Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.Nghiên cứu và ứng dụng xạ khuẩn trong phòng trị bệnh đạo ôn hại lúa do nấm Pyricularia oryzae Cav. trên vùng đất nhiễm mặn.
MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS SPECIALITY: PLANT PROTECTION Speciality ID: 62620112 DANG NGUYET QUE RESEARCH AND APPLICATION OF ACTINOMYCETES STRAINS FOR THE MANAGEMENT OF RICE BLAST DISEASE CAUSED BY Pyricularia oryzae Cav IN THE SALT-AFFECTED SOILS AREAS Can Tho, 2023 THE THESIS WAS COMPLETED AT CAN THO UNIVERSITY Scientific supervisors 1: PGS.TS Le Minh Tuong Scientific supervisors 2: PGS.TS Tran Thi Thu Thuy Reviewer 1: Assoc Prof, Ph.D Pham Van Du Reviewer 2: Assoc Prof, Ph.D Nguyen Thi Thu Nga The thesis is defended in front of the Doctoral Thesis Examination Council at Can Tho University Time: 8:00 am Date: September 3th, 2022 Further information of the thesis could be found at: Learning Resource Center of Can Tho University National library of Vietnam LIST OF PUBLISHED PAPERS Dang Nguyet Que, Le Minh Tuong and Tran Thi Thu Thuy, 2019 Determination of characterictic of actinomycetes isolates on Pyricularia oryzae causing rice blast disease on salt afected soils Science and Technology Journal of Agriculture and Rural Development Ministry of Agriculture and Rural Development, Vietnam May, 2019, 81-86 Dang Nguyet Que, Le Minh Tuong and Tran Thi Thu Thuy, 2019 Identification of actinomycetes as promissing biocontrol against rice blast disease in salt affected soils Journal of Vietnam Agriculture Science and Technology Vietnam Academy of Agricultural Sciences Vol (103)/2019, 125-129 Dang Nguyet Que, Le Minh Tuong and Tran Thi Thu Thuy, 2021 Determination biocontrol of actinomycetes isolates on rice blast disease on salt affected soils in the net house condition Đánh giá khả phịng trị bệnh đạo ơn hại lúa canh tác vùng đất nhiễm mặn số chủng xạ khuẩn điều kiện nhà lưới Journal of Plant Protection Vol (298)/2021, 21-27 Chapter 1: INTRODUCTION 1.1 The urgency of thesis Blast disease caused by the fungus Pyricularia oryzae (P oryzae) is one of the three major diseases that significantly affect the yield and quality of rice worldwide (Dean et al., 2012; Gao et al., 2019; Chen et al., 2021) Because the fungus P oryzae attacks all parts of the rice plant, it causes losses of up to hundreds of millions of tons of rice each year (Wang, 2009) In Bac Lieu, rice blasts caused widespread damage in all seasons with varying degrees of severity Especially in salt-affected rice-shrimp fields, blast disease occurs relatively early and spreads quickly if not sprayed in time Meanwhile, chemical drug use also affects the giant freshwater shrimp intercropping in rice fields and the next shrimp rotation crop To maintain and develop an effective and sustainable rice-shrimp farming model and to adapt to climate change towards safe rice and shrimp production that meets export standards, it is necessary to research and find out biological solutions These must effectively manage rice blast disease, progressing to developing an integrated rice blast disease process on saline soil in the rice-shrimp farming system Regarding biological measures, the past has shown that the study of the application of actinomycetes is considered a promising solution and has great potential to replace fungicide to prevent blast disease towards safe agriculture and sustainability Many valuable scientific works on the application of actinomycetes to control rice blast disease have been published in national and international journals (Tuong & Em, 2014; Khalil et al., 2014; Lan and ctv., 2015, 2016; Awla et al., 2016, 2017; Law et al., 2017; Gao et al., 2019) However, most of the studies about actinomycetes were conducted in the laboratory Some studies were carried out in net houses and the field However, there is no research on the application of actinomycetes to prevent and treat rice blast disease in saline soil Therefore, the thesis "Research and application of actinomycetes strains for the management of rice blast disease caused by Pyricularia oryzae in the salt-affected soils area" is essential 1.2 Research objectives and requirements (i) Selecting salt-tolerant actinomycete strains’s ability against pathogen P oryzae in the laboratory (ii) Study on the antagonistic ability of actinomycetes through the ability to secrete chitinase and β-1,3-glucanase enzymes (iii) Identifying the species of promising actinomycetes strains (iv) Evaluation capable of managing rice blast disease in the net house conditions (v) Evaluating the effectiveness of managing rice blast disease of promising salt-tolerant actinomycetes strains in field conditions 1.3 Scientific and practical significance of the thesis The topic has high scientific and practical significance; the project's results are to find actinomycetes strains capable of managing rice blast disease caused by the fungus P oryzae on salt-affected soils The results of this study serve as a premise for the following studies to find probiotics from salt-tolerant actinomycetes and apply them to the integrated management of rice blast disease on salt-affected soils Both are effectively and environmentally friendly, contributing to developing a sustainable riceshrimp farming model 1.4 The new results of the dissertation - Three salt-tolerant actinomycetes