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Effect of inoculum type and placement on nodulation and growth of Casuarina cunninghamiana seedlings
Forest Ecology and Management, 36 (1990) 135-147
135
Elsevier Science Publishers B.V., Amsterdam
Effect of inoculum type and placement on nodulation and growth of Casuarina cunninghamiana seedlings P.A. Rosbrook Division of Soils, CSIRO, P.M.B., P.O. Aitkenvale, Townsville, Qld. 4814 (Australia) (Accepted 11 July 1989)
ABSTRACT Rosbrook, P.A., 1990. Effect of inoculum type and placement on nodulation and growth of Casuarina cunninghamiana seedlings. For. Ecol. Manage., 36:135-147. Seedlings of Casuarina cunninghamiana grown in peat/vermiculite under nursery conditions were inoculated with (a) a crushed-nodule suspension of Frankia strain KB by three methods: watering the inoculum onto the base of seedlings, syringing inoculum around the root system or mixing dried, ground nodules through the potting mix, or (b) an isolate of the actinomycete Frankia (strain JCT287 ) by two methods: watering the inoculum onto the base of the seedlings, or mixing the isolate through the potting mix. No differences in shoot dry-weight, nodule number, or nodule dry-weight per plant were found between the two methods of application when an isolate of Frankia was employed. When crushed nodules were used as inocula, the response to each inoculum method depended on the level applied. At high levels of application (0.2 g crushed nodule per seedling), no differences were found in shoot dry-weight, nodule number or nodule dry-weight per plant between the three inoculation methods. At low levels of inoculum, the fastest nodulation and largest plant-growth response to inoculation occurred where the inoculum was placed close to the root system.
INTRODUCTION
Species of Casuarina sensu stricto have potential for soil conservation and rehabilitation, fuelwood production and agroforestry in many warm-temperate and tropical climates. The survival and growth of Casuarina seedlings on adverse sites that are low in nitrogen is often dependent on symbiotic N2 fixation with the actinomycete Frankia. Differences in the effectiveness of Frankia/Casuarina symbioses have been shown (Reddell and Bowen, 1975; Sellstedt, 1988), and hence it is desirable to develop inoculation techniques in order to introduce selected Frankia in the nursery or field. The development of reliable procedures for the routine inoculation of Casuarina in forest nurseries has, until recently, been limited by the difficulties 0378-1127/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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in isolating and cultivating Frankia in vitro. Previously, Casuarina seedlings have been inoculated using either nodule suspensions or soil or leaf litter collected from the immediate vicinity of effectively nodulated plants (McCluskey and Fisher, 1983; Torrey, 1983 ). However, these methods present problems in that they are not fully reproducible (with nodulation often being erratic), they require large amounts of inoculant materials and there is a danger of accidental introduction of pathogenic micro-organisms into the forest nursery (McCluskey and Fisher, 1983; Perinet et al., 1985 ). Little is known about how to introduce Frankia successfully into the root environment of Casuarina seedlings such that rapid nodulation takes place and an effective symbiosis is established. The present study compared methods for inoculating nursery-grown seedlings of C. cunninghamiana. Two sources of Frankia were used, a suspension prepared from C. cunninghamiana nodules from southeast Queensland (strain KB; Reddell and Bowen, 1985 ) and an isolate of Franlda originating from nodules of C. equisetifolia from Townsville, north Queensland (strain JCT287; Shipton and Burggraaf, 1983). The effect of amount of inoculum applied on nodulation was also assessed. MATERIALSAND METHODS
General nursery procedures Seeds of C. cunninghamiana were germinated on trays of sterile potting mix (1:2 peat:vermiculite with 1.25 g CaCO3 1-~ to bring the pH to 6.0). After 4 weeks, seedlings were transplanted into 70-ml free-draining plastic pots ( 1 seedling per pot) containing the potting mix described above, with a nitrogen-free nutrient formulation, consisting of superphosphate 9.5 g ( 19.2% P, 16% Ca, 1.6% S), KC1 2.6 g, MgSO4 6.9 g, mixed micronutfient fertilizer 1l0 m g ( 12% Fe, 2.5% Mn, 1.0% Zn, 0.5% Cu, 0.1% B, 0.05% Mo, 15% S), ZnSO4-7H20 1.4 rag, MnC12.4H20 46 rag, COSO4-7H20 2.4 mg, Na2MoO4-2H20 6.4 mg and H3BO3 34 mg, added to each 1. No nitrogen was added, as peat supplies sufficient N for the first 3-4 weeks of growth. The pots were placed in styrofoam trays (84 pots per tray) in a sheltered part of the greenhouse. As signs of toxicity had been observed in an adjacent experiment using the above nutrient mix, pots in experiments 1A and 1B were heavily watered prior to planting to remove any excess nutrients. For two of the experiments (2A and 2B) the fertilizer levels were reduced so that superphosphate was applied at one third of the original level and the rates of KC1 and MgSO4 were halved. The micronutrient fertilizers were unchanged. After inoculation, seedlings remained in the greenhouse for 2 weeks. They were then transferred outside (open air) with trays of normal nursery stock
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placed between experimental trays to reduce the risk of cross-contamination. Plants were watered three times daily by an automatic watering system.
