Abstract Mycorrhizal (Lactarius rufus Fr.) and non- mycorrhizal Norway spruce seedlings (Picea abies Karst.) were grown in a sand culture and inoculated with ...
Biol Fertil Soils (1995) 20:263-269
9 Springer-Verlag 1995
Georg Jentschke 9 Michael Bonkowski Douglas L. Godbold 9 Stefan Scheu
Soil protozoa and forest tree growth: non-nutritional effects and interaction with mycorrhizae
Received: 5 April 1994
Abstract Mycorrhizal (Lactarius rufus Fr.) and nonmycorrhizal Norway spruce seedlings (Picea abies Karst.) were grown in a sand culture and inoculated with protozoa (naked amoebae and flagellates) extracted from native forest soil or with protozoa grown on agar cultures. A soil suspension from which the protozoa were eliminated by filtration or chloroform fumigation was used as a control. After 19 weeks of growth in a climate chamber at 2 0 - 2 2 ~ the seedlings were harvested. Protozoa re: duced the number of bacterial colony-forming units extracted from the rhizoplane of both non-mycorrhizal and mycorrhizal seedlings and significantly increased seedling growth. However, concentrations of mineral nutrients in needles were not increased in seedlings with protozoan treatment. It is concluded that the increased growth of seedlings was not caused by nutrients released during amoebal grazing on rhizosphere micro-organisms. The protozoa presumably affected plant physiological processes, either directly, via production of phytohormones, or indirectly, via modification of the structure and performance of the rhizosphere microflora and their impact on plant growth. Mycorrhizal colonization significantly increased the abundance of naked amoebae at the rhizoplane. Our observations indicate that protozoa in the rhizosphere interact significantly with mycorrhizae.
Introduction
A significant part of photosynthetically fixed C is lost by the living root and reaches the soil via root exudates or deposition of non-soluble cell material (Lynch and Whipps 1990). As a result, much more bacteria and fungi are found near roots than in the bulk soil (Newman 1985). This local increase in microflora productivity provides food resources for a number of faunal grazers, including nematodes and protozoa (Coleman et al. 1988), which may therefore be concentrated in the rhizosphere. High densities of protozoa in the rhizosphere of many plant species have been demonstrated repeatedly (Darbyshire and Greaves 1967; Clarholm 1989; Griffiths 1990; Foster and Dormaar 1991). Since grazing on the microflora intensifies mineralization processes (Woods et al. 1982; Coleman 1985; Coleman et al. 1988; Rutherford and Juma 1992), nutrient availability in the rhizosphere soil may be increased, leading to an increase in plant growth. It has been concluded that this series of events is mainly responsible for the stimulation of plant growth in the presence of protozoa (Elliott et al. 1979; Clarholm 1985; Kuikman and van Veen 1989; Kuikman et al. 1990). However, on the basis of simulation models, the significance of this process for plant nutrient supply has been quesKey words Ectomycorrhiza Lactarius Mineral tioned (Griffiths and Robinson 1992). In addition, there nutrition 9 Picea abies 9 Plant growth 9 Protozoa 9 is experimental evidence (Nikolyuk 1969) that non-nutriRhizosphere tional effects may contribute to the observed growth stimulation. Although mycorrhizae play a key role in rhizosphere processes (Harley and Smith 1983) and interact with the soil fauna (Coleman 1985; Paulitz and Linderman 1991; Fitter and Sanders 1992), most work concerning the influence of protozoa on plants has been carried out with G. Jentschke (~) 9 D.L. Godbold non-mycorrhizal seedlings. Little is known about the inForstbotanisches lnstitut, Universit~t GOttingen, Biisgenweg 2, teractions of protozoa with soil (Bamforth 1988) and, D-37077 G6ttingen, Germany particularly, mycorrhizal fungi (Paulitz and Linderman M. Bonkowski 9 S. Scheu 1991). II. Zoologisches Institut, Universit~tt GOttingen, The aim of the present study was to test the effects of Abteilung Okologie, Berliner StraBe 28, D-37073 GOttingen, Germany protozoa on plant growth. In order to assess them under
264
more realistic conditions, and to study interactions between protozoa and mycorrhizal fungi, mycorrhizal treatments were included in the experiment.
Materials and methods Culture of protozoa Cultures of naked amoebae and flagellates were obtained from a diluted soil suspension of the A h horizon of a Norway spruce stand at Lange Bramke, Harz mountains, Germany. The cultures were grown at 15 ~ on soil extract agar (Singh 1946, 1955). The bacterial flora inoculated with the soil suspension served as a food source for the protozoa.
