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Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill. Author(s): C. Van Dijk Source: New Phytologist, Vol. 81, No. 3 (Nov., 1978), pp. 601-615 Published by: Wiley on behalf of the New Phytologist Trust Stable URL: http://www.jstor.org/stable/2433853 . Accessed: 12/06/2014 09:43 Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp . JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected]. . Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to New Phytologist. http://www.jstor.org This content downloaded from 193.105.154.4 on Thu, 12 Jun 2014 09:43:39 AM All use subject to JSTOR Terms and Conditions

Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill

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Page 1: Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill

Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill.Author(s): C. Van DijkSource: New Phytologist, Vol. 81, No. 3 (Nov., 1978), pp. 601-615Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2433853 .

Accessed: 12/06/2014 09:43

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .http://www.jstor.org/page/info/about/policies/terms.jsp

.JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range ofcontent in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new formsof scholarship. For more information about JSTOR, please contact [email protected].

.

Wiley and New Phytologist Trust are collaborating with JSTOR to digitize, preserve and extend access to NewPhytologist.

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Page 2: Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill

New Phytol (1978) 81, 601-615.

SPORE FORMATION AND ENDOPHYTE DIVERSITY IN ROOT NODULES OF ALNUS GL UTINOSA (L.) VILL.

By C. VAN DIJK*

Department of Botany, Research Group on BiologicalNitrogen Fixation, University of Leiden, Leiden, The Netherlands

(Received 30 March 1978)

SUMMARY

Analysis of root nodules of Alnus glutinosa sampled in a natural alder vegetation revealed two types, one containing clumps of endophytic spores and one without spores. The distri- bution pattern of both types, but especially the latter, showed considerable clustering. Both types of nodule sometimes occurred on the same tree, and some information on the dynamics of the distribution pattern was obtained from age analysis of nodules of both types. Since cross-inoculation experiments showed a genetic basis for the presence or absence of spores in root nodules, the existence of two endophytic strains di-ffering in their ability to produce spores seems likely. Different concentrations of ammonium and nitrate incorporated into the nutrient solution of nodulated test plants had only a slight influence on spore-clump abundance in root nodules, and complete inversion of the spore type was not observed. Attempts to find additional strain differences led to the comparison of isomers of diamino- pimelic acid (DAP) from the cell wall of both types of endophyte. DAP isomer analysis of genetically more distinct 'A lnus-type' root nodules, i.e. those of Myrica gale and Hippophae rhamnoides, were used as reference. No additional taxonomic differences were found between the nodule types in Alnus glutinosa, but spore-free nodules of Hippophae rhamnoides and Myrica gale differed with respect to the DAP isomer composition. Test plants of Alnus glutinosa provided artificially with either spore-rich or spore-free nodules did not differ significantly in acetylene reduction rate.

INTRODUCTION

The root-nodule endophyte of Alnus glutinosa (L.) Vill. has been the subject of morpho- logical and cytological, studies in which hyphae, 'vesicles', and spores were recognized as distinct stages in the life cycle of this actinomycete.

Spores have been referred to as bacteroids (Schaede, 1933), bacteria-like cells or poly- hedral-shaped cells (Becking, de Boer and Houwink, 1964; Becking, 1970), and granula (Akkermans and van Dijk, 1975), which illustrates the obscurity of this stage as far as the mode of development and the function within the life cycle of the endophyte are con- cerned. Since van Dijk and Merkus (1976) have shown that these cells must be regarded as spores, the term spore will be used here.

Hyphae and vesicles are persistent endophytic stages in A. glutinosa root nodules, but Schaede (1933) noted that spores were sometimes absent and suggested that their forma-

*Present address: Institute for Ecological Research, Department of Dune Research, Weevers' Duin, Oostvoorne, The Netherlands.

0028-646X/78/1100-0601$02.00 ?1978 Blackwell Scientific Publications

601

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602 C. VAN DIJK

tion depends on the strength of the host-endophyte interaction. Kappel and Wartenberg (1958) showed that nitrate-nitrogen added to the nutrient solution repressed spore forma- tion in newly developed root nodules. However, environmental control of spore formation was questioned by van Dijk and Merkus (1976) on the basis of microscopical studies on spore development. Wheeler and Bowes (1974) showed that the abundance of spores in root nodules of A. glutinosa was not significantly affected by plant dormancy or by a long period of continuous darkness resulting in nodule decay. A study on nitrogen-fixation activity of field-sampled spore-free and spore-rich nodules by Akkermans (1971) did not reveal signi- ficant differences.

The widespread existence of nodules with and without spores was established by pre- liminary investigations, but the cause and significance of the divergence still remained obscure.

