tóm tắt luận án tiếng anh: Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).

Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).Nghiên cứu một số đáp ứng sinh lý, hóa sinh liên quan đến phản ứng tự bảo vệ của cây đậu tương Nam Đàn (Glycine max (L.) Merr.) đối với rệp muội đen (Aphis craccivora Koch).

TRAN NGOC TOAN

STUDY ON PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES RELATE TO DEFENSE RESPONSE

OF SOYBEAN (Glycine max (L.) Merr cv “Nam Dan”)

TO COWPEA APHID (Aphis craccivora Koch)

Speciality: Plant Physiology

No: 9420112

SUMMARY OF DOCTORAL THESIS

HA NOI - 2023

Supervisors: 1 Associate professor Tran Thi Thanh Huyen

2 Associate professor Mai Van Chung

Review 1: Associate professor Nguyen Van Dinh, Hanoi Pedagogical University 2 Review 2: Associate professor Cao Phi Bang, Hungvuong University

Review 3: Associate professor Bui Minh Hong, Hanoi National University of Education

This thesis will be defensed in front of Thesis Judging Committee at Hanoi

National University of Education at ………, 2023

This thesis can be found at: National Library, Hanoi

Or Hanoi National University of Education’s Library

1 Tran Ngoc Toan, Tran Thi Thanh Huyen, Mai Van Chung (2016), “Antioxidative

defense responses to aphid-induced oxidative stress in Glycine max (L.) Merr.cv.

“Nam Dan”, Vietnam Journal of Science and Technology, 54(6), tr 719-728, DOI:

10.15625/0866-708X/54/6/7952, (ISSN 0866-708x)

2 Ngoc Toan Tran, Thi Thanh Huyen Tran, Ngoc Dai Do, Van Chung Mai (2017),

“The accumulation of SA- and JA-signaling pathways in the response of Glycine max cv “Nam Dan” to infestation by Aphis craccivora” Journal of Plant Protection Reseacrh, 57 (4), tr 321–330, DOI: 10.1515/jppr-2017-0043, (ISSN:

1427-4345)

3 Tran Ngoc Toan, Ngo Thi Lien, Nguyen Thi Hoang Anh, Tran Thi Thanh

Huyen and Mai Van Chung (2019), “Effect of Cowpea Aphid on the

biosynthetic pathway of Salicylic acid in Glycine max cv Namdan at reproductive growth stages”, Journal of Science Vinh University , 48 (2A) tr.

29-38, (ISSN: 1859 – 2228)

1 Regarding Rationale of the study

Plant species is affected by many different biotic and abiotic stress fromenvironment in its life cycle Although always suffer a certain amount of damage,most of plant still survive and develop well This is because plants develop differentself-defense mechanisms that are compatible with each effector In the ability toactively protect plants, biochemical defense mechanisms have been attracting theattention of many scientists According to this mechanism, fungi, bacteria, viruses,insects are groups pathogens known to produce elicitors that induce the formation ofmetabolic products of different origins: hormones, enzymes, genes, proteins,secondary exchange products, free radicals ; initiate special protective responsessuch as hypersensitive response (HR); stimulates systemic acquired resistance (SAR)

At the same time, different signal transduction pathways, for example: biosynthesis

of plant hormones salicylic acid (SA), jasmonic acid (JA), ethylene (ET); Theactivated oxygen signaling pathway… is also activated All form defensemechanisms specific to each type of plant for each impact factor

In the interaction between legumes (Fabaceae) and insect, there have beenstudies around the world evaluating the machinery and protective mechanisms of

soybean Glycine max (Diaz-Montano et al 2007; Li) et al 2008), clover Medicago truncatula (Klingler et al 2007; Gao et al 2008), lentils Lens culinaris (Andarge and Westhuizen 2004), alfalfa Medicago sativa (Julier 2004), peas Pisum sativum L (Mai

et al 2014; Morkunas et al 2015) These studies reported that, when under theinfluence of aphids, some plant hormones (SA, JA, ET, etc.), active ingredients such

as pisatin, flavonoids , activated forms of oxygen/nitrogen Endogenous substancessuch as H2O2, NO, as well as superoxide anion radical (superoxide O2., semiquinone),biosynthetic enzymes as well as genes encoding the aforementioned enzymes havedifferent synthesis inducers to participate in protective reactions of plants againstaphids (Morunas et al 2011)

Nam Dan soybean is a local variety associated with the brand "Nam Dan soysauce" of Nghe An province, which was successfully restored in 2009 and started to

be produced widely On this soybean, cowpea aphid (Aphis craccivora Koch) often

arises, causing damage from the end of stem and leaf development, beginning to formflower buds and lasting throughout the fruiting period, and seed filling The assessment

of the self-defense mechanism of plants against aphids will provide an importantdatabase on the physiology of plant tolerance to adverse agents.

