Wolfram Köller

The Plant Cuticle

Most plant-pathogenic fungi gain access into their host by penetration of unwounded tissue. Some pathogens such as rusts invade the host via stomata (Hoch and Staples, 1987 and Chapter 2), whereas others penetrate the intact leaf surface without the requirement of natural openings (Aist, 1976; Emmett, 1975; Kunoh, 1984). The latter type of direct penetration encounters the plant cuticle, a noncellular hydrophobic structure covering the layer of epidermal cells. The cuticle thus serves as the first surface barrier that the pathogen has to breach. There is little evidence for the mere physical strength of the plant cuticle as a major factor in plant defense against pathogens. In some cases, the thickness of plant cuticles has been correlated with an increased passive resistance against fungal attack. This correlation, however, appears to be an exception rather than the rule (Martin, 1964). Furthermore, the cuticle has not been considered to play a major role in the active defense mechanisms of disease-resistant cultivars. There is good evidence that the cuticle is penetrated by the attacking pathogen before the sequential steps of disease development are halted by the active defense reactions of the challenged plant. Recent examples for this lack of cuticle involvement in cultivar resistance are the host—pathogen interactions of Venturia ivaequalis—apple (Valsangiacomo and Gessler, 1988) or Phytophthora infestans—potato (Gees and Hohl, 1987). The breaching of the cuticle can also be accomplished in many interactions of pathogens with nonhost plants (Heath, 1987).

Characterization of laboratory mutants of Venturia inaequalis resistant to the strobilurin-related fungicide kresoxim-methyl

Abstract Several agricultural fungicides related to the antifungal strobilurins act as inhibitors of respiration by binding to mitochondrial cytochrome b. Two types of laboratory mutants resisting higher doses of the strobilurin-related inhibitor kresoxim-methyl were characterized for Venturia inaequalis, the causal agent of apple scab. Selection of mutagenized conidia by kresoxim-methyl yielded mutants altered in the expression of alternative respiration during the stage of conidia germination. Cytochrome b sequences were not affected in the respective mutants. Selection of conidia on media containing the alternative oxidase inhibitor salicylhydroxamic acid in addition to kresoxim-methyl yielded a highly resistant mutant distinguished by a G143A exchange in cytochrome b. The status of mitochondrial cytochrome b genes remained heteroplasmic, and mitochondria containing wild-type cytochrome b returned to high frequencies during cultivation on inhibitor-free medium. However, continuation of the selection process led to a more pronounced replacement of sensitive by mutated mitochondria. The G143A mutation of cytochrome b causing resistance of V. inaequalis to a strobilurin-related inhibitor has been reported previously for mouse mitochondria; and a permanent G143A exchange rendering naturally resistant mitochondria has been reported for the strobilurin-producing basidiomycete Mycena galopoda and for the sea urchin Paracentrotus lividus. At the corresponding position, alanine was also present in chloroplast cytochrome b6 exhibiting low binding of strobilurin-related inhibitors. The mutation of cytochrome b reported here for V. inaequalis describes the first example of a mutation in filamentous ascomycetes and is part of an assessment of resistance risks inherent to strobilurin fungicides.

Characterization of the mitochondrial cytochrome b gene from Venturia inaequalis

Abstract A new class of agricultural fungicides derived from the group of antifungal strobilurins acts as specific respiration inhibitors by binding to mitochondrial cytochrome b. The cytochrome b gene was cloned and sequenced from the mitochondrial genome of Venturia inaequalis, the causal agent of apple scab. The gene was 10.65 kbp in size and contained seven exons and six introns. The exons encoded a protein of 393 amino acids. Comparison of the deduced amino-acid sequence with cytochrome b proteins from other fungi revealed highest homologies to the respective proteins of Aspergillus nidulans, Podospora anserina and Neurospora crassa. All amino acids of the V. inaequalis cytochrome b at positions altered in mutants of Saccharomyces cerevisiae resistant to strobilurins, and other fungi with reduced sensitivities to strobilurins, were identical to wild-type isolates of several fungi. The cloning and characterization of the V. inaequalis cytochrome b gene is the initial step in the assessment of resistance risks inherent to the strobilurin fungicides.

