Pathogen Ingress Research Articles

Cytology of Infection of Maize Seedlings by Fusarium moniliforme and Immunolocalization of the Pathogenesis-Related PRms Protein

ABSTRACT We have investigated the histology of infection of maize seedlings by Fusarium moniliforme in association with a biochemical host defense response, the accumulation of the PRms (pathogenesis-related maize seed) protein. Light microscopy of trypan blue-stained sections and scanning electron microscopy revealed direct penetration by F. moniliforme hyphae through the epidermal cells of the seedling and colonization of the host tissue by inter- and intracellular modes of growth. Pathogen ingress into the infected tissue was associated with the induction of defense-related ultrastructural modifications, as exemplified by the formation of appositions on the outer host cell wall surface, the occlusion of intercellular spaces, and the formation of papillae. Cellular and subcellular immunolocalization studies revealed that PRms accumulated at very high levels in those cells types that represent the first barrier for fungal penetration such as the aleurone layer of germinating seeds and the scutellar epithelial cells of isolated germinating embryos. A highly localized accumulation of PRms within papillae of the inner scutellar parenchyma cells also occurred, suggesting that signaling mechanisms that lead to the accumulation of PRms in papillae of cell types that are distant from the invading pathogen must operate in the infected maize tissues. Our study also revealed the presence of a large number of fungal cells with an abnormal shape that showed PRms-specific labeling. PRms was found to accumulate in clusters over the fungal cell wall. Taken together, the occurrence of PRms in cell types that first establish contact with the pathogen, as well as in papillae, and in association with fungal cell walls suggests that PRms may have a function in the plant defense response.

OLIGOAGARS ELICIT A PHYSIOLOGICAL RESPONSE IN GRACILARIA CONFERTA (RHODOPHYTA)

Gracilaria conferta (Schousboe ex Montagne) J. et G. Feldmann responded with an oxidative burst and rapid increases in respiration and halogenating activity when agar, agarose, or the agarose degradation products neoagarotetraose and neoagarohexaose were added to the growth medium. In contrast, carrageenan, oligocarrageenans, neoagarobiose, l‐galactose, d‐galactose, and several other mono‐ and oligosaccharides did not have any effect. Sixfold increases in respiration were observed 3 min after addition of neoagarohexaose. The response could only be induced in species of the genera Gracilaria and Gracilariopsis. Neoagarohexaose also elicited a release of hydrogen peroxide in less than 15 min, resulting in an immediate increase in algal brominating activity. Bleached thallus tips appeared a few hours after the addition of neoagarohexaose. This effect was dependent on the release of hydrogen peroxide and exposure to light. Exposure to light and oligosaccharide elicitors increased the production of reactive oxygen species, which reached destructive concentrations when both mechanisms were simultaneously active. Concentrations of 0.1 to 3.3 μM agarose or agars were sufficient to trigger an increase in respiration, an oxidative burst response, and tip bleaching. However, higher concentrations of neoagarohexaose and neoagarotetraose were necessary to elicit the responses, indicating that the alga is more sensitive to oligoagars with degrees of biose‐polymerization > 3. The extremely short reaction time and high specificity indicate that intermediates of agar degradation are recognized by Gracilaria as messengers when microbial degradation of its cell wall occurs. The physiological responses may represent the early stages of algal defense mechanisms involved in repression of pathogen ingress.

Benzothiadiazole-mediated induced resistance to fusarium oxysporum f. sp. radicis-lycopersici in tomato

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), a synthetic chemical, was applied as a foliar spray to tomato (Lycopersicon esculentum) plants and evaluated for its potential to confer increased resistance against the soil-borne pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In nontreated tomato plants all root tissues were massively colonized by FORL hyphae. Pathogen ingress toward the vascular stele was accompanied by severe host cell alterations, including cell wall breakdown. In BTH-treated plants striking differences in the rate and extent of fungal colonization were observed. Pathogen growth was restricted to the epidermis and the outer cortex, and fungal ingress was apparently halted by the formation of callose-enriched wall appositions at sites of fungal penetration. In addition, aggregated deposits, which frequently established close contact with the invading hyphae, accumulated in densely colonized epidermal cells and filled most intercellular spaces. Upon incubation of sections with gold-complexed laccase for localization of phenolic-like compounds, a slight deposition of gold particles was observed over both the host cell walls and the wall appositions. Labeling was also detected over the walls of fungal cells showing signs of obvious alteration ranging from cytoplasm disorganization to protoplasm retraction. We provide evidence that foliar applications of BTH sensitize susceptible tomato plants to react more rapidly and more efficiently to FORL attack through the formation of protective layers at sites of potential fungal entry.