Experiments with crushed nodule inocula Experiment 1A Three methods of inoculation - 'water-on', 'syringe', and 'soil-mix' - were compared. A fourth treatment consisted of uninoculated control plants. The design was a randomized complete block with two blocks, in which each treatment was applied to 140 seedlings contained in two separate trays (70 seedlings per tray). Trays were randomized within blocks. Each inoculation treatment used 5.25 g of dried nodules for the 140 seedlings (i.e. 0.04 g dried nodule per seedling). Two weeks prior to inoculation the young, flesh-coloured nodules had been collected from 2-year-old plants growing vigorously in the field and had been dried at room temperature. In all cases the nodules were surface-sterilized with 50% ethanol for 1 min, rinsed in deionised water, air-dried for 24 h, and ground (Braun Aromatic KSM2 ) for 1 min. For the water-on treatment, the ground nodules were further crushed with a mortar and pestle with a 1% sucrose solution. The nodule suspension was then made to volume (700 ml) with 1% sucrose (in deionised water) and 5 ml of the frequently stirred suspension was applied to the base of each seedling. For the syringe treatment, two aliquots of 2.5 ml each of the nodule suspension prepared as above (including further crushing with the mortar and pestle) were syringed approximately 2 cm into the potting mix either side of each seedling, with the suspension being directed towards the root system. The soil-mix treatment consisted of mixing the dried, ground inoculum through the potting mix using a cement mixer. Seedlings were then transplanted into mix containing the inoculum. Experiment 1B To check that the level of inoculum used in experiment 1A was optimal, a factorial arrangement of the three inoculation methods described above was used with twelve inoculum levels and seven replicate plants for each. The levels ofinoculum applied were × 5 (i.e. 0.19 g dried nodule per seedling), 1, 0.5, 0.2, 0.1, 0.02, 0.01, 0.005, 0.002, 0.001, 0.0001 of that used in experiment 1A. Seedlings inoculated with the same dilution level were placed together in styrofoam trays. Single or double empty rows separated dilution treatments and the four trays were placed randomly within experiment 1A. Experiments with an isolate of Frankia Experiment 2A This experiment, established 8 weeks after experiment 1, compared two methods of inoculation with a Frankia isolate: "water-on' or 'soil-mix', each
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with two levels of inoculum (nil, applied). The Frankia isolate (JCT287) was grown in stationary culture on a liquid propionic acid medium (Shipton and Burggraaf, 1983). After 12 weeks, numerous colonies were visible in the culture, which was then ground aseptically in a Sorvall homogenizer (0.1-1 quantities for 10 s) to break up the colonies and increase the number of propagules. Seedlings in the water-on and soil-mix treatments received equal quantities ofinoculum. The water-on treatment was applied as for experiment 1A, whilst the soil-mix treatment involved diluting the isolate suspension so that when mixed through the potting media the mix was completely moistened (gravimetric moisture of 270%), but not saturated. Seedlings were then transplanted into the potting mix as for experiment 1A. Experiment 2B A dilution series was established using both methods of inoculation used in experiment 2A. The levels ofinoculum applied and general layout were as for experiment 1B except that six replicates were used and the × 5 and 0.002 levels were replaced by × 2 and 0.00001 respectively. Harvest and assessments In experiment 1A, six replicate plants were harvested from each treatment at 80, 87, 97, 111,125, 139 and 153 days after inoculation, whilst for experiment 2A the harvest times were 33, 47, 61, 75, 90 and 117 days after inoculation. Experiments 1B and 2B were harvested 153 and 117 days after inoculation, respectively. For experiments 1A and 2A, nodule number, nodule dryweight and shoot dry-weight were determined at each harvest. For experiment 2B, shoot dry-weight, nodule number and nodule dry-weight were measured for all dilution treatments. For experiment 1B, nodule dry-weight was measured for the five highest inoculum levels ( × 5-0.1 ), nodule number for the ten highest levels ( × 5-0.002 ) and shoot dry-weight for all treatments. Analysis of variance was performed on log-transformed data for each parameter. RESULTS
Comparison of inoculum application methods Experiment 1A - crushed nodules A small but insignificant amount of contamination occurred in this experiment, with one nodule present on one of the six uninoculated plants at 125 days and two nodules present on one of the six uninoculated plants at the final harvest.
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Differences were found in nodule number, nodule dry-weight and shoot dry-weight per plant between the syringe and soil-mix methods. The syringe treatment resulted in the most prolific nodulation, giving significantly more nodules than the soil-mix treatment at all harvests from 87 days onwards (P= 5%; Fig. 1 ). The difference between the syringe and soil-mix treatments was largest during the early stages of nodulation: at 87 days plants inoculated by syringe had 16 times the number of nodules of plants inoculated by soil mix, whereas by 153 days the difference had been reduced to 2.8 times. Although syringe-inoculated plants consistently gave larger numbers of nodules per plant than the water-on treatment, the difference was only significant at 111 days after inoculation (P= 5%; Fig. 1 ). The trends in nodule dry-weight per plant were similar to those described for nodule number, with syringe-inoculated plants having the greatest nodule dry-weight throughout the experiment (Fig. 2 ). The nodule dry-weight formed by syringe inoculation was consistently greater than that formed by wateringon the inoculum (Fig. 2 ), but the differences were not significant. Inoculated plants showed no increase in shoot dry-weight compared with uninoculated plants until 111 days after inoculation. At 111 days, the shoot dry-weight of inoculated plants was nearly twice that of uninoculated plants, and by 153 days the increase was 19 times (Fig. 3). Plants inoculated by syringe showed the greatest increase in shoot dryweight, reaching 27-fold that of uninoculated plants at the final harvest (Fig. 3 ). The shoot dry-weight of plants inoculated by water-on was consistently
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less than that of plants inoculated by syringe (Fig. 3 ); however, the difference was significant only at the 125-day harvest (P= 5%). Plants inoculated by the soil-mix method showed the poorest growth, with shoot dry-weights being
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10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Noduledry-weight(mg plant1) Fig. 4. T h e r e l a t i o n s h i p b e t w e e n s h o o t d r y - w e i g h t a n d n o d u l e d r y - w e i g h t p e r p l a n t for Casuar-
ina cunninghamiana seedlingsinoculated with a crushed-noduleinoculum by three methods:
syringe (SYR), water-on (WO) and soil-mix (SM). significantly less than those of syringe-inoculated plants from 125 days after inoculation until the final harvest (Fig. 3 ). The relative growth rates (as estimated from the slope of the curves in Fig. 3 ) of the three inoculation treatments were similar from 111 days onwards. Thus the differences in dry-weight were due to the time it took to attain this growth rate. When shoot dry-weight was plotted against nodule dry-weight for each treatment and harvest time (Fig. 4), the relationship between these two parameters was found to contain a lag phase, with increasing low nodule weights not resulting in increased shoot weight, and then a linear phase, where shoot dry-weight was directly proportional to nodule dry-weight. Thus treatment effects on shoot dry-weight would be due to effects on the rate of nodule formation. E x p e r i m e n t 2A - F r a n k i a isolate
Inoculated plants were nodulated by the first harvest, 33 days after inoculation. By 75 days after inoculation, the shoot dry-weight of inoculated plants was twice that of uninoculated plants ( P = 5%; Fig. 5 ). By the final harvest the difference was 12-fold. The method of inoculation did not affect nodule number except at day 90, when the nodule number on plants inoculated by the soil-mix method was significantly greater than on plants inoculated by the water-on method ( P = 5%; Fig. 6 ). Inoculation method had no effect on nodule dry-weight per plant. Uninoculated plants remained unnodulated. The trends for shoot dry-weight were similar to those found for nodule number and nodule dry-weight. The method of inoculation did not affect shoot
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Fig. 6. The change in the number of nodules per plant over time for Casuarina cunninghamiana seedlings inoculated with an isolate of Frankia by two methods: water-on (WO) or soil-mix (SM). LSD for log~o nodule number 5%=0.25, 1%=0.38.
dry-weight ( P = 5%) except at day 90, when the shoot dry-weight of soil-mix plants was just significantly greater than that of plants inoculated by the wateron procedure (P= 5%; Fig. 5 ).
