Citrus tristeza virus Promotes the Acquisition and Transmission of ‘Candidatus Liberibacter Asiaticus’ by Diaphorina citri

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.


Introduction
Citrus Huanglongbing (HLB) is a bacterial disease caused by the fastidious "Candidatus Liberibacter spp." [1]. It is one of the most devastating diseases in the worldwide citrus industry [2]. The citrus plants affected by HLB gradually lost their economic value due to fruit malformation, leaf yellowing, root rot, rapid tree decline, and finally, reduction of the fruit quantity and quality. Of the reported "Ca. L. spp.", "Ca. L. asiaticus" (CLas) is the most prevalent and predominant species [3]. It is a significant scientific challenge toward effective HLB management because CLas is a noncultured phloem-inhabiting bacterium and is readily graft-transmissible and easily transmitted by its insect vector.
Diaphorina citri Kuwayama (Hemiptera) (D. citri) causes damage to most plants within the family Rutaceae [4], especially citrus. Both adult and nymph D. citri insects feed on the tender shoots and mature leaves of the host plants, which causes gradual withering of the tender shoots and the curling and falling of leaves, thus inhibiting the growth of the host plants. In addition, D. citri excretes sticky white honeydew after feeding, which leads to sooty mold in host plants and affects plant photosynthesis [5]. In the HLB epidemic areas, the indirect harm caused by D. citri as the vector transmitting CLas is significantly heavier than the direct harm caused by feeding [2]. In the field, 9-16 overlapping generations of Plants were kept in the greenhouse ambient with seminatural light and temperature conditions until being transferred to the growth chambers when necessary. The plants were watered every other day, fertilized using compound fertilizer biweekly, and pruned monthly to generate sufficient new shoots. Conditions in the secure quarantine facilities (growth chambers) for plant and psyllid colony maintenance were set as follows: illumination for 14 h at a temperature of 27 ± 1 • C, dark for 10 h at 25 ± 1 • C and the relative humidity (RH) of 70-80%. All plants were initially screened by RT-qPCR using specific primers and ELISA (direct triple antibody sandwich protocol, TAS-ELISA) to be free of CTV, CLas, and other common pathogens, e.g., Citrus exocortis viroid, Citrus chlorotic dwarf-associated virus, Citrus tatter leaf virus, and Citrus yellow vein clearing virus [34]. Antibody (Catalog No: CAB 78900; Lot No: 00805) specific to the CTV capsid protein was purchased from Agdia Company (Elkhart, IN, USA).

Diaphorina citri Samples
Diaphorina citri collected from citrus orchards were all from the above-mentioned trees from three orchards (Table A1). A sufficient number of D. citri at different ages were collected from four branches of each citrus tree in four directions (Table A2). All D. citri samples from a tree were kept in a collection tube separately and temporarily maintained at low temperatures for subsequent RNA extraction and pathogen (CLas and CTV) detection individually in the lab. Breeding and feeding conditions of D. citri colony: Adult D. citri were collected from the orange jasmine plants on the campus of SCAU. After one-quarter of them were randomly selected and detected to be free of CLas and CTV, the other D. citri were raised on healthy orange jasmine plants in a temperature-controlled chamber. Twenty D. citri were tested (with 2 adults and 2 nymphs mixed as one sample) by RT-qPCR biweekly to ensure the colony was free of CTV or CLas. When D. citri had multiplied to more than 10 generations, the population of D. citri was further used in the following experiments.
For CTV and/or CTV acquisition and transmission experiments, the third-instar D. citri nymphs or 7-day-post-emergence adult insects were collected and enclosed in meshed plastic gauze bags of 250 mm × 180 mm on branches without touching the leaves. Populations of D. citri carrying CTV and/or CLas were generated by rearing the thirdinstar nymph D. citri insects on 'Shatangju' plants carrying CLas and/or CTV (CLas + CTV + , CLas + CTV − , and CTV + CLas − ) and healthy (CTV − CLas − ) "Shatangju" trees for days, and recorded as CLas + CTV + , CLas + CTV − , CTV + CLas − , and CTV − CLas − D. citri populations, respectively. Thereafter, the emerged D. citri adults were used in the acquisition and transmission experiments.

RNA Extraction and cDNA Synthesis
Total plant RNA was extracted from the midrib of either citrus leaves or orange jasmine leaves using plant tissue E.Z.N.A. ® HP plant RNA extraction kit (Omega Biotek., Norcross, GA, USA). Genomic DNA contamination was eliminated by digestion with RNase-free DNase I (TaKaRa Bio-tek., Shuzo, Kyoto, Japan). Total insect RNA was extracted from tissues of a single D. citri using TRlzol ® Reagent (Life Technologies, Guangzhou, China). The concentration and purity of total RNA were determined by absorbance using NanoDrop™ One (Thermo Scientific, Shanghai, China). RNA samples with OD260/OD230 of 2.0-2.4 and OD260/OD280 of 1.8−2.2 were selected and stored at −80 • C for further use. The total RNA samples were individually used for reverse transcription by Verso cDNA Synthesis (TransScript) kit (TransGen Biotech, Beijing, China).