Distribution of Zimbabwean Armillaria groups and their pathogenicity on cassava

Disease surveys carried out between 1989 and 1992 showed Armillaria to be restricted to the northern and eastern parts of Zimbabwe and to be absent from the western and southern parts. Armillaria disease is of local economic significance especially in fruit and clonal pine seed orchards. Pathogenicity studies showed significant differences in infection of cassava (Manihot esculenta) variety Zanaga (Ccv1) between isolates belonging to the three Zimbabwean Armillaria groups (I, II and III), but there were no significant differences in the infection of another cassava variety, Zcv1, from Zimbabwe. The group II isolates failed to cause any infection on the cassava clones. Rhizomorphs were the main means of infection in the pathogenicity studies, although they are rare in nature in Zimbabwe. Results using detached cassava tubers to assess pathogenicity proved to be inconsistent. Periderm restoration responses, which have been observed in woody plants and are presumed to function by restricting pathogen ingress, were observed in cassava infected with Armillaria.

Induction of systemic resistance to Pythium damping‐off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response

Benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH, CGA 245704), a non-toxic, synthetic chemical, was applied as a foliar spray to cucumber plants and evaluated for its potential to induce defense mechanisms in root tissues infected by the soilborne pathogen, Pythium ultimum Trow. In non-treated cucumber plants, fungal colonization was intense and paralleled marked host tissue damage, whereas in BTH-treated plants, pathogen ingress towards the vascular stele was apparently halted by the massive deposition of a phenolic-enriched material which occluded a large number of cortical and vascular parenchyma cells. This considerable increase in the accumulation of phenolics was accompanied by cytological disorders of the invading pathogen at a time when the wall-bound cellulose component was preserved. In addition to phenolic compounds, the occluding material contained large amounts of beta-glucoside residues. These residues gradually decreased in the areas neighboring fungal cells whereas phenolic deposition appeared to be more uniformly distributed throughout the occluded host cells. Pathogen penetration in non-occluded cucumber root cells coincided with other changes, mainly characterized by both the deposition onto the inner surface of the cell walls of some heterogeneous wall appositions and the coating of some intercellular spaces with an electron-opaque material. Evidence is provided in this study that BTH has the ability to induce SAR in cucumber. Exogenous, foliar applications of the chemical sensitize susceptible cucumber plants to react more rapidly and more efficiently to P. ultimum attack, mainly through the massive accumulation of phenolic compounds at sites of attempted pathogen penetration.

Ultrastructural and Cytochemical Investigation of Asymptomatic Infection by Pythium spp.

ABSTRACT The influence exerted by Pythium group F (a minor pathogen ubiquitous in soilless cultures) and P. uncinulatum (a nonpathogenic species) colonization on tomato roots was investigated. In both interactions, infected roots did not exhibit obvious symptoms; however, major physiological changes occurred within the host tissues colonized by Pythium group F compared to P. uncinulatum. According to our cytological observations, Pythium group F colonization involved a series of events: first, development and growth of the fungus in the epidermis and outer cortex tissues, which was associated with marked host cell disorganization and even breakdown. In colonized roots, symptoms were not easily discernible because alterations were restricted to the epidermis and outer cortex tissues. Second, pathogen ingress in the inner cortex and stele tissues was associated with massive induction of host defense reactions and alteration of invading hyphae. In a complex interaction that involved major metabolic changes in root cells, an array of defense-related reactions was produced, as exemplified by the formation of wall appositions and plugging of host cells with osmiophilic, electron-dense, granular, or fibrillar materials. P. uncinulatum growth was restricted to the epidermis and outer cortex tissues and associated with relatively minor damage to host cells. P. uncinulatum colonization of root tissues did not result in defense events similar to those observed in Pythium group F-infected inner cortex and stele tissues. Moreover, most of the invading P. uncinulatum hyphae were moribund. The results of the current study indicated that symptomless infections can be insignificant in terms of pathology for the plant (i.e., P. uncinulatum) or can be potentially dangerous (i.e., Pythium group F). Our results clearly showed a striking difference between nonpathogenic and minor pathogenic Pythium spp. attacks. The description of the Pythium group F infection process suggests that this interaction is unique, because the fungus behaves as a necrotrophic fungus in the epidermis and outer cortex tissues, whereas it is a potential inducer of plant defense reactions in the inner cortex and stele tissues. To our knowledge, cytological events similar to those observed with Pythium group F have not been described until now, even though descriptions of these events could help elucidate several aspects of the relationships between plants and minor pathogens.