EFFECT OF INOCULUM ON CASUARINA
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Effect o f inoculum level
Experiment 1B - crushed nodules No significant difference in nodule number was found between the × 5 and X 1 level for syringe-inoculated plants, implying that the number of infective propagules was not limiting for this treatment (Table 1 ). For both the soilmix and water-on treatments, nodule number significantly declined between the X 5 and X 1 levels. Nodule dry-weight was unaffected ( P = 5%; Table 1 ) at the five highest levels of inoculum when the inoculum was syringed into the potting mix. For the water-on treatment, there was a rapid decline in nodule dry-weight with increasing dilution level, with nodule dry-weight at the X 5 dilution level being significantly greater than that at the X0.5 level ( P = 1%; Table 1 ). A similar decline in nodule dry weight was observed for the soil-mix treatment (Table
1). The above trends were reflected in shoot dry-weights. With syringe-inoculated plants, dilution of the inoculum did not significantly decrease shoot dryweight until the 0.02 level ( P = 5%), implying that the level of inoculum applied in experiment 1A was optimal (Table 1 ). However, for both the wateron and soil-mix treatments, there was a rapid decline in shoot dry-weight at inoculation levels lower than × 5, with a significant decrease occurring at the × 1 level ( P = 5%; Table 1 ). Thus for the two last-named methods of inocuTABLE 1
Nodule number, nodule dry-weight and shoot dry-weight per plant for Casuarina cunninghamiana seedlings 153 days after inoculation with dilutions of a crushed-nodule inoculum by syringe ( S Y R ) , water-on (WO) or soil-mix (SM) method (experiment I B ) Inoculum level t
5 1 0.5 0.2 0.1 0.02 0.01 0.005 0.002 0.001 0.0001
Nodule number
Nodule dry-weight (mg)
Shoot dry-weight (g)
SYR
WO
SM
SYR
WO
SM
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WO
SM
41a 2 25ab 14bc 28ab 12c 2d 2d 2d ld . .
41a 13b 6c 6bc 5c 0.7d ld 0.5d 0.3d . .
26a 4b 4b 4b 0.3c 0.8c 0.7c 0.3c 0.3c . .
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85a 18bc 44ab 13c 0.33d -
0.86a 1.10a 0.76a 1.19a 0.87a 0.18bc 0.29b 0.28b 0.26bc 0.16bc 0.10c
1.13a 0.48b 0.14cd 0.23bc 0.24bc 0.12cd 0.15cd 0.20bc 0.10d 0.15cd 0.11cd
0.73a 0.16bc 0.36b 0.13bc 0.08c 0.13bc 0.20b 0.10bc 0.10bc 0.12bc 0.17bc
. .
. .
qnoculum levels are expressed as multiples of the level applied in experiment IA. 2For each inoculum treatment, numbers with the same letter are not significantly different at P = 5%. 3_, not determined.
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P.A. ROSBROOK
TABLE 2 Nodule number, nodule dry-weight and shoot-dry weight per plant for Casuarina cunninghamiana seedlings 117 days after inoculation with dilutions of an isolate of Frankia in a liquid medium by water-on (WO) or soil-mix (SM) method (experiment 2B) Inoculum level
2 1 0.5 0.2 0.1 0.02 0.01 0.005 0.001 0.0001 0.00001
Nodule number
Nodule dry weight (mg)
Shoot dry weight (g)
WO
SM
WO
SM
WO
SM
47a 2 30ab 29abc 27abc 26abc 16c 19bc 0.3e 8.9d 1.9e 1.4e
37ab 3lab 39a 30ab 20b 0.1c lc 0.3c 0.1c 0c 0.7c
56a 69a 52a 72a 43a 54a 46a 7b 35a 14b 4b
49a 45a 59a 48a 39a lc 6b lc 3bc 0c 3bc
0.59a 0.52a 0.46ab 0.53a 0.38ab 0.43a 0.47a 0.10c 0.31b 0.06c 0.04c
0.42a 0.32a 0.49a 0.41a 0.34a 0.05b 0.05b 0.05b 0.05b 0.03b 0.04b
qnoculum levels are expressed as multiples of the level applied in experiment lB. 2For each inoculation treatment, numbers with the same letter are not significantly different at P = 5%.
lation, greater quantities of inoculum than the × 1 level were required for optimal plant growth. The X 5 inoculum level gave equivalent shoot dryweights for each inoculation method, suggesting that this level was optimal for plant growth for each method. E x p e r i m e n t 2 B - F r a n k i a isolate
The first significant decrease in nodule number per plant occurred at the 0.02 dilution level for the water-on treatment and the 0.1 dilution level for the soil-mix treatment (Table 2). Both shoot dry-weight and nodule dryweight per plant were maintained to the 0.01 dilution level for the water-on treatment, whilst for the soil-mix treatment, shoot dry-weight and nodule dryweight per plant were maintained only to the 0.1 level (Table 2 ). DISCUSSION
T i m e to nodulation
Plants inoculated by the crushed-nodule and isolate inocula showed large differences in the time between inoculation and the appearance of nodules, the times being approximately 80 and 33 days, respectively. It is unlikely that the delay in nodulation that occurred with crushed-nodule inocula was due to low numbers of propagules, as the results for the dilution experiment (Table 1 ) show nodule number, nodule weight and shoot weight per plant for sy-
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ringe-inoculated plants did not decline significantly until the 0.1 or 0.05 level, indicating that the higher levels of inoculum applied were optimal. For the water-on and soil-mix treatments (Table 1 ) lack of infective propagules was a problem, yet these treatments began to nodulate at approximately the same time as the syringe treatment. The greater length of time that seedlings inoculated with crushed-nodule inocula need to nodulate might be due to differences between strains or the presence of polyphenols released from the ground nodules (Torrey, 1983 ), which could limit growth of Frankia in the potting medium (Perradin et al., 1983). Baker et al (1979) found separation of Frankia from nodules of Elaegnus umbellata by sephadex fractionation could result in a 5-fold increase in nodulation capacity over the crude crushed-nodule extract. Phenolics and pigments, which may act as inhibitors, are thought to be removed from the nodule extract by sephadex fractionation. If crushed nodules are to be used to inoculate seedlings of Casuarina, techniques to remove inhibitors released upon grinding should be investigated.
