BIOTIC AND BIOTECHNICAL FACTORS INHIBITING THE GROWTH AND DEVELOPMENT OF TOPOSPORA MYRTILLI (FELTG.) BOEREMA

Mariusz Szmagara
Institute of Ornamental Plants and Landscape Architecture, Agricultural University of Lublin, Poland

ABSTRACT

The experiment studied the effect of 14 fungi species isolated in the years 2001-2003 from the shoots of highbush blueberry (Vaccinium corymbosum L.) on the growth of Topospora myrtilli. Besides, the effect of biopreparations of animal and plant origin: Biochikol 020 PC and Biosept 33 SL, was examined on T. myrtilli. Trichoderma harzianum and T. koningii belong to the fungi that inhibited the growth of the pathogen in the strongest manner. The studied species from genus Trichoderma caused degeneration of the hyphae and the dying out of the pathogen’s colonies already eight days after their common growth. The fungi inhibiting the growth of T. myrtilli also included: Altenaria alternata, A. raphani, Epicoccum purpurascens and Penicillium decumbens. Among those, only Epicoccum purpurascens formed an inhibition zone in common growth with T. myrtilli. The studies conducted on the effect of biopreparations on T. myrtilli showed that both Biochikol 020 PC and Biosept 33 SL considerably limited the growth and development of the pathogen. Biosept in the concentration of 0.3% caused very strong inhibition of the growth of the hypha and their deep degeneration.

Key words: antagonistic fungi, biopreparation, Topospora myrtili, Vaccinium.

INTRODUCTION

Fungi occupy a significant place among the microorganisms causing the diseases of highbush blueberry (Vaccinium corymbosum L.). Very dangerous pathogens of this plant include Topospora myrtilli (Feltg.) Boerema, which causes shoot necrosis [7, 22, 28]. Disease symptoms occur mainly in the lower part of the shoots in the form of characteristic ellipsoidal necrotic spots of chestnut-brown colour with a brighter center and a purple edge. Later, the shoot usually dies out above the place of the infection. The pathogen is especially dangerous due to the perennial character of this plant’s cultivation. It is very dangerous to young plants since its destructive effect leads to the whole shrubs’ dying out [2,24,25,26].

Besides pathogenic species, numerous species of non-pathogenic fungi occur on the overground parts of highbush blueberry. They can be neutral towards the pathogens, but some can inhibit their growth, which is of big importance for the natural control of the size of the population of pathogenic factors.

Antagonistic properties of microorganisms can be used for biological control of the populations of pathogens. Studies are conducted on the possibility of using these organisms as components of microbiological preparations applied in plant protection [3]. Information in literature points to an effective action of biopreparations on the basis of fungi from the genera of Trichoderma and Gliocladium in the protection of different plant species from infection by a large spectrum of pathogens. Fungi from the genera of Epicoccum and Penicillium display the ability to inhibit the growth of certain microorganisms [9,13,19,27,31].

Biopreparations based on organic compounds which are, for instance, Biochikol 020 PC and Biosept 33 SL, are considered promising. The active substance of Biochikol 020 PC is chitosan, which limits the development of a number of bacteria and fungi infecting cultivated plants [10,21,30]. Similarly, the endogenous flavonoids and aliphatic aldehydes, monoterpenes and nutkakon contained in Biosept 33 SL have the ability to inhibit the development of bacteria and fungi, such as Botrytis cinerea, Colletotrichum spp. and Penicillium spp. [21,29].

In view of the lack of information in literature on biotic factors inhibiting the growth and development of T. myrtilli, studies were undertaken on the antagonistic effect of this pathogen on phyllosphere fungi inhabiting the shoots of highbush blueberry and on the possibility to limit it by means of biopreparations.

MATERIAL AND METHODS

The study material consisted of Topospora myrtilli Bw 1878 strain and 14 selected fungi species that were isolated in the years 2001-2003 from highbush blueberry shoots [25, 26]. The following species belonged to them: Altenaria alternata, Altenaria raphani, Fusarium avenaceum, Fusarium culmorum, Phoma capitulum, Phoma glomerata, Trichoderma harzianum, Trichoderma koningii, Cladosporium cladosporoides, Epicoccum purpurascens, Penicillium decumbens, Botrytis cinerea, Cytospora sp. and Phomopsis archeri. The growth of the pathogenic species of T. myrtilli was studied in common culture with particular species of accompanying fungi [14]. An estimation of the effect of biopreparations was performed according to an eight-grade scale prepared by Mańka and Kowalski [15]. The effect of the fungi tested on T. myrtilli was expressed by means of the individual biotic effect (IBE) and the general biotic effect (GBE), which is the ratio of the value of an individual effect and the multiple of the occurrence of the fungus tested in fungi communities obtained in a given year from highbush blueberry shoots. In the years 2001, 2002 and 2003 the total biotic effects were established [14].

