Glycosylation and oxidative dimerization of resveratrol are respectively associated to sensitivity and resistance of grapevine cultivars to downy mildew

Levels of resistance to Plasmopara viticola, from susceptible to highly resistant, of different grapevine cultivars were observed in vineyards and confirmed by the symptoms developed after inoculations. On the abaxial surface of infected leaves, P. viticola developed abundant sporangiophores on susceptible cultivars (Chasselas, Gamay, Gamaret and Pinot Noir), whereas on less susceptible cultivars (Seyval Blanc and Johanniter) the parasite produced few sporangiophores and some necrotic spots at the site of infection. On resistant cultivars (Bronner, Solaris, IRAC 2091), P. viticola induced a hypersensitive response and only necrotic spots were visible and the disease ceased to develop. Stilbenes were analyzed 4, 7, 24, 48 h post-infection (hpi) on small leaf samples cut from the site of infection. Large differences were observed between the cultivars at 24 and 48 hpi. Susceptible grapevines produced resveratrol and its glycoside, piceide. In contrast, resistant plants produced high concentration of ε- and δ-viniferin. Resveratrol and piceide have little or no toxicity activity against P. viticola, whereas δ-viniferin is highly toxic and can be considered an important marker for resistance of grapevine to downy mildew. The importance of oxidative dimerization of resveratrol in comparison to the extent of its glycosylation in defense reaction of grapevines against P. viticola is discussed.     

Ultrastructural study on scab resistance expressed in epidermal pectin layers of pear leaves

Proliferation and collapse of subcuticular hyphae of Venturia nashicola race 1 were studied ultrastructurally, after inoculation of susceptible Japanese pear cv. Kousui, resistant Japanese pear cv. Kinchaku, resistant Asian pear strain Mamenashi 12 and nonhost European pear cv. Flemish Beauty leaves, to understand the nature of the resistance mechanism. After cuticle penetration by the pathogen, the hyphae were observed at lower frequency in epidermal pectin layers and middle lamellae of leaves of the three resistant plants than in those of susceptible ones. This result suggested that fungal growth was suppressed in the incompatible interaction between pear and V. nashicola race 1. In the pectin layers of all inoculated plants, some hyphae had modifications such as breaks in the plasmalemma with plasmolysis, necrotic cytoplasm and degraded cell walls. More hyphae had collapsed in the leaves of the three resistant plants than in those of the susceptible cv. Kousui. In collapsed hyphae, the polymerized cell walls broke into numerous fibrous and amorphous pieces, showing that the scab resistance might be associated with cell wall-degrading enzymes from pear plants.     

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.     

46份野生葡萄株系对霜霉病的抗性鉴定及其相关基因的表达

为明确不同野生葡萄株系对霜霉病的抗性差异,以18个种的46份野生葡萄株系为试材,采用叶盘法鉴定其对霜霉病的抗性,并利用实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT-qPCR)技术对部分关键基因进行定量分析,探讨其在不同抗病株系中的表达模式差异。结果表明,46份野生葡萄株系的病情指数为0～34.72,其中17份为感病株系,病情指数在25.93～34.72;21份为抗病株系,病情指数在5.32～24.35;5份圆叶葡萄株系均表现为免疫,病情指数为0.00;云南-元谋2、云南-2和木扎岭-3为高抗株系,病情指数分别为1.81、4.40和1.62。当被霜霉病菌侵染后,抗病株系和感病株系中的PAL、PR1、TLP和NPR1基因的诱导表达模式不同;与感病株系相比,抗病株系中的TLP、PR1和NPR1基因有强烈的诱导表达,PAL基因在感病株系比在抗病株系中表达量高。在免疫株系普莱德和高抗株系云南-元谋2中,NPR1与其它3个基因的表达模式差异最大;TLP在抗病株系蘡薁-林县中与其它3个基因的表达模式差异最大;在感病株系秋-嵩县中,NPR1与TLP表达模式相近,PAL和PR1表达模式相近。研究表明,在中国野生葡萄种质中,云南-元谋2、云南-2和木扎岭-3对霜霉病有良好的抗性,可作为抗病育种的原始材料;抗性基因可能在抗病株系中发挥着重要作用。     

First report of oospore formation in Plasmopara viticola,the causal agent of grapevine downy mildew,in highland regions of southern Brazil