isolates, including S06-MBL, S09MBL and S17-MBL, have been identified to prevent rice blast disease under net house conditions Two strains of Streptomyces bikinensis (S09-MBL) and Streptomyces lavendulate (S17-MBL) could prevent rice blast disease in saline soil field conditions in Bac Lieu - The biocontrol ability of actinomycetes S06-MBL, S09-MBL and S17-MBL against rice blast disease caused by P oryzae was related to the ability to secrete chitinase and β-1,3-glucanase enzymes - Species of actinomycete strains have been identified: S06-MBL is Streptomyces fradiae, S09-MBL is Streptomyces bikiniensis, and S17-MBL is Streptomyces lavendulae These species differ from previous actinomycete species studied in managing blast disease in Mekong Delta Chapter 2: RESEARCH METHODS 2.1 Time and place of the thesis - Time: from October 2015 to February 2019 - Place: the laboratory and the nethouse of the Plant Protection Department, Can Tho University; Bạc Liêu University saline rice fields of farmers in Hồng Dân district, Bạc Liêu province 2.2 Experimental Materials: fungus P oryzae causing rice blast disease and actinomycetes 2.3 Experimental methods 2.3.1 Content 1: Collect, isolate and evaluate the pathogenicity of P oryzae strains causing rice blast disease - Collect and isolate P oryzae causing rice blast disease in the Mekong Delta salt-affected soils area: Investigate and collect rice samples with blast disease in, rice-shrimp fields in Bac Lieu, Ca Mau, Kien Giang and Hau Giang provinces; Isolation and culture of P oryzae fungus were carried out at the Plant Diseases laboratory of Plant Protection Department, Can Tho University according to the method of Burgess et al (2009) Identification of P oryzae fungus was based on morphological characteristics of fungal strains compared with those of P oryzae described by Agrios (2005) and Zhang et al (2014) - Evaluation of pathogenicity of P oryzae isolates under the nethouse condition: In the period of 18 days after planting, the rice plants were artificially infected Spraying a suspension of P oryzae fungi spores evenly wet the foliage with a spore density of 105 spores/ml in the fantastic afternoon; each spraying pot is 10 ml Pots of rice after artificial inoculation were placed in the incubation room and covered in darkness for 24 hours (the temperature at 260C and humidity at 96-98%) After that, the pot of rice was transferred to the net house with a shading system to reduce 50% of light conditions, misted to create the right humidity for fungal growth (incubation net house) The recorded indicators were the ratio of infected leaf area and disease index at the time of 4, 6, 8, 10, 12 and 14 days after artificial infection 2.3.2 Content 2: Collect, isolate and evaluate the antagonistic ability of actinomycete isolates derived from rice saline soil against the fungus P oryzae strains causing rice blast disease under laboratory condition - Collecting and isolating actinomycetes from rice saline soil: Soil samples were taken around the root zone of rice, 10-25 cm from the surface and isolated actinomycetes at the laboratory of the Department of Plant Protection Can Tho University according to the method of Hsu and Lockwood (1975) - Evaluation of the antagonistic ability of actinomycetes against P oryzae causing rice blast disease under laboratory condition: Put the sterile absorbent paper rolls into the actinomycete suspension solution (108cfu/ml), shook the solution well Use specialized clamps to put the absorbent paper on the wall, let it dry Place the blotting paper (Ø = mm) impregnated with actinomycetes opposite the P oryzae and cm from the sides of the plate In the control treatment, the paper strips impregnated with actinomycetes were replaced with ones impregnated with sterile distilled water The follow-up indicator was to measure the radiuses of inhibition zones and calculate the antagonistic efficiency at the time of 3, 5, and days after inoculation (DAI) - Investigation of the antagonistic ability of promising actinomycete strains against P oryzae under the conditions of addition of NaCl salt: perform the same experiment as above, except that the experimental medium is supplemented with NaCl salt (2g/l) The follow-up indicator was to measure the radiuses of inhibition zones and calculate the antagonistic efficiency at the time of 3, 5, 7, and 11 days after inoculation (DAI) - Investigation of the ability to inhibit the growth of spores of P oryzae of promising actinomycetes: Treatment of actinomycetes: Add 500 µl of actinomycete suspension (108 cfu/ml) + 500 µl of fungal suspension (5x105 spores/ml) into eppendorft tubes and keep at 28oC, each treatment with times of replicationt Control treatment: Put 500 µl of sterile distilled water + 500 µl of fungal spore suspension (5x105 spores/ml) into eppendorft tube and keep at 25oC Recording indicators: The percentage of spores sprouting at the time of 6, 12 and 24 hours after treatment (HAT) by observation under the microscope 2.3.3 Content 3: Investigate a number of mechanisms related to the antagonistic ability of promising actinomycetes - Investigate the ability to secrete chitinase-degrading enzyme chitinase of actinomycete strains on agar: The experiment was arranged in a completely randomized design with one promising actinomycete strain