First Report of Physico-Chemical Agents of Viable But Non Culturable (VBNC) forms of Ralstonia pseudosolanacearum

The germicidal efficacy of various physico-chemical agents against culturable bacterial cells and bacterial spores is well studied and reviewed; except for the dormant Viable But Non Culturable state (VBNCs). VBNC is a special physiological state, where the bacterium is not culturable in culture media, but remains alive, infective and virulent. The aim of this study is to evaluate the germicidal efficacy of three physico-chemical agents against the VBNC forms of destructive phytopathogen Ralstonia pseudosolanacearum. Effect of thermotherapy Formalin and Sodium Hypochlorite on these forms was evaluated using standard plate count and reverse culturability tests. The disinfectants were found equally effective against the culturable cells and VBNC forms at recommended concentrations.

3) is a soil, seed and waterborne bacterial phytopathogen which causes the bacterial wilt disease across 53 different botanical families. 1,2he bacterium infects through the root epidermis and colonizes the xylem, resulting in wilting and subsequent plant death.In the environment, the bacterium survives in reservoir plants, soil and water through diverse strategies, including the formation of Viable But NonCulturable (VBNC) state. 3any bacterial species are known to enter the VBNC state during the logarithmic growth phase (stochastic transformation) and stressed phase triggered by chemical and environmental factors.5][6][7][8] R. pseudosolanacearum enters into VBNC state under nutrient starvation, low temperature and heavy metal exposure and are capable of causing disease outbreaks when environmental conditions turn favourable for infection. 9-11R. pseudosolanacearum is a soil, water and seed-borne pathogen. 12To reduce its load in seeds and other planting materials, laboratories perform various phytosanitary measures like thermotherapy at 60°C for two hours 13 treatment with 50% sodium hypochlorite 14 and 0.5% formalin. 15The efficacy of these chemical and physical agents on culturable cells of different microcosms have been extensively published and validated through laboratory protocols.But, to our knowledge, no scientific publication has reported the efficacy of the above mentioned phytosanitary measures and disinfectants on VBNC forms of R. pseudosolanacearum or in any other phytopathogen.In this study, we have evaluated the germicidal efficacy of physico-chemical agents on two strains of R. pseudosolanacearum culturable cells and VBNC forms.

Preparation of microcosms
R. pseudosolanacearum isolates used in this study are DIBER115 (NCBI Accession number MG266193) and DIBER118 (NCBI Accession number MG266203).These isolates collected from different locations in the state of Uttarakhand, India was confirmed by 16s rDNA sequencing and pathogenicity assays.The phylotype of the isolates were found to be Phylotype I of R. solanacearum species complex 16 which are taxonomically reclassified as R. pseudosolanacearum. 1 The other strains used in this study were Ralstonia pickettii (ATCC 648) and E. coli (ATCC 10536).R. pseudosolanacearum strains were grown and counted in mSMSA Modifed Semi Selective South Africa Media (mSMSA) broth and mSMSA plates, respectively. 17Other strains were grown in Nutrient broth and Nutrient agar.Dead cells of R. pseudosolanacearum (DIB115D and DIB11 8D) for controls were prepared by exposing live cells in logarithmic phase with UV at 2,000 µJ/ cm 2 .For all Physico-Chemical treatments, the concentration of culturable cells and VBNC forms were maintained between 1-5X10 6 Cfu/ml.For concentration of VBNC forms, was fixed after counting the resuscitated cells.

Preparation and confirmation of R. pseudosolanacearum VBNC forms
VBNC forms of R. pseudosolanacearum (DIB115V and DIB118V) were prepared by incubating water washed cells at 4°C in a refrigerator for 180 days. 18Generated VBNC forms were confirmed by direct viable count and reverse culturability assay. 19Before proceeding for physical and chemical treatments, the VBNCs were normalised to a concentration of 1-5X10 6 Cfu/ml using sterile water.
The data presented in this study are the means of two experiments.The difference between the samples duplicates were analysed by two-tailed "Student's t" test.P-Value ≤ 0.05 denotes significant variation between the sample duplicates.

Physical and Chemical treatment
Thermotherapy was carried out at 60°C for 2 hrs by placing 1 ml of microcosms in glass test tubes in a heating water bath as per the protocol given by Grondeau and coworkers.After treatment, the viability, reverse culturability and Pathogenicity index were determined after washing the microcosms twice with sterile distilled water.For Chemical treatment, pelleted microcosms were exposed to different v/v concentrations of Formalin and NaOCl.Sauer and Burroughs in 1986 noticed the differences in disinfection efficacy among different brands of NaOCL.] The chemical agents were allowed to react with microcosms for 5 minutes and 10 minutes.After physical and chemical treatments, the microcosms were washed twice with sterile distilled water, thereafter processed for viability and reverse culturability assay and pathogenicity determination.Appropriate controls were also taken.

Reverse Culturability Assay
Reverse culturability otherwise known as resuscitation is used to confirm VBNC forms.All stress exposed Microcosms were resuscitated 22 in sterile distilled water supplemented with 1000u/ml of catalase (HiMedia Labs, Mumbai).Resuscitation was carried at 28°C for five days and the resultant growth was counted on mSMSA plates after washing twice with sterile distilled water.

Pathogenicity assay
Pathogenicity was performed on bacterial wilt susceptible Tomato variety seeds ICAR H-86 as per the protocol developed by Singh groups 23 The disease severity scale was determined on 7 th day according to the key: 0 = No symptoms; 1 = One leaf wilted; 2 = Two leaves wilted; 3= Plant dead.The Disease index (DI) was calculated by the formula DI = DI=ΣRT×100/4N.Where: R = disease severity scale (0, 1, 2, 3); T = number of wilted plants in each category and N = total number of tested plants.Pathogenicity tests were not performed for E. coli and R. pickettii.

