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Plant and Soil 127, 107-121, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 8231 An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner C. VAN DIJK and A. SLUIMER-STOLK Institute for Ecological Research, Weevers' Duin, Duinzoom 20a, 3233 EG Oostvoorne, The Netherlands Received 1 May 1989. Revised January 1990 Key words: Alnus glutinosa, competition, dune slack, ecology, Frankia, hydroculture, ineffective symbioses, inundation, root nodules Abstract An ineffective strain type of Frankia of unknown strain composition, coded AgI-WD1 was discovered in the soil of wet dune slacks where A. glutinosa was the dominant tree species. Strain type AgI-WD1 was recognized by the development of slow growing root nodules on A. glutinosa testplants inoculated with soil suspensions. Microscopical examination of these nodules showed extremely reduced development of vesicles, normal development of intracellular clusters of hyphae and absence of sporangia. The stability of characteristics of this strain type such as the expression of root nodule symbiosis and ineffectivity of symbiontic N-fixation was demonstrated through 'subculture' of ineffective root nodules in successive hydrocultures of A. glutinosa. The nodulation process also differed from normal effective root nodules by the occurrence of resistance to strain type AgI-WD1 among part of the half-siblings of A. glutinosa used in the nodulation tests. Strain type AgI-WD1 was detected in the soil of different dune slacks which are inundated for a large part of the year and in a nearby peatbog covered with alder. The contribution of this strain type to soil populations of Frankia was demonstrated by nodulation potentials that were up to 500 times higher than that of the concurrent effective strain type AgSp-. The distribution of strain type AgI-WD1 appeared to be restricted to sites with water-logged soil conditions. Nodulation experiments pointed to potentials for competitive interactions between effective and ineffective strain types, especially to a density dependent reduction of nodule type AgI-WD1 by strain type AgSp-. The impact of competitive interactions is also affected by host trees that are resistant to AgI-WD1. The occurrence of resistance in the study areas was suggested by resistance among seedlings of a local seedbatch (_+70% of the half-siblings) and by the absence of ineffective root nodules at site VD7-1, despite a high nodulation potential of the soil population of strain type AgI-WD1. Introduction A prerequisite for the development and mainte- nance of nitrogen fixing root nodules in actino- rhizal plant species is the presence of the mi- crosymbiont Frankia in the root environment. The widespread distribution of root nodules that are effective in nitrogen fixation in natural and semi-natural stands of Alnus glutinosa (Akker- roans and van Dijk, 1981; Bond, 1976; Van Dijk, 1984) reflects the distribution of effective Fran- kia strains in the soil of host stands. Strains of Frankia which are persistent in par- tial compatibility (infective, but highly ineffec- tive in symbiotic N2-fixation ) with appropriate host species were isolated from effective root nodules (Baker et al., 1980; Hahn et al., 1988, Lechevalier et al., 1983). The origin of these

An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

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Page 1: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

Plant and Soil 127, 107-121, 1990. © 1990 Kluwer Academic Publishers. Printed in the Netherlands. PLSO 8231

An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

C. VAN DIJK and A. SLUIMER-STOLK Institute for Ecological Research, Weevers' Duin, Duinzoom 20a, 3233 EG Oostvoorne, The Netherlands

Received 1 May 1989. Revised January 1990

Key words: Alnus glutinosa, competition, dune slack, ecology, Frankia, hydroculture, ineffective symbioses, inundation, root nodules

Abstract

An ineffective strain type of Frankia of unknown strain composition, coded AgI-WD1 was discovered in the soil of wet dune slacks where A. glutinosa was the dominant tree species.

Strain type AgI-WD1 was recognized by the development of slow growing root nodules on A. glutinosa testplants inoculated with soil suspensions. Microscopical examination of these nodules showed extremely reduced development of vesicles, normal development of intracellular clusters of hyphae and absence of sporangia. The stability of characteristics of this strain type such as the expression of root nodule symbiosis and ineffectivity of symbiontic N-fixation was demonstrated through 'subculture' of ineffective root nodules in successive hydrocultures of A. glutinosa. The nodulation process also differed from normal effective root nodules by the occurrence of resistance to strain type AgI-WD1 among part of the half-siblings of A. glutinosa used in the nodulation tests. Strain type AgI-WD1 was detected in the soil of different dune slacks which are inundated for a large part of the year and in a nearby peatbog covered with alder. The contribution of this strain type to soil populations of Frankia was demonstrated by nodulation potentials that were up to 500 times higher than that of the concurrent effective strain type AgSp-. The distribution of strain type AgI-WD1 appeared to be restricted to sites with water-logged soil conditions. Nodulation experiments pointed to potentials for competitive interactions between effective and ineffective strain types, especially to a density dependent reduction of nodule type AgI-WD1 by strain type AgSp-. The impact of competitive interactions is also affected by host trees that are resistant to AgI-WD1. The occurrence of resistance in the study areas was suggested by resistance among seedlings of a local seedbatch (_+70% of the half-siblings) and by the absence of ineffective root nodules at site VD7-1, despite a high nodulation potential of the soil population of strain type AgI-WD1.

Introduction

A prerequisite for the development and mainte- nance of nitrogen fixing root nodules in actino- rhizal plant species is the presence of the mi- crosymbiont Frankia in the root environment. The widespread distribution of root nodules that are effective in nitrogen fixation in natural and semi-natural stands of Alnus glutinosa (Akker-

roans and van Dijk, 1981; Bond, 1976; Van Dijk, 1984) reflects the distribution of effective Fran- kia strains in the soil of host stands.

Strains of Frankia which are persistent in par- tial compatibility (infective, but highly ineffec- tive in symbiotic N2-fixation ) with appropriate host species were isolated from effective root nodules (Baker et al., 1980; Hahn et al., 1988, Lechevalier et al., 1983). The origin of these

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108 van Dijk and Sluimer-Stolk

strains and their impact on the establishment of effective actorhizal symbioses is still obscure. The occurrence of ineffective root nodules in field populations of A. glutinosa has not yet been recorded. The occurrence of host-induced inef- fectivity in soil populations of Frankia has only been observed with strain type AiSp ÷ Finland which is phenotypically effective on Alnus in- cana, but ineffective on A. glutinosa (Van Dijk et al., 1988). The term strain type was introduced to designate groups of specified and unspecified strains of Frankia, which share one or more distinctive characteristics.

