Rapid Detection By Loop-Mediated Isothermal Amplification (LAMP) And Biocontrol Of Citrus Canker

Citrus canker is one of the most important diseases affecting citrus production worldwide caused by the phytopathogenic bacterium Xanthomonas citri subsp.citri.The early and accurate detection of this bacterium is essential for the management of the citrus canker.Previously,detection methods based on bacterial isolation,antibodies and polymerase chain reaction(PCR)have been developed;however,these detection methods are time consuming,laborious and require expensive laboratory equipment.Loop-mediated isothermal amplification(LAMP)is a novel isothermal DNA amplification technique;LAMP is fast,specific,sensitive and requires no specialized laboratory equipment.Recently,LAMP has been used for rapid and accurately identification of suspected diseased samples in local plant inspection and quarantine.In this study,we designed six LAMP primers and established a LAMP system for detection of citrus canker according to the specific and conserved nucleotide sequence from X.citri subsp.citri.LAMP detection method can be effectively used for accurately detection based on genomic DNA from bacterium X.citri subsp.citri or citrus canker leaves respectively;however,no products of LAMP can be detected from genomic DNA from X.oryzae pv.oryzae,Acidovorax avenae subsp.avenae,Pseudomonas and Pantoea,consistently with PCR detection method.This LAMP method could detect as low as 100 pg DNA of X.citri subsp.citri,and 4×103 cfu mL-1 ofX.citri subsp.citri;the sensitive of LAMP was 1,250 times higher than PCR.Furthermore,LAMP could specifically distinguish between citrus canker and citrus scab that these two diseases are easily confused according to symptoms in field.The advantage of this LAMP detection method is simple,rapid,sensitive and specific.Therefore,it could be applied for rapid detection of citrus canker in the fields.The main strategy for management of citrus canker in endemic areas is chemical control with preventive sprays;the significant disadvantage of chemical control is selection for fungicide resistant X.citri subsp.citri strains and the subsequent serious environmental problems due to fungicide toxicity.In order to investigate the antibacterial potential of chitosan,and biocontrol using Bacillus against X.citri subsp.citri,the plate colony count method was performed to evaluate the antibacterial effects of chitosan with three different viscosities against X.citri subsp.citri,and modified paper disk method was performed to measure the antibacterial effects of fermentation fluids of two strains of B.subtilis against X.citri subsp.citri.The results of chitosan treatment for 0.5h,3h and 6h together show that the antibacterial ability of chitosan with three different viscosities against X.citri subsp.citri is:viscosity 12>viscosity 100>viscosity 20.The fermentation of B.subtilis strains BS101 and 54-6 were obtained by shaking culture in YPG media for 3 d.Inhibition zones generated by fermentation from two Bacillus strains was measuredin diameters,the Inhibition zone diameter of BS101 and 54-6 were 3.45 cm and 3.21 cm respectively,which are 45%and 35%higher than that generated by 0.1 gml-1Ampicillin.We speculated that there are antibacterial activity chemical substances against citrus canker in the fermentation of the two Bacillusstrains.Thus,chitosan with 12 viscosity and two Bacillus strains BS101 and 54-6have the potential for management of citrus canker in the fields.