A fungal powdery mildew pathogen induces extensive local and marginal systemic changes in the Arabidopsis thaliana microbiota

Powdery mildew is a foliar disease caused by epiphytically growing obligate biotrophic ascomycete fungi. How powdery mildew colonization affects host resident microbial communities locally and systemically remains poorly explored. We performed powdery mildew (Golovinomyces orontii) infection experiments with Arabidopsis thaliana grown in either natural soil or a gnotobiotic system and studied the influence of pathogen invasion into standing natural multi-kingdom or synthetic bacterial communities (SynComs). We found that after infection of soil-grown plants, G. orontii outcompeted numerous resident leaf-associated fungi while fungal community structure in roots remained unaltered. We further detected a significant shift in foliar but not root-associated bacterial communities in this setup. Pre-colonization of germ-free A. thaliana leaves with a bacterial leaf-derived SynCom, followed by G. orontii invasion, induced an overall similar shift in the foliar bacterial microbiota and minor changes in the root-associated bacterial assemblage. However, a standing root-derived SynCom in root samples remained robust against foliar infection with G. orontii. Although pathogen growth was unaffected by the leaf SynCom, fungal infection caused a twofold increase in leaf bacterial load. Our findings indicate that G. orontii infection affects mainly microbial communities in local plant tissue, possibly driven by pathogen-induced changes in source-sink relationships and host immune status.     

Inoculum production and long-term conservation methods for cucurbits and tomato powdery mildews

The behaviour of cucurbit powdery mildews (Podosphaera xanthii and Golovinomyces cichoracearum) and tomato powdery mildew (Oidium neolycopersici) infesting detached cotyledons of Lagenaria leucantha cv. ‘Minibottle’ was studied in order to develop an easy culture method for pure inoculum production. High spore production was found with a combination of mannitol (0.1 m), sucrose (0.02 m) and agar (8 g l⁻¹) in the cotyledon survival medium. Sporulation on cotyledons and viability of conidia were affected by the age of culture for the three species of powdery mildew tested. The age of cotyledons had also an impact of the spore production. This method was used to produce large amounts of inoculum for P. xanthii, G. cichoracearum and O. neolycopersici and enable the development of other species of powdery mildew like Leveillula taurica. Freezing conidia in liquid nitrogen enabled the long-term conservation of P. xanthii without any loss of virulence. The same method was unsuccessful with G. cichoracearum, and L. taurica and partly successful with O. neolycopersici.     

Distribution,Host Range and Disease Severity of Pseudoperonospora cubensis on Cucurbits in the Czech Republic

Cucurbit downy mildew, caused by Pseudoperonospora cubensis, is a major cucumber disease in the Czech Republic. Disease prevalence, host range and disease severity were evaluated from 2001 to 2009. The geographical distribution of P. cubensis was assessed on ca 80–100 locations per year in two main regions of the Czech Republic (central and southern Moravia, and eastern, northern and central Bohemia). Infection by P. cubensis was observed primarily on cucumber (Cucumis sativus) but only on the leaves. During the study, disease prevalence ranged from 66 to 100%. The majority of C. sativus crops were heavily infected at the end of the growing season (second half of August). Generally, P. cubensis was present at high or very high disease severity. The loss of foliage results in the reduction in the quality and quantity of marketable yield of fruit. Pseudoperonospora cubensis was widespread across the whole area of the Czech Republic studied. Very rarely, infection was recorded in muskmelon (Cucumis melo) and Cucurbita moschata. Of other pathogens, the most frequently recorded was the cucurbit powdery mildew (Golovinomyces cichoracearum and Podosphaera xanthii).     

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.     