This paper presents a study on the occurrence of soil populations of an ineffective strain type of Frankia in dune slack vegetation with stands of A. glutinosa. In nodulation experiments poten- tials for competition between effective and inef- fective Frankia were studied. The results are discussed with reference to the ecological signifi- cance of this strain type and occurrence of host resistance.

Materials and methods

Description sampling sites

On the Isle of Voorne, the Netherlands, loca- tions Voorne's Duin (VD7, VD8), Quackjeswa- ter (Q) and Meertje de Waal (M) were selected because of the presence of natural stands of A. glutinosa and water-logged soil conditions. All locations have been managed as nature reserves and human influence has been negligible for at least 40 years. Co-ordinates of the Dutch State Survey Grid (S.S.G.) indicate the geographical position of the study areas.

Locations VD7, VD8, and Q represent wet dune slacks of the coastal sanddune area where natural fluctuations of the groundwater table are considerable and cause annual inundation of long duration. The root zone at these locations consists of calcareous mineral sand covered with 5-15 cm of organic material. Location M is part of a floating peat bog area and situated on the land side of the sanddune area. The mineral subsoil is at least 4 m below the rootzone. The pH of the upper 20-cm of soil in the study areas ranged between 6.2-7.3.

Location VD7 (S.S.G. 64.2; 436.8) is situated in a 60-years-old primary dune valley covered with a dense vegetation of woody species and herbs. A. glutinosa was dominant in the lowest parts. Four sampling sites were selected within an area of 50 x 50 m.

Sampling sites VD7-1 and VD7-2 were situated in the lowest parts of the valley which was inundated for 8---10 months annually and had almost permanently water-logged soil. At these sites A. glutinosa was the dominant tree species (70% crown-cover) with a sparse under- story of Mentha aquatica (perc. cover <5%).

The soil at site VD7-4 is usually water-logged in winter and early spring, but in summer the upper 20---30 cm of the soil are usually well drained. Both Betula pendula and A. glutinosa were present with a dense understory of M. aquatica, Cardamine pratensis, L ycopus europaeus, Hydrocotyle vulgaris, and Iris pseudacorus (perc. total cover: 60-90%).

Sampling site VD7-3 had an intermediate posi- tion between VD7-2 and VD7-4 with respect to soil humidity and understory.

Location VD8 (S.S.G. 64.6; 436.5), with one sampling site, was originally shaped as a wide ditch, but has not been maintained as such for the last 40 years. A. glutinosa was present along the ditch and gave 100% crown cover. Natural ground water movement caused inundation in winter and early spring and drier conditions in summer. The soil humidity was comparable to site VD7-3. A sparse herb layer (10% vegetation cover) was composed mainly of M. aquatica, Scutellaria galericulata and Galium palustre.

Location Q (S.S.G. 64.7; 429.9) is part of a late-medieval dune lake, with an old stand of A. glutinosa. Sampling sites Q1-Q4 were situated in permanently inundated sites with a dominant vegetation of I. pseudacorus. Sites were chosen at random within an area of 25 x 72 m 2.

Location M (S.S.G. 64.3; 432.9) had a dense vegetation of young alder with a high natural turn-over of trees. The soil surface showed strong heterogeneity due to a high density of old tufts of Carex spec. The peat layer between the tufts was 20-50 cm thick and floating on aqueous mud. Sampling sites M1-4 were chosen at ran- dom within an area of 10 x 15 m. Soil pH varied between 6.2 and 6.7. Local abundance of Rubus

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spec. indicated eutrophic soil conditions. Studies by Blauw (1917) suggest that a very similar situation with abundantly nodulated alder has been present for at least 70 years.

Culture of testplants

A seedlot of A. glutinosa collected from one tree at location Voorne coded V22 was used for hydroculture of testplants. For the third sub- culture of strain type AgI-WD1 testplants of A. glutinosa were raised from a highly susceptible half-sib seedlot coded $3-531 from experimental alder stands in G6ttingen, FRG (kindly provided by Dr Linares). Test plants of Alnus nitida Endl. were raised from a seedlot from Pakistan (kindly provided by Dr A H Chaudhary).

Seed was surface sterilized with 0.1% bromine solution for three minutes and left to germinate on 3 mm glass beads in demineralized water. After two weeks the plants were transferred to a half-strength modified Hoagland solution pH 6.8 (Quispel, 1954). This nutrient solution contained trace elements according to Allen and Arnon (1955) and 0.02 mM Fe-citrate. Six weeks after germination plants were transferred to 5-L jars containing full strength modified Hoagland solu- tion with reduced N-content (0.375 mM NO 3 as the sole source of N). Plants were raised in Conviron growth cabinets at 25°C, illuminated for 16 h with Sylvania VHO (45 W m-2, PI) at a relative humidity of 80%. After selection and redistribution plants were inoculated and were subsequently grown in a glasshouse at 23--+ 2°C. Additional illumination with Philips HLRG 400W at an irradiance (PI) of 3 0 W m -2 pro-

- I vided a minimum photoperiod of 14h day Contamination with extraneous Frankiae was avoided by strict hygienic measures and use of plastic shelters. Details on numbers of jars, test plants etc. are given in the Tables and Figures.

Preparation of inocula

At each sampling site, after removal of surface litter, 15 cores of 5 cm ~b were collected at ran- dom from the upper 20cm of the soil. Soil samples were sealed in plastic bags and tools were disinfected between the sampling proce- dures. All cores per sampling site were mixed

Ineffective Frankia in Alder stands 109

thoroughly and thick roots and occasional nodule fragments were removed.

Of each mixed sample 400 mL soil was sus- pended in 1.5 L of the modified Hoagland nu- trient solution with reduced N-content. The sus- pension was stirred for 30 minutes and sub- sequently filtered through 2- and 1-mm mesh filters. Filters were washed with an equal volume of the modified Hoagland nutrient solution. The quantity of soil suspension added to the jars with test plants is expressed in terms of the volume of soil used initially for preparation of the suspen- sion. The volume of soil lost during filtration was not determined. Unless otherwise stated soil sus- pensions representing 20 mL fresh weight of soil were added to 1-L jars with 12 testplants per jar.

Root nodule homogenates were prepared from fresh field sampled AgSp- type nodules and from fresh ineffective nodules grown in hydro- culture. Nodule lobes were surface sterilized with 0.1% v/v bromine solution for 1 min and were homogenized in respectively 80 and 5 mL of the modified Hoagland nutrient solution, with reduced N-content using a 'Virtis 45' homogen- izer operated at 30.000rpm for 2min. The homogenate was filtered to remove particles >100/~m. The filtrate was diluted and added to hydrocultures with testplants.

