Wall Appositions Research Articles

Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

BackgroundPlant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle.ResultsWithin 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed.ConclusionOur results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the pathogen as it attempts to invade the epidermal cell surface.

Root inoculation with a forest soil streptomycete leads to locally and systemically increased resistance against phytopathogens in Norway spruce

Soil streptomycetes are commonly antagonistic against plant pathogens. However, interactions involving increased defense responses in the host plant, leading to suppression of plant disease development, have not yet been detailed. Here, the mechanisms were studied of disease suppression by Streptomyces sp. GB 4-2 against Heterobasidion root and butt rot in Norway spruce (Picea abies) seedlings. GB 4-2 promoted mycelial growth of the phytopathogenic fungus, germination rate of fungal spores, extension of germ tubes and early colonization of outer cortical layers of the plant root. Reduced colonization of the inner cortical cell layers was accompanied by the induction of cell wall appositions, and increased xylem formation in the vascular cylinder emerged after bacterium-fungus coinoculation. Bacterial treatment led to decreased water content in roots and needles and increased photosynthetic yield (F(v)/F(m)) and peroxidase activities in needles. The infection of needles by Botrytis cinerea was reduced by bacterial pretreatment. Complex interactions of GB 4-2 with Norway spruce and Heterobasidion abietinum were discovered. The bacterium promoted the growth of the phytopathogenic fungus but induced plant defense responses. Host responses indicate that GB 4-2 induces both local and systemic defense responses in Norway spruce.

Cytological and immunocytochemical studies on responses of wheat spikes of the resistant Chinese cv. Sumai 3 and the susceptible cv. Xiaoyan 22 to infection by Fusarium graminearum

Fusarium head blight (FHB), predominantly caused by Fusarium graminearum and F. culmorum, is one of the most destructive diseases of wheat, reducing grain yield and quality of kernels. In diseased kernels trichothecne mycotoxins accumulate, which are harmful to human and animal health. Pathogen development and host responses to infection by F. graminearum were investigated in wheat spikes of the resistant cv. Sumai 3 and susceptible cv. Xiaoyan 22 to infection by means of electron microscopy and immunogold labelling techniques. The infection process of the pathogen in wheat spikes was similar in both the resistant and susceptible cultivars, but the pathogen developed more slowly in the resistant cv. Sumai 3 compared to the susceptible cv. Xiaoyan 22, indicating that fungal spread was restricted to the spike tissues of the resistant cultivar. The formation of thick-layered appositions and papillae was essentially more pronounced in the infected host tissues of the resistant cultivar than in the susceptible one. s-1,3-glucan was detected in the appositions and papillae. Immunogold labelling studies demonstrated that labelling densities for lignin, thionins and hydroxyproline-rich glycoproteins (HRGP) over the cell walls of the infected tissues of the susceptible wheat cv. Xiaoyan 22 only slightly increased whereas these compounds intensely accumulated in the host cell walls of the infected wheat spikes of the resistant cv. Sumai 3. The labelling densities for the two plant hydrolases, s -1,3-glucanase and chitinase, increased slightly in the infected wheat spike tissues of the susceptible cv. Xiaoyan 22, whereas higher labelling densities of both enzymes were found in the infected wheat spikes from the resistant cv. Sumai 3. Immunogold labelling of the Fusarium toxin DON in the infected wheat spike tissues showed that labelling densities in spike tissues for DON in the resistant cv. Sumai 3 were significantly lower than those in the susceptible cv. Xiaoyan 22. The significance of the induced morphological (e.g. thick-layered wall appositions and papillae) and chemical defence constituents (e.g. s -1,3-glucanase, chitinase, lignin, thionin and HRGP) in resistance to FHB as well as the possible role of DON as an aggressiveness factor in translation of transcripts of defence response genes and spreading of F. graminearum and F. culmorum are discussed.