New records of microcyclic conidiogenesis in some powdery mildew fungi

Microcyclic conidiogenesis (MC) was recently described in several species of powdery mildew fungi. This process, defined as the production of conidia on a fungal spore without any, or only a minimal, involvement of hyphal growth, was observed on powdery mildew conidia that have already germinated on host plant surfaces and have been attached to the epidermal cells. Most probably, MC contributes to a quick propagation of young powdery mildew colonies because new conidia are sometimes produced in a shorter time on microcyclic conidiophores than on the hyphae of the young mycelium. This article reports MC in Erysiphe necator ex grapevine, Podosphaera leucotricha ex apple, Golovinomyces orontii ex tobacco, and Neoerysiphe galeopsidis ex Lamium purpureum based on light and low-temperature scanning electron microscopic studies.     

Evaluation of biocontrol agents and potassium silicate for the management of powdery mildew of zucchini

Investigations were conducted under greenhouse and field conditions to evaluate the effects of potential biocontrol agents (BCAs) and soluble silicon (Si) on powdery mildew of zucchini caused by Podosphaera xanthii. Five BCAs were applied as foliar sprays to zucchini leaves and Si was drenched weekly into the rhizosphere of these plants.In the greenhouse, all BCAs provided significant control of powdery mildew with fungal isolates, reducing disease levels by up to 90%. Si alone reduced powdery mildew by as much as 35% and improved the efficacy of most of the biocontrol agents. Higher disease pressure reduced the efficacy of Si on powdery mildew but did not affect the performance of the BCAs. In the field, a disease reduction of 10–70% was achieved by BCAs and Si. Lower temperatures and high humidity ranges were suitable for optimal performances. The efficacy of the bacterial BCA, Serratia marcescens – B15 and silicon diminished at temperatures above 25 °C. The fungal BCAs (Clonostachys rosea – EH and Trichothecium roseum – H20) were better suited to higher temperatures (25–30 °C) and were tolerant of low RH values. Application of K₂SiO₂ to zucchini roots increased the level of Si in the leaves, which was responsible for suppression of the disease.     

瓜类白粉病的生物防治研究进展

白粉病是危害瓜类作物最为严重的一种气传病害,引起该病的病原菌为单囊壳白粉菌Podosphaera xanthii(synonym Podosphaera fusca)和二孢白粉菌Golovinomyces cichoracearum(synonym Erysiphe cichoracearum),其中对Podosphaera xanthii的报道较为常见。主要概述了瓜类白粉病病原菌的分类地位、病理特征和生物防治方面的研究进展,重点阐述了微生物源生防制剂和植物源生防制剂对瓜类白粉病的防治成果,并对当前研究与应用中存在的问题进行了探讨,为该病的深入研究和有效防治提供参考。     

Gaps and perspectives of pathotype and race determination in <Emphasis Type="Italic">Golovinomyces cichoracearum</Emphasis> and <Emphasis Type="Italic">Podosphaera xanthii</Emphasis>

Golovinomyces cichoracearum and Podosphaera xanthii (family Erysiphaceae) are the most important species causing cucurbit powdery mildew (CPM), a serious disease of field and greenhouse cucurbits. Both species are highly variable in their pathogenicity and virulence, as indicated by the existence of large number of different pathotypes and races. Various independent systems of CPM pathotype and race determinations and denominations are used worldwide. CPM pathotype identification is based on intergeneric and interspecific differences in host-CPM interactions. The most commonly used set of CPM pathotype differentials includes one genotype from four species representing three agriculturally important cucurbit genera plus two genotypes from a fifth species, melon Cucumis melo L. CPM races are characterized by specialization on different cultivars or lines of one host species and have, to date, been differentiated only on melon (C. melo L.). The most frequently used set of melon differentials includes 11 genotypes that can differentiate CPM races originating from melon and other cucurbits, e.g., cucumber, Cucurbita spp., and watermelon. In this paper, we critically review the current state, gaps, and perspectives in our understanding of pathogenicity variation in these two CPM pathogens at the pathotype and race levels.     

First report of powdery mildew caused by Podosphaera xanthii on Luffa acutangula in Odisha State,India

Abstract

Ridge gourd (Luffa acutangula) is an herbaceous perennial twining vine cultivated globally as vegetable and medicinal plant. During October to January in 2014 and 2015, 40% powdery mildew disease incidence was observed in different areas of Odisha state, India. The pathogenicity experiments confirmed the powdery mildew disease symptoms on artificially inoculated L. acutangula seedlings. Causal organism was identified as Podosphaera xanthii on the basis of morphological and molecular studies. This is the first report of powdery mildew disease on L. acutangula caused by P. xanthii.     

Simultaneous modification of three homoeologs of TaEDR1 by genome editing enhances powdery mildew resistance in wheat

Wheat (Triticum aestivum L.) incurs significant yield losses from powdery mildew, a major fungal disease caused by Blumeria graminis f. sp. tritici (Bgt). enhanced disease resistance1 (EDR1) plays a negative role in the defense response against powdery mildew in Arabidopsis thaliana; however, the edr1 mutant does not show constitutively activated defense responses. This makes EDR1 an ideal target for approaches using new genome‐editing tools to improve resistance to powdery mildew. We cloned TaEDR1 from hexaploid wheat and found high similarity among the three homoeologs of EDR1. Knock‐down of TaEDR1 by virus‐induced gene silencing or RNA interference enhanced resistance to powdery mildew, indicating that TaEDR1 negatively regulates powdery mildew resistance in wheat. We used CRISPR/Cas9 technology to generate Taedr1 wheat plants by simultaneous modification of the three homoeologs of wheat EDR1. No off‐target mutations were detected in the Taedr1 mutant plants. The Taedr1 plants were resistant to powdery mildew and did not show mildew‐induced cell death. Our study represents the successful generation of a potentially valuable trait using genome‐editing technology in wheat and provides germplasm for disease resistance breeding.     

Isolation and evaluation of antagonistic bacteria towards the cucurbit powdery mildew fungus<Emphasis Type="Italic"> Podosphaera fusca</Emphasis>

Powdery mildew is one of the most important limiting factors for cucurbits production in Spain, its management being strongly dependent on chemicals. The aim of this work was to evaluate the possibility of exploiting antagonistic bacteria in the biological control of the cucurbit powdery mildew fungus Podosphaera fusca (syn. Sphaerotheca fusca). Among a collection of bacterial strains isolated from distinct cucurbit powdery mildew diseased plants and rhizospheric soils, four isolates were selected, by means of a screening method based on antibiotic production, and identified as Bacillus spp. These isolates proved to be efficacious in the control of cucurbit powdery mildew in in vitro detached leaves and seedling biocontrol assays, where reductions of disease severity of up to 80% were obtained. Furthermore, bacterial populations on melon leaves remained at similar levels (10⁵ cfu cm^–2) over the 16-day period studied and, as observed by scanning electron microscopy analysis, they were able to establish microcolonies associated with an extracellular matrix, which reveals that these isolates efficiently colonize melon phylloplane. These results indicate that the bacterial isolates selected are promising candidates for biological control agents of cucurbit powdery mildew in southern Spain.     

Transient transformation of Podosphaera xanthii by electroporation of conidia

Background

Powdery mildew diseases are a major phytosanitary issue causing important yield and economic losses in agronomic, horticultural and ornamental crops. Powdery mildew fungi are obligate biotrophic parasites unable to grow on culture media, a fact that has significantly limited their genetic manipulation. In this work, we report a protocol based on the electroporation of fungal conidia, for the transient transformation of Podosphaera fusca (synonym Podosphaera xanthii), the main causal agent of cucurbit powdery mildew.

Results

To introduce DNA into P. xanthii conidia, we applied two square-wave pulses of 1.7 kV for 1 ms with an interval of 5 s. We tested these conditions with several plasmids bearing as selective markers hygromycin B resistance (hph), carbendazim resistance (TUB2) or GFP (gfp) under control of endogenous regulatory elements from Aspergillus nidulans, Neurospora crassa or P. xanthii to drive their expression. An in planta selection procedure using the MBC fungicide carbendazim permitted the propagation of transformants onto zucchini cotyledons and avoided the phytotoxicity associated with hygromycin B.

