I. A.

Initial Identification of Common Genera N. W. Schaad INTRODUCTION Relatively few differential characters are required to identify the predominant genera of plant pathogenic bacteria. They can be conveniently divided into 1) those that are isolated easily on standard bacteriological media (Table 1) and those that are not (Table 2).

+ !"# or more strains positive after five days$ % between 21&'(# of strains positive$ & !"# or more strains negative$ )* not determined.
a b c

& +olonies ofPantoea citrea and some strains off. agglomerans are generally white. & +olonies of X. campestris pathovars manihotis and mangiferaeindicae are white. -Xylophilus grows very slowly on these media but somewhat better on *ifco nutrient agar.

d& +olonies of Clavibactermichiganensis subsp. sepedonicus are generally white e& Enrinia nigrifluens is positive.
f

& Burkholderia andropogodis is o,idase negative. -&Burkholderia andropogodis and B. glumae pv. agricola are negative.

.enera within the first group are relatively easy to differentiate from one another on the basis of the characters presented in Table 1 and the simplified flow chart presented in /ig. 1 (see p. 0). The .ram reaction (see 1 p. ') is related to structural and chemical properties of the cell wall and this characteristic serves as a rapid and very basic step in the initial identification of un1nown plant pathogenic bacteria. 2t is important when doing the .ram reaction to use rapidly growing freshly prepared cells (see la p. '). The anaerobic test is a 1ey test for identification of the genera Erwinia. Pantoea. and Clostridium. 3lso the presence of spores is a 1ey test for differentiation of .ram&positive bacteria. +olony morphology and pigmentation growth at 4" 5+ and the o,idase reaction are 1ey tests for differentiation of Acidovora ! Burkholderia! "alstonia! and Pseudomonas. 3lways be sure to include 1nown cultures as positive and negative controls for each of the tests. 6hen identifying an un1nown bacterium it is absolutely essential that you use a pure culture of the organism being identified. 2t should be cloned at least twice by strea1ing onto a nonselective agar medium. 7e sure that the selected colony is well separated and is allowed to grow for 4&8 days to be sure other colonies do not appear. 9ore rapid commercial automated techniques (see 3ppendi, +) such as fatty acid analysis 7iolog and :;2<3 1its are useful for presumptive identification of a large number of bacteria isolated from soil or plant leaves however they should not be relied upon for final identification. *etails for molecular and serological&based techniques are presented in 3ppendi, 3 and 7 respectively. 9embers of the second group (Table 2) are relatively new to plant pathologists and are initially identified on the basis of 1) growth on <&medium (see h p. 8) 2) growth on =6 (see g p. 8) and other comple, media (see a) p. 2"4&2"8) >) presence or absence of a cell wall (see p. 12 p. 1>) and 4) helical morphology (see 1 p. 2(>). 2f no growth occurs on any of the three agar media it is advisable to prepare samples for the electron microscope and use serological (see p. >1") and?or =+R&based techniques (see p. >">). Table 2. +haracters used to differentiate fastidious and non&culturable plant pathogenic bacteria. Xyella +haracter .rowth on <&medium .rowth on =6 agar =ossess cell wall .rowth on serum agar @elical morphology
a

#piroplasma & & & + + &

Phytoplasma-like & & & & & & &

Phloemlimited%

"hi$omonas + & + &

fastidiosa & + + -

2ncludes proposed &iberobacter spp. and ric1ettsia&li1e (papaya bunchy top) bacterium (see p. 2'8).

ISOLATION TECHNIQUES USING DI !EDIA #. $lant !aterial

ERENTIAL AND SE!ISELECTI"E

6hen isolating an un1nown plant pathogenic bacterium it is a good practice to use several different common agar media. 9ost Erwinia! Xanthomonas! Acidovora ! Burkholdera! "alstonia! and Agrobacterium spp. grow well and produce characteristic colonies on nutrient broth&yeast e,tract agar ()7A) (18) or yeast e,tract&de,trose& calcium carbonate (A*+) (10) agar. 6ithout e,perience it is advisable to strea1 cultures of 1nown strains for comparing the colony characteristics. 3 few species or pathovars do not grow well on A*+. /or e,ample P. stewartii! X. ory$ae! X. albilineans! and X. fragariae grow poorly on A*+ but quite well on )7A. Xylophilus ampelimis grows poorly on A*+ or )7A but much better on nutrient agar. 9ost pseudomonads and Clavibacter spp. grow well and are very characteristic on Bing et al-s medium 7 (B7) (0) and )7A agar respectively. 2f Bacillus or Clostridium is suspected casein agar plus glucose or =2 medium (see a p. 20!) should be included. Cne plate should be incubated in an anaerobic chamber (see 2 p. 202). 2f #treptomyces is suspected include water agar (1"). /or isolation remove a small amount of tissue from the advanced portion of the lesion using a sterile scalpel. The tissue should be washed with sterile water or surface steriliDed for > min. in a 1E1" dilution of a household bleach (8.28# active sodium hypochlorite). 3fter rinsing in sterile water the tissue should be chopped up with a sterile scalpel in a droplet of water in a plastic =etri dish. 2f the tissue is too difficult to cut a sterile mortar and pestle can be used. 3fter sitting for 2 or > minutes the macerate is strea1ed onto agar media with a platinum wire loop. 2f a particular nomenspecies is suspected to be the causal agent follow the isolation techniques suggested for that particular organism. <trea1s should be made in four right angle directions flaming the loop after each directional strea1. 3 single colony (well spaced from other colonies) should be restrea1ed on one of the media to be sure the bacterium is pure. 2f no colonies result from strea1ing onto a common medium determine if the causal organism is anaerobic 'Clostridium spp.) a fastidious organism (such as Xylella fastidiosa! or "hi$omonas(! or a non&culturable phloem&limited bacterium such as @uanglongbin (citrus greening) bacterium )&iberobacter(. /or the latter organisms =+R techniques should be used (see / p. 2'!). %. Reci&e' for common and 'emi'electi(e media N)trient a*ar +NA, and n)trient -roth +N.,/ per; 7eef e,tract (*ifco) >." g =eptone 8." g 3gar 18." g
3

a.

)utrient agar ()3) or nutrient broth may be purchased in dehydrated form from *ifco. F*o not add agar if nutrient broth is desired. -. 0ea't e1tract2de1tro'e2CaCO3 l0DC4 5#64 per; Aeast e,tract 1"." g *e,trose (glucose) 2"." g +alcium carbonate G<= light powder 2"." g 3gar 18." g To obtain an even mil1y white medium the +a+"> must be of the finely ground form otherwise it will precipitate to the bottom. 3utoclave at 1" =<2 for 1 h or autoclave de,trose in 1"" ml of water separately. The autoclaved medium should be cooled to 8"5 + in a waterbath and +a+"> suspended by swirling before pouring the plates. /or tubes a vorte, mi,er should be used to mi, thoroughly the +a+"> after removing tubes from the waterbath. N)trient2-roth 7ea't e1tract a*ar +N.0, +#8, per; )utrient broth (*ifco) !." g Aeast e,tract 2." g B2@="4 2." g B@2="4 ".8 g .lucose 2.8 g 3gar 18." g 3fter autoclaving add 1." ml of a sterile solution of 19 9g<"4&'@2". d. 9in* et al.:' medi)m . a*ar 5+9. +6,# per; =roteose peptone H> (*ifco) 2"." g B2@="4 1.8g 9g<"4.'@2" 1.8g .lycerol 18." ml 3gar 18."g c.

e.

Ca'ein a*ar &l)' *l)co'e per; 1"." g 8." g 8." g 4." g 1'." g per; 2" g per; 4." g 1." g 1"." mgF 1"." ml ".4 g 1.2 g 1." g 4." g 18." g >"." mlFF

+asein acid hydrolysate Aeast e,tract .lucose B2@="4 3gar f. 3gar *. $Wa*ar+3, Water a*ar

<oytone Tryptone @emin chloride =henol red (".2# aqueous) 9g<+y'@2" B2@="4 B@2@="4 ;&glutamine 3gar 7ovine serum albumin %

F Cr add 1" ml of a ".1# solution in "."8 n )aC@ FF3dd 2"# filter steriliDed (".2 um filter) stoc1 solution after autoclaving h. S2medi)m +#;,

"hi$omonas is isolated from soil and plant tissue using <&medium. per; +asein acid hydrolysate 8." g .lucose 2.8 g B2 @="4 .>@2" 1.> g B)C> ".8 g 9g<"4 .'@2" ".8 g +a()">)2 .4@2" 0"." mg <treptomycin sulfate 1." mlF 3gar )oble 11." g I<treptomycin sulfate (aqueous filter&steriliDed 18 mg?ml stoc1 solution) is added to the medium after autoclaving and has cooled to about 8"5+.

+.

