Brazilian Journal of Microbiology (2007) 38:71-77 ISSN 1517-8283 MORPHOLOGICAL AND MOLECULAR DIFFERENTIATION OF THE PECTINASE PRODUCING FUNGI PENICILLIUM EXPANSUM AND PENICILLIUM GRISEOROSEUM Patrícia Gomes Cardoso; Marisa Vieira de Queiroz; Olinto Liparini Pereira; Elza Fernandes de Araújo* Departamento de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa-MG, Brazil Submitted: May 15, 2006; Returned to authors for corrections: August 28, 2006; Approved: January 18, 2007. ABSTRACT Two species from the genus Penicillium, Penicillium expansum and P. griseoroseum (Brasilian isolates) were characterized morphologic and molecularlly. Morphological variability was detected among isolates in regard to colony morphology and to conidia coloration. The molecular characterization was based on the RAPD markers, telomeric fingerprinting and ITS sequencing. A total of 78 RAPD primers were used and 8 presented differences in band patterns with 54% of the amplified polymorphic fragments. The monomorphic fragments of 600 bp (P. expansum) and 594 bp (P. griseoroseum) were amplified. The only internal transcribed spacer region variation detected between the two species was the additional six initial nucleotides. The analysis by telomeric fingerprinting showed polymorphism between both species and the chromosome minimal numbers estimated were three. The polymorphism observed in the organization of the subtelomeric region in the genome of two Penicillium species within the high homogeneous Penicillium subgenus is for the first time reported and perhaps can be employed in future phylogenetic studies. Key words: Ribossomal DNA, Internal Transcribed Spacer, Subtelomeric Region, Penicillium spp. INTRODUCTION Furcatum) determined by the number of branch points between phialide and stipe, down the main axis of the penicillus and The genus Penicillium is worldwide known for production others characters, like ratio of metula length to phialide, length of secondary metabolites and extracellular enzymes of and colony diameter on G25N, when the number of branch commercial value, including the pectinases, utilized in fruit points is the same (31). juice industry during the stage of pulp maceration, juice Two species from the genus Penicillium, P. expansum and P. liquefaction or depectinization (19). Most Penicillium species griseoroseum have been selected in our laboratory and they were are considered ubiquitous, opportunistic saprophytes. shown to be excellent pectinase producers (4). These species Nutritionally, they are supremely undemanding being able to have been extensively studied physiologically, showing clear grow in almost any environment with a sprinkling of mineral differences in their answers to the influence of inoculum age and salts, any but the most complex forms of organic carbon, and a concentration, as the effect of some cultural conditions (growth wide range of physical-chemical environments, aW, temperature, period, substrate concentration and different carbon sources) pH and redox potential. The taxonomy of this genus is hard as on the production of pectin lyase (PL) and polygalacturonase its classification is based mainly on conidiophore and conidia (PG). The P. griseoroseum PL activity was highest with 9-day-old structure, although the colony diameter after incubation under spore, 5x104 spores/mL after 48 h of growth and the P. expansum standardized conditions has greater importance for PL activity with 5-day-old spore, 106 spores/mL after 72 h of classification (31). The genus Penicillium is subdivided in four growth (1-3,10,11,26,27). Several genetic studies have been carried subgenera (Aspergilloides, Penicillium, Biverticillium and out in our laboratory in order to isolate strains with enhanced *Corresponding Author. Mailing address: UFV - BIAGRO - Dep. de Microbiologia 36570-000. Viçosa, MG - Brasil. Tel.: (31) 3899-2553 ou (31) 3899-2573. E-mail: [email protected] 71 Araújo, E.F. et al. pectinolytic enzyme activities. Different methodologies which Morphological characterization of P. expansum and have been used to achieve these goals include protoplast P. griseoroseum production and regeneration, mutant isolation and Fungi were grown in specific medium to differentiate species characterization, protoplast transformation and isolation and of Penicillium, Czapek Yeast Extract Agar (CYA) and Neutral characterization of genes coding pectinolytic enzyme in P. Creatine Sucrose Agar (CSN), as described by Pitt and Hocking griseoroseum and P. expansum. (5,9,12,13,16,21,33,34-36,38,41). (31). Colors of colony were recorded under bright tungsten Incompatibility was observed between these two species for the illumination and designated according to Munsell Color Charts formation of heterokaryons and diploids when conidia were