Molecular characterization and endophytic nature of the root-associated fungus Meliniomyces variabilis (LtVB3)

The root-associated fungus LtVB3 was formerly reported as a potential, new biocontrol agent of Verticillium yellows of Chinese cabbage and Fusarium wilt of tomato. According to molecular phylogenetic analysis of the ITS 1-5.8S rDNA-ITS 2 gene regions and morphological characteristics, LtVB3 is here identified as Meliniomyces variabilis, a recently described species [formerly called “Variable White Taxon” (VWT)] in a new genus erected to accommodate sterile fungi with phylogenetic affinities to Rhizoscyphus ericae, an ericoid mycorrhizal fungus. In vitro inoculation experiments showed that LtVB3 colonized the roots of torch azalea and eucalyptus; however, growth was enhanced only in azalea but not in eucalyptus. Hyphae of M. variabilis formed intracellular structures typical of ericoid mycorrhizas in epidermal cells of azalea roots but did not produce any typical ectomycorrhizal structures with or within root cells of either host. In LtVB3-treated eucalyptus roots, some cells in the epidermal and cortical layers had wall appositions and thickenings, which appeared to restrict fungal growth.

Cytoskeleton and cell wall function in penetration resistance

Plants successfully repel the vast majority of potential pathogens that arrive on their surface, with most microorganisms failing to breach the outer epidermal wall. Resistance to penetration at the epidermis is a key component of basal defence against disease and critically depends on fortification of the cell wall at the site of attempted penetration through the development of specialised cell wall appositions rich in antimicrobial compounds. Formation of cell wall appositions is achieved by rapid reorganisation of actin microfilaments, actin-dependent transport of secretory products to the infection site and local activation of callose synthesis. Plants are finely tuned to detect the presence of pathogens on their surface, perceiving both chemical and physical signals of pathogen origin. In the on-going evolution of interaction strategies, plants must continually monitor and out manoeuvre pathogen avoidance or suppression of plant defences in order to preserve the effectiveness of penetration resistance.

Histopathology of Infection and Colonization of Susceptible and Resistant Port-Orford-Cedar by Phytophthora lateralis

ABSTRACT Port-Orford-cedar (POC) root disease, caused by Phytophthora lateralis, continues to kill POC in landscape plantings and natural forests in western North America. POC trees resistant to P. lateralis have been identified and propagated. Cytological observations of P. lateralis in susceptible and resistant roots and stems were made with light and transmission electron microscopy to identify resistance mechanisms. No differences in infection pathway and initial colonization were observed between susceptible and resistant roots, although there were differences in the rate and extent of development. Germ tubes formed appressoria, and penetration hyphae grew either between or directly through epidermal cell walls; inter- and intracellular hyphae colonized the root cortex. In susceptible roots, hyphae penetrated into the vascular system within 48 h of inoculation. In contrast, hyphae in roots of resistant seedlings grew more slowly in cortical cells and were not observed to penetrate to the vascular tissues. In resistant roots, infection was marked by general thickening of cortical cell walls, wall appositions around penetrating hyphae, collapse of cortical cells, and accumulation of osmophillic granules around hyphae. In susceptible stems, hyphae grew inter- and intracellularly in all cells of the secondary phloem except fiber cells, but were concentrated in sieve and parenchyma cells in the functional phloem. The pattern of penetration and colonization of hyphae was similar in the resistant stems, except that hyphae were found in the fiber cells of the xylem. In resistant stems, there were fewer hyphae in the functional phloem, and cytological changes such as damaged nuclei and disintegrated cytoplasm were evident. Structural changes in resistant stems included collapsed cells, wall thickening, secretory bodies, apposition of electron dense materials, and crystals in cell walls.

Targeted alterations in iron homeostasis underlie plant defense responses

Iron (Fe) is a ubiquitous redox-active element essential for most life. The formation of localized cell wall appositions, the oxidative burst and the production of pathogenesis-related proteins are hallmarks of plant defense responses. Here, we report that iron is a central mediator linking these three phenomena. We show that in response to pathogen attack, reactive Fe3+, but not Fe2+, is deposited at cell wall appositions where it accumulates and mediates the oxidative burst. We provide evidence that the bulk secretion of Fe3+ provoked by pathogen attack leads to intracellular iron depletion, and that H2O2 itself induces ATP-dependent intracellular iron efflux. Finally, we demonstrate that this intracellular iron depletion promotes the transcription of pathogenesis-related genes in concert with H2O2. This work identifies iron as an underlying factor associated with the oxidative burst and regulating cereal defenses, and establishes links between disease-related iron homeostasis in plants and animals.

