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.     

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.     

Long-term Preservation of Podosphaera fusca Using Silica Gel

Podosphaera fusca is the main causal agent of cucurbit powdery mildew in Spain and one of the most important limiting factors for cucurbit production worldwide. As an obligate biotrophic parasite, this fungus has been traditionally cultured and conserved by periodical transfers of conidia to fresh plant material. Here we describe a simple protocol for preservation of P. fusca isolates in absence of living tissue based on the dry spore, slow‐freezing technique, and demonstrate that storage of silica gel desiccated conidia at ?80°C is an efficient method for the long‐term preservation of the pathogen.     

The antagonistic strain Bacillus subtilis UMAF6639 also confers protection to melon plants against cucurbit powdery mildew by activation of jasmonate-and salicylic acid-dependent defence responses

Biological control of plant diseases has gained acceptance in recent years. Bacillus subtilis UMAF6639 is an antagonistic strain specifically selected for the efficient control of the cucurbit powdery mildew fungus Podosphaera fusca, which is a major threat to cucurbits worldwide. The antagonistic activity relies on the production of the antifungal compounds iturin and fengycin. In a previous study, we found that UMAF6639 was able to induce systemic resistance (ISR) in melon and provide additional protection against powdery mildew. In the present work, we further investigated in detail this second mechanism of biocontrol by UMAF6639. First, we examined the signalling pathways elicited by UMAF6639 in melon plants, as well as the defence mechanisms activated in response to P. fusca. Second, we analysed the role of the lipopeptides produced by UMAF6639 as potential determinants for ISR activation. Our results demonstrated that UMAF6639 confers protection against cucurbit powdery mildew by activation of jasmonate- and salicylic acid-dependent defence responses, which include the production of reactive oxygen species and cell wall reinforcement. We also showed that surfactin lipopeptide is a major determinant for stimulation of the immune response. These results reinforce the biotechnological potential of UMAF6639 as a biological control agent.     

Seasonal variations in the occurrence of Golovinomyces orontii and Podosphaera xanthii,causal agents of cucurbit powdery mildew in Northern Italy

Powdery mildew, caused by Golovinomyces orontii and Podosphaera xanthii, is a widespread disease that causes important losses in cucurbit production. To determine the aetiology and the epidemiology of cucurbit powdery mildew disease in the North of Italy, observations on the occurrence of the main disease‐causing fungal species were conducted during the 2010, 2011 and 2012 growing seasons. Samples of infected leaves of zucchini, melon and pumpkin plants, either from field or greenhouse crops, were collected every 15–18 days from May to September/October. To identify the fungal species, both morphological observations based on the asexual stage and molecular identifications by a Multiplex‐PCR reaction with species‐specific primers were performed. Climatic parameters of temperature and relative humidity were also monitored. Pearson's correlation coefficient and Principal Component Analysis showed a negative significant correlation between the two species, and a peculiar epidemiological behaviour was also observed: the earlier infections were caused by G. orontii, which was the predominant species till the end of June–middle of July. At this time, this species progressively decreased in frequency and was replaced by P. xanthii that became the main species infecting cucurbits till the end of the growing season. As the two species have different ecological requirements, these seasonal variations in the cucurbit powdery mildew species composition could possibly be explained by the influence of temperature and relative humidity on the pathogen epidemiology during the growing season but also by the different overwintering strategies adopted by the two species.     

Isolation and selection of plant growth-promoting rhizobacteria as inducers of systemic resistance in melon

Backgroud and aims

Powdery mildew elicited by Podosphaera fusca is an important threat to cucurbits. In order to find alternatives to the current use of chemicals, we examined the potential use of plant growth-promoting rhizobacteria (PGPR) for controlling the disease by induction of systemic resistance in the host plant.

Methods

A collection of Bacillus and Pseudomonas strains from different origins was studied, including strains isolated from roots of disease-free melon plants obtained from a greenhouse plagued by powdery mildew. The selection of best candidates was based on the evaluation of different traits commonly associated with PGPR, such as antifungal and siderophore production, swimming and swarming motilities, biofilm formation, auxin production and promotion of root development.

