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Mycologia, 94(6), 2002, pp. 1078-1086.
© 2002 by The Mycological Society of America

Interspecific mitochondrial DNA restriction fragment length polymorphisms in Aspergillus section Flavi


Jeffrey T. Quirk

     Edinboro University of Pennsylvania, Department of Biology and Health Service, Edinboro, PA 16444

John M. Kupinski 1

     Department of Biology, St. Bonaventure University, St. Bonaventure, New York 14778

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

Mitochondrial DNA restriction fragment length polymorphisms (RFLPs) are described for 64 isolates, representing 11 species of Aspergillus section Flavi. Mitochondrial DNA haplotypes were identified following digestion of total cellular DNA with the restriction enzymes HaeIII, AseI, or DraI. In general, isolates of the same species possessed identical mitochondrial DNA haplotypes. Three haplotypes were found in multiple, closely related species: one in A. flavus, A. oryzae, and A. subolivaceus; a second in A. parasiticus and A. sojae; and a third in A. tamarii and A. flavofurcatis. Four distinct haplotypes were each associated with a single species: A. nomius, A. avenaceus, A. leporis, and A. zonatus. Mitochondrial DNA haplotypes complement traditional morphological and growth criteria in making taxonomic decisions within Aspergillus section Flavi.

Key words: fungal identification, haplotype, taxonomy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The members of the anamorphic Aspergillus section Flavi (Gams et al 1985Citation) are economically important species used in biotechnological applications and food fermentations. Certain species are also involved in food spoilage and aflatoxin production, while others are known plant and animal pathogens. At the present time, accurate species identifications and taxonomic decisions within this section remain difficult due to overlapping morphological and biochemical characteristics.

Recent efforts to improve species identification and to increase our understanding of the phylogenetic relationships among Aspergillus taxa have focused on direct comparisons of DNA (Kozlowski and Stepien 1982Citation, Kurtzman et al 1986Citation, 1987Citation, Klich and Mullaney 1987Citation, Klich and Pitt 1988Citation, Gomi et al 1989Citation, Moody and Tyler 1990aCitation, bCitation, Keller et al 1992Citation, Klich et al 1993Citation, Chang et al 1995Citation, McAlpin and Mannarelli 1995Citation, Yuan et al 1995Citation). Restriction fragment length polymorphisms (RFLPs) can be used as fingerprints to distinguish between closely related organisms and to infer phylogenetic relationships. RFLP data for the genus Aspergillus are limited, but the utility of this approach for distinguishing between taxa has been demonstrated (Croft and Varga 1994Citation, Klich and Mullaney 1987Citation). For example, RFLPs generated following digestion with PstI can differentiate A. caespitosus Raper and Thom, A. versicolor (Vuill.) Tiraboschi, and A. sydowii (Bain. and Sart.) Thom and Church (Klich et al 1993Citation), and nuclear and mitochondrial RFLPs can divide the A. niger aggregate into two distinct groups represented by A. niger van Tieghem and A. tubingensis (Schöber) Mosseray (Varga et al 1994Citation).

Mitochondrial RFLPs have been used extensively in fungal systematics (Bruns et al 1991Citation) and restriction enzyme analysis of mitochondrial DNA has clarified taxonomic relationships of Aspergillus species (Kozlowski and Stepien 1982Citation). It has been demonstrated that restriction profiles of purified mitochondrial DNA can distinguish A. flavus Link, A. parasiticus Speare, and A. nomius Kurtzman et al (Moody and Tyler 1990aCitation). However, for routine identification of Aspergillus isolates it is desirable to detect mitochondrial DNA RFLPs without first separating the mitochondrial DNA from the nuclear DNA (Bruns et al 1991Citation).

