FUNGI OCCURRING IN THE IN VITRO CULTURES

Maria Kowalik
Department of Plant Protection , Agricultural University of Cracow, Poland

ABSTRACT

The goal of this paper was to define the species composition of the populations of fungi contaminating plant material in the in vitro cultures. The practice of micropropagation indicates that fungi growing on explants cause their elimination. The colonies of fungi in the number of 5 000 were obtained from the explants of ornamental plants, orchard plants and vegetables. Fungi occurring in the in vitro cultures belonged to 195 species, within 76 genera. Fungi of genera: Acremonium, Aspergillus, Alternaria, Botrytis, Chaetomium, Cladosporium, Cylindrocarpon, Epicoccum, Humicola, Monilia, Paecilomyces, Penicillium and Scopulariopsis were represented in great quantities.

Key words: fungi, contamination, in vitro cultures, ornamental plants, orchard plants, vegetables.

INTRODUCTION

In vitro cultures are used in micropropagation of many plant species, which enables us to obtain many healthy plants in a short time, compared to traditional ways of proliferation. In practice there are, however many difficulties connected with the obtaining of regeneratively active and microorganism-free explants. One of the problems is the contamination of explants caused by fungi, drawing into the elimination of explants from further micropropagation techniques. The results of done so far work Leifert et al. [7], Debergh [4], Leifert and Woodward [8], Williamson et al. [16], Kowalik et al. [10] present a wide variety of fungi species, appearing in the in vitro cultures, on the subsequent stages of micropropagation. The identification of the contaminating fungi enables to determine the frequency of taxi on the micropropagated ornamental plants, orchard plants, vegetables in the laboratories of tissue cultures.

The purpose of the paper was to define species composition of the populations of fungi contaminating plant material in the in vitro cultures.

MATERIAL AND METHODS

Material for the studies consisted of the fungi occurring on plant material in the in vitro cultures. Colonies of fungi were obtained from explants of ornamental plants, orchard plants and vegetables kept in the containers with the Murashige-Skoog medium. Explants originated from the micropropagation on the stage of the stabilization of cultures (I), multiplication (II) and rhizogenesis (III). Explants, in the number of 5000 were collected in the period of 1996-2005, from twelve commercial and scientific laboratories.

Fungi were identified according to mycological keys: Booth [1,2], Rifai [14], Domsch et al. [5], Ramirez [12] and Ellis and Ellis [6].

RESULTS AND DISCUSSION

A wide spectrum of fungi contaminating micropropagation of ornamental plants, orchard plants and vegetables was found.

The greatest number of fungi colonies was obtained from the explants of orchard plants; it was 48.84 per cent of the total. Slightly less was obtained from ornamental plants – 42.08 per cent. Majority of contamination came from the multiplication stage (II) and the stabilization explants (I) – 50.76 and 40.14 per cent, respectively.

The frequencies of the contamination of explants in respective groups of plants and on different stages of micropropagation are put together and compared in table 1.

Fungi contaminating the micropropagated plants belonged to 195 species, within 76 genera. The most frequent were: Aspergillus niger, A. versicolor and Paecilomyces fumosoroseus. These fungi made more than 23 per cent of all the derived colonies. In the group of fungi making 1-5 per cent of total colonies there were: Acremonium kiliense, Alternaria alternata, Botrytis cinerea, Chaetomium globosum, Cladosporium cladosporioides, C. herbarum, C. macrocarpum, C. sphaerospermum, Cylindrocarpon destructans, Epicoccum purpurascens, Humicola fuscoatra v. fuscoatra, Monilia geophila, Penicillium expansum, P. funiculosum, P. steckii, P. verrucosum v. corymbiferum, P. verrucosum v. verrucosum and Scopulariopsis brumptii (tab. 2).

The estimated species composition of the populations of fungi in the in vitro cultures indicates that one in four species contaminated explants in three groups of plants (ornamental, orchard, vegetables). 48 species were put into this group and the following species occurred in a considerable number (more than 20 colonies): Acremonium furcatum, A. kiliense, Alternaria alternata, Aspergillus niger, A. versicolor, Botrytis cinerea, Chaetomium globosum, Cladosporium cladosporioides, C. herbarum, C. macrocarpum, C. sphaerosperum, Cylindrocarpon destructans, Epicoccum purpurascens, Fusarium oxysporum, Humicola fuscoatra v. fuscoatra, Memnoniella echinata, Monilia geophila, Mortierella alpina, M. isabellina, M. parvispora, Paecilomyces farinosus, P. fumoroseus, Penicillium brevicompactum, P. citrinum, P. expansum, P. funiculosum, P. lanosum, P. steckii, P. verrucosum v. corymbiferum, P. verrucosum v. verrucosum, Phialophora cyclaminis, Trichoderma harzianum, Verticillium dahliae and V. nigrescens. As single colonies 36 species of fungi were isolated from the explants, and 19 species appeared twice (tab. 2).

