Growth of ‘Candidatus Liberibacter asiaticus’ in Commercial Grapefruit Juice-Based Media Formulations Reveals Common Cell Density-Dependent Transient Behaviors
- Marcus V. Merfa
- Eber Naranjo
- Deepak Shantharaj
- Leonardo De La Fuente †
- Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849
The phloem-restricted, insect-transmitted bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. The inability to culture CLas impairs the understanding of its virulence mechanisms and the development of effective management strategies to control this incurable disease. Previously, our research group used commercial grapefruit juice (GJ) to prolong the viability of CLas in vitro. In the present study, GJ was amended with a wide range of compounds and incubated under different conditions to optimize CLas growth. Remarkably, results showed that CLas growth ratios were inversely proportional to the initial inoculum concentration. This correlation is probably regulated by a cell density-dependent mechanism, because diluting samples between subcultures allowed CLas to resume growth. Moreover, strategies to reduce the cell density of CLas, such as subculturing at short intervals and incubating samples under flow conditions, allowed this bacterium to multiply and reach maximum growth as early as 3 days after inoculation, although no sustained exponential growth was observed under any tested condition. Unfortunately, cultures were only transient, because CLas lost viability over time; nevertheless, we obtained populations of about 105 genome equivalents/ml repeatedly. Finally, we established an ex vivo system to grow CLas within periwinkle calli that could be used to propagate bacterial inoculum in the lab. In this study we determined the influence of a comprehensive set of conditions and compounds on CLas growth in culture. We hope our results will help guide future efforts toward the long-sought goal of culturing CLas axenically.
The ability to grow bacteria in pure culture has been the foundation of bacteriology, enabling a wide range of studies elucidating their biology (Austin 2017; Lagier et al. 2015). However, most bacterial species have not been successfully established in axenic in vitro cultures (Zengler et al. 2002), hindering the assessment of their characteristics and elucidation of their role in the environment or within their hosts. One relevant hitherto unculturable group of emergent plant pathogenic bacteria is comprised by ‘Candidatus Liberibacter spp.’, which are psyllid-transmitted, phloem-limited, fastidious gram-negative bacteria of the α subdivision of Proteobacteria (Jagoueix et al. 1994; Killiny 2022; Wang and Trivedi 2013). In addition, they are plant pathogens and endophytes that harm a wide range of economically important crops worldwide, including citrus, potato, tomato, carrot, and pear (Bové 2014b; Merfa et al. 2019; Nelson et al. 2012; Thompson et al. 2013).
The citrus huanglongbing (HLB) disease (also known as citrus greening) is currently the most single devastating disease of citrus worldwide, leading to significant economic losses in the Americas, Asia, and Africa (Bové 2014b; Gottwald 2010). The disease is characterized by reductions in yield and tree lifespan and lopsided, bitter fruits (Bové 2006; Wang and Trivedi 2013). In addition, although an ultimate solution to control HLB in the field is yet to be developed (Bové 2006; Wang and Trivedi 2013), substantial progress has recently been made by different studies toward this goal. These include the identification of a stable antimicrobial peptide that both treated and prevented HLB in greenhouse conditions (Huang et al. 2021), a region-wide implementation of roguing of infected trees followed by tree replacement that significantly reduced the incidence of HLB in the Gannan region of China (Yuan et al. 2021), application of streptomycin through trunk injection to reduce the in planta population of the HLB-associated bacterium (Li et al. 2021), assessment of the use of thermotherapy to treat infected trees (Thapa et al. 2021), and the development of an assay for early detection of the HLB-associated bacteria in asymptomatic tissues to aid the management of the disease (Wheatley et al. 2021). In the United States, and in most of the major citrus producing areas globally, HLB is associated with ‘Ca. Liberibacter asiaticus’ (CLas). Nonetheless, different ‘Ca. Liberibacter’ species (‘Ca. L. americanus’ and ‘Ca. L. africanus’) are also associated with the disease in other parts of the world (Bové 2006, 2014b).
The recalcitrance of CLas to grow axenically has greatly impaired our ability to understand important characteristics such as virulence mechanisms, host–pathogen interactions, and even fulfillment of Koch’s postulates for HLB (Bové 2006; Merfa et al. 2019; Wang and Trivedi 2013). These limitations hamper the development of efficient management approaches to control this disease. Because having CLas grown in laboratory conditions is fundamental to advance its research, in the past decade several efforts have been made to culture this bacterium. Even though a reliable and reproducible method to culture CLas in axenic conditions is yet to be developed, these studies have significantly advanced our knowledge about the nutrient and environmental requirements of CLas. Research approaches have included formulating culture media to mimic the natural environments where CLas lives (Parker et al. 2014; Sechler et al. 2009), coculturing it with one or more bacterial species (Davis et al. 2008; Fujiwara et al. 2018; Ha et al. 2019) and using explants of CLas-infected plant tissues to establish ex vivo systems to grow this bacterium (Attaran et al. 2020; Irigoyen et al. 2020). Data suggest that CLas is able to grow in conditions close to its natural environments, and it may also obtain additional nutrients or chemical signals by establishing mutualistic relationships with other cohabiting bacteria. However, these approaches do not allow CLas to be studied directly due to either the nature of mixed cultures, transient growth of CLas, low number of CLas cells in comparison with the overall population, or growth within plant tissues. Thus, further investigation is needed to elucidate factors that are necessary to reach the long-sought goal of obtaining pure cultures of CLas.
Previously, our research group established transient cultures of CLas with other microflora by using commercial grapefruit juice (GJ) as culture medium (Parker et al. 2014). Therefore, in this study we aimed at amending GJ by using compounds with potential to help CLas growth based on the current literature (Merfa et al. 2019), in addition to testing different incubation settings to define optimized growth conditions for our system. Compounds and conditions that were tested were chosen based on studies performed with the surrogate bacterium Liberibacter crescens, the only culturable species of the genus Liberibacter (Cruz-Munoz et al. 2019; Fagen et al. 2014b; Jain et al. 2017a) and CLas itself (Duan et al. 2009; Vahling et al. 2010; Wang and Trivedi 2013) and considering the composition of the original GJ (Parker et al. 2014). We were able to repeatedly establish transient cultures of CLas, which survived sequential subcultures. However, CLas growth was inversely proportional to the concentration of cells present in the initial inoculum, because higher growth ratios were obtained when the initial inoculum had fewer cells. Unfortunately, most tested compounds and conditions did not improve CLas growth in our system.
