Hyphal Penetration Research Articles

Morpho-Pathological and Global Transcriptomic Analysis Reveals the Robust Nonhost Resistance Responses in Chickpea Interaction with Alternaria brassicae.

Alternaria blight, caused by Alternaria brassicae, causes considerable yield loss in Brassica crops. While several blight-resistant varieties have been developed using resistance sources from host germplasm, none of them are entirely successful in imparting durable resistance. This has prompted the exploration of novel gene pools of nonhost plant species. Nonhost resistance (NHR) is a durable form of resistance, comprising pre- and postinvasion layers of defense. We aimed to identify the molecular basis of NHR to A. brassicae and identify the layers of NHR operating in a nonhost, chickpea (Cicer arietinum). To elucidate the layers of NHR operating against A. brassicae, we compared the histopathology and infection patterns of A. brassicae in C. arietinum and Brassica juncea. Delayed conidial germination, impeded hyphal growth, suppressed appressorium formation, and limited hyphal penetration occurred in the nonhost plant compared with the host plant, implying the involvement of the preinvasion layer of NHR in C. arietinum. Next, we investigated the molecular basis of robust NHR, in C. arietinum challenged with A. brassicae, by microarray-based global transcriptome profiling. Genes involved in stomatal closure, cuticular wax biosynthesis, cell-wall modification, and secondary metabolite production (contributing to preinvasion NHR) as well as reactive oxygen species (ROS) and cell death (contributing to postinvasion NHR) were found to be upregulated. Consistent with transcriptomic analysis, the morpho-pathological analysis revealed stomatal closure, ROS accumulation, and localized cell death in C. arietinum as the defense strategies against A. brassicae. Thus, we identified NHR-contributing genes with potential applications in blight resistance gene transfer to B. juncea.

MoGT2 Is Essential for Morphogenesis and Pathogenicity of Magnaporthe oryzae.

Magnaporthe oryzae causes the rice blast disease, which is one of the most serious diseases of cultivated rice worldwide. Glycosylation is an important posttranslational modification of secretory and membrane proteins in all eukaryotes, catalyzed by glycosyltransferases (GTs). In this study, we identified and characterized a type 2 glycosyltransferase, MoGt2, in M. oryzae Targeted gene deletion mutants of MoGT2 (mogt2Δ strains) were nonpathogenic and were impaired in vegetative growth, conidiation, and appressorium formation at hyphal tips. Moreover, MoGT2 plays an important role in stress tolerance and hydrophobin function of M. oryzae Site-directed mutagenesis analysis showed that conserved glycosyltransferase domains (DxD and QxxRW) are critical for biological functions of MoGt2. MoGT2 deletion led to altered glycoproteins during M. oryzae conidiation. By liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified several candidate proteins as potential substrates of MoGt2, including several heat shock proteins, two coiled-coil domain-containing proteins, aminopeptidase 2, and nuclease domain-containing protein 1. On the other hand, we found that a conidiation-related gene, genes involved in various metabolism pathways, and genes involved in cell wall integrity and/or osmotic response were differentially regulated in the mogt2Δ mutant, which may potentially contribute to its condiation defects. Taken together, our results show that MoGt2 is important for infection-related morphogenesis and pathogenesis in M. oryzaeIMPORTANCE The ascomycete fungus Magnapothe oryzae is the causal agent of rice blast disease, leading to severe loss in cultivated rice production worldwide. In this study, we identified a conserved type 2 glycosyltransferase named MoGt2 in M. oryzae The mogt2Δ targeted gene deletion mutants exhibited pleiotropic defects in vegetative growth, conidiation, stress response, hyphal appressorium-mediated penetration, and pathogenicity. Furthermore, conserved glycosyltransferase domains are critical for MoGt2 function. The comparative transcriptome analysis revealed potential target genes under MoGt2 regulation in M. oryzae conidiation. Identification of potential glycoproteins modified by MoGt2 provided information on its regulatory mechanism of gene expression and biological functions. Overall, our study represents the first report of type 2 glycosyltransferase function in M. oryzae infection-related morphogenesis and pathogenesis.

