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Biol Fertil Soils (1996) 21:19 7-200 9 Springer-Verlag 1996

F a b i o L . O l i v a r e s 9 V e r a L . D . B a l d a n i 9 V e r o n i c a M . R e i sJ o s e I . B a l d a n i 9 J o h a n n a D i ~ b e r e i n e r

O c c u r r e n c e o f t h e e n d o p h y t ic d i a z o t ro p h s H e r b a s p i r i l l u m s p p .in r o o t s , s t e m s , a n d le a v e s , p r e d o m in a n t ly o f G r a m in e a e

Received: 23 September 1994

A b s t r a c t S i n c e t h e f i r s t d e s c r i p t i o n o f Herbaspir i l lumseropedicae i n 1 9 8 6 , f e w d a t a h a v e b e e n p u b l i s h e d o n t h i sd i a z o t r o p h , p o s s i b l y d u e t o d i f f i c u l t i e s i n i s o l a t i n g i t f r o ms o i l . I n t h e p r e s e n t s t u d y w e f o u n d t h a t t h i s b a c t e r i u ms e e m s t o b e a n o b l i g a t e e n d o p h y t e w h i c h h a s b e e n i s o -l a t e d f r o m r o o t s , s t e m s , a n d l e a v e s o f a l a r g e n u m b e r o fs a m p l e s o f m o r e t h a n 1 0 d i ff e r e n t s p e c i e s o f t h e G r a m i -n e a e f a m i l y , b u t o n l y e x c e p t i o n a l l y f r o m o t h e r p l a n t s . H .rubrisubalbicans, p r e v i o u s l y m i s n a m e d a s " P s e u d o m o n a s "rubrisubalbicans, a n d k n o w n a s a m i l d p a t h o g e n o f su g a r -c a n e c a u s i n g m o t t l e d s t r ip e d i s e as e , c o n f i r m s t h e e n d o p h y -t i c h a b i t a t o f t h i s g e n u s . T h i s s p e c i e s o c c u r s i n ro o t s ,s t e m s , a n d l e a v e s o f s u g a r c a n e a n d s e e m s t o b e r e s t ri c t e dt o th i s c r o p . I n o c u l a t i o n o f st r a in s f r o m b o t h s p e c i e s i n t os o i l i n h i g h n u m b e r s r e s u l t e d i n a r a p i d d e c l i n e i n t h e i rn u m b e r s . I n o n l y 3 0 d a y s t h e p o p u l a t i o n o f Herbaspiril-lu m s p p . i n s o i l d e c r e a s e d b e l o w d e t e c t i o n l i m i t s ( < 1 0 0c e l l s g - Z ) . W h e n s o r g h u m w a s p l a n t e d i n t h i s s o i l , t h e b a c -t e r i a r e a p p e a r e d a n d m u l t i p l i e d w i t h i n t h e p l a n t t i s s u e s .

K e y w o r d s Herbaspir i l lum 9 E n d o p h y t e s o f G r a m i n ea e 9D i a z o t r o p h s S u r v i v a l i n s o i l N i t r o g e n f i x a t i o n 9S u g a r c a n e

IntroductionA l t h o u g h H. seropedicae w a s d e s c r i b e d 8 y e a r s a g o ( B a l -d a n i e t a l . 1 9 8 6 ) , t h e r e i s n o a d d i t i o n a l i n f o r m a t i o n o n t h eo c c u r r e n c e o f t h i s d i a z o t r o p h i n c o u n t r i e s o t h e r t h a n B r a z i la n d H a w a i i ( J . D 6 b e r e i n e r , p e r s o n a l c o m m u n i c a t i o n 1 9 9 5 ) .T h i s d i a z o t r o p h w a s i n i t i a l l y t h o u g h t t o b e a n a d d i t i o n a lr h i z o s p h e r e b a c t e r i u m l i ke Azospir i l lum spp . , ab l e t o i n f ec t

