A rapid multiplication system for 'Candidatus Liberibacter asiaticus'through regeneration of axillary buds in vitro

LEI Tian-gang,HE Yong-rui,ZOU Xiu-ping,WANG Xue-feng,FU Shi-min,PENG Ai-hong,XU Lan-zhen,YAO Li-xiao,CHEN Shan-chun,ZHOU Chang-yong

Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.China

Abstract‘Candidatus Liberibacter asiaticus (CLas)’,which causes citrus Huanglongbing (HLB) disease,has not been successfully cultured in vitro to date. Here,a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG).This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration,CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture,the titers of CLas in regenerated shoots rapidly increased from 7.5×104 to 1.4×108 cells μg-1 of citrus DNA during the first 40 DAG. The percentages of shoots with ＞1×108 CLas cells μg-1 DNA were 30 and 40% at 30 and 40 DAG,respectively. Direct tissue blot immunoassay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back,stunted growth,leaf necrosis/yellowing,and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in citrus plantlests in vitro.This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.

Keywords:Citrus,‘Candidatus Liberibacter asiaticus’,multiplication,in vitro citrus plantlets

1.Introduction

Citrus Huanglongbing (HLB),also known as greening disease,is the most devastating disease in the citrus industry (Bové 2006). The disease is caused by at least three species of ‘CandidatusLiberibacter’:CaL.asiaticus (CLas),CaL.africanus (CLaf) andCaL.americanus (CLam) (Jagoueixet al.1994;Teixeiraet al.2005),of whichCLas is the most destructive strain. The pathogen is spread by grafting and by psyllid insect vectors (DiaphoriancitriorTriozaerytreae) in the field(Bové 2014). This disease is now widely distributed in the major citrus-producing areas of the world and causes substantial economic losses in Asia,Africa,and America(Bové 2014;Wanget al.2017). There are no resistant commercial citrus varieties and no effective cures for this disease (Zhou 2020).

The inability to culture the causative agent of HLBinvitrohas hampered research on its etiology and pathogenic mechanism,and the development of efficient control methods (Stewart 2012;Merfaet al.2019). In the past 20 years,the many attempts to isolate and culture HLB pathogensinvitrohave achieved little success (Daviset al.2008;Sechleret al.2009;Parkeret al.2014;Fujiwaraet al.2018). Recently,Haet al.(2019) developed a host-free biofilm method to cultureCLas,in whichCLas was able to survive,but at a very low titer,in cultured biofilms derived from infected citrus tissue. Metagenomic analyses have shown that all of the ‘Ca.Liberibacter spp.’ have a reductive genome that has lost multiple genes encoding essential enzymes and proteins in biosynthetic and metabolic pathways (Duanet al.2009;Linet al.2013,2015;Wulffet al.2014).Therefore,the growth ofCLas depends on both other phloem microbiota and nutrients (Hijaz and Killiny 2014;Fujiwaraet al.2018). Previous studies investigated the chemical composition of the phloem sap of citrus and periwinkle plants and psyllid hemolymph to identify the essential nutrients forCLas growth ininvitroculture (Hijaz and Killiny 2014;Killiny 2016,2017;Killinyet al.2017).However,invitroreproduction of the natural environment of ‘Ca.Liberibacter spp.’ is still a challenge.

