Masikane, Siyethemba

Thesis (MScAgric)--Stellenbosch University, 2019.ENGLISH ABSTRACT: Phytophthora root rot (PRR) is a very destructive soil-borne disease of avocado caused by
Phytophthora cinnamomi (Pc) Rands. The disease can be effectively managed using
phosphonate fungicides, which breaks down to phosphite anions in plants. Limited information
is available on the effect of phosphite on Pc root quantities. Most studies have investigated
the suppression of Pc in stems and stem phosphite concentrations in native Australian plant
species. This is due to difficulties in quantifying Pc from roots.
Phosphate (analogue of phosphite) is an important macronutrient in plants, of which
the concentrations might be negatively influenced by phosphite. Knowledge on in planta
phosphate concentrations is required, since in vitro it has been reported that phosphate has
the potential to negatively influence the toxicity of phosphite against Phytophthora spp.
The current study developed a Pc-avocado seedling bioassay system, which was used
to identify three effective Pc root quantification methods. The two best quantification
techniques were Root-baiting-plating (assesses asexual reproduction from roots) and Root-
plating (assesses growth in roots) analyses, where lemon leaf disk baits obtained from root
baitings, or roots, were respectively plated onto a semi-selective medium. The third
quantification method involved qPCR amplification of Pc from root DNA extracts (Root-qPCR).
A fourth technique, which amplified Pc from lemon leaf disk baits obtained from root baitings,
was deemed ineffective. The efficacy of the quantification methods was determined based on
their ability to differentiation between roots inoculated with a range of zoospore concentrations,
Pc quantity correlations with zoospore concentrations, the variability in the quantification data
and the presence of false negatives.
The Pc-avocado seedling bioassay system was also used to show that phosphite
inhibited asexual reproduction of Pc from roots (Root-baiting-plating analysis), but not growth
in roots (Root-plating and Root-qPCR) in two avocado seedling rootstocks (Hall and Booth 6).
Modeling of the percentage control versus root phosphite concentrations using the Booth 6
Root-baiting-plating data yielded an EC90 value of 48.50 μg/gFresh Weight. For the Hall rootstock,
no model could be fitted to the data. Root-plating analysis showed that Pc growth was not
suppressed (< 10% control) by phosphite, whereas Root-qPCR analysis yielded highly
variable levels of control (-943% to 100%). The latter was unrelated, and not significantly
correlated with root phosphite concentrations.
The study showed that under orchard conditions, just following phosphonate
applications, Pc growth in roots and asexual reproduction were significantly reduced relative
to the control by all treatments to a similar level. This was irrespective of root phosphite
concentrations differing significantly between treatments, and over the course of the season.
At the end of the trial, Pc growth and asexual reproduction was differentially suppressed; a
treatment that yielded the lowest root phosphite concentrations resulted in a significant
reduction in the asexual reproduction of Pc relative to the untreated control, whereas the
growth of Pc in roots was not suppressed. A high yielding root phosphite phosphonate
treatment yielded the opposite result. In avocado orchard trials, highly significant positive
correlations were identified between root phosphite concentrations and root phosphate
concentrations. Furthermore, root phosphate concentrations fluctuated over the course of the
season (5 to 21 mMFW).
This study increased our knowledge on and identified three quantification methods that
can be used for investigating the extent of Pc growth and asexual reproduction in avocado
roots. A fourth method will require further optimization. The quantification methods are also
useful for investigating the effect of phosphite towards Pc in roots under glasshouse and
orchard conditions. Further research is required to evaluate why certain treatments resulted
in a differential suppression in Pc growth and asexual reproduction in roots. The root
phosphate quantities established in the study can be used for assessing the in vitro toxicity of
phosphite against Pc.AFRIKAANSE OPSOMMING: Phytophthora wortelverrotting (PWV) word veroorsaak deur Phytophthora cinnamomi (Pc)
Rands en is ‘n baie vernietigende grondgedraagde siekte op avokadopeer. Hierdie siekte kan
effektief bestuur word deur fosfiet swamdoders, wat fosfietanione in plante afbreek. Min
inligting is beskikbaar oor die effek van fosfaat op Pc se wortelhoeveelhede. Die meerderheid
