Mycosphere Doi 10.5943/mycosphere/2/6/4/ Physarella oblonga-centered bioassays for testing the biological activity of myxomycetes 1* 2 3 4 1 Herrera NA , Rojas C , Franco-Molano AE , Stephenson SL and Echeverri F 1 Laboratorio de Química Orgánica de Productos Naturales, SIU, Universidad de Antioquia, Medellín, Colombia 2 Instituto de Investigaciones en Ingeniería, Universidad de Costa Rica, San Pedro de Montes de Oca 11501-2060, Costa Rica 3 Laboratorio de Ecología y Taxonomía de Hongos, Universidad de Antioquia, Medellín, Colombia 4 Department of Biological Sciences, University of Arkansas, Fayetteville AR 72701-1201, USA Herrera NA, Rojas C, Franco-Molano AE, Stephenson SL, Echeverri F. 2011 – Physarella oblonga- centered bioassays for testing the biological activity of myxomycetes. Mycosphere 2 (6), 637–644, Doi 10.5943/mycosphere/2/6/4/ To study the trypanocidal, antibacterial, antifungal activity and cytotoxicity of myxomycetes, a rapid assessment focused on the species Physarella oblonga was carried out. Optimum conditions for culturing were utilized to develop a protocol that was adequate for bioanalysis of chemical compounds. Nuclear magnetic resonance (NMR) and mass spectroscopy (MS) detected the presence of stigmasterol and fatty acids in plasmodial extracts of Ph. oblonga through H1 analysis. These plasmodial extracts showed low toxicity and positive activity against epymastigote forms of Trypanosoma cruzi. This activity was significantly higher than the activity shown by one of the controls used. Similarly, the extracts from an unidentified species of myxomycete showed strong antimicrobial and antifungal activities against isolated strains of Bacillus cereus, Fusarium oxysporum and Rhizoctonia solani, whereas the myxomycete Physarum melleum displayed growth inhibition of the phytopathogen F. oxysporum. These results showed that with the use of an appropriate methodology, bioprospective analysis can be carried out on myxomycetes. In addition, this is apparently the first report on the antifungal and antiparasitic potential of myxomycetes. Key words – antibacterial activity – antifungal activity – Chagas disease – myxogastrids – secondary metabolites – slime molds Article Information Received 02 November 2011 Accepted 24 November 2011 Published online 26 December 2011 *Corresponding author: Carlos Rojas – e-mail – crojas@fungica.com Introduction referred to as a myxoamoeba or swarm cell, The myxomycetes, also known as slime depending on the presence or absence of a molds or myxogastrids, are a group of flagellum, and a macroscopic, multinucleate approximately 850 amoeboid protists (Lado structure known as a plasmodium (Martin & 2005-2011) that occur in all terrestrial Alexopoulos 1969). ecosystems but are poorly studied in some Myxomycetes are unique organisms regions of the world. Myxomycetes are heterot- from a biochemical point of view (Steglich rophic organisms that resemble certain micro- 1989). This is partially due to the presence of a scopic fungi and produce spores as their large group of secondary metabolites, some of primary dispersal mechanism (Stephenson & which have been isolated from several of the Stempen 1994). Their life cycle includes two more commonly cultured species. Among these different trophic stages, a uninucleate cell often metabolites, a number of fatty acid derivatives, 637 alkaloids, aromatic compounds, terpenoids and there is a need and responsibility to try new amides have been identified (Dembitsky et al. alternatives for its treatment. In this sense, an 2005). Interestingly, the derivates from fatty answer to the question as to whether or not acids are known for their antimicrobial activity, myxomycetes can be potential candidates for which exhibits a reversal effect of multidrug research against Chagas-like diseases is still resistance (Ishibashi et al. 2001, Misono et al. unknown, since these organisms have not yet 2003, Nakatani et al. 2005). These facts cause been studied in this way. However, as a myxomycetes to be a very interesting and inno- relatively new biological group for bioprospe- vative group to be analyzed from a biochemical cting research, myxomycetes provide a wide perspective. spectrum of possibilities due their unique Similarly, their secondary metabolic biochemistry. pathways produce compounds with unique For those reasons, the project