Dissertations / Theses on the topic 'Bacillus mycoides'

The objective of this study was to investigate the plant defense pathways induced by Bacillus mojavensis isolate 203-7 (203-7) and B. mycoides isolate BmJ (BmJ) and to test their ability to control fungal pathogens on tomato and cucumber by means of systemic acquired resistance (SAR). An Arabidopsis thaliana mutant - Botrytis cinerea pathosystem was used to investigate plant defense pathways activated by 203-7 and BmJ. A. thaliana wild type (Col-0), ein2-1, jar1-1, NahG, ndr1-1/npr1-2, and npr1-1 mutants were induced by application of bacilli, distilled water, or chemical inducers. Both bacilli reduced disease severity on wild type and NahG mutants, but provided no reduction on jar1-1, indicating that induction was salicylic acid (SA) independent but jasmonic acid (JA) dependent. 203-7 induced plants had lower disease severity on npr1-1 and ein1-2 mutants but were equivalent to buffer controls on jar1-1 mutants. BmJ did not decrease disease severity on npr1-1, jar1 or ein2-1 mutants. Enzyme assays confirmed the induction of chitinase, beta-1,3-glucanase, and superoxide dismutase by 203-7 and BmJ. These results demonstrate that induction by 203-7 is JA dependent and NPR1 independent. BmJ is SA independent but NPR1 and JA/ethylene dependent. Bacilli were tested for their ability to control B. cinerea grey mold on hydroponically grown greenhouse tomatoes and Glomerella cingulata var. orbiculare on cucumber by means of SAR. Weekly foliar applications of bacilli were able to significantly (a=0.05) reduce the severity of grey mold leaf lesions and to reduce the area under the disease progress curve (AUDPC) calculated for seven Botrytis stem canker ratings. Chitinase, beta-1,3-glucanase, and SOD activity of apoplastic fluids were not significantly (a=0.05) increased by the treatments. Bacilli reduced total and live spore production of G. orbiculare per mm 2 of lesion and increased beta-1,3-glucanase activity of cucumber apoplastic fluids. Applications of BmJ compared to fungicides for the control of anthracnose in cucumber (var. 'General Lee') and cantaloupe (var. 'Athena') were evaluated in 2004 and 2005 field experiments. BmJ applied seven days before inoculation provided disease reduction in cucumber and cantaloupe statistically equal to the fungicide treatments.

This study is focused on biosynthesis and characterization of Selenium nanoparticles (SeNPs) by Bacillus mycoides SelTE01 (biogenic SeNPs), evaluating their ability as antimicrobial agents. In so doing, a comparison between biogenically and chemically synthesized SeNPs was carried out, in order to stress differences and similarities. During my project, I worked in the Environmental Microbiology Laboratory at University of Verona and in Biofilm Research Group at University of Calgary (Canada). At University of Verona, I synthesized biogenic SeNPs by Bacillus mycoides SelTE01 grown with Na2SeO3 and chemical SeNPs using L-cysteine, ascorbic acid or a mix of SDS and Na2S2O3. I also characterized both SeNPs using Dynamic Light Scattering (DLS), Z potential measurement, Scanning Electron Microscopy (SEM) analysis and Energy Disperse Spectrometer (EDS) analysis. In Biofilm Research Group in Calgary, I evaluated SeNPs antimicrobial activity against pathogenic biofilms, normally resistant to conventional methods of disinfection. I investigated SeNPs ability to inhibit biofilm formation, exposing pathogenic biofilms to different concentrations of SeNPs and using Minimum Biofilm Eradication Concentration (MBEC) test and Calgary Biofilm Device (CBD). MBEC test is a high throughput screening assay used to determine the efficacy of antimicrobials against biofilms. It’s based on use of CBD: particular 96-well plate in which one batch culture apparatus allows multiple species biofilms to be tested against a lot of variables. In particular, I used CBDs coated of hydroxyapatite (HA), component of bones and teeth. In so doing, I was able to verify that biogenic SeNPs have stronger antimicrobial activity than those chemically synthetized.

Fungal diseases pose significant challenges for grapevine producers in Kentucky due to the region’s abundant moisture and relative humidity. Methods to reduce fungicide application frequency would prove both economically and temporally valuable to producers. A field experiment was established in Bowling Green, KY in 2017 to investigate Bacillus mycoides isolate J (LifeGard) as a supplement to a fungicide program for systemic acquired resistance (SAR). Three fungicide treatment regimens were implemented consisting of a program modelled from the Midwest Fruit Pest Management Guide (2017) and an identical program supplemented with 140 g ha-1 LifeGard per application (both applied on 14 day intervals), a reduced frequency application every 28 days supplemented with 140 g ha-1 LifeGard, and an untreated control. Treatments were applied to 9-year-old French-Hybrid grapevines (cv. Chambourcin); each treatment was replicated 3 times in a randomized complete block design. All treatments were applied with a backpack sprayer delivering 150 L ha-1 at 2 Bar pressure. Canopy management, fertility, herbicide, and insect management were standardized across treatments and no supplemental irrigation was applied. Data collected included fruit yield, pH, ºBrix, and titratable acidity (TA). Data were analyzed with SAS PROC GLIMMIX; differences in means were determined at  < 0.05. Plots supplemented with B. mycoides had lower fruit pH than untreated plots but higher fruit pH than the traditional fungicide program. Treatment regime did not influence Brix, TA, or total yield; however, all treated plots yielded more high quality fruit than the untreated control.

