FUNDAMENTALS OF TESTING OF FUNGAL PHYSIOLOGICAL REQUIREMENTS Prof. Dr Milenko Mirić, Dipl. eng. Snežana Ivković milenko.miric@sfb.rs University in Belgrade, Forestry Faculty, Kneza Višeslava 1, 11030 Belgrade, Serbia Abstract: The essential fungal requirements for growth besides the water, as a medium in which all metabolic proceses are performing, are appropriate temperature, ph of substrate and available sources of basic nutrients. Investigation of impact of these factors should be methodically established very carefully in order that gained results could be usefull for further research of bioecological characteristic of certain fungi. That is why it is necessary that investigating methods have to be very precise and carefully performed. The optimal temperature for the growth of mycelia should be performed in standard Petri dishes containing i.e. 2 % Malt / 2 % Agar. This relatively simple test should show results which one must respect for certain strain, using its own optimal temperature for each fungi in different tests. If one intend to compare results gained for different strains, than each single fungi have to grow on its` optimal temperature, but not as always and usually on standard 21 C. Concentration of H ions in nutritive media is of extraordinary significance for fungal growth and all other metabolic activities, as like for consuming of nutrients or decomposition of substrate. The test have to be run with unbuffered media to define the impact of the fungus on the changes of initial ph value of liquid media during the test, while the test with buffered media should show the influence of stabile ph value of substrate on the fungal growth. Nutritional requirements should be the most corectly investigated in liquid media as convenient one for measuring of mycelial mass yielded after i. e. 30, 60 and 90 days of incubation. The basal media should contain: glucose, L glutamic acid, KH 2 PO 4, MgSO 4, CaSO, solution of oligoelements: B, Mn, Zn, Cu, Mo, Thiamine HCl and Fe EDTA in distilled water solution. Certain substances which provide necessary amount of carbon, nitrogen and phosphorus, as the most important elements for fungal growth, should be excluded from test series and substituted with tested substances sources of certain nutrients. In C test necessary quantity of nitrogen in basal media should be provided by L glutamic acid, while in N test necessary quantity of carbon should provide glucose. The appropriate quantities of tested sources of nutrients should be calculated based on molecular and atomic weights of substances and elements, so that in all test series exactly the same concentration of nutritive elements should be provided. By using prescribed model one could gain very usefull and correct results, what exactly is the main target of any scientific investigation. Original methods are described precisely in article itself with the aim to be helpful to researchers. Key words: fungi, nutrients, physiology, method, ph, temperature, carbon, nitrogen, phosphorus 1723
Introduction Wood decaying fungi, depending to their enzymatic activity, can utilize each cellulose, lignin and hemicelluloses, but in amount which in quantitative and qualitative sense define them as causers of brown-, soft- or white rot (Schmidt, Kerner - Gang, 1986; Karadžić, 1990). Among this group of fungi extremely important are those species with the both parasitic and saprophytic way of nutrition. Their destructive action can start in stems and continue (or start) it in felled timber. All wood anatomical elements play their own role in wood structure, and depending to their quantity and organization the mechanical and physical properties of different wood species are specifically characterized. From the other side, depending to fungal enzymatic systems, all of these elements are endangered in different amount. The effects of fungal activity are previously visible through the changes on the elements of anatomical structure of wood. In that process, as a consequence of fungal development it appears the changes of chemical, physical, mechanical and technological wood properties, what leads to the last effect - death of stem and decomposition of wood mass. The differences in chemical structure of the most important anatomical elements of certain wood species, as well as enzymatic systems of wood decaying fungi, are responsible for appearance of different types and different amount of damages. The content of available and easy - assimilable nutrients inside the wood for shore plays the most important role in the very first stage of fungal attack, just before development of active wall - destroying process, while fungus use to consume nutrients from vessels containing sap i.e. minerals, vitamins, proteins etc. dissolved in water (Fig 1 2.). Fig. 1: Hyphens of wood decaying fungi inside vascular tissue of Oak (SEM 800 x) (original photo) Fig. 2: Penetrating of wood decaying fungi threw wood cell wall to the neighboring vessel (SEM 720 x) (original photo) 1724
