Interaction Of "Candidatus Liberibacter Asiaticus" And "Candidatus Phytoplasma Asteri" In Periwinkle

Citrus Huanglongbing(HLB)is one of the most devastating diseases in citrus production,which caused the large economic losses in citrus industry worldwide.The HLB was known to be associated with a fastidious,unculturable phloem-limitedα-Proteobacteriacea Gram-negative bacterium in the genus “Candidatus Liberibacter spp.”,and Koch’s postulates have not been fulfilled.Early studies found that “Candidatus Liberibacter asiaticus”(CLas)and phytoplasma(“Candidatus Phytoplasma asteri”,CPa)can be simultaneously detected in HLB-infected citrus,however,the relationship between CLas and CPa is still unknown.To data,Real Time PCR has been widely used in detection of CLas,but not applied for CPa detection yet.Based on the specific region of CPa’s 16 S rDNA sequence,the primer set for specific detection of CPa was deigned in this study.In this study,the periwinkle(Catharanthus roseus),an optimal plant for research of phloem-limited fastidious bacteria,were used as experimental materials.To explore a faster and more efficient grafting method,two grafting materials,CLas-infected periwinkle leaves and buds,were used as sources to compared the transmission efficiency of CLas.To understand the interaction between CLas and CPa in periwinkle,the infection rate,the transfer direction,the propagation and distribution of the CLas and CPa were analyzed through different grafting combinations.Ethidium Monoazide Bromide(EMA)is a specific nucleic acid dye that selectively enters dead cells and binds tightly with its DNA to form a covalent complex,which led to the inhibition of the PCR amplification of DNA.In this study,the EMA-PCR detection technique was used to determine the proportion of viable cells in leaves of CLas-infected periwinkle and citrus,which can accurately understand the content of viable CLas during different infection periods.The main results were following :1.Comparion of the 16 S rDNA sequence of CPa with 16 S rDNA sequences from six related phytoplasma and plant chloroplast,based the specific region of CPa’s 16 S rDNAsequence,revealed a specific primer set CPa1-F/CPa1-R and probe CPa-P for detection of CPa.2.An innovative individual leaf grafting was invented to compare with trditional bud grafting,showed that the infection rate of CLas was 79.00%(79/100)by individual leaf grafting indicating a higher efficiency of CLas infection,which was higher than the bud grafting(49.17%,59/120).In addition,more leaves than buds in the periwinkle also revealed the superior of individual leaf grafting.3.The grafting result with different combinations revealed that the propagation of CLas was inhibited by infection of CPa.Periwinkle plant showed the phyllody and internode shorten but not led to the death of plant when only infected with CPa.The CLas-infected periwinkle showed mottled leaves and cannot survival more than two months after grafting.When the periwinkle was infected with CLas and CPa,the plant showed both mottled leaves and phyllody and cannot be survival more than three months after grafting,however,this was longer than the survival time of periwinkle only infected with CLas.In addition,our results also showed that the movement speed of CLas was faster than CPa,but the propagation rate of CLas was slower than CPa.4.A large number of CLas and CPa pathogens were observed in the paraffin sections of infected periwinkle by TEM.Although both of CLas and CPa were round,short ellipse and rod-shaped,the CPa were mostly round-shape.Both CLas and CPas showed the structure with three-layer membrane.The size of CLas and CPa was 90~420×320~1700 nm and 57~307×300~700 nm,respectively.And the thickness of cell film thickness of CLas and CPa were 20 nm and 9 nm,respectively.This indicated the small of CPa compared with CLas in both cell size and thickness of cell film.The CLas and CPa can be existed in the same sieve tube cells with the higher concentration of CPa than CLas due to the possible suppression of CLas propagation by CPa.5.Detection result by EMA-PCR technology revealed that the best inhibitory effect to death bacteria in the periwinkle sample by PCR was 50 μg/mL concentration of EMA.The concentration of viable CLas in the leaves of the periwinkle was significantly higher in the early infection stage than others infection stage(p<0.01),even symptoms was not obvious in the early stage.However,the concentration of CLas in the middle and late infection stagewas lower than it in early stage,but the symptoms of citrus leaves were more obvious in the middle and late infection stage.