Soil Contamination by Crude Oil_impact on the Mycorrhizosphere and on the Revegetation Potential of...

7/30/2019 Soil Contamination by Crude Oil_impact on the Mycorrhizosphere and on the Revegetation Potential of Forest Trees

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ENVIRONMENTALPOLLUTIONE L S E V 1 E R En v i r o n m en ta l P o l lu t io n 9 9 ( 19 9 8) 3 7 - 4 3

S o i l c on tami n at i on b y c r u d e o i l : i mp ac t on th e myc or r h i z osp h e r eand on the revegetat ion potent ia l o f forest treesG i o v a n n i N i c o l o t t i a , S i m o n E g li b

aUniversity of Torino, DL VA.P.R.A.--Plant Pathology, 10095, Grugliasco, ( Torino), ItalybSwiss Federal Institute for Forest, Snow and Landscape Research WSL, 8903, Birmensdorf, (Zurich), SwitzerlandR ece iv ed 7 A p r i l 1 9 97 ; accep ted 2 9 O c to b e r 1 9 9 7

A b s t r a c t

In vitro and g reenh ous e b io t e s t s w ere ca r r i ed ou t t o s t udy th e e f fec ts o f va r ious concen t r a t i ons o f c rude o i l on t h e m y cor rh i z o -s ph e re and t h e ab i l i t y o f ec tom y co r rh i z a l fung i t o co lon is e N orw ay s p ruce and pop l a r s eedl ing s g row n on co n t am ina t ed s o il .E c tom y co r rh i z a l fung i g row n in pu re cu l t u r e s s h ow ed a v a r i e ty o f r eac t i ons t o c rude o i l, r ang ing f rom g row th s t im u la t i on t o t o t a linh ibi t ion o f g rowth , depen ding on the species of fung i. G erm inat i on o f popla r and spruce seeds was not s ignif icantly affected. T hegrow th of spruce seedl ings was no t affected by crude oi l , whereas tha t of pop lar seedl ings was s igni ficant ly reduced a t h igh con-cen t r a t i ons . N one o f t h e concen t r a t i ons h ad any e f fec t on t h e deg ree o f ec tom y cor rh i z a l and endom y cor rh i z a l co lon i s a t i on o fpoplar . With spruce, however , the ectomycorrh izal fung i showed species -speci f ic react ions to increas ing concentra t ions , in accor-danc e wi th the resul ts of the pure cul ture tes t. T he leng th of t ime between soi l contam inat io n and seeding affects bot h seedlingg row th a nd t h e m y cor rh i z a l i n fec t ion po t en t i a l o f t h e s o il . T h e r e s u l ts conf i rm th e im po r t ance o f m y cor rh i z a l fung i i n t h e b io r -eme diat ion o f soi ls conta min ated by crude oi l . 1998 E lsevier Science L td. Al l r igh ts reserved.In L abo r - und G ew ~ ich sh aus ver suchen w urde d i e W i rk ung v on E rd6 1 au f d i e M y k o r rh i z a f lo ra und i h r P o t en ti a l , F e inw urz e lnvon F ich ten und Pappels / iml ingen zu bes iedeln, durchgeff ih r t . In Reinkul tur zeig ten die 10 untersuch ten Ektomykorrh izapi lzeun t e r sch i ed li ch e R ea k t ion a u f E rd6 1k on tam ina t i on , vo n S t im ula t i on b i s zu t o t a l e r H em m u ng des Wach s tum s . E in E in flus s vonErd61 au f die Same nkeim ung war bei keiner der beiden Ba um arten fes tzus te l len. Das S~imlingswachs tum wurde bei Pappel durchhohe Erdf lkonzentra t ionen s igni f ikant gehemmt, w~ihrenddem es bei F ich te prak t isch unbeeinf luss t b l ieb. Die Bes iedlung derFeinwurzein du rch En do- un d E ktom ykorr h izapi lz e wurde bei Pappel durch Erd 61 nich t beeinf lusst , bei F ich te zeig ten E ktom y-k or rh i z ap i l z e - - i n L rbe re ins t im m un g m i t den R es u l t a ten de r R e ink u l tu rve r s uch e - - a r t s pez if i s ch e R eak t ionen au f E rd6 1k on tam ina -t ion. Die Wartezei t zwischen der Erdf lkontaminat ion des Bodens und der Aussaat beeinf luss t sowohl das S~imlingswachs tum wiedas M y k or rh i z a - In fek t i ons po t en t i a l des B odens. D ie R es u l t a t e bes t~ it ig en d i e B edeu tung de r M y k or rh i z ap i l z f l o r a im Z u s am m en-hang m i t der W iederbes iedlung v on erd61ko ntaminier ten B6den. 1998 E lsevier Science L td. Al l r igh ts reserved.Keywords: C rude o i l; E c tom y cor rh i z a ; E ndom y cor rh i z a ; P o l l u t ion ; In fec t i on po t en ti a l ; Picea abies; Populus nigra

