Derek W. Hollomon

Fungal respiration: a fusion of standard and alternative components.

In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.

Occurrence and molecular characterization of strobilurin resistance in cucumber powdery mildew and downy mildew.

ABSTRACT Between 1998 and 1999, control failure of powdery mildew (Podosphaera fusca) and downy mildew (Pseudoperonospora cubensis) by the strobilurin fungicides azoxystrobin and kresoxim-methyl was observed in cucumber-growing areas of Japan. Results from inoculation tests carried out on intact cucumber plants and leaf disks clearly showed the distribution of pathogen isolates highly resistant to azoxystrobin and kresoximmethyl. Fragments of the fungicide-targeted mitochondrial cytochrome b gene were polymerase chain reaction amplified from total pathogen DNA and their sequences analyzed to elucidate the molecular mechanism of resistance. A single point mutation (GGT to GCT) in the cytochrome b gene, resulting in substitution of glycine by alanine at position 143, was found in resistant isolates of downy mildew. This substitution in cytochrome b seemed to result in high resistance to strobilurins in this pathogen. The same mutation was found in some but not all resistant isolates of powdery mildew. This study suggests that a mutation at position 143 in the target-encoding gene, resulting in an amino acid substitution, was probably a major cause of the rapid development of high strobilurin resistance in these two pathogens.

Rapid detection and diagnosis of Septoria tritici epidemics in wheat using a polymerase chain reaction/PicoGreen assay

In order to detect and quantify Septoria tritici infection levels in wheat leaves, a polymerase chain reaction (PCR) assay was developed using the β‐tubulin gene as target. Specific PCR primers were designed by aligning and comparing β‐tubulin sequences from other fungi. The final primer set was selected after being tested against several fungi, and against S. tritici‐infected and uninfected wheat leaves from different localities. A single DNA fragment (496 bp) was amplified from S. tritici, whereas no products were generated from DNA of the host plant or other micro‐organisms associated with wheat leaves. Using agarose gel analysis, approximately 2 pg S. tritici genomic DNA could be detected in each assay. However, for rapid quantification of PCR‐amplified products, a fluorometric microtitre plate‐formatted PicoGreen assay was used; this could detect as little as 10 pg S. tritici DNA in the presence of 200 ng wheat leaf DNA. The PCR/PicoGreen assay was applied successfully to study the colonization, infection and subsequent disease development of S. tritici on wheat, both under controlled conditions in the glasshouse and in the field.

PCR-based Assays to Assess Wheat Varietal Resistance to Blotch (Septoria Tritici and Stagonospora Nodorum) and Rust (Puccinia Striiformis and Puccinia Recondita) Diseases

A multiplex Polymerase Chain Reaction (PCR) assay was developed to detect and quantify four fungal foliar pathogens in wheat. For Septoria tritici (leaf blotch) and Stagonospora nodorum (leaf and glume blotch), the β-tubulin gene was used as the target region. Diagnostic targets for Puccinia striiformis (stripe or yellow rust) and P. recondita (brown rust) were obtained from PCR products amplified with β-tubulin primer sequences. Final primer sets were designed and selected after being tested against several fungi, and against DNA of infected and healthy wheat leaves. For detection of the four pathogens, PCR products of different sizes were amplified simultaneously, whereas no products were generated from wheat DNA or other non-target fungi tested. The presence of each of the diseases was wheat tissue- and cultivar specific. Using real-time PCR measurements with the fluorescent dye SYBR Green I, PCR-amplified products could be quantified individually, by reference to a standard curve generated by adding known amounts of target DNA. Infection levels for each of the diseases were measured in the flag leaf of 19 cultivars at Growth Stage (GS) 60–64 in both 1998 and 1999. The infection levels for the cultivars were ranked, and showed, with a few exceptions, a good correlation with the NIAB Recommended List for winter wheat, which is based on visual assessment of symptoms. With PCR, the presence of the different pathogens was accurately diagnosed and quantification of pre-symptomatic infection levels was possible. Although sampling and DNA detection methods need further optimisation, the results show that multiplex PCR and quantitative real-time PCR assays can be used in resistance screening to measure the interaction between different pathogens and their hosts at different growth stages, and in specific tissues. This should enable an earlier identification of specific resistance mechanisms in both early-stage breeding material and field trials.

Cross-resistance to polyene and azole drugs in Cryptococcus neoformans.

Fluconazole was observed to inhibit sterol 14 alpha-demethylase in the human pathogen Cryptococcus neoformans, and accumulation of a ketosteroid product was associated with growth arrest. A novel mechanism(s) of azole and amphotericin B cross-resistance was identified, unrelated to changes in sterol biosynthesis, as previously identified in Saccharomyces cerevisiae. Reduced cellular content of drug could account for the resistance phenotype, indicating the possible involvement of a mechanism similar to multidrug resistance observed in higher eukaryotes.

Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression.