studies het die onderdrukking van Pc in stamme en stamfosfietkonsentrasies in inheemse
Australiese plantspesies ondersoek. Dit is as gevolg van die stryd om Pc te kwantifiseer vanaf
wortels.
Fosfaat (analoog van fosfiet) is 'n belangrike makronutriënt in plante, waarvan die
konsentrasies negatief deur fosiet beïnvloed kan word. Kennis oor fosfaatkonsentrasies in in
planta studies word vereis, aangesien in vitro studies rapporteer dat fosfaat die potensiaal het
om die toksiese effek van fosfiet teen Phytophthora spp. te beïnvloed.
Die huidige studie het ‘n Pc-avokadopeer saaling biotoets-stelsel ontwikkel om drie
effektiewe Pc wortel kwantifieringstegnieke te identifiseer. Die twee beste
kwantifiseringstegnieke was wortel-lokaas-uitplating (assesseer ongeslagtelike voortplanting
vanaf wortels) en wortel-uitplating (assesseer die groei in wortels). Onderskeidelik was skyfies
suurlemoenblare verkry vanaf die wortel-lokaas eksperiment en wortels vanaf die wortel
eksperiment en uitgeplaat op 'n semi- selektiewe medium. Die derde kwantifiseringstegniek
het qPKR-amplifikasie van Pc vanaf wortel-DNA (Root-qPKR) behels. Die vierde tegniek, was
onsuksesvol en het gepoog om Pc te amplifiseer vanaf die suurlemoenblaarskyfies. Die
doeltreffendheid van die kwantifiseringstegniek was bepaal eerstens op grond van hul vermoë
om te onderskei tussen wortels wat geinokuleer is met verskillende konsentrasies soëspore,
tweedens die korrelasie van Pc-hoeveelheid en soëspoorkonsentrasies, derdens die
veranderlikheid tussen die kwantifiseringsdata en laastens die teenwoordigheid van vals
negatiewes.
Die Pc-avokadopeer saailing biotoets-stelsel was ook gebruik om te wys dat fosfiet die
ongeslagtelike voortplanting van Pc vanaf wortels inhibeer (wortel-lokaas-uitplatings-analise),
maar nie die groei in wortels inhibeer nie (wortel-uitplating en wortel-qPKR) vir twee avokado-
saailinge onderstamme ( Hall en Booth 6).
Modellering van die persentasie beheer teenoor wortel fosfietkonsentrasies met
gebruik van die “Booth 6 Root-baiting-plating” data, het 'n EC90 waarde van 48.50 μg / gvarsgewig
opgelewer. Vir die “Hall” onderstam, kon geen model aangebring word op hierdie data nie.
Wortel-uitplating analise het getoon dat Pc-groei nie onderdruk word (<10% beheer) deur
fosfiet nie, terwyl wortel-qPKR-analise hoë veranderlike beheervlakke (-943% tot 100%)
getoon het. Laasgenoemde was nie verwant nie, en was ook nie beduidend gekorreleer met
die wortelfosietkonsentrasies nie.
Hierdie studie het gewys dat onder boordtoestande die groei van Pc in wortels asook
ongeslagtelike voorplanting beduidend verminder na die behandeling van fosfonaat teenoor
die kontrole vir alle behandelings teen ‘n soortgelyke vlak. Die resultaat was ongeag van die
wortelfosietkonsentrasies wat beduidend verskil het tussen die behandelings asook deur die
loop van die seisoen. Aan die einde van die eksperiment was die groei van Pc en
ongeslagtelike voortplanting verskillend onderdruk. Die behandeling wat die laagste
fosfietkonsentrasie gelewer het, het gelei tot ‘n beduidende vermindering in ongeslagtelike
voorsplanting van Pc relatief tot die onbehandelde kontrole maar die groei van Pc in wortels
is nie onderdruk nie. ‘n Hoë opbrengs wortelfosfiet-fosfonaat behandeling het gelei tot die
teenoorgestelde effek.
In avokadopeer-boordproewe was daar ‘n hoë beduidende positiewe korrelasie gevind
tussen wortelfosfietkonsentrasies en wortelfosfaatkonsentrasies. Verder die
wortelfosfaatkonsentrasies fluksueer deur die loop van die seisoen (5 to 21 mMFW).
Hierdie studie het ons kennis verbreed asook drie kwantifikasietegnieke geïdentifiseer,
wat gebruik kan word om die omvang van Pc groei en ongeslagtelike voorplanting in
adovokadopeerwortels te ondersoek.’n Vierde tegniek vereis verdere optimiseering. Die
kwantifiseeringstegnieke kan ook gebruik word om die effek van fosfiet teenoor Pc in wortels
te toets onder glashuis- asook boordkondisies. Verdere navorsing is nodig om te evalueer
hoekom sekere behandelings gelei het tot beduidende onderdrukking van Pc groei asook
ongeslagtelike voorplanting in wortels. Die wortelfosfonaat hoeveelhede wat gebruik is in
hierdie studie kan gebruik word om die in vitro toksisiteit van fosfiet teen Pc te evalueer.Master

Stellenbosch University SUNScholar Repository

Phytophthora cinnamomi quantification techniques, root phosphite concentrations and their response to phosphonate application in avocado

https://scholar.sun.ac.za:443/bitstream/10019.1/105609/1/masikane_avocado_2019.pdf

PHYTOPHTHORA CINNAMOMI QUANTIFICATION TECHNIQUES, ROOT PHOSPHITE CONCENTRATIONS AND THEIR RESPONSE TO PHOSPHONATE APPLICATION IN AVOCADO

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Stellenbosch University SUNScholar Repository