CASIROZ - 2nd meeting: Ectomycorrhizal studies in WP1 & WP2

CASIROZ - 2nd meeting: Ectomycorrhizal studies in WP1 & WP2

Tine GREBENC, Peter ZELEZNIK

& Hojka KRAIGHER

Slovenian Forestry Institute, Ljubljana, SloveniaSlovenian Forestry Institute, Ljubljana, Slovenia

Contribution to CASIROZ & interfaces with other participants & studies

Contribution to CASIROZ & interfaces with other participants & studies

New ECM characterisations & molecular databases Diversity of ECM & differences among plots &

containers / ozone impacts & sensitivities of whole mycorrhizal trees & seedllings

A contribution to studies of above & below-ground interactions / hormonal relationships, roots & ECM in young beech trees

A contribution to simulations of responses of the entire stand to ozone impact

Identification of types of ectomycorrhizae:Identification of types of ectomycorrhizae:

Anatomical characteristics (AGERER 1987-2000), Molecular methods (PCR-ITS-RFLP & sequencing) (GARDES & BRUNS 1993,

as descr. in KRAIGHER & al. 1995; sequencing.: MARTĺN 2000)

©T.Bruns http:/

•Contains ca 400 RFLPs from fruitbodies and ectomycorrhizae (on Norway spruce, European beech, silver fir, austrian pine & alder) from the same FRP. •some types will remain un-identified until GenBank is better filled in with sequencies, •more difficult - also the physiology of most identified types is largely unknown:

The PCR-ITS-RFLP library:

Right: Taxotron® DENDROGRAM from three RFLP patterns

CASIROZ - Ectomycorrhizal studies WP1: ECOPHYSIOLOGY & MODELING

Y1 - 3: •Mapping of fruitbodies & ECM on the whole plot•Identification & characterisation of types of ECM•Collection of herbarium material•Regular analyses, preparation of discussions & reports, presentations & inputs to WP2 & for other partnersY1: •Pilot study of roots & ECM of beech seedlings in containersY2:•ECM identification & quantification on the beech O3 & C plot

Y3:•ECM identification & quantification on the beech O3 & C plot•Study of roots & ECM of beech seedlings in containers

CASIROZ - Ectomycorrhizal studies WP2: BIOCHEMISTRY & MOLECULAR BIOLOGY

CASIROZ - Ectomycorrhizal studies WP2: BIOCHEMISTRY & MOLECULAR BIOLOGY

Y1 - 3: Preparation of PCR-ITS-RFLP database from fungal exicates & ECM

from the whole plot Regular analyses, preparation of discussions & reports, presentations &

inputs to WP1 & for other partnersY1: Build-up of the molecular databases (RFLP & sequencies)Y2: Molecular identifications of ECM Preparation of quantitative PCR for selected sp. / spp. (optional)Y3: Study of ECM of beech seedlings in containers

Methods: Trees in stand

•sporocarp sampling on and around the plot•anatomical-morphological analysis of ECM in soil cores (V=274 ml) - complete results for 3/5 samplings•molecular identification/confirmation of identification and build-up of PCR-RFLP database for ECM on beech

Methods: young beech trees in containers

•WinRhizo analysis of whole root system•anatomical-morphological analysis of ECM on whole root system•molecular identification / confirmation of identification (PCR-RFLP)

1x ozone, shaded

2x ozone, sun

Results: Anatomical-morphological analysis of ECM in soil cores

•in about 1/2 of soil samples beech and spruce roots were intermixed•21 different morphotypes were found and identified at least to the genus level (seven of them on spruce)•Cenococcum geophilum, Russula fellea and R. cyanoxantha were found in most samples•Lactarius pallidus, L. acris, Lactarius sp. and Russula sp.3 (on spruce) were recorded only in one soil sample

•Cenococcum geophilum Fr. •Cortinarius sp.•Cortinarius sp. 2•Genea hispidula Berk. et Br.•Laccaria amethystina Cooke•Lactarius acris (Bolton) Gray•Lactarius blennius/vellereus•Lactarius pallidus Pers. ex Fr.•Lactarius sp. (spruce)•Lactarius sp.2 (spruce)•Lactarius sp.3•Russula cyanoxantha (Schaeff.) Fr.•Russula fellea (Fr.:Fr.) Fr.•Russula illota Romagn.•Russula ochroleuca (Pers.) Fr.•Russula sp.(cf. densifolia Secr.ex. Gillat) (spruce)•Russula sp.2 (Emeticineae) (spruce)•Russula sp.3 (spruce)•Tuber cf. puberulum Berk&Broome (beech and spruce)•Xerocomus chrysenteron (Bull.) Quel.•unknown type (spruce)

Results: Sporocarp sampling on and around the plot

•No sporocarps of ECM species were collected on or around the plot in all five samplings during the year 2003 due to the extreme weather conditions. •For molecular analysis sporocarps collected on the plot in years 1998-2001(by S. Reidl) were used.

Results: Molecular identification and build-up of PCR-RFLP database for ECM on beech

•50 sporocarps from Herbarium in Munich, collected from research plot area were included in PCR-ITS-RFLP database•103 samples of ECM root tips from 3 samplings are to be included and analysed

Results: young beech trees in containers

8 seedlings were analysed by WinRhizo:

Sample codeOzon treatment

PositionLength of root system(cm)

Surface area of root system (cm2)

Volume of root system (cm3)

Root tips

c02p07 2x ambient Sun 8,219,326 1,456,459 2,054 1772c02p10 2x ambient Sun 14,782,978 2,712,535 3,961 3475c02p11 2x ambient Sun 10,214,862 1,954,580 2,976 2389c05p26 2x ambient Shadow 7,283,727 1,351,931 1,997 1557c05p27 2x ambient Shadow 7,792,402 1,115,510 1,271 2359c07p42 ambient Sun 3,603,674 942,973 1,964 1077c09p50 ambient Shadow 8,104,539 1,219,269 1,460 2040c09p54 ambient Shadow 6,999,586 1,169,619 1,555 1538

Not all seedlings were analysed so far!

WinRhizo - analysed root (partial)

Whole root system (tree c09p54)

Results: Analysis of ECM on whole root systems

•high percentage of non-mycorrhizal roots•low percentage of vital ECM root tips•usually one type dominates the root system of a single seedling•since not all seedlings have been completely analysed no correlation to the ozone treatment could be estimated so far.

Biodiversity indices (Atlas & Bartha 1981):Biodiversity indices (Atlas & Bartha 1981):

Species diversity: d = (S-1)/logN; S=# species, N=# individuals

Shannon-Weaver index: H= C/N(NlogN-logni);C=2.3, N=# individuals, ni=# individuals in i-th sp.

Evenness: relative abundance of individuals in each sp. e=H/logS;

Equitability: J=H/Hmax; Hmax= theoretical max. H index for a population, assuming that each sp. has only 1 member

& other.

Biodiversity & bioindication of stress (ecosystem level)

Biodiversity & bioindication of stress (ecosystem level)

Pollution and other anthropogenic stresses diminish biodiversity indices (biodiversity indices also indicate natural successions).

Theoretically the stressed populations with lower diversity are less adaptable to future changes in the environment.

Therefore some indication about impacts of the fumigation on the ecosystem level (soil ecosystem) are expected.

Discussion and problems

•drought in the year 2003 could mask the effect of ozone treatment on mycorrhizal level•there might be a significant human influence (tresspassing on the plot for several years) on the occurence and species composition of ECM•seedlings in containers: which other stresses could influence ECM development (drought, competition, availability of inoculum, time-scale-dependance for development of different ECM…)

Continuation

•According to the plan (more sporocarp sampling would be beneficial, if fruitbodies do appear in 2004)

•Organization of the database

•Publication of the molecular database and ECM descriptions