Bacteriology

Spatiotemporal Dynamics of ‘Candidatus Liberibacter asiaticus’ Colonization Inside Citrus Plant and Huanglongbing Disease Development

Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL 33850

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Fernanda Nogales da Costa Vasconcelos

https://orcid.org/0000-0002-1975-8597

Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL 33850

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, and

Nian Wang

^†Corresponding author: N. Wang;

E-mail Address: [email protected]

https://orcid.org/0000-0001-7743-0728

Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL 33850

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Affiliations

Authors and Affiliations

Sheo Shankar Pandey
Fernanda Nogales da Costa Vasconcelos
Nian Wang^†

Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL 33850

Published Online:19 Jul 2021https://doi.org/10.1094/PHYTO-09-20-0407-R

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Abstract

‘Candidatus Liberibacter asiaticus’ (CLas), the causal agent of citrus huanglongbing (HLB), colonizes inside the phloem and is naturally transmitted by the Asian citrus psyllid (ACP). Here, we investigated spatiotemporal CLas colonization in different tissues after ACP transmission. Of the nine plants successfully infected via ACP transmission, CLas was detected in the roots of all trees at 75 days postremoval of ACPs (DPR) but in the mature leaf of only one tree; this finding is consistent with the model that CLas moves passively from source to sink tissues. At 75 and 365 DPR, CLas was detected in 11.1 and 43.1% of mature leaves not fed on by ACPs during transmission, respectively, unveiling active movement to the source tissue. The difference in colonization timing of sink and source tissues indicates that CLas is capable of both passive and active movement, with passive movement being dominant. At 225 DPR, leaves fed on by ACPs during the young stage showed the highest ratio of HLB symptomatic leaves and the highest CLas titer, followed by leaves that emerged after ACP removal and mature leaves not fed on by ACPs. Importantly, our data showed that ACPs were unable to transmit CLas via feeding on mature leaves. It is estimated that it takes 3 years at most for CLas to infect the whole tree. Overall, spatiotemporal detection of CLas in different tissues after ACP transmission helps visualize the infection process of CLas in planta and subsequent HLB symptom development and provides evidence showing that young leaves should be the focus of HLB management.

Huanglongbing (HLB), also known as citrus greening, is currently the most devastating citrus disease. HLB has been reported in 51 of the 140 citrus producing countries in the world (Wang 2019). The predominant HLB pathogen is ‘Candidatus Liberibacter asiaticus’ (CLas), which colonizes in the phloem and is vectored by the Asian citrus psyllid (ACP; Diaphorina citri Kuwayama; Hemiptera: Liviidae) (Bové 2006; Graça et al. 2015; Grafton-Cardwell et al. 2013; Wang et al. 2017b). Typical HLB symptoms include yellow shoots, blotchy mottled leaves, corky veins, stunted growth and root decline, and twig diebacks (Bové 2006; Gottwald 2010; Wang et al. 2017a). CLas remains uncultured in artificial media (Davis et al. 2008; Merfa et al. 2019; Parker et al. 2014; Sechler et al. 2009), which significantly hinders genetic manipulation of the pathogen and understanding of CLas biology and pathogenesis (Prasad et al. 2016). HLB management is one of the most challenging tasks for citrus growers in HLB-endemic regions despite numerous advances in controlling ACP (Boina and Bloomquist 2015; Boina et al. 2010; Stansly et al. 2014), targeting CLas (Akula et al. 2011; Hu and Wang, 2016; Hussain et al. 2019; Zhang et al. 2014), treating HLB-diseased trees (Canales et al. 2016; Gottwald et al. 2012; Li et al. 2016, 2017, 2019; Wang et al. 2017a), improving CLas detection (Fujikawa and Iwanami 2012; Gottwald et al. 2020; Kim and Wang 2009), and developing tolerant or resistant citrus varieties using traditional and novel approaches (Deng et al. 2019; Jia and Wang 2014; Jia et al. 2019; Rawat et al. 2015).

Immature leaves are shown to be a key battleground in the CLas–citrus–ACP interaction that characterizes the HLB pathosystem (Sétamou and Alabi 2018). ACPs preferentially feed and exclusively reproduce on young shoots (Bové 2006; Pluke et al. 2008; Sétamou et al. 2016b; Tomaseto et al. 2016; Wenninger and Hall 2007; Yasuda et al. 2005), and citrus trees are usually infected by CLas when new flushes are present (Hall et al. 2016). Young shoots are also the most important tissue from which ACPs acquire CLas (Pelz-Stelinski et al. 2010; Sétamou et al. 2016a). However, how CLas establishes inside the plant after ACP transmission into the young flush remains elusive.