Method of application Crushed nodules The method of inoculum application was more critical when crushed-nodule inoculum was used. All three crushed-nodule methods of inoculation resulted in nodulation, but the rate at which infection took place and the resultant growth rate of plants varied between the different methods. Although no significant differences occurred in plant growth between the syringe and wateron treatments, syringe-inoculated plants tended to form nodules more quickly (Fig. 1 ) and grow faster (Fig. 3) than plants inoculated by the water-on method, suggesting that concentrating the inoculum close to the root system was advantageous. Syringe-inoculated plants had greater nodule number, nodule dry-weight and shoot dry-weight per plant (Figs. 1, 2, 3 ) than soil-mix inoculated plants at most harvest times. The dilution experiment (Table 1 ) showed the soilmix inoculation method resulted in significantly fewer nodules on the Casuarina plants than did the syringe inoculation method. This was probably due not only to the diluting effect of the application method, which results in fewer propagules coming into direct contact with the young root system, but also to the inoculum not being further ground by mortar and pestle. Fewer infective propagules may have been released from the nodules compared with the preparation methods of the syringe and water-on treatments. The dilution experiment showed all three application methods were equally effective at inducing nodulation and enhancing plant growth, as long as large quantities of inoculum are available. As the level of inoculum declined (Table 1 ), large differences were observed between methods, with the syringe
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method consistently giving the best plant growth and the soil-mix method the worst. Thus when using crushed-nodule inocula, large quantities of nodules should be used, particularly if inoculating by a method similar to the wateron and soil-mix techniques. Franida isolate Inoculation with the Franlda isolate resulted in rapid nodulation and enhanced plant growth compared with uninoculated plants, whether wateredon or mixed through the soil. The dilution results suggest that the inoculum could have been further diluted by a factor of 10 without a significant decrease in plant growth occurring (Table 2). At low levels ofinoculum (0.02) differences were observed in plant dry-weight and nodule dry-weight per plant between the inoculation methods, with the water-on treatment resulting in greater shoot dry-weights and nodule dry-weights per plant than the soil-mix treatment. A major problem with the use of Frankia isolates is the lack of a simple method for defining the quantity of inoculum that is applied. Microscopic examination of the inoculum revealed both sporangia and hyphae. Workers have used optical density (Lalonde and Calvert, 1979) and packed cell volume (Berry and Torrey, 1985; Stowers and Smith, 1985 ) to define inoculum levels. However, a given optical density or packed cell volume may contain different proportions ofhyphae and sporangia and thus may differ in infectivity. In addition, there are practical problems with large-scale use of liquid inocula. Thus the present study demonstrates that, for rapid nodulation of C. cunninghamiana seedlings in the nursery, use of crushed-nodule inocula requires relatively large amounts of nodule (>10,2 g dried nodule per seedling) and placement of the inoculum as close as possible to the root system. With isolates of Franlda, placement is generally less critical. ACKNOWLEDGEMENTS The statistical advice of Dr. R. Correll and the technical assistance of Ian Francis, Leanne Stuckey and Dave Taylor are greatly appreciated. The research was funded by a grant from ACIAR (Australian Centre for International Agricultural Research).
REFERENCES Baker, D., Kidd,G.H. and Torrey,J.G., 1979. Separationof actinomycetenoduleendophytes fromcrushednodulesuspensionsby sephadexfractionation.Bot.Gaz., 140:549-551.
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Berry, A.M. and Torrey, J.G., 1985. Seed germination, seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials. Plant Soil, 87:161-173. Lalonde, M. and Calvert, H.E., 1979. Production of Frankia hyphae and spores as an infective inoculant for Alnus species. In: J.C. Gordon, C.T. Wheeler and D.A. Perry (Editors), Symbiotic Nitrogen Fixation in the Management of Temperate Forests. Oregon State University Press, Corvallis, pp. 94-110. McCluskey, D.N. and Fisher, R.F., 1983. The effect of inoculum source on nodulation in Casuarina glauca. Commonw. For. Rev., 62:117-124. Perinet, P., Brouillette, J.G., Fortin, J.A. and Lalonde, M., 1985. Large scale inoculation of actinorhizal plants with Frankia. Plant Soil, 87:175-183. Perradin, Y., Mottet, M.J. and Lalonde, M., 1983. Influence of phenolics on in vitro growth of Frankia strains. Can. J. Bot., 61: 2807-2814. Reddell, P. and Bowen, G.D., 1985. Frankia source affects growth, nodulation and nitrogen fixation in Casuarina species. New Phytol., 100:115-122. Sellstedt, A., 1988. Nitrogenase activity, hydrogen evolution and biomass production in different Casuarina symbioses. Plant Soil, 105:33-40. Shipton, W.A. and Burggraaf, A.J.P., 1983, Aspects of the cultural behaviour of Frankia and possible ecological implications. Can. J. Bot., 61: 2783-2792. Stowers, M.D. and Smith, J.E., 1985. Inoculation and production of container-grown red alder seedlings. Plant Soil, 87:153-160. Torrey, J.G., 1983. Casuarina: Actinorhizal dinitrogen-fixing tree of the tropics. In: S.J. Midgley, J.W. Turnbull and R.O. Johnston (Editors), Casuarina Ecology, Management and Utilization. CSIRO, Melbourne, pp. 192-204.
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Effect of inoculum type and placement on nodulation and growth of Casuarina cunninghamiana seedlings P.A. Rosbrook Division of Soils, CSIRO, P.M.B., P.O. Aitkenvale, Townsville, Qld. 4814 (Australia) (Accepted 11 July 1989)
ABSTRACT Rosbrook, P.A., 1990. Effect of inoculum type and placement on nodulation and growth of Casuarina cunninghamiana seedlings. For. Ecol. Manage., 36:135-147. Seedlings of Casuarina cunninghamiana grown in peat/vermiculite under nursery conditions were inoculated with (a) a crushed-nodule suspension of Frankia strain KB by three methods: watering the inoculum onto the base of seedlings, syringing inoculum around the root system or mixing dried, ground nodules through the potting mix, or (b) an isolate of the actinomycete Frankia (strain JCT287 ) by two methods: watering the inoculum onto the base of the seedlings, or mixing the isolate through the potting mix. No differences in shoot dry-weight, nodule number, or nodule dry-weight per plant were found between the two methods of application when an isolate of Frankia was employed. When crushed nodules were used as inocula, the response to each inoculum method depended on the level applied. At high levels of application (0.2 g crushed nodule per seedling), no differences were found in shoot dry-weight, nodule number or nodule dry-weight per plant between the three inoculation methods. At low levels of inoculum, the fastest nodulation and largest plant-growth response to inoculation occurred where the inoculum was placed close to the root system.