Currently, there is no research has been done for this issue on Nam Dan soybean

Due to that, the implementation of " Study on physiological and biochemical responses

relate to defense response of soybean (Glycine max (L.) Merr cv “Nam Dan”) to cowpea aphid (Aphis craccivora Koch)” is needed.

2 Objectives of the study

Analysis and evaluation of a number of physiological and biochemicalresponses related to the self-defense response of Nam Dan soybean when blackaphid attacked at the vegetative growth stages V1, V3 and V5

- Specific goals:

Evaluation of some damage at the cellular level in soybean leaves caused

by aphids through indicators, and lipid peroxidation;

Analysis and evaluation of biosynthesis and pathway of signal molecules such

as JA, SA, H2O2 and superoxide radical O2.- in Nam Dan soybean leaves duringvegetative growth under the influence of cowpea aphids;

Evaluation of the activity of some enzymes involved in the synthesis of theabove signaling molecules These are the LOX enzymes in JA synthesis; PAL andBA2H participate in SA synthesis; SOD and CAT are endogenous H2O2

metabolism enzymes in Nam Dan soybean leaves during vegetative growth underthe influence of cowpea aphid

3 Scientific and practical significance of the study

Studying protective responses of plants to insect attack is a new directionwhich have not done yet in Vietnam so far The current study is the first study onphysiological and biochemical responses related to the self-protective response ofNam Dan soybean in Vietnam

The research results aim to provide scientific data on the self-protectionmechanism of Nam Dan soybean plant against cowpea aphid, contributing to clarifythe theoretical basis of the physiological resistance of the plant to the disease, adverseenvironmental factors, including biotic factors

The project findings were the premise for further studies on the relationshipbetween soybean plants and aphids, thereby finding solutions to prevent aphids onNam Dan soybean variety

4 Contribution of the study

This is the first work on defense ability of Nam Dan soybean plant againstcowpea aphid The findings in this study will be the first scientific data on the self-protection mechanism of Nam Dan soybean plant against cowpea aphid, including:

- Effect of cowpea aphids on Nam Dan soybean leaf cells (Rate of damage incells; Lipid peroxidation of membrance);

- Content of signal molecules pathway in Nam Dan soybean leaves under theinfluence of cowpea aphid (H2O2, O2.-, SA, JA);

- Induction of activity of some enzymes that synthesize/metabolize signalmolecules in Nam Dan soybean leaves under the influence of cowpea aphid (SOD,CAT, PAL, BA2H, LOX)

The results obtained in this study will be important, scientifically meaningful,and valuable references to plant physiology, especially plant tolerance physiology.Moreover, the study will also provide an important database for plant protection,suggestions for research, and application of solutions to enhance the resistance ofNam Dan soybean

5 Thesis structure

Preface

Chapter 1 Introduction

Chapter 2 Material and Methodology

Chapter 3 Result and Discussion

Conclusion and Suggestion

In Vietnam, studies on the interaction between aphids and plants mainly focus

on composition, biological characteristics, pest density and aphid control measures ondifferent crops Some studies have focused on substances with aphrodisiac activityfrom insect parasitic fungi Regarding the self-protection mechanism of plants, thereare studies under the influence of heavy metals Thereby, it suggests that, in Vietnam,there have not been in-depth studies on the self-protection mechanism of plantsagainst the attack of aphids, including soybean

CHAPTER 2 MATERIAL AND METHODOLOGY

2.1 Material

2.1.1 Nam Đan soybean

The plant used in this study is Nam Dan soybean variety (Glycine max (L.)

Merr.), provided by the Agricultural Extension Station of Nam Dan district (Nghe Anprovince)

Sterilized seeds were incubated for germination, then planted in the laboratory.Soybean plants at the stages V1, V3 and V5 (corresponding to the vegetative growthstages, with 1, 3 and 5 compound leaves) were used as research materials

2.1.2 Cowpea aphid

The aphids used in the experiments were cowpea aphid (Aphis craccivora

Koch) cultured by the Department of Applied Entomology (Institute of Ecology andBiological Resources, Vietnam Academy of Science and Technology) Virus-freeindividuals were reared on their host soybean plant, in a grow-chamber at 23 – 250C,relative humidity 70–75 %, light intensity 110–130 μM photons mM photons m2, and the timeperiod 14 hours light/10 hours dark