described structures, and these compounds resemble a herein was designed with two main objectives. series of metabolites from both plants and The first was to rapidly assess the biological fungi. However, a standard procedure for activity and cytotoxicity of some myxomycete obtaining cultures of these microorganisms has plasmodia and their secondary metabolites, and not yet been standardized. Therefore, the the second was to develop a suitable protocol availability of these chemical compounds is for the production of large amounts of myxo- very low. In fact, most of the studies of spore mycete plasmodial mass. The ultimate goal of germination, culturing and fruiting body this project is to make available, at some point formation in myxomycetes were carried out in the future, the material required to evaluate more than 40 years ago (Gray 1949) and few, if active chemical compounds from myxomycetes any, studies on secondary metabolites are still that might act as bioactive agents against micr- being carried out in the Western Hemisphere. oorganisms. In spite of this, one group of myxomycetes that has been successfully cultu- Methods red is the order Physarales (Gray & This study was carried out in the Alexopoulos 1968, Lado et al. 2007). Some Laboratory of Organic Chemistry of Natural species in this group, such as Physarum Products at the University of Antioquia in polycephalum, have even become model Medellin, Colombia, during 2007 and 2008. organisms. However, most of the species in The morphological concept of species was used this group are still understudied at the to identify myxomycetes, following the laboratory level. One of the species in the taxonomic treatment of Lado (2005-2011). group, Physarella oblonga, has been cultured Plasmodia were first cultivated in moist for a number of different purposes (e.g. Franke chamber cultures following a modification of 1967, Bechtel 1975, Ribeiro et al. 2003) but the protocol provided by Stephenson & Stem- apparently not yet examined for the presence of pen (1994). potentially useful active substances. The same In all cases, plasmodia were placed on can be said for the majority of myxomycetes. filter paper (Whatman #1, 90 mm) in a This fact does not really represent a standard Petri dish (150 x 15 mm), and a small problem in relation to the search of alternative amount of distilled water was added to the cures for diseases, but it does represent a gap in dish. After approximately 2 weeks, small secti- a line of research with a potentially high social ons of the plasmodia were cut off and placed impact. For instance, diseases such as the on 1.6% water agar plates. The first fruiting American Trypanosomiasis, also known as bodies obtained in this study were deposited in Chagas disease, have been traditionally treated the herbarium of the University of Antioquia with potent chemicals, but it has been poorly (HUA). investigated with respect to the use of more For all bioassays, the myxomycete natural medicines (e.g. Ferraz et al. 2009). Physarella oblonga (Berk. & M.A. Curtis) Since more than 40 million people are at risk of Morgan was used. However, to test the being infected with this disease (Schofield antimicrobial activity, two more taxa, Physar- 2006) and its eradication has been difficult, um melleum (Berk. & Broome) Massee and an 638 Mycosphere unidentified species also were utilized. Cultur- chromatography using Merck silica gel 60, ing conditions were established for the first eluted with 1000 ml of a mixture of species and then applied to the other two dichloromethane:methanol (4:1 v/v). Fractions myxomycetes as a way of testing the viability were obtained and monitored using thin layer of the obtained protocol for generating an chromatography (TLC) on silica gel 60 chrom- adequate amount of plasmodial extracts for atoplates using dichloromethane:methanol 4:1 bioassay purposes. v/v. From the latter plates, candidate fractions were recovered and purified using subsequent Culturing TLC plates (dichloromethane:methanol 4:1 Seven days after the initial growth on v/v) and column separation, using Merck silica water agar, an evaluation of the best type of gel 60, eluted with 500 ml of a mixture of media and amount of food for the plasmodial hexane:ethyl acetate (3:1 v/v). cultivation of Ph. oblonga was carried out. For Isolated compounds were analyzed by the