INTRODUCTION
Species of Casuarina sensu stricto have potential for soil conservation and rehabilitation, fuelwood production and agroforestry in many warm-temperate and tropical climates. The survival and growth of Casuarina seedlings on adverse sites that are low in nitrogen is often dependent on symbiotic N2 fixation with the actinomycete Frankia. Differences in the effectiveness of Frankia/Casuarina symbioses have been shown (Reddell and Bowen, 1975; Sellstedt, 1988), and hence it is desirable to develop inoculation techniques in order to introduce selected Frankia in the nursery or field. The development of reliable procedures for the routine inoculation of Casuarina in forest nurseries has, until recently, been limited by the difficulties 0378-1127/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
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in isolating and cultivating Frankia in vitro. Previously, Casuarina seedlings have been inoculated using either nodule suspensions or soil or leaf litter collected from the immediate vicinity of effectively nodulated plants (McCluskey and Fisher, 1983; Torrey, 1983 ). However, these methods present problems in that they are not fully reproducible (with nodulation often being erratic), they require large amounts of inoculant materials and there is a danger of accidental introduction of pathogenic micro-organisms into the forest nursery (McCluskey and Fisher, 1983; Perinet et al., 1985 ). Little is known about how to introduce Frankia successfully into the root environment of Casuarina seedlings such that rapid nodulation takes place and an effective symbiosis is established. The present study compared methods for inoculating nursery-grown seedlings of C. cunninghamiana. Two sources of Frankia were used, a suspension prepared from C. cunninghamiana nodules from southeast Queensland (strain KB; Reddell and Bowen, 1985 ) and an isolate of Franlda originating from nodules of C. equisetifolia from Townsville, north Queensland (strain JCT287; Shipton and Burggraaf, 1983). The effect of amount of inoculum applied on nodulation was also assessed. MATERIALSAND METHODS
General nursery procedures Seeds of C. cunninghamiana were germinated on trays of sterile potting mix (1:2 peat:vermiculite with 1.25 g CaCO3 1-~ to bring the pH to 6.0). After 4 weeks, seedlings were transplanted into 70-ml free-draining plastic pots ( 1 seedling per pot) containing the potting mix described above, with a nitrogen-free nutrient formulation, consisting of superphosphate 9.5 g ( 19.2% P, 16% Ca, 1.6% S), KC1 2.6 g, MgSO4 6.9 g, mixed micronutfient fertilizer 1l0 m g ( 12% Fe, 2.5% Mn, 1.0% Zn, 0.5% Cu, 0.1% B, 0.05% Mo, 15% S), ZnSO4-7H20 1.4 rag, MnC12.4H20 46 rag, COSO4-7H20 2.4 mg, Na2MoO4-2H20 6.4 mg and H3BO3 34 mg, added to each 1. No nitrogen was added, as peat supplies sufficient N for the first 3-4 weeks of growth. The pots were placed in styrofoam trays (84 pots per tray) in a sheltered part of the greenhouse. As signs of toxicity had been observed in an adjacent experiment using the above nutrient mix, pots in experiments 1A and 1B were heavily watered prior to planting to remove any excess nutrients. For two of the experiments (2A and 2B) the fertilizer levels were reduced so that superphosphate was applied at one third of the original level and the rates of KC1 and MgSO4 were halved. The micronutrient fertilizers were unchanged. After inoculation, seedlings remained in the greenhouse for 2 weeks. They were then transferred outside (open air) with trays of normal nursery stock
EFFECT OF INOCULUM ON CASUARINA
137
placed between experimental trays to reduce the risk of cross-contamination. Plants were watered three times daily by an automatic watering system.
Experiments with crushed nodule inocula Experiment 1A Three methods of inoculation - 'water-on', 'syringe', and 'soil-mix' - were compared. A fourth treatment consisted of uninoculated control plants. The design was a randomized complete block with two blocks, in which each treatment was applied to 140 seedlings contained in two separate trays (70 seedlings per tray). Trays were randomized within blocks. Each inoculation treatment used 5.25 g of dried nodules for the 140 seedlings (i.e. 0.04 g dried nodule per seedling). Two weeks prior to inoculation the young, flesh-coloured nodules had been collected from 2-year-old plants growing vigorously in the field and had been dried at room temperature. In all cases the nodules were surface-sterilized with 50% ethanol for 1 min, rinsed in deionised water, air-dried for 24 h, and ground (Braun Aromatic KSM2 ) for 1 min. For the water-on treatment, the ground nodules were further crushed with a mortar and pestle with a 1% sucrose solution. The nodule suspension was then made to volume (700 ml) with 1% sucrose (in deionised water) and 5 ml of the frequently stirred suspension was applied to the base of each seedling. For the syringe treatment, two aliquots of 2.5 ml each of the nodule suspension prepared as above (including further crushing with the mortar and pestle) were syringed approximately 2 cm into the potting mix either side of each seedling, with the suspension being directed towards the root system. The soil-mix treatment consisted of mixing the dried, ground inoculum through the potting mix using a cement mixer. Seedlings were then transplanted into mix containing the inoculum. Experiment 1B To check that the level of inoculum used in experiment 1A was optimal, a factorial arrangement of the three inoculation methods described above was used with twelve inoculum levels and seven replicate plants for each. The levels ofinoculum applied were × 5 (i.e. 0.19 g dried nodule per seedling), 1, 0.5, 0.2, 0.1, 0.02, 0.01, 0.005, 0.002, 0.001, 0.0001 of that used in experiment 1A. Seedlings inoculated with the same dilution level were placed together in styrofoam trays. Single or double empty rows separated dilution treatments and the four trays were placed randomly within experiment 1A. Experiments with an isolate of Frankia Experiment 2A This experiment, established 8 weeks after experiment 1, compared two methods of inoculation with a Frankia isolate: "water-on' or 'soil-mix', each
138
P.A. ROSBROOK
