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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]
(1)MINISTRY OF EDUCATION AND
TRAINING
VIETNAM ACADEMY 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: 42 02 01
SUMMARY OF BIOTECHNOLOGY DOCTORAL THESIS
(2)The thesis was completed at: Graduate University of Science and 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
(3)INTRODUCTION
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
(4)the 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
(5)and C truncatum causing anthracnose disease in hot chili in greenhouse and field conditions
Chapter 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 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, 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
(6)soybeans (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
(7)bacteria, pests, herbivore, chemicals) Studies of molecular biology report that elicitor hormones such as acid salicylic (SA), acid jasmonic (JA) 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
(8)Figure 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
(9)(10)was 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
(11)Chapter 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 strains in vitro
- Experiment 2: Evaluate the pathogenicity of isolated fungal strains in vivo
- Identify morphology and molecular biology of isolated pathogenic fungal strains
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 C truncatum causing anthracnose disease in hot chili in in vitro condition
- Experiment and 4: Evaluate the effect of oligochitosan on resistance against C gloeosporioides and C truncatum
- Experiment and 6: Evaluate the effect of nano silica on resistance against C gloeosporioides and C truncatum
- Experiment and 8: Evaluate the effect of oligochitosan silica on resistance against C gloeosporioides and C truncatum
(12)- Experiment 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
Content
Result of contents 1, Content
(13)The 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 (SiO2)
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 H2O2
Methods for preparing oligochitosan segments have low molecular weight (2.5 kDa-10 kDa), investigating some characteristics of segmented properties (IR 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
(14)2.2.3 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 treatments x 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 RESULTS AND DISCUSSION
3.1 Isolation, investigation of infection and identification of
Colletotrichum spp on chilli (Capsicum frutescens L.)
(15)mycelial structure, spore’s colour and fungal appressoria under a microscope According to the species classification criteria of Sutton (1992), 20 samples were identified as Colletotrichum with the following characteristics: the mycelium growing on or close to the agar surface, mycelia was in the form of flowers or circles and the colour was white or light orange to pink or drab to umber, with reppled or round edge The micro-sclerotia appear on the surface of mycelia Spores were from cylindrical, circle, one spike and one round head or two round heads to two pointed ends or sickle shaped The sporangium was orange to black colour in drop shaped The acervuli had fur or not Spores were formed after 12 hours, then the appressoria were formed after 24 hours, which had round, cylindrical, lobed, oval shape or variable shape In the beginning, appressoria were colourless, then change to brown or umber with smooth or rough surface Based on the morphological classification, 10 Colletotrichum species were indentified as C.gloeosporioides (Tr1; TN-Tr2; TN-Tr3; TN-L1; TN-L2; HCM-Tr1; HCM-TN-Tr2; HCM-Tr4; ĐT-Tr1; DT-Tr3) and the others were C.truncatum (TN-Tr4; TN-L3; TN-Th1; TN-Th2; HCM-Tr3; HCM-Tr5; HCM-L1; HCM-L2; ĐT-Tr2; ĐT-Th1)
TN-Tr2 HCM-Tr2
(16)TN-Th1 HCM-L2 ĐT-Th1
Figure 3.3 Morphological of Colletotrichum truncatum