Numerous contaminations in the cultures of orchard plants could be explained by the difficulties in the elimination of endophytes, which live in the organs that are difficult to access during the surface disinfection [3,8,11]. This could be confirmed by the isolation of fungi from different explants. These fungi belonged to the following genera: Botrytis, Cylindrocarpon, Emericella, Fusarium, Humicola, Phialophora and Verticillium.

In case of the explants of leaf-obtained ornamental plants cultivated from leaves. Epiphytic fungi, growing on phyllosphere could be preserved among hair and scales, in stomata, in follicles with volatile oil and in the area of ageing tissues [17]. The problem of epiphytic saprotrophs and pathogens in the contamination of explants was presented by Debergh [4], Williamson et al. [16], Cassells [3] and Zenkteler [17]. Among the identified in the in vitro cultures fungi the following genera belonged to epiphytic saprotrophs and pathogens: Acremonium, Aspergillus, Chaetomium, Cladosporium, Epicoccum, Monilia, Paecilomyces, Penicillium, Phoma, Scopulariopsis, Trichoderma and other (tab. 2).

As stated in Leifert et al. [7] and Kritzinger et al. [11] the source of persistent contamination are explants taken from underground organs, containing the microorganisms difficult to eliminate during surface disinfection. This was confirmed by the isolation of fungi from the following genera: Alternaria, Botrytis, Fusarium, Sclerotinia and Torula from the explants of vegetables and some ornamental plants, the maternal material of which were underground organs.

Leifert and Woodward [8] state that in the stadium of the stabilization of explants (I) up to 70 per cent of contamination brought with the initial explant is revealed, while at the multiplication stage (II) most contamination is of secondary character, caused by (among others) failing to obtain aseptic conditions in the laboratory. In the analysis of the results referring to the identification of fungi, it was shown that the spectrum of fungi in vitro is changing with the age of the culture, thus the earliest (at the stabilization stage) cultures were contaminated by fungi such as: Alternaria alternata, A. radicina, Botrytis cinerea, Cylindrocarpon destructans, C. didymum, Fusarium avenaceum, F. culmorum, F. oxysporum, F. solani, F. trichothecioides, Nectria inventa, N. radicicola, Phialophora cyclaminis, Ph. fastigiata, Rhizoctonia carotae, R. solani, Sclerotinia sclerotiorum, Torula allii, Vericillium albo-atrum, V. nigrescens and other, living on and contaminating the initial maternal plant material.

Some fungi caused intra-laboratory contaminations. They were related to the specific of the laboratory, proliferated material, lack of sterility and the possibility of spreading of dermatophytes by the laboratory staff [15,17]. This referred to a fungus occurring in great quantities – A. niger, isolated from one commercial laboratory of tissues.

Secondary contaminants include fungi occurring in the ambient air of the laboratories. As it is said in Reby and Kowalik [13] the spores of fungi from genera: Alternaria, Aspergillus, Cladosporium, Paecilomyces, Penicillium and Trichoderma could make the source of the contamination of plant material.

Analysing the populations of microrganisms in the in vitro cultures it is difficult to say strictly which of them should be included into latent contaminants, being revealed only at stage III of micropropagation [9]. At this stage from the explants of orchard plants mostly the fungi from the following genera were found: Acremonium, Monilia and Mortierella.

In literature the problem of “vitro-pathogens” has been presented [7,17]. It seems that it can only refer to few species of fungi living on explants and showing pathogenic properties. Vast majority of fungi contaminate explants with the mycelium and its hyphae, without parasite-like penetration of explants. As stated in Leifert and Woodward [8] most microorganisms, including fungi, shortly after the introduction to in vitro cultures destroy the material due to the secretion of metabolites changing pH and osmotic potential of the medium, as well toxins poisoning the explant and enzymes macerating the tissues.

To elaborate efficient methods eliminating contaminations from the micro-propagated plant material it is necessary to know the fungi occurring in the in vitro cultures.

CONCLUSIONS

1. The isolation 195 species of fungi belonging to 76 genera from in vitro cultures allows describing this population as complex and diverse.

2. Among the contaminants the following dominated: Aspergillus niger, Aspergillus versicolor and Paecilomyces fumosoroseus, also numerous were the fungi belonging to genera: Acremonium, Aspergillus, Alternaria, Botrytis, Chaetomium, Cladosporium, Cylindrocarpon, Epicoccum, Humicola, Monilia, Paecilomyces, Penicillium and Scopulariopsis.

3. The carried out research shows that fungi of genera: Botrytis, Cylindrocarpon, Emericella, Fusarium, Humicola, Phialophora and Verticillium, should be considered endophytes, which were brought to the cultures with the propagated in vitro material.

4. Epiphytes include fungi from genera: Acremonium, Aspergillus, Chaetomium, Cladosporium, Epicoccum, Monilia, Paecilomyces, Penicillium, Phoma, Scopulariopsis and Trichoderma.

5. Fungi of genera Acremonium, Monilia and Mortierella play the role of latent contaminants in the in vitro cultures.

REFERENCES

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Accepted for print: 16.01.2007

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