9
PlantandSoil 176: 161-169, 1995. Q 1995 KluwerAcademic Publishers. Printed in theNetherlands. The effect of phosphorus on nodule formation and function in the Casuarina- Frankia symbiosis Yun Yang CSIRO, Davies laboratory, P.O. Aitkenvale, Townsville, QLD 4814, Australia and Department of Veterinary and Biomedical Sciences, James Cook University, Townsville, QLD 4814, Australia Received7 November1994.Accepted in revisedform20 March 1995 Key words: Casuarina cunninghamiana, Frankia, nitrogen fixation, nodulation, phosphorus Abstract A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina- Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 #M significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 #M. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth. Introduction Phosphorus deficiency is a major limiting factor to growth and nitrogen fixation in Casuarina species in many tropical soils (Reddell, 1986, 1990). Develop- ment of management approaches to minimize or alle- viate the impacts of soil P limitations (and maximize the benefits of N-fixation) on plantation productivity first requires an understanding of the mechanisms by which P affects plant growth and symbiotic N-fixation. In particular it is necessary to identify the stage of plant growth or symbiotic N-fixation which is most sensitive to P deficiency. As outlined by Loneragan (1972) and Robson (1978), there are three approaches: 1) evalu- ating the interaction between combined nitrogen and supplied nutrients and their relative impact on growth and nitrogen fixation; 2) determining the effect of a nutrient supply on nitrogen concentrations in plants supplied with combined nitrogen and inoculated with nitrogen fixing microorganisms and 3) measuring the effect of a nutrient supply on nodulation parameters (e.g. nodule mass, nodule size and nodule numbers) and nodule function (e.g. nitrogenase activity). Based on these criteria, studies have been conduct- ed to examine various roles of P in plant growth and symbiotic nitrogen fixation processes (Israel, 1987; Robson et al., 1981). However, there may be spe- cific effects of P on the microorganism populations in the rhizosphere and nitrogen fixing ability of these microorganisms (Mullen et al., 1988). This could sig- nificantly affect nodule formation and N-fixation activ- ity of nodules and mask a specific P requirement by a nitrogen fixing plant. Therefore, in an attempt to iden- tify which component of the Casuarina-Frankia sym- biosis is sensitive to P deficiency, the following aspects were examined in this study:

The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis

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Plant andSoil 176: 161-169, 1995. Q 1995 KluwerAcademic Publishers. Printed in the Netherlands.

The effect of phosphorus on nodule formation and function in the Casuarina- Frankia symbiosis

Yun Yang CSIRO, Davies laboratory, P.O. Aitkenvale, Townsville, QLD 4814, Australia and Department of Veterinary and Biomedical Sciences, James Cook University, Townsville, QLD 4814, Australia

Received 7 November 1994. Accepted in revised form 20 March 1995

Key words: Casuarina cunninghamiana, Frankia, nitrogen fixation, nodulation, phosphorus

Abstract

A study was conducted to investigate the effects of phosphorus on nodule formation and function in the Casuarina- Frankia symbiosis. The effects of P on growth and survival of Frankia in the rhizosphere was assessed by examing Frankia growth and survival in flasks of basal nutrient solution. There was no growth in the nutrient solution during the experimental period. However, the viability of Frankia in the nutrient solution without P supply was half that of the initial level, whereas, with P supply, there was only a minor decline during the first week. In a growth pouch experiment, supplying P increased plant and nodule growth, irrespective of P status of the inoculant Frankia culture. There were no effects of P status on any growth or nodulation parameters measured when the inoculants had been standardized on the basis of viability. In a split root experiment, Frankia inoculation and application of P together or separately did not cause any significant difference. This suggests that growth and nodulation respond only to total P supply. Increasing P from 0.1 to 10 #M significantly increased plant growth but not N concentrations. Both nitrogen-fixation and nitrate supported growth were strongly increased as P increased from 0.1 to 1.0 #M. This study indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes and P deficiency on nodulation and symbiotic nitrogen fixation in Casuarina cunninghamiana operated indirectly via reducing host plant growth.