The infection results of 20 species Colletotrichum spp isolated from chilli fruits in caused and non-caused wound showed that all species caused anthracnose disease on fruits in wounds condition, species caused disease in wound free condition after days infection (NSLN), including Tr2 TN-L3, TN-Th1, and ĐT-Th1 All 20 species Colletotrichum spp isolated from chilli leaves could cause anthracnose disease in wound condition but not in wound free condition The results showed that TN-Tr2, TN-L3, TN-Th1, TN-Th2, HCM-Tr1, HCM-Tr2 and ĐT-Th1 were truly remarkable The results showed that Tr2, HCM-Tr2 (C gloeosporioides ) and TN-L3, TN-Th1, HCM-L2, ĐT-Th1 (C truncatum) had high toxicity levels
Table 3.6 The results of identification of isolated fungus
Product/ Genome
region
TN-Tr2 (1)
HCM-Tr2 (2)
ĐT-TH1 (3)
TN-TH1 (4)
HCM-L2 (5)
TN-L3 (6) ITS (I) C.gloeosporioides C scovillei C truncatum C truncatum C truncatum C truncatum ACTIN (A) C siamense C siamense C truncatum C truncatum C truncatum C truncatum GAPDH (G) C siamense C scovillei C truncatum C truncatum C truncatum C truncatum
TUBULIN
(17)3.2 Perfecting the technology for making oligochitosan-silica nano
3.2.1 Preparation of low molecular weight oligochitosan by gamma Co-60 irradiation combine with H2O2
When irradiation dose was increased, the molecular weight (Mw) of chitosan decreased in samples of chitosan solution (CTS, 4%) with and without hydrogen peroxide (0.5%) (Table 3.7) The addition of H2O2 led to a rapid decrease in the
Mw of the chitosan product, compared to its without addition H2O2, and the reduction of Mw chitosan product increased
when using high concentration H2O2, from 0.5% to 1% The
results of Table 3.7, two samples of oligochitosan were selected from chitosan solution (4%) / hydrogen peroxite (1%) at dose 10.5 and 17.5 kGy with the Mw of the oligochitosan were about 7.7, and 4.6 kDa, respectively, used to test inhibition in pepper plants
The chitosan solution 4% / H2O2 1% with irradiation dose 21
kGy, DDA decreased from 91.3% to 85.6% (table 3.8) The less Mw of the chitosan or oligochitosan product was, the lower likely PI index was, the narrower and more homogeneous dispersion of the original chitosan sample was (PI = 3.37) With lower chitosan concentration 2%, Mw was decreased faster than in high concentration chitosan solution (4%)
Table 3.7 Change in the Mw of chitosan (4%) by gamma
irradiation dose with and without the presence hydrogen peroxide
Dose, kGy CTS 4% CTS 4%/H2O2 0.5% CTS 4%/H2O2 1%
0 44.500 44.500 44.500
3.5 19.000 17.900 16.700 7.0 14.800 12.600 10.500
10.5 12.300 9.000 7.700
14.0 10.500 6.600 5.500
17.5 9.100 5.500 4.600
(18)Table 3.8 DDA index (%) and PI index of chitosan
Dose, kGy
CTS 4% CTS 4%/H2O2 0.5% CTS 4%/H2O2 1%
DDA % PI DDA % PI DDA % PI 91.3 3.37 91.3 3.37 91.3 3.37 3.5 90.2 2.63 89.9 2.78 89.6 2.69 7.0 89.4 2.60 89.1 2.52 88.5 2.55 10.5 89.0 2.56 88.6 2.48 87.7 2.31 14.0 88.7 2.49 88.0 2.18 86.2 1.95 17.5 88.5 2.43 87.6 1.97 85.9 1.81 21.0 88.3 2.30 87.2 1.88 85.6 1.71
Table 3.9 Mw, PI and DDA of chitosan solution (2%) with
hydrogen peroxide 0.5%
Dose, kGy Mw DDA % PI
0.0 44.500 91.3 3.37
3.5 11.900 90.7 2.90
7.0 6.300 89.4 2.33
10.5 4.400 88.1 1.63
14.0 3.500 87.6 1.40
17.5 2.900 86.4 1.32
21.0 2.500 85.9 1.25
Sample of oligochitosan were selected with the Mw 2.5 kDa, irradiation dose 21 kGy, this sample was used to test the resistant stimulation on pepper plants in Table 3.9
(19)Figure 3.6 The FT-IR spectrum (IR) and the X-ray diffraction
spectra (XRD) of chitosan (a) and oligochitosan fraction with Mw 7.7 kDa (b); 4.6 kDa (c) 2.5 kDa (d)
3 samples of oligochitosan were selected with Mw: 7.7; 4.6 and 2.5 kDa were used to test disease resistant and growth promotion in pepper plants Oligochitosan fractions was used to prepare oligochitosan-silica nano from rice husk (SiO2) The
results showed that oligochitosan fractions (2.4 kDa) with 0.1% concentration inhibited Colletotrichum spp was the most suitable
