- Email: [email protected]
Effect of dose and formulation of Laccaria laccata inoculum on mycorrhizal infection and growth of Douglas fir in a nursery
351
Agriculture, Ecosystems and E n v i r o n m e n t , 28 Elsevier Science Publishers B.V., A m s t e r d a m P r i n t e d in C z e c h o s l o v a k i a
(1989)
351-354
EFFECT OF DOSE AND FORMULATION OF L,41~,~IA LACCATA INOCULUM ON MYCORRHIZAL INFECTION AND eROWTH OF DOI,16LA,5FIR IN ^ NURSERY F. Mortier, F. Le l'aconandJ. Garbaye INRA, Centre de Recherches Forostii'~resde Nancy, Champenoux, F-54280 Seichamps
Abstract A fumigated nursery bed on a sandy loam was inoculatedwith the ectomycerrhizalfungus Lapcar/a lactate and seeded with Douglas fir.Two types of inoculum were compared: mycelium grown in a vermicuIitelpeat mixture, and myceIium grown in liquid medium and entrapped in a calcium alginote gel with differentquantitiesof myceIium. At the end of the first growing season, the alginatemocuium at the dose of 5 g mycelium (dry weight) per m 2 proved to be the most efficient The top dry weight of the seedlings in this treatment was 2.3 fold that of the non-inoculated fumigatedcontrols.This inoculationtreatmentalso ensured hearty totalmycerrhizal infectionby L. lactate
Introduction The ectomycorrhizalfungus Laccarla laccata(Scop.ex Fr.) Cke. has proved to be very efficientfor promoting the growth of coniferplantingstocks in temperate nurseriesand plantationsin acidsoils (MOLINA, 1982; MOLINA and CHAMARD, 1983; .THOMAS and JACKSON,1983; LE TACON and 80UCHARD, 1986) ,~commercial inoculum of L. lactateproducedon a vermicuIlte-peatmixture sealed in plasticbags is now availablein France (LE TACON eta/, 1988). However, LE TACON etal ( 1983 and1985) and MAUPERIN ete/(1987) have shown with other ectomycorrhizalfungi that mycelium grown in a liquidmedium and entrapped in calcium alginategel (JUNG eta/, 1981 ) is a very efficientinoculum for mycorrhizal development,and hence can be used as an alternativeto the classicalvermiculite-peat mixture (MARX and BRYAN, 1975). In order to improve the techniques of mycorrhizal inoculationwith L. lactate, the experiment which is described here compares the efficiency of different doses of the two types of inocuium for promoting ectomycorrhizaldevelopmentand growth of Douglas fir seadIin~
35Z
Material and Hethods
Douglas fir seedlings were grown from May to October in a forest nursery in North-Eastern France. The soil (a sandy loam with pH 5,5 and 4% orojanicmatter) had been previously fumigated with methyl bromide. Five treatments were compared: non inoculatedand inoculatedwith L ~ r i a laccataas standard, non washed, vermiculite-peat inocu!um or as mycelium grown in liquidculture and entrapped in calcium alginatebeads with powdered sphagnum peat as a stabilizing agent (Mauperin eta/, 1987). Two liters of inoculum were applied per .square meter in each treatment, corresponding to approximatively 2 g of mycelium for the vermiculite-peat mixture and to 2 g, 5 g or I0 g of myceIium (dry weight) for the alginateinoculum The inoculum was mixed into the soil ( IO- 15 cm deep) before seeding. The experimental plots were randomly ditributed in a 4 blocks design. The mean heights of the seedlings in each pK,t were measured on weeks 8, I I, 15 and 25 Four seedling with heights equal to the mean value were liftedand the dry weights of the tops were measured. The root systems were gently washed and the per cent of mycorrhizal short roots was determined, Datawere treated by two-ways analysis of variance and the treatments were compared with I.s d 5%
