| Citrus Huanglongbing(HLB)is one of the most serious diseases on citrus,causing huge economic losses to citrus production worldwide.The causal pathogen is a phloem-limited fastidious α-proteobactericea Gram-negative bacteria,(“Candidatus Liberibacter spp.”).Due to the inability of in vitro culture,therefore limited the progress of“Ca.L.asiaticus”(CLas),research.So CLas detection plays an important role in prevention and control of HLB,expecially for the HLB-free and in early-infected citrus growing regions.With the development of high-throughput sequecing technology,providing a better method for CLas genome sequencing and more comprehensive biological information for the research of CLas.This study analyzed the whole genome sequences of CLas to improve the sensitivity and accuracy for CLas detection,identified high copy number gene as target detection gene for CLas detection,and identified different type prophages in all CLas whole genomes,to exporing the pathogenicity difference between different CLas strains.Moreover,phloemlimited “Candidatus Phytoplasma” caused Texas Phoenix Palm Decline(TPPD)is very serious on palm plants.Until now,there was no available whole genome of TPPD-related phytoplasma,this study sequenced TPPD strain ACPD using Illumina Miseq and Nanopore.The main conclusions of this study are as follows:1.We found the nucleotide "G" missed in the signature oligonucleotide OI1 region of16 S r RNA gene sequence,and this base was in the position 14-15 of standard Real-time PCR primer HLBas for CLas detection.Therefore,the base "G" was added and adjustment was made in HLBas at 5’ and 3’ to obtain a serial new forward primers,while the reverse primer HLBr and the probe HLBp remained.Among them,primer set CLas-4G/HLBr was tested to be the most specific and sensitive one.And analysis the detection sensitivity of primer CLas-4G/HLBr compared with other commonly used detection primers.The results showed that the detection sensitivity and accuracy of the primer CLas-4G/HLBr was higher than primer HLBas/HLBr,with a significant difference(P < 0.05).2.In this study,four copies of the hypothetical gene 4CP was identified,and designed primers on the conserved regions of the 4CP gene,the primers 4CPf/4CPp/4CPr based on the 4CP gene were more sensitive to detect CLas than the primers HLBas/HLBr and CLas-4G/HLBr,it can be used as another primer for CLas detection.3.By performing metagenomic sequencing and analysis on false positive(high Ct value)psyllid sample from Temecula,California,and the result indicated that endophytic bacteria in psyllid might be the reason of false positive.4.Exploring the pathogenicity difference between different CLas strains by grafting method.The results showed that different citrus genotypes had different symptoms responsed to the same CLas strain.The same citrus genotype showed different symptoms responsed to different CLas strains.However,on the Nianju plants,showed yellowing,leaves curled back accompanied by severe defoliation after infected by CLas strain harboring type 1 prophage;while showed asymmetric lumps and leaves,slightly defoliated infected by CLas strain associated with Type 2 prophage;When plants infected by CLas strain containing both Type1 and Type 2 prophages,the titer of Type 1 is higher than Type 2 proohage with the whole infection period,so the pathogenicity of Type 1 CLas strain is stronger than Type 1 CLas strain.5.Whole-genome sequencing and assembling of Texas Phoenix Palm Decline(TPPD)related phytoplasma strain ACPD using Illumina Miseq and Nanopore technologies,successfully obtained the first phytoplasma whole genome sequencing of TPPD. |
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