The Total Population Size of ‘Candidatus Liberibacter asiaticus’ Inside the Phloem of Citrus Trees and the Corresponding Metabolic Burden Related to Huanglongbing Disease Development
- Fernanda N. C. Vasconcelos1
- Jinuyn Li1
- Zhiqian Pang1
- Christopher Vincent2
- Nian Wang1 †
- 1Citrus Research and Education Center (CREC), Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL 33850
- 2Citrus Research and Education Center (CREC), Department of Horticultural Sciences, University of Florida, Lake Alfred, FL 33850
‘Candidatus Liberibacter asiaticus’ (CLas) is the predominant causal agent of citrus huanglongbing, the most devastating citrus disease worldwide. CLas colonizes phloem tissue and causes phloem dysfunction. The pathogen population size in local tissues and in the whole plant is critical for the development of disease symptoms by determining the load of pathogenicity factors and metabolic burden to the host. However, the total population size of CLas in a whole plant and the ratio of CLas to citrus cells in local tissues have not been addressed previously. The total CLas population size for 2.5-year-old ‘Valencia’ sweet orange on ‘Kuharske’ citrange rootstock trees was quantified using quantitative PCR to be approximately 1.74 × 109 cells/tree, whereas 7- and 20-year-old sweet orange trees were estimated to be 4.3 × 1010 cells/tree, and 6.0 × 1010 cells/tree, respectively. The majority of CLas cells were distributed in leaf tissues (55.58%), followed by those in branch (36.78%), feeder root (4.75%), trunk (2.39%), and structural root (0.51%) tissues. The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were within approximately 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively, representing the metabolic burden of CLas in different organs. It was estimated that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. Approximately 0.01% of the total citrus phloem volume was estimated to be occupied by CLas, explaining the difficulty to observe CLas in most tissues under transmission electron microscopy. The CLas titer inside the leaf was estimated to be approximately 1.64 × 106 cells/leaf or 9.2 × 104 cells cm–2 in leaves, approximately 104 times less than that of typical apoplastic bacterial pathogens. This study provides quantitative estimates of phloem colonization by bacterial pathogens and furthers the understanding of the biology and virulence mechanisms of CLas.
Citrus huanglongbing (HLB, also called “greening”) is the most devastating citrus disease worldwide (Bové 2006; Gottwald 2010). ‘Candidatus Liberibacter asiaticus’ (CLas), ‘Ca. L. africanus’ (CLaf), and ‘Ca. L. americanus’ (CLam) are causal agents of HLB (Garnier et al. 1999; Jagoueix et al. 1997; Teixeira et al. 2005). CLas and CLam are mainly transmitted by Asian citrus psyllid (Diaphorina citri), whereas CLaf is transmitted by African citrus psyllid (Trioza erytreae; Bové 2006). All three HLB pathogens are also transmitted by grafting (Bové 2006; Lin 1956). Among the three HLB pathogens, CLas is the most prevalent worldwide. Typical HLB symptoms include blotchy mottled leaves; small upright leaves; branch dieback; thin canopy; off-season flowering; root decline; and smaller fruit that do not color normally, staying green for most varieties or turning red for certain varieties, such as naval sweet orange (Bové 2006; Wang et al. 2017a; Zheng et al. 2018). HLB-diseased citrus groves are usually devastated or become unproductive in 5 to 8 years (Bové, 2006).
