Wu, Fengnian

Abstract Citrus Huanglongbing and Citrus tristeza are two diseases that affect the citrus industry worldwide. The pathogens causing these diseases are the phloem-limited bacteria ‘Candidatus Liberibacter spp.’ (mainly Ca. L. asiaticus, CLas) and citrus tristeza virus (CTV). We recently found that both CLas and CTV could be acquired and retained by the Asian citrus psyllid Diaphorina citri. However, the mechanism through which CLas and CTV interact with the insect vectors and plant hosts has not been defined. In this study, an electrical penetration graph was used to study the feeding behavior of D. citri adults on four groups of Citrus reticulata Blanco cv. Hongjü plants: healthy, CLas-infected, CTV-infected, and CTV-CLas coinfected plants. Liquid chromatography with tandem mass spectrometry (LC–MS/MS) was applied to analyze the metabolites of the four groups of plants. The combined results are as follows: (1) The lowest number of metabolites were enriched in CTV-infected plants, which hardly influenced the feeding behavior of D. citri, suggesting that mild CTV strain (CT31) infection caused limited disorders in citrus plants compared with CLas infection; (2) Increased levels of L-arabinose and kaempferol in CTV-infected and CLas-CTV coinfected plants were suggested to contribute to increased penetration time during feeding of D. citri. CLas-infection increases the difficulty of finding appropriate feeding sites by the vector and results in xylem feeding for certain duration; (3) A significant reduction in α-linolenic acid metabolism in CLas-infected plants was found to be related to methyl jasmonate signaling, which induced resistance to D. citri and increased the duration of salivation. This effect was reversed by coinfection with CTV and was consistent with the phloem structure and carbohydrate accumulation alteration; (4) Stress response-associated 2'-hydroxygenistein and sakuranetin were highly upregulated flavonoid in CTV-CLas coinfected plants. This combinged with the anatomical alterations might interfere with D. citri feeding in the citrus phloem, as reflected by the time reduction of sap-sucking there. These findings will provide new insights into the interactions between CTV and CLas in citrus and the insect vector D. citri that transmiting these pathogens.

‘Candidatus Liberibacter asiaticus’ is the bacterium associated with the citrus disease known as huanglongbing (HLB). This study evaluated the influence of ‘Ca. L. asiaticus’ infection on a number of key plant physiological variables concerning photosynthesis, cell integrity, reactive oxygen species scavengers’ activity, and osmoregulation of two different species of citrus—the pomelo Citrus maxima and the mandarin C. reticulata ‘Tankan’—relative to their measured ‘Ca. L. asiaticus’ infection load. Results indicated that all measured physiological variables except soluble sugar were affected by increased ‘Ca. L. asiaticus’ infection titers, wherein the variety C. maxima proved overall more resistant than C. reticulata. ‘Ca. L. asiaticus’ infection was linked in both plants to decrease in chlorophyll concentration, cell membrane permeability, and malondialdehyde, as well as increased free proline and starch contents. Chlorophyll fluorescence measurements taken 9 months after grafting the mandarin C. reticulata with ‘Ca. L. asiaticus’ scions revealed a significant decrease in the photosynthesis variables maximum photochemical quantum yield of photosystem II (PSII), effective photochemical quantum yield of PSII, and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae, whereas nonphotochemical fluorescence quenching increased significantly; C. maxima plants, on the other hand, did not show significant differences until the 12th month from infection exposure. The variables superoxide dismutase, catalase, peroxidase, and soluble protein initially increased and later decreased. In addition, progression of ‘Ca. L. asiaticus’ replication in both citrus species was accompanied by rapid changes in three reactive oxygen species scavenging enzymes in C. maxima, while the pattern was different in C. reticulata. We hypothesize that the observed interspecific differences in physiological change are related to their relative resistance against ‘Ca. L. asiaticus’ infection. These results provide a scaffold for better describing the pathogenesis, selecting the most resistant breeds, or even validating pertaining omics research; ultimately, these detailed observations can facilitate the diagnosis of ‘Ca. L. asiaticus’ infection.