I . I n t r o d u c t i o n

C r u d e o i l is e x t r a c t e d w o r l d - w i d e a t t h e r a t e o f m o r et h a n 6 5 m i l l i o n b a r r e l s p e r d a y a n d m e e t s t h e b u l k o ft h e w o r l d ' s e n e r g y r e q u i r e m e n t s . A t t h e s a m e t i m e ,h o w e v e r , i t i s a p o t e n t i a l h a z a r d a n d c a n c a u s e s e v e r ea n d l a s t in g d a m a g e i f i t i s a c c i d e n t a l l y d i s c h a r g e d i n t ot h e e n v i r o n m e n t . S u c h a c c i d e n t s h a p p e n d e s p i t e t h e f a c tt h a t t h e d i s t r ib u t i o n a n d s t o r a g e n e t w o r k f o r c r u d e o i l i sc l o s e l y m o n i t o r e d .

A n a c c i d e n t o f t h is k i n d o c c u r r e d a t T r e c a t e , N . I t a l y ,i n 1 9 94 , w h e n a n o i l w e l l b e c a m e u n c o n t r o l l a b l e a n db l e w o u t s o m e 1 8 0 0 0 S m 3 ( s t a n d a r d m 3 , i ts g a s v o l u m ea t 1 5 C ) o f c r u d e o il o v e r a n a r e a o f 1 5 0 0 h a ( A G I P ,0269-7491/98 /$19.00 1998 Elsevier Science Ltd . All r ights reserved.P I I : S 0 2 6 9 - 7 4 9 1 ( 9 7 ) 0 0 1 7 9 - 6

1 9 95 ). T h e a r e a c o n s i s t s m a i n l y o f f a r m l a n d a n d f o r e st .S i n ce o i l sp i ll s h a v e r a r e l y a f f e c t ed a r e a s w i t h a b u n d a n tt e r r e s t r i a l v e g e t a t i o n , n e w m e t h o d s f o r a s s e s s i n g t h eh a r m c a u s e d t o t h e e c o s y s t e m s i n T r e c a t e ' s a f f e c t e d a r e ah a d t o b e s o u g h t .

M o s t o i l- w e l ls a re s i t u a t e d i n d e s e r t s a n d o c e a n s ,w h i c h i s w h e r e t h e m a j o r i t y o f th e w o r l d ' s d o c u m e n t e db l o w - o u t s h a v e o c c u r r e d . T a n k e r a c c i d e n t s h a v e b e e nt h e m o s t f r e q u e n t c a u s e o f o i l sp i ll s, r e s u l t in g i n p o l l u t i o ni n c o a s t a l r e g io n s . K n o w l e d g e a b o u t t h e e f fe c t s o f o ilp o l l u t i o n o n v e g e t a t i o n h a s , t h e r e f o r e , b e e n l a r g e l yb a s e d o n a q u a t i c b i o c o e n o s e s , s u c h a s s a l t m a r s h e s( B a k e r e t a l . , 1 9 9 3 ) , m a n g r o v e f o r e s t s ( W a r d r o p e t a l . ,1987; T ea s e t a l ., 1989 ; G ar r i t y e t a l. , 1993) o r o th e r


