Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long

Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.Nghiên cứu khả năng và cơ chế kích thích tính kháng bệnh cháy bìa lá lúa của hợp chất trong các loài thực vật tại Đồng bằng Sông Cửu Long.

MINISTRY OF EDUCATION AND TRAINING

CAN THO UNIVERSITY

SUMMARY OF DOCTORAL THESIS

Majors: Biotechnology Code: 9420201

THIS STUDY WAS ACHIEVED

AT CAN THO UNIVERSITY

Scientific advisor: Assoc Prof Nguyễn Đắc Khoa

Confirmation of review by the Chairman of the Board

The thesis can be found at the library:

- Learning Resource Center, Can Tho University

- Vietnam National Library

PUBLICATIONS RELATED WITH THE PhD THESIS

1 Truong Van Xa, Tran Kim Thoa, Thai Tran Anh Thu, Nguyen Dac

Khoa (2023) Effects of seed soaking and foliar spraying of Kalanchoe

pinnata aqueous leaf extracts against rice bacterial leaf blight CTU Journal of Innovation and Sustainable Development, 15(3), 12-22 DOI:

10.22144/ctujoisd.2023.047

2 Truong Van Xa, Tran Kim Thoa, Nguyen Duc Do, Nguyen Dac

Khoa (2023) Seed soaking using methanol Kalanchoe pinnata leaf

extracts induces rice resistance against bacterial leaf blight

https://doi.org/10.3390/ijpb14040084

3 Truong Van Xa, Tran Kim Thoa, Nguyen Dac Khoa (2024)

Aqueous Chromolaena odorata leaf extracts induce rice resistance against bacterial leaf blight International Journal of Phytopathology

Accept 31.08.2024

4 Truong Van Xa, Nguyen Duc Do, Nguyen Dac Khoa 2024 Seed

soaking using fractional leaf extracts of Kalanchoe pinnata (Lam.) Pers induces rice resistance against bacterial leaf blight (Xanthomonas

oryzae pv oryzae) Vietnam Journal of Agriculture and Rural Development, special Issue in Phytopathology (July 2024): 61-69

5 Nguyen Thi Thuy Ngan, Truong Van Xa, Nguyen Dac Khoa 2024

Investigation of induced resistance against rice bacterial blight of

aqueous Ageratum conyzoides leaf extracts using foliar spraying

Vietnam Journal of Agriculture and Rural Development, special Issue in

Phytopathology (July 2024): 44-51

6 Pham Thiet Trinh, Luu Minh Long, Truong Van Xa, Nguyen Dac

Khoa 2024 Foliar spraying using aqueous Kalanchoe pinnata leaf extracts induces rice resistance against bacterial leaf blight Vietnam

Journal of Agriculture and Rural Development, special Issue in

Phytopathology (July 2024): 52-60

Chapter 1: INTRODUCTION

1 1 Rationale of the study

Bacterial leaf blight (BB) is caused by Xanthomonas oryzae pv oryzae (Xoo) (Swings et al., 1990) In Viet Nam, the disease is more destructive during rice harvest where loses can reached 65% of yields (Dinh et al.,

2008; Son, 1993) In addition, the disease also causes serious damage to

the quality of rice, which affects the export of high-quality rice (Dinh et al.,

2008; Khoa, 2018; Son, 1993)

BB management has centered on methods which reduce the initial inoculum and subsequent development of the patroon rice plants Induced resistance is a sustainable and environmentally friendly way to control the

BB disease (Kloepper et al., 1992, Khoa et al., 2011, 2017; Lyon et al., 2007; Khoa, 2018; Pieterse et al., 2014; Walters et al., 2007) For this

method, the resistance does not directly affect the pathogens, but generates signals to stimulate the self-defense mechanism in the plants Here there is

an increased accumulation of phenolic compounds, phytoalexins and disease-related proteins (PR-proteins) such as peroxidase (POX), catalase (CAT), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL) to

prevent the infection and growth of pathogens (Van Loon et al., 1998; Vidhyasekaran et al., 1997)

Numerous chemicals, bacteria and plant extracts can induce rice

resistance against BB They include salicylic acid (Mohan Babu et al.,

2011), Pseudomonas fluorescens (Lingaiah et al., 2013), methanol extracts

of Datura metel (Kagale et al., 2004), aqueous and methanol extracts of

Adhatoda vasica (Govindappa et al., 2011), aqueous extracts of Vitex negundo (Nisha et al., 2012), and aqueous crude extracts of Kalanchoe pinnata (Khoa et al., 2017; Hương et al., 2018) The protection involved

induced resistance since activities of POX and CAT increased after extract

applications, particularly with the presence of Xoo, while those of PPO and PAL increased at an early stage after pathogen inoculation (Kagale et al.,

