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).
MINISTRY OF EDUCATION AND TRAINING HANOI NATIONAL UNIVERSITY OF EDUCATON 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 THIS STUDY HAS BEEN DONE AT HANOI NATIONAL UNIVERSITY OF EDUCATION Supervisors: Associate professor Tran Thi Thanh Huyen Associate professor Mai Van Chung Review 1: Associate professor Nguyen Van Dinh, Hanoi Pedagogical University 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 LIST OF PUBLICATIONS 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) 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) 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) INTRODUCTION Regarding Rationale of the study Plant species is affected by many different biotic and abiotic stress from environment 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 different self-defense mechanisms that are compatible with each effector In the ability to actively protect plants, biochemical defense mechanisms have been attracting the attention of many scientists According to this mechanism, fungi, bacteria, viruses, insects are groups pathogens known to produce elicitors that induce the formation of metabolic products of different origins: hormones, enzymes, genes, proteins, secondary exchange products, free radicals ; initiate special protective responses such 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); The activated oxygen signaling pathway… is also activated All form defense mechanisms specific to each type of plant for each impact factor In the interaction between legumes (Fabaceae) and insect, there have been studies 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 the influence of aphids, some plant hormones (SA, JA, ET, etc.), active ingredients such as pisatin, flavonoids , activated forms of oxygen/nitrogen Endogenous substances such as H2O2, NO, as well as superoxide anion radical (superoxide O 2., semiquinone), biosynthetic enzymes as well as genes encoding the aforementioned enzymes have different synthesis inducers to participate in protective reactions of plants against aphids (Morunas et al 2011) Nam Dan soybean is a local variety associated with the brand "Nam Dan soy sauce" 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 form flower buds and lasting throughout the fruiting period, and seed filling The assessment of the self-defense mechanism of plants against aphids will provide an important database 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 Objectives of the study Analysis and evaluation of a number of physiological and biochemical responses related to the self-defense response of Nam Dan soybean when black aphid 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 during vegetative growth under the influence of cowpea aphids; Evaluation of the activity of some enzymes involved in the synthesis of the above signaling molecules These are the LOX enzymes in JA synthesis; PAL and BA2H participate in SA synthesis; SOD and CAT are endogenous H2O2 metabolism enzymes in Nam Dan soybean leaves during vegetative growth under the influence of cowpea aphid Scientific and practical significance of the study Studying protective responses of plants to insect attack is a new direction which have not done yet in Vietnam so far The current study is the first study on physiological and biochemical responses related to the self-protective response of Nam Dan soybean in Vietnam The research results aim to provide scientific data on the self-protection mechanism of Nam Dan soybean plant against cowpea aphid, contributing to clarify the theoretical basis of the physiological resistance of the plant to the disease, adverse environmental factors, including biotic factors The project findings were the premise for further studies on the relationship between soybean plants and aphids, thereby finding solutions to prevent aphids on Nam Dan soybean variety Contribution of the study This is the first work on defense ability of Nam Dan soybean plant against cowpea aphid The findings in this study will be the first scientific data on the selfprotection mechanism of Nam Dan soybean plant against cowpea aphid, including: - 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 the influence of cowpea aphid (H2O2, O2.