Characterization and control of Phaeomoniella chlamydospora in grapevines
Date
2000-12
Authors
Groenewald, Michelle
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Petri grapevme decline, also known as black goo, slow die-back and
Phaeoacremonium grapevine decline, causes significant losses of young vines worldwide.
Species of Phaeoacremonium, Phaeomoniella chlamydospora and related
genera are associated with this grapevine disease. This study investigates the
Phaeoacremonium-complex and Phaeomoniella chlamydospora, focussing on the
species isolated from grapevines. Fungicide sensitivity of Pa. chlamydospora and the
possibility of employing molecular techniques for the detection of Pa. chlamydospora
in grapevines were also investigated.
In an overview of the literature on Petri grapevine decline the disease history
and the relatedness of Petri grapevine decline to esca is discussed. Petri grapvine
decline occurs in propagation material or young vines. Infected material can appear
asymptomatic and therefore the possibilities of molecular techniques for identification
were also investigated in the literature.
In South Africa Pa. chlamydospora is the dominant organism causing Petri
grapevine decline and therefore different fungicides were evaluated to control this
fungus. Six isolates of Pa. chlamydospora, from Stellenbosch, Wellington, Somerset
West and Malmesbury of Western Cape province, South Africa, were screened
against twelve fungicides testing their effect on mycelial inhibition in vitro. These
fungicides included benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione,
kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene,
tebuconazole and thiram. Results provided the base-line sensitivity of South African
isolates of Pa. chlamydospora. Benomyl, fenarimol, kresoxim-methyl, prochloraz
manganese chloride and tebuconazole were the most effective (with EC50 values
ranging from 0.01 to 0.05 ug/ml) for inhibiting mycelial growth of Pa.
chlamydospora in vitro. This in vitro test gave a good indication of which fungicides
could be selected for further studies in glasshouses and nurseries.
The molecular phylogeny of Phaeoacremonium and Phaeomoniella isolates
from grapevines of South Africa, or isolates obtained from the Centraalbureau voor
Schimmelcultures (CBS) in the Netherland, were investigated. Sequence data were
created from the rONA region and partial B-tubulin gene of 33 of these isolates using
the PCR technique. This sequence data were analysed with PAUP* version 4.Ob2a.
An analysis of the sequence data confirmed the genus Phaeomoniella to be distinct from Phaeoacremonium (Pm.) based on DNA phylogeny. Although morphologically
similar, the species status of Pm. aleophi/um and Pm. angustius was confirmed with
DNA phylogeny and cultural characteristics. Pm. aleophilum has an optimum growth
rate at 30°C and the ability to grow at 35°C, where as Pm. angustius has an optimum
growth rate at 25°C and cannot grow at 35°C_ Pm. viticola was shown to be
synonymous with Pm. angustius, and a new species, Pm. mortoniae, was newly
described from grapevine occurring in California. Futhermore, Pm. aleophilum was
newly reported from South Africa and grapevine isolates thought to be Pm. inflatipes
were all re-identified as Pm. aleophilum. These findings therefore also shed some
doubt on the possible role of Pm. inflatipes in Petri grapevine decline. It was
confirmed that Pa. chlamydospora, Pm. aleophilum and Pm. angustius are the species
involved in Petri grapevine decline. Pm. mortoniae was isolated from grapevines, but
its pathogenicity should still be confirmed and the role of Pm. injlatipes in Petri
grapevine decline remains unclear.
Pa. chlamydospora has been routinely isolated from symptomless propagation
and nursery material. Because the disease can take years to develop, it is crucial that
healthy propagation material is used at planting. Pa. chlamydospora is a slowgrowing
fungus, and positive identification from symptomless grapevine tissue can
take up to 4 wks. The possibility of employing molecular techniques for the detection
of Pa. chlamydospora in apparently healthy grapevines was investigated. Speciesspecific
primers (PCLI and PCL2) based on the regions ITSI and ITS2 were designed
for Pa. chlamydospora. These primers were highly sensitive and amplification was
achieved from genomic DNA of Pa. chlamydospora from as low as 16 pg.
Phaeoacremonium spp., related genera and common fungal taxa from grapevines
were tested with these primers, but positive amplification was achieved for Pa.
chlamydospora only. The presence of Pa. chlamydospora in symptomless grapevine
tissue culture plants was confirmed by PCR within 24 hours. These primers therefore
allow rapid and accurate identification of Pa. c~lamydospora. Testing on a larger
scale with nursery material should be conducted to determine the feasibility of using
these species-specific primers in the grapevine industry.
AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.
