Morphological, biological, molecular characterization and biological control of pythium cucurbitacearum causing root rot disease of citrus trees

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF AGRONOMY  UNDERGRADUTE THESIS TOPIC: MORPHOLOGICAL, BIOLOGICAL, MOLECULAR CHARACTERIZATION AND BIOLOGICAL CONTROL OF PYTHIUM CUCURBITACEARUM CAUSING ROOT ROT DISEASE OF CITRUS TREES Supervisor : Ph.D NGUYEN DUC HUY Department : PLANT PATHOLOGY Student : LUU THI VAN Class : K62KHCTT ID : 621836 HANOI-2022 COMMITMENT I here by declare that this graduation thesis is my own original work Data and results published in this essay completely truthful, accurate and not been published in any other works of research Further, I have acknowledged all sources used and have cited them in the reference section Hanoi, March 2021 Signature Luu Thi Van i ACKNOWLEDGEMENTS To complete my thesis, out of my best effort, I have had received help and enthusiastic attention from my teachers, friends, and family First and foremost, I wish to express my most sincere and profound gratitude to my supervisors Dr Nguyen Duc Huy, Department of Plant Pathology, Faculty of Agronomy, Vietnam National University of Agriculture for their guidance, valuable advice, and tremendous encouragement throughout this investigation I also would like to thank the staff and people working in the laboratory of Plant Clinic for sharing their experiences, time, and commitment during my work to complete my undergraduate thesis My sincere thanks to all teachers of Faculty of Agronomy (Vietnam National University of Agriculture) who supported and imparted knowledge during my 5year study in the university To all students of the Plant Pathology laboratory, I am grateful for their assistance in every way Last but not least, my special thanks go to my family and friends for their spiritual support for me throughout my study as well as completing the undergraduate thesis Hanoi, March 2021 Student Luu Thi Van ii TABLE OF CONTENT COMMITMENT i ACKNOWLEDGEMENTS ii TABLE OF CONTENT iii LIST OF TABLES vi LIST OF FIGURES vii LIST OF CHARTS viii LIST OF ABBREVIATIONS ix PART I INTRODUCTION 1.1 Preface 1.2 Objectives and requirements 1.2.1 Objectives 1.2.2 Requirements PART II LITERATURE REVIEW 2.1 Study in the world 2.1.1 The situation of citrus production in the world 2.1.2 The stiuation of citrus root rot in the world 2.2 Study in the Vietnam 2.2.1 The situation of citrus production in Vietnam 2.2.2 The situation of citrus root rot in Vietnam 12 2.3 Pathogenic fungus of citrus root rot 14 2.3.1 Phytophthora species 14 2.3.2 Pythium species 16 2.3.3 Phytopythium species 19 2.3.4 Comparison of Pythium species and Phytophthora species 20 2.4 Management Strategies for Root Rot 22 iii PART III MATERIAL AND METHOD 24 3.1 Objectives 24 3.2 Location and Time 24 3.3 Material 24 3.4 Methods 24 3.4.1 Collection of rhisosphere soil and root samples 24 3.4.2 Isolate collection 25 3.4.3 Molecular identification 26 3.4.4 Morphological identification 31 3.4.5 Virulence tests 34 3.4.6 Pathogenic tests 35 3.4.7 Assessing the inhibitory efficacy of antagonistic organism against fungal pathogen 38 3.5 Calculation formula 41 3.6 Statistical Analysis 41 PART IV RESULTS AND DISCUSSION 42 4.1 Isolation of Pythium species 42 4.1.1 Results of fungual isolation from collected samples 42 4.1.2 Identification of Ppythium species by Polymerase chain reaction (PCR) 44 4.2 Mophologycal and biological characteristics of isolates 46 4.2.1 Mophologycal characteristics of isolates 46 4.2.2 The effect of medium on growth of Phytopythium sp 48 4.2.3 Sporangia production for Pythium cucurbitacearum of different medium 51 4.2.4 Sporulation in different kinds of liquid cultures 53 4.3 Virulence tests – The lesions on Lime fruits, Orange fruits and Pomelo fruits caused by Pythium cucurbitacearum 55 4.4 Pathogenicity test 