HLB-positive plants display highly variable and uneven CLas distribution in different tissues, including leaves, roots, fruit, flowers, seed coat, and stems (Ding et al. 2015; Li et al. 2018; Tatineni et al. 2008). Citrus plants remain asymptomatic for several months to years after CLas transmission by grafting of CLas-positive budwood or ACP feeding (Hilf and Lewis 2016; Lee et al. 2015; Lopes and Frare 2008; Pandey and Wang 2019). CLas remains in the young flush after ACP transmission until it turns into the source for photoassimilate export, which helps devise a strategy for early detection of CLas before appearance of HLB symptoms (Andrade et al. 2020; Pandey and Wang 2019). Grafting of CLas-infected budwood to Hamlin/Swingle shows that CLas quickly colonizes the roots after grafting and causes root decline before the appearance of any visible leaf symptoms (Graham et al. 2013; Johnson et al. 2014). The presence of Ca. Liberibacter in the roots and its effect on tree health explain the ineffectiveness of pruning to control HLB (Lopes et al. 2007). Several microscopy analyses suggest a solitary free-floating lifestyle of CLas cells in sieve elements of host plants without forming any biofilm (Hilf 2011; Kim et al. 2009; Koh et al. 2012), supporting a passive movement model with the phloem sap (Andrade et al. 2020). Achor et al. (2020) reported that CLas passes through phloem pores in an elongated form. CLas was shown to attach to the plasma membrane at sieve plates through unknown extracellular filaments that assist the orientation (Achor et al. 2020). Apparently, CLas moves at a slower speed than the flow of phloem sap inside the citrus host (Etxeberria et al. 2016; Wang et al. 2017a).

In this study, we investigated the spatiotemporal colonization of CLas inside the citrus plant after transmission by ACPs over a period of 1 year. This study provides a dynamic depiction of CLas establishment in the leaves fed on and not fed on by ACPs, in leaves that emerged after ACP feeding and removal, and in the roots. One important finding of this study is the dynamic quantification of CLas in mature leaves not fed on by ACPs, suggesting an active movement of CLas to the source tissue in citrus. This study unveils vital information regarding how CLas moves and establishes inside the plant after ACP transmission by feeding on young leaves. The findings are discussed in light of HLB symptom development and management.

MATERIALS AND METHODS

Plant materials.

The 2-year-old trees used were Valencia sweet orange on Cleopatra rootstock. These plants were kept in a greenhouse under controlled conditions (28 ± 2°C with 50 ± 5% relative humidity and natural light). CLas-infected ACPs were maintained on HLB-positive citrus trees in a greenhouse at 24 ± 2°C with 50 ± 5% relative humidity and controlled light (8 h of darkness and 16 h of light).

ACP feeding experiment.

For ACP transmission of CLas to young leaves of healthy 2-year-old Valencia sweet orange on Cleopatra mandarin rootstock trees in the greenhouse with controlled conditions, a few branches of each tree were trimmed to induce growth of new flushes. The new shoot with young leaves (Supplementary Fig. S1A) was caged with 30 ACPs maintained on HLB-positive citrus trees in a sleeve cage. The trees were kept in ACP-proof screen cages to avoid exposure of the mature leaves to ACPs. The mature leaves were marked.

**Fig. 1.** Huanglongbing symptom development and ‘*Candidatus* Liberibacter asiaticus’ (CLas) titer in leaves after Asian citrus psyllid (ACP) transmission of CLas to young leaves. A, Ratio of symptomatic leaves among different groups of leaves. B, CLas titers in asymptomatic and symptomatic leaves after ACP transmission. Data are presented as means ± SE (n = 7 in A and n = 3 in B). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. P values were calculated using the paired Student t test. FL = leaves fed on by ACPs, UFML = leaves not fed on by ACPs, YFL₃₀ = 30 days and older young leaves emerged after ACPs removal, YFL_t = all young leaves emerged after ACPs removal, FL-Asym = asymptomatic leaves derived from young leaves fed on by ACPs, FL-Sym = symptomatic leaves derived from young leaves fed on by ACPs, UFML-Asym = asymptomatic mature leaves not fed on by ACPs, UFML-Sym = symptomatic mature leaves not fed on by ACPs, YFL-Asym = asymptomatic leaves that emerged after ACP removal, and YFL-Sym = symptomatic leaves that emerged after ACP removal.

For the mature leaf feeding experiment, fully mature leaves with hardened textures (Supplementary Fig. S1B) were caged with 30 ACPs maintained on HLB-positive citrus trees in a sleeve cage. The ACP feeding experiments were performed in the greenhouse with controlled conditions as described above.

ACPs were allowed to feed for 1, 2, 3, and 4 weeks in the greenhouse with controlled conditions as aforementioned. The feeding experiments were staggered so that ACP removal was conducted for all trees at the same time. After removal of ACPs, plants were cleaned thoroughly to remove any immature psyllids (eggs and nymphs) and moved to another greenhouse under natural light as mentioned above. The experiments were conducted in two separate batches. Trees 1 to 6 and 13 to 15 were used in the first experiment, whereas trees 7 to 12 were used in batch 2. The day ACPs were removed was considered 0 days postremoval of ACPs (DPR). The plants were kept for >1 year for tests at 0, 75, 150, 225, and 365 DPR.

CLas genomic DNA extraction.

