Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most
important diseases of this cereal in the south Western Cape Province of
South Africa. This fungus exists as two different types (forms), namely a nettype
and a spot-type that are distinguished by differential symptom expression
on barley leaves. Based on this specific plant pathological difference a series
of studies of agricultural importance were executed to investigate the effects
of sexual recombination between these two types. In addition, studies were
done to determine the difference between local net- and spot-type populations
with regards to population structure and fungicide sensitivity. This dissertation
therefore, consists of a collection of separate publications and as a result a
certain degree of redundancy has been unavoidable.
Recombination is one of the most important evolutionary forces
involved with sexual reproduction. In plant-fungal agricultural ecosystems this
may result in pathogenic fungal populations adapting more rapidly to control
programs such as fungicide applications. The first section of the review in
part 1 of this dissertation covers different aspects of sexual reproduction in
ascomycetes, specifically focussing on mating-type genes, vegetative
incompatibility and recombination. The major part of the review is then
dedicated to various plant pathological aspects of P.teres, specifically
addressing the differences between the two types, and in various cases
highlighting the significance of sexual recombination within and between the
net- and spot-type.
Using morphological criteria for identification purposes there have been
many conflicting reports concerning the identity of leaf spot isolates in the
Western Cape Province of South Africa. In part 2, the correct identity was
eventually achieved employing mating studies and molecular markers .: This
was accomplished after single ascospores were obtained from pseudothecia
after in vitro mating had occurred between a verified P. teres net-blotch isolate
from Denmark and a representative Pyrenophora leaf spot isolate from South
Africa. Using amplified fragment length polymorphism (AFLP) and RAPD
markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials
also confirmed that recombination had taken place during mating.
Inoculations were conducted on the differential cultivars susceptible to the
net-blotch and leaf spot forms. The two parents induced typical net-blotch or
leaf spot symptoms whereas the progeny mostly induced a jagged spot
symptom on each cultivar. Fungicide sensitivity tests using the ergosterol
biosynthesis inhibitors showed that, due to recombination, some progeny
could have increased resistance to these fungicides. Due to mating and
subsequent recombination between a net blotch isolate of P. teres and a
representative leaf spot isolate, it was concluded that the latter was P. teres f.
maculata.
Fifteen of the net-spot hybrid progeny (F1) produced from the mating
study in Part 2 were screened in Part 3 to assess their viability and genetic
stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of
the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and
Clipper (both differentially susceptible to spot-type isolates) produced
intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from
foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a
healthy set of seedlings produced similar intermediate symptoms. RAPDs
conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny
revealed profiles similar to those obtained for F1 isolates. RAPD molecular
data, therefore, indicated that hybrid progeny of this net x spot mating were
genetically stable after having been subjected to two repetitive inoculation and
reisolation cycles. Phylogenetic analysis of DNA sequences of the internal
transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA
gene and the 5' end partial histone-3 gene confirmed the genetic stability of
the hybrid progeny. These results also indicated that the hybrid progeny
produced consistent symptoms throughout the series of experiments, and
maintained their virulence to the differential cultivars screened.
Both types of P. teres are prevalent in the south Western Cape
Province of South Africa, found on susceptible cultivars often grown within
close proximity of each other. In Part 4, a net- and spot-type population were
characterised in terms of their population structure using RAPD markers.
Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields,
diagonal to one another. A total of 65 loci were produced of which 54 were
polymorphic. Total gene diversities determined for all loci resulted in mean
indices of 0.063 and 0.082 being obtained respectively for the net- and spottype
populations. A coefficient of genetic differentiation (Gs) of 0.0149 was
obtained between sites within populations while a coefficient (GT) of 0.63 was
obtained between the two populations. Genotypic variation revealed 13
distinct multilocus genotypes (haplotypes) in the net-type population while
there were 12 in the spot-type population. UPGMA cluster analysis done on
the two populations together with six progeny from the mating between a netand
spot-type isolate resulted in three main clusters being produced, one for
each population and one for the progeny. One isolate collected from the nettype
population also contained a unique spot-type RAPD fragment. This
suggested that sexual recombination may be taking place between isolates of
the net- and spot-type under field conditions.
Fungicide application is the most important method used in the control
of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values)
of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol
demethylation inhibiting fungicides were determined, based on the inhibitory
effect on radial mycelial growth. The fungicides evaluated were triadimenol,
bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and
spot-type isolates revealed strong resistance to triadimenol while flusilazole
was shown to be the strongest inhibitor of fungal growth. Spot-type isolates
showed a higher resistance than net-type isolates to all five fungicides
screened. The ICsD values indicated significant differences between four of the
fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The
ICsD values between propiconazole and bromuconazole were not significant.
This study suggested that spot-type isolates showed a higher degree of
resistance to commercially used fungicides than net-type isolates.
