Enrichment Of "Candidatus Liberibacter Asiaticus" And Its Evaluation

Citrus Huanglongbing(HLB)is one of the most devastating diseases in citrus production,which have caused large economic losses in citrus industry worldwide.Currently,there is borad agreement that HLB is associated with “Candidatus Liberibacter spp.”.Due to the current incapcity of in vivo culture of CLas,it is difficult to study the CLas biology and morphology.In recent years,genomics has been widely used and promoted the research of CLas.However,the key to obtain the high quality CLas genome is to get the host materials with high CLas concentration.In this study,we performed two methods,the dodder-mediated CLas enrichment and the in vivo incubation of CLas-infected fruits,to enrich CLas by increasing the CLas concentration in host.The CLas enrichment was further evaluated and the results were showed as follow:1.Dodder-mediated CLas enrichment assay: Among 55 experimental sets,the appearances of dodder grown on citrus shoots can be simply classified into four types: Type I(7 groups / 12.6 %),Type II(21 groups / 38.2 %),Type III(17 groups / 30.9 %)and Type IV(10 groups / 18.2 %).A total of 48 out of 55 dodder successfully formed the haustoria and parasitized in 48 CLas-infected citrus shoots.Quantitative comparison result revealed the higher concentration of CLas was observed in 30 dodders samples(30/48,62.50 % of survived dodder)than the corresponding source citrus leaves midribs with increasing by 2-to 419-fold.The growth types of dodder which had better enrichment effect on CLas were mostly Type III and Type IV.When the parasitism time of dodder was between 13 and 17 days,the enrichment effect can reach a good level and the enrichment level was relatively stable and concentrated.In addition,among the four cultivars,the CLas enrichment effect was better on the citrus shoots,most of which were nianju and shatangju.2.Three sets of CLas-infected citrus leaves midrib and their parasitized dodder DNA samples were selected for Hi Seq sequencing.The assembly quality of CLas genome sequencing from different hosts(citrus and dodder)was compared.The results showed that the total number of CLas reads in dodder Hi Seq data was 6 to 33 times higher than those in Hi Seq data using citrus as material.De novo assembly results showed that less number but longer N50 of CLas contigs was obtained by using dodder Hi Seq data than the corresponding citrus Hi Seq data,indicating the high quality of CLas genome obtained by using CLas-enriched dodder Hi Seq data and without induce of genomic variation during a short period of parasitism.3.To evaluate whether CLas multiplied in dodder after transmission from citrus,five house-keeping genes that involved with cell division and DNA replication were selected to determine the expression level of CLas at different period after transmitted into dodder,and compared with their expression level in its host citrus.The results showed that the expression level of five CLas house-keeping genes in dodder increased gradually after parasitizing in citrus shoot,and was significantly higher than that in Citrus(P < 0.05)after15 days.This indicated that a rapid reproduction of CLas in dodder tendrils.4.The CLas-infected fruits were collected and maintained under constant temperature and moisture to increase the concentration of CLas in fruit.The content of total DNA,host DNA and CLas DNA were decreased after 14 days,but the ratio of CLas DNA in the total DNA and the copy number of CLas were increased.After 28 days,comparing with diseased fruit on the tree,the excised diseased fruits showed the almost completely color changed but the color of diseased fruit on the tree was still green.5.Both the CLas-infected fruit pith DNA sample and midrib DNA samples were used as DNA source for CLas genomic sequence and assembly.When the CT values were similar,comparesion with the midrib-origin CLas-infected DNA samples,the total number of CLas reads in the pith-origin Hi Seq data was improved,the less number but longer CLas contigs was obtained by using midrib-origin Hi Seq data than the corresponding the pith-origin Hi Seq data,DNA sequence and assembly(whatever de novo assembly or reference-based assembly)based on the CLas-infected fruit pith DNA can improve the CLas genome quality after sequencing and assembly.In conclusion,both dodder-mediated and the excised infected citrus frut-mediated CLas-enrichment can increase the concentration of CLas in host,which provided the material source for the subsequent CLas genomics research and an important reference method for obtaining high concentration of CLas.