DI ERENTIATION O CO!!ONL0 ISOLATED GENERA The following flow chart (/ig. 1) should be used as a quic1 guide for identifying a particular species or pathovarE

a +olonies ofPantoea citrea and some strains of =. agglomerans are generally white.
b c

Clostridia cells with spores are swollen whereas Bacilli are not. Burkholderia andropogonis is negative for arginine and betaine. @owever it is o,idase

+olonies of Xanthomonas cassavae and two pathovars ofJ campestris are white.

negative whereas "alstonia and Acidovora are positive.

DIAGNOSTIC !EDIA AND TESTS #. Gram reaction This test is essential for differentiating plant pathogenic bacteria into two broad groupsE .ram&positive and .ram&negative. The simple BC@ technique can be used as a rapid test for presumptive identification (1>). @owever the .ram&stain is very important when identifying a newly described bacterium and should not be omitted. a. BC@ test (1>)E 9i, loopful of bacterial with 2 drops of ># BC@. .ram&negative bacteria will become gummy upon mi,ing with a loop while .ram&positive bacteria will not. 2f questionable results are obtained the .ram stain should be used. b. .ram stain (1 4 11 3. %idaver =ersonal communication) .ram&positive bacteria retain the primary dye giving a purple to blue&blac1 appearance. .ram&negative bacteria ta1e up the color of the counter stain. Cnly a few bacteria cannot be readily grouped into either category. The maKor points in successful staining are as followsE a. b. c. d. Gse reagents less than one year old. =articular attention is needed for the iodine solution. 2f the solution is not put in a brown bottle or 1ept in the dar1 it will decoloriDe and become ineffective. The reagents may also brea1 down. Gse freshly grown bacteria ideally a liquid overnight culture in e,ponential phase. Older 'tationar7 &ha'e cell' ma7 *i(e a Gram2(aria-le reaction. Gse a 1nown positive and 1nown negative for controls. The bacterial smears must show some well&separated groups of bacteria. +lumps are to be avoided.

<taining procedureE a. b. c. d. e. f g. Cn a clean slide dry a thinly spread bacterial film in air without heat. Then lightly flame the underside of the slide twice to fi, the bacteria to the slide. /lood the smear with crystal violet solution for 1 minute. 6ash in tap water a few second. *rain off e,cess water and lightly blot dry on a paper towel. /lood the smear with iodine solution for 1 minute 6ash in tap water a few seconds$ blot dry. *ecoloriDe with solvent e.g. ethyl alcohol until the solvent flows colorlessly from the slide (about >" seconds). 7lot dry. (2f decoloriDer is used longer the .ram& positive bacteria may lose color.) Rinse in tap water for about 2 seconds.
7

h. i.

+ounterstain for about 1" seconds with safranin solution. 6ash briefly in tap water. 7lot dry and e,amine.

ResultsE .ram&positive bacteria stain purple to blue&blac1$ .ram&negative bacteria stain red (=late l /igl). <olutionsE a. @uc1er-s ammonium o,alate crystal violet per; <olution 3 +rystal violet :thyl alcohol ((8#) <olution 7 3mmonium o,alate *istilled water 2." g 2"." ml ".! g !"." ml

9i, solutions 3 and 7. <tore 24 hours before use. /ilter through paper into storage bottle. b. .ram-s modification of ;ugol-s solution 2odine =otassium iodide *istilled water per; 1." g 2." g >""." ml

3llow iodine solution to dissolve several hours or overnight in a dar1 place. 3lternately grind the dry iodine and B2 in a mortar adding water slowly. +ontinue grinding until 2 and B2 are in a solution. Rinse the solutions with remaining water into a dar1 bottle. c. *ecoloriDers 1. 2. 1. :thyl alcohol (8# slowest agent 3cetoneE fastest agent 3cetone&alcoholE intermediate ((8# ethyl alcohol 1"" ml$ acetone 1"" ml.).

6ith practice any of the three decoloriDing agents will yield good results. d +ounterstain

per; <toc1 solution <afranin " :thyl alcohol (8# 6or1ing solution <toc1 solution *istilled water %. S&ore determination +;, (also see c p. 11) <uspend colony of bacteria growing on agar medium in a drop of water on a slide and air dry. /lood slide with 8."# (w?v) aqueous malachite green and stain for 1" minutes. 6ash thoroughly under running water and dry briefly. +ounter stain by flooding slide with ".8# (w?v) aqueous safranin for 18 seconds. Rinse thoroughly with water and blot dry. Cbserve cells at 4",. 7acterial cells stain red and spores green. per; 2." g 8." g ".> g >" g >." 2.8 g 1""." ml 1"." ml ("." ml

3.

Anaero-ic *ro<th +H)*h and Leif'on 584, =eptone )a+l B@2="4 3gar 7romthymol blue (1# aqueous solution)

*issolve ingredients and adKust the p@ to '.1. 3dd 8 ml of basal medium to 1> cm diameter test tubes and steriliDe at 121 5+ for 2" min. =repare a 1"# aqueous solution of glucose and steriliDe by filtration. 3dd ".8 ml of the sterile glucose aseptically to each tube of basal medium. 2noculate two tubes with each strain of the organism to be tested. +over one tube with a layer of sterile melted %aseline or paraffin to a depth of about 8mm. 3 color change from blue to yellow in both tubes is recorded as positive for anaerobic growth (fermentation). ;. Gro<th on DI! a*ar +#%,. +ellobiose )@4+1 )a@2="4 B2@="4 9g<"4L'@2" 9alachite green 3gar per; 8." g 1." g 1." g 1." g >." g 1"." mg 18." g

The medium is adKusted to p@ '." before autoclaving.

Gseful for differentiating Agrobacterium from Acidovora ! Burkholderia! and "alstonia. 8 .Gro<th at 33 or ;=>C .row culture overnight in 8 to 1" ml of liquid )7A in a 28 cm diameter test tube on a slanted rac1 on a rotary sha1er at 28 & 2' 5+ . Remove 8" ??l and add to a new tube of )7A and incubate at 4"5 + on a rotary sha1er. Record growth after 18 and 24 h 6.Urea'e +'ee e? &. ;@, @.O1ida'e te't (') /or inoculum use 24&hour&old cultures grown on ).3 (false negatives may result if greater than ".28# glucose is used). Rub a small loopful of the inoculum on a filter paper impregnated with 1# (w?v) aqueous tetramethyl&p&phenylenediamine dihydrochloride solutionF (freshly made). The strain is rated o,idase&positive if a purple color develops within 1" seconds (=late 1 /ig. 2) delayed positive if coloration develops within 1"&0" seconds$ and negative if no color develops after 0" seconds. 3 platinum or plastic loop or a toothpic1 is recommended since traces of iron can catalyDe the o,idation of the phenylenediamine compound. F +ommercial o,idase strips are available from *ifco$ some investigators prefer dimethyl to tetramethyl. A.Colon7 coloration and con'i'tenc7 on 0DC a*ar. <trea1 onto agar plate and incubate at >"5+. B. l)ore'cent and diff)'i-le non2fl)ore'cent &i*ment' on 9.. <trea1 onto agar plate and incubate at 285+. 3fter 4! h observe for non&fluorescent pigment under normal light and in dar1 with long wave length uv light (>00 nm) for fluorescent pigment. #=. la*ella a.. <ilver impregnation (2) =reparation of bacteria and precautionsE 1) .row bacteria on agar plates of a standard medium such as A*+ at 2' 5+ for 24&4! h. 2) Gse a 1nown peritrichous species such as E. carotovora or a polar species such as P. marginalis as a control. >) =repare a faintly cloudy suspension by carefully adding 1." ml of distilled water to butt of slant. 4) *o not mechanically agitate slant. 8) =lace a loopful of distilled water on an alcohol&cleaned slide. 0) 3 loopful of bacterial suspension is placed Kust touching the distilled water. ') 3llow slides to air dry. 1"

<taining procedureE 1) 2) >) +over the smear with reagent 3 for 2 to 4 mins then rinse in distilled water. 3dd reagent 7 (p@ 1".") for about >" sees and immediately wash in distilled water. 3ir dry and e,amine under oil immersion.

ResultsE 7acteria and flagella are stained dar1&brown to blac1 on a light to golden bac1ground. per; ReagentsE 3. Tannic acid solution Tannic acid 8." g /erric chloride 1&8 g /ormalin (18#) 2." ml <odium hydro,ide (1#) 1." ml 7ring to 1"" ml with distilled water. 7. 3mmoniated silver nitrate solution <ilver nitrate (2#) 1""." ml

Remove and save 1" ml of silver nitrate. Then add ammonium hydro,ide to remaining (" ml until a heavy precipitate is formed. +ontinue adding ammonium hydro,ide until precipitate is dissolved. /rom the 1" ml of silver nitrate saved bac1 titrate until a slight clouding appears and persists. 3dKust p@ to 1"." with ammonium hydro,ide and silver nitrate. Gse 6ithin 4 h of =reparation. b. :lectron microscopy (.. .aard Gniversity of 6isconsin 9adison 6isconsin personal communication.) =repare a test tube agar slant culture of the test organism on an optimal growth medium. +ulture should be sampled when it is in the logarithmic growth phase (18&'2 h). @owever it is also helpful to incubate the culture at a temperature 2 &>5+ below the optimum for growth. =repare the culture for viewing by the following procedureE 1) Rinse the base of the agar slant about 8 times with double distilled water and discard water. This is to remove the cells that accumulate at the base of the slant. 2) .ently rinse bacteria from surface growth of the agar slant with about 1 ml of double distilled water. >) =repare a dilution series using a suspension of about 1"( cells as the initial concentration. 9i, dilutions very gently to avoid loss of flagella. *ilute 11

4)

8)

this suspension by 1?2 1?4 1?10 and 1?>2 then put a small droplet of each on different grids. /or optimal viewing the final concentration on the grid should be 8 cells?grid hole on 2"" mesh grids. Gse of a spreading agent (detergent) is normally not necessary. <hadow or negative stain. a) To shadow freeDe dry grids at&1"5+ina desiccator overnight and shadow with carbon&platinum at an angle of !5. b) To stain place grid on a drop of 1# aqueous uranyl acetate for 1&2 min or 2# potassium phosphotungstate p@ 0.8 for >" sees. Remove stain by touching the edge of the grid to filter paper. Cbserve under an electron microscope (/ig 2).