58 iv 4.4.1 Evalution lesions caused by Pythium cucurbitacearum test isolates on citrus plants 59 4.4.2 Phathogenicity test on Pomelo detached leaves of Pythium cucurbitacearum 61 4.4.3 Phathogenicity test on Pomelo germinating seeds of Pythium cucurbitacearum 63 4.4.4 The influence of antagonistic organism on the causal agents 64 PART V CONCLUSTION AND RECOMMENDATION 75 5.1 Conclusion 75 5.2 Recommendation 76 REFERENCES 77 APPENDIX 82 v LIST OF TABLES Table 2.1 The planted area and total citrus production in Viet Nam from 2015 to 2019 Table 4.1 Results of collection and isolation of Pythium sp on citrus trees in and some provinces of Vietnam 43 Table 4.2 Results of PCR and ITS sequences of isolated LNCC2, HG11, UHD 45 Table 4.3 Morphological characteristics of Pythium cucurbitacearum 46 Table 4.4 The diameter (mm) mycelium on growth of Pythium cucurbitacearum on different medium 49 Table 4.5 Sporulation in water of agar disk of isolates growing in different culture media 52 Table 4.6 Sporulation in different kinds of liquid cultures of three sources on V8 medium 53 Table 4.7 Diameter of the lesion (mm) after to days on Lime fruits, Orange fruits and Pomelo fruits caused by Pythium cucurbitacearum 55 Table 4.8 The result of artifial inoculation of Pythium cucurbitacearum on pomelo seedlings 60 Table 4.9 The result of artificial inoculation of Pythium cucurbitacearum on Pomelo detached leaves 61 Table 4.10 The result of artificial inoculation of Pythium cucurbitacearum on Pomelo seeds germination 63 Table 4.11 Growth inhibition of Bacillus velezensis, Bacillus velezensis, Bacillus sp CLNA, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9 during in vitro with Pythium cucurbitacearum HG11 65 Table 4.12 Growth inhibition of Trichoderma asperellum on the Pythium cucurbitacearum during in vitro 69 Table 4.13 Evalution of Trichoderma asperellum to control citrus root rot in the greenhouse 72 vi LIST OF FIGURES Figure 2.1 World production of citrus fruits in 2020, by region (in thousand metric tons) Figure 2.2 Life cycle of Phytophthora species 16 Figure 2.3 Life cycle of a typical root Pythium species 18 Figure 3.1 The way to inoculated line of antagonistic organism 38 Figure 3.2 The way to inoculate point Trichoderma asperellum 39 Figure 4.1 Isolation method 44 Figure 4.2 Electrolysis results of isolateted samples after PCR 45 Figure Morphological characteristics of Pythium cucurbitacearum 47 Figure 4.3 Colony morphology of different isolateds on PDA media 48 Figure 4.4 Colony morphology of Pythium cucurbitacearum LNCC2 51 Figure 4.5 Sporulation in different kinds of liquid cultures on V8 medium 54 Figure 4.6 The brown necrotic lesions on citrus fruits causing by Pythium cucurbitacearum 58 Figure 4.7 Evalution lesions caused by Pythium cucurbitacearum test isolates on pomelo seedlings 61 Figure 4.8 Phathogenicity test on Pomelo detached leaves of Pythium cucurbitacearum 62 Figure 4.9 Phathogenicity test on Pomelo germinating seeds of Pythium cucurbitacearum 64 Figure 4.10 Growth inhibition of Bacillus velezensis, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9 during in vitro with Pythium cucurbitacearum HG11 67 Figure 4.11 Growth inhibition of Trichoderma asperellum on the Pythium cucurbitacearum during in vitro 71 Figure 4.12 Evalution of Trichoderma asperellum to control citrus root rot in the greenhouse 74 vii LIST OF CHARTS Chart 4.1 The diameter (mm) mycelium on growth of Pythium cucurbitacearum on different medium 49 Chart 4.2 The diameter of the lesion (mm) after days on Lime fruits, Orange fruits and Pomelo fruits caused by Pythium cucurbitacearum 56 Chart 4.3 The percentage of inhibition of radial growth ( antagonistic effect) of B velezensis, Bacillus sp TN1-KL1, Bacillus sp D1, Bacillus sp YB12, Bacillus sp YB9 during in vitro with Pythium cucurbitacearum HG11 66 Chart 4.4 The percentage of