Fungal Diversity An integrated approach to taxonomical identification of the novel filamentous fungus strain producing extracellular lipases: morphological, physiological and DNA fingerprinting techniques Konstantin A. Lusta1, Galina A. Kochkina1, Ill Whan Sul2, Il Kyung Chung3, Hee Sung Park3 and Dongill Shin3* 1Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino Biological Center, Moscow Region, Russia 2Daegu Polytech College, Daegu 706-020, Republic of Korea and 3Faculty of Life Resources, Catholic University of Daegu, Hayang, Kyungsan, Kyungpook, 712-702, Republic of Korea Lusta, K.A., Kochkina, G.A., Sul, I.W., Chung, I.K., Park, H.S. and Shin, D. (2003). An integrated approach to taxonomical identification of the novel filamentous fungus strain producing extracellular lipases: morphological, physiological and DNA fingerprinting techniques. Fungal Diversity 12: 135-149. A new filamentous fungal strain was isolated into pure culture and initially named as strain L- 1. The strain was found to secrete a high level of extracellular lipase at high temperatures. The identification of the isolate was performed by the combination of conventional morphological- physiological methods, scanning electron microscopy and RAPD. Phenotypic and genotypic characteristics of the L-1 strain were compared to reference strains. The morphological characteristics, radial growth rate at different temperatures and surface ornamentation of sporangiospores of the isolate almost completely match the reference strain Rhizopus [= microsporus var. rhizopodiformis VKM F-3693. The strain L-1 was characterized by high growth rate and the spore maturation abilities at 50°C. These characteristics are unique among all other strains of Rhizopus. The results of RAPD-diagnosis indicate the high degree of genetic similarity between strains L-1 and F-3693. We therefore identified strain L-1 as Rhizopus microsporus var. rhizopodiformis. The strain has been submitted and included in the All- Russian Collection of Microorganisms as VKM F-3688. Key words: fungal taxonomy, identification, lipase, RAPD, Rhizopus microsporus-group, SEM. Introduction The extent of biological diversity has generated the need to isolate and culture the large numbers of microorganisms that remain to be studied. The number of fungal species presently described is only a small proportion of those that actually exist (Hawksworth, 1991, 1997, 2001). Accordingly the * Corresponding author: D. Shin; e-mail: [email protected] 135 isolation and correct identification of fungi is of great practical importance. The fungi of Rhizopus genus are widespread in nature, and many species of this genus have significant industrial applications, particularly the production of lipases (Iwai and Tsujisaka, 1984). For the efficient lipase production and functioning, it is strongly desirable to have thermophilic producers and thermostable lipolytic systems. Previously we reported the isolation of a new thermophilic lipase-producing strain (Lusta et al., 1999). Here we discuss the identification of this isolate. Different concepts have been used to define the fungal species. The phenotypic concept is the classic approach based on the morphological characteristics (Inui et al., 1965). The polythetic concept is based on a combination of characters (Guarro et al., 1999). Identifications based on colour and physical appearance of growing colonies and microscopic detail of morphological structures in some fungi may be equivocal because the shapes and sizes of different fungal organs are variable (Schipper and Stalpers, 1984). Current methods to identify Zygomycetes are incorporate sporangiospore topography. SEM is capable of allowing the surface ornamentation of spores to be defined and these characteristics appeared to be distinctive for individual species (Ellis, 1981). A variety of biochemical and physiological methods for fungal identification have been also devised (Kohn, 1992; Carlile and Watkinson, 1994; Guarro et al., 1999). These alternative approaches to identifying fungi are labourious, time-consuming and provide insufficient taxonomic resolution. The major disadvantages are that all the assays based on phenotypes are too sensitive to growth conditions and depend on gene expression (De Bernardis et al., 1998). These problems may be obviated by adopting DNA-based methods. The basic DNA sequences