On the other hand, the use of surrogate organisms is a valuable tool that advances our knowledge about fastidious bacteria. In this context, periwinkle (Catharanthus roseus) has been used as a surrogate host to study the HLB pathosystem because it carries a higher population of CLas in comparison with citrus plants and produces HLB-like symptoms in a short period of time (Bové 2014a). Studies with this plant host include determining the distribution of CLas in planta (Ding et al. 2015; Li et al. 2018), screening of antimicrobial compounds (Zhang et al. 2010), visualization of CLas-associated phage particles within the phloem (Zhang et al. 2011), analysis of the nutritional requirements of CLas by evaluating the phloem sap composition (Killiny 2016), and assessment of the plant response against CLas by performing transcriptome profiling of infected plants (Liu et al. 2019).Therefore, we established an ex vivo system to grow CLas in calli produced from infected periwinkle leaves to propagate inoculum of CLas in laboratory conditions. Overall, we believe our results demonstrate a set of growth conditions and nutrient supplements, as well as the establishment of an ex vivo system that could increase the axenic growth of CLas.
MATERIALS AND METHODS
Bacterial strains and growth conditions.
CLas-infected material was used as initial inoculum in all assays performed in this study. Inoculum was obtained from either CLas-infected sweet orange seeds, periwinkle plants, or excised Asian citrus psyllid (ACP; Diaphorina citri) guts. Growth assessment was performed in broth media containing grapefruit juice (Simply Grapefruit; Simply Orange Juice Company, Apopka, FL) as the base culture medium. L. crescens strain BT-1 was grown on BM7 agar plates at 28°C (Fagen et al. 2014a). L. crescens cryopreserved stocks (BM7 with 20% glycerol, −80°C) were plated on BM7 and grown for 7 days. Then, cultures were restreaked on new BM7 agar plates for a second passage and grown for an additional 7 days before the start of the experiments. Propionibacterium acnes was grown on Reinforced Clostridial Agar plates (HiMedia, Mumbai, India) at 28°C in an anaerobic chamber for 4 days before use. Escherichia coli bearing the pLas16S plasmid, which is used as standard to quantify the CLas population by quantitative PCR (qPCR) (Parker et al. 2014), was grown overnight at 37°C on Luria-Bertani medium (Becton, Dickinson and Company, Sparks, MD) amended with kanamycin at a final concentration of 50 µg/ml.
CLas inoculum preparation.
Sweet orange fruits (Citrus sinensis [L.] Osbeck ‘Hamlin’ and ‘Valencia’) were collected from HLB-symptomatic trees in a grove located in Vero Beach, Florida (June 2017 to October 2020) and shipped overnight to Auburn University, Alabama. Experiments started about 24 h after sample collection; however, fruits were stored at 4°C in a cold chamber for up to a month and used regularly in assays. No clear differences in CLas growth behavior were observed when fruits were used at time of arrival or after storage at 4°C (data not shown). Symptomatic, greenish lopsided fruits were used to obtain CLas inoculum. We surface sterilized fruits by submerging them in 1.2% sodium hypochlorite solution for 1 min, followed by 70% ethanol solution for 1 min and two subsequent wash steps with sterile deionized water for 1 min each. Seeds were collected aseptically with sterile tweezers and scalpel and weighed. About 200 mg of seed material was used in each assay, and aborted seeds were preferentially collected. When there were not enough aborted seeds, only the seed coat of normally developed seeds was collected, because CLas is more abundant in this tissue in citrus seeds (Achor et al. 2020; Hilf 2011). The seed material was thoroughly ground with a sterile mortar and pestle in 5 ml of broth media and suspended to a final volume of 10 ml. The suspension was then vortexed for 1 min, filtered through a 100-µm nylon net Steriflip Filter Unit (EMD Millipore, Billerica, MA) to remove large seed particles, and used as CLas inoculum.
CLas-infected periwinkle plants (surrogate host) were shipped overnight to Auburn University from the U.S. Department of Agriculture (Fort Piece, FL) and kept in greenhouse conditions (day/night temperatures of 28/22°C, 60 to 80% relative humidity and seasonal day/night photoperiod) at the Plant Science Research Center (Auburn University, Auburn, AL). To propagate infected plants, healthy periwinkle seeds (Catharanthus roseus Pacifica XP White; Harris Seeds, Rochester, NY) were germinated in 1206 inserts (Grower’s Solution, LLC, Cookeville, TN) with Sunshine Mix #8 (Sun Gro Horticulture Canada Ltd., Vancouver, Canada), then transferred to 8-inch round pots and regularly fertilized with Osmocote 19-6-12 (The Scotts Company, Marysville, OH) as needed. After about 1 month of transplant, healthy plants were pruned and grafted with branches of CLas-infected periwinkle plants (one branch per healthy plant). Grafted plants were bagged for 7 days to retain humidity and then grown until symptoms appeared. Only blotchy mottled leaves were used for inoculum preparation in each assay. Periwinkle leaves were surface sterilized as described previously for citrus fruits, and the petioles and midribs were collected aseptically with tweezers and scalpel. Leaf material (about 200 mg) was thoroughly ground in 5 ml of broth media with a sterile mortar and pestle and suspended to a final volume of 10 ml. Then, the suspension was vortexed for 1 min, filtered through a 100-µm nylon net Steriflip Filter Unit (EMD Millipore, Billerica, MA) to remove large leaf particles and finally used as CLas inoculum.
