우리는 그들의 기술 지원을 위해 브랜든 Hassett과 카를로스 오르 티즈 감사드립니다. 이 작품은 박사 마이클 Kolomiets에 NSF 교부금 IOB-0544428, IOS-0951272, 그리고 IOS-0925561에 의해 지원, 그리고 식량 농업 USDA의 국립 연구소 (NIFA), AFRI 공장 사육 및 교육 그랜트 # 2010-85117하여 -20539 Drs 있습니다. 세스 머레이, 토마스 Isakeit, 그리고 마이클 Kolomiets.

1. 옥수수 커널 Bioassay <ol><li> 두 주 전에, 문화 28의 감자 덱스 트로 오스 한천 배지 (PDA)에서 곰팡이 병원균 ° C. </li><li> 그들이 bioassay의 튜브의 바닥과 수준, 50 ML 팔콘 튜브의 위치를​​ 누워 있도록 선호 평평 비슷한 크기와 모양으로 커널을 선택합니다. 선택된 커널은 유사한 종자 연령 및 신진 대사 조성을 위해 동일한 환경에서 동시에 생산된 것이어야합니다. </li><li> 6 % 나트륨 차아 염소 산염과 70 % 에탄올, 멸균 물 1 분, 10 분 : 5 분 동안 실온에서 튜브를 흔들어하여 커널을 소독 표면 이군. 계속 떨고 동안 멸균 물을 때마다 5 분, 세 번 씻어. autoclaved 수건에 건조한 팻 커널. </li><li> Aspergillus flavus의 감염을 촉진하기 위해서, 0.5 cm의 깊이에 18 G 바늘로 배아 사이드에 작은 상처를 만듭니다. 그러나 우리의 경험에 큰 상처가 Fusarium의 verticillioides 감염 필요합니다. 일erefore, 0.5 mm의 깊이에서 배아 사이드로 잘라 면도날을 사용합니다. </li><li> 포자를 해방하고 autoclaved 일종의 투박한 무명의 최소 4 레이어를 통해 필터링 중력에 균사체를 제거하는 autoclaved 0.01 % 십대 초반-20 솔루션의 약 5 ML에 교양 번호판을 위해서 힘든하여 inoculum 서스펜션을 준비합니다. hemocytometer로 포자 농도를 계산하고 10 6 포자 / A 용 ML 조정 flavus 또는 F. verticillioides. 다른 종의 병원균이나 호스트를 사용하는 경우, 적절한 감염 inoculum의 농도를 평가하는 것이 중요합니다. </li><li> 종이 타올 다섯 시트 및 멸균 물 100 ML 줄지어 플라스틱 컨테이너 (29.2 cm X 18.7 cm X 8.3 ㎝)의 습도 챔버를 만듭니다. 상공 회의소가 아닌 물 또는 공기 꽉 및 추가 물은 종이 타올은 습기가 유지하는 실험을하는 동안 추가되어야합니다. </li><li> 장소 네 autoclaved 20 ML 유리 섬광 유리병에 커널과 그 질량을 기록합니다. 200 μl 포자 현탁액과 예방모자 및 정지로 고르게 코트 커널에 소용돌이. 자유롭게 습도 챔버로 공기 교환 및 장소를 허용하도록 뚜껑을 풀어. </li></ol> 주 : 씨앗 또는 이러한 방법에서 설명한 이외의 진균류의 경우 inoculum 필수의 금액이 다를 수 있으며 실험적으로 도출되어야한다. <ol start="8"><li> ° C에서 7 일간이나 28이나 원하는까지 12-h-light/12-h-dark photoperiod 미만의 커널을 품어. </li></ol> 2. Conidia의 열거 <ol><li> 감염 기간 후 1 분 철저하게 감염된 커널과 소용돌이를 포함하는 섬광 튜브에 autoclaved 0.01 % 십대 초반-20의 5 ML에 추가합니다. </li><li> 넓은 구멍 피펫 팁 사용하면, 즉시 0.01 % 십대 초반-20 (또는 감염에 따라 필요에 따라 희석)의 1.8 ML을 포함하는 2.0 ML Eppendorf 튜브에 포자 현탁액의 두 개의 서로 다른 200 μl aliquots를 전송하여 희석. </li><li> hemocytometer 4 회 각 200 μl 나누어지는을 열거그리고 트리 트먼트 사이 conidia의 수준을 비교합니다. </li></ol> 3. 아플라톡신의 부량 <ol><li> 80 % 메탄올 및 0.05 g NaCl, 1 분 고속으로 표지 및 조화 20 ML : 1 개 시료 50 ML 믹서 컵에 커널을 추가합니다. </li><li> 깨끗한 수거 용기를 통해 홈 붙이 필터 용지를 삽입하고 필터로 추출을 붓는다. </li><li> 깨끗한 그릇에 여과액 10 ML 전송 20 ML 증류수 H 2 O를 추가하고 잘 섞는다. </li><li> 깨끗한 컵에 1.5 μm의 유리 microfibre 필터를 통해 필터 샘플. </li><li> Afla 테스트 열에 필터링된 추출물의 1 ML을 적용하고, 압축 공기를 사용하는 비운까지 1-2 드랍스 / 초의 속도로 칼럼을 통해 여과 추출물을 강제. </li><li> 증류수 H 2 O 1 ML에 두 번 칼럼을 씻어 놓기 / 초 공기를 넘지 때까지 1-2에서 컬럼을 통과. </li><li> 드롭 / 유리 cuvette에 수집된 초 속도 1-2에서 1ml 100 % HPLC 급 메탄올과 Elute 칼럼. </li><li> 추가한 eluate로 Afla 시험 개발자의 ML과 잘 섞는다. 