gloeosporioides fungus through the expenditure of disease rate, disease index and the constituent factors of yield and yield when using preparations with different concentrations[r]
Trang 1EDUCATION AND
TRAINING
OF SCIENCE AND TECHNOLOGY
GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY
-
PHAM DINH DUNG
Study to produce oligochitosan-silica nano and investigate the induced systemic resistance against anthracnose
disease caused by Colletotrichum spp on hot chilli
(Capsicum frutescens L.)
Major: Biotechnology
Code: 9 42 02 01
SUMMARY OF BIOTECHNOLOGY DOCTORAL THESIS
Ho Chi Minh City – 2020
Trang 2and Technology - Vietnam Academy of Science and Technology
Supervisor 1: Assoc Prof., Dr NGUYEN TIEN THANG Supervisor 2: Assoc Prof., Dr BUI VAN LE
Reviewer 1:
Reviewer 2:
Reviewer 3:
The thesis shall be defended in front of the Thesis Committee
at Academy Level at Graduate University of Science and Technology - Vietnam Academy of Science and Technology At hour date month year 20
The thesis can be found at:
- The National Library
- The Library of Graduate University of Science and
Technology
Trang 3INTRODUCTION
1 The necessity of the thesis
Hot chili (Capsicum sp.) is a spice plant grown in the
tropics and consumed around the world due to its high economic value However, diseases caused by fungi, viruses and bacteria are the major constraints to hot chili production Among the diseases on hot chilli, pathogenic fungus is one of the main reasons cause of loss 10-80% hot chilli production in Vietnam, India, Thailand, Korea… Common diseases on hot
chili are caused by Rhizoctonia solani, Colletotrichum spp., Botrytis cinerea, Fusarium oxysporum, Phythopthora capsica,…, anthracnose disease, caused by Colletotrichum spp
is one the most destructive diseases restricting hot chili
production Colletotrichum spp mainly causes anthracnose
disease in hot chili This fungus damages on branches, leaves, flowers and fruits The symptoms of anthracnose are circular or angular sunken lesions on hot chili fruits and shaped brown spots with dark brown edges on leaves, with concentric rings of acervuli When symptoms are more serious, hot chili fruits become shrinkage and inedible or drop, which can cause 70-80% yield loss
Nowadays, many kinds of fungicides are used to control pathogenic fungi in plants However, the utilization of high concentration of fungicide disrupts the balance of useful microorganisms in soil This condition can become good environment for the growth of pathogens and existence of chemical resistant insect pests The fungicidal residues in agricultural products and soil cause the groundwater pollution, environmental pollution, especially health hazard of human and animals To prevent intentionally using of fungicides, disease tolerant plants are grown but the result is not effective These strains are low productivity and instability Moreover, plant elicitors, used as biological solutions, activate the disease resistance mechanism in plants This solution is becoming a trend in sustainable and green agriculture development With
Trang 4the aim of reducing the use of toxic chemicals and genetically modified plants Chitin and silicon are two common ingredients
in nature Many studies reported that chitosan, oligochitosan (chitin derivatives), along with silicon and nano silica have biological activities such as antimicrobial, antifungal and increasing disease resistance ability in most of plants, they help plant to secrete enzymes, bioagents for pathogenic prevention, and promote the plant growth and development
Based on the above mentions, “Study to produce oligochitosan-silica nano and investigate the induced systemic
resistance against anthracnose disease caused by Colletotrichum spp on hot chilli (Capsicum frutescens L.)” was carried out
2 The objectives of the thesis
- Identify the pathogenicity potential and classificate at
species level of Colletotrichum spp isolated from hot chilli in
Vietnam
- Produce stable oligochitosan-silica nano, which potentially highly induced effectively the systemic resistance against anthranose disease on hot chilli, based on the combination of low molecular weight chitosan with nano silica
- Detect the potential control the anthracnose disease
caused by C gloeosporioides and C truncatum on hot chilli in vitro, greenhouse and opened-field conditions of oligochitosan-
silica nano created
3 The main contents of the thesis
- Content 1: Isolation, investigation of pathogenicity
and morphological and molecular identification of
Colletotrichum spp causing anthracnose disease in hot chilli
- Content 2: Improving technology for making
oligochitosan-silica nano (SiO2)
- Content 3: Evaluation of the ability of
oligochitosan-silica nano stimulating resistance against C gloeosporioides and C truncatum causing anthracnose disease in hot chili in in vitro condition
