Effect of inoculation and substrate disinfection method on rooting and ectomycorrhiza formation of Douglas fir cuttings

HAL Id: hal-00883250

https://hal.archives-ouvertes.fr/hal-00883250

Submitted on 1 Jan 1999

HAL is a multi-disciplinary open access

archive for the deposit and dissemination of

sci-entific research documents, whether they are

pub-lished or not. The documents may come from

teaching and research institutions in France or

abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire HAL, est

destinée au dépôt et à la diffusion de documents

scientifiques de niveau recherche, publiés ou non,

émanant des établissements d’enseignement et de

recherche français ou étrangers, des laboratoires

publics ou privés.

Effect of inoculation and substrate disinfection method

on rooting and ectomycorrhiza formation of Douglas fir

cuttings

Javier Parladé, Joan Pera, Isabel F. Álvarez, Daniel Bouchard, Benoit Généré,

François Le Tacon

To cite this version:

Original

article

Effect of inoculation and

substrate

disinfection method

on

rooting

and

ectomycorrhiza

formation

of

_Douglas

fir

_cuttings

Javier Parladé

Joan Pera

Isabel

F. Álvarez

a

Daniel

Bouchard

b

Benoit Généré

c

_François

Le

Tacon

b

a

Departament de _{Patologia Vegetal,} Ctra de Cabrils _{s/n, IRTA,} 08348 _{Cabrils, Barcelona, Spain}

b

Centre de recherches forestièrès de

_Nancy,

_Inra, 54280

_Champenoux,

France

c

Cemagref,

Domaine des Barres, 45290

_{Nogent-sur-Vernisson,}

France

(Received 2

_February

1998; accepted 11

May

1998)

Abstract -

_{Ectomycorrhizal}

inoculation

techniques

were

_applied

to

_Douglas

fir

_(Pseudotsuga

menziesii (Mirb) Franco)

cuttings,

dur-ing

the

_rooting

_phase, to determine the effect of inoculation on _rooting and _mycorrhizal colonization. Two

_experiments

were

con-ducted: 1) an inoculation

_experiment,

established at three different locations with the same _treatments, to determine the effect of inoc-ulation with Laccaria bicolor,

Melanogaster ambiguus

and

_Rhizopogon

subareolatus; and 2) a local factorial

_experiment

to determine

the effect

_{of fungal}

_inoculation, substrate disinfection methods, and their

_possible

interactions. At

_{Peyrat-le-Château}

and

_Champenoux

(France), there were no differences in

_rooting

due to the inoculation treatment. At Cabrils

_(Spain),

the

_cuttings

inoculated with L.

bicol-or and R. subareolatus rooted

_{significantly}

better than non-inoculated

_{cuttings. Vegetative}

inoculum of L. bicolor and spores of R. subareolatus were effective in

_{forming ectomycorrhizas}

in the three _locations, whereas _spores of M.

_ambiguus

were effective

_only

at

Cabrils. Substrate disinfection with

_methyl

bromide

_{significantly}

decreased the _percentage of rooted

_{cuttings compared}

to steam-dis-infected substrate.

_Methyl

bromide

_application

also lowered the percentage of

_{mycorrhizal cuttings}

after inoculation with L. bicolor and R. subareolatus. The disinfection method did not alter

_{significantly}

_{the colonization level among the}

_{mycorrhizal plants.}

(@ Inra/Elsevier, Paris.)

Laccaria bicolor /

_{Melanogaster ambiguus}

/

_Rhizopogon

subareolatus /

_propagation

/

_mycorrhiza

Résumé - Effet de méthodes d’inoculation et de désinfection du substrat sur l’enracinement de boutures de _douglas et sur la formation de

_mycorhizes.

La

_{mycorhization}

contrôlée de boutures de

_{douglas (Pseudotsuga}

menziesii (Mirb) Franco) a été étudiée

durant la

_phase

d’enracinement, afin de mesurer son effet sur l’enracinement et sur l’état

_mycorhizien

final des

_plants.