Genomic DNA of CLas from ACPs was extracted as described previously (Pandey and Wang 2019). Briefly, four to five ACPs were pooled together after the feeding experiment to extract the genomic DNA. ACP samples were homogenized into powder using a TissueLyser II system (Qiagen, Germantown, MD) and transferred to 200 µl of extraction buffer (50 mM of Tris⋅Cl, pH 8.0, 10 mM of EDTA, and 100 µg/ml of RNase A). The solution was incubated at 50°C for 1 h after the addition of 0.25 mg/ml of lysozyme and 80 μl of 10% SDS. Then samples were treated with 100 µM of proteinase K overnight at 37°C. Samples were incubated at 65°C for 30 min with 400 μl of preheated cetrimonium bromide (CTAB)/NaCl (10% CTAB, 0.7 M of NaCl). Afterward, samples were treated with 1 ml of chloroform and isoamyl alcohol (24:1) and 1 ml of phenol, chloroform, and isoamyl alcohol (25:24:1). DNA from the aqueous phase was precipitated using 170 μl of sodium acetate (3 M, pH 7) and 700 μl of isopropanol at –20°C for 1 h. The DNA pellet was washed with 200 μl of 70% ethanol and dissolved in water. Genomic DNA from plants was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The midrib tissues from two to three leaves were collected and homogenized into powder using a TissueLyser II system (Qiagen). Then these powders were used to extract the genomic DNA. Genomic DNA from root samples was extracted using a DNeasy PowerSoil Kit (Qiagen) following the manufacturer’s instructions.

CLas detection using quantitative real-time PCR and conventional PCR.

CLas detection was conducted using quantitative real-time PCR (qPCR) as described previously (Wang et al. 2006). Quantitect Probe PCR Master Mix (Qiagen) was used to perform qPCR. Molecular probe CQULAP10 and primers CQULA04F and CQULA04R were used as described by Wang et al. (2006). The molecular probe was labeled with 6-carboxy-fluorescein amidite reporter fluorescent dye at the 5′ end and 6-carboxy-N,N,N′,N′-tetramethylrhodamine quencher dye at the 3′ end. The qPCR assay was performed using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). qPCR assays were used for detection of CLas, with cycle threshold (Ct) values ≥35 considered CLas negative and ≤32 considered CLas positive; values between ≤32 and ≥35 were considered questionable and confirmed with conventional PCR. Conventional PCR was performed using primers LAA2 and LAJ5 (Wang et al. 2006). The CLas titer was estimated using the qPCR data as described previously (Trivedi et al. 2009; Vasconcelos et al. 2021).

RESULTS

Spatiotemporal analysis of CLas colonization after ACP feeding on the young flush.

Numerous previous studies investigated CLas colonization in planta after grafting (Ding et al. 2015; Johnson et al. 2014; Tatineni et al. 2008). However, CLas is predominantly transmitted by ACPs in the environment. Here, we investigated the spatiotemporal colonization of CLas after ACP transmission. Healthy 2-year-old Valencia/Cleopatra trees were trimmed to induce the growth of young flushes. One young flush from each tree was caged with CLas-infected ACPs that were allowed to feed for different time intervals. The mature leaves were not exposed to ACPs and were marked to differentiate them from the young leaves fed on by ACPs. After removal of ACPs, the trees were cleaned to remove psyllid nymphs and eggs and moved to an insect-free greenhouse.

At 0 DPR, CLas was not detected in young-stage leaves fed on by ACPs based on qPCR assays (Table 1). ACPs collected after feeding showed high CLas titers, with a mean Ct value ranging from 19.95 to 22.43 (Table 1). At 75 DPR, CLas was detected in young-stage leaves fed on by CLas-positive ACPs in 9 of 15 trees, with mean Ct values ranging from 22.81 to 26.49. CLas was detected in the roots of seven of the nine trees, with Ct values ranging from 22.01 to 28.52. Of note, CLas was also detected in the mature leaves of one tree, which were not fed on by ACPs during transmission, with a Ct value of 28.82, representing an infection rate of 11.1% (Table 1).

