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1 MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES NGUYEN NAM DUONG STUDY ON DISEASES CAUSED BY PHYTOPHTHORA SPP ON CITRUS TREE[.]
MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES NGUYEN NAM DUONG STUDY ON DISEASES CAUSED BY PHYTOPHTHORA SPP ON CITRUS TREES AND TOWARD BIOLOGICAL MANAGEMENT IN CAO BANG PROVINCE Branch of study: Code: Plant Protection 9.62.01.12 SUMMARY OF AGRICULTURAL DOCTORAL THESIS HA NOI - 2021 Project completed at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Scientific supervisors: Dr Ha Minh Thanh Dr Đang Vu Thi Thanh Reviewer 1: Reviewer 2: Reviewer 3: The doctoral thesis was defended in front of the Institute-level Thesis Judging Committee meeting at the Vietnam Academy of Agricultural Sciences At… hour… , day … month … year 20… The thesis can be found at: National Library of Vietnam Library of Vietnam Academy of Agricultural Sciences Library of Plant Protection Research Institute LIST OF WORKS HAS BEEN PUBLISHED Nguyen Thi Bich Ngoc, Nguyen Nam Duong, Pham Thi Dung, Le Mai Nhat, Đo Duy Hung, Ngo Thi Thanh Huong (2016), “Management of foot and root rot disease on Tra Linh mandarin in Cao Bang province”, Plant Protection Magazine, No (297), pp 3-8 Nguyen Nam Duong, Ngo Thi Thanh Huong, Vu Duy Minh, Ha Minh Thanh, Ha Viet Cuong (2021), “Results of research on antagonistic microorganisms to control Phytophthora spp caused diseases on citrus trees in Cao Bang province”, Plant Protection Magazine, No (264), pp 39-45 Nguyen Nam Dương, Ha Minh Thanh, Nguyen Thị Bich Ngoc, Ngo Thi Thanh Huong, Vu Duy Minh, Ha Viet Cuong, Pham Bich Hien (2021), “Identification of the Phytophthora spp causing root rot and gum disease on citrus trees in Cao Bang province”, Vietnam Journal of Agricultural Science and Technology, No 11 (132), pp 82-92 Nguyen Nam Dương, Ha Minh Thanh, Nguyen Thi Bich Ngoc, Ngo Thi Thanh Huong, Vu Duy Minh, Ha Viet Cuong, Pham Bich Hien (2021), “Possibility of using a combination of antagonistic microorganisms CB-1 to prevent Phytophthora root rot and gum disease on citrus trees in Cao Bang province”, Vietnam Journal of Agricultural Science and Technology, No 11 (132), pp 92 – 99 INTRODUCTION Rational Cao Bang is a mountainous province in the North, with very suitable soil and climate conditions for citrus fruit trees In Cao Bang province, the Tra Linh mandarin and Trung Vuong orange with high quality and special flavor have brought significant income to farmers With the orientation of developing some indigenous crops to replace some inefficient ones, Citrus trees are the choice in the current crop structure of many localities in Cao Bang However, in recent years, the area, output as well as quality of Cao Bang citrus have declined sharply, many gardens are heavily degraded The expansion of the area and the promotion of intensive farming to increase productivity are suitable conditions for many insect pests and diseases to arise and cause damage on citrus trees In addition to greening disease, root rot, gum disease, fruit rot, etc have been detected in most citrus growing areas in Cao Bang When infested, citrus trees become stunted, the foliage turns yellow, on the trunk and especially at the base of the tree, there are gum bleeding, the wood becomes dark brown, the roots are black rotten and easy to peel off, the fruit is few, when the disease is severe, the tree does not give fruit and may die The citrus orchards infected are called "sad orange" in Cao Bang The disease has been causing damage on many citrus orchards in Hoa An, Tra Linh districts, Cao Bang provinces Root rot and gum diseases on citrus in Cao Bang had only been preliminary studied as Phytophthora spp causes, in-depth studies of the disease have not been conducted Therefore, this project of “Study on diseases caused by Phytophthora spp on citrus trees and biological control strategies in Cao Bang province” was carried out for a scientific foundation of effective diseased management measures for a sustainable citrus production in Cao Bang Objectives and requirements of the project 2.1 Objectives Identification of Phytophthora species as pathogens on citrus fruit trees in Cao Bang as well as biological characteristics of fungal species and factors affecting the development and damage of root rot and gum disease of citrus fruit trees and effective disease control in Cao Bang 2.2 Requirements - Assess status of root rot and gum bleeding disease on citrus fruits, - Identify Phytophthora species as the causal agents and biological & ecological features of fungi causing disease on citrus fruit trees in Cao Bang - Isolate and select highly active strains of Phytophthora antagonistic microorganisms Testing the ability to use antagonistic microorganisms to control Phytophthora fungus that causes disease on citrus fruit trees Proposing effective control