Biology of the ectomycorrhizal genus, Rhizopogon - Wiley Online Library

Neiu Phytol. (1994), 126, 677-690

Biology of the ectomycorrhizal genus, Rhizopogon II. Patterns of host-fungus specificity following spore inoculation of diverse hosts grown in monoculture and dual culture MASSICOTTE^*, RANDY MOLINA^f, DANIEL L. LUOMA^ AND JANE E. SMITH'

BY H U G U E S B.

^Department of Forest Science, Oregon State University, Corvallis, OR 97331, USA ^Department of Agriculture, Forest Service, Pacific Northwest Research Station, Forestry Sciences Laboratory, 3200 Jefferson Way, Corvallis, OR 97331, USA {Received 27 January 1993; accepted 30 September 1993) SliMMARY

Seedlings of Abies grandis, Alnus rubra, Pinus ponderosa, Picea sitchensis, Pseudotsuga menziesii and Tsuga heterophylla were grown in monoculture and dual culture in the greenhouse and inoculated with spore slurries of 20 isolates representing 15 species of ectomycorrhizal hypogeous fungi {11 Rhizopogon species, Alpoz'a diplophloeus, Truncocolumella citrina, Melanogaster euryspermus and Zetleromyces gilkeyae). The primary objectives were to assess and compare the pattern of bost specificity between symbionts and to study the influence of neighbouring plants on ectomycorrhiza development. None of the fungai species had broad host range affinities. A variety of specificity responses were exhibited by the different fungal taxa, rangmg from genus-restricted to intermediate host range. In monoculture, nine species oi Rhizopogon (R. arctostaphyli, R. ellenae, R. flavofibrillosus, R. occidentalis, R. rubescens, R. smithii, R. suhcaerulescens, R. truncatus and R. vulgaris) formed ectomycorrhizas on Pinu.'! ponderosa whevess three Rhizopogon species {R. parksii, R. vinicolor and R. suhcaerulescens) formed ectomycorrbizas on Pseudotsuga menziesii. Truncocolumella citrina associated with Pseudotsuga menziesii and Alpozia diplophloeus with Alnus ruhra. Melanogaster euryspermus and Z. gilkeyae did not form ectomycorrhizas with any hosts. None of the fungi tested developed ectomycorrhizas on Ahies grandis, Tsuga heterophylla or Picea sitchensis in monoculture. In dual culture, the same nine Rhizopogon species that formed abundant ectomycorrhizas on Pinus ponderosa formed some ectomycorrhizas on secondary hosts such as Ahies grandis., Tsuga heterophylla, Pseudotsuga menziesii and Picea sitchensis. Similarly, Truncocolumella citrina formed abundant ectomycorrhizas on Pseudotsuga menziesii and low levels on the secondary hosts Abies grandis, Tsuga heterophylla and Picea sitchensis. Rhizopogon parksii and R. vinicolor only formed ectomycorrhizas on Pseudotsuga menziesii, and Alpova diplophloeus only formed ectomycorrhizas on Alnus rubra. The specificity pattern obtained by using this dual-culture approach is contrasted with previous pure-culture synthesis data and is discussed in terms of potential interplant linkages and community dynamics. Key words; Rhizopogon, ectomycorrhizas, specificity, spore, inoculation.

where Pinaceae are widespread dominants, parINTRODUCTION

The genus Rhizopogon comprises a diverse assemblage of hypogeous ectomycorrhizal fungi that are mostly restricted to the Pinaceae. Most species occur in the coniferous forests of Western North America * Current address: University of British Columbia, Department of Forest Sciences, Faculty of Forestry, MacMillan Building, 193-2357 Main Mall, Vancouver, B.C., Canada V6T t To whom correspondence should be addressed.

i

i

n

..j ^

•

,i\/f

u \ c

j

ttcularly Pseudotsuga menziesn {Mirb.) rranco and Pinus spp. A few Rhizopogon spp. are found in Europe (Smith & Zeller, 1966) and Asia (Bakshi, 1974; Khan, 1980; Hosford & Trappe, 1988), and several species are found in exotic pine plantations worldwide (Molina & Trappe, 1994). In a pureculture synthesis study, MoHna & Trappe (1994) tested 29 isolates from 20 Rkizopogon species for ^j.^orrhizal formation wjth Pseudotsuga menziesii, Tsuga heterophylla (Raf.) Sarg., and Pinus contorta

678

H. B. Massicotte and others

Laws. They report three general responses: strong specificity to Pseudotsuga menziesii, specificity or strongest development on Pinus contorta, and an intermediate response where mycorrhizas were formed on two or three of the hosts. Some of the synthesized ectomycorrhizas, however, were weakly formed or present only in limited numbers; the presence of glucose in the substrate may have contributed to these resu]ts (Duddridge & Read, 1984; Duddridge, 1986a, 6). Molina & Trappe (1994) stressed the need to confirm patterns of host specificity in Rhizopogon by examining mycorrhiza formation in natural soil conditions.

