Xiaoling Deng

Feeding behaviour of the Asiatic citrus psyllid, Diaphorina citri, on healthy and huanglongbing-infected citrus

Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psyllidae) is a vector of huanglongbing, a disease of citrus that in Asia is caused by ‘Candidatus Liberibacter asiaticus’ (α‐Proteobacteria) (Las). Acquisition of Las by D. citri appears to be variable, and this variability may be due to the suitability of the host plants and their tissues for acquisition. Therefore, this study aimed to determine the effect of symptom severity of the disease on the feeding behaviour of D. citri. Use of an electrical penetration graph showed that the pathway phase of D. citri consisted of four waveforms, A, B, C, and D; waveforms A and B have not been reported for D. citri before. The remaining waveforms, E1, E2, and G, conform to those described before for D. citri. The duration of the non‐penetration period did not differ between healthy or infected plants. However, in moderately and severely symptomatic plants, the duration of the pathway phase increased, whereas the phloem phase was shorter. In all diseased plants, the times to first and sustained salivation in the phloem were longer than those in control plants, with the times being related to symptom severity. As symptom expression increased, the percentage of time spent by psyllids salivating during the phloem phase increased; however, the percentage of time spent in phloem activities reduced gradually from ca. 74% in the control plants to ca. 8% in the severely symptomatic plants. In contrast, the percentage of time spent on xylem activities increased, as did the proportion of psyllids feeding from xylem. The differences in the durations of the E waveforms on plants showing different levels of symptom expression may account for differences in acquisition found amongst studies; therefore, future work on the acquisition and transmission of Las needs to carefully document symptom expression.

DETECTION OF 'CANDIDATUS LIBERIBACTER ASIATICUS' IN CACOPSYLLA (PSYLLA) CITRISUGA (HEMIPTERA: PSYLLIDAE)

ABSTRACT Nymphs of the pomelo psyllid, Cacopsylla (Psylla) citrisuga Yang & Li, were collected from huanglongbing (HLB) symptomatic lemon trees, Citrus limon (L.) in Yunnan Province, China. DNA samples extracted from groups of 2–10 pomelo psyllid nymphs of all stages and from leaves of lemon plants were analyzed with nested-PCR in order to detect ‘Candidatus Liberibacter asiaticus’ (Las). The results showed that 24 out of the 49 lemon trees were Las-positive. Also psyllid nymphs collected from 10 out of the 24 Las-positive plants were Las-positive. DNA extracted from individual late stage nymphs (3rd to 5th instars) collected from Las-infected trees were then subjected to nested-PCR trials. Twelve out of the 29 nymphs proved to be Las positive. All nymphs collected from Las-negative lemon trees were also Las-negative. Most psyllid samples which showed positive in nested-PCR were also positive in conventional-PCR detection. The amplified fragment of the 16S rRNA gene of ‘Ca. Liberibacter spp.’ from positive psyllid samples was 99% similar to those of Las strain psy62 in GenBank. These results demonstrate that Cacopsylla (Psylla) citrisuga is another Las carrier insect. Transmission studies are underway to determine whether Las-positive psyllids can transmit Las to healthy citrus.

Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

“Candidatus Liberibacter asiaticus” (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the “Ca. Liberibacter” genera.

Whole-Genome Sequence of “Candidatus Liberibacter solanacearum” Strain R1 from California

ABSTRACT The draft whole-genome sequence of “Candidatus Liberibacter solanacearum” strain R1, isolated from and maintained in tomato plants in California, is reported. The R1 strain has the genome size of 1,204,257 bp, G+C content of 35.3%, 1,101 predicted open reading frames, and 57 RNA genes.

Analyses of Mitogenome Sequences Revealed that Asian Citrus Psyllids ( Diaphorina citri ) from California Were Related to Those from Florida

Asian citrus psyllid (ACP, Diaphorina citri Kuwayama) transmits “Candidatus Liberibacter asiaticus” (CLas), an unculturable alpha-proteobacterium associated with citrus Huanglongbing (HLB). CLas has recently been found in California. Understanding ACP population diversity is necessary for HLB regulatory practices aimed at reducing CLas spread. In this study, two circular ACP mitogenome sequences from California (mt-CApsy, ~15,027 bp) and Florida (mt-FLpsy, ~15,012 bp), USA, were acquired. Each mitogenome contained 13 protein coding genes, 2 ribosomal RNA and 22 transfer RNA genes, and a control region varying in sizes. The Californian mt-CApsy was identical to the Floridian mt-FLpsy, but different from the mitogenome (mt-GDpsy) of Guangdong, China, in 50 single nucleotide polymorphisms (SNPs). Further analyses were performed on sequences in cox1 and trnAsn regions with 100 ACPs, SNPs in nad1-nad4-nad5 locus through PCR with 252 ACP samples. All results showed the presence of a Chinese ACP cluster (CAC) and an American ACP cluster (AAC). We proposed that ACP in California was likely not introduced from China based on our current ACP collection but somewhere in America. However, more studies with ACP samples from around the world are needed. ACP mitogenome sequence analyses will facilitate ACP population research.

The Complete Mitochondrial Genome Sequence of Bactericera cockerelli and Comparison with Three Other Psylloidea Species.