碩士<br>國立中興大學<br>化學工程學系所<br>100<br>In recent years, microbial pesticides for biocontrol control has become the goal of agricultural development. In this study, the production of biosurfactant by Bacillus mycoides NP02 under peptone-based medium was found to reached 71.27±1.93 mg/l. Various types of additives were added into main culture. It was observed that corn oil could reduce the broth tension to 33 mN/m with peptone-based medium, and its biosurfactant production could reach 1744.81±144.89 mg/l, which was approximately 20-fold to that of the control. This result demonstrates oil addition to medium in submerged culture could enhance the biosurfactant production. The biosurfactant produced by B. mycoides NP02 was analyzed with HPLC, and its retention time of 14.6 min is different from that of the surfactin standard. This unknown biosurfactant in broth is expected to be a surfactin isoform, which need further identification. On the other hand, the optimizing medium enhanced biosurfactant production by Bacillus subtilis CWS1. In carbon source study, sucrose gave the best biosurfactant production, reaching 2283.09±86.85 mg/l. In nitrogen source study, ammonium sulfate was identified to be the most favorable source for biosurfactant production, reaching 2613.16±49.69 mg/l. In the medium optimization, carbon source, nitrogen source and corn oil were chosen as designing factor. According to the response surface methodology, the optimal concentrations of sucrose, ammonium sulfate and corn oil were 50 g/l, 3 g/l and 5 g/l, respectively, where the best biosurfactin production reached 8057.57 mg/l.

碩士<br>國立中興大學<br>植物病理學系所<br>100<br>Bacillus mycoides CHT2402 and NP02 were effective in respectively reducing 48.1% and 46.2% disease severity of Fusarium wilt of tomato plants caused by Fusarium oxysporum f. sp. lycopersici Fol-04 in the greenhouse. In order to explore related mechanisms for inducing tomato plants resistant to Fusarium wilt disease by the biocontrol agent, a platform was set up for simultaneously culturing tomato seedlings, B. mycoides, and F. oxysporum f. sp. lycopersici Fol-04 in the flask cultivation system. Biolog GP Microplate was used to analyze the utilization of carbon and nitrogen sources by F. oxysporum f. sp. lycopersici Fol-04, B. mycoides CHT2402 and NP02. The results showed that sucrose concentration of Murashige’s and Skoog’s (MS) medium was adjusted to 1% (w/v) had more suitable interaction among tomato seedlings, the pathogen and biocontrol agents. The roots of tomato seedlings could be completely colonized by B. mycoides CHT2402 and NP02 nine days after the seeds were treated with cell suspension of the biocontrol agents and grown in the flask and greenhouse cultivation systems. Tomato seeds were incubated in the cell suspension (108 cfu/ ml) of B. mycoides CHT2402 and NP02 for 3 days, and then they were transplanted to the modified MS medium in the flask. Two weeks later, each of tomato seedlings was inoculated with single spore of F. oxysporum f. sp. lycopersici Fol-04 near the root. It was found that tomato seedlings could be protected from the pathogen by B. mycoides CHT2402 and NP02 for five days in the flask cultivation system. To study mechanisms for controlling tomato Fusarium wilt by the biocontrol agent, the root tissues of tomato plants treated with B. mycoides CHT2402 and NP02 were analyzed by tissue section and qPCR techniques. The results of Spurr’s resin block section indicated that the cell wall thickness of epidermis cells of tomato plant treated with B. mycoides CHT2402 and NP02 did increase 0.1-0.16 μm and 0.12-1.17 μm, and the cell wall thickness of cortex cells increased 0.05-0.1 μm and 0.12-0.18 μm. In addition, the results of qPCR showed that expression of LOX and PAL genes of tomato plant were induced by B. mycoides CHT2402 and NP02 in both cultivation conditions. According to above results, it was found that B. mycoides CHT2402 and NP02 were able to control tomato Fusarium wilt if they could colonize the roots and vascular tissues of tomato plants prior to the pathogen infection.