of an organism are insensitive to short-term environmental change and thus should provide a more stable alternative for strain identification (Kohn, 1992; Weising et al., 1995; Liew et al., 1998; Soll, 2000). Methods that directly detect DNA differences among species and strains are referred to as DNA fingerprinting methods (Soll, 2000). One approach uses a random amplified polymorphic DNA (RAPD) technique to generate markers for any specific genome (Williams et al., 1990). This RAPD assay is genotypic method based on PCR technique in combination with single short arbitrary primers and requires no post-amplification sample manipulation (Poonwan et al., 1998). A single random primer hybridizes to homologous sequences in the genome, and DNA region between the two hybridization sites will be amplified using Taq polymerase. Each random primer gives a different pattern of PCR products (Brandt et al., 1998). The data 136 Fungal Diversity generated by RAPD can be used to determine the degree of genetic relatedness between different strains on the basis of similarity coefficients (Nei and Li, 1987; Soll, 2000). The present paper describes the identification and species assignment of a new fungal isolate that makes use of several different methods: traditional morphological-physiological techniques, SEM and the PCR-based RAPD. In this study, twenty random primers were tested to identify individual fingerprints by PCR. The results of the analysis performed on the new isolate, strain L-1, by conventional and RAPD methods are compared with those of several reference strains from Aspergillus and Rhizopus and particularly the Rhizopus microsporus-group. Materials and methods Microorganisms The following reference strains have been used for comparison by morphological-physiological techniques or RAPD: Rhizopus microsporus var. rhizopodiformis VKM F-3692 (= CBS 343.29); R. microsporus var. rhizopodiformis VKM F-3693 (= CBS 607.73); R. microsporus var. chinensis VKM F-1218, MT- (= ATCC 1227b); R. microsporus var. chinensis VKM F- 1360, MT- (= CBS 262.28; ATCC 52812); R. microsporus var. chinensis VKM F-1361, MT+ (= CBS 261.28; ATCC 52811; DSM 2193); R. microsporus var. microsporus VKM F-773, MT+ (= CBS 699.68; ATCC 52813; CCRC 31140); R. microsporus var. microsporus VKM F-774, MT- (= CBS 700.68; ATCC 52814; CCRC 31141; CCF 1570); R. microsporus var. microsporus VKM F- 1063; R. stolonifer var. stolonifer VKM F-601; R. oryzae VKM F-605 and Aspergillus terreus VKM F-3687. Strains were obtained from the All-Russian Collection of Microorganisms at the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences. Strain L-1 was isolated from soil. Strain isolation The combined technique of dilution plating and direct isolation were used for fungal strain isolation into pure culture on selective media. A mold growing in the soil around a hot spring in Ashkhabad City (Turkmenistan) was maintained in a moist chamber with the soil until sporulation. Spores were dispersed in sterile water and after following serial dilutions were inoculated into selective liquid medium: 0.3% (w/v) (NH ) SO and 0.1% (w/v) CaCl 4 2 4 2 which contained 1% (v/v) of olive oil as the sole carbon and energy source (Lusta et al., 1999). Cultures were grown on a shaking incubator at 40°C, over 137 two days, and then transferred evenly onto the surface of malt agar medium (MEA). After maturation of the spores (~ four days at 40°C), spores from a particular fungus were transferred to sterile culture medium using a stereomicroscope and an inoculating sterile needle. Isolated strains were maintained on slants of MEA medium and stored at 4°C. Strain cultivation and description The MEA used as the basal medium for morphological and growth studies was contained in 90 mm Petri dishes (20 ml in each plate). Growth/temperature relationships were established as the average radial growth rate (cm/h) of the fungal strain. Petri dishes with nutrient medium were inoculated with equal amounts of spore suspension centrally and were cultivated at different temperatures. Measurements of colony diam. were made after 4, 24, 48 and 120 hours (Kochkina et al., 1978). The length and colour of the sporangiospores and rhizoids, and sizes of sporangia, columellae, and sporangiospores were determined using light microscopy (magnification ×160, ×400 and ×640). Electron microscopy techniques For