Patent Application: US-201414774255-A

Abstract:
citrus canker caused by the bacterium xanthomonas citri subsp . citri is an economically important disease of citrus worldwide . biofilm formation plays an important role in early infection of xac on host leaves . in this study , we assessed the hypothesis that small molecules able to inhibit biofilm formation reduce xac infection and enhance the control of citrus canker disease . d - leucine and 3 - indolylacetonitrile were found to prevent biofilm formation by xac on different abiotic surfaces and host leaves at a concentration lower than the minimum inhibitory concentration . quantitative real

Description:
in other embodiments , the present invention relates to methods for reducing the population of a crop - related microbe . the method includes applying to an object a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent . the method includes applying the agents together in one composition or separate compositions in amount and for time sufficient to reduce the microbial population . the metal antimicrobial agent may include copper or copper containing compounds . in one embodiment , provide is a composition that includes a carrier ( e . g ., water ), and optionally a surfactant ( e . g ., triton x - 100 ( octyl phenol alkoxylate n9 . 5 ) and tectronic 1107 ( alkoxylated ethylene diamine with , for example an average molecular weight of about 15 , 000 ), solvent ( e . g ., 1 - methyl - 2 - pyrrolidinone ), and optionally alcohol ( e . g ., ethanol ). in a specific embodiment , the crop is citrus , and the microbe is a canker microbe ( e . g ., citrus canker microbe ) or microbes . the method can include applying the biofilm reducing agent , or a metal antimicrobial agent and biofilm reducing agent , to any of a variety of objects , such as a citrus tree . the method can include applying the agents to citrus fruit . the citrus fruit can be on the tree or can be off the tree ( i . e ., it can already have been picked ). the method can include applying the agents to inanimate objects , such as equipment . the equipment can be equipment used in a citrus orchard , equipment used for transporting or processing produce , such as citrus fruit , equipment used for transporting or processing a plant , or the like . in a particular embodiment , provided is a method of reducing a population of a microbe on an object by applying a biofilm reducing agent and a metal antimicrobial agent to the object , such that efficacy of the metal antimicrobial agent is enhanced . enhancement of the metal antimicrobial agent involves the achievement of efficacy of a lower dose of the metal antimicrobial agent in the presence of the biofilm reducing agent that requires a higher dose of the metal antimicrobial agent in the absence of the biofilm reducing agent . this embodiment allows for a reduction in the amount of metal antimicrobial agents needed to control microbe populations , which , in turn , is less harmful to the environment , less toxic , and reduces costs involved in controlling microbes . embodiments also relate to methods for treating citrus canker . the method may include applying to a citrus tree a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent . the method includes applying the antimicrobial composition in amount and for time sufficient to reduce the microbial population . embodiments also relate to methods and compositions for reducing the population of canker microbe ( e . g ., citrus canker microbe ). such a composition includes a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent . the composition can be for applying to an object subject to contamination with canker microbe ( e . g ., citrus canker microbe ). in another embodiment , a biofilm reducing agent is applied alone or separate to a metal antimicrobial agent . for example , a composition that includes biofilm reducing agent to reduce population of a canker microbe without an antimicrobial agent ( such as a metal antimicrobial agent is applied to a citrus tree . embodiments also relate to articles of manufacture . such an article of manufacture can include a composition including a biofilm reducing agent or a metal antimicrobial agent and a biofilm reducing agent . such an article of manufacture can include a sprayer configured for spraying citrus and a composition including metal antimicrobial agent and a biofilm reducing agent . suitable sprayers configured for spraying citrus include those large enough to be towed behind a truck and that , for example , use air in forming a spray from a composition in a tank or other container . suitable sprayers include electrostatic sprayers . such an article of manufacture can include composition including a biofilm agent , or a metal antimicrobial agent and a biofilm reducing agent , and instructions for applying the composition to citrus . such an article of manufacture can include composition including metal antimicrobial agent and a biofilm reducing agent and instructions for applying the composition to object subject to contamination with canker microbe ( e . g ., citrus canker microbe ). any of a variety of known methods can be employed for testing for activity against a canker microbe ( e . g ., citrus canker microbe ). for example , a composition can be tested in a laboratory test ( e . g ., in vitro ) or a nursery . embodiments of such methods are described in the examples . for example , a composition can be tested in a prevention fruit protocol . a prevention fruit protocol can employ citrus fruit ( non - waxed ) treated with 50 ppm sodium hypochlorite and rinsed with sterilized milli - q water . the method can include treating a fruit surface by spraying a solution of the test substance over the fruit surface with a spray bottle several times over one week . infecting the fruit can be carried out by misting