2004; Govindappa et al., 2011; Nisha et al., 2012; Khoa et al., 2017) As

bioactive compounds have different characteristics and polarities, their contents and antioxidant power varied when extraction was conducted

using different solvents (Jaiswal et al., 2012) It could be argued that

different solvents and/or extraction methods could extract different resistance-inducing compounds against BB

This study aims at testing for disease-reducing effects of bioactive compounds in extracts of indigenous plants in the Mekong Delta of Vietnam and investigating the involvement of induced resistance in the observed disease reduction This helps make an eco-friendly strategy to control the disease thus improve rice yield and quality

(1) Selection of plant extracts in the Mekong Delta that can help

reduce rice leaf disease caused by Xoo bacteria;

(2) Proving the ability of selected plant extracts to reduce disease

related to the mechanism of rice leaf blight resistance;

(3) Investigate the ability of a group of compounds contained in

selected plant extracts to stimulate resistance to rice leaf blight disease;

(4) Identify a group of compounds capable of stimulating rice leaf

blight disease resistance

1.3 Research content

Content 1: Selection of plant extracts capable of stimulating resistance to rice leaf blight disease

Content 2: Investigating the ability to stimulate resistance to rice leaf blight

disease of the group of compounds contained in Kalanchoe pinnata leaves Content 3: Identify compounds in Sống đời (Kalanchoe pinnata) that have

the ability to stimulate resistance to rice leaf blight disease

1.4 Meaning of the study

1.4.1 Scientific significance

The thesis contributes to providing complete and systematic scientific data on promoting disease resistance in rice plants using biological agents, including: selecting, surveying the ability to reduce disease, proving the mechanism and identifying compounds that have resistance ability

The results of the thesis determine the ability and mechanism to stimulate resistance to rice leaf blight disease by water extracts of cỏ cứt

heo (Ageratum conyzoides), cỏ hôi (Chromolaena odorata), and Sống đời (Kalanchoe pinnata) Preparation and identification of compounds in

Kalanchoe pinnata leaves that have the ability to stimulate resistance to

rice leaf blight disease

1.4.2 Practical significance

Using plants containing natural compounds that help stimulate resistance to rice leaf blight disease is an economically effective and environmentally friendly solution, non-toxic to humans, easy to use, and can be utilized Local raw material sources should help reduce production costs;

Besides, disease prevention and treatment measures using plant extracts are completely consistent with the orientation of building organic agriculture

1.5 The new contributions of the thesis include

Selected and identified three plant extracts that have the potential to

help reduce rice leaf blight disease such as: A conyzoides, C odorata, and

pinnata (Lam.) Pers using organic solvents with different polarities can

help reduce leaf blight disease;

Proving that the ability of K pinnata extract to reduce BLB is related to

the resistance mechanism through increasing the activity of POX, CAT, PPO, and PAL enzymes;

Identifying 27 plant compounds in total MeOH extract, 34 plant

resistance to rice leaf blight disease It has been proven that two plant compounds, 5-Oxotetrahydrofuran-2,3-dicarboxylic acid, dimethyl ester

and 3-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester, in K pinnata

have the ability to stimulate resistance to leaf blight disease

Chapter 2: LITERATURE REVIEW 2.1 Bacterial leaf blight

2.2 Disease resistance of plants

2.3 Studies on resistance mechanisms in plants

2.3.1 Plant resistance mechanism through physical barriers

2.3.2 Plant resistance mechanism through biochemical reactions

Biochemical reactions of plants such as increased synthesis of phenolic

compounds (Fan et al., 2017); phytoalexins (Cho et al., 2015; Roop Singh

et al., 2017); hypersensitive reaction (Hammerschmidt et al., 2000);

pathogenesis-related proteins (Van Loon et al., 2006)

Disease resistance responses of plants often appear in groups of

contribute to limiting the growth of pathogens (Apel et al., 2004; Shetty et

al., 2008) The action of a group of substances with high antioxidant

activity as antibacterial agents, participating in part of lignin, suberrin and other cell wall constituents, stimulates genes related to protection, stimulating Synthesize phytoalexins that are toxic to pathogens (Nicholson

et al., 1992; Apel et al., 2004; Edreva, 2005; Cho et al., 2015) In addition,

a group of substances with high antioxidant activity plays a role in

detoxifying cells (Rao et al., 1997; Govrin et al., 2000) ao et al., 1997; Govrin et al., 2000) Therefore, to maintain a balanced state in plant cells,

the detoxification mechanism is activated at the same time as the increase

in highly oxidized groups, in which the two enzymes POX and CAT

often present in forms such as lignin peroxidase, manganese peroxidase

and peroxidase (Hammerschmidt et al., 1982)