Ca. Liberibacter spp. are alphaproteobacteria and phylogenetically related to Rhizobium and Sinorhizobium that are capable of endocytosis, a phenomenon through which bacteria enter the cytoplasm of plant cells (Verma 1992). It was suggested that ancestors of Liberibacter first established their presence inside the phloem elements of plant hosts via endocytosis (Wang 2020). Ca. Liberibacter species have a much-reduced genome size of approximately 1.2 to 1.5 Megabits (Mb) compared with their relatives, probably resulting from reductive evolution owing to living in the nutrient-rich phloem tissues (Duan et al. 2009; Leonard et al. 2012; Lin et al. 2009; Wulff et al. 2014). CLas, Clam, and CLaf are yet to be cultivated in pure culture (Merfa et al. 2019), which has significantly hindered our understanding of their pathogenesis mechanisms (Wang 2019). It was reported that CLas suppresses plant immune responses by degrading salicylic acid (Li et al. 2017) and by using effectors such as SDE1, which interacts with papain-like cysteine proteases (Clark et al. 2018), and SDE15, acting on CsACD2, a homolog of Arabidopsis ACCELERATED CELL DEATH2 (ACD2) (Pang et al. 2020). It was suggested that SC2-gp095, a CLas prophage gene that encodes a reactive oxygen-scavenging peroxidase, assists CLas to evade plant immune responses (Jain et al. 2015). Another prophage gene, LASP235 (CLIBASIA_05525), was reported to contribute to HLB-like symptoms, including leaf chlorosis and plant growth retardation (Hao et al. 2019).
CLas colonizes phloem in bark tissue, leaf midrib, root, and different floral and fruit parts (Tatineni et al. 2008). CLas causes phloem dysfunction (Wang and Trivedi 2013) and root decline (Johnson et al. 2014). Together these effects explain many of the HLB symptoms, although molecular mechanisms remain poorly understood. HLB primarily affects source–sink relationships, hormone pathways, and nutrient distribution within plants, probably the result of reduced phloem function (Albrecht and Bowman 2008; Fan et al. 2010; Martinelli et al. 2012). Root decline significantly affects both nutrients and water absorption, contributing to overall decline of the tree and a reduced tolerance to heat stress. It was reported that CLas causes callose deposition and accumulation of phloem proteins, leading to phloem blockage, but not directly blocking sieve pores per se (Kim et al. 2009).
The population size of a pathogen in local tissues and its overall population size in a host correlate with the development of disease symptoms. Excessive proliferation of pathogenic microbes can lead to plant diseases (Nobori et al. 2018). It was reported that a minimal CLas titer is required for HLB symptom development (Trivedi et al. 2009). In addition to the pathogenic effects on phloem tissues and roots, as a biotrophic pathogen, Ca. Liberibacter is a metabolic burden to the plant host, which disrupts the metabolism of neighboring host cells; and source-sink relationships, contributing to tree decline. It was reported that the number of CLam in 1 gram (g) of fresh midribs from blotchy mottled sweet orange leaves ranges from 4.6 × 105 to 1.1 × 108 (Teixeira et al. 2008). Similarly, 6.74 × 105 to 3.38 × 108 CLas cells/g of tissue were reported in sweet orange (Trivedi et al. 2009). In addition, up to 2 × 1011 genome equivalents per gram of tissue were also reported in sweet orange (Li et al. 2006). Despite these previous studies on CLas titers in planta, the total population size of CLas in a whole plant and the ratio of CLas to host cells in local tissues have previously not been addressed. Here, we estimated the total CLas cell numbers in citrus trees and different organs and the ratio of citrus cells to CLas cells in different tissues. We also aimed to quantify CLas cells in individual leaves, and by leaf area and phloem volume to help understand CLas biology and pathogenicity.
MATERIALS AND METHODS
Plant material and DNA extraction.
For quantification of CLas in young plants, five 2.5-year-old HLB-positive ‘Valencia’ sweet orange on ‘Kuharske’ citrange rootstock trees planted in 2017 in a citrus grove at Lake Alfred, Florida were selected for a destructive assay (Fig. 1A and B). Trees were similar in size and had been previously determined to be CLas-positive. Canopy volume was estimated for each tree using the following equation: V = 0.524 hd2, where h is the tree height and d is the average diameter of the tree (Pons et al. 2012).
To estimate the total number of leaves, we used the following approach: A square frame, measuring 0.263 m2, was placed along the edge of the canopy. All leaves found within this frame were counted. This was repeated three times per tree. The total number of leaves was calculated by calculating the surface area of the canopy as a sphere (using the height and width as mean diameters): 4πr2. The number of leaves was then calculated by dividing the mean number of leaves per frame by the fraction of the canopy surface area occupied by the frame.