Conclusion

This is the first report on the transformation of P. xanthii and the transformation of powdery mildew fungi using electroporation. Although the transformants are transient, this represents a feasible method for the genetic manipulation of this important group of plant pathogens.     

The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes

Plants employ multiple cell‐autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre‐invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane‐localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence‐related vesicle‐resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant–fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle‐mediated trafficking of RPW8.2–yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2–YFP within the EHM exceeds vesicle‐mediated replenishment of RPW8.2–YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre‐invasive defense at the cell periphery and post‐invasive defense at the EHM.     

The Podosphaera fusca TUB2 gene,a molecular “Swiss Army knife” with multiple applications in powdery mildew research

The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii) is the main causal agent of cucurbit powdery mildew and one of the most important limiting factors for cucurbit production worldwide. Despite the fungus' economic importance, very little is known about the physiological and molecular processes involved in P. fusca biology and pathogenesis. In this study, we isolated and characterised the β-tubulin-encoding gene of P. fusca (PfTUB2) to develop molecular tools with different applications in powdery mildew research. PfTUB2 is predicted to encode a protein of 447 amino acid residues. The coding region is interrupted by six introns that occur at approximately the same positions as the introns present in other fungal TUB2-like genes. Once cloned, the PfTUB2 sequence information was used in different applications. Our results showed that the TUB2 gene is a good marker for molecular phylogenetics in powdery mildew fungi but it is unsuitable for the analysis of intraspecific diversity in P. fusca. The expression of PfTUB2 was proven to be stable in different temperature conditions, supporting its use as a reference gene in quantitative gene expression studies. Furthermore, an allele-specific PCR assay for the detection of resistance to methyl-2-benzimidazole carbamate (MBC) fungicides in P. fusca was developed based on the correlation between the single amino acid change E198A in β-tubulin and the MBC resistance phenotype. Lastly, PfTUB2 was used as a target gene in the development of a high-throughput method to quantify fungal growth in plant tissues.     

Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic fungi

Understanding the mechanisms responsible for divergence and specialization of pathogens on different hosts is of fundamental importance, especially in the context of the emergence of new diseases via host shifts. Temporal isolation has been reported in a few plants and parasites, but is probably one of the least studied speciation processes. We studied whether temporal isolation could be responsible for the maintenance of genetic differentiation among sympatric populations of Ampelomyces, widespread intracellular mycoparasites of powdery mildew fungi, themselves plant pathogens. The timing of transmission of Ampelomyces depends on the life cycles of the powdery mildew species they parasitize. Internal transcribed spacer sequences and microsatellite markers showed that Ampelomyces populations found in apple powdery mildew (Podosphaera leucotricha) were genetically highly differentiated from other Ampelomyces populations sampled from several other powdery mildew species across Europe, infecting plant hosts other than apple. While P. leucotricha starts its life cycle early in spring, and the main apple powdery mildew epidemics occur before summer, the fungal hosts of the other Ampelomyces cause epidemics mainly in summer and autumn. When two powdery mildew species were experimentally exposed to Ampelomyces strains naturally occurring in P. leucotricha in spring, and to strains naturally present in other mycohost species in autumn, cross‐infections always occurred. Thus, the host‐related genetic differentiation in Ampelomyces cannot be explained by narrow physiological specialization, because Ampelomyces were able to infect powdery mildew species they were unlikely to have encountered in nature, but instead appears to result from temporal isolation.     