For nodulation experiments in water-logged soil 2 glass jars of 2L were each filled with 1900mL of a mixed soil sample (0-20cm) of location VD7-1, thus providing a soil column of 19 cm high (~b 11 cm). In each jar 100mL of a 3 x concentrated Hoagland nutrient solution with reduced N was mixed through the upper 10-cm of the soil. Water level was maintained at 0.5 cm above the soil surface. After 1 week of equilibration 8 seedlings of A. glutinosa with emerging first leaf were placed on each jar. Similar jars with Hoagland solution inoculated with 40 mL of suspended soil were used as hy- droculture reference. Nodules were counted 10 weeks after planting.

Microscopy

Hand cut nodule sections, 30-50/zm thick, were stained with Fabil reagent and screened under the light-microscope for the occurrence of vesi- cles and spores (Van Dijk and Merkus, 1976).

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110 van Dijk and Sluimer-Stolk

Nodulation capacity

Calculation of nodulation capacities, viz. the number of nodulating units per unit quantity of inoculum, has been previously described (Van Dijk, 1984; Van Dijk et al., 1987). Analysis of Variance was applied for data obtained from serial dilutions (N van der Hoeven, pers. comm.).

Results

Description of ineffective root nodules

In nodulation tests most soil samples that were collected near alder trees at different sites in the dune area of Voorne, developed low numbers of fast growing actinorhizal root nodules. The latter were distributed according to a random pattern among all test plants. However, in two nodula- tions tests, with soil samples from locations VD7 and VD8 some test plants also showed a large number of slow growing root nodules.

Microscopical examination of these slow grow- ing root nodules showed that their anatomical structure and the initial stages of actinomycetous cortex infections were not different from com- mon effective actinorhizal root nodules of A. glutinosa. However, the development of terminal vesicles upon proliferation of intracellular hy- phae was almost completely blocked. The abundance of tight clusters of intracellular hy- phae without vesicles suggested that the propor- tion of infected cells and hyphal proliferation was not different from normal effective root nodules with abundant vesicle clusters. A small portion of the mature clusters of hyphae, less than 10%, showed incidentally the presence of few immature vesicles, but the development of full-grown clusters of vesicles which are charac- teristic of effective root nodules in Alnus were not observed in any of these ineffective nodules. Endophytic sporangia which are characteristic of spore-positive (Sp + ) strain types of Frankia were not found in any of the ineffective nodules from the Voorne region.

The nodule type was coded AgI-WD1 and pure cultures of the endophyte of these ineffec-

tive nodules, isolated by Dr A D L Akkermans (Agricultural University of Wageningen, The Netherlands) showed undifferentiated hyphae as has been observed in other non-N-fixing (ineffec- tive) strains of Frankia (Hahn et al., 1988). Hence root nodule type and Frankia strain type were both coded AgI-WD1.

Distribution of Frankia strain types AgI-WD1 and AgSp- in the soil of dune slacks VD7 and VD8

The occurrence of Frankia strain types AgSp- and AgI-WDI in the soil of dune slacks VD7 and VD8 was confirmed in nodulation tests with hydrocultures of A. glutinosa and soil suspen- sions as inocula. Numbers of nodules of both strain types were scored 30 and 60 days ~/fter inoculation of the test plants. The results pre- sented in Table 1. showed that a minor part (n) of the 12 testplants in each jar (20-40%) was abundantly nodulated with nodule type AgI- WD1 while the remaining testplants, exposed to the same inocula, were completely free from this nodule type. It is expected that the distribution of nodules among testplants from the same jar follows a Poisson distribution. However at mean numbers of I>22 nodules of type AgI-WD1 per plant (jars A-D, non-nodulated plants included) non-nodulated plants are not expected to occur (p~0.001) . The same holds for jars E-F and G-H inoculated with soil VD8, which had much lower arithmetic means of 3.8 and 5.8 nodules of type AgI-WD1 per plant (non-nodulating plants included). It is concluded that in these experi- ments plants without nodules of type AgI-WD1 were not susceptible to the soil population of Frankia strain type AgI-WD1. As a consequence plants without nodule type AgI-WD1 were ex- cluded from calculation of nodulation response (mean number of nodules per plant) to the soil samples.

Thirty days after inoculation the soil sample from location VD7 had produced a mean num- ber of 112 nodules per plant of type AgI-WD1 on 6 (n) out of 24 testplants (jars A, B). Sixty days after inoculation there were 104 nodules of type AgI-WD1 per nodulated plant (n = 9; jars C,D).

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Ineffective Frankia in Alder stands l 11

Table 1. Nodulation response of Alnus glutinosa inoculated with soil populations of Frankia from dune slacks VD7 and VD8. Numbers of root nodule types AgI-WDI and AgSp- were recorded 30 and 60 days after inoculation

Nodulation period (days)

Inoculum VD7 Inoculum VD8

No. of nodules plant 1 No. of nodules plant l

Jar" Aglb n AgSp " Jar" AgIb n AgSp •

30 A 135 (3) 0 E 18 (2) 0.4 B 90 (3) 0 F 22 (2) l)

60 C 133 (4) 0.08 G 7 (2) 0 D 82 (5) (LOS H 63 (2) 0.8

~' 12 plants per jar. mean numbers of nodules (type AgI-WD1) per plant, calculated only from nodulated plants (n). mean number of nodules (type AgSp ) per plant determined from all plants (12) per jar.

Soil from location VD8 produced 18 (n=5 ; jars E, F) and 35 (n = 4; jars G, H) nodules of type AgI-WD1 per susceptible plant after 30 and 60 days of nodule development, respectively.

In the same series of testplants very low num- bers of nodules were produced by the effective Frankia strain type AgSp in samples VD7 and VD8 (Table 1) and non-susceptibility among testplants could not be judged properly. How- ever, experience with this strain type in previous nodulation experiments and the distribution of Sp nodules among testplants in the present paper made us to assume that all testplants were susceptible to strain type AgSp . A mean nodu- lation response of 0.2 nodules per plant (VD7) and 0.7 nodules per plant (VD8) was observed 60 days after inoculation. As a consequence the nodulation response of nodule types AgSp- and AgI-WD1 had ratios of 1:520 for soil VD7 and 1:50 for soil VD8.