It is difficult to study the biology of ‘Ca.Liberibacter’and its pathogen-host interactions because of the low titer of the pathogen and its uneven distribution within the tissues of infected citrus plants (Tatineniet al.2008;Liet al.2009). Compared with citrus as the host,infected periwinkle and dodder and insect host psyllids often carry a higher titer ofCLas (Duanet al.2009;Linet al.2013;Zhenget al.2016;Liet al.2018;Liuet al.2019).However,the titer ofCLas is variable and difficult to predict within a psyllid population (Walteret al.2012;Ukuda-Hosokawaet al.2015;Wuet al.2018),and the spatial distribution ofCLas in periwinkle and dodder is also uneven (Liet al.2018). Recently,a dodder-mediatedCLas enrichment system was successfully established and was shown to be suitable forCLas genomic and transcriptomic analyses (Liet al.2021).CLas is restricted to the phloem sieve tubes of plants,where it multiplies(Bové and Garnier 2003;Killiny 2017). Sufficient nutrition is considered to be conducive to the colonization and rapid replication ofCLas (Huber and Haneklaus 2007).Relatively high levels ofCLas were detected in the locular membranes and septa of citrus fruit (Liet al.2009). It has also been found that phloem-limited bacteria move freely through the sieve pores along with nutrients to the sugar-consuming plant tissues (Christensenet al.2004;Jianget al.2004). Large numbers ofCLas cells were detected in phloem sieve tubes in tissue samples from pre-symptomatic young flushes (Folimonova and Achor 2010).Invitroculture is a rapid propagation method for many plant species. Multiple shoots can be rapidly induced from explants ininvitroculture. Like the tissue samples obtained from pre-symptomatic young flushes,in vitroregenerated shoots could be a platform for the rapid enrichment ofCLas cells in citrus tissue.

In this study,aninvitroculture method was established for the regeneration of shoots fromCLas-infected sweet orange explants throughinvitroregeneration of axillary buds. The characteristics ofCLas growth in the regenerated shoots were investigated by quantitative PCR (qPCR). The HLB symptoms in the regenerated shoots were compared with those of infected plants growing in a greenhouse. The localization ofCLas within citrus plantletsinvitrowas visualized using direct tissue blot immunoassay (DTBIA) with anti-outer membrane protein A (anti-OmpA) antibody. Our results show that shoots regenerated fromCLas-infected explantsinvitrocan support the rapid growth ofCLas to titers much higher than those in the midribs of leaves from infected plants growing in a greenhouse.

2.Materials and methods

2.1.Source of plant materials and preparation of explants

Two-year-old ‘Changyecheng’ sweet orange (Citrus sinensisOsbeck) seedlings were graft-inoculated with budwoods from HLB-affected Miyagawa satsuma(C.unshiu) trees (collected from Jiangxi Province) and maintained in a greenhouse at the Citrus Research Institute,Southwest University,Chongqing,China. Stems and leaves were collected from the HLB symptomatic trees approximately 10 months after inoculation. The presence ofCLas in the seedlings was confirmed by PCR as described previously (Teixeiraet al.2005). Branches were collected from HLB-infected ‘Changyecheng’ sweet orange seedlings and used as explant materials.

Leaves on theCLas-infected branches were numbered according to nodal segments. There is a one-to-one correspondence between the number of each leaf and stem segment with a single axillary bud. Leaf midrib material (0.2 g) was excised from infected branches and stored at ?80°C until DNA extraction. TheCLas-infected branches were scrubbed with household detergent and washed thoroughly under running tap water. Then,the branches were surface-sterilized for 1 min in 75% ethanol,washed three times with sterile distilled water,immersed in 0.1% HgCl2solution for 10 min,and then rinsed eight to ten times with autoclaved distilled water.

The basal medium was MS medium (Murashige and Skoog 1962) containing 30 g L-1sucrose and 8 g L-1agar powder (TaKaRa,Dalian,China). The pH of the medium was adjusted to 5.8 before autoclaving (121°C,115 kPa,25 min).

2.2.In vitro culture of CLas-infected citrus stem segments

Different concentrations of gibberellic acid (GA) (0.1 or 0.2 mg L-1),indoleacetic acid (IAA) (0.2 or 0.5 mg L-1),and 6-benzyladenine (BA) (1 mg L-1) were added to the basal medium. The disinfected branches were cut into stem segments with a single axillary bud (1.5-2 cm) and placed on the media. Each group consisted of 36 singlebud stem segments and three replicates. Cultures were maintained at 28°C in darkness for 10-14 d to promote germination. Then,the cultures were incubated at 28°C under a 16-h light/8-h dark photoperiod,with light supplied by Panasonic lamps. The germination rate and shoot length in each treatment were recorded at 20 days of culture. Theinvitroculture ofCLas-infected‘Changyecheng’ sweet orange stem segments was performed by utilizing the optimal medium. The midrib of each leaf was excised from theCLas-infected branches for DNA extraction. Forty single-bud stem segments were cultured and the experiment was repeated twice. At 30 days after germination (DAG),CLas in the regenerated shoots was detected by PCR and the titers ofCLas in shoots regenerated from infected stem segments and in the original leaf midribs were determined by qPCR.

