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Use of flow cytometry, fluorescence microscopy, and PCR-based techniques to assess intraspecific and interspecific matings of Armillaria species

Published online by Cambridge University Press:  18 April 2001

Mee-Sook KIM ,
Ned B. KLOPFENSTEIN ,
Geral I. McDONALD ,
Kathiravetpillai ARUMUGANATHAN  and
Anne K. VIDAVER
Mee-Sook KIM
Affiliation:
Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722, USA. E-mail: meeskim@uidaho.edu Present address: Department of Forest Resources, University of Idaho, Moscow, ID 83844, USA.
Ned B. KLOPFENSTEIN
Affiliation:
USDA Forest Service, RMRS, Forestry Sciences Lab, 1221 South Main, Moscow, ID 83843-4298, USA.
Geral I. McDONALD
Affiliation:
USDA Forest Service, RMRS, Forestry Sciences Lab, 1221 South Main, Moscow, ID 83843-4298, USA.
Kathiravetpillai ARUMUGANATHAN
Affiliation:
Center for Biotechnology, University of Nebraska, Lincoln, NE 68588-0665, USA.
Anne K. VIDAVER
Affiliation:
Department of Plant Pathology, University of Nebraska, Lincoln, NE 68583-0722, USA. E-mail: meeskim@uidaho.edu
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Abstract

For assessments of intraspecific mating using flow cytometry and fluorescence microscopy, two compatible basidiospore-derived isolates were selected from each of four parental basidiomata of North American Biological Species (NABS) X. The nuclear status in NABS X varied with basidiospore-derived isolates. Nuclei within basidiospore-derived isolates existed as haploids, diploids (doubled haploids), or a mixture of haploids and diploids (doubled haploids). Depending on the nuclear status of the basidiospore-derived lines of NABS X, intraspecifically mated cultures can exist as diploids or tetraploids, and possibly triploids or aneuploids under in vitro conditions. Based on previous in vitro mating studies, seven basidiospore isolates were specifically selected to assess rare, interspecific mating among Armillaria cepistipes, A. sinapina, NABS X, and NABS XI. Cultures from basidiospore-derived isolates were paired to produce four interspecifically paired cultures, and matings were assessed using flow cytometry and restriction fragment length polymorphism (RFLP) analyses. Based on flow cytometric analysis, the A. cepistipes isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolate exhibited compatibility with a NABS X isolate, and the A. sinapina isolates were individually compatible with isolates of NABS X and NABS XI. Mean fluorescence intensities of A. cepistipes × NABS X, A. sinapina × NABS X, and A. sinapina × NABS XI mated cultures revealed a triploid or tetraploid nuclear status compared to the haploid or diploid (doubled haploid) nuclear status of initial basidiospore-derived isolates. Polymerase chain reaction (PCR) and RFLP of the intergenic spacer (IGS) region generated banding patterns for basidiospore-derived isolates and mated cultures. Four species-specific RFLP banding patterns were observed in basidiospore-derived isolates of A. cepistipes, A. sinapina, NABS X, and NABS XI. PCR-RFLP analysis showed combined banding patterns from mated cultures. Flow cytometry and PCR-RFLP analysis are effective tools to assess matings of Armillaria species.

Type
Research Article
Information
Mycological Research , Volume 105 , Issue 2 , February 2001 , pp. 153 - 163
Copyright
© The British Mycological Society 2001

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