Biocontrol potential of cell-free supernatant of Paenibacillus chitinolyticus against Plasmodiophora brassicae in two important Brassica species

Biocontrol potential of cell-free supernatant of Paenibacillus chitinolyticus against Plasmodiophora brassicae in two important Brassica species | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biocontrol potential of cell-free supernatant of Paenibacillus chitinolyticus against Plasmodiophora brassicae in two important Brassica species Maryam Khodashenas Rudsari, Miloslav Zouhar, Marie Manasova, Tongda Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3769218/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Plasmodiophora brassicae is a serious threat to Brassica crops worldwide, resulting in substantial economic losses for growers, with the challenge of control persisting. Biocontrol with chitinolytic bacteria producing chitinase is gaining attention as a natural alternative to chemicals. This approach is favored due to the essential role chitinases play in protecting against chitin-containing pathogens. Given that chitin is a major component in the resting spores of P. brassicae and plays a crucial role during pathogenesis, it is probable that Paenibacillus chitinolyticus , producing a high level of chitinase, could suppress P. brassicae by targeting chitin in a critical stage of this pathogen’s life cycle. Our research aimed at evaluating the effect of various applications of P. chitinolyticus on clubroot suppression in two economically important Brassica species: Chinese cabbage and rapeseed. The effectiveness of the cell-free supernatant (CFS) of an endemic strain of P. chitinolyticus from the Czech Republic at five different time points was studied in the greenhouse by measuring the disease severity index. The results showed that early application of P. chitinolyticus decreased the disease index significantly within both plants. Additionally, in both plants, P. chitinolyticus increased shoot dry weight to a great extent. In conclusion, the CFS of P.chitinolyticus has significant antagonistic activity against clubroot in Chinese cabbage and rapeseed in the early developmental stages of clubroot occurrence and holds the potential as a biofertilizer as well as bioprotectant agent in clubroot management of P. brassicae . Chitinolytic bacteria Clubroot Rapeseed Chinese cabbage Paenibacillus chitinolyticus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Chinese cabbage ( Brassica rapa L.) is one of the most important green leafy vegetables in the family Brassicaceae with increasing popularity in the world (Sivanandhan et al., 2021 ; Vanjildorj et al., 2009 ). Rapeseed ( Brassica napus L.) is also one of the most widely grown crops of Brassicas in the world and is the second most important crop after cereal in the Czech Republic (Anonymous, 2018 ). Both rapeseed and Chinese cabbage can be infected by Plasmodiophora brassicae severely (Ma et al., 2022 ; ŘičaŘoVá et al., 2016 ). P. brassicae is a soilborne pathogen that induces clubroot disease across all Brassicaceae species, resulting in significant yield losses (Woronin, 1878 ; Zhu et al., 2020 ). Managing P. brassicae proves challenging due to persistent resting spores in the soil, a broad host range, and pathotypes capable of overcoming resistance in cultivars (Diederichsen et al., 2014 ). Cultural strategies, such as extended rotations and equipment sanitization, show efficacy but lack widespread adoption (Hwang et al., 2014 ; Strelkov et al., 2011 ). In the ongoing challenge of managing clubroot disease, the use of microbial antagonists like Trichoderma spp., Bacillus spp., Saccharomyces cerevisiae , and Acremonium spp. has emerged as a promising alternative (Gao and Xu, 2014 ; Jäschke et al., 2010 ; Zhu et al., 2020 ). However, a few numbers of these candidates were highly effective in the high inoculum pressure, especially in the field, or have been registered for commercial uses (Narisawa et al., 1998 ; Peng et al., 2011 ). Given the diverse mechanisms of action of biocontrol agents and the complex life cycle of P. brassicae , a strategic approach focusing on correlation between antagonist’s biocidal substances and critical stages of P. brassicae life cycle was proposed in current study. It has been reported that the critical stage in the life cycle of P. brassicae , where it is most volnurable, is the brief interval between germination of resting spore and penetration into the host (Dixon, 2009 ). P. brassicae resting spore has also a key role to commence pathogenesis (Kageyama and Asano, 2009 ) and its walls contain 25% of chitin (Moxham and Buczacki, 1983 ). Notably, chitin synthase-encoding genes are expressed during P. brassicae infection process (Schwelm et al., 2015 ). Therefore, it is probable that chitin degrader like chitinolytic bacteria, through the breakdown of chitin and decreasing resting spores germination, may serve as potent antagonists of this pathogen (Dixon, 2009 ; Hjort et al., 2007 ). Furthermore, chitinolytic bacteria producing chitinases were recognized as a promising alternative to synthetic chemicals. This approach is favored due to the essential role chitinases play in protecting against chitin-containing pathogens (Chen et al., 2018 ; Kumar et al., 2022 ; Prapagdee et al., 2008 ; Prasanna et al., 2013 ; Subbanna et al., 2018 ). Paenibacillus bacteria are gaining attention for their effectiveness against soil-borne pathogens, produce a variety of metabolites, including antimicrobial peptides and chitinolytic enzymes (Akhtar and Siddiqui, 2007 ; Budi et al., 2000 ; Grady et al., 2016 ; Jung et al., 2003 ; Li et al., 2021 ; Seo et al., 2016 ; Singh et al., 2019 ; Xu et al., 2014 ). Previous studies highlighted the role of chitinase enzymes in Paenibacillus cell-free supernatant for biocontrol against phytopathogens (Loni et al., 2014 ; Seo et al., 2016 ). Given the well-known chitinase production ability of Paenibacillus chitinolyticus (Liu et al., 2020a ; Song et al., 2015 ), we hypothesized its potential as an effective biological agent for clubroot management. Additionally, the genus Paenibacillus exhibits noteworthy traits to promot plant growth (Grady et al., 2016 ; Li et al., 2021 ), which was of interest in current study. Considering the significant impact of clubroot infection on globally essential crops, including Chinese cabbage and rapeseed, leading to substantial economic losses for farmers, this study was conducted to assess the efficacy of P. chitinolyticus , an endemic chitinolytic bacterium in the Czech Republic, in combating clubroot in Chinese cabbage and rapeseed. The research also aimed to evaluate the effectiveness of different time points of P. chitinolyticus application against clubroot in greenhouse conditions. This work may contribute to the development of effective strategies for clubroot biocontrol and the facilitation of sustainable agriculture. Materials and methods Plant materials Seeds of Brassica rapa subsp. pekinensis (Chinese cabbage cv. ‘Granaat’) considered a universally susceptible host to clubroot (Buczacki et al., 1975 ) and seeds of Brassica napus subsp. napus (rapeseed var. inspiration) were obtained from Osiva Moravia, Czech Republic. The substrate was Gramoflor (final pH 6.2) and was obtained from Germany. The seeds were sown in 2 cm deep holes in the sterilized substrate in 9×9×10 cm plastic pots with 2 seeds per pot to ensure uniform growth and thinned to one seedling per pot before inoculation. Pots were kept in a greenhouse at Czech university of life science, Prague, at 24_+2°C, and a 16 h photoperiod for 45 days. Plants were irrigated to maintain soil moisture but were not water saturated. Pathogen material Mature clubroot galls were collected from infested rapeseed fields in the Czech Republic, dried at room temperature, and stored at -20°C until use. Following the method described by Castlebury et al. ( 1994 ), crude resting spores were extracted (Castlebury et al., 1994 ). Approximately 5 g of dried galls were soaked in 50 ml sterile deionized water (SDW) and then blended at high speed for 2 minutes. The resulting suspension was filtered through eight layers of cheesecloth. Concentrations of resting spores were estimated using a hemocytometer, and spore suspensions were diluted with SDW to 1 × 10 7 resting spores per ml for plant inoculation. Bioagent material (Bacterial inoculum) Endemic strain of P. chitinolyticus CCM 4527, was provided by the national collection of microorganisms in Brno, Czech Republic. To produce P. chitinolyticus inoculum, CFS was obtained after the growth of P. chitinolyticus in a liquid medium, followed by centrifugation and filtration through a small pore-size filter to remove all bacterial cells. For this purpose, single colonies of P. chitinolyticus were cultured on Luria- Bertani (LB) Agar medium, transferred to LB liquid medium, and incubated at 30°C for 3 days at 180 RPM to prepare bacterial fermentation (Zhu et al., 2020 ). The optical density of bacterial fermentation was measured using nanodrop (OD600 = 0.8). The fermentation filtrate of the bacterial isolate was centrifuged at 8000 RPM for 15 min, and the supernatant was filtered through a 0.22 mm cellulose nitrate filter to obtain bacterial CFS (Zhu et al., 2020 ). Plants Inoculation Inoculation with the pathogen ( P. brassicae ) was conducted 10 days after planting when all plants had developed two true leaves. This involved applying 2 ml of 1×10 7 resting spores ml -1 per plant by pipetting into the soil. For bioagent inoculation, each seedling received 10 ml of P. chitinolyticus CFS. Given the critical stages of P. brassicae life cycle, including resting spore germination, primary infection, and secondary infection, we investigated the efficacy of the bioagent against clubroot at 