| Summary: | Dissertation (PhD (Agric))--University of Stellenbosch, 2005. === ENGLISH ABSTRACT: During the past few years a drastic reduction has been noted in the survival rate of
grafted grapevines in nurseries, as well as in young vineyards in the Western Cape Province of
South Africa. Circumstantial evidence suggested that Cylindrocarpon spp., which cause black
foot disease of grapevine, were associated with this decline. Black foot disease of grapevine is a
relatively new, and as yet poorly known disease affecting vines in various countries where
grapevines are cultivated. Primary aims of this research have been (1) to conduct nursery
surveys in order to determine which fungi are involved in the decline phenomenon, with special
reference to the involvement of Cylindrocarpon spp., (2) to identify and characterise the
organisms believed to be the causal organisms of black foot disease, and (3) the development of
control and/or management strategies to prevent or eradicate Cylindrocarpon infections.
Nursery grapevines were sampled at different stages from three commercial nurseries in
the Wellington area of the Western Cape Province and were investigated during the 19992000
season by means of destructive sampling. The first samples were taken in September from
callused cuttings prior to planting in nurseries. After planting, asymptomatic rooted cuttings
were selected from nurseries after 3, 6 and 9 months. Isolation studies clearly demonstrated that
different “Cylindrocarpon spp.” infected cuttings from nursery soils. These species rarely
occurred in rootstock propagation material prior to planting. At the time of planting, the
susceptible basal ends (especially the pith area) of most of the nursery cuttings are partly or even
fully exposed. Callus roots also break during the planting process, resulting in small wounds
susceptible to infection by soilborne pathogens. The isolation studies revealed that the first
infections occurred in the roots, followed by infections of the rootstocks. These infections
increased progressively during the course of the growing season.
Substantial variation in cultural and morphological characters was observed among the
Cylindrocarpon isolates obtained from the nursery survey, as well as from isolations that were
made from diseased grapevines. Morphological and phylogenetic studies were conducted to
identify these “Cylindrocarpon spp.” and to establish their association with black foot disease.
Sequences of the partial nuclear large subunit ribosomal DNA (LSU rDNA), internal transcribed spacers 1 and 2 of the rDNA including the 5.8S rDNA gene (ITS), and partial β-tubulin gene
introns and exons were used for phylogenetic inference. Phylogenetic analyses confirmed the
diversity observed among the isolates and four Cylindrocarpon-like species were identified.
One of these species was initially identified as Cylindrocarpon destructans. However, further
research revealed C. destructans to represent a species complex. Grapevine isolates of “C.
destructans” proved to be identical to the ex-type strain of Cylindrocarpon liriodendri, which
also produced a teleomorph, Neonectria liriodendri in culture. A second species was newly
described in this study as Cylindrocarpon macrodidymum (Neonectria macrodidyma). The two
remaining Cylindrocarpon-like species were placed in a new genus, Campylocarpon. The two
species were named Campylocarpon fasciculare and Campylocarpon pseudofasciculare.
Pathogenicity studies confirmed that all four species were able to reduce root and shoot mass
significantly.
Knowledge obtained pertaining to the disease cycle of black foot disease suggest that suitable
management strategies should focus on prevention of primary infection in nurseries. However,
at present, no fungicides are registered for control of this disease in South African vineyards or
nurseries. Thirteen fungicides were screened in vitro for mycelial inhibition of these pathogens.
Prochloraz manganese chloride, benomyl, flusilazole and imazalil were the most effective
fungicides tested, and were subsequently included in semi-commercial field trials. Basal ends of
grafted cuttings were dipped (1 min) in various chemical and biological treatments prior to
planting in open-rooted nurseries. Black foot pathogens were not isolated from grafted cuttings
prior to planting in nurseries. Additional treatments involved soil amendments with
Trichoderma formulations and hot water treatment (50°C for 30 min) of dormant nursery
grapevines. Field trials were evaluated after a growing season of eight months. The incidence of
black foot pathogens was not significantly and/or consistently reduced by the majority of
chemical or biological treatments. However, these pathogens were not isolated from uprooted
plants that were subjected to hot water treatment. It is therefore recommended that hot water
treatment of dormant nursery plants be included in an integrated strategy for the proactive
management of black foot disease in grapevine nurseries. === AFRIKAANSE OPSOMMING: Gedurende die afgelope paar jaar is ‘n drastiese afname waargeneem in die sukses van
geënte wingerdplante in kwekerye, sowel as jong wingerde van die Wes-Kaap.
Omstandigheidsgetuienis dui daarop dat Cylindrocarpon spp., wat die wingerdsiekte swartvoet
veroorsaak, geassosieer word met hierdie agteruitgang. Swartvoet is ‘n relatiewe nuwe siekte
waarvan daar baie min inligting bekend is, alhoewel dit voorkom in verskeie lande waar wingerd
verbou word. Die primêre doel van navorsing was (1) om opnames in wingerdkwekerye uit voer
om te bepaal watter swamme betrokke is by die verskynsel van agteruitgang, met spesiale
verwysing na die betrokkenheid van Cylindrocarpon spp., (2) om die organismes te identifiseer
en te karakteriseer wat daarvan verdink word dat hulle die siekte swartvoet veroorsaak, en (3)
om beheer en/of bestuurspraktyke te ontwikkel om Cylindrocarpon infeksies te voorkom of uit
te wis.