TABLE 1. Spatiotemporal ‘*Candidatus* Liberibacter asiaticus’ (CLas) detection in different tissues of Valencia sweet orange on Cleopatra rootstock trees after Asian citrus psyllid (ACP) transmission of CLas to young leaves^a
FP (week)	Tree number	Ct values for CLas detection determined by qPCR
		0 DPR		75 DPR			150 DPR
		ACPs	FL	FL	UFML	Root	FL	UFML	YFL	Root
1	1	22.04 ± 0.32	38.25 ± 0.68	ND	40.84 ± 1.11	38.49 ± 0.20	37.83 ± 0.78	40.30 ± 1.86	ND	39.02 ± 0.04
1	2	21.33 ± 0.07	38.38 ± 0.27	40.21 ± 0.09	ND	39.14 ± 1.52	NA	34.53 ± 0.26	38.14 ± 0.70	38.60 ± 0.52
1	3	22.05 ± 0.37	40.12 ± 0.73	22.81 ± 0.19	ND	28.14 ± 1.09	21.93 ± 0.27	31.21 ± 0.08	NA	25.14 ± 0.30
2	4	21.35 ± 0.4	35.16 ± 0.63	23.64 ± 0.17	28.82 ± 0.53	28.52 ± 0.72	21.57 ± 0.16	24.79 ± 0.29	NA	23.97 ± 0.03
2	5	22.36 ± 0.37	36.51 ± 0.39	24.06 ± 0.08	ND	32.30 ± 0.97	22.77 ± 0.06	37.42 ± 0.78	24.90 ± 0.14	25.45 ± 0.17
2	6	22.32 ± 0.52	35.61 ± 0.68	26.49 ± 0.14	36.61 ± 0.38	33.74 ± 0.46	23.91 ± 0.22	30.05 ± 0.13	23.80 ± 0.13	29.74 ± 0.11
3	7	20.07 ± 0.56	36.95 ± 0.44	38.54 ± 1.23	34.06 ± 0.29	37.98 ± 0.89	ND	ND	NA	37.31 ± 0.51
3	8	22.11 ± 0.64	35.86 ± 0.19	24.14 ± 0.25	38.83 ± 1.47	23.40 ± 0.12	31.89 ± 0.37	29.33 ± 0.54	ND	27.66 ± 0.15
3	9	20.38 ± 1.39	39.91 ± 0.58	24.60 ± 0.08	37.74 ± 1.01	24.76 ± 0.05	25.25 ± 0.44	37.73 ± 0.05	25.45 ± 1.78	25.88 ± 0.01
3	10	22.43 ± 0.53	35.61 ± 0.94	22.93 ± 0.35	33.84 ± 0.08	22.01 ± 0.04	22.68 ± 1.27	25.15 ± 1.75	ND	28.23 ± 0.08
3	11	20.45 ± 1.1	38.30 ± 1.02	24.46 ± 0.04	38.19 ± 0.56	22.74 ± 0.05	26.41 ± 0.85	36.13 ± 0.97	38.46 ± 1.50	28.64 ± 0.28
3	12	21.04 ± 0.18	39.56 ± 1.33	ND	38.34 ± 0.59	ND	ND	ND	ND	38.55 ± 0.09
4	13	19.95 ± 0.25	35.63 ± 0.63	24.28 ± 0.34	39.12 ± 0.85	26.76 ± 0.65	22.31 ± 0.21	38.49 ± 1.21	NA	24.10 ± 0.04
4	14	20.00 ± 0.14	36.17 ± 0.84	39.15 ± 0.44	40.96 ± 0.79	37.92 ± 0.45	37.89 ± 0.19	37.31 ± 0.33	39.07 ± 0.48	37.36 ± 0.27
4	15	21.89 ± 0.5	35.04 ± 0.36	ND	ND	37.28 ± 0.50	38.33 ± 0.67	39.03 ± 0.72	37.56 ± 0.90	38.03 ± 0.96
	Negative control	ND	38.61 ± 1.04	39.94 ± 0.45	ND	ND	ND	–^b	–	40.43 ± 4.01

^a Data are presented as mean ± SD cycle threshold (Ct) values for three technical replicates. A healthy plant was used as the negative control. Quantitative real-time PCR (qPCR) assays were used for detection of CLas, with Ct values ≥35 considered CLas negative and ≤32 considered as CLas positive; values between ≤32 and ≥35 were considered questionable and further confirmed using conventional PCR. FP, feeding period; DPR, days postremoval of ACP; FL, leaves fed on by ACPs during the flush stage; UFML, mature leaves not fed on by ACPs; YFL, leaves that emerged after ACP removal; ND, nondetected, and NA, not available.

^b Dashes indicate not done.

TABLE 1. Spatiotemporal ‘*Candidatus* Liberibacter asiaticus’ (CLas) detection in different tissues of Valencia sweet orange on Cleopatra rootstock trees after Asian citrus psyllid (ACP) transmission of CLas to young leaves^a

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As expected, CLas was detected at 150 DPR in leaves developed from the young shoot fed on by ACPs for the nine trees that were CLas positive in young leaves at 75 DPR. CLas was also detected in the roots of all nine plants, with Ct values ranging from 23.97 to 29.74 (Table 1). Importantly, CLas was detected in mature leaves not fed on by ACPs during transmission in five of the nine trees, with Ct values ranging from 24.79 to 31.21, representing an infection rate of 56% for mature leaves. In addition, CLas was detected in leaves that emerged after ACP removal in three of the nine trees, with Ct values of 23.8, 24.9, and 25.45 (Table 1).

It was suggested that CLas moves passively with the phloem sap from source to sink tissues (Andrade et al. 2020; Wang et al. 2017a). The detection of CLas in mature leaves, which CLas-positive ACPs did not feed on during transmission, at both 75 and 150 DPR was unexpected (Table 1). To understand the uniformity of CLas movement into mature leaves, the presence of CLas was evaluated in three mature leaves per plant from different locations in seven of the nine CLas-positive plants at 225 DPR. CLas was detected in 11 of the 17 tested mature leaves, representing an infection rate of 64.7% for mature leaves (Supplementary Table S1).

HLB symptom development and CLas colonization after ACP transmission.