method to prevent Phytophthora disease affecting citrus fruit trees in Cao Bang Scientific and practical significance of the project In science: - Supplement the scientific data of Phytophthora species that caused root rot and gum disease on citrus trees in Cao Bang province, influent factors and solutions to prevent disease - Isolate and assess antagonistic microorganisms in Cao Bang which could be used as bio-agent to produce bioproduct to manage disease caused by Phytophthora fungi In practical: This topic contains a proposal of general management procedure on the said disease with the spirit of effectiveness, environmentally friendly and a sustainable citrus industry development in Cao Bang Object and scope of research - Phytophthora spp fungi caused disease on citrus trees in Cao Bang - Microorganisms are antagonistic to Phytophthora spp - Varieties of citrus fruit trees in Cao Bang (Trung Vuong orange, Ha Tri and Tra Linh mandarin, and Phuc Hoa grapefruit) Contributions of the project - Three species of fungi including Phytophthora palmivora, P nicotianae and P citrophthora have been identified as the causative agents of root rot and gum disease on citrus fruit trees in Cao Bang and added the fungus Phytophthora palmivora to the list of fungi that damage citrus fruit in the northern mountainous provinces - Three species of antagonistic microorganisms such as Bacillus amyloliquefaciens, B methylotrophicus and Streptomyces misionensis have been isolated and selected with high effect (82.2-85.7% in culture medium) on inhibitory of Phytophthora fungi - The bioproduct named CB-1 (mixture of Bacillus amyloliquefaciens, B methylotrophicus and Streptomyces misionensis) was highly against Phytophthora fungi with effect ranged from 75.85 - 83.18% in greenhouses and 73.1% in fields - Proposing the procedure of using CB-1 bioproduct to prevent root rot and gum disease on citrus fruit trees in Cao Bang CHAPTER I SCIENTIFIC FOUNDATION AND LITERATURED REVIEW 1.1 Scientific foundation The fungal species of the genus Phytophthora are causative agents of root rot and gum disease, and an economic disease of citrus growing regions around the world In this genus, ten species of fungi have been identified that caused diseases of citrus fruit trees, in which P palmivora, P nicotianae and P citrophthora are the most common and important fungi The rule of emergence of Phytophthora fungi and effective control strategies have been studied quite comprehensively and systematically in citrus growing regions around the world In Vietnam, species of Phytophthora fungi have been found to cause harm on agricultural crops Among them, P palmivora, P nicotiane, P cinamomi, P capsisi and P infestans are important species of high economic value crops durian, pineapple, black pepper, rubber and food plants of the family Solanaceae (Đang Vu Thi Thanh, Ngo Vinh Vien, Drenth, 2004) Studies on the rule of the generation of harm and Phytophthora disease management in durian, pepper, pineapple and other food crops have been systematically studied by many authors in Vietnam (Mai Văn Tri cs (2001), Nguyen Vinh Truong (2002), Pham Ngoc Dung cs (2007) The P citrophthora was first detected on oranges in Vietnam in the 50s of the 20th century (Roger, 1951) After that, the P nicotianae continued to be discovered in all citrus growing areas in Vietnam (Đang Vu Thi Thanh, Ngo Vinh Vien, Drenth, 2004) However, studies on Phytophthora disease on citrus in Vietnam are fragmentary and discontinuous and is often limited to the prevalence of disease surveys There is no research to develop disease management strategies, nursery management, disease resistance breeding Especially in Cao Bang, there have not been any studies on the causes of the disease and measures to manage root rot and gum disease on specialty citrus trees Research on Phytophthora fungi damaging citrus fruit trees and the use of biological products for disease management in Cao Bang is a necessary to meet the needs of developing specialty citrus fruit areas of Cao Bang province as well as citrus growing areas in Vietnam 1.2 An overview of citrus fruit trees Currently, citrus is one of the most important trees in the world The world production of citrus fruit is estimated at 124,246,000 tons (Citrus Fruit – Fresh and Processed Statistical Bulletin 2016, FAO) Citrus trees are capable of growing in latitudes 40o N to 40o S (Davies and Albrigo, 1994) However, the commercial citrus growing areas are mainly restricted to two subtropical belts, about 20 to 400N and S of the equator (Castle, 1987; Saunt, 1990) In Vietnam, according to statistics from the Department of Crop Production (Ministry of Agriculture and Rural Development, 2020), the total area of citrus trees is 235,216 In which, the area planted to citrus in the northern provinces is 106,125 ha, the North Central provinces are 29,630 ha, The South