Analyses Of Endo-prokaryotic And Mitochondrial Genomes Of Two "Candidatus Liberibacter Spp." Psyllid Vectors

The Asian citrus psyllid, Diaphorina citri Kuwayama and Potato psyllid(Bactericera cockerelli) are the important pests of agricultural production. D. citri is the vector of “Candidatus Liberibacter asiaticus”(CLas) and “Ca. L. americanus,” associated with Citrus Huanglongbing(HLB, citrus greening disease), which threatens the viability and production of citrus worldwide. B. cockerelli is a pest of potato, tomato, pepper and other Solanaceae plants. B. cockerelli can cause serious phytotoxemia in some host plants called “Psyllid yellows disease,” and can also transmit “Ca. L. solanacearum”(CLso), the putative bacterial pathogen of Zebra chip(ZC) disease. Because both pathogens are unculture and incurable, research on the two vectors is critical for HLB and ZC control. In this study, the surviving ability of these psyllids on different host plants and tissues was investigated and the efficiency of psyllids acquiring CLas or CLso was determined. Also, the endo-prokaryotic and mitochondrial genomes of were characterized via next-generation sequencing. The main details of the researches are described below:(1) Three host species including Murraya exotica, Citrus sunki and Citrus reticulate at different stages of maturity were used to compare the effects of survival percentage of D. citri nymphs and adults. The result showed young shoots were essential for the surviving of nymphs while the survival rates of psyllid adults on mature shoots were higher than those on young shoots, and the survival rate on Citrus sunki was higher than on the other 2 hosts.The biological characteristics of B. cockerelli were compared between 2 tomato species(“Big boy” and “Yellow pearl”). The result indicated the 2nd nymph period and preoviposition period were shorter than others, survival rates of nymph and adult fed on “Yellow pearl” were higher.(2) The infected rates Ca. L. spp of D. citri and B. cockerelli in nymph life stage were higher than those at the adult stage, especially at 6 and 12 d respectively. The infected rate of B. cockerelli was much higher than D. citri at the adult stage, the proportion of which could reach 90% and CT value significantly decreased on 18 d. Nevertheless, only 33.33% of D. citri can acquire Ca. L. spp with no significant dynamic change of CT value. Meanwhile, the copy number of 16 S r RNA of CLso in B. cockerelli was three times that of CLas in D. citri.(3) The endo-prokaryotic genomes of 2 psyllids were obtained after analyzing both psyllid sequencing libraries. The genomes of HLB pathogen(CLas strain YCPsy), primary endosymbiont of D. citri(“Ca. Carsonella ruddii” strain YCCR) and the secondary endosymbiont(“Ca. Profftella armature”) were obtained from D. citri. In addition, 291 contigs from D. citri assembly result were similar to Wolbachia sp.The genomes of CLso strain RSTM(pathogen of ZC), “Ca. Carsonella ruddii” strain BC(primary endosymbiont genome), and Wolbachia sp.(secondary endosymbiont), were obtained from B. cockerelli.(4) This report provides the first complete mitogenome sequence of these two species of psyllids. The complete B. cockerelli mitogenome had a size of 15220 bp, the circular D. citri mitogenomes from Guangdong, China and California, USA were 14996 and 15013 bp respectively. All of the three genomes had 13 protein-coding gene(PCGs), 2 ribosomal RNA genes(r RNAs), 22 transfer RNA genes(t RNAs), and a non-coding region. The overall gene order of the mitogenomes is identical to a common ancestral mitogenome.Compared of nucleotide diversity of 13 PCGs among known Psylloidea species from Gen Bank(Paratrioza sinica, Cacopsylla coccinea and Pachypsylla venusta), cox1 is the most conserved. Sequence analyses revealed differences between and among the insect families, in particular a unique region that can be folded into three stem-loop secondary structures presents only within the B. cockerelli mitogenome. A phylogenetic tree based on the 13 protein coding genes matched an existing taxonomy scheme that was based on morphological characteristics.Compared between D. citri mitogenomes collected from Guangdong and California, 52 SNPs were found in the whole genome. Three methods were used for psyllid population detection(cox1, trn Asn and conventional PCR using 3 nad genes). After detection of D. citri samples collected from China and USA, D. citri in California was likely introduced from eastern USA, which is critical for HLB control in California. In addition, 20 primers were designed using the conserved regions of mitogenomes, to amplify the complete mitogenomes of D. citri from different areas, and multiple mitogenomes were likely found in the control region. Acquisition of the complete mitogenomes of psyllids will possibly use all the mitochondrial genes for analysis and evaluation of population diversity.