5 different time points. Plants were inoculated with P. chitinolyticus CFS on the same day as the pathogen was inoculated (0), on day 5, day 9, day 14, and day 18 after the pathogen inoculated. Evaluation of biocontrol potential of P. chitinolyticus on clubroot Disease severity was determined 35 days after plants inoculation. The evaluation involved removing all the plants and washing the roots under tap water to eliminate soil particles. Clubroot severity was visually assessed based on a scale of 0 to 3 (0 = no galling, 1 = a few small galls, 2 = moderate galling, and 3 = severe galling) (Kuginuki et al., 1999 ). The disease severity index (DSI) was calculated using the following equation: \(\text{D}\text{S}\text{I} \text{\%}=\frac{{\Sigma } (\text{n}\times 0+\text{n}\times 1+\text{n}\times 2+\text{n}\times 3)}{\text{N} \times \text{N}\text{o}. \text{o}\text{f} \text{c}\text{l}\text{a}\text{s}\text{s}\text{e}\text{s} \text{w}\text{i}\text{t}\text{h} \text{s}\text{y}\text{m}\text{p}\text{t}\text{o}\text{m}\text{s}}\) × 100 Here ‘n’ is the number of plants in each class; ‘N’ is the total number of plants; and the values 0, 1, 2, and 3 represent the respective symptom severity classes. Disease control effect (DCE) was calculated using the following formula: \(\text{D}\text{C}\text{E} \text{\%}=(1-\frac{ \text{D}\text{S}\text{I} \text{i}\text{n} \text{t}\text{r}\text{e}\text{a}\text{t}\text{m}\text{e}\text{n}\text{t}}{\text{D}\text{S}\text{I} \text{i}\text{n} \text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}})\) × 100 Measurement of shoot dry weight Shoot fresh and dry weight of both plants was measured at the end of experiments to assess the effect of the antagonist on plants growth. Shoot fresh weight of all plants for each treatment was estimated by a scale, then a sub-sample of them were put in paper bags and dried in an oven at 70° C, for 2 days. Dry weight was calculated by humidity percentage measured after oven-drying of the plant sub-samples (Mousavi Shalmani et al., 2017 ). Experimental design and Statistical analysis The experimental design employed a split plot design based on completely randomized design (CRD) with four replicates per treatment, and each experimental unit consisted of 10 plants. The first level comprised two Brassica species: Chinese cabbage and rapeseed. The second level included six treatments, encompassing five different time points of P. chitinolyticus CFS, and one control. Control was inoculated with P. brassicae and treated by 10 ml of LB liquid medium on the same day as the pathogen inoculated. The treatments with P. chitinolyticus CFS at five different time points were as follows: 0, 5, 9, 14, and 18 day after pathogen inoculation (DAPI). Statistical analyses, including ANOVA and multiple comparisons (Duncan Test), were conducted using GenStat-14 (General Statistics software). Results Biocontrol effects of P. chitinolyticus on clubroot disease The average clubroot severity index in Chinese cabbage by treatments of P. chitinolyticus CFS at different time points was presented in Fig. 1 . The analysis of the effect of P. chitinolyticus on the clubroot severity index showed differences in DSI values among the different time points. Clubroot severity index had a value of 96% in control which was inoculated just with P. brassicae . When P. chitinolyticus was applied at 0 DAPI, the highest reduction in clubroot severity index (49%) was achieved. Moreover, clubroot severity index was significantly lower than control when P. chitinolyticus applied at 5 DAPI and 9 DAPI, which were not significantly different from 0 DAPI (Fig. 1 ). Clubroot progressed at the highest rate when P. chitinolyticus inoculated at 18 DAPI. Application of P. chitinolyticus at 14 DAPI provided almost same clubroot severity index with 18 DAPI and there were no significant reductions in Clubroot severity index of these two treatments compared to the control (Fig. 1 ). In case of rapeseed, DSI had a value of 91% in control which was inoculated just with P. brassicae (Fig. 2 ). Significant reductions in clubroot DSI were found with inoculations of P. chitinolyticus at 0, 5 and 9 DAPI, whereas there were no significant reductions in DSI with delay in application of P. chitinolyticus at 14 DAPI and 18 DAPI. The highest reduction in clubroot DSI in rapeseed was observed at 0 DAPI which was 2.5 times less than control and followed by its application at 5 DAPI and 9 DAPI. Treatment with P. chitinolyticus at 9 DAPI lowered clubroot to 50% compared to control that was less efficient than that of at 0 DAPI but was not significantly different from 5 DAPI. The results showed that DSI with a value of 85% and 90.8% were observed with inoculations of P. chitinolyticus on 14 DAPI and 18 DAPI respectively, which had no significant difference when compared to that in control. Application of P. chitinolyticus at 14 DAPI was not significantly different from 18 DAPI, while were significantly different from treatment at 0, 5, and 9 DAPI (Fig. 2 ). The average clubroot control effect in Chinese cabbage and rapeseed by treatments of P. chitinolyticus at different time points was presented in Fig. 3 . The highest clubroot control effect on Chinese cabbage was 49.2% that achieved with application of this strain at 0 DAPI, in which pathogen and antagonistic bacteria have been used at the same day. Treatment with P. chitinolyticus at 5 DAPI and 9 DAPI controlled clubroot by 43.5% and 37.4% respectively and there was not found to be a significant difference between these treatments (Fig. 3 ). In rapeseed, comparing all treatments of P. chitinolyticus , its application at 0, 5, and 9 DAPI could control clubroot up to 60.1%. Multiple comparisom of Chinese cabbage and rapeseed showed that in both plants, P. chitinolyticus CFS at 0, 5 and 9 DAPI controlled clubroot. However, no significant differences were observed in the ability of P. chitinolyticus CFS to control clubroot between Chinese cabbage and rapeseed (Fig. 3 ). Impact of P. chitinolyticus on dry matter production The effect of P. chitinolyticus on the shoot dry weight of Chinese cabbage was shown in Fig. 4 . The results showed a significant increase in dry matter production across all treatments with P. chitinolyticus compared to the control, where plants were only inoculated with the pathogen. No significant differences were observed within P. chitinolyticus treatments at 0, 5, 9, 14 and 18 DAPI. The maximum shoot dry weight increased by 1.47 times compared to the control (Fig. 4 ). The impact of P. chitinolyticus on the shoot dry weight of rapeseed was outlined in Fig. 5 . Similar to its effect on Chinese cabbage, the application of P. chitinolyticus showed consistent enhancement in rapeseed dry matter at various time points. No significant differences were observed among treatments with P. chitinolyticus at 0, 5, 9, 14 and 18 DAPI. The maximum shoot dry weight saw a 1.46-fold increase compared to the control. (Fig. 5 ). In both plants, dry matter production was significantly influenced by P. chitinolyticus treatments at all time points. Moreover, the efficiency of P. chitinolyticus was similar when comparing between Chinese cabbage and rapeseed and the maximum shoot dry weight increase, reaching almost 45%, was observed in both plants (Fig. 6 ). Discussion Our research aimed for evaluating the effectiveness of various application of P. chitinolyticus CFS on clubroot suppression in Chinese cabbage and rapeseed in greenhouse. The result showed that P. chitinolyticus CFS was capable of suppressing clubroot (based on clubroot control effect) up to 49.2% in Chinese cabbage and 60.1% in rapeseed. However, no significant difference was observed in the efficiency of P. chitinolyticus to control clubroot on Chinese cabbage and rapeseed. In addition, P. chitinolyticus was able to increase shoot dry weight to a great extent simultaneously (in average 40%), which offers significant potential as a bioagent in Chinese cabbage and rapeseed. Although Paenibacillus species have been emphasized to control many disease (Akhtar and Siddiqui, 2007 ; Grady et al., 2016 ; Singh et al., 2019 ; Xu et al., 2014 ), only a few of them have been reported to reduce clubroot severity. Liu et. al. ( 2014 ) have reported that P. polymyxa was effective in clubroot suppression on cabbage up to 71. 09% (Liu et al., 2014 ). In their study, the fermented liquid of P. polymyxa showed more control efficacy than Bacillus subtilis. Additionally, P. kribbensis has been reported to inhibit clubroot in Chinese cabbage in greenhouse, achieving control rates ranging from 89–99.2% (Xu et al., 2014 ). These two prior studies using different species of Paenibacillus have showed that this genus was effective in clubroot suppression, and this was seen in our experiment, albeit to a lower proportion. To our knowledge it is a first report of usage of P. chitinolyticus to control clubroot in rapeseed and Chinese cabbage. Despite the availability of two previous studies on using Paenibacillus for clubroot control, none of them focused on the effect of timing of Paenibacillus application against the pathogen. This study filled this gap and results showed that delayed application of this antagonist in Chinese cabbage and rapeseed, independently of the host, could not control clubroot. This indicated the bacterium’s effectiveness in the early stages of clubroot development but lack of suppressive effects in the later stages. Our study supported an earlier study (He et al., 2019 ) that early application of B. subtilis was the best way to reduce the clubroot severity on Chinese cabbage. Our results agreed with their conclusion that time of application of bacterial antagonist had an important effect on clubroot suppression. Such results suggested that early-stage interaction between