Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Streptomyces spp. are responsible for a large proportion of the world-wide quality deterioration of
potatoes causing a potato tuber disease called cornmon scab. Determining the genetic diversity of the
Streptomyces spp., especially the main pathogen, S. scabies, has been a prerequisite for the ultimate
control of common scab. Techniques responsible for the classification and determination of genetic
diversity 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 and
melanin production, but the classification of S. scabies using DNA techniques has not yet been
explored.
In this study various DNA techniques were screened for optimal use in determining the genetic
diversity within and among isolates of S. scabies. Bacteria had been sampled from the main potato
producing 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 be
abandoned due to non-reproducibility between the same isolate extracted on separate occasions and
ITS 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 reproducible
results. BOX-PCR and 16S rDNA sequencing were therefore ultimately selected as the methods to
analyse the genetic diversity of the S. scabies isolates.
Information concerning the pathogenicity of the isolates was supplied by the Vegetable and
Ornamental 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 PCR
technique. Presence of the necJ gene was previously shown to be an indication of the pathogenicity
within 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 the
test for pathogenicity lacked specificity and sensitivity and some of the problems experienced included
non-reproducibility between PCR reactions and the presence of the pathogenic fragment in the nonpathogenic
isolates (as designated by VOPI, ARC). These observations led to the conclusion that this
technique 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's
genetic distance calculation (Nei M, 1975). Clusters from these matrices were constructed using the
unweighted pair group average (UPGMA) with the PAUP4 package. The clusters for the 16S rDNA
sequences were formed with the Neighbor Joining (NJ) method and the PAUP4 package. The NJ trees
do not take small sequencing differences into account, therefore a Parsimony Network had to be
constructed.
The trees obtained with the 16S rDNA sequencing techniques grouped most S. scabies isolates into one
major group with a 100% bootstrap robustness of this group. More genetic diversity was illustrated by
the BOX-PCR technique and the isolates were generally grouped according to their different regions of
origin. However, the bootstrap values were low, indicating a lack of robustness regarding the BOXPCR
clustering. This was not unexpected as the number of data points employed in the BOX technique
is very limited. Both techniques revealed unexpected grouping of a few isolates. Their isolated
positions could be attributed to possible misclassification or to the fact that they could be genetically
different S. scabies isolates. Streptomyces spp. (other than S. scabies) displayed enough differences to
place them in their own distinct groups using both techniques. Comparison of the cluster results
obtained 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. scabies
isolates within their respective regions.
The lack of diversity displayed by the 16S rDNA technique can be attributed to the fact that only a
limited section of the genome is involved making it inappropriate for intra-species genetic diversity
analysis. The BOX technique takes various loci within the genome but is still not ideal for a thorough
genetic diversity analysis. This study represents the first attempt to determine the genetic diversity of S.
scabies in S.A. on DNA level. / AFRIKAANSE OPSOMMING: Streptomyces spp. is verantwoordelik vir 'n _groot deel van die wereld afname in aartappel kwaliteit as
gevolg van die aartappelknol siekte bruinskurf. Die bepaling van die genetiese diversiteit tussen die
Streptomyces spp., varal die hoof patogeen in die groep, S. scabies, is 'n vooreiste vir die uiteindelike
beheer van bruinskurf. Tegnieke verantwoordelik vir die klassifikasie en bepaling van genetiese
diversiteit het verbeter met vooruitgang in DNA tegnologie. Analise van Suid Afrika (S.A.) se S.
scabies isolate konsentreer op die organisme se morfologie, fisiologie, patogenisiteit en malanien
produksie, maar die klassifikasie van S. scabies met die behulp van DNA tegnieke is nog nie uitgevoer
me.