Confirmation of VBNC state in R. pseudosolanacearum
In sterile water, R. pseudosolanacearum strains DIBER115 and DIBER118 lost their culturability within 180 days as determined by direct plate counting.Hence, the 'Alive' statuses of the cells were confirmed by reverse culturability, where about 80-90% of the cells were resuscitated following catalase treatment (Table 1).This confirms that a substantial culturable microcosms has transformed into VBNC forms.The VBNC forms were rechristened as DIB115V and DIB118V.Hereafter, experiments were performed to determine the germicidal efficacy of physicochemical agents against R. pseudosolanacerum cells and its VBNC forms.

Efficacy of Physical Treatment
The Phytosanitary method evaluated was thermotherapy, which was performed at 60°C for 2 hrs.The prescribed temperature and time were found sufficient for total inactivation of the pathogen as revealed by direct plate count, reverse cultivability, pathogenicity tests.Meanwhile, some culturable cells of E. coli and R. pickettii cells showed resistance towards thermotherapy (Table 2).

Effect of Chemical Treatment
All the chemical disinfectants at concentrations recommended for phytosanitation were effective in inactivating all the culturable and VBNC microcosms within 5 minutes.None of the treated microcosms were successful in causing bacterial wilt in host plants.The effect of different concentrations of formalin and NaOCl is summarized in (Table 2).

disCussiON
To prevent seed-borne infections, various phytosanitary measures like thermotherapy, washing with chemical disinfectants like Sodium Hypochlorite (NaOCl) and formalin 15 are widely used.In this work, we evaluated the efficacy of these phytosanitary measures.Cells that are metabolically or physiologically active (Viable) but cannot be cultured on microbial media are known as Viable But Nonculturable Cells (VBNC).To confirm the live/dead status of microcosms, we used direct plate count and reverse culturability (plate count after resuscitation) with catalase, as reported. 24BNC forms of two R. pseudosolanacearum strains were prepared by incubating culturable cells at refrigerating temperature for 180 days.The transformation was periodically monitored by direct plate count and reverse culturability assay.Both the strains DIBER115 and DIBER118 reached nonculturable state in 180 days.The fundamental property of VBNC forms is resuscitation. 25o confirm VBNC transformation on 180 th day, reverse culturability assay using catalase was performed.In 180 days, 90% of the cells were reverse culturable.Previous studies with Log 6.78 R. solanacearum cells have reported loss of pathogenicity within 125 days. 18This variation between ours and Leo et al is due to strain to strain variation. 26hermotherapy, at 60°C for 2 hrs is widely followed approach for phytosanitation.The approximate maximal growth temperature of R.pseudosolanacearum is 37-39°C with optimum growth temperature between 27-35°C. 27Increasing the temperature beyond the optimal value hampers the cells survivability.Thermotherapy to inactivate pathogens depends on incubating microcosms at temperatures beyond the maximal growth temperature. 13Previously, a 7 log reduction of R. solanacearum culturable cells in infested soil using thermotherapy at 60°C for 2 hrs was achieved. 28We are of the view, that the effect of heat on culturable cells as revealed by Jirasak and Siricha should not be generalized to VBNC forms.Hence, we exposed culturable cells and VBNC forms to 60°C for 2 hrs and observed total elimination of R. pseudosolanacearum culturable cells and VBNC forms as confirmed by zero colony counts (Table 2).
Sodium hypochlorite is one of the best Chlorine releasing agents, which forms hypochlorite ion, OCl-and hypochlorous acid (HOCl), which are known oxidizers of various biomolecules. 20In our laboratory, we routinely treat tomato seeds with 50% v/v NaOCl (which corresponds to 0.5% of active Chlorine) for 10 minutes and achieve seed germination efficacy of >90%.When exposed to 0.5% of active Chlorine, all microcosms, culturable cells and VBNCs were totally inactivated within 5 mins of exposure.To further investigate the effect at lower concentrations, NaOCl was diluted to active chlorine concentrations of 0.1%, 0.05% and 0.035% (0.035% of active Chlorine equals 10% v/v NaOCl).Previous studies with NaOCl 29 have established that, disinfection of seeds is incomplete at 5% v/v of NaOCl, hence we didn't continue with concentrations below 10% v/v dilutions.In this study NaOCl, was found effective upto 0.035% of active chlorine.Hence, it can be confirmed that, NaOCl can effectively sanitize seeds contaminated with R. pseudosolanacearum cells and VBNC forms at active chlorine concentration above 0.05%.
We noticed the same disinfection with formalin as in NaOCl.At concentrations used for seed sanitization (0.5% v/v), all microcosms were inactivated within 5 mins of exposure.This trend was also observed at treatments below 0.25% v/v.It was observed that unlike thermotherapy, both the chemical disinfectants didn't discriminate between the studied microcosms.No CFU count indicative of resuscitation was observed at tested concentrations, indicating that NaOCl with active chlorine concentration between 0.5-0.035%and formalin concentration between 0.5-0.25%can be used for germplasm sanitization within 5 minutes.However, it is recommended to treat the seeds for 10 minutes.

CONClusiONs
The germicidal action of phytosanitary methods against culturable cells has been described and endorsed previously.In this work, we evaluated the efficacy of phytosanitation method against VBNC.It was observed that the methods are as equally effective against VBNC as for culturable cells.
Journal of Pure and Applied Microbiology Faridi et al. | J Pure Appl Microbiol | 16(2):943-949 | June 2022 | https://doi.org/10.22207/JPAM.16.2.16 iNtROduCtiON R a l sto n i a p s e u d o s o l a n a c e a r u m (Previously R. solanacearum Phylotype 1 &