P. chitinolyticus and P. brassicae is critical in clubroot inhibition. Our findings about the most effective time point for P. chitinolyticus application demonstrated that P. chitinolyticus suppressed P. brassicae significantly when applied on the same day as P. brassicae inoculated, on day 5 and 9 after P. brassicae inoculation, while no suppression occured in latter treatment on day 14 and 18 after P. brassicae inoculation. The critical stage in the life cycle of P. brassicae , where it is most susceptible, is the brief interval between germination and penetration of resting spore into the host root hair. Understanding the factors affecting resting spores germination, which is essential for P. brassicae pathogenicity, is key to controling clubroot disease (Dixon, 2009 ). Consistent with Dixon’s insights, the observed suppression on the same day as P. brassicae inoculated in our study, seems attributable to the inhibitory effect of P. chitinolyticus CFS on the germination of resting spores as an essential component within the most vulnerable phase of the P. brassicae life cycle. These findings emphasized the potential of P. chitinolyticus as an effective antagonist, strategically targeting chitin in a critical stage in P. brassicae development and disrupting the pathogen life cycle . Inhibitory effect of P. chitinolyticus CFS on 5 and 9 DAPI might be due to its antagonistic effect on primary infection of P. brassicae including primary zoospores, primary plasmodium and zoosporangium which were produced before day 9. Ineffectiveness on 14 and 18 DAPI is probabely due to inability of this bacterium on suppression of secondary infection of P. brassicae which was initiated around day 9 and subsequently developed till 18 DAPI in the inner tissue of the cortex. This assumption was further supported by findings about developmental stages of P. brassicae . Resting-spore germination is the first step in the life cycle of P. brassicae (Kageyama and Asano, 2009 ). Under favorable conditions the spores of P. brassicae can germinate immediately and releases zoospores, which form primary infection in the host. Liu et al. ( 2020c ) reported that P. brassicae initiates the primary infection on Arabidopsis root hairs and epidermal cells at 1 day after Plasmodiophora inoculation, with the establishment of secondary infection occuring in cortical cells at 8 day after Plasmodiophora inoculation (Liu et al., 2020c ). Zhu et al. ( 2020 ) reported a slightly adjusted timeframe of 2 days after Plasmodiophora inoculation for primary infection and 9 days after Plasmodiophora inoculation for secondary infection in a susceptible oilseed rape cultivar, aligning with similar data reported for susceptible Chinese cabbage cultivars (Liu et al., 2020b ). It can therefore be assumed that Paenibacillus should be considered as an antagonist against resting spores germination and primary infection of P. brassicae in Chinese cabbage and rapeseed. Interestingly, P. chitinolyticus efficacy on day 5 and 9 after P. brassicae inoculation, suggested that this antagonist can compete with established P. brassicae resting spores to some extent. This partial curative potential may be a favourable aspect for use in the field which requires further study. In the present study, CFS of P. chitinolyticus has been used. CFS are rich in metabolites with antimicrobial activity. Notably, the potential of this resource to control phytopathogens could be further enhanced by optimizing the condition and culture (Mani-López et al., 2022 ; Singh, 2010 ). This optimization strategy holds promise in revealing a broader spectrum of antimicrobial compounds in CFS of P. chitinolyticus , exhibiting enhanced ability to combat Plasmodiophora. This warrants further examination in the future. Our results revealed the significant ability of CFS of P. chitinolyticus to suppress clubroot. We utilized P. chitinolyticus , a strain that produces a high level of chitinase, which plays a pivotal role in the antagonistic ability of this species against plant pathogens (Ahmadi et al., 2008 ; Budi et al., 2000 ; Liu et al., 2020a ; Song et al., 2015 ; Von der Weid et al., 2003 ). This finding aligns with the recognized role of chitinase enzymes in the CFS of various Paenibacillus species for biocontrol, targeting specific pathogens such as Collectotrichum gloeosporioides , Rhizoctonia solani , and Aspergillus sp. (Loni et al., 2014 ; Seo et al., 2016 ). Microbial chitinases are believed to act against chitin-containing pathogens by degrading chitin, thereby exhibiting biocontrol potential (Chen et al., 2018 ; Gomaa, 2021 ; Heustis et al., 2012 ; Sharma et al., 2020 ). Given that chitin is a major component in the resting spores of P. brassicae (Moxham and Buczacki, 1983 ) and various chitin synthesis genes are significantly upregulated during the infection process of this pathogen (Schwelm et al., 2015 ), we hypothesized that chitinase in P. chitinolyticus CFS could target and degrade the chitin in P. brassicae . Our suggestion regarding the potential link between clubroot suppression and the chitinases present in the CFS of P. chitinolyticus can be further supported by up-regulation of chitinase-related genes in Chinese cabbage in response to P. brassicae infection (Chen et al., 2016 ; Chen et al., 2018 ). They inferred that the induced chitinase might provide the evidence for the degradation of chitin secreted by P. brassicae . Moreover, the discovery of chitin-binding effectors in P. brassicae , which can bind to resting spores' chitin for the suppression of chitin-triggered immunity in rapeseed (Muirhead and Pérez-López, 2022 ), further strengthened our hypothesis. Chitinases, as shown in their study, prevent these chitin-binding effectors from binding to resting spores, thereby increasing plant immunity. While these findings supported our results and hypothesis regarding the inhibitory roles of P. chitinolyticus chitinase in clubroot suppression through chitin targeting, further studies are necessary to elucidate the specific functions of chitinase in P. chitinolyticus CFS and the underlying mechanisms through which it suppresses P. brassicae . Potential experiments could involve generating mutants of P. chitinolyticus lacking chitinase activity and assessing their bioprotective efficacy against clubroot. Furthermore, a transcriptomic analysis may be conducted for both mutant and original strains. We demonstrated that P. chitinolyticus effectively enhances the growth of Chinese cabbage and rapeseed, leading to significant increases in shoot dry weight. This positive impact remained consistent across various treatments of P. chitinolyticus , including both early and late inoculations. The ability of P. chitinolyticus to enhance shoot dry weight may be attributed to several mechanisms, such as phosphate solubilization, nitrogen fixation, and hormone production. This aligns with the mechanisms of many other species of Paenibacillus , known for their capacity to promote plant growth (Grady et al., 2016 ; Li et al., 2021 ). The significant influence of P. chitinolyticus on growth, coupled with its bioprotective ability, positioned it as a promising bioagent for managing clubroot in Chinese cabbage and rapeseed. This statement became more important when considering the decrease in plant yield due to clubroot in previous studies. Greenhouse experiments revealed canola yield was reduced between 0.26% and 0.49% for each 1% increase in the clubroot disease index (Botero-Ramírez et al., 2022 ). Furthermore, field experiments demonstrated yield losses of approximately 0.03 tonne per hectare for every 1% increase in clubroot severity in rapeseed (McGrann et al., 2016 ). The observed growth-promoting effects of P. chitinolyticus in our study suggested a promising approach to mitigate yield losses in brassica fields at risk of clubroot, which should be examined in future studies. Conclusion In conclusion, our findings demonstrated the antagonistic activity of P. chitinolyticus CFS against clubroot in Chinese cabbage and rapeseed during the early stages of clubroot occurrence. Chitinase enzymes play important roles in Paenibacillus CFS, combating chitin-containing phytopathogens. Considering the remarkable chitinase production capability of P.chitinolyticus , its efficacy against clubroot likely stems from a precise targeting of chitin during critical stages of the pathogen's life cycle, but additional research is needed to prove this assumption, and elucidate the mechanism of action of P. chitinolyticus CFS. This can clarify the role of antimicrobial compounds such as chitinas derived from P. chitinolyticus CSF and finding novel compounds to replace conventional toxins for suppressing Plasmodiophora in Brassicaceae. The positive impact of P. chitinolyticus CFS on shoot dry weight in Chinese cabbage and rapeseed underscores its potential as both a biofertilizer and a bioprotectant agent in clubroot management. However, field trials should be the next step to verify the the efficacy of P. chitinolyticus CFS in clubroot control. Declarations Funding This study was funded by Czech University of Life Sciences, Prague. Confict of interest: The authors have no financial or proprietary interests in any material discussed in this article. References Ahmadi, K., Yazdi, M. T., Najafi, M. F., Shahverdi, A., Faramarzi, M., Zanini, G., & Behrava, J. (2008). Isolation and characterization of a chitionolytic enzyme producing microorganism, Paenibacillus chitinolyticus JK2 from Iran. 