Results and Discussion Table I shows that the two types of inoculum behaved very differently: with the vermiculite-peat mixture, infectionwas rapid with littlesubsequent increase in the number of mycorrhizal roots, while with the two lowest doses of alginate inoculum infectionwas slower but reached a higher level at the end of the growing season The highest dose of alginate inoculum induced both a quick and intense infection. At the end of the growing season, the mean dry weight of the seedlingswas significantlyhigher in the treatment with alginate inocuIum ( 5 g myceIium per m 2 than in the non mycorrhiza] control or in the vermiculite-peat treatment (1'able2). In conclusion, alginate entrapped mycolium of Laccar10laccata proved to be very efficient.The lowest amount of mycelium ( 2 g dry weight per square meter) was enough for a complete infection of Douglas fir seedlings during the firstyear in a barefoot nursery. This efficiency seems to be mestIv due to a latebut sustainedactivityof the entrapped mycelium, compared with an earlier but shorter activity of the myceIium grown on the vermiculite-peat mixture and used at approximativeIy the same dose. MAUPERIN eta/ (1987) already noticed the protective and stablIizlngeffectof powdered peat added to the gel The fact that this type of ineculum has already proved to be efficientin another nursery and with another fungus ( LE TACON d#/, 1985) suggests that it could be adapted to a wide range of conditions. A commercial inoculum of this type is presently being developed in France
353
Table I _ Effect of the inoculum type on myoorrhizal development of Douglas fir seedlings'with L,~:~ria ,I,~:~ta in a fumigated nursery bed (per cent myoorrhizal short roots). For each date, values followed by the same letter are not slngnificantly different at the 5% probability level. treatment
8 weeks
11 weeks
15 weeks
25 weeks
non inoculated
O
0
0
0
vermiculite-peat
0
43 a
70 a
70 a
alginate 2 g myoelium/m2
0
5 b
46 b
90 b
olginate5 g.mycaliumlm 2
0
13 b
66 a
92 b
alglnate 10 g mycallum/m2
0
89 c
82 a
95 b
Table 2 _ Dry weight of the aerial parts of the seedlings (rag). For each date, values followed by the same letter are not significantly different at th 5 % probability level. treatment
8 we~s
I I weeks
I 5 weeks
25 weeks
non inoculated
44 a
84 a
IO0 a
140 a
vermiculite-peat
24 e
68 a
108 a
220 ab
alginate2 g mycaliumlm 2
24 a
66 a
132 ab
276 abc
alginate5 g myceliumlm 2
36 a
84 a
208 b
356 c
alginate I0 g myceliumlm 2
16a
1080
124ap
260 abc
References dUNG G., MUGNIER d., DOMMEROUES
Y., DIEM
H.G., 1981. Proclkle d'inclusion des
microorganismes du groupe des actinorhlzes el des myoorhizes. Brevet n° 81.04474. LE TACON F., dUNG G., MICHELOT P., MUGNIER d., 1983. Efficaciteen pepiniere forestiered'un inoculum de champignon ectomycarhizienproduit en fermenteur et inclus dens une matrice de polymeres. Ann ,~.?iF~, 40(2), 165- 176. LE TACON F., dUNG G., MUGNIER J., MICHELOT P, MAUPERIN C., 1985. Efficiencyin a forest nur~ry of an ectomycorrhizalinoculum produced in a fermenter and entrapped in polymeric gels. ~2.-vzJ BoL, 63(9), 1664- 166.
354
LE TAOON F., BOUCHARD D., 1986. Effectsof differentectomycorrhizal funQi.ongrowth of larch, Douglas fir, Scots pine and Norway spruce seedlings in fumigated nursery soil ~9oo/ Applic., 7(4), 389-402. MAUPERIN Ch., MORTIER F., C~RBAYE J., LE TAOON F., CARR 8., 1987. Viabilitv of an ectomycerrhizal inoculum produced in a liquidmedium and entrapp~ in a calcium alginategel.Ca~ d 80L, 65. 2326-2329 MARX D., BRYAN W.C., 1975. Growth and ectomycorrhizal development of LobloIIvPine seedlings in fumigatedsoll infestedwith the fungal symbiont Pldolithus tinctooius Forest 5cleric, 22(3), 245-254. MOLINA R, 1982. Use of the ectomyoorrhizal fungus La~cama l~ta in forestry. I.C,onsisten~ between isolatesin effectivecelonizatianof containerizedconifer soedlings.~ J. ;Coo Re~, 12, 469-473. MOLINA R., CHAMARD J., 1983. Use of lhe eclomycorrhizal fungus Lac~ria laocat8 in forestry.II. Effectsof fertilizerforms and IBvelson growth of container-~own Douglas firand ponderosa pine seedlings.~
J. Foo R6~., 13, 89-95.
THOMAS O.W., JACKSON R.M., 1983.0rowth response of Sitka spruce seedlings to mycerrhizal ineculation.IVewPhytol., 95 (2), 223-229.
Mortier, F., Le Tacon, F. and Garbaye, J., 1989: Effect of dose and formulation of Laccaria laccata inoculum on m y c o r r h i z a l infection and growt of Douglas fir in a nursery. Agric., E c o s y s t e m s Environ., 28: 351-354.
