SỬ DỤNG PHÂN hữu cơ VI SINH từ NGUỒN nấm PHÂN lập TRONG cải THIỆN bạc màu đất và NĂNG SUẤT CAM SÀNH tại HUYỆN TAM BÌNH, TỈNH VĨNH LONG TT TIENG ANH

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DORTORAL DISSERTATION Major in Soil Science Identification: 62620103 NGUYEN NGOC THANH USING BIO-ORGANIC FERTILIZERS FROM ISOLATED FUNGUS STRAINS IN IMPROVING SOIL DEGRADATION AND CITRUS FRUIT YIELD IN TAM BINH DISTRICT, VINH LONG PROVINCE Can Tho, 2021 THE DISSERTATION WAS COMPLETED AT CAN THO UNIVERSITY Supervisor: Prof Dr Vo Thi Guong Co-supervisor: Dr Duong Minh Vien The dortoral dissertation was defended at the University Examination Council At:………………………………………………… At:…….hour……day……month…….year…… Reviewer 1:………………………………… Reviewer 2:………………………………… Reviewer 3:………………………………… Confirmation of review by the Chairman of the Board The dissertation is available at: Learning Resource Center, Can Tho University National Library of Viet Nam PUBLICATIONS RELATED WITH THE DISSERTATION [1] Nguyen Ngoc Thanh, Tat Anh Thu, Vo Thi Van Anh, Nguyen Van Loi, Vo Thi Guong 2018 Present situation of King mandarin technical cultivation in Tam Binh district, Vinhx Long province Journal of Vietnam Agricultural Science and Technology, (89): 38-44 [2] Nguyen Ngoc Thanh, Tat Anh Thu, Vo Thi Guong 2018 Effect of microbial organic fertilizers combined with Trichoderma fungi on soil nutrients and control Fusarium spp density in Citrus orchards Journal of Vietnam Agricultural Science and Technology, (89): 70-78 [3] Nguyen Ngoc Thanh, Duong Minh Vien, Tat Anh Thu, Mai Thi Cam Trinh, Vo Thi Guong 2018 Evaluation of some selected physical, chemical and biological soil properties on Citrus nobilis infected by dry root rot disease in Tam Binh district, Vinh Long province Can Tho University Journal of Science, 54(6B): 72-81 [4] Nguyen Ngoc Thanh, Tat Anh Thu, Duong Minh Vien, Vo Thi Guong 2018 The efficiency of microbial organic fertilizer in improving soil fertility and fruit yield of King Mandarin Vietnam Soil Science Journal, 53: 43-47 [5] Nguyen Ngoc Thanh, Tat Anh Thu, Duong Minh Vien, Nguyen Van Nam, Vo Thi Guong 2018 Effect of microbial organic fertilizers on improvement of physical, chemical and biological properties in citrus orchards Journal of Vietnam Agricultural Science and Technology, (94): 90-98 CHAPTER INTRODUCTION 1.1 Introduction Citrus nobilis is one of the major crops in agricultural production in Vinh Long province However, average yield of citrus orchards is variable among difference cultivated areas A plant growth of the citrus orchards only lasts from to years with low yield The fruit yield in Tam Binh district is only 50% of the fruit yield in other areas Previous study showed that soil characteristics on citrus orchards with raised beds older than 15 years were degraded as soil compaction, reduced soil available nutrients and poor microbiological activity (Vo Thi Guong et al., 2010, Pham Van Quang et al., 2013; Vo Thi Guong et al., 2016) The Tam Binh district has a history of growing the citrus orchards on raised beds with average of 18 years old Therefore, soil degradation can be the cause of fruit yield reduction In addition, root rot disease increases up to 58%, based on our survey data This factor also effects to fruit yield Therefore, improving soil fertility, reducing the root rot disease and improving fruit yield of the citrus orchards is rather necessary to be studied to support farmers in Citrus orchards 1.2 Objectives The objectives of this study were to improve soil fertility, to reduce the root rot disease, and to enhance fruit yield on Citrus nobilis orchards by applying bio-organic fertilizers containing isolated native microbes as antagonistic agents against the root rot disease 1.3 Research contents Content 1: Evaluating the current farming and soil characteristics of the citrus orchards in Vinh Long Content 2: Identifying agent of the root rot disease on citrus orchards Content 3: Isolating and selecting of fungal strains as Trichoderma fungi from citrus rhizosphere soil for producing the bio-organic fertilizers with the ability of resistant to the root rot disease Content 4: Evaluating the effect of the bio-organic fertilizer amendment to improve the soil fertility, to control root rot disease and to improve fruit yield 1.4 New contributions of the thesis The results from surveying and evaluation of soil properties on Citrus nobilis orchards in Tam Binh district identified: (1) Soil degradation on citrus orchards: low pH, less soil organic matter, unbalanced application of N, P and K