Luận văn thạc sĩ HUS nghiên cứu đặc điểm sinh học chủng xạ khuẩn streptomyces toxytricini (VN08 a12) kháng bệnh bạc lá lúa do xanthomonas oryzae

ĐẠI HỌC QUỐC GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN - NGUYỄN THỊ VÂN NGHIÊN CỨU ĐẶC ĐIỂM SINH HỌC CHỦNG XẠ KHUẨN Streptomyces toxytricini (VN08 - A12) KHÁNG BỆNH BẠC LÁ LÚA DO Xanthomonas oryzae LUẬN VĂN THẠC SĨ KHOA HỌC Hà Nội - 2014 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com ĐẠI HỌC QUỐC GIA HÀ NỘI TRƯỜNG ĐẠI HỌC KHOA HỌC TỰ NHIÊN - NGUYỄN THỊ VÂN NGHIÊN CỨU ĐẶC ĐIỂM SINH HỌC CHỦNG XẠ KHUẨN Streptomyces toxytricini (VN08 - A12) KHÁNG BỆNH BẠC LÁ LÚA DO Xanthomonas oryzae Chuyên ngành: Vi sinh vật học Mã số: 60420107 LUẬN VĂN THẠC SĨ KHOA HỌC NGƯỜI HƯỚNG DẪN KHOA HỌC: PGS TS Dương Văn Hợp TS Phạm Thế Hải Hà Nội - 2014 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com LỜI CẢM ƠN Lời đầu tiên, em xin gửi lời cảm ơn chân thành tới PGS TS Dương Văn Hợp TS Phạm Thế Hải, người thầy trực tiếp hướng dẫn em tận tình trình thực đề tài, giúp em vượt qua khó khăn hồn thành tốt luận văn Em xin gửi lời cảm ơn tới thầy cô giáo Khoa Sinh học, trường Đại Học Khoa học Tự nhiên nhiệt tình giảng dạy, hướng dẫn cung cấp cho em kiến thức bổ ích suốt hai năm học vừa qua giúp đỡ em nhiều việc nắm bắt kiến thức động viên em lớn mặt tinh thần Em xin gửi lời cảm ơn sâu sắc tới TS Nguyễn Kim Nữ Thảo Viện Vi sinh vật Công nghệ Sinh học, Đại học Quốc gia Hà Nội tận tình giúp đỡ, bảo, động viên tạo điều kiện thuận lợi để em hoàn thành tốt luận văn Em xin chân thành cảm ơn TS Phan Thị Phương Hoa (chủ nhiệm đề tài Nafosted số 106.03 - 2010.34 “Sàng lọc chất có hoạt tính sinh học từ xạ khuẩn dùng cho đấu tranh sinh học diệt vi khuẩn Xanthomonas oryzae pv oryzae gây bệnh bạc lúa Việt Nam”) thành viên tham gia đề tài hỗ trợ cho em kỹ thuật chuyên môn để em hoàn thành luận văn Em xin gửi lời cảm ơn Ban lãnh đạo anh chị Viện Vi sinh vật Công nghệ sinh học chia sẻ, giúp đỡ tạo điều kiện lớn suốt trình em thực luận văn Em xin cảm ơn TS Kyung Sook Bae, TS Song Gun Kim cử nhân Hyangmi Kim Bảo tàng giống chuẩn (KCTC), Viện Khoa học Công nghệ sinh học Hàn Quốc (KRIBB) tạo điều kiện thuận lợi giúp đỡ em thời gian học tập ngắn hạn Hàn Quốc để em hoàn thành luận văn Em xin cảm ơn TS Hiroshi Kinoshita trường Đại học Osaka Nhật Bản hỗ trợ giúp đỡ em q trình nghiên cứu để em hồn thành luận văn i LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Xin cảm ơn sinh viên Vũ Thị Kim Chi hỗ trợ em số cơng việc thí nghiệm q trình nghiên cứu để em hồn thành luận văn Cuối cùng, em xin gửi lời cảm ơn đến gia đình thân u bạn bè ln bên, động viên, giúp đỡ em suốt thời gian học tập thực luận văn Hà Nội, ngày tháng 10 năm 2014 Học viên Nguyễn Thị Vân ii LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com MỤC LỤC MỞ ĐẦU MỤC TIÊU NGHIÊN CỨU CHƢƠNG I TỔNG QUAN 1.1 Bệnh bạc lúa tác hại bệnh bạc lúa 1.1.1 Giới thiệu chung 1.1.2 Tác nhân gây bệnh bạc lúa 1.2 Các cách phòng trừ bệnh bạc lúa 1.2.1 Sử dụng thuốc hóa học 1.2.2 Chọn giống lúa kháng bệnh 1.2.3 Khống chế sinh học 11 1.3 Xạ khuẩn 12 1.3.1 Giới thiệu chung xạ khuẩn 12 1.3.2 Khả sinh chất kháng sinh xạ khuẩn 14 1.3.3 Sử dụng xạ khuẩn khống chế sinh học .16 1.4 Tình hình nghiên cứu xạ khuẩn kiểm soát bệnh bạc lúa 17 CHƢƠNG NGUYÊN LIỆU VÀ PHƢƠNG PHÁP NGHIÊN CỨU 19 2.1 Nguyên liệu hóa chất 19 2.1.1 Nguyên liệu 19 2.1.1.1 Các chủng Xoo 19 iii LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 2.1.1.2 Các chủng xạ khuẩn 20 2.1.1.3 Vi sinh vật kiểm định 20 2.1.2 Hóa chất, dụng cụ thiết bị .20 2.1.2.1 Hóa chất 20 2.1.2.2 Dụng cụ thiết bị 21 2.1.2.3 Môi trƣờng nghiên cứu 21 2.2 Phƣơng pháp nghiên cứu 22 2.2.1 Phƣơng pháp thử hoạt tính kháng vi sinh vật 22 2.2.1.1 Phƣơng pháp thỏi thạch 22 2.2.1.2 Phƣơng pháp đục lỗ thạch 23 2.2.2 Phân loại đặc điểm hình thái .23 2.2.2.1 Hình thái khuẩn lạc 23 2.2.2.2 Hình thái chuỗi sinh bào tử bề mặt bào tử 23 2.2.3 Hóa phân loại .24 2.2.3.1 Phân tích thành phần axit amin thành tế bào 24 2.2.3.2 Phân tích thành phần menaquinone 24 2.2.3.3 Phân tích thành phần axit béo 25 2.2.3.4 Phân tích thành phần G+C ADN 25 2.2.4 Phân loại sinh học phân tử giải trình tự 16S - rADN 25 2.2.5 Tối ƣu hóa điều kiện nuôi cấy xạ khuẩn 27 2.2.5.1 Lựa chọn mơi trƣờng ni cấy thích hợp 27 2.2.5.2 Lựa chọn thời gian ni cấy thích hợp 27 2.2.5.3 Lựa chọn thể tích ni cấy thích hợp 28 2.2.5.4 Lựa chọn nhiệt độ nuôi cấy 28 iv LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 2.2.5.5 Lựa chọn cách cấy giống 28 2.2.6 Tinh hoạt chất kháng Xoo 28 2.2.6.1 Tách dịch chiết thô 28 2.2.6.2 Tinh silica gel 28 2.2.6.3 Tinh HPLC 29 2.2.7 Xác định khối lƣợng phân tử khối phổ (MS) 29 CHƢƠNG KẾT QUẢ VÀ THẢO LUẬN 31 3.1 Khả kháng vi sinh vật chủng xạ khuẩn VN08 - A12 31 3.2 Phân loại chủng VN08 - A12 32 3.2.1 Phân loại hình thái 32 3.2.2 Hóa phân loại 33 3.2.2.1 Thành phần axit amin thành tế bào 33 3.2.2.2 Thành phần menaquinone 34 3.2.2.3.Thành phần axit béo 35 3.2.2.4 Thành phần G + C ADN 37 3.2.3 Trình tự 16S rADN 38 3.3 Tối ƣu hóa điều kiện nuôi cấy chủng VN08 - A12 39 3.3.1 Mơi trƣờng ni cấy thích hợp 39 3.3.2 Thời gian ni cấy thích hợp .40 3.3.3 Thể tích ni cấy thích hợp .41 3.3.4 Nhiệt độ ni cấy thích hợp 41 3.3.5 Cách cấy giống thích hợp 42 3.4 Tinh phân tích hoạt chất kháng Xoo chủng VN08 - A12 43 3.4.1 Tinh hoạt chất kháng Xoo chủng VN08 - A12 43 v LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 