Seedling and fungal culture Seeds of Picea abies (L.) Karst. collected from the Black Forest Mountains, Germany, were surface-sterilized and germinated on 1.5% (w/v) water agar, pH 45, as described previously (Godbold et at. 1988). L. rufus Fr. was isolated from sporocarp tissue collected at a Norway spruce stand at Hils, Germany (Schlechte 1986), and grown on modified Melin-Norkrans agar (Marx 1969), pH 4.5. Liquid cultures were grown on a medium according to Palmer and Hacskaylo (1970). After 3 weeks, the seedlings were transplanted to an axenic sand culture and some of the seedlings were inoculated with ca. 5 ml homogenized mycelium from liquid culture.
Growth conditions The sand culture system described previously (Jentschke et al. 1991) was modified so that the seedlings were grown in glass tubes (ca. 60ml) filled with quartz sand ( 0 . 3 - 0 . 9 m m particle size). Upon transplanting (one seedling per glass tube), the root of each seedling was placed in the sand under axenic conditions. The shoot was exposed to ambient air. The top of each tube with the stem of the seedling passing through a hole in a silicon septum was sealed with non-toxic silicon glue (Kettenbach, Eschenburg, Germany). Growing conditions were kept constant at 22/20~ day/night temperature, a 16-h photoperiod (L18W/21 lamps, Osram), and a photosynthetically active photon flux density of 220 ~tmol m -2 s-1. To reduce algal growth after inoculation with the soil suspension the growth tubes were kept in darkness. The tubes were automatically supplied with 1.25 ml sterile nutrient solution every 2 h. The nutrient solution contained 300 g M NH4NO3, 50 g M Na2SO 4, 100 ~ KzSO 4, 30 g M KH2PO 4, 60 p M MgSO4, 130 g M CaSO 4, 5 g M MnSO 4, 5 ~tM FeC13, 5 g M H3BO 3, 0.1 ~tM Na2MoO 4, 0.1 IxM ZnSO 4, 0.1 g M CuSO 4, and 120 p M HCL The pH was 3.9. Each growth tube was drained using ceramic tensiometer candles (P80; KOnigliche Porzellanmanufaktur Berlin, Germany) and a negative pressure of 5 0 - 9 0 kPa. In each growth tube, the roots were continuously supplied with approximately 0.5 cm 3 s -1 of sterile filtered air.
Experimental design The seedlings were grown in the sand culture for 4 weeks to ensure adaptation after transplanting and establishment of the mycorrhiza (Jentschke et al. 1991). Then differently treated soil extracts (with or without protozoa) or protozoa from agar cultures were added as suspensions (10 ml per tube) to the rhizosphere of the spruce seedlings: (1) A protozoa-free soil extract (control) was obtained by passing a soil suspension of untreated humus material (Of and O h
horizons) from an acidic Norway spruce stand at Lange Bramke (Harz mountains, northern Germany) through a membrane filter (pore size 3 ~m, Schleicher und Schfill, Dassel, Germany). The filtrate was checked for protozoan contamination and no protozoa were detected. (2) A soil suspension of untreated humus material (Lange Bramke) containing native soil protozoa was passed through a 45-gm nylon sieve. It contained no animals other than protozoa (J Alphei, personal communication). (3) A suspension was prepared as for the control, but with the addition of protozoa from agar cultures (naked amoebae and flagellates). Since the cultures of soil protozoa contained bacteria, precautions were taken to reduce possible side effects of these bacteria on plant performance. A suspension of the protozoan cultures was passed through a membrane filter (pore size 3 gm, Schleicher und Schtill, Dassel, Germany) and the resulting suspension containing bacteria was checked for protozoan contamination. No protozoa were detected. The suspension was added to the control and the native protozoa treatment. In addition, to increase the diversity of the microbial flora in each of the treatments, a soil suspension obtained from defaunated (chloroform fumigation, Alphei and Scheu 1993) humus material (Lange Bramke) was added to the control and both protozoan treatments. The quantities of nutrients added with the different inocula were small compared to the quantities added during the experiment; no corrections were made to counter differences in nutrient contents added in inocula. A total of 72 tubes with spruce seedlings were set up. At the start of the experimental period (0 weeks of protozoan treatment; 4 weeks after the tubes with seedlings had been set up) 12 tubes each of mycorrhizal and non-mycorrhizal seedlings were harvested. Nineteen weeks after inoculation with protozoa, all other seedlings were harvested. This final harvest comprised a complete factorial design. The factors were protozoa (without, native, cultured) and mycorrhiza (without and with L. rufus). The seedlings were separated into needles, stems and roots. Coarse roots (diameter > 1 mm, usually the main root axis) were removed from the root systems and processed separately. Fine roots were cut into pieces 2 - 3 cm long and thoroughly mixed. Half of the material was used to determine root length (method according to Tennant 1975), the number of root tips, the degree of mycorrhizal colonization (percentage of mycorrhizal root tips in total number of root tips), and nutrient contents. The total number of root tips and the root length per seedling were calculated in terms of the dry weight proportions of the root sample examined and the total dry weight of fine roots per seedling.