(3) determine the infiuence of primary hosts on development on neighbouring host plants; and (4) evaluate the potential for interplant linkages via compatible mycorrhizal fungi. MATERIALS AND METHODS

Seedling preparation and growth conditions

Seeds of grand fir [Abies grandis (Dougl.) Lindl,], red alder {Alrms rubra Bong.), Sitka spruce [Picea sitchensis (Bongard) Carriere], ponderosa pine (Pinus ponderosa), Douglas fir {Pseudotsuga menziesii) and western hemlock {Tsuga heterophylla) were soaked The concept of ecological specificity was de- overnight in distilled water, spread to dry on paper veloped to emphasize that ectomycorrhizal fungus towels and cold stratified at 4 °Q. for 37 d. Seeds were host ranges seen in nature may differ from those then planted in either "Pine Cells' {60-mI capacity, experimentally determined in pure-culture syntheses 25 mm top diameter, 165 mm long) or ' Super Cells' (Harley & Smith, 1983). In a recent review, Molina, (160-ml capacity, 38 mm top diameter, 210 mm Massicotte & Trappe (1992) point out that ecological long) (Cone-Tainer Nursery, Canby, OR, USA) specificity probably involves a complex set of biotic containing a mixture of equal parts by volume of and environmental factors, many of which are poorly peat and vermiculite, filled to 2 5 cm frotn the top of studied. One such factor, the matrix of neighbouring the container. The smaller containers were used for plants, may afFect the development of certain single tree species (monoculture) and the larger mycorrhizal fungi on particular hosts. Understand- containers for a mixture of two tree species (dual ing vegetation matrix effects becomes important culture). when evaluating the potential for plants to be linked Seeds were planted in each container and covered by shared, compatible fungi in natural ecosystems with a thin layer of w^hite quartz sand (8 grade) to (Molina et al., 1992). reduce splash during watering. Most seeds Several seedling bioassays demonstrate the wide- germinated within 10 d. Abies grandis and Tsuga spread occurrence of Rhizopogon spp. in disturbed heterophylla germinated over a period of one month. and undisturbed forest habitats in Oregon Seedlings were grown in the greenhouse under a (Schoenberger & Perry, 1982; Pilz & Perry, 1984; combination of sunlight and artificial light (280 /(mol Amaranthus & Perry, 1989a, b; Borchers & Perry, m ^' s""^) provided by sodium-vapour lamps. Air 1990; Miller, Koo & Molina, 1992). These studies temperature fluctuated from 21 to 32 °C. Seedlings indicate that some Rhizopogon t>'pes form only on were watered at least twice weekly with tap water. Pseudotsuga menziesii and others are restricted to Each seedling was fertilized monthly with 5 ml of Pinus ponderosa Dougl. How^ever, the soils were Peter's fertilizer (N-P-K/473-449-426 ppm plus bioassayed with single plant species grown in trace elements) applied at half strength. This monoculture. This tests the ability of host seedlings amounts to 11-9 mgN, 113 mgP and 10 7 mgK, to form ectomycorrhizas with fungal propagules applied per seedling over the length of the ex(spores or hyphal fragments) in the disturbed soils, periment. but does not test ectomycorrhiza formation by vegetative mycelium attached to other hosts. Inoculation and host combinations T o determine the potential of hosts to be connected by connpatible mycorrhizal fungi, the host's Hypogeous sporocarps of the test fungi were colresponse to ectomycorrhiza] fungi already physio- lected from different habitats in the Pacific logically associated with other host species can be Northwest over a 6 month period and stored in tap examined. The present study used spore inoculation water at 4 °C until used. Voucher specimens for each and a combination of monoculture and dual culture fungus were deposited with the Oregon State host bioassays to examine the initiation and es- University Herbarium (OSC). Voucher numbers, tablishment of ectomycorrhizal fungi from spore associated host trees and collection numbers are propagules and the influence of neighbouring plants listed in Table 1. Sporocarp characteristics including and their associated fungi on mycorrhizal devel- colour, potassium hydroxide (KOH) reaction, and opment. Our specific objectives were to (1) assess rhizomorph structure were noted for later comhost-fungus specificity and compare it to the results parison with ectomycorrhizal characters. of previous pure culture syntheses with Rhizopogon; Overall, 2,440 containers (monocultures and dual (2) broaden the scope of Pinaceae tested to include cultures of 3,880 seedlings) were inoculated with Picea and Abies (both are widespread forest trees that spores 18 and 21 wk after planting. Of these, 3,737 often associate with Pseudotsuga, Pinus and Tsuga); (96-3%) plants survived for analysis. Each spore

Biology of Rhizopogon //.