MaPacC, a pH-responsive transcription factor, negatively regulates thermotolerance and contributes to conidiation and virulence in Metarhizium acridum

PacC is a pH-responsive transcription factor gene highly expressed at alkaline pH and plays distinct roles in environmental fitness, conidiation and virulence of different fungi. Here, we show biological functions of orthologous MaPacC in the locust-specific fungal pathogen Metarhizium acridum. Disruption of MapacC slowed down the fungal growth only under alkaline conditions. Intriguingly, the fungal thermotolerance was enhanced by the MapacC deletion, accompanied by transcriptional upregulation of some heat shock-responsive genes. The disruptant suffered a reduction in conidial yield and a change in conidial surface structure, but showed little change in cell wall integrity. The virulence of the disruptant against a locust species was markedly attenuated due to delayed appressorium formation, repressed expression of some insect cuticle hydrolases and slowed growth in locust hemolymph. The phenoloxidase activity and nodules of the locusts infected by the disruptant were also boosted. All of these phenotypic changes were restored by targeted gene complementation. Our results indicate that MaPacC acts a negative regulator of thermotolerance and contributes to the virulence of M. acridum by an involvement in hyphal penetration through insect cuticle and evasion from insect immunity.

Direct and Indirect Bioleaching of Cobalt from Low Grade Laterite and Pyritic Ores by Aspergillus niger

The bioleaching efficiency and mechanism of recovery of cobalt (Co) and nickel from laterites and pyritic ores by Aspergillus niger were investigated. Recoveries of Co from laterites and pyritic ores by direct bioleaching were 65.9 ± 1.8% and 4.9 ± 2.7%, respectively, while 30.9 ± 0.6% and 10.9 ± 6.2% recovery of Ni were obtained from laterites and pyritic ores, respectively. Recovery of Co via indirect bioleaching in the absence of the fungal biomass from laterite was significantly lower when compared with Co released by direct bioleaching. In the latter, hyphal penetration and colonization of the laterites were clearly observed by scanning electron microscopy (SEM). X-ray powder diffraction (XRPD) analysis of mineral phases before and after bioleaching indicated that cobalt-bearing goethite was the main phase bioleached in the laterites. No significant difference was found between Co recoveries from synthesized cobalt-bearing goethite by both direct and indirect bioleaching. Therefore, we propose that two processes are involved in bioleaching from laterites: (1) cobalt-bearing goethite was exposed via direct interactions between the fungus and the minerals and (2) cobalt-bearing goethite was dissolved by released metabolites of A. niger, such as organic acids. An incongruent pattern of Co and Fe bioleaching from the laterites was also a feature of the metal recovery process.

BrlA and AbaA Govern Virulence-Required Dimorphic Switch, Conidiation, and Pathogenicity in a Fungal Insect Pathogen.

