Masters Degrees (Plant Pathology)

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Browsing Masters Degrees (Plant Pathology) by browse.metadata.advisor "Adele, Mcleod"

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    Phosphite sensitivity of phytophthora cinnamomi and methods for quantifying phosphite from avocado roots
    (Stellenbosch : Stellenbosch University, 2016-03) Jing, Ma; Adele, Mcleod; Stellenbosch University. Faculty of AgriSciences. Dept. of Plant Pathology.
    ENGLISH ABSTRACT: Phytophthora root rot caused by Phytophthora cinnamomi threatens the production of avocado worldwide, but the disease can be effectively managed using phosphonates. The mode of action of phosphonates is controversial and can include a direct fungistatic action and/or an indirect action involving host defence responses. In South Africa, in vitro radial growth inhibition studies, which can be indicative of a direct mode of action, have only been conducted on isolates collected in one orchard in previous studies, more than a decade ago. In in vitro studies, phosphate in the test medium can influence the in vitro toxicity of phosphite (H2PO3-), but this has not been studied in large P. cinnamomi populations. The in vivo phosphite sensitivity of P. cinnamomi isolates in avocado, which is indicative of host defence responses, has only been investigated in two non-peer reviewed studies in South Africa. Quantification of phosphite in roots is required for elucidating the mode of action of phosphonates, but no commercial analytical laboratory in South Africa can conduct these analyses due to the lack of a validated analytical method. The current study optimized a liquid chromatography-mass spectrometry (LC-MS/MS) method for quantification of phosphite, the breakdown product of phosphonates in plants, using avocado roots collected in orchards. Phosphite recovery rates were good (78 - 124%) and the precision was excellent with the percentage coefficient of variation (CV%) being between 1.9 to 9.7. Although the incurred sample reanalyses (ISR) precision for the method was unacceptable for samples with phosphite concentrations lower than 27 μg/gDW (~6.75 μg/gFW), it was acceptable for samples with higher phosphite concentrations (0.4 and 11 CV%; 0.6 - 21% difference [%DF]). The other investigated analytical methods, ion chromatography and an enzymatic fluorescent assay, were unreliable due to unacceptable ISR precision values. The in vitro phosphite (H2PO3-) sensitivities of 42 P. cinnamomi isolates from avocado in South Africa were investigated, as influenced by phosphate (HPO42-), using radial growth inhibition assays. Based on the response of isolates to all the evaluated phosphite concentrations (30 and 100 μg/ml) and phosphate concentrations in the test medium (1, 7 and 15 mM), the isolates could be grouped into a sensitive (11.04 - 89.21% inhibition), intermediate (11.26 - 66.75% inhibition) and tolerant group (3.9 - 19.09% inhibition). The inhibition of isolates as influenced by phosphate concentration was dependent on the phosphite sensitivity of isolates. In general, inhibition by phosphite for the sensitive and intermediate groups decreased as phosphate concentration increased, whereas inhibition of the tolerant group was not influenced significantly by phosphate at a phosphite concentration of 30 μg/ml. The in vivo sensitivities of one isolate from each of the sensitive and tolerant groups were investigated using an excised root bioassay. The two isolates responded similar to all phosphonate treatments, but the tolerant isolate tended to be more virulent, making it difficult to differentiate between phosphite- and virulence responses. The roots from seedlings that received phosphonate treatments that yielded root phosphite concentrations of 9.82 μg/gFW or higher, resulted in significant control. The only exception was one treatment, in one of two experiments, which contained 1.92 μg/gFW that unexpectedly caused significant control. Increasing phosphite root concentrations from 9.82 to 19.30 μg/gFW did not significantly improve control. Both isolates were inhibited to a greater extent (> 40%) in vivo than in vitro. Altogether, the data supports the involvement of host defence responses in suppression of P. cinnnamomi by phosphite. The study has improved our knowledge on the in vitro and in vivo response of P. cinnamomi isolates from avocado to phosphite, and consequently its mode of action. The data suggested that the mechanism of action is most likely host defence induction. Phosphonates thus seem to be true resistance inducing crop protection products. However, further trials are required to proof this hypothesis, since limited data and some controversial data were obtained in the current study. The in vivo data provided preliminary indications that in phosphonate application trials root phosphite concentrations should be above 10 μg/gFW to suppress P. cinnamomi. However, more in vivo trials must be conducted to confirm this. The developed LC-MS/MS method, is the only method available for root phosphite quantifications in South Africa, and is crucial for investigating the P. cinnamomi-avocado-phosphonate system.