• The Plant Pathology Journal
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• 제15권1호
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• pp.21-27
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• 1999

Nonpathogenic mutants of Xanthomonas campestris pv. glycines were generated with Omegon-Kim to isolate genes essential for pathogenicity and inducing hypersensitive response (HR). Three nonpathogenic multants and two mutants showing slow symptom development were isolated among 1,000 colonies tested. From two nonpathogenic mutants, 8-13 and 26-13, genes homologous to hrcC and hrpF of X. campestris pv. vesicatoria were identified. The nonpathogenic mutant 8-13 had a mutation in a gene homologous to hrpF of X. campestris pv. vesicatoria and failed to cause HR on pepper plants but still induced HR on tomato leaves. The nonpathogenic mutant 26-13 had an insertional mutation in a gene homologous to hrcC of X. campestris pv. vesicatoria and lost the ability to induce HR on pepper leaves but still caused HR on tomato plants. Unlike other phytopathogenic bacteria, the parent strain and these two mutants of X. campestris pv. glycines did not cause HR on tobacco plants. a cosmid clone, pBL1, that complemented the phenotypes of 8-13 was isolated. From the analysis of restriction enzyme mapping and deletion analyses of pBL1, a 9.0-kb Eco RI fragment restored the phenotypes of 8-13. pBL1 failed to complement the phenotypes of 26-13, indicating that the hrcC gene resides outside of the insert DNA of pBL1. One nonpathogenic mutant, 13-33, had a mutation in a gene homologous to a miaA gene encoding tRNA delta (2)-isopentenylpyrophosphate transferase of Escherichia coli. This indicated that tRNA modifications in X. campestris pv. glycines may be required for expression of genes necessary for pathogenicity. The mutant 13-33 multiplied as well as the parent strain did in the culture medium and in planta, indicating that loss of pathogenicity is not due to the inability of multiplication in vivo.

• The Plant Pathology Journal
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• 제25권3호
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• pp.256-262
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• 2009

The potential of. nonpathogenic Fusarium oxysporum strain Avr5, either alone or in combination with chitosan and Bion, for inducing defense reaction in tomato plants inoculated with F. oxysporum f. sp lycopersici, was studied in vitro and glasshouse conditions. Application Bion at concentration of 5, 50, 100 and $500{\mu}g$/ml, and the highest concentration of chitosan reduced in vitro growth of the pathogen. Nonpathogenic F. oxysporum Avr5 reduced the disease severity of Fusarium wilt of tomato in split plants, significantly. Bion and chitosan applied on tomato seedlings at concentration $100{\mu}g$ a.i./plant; 15, 10 and 5 days before inoculation of pathogen. All treatments significantly reduced disease severity of Fusarium wilt of tomato relative to the infected control. The biggest disease reduction and increasing tomato growth belong to combination of nonpathogenic Fusarium and Bion. Growth rate of shoot and root markedly inhibited in tomato plants in response to tomato Fusarium wilt as compared with healthy control. These results suggest that reduction in disease incidence and promotion in growth parameters in tomato plants inoculated with nonpathogenic Fusarium and sprayed with elicitors could be related to the synergistic and cooperative effect between them, which lead to the induction and regulation of disease resistance. Combination of elicitors and non-pathogenic Fusarium synergistically inhibit the growth of pathogen and provide the first experimental support to the hypothesis that such synergy can contribute to enhanced fungal resistance in tomato. This chemical could provide a new approach for suppression of tomato Fusarium wilt, but its practical use needs further investigation.

• 한국식물병리학회지
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• 제12권2호
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• pp.137-141
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• 1996

오이에서 분리한 비병원성 Fusarium oxysporum 균주 4-1은 온실에서 오이에서 선접종하였을 때 덩굴쪼김병에 안정적인 방제효과를 보여 세 번의 시험에서 그 방제가가 67~100%에 달하였다. 이 균주는 접종후 90일에도 오이 뿌리에서 높은 빈도로 재분리 되었으며 g 토양당 분생포자 농도가 10\ulcorner개 이상의 높은 농도로 접종하였을 때는 뿌리의 갈변현상을 초래하였다. 1993년부터 1995년에 걸친 세 번의 포장시험에서 이 균주는 오이덩굴쪼김병의 발생을 무처리 발병율 56%, 11%, 35%에 비해 18%, 1%, 8%로 각각 억제하였다.

