Endophytic Stage Research Articles

Texas microfungi: a new species of Corynesporopsis (Xylariales) associated with the trifoliate orange

Corynesporopsis ponciri sp. nov. is described from dead twigs, branches, and thorns still attached to trees of Poncirus trifoliata, the trifoliate orange, in Texas, U.S.A. The fungus is characterized by black, punctiform at first, later caespitose and hairy colonies forming large superficial patches covering almost entirely the surface of colonized parts of the host, unbranched conidiophores, solitary or fasciculate and arising from substomatal, compact stromata in loose to dense fascicles and bearing monotretic, mostly determinate, rarely percurrent conidiogenous cells. Conidia are ellipsoidal or fusiform to naviculiform, smooth or verruculose, brown, often with the apical cell light brown, euseptate, with 2, rarely 1 or 3 septa, usually with an associated dark band and forming short, unbranched, acropetal chains of up to four conidia. Multilocus phylogenetic analyses suggested that C. ponciri belongs within the Xylariales and forms a strongly supported monophyletic lineage sister to members of Vamsapriya in Vamsapriyaceae. Corynesporopsis is revealed as polyphyletic based on this and the other two species with available molecular data, C. iberica and C. acaciae. The newly described fungus is compared with morphologically similar species of Corynesporopsis and Heteroconium having 1- or 2-septate conidia and phylogenetic affinities of these polyphyletic anamorphic genera are discussed. The mode and successful colonization of dead parts of the host with conidiophores arising from substomatal stromata suggest that C. ponciri may have a putative endophytic stage and it is capable of switching to a saprobic lifestyle upon senescence or death of the host.

Prevention of Stomatal Entry as a Strategy for Plant Disease Control against Foliar Pathogenic Pseudomonas Species.

The genus Pseudomonas includes some of the most problematic and studied foliar bacterial pathogens. Generally, in a successful disease cycle there is an initial epiphytic lifestyle on the leaf surface and a subsequent aggressive endophytic stage inside the leaf apoplast. Leaf-associated bacterial pathogens enter intercellular spaces and internal leaf tissues by natural surface opening sites, such as stomata. The stomatal crossing is complex and dynamic, and functional genomic studies have revealed several virulence factors required for plant entry. Currently, treatments with copper-containing compounds, where authorized and admitted, and antibiotics are commonly used against bacterial plant pathogens. However, strains resistant to these chemicals occur in the fields. Therefore, the demand for alternative control strategies has been increasing. This review summarizes efficient strategies to prevent bacterial entry. Virulence factors required for entering the leaf in plant-pathogenic Pseudomonas species are also discussed.

Unraveling the Secrets of a Double-Life Fungus by Genomics: Ophiocordyceps australis CCMB661 Displays Molecular Machinery for Both Parasitic and Endophytic Lifestyles.

Ophiocordyceps australis (Ascomycota, Hypocreales, Ophiocordycipitaceae) is a classic entomopathogenic fungus that parasitizes ants (Hymenoptera, Ponerinae, Ponerini). Nonetheless, according to our results, this fungal species also exhibits a complete set of genes coding for plant cell wall degrading Carbohydrate-Active enZymes (CAZymes), enabling a full endophytic stage and, consequently, its dual ability to both parasitize insects and live inside plant tissue. The main objective of our study was the sequencing and full characterization of the genome of the fungal strain of O. australis (CCMB661) and its predicted secretome. The assembled genome had a total length of 30.31 Mb, N50 of 92.624 bp, GC content of 46.36%, and 8,043 protein-coding genes, 175 of which encoded CAZymes. In addition, the primary genes encoding proteins and critical enzymes during the infection process and those responsible for the host-pathogen interaction have been identified, including proteases (Pr1, Pr4), aminopeptidases, chitinases (Cht2), adhesins, lectins, lipases, and behavioral manipulators, such as enterotoxins, Protein Tyrosine Phosphatases (PTPs), and Glycoside Hydrolases (GHs). Our findings indicate that the presence of genes coding for Mad2 and GHs in O. australis may facilitate the infection process in plants, suggesting interkingdom colonization. Furthermore, our study elucidated the pathogenicity mechanisms for this Ophiocordyceps species, which still is scarcely studied.