In the present study, we digested total cellular DNA of 64 Aspergillus isolates with restriction enzymes and detected DNA polymorphisms with ethidium bromide staining and hybridization to mitochondrial DNA sequences. Our objective was to determine if mitochondrial DNA polymorphisms could assist in species identification in Aspergillus section Flavi.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Fungal strains and culture conditions – The Aspergillus isolates used in the study are listed in Table I . Fungi were maintained on Czapek-Dox agar and species designations were confirmed on the basis of morphological and growth characteristics (Raper and Fennell 1965Citation, Christensen 1981Citation, Kurtzman et al 1987Citation, Horn 1997Citation, Feibelman et al 1998Citation). Spores were harvested from cultures grown on sporulation medium: 0.2 mM MgSO4, 0.4 mM K2HPO4, 5.5 mM KNO3, 0.25 mM FeSO4, 0.1% Bacto-Peptone, 18% glucose, and 2.5% agar. Cultures were initiated by inoculating with spores to a final concentration of approximately 106 per mL and incubated on a rotary shaker at 200 rpm for 1 to 2 d at 22–25 C. We grew mycelia for DNA purification in 250 mL of liquid medium containing: 50 mM NaCl, 6.7 mM KCl, 2.0 mM MgSO4, 5.7 mM K2HPO4, 35 µM FeSO4, 1.5% acid hydrolysate of casein (Sigma Chemical Co., St. Louis, Missouri), and 1% sucrose.


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  		TABLE I. Aspergillus strains used in mitochondrial DNA RFLP analysis

 

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  		TABLE I. Continued

 
Preparation of total cellular DNA – We used the method of Klich and Mullaney (1987)Citation to purify total cellular DNAs from 0.5 g of freeze-dried mycelia. DNA was dissolved in TE buffer [10 mM Tris, 1 mM EDTA (pH 8.0)] and concentrations were determined by a fluorometric procedure (Cesarone et al 1979Citation) using the dye Hoechst 33258 (Sigma Chemical Co.). DNA integrity was assessed by electrophoresis in 1% agarose gels; in all cases the DNA migrated as a single band with a size greater than 20 000 bps.

Separation of mitochondrial DNA from total cellular DNA – We isolated mitochondrial DNA using CsCl gradient ultracentrifugation (Garber and Yoder 1983Citation). Briefly, 200 to 300 µg of cellular DNA in 30 mL of TE buffer was mixed with 28.4 g of CsCl and 5 mg of Hoechst 33258. After centrifugation at 110 000 x g for 48 h, the mitochondrial DNA and nuclear DNA bands were located by illumination with UV light (365 nm) and collected. After dye removal by extraction with water-saturated n-butanol, the DNA was concentrated by ethanol precipitation.

Restriction digestions – Restriction enzymes AseI (AT/TAAT), DraI (TTT/AAA), Eco RI (G/AATTC), HaeIII (GG/CC), and HindIII (A/AGCTT) were obtained from either Sigma Chemical Co. or New England Biolabs (Beverly, Massachusetts). Two µg of total cellular DNA or 0.5 µg of purified mitochondrial DNA were incubated for 2 to 4 h at 37 C with 5 to 10 units of restriction enzyme. Reactions were terminated by freezing.

Agarose gel electrophoresis – We separated DNA restriction fragments by electrophoresis in 1% agarose gels buffered with 0.1 M Tris, 0.05 M acetate, and 2.5 mM EDTA [pH 7.8]. Gels were stained with 0.5 µg/mL ethidium bromide, destained in deionized water, and then photographed on a UV transilluminator.

