Elucidation of biochemical defence mechanisms in wheat (Triticum aestivum L.) against root-lesion nematode (Pratylenchus thornei)

Elucidation of biochemical defence mechanisms in wheat (Triticum aestivum L.) against root-lesion nematode (Pratylenchus thornei)

Pratylenchus thornei is an economically damaging root-lesion nematode that has a worldwide distribution. It is one of the major threats for wheat production in Australia and is particularly damaging in the northern grains region of the country. This nematode causes nutrient deficiency and water stress in wheat, which results in yield loss. Recent studies suggest that resistance in wheat occurs post penetration of the nematodes into the roots. Little is known about the biomolecules responsible for providing defence against P. thornei in wheat. In this thesis, histopathology, comparative enzyme profiling and metabolomics studies were conducted to elucidate the potential defence mechanisms in wheat against P. thornei infestation. Mainly, two sources of resistance against P. thornei were used in this study for different experiments (i) GS50a and its derived lines (ii) synthetic hexaploid CPI133872 and its derived lines. These were compared with susceptible wheat genotypes that were parents of the resistant derivatives.

Histopathological analysis was performed on one moderately resistant wheat genotype (QT8343; a GS50a derived line) and two susceptible wheat genotypes (Gatcher and Janz) to understand critical time points for reduced nematode reproduction in moderately resistant wheat cultivars. The significantly reduced nematode numbers were recorded inside QT8343 at 4 to 12 weeks post nematode inoculation (PNI). Clear differences were observed in both P. thornei nematode numbers and egg depositions at 8 weeks post nematode inoculation (PNI), with significantly (P ≤ 0.05) fewer nematodes and eggs inside the roots of the moderately resistant genotype (QT8343) compared with the susceptible wheat genotypes (Gatcher and Janz). The results have suggested that 8 weeks PNI could be a critical time point for changes in nematode reproductions inside resistant wheat genotypes. No effect of nematode inoculation was found on total protein content, cell-wall bound phenolics and lignin, plant height, shoot and root biomass of moderately resistant and susceptible wheat genotypes, in this histopathological study.

The time point 8 weeks PNI was selected for further biochemical profiling of the wheat roots, namely, total phenol estimation, estimation of phenol oxidase activities and detailed metabolic profiling. The effects of total phenol and phenol oxidases in wheat defence against P. thornei were evaluated in 21 wheat genotypes ranging in susceptibility and resistance to P. thornei. Polyphenol oxidase (PPO) and peroxidase (POD) enzyme assays were optimised as there was no standardised protocol to test multiple samples at a time using a microplate reader. Higher constitutive levels of total phenols were found in resistant synthetic hexaploid wheats CPI133872 (576 μg gallic acid equivalent (GAE)/g root) and CPI133859 (518 μg GAE/g root) at 8 weeks PNI, compared with moderately resistant and susceptible bread wheat genotypes (192 to 390 μg GAE/g root). The activity of PPO was induced in response to P. thornei in resistant (CPI133872) and moderately resistant bread wheat genotypes (GS50a and its derivate QT8343), becoming maximal at 4 weeks PNI. The activity of POD was similarly induced in response to P. thornei in CPI133872 at 6 weeks PNI. Different genetic sources of resistance to P. thornei showed diverse defence mechanisms and differences in timing of responses. The results have suggested both higher levels of total phenol and phenol oxidases could be responsible for superior resistance in the synthetic hexaploidy CPI133872. In contrast, although total phenol contents in moderately resistant GS50a and its derived lines were comparable to susceptible wheat genotypes (Gatcher and Janz), the oxidised phenolic molecules due to higher level of phenol oxidases in GS50a and its derived lines than in Gatcher and Janz could be responsible for providing defence against P. thornei.

Metabolomic profiling was performed with resistant (QT16528; an advanced breeding lines derived from the synthetic hexaploid CPI133872) and susceptible wheat genotypes (including Janz) to understand the role of wheat metabolites in resistance and susceptibility to P. thornei. Detailed untargeted metabolic profiling using high performance liquid chromatography (HPLC) mass spectrometry (MS) was performed on the wheat roots at 8 weeks PNI. The majority of metabolites potentially responsible for resistance in QT16258 were found to be constitutively expressed. Gossypetin-8-glucosides, desoxypeganine, and hirsutine metabolites which were significantly (P ≤ 0.01) higher in concentration in QT16258 than Janz, could potentially act as acetyl choline esterase inhibitors of P. thornei to damage neural connections and restrict nematode motility inside QT16258 root tissue. Significantly expressed flavonoid metabolites such as quercetin-3,4'-O-di-beta-glucoside, myricetin-xyloside in QT16258 could have important roles in reducing P. thornei reproduction and egg deposition. Resistance in QT16258 could also be due to increased deposition of cutin, suberin and wax on the root cell walls to impede penetration of P. thornei and its movement inside the root. Some metabolites occurring at higher concentrations in susceptible Janz, including indole acetic acid and vanillin acetate conjugates could be attractants for P. thornei and phenolics, including coniferyl alcohol could be part of a hypersensitive browning reaction resulting from P. thornei invasion.

These findings suggest that phenolics in the presence of phenol oxidases can have important roles in wheat defence against P. thornei. Eight weeks post nematode inoculation is a critical time point for detailed biochemical studies as there were highly significant (P ≤ 0.05) differences both in egg deposition and nematode numbers inside roots of resistant wheat genotypes. The defence in wheat against P. thornei is mostly constitutive and several biomolecules including metabolites and enzymes are likely to be acting together. Understanding the biochemical defence mechanisms in wheat against P. thornei could lead to novel nematode management tools to minimise plant damage and consequent loss in wheat yield from this nematode species.