碩士<br>國立中興大學<br>植物病理學系所<br>100<br>Watermelon is one of the important fruits consumed in the world. In fact, the cultivation area for watermelon is the second largest vegetable worldwide. Among the fungal diseases, Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (Fon), limits watermelon production. This pathogen colonizes in vascular bundle which causes the difficulty of water transport and leads to wilting of plants. Currently, one of the most effective ways to control this disease is the resistant line breeding. However, this method is time consuming. In this experiment, Bacillus mycoides CHT2401 and CHT2402, isolated from land field, were cultured respectively on TSA (tryptic soy agar), NA (nutrient agar), and KB (King’s B medium) media, to evaluated the inhibition of mycelia growth and spore germination of Fon by the volatile compounds released by these bacteria. The volatile compounds released by B. mycoides cultured on TSA medium had better inhibition on mycelial growth and spore germination of Fon. Results of spore production of Fon in the presence of volatile compounds released by B. mycoides CHT2402 cultured on NA and KB media for 7 days showed significant difference compared to the control. But there was no difference in mycelial growth and spore germination of Fon between the control and the treatments with B. mycoides volatile compounds. The volatile compounds released by B. mycoides did not cause irretrievable damage to mycelia. Gas chromatography-mass spectrometry analysis showed that the major volatile compound was dimethyl disulfide (DMDS) released by CHT2401 and CHT2402 cultured on both TSA and NA media. The results of mycelial inhibition were similar among treatments of 5.15 μg DMDS and the volatile compounds produced by the treated B. mycoides isolates cultured on TSA medium. Nevertheless, the mycelial inhibition by the volatile compounds produced by B. mycoides in both NA and KB media was lower than that by the 5.15 μg DMDS treatment. For spore germination and the mycelial growth after spore germination, the corresponding DMDS amounts for inhibition were larger than that for mycelial growth. The possible reason for the higher concentrations of DMDS in spore suspension is that the volume of spore suspension is smaller than that of the medium. The Fon spores treated with 5.15 μg DMDS swelled and branched earlier comparing to the spores treated with bacterial volatile compounds. This indicates that the concentration of DMDS in B. mycoides volatile compounds used in our experiments was less than 5.15 μg. In survival test of chlamydospore treated with DMDS, treating infested soil with 206 μg DMDS for 13 days and treating infested soil with 515 μg DMDS for 3 days both decreased the survival rate of chlamydospore to 53%; whereas treating infested soil with 772.5 μg DMDS for 15 days decreased the survival rate of chlamydospore to 20%. According to our results, DMDS in the volatile compounds released by B. mycoides could reduce the number of chlamydospores in infested soil. SEM observation revealed that the Fon mycelia treated with B. mycoides volatile compounds and DMDS showed surface shrinkage. Furthermore, needle structures were observed on the Fon mycelia treated with volatile compounds released by B. mycoides cultured on TSA medium and DMDS. This structure could be caused by excess DMDS.

碩士<br>淡江大學<br>化學學系碩士班<br>104<br>The objectives of this investigation were to produce a novel chitosanase for application in industries and waste treatment. The conversion of chitinous biowaste into bioactive chitooligomers (COS) is one of the most promising applications of chitosanase. A chitosanase-producing strain was isolated form Taiwan soil and identified as Bacillus mycoides. The chitosanase was produced using 0.5% (w/v) squid pen powder (SPP) as the sole carbon/nitrogen source, and these enzymes was purified from the culture supernatant by column chromatography. Extracellular chitosanase was purified to 130-fold with a 35% yield, and its molecular mass was approximately 48 kDa. The purified chitosanase exhibited optimum activity at 50 °C, pH 6, 10 and was stable at 30-50 °C, pH 4-10. The chitosanase was significantly inhibited by Cu2+, Ba2+, Zn2+, Fe2+, Mn2+, EDTA and PMSF. The value of Km and Vmax for chitosanase were 0.098 mg/mL and 1.336 U/mL, respectively. A combination of the HPLC and MALDI-TOF MS results showed that the chitosan oligosaccharides obtained from the hydrolysis of water-soluble chitosan by TKU038 comprise oligomers with various degrees of polymerization (DP), ranging from 3 to 9. The TKU038 culture supernatant and COS mixture exhibited 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging activities. The COS with high DP exhibited enhanced DPPH· radical scavenging compared with COS with low DP. The COS also had anti-inflammatory activity and enhanced the growth of L. paracasei TKU010 and L. paracasei TKU012. TKU038 has potential applications in SPP waste treatment and industries for COS production as a medical prebiotic.