The ratios of AgI-WD1 nodulated and non- nodulated plants per jar (n = 12) were not signifi- cantly different between the treatments nodula- tion period and location (chi-square 3.111, p = 0.375, d f= 3). The percentage of AgI-WD1 re- sistant plants among all testplants (n=96) amounted to 75%.

Plants that were well nodulated with AgI-WD1 but lacking AgSp nodules showed decreased growth rate and leaves turned yellow, whereas plants which had also AgSp type root nodules had a high growth rate and no symptoms of N-deficiency. This indicated the absence of any substantial N-fixation activity in root nodule type AgI-WD 1.

D&tribution of AgI-WD1 in dune slack VD7 related to water-logging of the soil

In the previous experiment the abundance of Frankia strain type AgI-WD1 in the soil of loca- tion VD7 was demonstrated for one sampling site that covered 4 m 2 of the lowest part of this valley. In the following experiment the distribu- tion of AgI-WD1 in valley VD7 was studied in greater detail by the choice of 4 different sam- pling sites, coded VD7-1, 2, 3 or 4, with slightly different elevation and inundation charac- teristics.

Nodulation capacities of Frankia strain types AgSp- and AgI-WD1 were determined for each soil sample by means of nodulation tests with hydrocultures of A. glutinosa and dilution series of soil samples as inocula. In all soil samples strain type AgI-WD1 caused high to moderate nodule numbers among a small number of the test plants, but strain type AgSp- caused low numbers of nodules and the majority of plants without this nodule type could be explained by Poisson distribution. In Table 2 the mean nodule numbers per plant (all plants per treatment, n=36 ; VD7-2, 20mL, n = 2 4 ) are presented together with the percentages of non-nodulated testplants in excess of the Poisson probability. At location VD7-4 strain type AgI-WD1 was absent (not included in Table 2). A share of 68% (S.D. = 9%) of the testplants (seedbatch V22) was found to be non-nodulated by Frankia strain type AgI-WD1, irrespective of sampling site and of inoculum concentrations that produced 3.6-55 nodules per plant. These results are in agree-

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112 van Dijk and Sluimer-Stolk

Table 2. The occurrence of non-nodulated testplants of seedbatch Voorne22 in excess of expectation at random (Poisson) distributions of nodule types AgI-WD1 and AgSp-. Both nodule types developed from common exposure of testplants to dilution series of soil samples from location VD7. The absence of nodule development per plant refers only to the nodule type under study.

Soil Inoculum AgI-WD 1 AgSp-

sample density Mean No. of Surplus a of Mean no. of Surplus a of mL jar 1 nod. plant -1 non-nod plants nod. plant 1 non-nod plants

(n = 36) b (% of n) (n = 36) 8 (% of n)

VD7-1 20 55 58 0.9 17 2 44 64 0.3 6 0.4 4.3 83 0.2 3

VD7-2 20 58 c 63 6.8 c 0 2 31 66 2.2 8 0.4 6.8 78 0.7 14

VD7-3 20 20 78 2.8 8 2 11 61 0.6 14 0.4 3.6 64 0.2 3

a non-nodulated plants in excess of Poisson distribution. b 3 jars with 12 plants each. c n = 24 plants.

ment with experiments ment ioned above. In the same groups of testplants the distribution of nodule type A g S p - is different with respect to low mean numbers of nodules and only a small share of non-nodulated testplants (8%) in excess of the Poisson distribution. This excess is not explained by non-susceptibility of testplant genotypes and tends to disappear with increasing nodule numbers (Table 2, sample VD7-2, 20 mL; Fig. 1). In this case the slight excess of non- nodulating plants is most likely explained by clumping effects due to uncontrolled variation in the nodulation test (plant size, inoculum distri- bution). The distribution of nodule type A g S p - among the testplants was independent of non- susceptibility to strain type AgI-WD1.

The nodulation response to each strain type, presented in Table 3, is given as the total num- ber of root nodules per jar ( three jars per treat- ment) at successive inoculum dilutions. The nodulation response of Frankia AgI-WD1 was corrected for the presence of non-susceptible testplants by multiplication of the number of AgI-WD1 nodules per jar with a factor ntot/nsus (total number of plants j a r - I / n u m b e r of AgI- WD1 susceptible plants j a r - l ) . In all jars the numbers of AgI-WD1 nodules per susceptible plant were high enough not to expect Poisson distributed scores in nodule class 0. Clearly, the nodulation response of strain type A g S p - did not

need such corrections. Da ta for calculation of the nodulation capacity were selected on the basis of linearity between dilution series and nodule numbers. Nodulat ion capacities of Fran- kia strain types AgI-WD1 and A g S p - in the 4 soil samples are presented in Table 4.

The nodulation capacities of strain type AgI- WD1 showed a positive correlation with the period of inundation of the soil, and were most prominent for the wet sites VD7-1 and VD7-2. Absence of this strain type (density below detec- tion level) was recorded for the least inundated site VD7-4 with some aerat ion of the upper 20-30 cm soil during summer and early autumn. The intermediate position of sample site VD7-3 with respect to inundation and water saturation of the soil was also expressed by the modera te nodulation response of strain type AgI-WD1. Strain type AgSp- had low nodulation capacities at all sampling sites. The ratio of nodulation capacities of strain types AgSp- and AgI-WD1 was 1:140 at location VD7-1, 1:45 at locations VD7-2 and VD7-3 but in VD7-4 this ratio was est imated as >40:1, due to the absence of nodule type AgI-WD 1.

Nodulation potential of AgI-WD1 in symbiotic association

Homogena tes of ineffective nodules grown in the

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Ineffective Frankia in Alder stands 113

nodulation experiment with soil sample VD7-1 were used to initiate successive subcultures of strain type AgI-WD1 in symbiotic assocation with testplants of A. glutinosa in hydroculture. Three subcultures of individual nodules thus pro- vided 4 successive 'generations' of strain type AgI-WD1.

The first two subcultures were made with plants from seedbatch V22, with -+70% of non- susceptibility to strain type AgI-WD1 from soil

suspensions. In order to reduce the number of testplants required to obtain nodule develop- ment, the third subculture was carried out with testplants from a different seedbatch, viz. $3-513 G6ttingen, with only 20% of non-susceptibility to soil productions of AgI-WD1 (data not pre- sented).