Do Plant Cells Secrete Exosomes Derived from Multivesicular Bodies?

Multivesicular bodies (MVBs) are spherical endosomal organelles containing small vesicles formed by inward budding of the limiting membrane into the endosomal lumen. In mammalian red cells and cells of immune system, MVBs fuse with the plasma membrane in an exocytic manner, leading to release their contents including internal vesicles into the extracellular space. These released vesicles are termed exosomes. Transmission electron microscopy studies have shown that paramural vesicles situated between the plasma membrane and the cell wall occur in various cell wall-associated processes and are similar to exosomes both in location and in morphology. Our recent studies have revealed that MVBs and paramural vesicles proliferate when cell wall appositions are rapidly deposited beneath fungal penetration attempts or during plugging of plasmodesmata between hypersensitive cells and their intact neighboring cells. This indicates a potential secretion of exosome-like vesicles into the extracellular space by fusion of MVBs with the plasma membrane. This MVB-mediated secretion pathway was proposed on the basis of pioneer studies of MVBs and paramural vesicles in plants some forty years ago. Here, we recall the attention to the occurrence of MVB-mediated secretion of exosomes in plants.

CYTOLOGICAL AND IMMUNOCYTOCHEMICAL STUDIES ON THE EFFECTS OF THE FUNGICIDE TEBUCONAZOLE ON THE INTERACTION OF WHEAT WITH STRIPE RUST

Effects of the fungicide tebuconazole, applied as a leaf spray after inoculation of wheat leaves with stripe rust were examined by means of electron microscopy and immunogold labelling Tebuconazole strongly inhibited the development of Puccinia striiformis in host leaf tissues, and also markedly altered the structures of the hyphae and haustorium. These changes included increased vacuolation, irregular thickening of cell walls, necrosis or degeneration of cytoplasm, and collapse of the hypha and haustorium. Large differences were detected in P. striiformis-infected wheat leaves treated with fungicide compared with the untreated control. A very thick layer of wall appositions and large encasements were usually formed in the infected fungicide-treated leaves whereas such structures were never observed in the infected wheat leaves without fungicide treatment. In addition, chitinase, s-1,3-glucanase and lignin were detected in wheat leaf tissues by immunogold labelling. Accumulation of the two enzymes and lignin increased slightly in infected leaves compared with uninoculated healthy leaves, while a much higher accumulation of the two enzymes and lignin was detected in infected wheat leaves after fungicide treatment. Our results suggest that tebuconazole not only inhibited development of P. striiformis in wheat leaves, but also enhanced structural and biochemical host defense reactions in the infected host.

Respiratory Burst Oxidase Homologue A of barley contributes to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei

Reactive oxygen intermediates (ROI) are closely related to defence reactions of plants against pathogens. A prominent role in the production of ROI has been attributed to the plant respiratory burst oxidase homologues (RBOH) of the human phagocyte GP91(phox). A barley RBOH, which encodes a putative superoxide (O2*-)) producing NADPH oxidase, is described here. Histochemical analysis of the barley-Blumeria graminis f. sp. hordei (Bgh) interaction showed that O(2*-) is produced locally at the site of penetration. In contrast, hydrogen peroxide (H2O2) is produced in non-penetrated cell wall appositions. A barley RBOHA cDNA was isolated and a minor induction of expression of RBOHA was observed during the interactions of barley with Bgh. Transient RNA interference-mediated gene silencing of HvRBOHA during the penetration process of Bgh led to an increase of basal penetration resistance. The results support a potential role of HvRBOHA in cellular accessibility to Blumeria graminis.