碩士<br>國立中興大學<br>植物病理學系所<br>96<br>Damping-off caused by Rhizoctonia solani kühn AG-4 or Pythium aphanidermatum Edson is a serious disease of cabbage seedlings, especially in culture medium in Taiwan. Thus, preventing culture medium from contamination of these pathogens should be an important method for controlling the disease. Effective evaluations of the volatile compounds produced by Bacillus mycoides isolates CHT2401 and CHT2402 grown on plates of King’s medium B, Luria-Bertani medium, nutrient agar, potato dextrose agar, soy powder milk agar, and tryptic soy agar for suppression of mycelial growth of R. solani RST-04 and P. aphanidermatum Pa01 were conducted. The two bacterial isolates grown on soy powder milk agar and tryptic soy agar expressed better effectiveness in inhibiting the pathogens. The volatile compounds produced by the two bacterial isolates were identified using ammonia detector tube and Gas Chromatography – Mass Spectrometry (GC-MS). The major volatile compounds produced by both isolates were identified as ammonia and dimethyl disulfide that were much more effective in inhibiting mycelial growth of R. solani RST-04 and P. aphanidermatum Pa01. The mycelial morphology of R. solani RST-04 and P. aphanidermatum Pa01 treated with dimethyl disulfide and the volatile compounds produced by B. mycoides CHT2402 respectively were observed under scanning electron microscope and transmission electron microscope. The hyphal structures of R. solani RST-04 and P. aphanidermatum Pa01 after treatments were malformed and the numbers of the organelles in their hyphal cells significantly decreased. These results demonstrated that dimethyl disulfide, ammonia, and the volatile compounds produced by B. mycoides CHT2402 could be harmful to the mycelia of the pathogens. In the greenhouse, the culture media were individually treated with the culture solutions of B. mycoides CHT2401and CHT2402 grown in soy powder milk. The culture solutions of the Bacillus isolates could increase the plant seedlings biomass of asparagus bean, cabbage, edible rape, lettuce, and tomato compared to the untreated control. Furthermore, the culture solutions of the Bacillus isolates were separately tested for controlling damping-off of cabbage seedlings caused by R. solani RST-04 or P aphanidermatum Pa01. The culture solutions of the two bacterial strains could respectively reduce 28% and 27% disease incidence of cabbage seedlings caused by P. aphanidermatum Pa01, but not significantly reduce the disease incidence of seedlings damping-off caused by R. solani RST-04. The results suggested that B. mycoides isolates CHT2401 and CHT2402 were potential biocontrol agents on controlling damping-off of cabbage seedlings caused by P. aphanidermatum Pa01.

碩士<br>國立中興大學<br>國際農學研究所<br>102<br>Green mould disease, caused by several Trichoderma spp. is a major disease of Agaricus mushroom around the world. In severe cases, green mould infection inhibited the mushroom fruiting formation resulting to no production at all. Control method widely used against green mould is strict hygiene and sanitation within the growing area accompanied by application of commercial chemicals. However, continuous usage of chemical control may lead to the development of resistance of the fungal pathogen. Apart from that, rampant chemical application has adverse effect to the environment and human health. In the present study, soil sample of green mould disease was isolated from an infected growing substrate medium of Agaricus bisporus. After morphological characterization, the isolate was identified as Trichoderma virens. In vitro study of the causal agent of green mould disease showed aggressive growth at 28°C. Fresh mushroom cap of A. bisporus turned brown and decreased in size when artificially infected with 10 ?l of T. virens containing 103 cfu/ml. Same aliquot containing 105 cfu/ml caused green sporulation of the mushroom caps. In this study, the possibility of using bacterial strains as biocontrol agent and as alternative control method was carried out. Under laboratory conditions, thirteen Bacillus mycoides strains including NP02, BM02, RA-01, RA-08, RA-11, GS02, LG-01, NRHG-04, NRCL-03, TSFA-01, TSO-05, WT-15, and WT-16 were evaluated for suppressive effects against T. virens, the identified green mould mushroom pathogen isolated from local mushroom growing substrate. All the tested B. mycoides strains exhibited varied antagonistic effects on mycelial growth of the mushroom pathogen. Among the antagonistic bacterial strains, BM02 was the most effective fungal suppressant. Infection of T. virens was on mushroom cap was reduced upon exposure to BM02. Test revealed that 100 x dilute BM02 greatly reduced the population density of T. virens in an artificially infected casing soil. Application of BM02 suspension in growing media of white fleshed button mushroom (ARI-1H11) increased yield by over 60% whereas the same suspension reduced yield of brown fleshed button mushroom (CTB-10) by 40%. Contrasting result revealed that the effect of B. mycoides in relation to growth and yield of button mushroom depends on the strain level. Nevertheless, field tests confirmed that BMO2 applied on mushroom growing media does not have adverse impact on button mushroom.

