Antifungal activity of ethanolic extract of Archu (Rheum emodi) on powdery mildew (Erysiphe cichoracearum) and its role in the induction of resistance in balsam (Impatiens balsamania)

Rheum emodi, vernacularly known as Archu, is one of the important high altitude medicinal plants widely distributed in Himalayan regions. Though widely used in Ayurveda for curing various human diseases, its use in plant diseases is limited. Ethanolic extract of Rheum rhizome was assayed against spore germination of Alternaria solani, Heliminthosporium penniseti and Curvularia palliscens. The inhibition of spore germination was concentration dependent. Maximum inhibition was obtained at 4000 and 5000 ppm followed by 3000, 2000 and 1000 ppm. However, the extract was highly effective in the pre-inoculation treatment against powdery mildew (Erysiphe cichoracearum) of balsam (Impatiens balsamania) under field conditions. High performance liquid chromatographic (HPLC) analysis of balsam leaves showed increased synthesis of phenolic acids, which has been correlated with induced resistance in inhibiting the disease intensity of balsam powdery mildew.     

Intraspecies differentiation in the powdery mildew Erysiphe cichoracearum determined with rDNA RFLPs

The powdery mildew species Erysiphe cichoracearum has a described host range of over 300 plant species from among several families. Host-range testing indicates host-specialized subdivision within this taxonomic species. However, the extent of subdivision remains largely undetermined among host-limited forms. We have characterized diversity among field collections of E. cichoracearum from a variety of hosts, and from other powdery mildew species, with RFLPs from a PCR amplified ribosomal DNA (rDNA) segment The E. cichoracearum samples expressed six distinct RFLP haplotypes. Each haplotype was specific to either a single host or to a set of related host species. These haplotypes formed a continuum of divergence ranging from about 18–35% average pairwise distance from one another, while those from other mildew species clustered at consistently higher average pairwise distances from E. cichoracearum and from each other. Our findings support earlier suggestions, based on host-range and morphological characterizations, that E. cichoracearum is a complex of morphologically similar, but host-limited forms. Also, comparisons of rDNA haplotype distance between E. cichoracearum and Blumeria (Erysiphe) graminis were consistently greater than between E. cichoracearum and Sphaerotheca fulginea. This result supports earlier questions concerning the monophyletic nature of Erysiphe.     

First record of dill powdery mildew caused by Erysiphe heraclei DC in Egypt

In Egypt, powdery mildew was observed for the first time on dill plants, during annual disease surveys of March–May 2003 and 2005. Typical symptoms of powdery mildew of dill plant (Anethum graveolens L.) were observed in Gharbeia Governorate. Symptoms of powdery mildew became common on leaves, stems inflorescences and fruits as white irregular areas. These symptoms appeared at vegetative and early flowering stages then gradually increased through fruiting and pre-maturity stages. Samples of infected leaflets, stem, inflorescences and fruits were collected for examination by light and scanning electron microscope (SEM). Microscopic examination revealed that conidiophores were short, erect–69 × 6–10 μm in dimension, conidia were observed without conspicuous fibrosin bodies singly, elliposid to ovoid 25–33 × 10–16 μm in dimension, and the length to width ratio of conidia ranged from 1.7 to 2.0 and were produced singly. Cylindrical foot cells (22.0 × 8.0 μm) were followed by one or two shorter cells (12.5 × 7.5 μm). In spring, the sexual stage (cleistothecia) appeared on infected leaves and stems in spherical, gregarious measures 105–117 (111) × 100– 87.5 μm in diameter. Each cleistothecium contained (2–4) round to ovoid asci, 45–55 (50) × 45–25 (35) μm in dimension. The ascus contained (3–4) ellipsoid to ovoid ascospores, 20–17.5 × 15–10 (13.2) μm. Cleistothecia appendages are simple myceloid branched tips measuring 80–200 (140) μm in length and 3–5 (4) μm in diameter. Based on the observations of the morphology of its anamorph and teleomorph stages, the causal agent of dill powdery mildew was identified as Erysiphe heraclei which is reported for the first time in Egypt.     

