Evaluation of Enrichment to Improve qPCR Detection of <i>Ralstonia solanacearum</i> in Latently Infected Geranium Plants

Ralstonia solanacearum race 3 biovar 2 (r3b2), a U.S. select agent pathogen, has repeatedly entered the U.S. via latently infected geranium cuttings. To improve molecular detection in asymptomatic tissue, bacterial enrichment is often necessary. This study first evaluated five enrichment media, selecting three (mCPG, mSMSA, and mCPG with antibiotics) for further testing. Twenty four-hour enrichment protocols using these media were assessed for their ability to improve qPCR detection of r3b2 strain UW551 and non-r3b2 strain RUN302 in 51 UW551- and 28 RUN302-inoculated geranium plants. No significant difference was observed among the media, indicating antibiotics did not affect qPCR detection when using specific primers and probes. RUN302, a phylotype IIB-4 strain potentially emerging on ornamentals, was less aggressive on geranium than UW551. In symptomatic plants (n=14), initial qPCR Cq values were consistently below 25 (range of 17.59-24.53). In asymptomatic plants (n=65), Cq values ranged from 20.43 to undetectable. Enrichment significantly lowered Cq values in samples with initial Cq &lt; 34. Among samples with initial Cq ≥ 34, results were variable: 45% showed Cq reductions of at least 2 with at least one medium, up to 13.65 for UW551 and 16.43 for RUN302, while 55% showed minimal or no reduction in Cq values. Overall, although 24-hour enrichment significantly enhanced qPCR detection of both r3b2 and non-r3b2 strains in symptomatic and asymptomatic geraniums, its effectiveness was inconsistent in samples with very low initial bacterial populations (Cq≥34). These findings highlight limitations of enrichment in improving detection of very low pathogen levels.