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38 G. Nicolotti , S. Egli/Environmental Pollution 99 (1998) 37-43aqua t ic o rgan isms (Os tgaard e t a l . , 1987 ) . Jenk ins e t a l .( 1978) , how ever , r ep o r t on th e e f f ec t s o f l a rge , ho tc rude-o i l sp i l l s upon a b lack sp ruce (Picea mariana) si te .Inves t iga t ions in to the e f f ec t s o f c rude o i l on p la n tsh a v e c o n c e n t r a t e d o n t h e d i r e c t t o x i c e ff e c ts o f oi l o nt h e u p p e r p a r t s o f p l a n t s , a n d a c o m p l e t e o v e r v i e w o fthese i s g iven by Baker (1970) . However , ve ry l i t t l e i sk n o w n a b o u t t h e e f fe c t s o f o i l o n t h e s o i l a n d i t s m i c r o -o rgan isms , such as myco r rh iz a l fung i , and the r esu l t ingind i r ec t e f fec t s on p lan ts . I n a dd i t ion , owin g to the g rea tv a r i e t y a n d c o m p l e x i t y o f t h e b i o l o g i c a l s y s te m s a f f e c t e db y t h e r e l e a s e o f c r u d e o i l i n t o t h e e n v i r o n m e n t , i n v es -t iga t ions in to i t s e f f ec t s have u s ua l ly been con f ined tol a b o r a t o r y t e s t s t h a t h a v e f o c u s e d o n ju s t a f e w o f t h em a n y c h e m i ca l c o m p o u n d s t h a t m a k e u p c r u d e o il .T h e d e g r a d a t i o n r a t e o f h y d r o c a r b o n s i s d e t e r m i n e db y t h e c o m p l e x i t y o f t h ei r m o l e c u l e s. L i n e a r c o m p o u n d su p t o C 3 0 , f o r e x a m p l e , m a y d i s a p p e a r i n a m o n t h ,w h e r e a s t h o s e u p t o C 3 7 n e e d a t l e a s t 2 00 d a y s . I n d e e d ,the en t i r e t a r f r ac t ion can per s i s t fo r 4 year s o r longer int h e n o r t h e r n t e m p e r a t e z o n e ( G u d i n , 1 97 8) .T h e C / N r a t i o t e n d s t o f a v o u r c a r b o n i n o i l-c o n t a m i n a t e d s o il s . T h i s p r o m o t e s in t e n s iv e m e t a b o l i ca c t i v i ty o n t h e p a r t o f a ll t h e m i c r o - o r g a n i s m s n o t i n h ib -i t e d d i r e c tl y b y t h e o i l . T h e s e v e r it y o f t h e d a m a g e c a u s e dt o t h e v e g e t a t i o n b y t h e r u p t u r e o f th e l o n g h y d r o c a r b o nc h a i n s a n d b y t h e m o b i l i s a t i o n o f al l th e n i t r o g e n p r e s e n ti n c r ea s e s a s t h e a m o u n t o f n i tr o g e n a v a i l a b l e d e c re a s e s .O n l y n i t r o g e n -f i x in g p l a n t s c a n s u r v iv e u n d e r t h e s e c o n -d i t ions . The r esu l t i s a de acto n a t u r a l s e l e c ti o n o f c r u d e -to le r an t spec ies (An ton iewsk i a nd Schaefe r , 1972) . Ox i -d a t i v e d e g r a d a t i o n a l s o c h a ng e s t h e c o m p o s i t i o n o f th es o i l b a c t e r i a p o p u l a t i o n , s o t h a t a e r o b i c c e U u l o s o l y t i ca n d p r o t e o l y t i c s p e c ie s d e c r e a s e a n d a n a e r o b i c n i t ro g e n -f ix ing spec ies increase (And er son e t a l . , 1993 ).S imi la r e f f ec t s , namely a d r as t ic r educ t ion in thee c t o m y c o r r h i z a l b i o m a s s a n d i n i t s i n f e c t i o n p o t e n t i a l ,h a v e b e e n n o t e d i n t h e a e r o b i c m i c r o f l o r a o f t h e A r c t i ctund ra (Mi l le r e t a l . , 1978 ) , whereas some fung i appearto be a lmos t fu l ly r es i s tan t to gas o i l . I ndeed , Tricho-derma harz ianum ac tua l ly benef i t s f rom i t s p r esence(Gud in and Chate r , 1977 ) .

T h i s p a p e r d e s c r i b e s a s t u d y o f t h e e f fe c t s o f c r u d e o i lo n t h e m y c o r r h i z o s p h e r e a n d t h e a b i l i t y o f 1 0 d i f fe r e n te c t o m y c o r r h i z a l f u n g i t o r e c o l o n i s e N o r w a y s p r u c e(Picea abies) a n d b l a c k p o p l a r (Populus nigra) seed l ingsg r o w n o n c o n t a m i n a t e d s o i l s .

2 . M a t e r i a l s and m e t ho d s

2.1. In v i t ro growth tests with pure cu ltures ofectomycorrhizal fun giT h e r e a c t i o n t o c r u d e o i l o f 1 0 e c t o m y c o r r h i z a l f u n g i(Amanita pantherina, Amphinema bissoides, Armillaria

mellea, Ceno coccu m geop hilum, Hebeloma crustul ini-forme, Laccaria amethystea, L. bicolor, L. laccata, Piso-lithus tinctorius, Tricholoma vaccinum) w a s t e s t e d b yin ser t ing agar d i sks wi th the i r pu re cu l tu r es in Pe t r id i shes con ta in ing e i ther no c rude o i l o r s ix d i f f e r en tconc en t r a t ion s o f o i l ( 0.1, 1 , 5 , 10, 20 o r 50g kg- I ) . Theo i l was added j u s t befo re the d i shes were f i l l ed wi thn e a r l y s o l i d M M N - a g a r ( p r e p a r e d a c c o r d i n g t o M a r xand Bryan , 1971 ) to p r even t the o i l f rom r i s ing to thesu r face and to ensu re tha t i t was wel l -d i s t r ibu ted wi th int h e m e d i u m . T h e P e t r i d i s h e s w e r e k e p t i n t h e d a r k a t1 8 C. F u n g u s g r o w t h w a s m e a s u r e d w e e k l y f o r a m a x i -m u m o f 3 5 d a y s o r u n t i l t h e e d g e o f t h e d i s h w a sreached . Ten r ep l ica tes were taken fo r each funga l spe-c i es a n d e a c h c o n c e n t r a t i o n .2.2. Biotest w ith contaminated soil