Psyllids (D. citri Kuwayama) were reared on CLas-infected citrus plants in the Citrus Research and Education Center (University of Florida, Lake Alfred, FL) and shipped overnight to Auburn University. Experiments started about 24 h after psyllids collection, and 20 to 40 ACPs were used in each assay. Before inoculum preparation was started, the insects were inactivated temporarily by incubation at −20°C for 1 min. Then, we transferred ACPs to a 1.5-ml tube on ice and surface sterilized them by adding 1 ml of ice-cold 70% ethanol and vigorously inverting the tube for few seconds. Next, the ethanol was discarded, and we washed psyllids 20 times with 1 ml of ice-cold sterile deionized water by vigorously inverting the tube for few seconds. The insects were then individually placed on 10-µl drops of sterile deionized water on a Petri dish lid and dissected with the aid of an Olympus SZ3060 stereo microscope (Olympus, Tokyo, Japan) with a pair of surface sterilized Dumont tweezers 4 and 5 (Electron Microscopy Sciences, Hatfield, PA) to recover their guts. Guts were placed in 1 ml of broth media and vortexed twice for 1 min to release CLas cells. Finally, the inoculum was briefly centrifuged at 5,000 rpm for 2 s to pellet guts, and only the supernatant was used in experiments.
All inocula used in this study were tested by qPCR to determine the presence and total population of CLas at the beginning and throughout development of assays.
Commercially available GJ was used as the base culture medium throughout all assays described here (Parker et al. 2014), purchased at different times during the study. Before being used as culture medium, GJ was centrifuged at 20,000 rpm for 30 min to pellet solid particles, filtered with a 0.45-µm polyethersulfone (PES) membrane (Pall Corporation, Port Washington, NY), and then filter sterilized with a 0.2-µm PES filter Unit (VWR, Radnor, PA). This processed GJ was stored at −20°C as 45-ml aliquots and thawed at room temperature before use. When needed, the pH of GJ was adjusted to 5.85 with 5 M NaOH (the regular pH of the processed GJ is about 3.2 to 3.5). In addition, solid GJ plates were prepared by autoclaving Gelrite (10 g/liter; Research Products International Corp., Mt. Prospect, IL) in deionized water and supplementing with sterile GJ to a final concentration of 50%. The autoclaved Gelrite and GJ were brought to 40°C before mixing.
The most common amendments used here to modify GJ broth medium were α-ketoglutaric acid (2 g/liter), L-asparagine (1 g/liter), L-histidine (0.05 g/liter), monosodium phosphate monohydrate (1.5 g/liter), and cycloheximide (200 µg/ml), with pH adjusted to 5.85. This culture medium was hereafter named modified grapefruit juice (mGJ). We chose to add these compounds to GJ based on studies about the nutritional requirements of CLas and Liberibacter crescens and considering the composition of GJ itself (Cruz-Munoz et al. 2019; Duan et al. 2009; Fagen et al. 2014a; Parker et al. 2014; Wang and Trivedi 2013). Cycloheximide was added to mitigate eukaryotic (mainly fungal) contamination, observed mostly when ACP guts were used as source of CLas inoculum. Other growth conditions and compounds added to GJ or mGJ, as well as the concentrations used and rationale for testing them, are reported throughout this study (Table 1). All compounds mentioned here were dissolved directly in GJ, and the broth medium was once again filter sterilized with a 0.2-µm PES filter unit (VWR, Radnor, PA).
CLas culturing conditions.
To assess CLas growth under different media formulations and growth conditions, we obtained CLas suspensions from the different inoculum sources and inoculated them (1.8 ml of broth media + 200 µl of CLas inoculum) into GJ, mGJ, or other amended GJ formulations in polystyrene 24-well plates (VWR, Radnor, PA) as 10-fold serial dilutions. Plates were incubated at 28°C for 21 days in the dark without agitation. Then, samples were subcultured into fresh broth medium, and 200-µl aliquots from each sample were collected to assess the total CLas population by qPCR. The optical density at 600 nm of cultures was also measured at initial and final timepoints of assays with a plate reader (Cytation 3 Image Reader spectrophotometer; Biotek Instruments Inc., Winooski, VT). However, data from these readings were not informative about CLas growth, because the numbers of cells were usually below the detection limit, which also indicates a lack of contamination with fast-growing microflora (data not shown). Thus, only qPCR was used to evaluate CLas growth over time in the various assessed culturing conditions. Three or four subcultures were performed in each assay at 21-day intervals. In all experiments conducted in 24-well plates, samples were diluted by 10-fold serial dilutions with broth media to 10−4. All dilutions, including the concentrated suspension, were inoculated into 24-well plates to assess CLas growth in each tested condition. During the time series assays, we regularly inspected plates for visual presence of cocultured bacteria (non-CLas) by assessing any sudden increase in the turbidity of the medium. These were presumed to be contaminating fast-growing bacteria.
CLas growth was also evaluated in a condition that allowed constant broth medium replenishment over time by incubating cells in a flow system (Supplementary Fig. S1). For that, broth culture medium was put in a 1-ml plastic syringe (Becton, Dickinson and Company, Franklin Lakes, NJ) while 1 ml of CLas inoculum was placed in a 5-ml plastic syringe barrel (Henke Sass Wolf, Tuttlingen, Germany), which served as a collection tube and was capped with autoclaved cotton plugs. Both syringes were connected to each other with a polytetrafluoroethylene tube with a nominal inside diameter of 0.022 inches (Weico Wire & Cable, Inc., Edgewood, NY). The loaded 1-ml syringes were then inserted into an automated syringe pump (11 Plus; Harvard Apparatus, Holliston, MA), and the broth medium was supplied at a constant flow rate of 0.25 µl/min. During the experiments, broth culture medium was replenished in 1-ml syringes as needed (usually every 3 days). Samples were incubated for 12 days at room temperature, and 200-µl aliquots were taken every 3 days to assess CLas population by qPCR. Finally, in experiments that required incubation of cells at a constant temperature, samples were placed inside a Styrofoam box filled with deionized water in which the temperature was kept at 28°C by a 50-W aquarium heater (Aqueon, Franklin, WI).
Subculturing at 3-day intervals.
To evaluate whether CLas could grow in a shorter period than 21 days, subculturing of samples was performed at 3-day intervals, as described previously for assays in 24-well plates. Accordingly, samples were incubated in 24-well plates at 28°C for a total of 15 days, and at every 3 days subcultures were performed and 200-µl aliquots were taken to assess CLas population by qPCR. The CLas population was assessed in each subculture only after the 3-day incubation period and not throughout the entire 15 days. Inocula used in these assays were taken from CLas samples previously incubated in 24-well plates for 21 days. Only samples that had previously presented growth were used to perform assays with subculturing at 3-day intervals.