Introduction

Phosphorus deficiency is a major limiting factor to growth and nitrogen fixation in Casuarina species in many tropical soils (Reddell, 1986, 1990). Develop- ment of management approaches to minimize or alle- viate the impacts of soil P limitations (and maximize the benefits of N-fixation) on plantation productivity first requires an understanding of the mechanisms by which P affects plant growth and symbiotic N-fixation. In particular it is necessary to identify the stage of plant growth or symbiotic N-fixation which is most sensitive to P deficiency. As outlined by Loneragan (1972) and Robson (1978), there are three approaches: 1) evalu- ating the interaction between combined nitrogen and supplied nutrients and their relative impact on growth and nitrogen fixation; 2) determining the effect of a nutrient supply on nitrogen concentrations in plants

supplied with combined nitrogen and inoculated with nitrogen fixing microorganisms and 3) measuring the effect of a nutrient supply on nodulation parameters (e.g. nodule mass, nodule size and nodule numbers) and nodule function (e.g. nitrogenase activity).

Based on these criteria, studies have been conduct- ed to examine various roles of P in plant growth and symbiotic nitrogen fixation processes (Israel, 1987; Robson et al., 1981). However, there may be spe- cific effects of P on the microorganism populations in the rhizosphere and nitrogen fixing ability of these microorganisms (Mullen et al., 1988). This could sig- nificantly affect nodule formation and N-fixation activ- ity of nodules and mask a specific P requirement by a nitrogen fixing plant. Therefore, in an attempt to iden- tify which component of the Casuarina-Frankia sym- biosis is sensitive to P deficiency, the following aspects were examined in this study:

162

• survival and growth of Frankia in plant nutrient solution with and without P supply as an indication of P status in the rhizosphere

• impacts of P status in Frankia on nodule formation

• the external and internal P requirement for nodula- tion and growth of C. cunninghamiana Miq.

• interactions in relation to increasing P supply between seedlings either (I) provided with com- bined N at a rate adequate to support maximum growth or (ii) reliant on symbiotic N-fxation to satisfy their N demands

Materials and methods

Experiment 1: In vitro growth and survival of Frankia in flasks of plant nutrient solution

This experiment aimed to investigate the survival and growth of Frankia inoculum introduced into the basal plant nutrient solution which was prepared for the solu- tion culture studies. There were two treatments (basal nutrient solution and basal nutrient solution with 200 #M KH2PO4) each replicated five times with complete randomized design.

The concentrations of a basal nutrient solution were as follows (#M): K2SO4 1000; CaC12.2H20, 500; Ca(NO3)2.2H20, 50; MgSOa.7H20, 1000; NaC1, 10; ZnSO4.7H20, 2.5; CuSOa.5H20, 0.5; MnSOa.H20, 0.5; COSO4.7H20, 0.2; Na2MoO4.2H20, 0.1; HaBO3, 15; FeEDTA, 100. Thirty mL of this solution was dis- pensed into 50 mL medicine flask prior to inoculation with Frankia.

Colonies of Frankia JCT287 grown in the stan- dard propionate-based media (Shipton and Burggraaf, 1983) were washed three times with sterilized dis- tilled water and homogenized by aspirating 20 times through a 10 mL syringe fitted with an 18 gauge nee- dle. Four mL of this homogenate (with protein con- tent of about 40 #g mL -1) was introduced into each medicine flask. The inoculated flasks were incubat- ed in a darkened container in a glasshouse (temper- ature range 18°C to 34°C). Five replicates of each treatment were harvested immediately after inocula- tion and then weekly for five weeks, Frankia sur- vival and growth were estimated using the protein and dehydrogenase (2-(4-iodophenyl)-3(4-nitrophenyl)-5- phenyltetrazolium chloride, INT) and protein assays described by Prin et al. (1990) and Shipton and Burggraaf (1983), respectively.

