Detection and quantification of ‘Candidatus Phytoplasma asterisʼ and ‘Candidatus Liberibacter asiaticusʼ at early and late stages of Huanglongbing disease development

ʻCandidatus Phytoplasma asterisʼ (CPa) has been identified along with ʻCandidatus Liberibacter asiaticusʼ (CLas) from citrus trees showing Huanglongbing (HLB) symptoms in Mexico. The low titre of CPa and CLas in infected plants is the major constraint to developing an accurate and sensitive method for their detection that would support an effective HLB diagnosis and management programme. This investigation reports the development and validation of a new TaqMan quantitative PCR to detect low titres of CPa in citrus trees with HLB symptoms based on the phytoplasma 16S rRNA gene region. The assay was used to monitor the titre of CPa at early and late stages of disease development in symptomatic and asymptomatic Mexican lime trees in Colima, Mexico. The concentration of CLas was determined by nested-qPCR. Only eight out of 39 sampled branches showed HLB symptoms at the early infection stage. All 39 branches were symptomatic at the late infection stage. The qPCR yielded an increase in the branch incidence and concentration of CLas (49 to 100% and 5 × 10³ to 12 × 10³ cells/100 ng of total DNA), while CPa showed a decreased incidence and concentration (72to 41% and 199 to 19 cells/100 ng of total DNA) throughout the disease development. Results confirm the occurrence of mixed infection of CLas and CPa in HLB-affected citrus trees, although their epidemiological role in co-infecting remains unknown.