PMR5, an acetylation protein at the intersection of pectin biosynthesis and defense against fungal pathogens

Powdery mildew (Golovinomyces cichoracearum), one of the most prolific obligate biotrophic fungal pathogens worldwide, infects its host by penetrating the plant cell wall without activating the plant's innate immune system. The Arabidopsis mutant powdery mildew resistant 5 (pmr5) carries a mutation in a putative pectin acetyltransferase gene that confers enhanced resistance to powdery mildew. Here, we show that heterologously expressed PMR5 protein transfers acetyl groups from [¹⁴C]‐acetyl‐CoA to oligogalacturonides. Through site‐directed mutagenesis, we show that three amino acids within a highly conserved esterase domain in putative PMR5 orthologs are necessary for PMR5 function. A suppressor screen of mutagenized pmr5 seed selecting for increased powdery mildew susceptibility identified two previously characterized genes affecting the acetylation of plant cell wall polysaccharides, RWA2 and TBR. The rwa2 and tbr mutants also suppress powdery mildew disease resistance in pmr6, a mutant defective in a putative pectate lyase gene. Cell wall analysis of pmr5 and pmr6, and their rwa2 and tbr suppressor mutants, demonstrates minor shifts in cellulose and pectin composition. In direct contrast to their increased powdery mildew resistance, both pmr5 and pmr6 plants are highly susceptibile to multiple strains of the generalist necrotroph Botrytis cinerea, and have decreased camalexin production upon infection with B. cinerea. These results illustrate that cell wall composition is intimately connected to fungal disease resistance and outline a potential route for engineering powdery mildew resistance into susceptible crop species.     

Oleozon: A novel control strategy against powdery mildew in cucumber

Ozonized sunflower oil (oleozon) is an effective agent for controlling powdery mildew in cucumber. In this study, the mechanisms of oleozon in the control of powdery mildew were determined. The development of Podosphaera xanthii on cucumber leaves treated with oleozon (2%) and water was investigated at different times after inoculation. The germinating conidia, hyphae and conidiophores of the pathogen were severely damaged by oleozon. No visible phytotoxic effect was observed on cucumber after the application of oleozon. This compound had highly preventive effects as well as curative effects against powdery mildew based on in vivo potted seedling assays. The control effects of oleozon were further confirmed in a greenhouse trial. These results may provide a basis for further development of a natural fungicide against cucumber powdery mildew.     

Genetic linkage map of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.) and localization of a major QTL for powdery mildew resistance

Powdery mildew caused by Podosphaera xanthii has become a major problem in melon since it occurs all year round irrespective of the growing system. The TGR-1551 melon genotype was found to be resistant to several melon diseases, among them powdery mildew. However, the corresponding resistance genes have been never mapped. We constructed an integrated genetic linkage map using an F2 population derived from a cross between the multi-resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’. The map spans 1,284.9 cM, with an average distance of 3.6 cM among markers, and consists of 354 loci (188 AFLP, 39 RAPD, 111 SSR, 14 SCAR/CAPS/dCAPS, and two phenotypic traits) distributed in 14 linkage groups. QTL analysis identified one major QTL (Pm-R) on LG V for resistance to races 1, 2, and 5 of powdery mildew. The PM4-CAPS marker is closely linked to the Pm-R QTL at a genetic distance of 1.9 cM, and the PM3-CAPS marker is located within the support interval of this QTL. These codominant markers, together with the map information reported here, could be used for melon breeding, and particularly for genotyping selection of resistance to powdery mildew in this vegetable crop species.     

Molecular phylogenetic status of korean strain of Podosphaera xanthii, a causal pathogen of powdery mildew on Japanese thistle (Cirsium japonicum) in Korea

Powdery mildew diseases are sensitive to climate change and spread can be favored by increased temperature and low moisture. During 2011 to 2012, a powdery mildew disease by a Podosphaera species was observed on the leaves of Japanese thistle (Cirsium japonicum) in Korea. The initial sign of this disease included scattered superficial white mycelia on leaves. As the disease progressed, abundant necrotic black spots exhibiting chasmothecia were formed on the leaves. rDNA ITS and 28S homologies of the fungus (EML-CSPW1) showed 100% identity values with those regions from many strains of P. xanthii (syn. P. fusca) via NCBI BLASTN search.