The present study was designed to find out whether the absence or presence of spore production in root nodules is gene-controlled by the host or the endophyte or is dependent on environmental factors. For this purpose, analysis of the distribution pattern of the two nodule types in the field was followed by cross-inoculation experiments. Since genetic dif- ferences proved to be involved, as will be shown below, the influence of combined nitrogen on spore production (Kappel and Wartenberg, 1958) was reinvestigated. The very small amounts of spores occasionally found in spore-free nodules (van Dijk and Merkus, 1976), were quantified to test the validity of a 'two-strain' concept. In addition, we compared cell-wall diaminopimelic acid isomers of both types of endophyte and the acetylene reduction activity of whole plants provided with one or the other of the two types of nodule, because of the taxonomical and physiological importance of these characters.

MATERIALS AND METHODS

Microscopical determination of nodule types The presence or absence of spores in root nodules was determined by microscopical in-

vestigation of fresh sections cut about 30 ,m thick, stained with diluted Fabil reagent (Noel, 1964), and examined immediately in connection with optimal colour development of spore clumps. Spore clumps stain bright red. For the identification of spore type, all sections of 3 peripheral lobes per nodule were screened. For further details, see van Dijk and Merkus (1976).

Microscopical estimation of spore-clump density Spore-clump density was assessed microscopically in serial sections of diglycolstearate-

embedded nodules. Sections cut 15 gim thick were stained with Toluidine Blue (0.1% solu- tion). Two methods were used as follows.

Method a. Every fourth section in a series from one nodule was examined. The series was restricted to the part of the nodule where the most spore clumps could be expected (region where vesicle decay starts). Spore-clump density was calculated as the number of spore clumps per microscopical field established with X 10 eyepieces and a X40 objective (Olympus) and covering about eighty cortical cells. Per section, four to six fields were chosen at random within the area of the cortex where vesicle decay had started. Spore-clump density per nodule was calculated as the average value of all fields counted per nodule.

Method b. Three successive sections of every fifteen of a series were examined. Spore-

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Endophytes of Alnus 603

clump density was determined on the basis of the total number of spore clumps per section, expressed as a percentage of the total number of spore clumps + vesicle clusters in the same section. Of all the sections counted per nodule, only the one with the highest value for spore-clump density was considered representative of the nodule.

Test plants for the nodulation experiments Test plants of Alnus glutinosa were raised from seed collected from native trees. Test

plants of A. cordata (Lois.) Desf., and A. incana (L.) Vill. were raised from seed collected from trees in the Botanical Garden in Leiden (The Netherlands). Seeds were surface-sterilized with a 0.1% bromine solution and left to germinate on wet gravel. After 10 days, a modified Hoagland solution (Quispel, 1954) reduced to half strength was added to the gravel and the seedlings were exposed to 220C, 70-80% relative humidity, and 17 h illumination with 30 klux (Philips TL65/33 or TL140/33) alternative with 7 h darkness. After 3 weeks, plants were transferred to jars filled with Crone's solution containing KNO3 (Bond, 1951), Fe-EDTA, and trace elements, according to Allen and Arnon (1955). Two days before inoculation, the nutrient solution was replaced by Crone's solution without nitrogen.

Inoculation of test plants Surface-sterilized (0.1% bromine solution for 3 min) nodule lobes were homogenized in

a Virtis homogenizer at 30,000 rpm together with nutrient solution. Large particles were removed by filtration through 100-gm mesh nylon netting. Suspensions for inoculation corresponding to 30 mg (fresh weight) nodule lobes were added to 250-ml jars each contain- ing 12 test plants. Inoculation of test plants and nodule growth took place in the nitrogen- free nutrient solution, which was renewed weekly. Care was taken to avoid undesired spon- taneous nodulation in the test plants. Deviations from these methods are mentioned at the appropriate place in the description of the experiment.

Sample preparation, extraction procedure, and paper chromatography for DAP analysis Endophytic vesicle clusters were liberated from the nodule tissue in a Virtis homogenizer

operated at 20,000 rpm for 2 min. The nodule suspension was repeatedly filtered over 90- pm mesh nylon netting to remove undisrupted plant cells and tissue fragments from the suspension. In the next step vesicle clusters were collected on 50-gm mesh nylon netting. The abundance of vesicle clusters was determined microscopically with the aid of a cell- counting device. The same procedure was used for the preparation of uninfected root samples. Pure cultures of Streptomyces prasinopilosus (strain CBS 551.68) and Geodermato- philus obscurus (strain CBS 237.69), obtained from the Centraal Bureau voor Schimmel- cultures, Baam, The Netherlands were harvested from a liquid culture. All specimens were washed with distilled water and 96% ethanol and were air-dried.