Deux essais ont

été suivis: ₁₎ un essai commun à trois sites _comparant l’effet de souches

_fongiques

de Laccaria bicolor, Melauogaster ambiguus et

Rhizopogon

subareolatus, et ₂₎ un essai factoriel local testant l’effet de l’inoculation, du mode de désinfection du substrat et leurs interactions. Contrairement à

_{Peyrat-le-Château}

et à

_Champenoux

où le taux _{d’enracinement n’a pas été modifié par les}

_symbiotes

inoculés, les boutures inoculées _{par L.} bicolor and R. subareolatus se sont mieux enracinées que les témoins à Cabrils. Le

mycélium

de L. bicolor et _{les spores de R. subareolatus} ont

_permis

la formation de

_mycorhizes

sur les trois sites, alors que les spores de

M. _ambiguus ne l’ont permis

qu’à

Cabrils. La désinfection du substrat au bromure de

_méthyle

a réduit

_{significativement}

le taux de boutures racinées par rapport à celle effectuée à la vapeur.

L’application

de bromure de

_méthyle

a diminué

_également

le taux de

bou-tures

_{mycorhizées, après}

_{inoculation par L. bicolor} et R. subareolatus. En revanche, la _{méthode de désinfection n’a pas} modifié

signi-ficativement le taux de

_{mycorhization}

des

_plants

effectivement

_mycorhizés.

(© Inra/Elsevier, Paris.)

Laccaria bicolor /

_{Melanogaster ambiguus}

/

_Rhizopogon

subareolatus /

_{multiplication végétative}

/

_mycorhize

*

Correspondence

and

_reprints

1. INTRODUCTION

Vegetative

propagation

of forest trees is an alternative

to the lack of seeds of

_high

_genetic

value when a

breed-ing

programme has been concluded. Several

species

of forest trees can be

_propagated

as rooted

_cuttings

from

selected mother

_plants

_[22].

The

_increasing

_availability

of

genetically improved Douglas

fir

_(Pseudotsuga

menziesii

(Mirb) Franco) seed,

its successful

_propagation

from

cut-tings

in North America

_[23],

and its

_increasing

impor-tance as a reforestation

_species

in

_Europe

[17]

make this

species

a

_good

candidate to be

_propagated

as rooted

cut-tings.

Since this

_propagation

method is more

_expensive

than

_seedling

_production,

the effort to

_improve

the

root-ing

process and the

performance

of the rooted

cuttings

in the nursery and after field

transplantation

is of

_great

inter-est. The critical

_stages

_{during cutting propagation}

are the

rooting

phase

and the

_{transplantation}

to _nursery beds and

to the field. Inoculation of

_Douglas

fir

_seedlings

with selected

_{ectomycorrhizal fungi,}

in both containerized and

bareroot

_nurseries,

can increase survival and/or

_growth

of

the

_plants

in the _nursery

_phase

_[13]

and when

_outplanted

in the field in

_European

trials

_{[1, 2, 9, 15, 27].}

On

cut-tings,

the

_fungal

inoculum is

_{generally supplied}

when the rooted

_cuttings

are

_transplanted

in

_fumigated

beds

[10].

In this

_study,

we inoculated

_{directly during}

the

_rooting

phase

to determine the effect of inoculation on

_rooting

and

_mycorrhizal

colonization. Available inoculation

tech-niques

with

_{ectomycorrhizal}

_fungi

used for containerized

seedlings

were

_applied

and

_adapted

to

_Douglas

fir

cut-tings.

Two

_experiments

were

_designed:

1)

an inoculation

experiment,

established at three different locations with the same _treatments, to determine the effect of inocula-tion with Laccaria

_bicolor,

_{Melanogaster ambiguus}

and

Rhizopogon

subareolatus;

and

₂₎

a local factorial

experi-ment to determine the effects of inoculation with the above

_fungi

and the substrate disinfection method.

2. MATERIALS AND METHODS 2.1. Inoculation

_experiments

at three different locations

The

_experiments

were set _up in

_greenhouses

at three

experimental

locations: _{the nursery} of

_{Peyrat-le-Château}

located in central

_France,

and the research centres of

Champenoux

in northeast France and Cabrils in northeast

Spain.

