Antagonistic process of Dicyma pulvinata against Fusicladium macrosporum on rubber tree Sueli C. M. de Mello1, Carlos Eduardo Estevanato1, Leonardo M. Braúna2, Guy Capdeville1, Paulo Roberto Queiroz & Luzia C. Lima1. (1Embrapa Genetic Resources and Biotechnology, Parque Estação Biológica, Cx. Postal 02372, CEP 70770-900, Brasília, DF, e-mail: smello@cenargen.embrapa.br; Departamento de Fitopatologia, Universidade de Brasília). (Accepted to for publication on ...) Corresponding author: Sueli C. M. de Mello _________________________________________________________________________ Estevanato, C.E., Braúna, L.B. & Mello, S. C. M, Capdeville, G. Antagonistic process of Dicyma pulvinata against Fusicladium macrosporum on rubber tree. ABSTRACT The Dicyma pulvinata and Fusicladium macrosporum interaction was studied by scanning electron microscopy. Spores of D. pulvinata germinated on the surface of F. macrosporum lesions induced on rubber plants artificially infected, fixed 8 h after inoculation. Germ tubs seemed to elongate toward F. macrosporum. The penetration into the F. macrosporum spores was verified 24 h after D. pulvinata inoculation. In the end of the process, the F. macrosporum spores looked disintegrated and devoid of content. The antagonist completely overgrew the pathogen. Six to seven days after the inoculation with the antagonistic fungus, it was observed D. pulvinata conidiophores emerging from F. macrosporum structure, with profuse sporulation. Also, studies have appointed the ability of D. pulvinata to produce hydrolytic enzymes, which could be associated to the control of plant pathogens. This information may help elucidate the mode of action of D. pulvinata, a potential biological control agent to the South American Leaf Blight of Hevea rubber. RESUMO Estudou-se a interação entre Dicyma pulvinata e F. macrosporum ao microscópio eletrônico de varredura. Esporos de D. pulvinata germinaram na superfície das lesões induzidas por F. macrosporum em plantas de seringueira (Hevea brasiliensis), infectadas artificialmente, fixadas 8 h após a inoculação do antagonista. Aparentemente, os tubos germinativos se alongaram em direção ao patógeno. Penetração foi verificada em amostras fixadas 24 h a após inoculação de D. pulvinata. Ao término do processo, os esporos de F. macrosporum invadidos pelo antagonista mostraram-se desintegrados e esvaziados de seu conteúdo. D. pulvinata cresceu sobre as lesões, sobrepondo totalmente o patógeno. Seis dias após a inoculação, conidióforos do fungo antagonista foram observados emergindo das estruturas do patógeno e produção de esporos em grande quantidade. Verificou - se, também, um possível envolvimento de enzimas hidrolíticas na associação antagonística entre D. pulvinata e o patógeno. Estas informações podem contribuir para elucidar o modo de ação de D. pulvinata, um potencial agente de controle biológico para o mal das folhas da seringueira. INTRODUCTION The South American Leaf Blight of Hevea rubber (SALB), caused by Microcyclus ulei (P. Henn.) Arx, is one of the world’s five most threatening plant diseases and it is still epidemic to Central and South American. It was first recorded in 1900 on rubber trees in Brazil. Currently this disease extends from Southern Mexico (180 latitude North) to Sao Paulo State in Brazil (240 latitude South), covering Brazil, Bolivia, Colombia, Peru, Venezuela, Guiana, Trinidad, Tobago, Haiti, Panama, Costa Rica, Nicaragua, Salvador, Honduras, Guatemala and Mexico. This disease has been the main restraint to the development of rubber cultivation in Latin American countries. Studies have appointed that usually the epidemiological process begins from conidia germinating, in an imperfect stage of the pathogen (Fusicladium macrosporum Kuyper, mitosporic), which occurs within 1 hr (optimum temperature near 24 C). Four – five hours leaf-wetness is required for hosp penetration wich is through the immature cuticle. Conidia are viable a few days under ambient temperature and shade. Sporulation begins 5-6 days after infection; pycnidia of Aposphaeria ulei P. Henn., another imperfect stage of the fungus, are formed after 3-5 weeks, and M. ulei ascocarps, after a further 4-6 weeks (Holliday, 1970). In spite of the recommended control strategy of planting Hevea brasiliensis (Willd ex. A. Juss) Muell. in areas where the climatic conditions are unfavorable to the epidemic development of the disease (escape zones), experiments conduced by Gasparotto and Junqueira (1994) showed evidences on the existence of ecological races of M. ulei, better adapted in adverse climatic conditions. This information was confirmed later (Rivano, 1997; Mattos et. al., 2003; Romero, et. al, 2006). Hence, it is predictable difficulties for controlling of this disease even in escape zones. All improved H. brasiliensis clones, worldwide, are susceptible to SALB, although the disease is confined to South America. However, the possibility of the future spread of the disease should always be considered, even though natural rubber producing countries have now adopted appropriate measures to prevent the introduction of the disease into their territories. It has been shown that two types of spores (conidia and ascospores) are responsible for disease dissemination, and was predicted that parts of the host plant (Hevea), infected can spread the disease over long distances. Efforts have been made in order to control this disease, including the use of Dicyma pulvinata (Berk. & Curt.) Arx [Hansfordia pulvinata (Berk. & Curt.) Hughes]. This fungus at first observed in the Amazon Region, colonizing stromatic lesions produced by M. ulei spread up to different geographic areas from Brazil. Results obtained from field trials (Junqueira and Gasparotto, 1991) have demonstrated the action of D. pulvinata against SALB in decreasing the inoculum potential of the parasite by death of hyperparasitized conidia on colonized lesions. The mitosporic fungus D. pulvinata, which was first reported mycoparasitic on Isariopsis indica (Rathaiah and Pavgi, 1971) and Cercospora spp. in India (Krishna and Singh (1979), has been studied as a parasitic of Cladosporium fulvum Cooke and Cercosporidium personatum Earle, causal agents of tomato (Lycopersicon esculentum L.) leaf mould, and late leaf spot of peanut (Arachis hypogaea L.), respectively (Peresse and Le Picard, 1980; Tirilly et al. 1983; Mitchell and Taber, 1986; Mitchell et al.,1986; Mitchell et al.,1987; Tirilly, 1991). Peresse and Le Picard (1980) suggested that this fungus could be used in the biological control of C. fulvum in grasshouse-grown tomatoes. Tirilly et al. (1983) isolated a fungitoxic metabolite (13-desoxyphomenome) from liquid cultures of D. pulvinata obtained from C. fulvum lesions in tomato. More recently, D. pulvinata was reported colonizing tissue of fruit bodies of Aphyllophorales (Basidiomycetes) in Japan (Watanabe et. al. (2003). According to Sharma and Sankaran (1986), organisms adapted to the same habitat as the pathogen are generally preferred over those from other habitats, as the latter are less likely to survive for long in the ecosystem and consequently would have to be reapplied to foliar surfaces more frequently. Based in this aspect, we have considered D. pulvinata as a potential candidate for biocontrol of SALB. A survey was carried out from late February to late December of 1999, in different geographic areas across the country. D. pulvinata isolates were harvest from lesions of M. ulei on leaves of Hevea rubber and incorporated to the Embrapa’s collection of fungi for biological control of plant pathogen (Mello et. al., 2006). A performance comparision of several of these D. pulvinata isolates showed that at least the isolates CG774, CG801, CG773, CG790, CG679, CG826 and CG682 could be used to control the disease (Mello et. al., 2005). Antagonism may be accomplished by competition, parasitism, antibiosis or by a combination of these modes of action. (Whipps, 1992). The present study is the first report on the interaction by scanning electronic microscopy and to elucidate the possible involvement of hydrolytic enzymes in the antagonistic association between D. pulvinata and the plant pathogens. MATERIALS AND METHODS Healthy potted plants of rubber (H. brasiliensis, clone GT1) were inoculated by spraying a conidia suspension (106 conidia mL-1) of F. macrosporum on the leaflet surface. The leaflet age was 6-8 days, which correspond to the B1 and B2 stage (Hallé et al., 1978). The conidia were originally obtained from rubber plants artificially infected, by washing lesions with sterile water and rubbing gently with a soft camel’s hair brush. Conidia concentrations were determined by neubauer chamber counts before use. The inoculated plants were kept inside a growth clamber (Lab-line Instruments, inc.) adjusted for 24-h darkness (100% RH; 25 oC). After that, the chamber was adjusted for 12-h darkness provided by fluorescent lamps. Five days after