Experiment 2: Effect of P status ofFrankia inoculant on nodulation of C. cunninghamiana under P-adequate and P-deficient conditions

This experiment was conducted to examine the influ- ence of prior P status of the inoculant Frankia strain on speed and intensity of nodulation. A randomized block design was used with four replicates of each treatment combination.

Commercial plastic growth pouches (Fortin et al., 1980) were used for this experiment. Two seedlings were transferred to each pouch by inserting their roots through holes made in the paper wicks within each pouch. The pouches were then stapled at three points along their upper side (one between the two seedlings, one on each outside edge) to reduce evaporation and contamination through dust input. Growth pouches containing seedlings were suspended in folder paper on a file rack in an air-conditioned glasshouse (tem- perature range 24 ° to 33°C).

Ten mL of a sterilized, low N (as 50 ~M Ca(NO3)2.4H20), minus P nutrient solution contain- ing either adequate P or no P was added to each pouch immediately after seedlings were transplanted. This quantity of nutrient solution was sufficient to moisten the paper wick. Nutrient solutions in each pouch were drained and replaced every two weeks. In between renewal of the nutrient solution the wick was kept moist by adding sterilized deionized water whenever required.

Pouches were inoculated 14 days after seedlings were transplanted. Inoculum was prepared from 25- day-old cultures of Frankia JCT287 grown in modified P media containing either low (1/~M) to adequate P or luxury P (1 #M). Prior to inoculation, inoculum 'con- centration' was standardized to either the same protein density (as a measure of total Frankia biomass) or the same total viability (using INT reduction activity) to ensure that similar amounts of inoculum were applied, irrespective of the original P status of the cultures. The standard concentrations used in this experiment were: 3.3 #g protein mL -1 of culture and 0.7 nmol INT formazan (INTF) mL- 1 of culture. Pouches were then inoculated by using a syringe to apply of homog- enized inoculum over the whole root system of each individual seedling.

Nodulation was assessed weekly, for nine weeks after inoculation, by scoring the numbers of mature nodules on the root systems with the aid of a dissect- ing microscope (Wild M3Z). Although nodule initia- tions were observed two weeks after inoculation, only

aeration ~ _ _ system

solution ~¢:~ ~ ................. ~. . . . . . . . . lever

inner compartment

outer compartment -

Fig. 1. The vertical split root system devised in Experiment 3 for studying growth and nodulation of Casuarina cunninghamiana seedlings.

mature (probably functional) nodules were scored. Nine weeks after inoculation, plants were harvest- ed and shoots, root, and nodules were separated for biomass determinations following drying at 60°C for two days.

Experiment 3: Is the P requirement for nodulation internally or externally mediated?

A split-root experimental system using solution culture was devised in an attempt to separate any direct effects of P on infection and nodule development processes from indirect effects mediated via host-plant growth. There were seven treatments involving Frankia inocu- lation and/or P application to each of the two compart- ments in the split-root system used. A low N, minus P basal solution was used in all compartments in all treatments. Each treatment was replicated four times in a randomized complete block design. The treatments applied to the basal nutrients in each compartment are summarized in Table 2,

Individual seedlings (described in Exp. 2) were transplanted into 1L pots over an aerated, low-N nutri- ent solution (as described previously) for four weeks to allow the root systems to develop to the minimum length of 10 cm. These established seedlings were then transferred gently into a vertical split-root sys- tem which consists of two different sized containers (Fig. 1). A small container was positioned inside a larger vessel, thus constructing two compartments of identical volume (500 mL). The seedling was support- ed by the lid over the larger compartment. The lower portion of the root system of each seedling was then gently threaded through a hole in the wall of the inner

163

compartment which was then sealed with waterproof silicon sealant. The pots were placed in controlled tem- perature water tanks to maintain root temperatures at approximately 25°C throughout the experimental peri- od. Nutrient solutions in each compartrnent were aer- ated continuously.

