Temporal And Spatial Distribution Of "Candidatus Libribacter Asiaticus" On Citrus Branches With Fruits

Citrus Huanglongbing(HLB)is one of the most serious diseases of the citrus industry in recent 100 years.Countries around the world have not found any practical and effective method toward this disease.HLB is caused by "Candidatus Liberibacter asiaticus",CLas in China.The distribution of CLas is uneven in the infected trees,and the titers of CLas varies with citrus varieties and different growth periods.In this study,we compared the DNA quality extracted by the OMEGA kit method,the BIOPURE kit(magnetic beads method)and the Genesee column method,and determined a DNA extraction method for detection of CLas.Based on the research,real-time PCR was used to measure the CLas on the infected Shatangju(Citrus reticulate Blanco cv.Shatang ju),Red Pomelo(Citrus maxima(Burm.)Merr.cv.Red Pomelo),Shatian Yu(Citrus maxima(Burm.)Merr.cv.Shatian Yu.),and W.Murcott(Citrus reticulata cv.Murcott Afourer)fruit branches.Temporal and Spatial analysis of CLas in different citrus species has came out the following results:1.We compared the quality of DNA in the citrus pericarp and the midribs extracted by the three methods mentioned above.The OMEGA kit method has confirmed to be the optimal DNA extraction method for the detection of CLas in the laboratory.2.The CLas-positive rates in different parts of citrus branches were different.The total detection rates in different parts of infected Red Pomelo and infected Shatian Yu branch were 100%.However,the rates were 96.2% and 96.4% in infected W.Murcott and infected Shatangju.Among the infected branches of all varieties,CLas detection rate was100% in the white pith of oranges.3.There was a significant difference in the number of pathogens in different tissues on the same branch in the mature season of citrus: the concentration of CLas in the symptomatic fruits was much higher than that in the leaves.The highest CLas titers were found in the white pith of Shatangju and W.Murcott or at the fruit axis of Shatian Yu andRed pomelo.4.The concentration of CLas was dynamically distributed in the branches of different seasons or different citrus growth periods.On the whole,with the increase of fruit maturity,CLas gradually accumulated in the fruit(especially in the the white pith of oranges the fruit central axis),while the CLas concentration on the leaves showed a decreasing trend.5.There are differences in the symptoms of infected plants of different varieties and different periods.The symptoms on leaves of infected Shatangju,Red pomelo,and Shatian Yu were more obvious than those on leaves of W.Murcott.The symptom was the most obvious at fruit ripening stage of infected Shatangju.The red nose fruits of Shatangju and asymmetric malformation fruits of Red Pomelo and Shatian Yu can be used as a basis for rapid diagnosis in the field.Due to the lack of an effective method to control Citrus HLB and the unsuccessful of in vitro culture of the pathogen,the control and the pathogenic mechanisms of HLB have progressed slowly.Therefore,this study confirmed a better DNA extraction method for the detection of CLas in the laboratory and analyzed the temporal and spatial distribution of Candidatus Libribacter Asiaticus on citrus branches with fruits systematically.The outcomes of this study would provides some ideas for finding a suitable material to isolation and cultivation of the pathogen,and providing a guidance for the early detection of HLB,thus guiding the prevention and control of the disease.