Hydrolysis of the samples was carried out according to Becker, Lechevalier and Lechevalier (1965), using 1 ml HCI 6 N per 10 mg air-dried sample. Hydrolysates were cleared by filtra- tion and HCI was damped off. Aqueous hydrolysates were analysed for isomers of 2,6-di- aminopimelic acid (DAP) by descending paper chromatography with methanol-H20-HCl- pyridine as solvent mixture (Rhuland et al., 1955). Large quantities of hydrolysates (? 20 mg air-dried sample) were chromatographed in two successive runs. Chromatograms were developed with 0.1 % ninhydrine in acetone followed by heating over 1 00?C.

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604 C. VAN DIJK

RESULTS

The distribution of spore-rich and spore-free root nodules in a natural alder vegetation

Description of the area The alder vegetation under investigation is part of a peat bog covered mainly with reed,

alder, and willow. The area, known as the Beulaker Wijde and situated in the province of Overijssel (The Netherlands), is managed as a reserve by the Society for the Preservation of nature Reserves in The Netherlands.

The distribution of alder trees on 1000 m2 of the peat bog is shown in Fig. 1. The position of each tree (both dots and circles) is characterized by x:y coordinates which represent the distance in metres from an arbitrarily chosen point. Trees represented by dots were not examined for nodulation. All trees above y = 25 m are situated in a grove in which Salix, Betula, and Rubus species are also present and where human influence is negligible. Local remnants of reed roots indicate that a reed vegetation was present prior to shrub develop- ment. The age of the alder trees varies between 5 and 25 years. The ground-water level is about 15 cm below the soil surface. Below this level the soil is highly anaerobic and root nodules do not occur. The pH of the top 15 cm of the soil varies between 4.0 and 4.8. Alder trees with y values below 25 m are situated in a reed stand which is cut every year. Except for one 25-year-old alder tree (38;18, Fig. 1), the alder shrubs in the reed stand have been cut together with the reed. The age of the root systems of these shrubs varies from 2 to 5 years.

Nodule sampling and distribution pattern Nodules were collected from thirty-three trees out of 130. These trees were randomly

chosen all over the area with the restriction that, for practical reasons, only trees older than 10 years were considered for sampling. Per tree, ten to twenty-five root nodules were collected.

Each nodule was screened for the presence or absence of spores and was accordingly recorded as spore-rich (Sp(+)) or spore-free (Sp(-)), respectively. Nodule age was deter- mined by microscopical counting of the number of annual rings at the base of the root nodule.

The distribution pattern of both types of nodules is shown in Fig. 1. Of thirty-three trees investigated, ten had only Sp(-) nodules, eleven had only Sp(+) nodules, and twelve had nodules of both types in different ratios. On trees with both types, Sp(+) and Sp) nodules were frequently found close together on the same root. Sp(-) nodules showed a high degree of clustering between coordinates x: 147-170 and y: 25-40. Within this area, all trees with only Sp(-) nodules are concentrated. Outside the Sp(-) area, Sp(-) nodules are sparsely distributed over the remaining part of the study area, where Sp(+) nodules are dominant. A kind of transition area is present between the coordinates x: 117-147;y: 25- 35, where roughly equal numbers of Sp(+) and Sp) nodules are distributed in varying ratios over five trees included in the study.

The distribution pattern of Sp(+) and Sp) nodules showed no relationship with the composition of the vegetation, macroscopical soil marks, or the ground-water level. A picture of the dynamic behaviour of the distribution pattern of Sp(+) and Sp(-) nodules can only be obtained by successive nodule analyses over a period of years, but some information was provided by comparison of the age distribution of both types of nodule collected from trees

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Endophytes of Alnus 605

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Page 7: Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill

606 C. VAN DIJK

Table 1. Age distribution of a random nodule sample collected from ten trees

No. of nodules per age class (class interval: 2 years) 1-2 years 3-4 years 5-6 years

74 43 10

Table 2. Age distribution of Sp(-) and Sp(+) nodules collected from trees with both types or only Sp(-) nodules

Position of tree No. of nodules per age class (class interval: 2 years)

(coordinates Sp(-) Sp(+) Fig. 1) 1-2 yr 3-4 yr 5-6 yr 1-2 yr 3-4 yr 5-6 yr

6;31 0 0 2 6 4 0 38;27 0 0 2 8 4 0

104;37 0 1 0 6 2 0 119;37 0 2 3 3 2 0 127;37 4 9 1 2 0 0 133;30 0 3 2 10 1 0 141;33 4 9 0 3 0 0 146;29 2 2 0 3 1 0 169;30* 12 2 0 166;36* 1 1 4 0