Studies on the secondary metabolism of Rosellinia and Dematophora strains (Xylariaceae) from Iran

The xylariaceous genus Dematophora has recently been resurrected and segregated from Rosellinia based on a molecular phylogeny and morphological characters. This was an important taxonomic change because Dematophora in the current sense contains several important pathogens, while Rosellinia is limited to mainly saprotrophic species that have an endophytic stage in their life cycle and may even have beneficial effects on the host plants. During our ongoing work on the functional biodiversity of the Xylariales, we have encountered new strains of rosellinoid Xylariaceae from Iran and have studied their mycelial cultures for secondary metabolites in an attempt to establish further chemotaxonomic affinities. In the process, we isolated and identified 13 compounds, of which rosellisteroid (1), the cichorine derivative 2, and the alkaloid 3 are new. Out of these, nine were tested for their antimicrobial affinities with cytochalasin E (6) exhibiting weak activity against Schizosaccharomyces pombe. The cytotoxicity of three cytochalasin derivatives was examined and their effects on the F-actin cytoskeletal organization studied by fluorescence microscopy using fluorescent phalloidin. Cytochalasin E (6) and Δ6,12-cytochalasin E (7) showed strong and irreversible action on actin, while cytochalasin K (8) exhibited weaker, reversible effects.

Stem-end rot in major tropical and sub-tropical fruit species

Stem-end rot (SER) is one of the most destructive postharvest diseases affecting some tropical and sub-tropical fruits. The disease is caused by several fungal pathogens, including Botryosphaeriaceae species. In mango, Dothiorella dominicana, D. mangiferae, Lasiodiplodia theobromae, Phomopsis mangiferae, Cytosphaera mangiferae, Pestalotiopsis sp. endophytically colonize the inflorescence in the orchard or field. Fungi may occur endophytically in the stem tissue of the trees prior to inflorescence emergence. During the endophytic stage, the fungi colonize the phloem and xylem at stem-end and, after fruit ripening, some fungi become necrotrophic and colonize the fruit pulp causing SER. The SER pathogens in other fruit species, including ripe avocado or papaya, are not known to undergo an endophytic phase. Stem-end rot is a disease that is difficult to control. Preharvest cultural practices and selection of resistant varieties are beneficial to reduce field infections. Studies have shown that numerous control options, involving cultural, chemical (fungicides, essential oils) and biological approaches, are available to manage SER through either direct inhibition of fungi or induction of host defenses. These may be used individually or integrated manner. The most common and effective practice for controlling the SER is pre- and postharvest application of synthetic fungicides. With the new tools that are currently available, and expected to emerge, there is a possibility of a better understanding the host–pathogen or host-microbiota interactions within the stem-end region, leading to newer technologies as alternatives to toxic synthetic fungicides for the management of SER in fruits.

The entomophagous caterpillar fungus Ophiocordyceps sinensis is consumed by its lepidopteran host as a plant endophyte

Abstract Endophytic insect pathogenic fungi of the order Hypocreales reside in plant tissues as mutualistic partners that protect plants against insect herbivores. Caterpillar fungus Ophiocordyceps sinensis (order: Hypocreales, family: Ophiocordycipitaceae) is often described as an entomophagous fungus that parasitizes lepidopteran larvae of the ghost moth genus Thitarodes (family: Hepialidae), but recent evidence suggests involvement of a plant-endophytic stage in O. sinensis infection of insect larvae. In this study, we screen sample plant species in a caterpillar fungus collection site at Mt. Gongga, Sichuan, China, for endophytic O. sinensis and analyze the diet of Thitarodes larvae living in the soil of the same habitat. We show that the entomophagous caterpillar fungus O. sinensis is a prevalent plant endophyte (present in 52.6% of all plant genera and 66.7% of plant families) and that the presence of endophytic O. sinensis in plant leaf tissue is significantly correlated with its presence in plant root tissue. Our analysis of larval diet content reveals a high diversity generalist diet and confirms that the plant families harboring endophytic O. sinensis are consumed by Thitarodes larvae, although the composition and diversity of an individual's diet do not predict its level of fungal infection. These results suggest a reproductive strategy of O. sinensis involving a plant endophytic stage that facilitates infection of root-eating host larvae. Our study highlights the role of resident plant communities as a third participating partner in this complex symbiosis.

Pest categorisation of Gremmeniellaabietina.