3.2.2 Preparation of nanosilica (SiO2) from rice husk
The rice husk was treated with acids and incinerated to obtain white nanosilica (SiO2) with the yield of 10.21 ± 0.38 %
(Table 3.15) The size of prepared nanosilica was 10-30nm
Table 3.15 The yield of silica nano from rice husk treated with
acid 5%
Sample Rice husk (g) Nano-SiO2 (g) Yield (%)
1 0.5105 10.33
2 0.5022 10.04
3 0.5128 10.26
The results showed that the size of nanosilica was synthesized by incineration of RH powder at 700oC for h was
(20)Figure 3.8 Figure TEM (A, B); the size distribution by laser
diffraction method (C); XRD (D) and EDX (E) of nano silica praticle
The XRD pattern of the nanosilica was shown in Figure 3.8D, the only one peak at 2 ~22o confirmed the purity and
amorphous structure of nanosilica generated from acid treated rice husks powder In this study, rice husks (not rice husks ash) was treated with HCl before incineration, so the metallic impurities were efficiently removed Only Al2O3 (kα at 1,486
keV) still remained with small amount of 0.7% calculated as atomic percentage Value ka of silicon (Si) and oxygen (O) in EDX spectrum were 1,739 and 0.525 keV, respectively in Figure 3.8E
In conclusion, the rice husks were treated with acids and incinerated to obtain nanosilica The size of nano silica particled was about 10-30nm, high purity and amorphous structure with a peak at 2 ~22o Moreover, the nano silica
(21)3.2.3 Preparation of oligochitosan-silica nano hybrid material
When mixing silica nano and oligochitosan, a homogenous gel was produced in Figure 3.9A However, the stability of gel was different and depended on pH of oligochitosan-silica nano solution The results showed that at pH 5.0 and 6.5, gel was clearly precipitated whereas gel was stable and homologous at pH 7.5 The oligochitosan-silica nano solution sample with pH 7.5 not only did not precipitate over 24 h at room temperature, or even for longer time, but also agglomeration did not occur Thus, pH 7.5 is suitable for obtaining a stable oligochitosan-silica nano solution gel (Figure 3.9D) These results are also in good agreement with Tiraferri et al 2014 studied about chitosan adsorption on the silica surface HEC was added at concentration of 1% (w/v) (Figure 3.9E) to increase the stability of oligochitosan-silica nano solution gel for practical application purposes The oligochitosan-silica nano solution with HEC 1% was more stable than the one without HEC
The particle size was about 8-10 nm, and the silica particle shape differed from the original particles in TEM (Figure 3.9G) The FT-IR spectra of silica nanoparticles in Figure 3.9B showed characteristic absorptions: peaks 3416- 3454 cm-1
assigned to formation of H bonds between the silanol groups (Si-O-H) due to water absorption of silica; the bending vibration asymmetric stretching was at peaks 1,101 cm-1; symmetric
stretching vibration was at peak 820 cm-1; bending vibration was
at peak 467 cm-1 In addition, the FT-IR spectra shows peak of
1,635 cm-1 appointed to bending vibration of H-O-H groups of
the with trapped water molecules in matrix network of silica nano
The FT-IR spectra in Figure 3.9C showed characteristic absorptions: peak 1,647 cm-1 assigned to the elasticing vibration
of C-O; peak 1,539 cm-1 assigned to the elasticing vibration of
C-N; peak 771 cm-1 gave to the bending vibration (N-H); peaks
1,153 cm-1 allocated to the asymmetric stretching vibration
(22)vibration (C-O); peaks 3,420 cm-1, 3,450 cm-1 designated to the
-OH
Figure 3.9 Gel-forming mix for inoculants (A), investigation
of gel strength according to pH 5; 6.5; 7.5; 8.5 (D) final products pH 7.5 and 1% HEC (E) The characteristics such as TEM (G) and TEM pH (H) and initial FTIR spectrum of silica nanoparticles (B) oligochitosan (C) when mixed (F) and final product (I)
The FT-IR spectra in Figure 3.9F showed characteristic absorptions: peak 1,083 cm-1 and 781 cm-1 assigned to Si − O −