Preparation of DNA probes – Mitochondrial DNA from Aspergillus nidulans (Eidam) G. Winter (strain FGSC 93, Fungal Genetics Stock Center, Kansas City, Kansas) was cut with HindIII and cloned into plasmid cloning vector pUC18 (Sigma Chemical Co.). Recombinant plasmids containing fragments H3 (4.2 kb), H5 (2.0 kb), and H8 (1.4 kb) were recovered (Stepien et al 1978Citation). The identity of these fragments was confirmed by hybridizing each to Southern blots containing purified mitochondrial DNA of A. nidulans digested with either EcoRI or HindIII. Fragment H3 encodes the 16S rRNA, cytochrome c oxidase subunit III, and NADH dehydrogenase subunit 6 genes; fragment H5 encodes the ATPase subunits 6 and 8 and a portion of the NADH dehydrogenase subunit 4 genes; fragment H8 encodes part of the 23S rRNA gene (Brown et al 1985Citation, Brown 1990Citation). We prepared probes for Southern blot hybridizations by excising the cloned mitochondrial fragment from the plasmid with HindIII, separating the fragment from the cloning vector by agarose gel electrophoresis, and extracting the fragment from the gel with the QIAquick Gel Extraction System (Qiagen, Valencia, California). Purified H3, H5, and H8 DNA fragments were labeled by the random-primer method using a digoxigenin (DIG) DNA Labeling Kit (Roche Molecular Biochemicals, Indianapolis, Indiana).

Southern blot analysis of total cellular DNA – Following electrophoresis, DNA in agarose gels was denatured in 1.5 M NaCl, 0.5 M NaOH for 30 m, neutralized in 1.5 M NaCl, 1.0 M Tris-HCl [pH 7.0] for 30 m and then vacuum blotted onto Nytran nylon membranes (Schleicher & Schuell, Keene, New Hampshire). The DNA was covalently linked to the membrane by exposure to 150 mJoules of UV light (254 nm). Prior to hybridization, the membranes were blocked with hybridization solution—0.25 M NaH2PO4, 20%SDS, 1mM EDTA [pH 7.2]—containing 1% Blocking reagent (Roche Molecular Biochemicals), and then incubated overnight at 68 C with DIG-labeled probe. DIG-labeled bands were detected by enzyme-linked immunoassay using an anti-DIG antibody-alkaline phosphatase conjugate and the chemiluminescent substrate disodium 3-(4-meth-oxyspiro{1 , 2 - dioxetane - 3 , 2 ' -(5'-chloro)tricyclo[3.3.1.13,7]-decan}-4-yl)phenyl phosphate (CSPD) (Roche Molecular Biochemicals).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
We digested total cellular DNA of 64 Aspergillus isolates (Table I) with HaeIII and identified seven distinct HaeIII restriction patterns with bands 2 to 20 kb in size (Fig. 1, Table II). Of the 22 isolates of A. flavus examined, 19 shared an identical four-band pattern. The common A. flavus HaeIII RFLP was also identified in 22 of 23 isolates of A. oryzae (Ahlb.) Cohn, A. parasiticus, A. sojae Sakaguchi and Yamada, and A. subolivaceus Raper and Fennell. The HaeIII RFLP for A. nomius was similar to the common A. flavus-type; however, three of the four bands for A. nomius were slightly larger than the corresponding bands for A. flavus. Seven isolates of A. tamarii Kita and and 2 isolates of A. flavofurcatis Batista and Maia shared a distinctive five-band pattern. Three species, A. leporis States and M. Christensen, A. avenaceus Smith, and A. zonatus (Kwon and Fennell) Raper and Fennell, were clearly differentiated on the basis of their unique HaeIII fingerprints. Varietal status of A. flavus and A. oryzae isolates had no effect on the HaeIII RFLPs.



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  		 FIG. 1. HaeIII digests of total cellular DNA separated by agarose gel electrophoresis and stained with ethidium bromide. Lane 1, Lambda EcoRI–HindIII DNA fragments; lane 2, A. flavus var. flavus (ATCC 16883); lane 3, A. flavus var. columnaris (ATCC 16870); lane 4, A. oryzae (NRRL 466); lane 5, A. oryzae var. effuses (ATCC 1010); lane 6, A. oryzae var. viridis (ATCC 22788); lane 7, A. parasiticus (ATCC 15517); lane 8, A. nomius (ATCC 15546); lane 9, A. flavofurcatis (ATCC 16864); lane 10, A. subolivaceus (ATCC 16862); lane 11, A. leporis (ATCC 16490); lane 12, A. avenaceus (ATCC 16861); lane 13, A. zonatus (ATCC 16867)