Phosphorus application (200 / IMP as KH2PO4) and Frankia inoculation treatments were imposed one week after the roots were separated into the two com- partments. For the inoculation treatment, 4mL of Frankia JCT287 (at 9 #g protein mL- l culture) was added into the appropriate compartment. The nutrient solution in each compartment was renewed every two weeks.

Plants were harvested eight weeks after inocula- tion and imposition of P treatments. Shoots, roots and nodules presented in each split-root compartment were separated. All tissues were dried at 60°C for 48 hours. N and P concentrations in shoots were determined using an auto-analyzer (McLeod, 1982).

Experiment 4: Initial calibration of N and P supply rates for growth of C. cunninghamiana in solution culture

The aims of this preliminary experiment were to deter- mine the growth rate of C. cunninghamiana in solution culture in response to a range of rates of P supply and to identify the order of magnitude of an appropriate com- bined N fertilizer treatment to act as a comparison with plants reliant on symbiotically fixed N. A randomized block design with a complete factorial combination of the following treatments was used.

Three rates of 0.1/zM KH2PO4

phosphorus supply: 10~uM KH2PO4

100/~M KH2PO 4

Three nitrogen: 50#M Ca(NO3)2.4H20,

nitrogen treatments uninoculated (100~M N)

500uM Ca(NO3 )2.4H20,

uninoculated (1000#M N)

50/IM Ca(NO3 )2.4H20,

inoculated with Frankia JCT287. There were eight replicates of each treatment combi- nation.

An aerated, intermittently-renewed solution culture system was used. Black plastic pots (21 cm in diame- ter) were each filled with 5 L of deionized water and a basal nutrient solution (as described in Exp. 1) was added as concentrated stock solutions of each nutri-

164

ent salt. The pH of the nutrient solution was adjusted to 6.5 using 1 mol KOH. Additional salts were added for the appropriate P and N treatments. Each pot was covered with an opaque, grey PVC lid in which five 2.5 cm-diameter holes had been drilled at regular spac- ings. Individual seedlings were suspended in 'baskets' which fitted tightly into the holes in each lid. The bas- kets were produced by removing the base from plastic 'Nalgene' stoppers, annealing a coarse nylon mesh, placing one seedling in each and filling the basket with high density, black PVC beads to support the seedling. Each pot was aerated using a disposable glass pasteur pipette attached via plastic tubing to a hypodermic nee- dle inserted into a central airline of Silastic tubing.

Four C. cunninghamiana seedlings (prepared as described in Exp. 2) were transplanted into each black plastic pot. In treatments involving inoculation with Frankia, thirty-day-old Frankia JCT287 colonies were disrupted gently for a few seconds using a homogeniz- er (Omni-Mix). Thirty mL of this Frankia suspension was transferred to a beaker (50 mL capacity) and the root system of each seedling was dipped into the sus- pension for a few seconds prior to transplanting into the solution culture pots. To ensure that the Frankia inocu- lum was not dispersed from the roots immediately after inoculation, the air supply to all pots was turned off for the first four days after transplanting. Pots were placed in a root temperature tank maintained at 25°C in an air conditioned glasshouse (temperature range 18°C to 34°C) for the course of this experiment. Nutrients and concentrations used were similar to those described in Experiment 1 and renewed by adding the same concen- trations (see Exp. 1 and 2) to the pots weekly. All nutri- ent solutions were discarded and completely replaced every four weeks.

Four months after transplanting, the plants were harvested. The number of nodules on the roots from each pot were recorded. Shoots, roots and nodules were separated and dried at 65°C for 48 hours for dry weight assessment. Materials were then digested by the Kjeld'ahl digestion method and N and P concentra- tions were determined using an autoanalyzer (McLeod, 1982).

