Abstract
The results of the post-culture fungicidal medium from Trichoderma viride Pers. and Alternaria alternata (Fr.) Keissl. mold fungi and Acetobacter xylinum bacteria were studied relative to selected fungi belonging to Basidiomycetes, which cause wood decay. The obtained results confirmed that post-culture liquids derived from the cultivation of various microorganisms might have a differentiated fungicidal effect on wood-decaying fungi. The lowest concentration of fluid from A. xylinum culture added to the growth medium of the studied fungi that completely inhibited the growth was 5mL/100mL.The fungicidal effect of the liquid from the mold fungus culture on the tested wood-decaying fungi turned out to be definitely low. Trametes versicolor (L.) Lloyd proved to be the most sensitive species. Pleurotus cornucopiae (Paulet) Rolland showed complete resistance to the liquid added to the growth medium, derived from mold fungi. The A. xylinum bacterial culture-fluid may be subject to further analysis as a natural biocide in protecting wood against wood-decaying fungi.
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Evaluation of Fungicidal Effects of Post-culture Medium of Selected Mold Fungi and Bacteria in Relation to Basidiomycetes Fungi, Causing Wood Destruction
Izabela Betlej, Andres Bogusław,* and Krzysztof J. Krajewski
The results of the post-culture fungicidal medium from Trichoderma viride Pers. and Alternaria alternata (Fr.) Keissl. mold fungi and Acetobacter xylinum bacteria were studied relative to selected fungi belonging to Basidiomycetes, which cause wood decay. The obtained results confirmed that post-culture liquids derived from the cultivation of various microorganisms might have a differentiated fungicidal effect on wood-decaying fungi. The lowest concentration of fluid from A. xylinum culture added to the growth medium of the studied fungi that completely inhibited the growth was 5mL/100mL.The fungicidal effect of the liquid from the mold fungus culture on the tested wood-decaying fungi turned out to be definitely low. Trametes versicolor (L.) Lloyd proved to be the most sensitive species. Pleurotus cornucopiae (Paulet) Rolland showed complete resistance to the liquid added to the growth medium, derived from mold fungi. The A. xylinum bacterial culture-fluid may be subject to further analysis as a natural biocide in protecting wood against wood-decaying fungi.
Keywords: Fungicides; Post-culture liquid medium; Basidiomycetes
Contact information: Institute of Wood science and Furniture, Warsaw University of Wood Science – SGGW, 159 Nowoursynowska St., 02-776 Warsaw, Poland;
* Corresponding author: boguslaw_andres@sggw.pl
INTRODUCTION
The durability and preservation of wood intended for specific use can be guaranteed by appropriate selection of protection means and impregnation methods. Wood preservatives are chemical compounds, whose market value and usage are subject to the restrictive provisions of Regulation EU 528/2012 on the supply and use of biocidal products on the market. The restrictive biocidal law does not allow the market placement of compounds with proven harmfulness to human, animal, and the environment. Changes in the biocidal law have resulted in the withdrawal of many effective impregnants formerly used in the protection of wood. Notably, products containing chromium and arsenic compounds, phenols, petroleum compounds, and fluorine compounds were permanently withdrawn from the market. The current market situation of wood preservatives includes a limited group of active substances, mainly copper compounds, boron, quaternary ammonium salts, HDO complexes, propiconazole, tebuconazole etc. Based on the data provided by the European Chemicals Agency (ECHA 2019), it is allowed to use biocidal products for wood impregnation containing 39 types of substances. The list of active substances is not closed, because Regulation EU 528/2012 provides the opportunity to conduct scientific research in the selection of new active substances with high fungicidal effectiveness and a guaranteed level of toxicological and eco-toxicological safety. Bearing in mind the safety in the use of biocides, research is being carried out to assess the fungicidal activity of substances of natural origin, such as plant extracts, alkaloids, essential oils, but also substances produced by microorganisms (Kundzewicz and Ważny 1994; Cofta et al. 2014; Adedeji et al. 2017). Many substances of natural origin in fact have fungicidal activity, which was confirmed from in vitro tests (Barbero-López et al. 2018).