Dimorphic plant and human mycopathogens require a switch from the usual yeast growth to filamentous growth for host tissue penetration, and the switch is controlled by multiple signaling systems other than the central developmental pathway. Unlike these fungi, dimorphic insect mycopathogens usually grow by hyphal extension, infect the host by hyphal penetration through the insect cuticle, and switch to unicellular blastospores from the penetrating hyphae only after entry into the host hemocoel, where blastospore propagation by yeast-like budding accelerates host mummification. Here, we report a dependence of the virulence-required dimorphic transition on the central pathway activators BrlA and AbaA in Beauveria bassiana Deletion of brlA or abaA abolished both aerial conidiation and submerged blastospore formation in vitro despite no negative impact on hyphal growth in various media, including a broth mimic of insect hemolymph. The hyphae of either deletion mutant lost insect pathogenicity through normal cuticle penetration, contrasting with a high infectivity of wild-type hyphae. The mutant hyphae injected into the host hemocoel failed to form blastospores, resulting in slower lethal action. Uncovered by transcriptomic analysis, several genes involved in host adhesion and cuticle degradation were sharply repressed in both deletion mutants versus wild type. However, almost all signaling genes homologous to those acting in the dimorphic switch of other fungi were not differentially expressed at a significant level and hence unlikely to be involved in shutting down the dimorphic switch of each deletion mutant. Therefore, like aerial conidiation, the submerged dimorphic switch in vitro and in vivo is a process of asexual development governed by the two central pathway activators in B. bassiana IMPORTANCE Dimorphic insect mycopathogens infect the host by hyphal penetration through the host cuticle and switch from the penetrating hyphae to unicellular blastospores after entry into the host hemocoel, where blastospore propagation by yeast-like budding accelerates host mummification to death. The fungal virulence-required dimorphic switch is confirmed as a process of asexual development directly regulated by BrlA and AbaA, two key activators of the central developmental pathway in an insect mycopathogen. This finding unveils a novel mechanism distinct from the control of the dimorphic switch by multiple signaling systems other than the central developmental pathway in dimorphic plant and human mycopathogens, which switch from the usual yeast growth to filamentous growth required for pathogenicity through host tissue penetration.

Ovicidal potential of Pochonia chlamydosporia isolate Pc-10 (Ascomycota: Sordariomycetes) on egg masses of the snail Pseudosuccinea columella (Mollusca: Gastropoda)

Snails of the species Pseudosuccinea columella are considered intermediate hosts of Fasciola hepatica, a digenetic trematode that infects bile ducts of ruminants and humans, causing economic damage and serious problems for public health. These gastropods inhabit ponds, have high reproductive capacity, and lay their egg masses in submerged substrates on pond edges where they are exposed to desiccation and microbes, including fungi, that may exert pathogenic effects on the snail and its embryos. This information is relevant for control of the intermediate host and therefore of fasciolosis. With the objective of evaluating ovicidal potential of Pochonia chlamydosporia (Pc-10 isolate), a nematophagous fungus used as antagonistic agent for a wide variety of helminths of medical and veterinary importance, on egg masses of P. columella, we compared a treated group, where the egg masses were exposed to Pc-10 for a period of 25 days, and a control group, in which there was no exposure to the fungus. The results indicated that the embryogenesis process was significantly inhibited (93.15%) by Pc-10, suggesting its applicability in biological control programs of lymnaeid snails. In addition, ultrastructure showed the occurrence of different types of interactions between the egg masses with the mycelia of Pc-10: type 1, biochemical effects by the adherence of hyphae; type 2, morphological alterations, but without hyphal penetration; and type 3, lytic effect, morphological damage caused by penetration of hyphae by the fungus, resulting in some important structural modifications, thus compromising the viability of the eggs. The results demonstrate the susceptibility of P. columella egg masses to an isolate of P. chlamydosporia under laboratory conditions, providing valuable information for the biological control of this intermediate host.

Defensin AFP1 inhibits appressorium formation and penetration of rice cells by the rice blast fungus Magnaporthe oryzae

The plant defensin antifungal peptide 1 (AFP1) is composed of only 51 amino acids and has potent antifungal activity against the rice blast fungus Magnaporthe oryzae. To elucidate the antifungal mechanisms of AFP1, we assessed the suppression of appressorium formation and hyphal penetration by M. oryzae. AFP1 interfered with appressorium formation and host cell penetration following to invasion in a dose-dependent manner. This is the first report of the prevention of M. oryzae infection in rice by AFP1 and our observations could help to develop resistant cultivars and antifungal formulations for use in agriculture.

