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 ìg/ml for P. cinnamomi, to 13 ìg/ml for P. nicotianae, to 27 ìg/ml for P.
citricola, to 24 ìg/ml for P. palmivora and to 49 ìg/ml for P. capsici.
Phosphite concentrations from 5 to 100 ìg/ml caused different degrees of morphological
changes. Mycelial growth of all species was significantly suppressed by phosphite at 5
ìg/ml while at 100 ìg/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 (ÄÄCT) 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.
| Identifer | oai:union.ndltd.org:ADTP/221857 |
| Date | January 2006 |
| Creators | 30365216@student.murdoch.edu.au, Mee-Hua Wong |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Mee-Hua Wong |
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