EL. Olivares 9 V.L.D. B aldani 9 V.M. Reis 9 J.I. BaldaniJ. D6bereiner (~e)Embrapa-Centro Nacional d e Pesquisa de Agrobiologia,Serop6dica CEP 23851-970, Rio de Janeiro, Brazil

c e r e a l s a n d c o l o n i z e t h e r o o t i n t e r i o r a n d a e r i a l p a r t s o fg r a m i n e o u s p l a n t s . H o w e v e r , m a n y a t t e m p t s t o i s o l a t e H .seropedicae f r o m u n c r o p p e d s o il w e r e u n s u c c e s s f u l ( B a l -d a n i e t a l. 1 9 9 2 a ) , s u g g e s t i n g a n e n d o p h y t i c m o d e o f li v -i n g a c c o r d i n g t o t h e d e f i n i t i o n b y K l o e p p e r e t a l . ( 1 9 9 2 ) .T h e i n d i c a t i o n o f t h is c h a r a c t e r i s t i c w a s r e i n f o r c e d b y t h ei d e n t i f i c a ti o n o f a s e c o n d Herbaspir i l lum sp. , H. rubrisu-balbicans, p r e v i o u s l y m i s c l a s s i f i e d a s "Pseudomonas" ru -brisubaIbicans ( G i l l i s e t a l . 1 9 9 1 ) . T h i s m i l d p a t h o g e n o fs u g a r c a n e c a u s e s m o t t l e d s t r i p e d i s e a s e i n s o m e s u g a r c a n eg e n o t y p e s g r o w n i n c o u n t r i e s w h e r e h i g h N f e r t i l i z e r l e -v e l s a r e u s e d , b u t n o t o n t h e B r a z i l i a n s u g a r c a n e g e n o -t y p e s t e s t e d a t l o w N f e r t i l i z e r l e v e l s ( P i m e n t e l e t a l .1991) .

I n c o n t r a s t t o r h i z o s p h e r e d i a z o t r o p h s , w h i c h h a v e t oc o m p e t e w i t h t h e s o i l m i c r o f l o r a f o r C s u b s t r a te s , a n d r e -l e a s e f i x e d N t o t h e p l a n t o n l y a f t e r t h e i r d e a t h , e n d o p h y -t i c d i a z o t r o p h s l i k e Herbaspir i l lum s p p . c a n b e f o u n dw i t h i n p l a n t t i s s u e s , a m o r e u n i f o r m a n d p r o t e c t i v e e n v i r -o n m e n t w h i c h m a y f u r t h e r e x p l a i n t h e h i g h c o n t r i b u t i o nf r o m b i o l o g i c a l N f i x a t i o n i n s u g a r c a n e ( U r q u i a g a e t a l.1992) .

I n th e p r e s e n t w o r k w e c o n f i r m e d t h a t H. seropedicaeand H. rubrisubalbicans, l ike Acetobacter diazotrophicus,a r e p l a n t e n d o p h y t e s a n d c o l o n i z e ro o t s , s t e m s , a n d l e a v e so f v a r i o u s g r a m i n a c e o u s p l a nt s .

Materials and methodsMethods of isolation and identificationThe m ethod used to isolate both sp ecies of Herbaspirillum was thatdescribed by Baldani et al. (1992a) with a few modifications. Sam-ples o f washed or surface-sterilized roots, stem s, and leave s werehomogenized, diluted in salt solution of JNFb medium, and 0.1 ml ofthe suspension w as inoculated into vials with 5 ml sem isolid JNFbmedium with the following comp osition per litre: malic acid (5 g);K2HPO4 (0 .6g) ; KH 2PO4 (1 .8 g) ; MgSO4.7H20 (0 .2g) ; NaC1(0.1 g); CaC12.2H20 (0 .02 g); bromothymol blue 0.5% in KOH0.2 N (2 ml); vitamin solution (1 ml); micronutrient solution (2 ml);1.64% Fe-ethylene diaminetetraacetic acid solution (4 ml); K OH