inoculation, when the leaf lesions had formed, the plants were taken to the greenhouse for inoculating with the antagonistic fungus. The D. pulvinata antagonist used in this study, isolate CG 774, was obtained from a survey (Mello et al., 2006) and stored at -180 oC on the Embrapa Recursos Genéticos e Biotecnologia fungus collection. Current cultures were grown at 25 to 27 oC on potato dextrose agar (PDA) home medium and storage at 4 oC. In order to produce of sporulating cultures for trials, mycelium disc from these stock cultures were inoculated on PDA plates and incubated under 12 h of alternating dark and light at 25 oC. The inoculum was obtained from 15-day-old cultures. It was prepared by adding 2 mL of sterile distilled water + Tween 20 (0.02%) solution to each plate that then was swept with a soft camel’s hair brush to dislodge conidia. Conidia concentration was adjusted for 106 conidia mL-1 and the suspension obtained thus was sprayed on the surface of rubber leaves presenting F. macrosporium lesions. Post inoculated, the plants were placed into plastic bag overnight. Bags were moistened by spraying water inside prior to insertion of plants. Leaf samples were collected at 4, 8, 12 and 24 hours and 3, 4, 5, 6, 7 and 8 days after inoculation. The samples were fixed with a modified Karnovsky solution (2% glutaraldehyde, 2% paraformaldehyde in 0.05M cacodylate buffer, pH 7.2), post-fixed in 1% osmium tetroxide in the same buffer for 2 hours (Bozzola & Russel, 1992) and dehydrated in a graded acetone series. The specimens were then dried in an Oryer Emitech Critical Point K 850, using CO2 as transition fluid. The dried samples were glued onto specimen stubs and coated with gold in an Emitech K 550 Sputter Coater. ZEISS DSM 962 AT scanning electron microscope at 20KV was used to examine the samples. For enzyme production essays, the D. pulvinata isolate was cultured in 50 mL of liquid medium (25 g L-1 glucose, 5 g L-1 yeast extract) at 28o C under agitation (150 rpm) and after 72 hours it was collected in sterile distilled water and transferred to 50 mL of liquid culture medium contained (g L-1) MgSO4.7H2O, 0.2; K2HPO4, 0.6; KCl, 0.15; NH4NO3, 1.0;FeSO4.7H2O, 5.0 mg L-1; MnSO4.H2O, 6.0 mg L-1; ZnSO4.H2O, 4.0 mg L-1; CoCl2, 2.0 mg L-1; crab shell chitin (0.5% and 0.1% (v/v) trace elements (Fe2+, Mn2+ and Co2+), adjusted to pH 5.5. Cultures were then incubated for 24 h, 48 h and 72 h, at 28o C under agitation (150 rpm), in order to obtain enzyme production. After incubation for time periods, culture filtrates were collected by filtration (Whatman No. 1 paper) and stored at -20oC with sodium azide (0.02%). Enzyme assays - β-1,3-Glucanase (EC 3.2.1.39) was assayed based on the release of reducing sugar from laminarin as described by Santos et al. (1977). Briefly, the reaction mixture contained 100 µL of laminarin dissolved in 50 mM sodium acetate buffer, pH 5.0 and a 100 µL substrate of enzyme solution. The reaction was allowed to proceed for 30 min at 37oC, after which the liberated reducing sugars were determined by dinitrosalicilic acid method (Miller, 1959) using a reference curve constructed with glucose as the standard. Enzyme and substrate blanks were also included. One unit of enzyme activity (U) was defined as the amount of enzyme that catalyzes the equivalent release of one µmol of glucose per minute under the described assay conditions. Chitinase activity (EC 3.2.1.14) was assayed using the colorimetric method described by Molano et al. (1977) with minor modifications (Ulhoa & Peberdy, 1992). The assay mixture contained 1 mL of 0.5% regenerated chitin (suspended in 0.05 M acetate buffer pH 5.2) and 1 mL of enzyme solution. The reaction mixture was incubated for a minimum of 6 h under agitation at 37oC and the reaction was blocked by centrifugation (5000 rev/ min) for 10 min and the addition of 1 mL of dinitrosalicylate reagent (Miller, 1959). The amount of reducing sugar produced was estimated using a reference curve constructed with N-acetylglucosamine (GlcNAc) as standard. One unit of enzyme activity (U) corresponded to the amount of protein necessary to release 1 µM of GlcNAc equivalent in 1 h at 37oC. Alternatively, the presence of GlcNAc as a product of chitinase activity was determined according to Reissing et al. (1959) using the reagent p-dimethylaminobenzaldehyde (DEMAB). The N-acetylglucosaminedase (NAGase) activity (EC 3.2.1.30) was measure as described by Yabuki et al. (1986) using p-nitrophenyl-β-N-acetylglucosaminide (Np-GlcNAc) as the substrate. One unit of enzyme activity (U) was defined as the amount of the enzyme that releases one µmol of p-nitrophenol per minute under the described assay conditions. Protein estimation was performed by a simplification of the Lowry method (Peterson, 1977) and proteases assay was based on the written paper by Haran et al. (1996). In general, all assays were run in triplicates. RESULTS Typical symptoms of the SALB appeared on the abaxial surface of rubber leaves three days after F. macrosporum inoculation, as small light green spots, becoming dark and larger subsequently. Samples of the lesions taken to examine under light microscopy showed sporulation profuse just before D. pulvinata inoculation. Conidial germination and germ tub growth of the antagonistic fungus was observed 8 h after inoculation on all leaf surface tissues examined. During the incubation, D. pulvinata mycelium expanding from germ tubs reached F. macrosporum structures, attacking and invading them despite none perforations in the host cells were observed. Frequently, D. pulvinata hyphae grew to the host structures (mycelium, conidiophores and conidia), surround and held them (Figs). D. pulvinata hiphae once on contacting F. macrosporum conidia sometimes produced appressorium-like structures which penetrated them, and a peg was visualized (Figs……). Most of F. macrosporum conidia were penetrated three days after inoculation. D. pulvinata colonization into conidia was not studied, although it could be seen to be growing inside the host spores (Fig). Conidiophores with conidia emerged from the pathogen structures was observed in the samples fixed six days pos inoculated with the antagonistic. After seven days, entire foliar lesions induced by F. macrosporum were covered by the typical growth of D. pulvinata expressed as a peculiar whitish, downy growth (Fig. ). Aiming to elucidate the possible involvement of hydrolytic enzymes in the antagonistic association between D. pulvinata and the plant pathogens, we have undertaken studies on characterization of the enzymes produced by this antagonist. The determination of the total proteins secreted during a period of one week demonstrated growing liberation of proteins during the whole induction period. Substantial amounts of hydrolytic enzymes as NAGase (maximum in 48 h / 0.11 U) and Glucanases was produced during the induction period, containing chitin (0.5%). The endoglucanases indicated the highest activity in 48 h (0.295 U) and after that, in 96 h (0,129 U), staying unaffected until a week of induction. The exoglucanases indicated the highest activity in 48 h (0.037 U) and in 72 h (0.023 U). After the reduction in the activity, this stayed constant until the end of the enzymatic induction. The chitinase enzyme did not reveal activity, therefore, it was detected a high proteolytic activity in the period of a week (0.075 U), at the end of induction. DISCUSSION Conidia germination and appressorium formation are important antagonism determinants in pathogenic fungus and should also receive special attention in the studies, involving the action mode in hyperparasitic interaction. Here we present experimental results showing germination and formation of these kinds of infective structures in D. pulvinata, a hyperparasite of the foliar pathogen F. macrosporium. The above-described in controlled system is a very useful and rapid method to study the antagonistic interaction process and may help elucidate the mode of action of D. pulvinata, a potential biological control agent to the South American Leaf Blight of Hevea rubber. Antagonism may be accomplished by different modes of action, as competition, parasitism and antibiosis which can act each alone or combined (Whipps, 1992). Ours observations suggest that the efficiency of D. pulvinata can be from a direct effect traduced by the attack to the pathogens destroying its spores. The aspects of the cell surface beneath the penetrated area do not showed points of degradation in the host cell wall. However, fungal cell wall-degrading enzymes have been associated with degradation of hyphae of many pathogens (Berto et. al., 2001) and can be a mechanism involved in the digestion of wall-layers of F. macrosporum spores at the penetration point. By using assays on liquid medium containing chitin, D. pulvinata revealed considerable activity of extracellular enzymes such as Glucanase, N-acetylglucosaminedase (NAGase), and proteases. The