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Genetic variation of Phymatotrichum omnivorum was analyzed by restriction fragment length polymorphisms (RFLPS) and random amplified polymorphic DNA (RAPD). Two populations have been extensively surveyed. One population consist of cultures of P. omnivorum isolates originally from different hosts and/or different geographic location. The other population is a collection of many isolates from within a single site of infestation in the Blacklands of Texas. For RFLP analysis the DNA was probed with radioactive probes: a Saccharomyces cerevisiae ribosomal DNA (rDNA) probe, a Neurospora crassa rDNA probe, a N. crassa beta-tublin gene probe, and over 50 mitochondrial-DNA fragments from a P. omnivorum mitochondrial-DNA (mtDNA) library, along with several probes from a P. omnivorum nuclear-DNA (nDNA) library. An isolate from P. omnivorum from cotton in south central Texas constantly differed from other isolates with respect to RFLP patterns obtained from DNA digested with six restriction enzymes (BAM Hi, DRA 1, ECO RI, HIND III, MSP 1, and PVU 11) and probed with the S. cerevisiae rDNA and P. omnivorum mtDNA. This culture has the distinctive morphological characteristics of P. omnivorum. No variation was discovered within the population from the single infestation locus using RFLPS. Since little genetic variation was detected by RFLPS, another molecular tool, RAPD, was used to determine if variation could be found within or among the populations of P. omnivorum. Initially, 25 random 10-mers were screened for banding patterns and clarity; from this group, seven primers were chosen to screen both populations. Each isolate received a seven digit identification number based on banding patterns per primer. Based on these numbers, eight groups of P. omnivorum isolates were determined. The following observations could be concluded from these numbers. First, every isolate from a host other than cotton falls into one single group, void of any cotton isolates. Second, three cotton isolates from different geographic regions fall into three separate groups based on banding patterns which are unique only to themselves. Thirdly, the cotton isolates fall into seven groups which are not correlated to broad geographic location. Finally, the variation within a population from a single year appears to be small. A separate experiment checked for variation within each isolate. Every isolate, whether from cotton or from another host, was placed in the greenhouse and tested for pathogenicity on cotton. Once pathogenicity was determined, P. omnivorum was reisolated and compared to the starting isolate. In each instance using the seven primers from the RAPD experiments, no variation was detected between the two cultures of the original isolate. There are differences among isolates of P. omnivorum based on screening of the total genomic DNA with random oligonucleotide primers. However, the rate of change appears to be slow, at least greater than one life cycle of this soilborne fungus. |
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©National Cotton Council, Memphis TN |
Document last modified Sunday, Dec 6 1998
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