Production of Metal‐Chelating Compounds by Species of Heterobasidion annosum sensu lato

The role of iron and compounds that chelate iron in the development of fungal diseases and wood degradation is not well understood, and their involvement in the simultaneous pathogenic and wood‐decomposing capabilities of Heterobasidion annosum s.l. is unknown. In the current study, the production of low‐molecular‐mass compounds that can chelate iron, such as catecholate, hydroxamate and oxalate, by H. annosum s.l. was correlated positively with supplementation of the medium with iron. In contrast, iron supplementation did not increase the Fe³⁺‐reducing ability of H. annosum s.s. and H. abietinum hyphae. Indeed, H. annosum s.s. is known to cause higher mortality of the plant host, but produced a lower quantity of siderophores than H. abietinum or H. parviporum. Under iron supplementation, siderophore production was correlated with phenoloxidase activity in the low‐molecular‐mass fraction, which might have consequences for cell wall decomposition.     

WS-5995 B,an antifungal agent inducing differential gene expression in the conifer pathogen <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> but not in <Emphasis Type="Italic">Heterobasidion abietinum</Emphasis>

The mycorrhization helper bacterium Streptomyces sp. AcH 505 inhibits Norway spruce root infection and colonisation by the root and butt rot fungus Heterobasidion annosum 005 but not by the congeneric strain Heterobasidion abietinum 331 because of higher sensitivity of H. annosum 005 towards the AcH 505-derived naphthoquinone antibiotic WS-5995 B. Differences in antibiotic sensitivity between two isolates belonging to two species, H. annosum 005 and H. abietinum 331, were investigated by comparative gene expression analysis using macroarrays and quantitative RT-PCR after WS-5995 B, structurally related mollisin and unrelated cycloheximide application. Treatment with 25 μM WS-5995 B for 2 h resulted in a significant up-regulation of expression of inosine-5′-monophosphate dehydrogenase, phosphoglucomutase and GTPase genes, while the expression of genes encoding for thioredoxin and glutathione dependent formaldehyde dehydrogenase was down-regulated in the sensitive fungal strain. No differential expression in the tolerant strain was detected. Application of WS-5995 B at higher concentrations over a time course experiment revealed that H. annosum 005 and H. abietinum 331 responded differently to WS-5995 B. The fungal gene expression levels depended on both the concentration of WS-5995 B and the duration of its application. The WS-5995 B-unrelated cycloheximide caused highly specific changes in patterns of gene expression. Our findings indicate considerable variations in response to bacterial metabolites by the isolates of the conifer pathogen.     

An ectomycorrhizal symbiosis differently affects host susceptibility to two congeneric fungal pathogens

The role of ectomycorrhizal fungi in modulating host susceptibility to fungal pathogens is poorly known. Pinus sylvestris susceptibility to two congeneric fungal pathogens was compared between mycorrhizal and non-mycorrhizal plants. The ectomycorrhizal fungus was Suillus luteus, while the pathogens were Heterobasidion irregulare and H. annosum. H. irregulare is native to North-America and invasive to Europe, whereas S. luteus and H. annosum are native to Eurasia. Non-mycorrhizal plants were equally susceptible to both pathogens. Mycorrhizal plants were significantly less susceptible to H. annosum than non-mycorrhizal plants, whereas there were no differences when considering H. irregulare. The abundance of ectomycorrhizas was negatively and significantly correlated to the level of host susceptibility only to H. annosum. This study shows that the protective role of ectomycorrhizal fungi may differ when the host is challenged by congeneric fungal pathogens. Results add a further clue to explain and predict the invasiveness of H. irregulare in Europe.     

Does temperature regime govern the establishment of <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> in Scandinavia?