2.3. In vitro culture of CLas-infected citrus plantlets

Fruits were harvested from ‘Changyecheng’ sweet orange trees growing in the citrus orchard of the National Center for Citrus Varieties Improvement,Chongqing,China. The fruits were immersed in 75% ethanol for 3 min,then the seeds were removed and placed on solid MS medium under aseptic conditions. The seedlings were used asinvitrorootstocks (Penget al.2021). Axillary buds were cut from the surface-sterilized branches fromCLasinfected seedlings and grafted onto theinvitrorootstocks(Heet al.2011). Then,the grafted citrus plantlets were cultured in liquid MS medium containing 30 g L-1sucrose.Cultures were maintained at 28°C in darkness for 14 days to promote the germination of axillary buds. The citrus plantlets were then maintained at 28°C under a 16-h light/8-h dark photoperiod until the regenerated shoots reached 0.5-1 cm in length. Shoots (20 samples) were collected at 10,15,20,25,30,and 40 DAG,and stored at?80°C until analysis.

2.4.DNA extraction

The DNA was extracted from the leaf midribs and regenerated shoots with the Biospin Omni Plant Genomic DNA Extraction Kit (Bioer,Hangzhou,China). The extracted DNA was quantified and checked for purity at A260/A280(Nanodrop,Thermo Fisher Scientific,Waltham,MA,USA). The concentration was adjusted to 20 ng μL-1and the DNA samples were stored at ?20°C until PCR and qPCR analysis.

2.5.CLas detection

The presence ofCLas was detected by PCR. The specific 16S rDNA amplicon (1 160 bp) was amplified using the primer pair OI1 (5′-GCG CGT ATG CAA TAC GAG CGG CA-3′) and OI2c (5′-GCC TCG CGA CTT CGC AAC CCA T-3′). The PCR solution contained 3 μL total DNA,12.5 μL 2× PCR mix (TaKaRa,Dalian,China),0.2 mmol L-1each primer,and double-distilled H2O to yield a final volume of 25 μL. PCR amplification was carried out on a Thermocycler (Biometra,Goettingen,Germany) with the following program:95°C for 5 min;39 cycles of 94°C for 30 s,62°C for 30 s,and 72°C for 1 min;with final extension at 72°C for 10 min. All reactions were performed with a positive control and a negative control.Amplified products were electrophoresed on a 1.5%(w/v) agarose gel with ethidium bromide (EB) and visualized using a BioSpectrum?300 Imaging System(UVP LLC,Upland,CA,USA).

TheCLas bacterial populations in samples were also tested using the protocol of Tatineniet al.(2008) with some modifications. The qPCR assays were performed using the 7500 Fast Real-Time PCR System (Applied Biosystems,Foster City,CA,USA). The specific primers qClas-f/qClas-r and Ct18s-f/Ct18s-r (Zouet al.2017) were used to amplify the 16S rDNA gene ofCLas and the 18S gene of citrus,respectively. The qPCR reactions were performed using 1×TaqUniversal SYBR Green Supermix(Bio-Rad,Hercules,CA,USA),0.2 μmol L-1each primer,and 2 μL (40 ng) template DNA in a 20-μL reaction volume. The PCR conditions were as follows:94°C for 5 min;followed by 40 cycles at 94°C for 20 s,and 60°C for 1 min. Three technical replicates were analyzed for each sample. The following equation was used to calculate the size of the bacterial population (CLas cells μg-1citrus DNA):[10(-0.2718×Ct16S+10.624)/10(-0749×Ct18S+4.0531)]×103(12.7＜Ct16S＜32.0 and 8.4＜Ct18S＜26.5) (Zouet al.2017).