All phloem-containing tissues from each tree—leaves, feeder roots, and peeled bark tissues of branches, trunks, and structural roots—were collected, pooled, weighed, and ground together for the same tissue types. For branches, trunks, and structural roots, the woody tissues, which do not contain phloem, were excluded. (The general features of the citrus trees used are shown in Supplementary Table S1.) The tissue was used to measure the weight and for DNA extraction. For each tissue type, four samples of 100 milligrams (mg)/sample were used for DNA extraction, totaling 20 samples per tree.
To quantify CLas in individual leaves from trees of different ages, five 2.5-year-old trees of ‘Valencia’ sweet orange (Citrus × sinensis L.) scion on ‘Kuharske’ citrange (C. sinensis × Poncirus trifoliata L.) trees, five 7-year-old ‘Hamlin’ sweet orange on ‘Swingle’ citrumelo (× Citroncirus spp.) rootstock planted in 2012 in a grove in Auburndale, Florida, and five 20-year-old ‘Hamlin’ on ‘Swingle’ citrumelo trees planted in 1999 in Lake Alfred, Florida, were sampled. All samples were collected in September 2019. A total of 59 leaves from 2.5-year-old trees, 30 leaves from 7-year-old trees, and 28 leaves from 20-year-old trees were collected. Six 27.3-mm2 leaf discs taken along the midrib from each leaf were used for DNA extraction and quantification of CLas.
In August 2020, another batch of samples were taken from the same three groves as mentioned above with five trees from each age group. From each tree, four leaves showing nutrient deficiency (i.e., zinc deficiency), four asymptomatic leaves, and four HLB symptomatic (i.e., blotchy mottle) leaves were taken. Leaves were individually weighed and homogenized, and DNA was extracted from 100 mg of tissue of each leaf.
DNA was extracted using a DNeasy Plant Pro kit (Qiagen, Valencia, CA), following the manufacturer’s instructions, and eluted in 100 μl of nuclease-free water. DNA concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE).
CLas quantification using quantitative PCR.
Quantitative PCR (qPCR) was carried out with primers and probe for CLas (Wang et al. 2006). Briefly, qPCR assays were performed with the software QuantiStudio3 (Thermo Fisher Scientific, Waltham, MA) using the Quantitec Probe PCR Master Mix (Qiagen, Valencia, CA) in a 25-µl reaction. The standard amplification protocol was 95°C for 10 min followed by 40 cycles at 95°C for 15 s and 60°C for 60 s. All reactions were conducted in triplicate or duplicate with CLas-positive plants and water controls. Quantification of CLas was conducted using the equation Y = −0.288 × (CLas cycle threshold [Ct]) + 11.607 for leaf, branch, and trunk samples, and Y = −0.2768 × (CLas Ct) + 11.677 for root samples (Trivedi et al. 2009).
Measure of leaf morphology and calculation of the phloem volume of citrus trees.
To measure the leaf area, 152 representative citrus leaves were collected from the field and scanned together with a ruler on a white background to generate the digital images. The leaf area was calculated using the software ImageJ (National Institutes of Health, Bethesda, MD) as described in Abramoff et al. (2004). Because the citrus phloem volume is unknown, here we estimated the citrus phloem volume using the equation calculated for aspen trees (Populus tremulus L.): Vphloem = 9.0 × 10–5 × L2.31 (L indicates tree height) (Hölttä et al. 2013). Although this relationship is from a different species, other studies have shown that allometric relationships of tree vasculature are broadly applicable across species, including across groups of different growth habits and those adapted to the gradient of tropical to temperate climates (Olson et al. 2014).
Calculation of the ratio of citrus cells to CLas cells.
To quantify the citrus cells in the samples, CLas genomic DNA was subtracted from the total DNA. The following equation was used to calculate the weight of CLas genomic DNA and copy numbers of citrus genomic DNA: number of copies = (amount [nanograms, ng] × 6.022 × 1023)/(length [in basepairs] × 109 × 650). For this calculation, the CLas genome size of 1.23 Mb (Duan et al. 2009) was used, whereas 367 Mb was used as the genome size of sweet orange (Wu et al. 2014, 2018; Xu et al. 2013). The ratio of plant cells to CLas cells was calculated as the ratio between the CLas calculated number and the citrus cells calculated number in each sample. To further verify the result, we also estimated the total citrus cell number based on the cell weight. A plant cell weight is approximately 1 ng, whereas a bacterial cell weight is approximately 1 picogram (pg) (Wayne 2009).
