Comparison of whole-tissue maceration and sap extraction for isolation of ‘ Candidatus Liberibacter asiaticus’

‘ Candidatus Liberibacter spp.’ are fastidious alphaproteobacteria associated with Huanglongbing (HLB), a highly destructive disease for citrus production around the world. So far, no axenic culture of these bacteria have been established, limiting its study. However, the first step to cultivate plant pathogenic bacteria is to obtain viable cells. So, the aim of this study was to compare two methods to extract viable cells of ‘ Ca . L. asiaticus’ from diseased plant tissue of infected citrus, periwinkle and dodder by using the PMA-qPCR technique. We applied two methods for extraction of bacterial cells, the whole-tissue maceration procedure for citrus, periwinkle and dodder and the sap extraction method by using the Scholander pressure pump for citrus and periwinkle. The extraction of ‘ Ca . L. asiaticus’ cells by tissue maceration method provided a larger number of viable and non-viable bacterial cells when compared to the Scholander pressure pump method in both, citrus and periwinkle. The largest amount of bacteria extracted by the Scholander pressure pump was in periwinkle plants. However, the amount of cells obtained from dodder plants using maceration was higher than those obtained from the other plants by the same method, pointing out that dodder could be an excellent inoculum source for ‘ Ca . L. asiaticus’ isolation tests. In this study, the two methods provided sufficient amounts of ‘ Ca. L. asiaticus’ to perform bacterial isolation tests, and may be used in further studies on HLB bacteria and its disease

from host tissues can irreparably compromise the integrity of the pathogen cells and, thereby, preclude isolation and cultivation of bacteria in pure culture.
A technique for HLB bacteria extraction from host tissues should allow to obtain sufficient amount of viable bacterial cells to establish cultures in artificial culture medium.Here, we analyze the viability of 'Ca.L. asiaticus' cells present in saps extracted from tissue of plants infected with HLB bacteria by using propidium monoazide (PMA) associated with quantification by qPCR (real-time quantitative Polymerase Chain Reaction) technique.PMA is a dye that binds only to the DNA of cells that have damaged membrane (Nocker et al., 2006;Hu et al., 2013Hu et al., , 2014) ) and, thus inhibits DNA amplification by PCR from non-viable cells.Furthermore, the qPCR technique associated with PMA allow us to quantify the amount of viable 'Ca.L. asiaticus' cells present in the extracts used for bacteria isolation.Thus, the objective of this study was to establish efficient protocols to extract the largest number of viable cells of 'Ca.L. asiaticus' from tissues of different host plants.

Plant material
In this study, we used samples of HLB symptomatic citrus (Citrus sinensis (L.) Osbeck cv.'Pera'), periwinkle (Catharanthus roseus) and dodder (Cuscuta sp.) infected with 'Ca.L. asiaticus' bacterium.The presence of bacteria in samples of all three plants was confirmed by PCR using the A2/J5 primers (Hocquellet et al., 1999).The HLB bacteria was extracted from the different plant tissues by two methods, the tissue maceration procedure and the sap extraction by using the Scholander pump.The bacterium was extract from dodder samples only by the tissue
The first attempts to isolate the HLB organisms were by the 1970s and 1980s (Ghosh et al., 1971;Garnett, 1984).More recently, studies concerning the isolation and cultivation of 'Ca.Liberibacter spp.' have been published (Davis et al., 2008;Sechler et al., 2009).However, these results could not be reproduced independently and the Koch's postulates were also not fulfilled, thus the HLB bacteria remain considered as a non-cultivable organism.
The establishment of 'Ca.Liberibacter spp.' in pure cultures is of utmost importance to carry out several different studies on bacteria and its interactions with host plants and insect vetors.However, the bacteria isolation require several steps, such as the establishment of appropriate methods for extracting viable bacteria from plant and insect vetors tissues, development of culture media for isolation and cultivation, and suitable storage conditions.Nevertheless, inappropriate methods for bacteria extraction maceração de tecido para plantas de citros, vinca e cuscuta.O método de extração de células de 'Ca.L. asiaticus' por maceração apresentou o maior número de células bacterianas, viáveis e não viáveis, quando comparado ao método da bomba de pressão de Sholander tanto em citros quanto em vinca.A maior quantidade de bactérias extraídas pela bomba de pressão de Sholander foi em plantas de vinca.Todavia, a quantidade de células obtidas de plantas de cuscuta utilizando a maceração foi superior à obtida nas demais plantas pelo mesmo método, podendo a cuscuta ser excelente fonte de inóculo para testes de isolamento de 'Ca.L. asiaticus'.Os dois métodos de extração forneceram quantidades suficientes de células de 'Ca.L. asiaticus' para realização de estudos de isolamento da bactéria, podendo também ser empregados em diferentes estudos sobre a bactéria e a doença por ela causada.Termos de indexação: propídio monoazida, greening, método de extração, bomba de pressão.