Ultrastructural and cytochemical investigations of pathogen development and host reactions in susceptible and partially-resistant carrot roots infected by Pythium violae, the major causal agent for cavity spot

Two carrot genotypes, cultivar Nanco and line 24, susceptible and partially- resistant respectively to cavity spot, were compared ultrastructurally and cytochemically 24 h, 48 h and 72 h after root inoculation with a virulent Pythium violae isolate. The extent of pathogen ingress and the response of the host differed markedly with the two genotypes. In cv Nanco, growth of fungal hyphae was predominantly intracellular and was accompanied by pronounced damage; by 48 h after inoculation, pericycle and the first cell layers of the phloem parenchyma were invaded, resulting in host wall dissolution and cytoplasm aggregation. The growth of P. violae in line 24 was limited to the pericycle, even up to 72 h after inoculation; fungal colonization was accompanied by retraction of cytoplasm and in the appearance of granular or fibrillar material in the host cell lumen. Some affected host cells were filled with structureless osmophilic material. In cultivar Nanco, invading fungal hyphae were unaffected; by contrast in line 24, the cytoplasm of invading hyphae, particularly those inside the cell host, was disorganised and structureless. Infection and host response in the two cultivars were studied with two specific labels: Aplysia gonad lectin (AGL), a polygalacturonic acid-binding lectin, and an exoglucanase complexed to colloidal gold were used to locate pectin and cellulosic β-(1,4)-glucans respectively in infected tissues. The decrease of cytochemical labeling beyong fungal penetration showed clearly hydrolysis of pectin and cellulose in cell walls of the cv Nanco. By contrast, the cell wall of line 24 remained largely intact, although, unlabeled amorphous and electron-dense material was observed inside the wall. Fibrillar or electron dense material commonly observed in infected tissue of line 24 apparently did not contain pectic or cellulosic substances. Moreover, material observed in host cells or fungal hyphae was also free of labeling. The origin and the chemical composition of these compounds as well as their possible role in the defence mechanisms of carrot against P. violae are discussed.

Increased resistance toFusarium oxysporumf. sp.radicis-lycopersiciin tomato plants treated with the endophytic bacteriumPseudomonas fluorescensstrain 63-28

The potential ofPseudomonas fluorescensstrain 63-28 for inducing defence reactions in tomato plants inoculated with the vascular fungusFusarium oxysporumf.sp.radicis-lycopersiciwas studied by light and transmission electron microcopy, and further investigated by gold cytochemistry. In non-bacterized tomato plants the pathogen multiplied abundantly through much of the root tissues, causing severe damage including cell disorganization and cell wall alterations. Marked changes in the rate and extent ofFusariumcolonization were observed in bacterized plants. The intensity of colonization was appreciably reduced compared to that observed in control plants, and pathogen growth was restricted to the outer root tissues with preferential localization in the intercellular spaces. The accumulation of an electron-dense material in epidermal and outer cortical cells and the coating of most intercellular spaces with similar substances were among the most typical features of host reactions. Cytochemical labelling of chitin showed that disorganization ofFusariumhyphae colonizing bacterized roots correlated with chitin degradation. These observations provided indirect evidence for the production of plant chitinases. Another noticeable change occurring during pathogen ingress in the outer root cortex was the elaboration of callose-enriched wall appositions at sites of attempted fungal penetration. These appositions were slightly labelled by the gold-complexed β-glucosidase, whereas gold particles were mainly concentrated over the more opaque regions lining the host cell walls and at the periphery of the electron-dense deposits accumulating in the intercellular spaces. The present ultrastructural observations confirm thatP. fluorescensstrain 63-28 may function as an inducer of plant disease resistance.