Central Coast and Central Highlands provinces are 7,761 and the southern provinces are 91,702 Orange is the most commonly grown citrus tree with an area of over 130,000 ha, followed by pomelo trees with an area of over 85,500 1.3 Studies on Phytophthora fungi that damage plants 1.3.1 General information about Phytophthora Phytophthora was first described by Heinrich Anton de Bay in 1875 Today, 170 species have been discovered More than 60 species of Phytophthora have been described as plant pathogens Each species can cause disease from a few to more than 1000 different plant species (David and André Drenth, 2004) Phytophthora species damage plants by destroying cells, causing necrosis of leaves, stems or roots The fungus may cause the burn leaves (blight), the fungus cracks the bark on the stem or branches, causing gum bleeding and ulcers (canker), at a point or around the trunk, branches Twig or branch can be withered (dieback) Phytophthora penetrates the xylem conduits, causing part or all of the canopy to lack water, and the plant will wilt Many species of Phytophthora infect plant roots causing root rot, in some cases, the entire hairy roots are damaged, some cases cause necrosis and lead to root tip rot (crown rot) Necrotic hairy roots will lead to a lack of water for the plant, in the long-term, the plants will wilt (decline) Root rot will lead to the main root and stem wither and the tree will die quickly (sudden death) Diseases from orchid roots damage the body close to the face causing stem rot (foot rot) or root neck rot (collar rot), brown rot fungus (brown rot) 1.3.2 Research on Phytophthora fungus damaging citrus fruit trees 1.3.2.1 Studies on Phytophthora fungi in the world Phytophthora exist in soil and water, causing diseases of citrus trees (Feichtenberger 2001; Feichtenberger et al., 2005; Graham and Menge, 1999, 2000) The fungi are distributed worldwide and cause significant yield losses for citrus in the subtropical regions with high rainfall (Graham Feichtenberger, 2015) Among the 10 species of Phytophthora reported to cause damage to citrus trees worldwide, the three most serious pests causing gum disease, root rot and fruit rot are Phytophthora citrophthora, P nicotianae and P palmivora (Erwin and Ribeiro, 1996; Graham and Menge, 2000) The fungi P citrophthora and P parasitica (P nicotianae) have been identified as the main causes of gum disease and root rot disease in citrus fruits worldwide (Timmer and Menge, 1993; Martin et al., 2001; Siviero et al., 2002; Timmer et al., 2003) The fungus P nicotianae causes neck rot and root rot but rarely causes damage in parts far from the ground The fungus P palmivora causes brown rot, P citrophthora causes root rot and pus, and also on trunks and large branches (Feichtenberger 2001) Phytophthora fungi survive in soil when humidity and temperature are high, infected roots produce sporangia and will release zoospores Zoospores swim in water for short distances or are moved by rain or irrigation water Once contact with roots, spores form cysts, germinate, and then infect the root tip (Duniway, 1983) Identification of Phytophthora species based on morphological characteristics: This method is based on the characteristics of the sporangium, sexual reproductive structures such as antheridium, oogonium, oospore, form of sexual reproduction (homothallic or heterothallic), with or without chlamydospores to identify the species of fungus Identification of Phytophthora species by molecular biology method: Currently, nucleotide sequencing of ITS often applied to identify and evaluate the relationships of Phytophthora species (Blair et al., 2008) Appiah (2004) used ITS sequencing to evaluate 88 isolates of P palmivora collected from cocoa trees from different regions of the world Cacciola also reported the use of ITS sequencing to identify P palmivora species harmful to the palm tree (Trachycarpus fortunei) Phytophthora disease management There are basic factors closely related to disease manage: (1) cultural techniques, (2) breeding resistant plants, (3) biological control, (4) chemical control (David and André Drenth, 2004) Cultural techniques: The application of integrated farming methods significantly reduces the source of Phytophthora fungi in the soil Control with chemical products: The use of chemical fungicides for the control of Phytophthora diseases has been widely used since the production of highly effective rhizogenic fungicides such as metalaxyl and Al ethyl-phosphyte (or fosetyl-Al) (Farih et al., 1981; Davis, 1982; Timmer & Castle, 1985) Biological control: In the world, many antagonistic microorganisms have been selected, used for the production of probiotics that control the fungus Phytophthora, typical are some species of Streptomyces, Bacillus, Trichoderma, … 1.3.2.2 Research situation on Phytophthora fungus on citrus fruit trees in Vietnam