Diversity of kresoxim‐methyl sensitivities in baseline populations of Venturia inaequalis

Several strobilurin fungicides inhibiting fungal respiration by binding to cytochrome b have been introduced recently. A mechanism of strobilurin resistance identified as active in several plant pathogenic fungi is based on the activation of alternative respiration. Thus far, respective studies have been restricted to single isolates of respective pathogens. Here, we report a study on 250 Venturia inaequalis baseline isolates to the strobilurin kresoxim-methyl having a broad sensitivity distribution characterized by a 50-fold difference in sensitivity of the most- and least-sensitive isolates. For the majority (62%) of these isolates, differences in sensitivity were not caused by the interference of alternative respiration with the full inhibitory potency of kresoxim-methyl. Rather, variable dose-responses with largely different degrees of inhibition achieved at a low dose of kresoxim-methyl were found to be responsible. For 38% of the baseline isolates, alternative respiration was already active during the stage of conidia germination. Activation of this pathway was, again, dependent on the strobilurin dose. Selection of sub-populations of isolates resisting low doses of kresoxim-methyl by multiple mechanisms and the recombination among isolates expressing such mechanisms singly can be expected to be slowed by an anti-resistance strategy based on high strobilurin doses. © 1999 Society of Chemical Industry

Disruption of the alternative oxidase gene in Magnaporthe grisea and its impact on host infection.

Plants and numerous fungi including Magnaporthe grisea protect mitochondria from interference by respiration inhibitors by expressing alternative oxidase, the enzymatic core of alternative respiration. The alternative oxidase gene AOXMg of M. grisea was disrupted. Several lines of evidence suggested that the disruption of AOXMg was sufficient to completely curb the expression of alternative respiration. In the infection of barley leaves, several AOXMg-minus and, thus, alternative respiration-deficient mutants of M. grisea retained their pathogenicity without significant impairment of virulence. However, differences between the wild-type strain and an AOXMg-minus mutant were apparent under oxidative stress conditions generated by the treatment of infected barley leaves with the commercial respiration inhibitor azoxystrobin. Symptom development was effectively suppressed on leaves infected with the alternative respiration-deficient mutant, while lesions on leaves infected with the wild-type strain continued to develop at much higher inhibitor doses. However, respective lesions rarely developed to the stage of full maturity. The results did not conform to a previous model implying that expression of alternative respiration is silenced during pathogenesis by the presence of constitutive plant antioxidants. Rather, alternative respiration provided protection from azoxystrobin during both saprophytic and infectious stages of the pathogen. The nature of similar oxidative stress conditions in the ecology of M. grisea remains an open question.

Diversity of cutinases from plant pathogenic fungi: Evidence for a relationship between enzyme properties and tissue specificity

Abstract Three fungal pathogens with different tissue specificities were cultivated on cutin as the sole carbon source and assayed for cutinase activity over a wide range of pH values. Cutin hydrolysis was optimal at pH 6·5 for the leaf pathogen Cochliobolus heterostrophus , and at pH 8·5 for the stem-base pathogen Rhizoctonia solani (AG 2-2). The pH-profile observed for Altemaria brassicicola , a pathogen that infects both leaves and stems, exhibited two distinct optima. Two cutinases from this organism were separated by chromatofocusing, one with a pH optimum at 7·0 and the other with an optimum at pH 9·0. The presence of different types of cutinase secreted by these pathogens was substantiated by treatment of the culture filtrate with [ 3 H] diisopropyl fluorophosphate, an active-site probe for serine esterases. Electrophoresis of labelled proteins under denaturing conditions revealed two types of serine esterases in the molecular weight range of cutinase. Serine esterases with cutinolytic pH optima in the slightly acidic range had molecular weights of 23·0 kDa ( A. brassicicola ) and 22·0 kDa ( C. heterostrophus ). Esterases with cutinolytic pH optima in the alkaline range appeared as doublets, as previously observed in Fusarium spp. The esterase doublet from A. brassicicola had molecular weights of 19·0 and 21·0 kDa, and that from R. solani had molecular weights of 17·0 and 18·5 kDa. These results, together with evidence from the literature, indicated a relationship between cutinase properties and the expression of tissue specificity by directly penetrating fungi. To substantiate this hypothesis, inoculum from a stem specific isolate of R. solani nonpathogenic on leaves was amended with cutinases purified from either Venturia inaequalis or Fusarium solani f. sp pisi . Inoculum amended with cutinase from the leaf-specific pathogen penetrated the bean leaf cuticle and produced disease symptoms, whereas inoculum amended with cutinase from the stem-base pathogen remained nonpathogenic on bean leaves. These results are the first evidence for a role of cutinase in the expression of tissue specificity by fungal pathogens.