and five replications for each treatment according to the method of Dai et al (2011) and Ha (2012) - Determination of secreted chitinase enzyme content of experimental actinomycetes: The experiment was arranged in a completely randomized design with replications and performed according to the method of Dai et al (2011) and Ha (2012) - Investigation of the ability to secrete β-1,3-glucanase enzyme to degrade β-1,3-glucan of actinomycete strains on agar: The experiment was arranged in a completely randomized design with each treatment being a promising actinomycete strain with replicates The experiment was carried out according to the method of Renwich et al., (1991) - Determination of the secreted β-1,3-glucanase enzyme content of actinomycete strains: The experiment was arranged in a completely randomized design with one factor, the number of treatments was the number of actinomycete strains tested with replicates and was performed according to the method of Renwich et al (1991) 2.3.4 Content 4: Identify promising actinomycetes species by observation biological characteristics methods and molecular biology methods - Identification of actinomycetes based on culture, morphological and biochemical characteristics * Observation of the color of biogas, substrate fibers and soluble pigments which were conducted according to the method of Shirling and Gottlieb (1966) * Observation of spore stalk and spore surface shape which were conducted according to the method of Tresner et al (1961) * Protease secretion ability of actinomycete strains: was performed according to the method of Mitra and Chakrabartty (2005) * The ability to secrete lipase enzyme of actinomycete strains: was performed according to the method of Ertuğrul et al., (2007) * The ability to secrete amylase enzyme of actinomycete strains: was performed according to the method of Santos (2012) * The melanin pigment formation of actinomycete strains: was performed according to the method of Shirling and Gottlieb (1966) - Identification to species of actinomycete strains by molecular biology method DNA extraction of actinomycetes was performed according to the method of Weisburg et al., (1991) The primer pairs were used to amplify 16S-rRNA gene segments of actinomycete strains in the study were: 1492R: 5'-TACGGTTACCTTGTTACGACT-3' and 27:5'AGAGTTTGATCCTGGCTC-3' (Weisburg et al., 1991) Analytical samples were sequenced on the ABI 3130XL system The results were anylized by using sequecing analysis 6.0 software and compared with the results on the gene bank to determine the name of actinomycetes 2.3.5 Content 5: Evaluation of the ability to control rice blast disease with promising actinomycetes strains under nethouse condition - Location: the experiment was carried out at the nethouse of the Plant Protection Department, Bac Lieu University - Experimental arrangement: The experiment was arranged in completely randomized design with 14 treatments (12 treatments using actinomycetes and controls) with replications - Monitoring criteria: count the number of leaf blast lesions according to Pinnschmidt et al (1993), measure length and width leaf, and calculate the percentage of infected leaf area, disease index and disease suppression efficiency at the time of 4, 6, 8, 10, 12 and 14 days after artificial infection (DAAI) 2.3.6 Content 6: Evaluation of the ability to control rice blast disease with promising actinomycetes strains under the field condition Field experiment was carried out in seasons: 2017 season in farmer's fields in Ninh Thanh Loi commune, Hong Dan District, Bac Lieu province from September 2017 to December 2017 with an area of 2,200 m2, block layout completely random, factor, repetitions The 2018 season was carried out in Ninh Hoa Commune, Hong Dan District, Bac Lieu province from October 2018 to January 2019 with an area of 2,700 m2, arranged like the 2017 province, with repetitions Each experimental unit has a size of 30 m2 (6 m x m) Experimental units are arranged 0.5 m apart to limit the interaction when handling actinomycetes The experimental field is m away from the farmer's field in order to limit the influence of fertilizers and pesticides of next rice fields Experimental treatments as Table 3.5 ò Record disease parameters: Each experimental plot placed five frames (40x50) cm fixed on two diagonal lines On each frame, the indicators of leaf blast disease (27, 34, 41, 48 and 55 days after sowing) and blast disease were recorded collar blast (67, 74 and 81 days after sowing), then calculate the following indicators: (1) Disease rate; (2) Disease index; (3): Reduce disease severity ò Record productivity targets + The number of panicle /m2: in each experimental plot of 30 m2, count all panicle in plots to record the criteria (40x50 cm2), from which the number of panicle/m2 is calculated + The number fill grain/ panicle: randomly select 50 flowers in the target recording box (40x50 cm2) Count the number fill grain in each panicle, and then calculate the average number of fill grain for each of the 50 panicles + Actual yield (ton/ha): harvest all rice within a frame of 5m2 Weigh and measure grain moisture at the time of weighing, then convert to 14% moisture From there calculate the actual yield Table 3.5 Treatments used in the experiment (Days after treatment: DAT) Treatment