1a. Objectives (from AD-416):
To distinguish live from dead cells of Candidatus Liberibacter asiaticus, presumed causal agent of citrus huanglongbing disease, in citrus tissue.
1b. Approach (from AD-416):
The use of two DNA intercalating dyes (Ethidium Monoazide and Prodidium Monoazide) will be used to degrade DNA from dead cells while not affecting DNA contained in live cells, following treatment, DNA will be extracted and used in qPCR analysis; only DNA from live cells should amplify. Results with and without dye will be compared, parameters optimized and tested in citrus samples expressing various levels of HLB symptoms.
3. Progress Report:
This project is related to Objective 1 of the parent project: Evaluate citrus scion cultivars and advanced selections with potential for commercial use, identifying genotypes that will survive to bear age and produce high quality fruit for the fresh and juice markets. During months 1-4 of the project our objectives were to: 1)Determine suitability of Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) for distinguishing between living and dead Candidatus Liberibacter asiaticus (CLas) cells in citrus; and 2)validate and compare results of PMA-qPCR with Ethidium Monoazide (EMA)qPCR. We have made significant progress towards meeting each of these objectives. Specificity and efficacy of EMA- and PMA-qPCR were determined using both purified plasmid Deoxyribonucleic Acid (DNA) containing the Candidatus Liberibacter (CLas) DNA target sequence and E. coli cells transformed with the same plasmid. Results with this model system confirm that both EMA and PMA treatments are specific for the CLas target sequence. Amplification of plasmid DNA in qPCR was inhibited 100% by both EMA and PMA. Estimates of live cells using E. coli with EMA or PMA gave similar results of ca. 10% live cells. If cells are heat killed prior to dye treatment, amplification is inhibited 100%. In the course of these experiments we also optimized variables in the protocol to give greatest sensitivity in the assay and the widest working range. We have conducted preliminary tests of EMA- and PMA-qPCR for distinguishing between live and dead cells in citrus seed coat vascular bundles, a tissue known to contain high titers of CLas, and in citrus leaves, both from CLas-inoculated trees in the greenhouse and from Huanglongbing symptomatic trees in the field. DNA extracted from seed coat vascular bundles that had been treated with EMA prior qPCR protocol showed about 25% of the CLas copy number of that in DNA from non-treated seed coat vascular bundles. We compared results of EMA- and PMA-qPCR with citrus leaf samples. We used leaves that expressed a range of Huanglongbing symptoms for these experiments. Samples were collected both from the greenhouse and from the field. Estimates of the number of live CLas cells in leaves treated with EMA were typically less than those obtained using PMA. Over a range of total CLas titers, estimates of live cells averaged 15% based on EMA-qPCR and 50% based on PMA-qPCR.