Following a request from the European Commission, the EFSA Plant Health (PLH) Panel performed a pest categorisation of Gremmeniella abietina, a well‐defined species and distinguishable fungus of the family Godroniaceae. The species G. abietina includes several varieties, races and biotypes that are found in different geographical locations, on different hosts and that vary in aggressiveness. The pathogen causes diseases on Pinus species and other conifers such as Abies spp., Picea spp., Larix spp. and Pseudotsuga spp. known as Scleroderris canker in North America and Brunchorstia dieback in Europe. G. abietina has been reported from 19 EU Member States, without apparent ecoclimatic factors limiting establishment. The pathogen is a protected zone (PZ) quarantine pest (Annex IIB) for Ireland and the UK (Northern Ireland). The main European hosts are widespread throughout most of the EU and have been frequently planted in the PZ. The main means of spread are wind‐blown ascospores, rain‐splashed conidia, plants for planting and traded Christmas trees. Given that G. abietina is most damaging to species that are grown towards the limit of their range, impacts can be expected in the PZ, should the pathogen be introduced there. Risk reduction options include selection of disease‐free planting material, nursery inspections, selection of planting sites at some distance from infested plantations, appropriate spacing between plants and thinning. The main uncertainties concern the indeterminate endophytic stage of the fungus, the pathogen distribution and the future taxonomic status of G. abietina, given its intraspecific diversity. All the criteria assessed by the Panel for consideration as potential PZ quarantine pest are met. The criterion of plants for planting being the main pathway for spread for regulated non‐quarantine pests is not met: plants for planting are only one of the means of spread of the pathogen.

Host-synthesized cysteine protease-specific inhibitor disrupts Cuscuta campestris parasitism in tomato

Dodder (Cuscuta campestris) is one of the most important pests of tomato causing severe losses in yield. Cuscutain is a pre-pro-protein produced by dodder that has a cysteine proteinase function essential for normal development of the haustoria and parasitism, which involves the secretion and activation of cuscutain cysteine protease in the host plant tissue. The propeptide subunit of this enzyme has an inhibitory function and restricts the enzymatic activity of cuscutain. Here, we transformed the inhibitory propeptide segment of this enzyme into tomato and examined the tomato resistance to C. campestris. We demonstrate the expression of inhibitory propeptide in transgenic plants and find that it effectively interrupted cuscutain enzyme activity and haustoria development at the endophytic stage. Mature haustoria infecting transgenic hosts showed defects in searching hyphae development and these structures were not elongate, and in most cases no functional haustoria were formed due to inhibitor expression in the transgenic plants after prehaustoria contact. Dodder grown on transgenic lines showed an overall reduction in vigor and fecundity due to defective attachment of haustoria. The increased growth of dodder-challenged transgenic plants relative to controls, demonstrates the efficacy of cysteine protease inhibition in parasite plant control.

An HPLC-based Method to Quantify Coronatine Production by Bacteria.

Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.

Genetic variation between and within two populations of Rhabdocline pseudotsugae in Germany

Knowledge of the ecology and biology of Rhabdocline pseudotsugae Sydow is still at a very early stage. Recent results indicate the existence of an endophytic stage of the fungus in the form of symptomless infections in various Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) tissue types. This study represents the first description of genetic variation between and within two populations of R. pseudotsugae. Needles featuring fruiting bodies of R. pseudotsugae were collected from sample areas in Saxony and North Rhine-Westphalia (in Germany), with different fungal genotypes distinguished using the start codon targeted polymorphism (SCoT) and branch point signal sequences (BPS) techniques. Five of the 20 primers tested could be selected for further investigation. A total of 349 fragments were amplified at an average of 69.8 fragments per primer. Results showed the two sample areas to exhibit a high degree of both genetic variability and genetic diversity. A clear differentiation between the sample areas was not observed. There was nevertheless a clear connection between the genetic distance and spatial distribution of the fruiting bodies of R. pseudotsugae within the sample areas themselves.

Comparative genome analysis of entomopathogenic fungi reveals a complex set of secreted proteins.