Handling Processing Dosage/ Treatment (T) agent measures time density Seed coating, Actinomycetes S17-MBL -A NT1 12 GTKS S17-MBL suspension: S17-MBL-A, 20 NT2 20 NSS 108 cfu/ml Seed coating 40 NSS S17-MBL-A, 40 NT3 + S17-MBL-A, 60 NT4 60 NSS * Seed coating: Folia Just enough S17-MBL-A, 20, 40 NT5 20 & 40 NSS Spraying (12 H) S17-MBL-A, 20, 60 NT6 20 &60 NSS S17-MBL S17-MBL-A, 40, 60 NT7 40 & 60 NSS * Folia S17-MBL-A,20,40,60 NT8 20, 40 & 60 NSS Spraying Coating + Tillering S09-MBL -A, Áo NT9 12 GTKS S09-MBL stage: S09-MBL-A, 20 NT10 20 NSS 25 lít/1000 m2 Coating + S09-MBL-A, 40 NT11 40 NSS + Flowering Folia stage: S09-MBL-A, 60 NT12 60 NSS Spraying 40 lít/1000 m2 S09-MBL-A, 20, 40 NT13 20 & 40 NSS S09-MBL S09-MBL-A, 20, 60 NT14 20 &60 NSS S09-MBL-A, 40, 60 NT15 40 & 60 NSS three actinomycetes strains (S06-MBL, S09-MBL and S17-MBL) with the highest radiuses of inhibition zones and antagonist efficacy at 11 DAT Table 3.7 Antagonistic ability (%) of actinomycetes against P oryzae in laboratory condition (added NaCl 2‰) over the survey times Treatment S06-MBL S09-MBL S17-MBL S09-MTV S03-MST S18-MCM S31-MCM Significance level CV (%) Radiuses of inhibition zones over times (mm) DAT DAT DAT DAT 11 DAT a a a 18.0 17.0 15.6 13.4a 12.2a 17.2ab 15.8ab 14.8a 13.6a 12.0a 17.2ab 15.8ab 14.6a 13.6a 12.3a abc b b b 16.4 11.8 6.2 6.0 2.6c bc bc bc c 15.4 9.6 4.6 1.8 1.6c ab ab ab a 17.4 15.0 13.8 12.0 9.4b c c c c 15.0 8.2 2.8 1.6 1.6c * * * * * 7.79 10.03 14.25 15.03 17.76 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test *: statistically significant difference at 5% level; Days after Testing (DAT) Table 3.8 Antagonist efficacy (%) of actinomycetes with P oryzae in laboratory condition (added NaCl 2‰) over the survey times Treatment S06-MBL S09-MBL S17-MBL S09-MTV S03-MST S18-MCM S31-MCM Significance level CV (%) DAT 46.15a 36.59ab 36.59ab 29.27b 31.71b 35.34b 9.76c * 14.60 Antagonist efficacy over times (%) DAT DAT DAT 63.75a 73.04a 75.84a 62.50a 73.04a 76.51a a a 62.50 69.57 75.17a 47.50b 40.00b 53.69b c c 36.25 31.30 43.62c 60.75a 72.17a 76.51a d d 16.25 22.61 38.26d * * * 6.39 4.91 3.64 11 DAT 78.50a 78.51a 78.57a 56.59b 54.95b 76.35a 49.45c * 3.49 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data are converted to arcsin x for statistical analysis *: statistically significant difference at 5% level; Days after Testing (DAT) Thus, the experimental results in Table 3.7 and Table 3.8 show that actinomycete strains exhibit different antagonisms against P.oryzae under the addition of NaCl (2g/l) compared with the conditions without NaCl In which strain S06-MBL always showed more potent antagonism against P oryzae than strain S31-MCM, the difference was statistically significant at a 5% significance level (Figure 3.6) 14 S17MBL S18-MCM S09-MTV S09-MBL S31MCM S06MBL ĐC Figure 3.6 Resistance against P oryzae fungi of actinomycetes in the medium supplemented with NaCl 2‰ at 11 DAT 3.2.4 The ability to inhibit the growth of P oryzae fungal spores of promising actinomycetes The results of the inhibition of P oryzae spore germination of the experimental actinomycetes are presented in Table 3.9 The results showed that all seven experimental actinomycetes strains showed the ability to inhibit the growth of P oryzae fungus to different extents Strain S17-MBL showed high inhibition of fungal spore germination with a low spore germination rate from hours after the experiment until 24 hours after the experimental setup Table 3.9 Rate of spore of P oryzae sprouted over the survey time points (%) Treatment Rate of sporulation over times of survey (%) HAT 12 HAT 24 HAT S06-MBL 5.14d 17.6c 20.1cd cd c S09-MBL 6.94 16.4 17.5d e c S17-MBL 0.00 14.2 15.9d S09-MTV 9.31b 24.5b 27.4b S03-MST 8.61bc 18.6bc 24.3bc bc bc S18-MCM 8.61 18.6 25.8b d c S31-MCM 6.11 16.1 17.6d a a Control 14.4 31.5 35.4a Significance level * * * CV (%) 27.8 12.1 9.6 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data HAT converted to x + 0,5 , 12 24 HAT converted to arcsin x for statistical analysis.*: statistically significant difference at 5% level; Hours after Testing (HAT) 15 Over the survey, it was found that the treatments with four actinomycete strains, S06-MBL, S09-MBL, S17-MBL and S31-MCM, maintained a stable low germination rate, demonstrating the ability to inhibit P oryzae better than the rest of the strains However, strain S06-MBL has BKVVK and higher antagonistic efficiency than strain S31-MCM when tested in the saline environment (NaCl 2g/l) Thus, three actinomycete strains, S06-MBL, S09-MBL and S17-MBL, were selected for the follow-up experiments 3.3 CONTENT 3: INVESTIGATE A NUMBER OF MECHANISMS RELATED TO THE ANTAGONISTIC ABILITY OF PROMISING ACTINOMYCETES 3.3.1 The ability to secrete chitinase enzyme to degrade chitin of actinomycetes under laboratory condition The ability to secrete chitinase enzymes to break down chitin is shown by the chitin lyse halo radius (Table 3.10) and the amount of chitinase enzyme secreted by actinomycetes was presented in Table 3.11 Table 3.10 Chitin lyse halo radius (mm) of the actinomycetes tested over survey times No Actinomycetes Chitin lyse halo radius (mm) strain NSTN 3NSTN 5NSTN 7NSTN S06-MBL 4.2b 101c 15.3b 18.5b S09-MBL 5.2a 13.5a 18.9a 23.3a a b a S17-MBL 5.1 12.8 18.3 23.2a Significance level * * * * CV (%) 6.22% 5.57% 5.3% 