*Trees with Sp(-) nodules only.

with mixed nodule populations (Table 2) with the age distribution of a large random nodule sample (Table 1). Table 1 shows a steady decline in the number of nodules classified according to increasing age class in a random nodule sample. The same type of age distribution of a large nodule sample was found by Akkermans and van Dijk (1975). Analysis of the Sp(+) and Sp(-) fractions of nodule samples from trees with mixed nodule populations (Table 2) showed that the two types are not equally distributed over the successive age classes. The trees farthest from the Sp(-) area (up to x: 127) have no Sp(-) nodules in the lowest age class and only a few in the age class of 3-4 years. In these classes, Sp(+) nodules are distri- buted as could be expected from Table 1, i.e. they show a direct inverse correlation between number and age. The 5-6-year age class, on the contrary, has only Sp(-) nodules. The 'complementary' age distributions of Sp(-) and Sp(+) nodules from these trees suggest a recent recession of Sp(-) nodules in favour of Sp(+) nodules in that area. The recession of Sp(-) nodules seems to decrease toward the Sp(-) area. For the sake of completeness, Table 2 shows the data which indicate that Sp) nodules collected from the Sp) area (trees 169;30 and 166;36) have roughly the same age distribution as that in Table 1. Small- scale nodule analysis in various alder stands in The Netherlands showed that the age of both Sp(+) nodules and Sp(-) nodules can be up to about 10 years.

The foregoing results donot explain the existence of both nodule types but merely restrict the number of possible causative mechanisms. On the basis of the phenotypical determination of spore type postulated by Schaede (1933) and Kappel and Wartenberg (1958), the environ- mental pressure would be exerted via the host plant. Because root nodules of both types occur together on the same host plant, it must be excluded that either the host plant geno- type or the phenological status of the plant as a whole is responsible for spore-type deter-

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Page 8: Spore Formation and Endophyte Diversity in Root Nodules of Alnus glutinosa (L.) Vill

Endophytes ofAlnus 607

mination. However, the distribution pattern and age distribution might be explained in terms of endophytic strain differences.

Factors affecting spore formation in root nodules

Involvement of endophytic strain differences in spore-type expression and the possible action of environmental factors including growth conditions and host-plant genotype were studied in nodulation experiments.

Intraspecific cross-inoculation In the first set of experiments seedlings of Alnus glutinosa originating from trees with

either Sp(+) or Sp(-) nodules were inoculated with homogenates of Sp(+) or Sp(-) nodules to determine whether the host origin or spore type of the inoculum influences the spore type of newly developed nodules. Both the seeds for test plants and root nodules for the preparation of inoculi were collected from tree 38;18 (Fig. 1),which had only Sp(+) nodules, and from trees 157;27 and 151 ;30 which had exclusively Sp(-) nodules. The inoculi and test plants were used for 6 cross-inoculation combinations (Table 3). The spore type of newly developed nodules was microscopically determined 6 weeks after inoculation.

Table 3. Spore type of nodules developing from Alnus glutinosa Sp(+) and Sp(-) inoculi and A. glutinosa test plants from different seed

samples

Combinations of cross-inoculation Sp(+)/Sp(-) ratio of nodule Test plants: samples obtained by

Inoculum: Spore type and cross-inoculation Spore type origin mother No. of nodules and origin tree Ratio examined

Sp(+), 38;18* X Sp(+), 38;18 9/1 10 X Sp(-), 157;27 10/0 10 X Sp(-), 151;30 10/0 10

Sp(-), 157;27 X Sp(+), 38;18 0/9 9 X Sp(-), 157;27 0/10 10

Sp(-), 151;30 X Sp(-), 151;30 0/10 10

*Coordinates give location of trees (Fig. 1) sampled for nodules and seed.

As can be seen from Table 3, the spore type of newly formed nodules proved to be the same as that of the inoculi from which these nodules originated. These results suppose a genetic determination of the spore type, and can be explained in terms of endophytic strain differences. In all likelihood, the exceptional Sp(-) nodule from the Sp(+) X Sp(+) combination arose from a contamination, and so far there have been no cases pointing to the possibility of low-rate transformation of one spore type into the other. Environmental con- ditions prevailing during infection and nodule growth (see under Materials and methods) had no influence on spore-type expression and the same holds- for host plant origin at the intraspecific level.