with two levels of inoculum (nil, applied). The Frankia isolate (JCT287) was grown in stationary culture on a liquid propionic acid medium (Shipton and Burggraaf, 1983). After 12 weeks, numerous colonies were visible in the culture, which was then ground aseptically in a Sorvall homogenizer (0.1-1 quantities for 10 s) to break up the colonies and increase the number of propagules. Seedlings in the water-on and soil-mix treatments received equal quantities ofinoculum. The water-on treatment was applied as for experiment 1A, whilst the soil-mix treatment involved diluting the isolate suspension so that when mixed through the potting media the mix was completely moistened (gravimetric moisture of 270%), but not saturated. Seedlings were then transplanted into the potting mix as for experiment 1A. Experiment 2B A dilution series was established using both methods of inoculation used in experiment 2A. The levels ofinoculum applied and general layout were as for experiment 1B except that six replicates were used and the × 5 and 0.002 levels were replaced by × 2 and 0.00001 respectively. Harvest and assessments In experiment 1A, six replicate plants were harvested from each treatment at 80, 87, 97, 111,125, 139 and 153 days after inoculation, whilst for experiment 2A the harvest times were 33, 47, 61, 75, 90 and 117 days after inoculation. Experiments 1B and 2B were harvested 153 and 117 days after inoculation, respectively. For experiments 1A and 2A, nodule number, nodule dryweight and shoot dry-weight were determined at each harvest. For experiment 2B, shoot dry-weight, nodule number and nodule dry-weight were measured for all dilution treatments. For experiment 1B, nodule dry-weight was measured for the five highest inoculum levels ( × 5-0.1 ), nodule number for the ten highest levels ( × 5-0.002 ) and shoot dry-weight for all treatments. Analysis of variance was performed on log-transformed data for each parameter. RESULTS
Comparison of inoculum application methods Experiment 1A - crushed nodules A small but insignificant amount of contamination occurred in this experiment, with one nodule present on one of the six uninoculated plants at 125 days and two nodules present on one of the six uninoculated plants at the final harvest.
EFFECTOF INOCULUMON CASUARINA
139
Differences were found in nodule number, nodule dry-weight and shoot dry-weight per plant between the syringe and soil-mix methods. The syringe treatment resulted in the most prolific nodulation, giving significantly more nodules than the soil-mix treatment at all harvests from 87 days onwards (P= 5%; Fig. 1 ). The difference between the syringe and soil-mix treatments was largest during the early stages of nodulation: at 87 days plants inoculated by syringe had 16 times the number of nodules of plants inoculated by soil mix, whereas by 153 days the difference had been reduced to 2.8 times. Although syringe-inoculated plants consistently gave larger numbers of nodules per plant than the water-on treatment, the difference was only significant at 111 days after inoculation (P= 5%; Fig. 1 ). The trends in nodule dry-weight per plant were similar to those described for nodule number, with syringe-inoculated plants having the greatest nodule dry-weight throughout the experiment (Fig. 2 ). The nodule dry-weight formed by syringe inoculation was consistently greater than that formed by wateringon the inoculum (Fig. 2 ), but the differences were not significant. Inoculated plants showed no increase in shoot dry-weight compared with uninoculated plants until 111 days after inoculation. At 111 days, the shoot dry-weight of inoculated plants was nearly twice that of uninoculated plants, and by 153 days the increase was 19 times (Fig. 3). Plants inoculated by syringe showed the greatest increase in shoot dryweight, reaching 27-fold that of uninoculated plants at the final harvest (Fig. 3 ). The shoot dry-weight of plants inoculated by water-on was consistently
l°°r =~" lO
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80
100 120 140 Time after inoculation (days)
160
Fig. 1. The change in the number of nodules per plant over time for Casuarina cunninghamiana seedlings inoculated with a crushed-nodule inoculum by three methods: syringe (SYR), wateron (WO) and soil-mix (SM). LSD for loglo nodule number 5%=0.26, 1%=0.38.
140
P.A. ROSBROOK
i] ~ .
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o
o
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Fig. 2. The change in nodule dry-weight per plant over time for Casuarina cunninghamiana seedlings inoculated with a crushed-nodule inoculum by three methods: syringe (SYR), wateron (WO) and soil-mix (SM). LSD for logm nodule weight 5%=0.29, 1%=0.43.
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Fig. 3. The change in shoot dry-weight over time for Casuarina cunninghamiana seedlings left uninoculated (Uninoc) or inoculated with crushed nodules by three methods: syringe (SYR), water-on (WO) and soil-mix (SM). t.SD for Ioglo shoot weight 5%=0.24, 1%=0.36.
less than that of plants inoculated by syringe (Fig. 3 ); however, the difference was significant only at the 125-day harvest (P= 5%). Plants inoculated by the soil-mix method showed the poorest growth, with shoot dry-weights being
EFFECT OF INOCULUM ON CASUARINA
141
2.2
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10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 Noduledry-weight(mg plant1) Fig. 4. T h e r e l a t i o n s h i p b e t w e e n s h o o t d r y - w e i g h t a n d n o d u l e d r y - w e i g h t p e r p l a n t for Casuar-
ina cunninghamiana seedlingsinoculated with a crushed-noduleinoculum by three methods:
syringe (SYR), water-on (WO) and soil-mix (SM). significantly less than those of syringe-inoculated plants from 125 days after inoculation until the final harvest (Fig. 3 ). The relative growth rates (as estimated from the slope of the curves in Fig. 3 ) of the three inoculation treatments were similar from 111 days onwards. Thus the differences in dry-weight were due to the time it took to attain this growth rate. When shoot dry-weight was plotted against nodule dry-weight for each treatment and harvest time (Fig. 4), the relationship between these two parameters was found to contain a lag phase, with increasing low nodule weights not resulting in increased shoot weight, and then a linear phase, where shoot dry-weight was directly proportional to nodule dry-weight. Thus treatment effects on shoot dry-weight would be due to effects on the rate of nodule formation. E x p e r i m e n t 2A - F r a n k i a isolate
Inoculated plants were nodulated by the first harvest, 33 days after inoculation. By 75 days after inoculation, the shoot dry-weight of inoculated plants was twice that of uninoculated plants ( P = 5%; Fig. 5 ). By the final harvest the difference was 12-fold. The method of inoculation did not affect nodule number except at day 90, when the nodule number on plants inoculated by the soil-mix method was significantly greater than on plants inoculated by the water-on method ( P = 5%; Fig. 6 ). Inoculation method had no effect on nodule dry-weight per plant. Uninoculated plants remained unnodulated. The trends for shoot dry-weight were similar to those found for nodule number and nodule dry-weight. The method of inoculation did not affect shoot
142
P.A. ROSBROOK
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Fig. 5. The change in shoot dry-weight over time for Casuarina cunninghamiana seedlings left uninoculated (Uninoc) or inoculated with an isolate of Frankia by two methods: water-on (WO) and soil-mix (SM). LSD for loglo shoot weight 5% = 0.21, 1%= 0.32.