The overall conclusion of this study is that the spot-type of P. teres is
the pathogen associated with leaf spots of barley in the south western Cape
province of South Africa and not P. japonica as earlier reported. Together
with the net-type, both types exist as genetically variable populations in this
barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to
one another in which either net- or spot-type susceptible cultivars are being
cultivated may lead to sexual progeny being produced. This in turn may lead
to an increased rate at which fungal populations may become resistant to
commercially used fungicides. It is furthermore suggested that an alternative
fungicide seed treatment is used instead of triadimenol due to high resistance
of P. teres to this fungicide. / AFRIKAANSE OPSOMMING: Netvlek op gars is een van die belangrikste siektes van hierdie graansoort in die
suidelike deel van die Westelike Kaapprovinsie. Dié siekte word veroorsaak deur
die swam Pyrenophora teres. Hierdie swam kom voor as twee verskillende tipes,
naamlik 'n net-tipe en 'n kol-tipe wat onderskei word op grand van die voorkoms
van hulle simptome op garsblare. Hierdie planpatologiese verskil in ag genome,
is 'n reeks studies van landboukundige waarde uitgevoer om die effek van
geslagtelike rekombinasie tussen die twee tipes te ondersoek. Daarbenewens is
ook studies uitgevoer om om die verskil te bepaal tussen plaaslike net- en koltipe
populasies ten opsigte van populasiestruktuur en fungisiedsensitiwiteit.
Hierdie verhandeling bestaan dus uit 'n versameling afsonderlike publikasies en
as gevolg daarvan is daar onvermydelik'n mate van oorvleueling.
Rekombinasie is een van die belangrikste evolusionêre kragte betrokke by
geslagtelike voortplanting. In plant-swam landboukundige ekostelsels kan dit
veroorsaak dat patogene swampopulasies vinniger aanpas by beheerpragramme
soos fungisiedtoediening. Die eerste gedeelte in deel 1 van hierdie verhandeling
dek die verskillende aspekte van geslagtelike voortplanting van ascomycetes,
met spesifieke verwysing na paringstipe gene, vegetatiewe onverenigbaarheid
en rekombinasie. Die grootste gedeelte van die oorsig word gewyaan verskeie
plantpatologiese aspekte van P. teres,en wys veralop die verskille tussen die
twee tipes. In verskeie gevalle word die betekenis van geslagsrekombinasie
binne en tussen die net- en koltipe uitgelig.
Deur morfologiese kenmerke vir identifikasiedoeleindes te gebruik, is daar
baie teenstrydige verslae rakende die identifikasie van blaarvlekisolate in die
Westlike Kaapprovinsie van Suid-Afrika. In deel 2 is die korrekte identifikasie
eventueel verkry deur gebruik te maak van paringstudies en molekulêre merkers.
Dit is bereik nadat enkel ascospore verkry is uit pseudothecia gevorm na in vitro
paring plaasgevind het tussen 'n bevestigde P. teres netvlek isolaat uit
Denemarke en 'n verteenwoordigende Pyrenophora blaarvlekisolaat van Suid-
Afrika. Deur gebruik te maak van versterkte fragmentlengte polimorfisme [AFLP] en RAPD merkers, is rekombinasie gedemonstreer in die nasate wat DNA
bandpatrone gehad het wat verskil het van dié van die "ouer" isolate.
Patogenisiteitstoetse het ook bevestig dat rekombinasie tydens paring
plaasgevind het. Inokulasies is uitgevoer op die verskillende cultivars wat
vatbaar is vir die netvlek en blaarvlek vorme. Die twee ouers het tipiese netvlek
of blaarvlek simptome veroorsaak, terwyl die nasate hoekige vlekke veroorsaak
het op elke cultivar. Toetse vir fungisiedsensitiwiteit deur gebruik van die
ergosterol biosintese inhibeerders het gewys dat a.g.v. rekombinasie sekere
nasate verhoogde weerstand teen hierdie fungisiedes het. As gevolg van paring
en daaropvolgende rekombinasie tussen 'n netvlek isolaat van P. teres en 'n
verteenwoordigende blaarvlek isolaat is afgelei dat laasgenoemde P. teres f.
maculata is. Vyftien van die netvlek hibried nakomelinge (F1) verkry van die
paringstudie in deel 2 is ondersoek in deel 3 om hul lewensvatbaarheid en
genetiese stabiliteit te bepaal. Hibried nasate (F1) geïnokuleer op garssaailinge
bestaande uit die volgende cultivars: Stirling (soms vatbaar vir net-tipe isolate) ,
B87/14 en Clipper (albei soms vatbaar vir kol-tipe isolate) het intermediêre
simptome op al die cultivars veroorsaak. Akseniese kulture (F1-1) geïsoleer uit
blaarletsels gevolg deur herhaalde inokulasie en isolasie (F1-2) op 'n gesonde stel
saailinge het dieselfde intermediêre simptome veroorsaak. RAPDs uitgevoer
met twee 10-mer inleiers op al die isolate van F1-1 en F1-2 nasate het profiele
opgelewer soortgelyk aan dié wat vir F1 isolate verkry is. RAPD molekulêre data
het dus gewys dat die hibried nasate van hierdie net x kol paring geneties stabiel
was nadat dit onderwerp is aan twee inokulasie en reïsolasie siklusse.
