Comparisons of levels of genetic diversity among Streptomyces scabies isolates of South Africa using various DNA techniques

[摘要] ENGLISH ABSTRACT:Streptomyces spp. are responsible for a large proportion of the world-wide quality deterioration ofpotatoes causing a potato tuber disease called cornmon scab. Determining the genetic diversity of theStreptomyces spp., especially the main pathogen, S. scabies, has been a prerequisite for the ultimatecontrol of common scab. Techniques responsible for the classification and determination of geneticdiversity have improved with advances in DNA technology. Analysis of South African (S.A.)S. scabies isolates has been focusing on the organisms' morphology, physiology, pathogenicity andmelanin production, but the classification of S. scabies using DNA techniques has not yet beenexplored.In this study various DNA techniques were screened for optimal use in determining the geneticdiversity within and among isolates of S. scabies. Bacteria had been sampled from the main potatoproducing regions in S.A. and a few other regions. The techniques explored included RAPDs, AFLPs,RAMS, Rep-PCR, 16S rDNA sequencing and ITS analysis. The first three techniques had to beabandoned due to non-reproducibility between the same isolate extracted on separate occasions andITS analysis was abandoned due to sequencing difficulties. Of the three Rep-PCR techniques tested(BOX, ERIC and REP), BOX was selected because it produced the clearest and most reproducibleresults. BOX-PCR and 16S rDNA sequencing were therefore ultimately selected as the methods toanalyse the genetic diversity of the S. scabies isolates.Information concerning the pathogenicity of the isolates was supplied by the Vegetable andOrnamental Plant Research Institute of the Agricultural Research Council (VOPI, ARC, Roodeplaat).A brief analysis of the pathogenicity prediction of the isolates in this study was explored with the PCRtechnique. Presence of the necJ gene was previously shown to be an indication of the pathogenicitywithin the Streptomyces spp. group. PCR analysis is based on the amplification of a O.72kb fragment(necl) in pathogenic isolates which was absent in non-pathogenic isolates. However, in this study thetest for pathogenicity lacked specificity and sensitivity and some of the problems experienced includednon-reproducibility between PCR reactions and the presence of the pathogenic fragment in the nonpathogenicisolates (as designated by VOPI, ARC). These observations led to the conclusion that thistechnique is not an ultimate test for pathogenicity of S. scabies isolates in a South African context. The genetic distances and similarity matrices of the Rep-PCR results were calculated using Nei'sgenetic distance calculation (Nei M, 1975). Clusters from these matrices were constructed using theunweighted pair group average (UPGMA) with the PAUP4 package. The clusters for the 16S rDNAsequences were formed with the Neighbor Joining (NJ) method and the PAUP4 package. The NJ treesdo not take small sequencing differences into account, therefore a Parsimony Network had to beconstructed.The trees obtained with the 16S rDNA sequencing techniques grouped most S. scabies isolates into onemajor group with a 100% bootstrap robustness of this group. More genetic diversity was illustrated bythe BOX-PCR technique and the isolates were generally grouped according to their different regions oforigin. However, the bootstrap values were low, indicating a lack of robustness regarding the BOXPCRclustering. This was not unexpected as the number of data points employed in the BOX techniqueis very limited. Both techniques revealed unexpected grouping of a few isolates. Their isolatedpositions could be attributed to possible misclassification or to the fact that they could be geneticallydifferent S. scabies isolates. Streptomyces spp. (other than S. scabies) displayed enough differences toplace them in their own distinct groups using both techniques. Comparison of the cluster resultsobtained in this study did not correlate to the data supplied by the VOPI, ARC (morphology,physiology, pathogenicity and melanin production) which revealed differences between the S. scabiesisolates within their respective regions.The lack of diversity displayed by the 16S rDNA technique can be attributed to the fact that only alimited section of the genome is involved making it inappropriate for intra-species genetic diversityanalysis. The BOX technique takes various loci within the genome but is still not ideal for a thoroughgenetic diversity analysis. This study represents the first attempt to determine the genetic diversity of S.scabies in S.A. on DNA level.

[发布机构] Stellenbosch University