The fungus Phytophthora citrophthora was discovered on orange trees in the Mekong Delta in 1950 (Roger, 1951) In 1970, the fungus was discovered to cause damage on orange trees in the North and Central regions Since then, the disease has quickly spread in all citrus growing areas The results of the basic survey of pests and diseases on citrus by the Plant Protection Institute in the years 2006-2010 have found that Phytophthora was harmful to citrus in the regions of Ha Giang, Tuyen Quang, Phu Tho and Hoa Binh, Nghe An, Ha Tinh belong to species P citrophthora P nicotiana Both of these fungi were found on oranges, Only P citrophthora was detected on pomelo trees, on mandarin and lemon, only P nicotiana was detected (Dang Vu Thi Thanh & nnk, 2010) Gum bleeding disease on citrus trees can be controlled with Ridomil MZ 72WP and Aliette 80WP to sweep the roots, branches and water around the canopy for high efficiency (Nguyen Thi Kim Son, 2003) Additional foliar application of fertilizers in combination with Kasuran 47WP 0.2%, Kocide 51.8DF 0.2% and Caltex DC Tron Plus 0.5% oil is effective in limiting oil spot disease on citrus trees (Nguyen Thi Bich Ngoc, 2009) Bioproducts such as SH-1, Phyto-M and FusaT of the Plant Protection Institute have been used to limit the damage of nematodes and some fungi that damage the root zone of pepper, coffee, durian, mangoes, citrus fruits and vegetables (Le Thu Hien et al 2014; Ha Minh Thanh et al 2013) 1.4 Production of citrus fruit trees in Cao Bang Cao Bang has harsh climatic conditions, cold winters and high average temperature difference between day and night, but it is that harshness, along with natural evolution, that has contributed to the flavor of many citrus varieties of the province such as Trung Vuong orange and Ha Tri mandarin, Tra Linh mandarin , become a spearhead crop of many localities in the province In 2003, Cao Bang Plant Protection Department carried out a survey to assess pests and diseases on citrus in Cao Bang 12 types of diseases have been recorded, in which there are diseases caused by viruses and bacteria, diseases cause by fungi Four important harmful diseases that clearly affect the yield and quality of Trung Vuong oranges and Ha Tri mandarins were greening, tristeza, leaf yellowing, root rot and gum diseases Citrus trees in areas of the province are being severely degraded, the risk of yellow leaf disease is increasing 1.5 General comments and matters of interest Phytophthora is one of the important fungus that damage citrus fruits Fungi species P nicotianae, P citrophthora, P.palmivora were identified as the main causative agents of root rot, gum rot and fruit rot on citrus fruits and have been comprehensively studied for their taxonomic position, biological characteristics and control measures in citrus producing regions of the world However, studies on Phytophthora disease on citrus in Vietnam are fragmentary and often limited to investigating the prevalence of the disease There is no research to develop disease management strategies, nursery management, disease resistance breeding Especially in Cao Bang, there have been no studies on the causes of the disease and methods of managing root rot and gum rot on specialty citrus trees The research on using antagonistic microorganisms to produce probiotics to prevent Phytophthora disease on major crops in our country has had initial results but only tests in the laboratory or on a small garden have not met the requirements of the production The direction of Phytophthora study that damages citrus fruit trees and the use of biological products for disease management is necessary to meet the development needs of the specialty citrus fruit area of Cao Bang CHAPTER II CONTENTS AND METHODS OF THE RESEARCH 2.1 Research content - Investigation of the current status of Phytophthora diseases on citrus trees in Cao Bang Research on biological characteristics, biological rule of Phytophthora spp on citrus trees in Cao Bang - Isolation and selection of antagonistic microorganisms to Phytophthora spp in Cao Bang - Evaluation of the ability to use antagonistic microorganisms to control Phytophthora disease on citrus trees in Cao Bang in integrated crop management 2.2 Location and time of study 2.2.1 Study location - The citrus growing areas of Cao Bang - Nethouse, Laboratory of Plant Disease Department - Plant Protection Institute 2.2.2 Study time - From 2014 - 2020 2.3 Research materials - Phytophthora sources isolated from citrus fruit trees in Cao Bang and sources of antagonistic microorganisms - Microbial culture media, chemicals used in PCR methods, ITS4 and ITS5 primer pairs Some chemical fungicides and probiotics - PCR machine, electrophoresis machine, optical microscope, stereo magnifying glass, pH meter, drying oven, autoclave, other laboratory tools and materials, necessary 2.4 Research Methods 2.4.1 