Quantification of CTV and CLas by RT-qPCR
The primers used to detect CTV and CLas were named cquctv1/2, designed based on the p20 gene of CTV [35], and named CLas4G/HLBr based on the three copies of the 16srRNA genes [36], respectively. RT-qPCR was performed using Bestar ® DBI SYBR Green PCR Reagent Kits (DBI Bioscience, Shanghai, China) according to the manufacturer's instructions. The 20 µL reaction system consists of 10 µL of SYBR Green Mix, 8 µL of dd H 2 O, 0.5 µL of forward and reverse primers (10 pM), respectively, and 1 µL of DNA (cDNA). RT-qPCR reaction conditions were set as pre-denaturation at 95 • C for 2 min, 40 cycles of denaturation at 95 • C for 15 s, and extension at 60 • C for 20 s. cDNA samples with Ct values less than 33 were considered CTV-positive. Concomitantly, to relatively quantify the pathogen titers, standard curves were drawn using pEASY-T1 (TransGen Biotech, Beijing, China) recombinant cloning plasmids containing the target fragments and the relative primers, with 8 gradients (10(7)-fold). CTV and CLas concentrations were assessed by the copy number of CTV or CLas in per ng cDNA using a formula referencing to the study of Ruiz-Ruiz et al. [15].

Sample Collection and Experimental Designs under Laboratory Conditions
All plants and psyllid used in AAP and IAP were maintained in plant growth chambers under the conditions mentioned above.
CLas and CTV acquisition by D. citri in trees co-infected by the two pathogens: This experiment was carried out after confirming the eggs of D. citri from the field's CTVpositive (detected by RT-qPCR) trees were without detectable CTV. More than 100 healthy adult D. citri (with a male-to-female ratio of about 1:1) were collected into a mesh bag and transferred onto the young shoots of "Shatangju" plants carrying both CLas and CTV (CLas + CTV + ) for AAPs. After laying a large number of eggs on the buds, adult D. citri insects were removed. The eggs hatched gradually until the number of different stages of nymph and adult insects was more than 30 on the plants. Forty individuals of each 3rd, 4th, and 5th instars, 10 1-min-old postemergence (emerged on CTV − "Shatangju" plants) adults, and 24 6-hour-old postemergence adult insects (moved to the healthy 'Shatangju' plants for emergence) were collected respectively for pathogen detection.
CTV acquisition by D. citri with and without CLas: This experiment was done with six replications. According to the study of Inoue et al. [7] and Pelz-stelinski et al. [8], D. citri nymphs were used to acquire the CLas. More than 70 healthy third-instar nymphs were transferred onto the young shoots of CLas + CTV − Citrus tangerine Hort. plants for a 15-day AAP to acquire CLas. On the 7th and 14th days of bagging rearing, five D. citri were collected from each plant for CLas detection individually. At 15-day AAP, adult D. citri populations (about 7-day postemergence) with more than 50% individuals carrying CLas (named CLas + CTV − ) were obtained. Meanwhile, more than 70 healthy third-instar nymphs were placed on a healthy tree and bagged for 15 days' rearing as control (named CLas − CTV − population). Both the emerged CLas + CTV − population and the CLas − CTV − population were enclosed in meshed plastic gauze bags and transferred to 6 CTV + CLas − Citrus grandis Osbeck plants for another 10-day AAP to acquire CTV. Ten adult CLas + CTV − insects or 10 adult CLas − CTV − insects were assigned to each plant in a bag. On the 1st, 3rd, 7th, and 10th day after CTV AAP, 10 D. citri adults from each population were randomly collected from the plants, and RNA was extracted individually for the pathogen (CLas and CTV) detection. The CTV acquisition efficiency and CTV titers were compared between the two D. citri populations with and without CLas.
CLas acquisition by D. citri with and without CTV: Likewise, the CLas − CTV + psyllid population was generated in Citrus grandis Osbeck plants and subsequently used to compare the CLas acquisition efficiency by D. citri with and without CTV. Both the CLas − CTV + and the CLas − CTV − psyllid populations were transferred to CTV − CLas + Citrus tangerine Hort. Ex Tanaka plants for CLas acquisition. D. citri samples were collected for pathogen detection. CLas and CTV transmission by different D. citri: Four psyllid populations (more than 120 psyllids in each population), namely the CLas − CTV + , CLas + CTV − , CLas + CTV + , and the CLas − CTV − , were generated separately from the corresponding citrus plants and healthy orange jasmine seedlings, respectively. Three experimental replications were conducted. Specifically, more than 40 healthy third-instar nymphs were reared on each plant enclosed by meshed plastic gauze bags. On the 14th day of AAP, 10 D. citri were randomly collected from each plant for RNA extraction and pathogen (CLas and CTV) detection individually. The other 30 psyllids in each population were transferred to 3 healthy Citrus reticulata Blanco "Shatangju" plants that were in good growing conditions and with sufficient new shoots, with 10 insects on each plant, to transmit CTV and/or CLas or used as control. To avoid the influence of secreted honeydews on pathogen detection, all D. citri and eggs, together with the enclosing bags, were removed from the 12 "Shatangju" plants after 15-days-IAP. At this time, plants were recorded as 0 days post inoculation (0 dpi) and were transferred to the insect-proof screenhouse. Fully expanded new leaves of approximately the same age were sampled at 0 dpi, 15 dpi, 30 dpi, 45 dpi, 60 dpi, and 150 dpi for RNA extraction and pathogens detection. Three leaves from each of the three shoots, with one fed by the psyllids, from each plant were sampled. One leaf equals one replicate in the pathogen detection. The symptoms of "Shatangju" trees were photographed at the same time. The transmission effects were defined according to the RT-qPCR results and symptoms.

