Nghiên cứu nấm colletotrichum spp gây bệnh thán thư trên cây cà phê chè và biện pháp quản lý bệnh tại tỉnh sơn la tt tiếng anh

MI NISTRY OF EDUCATION AND TRAI NI NG MI NISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES HOANG VAN THANH RESEARCHING ON COLLETOTRICHUM SPP CAUSING ANTHRACNOSE ON COFFEA ARABICA AND DISEASE MANAGEMENT MEASURES IN SON LA PROVINCE Major: Plant protection Code: 9.62.01.12 SUMMARY OF AGRICULTURAL DOCTORAL THESIS Ha Noi – 2020 The research work is published at: VIETNAM ACADEMY OF AGRICULTURAL SCIENCES Supervisors: 1) Dr Trinh Xuan Hoat 2) Prof Nguyen Van Tuat Reviewer 1: Reviewer 2: Reviewer 3: The thesis is defended by the Academy Assessment Council at: Vietnam Academy of Agricultural Sciences Date ……… month……… year ……… It is possible to learn more about the thesis at: National Library of Vietnam Library of Vietnam Academy of Agricultural Sciences Library of Plant Protection Research Institute INTRODUCTION The urgency of the thesis Coffee (Coffea) plants play an important role in the production and trading of agricultural products on domestic and foreign markets In the world today, there are 80 countries planting coffee with a total area of over 10 million hectares and an export value of over 10 billion USD In Vietnam, coffee trees are grown mainly in the hilly areas of the North and the Central Highlands In the crop year 2016/2017, the whole country has a total area of 592,000 of Robusta coffee, the production reached 1,536,000 tons of coffee bean Coffea arabica is grown mainly in Lam Dong, Son La, Dien Bien and Quang Tri with an area of about 46,000 and modest production In recent years, Vietnam has exported stable coffee beans about 24-25 million bags /year In Son La, the coffee growing area in Son La has tended to increase sharply in recent years and along with the increase in the area and production, coffee trees pests have been growing, causing significant damage in main planting coffee regions, causing reduces yield and quality of coffee According to the Son La Crops and Plant Protection Department, pests and diseases on coffee trees in this region are common species: Colletotrichum sp.; Cercospora coffeicola Berk & Cooke; Hemileia vastatrix B & Br; Pseudomonas syringae, Pseudococcus mercaptor; Planococcus citri; Xylotrechus quadripes; Hypothenemus hampei In particular, the object of serious damage on coffee trees in Son La is anthracnose caused by some species of the fungus Colletotrichum In order to contribute to the effective management of pests on coffee trees, implementing the project "Researching on Colletotrichum spp causing anthracnose on Coffea arabica trees and disease management measures in Son La province ” is urgently theoretical and practical Aims of the thesis Identify the composition of species Colletotrichum sp causing anthracnose on cofea arabica trees; study the characteristics of Colletotrichum and disease control measures to contribute to improving productivity, quality, increasing economic efficiency, protecting the environment in coffee growing areas in Son La The new findings and contributions of the thesis - Identified 05 species of Colletotrichum fungus causing anthracnose on coffee trees, some biological characteristics of fungi - Evaluate the percentage of coffee cherries lost due to anthracnose (Colletotrichum sp.) accounting for 42.63-52.83% of the total number of berries in Son La - Research to identify technical methods of pruning to create canopy, balanced fertilizer, weeding in combination with collecting and destroying diseased branches with high efficiency in order to eliminate coffee anthracnose - Anvil 5SC (hexaconazole active ingredient), Antracol 70WP (proneb active ingredient), and CFO (curcumin active ingredient extracted from yellow turmeric) were evaluated to have a effectiveness of 72.53-79.14% anthracnose control (Colletotrichum sp.) on tea plants Significance of the thesis 4.1.Science significance - Research results on the composition of 05 species of Colletotrichum fungus causing anthracnose on coffee trees as a basis for disease management; basis for research to select and use suitable coffee varieties with high tolerance to anthracnose - The research data of the topic is the reference for the follow-up studies related to harmful anthracnose on coffee plants in other production areas 4.2 Practical significance - The results of the research on identification of species of Colletotrichum fungus causing anthracnose on coffee trees, the degree of harm and some measures to prevent anthracnose in Son La region have contributed to the help of specialized agencies in making recommendations, guide sustainable and efficient coffee cultivation; It emphasizes the main methods such as pruning, fertilization, weeding, weed management and diseased crop residues to control coffee berry disease - Coffee berry disease management model initially affirms the results of research on pathogens, some ecological biological characteristics and disease control measures and serves as a basis to build a complete process