Low Temperature Induces Infectious Nonresuscitatable VBNC Cells in Ralstonia Pseudosolanacearum

Ralstonia pseudosolanacearum and other members of Ralstonia solanacearum species complex (RSSC) causes the disease bacterial wilt in many crops of economic importance. The organism is known to form Viable But Non Culturable cells (VBNC). VBNCs resuscitate invitro during the “resuscitation window” period and are infectious Previous studies have identied nonresuscitatable VBNCs in various bacterial genus including RSSC, however their infectivity was not elucidated and described. In this work, VBNCs of two Ralstonia pseudosolanacearum strains were generated by exposing the microcosms to psychrophilic stress, UV-C radiation and 70% isopropanol. Both resuscitatable and nonresuscitatable VBNCs were observed in psychrophilic and UV-C stressed microcosms. The nonresuscitatable VBNCs generated at psychrophilic temperature were found infective. Based on resuscitation properties, nonresuscitatable VBNCs can be considered as a different VBNC type from resuscitatable VBNCs.


Introduction
Ralstonia solanacearum species complex (RSSC) is a soil, seed and water borne bacterial phytopathogen which causes the disease bacterial wilt across different botanical families (Hayward 1994). Based on genomic differences, RSSC has been reclassi ed into three different species: Ralstonia solanacearum, Ralstonia pseudosolanacearum and Ralstonia syzygii (Safni et al. 2014). Ralstonia pseudosolanacearum (R. pseudosolanace arum) infects the host through root epidermis and colonizes xylem, resulting in wilting and subsequent plant death. In the environment, the bacterium survives in reservoir plants, soil Resuscitation or reverse culturability is the most important phenotypic character used to differentiate culturable cells from VBNCs. Resuscitation happens during the "resuscitation window" period under the in uence of external resuscitating agent like catalase. Beyond this period, VBNCs may remain viable, but are considered nonresuscitatable, noninfectious and nonpathogenic (Pinto et al. 2011;Pinto et al. 2015).
VBNCs nonresuscitatable with catalase has been observed in members of RSSC incubated in sterile arti cial soil (Kong et al. 2014). However, their infectivity is unknown. The existence of such infectious VBNCs will have profound implications on understanding the pathogen's life cycle and its detection. In this work, the formation of stress induced nonresuscitatable VBNCs in R. Pseudosolanacearum and their infectivity has been evaluated.

Materials And Methods
Bacterial strains and growth conditions Two R. Pseudosolanacearum isolates DIBER115 (NCBI Accession number MG266193) and DIBER118 (NCBI Accession number MG266203) isolated from tomato plants were used in this study. They belong to Phylotype-I of RSSC which are taxonomically reclassi ed as R. pseudosolanacearum (Prior and Fegan 2005;Safni et al. 2014). Both the strains were grown at 28 0 C in casamino acid peptone glucose (CPG) broth for 48 hours and counted in CPG agar (Kelman A. 1954).

Induction of VBNC state at psychrophilic temperature
To induce VBNC state, CPG grown microcosms were pelleted, water washed and resuspended in sterile distilled water and aliquoted into batches of 10 ml to prevent contamination during periodical investigations. The microcosms were given psychrophilic stress at 4 0 C for 480 days. The transformation was monitored periodically by direct viable plate count (DVC) and resuscitation assays. Whereas, the viability status and infectivity were monitored by quantifying the 16S rRNA transcriptomes and inoculation assays respectively (van Overbeek et al. 2004;Lahtinen et al. 2008).

UV-C radiation and isopropanol treatment
To induce VBNCs by UV-C and isopropanol, water washed CPG grown cells were resuspended in 10 ml of sterile distilled water or 70% isopropanol. For UV-C treatment, the microcosms were irradiated with different UV-C uencies using UV strata linker 2400 in "energy" mode (Strata gene, USA). The instrument emits radiation at a wavelength of 254 nm with a total power output of 75 watts. Brie y, 10 ml of microcosms in open petriplates were exposed to 200 mJ/cm 2 ,1000 mJ/cm 2 and 2500 mJ/cm 2 of UV-C uencies. During the exposure, the suspension was stirred slowly using magnetic stirrer to facilitate uniform radiation exposure.
In another set of experiment, alcohol stress was provided using 70% isopropanol for 5 and 10 minutes using standard microbiological methods. After exposing the microcosms to different stresses, the microcosms were washed once and resuspended in equal amount of sterile distilled water and processed for different examinations.
Con rmation of VBNC state VBNC cells are distinguished from culturable cells based on two key characteristics: nonculturability, resuscitatability and viability. In this work, nonculturability was con rmed by the absence of visible colonies in direct plate counting (DVC) technique. The resuscitatability and cell viability was determined by resuscitation assay using catalase and by quantifying 16S rRNA using Reverse-transcription quantitative polymerase chain reaction. 16S rRNA is a potential viability marker for psychrophilic stressed and UV-C exposed microcosms ( Brie y, 1 ml of the sample aliquots was pelleted at 5000g for 10 minutes and processed separately for DVC, resuscitation assay, inoculation assay and cell viability assays. Direct viable count was performed as follows. The cell pellet was resuspended in 1ml of sterile distilled water and serially diluted in sterile water. 100µl of the dilutions were spread plated on CPG agar plates and the resultant colonies were counted and expressed in logarithmic numbers. Untreated control cells were also pelleted and processed by the same method as test microcosms.