Pre-treatment with PMA
The extracts obtained by each procedure were treated with PMA (Biotium Inc., Haward, CA, USA), according to the protocol described by Hu et al. (2013).Initially, a stock solution of 10 mg mL -1 of PMA (dissolved in sterile distilled water) was prepared and this solution was kept protected from light at 4 °C.The PMA solution (50 μM) was added to the tubes containing plant extracts samples at a final concentration of 25 μg mL -1 .The tubes were incubated in the dark for 5 min at room temperature, with occasional inversions to allow PMA to penetrate into the cells with damaged membrane and to bind to the bacterial DNA.After the incubation period, the tubes were briefly vortexed for 5 sec and placed on ice.To activate the PMA reaction, the tubes were opened and subjected to halogen light (650 W) at 20 cm of distance for 2 min.After treatment with PMA, samples were centrifuged for 5 min at 12,000 g.The supernatant was discarded and 700 μL of CTAB buffer was added to the pellet.DNA extraction was performed following the protocol of Murray & Thompson (1980) and resuspended in 50 μL of Milli-Q water.

qPCR test for HLB bacterium quantification
Samples treated and not treated with PMA, obtained by the different extraction methods, were submitted to quantification of 'Ca.L. asiaticus' bacterium by qPCR technique.A TaqMan  probe detection system for a specie-specific 16S rDNA gene region of bacterium was used.The qPCR reaction was prepared for a final volume of 15 μL, containing 7.5 μL of 2× ABI TaqMan Universal Master Mix (Life Technologies), 10-20 ng of DNA, 0.25 μM of the HLBas and HLBr primers, 0.15 μM of the probe HLBp labeled at the 5' terminus with the reporter dye 6-carboxyfluorescein (FAM) and at the 3' terminus with Quencher (BHQ), and nuclease free water (Ambion™, Invitrogen, Carlsbad, CA, USA) to adjust the volume.The reaction conditions for amplification were 2 min of incubation at 60 °C, 10 min of denaturation at 95 °C, followed by 40 cycles of 15 sec at 95 °C and 1 min at 58 °C.Fluorescence signals were collected after one minute at 58 °C during each cycle (Li et al., 2006).Amplification and detection were performed on an ABI Viia 7 qPCR apparatus (Applied Biosystem, Foster City, CA, USA).All reactions were performed in duplicate and, at each run, negative and positive controls were maceration procedure due to technical difficulties to use the Scholander pump in this case.

Tissue maceration procedure
Three samples of citrus, periwinkle and dodder plants were processed by using the protocol described by Trivedi et al. (2009), with minor modifications, to evaluate the extraction of viable HLB bacterial cells by the tissue maceration procedure.Bark of citrus or periwinkle stems with, approximately, 5 mm in diameter were removed and scraped with an aid of a scalpel.These tissue pieces were macerated in a mortar with 15 mL of sterile distilled water.The dodder tendrils were also macerated in 15 mL of sterile distilled water.The macerated tissue was transferred to a 50 mL tube and centrifuged for 2 min at 1,200 g.The supernatant was aliquoted into 1.5 mL tubes and centrifuged for 5 min at 12,000 g and then discarded.The resulting pellet was weighed and about 40 mg was resuspended in 1 mL of sterile distilled water.After this step, half of the samples were subjected to PMA treatment as described below.

Sap extraction by using the Scholander pump
Three samples of citrus or periwinkle were used to evaluate the extraction of viable cells of 'Ca.L. asiaticus' from infected plant shoots using the Scholander pressure pump (Soil Moisture Equipment Corp., Santa Barbara, CA, USA).For higher efficiency in the extraction process with the Scholander pump, considerable care was taken to maintain the integrity of the stems, avoiding the removal of leaves and the use of broken shoots.Stems of approximately 15 cm in length and maximum thickness of 4 mm in diameter were individually placed inside the Scholander pump, leaving around 3 cm of the end out of the pump.The total phloem and xylem saps of plant samples were forced out of the branch by applying a pressure of approximately 3,000 to 4,000 kPa.The sap was collected with micropipette and deposited in 1.5 mL microcentrifuge tube.For each sample, 2 mL of sap were collected and equally distributed in two microcentrifuge tubes.After this step, half of samples were subjected to PMA treatment as described below.The other half of samples were used for DNA extraction according to Murray & Thompson (1980).The DNA obtained from each sample was resuspended in 50 μL of Milli-Q water.
in citrus, periwinkle and dodder samples ranging from 1.21 × 10 11 to 5.88 × 10 12 , 5.86 × 10 10 to 1.80 × 10 14 and 1.21 × 10 15 to 3.43 × 10 15 cells per milligram of tissue, respectively (Table 1).Therefore, dodder plants provided the recovery of the largest number of total and viable cells of 'Ca.L. asiaticus' compared to periwinkle and citrus (Table 1).Further, more than 90% of the cells of 'Ca.L. asiaticus' recovered were viable ones for all kind of plant tissue tested (Table 1).