of coffee berry disease control in Son La region in particular and the Northwest area in general Objectives and research area 5.1 Objectives The object of the research was the fungus causing harmful anthracnose on coffee trees (Coffea arabica) grown in Son La 5.2 Research area - Identification of harmful Collectotrichum fungi on coffee trees in Son La Experimental study of plant protection fungicides against harmful anthracnose on coffee trees in Son La Surveying and evaluating the level of harmful anthracnose on coffee trees in Son La province - Research period: from 2015-2019 CHAPTER OVERVIEW 1.1 General introduction about coffee trees Coffee is an industrial tree with high economic value and an important commercial product in the international market In the world today, coffee is grown in over 80 countries in Central and South America, Africa and Asia Annual value of goods from coffee reaches about 70 billion US dollars In the 20152016 inter-season, coffee production of 53 countries producing and exporting coffee reached 143,306 thousand bags (60 kg / bag); of which, Vietnam reached 28,737 thousand bags (Man, 2013; FAO, 2016) 1.1.1 Some key features in the classification of coffee plants Coffee of the Class: Dicotyledoneae; Sub-class: Sympetalae or Metachlamydeae; Department: Rubiales; Family: Rubiaceae; genus: Coffea Chevalier (1947) grouped Coffea species into four main groups: Agrocoffea, Paracoffea, Mascarocoffea and Eucoffea The Eucoffea group is divided into subgroups based on a number of very diverse criteria such as tree height (Nanocoffea), leaf thickness (Pachycoffea), fruit color (Erythrocoffea) and geographical distribution area (Mozambicoffea) ( Chevalier, 1947) 1.2 Situation of coffee production and consumption in the world and in Vietnam 1.2.1 Situation of coffee production and consumption in the world Over the past 50 years, both coffee production and consumption have increased significantly Consumers have gained some profit through the variety of coffee products, improved quality and reduced prices Currently, more than 50% of production comes from the three countries Brazil, Vietnam and Colombia From the 2013/14 to 2017/18 inter-season, the total output of coffee exporting countries was quite stable, ranging from 148,559-159,047 bags, total export turnover from 105,492-114,596 bags 1.2.2 Situation of coffee production and export in Vietnam and Son La The country's coffee area is most concentrated in the Central Highlands, in provinces like Dak Lak, Dak Nong, Gia Lai, Kon Tum and Lam Dong accounting for 72% of the total area and 92% of the total coffee output of the whole country, and the varieties are mostly Robusta coffee According to the statistics of FAO (2018), Vietnam's total green coffee output from the 2013/14 to 2016/17 crop tended to decrease from 27,610 bags to 25,540 bags In the 2017/18 crop, the output increased to 29,500 bags In Vietnam, arabica coffee production accounts for about 4% of total coffee production and about 6% of the national coffee area In Son La and Dien Bien provinces, the area of arabica coffee has increased in recent years from 16,000 to over 18,000 hectares (USDA, 2018), Catimor arabica coffee varieties are grown mainly 1.3 Situation of research on coffee berry disease in the world and Vietnam 1.3.1 Harm of coffee berry disease For coffee, anthracnose caused by fungus Colletotrichum sp causing is the second most important disease after rust disease The disease causes fruit to dry, branches, leave, flower blight and flower death The disease is a major cause of reduced productivity in African coffee In Vietnam, according to the Notice of Plant Protection Department in January 2017, the country had 14,195 hectares of coffee trees infected with anthracnose, 240 hectares of heavily infected areas, and 867 hectares of control in Dien Bien Son La, Quang Tri, Gia Lai, Dak Nong, Binh Phuoc Dong Lai, and Lam Dong (Plant Protection Department, 2017) 1.3.2 Symptoms of the disease Colletotrichum pathogenic fungi on coffee plants often produce symptoms such as brown or dark brown marks on flowers and fruits; creates irregularly shaped dead spots on the leaf margins and if infected is severe, the leaves will fall off; create black stains on branches and if seriously infected make branches dry (Waller et al., 2007) On fruits, symptoms are necrotic spots with brown contours; causing rot, failure of fruits, fast-falling fruits when infected In the fruiting season, fungi survive on plant parts such as leaves and branches and are non-invasive and cause harm (Graaff, 1992) 1.3.3 Research on the causes of coffee berry disease 1.3.3.1 Classification location of Colletotrichum Colletotrichum (sexual phase called Glomerella) belongs to Ascomycota, Sordariomycetes, Glomerellales and Glomerellaceae families are important pests of many crops in general and coffee in particular in the world and Vietnam at pre- and post-harvest stages (Corda, 1831) 1.3.3.2 Species of the fungus Colletotrichum causing coffee berry disease Among the species of the