صاحب البلاغ وذلك بفضل سوزان ويتفيلد لإعداد الأرقام ومارتن كميل وهيلاري ساماجايو المساعدة مع بعض التجارب.

<ol>
	<li>Heaton, J. C., Jones, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+contamination+of+fruit+and+vegetables+and+the+behaviour+of+enteropathogens+in+the+phyllosphere:+a+review.">Microbial contamination of fruit and vegetables and the behaviour of enteropathogens in the phyllosphere: a review.</a> J. Appl. Microbiol. 104, 613-626 (2008).</li><li>Beuchat, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+factors+influencing+survival+and+growth+of+human+pathogens+on+raw+fruit+and+vegetables.">Ecological factors influencing survival and growth of human pathogens on raw fruit and vegetables.</a> Microbes Infect. 4, 413-423 (2002).</li><li>Berger, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fresh+fruit+and+vegetables+as+vehicles+for+the+transmission+of+human+pathogens.">Fresh fruit and vegetables as vehicles for the transmission of human pathogens.</a> Environ. Microbiol. 12, 2385-2397 (2010).</li><li>Lugtenberg, B. J., Chin, A. W. T., Bloemberg, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbe-plant+interactions:+principles+and+mechanisms.">Microbe-plant interactions: principles and mechanisms.</a> Antonie Van Leeuwenhoek. 81, 373-383 (2002).</li><li>Preston, G. M., Haubold, B., Rainey, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+genomics+and+adaptation+to+life+on+plants:+implications+for+the+evolution+of+pathogenicity+and+symbiosis.">Bacterial genomics and adaptation to life on plants: implications for the evolution of pathogenicity and symbiosis.</a> Curr. Opin. Microbiol. 1, 589-597 (1998).</li><li>Compant, S., Duffy, B., Nowak, J., Clement, C., Barka, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Plant+Growth-Promoting+Bacteria+for+Biocontrol+of+Plant+Diseases:+Principles,+Mechanisms+of+Action,+and+Future+Prospects.">Use of Plant Growth-Promoting Bacteria for Biocontrol of Plant Diseases: Principles, Mechanisms of Action, and Future Prospects.</a> Appl. Environ. Microbiol. 71, 4951-4959 (2005).</li><li>Mathews, S. L., Smith, R. B., Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+retention+of+pathogenic+Escherichia+coli.+O157+by+sprouts,+leaves+and+fruits.">A comparison of the retention of pathogenic Escherichia coli. O157 by sprouts, leaves and fruits.</a> Microb. Biotechnol. 7, 570-579 (2014).</li><li>Fuqua, C., Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+bacterial+biofilms+associated+with+plants.">Methods for studying bacterial biofilms associated with plants.</a> Methods Enzymol. 337, 3-18 (2001).</li><li>Torres, A. G., Jeter, C., Langley, W., Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+binding+of+Escherchia+coli.O157:H7+to+alfalfa,+human+epithelial+cells,+and+plastic+is+mediated+by+a+variety+of+surface+structures.">Differential binding of Escherchia coli.O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures.</a> Appl. Environ. Microbiol. 71, 8008-8015 (2005).</li><li>Matthysse, A. G., Deora, R., Mishra, M., Torres, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharides+cellulose,+poly-beta-1,6-n-acetyl-D-glucosamine,+and+colanic+acid+are+required+for+optimal+binding+of+Escherichia+coli+O157:H7+strains+to+alfalfa+sprouts+and+K-12+strains+to+plastic+but+not+for+binding+to+epithelial+cells.">Polysaccharides cellulose, poly-beta-1,6-n-acetyl-D-glucosamine, and colanic acid are required for optimal binding of Escherichia coli O157:H7 strains to alfalfa sprouts and K-12 strains to plastic but not for binding to epithelial cells.</a> Appl. Environ. Microbiol. 74, 2384-2390 (2008).