Results

Experiment 1

Irrespective of P treatment, as estimated by protein concentration in the plant nutrient solution, the growth

.~ 12

~ 8

l.k

-~ 4

0 , , , , ,

0 1 2 3 4 5

12

lo I

$ 4

e 2 Z

v

1 2 3 4 5

Incubation time (weeks)

Fig. 2. Growth and survival of Frankia JCT287 in flasks of plant nutrient solution with and without P present, a: growth; b: viability. symbols: • - P supplied in plant nutrient solution; • - no added Pin plant nutrient solution. Bars represent standard errors of 5 replicates.

of Frankia remained constant at about 7 /zg mL-] nutrient solution over all harvests (Fig. 2a). Although there was some variations of protein concentration in nutrient solution, they were not significantly different over the experimental period (p<0.01). This indicates that no Frankia growth occurred during the five week period following inoculation of the Plant nutrient solu- tion. In contrast there was a very rapid decline in via- bility of Frankia in the plant nutrient solution with no viable Frankia detected in either of the two P treat- ments at five weeks after inoculation (Fig. 2b). How- ever, the presence or absence of P in nutrient solution strongly affected the survival of Frankia during the first week following inoculation. During this period, there was only a minor decline in viability of Frankia in the nutrient solution with P present whereas viabil- ity of Frankia in nutrient solution without P was half that of the initial level (p<0.01). In the second and subsequent weeks no differences were found between the P treatments in survival of Frankia (p<0.01).

165

Table 1. Effect of P status in Frankia on nodulation and growth of Casuarina cunninghamiana seedlings in nutrient solutions with and without P supply 9 weeks after inoculation in Experiment 2. Note that inoculum density was standarized by using either protein concentration (gg m L - t culture) or INTF 2 concentration (nmol INTF m L - t culture) in Franl~ia. Values with same superscript letter(s) in each row are not significantly different on the basis of LSD (p<0.01)

P supplying in 200 tzM P No added P

plant nutrient

solution

Inoculum source --* Frankia cultured in media Frankia inoculum cultured Frankia cultured in media Frankia cultured in media

with O.OO1 m M P in media 1.O m M P with O.OO1 mM P with 1.0 m M P

Inoculum standardized by Inoculum standardized by

Protein INTF Protein INTF Protein INTF Protein 1NTF

Total nodule dwt v 50.5 a 38.1 ° 42.14 35.9 ° 8.1/' 8.7/' 5.9/' 7.2/'

(rag/pouch)

Total nodule numbers 17 ° 21 ° 19 a 22 a 9/' 12/' 9/' 9/'

per pouch Average nodule size 3.0 ~ 1.9 abe 2.34b 1.8 ~'¢(/ 0.9 ~d 0.8 ¢4 0.74 0.8 c4

(rag/nodule)

Shoot dwt (mg/pouch) 147.4 bc 214.9" 223.4 ~ 191.64b 75.7(/ 89,V 51.9 (/ 83.2 c(/

Root dwt(mg/pouch) 113.0 a 110.7 ~ 102.1 a 87.0 '~ 90.4 a 105.1 '~ 78.84 88.9 a

Totaldwt(mg/pouch) 260.44be 324.7 '~ 325.5 ° 278.69 b 166.1 c4 194.2 ~¢4 130.7 a 172.1 c4

Root/shoot ratio 0.77 at' 0.52 ° 0.46 a 0.45 ~ 1.19/' 1.18b 1.53/' 1.06 b

Nodule dwt/total 17.6 ° 12.4 a 12.8 a 12.7 '~ 4.8/' 4.6 b 4.6 b 3.9 b

z INTF = 2-(4-iodopbenyl)-3(4-nitrophenyl)-5-phenyltetrazolium chloride formazan. Vdwt = dry weight.