-, SA, JA); - Induction of activity of some enzymes that synthesize/metabolize signal molecules 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 of Nam Dan soybean Thesis structure Preface Chapter Introduction Chapter Material and Methodology Chapter Result and Discussion Conclusion and Suggestion Publications References Supplements CHAPTER INTRODUCTION There are many works dealing with defense mechanisms at the molecular level, related to the activation of plant signaling pathways, the reception of molecular signals, the exchange relationship between plants and animals crossover of these molecules in the defense signaling network Signals mediating various defensive responses are of interest to plant scientists not only because of their biological nature but also because of their important role in agriculture In Vietnam, studies on the interaction between aphids and plants mainly focus on composition, biological characteristics, pest density and aphid control measures on different crops Some studies have focused on substances with aphrodisiac activity from insect parasitic fungi Regarding the self-protection mechanism of plants, there are 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 plants against the attack of aphids, including soybean CHAPTER 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 An province) Sterilized seeds were incubated for germination, then planted in the laboratory Soybean plants at the stages V1, V3 and V5 (corresponding to the vegetative growth stages, with 1, and 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 and Biological Resources, Vietnam Academy of Science and Technology) Virus-free individuals 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 time period 14 hours light/10 hours dark 2.2 Area of study Study on some physiological and biochemical responses related to selfprotection 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 of Plant Physiology and Laboratory of Plant Science, Vinh University The concentrations of plant hormones SA and JA were analyzed in cooperation with the Institute of Natural Compound Chemistry (Vietnam Academy of Science and Technology) The expression of the gene encoding PAL was analyzed in collaboration with 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 the influence of cowpea aphid: - Active oxygen + Superoxide free radicals + Hydrogen peroxide - Salicylic acid - Jasmonic acid * 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 + Enzyme Lipoxygenase 2.4 Methodology 2.4.1 Experiment design Soybean plants at the vegetative growth stages V1, V3 and V5 were used to set up the experiments At each growth stage, the experiment was arranged in a completely randomized block design (RCBD) with treatments, and replications: Treatment (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 the experimental soybean plants with a fine-bristled brush Pupation or dead individuals were removed every day and supplemented with new individuals to limit the effect of density on aphid performance so that the number of black aphids remained constant in each experiment Soybean plants in the aphid-infested and control treatments were placed 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 lighting time is 14 hours light /10 hours dark At the growth stages V1, V3 and V5, on the experimental treatments, soybean leaves were collected at h, 24 h, 48 h, 72 h and 96 h, respectively, after aphid infestation 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) were determined 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 injury percentage of the plasma membrane (Sullivan et al., 1971) Lipid peroxidation The level of lipid peroxidation was determined by thiobarbituric acid reactive substances (TBARS) assay (Heath R L Packer L, 1968) Determination of superoxide anion radical content The determination of superoxide anion radical (O2.