Resuscitation assay
Resuscitation or reverse culturability is considered as a fundamental property of pathogenic VBNCs.  Kong et al. 2004). After incubation, serial dilutions were performed and 100µl of the microcosms were plated onto CPG plates and monitored for colony formation. The resultant colonies were counted and expressed in logarithmic numbers.

RT-qPCR
As nonresuscitatable VBNCs cannot be grown and quanti ed in microbial culture media, reversetranscription quantitative polymerase chain reaction (RT-qPCR) was performed to determine the viability status of microcosms and quantify them. Brie y, pellet from the 1ml samples was resuspended in 1ml of TRIZOL reagent (Sigma Chemicals) and RNA extraction was performed as per manufacturer's instructions. The qPCR involving BRYT Green® dye chemistry was performed using 1-step RT-qPCR reagents (Promega Corporation, USA) after removing residual DNA using DNAse. The primer pairs Rp16F Epi uorescence microscopy Epi uorescence microscopy was performed to determine the membrane integrity of isopropanol treated microcosms using Live/Dead BacLight bacterium viability kit containing the dyes SYTO9 and Propidium iodide (PI). The staining was performed as per the manufacturer's instructions. The stained cells were viewed in Olympus BX63 microscope using image analysis software Olympus Cellsens Dimension (Version 1.16). The SYTO9 and PI channels were super imposed using the same software to view membrane integrity.

Data analysis
The data presented in this study are the means of two experiments. The difference between sample duplicates were analysed by two-tailed Student's t test. The percentage of nonresuscitatable VBNCs were calculated by comparing the16S rRNA copy number against the control by the formula: [(RNA copies of N th day/sample treatments X 100)/ RNA copies of N th day/control].

Results
Con rmation of VBNCs after psychrophilic stress  (Fig.1). These results con rm the existence of infectious nonresuscitatable VBNCs and their survival over a period of time.
To the best of our knowledge, the infectivity of nonresuscitatable VBNCs was not reported previously in any bacterial genera. In this work, varying degrees of wilting symptoms were observed on tomato seedlings inoculated with nonresuscitatable VBNCs. On 360 th day, the disease index was 65.3% and 33.3%, and on 480 th day it was 33.3% and 40% respectively for the strains DIBER115 and DIBER118 ( Fig.1). The infection was double con rmed by reisolating the pathogen from all wilted seedlings. These results con rm that nonresuscitatable VBNCs can also remain infectious over a period of time.

Con rmation of VBNCs after UV-C treatment
To investigate, whether the formation of nonresuscitatable VBNCs is restricted to psychrophilic stress or in generally associated with other stresses, we choose two stress inducers, UV-C and isopropanol.
Most of the studies on UV-C were performed below the uence of 200 mJ/cm 2 for water disinfection studies (Hinjen et al. 2006). At uence of 200 mJ/cm 2 , no colonies were observed in DVC (Table 1). However, log 6.389 (P=0.1) and log 6.34 (P=0.4) cfu/ml of cells resuscitated, con rming the transformation of culturable microcosms into resuscitatable VBNCs. These resuscitatable VBNCs were found infectious, which was con rmed by reisolating the pathogen from all wilted seedlings (Disease Index; DIBER115 -40%, DIBER118 -33%). These results con rm that UV-C induced resuscitatable VBNCs remain infectious. At uence of 1000 mJ/cm 2 , both the strains failed to resusucitate and infect the seedlings. To explore the possible formation of nonresuscitatable VBNC cells at this uence, the live/dead status of the microcosms were investigated by RT-qPCR. Presence of modest number of viable cells (0.37 % in DIBER115 and 8.5 % in DIBER118) was noticed, con rming the existence nonresuscitatable VBNC state. At higher uence of 2500mJ/cm 2 , all microcosms were eliminated as no transcriptomes were detected (Table 1).