Table 2. Growth and nodulation of Casuarina cunninghamiana seedlings grown in a vertical split root experimental system in response to P supply and Frankia inoculation in Experiment 3. Mean values in the columns followed by same superscript letters are not significantly different from each other on the based of LSD at p<0.05

Treatments Growth and nodulation parameters

Root dwt (g/pot)

Inner Outer Inner root Outer root Nodule Total dwt Nodule

compartment compartment dwt z (g/pot) dwt(g/pot) number (g/pot) dwt/total

per pot dwt (%)

% shoot N % shoot P

Fr+pU nil 0.344 0.124 150 ° 2.394

nil Fr+p 0.50 b 0.17 a 83 b 2.22 a

Fr P 0.26 ~ 0.15 ~ 152 a 1.84 b~

P Fr 0.58/' 0.11 ~ 156 a 2.11 ab

P nil 0.54 b 0.16 ̀~ 0 ~ 1.64 ¢

nil P 0.57 b 0.19 a 0 c 1.64 c

nil nil 0.21 a 0.144 0 c 0.844

7.7 a

3.5 b

5.2 b

4.5 b

2.4 a 0.31 °b

2.0 a 0,19 c(/

2.3 ~ 0.23 bc

1.9 b 0.31 ~b

1.4 ¢ 0.13 d

1.7 c 0.31 ab

1.7 ¢ 0 . 1 4 a

dwt = dry weight. UFr = Frankia inoculation; P = supplied with 200/zM KH2PO4.

Exper iment 2

I r r e s p e c t i v e o f P s t a t u s o f t h e i n o c u l a n t Frankia

c u l t u r e , s u p p l y i n g P in t h e p l a n t n u t r i e n t s o l u t i o n

i n c r e a s e d s h o o t a n d n o d u l e d r y w e i g h t s , n o d u l e n u m -

b e r p e r p o u c h , a v e r a g e n o d u l e s i ze a n d % o f to t a l p l a n t

d r y w e i g h t in n o d u l e s ( T a b l e 1). P s u p p l y in t h e n u t r i e n t

s o l u t i o n a l so d e c r e a s e d t h e r o o t / s h o o t r a t io b y b e t w e e n

3 5 % a n d 7 0 % ( p < 0 . 0 1 ) .

166

Table 3. Effect of N treatment and P supply on growth, nodulation and N and P concentrations in different segments of C. cunninghamiana seedlings in Experiment 4. Values in column with same superscript letter(s) am not significantly different on the basis ofLSD (p<0.01)

N treatment P rates Total dwt ~ pot- l Shoot N% Shoot P% Nodule Nodule Nodule N% Nodule P%

(#M) number pot- 1 dwt/total dwt

(X 100)

100#MN

1000#MN

0.1 0.41 a 0.55 a 0.05 a

10 2.67 ca 1.22 ~b 0.16 ab

100 2.32 ca 0.93 ab 0.29 bc

0.1 0.20 a 2.44 a 0.03 a

I0 5.35 bc 1.41 abca 0.04 a

100 3.36 ed 1.92 abed 0.33 c

Frankia+ 0.l 1.59 ca 2.10 d 0.02 a

100#MN 10 8.05 ab 1.66 abca 0.04 a

100 11.16 ~ 2.29 a 0.28 bc

113 a 2.59 a 1.91 a 0.05 a

145 a 3.06 a 2.32 ~ 0.08 a

381 b 6.30 b 2.56 ~ 0.16 b

z dwt=dry weight.

o

c 0>

25

20

15

10

0 I 1

( I, I I I I I I I I 2 3 4 5 6 7 8 9 10

w e e k s after inoculation

Fig. 3. Effect of P status of Frankia on nodulation and growth of Casuarina cunninghamiana seedlings grown with and without P in the plant nutrient solution. Inoculum densities were standardized by inoculating with equal protein concentrations. Symbols: • - Frankia grown in a P-luxury medium and seedlings supplied with an adequate P in nutrient solution; • - Frankia grown in a P-luxury medium and seedlings without added P; • - Frankia grown in a medium without added P and seedlings supplied with an adequate P in nutrient solution; • - Frankia grown in a medium without added P and seedlings without added P. Bars represent standard errors of 4 replicates.