Individual nodules used for subculture were 8 weeks old and had fresh weights ranging from 6 to 42mg. Surface sterilization (0.1% Br~ for

Table 3. Nodulation response of Alnus glutinosa inoculated with serial dilutions of soil populations of Frankia from 4 sites at location VD7. Inoculum density in ml fresh soil added per jar. Numbers of nodule types AgI-WD1 and AgSp- per jar (12 plants), with corrections for the presence of Agl-WD1 resistant host plants

Soil lnoculum AgI -WDI AgSp-

sample density No. of nodules No. of nodules jar ~ No. of nodules jar mL jar 1 jar -~ (p" plants) p" (12 plants) b (12 plants)

VD7-1 20 483 5 1159 12 20 1044 6 2088 8 20 466 3 1864 13

2 644 5 1546 *c 4 2 783 5 1880" 3 2 182 3 732* 1 0.4 60 2 360* 0* 0.4 22 1 264* 3* 0.4 74 3 296* 3*

VD7-2 20 636 4 1908 68 20 758 5 1819 94 20 . . . .

2 792 6 1584 33 2 200 3 800 18 2 128 3 512 29 0.4 59 2 354* 9* 0.4 136 4 408* 6* 0.4 52 2 312" 11"

VD7-3 20 357 3 1428 44 20 92 2 552 24 20 253 3 1012 31

2 101 5 242 3 2 182 5 437 14 2 96 4 288 6 0.4 44 5 106" 1" 0.4 42 3 168" 1" 0.4 45 5 108" 6*

VD7-4 20 0 0 0 11 20 0 0 0 4 20 0 0 0 35

2 0 0 0 6* 2 0 0 0 2* 2 0 0 0 1" 0.4 0 0 0 1 * 0.4 0 0 0 1" 0.4 0 0 0 0*

" Number of AgI-WDI susceptible plants per jar. b Estimated nodule number in case of 12 (all) susceptible plants c data (*) selected for calculation of the nodulation capacities.

per jar.

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114 van Dijk and Sluimer-Stolk

Table 4. Nodulation capacities (numbers of nodules produced per ml soil) of Frankia strain type AgI-WD1 and AgSp in soil samples from 4 sites at location VD7. Calculated from * labelled data in Table 3

Soil Elevation a pH Nodulation capacity (number mL 1 soil) sample soil surface

(cm) H20 KC1 AgI-WD1 s.e. AgSp- s.e.

VD7-1 0 6.7 6.5 700 a b 100 5 d 2.0 VD7-2 +14 6.8 6.6 900 a 60 20 e 3.0 VD7-3 +21 7.3 7.1 320 b 40 7 d 3.4 VD7-4 +25 6.6 6.2 0 c c - 2 d 0.5

a Relative to elevation of sampling site VD7-1. b Different lettercodes represent significant differences at P >/0.05. c Detection level 0.05 nod. unit mL -1 soil.

1 min) prior to homogenization reduced risks of exterior contaminations. Ineffective root nodules thus produced had all morphological characteris- tics of nodule type AgI-WD1 as primarily pro- duced by the soil inocula. Nodulated plants tur- ned yellow by the lack of N-fixation activity. Effective root nodules were not present in any of the subcultures.

Twelve homogenates of individual nodules from 3 successive generations of hydrocultures of Alnus glutinosa (both seedbatch V22 and $3-531) produced 10.4 (sd7.8) nodules mg -1 fresh nodule homogenate on the testplants. These data include corrections for the presence of non- susceptible plants in each of the seedbatches. Non-susceptibility among part of the test plants was evident, but especially in seedbatch V22 the combination of low numbers of test plants and low numbers of nodules did not allow appropri- ate tests for Poisson distribution. In the nodula- tion tests with homogenates of individual nodules from the third generation and testplants from seedbatch S3-531-G6ttingen a surplus of 18-44% of non-nodulated plants in excess of the Poisson distribution was observed among a total of 44 testplants. These data cover the 20% non- susceptibility of this seedbatch, as determined with soil suspensions from VD7 and suggest that compatibility of tests plants is not affected by a different origin (symbiotic- or soil population) of the Frankia strain type AgI-WD1. As a con- sequence 70% of the testplants of seedbatch V22 was assumed non-susceptible to the nodule homogenates, conform to the results obtained with soil populations of AgI-WD1 from locations VD7 and VD8.

The small dimensions and low infectivity of

AgI-WD1 nodules grown in hydroculture, as well as the absence of perennial nodules of this type in the field prevented the use of nodule homoge- hates in the quantitative studies on nodulation characteristics of this strain type. Instead, soil homogenates of location VD7-1 that produced high numbers of almost exclusively AgI-WD1 type nodules offered a satisfactory alternative as a source of AgI-WD1 inoculum in large scale nodulation experiments.

Interactions between strain types AgSp- and AgI-WD1

In order to compare nodulation patterns of Fran- kia strain types AgSp- and AgI-WD1 and pos- sible interactions between them, seedlings of A. glutinosa were inoculated with 20 g of a soil suspension that contained almost exclusively strain type AgI-WD1 (location VD7), either pure (series 00-20) or premixed with 1 or 10 mg of an AgSp- nodule homogenate (series 01-20 and 10-20 respectively) from the same location. Series 50k-20 (50mg heat-inactivated nodule homogenate, 20 g soil) served as a reference for the influence on nodule development of non-vital substances present in the nodule homogenates. The numbers of nodule types AgI-WD1 and AgSp- were recorded 8 weeks after inoculation of the testplants.

The frequency distributions of both types of nodules formed on the testplants of series 10-20 are presented in Figure 1. The distribution of nodule type AgSp- (21.9 nodules per plant) fits a Poisson distribution (G-test Williams, 0 . 5 > p > 0.1) and the absence of non-nodulated plants confirmed that all testplants were susceptible to

Page 9: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

Frankia strain type AgSp-. Contrary to this, the distribution of nodule type AgI-WD1 showed an aberrant peak in class 0 which is covered by 52 testplants (n = 60) at an arithmetic mean of 5.3 nodules per plant. In case of a Poisson distribu- tion the expected frequency in class 0 at n = 60 would be <1 plant (rei. expected frequency of Poisson distr.: 0.0049 at u = 5.3 and y = 0). The fraction of plants that were present in class 0, beyond expectation of the Poisson distribution (87% of the testplants in series 10-20) were considered non-susceptible to strain type AgI- WD1.

In series 00-20, Figure 2, the low nodulation response of strain type AgSp- relative to AgI- WD1 in the soil of location VD7 is illustrated by a ratio of nodule numbers of 1:512, which is in close agreement with the results of sample VD7.