2.2 Area of study

Study on some physiological and biochemical responses related to

self-protection response of Nam Dan soybean (Glycine max (L.) Merr.) at the vegetative growth stage (V1, V3 and V5) to with cowpea aphids (Aphis craccivora Koch) was

carried out from 2015 to 2019 The experiments were arranged in the Laboratory ofPlant Physiology and Laboratory of Plant Science, Vinh University

The concentrations of plant hormones SA and JA were analyzed in cooperationwith the Institute of Natural Compound Chemistry (Vietnam Academy of Science and

Technology) The expression of the gene encoding PAL was analyzed in collaborationwith the Institute of Biotechnology (Vietnam Academy of Science and Technology).

Other indicators were analyzed at the Plant Physiology Laboratory and the Plant Science Laboratory, Vinh University

2.3 Contents of study

* Effect of cowpea aphids on Nam Dan soybean leaf cells

- Rate of damage in cells

- Lipid peroxidation of membrance

* Content of signal molecules pathway in Nam Dan soybean leaves under theinfluence of cowpea aphid:

* Induction of activity of some enzymes that synthesize/metabolize signal molecules

in Nam Dan soybean leaves under the influence of cowpea aphid:

- Enzymes that convert active oxygen species

+ Enzyme Superoxide Dismutase

+ Enzyme Catalase

- Salicylic acid biosynthetic enzyme

+ Enzyme Phenylalanine ammonia-lyase

+ Enzyme Benzoic 2-hydroxylase

- Jasmonic acid biosynthetic enzyme

Treatment 1 (control): no infestation

Treatment 2: infecting 10 aphids/plant

Treatment 3: infecting 20 aphids/plant

Treatment 4: infecting 30 aphids/plant

Selected aphids (adults, wingless) were carefully transferred to the leaves of theexperimental soybean plants with a fine-bristled brush Pupation or dead individualswere removed every day and supplemented with new individuals to limit the effect ofdensity on aphid performance so that the number of black aphids remained constant

in each experiment Soybean plants in the aphid-infested and control treatments wereplaced separately in net cages (50 × 50 × 50 cm) and placed in a growing chamber at

23 – 25 oC, 70-75% humidity, 110-130 µM photons.m-2.s-1 light intensity and lightingtime is 14 hours light /10 hours dark

At the growth stages V1, V3 and V5, on the experimental treatments, soybeanleaves were collected at 0 h, 24 h, 48 h, 72 h and 96 h, respectively, after aphidinfestation Leaves were taken and equilibrated with electronic analytical balance,then frozen with liquid nitrogen and kept at-70 °C for further analyses Particularly,the content of superoxide anion radicals (O2.-) and hydrogen peroxide (H2O2) weredetermined in fresh raw materials according to the specific contents of the study

2.4.2 Analysis methods

Injury percentage of the plasma membrane

Electrolyte leakage was conductometrically measured to assess the injurypercentage of the plasma membrane (Sullivan et al., 1971)

Lipid peroxidation

The level of lipid peroxidation was determined by thiobarbituric acidreactive substances

(TBARS) assay (Heath R L và Packer L, 1968)

Determination of superoxide anion radical content

The determination of superoxide anion radical (O2.-) content in biologicalsamples was based on its ability to reduce nitro blue tetrazolium (NBT) (Doke N.,1983)

Determination of hydrogen peroxide content

The content of hydrogen peroxide (H2O2) was determined following thespectrophotometric

method (Becana M và cs 1986)

Determination of enzyme superoxide dismutase, the enzyme catalase

The activity of SOD (EC 1.15.1.1) was spectrophotometrically assayed bymeasuring its

ability to inhibit the photochemical reduction of NBT (Beauchamp C và cs.1971)

Salicylic acid measurement

Free salicylic acid (SA) was extracted and quantified following the performance liquid chromatography (HPLC) method described by Yalpani et al.(1993)

high-Phenylalanin amoniac-lyase measurement

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activity was determinedusing the spectrophotometric method (Cahill và McComb, 1992)

Benzoic acid 2-hydroxylase measurement

Benzoic acid 2-hydroxylase (BA2H) was measured by using the HPLC methodaccording to León et al (1995)

Jasmonates measurement

Jasmonic acid (JA) and its metyl este (MeJA) was measured following methoddescribed by GC-MS (Fan et al., 1998)

Enzyme lipoxygenase measurement

Activity of lipoxygenase (LOX, EC 1.13.11.12) was determined using thespectrophotometric method (Sekhar and Reddy, 1982)

RESULT AND DISCUSSION

3.1 Effect of cowpea aphids on Nam Dan soybean leaf cells

3.1.1 The percentage of injury in soybean “Nam Dan” leaves’ cells

Table 3.1 Injury in Nam Dan soybean leaf cells when affected by cowpea aphid

(Pisum sativum L.) when damaged by aphids (Acyrthosiphon pisum Harris).