In the case of Trichiderma spp. studies on their effect on the viability of T. myrtilli were conducted if the area of the pathogen colony was occupied by a micro-parasite [11].

The same cycle of studies also determined the effect of biopreparations on Topospora myrtilli. The following were taken into consideration: Biochikol 020 PC and Biosept 33 SL as well as 3 isolates of T. myrtilli: Bw 1535, Bw 1577 and Bw 2462 obtained from the shoots of highbush blueberry in the years 2001-2003. Biochikol 020 PC was tested in four concentrations: 0.1%, 0.025%, 0.05% and 0.1% on poor maltose medium (Difco) with half reduced quantity of maltose to 1 liter [17]. The concentrations of 0.05%, 0.1%, 0.2% and 0.3% as well as a standard PDA medium were used in the experiment with Biosept 33 SL. Each of the preparations was added to the sterilized and cooled medium in proper quantities. Fungus inocula, which were the rings of particular isolates cut out from 10-days’ old mother cultures were placed on the settled medium. The cultures were kept at the temperature of 22°C. 4 repetitions were considered for each fungus isolate and concentration of the biopreparation. The control were the colonies growing on the maltose without an addition. After 4 and 8 days the measurements of the diameter were made and the percentage of colony inhibition [8] was calculated, and the numerical data were submitted to a statistical analysis. Next, the material for microscopic examinations was taken from each dish.

RESULTS

The studies showed that out of 14 tested fungi species, 12 inhibited the growth of the pathogen, which is testified to by the positive values of individual biotic effect (Table 1). Among them, Trichoderma harzianu and, T. koningii completely inhibited the growth of T. myrtili colonies since the values of the individual biotic effect was +8 (Table 1, Photo 1). The colonies of Trichoderma spp. grew on ¼ of the pathogen’s colony as early as after two days of common growth, and after three days they occupied the whole area of the colony. After 8 days of common growth they caused degeneration of the hypha, which was manifested in the shrinking and sticking out of cytoplasm from the cell wall, lysis and degradation. The observed changes caused by T. harzianum and T. koningii (Photo 2) led to the necrosis of the pathogen colony.

The tested fungi that considerably inhibited the growth of T. myrtilli colonies included Botrytus cinerea. It inhibited the growth of the pathogen colonies very strongly already in the initial period of common growth, and the individual biotic effect after eight days was +7 (Table 1).

The species of Phomopsis archeri, Fusarium culmorum and Penicillium decumbens inhibited the growth of T. myrtilli colonies, as an effect giving the individual biotic effect of +6 after eight days of common growth (Photo 1). On the other hand, F. avenaceum, Phoma glomerata, Altenaria alternate and A. raphani inhibited the growth of the pathogen colonies, with the unit biotic effect of +5 (Table 1).

The growth of T. myrtilli inhibited Epicoccum purpurascens. There occurred a slight inhibition zone between these species, which was formed by E. purpurescens, and the value of the individual biotic effect was +4 (Table 1, Photo 1). The growth of the pathogen colony was slightly inhibited by Cytospora sp., and the individual biotic effect was only +1 (Table 1). Cladosporium cladosporoides and Phoma capitulum showed a neutral effect towards T. myrtilli, which is testified to by the values of the individual biotic effect, which were 0 (Table 1).

Among the tested species in the fungi communities obtained from highbush blueberry in 2001, the following occurred in the greatest numbers: A. alternata, E. purpurascens, F. avenaceum, P. archeri and A. raphani. The general biotic effects for these species and for T. myrtilli were positive and ranged from +505 to +2680 (Table 1).

In 2002 the most numerous were A. alternate, E. purpurascens and B. cinerea, and their general biotic effects for T. myrtilli ranged from +620 to +2480. In the case of T. koningii it was +504, T. harzianum +312, Penicilllium decumbens +234, and E. purpurascens +620 (Table 1).

In the year 2003 the most numerous in the fungi community from the shoots of highbush blueberry were A. alternate, E. purpurascens, B. cinerea, Cytospora sp. and F. avenaceum. Their general biotic effects towards T. myrtilli were positive and had the values ranging from +131 to +2315 (Table 1).

In each of the successive years of the studies the total biotic effect, determining the effect of the fungi occurring on highbush blueberry shoots on T. myrtilli was positive and in 2001 was +7462, while in 2002 + 6908, and in 2003 +5961 (Table 1).