The Acquisition and Persistence Mode of CTV by D. citri
The complete midguts and salivary glands of 90 (3 replications) adult D. citri insects from the population with 66.67% CTV-positive rate were individually dissected by using Insect anatomy stereo microscope Gmbh 37,081 (Carl Zeiss Jena GmbH, Gottingen, Germany) and 0.05 mm dissection forceps. RT-qPCR was used to detect the CTV titers in these two parts. After confirming the enrichment of CTV in gut tissues, midgut samples from psyllid on CTV-positive trees were further used to investigate the CTV propagation status within three days' AAP. Specifically, more than 300 adult D. citri were collected from orange jasmine trees and enclosed in mesh bags for 10 h of starvation treatment. Then CTV acquisition was evaluated by confining three groups of 100 adult D. citri on CT31-infected 'Shatangju' shoots (Ct = 21.68 ± 1.93). Ten D. citri insects on 0 h AAP, 6 h AAP, 24 h AAP, 48 h AAP, and 72 h AAP were collected separately and subsequently dissected individually to obtain the midgut (n = 3 × 10 psyllids per AAP). CTV titers in the midgut tissues were evaluated by RT-qPCR. For the CTV persistence assay, groups of the CTV + D. citri adults (with more than 80% individuals infected) were transferred onto CTV − orange jasmine trees for feeding. The orange jasmine trees were replaced with new ones every 4 days. Samples were collected at 3 d AAP, 6 d AAP, 12 d AAP, and 24 d AAP, with n = 6 × 10 psyllids per AAP for CTV detection.

Midgut Dissection and Transmission Electron Microscopic Observation
The transmission electron microscopy (TEM) (Model: FEI/Talos L120C, Thermo Fisher Scientific, OR, USA) was used to further analyze the CTV-infected midgut at the ultrastructural level. Samples were fixed with 2.5% (v/v) glutaraldehyde in 0.1 M of potassium phosphate buffer at PH 7.4 for 4 h at room temperature (RT) and then temporarily stored at 4 • C. After fixation, samples were rinsed with 0.1 M phosphate buffer (PB, pH = 7.4) four times. Postfixtion was then done by transferring samples into 2% osmium tetroxide (w/v) for another 2 h at RT and then in 1% uranyl dioxyacetate solution at 4 • C overnight. The samples were further rinsed with ddH 2 O four times at intervals of 10 min, dehydrated in 30%, 50%, 70%, and 90% ethanol successively for 10 min each, in 100% ethanol and then 100% acetone for 15 min twice, respectively. Samples were infiltrated and embedded in acetone resin solution (acetone: resin = 1:1) for 2 d, followed by overnight incubation in acetone resin solution (acetone: resin = 1:3). Sample sections were embedded with pure resin and mounted on 200-mesh form var-coated copper grids, sectioned at 60 nm thickness, viewed and photographed using the TEM.

Data Analysis
CTV and CLas concentrations (copy numbers) in samples of D. citri were calculated according to the standard curve. CTV and/or CLas carried by D. citri at different developmental stages were compared and analyzed. The results were statistically analyzed and diagram plotted using Microsoft Excel 2019, IBM SPSS Statistics 26, and Origin 2018. Kruskal-Wallis test, independent t-test with Dunn's multiple comparisons, or the Student's t-test was used for significant difference analysis of pathogen copy numbers in different D. citri populations (p < 0.05). Data were subjected to statistical analysis by one-way analysis of variance (ANOVA) followed by Duncan's new multiple range test. Different letters in the figure indicate significant differences at p < 0.05 level. A2), were collected from the 31 CTV CLas trees (Figure 1b). Neither CTV nor CLas were detected in the eggs, with 5 eggs pooled into 11 samples. The 1st instar nymphs did not carry CLas, but 38.46% of them were CTV-positive. For the 2nd to the 5th instar nymphs and adults, 61.25% to 85.71% of individuals were CTV-positive, while the CLas infection rates were relatively lower (from 28.13% for the 2nd instars to 60% for the adults). Relative high CTV-positive rates were observed in the 3rd and 4th instars. By contrast, the CLasinfection rates showed an increment with the age of the psyllid. For the pathogen titers in the 711 psyllid samples, the average Ct values for CLas detection of CLas + samples of 2nd to 5th instar nymphs and adults were 29.36 ± 0.62, 29.46 ± 0.52, 28.56 ± 0.42, 27.84 ± 0.37, and 26.51 ± 0.43, respectively. The Ct values of CLas + adults were significantly lower (with CLas titers significantly higher) than those of the nymphs (p < 0.05) (Figure 2a). By contrast, no significant difference was in the CLas titers acquired by nymphs of different stages. Comparatively, the average Ct values of CTV + samples of 1st-5th instar nymphs and adults were 29.90 ± 1.20, 29.46 ± 0.45, 29.20 ± 0.47, 28.40 ± 0.31, 29.02 ± 0.32, and 28.46 ± 0.36, respectively. The Ct values of the CTV + 4th instar nymphs and adults were significantly lower than those of the 1st to 3rd instar nymphs and the 5th instar nymphs (p < 0.05). Likewise, there was no significant difference in the CTV concentration obtained by nymphs of different instars.