Douglas

fir

_cuttings

were obtained from a total of 200

3-year-old

mother

_plants

_(seed

orchard 24 established at

Bout,

Allier, France)

in

_February

1995. The

_cuttings

were

mixed

_{homogeneously}

and cold stored at 2 °C for 4

weeks to

_{improve rooting.}

After _storage, the bases of the

cuttings

were

_dipped

in a solution of

Exuberone® (Rhone

Poulenc, 4 g

L

-1

_{indole-butyric}

_{acid, IBA)}

for 24 h. The

substrate,

consisting

of a mixture of

_peat

and vermiculite

(1:1, v:v),

was disinfected with 60 _g

L

-1

_methyl

bromide

(2

%

_{chloropicrine)}

at a _temperature over 13 °C. The

con-tainers were

_plastic

horticultural _trays

(510

x 365 x 180

mm)

at

_Peyrat-le

Château, HIKO®

plates

with 40 alveoles of 90 cc each at

_Champenoux,

and

_forest-pot

containers,

150 cc

_each,

at Cabrils. Insertion of the

_cuttings

was done

during

the first week of March

1995,

2 weeks after

sub-strate disinfection.

Laccaria bicolor

_{(R Mre)}

Orton strain S-238 N was

inoculated at insertion as

vegetative inoculum grown in

peat:vermiculite

as described

_by

Marx and

_Bryan

[16]

at

the rate of 1:20

_{(v:v, inoculum:substrate).}

_Sporocarps

of

Melanogaster ambiguus

(Vitt)

Tul & Tul and

_Rhizopogon

subareolatus Smith were collected in the

_Montseny

range, Girona, northeast

_Spain,

in autumn 1994.

_Spore

inoculum of both

_fungi

was

_prepared

as described

_by

Castellano et al.

_[6]

and

_applied

2 months after insertion

at the rate of

10

6

spores per

cutting

as described in

Parladé et al.

_[21].

For each

location,

the

_{experimental design}

consisted of four

_replications

of 40

_cuttings

_{(experimental}

_unit)

for each inoculation treatment

_including

a

_control,

non-inoc-ulated treatment. The _trays were distributed

_randomly

in

a

_greenhouse

and maintained under mist

_{irrigation during}

the

_{rooting process and}

afterwards,

under

_regular

irriga-tion.

_{Fertirrigation}

was started 4 months after insertion

_by

using

the N-P-K soluble fertilizer Plant Prod 20-20-20

sprayed weekly

at the rate of 1.5 _g

m

-2

.

2.2. Factorial

_experiment

at Cabrils location The factorial

_{experiment design}

included two factors: inoculation and the substrate disinfection method with four

_replications

of 40

_{cuttings (experimental}

_unit)

for each treatment.

_Fungal

inocula,

inoculation levels and

growing

conditions were

_exactly

the same as described earlier. The disinfection levels were

_methyl

bromide

applied

as in the

_previous

_experiment,

and steam was

applied

twice at 90 °C for I h with a 24-h

_period

between

applications.

Eight

months after

_insertion,

all the

_cuttings

from both

experiments

were assessed

_visually

for

_rooting

_(root

sys-tem

_fully

_developed)

and

_mycorrhizal

colonization.

_Forty

cuttings

taken

_randomly

from each treatment were

exam-ined for root colonization

_{by counting}

at least 200 short

roots per

plant

under a

_{stereomicroscope.}

_Only

mycor-rhizal

_plants

were considered to determine the coloniza-tion level.

_Rooting

and

_mycorrhizal

colonization data

were

_analyzed

_by

_analysis

of variance

_(ANOVA)

on the

Colonization percentages were transformed with the arc

sinus function to reduce the error variance

_[26]

before

performing

the ANOVA. Differences _among means were

detected

_{by Tukey’s}

test

(P ≤ 0.05).