Sap extraction by using the Scholander pump
Extraction of the 'Ca.L. asiaticus' by using the Scholander pump was only carried out for citrus and periwinkle samples, as it was not possible to test this method in dodder due to the structure of the tendrils.The amount of total cells of bacteria obtained from the sap of citrus and periwinkle tissue ranged from 2.34 × 10 7 up to 1.15 × 10 11 cells per milliliter of sap, respectively (Table 1).Regarding the amount of viable cells of 'Ca.L. asiaticus', we found it ranging from 1.36 × 10 7 up to 3.18 × 10 10 cells per milliliter of sap of citrus and periwinkle, respectively, based on the PMA-qPCR test (Table 1).The amount of total and viable bacterial cells in the sap of citrus and periwinkle plants obtained using the Scholander pump did not differ significantly (Table 1).For citrus, approximately 94% of 'Ca.L. asiaticus' cells were viable ones based on the PMA-qPCR test, whereas for periwinkle cell viability was 97% (Table 1).included.To generate a standard curve, DNA fragments of 16S rDNA of 'Ca.L. asiaticus' obtained by previous PCR were cloned into competent Escherichia coli cells with TOPO TA plasmid insert (Invitrogen, Carlsbad, CA, USA).Known concentrations of these plasmids carrying the target DNA, obtained from cloned libraries, were diluted serially, starting from the concentration of 10 ng μL -1 under ten serial dilutions in the ratio of 1:2 for higher resolution and encompassing most unknown samples concentration matches in the standard curve midpoints (Sow et al., 2009;Gallup & Ackermann, 2006).The threshold cycle numbers (Ct) reported were used to establish a linear regression based on copy number of DNA samples to generate a standard curve.The Ct values of the samples were interpolated in a standard curve to determine the number of copies of the gene in the samples (Li et al., 2006).The data were submitted to analysis of variance using the SASM-Agri software (Canteri et al., 2001).The means were compared by the Tukey test with a significance level of 5%.

Tissue maceration procedure
The tissue maceration method yielded extracts with total 'Ca.L. asiaticus' concentrations ranging from 5.77 × 10 11 bacterial cells per milligram of tissue of periwinkle and up to 1.57 × 10 16 bacterial cells per milligram of tissue of dodder (Table 1).Using PMA-qPCR test to estimate viable cells of 'Ca.L. asiaticus', we found concentrations  Comparison of whole-tissue maceration… inoculum source.Therefore, the amount of inoculum could be insufficient to establish pure culture of the bacteria.A large amount of viable cells of 'Ca.Liberibacter spp.' is, for certain, the first step to succeed in isolation and establishment of axenic culture of HLB bacteria.
'Ca.L. asiaticus' is not uniformly distributed in the host plant tissues (Tatineni et al., 2008;Li et al., 2009;Hartung et al., 2010), however, the bark of the stems of citrus and periwinkle and the tendrils of dodder showed a high concentration of the bacterium (Table 1).For that reason, these tissues seem to be appropriate as an inoculum source for the isolation of the bacteria.In regard to dodder, the highest concentration of viable cells of 'Ca.L. asiaticus' were obtained by using the tissue maceration extraction method from this plant (Table 1).Our findings confirm other studies, in which differences in 'Ca.L. asiaticus' concentration were observed among different plant species (Trivedi et al., 2009;Hartung et al., 2010;Hu et al., 2013).Another interesting aspect in the use of dodder for extraction of 'Ca.L. asiaticus' cells is the ease in the process of tissue maceration, besides the small amount of contaminating organisms (data not shown).
The PMA-qPCR technique used to determine the viability of uncultured bacterial cells is relatively recent (Hu et al., 2013;Hu et al., 2014).The results obtained in this study revealed that the PMA-qPCR technique, used to evaluate the viability of 'Ca.L. asiaticus' present in different host plants, is a valuable technique to monitor the amount and quality of the initial inoculum in studies on bacterial isolation, as well as in other studies regarding the HLB bacterium.Further, among the studied plants, dodder seems to be an excellent inoculum source for 'Ca.L. asiaticus' isolation.
± 0.5 a (3) 13.73 ± 0.9 a (3) 12.55 ± 0.5 a (3) 13.46 ± 0.6 a (3) 5.22 9.92 1 CV(%), coefficient of variation; 2 Values represent mean and standard deviation (n = 3 or n = 9, different sample size due to the amount of infected tissue obtained); 3 Means followed by the same letter in the line do not differ statistically by the Tukey test at the 5% level of significance

Table 1 .
PMA-qPCR analysis and quantification of 'Candidatus Liberibacter asiaticus' extracted by different methods from different host plants.