first part, three sections of plasmodia means of nuclear magnetic resonance (NMR) 2 approximately 1 cm in extent were transferred analyses, using a Bruker AMX at 300 MHz for onto sets of three plates prepared with eight 1H and 75 MHz for 13C. In addition, mass different types of media, for a total of 24 spectroscopy (MS) analyses were performed on different cultures. The media types evaluated isolated compounds in order to evaluate the were 1.6% water agar, oatmeal agar, malt chemical composition and possible identity of extract agar, potato dextrose agar (PDA), the metabolites. oxytetracycline glucose yeast agar (OGY), sabouraud agar, mixed-bark infusion agar and a Cytotoxicity mushroom (Flammulina velutipes) infusion For this essay, Jurkat Lat GFP cells agar. were used. Approximately 500,000 cells were To evaluate the amount of food deposited in individual 1 ml wells on a 24-well necessary to obtain the largest plasmodia, four plate. Samples were activated with a concentra- quantities (0.1, 0.2, 0.5 and 1 g) of oatmeal tion of 100 µg/ml from the fractions of flakes previously autoclaved at 121ºC and 15 plasmodial extracts of Ph. oblonga, a lb/atmospheric pressure for 20 minutes, were polyunsaturated fatty acid and a yellow used in bark infusion agar plates. A set of three compound from Ph. oblonga plasmodia replicates was prepared for each of the four recovered after chromatrography. A negative quantities, for a total of 12 different cultures. control was also used. Samples were incubated The inoculated plasmodia on bark infusion agar for 12 hours at 37 ºC in an atmosphere of 5% were subjected to temperature of 26°C. For CO2. The liquid in each well was transferred to extraction of secondary metabolites, an additi- a microcentrifuge tube in which propidium onal experiment was carried out to determine iodide at a concentration of 1 μl/100 μl was the kinetic plasmodium growth. During six added. Immediately after this, samples were consecutive days, the weight of the plasmodial analyzed using a flow cytometer by collecting mass was determined on three cultures per day ten thousand events per sample (see Zaritskaya after an initial inoculation of eighteen plates of et al. 2010). bark infusion agar with plasmodia, following the best combination of parameters as determi- Trypanocidal activity ned from the previous experiments. The activity against Trypanosoma cruzi was evaluated by first cultivating a series of Extraction of chemical compounds samples corresponding to the epimastigote For Ph. oblonga, the chemical forms of the Tulahen strain of the parasite at extraction of secondary metabolites was carried 26˚C in a liver infusion-tryptose medium out by starting with a mixture consisting of 1.0 supplemented with 5% of heat-inactivated fetal L of 95% ethanol and approximately 50 g of bovine serum. The parasites in logarithmic fresh plasmodial mass. This step was followed growing phase were distributed by triplicate in by filtration and evaporation of the solvent and 96-flat bottom-well microplates at a concentra- 4 fractioning of the crude extract (5 g) by column tion of 5 x 10 cells/ml. Each well was exposed 639 to different concentrations of crude plasmodial data structure. On the other hand, the cytotox- extract and two fractions isolated from crude icity and trypanocidal activity were tested extract of Ph. oblonga at a concentration of using analysis of variance due the more norm- 100, 50 and 25 g/ml, respectively, for a period ally structured nature of the datasets. In the of 72 hours. latter instances, effect-size correlation values The anti-trypanosome activity was were also calculated to test variability due to determined by optical count, using an inverted biological variation (population-based differe- microscope. For this, the reference drugs nces). In all cases, the probability value associ- utilized correspond to Benznidazole and ated with the rejection of the null hypothesis Amphotericin B at a concentration of 10 μg/ml. was 0.05 and all tests were performed using the The results of the antiparasite activity are exp- software JPM, version 7.0.2. (SAS 2007). ressed in terms of IC50, the drug concentration that inhibits 50% of the development in the Results parasite. These values were obtained by As result of the experiments described creating a dose-response curve which corresp- above, a successful