- Email: [email protected]
Influence of acid-producing agrobacterium and Laccaria laccata on pine and beech growth, nutrient uptake and exudation
313
Agriculture, Ecosystems and Environment, 28 (1989) 313-319 Elsevier Science Publishers B.V., Amsterda~ Printed i n Czechoslovakia
INFLUENCE OF ACID-PRODUCING A G R O B A C T E R I U M A N D LACCARIA L A C C A T A ON PINE .~ND BEECH GROWTH, NUTRIENT U P T A K E A N D EXUDATION
LEYVAL, C. and BERTHELIN, J.
Centre de l~dologie Biologique- C.N.R.S. B.P.5, 17. rue N,D. des Pauvres. 54500 Vandoeuvre-les-Nancy, FRANCE
A b s t r a c t - Two scid-producmg bscteria ( ~,ro~uu'~rJ.m n ~ b b ~ ' ~ r a n d Agrobscterium sp )
and/or an ectomycorrhizal fungi ( L~ccJri~ lacca~ ) vero iaoculsted in the rhizospheros of beech and pi-e seediinEs,in lysimetricdevices in • g~nhouse. Plants were grown on sand mixed with rock phosphate and phlogopite (iron-magnesian mica ) Influence of these microorganisms on plant growth, plant nutrition and plant exudation was observed after two years. A~ro~cterium r~'o~nc~r, or l~cci~ri&lacca~ root inoculation incrossed beech growth and nutrientuptake ( P. Cs, K. Fe. M8 } by beech seediia4|s.But dual inoculationby both micro-orgshi•ms had no significanteffect on plant growth sad plant nutrition With pine se~lliags,the only growth and nutrient uptake promoting effect was observed with Agrohacterium sp inoculation and concerned only the root system. Bacterial inoculation increased the amount of organic scids ( malic, lactic, fumsric, citric ) released in the rh/zosphere, which promoted mineral element solubiIization and, thus, plant mineral nutrition. Mycorrhizal plan~ did not released greater amounts of organic •cids than non mycorrhizal plants. The effect of Laccaria/accata on beech growth and nutrition could be attributed to a better soil exploration, as shown by the increased rh/zospheric volume, Introduction Mycorrhizal plantsusually ttke up more phosphorous than non mycorrhizal plants.The abilityof"ectomycorrhizal fungi to solubilizeinsolublephospha~s in non symbiotic conditions,as
314
many soilbacteriado, has been shown ( Leyval and Berthelin, 1986 .),Some works on the:interactions between mycorrhizal fungi and other soilmicroorganisms have been reported by Leyval (198~) and Leyval and Berthelin (submitted) but few of them concerned the combined effectsof ectomycorrhizal fungi and phosphate-dissolving bacteria in the rhizosphere The aim of this study, was to observe these effects, in pine and beech rhizospheres, on the solubilizationof phosphorous from rock phosphate but also from mineral elements of a mica {phlogopite ).These minerals were the only sources of elements for plant nutrition and, thus, their solubilization influenced plant growth. Materials a n d methods Six month-old beech ( Fagussyiva~'c#L ) and pine ( PinussylwstrisL, ) seedlingswore grown in PVC cylinders in a greenhouse for two years ( Leyval, 1988. in press ). The cylinders were filledwith sand mixed with rock phosphate and phlogopite ( iron-magnesian mica ),Plants were automatically fed with a nutrient solution devoid of soluble P, Fe. Mg, K and .Ca, which were brought as insoluble forms by the two minerals. Plants were inoculated five times with an ectomycorrhizal fun gi, Laccaria lacca~, with a phosphate-dissolvingbacteriaisolatedfrom their rhizospheres, or with both microorganisms. Beech seedlings were inoculated with an
Agrobacterium rad~'obacterand pine ones with an Agrobacterium sp.. Control plants were maintained non inoculated. There were ten replicates in each treatment, After two years of experiment, mycorrhizal infection rate was evaluated and bacteria number in the rhizospheres was counted ( Leyval, 1988 ) Dry weight and mineral element amounts of plantswere determined. Rhizospheric soil ( ie adhering to the rootsafter being shaken by hand ) was evaluated ( Leyval, ~n press ) and rinsed in distilledwater for organic acid content analysis ( HPLC, Aminex HPX-$7 H Column )
Results a n d discussion The inoculation of beech roots with Agrobacterium radiobacter on one hand, and with
Laccarin/accataon the other hand, significantlyincreased shoot and root growth ( Fig. I ), It also significantlypromoted P. K, Ca, Fe and M g uptake by plant ( Tabl ) But dual inoculationwith both microorganisms did not significantlymodify beech growth and mineral element uptake.
