CLADOSPORIUM CLADOSPOROIDES - HOUBOVÝ PATHOGEN NA OVSU

Cladosporium cladosporoides - houbový pathogen na ovsu

Cladosporium cladosporoides - a fungal pathogen on oat

Kamila Vrátilováa), Jan Moudrýa), and Alexandr Jegorovb)

a) Faculty of Agriculture, South Bohemian University

b) Galena a.s., Research Unit

Souhrn, klíčová slova

Mykologický průzkum 4 odrůd ovsa sklizeného v roce 1999 na 2 lokalitách v Čechách a 2 v severním Rakousku ukázal na velmi významné zastoupení fytopathogenní houby C. cladosporoides izolované z vnitřní části obilek. Zatímco je tento pahogen běžný v zemědělské praxi, o potencialní toxicitě jeho případných sekundarních metabolitů není prakticky nic známo. Submerzní in-vitro kultivace izolátů C. cladosporoides z ovsa z různých lokalit neprokázala významný rozdíl v jejich schopnosti produkovat sekundární metabolity. Souhrn sekundárních metabolitů byl extrahován organickými rospouštědly a jednotlivé látky byly dále čistěny sloupcovou chromatografií a preparativní chromatografií na vysokotlakých kolonách. Předběžná identifikace látek ukázala jejich blízkou příbuznost s AM-toxiny, mykotoxiny produkovanými fytopathogenní houbou Alternaria mali.

Oves, fytopathogenní houby, Cladosporium, mykotoxiny

Summary, Key words

Mycological screening of fungi from 4 varieties of oat from 2 localities in Bohemia and 2 in northern Austria harvested in 1999 revealed a significant occurrence of phytopathogenic fungus C. cladosporoides grown inside the oat grains. Whereas this pathogen is well known in agricultural experience, the potential toxicity of its secondary metabolites remains largely unknown. Submerged in-vitro cultivation of various strains of C.cladosporoides isolated from the oat from different areas did not shown any significant difference of their pattern of secondary metabolites. Crude secondary metabolites were obtained by extraction of cultures by organic solvents. Individual compounds were obtained by a combination of column chromatography and preparative high-performance liquid chromatography. Preliminary identification indicates that metabolites of C. cladosporoides are related to AM-toxins, the mycotoxins produced by phytopathogenic fungus Alternaria mali.

Oat, phytopathogenic fungi, Cladosporium, mycotoxins

Introduction

Coming into the European Community, the quality of crop, much likely than the quantity, has become the key criterion of agricultural production. No doubt that the absence of fungal pathogens and their mycotoxins are appreciated at the common market. Traditional approach had focused on the analysis of presence of various "mycotoxins", considering simply their direct effect on human or animal health. Nowadays, however, is well recognised that fungi can produce, e.g., such metabolites which can influence the immune system and thus to cause weakness of organism. Such effect can be even more important than the direct toxicity. Thus besides being important phytopathogens, some are even opportunistic human pathogens.1,2 C. cladosporoides itself is listed among 96 species of the dematiaceous fungi verified as causing phaeohyphomycosis and belongs also to "top five" fungal allergens.3 It is obvious, from this point of view that secondary metabolites of C. cladosporoides can play a significant role in the fungal pathogenesis both in plants as well as in humans. Finally, it cannot be forgotten that the presence of phytopathogenic fungi affects also the quality of seeds.

So far, only a few fungal secondary metabolites, like Fusarium and Aspergillus mycotoxins, are analysed routinely. There are, however, a large number of fungi, which are widespread, but our knowledge of their secondary metabolites remains obscure. Our representative screening of various samples of oat revealed a significant occurrence of C.cladosporoides. A number of biologically active secondary metabolites were described so far from the fungi of the genus Cladosporium. C. cladosporoides was described to produce perylenequinones calphostins,4 inhibitors of protein kinase C, epoxy alcohol cladosporol,5 which was described as inhibitor of a â-1,3-glucan synthesis, as well as octaketide cladosporin6 (produced also by various Aspergillus sp.).

These findings prompted us to investigate the ability of C. cladosporoides to produce various secondary metabolites, to elucidate in more detail their nature, and in future, to evaluate their presence and significance in some agricultural products. This contribution deals with the screening scheme, isolation of fungal strains, cultivation of C. cladosporium and isolation of its secondary metabolites. Since the determination of structure of metabolites is still in progress it will be reported in detail elsewhere.

Experimental

Oat samples and fungal screening

Six varieties of oat (ABEL, IZAK, SALOMON, AURON, EDMUND, EXPANDER) were grown in 1999 each in 2 localities in Bohemia (Humpolec, České Budějovice) and 2 localities in Austria (Lambach, Freistadt) giving thus 24 grain samples. Ten random individual grains were used for the screening. The surface of each grain was sterilised with aqueous formaldehyde (1 %, 5 min), than with ethanol (96 %, 30 s). Attention was paid particularly to the thorough surface sterilisation of oat grains in order to obtain fungal pathogens from inside of the grains and to minimise any potential airborne contamination. Grains were cut, placed into Petri dishes on Saburaud agar and incubated up to 10 d at 25 oC. Several isolates of one fungus from the same series of grains giving the same morphology and appearance of monosporic colonies were considered as identical and calculated as single isolate into the statistical evaluation. Yeasts or bacteria were not included into the statistics. Identification of key fungal isolates was provided at the Prague Culture Collection of Fungi (CCF).