fertilizers (excess application of N, P but K deficiency in the soil); (2) a very high rate of the root rot disease on citrus orchards; (3) a low decrease of fruit yield The study results showed that the agent of the root rot disease on the citrus orchards caused by Fusarium solani The study identified that effectiveness of the bio- bio-organic fertilizers containing antagonistic microbes with Trichoderma asperellum (isolated at citrus rhizosphere soil), Gongronella butlerium (fungi with increasing the ability to decompose organic matter) showed an effective improvement of soil chemistry (soil pH, SOM, labile organic C, labile organic N, available N, available P, exchangeable K, CEC and base saturation), increased total soil microbiological density, decreased Fusarium sp density The impact of the bio-organic fertilizers reduced the root rot disease on the citrus orchards The fruit yield increased significantly Bases on the study results, a recommended dose of the bioorganic fertilizers by kg per tree combined with recommended inorganic fertilizers improved effectively soil fertility, reduce of the root rot disease, increase the fruit yield on the citrus orchards 1.5 Scope and research subjects The bio-organic fertilizer amendment containing antagonistic strains of the native fungus isolates to improve soil fertility, reduce the root rot disease and increase the fruit yield of the citrus orchards in Tuong Loc commune, Tam Binh distric, Vinh Long province 1.6 Scientific and practical significance The research showed the ability of using available rice straw locally and the beneficial native fungi has an effective impact on improvement of soil physical, chemical and biological characteristics and control of Fusarium sp density in soil that resulted in reducing the root rot disease on the citrus orchards The study provided the scientific basic for producing the biofertilizers with isolated fungus from citrus rhizosphere soil (Trichoderma asperellum and Gongronela butleri) that improved effectively soil degradation on the citrus orchards, reducing the root rot disease and increasing the fruit yield CHAPTER METHODOLOGY 2.1 Evaluating the current farming systems of the citrus orchards in Tam Binh district, Vinh Long province Investigate of current farming systems through direct interviews of farmers by printed questionnaires and a filed survey of the citrus orchards with an area of 0.1 or more A total of 75 households were interviewed including information of households, cultivars, densities, farming systems, age of bed, age of citrus orchards, using of fertilizers, root rot disease, fruit yield The rate of the root rot disease was evaluated according to the classification of disease index by Jones (1998), changed into three groups: C0-1: the number of the root rot disease trees per a citrus orchard accounting for 0-5%; C2-3: the number of the root rot disease trees per a citrus orchard accounting for 6-50%; C4-5: the number of the root rot disease trees per a citrus orchard accounting for 51% or more 2.2 Evaluating soil characteristics of the citrus orchards in Vinh Long province - The study was conducted from November 2014 to May 2015 from 16 citrus orchards of the 75 surveyed citrus orchards, dividing into two groups of different citrus orchards: the citrus orchards of the bed soil with age of 15 years or less and the other with age of over 15 years - 16 soil samples of 16 the citrus orchards were collected at a depth of – 20 cm, at the location of each tree and below the canopy, at rhizosphere system with most developed root, collecting the soil samples for each replicate and mixing them into a sample for analysing some soil properties such as soil pH, SOM, available N, available P, exchangeable K and base saturation 2.3 Evaluating soil characteristics related to the root rot disease on citrus orchards Forty soil samples according to two groups of non-infected and infected disease with the root rot disease was collected at the depth of – 20 cm, at the location of each tree and below the canopy, at rhizosphere system with most developed root, collecting the soil samples for each replicate and mixing them into a sample for analyzing some soil characteristics such as soil physical (soil moisture); soil chemical (soil pH, SOM); soil biological properties (total soil microbiological, Fusarium sp and Trichoderma sp density) Methods of soil analysis - Soil physical and chemical properties + Soil moisture: soil moisture is calculated by moisture content, soil samples are dried at 105oC until the weight