 

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  		TABLE II. DNA restriction patterns of total cellular DNA detected by ethidium bromide staining and hybridization of mitochondrial DNA probes

 
The sum of the high molecular weight HaeIII fragments for both the A. flavus-type and A. tamarii-type RFLPs was approximately 30 kb, suggesting that the bands were mitochondrial DNA. Electrophoretic analysis of HaeIII digests of purified nuclear and mitochondrial DNA from A. sojae and A. tamarii showed that the high molecular weight bands observed in HaeIII digests of total cellular DNA were of mitochondrial origin (Fig. 2).



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  		 FIG. 2. HaeIII digests of purified mitochondrial and nuclear DNAs separated by agarose gel electrophoresis and stained with ethidium bromide. Lane 1, A. tamarii mitochondrial DNA; lane 2, A. tamarii nuclear DNA; lane 3, A. sojae mitochondrial DNA; lane 4, A. sojae nuclear DNA

 
Detection of mitochondrial RFLPs by ethidium bromide staining was not successful with the restriction enzymes AseI or DraI due to comigration of mitochondrial and nuclear DNA fragments. We hybridized cloned HindIII fragments of A. nidulans mitochondrial DNA to Southern blots of total cellular DNA. We found significant variation when AseI digests were hybridized to mitochondrial DNA fragment H3 (Fig. 3). Nineteen of 22 A. flavus and six of seven A. oryzae isolates gave the same H3 fingerprint. A single isolate of A. subolivaceus was similar to the prevalent A. flavus and A. oryzae RFLP profile. Two A. flavofurcatis isolates gave H3 fingerprints that were identical to those given by all eight A. tamarii isolates. Aspergillus leporis, A. avenaceus, and A. zonatus gave species-specific AseI RFLPs. AseI digests of section Flavi isolates probed with H5 gave the same strain groupings as those identified with probe H3. Four isolates of A. nomius were differentiated from A. flavus and A. parasiticus with mitochondrial probe H8 used in combination with the restriction enzyme DraI.



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  		 FIG. 3. Composite Southern blot of AseI digests probed with H3 fragment of A. nidulans mitochondrial DNA. Lane 1, A. flavus var. flavus (ATCC 16883); lane 2, A. flavus var. columnaris (16870); lane 3, A. oryzae (ATCC 14895); lane 4, A. oryzae (NRRL 466); lane 5, A. oryzae (47Sc); lane 6, A. parasiticus (ATCC 15517); lane 7, A. parasiticus (NRRL 502); lane 8, A. sojae (ATCC 11906); lane 9, A. sojae (ATCC 42251); lane 10, A. nomius (ATCC 15546); lane 11, A. flavofurcatis (55Sc); lane 12, A. flavofurcatis (ATCC 16864); lane 13, A. tamarii (ATCC 10836); lane 14, A. tamarii (ATCC 16865); lane 15, A. subolivaceus (ATCC 16862); lane 16, A. leporis (ATCC 16490); lane 17, A. leporis (ATCC 44565); lane 18, A. avenaceus (ATCC 16861); lane 19, A. zonatus (ATCC 16867)

 
We assigned each mitochondrial RFLP an arbitrary letter designation (Table II) and defined mitochondrial DNA haplotypes by unique combinations of HaeIII, AseI, and DraI RFLP profiles. Eight haplotypes encompassed the 64 isolates representing 11 species of section Flavi (Table III). In general, isolates of the same species possessed the same mitochondrial haplotype. Three haplotypes included more than one species: (1) A. flavus, A. oryzae and A. subolivaceus; (2) A. parasiticus and A. sojae; and (3) A. tamarii and A. flavofurcatis. Five isolates, A. tamarii (NRRL 428), A. flavus (ARSEF 2157), A. flavus (UAMH 362), A. flavus (NRRL 425), and A. oryzae (ATCC 14895) had RFLP profiles that did not conform to the consensus profiles of their respective species.