fungus Colletotrichum, only a few are harmful in coffee (Stephen, 1991) Waller (1993) described the pathogen on coffee cherries in Africa and named it C kahawae Species such as C gloeosporioides, C acutatum and C coffeanum cause anthracnose on coffee plants in Asia and South America (Chen et al., 2003; Prihastuti, 2009a) In Vietnam, two species of C coffeanum and C capsici are thought to be the main causes of anthracnose on coffee plants (Tran et al., 1998) Nguyen Thi Hang Phuong (2010) recorded C gloeosporioides, C acutatum, C capsici C boninense on coffee trees 1.3.3.3 Identify the causative agents of coffee berry disease based on the morphological characteristics of the fungus According to Prihastuti's (2009) study of 34 Colletotrichum fungus samples collected from some coffee growing locations in Northern Thailand and C acutatum, C gloeosporioides and C kahawae Morphological differences of spores, fungal characteristics and growth among isolates allow them to be classified into different groups In Vietnam, 46 isolate has been isolated from different ecological regions On artificial media, the hyphae of the isolate are dark gray to light gray, some isolates produce many sclerotia, some other isolates form the sexually active spore All isolate is divided into groups based on spore shape (Phuong, 2010) 2.3.3.4 Methods to identify anthracnose agents using molecular techniques Molecular techniques have been successfully applied to distinguish between species and genetic characteristics of Colletotrichum obtained from host plants Based on the sequence comparison of genomic regions, the method is being widely used to detect and classify species of Colletotrichum (Cannon et al., 2008; Hyde et al., 2009; Than, 2006) A combination of morphological characteristics, specific primers and sequence analysis of mtSSU and ITS areas have recorded C gloeosporioides, C acutatum, C capsici and C boninense together causing anthracnose on coffee trees in Vietnam (Phuong, 2010) 1.3.3.5 Assess the toxicity of species belonging to Colletotrichum The pathogenicity test of Colletotrichum fungi isolated on coffee trees in Northern Thailand shows that both wound and non-wound pathogens using green and ripe berries have infected results disease (Prihastuti, 2009a) Most Colletotrichum isolates of Vietnam are of medium toxicity, mainly causing lesions with smaller severity and lesions than anthracnose fungi (C kahawae) 1.3.3.6 Conditions arising and causing harm of the disease Occuring of anthracnose on coffee plants vary among regions and seasons depending on the susceptibility of the coffee plant, disease inoculum, weather conditions Humidity near saturation and temperature in the range of 20-22o C is a favorable condition for germination of spores and pressure plate (Gibbs, 1969; Masaba and Waller, 1992) If the protein content of the leaves is 4% or more, the coffee tree is not infected by Colletotrichum sp 1.3.3.7 Solutions to control coffee berry disease On coffee trees in the western region of Cameroon, a major coffee area has been affected by anthracnose, farmers often take measures to prune and remove dead berries from the branches before the coffee trees has flowers, in dry season Besides this measure, a number of other measures are applied such as intercropping of fruit trees (mango, guava, banana, ) and intercropping food crops (beans, potatoes, corn, ) with coffee plants So far, many varieties of coffee resistant to anthracnose such as: Geisha, Geisha 10 generations of Hibrribon Timor, "K7", "Blue Mountain" and "Rume Sudan" and some not completely resistant varieties such as: Bourbon, Selections and Sl In Kenya, to control anthracnose, people spray fungicides once a month during the rainy season CHAPTER MATERIALS, CONTENTS AND METHODS 2.1 Materials - Colletotrichum fungi collected and isolated on coffee berries of Coffea arabica, Catimor varieties - Chemical fungicides, bio-fungicides: active ingredient propineb (Antracol 70WP); active ingredient hexaconazole (Anvil 5SC); ningnanmycin (Supercin 20SC); curcumin, extracted from Curcuma longa (CFO); Ethyl 2,4,6trihydroxybenzoate, kaempferol, epiafzelechin, kaempferol-3O-glucoside, kaempferol-3-O- gentiobioside Derived from Cassia alata (MBG), O-coumaric extracted from Eupatorium fortune (MANTU) 2.2 Contents Content Studying species composition of Colletotrichum fungus causing anthracnose on arabica coffee trees Content Studying the severity of anthracnose (Colletotrichum spp.) on arabica coffee trees and its influence factors Content Research on some of chemical fungicides/ biofungicides to against coffee anthracnose disease Content Carry out 02 demonstrations for anthracnose integrated management in Son La 2.3 Research area The research is carried out in major coffee growing areas of Son La province and in laboratories - Northwestern University, Laboratory - Plant Protection Research Institute 2.4 Methodology 2.4.1 Surveying the situation of coffee production in Son La 2.4.1.1 Current situation of coffee production in Son La - Collecting primary