C bond, these peaks did not appear on FT-IR spectrum of oligochitosan and silica nano particle In addition, The FT-IR spectra of oligochitosan-silica nano solution in Figure 3.9I showed new peak at 927 cm-1 assigned to Si-O-H bond of
hydrogen bond between nano-silanol groups and -NH2 groups,
(23)lower than oilgochitosan solution due to the surface interaction of oilgochitosan with neutral phase
3.3 Evaluation of immune stimulation against
Colletotrichum gloeosporioides và C truncatum of oligochitosan-silica nano on chilli in vitro
The results showed that oligochitosan was used at dose from 25 to 100 ppm, the disease rate and disease index are lower than the controls Oligochitosan at concentration 25 ppm after 14 days showed the best result, but after 21 days, disease rate and disease index were increased slightly Nano silica and oligochitosan-silica nano reduced disease rate and disease index with concentration at 50-100 ppm and 25-100 ppm, respectively This result confirmed that oligochitosan-silica nano effected on disease rate and disease index, but the effective result were different depending on various treatment stages
3.4 Evaluation of the anthracnose resistance stimulation induced by Colletotrichum gloeosporioides and C truncatum of oligochitosan-silica nano on hot chili in greenhouse and field conditions
Evaluation of the anthracnose resistance stimulation induced by Colletotrichum gloeosporioides C truncatum of oligochitosan-silica nano on hot chili in greenhouse and field conditions was essential Especially in fruiting stage, the yield and quality of hot chili product after harvest were determined 3.4.1 Evaluation of the anthracnose resistance stimulation induced by Colletotrichum gloeosporioides of oligochitosan-silica nano on hot chili in greenhouse
(24)25-100 ppm had disease resistance and affect the yield of hot chili after harvest, in which, the best result was spraying oligochitosan-silica nano at concentration 50 ppm
3.4.2 Evaluation of the anthracnose resistance stimulation induced by Colletotrichum truncatum of oligochitosan-silica nano on hot chili in greenhouse
Spraying oligochitosan has an impact on anthracnose diseases and productivity of hot chili In particular, spraying oligochitosan at concentration of 25 ppm gave better results through criteria For nano silica, there was an impact on anthracnose and growth and development of chilli plants when spraying with nano silica In particular, spraying nano silica at a concentration of 100 ppm indicated the best results Spraying oligochitosan-silica nano impacted on anthracnose status on fruit and growth and development of pepper plants In particular, spraying oligochitosan-silica nano at concentration of 50 ppm gave the best results
3.4.3 Evaluation of the anthracnose resistance stimulation induced by Colletotrichum gloeosporioides of oligochitosan-silica nano on hot chili in field
Oligochitosan had a clear impact on anthracnose resistance as well as on the growth and development of hot chili Different concentrations of oligochitosan had different effects, spraying oligochitosan at a concentration of 25 ppm gave the best results
(25)3.4.4 Evaluation of the anthracnose resistance stimulation induced by Colletotrichum truncatum of oligochitosan-silica nano on hot chili in field
Nano silica and nano-silica oligochitosan all have an impact on the growth and development of peppers In particular, spraying oligochitosan at a concentration of 25 ppm gave the best results For nano silica, there was also an impact on anthracnose resistance and pepper growth and development In particular, spraying nano silica at concentrations of 50 and 100 ppm showed the best results For oligochitosan-silica nano, all experiments had an impact on anthracnose diseases resistance on fruit and the growing growth of hot chili; spraying oligochitosan- silica nano at a concentration of 50 ppm indicated best results
