Bangladesh J. Plant Taxon. 30(2): 255-261, 2023 (December) DOI: https://doi.org/10.3329/bjpt.v30i2.70501 © 2023 Bangladesh Association of Plant Taxonomists NEW RECORD OF SCENEDESMUS VACUOLATUS FROM SOIL IN VOJVODINA, SERBIA TIMEA HAJNAL JAFARI*, VLADIMIRA ŽUNIĆ, SIMONIDA DJURIĆ AND DRAGANA STAMENOV University of Novi Sad, Faculty of Agriculture, Dositeja Obradovića Sq.8, Novi Sad, Serbia Keywords: Soil algae; Scenedesmus vacuolatus; DNA extraction; ITS2. Abstract Soil algae are photosynthetic microorganisms showing huge differences in morphology depending on the soil type, agricultural practices, and environmental factors. The aim of this research was to compare the results of classical and molecular protocols for microalgal identification. The green microalgae were isolated from acidic arable soil of dystric cambisol type, using BG11 medium and the agar plate method. Firstly, the identification of the microalgae was performed based on its morphological characteristics using light microscopy (LM) and taxonomic monographs and reference books. The isolate was initially annotated on genus level as Coelastrella sp. Gene marker (ITS2) was used for molecular identification. According to this protocol, the microalga was identified as Scenedesmus vacuolatus. Combination of morphology and DNA-based approach proved to be the most effective for obtaining a consistent species level identification. Moreover, this is the first record of Scenedesmus vacuolatus identification from soil in Vojvodina, Serbia. Introduction Microalgae are microscopic organisms (1-900 µm) that can be either prokaryotic or eukaryotic. They can grow and reproduce in freshwater and marine environments, as well as in soil (Nabti et al., 2017). It is estimated that out of existing 800.000 microalgae species, around 50.000 are described (Ronga et al., 2019), but only a few species are used for different purposes, such as food or animal feed, in the pharmaceutical industry, agriculture, etc (Seman et al., 2021). Due to various application potential, microalgae have attracted considerable interest worldwide. The most examined microalgal species originate predominantly from various freshwater environments and belong to the branch of green algae (Chlorophyta) and cyanobacteria (Bumandalai and Tserennadmid, 2019; Hernandez et al., 2009). However, soil algal diversity is also enormous, yet nothing or little is done to uncover their importance in the ecosystems they inhabit. Underestimating the algal diversity in these ecosystems is due to, first of all poor knowledge about their role in soil ecosystem maintenance as well as lack of significant scientific interest by researchers in algal taxonomy and phylogeny (Trbojević and Predojević, 2022). Though the importance and application of specific soil microalgae are acknowledged by many researchers (Hajnal Jafari et al., 2016; Kholssi et al., 2019; Žunić et al., 2022), there are no published articles about the diversity and taxonomy of green soil microalgae in Serbia. Generally, studies on the identification of microalgae are done using a classical approach based upon the description of cell and colony structures. This is highly subjective since it requires a microscope of high magnification and a skilled observer. Zou et al., (2016) even argued that the morphological *Corresponding author. E-mail: mikrobiologija@polj.uns.ac.rs mailto:mikrobiologija@polj.uns.ac.rs 256 JAFARI et al. identification of some species is impossible. Moreover, there are morphological adaptation and endemism among many microalgae species which make them difficult to correctly identify (Coleman, 2002). That is why the combination of morphology and DNA-based approach proved to be the most effective for obtaining a consistent species level identification. Hence, in this study, the aim was to compare the results of classical and molecular identification of soil microalgae using gene marker (ITS2). The identity of the microalgae was initially identified using its morphological characteristics. Materials and Methods Isolation and growth conditions The green microalgae species was isolated from acidic arable soil (pH 4.31) in