data through collecting documents - Survey by interviews and questionnaires - Evaluation criteria: general situation of coffee production, influencing factors, farming techniques, pests and diseases, productivity/production of coffe, market information, etc 2.4.1.2 Situation of pests on coffee trees in Son La Each area selected representative fields, with 0.5-1 Random survey, each field survey at least 20 points according to the chessboard, periodically surveying 14 days/time and diagnosis pests 2.4.2 Identify species of Colletotrichum fungus causing anthracnose on arabica coffee trees in Son La 2.4.2.1 Collecting and isolating fungi causing anthracnose disease on arabica coffee trees in Son La Survey, collect anthracnose samples in main coffee growing areas of Son La including Thuan Chau district, Mai Son district and Son La city Survey gardens (>1 ha/garden)/district, collecting 50 diseased coffee berries/garden Collecting time is from June to August 2016; describe symptoms and set symbols for each sample collected 2.4.2.2 Assess the pathogenicity of Colletotrichum fungus * Pathogenic characteristics of the Colletotrichum fungus on arabica coffee tree stems at seedling stage Stem infection is done by follow Wokocha (2010) method Cartimor coffee variety seedlings are grown in plastic bags, nursed in net houses, ensuring disease free, conducting artificial infection when the plants reach 6-7 leaves/tree Experiment was arranged repilcations , 10 plants / isolate samples/replication After infection, record and assess the incubation period After the first 20, 25, 30, and 35 days of transmission, carry out a 5level inspection to assess the extent of damage (Waller, 1998) * Pathogenic characteristics of the Colletotrichum fungus on coffee berries The experiment was arranged replicates, 10 green fruits or 10 ripe fruits /fungus samples/replications The experiment was conducted on berries with wounded and non-wounds, conducted a small µl solution containing fungal spores (106 spores / ml), and a small µl sterile distilled water on the control berries Infected berries are placed in sterilized plastic containers at laboratory temperature of 28 ± 2o C Classification of diseased berries according to the 9th scale according to Vietnam Standard No 01-38: 2010 / BNNPTNT (MARD, 2010); assess the incidence (%) of infected berries, symtom and disease severity (%) within 1-20 days after infection (Than, 2008) 2.4.2.3 Identify species of Colletotrichum fungus that causes anthracnose on coffee trees initial number of berries (Btot1) This represents the total diseased berries observed throughout the year of observations - The % losses not due to CBD (Pfall) which was expressed by the difference between the total losses and losses due to anthracnose (Ptot–Pdis) 2.4.3.3 The influence of cultivation techniques on the percentage of coffee cherries dropped by coffee berry disease The experiment was divided into large plots: The experimental plot applied advanced techniques to prevent anthracnose; Control plots use traditional cultivation methods of farmers (apply methods such as not pruning branches, removing diseased fruits, weeding by hand, applying chemical fertilizers or manure according to experience) Each experimental plot consists of 100 coffee trees in the period of high yield (9 years old), 200 trees under shade under plum trees (9 years old) planting density of × 10 m, the remaining 200 trees not have shade trees; Observing the rate of fruit loss by the method of Bedimo et al., (2007) in Section 2.4.3.2 2.4.4 Surveying the effectiveness of some pesticides to prevent anthracnose fungus on arabica coffee trees 2.4.4.1 Study the effect of some fungicides against Colletotrichum spp on the artificial medium After isolating and identifying 05 fungal species: C gloeosporioides (CBMS5), C siamence (CBMS13), C fragariae (CBMS16), C theobromicola (MNTC11), C acutatum (MNTC14) cause disease anthracnose, conducting drug trials for each species The experiment consisted of one factor with replicates and petri dish /replicate for each isolate representative of each species After cooking, the medium is mixed into the medium according to the recommended ratio, then pour into 80 mm diameter Petri dishes Fungicides used: Antracol 70WP, Anvil 5SC, Supercin 20SC, CFO, MBG, MANTU, Control (no using fungicide) Monitoring criteria: measuring the diameter of fungal colonies, calculating the effectiveness of the drug according to Abbott formula, after 3, days of drug treatment In which: C is the diameter of fungal colonies in the control (mm); T is the diameter of fungal colonies in the experiments (mm) 11 2.4.4.2 Surveying the effectiveness of fungicides to control anthracnose on arabica coffee trees in the fields The experiment was arranged with one factor, replications in full random complete block (RCB) style Each fungicide is 15 plants/time, The plots are separated by a coffee tree row Spray times days apart Experiment to evaluate the effect of disease elimination on berries, arranged on 6-year-old coffee