All statistical analyses were performed using the software SAS V9.4 (SAS Institute Inc., Cary, NC). The data were first tested for normality and homogeneity of variance using the Shapiro-Wilk’s test and Levene’s test, respectively. CLas titer data were log10-transformed to satisfy assumptions of normality and homoscedasticity. A one-way analysis of variance (ANOVA) was performed to determine any differences in tissue effects and the means for different tissues were separated using Tukey’s honestly significant difference test (α = 0.05). When the sample sizes were unequal, Welch’s ANOVA was performed to determine any differences in treatment effects, and the treatment means were separated using the Games-Howell post hoc test (P < 0.05).
Quantification of CLas population in planta.
CLas was reported to be widely present in plant organs including leaf, trunk, and root (Tatineni et al. 2008). To quantify CLas in planta, we first characterized the titers of CLas in leaf, branch, trunk, structural root, and feeder root samples collected from 2.5-year-old ‘Valencia’ sweet orange on ‘Kuharske’ citrange rootstock trees (Fig. 1) from citrus groves in Lake Alfred, Florida in September 2019. For this purpose, the five trees were dug out and leaf, branch, trunk, structural root, and feeder root samples were pooled together for each tree for DNA extraction. CLas titers in the branch (9.9 × 106 cells g–1 of tissue) and leaf samples (8.3 × 106 cells g–1 of tissue) were highest, followed by that in feeder root (1.5 × 106 cells g–1 of tissue), trunk (9.1 × 105 cells g–1 of tissue), and structural root (3.2 × 105 cells g–1 of tissue) samples (Table 1).
The average CLas populations in leaf, branch, trunk, structural root, and feeder root samples from individual 2.5-year-old citrus trees in September 2019 were approximately 9.7 × 108, 6.4 × 108, 4.2 × 107, 8.8 × 106, and 8.3 × 107 cells (Table 2). The total CLas populations in the five 2.5-year-old trees ranged from 1.3 × 109 to 2.9 × 109 (Table 2).
The majority of CLas were distributed in the leaf samples (55.58%), followed by that in the branch samples (36.8%). The CLas populations were relatively small in the feeder root (4.8%), trunk (2.4%), and structural root (0.5%) samples (Table 2).
To understand the metabolic burden that CLas imposes on different tissues, we calculated the ratio of citrus cells to CLas cells in different tissues. Using the qPCR-based method, the ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were calculated to be approximately 39, 44, 153, 191, and 561, respectively (Table 1). Using the cell-weight-based method, the ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were calculated to be approximately 79, 124, 1,355, 1,054, and 3,760, respectively (Table 1). The ratios calculated based on cell weight were 2.0- to 8.8-fold higher than that based on qPCR. It is important to note that the estimate for the ratio in leaf tissues was only 2.8-fold higher.
CLas detection in individual leaves.
Here, we analyzed CLas titers in representative leaves from citrus trees planted in 1999, 2012, and 2017. For trees planted in 2012 and 2017, there were no statistically significant differences among the CLas titers in asymptomatic leaves, symptomatic leaves, and leaves showing nutrient deficiency in samples collected in August 2020 and September 2019 (Table 3 and Supplementary Table S2). However, for trees planted in 1999, the CLas titers in symptomatic leaves and leaves showing nutrient deficiency were significantly higher than that of asymptomatic leaves in samples collected in August 2020 (Table 3). For samples collected in September 2019, the CLas titers in symptomatic leaves were significantly higher than those of asymptomatic leaves, whereas those of leaves showing nutrient deficiency were not significantly different from the other two categories. In all three age groups, CLas titers varied dramatically in different leaves (Fig. 2).
Next, we calculated the total CLas population in individual leaves. Based on the total CLas population size in leaves (Table 2), total leaf weight, and individual leaf weight (Supplementary Table S3), it was estimated that CLas population was approximately 1.6 × 106 cells/leaf in September 2019.