4.26% Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test *: statistically significant difference at 5% level; Days after Testing (DAT) Bảng 3.11 Concentration of chitinase (IU/ml) of 03 actinomycetes over survey times Concentration of chitinase (IU/ml) Actinomycetes No strain NSTN 3NSTN 5NSTN 7NSTN S06-MBL 0.15c 0.20c 0.26b 0.28c S09-MBL 0.27a 0.38a 0.44a 0.51a bc b b S17-MBL 0.18 0.27 0.29 0.33b Significance level * * * * CV (%) 5.81 6.43 6.95 5.36 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test *: statistically significant difference at 5% level; Days after shaking (DAS) 16 The results of Table 3.10 and Table 3.11 show that all tested actinomycete strains have the ability to secrete chitinase enzymes, in which strains S09-MBL and S17-MBL had the highest ability to secrete chitinase enzyme with the highest radiuses of inhibition zones and enzyme content secretion was highest until days after the testing 3.3.2 The ability to secrete β-1,3-glucanase enzyme to degrade β-1,3glucan of 03 actinomycetes strains under laboratory condition The ability to secrete β-glucanase enzyme to break down β-glucan was shown by the radiuses of inhibition zones (Table 3.12) and the content of βglucanase enzyme secreted by actinomycetes was presented in Table 3.13 Table 3.12 Radiuses of inhibition zones β-1,3-glucan (mm) of the tested actinomycetes over the survey times Radiuses of inhibition zones β-1,3-glucan No Treatment (mm) 10 NSTN 12 NSTN 14 NSTN b b S06-MBL 9.6 10.6 11.6c c a S09-MBL 9.0 12.2 12,8bc S17-MBL 9.8a 12.0ab 14,0a Significance level * * * CV (%) 6.03 4.12 4.8 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test *: statistically significant difference at 5% level; Days after Testing (DAT) Table 3.13 Concentration of β-1,3-glucanase (IU/ml) of 03 actinomycetes over survey times Concentration (IU/ml) of β-glucanase over survey time 10 NSTN 12 NSTN 14 NSTN b bc S06-MBL 0.28 0.21 0.07b c c S09-MBL 0.21 0.18 0.05b a a S17-MBL 0.46 0.47 0.15a Significance level * * * CV (%) 6.70 13.76 7.50 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test *: statistically significant difference at 5% level; Days after shaking (DAS) No Treatment The results showed that all experimental actinomycete strains were able to secrete β-1,3-glucanase to degrade β-glucan, in which strain S17MBL showed the highest ability to secrete β-1,3-glucanase enzyme with the 17 highest radiuses of inhibition zones and the highest secreted enzyme content was 14 days after testing 3.4 CONTENT 4: IDENTIFY PROMISING ACTINOMYCETES SPECIES BY OBSERVATION BIOLOGICAL CHARACTERISTICS METHODS AND MOLECULAR BIOLOGY METHODS 3.4.1 Identification of actinomycetes based on culture, morphological and biochemical characteristics The survey results on the culture characteristics, morphological characteristics and biochemical characteristics of the three experimental actinomycetes are presented in Table 3.14, Table 3.15 and Table 3.16 Table 3.14 Morphological and biochemical characteristics of experimental actinomycetes strains Characteristics Chain of spores Spore stem Spore surface Color of aerial hypha Color Melanin pigment Salt tolerance Enzym secrection Gram Experimental actinomycetes strains S06-MBL S09-MBL S17-MBL Straight Straight Hook Flexous Straight -Flexous Straight -Flexous Sleek Sleek Sleek White White Grey No No ≤ 6% Protease, Amylase, Lipase, Cellulase Positive No Yellow-brown ≤ 8% Protease, Amylase, Lipase, Cellulase Positive No No ≤ 8% Protease, Amylase, Lipase, Cellulase Positive Figure 3.10 Shape of straight spore stem (A; S17-MBL), hook spore chain (A, S17MBL), straight chain of spore (B; S09-MBL), spore surface smooth (C; S17-MBL) 18 Table 3.15 The secretion of amylase, protease and lipase enzymes of actinomycetes Radius of substrate resolution zone (mm) of actinomycete strains at days after experiment (*) Amylase Protease Lipase 9,5 ± 1,29 8,3 ± 0,50 10,8 ± 0,63 10,8 ± 1,16 11,5 ± 0,57 11,2 ± 0,65 8,8 ± 0,96 13,5 ± 0,58 9,8 ± 0,96 Actinomycetes strain S06-MBL S09-MBL S17-MBL (*) Number is average of three times repeat ± SD Bảng 3.16 Salt tolerance test of actinomycete strains Actinomycetes strain The concentration of salt NaCl (%) S06-MBL S09-MBL S17-MBL +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ ++ ++ ++ ++ ++ ++ + ++ + - Note: (+++) Good; (++) Modrerate; (+) Poor ; (−) No growth Morphological characteristics (Spore stem, chain of spores, spore surface), biochemical characteristics (color, melanin pigment, enzyme, gram secretion and salt tolerance ability) of the actinomycete strains S06-MBL, S09-MBL, S17-MBL are presented in Table 3.14, Table 3.15, Table 3.16 and compared with the key actinomycetes of the International Streptomyce Project (Shirling and Gottlieb, 1972; Pridham et al., 1958 ; Waksman, 1961) showed that these three actinomycetes were classified in the genus Streptomyces 3.4.2 Identification of actinomycetes by molecular biology method Based on the results of Table 3.20, it shows that the strains of actinomycetes tested have similarity from 98 to 100% when compared with the standard species based on the 16S - rRNA gene sequence Specifically, strain S06-MBL has 99% similarity with Streptomyces fradiae species; strain S09-MBL has 99% similarity with Streptomyces bikiniensis species and strain S17-MBL has 98% similarity with Streptomyces