Ascocarp formation and survival and primary inoculum production in Erysiphe (sect. Microsphaera) pulchra in dogwood powdery mildew

Development of powdery mildew Erysiphe (sect. Microsphaera) pulchra in dogwood (Cornus florida) was assessed over a 5‐year period (1996–2000). Variations in the timing of initial infection, disease severity, ascocarp formation, and primary inoculum density were evaluated. Ascocarps formed late in the growing season (September‐November) when relatively low temperatures (< 27°C) persisted for at least 2 weeks, but ascocarp abundance was not influenced by disease severity. Studies conducted in a controlled environment showed that low temperatures triggered ascocarp formation and neither day length nor host plant age affected ascocarp formation. Ascocarps formed within 12–14 days at 18°C/ 10°C (day/night) and 23°C/15°C, but required 25 days at 26°C/18°C; no ascocarps formed at 28°C/ 20°C. Because ascocarps are an important source of primary inoculum for dogwood powdery mildew, ascocarp survival was evaluated in a 2‐year study (1998–2000). 60–80% of mature, dark‐coloured ascocarps survived at ‐10°C and ‐20°C and maintained viable spores for 4 months, but only 4–12% of partially developed, light brown ascocarps survived at ‐10°C and ‐20°C in the first experiment and only 30–40% survived in the second experiment. Immature ascocarp initials (cream‐yellow in colour) withered and disintegrated at all temperatures (24°C/20°C, 4°C, ‐10°C, and ‐20°C). Because ascocarps need time to mature, the timing of ascocarp initiation affects ascocarp maturity and thus winter survival and primary inoculum density. The evaluation of spring inoculum dispersal to spore traps and trap plants in 1999 and 2000 showed that rainfall patterns in early spring influenced primary inoculum and thus the timing of initial infection.     

Loss of actin cytoskeletal function and EDS1 activity,in combination,severely compromises non-host resistance in Arabidopsis against wheat powdery mildew

Plant immunity against the majority of the microbial pathogens is conveyed by a phenomenon known as non-host resistance (NHR). This defence mechanism affords durable protection to plant species against given species of phytopathogens. We investigated the genetic basis of NHR in Arabidopsis against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt). Both primary and appressorial germ tubes were produced from individual Bgt conidia on the surface of the Arabidopsis leaves. Attempted infection occasionally resulted in successful penetration, which led to the development of an abnormal unilateral haustorium. Inoculation of a series of Arabidopsis defence-related mutants with Bgt resulted in the attenuation of reactive oxygen intermediate (ROI) production and salicylic acid (SA)-dependent defence gene expression in eds1, pad4 and nahG plants, which are known to be defective in some aspects of host resistance. Furthermore, Bgt often developed bilateral haustoria in the mutant Arabidopsis lines that closely resembled those formed in wheat. A similar decrease in NHR was observed following treatment of the wild-type Arabidopsis plants with cytochalasin E, an inhibitor of actin microfilament polymerisation. In eds1 mutants, inhibition of actin polymerisation severely compromised NHR in Arabidopsis against Bgt. This permitted completion of the Bgt infection cycle on these plants. Therefore, actin cytoskeletal function and EDS1 activity, in combination, are major contributors to NHR in Arabidopsis against wheat powdery mildew.     

