Manipulation of plant root exudation for soil-borne disease control

<p dir="ltr">Potatoes, the world’s fourth-largest food crop in the world, are grown on over 20 million hectares in 150 countries with a total global output of 359 million tonnes in 2020, and in Australia accounts for 20% of total vegetable production. The economic potential for this crop is extensive; yet, several diseases, both soil, and air-borne can result in significant production losses. Around 40 soil-borne diseases harm potatoes worldwide, causing serious damage, particularly to tubers, the plant’s most commercially important component. Powdery scab, caused by <i>Spongospora subterranea f. sp. subterranea</i> (Sss), is one of the most serious soil-borne diseases impacting potato crops. Sss not only produces characteristic tuber lesions, and powdery scabs, resulting in reduced value across the processing, retail, and seed markets, but also root infection that can limit plant growth and commercial tuber production. This pathogen can be found infesting both seed tubers and cropping soils and can persist in soil for an extended period (>10 years). Disease progression is influenced by inoculum levels in the soil or on seed tubers, soil, and climatic conditions, cropping history, and potato cultivar. There are currently no fully effective and environmentally sustainable control methods for this disease. Powdery scab lesions can also become infection sites for other pathogens including tuber late blight (<i>Phytophthora infestans</i>), pink rot (<i>Phytophthora erythroseptica</i>), dry rot <i>(Fusarium caeruleum</i>), and tuber rot (<i>Colletotrichum atramentarium</i>).<br>The extent to which root exudation contributes to the development of powdery scab disease is not yet well understood. L-Glutamine (Gln) and Tyramine (Tyr), both present in potato-root exudates, have previously been demonstrated to trigger germination of dormant Sss spores and act as chemoattractants for the released motile zoospores. We hypothesized that adding a bacterial degrader of key root exudate chemicals into the rhizosphere would alter pathogen germination and zoospore chemotactic attraction to roots, resulting in lower infection and germination. We also hypothesized that a well-adapted rhizosphere bacterium may possess additional traits including plant growth promotion and mitigation on impact of disease on root development and function. Numerous studies have shown that inoculating plants with microbes that putatively increase defensive responses results in the activation of a variety of metabolic processes, including primary metabolite alteration and secondary metabolite production. Taking this into consideration, breakthroughs in metabolomics techniques have been effectively exploited, providing critical information about system?wide changes in plant metabolism during pathogen infection. As a result, targeted metabolic profiling using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) in combination with statistical techniques was used in this study to determine the role of a bacterial isolate in disrupting stimulatory chemicals involved in Sss resting spore activation and to acquire a better understanding of the biochemical events that occur in relation to pathogen infection. In addition, this study investigated the potential role of the isolate in disease prevention as well as its role in plant growth promotion.<br>Two rhizosphere bacteria identified as <i>Pantoea</i> isolate RR15 and <i>Rhodococcus</i> isolate RR09, were found capable of using and degrading both Gln and Tyr as their sole carbon source. Isolate RR15 degraded Gln and Tyr to nearly undetectable levels within 48 h, whereas isolate RR09 took 96 h to achieve the same levels of substrate degradation. Similarly, when established within the potato root rhizosphere, both isolates were able to reduce detectable levels of Gln and Tyr. However, <i>Pantoea</i> RR15 had a greater impact on reducing the target compounds and has been demonstrated to drastically affect the upregulation and downregulation of amino acids and organic compounds composition of potato root exudates.<br>Quantitative PCR was used to measure the ability of isolates to colonise potato roots, and the results show that <i>Pantoea</i> RR15 colonised roots well, with better rhizosphere re-establishment than <i>Rhodococcus</i> RR09. The former isolates’ cell numbers increased over time when roots were present, and it maintained its establishment and activity even when roots were absent. Consequently, it was shown that <i>Pantoea-</i>RR15-treated potatoes demonstrated a root system significantly improved. Additionally, more beneficial bacteria were observed, such as <i>Bacillus</i> and <i>Streptomyces</i>, both of which are known to produce a variety of compounds that may aid plant growth and disease resistance.<br>Metabolic alterations related to the establishment of <i>Pantoea</i> RR15 in the potato rhizosphere were evaluated during Sss infection. The number of critical compounds involved in Sss attraction and chemotaxis was reduced considerably. In both healthy and diseased potatoes, Pantoea RR15 reduced the levels of the key stimulant components glutamine and tyramine as well as other stimulatory substances including serine, aspartic acid, and nicotinamide. The non-stimulatory compounds spermine and choline were enhanced by the presence of RR15 but then reduced when the pathogen was present.<br>As a result of the significant growth promotion observed in pot experiment studies, we initially developed isolate RR15 into a formulation, testing two frequently used microbial inoculant carriers, talcum powder and vermiculite with a modified composition of a bacterial suspension by adding xanthan gum as a cellular protectant. In a pot study, bio formulations applied to potato seed tubers enhanced root growth by 42.8% and 24.2% in uninfected and infected soil, respectively, with the vermiculite?based formulation providing the greatest advantages. To investigate the effectiveness of vermiculite formulation under field conditions, a preliminary field trial was conducted, revealing a similar trend of higher root growth, greater processable tuber weight, and increased processable tuber number, indicating that vermiculite formulations were an effective carrier for <i>Pantoea</i> RR15 formulations; nevertheless, more formulation testing is required.<br>Overall, the findings of this thesis showed that the root exudation profile in potatoes was diverse and altered in a completely different manner after inoculation with two bacterial isolates, <i>Pantoea</i> RR15 and<i> Rhodococcus</i> RR09. Moreover, RR15 shown the most effective in lowering the target chemicals Gln and Tyr, as well as other known stimulatory compounds. Given that the mechanisms behind changes in these below-ground chemical signals are yet unclear, our findings suggest that <i>Pantoea</i> RR15 inoculation alters the exudate profile in both healthy and diseased potatoes. Interestingly, evidence of plant growth promotion activity by selected rhizosphere microbes suggests that the strategy can also stimulate the formation of stronger roots, which may compensate for the loss of root function following infection while also potentially increasing yield. To the best of our knowledge, there is no information or research exist regarding the use of rhizosphere bacteria to reduce the potato metabolome involved in the germination of <i>Sss</i> spores, hence reducing the incidence of powdery scab disease. The idea of employing rhizosphere bacteria to modify pathogen-activating compounds as a method of biological root disease management is unique. In addition, these rhizosphere inoculants have the ability to promote root growth and so compensate for the loss of root mass and function caused by pathogen attack of the root. In practise, our findings could help to develop novel rhizosphere bacterial bio-formulations for the management of powdery scab disease and the enhancement of potato health and tuber yields.</p>