To investigate the pattern of HLB symptom development, we monitored HLB symptoms at 225 DPR in leaves fed on by ACPs, leaves that emerged after ACP removal, and mature leaves not fed on by ACPs. As described above, only 9 of 15 trees fed on by ACPs showed successful ACP transmission of CLas (60% transmission efficacy). The transmission rate was 33, 100, 67, and 33% for 1, 2, 3, and 4 weeks of ACP feeding periods, respectively (Table 1). At 225 DPR, two of the nine CLas-transmitted trees exhibited diebacks; tree 13 died completely, whereas the roots of tree 4 declined with high CLas titer (Supplementary Table S1). We observed both asymptomatic and symptomatic leaves. Leaves directly fed on by ACPs displayed a high frequency of HLB symptoms (65.83%), followed by leaves that emerged >30 days after ACP removal (39.24%), all leaves that emerged after ACP removal (20.49%), and mature leaves not fed on by ACPs during the feeding assay (8.28%) (Fig. 1A). The CLas titer in asymptomatic leaves was significantly lower than that in symptomatic leaves (Fig. 1B). The CLas titer in symptomatic mature leaves not fed on by ACPs during the feeding assay was lower than that in symptomatic leaves that were directly fed on by ACPs during the flush stage and that emerged after ACP removal; a similar pattern was also observed for asymptomatic leaves (Fig. 1B). The CLas titer in symptomatic leaves that were directly fed on by ACPs and that emerged after ACP removal was approximately 14-fold and sixfold higher than in mature symptomatic leaves not fed on by ACPs during the feeding assay, respectively (Fig. 1B).

Systemic CLas colonization inside the citrus tree 1 year after ACP transmission.

We investigated the systemic colonization of CLas inside the citrus tree 1 year after ACP transmission of CLas to young leaves. Only seven trees infected by CLas via ACP transmission remained alive at 1 year. CLas was detected in the roots from all four directions with similar Ct values (Table 2). Furthermore, all remaining mature leaves not fed on by ACPs during the feeding assay were collected and analyzed for the CLas titer (Fig. 2). CLas was detected in approximately 43.1% of mature leaves collected from six of the seven trees (Fig. 2; Table 3) because tree 3 did not have any mature leaves left. However, no obvious patterns were observed between CLas appearance in mature leaves not fed on during the feeding assay with respect to the branch directly exposed to ACPs. We also tested the CLas titer in leaves that emerged after ACP removal collected from different branches (Fig. 2). Three leaves (≥30 days old) were collected from each branch and CLas was detected in 91.5% of these leaves (Table 4). Of note, leaves that emerged after ACP removal but were <30 days old were not tested.

**Fig. 2.** Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in leaves of Valencia sweet orange on Cleopatra rootstock trees 1 year after Asian citrus psyllid (ACP) transmission of CLas to young leaves. The CLas titer is shown for each branch. The number of CLas-positive versus that of total mature leaves of the tested branch and the cycle threshold (Ct) values of CLas-positive leaves are shown in maroon. The number of CLas-positive versus that of total leaves (≥30 days old) that emerged after ACP removal and Ct values of CLas-positive leaves are shown in blue. FB = branch directly exposed to ACPs, MB = branch with mature leaves not fed on by ACPs, YB = leaves and branches that emerged after ACP removal, ML = mature leaves not fed on by ACPs, and YL = leaves (≥30 days old) that emerged after ACP removal.

TABLE 2. Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in the roots of Valencia sweet orange on Cleopatra rootstock trees 1 year after Asian citrus psyllid (ACP) transmission of CLas to young leaves^a
FP (week)	Tree number	SS	OS	L1	L2
1	3	24.70 ± 1.33	NA^b	NA^b	NA^b
2	5	27.35 ± 1.08	27.11 ± 1.1	27.73 ± 1.89	27.60 ± 0.07
2	6	28.01 ± 1.1	27.83 ± 1.61	NA^b	NA^b
3	8	29.33 ± 0.92	30.95 ± 0.19	29.76 ± 0.28	28.51 ± 0.27
3	9	27.26 ± 0.14	26.25 ± 0.12	26.14 ± 0.28	26.76 ± 0.5
3	10	29.87 ± 0.32	30.66 ± 0.78	31.44 ± 2.62	30.38 ± 0.55
3	11	30.64 ± 1.79	30.21 ± 0.98	29.20 ± 1.5	29.95 ± 0.28

^a Data are presented as mean ± SD cycle threshold (Ct) values for three technical replicates. Quantitative real-time PCR assays were used for detection of CLas, with Ct values ≥35 considered CLas negative and ≤32 considered CLas positive; values between ≤32 and ≥35 were considered questionable and further confirmed using conventional PCR. FP, feeding period; SS, roots collected from the same side as leaves fed on by ACPs; OS, roots collected from the opposite side of leaves fed on by ACPs, and L1 and L2, roots collected from the other two directions perpendicular to the SS and OS.

^b NA, not available. The feeder roots in these directions were not available because of heavy deterioration caused by huanglongbing.