3.4.2 Phân tích trọng lƣợng phân tử 47 KẾT LUẬN VÀ KIẾN NGHỊ 51 4.1 Kết luận 51 4.2 Kiến nghị 51 TÀI LIỆU THAM KHẢO 52 PHỤ LỤC - - vi LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com DANH MỤC CÁC BẢNG Bảng Đặc điểm nguồn gốc gen Xa kháng bệnh bạc lúa [37] Bảng Trình tự đoạn mồi đặc hiệu để phát gen Xa lúa [15] Bảng Danh sách 10 chủng Xoo phân lập đƣợc miền Bắc Việt Nam đƣợc sử dụng nghiên cứu 19 Bảng Chƣơng trình phân tích HPLC 29 Bảng Hoạt tính kháng 10 chủng Xoo chủng xạ khuẩn VN08 - A12 31 Bảng Hoạt tính kháng vi sinh vật chủng xạ khuẩn VN08 - A12 31 Bảng Hoạt tính kháng vi sinh vật có lợi chủng xạ khuẩn VN08 - A12 32 Bảng : Kết phân tích thành phần menaquinone chủng VN08 - A12 35 Bảng Kết phân tích thành phần menaquinone chủng VN08 - A12 36 Bảng 10 Kết phân tích thành phần GC chủng VN08 - A12 38 Bảng 11 Hoạt chất VN08 - A12 thu đƣợc từ phân đoạn tinh silica gel 44 Bảng 12 Hoạt chất VN08 - A12 thu đƣợc từ HPLC từ phân đoạn 4.1 45 Bảng 13 Chƣơng trình chạy HPLC phân tích thành phần axit amin - Bảng 14 Phân tích thành phần menaquinone chuẩn - Bảng 15 Phân tích thành phần axit béo chuẩn - Bảng 16 Trình tự mồi dùng cho phản ứng đọc trình tự ADNr 16S - - vii LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com DANH MỤC CÁC HÌNH Hình Lúa bị nhiễm bệnh bạc vi khuẩn Xoo Hình Khuẩn lạc tế bào vi khuẩn Xoo [9] Hình Tổn thƣơng giống lúa IRBB21 chứa gen Xa21 bệnh bạc lúa Ấn Độ [19] 10 Hình Khuẩn lạc số chủng xạ khuẩn 14 Hình Cuống sinh bào tử số chủng xạ khuẩn 14 Hình Bản đồ phân bố 10 chủng Xoo miền Bắc Việt Nam đƣợc sử dụng nghiên cứu 20 Hình Khuẩn lạc chuỗi bào tử chủng VN08 - A12 33 Hình Chuỗi bào tử bào tử chủng VN08 - A12 33 Hình Kết chạy sắc ký mỏng thành phần axit amin thành tế bào chủng VN08 - A12 34 Hình 10 Kết phân tích LC thành phần menaquinone chủng VN08 - A12 35 Hình 11 Sắc ký đồ thành phần axit béo chủng VN08 - A12 36 Hình 12 Sắc ký đồ thành phần GC chủng VN08 - A12 37 Hình 13 Cây phân loại chủng VN08 - A12 dựa trình tự gen 16S – rADN 39 Hình 14 Ảnh hƣởng mơi trƣờng ni cấy khác đến sản sinh hợp chất kháng sinh ức chế vi khuẩn Xoo chủng VN08 - A12 40 Hình 15 Ảnh hƣởng thời gian cấy khác đến sản sinh hợp chất kháng sinh ức chế vi khuẩn Xoo chủng VN08 - A12 40 Hình 16 Ảnh hƣởng thể tích mơi trƣờng ni cấy khác đến sản sinh hợp chất kháng sinh ức chế vi khuẩn Xoo chủng VN08 - A12 (S toxytricini) 41 Hình 17 Ảnh hƣởng nhiệt độ môi trƣờng nuôi cấy khác đến sản sinh hợp chất kháng sinh ức chế vi khuẩn Xoo chủng VN08 - A12 42 Hình 18 Ảnh hƣởng cách cấy giống đến sản sinh hợp chất kháng sinh ức chế vi khuẩn Xoo chủng VN08 - A12 43 Hình 19 Quy trình tách dịch chiết thô chủng VN08 - A12 44 Hình 20 Phổ HPLC hoạt chất chủng VN08 - A12 46 viii LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Table Abilities of actinomycete strains to inhibit races of Xoo Number of actinomycete isolates 77 (85.6%) No of actinomycete isolates inhibit No of actinomycete isolates each Xoo race that inhibited each Xoo race Number of Xoo races (0%) (1.1%) R2 R3 (0%) (1.1%) (0%) (0%) (5.6%) (2.2%) (1.1%) 10 (3.3%) 14 12 10 R1 R4 R5 R6 Xoo races R7 R8 R9 R10 Xoo races Figure Inhibition of Xoo races by actinomycete isolates Figure Inhibition of actinomycete isolates to Xoo races seeded by R2 race in triplicate After incubation for 24 to 72 h, results were read by the diameter of the inhibition zone pH and heat stability test A volume of ml cell extract/1.5 ml-Eppendorf tube was boiled at 100°C for 10 On the other hand, the cell extract solution was adjusted to pH 2, and 10 100 μl of the treated solutions were used for antagonistic activity assays in the R2 race 16S rRNA gene (1300 to 1400 bp) was amplified by PCR as reported by Tamura et al (1999) and directly sequenced using an ABI Prism BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and an ABI Model 3730 automatic DNA sequencer The 16S rRNA gene sequence was compared with other sequences in the EMBL/GenBank/DDBJ database using BLAST searches and in the EzTaxon16 database, which includes only type strain sequences RESULTS Solvent extraction test The cell extract by acetone was extracted again by ethyl-acetate and n-butanol at pH and 10 100 μl of the treated solution (top and bottom of ethyl acetate extract at pH and 10; top and bottom of n-butanol extract at pH and 10) were used for antagonistic activity assays in the R2 race Identification of VN 10-A44 Identification of VN-A-44 was based on morphological, chemical and molecular approaches (Sakiyama et al., 2009) The VN10-A-44 was identified by 16S rRNA gene sequencing Genomic DNA extraction was carried out using a Promega (Madison,WI, USA) extraction kit according to the manufacturer’s protocol The 16S ribosomal RNA (rRNA) gene was amplified by PCR using TaKaRa Ex Taq (Takara Bio, Otsu City, Shiga, Japan) with the primers, 9F (5'-GAGTTTGATCCTGGCTCAG-3') and 1541R (5'AAGGAGGTGATCCAGCC-3'), or occasionally 1510R (5'GGCTACCTTGTTACGA-3') Almost all the entire sequence of the Screening for the actinomycete strains capable of inhibiting Xoo races Results are summarized in Table and Figure We found that 13 among 90 isolates (14.4%) investigated were able to inhibit at least two races of Xoo Three (3.3%) isolates were able to inhibit all the 10 races of pathogen (Tables and 3) All the 13 (100%) selected strains were able to inhibit pathogen