S o i l c o l l e c te d a t a d e p t h o f 0 3 0 c m f r o m t h e T r e c a t ea r e a ( N o v a r a , I t a l y ) a n d a t a d e p t h o f 0 - 1 0 c m f r o m as p r u c e s t a n d i n S c h w a n d e n ( C a n t o n G l a r u s , S w i t z e r -l a n d ) w a s p a s s e d t h r o u g h a 2 - r a m s i e v e , h o m o g e n i s e dw i t h a g a r d e n i n g m i x e r a n d t h e n c o n t a m i n a t e d w i t hc r u d e o i l f r o m t h e A G I P O i l s C e n t r e T r e c a t e b y h a n ds p r a y i n g a n d c o n t i n u o u s m i x in g i n u n t il t h e a p p r o p r i a t econc en t r a t io n leve l (0 .1 , 1 , 5, 10 , 20 o r 50 g kg -1 ) wasr e a c h e d . T h e c h e m i c a l c o m p o s i t i o n o f t h e c r u d e o i l i sd e s c r i b e d i n T a b l e 1 . A n u n c o n t a m i n a t e d c o n t r o l w a sse t up fo r bo th so i l s . The so i l was then p laced in to' S p e n c e r L e M a i r e ' p l a s t ic r o o t r a i n e r s d i v i d e d i n t o 3 2p o t s m e a s u r in g 3 . 5 3 . 5 x l l c m ( 1 3 5 c m 3 s oi l p e r p o t )a n d b e d d e d d o w n b y w a t e ri n g.S e e d s o f b l a c k p o p l a r (Populus nigra) f r o m t h e s e e db a n k o f t h e C a s a le M o n f e r r a t o E x p e r i m e n t a l P o p l a rR e s e a r c h S t a ti o n a n d N o r w a y s p ru c e (Pieea abies) seedsf r o m W S L s e e d b a n k w e r e s u r f a c e- s te r i li s e d f o r 2 r a in( p o p l a r ) o r 3 0 m i n ( s p r u c e ) i n 3 0 % H 2 0 2 . T h e n 1 0p o p l a r s e e d s w e r e s o w n i n e a c h p o t o n t h e T r e c a t e s o i la n d 5 s p ru c e s e e d s o n t h e S c h w a n d e n s o il . T h e n u m b e rT a bl e 1C om pos i t i on o f t h e c rude o i l o f T re c a t e (AG IP , 1995)W a t e r 0 .10 %H2S 0 .01%H y d r o c a r b o n s 8 8 %G a se ous f r a c t i on 11% (4% me t h a ne ,

7 % C 2 - C 4 c o m p o u n d s )L i q u i d f r a c t i on 77%Pol y c y c li c Aro ma t i c H y droc a rb ons 8%Sul fur o rg a n i c c omp ounds 0 .16 %Light fue l 6 .5 %T ot a l fue l 32 .5 %K e rose ne 20 .0 %Diese l fue l 11.0%Lub rica t ing oi l s 16 .5 %Residue 18 %L oss 2%


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G. Nico lotti, S. Egli/Environmental Pollution 99 (199 8) 37-4 3 39of the seedlings was reduced later to 3 per pot. Seedingwas performed at four different times: Time 0 (momentof oil contamination), Time I (7 days after contamina-tion), Time II (21 days) and Time III (42 days). Eightreplicates (pots) were performed for each treatment andplaced in a greenhouse with the temperature maintainedat 20C with a relative humidity of 80% in daylight.Germination percentages were determined 7 days and14 days after sowing. After 4 months, the height and dryweight of the seedling shoots were measured. The root-lets were washed carefully and fixed in 6% formal in formicroscopic examination. On spruce roots, ectomycor-rhizal morphotypes were determined and quantified.Poplar roots were stained according to Rajapakse andMiller (1991) to assess the percentage of ectomycor-rhizal tips and the percentage of roots with arbuscularmycorrhizas. Student's two-sample t-test was used toevaluate the effects of each treatment.

3. Results3.1. In vitro g r o w t h t e s ts o f p u r e c u l tu r e s o fe c t o m y c o r r h i z a l f u n g i

Three types of reaction were noted: (1) the growth ofA . p a n t h e r i n a , T . v a c c i n u m and P . t i nc t or i us was reducedby increasing crude-oil concent rations, and 50g kg -1was lethal for A . p a n t h e r i n a and T . vacc i num (Fig. 1);(2) the growth of L . b i co l or , L . ame t hys t ea and L. lac-c a t a increased with oil concentrations between 1 and20 g kg - 1, and decreased slightly at 50 g kg -1 (but it wasstill more than the control up to 5g kg-l); (3) thegrowth o f the other four species was unaffected and they

displayed no conspicuous response to the presence ofcrude oil.3 . 2 . B i o t e s t w i t h c o n t a m i n a t e d so i l3 .2 .1 . P op l arThe germination percentages of the poplar seeds wererather low, even in the control, and their great vari-ability made it impossible to establish any statisticallysignificant differences. Seedling shoot lengths wereseverely affected by concentrat ions higher than 5 g kg- Iand growth was greatly reduced at all seeding times bythose concentrat ions higher than 10 g kg -l . Significantgrowth and shoot biomass (dry weight) differencesbetween Time 0 and Time III were found only forconcentrat ions up to 1 g kg -1 (Table 2). At lower con-centrations, growth was actually stimulated, especiallybetween Time 0 and Time I.