We further estimated the CLas density in individual leaves based on the leaf area using the software ImageJ. The citrus leaf area (mean ± significant error) was estimated to be 17.7 ± 0.6 cm2. Consequently, CLas density was quantified to be 9.2 × 104 ± 2.3 × 104 cells cm–2.
Estimate of the total CLas population size in citrus trees and CLas occupation of citrus phloem volume.
Because approximately 56% of CLas were distributed in the leaf samples (Table 1), we decided to estimate the total CLas population size per tree based on leaf count. The total number of leaves (mean ± significant deviation) was estimated to be 25,704 ± 4,361 and 36,133 ± 5,774 for 7- and 20-year-old trees, respectively. It was reported that the total number of leaves for 6-year-old sweet orange trees is approximately 37,257 based on counting (Turrell et al. 1969), consistent with our estimates. The reduced leaf number in HLB diseased trees probably resulted from leaf drop caused by HLB. Thus, the total CLas population size in September 2019 was estimated to be approximately 4.3 × 1010, and 6.0 × 1010 for 7- and 20-year-old citrus trees, respectively.
Next, we estimated the total volume of phloem that is occupied by CLas. Citrus phloem volume was estimated to be approximately 4.16 × 1013 μm3 for the 2.5-year-old trees. CLas was reported to be in both round and elongated bacilliform-like shapes (1.7- to 6.3-μm long, 0.33- to 0.66-μm diameter; Ammar et al. 2011; Hartung et al. 2010) with an estimated bacterial volume in the order of 1 μm3. Because the total CLas populations in the 2.5-year-old trees are in the order of 109 cells, it was estimated that approximately 0.01% of the total citrus phloem volume was occupied by CLas in September 2019.
The total CLas population for 2.5-, 7-, and 20-year-old citrus trees were estimated to be 1.74 × 109, 4.3 × 1010, and 6.0 × 1010, in September 2019, respectively. This estimate of CLas population in the phloem of citrus trees should be considered in the knowledge of dynamic seasonal population changes (Hu et al. 2014; Ibanez and Stelinski 2020; Sauer et al. 2015), stage of HLB disease, and uneven distribution (Tatineni et al. 2008) of CLas in planta. CLas titers were reported to vary by 10 to 100 times between the peak season and seasons with low CLas populations (Hu et al. 2014; Ibanez and Stelinski 2020; Sauer et al. 2015). In addition, citrus trees in the early stages of CLas infection have more leaves than later stages because HLB is known to cause leaf drop and thin canopy with disease progression. Thus, CLas population in the phloem tissues of mature trees can be up to 1011 and 1012 cells per tree. It is important to note that we did not include fruit and flower tissues in our analysis because we focused on vegetative tissues that are more relevant to disease development. Given that HLB disease results in progressively less dense canopies with smaller leaves, the actual total CLas population in the whole plant of severely affected trees might be much less than these estimates.
Previous studies demonstrated that CLas is the only bacterium colonizing the citrus phloem in Florida (Sagaram et al. 2009; Tyler et al. 2009). The total bacterial population in the phloem tissues of mature citrus trees is approximately 10 to 100 times lower than the mixed bacterial population in the human gut (Sender et al. 2016). This is reasonable because of the small size of sieve elements, despite the large size of citrus trees and the systemic distribution of CLas throughout the tree. The reported diameters of phloem sieve elements are on the order of hundreds of micrometers (Jensen et al. 2016). Here we estimate that 0.01% of the total citrus phloem volume is occupied by CLas, but this could be up to 1% during CLas peak seasons. This difference could explain the low probability of observing CLas in most tissues under transmission electron microscopy. In our study and other studies, CLas is rarely observed in plant tissues except in the seed coat (Achor et al. 2020; Kim et al. 2009).