races R1 and R3; whereas 12/13 (92.3%) inhibited R4 and R5; and 11/13 (84.6%) inhibited R7 and R8 Six among 13 selected strains inhibited R6 and four inhibited R9 (Figure 2) Only 3/13 actinomycete strains could inhibit the pathogen race R2 The actinomycete strains that inhibited race R2 (VN10-A-23, VN10-A-44 and VN10-A-54) were also able to inhibit race R9 (Table 3) LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Table Antagonistic activities of actinomycete-tested isolates Actinomycete strain VN10-A-15 VN10-A-16 VN10-A-19 VN10-A-23 VN10-A-24 VN10-A-30 VN10-A-38 VN10-A-39 VN10-A-44 VN10-A-58 VN10-A-74 VN10-A-76 VN10-A-77 R1 2.0 0.9 0.9 0.9 2.4 1.1 1.8 1.1 2.5 2.6 0.5 3.5 2.0 R2 0.3 0.1 0.1 - Inhibition zones against to a Xoo race (D-d) cm R3 R4 R5 R6 R7 R8 1.2 2.8 3.5 2.0 2.6 1.5 1.2 1.9 0.2 0.3 1.2 1.1 0.4 0.3 2.3 1.1 1.8 0.1 0.9 1.2 2.7 2.5 1.7 0.1 1.0 1.5 1.2 1.1 1.5 0.7 0.5 1.8 1.5 1.7 1.3 1.3 0.7 0.7 1.3 0.8 0.8 2.5 1.5 2.5 0.2 1.9 1.9 0.1 1.8 1.8 0.5 1.7 1.3 1.8 0.8 1.1 0.9 0.1 1.5 0.8 1.1 1.8 2.3 1.1 1.1 R9 0.1 0.1 0.3 0.1 - R10 2.0 0.1 0.2 2.0 1.0 1.3 + 2.0 0.1 0.9 + The inhibition zones were measured by (D-d) cm; the plus denotes that actinomycete strains have positive results but these were not large enough to be measured; the minus denotes that actinomycete strains did not inhibit the pathogen races Table Antagonistic activities of the selected strains Microorganism B subtilis E coli S cerevisiae M luteus Actinomycete strain VN10-A-44 (D-d) cm 1.4 ± 0.2 - VN10-A-23 (D-d) cm 1.3 ± 0.4 + 4.7 ± 0.3 + VN10-A-58 (D-d) cm 2.1 ± 0.1 + 0.9 ± 0.1 3.0 ± 0.2 The inhibition zones were measured by (D-d) cm; the plus denotes that actinomycete strains have positive results but these were not large enough to be measured; the minus denotes that actinomycete strains did not inhibit the microorganisms; ±, indicates standard deviation Primary tests for toxicity producer We selected strains that inhibited all the 10 races of Xoo for primary toxicity production test The strains were tested for their inhibition of indicator microorganisms (B subtilis, M luteus, E coli and S cerevisiae) We found that VN10-A-23 and VN10-A-58 could inhibit all the tested microorganisms; whereas VN10-A-44 inhibited B subtilis only and was therefore chosen for further study (Table 4) Enhancing antibiotic production of VN10-A-44 by replacing soybean meal with waste of tofu in antibiotic producing medium VN10-A-44 inhibits all the 10 pathogen races; however, the inhibition zone for R2 was very small (Figure 3) Tofu waste is abundant in Vietnam and some other countries in Asia The APM with tofu waste is called modified APM (MAPM) and it is used to test whether tofu waste can be recycled for producing antibiotics of VN10-A-44 If so, tofu waste may be used for large-scale production of the antibiotics Result shows that the inhibition zone was improved (Table 5) when VN10-A-44 was cultured in broth APM (Figure 3B), and largest in broth MAPM (Figure 3C) The amount of tofu waste for replacing soybean meal was determined (Table 6) We found that 20 and 30 g of tofu waste/litter gave the highest inhibition zones for R2 On a daily basis, large amounts of tofu waste were excreted at tofu production sites in Vietnam We examined the influence of the life-time of tofu waste on the results of R2 inhibition The results are recorded in Figure We found that 4-day old tofu waste was the best to produce antibiotics against race R2 Research on preliminary extraction tests of antibiotics produced by VN 10-A-44 showed that the active compound was in n-butanol cells and a pH of was the best conditions for extract The active compound lost its LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com (B) (A) (C) Figure The inhibition ofFigure Xoo R23 byThe VN10-A-44 A, Broth medium; B, broth APM; inhibition of YS VN10-A-44 to Xoo R2C, broth MAPM A, broth YS medium; B, broth APM; C, broth MAPM Table Inhibition zones (D-d) cm of VN10-A44 against 10 Xoo races in YS, APM and MAPM Medium YS APM MAPM R1 2.5 1.9 2.5 R2 0.1 0.6 1.4 R3 2.5 1.5 2.2 R4 1.5 1.8 2.0 R5 2.5 2.2 2.5 R6 0.2 1.0 1.0 R7 1.9 1.3 2.0 R8 1.9 1.7 2.0 R9 0.3 1.0 1.2 R10 2.0 1.7 2.0 YS, Yeast-extract medium; APM, antibiotic-producing medium; MAPM, modified antibiotic producing medium Table Antagonistic activities of VN10-A-44 against Xoo R2 with different amounts of tofu waste in broth MAPM Medium and different amounts of tofu waste YS APM (10 g/ littler or 1% soybean meal) MAPM (10 g/ littler or 1% tofu waste) MAPM (20 g/ littler or 2% tofu waste) MAPM (30 g/ littler or 3% tofu waste) MAPM (40 g/ littler or 4% tofu waste) MAPM (50 g/ littler or 5% tofu waste) MAPM (60 g/ littler or 6% tofu waste) MAPM (70 g/ littler or 7% tofu waste) MAPM (80 g/ littler or 8% tofu waste) MAPM (90 g/ littler or 9% tofu waste) MAPM (100 g/ littler or 10% tofu waste) MAPM (130 g/ littler or 13% tofu waste) MAPM (150 g/ littler or 15% tofu waste) MAPM (200 g/ littler or 20% tofu waste) inhibitionactivity against the pathogen when being treated at 100°C Identification of VN-A-44 by analyzing the 16S- DNA sequence showed that the VN10-A-44 strain was closest to Streptomyces virginiae (100%, 1464/1464 bp, identity to accession number AB184175) (Figure 6) Morphology of VN10-A-44 was recorded and is shown in Figure It belongs to Streptomyces with spore chains Test 0.1 0.7 1.3 