Incidence of CTV and CLas in D. citri from Citrus Orchards
In total, 161 D. citri samples were collected from CLas -CTV + trees in the field. The CTV positive rates of 1st to 5th instars nymphs and adults are shown in Figure 2a. The mean Ct values of the CTV + samples were 28.98 ± 0.00, 28.13 ± 1.45, 27.15 ± 1.39, 27.46 ± 1.11, 27.97 ± 0.56, and 28.60 ± 0.66, respectively. There was no significant difference in the In total, 711 psyllid samples, including 55 eggs, 576 nymphs, and 80 adults (Table A2), were collected from the 31 CTV + CLas + trees (Figure 1b). Neither CTV nor CLas were detected in the eggs, with 5 eggs pooled into 11 samples. The 1st instar nymphs did not carry CLas, but 38.46% of them were CTV-positive. For the 2nd to the 5th instar nymphs and adults, 61.25% to 85.71% of individuals were CTV-positive, while the CLas infection rates were relatively lower (from 28.13% for the 2nd instars to 60% for the adults). Relative high CTV-positive rates were observed in the 3rd and 4th instars. By contrast, the CLas-infection rates showed an increment with the age of the psyllid.
For the pathogen titers in the 711 psyllid samples, the average Ct values for CLas detection of CLas + samples of 2nd to 5th instar nymphs and adults were 29.36 ± 0.62, 29.46 ± 0.52, 28.56 ± 0.42, 27.84 ± 0.37, and 26.51 ± 0.43, respectively. The Ct values of CLas + adults were significantly lower (with CLas titers significantly higher) than those of the nymphs (p < 0.05) (Figure 2a). By contrast, no significant difference was in the CLas titers acquired by nymphs of different stages. Comparatively, the average Ct values of CTV + samples of 1st-5th instar nymphs and adults were 29.90 ± 1.20, 29.46 ± 0.45, 29.20 ± 0.47, 28.40 ± 0.31, 29.02 ± 0.32, and 28.46 ± 0.36, respectively. The Ct values of the CTV + 4th instar nymphs and adults were significantly lower than those of the 1st to 3rd instar nymphs and the 5th instar nymphs (p < 0.05). Likewise, there was no significant difference in the CTV concentration obtained by nymphs of different instars.
In total, 161 D. citri samples were collected from CLas − CTV + trees in the field. The CTV positive rates of 1st to 5th instars nymphs and adults are shown in Figure 2a. The mean Ct values of the CTV + samples were 28.98 ± 0.00, 28.13 ± 1.45, 27.15 ± 1.39, 27.46 ± 1.11, 27.97 ± 0.56, and 28.60 ± 0.66, respectively. There was no significant difference in the average Ct value of CTV carried by the D. citri at different developmental stages. In general, the content of CTV contained by 1st to 4th instar D. citri increased with its developmental stages. Comparatively, the titers of CTV acquired by the nymphs from the 2nd to 5th instars and adults from the CLas -CTV + citrus trees were higher than those of the same developmental stages fed on the HLB-affected (CLas + CTV + ) trees. Herein, the titers of CTV acquired by the 3rd instar nymphs feeding on CLas-negative trees were significantly higher than those of the 3rd instar nymphs feeding on CLas-infected trees (p < 0.05) (Figure 2b). Hence, we concluded that CLas in plants might inhibit CTV acquisition by D. citri.