1种小麦白粉病菌DNA基因组的微量简捷提取方法

为了寻找适合小麦白粉菌基因组DNA微量提取的方法,分别采用改进破壁法,液氮研磨法和溶菌酶消化法进行破壁,提取专性寄生菌小麦白粉菌DNA。结果表明,用改进的破壁方法,仅用3~10 mg的分生孢子粉所获得DNA的收率为（12.23±3.46）~（40.32±5.67）ng/mg,且OD260/OD280比值为1.71~1.92之间,说明该破壁方法获得的DNA收率大且纯度高。通过PCR反应获得了良好的效果。同时该方法也适用于小麦条锈菌和大麦白粉菌专性寄生菌DNA的提取。     

Management of cucumber powdery mildew by certain biological control agents (BCAs) and resistance inducing chemicals (RICs)

The efficiency of some commercial biological control agents (BCAs), Bio Zaid and Bio Arc, and resistance inducers chemicals (RICs) (salicylic acid and Bion) on management of cucumber powdery mildew, caused by Sphaerotheca fuliginea, was evaluated in vitro and in vivo. In vitro experiments revealed that application of the tested BCAs and RICs significantly reduced the disease severity, number of conidia per leaf area unit (cm) and reduced germination of conidiospores of the causal pathogen. Under field conditions, application of BCAs and RICs caused significant decrement in the disease severity with significant increment in the fruit yield compared with check treatments. On the average, application of Bion recorded the highest values, either in reducing the disease severity or in raising the average fruit yield followed by Bio Arc. On the other hand, applying salicylic acid (SA) recorded, on the average, the lowest efficiency in reducing disease severity and low values of fruit yield in both growing seasons.     

Identification of powdery mildew (Erysiphe graminis sp. tritici) and take-all disease (Gaeumannomyces graminis sp. tritici) in wheat (Triticum aestivum L.) by means of leaf reflectance measurements

The ability to identify diseases in an early infection stage and to accurately quantify the severity of infection is crucial in plant disease assessment and management. A greenhouse study was conducted to assess changes in leaf spectral reflectance of wheat plants during infection by powdery mildew and take-all disease to evaluate leaf reflectance measurements as a tool to identify and quantify disease severity and to discriminate between different diseases. Wheat plants were inoculated under controlled conditions in different intensities either with powdery mildew or take-all. Leaf reflectance was measured with a digital imager (Leica S1 Pro, Leica, Germany) under controlled light conditions in various wavelength ranges covering the visible and the near-infrared spectra (380–1300 nm). Leaf scans were evaluated by means of L*a*b*-color system. Visual estimates of disease severity were made for each of the epidemics daily from the onset of visible symptoms to maximum disease severity. Reflectance within the ranges of 490₇₈₀ nm (r² = 0.69), 510₇₈₀nm (r² = 0.74), 516₁₃₀₀nm (r² = 0.62) and 540₁₃₀₀ nm (r² = 0.60) exhibited the strongest relationship with infection levels of both powdery mildew and take-all disease. Among the evaluated spectra the range of 490₇₈₀nm showed most sensitive response to damage caused by powdery mildew and take-all infestation. The results of this study indicated that disease detection and discrimination by means of reflectance measurements may be realized by the use of specific wavelength ranges. Further studies have to be carried out, to discriminate powdery mildew and take-all infection from other plant stress factors in order to develop suitable decision support systems for site-specific fungicide application.     

Involvement of lipid transfer proteins in resistance against a non-host powdery mildew in Arabidopsis thaliana

Non-specific lipid transfer proteins (LTPs) are involved in the transport of lipophilic compounds to the cuticular surface in epidermal cells and in the defence against pathogens. The role of glycophosphatidylinositol (GPI)-anchored LTPs (LTPGs) in resistance against non-host mildews in Arabidopsis thaliana was investigated using reverse genetics. Loss of either LTPG1, LTPG2, LTPG5 or LTPG6 increased the susceptibility to penetration of the epidermal cell wall by Blumeria graminis f. sp. hordei (Bgh). However, no impact on pre-penetration defence against another non-host mildew, Erysiphe pisi (Ep), was observed. LTPG1 was localized to papillae at the sites of Bgh penetration. This study shows that, in addition to the previously known functions, LTPGs contribute to pre-invasive defence against certain non-host powdery mildew pathogens.     