Solid GJ plates.
CLas samples were plated in solid GJ plates to assess for colony or microcolony growth. For this purpose, 100 µl of CLas inocula obtained from citrus seeds or periwinkle plants was spread on GJ Gelrite plates and incubated at 28°C. Growth of bacteria was visually assessed at weekly intervals for the presence of colony-forming units. At the final timepoint of assays (14, 21, and 28 days after incubation), we harvested cells by scrapping the medium with 500 µl of sterile GJ and transferring them to 2-ml microcentrifuge tubes. CLas growth was then analyzed by qPCR.
Periwinkle callus culture.
Induction and propagation of periwinkle calli were performed according to well-established protocols (Pietrosiuk et al. 2007; Singh 2011). In summary, leaves from CLas-infected periwinkle plants were collected and surface sterilized as described perviously. Then, cross-sections of leaves were taken with a sterile blade and transferred to culture media for callus induction and propagation. Three different culture media that differ in ammonium and nitrate concentration ratios were tested: Murashige & Skoog basal medium (Murashige and Skoog 1962), Gamborg’s basal medium (Gamborg et al. 1968) and Nitsch & Nitsch (Nitsch and Nitsch 1969). Basal media were supplemented with Gamborg’s B5 vitamins, indole-3-acetic acid (1 mg/ml), benzyl adenine (0.1 mg/ml), sucrose (30 g/liter), and 0.8% phytagel (chemicals were purchased from PhytoTech Labs, Lenexa, KS). Inoculated plates (containing about 10 explants each) were incubated at room temperature and humidity conditions under a 12:12 h (light/dark) photoperiod with fluorescent tube light (intensity of light about 200 lux). Plates were placed about 1.5 m away from the light source. Controls were prepared with healthy periwinkle leaves. Periwinkle cell proliferation and callus formation were monitored weekly for 6 weeks from the initial date after the explant transfer to the culture media and eventually subcultured by being transferred to new culture medium plates. When contamination was present, noncontaminated explants were transferred to new plates, and contaminated samples were discarded. At select timepoints, as well as in the source plant material that contains the initial inoculum, we determined the total CLas population by extracting DNA from about 200 mg of callus material and submitting samples to qPCR. The presence of CLas within samples was also analyzed by immune tissue printing (Ding et al. 2017a).
CLas detection within periwinkle calli through immune tissue printing.
Detection of CLas via immune tissue printing was performed as described elsewhere (Ding et al. 2017a). Briefly, 5- to 6-week-old periwinkle calli obtained as described previously, including calli obtained from both CLas-infected and healthy leaves, were cut aseptically into slices and imprinted onto activated 0.2-µm polyvinylidene difluoride membranes (Bio-Rad Laboratories Inc., Hercules, CA; protein binding capacity of 150 to 160 µg/cm2) until dried. Membranes were processed for immunodetection at 37°C with a reciprocal shaker (80 rpm) incubator (Thermo Scientific, MaxQ 6000, Swedesboro, NJ). Initially, membranes were reactivated in 95% methanol for 1 min and transferred to phosphate-buffered saline + 0.05% Tween-20 (PBST) at room temperature and washed twice (5 min each). Later, membranes were blocked in PBST + 5% fat-free skim milk (SACO Foods Inc., Middleton, WI) for 1 h. Membranes were then treated with rabbit anti-OmpA primary antibody (Abnova, Taipei City, Taiwan) (1:500 dilution in PBST), which detects the outer membrane protein OmpA of CLas (Ding et al. 2015), by overnight incubation, washed three times with PBST (10 min each), treated with goat anti-rabbit secondary antibody coupled to horseradish peroxidase (Promega Corporation, Madison, WI) at a 1:10,000 dilution in PBST for 1 h, and washed again three times with PBST (10 min each). Finally, membranes were developed with 1 ml of 3,3′,5,5′-tetramethylbenzidine substrate (Agdia, Elkhart, IN) added at room temperature. Incubation was stopped when the blue-purple color developed on the prints by washing in sterile deionized water. In addition, total proteins in each sample were visualized by membrane staining with Ponceau S (0.1% wt/vol in 0.5% acetic acid) (Amresco, Solon, OH) for 5 min and destaining in 0.5% acetic acid.
DNA from all samples was extracted via a modified cetyltrimethylammonium bromide procedure (Doyle and Doyle 1987). Then, qPCR was performed via the previously described HLBas/HLBr/HLBp pair of primers and TaqMan probe set (Li et al. 2006) to quantify CLas population in each sample as genome equivalents. DNA amplifications were performed in 20-µl reactions containing 1× ABsolute Blue QPCR ROX Mix (Thermo Scientific, Waltham, MA), 250 nM of each primer, 150 nM of the probe (labeled 5′-6FAM, 3′-BHQ1), and 1 µl of DNA template, and carried out with a C1000 thermal cycler base with a CFX96 real-time system (Bio-Rad Laboratories Inc., Hercules, CA). Each DNA amplification was repeated in triplicate. Cycling parameters were the same as described by Parker et al. (2014). Reaction efficiencies of 90 to 105% were confirmed for each qPCR run. The number of CLas genome equivalents in each sample was calculated by coamplifying a four-point standard curve (Supplementary Fig. S2) made from 10-fold serial dilutions of the pLas16S plasmid (Parker et al. 2014). Aliquots of the standard curve were used once, then discarded. Samples with a threshold cycle (CT) value <40 were considered CLas-positive (considering that our cycling parameters had a total of 44 amplification cycles), which corresponded to ≥12 genome equivalents of CLas according to the standard curve (Supplementary Fig. S2). DNA extracted from noninoculated sterile GJ (or the corresponding used medium formulation) was included as negative control in our qPCR runs. If amplification was observed in these samples, their CT values were considered as cutoff value for positive samples, meaning that any sample with a CT value close (with less than one cycle difference) or above that of the noninoculated GJ was considered negative and treated as CLas-negative.