CLas and CTV Acquisition on Citrus Trees Infected by Both Pathogens
After rearing from eggs to a population with all five stages' nymph and adults on the CLas and CTV positive trees, pathogen acquisition results of 120 nymphs from 3rd instar to 5th instar (with 40 in each group) and 61 adults are shown in Figure 3a,b. The CLas positive rate of the 3rd to 5th instar nymphs was from 45.00% to 60%, respectively, with average Ct values of CLas + samples of 23.52 ± 0.96, 19.93 ± 0.93, and 24.09 ± 1.06, respectively. The CTV positive rate of the 3rd to 5th instar nymphs were all higher than 80%, while the average Ct values of CTV + samples were 26.85 ± 0.47, 26.91 ± 0.53, and 26.91 ± 0.94, respectively. There were 62.96% (Ct = 21.65 ± 1.47) and 70.73% (Ct = 26.43 ± 0.41) newly emerged adult individuals (recorded as 1 min post-emergence) found to be positive in CLas detection and CTV detection, respectively. After 6 h on CLas -CTVtrees, 54.17% (Ct = 21.53 ± 0.78) and 100.00% (Ct = 26.99 ± 0.24) of them were positive for CLas and CTV, respectively. The D. citri kept carrying CTV and CLas after emergence on healthy 'Shatangju' plants. There was no significant difference between the average Ct values of CLas and CTV carried by all stages' psyllids. No CTV particles were detectable in the exuviates, whereas they could be excreted, as the honeydew samples were detected as CTV-positive (Figure 3d). Comparatively, the titers of CTV acquired by the nymphs from the 2nd to 5th instars and adults from the CLas − CTV + citrus trees were higher than those of the same developmental stages fed on the HLB-affected (CLas + CTV + ) trees. Herein, the titers of CTV acquired by the 3rd instar nymphs feeding on CLas-negative trees were significantly higher than those of the 3rd instar nymphs feeding on CLas-infected trees (p < 0.05) (Figure 2b). Hence, we concluded that CLas in plants might inhibit CTV acquisition by D. citri.

CLas and CTV Acquisition on Citrus Trees Infected by Both Pathogens
After rearing from eggs to a population with all five stages' nymph and adults on the CLas and CTV positive trees, pathogen acquisition results of 120 nymphs from 3rd instar to 5th instar (with 40 in each group) and 61 adults are shown in Figure 3a,b. The CLas positive rate of the 3rd to 5th instar nymphs was from 45.00% to 60%, respectively, with average Ct values of CLas + samples of 23.52 ± 0.96, 19.93 ± 0.93, and 24.09 ± 1.06, respectively. The CTV positive rate of the 3rd to 5th instar nymphs were all higher than 80%, while the average Ct values of CTV + samples were 26.85 ± 0.47, 26.91 ± 0.53, and 26.91 ± 0.94, respectively. There were 62.96% (Ct = 21.65 ± 1.47) and 70.73% (Ct = 26.43 ± 0.41) newly emerged adult individuals (recorded as 1 min post-emergence) found to be positive in CLas detection and CTV detection, respectively. After 6 h on CLas − CTV − trees, 54.17% (Ct = 21.53 ± 0.78) and 100.00% (Ct = 26.99 ± 0.24) of them were positive for CLas and CTV, respectively. The D. citri kept carrying CTV and CLas after emergence on healthy 'Shatangju' plants. There was no significant difference between the average Ct values of CLas and CTV carried by all stages' psyllids. No CTV particles were detectable in the exuviates, whereas they could be excreted, as the honeydew samples were detected as CTV-positive (Figure 3d).  Collectively, the CTV titers increased over time in the 10 d acquisition period for CLas -D. citri but not for the CLas + D. citri. CTV concentration acquired by CLas -D. citri was significantly higher at 10 d AAP compared with those of 1 d and 3 d AAPs according to the Ct values (Figure 4c). However, the abundance of CTV acquired by the CLas + D. citri was higher than that by the CLas -D. citri, especially when adults were given access to the CTV + plants for 3 days (p < 0.05) (Figure 4d). This result suggests that CLas facilitated the CTV acquisition by D. citri in the early stage (Figure 4d). However, the CTV acquisition efficiency was inhibited thereafter.

CLas Acquisition by D. citri was Promoted by CTV
After 15-day AAP of CTV, the 3rd instar D. citri nymphs had emerged to adults for about one week. This psyllid population (CTV + CLas − ) was tested 100% (n = 20) positive for the presence of CTV (Ct = 25.54 ± 0.28). The population and the CTV -CLas − population of the same age were placed on CLas + CTV − Tanaka seedlings for CLas acquisition. Noteworthily, although feeding on the CTV-free trees, the CTV titers in the first population remained at a stable level during the 10-day CLas acquisition.
CLas-positive rates increased for both D. citri populations in the 10 d AAP period. Wherein the rates of CLas + psyllids in the CTV + CLas − D. citri population at all four testing stages (From 46.67% to 83.33%) were all higher than those in the CTV -CLas -D. citri population (from 13.33% to 66.67%) (Figure 5a). Additionally, a significantly higher level of Collectively, the CTV titers increased over time in the 10 d acquisition period for CLas − D. citri but not for the CLas + D. citri. CTV concentration acquired by CLas − D. citri was significantly higher at 10 d AAP compared with those of 1 d and 3 d AAPs according to the Ct values (Figure 4c). However, the abundance of CTV acquired by the CLas + D. citri was higher than that by the CLas − D. citri, especially when adults were given access to the CTV + plants for 3 days (p < 0.05) (Figure 4d). This result suggests that CLas facilitated the CTV acquisition by D. citri in the early stage (Figure 4d). However, the CTV acquisition efficiency was inhibited thereafter.