BackgroundMetarhizium anisopliae is an entomopathogenic fungus used in the biological control of some agricultural insect pests, and efforts are underway to use this fungus in the control of insect-borne human diseases. A large repertoire of proteins must be secreted by M. anisopliae to cope with the various available nutrients as this fungus switches through different lifestyles, i.e., from a saprophytic, to an infectious, to a plant endophytic stage. To further evaluate the predicted secretome of M. anisopliae, we employed genomic and transcriptomic analyses, coupled with phylogenomic analysis, focusing on the identification and characterization of secreted proteins.ResultsWe determined the M. anisopliae E6 genome sequence and compared this sequence to other entomopathogenic fungi genomes. A robust pipeline was generated to evaluate the predicted secretomes of M. anisopliae and 15 other filamentous fungi, leading to the identification of a core of secreted proteins. Transcriptomic analysis using the tick Rhipicephalus microplus cuticle as an infection model during two periods of infection (48 and 144 h) allowed the identification of several differentially expressed genes. This analysis concluded that a large proportion of the predicted secretome coding genes contained altered transcript levels in the conditions analyzed in this study. In addition, some specific secreted proteins from Metarhizium have an evolutionary history similar to orthologs found in Beauveria/Cordyceps. This similarity suggests that a set of secreted proteins has evolved to participate in entomopathogenicity.ConclusionsThe data presented represents an important step to the characterization of the role of secreted proteins in the virulence and pathogenicity of M. anisopliae.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-822) contains supplementary material, which is available to authorized users.

Rhabdocline needle cast—investigations on various Douglas fir tissue types

Douglas fir (Pseudotsuga menziesii) is one of the most important non-indigenous tree species in Germany. The species is characterized by a wide amplitude of growing conditions, and is of increasing interest, in particular from the perspective of climate change. Douglas fir is nevertheless particularly susceptible to fungal pathogens, such as Rhabdocline pseudotsugae . The aim of the present study was therefore to develop an early detection method for R. pseudotsugae based on the polymerase chain reaction (PCR). Existing molecular techniques were adapted and optimized to detect the pathogen in small sample volumes. Both healthy and infected Douglas firs were examined, with various tissue types (buds, cambial tissue, needles) and seeds tested for the presence of R. pseudotsugae. Non-infected Douglas firs did not give positive responses in the molecular analyses, but the pathogen was clearly detected in buds, cambial tissues, needles and seeds of infected trees. To date, the fungus has been considered an obligate biotrophic needle parasite. The present results provide clear evidence, however, for the existence of an endophytic stage in the life cycle of R. pseudotsugae. In contrast with previous studies, this paper investigates the dispersal of the fungus via seeds.

Xerotolerant foliar endophytic fungi of Populus euphratica from the Tarim River basin, Central China are conspecific to endophytic ITS phylotypes of Populus tremula from temperate Europe

Fungal research on non-lichenized taxa in xeric and other unusual habitats for fungi is comparatively scarce but constantly reveal an enormous ecological and functional adaptiveness of these organisms to extremes. This holds true in the topmost forest canopies as well as in marine, arctic, antarctic or desert habitats. In the present study dried leaves of Populus euphratica from a continental desert area in Central China were investigated for endophytic fungi 6 weeks after sampling by cultivation of small leaf fragments. Thirty-five species, many of them well-known from extreme environments, were delimitated from as few as 58 isolates by a combined analysis of morphotypes, micromorphology and ITS sequences. Meta-analyses revealed pronounced conspecificity to endophytes from Populus tremula leaves, but significant differences to foliar endophytic assemblages in Pinus monticola and Fagus sylvatica. The deduced high similarity of endophytic communities on both Populus species compared with that on Pinus and Fagus suggests that many fungal phyllosphere species show a well-pronounced host preference, at least for their endophytic stages. In addition, it demonstrates that leaving leaves as a microhabitat might provide similar conditions such as constantly high relative humidity, regardless of the surrounding climatic conditions. The evolution of extremotolerance might be of particular importance for retaining competitiveness of foliar endophytes in deciduous host trees after loss of pathogenicity.

