A combined cryo-scanning electron microscopy/cryoplaning approach to study the infection of Meloidogyne incognita eggs by Pochonia chlamydosporia

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The fungus Pochonia chlamydosporia is a saprophytic soil-dwelling fungus and is also a parasite of the eggs of the root-knot plant-parasitic nematodes (Meloidogyne spp.). Studies on its molecular characterisation, diversity, tritrophic interactions and ecology have been carried out. However, to elucidate the role in soil and rhizosphere ecology of this and other nematophagous fungi used in biological control, and to enhance their exploitation, it is necessary to improve the understanding of the biology and fungus-nematode infection process using different approaches, including microscopy. Low-temperature Scanning Electron Microscopy (cryo-SEM) techniques allow the examination of frozen, fully hydrated samples that can reveal important ultrastructural features occurring through fungus-nematode interactions. A method that combined cryo-SEM with cryoplaning was developed to examine samples of eggs, gelatinous matrix and females of Meloidogyne incognita colonised by P. chlamydosporia. The fungal samples were produced in potato dextrose agar to which different nematode stages were added and processed for cryo-SEM and cryoplaning within a period of 0-72 h post inoculation. The method was found to be rapid and economical, provided clear and detailed external images of the infection process and allowed viewing of sections through structures with minimal processing in comparison to other cryo-SEM techniques and it could be adapted to study other fungus-nematode interactions.


International Journal of Fundamental and Applied Nematological Research



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  • Cryo-SEM micrographs of Meloidogyne incognita egg samples on Pochonia chlamydosporia colonised Potato Dextrose Agar (PDA) plugs. A, B: Eggs in different stages of embryogenesis added to fungal colonised PDA plugs. A: Eggs in early stage of development; B: Second-stage juvenile ready to hatch; C: Hyphae (arrow) in contact with the egg surface (24 h post inoculation); D: Hyphae, conidia and egg extracellular matrix (ecm) coated surfaces. Egg surface showing close contact between hyphae and conidia (24 h post inoculation). (Scale bars: A, B = 10 μm; C = 10 μm; D = 5 μm.)

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  • Cryoplaning micrographs of Pochonia chlamydosporia infection of Meloidogyne incognita adult female, gelatinous matrix (gm) and eggs at 72 h post inoculation (pi). A: Female body showing external (eh) and internal (ih) colonisation by the hyphae; B: Fine grain texture of the gm egg sac surrounding the hyphae (arrow); C: Egg sac showing the mesh-like texture of the gm surrounding hyphae and eggs; D: Egg sac internal colonisation by hyphae at 24 h post inoculation (pi). Note the packed eggs (arrow heads) within the colonised gm; E: Multi-layered egg shell and internal details of the second-stage juvenile (J2) before fungal infection. In the cryoplaned image the distinct multi-layered structure (mes) and the annulations (a) on the cuticle of the developing J2 can be seen; F: Cryoplaned image of M. incognita egg showing P. chlamydosporia hyphae colonisation inside (ih) and outside the egg (eh) by hyphae at 72 h pi. The eggshell is represented by a single thin membrane and the inset at higher magnification illustrates how cryoplaning captures the position of the eggshell breached by the hyphae. (Scale bars: A = 5 μm; B = 2 μm; C = 5 μm; D = 10 μm; E = 4 μm; F = 10 μm, inset bar = 1 μm.)

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  • Cryoplaning and cryofracture micrographs of Pochonia chlamydosporia infection of Meloidogyne incognita eggs and gelatinous matrix (gm) at 72 h post inoculation (pi). A: Cryoplaning micrograph of an egg containing a developing non-infected second-stage juvenile (J2); B, C: Cryoplaning micrographs of infected eggs showing body contents of developing J2 and surrounding gm fully colonised by the fungus (h); D: Cryofracture micrograph of a colonised egg at 24 h pi. Note irregular borders of the eggshell (arrow heads) and egg contents in contrast to the flat, smoother surfaces obtained when cryoplaning; E: Cryofracture of hyphae colonising the gm; F: Cryofracture micrograph of gm and eggs colonised by the fungus. The gm texture is finer than in Figure 3E. Note the mesh-like texture of the gm. (Scale bars: A = 10 μm; B, C = 10 μm; D = 5 μm; E = 1 μm; F = 4 μm.)

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