硅和白粉菌诱导接种对黄瓜幼苗白粉病抗性影响的研究

研究了硅酸盐和诱导接种白粉菌对黄瓜活性氧代谢、SiO2含量和抗病性的影响．结果表明,诱导接种能使叶片的超氧自由基(O2^-)产生速率、H2O2和丙二醛(MDA)含量升高,加硅接种处理的O2^-产生速率、H202和MDA含量明显低于不加硅接种处理．诱导接种能使叶片的过氧化氢酶(CAT)、过氧化物酶(POD)活性升高,超氧化物歧化酶(SOD)活性降低．加硅接种处理植株叶片的CAT、POD和SOD活性明显高于不加硅接种处理．诱导接种提高叶片的抗坏血酸(AsA)和还原型谷胱甘肽(GSH)含量,加硅处理的AsA含量明显低于不加硅处理,GSH含量高于不加硅处理．无论接种与否,加硅处理的SiO2含量显著高于不加硅处理,病情指数明显低于不加硅处理．     

Transgenic Pm3 multilines of wheat show increased powdery mildew resistance in the field

Resistance (R) genes protect plants very effectively from disease, but many of them are rapidly overcome when present in widely grown cultivars. To overcome this lack of durability, strategies that increase host resistance diversity have been proposed. Among them is the use of multilines composed of near-isogenic lines (NILs) containing different disease resistance genes. In contrast to classical R-gene introgression by recurrent backcrossing, a transgenic approach allows the development of lines with identical genetic background, differing only in a single R gene. We have used alleles of the resistance locus Pm3 in wheat, conferring race-specific resistance to wheat powdery mildew (Blumeria graminis f. sp. tritici), to develop transgenic wheat lines overexpressing Pm3a, Pm3c, Pm3d, Pm3f or Pm3g. In field experiments, all tested transgenic lines were significantly more resistant than their respective nontransformed sister lines. The resistance level of the transgenic Pm3 lines was determined mainly by the frequency of virulence to the particular Pm3 allele in the powdery mildew population, Pm3 expression levels and most likely also allele-specific properties. We created six two-way multilines by mixing seeds of the parental line Bobwhite and transgenic Pm3a, Pm3b and Pm3d lines. The Pm3 multilines were more resistant than their components when tested in the field. This demonstrates that the difference in a single R gene is sufficient to cause host-diversity effects and that multilines of transgenic Pm3 wheat lines represent a promising strategy for an effective and sustainable use of Pm3 alleles.     

Inheritance of powdery mildew (Erysiphe polygoni DC) resistance in mungbean (Vigna radiata L. Wilczek)

Detached mungbean (Vigna radiata L.Wilczek) leaves were inoculated with a conidial suspension of a local isolate (TI-1) of the powdery mildew pathogen (Erysiphe polygoni DC) under controlled environment conditions. Based on the latent period and severity of the infection, a rating scale of 0–5 was used to classify the host pathogen interactions. Reactions 0, 1 and 2 were considered resistant and referred to as R0, R1 and R2 while 3, 4 and 5 were classified as susceptible (S). RUM lines (resistant to powdery mildew) and their derivatives are crossed with several susceptible (reaction types 3–5) genotypes and the inheritance of the resistance was studied in the F₁, F₂ and F₃ generations. The results showed that powdery mildew resistance in mungbean is governed by two dominant genes designated as Pm-1 and Pm-2. When both Pm-1 and Pm-2 were present, an R0 reaction was observed after inoculation with TI-1. The resistant reaction was R1 when only Pm-1 was present and R2 in the presence of Pm-2. In the absence of both Pm-1 and Pm-2, susceptible reactions 3, 4 and 5 were observed.     