protocol for the laboratory onds to the logarithm of the drug concentration cultivation of myxomycete vegetative (plasmo- versus the percentage of inhibition. dial) mass was obtained. This was based on the performance of Ph. oblonga plasmodial gro- Determination of antimicrobial activity wth, which was observed to take place only on For these series of assays, three species water agar, mushroom infusion agar and bark of myxomycetes—Ph. oblonga, P. melleum infusion agar. Five days after inoculation the and one unidentified species—were used to test amounts of fresh plasmodial mass for the three biological activity of the group on pathogenic positive media types were 0.041, 0.312 and microorganisms (see Table 1). The cultures of 0.980 g, respectively. The ranks of plasmodial these organisms were obtained from the growth were significantly different on both Microbiology laboratory at the Universidad de water agar and mushroom infusion agar than Antioquia. The inhibitory effects of the extracts on bark infusion agar (H=7.20, d.f.=2, P<0.05). produced were tested by performing a disc Based on these results, it was determined that diffusion method (Thitilertdecha et al. 2008). bark infusion agar was a suitable medium for For this, 50 µl of sample solutions at a the cultivation of Ph. oblonga. In a similar concentration of 0.5 mg/ml (dry residue/vo- way, the largest plasmodial fresh weight rank lume of dimethyl sulfoxide, DMSO) were used. differences were obtained by using the smallest Three repeats were tested in every inhibition amount of food (H=8.33, d.f.=3, P<0.05). experiment. The magnitude of the antimicro- The average fresh weight obtained for bial action was assessed by measuring the each day was determined to be 0.039, 0.186, diameter (in mm) of the inhibition zones, in 0.528, 0.932, 0.856 and 0.715 g from the first relation to those of positive and negative to the sixth day. There was a progressive controls. The latter were co-assayed by using increase in plasmodial mass until the fourth 10 μg of Gentamicine and 1g/L of Benomil as day, after which the plasmodia seemed to positive antimicrobial references and plain decrease in mass. Interestingly, the fresh DMSO as negative control. weight of the last three days was not significantly different (H=5.68, d.f.=2, P>0.05) Statistical analysis in spite of obvious differences for the entire In order to evaluate the performance of period of time (H=15.9, d.f.=5, P <0.05). plasmodial growth under the different culturing Chromatographic fractioning, NMR and conditions tested, a series of Kruskal-Wallis MS analyses allowed the isolation of a large tests was carried out on the respective data. number of triglycerides and several polyunsa- This non-parametric test uses ranks to evaluate turated fatty acids. However, only one comp- the independence of three or more groups that ound (stigmasterol) could be identified succe- do not necessarily meet the normality ssfully using these techniques. The cytotoxi- requirements of similar tests for heterogeneous city test showed that both of the plasmodial 640 Mycosphere Table 1 Antimicrobial activity of crude extracts and fractions from myxomycetes and positive and negative controls. Sample type Organism used Bs Sa Ec Fo Rs Bc Crude extract Physarella oblonga - - - - - - Crude extract Physarum melleum - - - ++ +++ - Crude extract Unidentified +++ - - +++ +++ - Fraction 1 - - - - - - Fraction 4 ++ - - - - - Gentamicine (0.1 mg/ml) +++ +++ +++ - - - Benomil (1 mg/ml) - - - +++ - +++ DMSO (50 µl) - - - - - - Symbols: – no activity, + inhibition zone less than 10 mm, ++ inhibition zone 10-20 mm, +++ inhibition zone greater than 20 mm. Abbreviations: Bs = Bacillus subtilis, Sa = Staphylococcus aureus, Ec = Escherichia coli, Fo = Fusarium oxysporum, Rs = Rhizoctonia solani, Bc = Botrytis cinerea. fractions isolated from the crude extract from case of both antibacterial and antifungal plasmodia of Ph.oblonga have low toxicity activity, the positive controls showed a strong when compared to the negative control (Table effect on the target organisms. Interestingly, 2) at a concentration of 100 µg/ml. The highest the strain of R. solani used in this study seemed percentage of inhibition was observed for one to be very resistant to the different antifungal of the plasmodial fractions, designated as agents used, except in the case of