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19 8(4.5 g). The pH was adjusted to 5.8 and 1.9 g 1-1 of agar was added.The m icronu tr ien t so lu t ion con ta ined (quan t i t ies per l it re ) :CuSO 4.5H20 (0 .4 g ) ; ZnSO 4-7H20 (0 .12 g ) ; H2 BO 3 (1.4 g ) ;Na2Mo Oa.2H20 (1 .0 g ) ; MnSO 4.H20 (1 .5 g ) . The v i tam in so lu t ioncontained (in 100 ml): biotin (10 mg); pyridoxol-HC1 (20 mg).Two to five days after ino culation , veil-l ike pellicles identical tothose o f AzospiriUum spp . appeared in the sem iso l id JNFb m edium .They were transferred into a fresh JNFb medium and after 2 dayswere s t reaked ou t on NFb agar p la tes (Drbere iner 1992) con ta in ingth ree t im es the b rom o thym ol b lu e concen t ra t ion , and 50 m g 1-1 o fyeast extract, and the pH was adjusted to 6.5. On this medium, theco lon ies o f bo th Herbaspirillum spp . becam e sm ooth and whi t i shwi th b lue o r g reen cen te rs a f te r one week , which a re qu i te d is t inc tf rom Azospirillum spp . co lon ies . Microscop ic . observa t ions he lped toconfirm these species on the basis of the smaller cell s ize of Herbas-pirillum spp. F ina l pur i f ica t ion was ob ta ined by s t reak ing the co lon ieson po ta to agar p la tes (D6bere iner 1992) . The ce l l s on th is m ediumand o n o ther r ich m edia were longer and typ ica l ly sp i ra lo id.The two species of HerbaspiriUum were d is t ingu ished by the i rg r o w t h i n s e m i s o l i d J N F b m e d i u m c o n t a i n i n g 0 . 5 % N - a c e ~ l g l u c o sa -m i n e (H. seropedicae) or 0.5% rneso-erythritol plus 1 g F of NH4C1(H. rubrisubalbicans) as C sources instead of malate (Baldani et al .in preparation).

Surv iva l o f HerbaspiriUum spp. in soil and rootsTwo exper im ents were per fo rm ed to s tudy the su rv iva l o f Herbaspir-ilium spp. in soil . The first was carried out in red-yeUow podzolicsoil ( typic Hapludult; pH 4.9) collected from a site with natural vege-ta t ion , and the second f rom a sandy so i l (pH 4 .7 ) cu l t iva ted fo r 13years w i th sugarcane .In the f i r s t exper im ent H. seropedicae s t ra in Z67 and H. rubrisu-balbicans s t ra in M4, w hich a re na tu ra l ly res is tan t to na l id ix ic ac id(30 Ixg ml I , and Azospirillum brasilense strain SP109, which is resis-tan t to s t rep tom ycin (100 ~ tg m l l ) , were g rown in l iqu id nu t r ien tb ro th (D ifco) m ed ium con ta in ing 10 g F ~ o f g lycero l fo r 36 h a t30~ The cells were harvested, centrifuge d, washed , and the opticaldens i ty ad jus ted to 0 .65 a t 610 nm . Sam ples o f 1 m l (approx im ate ly107 cells) were inoculated into plastic vials containing 10 g of eitherthe red-ye l low podzo l ic o r the sandy so i l and m ain ta ined a t 60% f ie ldcapacity. A t various intervals serial dilutions of 10-g soil samp leswere per fo rm ed and inocu la ted in to th ree rep l ica te v ia ls con ta in ing5 m l o f sem iso l id JNFb m edium . Sev en days la te r , the m os t p robab lenum bers were es t im ated by a typ ica l ve i l - l ike pe l l ic le us ing theMcCrady tab le (Pochon and Tard ieux 1962). U uinocu la ted so il s m ain-ta ined under the sam e cond i t ions were used as con t ro l .In the second exp er im ent the sam e inocu la t ion p rocedure wasused b u t the po ts con ta ined 300 g o f the typ ic H apludu l t so i l(pH 4.9). Th ere were three treatmen ts: uncroppe d soil , soil croppedwith so rghum f rom the b eg inn ing , and sorghum p lan ted on ly 76 daysafter inoculation. Seeds of Sorghum vulgare var. BR005 were steri-l ized by therm al t rea tm ent at 65~ fo r 2 h , r insed tw ice w i th s te ri lewate r , then im m ersed fo r 30 s in 70% a lcoho l and fo r 1 ra in inHgCI2 (1:500), and rinsed three times with sterile water. The effi-c iency o f the s te r i liza t ion p rocess was de te rm ined by the inocu la t ionof 200 ind iv idu a l c rushed seeds in to sem iso l id JNFb m edium andeva lua t ion by pe l l ic le fo rm at ion . Only 3 o u t o f 10 seed l ings were le f tper po t and each seed was t rea ted w i th 2 m l inocu lum . P lan ts andso i l f rom un inocu la ted po ts were used as con t ro ls. There were th reereplicates per treatment. At var ious intervals, samples of soil , root-sur-face so i l , and washed roo ts were assayed as descr ibed above .

