Tom Tat English Nghiên Cứu Sử Dụng Các Hợp Chất Thiên Nhiên Trong Phòng Trừ Bệnh Thán Thư Trên Trái Xoài Cát Chu (Mangifera Indica Var Chu) Sau Thu Hoạch.pdf

Application of Essential Oils Extracted from Peel Wastes of Four Orange Varieties to Control Anthracnose Caused by Colletotrichum scovillei and Colletotrichum gloeosporioides on Mangoes

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

SUMMARY OF DOCTORAL THESIS

Major: Crop science Industry code: 62620110

CHAU TRUNG DUONG

RESEARCH ON THE USE OF NATURAL COMPOUNDS IN PREVENT ANTHRACNOSE

DISEASE ON CAT CHU MANGO

(Mangifera indica var Chu)

POSTHARVEST

Can Tho, 2024

WORK WAS COMPLETED AT CAN THO UNIVERSITY

Scientific supervisor: Dr Luu Thai Danh

Scientific supervisor: Dr Nguyen Thi Ngoc Truc

The thesis will be defended in Can Tho University At:………date………Month…… year………

Reviewer 1:……… Reviewer 2:………

Confirmation of Chairman

The thesis can be found at the library:

- Learning Resource Center, Can Tho University

- Vietnam National Library

LIST OF PUBLISHED PROJECTS

1 Chau Trung Duong, Nguyen Thi Nhu Y, Nguyen Thi Thu Nga,

Doan Thi Kieu Tien, Nguyen Thi Ngoc Truc and Luu Thai

Danh (2022) The fungus species (Colletotrichum spp.) that

causes anthracnose is highly toxic on Cat Chu mango in the

Mekong Delta Journal of Agriculture and Rural Development

No 18/2022, Ministry of Agriculture and Rural Development (ISSN 1859-4581), p 50 – 57 2

2 Chau Trung Duong, Huynh Thi Phuong Thao, Nguyen Thi Nhu Y,

Doan Thi Kieu Tien, Nguyen Thi Thu Nga, Tran Chi Nhan, Bui Thi Cam Huong, Sezai Ercisli, Nguyen Thi Ngoc Truc and Luu

Thai Danh (2023) Application of Essential Oils Extracted from

Peel Wastes of Four Orange Varieties to Control Anthracnose

Caused by Colletotrichum scovillei and Colletotrichum gloeosporioides on Mangoes Plants, 12(15), 2761

3 Chau Trung Duong, Nguyen Thi Nhu Y, Huynh Thi Phuong Thao,

Tran Chi Nhan, Nguyen Thi Ngoc Truc and Luu Thai Danh (2023) Effect of Lemongrass, Cinnamon Leaf essential oils and

a mixture of controls on anthracnose disease on post-harvest Cat

Chu mangoes Journal of Agriculture and Rural Development

No 16/2023, Ministry of Agriculture and Rural Development (ISSN 1859-4581), p 20 – 28

4 Chau Trung Duong, Huynh Thi Phuong Thao, Doan Minh Khang,

Nguyen Thi Nhu Y, Tran Chi Nhan, Bui Thi Cam Huong, Nguyen Trong Tuan, Nguyen Thi Ngoc Truc, Duong Minh Tue and Luu Thai Danh (2024) Control of mango anthracnose

(Colletotrichum gloeosporioides) by essential oil mixture Turkish Journal of Agriculture and Forestry (Accepted on 14

June 2024)

CHAPTER 1 INTRODUCTION 1.1 The necessity of the thesis

Mango (Mangifera indica L.) is one of commonly grown fruit trees

in tropical and subtropical regions In Vietnam, Cat Chu mango is grown popularly in the Mekong Delta, with good quality, moderate sweetness (Brix level 14.4%), and a highly edible rate of 76.5% (Nguyen Bao Ve and Le Thanh Phong, 2011) Mango is a highly valued fruit, however, it is susceptible to quick spoilage after harvest due to pathogenic microorganisms (Ploetz, 2003) Among them, anthracnose

caused by Colletotrichum spp., is one of the major causes of damage

and loss of commercial fruits after harvest

The usage of synthetic chemicals to control and reduce post-harvest diseases raises concerns due to residues in agricultural products and the

developement of drug-resistant fungi (Mari et al., 2014; Benítez et al., 2020) These limitations have promoted the development