Bacterial and protozoan numbers Rhizoplane bacteria and protozoa were extracted from the second part of the fine root material. The extraction was carried out in 50 ml sterile nutrient solution (composition as in the experiment) on a horizontal shaker (170 rpm, 10 min). The number of protozoa was determined in 10 ml of the suspension by a most probable number method using fourfold dilutions with Prescott's and James' solution (Page 1976). Aliquots of the diluted suspensions (six replicates) were incubated in six-well microtiter plates with soil extract agar (Singh 1946, 1955). Naked amoebae, flagellates, and ciliates were recorded after 4, 7, 10, and 14 days of incubation at 15 ~ Dilution series with homogeneous distribution of positive replicates only were used for the calculation of most probable numbers. For the extraction of bacteria, 10ml Na phosphate buffer (pH 7.5) was added to the remaining "root suspension" at a final concentration of 1 0 m M phosphate. The solution was shaken for another 10 min and bacterial numbers were determined by a platedilution technique. The number of colony-forming units was determined (three replicates) using yeast tryptone soybean peptone agar (Hensel et al. 1990). The agar plates were incubated at 24 ~ and colonies were counted after 1 and 2 weeks.
265 Determination of mineral elements
O ,v-
Plant material was dried at 70~ to a constant weight and wetashed using 65~ (w/w) HNO 3 in closed teflon vessels under high pressure at 180 ~ Concentrations of metals, P, and S were determined by inductively coupled plasma emission spectroscopy. Concentrations of N were determined after dry combustion in a carbon/nitrogen analyser. The total element uptake by plants during the experimental period was calculated as the difference between the total element content at the final harvest and the content at the start of the protozoan treatments.
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Statistical analysis
E" 25
Data on mycorrhizal colonization (percent mycorrhizal root tips to total number of root tips) were arcsin-transformed. All other data were transformed into common logarithms before statistical analysis. The general linear model procedure of the SAS software (SAS Institute 1987) was used and the data were analysed by a two-way analysis of variance with factors protozoa and mycorrhiza. Calculation of F values was based on type 3 sums of squares (SAS Institute 1987). Comparison among means was carried out using the ~ihkeyKramer method for unequal cell sizes (SAS Institute 1987).
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Results
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P l a n t growth a n d r o o t d e v e l o p m e n t
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"6 10 S h o o t d r y weight was significantly increased in p r o t o z o a n t r e a t m e n t s in b o t h the m y c o r r h i z a l a n d n o n - m y c o r r h i z a l seedlings (Fig. 1, Table 1). R o o t d r y weight was also increased, b u t the effect was n o t significant. I n n o n - m y c o r rhizal spruce seedlings p r o t o z o a significantly increased the m e a n n u m b e r o f r o o t tips a n d the m e a n r o o t length b y 124 a n d 115% in the native a n d cultured p r o t o z o a n
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400
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O'1
300
0 Ctr P1 P2 nonmycorrhizal
Ctr P1 P2 mycorrhizal
Fig. 2 Effects of protozoa on number of root tips (a), root length (h), and specific root length (root length per unit of root dry weight; e) of non-mycorrhizal and mycorrhizal (Lactarius rufus) Picea abies seedlings. For further explanations, see Fig. l
treatments, respectively (Fig. 2, Table 1). Generally, the effects o f p r o t o z o a f r o m the soil s u s p e n s i o n a n d f r o m a g a r cultures were very similar. T h e effect o f p r o t o z o a o n r o o t length a n d the n u m b e r o f r o o t tips a p p e a r e d to be less p r o n o u n c e d in t r e a t m e n t s with m y c o r r h i z a ( P < 0.t for the i n t e r a c t i o n terms). M y c o r r h i z a d i d n o t affect the s h o o t a n d r o o t b i o m a s s b u t decreased t h e n u m b e r o f r o o t tips a n d t h e r o o t length significantly (Figs. 1, 2, Table 1).
600
500
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300 200
Table 1 Effects of protozoa and mycorrhizae on shoot and root dry weight, number of root tips, root length and specific root length (root length per unit of root dry weight) of Picea abies seedlings. F values of the two-factorial analysis of variance. All data log-transformed before analysis
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i
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Fig. 1 Effects of protozoa (Ctr control, P1 with protozoa from soil suspension, P2 with protozoa from cultures) on shoot (a) and root (b) dry weight of non-mycorrhizal and mycorrhizal (Lactarius rufus) Picea abies seedlings. Mean values of non-mycorrhizal and mycorrhizal seedlings on the level of main effects (analysis of variance, see Table 1). Bars of each graph that do not have the same letter are significantly different (P_