However, P status of the inoculant Frankia cul- ture did have some influence on plant growth, but only when inoculum was standardized using total pro-

tein concentration. This effect was most pronounced in reducing shoot dry weight at harvest. There were no effects (p<0.01) of Frankia P status on any growth or nodulation parameters measured over the experimen- tal period when the inoculant had been standardized on the basis of viability (Table 1, Fig. 3).

Experiment 3

Irrespective of the P supply or Frankia inoculation, there was greater root growth in the inner compartment of the split-root system than in the outer compartment (Table 2). Within the treatments involving Frankia inoculation, application of Frankia and P together in the inner compartment increased total plant dry weight by about 120% and the percent of total plant dry weight as nodules by 180% (p_<0.05) when compared with treatments where Frankia and P were applied separate- ly (Table 2). However, nodule numbers were similar in these treatments, except that there were lower nodule numbers and percent of total dry weight as nodules when Frankia and P were applied together in the outer compartment (p_0.05). Shoot N concentrations were not significantly different in these treatments. Howev- er, shoot P concentrations in seedlings supplied with P in the inner compartment were significantly higher than those supplied with P in the outer compartment (p<0.05).

Experiment 4

The responsiveness of C. cunninghamiana seedlings to increasing P supply from 0.1 #M to 100 #M was depen- dent upon the N treatment applied (Table 3). Growth of seedlings inoculated with Frankia increased with increasing P supply (p<0.01). Similarly, nodule num- ber and the proportion of total plant dry weight in nodules increased with increasing P supply. In addi- tion, there was a strong positive relationship (r2=0.82) between total plant dry weight and nodule dry weight over all rates of P supplied.

In contrast to the inoculated plants, the growth of seedlings supplied with 1000 #M N only responded to increasing P supply between 0.1 and 10 #M. Increasing P supply from 10 to 100 #M had no effect on plant dry weight (p<0.01). At the lowest rate of P supply (0.1 #M), seedlings provided with the 'standard' 100 #M N in the basal nutrient solution had similar dry weight to those provided with 1000 ~M N (p<0.01). However, at 10 #M P, seedlings with 1000 #M N produced almost twice the biomass of the low N plants (p<0.01).

At the lowest rate (0.1 #M) of P supply, total dry weight of inoculated seedlings was slightly higher than that of uninoculated plants, but the difference was not significant according to analysis of variance and LSD (p< 0.01). At the intermediate P rate (10 #M), seedlings dependent on symbiotic N fixation and those supplied with the higher level of combined N produced equiv- alent amounts of dry matter. At the highest rate of P supply, inoculated plants produced between three and four times more dry matter than did plants supplied with combined N only.

In seedlings supplied with 1000 #M N, increasing P supply did not increase shoot %N, but significantly increased shoot %P (p<0.01). Similarly, in plant inoc- ulated with Frankia plus 100 #M N, P supply had no effect on shoot %N and nodule %N, but significantly increased shoot %P, and nodule %P (Table 3).

Discussion

The lack of Frankia growth in flasks of plant nutrient solution was not unexpected as no carbon and vita- min sources were present. However, the rapid decline in viability of inoculum in the nutrient solution was more surprising, as was the effect of P in maintaining viability for the first week after inoculation. In Rhi- zobium spp. the growth and survival in liquid defined media culture were limited when P supply was low-

167

er than 1 #M (Keyser and Munns, 1979) or 0.06 #M (Beck and Munns, 1984). Some rhizobia strains have the capacity to store P and then utilize this to maintain the growth for several generations (Beck and Munns, 1984; Cassman et al., 1981 a,b; Smart et al., 1984). Results from the present study suggest that the sensitiv- ity of Frankia growth to P stress may be similar to that of Rhizobium. With the presence of appropriate levels of P, the growth and survival of Frankia in the rhizo- sphere could be secured. As Frankia colonization and infection processes commence at the first week after inoculation (Callaham et al., 1979), the probability of infection by Frankia depends on its effective abun- dance in the rhizosphere over this period. Thus, the P status of rhizosphere may signifcantly influence the resultant pattern of nodulation (e.g. number of nodules formed). Further research is needed on quantifying the minimum P requirement for growth and survival of Frankia in the rhizosphere.