L. crescens qPCR analysis was performed with the Lcr-F/Lcr-R/Lcr-P pair of primers and TaqMan probe set as described elsewhere (Naranjo et al. 2020). In summary, DNA amplifications were carried out in 20-µl reactions containing 1× PerfeCTa qPCR FastMix II Low ROX (QuantaBio, Beverly, MA), 250 nM of each primer, 150 nM of the probe (labeled 5′-6FAM, 3′-BHQ1), and 1 μl of DNA template. Each DNA amplification was repeated in triplicate, and cycling parameters were 95°C for 2 min, followed by 39 cycles of 95°C for 5 s and 60°C for 30 s. Reaction efficiencies of 90 to 105% were confirmed for each qPCR run. We calculated the number of Liberibacter crescens genome equivalents in each sample by coamplifying a five-point standard curve made from 10-fold serial dilutions of the Liberibacter crescens genomic DNA. Aliquots of standard curves were used once before discarding.
The overall trend of CLas cell population in the different experimental conditions evaluated in this study was analyzed by two-tailed Student’s t test, except when cells were incubated via the flow system. The CLas population obtained after the incubation period, calculated as genome equivalents, was always compared with the population in the initial inoculum to determine whether there was significant growth or death of cells. The growth ratio was also determined, when possible. This was calculated as the ratio of genome equivalents obtained after the incubation period by the number of genome equivalents in the initial inoculum of each sample. For assays performed in flow conditions and for the CLas growth ex vivo in the periwinkle callus system, the CLas population in the different timepoints of evaluation and in the different analyzed media was compared by one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference multiple comparisons of means in R 4.0.0 with the package multComp (Ihaka and Gentleman 1996). Because of the variable nature of CLas growth observed in the assays described here and the variation in CLas population in the initial inocula, which was influenced by the source material and time of the year collected, representative results from independent experiments are shown. In other words, all results included here are from a single experiment selected from at least two independent experiments (Supplementary Table S1) that showed the same trend. Variation in population numbers between experiments precluded the consideration of data from multiple experiments for statistical analysis.
CLas reaches higher growth ratios in GJ when starting from low cell numbers, a behavior repeated among subcultures.
To reproduce our previous findings and establish transient cultures of CLas (Parker et al. 2014), samples were inoculated into GJ and incubated at 28°C, and CLas growth was monitored by qPCR. However, instead of growing cells in Erlenmeyer flasks and evaluating growth every other day, as done in the past (Parker et al. 2014), in this study we diluted samples by 10-fold serial dilutions, incubated them in 24-well plates, and assessed growth after 21 days of inoculation. As described previously, different sources of CLas inoculum were used throughout this study (citrus seeds, periwinkle leaves, and ACP guts), but no clear differences in CLas growth behavior were observed among these sources (data not shown). Thus, the source of inoculum used in each assay will not be mentioned here. Nonetheless, the number of CLas cells in citrus seeds used as inoculum source to perform the assays described in this study was quantified to assess the distribution of its population throughout the years. No obvious annual trend was observed for the CLas population within citrus seeds; however, its concentration was higher in 2018 (Supplementary Fig. S3), although not statistically significant. When periwinkle leaves and ACP guts were used as the inoculum source, the CLas concentration was more uniform (usually about 106 cells/ml), possibly because these samples were produced under controlled conditions (data not shown).
Curiously, when CLas was grown in GJ, results showed that decreasing the initial inoculum by 10-fold serial dilution was associated with a significant increase in CLas growth (Fig. 1). In other words, CLas did not grow in GJ when its titer was high in the initial inoculum (>105 genome equivalents/ml), whereas decreasing the initial number of cells by performing 10-fold serial dilutions allowed samples to grow. This association was observed consistently in most of the various experiments (Supplementary Table S1) performed in this study (about 88% out of 17 independent experiments that were performed, including different medium formulations) and suggests that the ability of CLas to grow is affected by its initial population, at least in GJ and related media tested here.
Next, to evaluate whether CLas could survive passages in fresh culture medium and to determine its growth behavior, samples were incubated as described previously, but three subcultures at 21-day intervals each were performed (Fig. 2). In this assay, mGJ, which contains compounds selected for their potential aid for the in vitro growth of CLas, was used as broth medium. In the first inoculated plate, similarly to the previous result, CLas numbers increased only when the initial inoculum was diluted until 10−4 (about 103 genome equivalents/ml) (Fig. 2A), although not significantly (P = 0.15). However, when samples were diluted for the first subculture, all presented significant growth after the 3-week incubation period (Fig. 2B), and an additional subculture led to a significant decrease in CLas population in almost every sample (Fig. 2C). Finally, no CLas was detected at the final timepoint of the last subculture (Fig. 2D). This result was also consistent among independent replicates and indicates a highly repeatable transient growth behavior of CLas in which cells grow better starting from a low inoculum concentration, and although the first subculture seems to support CLas growth, further subculturing attempts do not improve or even impair the in vitro viability of this bacterium over time. Interestingly, some samples presented CLas growth even though bacterial titers were too low to be detected in the initial inoculum by qPCR (Fig. 2B). We stress that this finding was a common trend among independent replicates. It is worth noting that in both results presented previously (Figs. 1 and 2) there was no visible growth of fast-growing cocultured bacteria with CLas, as analyzed by turbidity. Nonetheless, a few of the replicates with similar CLas growth trend results did show contamination with fast-growing bacteria (data not shown).
CLas shows quick but limited growth when incubated in a flow system.
Because we obtained consistent growth behavior of CLas by diluting samples between subcultures, we attempted to grow this bacterium in flow conditions with a constant increase of culture volume. We designed a system that allows constant broth culture medium replenishment in CLas cultures for 12 days by introducing inocula to a reservoir that is continually supplied with medium via a syringe pump (Supplementary Fig. S1). This experiment was carried out to minimize the density of CLas while seeking to maximize its growth. Although results were somewhat variable, in most assays CLas presented a significant increase in bacterial cell numbers as early as 3 and 9 days postinoculation (dpi) in mGJ (Fig. 3). On the other hand, this bacterium usually reached nondetectable levels after 12 days of incubation, evidencing that this quick increase in CLas population is not sustainable for prolonged periods in this system. Noteworthy is that an oscillating behavior of growth was evidenced here as well, similar to our results in 24-well plates subcultures and our previous study (Parker et al. 2014). Likewise, as in our previous results in 24-well plates, there was no visual presence of fast-growing cocultured bacteria with CLas in the flow system results shown here (Fig. 3). When cocultured bacteria were present in this system, they were mostly harmful to CLas and decreased its population (data not shown).