Agriculture, Ecosystems and E n v i r o n m e n t , 28 Elsevier Science Publishers B.V., A m s t e r d a m P r i n t e d in C z e c h o s l o v a k i a
(1989)
351-354
EFFECT OF DOSE AND FORMULATION OF L,41~,~IA LACCATA INOCULUM ON MYCORRHIZAL INFECTION AND eROWTH OF DOI,16LA,5FIR IN ^ NURSERY F. Mortier, F. Le l'aconandJ. Garbaye INRA, Centre de Recherches Forostii'~resde Nancy, Champenoux, F-54280 Seichamps
Abstract A fumigated nursery bed on a sandy loam was inoculatedwith the ectomycerrhizalfungus Lapcar/a lactate and seeded with Douglas fir.Two types of inoculum were compared: mycelium grown in a vermicuIitelpeat mixture, and myceIium grown in liquid medium and entrapped in a calcium alginote gel with differentquantitiesof myceIium. At the end of the first growing season, the alginatemocuium at the dose of 5 g mycelium (dry weight) per m 2 proved to be the most efficient The top dry weight of the seedlings in this treatment was 2.3 fold that of the non-inoculated fumigatedcontrols.This inoculationtreatmentalso ensured hearty totalmycerrhizal infectionby L. lactate
Introduction The ectomycorrhizalfungus Laccarla laccata(Scop.ex Fr.) Cke. has proved to be very efficientfor promoting the growth of coniferplantingstocks in temperate nurseriesand plantationsin acidsoils (MOLINA, 1982; MOLINA and CHAMARD, 1983; .THOMAS and JACKSON,1983; LE TACON and 80UCHARD, 1986) ,~commercial inoculum of L. lactateproducedon a vermicuIlte-peatmixture sealed in plasticbags is now availablein France (LE TACON eta/, 1988). However, LE TACON etal ( 1983 and1985) and MAUPERIN ete/(1987) have shown with other ectomycorrhizalfungi that mycelium grown in a liquidmedium and entrapped in calcium alginategel (JUNG eta/, 1981 ) is a very efficientinoculum for mycorrhizal development,and hence can be used as an alternativeto the classicalvermiculite-peat mixture (MARX and BRYAN, 1975). In order to improve the techniques of mycorrhizal inoculationwith L. lactate, the experiment which is described here compares the efficiency of different doses of the two types of inocuium for promoting ectomycorrhizaldevelopmentand growth of Douglas fir seadIin~
35Z
Material and Hethods
Douglas fir seedlings were grown from May to October in a forest nursery in North-Eastern France. The soil (a sandy loam with pH 5,5 and 4% orojanicmatter) had been previously fumigated with methyl bromide. Five treatments were compared: non inoculatedand inoculatedwith L ~ r i a laccataas standard, non washed, vermiculite-peat inocu!um or as mycelium grown in liquidculture and entrapped in calcium alginatebeads with powdered sphagnum peat as a stabilizing agent (Mauperin eta/, 1987). Two liters of inoculum were applied per .square meter in each treatment, corresponding to approximatively 2 g of mycelium for the vermiculite-peat mixture and to 2 g, 5 g or I0 g of myceIium (dry weight) for the alginateinoculum The inoculum was mixed into the soil ( IO- 15 cm deep) before seeding. The experimental plots were randomly ditributed in a 4 blocks design. The mean heights of the seedlings in each pK,t were measured on weeks 8, I I, 15 and 25 Four seedling with heights equal to the mean value were liftedand the dry weights of the tops were measured. The root systems were gently washed and the per cent of mycorrhizal short roots was determined, Datawere treated by two-ways analysis of variance and the treatments were compared with I.s d 5%