</li><li>Matthysse, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cellulose+overproduction+on+binding+and+biofilm+formation+on+roots+by+Agrobacterium+tumefaciens.">The effect of cellulose overproduction on binding and biofilm formation on roots by Agrobacterium tumefaciens.</a> Mol. Plant Microbe Interact. 18, 1002-1010 (2005).</li><li>Matthysse, A. G., Kijne, J. W., Spaink, H. P., Kondorosi, A., Hooykaas, P. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Rhizobiaceae+to+plant+cells.">Attachment of Rhizobiaceae to plant cells.</a> The Rhizobiaceae. , 235-249 (1998).</li><li>Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditioned+medium+promotes+the+attachment+of+Agrobacterium+tumefaciens.strain+NT1+to+carrot+cells.">Conditioned medium promotes the attachment of Agrobacterium tumefaciens.strain NT1 to carrot cells.</a> Protoplasma. 183, 131-136 (1994).</li><li>Matthysse, A. G., McMahan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+the+Agrobacterium+tumefaciens.+attR+mutation+on+attachment+and+root+colonization+differs+between+legumes+and+other+dicots.">The effect of the Agrobacterium tumefaciens. attR mutation on attachment and root colonization differs between legumes and other dicots.</a> Appl. Environ. Microbiol. 67, 1070-1075 (2001).</li><li>Jeter, C., Matthysse, A. G. <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+the+binding+of+diarrheagenic+strains+of+E.+coli.to+plant+surfaces+and+the+role+of+curli+in+the+interaction+of+the+bacteria+with+alfalfa+sprouts.">Characterization of the binding of diarrheagenic strains of E. coli.to plant surfaces and the role of curli in the interaction of the bacteria with alfalfa sprouts.</a> Mol. Plant Microbe Interact. 18, 1235-1242 (2005).</li><li>Matthysse, A. G., McMahan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root+colonization+by+Agrobacterium+tumefaciens+is+reduced+in+cel,+attB,+attD,+and+attR+mutants.">Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD, and attR mutants.</a> Appl. Environ. Microbiol. 64, 2341-2345 (1998).</li><li>Morton, E. R., Fuqua, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+analyses+of+Agrobacterium.">Phenotypic analyses of Agrobacterium.</a> Curr. Protoc. Microbiol. , (2012).</li><li>Brandl, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+lesions+promote+the+rapid+multiplication+of+Escherichia+coli+O157:H7+on+postharvest+lettuce.">Plant lesions promote the rapid multiplication of Escherichia coli O157:H7 on postharvest lettuce.</a> Appl. Environ. Microbiol. 74, 5285-5289 (2008).</li><li>Franz, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+contamination+of+lettuce+by+GFP-expressing+Escherichia+coli+O157:H7+and+Salmonella+enterica+serovar+Typhimurium.">Quantification of contamination of lettuce by GFP-expressing Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.</a> Food Microbiol. 24, 106-112 (2007).</li><li>Morton, E. R., Fuqua, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+maintenance+of+Agrobacterium.">Laboratory maintenance of Agrobacterium.</a> Curr. Protoc. Microbiol. , (2012).</li><li>Lugtenberg, B. J., Kravchenko, L. V., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomato+seed+and+root+exudate+sugars:+composition,+utilization+by+Pseudomonas+biocontrol+strains+and+role+in+rhizosphere+colonization.">Tomato seed and root exudate sugars: composition, utilization by Pseudomonas biocontrol strains and role in rhizosphere colonization.</a> Environ. Microbiol. 1, 439-446 (1999).</li><li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Revised+Medium+for+Rapid+Growth+and+Bio+Assays+with+Tobacco+Tissue+Cultures.">A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures.</a> Physiologia Plantarum. 15, 473-497 (1962).</li><li>Danhorn, T., Fuqua, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+by+plant-associated+bacteria.">Biofilm formation by plant-associated bacteria.</a> Annu. Rev. Microbiol. 61, 401-422 (2007).</li><li>Branda, S. S., Vik, S., Friedman, L., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+the+matrix+revisited.">Biofilms: the matrix revisited.</a> Trends Microbiol. 13, 20-26 (2005).</li><li>O'Toole, G., Kaplan, H. B., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+as+microbial+development.">Biofilm formation as microbial development.</a> Annu. Rev. Microbiol. 54, 49-79 (2000).</li><li>Charkowski, A. O., Barak, J. D., Sarreal, C. Z., Mandrell, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+growth+of+Salmonella+enterica+and+Escherichia+coli+O157:H7+on+alfalfa+sprouts.">Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts.</a> Appl. Environ. Microbiol. 68, 3114-3120 (2002).</li><li>Loper, J., Suslow, T., Schroth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lognormal+distribution+of+bacterial+populations+in+the+rhizosphere.">Lognormal distribution of bacterial populations in the rhizosphere.</a> Phytopathology. 74, 1454-1460 (1984).</li><li>Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Agrobacterium+to+plant+surfaces.">Attachment of Agrobacterium to plant surfaces.</a> Front Plant Sci. 5, 252 (2014).</li><li>Douglas, C. J., Halperin, W., Nester, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+mutants+affected+in+attachment+to+plant+cells.">Agrobacterium tumefaciens mutants affected in attachment to plant cells.</a> J. Bacteriol. 152, 1265-1275 (1982).</li><li>Tomlinson, A. D., Fuqua, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+regulation+of+polar+surface+attachment+in+Agrobacterium+tumefaciens.">Mechanisms and regulation of polar surface attachment in Agrobacterium tumefaciens.</a> Curr. Opin. Microbiol. 12, 708-714 (2009).</li><li>Matthysse, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attachment+of+Agrobacterium+to+plant+surfaces.">Attachment of Agrobacterium to plant surfaces.</a> Front Plant Sci. 5, (2014).</li><li>Wright, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endophytic+lifestyle+of+Escherichia+coli+O157:H7:+quantification+and+internal+localization+in+roots.">The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.</a> Phytopathology. 103, 333-340 (2013).</li><li>Berger, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fresh+fruit+and+vegetables+as+vehicles+for+the+transmission+of+human+pathogens.">Fresh fruit and vegetables as vehicles for the transmission of human pathogens.</a> Environ. Microbiol. 12, 2385-2397 (2010).</li><li>Erickson, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internalization+and+Fate+of+Escherichia+coli+O157:H7+in+Leafy+Green+Phyllosphere+Tissue+Using+Various+Spray+Conditions.">Internalization and Fate of Escherichia coli O157:H7 in Leafy Green Phyllosphere Tissue Using Various Spray Conditions.</a> J. Food Prot. 77, 713-721 (2014).</li><li>McKellar, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+different+approaches+for+modeling+Escherichia+coli+O157:H7+survival+on+field+lettuce.">Evaluation of different approaches for modeling Escherichia coli O157:H7 survival on field lettuce.</a> Int. J. Food Microbiol. 184, 75-85 (2014).</li><li>Wu, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+detection+and+enumeration+of+Escherichia+coli+O157:H7+on+alfalfa+seeds.">Factors influencing the detection and enumeration of Escherichia coli O157:H7 on alfalfa seeds.</a> Int. J. Food Microbiol. 71, 93-99 (2001).</li></ol>