Res ults and discussionT h e r e s u l t s s h o w e d t h a t H . s e r o p e d i c a e o c c u r r e d i n m a n yg r a m i n e o u s p l a n t s , i n ro o t s , s t e m s , a n d l e a v e s ( T a b l e 1) .T h e o c c u r r e n c e i n r h i z o s p h e r e s o i l r e p o r t e d e a r l i e r ( B a l d a -n i e t a l . 1 9 8 6 ) w a s p r o b a b l y d u e t o t h e i n c l u s i o n o f r o o t

Tab le 1 Occurrence o f Herbaspirillum seropedicae and H. rubrisu-balbicans in different species of Gramineae. Plant species consistedof 5 i roo t sam ples o f n ine genera in seven fam i l ies o ther than whichGram ineae d id no t con ta in Herbaspirillum spp. , except one sample ofCajanus cajan grown in a field with a 10-year history of maizem onocu l tu re , and one o f Cyperus rotundus grown in a sugarcane f ie ld(N I not isolated, a only roots samp led)Plan t species H. seropedicae H. rubrisubalbicansSaccharum sp. hyb rid Roots, stems Roots, stems, leavesWeed p lan ts in sugarcane f ie ldsDigitaria insularis NI RootsEchinola crusgalIi Roots NIF ive un iden t i f ied spp . Roots NIOryza sativa Roots, stems, leaves NIZea mays Roots, stems, leaves NISorghum bicolor Roots a NIPennisetum purpur eum Roots a NIPanicum maximum Roots a NIDig#aria decumbens Roots a NIBrachiaria decum bens Roots a NIMelinis minutiflora Roots a NI

h a i r s o r s m a l l r o o t p i e c e s i n th e s a m p l e s b e c a u s e o f th em e t h o d u s e d , w h e r e b y s o i l a d h e r i n g to t h e ro o t s w a sw a s h e d o f f b y i m m e r s i o n i n th e d i l u t i o n w a t e r w i t h s h a k -i n g . T h e o n l y s a m p l e o f a l e g u m e s p e c i e s ( C a j a n u s c a j a n )t h a t c o n t a i n e d t h e o r g a n i s m w a s f r o m a w e e d g r o w i n g i n af i e ld w i t h a 1 0 - y e a r h i s t o ry o f m a i z e m o n o c u l t u r e , a n ds m a l l p i e c e s o f m a i z e r o o t m a y h a v e b e e n i n c l u d e d i n t h es a m p l e ( T a b l e 1 ). T h e o n e o u t o f t h r e e s a m p l e s o f a C y-p e r a c e a e s p . t h a t c o n t a i n e d H. sero p ed i ca e w a s c o l l e c t e di n a s u g a r c a n e f i e l d a n d t h e re f o r e c o u l d a l so h a v e b e e n o nG r a m i n e a e r o o t s ( T a b l e 1) . T h i s w a s c o n f i r m e d b y t h e a b -s e n c e o f t h e b a c t e r i u m i n s a m p l e s o f Cyp eru s ro tu n d u sc o l l e c t e d f r o m f i e l d s o t h e r t h a n s u g a r c a n e .

I n c o n t r a s t t o H. sero p ed i ca e , H . ru b r i su b a lb i ca n s w a si s o l at e d o n l y f r o m r o o ts , s t e m s , a n d l e a v e s o f s u g a r c a n e( T a b l e 1 ). T h e o n l y e x c e p t i o n w a s a n i s o l a t e f r o m Dig i ta r -ia insu taris w h i c h w a s t a k e n f r o m a s u g a r c a n e f ie l d . T h i sa g r e e s w i t h t h e d e s c r i p t i o n o f th i s o r g a n i s m a s a m i l d s u -g a r c a n e p at h o g en , k n o w n a s P s e u d o m o n a s r u b r i s u b a l b i -c a n s b u t r e c e n t l y r e c o g n i z e d a s a n a d d i t i o n a l Herb a sp i r i l -lu m s p . ( G i l l i s e t a l. 1 9 9 1 ) . I n c o n t r a s t , a l ar g e n u m b e r o fs a m p l e s f r o m s e v e n d i f f e r en t p l a n t fa m i l i e s (Co mp o s i t a e ,M o l lu g in a cea e , S t ercu l i a cea e , Cyp era cea e , Po r tu la ca cea e ,L e g u m i n o s a e , a n d C u c u r b it a c e a e ) d i d n o t c o n t a i n H e r b a s -p i r i l l u m s p p . ( T a b l e 1 ). I n a d d i t i o n , n o n e o f t h e 1 0 s o i ls a m p l e s t a k e n f r o m v a r i o u s s i te s c o n t a i n e d t h is o r g a n i s m ,c o n f i r m i n g i t s e n d o p h y t i c h a b i t a t (d a t a n o t s h o w n ) .