Similar numbers of nodule type AgI-WD1 in series 00-20 and 50k-20 indicated that the addi- tion to the soil suspensions of different quantities of non-vital fragments and soluble substances from nodule homogenates did not affect the nodulation response in series 01-20 and 10-20.

Ineffective Frankia in Alder stands 115

The addition of strain type AgSp to the soil suspension, significantly reduced the average number of ineffective (AgI-WD1) nodules per susceptible plant from 89 (series 00-20) to 53 (series 01-20) and 40 nodules (series 10-20; Rank sum test, p = 0.028).

Adversely, in series 01-20, Figure 3, the num- ber of AgSp nodules was slightly lower in test- plants that had also developed nodule type AgI- WD1 (AgI-WDl-susceptible plants) than in the testplants without this nodule type (AgI-WD1- resistant plants). This reduction was expressed by average numbers of 3.2 and 5.4 nodules of type AgSp- per plant, respectively (Rank sum test, p = 0.021). In series 10-20 the number of AgSp- nodules was not affected by the presence of nodule type AgI-WD1.

The ratios of testplants that were susceptible and resistant to strain type AgI-WD1 were not clearly different between the treatments (chi- square test for heterogeneity, chi-square 7.771, d f = 3 , p =0.051). The over-all percentage of AgI-WD1 resistant test plants (seedbatch V22, n = 238) amounted to 73%.

>, 0

(1)

O- (D

q -

100

9 0

8 0

7 0

nodule t y p e "

I I A g S p -

A g I - W D 1

6 0

5 0

4 0

3 0

2 0

10

0 FL 0 1-10

21 .9

11"-20 21--30 31--40 41--50

number of nodules

r - - ] r ' r l

5t--60 61-70 71--80 81--90

per plant Fig. 1. Frequency distributions of nodule numbers per plant between 60 test plants of A. glutinosa inoculated with a mixture of Frankia strain types AgSp- and AgI-WD1 (series 10-20). Arrows indicate over-all mean number of root nodules per susceptible plant (nodule type AgSp n = 60; nodule type AgI-WD1, n = 8).

Page 10: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

116 van Dijk and Sluimer-Stolk

r- e 1 0 0 0.. b

9 0

__e 8 O

'u 70 0 "- 6 0 4-' o 5 o L .

4- 4 0 0 ~- 3 0

2 0

10 a C 0 ¢ E 0 0 - 2 0

n: 5 9 18

b

a.

nodule type"

J J AgSp-

J ~ Ag T - W D 1

d

c

I

d

5 0 k - 2 0 60 20

0 1 - 2 0 1 0 - 2 0 6 0 18 6 0 8

inoculum combinations ( A g S p - - A g E ) Fig. 2. The nodulation response of A. glutinosa inoculated with mixtures of Frankia strain types AgSp and AgI-WD1. Inoculum combinations contain 20 mL soil sample VD7-1 as a source of strain type AgI-WD1 (coded 20), respectively mixed with 0 mg (coded 00), 50 mg heat-killed (coded 50k), 1 mg (coded 01) or 10 mg (coded 10) AgSp- nodule homogenate, n: number of test plants used for calculation of the nodulation response. Significant differences between mean values are indicated by different letters.

Distribution of strain type AgI-WD1 in locations Q and M

The previous experiments suggested that abund- ance of AgI-WD1 might be a more general phe- nomenon in long-term inundated alder vegeta- tion. To test the validity of this suggestion the distribution of strain type AgI-WD1 was also investigated in the soil at 4 permanently sub- merged sites Q 1 - Q 4 of an old dune slack, Quack- jeswater, and at 4 sites M1-4 of a peak bog, location Meert je de Waal. Nodulation tests were carried out with seedlings of Alnus nitida. This species had proven to be approximately 100% susceptible to AgI-WD1 and was therefore very suitable to study the distribution of this strain type.

Contrary to nodulation experiments with soil from VD7, differences in growth rate between nodule types AgSp- and AgI-WD1 from Q1-4

were small enough to cause significant overlap in the size of both nodule types. This was because of the slower growth rate of AgSp- nodules due to relatively high numbers of this nodule type per plant. Therefore the share of each nodule type was estimated entirely by microscopical ex- amination of fresh sections of 10% of the num- bers of nodules in each jar.

The results are presented in Table 5. The samples Q 1 - Q 4 showed a significant nodulation response of both strain types. Strain type AgSp- was dominant at sites Q1-3 . The nodulation tests with soil samples M1 and M2 were not successful due to severe root rot of testplants within 2 weeks after inoculation. The causative organism was determined as a Pythium species. In sample M3 the nodulation response was nega- tive despite good growth of testplants. Soil from site M4 caused a high nodulation response of exclusively AgI-WD1 nodules. Effective AgSp-

Page 11: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

Ineffective Frankia in Alder stands 117

"4--' f.-

Q_

cO ¢

(3 0 f-

I Q_

03 03 <f. q..-

0 k - -

(19 _O E (-

25

20

15

10

5

0

p l a n t s r e s i s t a n t

to A g Z - W D 1

plants susceptible to A g T - W D 1

8 a

b c

d i / i / 1 1 / I U/// / /Z

~'//I///5 Y / / / / /A . . . . . . . . g / / / / 1 1 1 1 1 / ~ "//////Z / / / / . H . H . . / / / / ~ 4 ~ / / / /

/ / i / i / / ~ # . . . i / / i / i / I . . . .

. . . . . . . . I///

11/i ;;;~;~//// ///////~ / ~

////

"///////,I/// / / 1 1 1 1 1 / 1 1 1 1

¢z//zzz~ll// "/.,'///,/~.,. ~ ~..

/ l / I l l / , / / / / ~ / / / / / / / k / / / / I / i l l / X / l / l /

~'/////A / / / /

. . . . . . . . IIII

rq:

0 0 - 2 0 5 0 k - 2 0 0 1 - 2 0 10 -20 41 18 40 20 42 18 52 8

inoculum combinations (AgSp- - A g I ) Fig. 3. T h e nodulat ion response of AgI -WDl- res i s tan t and susceptible seedlings of A . glutinosa to strain type AgSp added as an inoculum in mixture with strain type A g I - W D I (see legend to Fig. 2).

nodules were not obtained from M3 and M4, although the local alder population was well provided with this nodule type. The narrow range of soil pH at the sites M and Q suggest that differences in the nodulation response be- tween the sites were not related to soil pH.