Impact of a beneficial and of a pathogenic Fusarium strain on the fingerprinting-based structure of microbial communities in tomato ( Lycopersicon esculentum Milll.) rhizosphere

Fusarium solani strain FsK (FsK), isolated from a plant pathogen-suppressive compost, grows endophytically in tomato roots and controls infestations by Fusarium oxysporum f.sp . radicis- lycopersici (FORL). The effect of root colonization by the two fungi on the diversity of rhizosphere microbial community was studied. Tomato plants were inoculated with FsK and/or FORL and rhizosphere soil was collected 8, 15 and 30 days post inoculation (dpi) and analyzed by denaturating gradient gel electrophoresis (DGGE) of PCR-amplified internal transcribed spacer (ITS) sequences of fungi and 16S rRNA gene sequences of common rhizosphere bacterial guilds like alpha-proteobacteria and pseudomonads. Cluster analysis of DGGE fingerprints showed that FsK had a transient impact on the fungal and alpha-proteobacterial community only during its endophytic stage (15 dpi), while FORL had a readily distinguished and persistent effect on the fungal community. The changes observed in the rhizosphere fungal and bacterial communities may depict the interactions of the two fungal inocula with the plant. Cloning of selected DGGE bands stimulated by FsK showed that the responsive bacteria were closely related to species known to include biological control agents (BCA). Overall, the inoculation of FsK in tomato rhizosphere did not appear to have a significant impact on the diversity of non-target microbial groups inhabiting plant rhizosphere.

The fungus Epichloë typhina in populations of a halophyte Puccinellia distans: salinity as a possible inhibitor of infection

<em>Puccinellia distans</em> is a non-agricultural halophytic grass that has become another host plant for <em>Epichloe typhina</em>, hitherto not reported from Poland. In 1992 we noticed the first symptoms of choke disease in a single population of <em>P. distans</em> in central Poland. Since then we have observed choke disease in 5 populations of <em>P. distans</em> only in man-made habitats. These habitats are strongly anthropogenically salinated but they exhibit the pattern of species composition characteristic of natural salines. In this paper we test the hypothesis that the level of salinity affects the infection of <em>P. distans</em> by the fungus <em>E. typhina</em>. Seven plots were established in the field and each plot was divided into 25 subplots. Within each plot the level of infection in a spring generation of shoots was negatively correlated with salinity (common regression within the plots, beta = -0.674, df = 117, p < 0.001). Negative correlation was also found in an autumn generation within the plots (beta = -0.682, df = 94, p < 0.001) after excluding plot P in which the frequency of infected individuals was the lowest and equal only to 0.05. The proportion of individuals infected by the endophytic stage of the fungus in the populations was assessed using diagnostic polymerase chain reaction. The greatest percentage (98.3%) of infected individuals was found in the population growing in the habitat of the lowest salinity. The high salinity reduces the chance of <em>P. distans</em> to become infected, but may promote the stroma formation of <em>E. typhina</em> twice in the season. Disease expression in autumn clearly represents a misadaptation which could be explained by the fact that the species interactions described here appeared relatively recently as a result of human activity. This hypothesis requires further experimental verification.

Linking ex planta fungi with their endophytic stages: Perisporiopsis, a common leaf litter and soil fungus, is a frequent endophyte of Hevea spp. and other plants

Abstract It has been hypothesized that endophytic fungi originate from local environmental sources, and endophyte identity and composition can be predicted based on surrounding ex planta fungi. As part of a pilot study to characterize endophytes and ex planta fungi (e.g. saprotrophs) of wild Hevea trees, endophytes were isolated with almost identical ITS sequences, but these were unidentifiable using GenBank database. Along with the endophytes, ex planta fungi on Hevea leaves were also collected at the sampling sites. The objective of this study was to determine if selected unknown Hevea endophytes could be linked with the ex planta fungi using DNA sequences. Methods included BLAST searches, phylogenetic analysis, and morphological characterizations of the endophytes and saprotrophs. Results show that the BLAST-unidentifiable Hevea endophytes belong in the fungal genus Perisporiopsis (Parodiopsidaceae, Dothideomycetes, Ascomycota). In addition, this Hevea endophyte is at least 98 % identical to >20 other sequences from GenBank, demonstrating that Perisporiopsis is a common endophyte from various plants, and a leaf litter and soil fungus. Connecting a DNA sequence with a name that has biological meaning enables microbial (i.e. fungal) biologists to make more accurate inferences about the functional ecology of a particular species in a community or ecosystem.