Multivesicular compartments proliferate in susceptible and resistant MLA12‐barley leaves in response to infection by the biotrophic powdery mildew fungus

There is growing evidence that multivesicular bodies and cell wall-associated paramural bodies participate in the enhanced vesicle trafficking induced by pathogen attack. Here, we performed transmission electron microscopy in combination with cytochemical localization of H2O2 to investigate multivesicular compartments during establishment of compatible interaction in susceptible barley (Hordeum vulgare) and during hypersensitive response in resistant MLA12-barley infected by the barley powdery mildew fungus (Blumeria graminis f. sp. hordei). Multivesicular bodies, intravacuolar vesicle aggregates and paramural bodies proliferated in the penetrated epidermal cell during development of the fungal haustorium. These vesicular structures also proliferated at the periphery of intact cells, which were adjacent to the hypersensitive dying cells and deposited cell wall appositions associated with H2O2 accumulation. All plasmodesmata between intact cells and hypersensitive cells were constricted or blocked by cell wall appositions. These results suggest that multivesicular compartments participate in secretion of building blocks for cell wall appositions not only to arrest fungal penetration but also to contain hypersensitive cell death through blocking plasmodesmata. They may also participate in internalization of damaged membranes, deleterious materials, nutrients, elicitors and elicitor receptors.

Seed-transmitted beneficial endophytic Stagonospora sp. can penetrate the walls of the root epidermis, but does not proliferate in the cortex, of Phragmites australis

Stagonospora sp. (4/99-1) is a beneficial endophytic fungus frequently transmitted by seeds of Phragmites australis [Cav.] Trin. ex Steudel. Here we show that this fungus also penetrates the root epidermis. At first, hyphae were attracted by the root and proliferated on the root surface, preferably over the anticlinal walls. Penetration occurred directly by undifferentiated hyphae or was facilitated by hyphopodia. Hyphal growth within the root was restricted to the walls of epidermal cells and the walls of the cells of the outermost cortical layer. Deeper growth by the fungus elicited wall appositions and ingress into the cytoplasm of cortical cells was blocked by papillae. In the rare cases, the fungus managed to penetrate into cortical cells, these reacted with necrosis. Immunological studies suggested that fungal material reached the host plasmalemma and may have been taken up by endocytotic events. Our observations explain the endophytic lifestyle of hyphae close to the epidermis and the restricted development within the cortex.

Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus

Localized cell wall modification and accumulation of antimicrobial compounds beneath sites of fungal attack are common mechanisms for plant resistance to fungal penetration. In barley (Hordeum vulgare) leaves, light-microscopically visible vesicle-like bodies (VLBs) containing H(2)O(2) or phenolics frequently accumulate around cell wall appositions (syn. papillae), in which the penetration attempt of the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) is halted. By ultrastructural analyses, we demonstrated that the Bgh-induced VLBs represent different structures. VLBs intensively stained by H(2)O(2)-reactive dyes were actually small papillae instead of cytoplasmic vesicles. Other VLBs were identified as osmiophilic bodies or multivesicular compartments, designated paramural bodies (PMBs) and multivesicular bodies (MVBs). MVBs seemingly followed two distinct pathways: either they were engulfed by the tonoplast for degradation in the vacuole or they fused with the plasma membrane to release their internal vesicles into the paramural space and hence could be the origin of PMBs. MVBs and PMBs appeared to be multicomponent kits possibly containing building blocks to be readily assembled into papilla and antimicrobial compounds to be discharged against fungal penetration. Finally, we propose that released paramural vesicles might be similar to exosomes in animal cells.