Studies on the effect of catechins, in particular 1,2-benzenediol (catechol), 1,3,5-benzenetriol, and dimethyl terephthalate on the growth of the Schizophyllum commune Fr. were conducted by Nandika et al. (2019). These researchers confirmed that a 12% concentration of compounds effectively inhibits the development of wood-decaying fungus. The fungicidal properties of teak extracts against fungi causing white and brown wood decay were the subject of research by Brocco et al. (2017). The authors of the study observed that wood samples impregnated in a vacuum method with extracts from 20-year-old teak were resistant to biodegradation processes caused by test fungi. Silveira et al. (2017) obtained similar positive results from the use of tannins to protect acacia wood against white wood rotting fungi.Substances derived from wood as fungicide were the subject of research by Salido et al. (2015), Alejo-Armijo et al. (2017), and Kadir and Hale (2019).
Fungicidal or fungistatic properties are also attributed to certain groups of microorganisms and their metabolites. Fidanza and Caneva (2019) reported that selected species of Bacillus sp. and numerous mold fungi belonging to the genera: Aspergillus, Penicillium, Candida, Fusarium or Trichoderma exhibited fungicidal and bactericidal activities against a wide group of microorganisms. At the same time, the data provided by ECHA that microorganisms such as Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus thuringiensis subsp. israelensis, Strain SA3A and Serotype H14, Strain AM65-52 are listed as active substances and authorized for use in biocidal products intended for disinfection in the veterinary area and for insect control (ECHA 2019).
The analysis of the possibility of using metabolites from the Trichoderma viride culture for biological protection of wood against decomposition was already carried out in early 90s by Kundzewicz and Ważny (1994).
In this study, an attempt was made to assess the action of fungicide post-breeding medium from the culture of selected mold fungi: Trichoderma viride Pers., Alternaria alternata (Fr.) Keissl. and bacteria: Acetobacter xylinum against fungi causing decay of wood. The selection of the indicated microorganisms was dictated by the following criteria: selected mold fungi exhibit high growth expansion and are relatively resistant to wood preservatives, suggesting that they can produce metabolites that protect fungal cells from biocide toxicity. Acetobacter xylinum is a component of Kombucha biofilm, which produces many chemical substances with antimicrobial or antioxidant activities that could also be tested as biocides acting against wood-decaying microorganisms. The presented results have the nature of screening research, the purpose of which is preliminary and approximate determination of fungicidal activity as potential factors supporting the resistance of biomaterials, such as wood and wood-based materials against fungi. From the point of view of assessing the effectiveness of biocides, this type of research is important, and in addition it allows for a relatively quick assessment of the impact of the test substances on the development of microorganisms. The research was carried out on media having an increasing proportion of influence with respect to potential bioprotective properties. Preliminary assessment of the fungicidal activity of the culture media derived from the cultivation of mold fungi and bacteria made it possible to determine the lowest concentration (MIC), completely inhibiting the growth of the tested fungi able to decay wood. The results obtained are the basis for planning further research work aimed at improving the bioprotective properties of various materials (wood, wood-based materials) based on the use of natural resistance factors obtained from the cultivation of microorganisms.
EXPERIMENTAL
Materials
The material that was used to assess fungicidal properties was post-culture liquid medium, derived from a 14-day-old culture of mold fungi Trichoderma viride Pers., strain A-102, Alternaria alternata (Fr.) Keissl., strain A-166, and bacteria Acetobacter xylinum, strain ATCC 23767. Mold fungi were grown on a liquid maltose medium containing 2.5% maltose extract (Biomaxima, Lublin, Poland). Bacteria were grown on liquid medium containing 2.5% mannitol (Pol-Aura, Zabrze, Poland), 0.3% peptone (Biomaxima, Lublin, Poland), and 0.5% yeast extract (Biomaxima, Lublin, Poland). Cultivation of microorganisms was carried out for 14 days in a heat-incubator under the following conditions: mold fungi at 22 °C, and bacteria at 26 °C. The relative humidity at the test site was 66% ± 2%. Only liquid post-culture medium was used to evaluate the fungicidal effect, and it was not modified. For this purpose, the liquid was separated from the microorganisms with a paper filter. Then, in order to eliminate the presence of microorganisms in the prepared post-culture liquid medium, it was sterilized using the syringe filter method. To eliminate all microbiological structures, 0.25 µm pore diameter filters (Nalge Company, Rochester, New York, USA) were used. The fluid prepared in this way was used as research material to assess the biocidal effect. The evaluation of fungicidal properties of the post-culture liquid from mold fungi and bacteria was carried out against strains of Basidiomycetes fungi causing deep wood decomposition: Trametes versicolor (L.) Lloyd strain 30, Coniophora puteana (Schumach.) P. Karst. strain EB 97, and Pleurotus cornucopiae (Paulet) Rolland (n.d.). Basal fungal cultures were grown on a maltose-agar medium containing 2.5% maltose extract (Biomaxima, Lublin, Poland) and 2.5% agar (Difco, New Jersey, USA). The strains of fungi used in the study were obtained from the collection of the Department of Wood Science and Wood Protection, Warsaw University of Life Science – SGGW in Warsaw.