We explored the reasons underlying the biogeographic distribution patterns of the economically important, wood-rotting basidiomycete Heterobasidion annosum in Sweden. Despite the commonness of suitable host trees, Heterobasidion annosum has not been recorded in the north of Sweden, whereas its relative, H. parviporum, is present throughout the country. To test the hypothesis that H. annosum has not spread to the north because of the effect of climate, mainly differences in the general temperature regime, we inoculated Norway spruce stumps and standing trees with H. annosum and H. parviporum at six field sites, three in the south and three in the north of Sweden. Three strains of both species were used in random combinations, so that each selected stump and tree was inoculated with both species at the same time. At 2 and 10 months after the inoculations, we compared the frequencies of detection of H. annosum and H. parviporum colonies at different distances from inoculation points in the stumps and in trees. The H. annosum colonies were detected only infrequently on disks cut from the inoculated stumps (0–4% of re-isolations) in both areas, whereas H. parviporum was detected much more frequently (26–47% of re-isolations). In standing trees, colonies belonging to H. annosum could be detected up to 210 cm (south) and 80 cm (north) and those belonging to H. parviporum up to 210 cm (south) and 140 cm (north) above the inoculation points. Our results suggest that difference in temperature regime does not provide an explanation for the distribution limit of H. annosum.     

Congruent population genetic structures and divergence histories in anther‐smut fungi and their host plants Silene italica and the Silene nutans species complex

Study of the congruence of population genetic structure between hosts and pathogens gives important insights into their shared phylogeographical and coevolutionary histories. We studied the population genetic structure of castrating anther‐smut fungi (genus Microbotryum) and of their host plants, the Silene nutans species complex, and the morphologically and genetically closely related Silene italica, which can be found in sympatry. Phylogeographical population genetic structure related to persistence in separate glacial refugia has been recently revealed in the S. nutans plant species complex across Western Europe, identifying several distinct lineages. We genotyped 171 associated plant–pathogen pairs of anther‐smut fungi and their host plant individuals using microsatellite markers and plant chloroplastic single nucleotide polymorphisms. We found clear differentiation between fungal populations parasitizing S. nutans and S. italica plants. The population genetic structure of fungal strains parasitizing the S. nutans plant species complex mirrored the host plant genetic structure, suggesting that the pathogen was isolated in glacial refugia together with its host and/or that it has specialized on the plant genetic lineages. Using random forest approximate Bayesian computation (ABC‐RF), we found that the divergence history of the fungal lineages on S. nutans was congruent with that previously inferred for the host plant and probably occurred with ancient but no recent gene flow. Genome sequences confirmed the genetic structure and the absence of recent gene flow between fungal genetic lineages. Our analyses of individual host–pathogen pairs contribute to a better understanding of co‐evolutionary histories between hosts and pathogens in natural ecosystems, in which such studies remain scarce.     

The capacity in Heterobasidion annosum s.l. to resist overgrowth by the biocontrol agent Phlebiopsis gigantea is a heritable trait

The necrotrophic pathogen Heterobasidion annosum sensu lato (Fr.) Bref. causes severe root rot on coniferous trees in the boreal and temperate forests. The annual economic losses caused by this fungus in Europe are estimated to at least 790 million €. In managed forests, the major route of infection is via stump surfaces from which the H. annosum s.l. grows through the roots and attacks adjacent healthy trees. A biocontrol method to reduce H. annosum s.l. infection is to apply the wood degrading fungus Phlebiopsis gigantea in a spore solution (Rotstop) directly on the freshly cut stumps immediately after cutting. We investigated the potential risk for a build-up in the capacity of H. annosum s.l. to resist overgrowth by P. gigantea. Wood blocks of Picea abies, precolonized with the two fungal species, were juxtaposed on top of agar and the overgrowth of the P. gigantea strain (Rotstop) on the H. annosum s.l. was measured periodically. We found a natural variation in Heterobasidion parviporum to resist overgrowth by P. gigantea. There was no difference between homo- and heterokaryotic strains. In a mapping population of 91 progenies from a H. annosum hybrid strain we were able to identify one quantitative trait locus (QTL) which controls the examined resistance capacity. We estimated the broad sense heritability to 0.336 for the capacity to resist the P. gigantea overgrowth. We conclude that there exists a theoretical risk for resistance build-up in the H. annosum s.l. population towards its biological control agent P. gigantea.     