Lichens and other lithobionts on the carbonate rock surfaces of the heritage site of the tomb of Lazarus (Palestinian territories): diversity, biodeterioration, and control issues in a semi-arid environment

Investigations on the lithobiontic colonization of the stone cultural heritage in (semi-)arid regions are needed to address conservation strategies. In this work, lithobiontic communities were examined on the carbonate rock surfaces of the heritage site of the Tomb of Lazarus. We aimed to evaluate their distribution and interaction with the lithic substrate, together with the efficacy of biocidal treatments for their control. Diversity and abundance of lithobionts were surveyed on the Jerusalem stone blocks of three architectural elements. Observations at the lichen-rock interface were carried out by reflected light and scanning electron microscopy. The efficacy against lichens of the widely used biocide benzalkonium chloride (BZC) was compared for different concentrations and application methods, and evaluated by epifluorescence microscopy. Chlorolichens were the dominant component of lithobiontic communities, more thoroughly adapted to the semi-arid conditions of the site than mosses and black biofilms of cyanobacteria and dematiaceous fungi. A different structural organization, in terms of thallus thickness and depth of the hyphal penetration component, characterized epilithic and endolithic lichen species, responsible for different deteriogenic activities. Biocidal assays showed that even the methodologies that are usually effective in temperate conditions (as the application of BZC 1.5% by poultice) may not completely devitalize lichens adapted to the stress conditions of semi-arid climates, unless a pervasive biocide diffusion through metabolically active thalli is carefully guaranteed. Lithobionts act as biodeteriogens on the semi-arid surfaces of the investigated heritage site. Their removal is thus recommendable, but it needs to be adequately supported with a careful calibration of devitalization strategies.

Comparative Analysis of Inflammatory Cytokine Release and Alveolar Epithelial Barrier Invasion in a Transwell® Bilayer Model of Mucormycosis.

Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.

Microenvironmental features drive the distribution of lichens in the House of the Ancient Hunt, Pompeii, Italy

On the stone cultural heritage, the influence of architecture-related microenvironmental features on lichen diversity, abundance and consequent threats for conservation has been still poorly characterized to support management plans. Such relationships were here investigated on the vertical surfaces of the House of the Ancient Hunt in Pompeii, archaeological site in S-Italy where the variability of lichen saxicolous communities has been still completely neglected despite their widespread occurrence. Lichen colonization in semiconfined rooms was sporadic and limited to Dirina massiliensis, while a remarkable turnover of six communities, encompassing 22 species, characterized mortar, painted and plastered surfaces in outdoor environments, with local covers up to 80%. Microscopic and spectroscopic analyses displayed the deteriogenic potential of three dominant species, due to hyphal penetration within paint and plaster layers (Verrucaria macrostoma) and the release of oxalic acid and/or secondary metabolites with acidic and chelating functions (D. massiliensis, Lepraria lobificans). A higher vertical distance of surfaces from the ground and a larger room dimension were the main conditional factors related to a higher lichen abundance and the distribution of the different communities. Such knowledge on architecture-related microenvironmental features driving lichen distribution and biodeterioration threats may contribute to address restoration priorities and conservation strategies.

Histopathology of charcoal rot disease (Macrophomina phaseolina) in resistant and susceptible cultivars of soybean

Charcoal rot caused by the fungus, Macrophomina phaseolina, is one of the most important fungal diseases affecting soybean (Glycine max (L.) Merr.) production worldwide, especially under climate change scenarios. The infection events including microsclerotia germination, hyphal penetration, and pathogen colonization into the infected root tissues of two soybean cultivars, Williams (susceptible) and Hadgeston (resistant), with reported differences in the level of resistance to M. phaseolina, were investigated by histological analyses. The soybean plants were infected by immersing the roots in microsclerotia suspension (1 g of microsclerotes in 300 ml of agarose 0.015%). Pathogen penetration took place through the roots epidermal cells 3 days after inoculation. The observations revealed that pathogen's pre-penetration steps, including microsclerotia germination and hyphae development, are not linked to resistance as these events occurred as the same in both cultivars. However, in the post-penetration steps, there was a significant difference between two cultivars in terms of root colonization by M. phaseolina and disintegration of root tissues. The appearance of adventitious roots in the resistant cultivar in response to the pathogen, and infection of secondary roots at the cell differentiation stage as well as the inability of the pathogen to complete its life cycle in the resistant cultivar, are the most significant findings of this study.

