Catching Endophytic Fungi
Comparison of Histochemical and Immunological Methods
- Beauveria bassiana on a Brassica napus leaf: Immunofluorescence labelling with rabbit primary polyclonal and FITC conjugated secondary fluorescent antibodies. Image centre: germinating spores growing through stomata.
- Fig. 1: Beauveria bassiana on a Brassica napus leaf: stained with safranin/solophenyl 7GFE, 7 dpi.
- Fig. 2: Germinating conidia of Beauveria bassiana on a Brassica napus leaf: stained with trypane blue, 6 dpi.
- Fig. 3: Beauveria bassiana hyphae in the intercellular space of the tissue of a young shoot of Vicia faba: Immunolocalization with rabbit primary polyclonal and FITC conjugated secondary fluorescent antibodies.
Entomopathogenic fungi are efficiently used in biological control of economically important herbivorous insects. Efforts have been made to establish entomopathogenic fungi directly in crop plants to control both insect pests and plant pathogens. However, establishment and unequivocal detection of systemically growing endophytes in most dicots is still problematic. Optimization of histochemical and immunofluorescent localization of the applied fungi in plant sections is essential.
Biological control of herbivorous insects is gaining more and more importance in production of healthy food. The damage of crop plants by insects causes considerable economical loss, e.g. of Canola (Brassica napus) by several beetles, e.g. the rape pollen beetle (Brassicogethes aeneus), the cabbage stem flea beetle (Psylliodes chrysocephalus) and the rape stem weevil (Ceutorhynchus napi), and of faba bean plants (Vicia faba) by the black bean aphid (Aphis fabae). One strategy to control those pests biologically is the application of entomopathogenic fungi, commonly as spore suspensions. Insects coming into contact with these fungi are colonized and killed by fungal growth inside the insect and by insecticidal toxins. However, formulation of entomopathogenic fungi is still critical, because propagules not adhering well to the plant surface may be easily washed off or inactivated due to desiccation or UV light. A much more elegant solution would be plant protection by endogenously growing entomopathogenic fungi. Many plant species have been shown to harbor fungal endophytes, of which some genera like Beauveria, Lecanicillium or Isaria include fungal entomopathogens . Encouraged by these apparently harmless endophytes, attempts have been made to experimentally introduce these into crop plants. For monitoring the presence and distribution of endophytes in plants, three principle methods can be applied: 1. direct localization by microscopical methods, 2. detection with diagnostic tools like PCR or ELISA, and 3. isolation of the endophytes from the tissue using agar media.
Light and fluorescence microscopical techniques were established in the present work to investigate inoculated canola and faba bean plants by different entomopathogenic fungi and to follow fungal growth on the plant surface and inside the leaf tissue. The fungal structures were visualized using common dyes as well as polyclonal antibodies labelled with a fluorescence marker.
Experimental plants, Brassica napus L. and Vicia faba L., were treated with Beauveria bassiana ATTC74040, Isaria fumosorosea, Lecanicillium muscarium and Metarhizium anisopliae from the fungal culture collection of the Institute for Biological Control of the Julius Kühn-Institut. Suspensions were applied to the plants, either by infiltration of blastospores from submerged cultures into leaves or stems with a syringe or by spraying on leaves, flowers, and seeds. Between 4 hpi and 27 dpi, plant samples were taken and submitted to various staining methods. Sections of inoculated leaves were cleared with 100% chloralhydrate/90% lactic acid (2:1) for one to three days, and after thoroughly rinsing with H2Odest., sections were stained with the following dyes: Trypane blue, 0.01%; Blankophor, 0.01% in 0.1 M Tris buffer pH 9.0; Solophenyl flavin 7GFE, 0.1% in 0.1 M Tris/HCl pH 8.5. To diminish the tissue fluorescence, sections were pre-stained with 0.05% safranin; Immunofluorescence labelling: the IgG fraction of polyclonal antibodies raised against B. bassiana in rabbit was used in combination with FITC-labelled goat anti rabbit secondary antibodies.
Samples were viewed under UV (Leica-filter block A, excitation 340-380 nm, emission ≤ 430 nm) and under fluorescent blue light (Leica-filter block I3, excitation 450-490 nm, emission ≤ 515 nm) with an Aristoplan epifluorescence microscope (Leica, Wetzlar, Germany). Digital images were taken with a CCD camera (ColorView II, Olympus) using the software AnalySIS Five.
Results and Discussion
All dyes tested did stain the examined entomopathogenic fungi, i.e. B. bassiana, I. fumosorosea, L. muscarium and M. anisopliae. Blankophor efficiently bound to the hyphae on the leaf surface of B. napus. Solophenyl flavone 7GFE, binding unspecifically to glucanes, stained B. bassiana spores and hyphae, yielding the best contrast to the plant tissue after pre-staining with safranin, which suppresses plant cell wall fluorescence (fig. 1). Comparable patterns were obtained with M. anisopliae on B. napus leaves. Also trypane blue contrasted well spores and hyphae on B. napus leaf surfaces (fig. 2). Polyclonal antibodies, raised against B. bassiana and I. fumosorosea protein detected B. bassiana, I. fumosorosea, L. muscarium and M. anisopliae (e.g. fig. 3). Though the spore suspension was visibly infiltrated into the intercellular space of B. napus leaves through the stomata, B. bassiana hyphae were detected only on the lower leaf surface, the site of injection. The spongy and palisade parenchyma were clearly devoid of hyphae in B. napus, as well as the vascular bundles. However, inside the tissue, in cross sections no hyphae were detected. Safranin/7GFE-staining of the tissue allowed a view into the spongy parenchyma. With this method, spores and small hyphae could be detected in the tissue of V. faba 2 dpi. In contrast to B. napus, within the bean tissue spores germinated and B. bassiana hyphae were detected abundantly growing within the intercellular space along the cell walls (fig. 3).
The family Brassicaceae is known to be highly defensive against many fungi and, in contrast to nearly all other plant classes and families are devoid of mycorrhizal fungi . One reason probably is that the Brassicaceae produce toxic glucosinolates . Fabaceae roots are known for their symbiosis with rhizobia, so they may be more predispositioned to other microorganisms. In our experiments, hyphae of B. bassiana, I. fumosorosea and L. muscarium could, indeed, be found in faba bean leaves and young stems, best documented by detection with antibodies and enhancement by secondary FITC-conjugated antibodies (fig. 3).
The problem of weak endophytic fungal life in crop plants is suggested to be due either to defense reactions within the plant tissue, as in the case of B. napus, or to lack of organic nutrients within the intercellular space of the plant tissue, such as leaves, stem, vascular bundles, and shoot apical meristem.
The authors thank Helga Radke and Ursula Apel for valuable technical assistance.
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 Barbara A. Halkier, Jonathan Gershenzon: Biology and Biochemistry of Glucosinolates, Annual Review of Plant Biology 57, 303–333 (2006) DOI 10.1146/ annurev.arplant.57.032905.105228
Cornelia I. Ullrich1
Regina G. Kleespies1
1Julius Kühn-Institut (JKI), Institute for Biological Control, Darmstadt, Germany
2Julius Kühn-Institut (JKI), Institute for Epidemiology and Pathogen Diagnostics, Quendlinburg, Germany