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

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.

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Differentiation of infection structures of the powdery mildew fungusUncinula necatorand adhesion to the host cuticle

Canadian Journal of Botany ◽

10.1139/b00-016 ◽

2000 ◽

Vol 78 (3) ◽

pp. 409-421 ◽

Cited By ~ 1

Author(s):

J Rumbolz ◽

H -H Kassemeyer ◽

V Steinmetz ◽

H B Deising ◽

K Mendgen ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Low Temperature ◽

Powdery Mildew ◽

Magnaporthe Grisea ◽

Post Inoculation ◽

Infection Structures ◽

Grapevine Powdery Mildew ◽

Temperature Scanning ◽

Scanning Electron

Development and adhesion of infection structures of the grapevine powdery mildew fungus, Uncinula necator (Schw.) Burr., were investigated during the early stages of leaf colonization. Light microscopy showed that primary appressoria occurred 3.5 h post inoculation (p.i.) and that hyphae on the leaf surface, indicative of successful host colonization, appeared 14 h p.i. Low temperature scanning electron microscopy revealed deposits of extracellular material at the contact zone of fungal structures and plant cuticle, suggesting firm attachment of the pathogen. To investigate whether or not esterase or cutinase activity is involved in establishing the fungus on the host cuticle, histochemical assays and inhibitor studies were performed. Results indicated that esterase activity was associated with conidia and infection structures. A single fungal extracellular protein was identified as a cutinase by its ability to hydrolyze3H-cutin. Probing Southern blots of genomic DNA of U. necator, Magnaporthe grisea, and Fusarium solani f.sp. pisi with the cutinase gene of F. solani f.sp. pisi suggested that the cutinase gene of U. necator shares only limited sequence similarities with the cutinase genes of the other fungi investigated. Adhesion assays showed that the presence of esterase-cutinase inhibitors on the cuticle did not significantly affect adhesion. The role of the enzyme in fungal adhesion is discussed.Key words: grapevine powdery mildew, Vitis vinifera, cutinase, extracellular matrix, cryofixation, low temperature scanning electron microscopy.

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Intracellular structures of rapid frozen biological samples observed by high-resolution low-temperature Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100155657 ◽

1989 ◽

Vol 47 ◽

pp. 736-737

Author(s):

T. Inoué ◽

H. Koike

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Low Temperature ◽

Liquid Nitrogen ◽

Specimen Preparation ◽

Chemical Fixation ◽

Brown Adipose ◽

Critical Point Drying ◽

Temperature Scanning ◽

Scanning Electron

Low temperature scanning electron microscopy (LTSEM) is useful to avoid artifacts such as deformation and extraction, because specimens are not subjected to chemical fixation, dehydration and critical-point drying. Since Echlin et al developed a LTSEM, many techniques and instruments have been reported for observing frozen materials. However, intracellular structures such as mitochondria and endoplasmic reticulum have been unobservable by the method because of the low resolving power and inadequate specimen preparation methods. Recently, we developed a low temperature SEM that attained high resolutions. In this study, we introduce highly magnified images obtained by the newly developed LTSEM, especially intracellular structures which have been rapidly frozen without chemical fixation.[Specimen preparations] Mouse pancreas and brown adipose tissues (BAT) were used as materials. After the tissues were removed and cut into small pieces, the specimen was placed on a cryo-tip and rapidly frozen in liquid propane using a rapid freezing apparatus (Eiko Engineering Co. Ltd., Japan). After the tips were mounted on the specimen stage of a precooled cryo-holder, the surface of the specimen was manually fractured by a razor blade in liquid nitrogen. The cryo-holder was then inserted into the specimen chamber of the SEM (ISI DS-130), and specimens were observed at the accelerating voltages of 5-8 kV. At first the surface was slightly covered with frost, but intracellular structures were gradually revealed as the frost began to sublimate. Gold was then coated on the specimen surface while tilting the holder at 45-90°. The holder was connected to a liquid nitrogen reservoir by means of a copper braid to maintain low temperature.

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Criteria for the evaluation of low-temperature Scanning Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100145820 ◽

1986 ◽

Vol 44 ◽

pp. 902-903

Author(s):

Alan Beckett

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Low Temperature ◽

Conformational Changes ◽

Chemical Vapour ◽

List Type ◽

Dimensional Changes ◽

Critical Point Drying ◽

Temperature Scanning ◽

Scanning Electron

Low temperature scanning electron microscopy (LTSEM) has been evaluated with special reference to its application to the study of morphology and development in microorganisms. A number of criteria have been considered and have proved valuable in assessing the standard of results achieved. To further aid our understanding of these results, it has been necessary to compare those obtained by LTSEM with those from more conventional preparatory procedures such as 1) chemical fixation, dehydration and critical point-drying; 2) freeze-drying with or without chemical vapour fixation before hand.The criteria used for assessing LTSEM for the above purposes are as follows: 1)Specimen immobilization and stabilization2)General preservation of external morphology3)General preservation of internal morphology4)Exposure to solvents5)Overall dimensional changes6)Cell surface texture7)Differential conformational changes8)Etching frozen-hydrated material9)Beam damage10)Specimen resolution11)Specimen life

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