Investigation of the current status of Phytophthora diseases on citrus in Cao Bang Investigate and collect samples of root rot, gum rot and fruit rot in citrus production areas in Hoa An, Tra Linh, Thach An, Nguyen Binh and Phuc Hoa districts According to the research method of Plant Protection, 1997, National Technical Regulation (QCVN 01 - 119: 2012/BNNPTNT) 2.4.3.1 Research on factors affecting the growth and development of antagonistic microorganisms Including studies on the effects of temperature, pH on growth and development of antagonistic microorganisms Determination of physiological and biochemical reactions of antagonistic microorganisms - Media used in physiological and biochemical reactions: + Selective medium for anaerobic determination of sources of bacteria and actinomycetes: Peptone 2g; NaCl 5g; KH2PO4 0.3g; Agar 3g; Bromthymol blue (1%) 3ml; distilled water 1000 ml; pH = 7.0 + Selective medium for testing for nitrate reduction: pepton gravy 1000 ml; KNO3 10 g; pH = 7.0 - 7.6 + Basic mineral medium capable of assimilating carbon sources from Glucose and Saccharose of promising bacteria and actinomycetes: NH4)2SO4 g MgSO4.7H2O 0.2 g; NaH2PO4.H2O 0.5 g; CaCl2.2H2O 0.1 g; K2HPO4 0.5 g; Distilled water 1000 ml The activities of chitanase, -glucanase and cellulase enzymes of bacteria and actinomycetes were quantified by measuring the resolution ring diameter on the synthesis induction medium of each enzyme biology by Nguyen Duc Luong, 2004) 2.4.3.2 Ability of antagonistic microorganisms in greenhouse soil conditions The experiment consisted of 04 treatments, each with plants, repeated times Treatment 1: B amyloliquefaciens Treatment 4: S misionensis (STL2.7) (BHA12.2), Treatment 2: B.methylotrophicus Treatment 5: Distilled water (control) (BNB3.8), Treatment 3: S misionensis (STL2.7) Test for Phytophthora in soil after 15 days, month, months and months with each rose petal trap 2.4.3.3 Ability of Phytophthora inhibition of CB-1 product in the greenhouse Treatment P palmivora (Phyto-1) P nicotianae (Phyto-3) P citrophthora (M2) 2,5 2,5 2,5 5,0 5,0 5,0 10 10 10 Đ/C Đ/C Đ/C Test the density of Phytophthora fungi in the soil after month, months and months with rose petal traps The control effectiveness was calculated according to the Abbott formula 2.4.3.4 Ability of Phytophthora inhibition of CB-1 and other products in field condition The experiment was carried out on 7-8 years old orange trees in Trung Vuong commune, Hoa An district with the biological fungicide Actinovate 1SP (Streptomyces lydicus WYEC 108), probiotics SH-BV of the Institute of Plant Protection, Trico preparations of CTU of Can Tho University and CB-1 preparations 10 Treatment 1: Actinovate 1SP SH-BV Concentration Treatment Concentration 2,5 g/ plant Treatment CB-1 80 g/ plant 80 g/ plant Treatment Bio80 g/ plant VAAS.1 Phyto-M 80 g/ plant Treatment Control (untreated) Tricô ĐHCT 2,5 g/ plant Criterial evaluation: Diseased incidence, diseased index before and 3, months after treatment and effectiveness of disease reduction, fruit yield of the experimental treatments 2.4.3.5 Effectiveness of pesticides against root rot and gum disease The small-scale trial was arranged on a 10-year-old mandarin garden in Quang Han commune, Tra Linh district with 05 recipes, 10 trees each, repeated times Follow: TLB (%) and CSB (%)at 1, months after 2nd treatment and drug efficacy (%) 2.4.4 Study on the possibility of using antagonistic microorganisms in the prevention of root rot and gum disease caused by Phytophthora on citrus in integrated crop management 2.4.4.1 Research on the use of CB-1 product The large-scale trial includes 04 recipes on the 10-year-old Tra Linh mandarin garden, each recipe has 30 trees Each recipe collects points, each point collects 03 acres of soil around the canopy Evaluation of the percentage of petals losing color at 1, and months after treatment 2.4.4.2 Testing of times to use CB-1 product The large-scale trial includes 04 recipes on the 10-year-old Tra Linh mandarin garden, each recipe has 30 trees Each recipe collects points, each point collects 03 acres of soil around the canopy Evaluation of the percentage of petals losing color at 1, and months after treatment 2.4.4.3 Combination of CB-1 product and fertilizers The experiment consisted of treatments, replicates trees each time Monitoring indicators: Disease rate and disease index after 1, and months of fertilizer application Using CB-1 preparations in combination with pruning, creating canopy and cleaning the field 2.4.4.4 Combination of CB-1 product and cultivation methods The large-scale experiment was arranged sequentially on 10-year-old orange orchards in Hoa An district, 30 plants/recipe The trial was started after the fruit was harvested Each recipe collects points, each point collects 03 soil samples around the canopy Evaluation of the percentage of petals losing