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.     

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.     

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.     

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.     

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.     

Sensibilité du Melon àSphaerotheca fuliginea en Fonction de L'Etage Foliaire

Effect of leaf position on the susceptibility of melon plants to artificial infection with powdery mildew, Sphaerotheca fuliginea The leaf position of melon plants seems to play a role on their susceptibility when they are artificially infected with powdery mildew Sphaerotheca fuliginea. The cotyledons are generally very susceptible, while the first leaf relatively resistant; the susceptibility again continues up to the 4th–5th leaf (but less susceptible than the cotyledons) and then after it decreases; these results can be obtained on plants in greenhouse or on detached leaves in Petri dishes. From this observation, we think that the screening of melon genotypes for resistance to powdery mildew can be evaluated neither on the cotyledons which are very susceptible nor on the first leaf which is resistant, but on the third leaf which is moderately susceptible. In fact, there is a good correlation between the reaction of the third leaf and the resistance or susceptibility of genotypes.     

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.     

The iturin-like lipopeptides are essential components in the biological control arsenal of Bacillus subtilis against bacterial diseases of cucurbits

The antibacterial potential of four strains of Bacillus subtilis, UMAF6614, UMAF6619, UMAF6639, and UMAF8561, previously selected on the basis of their antifungal activity and efficacy against cucurbit powdery mildew, was examined. Among these strains, UMAF6614 and UMAF6639 showed the highest antibacterial activity in vitro, especially against Xanthomonas campestris pv. cucurbitae and Pectobacterium carotovorum subsp. carotovorum. These strains produced the three families of lipopeptide antibiotics known in Bacillus spp.: surfactins, iturins, and fengycins. Using thin-layer chromatography analysis and direct bioautography, the antibacterial activity could be associated with iturin lipopeptides. This result was confirmed by mutagenesis analysis using lipopeptide-defective mutants. The antibacterial activity was practically abolished in iturin-deficient mutants, whereas the fengycin mutants retained certain inhibitory capabilities. Analyses by fluorescence and transmission electron microscopy revealed the cytotoxic effect of these compounds at the bacterial plasma membrane level. Finally, biological control assays on detached melon leaves demonstrated the ability of UMAF6614 and UMAF6639 to suppress bacterial leaf spot and soft rot; accordingly, the biocontrol activity was practically abolished in mutants deficient in iturin biosynthesis. Taken together, our results highlight the potential of these B. subtilis strains as biocontrol agents against fungal and bacterial diseases of cucurbits and the versatility of iturins as antifungal and antibacterial compounds.     

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.     

Identification and mapping of two powdery mildew resistance genes in <Emphasis Type="Italic">Triticum boeoticum</Emphasis> L.

Powdery mildew (PM) caused by Blumeria graminis f. sp. tritici (Bgt), is one of the important foliar diseases of wheat that can cause serious yield losses. Breeding for cultivars with diverse resources of resistance is the most promising approach for combating this disease. The diploid A genome progenitor species of wheat are an important resource for new variability for disease resistance genes. An accession of Triticum boeoticum (A^bA^b) showed resistance against a number of Bgt isolates, when tested using detached leaf segments. Inheritance studies in a recombinant inbred line population (RIL), developed from crosses of PM resistant T. boeoticum acc. pau5088 with a PM susceptible T. monococcum acc. pau14087, indicated the presence of two powdery mildew resistance genes in T. boeoticum acc. pau5088. Analysis of powdery mildew infection and molecular marker data of the RIL population revealed that both powdery mildew resistance genes are located on the long arm of chromosome 7A. Mapping was conducted using an integrated linkage map of 7A consisting of SSR, RFLP, STS, and DArT markers. These powdery mildew resistance genes are tentatively designated as PmTb7A.1 and PmTb7A.2. The PmTb7A.2 is closely linked to STS markers MAG2185 and MAG1759 derived from RFLP probes which are linked to powdery mildew resistance gene Pm1. This indicated that PmTb7A.2 might be allelic to Pm1. The PmTb7A.1, flanked by a DArT marker wPt4553 and an SSR marker Xcfa2019 in a 4.3 cM interval, maps proximal to PmT7A.2. PmTb7A.1 is putatively a new powdery mildew resistance gene. The powdery mildew resistance genes from T. boeoticum are currently being transferred to cultivated wheat background through marker-assisted backcrossing, using T. durum as bridging species.     

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.     

Identification and Analysis of Powdery Mildew‐Responsive miRNAs in Wheat

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most consistently damaging diseases of common wheat worldwide and greatly affects crop productivity. Recently, several plant microRNAs (miRNAs) have been reported as gene expression regulators related to various adverse environments. However, up to now, less is known on the roles of miRNAs in powdery mildew infection response of wheat. In this study, miRNA expression patterns were investigated for identifying Bgt‐responsive miRNAs in wheat leaves using a plant miRNA microarray platform. A total of 79 miRNAs from 24 families were detected in wheat leaves. Among those, seven miRNAs were further validated to be involved in wheat powdery mildew response and two of them have never been reported. In addition, their target expression profiles showed a negative correlation with that of the seven miRNAs in mock‐ and Bgt‐infected samples furtherly proved, which in turn as the robust evidence, that those seven powdery mildew‐responsive miRNAs are highly reliable. These findings could extend the current view about miRNAs as ubiquitous regulators under stress conditions.     