• 한국식물병리학회지
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• 제13권3호
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• pp.179-183
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• 1997

십자화과 식물에 검은썩음병을 일으키는 Xanthomonas campestris pv. campestris에 대하여 담배 18품종 중 가장 과민반응을 강하게 나타내는 품종(Nicotiana tabacum cv. TC500)을 과민반응 비기주식물로 선발하였다. NTG 돌연변이를 실시하여 얻은 약 4,500개의 mutant들을 $10^{8}$ cells/ml의 농도로 주사 접종하여 비기주식물인 담배 잎에서 과민반응을 유도하지 않는 HR-mutans(XHN 514-774, XHN 620-831)를 선발하였고, 기주식물인 양배추 잎에서 병원성을 나타내지 않는 비병원성변이주(XPN 1001)를 선발하였다. HR mutant를 기주식물인 양배추에 접종한 결과 병원성을 나타내었고, nonpathogenic mutant를 비기주식물인 담배잎에 접종한 결과 과민반응을 유도하였다. 담배 잎에서 HR-mutant와 wild type의 population를 조사한 결과 wild type은 24시간 이후 급격히 감소하는 반면, HR mutant는 일정수준을 유지하였으나 72시간 이후에 감소하였다. 그리고 mutant들의 protease, cellulase, amylase, polygalacturonate lyase 분비여부를 조사한 결과 wild type과 비슷하게 분비하였다.

• 식물병연구
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• 제16권2호
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• pp.136-140
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• 2010

Pectobacterium carotovorum subsp. carotovorum은 채소 무름병의 원인균이다. 박테리오신인 carocin D는 Pectobacterium carotovorum subsp. Carotovorum Pcc21 균주가 생산한다. Pcc21 균주의 비병원성인 돌연변이주를 선발하기 위해 Tn5을 이용하여 무작위적 삽입 돌연변이체들을 대상으로 병원성 검사를 하였다. 실험 결과 선발된 P. carotovorum subsp. carotovorum Pcc21-M15과 carocin D 유전자의 형질전환체인 E. coli(pRG3431)은 비병원성인 동시에 carocin D를 생산하여 무름병균인 P. carotovorum subsp. carotovorum Pcc3의 생장을 억제함을 확인하였다. Pcc21-M15와 E. coli (pRG3431)를 무름병균인 P. carotovorum subsp. carotovorum Pcc3과 함께 미네랄 오일법으로 상추에 처리한 결과 Pcc3 균주를 단독 처리했을 경우에는 3일만에 90%가 무름병에 걸린 반면, Pcc21-M15와 E. coli(pRG3431)를 함께 처리한 경우는 6일 후에도 25%의 상추에서만 무름병 증상을 보였다.

• Journal of Microbiology and Biotechnology
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• 제11권2호
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• pp.222-228
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• 2001

Vibrio metschnikovii strain RH530 is a non-pathogenic, industrially-important alkaline protease producer which has been isolated from wastewater. In this paper, we report on the transformation of this strain by using the method of electroporation. A field strength of $7.5\;kVcm^{-1}$ and $25\;{\mu}F$, and using a 0.2-cm cuvette, appeared to be the optimal conditions for electroporation of the cells with the recombinant pSBCm plasmid carrying the vapK alkaline protease gene and the ColE1 replicon. Cells were subjected to osmotic shock in order to remove extracelluar DNase, and adding 200 mM of sucrose to electroporation buffer cells showed an increased transformation efficiency. Maximum efficiency of transformation was obtained at an early exponential growth phase. Using all of the conditions mentioned above, we routinely obtained a transformation efficiency of more than $10^4{({\mu}g\;plasmid\;DNA)}^{-1}$. The stability of the plasmid pSBCm in V. metschnikovii RH530 was 25% after 18h of growth (27 generations) in the medium without antibiotic selection. The insertion of the par locus to the pSBCm increased the stability of the plasmid up to 42% without selective pressure. The increase in plasmid stability was accompanied by the increase in the productivity of alkaline protease in the recombinant V. metschnikovii strain RH530. Determining optimal conditions for the transformation of the industrially-important, nonpathogenic Vibrio strain, and the improvement of plasmid stability by introducing the par locus into the high copy number plasmid vector, will allow the development of procedures involved in the genetic manipulation of this strain, particularly for its use in the production of industrial enzymes such as alkaline protease.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.330-336
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• 2004

Fifty-two pathogenic Vibrio parahaemolyticus strains were isolated from the environments of Busan and Yeosu, Korea. Forty-three of these strains showed protease activities, whereas 4 strains showed $\alpha / \beta$ hemolysin activities and 6 strains had urease activities. Their pathogenic factors were not overlapping except one strain, which had both protease and hemolysin activities. The 6 urease-positive strains (V. parahaemolyticus YKB4, YKB14, S25, YFB20, YFO21, and YFO22) showed the same biochemical characteristics as a reference strain [V. parahaemolyticus KCTC 2471 (urease-negative)], except for urease production. The 6 urease-positive strains showed different urease activities in their culture supernatant during the growth. The urease activity of S25 increased sharply at the late exponential phase, and was the highest at the initial stationary phase and was kept until the late stationary phase. The other 5 isolates, except C25, showed urease activities at the mid-stationary phase and increased steadily until the late stationary phase, when the urease activity was maximal. To compare the degree of virulence of V. parahaemolyticus with different pathogenic factors, hemolysin, protease, or urease-positive strains were injected into groups of 10 each of ICR mice (7- to l0-week-old males). The lethal rates of urease-positive V. parahaemolyticus, YKB14, YKB4, and S25, were significantly high, being 50, 70, and 80%, respectively. Protease-positive V. parahaemolyticus strains FM39 and FM50 showed 40% and 60% of lethal rate, respectively. Hemolysin-positive V. parahaemolyticus strains S34 and S72 had no mortality, similar to nonpathogenic V. parahaemolyticus FM12.

• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.155-157
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• 2001

Bifidobacterium spp. is nonpathogenic, gram-positive and anaerobic bacteria, which inhabit the intestinal tract of humans and animals. In breast-fed infants, bifidobacteria comprise morethan 90％ of the gut bacterial population. Bifidobacteria spp. are used in commericial fermented dairy products and have been suggested to exert health promoting effects on the host by maintaining intestinal microflora balances, improving lactose tolerance, reducing serum cholesterol levels, increasing synthesis of vitamins, and aiding the immune enchancement and anticarcinogenic activity for the host. These beneficial effects of Bifidobacterium are strain-related. Therefore continued efforts to improve strain characteristics are warranted. in these respect, development of vector system for Bifidobacterium is very important not only for the strain improvement but also because Bifidobacterium is most promising in serving as a delivery system for the useful gene products, such as vaccine or anticarcinogenic polypeptides, into human intestinal tract. For developing vector system, we have characterized several bifidobacterial plasmids at genetic level and developed several shuttle vectors between E. coli and Bifidobacterium using them. Also, we have cloned and sequenced several metabolic genes and food grade selection marker. Also we have obtained bifidobacterial surface protein, which will be used as the mediator for surface display of foreign genes. Recently we have succeeded in expressing amylase and GFP in Bifidobacterium using our own expression vector system. Now we are in a very exciting stage for the molecular breeding and safe delivery system using probiotic Bifidobacterium strains.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.459-466
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• 2013

The lactic acid bacterium Streptococcus thermophilus is widely used as a starter culture for the production of dairy products. Whole-genome sequencing is expected to utilize the genetic basis behind the metabolic functioning of lactic acid bacterium (LAB), for development of their use in biotechnological and probiotic applications. We sequenced the whole genome of Streptococcus thermophilus MTCC 5461, the strain isolated from a curd source, by 454 GS-FLX titanium and Ion Torrent PGM. We performed comparative genome analysis using the local BLAST and RDP for 16S rDNA comparison and by the RAST server for functional comparison against the published genome sequence of Streptococcus thermophilus CNRZ 1066. The whole genome size of S. thermophilus MTCC 5461 is of 1.73Mb size with a GC content of 39.3%. Streptococcal virulence-related genes are either inactivated or absent in the strain. The genome possesses coding sequences for features important for a probiotic organism such as adhesion, acid tolerance, bacteriocin production, and lactose utilization, which was found to be conserved among the strains MTCC 5461 and CNRZ 1066. Biochemical analysis revealed the utilization of 17 sugars by the bacterium, where the presence of genes encoding enzymes involved in metabolism for 16 of these 17 sugars were confirmed in the genome. This study supports the facts that the strain MTCC 5461 is nonpathogenic and harbors essential features that can be exploited for its probiotic potential.

• Mycobiology
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• 제38권4호
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• pp.286-294
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• 2010

Epicoccum purpurascens stain 5615 AUMC was investigated for its biocontrol activity against root rot disease caused by Pythium irregulare. E. purpurascens greenhouse pathogenicity tests using three leguminous plants indicated that the fungus was nonpathogenic under the test conditions. The germination rate of the three species of legume seeds treated with a E. purpurascens homogenate increased significantly compared with the seeds infested with P. irregulare. No root rot symptoms were observed on seeds treated with E. purpurascens, and seedlings appeared more vigorous when compared with the non-treated control. A significant increase in seedling growth parameters (seedling length and fresh and dry weights) was observed in seedlings treated with E. purpurascens compared to pathogen-treated seedlings. Pre-treating the seeds with the bioagent fungus was more efficient for protecting seeds against the root rot disease caused by P. irregulare than waiting for disease dispersal before intervention. To determine whether E. purpurascens produced known anti-fungal compounds, an acetone extract of the fungus was analyzed by gas chromatography mass spectrometry. The extract revealed a high percentage of the cinnamic acid derivative (trimethylsiloxy) cinnamic acid methyl ester. The E. purpurascens isolate grew more rapidly than the P. irregulare pathogen in a dual culture on potato dextrose agar nutrient medium, although the two fungi grew similarly when cultured separately. This result may indicate antagonism via antibiosis or competition.