Diverse DNA extraction methods and PCR primers for detection of Huanglongbing-associated bacteria from roots of ‘Valencia’ sweet orange on sour orange rootstock

Abstract

Fibrous root samples from Huanglongbing symptomatic (S) and asymptomatic (AS) ‘Valencia’ sweet orange trees (Citrus sinensis) grafted onto sour orange (C. aurantium) rootstock were analyzed using different DNA extraction procedures and primers to detect Candidatus Liberibacter asiaticus. Root sampling procedure was modified from the conventional use of soil probe to reduce fatigue, and to facilitate sample collection. DNA extraction kits including Qiagen DNeasy, Mo Bio PowerSoil and PowerPlant generated root DNA suitable for polymerase chain reaction (PCR) diagnostic assays. Quantitative PCR (qPCR) using HLBaspr primers and probe on root DNA extracts from a total of 206 samples from 60 AS trees showed unreliable results for the presence of Ca. L. asiaticus. Except for one sample, all the remaining samples gave negative qPCR result using LJ900fpr primers and probe. Further analysis of roots samples from S and AS trees indicate that HLBaspr is not suitable for Ca. L. asiaticus diagnosis in root samples. Leaf samples from all 60 AS trees showed negative qPCR result with HLBaspr. Conventional PCR (cPCR) using OI1/OI2c primers on 10 randomly selected root samples, from the 206 samples, produced non-specific amplification products which were easily distinguished from products specific to Ca. L. asiaticus seen in the positive control. The same root samples did not produce any amplification product in cPCR using A2/J5 primers. Any of the above mentioned cPCR or qPCR assays could accurately detect Ca. L. asiaticus in S leaf samples of Ca. L. asiaticus-infected trees. Root samples from 10 (5 S and 5 AS) additional trees analyzed by cPCR using A2/J5 and Las606/LSS primers and qPCR using LJ900fpr and CQULA (CQULA04F/CQULA04R, and TaqMan probe CQULAP10) clearly showed that all these primers are efficient in detecting Ca. L. asiaticus in root samples. There was no difference in frequency and consistency of Ca. L. asiaticus detection for root samples collected near the tree trunk compared to the roots collected about 1.8m from the trunk. Ca. L. americanus was not detected in either root or leaf samples.