Accordingly, electrolyte leakage in all 10-day-old seedling leaves infected bydifferent numbers of aphids increased from 0 to 72 h, and then decreased to 96 h Celldamage in all treatments with aphids was highest at 72 h (3.19%, 4.58% and 7.51%respectively for 10, 20 and 30 aphids) higher than the control plants (Van Chung Mai

et al., 2014)

The effect of Pb2+ also resulted in damage to DT84 soybean root cells, this rateincreased after 24 hours and peaked at 48 hours, the highest was 25.03% in the1.0mM Pb2+ formula, up to 96 hours the index decreased to 9.59 - 12.14% (Mai VanChung, Tran Ngoc Toan, 2015) The results obtained in our experiments showed thatthere were some similarities in the increase in cell damage at the time points afterPb2+ treatment or aphid treatment (increased after 24 h of treatment, followed bytreatment continued to increase or maintain, and then fell to a low at 96 hours)

However, the recorded damage rate was completely different, according to whichDT84 soybean root cells were damaged at a very high level (the highest was 25.03%

at 48 hours), while Nam Dan soybean leaf cells had a damage level of 2.71 timessmaller (the highest was 9.23% at 72 hours in phase V1)

When the aphids were affected, the TBARS content in Nam Dan soybean leaves

in the treatments with aphids in the growth stages V1, V3, V5 all increased and werehigher than the control and the baseline time For the low density (10 individuals),TBARS content increased and reached a high level, then maintained or decreased at

72 – 96 hours While the treatments were infested at higher densities (20 individualsand 30 individuals), TBARS concentrations increased and peaked between 48 and 72hours, but they all decreased at 96 hours

The results of our study on Nam Dan soybean are in agreement with previousreports, all of which confirmed that the increase in lipid peroxidation in crops (such

as beans) is directly caused by attack of bed bugs (Capinera J L, 2001) Thus, withthe invasion of black aphid, oxidation caused destruction of membrane integrity by

increasing cell membrane damage and activating lipid peroxidation in Nam Dansoybean The percentage of cell damage tended to increase 24 hours after the aphidattack and reached a high level between 48 and 72 hours, however, they tended todecrease at 96 hours after the aphid attack.

The effect of aphid on Nam Dan soybean leaf cells stimulated strong production

of ROS (O2.- và H2O2) in Nam Dan soybean leaf cells and induced this oxidation Thisprocess will be clarified in the next section

3.2 Content of signal molecules pathway in Nam Dan soybean leaves under the influence of cowpea aphid

3.2.1 Reactive oxygen

3.2.1.1 Superoxide anion radical

Table 3.3 Superoxide anion radical content in Nam Dan soybean leaves

when affected by aphid

The results obtained from our study showed that all 3 growth stages V1, V3,V5 when attacked by aphids led to changes in the content of free radicals O2.- in theleaves of Nam Dan soybean tends to increase rapidly in the early stage (in which, O2-

concentration reaches the highest level after 24 hours since the aphid damage) andthen decreases in about 48-72 hours and reaches the lowest level at the time of pestinfestation 96 hours after bedbug infestation From these results, it is shown that O2-radicals are generated when plants are stressed not only by drought, salinity, hightemperature, cold, heavy metals, ozone, lack of oxygen, aluminum but also byaphids attack.

3.2.1.2 Hydrogen peroxide in Nam Dan soybean leaves when aphid affects

Table 3.4 Hydrogen peroxide content in Nam Dan soybean leaves when

The increase in H2O2 associated with cell death in aphid-infested areas isknown as the oxidative stress in the plant defense mechanism (Radville et al., 2011)and may be the central component in mechanism of cross tolerance in plants (Maffei

et al., 2007) H2O2 also functions as a deterrent to insect feeding (Apel and Hirt2004) The production of H2O2 has been known as a phytoprotective response inmany plant-aphid interactions (Argandona et al 2001; Moran et al., 2002; Zhu-Salzman et al., 2004; Divol and colleagues) et al., 2005; Moloi and van derWesthuizen, 2006; Boyko et al., 2006; Kusnierczyk et al., 2008)