- Content 4: Evaluation of the ability of
oligochitosan-silica nano stimulating resistance against C gloeosporioides
Trang 5and C truncatum causing anthracnose disease in hot chili in
greenhouse and field conditions
Chapter 1 LITERATURE REVIEW
1.1 Introduction of hot chili (Capsicum sp.)
Hot chili (Capsicum sp.) belongs to the family Solanaceae,
originating from the Americas Hot chili plants usually grow in clump, 60-80 cm or 1 m high, having many branches and smooth body; the leaves alternate, oblong shaped, apex pointed The flowers grow alone in the leaves Peppers are easy to grow and suitable for many kinds of soil and ecological areas Hot chili peel contains alkaloid-capsaicin Hot chili grows in warm condition and high humidity but dry condition for maturation The suitable temperature for growth and development of hot chili is 18-30oC Hot chili can not grow well in high temperature- above 32oC or low temperature- below 15oC (Tripodi and Kumar 2019) Hot chili genus has about 25-30
species, 5 species (Capsicum frutescens L., C annuum L., C chinense Jacq, C pubescens Keep and C baccatum L.) have been domesticated and cultivated (figure Nowadays, C frutescens is the most popular strain, then C annuum L.(Jaret et
al 2019)
1.2 Introduction of Colletotrichum spp and anthracnose
disease of chilli
1.2.1 Introduction of Colletotrichum spp
Colletotrichum species belong to Kingdom-Fungi, Division
- Ascomycota, Class-Sordarriomycetes, Order-Phyllachorales,
Family-Phyllachoraceae Disease caused by Colletotrichum spp
very popular on plants (ornamental plants, leafy plants and fruity trees) According to the ranking of Molecular Plant
Pathology Journal (2012), Colletotrichum spp was ranked No
8 among the top 10 pathogenic fungi and caused anthracnose, which seriously affects growth, development and productivity
on many valuable crops such as citrus, hot chili (Capsicum sp.),
Trang 6soybeans (Glycine max (L.) Merr.), and tomatoes (Solanum lycopesicum) (Dean et al 2012; Cannon et al 2012) Fig 1.1
Figure 1.1 Disease cycle of Colletotrichum spp
1.3 Resistance mechanism and stimulation of disease resistance in plants
1.3.1 Resistance mechanism
There are two resistance mechanisms in plants: the first mechanism is based on histological structure and the second one is chemical biology (Andersen et al 2018) In histological structure mechanism, there are four mechanisms, depending on the way of invasion of pathogen (Shen et al 2017) In chemical biology, the resistance mechanism starts at the infected point ((Localized Acquired Resistance or LAR), then spreading through plant system (Systemic Acquired Resistance or SAR) (Gao et al 2015) SAR is interacted with pathogen induced PAMPs (Pathogen-Associated Molecular Patterns) or DAMPs (Associated Molecular Patterns) whereas MAMPs (Microbes-Associated Molecular Patterns) is associated with induced resistance or ISR ((Induced Systemic Resistance) SAR and ISR support plants against the attack of pathogens (virus, fungi,
Trang 7bacteria, pests, herbivore, chemicals) Studies of molecular biology report that elicitor hormones such as acid salicylic (SA), acid jasmonic (JA) và Ethylen (ET) play an important role in controlling signaling network of resistance, SA has a role in SAR whereas ET is in ISR way (Imran and Yun 2020)
1.3.2 Elicitor in plant
Elicitors have been recognized recently with the role of resistance When elicitors are used to treat in seed, leave, root or soil, plant defenses are triggered, which help plants resist pathogens or reduced external lesions Elicitors do not directly destroy pathogens but induce plant defense The number and origin of elicitors are diverse Elicitors attach to receptors and perception and signal transduction, then stimulate plant innate immunity Elicitors are divided into two groups: general elicitors and specific elicitors General elicitors are able to induce defense response in host and nonhost plants, while specific elicitors trigger defense in specific host General elicitors activate primary innate immunity and PTI (PAMP triggered immunity) Specific elicitors are produced by some pathogens (fungi, bacteria) which release effectors (Avr protein), inhibiting plant primary innate immunity, thus some plants become sensitivity with pathogen In this case, plants activate second resistance mechanism to induce secondary innate immunity or ETI (Effector-triggered-immunity) via resistance genes, these genes neutralize avirulence genes Chemical structures of elicitors are different, containing different functional groups such as oligosaccharide, peptide, protein, glycoprotein and lipid Oligosaccharide elicitors include oligoglucan, oligochitin, oligochitosan and oligogalacturonic They are molecules from cell wall (glucan, chitin, microbial flagellin or lipopolysaccharide (LPS)), or molecules from pathogens Some functions of elicitors are still unknown In plant pathogen interactions, elicitors induce production of enzymes that degrade cell wall, release pectic segments, oligogalacturonides (OGAs) play the role of internal elicitor (Abdul-Malik et al 2020) Fig.1.4
Trang 8Figure 1.4 Characteristics of resistance mechanism in plants
(A) and PTI pathway of chitin (B) Elicitors produced by virus or insects can be fatty acid amino acid conjugates They lead to the formation of volatile compounds that attract or activate insect resistance genes Chemical elicitors activate resistant as well as accumulate phytoalexin Elicitors are abiotic agents such as metal ions and inorganic compounds, or metabolites from other organisms such
as chemicals released from an attack site or accumulating in the system due to disease or insects (Tawasaki et al 2017; Jamiolkowska 2020)
1.4 Chitin/ Chitosan and Silic in disease resistance stimulation
1.4.1 Role of Chitin / chitosan in disease resistance
Chitin (poly N-acetylglucosamin) is macromolecule composed of repeating N-acetyl-D-glucosamine units linked by β-(1-4) glucoside, high molecular weight Chitosan is polyglucosamin metabolized from chitin after deacetylation The level of deacetylation effects on solubility of chitosan in diluted acid solution A special function of chitosan chemical structure is the presence of oxidize amin group This group
Trang 9becomes cation in acid medium, forcing the solubility of chitosan into poly-electrolyte in solution This is natural products, non-toxic, environment friendly and applied widely (Katiyar et al 2015) Studies of chitin and its hydrolyzed fragments showed that they have ability to effect directly on pathogens such as fungi and oomycete through mechanisms to increase resistance of plants based on pathway the PAMP triggered Immunity (PTI), which helps plant release substances that resist pathogen invasion (Imran et al.2020) In addition, when chitin penetrates into plant tissue, it usually binds around intrusive sites and has three main effects: firstly, constructing an isolated barrier to prevent pathogens spreading from intrusive site and protecting other healthy cells At the isolated site, the plant will recognize to stimulate the sensitive reaction, then release reactive oxygen species (ROS) to help strengthen cell walls and alert adjacent cells Chitin has a positive charge and is able to adhere to biofilms, chitin provides the ability to quickly heal wounds when mechanics are damaged or pathogens attack Chitin is capable of activating plant defense mechanisms, chitin interacts with plant tissues and stimulates secretion of protective enzymes such as chitinase, glucanase, disease-resistant proteins
or phytoalexin compounds, from that pathogens are killed and plants are resisted (Jamiolkowska 2020) In some researches, chitin and oligochitin were used to resist pathogen in plants These studies showed that the mechanism of chitin resistance through the PTI pathway, chitin is role of kinase receptor in chitin and plant interactions (Fig 1.4B) Chitin associated with receptor subunits including CEBiP (Chitin elicitor binding protein, motif lysine or LYM) and CERK1 (Chitin elicitor receptor kinase 1) on the cell membrane that initiates the RLCK signaling pathway (Receptor Like cytoplasmic Kinase) transmitted to RLCK185 via MAPKK phosphorylation (Mitogen activated ptotein kinase) in order to induce plant disease resistance from chitin A similar mechanism was also