Ultrastructural and cytochemical studies of cucumber roots infected by two Pythiumspecies with different modes of pathogenicity

Two Pythiumspecies showing different patterns of pathogenicity, PythiumF and Pythium aphanidermatum, were inoculated into the roots of Cucumis sativusand their processes of infection investigated and compared. PythiumF, a ubiquitous minor pathogen of cucumber in soilless cultures, induces marked damage to the epidermal and cortical tissues but it never penetrated further than the endodermis. Host cell wall labelling with specific gold-complex probes (AGL-and exoglucanase-gold complexes) revealed pectin breakdown and cellulose alterations which were mainly restricted to sites of fungal penetration. Invasion was also associated with striking host cell reactions, including the formation of papillae, and wall appositions lining the host cell walls. These newly-formed materials were highly heterogeneous with respect to chemical composition as judged by the differential labelling obtained with an exoglucanase probe and with an RcA gold complex used to localize galactose molecules. Plugging of the intercellular spaces in the cortex with an electron opaque material, presumably enriched with phenolic compounds, was also frequently observed. Some cortical cells were also completely filled with opaque flecks of amorphous or osmiophilic material and PythiumF hyphae growing in such occluded cells were often moribund. Inoculation of cucumber roots with P. aphanidermatumresulted in a rapid and massive colonization of all root tissues, including those of the stele. Pathogen ingress was associated with extensive host cell alterations with cellulose and pectin breakdown occurring at a distance from the fungus. The main host reaction was the occlusion of parenchyma cells in the stele and cortex with an amorphous material but the deposition of this material appeared to occur too late to be effective in blocking P. aphanidermatumattack. Other host reactions were seldom observed in P. aphanidermatuminfected roots.

Localized stimulation of anthocyanin accumulation and delineation of pathogen ingress in maize genetically resistant to Bipolaris maydisrace O

The present investigation is a continuation of our characterization of phenolic compound metabolism that occurs in the expression of resistance by maize with the recessive rhmgene for resistance to Bipolaris maydis. We show that a final stage of resistance expression involves the accumulation of a pigment in uninfected, healthy epidermal cells that surround restricted lesions on leaves of the resistant cultivar. The pigment was characterized by plasma desorption mass spectrometry and found to have a structure consistent with that of cyanidin 3-dimalonyl glucoside. The function of this zwitterionic anthocyanin is proposed to involve the protection of uninfected, healthy plant tissue from the toxic, oxidative metabolites that accumulate during the expression of resistance. Microscopic examination of leaf tissue showed that the pathogen ceased to grow in the resistant cultivar at 18 h post-inoculation, a time considerably prior to pigment accumulation. Thus, the pigment accumulates in cells that are affected but not infected by the pathogen.

Accumulation of diferulic acid during the hypersensitive response of oat leaves to Puccinia coronataf.sp. avenaeand its role in the resistance of oat tissues to cell wall degrading enzymes

The relationship between diferulic acid formation and locally induced resistance was investigated in the interactions of two physiological races of Puccinia coronataf.sp. avenaewith Shokan 1 oat. Diferulic acid was detected in the hemicellulosic fraction extracted with oxalic acid from the cell wall. The amount of ferulic acid bound in the cell wall matrix polysaccharides did not vary in uninoculated and inoculated primary leaves of oats during the time course of the experiment. In contrast, the formation of diferulic acid bound to wall polysaccharides became significantly higher in the incompatible than the compatible interaction at approx. 36h after inoculation. The timing of increased formation of diferulic acid was correlated with the accumulation of avenalumins and inhibition of protoplast liberation from infected primary leaves. Activities of soluble, and of ionically- and covalently-bound cell wall peroxidases from uninoculated and inoculated leaves were measured. Ionically-bound cell wall peroxidase activity increased in the incompatible interactions at about 36h after inoculation. This was the same time that increased levels of diferulic acids were observed. This may indicate that the cross-linking of matrix polysaccharides by ferulic acids bound in cell walls is caused by a coupling reaction initiated by ionically-bound cell wall peroxidases. It is postulated that the diferulic acid formation occurs in oat cell walls at the infection sites in incompatible interactions and may contribure to locally induced resistance in oats by providing a barrier to pathogen ingress.

Two classes of cis sequences contribute to tissue-specific expression of a PAL2 promoter in transgenic tobacco.