Cercospora leaf spot in Toona ciliata: Epidemiology and infection process of Cercospora cf. alchemillicola

AbstractThe biology of Cercospora leaf spot (Cercospora cf. alchemillicola) of Toona ciliata was examined in terms of the effects of temperature and duration of foliage wetting on the progress of disease and the mechanism of pathogen infection. To evaluate the effect of temperature on mycelial growth, cercosporin production and conidial germination, experiments were conducted in a 4 × 5 factorial design, with four isolates (HR01, HR02, HR03 and CML3059) at five temperatures (15, 19, 23, 26 and 30°C). The area under the disease progress curve (AUDPC) was calculated in a 3 × 5 factorial design at three temperatures (20, 23 and 29°C) and five leaf wetness durations (0, 12, 24, 48 and 72 hr). Penetration of C. cf. alchemillicola into leaves was examined by scanning electron microscopy. Maximum mycelial growth of all isolates occurred at 24.8°C. HR02 and HR01 produced higher quantities of cercosporin compared with CML3059 and HR03. Temperatures between 21.6 and 23.0°C favoured production of the toxin among isolates. Higher conidial germination rates occurred in isolates HR02 and HR01 compared with other isolates, with optimum germination occurring at 24.2–26.3°C. A leaf wetting period of 72 hr at 23.3°C gave the highest AUDPC value. No hyphal penetration was observed through natural openings. However, germ tubes grew inside wounds on the leaf epidermis. In addition, Phyllachora balansae (cause of tar spot) opened fissures on the leaf epidermis, thus creating wounds enabling C. cf. alchemillicola to infect.

Influences of light on growth, reproduction and hypocrellin production by Shiraia sp. SUPER-H168.

Light is a very important signal for fungi since it influences many different physiological responses. The effects of dark or light at different wavelengths on growth, reproduction and hypocrellins of Shiraia sp. SUPER-H168 were studied: dark, white, red, yellow, green, blue and purple. All incubations under different light conditions had significant stimulating effects on aerial hyphae and suppressing effects on hypocrellin biosynthesis compared with dark incubation. Under blue and purple light especially blue light, the colonies with profuse growth of aerial mycelium were formed. Hypocrellin production reached 13.73mg per dish under dark condition, and decreased to 4.01mg and 2.83mg per dish under white and blue light, respectively. Light condition not only influenced hypocrellin production but also influenced the composition of hypocrellins. Four types of hyphae, namely surface, aerial, biofilm and penetrative hyphae, were observed by light microscopy and SEM. This study found that biofilm hyphae was so closely connected with production of secondary metabolites, and hypocrellins were only produced by biofilm hyphae. Light promoted sexual development and inhibited asexual reproduction, especially blue light strongly inhibited asexual development.

Ultrastructures of Colletotrichum orbiculare in Cucumber Leaves Expressing Systemic Acquired Resistance Mediated by Chlorella fusca.

Chlorella, one single-cell green algae organism that lives autotrophically by photosynthesis, can directly suppress some plant diseases. The objective of this study was to determine whether pre-spraying with Chlorella fusca suspension could induce systemic acquired resistance (SAR) in cucumber plants against anthracnose caused by Colletotrichum orbiculare. In order to illustrate SAR induced by algae, infection structures in host cells were observed under a transmission electron microscope (TEM). Cytological changes as defense responses of host mesophyll cells such as accumulation of vesicles, formation of sheath around penetration hyphae, and thickness of cell wells adjoining with intracellular hyphae were demonstrated in cucumber leaves. Similar defense responses were also found in the plant pre-treated with DL-3-aminobutyric acid, another SAR priming agent. Images showed that defense response of host cells was scarcely observed in untreated leaf tissues. These cytological observations suggest that C. fusca could induce SAR against anthracnose in cucumber plants by activating defense responses of host cells.