The distribution ofCLas in regenerated shoots was detected by DTBIA with an anti-OmpA antibody (provided by USDA ARS Molecular Plant Pathology Laboratory,Beltsville,Maryland,USA). Sections were cut from the petioles,stems (shoot and rootstock),and roots ofCLasinfected citrus plantletsinvitroat 30 DAG and petioles,leaf midribs,and stems of new autumn shoots from trees growing in the greenhouse using separate razor blades.Samples were pressed onto nitrocellulose membranes(Whatman Inc.,New Jersey,USA) to make tissue imprints,and DTBIA was performed as described by Dinget al.(2015).

2.6.Data analysis

Data were analyzed with SPSS Statistics 22 Software(SPSS Inc.,Chicago,IL,USA). Data are presented as the means±standard deviation (SD). Significant differences were subjected to Duncan’s test (P＜0.05).

3.Results

3.1.Optimization of phytohormones for germination of CLas-infected axillary buds

In previous experiments,we found that the regeneration ofCLas-infected axillary buds was very poor and the regenerated shoots grew slowly,compared with healthy controls. Hence,preliminary tests were carried out to determine the optimal combination and concentration of phytohormones to promote the regeneration ofCLasinfected axillary buds from ‘Changyecheng’ sweet orange. The highest germination rate ofCLas-infected axillary buds was on medium containing 1.0 mg L-1BA,0.2 mg L-1GA,and 0.2 mg L-1IAA (Table 1). Under these conditions,the germination rate ofCLas-infected axillary buds was 85.2% and the length of regenerated shoots was 12.5 mm at 20 days of culture. Compared with IAA,GA significantly increased the germination rate and shoot elongation.

Table 1 Effects of hormones on the germination of axillary buds from ‘Candidatus Liberibacter asiaticus’ (CLas)-infected‘Changyecheng’ sweet orange (Citrus sinensis Osbeck)1)

3.2.Characteristics of CLas growth in shoots regenerated from axillary buds in vitro

At 30 DAG,PCR analyses revealed thatCLas was present in 72.4% (a total of 58 regenerated shoots were obtained,of which 42 were PCR positive) of the regenerated shoots (Fig.1-A). To explore the characteristics ofCLas growth in regenerated shoots,we compared theCLas titers between the shoots regenerated from axillary buds and the midribs of leaves of infected plants growing in the greenhouse. As determined by qPCR assays,theCLas titers in regenerated shoots ranged from 1.1×105to 2.3×108cells μg-1DNA (average,6.6×107). On average,theCLas titer in regenerated shoots was 28.2-fold higher than that in the leaf midribs,and the largest difference was 482.2-fold (Fig.1-B). Thus,CLas was able to proliferate rapidly in the shoots regenerated from axillary buds of infected ‘Changyecheng’ sweet orange ininvitroculture.

Fig.1 Detection of ‘Candidatus Liberibacter asiaticus’ (CLas) in the shoots regenerated from axillary buds of CLas-infected‘Changyecheng’ sweet orange (Citrus sinensis Osbeck) stem segments in vitro by PCR and qPCR. A,presence of CLas in shoots regenerated from axillary buds of infected ‘Changyecheng’ sweet orange trees as detected by PCR. M,DL2000 DNA marker;1-20,samples;21,positive control;22,negative control. B,comparison of CLas titers between in vitro regenerated shoots and the original leaf midribs of ‘Changyecheng’ sweet orange trees growing in the greenhouse by qPCR. Values are mean±standard deviation from three different tests. ＊ indicates significant difference in titer from that in the original leaf midribs (Duncan’s test,P＜0.05).