does not change + Soil pH is measured by using a pH meter with a ratio of soil : water (1:2.5) + Soil organic matter is measured according to method of Walkley – Black (Nelson Sommers, 1982) + Soil available nitrogen (including NH4+ NO3-) is extracted by KCl 2M salt with a ratio of soil: extracted solution (1:10 w/v) Content of the soil available nitrogen after extraction is calculated by a colorimetric analysis on spectrophotometer at 650 nm for NH4+ and 540 nm for NO3- (Rhine et al., 1998; Miranda et al., 2001) + Soil available phosphorus is analysed by the Bray The solution after extraction is calculated by a colorimetric analysis on spectrophotometer at 880 nm (Bray Kurtz, 1945) + Exchangeable potassium in soil is extracted by unbuffered solution of BaCl2 0,1M (Hendershot et al., 1986; Rhoades, 1982) The solution after extraction is measured on an atomic absorber at 766 nm + Exchangeable cations in soil and CEC are extracted by BaCl2 0,1M and titrated by EDTA 0,01M, after measuring on an atomic absorber (Kariuki et al., 2010) for calculating base saturation in the soil - Soil biological properties Density of microorganisms is calculated by counting the number of living colonies on agar plates of culture medium, combining with appearance of fungus spores under microscope in order to identify species of fungi PDA medium (Potato Dextrose Agar) is used to screen total soil microbiological density (Gupta et al., 2010; El-Mohamedy et al., 2012), Fusarium sp (Gupta et al., 2010; El-Mohamedy et al., 2012) and Trichoderma sp density (Elad et al., 1981) respectively 2.4 Investigated of the causing the root rot disease by Fusarium solani in greenhouse condition - Thirteen isolated strains of Fusarium solani fungi were cultured on PDA medium at room temperature until spore formation The spore suspension with Fusarium solani fungi with density of x 106 spores/mL was injected in citrus sleedings (Duong Minh et al., 2010) - After the citrus sleedings grown well in the potting medium, pouring the fungal suspension with of x 106 spores/mL into the pottings After 30 days of inoculation, assessing disease index, mortality rate and disease level (Duong Minh, 2010) At this time, collecting and isolating presence of Fusarium sp density in thirdteen treatmeats at 60 days after inoculation 2.5 Isolating and selecting of fungal strains that resistant to the fungal pathogen by Fusarium solani An experiment was conducted at Lab of soil microbiology – Department of Soil Science, College of Agriculture, Can Tho University, from November 2015 to May 2016 2.5.1 Isolation of Trichoderma fungus from rhizosphere soil that antagonistic potential for Fusarium solani fungus A total of 20 soil samples were collected in rhizosphere soil of 20 non-infected citrus orchards, at a depth of - 20 cm, each stump collected soil samples, mixed them into a replicate Soil samples were extracted with sterile solution of Sodium pyrophosphate 0,2% (w/v) at the rate of 1:10 (Junghanns et al., 2008), diluted 10-1 to 10-5 the extraction and put 100 µL of the dilution onto plates of TSM medium and cultivated at room temperature Isolating of Trichoderma sp at 2-4 days after incubation Identification of Tricohderma sp strains according to species collection of the USDA (Farr and Rosman, 2017) 2.5.2 Evaluate the ability of the isolated fungal strains to inhibit the fungus pathogen of the root rot disease Fusarium solani was isolated from the rhizosphere of citrus orchards, identifying agent of the root rot disease on citrus orchards Trichoderma sp was isolated from rhizosphere soil of citrus orchards (19 fungal strains) Gongronella butleri was isolated from paddy rice soil Malt extract agar (MEA) 1% medium was used to evaluate the ability of inhibitting the growth of fungal strains (Royse and Ries, 1978) Put each two-agar block (diameter mm) contained antagonistic strains to exhibit the fungal pathogen of Fusarium solani on 1% MEA culture respectively Each block had area of 0.25 cm2 The 2-agar blocks was cm apart on the petri dish, each one was 2.5 cm from the edge of the dish (9 cm diameter of the Petri dish) Each combination was designed with replicates Mycelial growth of fungi was recorded about four weeks (Suciatmih and Rahmansyah, 2013; Chand and Logan, 1984; Skidmore and Dickinson, 1976) Evaluating the growth exhibition of the fungal pathogens by calculating the percentage of growth limitation by formula of Royse and Ries (1978) at 2, and 12 days after cultivation [100 x (r1-r2)/r1] r1: The range of the mycelium growing farthest to the pathogen