Interspecific cross-inoculation In the foregoing experiment genetic variation between host plants was probably very low,

and the results do not permit general conclusions about host plant influence on spore-type expression. Additional information on this point was obtained from nodulation experiments

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608 C. VAN DIJK

with host plants belonging to different species. Successful cross-inoculation experiments with inoculi and host plants of European alder species have already been described by Roberg (1938), Becking (1966), and Rodriguez-Barrueco and Bond (1968), but no attention has been paid to the spore type of inoculi and newly developed root nodules. The cross-inocula- tion experiments described below were carried out with seedlings of A. cordata and A. incana as host plants and with crushed Sp(+) and Sp(-) nodules originating from A. glutinosa as inoculi.

The spore types of 6-week-old nodules developed from four cross-inoculation combina- tions are shown in Table 4. European alder species proved to be susceptible to both Sp(+) and Sp(-) inoculi of A. glutinosa origin. Here too, the spore-type expression of nodules developed from crossinoculation was completely consistent with the spore types of the inoculi used, and genetic host plant differences did not alter spore-type expressions.

Table 4. Spore type of nodules developing from Alnus glutinosa Sp(+) and Sp(-) inoculi and A. cordata and A. incana test plants

Sp(+)/Sp(-) ratio of Combinations of inoculation: nodules obtained by

Inoculum inoculation (origin and Test plant No. of nodules spore type) species Ratio examined

A. glutinosa Sp(+) X A. cordata 14/0 14 X A. incana 16/0 16

A. glutinosa Sp(-) x A. cordata 0/14 14 XA. incana 0/15 15

Validity of a 'two-strain 'concept based on endophytic spore formation Cross-inoculation experiments demonstrated that spore formation is controlled by the

endophyte. The postulation of two endophytic strains is based on the classification of nodules into two categories: Sp(+) and Sp(-). These two categories are usually very distinct, and dubious intermediate forms are very rare. Careful microscopical examination of microtome sections showed that Sp(-) nodules sometimes contain very low numbers of smaRl inter- cellular spore clumps (van Dijk and Merkus, 1976) and that Sp(+) nodules vary to some degree in the number of spore clumps.

To test the two-strain concept the variations in spore clump abundance had to be quanti- fied. The Sp(+) and Sp(-) nodules used for quantitative spore-clump analyses were obtained from a nodulation experiment in which A. glutinosa seedlings were used as host plants and Sp(+) and Sp) nodule homogenates as inoculi. Eight weeks after inoculation, fifteen nodules that had developed from each of the two inoculation combinations were harvested. The spore-clump density of each nodule was determined by counting the number of spore clumps per microscopical field in serial microtome sections (method a, see under Materials and methods), and was calculated from 20-35 counts per nodule. The mean values of fifteen nodules of each spore type and standard errors are given in Table 5. There is a highly significant difference in spore-clump abundance between newly developed Sp(+) and Sp) nodules (Mann-Whitney U-test, P < 0.001). Also, the number of spores per clump in Sp(-) nodules is on average at least ten times lower than in Sp(+) nodules. Therefore, the actual difference in spore content between Sp(+) and Sp) nodules must be much larger than is indicated by the counts in Table 5. It can be concluded that Sp) nodules may contain

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Endophytes of Alnus 609

Table 5. Spore-clump density in newly developed nodules of Sp(+) and Sp(-) origin

Spore type Mean number of spore clumps of No. of nodules per microscopical field per

inoculum examined nodule (mean of 15 nodules) Sp(+) 15 7.6 ? 1.0* Sp(-) 15 0.15 ? 0.18

*Standard error.

small amounts of spores but these numbers are in no way comparable to the amounts ob- served in Sp(+) nodules.

The intercellular location of small spore clumps in Sp(-) nodules and the absence of particular intracellular stages in spore-clump development (spindles) are in agreement with earlier observations (van Dijk and Merkus, 1976). The existence of two endophytic strains which differ in their ability to produce spores is supported by the stability of the spore-type expression in two successive generations of nodules.

The influence of nitrate and ammonium on spore-clump density during nodule development A genetic basis for differences in spore-type expression has been shown, but additional

modification by environmental factors cannot be excluded. Kappel and Wartenberg (1958) observed inhibition of spore formation in alder root nodules when nitrate was added to the nutrient solution of nodulated alder plants. These observations are not in agreement with the results of our cross-inoculation experiments, all of which were carried out with a nitro- gen-free nutrient solution. Because the observations of Kappel and Wartenberg (1958) represent the only serious indication of environmental influence on spore formation, we investigated the possible influence of nitrate and of ammonia on spore formation in more detail.