CLas Acquisition by D. citri was Promoted by CTV
After 15-day AAP of CTV, the 3rd instar D. citri nymphs had emerged to adults for about one week. This psyllid population (CTV + CLas − ) was tested 100% (n = 20) positive for the presence of CTV (Ct = 25.54 ± 0.28). The population and the CTV − CLas − population of the same age were placed on CLas + CTV − Tanaka seedlings for CLas acquisition. Noteworthily, although feeding on the CTV-free trees, the CTV titers in the first population remained at a stable level during the 10-day CLas acquisition.
CLas-positive rates increased for both D. citri populations in the 10 d AAP period. Wherein the rates of CLas + psyllids in the CTV + CLas − D. citri population at all four testing stages (From 46.67% to 83.33%) were all higher than those in the CTV − CLas − D. citri population (from 13.33% to 66.67%) (Figure 5a). Additionally, a significantly higher level of CLas was acquired at the early stage (3 d AAP) than that of the later stage (7 d AAP and 10 d AAP) by the CTV + psyllid (Figure 5b,c). Although relatively lower Ct values for CLas detection were detected for the CTV − psyllid at 10 d following CLas AAP, there was no significant difference among the testing stages in CLas titers within them. When comparing the effect of CLas acquisition between the two psyllid groups, CTV + D. citri adults acquired much more CLas at 1 d AAP and 3 d AAP. However, the differences were not significant after 7 d AAP. Consequently, the CTV + psyllid was more efficient in CLas acquisition according to CLas-positive proportion and CLas titers.
Viruses 2023, 15, x FOR PEER REVIEW 11 of 19 CLas was acquired at the early stage (3 d AAP) than that of the later stage (7 d AAP and 10 d AAP) by the CTV + psyllid (Figure 5b,c). Although relatively lower Ct values for CLas detection were detected for the CTV − psyllid at 10 d following CLas AAP, there was no significant difference among the testing stages in CLas titers within them. When comparing the effect of CLas acquisition between the two psyllid groups, CTV + D. citri adults acquired much more CLas at 1 d AAP and 3 d AAP. However, the differences were not significant after 7 d AAP. Consequently, the CTV + psyllid was more efficient in CLas acquisition according to CLas-positive proportion and CLas titers.

CTV Contributed to the Transmission of CLas by D. citri
Four groups of the same aged D. citri, namely CLas + CTV − , CLas + CTV + , CLas -CTV + , and CLas -CTV − , were used for the CTV and CLas transmission. For the first and second groups, the CLas-positive proportions were 50%, while for the second and third groups, the CTV-positive rates were 90%. All selected "Shatangju" trees used for transmission were confirmed to be negative in CTV and CLas after detecting by RT-qPCR.
Firstly, we compared the influence of CTV in psyllid on CLas transmission. All plants tested positive for CLas at 30 and 60 days after IAP by the CLas + CTV + adult D. citri. The average Ct value for the CLas detection was 23.70 ± 3.88 at 30 d after IAP. Moreover, the mean Ct values of plants at 45 d and 60 d IAPs were significantly lower than those feeding-inoculated by the CLas + CTV − D. citri adults. These results suggest that D. citri carrying CTV can transmit CLas to "Shatangju" plants more quickly, and CLas + CTV + D. citri transmitted more CLas inoculum (Figure 6a).

CTV Contributed to the Transmission of CLas by D. citri
Four groups of the same aged D. citri, namely CLas + CTV − , CLas + CTV + , CLas − CTV + , and CLas − CTV − , were used for the CTV and CLas transmission. For the first and second groups, the CLas-positive proportions were 50%, while for the second and third groups, the CTV-positive rates were 90%. All selected "Shatangju" trees used for transmission were confirmed to be negative in CTV and CLas after detecting by RT-qPCR.
Firstly, we compared the influence of CTV in psyllid on CLas transmission. All plants tested positive for CLas at 30 and 60 days after IAP by the CLas + CTV + adult D. citri. The average Ct value for the CLas detection was 23.70 ± 3.88 at 30 d after IAP. Moreover, the mean Ct values of plants at 45 d and 60 d IAPs were significantly lower than those feeding-inoculated by the CLas + CTV − D. citri adults. These results suggest that D. citri carrying CTV can transmit CLas to "Shatangju" plants more quickly, and CLas + CTV + D. citri transmitted more CLas inoculum (Figure 6a).
Secondly, the influence of CLas in psyllid on CTV transmission was analyzed. As shown in Figure 6b Secondly, the influence of CLas in psyllid on CTV transmission was analyzed. As shown in Figure 6b, the two groups of 1-week-post-emergence D. citri (CLas + CTV + and CLas -CTV + ) transmitted the virus to healthy 'Shatangju' plants at a rate of 100% after 30 days following the IAP. The average Ct values of CTV detection by RT-qPCR were 30.40 ± 0.84 and 32.45 ± 0.24, respectively, for the trees fed by the two psyllid populations at 30 d after IAP, showing no significant difference. These results indicated that the CLas carried by psyllid had no significant effect on the efficiency of CTV transmission to Citrus reticulata Blanco 'Shatangju'.
On all 'Shatangju' plants fed by D. citri, the leaves curled and showed less luster after IAP (recorded as 0 dpi) ( Figure A1 a1-1 to d3-1). The leaves were slightly withered at 0-30 dpi, with some falling off. However, the situation was improved after the booting of new shoots ( Figure A1 a1-1 to d3-3). After inoculation by the CLas + D. citri, the leaves of the new shoots of "Shatangju" showed slight chlorosis at 15 dpi, although no CLas was detected until 60 dpi. No blotchy mottled leaves were observed even at 60 dpi ( Figure A1 a1-2 to a3-5). With both pathogens at a detectable level from 30 to 60 dpi by CLas + CTV + D. citri, the young leaves of new shoots showed no typical symptoms of HLB or apparent variegated yellowing. However, the number of new shoots decreased ( Figure A1 c1-3 to c3-5). Although no obvious symptoms of recession or dwarfing were observed, the mature leaves of CTV-positive "Shatangju" trees were slightly yellow in veins, cured, and fell easily ( Figure A1 c1-3 to d3-5).