None of the oil concentrations had any effect on thedegree of ectomycorrhizal and endomycorrhizal infec-tion, but the length of time between contamination andseeding did make a difference. The percentages of ecto-mycorrhizal colonisat ion decreased between Time 0 andTime III, and those of endomycorrhizal colonisationincreased (Fig. 2).3 . 2 .2 . N o r w a y s p ru c e

The germination rate of Norway spruce seeds wasreduced significantly only at the highest oil concentra-tion (50g kg-1). At lower concentrations there was noeffect. Seedling growth was not influenced by the oilconcentration (Table 2), but it was by the seeding time.Shoot length and biomass were significantly reducedwhen seeded 42 days after contamination (Time III)

7 0 L a c c a r i a a m e t h y s t e a- - - - - 0 L a c c a r i a b i c o l o r

, ~, L a c c a r i a l a c c a t a- - - - P i s o l i t h u s t i n c t o r i u s- - " ~ - - A m a n i t a p a n t h e r i n a- - - dk - - T r i c h o l o m a v a c c i n u m

5 0

40v

E 3 0

60 I .

20 ~ - - - . " " . . . . - I

1 0 " . " , , . . . A . . . . ~ . . . . . . ~ . . . . . . @ . .

0 ,, " ' ' A . . . . . . ~ . . . . . . . . . . . . . L _ _ ' . ' . ' . .c o n t r o l O . 1 1 5 1 0 2 0 5 0

o i l c o n c e n t r a t i o n ( g / k g )Fig. 1. In vitro growth of some ectomycorrhizal ungi at differentconcentrationsof crude oil (35 days after inoculation).


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40 G. Nicolotti, S. Egli/Environmental Pollution 99 (1998) 37-43Table 2Growth response of poplar and spruce seedlings to crude-oil concentration and time of sowing after contamination

Time of so wi ng Control Crude oil concentration (g kg 1 soil)0.1 1 5 10 20 50

PoplarShoot length (cm)

Shoot biomass (mgplant 1)

SpruceShoot length (cm)

Shoot biomass (mg plant I)

Time 0 6.1 6.0 7.2 5.7 2.4** 1.7"* 2.0**Time I 5.2 6.9** 7.1 * 4.8 2.3** 1.7* 1.7"*Time II 5.3 4.6 5.0 4.7 2.5** 2.0** 2.2**Time III 3.7 4.1 4.7* 4.6 3.1 2.1"* 2.4Time II1-Time 0 -2.4** -1.9"* -2.5** -1.1 0.6 0.4* 0.4Time 0 41.4 40.1 61.9" 40.2 8.3** 4.6** 4.6**Time I 29.5 48.3** 46.6* 24.6** 7.1"* 4.7** 3.9**Time II 31.9 26.6 36.2 35.8 8.6** 4.1"* 4.4**Time II I 15.9 23.8** 24.8** 30.3 14.8 6.2** 3.3**Time Ill -T ime 0 -25.5** -16.3"* -37 .1'* -9.9 6.5* 1.6" -1.3"

Time 0 3.1 3.3 3.2 3.2 3.3 3.3 2.9Time I 3.8 2.8 ** 3.2 3.2 3.3 3.3 3.1"Time II 2.7 2.4 2.4 2.5 2.4* 2.4 2.3Time III 2.4 2.1 2.1 2.6 2.4 2.2 2. 5Time III -Tim e 0 -0.7** -1.2"* -1.1"* -0.6** -0.9** -1.1"* -0.4**Time 0 15.5 16.3 18.1 12.4 19.5" 18.5 9.1"*Time I 13.9 15.9 15.7 14.2 17.3 17.2 9.2Time II 13.7 13.3 14.7 14.0 15.1 15.0 8.7**Time II1 10.1 8.7 8.3 12.1 11.0 9.4 8.0Time III -Ti me 0 -5.4* -4.6** -9.8** -0.3 -8.5** -9.1"* -1.1

Means of 8 (biomass) or 24 (length) replicates. Significant differences (t-test) between concentrations and the control and between Time 0 and TimeIII are indicated by *p < 0.05 and **p < 0.01.

compared with when they were seeded at the t ime ofconta minat io n (Time 0).Plants sown at Ti me 0 were extensively colonised byectomycorr hizal fungi (Fig. 3). More th an 90% of theroot t ips were mycorrhizal . Wi th increasing lapses oft ime between conta mina t ion and seeding, this percen-

endo mycorrh iza l in fec t ion (% co lon izat ion)

tage went down to 60% (Time III). Six morphot ypes (C,D, E, F, G, H) were dis t inguished and describedaccording to their macroscopic an d microscopic features(Table 3). Types C an d E were identified as C . g e o p h i l u mand L a c c a r i a sp., respectively. Types C and D becameless frequent with increasing oi l concentrat ion. Type D

ectomycorrhizal infect ion (% colon ized roo t Ups)30 80

1"10 702 5 [ ] I 6 0

[ ] I I ]20 I1 5 4 0

301020

5 100 O ,

control 0.1 1 5 10 20 50 control 0.1 1 5c r u d e oil (g/kg)

1 0 2O 50crude oi l (g/kg)Fig. 2. Endo- and ectomycorrhizal colonisation of poplar roots.