장소 보정 fluorometer로 cuvette 60 초에서 읽어보세요. </li></ol> 참고 : Aflatest FGIS 프로토콜을 사용하는 경우 fluorometer의 측정 100 ML에서 추출한 시료의 초기 50g를 기반으로 계산됩니다. 네가 물과 시료의 희석을 고려하면 한 ML이 샘플의 0.166 g을 나타냅니다 칼럼에 적용. 그래서 프로토콜을 수정할 때, 당신은 계정으로 표본의 크기의 차이와 초기 추출 솔루션을해야합니다. 예를 들어, 커널 2g 20 ML 80 % 메탄올로 추출됩니다. 이것은 0.1 g / ML입니다. 이 샘플 1 ML은 2 ML 워터와 혼합되면 액체의 시료의 비중은 이제 0.033 g / ML입니다. 이 샘플은 100 ppb의 fluorometer 읽기를 제공하는 경우 실제 농도는 ppb = (0.166 g X 100 ppb) / 0.1 g), 또는 166 ppb는 0.166으로 시료의 비중에 따라 달라집니다. 대안 아플라톡신 quantifi위한양이온 방법, 참고 7 참조하십시오. 4. Fumonisin B1 (FB1) 분석 <ol><li> 곰팡이가 옥수수 커널에서 재배되면 교반하지 않고 상온에서 acetonitrile / 물 (50분의 50, V / V)의 10 ML과 하룻밤 샘플을 추출합니다. 그 후, 탈이온수 (6 ML)으로 추출 (2 ML)를 섞어, 그리고 C-18 고체 상 추출에 직접 (8 ML)이 믹스를 적용합니다. </li><li> 샘플을 로딩하기 전에 전제 조건 물 2 ML 다음 acetonitrile 2 ML과 rinsing에 의한 C-18 고체 상 추출 칼럼. </li><li> 샘플을 로딩 후 열은 acetonitrile / 물 (85분의 15, V / V) 2 ML 뒤에 물을 2 ML로 씻어 있습니다. HPLC 분석 (FB1를 포함)에 대한 샘플은 acetonitrile / 물 (30분의 70, V / V) 2 ML로 eluted있다. </li><li> 0.1 ML borate 완충액 (0.05 M 붕소의 acid/0.05 M 나트륨 borate는 [50분의 50, V / V], 산도 8.5)과 0.1가 담긴 병을로 열 eluate의 0.1 ML를 전송하여 O-phthaldehyde (오파)으로 FB1를 Derivati​​ze ML 오파 (aceton에서 0.1 밀리그램 / ML메르 캅 토 에탄올) - 0.5 %로 itrile. </li><li> acetonitrile/0.01 M 붕산 (60분의 40, V / V) 0.5 ML을 추가하여 10 분 후에 반응을 중지합니다. </li><li> 분석 Zorbax ODS 컬럼 (4.6 150mm)과 가변 파장 Shimatzu RF-10Axl 형광 검출기 (여기 335 nm의 / 방출 440 nm의)이 장착된 HPLC Shimadzu LC-20AT 시스템에 FB1를 분석합니다. </li><li> 선형 그라데이션을 사용 (용매 : acetonitrile/0.1 M의 인산 나트륨 (60분의 40), 산도 3.3, 용매 B : acetonitrile / 0.1 M의 인산 나트륨 (40분의 60), 산도 3.3)되어 다음과 같이하고 기울기 프로그램입니다 : 100 % 5 분, 10 분, 100 % B의 100 % B에. </li><li> HPLC에 의해 FB1 기준을 분석하고, 피크 면적 측정은 표준 곡선을 생성하는 데 사용됩니다. 그 후, FB1 표준 곡선과 피크 영역을 비교하여 시료의 FB1 수준을 계량. </li></ol> 5. Ergosterol 분석 <ol><li> 7~14일을위한 옥수수 커널의 문화 균류. </li><li> 필로폰 : 잠복기 후, 클로로포름 10 ML 추가각 유리병에 anol (2시 1분, V / V). 