3. RESULTS

3.1. Inoculation

_experiments

at three different locations

At

_{Peyrat-le-Château}

and

_Champenoux,

there were no

differences in

_rooting

due to the inoculation treatment. At

Cabrils,

the

_cuttings

inoculated with L. bicolor and R.

subareolatus rooted

_{significantly}

better than non-inocu-lated

_cuttings.

The _average

_rooting

_percentage,

_however,

was lower than in the other two locations

_{(table I).}

The

_cuttings

inoculated with L. bicolor became myc-orrhizal in a

_{proportion varying}

from 20 to 89

%,

depend-ing

on the location

_{(table II).}

The

_mycorrhizal

short roots rate

_{(colonization}

level)

among the

mycorrhizal

plants

ranged

from 40 to 59 %

_{(table III).}

The

_proportion

of

mycorrhizal

cuttings

with R. subareolatus varied from 31

to 88 % _{among locations}

_{(table II).}

The colonization level

ranged

from 14 to 39 % of the short roots

_{(table III).}

The

inoculation with M.

_ambiguus

was effective

_only

at

Cabrils.

_Only

8 % of the

_plants

became

_mycorrhizal

(table II)

with an _average of 31 % of the short roots

col-onized

_{(table III).}

3.2. Factorial

_experiment

at Cabrils

The _percentage of rooted

_cuttings

was

_{significantly}

affected

_by

both inoculation

_(P

=

0.0006)

and disinfection

(P

=

_0.0000)

factors. _No

significant

interactions were

detected between them

_(P

=

0.7223).

Inoculation with L.

bicolor and R. subareolatus

_{significantly}

increased the

percentage

of rooted

_cuttings

over non-inoculated

cut-tings

(table

IV).

No

_significant

effect on

_rooting

occurred when the

_cuttings

were inoculated with M.

_ambiguus.

Substrate disinfection with

_methyl

bromide

signifi-cantly

decreased the _percentage of rooted

_cuttings

com-pared

to the

_rooting

_percentage

obtained on steam-disin-fected substrate

₍₄₃

and 70

_%,

_{respectively).}

_Methyl

bro-mide

_application

also decreased the

_percentage

of

mycor-rhizal

_cuttings

after inoculation with L. bicolor and R.

subareolatus

_(table

V).

The disinfection method did not

_{significantly}

affect the

colonization level of the

_plants

for _any of the inoculation

treatments

_{(table V).}

4. DISCUSSION

The substrate disinfection with

_methyl

bromide

probably

because of the

_persistence

of

_phytotoxic

residu-als in the substrate.

_{Although methyl}

bromide is

consid-ered a short-residual sterilant when

_{applied properly}

in

the soil

_[4],

it _may

_persist

in

_organic

or

_clayey

_soils

pro-ducing phytotoxic

effects

_[24].

_Methyl

bromide has been

widely

used in bareroot nurseries in

_mycorrhizal

inocula-tion _programmes to reduce

_competition

between wild and inoculated

_fungi,

as well as to control soil-borne root

pathogens

[7]. However,

steam has been recommended

for the disinfection of artificial

_growing

media in

con-tainer nurseries

_[5].

Chemicals such as

_{chloropicrin,}

methyl

bromide and

_Vapam®

are also used for

steriliza-tion of

_growing

media,

but

_they

_may bind with the

expanded

vermiculite

_{causing phytotoxicity}

even after

prolonged

aeration

[28].

Problems caused

_{by methyl}

bro-mide as a disinfectant were not consistent _among loca-tions

_although

the

_{application methodology}

was the same.

As Molina and

_Trappe

stated

[19],

a

_given

chemical will

not

_{necessarily produce}

the same _{response in all}

_species

of

_fungi

or hosts or in all nurseries. _{In any} case, the use of

steam can avoid the

_{possible phytotoxicity problems}

of

residuals connected with local

_growth

conditions

(sub-strate

_compaction,

container

_{type, temperature,}

_humidity,

etc.).