color at 1, and months after treatment 2.5 Experimental data processing: Statistical data were processed by MS.Exel, 2007 and IRRISTAT VERSION 5.0 11 CHAPTER III RESULTS AND DISCUSSION 3.1 Current status of citrus fruit production and root rot and gum disease on citrus fruit trees in Cao Bang 3.1.1 Current status of citrus fruit production in Cao Bang Area of citrus fruit trees in Cao Bang: Cultivation area by 2014 of districts reached 208 with species of special citrus fruit trees of Cao Bang Tra Linh tangerine area in 2019 reached 164.5 hectares, accounting for 62.5% of the province's citrus area Hoa An is a suitable area for both oranges and tangerines to grow Current status of seedling: In Hoa An, new plants are grown by seeds (50%), by cuttings (23.3%), 10% of households grow grafted trees on pomelo stumps and 16.7% of households grow grafted seedlings on grafted stumps In Tra Linh: In the past, people used to grow by seeds and cuttings, in recent years, many households have propagated themselves by grafting on pomelo root , reaching 53.3% In Phuc Hoa, the percentage of households growing by cuttings was higher (66.7%) than those growing by grafting on pomelo stumps (33.3%) Current status of tree age: The citrus fruit orchards in Cao Bang are mainly in the age group of 5-15 years, in which orchards under years old account for 6.7 - 33.3%, trees from to 10 years old are the most, reaching 33.3 - 66.6% and dominant in Hoa An, Phuc Hoa, Nguyen Binh and Thach An regions There are 40% of orchards from 10 to 15 years old in Tra Linh tangerine area Garden over 15 years old only reached the highest 13.3% in Phuc Hoa pomelo growing area In the areas of Hoa An and Tra Linh, citrus fruit orchards over 15 years old no longer exist or are very few, due to pests and diseases that cause severe damage and people often have to cut down and replant many times Cultivational technique * Fertilizing: 100% of households growing tangerines in Tra Linh have fertilized the trees, in Hoa An is 86.7%, Phuc Hoa, Thach An and Nguyen Binh districts all have a low rate of only 13.3 - 26.7% of households fertilizing citrus trees * Pruning: The highest number of households applying pruning techniques is in Tra Linh (53.3%), Hoa An, Nguyen Binh and Thach An districts have the rate from 26.7 to 33.3%, in Phuc Hoa 100% of households no pruning of trees * Watering: Only 66.7% of households growing tangerines in Tra Linh have additional watering for trees in the dry season * Pest control: The percentage of households using pesticides to control pests and diseases is highest in Tra Linh, 100%, followed by Hoa An 66.7%, Nguyen Binh and Thach An districts have a low rate from 6.7 – 20.0%, in Phuc Hoa 100% of households not use pesticides to prevent pests 12 3.1.1.2 Disease composition on citrus trees in Cao Bang Detected on citrus fruit trees in Cao Bang, there are 13 types of harmful attack organisms Among these harmful organisms, viruses have species, bacteria have species, fungi have species and algae species 3.1.1.3 Symptoms of root rot and gum disease on citrus fruit trees in Cao Bang The disease affects all stages of plant growth from nurseries to production gardens and caused damage on roots, stems, branches, leaves and fruits When the fungus invades and causes disease, the symptoms of diseased plants of orange, mandarin and pomelo are quite similar On the commercial garden due to damaged plant roots, the poor ability to absorb water and nutrients does not provide enough for the growth needs of the plants The tree is stunted, growth is poor and uneven, bark, branches are cracked, partially gummed, foliage is pale or yellow, leaves are falling, dead branches are dead Infected fruits have brown lesions, hard skin at the initial stage, then the lesions are rotten and cracked, wet weather has a white fungal layer on the surface of the disease 3.1.2 Determining the cause of root rot and gum disease 3.1.2.1 Isolation of citrus pathogens caused by Phytophthora From 375 soil and diseased plant samples, 142 Phytophthora samples were isolated Isolation results presented in Table 3.6 showed that Phytophthora was present in all growing areas and caused damage on all citrus species of Cao Bang 59.2% of soil samples collected from diseased orchards were able to isolate Phytophthora from diseased stems, leaves and fruits, 33.6% and from diseased roots, 20.8% The number of Phytophthora mushroom samples isolated from samples collected in Tra Linh was 35 samples, in Hoa An and Phuc Hoa each was 29 samples, in Nguyen Binh was 25 samples and finally in Thach An was 24 samples 3.1.2.2 Identification of Phytophthora species based on morphological characteristics The growth of 142 sources of Phytophthora fungi on PDA medium was divided into different groups Group (1) Fungal blooms develop in the form of cotton, the color is slightly clear white on V8 medium, on