-) content in biological samples 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 the spectrophotometric method (Becana M cs 1986) Determination of enzyme superoxide dismutase, the enzyme catalase The activity of SOD (EC 1.15.1.1) was spectrophotometrically assayed by measuring its ability to inhibit the photochemical reduction of NBT (Beauchamp C cs 1971) Salicylic acid measurement Free salicylic acid (SA) was extracted and quantified following the highperformance liquid chromatography (HPLC) method described by Yalpani et al (1993) Phenylalanin amoniac-lyase measurement Phenylalanine ammonia-lyase (PAL, EC 4.3.1.24) activity was determined using the spectrophotometric method (Cahill McComb, 1992) Benzoic acid 2-hydroxylase measurement Benzoic acid 2-hydroxylase (BA2H) was measured by using the HPLC method according to León et al (1995) Jasmonates measurement Jasmonic acid (JA) and its metyl este (MeJA) was measured following method described by GC-MS (Fan et al., 1998) Enzyme lipoxygenase measurement Activity of lipoxygenase (LOX, EC 1.13.11.12) was determined using the spectrophotometric method (Sekhar and Reddy, 1982) Determination of protein In all the enzyme preparations, the protein in soybean seedlings’ leaves was determined following the method of Bradford (1976) using bovine serum albumin (Sigma-Aldrich) as a standard 2.4.3 Statistical analysis Analysis of variance (ANOVA) was applied to verify whether means from the given experimental variants were significantly different with the level of significance as P-value < 0.05 on SPSS 20.0 software CHAPTER 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 12 H2O2 is a relatively stable, partially reduced form of ROS, and its ability to freely diffuse allows H2O2 to play a controlling role in plant defense responses (Bo´ka et al., 2007) H2O2 induces the intracellular release of Ca2+, which is a very important, ubiquitous signaling molecule involved in many signaling pathways in plants (Tuteja and Mahajan, 2007) H2O2 also activates several members of the mitogen-activated protein kinase (MAPK) family, which are central to the regulation of plant cell responses to stress, for example, AtMPK3 and AtMPK6 in Arabidopsis ( Desikan et al., 2005) As a signaling molecule in plant defense mechanisms, H2O2 accumulation may be the start of a chain of events that trigger physiological and molecular responses to reduce aphid attacks (Argandona et al et al., 2001) In a recent study by Mai et al 2013, a significant enhancement of H2O2 was observed to activate the defense mechanism of legume (Pisum sativum cv Cysterski) against attack by aphid (Acyrthosiphon pisum) Similar to the above results, the increase of H2O2 content in Nam Dan soybean leaves at growth stages V1, V3, V5 infected with aphids within 0-24 hours after aphid invasion was evaluated as closely related to the defensive aspects of the plants In contrast, high levels of H2O2 involved in oxidation can have toxic effects, and uncontrolled generation often causes damage to cellular components such as proteins, lipids, and proteins nucleic acids (Ahmad et al., 2008) The results of our study showed that the invasion of aphid stimulated the strong production of O2.- and H2O2 in the cells of Nam Dan soybean leaves, causing oxidation that destroys the integrity of the leaves membrane due to an increase in the percentage of membrane damage and activation of lipid peroxidation 13 3.2.2 Salicylic acid in Nam Dan soybean leaves when aphid affected Table 3.5 Salicylic acid content in Nam Dan soybean leaves when the aphis affects TG 0h CT V1 growth stage Control 34,28 ± 0,39a B 10 aphids 34,28 ± 0,39a A 20 aphids 34,28 ± 0,39a A 30 aphids 34,28 ± 0,39a A V3 growth stage Control 32,76 ± 0,45a A 10 aphids 32,76 ± 0,45a A 20 aphids 32,76 ± 0,45a A 30 aphids 32,76 ± 0,45a A V5 growth stage Control 30,70 ± 0,48a A 10 aphids 30,70 ± 0,48a A 20 aphids 30,70 ± 0,48a A 30 aphids 30,70 ± 0,48a A Salicylic acid content (ng/g FW) 24h 48h 72h 96h 30,46 ± 0,37a A 78,34 ± 0,53b D 106,42 ± 0,99c D 111,40 ± 0,65d E 35,76 ± 0,44a C 77,58 ± 0,38b D 90,64 ± 0,46c C 97,38 ± 0,41d D 36,24 ± 0,46a C 58,13 ± 0,47d C 50,59 ± 0,41b B 54,63 ± 0,68c C 30,57 ± 0,46a A 54,08 ± 0,43d B 49,59 ± 0,47c B 48,64 ± 0,50b B 48,08 ± 0,53a C 79,32 ± 0,57b D 133,07 ± 0,48c D 151,38 ± 0,46d E 52,65 ± 0,59a E 90,03 ± 0,42b E 137,96 ± 0,76c E 141,24 ± 0,51d D 46,24 ± 0,47a B 62,47 ± 0,43b C 68,13 ± 0,42c C 112,21 ± 0,67d C 49,63 ± 0,50a D 60,39 ± 0,38b B 63,04 ± 0,50c B 63,09 ± 0,57c B 38,48 ± 0,42a B 49,10 ± 0,57b B 79,25 ± 0,41c E 86,15 ± 0,43d D 41,25 ± 0,27a C 68,05 ± 0,55b E 72,48 ± 0,41c D 91,19 ± 0,55d E 41,09 ± 0,31a C 60,45 ± 0,47c D 54,26 ± 0,28b C 60,76 ± 0,76c C 43,04 ± 0,38a D 56,64 ± 0,52c C 50,41 ± 0,47b B 58,80 ± 0,48d B (Note: Considering each growth stage separately, in the same column there are different exponential letters (lowercase letters) with p