Diaphorina citri Kuwayama (D. citri) is an insect vector of phloem-limited ‘Candidatus Liberibacter asiatus’ (CLas), the presumed pathogen of citrus Huanglongbing (HLB). Recently, our lab has preliminarily found it acquired and transmitted Citrus tristeza virus (CTV), which was previously suggested to be vectored by species of aphids. However, the influences of one of the pathogens on the acquisition and transmission efficiency of the other pathogen remain unknown. In this study, CLas and CTV acquisition and transmission by D. citri at different development stages under field and laboratory conditions were determined. CTV could be detected from the nymphs, adults, and honeydew of D. citri but not from the eggs and exuviates of them. CLas in plants might inhibit CTV acquisition by D. citri as lower CTV–positive rates and CTV titers were detected in D. citri collected from HLB-affected trees compared to those from CLas–free trees. D. citri were more likely to obtain CTV than CLas from host plants co-infected with the two pathogens. Intriguingly, CTV in D. citri facilitated the acquisition and transmission of CLas, but CLas carried by D. citri had no significant effect on the transmission of CTV by the same vector. Molecular detection and microscopy methods confirmed the enrichment of CTV in the midgut after a 72-h acquisition access period. Collectively, these results raise essential scientific questions for further research on the molecular mechanism of pathogen transmission by D. citri and provide new ideas for the comprehensive prevention and control of HLB and CTV.

AbstractBACKGROUNDDiaphorina citri is a vector of ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus huanglongbing (HLB). In this study, the adaptability and CLas titres of D. citri adults on three weed species, namely, Ageratum conyzoides, Solanum nigrum and Praxelis clematidea, which are widely distributed in citrus orchards in China, were determined.RESULTSD. citri selected S. nigrum preferentially over the other weed species. The longest survival times of D. citri adults on A. conyzoides, S. nigrum and P. clematidea were 48, 41 and 11 days, respectively. On S. nigrum, the survivorship of females was significantly higher than that of males. The CLas titres of D. citri adults feeding on the three weeds for 1 week increased significantly during autumn but did not change significantly during spring. Conversely, the titres of adults on citrus increased significantly during spring but did not change significantly during autumn.CONCLUSIOND. citri exhibited different adaptabilities to the three weed species, but the CLas titres of D. citri adults did not decrease on any of the weeds. These results suggested that the three weed species could help dispersing of D. citri and transmission of CLas while ideal host conditions were scarce or absent. © 2021 Society of Chemical Industry.

Abstract Background Diaphorina citri is a vector of ‘ Candidatus Liberibacter asiaticus’ (CLas) associated with citrus Huanglongbing. In this study, the infection and titers of CLas in the psyllid, were monitored for life cycle stage, sex, host‐plant CLas titer, host‐plant genotype, and ambient temperature. Results Acquisition efficiency of CLas by D. citri was highest in nymphs reared at 25 °C on a host plant with high CLas titers but was independent of the host genotypes assessed and of vector sex. We further observed that D. citri nymphs acquired CLas more rapidly than adults based on acquisition access periods (AAPs). CLas did not multiply in the alimentary canal, hemolymph, and salivary glands of adults for 18 days after a 3‐day AAP as adult. However, CLas multiplication was detected in hemolymph and salivary gland of adults after the bacterium was acquired by nymphs. Eighty percent of salivary glands of adults contained CLas 18 days after a 3‐day AAP as nymph compared to 10% 18 days after a 3‐day AAP as adults. Conclusion Different factors tested herein influenced CLas acquisition efficiency of D. citri , CLas multiplication and spread inside the psyllid. These observations serve to better understand mechanisms of CLas infection in D. citri . © 2018 Society of Chemical Industry

Prophages, the lysogenic form of bacterial phages, are important genetic entities of ‘Candidatus Liberibacter asiaticus’ (CLas), a nonculturable α-proteobacterium associated with citrus Huanglongbing. Two CLas prophages have been described, SC1 (NC_019549.1, Type 1) and SC2 (NC_019550.1, Type 2), which involve the lytic cycle and the lysogenic cycle, respectively. To explore the prophage repertoire, 523 CLas DNA samples extracted from leaf petioles of CLas-infected citrus were collected from southern China and surveyed for Type 1 and Type 2 prophages by specific PCR. Eighteen samples were found lacking both prophages. One sample, JXGC, sequenced using Illumina HiSeq, generated >100 million short sequence reads (150 bp per read). Read mapping to known prophage sequences showed a sequence coverage of 46% to SC1 and 50% to SC2. BLAST search using SC1 and SC2 as queries identified three contigs from the JXGC de novo assembly that form a circular P-JXGC-3 (31,449 bp), designated as a new Type 3 prophage. Chromosomal integration of P-JXGC-3 was detected to occur within a helicase gene, resulting in a duplication of this gene. P-JXGC-3 had 36 open reading frames (ORFs), 10 of which were not found in Type 1 or Type 2 prophages, including four genes that encoded a restriction-modification (R-M) system (hsdR, hsdS, hsdM1, and hsdM2). Typed by prophage-specific PCR, the CLas strains in southern China contained all combinations of the three prophage types with the exception of a Type 2−Type 3 combination, suggesting active ongoing prophage−phage interactions. Based on gene annotation, P-JXGC-3 is not capable of reproduction via the lytic cycle. The R-M system was speculated to play a role against Type 1 prophage−phage invasion.

Abstract“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.