In hierdie studie is verskeie DNA tegnieke ondersoek vir optimale bepaling van genetiese diversiteit
binne en tussen S. scabies isolate van S.A Bakteriee is verkry van die hoof aartappel-produserende
areas in S.A. en ook van 'n paar ander areas. Die tegnieke wat in die studie gebruik is, het RAPDs,
AFLPs, Rep-PKR, 16S rDNA volgordebepaling en ITS analise ingesluit. Die eersgenoemde drie
tegnieke is uitgesluit as gevolg van nie-herhalende resultate tussen dieselfde isolaat geisoleer op
verskillende geleenthede. ITS analise is uitgesluit as gevolg van probleme met volgordebepaling. Rep-
PKR en 16S rDNA volgordebepaling is uiteindelik gekies as die mees geskikte metodes vir die analise
van genetiese diversiteit tussen S. scabies isolate in hierdie studie omdat albei skynbare herhaalbare
resultate gelewer het.
Inligting met betrekking tot die patogenisiteit van die isolate is voorsien deur die Groente en Sierplant
Instituut van die Landbou Navorsinsraad (YOPI, LNR). 'n Vinnige analise van die patogenisiteits
voorspelling van die isolate is uitgevoer met die PKR tegniek. Dit is voorheen aangetoon dat die
teenwoordiheid van die necJ geen dui op die patogenisiteit in die Streptomyces sp _groep. PKR analise
het 'n O.72kb fragment (necl) in patogeniese isolate geamplifiseer wat nie teenwoordig was in niepatogeniese
isolate nie. Hierdie toets vir patogenisiteit soos gebruik in hierdie studie was egter
onspesifiek en onsensitief en sommige van die probleme wat ondervind is sluit in nie-herhaalbaarheid
tussen PKR reaksies en die teenwoordigheid van die patogeniese fragment in die nie-patogeniese
isolaat (soos beskryf deur YOPI, LNR). Uit hierdie waarnemings word afgelei dat die tegniek nie 'n
geskikte toets vir patogenisiteit van S. scabies isolate in 'n S.A konteks is nie. Die genetiese afstande en ooreenkomstige matrikse van die Rep-PCR resultate is bereken met die
genetiese afstand bepaling van Nei (Nei M, 1975). Groepe is gevorm met die "unweighted pair group
average" (UPGMA) en PAUP4 pakket. Die "Neighbor Joining" (NJ) groepe is gevorm met die 16S
rDNA volgordebepaling data mbv die PAUP4 pakket. Die NJ groepering neem nie klein volgorde
verskille in ag nie en gevolglik moes'n "Parsimony Network" opgestel word.
Die groepering met die 16S rDNA volgordebepaling het meeste van die isolate in een hoof groep
geplaas met 'n 100% "bootstrap" waarde. Meer genetiese diversiteit is met die BOX-PCR tegniek
gevind en isolate was oor die algemeen gegroepeer vol gens van hul oorsprong. Die "bootstrap"
waardes vir die BOX tegniek was baie laag. Dit was nie onverwags nie, want die hoeveelheid data
punte was beperk met die BOX tegniek. Albei tegnieke het 'n aantal afwykende isolate vertoon. Hul
ge-isoleerde posisies kan toegeskryf word aan moontlike misklassifikasies van die isolaat. Die
moontlikheid dat daar wel genetiese verkille tussen die isolate is, kan egter nie uitgesluit word nie.
Streptomyces spp. (uitgesluit S. scabies) het genoeg variasie vertoon om hulle in hul eie groepe met die
gebruik van beide tegnieke te plaas. Vergelyking tussen die groepe in die studie stem nie ooreen met
die data verkry vanaf YOPl, LNR (morfologie, fisiologie, patogenesiteit en melanien produksie) nie
wat verskille tussen S. scabies isolate binne 'n sekere gebied vertoon.
Die gebrek aan diversiteit soos vertoon deur die 16S rDNA tegniek kan toegeskryf word aan die feit dat
slegs 'n beperkte gedeelte van die genoom ondersoek word, wat dit ongeskik vir intra-species genetiese
diversiteit analise maak. Die BOX tegniek neem verskeie loci in die genoom in ag, maar is steeds nie
ideaal vir deeglike genetiese diversiteit analise nie. Hierdie studie verteenwoordig die eerste poging om
die genetiese diversiteit van S. scabies in S.A. op DNA vlak te bepaal.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51658 |
| Date | 12 1900 |
| Creators | Lynch, Alisson |
| Contributors | Warnich, L., Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 124 p. : ill. |
| Rights | Stellenbosch University |
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