r2: The opposite distance between two fungus strains of antagonistic and pathogen species 2.6 Production of bio-organic fertilizers The experiment was conducted at experimental area, Lab of soil microbiology – Department of Soil Science, College of Agriculture, Can Tho University, from November 2015 to May 2016 Source of organic matter: 1.2 tons of rice straw was used for composting according to method of using only rice straw as the composting material (Goyal and Sindhu, 2011) Antagonistic strains were inoculated into the compost for producing types of the bio-compost as in Table 3.2 Table 2.1: The role of fungal strains in the bio-compost Name of fungi Types of bio-organic Source of isolated fungi strains fertilizers strains Trichoderma sp Bio-organic fertilizer Rhizosphere soil of citrus orchards Gongronella butleri Bio-organic fertilizer Paddy rice soil Trichoderma sp + Rhizosphere soil of citrus Gongronella butleri Bio-organic fertilizer orchards and Paddy rice soil Trichoderma sp Bio-organic fertilizer Commercial product The density of beneficial fungus strains was inoculated into the substrate of organic matter corresponding to the bio-fertilizers with the moisture and lasting for a certain period often caused lack of oxygen, respiration of roots under anaerobic conditions that leaded to poor activity of root systems in soil Under anaerobic conditions, production of polyphenol compounds in the soil damaged cells of young roots that facilitated easily Fusarium sp to infect root systems of citrus trees (Ownley and Benson, 1991; Nguyen Minh Hieu et al., 2013) According to the research of Kunta et al (2015), the above unfavorable conditions that facilitated Fusarium solani to penetrate inside root systems Thus, growth speed of the root rot disease increased higher under conditions of high soil moisture than under conditions of low soil moisture Figure 3.5 Evaluation of soil moisture related to the root rot disease 3.3.2 Soil organic matter and the root rot disease The results showed that, content of soil organic matter in the group of infected citrus orchards was evaluated poorly in soil (2.48%) (Landon, 1984) Generally, the content of soil organic matter was low the both groups of the citrus orchard However, the group of noninfected citrus orchards had higher content of soil organic matter (3.25%), different statistically compared with the group of infected citrus orchards (Figure 3.6) 14 Figure 3.6 Evaluation of soil organic matter and the root rot disease Notes: The letters a,b,c in each column represent the significant difference by Tukey 5%(*) test and the error bar on the graph represents the standard deviation (SD) 3.3.3 Assessment of available nitrogen and the root rot disease The study in Figure 3.7 presented that the concentration of available nitrogen in the non-infected orchards was quite high (126.6 mg/kg), three times higher and different statistically compared to the infected orchards (36 mg/kg) According to research results of Robert (2015), content of the available nitrogen was sufficient for plants from 30-50 mg/kg The group of infected orchards showed a low content of the available nitrogen in soil for growth of citrus trees Figure 3.7 Evaluation of available nitrogen and the root rot disease 15 3.3.4 Evaluation of exchangeable potassium and the root rot disease The study in Figure 3.8 showed that the content of exchangeable potassium in the group of the non-diseased citrus orchards was higher, different statistically compared to the group of the diseased citrus However, content of the exchangeable potassium on both groups of citrus orchards varied from 0.25 to 0.30 meq/100g that was assessed at a low level of exchangeable potassium in soil (Landon, 1984) Figure 3.8 Assessment of exchangeable potassium and the root rot disease 3.3.5 Evaluation of total soil microbiological related to the rate of root rot disease The results in Figure 3.9 showed that the total soil microbiological density the group of the non-diseased citrus orchards was high (2.35 x 106 cfu g-1), different statistically compared to the group of the diseased citrus orchards Activity of soil microorganism affected greatly soil quality and plant growth (Hill et al., 2000; Araújo et al., 2009) 16 Figure 3.9 Evaluation of total microbiological density and the root rot disease Notes: The letters a,b,c in each column represent the significant difference by Tukey 5%(*) test and the error bar on the graph represents the standard deviation (SD) 3.3.6 Assessment of density of Fusarium sp in soil related to the