Results

Three Bacillus strains, B. subtilis UMAF6614 and UMAF6639 and B. cereus UMAF8564, and two Pseudomonas fluorescens strains, UMAF6031 and UMAF6033, were selected after ranking the strains using a nonparametric statistics test. Applied to melon seedlings, the selected strains were able to promote plant growth, increasing fresh weight up to 30%. Furthermore, these strains provided protection against powdery mildew and also against angular leaf spot caused by Pseudomonas syringae pv. lachrymans, with disease reductions of up to 60%.

Conclusions

These results suggest that the use of ISR-promoting PGPR could be a promising strategy for the integrated control of cucurbit powdery mildew and other cucurbit diseases.     

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。     

Cucurbit Powdery Mildew: First Insights for the Identification of the Causal Agent and Screening for Resistance of Squash Genotypes (Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir.) in Mendoza,Argentina

The cucurbit powdery mildew (CPM) caused by different fungal species is a major concern for cucurbit crops around the world. In Argentina CPM constitutes the most common and damaging disease for cucurbits, especially for squash crops (Cucurbita moschata). The present study displays initial insights into the knowledge of the disease in western Argentina, including the determination of the prevalent species causing CPM, as well as the evaluation of the resistance of squash cultivars and breeding lines. Fungal colonies were isolated from samples collected in Mendoza province, Argentina. A field trial was also performed to assess the resistance of five squash accessions, including commercial cultivars and breeding lines. The severity of CPM was analyzed and epidemiological models were built based on empirical data. The morphological determinations and analysis with specific molecular markers confirmed Podosphaera xanthi as the prevalent causal agent of CPM in Mendoza. The results od the field trial showed differences in the resistance trait among the squash accessions. The advanced breeding line BL717/1 showed promising results as source of CPM resistance for the future development of open pollinated resistant cultivars, a crucial tool for an integrative control of the disease.     

Synthesis and antifungal activity of two novel spermidine analogues

Two spermidine analogues were synthesised and examined for antifungal activity. Both compounds used as 1 mM post-inoculation sprays reduced infection of barley seedlings by the powdery mildew fungus, Erysiphe graminis f.sp. hordei, infection of broad bean seedlings by the rust fungus, Uromyces viciae-fabae, and infection of apple seedlings by the powdery mildew fungus, Podosphaera leucotricha. Since these fungal pathogens cannot be cultured axenically, the effects of the two spermidine analogues on mycelial growth in vitro, as well as preliminary investigations on polyamine biosynthesis, were undertaken using the oat stripe pathogen, Pyrenophora avenae. Although neither compound affected radial growth of the fungus on plates, both analogues reduced fungal biomass in liquid culture substantially. The two spermidine analogues, used at a concentration of 1 mM, had no significant effect on the conversion of labelled ornithine into polyamines in P. avenae.     

Mapping of novel powdery mildew resistance gene(s) from <Emphasis Type="Italic">Agropyron cristatum</Emphasis> chromosome 2P

Key message

A physical map of Agropyron cristatum 2P chromosome was constructed for the first time and the novel powdery mildew resistance gene(s) from chromosome 2P was(were) also mapped.

Abstract

Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), a wild relative of common wheat, is highly resistant to powdery mildew. Previous studies showed that wheat-A. cristatum 2P disomic addition line II-9-3 displayed high resistance to powdery mildew, and the resistance was attributable to A. cristatum chromosome 2P. To utilize and physically map the powdery mildew resistance gene(s), 15 wheat-A. cristatum 2P translocation lines and three A. cristatum 2P deletion lines with different chromosomal segment sizes, obtained from II-9-3 using ⁶⁰Co-γ ray irradiation, were characterized using cytogenetic and molecular marker analysis. A. cristatum 2P chromosomal segments in the translocations were translocated to different wheat chromosomes, including 1A, 4A, 5A, 6A, 7A, 1B, 2B, 3B, 7B, 3D, 4D, and 6D. A physical map of the 2P chromosome was constructed with 82 STS markers, consisting of nine bins with 34 markers on 2PS and eight bins with 48 markers on 2PL. The BC₁F₂ populations of seven wheat-A. cristatum 2P translocation lines (2PT-3, 2PT-4, 2PT-5, 2PT-6, 2PT-8, 2PT-9, and 2PT-10) were developed by self-pollination, tested with powdery mildew and genotyped with 2P-specific STS markers. From these results, the gene(s) conferring powdery mildew resistance was(were) located on 2PL bin FL 0.66–0.86 and 19 2P-specific markers were identified in this bin. Moreover, two new powdery mildew-resistant translocation lines (2PT-4 and 2PT-5) with small 2PL chromosome segments were obtained. The newly developed wheat lines with powdery mildew resistance and the closely linked molecular markers will be valuable for wheat disease breeding in the future.