Con rmation of VBNC state after isopropanol treatment
Alcohols are used at a concentration of 60 -90% (v/v) to eliminate spores, bacteria and viruses (McDonnell and Russell 1999). In this work, isopropanol, the common laboratory disinfectant was tested at a concentration of 70% (v/v). After 5 minutes of exposure, the average plate count of R. pseudosolanacearum strains DIBER115 and DIBER118 respectively dropped from log 6.39 (P=0.51) and log 6.28 (P=0.10) CFU/ml to below detection. However, when resuscitated with catalase, log 4.16 (P=0.07) and log 4.21 (P=0.33) cfu/ml of cells formed colonies, con rming the transformation of culturable microcosms into resuscitatable VBNCs. However, these VBNCs failed to infect the seedlings, concluding the loss of pathogenicity after isopropanol exposure (Table 1). In epi uorescence microscopy, most of the 5 min exposed cells were found double stained (>95%), where SYTO9 and PI simultaneously stain individual cells con rming damage to membrane integrity (Berney et al. 2007) (Fig. 2). The loss of infectivity observed was due to this membrane damage. After 10 minutes of exposure, both the strains failed to resuscitate and extensive loss of membrane integrity con rming cell death. However, the infectivity of these strains was not elucidated. The crucial observation in our study is the infectivity exhibited by UV-C (200 mJ/cm 2 ) exposed microcosms (Table 1). Although >99% of culturable microcosms transformed into VBNCs, only few cells were in a capacity to induce infection as evidenced by the low disease index observed (≤40%). UV-C damages nucleic acids and cellular membranes, which impairs the pathogens ability to infect their host (  (Table 1). Similarly, a subpopulation of cells is known to survive higher uencies of UV-C (800mJ/cm 2 ) irradiation (Guo et al. 2019). In that capacity, it can be hypothesized that some cells surviving 800 mJ/cm 2 can also survive 1000 mJ/cm 2 uence due to tailing effect (Schenk et al. 2011). Accordingly, in our experiments ≤0.3% of cells survived as nonresuscitatable VBNCs (Table 1).

Discussion
At higher uences of 1000 mJ/cm 2 , the acquired DNA and membrane damage may have crossed a critical threshold which is beyond the competence of cellular repair mechanisms (Rastogi et al. 2010). This accounts for the loss of infectivity observed at higher doses observed.
Our results raise a crucial question; why 16S rRNA transcriptomes were detected from cells having heavily In such a scenario the probability of 16S rDNA and its transcriptomes surviving a lethal UV-C is more than a gene having a single copy. Secondly, a preferential DNA damage prevention system equivalent to AidB protein (protects and repair housekeeping genes from alkalyting agents) can also be proposed (Rippa et al. 2011). To our knowledge, such selective mechanism for high energy radiation has not been elucidated. In the light of the above, we assume that the phenomenon encountered is more likely due to the presence of multiple copies of 16S rDNA genes.
Isopropanol is wildly used as sanitizer and disinfectant in laboratory, domestic and hospital settings. Immersing scalpels, scissors and other tools used for nursery operations in aqueous solution containing 70% isopropanol (v/v) for 10 minutes is an established phytosanitising procedure (Gebel et al. 2019;Wilkinson et al. 2014). Several disinfectants containing surfactants and salts were known to induce VBNC formation in many bacterial genera (Robben et al. 2018). However, the status of isopropanol is unknown. In our investigations, we observed 0.6% of culturable cells survived isopropanol by transforming into resuscitatable VBNCs after 5 minutes of exposure. However, these resuscitatable VBNCs failed to initiate any infection in tomato seedlings. This is due to the membrane damage caused by isopropanol, as evidenced by epi uorescence microscopy ( Fig.2) (Table 1). Meanwhile, cells exposed to low temperature retain membrane integrity and other molecular mechanisms involved in pathogenesis (Pawlowski et al. 2011 Therefore, to differentiate it from the classical resuscitating VBNCs belonging to "resuscitation window" type, these VBNCs can be considered as "nonresuscitatable VBNCs". Brie y the terms "resuscitatable VBNCs" and "nonresuscitatable VBNCs" denotes two different phenotypic variations revealed during catalase treatment. For practical purposes, they can be rechristened as Type-1 VBNC, which are resuscitatable and Type-2, which are nonresuscitatable. In such a scenario, a crucial question remains unanswered; whether Type-2 VBNCs are continuum of Type-1 VBNCs or stochastically formed within growing cultures? This can only be answered after unlocking the rudimentary molecular mechanisms associated with these phenotypic variations. The formation of nonresuscitatable VBNCs (Type-2) in response to various stresses and their ability to infect and initiate infections emphasise the importance of these cells in the life cycle and survival of R. pseudosolanacearum. Previous studies and our limited experiments emphasise the possibility of infective nonresuscitatable VBNCs existing in the environment (Caruso et al. 2005). This also incites the necessity to develop alternative VBNC detection strategies for environmental samples as classical resuscitation assays and RT-PCR targeting 16s RNA are indeed nonspeci c procedures when dealt with environmental samples having heterogenous microbial populations. In such scenario it is essential to identify and validate speci c R. pseudosolanacearum transcriptomes for identifying VBNCs from the environment.