SEM (Goldstein et al., 1992) the specimens were fixed in glutaraldehyde-paraformaldehide and then in OsO solutions, dehydrated in 4 ethanol and dried using a Critical Point Dryer BAL-TEC CPD 030 (Switzerland). Gold sputtering was performed using BAL-TEC SCD 050 Sputter Coater (Switzerland). Specimens were examined and photographed with Zeiss Scanning Electron Microscope DSM 962 (Germany). DNA preparation Two gram (wet weight) of fresh mycelium of each strain was frozen with liquid nitrogen and then ground with a -20°C cooled mortar and pestle. The powder (0.4 ml) was dispersed in 0.6 ml of extraction buffer (Tris HCl 100 mM, Na EDTA 50 mM, NaCl 700 mM, Na (SO ) 100 mM, 1% (w/v) SDS, 2 2 3 pH 7.5) and heated to 65°C for 20 minutes. Then 0.6 ml of chloroform/isoamyl alcohol was added and incubated on ice for 30 minutes. After centrifugation at 12000 × g for 20 minutes the aqueous phase was mixed with an equal volume of isopropanol. The mixture was incubated at room temperature for 10 minutes and then centrifuged at 2000 × g for 5 minutes. The pellet was air dried, resuspended in 200 µl of double distilled water, incubated at 37°C for 15 minutes, mixed with 100 µl of ammonium acetate and incubated on ice for 1 hour. Following centrifugation at 12000 × g for 20 minutes the supernatant was mixed with 0.54 volumes of isopropanol, incubated at room temperature for 10 138 Fungal Diversity minutes and recovered by centrifugation at 12000 × g for 5 minutes. The pellets were rinsed in 500 µl of 70% ethanol prior to air drying. The DNA pellets were then resuspended in 100 µl of TE buffer (Tris-HCl 10 mM, Na EDTA 1 mM), pH 7.5 containing of 100 µg ml-1 of RNase A, incubated at 2 37°C for 60 minutes, examined for DNA concentration using 0.8% agarose gel electrophoresis and then stored at -20°C until use. RAPD assay Amplification reactions were carried out in 25 µl volume containing KCl 50 mM, Tris-HCl 10 mM (pH 9.0), MgCl 15 mM and 0.1% (v/v) TritonX-100, 2 200 µM each of dNTPs, 25 pmole of each random primer (Table 1), 1.5 U of Taq DNA polymerase and 50 ng of template DNA. The reaction mixture was overlaid with approximately 40 µl of mineral oil. Amplifications were performed by Minicycler (MJ Research). The temperature cylces were 95°C for 5 minutes, followed by 40 cycles of 40 seconds at 94°C, 40 seconds at 36°C and 1 minute at 74°C with a final extension of 5 minutes at 74°C. The amplified products were separated by electrophoresis on the 0.8% agarose gel in 1 × TBE buffer at 10 V cm-1 for 90 minutes and visualized by staining with ethidium bromide on an uv transilluminator. Analysis of RAPD data Amplified DNA fragments, reproducible in three reactions, were scored by the binary values on two possible character states 0 and 1. Estimation of genetic relationships between all pairs of strains were performed using Jaccard’s coefficients calculated from the following formula (Nei and Li, 1987): S = 2C /(U + U + 2C ) j xy x y xy in which C is the number of bands common in lanes x and y, and U and U xy x y represent the number of unique bands in each sample. The total number of mismatches is U + U . S values range from 0 to 1. A measure of 0 reflects no x y j common bands and lowest degree of genetic similarity indicated, while a measure of 1 reflects all common bands and highest degree of genetic similarity. Measures of 0.01 to 0.99 represent increasing degrees of commonness (Soll, 2000). Results Morphological identification Strain L-1 formed very fast growing dark-grayish colonies, which became slightly brown at higher temperatures. Hypha were not septate. The 139 Table 1. RAPD primer sequences used for the experiments (OPERON company, kit F). Primer code Seqeunces Primer code Seqeunces F01 ACGGATCCTG F11 TTGGTACCCC F02 GAGGATCCCT F12 ACGGTACCAG F03 CCTGATCACC F13 GGCTGCAGAA F04 GGTGATCAGG F14 TGCTGCAGGT F05 CCGAATTCCC F15 CCAGTACTCC F06 GGGAATTCGG F16 GGAGTACTGG F07 CCGATATCCC F17 AACCCGGGAA F08 GGGATATCGG F18 TTCCCGGGTT F09 CCAAGCTTCC F19 CCTCTAGACC F10 GGAAGCTTGG F20 GGTCTAGAGG Table 2. Comparative average data on the dimensions of different organs (diagnostic characters) in various Rhizopus isolates. Strains Diagnostic characters Sporangia, µm Columellae, µm Sporangiospores, µm VKM F-1218 (63.8-)80.2(-89.9) Pyriform, Globose,-ellipsoidal, (52.2-)60.2(-72.5) × (4.6-)4.8(-5.4) × (46.4-)52.3(-60.9) (3.6-)4.1(-4.6) VKM F-1063 (72.5-)90.3(-107.3) Subglobose, Broadly- ellipsoidal, (52.2-)77.9(-95.7) × (5.1-)5.5(-6.0) × (49.3-)68.1(-78.3) (3.5-)3.9(-4.4) VKM F-773 (72.5-)87.9(-104.4) Subglobose, Broadly-ellipsoidal, (46.4-)55.2(-69.6) × (4.2-)4.9(-5.6) × (37.7-)45.0(-49.3) (2.4-)2.8(-3.0) VKM F-3693 (55.5-)76.4(-92.5) Ellipsoidal, Globose, subglobose, (51.8-)66.2(-81.4) × (3.8-)4.1(-4.7) × (44.4-)57.6(-66.6) (3.0-)3.3(-3.6) L-1 (55.0-)78.2(-100.5) Subglobose, ellipsoidal, Globose, subglobose, (49.3-)70.2(-81.2) × (3.9-)4.2(-4.8) × (46.4-)57.9(-72.5) (3.0-)3.3(-3.6) strain was characterized as bearing stolons and rhizoids, forming globular sporangia with columellae and apophyses. Unbranched sporangiophores were borne on stolons singularly or in clusters. They were straight or slightly curved, with smooth walls, and ranged from about 400 to 700 µm in height and 5-8 µm diam. Grayish-black sporangia were globular. Cylindrical sporangiophores were slightly and gradually swollen near the columellae to form funnel-shaped apophysis of about 10-25 µm diam. Comparative data on different organs in strain L-1 and reference Rhizopus microsporus-group strains are presented in Table 2. 140 Fungal Diversity Figs. 1-8. Scanning electron micrographs of sporangiospores of different Rhizopus microsporus strains: 1. Rhizopus sp. L-1. 2. R. microsporus var. rhizopodiformis VKM F-3693. 3. R. microsporus var. chinensis VKM F-1218. 4. R. microsporus var. microsporus VKM F- 1063. 5. R. microsporus var. microsporus VKM F-773. 6. R. microsporus var. microsporus VKM F-774. 7. R. microsporus var. chinensis VKM F-1360. 8. R. microsporus var. chinensis VKM F-1361. Spores of both strains (VKM F-1360 and 1361) are ellipsoidal and striated ranging from 3.5 to 6 µm. Bars = 1 µm. 141 Table 3. Radial growth rate (cm/h) of Rhizopus microsporus strains at different temperatures. Temperature Strains (°C) VKM F-1218 VKM F-1063 VKM F-773 VKM F-3693 L-1 8 – – – – – 16 0.025+0.005 0.033+0.003 0.010+0.003 0.016+0.004 0.028+0.003 20 0.032+0.007 0.062+0.009 0.056+0.008 0.057+0.007 0.045+0.005 26 0.063+0.009 0.062+0.009 0.083+0.008 0.087+0.011 0.054+0.005 30 0.120+0.014 0.125+0.019 0.100+0.010 0.125+0.013 0.131+0.0015 37 0.138+0.022 0.162+0.024 0.137+0.014 0.138+0.018 0.136+0.008 45 0.089+0.011 0.098+0.009 0.130+0.010 0.156+0.018 0.150+0.010 50 – 0.001+0.001 – 0.034+0.009 0.067+0.007 52 – – – – – –: No growth. Morphological parameters of strain L-1 were dependent on growth temperature. As temperatures increased the mycelium becomes more closely adhered to the agar surface and all asexual reproductive structures were smaller. For instance, the average size of sporangium reduced by 26% at a temperature of 45°C and by 39% at 50°C, as compared to those grown at 20- 26°C. At higher temperatures the size and shape of the columellae changed; these became smaller (30-40%) and more globose without collars. It is important to emphasize that strain Rhizopus sp. L-1 did not to produce zygospores in the contact with any strains from the Rhizopus microsporus-group tested. The morphological characteristics of Rhizopus microsporus var. rhizopodiformis VKM F-3693 reference strain, were mostly close to strain L-1, and are as follows: colonies were dark-grayish; rhizoids were simple and unbranched; sporangiophores varied from 200-600 µm high, 7-11 µm wide. Sporangia were grayish-black. Characteristics of sporangiospores Strain L-1 has one-celled black, globose to slightly elliptical spores, which have distinctive surface warts or spines (Fig. 1). Comparative average data on shape and size of strain L-1 spores and some reference strain spores are presented at Table 2. Ultrastructural characteristics of these spores obtained by SEM are shown in Figs. 1-8. Spores of strain L-1 are very similar to that of the VKM F-3693 reference strain (Fig. 2), but are distinctly different from spores of the other strains examined (Figs. 3-8). These features provide convincing evidence that strain L-1 is Rhizopus microsporus var. rhizopodiformis. 