Ulrich Gisi

Large-Scale Gene Discovery in the Oomycete Phytophthora infestans Reveals Likely Components of Phytopathogenicity Shared with True Fungi

To overview the gene content of the important pathogen Phytophthora infestans, large-scale cDNA and genomic sequencing was performed. A set of 75,757 high-quality expressed sequence tags (ESTs) from P. infestans was obtained from 20 cDNA libraries representing a broad range of growth conditions, stress responses, and developmental stages. These included libraries from P. infestans-potato and -tomato interactions, from which 963 pathogen ESTs were identified. To complement the ESTs, onefold coveragethe P. infestans genome was obtained and regions of coding potential identified. A unigene set of 18,256 sequences was derived from the EST and genomic data and characterized for potential functions, stage-specific patterns of expression, and codon bias. Cluster analysis of ESTs revealed major differences between the expressed gene content of mycelial and spore-related stages, and affinities between some growth conditions. Comparisons with databases of fungal pathogenicity genes revealed conserved elements of pa...

Mode of resistance to respiration inhibitors at the cytochrome bc1 enzyme complex of Mycosphaerella fijiensis field isolates.

Field isolates of Mycosphaerella fijiensis, causing black Sigatoka of banana, were characterised for their sensitivity to different inhibitors of the cytochrome bc1 enzyme complex (Qo respiration inhibitors, strobilurin fungicides), using physiological, biochemical and molecular genetic methods. Strobilurin-resistant isolates exhibited very high resistance factors both in mycelial growth inhibition and NADH consumption assays. Cross-resistance was observed among all Qo inhibitors, including trifloxystrobin, azoxystrobin, famoxadone, strobilurin B and myxothiazol. However, the Qi and the cytochrome aa3 inhibitors, antimycin A and potassium cyanide, respectively, were not cross-resistant to Qo inhibitors. In sensitive but not in resistant isolates, mixtures of Qo inhibitors and SHAM, an inhibitor of the alternative oxidase (AOX), were more active than the components alone, indicating that the alternative pathway is essential in metabolism, but not causal for resistance. In the cell-free NADH-consumption assay, the Qo inhibitors affected the sensitive but not the resistant isolates, suggesting that AOX was not active in sub-mitochondrial particles. In whole cells, however, the AOX has a basic expression level and is probably not inducible by trifloxystrobin. Sequencing of the cytochrome b gene of sensitive and resistant M fijiensis isolates revealed a difference in the nucleotide sequence leading to a single amino acid change from glycine to alanine at position 143 in the resistant isolate. This change is known to occur also in the naturally tolerant basidiomycete Mycena galopoda. It is suggested that the field isolates of M fijiensis can acquire resistance to Qo inhibitors due to a target site alteration with a single base pair change. Resistant isolates do not seem to contain a mixture of mutated and non-mutated DNA, indicating a complete selection of resistant mitochondria and a maternally donated mode of resistance. © 2000 Society of Chemical Industry

Recent developments in elucidating modes of resistance to phenylamide, DMI and strobilurin fungicides

Abstract Resistance risk assessment, analysis of resistance events and risk management are based on elements such as the mode of resistance in target organisms, similarity of inhibitors, detection methods for resistant individuals, and design and validation of product use strategies. The mode of resistance may be analysed in several steps including the molecular, genetic, biochemical, physiological, and population level. Historical and recent findings are described for phenylamide fungicides (PAFs), demethylation inhibitors (DMIs) in sterol biosynthesis and fungicides of the strobilurin-type action and resistance (STAR) group (Qo respiration inhibitors, QoIs) with regard to the site of resistance in target pathogens and the presence of resistant individuals in populations.

Systemic resistance of potato plants against Phytophthora infestans induced by unsaturated fatty acids

Five unsaturated fatty acids were tested for their ability to induce systemic resistance in potato plants (cv. Bintje) to the late blight fungus Phytophthora infestans . Arachidonic acid and eicosapentaenoic acid applied to leaves 1–3 of potato plants at a dose of about 1 mg per plant induced 94% to 97% protection in leaves 4 to 11, respectively. Linoleic acid, linolenic acid and oleic acid provided 82%, 39% and 42% protection, respectively. Protection was evident as a reduction in lesion number and lesion diameter. It was maximal in plants challenged 5 days after induction and lasted for at least 12 days. A prior infection of lower leaves with P. infestans also induced up to 88% protection in the upper leaves. The mechanism involved in the induction of systemic resistance of potato plants by fatty acids is not understood.