xanthomonas ( e . g ., x . citri ) or a model microorganism over surface or by spot inoculation ( especially to vulnerable areas ). this can be followed by allowing bacteria to sit on the fruit overnight . the fruit can be treated with the test substance . the fruit can be incubated in a hood for about 2 to about 3 weeks to determine if there is growth . the fruit can be sampled after the incubation period by putting the fruit into neutralizer ( bag ), massaging for one minute , and plating . controls can include fruit treated with chemicals for the first treatment period , inoculated with no follow - up treatment , and fruit untreated with chemicals but inoculated . according to other embodiments , provided are methods of treating and using water - based systems for transporting , processing , and / or washing citrus . also provided are methods for transporting or processing citrus using an aqueous medium to transport the citrus through , for example , one or more processing steps and environments . according to one embodiment , the aqueous medium includes a metal antimicrobial agent and a biofilm reducing agent . in addition , provided is a method for reducing the population of microbes in aqueous streams by applying or incorporating a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent , to or into the aqueous stream . generally , the aqueous streams used in any number of applications such as the application of streams for the transport of citrus into the processing environment and through the various steps of processing . in a further method embodiment , after picking , the method includes transporting and / or washing citrus in a stream of a biofilm reducing agent composition , or a metal antimicrobial agent and a biofilm reducing agent composition . for example , an aqueous metal antimicrobial agent and biofilm reducing agent composition can be used to support or transport the citrus from an unloading site to a storage , packing , or processing location . the method can include introducing the citrus into a flume containing an aqueous metal antimicrobial agent and a biofilm reducing composition . in a further embodiment , provided is a method that includes transporting fresh citrus in and to food handling equipment used at a processing plant using a stream of a biofilm reducing agent , or a metal antimicrobial agent and biofilm reducing agent composition . for example , the method can include transporting a food item using or in a biofilm reducing agent composition , or a metal antimicrobial agent and biofilm reducing agent composition , from an initial location through a series of individual processing stages to a station where the citrus is removed from the water and packed . the method can include recycling the aqueous biofilm reducing agent composition , or aqueous metal antimicrobial agent and biofilm reducing agent composition used for transporting or processing citrus . in a further embodiment , provided is a method of cleaning ( e . g ., washing ), cooling ( e . g ., in a bath ), heating , cooking , or otherwise processing the citrus before packaging using a biofilm reducing agent composition , or an metal antimicrobial agent and a biofilm reducing agent . in an embodiment , the present method includes transporting and processing the citrus using the same stream . in a specific embodiment , the present method includes transporting the citrus in a first aqueous stream and processing the citrus in a second aqueous composition distinct from the transport stream . the present invention includes recycling the aqueous metal antimicrobial agent and biofilm reducing agent employed in methods for cleaning , cooling , heating , cooking , or otherwise processing the citrus . in another embodiment , disclosed is a method of reducing the population of microbes on or in the water , flume , or other transport or processing equipment employed with the citrus . the method includes contacting the water , flume , or other transport or processing equipment with a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent . in an embodiment , the present invention includes reducing or preventing the buildup of slime or biofilm on surfaces of the flume or other transport or processing equipment employed with the citrus . the method includes contacting the surfaces of the flume or other transport or processing equipment with a metal antimicrobial agent and biofilm reducing agent . the present invention also includes methods for packaging citrus . in an embodiment , the present method can reduce the microbial population on citrus or packaging material before or during the packaging operation . the method includes contacting the citrus or packaging material with a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent before or during the packaging operation . in an embodiment , the present method can reduce the microbial population on packaged citrus . the method includes contacting the package of citrus with metal antimicrobial agent and biofilm reducing agent . embodiments include transporting or processing packaged citrus using the biofilm reducing agent , or a metal antimicrobial agent and biofilm reducing agent composition . in an embodiment , the present method includes heating , cooling , or otherwise processing packaged citrus using a metal antimicrobial agent and biofilm reducing agent . in an embodiment , the present disclosure includes a method of reducing the population of microbes on citrus . the method can include contacting the citrus with a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent . contacting can include applying the present composition to the citrus . applying can occur at any step of the life cycle , production cycle , or marketing of the citrus . for example , the present composition can be applied to the citrus in the field , in or on any apparatus ( e . g ., harvester ), in a transport apparatus or during transport , in a warehouse , in a processing facility , in a wholesaler , in a retail establishment ( e . g ., a grocer ), in a home , or in a restaurant . in a specific embodiment , applying a biofilm reducing agent , or a metal antimicrobial agent and a biofilm reducing agent , involves injecting into a trunk of a tree , administering to bark of a tree , administering to soil proximate to a tree , or irrigating a tree . proximate to the tree involves a distance immediately adjacent to the tree or at distance from the tree no more than what is sufficient to exact benefits of the composition . in one example , proximate means within ten feet , within 20 feet , within 30 feet , within 40 feet , within 50 feet or within 60 feet from the tree . once the biofilm agent , or metal antimicrobial agent and biofilm reducing agent , are applied to any given transport stream , the biofilm agent or antimicrobial agent will be subjected to a demand resulting from microbes present in the stream as well as optionally added organic or inorganic material present in the stream . as a general guideline , not limiting of the invention , the present invention includes the concentrations of metal antimicrobial agent and biofilm reducing agent containing composition found after demand . embodiments of the methods of the present invention can include agitation or sonication of the use composition , particularly as a concentrate is added to water to make the use composition . in an embodiment , the present methods include water systems that have some agitation , spraying , or other mixing of the solution . the citrus can be contacted with the compositions of the invention effective to result in a reduction significantly greater than is achieved by washing with water , or at least a 50 % reduction , at least a 90 % reduction , or at least a 99 % reduction in the resident microbial preparation . the present methods can employ a certain minimal contact time of the composition with of citrus for occurrence of significant antimicrobial effect . the contact time can vary with concentration of the use composition , method of applying the use composition , temperature of the use composition , amount of soil on the citrus , amount of soil in the aqueous stream , number of microorganisms on the citrus , number of microorganisms in the aqueous stream , or the like . contact time in the field can be for as long as nature allows , for example , until the next rain or heavy rain . in an embodiment , the exposure time is at least about 5 to about 60 seconds . in a further embodiment , exposure time is at least 5 minutes , at least 1 hour , at least 6 hours , or at least 24 hours . in another alternative embodiment of the present invention , the citrus or other plants can be treated with an electrostatically charged spray of a composition including a biofilm agent , or a metal antimicrobial agent and a biofilm reducing agent or coadministration of separate compositions containing one or the other agents . the composition can be spray applied as charged droplets by using conventional electrostatic spray technologies including inductively charged methodologies . as charged droplets , the composition will be attracted to opposite or differentially charged surfaces such as the surface of the citrus or other plants . as a result , more composition can be applied to the citrus and less solution will miss the intended target , commonly called over - spray . the charged droplets can provide an evenly distributed solution layer on the plants . the charged droplet size can range from about 10 microns to about 500 microns . the antimicrobial composition of the invention can also include any number of adjuvants . specifically , the composition of the invention can include additional antimicrobial agent , wetting agent , defoaming agent , thickener , a surfactant , foaming agent , aesthetic enhancing agent ( i . e ., colorant ( e . g ., pigment ), odorant , or perfume ), among any number of constituents which can be added to the composition , or combinations of the foregoing . in exemplary embodiments , an adjuvant is any material that when added to a spray solution enhances or modifies the action of a pesticide . a surfactant is a class of adjuvant including any compound which possesses distinct hydrophilic and lipophilic regions , which allow it to reduce the surface tension when mixed with water . example chemical classes include , but are not be limited to : alcohol alkoxylates , alkylaryl ethoxylates , fatty amine ethoxylates , organosilicones , some surfactants include multiple active constituents . in addition to surfactants , other types of adjuvants would include oils ( petroleum and crop based ), acidifiers , buffers , and others . adjuvants can be preformulated with the antimicrobial composition of the invention or added to the system simultaneously , or even after , the addition of the antimicrobial composition . composition embodiments can also contain any number of other constituents as necessitated by the application , which are known and which can facilitate the activity of the present invention . the antimicrobial compositions of the invention can contain an additional antimicrobial agent . additional antimicrobial agent can be added to use compositions before use . suitable antimicrobial agents include , but are not limited to , peroxycarboxylic acid ( e . g ., medium chain ( e . g ., c5 - c12 , c6 to c10 , or c8 ) peroxycarboxylic acid or mixed medium chain and short chain ( e . g ., c2 - c4 ) peroxycarboxylic acid ( e . g ., c2 and c8 )), carboxylic esters ( e . g ., p - hydroxy alkyl benzoates and alkyl cinnamates ), sulfonic acids ( e . g ., dodecylbenzene sulfonic acid ), iodo - compounds or active halogen compounds ( e . g ., elemental halogens , halogen oxides ( e . g ., naocl , hocl , hobr , clo 2 ), iodine , interhalides ( e . g ., iodine monochloride , iodine dichloride , iodine trichloride , iodine tetrachloride , bromine chloride , iodine monobromide , or iodine dibromide ), polyhalides , hypochlorite salts , hypochlorous acid , hypobromite salts , hypobromous acid , chloro - and bromo - hydantoins , chlorine dioxide , and sodium chlorite ), organic peroxides including benzoyl peroxide , alkyl benzoyl peroxides , ozone , singlet oxygen generators , and mixtures thereof , phenolic derivatives o - phenyl phenol , o - benzyl - p - chlorophenol , tert - amyl phenol and c 1 - c 6 alkyl hydroxy benzoates ), quaternary ammonium compounds ( e . g ., alkyldimethylbenzyl ammonium chloride , dialkyldimethyl ammonium chloride and mixtures thereof ), aminoglycosides ( streptomycin , kasugamycin ), tretracyclines ( oxytetracycline ), bacillus biologicals ( bacillus subtilis , bacillus amyloliquefaciens ), pantoea biologicals ( pantoea agglomerans ), pseudomonas biologicals ( pseudomonas fluorescens ), bacteriophages ( many phage strains ), and mixtures of such antimicrobial agents , in an amount sufficient to provide the desired degree of microbial protection . the present composition can include an effective amount of additional antimicrobial agent , such as about 0 . 001 wt -% to about 60 wt -% antimicrobial agent , about 0 . 01 wt -% to about 15 wt -% antimicrobial agent , or about 0 . 08 wt -% to about 2 . 5 wt -% antimicrobial agent . the present compositions may include concentrate compositions and use compositions . for example , a concentrate composition can be diluted , for example with water , to form a use composition . in an embodiment , a concentrate composition can be diluted to a use solution before to application to an object . for reasons of economics , the concentrate can be marketed and an end user can dilute the concentrate with water or an aqueous diluent to a use solution . the level of active components in the concentrate composition is dependent on the intended dilution factor and the desired activity of the composition components . generally , a dilution of about 1 fluid ounce to about 20 gallons of water to about 5 fluid ounces to about 1 gallon of water is used for aqueous antimicrobial compositions . higher use dilutions can be employed if elevated use temperature ( greater than 25 ° c .) or extended exposure time ( greater than 30 seconds ) can be employed . in the typical use locus , the concentrate is diluted with a major proportion of water using commonly available tap or service water mixing the materials at a dilution ratio of about 3 to about 20 ounces of concentrate per 100 gallons of water . for example , the use composition can include surf acme diluted 1 : 2 , 1 : 4 or 1 : 8 . for example , a use composition can include about 0 . 01 to about 4 wt -% of a concentrate composition and about 96 to about 99 . 99 wt -% diluent ; about 0 . 5 to about 4 wt -% of a concentrate composition and about 96 to about 99 . 5 wt -% diluent ; about 0 . 5 , about 1 , about 1 . 5 , about 2 , about 2 . 5 , about 3 , about 3 . 5 , or about 4 wt -% of a concentrate composition ; about 0 . 01 to about 0 . 1 wt -% of a concentrate composition ; or about 0 . 01 , about 0 . 02 , about 0 . 03 , about 0 . 04 , about 0 . 05 , about 0 . 06 , about 0 . 07 , about 0 . 08 , about 0 . 09 , or about 0 . 1 wt -% of a concentrate composition . amounts of an ingredient in a use composition can be calculated from the amounts listed above for concentrate compositions and these dilution factors . the administration of a metal antimicrobial agent and biofilm reducing agent can be conducted by application of a composition containing both components or by coadministration of two or more compositions containing either of the agents . typically , administration will involve the application of the metal antibiotic agent and the biofilm reducing agent such that they are both present on the intended object contemporaneously . alternatively , as noted above , administration involves application of a biofilm reducing agent without administration of a metal antimicrobial agent . the present invention may be better understood with reference to the following examples . these examples are intended to be representative of specific embodiments of the invention , and are not intended as limiting the scope of the invention . xac strain 306 [ rifamycin ( rif ) resistant ] ( rybak et al ., 2009 ) was used . nutrient agar ( na ) and nutrient broth ( nb ) were as the media for the growth of xac . the bacterium was initially streaked from − 80 ° c . glycerol stock on a na plate and a fresh single colony was inoculated in nb ( 40 ml ) in 150 ml flasks and cultured at 28 ° c . with agitation at 200 r . p . m . the overnight cultures were diluted in nb to standardize the cultures to obtain an optical density at 600 nm ( od 600 ) of 1 . 0 prior to setting up the biofilm assay and cell growth measurements . when necessary , rifamycin was added in the medium at a final concentration of 50 mg / ml . d - amino acids ( d - alanine , d - leucine , d - methionine , d - serine , d - tryptophan , and d - tyrosine ) and indole derivatives [ 3 - indoleacetic acid ( iaa ), 3 - indolylacetonitrile ( ian ) and indole - 3 - propioninc acid ( i3pa )] from plant sources were purchased from sigma - aldrich co . ( missouri , usa ). the other chemicals including crystal violet ( cv ), ethyl alcohol , dimethyl sulfoxide ( dmso ) were purchased from fisher scientific co . ( pittsburgh , usa ). stock solutions ( 500 mm in double distilled water for d - amino acids ; 5 mg / ml in 1 % dmso for indole derivatives ) were filter sterilized and stored at − 20 ° c . and diluted in sterilized distilled water ( sdw ) or dmso for the initial test concentrations . evaluation of d - amino acids and indole derivatives for potential biofilm inhibition ability . a static biofilm formation assay in 96 - well polystyrene plates coupled with cv staining was performed as previously reported with modifications ( li and wang , 2011 ). briefly , a 96 - well polystyrene plate ( nunclon surface , nuncbrand , denmark ) was prepared with 170 μl nb plus 1 . 