lavendulae species 19 Hình 3.13 Sản phẩm PCR khuếch đại với cặp mồi thuộc vùng 16S-rRNA chủng xạ khuẩn S06-MBL, S09-MBL S17MBL Table 3.20 Results of identification of actinomycetes based on 16S-rDNA region sequences Base pair (bp) Sample S06-MBL S09-MBL S17-MBL 1462 Degree of similarity (%) 99 Code of the same strain coin on GenBank AB184253.1 1476 99 NR_112436.1 1515 98 DQ645958.1 Species Streptomyces fradiae Streptomyces bikiniensis Streptomyces lavendulae 3.5 CONTENT 5: EVALUATION OF THE ABILITY TO CONTROL RICE BLAST DISEASE WITH PROMISING ACTINOMYCETES STRAINS UNDER NETHOUSE CONDITION The ability to control rice blast disease caused by P oryzae of actinomycete strains under net house conditions is shown by disease rate (Table 3.21), disease index (Table 3.22) and reduced disease severity (Table 3.23) Results Table 3.21, Table 3.22 and Table 3.23 show the treatment of actinomycete strains (S06-MBL, S09-MBL and S17-MBL) when combined treatment at days before and days after artificial inoculation has low disease rate, low disease index and the highest disease reduction effect lasts up to 14 days after artificial infection Table 3.21 Rate of rice leaf blast disease area (%) of actinomycetes strains under net house condition over the survey periods Rate of rice leaf blast disease area (%) over the survey periods Treatment 4DAI DAI DAI 10 DAI 12 DAI 14 DAI a cd b S06-MBL-A 4.51 7.63 6.98 6.12b 5.41c 7.02b c def b d bc S06-MBL-A-T 2.04 6.88 6.87 4.62 5.17 5.84cd S06-MBL-A-S 1.98c 8.89b 6.06cd 5.88bc 5.16bc 5.88cd c def de d de S06-MBL-A-TS 2.19 6.49 5.49 4.54 4.67 5.16e S09-MBL-A 3.13b 7.02de 6.16c 5.89bc 5.32c 6.24c 20 S09-MBL-A-T S09-MBL-A-S S09-MBL-A-TS S17-MBL-A S17-MBL-A-T S17-MBL-A-S S17-MBL-A-TS Beam 75WP Control Significance level CV(%) 2.12c 2.12c 1.14d 2.45bc 1.96c 2.15c 1.28d 0.64e 4.80a ** 10.73 6.09ef 6.06f 2.52gh 7.26de 6.50def 6.60def 3.16g 1.43i 9.98a ** 6.70 5.13e 5.12e 4.21f 7.42b 5.85cd 5.18e 4.57f 4.29f 9.52a ** 3.36 4.84d 4.59d 2.86f 6.22b 5.75bc 5.48c 4.08e 2.35g 12.08a ** 3.28 4.61de 4.25e 3.33f 6.95b 4.81bcd 4.88bcd 4.22e 3.48f 12.69a ** 3.62 5.29de 4.25f 2.88h 6.53bc 6.14c 5.90cd 3.48g 3.04h 11.09a ** 3.62 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data are converted to x + 0,5 for statistical analysis *: statistically significant difference at 5% level; Days after Inoculation (DAI) Table 3.22 Index of rice blast disease of actinomycetes strains under net house condition over the survey periods Disease index (%) over time of survey Treatment DAI DAI DAI 10 DAI 12 DAI 14 DAI a c cd b S06-MBL-A 13.08 22.63 20.38 20.19 18.11c 24.82b S06-MBL-A-T 5.92c 18.14e 21.06c 17.24cde 17.56cde 20.45cde c b def bc S06-MBL-A-S 5.74 26.67 18.77 19.41 17.29cde 20.57cd S06-MBL-A-TS 6.35c 19.45 ef 17.02fg 14.98efg 15.64def 18.06e b cde de bc S09-MBL-A 8.81 21.07 19.11 19.44 17.81cd 21.84c c cd gh def e S09-MBL-A-T 6.15 22.28 15.89 15.96 15.43 18.53de S09-MBL-A-S 5.74c 18.17f 15.87gh 15.14efg 14.23f 14.89f e h i i g S09-MBL-A-TS 3.31 7.57 13.05 9.3h 11.17 10.08i S17-MBL-A 7.12bc 21.29cde 23.01b 20.52b 23.29b 22.85bc cd ef efg bc de S17-MBL-A-T 5.17 19.49 18.13 18.97 16.10 21.51c S17-MBL-A-S 6.21c 19.80def 16.07gh 18.08bcd 16.34de 20.67cd de g h fg f S17-MBL-A-TS 3.70 11.99 14.18 13.46 14.14 12.18gh Beam 75WP 1.86f 4.30i 13.31i 7.74i 11.61g 10.63hi a a a a a Control 14.37 32.88 33.31 37.72 39.04 39.36a Significance level ** ** ** ** ** ** CV% 11,04 4,73 4,70 5,53 4,30 4,39 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data DAI are converted to x + 0,5 , other datas are converted to arcsin x for statistical analysis *: statistically significant difference at 5% level; Days after Inoculation (DAI) 21 Table 3.23 Reduce disease severity (%) of actinomycetes under net house condition over the survey periods Antagonistic efficacy (%) over time of survey Treatment DAI DAI DAI 10 DAI 12 DAI 14 DAI f g fg g e S06-MBL-A 261 28.89 30.57 48.08 55.01 35.87e d d gh fg de S06-MBL-A-T 55.87 43.00 28.29 55.68 56.36 47.18cd S06-MBL-A-S 57.26d 16.20h 36.06def 50.11gh 57.04de 46.84cd d ef cde cd cd S06-MBL-A-TS 52.70 38.80 42.05 61.49 61.15 53.34c e efg efg gh de S09-MBL-A 34.40 33.78 34.92 50.02 55.74 43.55d d fg bc ef cd S09-MBL-A-T 54.16 30.00 45.88 58.98 61.66 52.12c S09-MBL-A-S 54.58d 42.92d 45.96bc 61.08def 64.37bc 61.53b b b a ab a S09-MBL-A-TS 75.33 76.23 55.52 75.76 72.25 73.94a de efg i h f S17-MBL-A 46.99 33.10 21.64 47.25 42.13 40.97de S17-MBL-A-T 61.49cd 38.75ef 38.26def 51.22gh 60.00cde 44.43d d ef bc fgh cde S17-MBL-A-S 53.76 37.77 45.26 53.50 59.40 46.60cd bc c ab bc bc S17-MBL-A-TS 72.48 62.31 51.72 65.40 64.85 68.53ab a a a a ab Beam 75WP 88.71 86.29 54.21 80.44 68.79 71.72a Significance level ** ** ** ** ** ** CV% 11.69 7.05 8.82 6.19 3.52 5.39 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data are converted to arcsin x for statistical analysis *: statistically significant difference at 5% level; Days after Inoculation (DAI) 3.6 CONTENT 6: EVALUATION OF THE ABILITY TO CONTROL RICE BLAST DISEASE WITH PROMISING ACTINOMYCETES STRAINS UNDER THE FIELD CONDITION ON 2017 AND 2018 SEASONS 3.6.1 Height and number of shoots of experimental rice The results of field experiments in saline fields showed that the treatment of seed coat and spraying actinomycetes suspension on leaves did not affect the growth of height and number of