Comparative pathobiology of Heterobasidion annosum during challenge on Pinus sylvestris and Arabidopsis roots: an analysis of defensin gene expression in two pathosystems

Heterobasidion annosum is widely known as a major root and butt rot pathogen of conifer trees, but little information is available on its interaction with the roots of herbaceous angiosperm plants. We investigated the infection biology of H. annosum during challenge with the angiosperm model Arabidopsis and monitored the host response after exposure to different hormone elicitors, chemicals (chitin, glucan and chitosan) and fungal species that represent diverse basidiomycete life strategies [e.g., pathogen (H. annosum), saprotroph (Stereum sanguinolentum) and mutualist (Lactarius rufus)]. The results revealed that the tree pathogen (H. annosum) and the saprotroph (S. sanguinolentum) could infect the Col-8 (Columbia) ecotype of Arabidopsis in laboratory inoculation experiments. Germinated H. annosum spores had appressorium-like penetration structures attached to the surface of the Arabidopsis roots. Subsequent invasive fungal growth led to the disintegration of the vascular region of the root tissues. Progression of root rot symptoms in Arabidopsis was similar to the infection development that was previously documented in Scots pine seedlings. Scots pine PsDef1 and Arabidopsis DEFLs (AT5G44973.1) and PDF1.2 were induced at the initial stage of the infection. However, differences in the expression patterns of the defensin gene homologs from the two plant groups were observed under various conditions, suggesting functional differences in their regulation. The potential use of the H. annosum–Arabidopsis pathosystem as a model for studying forest tree diseases is discussed.     

Early defence reactions in Norway spruce seedlings inoculated with the mycorrhizal fungus <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis> (Persoon) Coker &; Couch and the pathogen <Emphasis Type="Italic">Heterobasidion annosum</Emphasis> (Fr.) Bref.

The activities of the enzymes responsible for cell-wall strengthening and salicylic acid (SA) content in Norway spruce seedlings were investigated after inoculation with the ectomycorrhizal fungus Pisolithus tinctorius or the pathogen Heterobasidion annosum, and after treatment with elicitors from both of these fungi. Inoculation with both fungi increased guaiacol peroxidase (POD) activity in the roots of the pathogen-inoculated seedlings during the earliest phases of colonisation, and induced the activities of several POD isoforms. Two of these were only seen in pathogen-inoculated seedlings and corresponded with increased POD activity against ferulic acid. Colonisation with H. annosum triggered an increase in phenylalanine ammonia lyase (PAL) activity in the roots of the spruce seedlings, which was followed by an accumulation of free SA. One month after inoculation levels of free SA were increased also in the shoots of H. annosum-inoculated seedlings. In contrast increase in free SA content in the roots of P. tinctorius-inoculated seedlings was only transient. Similarly to inoculation, treatment with elicitors of H. annosum increased the PAL and POD activity, as well as SA content in the roots of spruce seedlings. A positive correlation between PAL activity and SA content in the H. annosum-inoculated seedlings and accumulation of SA precursors in the phenylpropanoid pathway indicate that the plant defence mechanisms, during which SA is synthesised through the PAL pathway, are exploited by H. annosum for facilitation of colonisation.     

Interactions between embryogenic callus of Abies alba and Heterobasidion spp. in dual cultures

Interactions between three genotypes of a silver fir (Abies alba Mill.) embryogenic callus and Heterobasidion abietinum, H. parviporum, and H. annosum were examined in dual cultures. The aim of this study was to determine whether dual cultures can be used to evaluate the degree of fungal virulence at an embryogenic level, and whether different genotypes of a callus show different susceptibility. The dual cultures were performed on Schenk and Hildebrandt medium. Mycelial growth of H. parviporum and H. annosum was significantly stimulated in the presence of the callus but was not directional in nature. The embryogenic callus died between six and nine days after being colonized by H. parviporum suggesting that this was the most virulent species. By contrast, the callus remained healthy for up to 50 d after colonization with H. abietinum suggesting that this was the least virulent species. The callus of the A. alba genotype which originated in the mountain region of Poland remained healthy and alive for a significantly longer period than the other two genotypes in the dual cultures with all three Heterobasidion species even though overgrown by mycelium suggesting that the mountain genotype had the strongest defence response to Heterobasidion infection.     

