Reação de folhas de mandioca ao Microcyclus ulei, agente causal do mal-das-folhas da seringueira

Reaction of cassava leaves to Microcyclus ulei, causal agent of South American leaf blight of rubber tree

ARTIGOS / ARTICLES

Reaction of cassava leaves to Microcyclus ulei, causal agent of South American leaf blight of rubber tree

Reação de folhas de mandioca ao Microcyclus ulei, agente causal do mal-das-folhas da seringueira

Jens Hagen^I; Luadir Gasparotto^II; Vicente H. F. Moraes^II; Reinhard Lieberei^I

^IInstitute of Applied Botany, University of Hamburg, Ohnhorststr. 18, 22609 Hamburg, Germany

^IIEmbrapa Amazônia Ocidental, Cx. Postal 319, CEP 69011-970, Manaus, AM, e-mail: [e-mail]

ABSTRACT

Young cassava (Manihot esculenta) leaves inoculated with conidiospores or treated with an autoclaved conidiospore suspension of Microcyclus ulei, a pathogen to several species of the genus Hevea, react strongly with induction of blue fluorescing compounds in the contact areas, demonstrating local cell growth and irregular tissue formation, leaf distortion and, occasionally, leaf abscission. Restricted hyphal growth also occurs in areas inoculated with living spores, but without formation of new conidiospores. The plant reaction resembles a complex resistance reaction to a biotrophic pathogen rather than a typical non host defense reaction.

Additional keywords: host range, defense reactions, Manihot esculenta, Hevea spp.

RESUMO

Folhas jovens de mandioca (Manihot esculenta) inoculadas com uma suspensão de conídios viáveis ou autoclavados de Microcyclus ulei, um patógeno de diversas espécies do gênero Hevea, reagiram fortemente com indução de compostos azuis fluorescentes nas áreas de contato, com a morte das células locais e a formação irregular dos tecidos, distorção das folhas e, ocasionalmente, com a abscisão da folha. Também ocorreu crescimento restrito das hifas, mas sem produção de novos conídios. A reação das plantas foi mais semelhante a uma reação de resistência complexa a um patógeno biotrófico do que uma reação típica de defesa de uma planta não hospedeira.

INTRODUCTION

The ascomycete Microcyclus ulei (P.Henn.) v. Arx reveals a very narrow host range. According to Chee (1976), who used data of field observations of large rubber (Hevea spp.) experimental fields in Trinidad, the specialized fungus attacks only plants of the genus Hevea and within the genus only the species or the respective hybrid genotypes of the species H. brasiliensis (Wild. ex. A. Juss) Muell. Arg., H. guianensis Aub., H. benthamiana Muell. Arg., H. spruceana (Bth.) Muell. Arg., Observations in gene pool collections of Hevea in Manaus, AM, Brazil, revealed, that the genotypes of H. camargoana Pires and H. camporum Ducke are also severely attacked by M. ulei (Junqueira et al., 1989). Unfortunately it is not possible to decide if the latter species were attacked by new races of the fungus occurring in Manaus or if the plant material had not been available for testing according Chee (1976). Gasparotto & Junqueira (1994) described a high physiological variability of a number of isolates of M. ulei, derived from different agroclimatic regions of Brazil, ranging from the hot and humid environment of central Amazonas to the temperate, semidry areas in the southern areas of Brazil. Changes of host ranges combined with the changes in adaptation to a wide range of abiotic environments cannot be excluded for this economically important fungus. In addition to this fact, an enormous amount and variability of potential host genotypes were provided to the pathogen during the colonization of Amazonas with rubber being one of the most important crop plants for small landholders. During this phase (1972 to 1989) under the auspices of the SUDHEVEA (1971), more than 75,000 ha were planted with rubber (Gasparotto et al., 1997), which turned out to be completely susceptible to M. ulei.

Lieberei et al. (1989) reported on field observations and preliminary tests, in which two isolates of the M. ulei-collection of the National Brazilian Rubber Research Institute (Junqueira et al., 1986) were applied in greenhouse studies to cassava (Manihot esculenta Crantz) leaves, which led to a strong morphological reaction against spores of M. ulei including leaf deformation and premature leaf fall. In order to evaluate the potential threat of this fungus to the tropical staple food plant cassava, experiments were carried out to describe in more detail the reactions of cassava leaves to spore samples of M. ulei.