In

_addition,

_methyl

bromide has been listed under

the Montreal Protocol on Substances that

_Deplete

the

Ozone

_Layer

and is to be banned in the future. The use of steam as soil disinfectant has been cited in some case

studies

_[3]

as a

_{relatively inexpensive}

and efficient

alter-native to

_methyl

bromide.

Inoculation with L. bicolor and R. subareolatus

_only

increased

_{significantly}

the

_{rooting performance}

of the

cuttings produced

at Cabrils. At the other two

locations,

the average

rooting

percentages were

_higher

than those in

Cabrils and no inoculation effect was detected. The

mechanism

_underlying

the

_{fungal ability}

to

_improve

the

rooting performance

is unclear. It has been demonstrated

that several

_fungi

_produce

indole-acetic

_{acid, (IAA)}

in

variable amounts, which

_play

a role in the stimulation of

root

_{branching during}

the

_mycorrhizal

colonization

_[8,

25].

Inoculation with

_vegetative

inoculum of L. bicolor at

insertion was successful at the three locations. The

vari-ability

observed in the colonization data is consistent with

the results obtained with

_Douglas

fir

_seedlings

in

previ-ous inoculation

_experiments

with the same

_{fungal species}

[11, 12, 14, 18, 20].

Inoculations with _spores of M.

_ambiguus

and R.

sub-areolatus made at Cabrils resulted in root colonization

percentages similar to those obtained with

_seedlings

in

previous

experiments

[21 ]. At

the other two

locations,

only

spore inoculations with R. subareolatus were

effec-tive. Since

_fungal

inoculum,

plant

material and substrate

were the same in the three

_locations,

the failure of M.

ambiguus

in France could be due to the different

envi-ronmental

_growth

conditions

_{(temperature,}

_humidity,

light)

between France and

_Spain.

The

_methyl

bromide disinfection method

_applied

at

Cabrils also lowered the _percentage of

_{mycorrhizal plants}

obtained after inoculation with L. bicolor and R. subare-olatus. The _presence of toxic residuals in the substrate could alter the

_{fungal development}

after their inoculation.

The lack of effect on M.

_ambiguus

_{may be due} to the

delay

in its

_development

_compared

with that of the other

two

_fungi.

In

this,

and other

_experiments

carried out with

seedlings,

it has been observed that this

_fungus

takes much

_longer

than L. bicolor and R. subareolatus to form

ectomycorrhizas

[21]. It

can be

_hypothesized

_{that spore}

germination

takes

_place

when the toxic effect

_remaining

in the substrate has

_disappeared.

For the three

_fungi,

the

mycorrhizal

short root rate _{among the}

_{mycorrhizal plants}

was not affected

_by

the disinfection

method,

indicating

that the toxic effect on the

_fungi

was

_produced

before the

fungal penetration

into the root.

From these

_experimental

results,

it is

_possible

to use

the available inoculation

_techniques

_during

the

_rooting

phase

to obtain

_{mycorrhizal Douglas}

fir

_cuttings.

Inoculation with certain

_{ectomycorrhizal fungi improved}

rooting performance only

at Cabrils where the

_rooting

percentage of non-inoculated

_cuttings

was

_clearly

under

that obtained at the other two locations. In _any case, sub-strate treatment with steam seems to be the best

disinfec-tion method to

_optimize

both

_{rooting performance}

and short root

_mycorrhizal

colonization. Further research is needed to test the field

_performance

of the

_mycorrhizal

rooted

_cuttings

and to _{compare the} results with those

Acknowledgements:

Financial

_support

for this research was

_{provided by}

the

_European

Union contract

AIR 3 CT 93-1742. The authors also wish to thank A.

Rodriguez,

G.

_Vega

and A. Thivolle-Cazat for their tech-nical _support on

_{rooting techniques.}

REFERENCES

[1] Alvarez I.F., Duñabeitia M.,

Espinel

S., Hormilla S.,

Parladé J., Peña J.I., Pera J., Field

_performance

of containerized

Douglas

fir

_seedlings

inoculated with three

_{hypogeous fungi,}

in:

Azcon-Aguilar

C., Barea J.M. _(Eds.),

_Mycorrhizas

in

_Integrated

Systems

from Genes to Plant

_{Development, European}

Commission

_Report

EUR 16728 EN, Brussels,

Luxembourg,

1994, pp. 413-416.