1.3 1.3 1.4 1.2 1.2 1.2 1.2 1.4 0.9 0.9 1.0 0.9 Inhibition zone (D-d) cm Test Test 0.1 0.1 0.8 0.8 1.3 1.4 1.4 1.4 1.4 1.5 1.4 1.4 1.3 1.4 1.2 1.3 1.3 1.3 1.2 1.2 1.3 1.3 0.9 1.0 0.9 1.2 1.0 1.0 0.9 1.0 Average 0.1 0.8 1.3 1.4 1.4 1.4 1.3 1.2 1.3 1.2 1.3 0.95 1.0 1.0 0.9 DISCUSSION Biocontrol of Xoo was recently reported in Asia (Gnanamanickam 2009; Ji et al., 2008; Park et al., 2011) With an attempt to select a biological agent from strains of actinomycete, in this study, we screened for actinomyce strains inhibiting all the 10 races of Xoo, whereas at the same time selecting for strains that were LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Inhibition zones (cm) 2.5 2.0 1.5 1.0 0.5 Days Days Antagonisticactives actives of depending on life-time points of tofu waste afterwaste production FigureFigure Antagonistic ofVN10-A-44 VN10-A-44 depend on life-time points of tofu after production a) b) Figure Morphology of VN10-A-44 a, colony; b, spore Figure Morphology of VN10-A-44 a, Colony; b, spore not harmful to other micro-biota and humans Among 90tested strains, we selected 13 actinomyces isolates that were able to inhibit from one to 10 races of Xoo This number of actinomyces strains inhibiting races of Xoo (14.4%) was not high, and this result is in agreement with another study showing lower numbers of Streptomyces strains capable of inhibiting Xoo; whereas most of streptomyces isolates inhibited Xanthomonas vascatoria and Clavibacter michiganensis ssp michiganensis (Ndonle and Semu, 2000) In the present study, we found that the R1 and R3 races of Xoo were inhibited by 13 actinomyces strains; whereas R2 and R9 were the most difficult to inhibit Most of the actinomyces strains can inhibit at least races of Xoo (R1 and R3), however only strains (VN10-A-23; VN10-A-44; VN10-A-58) were able to inhibit all the 10 races Antibiotic production of actinomycete-inoculated soils was demonstrated in soils supplemented with a suitable organic source Stevenson (1959) found that fresh grass (3%), clover (3%) and soybean meal (2%) were suitable supplements for antibiotic production by the majority of the actinomycetes In this study, we showed that replacing soybean meal in APM with tofu waste (from to 4%) gave the largest inhibition zones (1.4 cm) against race R2; especially the 4-day old tofu waste (inhibition zones up to 2.0 cm) This could be due to the fact that the nutrient values obtained from fresh soybean found in tofu waste were much higher than those of soybean meal To make soybean meal, soybean was dried by heating and then made into powder This process reduced many vitamins and some essential amino acids which are very important for the development of actinomycetes from fresh soybean If tofu waste is used for fermentation of VN10-A-44, it may reduce the cost of large-scale LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 99 Streptomyces virginiae (AB184175) 0.005 98 VN-10A44 Streptomyces subrutilus (X80825) 90 Streptomyces setonii (D63872) Streptomyces bikiniensis (X79851) Streptomyces galbus (X79852) 73 Streptomyces lincolnensis (X79854) 72 Streptomyces diastatochromogenes (D63867) 99 Streptomyces bottropensis (D63868) 81 Streptomyces scabies (D63862) Streptomyces acidiscabies (D63865) Streptomyces griseocarneus (X99943) Streptomyces rimosus (X62884) Streptomyces mashuensis (X79323) Streptomyces sampsonii (D63871) Streptomyces glaucescens (X79322) Streptomyces pseudogriseolus (X80827) Streptomyces coelicolor (X60514) Streptomyces ambifaciens (M27245) Streptomyces caelestis (X80824) Streptomyces bluensis (X79324) Streptomyces espinosus (X80826) Streptomyces megasporus (Z68100) 92 80 90 76 Figure Phylogeny tree of VN10-A-44 and its relationship with some Streptomyces species antibiotic production and help recycle tofu waste Identification of VN10-A-44 by 16S-rDNA sequencing showed that VN10-A-44 is identical to S virginiae This species is reported to produce VirginiamycinM1 (Suzuki et al., 1998) and it has a high activity in vitro against Gram-positive and negative bacteria, yeast and fungi (Rifaat and Kansoh, 2005) ACKNOWLEDGEMENTS This work was supported by the National Foundation for Science and Technology Development, Vietnam and conducted as a joint research project between the Institute of Microbiology and Biotechnology, Vietnam National University Hanoi, Vietnam (VNUH-IMBT) and the Biological Resource Center, National Institute of Technology and Evaluation (NBRC), Japan REFERENCES Blackburn AS, Avery SV (2003) Genome-wide screening of Saccharomyces cerevisiae to identify genes required for antibiotic insusceptibility of eukaryotes Antimicrob Agents Chemother 47:676-681 Dai LY, Liu XL, Xiao YH, Wang GL (2007) Recent advances in cloning and characterization of disease resistance genes in rice J Int Plant Biol 49:112-119 Fredrickson JK, Elliott LF (1985) Effects on winter wheat seedling growth by toxin-producing rhizobacteria Plant Soil 83:399-409 Gnanamanickam SS (2009) Biological control of bacterial blight of rice, In Biological control of rice diseases Springer pp.67-78 Hastuti RD, Lestari Y, Saraswati R, Suwanto A, Chaerani (2012) Capability of Streptomyces spp In controlling bacterial leaf blight diseases in rice plants Am J Agric Biol Sci 7(2):217-223 Hayakawa M, Nonomura H (1989) A new method for the intensive isolation of actinomycetes