Vojvodina, Serbia, using BG11 medium. BG11 medium per one liter of distilled water contains: 1.5 g NaNO3, 0.04 g K2HPO4, 0.075 g MgSO4·7H2O, 0.036 g CaCl2·2H2O, 0.006 g Citric acid, 0.006 g Ferric ammonium citrate, 0.001 g EDTA, 0.02 g Na2CO3 and 1ml Trace metal solution A5. The composition of the trace metal mix solution is: 2.86 g H3BO3, 1.86 g MnCl2·4H2O, 0.22 g ZnSO4·7H2O, 0.39 g Na2MoO4·2H2O, 0.08 g CuSO4·5H2O, 0.05 g Co(NO3)2·6H2O. The pH of BG11 medium was adjusted to 7.1 and autoclaved at 121ºC. Microalgae isolation started with using agar plate method. Microalgae were grown in Petri dishes on solid BG11 medium under aseptic conditions at room temperature (24±2°C) and day: night photoperiod 14:10. Every 14 days, one colony was selected and transferred to fresh agar plates. Elimination of contaminants and pure culture was obtained by the agar streaking method and continued growth upon sub-culturing. Cell morphology and identification Taxonomic identification was performed on the basis of cell morphology. Morphological observations were done using a binocular light research microscope at 400× and 1000× magnification (Motic, BA210). Identification was based on taxonomic monographs and reference books for terrestrial algae and cyanobacteria (Komárek and Fott, 1983; Komárek and Anagnostidis, 2005; Bellinger and Sigee, 2010) Molecular identification Total genomic DNA was isolated from fresh algal biomass using the EURx DNA extraction protocol for environmental samples - soil (GeneMATRIX Soil DNA Purification Kit™). 50 ml of fresh algal biomass was centrifuged, and the pellet was used according to the protocol. PCR amplification protocol ITS2 region was attempted to PCR amplification using the ITS2 primers according to Hadi et al., (2016) and Liu et al. (2014): ITS-2_f, 5’-AGGAGAAGTCGTAACAAGGT-3’ (Tm=56.9°C) and ITS-2_r, 5’-TCCTCCGCTTATTGATATGC (Tm=61.5°C). Amplicon size varies (nucleotides span) 600-1000 bp, depending on algal strain. The 25 µL PCR reaction mix was composed of 10 µL of ultrapure water, 12.5 µL of MasterMix, 0.25 µL of each primer ITS2 forward and reverse. At the end, 2 µL of DNA template was added in the PCR reaction mixture in Eppendorf. The PCR reactions were performed in the PCR thermocycler TC-412 Thermal Cycler (United Kingdom) using a program designed for the amplification of ITS regions. The program starts with initial denaturation at 96°C, 40 cycles (96ºC - 1 min. of template denaturation, 52ºC - 1 min. primer annealing, and 72ºC – 1 min. for extension) and final extension at 72ºC for 5 min and hold at 4ºC. NEW RECORD OF SCENEDESMUS VACUOLATUS FROM SOIL 257 Gene visualization 10 µL of each PCR sample were mixed with 3µL 6x DNA Loading Dye (Thermo Fisher Scientific Baltics UAB, Lithuania) and loaded onto 1.5% agarose gel and electrophoresed using 1x TBE (Tris Borate EDTA) buffer (Sigma Aldrich Co., USA). Gene Ruler 100 bp DNA Ladder (Thermo Fisher Scientific Baltics UAB, Lithuania) was loaded onto the first and/or last lane. Agarose gel and electrophoresis was run at 100 W for 45 min. After that, gel was added to 1% ethidiumbromide solution (250ml dH2O + 20 µL ethidiumbromide) for 30 min. Bands were visualized with a UV lamp in BlueCube 300 (SERVA Electrophoresis GmbH, Germany). Genomic DNA was sent out for sequencing to Macrogen© (Seoul, South Korea) and the result was compared with sequences available on the NCBI website database through the BLAST bioinformatics tool. Results and Discussion Based on the morphology obtained using light microscopy (LM), the taxonomy was further performed using reference books for terrestrial algae and cyanobacteria. The isolated strain was annotated as a member of the genus Coelastrella. It is a single celled, non motile green algae with a round to coccoid shape of cell, size 4-12 µm (young and adult). Inside the cell, a cup-shaped chloroplast as well as a single pyrenoid can be easily observed (Fig. 1). The size and shape of microalgal cells depend on maturity, cell growth phase and origin (Darienko et al., 2019). Equal- sized autospores and autosporangia (mother cell) with autospores inside the cell were also present. The