TABLE 2. Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in the roots of Valencia sweet orange on Cleopatra rootstock trees 1 year after Asian citrus psyllid (ACP) transmission of CLas to young leaves^a

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TABLE 3. Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in the mature leaves of Valencia sweet orange on Cleopatra rootstock trees 1 year after Asian citrus psyllid (ACP) transmission of CLas to young leaves^a
FP (week)	Tree number	Branch number	Leaf 1	Leaf 2	Leaf 3	Leaf 4	Leaf 5	Leaf 6	Leaf 7
2	5		26.29 ± 0.03	25.29 ± 0.08	25.69 ± 0.15	27.47 ± 0.19	NA	NA	NA
2	6		27.08 ± 0.11	NA	NA	NA	NA	NA	NA
3	8	1	33.76 ± 0.19^b	34.05 ± 0.08	38.64 ± 0.61	NA	NA	NA	NA
		2	38.00 ± 1.18	ND	ND	31.69 ± 1.14	34.13 ± 0.25	36.93 ± 0.66	ND
		3	32.00 ± 0.25	34.36 ± 0.34	35.12 ± 0.53	31.74 ± 0.30	39.43 ± 0.61	ND	NA
		4	40.25 ± 0.98	33.01 ± 0.10^b	ND	23.03 ± 0.12	NA	NA	NA
		5	ND	36.90 ± 0.62	ND	ND	ND	37.52 ± 0.47	NA
3	9	1	23.83 ± 0.2	23.87 ± 0.03	23.52 ± 0.07	22.93 ± 0.03	23.67 ± 0.05	NA	NA
3	10	1	25.10 ± 0.06	35.25 ± 0.56	27.07 ± 0.03	25.91 ± 0.04	ND	29.21 ± 0.37	30.13 ± 0.33
		3	22.47 ± 0.95	21.30 ± 0.34	NA	NA	NA	NA	NA
3	11	1	35.12 ± 0.22	38.56 ± 0.84	38.07 ± 0.39	NA	NA	NA	NA
		2	ND	37.69 ± 0.41	ND	NA	NA	NA	NA
		3	35.61 ± 0.36	ND	37.08 ± 1.34	NA	NA	NA	NA
		4	ND	32.27 ± 0.7^c	26.05 ± 1.21	ND	NA	NA	NA

^a Data are presented as mean ± SD cycle threshold (Ct) values for three technical replicates. FP, feeding period; NA, not available; and ND, nondetected.

^b CLas presence was also confirmed by conventional PCR. Quantitative real-time PCR assays were used for detection of CLas, with Ct values ≥35 considered CLas negative and ≤32 considered CLas positive; values between ≤32 and ≥35 were considered questionable and further confirmed using conventional PCR.

^c Mature leaves were marked before ACP feeding on the young flush.

TABLE 3. Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in the mature leaves of Valencia sweet orange on Cleopatra rootstock trees 1 year after Asian citrus psyllid (ACP) transmission of CLas to young leaves^a

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TABLE 4. Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in leaves emerged after Asian citrus psyllid (ACP) removal of Valencia sweet orange on Cleopatra rootstock trees 1 year after ACP transmission of CLas to young leaves^a
FP (week)	Tree number	Branch number	Leaf 1	Leaf 2	Leaf 3
1	3	1	24.74 ± 0.08	24.70 ± 0.10	24.22 ± 0.07
		2	25.44 ± 0.45	23.48 ± 0.05	23.45 ± 0.24
		3	23.88 ± 0.01	NA	NA
2	5	1	23.76 ± 0.09	24.01 ± 0.07	25.22 ± 0.12
		2	24.65 ± 0.01	25.13 ± 0.09	23.80 ± 0.12
		3	24.52 ± 0.06	24.94 ± 0.01	24.75 ± 0.08
		4	23.83 ± 0.02	28.33 ± 0.001	25.75 ± 0.14
		5	23.05 ± 0.04	23.68 ± 0.25	NA
2	6	1	23.56 ± 0.34	24.41 ± 0.12	24.68 ± 0.04
		2	24.57 ± 0.55	24.81 ± 0.83	22.08 ± 0.11
		3	22.40 ± 0.14	22.19 ± 0.11	24.31 ± 0.27
		4	22.25 ± 0.14	24.52 ± 0.22	24.88 ± 0.09
		5	25.47 ± 0.25	24.85 ± 0.10	24.81 ± 0.54
3	8	1	24.51 ± 0.13	NA	NA
		2	27.85 ± 0.13	NA	NA
		3	ND	36.89 ± 0.19	NA
3	9	1	23.37 ± 0.41	22.55 ± 0.45	24.23 ± 0.84
		2	23.82 ± 0.08	23.95 ± 0.75	24.13 ± 0.62
		3	23.83 ± 0.76	24.01 ± 0.13	NA
3	10	1	25.55 ± 0.42	27.12 ± 0.39	27.66 ± 0.39
		2	ND	36.59 ± 0.76	23.78 ± 1.22
		3	25.44 ± 0.20	24.10 ± 0.27	NA
		4	27.28 ± 0.53	32.16 ± 0.49	24.70 ± 0.73
3	11	1	30.46 ± 0.50	23.91 ± 0.07	24.85 ± 0.03
		2	23.91 ± 0.06	23.68 ± 0.01	23.41 ± 0.045
		3	24.56 ± 0.07	23.61 ± 0.04	25.61 ± 0.33
		4	34.19 ± 0.51	35.11 ± 0.28	31.16 ± 0.27

TABLE 4. Detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in leaves emerged after Asian citrus psyllid (ACP) removal of Valencia sweet orange on Cleopatra rootstock trees 1 year after ACP transmission of CLas to young leaves^a

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ACPs do not transmit CLas via mature leaves.