CLas also presents a rapid but limited growth when subcultured at shorter intervals.
Because of the growth behavior of CLas in 24-well plates and in flow conditions that indicated a quick, limited growth when cultures initially had a low density of cells, CLas was inoculated into mGJ and grown in 24-well plates as previously described; however, subcultures were performed at 3-day intervals instead of 21 days. Similar to what was observed when we grew CLas in flow conditions, not all results were consistent between replicates. However, CLas presented an overall significant increase in cell numbers in the first subculture of most assays (Fig. 4). This demonstrates an ability to grow quickly within this system. However, CLas usually could not be detected in subcultures beyond the first subculture (Fig. 4). Possibly, subcultures beyond this point dilute the CLas inoculum to a level that is no longer detectable by qPCR because subcultures were done as 1/10 inoculations. The rapid increase in CLas population within 3 days was similar to what was observed when samples were incubated under flow conditions. It is worth noting that no visual presence of fast-growing cocultured bacteria was detected in any of the experiments conducted.
CLas does not grow on solid grapefruit juice culture medium.
CLas inocula were plated on solid GJ plates to evaluate the formation of colonies or microcolonies, either with or without cocultured bacteria. Culture medium was composed of 50% GJ + 50% autoclaved Gelrite aqueous solution. The concentration of GJ was chosen based on our previous results showing that CLas establishes transient cultures in 50% GJ broth (Parker et al. 2014). Plates were incubated at 28°C, and the total CLas population was determined weekly at 14, 21, and 28 dpi by qPCR. CLas presented neither an increase in genome equivalents nor formation of colonies or microcolonies in inoculated plates (Fig. 5). Instead, the CLas population declined progressively throughout the experiment, with no detection at the final two points of evaluation (21 and 28 dpi). This demonstrates an inability of CLas to grow when incubated in solid medium GJ Gelrite plates. Moreover, no growth of cocultured fast-growing bacteria was observed in the analyzed plates.
Assessing the contribution of other compounds and incubation conditions to CLas growth.
A wide range of additional compounds and incubation conditions, in addition to the results already presented here, were assessed to determine their contribution to CLas growth in our GJ system. These additional CLas culturing assays were performed in attempts to overcome the growth limitations we observed in our experiments. Changes to GJ and rationale to choose compounds and incubation conditions, as well as our main findings, are summarized in Table 1. Overall, adjusting the pH of GJ to 5.85, incubating cells in a nonshaking condition, and growing CLas at a constant temperature of 28°C in flow conditions increased CLas growth. Conversely, amending GJ or mGJ with adenosine triphosphate (ATP), Wolbachia repressor protein, or TNM-FH insect medium, among other compounds, as well as coculturing CLas with L. crescens or P. acnes, did not show a contribution to its growth, whereas incubating CLas at 37°C and coculturing it with SF9 insect cell line reduced its growth by decreasing the population of CLas to nondetectable levels by qPCR.
CLas presents substantial growth within periwinkle calli.
Looking to obtain an alternative approach to propagate inoculum of CLas in laboratory conditions, we developed a system to grow this bacterium ex vivo within calli from infected periwinkle leaves. In this experiment, explants from CLas-infected periwinkle leaves were inoculated into different culture media (Nitsch & Nitsch, Murashige & Skoog, and Gamborg’s media) to assess callus formation and propagation (Fig. 6A) as well as CLas growth. Analyses with 5-week-old calli demonstrated that CLas presented significant growth in periwinkle calli cultured in Murashige & Skoog and Gamborg’s media, with a growth ratio as high as 3,416-fold when calli were grown in Gamborg’s medium (Fig. 6B). Moreover, CLas also presented growth in 6-week-old calli that were subcultured from a previous assay (Fig. 6C). In this assay, significant growth was observed only in samples inoculated into Nitsch & Nitsch and Gamborg’s medium (Fig. 6C). Presence of CLas within periwinkle calli was also analyzed by immune tissue printing (Fig. 7), which confirmed its detection within infected calli (Fig. 7A) and absence in healthy calli (Fig. 7B).
Strategies to culture bacteria depend on the unique characteristics of each species (Overmann et al. 2017). This dependence is even more relevant when considering intracellular fastidious bacteria. Approaches to grow such organisms in axenic conditions may include mimicking their habitats and determining specific growth conditions by analyzing necessary nutrients, temperature, oxygen levels, period of incubation, use of reducing agents, need for signaling molecules, and coculture with one or more bacterial species that may provide metabolic goods to the community (Lagier et al. 2015; Overmann et al. 2017). CLas is an obligate intracellular pathogen of citrus plants with a reduced genome, lacking several enzymes and biosynthetic pathways (Duan et al. 2009; Merfa et al. 2019; Wang and Trivedi 2013). Thus, it probably depends on nutrients found in phloem sieve tubes, ACP cells, and hemolymph, as well as the coinhabiting microbial community to support its metabolic and physiological needs to grow (Killiny et al. 2017).
CLas has previously been obtained from citrus seeds and established in transient cultures with commercially available natural GJ used as culture medium (Parker et al. 2014). This was our first step in producing a host plant–based culture medium to grow CLas. A similar approach has also been performed by Sechler et al. (2009), who achieved limited success by using citrus vein extract as a carbon source in a medium formulation developed for CLas (for a recent comprehensive review of CLas culturing attempts, see Merfa et al. 2019). In the current study, we aimed at analyzing different compounds, molecules, and growth conditions to elucidate the growth needs of CLas. In our system, CLas survived a series of subcultures in GJ or mGJ, demonstrating that cell multiplication is caused by acquisition of nutrients from these media and not because of nutrients remaining from the inoculum source, as suggested elsewhere (Ha et al. 2019). However, growth depended mostly on the culture density. Significant growth ratios were obtained only when the initial inoculum had low number of cells (about 103 to 104 genome equivalents/ml), and its increase (>105 genome equivalents/ml) led to a decrease in the CLas population in subsequent subcultures, which sometimes resumed growth with follow-up subcultures. Even though CLas ultimately lost viability with the performance of subcultures, this intriguing oscillating growth behavior was observed in most assays. Curiously, it has been demonstrated that bacterial insect endosymbionts may restrict their growth and adapt their replication to coordinate with the development of their hosts (Baumann and Baumann 1994; Gil et al. 2004). However, because CLas is grown in the absence of host cells in our system, we hypothesize that this oscillating behavior, which was also previously observed by Parker et al. (2014), can be due to either (or a combination of) fast consumption of essential nutrients that are present in low quantities, production of toxic byproducts, or regulation by cell density–dependent mechanisms, such as quorum sensing or other signaling pathway, all of which are briefly discussed below.
