This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
One big threat for agriculture is bacterial wilt disease caused by Ralstonia solanacearum (Rs) – a quarantine pathogen; on the European Plant Protection Organization (EPPO) A2 list. This disease affects over 200 plant species worldwide. The bacteria is soil and water-borne and travels from the roots to the xylem where it multiplies, causing wilting and devastating crop harvests. Rs activates virulence factors in the plant such as cell wall degrading enzymes and the production of extracellular polysaccharide (EPS) that blocks the xylem. In the UK, during winter months, Rs has been found to reside in Solanum dulcamara (Sd) plants which grow near freshwater sources. Sd shows resistance to Rs, and can survive the infection, acting as a reservoir host. The resistance qualities of Sd have not been researched before which makes this work very novel.
My aim was to understand if this resistance is correlated to the soil microbiome of the plant. The roots of Sd grow in rivers so root exudates could be different in this weed compared with cultivated crops, and therefore they could attract plant growth-promoting bacteria which can potentially induce resistant mechanisms such as induced-systemic response (ISR). Previous research has suggested there are some existing key players in the field including Pseudomonas, Paraburkholderia, and Sphingomonas species. I have isolated and identified these taxa to test if they have an effect in plant growth or Rs inhibition so they can be used as agrochemical products to prime tomato plants (increase resistance) or directly kill the pathogen.
I homogenised the soil of 2-month-old Sd plants with water. This was diluted and plated on different media types. Genomic DNA of the colonies was extracted and PCR of the 16s rRNA region was amplified. I purified the PCR products and sent them to be sequenced using Sanger. The sequences were then input into BLASTn and Snapgene viewer to identify the species name of each colony. I identified 30 genera and tested a total of 127 colonies. Further analyses were performed with 4 species based on previous research: Sphingobium sp., Sphingopyxis ginsengisoli, Pseudomonas koreensis, and Paraburkholderia nodosa. I tested if they have an inhibitory role against Rs by using soft agar and liquid assays. Liquid assays were done in 96 well plates by growing non-fluorescent Rs with a GFP-tagged Rs or the species of interest with the GFP-tagged Rs. Optical density and GFP were measured as measuring optical density alone doesn’t allow the two bacteria growing together to be distinguishable from one another. With this analysis, I tested if the two bacteria aren’t just competing for nutrients, but that the bacteria of interest is inhibiting the growth of Rs.
Sphingobium sp. did not produce inhibition in soft agar or liquid assays. However, the other three bacteria showed inhibition rings in the soft agar assays. GFP-Rs in combination with a non-fluorescent Rs showed much higher GFP fluorescence compared to GFP-Rs grown with the other bacterial species, showing that the majority of bacterial species (except Sphingobium sp.) are inhibiting the growth of Rs in vitro. I used RStudio to analyse and plot the data which was highly significant.
Experiments were then completed in plantae to test if these bacteria are plant-growth-promoting bacteria. I found that Sphingopyxis ginsengisoli increases the growth of tomatoes significantly. A second experiment with the bacteria of interest was performed by inoculating tomato plants with the 3 bacteria able to inhibit Rs in vitro and with and without Rs. When monitoring the disease index of the positive control (Rs infected plants with no other bacteria), only 3 out of 8 Rs plants scored 4 (0 – no symptoms, 1 – 25% wilted, 2 – 50% wilted, 3 – 75% wilted and 4 – 100% wilted). Because of this, a kruskal-wallis test deemed that none of the 3 bacteria used as biocontrol agents against Rs significantly reduced disease symptoms. However, when comparing the plants visually, the impact that the bacteria had is undeniable. I believe the results are inconclusive, either because of the low number of replicates, or growing the tomatoes at 24℃ rather than the optimum 28℃ to reflect UK temperatures. If replicates were increased, the statistics would have shown significance in the results. In future experiments, combination treatments of the different bacteria should be tested to see whether the resistance to Ralstonia could be amplified further. Sphingopyxis could also be tested further by inoculating the plant prior to the addition of Rs to assess whether it may be influencing ISR, priming the plant to increase its resistance.
Jazmin Lye
University of York
IMAGE: Comparing Rs treatments on tomato plants. The wilted plants on the left have been treated with Rs alone. The green plants on the right have been treated with Rs and Paraburkolderia nodosa – a key bacteria of interest in my experiments.
This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
Click here to read more/apply for one yourself.