/ig. 2. =olar flagella of Pseudomonas marginalis shadowed with carbon&platinum and observed by an electron microscope. (=hotograph courtesy of .. .aard Gniv. 6is.) ##. Ser)m a*ar +5Liao and Chen medi)m4 5A4, per; ==;C broth base <ucrose @orse serum =henol red 3gar 1.8 g 12." g 2".2 g 2.8 mg 18 g

12

#%.

Cell <all determination This test is needed to identify a microorganism such as a mycoplasma or spiroplasma and should be performed by an e,perienced electron microscopist. The following method is that of M. 6orley G<*3?3R< 7eltsville 3gricultural Research +enter 7eltsville 9* 2"'"8 (personal communication)E <election of material is critical since some organisms are at low concentrations or limited to certain tissues. a. b. c. d. e. f. g. Remove pieces of tissue 1 to 2 mm2 using a raDor blade and place immediately into freshly prepared ># glutaraldehyde in ".19 phosphate buffer (p@ '."). 3fter infiltration in a vacuum desiccator for several hours (depending upon tissue) replace glutaraldehyde with fresh solution and continue fi,ation under vacuum for about 4 hr at room temperature. Remove glutaraldehyde and then wash tissue briefly three times in fresh phosphate buffer. =lace tissue pieces in 2# osmium tetrao,ide in the same buffer for 2&4 hr at room temperature or overnight a 45+. Rinse three times in fresh buffer. *ehydrate tissue in ethanol&propylene o,ide using single steps (18&>" min) of 2"# 8"# '"# and (8# ethanol two steps of absolute alcohol one step in absolute alcohol&propylene o,ide (1E1) and finally twice in propylene o,ide. :mbed in 3raldite resin ((). 3raldite8"2 1>." ml *odecanylsuccinic anhydride 1"." ml *9=&>" ".4 ml <tir mi,ture well before use. :mbed in propylene o,ide&resin mi,ture (2E1) for 1 hr then in propylene o,ide& resin mi,ture (1E2) for 4 hr covered and overnight uncovered. /inally embed in resin mi,ture for 4 hr. Remove tissue from resin blot dry and transfer to aluminum weighing dish containing fresh resin. 3rrange tissue in resin and cure at >85+ for !&12 hr 485+ for !&12 hr and 0"5+ for 4! hr. <aw tissue from bloc1 and mount on plastic cylinders. Trim and section transversely at a setting to obtain silver&to&gold (about 1"" nm) and purple&to& green (about >"" nm) sections. <pread the compressed ribbons by passing a cotton swab saturated with chloroform over the ribbons. 9ount on /ormvar & coated copper grids. <tain for 1" mins in saturated aqueous uranyl acetate wash thoroughly stain for 4& 8 mins in lead citrate wash thoroughly then dry and view in electron microscope (/ig.>). 1>

h. i. K. 1. 1. m.

/ig. >. <tereo pair of electron micrographs of a thic1 ultramicrotome section (cut at about ".> ??) of phloem tissue from a com stunt&infected com plant. < N helically coiled cell of com stunt spiroplasma. +6& plant cell wall. (/rom *avis and 6orley O1('>P Phytopathology 0>E4">&4"!). :. ;2T:R3TGR: +2T:* 1. 3nnette :. @. 3. 7aDows 6. M. @ausGer and @. M. <hadomy. 1(!8. 9anual of +linical 9icrobiology. 3merican <ociety for 9icrobiology 6ashington *.+. /ourth :dition. 2. 7lenden *. +. and @. <. .oldberg. 1(08. <ilver impregnation stain for leptospira and flagella. M. 7acterid. !(E!((&("". >. *avis 9. M. 6. M. /rench and ). 6. <chaad. 1(!1. 3,enic culture of the bacteria associated with phony disease of peach and plum leaf scald. +urr. 9icrobiol. 0E>"(&>14. 4. .erhardt =. (:d.). 1(!1. 9anual of 9ethods of .eneral 7acteriology. 3m. <oc. 9icrobiol. 6ashington *.+. 8. @ugh R. and :. ;eifson. 1(8>. The ta,onomic significance of fermentative versus o,idative metabolism of carbohydrates by various .ram negative bacteria. M. 7acteriol. 00E24&20. 0. Bing :. C. 9. B. 6ard and *. :. Raney. 1(84. Two simple media for the demonstration of pyocyanin and fluorescein. M. ;ab. +lin. 9ed. 44E>"1&>"'.

14

'. Bovacs ). 1(80. 2dentification of Pseudomonaspyocyanea by the o,idase reaction. )ature. 1'!E'">. !. ;iao +. @. and T. 3. +hen. 1(''. +ulture of corn stunt spiroplasma in a simple medium. =hytopathology 0'E!"2&!"'. (. ;uft M. @. 1(01. 2mprovements in epo,y resin embedding methods. M. 7iophys. 7iochem. +ytol. (E4"(&414. 1". 11. 12. 9enDies M. *. and +. :. *ade. 1(8(. 3 selective indicator medium for isolating #treptomyces scabies from potato tubers or soil. =hytopathology 4(E48'&48!. 9eynell .. .. and :. 9eynell. 1('". Theory and practice in e,perimental bacteriology. 2nd ed. +ambridge Gniversity =ress ;ondon. =erry B. ;. and +. 2. Bado. 1(!2. +haracteristics of Tiplasmids from broad&host&range and ecologically specific biotype 2 and > strains for Agrobacterium tumefaciens. M. 7acteriol. 181E>4>&>8". <uslow T. %. 9. ). <chroth and 9. 2sa1a. 1(!2. 3pplication of a rapid method for .ram differentiation of plant pathogenic and saprophytic bacteria without staining. =hytopathology '2E(1'&(1!. van 7ruggen 3. @. + R .. .rogan +. =. 7ogdanoff and +. 9. 6aters. 1(!!. +or1y root of lettuce in +alifornia caused by a .ram&negative bacterium. =hytopathology '!E11>(& 1148. %idaver 3 B. 1(0'. <ynthetic and comple, media for rapid detection of fluorescence of phytopathogenic pseudomonadsE :ffect of the carbon source. 3ppl. 9icrobiol. 18E182>& 1824. 6ilson :. :. /. 9. Qeitoun and *. ;. /redric1son. 1(0'. 7acterial phloem can1er a new disease of =ersian walnut trees. =hytopathology 8'E01!&021.

1>.

14.

18.

10. .

Chemical' Li't So)rce

Chemical'

Gnless stated otherwise all chemicals in this list were obtained from <igma +hemical +o.. =.C. 7o, 148"!. <t. ;ouis. 9C 0>1'!. 3gar 3mmonium o,alate 3rginine 3raldite 8"2 <igma *ifco 77; /isher ;add Research 2nc. 7urlington %T

18

7eef e,tract 7etaine 7ovine serum albumin fraction % 7romthymol blue & sodium salt +alcium carbonate precipitated light powder +a()">)2&4@2" +asein acid hydrolysate +ellobiose +rystal violet certified *9=&>" *odecanylsuccinic anhydride /erric chloride /ormalin (18#) .lucose .lutaraldehyde .lycerol @emin chloride & bovine @orse serum 2odine B2@="4 B2@=+y>@2" B@2="4 B@2="4 & anhydrous BC@ B)"> 9alachite green 9g<"4&'@2" )utrient agar or broth Csmium tetrao,ide =eptone =henol red =otassium iodide =otassium phosphotungstate ==;C broth base =roteose peptone H> =ropylene o,ide <afranin " certified <ilver nitrate (2#) <oytone <treptomycin sulfate Tannic acid Tetramethyl&p&phenylenediaminedihydrochloride Tryptone Granyl acetate Aeast e,tract

*ifco 9atheson +oleman 7ell 9allinc1rodt or M. T. 7a1er *ifco *ifco ;add Research 2nc. ;add Research 2nc.