Determining which nutrients are potentially limiting and impairing CLas growth in GJ is challenging. However, in an effort to overcome this limitation, GJ and mGJ were amended with different compounds that are predicted to be needed by CLas and incubated in different conditions. This approach is akin to the method used to culture the obligate intracellular bacterial human pathogen Coxiella burnetii, the causative agent of Q fever (Omsland et al. 2009). For C. burnetii, a systematic evaluation of its metabolic needs was performed via expression microarrays, genomic reconstruction, and metabolite typing, which led to the development of an optimal culture medium and defined the microaerophilic requirement of this bacterium (Omsland et al. 2009). These analyses were possible only because of the previous development of a culture medium that allowed C. burnetii to remain metabolically active in axenic conditions for ≥24 h (Omsland et al. 2008). In our study, on the other hand, the choice of compounds and conditions was based on studies performed by other research groups (Cruz-Munoz et al. 2019; Fagen et al. 2014a; Jain et al. 2019, 2017a; Killiny 2016; Parker et al. 2014; Sena-Vélez et al. 2019; Vahling et al. 2010), because a culturing condition that allows axenic metabolic activity of CLas is yet to be developed. However, even with this limitation, a large variety of compounds and conditions were screened and their contribution to CLas growth in GJ was established in the current study. Nonetheless, the recent development of a method to grow CLas ex vivo within hairy roots has allowed to validate prediction of nutrient requirements obtained from metabolic models and analyze changes in growth phenotypes of this bacterium (Zuñiga et al. 2020), constituting a powerful tool to advance our efforts to grow CLas in pure culture.
In relation to toxic byproducts, few candidates are predicted to be harmful to CLas. Because of its deficient glyoxalase system, it is believed that CLas is not able to detoxify methylglyoxal, a cytotoxic byproduct of glycolysis that causes carbonyl stress (Jain et al. 2017a). In addition to its defective glycolysis pathway, CLas may overcome these hurdles by directly importing ATP from its host cells through a functional ATP/adenosine diphosphate (ADP) translocase (Jain et al. 2017a; Vahling et al. 2010). This structure is present in plastids and few other obligatory intracellular prokaryotes related to Chlamydiae and Rickettsiae, and it catalyzes import of ATP from the host in exchange of bacterial ADP (Schmitz-Esser et al. 2004). Thus, to fulfill the energetic needs of CLas, as well as circumvent a possible methylglyoxal toxicity originated from insufficient ATP production, because CLas encodes ATP synthase nevertheless (Duan et al. 2009), ATP was added to culture medium. However, no improvement in CLas growth was observed, possibly because ATP is highly unstable in solution and readily degraded to ADP.
Another possible toxic effect of growing CLas is the expression of phage lytic cycle genes that may be activated when attempting to culture this bacterium axenically, because of lack of a repressor (Fleites et al. 2014; Jain et al. 2017b). Therefore, GJ was amended with a protein encoded by Wolbachia strain wDi (a bacterial endosymbiont of D. citri) called Wolbachia repressor protein (Wrp), which has been demonstrated to repress expression of a phage lytic cycle gene from CLas (Jain et al. 2017b). However, no increase in CLas growth was observed. It is difficult to determine whether this result was caused by a lack of protein activity in our culture medium, although Wrp has been used successfully in studies with the surrogate Lcr (Jain et al. 2017b), deficiency of other essential nutrients, or a combination of both. An alternative to bypass this hindrance is to use a prophage-free CLas strain in culturing studies, as recently performed by Fujiwara et al. (2018). However, these strains seem to be the minority and are not readily available everywhere.
Finally, there have been reports on cell density-dependent signaling mechanisms that may partially reduce specific bacterial populations. Allolysis is one of these mechanisms and refers to the phenomenon of cell lysis of a subpopulation of bacteria, which is induced by other cells of the same species or of closely related species of the same phylotype (Prozorov and Danilenko 2011). This quorum-sensing regulated mechanism, sometimes called cannibalism, is associated with the onset of biofilm formation and natural competence in some bacterial species, as well as temporarily helping overcome nutrient limitation (López et al. 2009; Prozorov and Danilenko 2011). Another quorum-sensing dependent mechanism is the production of extracellular DNA, which may involve autolysis of a subpopulation of cells and other nonlethal mechanisms (Ibáñez de Aldecoa et al. 2017). In both cases, CLas could benefit from the availability of nutrients and DNA from lysed cells to use in its own metabolism, and that may even help explain the oscillating growth behavior previously observed by our group (Parker et al. 2014), which at the time was called cryptic growth. Although we cannot ascertain whether the obtained cell density–dependent growth behavior of CLas in this study is caused by allolysis or other mechanism, the observed behavior may be signaling dependent. Unfortunately, attempts to “wash” our cultures with fresh medium to remove any signaling molecule that could impair CLas growth, and thus allow cells to grow to higher densities, failed. Nevertheless, CLas encodes a quorum-sensing LuxR transcriptional activator protein in its genome (Duan et al. 2009; Fujiwara et al. 2018), which is activated in a density-dependent manner upon interaction with a cognate N-acyl homoserine lactone (Tsai and Winans 2010). However, because CLas lacks a luxI gene that encodes an N-acyl homoserine lactone synthase, the mechanism behind the coordination of the gene expression of CLas through LuxR is still unknown (Duan et al. 2009; Fujiwara et al. 2018; Yan et al. 2013).