315
Agrobacterium sp significantly promoted pine growth and phosphorous uptake from rock phosphate, but these effects only concerned the root system, The increase in K, Ca and M g amounts after bacterial inoculation were not significant at p< 0.05 Laccari& l a c c ~ inoculation and dual inoculation with both micro-organisms had no significanteffect,in this experiment, on pine growth and mineral element amounts (Fig.l,Tab.l ) The effect of the two Agrobactet;ium on pine and beech nutrient uptake ( especially phosphorous uptake ) could be relatedto the release of organic acids in the rhizosphere. Figure2 shows that the rhizospttere of plants inoculated with the bacteria contained greater amounts of organic acids The organic acids detected were also qualitativelydifferent: mslic sud lacticacids appeared in beech rhizosphere and citricand fumaric acid in pine rhizosphere. These increased organic acid quantitiesmight have contributed to increase phosphorous solubilizationfrom rock phosphate and g, Fe, M g solubilizationfrom phlogopite. For mycorrhizal plants, the amount of organic acids in plant rhizosphere was not increased, Citric acid, which is very complexing, increased in beech rh~zosphere in comparison to non mycorrhizal beeches ( Leyval, 1988 ),and lacticacid appeared But the total amount of organic acids was not modified, In that case, the significant increase of the rhizospheric soil ( Tab,2 ), which suggests a bettor soilexploration, could explain l..¢ccaJ-ialacc#~aeffect on beech growth and nutrition In mycorrhizal pine rhizosphere, and in beech rhizosphere inoculated with both micro-organisms, no more organic acidswere detected ( Fig2 ),which could explain that P, K, Fe Ca and M g mobil~.ations were not increased. However, for mycorrhiza.l pines, rhizospheric soil percentage was higher than for non mycorrhizal pines ( Tab2 ), This results point out the importance of rhizospheric microorganisms and their interactions m
plant growth and nutrition. Thus. acid-producing micro-organisms
can increase the
availability of soilmineral elements for plant nutrition, and mycorrrhizal fungi can increase
316
soil exploration ( H a y m a n
and Mosse, 1972 ) But, if in some works reviewed by Leyval and
Berthelin ! submitted ) synergistic effects of microorganisms
were
observed, competition
between them m a y a/se occur, as the results of this experiment suggest it, and then, no effect on plant growth can be observed
Mineral element uptake
K
P
Ca
Fe
Beech non inoculated - bacteria + mycocrhiza - bacteria + mycorrhiza
5.97 a 8.82 b 8.51 b 3.84 a
5.95 a I0.26 b 10.04 b 4.56 a
11.53 a 20,09 b 19.83 b 8.59 a
Pine non inoculat~ + bacteria + mycorrhiza +bacteriaomycorrhiza
12.81 a
9.52 a
27.97 a
8.53 a
6.25 a
14.45 a 11.691 I0.$6a
11.20 b 8.061 ?.?la
30.91 a 19.991 21.32a
?,02 l 9.Sqa ll.31a
?.02 a 5.751 ?.24a
1.19 a 3.34 b 4A'~ b 1.69 a
MII
2.29 a '1.49b '1.50b 2.25 a
Tabl: In|luence o| bacterial and ectomycorrhizal inoculations o! pine and beech roots on mineral element u p t U e | tug,plant l Same letters in a same column indicate no l i l n i f i c l m t dlf/erenees at P< 0.05
Beech
Non inoculated + bacteria • mycorrhiza , b a c t ~ i a , mycorrhiza
1.6 a 3.2 a 5.0 b 3.0 a
Pine
5.8 a 5.2 a 9.2 b 11.0 b
Tab.2: Rhizospheric soil percentage ( ~ total soil dry tveigh ) ol pine and beech seedlings aJter 2 years, Same letters in a same column indicate no signi|icant differences at P~ 0.05
317
g
Pine
8 Beech
I[] []
I
roots shoots
I 6 a a
4
a
,/a/
NI
B
M
BM
NI
B
M
BM
Fig 1 Influence of the phosphate-dissolving bacteria ( B ) and/or of the ectomycorrhizal fungi (M) on pine and beech growth ( g dry weight/plant ) Same letters in histograms indicate no
significant differences at P0 05 NI non inocultted.