In plants, POX is a group of enzymes that are activated early in the process of resisting fungal and bacterial pathogens An increase in POX activity is associated with a decrease in the penetration and spread of

pathogens (Nicholson et al., 1992; Hammerschmidt et al., 2000) POX is

involved in a number of physiological and biochemical processes in plants such as cell growth and expansion, regulating defense responses when

plants are shocked by biotic or abiotic agents (Hammerschmidt et al.,

2000) In addition, POX plays a role in regulating oxidation reactions and

al., 2001; Shetty et al., 2008) n summary, POX activity is increased when

attacked by pathogens and is produced along with the content of oxidant active substances; is an important enzyme for the formation of defense

(Hammerschmidt et al., 2000; Hiraga et al., 2001; Van Loon et al., 2006)

Research results on rice resistance response against leaf blight caused

by Xoo bacteria show that POX activity in rice tissue increases when

infected with the pathogen; increased earlier and stronger when treated

with resistant agents (Song et al., 2001; Kagale et al., 2004; Govindappa et

al., 2011; Nisha et al., 2012; Khoa et al., 2017)

2.3.2.2 Enzyme catalase

In plants catalase (CAT) present in peroxysomes plays a role in

synthesis (Apel et al., 2004; Yu et al., 2016) esis (Apel et al., 2004; Yu et

al., 2016) CAT helps decompose H2O2 to help detoxify plant tissue when

necrotrophic pathogens invade (Apel et al., 2004); or after parasitic

pathogens in living tissue (biotrophic) rapidly multiply and spread in plant

tissue (Loprasert et al., 1996; Yu et al., 2016) When biotrophic pathogens

attack plant tissue, the superoxide dismutase enzyme present in chlorophyll

pathogen (Loprasert et al., 1996) At the same time, the POX enzyme in

plant tissue also participates in increasing H2O2 synthesis (Hiraga et al., 2001; Shetty et al., 2008) After that, CAT is increased in synthesis in the

oxygen molecules, helping plants detoxify (Apel et al., 2004; Yu et al.,

2016)

Seeds Soaking with leaf extract of Ocimum sanctum and Cymbopogan

citrus have the ability to stimulate resistance against Rhizoctonia solani

disease through a sharp increase in CAT activity from 0 to 96 hours after

inoculated (Pal et al., 2011) Similarly, seeds soaking with aqueous crude extracts of K pinnata helps stimulate rice plant resistance against rice leaf

blight disease through increased CAT enzyme from 1 to 6 days after

inoculation (Khoa et al., 2017)

2.3.2.3 Enzyme polyphenol oxidase

Polyphenol oxidase (PPO) is a copper (Cu) containing enzyme In the presence of oxygen, this enzyme catalyzes the hydroxylation of monophenol compounds to ortho-diphenols The generated ortho-diphenols are further oxidized to the corresponding ortho-diquinone compounds Diquinones have very strong activity, capable of participating in cross-

linking or alkylating proteins, forming brown pigments (Yoruk et al., 2003; Constabel et al., 2008) PPO participates in the function of protecting

plants against physical damage, attacks from pathogens and harmful insects

(Constabel et al., 2008) The plant's defense response to pathogen attack is

to increase the expression of the PPO enzyme to increase the ability to synthesize phenol and quinone compounds; These compounds are toxic to

pathogens such as germs and bacteria (Li et al., 2002; Constabel et al.,

2008) Besides, PPO also has the ability to activate genes encoding methyl jasmonate and oligogalacturonic acid signals in the plant's defense mechanism; Therefore, PPO also plays an important role in the resistance

mechanism that helps plants fight against microbial diseases (Constabel et

al., 2008)

In the study by Pal et al (2011), aqueous and ethanol extract of

Cymbopogan citrus and Ocimum sanctum have the effect of stimulating

rice resistance against sheath spot disease caused by Rhizoctonia solani,

shown by increasing PPO activity 3 times higher than rice plants whose

seeds are not treated with resistance According to Khoa et al., (2017) and Hương et al (2018), the resistance-stimulating ability of the extract of K

pinnata through activating and increasing PPO activity is higher than the

treatment without treatment of the extract

2.3.2.4 Enzyme phenylalanine ammonia lyase

Phenylalanine ammonia lyase (PAL) participates in the process of cell wall lignification and biosynthesis of flavonoids and phenylpropanoids in

plant defense reactions (Santiago et al., 2009) PAL catalyzes the

phenylpropanoid synthesis, leading to the synthesis of compounds involved

in plant defense such as lignin, isoflavonoids and coumarins (Solekha et

al., 2020)