籽用美洲南瓜叶片对白粉病菌的抗逆生理响应

以籽用美洲南瓜(Cucurbita pepo L.)白粉病抗病品系F2和感病品系M3为试材,在人工气候箱内接种白粉病生理小种2US孢子悬浮液,考察在接种白粉病菌后南瓜幼苗植株与白粉病菌的互作、叶片活性氧代谢及保护酶活性的变化,探讨南瓜抵御白粉病的生理机制。结果表明：(1)与感病品系M3相比,接种白粉病菌后,抗病品系F2叶片上病原菌发育缓慢,较难侵染叶片。(2)抗病品系F2在感病初期叶片H₂O₂、O₂^-·含量迅速升高后逐渐下降,而感病品系在感病初期H₂O₂、O₂^-·含量上升缓慢,在达最大值后始终保持较高水平,且感病品系叶片MDA含量始终高于抗病品系;组织化学染色分析发现,抗病品系叶片着色比感病品系快,之后着色面积有所减少并趋于较低水平。(3)抗病品系F2和感病品系M3叶片抗氧化酶CAT、SOD、POD活性及PAL、PPO活性在接种白粉病菌后均显著增加,但抗病品系的活性及其增幅均高于感病品系。研究发现,籽用美洲南瓜抗病品系叶片上白粉病菌发育缓慢,较难受到侵染,生成菌丝体后叶片上粉状斑点较小;抗病品系在被白粉病菌侵染初期依靠活性氧的增加抵御病原菌的入侵,随着活性氧含量增加抗病品系通过迅速增加自身抗氧化酶活性来防止氧化胁迫;与感病品系相比,抗病品系在受病原菌侵染后能迅速增加PAL、PPO活性以抵御病原菌侵染。     

Linkage of a RAPD marker with powdery mildew resistance <Emphasis Type="Italic">er-1</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis> L.

The aim of this study was to investigate the inheritance of powdery mildew disease and to tag it with a DNA marker to utilize for the marker-assisted selection (MAS) breeding program. The powdery mildew resistant genotype Fallon ^er and susceptible genotype 11760-3 ^ER were selected from 177 genotypes by heavy infestation of germplasm with Erysiphe pisi through artificial inoculation The F₁ plants of the cross Fallon/11760-3 indicated the dominance of the susceptible allele, while F₂ plants segregated in 3: 1 ratio (susceptible: resistant) that fit for goodness of fitness by χ² (P > 0.07), indicating monogenic recessive inheritance for powdery mildew resistance in Pisum sativum. A novel RAPD marker OPB18 (5′-CCACAGCAGT-3′) was linked to the er-1 gene with 83% probability with a LOD score of 4.13, and was located at a distance of 11.2 cM from the er-1 gene.     

Development of powdery mildew on leaves of several barley varieties at different growth stages

On detached leaves and intact plants of several barley varieties at different growth stages, lower percentages of germinated conidia of Erysiphe graminis f.sp. hordei penetrated the host and initiated infection on the abaxial than adaxial surface. More and larger E. graminis colonies developed on the adaxial surface and these comprised more densely packed hyphae and produced more conidiophores than did colonies on the abaxial surface. These results are consistent with the observation that there is usually more powdery mildew on the adaxial than abaxial surface of barley leaves in the field. Smaller proportions of germinated E. graminis conidia penetrated and infected the host on leaves of adult or near-adult plants than on those of seedlings or juvenile plants. Older plants also supported fewer, smaller and less dense colonies with less sporulation than young plants. The effects of growth stage of the host plant on development of powdery mildew were much greater in some barley varieties, and with some E. graminis isolates, than others.     

Evaluation of Lecanicillium longisporum, Vertalec for simultaneous suppression of cotton aphid, Aphis gossypii, and cucumber powdery mildew, Sphaerotheca fuliginea, on potted cucumbers

The commercial preparation of Lecanicillium longisporum, Vertalec^® was evaluated for simultaneous suppression of cotton aphid and cucumber powdery mildew on potted cucumber plants. Vertalec was applied onto cucumber plants that had been infested with either cotton aphid, spores of Sphaerotheca fuliginea or both. Irradiation-inactivated Vertalec (II Vertalec) was also applied to an identical series of cucumber plants as a control. The Vertalec was highly pathogenic against adult aphids with an LT₅₀ of 6.9 days. II Vertalec did not affect aphid survival. Application of either active or II Vertalec significantly suppressed spore production of S. fuliginea compared to the water control. For dual control assays, Vertalec applications were made one day after infestation of both aphid and S. fuliginea onto potted cucumbers. Fifteen days after the Vertalec treatments, the numbers of surviving aphids and the production of powdery mildew spores were significantly reduced compared with the water control. The presence of aphids also suppressed S. fuliginea spore production. Our results suggest the potential of a dual role for Vertalec as a microbial control agent of aphids and powdery mildew in cucumber.     

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.     

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。