Methods
The fungicidal properties of post-culture liquid medium from the cultivation of mold fungi and bacteria against Basidiomycetes were carried out by determining the percentage of growth inhibition in relation to the growth of reference fungi, i.e., in control cultures not containing fungicides in the medium. The sterilized post-culture medium was added to a sterile medium containing 2.5% maltose and 2.5% agar. The temperature of the medium was kept at 42±2 °C. The fluid medium from the microbial culture was added to the maltose-agar medium in an amount of 1, 5, 10, 15, 20, and 30 mL in such a way that the final volume of medium was 100 mL. After mixing the contents thoroughly, about 10 mL of the medium was added onto 90 mm diameter Petri dishes, where the test fungi were centrally inoculated. The inoculum size of each fungi was 5 mm. Cultivation was carried out under temperature and humidity conditions of 25°C and 66%±2%, respectively. The evaluation of the effect of post-culture liquid medium on the growth of test fungi was carried out by measuring the diameter of the growth of T. versicolor, C. puteana, and P. cornucopiae mycelium in two perpendicular directions. Height measurement was taken at 48 h intervals. Tests were completed on the day of complete growth of control medium. Each study was performed in triplicate.
Test Standards
To verify statistical hypotheses, analysis of variance was used for single classification using Snedecor statistics. Statistical inference was carried out for the significance level α = 0.05. In case of rejection of the null hypothesis, the next step was to compare the mean multiple comparison test – Tukey’s. The statistical hypothesis was as follows,
H0: Ø 0.5= Ø 1= Ø 5= Ø 10= Ø 15= Ø 20= Ø 30= Ø K
H1: There are at least two means that differ significantly.
RESULTS AND DISCUSSION
Analysis of the test results showed that the post-culture liquid medium, derived from the cultivation of T. viride and A. alternata mold fungi, inhibited the growth of T. versicolor, which causes white wood decay, but only at a specific concentration level. In the case of low share of post-culture fluid medium (1mL/100mL and 5mL/100mL) in the growth medium T. versicolor, no differences in growth diameter were observed compared to the control conditions, on the sixth day of observation. A clearly visible zone of inhibition of fungal growth appeared in the test in which the density of the post-culture medium in the growth medium was 10mL/100mL (Table 1). In contrast to T. versicolor, the fungus causing brown wood decay – C. puteana was more resistant to the fungicidal effect of the post-culture liquid medium, than T. versicolor, as indicated in the diameter of the fungus growth on the medium (Table2). Nevertheless, it was noted that fungicidal effects already appeared at the lowest concentrations of post-culture fluid in the growth medium. The results obtained during the last day of measurement, in which the complete mycelium growth of the medium was achieved in the control sample was analyzed. It was found that the inhibitory effect of the growth of C. puteana fungus by the ingredients contained in the post-culture liquid medium from mold fungi occurs in the test in whose share of post-culture medium was the smallest. A complete lack of growth inhibitory activity was found relative to the P. cornucopiae test fungus (Table 3). In the last day of observation, no significant differences were observed in the diameter of the mycelium growth of the medium under control conditions and in all research variants containing various shares of the media from T. viride and A. alternata cultures. Bruce and Highley (1991) observed a very similar effect of the effect of T. viride on the growth of T. versicolor, which may suggest that the indicated fungus causing white wood decay is relatively resistant to the potential fungicidal action of metabolites synthesized by T. viride. The low biocidal activity of the T. viride post-culture medium on the growth of C. puteana does not correlate with the results of other authors who at the cellular level obtained strong lysis of C. puteana cells due to the action of metabolites derived from mold fungal culture (de Vries and Wessels 1973). A much stronger action of the fungicide was obtained by examining the effect of the medium derived from A. xylinum bacterial culture on the growth activity of the studied fungi. The addition of 10% bacterial culture medium to the fungal growth medium practically completely inhibited the growth of T. versicolor. On the last day of breeding, a slight growth of the fungus was observed. In the case of the effect of A. xylinum post-culture liquid medium on the growth of P. cornucopiae and C. puteana, effective fungicidal activity was demonstrated for concentrations of 5% and higher. The fungicidal properties of A. xylinum cultures against fungi causing wood decay have not been the subject of analysis, unlike other organisms belonging to Actinobacteria such as Streptomyces exfoliatus or bacteria Pseudomonas spp., the use of which in the protection of wood has been evaluated by other researchers (Susi et al. 2011; Sharma et al. 2016).