This work evaluates the formation of oospores of Plasmopara viticola, the causal agent of grape downy mildew (DM), in highland regions in southern Brazil. Leaves of susceptible and resistant grape genotypes naturally infected with the pathogen were collected in the autumn of 2017, 2018, and 2019 from vineyards located in the highlands of Santa Catarina state. Leaf tissues were evaluated by light microscopy and scanning electron microscopy. Oospores of P. viticola were identified in both susceptible and resistant host genotypes. They were concentrated in the central regions of the DM lesions, close to the leaf veins, and exhibited a rounded shape, yellowish colour, thick wall, and a diameter ranging from 16.28 to 49.15 µm. The formation of oospores is strong evidence that sexual reproduction is occurring in P. viticola in the climatic conditions of the highlands of southern Brazil. Sexual reproduction contributes to the maximization of genetic diversity via meiosis. Populations with high genetic variability are more likely to break resistance mechanisms conferred by resistance genes and to develop resistance to fungicides applied for disease control. To our knowledge, this is the first scientific study to prove the formation of P. viticola oospores in Brazil. The results presented provide a solid basis for further studies on sexual recombination in P. viticola. Genetic improvement programmes for grapevines, disease management methods, and disease prediction models need to consider the sexual reproduction of this pathogen, otherwise their effectiveness may be compromised.     

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.     

Cytology of Cork Layer Formation of Citrus and Limited Growth of Elsinoe fawcettii in Scab Lesions

Ultrastructural aspects of host–parasite interactions were investigated in fruits and leaves of citrus (satsuma mandarin) infected with Elsinoe fawcettii. Fungal infection induced host tissues to form cork layers bordering the necrotic areas below the infected sites. The cork layers were composed of compact host cells with convoluted cell walls and alternating lamellations, indicating ligno–suberized tissues in the wound periderm. No host tissues below the cork layers were invaded by hyphae. Hyphae grew intercellularly and intracellularly, often causing hypertrophy and compartmentalization of infected host cells. Also, host cells adjacent to invading hyphae showed accumulation of electron-dense materials and the formation of host cell wall protuberances in intercellular spaces. Hyphae had concentric bodies that showed an electron-transparent core surrounded by an electron-dense layer with radiating filamentous structures on their surface. One or more intrahyphal hyphae were found in the cytoplasm of intercellular or intracellular hyphae. These results suggest that the ligno–suberized cork layers in the wound periderm of citrus act as a protective barrier, which leads to restricted growth of E. fawcettii in bordered scab lesions. The fungus is thought to form concentric bodies and intrahyphal hyphae as a survival mechanism against the water- and nutrient-deficient environments that occur in the cork layers of necrotic host parts.     

葡萄叶提取物对葡萄霜霉病的防治作用及有效成分分析

为探究葡萄叶提取物对葡萄霜霉病的防治作用,测定了葡萄叶提取物及其有效物质对葡萄生单轴霉Plasmopara viticola孢子囊和游动孢子的抑制作用以及对葡萄霜霉病的防治效果,观察了其对葡萄生单轴霉在叶片中扩展、产生孢子囊能力的影响,并对葡萄叶提取物组成成分进行了分析。结果表明,葡萄叶提取物对葡萄生单轴霉孢子囊的形成和萌发均有明显的抑制作用。当葡萄叶提取物浓度为10 mg/mL时,其对孢子囊形成的抑制率为82.4%,葡萄生单轴霉孢子囊和游动孢子的萌发率仅为7.6%和1.1%,均显著低于无菌水处理。荧光显微观察发现,葡萄叶提取物可以抑制葡萄生单轴霉在叶片内的扩展。当葡萄叶提取物浓度为10 mg/mL和50 mg/mL时,对葡萄霜霉病的室内防治效果分别为80.1%和100.0%;且浓度为10 mg/mL的葡萄叶提取物对葡萄霜霉病的田间预防效果为67.8%,与烯酰吗啉处理无显著差异。液质联用技术检测显示,葡萄叶提取物中含有白藜芦醇和紫檀芪,其对葡萄生单轴霉均有显著的抑制作用,且紫檀芪的抑制效果强于白藜芦醇。     

Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations

BACKGROUND: The occurrence of carboxylic acid amide (CAA)‐fungicide‐resistant Plasmopara viticola populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a method, which utilises PCR‐RFLP, for the rapid detection of resistance to the CAA fungicide mandipropamid in P. viticola populations. With this method, a glycine‐to‐serine substitution at codon 1105 of the cellulose synthase gene PvCesA3 of CAA‐fungicide‐resistant P. viticola was easily detected, although no resistant P. viticola was detected from 398 isolates in Japan. CONCLUSION: It is proposed that the PCR‐RFLP method is a reliable tool for the rapid detection of CAA‐fungicide‐resistant P. viticola isolates. Only 4 h was required from the sampling of symptoms to the phenotyping of fungicide resistance. Copyright © 2011 Society of Chemical Industry     

Effect of plant roots on the germination of microsclerotia ofVerticillum dahliae

Root observation boxes were used to study the effects of hosts and non-hosts on the germination of microsclerotia ofV. dahliae. The effects of roots on microsclerotia were examined within a radius of 1 mm around the root tip. Host plants such as potato and field bean induced a higher percentage of germination of the microsclerotia than a non-host such as barley. A susceptible potato cultivar stimulated germination more than a resistant cultivar. The germination percentage and the number of hyphae per microsclerotium decreased with distance from the root surface regardless of the plant species or cultivar.     

Histochemical and Cytochemical Investigations of Phenols in Roots of Banana Infected by the Burrowing Nematode Radopholus similis

ABSTRACT The burrowing nematode Radopholus similis is one of the most damaging pathogens on banana plantations. The role of phenolics in plant defense responses to the nematode was histochemically and ultrastructurally investigated in susceptible and partially resistant cultivars. Histochemical observations of healthy roots revealed that high levels of lignin, flavonoids, dopamine, cafeic esters, and ferulic acids were associated with a very low rate of nematode root penetration in the resistant cultivar. The presence of lignified and suberized layers in endodermal cells contributed to limit invasion of the vascular bundle by the pathogen. After infection, flavonoids were seen to accumulate early in walls of cells close to the nematode-migrating channel in both cultivars and in all tissues of the infected resistant roots including the vascular tissues. The labeling pattern obtained with the gold-complexed laccase and with anti-pectin monoclonal antibodies showed that phenolics were distributed in a loosened pectin-rich material surrounding the nematode. This study provides indications that constitutive phenolics in banana roots are associated with the limitation of host penetration and colonization by R. similis. Accumulation of flavonoids in response to infection was detected in the vascular tissues of susceptible plants and in all root tissues in the partially resistant plants.     

Host genotype- and temperature-dependent colonizationof In vitro carnation callus cultures byFusarium oxysporum f.sp.dianthi race 2

Differentin vivo resistance/susceptibility levels of 14 carnation cultivars toFusarium oxysporum f.sp.dianthi race 2, the causal agent of Fusarium wilt disease of carnation, were also expressed in anin vitro system and assayed as the degree of fungal colonization of callus cultures at 20° C. Temperature influenced thein vitro expression of carnation resistance. An incubation temperature of 27° C increased the colonization of calli derived from both the susceptible (‘Corrida’ and ‘Ambra’) and the resistant (‘Pulcino’ and ‘Pallas’) cultivars. At 15°C, the colonization of calli derived from Pulcino and Pallas diminished significantly more than for Ambra and Corrida. Inhibition of fungal growth on resistant calli was correlated to retardation in hyphal development. Both scanning electron microscopy and light microscopy observations showed that hyphae did not penetrate into carnation cells.     

Biological destruction of conidia ofVerticillium biguttatum

Because biological control ofRhizoctonia solani in potato with conidial suspensions of the mycoparasiteVerticillium biguttatum was often less successful in sandy soils than in loamy soils, we examined soils of potato fields for the presence of organisms destructive to conidia ofV. biguttatum.Representatives of conidiophagous testate amoebae were frequently present on sclerotium disks ofR. solani infected withV. biguttatum in all soils studied and were most active under moist conditions. Conidiophagous naked amoebae were also numerous, except for two loam soils, and were not sensitive to moist conditions. Conidiophagous ciliates were found in rather low numbers and were most frequently isolated from coarsely structured soils under moist conditions. Conidiophagous flagellates were very infrequently observed.A bacterial type, parasitizing and killing conidia and hyphae ofV. biguttatum, was observed in all soils studied. It produced clusters of cocci fixed to the outside of conidia and hyphae and was most active under moist soil conditions.The possible role of protozoan predators and bacterial parasites in the biological control ofR. solani in potato withV. biguttatum applied at planting is discussed.     

Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan

BACKGROUND: The increasing occurrence of QoI fungicide resistance in Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew. In Japan, the existence of QoI‐fungicide‐resistant P. viticola was reported in 2009. RESULTS: The QoI fungicide resistance in P. viticola samples collected from vineyards in Japan in 2008 and 2009 was monitored. Resistant P. viticola were detected in the regions where QoI fungicides have been introduced in accordance with the pest management programme, whereas in Hokkaido vineyards, where QoI fungicides have not yet been introduced, QoI‐fungicide‐resistant P. viticola were not found. CONCLUSION: Japan comprises thousands of islands and is physically isolated from other countries by the sea. Monitoring the emergence, incidence and distribution of QoI fungicide resistance in P. viticola populations in Japan is necessary to improve pest management strategies for downy mildew disease in Japanese vineyards. Copyright © 2010 Society of Chemical Industry     

Electron microscopic studies on the root hairs and cortex of a susceptible and a resistant variety of Brassica campestris infected with Plasmodiophora brassicae

The cortex of the roots of a susceptible and a resistant variety ofBrassica campestris var.rapa infected with sterile resting spores ofPlasmodiophora brassicae from senescent callus was studied at a stage prior to disease symptom development. Electron micrographs show the presence of amoeboid structures within the cortical cells of the susceptible variety 10 days after inoculation. Cell wall perforations, hypertrophied host cell nuclei, nucleoli and broken tonoplasts were frequently found in the susceptible variety. It has been concluded that amoeboid structures of the parasite penetrate the cell wall and disrupt the cortical cells.Electron micrographs of the resistant variety show the presence of zoosporangia with secondary zoospores in the root hairs nine days after inoculation. Two to four days later a large number of dead host cells can be observed in the outer cortical layer of the resistant variety, whereas no apparent changes are found in the inner cortex. The results suggest the occurrence of a hypersensitive host reaction which terminates further growth ofPlasmodiophora brassicae.Samenvatting Kiemplanten van een vatbare en een resistente variëteit vanBrassica campestris var.rapa werden geïnoculeerd met een suspensie van rustsporen vanPlasmodiophora brassicae. De rustsporen waren afkomstig uit verouderd ziek callusweefsel. Elektronenmicroscopisch onderzoek werd verricht aan de cortex van de hoofdwortel van vatbare planten in de periode die vooraf gaat aan de ontwikkeling van ziektesymptomen (10–13 dagen na inoculatie). Tien dagen na inoculatie werden amoebe-achtige structuren in de cortex gevonden (Fig. 1–4). Herhaaldelijk werden gaten in de celwand van de cortex gevonden (Fig. 5). De waardplantkern en kernlichaampje vertoonden een hypertrofische reactie terwijl de tonoplast vaak kapot was (Fig. 3). Deze resultaten vormen een aanwijzing dat de parasiet in de vorm van een amoebe door de celwand dringt en de celinhoud van de cortex verstoort. Dertien dagen na inoculatie werden de eerste secondaire plasmodiën in het binnenste gedeelte van de cortex of in de mergstraalcellen van de hoofdwortel gevonden (Fig. 6). Vanaf dat moment traden de typische knolvoetsymptomen op.Uit elektronen-microscopisch onderzoek van de resistente variëteit bleek dat negen dagen na inoculatie zoösporangien en secondaire zoösporen aanwezig waren in de wortelharen van de zijwortels (Fig. 7–9). Twee tot vier dagen later werden een groot aantal dode waardplantcellen in de buitenste cortexcellen van de hoofdwortel gevonden, terwijl het binnenste gedeelte van de cortex geen veranderingen vertoonde (Fig. 10 en 11). Deze resultaten wijzen er op dat in de resistente variëteit een overgevoeligheidsreactie in de cortex opgetreden is waardoor verdere groei vanPlasmodiophora brassicae verhinderd werd.     

Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations

BACKGROUND: The increasing occurrence of Qo inhibitor (QoI)‐fungicide‐resistant Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a rapid method for detecting resistance to a QoI fungicide, azoxystrobin, in P. viticola populations using the nested PCR‐RFLP method. With this method, a glycine‐to‐alanine substitution was discovered at codon 143 in the cytochrome b gene of P. viticola populations found in Japan. CONCLUSION: It is proposed that the nested PCR‐RFLP method is a high‐speed, sensitive and reliable tool for detecting azoxystrobin‐resistant P. viticola populations. Copyright © 2009 Society of Chemical Industry     

葡萄霜霉病菌对甲霜灵抗药性治理及其田间抗药菌株遗传稳定性分析

 为了明确葡萄霜霉病菌对甲霜灵的田间抗药性水平发展态势,于轮换用药前后,采用叶盘漂浮法测定了河北、山西、河南3省葡萄主要种植区11个葡萄园试验地葡萄霜霉病菌对甲霜灵敏感性变化动态。结果表明:田间采集的葡萄霜霉病菌对甲霜灵抗药的菌株其抗药性可以稳定遗传;不同地区轮换用药后,葡萄霜霉病菌对甲霜灵的抗药水平变化态势因用药流程的不同而发生相应的变化。采用不同作用机制的杀菌剂轮换或混合用药进行葡萄霜霉病菌对甲霜灵的抗药性治理时,需制定合理的施药流程,并根据抗药性治理的效果不断完善治理措施。     

吡虫啉胁迫对褐飞虱共生解脂假丝酵母抗性及敏感菌株生长的影响

为明确吡虫啉对褐飞虱共生解脂假丝酵母Candida lipolytica抗性及敏感菌株生长的影响,通过菌株培养、菌落观察和菌丝镜检等方法,比较了褐飞虱共生解脂假丝酵母不同菌株在含不同质量浓度吡虫啉的固体培养基上的菌落数,以及在液体培养基中的生长量差异。结果发现:不同质量浓度吡虫啉对褐飞虱共生解脂假丝酵母敏感和抗性菌株菌落生长均有抑制作用,且吡虫啉浓度越高,抑制作用越强。经500、1 000和2 000 mg/L吡虫啉处理4 d后,敏感菌株的菌落数量分别为对照的46.61%、27.58%和6.25%,均与对照差异显著;500和1 000 mg/L处理组抗性菌株菌落数量与对照无显著差异,而2 000 mg/L处理组与对照差异显著。经吡虫啉处理后,敏感菌株假菌丝形态变得不规则,部分假菌丝不舒展、萎缩或弯曲、顶端出现膨大,酵母出现空泡等,且吡虫啉浓度越高,不规则程度越明显;抗性菌株的假菌丝形态也有类似变化,但与敏感菌株相比,其菌丝体不规则形态的比例明显下降。500 mg/L吡虫啉处理对敏感和抗性菌株的生长量及菌丝干重均无明显影响,1 000和2 000 mg/L吡虫啉对不同菌株前期生长的抑制作用明显,但对后期生长影响不明显。研究表明,吡虫啉对褐飞虱共生解脂假丝酵母抗性菌株生长的影响显著小于对敏感菌株的影响。     

Benzothiadiazole and BABA improve resistance to <Emphasis Type="Italic">Uromyces pisi</Emphasis> (Pers.) Wint. in <Emphasis Type="Italic">Pisum sativum</Emphasis> L. with an enhancement of enzymatic activities and total phenolic content

Benzothiadiazole (BTH) and DL-β-aminobutyric acid (BABA) induced systemic resistance was investigated in susceptible and resistant pea genotypes against Uromyces pisi. Resistance was characterized by reduced infection frequency mainly due to decreases in appressorium formation, stomatal penetration, growth of infection hyphae and haustorium formation. Changes in β-1,3-glucanase, chitinase, phenylalanine ammonia-lyase and peroxidase activities and in total phenolics content, demonstrate that U. pisi resistance is induced by BTH and BABA treatments at early and late stages of the fungal infection process, but that the chemicals operate via different mechanisms. In fact, our study showed that BTH treatment primed the activity of pathogenesis related-proteins such as β-1,3-glucanase, chitinase and peroxidase in both susceptible and resistant genotypes. On the other hand, BABA treatment did not increase the enzymatic activities in the studied genotypes, but significantly increased their total phenolic contents.