Evidence for multiple ecological roles of Leptographium abietinum,a symbiotic fungus associated with the North American spruce beetle

The ophiostomatoid fungus Leptographium abietinum is symbiotic with the North American spruce beetle Dendroctonus rufipennis; however, the ecology of these interactions are not understood. Multiple functional hypotheses regarding beetle-symbiont interactions pervade the literature, especially the view that symbionts may provide nutrition, competitively exclude antagonistic microbes, or detoxify host plant compounds. Here, these three hypotheses are tested in an effort to discern whether the ecological profile of L. abietinum is consistent with bark beetle-fungus mutualisms. Three important findings emerged: (1) by comparison with conifer phloem, L. abietinum mycelia contained considerable quantities of N, P, and protein; (2) L. abietinum outcompetes a ubiquitous entomopathogen for growing space in a resource-limited environment and can maintain captured space; and (3) inoculation with L. abietinum significantly reduced concentrations of a tree defensive compound, (+)-3-carene, in growth media. Collectively, these findings indicate that L. abietinum fulfills multiple ecological functions that are potentially consistent with bark beetle-fungus mutualism.     

Molecular analyses identify hybridization‐mediated nuclear evolution in newly discovered fungal hybrids

Hybridization may be a major driver in the evolution of plant pathogens. In a high elevation Alpine larch stand in Montana, a novel hybrid fungal pathogen of trees originating from the mating of Heterobasidion irregulare with H. occidentale has been recently discovered. In this study, sequence analyses of one mitochondrial and four nuclear loci from 11 Heterobasidion genotypes collected in the same Alpine larch stand indicated that hybridization has increased allelic diversity by generating novel polymorphisms unreported in either parental species. Sequence data and ploidy analysis through flow cytometry confirmed that heterokaryotic (n + n) genotypes were not first‐generation hybrids, but were the result of multiple backcrosses, indicating hybrids are fertile. Additionally, all admixed genotypes possessed the H. occidentale mitochondrion, indicating that the hybrid progeny may have been backcrossing mostly with H. occidentale. Based on reticulate phylogenetic network analysis by PhyloNet, Bayesian assignment, and ordination tests, alleles can be defined as H. irregulare‐like or H. occidentale‐like. H. irregulare‐like alleles are clearly distinct from all known H. irregulare alleles and are derived from the admixing of both Heterobasidion species. Instead, all but one H. occidentale alleles found in hybrids, although novel, were not clearly distinct from alleles found in the parental H. occidentale population. This discovery demonstrates that Alpine larch can be a universal host favouring the interspecific hybridization between H. irregulare and H. occidentale and the hybridization‐mediated evolution of a nucleus, derived from H. irregulare parental species but clearly distinct from it.     

Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans

Non‐host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre‐ and post‐invasive resistance responses. Pre‐invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non‐host resistance to P. infestans, a genetic screen was performed by re‐mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2‐1 erp1‐3 and pen2‐1 erp1‐4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T‐DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen‐inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non‐autonomous defense responses against invasive filamentous pathogens.     

Efficient production of Al(OH)3‐immobilized laccase with a Heterobasidion annosum strain selected by microplate screening

Aims: Wild‐type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. Methods and Results: By using a newly developed 96‐well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l^?1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger‐scale liquid cultures. Aeration with a sparger lead to a 3·8‐fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l^?1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short‐range ordered Al(OH)₃ particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)₃‐adsorbed laccase was suitable for repeated decolourization of indigo carmine. Conclusions: Microplate cultivation allowed a large‐scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)₃ proved to be suitable as direct immobilization technique. Significance and Impact of the Study: The microplate screening method simplifies strain and medium development for laccase production. Two novel fungal strains suitable for laccase production were identified. Procedures for simple and efficient production of immobilized H. annosum laccase were established.     

