype linkage analysis showed that the genetic distance between the marker and the resistant locus was 14.9 cM.     

天津市混栽梨园中不同品种梨树上中国梨喀木虱的发生规律

为明确梨木虱在不同品种梨树上的发生规律,于2015-2016年对天津市混栽梨园中3个品种梨树上中国梨喀木虱Cacopsylla chinensis的周年发生情况进行调查。结果表明,中国梨喀木虱冬型成虫在早酥梨树上的出现日期为2月底,在黄冠和晚秋黄梨树上的出现日期为3月上旬至中旬。3月中下旬始见第1代卵,4月上旬达高峰期,4月下旬孵化为若虫并达到高峰期,4月底至5月初始见第1代夏型成虫,5月上旬至下旬达高峰期;5月下旬第2代若虫孵化,6月初达高峰期,中旬成虫羽化,下旬成虫达高峰期;6月底至7月初第3代若虫陆续出现,7月上旬至下旬达高峰期,下旬成虫羽化;8月上旬第4代若虫出现,中下旬达高峰期;9月中旬第5代若虫出现,月底达高峰期。早酥梨树上中国梨喀木虱发生高峰期的数量显著高于黄冠和晚秋黄梨树上的数量,晚秋黄梨树上1、2代的发生量显著高于黄冠梨树上的发生量。     

Ultrastructural and cell wall modifications during infection of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis> roots by a pathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain

Fusarium species are soil-borne fungal pathogens that produce a variety of disease symptoms when attacking crop plants. The mode of root colonization of Eucalyptus viminalis seedlings by a pathogenic F. oxyporum strain (Foeu1) at the ultrastructural level and changes in cell wall pectin during host pathogen interactions are described. Root systems of E. viminalis plants were inoculated with F. oxysporum in an in vitro model system. Hyphae of F. oxysporum adhered to the outer epidermal cell walls through fibrillar material, and after penetration they spread into the internal tissues. They developed intercellularly and intracellularly in the root cortex and invaded vascular tissues. Papillae were induced, and the host plasma membrane ruptured in colonized cells, causing rapid host tissue and cell damage. Changes in distribution and occurrence of nonesterified and methyl-esterified pectins were evaluated after root colonization by F. oxysporum using two monoclonal antibodies, JIM 5 and JIM 7, respectively. Nonesterified pectin in control roots was mainly localized in the epidermal cell walls and middle lamellae in parenchymal cortex, whereas methyl-esterified pectin accumulated more in primary cell walls of the cortex and phloem. Decreases in immunodetected nonesterified and methyl-esterified pectins were associated with extensive plant tissue degradation after root colonization by the pathogenic fungus.     

Hot water treatment of Japanese pear trees is effective against white root rot caused by <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> Prillieux

Hot water was dripped into the rhizosphere of Japanese pear trees (Pyrus serotina Rehd. grafted on P. betulifolia Bunge.) infested with the white root rot fungus Rosellinia necatrix Prillieux, to destroy the fungus. Isolates of R. necatrix from diseased roots of Japanese pear were vulnerable to water at temperatures above 35°C, and the fungus was eradicated from the colonized substrate when water at 35°C was provided for 3 days. The time required to eradicate R. necatrix decreased exponentially with increasing temperature. Japanese pear trees tolerated a temperature of 45°C without reduction in vigor. Field experiments demonstrated the practical use of hot water drip irrigation (HWD). HWD at 50°C completely destroyed white root rot mycelia on diseased roots, and many rootlets grew after the treatment. HWD at this temperature caused no injury to the trees. HWD of diseased orchard trees was assessed in Takamori and Iida in southern Nagano, Japan. The fungus recurred in two of four trees 28 months after treatment in Takamori and in two of ten trees 16 months after treatment in Iida. The new mycelia emerged on thick roots deep within the soil. Although there is a possibility of recurrence, HWD treatment is a practical control measure for white root rot.     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

梨和苹果种质对阿太菌果腐病菌的抗性评价及其防治药剂筛选

为明确不同梨和苹果种质对阿太菌果腐病菌Athelia bombacina的抗性以及筛选防治其有效杀菌剂,采用离体菌丝块有伤接种方法对40份梨种质和154份苹果种质进行病斑直径测定,通过聚类分析法和平均病斑直径法对不同种质进行了抗病性分级,并利用菌丝生长速率法测定了13种常用杀菌剂对阿太菌果腐病菌的毒力。结果表明,根据聚类分析法和平均病斑直径法均可将梨和苹果种质划分为高抗、抗、中抗、感和高感5类。与平均病斑直径法相比,梨和苹果种质分别以欧式距离为14和10作为最佳聚类分割点时进行聚类分析能够更加科学地划分类别,从40份梨种质中筛选出金锤子梨1个高抗种质,仅占总数的2.50%,从154份苹果种质中筛选出垂丝海棠、莫斯科透明、新疆苹果、路边石、伏帅等32个高抗种质,占总数的22.73%。药剂试验结果表明,戊唑醇、腈菌唑、咯菌腈和噻呋酰胺对阿太菌果腐病菌菌丝生长的抑制效果较好,抑制中浓度EC_(50)分别为0.027、0.048、0.054和0.095 mg/L。表明梨和苹果种质均可被阿太菌果腐病菌侵染,但不同种质间抗病性差异明显,戊唑醇是采前防治阿太菌果腐病菌菌丝生长的最佳药剂。     

Identification of putative defense-related genes in Japanese pear against <Emphasis Type="Italic">Alternaria alternata</Emphasis> infection using suppression subtractive hybridization and expression analysis

The Japanese pear pathotype of Alternaria alternata, a toxin-dependent necrotrophic pathogen, causes black spot of Japanese pear by producing the host-specific AK-toxin. Pre-inoculation with nonpathogenic A. alternata or pretreatment with an elicitor prepared from A. alternata reduced disease symptoms caused by the pathogen. Salicylic acid- and jasmonic acid-dependent signaling pathways are not involved in the induced resistance to infection by the pathogen. The expression of multiple defense-related genes in Japanese pear leaves inoculated with nonpathogenic A. alternata was examined using suppression subtractive hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database as accessions DC993229–DC993535.     