Genes encoding phenylalanine ammonia-lyase (PAL) are expressed in a complex pattern during plant development and in response to light, pathogen ingress, mechanical damage and other stresses. Analysis of the promoter of the bean PAL2 gene in transgenic tobacco has shown that some regions responsible for developmental expression are functionally compensatory. The minimum sequence containing all cis sequences necessary for developmental patterns of expression is within -254 bp of the transcription start site. Footprinting and electrophoretic mobility shift assay studies of this region revealed potential cis sequences which coincided with the functional domains defined by small deletions and promoter fusions. Mutations in these potential cis sequences in the context of the minimal -254 bp promoter altered tissue-specific expression patterns, confirming the importance of these sequences for expression in vivo. A functional model for the promoter is presented which predicts that three AC-elements, which are possible Myb protein binding sites, together with a G-box, interact to direct the complex patterns of tissue-specific expression observed.

Immunogold localization of THRGP-like epitopes in the haustorial interface of obligate, biotrophic fungi on monocots

Immunoelectron microscopy was used to determine the subcellular distribution of threonine-hydroxyproline-rich glycoprotein (THRGP) epitopes in host-parasite interactions between obligate, biotrophic fungi and cereals. Infection sites of stem rust (Puccinia graminis f. sp.tritici) and leaf rust (Puccinia recondita) on primary leaves of wheat (Triticum aestivum), as well as of powdery mildew (Erysiphe graminis f. sp.hordei) on coleoptiles of barley (Hordeum vulgare), wete probed with a polyclonal antiserum to maize THRGP. A few immunogold particles were found over the cell walls of wheat mesophyll tissue and barley coleoptile epidermis. Unlike previous examples in dicot plants, no enhanced accumulation of THRGP was observed in cereal cell walls adjacent to sites of pathogen ingress. Instead, the most pronounced accumulation of THRGP-like molecules occurred over the extrahaustorial matrix in both incompatible and compatible plant-pathogen interactions. For powdery mildew of barley, immunogold staining was distinctly increased over the center of the penetration sites; however, no labeling was found over papillae that formed during incompatible and compatible interactions. In addition, no cross-reactivity of the anti-THRGP antiserum with intercellularly growing rust pathogens was observed. The highly localized deposition of THRGP-like molecules in the extrahaustorial matrix suggests that the host plant establishes a modified barrier between itself and the pathogen.

The most abundant soluble basic protein of the stylar transmitting tract in potato (Solanum tuberosum L.) is an endochitinase

An abundant, pistil-specific basic protein has been purified and characterized from potato (Solanum tuberosum L.). A polymerase chain reaction (PCR) probe was generated for the corresponding gene using oligonucleotides based on internal peptide sequences of the protein, and the PCR probe was further employed to isolate cDNA and genomic clones. The sequence of the gene exhibits up to 70% similarity to previously described endochitinase class 1a protein sequences, and the purified protein possesses chitinase (poly[1, 4-(N-acetyl-beta-D-glucosaminide)] glucanohydrolase, EC 3.2.1.14) activity. The protein, termed SK2, has been located by immunocytochemistry to the intercellular matrix of the stylar transmitting tract. Immunoblot analysis has shown SK2 to be distinct from the wound-induced chitinases of potato. The SK2-class of chitinase is restricted in its distribution within the Solanaceae to the sub-family Solanoidae, which includes cultivated tomato and potato species. It was absent from the Cestroidae species tested (Petunia hybrida, Nicotiana tabacum). A role for SK2 endochitinase in protecting the ovary against pollen-tube-mediated pathogen ingress is proposed.

Chitinase gene expression in transgenic plants: a molecular approach to understanding plant defence responses

Recent studies suggest that the production of enzymes capable of degrading the cell walls of invading phytopathogenic fungi may be an important component of the defence response of plants. In this chapter, we summarize recent progress on the isolation and characterization of chitinolytic enzymes from higher plants. Emphasis is placed on experiments designed to study the regulation of chitinase gene expression in response to ethylene treatment or pathogen ingress and on determining the role of this enzyme in plant defence. The production of transgenic plants with enhanced resistance to attack by the fungal pathogen Rhizodonia solani is discussed.