LsGRP1, a class II glycine-rich protein of Lilium,confers plant resistance via mediating innate immune activation and inducing fungal programmed cell death

Defence-related LsGRP1 is a leaf-specific plant class II glycine-rich protein (GRP) involved in salicylic acid-induced systemic resistance against grey mould caused by necrotrophic Botrytis elliptica in lily (Lilium) cultivar Stargazer. The C-terminal region of LsGRP1 (LsGRP1^C) can inhibit fungal growth in vitro via a mechanism of inducing fungal apoptosis programmed cell death (PCD). In this study, the role of LsGRP1 in induced defence mechanism was investigated using LsGRP1-silenced Stargazer lily and LsGRP1-transgenic Arabidopsis thaliana. LsGRP1 silencing in lily was found to slightly inhibit plant growth and greatly increase the susceptibility to B. elliptica by suppressing callose deposition and early reactive oxygen species (ROS) accumulation. In contrast, LsGRP1-transgenic Arabidopsis showed higher resistance to Botrytis cinerea and also to Pseudomonas syringae pv. tomato DC3000 as compared to the wild type, accompanied with the enhancement of callose deposition and ROS accumulation. Additionally, LsGRP1 silencing increased plant cell death caused by B. elliptica secretion and reduced pathogen-associated molecular pattern (PAMP)-triggered defence activation in Stargazer lily. Consistently, LsGRP1 expression boosted PAMP-triggered defence responses and effector recognition-induced hypersensitive response in Arabidopsis. Moreover, fungal apoptosis PCD triggered by LsGRP1 in an LsGRP1^C-dependent manner was demonstrated by leaf infiltration with LsGRP1^C-containing recombinant proteins in Stargazer lily. Based on these results, we presume that LsGRP1 plays roles in plant defence via functioning as a pathogen-inducible switch for plant innate immune activation and acting as a fungal apoptosis PCD inducer to combat pathogen attack.     

Rapid linkage of indole carboxylic acid to the plant cell wall identified as a component of basal defence in Arabidopsis against hrp mutant bacteria

Changes occurring to plant cell walls were examined following inoculation of Arabidopsis leaves with pathogenic and non-pathogenic (hrpA mutant) strains of Pseudomonas syringae pv. tomato. We have targeted low molecular weight, cross-linked phenolic and indolic compounds that were released from wall preparations by alkaline hydrolysis at 70 °C and in a microwave bomb. Significantly higher concentrations of syringaldehyde, p hydroxybenzaldehyde and indole carboxylic acid were recovered from cell walls isolated from leaves 24 h after challenge with the hrpA mutant compared with wild-type DC3000. Time course experiments showed that the accumulation of indole carboxylic acid and the other group of differentiating metabolites had occurred within 12 h of inoculation. The callose synthase deficient mutant pmr4-1 was more resistant than wild-type Columbia plants to P. syringae pv. tomato. Restricted bacterial multiplication was associated with increased accumulation of indole carboxylic acid on the plant cell wall. In the absence of callose deposition in the pmr 4-1 mutant, indolic derivatives may serve as a structural scaffold for wall modifications following bacterial challenge.     

Comparing the influences of ecological and evolutionary factors on the successful invasion of a fungal forest pathogen

The fungal forest pathogen Heterobasidion annosum has been introduced from North America into Italy and is now associated with high mortality of Italian stone pines. Due to the presence of a closely related native H. annosum taxon, this pathosystem presents an unusual opportunity to test specific ecological and evolutionary factors influencing fungal invasions. Comparative inoculation experiments on Scots pine cuttings and on seedlings of European and North American pines failed to identify significant increased pathogenicity of North American genotypes on European hosts congruent with lack of host-pathogen co-evolution. However, spore trappings indicate that while reproductive potential of native H. annosum was significantly reduced in the dry season, that of the invasive taxon was consistently high regardless of season. Ecological differences between the native and exotic taxon may therefore facilitate this invasion. Understanding which factors enhance this emerging forest disease is important both for biotic invasion theory and for disease control.     

