Developing a culture medium for Liberibacter asiaticus through comparative multi ‘omics analysis with its closest cultured relative, L. crescens

Developing a culture medium for Liberibacter asiaticus through comparative multi 'omics analysis with its closest cultured relative, L. crescens

Report Date: 09/05/2017
Project: 15-027   Year: 2017
Category: CLas Bacteria
Author: Eric Triplett
Sponsor: Citrus Research and Development Foundation

Meida continued to be modified to improve the growth of L. crescens and use those results to develop better media for CLas culturing. During this period the Killiny lab provided an enormous amount of metabolomic data from the haemolymph of the Asian citrus psyllid to enhance our media formulations. Results from this work has suggested components that should be added or added at a higher concentration to the medium for the culturing of CLas including heptadecanoic acid, n-methyl-L-proline, methyl-maleic acid, homoserine, pyroglutamic acid, arabinopyranose, D-glucopyranoside, fucose, azaleic acid, arabinofuranose, iso-citric acid, n-acetylglucosamine, alpha-D galactoside, inositol-2-phosphate , trehalose, citrulline, ornithine, AMP, CMP, NADP, GMO, IMP, and FAD. In past media formulations, some of these molecules were in particularly low levels, particularly thiamine which was 100-fold below the level found in media. During this period, we continued to follow-up on our discovery that citrate is a preferred carbon source of L. crescens. We discovered that the optimal level of citrate required for growth was 2.5 g/l or 13mM. We also found that the pH of the medium increases dramatically during growth from 5.9 to 7.6. This increase in pH is expected from the metabolism of citric acid. We are now testing medium with more buffering capacity to maintain pH near 6.0 during culture growth. This is being tested with higher levels of phosphate buffer and ACES buffer. During this period we have considered ideas on whether CLas can be starved of citrate by modifying the nutrition of citrus saplings. The first experiment tested whether changes in nitrogen nutrition (potassium nitrate, sodium nitrate, or ammonium nitrate) can alter the levels of citrate in phloem. These nutritional changes are expected to alter the ionic balance in the plant. Sodium nitrate was expected to reduce the level of citrate in phloem. However, after 60 days of treatment in the greenhouse, we saw no difference in citrate phloem levels between these nutritional treatments. We are now testing the notion that adeqaute phosphate nutrition is required to reduce citrate levels in phloem. Under conditions of phosphorous deficiency, plants load citric acid into the phloem in order to exude citric acid from roots (Gaume et al. 2001. Plant and Soil 228:253-264). It is also known that the exuded citric acid can then solubilize rock phosphate (P2O5) or other forms of bound phosphate for use by plants (Chen and Liao 2016. J. Genetics & Genomics 43:631-638). Zhao et al. (2013, Molec. Plant 6:301-310) reported that certain small RNAs made in response to HLB infection were involved in P nutrition. They also found a 35% reduction of P in CLas-positive citrus trees compared to healthy trees. Applying phosphate to HLB-positive sweet orange trees in southwest Florida reduced HLB symptom severity and significantly improved fruit production in a 3-year field trial. So our hypothesis is that the severity of citrus greening disease can be greatly reduced with application of phosphate (not rock phosphate as is sometimes recommended). Added phosphate will improve the nutrition of the tree and lower citrate loading into the phloem. The reduced citrate levels in the phloem is expected to starve Liberibacter. These ideas will be proposed in a grant proposal to the USDA SCRI program with Killiny, Wang, and Vincent from the CREC as co-PIs with Triplett.

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