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Antimicrobial properties of the novel bacterial isolate Paenibacilllus sp. SMB1 from a halo-alkaline lake in India. Scientific reports , 9 (1), 11561. Sivanandhan, G., Moon, J., Sung, C., Bae, S., Yang, Z. H., Jeong, S. Y., Choi, S. R., Kim, S. G., & Lim, Y. P. (2021). L-Cysteine increases the transformation efficiency of Chinese cabbage (Brassica rapa ssp. pekinensis). Frontiers in Plant Science , 12 , 767140. Song, Y. S., Seo, D. J., Ju, W. T., Lee, Y. S., & Jung, W. J. (2015). Enzymatic properties of chitinase-producing antagonistic bacterium Paenibacillus chitinolyticus with various substrates. Microbial pathogenesis , 89 , 195–200. Strelkov, S. E., Hwang, S. F., Howard, R. J., Hartman, M., & Turkington, T. K. (2011). Progress towards the sustainable risk management of clubroot (Plasmodiophora brassicae) of canola on the Canadian prairies. Subbanna, A., Rajasekhara, H., Stanley, J., Mishra, K., & Pattanayak, A. (2018). Pesticidal prospectives of chitinolytic bacteria in agricultural pest management. Soil Biology and Biochemistry , 116 , 52–66. Vanjildorj, E., Song, S. Y., Yang, Z. H., Choi, J. E., Noh, Y. S., Park, S., Lim, W. J., Cho, K. M., Yun, H. D., & Lim, Y. P. (2009). Enhancement of tolerance to soft rot disease in the transgenic Chinese cabbage (Brassica rapa L. ssp. pekinensis) inbred line, Kenshin. Plant cell reports , 28 , 1581–1591. Von der Weid, I., Alviano, D., Santos, A., Soares, R., Alviano, C., & Seldin, L. (2003). Antimicrobial activity of Paenibacillus peoriae strain NRRL BD-62 against a broad spectrum of phytopathogenic bacteria and fungi. Journal of Applied Microbiology , 95 (5), 1143–1151. Woronin, M. (1878). Plasmodiophora brassicae, the cause of cabbage hernia. Translated by C. Chupp (1934). Phytopathological Classic No. 4. In: St. Paul, MN: American Phytopathological Society. Xu, S. J., Hong, S. J., Choi, W., & Kim, B. S. (2014). Antifungal activity of Paenibacillus kribbensis strain T-9 isolated from soils against several plant pathogenic fungi. The plant pathology journal , 30 (1), 102. Zhu, M., He, Y., Li, Y., Ren, T., Liu, H., Huang, J., Jiang, D., Hsiang, T., & Zheng, L. (2020). Two new biocontrol agents against clubroot caused by Plasmodiophora brassicae. Frontiers in microbiology , 10 , 3099. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision 01 May, 2024 Reviewers agreed at journal 01 Apr, 2024 Reviewers invited by journal 08 Jan, 2024 Editor invited by journal 22 Dec, 2023 Editor assigned by journal 20 Dec, 2023 First submitted to journal 19 Dec, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3769218","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":266004896,"identity":"cc4bff3c-fa4a-49d6-b1de-52f0e53d77cc","order_by":0,"name":"Maryam Khodashenas Rudsari","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0646-9841","institution":"Czech University of Life Sciences Prague Faculty of Agrobiology Food and Natural Resources: Ceska Zemedelska Univerzita v Praze Fakulta agrobiologie potravinovych a prirodnich zdroju","correspondingAuthor":true,"prefix":"","firstName":"Maryam","middleName":"Khodashenas","lastName":"Rudsari","suffix":""},{"id":266004897,"identity":"f70828f4-7a15-41e3-8c5e-f8af280d5e8d","order_by":1,"name":"Miloslav Zouhar","email":"","orcid":"","institution":"Czech University of Life Sciences Prague Faculty of Agrobiology Food and Natural Resources: Ceska Zemedelska Univerzita v Praze Fakulta agrobiologie potravinovych a prirodnich zdroju","correspondingAuthor":false,"prefix":"","firstName":"Miloslav","middleName":"","lastName":"Zouhar","suffix":""},{"id":266004898,"identity":"d2fbcd6b-3b95-4761-8f24-a9a88dfac080","order_by":2,"name":"Marie Manasova","email":"","orcid":"","institution":"Czech University of Life Sciences Prague Faculty of Agrobiology Food and Natural Resources: Ceska Zemedelska Univerzita v Praze Fakulta agrobiologie potravinovych a prirodnich zdroju","correspondingAuthor":false,"prefix":"","firstName":"Marie","middleName":"","lastName":"Manasova","suffix":""},{"id":266004899,"identity":"8853521f-7eb3-48c4-81bd-0edc699563e5","order_by":3,"name":"Tongda Li","email":"","orcid":"","institution":"Agriculture Victoria, Agribio, Centre for Agribioscience","correspondingAuthor":false,"prefix":"","firstName":"Tongda","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-12-18 00:19:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3769218/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3769218/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49402685,"identity":"8c1c8079-5508-46e5-9519-464a483c1cee","added_by":"auto","created_at":"2024-01-10 07:23:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3645463,"visible":true,"origin":"","legend":"\u003cp\u003eAverage disease severity index (mean ± standard deviation, n=4) in Chinese cabbage (a) and representative clubroot symptoms (b) by treatments of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS at different time points (0, 5, 9, 14 and 18 days after pathogen inoculation (DAPI)); control: inoculated just with \u003cem\u003eP. brassicae\u003c/em\u003e; similar lowercase letters indicate no significant differences at the 5% level (Duncan test) between each treatment\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/3cd254198a59e5e8ed44d85d.png"},{"id":49402946,"identity":"5f167b3c-cc61-4806-86ad-c3b43cb10aa4","added_by":"auto","created_at":"2024-01-10 07:31:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3399073,"visible":true,"origin":"","legend":"\u003cp\u003eAverage disease severity index (mean ± standard deviation, n=4) in rapeseed (a) and representative clubroot symptoms (b) by treatments of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS at different time points (0, 5, 9, 14 and 18 days after pathogen inoculation (DAPI)); control: inoculated just with \u003cem\u003eP. brassicae\u003c/em\u003e; similar lowercase letters indicate no significant differences at the 5% level (Duncan test) between each treatment\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/3645ba8cb9d4e902012274b2.png"},{"id":49402682,"identity":"361696e7-3fd3-4ea6-a984-ffbc60656e71","added_by":"auto","created_at":"2024-01-10 07:23:57","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63510,"visible":true,"origin":"","legend":"\u003cp\u003eDisease control effect (mean ± standard deviation, n=4) of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS at different time points in Chinese cabbage and rapeseed (Multiple comparisons). Five different time points of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS included 0, 5, 9, 14 and 18 days after pathogen inoculation (DAPI)); similar lowercase letters indicate no significant differences at the 5% level (Duncan test) between each treatment\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/8ce700a7a33798de477f82a2.jpeg"},{"id":49402947,"identity":"0d281978-1184-47a9-ab2c-18c2679c4195","added_by":"auto","created_at":"2024-01-10 07:31:57","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53805,"visible":true,"origin":"","legend":"\u003cp\u003eShoot dry weight (mean ± standard deviation, n=4) of Chinese cabbage at different time points of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS inoculation (0, 5, 9, 14 and 18 days after pathogen inoculation (DAPI)); control: inoculated just with \u003cem\u003eP. brassicae\u003c/em\u003e; similar lowercase letters indicate no significant differences at the 5% level (Duncan test) between each treatment\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/3ba074bfdb623537b285f785.jpeg"},{"id":49402684,"identity":"57495613-0a07-410d-8638-b27d057d73e8","added_by":"auto","created_at":"2024-01-10 07:23:57","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43383,"visible":true,"origin":"","legend":"\u003cp\u003eShoot dry weight (mean ± standard deviation, n=4) of rapeseed at different time points of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS inoculation (0, 5, 9, 14 and 18 days after pathogen inoculation (DAPI)); control: inoculated just with \u003cem\u003eP. brassicae\u003c/em\u003e; similar lowercase letters indicate no significant differences at the 5% level (Duncan test) between each treatment\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/efa59e73a8849e2e397f4e52.jpeg"},{"id":49402680,"identity":"5ecb5a08-b968-409e-94f9-fcedd8bf0950","added_by":"auto","created_at":"2024-01-10 07:23:57","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":79802,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple comparisons of shoot dry weight increase (mean ± standard deviation, n=4) in Chinese cabbage and rapeseed at different time points of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS application; (0, 5, 9, 14 and 18 days after pathogen inoculation (DAPI)); similar lowercase letters indicate no significant differences at the 5% level (Duncan test) between each treatment\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/55f2a699e55c461ee813a689.jpeg"},{"id":49403172,"identity":"8f5826ce-44a0-4839-a2be-ae93f3a417d6","added_by":"auto","created_at":"2024-01-10 07:40:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3623665,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3769218/v1/c6d9c1cb-fe10-4f39-9c9a-04ad872523f5.pdf"}],"financialInterests":"","formattedTitle":"Biocontrol potential of cell-free supernatant of Paenibacillus chitinolyticus against Plasmodiophora brassicae in two important Brassica species","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChinese cabbage (\u003cem\u003eBrassica rapa\u003c/em\u003e L.) is one of the most important green leafy vegetables in the family \u003cem\u003eBrassicaceae\u003c/em\u003e with increasing popularity in the world (Sivanandhan et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Vanjildorj et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Rapeseed (\u003cem\u003eBrassica napus\u003c/em\u003e L.) is also one of the most widely grown crops of \u003cem\u003eBrassicas\u003c/em\u003e in the world and is the second most important crop after cereal in the Czech Republic (Anonymous, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Both rapeseed and Chinese cabbage can be infected by \u003cem\u003ePlasmodiophora brassicae\u003c/em\u003e severely (Ma et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; ŘičaŘoV\u0026aacute; et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eP. brassicae\u003c/em\u003e is a soilborne pathogen that induces clubroot disease across all \u003cem\u003eBrassicaceae\u003c/em\u003e species, resulting in significant yield losses (Woronin, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1878\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Managing \u003cem\u003eP. brassicae\u003c/em\u003e proves challenging due to persistent resting spores in the soil, a broad host range, and pathotypes capable of overcoming resistance in cultivars (Diederichsen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Cultural strategies, such as extended rotations and equipment sanitization, show efficacy but lack widespread adoption (Hwang et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Strelkov et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the ongoing challenge of managing clubroot disease, the use of microbial antagonists like \u003cem\u003eTrichoderma\u003c/em\u003e spp., \u003cem\u003eBacillus\u003c/em\u003e spp., \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, and \u003cem\u003eAcremonium\u003c/em\u003e spp. has emerged as a promising alternative (Gao and Xu, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; J\u0026auml;schke et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, a few numbers of these candidates were highly effective in the high inoculum pressure, especially in the field, or have been registered for commercial uses (Narisawa et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the diverse mechanisms of action of biocontrol agents and the complex life cycle of \u003cem\u003eP. brassicae\u003c/em\u003e, a strategic approach focusing on correlation between antagonist\u0026rsquo;s biocidal substances and critical stages of \u003cem\u003eP. brassicae\u003c/em\u003e life cycle was proposed in current study. It has been reported that the critical stage in the life cycle of \u003cem\u003eP. brassicae\u003c/em\u003e, where it is most volnurable, is the brief interval between germination of resting spore and penetration into the host (Dixon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). \u003cem\u003eP. brassicae\u003c/em\u003e resting spore has also a key role to commence pathogenesis (Kageyama and Asano, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and its walls contain 25% of chitin (Moxham and Buczacki, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). Notably, chitin synthase-encoding genes are expressed during \u003cem\u003eP. brassicae\u003c/em\u003e infection process (Schwelm et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, it is probable that chitin degrader like chitinolytic bacteria, through the breakdown of chitin and decreasing resting spores germination, may serve as potent antagonists of this pathogen (Dixon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Hjort et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, chitinolytic bacteria producing chitinases were recognized as a promising alternative to synthetic chemicals. This approach is favored due to the essential role chitinases play in protecting against chitin-containing pathogens (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Prapagdee et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Prasanna et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Subbanna et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003ePaenibacillus\u003c/em\u003e bacteria are gaining attention for their effectiveness against soil-borne pathogens, produce a variety of metabolites, including antimicrobial peptides and chitinolytic enzymes (Akhtar and Siddiqui, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Budi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Grady et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Jung et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Seo et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Previous studies highlighted the role of chitinase enzymes in \u003cem\u003ePaenibacillus\u003c/em\u003e cell-free supernatant for biocontrol against phytopathogens (Loni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Seo et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Given the well-known chitinase production ability of \u003cem\u003ePaenibacillus chitinolyticus\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), we hypothesized its potential as an effective biological agent for clubroot management. Additionally, the genus \u003cem\u003ePaenibacillus\u003c/em\u003e exhibits noteworthy traits to promot plant growth (Grady et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which was of interest in current study.\u003c/p\u003e \u003cp\u003eConsidering the significant impact of clubroot infection on globally essential crops, including Chinese cabbage and rapeseed, leading to substantial economic losses for farmers, this study was conducted to assess the efficacy of \u003cem\u003eP. chitinolyticus\u003c/em\u003e, an endemic chitinolytic bacterium in the Czech Republic, in combating clubroot in Chinese cabbage and rapeseed. The research also aimed to evaluate the effectiveness of different time points of \u003cem\u003eP. chitinolyticus\u003c/em\u003e application against clubroot in greenhouse conditions. This work may contribute to the development of effective strategies for clubroot biocontrol and the facilitation of sustainable agriculture.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eSeeds of \u003cem\u003eBrassica rapa\u003c/em\u003e subsp. pekinensis (Chinese cabbage cv. \u0026lsquo;Granaat\u0026rsquo;) considered a universally susceptible host to clubroot (Buczacki et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) and seeds of \u003cem\u003eBrassica napus\u003c/em\u003e subsp. napus (rapeseed var. inspiration) were obtained from Osiva Moravia, Czech Republic. The substrate was Gramoflor (final pH 6.2) and was obtained from Germany. The seeds were sown in 2 cm deep holes in the sterilized substrate in 9\u0026times;9\u0026times;10 cm plastic pots with 2 seeds per pot to ensure uniform growth and thinned to one seedling per pot before inoculation. Pots were kept in a greenhouse at Czech university of life science, Prague, at 24_+2\u0026deg;C, and a 16 h photoperiod for 45 days. Plants were irrigated to maintain soil moisture but were not water saturated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePathogen material\u003c/h2\u003e \u003cp\u003eMature clubroot galls were collected from infested rapeseed fields in the Czech Republic, dried at room temperature, and stored at -20\u0026deg;C until use. Following the method described by Castlebury et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e), crude resting spores were extracted (Castlebury et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Approximately 5 g of dried galls were soaked in 50 ml sterile deionized water (SDW) and then blended at high speed for 2 minutes. The resulting suspension was filtered through eight layers of cheesecloth. Concentrations of resting spores were estimated using a hemocytometer, and spore suspensions were diluted with SDW to 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e resting spores per ml for plant inoculation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBioagent material (Bacterial inoculum)\u003c/h2\u003e \u003cp\u003eEndemic strain of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CCM 4527, was provided by the national collection of microorganisms in Brno, Czech Republic. To produce \u003cem\u003eP. chitinolyticus\u003c/em\u003e inoculum, CFS was obtained after the growth of \u003cem\u003eP. chitinolyticus\u003c/em\u003e in a liquid medium, followed by centrifugation and filtration through a small pore-size filter to remove all bacterial cells. For this purpose, single colonies of \u003cem\u003eP. chitinolyticus\u003c/em\u003e were cultured on Luria- Bertani (LB) Agar medium, transferred to LB liquid medium, and incubated at 30\u0026deg;C for 3 days at 180 RPM to prepare bacterial fermentation (Zhu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The optical density of bacterial fermentation was measured using nanodrop (OD600\u0026thinsp;=\u0026thinsp;0.8). The fermentation filtrate of the bacterial isolate was centrifuged at 8000 RPM for 15 min, and the supernatant was filtered through a 0.22 mm cellulose nitrate filter to obtain bacterial CFS (Zhu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePlants Inoculation\u003c/h2\u003e \u003cp\u003eInoculation with the pathogen (\u003cem\u003eP. brassicae\u003c/em\u003e) was conducted 10 days after planting when all plants had developed two true leaves. This involved applying 2 ml of 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e resting spores ml\u003csup\u003e-1\u003c/sup\u003e per plant by pipetting into the soil. For bioagent inoculation, each seedling received 10 ml of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS.\u003c/p\u003e \u003cp\u003eGiven the critical stages of \u003cem\u003eP. brassicae\u003c/em\u003e life cycle, including resting spore germination, primary infection, and secondary infection, we investigated the efficacy of the bioagent against clubroot at 5 different time points. Plants were inoculated with \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS on the same day as the pathogen was inoculated (0), on day 5, day 9, day 14, and day 18 after the pathogen inoculated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of biocontrol potential of\u003c/b\u003e \u003cb\u003eP. chitinolyticus\u003c/b\u003e \u003cb\u003eon clubroot\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDisease severity was determined 35 days after plants inoculation. The evaluation involved removing all the plants and washing the roots under tap water to eliminate soil particles. Clubroot severity was visually assessed based on a scale of 0 to 3 (0\u0026thinsp;=\u0026thinsp;no galling, 1\u0026thinsp;=\u0026thinsp;a few small galls, 2\u0026thinsp;=\u0026thinsp;moderate galling, and 3\u0026thinsp;=\u0026thinsp;severe galling) (Kuginuki et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The disease severity index (DSI) was calculated using the following equation:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{D}\\text{S}\\text{I} \\text{\\%}=\\frac{{\\Sigma } (\\text{n}\\times 0+\\text{n}\\times 1+\\text{n}\\times 2+\\text{n}\\times 3)}{\\text{N} \\times \\text{N}\\text{o}. \\text{o}\\text{f} \\text{c}\\text{l}\\text{a}\\text{s}\\text{s}\\text{e}\\text{s} \\text{w}\\text{i}\\text{t}\\text{h} \\text{s}\\text{y}\\text{m}\\text{p}\\text{t}\\text{o}\\text{m}\\text{s}}\\)\u003c/span\u003e \u003c/span\u003e \u0026times; 100\u003c/p\u003e \u003cp\u003eHere \u0026lsquo;n\u0026rsquo; is the number of plants in each class; \u0026lsquo;N\u0026rsquo; is the total number of plants; and the values 0, 1, 2, and 3 represent the respective symptom severity classes. Disease control effect (DCE) was calculated using the following formula:\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\text{D}\\text{C}\\text{E} \\text{\\%}=(1-\\frac{ \\text{D}\\text{S}\\text{I} \\text{i}\\text{n} \\text{t}\\text{r}\\text{e}\\text{a}\\text{t}\\text{m}\\text{e}\\text{n}\\text{t}}{\\text{D}\\text{S}\\text{I} \\text{i}\\text{n} \\text{c}\\text{o}\\text{n}\\text{t}\\text{r}\\text{o}\\text{l}})\\)\u003c/span\u003e \u003c/span\u003e \u0026times; 100\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of shoot dry weight\u003c/h2\u003e \u003cp\u003eShoot fresh and dry weight of both plants was measured at the end of experiments to assess the effect of the antagonist on plants growth. Shoot fresh weight of all plants for each treatment was estimated by a scale, then a sub-sample of them were put in paper bags and dried in an oven at 70\u0026deg; C, for 2 days. Dry weight was calculated by humidity percentage measured after oven-drying of the plant sub-samples (Mousavi Shalmani et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design and Statistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental design employed a split plot design based on completely randomized design (CRD) with four replicates per treatment, and each experimental unit consisted of 10 plants. The first level comprised two \u003cem\u003eBrassica\u003c/em\u003e species: Chinese cabbage and rapeseed. The second level included six treatments, encompassing five different time points of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS, and one control. Control was inoculated with \u003cem\u003eP. brassicae\u003c/em\u003e and treated by 10 ml of LB liquid medium on the same day as the pathogen inoculated. The treatments with \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS at five different time points were as follows: 0, 5, 9, 14, and 18 day after pathogen inoculation (DAPI). Statistical analyses, including ANOVA and multiple comparisons (Duncan Test), were conducted using GenStat-14 (General Statistics software).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eBiocontrol effects of\u003c/b\u003e \u003cb\u003eP. chitinolyticus\u003c/b\u003e \u003cb\u003eon clubroot disease\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe average clubroot severity index in Chinese cabbage by treatments of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS at different time points was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The analysis of the effect of \u003cem\u003eP. chitinolyticus\u003c/em\u003e on the clubroot severity index showed differences in DSI values among the different time points.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClubroot severity index had a value of 96% in control which was inoculated just with \u003cem\u003eP. brassicae\u003c/em\u003e. When \u003cem\u003eP. chitinolyticus\u003c/em\u003e was applied at 0 DAPI, the highest reduction in clubroot severity index (49%) was achieved. Moreover, clubroot severity index was significantly lower than control when \u003cem\u003eP. chitinolyticus\u003c/em\u003e applied at 5 DAPI and 9 DAPI, which were not significantly different from 0 DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Clubroot progressed at the highest rate when \u003cem\u003eP. chitinolyticus\u003c/em\u003e inoculated at 18 DAPI. Application of \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 14 DAPI provided almost same clubroot severity index with 18 DAPI and there were no significant reductions in Clubroot severity index of these two treatments compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn case of rapeseed, DSI had a value of 91% in control which was inoculated just with \u003cem\u003eP. brassicae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Significant reductions in clubroot DSI were found with inoculations of \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 0, 5 and 9 DAPI, whereas there were no significant reductions in DSI with delay in application of \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 14 DAPI and 18 DAPI. The highest reduction in clubroot DSI in rapeseed was observed at 0 DAPI which was 2.5 times less than control and followed by its application at 5 DAPI and 9 DAPI. Treatment with \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 9 DAPI lowered clubroot to 50% compared to control that was less efficient than that of at 0 DAPI but was not significantly different from 5 DAPI. The results showed that DSI with a value of 85% and 90.8% were observed with inoculations of \u003cem\u003eP. chitinolyticus\u003c/em\u003e on 14 DAPI and 18 DAPI respectively, which had no significant difference when compared to that in control. Application of \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 14 DAPI was not significantly different from 18 DAPI, while were significantly different from treatment at 0, 5, and 9 DAPI (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe average clubroot control effect in Chinese cabbage and rapeseed by treatments of \u003cem\u003eP. chitinolyticus\u003c/em\u003e at different time points was presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The highest clubroot control effect on Chinese cabbage was 49.2% that achieved with application of this strain at 0 DAPI, in which pathogen and antagonistic bacteria have been used at the same day. Treatment with \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 5 DAPI and 9 DAPI controlled clubroot by 43.5% and 37.4% respectively and there was not found to be a significant difference between these treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In rapeseed, comparing all treatments of \u003cem\u003eP. chitinolyticus\u003c/em\u003e, its application at 0, 5, and 9 DAPI could control clubroot up to 60.1%. Multiple comparisom of Chinese cabbage and rapeseed showed that in both plants, \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS at 0, 5 and 9 DAPI controlled clubroot. However, no significant differences were observed in the ability of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS to control clubroot between Chinese cabbage and rapeseed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of\u003c/b\u003e \u003cb\u003eP. chitinolyticus\u003c/b\u003e \u003cb\u003eon dry matter production\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe effect of \u003cem\u003eP. chitinolyticus\u003c/em\u003e on the shoot dry weight of Chinese cabbage was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The results showed a significant increase in dry matter production across all treatments with \u003cem\u003eP. chitinolyticus\u003c/em\u003e compared to the control, where plants were only inoculated with the pathogen. No significant differences were observed within \u003cem\u003eP. chitinolyticus\u003c/em\u003e treatments at 0, 5, 9, 14 and 18 DAPI. The maximum shoot dry weight increased by 1.47 times compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe impact of \u003cem\u003eP. chitinolyticus\u003c/em\u003e on the shoot dry weight of rapeseed was outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Similar to its effect on Chinese cabbage, the application of \u003cem\u003eP. chitinolyticus\u003c/em\u003e showed consistent enhancement in rapeseed dry matter at various time points. No significant differences were observed among treatments with \u003cem\u003eP. chitinolyticus\u003c/em\u003e at 0, 5, 9, 14 and 18 DAPI. The maximum shoot dry weight saw a 1.46-fold increase compared to the control. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn both plants, dry matter production was significantly influenced by \u003cem\u003eP. chitinolyticus\u003c/em\u003e treatments at all time points. Moreover, the efficiency of \u003cem\u003eP. chitinolyticus\u003c/em\u003e was similar when comparing between Chinese cabbage and rapeseed and the maximum shoot dry weight increase, reaching almost 45%, was observed in both plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur research aimed for evaluating the effectiveness of various application of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS on clubroot suppression in Chinese cabbage and rapeseed in greenhouse. The result showed that \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS was capable of suppressing clubroot (based on clubroot control effect) up to 49.2% in Chinese cabbage and 60.1% in rapeseed. However, no significant difference was observed in the efficiency of \u003cem\u003eP. chitinolyticus\u003c/em\u003e to control clubroot on Chinese cabbage and rapeseed. In addition, \u003cem\u003eP. chitinolyticus\u003c/em\u003e was able to increase shoot dry weight to a great extent simultaneously (in average 40%), which offers significant potential as a bioagent in Chinese cabbage and rapeseed.\u003c/p\u003e \u003cp\u003eAlthough \u003cem\u003ePaenibacillus\u003c/em\u003e species have been emphasized to control many disease (Akhtar and Siddiqui, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Grady et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Singh et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), only a few of them have been reported to reduce clubroot severity. Liu et. al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) have reported that \u003cem\u003eP. polymyxa\u003c/em\u003e was effective in clubroot suppression on cabbage up to 71. 09% (Liu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In their study, the fermented liquid of \u003cem\u003eP. polymyxa\u003c/em\u003e showed more control efficacy than Bacillus subtilis. Additionally, \u003cem\u003eP. kribbensis\u003c/em\u003e has been reported to inhibit clubroot in Chinese cabbage in greenhouse, achieving control rates ranging from 89\u0026ndash;99.2% (Xu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These two prior studies using different species of \u003cem\u003ePaenibacillus\u003c/em\u003e have showed that this genus was effective in clubroot suppression, and this was seen in our experiment, albeit to a lower proportion. To our knowledge it is a first report of usage of \u003cem\u003eP. chitinolyticus\u003c/em\u003e to control clubroot in rapeseed and Chinese cabbage.\u003c/p\u003e \u003cp\u003eDespite the availability of two previous studies on using \u003cem\u003ePaenibacillus\u003c/em\u003e for clubroot control, none of them focused on the effect of timing of \u003cem\u003ePaenibacillus\u003c/em\u003e application against the pathogen. This study filled this gap and results showed that delayed application of this antagonist in Chinese cabbage and rapeseed, independently of the host, could not control clubroot. This indicated the bacterium\u0026rsquo;s effectiveness in the early stages of clubroot development but lack of suppressive effects in the later stages. Our study supported an earlier study (He et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) that early application of B. subtilis was the best way to reduce the clubroot severity on Chinese cabbage. Our results agreed with their conclusion that time of application of bacterial antagonist had an important effect on clubroot suppression. Such results suggested that early-stage interaction between \u003cem\u003eP. chitinolyticus\u003c/em\u003e and \u003cem\u003eP. brassicae\u003c/em\u003e is critical in clubroot inhibition.\u003c/p\u003e \u003cp\u003eOur findings about the most effective time point for \u003cem\u003eP. chitinolyticus\u003c/em\u003e application demonstrated that \u003cem\u003eP. chitinolyticus\u003c/em\u003e suppressed \u003cem\u003eP. brassicae\u003c/em\u003e significantly when applied on the same day as \u003cem\u003eP. brassicae\u003c/em\u003e inoculated, on day 5 and 9 after \u003cem\u003eP. brassicae\u003c/em\u003e inoculation, while no suppression occured in latter treatment on day 14 and 18 after \u003cem\u003eP. brassicae\u003c/em\u003e inoculation. The critical stage in the life cycle of \u003cem\u003eP. brassicae\u003c/em\u003e, where it is most susceptible, is the brief interval between germination and penetration of resting spore into the host root hair. Understanding the factors affecting resting spores germination, which is essential for \u003cem\u003eP. brassicae\u003c/em\u003e pathogenicity, is key to controling clubroot disease (Dixon, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Consistent with Dixon\u0026rsquo;s insights, the observed suppression on the same day as \u003cem\u003eP. brassicae\u003c/em\u003e inoculated in our study, seems attributable to the inhibitory effect of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS on the germination of resting spores as an essential component within the most vulnerable phase of the \u003cem\u003eP. brassicae\u003c/em\u003e life cycle. These findings emphasized the potential of \u003cem\u003eP. chitinolyticus\u003c/em\u003e as an effective antagonist, strategically targeting chitin in a critical stage in \u003cem\u003eP. brassicae\u003c/em\u003e development and disrupting the pathogen life cycle .\u003c/p\u003e \u003cp\u003eInhibitory effect of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS on 5 and 9 DAPI might be due to its antagonistic effect on primary infection of \u003cem\u003eP. brassicae\u003c/em\u003e including primary zoospores, primary plasmodium and zoosporangium which were produced before day 9. Ineffectiveness on 14 and 18 DAPI is probabely due to inability of this bacterium on suppression of secondary infection of \u003cem\u003eP. brassicae\u003c/em\u003e which was initiated around day 9 and subsequently developed till 18 DAPI in the inner tissue of the cortex. This assumption was further supported by findings about developmental stages of \u003cem\u003eP. brassicae\u003c/em\u003e. Resting-spore germination is the first step in the life cycle of \u003cem\u003eP. brassicae\u003c/em\u003e (Kageyama and Asano, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Under favorable conditions the spores of \u003cem\u003eP. brassicae\u003c/em\u003e can germinate immediately and releases zoospores, which form primary infection in the host. Liu et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020c\u003c/span\u003e) reported that \u003cem\u003eP. brassicae\u003c/em\u003e initiates the primary infection on Arabidopsis root hairs and epidermal cells at 1 day after Plasmodiophora inoculation, with the establishment of secondary infection occuring in cortical cells at 8 day after Plasmodiophora inoculation (Liu et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020c\u003c/span\u003e). Zhu et al. (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported a slightly adjusted timeframe of 2 days after Plasmodiophora inoculation for primary infection and 9 days after Plasmodiophora inoculation for secondary infection in a susceptible oilseed rape cultivar, aligning with similar data reported for susceptible Chinese cabbage cultivars (Liu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e). It can therefore be assumed that \u003cem\u003ePaenibacillus\u003c/em\u003e should be considered as an antagonist against resting spores germination and primary infection of \u003cem\u003eP. brassicae\u003c/em\u003e in Chinese cabbage and rapeseed. Interestingly, \u003cem\u003eP. chitinolyticus\u003c/em\u003e efficacy on day 5 and 9 after \u003cem\u003eP. brassicae\u003c/em\u003e inoculation, suggested that this antagonist can compete with established \u003cem\u003eP. brassicae\u003c/em\u003e resting spores to some extent. This partial curative potential may be a favourable aspect for use in the field which requires further study.\u003c/p\u003e \u003cp\u003eIn the present study, CFS of \u003cem\u003eP. chitinolyticus\u003c/em\u003e has been used. CFS are rich in metabolites with antimicrobial activity. Notably, the potential of this resource to control phytopathogens could be further enhanced by optimizing the condition and culture (Mani-L\u0026oacute;pez et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Singh, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This optimization strategy holds promise in revealing a broader spectrum of antimicrobial compounds in CFS of \u003cem\u003eP. chitinolyticus\u003c/em\u003e, exhibiting enhanced ability to combat Plasmodiophora. This warrants further examination in the future.\u003c/p\u003e \u003cp\u003eOur results revealed the significant ability of CFS of \u003cem\u003eP. chitinolyticus\u003c/em\u003e to suppress clubroot. We \u003cem\u003eutilized P. chitinolyticus\u003c/em\u003e, a strain that produces a high level of chitinase, which plays a pivotal role in the antagonistic ability of this species against plant pathogens (Ahmadi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Budi et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e; Song et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Von der Weid et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This finding aligns with the recognized role of chitinase enzymes in the CFS of various \u003cem\u003ePaenibacillus\u003c/em\u003e species for biocontrol, targeting specific pathogens such as \u003cem\u003eCollectotrichum gloeosporioides\u003c/em\u003e, \u003cem\u003eRhizoctonia solani\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e sp. (Loni et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Seo et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Microbial chitinases are believed to act against chitin-containing pathogens by degrading chitin, thereby exhibiting biocontrol potential (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Gomaa, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Heustis et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sharma et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Given that chitin is a major component in the resting spores of \u003cem\u003eP. brassicae\u003c/em\u003e (Moxham and Buczacki, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1983\u003c/span\u003e) and various chitin synthesis genes are significantly upregulated during the infection process of this pathogen (Schwelm et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), we hypothesized that chitinase in \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS could target and degrade the chitin in \u003cem\u003eP. brassicae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eOur suggestion regarding the potential link between clubroot suppression and the chitinases present in the CFS of \u003cem\u003eP. chitinolyticus\u003c/em\u003e can be further supported by up-regulation of chitinase-related genes in Chinese cabbage in response to \u003cem\u003eP. brassicae\u003c/em\u003e infection (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). They inferred that the induced chitinase might provide the evidence for the degradation of chitin secreted by \u003cem\u003eP. brassicae\u003c/em\u003e. Moreover, the discovery of chitin-binding effectors in \u003cem\u003eP. brassicae\u003c/em\u003e, which can bind to resting spores' chitin for the suppression of chitin-triggered immunity in rapeseed (Muirhead and P\u0026eacute;rez-L\u0026oacute;pez, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), further strengthened our hypothesis. Chitinases, as shown in their study, prevent these chitin-binding effectors from binding to resting spores, thereby increasing plant immunity. While these findings supported our results and hypothesis regarding the inhibitory roles of \u003cem\u003eP. chitinolyticus\u003c/em\u003e chitinase in clubroot suppression through chitin targeting, further studies are necessary to elucidate the specific functions of chitinase in \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS and the underlying mechanisms through which it suppresses \u003cem\u003eP. brassicae\u003c/em\u003e. Potential experiments could involve generating mutants of \u003cem\u003eP. chitinolyticus\u003c/em\u003e lacking chitinase activity and assessing their bioprotective efficacy against clubroot. Furthermore, a transcriptomic analysis may be conducted for both mutant and original strains.\u003c/p\u003e \u003cp\u003eWe demonstrated that \u003cem\u003eP. chitinolyticus\u003c/em\u003e effectively enhances the growth of Chinese cabbage and rapeseed, leading to significant increases in shoot dry weight. This positive impact remained consistent across various treatments of \u003cem\u003eP. chitinolyticus\u003c/em\u003e, including both early and late inoculations. The ability of \u003cem\u003eP. chitinolyticus\u003c/em\u003e to enhance shoot dry weight may be attributed to several mechanisms, such as phosphate solubilization, nitrogen fixation, and hormone production. This aligns with the mechanisms of many other species of \u003cem\u003ePaenibacillus\u003c/em\u003e, known for their capacity to promote plant growth (Grady et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe significant influence of \u003cem\u003eP. chitinolyticus\u003c/em\u003e on growth, coupled with its bioprotective ability, positioned it as a promising bioagent for managing clubroot in Chinese cabbage and rapeseed. This statement became more important when considering the decrease in plant yield due to clubroot in previous studies. Greenhouse experiments revealed canola yield was reduced between 0.26% and 0.49% for each 1% increase in the clubroot disease index (Botero-Ram\u0026iacute;rez et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Furthermore, field experiments demonstrated yield losses of approximately 0.03 tonne per hectare for every 1% increase in clubroot severity in rapeseed (McGrann et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The observed growth-promoting effects of \u003cem\u003eP. chitinolyticus\u003c/em\u003e in our study suggested a promising approach to mitigate yield losses in \u003cem\u003ebrassica\u003c/em\u003e fields at risk of clubroot, which should be examined in future studies.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our findings demonstrated the antagonistic activity of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS against clubroot in Chinese cabbage and rapeseed during the early stages of clubroot occurrence. Chitinase enzymes play important roles in \u003cem\u003ePaenibacillus\u003c/em\u003e CFS, combating chitin-containing phytopathogens. Considering the remarkable chitinase production capability of \u003cem\u003eP.chitinolyticus\u003c/em\u003e, its efficacy against clubroot likely stems from a precise targeting of chitin during critical stages of the pathogen's life cycle, but additional research is needed to prove this assumption, and elucidate the mechanism of action of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS. This can clarify the role of antimicrobial compounds such as chitinas derived from \u003cem\u003eP. chitinolyticus\u003c/em\u003e CSF and finding novel compounds to replace conventional toxins for suppressing Plasmodiophora in Brassicaceae. The positive impact of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS on shoot dry weight in Chinese cabbage and rapeseed underscores its potential as both a biofertilizer and a bioprotectant agent in clubroot management. However, field trials should be the next step to verify the the efficacy of \u003cem\u003eP. chitinolyticus\u003c/em\u003e CFS in clubroot control.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding\u0026nbsp;This study was funded by Czech University of Life Sciences, Prague.\u003c/p\u003e\n\u003cp\u003eConfict of interest: The authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhmadi, K., Yazdi, M. T., Najafi, M. F., Shahverdi, A., Faramarzi, M., Zanini, G., \u0026amp; Behrava, J. (2008). Isolation and characterization of a chitionolytic enzyme producing microorganism, Paenibacillus chitinolyticus JK2 from Iran. \u003cem\u003eRes J Microbiol\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(6), 395\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkhtar, M., \u0026amp; Siddiqui, Z. (2007). 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Two new biocontrol agents against clubroot caused by Plasmodiophora brassicae. \u003cem\u003eFrontiers in microbiology\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e, 3099.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chitinolytic bacteria, Clubroot, Rapeseed, Chinese cabbage, Paenibacillus chitinolyticus","lastPublishedDoi":"10.21203/rs.3.rs-3769218/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3769218/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003ePlasmodiophora brassicae\u003c/em\u003e is a serious threat to \u003cem\u003eBrassica\u003c/em\u003e crops worldwide, resulting in substantial economic losses for growers, with the challenge of control persisting. Biocontrol with chitinolytic bacteria producing chitinase is gaining attention as a natural alternative to chemicals. This approach is favored due to the essential role chitinases play in protecting against chitin-containing pathogens. Given that chitin is a major component in the resting spores of \u003cem\u003eP. brassicae\u003c/em\u003e and plays a crucial role during pathogenesis, it is probable that \u003cem\u003ePaenibacillus chitinolyticus\u003c/em\u003e, producing a high level of chitinase, could suppress \u003cem\u003eP. brassicae\u003c/em\u003e by targeting chitin in a critical stage of this pathogen\u0026rsquo;s life cycle. Our research aimed at evaluating the effect of various applications of \u003cem\u003eP. chitinolyticus\u003c/em\u003e on clubroot suppression in two economically important \u003cem\u003eBrassica\u003c/em\u003e species: Chinese cabbage and rapeseed. The effectiveness of the cell-free supernatant (CFS) of an endemic strain of \u003cem\u003eP. chitinolyticus\u003c/em\u003e from the Czech Republic at five different time points was studied in the greenhouse by measuring the disease severity index. The results showed that early application of \u003cem\u003eP. chitinolyticus\u003c/em\u003e decreased the disease index significantly within both plants. Additionally, in both plants, \u003cem\u003eP. chitinolyticus\u003c/em\u003e increased shoot dry weight to a great extent. In conclusion, the CFS of \u003cem\u003eP.chitinolyticus\u003c/em\u003e has significant antagonistic activity against clubroot in Chinese cabbage and rapeseed in the early developmental stages of clubroot occurrence and holds the potential as a biofertilizer as well as bioprotectant agent in clubroot management of \u003cem\u003eP. brassicae\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Biocontrol potential of cell-free supernatant of Paenibacillus chitinolyticus against Plasmodiophora brassicae in two important Brassica species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-10 07:23:52","doi":"10.21203/rs.3.rs-3769218/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision","date":"2024-05-01T04:37:28+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-04-01T23:38:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-01-08T12:51:02+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"European Journal of Plant Pathology","date":"2023-12-22T05:23:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-20T13:32:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Journal of Plant Pathology","date":"2023-12-19T08:04:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-journal-of-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ejpp","sideBox":"Learn more about [European Journal of Plant Pathology](http://link.springer.com/journal/10658)","snPcode":"10658","submissionUrl":"https://www.editorialmanager.com/ejpp/default2.aspx","title":"European Journal of Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3b148e3e-b575-47d7-91db-d71b799195fc","owner":[],"postedDate":"January 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-17T04:20:19+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-10 07:23:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3769218","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3769218","identity":"rs-3769218","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}