Colonization of Fungi and Bacteria in Stumps and Roots of Scots Pine after Thinning and Treatment with Rotstop

The research areas were located in the Pisz Forest District, northeast Poland, in 10‐year‐old Scots pine (Pinus sylvestris L.) plantations, established in 2004 on a clear‐cut area. Reforestation was performed without a biological treatment against root pathogens, despite the presence of Heterobasidion annosum and Armillaria ostoyae in roots and stumps of trees growing previously. The aim of this research was to evaluate how thinning and treatment with the biological control agent Rotstop influences bacterial and fungal communities within roots and stumps. Twelve months after thinning, samples were collected from five stumps in each of two seasons, autumn and spring, from stands on two types of site, one previously forested and one agricultural (20 stumps in total). Wood samples were cultured on agar media, and (i) fungi in the upper part of the stump and (ii) in roots and (iii) bacteria in roots were genetically identified. Sequences were genetically identified by comparing sequences with records held in the GenBank database. We found great differences in the frequency of both fungi and bacteria in roots: they were more frequent (i) in healthy stumps compared to stumps infected with pathogens (H. annosum and A. ostoyae), (ii) in postagricultural soil than in forest soil and (iii) after spring rather than autumn biological treatment. The introduced species Phlebiopsis gigantea was only identified in the parts of the stumps which were above ground level. The bacterium Paenibacillus pini was associated with the presence of H. annosum infecting the stumps from the roots side. In areas seriously threatened by root pathogens, biological treatment can play only a limited role. It can spread to the upper part and impede the production of fruitbodies; however, it has no impact on the development of pathogens in deeper root areas.     

Race cultivar-specific differences in callose deposition in soybean roots following infection with Phytophthora megasperma f.sp. glycinea

Primary roots of soybean (Glycine max (L.), Merrill, cv. Harosoy 63) seedlings were inoculated with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea and total callose was determined at various times after inoculation. From 4 h onward, total callose was significantly higher in roots showing the resistant rather than the susceptible response. Local callose deposition in relation to location of fungal hyphae was determined in microtome sections by its specific fluorescence with sirofluor and was quantified on paper prints with an image-analysis system. Callose deposition, which occurs adjacent to hyphae, was found soon after inoculation (2, 3 and 4 h post inoculation) only in roots displaying the resistant response, and was also higher at 5 and 6 h after inoculation in these resistant roots than in susceptible roots. Early callose deposition in the incompatible root-fungus reaction could be a factor in resistance of soybean against P. megasperma.Abbreviation pi post inoculation     

Defining Plant Resistance to Phytophthora cinnamomi: A Standardized Approach to Assessment

Phytophthora cinnamomi is a soil‐borne plant pathogen that causes devastating disease in agricultural and natural systems worldwide. While a small number of species survive infection by the pathogen without producing disease symptoms, the nature of resistance, especially under controlled conditions, remains poorly understood. At present, there are no standardized criteria by which resistance or susceptibility to P. cinnamomi can be assessed, and we have used five parameters consisting of plant fresh weight, root growth, lesion length, relative chlorophyll content of leaves and pathogen colonization of roots to analyse responses to the pathogen. The parameters were tested using two plant species, Zea mays and Lupinus angustifolius, through a time course study of the interactions and resistance and susceptibility defined 7 days after inoculation. A scoring system was devised to enable differentiation of these responses. In the resistant interaction with Z. mays, there was no significant difference in fresh weight, root length and relative chlorophyll content in inoculated compared with control plants. Both lesion size and pathogen colonization of root tissues were limited to the site of inoculation. Following inoculation L. angustifolius showed a significant reduction in plant fresh weight and relative leaf chlorophyll content, cessation of root growth and increased lesion lengths and pathogen colonization. We propose that this technique provides a standardized method for plant–P. cinnamomi interactions that could be widely used to differentiate resistant from susceptible species.