추가한 후에는 잘 샘플을 흔들 후 24 시간 동안 실온에서 어둠 속에서 튜브를 품어. </li><li> 24 시간 후 샘플을 원심 분리기, 그리고 표면에 뜨는를 수집하고 0.45 음 나일론 막 통해 필터링합니다. </li><li> 직접 4.6 U ODS-C18 컬럼 (200, 250 ± 4.6 ㎜)와 Shimadzu SPD-20A의 UV / 282 NM에서 모니터링하도록 설정 VIS 검출기를 갖춘 HPLC Shimadzu LC-20AT 시스템으로 샘플을 주사. </li><li> 1.5 ML / 모바일 위상과 같은 분 유량에 메탄올 (100 %)를 사용합니다. HPLC 등급 ergosterol에서 생성된 표준 곡선에 샘플 피크 영역을 비교하여 샘플에 ergosterol의 부량를 결정합니다. </li></ol> 6. 대표 결과 2~3일위한 습도 챔버에 접종 및 배양 후, 곰팡이 성장은 커널에 표시하기 시작합니다. 이레 게시물 치료, 치료 식물에 대한 식물의 성장은 whil 명확하게 표시합니다온라인 모의 컨트롤 (그림 1B, 상단) 감염되지 않은 있어야합니다. 더 이상 부화 기간이 더 풍부한 식물 성장 (그림 1B, 아래)에 도움이됩니다. 조건에서 본 설명 (그림 2G), A. flavus 야생 형 NRRL 각각 8, 6, 8 일간의 식민지, 아플라톡신 축적 및 conidia 생산의 3,357 표시된 최대 값 (그림 2, AC). 그러나 진균 독소 오염과 conidiation은 ergosterol의 단위당 비교했을 때, 가장 큰 수준 (그림 2, DE) 각각 4, 6 일간에서 관찰되었다. 그림 층이 곰팡이 바이오 매스에 의존 최대 가치 관찰을 요약한 것입니다. 흥미롭게도 4-6일 포스트 접종 사이 커널은 같은 시간 코스 (그림 2 시간, 아래)에 대한 시료로부터 총 RNA에 의해 볼 핵산 저하를 받고있다. 우리 자신을 포함하여 몇몇 연구, 조사할을 가지고에드 F. verticilliodies는 옥수수 커널 2,8,9,10,11,12에 감염 성공적으로 시간을 관찰을위한 포인트로 7-13일 사용됩니다. 방법을 사용하면 본 설명한 3,500-8,000 NG / G 커널 4,13 및 ergosterol 수준 범위에서 fumonisin 레벨 범위 fumonisin (상단) 및 ergosterol (하단위한 5,000-10,000에서 NG / G 커널 14,13. 그림 3은 쇼를 대표하는 봉우리 ) HPLC chromatograms에서. ergosterol의 측정도 흡광 스펙트럼 15를 통해 진행하실 수 있습니다. <img alt="그림 1" src="/files/ftp_upload/3727/3727fig1.jpg" /> 그림 1. 곰팡이 바이오 매스, sporogenesis 및 진균 독소 생산과 커널 bioassays에서 식물 성장을위한 대표적인 결과의 부량을위한 플로우 차트. 옥수수 커널에 곰팡이 pathogenesis을 평가하는 데 사용되는 기본적인 생물 학적 매개 변수의 부량위한 설명한 방법을 요약) 플로우 차트. B) 대표커널 bioassays에 대한 검색 결과. 탑, A. flavus (NRRL3357) B73 유전자를 배경으로 옥수수 커널에서 식물 성장. 씨앗 10 6 포자 / ML 및 사진 200 μl 7 일 게시물 접종 찍은 사진으로 주사되었다. 아래에, F. verticillioides는 B73 배경 13일 포스트 감염에 커널을 주사. 커널은 10 6 포자 / ML 200 μl으로 주사되었다. <img alt="그림 2" src="/files/ftp_upload/3727/3727fig2.jpg" /> 그림 2. Aspergillus flavus의 커널 bioassay 시간 코스. B73 커널은 10 6 포자 / ML Aspergillus flavus conidial 정지 200 μl로 주사하고 2~8일 (G)에 incubated했다. (; 평균 ± SE N = 3-4) 모든 값은는 4 커널의 건조 중량 평균으로 결정됩니다. ) 콜로니 (ergosterol을 기준으로), (B) 아플라톡신, 그리고 (C) conidiation은 위에서 설명한 방법을 사용하여 계량했다. E) 아플라톡신 및 (F) conidiation이 표시됩니다H ergosterol를 통해 측정으로 곰팡이 바이오 매스의 함수로. F) ergosterol, 아플라톡신 축적, 그리고 곰팡이 바이오 매스의 함수로 conidiation위한 최대 값 - 각 수량에 대한 최대 값을 100 %로 설정되었다 관찰했다. 시간 코스의 총 RNA의 차선 당 (H) 1 μg. <img alt="그림 3" src="/files/ftp_upload/3727/3727fig3.jpg" /> 그림 3. fumonisin B1 (위)과 Fusarium의 verticillioides (M3125)에 감염된 커널과 분리돼 ergosterol (아래)를위한 대표적인 고성능 액체 크로마 토그래피 (HPLC) chromatograms.