I n o r d e r t o e x p l a i n t h e d i s t r i b u t i o n o f t h es e o r g a n i s m su n d e r f i e l d c o n d i t i o n s t w o h y p o t h e s e s w e r e e x a m i n e d :F i r st , t h e t r a n s m i s s i o n w i t h i n s t e m - c u t t i n g s u s e d t o p l a n tt h e s u g a r c a n e o r s o m e a d d i t i o n a l f o ra g e g r a s s e s p r o p a -g a t e d b y s t e m - c u t t i n g s a s o b s e r v e d f o r A c e t o b a c t e r d i a z o -t ro p h icu s ( R e i s e t a l . 1 9 9 4 ); s e c o n d , d i s s e m i n a t i o n w i t hs e e d s o f c e r ea l s. T h e t r a n s m i s s i o n o f H e r b a s p i r i l l u m s p p .v i a s t e m - c u t t i n g s o f s u g a r c a n e w a s c o n f i r m e d b y i ts o c c u r -

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Fig . 1 Surv iva l o f Herbaspirillum seropedicae strain Z67, H. rubri-subalbicans s t ra in M4, and Azospirillum brasilense strain SP 109 intyp ic Hapludu l t so i l (soil 1) and sandy soil cultivated for 13 yearswi th sugarcane (soil 2)

r e n c e i n 3 5 s a m p l e s o f f o u r m i c r o p r o p a g a t e d v a r i fi e s ( S P8 1 - 9 0 2 5 , R B 8 5 - 5 0 6 5 , R B 8 0 - 6 0 4 3 , I A C 8 2 - 2 0 4 5 ) o b -t a i n e d f r o m s t e ri l iz e d a p i c a l m e r i s t e m s o f 7 - 1 0 m m i n s i z eb u t n o t i n 3 3 samp les o f 1 1 v a r i e t i e s o b t a in ed f ro m s t e r i -l i z e d a p ic a l m e r i s t e m s o f a m a x i m u m 2 m m i n s iz e . I t i sk n o w n t h a t d e p e n d i n g o n t h e s i z e o f t h e m e r i s t e m e x -p l an t s , t h e re i s co n t ac t w i th t h e x y l em v esse l t h a t ca r r i e sb a c t e r i a ( K a r t h a a n d G a m b o r g 1 9 7 5 ) . T h e r e f o r e i t i s n o tsu rp r i s i n g t o f i n d Herbaspirillum spp . in these t i ssuess in ce t h i s g en u s i s k n o wn to co lo n i ze t h e x y l em o f su g ar -can e p l an t s (Ol iv a res e t a l . 1 9 9 3 ) .

T h e g e n u s h a s b e e n f o u n d i n r i c e s e e d s , a s d e m o n -s t r a t e d b y e l e c t r o n m i c r o s c o p y a n d i m m u n o a s s a y , w h i c hs h o w e d Herbaspirillum sp . i n t h e i n t e rn a l l ay er o f t h e t e -g u men t (Ba ld an i e t a l . 1 9 9 2 b ) . T ran smi ss io n t h ro u g h seed si s v e r y c o m m o n a m o n g p h y t o p a t h o g e n i c b a c t e r i a , w h i c hmay en t e r seed s d u r in g t h e f l o wer in g p ro cess o r v i a t h es t o m a t a o f t he s e e d c o a t a n d v i a t h e v a s c u l a r s y s t e m ( G o t o1 9 9 2 ). Th e o ccu r r en ce o f d i azo t ro p h s i n seed s h as a l sob e e n r e p o r t e d f o r Azospirillum s p p . ( S u n d a r a m a n d K l u c a s1988) .