碩士<br>淡江大學<br>化學學系碩士班<br>104<br>Chitosan has been more popular in recent years due to its good bio-compatibility, low toxicity and rarely no allergic reaction to human body. Chitosan also has other bioactivity like reduce blood pressure, lower cholesterol, increase immunity, and its antibacterial property can be used in many clinical applications. For the best resource of Chitin/Chitosan we extracted from the seafood wastes (squid pens, shrimp or crab shells),which is abundant of polysaccharides, and glucosamine that we can use enzyme hydrolysis to achieve it. In traditional way, using a lot of chemical to receive chitosan and its oligomer, but in nowadays, we use the natural way, chitosanase bacteria strain TKU039 identified as Bacillus sp. from the soil in New Taipei City Linkou mountain. TKU039 was culture in medium with the squid pen powder (SPP) and shrimp powder (SP).The SPP medium was found most suitable with 1.0% SPP as a sole carbon/nitrogen source optimized under cultured at 37°C with 150 rpm for 5 days in 150 mL of medium containing 1% SPP, 0.1% K2HPO4 and 0.05 % MgSO4·7H2O.

Selenium nanoparticles (SeNPs) are 10 to 400nm spheres composed of zero-valent selenium. SeNPs can be synthesized either chemically or biologically by microorganisms, plant extracts or enzymes. Biogenic SeNPs display a capping layer of organic molecules, which confer unique characteristics to such SeNPs, e.g. a major stability over time and a more efficient antimicrobial activity. Composition and role of the capping layer are mostly unknown and currently under investigation. In this study, environmental strains Bacillus mycoides SeITE01, Stenotrophomonas maltophilia SeITE02, Achromobacter sp. R2A, Ensifer sp. R2D and Lysinibacillus sp. R1E are considered, which are able to biosynthesize SeNPs. In the first section, SeNPs from the five bacteria are analyzed: microplate colorimetric assays are performed in order to quantify total carbohydrates, protein and lipids contents of such SeNPs capping layers. Moreover, SeNPs are treated with different protocols to remove part of the organic layer. Effect of such treatments on capping composition and SeNPs stability are studied and compared for all the five strains. In the second section, SeNPs produced by B. mycoides SeITE01 are analyzed from a proteomic point of view: biogenic SeNPs capping layer proteins are identified. Chemical SeNPs exposed to a SeITE01 cell free extract are analyzed as well. Identified proteins are compared in order to establish which proteins bind specifically biogenic SeNPs and are more probably involved in SeNPs formation. Finally, a model for SeNPs transport through SeITE01 cell wall is formulated, based on proteomic evidence. Native proteins activity assay and microscopy analysis are performed, in order to confirm the new model. In conclusion, studying the organic capping layer of biogenic SeNPs from different strains is of paramount importance to understand the effect of such molecules on SeNPs characteristics and formulate hypotheses on biosynthesis mechanism. SeITE01, SeITE02, R2A, R2D and R1E biosynthesized SeNPs show a different ratio of carbohydrates, proteins and lipids components of the capping layer and differently respond to treatments. Particularly, Gram-negative strains (SeITE02, R2A, R2D) show similar composition and respond to treatments in a similar fashion, while Gram-positive strains (SeITE01, R1E) show more variability. For SeITE01, proteomic and microscopy analyses led to a new model formulation for SeNPs transport outside the cell. Together with previous studies, this new hypothesis can contribute to a more complete vision of SeNPs synthesis in this aerobic strain.

碩士<br>國立中興大學<br>園藝學系所<br>104<br>The objective of this study was to research the effects of different nitrogen source and inoculation Bacillus mycoidies on Paphiopedilum orchids of nitrogen uptake and growth. Nitrogen source of media included different inorganic and organic nitrogen, in media of different treatment and inoculation beneficial microorganism regular to investigated into nitrogen compounds and nitrate reductase activity, and analyze of nutritious element on plant and media, to understand the effects of inoculation beneficial microorganism and different nitrogen source on Paphiopedilum orchid of nitrogen uptake and growth. Paphiopedilum orchids were grown in media with different rate of inorganic and organic nitrogen source for 90 days, plant treated with 60 mM organic nitrogen source had better morphological trait, significantly higher FAA in root and element contents in plant than other treatments. Paphiopedilum orchids were grown in media with different nitrogen type for 90 days, plant with peptone had better treated morphological trait, significantly higher fresh weight in shoot and root than other treatments, however, if plant treated with casein, they have significantly lower FAA in shoot and root、total nitrogen of plant、chlorophyll content of leaf and total nitrogen uptake in media. The other hand, plant treated with inorganic nitrogen source, significantly higher FAA in shoot and NRA in root. When inoculation Bacillus mycoidies on plant of in vitro Paphiopedilum cultivated in nitrogen medium with YE, plant growth will inhibition, media have IAA and NH4+ produce, if cultivated in nitrogen medium with inorganic nitrogen, non- significant in plant growth, media haven’t IAA and NH4+ produce, and population survival of BM increases as days decrease. These results show, if cultivated in nitrogen medium with peptone on the same nitrogen concentration, Paphiopedilum orchid were growth better. However media with different rate of inorganic and organic nitrogen, organic (YE) concentration increases as the plant growth increases. The other hand, inoculation Bacillus mycoidies on plant of in vitro Paphiopedilum cultivated in nitrogen medium with YE, high NH4+ produce by BM and media pH too high bring on plant growth inhibition.

