causing anthracnose on cofea arabica trees; study the characteristics of Colletotrichum and disease control measures to contribute to improving productivity, quality, increasing economi
Trang 1MI 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
Trang 2The research work is published at:
VIETNAM ACADEMY OF AGRICULTURAL SCIENCES
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:
1 National Library of Vietnam
2 Library of Vietnam Academy of Agricultural Sciences
3 Library of Plant Protection Research Institute
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INTRODUCTION
1 The urgency of the thesis
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 ha 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 ha 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
2 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
3 The new findings and contributions of the thesis
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- 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
4 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
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
Trang 5- 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 1 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 2015-
2016 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 5 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,
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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;
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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
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
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 3 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 3 groups based on spore shape (Phuong, 2010)
Trang 8Colletotrichum 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
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
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
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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 2 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,
2,4,6-trihydroxybenzoate, kaempferol, epiafzelechin, O-glucoside, kaempferol-3-O- gentiobioside Derived from
kaempferol-3-Cassia alata (MBG), O-coumaric extracted from Eupatorium fortune (MANTU)
2.2 Contents
Colletotrichum fungus causing anthracnose on arabica coffee
trees
Content 2 Studying the severity of anthracnose
(Colletotrichum spp.) on arabica coffee trees and its influence
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
- Evaluation criteria: general situation of coffee production, influencing factors, farming techniques, pests and diseases, productivity/production of coffe, market information, etc
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2.4.1.2 Situation of pests on coffee trees in Son La
Each area selected 5 representative fields, with 0.5-1 ha 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 3 main coffee growing areas of Son La including Thuan Chau district, Mai Son district and Son La city Survey 6 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 3 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 5-level inspection to assess the extent of damage (Waller, 1998)
* Pathogenic characteristics of the Colletotrichum fungus
on coffee berries
The experiment was arranged 3 replicates, 10 green fruits
or 10 ripe fruits /fungus samples/replications The experiment was conducted on berries with wounded and non-wounds,
control berries Infected berries are placed in sterilized plastic
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
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Total DNA extraction by CTAB (Cetyl Trimethyl Ammonium Bromide) based on descriptive material of Doyle & Doyle (1990) Use primers ITS4 (5'-CCT CCG CTT ATT GAT ATG C-3 ') and ITS5 (5'- GAA AGT AAA AGT CGT AAC
AAG G-3') (White et al., 1990) to amplify ITS region genes
(Internally Transcribed Spacer) were used in PCR with 35 cycles PCR products were electrophoretic with 1% agarose gel and photographed using Geldoc-ItTM Imaging System (USA) Directly sequence both directions with both ITS4 and ITS5 primers, using BigDye Terminator 3.1 Kit (Applied Biotech) on ABI3100 The order of samples was compared with Gen Bank
by online software http://blast.ncbi.nlm.nih.gov/Blast.cgi (S.F
et al., 1990) Genealogy tree is built by Neighbor-Joining method (N-J) by MEGA 6.0 software (Tamura, 2013)
2.4.2.4 Study on some biological characteristics of Colletotrichum species that causes anthracnose on coffee trees
in Son La
* Temperature effect on the growth of Colletotrichum spp
on the artificial media
Experiment with a factor of temperature, evaluate the growth of 5 representative isolates of Colletotrichum at the
threshold has 3 replications, 3 petri dishes/each replication; measuring the diameter of fungal colonies after 7 days of transplanting, 24 h measuring the colonization diameter once to
assess the growth rate of colonies in samples (Soltani et al.,
2014)
Colletotrichum spp
Add 10 µl of water containing the spores of each sample
to each end of the slide, which is then transferred to a humidifying chamber and placed in a incubator at 15, 20, 25,
28, 30, and 35° C Check the percentage of spores after 8, 14 and 24 h The number is counted as 50 spores per drop of liquid, observing germination when the sporula appears half the length of the spores, each isolate being repeated 3 times (Denner et al., 1986)
* Influence of light conditions on the growth of Colletotrichum spp on the artificial media
Experiment with a factor of light, assessing the growth of
5 representative isolates of Colletotrichum under the conditions:
12 light hours/12 dark hours and 14 light hours/10 dark hours and 0 light hours/ 24 dark hours, Lighting intensity is 600 lux
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Each light condition has 3 replicates, each replicate is 3 petri dishes with 85 mm diameter, the experiment was conducted at 28° C; measuring the diameter of fungal colonies after 7 days
of transplanting, assessing the growth rate of the mycelium in
samples (Soltani et al., 2014)
2.4.3 Study on the severity level of anthracnose (Colletotrichum spp.) on arabica coffee and its influence factors
2.4.3.1 Survey on the evolution of anthracnose coffee
Selecting 7-year-old coffee field, surveying 3 coffee fields representing coffee growing areas in Son La, with an area of 0.5-1 ha/field, fixing survey gardens according to VN01-38: 2010/ MARD (Ministry of Agriculture and Rural Development, 2010)
2.4.3.2 Survey on the percentage of coffee cherries that have been fail due to anthracnose
Survey on 3 representative fields (0.5-1 ha), 10 fixed points/field follow diagonally and distance 2 tree rows away from the bank; 4 directions × each direction 1 fruit branch/one tree/spot, every 1 week/times according to VN01-38: 2010/ MARD (Ministry of Agriculture and Rural Development, 2010) Firstly surveying on fruit development stage (6 weeks after the last flowering), counting a ll the fruits on the branches; count the number of healthy fruits, the number of diseased fruits, the number of new diseased fruits at each survey and wear a small card for each fruit to avoid confusion when
observed throughout the year of observations, which expressed all losses due to anthracnose or not, recorded over one year It was calculated by the formula:
Where: Ptot is total losses of berries; Btot1 is the total number of berries on the first observation; the expression (Btotn -Bmkn -Bdisn) is the number of healthy berries in the nth week of observations The terms Btotn, Bmkn and Bdisn are the total number of berries, the total number of old disease berries, and the total number of new infected berries on the nthobservation
The % diseased berries (Pdis) which was the ratio between the sum of new diseased berries counted weekly, from the first to the nth week of observations [Bdis(1_n)] and the
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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 4 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 7 × 10 m, the remaining 200 trees do 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 3 replicates and 1 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, 7 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)