from soil Actinomycetologica 3:95-104 Hayakawa M, Otoguro M, Takeuchi T, Yamazaki T, Iimura Y (2000) Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter Antonie Van Leeuwenhoek 78:171-185 Hop DV, Sakiyama Y, Binh CTT, Otoguto M, Hang DT, Miyadoh S, Luong DT, Ando K (2011) Taxonomic and ecological studies of actinomycetes from Vietnam: isolation and genus- level diversity J Antibiot (Tokyo):1-8 Ji GH, Wei LF, Wu YP, Bai XH (2008) Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1 Biol Control 45: 288-296 Ndonde MJM, Semu E (2000) Preliminary characterization of some Streptomyces species from four Tanzanian soils and their antimicrobial potential against selected plant and animal pathogenic bacteria W J Microb Biotech 16:595-599 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Park SB, Lee IA, Joo WS, Jeong GK, Lee HW (2011) Screening and Identification of Antimicrobial Compounds from Streptomyces bottropensis Suppressing Rice Bacterial Blight J Microbiol Biotechnol 21:1236-1242 Chithrashree A, Udayashankar C, Chandra NS, Reddy MS, Srinivas C (2011) Plant growth-promoting rhizobacteria mediate induced systemic resistance in rice against bacterial leaf blight caused by Xanthomonas oryzae pv Oryzae Biol Control 59:114-122 Rifaat HM, Kansoh AL (2005) Streptomyces verginiae: Taxonomic, Identification and biological activities Arab J Biotech 8:29-34 Rizk M, Abdel RT, Metwally H (2007) Screening of Antagonistic Activity in Different Streptomyces Species Against Some Pathogenic Microorganisms J Biol Sci 7:1418-1423 Sakiyama Y, Thao NKN, Giang NM, Miyadoh S, Hop DV, Ando K (2009) Kineosporia babensis sp nov., isolated from plant litter in Vietnam Int J Syst Evol Microbiol 59:550-554 Suzuki N, Lee CK, Nihira T, Yamada Y (1998) Purification and characterization of Virginiamycin M1 Reductase from Streptomyces virginiae Antimicrob Agents Chemother 42:2985-2988 Vasudevan P (2002) Isolation and characterization of Bacillus spp from the rice rhizosphere and their role in biological control of bacterial blight of rice caused by Xanthomonas oryzae pv oryzae Ph.D dissertation, University of Madras Velusamy P, Gnanamanickam SS (2003) Identification of 2,4diacetylphloglucinol (DAPG) production by plant-associated bacteria and its role in suppression of rice bacterial blight in India Curr Sci (Bangalore) 85:1270-1273 Velusamy P, Immanuel JE, Gnanamanickam SS, Thomashow LS (2006) Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol Can J Microbiol 52:56-65 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com Biocontrol Science, 2014, Vol 19, No 3, 103111 Original Biological Control of Xanthomonas Oryzae pv Oryzae Causing Rice Bacterial Blight Disease by Streptomyces toxytricini VN08-A-12, Isolated from Soil and Leaf-litter Samples in Vietnam DUONG VAN HOP1, PHAN THI PHUONG HOA1,4 , NGUYEN DUC QUANG2, PHAN HUU TON3, TRINH HOANG HA4, NGUYEN VAN HUNG3, NGUYEN THI VAN1, TONG VAN HAI3, NGUYEN THI KIM QUY1, NGUYEN THI ANH DAO1, AND VU THI THOM4 Institute of Microbiology and BiotechnologyIMBT , Vietnam National University, HanoiVNUH E2 Building, 144 Xuan Thuy road, Cau Giay District, Hanoi Province, Vietnam University of Engineering and Technology, Vietnam National University, Hanoi Faculty of Biotechnology, Hanoi University of Agriculture School of Medicine and Pharmacy, Vietnam National University, Hanoi Received 13 March, 2013/Accepted 13 December, 2013 A total of 2690 actinomycete strains were screened as potential biological control agents in controlling rice bacterial blightBB in Vietnam From these microorganisms, seventeen actinomycete strains were found to be capable of inhibiting all 10 major Xoo races isolated from Xoo-infected rice leaves One strain, namely VN08-A-12, contained effective characteristics in selectively inhibiting all 10 Xoo races in vitro, but did not inhibit most of the other tested microorganisms Therefore, VN08-A-12 was subsequently selected for rice field trials for two seasons on two rice cultivars SS1 and KD18 Results showed VN08-A-12 was not only able to reduce Xoo lesion lengths in the two rice cultivarslesion length reduction of up to 38.3 % , but it also significantly reduced Xoo-related yield loss in infected rice cultivars from the field yield loss reduction of up to 43.2 % Interestingly, the culture of this strain also increased the rice yield in healthy rice cultivars from 2.66 % to 16.98 % for SS1 and from 3.11 % to 5.94 % for KD18 cultivar The strain VN08-A-12 was shown to be identical to Streptomyces toxytricini To our knowledge, this is the first study reporting S toxytricini as a beneficial biological agent for the control of BB in rice Key words Xoo / Streptomyces toxytricini / Vietnam INTRODUCTION Bacterial blight BB of rice is caused by Xanthomonas oryzae pv oryzae Xoo , and is one of the most destructive diseases of this essential crop The disease can lead to yield losses of up to 50 or 60 % in tropical regions throughout Asia Adhikari et al., 1994; Dinh et al., 2008; Gnanamanickam, 2009 and it has occurred in many rice cultivating areas of Vietnam Dinh et al., 2008 In addition to using chemicals and Corresponding author Tel and Fax: +84-437547407, E-mail : phuonghoa a gmail.com varietal resistance, researchers have recently attempted to control BB in rice by using biological agents and their bioactive compounds