679

bc^

2 t:

OO

-^o 3 -r

00

— 00 —

3 o n— n. o V

Z

OS a:

O

-

u c o o

aa

„- ">" „• "H ^' 00 b 3 £ « £ O n; c c -. o p

"I

C

O Qj

us

u Cfi

O £

O

a:

II

0 0

B

D

3 C

II

O

•§• •G.'a "a "s - ^

©

tfl

rfl

l*j

?. N o o o

o

c

'^ a: ^ ^ ^

CO

u u a

^H

U

E

a a J= X • -

3^ S cfi

'2 -2 C ? «J ~ .-S

^"SJ

a ft. P 5

a

0 ;^

11

-c -=: -c -a QSa: a; a;

I CO

H

680

H, B. Massicotte and others

Figures 1-8. For legend see opposite.

Biology of Rhizopogon //.

681

slurry used for inoculation was prepared according (520-560 nm). Percentage viability was calculated by to Castellano & Molina (1989) from 1-2 sporocarps. counting the number of fluorescing spores out of a Each sporocarp was gently brushed to remove soil total of 500 (Table 1). and organic matter, cut into pieces (1-3 cc) and thoroughly blended in 200 ml of distilled water with Ectomycorrhiza assessment a blender at high speed for about 5 min. Spore density was calculated via haematocytometry, and Roots were examined over a 4 month period when the spore suspension was adjusted and divided so the seedlings were 10 to 14 months old. Inoculated that two inoculations of equal amount could be seedlings were removed carefully from the substrate, performed (Table 1). No attempt was made to their roots washed with tap water and ectomycorstandardize the inoculum concentration (number of rhizas examined with the aid of a dissecting microspores ml"') between the different fungi used. For scope. Ectomycorrhiza (and associated rhizomorphs) each inoculation, 10 ml of diluted spore suspension were characterized for form, colour, hyphal dimenwas deposited with a pipette on the top of the sions and morphology, mantle structure, presence of incrustations and crystals, presence of septa and substrate in each container. clamps, and 10"o KOH reaction. Colours were Depending on the availability of germinated recorded under bright tungsten illumination and seedlings, six to 10 replicate containers were designated according to ISCC-NBS standards inoculated with each fungus for each host mono- (Kelly & Judd, 1955; Kelly, 1965) except for culture and dual culture. In addition to the six occasional use of CIC-RBGE standards (Royal monocultures, nine dual cultures were tested (Table Botanic Garden, 1969). Photographs were on 2). To reduce cross-contamination and facilitate Kodachrome 64 film with a camera n:iounted with a growth, seedlings inoculated with the same fungus bellows, 55-mm macro or reversed 28-mm lens, were distributed on four neighbouring racks in the and a ring fiash. For each seedling, the number of following fashion: one rack with all dual cultures ectomycorrhizal root tips of each fungus species was except those containing Alnus rubra, one rack with estimated visually by the classes described in the dual cultures with A. rubra, one rack with A. rubra next section. The presence and abundance of monocultures and one rack with other monocultures. greenhouse ectomycorrhizal contaminants were Because A. rubra monocultures and dual cultures noted but not analyzed further. required frequent watering, they were separated from the other groups. Racks were rotated periodically in the greenhouse over the length of the Statistical analyses experiment. For each seedling and fungal type, the number of tips was converted into abundance classes as follows: Spore viability test 1: 1-5; 2: 6-25; 3: 26-100; 4: 101-1000; and 5: > Spore viability' was assessed with the vital stain 1000 tips. This tip abundance scale approximates a fluorescein diacetate (FDA) by using a modification logarithmic transformation. Examination of the data of methods by Ingham & KJein (1982, 1984) and did not reveal any reasons to question the assumpSoderstrom (1977). Fluorescein diacetate was dis- tions of normality and constant variance. Comsolved in acetone (2 mg ml"') and stored at —20 °C. parisons of mycorrhizal tip abundance among host One ml of FDA in acetone was diluted in 50 ml treatments were made for each inoculated fungus by potassium phosphate buffer (0-2 M, pH 7-6). A 1/10 using one-way analysis of variance (ANOVA). Host dilution of each spore slurry was prepared separately treatments with no ectomycorrhiza formation by in potassium phosphate buffer (0 2 M, pH 5). One ml inoculated fungi were excluded. When the mode! of the 1/10 spore dilution was added to 1 ml of the specified overall differences, between-treatment final FDA dilution. The spores were examined for differences were determined with Fisher's protected fluorescence at 1000 x magnification in non- least significant difference test at P ^ 0 05. Huorescent immersion oil with an epifluorescent microscope after 4 to 16 h of incubation in the dark. Illumination was provided by excitation filter blue RESULTS interference (455^90 nm) and chromatic beam Descriptions of ectomycorrhizas splitter green interference FT 510 and barrier filter Alpova diplophloeus {Ad). On Alnus rubra, ectoFigures 1-8. Ectomycorrhizas synthesized on to ponderosa pine or Douglas fir following spore inoculation with species of Rhizopogon or Truncocolumella citrina. Figure 1. R. occidentalis+ pon