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  		TABLE III. Summary of the mitochondrial DNA haplotypes of 64 Aspergillus isolates

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
We examined 11 species of Aspergillus section Flavi for mitochondrial DNA polymorphisms. HaeIII digests of total cellular DNA stained with ethidium bromide exhibited seven distinct, high molecular weight RFLP patterns. The large size of the mitochondrial DNA fragments generated by HaeIII is consistent with a paucity of GGCC recognition sequences in an AT-rich mitochondrial genome (Croft and Varga 1994Citation). The HaeIII restriction patterns generally correlate with the taxonomic status of the Aspergillus isolates examined. An aggregate of species including A. flavus, A. oryzae, A. parasiticus, A. sojae, and A. subolivaceus shared a readily identifiable four-band HaeIII RFLP profile that distinguished this group from the other species. The HaeIII profiles of A. flavus and A. nomius were similar, but three of the A. nomius bands were slightly larger than the corresponding bands in the A. flavus profile. Mitochondrial DNA of A. nomius has been reported to be about 1 kb larger than the 30 kb of A. flavus and A. parasiticus (Moody and Tyler 1990aCitation) and could account for this difference. Aspergillus tamarii and A. flavofurcatis shared a unique five-band mitochondrial DNA HaeIII pattern. The shared mitochondrial RFLP profile of A. tamarii and A. flavofurcatis is consistent with referenced, but unpublished nuclear DNA hybridization data, which indicates the unity of these two species (Samson 1994Citation). The identity of the DNA HaeIII profiles was established by examining restriction digests of purified mitochondrial DNA of A. sojae and A. tamarii. Other members of section Flavi examined, A. leporis, A. avenaceus, and A. zonatus, possessed unique HaeIII RFLPs; confirmation of a mitochondrial origin for these profiles was not attempted.

Species with the A. flavus-type HaeIII RFLP were differentiated when AseI and DraI digests of total cellular DNA were probed with cloned HindIII fragments of A. nidulans mitochondrial DNA. AseI polymorphisms split the A. flavus, A. oryzae, and A. subolivaceus cluster from the A. parasiticus, A. sojae, and A. nomius cluster, while DraI split A. nomius from A. parasiticus and A. oryzae. A total of eight DNA haplotypes encompassing 11 species of section Flavi were observed.

The similarity of the mitochondrial DNA RFLPs of A. flavus, A. oryzae, A. parasiticus, and A. sojae is consistent with the results of ribosomal DNA sequence data (Peterson 2000Citation), nuclear DNA hybridization studies (Kurtzman et al 1986Citation), and similarities in the morphological and growth characteristics of these species (Christensen 1981Citation, Klich and Pitt 1988Citation). Mitochondrial DNA polymorphisms detected with AseI are consistent with the reported close associations of A. sojae with A. parasiticus and of A. oryzae with A. flavus (Kurtzman et al 1986Citation). Previously described SmaI RFLPs that can differentiate these four species are thought to represent nuclear DNA polymorphisms (Klich and Mullaney 1987Citation, Gomi et al 1989Citation). Moody and Tyler (1990a)Citation observed species-specific AseI and DraI RFLPs for A. flavus and A. parasiticus when purified mitochondrial DNA was examined, but also detected significant intraspecific variation. Among the seven isolates of A. flavus they examined, three AseI and four DraI RFLP patterns were found, while five isolates of A. parasiticus produced two DraI RFLPs. One possible explanation for the relative lack of intraspecific AseI and DraI polymorphisms in our study may relate to the methodology that we used. Hybridization of a cloned mitochondrial fragment to a Southern blot can detect only a portion of the mitochondrial DNA. The intraspecific variability detected by Moody and Tyler (1990a)Citation could be within regions of the mitochondrial genome that are not represented by the probes that we used in our hybridization studies. Furthermore, since none of the A. flavus or A. parasiticus isolates examined by Moody and Tyler (1990a)Citation were used in this study, it is possible that sampling differences could account for the absence of AseI and DraI intraspecific mitochondrial DNA polymorphisms observed in the study.