Ultrastructure of CTV in Midgut
After assessing the CTV distribution in salivary glands and midguts using CTV-positive D. citri adults, we confirmed that CTV titers in the midguts of adult D. citri were significantly higher than those in salivary glands (p < 0.05). This indicates CTV can penetrate the inner body of D. citri adults. Therefore, the midguts of adult D. citri were selected as the experimental samples for subsequent TEM observation of CTV ( Figure A2). In total, 30 CTV-positive adult D. citri and 15 healthy adult D. citri were collected, and their midguts were dissected for TEM assay. A large number of long linear CTV particles were observed between microvilli and basal layer in TEM slices of midgut tissues of CTV-carrying On all 'Shatangju' plants fed by D. citri, the leaves curled and showed less luster after IAP (recorded as 0 dpi) ( Figure A1 a1-1 to d3-1). The leaves were slightly withered at 0-30 dpi, with some falling off. However, the situation was improved after the booting of new shoots ( Figure A1 a1-1 to d3-3). After inoculation by the CLas + D. citri, the leaves of the new shoots of "Shatangju" showed slight chlorosis at 15 dpi, although no CLas was detected until 60 dpi. No blotchy mottled leaves were observed even at 60 dpi ( Figure A1 a1-2 to a3-5). With both pathogens at a detectable level from 30 to 60 dpi by CLas + CTV + D. citri, the young leaves of new shoots showed no typical symptoms of HLB or apparent variegated yellowing. However, the number of new shoots decreased ( Figure A1 c1-3 to c3-5). Although no obvious symptoms of recession or dwarfing were observed, the mature leaves of CTV-positive "Shatangju" trees were slightly yellow in veins, cured, and fell easily ( Figure A1 c1-3 to d3-5).

Ultrastructure of CTV in Midgut
After assessing the CTV distribution in salivary glands and midguts using CTVpositive D. citri adults, we confirmed that CTV titers in the midguts of adult D. citri were significantly higher than those in salivary glands (p < 0.05). This indicates CTV can penetrate the inner body of D. citri adults. Therefore, the midguts of adult D. citri were selected as the experimental samples for subsequent TEM observation of CTV ( Figure A2). In total, 30 CTV-positive adult D. citri and 15 healthy adult D. citri were collected, and their midguts were dissected for TEM assay. A large number of long linear CTV particles were observed between microvilli and basal layer in TEM slices of midgut tissues of CTVcarrying adults (Figure 7a-c). Comparatively, no apparent CTV morphology was observed in slices of midgut tissues of healthy adults (Figure 7d). adults (Figure 7a-c). Comparatively, no apparent CTV morphology was observed in slices of midgut tissues of healthy adults (Figure 7d).

CTV Replicates in Midgut of D. citri
CTV could be detected at the midgut of 10% of adult D. citri individuals as early as 3 h AAP. The proportion of midgut samples detected as CTV-positive increased with confinement time, with 20%, 40%, 50%, and 80% at 6 h AAP, 24