3.3.In vitro regeneration of CLas-infected axillary buds using a micrografting method

AlthoughCLas proliferated in the regenerated shoots,we found that contamination was an issue when culturing stem segments. To solve this problem,we grafted theCLas-infected axillary buds onto ‘Changyecheng’ sweet orange seedlings by the micrografting method. This markedly reduced the contamination in the cultures. After about 2 weeks,micrografted axillary buds began to sprout and the regenerated shoots grew gradually. However,the germination rate was low,about 30%. Application of GA(100 mg L-1) to the micrografted axillary buds increased the germination rate to 70.7% (297/420).

3.4.Characteristics of CLas growth in in vitro regenerating micrografted axillary buds

After using the optimized micrografting culture method described above,the presence ofCLas in regenerated shoots was monitored using PCR. The percentages of PCR positive shoots were 10% (2/20),15% (3/20),15% (3/20),20% (4/20),55% (11/20),and 70% (14/20)at 10,15,20,25,30,and 40 DAG,respectively (Fig.2).The presence ofCLas in the shoots was detected as early as 10 DAG. The percentage of shoots containingCLas increased sharply at 30 DAG,and peaked at 40 DAG.

Fig.2 In vitro regeneration of ‘Candidatus Liberibacter asiaticus’ (CLas)-infected axillary buds using the micrografting method and CLas detection by PCR. A,CLas-infected citrus plantlets. B,PCR detection of CLas at 10,15,20,25,30 and 40 days after germination (DAG). M,DL2000 DNA marker;CK+,positive control;CK-,negative control;1-20,samples.

To quantifyCLas at different times during culture,the titer ofCLas in regenerated shoots was determined by qPCR. The titer ofCLas in the shoots increased gradually from 10 to 40 DAG (Table 2). The meanCLas titer at 10 DAG was 7.5×104cells μg-1DNA,whereas those at 15 and 20 DAG were 2.2×106and 1.4×107cells μg-1DNA,respectively. The mean titer ofCLas in shoots was 2.2×107cells μg-1DNA at 25 DAG,and 1.2×108and 1.4×108cells μg-1DNA at 30 and 40 DAG,respectively. The percentages of shoots with ＞108CLas cells μg-1DNA were 30% (6/20) at 30 DAG and 40% (8/20) at 40 DAG,respectively. Statistical analyses showed that theCLas populations at 30 to 40 DAG were significantly larger than those before 30 DAG,although there was no significant difference in the bacterial titer between 30 and 40 DAG. Thus,CLas could proliferate rapidly in citrus shoots regenerated from micrografted axillary buds and the bacterial population increased by eight orders of magnitude in a short time (30-40 days).

Table 2 Quantification of ‘Candidatus Liberibacter asiaticus’(CLas) in shoots regenerated from micrografted axillary buds of ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck)

3.5.Distribution of CLas in shoots regenerated from axillary buds in vitro

The localization ofCLas in shoots regenerated from axillary budsinvitrowas visualized using DTBIA with an anti-OmpA antibody,and compared with that in leaves of infected trees growing in the greenhouse. A dark purple color indicative ofCLas was observed in the phloem cells of plant tissue samples fromCLasinfected plantletsinvitroand trees,but not in phloem sieve cells of the healthy control (Fig.3). Strong and clearly dark purple staining was observed in the phloem sieve cells of petioles,stems (shoot and rootstock),and root tissues from infected plantletsinvitro,but not in the same tissues of infected trees growing in the greenhouse. Dark purple circles were also observed in the phloem cells of petioles,stems,and root tissues ofCLas-infected citrus plantletsinvitro(Fig.3-A-F). The distribution ofCLas was uneven in the petioles,midribs,and stems of infected samples from the greenhouse(Fig.3-I-K). The distribution ofCLas was much more uniform in the shoots regenerated from axillary budsin vitrothan in the tissues of infected citrus trees growing in the greenhouse.