Precise, simple screening for resistance in potato varieties to common scab using paper pots

Resistance to common scab pathogen Streptomyces turgidiscabies of seven potato varieties was compared in the field with a newly developed paper pot method. Seedlings raised in soil in paper pots containing inocula at 1 × 10³ to 10⁷cfu/g soil were transplanted into a scab-free field and grown for 3 months. The disease severity of the seven varieties in the field trials differed in iteration and from year to year, even though their resistance levels were approximately similar at the expected levels. With the paper pot method, the seven varieties had different resistance levels, which were almost completely consistent with the results of the field trials, at more than 1 × 10⁵cfu/g soil. Significant differences in disease severity between resistant and susceptible varieties were observed (P = 0.05) for 2 years, and the resistance level of the varieties was elucidated.     

A during-infection spray strategy using sulphur compounds,copper, silicon and a new formulation of potassium bicarbonate for primary scab control in organic apple production

In a field experiment conducted over two growing seasons, the effectiveness and phytotoxicity of inorganic fungicides such as sulphur, lime sulphur, copper, silicon and Armicarb (a new formulation of potassium bicarbonate) was compared with water for the control of primary apple scab infections in Belgium on high, medium and low scab-susceptible cultivars (cvs ‘Pinova’, ‘Pirouette’ and ‘Reinette des Capucins’, respectively). In order to drastically reduce the amount of fungicide applied in the orchard, two approaches were used: (1) a strategy involving spraying during the infection process, before fungal penetration and (2) a tunnel sprayer machine for treatment applications. Under field conditions highly favourable for disease, low rates of elemental sulphur (31.8 and 38.6 kg ha⁻¹ year⁻¹ in 2005 and 2006, respectively) combined with low rates of copper (2.1 kg ha⁻¹ year⁻¹ in both years) provided the best scab control and reduced scab severity on the fruits of cv. ‘Pinova’ by 97 and 98% compared with water control in 2005 and 2006, respectively. Lime sulphur was much more effective than wettable sulphur and appeared to be efficient at temperatures below 10°C, but its effectiveness against apple scab decreased if the treatments were applied 12–24 h later than in the ‘during-infection’ spray strategy. Armicarb used alone significantly reduced apple scab severity on the leaves and fruits of the three cultivars compared with the water control. Its effectiveness was as good as wettable sulphur applied using the same timing and dosage. Silicon reduced apple scab on fruits very slightly, but not on leaves. The amounts of wettable sulphur, lime sulphur, copper, silicon and potassium bicarbonate used in this experiment to control apple scab were not phytotoxic, did not increase fruit russet, did increase the yield of each cultivar and did not affect summer density of the beneficial Typhlodromus pyri. The potential and limitations of ‘during-infection’ spraying as a protection strategy against apple scab in organic farming are discussed.     

Induction of systemic resistance byPseudomonas fluorescens Pf1 againstXanthomonas oryzae pv.Oryzae in rice leaves

When lower leaves of rice plants were inoculated with powder formulation of a saprophytic strain ofPseudomonas fluorescens, Pfl, upper leaves, in addition to the inoculated lower leaves, showed resistance to the rice bacterial blight pathogenXanthomonas oryzae pv.oryzae. When the leaves were challenge-inoculated withX. oryzae pv.oryzae 4 days afterP. fluorescens application on lower leaves, the disease intensity in upper leaves decreased from 6.7 to 1.1. When rice seeds were treated with the formulation ofP. fluorescens Pfl and sown, 30-day-old seedlings showed resistance toX. oryzae pv.oryzae and the disease intensity decreased from 6.8 to 1.2. The induced resistance was transient; leaves sprayed withP. fluorescens Pfl at 30 days after treatment and leaves of 60-day-old seedlings fromP. fluorescens-treated seeds did not show resistance to the pathogen. In field trials, seed treatment followed by foliar application of the powder formulation ofP. fluorescens Pfl effectively controlled rice bacterial blight and increased the yield. In the induced resistant leaves a sharp increase in lignification and activities of peroxidase, phenylalanine ammonia-lyase and 4-coumarate: CoA ligase was observed when the leaves were challenge-inoculated withX. oryzae pv.oryzae. An approximately threefold increase in lignin content, peroxidase activity and phenylalanine ammonia-lyase activity and a fivefold increase in 4-coumarate: CoA ligase activity were observed 5 days after challenge inoculation withX. oryzae pv.oryzae in rice leaves pretreated withP. fluorescens for 5 days. A similar increase in defense-related activities was not observed in susceptible interactions or inP. fluorescens-treated plants at later stages of interactions when no resistance to the pathogen was observed.