Development of nodule types AgI-WD1 and AgSp- in water-logged soils

Greenhouse experiment In previous experiments nodulation tests were carried out with the aid of hydrocultures of A.

Table 5. Nodulat ion response of A l n u s nitida inoculated with soil suspensions from locations Quackjeswater ( Q 1 - Q 4 ) and Meert je de Waal (M1-M 4) . Mean total numbe r of root nodules (effective + ineffective) and share of nodule type AgI-WD1 as percentage of total numbe r of root nodules per jar (n = 3)

Sampling pH Total no. of s.e. Share of s.e. site H 2 0 root nodules nodule type

jar ~ AgI-WD1 (%)

Q1 6.8 102 17 18 4 2 6.6 160 21 31 5 3 6.2 102 40 18 8 4 6.2 52 17 50 4

M1 6.6 _a _ _ _ 2 6.3 a _ _ -- 3 6.7 0 0 0 0 4 6.3 125 b 54 100 100

"Plants el iminated by root rot. b n = 2 .

Page 12: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

118 van Dijk and Sluimer-Stolk

Table 6. Nodulation response of Frankia strain types AgI-WD1 and AgSp in hydroculture and in waterlogged soil planted with seedlings of Alnus glutinosa

Average AgI-WD1 AgSp

root Nodule Number of p Nodule Number of length depth nodules/p depth nodules / p (cm) (cm) susc. plants a (cm) susc. plants"

Hydroculture Waterlogged

soil

13 0-5 3.7 3 0-8 0.06 3.8 0-2 3.0 5 0-1.5 0.6

16 16

a Combined data of 2 jars.

glutinosa that were inoculated with a small amount of soil suspension. In order to compare the development of both nodule types under more natural conditions, nodulation tests were performed both in hydroculture inoculated with 20 g soil VD7-1 per L nutrient solution and in undiluted water-logged soil of site VD7-1, plan- ted with young seedlings of A. glutinosa. Test- plants on plain water-logged soil showed slow growth rate despite the addition of Hoagland nutrients. Nodule development and average root length after two months are presented in Table 6. Average root depth in water-logged soil was 3.8 cm and nodule development was present only in the 0-2-cm soil layer. In water-logged soil a 10 times higher AgSp- nodule number was present than in hydroculture, despite presumably lower mobility of Frankia and slower growth of test plants. In both treatments nodule numbers are low compared to previous experiments. This is most likely explained by the addition of Pre- vicur-N that prevents root rot by Pythium, but also causes a reduction of the nodule number (C van Dijk, unpublished). The small scale of the experiment does not permit detailed conclusions, other than the fact that both nodule types are formed in water-logged soil as well as in hydro- culture, without dramatic changes of their ratio. The significance of a 10 × lower ratio of nodule

Table 7. The distribution of nodule types AgI-WD1 and AgSp- VD7-1

types in water-logged soil cannot be determined without additional experiments.

Field survey Nodulation experiments in hydroculture and in water-logged soil suggested potentials for de- velopment of nodule type AgI-WD1 under the conditions present in the study areas. In a pre- liminary survey samples of root systems from three different trees situated around sample site VD7-1 were screened for the presence of effec- tive and ineffective actinorhiza. Of each tree to thick roots (3-4cm in diameter and 2-4m length) with intact branching and abundance of fine roots were removed from the mud. The perennial woody roots were inspected for the presence of actinorhiza. In addition all fine roots were scanned at 12 x magnification. In all root systems 50-70% of the fine roots thinner than 3 mm were brown and necrotic. Table 7 presents the total number of root nodules scored at differ- ent size classes. In each size class as sample of healthy root nodules was examined by micro- scope for strain types AgI-WD1 or AgSp-. Only one nodule of 0.5 mm had a low incidence of vesicle clusters that might point to some form of ineffectiveness. The remaining 68 nodules were all of type AgSp- with abundance of mature clusters of vesicles and lack of sporangia.

in root samples of three specimen of Alnus glutinosa at site

Size class Number of root nodules

(mm) Total Sample AgSp AgI-WD 1 size

0-3 71 36 35 1 a 4-10 37 21 21 0 >10 30 12 12 0

"Nodule size of 0.5 mm, identification doubtful.

Page 13: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

Discussion

Until now ineffective strains or strain types of Frankia have been isolated exclusively from root nodules (Baker et al., 1980; Hahn et al., 1988; Lechevalier et al., 1983), without reference to the occurrence in soil populations or to ecologi- cal implications. The widespread occurrence of an ineffective strain type of Frankia, coded AgI- WD1 in long-term water-logged soils of three dune slacks and a peat bog on the Isle of Voorne showed that ineffective strains can occur as major components of soil populations of Frankia in natural stands of A. glutinosa. In a survey on nodulation potentials of Scottish soils by Wheeler et al. (1981) the occasional develop- ment of very small nodules most likely refers to similar ineffective strains of Frankia. Strain type AgI-WD1 showed close similarity with effective frankiae with respect to root nodule structure and morphological characteristics of hyphae and of occasional vesicles. However, some typical characteristics of frankiae such as N-fixation ac- tivity and spore development were not observed. Root nodules grown in hydroculture were given to Dr A D L Akkermans (Agricultural Universi- ty of Wageningen, The Netherlands) for prepara- tion of pure cultures of strain type of AgI-WD1. Characteristics of these isolates will be published elsewhere.

In the Frankia-Alnus relationship effective strains of Frankia proved to be compatible with a wide range of host genotypes (Baker, 1987; Bond, 1976; Jiabin et al., 1985; Rodriguez Bar- rueco et al., 1981). Examples of reduced com- patibility, depending on host genotype have been documented (Dillon and Baker, 1982; Nesme et al., 1984; Van Dijk et al., 1988; Wheeler et al., 1981) but complete resistance among host genotypes has not been recorded. This suggests that the high incidence of complete resistance to strain type AgI-WD1 among half-siblings of A. glutinosa and similar observations by Hahn et al. (1988) represent a form of host resistance in Alnus that is restricted to the relationship with certain ineffective strains of Frankia. The rela- tionship between ineffective and effective strain types of Frankia is still unclear, and most likely not uniform. Phenotypically regulated expression of effectiveness or ineffectiveness, depending on

Ineffective Frankia in Alder stands 119

the host species, was strongly suggested for Fran- kia strain type AiSp ÷ from Finland (van Dijk et al., 1988; Weber et al., 1987) and, at the inter- generic level, for a Myrica gale endophyte de- scribed by Vandenbosch and Torrey (1983). In strain type AgI-WD1 ineffectiveness was ex- pressed as a stable characteristic during sub- culture of 4 generations of root nodules, but the restricted range of host species used in these studies left room for similar ambivalent poten- tials to express effectiveness depending on host genotype. The same holds for ineffective strains isolated by Hahn et al. (1988). It is noticed that the results of more extended studies with strain type AgI-WD1 and a range of host genotypes support the presumed stability of ineffectiveness of strain type AgI-WD1. These studies will be presented elsewhere.