博士<br>國立中興大學<br>植物病理學系所<br>106<br>Twenty isolates of Bacillus mycoides Flügge were obtained from crop rhizophere soils in central and southern Taiwan. Using inoculation method of cucumber seedlings cultured in the hydroponic plastic beaker, B. mycoides isolates BM02 and NP02 were able to effectively control the Pythium damping-off caused by Pythium aphanidermatum isolates Kpa and Potap1. The greenhouse experiments indicated that both cucumber seeds coated with cell suspension of each isolate and seedlings drenched with each fermentation broth of B. mycoides isolates BM02 and NP02 were also effective in controlling cucumber seedling damping-off. Especially, the isolate NP02 could reduce more than 30% of damping-off disease. Both cucumber seed coated with cell suspension and its seedlings drenched with each fermentation broth of B. mycoides isolates BM02 and NP02 could also enhance the growth of cucumber seedlings. Both isolates of B. mycoides increased fresh weight and height of cucumber seedlings by 25 % compared to the control. Foliar analyses of the treated cucumber seedlings indicated that B. mycoides had a stimulatory effect on nutrient uptake of cucumber seedlings in the greenhouse. NP02 promoted plant to uptake macro-element nutrients including N, K, Ca and Mg, except for P. BM02 promoted uptake of macro-element nutrients including N and Mg. Cucumber seedlings could uptake more concentrations of micro-element nutrients including Fe and Mn stimulated by NP02 (177ppm and 72 ppm) than by BM02 (115ppm and 27 ppm) compared to untreated control (130ppm and 18 ppm). In addition, B. mycoides isolates BM02 and NP02 were cultured in LB (Luria-Bertani-Miller) broth with tryptophan and showed the ability of producing auxin (IAA). NP02 produced the concentration of IAA at 36.2ppm more than BM02 at15.9 ppm. This study indicated that B. mycoides isolates BM02 and NP02 were more effective in controlling cucumber damping-off and promoting the growth of cucumber seedlings. It is suggested that isolates BM02 and NP02 of B. mycoides are potential biocontrol agents for developing biopesticide. Due to unique colony form, the colony size of Bacillus mycoides isolates BM02 and NP02 grown on the solid agar were used to evaluate their physiological conditions and sensitivities to pesticides. The optimal growth temperatures were at 28-32℃for BM02 and at 28-36℃ for NP02. Four different recipe media were used to evaluate the colony growth of isolates BM02 and NP02. The results showed that nutrient agar (NA) was the best for the colony growth of both isolates. However, colony growth of isolate BM02 could not grow on potato dextrose agar (PDA). Moreover, 31 carbon sources and 38 nitrogen sources were respectively added into modified Czapeck’s media without carbon and nitrogen compounds. The results indicated that colony growth of B. mycoides isolates BM02 and NP02 cultured on the solid media with polygalacturonic acid salt (PolyGA salt) and ornithine was significantly enhanced. In addition, three peptones from natural source were more suitable for colony growth of B. mycoides isolates BM02 and NP02. The combinations of PolyGA salt and five nitrogen sources were evaluated the colony growth of B. mycoides isolates BM02 and NP02. The result indicated that combination of PolyGA salt and ornithine was the best for the growth of isolates BM02 and NP02 in modified Czapeck’s medium. The sensitivity of B. mycoides isolates BM02 and NP02 to antibiotics and chemical pesticides was evaluated by using paper disc method. Among 7 antibiotics and 13 chemical pesticides tested, 6 antibiotics and 3 chemical pesticides, which included streptomycin, neomycin, kanamycin, ampicillin, cholortetracycline, chloramphenicol, paraquat, chlorothalonil, and mancozeb, inhibited B. mycoides isolates BM02 and NP02 for 12hrs on NA plates. In advance, B. mycoides isolates BM02 and NP02 were cultured on NA medium with each of 27 chemical pesticides. Three herbicides completely inhibited the colony growth of B. mycoides isolates BM02 and NP02. Among 17 fungicides and 5 insecticides, isolate BM02 could grow on NA medium with each of 15 chemical pesticides. However NP02 could grow on NA medium with each of 14 chemical pesticides. Bacillus mycoides isolates BM02 and NP02 were effective in controlling cucumber damping off. Volatile metabolites released from B. mycoides isolates BM02 and NP02 on different culture media showed antagonistic to Pythium aphanidematum isolates Kpa and Potap1. Especially, the inhibitory activity of volatile metabolites produced from isolate BM02 grown on tryptic soy agar (TSA) was more stronger. NP02 was dually cultured in potato sucrose agar (PSA) and V8 juice agar (V8A) and showed suppressive effect on the pathogen. Moreover, the experiments indicated that the lipopeptides produced by B. mycoides were able to inhibit zoospore production by P. aphanidermatum. Several natural matter media and synthetic media were used to culture B. mycoides isolates BM02 and NP02 and evaluated for inhibiting P. aphanidermatum. Co-application of fermentation broths of B. mycoides and P. aphanidermatum was markedly effective in inhibiting zoospore release and causing zoospore lysis. PSG-C fermentation broths of B. mycoides completely inhibited zoospore production by P. aphanidermatum and protected detached cucumber leaves from water soak lesion. The fermentation broths of B. mycoides were analyzed by salt-assisted homogeneous liquid-liquid micro-extraction coupled with high-performance liquid chromatography (SHLLME-HPLC). The results showed one isoform fraction of surfactin was extracted and eluted from PSG-C fermentation broths, 3 isoform fractions of surfactin and 3-5 isoform fractions of iturin A were extracted and eluted from SM fermentation broths of B. mycoides. Moreover, comparison of zoospores released from sporangia of P. aphanidermatum suppressed by lipopeptide fractions extracted from the culture broth of B. mycoides isolate NP02 and commercial surfactin was conducted. Inhibitory activity (over 99%) of lipopeptide fraction from B. mycoides isolate NP02 at 100 ppm was greater than commercial surfactin (750 ppm). This study indicated that B. mycoides could produce surfactin/ lipopeptide metabolites to inhibit zoospore production and lyse zoospores.