Our estimates of CLas titer inside the leaf are similar to previous studies by Teixeira et al. (2008) and Trivedi et al. (2009), but 103 to 105 times lower than that reported by Li et al. (2006). Based on our quantification, the ratio for citrus cells to CLas cell is approximately 44 in the leaf. However, approximately 100 CLas cells per leaf cell were estimated based on Li et al. (2006). We infer that estimates of CLas titers in the leaf in this study and by Teixeira et al. (2008) and Trivedi et al. (2009) are consistent with microscopic analyses (Kim et al. 2009), whereas that by Li et al. (2006) was probably overestimated.
The ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples were quantified using two different methods: qPCR-based method and cell-weight–based method. Intriguingly, the ratios of citrus cells to CLas cells in different tissues calculated based on cell weight were 2.0- to 8.8-fold higher than those based on qPCR, but within an order of 10. It remains unknown what factors are responsible for the different results of the two methods. It is probable that the ratios of citrus cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are within 39 to 79, 44 to 124, 153 to 1,355, 191 to 1,054, and 561 to 3,760, respectively. It was reported that phloem cells make up approximately 1% of the total cell population in a plant (Kaur et al. 2018; Knoblauch and Oparka 2012). Consequently, we can estimate that the ratios of phloem cells to CLas cells for branch, leaf, trunk, feeder root, and structural root samples are approximately 0.39 to 0.79, 0.44 to 1.24, 1.53 to 13.55, 1.91 to 10.54, and 5.61 to 37.60, respectively. This estimate is consistent with microscopic observations (Kim et al. 2009). However, this estimate is based on an average, and the ratios in individual tissues are complicated by the uneven distribution of CLas cells in planta (Tatineni et al. 2008) and different stages of CLas infection.
The CLas population inside the leaf is estimated to be approximately 1.64 × 106 cells per leaf or 9.2 × 104 cells cm–2 in September 2019. Interestingly, the populations of many apoplastic bacterial pathogens, such as Xanthomonas citri and Pseudomonas syringae, are commonly detected in the order of 109 cells cm–2 (Guo et al. 2010; Teper et al. 2019; Stavrinides et al. 2009). Hence, the ratio for apoplastic bacterial pathogens to citrus cells is approximately 104 times greater than that of CLas to citrus cells. This drastic difference in bacterial titers between phloem-colonizing CLas and apoplastic bacterial pathogens probably contributes to the different symptoms caused by those two types of pathogens in addition to that caused by different virulence factors. The high titer of apoplastic bacterial pathogens poses an intolerable metabolic burden for neighboring plant host cells to support, contributing to the development of necrotic lesions that are commonly associated with those intercellular pathogens. The low CLas titers in leaves are unlikely to pose an unbearable metabolic burden for host cells. In some rare cases, however, high titers of CLas were observed in aborted seeds (Achor et al. 2020), in which the metabolic burden by CLas probably contributes to the abnormality. The lower titer of CLas in planta than the apoplastic bacteria probably results from the longer generation time than its counterparts. The generation time for CLas and its close relative Liberibacter crescens was estimated to be 118 h (Fujiwara et al. 2018), and 11 to 14 h (Sena-Vélez et al. 2019), respectively, whereas the doubling time for apoplastic bacterial pathogens is usually within 2 h. It takes several months to several years to show HLB symptoms after CLas infection (Gottwald, 2010; Pandey and Wang 2019; Pandey et al. 2020; Wang et al. 2017b), whereas it usually takes several days for apoplastic bacterial pathogens to cause symptoms.
In summary, we calculated the total CLas population size in the phloem-containing tissues of different organs and whole citrus trees and estimated the ratio of citrus cells to CLas cells in different tissues. CLas was differentially distributed among organs with higher populations in aboveground tissues, and the majority of bacteria were found in the leaves and small branches. This study provides useful information regarding phloem colonization by bacterial pathogens and contributes to further understanding of the biology of CLas.
We thank Nicolle Collins and Myrtho Pierre for technical support, and Erica W. Carter for critical reading of this manuscript.
The author(s) declare no conflict of interest.
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The author(s) declare no conflict of interest.
Funding: This research was supported by the U.S. Department of Agriculture’s National Institute of Food and Agriculture under grants 2018-70016-27412, 2016-70016-24833, and 2019-70016-29796; the Florida Citrus Initiative; and the Florida Citrus Research and Development Foundation.