Molecular identification of fungi isolated from stem tissue of Upland cotton (Gossypium hirsutum)
L. Augusto Becerra-LopezLavalle A B , Jennifer A. Saleeba A and Bruce R. Lyon A CA Australian Cotton Cooperative Research Centre, School of Biological Sciences A12, The University of Sydney, Sydney, NSW 2006, Australia.
B Present address: CSIRO Division of Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia.
C Corresponding author. Email: brucel@bio.usyd.edu.au
Australian Journal of Botany 53(6) 571-578 https://doi.org/10.1071/BT04092
Submitted: 25 June 2004 Accepted: 17 January 2005 Published: 30 September 2005
Abstract
Molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, random amplification of polymorphic DNA (RAPD) fingerprinting, and DNA sequencing and database comparison, were employed to identify fungi isolated from field-grown cotton plants (Gossypium hirsutum L.). DNA fragments of between 510 and 590 bp, representing the two rDNA (rDNA) internal transcribed spacers (ITS1 and ITS2) and the intervening 5.8S rRNA gene, were amplified from the fungi with eukaryotic consensus primers. Subsequent digestion with the restriction endonucleases AluI, CfoI, HaeIII, HinfI and HpaII enabled the allotment of all 57 isolates to 13 different groups. Restriction analysis was supported by RAPD–PCR analysis of multiple isolates and rDNA sequencing of representative fungi from each group. Sequence alignment and comparison with rDNA sequences of other fungi available in GenBank allowed for putative identification of three different taxa of Fusarium, two taxa each of Cladosporium, Diaporthe and Nectria, and one taxon each of Alternaria, Ampelomyces, Bartalinia, Phaeosphaeria and Rhizoctonia. Many of the stem-colonising fungi identified in this study are either pathogenic on cotton or have elsewhere been found to act as biocontrol agents.
Acknowledgments
The authors acknowledge the financial support of The University of Sydney and the Australian Cotton CRC. We are indebted to Myong Hee Hong for technical assistance, and thank students of the senior advanced molecular genetics class in the School of Biological Sciences for their contributions to the DNA sequencing.
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