Studies have stimulated plant resistance against pathogens through increased PAL activity Specifically, treatment with acibenzolar-S-methyl

helps rice plants resist leaf blight (Pyricularia oryzae) by showing

increased synthesis of PAL and POX enzymes compared to the control

(Thieron et al., 1995) Methanol extract from Datura metel has the ability

to increase PAL enzyme activity in rice, contributing to resistance to leaf

blight and sheath spot disease in rice (Kagale et al., 2004) Aqueous extracts of Ocimum sanctum and Cymbopogan citrus have the ability to

increase PAL enzyme activity in rice plants, contributing to resistance to

sheath spot disease in rice (Pal et al., 2011) Extracts of Adhatoda vasica (Govindappa et al., 2011) Aqueous crude extracts of K pinnata has the

ability to help rice plants resist rice leaf blight disease due to an increase in

PAL enzyme activity (Khoa et al., 2017; Nguyễn Thị Thu Hương và ctv,

2018)

2.4 Plant compounds

2.5 Extraction methods of plant compounds

2.6 Plant species used in the study

Chapter 3: METHODOLOGY 3.1 Plant species used in the study

Plant species used in the study from plant sources in the Mekong Delta

is based on three criteria: (1) popular plants; (2) contains plant substances

capable of stimulating disease resistance in plants as described by Kuć

(2006) và (3) refer to research on stimulating resistance in different crops

3.2 Effects on rice seed germination and development

The experiment was performed based on the method of của Singh and

Rao (1997) and modifed to Khoa et al (2017) The experiment was

arranged in a completely randomized format with three repetitions

Germination rates (percentage of germinated seeds over total number of seeds), shoot and root lengths of the germinated seeds were recorded after

3, 5 and 7 days In addition, the vigor index after 7 days as a function of germination rate and seedling lengths was calculated using formula

proposed by Arumugam et al (2008) and modifed to Doni et al (2014)

3.3 Disease-reducing effects of plant extracts on BB under greenhouse conditions

The experiment was arranged in a completely randomized format, with

3 repetitions

Rice plants at 45 days after sowing are used to inoculate the disease

according to the method of Kauffman, (1973) and modifed to Khoa et al

(2017)

Lesion length were measured at 7, 14 and 21 days after inoculation

(DAI) (Mew, 1993; Khoa et al., 2017)

3.4 Assays of the induced defense-related and antioxidant enzymes

Four treatments were included in these assays, i.e (1) seeds soaked in

sterile distilled water, rice plants were not inoculated (water +

non-inoculated); (2) seeds soaked in sterile distilled water, rice plants were inoculated (water + inoculated); (3) seeds soaked in extract, rice plants

were not inoculated (extract + non-inoculated); and (4) seeds soaked in

extract, rice plants were inoculated (extract + inoculated)

Rice leaves were collected from 0 to 7 DAI (once a day) and quickly frozen in liquid For enzyme extraction, 10 g of rice leaves was ground in liquid nitrogen using a Retsch mixer mill (MM200, Retsch Co., Haan, Germany), and 0.1 g of the sample from each collection time point was subsequently homogenized in 1.5 mL of an extraction buffer solution corresponding to each enzyme Indeed, sodium potassium phosphate buffer 0.1 M (pH 6.5) was used for the extraction of POX and PPO, sodium potassium phosphate buffer 0.1 M (pH 7.0) for CAT, and sodium borate buffer 0.1 M (pH 8.7) for PAL The homogenates were then centrifuged at 10,000 rpm at 4 ◦C for 30 min Enzyme samples were always kept on ice during the assays Each assay was conducted with three replicates

3.4.1 POX enzyme assay

POX activity was expressed as changes in absorbance at 470 nm at 30s intervals during 2 minutes since the reaction occurred as the rate of conversion of tetraguaiacol and guaiacol using the method described by

Hammerschmidt et al (1982) and Khoa et al (2017) The mixture of 1.6

guaiacol solution, 0.15 mL of 0.1M sodium phosphate buffer solution and

pH 6.5 was used as blank The reaction mixture comprised 1.6 mL of 0.05

enzyme extract diluted two-fold with the extraction buffer The experiments were designed with three replications

3.4.2 CAT enzyme assay

CAT activity was recorded at 240 nm at 30s intervals during 2 minutes

described by Beers and Sizer (1952) and Khoa et al (2017) The

experiments were designed with three replications Blank sample was

mL of 0.1 M sodium potassium phosphate buffer, pH 7.0 The reaction