Groups of alder seedlings raised on Crone's nutrient solution and inoculated with a Sp(+) nodule suspension (5 mg fresh weight nodules/100 ml nutrient solution/twelve plants) were exposed to various concentrations of KNO3 or (NH4)2S04 incorporated into the nutrient solution as the sole source of nitrogen. Nitrogen treatments were started 5 days after inocula- tion, when the plants were transferred to 7-1 jars. The nitrogen concentrations applied are given in Table 6. All series except N3a were adjusted to pH 6.0 every 3 days. Series N3a, which had the same nitrate concentrations as series N3, was kept at pH 4.0. Eight weeks after the inoculation, nodules were harvested and serial sections were prepared for micro- scopical estimation of spore-clump density.

Spore-clump density might be influenced by the direct action of environmental factors but also by the degree of infection of the nodule tissue. The latter effect was ruled out by relating numbers of spore clumps to total numbers of infected cells in the same section. Consequently, in this experiment spore-clump density was calculated as the percentage of spore clumps in the total number of clumps and vesicle clusters per section and per nodule (method b, see under Materials and methods). For each nitrogen treatment, seven to ten nodules were examined with respect to spore-clump density. The mean spore-clump density found per treatment is shown in Table 6. The spore-clump densities were compared statis- tically by the Kruskal-Wallis one-way analysis of variance by ranks.

Nitrate concentrations ranging from 0 to 15 mEq at pH 6.0 did not influence spore-clump

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610 C. VAN DIJK

Table 6. Influence of NO- and NH4, added to Crone's nutrient solution on spore-clump density in root nodules of Alnus glutinosa

Combined N Mean nodule MEq/l Crone's Mean dry weight Mean plant

solution spore-clump per plant dry weight Series NOQ NH+ density (%) (mg)* (g)*

B1 0 0 8.5 78 3.5 Ni 0.5 0 8.1 215 8.8 N2 1.0 0 5.9 148 9.9 N3 7.5 0 9.8 109 15.4 N3at 7.5 0 3.5 22 7.6 N4 15.0 0 4.0 23 8.7 Al 0 0.75 3.5 -

A2 0 3.75 3.0 -

A3 0 7.5 6.0 -

A4 0 15.0 1.2 -

*Mean of twelve plants tpH of nutrient solution: 4.0.

density significantly (Kruskal-Wallis test for series Bi, NI, N2, N3, and N4: P= 0.2595). Optimum plant growth was found at 7.5 mEq NO_/l nutrient solution. Optimum nodule growth was found at 0.5 mEq NO-/l. A significant reduction of spore-clump density occurred in series N3a at low pH of the nutrient solution as compared with the corresponding nitrate series N3 at pH 6.0 (Kruskal-Wallis test: P = 0.0117).

Ammonium concentrations ranging from 0.75 to 15 mEq NH+/1 nutrient solution reduced spore-clump density compared with the nitrogen-free series Bi (Kruskal-Wallis test for series Bi, Al, A2, A3, and A4: P= 0.0028; for series Bi, Al, A2, and A3: P= 0.0228; for series Al, A2, A3, and A4: P= 0.0271; for series Al, A2, and A3: P= 0.1966). The strongest reduction of spore-clump density was seen in series A4, where plant and nodule growth were limited and the high ammonium concentration has no ecological importance.

Interpretation of the reduction of spore-clump density in the ammonium series is compli- cated by the repeated drop in pH of the nutrient solution from 6.0 to about 4.0 despite frequent readjustment. Therefore, the effects of low pH and of the ammonium treatment cannot be separated here. In all cases of spore-clump reduction easily detectable amounts of spore clumps remained, leaving no doubt about the Sp(+) status of the nodules under study.

Taxonomically and physiologically important properties of Sp(+) and Sp(-) endophytes

Isomeric forms of 2,6-diaminopimelic acid in Sp(+) and Sp(-) endophytes The extent of the genetic differentiation between Sp(+) and Sp(-) endophytes becomes

more evident when other taxonomically important characters of both types of endophyte are compared.

The composition of cell-wall amino acids has proven to be a useful characteristic in actinomycete taxonomy (Becker et al., 1965). Determination of cell-wall amino acids in obligate plant-actinomycete systems is disturbed by interference from plant amino acids, except for the taxonomically important isomeric forms of 2,6-diaminopimelic acid (DAP) which are usually absent in plant tissue.

