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Histological and Ultrastructural Characterization of Coffee
Resistance to Colletotrichum kahawae
A. LOUREIRO1, M.D.C. SILVA1, V. VÁRZEA1, P. MONCADA2, B. BERTRAND3, M. NICOLE3
1Instituto de Investigação Científica Tropical (IICT) - Centro de Investigação das Ferrugens do Cafeeiro (CIFC), Oeiras, Portugal
2Cenicafe – Chinchiná, Caldas, Colombia
3Unité Mixte de Recherche IRD-CIRAD-UM2, Montpellier, France
SUMMARY
The growth of Colletotrichum kahawae Waller and Bridge and the early responses it induced in hypocotyls with different levels of resistance were investigated by light and transmission electron microscopy. Fungal penetration occurred through melanized appressoria directly into the epidermal cell walls with formation of an infection vesicle. Plant susceptibility involved the intra- and intercellular ramification of the infection vesicle in the living host cells. This brief period of biotrophy was followed by the necrotrophic fungal growth and the production of symptoms (dark sunken lesions with sporulation). In the necrotrophic phase, colonization of host cells by the fungus was associated with severe walls alterations and death of the host protoplasts. The more resistant genotypes were characterized by a restricted fungal growth associated with hypersensitive-like host cell death, modifications in the cell walls (thickness and autofluorescence) and early accumulation of phenolic compounds, such as flavonoides and hydroxycinnamic acid derivatives.
INTRODUCTION
Coffee berry disease, caused by the fungus Colletotrichum kahawae Waller & Bridge is a major threat to the production of Coffea arabica in Africa. Selection of resistant coffee is an alternative to the use of chemical control since evidences has been obtained that Hibrido de Timor (HDT) derivatives may represent a good source of resistance (Silva et al., 2006).
Host resistance to Colletotrichum spp. have been usually associated to host cell wall modifications, namely the increase in the levels of hydroxyproline-rich glycoproteins (HRGPs), to an early accumulation of phenolic compounds in the infected host cells and in some cases with the rapid hypersensitive death of host infected cells characterized by a rapid loss of host cells membrane integrity and the accumulation of phenolic oxidation products (Esquerré-Tugayé et al., 1992; Goodman and Novacky, 1994; Skipp et al., 1995; Torregrosa et al., 2004).
The growth of a C. kahawae isolate from Malawi (CIFC - Mal2) and the sequence of responses it induced in resistant hypocotyls of HDT derivatives as well as on the susceptible cultivar Caturra were investigated by light and transmission electron microscopy.
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MATERIAL AND METHODS Hypocotyls inoculation
Resistant hypocotyls of HDT derivatives as well as susceptible hypocotyls of the cultivar Caturra were inoculated with the isolate Mal2 of C. kahawae, from Malawi. Hypocotyls at the soldier stage were inoculated according to the technique described by Van der Vossen et al. (1976) with slight modifications. The hypocotyls were placed on plastic trays lying down on a wet nylon sponge and then inoculated with a 5μl drop of a conidia suspension (2 x 106/ml). Covered trays were placed in a Phytotron 750 E at 22 ºC incubated the first 24h in the dark and then kept with a photoperiod of 12 hours.
Light microscope (LM) observation of fresh tissues
Cross sections of infected leaf fragments made with a freezing microtome were stained and mounted in blue lactophenol (Silva et al., 1999) to evaluate fungal post-penetration growth stages. Hyphal length inside hypocotyl tissues were estimated using a micrometric eyepiece. To detect autofluorescent cells, cross sections of infected hypocotyl fragments were placed in 0.07 M, pH 8.9 phosphate solution (K2HPO4), for 5 min and mounted in the same solution (Silva et al., 2002). Autofluorescence under blue light indicated accumulation of phenolic-like compounds. To detect callose deposition, cross sections of infected tissues were placed in 0.07 M, pH 8.9 phosphate solution (K2HPO4), for 10 min, and then transferred into a 0.01% solution of aniline blue in the phosphate solution, for 10 min before being mounted in the same solution (Silva et al., 2002). Callose deposition was identified by bright yellow fluorescence (Eschrich and Currier, 1964). To detect flavonoids, gallic and hydroxycinnamic derivatives the Neu’s Reagent (Neu, 1956) was used. Cross sections of infected hypocotyls were placed in 1% of 2-amino-ethyldiphenyl-borinate diluted in absolute methanol for 5 min and mounted in glycerine-water. Observations were made with a microscope Leitz Dialux 20 equipped with a mercury bulb HB 100W, u.v. light (excitation filter BP 340-380; barrier filter LP 430) and blue light (excitation filter BP 450-490; barrier filter LP 515).