Despite the stability of characteristics of inef- fectiveness it still can be questioned whether these ineffective strains have to be considered as independent genotypes or depend on frequent genetical conversion of effective strains. Isolates derived from the AgI-WD1 nodules were unable to grow with N 2 as a sole N-source, indicating the absence of nitrogenase in these Frankia strains (A D L Akkermans, Agricultural Uni- versity of Wageningen, the Netherlands, person- al communications). Hahn et al. (1988) isolated several ineffective strains from effective root nodules of A. glutinosa grown in peatland and in forest soil. None of these strains showed C2H 2 reduction activity in N-free medium. The ab- sence of nif genes has been confirmed by the lack of hybridization of DNA with nif K, H, D as DNA-probe (D Hahn, personal communica- tions). Recent analysis of the 16S ribosomal DNA of ineffective and effective Frankia strains showed significant differences (Hahn et al. 1989), indicating that ineffective isolates are not simply derived from effective strains by genetical con- version. In addition to this, the strong domi- nance of strain type AgI-WD1 among the soil population of Frankia at the locations VD7 and VD8 also points to a high degree of genetical independence from local Frankia populations.

The absence of spore development in root nodule type AgI-WD1 was consistent, and the microsymbiont thus behaved as a typical Sp strain type (Van Dijk, 1978; Van Dijk and Met-

Page 14: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

120 van Dijk and Sluimer-Stolk

kus, 1978). Further studies on isolates are neces- sary to evaluate the taxonomic position of strain type AgI-WD1 and the relationship with Frankia alni subspecies pommerii (Lalonde et al., 1988).

The results of nodulation experiments reflec- ted the capacity of Frankia populations in the soil to produce root nodules in nodulation tests but give no direct clue to actual population densities. The validity of ecological interpreta- tions is also restricted by the artificial conditions of the nodulation tests. However, nodulation experiments carried out in water-logged soil, thus imitating at least part of the natural en- vironment, did not differ essentially from the hydrocultures.

The positive correlation between the nodula- tion capacity of AgI-WD1 and degree of water- logging of the soil in location VD7 suggested that the distribution of this strain type in the soil was restricted to sites with long term waterlogging and anaerobiosis. This is in agreement with the absence of this strain type in alder vegetations with less severe waterlogging conditions as ob- served in previous studies (unpublished results; Van Dijk, 1984). Effective Sp- strain types which are compatible with alder had a much wider distribution and were usually found in a wide variety of habitats, either dry or wet and both in the presence or absence of host specimen (Van Dijk, 1984; Weber, 1986; Smolander and Sundman, 1987). However, the high nodulation capacity of strain type AgI-WD1 at location VD7-1 and VD7-2 suggests that AgI-WD1 is better adapted to the conditions of this location than strain type AgSp-.

Hence, competitive interactions of strain types AgSp- and AgI-WD1 are likely to be restricted to long-term inundated alder vegetations. It was speculated that at sites with high nodulation capacities of strain type AgI-WD1 (sites VD7- 1,2,3) competition with effective Frankia strains might reduce the number of effective root nodule symbioses. Potentials of competition for nodule development between strain types AgI- WD1 and AgSp were demonstrated (Figs. 2 and 3). The results suggested that strain type AgSp- was more repressive to the ineffective strain type AgI-WD1 than vice versa, thus favor- ing the establishment of effective symbiosis in situations of competition. The ecological inter-

pretation is due to limitations caused by the artificial conditions of the experiment and by the restricted number of Frankia sources of both strain types. Moreover, the effect of strain type AgI-WD1 on nodule development of strain type AgSp- could be biased by comparing different genotypes of testplants, i.e. AgI-WD1 resistant versus non-resistant plants. Simon et al. (1988) also concluded from dual inoculation experi- ments that an ineffective isolate of Frankia elaeagni had low competitive abilities when com- pared with three effective isolates.

An even more important factor that favours effective nodule development in potentially com- petitive situations is the occurrence of resistance to strain type AgI-WD1 among seedlings of A. glutinosa. The absence of ineffective root nodules in samples of three root systems of location VD7-1, where the nodulation potentials of AgI-WD1 were most dominant, was most likely explained by resistance of host trees in the study area. This view is supported by the high percentage of resistant half-siblings of the seed- batch V22 collected close to the study area and used in the nodulation experiments presented in this paper.

It is suggested that host resistance to strain type AgI-WD1 has developed in response to a parasitic behaviour of Frankia and that positive correlations between the distributions of Agl- WD1 and host resistance can be expected. Such correlation studies require host populations of considerable size, long-term succession of gener- ations and a consequent selection pressure. These conditions are hardly present in the Netherlands and were not present in the alder populations used in this study. From an evolu- tionary point of view it is speculated that ineffec- tive strain types such as AgI-WD1, with pathogenic rather than mutualistic characteris- tics, are more conservative than effective strain types involved in mutualistic relationships with osts plants.

The occurrence of strain type AgI at wet sites with more extreme pH conditions was not inves- tigated, but indications for their occurrence at low pH can be deducted from nodulation studies with some Scottish soils (Wheeler et al., 1981). Differences in abundance of each of the strain types between the sample sites in the peat bog

Page 15: An ineffective strain type of Frankia in the soil of natural stands of Alnus glutinosa (L.) Gaertner

area Meertje de Waal suggest a heterogeneous distribution of AgI-WD1 on a small scale. This was attributed to a high density of old tufts of Carex that caused very irregular patterns of well aerated and water-logged sites. This area was similar to the peatbog area 'Weerribben' investi- gated by Hahn et al. (1988), but was different with respect to a near neutral soil pH.

Acknowledgements

Field studies were made possible by the hos- pitality of the Foundation 'Het Zuidhollands Landschap' and of 'Natuurmonumenten', The Society for the Preservation of Nature Reserves. Dr A D L Akkermans is thanked for reading the manuscript.

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