results appointed that the time course of enzymes production of D. pulvinata in liquid medium containing chitin showed activity increased from low levels in early stages of cultivation to higher levels at latter stages. Nevertheless, the function of this enzymes activity enhancement remains unclear. It could rest on the direct interaction between the antagonist and the pathogens fungi, but could also result in a metabolic process, leading to a dead cell wall degradation of either M. ulei or D. pulvinata itself. However, the nature of lytic enzymes and determinants of host specificity are not known and deserve further study (Bastos, 1996). Probably, a chronological event of an antifungal activity is associated in a synergistic fashion of hydrolytic enzymes with the antagonistic properties (Lima et al. 1997). It is, therefore, likely that in nature the lytic enzymes act as a phytopathogen cell-wall-degrading factor following recognition and interaction of the antagonist with the phytopathogen and enzyme induction (Lima et al. 1999). On the other hand, a compound with fungitoxic activity have obtained from a D. pulvinata isolate colonizing C. fulvum late leaf spot lesions and was proposed the 13-desoxyphomenone structure for that metabolite. As reported, this toxin would be possibly a role in the tripartite system hyperparasite-parasite-host (Tirilly et al. (1983). Our work showed the death of the spores in advance of hyphal penetration that suggest the action of one or more fungitoxic compound. Epidemiologic studies of SALB have appointed that the infection process begin from F. macrosporum and conidial germination occurring 1 hr (optimum temperature near 24 C). Four – five hours leaf-wetness is required for hosp penetration which is through the immature cuticle. Conidia are viable a few days under ambient temperature and shade. Sporulation begins 5-6 days after infection; pycnidia (Aposphaeria ulei P. Henn.) are formed after 3-5 weeks and ascocarps after a further 4-6 weeks (Holliday, 1970). Our results confirmed the antagonistic effect of D. pulvinata destroying the spores on necrotic leaves. This effect destructor also can be observed in stromatic lesions (M. ulei) exams from material collected in field. Such reduction of inoculum by application of the antagonistic can contribute to slow down the SALB epidemy spread when the population of the pathogen is developing independently of exogenous inoculum. ACKNOWLEDGMENTS This work was supported in parte by grants from Conselho Nacional de Pesquisa – CNPq. We thank Rosana Falcão for her technical assistance. REFERENCES Berto, P., Haissam Jijakli, M., Lepoivre, P. 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Tirilly,Y., Kloosterman, J., Sipma, G., Kettenes-van den Bosch, J.J. (1983) A fungitoxic sesquiterpene from Hansfordia pulvinata. Phytochemistry 22:2082-2083. Tirilly,Y. The role of fosetyl- A1 in the potential integrated control of Fulvia fulva. Canadian Journal of Botanic 69:306-310. 1991. Watanabe, T.; Kawano, Y. New records of Ardhachandra, Dicyma, and Sibirina species from basidiomata of Aphyllophorales (Basidiomycetes) in Japan. Mycoscience 44: 411-414. 2003.  Fig. 1. (Fig 1.A) Germinacao do conidio de Dicyma pulvinata. (Fig 1.B) Enrrolamento das hifas de D. pulvinata no conídio do M. ulei. (Fig 1.C, seta branca) Penetração da hifa do D. pulvinata no conídio do M. ulei. (Fig 1.C, seta preta) Formação de apressório pela hifa do D. pulvinata no conídio do M. ulei. (Fig 1.D, seta preta) conidióforo do D. pulvinata. (Fig 1.D, seta branca) Formação de apressório pela hifa do D. pulvinata no conídio do M. ulei.  Fig 2. (Fig 2.A, seta branca) Produção de conidios de Dicyma pulvinata, após a colonização do M. ulei. (Fig 2.A, seta preta) Formação de conidióforo do D. pulvinata a partir da colonização do M. ulei. (Fig 2.B) Conídio de M. ulei destruído pelo D. pulvinata. (Fig 2.C, seta branca) Hifa do D. pulvinata internamente no conídio do M. ulei. (Fig 2.C, seta preta) Conídio do M. ulei destruido. (Fig 2.D) Superfície da folha de seringueira coberta por estruturas de Dicyma pulvinata após destruição total das estruturas do Microcyclus ulei.  Fig. 3 Lesions of Microcyclus ulei on leaf of rubber colonized by Dicyma pulvinata   Fig. 3 Eletron microscopy of Dicyma pulvinata on spores of Microcyclus ulei showing penetration (left) and conidiophores emerging from M. ulei structures (right), three and six days after the inoculation with the antagonist, respectively. PAGE 13