A Mycosphaerella graminicola strain transformed with the green fluorescent protein (GFP) downstream of either a carbon source-repressed promoter or a constitutive promoter was used to investigate in situ carbohydrate uptake during penetration of the fungus in wheat leaves. The promoter region of the acu-3 gene from Neurospora crassa encoding isocitrate lyase was used as a carbon source-repressed promoter. The promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase was used as a constitutive promoter. Fluorometric measurement of GFP gene expression in liquid cultures of acu-3-regulated transformants indicated that the N. crassa acu-3 promoter functions in M. graminicola as it does in N. crassa, i.e., acetate induced and carbon source repressed. Glucose, fructose, and saccharose triggered the repression, whereas mannitol, xylose, and cell wall polysaccharides did not. Monitoring the GFP level during fungal infection of wheat leaves revealed that acu-3 promoter repression occurred after penetration until sporulation, when newly differentiated pycnidiospores fluoresced. The use of GFP transformants also allowed clear visualization of M. graminicola pathogenesis. No appressoria were formed, but penetration at cell junctions was observed. These results give new insight into the biotrophic status of M. graminicola.

ALTERED P450 ACTIVITY ASSOCIATED WITH DIRECT SELECTION FOR FUNGAL AZOLE RESISTANCE

Azole antifungals inhibit CYP51A1‐mediated sterol 14α‐demethylation and the mechanism(s) of resistance to such compounds in Ustilago maydis were examined. The inhibition of growth was correlated with the accumulation of the substrate, 24‐methylene‐24,25‐dihydrolanosterol (eburicol), and depletion of ergosterol. Mutants overcoming the effect of azole antifungal treatment exhibited a unique phenotype with leaky CYP51A1 activity which was resistant to inhibition. The results demonstrate that alterations at the level of inhibitor binding to the target site can produce azole resistance. Similar changes may account for fungal azole resistance phenomena in agriculture, and also in medicine where resistance has become a problem in immuno‐compromised patients suffering from AIDS.

Changes in sensitivity to DMI fungicides in Rhynchosporium secalis

Abstract Laboratory tests on 2000 isolates of Rhynchosporium secalis from throughout the UK over a 4-year period revealed a decline in sensitivity to triadimenol and propiconazole but not to prochloraz. Change occurred throughout the UK irrespective of disease pressure, and was not correlated with fungicide use. Resistant isolates were no less pathogenic than sensitive ones. Selection with triadimenol generated a bimodal population distribution, whereas propiconazole produced a gradual shift of unimodal population towards a less sensitive mean. Some cross-resistance occurred between triadimenol, propiconazole and a third triazole, tebuconazole, although the change in sensitivity to tebuconazole was always less than to the other two triazoles. No cross-resistance was observed to the imidazole demethylation inhibitor (DMI), prochloraz. Field-trial data collected over several years showed that the performance of triadimenol, and to a lesser extent, of propiconazole, had declined. Control of Rhynchosporium with these fungicides could be improved by using mixtures with carbendazim. Tebuconazole, either alone or in mixtures with carbendazim or tridemorph, provided the best disease control, and did not appear to select for lower sensitivity. The findings emphasize that certain DMI fungicides may still be used in strategies where performance of other DMIs has been altered because of resistance.

Effect of Carbendazim Resistance on Trichothecene Production and Aggressiveness of Fusarium graminearum

Fusarium graminearum (teleomorph, Gibberella zeae) causes head blight of cereals and contaminates grains with trichothecene mycotoxins that are harmful to humans and domesticated animals. Control of Fusarium head blight relies on carbendazim (MBC) in China, but resistance to MBC in F. graminearum is now widespread. Sixty-seven strains were evaluated for trichothecene production in shake culture or in the field. The strains included 60 wild-type strains (30 MBC-resistant and 30 MBC-sensitive), three MBC-resistant site-directed mutants at codon 167 in beta(2)-tubulin, three MBC-sensitive site-directed mutants at codon 240 in beta(2)-tubulin, and their MBC-sensitive wild-type progenitor strain ZF21. The incidence of infected spikelets and the amount of F. graminearum DNA in field grain (AFgDNA) also were evaluated for all strains. MBC resistance increased trichothecene production in shake culture or in the field. Although MBC resistance did not change the incidence of infected spikelets, it did increase AFgDNA. Tri5 gene expression increased in MBC-resistant strains grown in shake culture. We found a significant exponential relationship between trichothecene production and Tri5 gene expression in shake culture and a linear relationship between the incidence of infected spikelets or AFgDNA and trichothecene production in field grain.

Functional diversity within the mitochondrial electron transport chain of plant pathogenic fungi

Mitochondrial transfer of electrons from NAD(P)H or FADH2 to the terminal electron acceptor, oxygen, follows a highly complex scheme, involving numerous redox components. Whilst electron transfer has been extensively studied over past decades in mammalian, plant and some fungal species, relatively little is known about the respiratory chain of phytopathogenic fungi. The recent identification of the electron transport chain as a viable target for effective control of fungal infections has contributed to a significantly increased research effort into this area of fungal biochemistry. A striking feature of the electron transport chain within phytopathogenic fungi is the presence of components not found in the classical (mammalian) chain. Recent research has suggested the presence of a plant-like ‘alternative’ oxidase, internal and external NAD(P)H dehydrogenases, and cyanide-insensitive cytochrome c oxidases. In this mini-review on electron transport in phytopathogenic fungi, the current status of research into the function and expression characteristics of these ‘alternative’ redox centres is discussed. © 2000 Society of Chemical Industry