Fungicide modes of action and resistance in downy mildews

Among oomycetes, Plasmopara viticola on grape and Phytophthora infestans on potato are agronomically the most important pathogens requiring control measures to avoid crop losses. Several chemical classes of fungicides are available with different properties in systemicity, specificity, duration of activity and risk of resistance. The major site-specific fungicides are the Quinone outside inhibitors (QoIs; e.g. azoxystrobin), phenylamides (e.g. mefenoxam), carboxylic acid amides (CAAs; e.g. dimethomorph, mandipropamid) and cyano-acetamide oximes (cymoxanil). In addition, multi-site fungicides such as mancozeb, folpet, chlorothalonil and copper formulations are important for disease control especially in mixtures or in alternation with site-specific fungicides. QoIs inhibit mitochondrial respiration, phenylamides the polymerization of r-RNA, whereas the mode of action of the other two site-specific classes is unknown but not multi-site. The use of site-specific fungicides has in many cases selected for resistant pathogen populations. QoIs are known to follow maternal, largely monogenic inheritance of resistance; they bear a high resistance risk for many but not all oomycetes. For phenylamides, inheritance of resistance is based on nuclear, probably monogenic mechanisms involving one or two semi-dominant genes; resistance risk is high for all oomycetes. The molecular mechanism of resistance to QoIs is mostly based on the G143A mutation in the cytochrome b gene; for phenylamides it is largely unknown. Resistance risk for CAA fungicides is considered as low to moderate depending on the pathogen species. Resistance to CAAs is controlled by two nuclear, recessive genes; the molecular mechanism is unknown. For QoIs and CAAs, resistance in field populations of P. viticola may gradually decline when applications are stopped.

A single point mutation in the novel PvCesA3 gene confers resistance to the carboxylic acid amide fungicide mandipropamid in Plasmopara viticola.

The grapevine downy mildew, Plasmopara viticola, is one of the most devastating pathogens in viticulture. Effective control is mainly based on fungicide treatments, although resistance development in this pathogen is reported for a number of fungicides. In this study we describe for the first time the molecular mechanism of resistance to a carboxylic acid amide (CAA) fungicide. We identified a family of four cellulose synthase (CesA) genes containing conserved domains that are found in all processive glycosyltransferases. Phylogenetic analysis revealed their close relationship to the cellulose synthases of Phytophthora sp. Sequencing of the CesA genes in a CAA- resistant and -sensitive field isolate revealed five single nucleotide polymorphisms (SNPs) affecting the amino acid structure of the proteins. SNP inheritance in F(1)-, F(2)- and F(3)-progeny confirmed resistance to be correlated with one single SNP located in PvCesA3. Only if present in both alleles, this SNP led to the substitution of a glycine for a serine residue at position 1105 (G1105S) in the deduced amino acid sequence, thus conferring CAA- resistance. Our data demonstrate that the identified genes are putative cellulose synthases and that one recessive mutation in PvCesA3 causes inheritable resistance to the CAA fungicide mandipropamid.

Chemical Control of Rhizoctonia Species

Ever since the destructive potential of Rhizoctonia pathogens on crop plants was realized, application of synthetic fungicides has been the most popular disease control measure employed by the farmers on various crops and the use of fungicides is practiced widely throughout the world. Even today many farmers will select disease-prone and high-yielding varieties and rely totally on fungicide treatments to maintain crop health. Disease control with fungicides has been largely successful against Rhizoctonia pathogens on many different crops in both temperate and tropical agro-ecological regions, attributable to targeted development of a variety of potent fungicides of diverse chemical nature and appropriate application methods against various Rhizoctonia diseases. But disease control with recommended anti-Rhizoctonia fungicides has not always been successful in some situations, and failures to control Rhizoctonia diseases on the same or different crop in an agro-ecological region are also documented in the literature, contrary to general belief that a potent fungicide shall curb the pathogen under all situations. The reasons for such diversity in results for satisfactory disease control are many and intricate, but have hardly been reasoned out scientifically. The purity of fungicides, their formulations and their improper usage have often been the alibi for extension workers to console the cultivators for poor disease control with the tested anti-Rhizoctonia fungicides. This contention is strongly discounted by the information generated from studies on quadrangular interactions of host-pathogen-fungicide-environment in the control of Rhizoctonia diseases. Recent progress in taxonomy and ecological diversity of population structures of Rhizoctonia pathogens in major cropping systems has greatly helped to understand the possible practical reasons for the effectiveness or ineffectiveness of fungicides to control Rhizoctonia in the fields.