AFRIKAANSE OPSOMMING: Petri-terugsterwing van jong wingerde, ook algemeen bekend as "black goo" en Phaeoacremonium-terugsterwing, veroorsaak wêreldwyd groot geldelike verliese in die wingerdbedryf. Spesies van Phaeoacremonium, Phaeomoniella chlamydospora en verwante genera word met hierdie wingerdsiekte geassosieer. In die tesis word In oorsig gegee van die geskiedenis van hierdie siekte, die verwantskap tussen Petriterugsterwing en esca, en moontlike maniere van siektebestuur. Swamme wat by die siektekompleks betrokke is, kan in simptoomlose plantweefsel voorkom en daarom is die moontlikhede van die gebruik van molekulêre tegnieke vir swamidentifikasie in oënskou geneem. In Suid-Afrika is Pa. chlamydospora die dominante swam wat met Petriterugsterwing geassosieerword, gevolglik is verskillende fungisiedes vir die chemiese beheer van Pa. chlamydospora geëvalueer. Ses isolate van Pa. chlamydospora, versamel vanaf verskillende areas in die Wes-Kaap provinsie, is in dié studie gebruik. Benomyl, chlorothalonil, fenarimol, fosetyl-Al, iprodione, kresoxim-methyl, mancozeb, metalaxyl, prochloraz manganese chloride, quintozene, tebuconazole en thiram se effek op miselium inhibisie van Pa. chlamydospora is in vitro geëvalueer. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole was die mees effektiewe middels. Die effektiewe konsentrasie waarby 50% van die miselium groei geïnhibeer is (EKso),was tussen 0.01 en 0.05 ug/ml vir die mees effektiewe groep middels. Benomyl, fenarimol, kresoxim-methyl, prochloraz manganese chloride en tebuconazole het in vitro goeie potensiaal getoon, en verder toetse moet in vivo uitgevoer word. 'n Molekulêre studie is van Phaeoacremonium en Phaeomoniella isolate; verkry uit Suid-Afrikaanse wingerde, of vanaf die "Centraalbureau voor Schimmelcultures" (CBS) van Nederland; gedoen. Deur van die PKR tegniek gebruik te maak, is die basispaaropeenvolgingsdata van 33 isolate, van die ITSl, 5.8S, ITS2 rDNA area en die gedeeltelike B-tubullen geen verkry. Gekombineerde molekulêre data het die teorie ondersteun dat Phaeomoniella (Herpotrichiellaceae) gedistansieerd is van Phaeoacremonium (Magnaporthaceae). Pm. aleophilum en Pm. angustius was morfologies moeilik onderskeibaar, maar kon op grond van molekulêre data en kulturele eienskappe onderskei word. Pm. aleophilum se optimum groeitemperatuur was by 30°C en die swam besit die vermoë om by 35°C te groei. Pm. angus/ius se optimum groeitemperatuur was by 25°C, maar het nie by 35°C gegroei nie. 'n Studie van molekulêre en kulturele eienskappe het getoon dat Pm. angus/ius en Pm. viticola sinoniem is. 'n Nuwe spesie, Pm. mortoniae, wat uit wingerde van Kalifornie geïsoleer is, is beskrywe. Verder is Pm. aleophilum die eerste keer in Suid-Afrikaanse wingerde aangetref en Pm. tnflatipes isolate, wat vanuit wingerde geïsoleer is, is almal met molekulêre data gewys om Pm. aleophilum te wees. Hierdie bevindinge trek die rol van Pm. inflatipes in Petri-terugsterwing van wingerde in twyfel. Phaeomoniella chlamydospora IS m voortplantingsmateriaal en kwekerystokkies opgespoor. Omdat dit jare kan duur voordat siektesimptome ontwikkel, is dit belangrik om vroegtydig te weet of jong stokkies met Pa. chlamydospora geïnfekteer is. Pa. chlamydospora groei baie stadig en positiewe identifikasie van simptoomlose infeksies duur tot vier weke. Die toepassing van molekulêre tegnieke vir die vinnige identifikasie van Pa. chlamydospora in wingerde is dus ondersoek. Spesie-spesifieke oligonukleotiedes (PCU en PCL2) is vir Pa. chlamydospora ontwerp. Hierdie oligonukleotiedes is uiters sensitief en genomiese DNA van Pa. chlamydospora is van so laag as 16 pg geamplifiseer. Phaeoacremonium spp., verwante genera en algemene swamme vanuit wingerdmateriaal is met die oligonukleotiedes getoets, maar positiewe amplifikasie was slegs met Pa. chlamydospora moontlik. Die teenwoordigheid van Pa. chlamydospora is binne 24 uur in asimptomatiese wingerd weefselkultuurplantjies bevestig. Hierdie oligonukleotiedes identifiseer Pa. chlamydospora vinnig en akkuraat en toetsing op 'n groter skaal moet vervolgens met kwekerymateriaal onderneem word.
Description
Thesis (MScAgric)--University of Stellenbosch, 2000.
Keywords
Grapes -- Diseases and pests, Fungal diseases of plants, Fungicide resistance