Dolores Fernández-Ortuño

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

UNLABELLED 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 x 15-22 microm, 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 microm 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.

Field resistance to QoI fungicides in Podosphaera fusca is not supported by typical mutations in the mitochondrial cytochrome b gene

BACKGROUND A single nucleotide polymorphism in the mitochondrial cytochrome b gene confers resistance to strobilurin (QoI) fungicides in phytopathogenic fungi. Recent studies have revealed worrying levels of resistance to strobilurins in Podosphaera fusca (Fr.) U Braun & N Shishkoff comb. nov. [ = Sphaerothecafusca (Fr.) S Blumer], the main causal agent of cucurbit powdery mildew in Spain. In the present study the underlying resistance mechanism to QoI fungicides in the Spanish populations of P. fusca was investigated. RESULTS Analysis of the Q(o) domains of cytochrome b in a collection of isolates revealed that none of the typical mutations conferring resistance to QoI, including the G143A and F129L substitutions, was present in the QoI-resistant isolates. Moreover, although different amino acid polymorphisms were observed in the two regions spanning the Q(o) site, none of them consistently distinguished QoI-resistant from QoI-sensitive strains. Exposure to salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, in the presence of trifloxystrobin did not have any effect on QoI resistance, ruling out alternative respiration as the mechanism of resistance. Sensitivity tests to a battery of respiration inhibitors revealed high levels of cross-resistance to all Qo-inhibitors tested but not to Qi-inhibitors, these features resembling those of a target-site-based resistance. CONCLUSIONS The results indicate that the mechanism responsible for QoI resistance in P. fusca is not linked to typical mutations in cytochrome b gene and that the absence of the G143A substitution cannot be explained by an intron following codon 143. These are important observations, especially in relation to the possible molecular diagnosis of resistance.

Comparative histochemical analyses of oxidative burst and cell wall reinforcement in compatible and incompatible melon–powdery mildew (Podosphaera fusca) interactions

The spatial-temporal expression patterns of oxidative burst and cell wall reinforcement were analyzed in leaves of resistant and susceptible melon (Cucumis melo L.) cultivars in response to Podosphaera fusca (Fr.) Braun & Shishkoff, the main causal agent of powdery mildew in cucurbits. Extensive development of powdery mildew mycelia and a progressive increase in haustorial count were recorded in the susceptible cultivar after 4d, while in the resistant cultivar powdery mildew failed to grow and small brownish and necrotic leaf areas were frequently observed. Rapid generation of the reactive oxygen intermediates hydrogen peroxide and superoxide radicals 4h after pathogen challenge, but before the fungal haustoria formation, stood upstream in the cascade of events induced during these interactions. This oxidative burst was followed by the accumulation of strengthening polymers of callose and lignin at the cell wall of attacked resistant plant cells. Interestingly, the transcriptional levels of phenylalanine ammonia-lyase (PAL), an important enzyme for phenylpropanoid metabolism, did not significantly change throughout the experiments. Although these physiological changes were observed in both cultivars, their faster kinetics and amplitude in the resistant line compared to the susceptible cultivar governed the differential visual response of these cultivars against P. fusca. These findings, along with data obtained in previous studies, have provided the bases for an integrated model in which the spatial-temporal response patterns of these resistance mechanisms have been arranged, which may ultimately lead to successful protection of melon plants against P. fusca.

Multiple displacement amplification, a powerful tool for molecular genetic analysis of powdery mildew fungi

Powdery mildew fungi (Erysiphales) are probably the largest group of plant pathogens that remain uncharacterized from genetic and molecular points of view, with the only exception of the powdery mildew of cereals, Blumeria graminis. Their nature as obligate biotrophic parasites and consequent inability to grow on culture media has significantly hampered research. A common bottleneck to the molecular genetic analysis of powdery mildew fungi is the availability of genomic DNA of suitable quality and in sufficient quantity. The so-called whole genome amplification technology has the potential to overcome this limitation. Here we present the application of phi29 DNA polymerase-mediated multiple displacement amplification (MDA) to amplify the whole genome of Podosphaera fusca, the main causal agent of powdery mildew in cucurbits, to address this problem. The genome coverage and fidelity of the MDA process was evaluated by PCR amplification and sequencing of two genetics markers: the nuclear rDNA internal transcribed spacer (ITS) regions and the mitochondrial cytochrome b gene (CYTB). Our results show that MDA is a valuable tool for molecular genetic analysis of powdery mildew fungi that can be used for a number of downstream applications in different fields, such as epidemiology and population genetics or systematics.

Sensitivities to DMI fungicides in populations of Podosphaera fusca in south central Spain.