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G. Nicolotti, S. Egli/Environmental Pollution 99 (1998) 3 7-4 3 41ec tom ycor rh i za l i n fec t i on (% co lon i zed root t i ps ) root t i ps (%)10 0

; 0 o

0 " " : :

control 0.1 1 5 10 20 50 control 0.1 1 5 10 20 50c rude o i l (g /kg) c rude o il (g/kg)

root t i ps (%) root t i ps (%)4 20

3 . 5 r l 0 C 1 8 r l 0 DD I 1 6 D I

3 [ ]11 14 rol l2 . 5 I I I I I 12 I l l l l1.5 8

6

O , O , , m ,contro l 0.1 1 5 10 20 50 control 0.1 1 5 10 20 50

c rude o i l (g /kg) c rude o i l (g /kg)

root t ips (% )30252 01510

50

I"10

l a l lI I I I I

con trol 0.1 1 5

E

1 0 20 50c rude o i l (g /kg)

root t ips (%)605O403 0 -2 0 -1 0 -

0cont ro l

rl0r'llgillmill

G

jil i0. 1 1 5 10 20 50

c rude o i l (g /kg)Fig. 3. Occurrenceof ectomycorrhizal morphotypes on spruce (M: ectomycorrhizas;PM: pseudomycorrhizas;C, D, E, G: morphotypes).

was the most sensitive: its frequency was reduced at allconcentrations compared with the control, types E andG were apparentl y favo ured by the presence of crudeoil: type G was only present at 5 gk g -1 and above,whereas type E displayed an optimum at intermediateconcentrations.

The percentage of pseudomycorrhizas did not seem tobe influenced by the presence of oil. Types F and H were

not present in sufficient quantities to enable any con-clusions to be drawn.

4 . Di scuss ion

Crude oil in the concentrations applied in this studyneither affects seed germination nor kills seedlings. It


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42 G. Nicolotti, S. Egli/Environmental Pollution 99 (1998) 37-43Table 3Ma cro- and microscopic descriptions of the ectom ycorrhizalmorpho types on spruce seedlings sown on the S chwanden soil (Rhiz= Rhizomorphs)Type Macroscopic features Microscopic features Remarks

Colour Mantle surface Rh iz Ramification Mantle structureC Bla ck Smooth to grainy - Simple,o f ten s i ng l e P l ec t ench y m at ous , Cenococcumstar-l ike arrangem ent geophilumD Yellow-bro wn Gelatinous - Simple PlectenchymatousE O p a q u ewhite to grey Sm oo th, i lvery,mat + Monopodial -pinnate Plec tench ym atou s Laccaria-liketo beigeF Da rk-br ow n o black No distinctiveman tle - Simple,of ten s ing le M an t le ackingG Red -brow n No distinctivemantle, - Simple Mantle la c k in g Pseudomycorrhiza-l ike,loose hyphae but thickenedH W hite Cottony, with emanating - Monopodial-pinnate Ple cte nc hym ato us Hebeloma-likehyphae

c a n , h o w e v e r , r e d u c e s e e d l i n g g r o w t h , a l t h o u g h t h i sv a r i e s a c c o r d i n g t o p l a n t s p e c ie s . I n t h e l i g h t o f t h er e s u lt s o f a n e a r l i e r s tu d y o n t h e s a m e s u b j e c t ( B a r o l o ,1 9 9 6 ) , i t s e e m s l i k e l y t h a t c r u d e o i l h a s a c a u s t i c o rl e t ha l e f f e ct o n l y w h e n i t c o m e s i n t o d i r e c t c o n t a c t w i t ht h e t i ss u e s o f a p l a n t , a s h a p p e n s w h e n a n o i l s l ic k f r o ma t a n k e r a c c i d e n t r e a c h e s t h e c o a s t . C r u d e o i l ' s i n d i r e c te f f e c t o n t h e s o i l i s c o n f i n e d t o a m o r e o r l e s s m a r k e dr e d u c t i o n i n p l a n t g r o w t h a n d b i o m a s s , p o s s i b l y a t t r i b -u t a b l e t o t h e c h a n g e s b r o u g h t a b o u t i n t h e s o i l m i c r o b ep o p u l a t i o n s . O n t h e o t h e r h a n d , t h e g r o w t h o f c e r t a i np l a n t s , s u c h a s Festuca rubra a n d Puccinellia maritima( B a k e r , 1 9 7 1 ) o r Trifolium rubra ( K l o k k , 1 9 8 4 ) , c a n a l s ob e s t i m u l a t e d b y c r u d e o i l . T h e m e c h a n i s m s f o r t h i sp h e n o m e n o n a r e u n c le a r , b u t g r o w t h s t i m u la t io n c o u l db e t h e r e s u l t o f t h e d i r e c t r e le a s e o f n u t r i e n t s o r g r o w t h -r e g u l a t i n g c o m p o u n d s f r o m t h e o i l o r t o t h e r e l e a s e o fn u t r i e n t s f r o m a n i m a l s o r b a c t e r i a k i l l e d b y t h e o i l( Bak e r , 1971) .T h e r e s u l t s o f t hi s s t u d y r e v e a l a v e r y h i g h v a r i a b i l i t yi n t h e d a t a , e s p e c i a l l y i n th e m y c o r r h i z a l d a t a . T h o s ew o r k i n g i n m y c o r r h i z a r e s e a r c h a r e q u i t e f a m i l i a r w i t ht h e p r o b l e m o f t h e l ac k o f h o m o g e n e i t y in m y c o r r h i z a ld a t a , b o t h i n f ie l d s t u d i e s a n d i n b i o a s s a y s . T h i s m a k e st h e i n t e r p r e t a t i o n o f s u c h d a t a m o r e d i f f i c u lt , b u t i t i ss ti ll p o s si b l e t o d r a w s o m e c o n c l u s i o n s f r o m t h e p r e s e n ts t u d y .