Ramírez-of safe and effective anthracnose control solutions that are safe for consumers and the environment One of which is the use of natural compounds (essential oils) Essential oil (EO) is a mixture of volatile organic compounds that are derived from a plant species, and responsible for the scent and flavor of that plant (Tisserand and Young, 2013) EOs are considered as secondary metabolites and are important for plant protection because they often have the antimicrobial activity

(Tajkarimi et al., 2010) EOs may not induce the development of

resistant strains of fungi due to synergism among their various chemical components (Varma and Dubey 1999) In addition, EOs are safe for human consumption as they are easily biodegradable and leave no toxic residues on fruits

Orange peel EO is one of the most abundant, available and inexpensive EOs on the market Oranges are one of the most popular citrus fruits, with a total world production of about 48 million tons/year

in the period 2022–2023, of which orange production in Vietnam is estimated at about 1.15 million tons per year (USDA, 2023) Oranges

are often consumed as a fresh product or can be a raw material for producing juices and concentrates Most orange waste is discharged into the environment (Manthey & Grohmann, 2001); only small amounts are

used to produce molasses, pectin, and EOs (Gavahian et al., 2019) In

the Mekong Delta, Vietnam (tropical climate), three varieties of

green-skinned oranges are commonly grown: Sanh orange (Citrus reticulata × sinensis) is the most popular, followed by Xoan orange (Citrus sinensis) and Mat orange (Citrus sinensis) Navel orange variety (Citrus sinensis)

is the most popular variety in the world, widely grown in subtropical climates (Aussie oranges, 2023) Many studies have demonstrated that

orange peel EO can inhibit C gloeosporiodes under in vitro conditions,

and orange essential oil has the ability to significantly reduce anthracnose disease on mango fruit (Abd-Alla & Haggag, 2013 ; Rabari

et al., 2018) To date, there have been no comparative studies on the

antifungal activity of EOs from green orange and yellow orange against

Colletrotrichum spp that causes mango anthracnose

In addition to orange EO, other EOs have been shown to have strong inhibitory activity against fungi that cause anthracnose on postharvest mango fruit EOs can be mixed together to increase the effectiveness of anthracnose control with limited or without effect on fruit quality As 2 or 3 EOs are combined together, the effective concentration of each EO in the mixture is very low and does not affect

the quality of the fruits (Nikkhah and Hashemi, 2020; Bounar et al., 2020; Nikkhah et al., 2017) The high antifungal activity in EO mixture

is due to the synergies of the chemical components in EOs The synergistic interactions are useful in postharvest disease management because pathogens do not readily exhibit resistance to mixture of chemical constituents from a variety of sources Several studies in recent years have demonstrated that blends of several EOs are more effective

in preventing the growth of pathogenic fungi on the postharvest fruits

than individual EOs (Sukatta et al., 2008; Hossain et al., 2016; Hassan

et al., 2020)

Currently, there are no studies comparing the effectiveness of

existing essential oils in Vietnam in inhibiting Colletotrichum spp

causing anthracnose disease on mango and the interactive effects of an

essential oil mixture on the growth of Colletotrichum spp Therefore, the

thesis was carried out “Research on the use of natural compounds in

prevent anthracnose disease on cat chu mango (Mangifera indica

var Chu) postharvest”

1.2 Objective of the thesis

- Determine the Colletotrichum spp extremely hazardous and

poisonous to Cat Chu mangos

- Determine the effectiveness of essential oil and essential oil

mixtures for inhibiting anthracnose-causing Collectotrichum spp on Cat

Chu mangoes

- Determine the effectiveness of essential oil and essential oil

mixtures for reducing anthracnose caused by Colletotrichum spp on Cat

Chu mangoes

1.3 Object and scope of the study

- The main research object of the thesis is essential oils of some plant species (Sanh orange, Xoan, Mat, Navel, cinnamon bark, cinnamon leaf, Lemongrass, Javanese lemongrass, ginger root, turmeric root, Da Xanh grapefruit, seedless lemon and basil) and fungi causing anthracnose on Cat Chu mangoes