PDA medium, opaque white color Group (2) fungal colonies develop fine, white, slightly transparent cotton on V8 medium and on PDA medium, slightly petal-shaped, slightly opaque white Group (3) fungal colonies have a slightly pinkish white color on V8 medium, on PDA media the fungal colonies develop a characteristic white petal shape According to the taxonomic key of Erwin and Ribeiro (1996), group includes two samples Phyt-01 and Phyt-02 with similar morphology to the fungus Phytophthora palmivora., group includes two samples Phyt-03 and Phyt-04 belonging to the fungus Phytophthora nicotianae Group included samples M1 M2, M3, M4, M5 and M6 similar to those described by Erwin and Ribero (1996) and Hamm and Hansen (1987) on Phytophthora citrophthora 13 3.1.2.3 Identification of Phytophthora species by PCR technique Using primer pairs ITS4 and ITS5 to identify samples of Phytophthora ITS4: 5’- TCCTCCGCTTATTGATATGC-3’ ITS5: 5’-GGAAGTAAAAGTCGTAACAAGG-3’ Table GenBank proximity search results of Phytophthora samples, Phyt-01 and Phyt-02, based on ITS sequences (2015) Percentage of Sequence No Defined species comparisons homogeneity GenBank Code (%) (%) P palmivora 100 99.9 MT052675 P palmivora 100 99.9 MK500842 P palmivora 100 99.9 MH401199 P palmivora 100 99.9 MF370566 P palmivora 100 99.9 MG434772 Note: only the closest sequences are shown Sequence analysis of two samples Phyt-01 and Phyt-02 showed that the closest species is P palmivora Table GenBank proximity search results of two Phytophthora samples, Phyt-03 and Phyt-04, based on ITS sequences (2015) Percentage of Sequence No Defined species comparisons GenBank Code homogeneity (%) (%) P nicotianae 99 99.9 KR827692 P nicotianae 99 99.9 KJ865230 P nicotianae 99 99.9 JF792541 P nicotianae 99 99.9 JF792540 P nicotianae 99 99.9 GU111681 Note: only the closest sequences are shown Sequence analysis of two samples Phyt-03 and Phyt-04 showed that the closest species is P nicotianae Table 10 GenBank proximity search results of Phytophthora samples (M1 – M6) based on ITS sequences (2015) Sequence Percentage of No Defined species homogeneity GenBank Code comparisons (%) (%) P citrophthora 100 100 GU111603 P citrophthora 100 100 GU111602 14 P citrophthora 100 100 GU111601 P citrophthora 100 100 GU111600 P citrophthora 100 99.6 GU133066 Note: only the closest sequences are shown Sequence analysis of samples showed that the closest species was P citrophthora 3.1.2.4 Results of artificial infection Artificial inoculations were conducted on physiologically ripen Trung Vuong oranges by applying a cloth cloth dipped in x 105 spores/ml of the with 03 species of fungi P palmivora, P nicotianae and P citrophthora to the surface of the orange, keep moist and place in the dark at a temperature of 26-28oC All fungi P palmivora, P nicotianae and P citrophthora infect fruit and young orange trees 100% of infected fruit samples were isolated Phytophthora fungus, the fungus has the same way of growing as the fungus of the sources used to spread the disease, the latent time of the disease is from 27 to 30 days after infection The results of artificial infection on fruit, stem and re-isolation showed that the fungal sources of P palmivora, P nicotianae and P citrophthora isolated from root rot and gum disease on citrus fruit trees in Cao Bang were the causative agent of gum rot on citrus fruit trees in Cao Bang 3.2 Study on the biological and culturing characteristics of Phytophthora 3.2.1 Study on the biological characteristics of the Phytophthora Effect of temperature on the ability of spore formation of 03 Phytophthora species The P palmivora Phyt-01, good conditions for the fungus to produce spore encapsulation in conditions 250C, the fungus P nicotianae Phyt-03 at temperature 300C is the most suitable, the fungus P citrophthora M2 suitable in temperature range from 15-250C Effect of culture medium on the growth of Phytophthora All three species of P palmivora, P nicotianae and P citrophthora were grown on types of media All species grow worst on PSA medium, best on V8A medium PDA, CRA and V8A media are suitable for all three species of fungi to grow Effect of pH on the growth of Phytophthora All three species of Phytophthora can grow and develop in a wide pH range from slightly acidic to slightly alkaline From pH 4.5 - 8.0 but pH level from to is suitable for the growth and development of all species 3.2.2 Rules of growth and damage of the Phytophthora spp on citrus trees in Cao Bang 3.2.2.1 Evolution of root rot and gum disease on citrus fruit trees in Cao Bang The root rot and gum disease appears, arises and damages citrus throughout the year In the dry season period (from November to April next year), the disease occurs but with a lower rate than in the rainy season period (from July to October) Orange and tangerine trees are more sensitive than grapefruit trees 15 Diagram of