root rot disease The results in Figure 4.10 showed density of Fusarium sp in the group of the diseased citrus orchards (2.41 x 104 cfu g-1), nearly times higher and different statistically compared to the group of non-diseased citrus orchards (1.39 x 104 cfu g-1) Fungi of Fusarium sp in soil with a level of high density secreted naphthazarins compounds and attacked the woody vessels of roots, causing root rot (Nemec et al., 1991; Derrick and Timmer, 2000; Janse van Rensburga et al., 2001) The root rot disease causing by Fusarium solani also caused loss of leaf pigmentation, leaves becoming yellow and caused significant damage to citrus yield and citrus growth (El-Mohamedy et al., 2016) 17 Figure 3.10 Assessment of Fusarium sp density in soil and the root rot disease in citrus orchards Notes: The letters a,b,c in each column represent the significant difference by Tukey 5%(*) test and the error bar on the graph represents the standard deviation (SD) The assessment of soil physical, chemical and biological properties for two groups of the citrus orchards showed that some soil properties related to the root rot disease In terms of soil chemical properties, the infected citrus orchards showed limitation of soil organic matter, available nitrogen, exchangeable potassium, base saturation in soil compared to the non-infected citrus orchards Density of Fusarium sp appeared highly in soil while the total microorganisms were low in the infected citrus orchards The results showed that soil beds of the infected citrus orchards were limited in terms of soil chemical and biological properties 3.4 Identifying agent of the root rot disease on citrus orchards Results showed that all treatments inoculated with a fungal suspension containing x 106 spores/mL resulted in citrus seedlings with exhibition of various degrees of the root rot disease, varying with a high rate of disease index from 40 to 84% in the period of 60 days after inoculating (Figure 3.11) Thus, the isolates of Fusarium solani from the rhizosphere of the infected citrus orchards showed a very high rate of causing the root rot in the citrus orchards under greenhouse conditions 18 In generally, the isolated fungal strains showed different levels of pathogenicity in citrus trees which depended the different periods after the inoculation, different statistically compared to the control treatment Thus, the isolated fungal strains at the citrus rhizosphere showed a high degree of infection in tested citrus seedlings under the greenhouse conditions, which were agent of causing the root rot disease in the citrus orchards by of Fusarium solani Figure 3.11 Index of the root rot disease in the citrus orchards over the period of 30, 40, 50 and 60 days after inoculating Notes: The treatments (T): T, T2, T3, T4, T5, T6, T7, T8, T9, T10, T11, T12 and T13 were inoculated with a suspension containing Fusarium solani with the density of x 106 cells/mL Control treatment (Control): no fungal strains, only treated with sterile distilled water 3.5 Isolation and selection of fungal strains from the citrus rhizosphere antagonistic potential against the fungus Fusarium solani The study showed that the strains of fungi that exhibited high resistance to Fusarium solani at 12-day period after cultivation was Gongronella butleri and Trichoderma asperellum, the percentage of inhibition ranged from 38.15% to 56.09% In particular, Gongronella butleri presented the highest inhibitory capacity, with an 56.1% Gongronella butleri was studied to produce chitosan with high levels (Teng et al., 2001; Chatterjee et al., 2005; Babu et al., 2015; Zhang et 19 al., 2020) Thus, the inhibitory ability of the promising fungal strains: Gongronella butleri and Trichoderma asperellum with a high inhibitory effect that againsted Fusarium solani on agar petri, were selected for the benefical groups of producing the bio-organic fertilizers 3.6 Evaluate the effectiveness of the bio-organic fertilizers on improvement of soil fertility 3.6.1 Soil aggregate The results in Figure 3.12 showed that soil aggregate increased significantly when the citrus soil was applied the bio-organic fertilizers over two crops in two treatments T4 (94.6) and T5 (76.9) compared to farming systems only applying chemical fertilizers (T1 and T2) According to research results of soil aggregate SQ> 85, evaluated that soil became high structural strength (Le Van Khoa and Nguyen Van Be Ty, 2013) In generally, the bio-fertilizer with beneficial fungi strains showed good efficiency in increasing soil aggregate Figure 3.12 Effect