In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

Abstract

The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g −1 of tissue, increased 10,000 times from 10 3 at 30 dpi to approximately 10 8 CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10 5 CN g −1 ). Blotchy mottle symptoms were observed in trees with 10 7 CN g −1 of bacteria after 180 dpi. Buds taken from infected, but nonsymptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.

Development of Disease and Growth on Six Scion/Rootstock Combinations of Citrus Seedlings under Huanglongbing Pressure

“…Almost all of rootstock/scion combination varieties contained CLas with different of CLas titers depending on the status of CLas inoculation, rootstock/scions varieties and duration of plant infection. Blotchy mottle symptom was first found at 180 days after inoculation on the Autumn and 240 days after inoculation in Spring (Coletta-Filho et al, 2009). …”

Infection Density Dynamics of the Citrus Greening Bacterium “Candidatus Liberibacter asiaticus” in Field Populations of the Psyllid Diaphorina citri and Its Relevance to the Efficiency of Pathogen Transmission to Citrus Plants

“…Liberibacter asiaticus" is noncultivable (6), most recent studies have adopted culture-independent approaches using conventional PCR and/or quantitative PCR techniques for the detection and quantification of "Ca. Liberibacter asiaticus" not only in citrus plants (15,(18)(19)(20)(21)(22)(23)(24)(25) but also in D. citri (9,13,(15)(16)(17)(18). Meanwhile, the biological relevance and consequence of the infection density of "Ca.…”

Reaction of sweet orange cultivars expressing the attacin A gene to 'Candidatus Liberibacter asiaticus' infection

“…Nevertheless, the inoculation method used in this study can be considered highly efficient: 80% for 'Hamlin', 79% for 'Natal', 75% for 'Pêra', and 86% for 'Valência' sweet orange. Although current research on HLB evaluation suggests Las inoculation by the vector (D. citri) feeding, the method used in this experiment was considered the most appropriate at the time of the research was carried out (Lopes & Frare, 2008;Folimonova et al, 2009;Coletta-Filho et al, 2010).…”

“…Las and Lam transmissions occur through the psyllid vector Diaphorina citri (Kuwayama) (Hemiptera: Psyllidae) (Bové, 2006). Las transmission can also be achieved artificially by grafting, using branch segments or buds removed from infected plants (Lopes & Frare, 2008;Coletta-Filho et al, 2010). Regardless of Liberibacter species, the management of HLB in commercial areas includes the planting of healthy nursery trees, the scouting and eradication of symptomatic trees, and the chemical control of insect vectors (Belasque Junior et al, 2009).…”