BACKGROUND Cucurbit powdery mildew elicited by Podosphaera fusca (Fr.) U Braun & N Shishkoff limits crop production in Spain. Disease control is largely dependent on fungicides such as sterol demethylation inhibitors (DMIs). Fungicide resistance is an increasing problem in this pathogen. To overcome such risk, it is necessary to design rational control programmes based upon knowledge of field resistance. The aim of this study was to investigate the state of DMI sensitivity of Spanish P. fusca populations and provide tools for improved disease management. RESULTS Using a leaf-disc assay, sensitivity to fenarimol, myclobutanil and triadimenol of 50 isolates of P. fusca was analysed to determine discriminatory concentrations between sensitive and resistant isolates. As no clearly different groups of isolates could be identified, discriminatory concentrations were established on the basis of maximum fungicide field application rate, 100 mg L(-1) for the three fungicides tested. Subsequently, a survey of DMI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates revealed that 23% were resistant to fenarimol and 7% to triadimenol, the provinces of Almería, Badajoz and Murcia being the locations with the highest frequencies of resistance. By contrast, no resistance to myclobutanil was found. CONCLUSION Results show that fenarimol and, to a lesser extent, triadimenol have become less efficient for controlling cucurbit powdery mildew in Spain. These are important observations that should lead to reconsideration of the current disease management programmes.

Characterization of Resistance to Six Chemical Classes of Site-Specific Fungicides Registered for Gray Mold Control on Strawberry in Spain

Botrytis cinerea, causal agent of the gray mold disease, is one of the most economically important fungal pathogens of strawberry worldwide. In Spain, as in other parts of the world, management of gray mold control primarily involves the application of fungicides. To determine the fungicide resistance of the Spanish strawberry field population, 367 B. cinerea isolates were examined from one organic and 13 conventional strawberry fields in Huelva (Spain) in 2014 and 2015. The sensitivities of these isolates to six fungicides used for gray mold management in Spain were examined using a spore germination assay based on previously published discriminatory doses. The frequency of resistance to pyraclostrobin, boscalid, cyprodinil, fenhexamid, iprodione, and fludioxonil was 74.6, 64.8, 37.0, 23.7, 14.7, and 0.8%, respectively. The majority of isolates (35.1%) were resistant to three different fungicides classes. Within these isolates, the most prevalent resistance profile (55.8%) was resistance to pyraclostrobin, boscalid, and cyprodinil, followed by the resistance profile (30.2%) of resistance to pyraclostrobin, boscalid, and fenhexamid. One isolate collected in 2015 was resistant to all six fungicide classes. Resistance to boscalid, fenhexamid, iprodione, and pyraclostrobin was found to be caused by amino acid substitutions on target proteins, including H272R/Y in SdhB, F412I/S/V in Erg27, I365 N/S in Bos1, and G143A in Cytb, respectively. The presence of multifungicide resistance phenotypes in B. cinerea isolates from strawberry fields in Spain must be considered in the development of future resistance management practices.

Resistance to the SDHI Fungicides Boscalid, Fluopyram, Fluxapyroxad, and Penthiopyrad in Botrytis cinerea from Commercial Strawberry Fields in Spain

Gray mold, caused by the necrotrophic fungus Botrytis cinerea., is one of the most economically important diseases of strawberry. Gray mold control involves the application of fungicides throughout the strawberry growing season; however, B. cinerea isolates resistant to multiple classes of site-specific fungicides have been recently reported in the Spanish gray mold population. Succinate dehydrogenase inhibitors (SDHI) constitute a relatively novel class of fungicides registered for gray mold control representing new alternatives for strawberry growers. In the present study, 37 B. cinerea isolates previously characterized for their sensitivity to boscalid and amino acid changes in the SdhB protein were used to determine the effective concentration that reduces mycelial growth by 50% (EC50) to fluopyram, fluxapyroxad, and penthiopyrad. The present study was also conducted to obtain discriminatory doses to monitor SDHI fungicide resistance in 580 B. cinerea isolates collected from 27 commercial fields in Spain during 2014, 2015, and 2016. The EC50 values ranged from 0.01 to >100 μg/ml for fluopyram, <0.01 to 4.19 μg/ml for fluxapyroxad, and, finally, <0.01 to 59.65 μg/ml for penthiopyrad. Based on these results, as well as findings from a previous publication, the discriminatory doses chosen to examine sensitivities to boscalid, fluopyram, fluxapyroxad, and penthiopyrad were 100, 15, 1, and 6 μg/ml, respectively. Over the course of the 3-year monitoring period, the overall frequencies of resistance to the four SDHI were 56.9, 6.9, 12.9, and 24.6%, respectively. The frequency of boscalid-resistant isolates decreased from 73 to 41% over the years; however, the fluopyram-resistant isolates increased from 5 to 10% after 1 year of registration. Four SDHI resistance patterns were observed in our population, which included patterns I (30%; resistance to boscalid), II (13.8%; resistance to boscalid and penthiopyrad), III (5.7%; boscalid, fluxapyroxad, and penthiopyrad), and IV (7.9%; resistance to boscalid, fluopyram, fluxapyroxad, and penthiopyrad). Patterns I and II were associated with the amino acid substitutions H272R and H272Y; pattern III was associated only with the H272Y mutation; and, finally, pattern IV was associated with the N230I allele in the SdhB subunit. For gray mold management, it is suggested that the simultaneous use of boscalid and penthiopyrad should be limited to one application per season; however, fluxapyroxad and, especially, fluopyram could be used as valid SDHI alternatives for gray mold control, although they should be applied with caution.