318
+B
/
29 pg/plant
=
Beech 8.5 ~ / p l e n t f n~i¢ I ~ I +M I ~ +B+M O.61~g/plent 0.0 pg/plont
Plne 49.3p!I/Plant
VN-1
)ni¢
+B
/
+
~ , ~
+B+M
60.2Jlg/plent 0,1~1 I/plent 0.0pg/plent citric fumaric
lactic medic
Fig2 Influence of the phosphate-dissolving bacteria ( .B ) and/or of the ectomycorrhizal fungi (-M) on the organic acid amounts ( Ug/plant ) in pine and beech rhizospheres after two years
319
References H A Y M A N , D S and MOSSE, B,; Plant growth responses to vesiculac-arbuscular mycorrhiza. Ill, Increase uptake of labileP from soil.-New Phytol.71: 41:47. 1972. LEYVAL, C. Lysimeters in a greenhouse as an help to study the interactions between microorganisms, minerals and forest tree roots.-In:"Fieldsmethods in terrestrial ecosystem nutrient cycling", Grange-over-Sand. U.K.. I-5 Dec. 1988. ( in press ). LEYVAL, C :Interactionsbacteries-mycorhizes darts la rhizosph(~re du pin sylvestre et du h6tre Incidences sur l'exsudationracinaire et l'alttrationdes mineraux - Doctoral thesis.Universit* de
Nancy I, 1988 LEYVAL, C and BERTHELIN, J.: Comparison between the utilization of phosphorus from insoluble mineral phosphates by ectomycorrhizal fungi and rhizobacteria.-In: GIANINAZZI-PEARSON,V and GIANINAZZI,S / e d . / : Physiolological and genetical aspects of mycorrhizae. Pp. 340-345.Inra, Paris 19fS6 LEYVAL C. and BERTHELIN,J.: Interactions between Imccaria laccata, Agrobacterium rmiiobacter and beech roots: influence on P, K. M g and Fe mobilization from minerals and plant growth,Plant and Soil ( submitted ).
L e y v a l , C. and B e r t h e l i n ,
J.,
1989t I n f l u e n c e o f a c i d - p r o d u c i n g A g r o b a c t e r l u m
and L a c c a r i a l a c c a t a on p i n e and b e e c h growth, n U t r i e n t uptake and e x u d a t i o n . &grit.,
Ecosystems E n v i r o n . , 28: 313-319,
Agriculture, Ecosystems and Environment, 28 (1989) 313-319 Elsevier Science Publishers B.V., Amsterda~ Printed i n Czechoslovakia
INFLUENCE OF ACID-PRODUCING A G R O B A C T E R I U M A N D LACCARIA L A C C A T A ON PINE .~ND BEECH GROWTH, NUTRIENT U P T A K E A N D EXUDATION
LEYVAL, C. and BERTHELIN, J.
Centre de l~dologie Biologique- C.N.R.S. B.P.5, 17. rue N,D. des Pauvres. 54500 Vandoeuvre-les-Nancy, FRANCE
A b s t r a c t - Two scid-producmg bscteria ( ~,ro~uu'~rJ.m n ~ b b ~ ' ~ r a n d Agrobscterium sp )
and/or an ectomycorrhizal fungi ( L~ccJri~ lacca~ ) vero iaoculsted in the rhizospheros of beech and pi-e seediinEs,in lysimetricdevices in • g~nhouse. Plants were grown on sand mixed with rock phosphate and phlogopite (iron-magnesian mica ) Influence of these microorganisms on plant growth, plant nutrition and plant exudation was observed after two years. A~ro~cterium r~'o~nc~r, or l~cci~ri&lacca~ root inoculation incrossed beech growth and nutrientuptake ( P. Cs, K. Fe. M8 } by beech seediia4|s.But dual inoculationby both micro-orgshi•ms had no significanteffect on plant growth sad plant nutrition With pine se~lliags,the only growth and nutrient uptake promoting effect was observed with Agrohacterium sp inoculation and concerned only the root system. Bacterial inoculation increased the amount of organic scids ( malic, lactic, fumsric, citric ) released in the rh/zosphere, which promoted mineral element solubiIization and, thus, plant mineral nutrition. Mycorrhizal plan~ did not released greater amounts of organic •cids than non mycorrhizal plants. The effect of Laccaria/accata on beech growth and nutrition could be attributed to a better soil exploration, as shown by the increased rh/zospheric volume, Introduction Mycorrhizal plantsusually ttke up more phosphorous than non mycorrhizal plants.The abilityof"ectomycorrhizal fungi to solubilizeinsolublephospha~s in non symbiotic conditions,as
314