- The scope of the study is to evaluate the inhibitory ability of single and essential oil mixtures on the fungus-causing anthracnose on Cat Chu mangoes in the Mekong Delta during the post-harvest period and help maintain the fruit quality

1.4 New contributions of the thesis

- Isolating and identifying Colletotrichum scovillei as a new agent

causing anthracnose disease on Cat Chu mangoes in the Mekong Delta

- Determining the content and the chemical composition of EO from different plant sources such as Sanh, Xoan, Mat and Navel orange, cinnamon bark, cinnamon leaf, Java lemongrass, Lemongrass, ginger, turmeric, grapefruit, lemon peel, mint, basil and coriander

- Identifying 3 EOs (lemongrass, cinnamon leaf and cinnamon bark)

with high antifungal activity against Colletotrichum spp Determining

the mixture concentrations of lemongrass EO with cinnamon leaf or cinnamon bark EO that are more effective than using EOs alone and commercial chemical fungicide in inhibiting fungi that cause anthracnose on Cat Chu mango The mixtures of EOs and EO has no effect on Cat Chu mango fruit's ensory values or quality

1.5 Scientific and practical significance of the research

The scientific contribution of the study is the demonstration of the

effectiveness of essential oils in suppressing Colletotrichumsp-causing

anthracnose on post-harvest mangoes and preserving Cat Chu mangoes while not affecting quality

Regarding practice: The results of the project can be applied to preserving post-harvest mangoes, limiting fungi that cause anthracnose, and maintaining fruit quality

CHAPTER 3 RESEARCH METHODS

3.1 Materials

3.1.1 Location and time

The thesis was carried out from February 2018 to October 2023 at Laboratory of Genetics and Plant Breeding Department, Laboratory of Plant Protection Department, Chemistry Laboratory of Faculty of Science, Can Tho University, and Horticulture Department Laboratory, Faculty of Agriculture, Can Tho Technical Economic College

3.1.2 Research materials

- Cat Chu mangoes used in the experiments were purchased from local farmers in Dong Thap, Hau Giang, Tien Giang, and An Giang

province Colletotrichum spp were isolated from infected Cat Chu

mangoes collected in Hau Giang, Tien Giang and An Giang provinces

- Lemongrass leaf stem, Java lemongrass leaf, orange peel (Sanh, Xoan, Mat, Navel), grapefruit peel, seedless lemon peel, cinnamon leaf, cinnamon bark, basil leaf and stem, turmeric root, and ginger root are used to extract essential oils Coriander and mint essential oils are

provided by Dalosa Vietnam Herbal Essential Oil Company Limited

3.2 Research Methods

3.2.1 Content 1 Isolation and identification of fungal species causing anthracnose on Cat Chu mango

3.2.1.1 Isolation and evaluation of harmful potential of

Colletotrichum spp causing anthracnose disease in Cat Chu mango

The infected Cat Chu mangoes were collected from gardeners at 3 locations: An Thoi Trung commune, Cai Be district, Tien Giang province (1); Hoa An commune, Cho Moi district, An Giang province (2); Chau Thanh district, Hau Giang province (3)

The fungal strains were isolated and verified their potential to cause

disease using Koch's postulates (Agrios, 2005; Burgess et al., 2009),

then identified highly toxic fungal strains based on morphological characteristics and molecular biology techniques

Experiment 1: Assessing the harmful potential of Colletotrichum

sp on Cat Chu mango

The experiment used a completely randomised design with nine treatments (9 fungal strains), each treatment having four replicates and five fruits per replicate

3.2.1.2 Identify fungal species by morphological characteristics combined with DNA sequencing of the ITS region

Identification of fungal species by morphological characteristics

Important morphological characteristics for classifying

Colletotrichum spp include shape, colour, growth rate, fungal structure,

and assessment of spores' shape and size from three-day-old and older fungi in PDA medium Spore shape and size were determined by culture

on slides according to the method of Waller et al (1998) The study

referred to and compared morphological observation using the taxonomic documents of Barnett & Hunter (1960) and Sutton (1980) to identify the fungal species