plasmodial number two, at 22.7%. In spite of the latter, the extracts from P. melleum and the unidentified cytotoxicity observed for fractions and extracts species. from Ph. oblonga plasmodia is not signifi- cantly different from the control (F(1,5)=3.07, Discussion 2 P=0.14, R =0.38). The production of fruiting bodies by The trypanocidal activity of Ph. Ph. oblonga only in water agar, mushroom oblonga is shown in Table 3. Both the crude infusion agar and bark infusion agar is not extract and the two fractions evaluated showed surprising. It is well known that myxomycete strong differences in the activity against the spores germinate when water is available parasite when compared to the Amphotericin B (Stephenson & Stempen 1994). Also, bark control group (for experimental versus control represents one of the best substrates on which 2 only, F(1,2)=1406, P=0.0007, R = 0.99) but no myxomycetes can grow (see Ing 1994) under differences when Benznidazole was considered natural conditions. This is probably related to 2 (F(1,3)=0.50, P=0.52, R =0.14). the chemical composition of bark from In the assays carried out on the different tree species and also to the capacity of antimicrobial activity (Table 3), the bacterium different types of bark to retain air-borne B. cereus was inhibited by the extract of the myxomycete spores. As such, it is very likely unidentified myxomycete. The diameter of the that some myxomycetes are well adapted to inhibition zone was about 20 mm at 500 g/ml. this corticolous environment and exploit its None of the other combinations of microresources efficiently. If this is the case, myxomycetes and bacteria tested yielded the Physarales, the order that contains Ph. positive results. On the other hand, when the oblonga, is likely to be one example of this antifungal activity was tested, both crude type of situation, as can be observed in its high extracts from P. melleum and the unidentified degree of specificity for bark (see Everhart & species showed growth inhibition of F. Keller 2008). Even though mushrooms are also oxysporum, although the inhibition zone of the known as a substrate for myxomycetes, the latter was larger than that of P. melleum. In the relationship does not seem to be that intricate 641 Table 2 Percentage of inhibition of Jurkat Lat GF cells shown by different fractions extracted from the myxomycete Physarella oblonga. Fraction Inhibition (%) Polyunsaturated fatty acid 22.7 Unidentified compound 12.4 Fraction 1 15.2 Fraction 2 27.0 Fraction 3 11.6 Fraction 4 16.7 Negative control 6.1 (Ing 1994). The fact that fatty acids and Similarly, Lycogala epidendrum has been stigmasterol were successfully isolated from reported to produce fatty acids that showed myxomycete plasmodia provides some inhibitory activities toward the growth of gram- evidence that these organisms can be used for positive bacteria (Rezanka & Dvorakova biosprospecting. Even though these types of 2003). What makes bioprospecting in metabolites already have been reported from myxomycetes more interesting is that it has myxomycete plasmodia (Dembitsky et al. been suggested that fatty acids can be used for 2005), this is the first time that stigmasterol has other important applications. For example, it been reported for Ph. oblonga. This compound seems that these compounds have a high has been found in other members of the potential for use against malaria, tuberculosis Physarales such as Physarum polycephalum and fungal diseases (Carballeira 2008). and Physarum flavicomum (Bullock & Dawson Interestingly, even though there are no studies 1976). The problem is that these sterols are of trypanocidal activity in any of these very common in plants as well (e.g. Shibuya compounds, the results obtained in the present 2001), which makes the evaluation of the investigation provide an alternative future nature of this compound difficult in the case of approach to study Chagas disease as well. the present study. Whether this compound is This alternative is of course highly being directly produced by the myxomycete or dependent on the properties of the system actively taken up from the woody substrates is analyzed. For example, whether or not a a question that warrants additional study. For system shows toxicity against human cells is example, stigmasterol is known to occur in oat something critical to be determined. In