According to results, the application of oligochitosan-silica nano materials in practice is feasible The use of resistant materials on plants is a trend in agriculture for green production The preventive effects of final product in this study are similar to those of traditional chemical drugs Therefore, the possibility of applying the products in this thesis could be applied in reality However, widespread testing should be carried out in the future
(26)CONCLUSIONS AND SUGGESTIONS CONCLUSIONS
- Isolation and identification 20 species of chilli anthracnose disease According to Sutton 1992, isolated samples were identified as Colletotrichum gloeosporioides and Colletotrichum truncatum Molecular identification of Colletotrichum with high toxicity levels (L3, Tr2, TN-Th1, HCM-Tr2, HCM-L2, ĐT-Th1) showed the diversity of this fungi
- Collection of low molecular weight oligochitosan segment (7.7 kDa; 4.6 kDa and 2.5 kDa) showed high antifungal activities (0.5-1%) and successfully prepared nano silica 30nm from husk, as a material for making oligochitosan-silica nano, which was stable at pH 7.5 when combining with HEC (1%) and investigation of characteristics of oligochitosan, nano silica and oligochitosan- silica nano
- Using oligochitosan-silica nano at concentrations of 50-100 ppm after 21 days helped to increase the disease resistance through enzyme activities of chitinase, 1,3 glucanase and accumulation of capsidiol, salycilic acid and jasmonic acid - Evaluation of resistance against Colletotrichum spp of oligochitosan-silica nano in greenhouse:
+ C gloeosporioides: : Through disease monitoring indicators as well as performance and yield constituent indicators, the best results were obtained when spraying oligochitosan 25 ppm or nano silica 100 ppm or oligochitosan-silica nano 50 ppm + C truncatum: Based on disease rate, disease index and the components of productivity and productivity, while using oligochitosan, nano silica and oligochitosan-silica nano at 25 ppm, 50 and 100 ppm, 50 ppm, respectively showed the best results
- Evaluation of resistance against Colletotrichum spp of oligochitosan-silica nano in field:
(27)oligochitosan, nano silica and oligochitosan-silica nano at 25 ppm, 50 and 100 ppm, 50 ppm, respectively showed the best results
+ C truncatum: Through the disease monitoring indicators as well as the constituent indicators of yield and yield, the best results were obtained when spraying oligochitosan 25 ppm; or nano silica 100 ppm; or oligochitosan-silica nano 50 ppm SUGGESTION
Applying oligochitosan-silica nano in large-scale hot chilli In greenhouse and field cultivation, the product should be sprayed at 50 ppm, using triple every days, the first spraying is when the plants are ready to bloom (25 days after planting – NST) to decrease anthracnose damage on leaves, on the fruit and increase hot chili productivity
NEW CONTRIBUTIONS OF THE THESIS
- Improved the process of oligochitosan-silica nano synthesis which can be applied in industrial scale
+ Preparation of oligochitosan having low molecular weight (7.7, 4.6 and 2.5 kDa) by gamma Co-60 irradiation with the different doses on chitosan/H2O2 4% chitosan/0.5% H2O2 and
2% chitosan/ 0.5% H2O2
+ Preparation of nano silica having high purity by mineral rice husks sintering method
+ Preparation of oligochitosan-silica nano having high stability in pH ~ 7.5
(28)LIST OF PUBLISHED WORKS
1 Pham Dinh Dzung, Dang Van Phu, Bui Duy Du, Le Si Ngoc, Nguyen Ngoc Duy, Hoang Dac Hiet, Dang Huu Nghia, Nguyen Tien Thang, Bui Van Le, Nguyen Quoc Hien Effect of foliar application of oligochitosan with different molecular weight on growth promotion and fruit yield enhancement of chili plant, Plant Production Science, 2017, 20 (1), 1-7
2 Pham Dinh Dzung, Le Thanh Hung, Le Si Ngoc, Hoang Dac Hiet, Bui Van Le, Nguyen Tien Thang, Dang Van Phu, Nguyen Ngoc Duy, Nguyen Quoc Hien Induction of anthracnose disease resistance on chili fruit by treatment of oligochitosan— nanosilica hybrid material, Agricultural Sciences, 2017, (10), 1105-1113