fields, like Cartimor coffee Fungicides used: Antracol 70WP, Anvil 5SC, CFO, Control (spraying water) The formula for calculating the effectiveness of the fungicides according to Henderson - Tilton: Ta  Cb  Effectiveness (%) =  1    100  Tb  Ca  In which: Ta is the disease severity in the fungicidal treatment after experiment; Tb is the disease severity in the fungicidal treatment before experiment; Ca is the disease severity in the control after experiment; Cb is the disease severity in the control before experiment 2.4.4.3 Evaluate the effect of using bio-fungicides on anthracnose on arabica coffee Tested on year old coffee tree Testing conducted using CFO preparations, control water spray Time to spray CFO when flowers last bloom weeks, 15 days / time The trial was arranged in the form of large plots, 0.25 / plot, the total area of the test field was 0.5 (including protection strip); monitor the rate of diseased fruits after spraying for the first time one week, then investigate once a month 2.4.5 Building integrated management demostration of anthracnose disease on arabica in Son La Two models were implemented in Chieng Doi and Chieng Den commune - Son La City The model was divided into two plots (0.25 ha), experimental plots applied advanced techniques to prevent anthracnose arising, control plots applied according to traditional farming techniques of farmer The model was conducted for consecutive years The plots applies the techniques according to the Technical Process (10 B.C 5272002) Control plots follow farmers' method; investigating the harmful occurrence of anthracnose on coffee berries; evaluate model economic efficiency 2.5 Data analytical methods The data was analyzed statistically using software MINITAB 16, Excel, by GLM model according to Tuckey 12 standard at significance level of 0.05 The % data such as disease index, germination rate of spores, fungicides efficacy were transferred to arcsin before statistical analysis CHAPTER RESULTS AND DISCUSSION 3.1 Current status of coffee production in Son La and situation of insect pests and diseases The coffee area of Son La by March 2018 amounted to 17,600 hectares, of which the coffee area for products was 14,781 hectares; The production of green coffee berries reaches 22,766.1 tons Coffee planting area is mainly concentrated in Son La city, Mai Son district, Thuan Chau district accounting for over 90% of the total coffee growing area of the province and Yen Chau and Sop Cop district 3.2 The identification of pathogenic Colletotrichum fungus in arabica coffee 3.2.1 Symptoms of anthracnose disease on coffee trees The disease appears and damages on all stages of plant growth, and is particularly damaging from coffee tree flowers until the fruit ripens: The disease on branches often appear near the branche nodes, creating a dark brown slightly sunken, leaves on diseased branches are yellow and gradually fade The lesions on the leaves are often round, brown to dark brown, the diseased tissue is concave, there are outer halos, the concentric surface has concentric circles, the diameter of 1.0-2.5 cm The lesions on fruits usually start from the position of the fruit part attached to the branch or at the point of contact between two fruits; The initial lesions are very clear with small spots on the skin of the fruit, which makes the shell indented and become dark; lesions spread gradually on the skin of the fruit and penetrate deep into the tree to dry black die fruits on branches or fall off 3.2.2 Some morphological characteristics of Colletotrichum fungus cause coffee berry disease Based on morphological characteristics, colony color and spores, Colletochitrum isolates were divided into groups Group I: the mycelium is white to brownish white, the hyphae are superficial and white; often appear greenish yellow spores, tiny, scattered on the surface of the culture medium; cylindrical spore-shaped spores, usually with a tapered tip and a sharp tip, with visible droplets of fluid; after days of culture, the diameter of the fungal colonies reached 68.00-82.33 mm, the spore size was 13.93ì4.69-18.47ì5.01 àm Group II: fungal colonies 13 with gray to grayish brown color, multiple mycelium on surface and grayish white; after days of cultivation, the diameter of the fungal colonies reached 68.67-81.67 mm; long cylindrical sporeshaped spores, usually with two heads, some with a head and a sharp tip, size 15.86ì4.68-18.90ì5.00 àm Group III: Mycelium is gray to dark gray, mycelia are small and brownish white; after days of cultivation, the diameter of the fungal colonies was 75.6781.33 mm; cylindrical spores with one tip, one pointed tip, some in the middle slightly bunched, size 14.69ì4.51-15.57ì4.73 àm Group IV: fungal colonies with pink-colored mushrooms; Mycelium surface less, short, pink white; after days of cultivation, the diameter of the fungal colonies reached 66.6777.53 mm; cylindrical spores, usually with two pointed tips, size 17.22 × 4.92-18.26 × 4.78 µm Group V: fungal colonies were gray to dark gray colonies, mycelium grows