Identification of fungal species using molecular biology techniques

PCR products were sequenced and compared with published sequences in the database using the NCBI bank’s BLAST search engine

to identify fungal species Branch diagrams comparing genetic relationships between species were established using the Neighbor-

Joining method in MEGA 6 software (Tamura et al., 2013)

3.2.2 Content 2 Evaluation of the ability to inhibit Colletotrichum

spp causing anthracnose disease on Cat Chu mango fruit from EO

alone and EO mixture in vitro and in vivo

3.2.2.1 Experiment 2.1: Extraction of EOs from fruit peels of 4 orange varieties (Sanh, Xoan, Mat and Navel varieties)

The experiment was completely randomised with four treatments (four orange varieties); each treatment had three replicates, with an EO sample per replicate

3.2.2.2 Analysing the chemical composition of orange peel essential oil using gas chromatography - mass spectrometry (GC-MS) and gas chromatography (GC)

EOs were analysed for chemical composition using gas chromatography-mass spectrometry (GCMS) The relative concentrations of chemical components in EOs were determined using gas chromatography (GC)

3.2.2.3 In vitro antifungal activity of orange oils against Colletotrichum spp

Experiment 2.2: Agar plate diffusion

The experiment was completely randomised with 6 treatments, each had five replicates with one paper dish per replicate The treatments included: (1) Sanh EO, (2) Xoan EO, (3) Mat EO, (4) Navel

EO, (5) Negative control (DMSO 5%), (6) Positive control (Probineb 4.2 mg/mL) EOs were diluted in 5% dimethyl sulfoxide (DMSO) solutions to obtain concentrations of 20, 40, and 80% v/v

Experiment 2.3: Minimum inhibitory concentration (MIC)

The experiment was completely randomised with 27 treatments (23 concentrations of oragen EOs and three control treatments including PDB, PDB + fungal spore suspension, PDB + fungal spore suspension + DMSO) Each had 5 replicates with 1 well per replicate The experiment

was set up for C gloeosporioides and C scovillei

Experiment 2.4: Minimum fungicide concentration (MFC)

The experiment was completely randomised with 10 treatments Each treatment had 10 replicates, with 1 petri dish per replicate The

experiment was set up for C gloeosporioides and C scovillei

3.2.2.4 Experiment 2.5: In vivo assay of orange essential oil against

anthracnose development in Cat Chu mango

The experiment was completely randomised with 7 treatments Each treatment had 10 replicates with one fruit per replicate The

experiment was set up for C gloeosporioides and C scovillei The

treatments included: (1) Xoan EO 4%, (2) Mat EO 4%, (3) Navel EO 8%, (4) Sanh EO 8%, (5) Sanh EO 16%, (6) Negative control distilled water, (7) Positive control (Probineb 4.2 mg/mL)

3.2.3.5 Experiment 2.6: Extraction of essential oils

The experiment was completely randomised with 10 treatments (ten EOs including: cinnamon bark, cinnamon leaf, lemongrass stem leaf, Java lemongrass leaf, roots ginger, turmeric root, Sanh orange peel, green grapefruit peel, seedless lemon peel and basil leaf stem) Each treatment had 3 replicates, with 1 extracted EO sample per replicate

3.2.2.6 Analysis of chemical composition of EOs using chromatography-mass spectrometry (GC-MS) and gas chromatography (GC)

Proceed similarly to analyse the chemical composition of orange EOs

3.2.2.7 In vitro antifungal activity of essential oils against Colletotrichum spp

Experiment 2.7: Agar plate diffusion

The experiment was completely randomised with 14 treatments (12 EOs at 10% concentration, negative control DMSO 5%, positive control Probineb 4.2 mg/mL) Each treatment had 5 replications with 1 paper

dish per replicate The experiment was set up for both C gloeosporioides and C scovillei

Experiment 2.8: Minimum inhibitory concentration (MIC)

Three EOs with high antifungal ring diameters were selected to determine MIC including: lemongrass (LG), cinnamon bark (CB), and cinnamon leaf (CL)