release of autospores was obtained by the rupture of the mother cell wall. Traditionally, taxonomic classification and subsequent identification depend on the morphological description of cell and colony features. This could generate mistakes in the taxonomic identification (Gour et al., 2016). Krienitz et al., (2003) also stated that the classical approaches using morphological characters for the circumscription of coccoid green algae do not adequately reflect the phylogenetic relationships. Though it is important to examine the culture and take images for any future record and publication, one should be aware of relying only on LM images because many different algae species may look alike. Having that in mind, the next step was using a DNA sequence for species-level identification. Fig. 1. Morphology of the soil microalgae Coelastrella sp. (magnification 40× and 1000×) Autospores 258 JAFARI et al. Considered as one of the frequently used molecular markers for distinguishing between eukaryotic species, the internal transcribed spacer 2 (ITS2) is a gene used in several applications among which molecular phylogenetic analyses can be included. The primary sequence of ITS2 region is highly conserved within species, but is highly divergent between species (Hoshina, 2014). Fawley and Fawley (2020) stated that for algae, and especially for green algae, the ITS2 region is an important region for species identification. Fig. 2 shows the band of sample 63s amplified using ITS2 rDNA primers. A total of 715 base pair sequences was obtained which was submitted to Genbank for BLAST search. The sequence got labeled as Scenedesmus vacuolatus 63 (Accession number: OP808227). The level of identity between the submitted sequence and a published sequence was 99.8%. Fig. 2. PCR amplification results of Scenedesmus vacuolatus (63s) ITS-2 region The phylogenetic tree (Fig. 3) shows the relationship of Scenedesmus vacuolatus 63 to other strains of microalgae based on their ITS2 sequences. The closest sequences were Scenedesmus vacuolatus S12, Coelastrella vacuolata FACHB-3315 and Coelastrella vacuolata FACHB-3314 with over 99% similarity. The other nearest group includes the strains S. vacuolatus SAG211-8c, S. vacuolatus SAG211-8e and S. vacuolatus UTEX252. The most distant branch is Desmodesmus regularis SAG24.95 (AM228925). The classification of Scenedesmus vacuolatus is given below: Domain: Eukarya; Phylum: Chlorophyta; Class: Chlorophyceae; Order: Sphaeropleales Family: Scenedesmaceae; Genus: Scenedesmus; Species: S. vacuolatus (Shihira & Krauss) E. Kessler, M. Schäfer, C. Hümmer, A. Kloboucek & V. Huss The molecular identification (BLAST search) showed that the studied strain had a different genus name compared to the one annotated based on morphology. However, that is not something unexpected in algal systematics. The demarcation of genera and species in the family Scenedesmaceae is rather difficult. Numerous phylogenetic studies revealed the existence of complex relationships among algal species since morphological and molecular identification often resulted in different outcomes (Lakshmana Senthil et al., 2019). Chlorella fusca var. vacuolata, Graesiella vacuolata and Coelastrella vacuolata (Kalina and Puncocharova, 1987; Hegewald and Hanagata, 2002) are the nomenclatural or homotypic synonyms for Scenedesmus vacuolatus that can be found in the literature covering algae identification and taxonomy (Darienko et al., 2010; NEW RECORD OF SCENEDESMUS VACUOLATUS FROM SOIL 259 Shetty et al., 2021). Basically, our morphology based identification matched the DNA bases, even though the genus names differed. This result is consistent with the initial idea that a combined classical and molecular method of identification has to be applied in order to perform an accurate genus/species level identification. Fig. 3. Maximum likelihood phylogenetic tree based on ITS2 sequences of Scenedesmus vacuolatus 63 gained by bootstrap analysis in 1000 repetition According to the literature survey, there is no report regarding the diversity of green soil microalgae in Serbia. 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