of the bio-organic fertilizers on soil aggregate after applying 15 months T1: Appling NPK fertilizer according to farmers 360 g N - 195 g P2O5 - 55 g K2O (control); T2: Appling NPK fertilizer as recommended 250 g N - 50 g P2O5 - 250 g K2O; T3: Applying NPK fertilizer according to T2 + Bio-organic fertilizer with Trichoderma asperellum; T4: Applying NPK fertilizer according to T2 + Bio-organic fertilizer with Gongronella butler; T5: Applying NPK fertilizer according to T2 + Bio-organic fertilizer with Trichoderma asperellum 20 and Gongronella butleri; T6: Applying NPK fertilizer according to NT2 + Bioorganic fertilizer with Trichoderma sp (commercial product) Notes: The letters a,b,c in each column represent the significant difference by Tukey 5%(*) test and the error bar on the graph represents the standard deviation (SD) 3.6.2 Available nitrogen in soil The results in Figure 3.13 showed that content of available nitrogen in the two treatments without applying the bio-organic fertilizers (T1 and T2) were low, 26.2 mg N/kg and 28.2 mg N/kg, respectively, different statistically compared to all the bio-organic fertilizer treatments According to the results of previous researches, the available nitrogen was enough for plant growth in a range of 30-50 mg/kg at soil pH from 6.5 to 8.0 (Robert, 2015; Nájera et al., 2015) Thus, decreasing inorganic nitrogen and supplementing of the the bioorganic fertilizers improved the available nitrogen effectively in soil for cultivation of the citrus orchards As a result, the citrus orchards with applying the bio-organic fertilizers reduced investment inputs because the previous results showed that excess fertilization of nitrogen influenced negatively growth, yield and quality of crops (Morgan and Connolly, 2013; Leghari et al., 2016) Figure 3.13 Effect of the bio-organic fertilizers on available nitrogen after applying 15 months 21 3.6.3 Available phosphorus in soil The analysis results of available phosphorus content in Figure 3.14 showed that the treatments with applying the bio-organic fertilizers improved an increase of available phosphorus in soil, ranging from 50.26 to 80.91 mg P/kg statistically compared to two treatments with only inorganic fertilizers (T1 and T2) According to previous results, content of available phosphorus was suitable for citrus growth at 65 mg P/kg or more (Obreza et al., 2008) In generally, supplement of the bioorganic fertilizers increased the available phosphorus content more than three times and different statistically compared to cultivation with only inorganic fertilizers Figure 3.14 Effect of the bio-organic fertilizers on available phosphorus after applying 15 months 3.6.4 Labile nitrogen in soil Results of labile nitrogen analysis after 15 months of applying the bio-organic fertilizers showed that the index of the labile nitrogen was the best for the treatments (T3, T4 and T5) while the bio-organic fertilizers containing two fungal isolates of Trichoderma asperellum and Gongronella butleri, with 35.41, 38.25 and 36.39 mg/kg respectively (Figure 3.15) Citrus soil with applying the bio-organic fertilizers improved the content of the labile nitrogen in soil by 20 mg N/kg (Vo Thi Guong et al., 2010) 22 Figure 3.15 Effect of the bio-organic fertilizers on available nitrogen after applying 15 months 3.6.5 Exchangeable potassium The results in Figure 3.16 showed that the treatments with applying the bio-organic fertilizers improved content of exchangeable potassium in soil, ranging from 0.18 to 0.24 cmol/kg statistically compared to the two treatments with only applying inorganic fertilizers According to the previous results showed that the exchangeable potassium in soil from 0.15 to 0.3 cmol/kg was evaluated that soil lacked less exchangeable potassium (Landon, 1984; Nájera et al., 2015) Thus, increasing of inorganic potassium fertilizer into soil at a dose of 250g K2O/tree in combination of the bio-organic fertilizers improve the exchangeable potassium in soil compared to farmers’ cultivation with the exchangeable potassium lowly in soil (0.0023 cmol/kg) Figure 3.16 Effect of the bio-organic fertilizers on exchangeable 23 potassium after applying 15 months 3.6.6 Total soil microbiological density The results in Figure 3.17 showed that the treatments with applying the bio-organic fertilizers increased the total soil microbiological density, ranging from 0.26 to 0.40 x 107 cfu g-1 significantly compared to the two treatments with only inorganic fertilizers (T1 and T2) The previous research results presented that the addition