inhibition of radial growth ( antagonistic effect) of Trichoderma asperellum on the Pythium cucurbitacearum 70 Chart 4.5 Evalution of Trichoderma asperellum to control citrus root rot in the greenhouse 73 viii LIST OF ABBREVIATIONS DI Disease incidence MARD Ministry of Agriculture & Rural Development FAO Food and Agriculture Organization of the United Nations PCR Polymerase Chain Reaction RT PCR Reverse Transcription Polymerase Chain Reaction WA Water agar PDA Potato Dextrose Agar PA Pea Agar SCDA Sweet Cassava Dextrose Agar CDA Carrot Dextrose Agar V8 V8 Juice Agar V8 - V8 Juice Agar without CaCO3 LSD Least Significant Diference μm Micrometer ( 10-6 meter) ix species causing dry root rot disease of citrus trees in Morocco J Plant Dis Prot 128, 431–447 14 Fry, W.E.; Goodwin, S.B (1997) Re-emergence of potato and tomato late blight in the United States Plant Dis., 81, 1349–1357 15 H John Webber (1967) History and Development of the Citrus Industry University of California Division of Agricultural Sciences Archived from the original on June 2004 16 Hung, P M., Wattanachai, P., Kasem, S., & Poeaim, S (2015) Efficacy of Chaetomium species as biological control agents against Phytophthora nicotianae root rot in citrus Mycobiology, 43(3), 288-296 17 Heffer V, Powelson ML, Johnson KB (2002) Oomycetes) The Plant Health Instructor 18 Ismail, M., & Zhang, J (2004) Post-harvest Citrus Diseases and their control Outlooks on Pest Management, 15(1), 29–35 doi:10.1564/15feb12 19 Ivana Puglisi, Alessandro De Patrizio, Leonardo Schena, Thomas Jung, Maria Evoli, Antonella Pane, Nguyen Van Hoa, Mai Van Tri, Sandra Wright, Mauritz Ramstedt, Christer Olsson, Roberto Faedda, Gaetano Magnano di San Lio, Santa Olga Cacciola (2017) Two previously unknown Phytophthora species associated with brown rot of Pomelo (Citrus grandis) fruits in Vietnam 20 Liberato JR, Sanhueza RMV, Miles AK, Horlock C (2011) White root rot (Rosellinia necatrix) Mar 2014 21 La Spada, F., Stracquadanio, C., Riolo, M., Pane, A., & Cacciola, S O (2020) Trichoderma counteracts the challenge of Phytophthora nicotianae infections on tomato by modulating plant defense mechanisms and the expression of crinkler, necrosis-inducing Phytophthora protein 1, and cellulose-binding elicitor lectin pathogenic effectors Frontiers in plant science, 1653 22 M Shahbandeh (2021) Global leading orange producers 2019/2020 Published 79 Aug 12, 2021 23 Mounde, L G., Ateka, E M., Kihurani, A W., & Wasilwa, L (2012) Morphological characterization and identification of Phytophthora species causing citrus gummosis in Kenya African Journal of Food, Agriculture, Nutrition and Development, 12(7) 24 Noireung P, Intaparn P, Maumoon R, Wongwan T, To–Anun C (2020) First Record of Pythium cucurbitacearum causing root rot on Mandarin (Citrus reticulate L cv Sainampueng) in Thailand Plant Pathology & Quarantine 10(1), 85–90, 25 Postma, J., Stevens, L.H., Wiegers, G.L., Davelaar, E and Nijhuis, E.H (2009) Biological control of Pythium aphanidermatum in cucumber with a combined application of Lysobacter enzymogenes strain 3.1 T8 and chitosan Bio Control 48(3): 301-309 26 Puglisi, I., De Patrizio, A., Schena, L., Jung, T., Evoli, M., Pane, A., & Cacciola, S O (2017) Two previously unknown Phytophthora species associated with brown rot of Pomelo (Citrus grandis) fruits in Vietnam PLoS One, 12(2), e0172085 27 Rossman, D.R., Rojas, A., Jacobs, J.L., Mukankusi, C., Kelly, J.D and Chilvers, M.I (2017) Pathogenicity and virulence of soil-borne oomycetes on Phaseolus vulgaris Plant Diseases 101: 1851-1859 Salmaninezhad 28 Schettini, T.M.; Legg, E.J.; Michelmore, R.W (1991) Insensitivity to metalaxyl in California populations of Bremia lactucae and resistance of California lettuce cultivars to downy mildew Phytopathology, 81, 64–70 29 Thompson, A H., Phillips, A J L., & Nel, E (1995) Phytophthora and Pythium