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Fig. 6. The change in the number of nodules per plant over time for Casuarina cunninghamiana seedlings inoculated with an isolate of Frankia by two methods: water-on (WO) or soil-mix (SM). LSD for log~o nodule number 5%=0.25, 1%=0.38.
dry-weight ( P = 5%) except at day 90, when the shoot dry-weight of soil-mix plants was just significantly greater than that of plants inoculated by the wateron procedure (P= 5%; Fig. 5 ).
EFFECT OF INOCULUM ON CASUARINA
143
Effect o f inoculum level
Experiment 1B - crushed nodules No significant difference in nodule number was found between the × 5 and X 1 level for syringe-inoculated plants, implying that the number of infective propagules was not limiting for this treatment (Table 1 ). For both the soilmix and water-on treatments, nodule number significantly declined between the X 5 and X 1 levels. Nodule dry-weight was unaffected ( P = 5%; Table 1 ) at the five highest levels of inoculum when the inoculum was syringed into the potting mix. For the water-on treatment, there was a rapid decline in nodule dry-weight with increasing dilution level, with nodule dry-weight at the X 5 dilution level being significantly greater than that at the X0.5 level ( P = 1%; Table 1 ). A similar decline in nodule dry weight was observed for the soil-mix treatment (Table
1). The above trends were reflected in shoot dry-weights. With syringe-inoculated plants, dilution of the inoculum did not significantly decrease shoot dryweight until the 0.02 level ( P = 5%), implying that the level of inoculum applied in experiment 1A was optimal (Table 1 ). However, for both the wateron and soil-mix treatments, there was a rapid decline in shoot dry-weight at inoculation levels lower than × 5, with a significant decrease occurring at the × 1 level ( P = 5%; Table 1 ). Thus for the two last-named methods of inocuTABLE 1
Nodule number, nodule dry-weight and shoot dry-weight per plant for Casuarina cunninghamiana seedlings 153 days after inoculation with dilutions of a crushed-nodule inoculum by syringe ( S Y R ) , water-on (WO) or soil-mix (SM) method (experiment I B ) Inoculum level t
5 1 0.5 0.2 0.1 0.02 0.01 0.005 0.002 0.001 0.0001
Nodule number
Nodule dry-weight (mg)
Shoot dry-weight (g)
SYR
WO
SM
SYR
WO
SM
SYR
WO
SM
41a 2 25ab 14bc 28ab 12c 2d 2d 2d ld . .
41a 13b 6c 6bc 5c 0.7d ld 0.5d 0.3d . .
26a 4b 4b 4b 0.3c 0.8c 0.7c 0.3c 0.3c . .
llla ll0a 96a 106a 100a _3 . .
ll0a 58ab 21b 32b 28b _ -
85a 18bc 44ab 13c 0.33d -
0.86a 1.10a 0.76a 1.19a 0.87a 0.18bc 0.29b 0.28b 0.26bc 0.16bc 0.10c
1.13a 0.48b 0.14cd 0.23bc 0.24bc 0.12cd 0.15cd 0.20bc 0.10d 0.15cd 0.11cd
0.73a 0.16bc 0.36b 0.13bc 0.08c 0.13bc 0.20b 0.10bc 0.10bc 0.12bc 0.17bc
. .
. .
qnoculum levels are expressed as multiples of the level applied in experiment IA. 2For each inoculum treatment, numbers with the same letter are not significantly different at P = 5%. 3_, not determined.
144
P.A. ROSBROOK
TABLE 2 Nodule number, nodule dry-weight and shoot-dry weight per plant for Casuarina cunninghamiana seedlings 117 days after inoculation with dilutions of an isolate of Frankia in a liquid medium by water-on (WO) or soil-mix (SM) method (experiment 2B) Inoculum level
2 1 0.5 0.2 0.1 0.02 0.01 0.005 0.001 0.0001 0.00001
Nodule number
Nodule dry weight (mg)
Shoot dry weight (g)
WO
SM
WO
SM
WO
SM
47a 2 30ab 29abc 27abc 26abc 16c 19bc 0.3e 8.9d 1.9e 1.4e
37ab 3lab 39a 30ab 20b 0.1c lc 0.3c 0.1c 0c 0.7c
56a 69a 52a 72a 43a 54a 46a 7b 35a 14b 4b
49a 45a 59a 48a 39a lc 6b lc 3bc 0c 3bc
0.59a 0.52a 0.46ab 0.53a 0.38ab 0.43a 0.47a 0.10c 0.31b 0.06c 0.04c
0.42a 0.32a 0.49a 0.41a 0.34a 0.05b 0.05b 0.05b 0.05b 0.03b 0.04b
qnoculum levels are expressed as multiples of the level applied in experiment lB. 2For each inoculation treatment, numbers with the same letter are not significantly different at P = 5%.