the development of root rot and gum disease on citrus trees in Cao Bang in 2015 and 2016 3.2.2.2 Effect of ecological factors on the development of root rot and gum disease The influence of topography on the prevalence of root rot and gum disease All three types of oranges, tangerines and pomelos had the highest disease rates in gardens located near the foot of rocky mountains (34.6 - 50.3%), followed by the areas planted in the foothills, along streams, and fields (32.1 - 41.3%) The disease rate is lowest in the area planted at the top of the hill (14.3 – 21.3%) Effect of varieties on the arising and development of root rot and gum disease Investigate the prevalence of root rot and gum disease of tangerine varieties Tra Linh, Ha Tri, Hoa Tham and Trong Con All varieties of mandarin oranges suffer from gum rot disease Tra Linh mandarin variety was infected with the highest number of infected plants with 42.5-45.1% of the investigated trees infected with severe disease, the disease index reached over 20% Hoa Tham mandarin variety was the lowest, the rate of diseased trees was from 10.4 to 12.5% and the disease index was from 6.3 to 6.8% (Table 3.21) Effect of tree age on the development of root rot and gum disease In Cao Bang, the rate of mandarin trees suffered from root rot disease was highest after 10 years (42.3% of trees were infected) and the lightest was pomelo trees with 24.1% of trees were infected 3.3 Isolation and selection of antagonistic microorganisms to Phytophthora from citrus fruit plantations in Cao Bang 3.3.1 Isolation and selection of antagonistic microorganisms From soil samples collected in Cao Bang, 112 microorganisms were isolated Of which, there are sources with strong resistance to Phytophthora, 31 sources with medium resistance and 30 sources with weak resistance 16 Selecting samples of microorganisms with high inhibitory ability against Phytophthora samples collected in different regions Sample STL2.7 has a high ability to inhibit the diameter of aseptic ring formed with samples of Phytophthora fungi, which are 28.4, 27.8 and 26.5 mm, respectively and sample BNB3.8 with aseptic ring diameters of 27.8, 28.6, and 27.2mm The average inhibitory effect with Phytophthora samples of samples BNB3.8, STL2.7 and BHA12.2 was 85.7, respectively; 84.8 and 82.2% 3.3.2 Identification of antagonistic microorganisms and biosafety Identification of antagonistic microorganisms according to biochemical characteristics The results of studying colony morphology, cells, Gram stain The ability to use oxygen and carbon sources has identified samples of VSV BHA12.2 and BNB3.8 belonging to genus Bacillus and sample STL2.7 belonging to genus Streptomyces Identification of antagonistic microorganisms by PCR method Primer pairs Phy1F and Phy1R were Used to identify samples BHA12.2, BNB 3.8 and STL2.7 The gene sequences of 03 samples BHA12.2, STL2.7, BNB3.8 were compared with those on the international gene bank The gene sequence of sample BHA12.2 is 99.62% similar to that of Bacillus amyloliquefaciens registered with Genbank code FN597644, sample BNB3.8 has 99.72 homology to Bacillus methylotrophicus species registered with Genbank number AB184285 and sample STL2.7 is 100% similar to Streptomyces misionensis species registered with Genbank code AB184285 Table 30 Results of identification of strains of antagonistic microorganisms to Phytophthora causing diseases on citrus fruits (2017) Microbial Sequence Product Percent GenBank Species samples code size (bp) identity Code BHA12.2 BNB3.8 STL2.7 13B5ZAA012 13B5ZAA013 13B5ZAA004 13B5ZAA005 13B5ZAA024 13B5ZAA025 1321 99,62 FN597644 1436 99,72 AB184285 1421 100 AB184285 Bacillus amyloliquefaciens Bacillus methylotrophicus Streptomyces misionensis Determination of biosafety levels of antagonistic microorganisms Contrast the names of antagonistic microorganisms with the US and European biosafety hierarchies All three species of these antagonistic microorganisms are at biosafety level No according to the biosafety level of the US and Europe, No danger to human health, animals and the ecological environment Thus, all three species of these antagonistic microorganisms can be used under normal production conditions to control the harmful Phytophthora fungus on citrus in Cao Bang 17 ... industry development in Cao Bang Object and scope of research - Phytophthora spp fungi caused disease on citrus trees in Cao Bang - Microorganisms are antagonistic to Phytophthora spp - Varieties of... of the current status of Phytophthora diseases on citrus trees in Cao Bang Research on biological characteristics, biological rule of Phytophthora spp on citrus trees in Cao Bang - Isolation and... antagonistic microorganisms to Phytophthora spp in Cao Bang - Evaluation of the ability to use antagonistic microorganisms to control Phytophthora disease on citrus trees in Cao Bang in integrated crop