associated with feeder root rot of citrus in the Transvaal Province of South Africa Journal of Phytopathology, 143(1), 37-41 30 Timmer, L W., and J A Menge "Phytophthora-induced 80 diseases." Compendium of citrus diseases (1988): 22-24 31 Tkaczyk, M (2020) origin, differences and meaning in modern plant pathology Folia Forestalia Polonica, 62(3), 227-232 32 Truong, N.V., Burgess, L.W & Liew, E.C.Y (2008) Prevalence and aetiology of Phytophthora foot rot of black pepper in Vietnam Australasian Plant Pathology 37, 431–442 33 UN Food and Agriculture Organization (2020) Citrus fruit, fresh and processed: Statistical Bulletin Retrieved February 2022 34 van West, P.; Appiah, A.A.; Gow, N.A.R (2003) Advances in research on oomycete root pathogens Physiol Mol Plant Pathol., 62, 99–113 35 Villa NO, Kageyama K, Asano T, et al (2006) Phylogenetic relationships of Pythium and Phytophthora species based on ITS rDNA, cytochrome oxidase II and beta-tubulin gene sequences Mycologia 98: 410–422 36 Wen-Wu, G U O., Chang-He, Z H O U., Hua-Lin, Y I., & Xiu-Xin, D E N G (2000) Intergeneric somatic hybrid plants between Citrus and Poncirus trifoliata and evaluation of their root rot resistance Journal of Integrative Plant Biology, 42(7), 668-672 37 Z Gloria Abad and Jorge A Abad (1997) Another Look at the Origin of Late Blight of Potatoes, Tomatoes, and Pear Melon in the Andes of South America Department of Plant Pathology, North Carolina State University, Raleigh 27695-7616 81 APPENDIX Phathogenicity test on Pomelo leaves of Pythium spp BALANCED ANOVA FOR VARIATE VAR01 FILE LEAF 5/ 3/22 16: :PAGE Phathogenicity test on Pomelo leaves 1 VARIATE V003 VAR01 LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 2356.89 785.630 ****** 0.000 * RESIDUAL 4.87317 609147 * TOTAL (CORRECTED) 11 2361.76 214.706 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE LEAF 5/ 3/22 16: :PAGE Phathogenicity test on Pomelo leaves MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 VAR01 5.00000 39.1000 39.4333 29.7667 SE(N= 3) 0.450609 5%LSD 8DF 1.46939 ANALYSIS OF VARIANCE SUMMARY TABLE FILE LEAF 5/ 3/22 16: :PAGE Phathogenicity test on Pomelo leaves F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE VAR01 GRAND MEAN (N= 12) NO OBS 12 28.325 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 14.653 0.78048 2.8 0.0000 Phathogenicity test on Pomelo germinating seeds of Pythium spp BALANCED ANOVA FOR VARIATE LENGHT FILE SEEDS 5/ 3/22 21: :PAGE VARIATE V003 LENGHT LN SOURCE OF VARIATION SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 84.0425 28.0142 94.96 0.000 * RESIDUAL 2.36000 295000 * TOTAL (CORRECTED) 11 86.4025 7.85477 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE SEEDS 5/ 3/22 21: :PAGE MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 DF LENGHT 0.000000 2.93333 82 3 7.33333 4.43333 SE(N= 3) 0.313581 5%LSD 8DF 1.02256 ANALYSIS OF VARIANCE SUMMARY TABLE FILE SEEDS 5/ 3/22 21: :PAGE F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE LENGHT GRAND MEAN (N= 12) NO OBS 12 3.6750 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 2.8026 0.54314 14.8 0.0000 The effect of mediums on growth of Pythium spp UHD BALANCED ANOVA FOR VARIATE 1NSC FILE UHD 3/ 3/22 11: :PAGE VARIATE V003 1NSC LN SOURCE OF VARIATION DF LN SOURCE OF VARIATION DF LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= CT 828.792 165.758 149.18 0.000 * RESIDUAL 12 13.3333 1.11111 * TOTAL (CORRECTED) 17 842.125 49.5368 BALANCED ANOVA FOR VARIATE 2NSC FILE UHD 3/ 3/22 11: :PAGE VARIATE V004 2NSC SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= CT 3092.46 618.492 ****** 0.000 * RESIDUAL 12 7.16661 597218 * TOTAL (CORRECTED) 17 3099.62 182.331 BALANCED ANOVA FOR VARIATE 3NSC FILE UHD 3/ 3/22 11: :PAGE VARIATE V005 3NSC SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= CT 3051.07 610.214 ****** 0.000 * RESIDUAL 12 4.00010 333342 * TOTAL (CORRECTED) 17 3055.07 179.710 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE UHD 3/ 3/22 11: :PAGE MEANS FOR EFFECT CT CT NOS 3 3 3 1NSC 35.6667 23.5000 20.1667 30.1667 16.3333 31.6667 2NSC 69.5000 44.1667 40.8333 60.6667 36.3333 67.0000 3NSC 90.0000 67.3333 67.6667 90.0000 58.8333 90.0000 SE(N= 3) 0.608580 0.446175 0.333337 5%LSD 12DF 1.87524 1.37482 1.02713 - 83 ANALYSIS OF VARIANCE SUMMARY TABLE FILE UHD 3/ 3/22 11: :PAGE F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE 1NSC 2NSC 3NSC GRAND MEAN (N= 18) NO OBS 18 26.250 18 53.083 18 77.306 STANDARD DEVIATION C OF V |CT SD/MEAN | BASED ON BASED ON % | TOTAL SS RESID SS | 7.0382 1.0541 4.0 0.0000 13.503 0.77280 1.5 0.0000 13.406 0.57736 0.7 0.0000 | | | | LNCC2 BALANCED ANOVA FOR VARIATE DAY FILE LNCC2 5/ 3/22 23:46 :PAGE VARIATE V003 DAY LN SOURCE OF VARIATION DF LN SOURCE OF VARIATION DF LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 616.278 123.256 177.49 0.000 * RESIDUAL 12 8.33335 694446 * TOTAL (CORRECTED) 17 624.611 36.7418 BALANCED ANOVA FOR VARIATE DAY FILE LNCC2 5/ 3/22 23:46 :PAGE VARIATE V004 DAY SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 2170.28 434.056 568.21 0.000 * RESIDUAL 12 9.16672 763893 * TOTAL (CORRECTED) 17 2179.44 128.203 BALANCED ANOVA FOR VARIATE DAY3 FILE LNCC2 5/ 3/22 23:46 :PAGE VARIATE V005 DAY3 SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 2566.94 513.389 577.55 0.000 * RESIDUAL 12 10.6670 888913 * TOTAL (CORRECTED) 17 2577.61 151.624 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE LNCC2 5/ 3/22 23:46 :PAGE MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 3 DAY 31.1667 21.1667 23.5000 29.8333 13.6667 26.3333 DAY 69.3333 46.0000 52.3333 62.3333 35.6667 58.0000 DAY3 90.0000 70.0000 79.3333 90.0000 57.0000 86.0000 SE(N= 3) 0.481126 0.504610 0.544338 5%LSD 12DF 1.48251 1.55487 1.67729 ANALYSIS OF VARIANCE SUMMARY TABLE FILE LNCC2 5/ 3/22 23:46 :PAGE F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE GRAND MEAN STANDARD DEVIATION 84 C OF V |TREATMEN| DAY DAY DAY3 (N= 18) NO OBS 18 24.278 18 53.944 18 78.722 SD/MEAN | BASED ON BASED ON % | TOTAL SS RESID SS | 6.0615 0.83333 3.4 0.0000 11.323 0.87401 1.6 0.0000 12.314 0.94282 1.2 0.0000 | | | HG11 BALANCED ANOVA FOR VARIATE DAY FILE HG11 5/ 3/22 23:51 :PAGE the effect of medium on growth of HG11 VARIATE V003 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 967.292 193.458 196.18 0.000 * RESIDUAL 12 11.8334 986118 * TOTAL (CORRECTED) 17 979.125 57.5956 BALANCED ANOVA FOR VARIATE DAY2 FILE HG11 5/ 3/22 23:51 :PAGE the effect of medium on growth of HG11 VARIATE V004 DAY2 LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 4007.17 801.433 749.38 0.000 * RESIDUAL 12 12.8336 1.06946 * TOTAL (CORRECTED) 17 4020.00 236.471 BALANCED ANOVA FOR VARIATE DAY FILE HG11 5/ 3/22 23:51 :PAGE the effect of medium on growth of HG11 VARIATE V005 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 4366.79 873.358 309.76 0.000 * RESIDUAL 12 33.8332 2.81943 * TOTAL (CORRECTED) 17 4400.63 258.860 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE HG11 5/ 3/22 23:51 :PAGE the effect of medium on growth of HG11 MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 3 DAY 33.3333 19.0000 23.8333 29.3333 11.3333 28.6667 DAY2 73.3333 42.0000 52.3333 67.3333 31.6667 66.3333 DAY 90.0000 60.8333 81.5000 90.0000 51.1667 90.0000 SE(N= 3) 0.573329 0.597066 0.969439 5%LSD 12DF 1.76662 1.83976 2.98717 ANALYSIS OF VARIANCE SUMMARY TABLE FILE HG11 5/ 3/22 23:51 :PAGE the effect of medium on growth of HG11 85 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE DAY DAY2 DAY GRAND MEAN (N= 18) NO OBS 18 24.250 18 55.500 18 77.250 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 7.5892 0.99303 4.1 0.0000 15.378 1.0341 1.9 0.0000 16.089 1.6791 2.2 0.0000 the lesions on Lime fruits, Orange fruits and Pomelo fruits LNCC2 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23: :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits LNCC2 VARIATE V003 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 524.278 104.856 22.47 0.000 * RESIDUAL 12 56.0000 4.66667 * TOTAL (CORRECTED) 17 580.278 34.1340 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23: :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits LNCC2 VARIATE V004 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 1287.11 257.422 42.51 0.000 * RESIDUAL 12 72.6668 6.05557 * TOTAL (CORRECTED) 17 1359.78 79.9869 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23: :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits LNCC2 VARIATE V005 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 2298.28 459.655 87.09 0.000 * RESIDUAL 12 63.3337 5.27780 * TOTAL (CORRECTED) 17 2361.61 138.918 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE VRUS 6/ 3/22 23: :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits LNCC2 MEANS FOR EFFECT TREATMEN TREATMEN SE(N= 5%LSD 3) 12DF NOS 3 3 3 DAY 5.00000 18.6667 14.3333 15.0000 16.3333 5.00000 DAY 5.66667 30.6667 18.0000 19.0000 21.6667 7.66667 DAY 6.66667 40.0000 24.6667 20.3333 32.3333 12.3333 1.24722 3.84311 1.42075 4.37780 1.32637 4.08701 86 ANALYSIS OF VARIANCE SUMMARY TABLE FILE VRUS 6/ 3/22 23: :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits LNCC2 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE DAY DAY DAY GRAND MEAN (N= 18) NO OBS 18 12.389 18 17.111 18 22.722 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 5.8424 2.1602 17.4 0.0000 8.9435 2.4608 14.4 0.0000 11.786 2.2973 10.1 0.0000 HG11 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:21 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - HG11 VARIATE V003 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 791.833 158.367 58.18 0.000 * RESIDUAL 12 32.6667 2.72222 * TOTAL (CORRECTED) 17 824.500 48.5000 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:21 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - HG11 VARIATE V004 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 1341.61 268.322 61.92 0.000 * RESIDUAL 12 52.0002 4.33335 * TOTAL (CORRECTED) 17 1393.61 81.9771 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:21 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - HG11 VARIATE V005 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 2318.94 463.789 64.71 0.000 * RESIDUAL 12 86.0002 7.16668 * TOTAL (CORRECTED) 17 2404.94 141.467 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE VRUS 6/ 3/22 23:21 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - HG11 MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 3 DAY 5.00000 23.6667 13.3333 12.3333 17.6667 DAY 6.00000 30.3333 17.6667 17.0000 26.3333 87 DAY 6.00000 40.6667 26.0000 20.6667 31.0000 5.00000 9.00000 13.3333 SE(N= 3) 0.952580 1.20185 1.54560 5%LSD 12DF 2.93522 3.70332 4.76253 ANALYSIS OF VARIANCE SUMMARY TABLE FILE VRUS 6/ 3/22 23:21 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - HG11 F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE DAY DAY DAY GRAND MEAN (N= 18) NO OBS 18 12.833 18 17.722 18 22.944 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 6.9642 1.6499 12.9 0.0000 9.0541 2.0817 11.7 0.0000 11.894 2.6771 11.7 0.0000 UHD BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:28 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - UHD VARIATE V003 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 402.944 80.5889 30.22 0.000 * RESIDUAL 12 32.0000 2.66667 * TOTAL (CORRECTED) 17 434.944 25.5850 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:28 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - UHD VARIATE V004 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 1294.44 258.889 56.83 0.000 * RESIDUAL 12 54.6668 4.55557 * TOTAL (CORRECTED) 17 1349.11 79.3595 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:28 