0 % glucose per well . aliquots of 20 μl each of the different concentrations of the tested compound were pipetted into eight wells containing 170 μl of nb plus 1 . 0 % glucose . to each of these wells was added 10 μl of the standardized overnight xac 306 culture ( od 600 = 1 . 0 ). control wells contained nb plus 1 . 0 % glucose and sdw or 0 . 1 % dmso as a sterility control , or standardized overnight xac 306 culture as a growth control . the plate was covered by a microwell lid ( nunclon ), sealed with parafilm to prevent evaporation and incubated at 28 ° c . for 48 h without shaking . after incubation , the planktonic growth was measured at od 600 . to quantify the amount of biofilms formed on the surfaces of the wells , the culture was removed from the wells . after drying , the wells were washed twice with 200 μl of sdw for 5 min , allowed to dry then 200 μl of 0 . 1 % cv added . after 30 minutes , the cv was removed . the wells were washed with excess sdw to remove unbound cv and air dried in an inverted position for 2 h . afterward , 200 μl of 95 % ethanol was added to the wells and incubated for 30 min at room temperature to elute bound cv . the eluted cv was 2 - fold diluted in double distilled water and the absorbance at 590 nm was measured . each data point was averaged from eight replicate wells . to gain further evidence of the effect of selected compounds on biofilm formation , biofilm formation in glass tubes and on leaf surfaces was examined as described previously ( li and wang , 2012 ). briefly , the standardized overnight culture of xac 306 ( od 600 = 1 . 0 ) were diluted 1 : 10 in fresh nb containing 1 . 0 % glucose and the selected compound . for biofilm formation assay in glass tubes ( fischer scientific , pittsburgh , pa . ), 1 ml of the diluted bacterial suspension was transferred into each sterilized borosilicate glass tube and incubated at 28 ° c . without shaking for 48 h . the planktonic growth was then discarded and the tubes were gently washed three times with sdw . the biofilm formed on the tubes was visualized by staining with 0 . 1 % cv . the stain remaining in cells on glass tubes was dissolved in 95 % ethanol and quantified by measuring the optical density at 590 nm . for biofilm formation assay on leaf surfaces , 20 μl of the diluted bacterial suspension was dropped onto the abaxial surface of citrus leaves . the leaves were kept in a humidified chamber at 28 ° c . for 24 h without shaking . the biofilm formed on the leaf surfaces was visualized by staining with 0 . 1 % cv . the biofilm assays were repeated three times with four replicates each time . xac strain 306 was grown in nb at 28 ° c . with shaking at 200 rpm for 7 h . the cultures were standardized to an od 600 of 0 . 03 ( 5 × 10 ′ colony forming unit ( cfu )/ ml ) in nb and then aliquoted into wells of a 96 - well plate , 190 μl per well . the initial test concentrations of the compounds were diluted ( 1 : 20 ) in the culture ( 10 μl of compound in 190 μl of culture ) and incubated at 28 ° c . under stationary conditions . the cultures were monitored at 24 and 48 h at od 600 , and the lowest concentration resulting in no growth after 48 h compared to the control samples was defined as the mic for xac strain 306 . all determinations were conducted in eight replicate wells and repeated three times . the level of copper resistance of biofilms was evaluated using a cell viability assays . briefly , xac biofilms were prepared using nb with stationary incubation in glass tubes as described above . after 48 h - incubation , the cultures were removed and bacterial cells attached to the tubes were gently washed three times with sdw . one milliliter of fresh nb with cuso 4 ( 1 . 0 mm ), d - leucine ( 10 mm ) or ian ( 100 μg / ml ), or a combination of these compounds was added to each tube . nb alone was used as control . tubes were kept at room temperature for 24 h and shaken vigorously for 5 min . the suspensions were diluted in 10 - fold series , and 10 μl of each dilution spotted in triplicate on na plate . plates were incubated at 28 ° c . for 48 to 72 h prior to assessing bacterial growth . colonies that grew near the dilution end - point were counted and bacterial populations in the initial suspensions prior to dilution were calculated . each treatment compromises four replicates and the experiment repeated three times . rna prepare and quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ). to investigate the mechanisms of d - leucine and ian inhibiting xac 306 biofilm formation , qrt - pcr analysis was used to determine differential gene expression for xac306 cells with and without ian ( 100 μg / ml ) or d - leucine ( 10 mm ). for this analysis , xac306 was cultured in nb medium with or without biofilm inhibitor at 28 ° c . without shaking . cells were collected after 48 h of incubation by centrifugation at 12 , 000 × g for 5 min at 4 ° c . and used for rna extraction . for the analysis of gene expression in xac 306 planktonic and biofilm cells with sub - mics cuso 4 concentrations , we followed the same procedure described for evaluation of resistance of xac biofilm cells to copper in glass tubes . both biofilm cells attached to the glass tube at the medium - air interface and planktonic cells in culture were used . cells collected after 48 h of incubation in the presence of cuso 4 were washed by centrifugation at 12 , 000 × g for 5 min at 4 ° c . with diethylpyrocarbonate treated water . cells collected from five tubes were combined and served