[2] Alvarez _I.F., Parladé J., Pera J.,

Espinel

S., Bouchard D.,

Le Tacon F., Performance of bareroot

_Douglas

fir

_seedlings

inoculated with Lacoaria bicolor S-238 five years after field

outplanting,

in:

_{Azcon-Aguilar}

C., Barea J.M. _(Eds.),

Mycorrhizas

in

_{Integrated Systems}

from Genes to Plant

Development, European

Commission

_Report

EUR 16728 EN, Brussels, Luxembourg, 1994, pp. 417-419.

[3] Banks H.J.,

Agricultural

Production without

Methyl

Bromide - Four Case Studies, CSIRO Division

_{of Entomology,}

CSIRO Australia, 1995.

[4] Bohmont B.L., The New Pesticide User’s Guide, Reston

Publishing Company

Inc., Reston, VA, 1983.

[5] Castellano M.A., Molina R.,

Mycorrhizae,

in: Landis

T.D., Tinus R.W., McDonald S.E., Barnett J.P. _(Eds.), The Container Tree

_Nursery

Manual

_Agric.

Handbook 674,

Washington

DC, US

_Department

of

_Agriculture,

Forest Service,

1989, pp. 101-167.

[6] Castellano M.A.,

Trappe

J.M., Molina R., Inoculation of

container-grown Douglas

fir with

_{basidiospores}

of

_Rhizopogon

vinicolor and R. colossus: effects of

_fertility

_{and spore}

applica-tion rate, Can. J. For. Res. 15 (1985) 10-13.

[7] Cordell C.E., Marx D.H., Effects of nursery cultural

practices

on _management of

_{specific ectomycorrhizae}

on

bare-root tree

_seedlings,

in:

_Pfleger

F.L., Linderman R.G. _(Eds.),

Mycorrhizae

and Plant Health, APS _Press, St. _{Paul, MN, 1994,}

pp. 133-151.

[8]

Gay

G., Gea L., Rôle de l’auxine

_fongique

dans la for-mation des

_{ectomycorhizes,}

Acta Bot. Gallica 141 ₍₁₉₉₄₎ 491-496.

[9] Généré B.,

Évaluation en jeune plantation

de deux _types de

_plants

de

_{douglas mycorhizés}

_{artificiellement par Laccaria} laccata S 238 _N, Ann. Sci. For. 52 ₍₁₉₉₅₎ 375-384.

[10] Généré B., Le Tacon F., Amirault J.M., Bouchard D.,

La

_{mycorhization}

contrôlée de boutures

_d’épicéa

commun en

pépinière,

Rev. For. Fr. 46 ₍₁₉₉₄₎ 49-58.

[11

] Hung

L.L., Molina R., Use of the

_{ectomycorrhizal}

fun-gus Laccaria laccata in

forestry.

III. Effects of

_commercially

produced

inoculum on

_{container-grown Douglas}

fir and

pon-derosa

_{pine seedlings,}

Can. J. For. Res. 16 ₍₁₉₈₆₎ 802-806.

[12]

Hung

L.L.,

Trappe

J.M.,

Ectomycorrhizal

inoculation of

_Douglas

fir

_transplanted

container

_seedlings

with

commer-cially produced

inoculum, New Forests _{1 (1987)} 141-152.

[13] Le Tacon F., Bouchard D., Effects of different

ectomy-corrhizal

_fungi

on

_growth

of larch,

Douglas

fir, Scots

_pine

and

Norway

spruce

seedlings

in

fumigated

nursery soil, Acta Oecol. Oecol.

_Appl.

7 (4) (1986) 389-402.