يعلن مقدم البلاغ أن لديها لا تضارب المصالح المالية.

من المهم أن يكون على بينه من جميع الأسطح التي يمكن أن تلتزم بها البكتيريا خلال التجربة. وبالتالي قد يمكن التقليل البكتيريا التي قادرة على الربط بالزجاج إذا فعلت عدد خلايا قابلة للحياة باستخدام أنابيب زجاجية والماصات. إذا كانت تزرع النباتات في أجار أو التربة وبعض من أجار أو يبقى التربة على النباتات قد ربط البكتيريا للمواد المنضمة إليها بدلاً من النباتات. من ناحية أخرى، الغسيل على سطح النبات، لا سيما في حالة الجذور، يمكن إزالة الطلاء السطحية الطبيعية مثل الأغشية المخاطية وهكذا تغير نتائج اختبارات الالتزام.
من المهم أن تكون على يقين من أن البكتيريا المضافة إلى خليط حضانة البقاء على قيد الحياة خلال التجربة. ومن ثم ينبغي بشكل روتيني عدد خلايا قابلة للحياة من البكتيريا الحرة فضلا عن المرفق. بعض العلاجات أو الطفرات البكتيرية قد خفض معدل النمو البكتيرية أو في الواقع سببا لوفاة جزء صغير من سكان الجرثومي. قد لا يمكن تمييزها بالمجهر البكتيريا الحية والميتة ما لم تستخدم البقع الخاصة. وهناك مجموعة من وصمة مفيدة للبكتيريا يعيش الميت الذي يعتمد على استبعاد الأصباغ من البكتيريا الحية. ومع ذلك، إذا كانت موجودة بعدد سكان مختلطة من الأنواع البكتيرية ثم عدد خلايا قابلة للبقاء من الأنواع ذات الأهمية يرجح أن أسهل طريقة لتحديد ما إذا كان الاحتضان أدى إلى وفاة البكتيرية.
التكوين المتوسط ستؤثر على بقاء البكتيرية والنمو. وستوفر الإفرازات الجذرية والمواد من الجرح وقطع مواقع الركيزة لدعم النمو البكتيري متواضعة. الفوسفات والنتروجين والحديد تميل إلى أن الحد في مثل هذه الظروف. وقد تؤثر divalent الكاتيونات مثل الكالسيوم والمغنيسيوم الالتصاق. وفي بعض الحالات يمكن أن تؤثر مصدر الكربون الالتصاق. أيضا المسائل الأس الهيدروجيني. بشكل عام الرقم الهيدروجيني رهيزوسفيري بين 5.5 و 6.5.
من الضروري أن نكون حذرين في استخدام البكتيريا المقاومة للمضادات الحيوية التي تحمل. أن المضادات الحيوية تستخدم في أغلب الأحيان هي الريفامبيسين وحمض الناليديكسيك. المقاومة لهذه المضادات الحيوية عموما بسبب الطفرات في الجينات الصبغية (بوليميريز الحمض النووي الريبي وجيرسي، على التوالي)، وهكذا لا يمكن بسهولة نقلها إلى سلالة أخرى خلال الاحتضان. لا يؤدي هذا النوع من المقاومة أيضا في تدهور أو تعديل للمضادات الحيوية. لا ينصح بوسم البكتيريا مع علامة تنقلها بلازميد جينات إلا بلازميد لا يمكن نقلها إلى أي بكتيريا أخرى. يجب أن لا مقاومة المضادات الحيوية بسبب تعديل التدهور أو الكيميائي للمضادات الحيوية البكتيريا الحساسة المضادات الحيوية ثم ستكون قادرة على النمو على ألواح المضادات الحيوية إذا كانوا على مقربة من البكتيريا المقاومة.
الطرق الموضحة في هذه الورقة مفيدة لأحجام عينة صغيرة و/أو تجارب فيها العينات تحتاج إلى ترد (على سبيل المثال، التجارب التي تنطوي على مسببات الأمراض البشرية). للعينات ذات الأحجام الكبيرة (فوق 100 غرام من المواد أو النباتات أكثر من 50) ستكون طرق أخرى أو تعديل جذري لهذه الأساليب المطلوبة10،،من1932،،من1933، 34 , 35 , 36-أن وجود عدد كبير من الكائنات الحية الدقيقة عدا الأنواع التي تجري دراستها يمكن أن تشكل أيضا مشاكل كبيرة. وتشمل الحلول الممكنة استخدام البكتيريا ملحوظ مع البروتين الفلورسنت أو المقاومة للمضادات الحيوية كما هو موضح في الخطوات 6.1.2 و 7-3. عندما تكون البكتيريا التي تهم الأفراد نادرة في عدد كبير من الكائنات الحية الدقيقة الأخرى ومع ذلك، قد لا تكون كافية للسماح بتقييم لا لبس فيها عدد من البكتيريا قيد الدراسة هذه العلامات.
جميع الأساليب الموصوفة هنا هي الطرق المختبرية على أساس. أنه يلزم إجراء تعديلات طفيفة لدراسات الاحتباس الحراري. التعديلات الرئيسية أكثر من المحتمل أن المطلوبة لإجراء دراسات ميدانية حيث البروتوزوا، والحشرات، وتباين اﻻفتراس والمناخ الأخرى الحيوانية تعقيد توفير شروط محددة لهذه التجارب. في المستقبل هذه الأساليب يمكن أن توسع لتشمل التفاعلات بين اثنين أو أكثر من الكائنات الدقيقة على سطح النبات.