found on PBL27 receptors of Arabidopsis thaliana (Kawasaki
et al.2017) In this experiment, the oligochitin fraction (DP: 7-8)
Trang 10was found to be suitable for signaling pathways in Arabidopsis thaliana Based on two model plants, results confirmed the role
of chitin in plant resistance
1.4.2 The role of Silic in plant disease resistance
Silicon (Si) is widely used in agriculture and many different fields Si increases the growth and productivity of plants In some plants, Si improves some morphology and mechanical properties (height, urea index, leaf exposure to light, resistance) Si reduces evaporation and increases strengthens resistance to drought-tolerant crops, salinity and metal toxicity and increases enzyme activity Si also participates in the regeneration of cell walls, an effective plant defense barrier Si protects plants against stress without affecting crop growth and productivity Moreover, Si has been shown to improve resistance in many plants to various pathogenic agents (fungal, virus) (Sakr 2016; Bhat et al 2019)
In theory, two hypotheses propose that Si enhances pathogenic resistance The first thing is the association with higher sedimentation of Si in the leaves to form physical barriers, then preventing invading pathogens (physical mechanism) The second thing is related to the role of biological activity in the expression of natural defense mechanisms (biochemical mechanism) with the increased activation of defense enzymes such as polyphenoloxidase, peroxidase, phenylalanine ammonialyase, chitinase, β-1,3-glucanase,…; the enhancement
of anti-fungi, phenolic metabolites (lignin), flavonoid, phytoalexin and disease related proteins in plants; and the activation of preventive gene in plants (Epstein 2009)
1.5 Synthesis of oligochitosan, nano silica and application in disease resistance
The combination of chitosan/oligochitosan with other metals such as gold, silver or zinc (nano particles) shows more effective results because they can combine and widely apply in many fields such as medical, food and agriculture
Trang 11Chapter 2 EXPERIMENTS AND METHODOLOGY 2.1 The research contents
Content 1: Isolation, investigation of pathogenicity and
morphological and molecular identification of Colletotrichum
spp causing anthracnose disease in chilli
- Isolating fungi causing anthracnose disease on chilli
- Experiment 1: Evaluate the pathogenicity of isolated fungal
Content 2: Improving technology for preparation
oligochitosan-silica nano (SiO2)
- Preparation of oligochitosan fractions
- Evaluation of inhibition of prepared oligochitosan on C gloeosporioides
- Preparation of nano-silica particles from rice husks
- Preparation oligochitosan-silica nano
Content 3: Evaluation of the ability of
oligochitosan-silica nano stimulating resistance against C gloeosporioides và
C truncatum causing anthracnose disease in hot chili in in vitro
condition
- Experiment 3 and 4: Evaluate the effect of oligochitosan on
resistance against C gloeosporioides and C truncatum
- Experiment 5 and 6: Evaluate the effect of nano silica on
resistance against C gloeosporioides and C truncatum
- Experiment 7 and 8: Evaluate the effect of oligochitosan silica
on resistance against C gloeosporioides and C truncatum
Content 4: Evaluation of the ability of
oligochitosan-silica nano stimulating resistance against C gloeosporioides và
C truncatum causing anthracnose disease in hot chili in
greenhouse and field condition
Trang 12- Experiment 9 and 10: Evaluate the effect of oligochitosan on
resistance against C gloeosporioides and C truncatum in
greenhouse
- Experiment 11 and 12: Evaluate the effect of nano silica on
resistance against C gloeosporioides and C truncatum in
greenhouse
- Experiment 13 and 14: Evaluate the effect of
oligochitosan-silica nano on resistance against C gloeosporioides and C truncatum in greenhouse
- Experiment 15 and 16: Evaluate the effect of oligochitosan on
resistance against C gloeosporioides and C truncatum in field
condition
- Experiment 17 and 18: Evaluate the effect of nano silica on