Potato psyllid (Bactericera cockerelli) is an important pest of potato, tomato and pepper. Not only could a toxin secreted by nymphs results in serious phytotoxemia in some host plants, but also over the past few years B. cockerelli was shown to transmit “Candidatus Liberibacter solanacearum”, the putative bacterial pathogen of potato zebra chip (ZC) disease, to potato and tomato. ZC has caused devastating losses to potato production in the western U.S., Mexico, and elsewhere. New knowledge of the genetic diversity of the B. cockerelli is needed to develop improved strategies to manage pest populations. Mitochondrial genome (mitogenome) sequencing provides important knowledge about insect evolution and diversity in and among populations. This report provides the first complete B. cockerelli mitogenome sequence as determined by next generation sequencing technology (Illumina MiSeq). The circular B. cockerelli mitogenome had a size of 15,220 bp with 13 protein-coding gene (PCGs), 2 ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and a non-coding region of 975 bp. The overall gene order of the B. cockerelli mitogenome is identical to three other published Psylloidea mitogenomes: one species from the Triozidae, Paratrioza sinica; and two species from the Psyllidae, Cacopsylla coccinea and Pachypsylla venusta. This suggests all of these species share a common ancestral mitogenome. However, 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 present only within the B. cockerelli mitogenome. A phylogenetic tree based on the 13 PCGs matched an existing taxonomy scheme that was based on morphological characteristics. The available complete mitogenome sequence makes it accessible to all genes for future population diversity evaluation of B. cockerelli.

The complete mitochondrial genome sequence of Diaphorina citri (Hemiptera: Psyllidae)

Abstract The first complete mitochondrial genome (mitogenome) sequence of Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) from Guangzhou, China, is presented. The circular mitogenome is 14,996 bp in length with an A + T content of 74.5%, and contains 13 protein-coding genes (PCGs), 22 tRNA genes (tRNAs), two rRNA genes and an AT-rich control region. Synteny was identical to the ancestral insect mitogenomes. All PCGs start with ‘ATN’ codons. Eleven PCGs use the complete termination codons (‘TAA’ or ‘TAG’), while the remaining two (cox2 and nad5) use a single ‘T’ as the stop codon. All tRNAs have the typical cloverleaf structure, except for trnTrp which lacks the variable arm and trnSer (AGN) which lacks the dihydrouridine arm. A phylogenetic tree, including 12 members of Sternorrhyncha, was constructed based on the 13 PCGs sequences. The resulting phylogenetic tree matched the established taxonomic scheme based on morphology.

Unusual Five Copies and Dual Forms of nrdB in “ Candidatus Liberibacter asiaticus”: Biological Implications and PCR Detection Application

“Candidatus Liberibacter asiaticus” (CLas), a non-culturable α-proteobacterium, is associated with citrus Huanglongbing (HLB, yellow shoot disease) currently threatening citrus production worldwide. Here, the whole genome sequence of CLas strain A4 from Guangdong of China was analyzed. Five copies of nrdB, encoding β-subunit of ribonucleotide reductase (RNR), a critical enzyme involving bacterial proliferation, were found. Three nrdB copies were in long form (nrdBL, 1,059 bp) and two were in short form (nrdBS, 378 bp). nrdBS shared >99% identity to 3′ end of nrdBL and had no active site. Sequences of CLas nrdB genes formed a distinct monophyletic lineage among eubacteria. To make use of the high copy number feature, a nrdB-based primer set RNRf/RNRr was designed and evaluated using real-time PCR with 262 HLB samples collected from China and USA. Compared to the current standard primer set HLBas/HLBr derived from the 16S rRNA gene, RNRf/RNRr had Ct value reductions of 1.68 (SYBR Green PCR) and 1.77 (TaqMan PCR), thus increasing the detection sensitivity three-fold. Meanwhile, RNRf/RNRr was more than twice the stability of primer set LJ900f/LJ900r derived from multi-copy prophage. The nrdB-based PCR thereby provides a sensitive and reliable CLas detection with broad application, especially for the early diagnosis of HLB.

De Novo Genome Sequence of "Candidatus Liberibacter solanacearum" from a Single Potato Psyllid in California.

ABSTRACT The draft genome sequence of “Candidatus Liberibacter solanacearum” strain RSTM from a potato psyllid (Bactericera cockerelli) in California is reported here. The RSTM strain has a genome size of 1,286,787 bp, a G+C content of 35.1%, 1,211 predicted open reading frames (ORFs), and 43 RNA genes.

Movement of Diaphorina citri (Hemiptera: Liviidae) Adults between Huanglongbing-Infected and Healthy Citrus

Abstract Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), is the vector of huanglongbing (HLB or citrus greening disease). Preferences of D. citri adults differed for HLB-infected and healthy citrus under different maturity conditions. The presence or absence of young shoots had a significant influence on the choice by D. citri adults between HLB-infected and healthy citrus hosts. When citrus plants had young shoots, infected plants were more attractive than healthy ones to the adults. Also, D. citri adults fed for a longer time on infected plants with young shoots than on their healthy counterparts. In the absence of young shoots, D. citri adults were at first also more attracted to infected mature leaves, but after 38 h they turned to healthy mature leaves. In a multiple choice experiment, infected young shoots and healthy young shoots were the most attractive, followed by shoots with infected mature-yellow leaves and physiologically mature-yellow leaves, and lastly by healthy or infected mature-green leaves. In an experiment to measure the relative attractiveness of yellow, green, and white boards, yellow color boards attracted more adults than green and white boards, indicating that the adults preferred the yellow color. The results suggest that D. citri adults when first confronted with a choice are more attracted to infected citrus because of the color, but subsequently they move to healthy citrus perhaps because of either the poor nutrition or a feeding barrier in the infected hosts. This behavior appears to facilitate the pathogens spread.