Diversity of cutinases from plant pathogenic fungi: cloning and characterization of a cutinase gene from Alternaria brassicicola

Alternaria brassicicola produced two cutinase isozymes in the presence of cutin monomers as specific inducers. A cDNA library was constructed from poly(A)+RNA isolated from mycelium incubated with cutin monomers. Specific cDNA clones were selected from the library according to their abilities to hybridize with first strand cDNA prepared from induced cultures, but not from glucose-grown cultures. Cutinase-specific cDNA clones were identified by Southern analysis of plasmid DNA, employing a mixture of two heterologous cutinase cDNAs and one cutinase gene as probes. The largest 984 bp insert found among positive clones contained the entire cutinase coding region composed of 209 amino acids. The amino acid sequence predicted from the cDNA nucleotide sequence contained amino acids and sequences highly conserved among fungal cutinases. The sequences of four additional positive cDNAs were identical and thus gave no indication for the presence of a second gene of origin. Southern analysis of genomic DNA of A. brassicicola yielded a similar result. The structural gene of cutinase (CUTAB1) was contained within a 1545 bp genomic DNA fragment. Nucleotide sequences of the cDNA and the gene were identical, with the exception of one intron of 56 bp. The location of the intron was identical with introns identified in other fungal cutinase genes. The potential role of CUTAB1 in pathogenicity will be determined by gene disruption.

Cutinase and other lipolytic esterases protect bean leaves from infection by Rhizoctonia solani

The results describe a novel activity of fungal cutinase, the protection of bean leaves from disease. Development of web blight symptoms on bean leaves infected with Rhizoctonia solani (AG-1) was prevented in the presence of cutinase purified from Venturia inaequalis. Instead of disease, small areas of tissue necrosis became visible, and the tissue in which the pathogen was restricted displayed strong autofluorescence beneath the inoculation sites. Mechanical wounding of the leaf surface had no effect on disease development and the permeability of the cuticle was not increased by cutinase action, indicating that surface wounding was not the cause for this novel activity of cutinase. A comparative study involving cutinase and other serine hydrolases revealed that the disease prevention resided in the lipolytic esterase activity rather than the cutinase activity. The pattern of expression of four pathogenesis-related (PR) protein genes provided no evidence for the modulation of known resistance responses of...

Biolistic transformation of conidia of Botryotinia fuckeliana

Botryotinia fuckeliana, the causal agent of grey mould, was biolistically transformed to hygromycin B resistance using a plasmid (pOHT) containing a bacterial hygromycin phosphotransferase gene fused to regulatory sequences from Aspergillus nidulans. Multiple copies of the plasmid, precipitated onto tungsten particles, were delivered into the conidia by a helium-driven gene gun. Southern analysis showed that the plasmid was integrated into the fungal genome at one single locus. After five subsequent transfers on selective medium, all transformants were mitotically stable. When propagated on non-selective medium, four out of eight transformants retained their resistance to hygromycin B. Southern analysis of the fifth generation of transformants showed that no genetic rearrangements occurred during vegetative growth of stable transformants.

Seasonal changes of sensitivities to sterol demethylation inhibitors in Venturia inaequalis populations

During three seasons and at three different orchard sites, the sensitivities of V. inaequalis populations to sterol demethylation inhibitors were reduced over the course of the summer and in the absence of exposure to these fungicides. A seasonal fluctuation was not observed for dodine with a mode of action unrelated to inhibition of sterol demethylation. Seasonal fluctuations of population sensitivities to sterol demethylation inhibitors are of theoretical and practical importance. They not only reflect a rarely reported population response to changing environmental conditions over a growing season, but also have implications regarding the development of anti-resistance strategies for the control of apple scab with sterol demethylation inhibitors. As the frequency of the least sensitive population segment increases in late summer, the performance of these fungicides is most likely decreased. The resulting effect on disease incidence and selection of a resistant subpopulation can be expected to accelerate the speed of resistance development.