قياس البكتيرية الملزمة لزرع السطوح أصبح هاما في ثلاث حالات متباينة. الحالة الأولى هي النظر في نقل مسببات الأمراض البشرية في مصنع السطوح1،،من23. والهدف هنا هو لمنع الربط البكتيرية أو لإزالة أو تقتل بكتيريا ملزمة، وبالتالي الحد من انتقال المرض من المواد النباتية. والحالة الثانية هي النظر في ربط مسببات أمراض النبات لزراعة الأسطح4. مرة أخرى والهدف هنا هو لمنع الربط أو لإزالة أو تقتل بكتيريا ملزمة، وبالتالي للحد من المرض. والحالة الثالثة هي دراسة الربط من البكتيريا التكافلية أو تشجع على نمو النبات5،6. والهدف هنا تعزيز البكتيريا ملزم، وبالتالي إلى زيادة صحة النباتات والمحاصيل غلة.
تقنيات لقياس البكتيرية الملزمة لزرع السطوح الموضحة في هذه المقالة رخيصة وسهلة نسبيا للقيام. المتطلبات الوحيدة هي مجهر والمواد التي توجد عادة في مختبر علم الجراثيم. سونيكاتور حمام مفيد لبعض التقنيات. صممت التقنيات الموضحة لربط التجارب التي أجريت باستخدام أحجام عينة صغيرة نسبيا. قياسات لربط مصنوعة في المختبر، على الرغم من أنه قد يكون من الممكن لتعديل بعض هذه التقنيات لاستخدامها في الدفيئة أو في الميدان.
وقد استخدمت هذه التقنيات لقياس ربط البكتيرية على جذور وبراعم وأوراق قص وقطع الفواكه والطماطم سليمة في المختبر7،،من89،10،11، 12،13،،من1415. أنها استخدمت أيضا قياس استعمار الجذر للنباتات التي تنمو في التربة أو الرمال في مختبر16. وقد استخدمت التقنيات مع العديد من أنواع الجراثيم بما في ذلك توميفاسينس المتبعة، الرايزوبيوم سينورهيزوبيوم، الإشريكيّة القولونية، انتيريكا السالمونيلا، و المتألقة Pseudomonas. يمكن الاطلاع على وصف مفيدة لأساليب لتقييم التفاعل بين A. tumefaciens مع الأسطح في مورتون وفوكوا (2012)17. وفي جميع الحالات كانت أحجام العينات المعنية صغيرة، عموما أقل من النباتات 25-50. التقنيات الموضحة مناسبة للاستخدام مع مسببات الأمراض البشرية التي تحتاج أن تبقى الواردة خلال التجارب.

<table><tbody><tr><td>light microscope</td><td>any</td><td>N/A</td><td>phase contrast or Nomarski optics are helpful, for studies using fluorescent markers a fluorescence microscope is required.</td></tr><tr><td>seeds</td><td>any&amp;nbsp;</td><td>N/A</td><td>make a note of the seed lot number and the cultivar</td></tr><tr><td>bleach</td><td>any</td><td>N/A</td><td></td></tr><tr><td>bath sonicator</td><td>any scientific supply company</td><td>N/A</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>nutrient agar&amp;nbsp;</td><td>Difco&amp;nbsp;</td><td>001-01-8</td><td></td></tr><tr><td>soytone</td><td>Difco&amp;nbsp;</td><td>24360</td><td></td></tr><tr><td>Sand</td><td>sea sand Fisher Scientific</td><td>S25</td><td></td></tr><tr><td>sand</td><td>&amp;nbsp;Sigma-Aldrich</td><td>S9887</td><td></td></tr><tr><td>conetainers</td><td>Stuewe &amp;amp; Sons, Inc.</td><td>Ray-Leach cone-tainers</td><td>many different sizes are available to suit the type of plant you wish to grow</td></tr><tr><td>parafilm</td><td>any scientific supply company</td><td>N/A</td><td></td></tr><tr><td>MS salts</td><td>Sigma-Aldrich</td><td>M5524</td><td></td></tr><tr><td>parrafin</td><td>any</td><td>N/A</td><td></td></tr><tr><td>centrigfuge</td><td>any scientific company</td><td>N/A</td><td>to pellet most bacteri only a small centrifuge with a max force of 10,000 XG is needed</td></tr><tr><td>vortex mixer</td><td>any scientific company</td><td></td><td></td></tr><tr><td>micrometer</td><td>ACCU-SCOPE Accessories</td><td>A3145</td><td></td></tr><tr><td>Sedgwick-rafter counting cell</td><td>Hauser Scientific</td><td>HS3800</td><td></td></tr><tr><td>probe-clip press-seal incubatin chamber</td><td>Sigma-Aldrich</td><td>Z359483</td><td></td></tr><tr><td>rifampicin</td><td>Sigma-Aldrich</td><td>R3501</td><td></td></tr><tr><td>naladixic acid</td><td>Sigma-Aldrich</td><td>n8878</td><td></td></tr><tr><td>Live/dead stain</td><td>In Vitrogen Molecular Bioprobes</td><td>L7007</td><td></td></tr></tbody></table>