     

A haustorial‐expressed lytic polysaccharide monooxygenase from the cucurbit powdery mildew pathogen Podosphaera xanthii contributes to the suppression of chitin‐triggered immunity

Podosphaera xanthii is the main causal agent of cucurbit powdery mildew and a limiting factor of crop productivity. The lifestyle of this fungus is determined by the development of specialized parasitic structures inside epidermal cells, termed haustoria, that are responsible for the acquisition of nutrients and the release of effectors. A typical function of fungal effectors is the manipulation of host immunity, for example the suppression of pathogen‐associated molecular pattern (PAMP)‐triggered immunity (PTI). Chitin is a major component of fungal cell walls, and chitin oligosaccharides are well‐known PAMP elicitors. In this work, we examined the role of PHEC27213, the most highly expressed, haustorium‐specific effector candidate of P. xanthii. According to different computational predictions, the protein folding of PHEC27213 was similar to that of lytic polysaccharide monooxygenases (LPMOs) and included a conserved histidine brace; however, PHEC27213 had low sequence similarity with LPMO proteins and displayed a putative chitin‐binding domain that was different from the canonical carbohydrate‐binding module. Binding and enzymatic assays demonstrated that PHEC27213 was able to bind and catalyse colloidal chitin, as well as chitooligosaccharides, acting as an LPMO. Furthermore, RNAi silencing experiments showed the potential of this protein to prevent the activation of chitin‐triggered immunity. Moreover, proteins with similar features were found in other haustorium‐forming fungal pathogens. Our results suggest that this protein is a new fungal LPMO that catalyses chitooligosaccharides, thus contributing to the suppression of plant immunity during haustorium development. To our knowledge, this is the first mechanism identified in the haustorium to suppress chitin signalling.     

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.     

The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits

Numerous vegetable crops are susceptible to powdery mildew, but cucurbits are arguably the group most severely affected. 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. Although great efforts have been invested in disease control, by contrast, many basic aspects of the biology of P. fusca remain unknown.
Taxonomy: Podosphaera fusca (Fr.) Braun & Shishkoff. Kingdom Fungi; Phylum Ascomycota; Subdivision Pezizomycotina; Class Leotiomycetes; Order Erysiphales; Family Erysiphaceae; genus Podosphaera ; species fusca.
Identification: Superficial persistent mycelium. Conidia in chains, hyaline, ellipsoid to ovoid or doliform, about 24–40 × 15–22 µm, with cylindrical or cone-shaped fibrosin bodies, which often germinate from a lateral face and produce a broad, clavate germ tube and cylindrical foot-cells. Unbranched erect conidiophores. Cleistothecia globose, mostly 70–100 µm in diameter, dark brown/black. One ascus per cleistothecium with eight ascospores.
Host range: Angiosperm species that include several families, such as Asteracea, Cucurbitaceae, Lamiaceae, Scrophulariaceae, Solanaceae and Verbenaceae.
Disease symptoms: White colonies develop on leaf surfaces, petioles and stems. Under favourable environmental conditions, the colonies coalesce and the host tissue becomes chlorotic and usually senesces early.
Control: Chemical control and the use of resistant cultivars. Resistance has been documented in populations of P. fusca to some of the chemicals registered for control.     

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.     

Molecular cloning,characterization and expression analysis of <Emphasis Type="Italic">Cm</Emphasis>APX

The RT PCR and RACE methods were used to obtain the cDNA sequence of an APX gene of muskmelon after the leaves were induced with powdery mildew. The cDNA length of the APX gene is 1,047 bp with a 750 bp ORF encoded a 249 amino acid and the molecular weight of APX protein is 27.3 kDa. The analysis showed that the CmAPX genomic DNA contained 10 extrons and 9 introns. The identity of the amino acid sequence deduced from the cDNA with the APX family of other homologous members was about 74–97%. A Full-length of ORF was sub-cloned into prokaryotic expression vector pET24a. The recombinant proteins had high expression level in E. coli. Analysis of expression at mRNA level showed that CmAPX exhibited highly tissue-specific patterns of expression. The mRNA level and enzyme activities assays showed that CmAPX might play an important role in the pathogenesis of powdery mildew.     