Transmission electron microscope (TEM) observations
Hypocotyl strips cut from healthy and infected tissues were fixed in glutaraldehyde and osmium tetroxide, embedded and polymerized (70 ºC, overnight) in Spurr´s resin (Sigma, Germany), as previously described (Rijo and Sargent, 1974). Semi-thin sections (2 μm) of the polymerized blocks were stained with 0.5% aqueous toluidine blue solution and observed using light microscopy. Ultrathin sections (80-90 nm) of the same selected blocks, collected on Formvar-coated nickel grids (200 mesh), were stained with uranyl acetate and Reynold´s lead citrate. The observations were made with a FEI transmission electron microscope operating at 70 kv.
RESULTS AND DISCUSSION
The early stages of C. kahawae development were essentially the same on the surface of resistant and susceptible coffee hypocotyls. Fungal conidia adhered to the cuticle and germinated producing germ tubes and melanized apressoria which penetrated the cuticle directly into the epidermal cell walls with the formation of infection vesicles that grew intra- and intercellularly (Figure 1).
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Figure 1. First stages of C. kahawae infection process on coffee hypocotyls. LM observations, blue lactophenol staining. Two infection sites showing melanized appressoria (A) and a infection vesicle (v) in one case and further intra- and intercellular growth (arrow) in the other, 41 h after inoculation (Scale bar = 11µm)
In the susceptible hypocotyls, the fungus pursued its growth on the living host cells. After a brief period of biotrophy, that lasted around 72 h, a necrotrophic fungal growth began culminating in the appearance of symptoms - dark sunken lesions with sporulation. The biotrophic phase was repeated as the fungus started the colonisation of new host cells. Consequently, it was possible to observe hyphal growth simultaneously in dead and living host cells. As shown by light and electron microscopic observations, the necrotrophic phase was associated with severe wall alterations and death of the host protoplasts (Figure 2 A and B) and the deposition of callose around some intracellular hyphae (Figure 2 C). These host responses in the susceptible tissues seemed to occur too late to prevent fungal growth and sporulation.
A B C
Figure 2. Necrotrophic growth of C. kahawae in susceptible hypocotyls, 7 days after inoculation. A - LM observation, toluidine blue staining. Fungal hyphae (arrows) in living and in necrotized (N) host cells (Scale bar = 9µm). B - TEM observation. Hypha (H) penetrating the cell wall between two cortex cells. Note the constriction of the hypha as it passes through the wall (arrows) and the disorganization of the cytoplasmic content of the invaded host cells and calose deposition around intracellular hyphae (arrow head) (Scale bar = 1µm). C - LM observation. Callose around intracellular hyphae (arrows) (Scale bar = 11µm).
In the resistant hypocotyls, the fungal hyphae were confined to epidermal cells or to the first layers of the cortex cells (Figure 3 A and B). As shown by light and electron microscopic observations this restricted fungal growth was associated with hypersensitive-like host cell death (membrane breakdown at the level of plasma membrane and in different organelles namely chloroplasts and mitochondria, change in the appearance of choroplasts and coagulation of cytoplasm), modifications in the cell walls (thickness and autofluorescence) and early accumulation of phenolic compounds, such as flavonoides and hydroxycinnamic acid derivatives (Figure 3 C). The hydroxycinnamic acid derivatives accumulation occurred mainly in the plant cell walls while flavonoids accumulation occurred in the cytoplasmic contents.
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A B C
Figure 3. Fungal growth inside resistant hypocotyls and host responses. A - LM observation, toluidine blue staining. Infection site showing a melanized appressorium (A) and an intracellular hypha (arrow) confined to the epidermal plant cell, 7 days after inoculation (Scale bar = 8 µm). B - TEM observation, 7 days after inoculation. Melanized appressorium (A), intracellular hypha (H) with cytoplasmic content totally disorganised and invaded host cell with thicker walls (Scale bar = 2 µm). C - LM observation. Infection site showing autofluorescence of the epidermal cell wall and cytoplasmic contents (arrow), 60h after inoculation. (Scale bar = 12.5 µm).
The host responses observed in the incompatible interaction between C. kahawae and Coffea were similar to other Colletotrichum-plant interactions such as C. lindemuthianum with Phaseolus vulgaris and C. trifolii with Medicago truncatula (Esquerré-Tugayé et al., 1992; Skipp et al., 1995; Torregrosa et al., 2004).
Cytochemical tests are currently under study to investigate the composition of the fungal - plant interface and also the involvement of cell wall-degrading enzymes in different stages of the infection.
REFERENCES
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Goodman R.N. and Novacky A.J. (1994). The hypersensitive reaction in plant to pathogen: a resistance phenomenon. APS Press, St. Paul, Minnesota.
Neu R. (1956). Naturwissenschaften 43:82.
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