many soilbacteriado, has been shown ( Leyval and Berthelin, 1986 .),Some works on the:interactions between mycorrhizal fungi and other soilmicroorganisms have been reported by Leyval (198~) and Leyval and Berthelin (submitted) but few of them concerned the combined effectsof ectomycorrhizal fungi and phosphate-dissolving bacteria in the rhizosphere The aim of this study, was to observe these effects, in pine and beech rhizospheres, on the solubilizationof phosphorous from rock phosphate but also from mineral elements of a mica {phlogopite ).These minerals were the only sources of elements for plant nutrition and, thus, their solubilization influenced plant growth. Materials a n d methods Six month-old beech ( Fagussyiva~'c#L ) and pine ( PinussylwstrisL, ) seedlingswore grown in PVC cylinders in a greenhouse for two years ( Leyval, 1988. in press ). The cylinders were filledwith sand mixed with rock phosphate and phlogopite ( iron-magnesian mica ),Plants were automatically fed with a nutrient solution devoid of soluble P, Fe. Mg, K and .Ca, which were brought as insoluble forms by the two minerals. Plants were inoculated five times with an ectomycorrhizal fun gi, Laccaria lacca~, with a phosphate-dissolvingbacteriaisolatedfrom their rhizospheres, or with both microorganisms. Beech seedlings were inoculated with an
Agrobacterium rad~'obacterand pine ones with an Agrobacterium sp.. Control plants were maintained non inoculated. There were ten replicates in each treatment, After two years of experiment, mycorrhizal infection rate was evaluated and bacteria number in the rhizospheres was counted ( Leyval, 1988 ) Dry weight and mineral element amounts of plantswere determined. Rhizospheric soil ( ie adhering to the rootsafter being shaken by hand ) was evaluated ( Leyval, ~n press ) and rinsed in distilledwater for organic acid content analysis ( HPLC, Aminex HPX-$7 H Column )
Results a n d discussion The inoculation of beech roots with Agrobacterium radiobacter on one hand, and with
Laccarin/accataon the other hand, significantlyincreased shoot and root growth ( Fig. I ), It also significantlypromoted P. K, Ca, Fe and M g uptake by plant ( Tabl ) But dual inoculationwith both microorganisms did not significantlymodify beech growth and mineral element uptake.
315
Agrobacterium sp significantly promoted pine growth and phosphorous uptake from rock phosphate, but these effects only concerned the root system, The increase in K, Ca and M g amounts after bacterial inoculation were not significant at p< 0.05 Laccari& l a c c ~ inoculation and dual inoculation with both micro-organisms had no significanteffect,in this experiment, on pine growth and mineral element amounts (Fig.l,Tab.l ) The effect of the two Agrobactet;ium on pine and beech nutrient uptake ( especially phosphorous uptake ) could be relatedto the release of organic acids in the rhizosphere. Figure2 shows that the rhizospttere of plants inoculated with the bacteria contained greater amounts of organic acids The organic acids detected were also qualitativelydifferent: mslic sud lacticacids appeared in beech rhizosphere and citricand fumaric acid in pine rhizosphere. These increased organic acid quantitiesmight have contributed to increase phosphorous solubilizationfrom rock phosphate and g, Fe, M g solubilizationfrom phlogopite. For mycorrhizal plants, the amount of organic acids in plant rhizosphere was not increased, Citric acid, which is very complexing, increased in beech rh~zosphere in comparison to non mycorrhizal beeches ( Leyval, 1988 ),and lacticacid appeared But the total amount of organic acids was not modified, In that case, the significant increase of the rhizospheric soil ( Tab,2 ), which suggests a bettor soilexploration, could explain l..¢ccaJ-ialacc#~aeffect on beech growth and nutrition In mycorrhizal pine rhizosphere, and in beech rhizosphere inoculated with both micro-organisms, no more organic acidswere detected ( Fig2 ),which could explain that P, K, Fe Ca and M g mobil~.ations were not increased. However, for mycorrhiza.l pines, rhizospheric soil percentage was higher than for non mycorrhizal pines ( Tab2 ), This results point out the importance of rhizospheric microorganisms and their interactions m