Other approaches to keep cell culture density low and thus promote growth involved incubating CLas in flow conditions and subculturing samples at 3-day intervals. Both had limited success, because although bacterial growth was rapid, with significant growth ratios as early as 3 dpi, cell viability was also quickly lost. A similar result was observed in an ex vivo system designed to grow CLas within citrus leaf discs, in which bacterial multiplication was observed 3 days after incubation of infected leaf discs in test conditions (Attaran et al. 2020). The CLas population in that study was not evaluated for longer periods, but this result, together with ours, demonstrates that CLas may present a rapid increase in its in vitro population when incubated in optimized conditions.
Attempts to grow CLas in solid GJ plates were unsuccessful. It has been demonstrated that autoclaving agar together with phosphate may harm microbes by producing reactive oxygen species (Tanaka et al. 2014). However, our solid GJ medium preparation used Gelrite instead of agar, which is not known to have any toxic effect and is solely autoclaved in deionized water before mixing with GJ. Thus, we hypothesize that availability of some compounds or leakiness of metabolic goods produced by cocultured bacteria, which may help survival of organisms with loss of function (Morris 2015), is probably compromised in solid medium. This effect may be caused by microbial interactions being restricted in the medium surface and suggests that all or at least most physiological and nutrient requirements must be fulfilled for fastidious bacteria to grow in solid plates. Nonetheless, a recent report indicates that CLas was able to form microcolonies with associated microbiota when grown in solid plates of other medium formulations (Fujiwara et al. 2018). Curiously, our group has grown the bacterial plant pathogen Xylella fastidiosa in PD3 broth amended with grapevine sap (1:1) (Kandel et al. 2016). However, as similarly observed for CLas in this study, agar plates of this same medium formulation do not support growth of X. fastidiosa (unpublished data).
To take advantage of the possible leakiness of metabolic goods, which may include ATP (Mempin et al. 2013), we tried to establish a coculture protocol that allows CLas to grow with other specific bacteria and share metabolic compounds but remain spatially separated by using plate inserts. Although the system we designed did not improve CLas culturability, the use of cocultures is a valuable method to isolate and grow bacteria deemed recalcitrant to axenic culturing (Stewart 2012). Many coculture methods have been used to isolate various fastidious and previous unculturable bacteria from distinct habitats (Ding et al. 2017b; Tanaka and Benno 2015; Vartoukian et al. 2016). For CLas specifically, various studies have established the importance of mutualistic relationships with coinhabiting bacteria for its survival and even determined which microbial taxa may be beneficial or harmful to its growth (Davis et al. 2008; Fujiwara et al. 2018; Ha et al. 2019; Molki et al. 2020; Parker et al. 2014). It is important to note that in our cultures growth of other bacteria was not visible in most assays. However, this phenomenon cannot be ruled out because it has a possible substantial role in aiding CLas growth.
Finally, we developed an ex vivo system to grow CLas within periwinkle calli. As mentioned previously, approaches to grow CLas ex vivo within infected tissues of plant hosts are valuable tools that allowed the analyses of CLas growth in different conditions and validation of metabolic models (Attaran et al. 2020; Zuñiga et al. 2020). In our assays, three different culture media were assessed for periwinkle callus formation and CLas growth. All three media include ammonium and phosphate, which have been demonstrated to be essential molecules in the Liberibacter–plant host interface by using the surrogate bacterium Lcr (Cruz-Munoz et al. 2019). However, Gamborg’s medium was the most suitable for both callus formation and CLas growth overall because samples inoculated into this medium consistently presented growth of this bacterium. It has been shown that a high concentration of ammonium may induce oxidative stress, ionic imbalances, and pH disturbances across cell membranes (Cruz-Munoz et al. 2019; Liu and von Wirén 2017) and that the alkalinization of the medium is harmful to the surrogate Lcr (Sena-Vélez et al. 2019). Thus, Gamborg’s medium possibly performed better than the other tested media because of its low ammonium concentration (in comparison with Murashige & Skoog medium). This shows that Gamborg’s medium has a great potential to be explored in ex vivo assays to assess growth requirements of CLas and plant–pathogen interactions and may constitute an important alternative tool to study this bacterium until axenic cultures are obtained.
In summary, this study has extended our knowledge of the suitability of using commercial GJ as a culture medium to grow CLas and determined the contribution of a wide range of compounds and conditions to CLas growth. Nevertheless, sustained exponential growth in vitro or development of colonies in solid media was not achieved with any of the conditions tested here. In addition, a highly conserved oscillating behavior of CLas growth was established, which is probably dependent on cell density levels. Many compounds and conditions analyzed here did not improve CLas growth. However, they still have the potential to increase CLas culturability if an optimal condition is achieved. We hope that this information is used in designing culture media for CLas and establishing optimal incubation conditions for this bacterium.
We thank Dean W. Gabriel and Mukesh Jain (Department of Plant Pathology, University of Florida, Gainesville, FL) for providing CLas-infected citrus fruits and the Wolbachia repressor protein, as well as helpful suggestions; Amit Levy and Nabil Killiny (Citrus Research and Education Center, University of Florida, Lake Alfred, FL) for providing CLas-infected psyllids; Yongping Duan (U.S. Department of Agriculture, Fort Pierce, FL) for providing CLas-infected periwinkle plants; Michael J. Davis (Citrus Research and Education Center, University of Florida, Lake Alfred, FL) for providing training to dissect Asian citrus psyllids guts; Laura M. Gomez (Auburn University, Auburn, AL) for helping with periwinkle grafting; Paul Cobine (Auburn University, Auburn, AL), John Beckmann (Auburn University, Auburn, AL), and Gwyn A. Beattie (Iowa State University, Ames, IA) for helpful suggestions; Adriana-Avila Flores (Auburn University, Auburn, AL) for providing cell-free spent media used to culture SF9 insect cell line; and Kathleen Martin (Auburn University, Auburn, AL) for providing SF9 insect cell line and SF-900 III SFM culture medium.
The author(s) declare no conflict of interest.
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Funding: Support was provided by the U.S. Department of Agriculture, National Institute of Food and Agriculture 2016-70016-24844, and Alabama Agricultural Experiment Station L.D.
The author(s) declare no conflict of interest.