The studies on the effect of biopreparations on T. myrtilli showed that the inhibition percentage for 4- and 8-days’ colonies in the case of Biochikol 020 PC and Biosept 33 SL differed significantly as compared to the control (Tables 2 and 3).

In the experiment with Biochikol 020 PC the greatest percentage of growth inhibition of 4-days’ old colonies, which was 59.76 and 54.89, was observed in the case of isolate Bw 2462 growing on the medium containing the biopreparation in the concentrations of 0.1% and 0.05%, and it was significantly higher as compared to the control (Table 2). On the other hand, in the case of isolate Bw 1535, growing on the medium containing 0.025% of the preparation, the studies found out the smallest percentage of inhibition of the pathogen growth, which was 16.78%, and this value differed significantly from the control value (Table 2).

The greatest percentage of inhibition of the growth of 8-days’ T. myrtilli occurred in isolate Bw 2462 at the concentration of 0.1%, and it was 75.39%. This value was significantly higher in comparison to the control value (Table 2). On the other hand, the smallest inhibition percentage was found for isolate Bw 1535 growing on the medium containing 0.01% and 0.025% of the biopreparation, and it was, respectively, 49.31% and 49.98%, and those values differed from the control in a significant way (Table 2).

The mean inhibition percentage of the studies T. myrtilli isolates increased proportionally to the concentration of Biochikol 020 PC, both after 4 and 8 days of the culture. After 4 days the mean inhibition percentage of the growth of the colonies growing with 0.1% and 0.05% of the preparation was significantly bigger than with the concentrations of 0.025% and 0.01%. After 8 days of the culture the mean inhibition percentage of the colonies’ growth with the concentration of 0.1% of the preparation was significantly higher than with the other tested concentrations (Table 2). The effect of Biochikol 020 PC within the examined concentrations was only fungistatic, and the diameter of the colonies of the studied isolates decreased only with the increased concentration of the biopreparation (Table 2).

With the application of Biosept 33 SL, complete inhibition of the growth of isolate Bw 1535 after 4 days of the culture on the medium containing 0.3% of the preparation took place, and it was significantly higher as compared top the control (Table 3). On the other hand, the smallest percentage of growth inhibition was also observed for the same isolate on the medium containing 0.05% of the biopreparation, and it was 38.66%, and that value significantly differed from the control value (Table 3).

The mean percentage of the growth inhibition of 4-days’ colonies of T. myrtilli growing on the medium containing 0.3% of the tested biopreparation was 73.44% and was significantly higher than with the concentrations of 0.05%, 0.1% and 0.2% (Table 3).

Similarly, after 8 days of the culture a complete, i.e. 100% inhibition of the growth of isolate Bw 1535, took place with the highest concentration, and that value differed significantly as compared to the control (Table 3). The smallest growth inhibition was also observed in that isolate on the medium containing 0.05% Biosept 33 SL and it was 69.03%, with that value significantly differing from the control (Table 3).

Besides, the mean percentage of growth inhibition for 8-days’ colonies was significantly higher than the mean percentage of growth inhibition for the colonies with the other values of the concentrations (Table 3).

The studies showed changes in the macro- and microscopic appearance of T. myrtilli colonies growing on the medium containing both biopreparations. It was observed that both 4- and 8-days’ fungus colonies, regardless of the concentration of the biopreparation, were dark grey, which differed from the lemon-olive coloured control colonies. The hypha of the air mycelium were considerably shortened, thickened and they formed a fluffy-hairy structure, while the structure of control colonies was tomentous (Photo 3). Besides, the hypha of the examined isolates growing in the presence of Biosept in the concentrations of 0.2% and 0.3% showed the signs of strong degeneration (Photo 3).

DISCUSSION

The studies showed big biodiversity of the fungi colonizing the shoots of highbush blueberry [26]. They also made it possible to get to know the effect of numerous fungi species on T. myrtilli, independently of the frequency of their occurrence. Among the phyllospheric fungi, the following had a large proportion: Altenaria alternata, Epicoccum purpurascens, Fusarium spp., Saccharomyces spp., Cladosporium sp. and Trichoderma spp.

