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
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.
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
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
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 25C, 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--+ 2C. Additional illumination with Philips HLRG 400W at an irradiance (PI) of 30Wm -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.
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).
110 van Dijk and Sluimer-Stolk
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.).
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).
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...