Chithrashree et al., 2011; Ndonde and Semu, 2000 The use of microorganisms for controlling Xoo has shown significant promise in being an ecologically-conscious and cost-effective solution in rice cultivationGnanamanickam, 2009; Park et al., 2011 Actinomycetes are gram-positive bacteria that are well-known as a source of natural products Anzai et al., 2008 It has been reported that of the approximately 10,000 antibiotics discovered from microorganisms, two-thirds were isolated from actinomycetes LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 104 P T PHUONG HOA ET AL Okami and Hotta, 1988 Actinomycetes, mainly the genus Streptomyces, have produced a wide variety of bioactive compounds, including antibioticsAnzai et al., 2008 In recent studies, Streptomyces activity against Xoo strains has been reported Ndonde and Semu, 2000; Yan et al., 2000; Park et al., 2011 and some bioactive compounds have been identifiedPark et al., 2011 , suggesting that actinomycetes are potential biological agents for the control of Xoo In the present study, from a collection of 2690 actinomycete strains, which had been isolated from 2005 to 2010 and preserved at the Vietnam Type Culture CollectionInstitute of Microbiology and Biotechnology, Vietnam National University Hanoi , we screened for actinomycete strains that would be able to inhibit all 10 major Xoo races occurring in northern Vietnam and Japan Ton et al., 2003; Hoa et al., 2012 An actinomycete strain named VN08-A-12 was selected as a possible biological control agent of BB This strain was selected for its potential to inhibit Xoo races, reduce Xoo lesion lengths in rice leaves and reduce Xoo-related yield losses in infected rice cultivars from field trials The strain was identified to the species level MATERIALS AND METHODS Experimental design A collection of 2690 actinomycete strains was used to test for inhibitory actions against Escherichia coli or Micrococcus luteus 167 strains that had high inhibition activity against E coli or M luteus were selected We then screened for the strains that inhibited all 10 Xoo races among those 167 strains 17 strains that inhibited all 10 Xoo races were selected Then, the antagonistic tests against indicator strainsM luteus NBRC 13867, E coli NBRC 14237, Bacillus subtilis NBRC 3134 and Saccharomyces cerevisiae NBRC 10217 and useful microogarnisms Azotobacter sp VTCC-B-106 and Pseudomonas putida VTCC-B-657 were done for the 17 selected strains The strain named VN08-A12 was selected as a potential strain for rice field trials over two cropping seasons; then we identified this strain to the species level Actinomycete strains During 2005 to 2010 at the Vietnam Type Culture CollectionInstitute of Microbiology and Biotechnology, Vietnam National University Hanoi , over 3000 actinomycete strains were isolated from soil and leaf-litter samplesHop et al., 2011; Hoa et al., 2012 by using four methods in conjunction with humic acid-vitamin agar as an isolation medium: the rehydration-centrifugation RC method, sodium dodecyl sulfate-yeast extract dilution method, dry-heating method and the TABLE Collection of actinomycete strains from 2005 to 2010 Name of strains VTCC VN07 VN08 VN10 Total Year of isolation 2005 2007 2008 2010 Obtained and tested strains 600 1000 500 90 2690 References Hop et al., 2011 Hoa et al., 2012 oil-separation methodHop et al., 2011 Among these strains, a total of 2690 actinomycete strains were used to screen for antibiotic-producers by assay for antagonism to M luteus NBRC 13867 and E coli NBRC 14237 The method was previously mentioned in other studiesIchikawa et al., 1971; Hoa et al., 2012 Xoo races In the present study, a collection of 10 pathogen races of Xoo were tested These were named R1, R2, R3, R4, R5, R6, R7, R8, R9 and R10T on et al., 2003; Hoa et al., 2012 Of the 10 races, eight were found from many provinces of northern Vietnam, with twoR9 and R10 from Japan The races were isolated from rice BB-infected leaves that were collected from the main rice-cultivating areas of northern Vietnam Among the 10 Xoo races, R2 and R3 were the most widely distributed Media In addition to the media Wakimoto medium WM , Yeast extract–soluble starch medium YS , Antibiotic producing mediumAPM , Yeast-extract malt medium YM , Muller Hinton agar MHA ; Ashby medium AM and Nutrient Agar NA described in our previous paper Hoa et al., 2012 , we used five other media Medium 2M, Medium 301, Medium A-16, Medium no 8, Medium A-3M in the present study Detection of actinomycetes capable of inhibiting Xoo races in vitro The antibacterial activity of actinomycetes against 10 pathogenic Xoo races was tested by the agar-piece method mm on Wakimoto medium as previously described Hoa et al., 2012 In brief, the actinomycete isolates were inoculated on YS medium at room temperature25 to 30 for to 14 days The pieces of agar6 mm that contained actively-growing actinomycete isolates were cut and placed onto the test plates culturing races of Xoo The plates were incubated at 28 for two to four days and the inhibitory effect of the actinomycete isolates on Xoo growth was evaluated by measuring inhibition zones cm To prepare the test plates, races of Xoo were cultured in Wakimoto broth to a concentration of ca 108 CFU/ml LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com BIOLOGICAL CONTROL OF