Phylogenic diversity of fungal endophytes in Spanish stands of Pinus halepensis

Fungal endophytes were collected from 168 Pinus halepensis trees sampled in 55 Spanish stands. In total, 229 endophytic isolates were grouped into 92 morphotypes according to their mycelium features. Thus, twigs enclosed 63.76% of the total endophyte isolates and needles 36.24%. Likewise, twig samples also yielded a higher endophyte species richness. Analysis of ITS rDNA region generated 38 different Ascomycota taxa and confirmed the endophytic stage of several pathogens previously associated with the Pinus halepensis decline in Spain. Naemacyclus minor, Brunchorstia pinea, Lophodermium pinastri, Phomopsis sp., Diplodia pinea, Pestalotiopsis besseyi and Truncatella angustata were isolated. Sequencing of LSU rDNA region verified the 38 taxa and contributed to infer their phylogenic relatedness using the Neighbour-Joining method. In the bootstrap consensus tree five classes were observed. Dothideomycetes resulted to be the dominant class because of its high isolation frequency (52.4%) and richest species composition (39.5%). On the contrary, class Eurotiomycetes was the least abundant (3.5%) and along with class Pezizomycetes harboured the lowest species richness (7.9%). Class Sordariomycetes and Leotiomycetes had intermediate abundance and species composition percentages. This study represents the first work concerning the taxonomy of the fungal endophytic community of the Mediterranean host species Pinus halepensis using molecular tools. The data provided here may help to establish the cause of Pinus halepensis decline.

Out of the rivers: are some aquatic hyphomycetes plant endophytes?

Ingoldian fungi, or aquatic hyphomycetes, are asexual microfungi, mostly ascomycetes, commonly occurring in running freshwater. They grow on dead plant material, such as leaves and twigs, and play a major role in nutrient flows in stream ecosystems (Barlocher, 1992). They were discovered and first extensively studied by Ingold (1942) and were thus named "Ingoldian" fungi. Ingold described their abundant multicellular asexual spores of sigmoid or, more typically, tetraradiate shape (Fig. 1). He recognized that they probably arose from multiple convergent evolutions, by secondary adaptation to aquatic life, as recently confirmed by molecular markers (Belliveau & Barlocher, 2005; Baschien et al., 2006). Because of their apparent lack of sexuality, Ingoldian species were placed in asexual genera, such as Tricladium or Tetracladium, based on conidial morphology and/or mode of conidiogenesis. Unexpectedly, several lines of evidence now suggest that some Ingoldian fungi are also plant endophytes, that is, they grow in plants without producing symptoms.[...]

Transcriptome analysis of root‐knot nematode functions induced in the early stages of parasitism*

Root-knot nematodes of the genus Meloidogyne are obligate biotrophic parasites able to infest > 2000 plant species. The nematode effectors responsible for disease development are involved in the adaptation of the parasite to its host environment and host response modulation. Here, the differences between the transcriptomes of preparasitic exophytic second-stage juveniles (J2) and parasitic endophytic third-stage juveniles (J3) of Meloidogyne incognita were investigated. Genes up-regulated at the endophytic stage were isolated by suppression subtractive hybridization and validated by dot blots and real-time quantitative polymerase chain reaction (PCR). Up-regulation was demonstrated for genes involved in detoxification and protein degradation, for a gene encoding a putative secreted protein and for genes of unknown function. Transcripts of the glutathione S-transferase gene Mi-gsts-1 were 27 times more abundant in J3 than in J2. The observed Mi-gsts-1 expression in the oesophageal secretory glands and the results of functional analyses based on RNA interference suggest that glutathione S-transferases are secreted during parasitism and are required for completion of the nematode life cycle in its host. Secreted glutathione S-transferases may protect the parasite against reactive oxygen species or modulate the plant responses triggered by pathogen attack.

Effects of <i>Fusarium</i> species on defence mechanisms in sorghum seedlings

Sorghum seedlings were inoculated with Fusarium proliferatum and F thapsinum 3 days after germination Seven days later infected seedlings were dissected into the coleoptile mesocotyl seed scutellum and root and defence responses measured Infection significantly increased the levels of soluble phenolics and anthocyanins the phytoalexins apigeninidin and luteolinidin and the enzymes peroxidase beta;13glucanase and chitinase in some tissues Constitutive levels of chitinase were high The strongest defence responses and highest levels of preformed defences were seen in the scutellum Responses to F thapsinum tended to be stronger than those to F proliferatum The findings supported the hypothesis that the plant is able to suppress the activity of these fungi during the endophytic stage of infection