as one biological replicate . the pellet was stored at − 80 ° c . until rna extraction . total rna of xac306 cells was isolated using rna protect bacterial reagent ( qiagen , valencia , calif .) and rneasy mini kit ( qiagen , valencia , calif . ), following the manufacturer &# 39 ; s instructions . the contaminated genomic dna was removed using a turbo dna - free kit ( ambion , austin , tex .). rna purity and quality were evaluated with a nanodrop nd - 1000 spectrophotometer ( nanodrop technologies , wilmington , del .). a one - step qrt - pcr was conducted using a 7500 fast real - time pcr system ( applied biosystems , foster city , calif .) with a quantitect sybr green rt - pcr kit ( qiagen , valencia , calif .) following the manufacturer &# 39 ; s instructions . the gene specific primers ( table 1 ) were designed based on the genome sequence of xac strain 306 ( da silver et al ., 2002 ). in the biofilm inhibition mechanism studies , those primers targeted fifteen genes that were previously identified to be related bioflim formation in xac strain 306 ( li and wang , 2011 ). in the copper resistance analysis , those primers targeted the gum genes gumb and gumd polysaccharides — related gene galu , and copper resistance - related genes copa and copb ( teixeira et al ., 2008 ; behlau et al ., 2012 ). the dna gyrase subunit a encoding gene gyra was used as endogenous control . the relative fold change in gene expression was calculated using the formula 2 − δδct ( livak and schmittgen , 2001 ). qrt - pcr was repeated twice with four independent biological replicates each time . the effect of selected biofilm inhibitors on xac infection / virulence - was investigated using 20 - week - old potted grapefruit ( citrus paradise cv . duncan grapefruit ) plants in a quarantine greenhouse at the citrus research and education center , lake alfred , fla . copper ( cuso 4 , 100 μg / ml ), d - leucine ( 10 mm ), ian ( 100 μg / ml ) and a combination of these compounds were individually prepared in 100 ml of sdw . bacterial inoculum was prepared by growing xac 306 on na plates at 28 ° c . for 48 h , suspending in sdw , adjusting concentration to approximately od 600 = 0 . 3 ( 5 × 10 8 cfu / ml ). the inoculation was performed by a spray method as described previously ( li and wang , 2012 ) with modifications . briefly , the abaxial surfaces of fully expanded , immature leaves of each plant were sprayed with the following treatments : sdw , xac 306 , and xac 306 combined with cuso 4 ( 100 μg / ml ), d - leucine ( 10 mm ), ian ( 100 μg / ml ), or a combination of these compounds . silwett - l77 ( silicone - polyether copolymer , fisher ) was added to each treatment at a 0 . 03 % ( v / v ) final concentration . after inoculation , the plants were covered with plastic bags for 24 h to maintain a high (& gt ; 90 %) relative humidity and then kept in a greenhouse ( approximately 60 % relative humidity ) for symptom development . all inoculations included a minimum of three immature leaves at a similar developmental stage from each plant , and each treatment comprised four plants . the experiments were repeated three times independently . sdw was applied as a negative control , and xac306 mixed with sdw as positive control . to determine whether the timing of application of biofilm inhibitors in relation to xac306 affected canker symptom suppression , the biofilm inhibitors were inoculated on leaves 6 h prior to or after inoculation with xac306 . for bacterial population assays , the leaves of grapefruit plants were inoculated as described above . two leaf discs randomly selected from each of three inoculated leaves were cut with a cork borer ( 0 . 8 cm in diameter ) and then ground in 1 ml of sdw . the suspensions were serially diluted and plated on na plates containing rifamycin . after incubation at 28 ° c . for 48 h , bacterial colonies were counted and the number of cfu per square centimeter of leaf tissue was calculated . the assays were repeated three times independently . six d - amino acids and three indole derivatives derived from plant sources were tested for their ability to inhibit biofilm formation of xac306 in 96 - well plate biofilm assays . in the absence of d - amino acid or indole derivative , xac306 formed robust biofilms ( fig1 a ). d - leucine , d - serine and ian significantly reduced xac306 biofilm formation by 55 to 70 %, whereas the other d - amino acids or indole derivatives tested did not affect xac306 biofilm formation ( fig1 a ). furthermore , the compounds d - leucine , d - serine and ian decreased xac306 biofilm in a dose - dependent manner from 0 to 20 mm for d - leucine and d - serine ( fig1 b ) and 0 to 150 μg / ml for ian ( fig1 c ). to test the toxicity of the three biofilm inhibitors , the mics were determined in nb medium . the results showed that d - serine had the lowest mic of 8 . 0 mm , followed by d - leucine and ian , with mics of 16 . 0 mm and 200 μg / ml respectively ( table 2 ). d - leucine and ian had relatively high mics but paradoxically demonstrated the potent decrease in biofilm mass ( 58 % decrease for d - leucine ; 65 % decrease for ian ) at lower concentrations than mics ( 10 mm for d - leucine and 100 μg / ml for ian ) ( fig1 a ). due to its low toxicity to xac306 and apparent potency against biofilm formation , d - leucine and ian were selected for further studies . d - leucine and ian reduce biofilm formation on abiotic surfaces and host leaves at sub - mic concentrations to further evaluate and confirm the antibiofilm properties of d - leucine and ian , biofilm formation of xac306 with the compounds at sub - mic ( 10 mm for d - leucine and 100 μg / ml for ian ), a concentration that does not significantly decrease planktonic cell density ( data not shown ), was examined on three different kinds of surfaces : polystyrene , glass and host leaves . the d - leucine or ian treated cultures exhibited a significant reduction in biofilm formation both on polystyrene surface and in glass tubes compared to the untreated control , where the level of biofilm formation were reduced to 50 % and 60 % of control , respectively ( fig2 a ; b ). similar to the observations on polystyrene surface and in glass tubes , the d - leucine or ian treated cultures showed declined biofilm formation on citrus leaf surfaces ( fig2 c ), suggesting that d - leucine and ian reduced biofilm formation of xac strain 306 on citrus leaves . these findings confirmed that d - leucine and ian had specific activity inhibiting biofilm formation by xac strain 306 . to obtain insight into the mechanisms by which d - leucine and ian inhibit biofilm formation , the effect of these two compounds was evaluated on expression of genes important for biofilm formation in xac 306 using qrt - pcr . the selected genes included the gum genes gumb and gumd , polysaccharides — related gene galu , o - antigen biosynthesis gene rfbc , chemotaxis and motility genes chea , chey , mcpa , and motb , type iv twitching motility gene pilb , flagellar biosynthesis genes flen and flic , and regulator genes colr , clp , rpfg and rpon . the results showed significantly ( p & lt ; 0 . 05 , student t - test ) repression of chemotaxis and motility genes chey , motb and pilb with ian , whereas no induction or repression of any of the tested genes by d - leucine ( fig3 ). suppression of xac 306 resistance to copper by d - leucine and ian the copper resistance levels of planktonic cells of xac306 were evaluated with and without biofilm inhibitor , respectively . in the nb medium , planktonic cells exhibited a mic of 0 . 50 mm cuso 4 without biofilm inhibitor . in the presence of d - leucine , ian or a combination of the two compounds at a sub - mic concentration ( 10 mm for d - leucine and 100 μg / ml for ian ), the mics of cuso 4 against xac 306 planktonic cells were decreased to 0 . 25 mm ( table 2 ). these results suggested that d - leucine and ian increased the susceptibility of xac 306 planktonic cells to copper under the applied conditions . in the cell viability assays , biofilms treated with d - leucine , ian or a combination of the two compounds at sub - mic concentrations ( 10 mm for d - leucine and 100 μg / ml for ian ) were about 10 times more susceptible to cuso 4 than the untreated control . the cell viability of xac biofilms exposed to cuso 4 ( 1 . 0 mm ) was significantly reduced by d - leucine or ian . biofilms exposed to cuso 4 ( 1 . 0 mm ) alone for 24 h contained an average of 3 × 10 7 cfu / ml , while the biofilms treated by d - leucine ( 10 . 0 mm ), ian ( 100 μg / ml ) or a combination of the two compounds contained an average of 1 . 1 × 10 6 , 4 . 3 × 10 6 , and 2 . 5 × 10 6 cfu / ml , respectively ( fig4 ). these findings indicated that d - leucine and ian increased the susceptibility of xac 306 biofilm cells to copper under the applied conditions . to obtain insight into the mechanisms by which d - leucine and ian increase the susceptibility of xac 306 cells to copper , evaluated wasthe effect of the two compounds on expression of the polysaccharides related gene galu , gum genes gumb and gumd , and copper resistance related genes copa and copb in xac306 planktonic and biofilm cells , respectively . the qrt - pcr results showed that either d - leucine or ian did not affect express of any of the gene tested in xac planktonic or biofilm cells in the presence of cuso 4 ( data not shown ). d - leucine and ian reduce canker symptom production and bacterial populations in planta plant inoculation by spray showed that both d - leucine ( 10 mm ) and ian ( 100 μg / ml ) were able to reduce canker symptom development on grapefruit leaves when applied along with or prior to the pathogen inoculation , as evidenced by decreased lesion numbers compared to the positive control ( pathogen inoculation alone ) ( fig5 a ). differences in lesion numbers were notable by 14 dpi and more distinctive over the remainder of the experiment . when applied after the pathogen inoculation , either d - leucine or ian did not affect the development of canker symptoms ( fig5 a ). both d - leucine and ian could reduce the level of canker lesions to that of copper spray ( cuso 4 , 100 μg / ml ). the leaves sprayed with d - leucine ( 10 mm ), ian ( 100 μg / ml ) or cuso 4 ( 100 μg / ml ) displayed a similar level of canker lesions , less than that sprayed with the pathogen alone ( fig5 a ). the combination of d - leucine ( 10 mm ), ian ( 100 μg / ml ) and cuso 4 ( 100 μg / ml ) were more effective in suppressing the development of canker symptoms than cuso 4 alone , with less lesions produced on the leaves ( fig5 a ). these visual observations correlated with bacterial populations recovered from the inoculated leaves after inoculation that revealed an approximately 10 to 100 - fold decrease in cfu / mm 2 leaf tissue for treatment with d - leucine , ian , or cuso 4 , alone or in combination , compared to the untreated control ( fig5 b ). treatment of the grapefruit leaves with d - leucine , ian or cuso 4 alone resulted in an approximate 1 . 0 log reduction in the number of cfu compared to the untreated control , while treatment with a combination of d - leucine or / and ian and cuso 4 resulted in an approximate 2 . 