9allinc1rodt or /isher

.rand 2sland 7iological 9allinc1rodt or /isher 9allinc1rodt or /isher

/isher M. T. 7a1er *ifco *ifco M. T. 7a1er *ifco *ifco ;add Research /isher *ifco

*ifco *ifco 77; 10

. 3. #.

GRA!2NEGATI"E .ACTERIA Agrobacterium L. W. !oore? H. .o)Car? and T. .)rr INTRODUCTION

Agrobacterium spp. are commonly 1nown as soil&borne bacteria that infect dicotylendonous plants from over (" different plant families (' 1>) including economically important fruit and nut crops grapes ornamental and landscape plants (e.g. rose euonymous dahlia chrysanthemum). Gpon infection of plants some strains ofAgrobacterium incite abnormal cell proliferation (hyperplasia) which result in tumor formation in the case of the crown gall disease and e,cessive adventitious roots in the hairy root disease. /inancial losses from the disease have been estimated in the millions of dollars per year (24) occurring primarily at nurseries where diseased plants are discarded (>"). +rown gall can also stunt mature plants by causing inferior development of the root system and?or disruption of vascular flow in the stem. 3 new cultivar of walnut has been especially susceptible in +alifornia where crown gall stunts and sometimes 1ills the tree and production is almost always affected. 3lmond trees in +alifornia with crown gall are more susceptible to blow&over during wind storms and the wood rotting fungi. =athogenic strains of Agrobacterium share a common feature$ they contain at least one large plasmid the tumor& or root&inducing (Ti and Ri respectively) plasmid (44). %irulence is determined by different regions of the plasmid including the transferred *)3 (T&*)3) and the virulence *yir( genes. The virulence genes mediate transfer of T&*)3 into infected plant cells where it integrates into the plant *)3 (11). RhiDogenic (root&inducing) agrobacteria T&*)3 contains rol (root locus) genes that render plant cells more sensitive to endogenous au,in (4"). 3lthough Ti and Ri plasmids vary considerably between strains they all carry similar vir genes (2"). The genus Agrobacterium belongs to the family "hi$obiaceae (2') which has been included in the alpha&2 subclass of Proteobacteria on the basis of ribosomal characteristics (48). 9embers of this family are aerobic and .ram&negative. The cells are normally rod&shaped (".0& 1." urn by 1.8&>." um) occur singly or in pairs without endospores and are motile by one to si, peritrichous flagella. +onsiderable e,tracellular polysaccharide slime is usually produced during growth on carbohydrate&containing media. The classification of gall& and root&inciting agrobacteria is in constant evolution. Gntil recently the species nomenclature was based primarily on plasmid&borne phytopathogenic characters and included Agrobacterium tumefaciens! causal agent of crown gall$ 3 rhi$ogenes! causal agent of hairy root disease$ and A. rubi! causal agent of cane gall on "ubus (2'). 3ll nonpathogens were lumped into A. radiobacter. @owever there was no correlation between this nomenclature based on pathogenicity characters and the ta,onomic structure based on 1'

phenotypical and genotypical traits of the genus (2'). 3nalysis of chromosome&encoded characters (>') chromosomal *)3 (11) and comparison of electrophoretic protein patterns (20) showed that most members of the genus Agrobacterium grouped into at least three ta,onomic groups not corresponding to pathogenicity. )ot including the five marine species recently described as a distinct subdivision within the genus Agrobacterium (>') the genus is composed of at least four distinct and separate ta,a (4 1! >!). *)3&*)3 homology studies showed that these ta,a corresponded to the genospeciesE Agrobacterium tumefaciens (formerly biovar 1) (8) A. rhi$ogenes (formerly biovar 2) (>!) A. vitis (formerly biovar >) (>2) and A. rubi. 3lthough this proposed speciation scheme appears to be a good solution to obKectively differentiate the agrobacteria it has been difficult for scientists to accept because of long historical usage of the previous names (Table 1). 6e recommend that the new speciation scheme be accepted. The phylogenetic relationships of members of the genus Agrobacterium have been determined by ribosomal R)3 sequencing (rrs) (>! 48). Two maKor lineages were distinguishableE one includes A. rhi$ogenes along with most "hi$obium species$ the second includes A. tumefaciens! A. vitis! A. rubi and a distinct strain )+==7 108" along with "hi$obium galegae. Thus biochemical traits permit the characteriDation of species in the genus Agrobacterium which in turn can be identified by rrs sequencing. 7ecause of the molecular tools now available for classification and phylogenetic studies it is predictable that in the future new species will be identified Oe.g. distinct strains have been isolated from +icus (0) and wild blac1berry (1")P. The genetic diversity within A. vitis has been recently studied. 2n addition to Ti plasmid groups chromosomal characteriDations of A. vitis strains were done by fingerprinting the region between the 10< and 2> < rR)3 genes. 3 high correlation was found between chromosomal type and the type of Ti plasmid carried by strains (>>).
Ta-le #. +omparison of old and new nomenclature for species of Agrobacterium )ew ta,onomyFRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR Old ta1onom7RRRRRRRRRRRRRR A. tumefaciens A! tumefaciens biovar 1 A. radiobacter biovar 1 A. rhi$ogenes biovar 1 A. rhi$ogenes A. tumefaciens biovar 2 A. radiobacter biovar2 A. rhi$ogenes biovar 2 A. vitis A. tumefaciens biovar > A. radiobacter biovar> RRRRRA. rubiRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRRR A. rubi I2ndividual strains in the species may be tumongenic rhiDogenic or nonpathogenic

% ISOLATION TECHNIQUES USING SE!ISELECTI"E !EDIA a. $lant !aterial Agrobacterium strains can be isolated from aerial and subterranean galls vascular fluids and from symptomless plants. 2solation of the pathogen is made most easily from white to cream&colored gall tissue of young actively growing galls. 6ash the gall surface

1!

phenotypical and genotypical traits of the genus (2'). 3nalysis of chromosome&encoded characters (>') chromosomal *)3 (11) and comparison of electrophoretic protein patterns (20) showed that most members of the genus Agrobacterium grouped into at least three ta,onomic groups not corresponding to pathogenicity. )ot including the five marine species recently described as a distinct subdivision within the genus Agrobacterium (>') the genus is composed of at least four distinct and separate ta,a (4 1! >!). *)3&*)3 homology studies showed that these ta,a corresponded to the genospeciesE Agrobacterium tumefaciens (formerly biovar 1) (8) A. rhi$ogenes (formerly biovar 2) (>!) A. vitis (formerly biovar >) (>2) and A. rubi. 3lthough this proposed speciation scheme appears to be a good solution to obKectively differentiate the agrobacteria it has been difficult for scientists to accept because of long historical usage of the previous names (Table 1). 6e recommend that the new speciation scheme be accepted. The phylogenetic relationships of members of the genus Agrobacterium have been determined by ribosomal R)3 sequencing (rrs) (>! 48). Two maKor lineages were distinguishableE one includes A. rhi$ogenes along with most "hi$obium species$ the second includes A. tumefaciens! A. vitis! A. rubi and a distinct strain )+==7 108" along with "hi$obium galegae. Thus biochemical traits permit the characteriDation of species in the genus Agrobacterium which in turn can be identified by rrs sequencing. 7ecause of the molecular tools now available for classification and phylogenetic studies it is predictable that in the future new species will be identified Oe.g. distinct strains have been isolated from +icus (0) and wild blac1berry (1")P. The genetic diversity within A. vitis has been recently studied. 2n addition to Ti plasmid groups chromosomal characteriDations of A. vitis strains were done by fingerprinting the region between the 10< and 2> < rR)3 genes. 3 high correlation was found between chromosomal type and the type of Ti plasmid carried by strains (>>). Ta-le 1. +omparison of old and new nomenclature for species of Agrobacterium )ew ta,onomyFRRRRRRRRRRRRRRRRRRRRRRRROld ta,onomyRRRRRRRRRRRRRRR
A. tumefaciens A! tumefaciens biovar 1 A. radiobacter biovar 1 A. rhi$ogenes biovar 1 A. tumefaciens biovar 2 A. radiobacter biovar 2 A. rhi$ogenes biovar 2 A. tumefaciens biovar > A. radiobacter biovar > A. rubi

3 rhi$ogenes

A. vitis A. rubi

L2ndividual strains in the species may be tumorigenic rhiDogenic or nonpathogenic ISOLATION TECHNIQUES USING SE!ISELECTI"E !EDIA a. $lant !aterial Agrobacterium strains can be isolated from aerial and subterranean galls vascular fluids and from symptomless plants. 2solation of the pathogen is made most easily from white to cream&colored gall tissue of young actively growing galls. 6ash the gall surface 1!