Submerged cultivation of C. cladosporoides

Submerged cultivation (15 days) of six isolates of C. cladosporoides, Table 1, was carried out in conical flasks containing 200 ml of medium (sucrose 100 g, fructose syrup 20 g, enzymatic casein hyhrolyzate 40 g, corn steep 4 g, (NH4)H2PO4 8 g, KCl 4 g, MgSO4·7H2O 0.2 g, water 1000 ml, pH 5.6); 6 d preculture (sucrose 30 g, corn steep 20 g, KH2PO4 2 g, water 1000 ml, pH 5.2) was used as inoculum (7 ml per one flask). The fermentation broth was filtered and mycelium was extracted with methanol (200 ml) a medium with dichloromethane (200 ml). Crude extracts were evaporated to dryness on vacuum evaporator. The patterns of secondary metabolites of individual strains were compared using TLC (HPTLC plates, Silica gel 60, dichloromethane/methanol, 19:1 v/v, detection at 254 nm or with iodine vapours).

Isolation of secondary metabolites of C. cladosporoides

The strain C. cladosporoides 8/20 (Lambach, Austria) was used. Cutlivation of the fungus in 8.6 l of media afforded roughly 1 g of crude dichloromethane extract. Crude secondary metabolites were prepared by LC on a silica gel (Merck 200 ěm, step gradient with dichloromethane containing 0 - 10 % of methanol). Individual cyclocladins were isolated by HPLC on a reversed phase column (Biospher SI C-18, 7 ěm, 250x25 mm I.D., from Labio a.s., Czech Republic) using isocratic elution (7 ml/min) with acetonitrile/water mixture (3:17, v/v) as an eluent. Analytical chromatography was carried out on a reversed phase column (SupelcosilTM LC-18-DB, ěm, 250x4.6 mm I.D., from Supelco, U.S.A.) using isocratic elution (1 ml/min) with acetonitrile/water mixture (either 3:17 or 1:5, v/v) as an eluent. Detector was set at 214 nm. Finally, about 100 mg of cyclodaladin A and 5-20 mg of cyclocladins B-D were available (>98 %, HPLC), which were crystallized from dichloromethane/n-heptane (2:1, v/v) mixture affording white well-crystalline solids.

Results

As expected, various Fusarium sp. were among the most frequent isolates of fungi from the oat (43 %). Among others, Cladosporium cladosporoides (22 %), various Penicillium sp. (18 %), and Alternaria alternata (11 %) created an additional 51 % of fungal isolates. C.cladosporoides was found on two-thirds of oat sorts (ABEL, SALOMON, EXPANDER, and AURON) at three of four localities (except of České Budějovice where Fusarium sp. dominated). Six isolates of C. cladosporoides provided a base for further evaluation, Table 1.

The fact that the screening revealed a significant occurrence of phytopathogenic fungus C. cladosporoides grown inside the oat grains has attracted our attention to the ability of this fungus to produce biologically active secondary metabolites. Individual fungal isolates were culltivated by submerged cultivation and the patterns of their secondary metabolites were compared by thin-layer chromatography. Based on the presence of various spots, their intensity and character, it was concluded that differences among individual strains are not significant. Hence, the strain C. cladosporoides 8/20 (Lambach, Austria) was chosen for further experiments.

The fungus was cultivated by the submerged cultivation and its secondary metabolites were extracted by organic solvents. Screening of secondary metabolites by TLC, HPLC and mass spectrometry revealed the presence of peptidic metabolites related to the AM-toxins described from the fungus Alternaria mali.7 Since these metabolites created a major part of extracellular metabolites extracted by dichloromethane from the medium, they were further purified by column chromatography and finally isolated in pure state by high-performance liquid chromatography on a reversed phase column. Individual cyclocladins were designated by capital letters according the order of their discovery and description without relationship to their chemical structure or their chromatographic properties. New HPLC method was developed for their analysis and purity control, Figure 1. Identification of isolated compounds by MS, NMR methods and X-ray crystal structure determination are currently in progress.

Figure 1: Chromatographic resolution of cyclocladins D-A (cyclocladin A, Rt 10.77 min), for chromatographic conditions see Experimental.

Discussion

Although mycological screening reflects the diversity of fungi on the oat rather than the actual fungal load, the present study clearly showed that C. cladosporoides belongs among important pathogens and possible source of contaminants on the oat grains. C. cladosporoides is known to produce biologically active compounds and our study has added to them a series of new potential mycotoxins.

Our future work will be dedicated to the characterization of individual isolated fungal metabolites, to the determination of their biological activity and to the development of sophisticated analytical methods making possible their direct determination in the grain samples.

References

Schell, W. A. 1995. Clin. Lab. Med. 15:365-87.

Perfect, J. R., and W. A. Schell. 1996. Clin. Infect. Dis. 22(Suppl 2):S112-8.

Mishra, S. K., L. Ajello, D. G. Ahearn, H. A. Burge, V. P. Kurup, D. L. Pierson, D. L. Price, R. A. Samson, R. S. Sandhu, B. Shelton, R. B. Simmons, and K. F. Switzer. 1992. J. Med. Vet. Mycol. 30(Suppl 1):287-305.

Miyayaki, Y., Y. Shinomura, S. Tsutsui, S. Kitamura, S. Hiraoka, and Y. Matsuzawa. 1998. Life Sci. 63:PL361-5.

Sakagami, Y., A. Sano, O. Hara, T. Mikawa, and S. Marumo. 1995. Tetrahedron lett. 36:1469-72.

Reese, P. B., B. J. Rawlings, S. E. Ramer, and J. C. Vederas. 1988. J. Amer. Chem. Soc. 110:316-8.

Ueno, T., T. Nakashima, Y. Hayashi and H. Fukami. 1975 Agric. Biol. Chem. 39:2081-9.

Correspondence address

Kamila Vrátilová, Faculty of Agriculture, South Bohemian University, Studentská 13, 370 05 České Budějovice, phone 038-7772716, e-mail: vratilova@atlas.cz, (Ph.D. fellowship at Galena a.s., R&D)