Mitigation of Fusarium Wilt Disease of Vicia faba Using Artemisia monosperma Extract

The antifungal activity of Artemisia monosperma aqueous extracts against Fusarium solani. sp. Phaseoli the causal agent of Vicia faba wilt and its ability in inducing disease resistance were studied in vivo using seed-soaking treatment before sowing. Plants were harvested at vegetative and flowering stages after sowing. Results showed that growth, yield parameters and phytohormone contents were markedly inhibited in bean plants in response to Fusarium wilt disease, whereas lipid peroxidation and the contents of non-enzymatic and enzymatic antioxidants were increased as compared with healthy control plants. Moreover, presoaking in A. monosperma extract increased all the mentioned parameters and decreased lipid peroxidation in both healthy and infected plants. Intracellular penetration of fungal hyphae was also observed. Moreover, transmission sections of roots showed other effects of Fusarium infection. Meanwhile, A. monosperma extract could alleviate the harmful effects of Fusarium infection via improving the above mentioned studied parameters. From the obtained results, it can be concluded that A. monosperma extract as a biocontrol agent played an important role in enhancing plant resistance against Fusarium wilt disease of bean plants.

Evaluation of the biodeterioration activity of lichens in the Cave Church of Üzümlü (Cappadocia, Turkey)

The Üzümlü Church is an unusual soft tuff formation located in central Turkey (Cappadocia) dated around the 7th century. The rocks on its external surfaces show a severe erosion with an evident surface detachment. Moreover, the widely distributed black-grey crusts mainly consisting of lichens point out the need of evaluating their deteriorative effects. This research was focused on lichen deterioration by indicating the biodeterioration processes and on selecting the most appropriate preservation treatments suitable for stone conservation. The interaction of lichens with stone was studied by evaluating through microscopic analyses, the spread and the depth of fungal hyphae penetration, and by applying the LPBA Index. The SEM analysis of all fragments shows a dramatic loss of the stone matrix and a dense network of fungal hyphae within the rock. Nevertheless, both the high penetration of fungal hyphae, and the consequent difficulty in their removal without a strong peeling effect, both the relevant loss of the stone matrix and finally the evident reduction of water penetration when crusts are present in the surfaces, advise against their removal. Further chemical treatments of consolidation should be carefully evaluated.

In vitro effect of Chrysosporium indicum and Chrysosporium keratinophylum on Toxocara canis eggs

The degree of antagonism exercised by fungi on geohelminth development varies according to the morphological alterations caused by different fungal species. Saprophytic fungi may exert ovicidal or ovistatic effects. The aim of this study was to apply scanning electron microscopy (SEM) to observe the action of two soil saprophytic species of Chrysosporium (C. indicum and C. keratinophylum) on Toxocara canis eggs. The fungal strains to be tested were incubated for 28 days at 28°C in 2% water agar with a suspension of unembryonated T. canis eggs. A suspension of T. canis eggs in 2% water agar was used as control group. The assay was done in triplicate for each fungus and the control group. SEM observations were performed on the 4th, 7th, 14th, 21st, and 28th day after inoculation. The effect of the fungi on eggs was evaluated in accordance with the alterations observed on the surface and the changes in the normal characteristics of the eggs. Hyphae around the eggs, appresoria penetrating the shell and changes in the typical egg membrane were observed in this assay. Type 3 effect (alterations that occur both in the embryo and the shell, and hyphal penetration of the eggs) was the prevalent effect. SEM allowed us to observe clearly the morphological alterations in T. canis eggs due to the effect of C. indicum and C. keratinophylum. Both saprophytic species of Chrysosporium alter the egg structure and alterations increase as exposure increases.