Fig.3 Distribution patterns of ‘Candidatus Liberibacter asiaticus’ (CLas) in phloem tissue from ‘Changyecheng’ sweet orange(Citrus sinensis Osbeck) plantlets in vitro and trees growing in the greenhouse visualized by direct tissue blot immunoassay (DTBIA)using an anti-OmpA antibody. A and E,petiole. B and F,stem (shoot). C and G,stem (rootstock). D and H,root tissue prints of CLas-infected plantlets in vitro. I-K,petiole,leaf midrib,and stem tissue prints of CLas-infected trees from the greenhouse,respectively. L,tissue print of a stem from a healthy plant. Bars=0.5 mm.

3.6.Symptoms of CLas-infected plantlets in vitro

The first symptoms observed in citrus trees infected withCLas are shoot yellowing and blotchy mottling of the leaves,followed by leaf yellowing,vein corking,stunted growth,defoliation,tree decline,and eventually death(Bové 2006;Dengatal.2012;Zhao 2017). In our study,a few infected plantletsinvitroshowed blotchy mottled leaves (Fig.4-A-D),while the characteristic symptoms of most infected plantletsinvitrowere die-back,stunted growth,leaf necrosis/yellowing,and defoliation (Fig.4-EH). Fibrous root rot was also observed in some plantlets(Fig.4-I). Rapid death was the main sign of disease in the infected sweet orange plantletsinvitro. The death of infected plantletsinvitroincreased rapidly after 40 DAG.The mortality rates were 17.9,51.6,82.0,and 89.7% at 40,50,60,and 70 DAG,respectively (Fig.5).

Fig.4 Symptoms of ‘Candidatus Liberibacter asiaticus’ (CLas)-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck)plantlets in vitro. A-D,blotchy mottled/yellow leaves. E and F,tip die-back. G and H,defoliation and eventual death. I,fibrous root rot. J,healthy plantlets in vitro. DAG,days after germination.

Fig.5 Mortality rate of ‘Candidatus Liberibacter asiaticus’(CLas)-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck) plantlets in vitro at different time points. DAG,days after germination. Values are mean±standard deviation from three different tests. ＊＊,significant difference in mortality rate between the 40 DAG and the other groups (t-test,P＜0.01).

4.Discussion

HLB is the most devastating disease of citrus worldwide(Bové 2006). The causative agent of HLB,CLas,has not been successfully culturedinvitrobefore now (Merfaet al.2019;Liet al.2021). Studies on this pathogen have been limited to its etiology and host-pathogen interactions,and it is time consuming and resource-intensive (Krystelet al.2019). There are several reasons why it is difficult to study theCLas pathogen:Its transmission rate is low in citrus trees after inoculation by grafting orviapsyllids,and its proliferation rate in the plant is also slow. In addition,the pathogen has a small population size and an uneven distribution in HLB-infected citrus trees (Duanet al.2009;Liet al.2021). In this study,we established a stable and rapid proliferation system forCLasviagrafted citrus plantletsinvitro.CLas was able to multiply quickly in citrus plantletsinvitroand reach a high titer in a short time (30-40 DAG). In contrast,for citrus trees growing in the field or the greenhouse,CLas can be usually detected by qPCR at 2 to 3 months after inoculation either by grafting or psyllids (Lopeset al.2009;Coletta-Filhoet al.2010;Folimonova and Achor 2010). Furthermore,the distribution ofCLas in the phloem sieve cells of the regenerated plantletsinvitrowas much more uniform than that in infected citrus trees in the greenhouse. This rapid multiplication system forCLas makes it an optimal source for further studies on the biological characteristics of the pathogen and rapid screening of bactericidal drugs.