shoots of rice plants in the experimental field in both seasons 3.6.2 The ability to control leaf blast disease 3.6.2.1 Rate of leaf blast disease Experimental results showed that both actinomycetes strain S09-MBL and S17-MBL managed blast disease caused by P oryzae The disease was lower than the control treatment in the 2017 and 2018 seasons On the other 22 hand, blast disease often appears early in the seedling stage, so treating actinomycetes before sowing and foliar spraying at the seedling stage (20 and 40 DAS) was more effective in limiting the damage of P oryzae 3.6.2.2 Index of leaf blast disease Similar to the disease rate, the index of leaf blast disease in the treatment treatments of actinomycetes was lower than that of the control treatments in both the 2017 and 2018 seasons Research results show that the treatment of actinomycete bacteria is lower than that of the control treatment in both 2017 and 2018 Seeds before sowing and foliar spraying at the 20 and 40 DAS stages brought about better effectiveness in limiting the damage of P oryzae and equivalent to chemical treatments In the 2018 season, two treatments treated with actinomycete strain S09-MBL (S09-MBL-A, 20, 40 and S09-MBL-A, 20, 40, 60) had a lower disease index than the two treatments with strain S17-MBL (S17-MBL-A, 20, 40 and S17-MBL-A, 20, 40, 60), statistically significant difference and treatment S09-MBL-A, 20, 40, 60 had disease index equivalent to fungicide treatment 3.6.2.3 Antagonistic efficacy of leaf blast disease Table 3.29 Antagonistic efficacy (%) on saline field in Ninh Thanh Loi Commune, Hong Dan District, Bac Lieu province, 2017 season No Treatment 10 11 12 13 14 15 16 17 S09-MBL-A S09-MBL-A,20 S09-MBL-A,40 S09-MBL-A,60 S09-MBL-A,20,40 S09-MBL-A,20,60 S09-MBL-A,40,60 S09-MBL-A,20,40,60 S17-MBL-A S17-MBL-A,20 S17-MBL-A,40 S17-MBL-A,60 S17-MBL-A,20,40 S17-MBL-A,20,60 S17-MBL-A,40,60 S17-MBL-A,20,40,60 Fungicide Significance level CV (%) Antagonistic efficacy (%) over survey time 27 DAS 34 DAS 41 DAS i 48 DAS i 55 DAS i 44.2bcd 37.2c 17.8b 15.2cd 15.4c 49.5abcd 44.2ab 14.2b 18.5bcd 20.6bc d c b b 41.5 37.4 18.0 23.5 23.5bc bcd c b d 43.8 36.6 17.8 14.7 16.6c 52.9ab 44.7ab 15.5b 23.3b 26.4ab 51.5abcd 46.0ab 15.9b 18.2bcd 19.7bc bcd c b b 43.9 36.7 16.8 24.9 23.5bc 51.9abc 42.9ab 12.4b 25.1b 26.7ab 43.6bcd 36.3c 17.0b 14.2d 15.0c abcd ab b bcd 50.3 43.9 13.1 18.0 17.6bc 42.4cd 36.1c 18.5b 25.7b 24.3abc 43.7bcd 36.4c 17.7b 13.4d 14.7c ab ab b b 53.0 44.4 15.8 23.8 26.3ab 47.5abcd 42.4b 14.9b 18.8bcd 20.9bc 42.5cd 35.6c 19.0b 24.8b 23.3bc abcd ab b bc 50.0 44.8 15.6 22.9 26.5ab 56.7a 47.9a 32.5a 35.8a 33.1a * * * * * 16.19 10.88 6.75 29.32 10.62 23 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data are converted to arcsin x (i) for statistical analysis; *: statistically significant difference at 5% level; %; A: seed coatings; 20,40,60: day after sowing (DAS) The results showed that most treatments that treated actinomycetes with seed coating and spraying on leaves at 20 and 40 DAS had higher efficiency in reducing leaf blast than those that treated only seed coating Treatment S09-MBL-A, 20, 40, 60 and S17-MBL-A, 20, 40, 60 showed high efficiency in reducing leaf blast disease, equivalent to fungicide in the 2017 and 2018 seasons (Table 4.29) The results show that two actinomycetes strains, S09-MBL and S17-MBL, have potential applications for managing rice blast disease in salt-affected soils 3.6.3 The ability to control collar blast disease 3.6.3.1 Rate of collar blast disease The results showed that all treatments with actinomycetes had a lower rate of collar blast than the control treatment in both seasons In the 2018 seasons, two treatments, S09-MBL-A, 20, 40, 60 and S17-MBL-A, 20, 40, 60, continued to have the lowest rate of blast disease, equivalent to the chemical treatment Thereby, two actinomycetes strains, S09-MBL and S17MBL, were effective in limiting collar blast compared with the adverse control treatment Thus, treating actinomycete suspension at the 60 DAS stage has reduced the incidence of collar blast disease in saline rice fields 3.6.3.2 Antagonistic efficacy of collar blast disease In general, two actinomycetes, S09-MBL and S17-MBL, contributed to the reduction of collar blasts in both seasons Especially in the 2018 season, the treatment was treated with actinomycetes S09-MBL with seed coating and spraying three times (20, 40 and 60 DAS) to reduce blast disease equivalent to the chemical treatment The results show the potential of collar blast disease management of two actinomycetes, S09-MBL and S17-MBL, under field conditions in saline cultivated fields (Table 3.31 Table 3.40) 24 Table 3.31 Table 3.40 Antagonistic efficacy of collar blast disease (%) on saline fields in Hong Dan District, Bac Lieu province 2017 and 2018 season No 10 11 12 13 14 15 16 17 Treatment S09-MBL-A S09-MBL-A,20 S09-MBL-A,40 S09-MBL-A,60 S09-MBL-A,20,40 S09-MBL-A,20,60 S09-MBL-A,40,60 S09-MBLA,20,40,60 S17-MBL-A S17-MBL-A,20 S17-MBL-A,40 S17-MBL-A,60 S17-MBL-A,20,40 S17-MBL-A,20,60 S17-MBL-A,40,60 S17-MBLA,20,40,60 Fungicide Significance level CV (%) Antagonistic efficacy (%) 2017 season 67 74 81 DASi DASi DASii 52.1e 46.2c 53.7d e bc 51.7 49.6 54.3d de bc 56.8 50.3 57.0bcd 57.5de 50.3bc 55.2bcd cd bc 62.9 50.3 56.9bcd de bc 57.8 49.9 53.9d 62.6cd 51.4bc 54.7cd b