<ol>
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Plant-Microbe Interact. 20, 922-933 (2007).</li><li>Brodhagen, M., Tsitsigiannis, D. I., Hornung, E., Goebel, C., Feussner, I., Keller, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reciprocal+oxylipin-mediated+cross-talk+in+the+Aspergillus+-+seed+pathosystem.">Reciprocal oxylipin-mediated cross-talk in the Aspergillus - seed pathosystem.</a> Mol. Microbiol. 67, 378-391 (2008).</li><li>Gao, X., Brodhagen, M., Isakeit, T., Brown, S. H., G&#246;bel, C., Betran, J., Feussner, I., Keller, N. P., Kolomiets, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+the+lipoxygenase+ZmLOX3+increases+susceptibility+of+maize+to+Aspergillus+spp.">Inactivation of the lipoxygenase ZmLOX3 increases susceptibility of maize to Aspergillus spp.</a> Mol. Plant-Microbe Interact. 22, 222-231 (2009).</li><li>Gao, X. Q., Kolomiets, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-derived+lipids+and+oxylipins+are+crucial+signals+in+modulating+mycotoxin+production+by+fungi.">Host-derived lipids and oxylipins are crucial signals in modulating mycotoxin production by fungi.</a> Toxin Rev. 28, 79-88 (2009).</li><li>Mukherjee, M., Kim, J. -. E., Park, Y. -. S., Kolomiets, M. V., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+G+protein+signaling+in+F.+verticillioides+mediate+differential+host-pathogen+responses+on+non-viable+versus+viable+maize+kernels.">Regulators of G protein signaling in F. verticillioides mediate differential host-pathogen responses on non-viable versus viable maize kernels.</a> Mol. Plant Pathol. 12, 479-491 (2011).</li><li>Zheng, M. Z., Richard, J. L., Binder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+rapid+methods+for+the+analysis+of+mycotoxins.">A review of rapid methods for the analysis of mycotoxins.</a> Mycopathologia. 161, 261-273 (2006).</li><li>Bacon, C. W., Bennett, R. M., Hinton, D. M., Voss, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+electron+microscopy+of+Fusarium+moniliforme+within+asymptomatic+maize+kernels+and+kernels+associated+with+equine+leukoencephalomalacia.">Scanning electron microscopy of Fusarium moniliforme within asymptomatic maize kernels and kernels associated with equine leukoencephalomalacia.</a> Plant Dis. 76, 144-148 (1992).</li><li>Munkvold, G. P., Hellmich, R. L., Rice, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+fumonisin+concentrations+in+kernels+of+transgenic+Bt+maize+hybrids+and+nontransgenic+hybrids.">Comparison of fumonisin concentrations in kernels of transgenic Bt maize hybrids and nontransgenic hybrids.</a> Plant Dis. 83, 130-138 (1999).</li><li>Sagaram, U. S., Shaw, B. D., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fusarium+verticillioides+GAP!,+a+gene+encoding+a+putative+glycolipid-anchored+surface+protein,+participates+in+conidiation+and+cell+wall+structure+but+not+virulence.">Fusarium verticillioides GAP!, a gene encoding a putative glycolipid-anchored surface protein, participates in conidiation and cell wall structure but not virulence.</a> Microbiol. 153, 2850-2861 (2007).</li><li>Shim, W. -. B., Flaherty, J. E., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Fumonisin+B1+biosynthesis+in+maize+germ+and+degermed+kernels+by+Fusarium+verticillioides.">Comparison of Fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides.</a> J. Food Protect. 66, 2116-2122 (2003).</li><li>Shim, W. -. B., Woloshuk, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+fumonisin+B1+biosynthesis+and+conidiation+in+Fusarium+verticillioides+by+a+cyclin-like+(C-type)+gene,+FCC1.">Regulation of fumonisin B1 biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1.</a> Appl. Environ. Micrbiol. 67, 1607-1612 (2001).</li><li>Christensen, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conversation with: Won-Bo Shim. , (2011).</li><li>Shin, J. -. H., Shim, W. -. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+PPR1+and+PPR2,+genes+encoding+regulatory+subunits+of+protein+phosphatase+2A+in+Fusarium+verticillioides.">Characterization of PPR1 and PPR2, genes encoding regulatory subunits of protein phosphatase 2A in Fusarium verticillioides.</a> Phytopathol. 99, S119 (2009).</li><li>Breivik, O. N., Owades, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+Semimicrodetermination+of+Ergosterol+in+Yeast.">Spectrophotometric Semimicrodetermination of Ergosterol in Yeast.</a> Yeast. Agric. and Food Chem. 5, 360-363 (1957).</li></ol>