The conditions in which fungus can express its` destructive activity are of the most importance affecting fungal metabolic process. Temperature and ph are among essential factors which influence fungal activity (Muntaňola Cvetković, 1987. Robinson, 1978). Physiological requirements i.e. nutritional requirements for essential nutrient containing C, N and P, are predominant for fungal growth. Fungi use to consume carbon, nitrogen and phosphorus both from organic and inorganic sources. For researchers it is of enormous significance to establish and use relevant and good investigating method which should gain appropriate exact results. Mistakes in investigations are mainly in the sphere of method errors. That is why one should carefully chose method for testing of fungal physiological requirements in all details. This article advices very precise step in this kind of testing, based on good practical experience in order to help researchers to gain useful scientific results. 1. MATERIJAL AND METHOD 1. 1. Temperature test In order to find out the optimal temperature for the growth of mycelia, a standard simple temperature test have to be done, but with the known strain of fungus dicariotic or homocariotic one, as like for all other investigations. Petri dishes containing 2 % Malt / 2 % Agar nutritive media should be inoculated (at the edge or in the center of the dish, depending to speed of mycelia growth) with fresh mycelia and incubated at different temperatures ranging i.e. 15, 20, 24, 26, 28, 30 and 34 C. Different rang of temperature one intend to investigate should be adjusted depending on certain fungus. Marking should be done daily, or each second day for slow growing cultures, while the measurements - at the end of the test. The average daily increment of mycelia should be calculated based on measurements in three directions in each Petri dish (angle of 45 have been measuring area), or in cross direction from center for slow growing cultures. At least each 5 replicates have to be used for each strains and each tested temperature. Estimation of optimal temperature for mycelia growth is of the essential importance, since the further planed tests should be run in conditions which should be optimal for certain strain. Serious mistakes could appear if one investigates some phenomena with different fungi in, so called - standard laboratorial conditions and on temperature of 21 ±1ºC for all tested fungi. In practice, that means that some fungi should grow on their optimal temperature, while some of tested fungi should be inhibited by temperature of 21 ±1ºC which is, for example, not the most convenient for their growth. Idea is that all tested fungi have to have optimal temperature in order to develop its` maximum metabolic activity, and just in that case one could compare results for different strains. The similar situation is also in the case of impact of ph value of substrate, but it is already well known that fungi are mostly capable to adjust the concentration of H ions towards their optimum, during metabolic process. So, one need not always calculate with negative impact of ph of substrate, except if it is so stabile that seems to behave as a buffer. 1725
1. 2. ph test Concentration of H ions in nutritive media is of extraordinary significance for fungal growth and all other metabolic activities, as like for consuming of nutrients or decomposition of substrate (Mirić, Schmidt, 1992.). Smart test should be run according to modified method, basically described by Schmidt and Liese (1987), with unbuffered- and buffered nutritive media. The first one should be used to define the impact of the fungus on the changes of initial ph value of liquid media during the test and to show the yield of mycelial mass at the end of incubation. The test with buffered media should show the influence of stabile ph value of substrate during the test on the fungal growth. Buffered system should be prepared according to method described by Wolpert (M i r i ć, 1993.) by using different quantities of phosphate solutions: H 3 PO 4, KH 2 PO 4 and K 2 HPO 4. On this way, according to sheme described by Wolpert, one could gain substrates with different ph values, but with the same quantity