Results and Discussion Table I shows that the two types of inoculum behaved very differently: with the vermiculite-peat mixture, infectionwas rapid with littlesubsequent increase in the number of mycorrhizal roots, while with the two lowest doses of alginate inoculum infectionwas slower but reached a higher level at the end of the growing season The highest dose of alginate inoculum induced both a quick and intense infection. At the end of the growing season, the mean dry weight of the seedlingswas significantlyhigher in the treatment with alginate inocuIum ( 5 g myceIium per m 2 than in the non mycorrhiza] control or in the vermiculite-peat treatment (1'able2). In conclusion, alginate entrapped mycolium of Laccar10laccata proved to be very efficient.The lowest amount of mycelium ( 2 g dry weight per square meter) was enough for a complete infection of Douglas fir seedlings during the firstyear in a barefoot nursery. This efficiency seems to be mestIv due to a latebut sustainedactivityof the entrapped mycelium, compared with an earlier but shorter activity of the myceIium grown on the vermiculite-peat mixture and used at approximativeIy the same dose. MAUPERIN eta/ (1987) already noticed the protective and stablIizlngeffectof powdered peat added to the gel The fact that this type of ineculum has already proved to be efficientin another nursery and with another fungus ( LE TACON d#/, 1985) suggests that it could be adapted to a wide range of conditions. A commercial inoculum of this type is presently being developed in France
353
Table I _ Effect of the inoculum type on myoorrhizal development of Douglas fir seedlings'with L,~:~ria ,I,~:~ta in a fumigated nursery bed (per cent myoorrhizal short roots). For each date, values followed by the same letter are not slngnificantly different at the 5% probability level. treatment
8 weeks
11 weeks
15 weeks
25 weeks
non inoculated
O
0
0
0
vermiculite-peat
0
43 a
70 a
70 a
alginate 2 g myoelium/m2
0
5 b
46 b
90 b
olginate5 g.mycaliumlm 2
0
13 b
66 a
92 b
alglnate 10 g mycallum/m2
0
89 c
82 a
95 b
Table 2 _ Dry weight of the aerial parts of the seedlings (rag). For each date, values followed by the same letter are not significantly different at th 5 % probability level. treatment
8 we~s
I I weeks
I 5 weeks
25 weeks
non inoculated
44 a
84 a
IO0 a
140 a
vermiculite-peat
24 e
68 a
108 a
220 ab
alginate2 g mycaliumlm 2
24 a
66 a
132 ab
276 abc
alginate5 g myceliumlm 2
36 a
84 a
208 b
356 c
alginate I0 g myceliumlm 2
16a
1080
124ap
260 abc
References dUNG G., MUGNIER d., DOMMEROUES
Y., DIEM
H.G., 1981. Proclkle d'inclusion des
microorganismes du groupe des actinorhlzes el des myoorhizes. Brevet n° 81.04474. LE TACON F., dUNG G., MICHELOT P., MUGNIER d., 1983. Efficaciteen pepiniere forestiered'un inoculum de champignon ectomycarhizienproduit en fermenteur et inclus dens une matrice de polymeres. Ann ,~.?iF~, 40(2), 165- 176. LE TACON F., dUNG G., MUGNIER J., MICHELOT P, MAUPERIN C., 1985. Efficiencyin a forest nur~ry of an ectomycorrhizalinoculum produced in a fermenter and entrapped in polymeric gels. ~2.-vzJ BoL, 63(9), 1664- 166.
354
LE TAOON F., BOUCHARD D., 1986. Effectsof differentectomycorrhizal funQi.ongrowth of larch, Douglas fir, Scots pine and Norway spruce seedlings in fumigated nursery soil ~9oo/ Applic., 7(4), 389-402. MAUPERIN Ch., MORTIER F., C~RBAYE J., LE TAOON F., CARR 8., 1987. Viabilitv of an ectomycerrhizal inoculum produced in a liquidmedium and entrapp~ in a calcium alginategel.Ca~ d 80L, 65. 2326-2329 MARX D., BRYAN W.C., 1975. Growth and ectomycorrhizal development of LobloIIvPine seedlings in fumigatedsoll infestedwith the fungal symbiont Pldolithus tinctooius Forest 5cleric, 22(3), 245-254. MOLINA R, 1982. Use of the ectomyoorrhizal fungus La~cama l~ta in forestry. I.C,onsisten~ between isolatesin effectivecelonizatianof containerizedconifer soedlings.~ J. ;Coo Re~, 12, 469-473. MOLINA R., CHAMARD J., 1983. Use of lhe eclomycorrhizal fungus Lac~ria laocat8 in forestry.II. Effectsof fertilizerforms and IBvelson growth of container-~own Douglas firand ponderosa pine seedlings.~
J. Foo R6~., 13, 89-95.
THOMAS O.W., JACKSON R.M., 1983.0rowth response of Sitka spruce seedlings to mycerrhizal ineculation.IVewPhytol., 95 (2), 223-229.
Mortier, F., Le Tacon, F. and Garbaye, J., 1989: Effect of dose and formulation of Laccaria laccata inoculum on m y c o r r h i z a l infection and growt of Douglas fir in a nursery. Agric., E c o s y s t e m s Environ., 28: 351-354.