the plants, but it also has been observed in present study, all the cytotoxicity tests showed examples of the Physarales cultured without that plasmodial extracts from Ph. oblonga are this as a food source (Bullock 1976). It is likely highly innocuous. This is an important aspect that the metabolic pathway used by to consider in biochemical systems when myxomycetes to produce this sterol is very potential medical uses are studied. It has been similar to the one found in plants (e.g. Schaller known for a number of years that 2004), but the true nature of the compound in myxomycetes do not have an adverse effect on the cultures we studied remains uncertain for humans (see Stephenson & Stempen 1994), but now. their properties at the cellular level have not On the other hand, even though been studied completely. The low levels of polyunsaturated fatty acids are known to cytotoxicity observed in this investigation are possess antibacterial activity (e.g. McGaw et important since they support the idea that this al. 2002), it is currently impossible to group of non-toxic organisms can be used for determine whether or not some of the bioprospecting research. compounds extracted from Ph. oblonga have The results from the trypanocidal such properties. However, it seems possible activity tests support the latter as well. Even that this is indeed the case. For example, though they do not show differences when the Chiappeta et al. (1999) reported antibacterial experimental treatments are compared to activity in Fuligo septica, a species that is Benznidazole, the significant differences with phylogenetically related to Ph. oblonga. Amphotericin B provide evidence for the 642 Mycosphere Table 3 Trypanocide activity of the crude extract and fractions from Physarella oblonga plasmodium on epimastigotes of Trypanosoma cruzi. Sample type Compound IC50- µg/ml Crude extract NH-MX-A 7.5 ± 0.14 Fraction 1 NH-MX-1A 7.8 ± 0.14 Fraction 2 NH-MX-2A 7.8 ± 0.07 Reference Amphotericin B 0.2 Reference Benznidazole 10 potential use of myxomycete plasmodia against tools. At the same time, the evaluation of T. cruzi. Of course, developing a more antimicrobial activity displayed by two other comprehensive research protocol is necessary species of myxomycetes provides an indication to evaluate this effect more carefully. However, of the potential use of the group. the indication of a possible positive effect against the parasite as shown herein is Acknowledgements important, since myxomycetes have not been We wish to thank the laboratory for reported previously to be active against any Eumycetozoan Research at the University of type of human parasite. Also, in the fight Arkansas for their collaboration during the against this disease, any new empirical data research process and the Universidad de supporting potential new lines of research are Antioquia (Colombia) for financial support. helpful for setting the stage for future investigations. References The unidentified myxomycete showed a Bechtel DB, Horner HT. 1975 – Calcium great inhibitory activity against some of the excretion and deposition during sporog- organisms tested. Interestingly, in a manner enesis in Physarella oblongga. similar to one of the other species, it seems that Calcified Tissue Research 18, 195-213. the crude extract is more bioactive than the Bullock E, Dawson CJ. 1976 – Sterol content fractioned one. The low level of biological of the Myxomycetes Physarum polyc- activity against bacteria and fungi in the case of ephalum and P. flavicomum. Journal of Ph. oblonga contrasts dramatically with the Lipid Research 17, 565-571. high level of activity against T. cruzi. However, Carballeira NM 2008 – New advances in fatty in the present study, it was not possible to acids as antimalarial, antimycobacterial evaluate this aspect completely, since a more and antifungal agents. Progress in Lipid detailed approached would have to have been Research 47, 50-61. followed. In any case, what seems likely is that Chiappeta A, de Sena KXFR, Nascimento SC, distinct species of myxomycetes have different Rocha CS, Cavalcanti L. 1999 – types and levels of biological activity, which Influence of pH on the extraction of may account for their individual strategies to bioactive substances of Fuligo septica. cope with different biotic elements of their Phyton (Buenos Aires) 65, 