碩士<br>國立中興大學<br>國際農學研究所<br>103<br>After the green revolution, a large amount of chemical fertilizers and pesticides have been applied to agriculture plantation to increase crop production. Overuse of chemical fertilizers and pesticides have raised numerous problems related to environmental hazards and food safety. To minimize these problems, scientists began to develop alternative methods in a hope to reduce the use of toxic chemicals. Plant oils and the combination with Bacillus mycoides can be ones of possible alternatives as a non-pesticide agent to control plant pests. The purpose of the study was to evaluate the efficacy of plant oils and the combination with Bacillus mycoides on controlling insect pests, including the green peach aphid (Myzus persicae) and the two-spotted spider mite (Tetranychus urticae), on strawberry. Effect of five plant oils, including sunflower seed oil (SF), peanut oil (PN), soybean oil (SY), moringa oil (MO) and coconut oil (CO) on the mortality of the green peach aphid and the two-spotted spider mite was evaluated with the oils sprayed or dropped on strawberry leaves. The Results showed that emulsified moringa oil, peanut oil and coconut oil at 200-fold dilution caused 95%, 65% and 55% mortality on the green peach aphid, respectively. Emulsified coconut oil at 200-fold dilution were able to reduce the offspring reproduction of the green peach aphid by 90%, while the moringa oil and peanut oil reduced the aphid offspring reproduction by 55%. Emulsified moringa oil at 100-fold, 200-fold and 500-fold dilution caused 100%, 76% and 64% mortality of the two-spotted spider mite, respectively. While the coconut oil at the same dilutions caused 60%, 88% and 44% of mite mortality, respectively. As the plant oils were sprayed to the strawberry leaves, and aphids allowed to contact with the oil film on the leaves, the moringa oil at 250-fold dilution also caused higher aphid mortality (75.52%) than the coconut oil (50.62%). Furthermore, combinations of emulsified moringa oil and the Bacillus mycoides isolates BM103, BM104 and BM105 were also tested for their synergistic effects on inducing the aphid mortality. Results showed that the combination of moringa oil and Bacillus mycoides isolate BM103 was the most effective one (causing 33.56% of aphid mortality) compared to the other combinations. This study indicated that plant oils such as moringa oil, peanut oil, coconut oil, and soybean oil have the potential as botanical insecticide for controlling the aphid and acarine pests on strawberry. However, further investigations under the field conditions are needed.