Table 1. Growth Diameter of T. versicolor on Media Containing Post-culture Liquid Medium from a T. viride, A. alternata, and A. xylinum Culture
Table 2. Growth Diameter of C. puteana on Media Containing Post-culture Liquid Medium from a T. viride, A. alternata, and A. xylinum Culture
Table 3. Growth Diameter of P. cornucopiae on Media Containing Post-culture Liquid Medium from a T. viride, A. alternata, and A. xylinum Culture
The percentage growth rate of fungi on the media containing various proportions of post-culture liquid medium, derived from the cultivation of mold fungi and bacteria is presented in Table 4 and Figs. 1, 2, and 3. In studies involving the fungicidal action of the post-culture liquid medium from mold fungi on the T. versicolor fungus, statistically significant differences were found between the control and the medium containing 5% and a higher concentration of post-culture liquid medium. In the case of the C. puteana fungus, statistically significant differences in growth compared to the control sample were found on a medium containing 15% and a higher concentration of post-culture medium. Similar analyzes of the effect of fungal extracts on the growth of C. puteana were conducted by Yildiz et al. (2019). The fungicidal effects of fungal extracts on many saprophytic and parasitic fungi have been studied by Okeke et al. (1992), Sudirman et al. (1992), and Highley (1997). This activity was attributed to the production of metabolites with antibiotic functions by many fungi, which inhibit the development of other organisms (Yang et al. 1993).
Table 4. Percentage Growth of Fungi on Medium, Containing a Post-culture Liquid Medium from Mold Fungi and Bacteria
None of the applied post-culture liquid medium concentrations from the mold fungi culture achieved complete inhibition of the growth of the test fungi. It is likely that, despite the use of high concentrations of post-culture medium, the proportion of active substances with fungicidal activity did not have to be high. It has been demonstrated that many factors can affect biocidal effectiveness. Both the bioavailability of given substances, and the extraction method and conditions may affect the results of toxicity obtained (Jo et al. 2014).
In the case of evaluation of the fungicidal effect of the post-culture liquid medium, derived from the culture of A. xylinum bacteria in relation to the tested fungi, statistically significant differences between the control and the tested samples were found at much lower concentrations than it was the case when assessing the effectiveness of the culture medium from mold fungi.
Fig. 1. Relative percentage of growth of T. versicolor on medium containing post-culture liquid medium from A. xylinum culture
Fig. 2. Relative percentage of growth of C. puteana on medium containing post-culture liquid medium from T. viride culture
Fig. 3. Relative percentage of growth of P. cornucopiae on medium containing post-culture liquid medium from A. alternata culture
CONCLUSIONS
- Results of this study showed that the tested fungi exhibited various levels of sensitivity to the toxic effects of medium from microbial culture.
- Post-cultural medium derived from the cultivation of molds A. alternata and T. viride did not cause as pronounced inhibition of fungal growth as it was in the experiment using liquid medium from bacterial culture.
- P. cornucopiae turned out to be a species insensitive to the toxic effects of post-cultural medium from mold fungi.
- The post-culture medium from A. alternata and T. viride fungi cultures did not show a clear fungicidal activity against the tested fungi causing deep wood decay.
- The post culture medium from A. xylinum bacteria showed strong fungicidal activity against tested wood-decaying fungi, which gives the basis for its further use in tests for determining bio-protective values under practical conditions of use in wood and wood-based materials.
ACKNOWLEDGMENTS
The authors thank Warsaw University of Life Sciences – SGGW for the financial support.
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Article submitted: November 29, 2019; Peer review completed: February 1, 2020; Revisions accepted: February 10, 2020; Published: February 12, 2020.
DOI: 10.15376/biores.15.2.2471-2482