O n e s t r i k i n g f i n d i n g is t h a t s e e d l i n g g r o w t h i s r e d u c e dt h e l o n g e r th e t i m e b e t w e e n c o n t a m i n a t i o n a n d s e e di n g .T h e r e v e r se w a s t o b e e x p e c t e d s i n c e s o il r e c o v e r s w i tht i m e a n d t h e c o n c e n t r a t i o n s o f p h y t o t o x i c v o l a t il e c o m -p o u n d s d e c r e a s e . O n e e x p l a n a t i o n c o u l d b e t h a t s e e d -l i n g g r o w t h i s a f f e c t e d b y m y c o r r h i z a l c o l o n i s a t i o n ,w h i c h r e a c t s c o n v e r s e l y . T h e e c t o m y c o r r h i z a l i n f e c ti o np o t e n t i a l o n s p r u c e a n d p o p l a r i s h i g h e s t i m m e d i a t e l ya f t e r c o n t a m i n a t i o n a n d d e c r e a s e s o v e r t im e . I t i s li k el yt h a t t h e i n c r e a s e d a c t i v it y o f e c t o m y c o r r h i z a l f u n g i j u s ta f t e r o i l c o n t a m i n a t i o n c o n t r i b u t e s t o t h e s e e d l i n g s 'b e t t e r g r o w t h .

T h e d e c r e a s e in t h e e c t o m y c o r r h i z a l i n f e c ti o n p o t e n -t i a l w i t h t i m e m a y b e d u e t o t h e d i r e c t e f f e c t o f t h e o i l

o n t h e h y p h a e o f s o m e m o r e s e n s i ti v e e c t o m y c o r r h i z a ls p ec ie s . O n t h e o t h e r h a n d , t h e e n d o m y c o r r h i z a l i n fe c -t i o n p o t e n t i a l o n p o p l a r i s h i g h e s t 4 2 d a y s a f t e r c o n -t a m i n a t i o n . T h i s c o u l d b e a t t r i b u t e d t o t h e f a c t t h a te n d o m y c o r r h i z a l fu n g i a r e e n d o w e d w i t h s u rv i v a ls p o r e s a n d a r e , t h e r e f o r e , m o r e r e s i s t a n t t o e n v i r o n -m e n t a l s t re s s f ac t o r s . W h e n t h e o il c o n t a m i n a t i o n h a sd e c r e a s e d s u ff i ci e n tl y o v e r t i m e t h r o u g h b i o d e g r a d a t i o n ,i t i s p o s s i b l e f o r n e w m y c e l i u m g e n e r a t e d b y g e r m i n a -t i o n o f su r v i v i n g s p o r e s t o d e v e l o p i n t h e s o i l a n d c o l o -n i s e p l a n t r o o t s .

S i n c e r a p i d a n d c o m p l e t e c o l o n i s a t i o n i s e s s e n t ia l f o rs u r v i v a l in t h e e a r l y s ta g e s o f p l a n t d e v e l o p m e n t , t h ed i f fe r e n t r e a c t i o n s o f e c t o - a n d e n d o m y c o r r h i z a l f u n git o o i l c o n t a m i n a t i o n c o u l d h a v e c o n s i d e r a b l e i n f l u e n c eo n t h e s u c c es s o f r e c o l o n i s a ti o n . R e c o l o n i s a t i o n w i t he c t o t r o p h i c p l a n t s c o u l d b e d i f f i cu l t a s i t w o u l d r e q u i r et h e m t o b e s e t o u t i m m e d i a t e l y a f t e r so i l c o n t a m i n a t i o nt o e n s u r e t h a t t h e y f i n d t h e i r s y m b i o n t s , s i n c e t h e s ec a n n o t s u r v i v e l o n g i n o i l - c o n t a m i n a t e d s o i l . T h i s ,h o w e v e r , i s i m p r a c t i c a b l e b e c a u s e r e c l a m a t i o n w o r kw h i c h d i s t u r b s t h e s o i l i s a l w a y s n e c e s s a r y a f t e r a b l o w -o u t . E n d o t r o p h i c p l a n t s , o n t h e o t h e r h a n d , c a n b e s e to u t m u c h l a t e r s i n c e t h e i r r e s i s t a n t s p o r e s c a n s u r v i v e i nt h e c o n t a m i n a t e d s o il .