0 log reduction in the number of cfu compared to the untreated control during 7 to 28 days after inoculation ( fig5 b ). preliminary study showed that both d - leucine and ian could reduce canker symptom production and x . citri subsp , citri populations on citrus fruit at a concentration lower than the mic . detached immature citrus fruit ( grapefruit , 20 - 40 mm in diameter ) inoculation by spraying x . citri subsp . citri ( 10 8 cfu / ml ) showed that both d - leucine ( 10 mm ) and ian ( 100 μg / ml ) were able to reduce canker symptom development on fruit surface when applied along with the pathogen inoculation , as evidenced by decreased lesion numbers compared with the positive control ( pathogen inoculation alone ) ( fig6 a ). the visual observations correlated with bacterial populations recovered from the surface of inoculated fruit that revealed an approximately 50 - fold decrease in cfu per square centimeter of fruit tissue for treatment with d - leucine or ian , compared with the untreated control ( fig6 b ). treatment of the fruit with d - leucine and ian resulted in an approximate 1 . 6 and 1 . 8 log reduction in the number of cfu per square centimeter respectively , compared with the untreated control at 11 days after inoculation ( fig6 b ). in the above examples , it was demonstrated that d - leucine and ian reduced biofilm formation by xac and increased the susceptibility to copper at a concentration without affecting its growth . canker control activity was evaluated as the ability to reduce the number of lesions and populations of xac on citrus leaves after applications . the results of this greenhouse assays support the use of foliar - applied biofilm inhibitors alone or combined with copper - based bactericides for the control of canker disease on citrus trees . d - leucine and ian appear to have distinct mechanisms of action in reducing biofilm formation by xac . in this study , it was found that both d - leucine and ian inhibit the biofilm formation of xac on different abiotic surfaces and host leaves at sub - mic concentrations ( fig2 ), while only ian repressed expression of several biofilm formation related genes ( chemotaxis / motility - related genes ) in xac ( fig3 ). flagellar - mediated chemotaxis / motility and type iv pili protein are necessary for xac biofilm formation ( li and wang , 2011 ). the observation of ian repressed expression of genes related to bacterial motility was consistent with previous reports for ian effect on biofilm formation in the human bacterial pathogen pseudomonas aeruginosa ( lee et al ., 2011 ). hence , ian probably reduces xac biofilm formation by repressing these chemotaxis / motility - related genes ( chey , motb and pilb ) ( fig3 ) and thus reducing its chemotaxis and motility . interestingly , it has been found that several d - amino acids including d - leucine inhibit biofilm formation in bacillus subtilis and staphylococcus aureus by preventing protein localization at the cell surface ( hochbaum et al ., 2011 ). whether d - leucine prevents protein localization at the cell surface of xac remains unknown . reduced sensitivity to copper in bacteria may result from genetic mutations and from the change of environmental factors influencing the ionic concentration or production of bacterial extracellular polysaccharides ( eps ) that can bind the biologically active ions of copper ( hasman et al ., 2009 ; hsiao et al ., 2011 ). it has also been shown that decreased sensitivity of bacteria to copper can be mediated by biofilms ( rodrigues et al ., 2008 ). in the experimental conditions described herein , the results showed that both d - leucine and ian increased the susceptibility of xac planktonic and biofilm cells to copper ( fig4 ), but did not affect expression of genes responsible for gum eps biosynthesis or related to copper resistance ( data not shown ). it is possible that d - 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microbe interact . 25 : 69 - 84 . it should be borne in mind that all patents , patent applications , patent publications , technical publications , scientific publications , and other references referenced herein and in the accompanying appendices are hereby incorporated by reference in this application to the extent not inconsistent with the teachings herein . it is important to an understanding to note that all technical and scientific terms used herein , unless defined herein , are intended to have the same meaning as commonly understood by one of ordinary skill in the art . the techniques employed herein are also those that are known to one of ordinary skill in the art , unless stated otherwise . for purposes of more clearly facilitating an understanding the invention as disclosed and claimed herein , the following definitions are provided . while a number of embodiments have been shown and described herein in the present context , such embodiments are provided by way of example only , and not of limitation . numerous variations , changes and substitutions will occur to those of skilled in the art without materially departing from the invention herein . for example , the present invention need not be limited to best mode disclosed herein , since other applications can equally benefit from the teachings . also , in the claims , means - plus - function and step - plus - function clauses are intended to cover the structures and acts , respectively , described herein as performing the recited function and not only structural equivalents or act equivalents , but also equivalent structures or equivalent acts , respectively . accordingly , all such modifications are intended to be included within the scope of this invention as defined in the following claims , in accordance with relevant law as to their interpretation . * the mics were examined using a 96 - well plate assay at 28 ° c . under stationary conditions as described in the materials and methods . the mic was defined as the lowest concentration resulting in no bacterial growth measured at od 600 after a 48 h - incubation , compared to the control samples . the determinations were repeated three times with eight replicate wells each time . l - serine from non - animal source , meets ep , usp testing specifications , suitable for cell culture , 98 . 5 - 101 . 0 % c 10 h 13 no 3 • c 6 h 8 o 3 s c 10 h 13 no 3 • c 8 h 6 o 3 s