plant growth and nutrition. Thus. acid-producing micro-organisms
can increase the
availability of soilmineral elements for plant nutrition, and mycorrrhizal fungi can increase
316
soil exploration ( H a y m a n
and Mosse, 1972 ) But, if in some works reviewed by Leyval and
Berthelin ! submitted ) synergistic effects of microorganisms
were
observed, competition
between them m a y a/se occur, as the results of this experiment suggest it, and then, no effect on plant growth can be observed
Mineral element uptake
K
P
Ca
Fe
Beech non inoculated - bacteria + mycocrhiza - bacteria + mycorrhiza
5.97 a 8.82 b 8.51 b 3.84 a
5.95 a I0.26 b 10.04 b 4.56 a
11.53 a 20,09 b 19.83 b 8.59 a
Pine non inoculat~ + bacteria + mycorrhiza +bacteriaomycorrhiza
12.81 a
9.52 a
27.97 a
8.53 a
6.25 a
14.45 a 11.691 I0.$6a
11.20 b 8.061 ?.?la
30.91 a 19.991 21.32a
?,02 l 9.Sqa ll.31a
?.02 a 5.751 ?.24a
1.19 a 3.34 b 4A'~ b 1.69 a
MII
2.29 a '1.49b '1.50b 2.25 a
Tabl: In|luence o| bacterial and ectomycorrhizal inoculations o! pine and beech roots on mineral element u p t U e | tug,plant l Same letters in a same column indicate no l i l n i f i c l m t dlf/erenees at P< 0.05
Beech
Non inoculated + bacteria • mycorrhiza , b a c t ~ i a , mycorrhiza
1.6 a 3.2 a 5.0 b 3.0 a
Pine
5.8 a 5.2 a 9.2 b 11.0 b
Tab.2: Rhizospheric soil percentage ( ~ total soil dry tveigh ) ol pine and beech seedlings aJter 2 years, Same letters in a same column indicate no signi|icant differences at P~ 0.05
317
g
Pine
8 Beech
I[] []
I
roots shoots
I 6 a a
4
a
,/a/
NI
B
M
BM
NI
B
M
BM
Fig 1 Influence of the phosphate-dissolving bacteria ( B ) and/or of the ectomycorrhizal fungi (M) on pine and beech growth ( g dry weight/plant ) Same letters in histograms indicate no
significant differences at P0 05 NI non inocultted.
318
+B
/
29 pg/plant
=
Beech 8.5 ~ / p l e n t f n~i¢ I ~ I +M I ~ +B+M O.61~g/plent 0.0 pg/plont
Plne 49.3p!I/Plant
VN-1
)ni¢
+B
/
+
~ , ~
+B+M
60.2Jlg/plent 0,1~1 I/plent 0.0pg/plent citric fumaric
lactic medic
Fig2 Influence of the phosphate-dissolving bacteria ( .B ) and/or of the ectomycorrhizal fungi (-M) on the organic acid amounts ( Ug/plant ) in pine and beech rhizospheres after two years
319
References H A Y M A N , D S and MOSSE, B,; Plant growth responses to vesiculac-arbuscular mycorrhiza. Ill, Increase uptake of labileP from soil.-New Phytol.71: 41:47. 1972. LEYVAL, C. Lysimeters in a greenhouse as an help to study the interactions between microorganisms, minerals and forest tree roots.-In:"Fieldsmethods in terrestrial ecosystem nutrient cycling", Grange-over-Sand. U.K.. I-5 Dec. 1988. ( in press ). LEYVAL, C :Interactionsbacteries-mycorhizes darts la rhizosph(~re du pin sylvestre et du h6tre Incidences sur l'exsudationracinaire et l'alttrationdes mineraux - Doctoral thesis.Universit* de
Nancy I, 1988 LEYVAL, C and BERTHELIN, J.: Comparison between the utilization of phosphorus from insoluble mineral phosphates by ectomycorrhizal fungi and rhizobacteria.-In: GIANINAZZI-PEARSON,V and GIANINAZZI,S / e d . / : Physiolological and genetical aspects of mycorrhizae. Pp. 340-345.Inra, Paris 19fS6 LEYVAL C. and BERTHELIN,J.: Interactions between Imccaria laccata, Agrobacterium rmiiobacter and beech roots: influence on P, K. M g and Fe mobilization from minerals and plant growth,Plant and Soil ( submitted ).
L e y v a l , C. and B e r t h e l i n ,
J.,
1989t I n f l u e n c e o f a c i d - p r o d u c i n g A g r o b a c t e r l u m
and L a c c a r i a l a c c a t a on p i n e and b e e c h growth, n U t r i e n t uptake and e x u d a t i o n . &grit.,
Ecosystems E n v i r o n . , 28: 313-319,