Bacteriomic Analyses of Asian Citrus Psyllid and Citrus Samples Infected With “Candidatus Liberibacter asiaticus” in Southern California and Huanglongbing Management Implications

Abstract

Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable α-proteobacterium “Candidatus Liberibacter asiaticus” (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid-associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, “Candidatus Profftella armature,” “Candidatus Carsonella ruddii,” CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

Bioprospecting Microbiome for Soil and Plant Health Management Amidst Huanglongbing Threat in Citrus: A Review

“…A metagenomic pipeline was developed for bacteriomic analysis of HLB and ACP using next-generation sequencing techniques ( Huang et al, 2021 ). The study identified bacteria in both, citrus as a host plant and vector psyllids, which included Buchnera , Bradyrhizobium , Burkholderia , “ Candidatus Carsonellaruddii,” “ Candidatus Profftella armature,” C Las, Pseudomonas, Mesorhizobium , Paraburkholderia , and Wolbachia .…”

“…The study identified bacteria in both, citrus as a host plant and vector psyllids, which included Buchnera , Bradyrhizobium , Burkholderia , “ Candidatus Carsonellaruddii,” “ Candidatus Profftella armature,” C Las, Pseudomonas, Mesorhizobium , Paraburkholderia , and Wolbachia . Such an outcome would be highly useful in understanding HLB biology and its management ( Huang et al, 2021 ). Profiling of endophytic microbes in HLB-infected and HLB-free leaf midribs of Shatangju mandarin, employing next generation sequencing, revealed 53 endophytic bacterial orders through 12 phyla and 24 endophytic fungal orders distributed through 2 phyla in healthy leaf midribs ( Yan et al, 2021 ).…”

Machine learning and analysis of genomic diversity of “Candidatus Liberibacter asiaticus” strains from 20 citrus production states in Mexico

“…Of particular interest was sample #9 from Hidalgo with a Ct of 36.99 by CLas4G-PCR. Such a high Ct value could have been attributed to the presence of non-CLas bacteria ( Huang et al., 2021 ). However, when sample #9 was cross-checked using the more sensitive RNRf-PCR, a lower Ct value confirmed the presence of CLas ( Bao et al., 2020 ).…”

“…1 in Table 1 ) and BCSMX (Term-A) from Baja California Sur (No. 37 in Table 1 ) were selected for whole genome sequencing following the procedure described previously ( Zheng et al., 2014 ; Huang et al., 2021 ). Briefly, 2–4 µl of sample DNA (mixture of bacteria + ACP) was enlarged (i.e., increased all DNA simultaneously) with GenomiPhiTM V2 DNA Amplification kit (GE Healthcare, Sigma-Aldrich Corp., St. Louis, MO, USA) following manufacturer’s instructions.…”

Candidatus Kirkpatrickella diaphorinae gen. nov., sp. nov., an uncultured endosymbiont identified in a population of Diaphorina citri from Hawaii

“…Despite having significant impacts on the citrus industry worldwide, there are limited 16S rRNA gene sequence surveys available on D. citri populations [1,2]. Outside of two core bacterial endosymbionts Candidatus Carsonella ruddii and Candidatus Profftella armatura, and in addition to the facultative bacterial endosymbiont Wolbachia wDi, no other symbionts are well characterized [1][2][3][4][5][6][7]. Like other hemipteran and sap-sucking insects, D. citri relies on nutritional supplementation delivered by one of its core endosymbionts, Ca.…”