[14] Le Tacon F.,

Garbaye

J., Bouchard _D., Chevalier G.,

Olivier J.M., Guimberteau J., Poitou N., Frochot N., Field results from

_{ectomycorrhizal}

inoculation in _France, in: Lalonde

M., Piché Y. _(Eds.),

_Proceedings

of the Canadian

_Workshop

on

Mycorrhizae

in

_Forestry,

1-4

_May

_1988, Centre de recherche en

biologie

forestière, Faculté de foresterie et de

_géodésie,

Université Laval,

Sainte-Foy,

Québec, 1988.

[15] Le Tacon F., Alvarez I.F., Bouchard D., Henrion B.,

Jackson R.M., Luff S., Parladé J., Pera J., Stenström E.,

Villeneuve N., Walker C., Variations in field response of forest

trees to _nursery

_{ectomycorrhizal}

inoculation in

_Europe,

in: Read

D.J., Lewis D.H., Fitter A.H., Alexander I.J. (Eds.),

Mycorrhizas

in Ecosystems, CAB,

Wallingford,

1992, pp. 119-134.

[16] Marx D.H.,

Bryan

W.C., Growth and

_{ectomycorrhizal}

development

of

_{loblolly pine seedlings}

in

_fumigated

soil infest-ed with the

_{fungal symbiont}

Pisolithus tinctorius, For. Sci. 21

(1975) 245-254.

[17] Matthews J.D., The Role of North-West American Trees in Western

_Europe,

H.R. McMillan

_Lectureship

in

Forestry,

Univ. _{BC, Vancouver, BC,} 1983.

[18] Molina R., Use of the

_{ectomycorrhizal fungus}

Laccaria laccata in

_forestry.

I.

_Consistency

between isolates in effective colonization of containerized conifer

_seedlings,

Can. J. For. Res. 12 ₍₁₉₈₂₎ 469-473.

[19] Molina _R.,

_Trappe

_J.M.,

_Mycorrhiza

_management in bareroot nurseries, in: _Duryea M.L., Landis T.D. _(Eds.),

Forestry Nursery

Manual: Production of Bareroot

_Seedlings,

Martinus

_Nijhoff/Dr

W. Junk Publishers, the

_Hague,

1984, pp. 211-223.

[20] Parladé J., Técnicas de inoculación de abeto de

Douglas [Pseudotsuga

menziesii (Mirb.) Franco] con

_hongos

ectomicorrícicos y su

_aplicación

en _{reforestación,} tesis doctoral,

Universidad Autónoma de Barcelona,

Spain,

1992.

[21] Parladé J., Pera J., Alvarez I.F., Inoculation of

con-tainerized

_Pseudotsuga

menziesii and Pinus _pinaster

_seedlings

with spores of five

_species

of

_{ectomycorrhizal fungi,}

Mycorrhiza

6 (1996) 237-245.

[22] Ritchie G.A., The commercial use of conifer rooted

cuttings

in

_forestry:

a world _overview, New Forests 5 ( 1992)

247-275.

[23] Ritchie G.A., Tanaka Y., Meade R., Duke S.D., Field survival and

_{early growth}

of

_Douglas

fir rooted

_cuttings:

rela-tionship

to stem diameter and root system

quality,

For. Ecol.

[24] Shurtleff M.C., How to Control Plant Diseases, Iowa State

_University

Press, Ames, IA, 1966.

[25] Slankis V., Hormonal

_relationship

in

_mycorrhiza,

in:

Marks G.C., Kozlowski T.T. (Eds.),

Ectomycorrhizae -

Their

Ecology

and

_Physiology,

Academic Press, New York, London,

1973, pp. 231-298.

[26] Snedecor G.W., Cochran W.G., Statistical _Methods, Iowa State

University

Press, Ames, IA, 1980.

[27] Stenström E., Ek M., Field

_growth

of Pinus

_sylvestris

following

nursery inoculation with

mycorrhizal fungi,

Can. J. For. Res. 20 (1990) 914-918.

[28] Tinus R.W., McDonald S.E., How to Grow Tree

Seedlings

in Containers in _Greenhouses, USDA Fr. Serv. Gen. Tech. _{Rep. RM-60,}

_Rocky

Mt. For. and

_{Range Exp.}

_Stn., For.