Dataset Information

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.

SUBMITTER: Huang J

PROVIDER: S-EPMC8283493 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) implicated as causative agent of citrus huanglongbing (citrus greening), currently the most serious citrus disease worldwide. Las is transmitted by D. citri in a persistent-circulative manner, but the question of replication of this bacterium in its psyllid vector has not been resolved. Thus, we studied the effects of the acquisition access period (AAP) by nymphs and adults of D. citri on Las acquisition, multiplication and inoculation/transmission. D. citri nymphs or adults (previously non-exposed to Las) were caged on Las-infected citrus plants for an AAP of 1, 7 or 14 days. These 'Las-exposed' psyllids were then transferred weekly to healthy citrus or orange jasmine plants, and sampled via quantitative polymerase chain reaction (qPCR) analysis 1-42 days post-first access to diseased plants (padp); all tested nymphs became adults 7-14 days padp. Our results indicate that following 1 or 7 day AAP as nymphs 49-59% of Las-exposed psyllids became Las-infected (qPCR-positive), whereas only 8-29% of the psyllids were infected following 1-14 day AAP as adults. Q-PCR analysis also indicated that Las titer in the Las-exposed psyllids (relative to that of the psyllid S20 ribosomal protein gene) was: 1) significantly higher, and increasing at a faster rate, following Las acquisition as nymphs compared to that following Las acquisition as adults; 2) higher as post-acquisition time of psyllids on healthy plants increased reaching a peak at 14-28 days padp for nymphs and 21-35 days padp for adults, with Las titer decreasing or fluctuating after that; 3) higher with longer AAP on infected plants, especially with acquisition as adults. Our results strongly suggest that Las multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for Las to reach higher levels in the vector. However, under the conditions of our experiments, only D. citri that had access to infected plants as nymphs were able to inoculate Las into healthy citrus seedlings or excised leaves. The higher probability of Las inoculation into citrus by psyllids when they have acquired this bacterium from infected plants during the nymphal rather than the adult stage, as reported by us and others, has significant implications in the epidemiology and control of this economically important citrus disease.

Project description:Genetic variability in insect vectors is valuable to study vector competence determinants and to select non-vector populations that may help reduce the spread of vector-borne pathogens. We collected and tested vector competency of 15 isofemale lines of Asian citrus psyllid, Diaphorina citri, vector of 'Candidatus Liberibacter asiaticus' (CLas). CLas is associated with huanglongbing (citrus greening), the most serious citrus disease worldwide. D. citri adults were collected from orange jasmine (Murraya paniculata) hedges in Florida, and individual pairs (females and males) were caged on healthy Murraya plants for egg laying. The progeny from each pair that tested CLas-negative by qPCR were maintained on Murraya plants and considered an isofemale line. Six acquisition tests on D. citri adults that were reared as nymphs on CLas-infected citrus, from various generations of each line, were conducted to assess their acquisition rates (percentage of qPCR-positive adults). Three lines with mean acquisition rates of 28 to 32%, were classified as 'good' acquirers and three other lines were classified as 'poor' acquirers, with only 5 to 8% acquisition rates. All lines were further tested for their ability to inoculate CLas by confining CLas-exposed psyllids for one week onto healthy citrus leaves (6-10 adults/leaf/week), and testing the leaves for CLas by qPCR. Mean inoculation rates were 19 to 28% for the three good acquirer lines and 0 to 3% for the three poor acquirer lines. Statistical analyses indicated positive correlations between CLas acquisition and inoculation rates, as well as between CLas titer in the psyllids and CLas acquisition or inoculation rates. Phenotypic and molecular characterization of one of the good and one of the poor acquirer lines revealed differences between them in color morphs and hemocyanin expression, but not the composition of bacterial endosymbionts. Understanding the genetic architecture of CLas transmission will enable the development of new tools for combating this devastating citrus disease.

Project description:BackgroundHuanglongbing (HLB) is one of the most destructive citrus diseases in the world. The disease is associated with the presence of a fastidious, phloem-limited α- proteobacterium, 'Candidatus Liberibacter asiaticus', 'Ca. Liberibacter africanus' or 'Ca. Liberibacter americanus'. HLB-associated Liberibacters have spread to North America and South America in recent years. While the causal agents of HLB have been putatively identified, information regarding the worldwide population structure and epidemiological relationships for 'Ca. L. asiaticus' is limited. The availability of the 'Ca. L. asiaticus' genome sequence has facilitated development of molecular markers from this bacterium. The objectives of this study were to develop microsatellite markers and conduct genetic analyses of 'Ca. L. asiaticus' from a worldwide collection. Two hundred eighty seven isolates from USA (Florida), Brazil, China, India, Cambodia, Vietnam, Taiwan, Thailand, and Japan were analyzed.ResultsA panel of seven polymorphic microsatellite markers was developed for 'Ca. L. asiaticus'. Microsatellite analyses across the samples showed that the genetic diversity of 'Ca. L. asiaticus' is higher in Asia than Americas. UPGMA and STRUCTURE analyses identified three major genetic groups worldwide. Isolates from India were genetically distinct. East-southeast Asian and Brazilian isolates were generally included in the same group; a few members of this group were found in Florida, but the majority of the isolates from Florida were clustered separately. eBURST analysis predicted three founder haplotypes, which may have given rise to three groups worldwide.ConclusionsOur results identified three major genetic groups of 'Ca. L. asiaticus' worldwide. Isolates from Brazil showed similar genetic makeup with east-southeast Asian dominant group, suggesting the possibility of a common origin. However, most of the isolates recovered from Florida were clustered in a separate group. While the sources of the dominant 'Ca. L. asiaticus' in Florida were not clearly understood, the less-pervasive groups may have been introduced directly from Asia or via Brazil. Notably, the recent outbreak of HLB in Florida probably occurred through multiple introductions. Microsatellite markers developed in this study provide adequate discriminatory power for the identification and differentiation of closely-related isolates, as well as for genetic studies of 'Ca. L. asiaticus'.

Project description:Prophages are highly dynamic components in the bacterial genome and play an important role in intraspecies variations. There are at least two prophages in the chromosomes of Candidatus Liberibacter asiaticus' (Las) Floridian isolates. Las is both unculturable and the most prevalent species of Liberibacter pathogens that cause huanglongbing (HLB), a worldwide destructive disease of citrus. In this study, seven new prophage variants resulting from two hyper-variable regions were identified by screening clone libraries of infected citrus, periwinkle and psyllids. Among them, Types A and B share highly conserved sequences and localize within the two prophages, FP1 and FP2, respectively. Although Types B and C were abundant in all three libraries, Type A was much more abundant in the libraries from the Las-infected psyllids than from the Las-infected plants, and Type D was only identified in libraries from the infected host plants but not from the infected psyllids. Sequence analysis of these variants revealed that the variations may result from recombination and rearrangement events. Conventional PCR results using type-specific molecular markers indicated that A, B, C and D are the four most abundant types in Las-infected citrus and periwinkle. However, only three types, A, B and C are abundant in Las-infected psyllids. Typing results for Las-infected citrus field samples indicated that mixed populations of Las bacteria present in Floridian isolates, but only the Type D population was correlated with the blotchy mottle symptom. Extended cloning and sequencing of the Type D region revealed a third prophage/phage in the Las genome, which may derive from the recombination of FP1 and FP2. Dramatic variations in these prophage regions were also found among the global Las isolates. These results are the first to demonstrate the prophage/phage-mediated dynamics of Las populations in plant and insect hosts, and their correlation with insect transmission and disease development.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets