CET 97 DOI: 10.3303/CET2297045 Paper Received: 6 May 2022; Revised: 20 July 2022; Accepted: 21 July 2022 Please cite this article as: Nguyen Q.N., Linh M.X., Nguyen T.L.P., Nguyen M.T., Pham T., 2022, Isolation of Antagonistic Rhizosphere Bacteria Toward Phytophthora capsici Induce Phytophthora Blight in Pepper (Piper nigrum), Chemical Engineering Transactions, 97, 265-270 DOI:10.3303/CET2297045 CHEMICAL ENGINEERING TRANSACTIONS VOL. 97, 2022 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Jeng Shiun Lim, Nor Alafiza Yunus, Jiří Jaromír Klemeš Copyright © 2022, AIDIC Servizi S.r.l. ISBN 978-88-95608-96-9; ISSN 2283-9216 Isolation of Antagonistic Rhizosphere Bacteria Toward Phytophthora capsici Induce Phytophthora Blight in Pepper (Piper nigrum) Quynh Nhi Nguyen a, Mai Xuan Linhb,c, Thi Lien Phuong Nguyen b,c, Nguyen Minh Thienb,c, Tien Phamd,* aFaculty of Biological Sciences, Nong Lam University Ho Chi Minh City, Ho Chi Minh City, Vietnam bFaculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Vietnam cVietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam dFaculty of Nursing and medical Technology, Van Lang University, Ho Chi Minh City, Vietnam tien.ptm@vlu.edu.vn Vietnam has been the largest exporter of pepper globally in recent years. However, the quick death disease caused by Phytophthora capsici is spearing rapidly, causing notable damage to many concentrated cultivation areas. The regular application of chemical pesticides to combat the diseases at pepper farms has increased the certain environmental problems. To reduce pesticide usage, biological methods for controlling P. capsici have been implied. In this study, rhizosphere bacterial strains were isolated from pepper plantations, and their chitinolytic and phytophthora antagonistic activities were evaluated. The chitinolytic activity was conducted on an agar medium supplemented with chitin (CM), and the antagonistic activity was done using a dual culture inhibition assay. As the result, 46 strains (signed NH1 - NH46) were isolated based on morphological distinctions in the CM medium. Out of the 46 isolated strains, 8 strains including NH7, NH10, NH11, NH27, NH31, NH32, NH33, and NH46, which accounted for 17 % of the isolates, showed high chitinolytic activity. In the dual culture assay, the strain NH7 showed the highest effectiveness that inhibiting the P. capsici mycelial growth with an antagonistic distance of 20.33 mm, followed by three strains NH27, NH32, and NH46 antagonistic distances of 13.67 – 15.33 mm. These strains were further identified as E. cloacae, S. flaveus, K. pneumoniae, and B. amyloliquefaciens through 16S rRNA sequencing. A phylogenetic tree showed a closed connection between the antagonistic isolated strains and antagonistic bacteria reported previously. 1. Introduction Black pepper (Piper nigrum L.) has been deemed the “king of spices”. Its unique spicy flavor makes some dishes become tastier, and easily reserved as well. This kind of spice has various good effects on our health. Black pepper has become the main export agricultural product of Vietnam in recent year. Pepper began to be introduced to our country and became popular during the postwar period in the 17th century (Ravindran, 2020). Vietnam is currently the world leader in pepper production. The central production areas are from Quang Tri to the Central Highlands, Southeast, and Phu Quoc Island (Thuy et al., 2012). Vietnam pepper production was always high and was expected to increase next year, accounting for 47 % of global production. New plantation area reached 16 % annually since 2011 and relatively high in the last 3 y (18 % - 28 %). The total pepper area of Vietnam was 140,000 ha, and production reached 287,000 t in 2019 (Thuy et al., 2012). Black pepper cultivation is not always going smoothly. Diseases on pepper have arisen and caused significant harm to many centralized production areas of Vietnam. Several pathogens comprising viruses, bacteria, fungi, and nematodes cause disorders in pepper’s normal metabolic pathway (Kang et al., 2022). Phytophthora blight caused by Phytophthora capsici is the most alarming disease that causes severe yield losses of about 2 % in pepper cultivation areas and 15 – 20 % production per year (Nguyen et al., 2020). The disease affects plants at any growth stage and the damping-off syndrome kills seedlings within 5 d of infection. 265 The pathogen also causes crown, leaf, and fruit blight, wilting of the whole plant and dark purplish discoloration of the stem (Babadoost et al., 2015). Blight control is dependent on cultural practices, utilization of resistant varieties, pesticide application, and combined with water management. These methods might not deliver significant results, as well as damage the environment even the food chain, and human survival (Hoang et al., 2021). Biological control has become the better method due to its eco-friendly feature and has become an important strategy to manage soil-borne diseases and reduce the application of chemical pathogens suppression methods (Hoang and My, 2021). Many microorganisms have been reported to suppress the growth of P. capsici including Streptomyces spp., Bacillus spp., Trichoderma sp., Paenibacillus spp., Aspergillus sp. The mechanisms of control include the production of antibiotics and lytic enzymes, physical or chemical interference, competition, induction of host resistance, hyperparasitism, and predation (Ozyilmaz, 2020). In recent years, rhizosphere bacteria have gained interest as biocontrol agents because of their abilities to colonize the rhizosphere of plants and their beneficial effects on plant growth (Khatun et al., 2018). In this study, bacterial strains from the pepper rhizosphere soil with a strong antagonistic potential to P. capsici were isolated, identified, and archived for further studies. 2. Materials and method 2.1 Materials Soil samples were collected from the rhizosphere of healthy plants at pepper plantations in the main production areas in Dong Nai province, Vietnam. The plantations had not been sprayed with pesticides less than 30 d. Phytophthora capsici strain was received from the Department of plant Biotechnology, Ho Chi Minh City of Biotechnology Center. Bacillus subtilis strain was received from the Department of Biotechnology, HCMUT-VNU. 2.2 Chitin solution 1 % w/v preparation The chitin powder was prepared from the shrimp shell using the method of base and acid treatment. The shrimp shell after washing several times to eliminate impurities was treated with NaOH 4 % at 70 – 75 °C for 4 h. The shell was treated with HCl 8 % at room temperature for 16 h and followed by water washing and centrifugation. The treated shrimp shell was ground into a powder that served as the chitin source. Gradually added 1 g chitin powder to 20 mL of concentrated HCl and kept at 4 °C overnight and stir vigorously. The mix was added to 200 mL of ice-cold ethanol 95 %, left overnight at 4 °C, and stirred rapidly. Precipitate was received using centrifugation at 4,000 rpm/min at 4 °C for 20 min, and then rinsed with sterile water until chitin colloidal evolved neutral (pH 7.0). The last volume was adjusted to 100 mL with 50 mM phosphate buffer (pH 6.5) (Dai et al., 2011). 2.3 Isolation of bacterial strains on chitin medium A volume of 100 µL of ten-fold serial dilutions (10-6 – 10-10) of each soil samples with sterile water was spread on Chitin Media (CM). CM components included chitin 0.5 %, yeast extract 0.01 %, KH2PO4 0.1 %, Na2HPO4 0.2 %, NaCl 0.05 %, MgSO4.7H2O 0.05 %, NH4Cl 0.1 %, KNO3 0.05 %, CaCl2.2H20 0.05 %, agar 2 %, and adjust pH to 7). After 5 d incubation at 30 °C, the strains that appeared halo zones around colonies were selected and checked for further experiments (Sophearenth et al., 2013). 2.4 Evaluation of the chitinolytic activity of bacterial isolates The diffusion method was used to evaluate the chitinolytic activities of isolates following by Dinh et al. (2018). Created a 9 mm diameter well in the center of a CM media Petri dish. Bacterial density was acquired by measuring the OD value (at OD 600 nm) in a spectrophotometer and controlling this value of all specimens to 1. Applied 100 µL of cultured broth into the well, incubated at 37 °C, 3 replicates for each isolate. B. subtilis was used as a positive control and fresh LB-broth media as a negative control. Stained the agar dish with Lugol, observed, and measured the halo diameter on the dish after 120 h of incubation. The diameter of the halo was used to evaluate the chitinolytic activity of isolates. 2.5 In vitro evaluation of Phytophthora capsici antagonistic ability Evaluating P. capsici antagonistic activity on Chitin Potato Dextrose Agar Media (CP) was taken from 4 d old PDA plate of P. capsici and placed at the center of the new CP plate. After 24 h of incubation, created 3 wells on the plate, 30 mm from the center on this CP plate. The bacterial culture LB broth at OD value = 1 was filled into the well (100 µL), 3 replicates for each strain. A similar agar disc of 4 d old cultured with P. capsici was placed at a fresh CP center and was used as a negative control and B. subtilis had been used as a positive control (Chung et al., 2008). 266 All the Petri plates were incubated at room temperature. After 5 d of incubation, antagonistic activity was acquired by measuring the radius (R) of the P. capsici colony in the direction of the antagonist colony (R2) and the radius of the P. capsici colony in the control plate (R1). The antagonistic activity was calculated as in Eq(1) (Zhao et al., 2022). 2.6 Identification of bacterial strains, phylogenetic tree, and data processing Identification based on 16S rRNA sequence and BLAST program for bacterial strains which showed the chitin degradation and suppression of P. capsici. The nucleotide sequences of 16S rRNA were compared with the 16S rRNA genes which published on NCBI using the BLAST. Phylogenetic tree based on 16S rRNA sequences with MEGA X software using UPMA method. All the data in study were processed using the one- way analysis of variance method (ANOVA) and post-hoc analysis by DUNCAN test. 3. Results 3.1 Isolation of bacterial strains on chitin medium There are 46 obtained isolates (NH1 – NH46) growing and creating halo zones around their colonies on the CM medium. They were distinguished by morphological characters. Most of the obtained isolates showed blight white, small, protruding, and smooth colonies. The colonies of three strains NH10, NH27, and NH46 were slightly different from the others. There was a morphology transformation from a milk-white, small and protruding colony to a colony spreading over an agar surface with more bacterial biomass. NH27 was observed at the time later in the culturing period with the small, wrinkled, rough, and ivory-colored colony. NH46 also had its colony spreading over the plate surface with the wrinkled biomass. 3.2 Evaluation of chitinolytic activity of bacterial isolates The chitin break-down ability of the 46 isolated strains was evaluated on the CM medium. Out of 46 strains, 8 strains (17 %) showed a decent ability to break down chitin (Table 1). The strains had chitin degradation diameters from 24 mm to 50 mm, NH46 strain showed the maximum chitin degradation activity with the largest halo zone diameter (50 mm) which was significantly different from other strains (P ≤ 0.05). NH27 strain was the smallest of the eight chosen strains (24 mm). The positive control showed strong chitinase activity (70 mm) and the negative control did not appear in any halo zone on the plate. Table 1: The chitinolytic activity of bacterial isolates 3.3 Evaluation of Phytophthora capsici antagonistic ability in vitro The antagonistic ability to P. capsici of eight isolates (NH7, NH10, NH11, NH27, NH31, NH32, NH33, and NH46) showing high chitinolytic degradation was assessed using a dual culture technique on CP medium. Four bacterial isolates including NH7, NH27, NH32, and NH46 inhibited the mycelial growth of pathogens on CP accounting for 50 % of total chitinolytic bacteria (Table 2 and Figure 1). The NH7 strain demonstrated the 𝑅𝑅 = 𝑅𝑅1 − 𝑅𝑅2 (1) Isolates Mean Diameter* (mm) NH7 26b NH10 25bc NH11 26bc NH27 24c NH31 31c NH32 25bc NH33 34d NH46 50e Bacillus subtilis (+) 70f (-) 0a 267 best capacity for the growth inhibition of hyphae with a resistance distance of 20.33 mm, which was statistically different from the other 3 strains. strains NH27, NH32, and NH46 had no difference in their ability to inhibit the growth of mycelium with resistance distances of 13.67 mm and 15.33 mm. Table 2: Phytophthora capsici antagonistic activity of bacterial isolates Figure 1: Soil bacterial isolation on CM media 3.4 Identification of bacterial strain Four bacterial strains (NH7, NH27, NH32, NH46) not only had high chitinolytic activity but also inhibited the growth of P. capsici. Gram staining results showed that strain NH7 and NH32 belonged to the group of gram- negative cocci, strain NH27 with filaments belonged to the gram-positive group, and strain NH46 belonged to the group of gram-positive Bacilli. Figure 2: Phylogenetic tree based on isolated strains 16S rRNA gene sequence Strains Antagonistic activity R (mm) Mean antagonistic activity 𝑅𝑅� (mm) 1st 2nd 3rd NH7 19 21 21 20.33a NH27 15 17 14 15.33b NH32 14 12 15 13.67b NH46 15 14 12 13.67b 268 Identification results based on 16S rRNA sequences showed that strains NH7, NH27, NH32, and NH46 were identified as Enterobacter cloacae, Streptomyces flaveus, Klebsiella pneumoniae, Bacillus amyloliquefaciens. The phylogenetic tree in Figure 2 showed the four isolates and the reference strains divided into 4 different groups. The strain NH32 belonged to group IA had a close genetic relationship with strains of Enterobacter sp., strain NH27 belonged to group II to other Streptomyces strains, strain NH46 belonged to group III had high genetic similarity with strain B. subtilis, and strain NH7 belonged to strain NH7 belonged to group 4 with low genetic similarity with the remaining strains. 4. Discussion The mechanisms for biological control of diseases could include competition for infection sites, nutrients, and spaces, parasitism on pathogens, destruction of fungal pathogens by the action of lytic enzymes (chitinase and β-1,3-glucanase), uncharacterized antifungal factors and many other metabolites produced by rhizobacteria (Rosier et al., 2018). The biocontrol activities of antagonistic bacteria could be related to their chitinase production (Zhang et al., 2016). This was a reason to explain chitin amendment of soil to stimulate the growth of chitinolytic microorganisms, increase the biocontrol efficacy and stimulate the expression of plant defense protein (Shao et al, 2018). Volatile factors formed in chitin-amended have been demonstrated to suppress chlamydospore formation such as NH3 release (Prigigallo et al., 2021). For all reasons above, isolation of antagonistic bacteria was conducted with the selective media which contains chitin as the main carbon source. As for the research of Toh et al. (2016), isolated strain E. cloacae had been reported with the highest antagonism against P. capsici mycelia with the percentage of inhibition up to 47.63 % and produced clear zones in spore germination test with radius measurements of 10 – 17 mm because of its produced volatile bioactive compounds (Toh et al., 2016). Prigigallo et al. (2021) reported that volatile compounds such as ammonia produced by E. cloacae were able the prevention of Pythium which caused disease in cotton. The increase of biological control was also shown in the release of ammonia during chitin degradation which subsequently reduces mycelia spreading (Prigigallo et al., 2021). It could be predicted that the use of selective media containing chitin was reasonable for E. cloacae isolation. During the bacterial growth, E. cloacae could utilize chitin provided, producing NH3 as a volatile active ingredient inhibiting P. capsici. Based on the phylogenetic tree, S. flaveus had very close relationship with S. halstedii whose culture broth suppressed the growth of P. capsici due to their low molecular faction (≤ 10 kDa) (Joo, 2005). A manumycin- type antibiotic from S. flaveus showed strong antifungal activity against P. capsici (Minh et al., 2015). NH32, K. pneumoniae, be in a relative correlation with E. cancerogenus which was reported as a phytophthora blight pathogen agent. K. pneumoniae was also a member of the family Enterobacteriaceae and known nitrogen-fixing bacterium, able to convert atmospheric nitrogen into ammonium. Subsequently, K. pneumoniae was considered to be a biological control agent, not only able to inhibit P. capsici but also to promote plant growth (Sopheareth et al., 2013). K. pneumoniae belonged to the group of bacteria that cause disease in humans, so evaluation was needed to confirm the safety of the strain. B. amyloliquefaciens was found to be a potential biocontrol agent for controlling the plant pathogen P. capsici (Zhang et al., 2016). The in vitro test demonstrated this strain to have antifungal properties with high efficiency and broad-spectrum. This bacterium could promote the growth of pepper seed, solubilize phosphate and produce indole acetic acid (IAA) and ammonia. 5. Conclusion In summary, the present study successfully isolated four bacterial strains that had a good inhibitory effect P. capsici growth suppression including E. cloacae, S. flaveus, K. pneumoniae and B. amyloliquefaciens which were potential strains to use as biological control agents for the rapid death on Pepper. These strains could have other beneficial properties that needed further research. Acknowledgments We would like to thank Ho Chi Minh City University of Technology (HCMUT), VNU-HCM for the support of time and facilities for this study. References Babadoost M., Pavon C., Islam S.Z., Tian D., 2015, Phytophthora blight (Phytophthora capsici) of pepper and its management, Acta Horticulturae, 1105, 61–66. 269 Chung S., Kong H., Buyer J.S., Lakshman D.K., Lydon J., Kim S.D., Roberts D.P., 2008, Isolation and partial characterization of Bacillus subtilis ME488 for suppression of soilborne pathogens of cucumber and pepper, Applied Microbiology and Biotechnology, 80(1), 115–23. Dai D.H., Hu W.L., Huang G.R., Li W., 2011, Purification and characterization of a novel extracellular chitinase from thermophilic Bacillus sp. Hu1, African Journal of Biotechnology, 10(13), 2476-2485. Dinh T.M., hayuki S., Dzung N.A., Takeshi W., Kazushi S., 2018, Identification and characterization of chitinolytic bacteria isolated from a freshwater lake, Bioscience, Biotechnology, and Biochemistry, 82(2), 343-355. Hoang A.H., Thien N.M., Anh P.T., Lai N.V., 2021, Efficacy of compost on the growth of some leafy vegetables: A case study in Vietnam, Chemical Engineering Transactions, 89, 457-462. Hoang A.H., My P.D.T, 2021, Phage cocktails to inactivate Edwardsiella Ictaluri, an infectious agent in striped catfish pangasianodon hypophthalmus, Chemical Engineering Transactions, 89, 535-540. Minh N.V., Woo E.E., Kim J.Y., Kim D.W., Hwang B.S., Lee Y.J., Lee I.K., 2015, Antifungal substances from Streptomyces sp. A3265 antagonistic to plant pathogenic fungi, Mycobiology, 43(3), 333-338. Kang W.H., Lee J., Kwon J.S., Park P., Kim Y.M., Yeom S.I., 2022, Universal gene co-expression network reveals receptor protein-like protein genes involved in broad-spectrum resistance in pepper (Capsicum annuum L.), Horticulture Research, 9, uhab003, DOI: 10.1093/hr/uhab003 Khatun A., Farhana T., Sabir A.A., Islam S.M.N., West H.M., Rahman M., Islam M., 2018, Pseudomonas and Burkholderia inhibit growth and asexual development of Phytophthora capsici, Zeitschrift für Naturforsch Journal of Biosciences, 73(3), 123–35. Nguyen S.D., Trinh T.H.T., Tran T.D., Nguyen T.V., Chuyen H.V., Ngo V.A., Nguyen A.D., 2020, Combined application of rhizosphere bacteria with Eendophytic bacteria suppresses root diseases and increases productivity of Black Pepper (Piper nigrum L.), Agriculture, 11(1), 15. Ozyilmaz U., 2020, Evaluation of the effectiveness of antagonistic bacteria against Phytophthora blight disease in pepper with artificial intelligence, Biological Control, 151, 104379. Rosier A., Medeiros F.H.V., Bais H.P., 2018, Defining plant growth promoting rhizobacteria molecular and biochemical networks in networks in beneficial plant-microbe interactions, Plant Soil, 428, 35-55. Prigigallo M.I., Stradis D.A., Anand A., Mannerucci F., L’Haridon F., Weisslopf L., Bubici G., 2021, Basidiomycetes are particularly sensitive to bacterial volatile compounds: Mechanistic insight into the case study of Pseudomonas protegens volatilome against Heteronasidion abietinum, Front Microbiol, 12, 684664. Shao Z., Li Z., Fu Y., Wen Y., Wei S., 2018, Induciton of defense responses against Magnaporthe oryzae in rice seedling by a new potential biocontrol agent Streptomyces JD211, Journal Basic Microbiology, 58 (8), 686-697. Sopheareth M., Chan S., Naing K.W., Lee Y.S., Hyun H.N., Kim Y.C., Kim K.Y., 2013, Biocontrol of late blight (Phytophthora capsici) disease and growth promotion of Pepper by Burkholderia cepacia MPC-7, the Plant Pathology Journal, 29(1), 67–76. Thuy T.T.T., Yen N.T., Tuyet N.T.A., Le L.L., Waele D.D., 2012, Plant - parasitic nematodes and yellowing of leaves associated with black pepper plants in Vietnam, Achives of Phytopathology and Plant Proteciton, 45(10), 1183-1200. Toh S.C., Samuel L., Awang A.S.A.H., 2016, Screening for antifungal-producing bacteria from Piper nigrum plant against Phytophthora capsici, International Food Reseach Journal, 23(6), 2616–2622. Ravindran P.N, 2020, Black pepper Piper nigrum, Harwood Academic Publishers, Reading, UK. Zhang M., Li J., Shen A., Tan S., Yan Z., Yu Y., Xue Z., Tan T., Zeng L., 2016, Isolation and identification of Bacillus amyloliquefaciens IBFCBF-1 with potential for biological control of phytophthora blight and growth promotion of Pepper, Journal of Phytopathology, 64(11–12), 1012–1021. Zhao X., Hou D., Xu J., Wang K., Hu Z., 2022, Antagonistic activity of fungal strains against Fusarium, Plants, 11, 225. 270 045.pdf Isolation of Antagonistic Rhizosphere Bacteria Toward Phytophthora capsici Induce Phytophthora Blight in Pepper (Piper nigrum)