The isoelectric point of proteins influences their translocation to the extrahaustorial matrix of the barley powdery mildew fungus

Many biotrophic fungal plant pathogens develop feeding structures, haustoria, inside living plant cells, which are essential for their success. Extrahaustorial membranes (EHMs) surround haustoria and delimit the extrahaustorial matrices (EHMxs). Little is known about transport mechanisms across EHMs and what properties proteins and nutrients need in order to cross these membranes. To investigate this further, we expressed fluorescent proteins in the cytosol of infected barley leaf epidermal cells after particle bombardment and investigated properties that influenced their localisation in the powdery mildew EHMx. We showed that this translocation is favoured by a neutral isoelectric point (pI) between 6.0 and 8.4. However, for proteins larger than 50 kDa, pI alone does not explain their localisation, hinting towards a more complex interplay between pI, size, and sequence properties. We discuss the possibility that an EHM translocon is involved in protein uptake into the EHMx.     

Solute transport into healthy and powdery mildew-infected leaves of pea and uptake by powdery mildew mycelium

The transport of sugars and amino acids into the mycelium of Erysiphe pisi DC. was investigated using two different systems, intact leaf discs and mycelial suspensions. Of the sugars tested, glucose was preferentially taken up by both uninfected and mildew-infected leaf discs, whereas glutamine was taken up by both tissues at a higher rate than lysine or aspartic acid. Leaf discs from infected tissue had a greater uptake capacity than those from healthy tissue for both sugars and amino acids. The uptake of glucose was inhibited more markedly than that of sucrose and fructose by 10 μ m carbonyl cyanide m -chlorophenylhydrazone (CCCP), 1 m m N -ethylmaleimide (NEM), 1 m m diethyl pyrocarbonate (DEPC) and 1 m m phenylglyoxal, whereas 1 m m PCMBS ( p -chloro-mercuribenzenesulphonic acid) inhibited sucrose uptake to the greatest extent. Uptake of glutamine, lysine and aspartic acid was inhibited similarly by CCCP (80%), NEM (20%), DEPC (70%) and PCMBS (60%). Additionally, leaf discs were used to determine which solutes could be taken up from leaf tissue by the fungus. The uptake of sugars into the mycelium was greater than that of amino acids.
Suspensions of powdery mildew mycelium accumulated glucose at about three times the rate of sucrose or fructose, and the amino acid glutamine was taken up at three times the rate of lysine or aspartic acid. Spores separated from the suspension had a low uptake capacity.
When the reducing sugar concentration of leaf apoplastic fluid was estimated, leaves infected by powdery mildew had much higher amounts in the apoplast, whereas the activity of acid invertase also appeared to be higher in apoplastic fluids from infected leaves. When apoplastic fluid samples were run on SDS gels, an invertase antibody detected two bands in samples from infected tissues that were not found in the uninfected samples.     

Phylogenetic analysis and predicted secondary structures of the rDNA internal transcribed spacers of the powdery mildew fungi (Erysiphaceae)

The nucleotide sequences of the internal transcribed spacer (ITS) regions of the ribosomal DNA including the 5.8S rRNA gene and the 5′ end of the 28S rRNA gene have been determined for 19 species in 10 genera of the powdery mildew fungi in order to analyze their phylogenetic relationship. These fungi were divided into two large groups based on the nucleotide length of the ITS regions, and this grouping was in line with that based on the morphological characters of the anamorphic stage rather than the teleomorphic stage. Although the variable ITS sequences were often ambiguously aligned, conserved sites were also found. Thus, a neighbor-joining tree was constructed using the nucleotide sequence data of the conserved sites of the ITS regions, the 5.8S rRNA gene, and the 5′ end of the 28S rRNA gene. The phylogenetic tree displayed the presence of four groups in the powdery mildews, which were distinguished by their morphology and/or host ranges. In the ITS2 region, the presence of a common secondary structure having four hairpin domains was suggested, in spite of the highly variable nucleotide sequences of this region. The predicted secondary structure was supported by the compensatory mutations as well as compensatory conserved sequences and high G+C content in the predicted stem regions. Contribution No. 142 from the Laboratory of Plant Pathology, Mie University.     