in concentric circular areas, short-surface mycelium and dark brown; after days of cultivation, the diameter of the fungal colonies was 79.33-82.00 mm; elongated conidia, usually with a prison head, a sharp tip, some with two heads, size 15.40 × 4.96-17.77 × 5.01 1m 3.2.3 Artificial infection of anthracnose disease on coffee 3.2.3.1 Result of artificial infection of anthracnose disease on arabica coffee seedling stage Under experimental conditions, the latent period of coffee anthracnose was from 15.70 to 23.23 days on stems of seedling with 6-7 leaves and can be divided into groups: i) isolate MNTC18 and MNTC 14 have a long latency period of 23 days; ii) isolate CBMS1, CBMS2, CBMS3, CBMS9, CBMS12, MNTC5, MNTC9, MNTC13 and MNTC15 have a short latency period of 15-16 days; and iii) the remaining isolates had an average latency of 17-19 days The latent period depends on many factors including the characteristics of fungi, different samples can be different species of fungi 3.2.3.2 Results of artificial infection on coffee berries Infectious results of Colletotrichum fungal isolated on coffee berries like Catimor 85 days old (green berries), 145 days old (ripe berries) showed that symptoms on the infected fruit had characteristics similar to those of harmful fruits on coffee berries in the field The berries in the control experiments did not show symptoms In the absence of a wound, the latency of the disease is longer than the case of wound formation from 3-5 days On green coffee berries with wound formation, the latent time was different among the isolates, the latent time of MNTC11 was the shortest, the longest MNTC14 was 8.13 days and 13.30 days, respectively 14 On green coffee berries with no wounds, the latent time of CBMS12 was shortest, the longest MNTC14 reached 13.30 days and 16.30 days, respectively On ripe berries with wound formation, the longest latent time of CBMS13 is 8.03 days, the longest MNTC8 reached 10.83 days On ripe cherries without wound, the shortest latent time of CBMS12 is 12.07 days, the longest CBMS15 and CBMS7 are 15.53 days The rate of infected fruits increases over time after the days of infection Ripe fruits have a higher rate of diseases than green ones, and fruits have a higher wound rate than no wound is produced 3.2.4 Results of the sequencing of fungal isolates by PCR method AJ301912.1 C fragariae KC411911.1 FJ172290.1 C fragariae C fragariae HM015855.1 C gloeosporioides AF451905.1 C gloeosporioides KR445682.1 C theobromicola KM505032.1 C theobromicola JX010285.1 KF877317 C theobromicola C theobromicola KT122929.1 C theobromicola KU498269.1 C musae JQ818203.1 C musae AJ301904.1 C musae AJ301945.1 KU498281.1 KP703373.1 100 C truncatum C siamense C siamense KR445677.1 C siamense KP703372.1 C siamense KC790975.1 C siamense CBMS2 CBMS13 MNTC18 MNTC7 CBMS12 87 MNTC10 CBMS5 MNTC11 0.10 Figure 3.20 Genealogy analysis of isolate CBMS 2, CBMS 5, CBMS 12, CBMS 13, MNTC7, MNTC10, MNTC11, MNTC18 belong to Group I on arabica coffee tree (Son La, 2017) CBMS14 KX227587.1C gloeosporioides CBMS16 CBMS9 AJ301988.1 C gloeosporioides KT122929.1 C theobromicola KJ883592.1 C theobromicola JX014401.1 Colletotrichum sp JX010285.1 C theobromicola KM505032.1 C theobromicola KP703373.1 C siamense KR445682.1 C theobromicola KF877317.1 C theobromicola KP703372.1 C siamense KJ131588.1 C siamense KR445677.1 C siamense KC790975.1 C siamense AJ301904.1 C musae 82 KU498269.1 C musae KC411911.1 C fragariae JX014401.1 Colletotrichum sp KC411911.1 C fragariae AJ301912.1 C fragariae AJ301912.1 C fragariae 78 KX227587.1 C gloeosporioides FJ172290.1 C fragariae AJ301977.1 C gloeosporioides KU498263.1 C asianum CBMS1 KX364718.1 C tropicale CBMS3 KX710116.1 Colletotrichum sp 84 KU881799.1 C acutatum KC845288.1 C fructicola AJ301904.1 C musae AJ301945.1 C truncatum 89 KU498269.1 C musae 0.0050 AJ301945.1 C truncatum 0.0050 Figure 3.21 Genealogy analysis of isolate Figure 3.22 Genealogy analysis of CBMS 9, CBMS 14, CBMS 16 belong to isolate CBMS 1, CBMS belong to Group II on arabica coffee tree (Son La, Group III on arabica coffee tree (S on 2017) La, 2017) 15 KU097210.1 C gloeosporioides KU097210.1 C.gloeosporioides AJ301988.1 C gloeosporioides CBMS15 84 KF877317.1 C theobromicola KJ883592.1 C theobromicola HM015855.1 C gloeosporioides AF451905.1 C gloeosporioides JX010285.1 C theobromicola MNTC15 KM505031.1 C theobromicola AJ301988.1 C gloeosporioides KM505032.1 C.theobromicola 87 KP703373.1 C siamense KR445677.1 C siamense KR445682.1 C theobromicola KC411911.1 C fragariae 97 FJ172290.1 C fragariae KU498281.1 C siamense 90 90 KU881799.1 C acutatum MNTC14 KJ131588.1 C siamense KU881799.1 C acutatum KC845288.1 C fructicola KP703373.1 C siamense JX014401.1 Colletotrichum sp KR445677.1 C siamense KX364718.1 C tropicale KP703372.1 C.siamense KX710116.1 Colletotrichum sp KX710116.1 Colletotrichum sp KU498263.1 C asianum KX364718.1 C tropicale AJ301904.1 C musae AJ301904.1 C musae 85 KU498269.1 C musae KU498269.1 C musae AJ301945.1 C truncatum AJ301945.1 C truncatum 0.0050 0.0050 Figure 