Characters of compost teas from different sources and their suppressive effect on fungal phytopathogens

Compost teas (CT) are fermented watery extracts of composted materials that are used to control plant diseases and on crop fertilization. In this work, aerated (ACT) and non-aerated compost teas (NCT) were obtained from four different composts: spent mushroom substrate compost, grape marc compost, greenhouse horticultural crop residues compost, and vermicompost. Physico-chemical and microbiological analysis were carried out to determine their properties. In vitro assays were performed to assess their suppressive effect on the mycelial growth of eight fungal phytopathogens. In vivo trials aimed to assess their effect on gummy stem blight (Didymella bryonae) and powdery mildew (Podosphaera fusca) in melon plants. Results showed that ACT and NCT filtrates inhibited the in vitro growth of all tested pathogens while autoclaved CT did not completely lose their inhibitory effect, and CT sterilized by microfiltration had no effect on the pathogen growth. The severity of powdery mildew was highly reduced by ACT and NCT from all sources, though in gummy stem blight assay only a delay in disease development was observed. In general, all compost teas showed a high level of microbial populations and nutrients. Results suggest that the efficacy of ACT and NCT firstly depend on the microbiota present in them. We consider compost teas from the four tested sources as a viable way to manage plant diseases and crop fertilization, throughout its integration in pest management programs and fertirrigation systems under different dilution rates.     

Effect of lipopeptides of antagonistic strains of Bacillus subtilis on the morphology and ultrastructure of the cucurbit fungal pathogen Podosphaera fusca

AIMS: To analyse the morphological and ultrastructural effects of lipopeptides of cell-free liquid cultures from the antagonistic Bacillus subtilis strains, UMAF6614 and UMAF6639, on the cucurbit powdery mildew fungus, Podosphaera fusca, conidial germination. METHODS AND RESULTS: Butanolic extracts from cell-free supernatants of B. subtilis cultures were tested for their ability to arrest P. fusca conidial germination using the zucchini cotyledon disc method. Previously, the occurrence of lipopeptide antibiotics fengycin, iturin/bacillomycin and surfactin in the extracts was verified by diverse chromatographic approaches. Conidial germination was strongly reduced by antifungal extracts obtained from liquid cultures of both B. subtilis strains. Scanning electron microscopy analysis showed morphological damage in conidia characterized by the presence of large depressions and loss of turgidness. Transmission electron microscopy analysis revealed severe modifications in the plasma membrane and disorganization of the P. fusca cell cytoplasm. CONCLUSIONS: The lipopeptides produced by the two strains of B. subtilis are able to reduce cucurbit powdery mildew disease by arresting conidial germination, which seems to result from the induction of important cytological alterations. SIGNIFICANCE AND IMPACT OF THE STUDY: We elucidated the mechanisms employed by these antagonistic strains of B. subtilis to suppress cucurbit powdery mildew disease and delineate the ultrastructural damages responsible for their suppressive effect.     

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.     

Molecular detection of a gene effective against powdery mildew in the wheat cultivar Liangxing 66

Since it was commercialized in 2008, Liangxing 66 is one of the most widely grown cultivars of wheat (Triticum aestivum L.) in winter and facultative wheat-producing regions in northern China. This cultivar displays broad-spectrum resistance to isolates of powdery mildew. To identify the powdery mildew resistance gene in Liangxing 66, genetic analysis and molecular mapping were conducted using the F₂ populations and F_2:3 families derived from the reciprocal crosses of Liangxing 66 and the susceptible cultivar Jingshuang 16. A single dominant gene, tentatively designated PmLX66, conferred resistance in Liangxing 66 to the powdery mildew isolate E09. The results of molecular mapping indicated that this gene was located on the short arm of chromosome 5D and flanked by SCAR203 and Xcfd81 at genetic distances of 0.4 and 2.8?cM, respectively, which is similar to the position of locus Pm2. However, PmLX66 and Pm2 showed different reactions to five of the 42 isolates of powdery mildew tested. Together, these results indicated that PmLX66 was most likely an allele of Pm2. Based on its superior yield and agronomic performance, in combination with powdery mildew resistance, Liangxing 66 is useful as a promising parent for control of powdery mildew and for the development of new disease-resistant cultivars.     

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.