in running water and remove dar1ened necrotic areas of tissue or surface steriliDe for 1"& 2" minutes in 1# sodium hypochlorite solution (or 2"# household bleach) prior to isolating. Rinse the tissues with several changes of sterile water before proceeding. Remove and combine two to three small subsamples from different locations within the gall. +ut the subsamples into small pieces and place in a culture tube with sterile distilled water or buffer vorte, and let stand for >" minutes or longer to allow the bacteria to diffuse into the liquid. <trea1 a loopful of the tissue suspension onto agar media (Table 2) and incubate at ,--,.C. <ome agrobacteria grow very slowly (e.g. wild 7lac1berry types) and colony morphology and pigmentation varies. <ome strains isolated from pecan produced a diffusible blue pigment while some isolated from grape produced a reddish diffusible pigment when cultured (7ouDar unpublished). 7ecause bacteria other than Agrobacterium can grow on the selective media it is helpful to plate 1nown strains on the same media to aid in identification. 7oth pathogenic and non&pathogenic agrobacteria are commonly isolated from galls. 6arningE many colonies loo1 morphologically correct on selective media following initial plating but may contain other bacteria hence the colonies should be cloned for purity before performing additional characteriDation. To clone for purity individual colonies from selective media are suspended uniformly in sterile distilled water or buffer and strea1ed onto =*3++a+"> (Table 2). <ingle colonies are then again selected suspended uniformly in buffer or sterile distilled water and strea1ed. Repeat this process until all the colonies on the plate appear homogeneous. Cn =*3++a+"> Agrobacterium strains grow well and their colonies are distinguishable from other species. They are conve, glistening circular with an entire edge and white to tannish& cream in color. <elective media for Agrobacterium include (Table 2)$ medium 13 (!) for A. tumefaciens (=late 1 /ig. >) medium 2: (!) for A. rhi$ogenes (=late 1 /ig. 4) and Roy and <asser-s (>0) medium for A. vitis (=late 1 /ig. 8). 7ecause more than one strain of Agrobacterium can cohabit the same tumor it may be necessary to use all three media when attempting to determine which species are present. -. Soil and Water

Gse of selective media is especially helpful for isolation of Agrobacterium from soil and water. :ven on selective media a few other bacterial species can grow. /or a qualitative determination of the presence of Agrobacterium! strea1 a loopful of the sample suspension onto the selective medium. /or quantitative determinations prepare 1"&fold serial dilutions and spread ".1 ml of each suspension over the entire surface of the selective medium with an ;&shaped glass rod. <ensitivity of the selective media is limited to about 1"4 colony forming units (+/G)?g soil. 2ncubate plates at 28&2'5+. GtiliDation of the pour plate method with selective media allows higher concentrations of soil suspensions to be assayed thus increasing the sensitivity of detection especially when combined with immunofluorescent colony detection?isolation (9oore and %an %uurde manuscript in review). =repare pour plates by adding ".1 to ".8 ml of a particular dilution series to an empty sterile =etri dish followed by 12 ml molten (cooled to 8"5+) selective 1(

Ta-le %. <elective media for isolation of the maKor species ofAgrobacterium SELECTI"E !EDIU! =atel (>4) !ADOR S$ECIES ISOLATED A. tumefaciens and A. rhi$ogenes CO!!ENTS *iscriminated poorly against the normal soil microflora$ however all agrobacteria tested by 7risbane and Berr grew (!). :,cluded ((# of other species growing on nonselective media$ efficiency of recovery was >!#. Reducing antibiotics increased efficiency of recovery$ some 3 rhi$ogenes grew when ammoniacal nitrogen was used$ use of this selective medium as a liquid enrichment medium allowed detection of nonpathogenic agrobacteria in 2! soils (41). Cmission of streptomycin helped improve recovery of some A. tumefaciens strains (!). A. rhi$ogenes also grew but poorly$ A. vitis was recovered as well. The manganese sulphate concentration had to be reduced by half to allow growth ofAgrobacterium strains in 3ustralia (!). A. vitis also grew on this medium (9oore ;.6. unpublished) (see 4 p. () )itrate was reported to prevent growth of 3 rhi$ogenes and "hi$obium trifolii. @owever 3nderson (1) found that 20?2' "hi$obium and 3 rhi$ogenes strains grew in the presence of nitrate. :ach of these media was reported as highly selective and efficient (((#) for the respective species. <elective for 3 vitis strains and effective when isolating from soil. +olony morphology is very distinct. (7urr T. unpublished and 9oore ;.6. unpublished).

<chrothetal. (41) A. tumefaciens

+lar1 (12)

A. tumefaciens

*29 (>8) )ew&Berr(>1)

A. tumefaciens and 3 rhi$ogenes A. rhi$ogenes

13 2: and >*. (!)

A. tumefaciens! A. rhi$ogenes and A. vitis A. vitis

Roy&<asser (>0)

medium from a culture tube. <wirl the contents of the dish to thoroughly mi, the aliquot of diluted suspension with the molten agar. 3llow to solidify and then incubate the poured plate upside down. +olonies buried in the agar layer appear lenticular in shape. +ompare colony morpholohogies in the dilution series to a similarly prepared pour plate containing a 1nown culture ofAgrobacterium.

20

c.

"a'c)lar Sa&

The presence of endophytic agrobacteria has been reported in grape (2!) and chrysanthemum (1(). A. vitis is a common inhabitant of the vascular tissues of grape and can be isolated from e,tracted ,ylem fluids. 2n the spring one can collect the e,uded sap and spread it to selective medium. 3lternatively a pressure chamber has been constructed which allows cane sections to be processed throughout the year (4>). <imilar procedures can be used to isolate Agrobacterium from other woody plants. +anes or branch sections are cut into 18&cm segments the ends dipped in (8# ethanol and flamed. /lamed segments are inserted through holes in rubber stoppers and the stopper and stem section are fitted into the lid of the sap&e,traction apparatus with the basal part of the cutting positioned in 1" ml of sterile water contained in a conical tube and the lid is secured tightly. =ressure is applied by pumping air into the chamber to force water through the vascular tissues. :,truded sap is collected at the apical end and ".1 ml is spread to selective media. +olonies from the selective medium are purified as described under 2a p. 1!. d. Reci&e' for 'emi'electi(e media #, !edi)m #A for A tumefaciens +formerl7 -(. #, +A, per; ;(&)arabitol >."4 g )@4)"> ".10 g B@2="4 ".84 g B2@="4 1."4 g <odium taurocholate ".2( g 9g<"4S'@2" ".28 g 3gar 18."" g +rystal violet ".1# (w?v) aqueous 2 ml 3utoclave cool to about 8"5+ then add filter&steriliDed cyclohe,imide (1." ml of 2# solution) and )aLeCK (0.0 ml of 1# solution). %, !edi)m %E for A rhizogenes +formerl7 -(. %, +A, per; )@4)"> ".10 g :rythritol >."8 g B@2="4 ".84 g B2@="4 1."4 g 9g<+y'@2" ".28 g <odium taurocholate ".2( g Aeast e,tract 1# (w?v) aqueous 1 ml ml 9alachite green ".1# (w?v) aqueous 8 ml ml g 3gar 18.""

21

3utoclave cool to 8"5+ then add filter&steriliDed cyclohe,imide (1." ml of 2# solution) and )aK<e+L (0.0 ml of 1# aqueous). 3, Ro7 and Sa''er medi)m for A vitis +formerl7 -(. 3, +36, per; 9g<"4&'@2" ".2 g B@2="4 ".'g g B2@="4 ".( g 3donitol 4." g Aeast e,tract ".14 g )a+l ".2 g @>7C> 1." g 3gar 18." g +hlorothalonil (7ravo 8"") 4# (w?v) aqueous ".8ml m1 ml 3dKust to p@ '.2 autoclave cool to 8"5+ and add aseptically the following (after dissolving separately in a few ml of distilled water and filter steriliDing)E Trimethoprim (with 1 drop @+1 to the distilled water) TriphenyltetraDolium chloride *&cycloserine 2" mg !" mg 2" mg

+olonies of A. vitis are countable after four days of incubation at 2'5+. They usually have dar1 red centers with white edges but some strains fail to develop the dar1 red centers. 2n all cases comparison to a 1nown strain plated at the same time is recommended. >. DI ERENTIATION O CO!!ONL0 ISOLATED S$ECIES

The following diagnostic tests (Table >) separate strains of Agrobacterium into the three most common species. 7ecause genetic mutation and recombination li1ely occurs among Agrobacterium strains in nature we e,pect to isolate some strains with phenotypic traits that are intermediate between the designated boundaries of the different species$ however their frequency of occurrence seems to be low. 3 simplified rapid procedure to putatively classify the three main species of Agrobacterium is presented in Table 4 and the authors request scientists who do comparative studies of the two procedures to inform them of their results. These diagnostic tests do not differentiate between pathogens and non&pathogens. 2noculation of susceptible plants is necessary to determine pathogenicity but beware that some strains have a limited host range (1). *)3 probes using gene sequences from a Ti plasmid provide a quic1 presumptive prediction of pathogenicity but suspect colonies should still be inoculated to plants to confirm pathogenicity.