Phloem sap contains all the essential nutrients and some unknown factors that favor pathogen growth and multiplication (Bové and Garnier 2003;Hijaz and Killiny 2014;Killiny 2017). Ininvitroculture,the roots take up sugars,amino acids,vitamins,and inorganic ions from the medium and transport these substances to the shoots to fuel growth. These substances may also be beneficial forCLas growth.CLas can grow in both citrus phloem sap and psyllid hemolymph (Bové and Garnier 2003). The average pH of phloem sap from sweet orange is about 6.0 (Hijaz and Killiny 2014),and the hemolymph of most insects is slightly acidic (Nation 2015).CLas can also grow inside the white cortex of acidic citrus fruit,and high titers ofCLas have been detected in the citrus fruit septa and locular membranes (Liet al.2009). Thus,slightly acidic culture conditions (pH 5.8) should be conducive to the growth ofCLas in citrus plantletsinvitro. In addition,the cultures were maintained at 27-28°C,which is suitable for the growth and reproduction ofCLas. In another study,the highest titer ofCLas in mature leaves of citrus plants was detected under night/day temperatures of 22/27°C (Gasparotoet al.2012). Thus,the conditions forinvitroculture may have contributed to the rapid proliferation ofCLas in regenerated shoots.

Surface disinfestation of the branches is very important to reduce contamination without killingCLas. We scrubbed the infected branches directly with a surfactant solution and cleaned the material thoroughly. Our method also required the removal of shoots from rootstock to increase the germination rate of infected buds and promote the rapid accumulation of pathogenic bacteria in the regenerated shoots. The germination rate was increased significantly by applying 100 mg L-1GA to theCLas-infected budwoods after the grafting site was completely healed.

The HLB pathogen is unevenly distributed in its hosts.Consequently,in previous studies,only certain shoots and parts of stems tested positive for the pathogen (Teixeiraet al.2008;Liet al.2009;Gottwald 2010;Dinget al.2015;Louzadaet al.2016;Fuet al.2019). Therefore,the spatial distribution ofCLas in citrus plantletsinvitrois a matter of concern. The DTBIA method is ideal for detectingCLas because it can reveal specific details regarding its localization and spatial distribution within plant tissues. Hence,it has been used to detectCLas in previous studies (Dinget al.2015;Fuet al.2019). In another study,an anti-OmpA polyclonal antibody was highly effective for detectingCLas in cells of infected citrus tissues (Dinget al.2017). In this study,DTBIA was used to monitor theCLas distribution using an anti-OmpA antibody. We detected strong hybridization signals in the phloem sieve cells of the petioles,stems,and root tissues of infected citrus plantletsinvitro. Furthermore,the distribution ofCLas in regenerated shoots was relatively uniform compared with that in the midribs of leaves of diseased citrus trees growing in the greenhouse. This may be because of the high titer ofCLas in the shoots and the small size of the plantletsinvitro. In addition,the characteristic symptoms of most infected plantlets differed from those of diseased trees. Shoot die-back was the main disease symptom in the infected plantletsinvitro.We speculated that this difference was due to theCLas population rapidly increasing and reaching a high level in the shoots,as well as the weak resistance of young citrus plantletsinvitro. Although theCLas-infected plantletsinvitrodied quickly,half of them survived for more than 50 DAG. Thus,invitroculturedCLas-infected citrus plantlets are a promising platform for research onCLashost interactions and the biological characteristics of the pathogen,and for screening antibacterial compounds.

5.Conclusion

In this study,we have constructed a rapid multiplication system forCLas in which the pathogen can proliferate efficiently and rapidly as early as 20 days after the start of culture. The titers ofCLas in regenerated shoots obtained using the micrografting method rapidly increased from 7.5×104to 1.4×108cells μg-1DNA during the first 40 DAG. Our analyses show that the pathogen is more uniformly distributed in the shoots of plantletsinvitrothan in infected citrus trees growing in a greenhouse. This system represents a new platform for research on the biology and pathogenesis ofCLas,and for screening antibacterial compounds againstCLas.

Acknowledgements

This work was supported by the National Key R&D Program of China (2018YFD0201500 and 2018YFD1000300),the National Natural Science Foundation of China (31972393),and the China Agriculture Research System of MOF and MARA (CARS-26). We thank Ph D Jennifer Smith,from Liwen Bianji (Edanz) (www.liwenbianji.cn/ac),for editing the language of a draft of this manuscript.

Declaration of competing interest

The authors declare that they have no conflicts of interest.