Differential Activity of Carboxylic Acid Amide Fungicides Against Various Developmental Stages of Phytophthora infestans

ABSTRACT Three carboxylic acid amide (CAA) fungicides, mandipropamid (MPD), dimethomorph (DMM) and iprovalicarb (IPRO) were examined for their effects on various asexual developmental stages of Phytophthora infestans in vitro and in planta. Germination of cystospores and direct germination of sporangia were inhibited with nanomole concentrations of MPD (0.005 mug/ml) and micromole concentrations of DMM (0.05 mug/ml) or IPRO (0.5 mug/ml). A temporary exposure of 1 h to CAAs was not detrimental to germination and infectivity of sporangia or cystospores. CAAs applied to cystospores at 1 h after the onset of germination did not prevent the emergence of germ tubes, but inhibited their further growth and deformed their shape. None of the fungicides affected discharge of zoospores from sporangia or the encystment (cell wall formation/assembly) of the zoospores. Mycelium growth in solid or liquid media was inhibited with micromole concentrations. CAAs mixed with sporangia and drop inoculated onto detached leaves strongly suppressed infection. Curative application at 1 day postinoculation (dpi) required higher concentrations of CAAs than preventive application to inhibit infection and lost its effectiveness at 2 dpi. When sprayed on established late blight lesions 4 days after inoculation, CAAs reduced sporangial production in a dose-dependent manner. Trans-laminar protection of potato or tomato leaves, although achieved with higher doses, was more effective with MPD than with DMM or IPRO. Shade house studies demonstrated superior control of late blight epidemics by MPD compared with the other molecules. The data suggest that germ tube formation by cystospores or sporangia is the most sensitive stage in the life cycle of P. infestans to CAAs. Of the three CAAs, MPD had the highest intrinsic activity against spore germination. This property, together with its better trans-laminar activity, makes MPD more effective than DMM or IPRO in controlling epidemics caused by P. infestans.

Isogamous, hermaphroditic inheritance of mitochondrion-encoded resistance to Qo inhibitor fungicides in Blumeria graminis f. sp. tritici.

A mutation of glycine to alanine at position 143 in the mitochondrial cytochrome b amino acid sequence of Blumeria graminis f. sp. tritici cosegregated with the QoI-resistant phenotype in a ratio of 1:1 in a cross between a sensitive and a resistant isolate. This mutation was used as a mitochondrial marker to determine whether mitochondrial inheritance in B. graminis was anisogamous, as in heterothallic Neurospora sp., or isogamous and hermaphroditic, as in Aspergillus nidulans. Segregation of mitochondrial genotypes in B. graminis f. sp. tritici was consistent with inheritance of mitochondria being hermaphroditic and isogamous, in that all ascospores from an individual cleistothecium had the same mitochondrial genotype and that either parent could act as the maternal parent of a cleistothecium. Within each cleistothecium, nuclear segregation occurred independently of mitochondrial inheritance, as shown by segregation of resistance to the fungicide triadimenol and by segregation of avirulences to the wheat cultivars Galahad (Pm2), Armada (Pm4b), and Holger (Pm6).

Mutagenesis of Phytophthora infestans for Resistance Against Carboxylic Acid Amide and Phenylamide Fungicides

The carboxylic acid amide (CAA) fungicides mandipropamid, dimethomorph, iprovalicarb, and the phenylamide fungicide mefenoxam (MFX, the active enantiomer of metalaxyl) are anti-oomycete fungicides effective against downy mildews and late blight. Resistance against MFX was reported in nature in several oomycetes including Phytophthora infestans and Plasmopara viticola, whereas resistance against CAAs was reported in P. viticola but not in P. infestans. In this study the mutability of P. infestans for resistance against CAAs and MFX (as a control) was explored under laboratory conditions. UV light or chemical mutagens (e.g., ethyl methan sulfonate [EMS]) were applied to sporangia, and the emergence of mutants resistant to CAAs or MFX, or with altered mating type, was followed. Many mutants resistant to CAAs developed at generation 0 after mutagenesis, but all showed erratic, instable resistance in planta, diminishing after 1 to 8 asexual infection cycles, and failed to grow on CAA-amended medium. In contrast, 19 mutants resistant to MFX were obtained: 6 with UV irradiation (in isolates 28 or 96) and 13 with EMS (in isolates 408, 409, and 410). In three experiments, a shift in mating type, from A1 to A2, was detected. To elucidate whether or not resistance to CAAs is recessive and therefore might emerge only after sexual recombination, A1 and A2 mutants were crossed and the F1 and F2 progeny isolates were tested for resistance. Offspring isolates segregated for resistance to MFX, with resistant isolates maintaining stable resistance in vitro and in planta, whereas all progeny isolates failed to show stable resistance to CAAs in planta or in vitro. The data suggest that P. infestans could be artificially mutated for resistance against MFX, but not against CAAs.