22

Ta-le3.!aEor -iochemical characteri'tic' differentiatin* '&ecie' of the *en)' Agrobacterium

Table >. 9aKor biochemical characteristics differentiating species of the genus Agrobacterium

)oteE 6e typically strea1 each isolate onto each selective medium as an additional test. <ome strains grow on more than one selective medium but they grow more slowly than on the medium designed specifically for another species (;. 9oore unpublished).
Ta-le ;. Sim&lified method for &)tati(e cla''ification of Agrobacterium '&ecie' +6,

*iagnostic Test >&1etolactose production 3cid&clearing on =*3 plus +a+"> 9otility at p@ '." =ectolytic at p@ 4.8

A. tumefaciens + & + &

A. rhi$ogenes & + + & & &

A. vitis

&

+ !"# or more strains positive$ % between 21&'(# of strains positive$ & !"# or more strains negative.

23

;.

DIAGNOSTIC !EDIA AND TESTS

The following general procedures apply to all of the following recipesE Gse distilled or deioniDed water to prepare the media. ;iquid test media are seeded with ".1 ml of a 4! h old culture. The culture is grown for 4"&4! h on ).3 or =*3 slants that were strea1ed from wor1ing cultures (wor1ing cultures are grown on either =*3 or A*+ slants and 1ept at 45+). The bacterial suspension is prepared by scraping bacterial cells from the slant washing once in ".!8# )a+l solution and diluting 1""&fold to provide 1"8to 1"0+/G per test medium. *iagnostic test responses are recorded after 14 days incubation at 2'5+ e,cept for tests performed in liquid medium or on slant media which are incubated an additional wee1. a. Acid clearin* on $DA &l)' CaCo3 =repare from *ifco dehydrated =*3 as recommended by manufacturer and supplement with ".8# +a+">. -. 32Fetolacto'e te't +3, <mear bacterial inoculum over about a 1." cm diameter spot on medium containing 1# L&lactose ".1# yeast e,tract and 2# agar. /our to si, strains can be applied to the same plate. 2ncubate the plate at 2' + for 2 days. Then flood the agar surface with a shallow layer of 7enedict-s reagent and leave at room temperature. 2f >& 1etolactose is present a yellow ring of +u " becomes visible around the cell mass in about one hour (=late 1 /ig. 0). 7enedict-s reagentE *issolve 1'.> g of sodium citrate and 1" g of anhydrous sodium carbonate in 0" ml of distilled water with heating (filter the resulting solution if a precipitate forms). *issolve 1.'> g of cupric sulfate in 18 ml distilled water. <lowly add the cupric sulfate solution into a large bea1er containing the sodium citrate& sodium carbonate solution while stirring constantly. *ilute to one liter. c. erric ammoni)m citrate -roth te't +#6,. /erric ammonium citrate 9g<"4&'@2" B2@="4 +a+l2 per; 1"." g ".8 g ".8 g ".2 g

3dKust to p@ '." before autoclaving. 2noculate the culture tubes containing the broth and incubate in a stationary position. A. tumefaciens strains produce a reddish brown pellicle at the surface of the medium (=late 1 /ig. '). 3u,otrophs may require media supplements of ".1# ;(&) glutamic acid or ".""1# yeast e,tract to grow.

24

d.

Gro<th in %G 'odi)m chloride +%3,. 2noculate ).3 slants containing ,/ (w?v) sodium chloride and chec1 for growth. Acid &rod)ction from er7thritol and D+H, meleCito'e +#8,.
)@4@2="4 B+1 Aeast e,tract 7romthymol blue 1# (w?v) in 8"# ethanol 9g2<"4&'@2" 3gar per; 1." g ".2 g 1." g >." ml ".2 g 1.8 g

f. *.

3dKust p@ to '.1 with 2) )aC@ before adding agar. 3fter autoclaving add 1 part of filter&steriliDed 1"# (w?v) erythritol meleDitose or sucrose solution to ( parts sterile and cooled basal medium$ then dispense about 4 ml of medium to sterile plugged tubes. *evelopment of a yellow color in the medium indicates production of acid from the o,idation of erythritol meleDitose or sucrose. O1ida'e te't +'ee @? &. #=, Citrate )tiliCation +;%, +ultures are inoculated to sodium citrate agar slants of the following compositionE per; <odium citrate (anhydrous) 2." g 9g<"4S'@2" ".2 g )@4@2="4 1." g B2@="4 1." g )a+l 8." g 3gar 2"." g 7romthymol blue 1# (w?v) in 8"# ethanol 18." ml *issolve the salts add 7romthymol blue and adKust the medium to p@ 0.!. 3dd the agar and heat the suspension to melt the agar. *ispense the medium in test&tubes autoclave and slant the tubes to cool. 3fter 24&4! hr the inoculated medium turns to a deep =russian blue if citrate has been utiliDed (=late 1 /ig. !).

h.

Action on litm)' milF. Gse *ifco dehydrated and prepare as recommended by manufacturer. 3dKust p@ to '." by addition of 2) )aC@ prior to steriliDing. /or preparation from scratch see e p. 1(". 3cid production turns the mil1 red whereas al1aline turns the mil1 blue (=late 1 /ig. ().

25

i.

AlFali from malonic add +%8,. per; ()@4)2<"4 2." g B@2="4 ".4 g B2@="4 ".0 g )a+l 2." g Aeast e,tract ".1 g 9alonic acid sodium salt >." g 7romthymol blue 1# (w?v) in 8"# ethanol 2.8 ml 3dKust to p@ '." dispense >&4 ml of medium in test tube before autoclaving. 3fter inoculation incubate at 2' 5+ for about two wee1s. The medium will turn blue when al1ali is produced (=late 1 /ig. 1").

E.

AlFali from m)cic acid and L2tartaric acid +%8,. per; )a)@4="4 ".8 g )a@2="4 ".1' g B+1 ".2 g 7romthymol blue 1# (w?v) in 8"# :thanol 2.8 ml 3dKust to p@ '." and dispense 4.8 ml basal medium to each test&tube before autoclaving. 3fter autoclaving add aseptically ".8 ml of a filter&steriliDed 1# solution of either ;&tartaric acid or mucic acid previously neutraliDed with )aC@. 3fter inoculation incubate at 2'5+ for about two wee1s. The medium will turn blue when al1ali is produced.

F. #. 8.

!otilit7 at &H @.=. +'eed? &. ;6, =ectolytic at &H ;.8 +'ee 3? medi)m A? &. A@,

$ATHOGENICIT0 TESTS

Agrobacteriwn pathogenicity is controlled primarily by genes on tumor&inducing (Ti) plasmids. 2ndividual strains of Agrobacteriwn typically infect a narrow range of hosts (1). )o host was infected by more than !1# of the pathogenic strains in the study by 3nderson and 9oore (1) nor was the host range of a strain predictably determined by the plant from which the pathogen was isolated. a. $re&aration of inoc)l)m

=repare an inoculum containing 1"0 to 1"' +/G?ml (see 8 p. 220) grown in liquid medium 82> (21).

20

#,

LiI)id medi)m 8%3 +%#, per; 1"." g !." g 4." g 2." g ".> gF

<ucrose +asein (acid hydrolysate) Aeast e,tract B2@="4 9g<"4. '@2"

F*issolve separately in 8" ml distilled water and add prior to autoclaving. -. Inoc)lation

3lthough the economics of time and space must be considered when testing the pathogenicity of an un1nown strain the following is recommendedE 1) 2) 2noculate two or more plants of each host species per strain. Tomato datura sunflower and bryophyllum are infected by a large number of Agrobacterium strains. Gsing combinations of these species pathogenicity of a strain was identified with '!&(4# certainty (1). <ome strains infect only the host species from which they are isolated and therefore must be tested on that host. 3lways include controls which consist ofE i) plants that are wounded but not inoculated ii) plants inoculated with a 1nown pathogenic strain of Agrobacterium and hi) plants inoculated with a 1nown nonpathogenic Agrobacterium strain.

>) 4)

Temperatures above >25+ inhibit pathogenicity (2). 6ounding is required for infection and can be done with a scalpel or raDor blade or by ma1ing multiple punctures with a thin sharp needle. Aoung and actively growing plants are recommended. 2noculum can be applied (before or after wounding) as a spray with a transfer loop hypodermic syringe micropipette sterile wooden toothpic1 or cotton swab. 3llow tumors to develop for at least > wee1s on herbaceous plants (=late 2 /ig. > 3) and for at least five wee1s on woody plants. 2ncubation periods of 4&0 months were required for tumor development on inoculated incense cedar (9oore ;. 6. unpublished) and roses may require 1! months. 2f tissue proliferation at the wound of datura plants is relatively small after 4 wee1s they are recorded as negative because 0atura plants often form callus in response to wounding (1'). +allus on datura usually ceases enlargement after 2&> wee1s of growth whereas agrobacteria&induced tumors grow.