MATERIAL AND METHODS

Cultivation of Microcyclus ulei

Isolate 40R7 was cultivated in 100 ml tubes on Potato-Sucrose-Agar (PSA) 0.5% Sucrose, pH 5.0. In a 2-month period the fungus (mycelium and spores) was transferred to fresh medium and was cultivated in the dark at 23 ºC. One week before transfer to the new tube the cultures were exposed two times to 45 min of light of a Osram DULUX 11 W / 21. This treatment enhanced induction of conidiophores and of spore production. Spore mass production was carried out according to Lieberei et al. (1983).

Plant cultivation

The experiments were carried out with non-defined material and with clone CG 165-7 of CIAT, Colombia. The plants were cultivated in pots of 90 cm diameter, at a day-night regime of 12 to 12 h, 70 to 90% relative humidity, 18 to 31 ºC, and 450 to 600 µE of light. Fertilization was carried out using 1.2 g Plantosan^® per plant per six weeks (Aglucon Spezialduenger GMBH, Kleinkaribach, Germany).

Inoculation and incubation

Conidia were isolated as an aqueous suspension by adding 5 ml of sterile tap water to a sporulating culture of M. ulei, grown in 250 ml Erlenmeyer flasks on 70 ml PSA (0.5% sucrose). The conidia were collected, the washing procedure was repeated two times and the three washings of 15 ml were pooled and submitted to low speed centrifugation (300 g, 5 min). The spores were washed two times using sterile tap water, the spore number was adjusted after microscopically counting to 1 x 10⁶ spores per ml and the suspension was applied to the leaves by a paintbrush type H, Paasch, USA. The distance of the paintbrush to the six days old leaves was 15 cm, at an initial pressure of 1.5 bar. Inoculated leaves were kept in humid polyethylene bags until harvest.

For control experiments, washed spore suspensions were autoclaved at 120 ºC, 15 min, 1 bar. Control solutions were tap water and diluted culture medium (PSA), which were applied in the same way.

Microscopy and staining

The microscopic studies were done with a BH-2 Research microscope of Olympus, which was equipped with a fluorescence device and interference contrast system.

Lignin was stained using phloroglucin-HCl according to Nultsch et al. (1979), phenolic cell wall-bound phenolics were detected with toluidin blue dye, according to Feder & O'Brien (1968) and fungal mycelium was detected applying the Pianese-stain (Gerlach, 1984).

RESULTS

Macroscopic and microscopic symptoms

Cassava leaves inoculated with living conidiospores or autoclaved conidiospore suspensions of M. ulei reacted in a similar way to the treatment, revealing leaf deformations and necrotic leaf areas (Figure 1a,b). Leaves treated with tap water or fresh culture medium did not show any visible change in leaf morphology and did not reveal any necrotic lesion.

 

The first day after inoculation there were no macroscopic changes visible. Using fluorescence microscopy with excitation 300 to 400 nm, and a 20 UB-W-2 filter, blue fluorescing spots were visible in the epidermal and subepidermal cell layers underneath the spores, regardless of whether living spores or autoclaved spores had been used for inoculation.

Four days after inoculation the blue fluorescence was less expressed, but still detectable.

On the lower leaf side, globose swollen structures developed directly in the contact zone of the leaf surface with the spores. The small swollen areas were water soaked and the epidermal cells were slightly enlarged. Within these areas of hypertrophic cell growth browning occasionally occurred (Figure 1c, d).

After ten days blue fluorescence was no longer detectable. The swollen areas revealed larger brown areas. Many cells collapsed and formed small necrotic lesions of less than 1 mm in size. Many leaves were abscised after about two weeks, but some remained attached to the plants. No spore formation was to be seen within the following two months of observation on the fallen leaves nor on the living leaves that remained attached to the treated plants.