To exclude the possibility that the detection of CLas in mature leaves resulted from ACP transmission, we investigated whether ACPs can transmit CLas via feeding on mature leaves. CLas-infected ACPs were caged on mature leaves of 13 healthy Valencia sweet orange on Cleopatra rootstock trees for 1 to 4 weeks (Supplementary Fig. S1B). Ct values for the ACPs used for transmission ranged from 20.34 to 25.02 (Table 5). CLas was not detected in mature leaves caged with ACPs at 0, 75, and 150 DPR. Similarly, CLas was not detected in mature leaves that were not caged with ACPs or in roots at 0, 75, and 150 DPR (Table 5). Importantly, CLas was also not detected in leaves that emerged after ACP removal at 150 DPR (Table 5). These data demonstrated that ACPs failed to transmit CLas via mature leaves.

TABLE 5. Spatiotemporal detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in different tissues of Valencia sweet orange on Cleopatra rootstock trees for which CLas-positive Asian citrus psyllids (ACPs) were caged with mature leaves^a
FP (week)	Tree number	Ct values for CLas detection determined by qPCR
		0 DPR		75 DPR			150 DPR
		ACPs	FML	FML	UFML	Root	FML	UFML	YFL	Root
1	1	24.05 ± 0.58	ND	40.43 ± 1.74	ND	ND	ND	ND	NA	ND
1	2	24.25 ± 0.88	41.61 ± 0.60	ND	38.61 ± 1.28	ND	ND	37.43 ± 1.37	NA	ND
1	3	25.00 ± 0.02	ND	ND	ND	39.54 ± 1.09	ND	ND	ND	ND
2	4	24.37 ± 0.02	42.24 ± 0.27	ND	ND	ND	ND	37.86 ± 1.12	38.24 ± 0.67	39.94 ± 0.65
2	5	25.02 ± 0.01	42.02 ± 0.67	ND	ND	ND	ND	ND	ND	ND
2	6	24.38 ± 0.03	42.31 ± 0.2	38.85 ± 1.14	ND	ND	39.45 ± 0.02	37.22 ± 0.91	NA	40.21 ± 0.96
3	7	20.34 ± 0.04	ND	ND	ND	ND	ND	ND	39.62 ± 0.17	ND
3	8	22.24 ± 0.01	40.99 ± 0.43	ND	ND	ND	38.95 ± 0.72	37.36 ± 0.52	NA	ND
3	9	22.34 ± 0.09	40.92 ± 1.39	ND	ND	ND	ND	37.28 ± 0.44	ND	ND
3	10	23.22 ± 0.04	39.64 ± 0.97	ND	ND	ND	38.99 ± 0.63	39.63 ± 0.85	NA	39.55 ± 0.06
4	11	22.27 ± 0.02	40.00 ± 0.08	ND	39.49 ± 0.56	ND	39.47 ± 0.01	36.96 ± 0.02	NA	ND
4	12	22.24 ± 0.04	38.48 ± 0.49	38.97 ± 0.03	39.55 ± 0.57	39.57 ± 0.76	ND	ND	39.87 ± 0.68	ND
4	13	20.36 ± 0.01	39.21 ± 0.64	ND	ND	38.58 ± 0.94	ND	37.85 ± 0.76	NA	ND
	Negative control	ND	–^b	ND	–	–	ND	39.79 ± 0.42	–	–

^a Data are presented as mean ± SD cycle threshold (Ct) values for three technical replicates. Healthy plant samples were used as the negative control. Quantitative real-time PCR (qPCR) assays were used for detection of CLas with Ct values ≥35 considered CLas negative and ≤32 considered CLas positive; values between ≤32 and ≥35 were considered questionable and further confirmed using conventional PCR. FP, feeding period; DPR, days postremoval of ACPs; ND, nondetected; NA, not available; FML, mature leaves caged with CLas-positive ACPs; UFML, mature leaves not exposed to ACPs; and YFL, leaves that emerged after ACP removal.

^b Dashes indicate not done.

TABLE 5. Spatiotemporal detection of ‘*Candidatus* Liberibacter asiaticus’ (CLas) in different tissues of Valencia sweet orange on Cleopatra rootstock trees for which CLas-positive Asian citrus psyllids (ACPs) were caged with mature leaves^a