XANTHOMONAS ORYZAE 150 ml of Wakimoto agar was autoclaved and cooled down to 45 to 50 and then mixed with 10 ml of the Xoo cultures This mixture was poured into Petri dishes that were used as the test platesHoa et al., 2012 Test for antibiosis to microorganisms The actinomycete isolates inhibiting all 10 Xoo races were tested for their antibiosis to four indicator microogarnisms, M luteus NBRC 13867, E coli NBRC 14237, B subtilis NBRC 3134 and S cerevisiae NBRC 10217 indicator microorganisms were provided by NITE, Japan , and to two beneficial bacteria, Azotobacter sp VTCC-B-106 , which is well-known in nitrogen fixation, and P putida VTCC-B-657 which degrades phosphors into accessible forms for plants Antibiosis tests were performed by using the agar-piece method as mentioned previously Ichikawa et al., 1971; Fredrickson and Elliott, 1985; Blackburn and Avery, 2003 Selection of medium for antibiotic production of the potential actinomycete strain Seven media Antibiotic producing medium APM , Modified-APM MAPM , Medium 2M, Medium 301, Medium A-16, Medium no 8, Medium A-3M were used for selection of a suitable antibiotic inducing medium of actinomycetes The method was described previouslyHoa et al., 2012 Biocontrol of BB in rice by actinomycetes in field trials Artificial infection of Xoo races R2 and R3 on rice leaves The rice cultivars Oryza sativa L SS1 and Oryza sativa L KD18 were chosen for field trials for two cropping seasons in 2011 because they are widely cultivated across Vietnam and have susceptibility to the Xoo races The BB pathogens R2 and R3, being the most prevalent in northern Vietnam, were inoculated in Wakimoto medium flasks and incubated at 28 for 48 hours The Xoo bacterial cells were then diluted in sterilized water to reach a concentration of 108 CFU/ml to infect the two rice cultivars This method was described by Ji et al.,2008 The two rice cultivars were planted in the field Artificial infection was conducted at the rice booting stage because this stage is the most susceptible to BB and infection during this stage subsequently impacts rice yields Infection was performed by dipping the scissors into a Xoo bacterial suspension and using them to clip the tip of the rice leaves Kauffman et al., 1973 After 14 days, the length of lesions on 15 leaves per rice cluster was measured at the field Three rice clusters per treatment formula were examined Xoo treatment by actinomycetes 105 Actinomycetes were cultured in MAPM broth with shaking at 150 cycles/minute for five days From this, 250 ml of the cultured solution was used for spraying over an area of 1m2 in each experimental square Treatments Each of the two rice cultivars Oryza sativa L SS1 and Oryza sativa L KD18 was planted in 45 experimental blocks within an experimental rice field at Hanoi University of Agriculture The biocontrol trials at the rice field were conducted during two growing seasons in 2011, which consisted of planting in October and harvest in May and another planting in the end of May and harvest in November Three squares were used for each treatment as follows Treatment 1: Positive control rice cultivars were not infected by the Xoo races and were not treated with VN08-A-12 This was done to measure the noninfected/non-treated baseline yield of each paddy for each rice cultivar Treatment 2: Rice cultivars were not infected by the Xoo races, but they were treated with VN08-A-12 This experiment was conducted to know the effect of VN08A-12 on the rice yield of each rice cultivar Treatment 3: Infected rice cultivars were treated as follows: CT1 denotes spraying VN08-A-12 after 1-day infection by Xoo races; CT2 denotes spraying VN08A-12 after 3-day infection by Xoo races; CT3 denotes spraying VN08-A-12 after 7-day infection by Xoo races; and CT4 denotes spraying VN08-A-12 times: after 1, and 7-day infection by Xoo races Treatment 4: Rice cultivars were infected by Xoo races but were not treated with the VN08-A-12 broth culture This was done to measure the effect of Xoo races on the host rice cultivars Measurement of the yield of a paddy T o measure and estimate paddy yield for each treatment, the rice grain dry weight from three individual rice plants per rice cluster was measuredT on et al, 2003 Identification of VN08-A-12 Identification of VN08-A-12 was based on morphological, chemical and molecular methods as mentioned by Sakiyama et al.2009 Statistical analysis Data from laboratory and rice field experiments were analyzed and subjected to analysis of Variance ANOV A and correlationSPSS Sofware The significance of each treatment through two rice seasons and for each rice cultivar were determined by ANOVA T ukey's posthoc test P values ≤ 0.05 LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com 106 P T PHUONG HOA ET AL TABLE Ability of 167 actinomycete strains to inhibit tested Xoo races Number of Xoo strains which actinomycete strains can inhibit Percentage of actinomycete 17.9% 8.4% Strains 7.8% 5.9% 10.2% 2.4% FIG proportion of the actinomycete strains that inhibits each Xoo race RESULTS Screening for actinomycete isolates capable of inhibiting Xoo races in vitro From 2690 actinomycete strains, a total of 167 strains showed high activity against M luteus or E coli and were subsequently chosen for further antagonistic tests against the 10 Xoo races The inhibition