Histological reactions

Normally the cells that were in direct contact with the spore material reacted with cell enlargement, but in some cases the neighboring cell layers were also modified. The deformation never passed over the contact area between palisade to spongy parenchyma. The morphological aspects of other side of the leaf, which did not come into contact with the spores, remained unchanged. Within the malformed areas, some cells turned brown and revealed an accumulation of wall-bound phenolic compounds in the cell walls next to them. Fungal mycelium was detected in small tissue areas after inoculation with living spores. This tissue did not grow out of the lesion area and was stopped by necrotic response. Mycelium did not develop, and intercellular mycelium growth or formation of conidiophores or spores did not occur.

DISCUSSION

Inoculated cassava leaves react very strongly to M. ulei. The reaction is independent of whether the spores used for inoculation are infective, living spores, or if autoclaved spore material is used. The response is complex and bound to signal compounds which induce morphogenetic reactions. It comprises the induction of phytoalexin-like compounds such as the blue fluorescing substance(s), which occur(s) in areas which are in direct contact with the inoculation material as well as cell growth stimulation and, later on, necrotic responses.

This reaction type is different from typical non-host resistance reactions, in which generally fast responses are induced by pathogen signals, leading to an oxidative burst (e.g. Lamb and Dixon, 1997) and subsequent necrosis of a limited number of cells which are in tight contact to the signal substances. The same symptom, the hypersensitive response with restricted tiny necroses, is known in host specific reactions of highly resistant hosts to avirulent or weakly virulent pathogen races (Scheel, 1998).

In contrast to hypersensitive reactions, the responses of cassava leaves to autoclaved inoculum include synthesis dependent resistance responses like phenolic compound formation in cell wall regions and the formation of fluorescing phytoalexins. In addition, cell growth and tissue reactions are visible. It cannot be excluded that the typical hypersensitive response was initially induced but subsequently inhibited by liberation of HCN from dead cells, as shown by Lieberei et al. (1989) for Hevea leaves, which like cassave leaves are also cyanogenic. It is known that HCN inhibits peroxidases and phenolase (Bolwell et al., 2002) and gives rise to additional reactions, leading to the lesion development reported here. Within about one day after cell death, at the latest the HCN content of the tissue is below any inhibitory concentration, synthesis dependent reactions to take place (Lieberei et al., 1996), and phytoalexins and phenolics can be produced. It is of great interest to study the factors released by M. ulei, which give rise to the expressed reaction of cassava leaves.

Using living spores as inoculum, a restricted growth of hyphae takes place in the intercellular spaces between the epidermal and the subepidermal layer. This is a first indication of the development of a potential colonization phase in this tissue by the biotrophic non host fungus. It may be a response that is again enabled by the occurrence of HCN, liberated from dying cells. It has been shown that the fungus is highly tolerant to HCN (Lieberei et al., 1983) whereas the host plant is inhibited in its defense reactions.

In the case studied here, the reaction pattern gives rise to the assumption, that in the course of this intensive interaction, a biochemical selection process to a new fungus-plant relationship may be on its way, which may result in the development of cassava as a new host of the biotrophic pathogen M. ulei.

This assumption may be premature, but as already pointed out, the pathogen M. ulei is physiologically highly variable (Junqueira et al., 1986; Gasparotto & Junqueira, 1994), and the enlargement of the host range to other members in the family of Euphorbiaceae which reveal the same biochemical properties of cyanogenesis seems to be possible.

Cassava is a very important staple food. In order to be prepared and to avoid losses of cassava production, research on this plant – fungus interaction should be continued using a broad base of cassava genotypes and a representative range of M. ulei isolates.

ACKNOWLEDGEMENTS

The authors thank Mr. Ricardo Pessoa Rebello for his continuous help and Mrs. Helga Schween and Mrs. Claudia Maehlmann for their technical support. Part of this study has been supported by the Ministry of Research and Education, BMBF, Germany and the CNPq, Brazil who funded the SHIFT-Project ENV-23, project number 0339457A.

The receipt of defined cassava clones from Dr. J.C. Lozano, CIAT, is also greatly appreciated.

LITERATURE CITED

Accepted for publication on 06/05/2003

Corresponding author: Luadir Gasparotto

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Datas de Publicação

• Publicação nesta coleção
  27 Nov 2003
• Data do Fascículo
  Out 2003