Determination of DAP isomers in a refined endophyte preparation of A. glutinosa root

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nodules revealed the presence of meso-DAP and the absence of LL-DAP (Angulo, van Dijk and Quispel, 1975; Becking, 1976). Although DAP isomers are normally identical where intrageneric actinomycete species are concerned, Lalonde, Knowles and Fortin (1975) reported the presence of both meso-DAP and LL-DAP in extracts of A. crispa var. mollis root nodules, which contain an endophytic actinomycete very similar to that of the A. glutinosa rootnodules.The rootnodulesusedby Lalonde etal. (1975) did not contain spores, but the A. glutinosa nodules used by Angulo et al. (1975) probably did. Therefore, we re-examined the DAP isomeric form(s) of extracts of both Sp(+) and Sp(-) nodules of A. glutinosa by paper chromatography (see under Materials and methods). Sp(-) nodule endophytes of Hippophae rhamnoides and Myrica gale, which belong to different cross- inoculation groups, served as genetically distinct references, and extracts of pure cultures of Streptomyces prasinopilosus and Geodermatophilus obscurus were used as references for LL-DAP and meso-DAP, respectively.

In refimed preparations of root-nodule endophyte, 30-40% of the particles were endo- phytic clusters, the remainder consisting of plant debris. The paper-chromatographic results are shown in Table 7. Both Sp(+) and Sp(-) nodule hydrolysates of Alnus glutinosa origin

Table 7. DAP-isomer composition of Sp(+)andSp(-) root nodules ofAlnus glutinosa and of Sp(-) nodules of Hippophae rhamnoides and Myrica gale (analysis by paper chromatography)

Maximum Unidentified sample

Meso-DAP pale brown LL-DAP quantityt (R s*t 1 .0) (Rs*t 1.0-1.1) (Rs*t 1-3) (mg)

Streptomyces prasinopilosust - - + 1.0 Geodermatophilus obscurus ? + - - 1.3 Alnus glutinosa Sp(+) nodules + - - 4.0 A. glutinosa Sp(-) nodules + - - 4.0 A. glutinosa roots - - - 3.5 Hippophae rhamnoides Sp-) nodules - + - 25.0 H. rhamnoides roots - + - 20.0 Myrica gale Sp(-) nodules - - 25.0 M. gale roots - - 20.0

*Run distance of spot relative to meso-DAP. tLargest amount of hydrolysate per run, expressed as mg air-dried sample before hydrolysis. tReference for LL-DAP. ? Reference for meso-DAP. **Very weak tailing spot. Rst-value uncertain.

showed intense spots of meso-DAP, but LL-DAP was absent. Therefore, the difference between the DAP isomer composition of the A. glutinosa endophyte (Angulo et al., 1975) and the A. crispa endophyte (Lalonde et al., 1975) cannot be attributed to the use of nodules of different spore types. The actinomycetic origin of the DAP isomers was con- firmed by chromatography of hydrolysates of uninfected rootlets of the appropriate host plants.

DAP isomers were not found in the endophytes of Hippophae rhamnoides or Myrica gale, despite chromatography of large quantities of the extracts. Extracts of both roots and root nodules of Hippophae rhamnoides caused a weak yellowish-brown spot on the chro- matogram with a RAt-value almost identical to that of meso-DAP.

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612 C. VAN DIJK

Comparison of nitrogen fixation rates in Sp(+) and Sp(-) nodules To investigate possible major ecological consequences of genetic differences between the

-two endophyte strains, the nitrogen fixation rates of Sp(+) and Sp(-) nodules were com- pared under controlled conditions. Akkermans (1971) compared the acetylene reduction activity of individual field-sampled Sp(+) and Sp(-) nodules but the marked variation in activity among the nodules made it impossible to detect minor differences between the two types.

In this experiment, the acetylene reduction rates of both types of nodule were compared on the basis of measurements made in whole plants bearing either Sp(+) or Sp(-) nodules. For this purpose, 7-week-old plants grown on Hoagland's nutrient solution were inoculated with either Sp(+) or Sp(-) nodule homogenates in quantities such that maximum nodulation could be expected. Three weeks later, nodulated plants showing equal growth and nodule number were selected and transferred to perlite saturated with Hoagland's nutrient solution. Each pot contained two plants. The nitrogen-free nutrient solution was used during inocula- tion and nodule development. When the nodules were 10 weeks old, the acetylene reduction rate per pot was measured under the current climatic standard conditions (230C, 20 klux (Philips TL140/33), and in transparent containers.

As can be seen from Table 8, the mean acetylene reduction rates found for nine pots

Table 8. Acetylene reduction rates of plants with either Sp(+) or Sp(-) nodules

Mean acetyle reduction rates (,umoles ethylene/g dry nodule/h)

Sp(+) nodules Sp(-) nodules 39.3 + 10.4* 45.9 ? 22.5

nt=9 n=13

*Standard error. tNumber of pots measured.

with Sp(+) nodules and thirteen pots with Sp(-) nodules do not differ significantly under the climatic conditions of the experiment. The results are in accordance with the observations made by Akkermans (1971) in field-sampled nodules.