A two component chitin-binding protein from French bean – association of a proline-rich protein with a cysteine-rich polypeptide

A 42 kDa chitin-binding proline-rich protein (PRP) from French bean has been previously characterised through its involvement in plant–pathogen interactions. It is located at the plasmalemma-wall interface, intercellular spaces and binds to the pathogen Colletotrichum lindemuthianum in vitro and in planta. It is also present in cell wall appositions formed in response to an hrp mutant of Xanthomonas campestris. We now show that the 42 kDa protein is composed of two components, a 25 kDa polypeptide member of the PRP family of legumes and a 6.8 kDa cysteine-rich peptide with high similarity to snakin-2 from potato. Snakins bind to pathogens and are antimicrobial. Molecular cloning of the longest PRP corresponding to the N-terminal sequence of the purified protein and the 6.8 kDa component is reported. The cognate mRNAs show coordinate expression. The two-component protein complex has already been shown to be involved in binding and immobilising pathogens through oxidative cross-linking of the PRP components but could also function as a two-component chitin-receptor involved in plant–pathogen interactions through antimicrobial activity and/or signalling.

Loss of pectin is an early event during infection of cocoyam roots by Pythium myriotylum

Cocoyam (Xanthosoma sagittifolium) is an important tuber crop in most tropical zones of Africa and America. In Cameroon, its cultivation is hampered by a soil-borne fungus Pythium myriotylum which is responsible for root rot disease. The mechanism of root colonisation by the fungus has yet to be elucidated. In this study, using microscopical and immunocytochemical methods, we provide a new evidence regarding the mode of action of the fungus and we describe the reaction of the plant to the early stages of fungal invasion. We show that the fungal attack begins with the colonisation of the peripheral and epidermal cells of the root apex. These cells are rapidly lost upon infection, while cortical and stele cells are not. Labelling with the cationic gold, which binds to negatively charged wall polymers such as pectins, is absent in cortical cells and in the interfacial zone of the infected roots while it is abundant in the cell walls of stele cells. A similar pattern of labelling is also found when using the anti-pectin monoclonal antibody JIM5, but not with anti-xyloglucan antibodies. This suggests that early during infection, the fungus causes a significant loss of pectin probably via degradation by hydrolytic enzymes that diffuse and act away from the site of attack. Additional support for pectin loss is the demonstration, via sugar analysis, that a significant decrease in galacturonic acid content occurred in infected root cell walls. In addition, we demonstrate that one of the early reactions of X. sagittifolium to the fungal invasion is the formation of wall appositions that are rich in callose and cellulose.

Histologia da interação Stemphylium solani e tomateiro

Estudou-se a infecção de Stemphylium solani Weber em folíolos de tomateiros (Lycopersicon esculentum) resistente (Motelle) e suscetível (Moneymaker), usando-se técnicas histológicas e de microscopia eletrônica de varredura e transmissão. Inicialmente, foram quantificados os eventos de pré-penetração nas duas cultivares, os quais não diferiram significativamente entre si (teste F a 5%), indicando que a resistência é exercida em pós-penetração. A penetração ocorreu principalmente via estômatos e a hifa diferenciou-se na cavidade subestomática formando uma vesícula globosa ou irregular; a qual ramificou-se em hifas secundárias. Tanto a colonização intra quanto intercelular ocorreram no período de 24 e 36 h após a inoculação nas duas cultivares estudadas. Aposições foram observadas na parede das células do mesofilo resistente e, na maioria das células examinadas, aparentemente restringiram a colonização intracelular.

Model Wheat Genotypes as Tools to Uncover Effective Defense Mechanisms Against the Hemibiotrophic Fungus Bipolaris sorokiniana

ABSTRACT We investigated the interaction of several differentially resistant wheatwith the hemibiotrophic phytopathogenic fungus Bipolaris sorokiniana (teleomorph Cochliobolus sativus). Wheat genotypes Yangmai, M 3 (W7976), Shanghai 4, and Chirya 7 showed higher levels of resistancewith cv. Sonalika, used as a susceptible control. In amicroscopic inspection, we found that fungal penetration intoepidermal layer failed mostly through a cell wall-associated defense. In cases where the fungus successfully overcame epidermal, its spread within the mesophyll tissue (necrotrophic phase) wasin the more resistant genotypes. Epidermal cell wall-associated, spreading as well as the extent of electrolyte leakage of infected, correlated well with field resistance. We propose that cellular hostsuch as formation of cell wall appositions as well as the degreeearly mesophyll spreading of fungal hyphae are indicative of thepotential of the respective host genotype and, therefore, could befor the characterization of new spot blotch resistance traits in cereals.