142 Fungal Diversity 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Fig. 9. Comparative fingerprinting of L-1 strain and Aspergillus terreus VKM F-3687 DNAs obtained by RAPD with ten different primers (F01- F10). Lanes 1 and 22: marker (Promega’s 1kb DNA Ladder); Lane 2: VKM F-3687 with primer F01; Lane 3: L-1 (primer F01); Lane 4: VKM F-3687 (primer F02); Lane 5: L-1 (primer F02); Lane 6: VKM F-3687 (primer F03); Lane 7: L-1 (primer F03); Lane 8: VKM F-3687 (primer F04); Lane 9: L-1 (primer F04); Lane 10: VKM F-3687 (primer F05); Lane 11: L-1 (primer F05); Lane 12: VKM F-3687 (primer F06); Lane 13: L-1 (primer F06); Lane 14: VKM F-3687 (primer F07); Lane 15: L-1 (primer F07); Lane 16: VKM F-3687 (primer F08); Lane 17: L-1 (primer F08); Lane 18: VKM F-3687 (primer F09); Lane 19: L-1 (primer F09); Lane 20: VKM F-3687 (primer F10); Lane 21: L-1 (primer F10). Growth at various temperatures The growth rate/temperature relationships of various fungal strains at different temperatures are presented in the Table 3. Strain L-1 only displayed rapid growth at 50°C, and its growth optimum was 45°C. Reference strain VKM F-3693 is mostly similar to strain L-1 in this respect, providing evidence that it belongs to the Rhizopus microsporus var. rhizopodiformis -group. Differentiation of fungal isolate and reference strains using random primers Initially RAPD patterns were assessed in genomic DNA extracted from the L-1 fungal isolate and a reference strain, A. terreus VKM F-3687. On the basis of amplified DNA patterns the two fungal species were distinctive. Both strains had characteristic DNA fingerprinting patterns for each particular primer as shown in Fig. 9. The numbers of bands detected using 10 different primers (F01-F10) ranged from two to ten. The greatest number of PCR products was found in A. terreus VKM F-3687 genomic DNA (10 bands with F01 primer, followed by 9 bands with F06 and F09 primers). In general, the numbers of PCR fragments showed in the reactions with strain L-1 genomic DNA were fewer than in the respective reactions with Aspergillus DNA. Product sizes typically ranged between approximately 0.3 up to 6 kbp. The 143 Table 4. Jaccard’s coefficients of genetic relatedness between Rhizopus sp. L-1 strain and reference strains from Rhizopus genus. Reference strains S j R. microsporus var. rhizopodiformis VKM F-3693 0.89 R. microsporus var. rhizopodiformis VKM F-3692 0.89 R. microsporus var. chinensis VKM F-1218 0.67 R. microsporus var. microsporus VKM F-1063 0.60 R. microsporus var. microsporus VKM F-773 0.58 R. microsporus var. microsporus VKM F-774 0.53 R. microsporus var. chinensis VKM F-1361 0.51 R. microsporus var. chinensis VKM F-1360 0.43 R. stolonifer var. stolonifer VKM F-601 0.36 R. oryzae VKM F-605 0.33 banding patterns for strain L-1 DNA were markedly different from the patterns for the A. terreus VKM F-3687 DNA (Fig. 9). DNA fingerprinting patterns of strain L-1 were also compared to ten reference strains of the R. microsporus-group and other species of Rhizopus using different random primers. Using 20 decamer primers more than 200 different reproducible RAPD markers were generated from 11 Rhizopus strains. Amplification carried out with all the primers used in this study showed very similar banding pattern in strain L-1 and those of VKM F-3692 and VKM F-3693 strains (Figs. 10-12) suggesting they are genetically very similar. On the other hand the RAPD results presented in Figs. 10-12 indicate that banding patterns from strain L-1 and those of other eight strains (R. microsporus var. microsporus VKM F-773; R. microsporus var. microsporus VKM F-774; R. microsporus var. microsporus VKM F-1063; R. microsporus var. chinensis VKM F-1218; R. microsporus var. chinensis VKM F-1360; R. microsporus var. chinensis VKM F-1361; R. stolonifer var. stolonifer VKM F-601; R. oryzae VKM F-605) differ in various extent. Differences can be also recognised between RAPD electrophoretic patterns of two sexual forms of one strain. Figures 11, 12 shows that the banding patterns of R. microsporus var. microsporus VKM F-773, MT+ and R. microsporus var. microsporus VKM F-774, MT- are different with primers F 13 and F 14. Rhizopus microsporus var. chinensis VKM F-1361, MT+ and R. microsporus var. chinensis VKM F-1360, MT- have slightly different fingerprints as well when primers F 12, F 13 and F 14 (Figs. 10-12) are used. The degree of genetic variation within the strains of Rhizopus studied here were assessed using the average genetic distance values calculated as Jaccard’s similarity coefficients (S). The object of the pattern comparison is to j obtain a measure of commonness or difference between the gel banding patterns of the two isolates. S values have been computed between all pairs of j 144