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Bradyrhizobium isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for Candidatus Liberibacter asiaticus

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Abstract

Bradyrhizobium sp., a slow-growing nitrogen-fixing symbiotic bacterium of legumes and common root endophyte of other plants, is closely related to Candidatus Liberibacter asiaticus (Las), the uncultured putative pathogen associated with citrus huanglongbing (HLB). In attempts to isolate Las on a low-nutrient medium that had been used for the isolation of several uncultured bacteria of the alpha subclass of proteobacteria, slow-growing Bradyrhizobium spp. were isolated and identified by sequencing of 16S rDNA. The individual isolates tested weakly positive (Ct = 31.2–36.0) with the USDA primers commonly used in qPCR assays for Las in foliar tissues. Direct DNA extracts from roots of HLB symptomatic trees that contained sequences of Bradyrhizobium sp. had Ct values ranging from 31.2 to 36.5; sequences of Las were not present in those samples. Potential cross-reaction between DNA of members of the Rhizobiales and sequences amplified by the Las primers were tested in silico with the Primer-BLAST tool in NCBI. Similar to Las, Bradyrhizobium generated predicted 16S rDNA amplicon sizes of 78–79 bp with the qPCR primers and of 1167-1172 bp with the conventional PCR primers. Bradyrhizobium sequences of 16S rDNA had 1–7 mismatches and only 1 mismatch at the 3′ end of qPCR and conventional PCR primers confirming potential cross-reactivity. As Bradyrhizobium is usually not found in foliage, the USDA qPCR primers can be safely used to check leaves for the presence of Las, but a threshold value of 31.0 is recommended for Las detection in roots. Other primers should be tested for potential cross-reaction with members of the Rhizobiales.

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Acknowledgements

This research was funded primarily by the Esther B. O’Keeffe Foundation in Deerfield Beach, Florida. We are grateful to Brian O’Keeffe who noticed our work on HLB in the Proceedings of the National Academy of Sciences and decided to fund our HLB research. Additional funding was provided by the Florida Department of Agriculture and Consumer Services (HLB-Mac 14-8130-0469-CA). We thank Dr. Greg Hodges and Dr. Tim Schubert of the Division of Plant Industry for providing these funds to us. We are grateful to Ben McLean III, vice-president of Uncle Matt’s Organic in Clermont, FL, for providing citrus samples from the organically and conventionally managed groves. We thank Dr. Marina S. Ascunce for providing bacterial colony mixtures and DNA samples from Fort Meade and also thank Dr. Miaomaio He for help with the processing of citrus samples.

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  1. Department of Plant Pathology, University of Florida, Gainesville, FL, 32611, USA

    Keumchul Shin & Ariena H. C. van Bruggen

  2. Emerging Pathogens Institute, University of Florida, Gainesville, FL, 32610, USA

    Keumchul Shin & Ariena H. C. van Bruggen

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Shin, K., van Bruggen, A.H.C. Bradyrhizobium isolated from huanglongbing (HLB) affected citrus trees reacts positively with primers for Candidatus Liberibacter asiaticus. Eur J Plant Pathol 151, 291–306 (2018). https://doi.org/10.1007/s10658-017-1372-9

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