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Phosphite induces morphological and molecular changes in Phytophthora species

Wong, Mee-Hua (2006) Phosphite induces morphological and molecular changes in Phytophthora species. Masters by Research thesis, Murdoch University.

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The influence of the chemical phosphite on Phytophthora species was investigated by studying the morphological and molecular changes induced by phosphite.

In vitro experiments were conducted to study the effects of phosphite on five isolates of each of five species of Phytophthora grown in low phosphate defined medium. Sensitivity to phosphite varied greatly among the five isolates of each species and resulted in a significant interaction between isolate and phosphite effect. The EC50 values ranged from less than 5 to 10 mcg/ml for P. cinnamomi, to 13 mcg/ml for P. nicotianae, to 27 mcg/ml for P. citricola, to 24 mcg/ml for P. palmivora and to 49 mcg/ml for P. capsici.

Phosphite concentrations from 5 to 100 mcg/ml caused different degrees of morphological changes. Mycelial growth of all species was significantly suppressed by phosphite at 5 mcg/ml while at 100 mcg/ml there was hyphal lysis. Swelling of hyphae with stunted sidebranches and shrinking of cytoplasm from hyphal tips and hyphal walls were characteristic changes observed. Phosphite also retarded the development and caused distortion and lysis of chlamydospores, sporangia and zoospores. Zoosporogenesis was also adversely affected.

Differential display reverse transcription-PCR was used to study changes in gene expression in P. cinnamomi induced in response to phosphite stress. The differential conditions were simulated by growth on a defined medium with and without phosphite amendment. This technique resulted in the isolation of 34 putative differentially expressed cDNA fragments which were cloned and sequenced. Nucleotide sequences of 26 of these cDNA clones were generated. BLASTX analysis of these nucleotide sequences against the NCBI database revealed that 18 exhibited homology to gene sequences encoding known proteins involved in various biological processes. The remaining eight showed homology to either hypothetical or unknown or unnamed proteins.

The expression level of four of these cDNA clones were further analysed by real-time quantitative RT-PCR using SYBR Green 1 assay. Three candidate endogenous reference genes namely, tubulin, cyclophilin and actin were evaluated to determine their expression level under the influence of phosphite. None of these genes were significantly regulated by phosphite. As tubulin had the highest expression among the three, it was chosen as the endogenous reference gene. Amplification efficiencies between the reference gene and each of the target genes were validated and found to be approximately equal or within 5% of each other. The relative gene expression between the phosphite-treated and untreated samples can thus be determined using the comparative CT ([Delta][Delta]CT) method. One of the cDNA clones, CP6 which showed differential expression of three-fold was up-regulated. The remaining three were constitutively expressed. CP6 which encodes 1564 nucleotides showed sequence homology, at the amino acid level with proteophosphoglycans from Leishmania major.

This study demonstrated the growth inhibition and morphological deformities caused by phosphite in Phytophthora species. It also illustrated the use of a modified DDRT-PCR method to study genes expressed in phosphite stress regulation. The application of real-time quantitative RT-PCR with SYBR Green I assay facilitated the quantification of the expression level of some of these genes.

Item Type: Thesis (Masters by Research)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): O'Brien, Philip and Hardy, Giles
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