It was reported that infection of primary roots of soybean seedlings (Glycine max (L.) Merr.) was delayed when P-limited cells of Bradyrhizobium japonicum were applied (Mullen et al., 1988). In the present study, the presence of P in the nutrient solution that was added to the growth pouch was the primary determinant of plant growth and nodulation (Table 1, Fig. 3). This was related to the viability of Frankia inoculants as standardizing inoculation rates on the basis of Frankia viability, rather than biomass (e.g. protein content), eliminated any effects on speed of nodulation. Thus, P requirements derived in vitro may not necessarily be an accurate indication of P require- ment for Frankia growth and colonization of the host- plant rhizosphere.

In a vertical split-root experiment, attempts to sep- arate the direct effects of P supply on host-plant growth from those on nodulation processes were unfortunately confounded by consistently greater root growth occur- ring in the inner compartment of the split-root system (Table 2). Nonetheless, Frankia inoculation and pro- vision of adequate P together in the inner compart- ment apparently produced signifcantly higher total dry weights and proportion of total dry weight in nodules than any other treatments. Since nodulation was not affected by whether P was supplied in separate com- partment this suggests that the growth and nodulation of C. cunninghamiana is more responsive to total P supply.

Data from Experiment 4 demonstrated that growth of C. cunninghamiana seedlings are very responsive to P. Alleviation of P deficiency from 0.1 to 10 mM

168

significantly increased dry weight in both inoculated and uninocula[ed seedlings. At a higher P (100 mM) supply, the growth of seedlings supplied with high N was unexpectedly lower than those inoculated with Frankia. Perhaps this was caused by alkalinity devel- oping during nitrate assimilation. An alternative source of N such ammonium should be considered for further studies. However, the growth of seedlings reliant on symbiotic nitrogen fixation and supported by N had a similar trend. This growth pattern clearly indicates that P deficiency limits the growth of host plants more severely than nitrogen fixation processes, particularly under low P conditions. As a consequence of the host plant being P-deficient, plant N demand is also reduced and it is likey that the proportion of photosynthate par- titioned to nodules decreases. This could explain the reduction in the % of total dry weight in nodules when plants are supplied with low P.

It is noted that the limitation of nodulation, partic- ularly infection, by P deficiency may not occur when large amounts of inoculum were imposed. The ade- quate inoculum density required depends greatly on the quantity of functional microorganisms and host plants. In this study, the choice of inoculum density was based on the results .from a dose experiment which gave the optimum nodulation in Casuarina cunninghamiana, and eleminated the limiting effect of inoculum densi- ty.

These finding suggest that the effect of P on symbi- otic nitrogen fixation operates indirectly via host-plant growth. There are four lines of evidence to support this:

• P status in Frankia cultured in plant mineral nutri- ents (Exp. 1) and derived in vitro (Exp. 2) has no effect on Frankia growth and colonization of host-plant rhizosphere and nodulation, other than maintenance of Frankia viability for a short period.

• Nodule and plant growth respond only to total P supply (Exp. 3).

• There is a strong positive correlation between total plant dry weight and total nodule dry weight. Hence, nodule growth may be controlled via plant growth.

• The P requirements for Frankia and nitrate- supported growth are similar. Thus, it is concluded that host-plant growth has

a higher P requirement than symbiotic nitrogen fixa- tion in Casuarina cunninghamiana. Therefore, select- ing Casuarina genotype on the basis of its high P-use efficiency and development of P nutritional fertiliza- tion strategies to optimize the efficiency of acquisition

of any applied P are the key solutions to overcome the effect of P deficiency on symbiotic nitrogen fixing plantation of Casuarina.

Acknowledgements

Drs P Reddell, W Shipton and Y Prin provided help- ful advice on this study. This study was funded by the scholarship of Australian Centre for International Agricultural Research and a James Cook University Postgraduate Scholarship Research Award.

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