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Toward understanding why Phytophthora species can be so hard to isolate

Sarker, Suchana Rani (2021) Toward understanding why Phytophthora species can be so hard to isolate. PhD thesis, Murdoch University.

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Amplification of environmental DNA using Phytophthora and oomycete specific primers frequently detects many more Phytophthora species than traditional soil baiting methods. Species missed when baiting include known species previously detected in other studies, but others (based on their sequence data) have never been isolated and are potentially new species. Three factors that could affect isolation success were investigated: 1) isolation medium, 2) time of sporulation, and 3) sampling and baiting techniques.

Firstly, the effect on the growth of Phytophthora species, of the antimicrobials nystatin, ampicillin, rifampicin, hymexazol and chloramphenicol commonly used in isolation media was investigated. Growth of ten Phytophthora species from different clades was tested in a medium with antimicrobials in a range of concentrations, singly and in combination, and covering the range used in the isolation medium NARH: nystatin 12.5 – 100 ppm, ampicillin 62.5 – 500 ppm, rifampicin 5 – 40 ppm, hymexazol 12.5 – 100 ppm and in addition, and chloramphenicol 5 – 40 ppm. Based on these results, two combinations of antimicrobials supporting good growth of most species were selected. The growth of 47 Phytophthora species from all clades was then compared in these new formulations and standard NARH, in aseptic culture, and when used in plates for bait leaves from soils known to be infested with Phytophthora. Although the growth of some Phytophthora species was better with the new combinations in aseptic culture, suppression of competing microorganisms from lesions on baits was best in standard NARH. It was concluded that sensitivity to the NARH antimicrobials does not appear to be the reason for the failure of isolating many of the species detected by metabarcoding.

Secondly, whether differences between species in the timing and abundance of sporangial production and zoospore release could be a reason for the lower number of species isolated by baiting was investigated. Excised stems of Eucalyptus marginata were inoculated with ten Phytophthora species, and lesioned sections for each species were baited separately in water. There were significant differences between species in the timing of sporangia production and zoospore release. The species that released zoospores early successfully infected bait leaves and were isolated in the first few days after the start of baiting, while the species that produced zoospores only after 48 hours were only isolated after 5-7 days or not isolated at all. When species were paired in the same baiting tub, those that produced zoospores in the shortest time were isolated most frequently, while species slow to produce zoospores, or which produced them in lower numbers, were isolated from few baits or not at all. Thus, species differences in the timing of sporangia production and zoospore release may contribute to the ease of isolation of some Phytophthora species when they are present together with other Phytophthora species in an environmental sample.

Thirdly, modifications to soil sampling and baiting conditions were examined. Fifty small soil samples from each of four sites were baited separately in small tubs (200 ml), and the number of isolations compared with the number resulting from the use of the standard technique: bulking soil samples from a site and baiting a subsample in a standard sized tub (1000 ml). A higher number of Phytophthora species was recovered from baiting of non-bulked soils than bulked soils. The presence of Phytophthora species in asymptomatic baits remaining after seven days in both large and small tubs and root samples from the bulked soils were assessed by metabarcoding. As in other studies, more species were identified in the root samples using the molecular method than from baiting. However, the highest number of species was detected using a combination of baiting and metabarcoding analysis of asymptomatic baits. It was concluded that data from metabarcoding of asymptomatic baits is more valuable than that from roots, as it detects only the viable species in the sample.

In conclusion, antibiotics in the medium are not the main reason for the differences in the number of species detected using baiting and metabarcoding. However, differences in species biology, namely the speed of sporulation and competitive ability in reaching and infecting baits, are contributing factors. Modification to the protocols for handling soil samples and the mass of material baited in a tub will increase the number of species isolated from a site but not eliminate the difference in the number of species detected using baiting and metabarcoding.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): Environmental and Conservation Sciences
Supervisor(s): Hardy, Giles, Burgess, Treena and McComb, Jen
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