27

2n the laboratory dis1s of carrot can be used to test for pathogenicity of A. tumefaciens and A. rhi$ogenes. 6e prefer the carrot root assay (2() (=late 2 /ig. 27) to test for pathogenicity of rhiDogenic agrobacteria strains. /or best results avoid carrots held in prolonged storage. 3fter peeling and washing with (8# ethanol rinse the tissue with '"# ethanol and surface steriliDe in a freshly prepared 1E1" dilution of household bleach for 18 minutes. Rinse serially in three changes of sterile water and aseptically cut 8 mm&thic1 slices perpendicular to the a,is of the carrot. =lace the slices on moistened sterile filter&paper in petri&dishes and inoculate with a bacterial suspension. Cbserve tumor formation or root proliferation after > wee1s. The aba,ial side of the carot slice appears more susceptible to infection. *)3 probes with homology to 1ey gene sequences on the Ti plasmid offer a fast method of predicting the pathogenicity of a strain isolated from different hosts and?or environments ((). 6. !OLECULAR? SEROLOGICAL? AND CO!!ERCIAL AUTO!ATED TECHNIQUES a. !olec)lar techniI)e'.

%arious =+R&primer sets have been used to identify pathogenic Agrobacterium. <awada et al. (>() reported a universal primer set (Table 8) based on sequences from the vir+ operon. These primers detected '8 of '' pathogenic strains which represented the maKor three Agrobacterium species. @aas et al. (14) designed =+R primers (Table 8) to identify a wide variety of pathogenic Agrobacterium strains from a broad strain collection containing pathogenic and nonpathogenic strains of A. tumefaciens! A. rhi$ogenes! A. vitis. They chose oligonucleotides specific for the endonuclease&encoding portion of the vir*2 gene as potential universal primers capable of identifying all pathogenic strains of Agrobacterium. The primers based on vir0, identified pathogenic agrobacteria from all three virulent species tested whereas the ipt primers identified only tumor&inciting Agrobacterium strains. Recently an immunocapture cultivation (with selective medium >*.) followed by =+R (primer set derived from a 1b gene) was developed (22). This procedure proved effective for identification of A. vitis in tumors as well as e,tracts from systemically coloniDed symptomless plants. <ee 3ppendi, 3 for details.

28

Ta-le 8. Gniversal primer sets for identification of pathogenic Agrobacterium strains $rimer' Citation

=rimer 3E 8-&3T. +++ .3T +.3 .+T +33 .T&>=rimer +E 8-&T+. T+T ..+ T.3 +TT T+. T+3 T33&>- =rimer :-E 8-&++T .3+ ++3 33+ 3T+ T+. .+T .++ +3&>These oligonucleotides were used in two different pairs to produce =+R products of >>! base pairs (bp) (3&:-) and 224 bp (3&+).

@aas et al. (14)

=rimer %+/E 8"&3T+3TTT.T3.+.3+T&>" =rimer %+RE 8"&3.+T+333++T.+TT+&>" These primer sets produced a product of '>" bp.

<awada et al. (>()

a. b.

Serolo*7. 3 species&specific monoclonal antibody was developed for identification of 3. vitas strains ((). <ee 3ppendi, 7 for details. att7 Acid Anal7'i'. 7ouDar et al. (4) reported that the 92*2 (92*2 9icrobial 2* 2nc. )ewar1 *:) .+ system for analysis of fatty acids in A. tumefaciens! A. rhi$ogenes! A. rubi and A. vitis provided a qualitative and?or quantitative measure of differentiation between members of these four species. /atty acid analysis revealed that cis&vaccenic acid accounted for more than 0"# of the fatty acid content of agrobacteria. 3nother fatty acid shared by agrobacteria was palmitic acid and amounts of these two fatty acids differed among species. The presence or absence of an oncogenic plasmid did not seem to impact the classifciation. +luster analysis of fatty acid profiles showed a closer relationship between 3. vitis and 3. tumefaciens! even though these two species had fewer acids in common than did A. vitis and A. rhi$ogenes.

c.

Car-on 'o)rce )tiliCation'. 7ouDar et al.(4) tested 8( Agrobacterium strains using the 7iolog system (7iolog 2nc. @ay ward +3) and reported that metabolic fingerprints generated from utiliDation patterns of the different 7iolog carbon sources allowed them to distinguish between A. tumefaciens! A. rhi$ogenes! A. rubi and 3. vitis. This system subsequently proved useful in describing what is li1ely a new species of Agrobacterium isolated from aerial galls on weeping fig trees (0). Results from 7iolog analysis agreed with results from classical diagnostics. 3s with the 92*2 system careful attention should be given to standardiDation of temperature growth&medium and age of culture. Cne should be aware that natural strains will be isolated from time to time with carbon&utiliDation patterns that differ from those in the database. /or details see 3ppendi, +. 2(

@.

CULTURE $RESER"ATION

Agrobacterium strains can be maintained on =*3 (supplemented with ".8# +a+">) or 9.A for about 0 months at 45+. ;ong&term preservation can be achieved when cultures are lyophiliDed or stored at or below &'"5+. /or storage at &'"5+ grow the cells on slants of ).3 or 9.A for 4! h. 6ash the growth from the agar surface with a sterile >"# (v?v) glycerol solution (glycerol serves as a cryoprotectant). *ispense 1." ml of the cell suspension containing at least 1"! +/G?ml into sterile 1.! ml&cryotubes (3?< )unc Bamstrup *B&4""" Ros1ilde *enmar1) and store the cryotubes in a freeDer at &'"5+. ;ong&term preservation can also be achieved by freeDing Agrobacterium cultures at minus 1'(&1(05+ in liquid nitrogen (>"). 3n economical and simple alternative for long&term storage is the use of sterile&water blan1s. Two loopsful of fresh bacterial growth (agar medium) are placed at the bottom of test&tubes containing sterile distilled water. To minimiDe evaporation use screw caps with Teflon liners and wrap =arafilm (3merican +an +o. .reenwich +T) around the tube cap or place the tubes in a plastic bag. These cultures should be 1ept at 45+. A. LITERATURE CITED 1. 2. 3nderson 3. R. and ;. 6. 9oore. 1('(. @ost specificity in the genus Agrobacterium. =hytopathology 0(E>2"&>2>. 7anta ;. 9. M. 7ohne and <. *. ;oveKoy. 1((!. <tability of the Agrobacterium tumefaciens %ir7lC protein is modulated by growth temperature and periplasmic osmoadaption. M. 7acteriol. 1!"E08('&08"0. 7ernaerts 9. M. and M. *e;ey. 1(0>. 3 biochemical test for crown gall bacteria. )ature 1('E4"0&4"'. 7ouDar @. M. 7. Mones and ). +. @odge. 1((>. *ifferential characteriDation of Agrobacterium species using carbon&source utiliDation patterns and fatty acid profiles. =hytopathology !>E'>>&'>(. 7ouDar @. 1((4. Request for a Kudicial opinion concerning the Type <pecies of Agrobacterium. 2nt. M. <yst. 7acteriol. 44E >'>&>'4 (;etter to the :ditor). 7ouDar @. 6. <. +hilton J. )esme A. *essau, %. %audequin 3. =etit M. 7. Mones and ). +. @odge. 1((8. 3 new Agrobacterium strain isolated from aerial tumors on +icus ben2amina ;. 3ppl. :nviron. 9icrobiol. 01E08&'>. 7radbury M. /. 1(!0. .uide to =lant =athogenic 7acteria. /erry ;ane Bew <urrey :ngland. +.3.7 2nternational. >>2 pp.

>. 4.

8. 0.

'.

30

!. (.

7risbane =. .. and 3. Berr. 1(!>. <elective media for three biovars of Agrobacterium. M. 3ppl. 7acterid. 84E428&4>1. 7urr T. M. M. ;. )orelli 7. @. BatD and 3. ;. 7ishop. 1((". Gse of Ti plasmid *)3 probes for determining tumorigenicity of Agrobacterium strains. 3ppl. :nv. 9icrobiol. 80E1'!2&1'!8. +anfield 9. ;. and ;. 6. 9oore. 1(!(. Gnusual characteristics of Agrobacterium tumefaciens isolated from wild blac1berry. =hytopathology '(E11!1. (3bstr.). +hilton 9. *. R. B. <ai1i ). Aadav 9. =. .ordon and /. Tuetier. 1(!". T&*)3 from Agrobacterium is in the nuclear *)3 fraction of crown gall tumor cells. =roc. )atl. 3cad. <ci. G<3 ''E4"0"&4"04. +lar1 3. .. 1(0(. 3 selective medium for the isolation of Agrobacterium species. M. 3ppl. 7acterid. >2E>4!&>81. *e+leene 9. and M. *eley. 1('0. The host range of crown gall. 7ot. Rev. 42E>!(& 400. @aas M. @. ;. 6. 9oore 6. Ream and <. 9anulis. 1((8. Gniversal primers for detection of pathogenic Agrobacterium strains. 3ppl. :nviron. 9icrobiol. 0E2!'(& 2!!4. @ayward 3. +. 1(04. +haracteristics of Pseudomonas solanacearum. M. 3ppl. 7acteriol. 2'E208&2''. @endric1son 3. 3. 2. ;. 7aldwin and 3. M. Ri1er. 1(>4. <tudies on certain physiological characters of Phytomonas tumefaciens! Phytomonas rhi$ogenes and Bacillus radiobacter. 22. M. 7acteriol. 2!E8('&01!. @ildebrand *. + M. =. Thompson and 9. ). <chroth. 1(00. 7acterial enhancement of self&limiting outgrowth formation on 0atura. =hytopathology 80E>08&>00. @olmes 7. and =. Roberts. 1(!1. The classification identification and nomenclature of agrobacteria. M. 3ppl. 7acteriol. 8"E44>&40'. Mones M. 7. and 7. +. RaKu. 1(!!. <ystemic movement of Agrobacterium tumefaciens in symptomless stem tissue of Chrysanthemum morifolium. =lant *is. '2E81&84. Mouanin ;. 1(!4. Restriction map of an agropine&type Ri plasmid and its homologies with Ti plasmids. =lasmid. 12E(1&1"2. >1

1". 11.