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Fig . 2 Surv ia l o f Herbaspirillum rubrisubalbicans swain M4 (A) andH. seropedicae strain Z67 (B) in soil , rhizosphere soil, and w ashedro tos o f so rghum p lan ts

T h e e n d o p h y t i c h a b i t a t o f H. seropedicae a n d H. rubri-subalbicans w a s c o n f i r m e d i n t w o a d d i t i o n a l e x p e r i m e n t so n t h e su rv iv a l o f t h e b ac t e r i a i n so i l . Desp i t e t h e i n o cu l a -t i o n o f v e ry l a rg e n u mb e r s o f ce l l s , n e i th e r sp ec i es c o u ldb e r e - i so l a t ed f ro m so i l 3 0 d ay s a f t e r i n o cu l a t i o n (F ig . 1 ) ,i n ag reemen t wi th t h e r esu l t s o b t a in ed b y Ba ld an i e t a l .( 1 9 9 2 a) , u s in g two o th er s t r a i n s o f H. seropedicae in d i f -f e r en t so i l s . I n co n t r as t , wh en Azospirillum brasilenses t r a in S P 1 0 9 was i n o cu l a t ed i n to t h e same so i l s , i t was r e -i so l a t ed 4 0 d ay s a f t e r in o cu l a t i o n (F ig . 1 ). F u r th e rmo re ,so i l t y p e h ad n o e f f ec t o n t h e su rv iv a l o f t h e two Herbas-pirillum spp . (F ig . 1 ) , ind icat ing that the so i l f rom thei rn a tu ra l h ab i t a t ( su g arcan e f i e l d ) d id n o t p e rmi t su rv iv a l . I nad d i t i o n , t h ese so i l s were i n cu b a t ed 4 0 d ay s a f t e r i n o cu l a -t i o n wi th a Herbaspirillum s p . m e d i u m c o n t a i n i n g m a l a t eb u t t h e s u r v i v a l o f t h e b a c t e r i a d i d n o t i m p r o v e ( d a t a n o ts h o w n ) . T h i s t y p e o f b e h a v i o r i n s o il h a s b e e n o b s e r v e dfo r man y o th er p h y to p a th o g en i c b ac t e r i a , i n c lu d in g t h ev a s c u l a r p a t h o g e n , Xanthomonas albilineans, w h i c h c a u s e sl ea f sca ld i n su g arcan e (Hu g h es 1 9 6 8 ) .

W h e n a s o r g h u m p l a n t w a s s o w n i n s o i l i n o c u l a te dw i t h b o t h Herbaspirillum sp p . t h e b ac t e r i a were d e t ec t edfo r a mu ch l o n g er p e r i o d i n ro o t - su r f ace so i l (F ig . 2 ) .Ho wev er , n u m b er s i n t h e r an g e o f 1 0 4 ce l l s p e r g o f f r eshweig h t were main t a in ed o n ly i n ro o t s . I n t h i s case , H. ru-brisubalbicans s t r a in M 4 was a l so d e t ec t ed i n so rg h u m

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F i g . 3 Re-isolation of Herbaspirillum rubrisubalbicans strain M4(A) and H. seropedicae strain Z67 (B) from sorghum plants planted76 days after soil inoculation (arrow)

r o o t s f r o m w h i c h i t c o u l d n o t b e i s o l a t e d u n d e r n a t u r a lc o n d i t i o n s .

W h e n e i t h e r s p e c i e s w a s i n o c u l a t e d i n t o s o i l w i t h o u tp l a nt s , bo t h s t r a i ns a g a i n d i s a ppe a r e d a f t e r 3 0 da y s( F i g . 3 ) . H o w e v e r , w h e n s o r g h u m w a s p l a n t e d in t h e p o t s4 5 da y s l a t er , u s i ng s e e ds t ha t d i d n o t c o nt a i n t he ba c t e r ia ,b o t h s p e c i e s c o l o n i z e d t h e r o o ts ( F i g . 3 ) . T h i s f i n d i n g i n d i -c a t e s t h a t t h e o r g a n i s m h a d s u r v iv e d , b u t i n n u m b e r s t h a tw e r e t o o l o w t o b e r e c o v e r e d f r o m t h e s o i l b y t h e u s u a lc o u n t i n g m e t h o d s . S i m i l a r r e s u lt s w e r e o b t a i n e d w i t h t h ephytopathogenPseudomonas solanacearum (Nesmith andJenkins 1983). This bacterium could not be detected in thesoil 30 days after inoculat ion but it was recovered by intr o-ducin g tomato seedlings. The detection of bacteria only afterintroduction of the plant supports the concept of viable butnot culturable cells proposed by Roszak and Colwell(1987). In this case, the plant provides certain substances(C sources, amino acids, etc.) or inducers that switch onthe mechanisms of cellular division and consequently at-tract the bacteria to the root system.