De novo indol-3-ylmethyl glucosinolate biosynthesis,and not long-distance transport,contributes to defence of Arabidopsis against powdery mildew

Powdery mildew is a fungal disease that affects a wide range of plants and reduces crop yield worldwide. As obligate biotrophs, powdery mildew fungi manipulate living host cells to suppress defence responses and to obtain nutrients. Members of the plant order Brassicales produce indole glucosinolates that effectively protect them from attack by non-adapted fungi. Indol-3-ylmethyl glucosinolate is constitutively produced in the phloem and transported to epidermal cells for storage. Upon attack, indol-3-ylmethyl glucosinolate is activated by CYP81F2 to provide broad-spectrum defence against fungi. How de novo biosynthesis and transport contribute to defence of powdery mildew-attacked epidermal cells is unknown. Bioassays and glucosinolate analysis demonstrate that GTR glucosinolate transporters are not involved in antifungal defence. Using quantitative live-cell imaging of fluorophore-tagged markers, we show that accumulation of the glucosinolate biosynthetic enzymes CYP83B1 and SUR1 is induced in epidermal cells attacked by the non-adapted barley powdery mildew Blumeria graminis f.sp. hordei. By contrast, glucosinolate biosynthesis is attenuated during interaction with the virulent powdery mildew Golovinomyces orontii. Interestingly, SUR1 induction is delayed during the Golovinomyces orontii interaction. We conclude that epidermal de novo synthesis of indol-3-ylmethyl glucosinolate contributes to CYP81F2-mediated broad-spectrum antifungal resistance and that adapted powdery mildews may target this process.     

Inbreeding increases susceptibility to powdery mildew (Oidium neolycopersici) infestation in horsenettle (Solanum carolinense L)

Inbreeding is common in flowering plants, but relatively few studies have examined its effects on interactions between plants and other organisms, such as herbivores and pathogens. In a recent paper, we documented effects of inbreeding depression on plant volatile signaling phenotypes, including elevated constitutive volatile emissions (and consequently greater herbivore recruitment to inbred plants) but reduced emission of key herbivore-induced volatiles that attract predatory and parasitic insects to damaged plants. While the effects of inbreeding on plant-insect interactions have been explored in only a few systems, even less is known about its effects on plant-pathogen interactions. Here we report the effects of inbreeding on horsenettle susceptibility to powdery mildew (Oidium neolycopersici), including more rapid onset of infection in inbred plants, particularly when plants were not previously damaged. These data suggest that inbreeding may increase plant susceptibility to pathogen infection and, therefore, may potentially facilitate pathogen establishment in natural populations.     

QTL molecular marker location of powdery mildew resistance in cucumber (Cucumis sativus L.)

The cucumber lines, S94 (Northern China open-field type, powdery mildew (PM) susceptible) and S06 (European greenhouse type, PM resistant), and their F6:7 populations were used to investigate PM re-sistance under seedling spray inoculation in 2005/Autumn and 2006/Spring. QTL analysis was under-taken based on a constructed molecular linkage map of the corresponding F6 population using com-posite interval mapping. A total of four QTLs (pm1.1, pm2.1, pm4.1 and pm6.1) for PM resistance were identified and located on LG 1, 2, 4 and 6, respectively, explaining 5.2%-21.0% of the phenotypic variation. Three consistent QTLs (pm1.1, pm2.1 and pm4.1) were detected under the two test conditions. The QTL pm6.1 was only identified in 2005/Autumn. The total phenotypic variation explained by the QTLs was 52.0% and 42.0% in 2005/Autumn and 2006/Spring, respectively. Anchor markers tightly linked to those loci (<5 cM) could lay a basis for both molecular marker-assisted breeding and map-based gene cloning of the PM-resistance gene in cucumber.