T h e in vitro g r o w t h t e s t s h o w e d a s p e c i e s - s p e c i f i cr e s p o n s e . S o m e f u n g a l s p e c i e s a r e in h i b i t e d b y o i l, w h i leo t h e r s a c t u a l l y g r o w b e t t e r i n o i l - c o n t a m i n a t e d s o i l ,e v e n a t v e r y h i g h c o n c e n t r a t i o n s (Laccaria sp . ) . Ye to t h e r s d i s p l a y n o d e f i n i t e r e a c t i o n . A s i m i l a r b e h a v i o u rp a t t e r n w a s e v i d e n t i n t h e b i o t e s t : t y p e G f u n g i o n l yf o r m e c t o m y c o r r h i z a s a t v e r y h i g h c o n c e n t r a t i o n s ; t y p eE p r e f e r i n t e r m e d i a t e c o n c e n t r a t i o n s o f o il ; o t h e r t y p e ss e e m t o b e u n i n f lu e n c e d .T h e b e s t c o l o n i s a t i o n r a t e f o r t y p e E w a s a t i n t e r -m e d i a t e o i l c o n c e n t r a t i o n s , w h i c h c o r r e s p o n d s e x a c t l yt o t h e g r o w t h r e s p o n s e o f t h e t h r e e Laccaria spec i e s i np u r e c u l t u r e , i n d i c a ti n g t h a t t y p e E p r o b a b l y b e l o n g s t ot h is g e n u s. A c o m p a r i s o n o f t h e m o r p h o l o g y a n d a n a t -o m y o f t y p e E w i t h t h o s e o f th e Laccaria m y c o r r h i z a s asd e s c r i b e d i n t h e l i t e r a t u r e ( A g e r e r , 1 9 9 5 ) r e v e a l e d a v e r y


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G, N ic o lo t t i , S . E g l i /E n v i ro n me n ta l P o l lu ti o n 9 9 ( 1 9 9 8 ) 3 7 - 4 3 43close resemblance in that both had a plectenchymatousmantle structure and smooth, undifferentiated rhizoomorphs. From this it can be concluded that the prob-ability is very high that morphotype E represents aLaccaria species.The findings of this study suggest that a crude oilblow-out in a mixed agricultural and forest area doesnot cause long-term environmental damage of the kindassociated, for example, with coastal ecosystems. Thiscould be due to bioremediation on the part of themicroflora in the soil, as already reported for certainbacteria (Miller et al., 1978) and fungi (Barr and Aust,1994). Our results support this hypothesis. Moreover,they show that some of the ectomycorrhizal fungi thatsurvive in contaminated soil may actually use crude oilas a nutrient and thus make their own contribution tothe process of bioremediation.

AcknowledgementsWe wish to thank Ms B. Schneider for sectioning andstaining the ectomycorrhizas and Dr. D. Valentino forstaining and observing the endomycorrhizas.

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Hoc k, B. (Ed.). M ycorrh iza : St ruc ture , Fun ct ion, M olecu lar Biol -ogy and Biotechnology. Springer , Berl in, pp. 685-734.AG IP , 1995. B l ow -out T re c a t e 24 . P ia n i d i bon i f i c a e d i mon i t o ra g g i o .Ande rson , T .A. , G u t h r i e , E .A . , W a l t on , B .T . , 1993 . B i ore me di a t ion i nt h e rh i z osphe re . P l a n t ro o t s a nd a s soc i a te d mi c robe s c l e a n c on t a m-ina ted soi l. Env i ronm enta l Sc ience and Techn ology 27(13), 2630 -2636.Antoniewski , J . , Schaefer , R . , 1972. Recherches sur l es reac t ions descoenoses microbiennes de sot s impregn~s par des hydrocarbures.Mo di fica t ion de l ' ac t ivi t~ respi ra toi re . Ann ales de l ' Inst i tut Pasteur123, 805--819.Baker, J .M., 1970. The effec t s of oi l s on plants . Envi ronmenta l Pol lu-t ion 1, 27-44.B a k e r , J .M. , 1971 . G row t h s t i mul a t i on fo l l ow i ng o i l po l l u t i on . In :

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propert i es . M ethods in Micro biolog y 24, 301-316.T e a s , H . J . , L a sda y , A .H . , L uq ue , E . , M ora l e s , R .A. , D e D i e g o , M.E . ,Baker, J .M ., 1989. Mangr ove restora t ion afte r the 1986 RefineriaPana ma oi l spi ll . In: Proceedings, 19 89 Oi l Spi ll Conference ,Am e r i c a n Pe t ro l e um Ins t i tu t e , W a sh i ng t on D C , pp . 433-437 .

Wa rdro p, J .A . , But t l e r , A.J . , Johnso n, J .E , , 1987. A field s tudy of thet ox i c i t y o f t w o o i l s a nd a d i spe r sa n t t o t h e ma ng rove Avicen iamarina. M arine Biolo gy 96 , 151-156.

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