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DISCUSSION

Understanding of in planta CLas movement, colonization, and HLB symptom development remains utterly challenging because of the unculturable nature and lack of genetic manipulation of CLas. CLas movement in planta is considered passive and CLas migrates together with the flow of phloem sap (Folimonova and Achor 2010; Hilf 2011; Kim et al. 2009; Koh et al. 2012; Wang et al. 2017a). This is consistent with the detection of CLas in the roots of all trees, but only in the mature leaf that was not fed on during the transmission assay of only one tree, of the nine plants successfully infected via ACP transmission into young leaves at 75 DPR. Intriguingly, this study unveils a new mode of movement: active movement to the source tissue. CLas was detected in 11.1, 56, 64.7, and 43.1% of mature leaves, which were not fed on by ACPs during the transmission assay, at 75, 150, 225, and 365 DPR, respectively, after ACP transmission. The higher detection rates in mature leaves at 150 and 225 DPR than at 365 DPR probably resulted from overestimation attributable to random sampling, whereas all mature leaves were sampled at 365 DPR. Of note, we excluded the possibility that CLas in mature leaves is transmitted by ACPs. Detection of CLas in mature leaves, a typical source tissue, could not be explained by passive movement from source to sink tissues. Instead, these data suggest that CLas possesses the capacity to actively move to source tissues. The difference in colonization timing of sink tissues (e.g., roots) and source tissues (e.g., mature leaves) indicates that CLas is capable of both passive and active movement, with passive movement being the dominant form.

The structures responsible for the active movement of CLas remain unknown. A 2020 study demonstrated the flexible morphology of CLas cells, which could display attachment near sieve plates through unknown filaments (Achor et al. 2020). CLas contains most flagellar genes required for movement (Duan et al. 2009; Thapa et al. 2020). Several flagellar genes, such as the flagellin‐encoding gene flaA, were reported to be functional via ectopic expression studies in genus Agrobacterium (Andrade et al. 2020). However, no flagellum was observed for CLas in citrus and dodder although its close relatives Liberibacter crescens and Sinorhizobium meliloti contain flagella (Andrade et al. 2020; Wang et al. 2013). The expression of flagellar genes was higher in psyllids than in planta (Andrade et al. 2020; Yan et al. 2013). One putative explanation for this mystery is that CLas differentially regulates the flagellar genes in different stages. In the early stage of CLas infection of plants, CLas might suppress the expression of flagella, a typical pathogen-associated molecular pattern (PAMP), to avoid eliciting PAMP-triggered immunity (Andrade et al. 2020; Shi et al. 2018; Zou et al. 2012). During infection of mature leaves, a later stage of CLas infection, flagella might express temporarily in the upward movement of CLas from the roots. Such a hypothesis needs to be tested. In addition, CLas encodes type IVc tight adherence Tad pili, suggested to be involved in adherence but not motility (Andrade and Wang 2019). Further characterization of the type IV pili in CLas movement in planta is needed.

Spatiotemporal detection of CLas in different tissues allows us to visualize how CLas systemically establishes inside the plant. After ACP transmission of CLas into young leaves, CLas seems to move to roots first after leaf maturation, as suggested by Johnson et al. (2014). CLas then begins to move to the young flush following source to sink phloem sap movement, but it also actively moves to mature leaves. At 365 DPR, CLas was detected in 91.5% of the leaves (≥30 days old) that emerged after ACP removal; however, it was detected in only 43.1% of mature leaves that were mature during ACP feeding but not fed on by ACPs. The lifespan of citrus leaves is approximately 1.5 years on average, but some can live up to 3 years (J. P. Syvertsen, personal communication). Taken together, it is estimated that it takes at most 3 years for CLas to infect the whole tree or almost all leaves on a tree when all mature leaves are replaced. The slow infection of mature leaves explains in part the slow development (months to years) of HLB symptoms (Bové 2006; Gottwald 2010) and the chronic nature of the HLB pathosystem (Gottwald 2010). In addition, differences in symptom development and CLas titers among leaves directly fed on by ACPs, leaves that emerged after ACP removal, and mature leaves not fed on by ACPs during the feeding assay (Fig. 1) elucidate one common phenomenon in the early stage of the infection: few branches showing obvious HLB symptoms. This phenomenon probably results from few psyllid transmissions of CLas into one or several young shoots.

This study further emphasizes that young leaves should be the focus of HLB management. ACPs can acquire CLas from mature leaves but at reduced efficiency than nymphs and adults feeding on young leaves (Luo et al. 2015; Pelz-Stelinski et al. 2010; Sétamou et al. 2016a). Here, our data show that ACPs could not transmit CLas via feeding on mature leaves. Probing by ACPs is visible on mature leaves. Xylem ingestion was shown to occur more on mature leaves than the young flush (Ebert et al. 2018). Furthermore, electrical penetration graph analyses suggest that the sclerenchymatous ring in mature leaves hinders ACPs feeding over phloem (George et al. 2017), probably contributing to the inability of ACPs to transmit CLas to mature leaves. Precise spraying of young leaves might allow for more frequent and thorough protection of citrus trees from HLB than the current scheme of spraying the whole tree.

In summary, we conducted the spatiotemporal detection of CLas in different tissues after ACP transmission of CLas to young leaves. Our study reveals CLas infection of mature leaves, suggesting active movement of CLas to the source tissue. This study helps visualize the infection process of CLas in planta and consequent symptom development and provides useful information for HLB management.

The author(s) declare no conflict of interest.

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Funding: This study was supported by the Florida Citrus Initiative Program, the Citrus Research and Development Foundation, and the U.S. Department of Agriculture National Institute of Food and Agriculture (grants 2016-70016-24833 and 2018-70016-27412).

The author(s) declare no conflict of interest.