DISCUSSION

Cross-inoculation experiments showed that the presence or absence of spores in root nodules is caused by different genotypes of the endophyte. Hopwood, Wildermuth and Palmer (1970) showed that the absence of spore formation in particular mutants of Streptomyces coelicolor was caused by the mutation of only two or three genes, which were thought to control the synthesis of sporulation septa. Several of the mutants obtained by these authors showed different degrees of defective spore formation. The existence of more than two endophytic strains cannot be excluded either, because differences in spore-clump abundance occurring among Sp(+) nodules could also represent strain differences. The genetic stability of different strains under natural conditions may be influenced by the exchange or loss of genome. Conjugation was found to occur among members of the Actinomycetales (Hop-

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Endophytes of Alnus 613

wood, 1973), and plasmid involvement might facilitate the alteration of strain characters under natural conditions (Vivian, 1971; Dunican and Cannon, 1971). These considerations remind us once again that the presumption of the existence of two rather stable strains differing in their ability to produce spores may be a simplification of the actual situation. Nevertheless, this presumption is justified by the results obtained so far and is useful as a working hypothesis.

Unlike Kappel and Wartenberg (1958), we observed both types of nodule on the same tree. The absence of spore formation in young nodules of Alnus glutinosa described by Schaede (1933) might be ascribable to the accidental observation of only Sp(-) strain nodules, because in our nodulation experiments Sp(+) nodules could be distinguished from Sp(-) nodules as early as 3-4 weeks after inoculation.

As can be expected for closely related organisms, Sp(+) and Sp(-) endophytes show the same cell-wall DAP isomer composition. The presence of both meso-DAP and LL-DAP in the root-nodule endophyte of A. crispa var. mollis (Lalonde et al., 1975), might imply that endophytes of A. glutinosa and A. crispa are taxonomically more distinct than is suggested by the subdivision of Frankiaceae by Becking (1970), which was based on the concept of one Frankia species per cross-inoculation group.

In root nodules of Hippophae rhamnoides and Myrica gale both meso-DAP and LL-DAP seem to be absent, and the same was found for root nodules of species of Casuarina by Becking (1977). Because Alnus, Myrica, Hippophae, and Casuarina belong to different cross- inoculation groups, we suppose that the absence of DAP isomers is a common feature among root-nodule endophytes of non-leguminous plant species. The validity of the unique position of Alnus endophytes with respect to the presence of DAP isomers needs further verification.

The influence of combined nitrogen on spore formation described by Kappel and Warten- berg (1958) must be interpreted in terms of environmental modification or suppression of strain characters. The results of our experiments concerning the effect of combined nitrogen on spore formation are not in agreement with those of Kappel and Wartenberg. We never observed suppression of spore formation in Sp(+) strains to the level of the Sp(-) strain, and significant reduction was only observed in the low-pH series. Conversely, we did not observe stimulation of spore formation in Sp(-) nodules when the nitrogen-free nutrient solution was used in nodulation experiments.

The nodule distribution pattern presented in this paper shows that considerable clustering of nodules of either spore type may occur. It seems likely that close co-existence of Sp(+) and Sp(-) clusters eventually leads to complete mixture of the two types of nodule as a result of exchange between the appropriate extra-nodular endophyte populations, unless these populations behave differently with respect to certain environmental factors. Differen- tiating factors of this kind could not be found in the study area, but analyses of distribution patterns in other areas are in progress. The age distribution of Sp(+) and Sp(-) nodules in the transition zone between Sp(+) and Sp(-) clusters in the study area suggests some mobility of the clusters. Cluster dynamics probably depend on local environmental condi- tions and cannot be generalized, but preliminary results of current investigations in large undisturbed areas give the impression that large-scale cluster patterns do not alter signifi- cantly over periods of many years.

The ecological significance of the occurrence of two endophytic strains and of local spatial separation is still obscure. There is no evidence for direct ecological consequences of the strain difference as far as nitrogen fixation activities are concerned, but the occurrence of two strains with different ecological amplitudes for one or more environmental factors

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614 C. VAN DIJK

might create a wider range of possibilities for host-endophyte interactions. A study on the ecological consequences of differences in spore production between the two strains is in progress.

ACKNOWLEDGMENTS

These investigations were supported by the Netherlands Organization for the Advancement of Pure Research (ZWO). The field work was made possible by the hospitality of the Society for the Preservation of Nature Reserves in The Netherlands.

The author is greatly indebted to Professor A. Quispel for his stimulating interest and criticism. Mr P. J. M. van der Geest participated as a postgraduate student. The technical assistance of Mr T. Tak is gratefully acknowledged.

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