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DNA Fingerprinting Analysis of Petromyces Alliaceus (Aspergillus Section Flavi)

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DNA Fingerprinting Analysis of Petromyces Alliaceus (Aspergillus Section Flavi) 276 1039 DNA fingerprinting analysis of Petromyces alliaceus (Aspergillus section Flavi) Cesaria E. McAlpin and Donald T. Wicklow Abstract: The objective of this study was to evaluate the ability of the Aspergillus flavus pAF28 DA probe to pro­ duce DA fingerprints for distinguishing among genotypes of Petromyces alliaceus (Aspergillus section Flavi), a fun­ gus considered responsible for the ochratoxin A contamination that is occasionally observed in California fig orchards. P alliaceus (14 isolates), Petromyces albertensis (one isolate), and seven species of Aspergillus section Circumdati (14 isolates) were analyzed by DA fingerprinting using a repetitive sequence DNA probe pAF28 derived from A. flavus. The presence of hybridization bands with the DA probe and with the P alliaceus or P albertensis genomic DA in­ dicates a close relationship between A. flavus and P alliaceus. Twelve distinct DA fingerprint groups or genotypes were identified among the 15 isolates of Petromyces. Conspecificity of P alliaceus and P albertensis is suggested based on DA fingerprints. Species belonging to Aspergillus section Circumdati hybridized only slightly at the 7.0-kb region with the repetitive DA probe, unlike the highly polymorphic hybridization patterns obtained from P alliaceus and A. jZavus, suggesting very little homology of the probe to Aspergillus section Circumdati genomic DNA. The pAF28 DA probe offers a tool for typing and monitoring specific P alliaceus clonal populations and for estimating the genotypic diversity of P alliaceus in orchards, vineyards, or crop fields. Key words: Aspergillus alliaceus, Circumdati, DNA probe, genotypic diversity, hybridization patterns, ochratoxin, Southern blot. Resume: L'objectif de cette etude etait d'evaluer les capacites d'une sonde d'ADN pAF28, preparee a partir d'Asper­ gillus flavus, a generer des empreintes d' ADN permettant de distinguer les genotypes de Petromyces alliaceus (Asper­ gillus section Flavi), un champignon responsable de la contamination a l'ochratoxine A observee occasionnellement dans les figueries de Californie. Quatorze isolats de P alliaceus, un isolat de Petromyces albertensis et 14 isolats de sept especes d'Aspergillus section Circumdati ont ete analyses par empreinte a I'AD avec la sonde d'AD compre­ nant la sequence repetee de pAF28 derivee de A. flavus. La presence de bandes lors de l'hybridation de I' AD de la sonde avec I' AD genomique de P alliaceus ou P albertensis indique que A. jZavus et P alliaceus sont etroitement re­ lies. Douze groupes d'empreintes d' AD ou de genotypes distincts ont ete identifies parmi les 15 isolats de Petromy­ ces. La conspecificite de P alliaceus et P albertensis est suggeree par les empreintes a l' AD . Les especes appartenant a la section Circumdati d'Aspergillus s'hybrident faiblement avec la sonde repetitive d' AD dans une re­ gion de 7,0 kb, contrairement a P alliaceus et A. flavus qui generent des partons d'hybridation hautement polymor­ phes, suggerant qu'il existe peu d'homologie entre la sonde et I'AD genomique d'Aspergillus section Circumdati. La sonde d' ADN pAF28 offre un outil permettant Ie typage et la surveillance de populations clonales specifiques de P al­ liaceus ainsi que l'estimation de la diversite genotypique de P alliaceus dans les vergers, les vignobles ou les champs. Mots efes : Aspergillus alliaceus, Circumdati, sonde d' AD ,diversite genotypique, patrons d'hybridation, ochratoxine, buvardage par Southern. [Traduit par la Redaction] Introduction for several months, may contain one to several thin-walled, nonostiolate ascocarps containing asci with ascospores (Raper Petromyces alliaceus Malloch and Cain (anamorph: Asper­ and Fennell 1965; Malloch and Cain 1972). Aspergillus gillus alliaceus Thorn and Church) is characterized by colo­ alliaceus has been classified in the Aspergillus wentii group nies forming upright, ovate to ellipsoidal, gray-black (Thorn and Raper 1945; Kozakiewicz 1989), the Aspergillus sclerenchymatous stromata, which after developing slowly ochraceus group (Raper an Fennell 1965), and Aspergillus section Circumdati (Gams et al. 1985). Peterson (1995) was the first to show that P. alliaceus NRRL 4181 belongs in Received 16 December 2004. Revision received 2 August 2005. Accepted 16 August 2005. Published on the NRC Research Aspergillus section Flavi because the large subunit (lsu) ri­ Press Web site at http://cjm.nrc.ca on 6 December 2005. bosomal DA sequence, also reported as the D1-D2 region of 265 rRNA gene, is similar to that of Aspergillus flavus 1 e.E. McAlpin and D.T. Wicklow. Mycotoxin Research Link NRRL 1957. A more comprehensive classification of Unit, USDA, ARS, ational Center for Agricultural Aspergillus sp. based on phylogenetic analysis of lsu rDNA Utilization Research, Peoria, IL 61604, USA. sequences showed that P. alliaceus NRRL 315, NRRL 4181, lCorresponding author (e-mail: [email protected]). RRL 5108; Petromyces albertensis RRL 20602; and Can. J. Microbial. 