Cytology of infection of 35S‐bean chitinase transgenic canola plants by Rhizoctonia solani: cytochemical aspects of chitin breakdown in vivo

Transgenic canola plants containing high, constitutive levels of bean endochitinase have been shown to be more resistant to infection by the soil‐borne pathogen, Rhizoctonia solani, than are wild‐type plants that lack the chimeric chitinase gene. To determine whether the resistance of the 35S‐chitinase plants to Rhizoctonia infection results from an antimicrobial activity of the bean chitinase in planta, an ultrastructural and cyto‐chemical study was performed on infected control and transgenic canola plants. Analysis of root tissues of infected wild‐type canola plants revealed that R. solani was capable of extensive tissue colonization including the xylem vessels. Pathogen ingress towards the vascular system was associated with marked host cell wall alterations such as disruption of middle lamella matrices that occurred in advance of fungal penetration. Fungal hyphae colonizing these tissues appeared metabolically active as judged by their typical morphological features and their extensive multiplication. In infected transgenic plants, however, the pattern of fungal colonization was different to that observed in wild‐type plants. Penetration of the host cuticle and epidermis was frequently observed, but fungal colonization was usually restricted to the cortex although, in a few cases, some fungal cells could be seen in xylem vessels. In all samples examined, severe hyphal alterations ranging from increased vacuolization to cell lysis were seen. Hyphae occasionally seen in xylem vessels were markedly damaged and often reduced to convoluted wall fragments. Cytochemical labeling of chitin using the WGA/ovomucoid‐gold complex showed that hyphal alterations correlated with extensive chitin degradation. Thus, reduction in fungal biomass, increase in hyphal alterations leading to fungal lysis and chitin breakdown appear to be typical features observed in transgenic canola plants. Because these features were not seen in infected wild‐type plants, it is likely that constitutive expression of the bean endochitinase gene is, at least in part, responsible for the enhanced protection against fungal attack observed in these plants. It is not known, however, if other components of the host defense response contribute to the resistance phenotype.

Ultrastructure of the Colletotrichum trifolii-Medicago sativa pathosystem. I. Pre-penetration events

Light and electron microscopy were used to compare the development of compatible with incompatible interactions in the Colletotrichum trifolii-Medicago sativa pathosystem. The host responses to infection by races 1 or 2 of the pathogen were similar. Differential host reactions became evident only after the fungal penetration peg entered the epidermal cell wall. On both resistant and susceptible plants, papillae and wall thickenings formed in the initially infected epidermal cells. Although histochemically different, the papillae failed to block pathogen ingress in either case. In both resistant and susceptible plants there was an apparent increase in the cytoplasmic: vacuolar ratio; however, the difference was more dramatic in the latter where the central vacuole almost disappeared. Penetration of the host plasmalemma resulted in the rapid death of the epidermal cell in all tested alfalfa plants. In incompatible interactions the massive deposition of phenolic and related compounds in and around the necrotic cell resulted, simultaneously, in necrosis of the fungal infection vesicle within and protection of adjacent plant tissues outside the target cell. In compatible interactions this did not occur. The cytoplasm of the target cell and its neighbours was completely degraded by autolysis while the fungus continued to proliferate into adjacent degenerate epidermal and cortical tissues.

Ultrastructure of the Colletotrichum trifolii-Medicago sativa pathosystem. II. Post-penetration events

Light and electron microscopy were used to compare the development of compatible with incompatible interactions in the Colletotrichum trifolii-Medicago sativa pathosystem. The host responses to infection by races 1 or 2 of the pathogen were similar. Differential host reactions became evident only after the fungal penetration peg entered the epidermal cell wall. On both resistant and susceptible plants, papillae and wall thickenings formed in the initially infected epidermal cells. Although histochemically different, the papillae failed to block pathogen ingress in either case. In both resistant and susceptible plants there was an apparent increase in the cytoplasmic: vacuolar ratio; however, the difference was more dramatic in the latter where the central vacuole almost disappeared. Penetration of the host plasmalemma resulted in the rapid death of the epidermal cell in all tested alfalfa plants. In incompatible interactions the massive deposition of phenolic and related compounds in and around the necrotic cell resulted, simultaneously, in necrosis of the fungal infection vesicle within and protection of adjacent plant tissues outside the target cell. In compatible interactions this did not occur. The cytoplasm of the target cell and its neighbours was completely degraded by autolysis while the fungus continued to proliferate into adjacent degenerate epidermal and cortical tissues.