12. 1>. 14.

18. 10.

1'.

1!. 1(.

2".

21.

Bado :. 2. and 9. .. @es1ett. 1('". <elective media for isolation of Agrobacterium! Corynebacterium! Erwinia! Pseudomonas! and Xanthomonas. =hytopathology 0"E(0(&('0. Baufifinan 9. @. @. Bassemeyere and ;. Ctten. 1((0. 2solation of Agrobacterium vitis from grapevine propagating material by means of =+R after immunocapture cultivation. %itis. >8E181&18>. Beane =. M 3. Berr and =. 7. )ew. 1('". +rown gall of stone fruit. 22. 2dentification and nomenclature of Agrobacterium isolates. 3ust. M. 7iol. <ci. 2>E8!8&8(8. Bennedy 7. 6. 1(!". :stimates of G.<. crop losses to procaryote plant pathogens. =lant *is. 04'E0'4&0'0. Berr 3. and +. .. =anagopoulos. 1(''. 7iotypes of Agrobacterium radiobacter var. tumefaciens and their biological control. =hytopath. Q. ("E1'2&1'(. Bersters B. and M. *e ;ey. 1('8. 2dentification and grouping of bacteria by numerical analysis of their electrophoretic protein patterns. M. .en. 9icrobiol. !'E>>>&>42. Bersters B. and M. *. ;ey. 1(!4. .enus 222. Agrobacterium +onn 1(42. =ages 244& 284 inE ). R. Brieg and M. .. @olt ed. 7ergey-s 9anual of <ystematic 7acteriology vol. 1. 6illiams U 6il1ins +o. 7altimore. ;ehocD1y M. 1(0!. <pread of Agrobacterium tumefaciens in the vessels of the grapevine after natural infection. =hytopath. Q. 0>E2>(&240. ;ippincott M. 3. and 7. 7. ;ippincott. 1(0(. Tumor&initiation ability and nutrition in the genus Agrobacterium. M. .en. 9icrobiol. 8(E8'&'8. 9oore ;. 6. and *. 3. +oo1sey. 1(!1. 7iology of Agrobacterium tumefaciens. plant interactions. =ages 18&40 inE B. ;. .iles and 3. .. 3therly eds. 7iology of the RhiDobiaceae. 3cademic =ress )ew Aor1. )ew =. 7. and 3. Berr. 1('1. 3 selective medium for Agrobacterium radiobacter 7iotype 2. M. 3ppl. 7acterid. >4E2>>&2>. Cphel B. and 3. Berr. 1((". Agrobacterium vitis sp. nov. for strains of Agrobacterium biovar > from grapevines. 2nt. M. <yst. 7acteriol. 4"E2>0&241. Ctten ;. =. *. Ruftray 3. :. 9omol 9. T. 9omol and T. M. 7urr. 1((0. =hylogenetic relationships between Agrobacterium vitis isolates and their Ti plasmids.
32

22.

2>.

24. 28. 20.

2'.

2!. 2(. >".

>1. >2. >>.

9ol. =lant&9irobe 2nteract. (E'!2&'!0. >4. >8. =atel 9. B. 1(20. 3n improved method of isolating Pseudomonas tumefaciens. (<mith and Towsend). =hytopathology 10E8''. =erry B. ;. and +. 2. Bado. 1(!2. +haracteristics of Ti plasmids from broad&host& range and ecologically specific biotype 2 and > strains for Agrobacterium tumefaciens. M. 7acteriol. 181E>4>&>8". Roy 9. and 9. <asser. 1(!>. 3 medium selective for Agrobacterium tumefaciens biotype >. =hytopathology '>E!1" (3bstr.). Ruger @. M. and 9. . @ofle. 1((2. 9arine star&shaped aggregate&forming bacteriaE Agrobacterium atlanticum sp. now! Agrobacterium meteori sp. nov.$ Agrobacterium ferrugineum sp. nov. nom. rev.3 Agrobacterium gelantinovorum sp. nov. nom. rev.$ and Agrobacterium stellulatum sp. nov. nom. rev. 2nt. M. <yst. 7acteriol. 42E1>>&14>. <awada @. @. 2c1i @. CyaiDu and <. 9atumoto. 1((>. =roposal for reKection of Agrobacterium tumefaciens and revised descriptions for the genus Agrobacterium and for Agrobacterium radiobacter and Agrobacterium rhi$ogenes. 2nt. M. <yst. 7acteriol. 4>E0(4&'"2. <awada @. @. 2e1i and 2. 9atsuda. 1((8. =+R detection of Ti and Ri plasmdids from phytopathogenic Agrobacterium stains. 3ppl. :nviron. 9icrobiol. 01E!2!&!>1. <chmulling T. M. <chell and 3. <pena. 1(!!. <ingle genes from Agrobacterium rhi$ogenes influence plant development. :97C M. 'E2021&202(. <chroth 9. ). M. =. Thompson and *. +. @ildebrand. 1(08. 2solation of Agrobacterium tumefaciens-A. radiobacter group from soil. =hytopathology 88E048& 04'. <immons M. <. 1(20. 3 culture medium for differentiating organisms of typhoid& colon aerogenes groups and for islolation of certain fungi. M. 2nfect. *is. >(E2"(&214. Tarbah /. 3. and R. ). .oodman. 1(!0. Rapid detection of Agrobacterium tumefaciens in grapevine propagating material and the basis for an efficient inde,ing system. =lant *is. '"E800&80!. %an ;arebe1e ). +. .enetello M. <chell R. 3. <chil&eroort 3. B. @ermans 9. =. @ernalsteens and 9. %an 9ontagu. 1('8. 3cquisition of tumor&inducing ability by non&oncogenic agrobacteria as a result of plasmid transfer. )ature 288E'42&'4>.

>0. >'.

>!.

>(. 4". 41.

42. 4>.

44.

33

48. B. Chemical'

6illems 3. and 9. *. +ollins. 1((>. =hylogenetic analysis of rhiDobia and agrobacteriabased on 10< rR)3gene sequences. 2nt. M. <yst. 7acteriol. 4>E>"8&1>. Chemical' Li't
J

So)rce

<ourceE Gnless stated otherwise all the chemicals in this list were obtained from <igma +hemical +o. <t. ;ouis 9C. 3cetic acid glacial 3donitol agar 3mmonium nitrate 3mmonium phosphate 3mmonium sulfate ;(&) 3rabitol d&7iotin 7oric acid 7romcresol purple 7romthymol blue +alcium carbonate +alcium chloride +aseine acid hydrolysate +hloroform +hlorothalonil (7ravo 8"") <*< 7iotech +o. =ainesville C@ +rystal violet +upric sulfate +yclohe,imide *&+ycloserine :*T3 (:thylenediamine tetraacetic acid) disodium salt :rythritol :thanol (ethyl alcohol) :thidium bromide /erric ammonium citrate /errous ammonium sulfate /errous sulfate /errous sulfate .elrite Belco 9erc1 U +o. 2nc. <an *iego +3 *&.lucose ;(&) .lutamic acid .lycerol @ydrochloric acid ;&;actose ;auryl sulfate sodium salt 7iorad Richmond +3
34

;itmus mil1 *ifco ;aboratories *etroit 92 9agnesium sulfate 9alachite green 9alonic acid sodium salt 9anganese chloride 9anganese sulfate *&9annitol *(+) 9eleDitose 9ucic acid )opaline )utrient agar *ifco ;aboratories *etroit 92 Cctopine =otassium chloride =otassium phosphate dibasic =otassium phosphate monobasic =*3 (potato de,trose agar) *ifco ;aboratories *etroit 92 =eptone type 222 (from soybean) =ronase +albiochem&7ehring ;aMolla +3 =ropionic acid sodium salt <odium carbonate <odium chloride <odium citrate <odium hydro,ide <odium phosphate dibasic <odium phosphate monobasic <odium selenite <odium taurocholate <ucrose ;&Tartaric acid Tetramethyl&p&phenylenediamine *ihydrochloride Trimethoprim TriphenyltetraDolium chloride Tris (hydro,ymethyl)&aminomethand (TriDma base) Tris (hydro,ymethyl)&aminomethane hydrochloride (TriDama hydrochloride) ;&Tyroside Aeast e,tract *ifco ;aboratories *etroit 92 Qinc sulfate

35