mixed (23). (41). When the parasexual cycle of P. expansum was analyzed, it was noted that diploids were rather unstable and that recombinant DNA extraction and Southern blot sectors could easily be isolated (21). Nevertheless, was observed Penicillium expansum and P. griseoroseum total DNA was different results with P. griseoroseum, in which the parasexual extracted according to Specht et al. (40) and cleaved with cycle was not obtained spontaneously, and the diploids isolated restriction enzymes AluI, BamHI, EcoRI, HindIII and SmaI. by protoplast fusion were stable and produced few discrete The reactions were analyzed on 0.8% agarose gel, and then haploid sectors even when placed in complete medium transferred to Duralon membranes (Stratagene) according to supplemented with benomil (38). High identity in amino acid Sambrook et al. (37). The membrane was probed with the plasmid sequence and similar global organization in PL and PG genes pTel13 (22) containing 225 bp DNA fragment labeled with [α- from P. expansum and P. griseoroseum was showed (5,12,34-36). 32P]d-ATP, using “Random Primer-It II Labeling Kit” Penicillium expansum PLE1 shares 100% amino acids identity (Stratagene). Hybridization was carried out overnight at 60ºC in with PLG1 from P. griseoroseum and the organization of PL genes standard hybridization buffer, washed twice with 2 X SSC, showed same hybridization pattern what suggests that PL genes 0.1% SDS for 20 min, and 1 X SSC, 0.1% SDS for 10 min. have a similar global organization in the genome segment in which Autoradiographs were taken by three-day exposure of XOMAT they are found. Some doubts emerged from the genetics studies K film (Kodak), with an intensifying screen. with respect to the basic differences that are required for strains to be considered different species. PCR amplification of ribossomal DNA regions The objective of this study was to analyze the differentiation The ribossomal ITS1-5.8S rRNA gene-ITS2 region was of the pectinase-producing fungi Penicillium expansum and amplified with primers ITS1 (5’-TCCGTAGGTGAACCTGCGG- Penicillium griseoroseum employing both morphological and 3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) constructed molecular methods. The use of molecular techniques based on for molecular phylogenetic studies (43). PCR amplification was the random amplified polymorphic DNA (RAPD) and performed in 25 mL reaction mixtures containing 10 ng of sequencing of the internal transcribed spacer (ITS) region of genomic DNA template, 1.0 U of Taq DNA polymerase the ribosomal DNA (rDNA) have been useful for the (Promega), 40 pmol of each primer, 10 mM Tris-HCl (pH 8,3), 50 identification and classification taxonomy of filamentous fungi. mM KCl, 2.5 mM MgCl2, 0.1 mM of each dNTP (dATP, dCTP, dGTP e dTTP). Amplification was performed in a thermocycler MATERIALS AND METHODS PCT-100 (MJ Research, Inc.) for 40 cycles, each cycle consisting of a denaturation step (15s; 94ºC), an annealing step (30s; 35ºC), Strains and growth conditions and a extension step (60 s; 72ºC). After the 40th cycle, a final Penicillium expansum and P. griseoroseum isolates were extension step was performed (60s; 72ºC). ITS PCR products obtained from forest tree seeds collected at Universidade Federal were cloned into pGEM-T Easy Vector System (Promega). This de Viçosa, Minas Gerais, Brazil. The isolate of Penicillium vector was used to transform E. coli DH5a and the plasmidial griseoroseum was deposited at Fundação Tropical de DNA was extracted by alkaline lysis method (37). The primers Tecnologia e Pesquisas André Toselo (Campinas, São Paulo, M13 forward and reverse (Promega) and “BigDye™” kit Brazil), under the code register CCT 6241, and the P. expansum Terminator Cycle Sequencing Ready Reaction” (Applied isolate was deposited at VIC Herbarium (Viçosa, Minas Gerais, Biosystems) were used to sequence both strands. The ITS Brazil), under the code register VIC 28728. Cultures were also sequences were analysed and aligned using BLAST and kept in incubation on agar/H2O at 4ºC for long-term storage. CLUSTAL W software. Sequence data were deposited in the The inoculum was prepared from 7-day-old cultures grown at GenBank Nucleotide Database. 25ºC. Erlenmeyer flasks (125 mL) containing 50 mL of minimal medium (MM) (32) were inoculated for a final spore RAPD analysis concentration of 106 spores mL-1 and shaken (150 rpm; 25ºC). PCR amplification of DNA from P. expansum and P. Bacterial transformation was carried out using method proposed griseoroseum was performed with 78 random primers (Operon by Inoue et al. (20). Technologies Inc., Alameda, CA) as described by Williams et 72 Pectinase producing fungi al. (44). Amplification products were electrophoresed in 1.5% (w/v) agarose gels immersed in TEB running