Kwekeryplantjies in drie kommersiële kwekerye in die Wellington omgewing van die
Wes-Kaap is gedurende verskillende tye gedurende die groeiseisoen gemonitor. Die opnames
het plaasgevind gedurende die 19992000 seisoen deur middel van destruktiewe
monsterneming. Die eerste monsters is geneem in September nadat die stokkies geënt en
gekallus is en voordat dit in die kwekery geplant is. Na plant is asimptomatiese, gewortelde
plante vanuit die kwekerye na 3, 6 en 9 maande uitgehaal. Isolasiestudies dui duidelik daarop
dat verskillende “Cylindrocarpon spp.” plante vanuit die kwekerygrond geïnfekteer het. Hierdie
spesies het selde voorgekom in onderstok-voortplantingsmateriaal voor plant. Tydens plant is
die vatbare basale gedeelte, veral die pit, van die meeste geënte stokkies gedeeltelik of selfs
volledig blootgestel. Kalluswortels breek ook tydens plant wat wonde laat vir infeksie deur
grondgedraagde siektes. Die isolasiestudies dui ook daarop dat die eerste infeksies in die wortels
plaasgevind het, gevolg deur infeksies van die onderstokke. Hierdie infeksies het toenemend
voorgekom gedurende die verloop van die groeiseisoen.
Substansiële variasie in kultuur- en morfologiese eienskappe is waargeneem in die
Cylindrocarpon isolate wat tydens die kwekeryopnames versamel is, sowel as van isolasies wat
gemaak is uit siek plante. Morfologiese en filogenetiese studies is uitgevoer om hierdie “Cylindrocarpon spp.” te identifiseer en hul betrokkenheid by die siekte swartvoet uit te klaar.
Gedeeltelike DNS volgordes van die groot ribosomale subeenheid (“LSU rDNA”), interne
getranskribeerde spasiëerderarea (“ITS1, “ITS2”), insluitend die 5.8S rRNS geen, en
gedeeltelike β-tubilien geen introns and eksons is gebruik vir filogenetiese analise.
Filogenetiese analises het die diversiteit wat waargeneem is tussen die verskillende isolate
bevestig deurdat vier Cylindrocarpon-agtige spesies geïdentifiseer is. Een van hierdie spesies is
aanvanklik geïdentifiseer as Cylindrocarpon destructans. Verdere navorsing het egter daarop
gedui dat C. destructans ‘n spesie-kompleks verteenwoordig. “C. destructans” afkomstig van
wingerd blyk identies te wees aan die ex-tipe isolaat van Cylindrocarpon liriodendri, wat ook ’n
teleomorf, Neonectria liriodendri in kultuur vorm. ’n Tweede spesie is nuut beskryf in hierdie
studie as Cylindrocarpon macrodidymum (Neonectria macrodidyma). Die twee oorblywende
Cylindrocarpon-agtige spesies is geplaas in ‘n nuwe genus, Campylocarpon. Die twee spesies
staan bekend as Campylocarpon fasciculare en Campylocarpon pseudofasciculare.
Patogenisiteitstudies het bevestig dat al vier spesies die vermoë het om wortel- en lootmassa van
wingerdplant drasties te verlaag.
Kennis wat opgedoen is rakende die lewensiklus van swartvoet dui daarop dat
bestuurspraktyke daarop moet fokus om primêre infeksies in wingerdkwekerye te voorkom. Op
die oomblik is daar egter geen fungisiedes geregistreer vir die beheer van die siekte in Suid-
Afrikaanse wingerde of kwekerye nie. Dertien fungisiedes is in vitro geëvalueer om te bepaal of
dit miseliumgroei van hierdie swamme kan inhibeer. Prochloraz mangaan chloried, benomyl,
flusilasool en imazalil was die effektiefste fungisiedes wat ondersoek is, en is gevolglik ingesluit
in semi-kommersiële veldproewe. Die basale gedeelte van geënte stokkies is gedoop (1 min) in
verskeie chemies en biologiese behandelings voordat dit geplant is in die kwekerye. Patogene
wat geassosieer word met swartvoet is nie vanuit geënte stokkies geïsoleer voordat dit in die
kwekerye geplant is nie. Addisionele behandelings het bestaan uit grondtoevoegings met
Trichoderma formulasies, sowel as warmwaterbehandeling (50°C vir 30 min) van dormante
kwekeryplante. Die veldproewe is geëvalueer na ‘n groeiseisoen van 8 maande. Die voorkoms
van swartvoet patogene is nie betekenisvol/konstant verlaag deur die meeste chemies en
biologiese behandelings nie. Hierdie patogene is egter nie vanuit plante geïsoleer wat na uithaal
aan warmwaterbehandeling blootgestel is nie. Dit word dus aanbeveel dat
warmwaterbehandeling van dormante kwekeryplante deel word van ‘n geïntegreerde strategie vir
die pro-aktiewe beheer van swartvoet in wingerdkwekerye.
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