lation, greater quantities of inoculum than the × 1 level were required for optimal plant growth. The X 5 inoculum level gave equivalent shoot dryweights for each inoculation method, suggesting that this level was optimal for plant growth for each method. E x p e r i m e n t 2 B - F r a n k i a isolate
The first significant decrease in nodule number per plant occurred at the 0.02 dilution level for the water-on treatment and the 0.1 dilution level for the soil-mix treatment (Table 2). Both shoot dry-weight and nodule dryweight per plant were maintained to the 0.01 dilution level for the water-on treatment, whilst for the soil-mix treatment, shoot dry-weight and nodule dryweight per plant were maintained only to the 0.1 level (Table 2 ). DISCUSSION
T i m e to nodulation
Plants inoculated by the crushed-nodule and isolate inocula showed large differences in the time between inoculation and the appearance of nodules, the times being approximately 80 and 33 days, respectively. It is unlikely that the delay in nodulation that occurred with crushed-nodule inocula was due to low numbers of propagules, as the results for the dilution experiment (Table 1 ) show nodule number, nodule weight and shoot weight per plant for sy-
EFFECT OF INOCULUM ON CASUARINA
145
ringe-inoculated plants did not decline significantly until the 0.1 or 0.05 level, indicating that the higher levels of inoculum applied were optimal. For the water-on and soil-mix treatments (Table 1 ) lack of infective propagules was a problem, yet these treatments began to nodulate at approximately the same time as the syringe treatment. The greater length of time that seedlings inoculated with crushed-nodule inocula need to nodulate might be due to differences between strains or the presence of polyphenols released from the ground nodules (Torrey, 1983 ), which could limit growth of Frankia in the potting medium (Perradin et al., 1983). Baker et al (1979) found separation of Frankia from nodules of Elaegnus umbellata by sephadex fractionation could result in a 5-fold increase in nodulation capacity over the crude crushed-nodule extract. Phenolics and pigments, which may act as inhibitors, are thought to be removed from the nodule extract by sephadex fractionation. If crushed nodules are to be used to inoculate seedlings of Casuarina, techniques to remove inhibitors released upon grinding should be investigated.
Method of application Crushed nodules The method of inoculum application was more critical when crushed-nodule inoculum was used. All three crushed-nodule methods of inoculation resulted in nodulation, but the rate at which infection took place and the resultant growth rate of plants varied between the different methods. Although no significant differences occurred in plant growth between the syringe and wateron treatments, syringe-inoculated plants tended to form nodules more quickly (Fig. 1 ) and grow faster (Fig. 3) than plants inoculated by the water-on method, suggesting that concentrating the inoculum close to the root system was advantageous. Syringe-inoculated plants had greater nodule number, nodule dry-weight and shoot dry-weight per plant (Figs. 1, 2, 3 ) than soil-mix inoculated plants at most harvest times. The dilution experiment (Table 1 ) showed the soilmix inoculation method resulted in significantly fewer nodules on the Casuarina plants than did the syringe inoculation method. This was probably due not only to the diluting effect of the application method, which results in fewer propagules coming into direct contact with the young root system, but also to the inoculum not being further ground by mortar and pestle. Fewer infective propagules may have been released from the nodules compared with the preparation methods of the syringe and water-on treatments. The dilution experiment showed all three application methods were equally effective at inducing nodulation and enhancing plant growth, as long as large quantities of inoculum are available. As the level of inoculum declined (Table 1 ), large differences were observed between methods, with the syringe
146
P.A. ROSBROOK
method consistently giving the best plant growth and the soil-mix method the worst. Thus when using crushed-nodule inocula, large quantities of nodules should be used, particularly if inoculating by a method similar to the wateron and soil-mix techniques. Franida isolate Inoculation with the Franlda isolate resulted in rapid nodulation and enhanced plant growth compared with uninoculated plants, whether wateredon or mixed through the soil. The dilution results suggest that the inoculum could have been further diluted by a factor of 10 without a significant decrease in plant growth occurring (Table 2). At low levels ofinoculum (0.02) differences were observed in plant dry-weight and nodule dry-weight per plant between the inoculation methods, with the water-on treatment resulting in greater shoot dry-weights and nodule dry-weights per plant than the soil-mix treatment. A major problem with the use of Frankia isolates is the lack of a simple method for defining the quantity of inoculum that is applied. Microscopic examination of the inoculum revealed both sporangia and hyphae. Workers have used optical density (Lalonde and Calvert, 1979) and packed cell volume (Berry and Torrey, 1985; Stowers and Smith, 1985 ) to define inoculum levels. However, a given optical density or packed cell volume may contain different proportions ofhyphae and sporangia and thus may differ in infectivity. In addition, there are practical problems with large-scale use of liquid inocula. Thus the present study demonstrates that, for rapid nodulation of C. cunninghamiana seedlings in the nursery, use of crushed-nodule inocula requires relatively large amounts of nodule (>10,2 g dried nodule per seedling) and placement of the inoculum as close as possible to the root system. With isolates of Franlda, placement is generally less critical. ACKNOWLEDGEMENTS The statistical advice of Dr. R. Correll and the technical assistance of Ian Francis, Leanne Stuckey and Dave Taylor are greatly appreciated. The research was funded by a grant from ACIAR (Australian Centre for International Agricultural Research).
REFERENCES Baker, D., Kidd,G.H. and Torrey,J.G., 1979. Separationof actinomycetenoduleendophytes fromcrushednodulesuspensionsby sephadexfractionation.Bot.Gaz., 140:549-551.
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Berry, A.M. and Torrey, J.G., 1985. Seed germination, seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials. Plant Soil, 87:161-173. Lalonde, M. and Calvert, H.E., 1979. Production of Frankia hyphae and spores as an infective inoculant for Alnus species. In: J.C. Gordon, C.T. Wheeler and D.A. Perry (Editors), Symbiotic Nitrogen Fixation in the Management of Temperate Forests. Oregon State University Press, Corvallis, pp. 94-110. McCluskey, D.N. and Fisher, R.F., 1983. The effect of inoculum source on nodulation in Casuarina glauca. Commonw. For. Rev., 62:117-124. Perinet, P., Brouillette, J.G., Fortin, J.A. and Lalonde, M., 1985. Large scale inoculation of actinorhizal plants with Frankia. Plant Soil, 87:175-183. Perradin, Y., Mottet, M.J. and Lalonde, M., 1983. Influence of phenolics on in vitro growth of Frankia strains. Can. J. Bot., 61: 2807-2814. Reddell, P. and Bowen, G.D., 1985. Frankia source affects growth, nodulation and nitrogen fixation in Casuarina species. New Phytol., 100:115-122. Sellstedt, A., 1988. Nitrogenase activity, hydrogen evolution and biomass production in different Casuarina symbioses. Plant Soil, 105:33-40. Shipton, W.A. and Burggraaf, A.J.P., 1983, Aspects of the cultural behaviour of Frankia and possible ecological implications. Can. J. Bot., 61: 2783-2792. Stowers, M.D. and Smith, J.E., 1985. Inoculation and production of container-grown red alder seedlings. Plant Soil, 87:153-160. Torrey, J.G., 1983. Casuarina: Actinorhizal dinitrogen-fixing tree of the tropics. In: S.J. Midgley, J.W. Turnbull and R.O. Johnston (Editors), Casuarina Ecology, Management and Utilization. CSIRO, Melbourne, pp. 192-204.