碩士<br>國立中興大學<br>國際農學研究所<br>99<br>In the conventional agriculture system, high applications of pesticides and fungicides were used widely in order to obtain yields as much as possible. Nowadays, the serious pollutions caused by these synthetic chemicals motivated scientists to find other solutions. Based on previous studies, plant growth-promoting rhizobacteria (PGPR) had potential to be the alternatives. These non-pathogenic microbes not only enhance plant growth but may also indirectly trigger the defensive responses of plants. Through the induced systemic resistance (ISR), pests aboveground may be affected negatively and the better fitness of plants is promised. In this study, the effects of three strains of Bacillus mycoides, CHT2401, CHT2402, and CHR001, on plant growth promoting were evaluated. Additionally, the performance of tobacco cutworm (Spodoptera litura) and green peach aphid (Myzus persicae) fed on treated plants were assessed as well. Cabbage and sweet pepper were first sowed in the soil contained each strain, and the dry weight of plants were recorded 5 weeks later. As for insect performance, both cutworms and aphids were restrained on the specific leaves, after two days, the relative growth rate (RGR), nymph development time and final population size of each treatment were recorded, respectively. In the same time, leaves before feeding and leaves after feeding were collected to analyze enzymes activity such as polyphenol oxidase (PPO) and peroxidase (POD). Results demonstrated that plants under bacterial treatments had significant higher biomass and leaf area than those of the control treatment. However, insects fed on bacteria-treated plants did not show any negative effects. In contrast, the bigger population size was built when cabbage was inoculated with B. mycoides. Since there had no significant PPO or POD activity induced, it is assumed that the responses of insect to plant could be influenced by nutrition-related factors. Although these result did not meet the prediction in the beginning, the function of promoting plant growth of these three strains were still valuable. Possibly, with different operating practices, more promising results could be obtained. No matter these beneficial microbes act as biofertilizers or pesticides or both, they should play a significant role in the sustainable agriculture system for sure.

Bacillus mycoides SeITE01 and Stenotrophomonas maltophilia SeITE02 are environmental bacterial isolates that rely on detoxification processes to transform selenite (SeO32-), a highly toxic and bioavailable chemical species of selenium, into insoluble and virtually nontoxic elemental selenium (Se0) with the formation of biogenic selenium nanoparticles (Bio-SeNPs). In the last decade, Bio-SeNPs have attracted attention for their interesting applications in the nanotechnology, industrial and medical fields not only due to their special physico-chemical features, but also for their attractive antimicrobial activities and anticancer properties. These worthwhile biotechnological traits are related to the presence on Bio-SeNPs of an external organic coating, whose composition and role are mostly unknown and currently under investigation. In the first part of this thesis, FTIR (Fourier Transform Infrared) spectroscopy was applied to study the SeO32- bio-reduction process analysing the bio-molecular composition of both SeITE01 and SeITE02 cells. The analysis was conducted during the diverse cellular growth phases and in different conditions, namely untreated (growth without the presence in the medium of sodium selenite Na2SeO3) and Se-treated (exposure to the stress factor SeO32-). Moreover, along with FTIR spectroscopic analyses, the biogenic intracellular and extracellular SeNPs bio-produced by the bacterial strains and collected with two extraction methods (vacuum filtration and pelleting processing) were examined also using DLS (Dynamic Light Scattering) measurements and TEM (Transmission Electron Microscopy) imaging. In the second part of this research, metabolomics was used to investigate the biological reduction and effect of Na2SeO3 on SeITE01 and SeITE02 cells by a LC-MS (Liquid Chromatography Mass-Spectrometry) approach. Both intracellular and extracellular metabolites and their concentration fluxes during a defined time course and in response to the exposure of bacterial cells to SeO32- were studied. From the results obtained with all the investigation techniques it was possible to observe and underline two distinct behaviors and trends assumed by the bacterial strains. SeITE01 cells showed substantial changes when exposed to SeO32-, activating a series of macromolecular responses and biochemical pathways to defend the cells against the toxic action of both the oxyanion and the Se nanostructures. Moreover, different FTIR spectral trends were acquired with regard to the organic coatings present on the surfaces of the biogenic SeNPs synthesized by this bacterium, and the most marked differences were recorded according to their different localization (intracellular or extracellular). The Bio-SeNPs production in this Gram-positive microorganism was also described by the data collected with the DLS and the zeta-potential measurements. The analyzed Bio-SeNPs showed an average dimension between 637 and 393 nm for the intracellular Bio-SeNPs, and 147 nm for the extracellular ones. Low value of negative potentials suggest a lower stability of these nanostructures in solution. On the contrary, the Gram-negative bacterium SeITE02 did not present drastic changes in the macromolecular composition after SeO32- exposure, and the variations recorded were mainly due to the maintenance of vital cellular functions. DLS data revealed an average size of 315 nm for the intracellular Bio-SeNPs and 160 nm for the extracellular ones, whilst negative potential values at or below -30 mV were recorded, indicating a remarkable stability of these biogenic Se nanostructures and a lower tendency to form aggregates and thus to precipitate. Thus, the results obtained in the course of this investigation revealed two distinct attitudes and responses on the part of the two microorganisms SeITE01 and SeITE02 to the SeO32- exposure and Bio-SeNPs production.