Discussion
In this study, it was speculated that CTV could penetrate (Section 3.3) and replicate (Section 3.4) in the body of D. citri, which was different from the non-circulating semipersis-tent manner of aphids [16]. Further research is required to determine when or whether CTV moves from the gut to the salivary glands to become circulative. As CTV particles were enriched in midguts of D. citri, and there was no significant decrease of CTV titers during the 24-day persistence assay, the CTV transmission manner by D. citri was suggested to be the same as that for CLas transmission [37,38]. Although the conclusion that CTV would be transmitted by D. citri is still controversial, this does not hinder us from exploring the details of CTV-vector-CLas interaction.
Co-infection of CTV and CLas in the field trees was found to be common in this study and several previous studies [39][40][41][42]. Both CTV and CLas were vector-transmitted obligate pathogens of phloem, although they belong to bacteria and viruses, respectively. Most natural vector insects, such as Trialeurodes vaporariorum, Delphacodes kuscheli, and Bemisia tabaci, can carry, acquire and even transmit more than one virus, which causes co-infection of host plants [43][44][45][46][47]. D. citri is a pest feeding on phloem sap by piercing mouthparts. Hence, finding the co-infection status of the two pathogens in D. citri from the field is not surprising. Although the results presented herein showed higher CTV titers could be detected in the plant host than those in the vectors, CTV was found accumulated from nymphs to adults when feeding on CTV + citrus plants. The results strongly suggest differential adaptation of these pathogens to the D. citri or differential adaptation of CTV to different hosts. In this study, the CTV-carrying rates of D. citri were higher than CLascarrying rates in all developmental stages on the citrus plant co-infected by two pathogens. Combined with the previous studies [8,33,48,49], this difference indicated that D. citri was more likely to acquire and carry CTV from host plants compared to acquiring CLas, although with lower titers.
Vector-born disease epidemiology is impacted by interactions between pathogens and the pathogen accumulation efficiency in host plants [50]. In the field trial, we also found that the CLas in plants would inhibit CTV acquisition by D. citri. After being infected by insect-borne pathogens, pigments, nutrients, hormones, volatile organic compounds, and resistance levels of host plants change to varying degrees, thereby affecting the behavior and adaptability of vector insects and thus affecting the spread and prevalence of other pathogens [51][52][53]. On the one hand, the content of volatile organic compounds produced by citrus due to the infection of CLas may reduce the attraction of citrus plants to D. citri [54,55]. On the other hand, when citrus was infected with CLas, the phloem autoimmune response enhanced [56], thus influencing the CTV acquisition by D. citri. Whether changes in phloem cells or volatiles of citrus co-infected by CLas and CTV influence the attractiveness of host plants to the vector remains to be further explored scientifically.
In the vectors capable of acquiring or transmitting more than one pathogen, the acquisition or transmission of one pathogen may be influenced by the other pathogen. We speculated that CTV infection promoted the feeding of D. citri on HLB trees and alerted the acquisition and transmission of CLas (Sections 3.2.2 and 3.2.3). The relationship between the vector and the pathogen can significantly impact transmission efficiency. For example, the Tomato infectious chlorosis virus is much more efficiently transmitted by Trialeurodes vaporariorum than the Tomato chlorosis virus [56]. Similarly, Glaser et al. [57] proposed that bacterial infection of vector insects can enhance their resistance to viruses. Zouache et al. [58] found that secondary symbiotic bacteria levels in the body of insect vectors can be affected after being infected by a virus. Yan [59] proposed that pathogens could directly or indirectly change the feeding behavior of vector insects to promote their effective transmission. This study preliminarily verified the interaction between CLas and CTV in D. citri. Studying the relationship between plant viruses and bacteria in vector insects can provide an important reference for revealing the internal mechanism of pathogen transmission by vector insects that can carry two or more pathogens.
It should be noted that for the field and laboratory CLas/CTV carrying/acquisition assays, different stages' D. citri samples were collected simultaneously. To be rigorous, D. citri populations carrying different pathogens in different stages should be used for the pathogen acquisition, persistence, and transmission study to better elucidate this interaction among D. citri-CTV-CLas. Besides, the effects of CTV infection on vector-plant interaction, including development, host selection, and feeding behavior, are being studied in our lab. It is clear that the CTV-infected D. citri adults preferred to feed on citrus leaves infected by CLas. A direct-current electrical penetration graph (DC-EPG) study has suggested that penetration in the CTV + phloem was much easier than in the CTV − phloem. Further studies should determine whether CTV infection alters the attractiveness of the host plant to the vector and how the CTV affects the behavioral response of its vector from the molecular level.

Conclusions
In this study, the characteristics of CLas and CTV acquisition and transmission by D. citri were systematically analyzed for the first time based on the previous confirmation that D. citri would acquire and persist CTV. The results presented herein further suggested that the proliferation of CLas and CTV in D. citri was similar. The titers of CLas carried by adult D. citri were higher than those of nymphs, but the titers of CTV carried by the adult D. citri were not significantly different from those of the nymphs. CTV carried by D. citri is beneficial to the acquisition and transmission of CLas of this vector. This study combines CTV with the research hotspots of the 'citrus-D. citri-CLas' interactions 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 Citrus tristeza disease.    1  45  30  20  8  24  78  37  2  0  30  108  104  135  88  37  3  10  10  28  32  12  10  26  Total  55  70  156  144  171  176  100 Orchard numbers are the same as those in Table 1. Egg, 1st, 2nd, 3rd, 4th, 5th, and Adult indicate the developmental stages of Egg, 1st instar nymph, 2nd instar nymph, 3rd instar nymph, 4th instar nymph, 5th instar nymph, and adult, respectively. Orchard numbers are the same as those in Table 1. Table A2. Information on D. citri samples collected from three citrus orchards. 1st  2nd  3rd  4th  5th  Adult  1  45  30  20  8  24  78  37  2  0  30  108  104  135  88  37  3  10  10  28  32  12  10  26  Total  55  70  156  144  171  176  100 Orchard numbers are the same as those in Table 1. Egg, 1st, 2nd, 3rd, 4th, 5 th , and Adult indicate the developmental stages of Egg, 1st instar nymph, 2nd instar nymph, 3rd instar nymph, 4th instar nymph, 5th instar nymph, and adult, respectively.