3.23 Genealogy analysis of isolate MNTC14 belong to Group IV on arabica coffee tree (S on La, 2017) Figure 3.24 Genealogy analysis of isolate CBMS 15, MNTC15 belong to Group V on arabica coffee tree (S on La, 2017) Based on the morphological morphology and spores, the samples were divided into groups, each group took 50% of the isolates (16/32 samples) to conduct the gene sequencing Among 16 isolates, there are 08 isolates of C siamense accounting for 50%, 03 isolates of C theobromicola species accounting for 18.75%, 02 isolates of C gloeosporiodes species accounted for 12.50%, 02 isolates of C fragariae accounted for 12.50% and one isolates of C acutatum accounted for 6.25% 3.2.5 Some biological characteristics of Colletotrichum spp damaging coffee trees in Son La 3.2.5.1 Temperature effect on the growth of Colletotrichum spp on the artificial environment Five representative isolates of five species were tested to evaluate the growth on the PGA medium at different temperature conditions Table 3.26 Influence of temperature conditions on the growth of Colletotrichum spp on the PGA environment (Son La, 2016) o Diameter of the fungal colonies after days of culture (mm) C 15 20 25 28 30 35 CV (%) LSD0.05 C gloeosporioides C siamense C fragariae C theobromicola C acutatum ( CBMS15) e ( MNTC10) d ( CBMS3) d ( CBMS9) e ( MNTC 14) e 2.66 16.66d 54.67b 80.01a 77.00a 25.97c 5.29 4.03 8.33 25.69c 51.31b 78.68a 79.03a 31.99c 10.32 8.42 15.33 38.64c 53.97b 72.31a 57.47b 23.03d 10.80 8.35 16 2.66 30.31c 56.35b 76.02a 73.64a 24.15d 4.94 3.86 2.31 31.64c 46.69b 67.34a 69.02a 25.48d 8.66 3.86 Note: Numbers in columns with the same accompanying characters are different figures that are not significant at the 95% confidence level Thus, the fungus causing anthracnose on c offee trees in Son La grows well in the condition of about 25 – 30 o C, the temperature at 20 or 35 o C is not favorable for the growth of fungal colonies 3.2.5.2 Temperature effect on the germination of Colletotrichum spp on the artificial environment An experiment to evaluate the germination rate of coffee anthracnose fungi isolate in Son La was conducted at temperatures o of 20, 25, 28, 30 and 35 C Spores of Colletotrichum fungi were isolated from coffee trees in Son La to germinate well in the condition of about 25-30 o C, the temperature of 20 or 35 o C is not favorable for the germination of spores 3.2.5.3 Effect of light on the growth of Colletotrichum spp on artificial media Table 3.29 Diameter of Colletotrichum spp after days of culture under different lighting conditions (Son La, 2016) Diameter of the fungal colonies (mm) Lighting C C C C siamense C acutatum time gloeosporioides fragariae theobromicola ( MNTC10) ( MNTC 14) ( CBMS15) ( CBMS3) ( CBMS9) (hours) 12 14 CV(%) LSD0.05 77.00a 77.84a 76.30a 2.39 3.68 78.89a 80.01a 78.82a 1.05 1.67 70.35a 68.67a 67.97a 1.71 2.35 73.64a 69.86a 68.67a 3.73 5.27 69.02a 68.46a 67.34b 1.19 1.63 Note: Numbers in columns with the same accompanying characters are different figures that are not significant at the 95% confidence level Isolates of anthracnose fungi collected and isolated from arabica coffee trees in Son La could grow under the conditions of 12 hours of lighting, 14 hours of lighting and full darkness 3.3 Severity of anthracnose on arabica coffee trees and its influencing factors 3.3.1 Developing of anthracnose disease (Colletotrichum spp.) on Catimor coffee in Son La In order to assess the development of anthracnose on arabica coffee trees, we conducted periodic surveys on 7-year-old coffee trees in fields representing coffee growing areas in Son La from April to October - On the leaves, the disease appears early in the early rainy season (April) in the period of coffee flowering 17 and fruiting, but with a low incidence and disease severity During this period, the disease has almost no effect on berries By the end of June to mid-July, leaf disease increased both in the incidence and severity, this time the growth of coffee trees is the strongest in the year, the leaves grow much This is also the middle of the rainy season in Son La, heavy rainfall, average temperature of about 25-28 o C, average humidity of 7080% are favorable conditions for the disease to arise and cause harm 30 Disease severity (%) 20.00 Disease severity (%) 25 20 Berries Branches Leaves 15.00 15 Leaves Branches 10.00 10 5.00 Time Time - 10/17/ 424/ 41/ 8/ 515/ 522/ 529/ 54/ 611/ 618/ 625/ 622/ 629/ 65/ 712/ 719/ 726/ 72/ 9/ 816/ 823/ 830/ 86/ 913/ 920/ 927/4/ 10 18/ 425/ 2/ 9/ 16/ 523/ 530/ 6/ 13/ 620/ 627/ 4/ 11/ 718/ 725/ 1/ 8/ 16/ 823/ 830/ 6/ 13/ 920/ 927/ 94/ 10 Figure 3.25 Devel opment of anthracnose levels on leaves, branches and coffee berries in Son La (2016) Figure 3.26 Development of anthracnose levels on leaves, branches and coffee berries in Son La (2017) - On the branches: Through servey shows that anthracnose often appears on the branches In the late dry season and early rainy season in Son La, the disease occurred at a low level of harm, then gradually increased and peaked in the middle