Comparison of mechanical properties for mite cuticles in understanding passive defense of phytoseiid mite against fungal infection

Entomogenous fungi have evolved many strategies for overcoming the defense systems that insects/mites, in turn, continue to evolve to fend off the fungi's attempt at infection. Although the host immune systems have garnered much attention, the importance of the cuticular defenses remains under-examined. The present study compared the mechanical properties – hardness and elastic modulus of the cuticles between a phytoseiid mite, Neoseiulus barkeri and a pest spider mite, Tetranychus urticae using nanoindentation techniques. We determined that the dorsal cuticle of N. barkeri possesses significantly greater hardness and stiffness, while the ventral cuticles of N. barkeri has mechanical properties similar to that of T. urticae. Exposure of the lateral abdomen region of N. barkeri after being distended by feeding, however, increased the susceptibility to fungal infection. It is found that the fungus can breach the exposed part of abdominal cuticle that is softer and less stiff, followed by conidial adhesion. This suggests mechanical properties of the dorsal cuticle of N. barkeri contribute substantially to withstand the mechanical force exerted by the attempted penetration of fungal hyphae. This study expand our knowledge of fungus–predator interactions and provide new insights into the feasibility of applying multiple biocontrol agents in pest control strategies.

Localization of helotialean fungi on ectomycorrhizae of Castanopsis cuspidata visualized by in situ hybridization

Non-ectomycorrhizal fungi that associate with typical ectomycorrhizae often remain hidden, and their localization inside ectomycorrhizal (ECM) roots has remained uncharacterized. In this study, the fungal community associated with the ectomycorrhizae of Castanopsis cuspidata was investigated using a culture-dependent isolation technique. Additionally, the species composition and localization were determined using molecular techniques. The results of the isolation and identification of fungal species revealed the predominance of a few species belonging to the order Helotiales. Furthermore, the fungal community structures were significantly different depending on the taxa of the ectomycorrhiza-forming fungi. A taxon-specific probe was developed to analyze the localization of one dominant Hyaloscyphaceae (Helotiales) species in ECM tissues by in situ hybridization. Hybridization signals were detected on the surface of the fungal mantle and around the ECM fungal cells within the mantle. Hyphal penetration into ECM hyphal cells of fungal mantles was also observed. Signals were not detected in the Hartig net or plant tissues inside the mantle in healthy ectomycorrhizae. These findings suggest that the analyzed species interact not only with host plant as root endophyte but also directly with the ECM fungi.

Hirsutella thompsonii and Pochonia chlamydosporia (Syn. Verticillium chlamydosporium) Mycelia Growth and Predation on Panagrellus redivivus

This research aimed to evaluate the nematophagous ability of 4077-Verticillium chlamydosporium var. chlamydosporium and 4466-Hirsutella thompsonii isolates and relate mycelia growth to the influence provoked by movement of nematodes. Each fungus grew in PDA (potato, dextrose, agar) medium end up to pure colonization. Then, ten mycelia plugs of 8 mm diameter were removed from colony borders and transferred to the center of ten Petri plates containing water-agar 2% medium. These plates were previously divided into four quadrants that received a number of 25 individuals of free-living nematodes (Panagrellus redivivus), composing a total of 100 nematodes per plate. Evaluations started after 24 hours of interaction, considering predation percentage and mycelia growth as stimuli of nematodes presence. Results showed growing predation performance to both isolates, being higher for V. chlamydosporium var. chlamydosporium since from first evaluation time, controlling more than 50% of nematode population initially added. Its predation potential was 39.2%, 38.4% and 48.35% higher than H. thompsonii at first, second and third evaluation day, respectively. Generally, nematodes did not stimulate mycelia growth, unless for H. thompsonii at 72 hours of interaction compared to control plates (without nematodes). Stress resulting from isolates transference from PDA to water-agar 2% resulted in sparse mycelia growth and it could have affected the predation performance of H. thompsonii that controlled nematodes in low levels throughout experiment. Independently of predation level, pictures revealed that both isolates has ability to control P. redivivus through hyphae penetration.