The strong antagonistic effect of fungi form genus Trichoderma should be considered as very positive in the aspect of their practical application, as a factor supporting the biological control of the pathogen. Fungi from genus Trichoderma spp. should be included within the positive antagonists inhibiting the growth and sporulation of T. myrtilli. Fungi from this genus are known for their strong competitive abilities, as well as the ability for supra-parasitism and to form toxic metabolites and exo- and andoenzymes [5,19]. It is probably owing to these mechanisms of direct effect on the pathogen that the species of Trichoderma harzianum and T. koningii caused a very strong growth inhibition of T. myrtilli, degradation of the hypha and the dying out of the pathogen colonies. Among the tested fungi from genus Trichoderma, the species of T. harzianum and T. koningii proved to be the strongest antagonists towards T. myrtilli. A considerable frequency of their occurrence in 2002 points out that they had a big role in the high positive value of the general biotic effect. There have been numerous attempts to use these fungi, and especially T. harzianum, T. viride and T. koningii sp. protect plants from infection by different species of pathogenic fungi [3, 9, 13,19, 31]. The inhibition zone appeared with common growth of T. myrtilli and E. purpurescens. This implies that the latter species can exudate secondary metabolites to the medium which are toxic towards the pathogen, which is proved by a 2-3 mm inhibition zone. The ability of Epicoccum purpurascens to inhibit the growth and sporulation of Botrytis cinerea, Monilia laxa and M. coryli was earlier shown by other authors [6, 27, 31]. The now studied fungi inhibiting T. myrtilli also included fast growing species of pathogenic fungi such as Botrytis cinerea, Phomopsis archeri as well as toxin-forming fungi Fusarium spp. and Altenaria spp. However, the occurrence of the latter should be considered as undesirable since they are harmful to highbush blueberry and other cultivated plants [4,12,18,23], and their presence testifies to the unfavourable phytosanitary conditions in the studied cultivation. It can be seen that the created biotic series, so in the cultivation environment of highbush blueberry shrubs, lacked the positive antagonists of T. myrtilli, for example the fungi from genus Gliocladia, and the populations of Trichoderma spp., Epicoccum sp. and Penicillium sp. were relatively small in relation to the populations of competitive pathogenic fungi such as B. cinerea and Phomopsis sp. Hence, in the present studies the effect of positive antagonists on the positive general and total biotic effect was not significant.

The lack of competitive abilities of T. myrtilli shown in the present studies points out that the latter can be effectively forced out from the natural or artificial medium by other, fast growing fungi species. Its occurrence as the primary pathogen of the shoots can be masked by other fast growing species of saprophytic fungi, which secondarily develop on the died out plant tissue. Therefore, the isolation of T. myrtilli from plant tissues onto the artificial medium is possible only at the initial stage of the necrosis of highbush blueberry shoots. After the necrotic spots are colonized by saprophytic species of fungi, the isolation of T. myrtilli can be difficult.

The results obtained from the studies on the effect of biopreparations on T. myrtilli confirm the ability of Biochikol 020 PC and Biosept 33 SL to directly inhibit the growth of fungi and to cause macro- and microscopic changes of the fungus, which is consistent with the results of other authors [10,16,21,30]. The studies showed the existence of intra-species diversity of T. myrtilli. It was expressed in a differentiated reaction of certain isolates to the effect of Biochikol 020 PC and Biosept 33 SL. Among the tested isolates, Bw 1577 and Bw 2462 had a very strong reaction to Biochikol 020 PC, while isolate Bw 1535 reacted very strongly to Biosept 33 SL.

It was stated the effect of chitosan in vitro depends on the susceptibility of the species or the fungus isolate and on the degree of chitosan polymerization [1]. On the other hand, the effect of the in vitro tested biopreparations was exceptionally high for T. myrtilli isolate Bw 1535 with the highest concentrations, which was proved in reference to other pathogens [10, 16]. In the case of the other isolates a significant inhibition of the growth of their colonies was observed, independently of the concentration, which was found out by other authors in the case of chitosan and soil-borne fungi [20].

The compounds in grapefruit extract inhibit the germination of sporules and they inhibit the growth of the germ hypha through the dehydration of the cytoplasm. The preparation causes a decrease of the populations of soil-borne pathogens and deformation of their spores as well as degradation of the mycelium hypha [21]. It can be supposed that the mode of their effect caused a significant inhibition of the growth of T. myrtilli colonies and changes in the morphological structures of the fungus.

CONCLUSIONS

1. The positive antagonists inhibiting the growth and sporulation of T. myrtilli include the species from genus Trichoderma, which is very favourable in the aspect of practical application as a factor supporting the biological control of this pathogen.

2. Lack of competitive abilities, shown in the present studies, points out that it can be forced out from the natural or artificial medium by fast growing fungi species, and its occurrence as a primary pathogen can be masked by other fast growing species of saprophytic fungi.

3. The present studies showed a differentiated reaction of certain isolates to the biopreparations.

4. Inhibition of the pathogen growth in vitro by Biochikol 020 PC and Biosept 33 SL points to the need to conduct further research on the possibility of using these preparations to protect highbush blueberry shoots from infection by T. myrtilli.

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Accepted for print: 17.10.2007

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