resistance against C gloeosporioides and C truncatum in field
condition
- Experiment 19 and 20: Evaluate the effect of
oligochitosan-silica nano on resistance against C gloeosporioides and C truncatum in field conditions
The diagram is detailed as following
2.2 Methodology
2.2.1 Isolation, investigation of pathogenicity and identification of fungus causing Anthracnose disease by Colletotrichum spp on chilli
2.2.1.1 Method of isolating fungi causing anthracnose disease
on chilli
Isolation of fungi using PDA culture
2.2.1.2 Evaluatation the pathogenicity of isolated fungal strains
in vitro and in vivo condition
Content 2
Content 3
Result of contents 1, 2 Content 4
Content 1
Trang 13The pathogenicity of the fungal pathogens was assessed by the level of disease on leaves/fruits according to QCVN 01-160:2014/Ministry of Agriculture and Rural Development
2.2.1.3 Identification of morphology and molecular biology of pathogenic fungal strains
Identification the morphology of disease fungal strains by Sutton (1992) Identification of molecular biological by PCR, based on reference to the sequence of primers that amplify the specific gene regions of fungi ( ITS4,5; GPDH; TUB2; GS; CHS and CAL)
2.2.2 Improving technology for making oligochitosan-silica nano (SiO 2 )
2.2.2.1 Preparation of oligochitosan segments with low molecular weight by irradiation method to determine dose of irradiation ɣ Co60 ray combine with H 2 O 2
Methods for preparing oligochitosan segments have low molecular weight (2.5 kDa-10 kDa), investigating some characteristics of segmented properties (IR và XRD)
Assessing the ability of inhibiting fungal pathogens of oligochitosan segments by measuring the inhibitory activity of
diameter (mm) of Colletotrichum spp colonies growing on
PDA environment with or without supplementation of
oligochitosan modulation fraction
2.2.2.2 Preparation of nano-silica particles from husk source
Calcination method at high temperature 700oC with HCl 5-10% was used to prepare nano-silica particles from husk and characterized the properties of nano-silica particles (TEM,
XRD, EDX)
2.2.2.3 Preparation of oligochitosan-silica nano
Mixing materials between oligochitosan and silica nano were at suitable pH endurance (5; 6.5; 7.5; 8.5) in combination with HEC thickener and investigation of composition properties
(TEM, FT-IR)
Trang 142.2.3 Evaluation of the ability of Colletotrichum spp of oligochitosan-silica nano on hot chili plants in vitro condition 2.2.3.1 Evaluation of anthracnose disease caused by the fungus Colletotricum spp on hot chili
Factors affecting the effectivity of disease resistance were carried out on a porous type of 50 holes (55cm x 30cm x 5cm) The second-leaf chilli plants were transferred to plastic cups grown in the growth room, the condition was 16 am/ day with a temperature of about 28oC ± 2oC Hot chili plants were treated with pathogens and inoculants according to each treatment before analysis
2.2.4 Evaluation of resistance to Colletotrichum spp of oligochitosan-silica nano on hot chili plants in greenhouse and
field conditions
Experiments in greenhouses and fields were arranged randomly, one factor and three replications Each treatment was arranged with 30 hot chili plants/replication Number of experimental plots were 5 treatments x 3 replicates = 15 plots Each experimental plot had an area of 20m2, total experimental area was 900 m2
2.2.5 Data analysis
Data were analyzed with ANOVA and Duncan's classification test with a confidence probability of P <0.01 with SAS 9.1 program
Chapter 3 RESULTS AND DISCUSSION
3.1 Isolation, investigation of infection and identification of
Colletotrichum spp on chilli (Capsicum frutescens L.)
20 samples were collected from Tan Chau District-Tay Ninh Province (TN), Cu Chi District- Ho Chi Minh City (HCM) and Thanh Binh District-Dong Thap Province (ĐT) Chilli anthracnose samples were chosen based on typical symptons from shoots (Th), leaves (L) and fruit (Tr) Isolated fungi were cultured on PDA, then observed the colour and the mycelium after 7 days based on morphological characteristics such as