Genetiese stabiliteit van die hibried nageslag is bevestig deur filogenetiese
analise van die DNA volgorde van die interne getranskribeerde spasieerders
(ITS1 en ITS2) reg langs die 5.8S nukluêre ribosomale RNA geen en die 5' end
gedeeltelike histoon-3 geen. Hierdie resultate het ook gewys dat die hibried
nasate konstante simptome getoon het tydens die hele reeks eksperimente en
hulle virulensie behou het vir die kultivars wat getoets is.
Beide tipes van P. teres kom algemeen voor in die suidelike deel van die
Westelike Kaapprovinsie en word gevind op vatbare cultivars wat dikwels naby mekaar groei. In deel 4 is 'n net- en kol-tipe populasie gekarakteriseer in terme
van hulle populasiestruktuur deur gebruik van RAPD merkers. Monsters is
versamel van geïnfekteerde garsblare van twee aparte kwadrante in elke
saailand. Die twee kwadrante is geplaas in die hoeke van die saailand,
diagonaal tot mekaar. 'n Totaal van 65 lokusse is gevorm, waarvan 54
polimorfies was. Die algehele genetiese verskeidenheid bepaal vir alle lokusse,
het gelei tot gemiddelde indekse van 0.063 en 0.082 soos gevind vir die net- en
kol-tipe populasies. 'n Koëffisiënt van genetiese differensiasie (Gs ) van 0.0149
is gevind tussen gebiede tussen populasies, terwyl 'n koëffisiënt (GT) van 0.63
gevind is tussen die twee populasies. Genotipiese variasie het 13 duidelike
multilokus genotipes (haplotipes) getoon in die net-tipe populasie, terwyl daar
twaalf was in die kol-tipe populasie. UPGMA groeperingsanalises wat gedoen is
op die twee populasies tesame met ses nasate van die paring van 'n net- en koltipe
isolaat het tot gevolg gehad dat drie hoof groepe gevorm is, een vir elke
populasie en een vir die nasate. Een isolaat wat versamel is, van die net-tipe
populasie het 'n unieke kol-tipe RAPD fragment bevat. Dit wys daarop dat
geslagtelike rekombinasie in veldomstandighede mag voorkom tussen isolate
van die net- en kol-tipe.
Fungisiedtoediening is die belangrikste metode wat gebruik word om
netvlek in Suid-Afrika te beheer. In deel 5 is die fungisiedsensitiwteit (Ieso
waardes) van 89 enkelkonidiale isolate (46 net-tipe en 43 kol-tipe) van P. teres
teen sterol demetielasie inhiberende fungisiedes bepaal, op die basis van die
onderdrukkende effek op die radiale groei van die miselium. Die volgende
fungisiedes is geëvalueer: triadimenol, bromuconazole, flusilazole, propiconazole
en tebuconazole. Beide net- en kol-tipe isolate het 'n sterk weerstand teen
triadimenol openbaar, terwyl flusilazole gevind is as die sterkste onderdrukker
van swamgroei. Kol-tipe isolate het 'n hoër weerstand as die net-tipe isolate teen
al vyf fungisiedes wat getoets is, gehad. Die lesowaardes het aangedui dat daar
beduidende verskille tussen vier van die fungisiedes IS (triadimenol,
tebuconazole, flusilazole en propiconazole). Die leso waardes tussen
propiconazole en bromuconazole was nie beduidend nie. Die gevolgtrekking van hierdie studie is dus dat die kol-tipe isolate 'n hoër graad van weerstand teen
kommersiëel gebruikte fungisiedes as die net-tipe isolate gehad het.
Die algehele gevolgtrekking van hierdie studie is dat die kol-tipe van P.
teres, die patogeen is wat geassosieer word met blaarvlekke op gars in die
suidwestelike Kaapprovinsie van Suid-Afrika, en nie P. japonica soos voorheen
gerapporteer nie. Tesame met die net-tipe, kom altwee tipes voor as geneties
veranderlike populasies in hierdie gars verbouingstreek. Paring tussen die twee
tipes lei tot geslagtelike nasate wat geneties stabiel is. Dit impliseer dat
aangrensende garsvelde waarop net- óf kol-tipe vatbare kultivars verbou word,
mag lei tot die produksie van geslagtelike nasate. Dit kan weer lei tot 'n
verhoogde tempo waarteen swampopulasies weerstandbiedend teenoor
kommersiële fungisiedes raak. Daar word verder ook voorgestel dat alternatiewe
fungisied saadbehandelings gebruik word in plaas van triadimenol as gevolg van
verhoogde weerstand van P. teres teenoor laasgenoemde.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52286 |
| Date | 12 1900 |
| Creators | Campbell, Graham F. (Graham Findlay) |
| Contributors | Crous, P. W., Lucas, J. A., Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 158 p. : ill. |
| Rights | Stellenbosch University |
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