51: 1039-1044 (2005) dai: 10.1 139/W05-097 © 2005 RC Canada 1040 Can. J. Microbial. Vol. 51, 2005 Aspergillus lanosus Kamal & Bhargava NRRL 3648 have helm, Aspergillus ostianus Wehmer, Aspergillus sclero­ identical sequences and are grouped as synonymous species tiorum G.A. Huber, and Aspergillus sulphureus (Fresenius) in the phylogram of Aspergillus section Flavi (Peterson 2000). Wehmer). A culture of A..flavus NRRL 19997 was used as a Phylogenetic analysis of DNA sequence data from internal control to demonstrate that the DNA probe pAF28 could hy­ transcribed spacer (ITS) regions using a neighbor-joining bridize successfully. tree grouped P. alliaceus, P. albertensis, and A. lanosus as distinct species within the P. alliaceus clade (Rigo et al. Cultural conditions and DNA extraction 2002), after a majority-rule consensus tree showed P. alliaceus Mycelia of P alliaceus were grown on 200 mL of yeast and P. albertensis to be conspecific (Varga et al. 2000). extract - peptone - dextrose broth (YEPD) in 500 mL Erlen­ There is compelling evidence that A. alliaceus may be re­ meyer flasks using conidial and (or) hyphal suspension sponsible for the ochratoxin A contamination occasionally (approx. 105 CFU/mL) from 5- to 7-day-old cultures, then observed in California figs (Doster et al. 1996; Bayman et incubated at 32 DC for 24 h on a rotary shaker at 200 r/min. al. 2002). Ochratoxin A is an important nephrotoxic and Mycelial mats were collected after filtering through sterile nephrocarcinogenic mycotoxin that is produced by a rela­ Whatman No. 1 filter paper, rinsing twice with sterile dis­ tively small number of species in Aspergillus and tilled water, transferring to a 50 mL Sarstedt tube, freezing Penicillium, including A. alliaceus (Abarca et al. 1994; at -80 DC, and lyophilizing for at least 24 h. Total DNA was Ciegler 1972; Frisvad 1994; Heenan et al. 1998; Hesseltine isolated and purified based on the method of Raeder and et al. 1972; Larsen et al. 2001; Varga et al. 1996; Wicklow et Broda (1985) with modifications as reported by McAlpin al. 1996). The occurrence of ochratoxin A in wine and rai­ and Mannarelli (1995). sins due to the growth of fungi, such as Aspergillus niger V. Tiegh., on grapes (Majerus et a1. 2000) has invited greater DNA fingerprinting attention to the potential for ochratoxin A contamination of Genomic DNA was digested with Pst!, a restriction dried fruits (Gareis 2004). endonuclease from Providencia stuartii (Roche Molecular The pAF28 repetitive sequence DA probe has proven re­ Biochemicals, Indianapolis, Indiana), following the manu­ liable in classifying A. flavus strains according to their previ­ facturer's recommendations. Southern blots were performed ously determined vegetative compatibility group (VCG) by transferring DNA from electrophoresis gels to nylon (McAlpin and Mannarelli 1995; McAlpin et al. 2002). The membranes using a vacuum blotter (Model 785, BioRad probe has also been used to estimate the genotypic diversity Laboratories, Hercules, California). DNA fingerprints were of (i) Aspergillus section Flavi isolates, including A. flavus identified using the DIG Nonradioactive Nucleic Acid La­ and Aspergillus parasiticus Speare populations isolated from beling, Hybridization, and Detection System (Roche Molec­ a corn field in central lllinois (Wicklow et al. 1998; McAlpin ular Biochemicals). X-ray films (Biomax MR, Eastman et al. 1998) and Aspergillus caelatus Horn from peanut field Kodak Imaging Systems, Rochester, New York) were ex­ soil in Atlanta, Georgia (McAlpin et al. 2004), (ii) isolates posed at room temperature for 1-3 h. of Aspergillus bombycis Peterson et a1. from silkworm culti­ vation (Goto et a1. 2003), and (iii) the yellow-green koji DNA fingerprint analyses molds Aspergillus oryzae (Ahlburg) Cohn and Aspergillus The absence or presence of fragments at specific positions sojae Sakaguchi & Yamada used in traditional Oriental food based on the molecular weights of the DA hybridization fermentations (Wicklow et a1. 2002). The probe has also bands were observed. Banding profiles of the different been used to type clinical isolates of A. flavus from human strains were compared and similarity was calculated using sources (James et al. 2000). The goal of this study was to the Dice coefficient, following the TSYS Numerical Tax­ evaluate the ability of A. flavus pAF28 DNA probe to pro­ onomy and Multivariate Analysis (Rohlf 1997). The similar­ duce DNA fingerprints for distinguishing among genotypes ity matrix was obtained and cluster analysis was made with of
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