The requirement of plant tissue for survival seems tobe a specific characteristic developed by H e r b a s p i r i l l u mspp. and other endophyfic bacteria. Within the plant tis-

sues they can find microniches to fix N2 without having tocompete with other microorganisms and without being ex-posed to many soil constraints.

Acknowledgements We thank Dr. Robert M. Boddey for Englishcorrections. We also thank Geraldo Baeta for technical assistance andDr. T.S.G. Lee for providing micropropagated sugarcane plants. EL.Olivares and V.M. Reis wer e supported by EMBRAPA, CNPq,UFRRJ and FAPERJ. This work constitutes part of the PhD thesis ofthe first author.

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root cells of rice. An Acad Bras Ci~n 64:431Drbereiner J (1992) The genera Azospirillum and Herbaspirillum. In:Balows A, Trtiper HG, Dworkin M, Harder W, Schleifer KH (eds)The prokaryotes 2nd edn, vol HI. Springer Verlag, New York Ber-lin Heidelberg London Paris Tokyo Hong Kong Barcelona Buda-pest, pp 2236-2253Gillis M, Drbereiner J, Pot B, Goor M, Falsen E, Hoste B, ReinholdB, Kersters K (1991) Taxonomic relationship between (Pseudo-monas) rubrisubalbicans, some clinical isolated (EF group 1) Her-baspirillum seropedicae, and (AquaspirilIum) autotrophicum. In:Pollinelli M, Materassi R, Vicenzini M (eds) Nitrogen fixation.Kluwer Academic Press, Dordrecht, pp 292-294Goto M (1992) Fundamentals of bacterial plant pathology. AcademicPress, New YorkHughes CG (1968) Transmission of leaf scald by soil leachate andsugarcane. Pathol Newslett 1:27-28Kartha KK, Gamborg OL (1975) Elimination of cassava mosaic dis-ease by meristem culture. Phytopatology 65:826-828Kloepper JW, Schippers B, Bakker PAM (1992) Proposed eliminationof the term endorhizosphere. Phytopathology 82:726-727Nesmith WC, Jenkins SF Jr (1983) Survival of Pseudomonas solana-cearum in select ed Nor th Carolina Soil s. Phytopathology73:1300-1304Olivares FL, James EK, Reis VM, Baldani VLD, Baldani JI. Doberei-net J (1993) Colonizaqfio do tecido vascular por Herbaspirillumspp. em sorgo e cana-de-a~ucar. Fitopatol Bras 18:313Pimentel JP, Olivares FL, Pitard RM, Urquiaga S, Akiba F, Drberei-net J (1991) Dinitrogen fixation and infection of grass leaves byPseudomonas rubrisubalbicans and Herbaspirillum seropedicae.Plant and Soil 137:1-65Pochon J, Tardieux P (1962) Techniques d'~inalyse en microbiologiedu sol. ]Zdifionsde la Tourelle, St. Mand6 (Seine). 105pReis VM, Olivares FL, Drbereiner J (1994) Improved methodologyfor isolation of Acetobacter diazotrophicus and confirmation of itsendophytic habitat. World J Appl Microbiol Biotech 10:401-405Roszak DB, Colwell RR (1987) Survival strategies of bacteria in thenatural environment. Microbiol Rev 51:365-379Sundaram S, Klucas RV (1988) Characterization of azospirilla iso-lated from seeds and roots of turf grass. Can J Microbiol 34:212-217Urquiaga S, Cruz KHS, Boddey R (1992) Contribution of nitrogenfixation to sugar cane: Nitrogen-15 and nitrogen balance esti-mates. Soil Sci Soc Am J 56:105-114