7-11. environment. Dembitsky VM, Řezanka T, Spížek J, Hanus It is evident that future studies are LO. 2005 – Secondary metabolites of necessary to provide the evidence required to slime molds ( myxomycetes). fill in the information gaps that exist today. In Phytochemistry 66, 747-769. spite of this, the present investigation Everhart SE, Keller HW. 2008 – Life history represents a first step in the study of one strategies of corticolous myxomycetes: additional group of non-traditional organisms the life cycle, plasmodial types, fruit- with potential applications. The successful ing bodies, and taxonomic orders. evaluation of culturing conditions and the Fungal Diversity 29, 1-16. isolation, using NMR, of some secondary Ferraz M, Gazzinelli R, Alvez R, Urbina J, metabolites in Ph. oblonga represents a step Romanha A. 2009 – Absence of CD4 T forward in the bioprospecting study of lymphocytes, CD8 T lymphocytes, or myxomycetes with the use of modern research B lymphocytes has different effects on 643 the efficacy of Posaconazole and Journal of Natural Products 66, 999- Benznidazole in treatment of experime- 1001. ntal acute Trypanosoma cruzi infection. Nakatani S, Kamata K, Sato M, Onuki H, Antimicrobial Agents and Hirota H, Matsumoto J, Ishibashi M. Chemotherapy 53, 174-179. 2005 – Melleumin A, a novel Franke RG. 1967 – Preliminary investigation peptide lactone isolated from the of the double-diffusion technique as a cultured myxomycete Physarum mell- tool in determining relationships amo- eum. Tetrahedron Letters 46, 267- ng some myxomycetes, order Physara- 271. les. American Journal of Botany 54, Rezanka T, Dvorakova R. 2003 – 1189-1197. Polypropionate lactones of deoxysugars Gray WD 1949 – The laboratory cultivation glycosides from slime mold Lyc- and development of the myxomycetes ogala epidendrum. Phytochemistry 63, Physarella oblonga and Physarum 945-952. didermoides. Ohio Journal of Science Ribeiro SM, Cavalcanti LH, Pereira EC, Silva 49, 105-108. NH. 2003 – Plasmodium immobi- Gray WD, Alexopoulos CJ. 1968 – Biology of lization of Physarella oblonga (Berk. & the myxomycetes. The Ronald Press Curt.) Morgan (Myxomycetes) using Company, New York. kaolinite as a matrix of entrapment. Ing B 1994 – The phytosociology of Research in Microbiology 154, 55-58. myxomycetes. New Phytologist 126, SAS 2007 – JMP Statistical Discovery 175-201. Software. Statistical Analysis System Ishibashi M, Iwasaki T, Imai S, Sakamoto S, (SAS) Institute, Cary, North Carolina. Yamaguchi K, Itos A. 2001– Schaller H 2004 – New aspects of sterol Laboratory culture of the myxomy- biosynthesis in growth and cetes: formation of fruiting bodies of development of higher plants. Plant Didymium bahiense and its plasmodial Physiology and Biochemistry 42, 465- production of makaluvamine A. Journal 476. of Natural Products 64, 108-110. Schofield CJ, Jannin J, Salvatella R. 2006 – Lado C 2005-2011 – An on line nomenclatural The future of Chagas disease control. information system of Eumycetozoa. Trends in Parasitology 22, 583-588. Real Jardín Botánico de Madrid, Shibuya M 2001 – Biosynthesis of sterols and Spain. www.nomen.eumycetozoa.com triterpenes in higher plants. Natural Lado C, Mosquera J, Estrada-Torres A, Medicines 55, 1-6. Beltrán-Tejera E, Wrigley de Basanta Steglich W 1989 – Slime moulds D. 2007 – Description and culture of a (Myxomycetes) as a source of new new succulenticolous Didymium biologically active metabolites. Pure (Myxomycetes). Mycologia 99, 602- and Applied Chemistry 61, 281-288. 611. Stephenson SL, Stempen H. 1994– Martin GW, Alexopoulos CJ. 1969 – The Myxomycetes: a handbook of slime Myxomycetes. University of Iowa molds. Timber Press, Oregon. Press, Iowa City. Thitilertdecha N, Teerawutgulrag A, McGaw LJ, Jager AK, van Staden J 2002 – Rakariyatham N. 2008 – Antioxidant Antibacterial effects of fatty acids and and antibacterial activities of related compounds from plants. South Nephelium lappaceum L. extracts. LWT African Journal of Botany 68, 417-423. Food Science and Technology 41, Misono Y, Ishikawa Y, Yamamoto Y, Hayashi 2029-2035. M, Komiyama K, Ishibashi M. 2003 – Zaritskaya L, Shurin MR, Sayers TJ, Dihydrolindbladiones, three new Malyguine AM. 2010 – New flow naphthoquinone pigments from a cytometric assays for monitoring cell- myxomycete Lindbladia tubulina. mediated cytotoxicity. Expert reviews of vaccines 9, 601-616. 644