<p class="jove_content"&gt; 저는 공개할 게 없다.</p

<p class="jove_content"&gt; 여기에 설명된 방식은 광범위하게 테스트 및 곰팡이 식민지, sporogenesis 및 mycotoxins의 생산을위한 같지는 결과의 세대에 견고한 것으로 입증되었습니다. 또한, 이러한 방법은 mycotoxigenic 진균류 (예 : 땅콩, 밀, 면화, 피스타치오 등)과 오염에 민감한 다른 식물 종 씨앗에 적용할 수 있어야합니다. 유능한 식물 - 병원균 상호 작용 분석을 위해, 씨앗은 생명을 유지하는 것이 필수적입니다. 씨앗의 생?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> # 카탈로그 </td></tr><tr><td> 감자 덱스 트로 오스 한천 배지 </td><td> 피셔 Scientifc </td><td> S71659A </td></tr><tr><td> 십대 초반-20 </td><td> 피셔 Scientifc </td><td> BP337-100 </td></tr><tr><td> 플라스틱 배양 용기 </td><td> Sterilite </td><td> 1713LAB06 </td></tr><tr><td> 혼합하는 것 </td><td> Vicam </td><td> 20,200 </td></tr><tr><td> 24cm 세로 홈을 판 여과지 </td><td> Vicam </td><td> 31,240 </td></tr><tr><td> 1.5 μm의 유리 microfibre </td><td> Vicam </td><td> 31,955 </td></tr><tr><td> Afla 테스트 칼럼 </td><td> Vicam </td><td> G1024 </td></tr><tr><td> Afrla 테스트 개발자 </td><td> Vicam </td><td> 32,010 </td></tr><tr><td> 메탄올 </td><td> Vicam </td><td> 35,016 </td></tr><tr><td> Acetonitrile </td><td> 피셔 Scientifc </td><td> AC14952-0025 </td></tr><tr><td> 에탄올 </td><td> 피셔 Scientifc </td><td> AC39769-0025 </td></tr><tr><td> C-18 고체 상 추출 칼럼 (준비 9월 SPE C18 열) </td><td> 피셔 Scientifc </td><td> 60108-304 </td></tr><tr><td> O-phthalaldehyde (오파) </td><td> 시그마 화학 공동 </td><td> 79,760~5g </td></tr><tr><td> 붕산 </td><td> 피셔 Scientifc </td><td> BP168-500 </td></tr><tr><td> 나트륨 borate </td><td> 피셔 Scientifc </td><td> RDCS0330500 </td></tr><tr><td> 메르 캅 토 에탄올 </td><td> 피셔 Scientifc </td><td> 45-000-231 </td></tr><tr><td> Shimadzu HPLC LC-20AT (펌프) </td><td> Shimadzu 과학 인 스트 루먼트 주식 회사 </td><td> LC-20AT </td></tr><tr><td> Zorbax ODS 칼럼 (4.6x150mm) </td><td> 애질런트 테크놀로지 </td><td> 443905-902 </td></tr><tr><td> Shimatzu RF-10Axl 형광 검출기 </td><td> Shimadzu 과학 인 스트 루먼트 주식 회사 </td><td> RF-10AXL </td></tr><tr><td> 인산 나트륨 </td><td> 피셔 Scientifc </td><td> AC38987-0010 </td></tr><tr><td> FB1 기준 </td><td> 시그마 화학 (주) </td><td> F1147-1mg </td></tr><tr><td> 클로로포름 </td><td> VWR </td><td> MK444410 </td></tr><tr><td> 0.45 음 나일론 막 (HPLC)와 13mm의 주사기 필터 </td><td> 막 생활 과학 </td><td> 4426 </td></tr><tr><td> Ergosterol </td><td> 시그마 - 올드 리치 </td><td> 45480-50G-F </td></tr><tr><td> 섬광 튜브 </td><td> VWR </td><td> 66021-602 </td></tr><tr><td> 나트륨 염화물 </td><td> Vicam </td><td> G1124 </td></tr></tbody></table>

quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays

에 의해 입자의 썩어가는 씨앗 - 감염 곰팡이는 인간과 동물 건강에 심각한 위험을 언급하지 않기로, 시리얼 생산 세계적으로 가장 큰 경제적 어려움 중 하나가 포즈. 시리얼 생산 중에서 옥수수는 곡물 무결성 및 진균 독소의 종자 오염의 병원체 유발 손실로 인해 거의 틀림없이 가장 영향을받는 작물이다. 옥수수 재배자 및 식품 및 사료 프로세서를위한 두 가지 가장 널리 사용하고 문제가 mycotoxins는 각각 Aspergillus flavus와 Fusarium의 verticillioides 제작한 아플라톡신과 fumonisin, 수 있습니다. 분자 식물 - 병원균 상호 작용의 최근 연구에 곰팡이 감염 및 진균 독소 오염 1,2,3,4,5,6으로 식물 반응과 관련된 구체적인 메커니즘을 이해 약속을 증명하고있다. 많은 실험실이 식물 병원체 상호 작용을 연구하기 위해 커널 assays를 사용하고 있기 때문에, 서로 다른 생물 학적 매개 변수를 quantifying위한 표준화된 방법에 필요하므로,이다른 실험실에서 결과를 비교 해석될 수 있습니다. 씨앗에 대한 정량 분석​​을위한 강력하고 재현할 수단을 위해서는 곰팡이 성장, 바이오 매스 및 진균 독소 오염을 수치 인 - 실험실 커널 assays와 후속 방법을 개발했습니다. 네 소독 옥수수 커널은 곰팡이 서스펜션 (10 6)와 함께 유리 튜브 안에 주사하고 미리 정해진 기간 동안 incubated됩니다. 샘플 튜브 그러면 고성능 액체 크로마 토그래피 (HPLC), AflaTest fluorometer 방법을 사용하여 아플라톡신의 부량, 그리고 HPLC에 의한 fumonisin의 부량하여 hemocytometer, ergosterol 기반의 바이오 매스 분석하여 conidia의 열거를 위해 선택됩니다.

Watch this Scientific Journal Video about Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays at JoVE.com

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

종자 - 감염 곰팡이에 의한 시리얼 작물의 참화 더 나은 식물 병원체 상호 작용을 이해하기 위해 많은 연구 노력을하라는 메시지가있다. 실험실 환경에서 씨앗 - 곰팡이 상호 작용을 연구하기 위해, 우리는 곰팡이 복제, 바이오 매스, 그리고 커널 bioassays를 사용하여 진균 독소 오염의 부량위한 강력한 방법을 개발했다.