Mycoparasitism of Rhizoctonia solani by Endophytic Chaetomium spirale ND35: Ultrastructure and Cytochemistry of the Interaction

AbstractThe interaction between endophytic biocontrol agent Chaetomium spirale ND35 and the soil‐borne plant pathogen Rhizoctonia solani was studied by light microscopy and transmission electron microscopy (TEM), as well as further investigated by gold cytochemistry to assess the potential role of cell wall degrading enzymes (CWDEs) during the mycoparasitic process. Macroscopic observations of fungal growth in dual cultures revealed that pathogen growth inhibition occurred soon after contact with the antagonist, followed by the overgrowth of C. spirale on the colony of R. solani. The coiling of C. spirale around R. solani and intracellular growth of the antagonist in its host occurred frequently. Moreover, in advanced stage of interaction between the antagonist and the pathogen, The growth and development of C. spirale were associated with highly morphological changes of the host fungal cell, characterized by retraction of plasma membrane and cytoplasm disorganization. Further, TEM investigations through localization by gold immunocytochemistry showed that contact between the two fungi was mediated by an amorphous β‐1,3‐glucan‐enriched matrix originating from cell wall of the antagonist C. spirale and sticking to its host surface. At the same time, the hemispherical wall appositions which were intensely labeled by the antibodies of β‐1, 3‐glucan in cell wall of R. solani were induced to form at sites of potential antagonist entry. However, the antagonist was capable of penetrating this barrier, indicating that β‐1,3‐glucanases were produced during the mycoparasitic process. Localization of N‐acetylglucosamine residues (chitin) with the gold‐labelled wheat germ agglutinin (WGA) implicated that chitinases might be involved in the CWD of R. solani in this antagonistic process as well. This report is the first evidence about mechanisms of the interactions between C. spirale and R. solani in ultrastructural and cytochemical aspects.

Immunocytochemical localization of the pathogenesis-related PR-1 protein in barley leaves after infection by Bipolaris sorokiniana

A pathogenesis-related PR-1 protein accumulates in barley ( Hordeum vulgare L.) leaves upon pathogen attack. The hyphae of Bipolaris sorokiniana invades primarily the intercellular space of mesophyll tissue. Light microscopy revealed that infected leaves show PR-1 labeling on mesophyll cell walls and both infected and non-infected leaves show labeling in chloroplasts. Subcellularly, PR-1 was detected on the outer cell wall layer and cytoplasm of primary hyphae, on intercellular electron dense material in junctions between host cells and in host cell wall appositions. The role of PR-1, as a defense and/or senescence related protein, in relation to its subcellular localizations is discussed.

Visualization of Infection of an Endophytic Actinomycete Streptomyces galbus in Leaves of Tissue-cultured Rhododendron

Infection of a Streptomyces galbus strain R-5 in leaves of tissue-cultured seedlings of rhododendron was examined using transmission electron microscopy and a histochemical method. Mycelia invaded the host through stomatal openings but not through other sites. Mycelia colonizing leaf surfaces and those growing through stomatal openings were embedded in an electron-dense, thick, amorphous material, while single hyphae were not. Within host tissues, hyphae were observed individually or in colonies in intercellular spaces but not in epidermal or mesophyll cells. The mycelia colonizing in intercellular spaces were also embedded in a similar amorphous material. Wall appositions formed within the epidermal and mesophyll cells, the outer surfaces of which were attached to the intercellular mycelia, suggesting that those cells had recognized the presence of mycelia of R-5 and responded to it. On the leaf surfaces, the wax that covered the undulating cuticular layer was degraded beneath the growing mycelia. This observation was supported by the presence of indigo blue crystals that formed as hydrolytic products of mycelial nonspecific esterase along the mycelia on fresh leaves.