The experiment was completely randomised with 18 treatments (concentrations of EO mixture and three control treatments including PDB, PDB + fungal spore suspension, PDB + fungal spore suspension + DMSO) Each treatment had 10 replicates with 1 well per replicate The

experiment was set up for C gloeosporioides and C scovillei

Experiment 2.9: Minimum fungicide concentration (MFC)

The experiment was completely randomised with 13 treatments for

C gloeosporioides and 10 treatments for C scovillei Each treatment

had 10 replicates with one petri dish per replicate

3.2.2.8 Evaluating the interaction effects of selected essential oils on

their ability to inhibit fungi causing anthracnose in vitro

Experiment 2.10: Minimum inhibitory concentration (MIC) of interaction of a mixture of pairs of essential oils

The experiment was completely randomised with 43 treatments for

C gloeosporioides and 53 treatments for C scovillei In which, three

control treatments included PDB, PDB + fungal spore suspension, and PDB + fungal spore suspension + DMSO Each treatment had 10 replicates with 1 well per replicate

Experiment 2.11: Minimum fungicidal concentration (MFC) interaction of EO mixtures

The experiment was completely randomised with 16 treatments for

C gloeosporioides and C scovillei Each treatment had 10 replicates

with 1 petri dish per replicate

Evaluation of interactions of EO mixtures

The inhibitory concentration index (FICi) and the fungicidal concentration index (FFCi) were used to evaluate the interaction

effectiveness of EO mixture (Chin et al., 1997) Both FICi and FFCi

were explained as following: FICi or FFCi ≤ 0.5: has a synergistic effect; 0.5≤ FICi or FFCi ≤ 1 indicates a synergistic effect, 1< FICi or FFCi ≤ 4 has no interactive effect; and FICi or FFCi > 4 indicates an antagonistic effect between the two essential oils tested

3.2.2.9 Experiment 2.12: In vivo assay of single EO and EO

mixtures against anthracnose development in Cat Chu mango

The experiment was completely randomised with 16 treatments for

C gloeosporioides and C scovillei Each treatment had 10 replicates,

with 1 fruit per replicate

3.2.3 Content 3: Evaluate the effectiveness of reducing anthracnose

disease caused by Colletotrichum spp on Cat Chu mangoes of

essential oil alone and essential oil mixture

3.2.3.1 Experiment 3.1: Evaluate the impact of Sanh essential oil on the quality of Cat Chu mangoes

The experiment was completely randomised with 6 treatments (containing different concentrations of Sanh EO) Each treatment had 10 replicates, with one fruit per replicate The treatments included: (1) 2%

EO, (2) 4%, (3) 8%, (4) 16%, (5) control DMSO 5%, and (6) control distilled water

3.2.3.2 Experiment 3.2: Evaluate the effects of individual essential oils and essential oil mixture on the quality of Cat Chu mangoes

The experiment was completely randomised with 15 treatments (selected from experiment 2.12) Each treatment had 5 replicates with 1 fruit per replicate

3.2.3.3 Experiment 3.3: Evaluate the effectiveness of reducing anthracnose disease in Cat Chu mangoes post-harvest of each single

EO and EO mixture using by spraying fungal suspension

The experiment was completely randomised with 8 treatments (selected from experiment 3.3) Each treatment had 10 replicates with 1 fruit per replicate

3.3 Data analysis

The programme SPSS 22.0 was used to analyse experimental data Differences among treatments were detected by Analysis of variance (ANOVA) Mean values were ranked by using Duncan test at the 1% and 5% significance levels The percentages were transformed before statistical analysis

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Isolation and identification of Colletotrichum spp causes

anthracnose disease on Cat Chu mango fruit

4.1.1 Isolation and assessment of harmful potential of

Colletotrichum sp on Cat Chu mango

Nine strains of Colletotrichum spp were isolated from disease

lesions in Cat Chu mangoes collected in 3 provinces (Tien Giang, An Giang and Hau Giang) The fungal strains were named TG1-TG4, AG1-AG2 and HG1-HG3

The results in Table 4.1 show that there was a significant difference

in wound length in fruits among treatments over observation period