pattern against each Xoo race showed that races R9, R6 and 10 8.4% 10.8% 11.9% 5.9% 10.2% R2 expressed the highest levels of inhibition relative to the othersFigur e1 Only 25.7, 27.1 and 36.5% of the actinomycete strains inhibited races R9, R6 and R2, respectively Inhibition of the other races of actinomycete strains accounted for at least 49.7% Most of the tested actinomycete isolates73% were able to inhibit more than Xoo races A total of 17 strains 10.2% were found to be able to inhibit all 10 Xoo racesT able The inhibition zones of the 17 actinomycete strains inhibiting 10 Xoo races varied from 0.2 to 2.6 cm, depending on the strains and Xoo races data not shown Test of antibiosis to microorganisms The purpose of this study was to select actinomycete strains capable of inhibiting all 10 Xoo races while not being harmful to other microorganisms The 17 actinomycete strains capable of inhibiting all 10 Xoo races chosen above were tested for their inhibition activity against four indicator microorganisms M luteus, E coli, B subtilis and S cerevisiae and two useful microorganismsAzotobacter sp and P putida TABLE Inhibition activity of the 17 actinomycete strains against tested microorganisms No Name 10 11 12 13 14 15 16 17 VN06-A-353 VN06-A-379 VN06-A-1098 VN06-A-1457 VN06-A-1477 VN08-A-12 VN08-A-306 VN08-A-352 VN08-A-395 VTCC-A-99 VTCC-A-289 VTCC-A-363 VTCC-A-367 VTCC-A-378 VTCC-A-456 VTCC-A-465 VN10-A-44 M luteus 0.8 1.8 1.1 0.6 1.6 1.2 2.3 1.2 0.1 0.2 0.1 0.3 0.1 0.1 0.1 0.3 E coli 2.0 1.5 2.4 1.1 0.2 0.4 0.2 0.1 1.0 0.2 1.0 0.1 0.8 0.2 1.0 0.1 0.5 0.1 1.2 0.2 0.5 0.2 2.3 0.1 Inhibition zonescm S cerevisiae B subtilis 1.4 0.1 1.0 0.2 1.2 0.1 1.8 0.1 1.0 0.2 1.0 0.1 2.0 0.3 1.4 0.1 Azotobacter sp n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d P putida n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d n.d 1.0 0.1 Note: - indicates that the actinomycete strain did not inhibit the indicator microorganism; + indicates that the strain has antagonistic activity, but the inhibition zone was too small to measure N.d denotes not done Each value is an average of a time-repeated assay LUAN VAN CHAT LUONG download : add luanvanchat@agmail.com BIOLOGICAL CONTROL OF XANTHOMONAS ORYZAE 107 We found that strains VN06-A-353, VN08-A-12, VN08-A-306, VN08-A-352, VTCC-A-99 and VN10-A-44 only inhibited one of the four indicator microorganisms whereas the others inhibited more Table It was observed that VN08-A-12 and VN10-A-44 grew faster than the other strains After three days of cultivation, they showed high inhibition activity against Xoo races while the other strains required from five to seven days of cultivation to express this activitydata not shown Thus, only two strains VN08-A-12 and VN10-A-44 were checked for their inhibition potential against the two beneficial microorganisms such as Azotobacter sp VTCC-B-106 and P putida VTCC-B-657 The strain VN10-A-44 inhibited P putida; whereas VN08-A-12 did not inhibit either of the beneficial bacteria tested Therefore, VN08-A-12 was selected for further analysis and for the rice field trials Selection of the best medium for fermentation of VN08-A-12 Seven media were used to find the best medium for antibiotic production of VN08-A-12 From this, MAPM was found to be the best medium for VN08-A-12 to produce bioactive compounds The inhibition zones measured when cultured in MAPM were significantly higher than those cultured in the other mediadata not shown Biological control of Xoo races by VN08-A-12 in the rice field trials Xoo lesion length of rice cultivars SS1 and KD18 Inhibition efficacy of VN08-A-12 on rice cultivars was evaluated by Xoo-induced lesion length and rice yield following two rice growing seasons The Xoo lesion lengths on rice leaves are shown for two rice cultivars SS1 Figure 2A and KD18 Figure 2B Bacterial blight disease occurred differently in the two rice cultivars, with SS1 being more severely infected by Xoo races than KD 18, as shown in the Xoo lesion lengths that were from 15.2 to 25.5 cm for SS1 and from 7.2 to 12.9 cm for KD18 Treatment with VN08-A-12 significantly reduced the Xoo lesion length in rice leaves in both rice seasons For example, in the R2-infected SS1 cultivar, the treatment after a 1-day infection reduced the Xoo lesion length by 25.3 % and 33.4 % reductions in the spring and summer seasons, respectively; 3-time treatment by VN08-A-12 reached 32.6 % and 38.3 % reductions in the spring and summer seasons, respectively Lesion lengths in the R3-infected SS1 cultivar were reduced by 27.5 % after a 1-day infection in both rice seasons and by 15.2 % and 29.4 % after 3-time treatments by VN08-A-12 in the spring and summer seasons, respectively On the other hand, for the R2-infected KD18 cultivar, the treatment by VN08- FIG Effect of VN08-A12 on Xoo lesion length on leaves SS1 A and Khang dan B in spring and summer rice seasons The leaves were artificially infected by X oo R2R2 or X oo R3 R3 in summer and spring seasons without VN08-A12 treatmentCT0 , with a single treatment of VN08A12 after 1-day infection CT1 , 3-day infection CT2 and 7-day infectionCT4 or with three-time treatments of VN08A12 after 1, and 7- day infections I refers to R2 in Summer; II refers to R2 in Spring; III refers to R3 in Summer; IV refers to R3 in Spring ANOVA was tested following by Tukey s posthoc test p