adherence of bacteria to plant surfaces measured in the laboratory

هذه المخطوطة وصف أسلوب قياس ربط البكتيرية على أسطح النبات أكسينيك في المجهر الخفيفة، ومن خلال استخدام عدد خلايا قابلة للحياة. المواد النباتية المستخدمة تشمل جذور وبراعم وأوراق وقطع الفواكه. الأساليب الموصوفة رخيصة وسهلة ومناسبة لأحجام عينة صغيرة. يتم قياس ملزمة في المختبر ويمكن استخدام مجموعة متنوعة من الوسائط الحضانة وشروط. ويمكن تحديد تأثير مثبطات. ويمكن أيضا تقييم الحالات التي تعزز وتمنع ملزمة. من الممكن في بعض الحالات التمييز بين ما إذا كان تغيير مختلف الظروف ملزمة أساسا بسبب آثارها على النبات أو البكتيريا.

Watch this Scientific Journal Video about Adherence of Bacteria to Plant Surfaces Measured in the Laboratory at JoVE.com

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

هي طريقة سهلة لقياس ووصف التصاق البكتيريا إلى النباتات وخاصة الجذور وبراعم، الموضحة في هذه المقالة.

الانضمام للبكتيريا لزراعة الأسطح قياسها في المختبر

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell ...

adherence-of-bacteria-to-plant-surfaces-measured-in-the-laboratory

Universidad de Cartagena UDEC

Research

JoVE Journal

Environment

12.6K Views.  University of North Carolina at Chapel Hill. هي طريقة سهلة لقياس ووصف التصاق البكتيريا إلى النباتات وخاصة الجذور وبراعم، الموضحة في هذه المقالة.

Video: Adherence of Bacteria to Plant Surfaces Measured in the Laboratory

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

الفحص في المختبر من الالتصاق الجرثومي على الخلايا الظهارية الثدييات

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Immunology and Infection

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

الهندسة البكتيريا تمسكا من إنشاء واحدة الاصطناعية Curli OPERON

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Bioengineering

كشف الاستعمار الجذري على أرابيدوبسيس ثاليانا

Measuring Bacterial Colonization on Arabidopsis thaliana Roots in Hydroponic Condition

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized method for measuring bacterial colonization in the rhizosphere is necessary to improve reproducibility. We first cultured sterile A.thaliana in hydroponic conditions and then inoculated the bacterial cells in the rhizosphere at a final concentration of OD600 of 0.01. At 2 days post-inoculation, the root tissue was harvested and washed three times in sterile water to remove the uncolonized bacterial cells. The roots were then weighed, and the bacterial cells colonized on the root were collected by vortex. The cell suspension was diluted in a gradient with a phosphate-buffered saline (PBS) buffer, followed by plating onto a Luria-Bertani (LB) agar medium. The plates were incubated at 37 &#176;C for 10 h, and then, the single colonies on LB plates were counted and normalized to indicate the bacterial cells colonized on roots. This method is used to detect bacterial colonization in the rhizosphere in mono-interaction conditions, with good reproducibility.

تسليط الضوء على المؤلف: تعزيز استعمار الجذور على جذور النباتات لتحسين كفاءة الأسمدة الميكروبية

Colonization of plant growth-promoting rhizobacteria (PGPR) in the rhizosphere is essential for its growth-promoting effect. It is necessary to standardize the method of detection of bacterial rhizosphere colonization. Here, we describe a reproducible method for quantifying bacterial colonization on the root surface.