커널 Bioassays를 사용하여 곰팡이 콜로니, Sporogenesis 및 Mycotoxins 생산의 부량

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

quantification-fungal-colonization-sporogenesis-production-mycotoxins

Research

JoVE Journal

Immunology and Infection

18.0K 조회수.  Texas A&M University. 종자 - 감염 곰팡이에 의한 시리얼 작물의 참화 더 나은 식물 병원체 상호 작용을 이해하기 위해 많은 연구 노력을하라는 메시지가있다. 실험실 환경에서 씨앗 - 곰팡이 상호 작용을 연구하기 위해, 우리는 곰팡이 복제, 바이오 매스, 그리고 커널 bioassays를 사용하여 진균 독소 오염의 부량위한 강력한 방법을 개발했다.

비디오: Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Colonization of Euprymna scolopes Squid by Vibrio fischeri

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no fitness consequence (commensal), or be beneficial (mutualistic). While much attention has been given to pathogenic interactions, little is known about the processes that dictate the reproducible acquisition of beneficial/commensal bacteria from the environment. The light-organ mutualism between the marine Gram-negative bacterium V. fischeri and the Hawaiian bobtail squid, E. scolopes, represents a highly specific interaction in which one host (E. scolopes) establishes a symbiotic relationship with only one bacterial species (V. fischeri) throughout the course of its lifetime6,7. Bioluminescence produced by V. fischeri during this interaction provides an anti-predatory benefit to E. scolopes during nocturnal activities8,9, while the nutrient-rich host tissue provides V. fischeri with a protected niche10. During each host generation, this relationship is recapitulated, thus representing a predictable process that can be assessed in detail at various stages of symbiotic development. In the laboratory, the juvenile squid hatch aposymbiotically (uncolonized), and, if collected within the first 30-60 minutes and transferred to symbiont-free water, cannot be colonized except by the experimental inoculum6. This interaction thus provides a useful model system in which to assess the individual steps that lead to specific acquisition of a symbiotic microbe from the environment11,12.
Here we describe a method to assess the degree of colonization that occurs when newly hatched aposymbiotic E. scolopes are exposed to (artificial) seawater containing V. fischeri. This simple assay describes inoculation, natural infection, and recovery of the bacterial symbiont from the nascent light organ of E. scolopes. Care is taken to provide a consistent environment for the animals during symbiotic development, especially with regard to water quality and light cues. Methods to characterize the symbiotic population described include (1) measurement of bacterially-derived bioluminescence, and (2) direct colony counting of recovered symbionts.

Colonization of Euprymna scolopes Squid by Vibrio fischeri

The method outlines the procedure by which the Hawaiian bobtail squid, Euprymna scolopes and its bacterial symbiont, Vibrio fischeri, are raised separately and then introduced to allow for specific colonization of the squid light organ by the bacteria. Colonization detection by bacterially-derived luminescence and by direct colony counting are described.

의 식민지 Euprymna scolopes 오징어에 의한 장염 fischeri</em

Specific bacteria are found in association with animal tissue1-5. Such host-bacterial associations (symbioses) can be detrimental (pathogenic), have no ...

연구

면역학 및 감염병학

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

곰팡이 병원체 Phagocytosis의 라이브 셀 비디오 현미경

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

연쇄상 구균 폐렴으로 비강 내 식민지 화 중 염증 반응의 특성화

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or pathogenic effects depending on the inflammatory milieu. Nonetheless, despite the indispensable role of neutrophils in immunity, detailed understanding of the molecular factors that mediate neutrophils' effector and immunopathogenic effects in different infectious diseases and inflammatory conditions is still lacking, partly because of their short half life, the difficulties with handling of these cells and the lack of reliable experimental protocols for obtaining sufficient numbers of neutrophils for downstream functional studies and adoptive transfer experiments. Therefore, simple, fast, economical and reliable methods are highly desirable for harvesting sufficient numbers of mouse neutrophils for assessing functions such as phagocytosis, killing, cytokine production, degranulation and trafficking. To that end, we present a reproducible density gradient centrifugation-based protocol, which can be adapted in any laboratory to isolate large numbers of neutrophils from the bone marrow of mice with high purity and viability. Moreover, we present a simple protocol that uses CellTracker dyes to label the isolated neutrophils, which can then be adoptively transferred into recipient mice and tracked in several tissues for at least 4 hr post-transfer using flow cytometry. Using this approach, differential labeling of neutrophils from wild-type and gene-deficient mice with different CellTracker dyes can be successfully employed to perform competitive repopulation studies for evaluating the direct role of specific genes in trafficking of neutrophils from the blood into target tissues in vivo.