of the end of August, the disease index reached 16.67% (2016) and 17.78 % (2017) From the end of September to October, the harmful level of the disease decreases Under adverse environmental conditions during the dry season, stalks, dead bark and fruits are considered the main primary source of the disease Having favorable conditions, the disease infects branches and leaves, then on fruits - On fruits, the disease appears later than the leaves, the period after 6-7 weeks after flowering, only the occurrence and damage of the disease were recorded At the end of August and early September when the coffee fruit starts to ripen, the harmfulness of the disease increases markedly This is the period when the coffee tree changes its concentration of nutrients to set fruits and metabolizes the substances to create ripe fruits During this period, there are many continuous rains 18 in the days, the average temperature is 22-25 o C, the average humidity is over 80%, these are very favorable conditions for harmful diseases From May to September is the rainy season in Son La, the average temperature in these months ranges from 22-27 o C, the annual rainfall is mainly concentrated during this time Since the end of September every year, rainfall has decreased significantly The total amount of precipitation from January to April and from October to December every year is often very low (2016: 278.6 / 1,334.1 mm; 2017: 445.1 / 1,402 mm), the average temperature fluctuates from 14-22 o C Therefore, harmful anthracnose on coffee trees in Son La concentrates and causes major damage from May to September every year 3.3.2 Percentage of coffee berries losses by anthracnose (Colletotrichum spp.) in Son La Through serveying of the years 2016-2017 from the 7th week after the last flowering, it was recorded that the coffee berries were shed due to anthracnose but with a low rate from 3.32 to 4.12% After that, the rate of falling fruits increased gradually until the end of the season with the total rate of falling fruits accounting for 48.34-52.83%, of which the rate of fruit falling from diseases was from 42.63-45.39% In particular, 100% 100% 90% 90% 80% 80% 70% 70% Berry losses (%) Berry losses (%) the proportion of disease berries increased sharply from week 10 to week 12 (about early to middle of July) 60% 60% 50% 50% 40% 40% 30% 30% 20% 20% 10% 10% 0% 0% 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Disease-free berries (%) Physiological berries fall (%) 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Berry age (weeks) Berry age (weeks) Inflected berries Figure 3.27 Cumulated losses of coffee berries in S on La (2016) Disease-free berries (%) Physiological berries fall (%) Inflected berries Figure 3.28 Cumulated losses of coffee berries in S on La (2017) 3.3.6 Effect of cultivation conditions on the occurrence of the disease The cultivation experiment was conducted for years (2016-2017) In the non-shade and applying technique plot, the 19 rate of fruit drop caused by anthracnose was 25.17 ± 4.86% lower than the control (35.14 ± 4.37% ) By the second year, the difference in fruit drop rate between the two conditions was more evident, that was 19.42 ± 0.67% in the experimental plot and 31.92 ± 1.78% in the control plot This result was similar to the applied in combination with shade plot, the rate of falling fruit were lower than the control 3.4 Evaluating the againsted effect of some chemical fungicides and biological fungicides for anthracnose disease on coffee arabica trees in Son La 3.4.1 Effect of some active ingredients against Colletotrichum spp on the culture medium An experiment to evaluate the effectiveness of fungicides on culture media was conducted on chemical drugs and biological drugs for isolates representing anthracnose fungi on coffee in Son La After days of experimentation: the chemical drugs propineb (Antracol 70WP), hexaconazole (Anvil 5SC) and curcumin (CFO) had low effect on fungal growth The fungicidal efficacy of different drugs for each fungal isolate The efficacy of the active ingredient propineb (Antracol 70WP) except isolate of MNTC 14 (C acutatum) reached 91.64%, for the remaining isoaltes reached 70.2978.82% after days of testing; active ingredient hexaconazole (Anvil 5SC) was 91.78% effective for CBMS5 (C gloeosporioides), for other isolates reached 63.66-77.06%; the fungicidal efficacy of curcumin (CFO) for MNTC11 (C theobromicola) reached 62.76% and there was no difference compared to the two tested chemicals; active ingredients Ningnanmycin (Supercin 20SC) and probiotics extracted from Cassia alata and Eupatorium fortune have very low inhibitory effected against the isolates (reached 17.61-27.14%) 3.4.2 Effect of some fungicides on coffee berry disease in field conditions Experimental results in 2016 indicated that all three experimental drugs were effective against anthracnose diseases in coffee tree fields After 15 days after spraying, effect on coffee berry disease of Antracol 70WP chemicals reached 79.14% and there was no significal difference at P