One isolate of A. tamarii (NRRL 428) deviated from the common haplotype observed for the other seven isolates of A. tamarii. The loss of a single HaeIII restriction site in the mitochondrial DNA of this strain of A. tamarii could account for the absence of two restriction fragments that are observed in the consensus A. tamarii HaeIII RFLP profile, and the appearance of a unique fragment with a size approximately equal to the sum of the missing ones. The identity of isolate NRRL 428 as A. tamarii is further supported by ribosomal DNA sequence data (Peterson et al 2000Citation).

We observed four other instances of disagreement between an isolate's mitochondrial DNA haplotype and its current taxonomic designation. Aspergillus oryzae (ATCC 14895), in contrast to the other 6 isolates of A. oryzae examined, possesses a haplotype that places it with A. parasiticus and A. sojae. Aspergillus oryzae (ATCC 14895) possesses conspicuously roughened conidia and relatively short conidiophores, which are characteristics of A. parasiticus and A. sojae (Christensen 1981Citation, Klich and Mullaney 1987Citation). Three A. flavus isolates possess mitochondrial haplotypes that differ from the A. flavus consensus haplotype, but are identical to those of other section Flavi species. Aspergillus flavus (ARSEF 2157), which was deposited prior to the description of A. nomius (Kurtzman et al 1987Citation), has the A. nomius mitochondrial DNA haplotype and morphological characteristics consistent with A. nomius (Kurtzman et al 1987Citation). Aspergillus flavus (UAMH 362) could be an isolate of A. caelatus B.W. Horn as it possesses morphological and cultural characteristics such as olive green colonies, thick-walled conidia with a rough surface texture, and vesicle diameter compatible with the formal description of A. caelatus (Horn 1997Citation). The A. tamarii-type mitochondrial DNA of isolate UAMH 362 is consistent with the close taxonomic relationship of A. tamarii and A. caelatus. Aspergillus flavus (NRRL 425) possesses the standard taxonomic features of A. terricola Marchal, but mitochondrial DNA of the A. tamarii-type. Until recently, A. terricola was regarded as a member of section Wentii; however, analysis of its ribosomal DNA sequence has led to its proposed reassignment to section Flavi (Peterson 1995Citation, 2000Citation). An examination of the mitochondrial DNA of A. terricola isolates may help clarify the exact relationship of A. terricola to the other species of section Flavi. The apparent intraspecies variation of mitochondrial DNA of A. flavus observed in this study could represent three cases of species misidentification. However, it is also possible that this intraspecific mitochondrial DNA variation reflects the existence of cryptic A. flavus species (Geiser et al 1998Citation, Taylor et al 1999Citation).

The characterization of mitochondrial DNA is a useful adjunct to the standard morphological and physiological characteristics used to determine the taxonomic status of isolates in Aspergillus section Flavi. Mitochondrial DNA RFLPs allow a clear differentiation between the A. flavus–A. oryzae group and the A. parasiticus–A. sojae group. In addition, five other mitochondrial types were identified representing A. tamarii, A. leporis, A. nomius, A. avenaceus, and A. zonatus. The utility of mitochondrial DNA haplotypes in making taxonomic decisions will depend upon establishing more precisely the extent of intraspecific variation of member species of Aspergillus section Flavi.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. James White of St. Bonaventure University for assistance in identification of Aspergillus species. Appreciation is also expressed to Dr. Stephen Peterson of Agricultural Research Service Culture Collection (NRRL), Peoria, Illinois, and Dr. Richard Humber of USDA-ARS Collection of Entomological Fungal Cultures (ARSEF), Ithaca, New York, for providing many of the fungal isolates used in this study.


   FOOTNOTES
 
1 Corresponding author, jkupski{at}sbu.edu Back

Accepted for publication April 15, 2002.


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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