b 70.7 53.7 54.4 d Antagonistic efficacy (%) 2018 season 67 74 81 DASi DASi DASii 13.2hi 20.2gh 17.2f ghi fgh 21.2 28.2 25.1ef fghi ef 21.9 32.5 19.7f 35.9de 46.3cd 24.8ef de de 38.6 39.9 35.1cde cd cd 45.4 43.9 39.9bc 52.7bc 51.2bc 40.7bc ab a 64.2 62.1 62.9a 50.9e 52.8e 56.8de 57.2de 61.4cd 56.7de 61.1cd 66.3bc 45.4c 46.0c 51.3bc 50.9bc 50.5bc 52.1bc 50.1bc 51.8bc 56.1bcd 53.8 d 57.1bcd 55.7bcd 59.4b 59.2bc 56.4bcd 58.2bcd 17.9hi 32.7def 28.1efgh 31.9efg 30.7efg 35.2de 45.7cd 59.0ab 21.2h 24.8gh 31.9efg 39.3de 30.3de 39.1bcd 49.4bc 56.8ab 14.8f 26.0def 26.1def 37.7bcd 23.7ef 30.9cde 40.4bc 50.1b 74.9a 74.9a 77.2a 68.2a 63.4a 67.3a * 7.29 * 6.65 * 4.14 * * * 9.82 14.3 12.9 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test Data are converted to x + 0,5 (i) and arcsin x (ii) for statistical analysis; *: statistically significant difference at 5% level; %; A: seed coatings; 20,40,60: day after sowing (DAS) 3.6.4 Effect of actinomycetes on yeild components and the yield rice The results of the 2017 season showed that, although there was no difference in the number of panicles/m2 between treatments, there was a difference in the number of fill grains/panicle, leading to a statistically significant difference in actual yield, the significance level of 5% The number of fill grain/panicles of all treatments with actinomycetes was higher than the control Two treatments, S09-MBL-A, 20, 40, 60 and S17-MBL-A, 20, 40, 60, had the highest number of fill grain/panicle and the highest actual yield (6.41-6.45 tons/ha) 25 Table 3.32 Table 3.41 The yeild components and the yield rice on saline fields in Hong Dan District, Bac Lieu province 2017 and 2018 season The yeild components and the yield 2017 season The The The number number actual of fill yield panicles grain/ (tons/ /m2 panicle ha) The yeild components and the yield 2018 season The The The number number actual of fill yield panicles grain/ (tons/ /m2 panicle ha) No Treatment S09-MBL-A 283 87.4fgh 5.51c 236hi 91.5ef 5.13e S09-MBL- A, 20 287 96.7def 5.57c 262cd 91.7ef 5.10e 5.38c 249fg 90.0f 5.31e S09-MBL- A, 40 286 106cd S09-MBL- A, 60 291 89.6fgh 5.84c 262cd 102bcd 5.68cd 5.73c 252ef 92.5ef 5.20e S09-MBL- A, 20, 40 288 110c S09-MBL- A, 20, 60 290 106cd 5.39c 261cd 106b 5.57cd 285 5.85c 264c 104bcd 5.69c 296 129b 6.41b 285ab 115a 6.23ab S09-MBL- A, 40, 60 S09-MBL- A, 20, 40, 60 S17-MBL-A 109c 288 86.3gh 5.54c 232j 90.3f 5.07e 10 S17-MBL- A, 20 287 93.6efg 5.69c 245g 96.8cdef 5.15e 5.88c 243g 96.7cdef 5.12e 11 S17-MBL- A, 40 284 88.7fgh 12 S17-MBL- A, 60 286 885gh 5.83c 257cde 99.1bcde 5.35de 5.84c 242gh 95.5def 5.25e 13 S17-MBL- A, 20, 40 293 101cde 14 S17-MBL- A, 20, 60 288 108c 5.57c 256de 104.5bc 5.65c S17-MBL- A, 40, 60 S17-MBL-A, 20, 40, 60 282 106cd 5.81c 258cde 106.7b 5.60cd 291 125b 6.45b 280b 114a 6.01b Fungicide Control 294 141.6a 6.98a 289a 118a 6.40a 284 79.8h 4.69d 225k 80.7g 4.75f ns * * * * * 7.30 5.27 5.12 13.56 8.36 15 16 17 18 Significance level CV (%) 6.85 Note: in the same column numbers followed by the same letters are not statistically significant in Duncan's test; ns: non-significant difference; *: statistically significant difference at 5% level; %; A: seed coatings; 20,40,60: day after sowing (DAS) In the 2018 season, the number of panicles/m2 of all treatments with actinomycetes was higher than the control, ranging from 232-285 panicles/m2 The highest number of fill grain/panicle was found in two treatments of seed coating and leaf spraying of actinomycetes S09-MBL and S17-MBL at the density of 108 CFU/ml all three times of 20, 40 and 60 DAS, equivalent to fungicide spraying At the same time, the treatment of 26 actinomycetes also increased the actual yield compared with the negative control In which the two treatments with the highest yield were S09-MBLA, 20, 40, 60 (6.23 tons/ha) and S17-MBL-A, 20, 40, 60 (6.01 tons/ha); only treatment S09-MBL-A, 20, 40, 60 had a high yield equivalent to fungicide spraying treatment The application of two actinomycetes, S09-MBL and S17-MBL, helped manage rice blast disease on salt-affected soils and increased the actual yield compared to the untreated control (Table 3.32 Table 3.41) CHAPTER CONCLUSION AND PROPOSAL 4.1 CONCLUSION One hundred and twenty six actinomycetes strains were collected and isolated from saline rice soils in the Mekong Delta In which, there are actinomycete strains (S06-MBL, S09-MBL, S17-MBL, S09-MTV, S03-MST, S18-MCM, S31-MCM) with high antagonism against the fungus Pyricularia oryzae causing rice blast disease Three actinomycete strains (S06-MBL, S09-MBL and S17-MBL) showed high antagonism against the blast fungus Pyricularia oryzae in the medium supplemented with 2‰ concentration of NaCl salt; capable of inhibiting fungal spore germination, and exhibited high secretion of chitinase and β-1,3-glucanase enzymes Strain S06-MBL is Streptomyces fradiae, strain S09-MBL is Streptomyces bikiniensis and strain S17-MBL is Streptomyces lavendulae Two actinomycete strains S09-MBL and S17-MBL, showed high disease reduction efficiency equivalent to Beam 75WP fungicide when treating seed coating and spraying on leaves (density 108 CFU/ml) two days before and two days after artificial inoculation showed a high ability to prevent rice blast disease in net house condition, with low disease rate (< 4.22%), low disease index (