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

분리, 정제 및 기능 연구 및 입양 전송 실험 쥐의 골수 호중구의 라벨링

Neutrophils are critical effector cells of the innate immune system. They are rapidly recruited at sites of acute inflammation and exert protective or ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

설립<em&gt; 체외</em&gt; 시스템의 세포 내 행동을 연구하는<em&gt; 칸디다 글라 브라</em&gt; 인간 THP-1 식세포에

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사<em&gt; 체외</em&gt; 준수의 분석

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised patients, the elderly, and pregnant women. The most common manifestations of listeriosis in humans include meningitis, encephalitis, and fetal abortion. A significant but much less documented sequelae of invasive L. monocytogenes infection involves the heart. The death rate from cardiac illness can be up to 35% despite treatment, however very little is known regarding L. monocytogenes colonization of cardiac tissue and its resultant pathologies. In addition, it has recently become apparent that subpopulations of L. monocytogenes have an enhanced capacity to invade and grow within cardiac tissue. This protocol describes in detail in vitro and in vivo methods that can be used for assessing cardiotropism of L. monocytogenes isolates. Methods are presented for the infection of H9c2 rat cardiac myoblasts in tissue culture as well as for the determination of bacterial colonization of the hearts of infected mice. These methods are useful not only for identifying strains with the potential to colonize cardiac tissue in infected animals, but may also facilitate the identification of bacterial gene products that serve to enhance cardiac cell invasion and/or drive changes in heart pathology. These methods also provide for the direct comparison of cardiotropism between multiple L. monocytogenes strains.

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Listeria monocytogenes causes fetal infections in pregnant women and meningitis in susceptible populations. Subpopulations of bacteria can colonize cardiac tissue, causing myocarditis in patients and laboratory animals. Here we present a protocol that describes how to assess L. monocytogenes cardiac cell invasion in vitro and cardiac colonization in infected animals.

리스테리아 단세포 유전자를 사용하여 감염된 마우스에 있는 문화와 심혼 식민지에 있는 심장 세포의 세균성 침략 평가

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

그룹 B의 쥐 모델<em&gt; 연쇄상 구균</em&gt; 질 식민

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Histological Quantification to Determine Lung Fungal Burden in Experimental Aspergillosis

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse models of pulmonary fungal infection. Although multiple methods are used to assess fungal burden, quantitative polymerase chain reaction (qPCR) of fungal DNA has emerged as a technique with several advantages over previous culture-based methods. Currently, a comprehensive assessment of lung pathology, leukocyte recruitment, fungal burden, and gene expression in mice with invasive aspergillosis (IA) necessitates the use of a significant number of experimental and control animals. Here the quantification of lung histological staining to determine fungal burden using a reduced number of animals was examined in detail. Lung sections were stained to identify fungal structures with Gomori&#39;s modified methanamine silver (GMS) staining. Images were taken from the GMS-stained sections from 4 discrete fields of each formalin-fixed paraffin-embedded lung. The GMS stained areas within each image were quantified using an image analysis program, and from this quantification, the mean percentage of stained area was determined for each sample. Using this strategy, eosinophil-deficient mice exhibited decreased fungal burden and disease with caspofungin therapy, while wild-type mice with IA did not improve with caspofungin. Similarly, fungal burden in mice lacking &#947;&#948; T cells were also improved by caspofungin, as measured by qPCR and GMS quantification. GMS quantification is therefore introduced as a method for the determination of relative lung fungal burden that may ultimately reduce the quantity of experimental animals required for comprehensive studies of invasive aspergillosis.

Here we describe a protocol to determine pulmonary fungal burden in mice with invasive aspergillosis by quantification of Gomori&#39;s modified methanamine silver staining in histological sections. Use of this method resulted in comparable results with less animals compared to assessment of fungal burden by quantitative PCR of lung fungal DNA.

조직학 정량화 폐 실험 Aspergillosis에 곰 팡이 부담을 결정 하

The quantification of lung fungal burden is critical for the determination of the relative levels of immune protection and fungal virulence in mouse ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

실시간 이미징 및 2 단계 반복 흐름 시스템을 사용 하 여 곰 팡이 Biofilm 개발의 정량화

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...