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.)

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. 6 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 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 7 becomes 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) 8 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 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. 9 Chapter 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 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 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 10 - 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 11 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 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) 12 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 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 13 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 (TN-Tr1; TN- Tr2; TN-Tr3; TN-L1; TN-L2; HCM-Tr1; HCM-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 Figure 3.2. Morphological of Colletotrichum gloeosporioides 14 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, 4 species caused disease in wound free condition after 7 days infection (NSLN), including TN-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 TN-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 (T) C. siamense C. scovillei C. truncatum C. truncatum C. truncatum C. truncatum GS (S) C. gloeosporioides C. acutatum C. truncatum C. truncatum C. truncatum C. truncatum CHS (C) C. fructicola C. scovillei C. truncatum C. truncatum C. truncatum C. truncatum CAL (L) C. siamense C. scovillei C. siamense C. siamense C. siamense C. siamense 15 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 3 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 21.0 7.900 5.000 4.200 16 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 0 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. The FTIR spectra of figure 3.6 indicated that the Mw of separated chitosan into oligochitosan were from 2.5 to 7.7 kDa, Structures of these oligochitosan were the same as the initial one (Figure 3.6a). 17 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) và 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 2 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 5 0.5105 10.33 2 5 0.5022 10.04 3 5 0.5128 10.26 The results showed that the size of nanosilica was synthesized by incineration of RH powder at 700oC for 2 h was 10-30 nm. The results also showed the size of silica nanoparticles was Gaussian distribution in Figure 3.8A,B. 18 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 particles were used to test disease resistance and growth promotion in pepper plants as well as to prepare oligochitosan- silica nano hybrid material. 19 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 (C- O-C); peak 1,031 cm-1, 1,074 cm-1 distributed to the stretching 20 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 5 (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, - OH groups of oligochitosan. When the silica nano particles were added to oligochitosan solution, the peaks intensity of chitosan including group (N − H) decreased in number of waves 21 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 ant