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - UHD VARIATE V005 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 1626.67 325.333 44.70 0.000 * RESIDUAL 12 87.3333 7.27778 * TOTAL (CORRECTED) 17 1714.00 100.824 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE VRUS 6/ 3/22 23:28 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - UHD MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 DAY 5.00000 DAY 5.33333 88 DAY 5.33333 3 3 16.6667 12.3333 11.0000 16.3333 5.00000 30.3333 16.0000 13.3333 22.0000 7.66667 36.6667 20.0000 20.0000 25.0000 15.0000 SE(N= 3) 0.942809 1.23228 1.55754 5%LSD 12DF 2.90512 3.79708 4.79931 ANALYSIS OF VARIANCE SUMMARY TABLE FILE VRUS 6/ 3/22 23:28 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits - UHD F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE DAY DAY DAY GRAND MEAN (N= 18) NO OBS 18 11.056 18 15.778 18 20.333 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 5.0582 1.6330 14.8 0.0000 8.9084 2.1344 13.5 0.0000 10.041 2.6977 13.3 0.0000 Control BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:33 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits control VARIATE V003 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 0.000000 0.000000 0.00 1.000 * RESIDUAL 12 0.000000 0.000000 * TOTAL (CORRECTED) 17 0.000000 0.000000 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:33 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits control VARIATE V004 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 1.61111 322222 1.93 0.162 * RESIDUAL 12 2.00000 166667 * TOTAL (CORRECTED) 17 3.61111 212418 BALANCED ANOVA FOR VARIATE DAY FILE VRUS 6/ 3/22 23:33 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits control VARIATE V005 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 5.77778 1.15556 1.60 0.233 * RESIDUAL 12 8.66667 722222 * TOTAL (CORRECTED) 17 14.4444 849673 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE VRUS 6/ 3/22 23:33 :PAGE 89 the lesions on Lime fruits, Orange fruits and Pomelo fruits control MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 3 DAY 5.00000 5.00000 5.00000 5.00000 5.00000 5.00000 DAY 5.00000 5.33333 5.66667 5.00000 5.66667 5.00000 DAY 5.00000 6.33333 6.00000 5.00000 6.00000 5.00000 SE(N= 3) 0.000000 0.235702 0.490653 5%LSD 12DF 0.000000 0.726279 1.51187 ANALYSIS OF VARIANCE SUMMARY TABLE FILE VRUS 6/ 3/22 23:33 :PAGE the lesions on Lime fruits, Orange fruits and Pomelo fruits control F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE DAY DAY DAY GRAND MEAN (N= 18) NO OBS 18 5.0000 18 5.2778 18 5.5556 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 0.00000 0.00000 0.0 1.0000 0.46089 0.40825 7.7 0.1617 0.92178 0.84984 15.3 0.2332 Growth inhibition of Trichoderma on the Pythium spp during in vitro BALANCED ANOVA FOR VARIATE DAY FILE TRICHOVI 7/ 3/22 1:14 :PAGE Growth inhibition of Trichoderma on the Phytophythium spp during in vitro VARIATE V003 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 0.000000 0.000000 0.00 1.000 * RESIDUAL 0.000000 0.000000 * TOTAL (CORRECTED) 0.000000 0.000000 BALANCED ANOVA FOR VARIATE DAY FILE TRICHOVI 7/ 3/22 1:14 :PAGE Growth inhibition of Trichoderma on the Phytophythium spp during in vitro VARIATE V004 DAY LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 121.187 60.5934 18.55 0.003 * RESIDUAL 19.5954 3.26590 * TOTAL (CORRECTED) 140.782 17.5978 BALANCED ANOVA FOR VARIATE DAY FILE TRICHOVI 7/ 3/22 1:14 :PAGE Growth inhibition of Trichoderma on the Phytophythium spp during in vitro VARIATE V005 DAY LN SOURCE OF VARIATION DF SUMS OF 90 MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 10.8156 5.40781 3.55 0.096 * RESIDUAL 9.12719 1.52120 * TOTAL (CORRECTED) 19.9428 2.49285 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TRICHOVI 7/ 3/22 1:14 :PAGE Growth inhibition of Trichoderma on the Phytophythium spp during in vitro MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 DAY 0.000000 0.000000 0.000000 DAY 14.6205 22.1852 22.6070 DAY 21.4443 22.8910 20.2086 SE(N= 3) 0.000000 1.04338 0.712086 5%LSD 6DF 0.000000 3.60920 2.46322 ANALYSIS OF VARIANCE SUMMARY TABLE FILE TRICHOVI 7/ 3/22 1:14 :PAGE Growth inhibition of Trichoderma on the Phytophythium spp during in vitro F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE DAY DAY DAY GRAND MEAN STANDARD DEVIATION C OF V |TREATMEN| (N= 9) SD/MEAN | | NO BASED ON BASED ON % | | OBS TOTAL SS RESID SS | | 0.00000 0.00000 0.00000 0.0 1.0000 19.804 4.1950 1.8072 9.1 0.0032 21.515 1.5789 1.2334 5.7 0.0956 Growth inhibition of Bacillus velezensis, DT-K1, D1, YB12,YB9 during in vitro with fungal pathogen HG11 BALANCED ANOVA FOR VARIATE PIRG1 FILE HG11VK 7/ 3/22 2:21 :PAGE Growth inhibition of Bacillus velezensis, CLNA, YB12,YB9, D1, TN1KL1 during in VARIATE V003 PIRG1 LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 24.0370 4.80740 2.29 0.111 * RESIDUAL 12 25.2389 2.10324 * TOTAL (CORRECTED) 17 49.2759 2.89858 BALANCED ANOVA FOR VARIATE PIRG FILE HG11VK 7/ 3/22 2:21 :PAGE Growth inhibition of Bacillus velezensis, CLNA, YB12,YB9, D1, TN1KL1 during in VARIATE V004 PIRG LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 29.1507 5.83015 2.79 0.067 * RESIDUAL 12 25.0523 2.08770 * TOTAL (CORRECTED) 17 54.2031 3.18842 91 :PAGE Growth inhibition of Bacillus velezensis, CLNA, YB12,YB9, D1, TN1KL1 during in VARIATE V005 PIRG3 LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 332.799 66.5599 24.78 0.000 * RESIDUAL 12 32.2298 2.68582 * TOTAL (CORRECTED) 17 365.029 21.4723 BALANCED ANOVA FOR VARIATE PIRG4 FILE HG11VK 7/ 3/22 2:21 :PAGE Growth inhibition of Bacillus velezensis, CLNA, YB12,YB9, D1, TN1KL1 during in VARIATE V006 PIRG4 LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 349.956 69.9912 12.96 0.000 * RESIDUAL 12 64.8189 5.40158 * TOTAL (CORRECTED) 17 414.775 24.3985 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE HG11VK 7/ 3/22 2:21 :PAGE Growth inhibition of Bacillus velezensis, CLNA, YB12,YB9, D1, TN1KL1 during in MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 3 PIRG1 1.55039 2.32558 3.87597 1.55039 4.65116 3.10078 PIRG 6.64570 3.48008 4.10602 2.84516 5.38185 3.79754 PIRG3 14.4984 15.5750 13.4203 7.35951 16.0094 4.76765 PIRG4 16.4099 2.77778 8.76263 11.9108 8.56061 5.21465 SE(N= 3) 0.837305 0.834205 0.946188 1.34184 5%LSD 12DF 2.58002 2.57047 2.91553 4.13465 ANALYSIS OF VARIANCE SUMMARY TABLE FILE HG11VK 7/ 3/22 2:21 :PAGE Growth inhibition of Bacillus velezensis, CLNA, YB12,YB9, D1, TN1KL1 during in F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE PIRG1 PIRG PIRG3 PIRG4 GRAND MEAN (N= 18) NO OBS 18 2.8424 18 4.3761 18 11.938 18 8.9394 Evalution of greenhouse STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 1.7025 1.4503 51.0 0.1114 1.7856 1.4449 33.0 0.0672 4.6338 1.6388 13.7 0.0000 4.9395 2.3241 26.0 0.0002 Trichoderma asperellum to control citrus root rot in the BALANCED ANOVA FOR VARIATE LENGTH FILE TRICHO2 9/ 3/22 22:43 :PAGE the lenght of root rot 92 VARIATE V003 LENGTH LN SOURCE OF VARIATION DF SUMS OF MEAN F RATIO PROB ER SQUARES SQUARES LN ============================================================================= TREATMEN 4543.58 1514.53 218.97 0.000 * RESIDUAL 55.3339 6.91673 * TOTAL (CORRECTED) 11 4598.92 418.083 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TRICHO2 9/ 3/22 22:43 :PAGE the lenght of root rot MEANS FOR EFFECT TREATMEN TREATMEN NOS 3 3 LENGTH 52.3333 15.3333 32.0000 0.000000 SE(N= 3) 1.51841 5%LSD 8DF 4.95139 ANALYSIS OF VARIANCE SUMMARY TABLE FILE TRICHO2 9/ 3/22 22:43 :PAGE the lenght of root rot F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE LENGTH GRAND MEAN (N= 12) NO OBS 12 24.917 STANDARD DEVIATION C OF V |TREATMEN| SD/MEAN | | BASED ON BASED ON % | | TOTAL SS RESID SS | | 20.447 2.6300 10.6 0.0000 93