of phosphates so that their quantity should not have any influence on the results. Control of ph after sterilization should verify the stability of buffered systems. By using this method, one can prepare medium with standard concentration of 5 Bé sugar and 2 % of agar, with physiologically the same presence of buffers (0,15 M). Test should be run on solid media in order to estimate rate of mycelial growth in length and at the end of the test, stability of buffered system should be verify by measuring of ph of control liquid media with the same content of substances as like test series which should be incubating at the same conditions like as test series. The impact of test fungi on changing of ph value of nutritive substrate should be estimated by using unbuffered liquid media. This usefull method has been clearly described by S c h m i d t and L i e s e (according to M i r i ć, 1993). Unbuffered system should be established with 1M HCl and 1M NaOH, and different values should perform the initial values of running test. Nutritiv media should be prepared with malt extract and all test series should have sugar concentration of about 5 Bé. At the end of the test the changed ph values should be measured, and yild of dry mycelial mass should be estimated by vacuum filtration and drying and measuring of dry mass of harvested mycelia. 1. 3. Nutritional requirements Nutritional requirements should be investigated in liquid media as convenient one, for measuring of mycelial mass yielded after i.e. 30, 60 and 90 days of incubation (or some other more convenient period of time). The basal media (basically according to Beever, 1969) should be prepared as follows: glucose: 25 g; L glutamic acid: 2.72 g; KH 2 PO 4 : 1g; MgSO 4 x 7H 2 O: 0.5g; CaSO 4 x 2H 2 O: 0.3g; solution of oligoelements: 1.0 ml (0.5 ppm B from Na 2 B 4 O 7 ; 0.5 ppm Mn from MnSO 4 ; 0.05 ppm Zn from ZnSO 4 ; 0.02 ppm Cu from CuSO 4 ; 0.02 ppm Mo from Na 2 MoO 4 ; Thiamine HCl: 100 μg; Fe EDTA: 6 ppm;) filled up to 1 l with distilled water. The value of ph should be adjusted around 6 with 1M NaOH or 1M HCl. Certain substances which provide necessary amount of carbon, nitrogen and phosphorus, have to be excluded from test series and substituted with tested substances sources of certain nutrients. In C test necessary quantity of nitrogen in basal media should be provided by L glutamic acid, while in N test necessary quantity of carbon should be provided by glucose. The appropriate quantities of tested sources of nutrients have to be calculated based on molecular and atomic weights of substances and elements, 1726
so that in all test series should be provided exactly the same concentration of nutritive elements as follows: carbon: 10 g/l; nitrogen: 225 mg/l (just Dl phenylalanine: 450 mg/l) and phosphorus: 0.5 g/l. Sterilisation could be done in autoclave by standard procedure (20 min. /121 C) except for urea and similar products, which must be aseptically filtered (R max = 0.2 μ) due to its thermo lability. The test should be run in 300 ml Erlenmeyer flasks containing each 50 ml of media (Fig. 3 4.). Fig. 3: Yield of mycelia mass of different fungi growing on the same source of carbon (liquid test) (original photo) Fig. 4: Yield of mycelia mass of different fungi growing on the same source of nitrogen (liquid test) (original photo) After 30, 60 and 90 days (or some other chosen period of time) of incubation, developed mycelia should be harvested by vacuum filtration, dried till absolutely dried mass and measured. It should be tested some 6-10 sources of each carbon, nitrogen and phosphorus (organic as well as inorganic ones). The same experiment should also be performed, but on solid media with agar in order to estimate the speed of growth in length (Fig. 5 6.). The results that one gains from liquid test should give estimation of fungal capability to develop its` metabolic activity, since harvested mass of mycelia has direct impact on metabolic strength, while the results from solid test should show how fast fungus could colonize the inner parts of wood in dependence of its` status of nutrients. This approach is very logical since it is well known that status of nutritive substances inside the wood is different in different season threw the year (Rayner, Boddy. 1988). One should know that it is of major importance in very first stage of colonizing of wood, before development of real decay. Fig. 5: Test of fungal growth in length on different carbon sources (solid media test) (original photo) Fig. 6: Test of fungal growth in length on different nitrogen sources (solid media test) (original photo) 1727