of compost in citrus orchards improved activity of soil microorganisms for plants (Krull et al., 2004; Raviv, 2008; Tran Ba Linh et al., 2008; Gong et al., 2009; Liu et al., 2010; Vo Thi Guong, 2010; Zhong et al., 2010; Tat Anh Thu et al., 2014) Thus, total soil microbiological density was increased through the addition of the bioorganic fertilizers in soil Figure 3.17 Effect of the bio-organic fertilizers on total soil microbiological density after applying 15 months 3.6.7 Density of Fusarium sp in soil Fusarium solani is considered to be soil pathogen (Cho et al., 2001; Khlij et al., 2008; Chandran and Kumar, 2012; McMahon, 2012; Pham Van Kim, 2014; Kurt et al., 2020) The results in Figure 3.18 showed Fusarium sp density decrease lowly statistical significantly in the treatments with applying the bio-organic fertilizers, ranging from 1.39 and 3.27 x 104 cfu g-1 compared to the two treatments with applying only inorganic fertilizer (T1 and T2) In particular, the 24 treatments of T3 and T5 presented higher effectiveness in reducing Fusarium sp density in soil with 1.39 and 1.31 x 104 cfu g-1 respectively Therefore, the supplementary application of the bioorganic fertilizers in the citrus orchards decreased effectively the density of Fusarium sp in soil with a low rate compared to cultivation with only inorganic fertilizers Figure 3.18 Effect of the bio-organic fertilizers on Fusarium sp density after applying 15 months 3.6.8 Effect of the bio-organic fertilizers to improve the root rot disease in citrus orchards The rate of the root rot disease in citrus trees were evaluated by the percentage of infected leaves per tree The results in Figure 3.19 presented that all the treatments with the bio-organic fertilizers showed lower rates of the root rot disease, ranging from 6% to 25.8%, statistical significantly compared to farmers’ cultivation (T1) with a disease index of 40.4% in the period of 15 months after applying the bio-organic fertilizers Application of the bio-organic fertilizers with different fungal isolates leaded to reduce the root rot disease in the citrus orchards In which, the treatments of T3 and T4 showed a higher effectiveness for reducing the root rot disease (disease index less than 15%) compared to other treatments with the bio-organic fertilizers (Figure 4.54) Therefore, the density of Fusarium sp decreased lowly in soil which 25 may contribute to reduce the incidence of the root rot disease in the citrus orchards Figure 3.19 Effect of the bio-organic fertilizers on the root rot disease after applying 15 months The study results in Figure 3.20 showed that there was a positive correlation between the density of Fusarium sp in soil with incidence of the root rot disease Increasing of Fusarium sp density in soil resulted in a raise of the root rot disease in the citrus orchards with a correlation coefficient r = 0.74 These results presented that application of the bioorganic fertilizers resulted in a low reduction of Fusarium sp density in soil, contributing to reduce the root rot disease in the citrus orchards Figure 3.22 Correlation between Fusarium sp density in soil to the ratio the root rot after 15 months of applying the bio-organic fertilizers 26 3.6.9 Evaluating the effect of the bio-organic fertilizers to improve fruit yield in citrus orchards Fruit yield of the citrus orchards was collected through two harvests after 15 months of applying the bio-organic fertilizers The results in Figure 3.21 showed effect improving effectively the fruit yield of the citrus orchards after applying the bio-organic fertilizers The treatment T3, T4 and T6 presented an equal improvement of the fruit yield, varying from 13.82 to 18.81 tons per hectare and statistically significant differences compared to the two treatments with only inorganic fertilization (T1 and T2) The period of 15 months after applying the bio-organic fertilizers showed that the fruit yield was improved significantly compared to two treatments with only inorganic fertilizers (T1 and T2) Cultivating without the bio-organic fertilizers showed a very low fruit yield (less than 10 tons per hectare) Figure 3.21 Effect of the bio-organic fertilizers on fruit yield In generally, supplement of the bio-organic fertilizers on the citrus orchards improved the fruit effectively yield thanks to reducing the rate of the root rot disease and improving soil fertility 27 CHAPTER CONCLUSIONS The raised bed of over 15 years old showed a soil degradation in soil organic matter, available nitrogen, phosphorus, exchangeable potassium (P