قياس الاستعمار البكتيري على جذور أرابيدوبسيس ثاليانا في حالة الزراعة المائية

Measuring bacterial colonization on Arabidopsis thaliana root is one of the most frequent experiments in plant-microbe interaction studies. A standardized ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

بكتيريا ورقة تسلل الفحص لتوصيف غرامة الردود الدفاع النبات باستخدام<em&gt; نبات الأرابيدوبسيس thaliana-الزائفة syringae</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Biomimetic Materials to Characterize Bacteria-host Interactions

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The biochemical and functional characterization of adhesins mediating these initial bacteria-host interactions is often compromised by the presence of other bacterial factors, such as cell wall components or secreted molecules, which interfere with the analysis. This protocol describes the production and use of biomimetic materials, consisting of pure recombinant adhesins chemically coupled to commercially available, functionalized polystyrene beads, which have been used successfully to dissect the biochemical and functional interactions between individual bacterial adhesins and host cell receptors. Protocols for different coupling chemistries, allowing directional immobilization of recombinant adhesins on polymer scaffolds, and for assessment of the coupling efficiency of the resulting &#8220;bacteriomimetic&#8221; materials are also discussed. We further describe how these materials can be used as a tool to inhibit pathogen mediated cytotoxicity and discuss scope, limitations and further applications of this approach in studying bacterial - host interactions.

Bacterial attachment to host cells is a key step during host colonization and infection. This protocol describes the generation of polymer-coupled recombinant adhesins as biomimetic materials which allow analysis of the contribution of individual adhesins to these processes, independent of other bacterial factors.

المواد الجزيئية الحيوية لوصف التفاعلات البكتيريا في استضافة

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

رصد الاستعمار البكتيري والصيانة على جذور الثاليانية العربية في نظام مائي عائم

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA. FimH adhesin, however, is present as a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor. Here, we provide protocols to evaluate and compare the function of type 1 fimbrial subunits in F18ab fimbriae+ Shiga toxin-producing Escherichia coli (STEC). We found that both FimA and FimH are required for bacterial adhesion, invasion, and biofilm formation. Deleting fimA gene showed much more reduction in bacterial adhesion and invasion to porcine intestinal columnar epithelial cells IPEC-J2, than that of fimH mutant. Biofilm formation was significantly reduced in both mutants with an equal level. In addition, qPCR demonstrated that either fimA or fimH deletion down-regulated the bacterial flagella and F18 fimbriae genes expression, while up-regulated adhesin was involved in diffuse adherence-I (AIDA-I) gene expression, suggesting the co-regulation of cell surface-localized adhesins in F18ab fimbriae+ STEC.

<meta charset="utf8"></head><body>استخدام قضيب فيمبريال لاستعمار F18ab Fimbriae + STEC للخلايا المضيفة

Here we present a protocol to study the function of fimbriae in bacterial colonization.

استخدام قضيب فيمبريال لاستعمار F18ab Fimbriae + STEC للخلايا المضيفة

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily ...

الانضمام للبكتيريا لزراعة الأسطح قياسها في المختبر

Summary

Explore More Videos

Chapters in this video

الفحص في المختبر من الالتصاق الجرثومي على الخلايا الظهارية الثدييات

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

الهندسة البكتيريا تمسكا من إنشاء واحدة الاصطناعية Curli OPERON

قياس الاستعمار البكتيري على جذور أرابيدوبسيس ثاليانا في حالة الزراعة المائية

قياس الاستعمار البكتيري على جذور أرابيدوبسيس ثاليانا في حالة الزراعة المائية

قياس الاستعمار البكتيري على جذور أرابيدوبسيس ثاليانا في حالة الزراعة المائية

بكتيريا ورقة تسلل الفحص لتوصيف غرامة الردود الدفاع النبات باستخدام

المواد الجزيئية الحيوية لوصف التفاعلات البكتيريا في استضافة

رصد الاستعمار البكتيري والصيانة على جذور الثاليانية العربية في نظام مائي عائم

استخدام قضيب فيمبريال لاستعمار F18ab Fimbriae ⁺ STEC للخلايا المضيفة