2. CONCLUSION The essential fungal requirements for growth besides the water are appropriate temperature, ph of substrate and available sources of basic nutrients. The conditions in which fungus can express its` destructive activity are of the most importance affecting fungal metabolic process. Temperature and ph are among essential factors which influence fungal activity. Physiological requirements i.e. nutritional requirements for essential nutrient containing C, N and P, are predominant for fungal growth. Fungi use to consume carbon, nitrogen and phosphorus both from organic and inorganic sources. For researchers it is of enormous significance to establish and use relevant and good investigating methods which should gain appropriate and correct results. Mistakes in investigations occur mainly in the sphere of method - errors. That is why one should carefully chose methods for testing of fungal physiological requirements in all details. Estimation of optimal temperature for mycelia growth is of the essential importance, since the further planed tests should be run in conditions which should be optimal for certain strain. Serious mistakes could appear if one investigates some phenomena with different fungi in, so called - standard laboratorial conditions and on temperature of 21 ±1ºC for all tested fungi. In practice, that means that some fungi should grow on their optimal temperature, while some of tested fungi should be partly inhibited by temperature of 21 ±1ºC, which is for example not the most convenient for their growth. Idea is that all tested fungi have to have optimal temperature in order to develop its` maximum metabolic activity, and just in that case one could compare results for different strains or series. The similar situation is also in the case of impact of ph value of substrate, but it is already well known that fungi are mostly capable to adjust the concentration of H ions in substrate towards their optimum, during metabolic process. So, one need not always calculate with negative impact of ph of substrate, except if it is so stabile that seems to behave as a buffer. Nutritional requirements should be investigated in liquid media as convenient one, for measuring of mycelial mass yielded. The appropriate quantities of tested sources of nutrients have to be calculated based on molecular and atomic weights of substances and elements, so that in all test series should be provided exactly the same concentration of nutritive elements. The same experiment should also be performed, but on solid media with agar in order to estimate the speed of growth in length. The results that one gains from liquid test should give estimation of fungal capability to develop its` metabolic activity, since harvested mass of mycelia has direct impact on metabolic strength, while the results from solid test should show how fast fungus could colonize the inner parts of wood in dependence of its` status of nutrients. This approach is very logical since it is well known that status of nutritive substances inside the wood is different in different season threw the year. Smart approach in investigation, which takes care of details in investigation methods, always provides useful and correct results if one runs the test correctly and according to prescribed procedure in advance. 1728
3. REFERENCES 1. Beever, D. J. (1969): Nutrition of Silver-leaf pathogen Stereum purpureum (Pers.) Fr. New Zealand Journal of Science, p. 268 275. 2. Karadžić, D. (1990): Uticaj taninske i galne kiseline na porast micelije nekih gljiva prouzrokovača truleži hrastovog drveta. Glasnik Šumarskog fakulteta, Beograd (1989/90), pp. 129-139. 3. Mirić, M., (1993): Bioekološka istraživanja najvažnijih gljiva iz roda Stereum izazivača truleži hrastovog drveta, Doktorska disertacija, Šumarski fakultet Univerziteta u Beogradu, Beograd. 4. Mirić, M., Schmidt, O. (1992): Ishrana nekih stereoidnih gljiva. Glasnik Šumarskog fakulteta br. 74. Univerzitet u Beogradu, Beograd. pp. 111 115. 5. Muntaňola Cvetković, M. (1987): Opšta mikologija, NIRO Književne novine, Beograd. 6. Rayner, A. D. M., Boddy, L. (1988): Fungal decomposition of Wood Its Biology and Ecology. A Wiley Interscience Publication, Avon. 7. Robinson, P. M. (1978): Practical Fungal Physiology. John Wiley and sons Ltd., Chichester New York Brisbane Toronto. 8. Schmidt, O., Kerner - Gang, N., (1986): Natural Materials (in: Biotechnology, Vol 8: H. J. Rehm and G. Reed - Edit.), Weinheim. 9. Schmidt, O, Liese, W. (1987): Biological Variations within Schizophyllum commune, Material und Organismen, 13. Bd., Heft 3. p. 169 185., Berlin. 1729