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Laboratory tests can predict beneficial effects of phosphate-solubilising bacteria on plants
ARTICLE IN PRESS
Soil Biology & Biochemistry 38 (2006) 1521–1526 www.elsevier.com/locate/soilbio
Laboratory tests can predict beneficial effects of phosphate-solubilising bacteria on plants Jodie N. Harris, Peter B. New, Peter M. Martin Received 16 April 2004; received in revised form 15 November 2005; accepted 22 November 2005 Available online 7 April 2006
Abstract Phosphorus is important in plant growth but in the soil it reacts readily to form insoluble compounds, which are not readily available for plant utilisation. Although the presence of soil microorganisms capable of solubilising phosphate has been known for many years, their isolation and use as crop inoculants have met with only limited success. This project aimed to develop a more robust method of selecting strains, which would give a more reliable indication of their usefulness for Australian crops, with a particular focus on wheat. Bacteria were isolated from a typical black wheat growing soil from the North-Western wheat belt of New South Wales using a differential medium containing insoluble phosphate. In all, 48 isolates were tested in the laboratory for their ability to colonise the roots of wheat seedlings in sterile perlite and to solubilise phosphate in the rhizosphere of plants grown on agar plates. To establish whether strains selected for their laboratory performance could benefit wheat grown in unsterilised field soil, a pot trial was carried out in a highillumination growth chamber. Seven strains of varying capabilities were used to determine the effects of inoculation on releasing unavailable phosphorus and improving growth and yield, in comparison with conventional phosphorus fertiliser. Strains that were good phosphorus solubilisers in laboratory tests resulted in increased grain yield and grain phosphorus content in the presence or absence of applied dicalcium phosphate. It was also seen that the ability to colonise plant roots in high numbers was not always necessary for a positive effect. Strains that were predicted in the laboratory to be either poor or highly variable phosphorus solubilisers performed worst in the pot trial. The laboratory tests were found to be useful tools for quickly ranking isolates in terms of their ability to supply the phosphorus requirements of wheat grown in pots of non-sterile soil. r 2006 Elsevier Ltd. All rights reserved. Keywords: Phosphate solubilisation; Rhizosphere; Laboratory screening; Wheat
1. Introduction Phosphorus is an essential part of the energy transfer system in plants. The element is generally abundant in soil but, due to its highly reactive nature, the amount of phosphorus in a form available to plants is a limiting factor; deficiencies lead to slower and often stunted growth and decreased yields. Phosphorus is sequestered mainly through the mechanisms of precipitation and adsorption. It is capable of adsorption to the surface of soil particles and also reacts readily with soil cations, particularly iron, aluminium and calcium, to form insoluble compounds, which plants are unable to utilise (Wild, 1988). Corresponding author. Tel.: 61 2 9351 2540; fax: 61 2 9351 4571.
E-mail address: [email protected] (P.B. New). 0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.11.016
The conventional approach to improving phosphorus nutrition for optimum crop yields is to apply a chemical fertiliser containing soluble phosphate. However, the applied phosphate is also subject to the same fixation processes, resulting in a large fraction of the fertiliser becoming unavailable for plant use. It has been estimated that the proportion of phosphorus fertiliser used by plants is in the order of only 5–25% (Wild, 1988). In order to maintain high yields, farmers need to continually apply more fertiliser than is used by crops (Goldstein, 1986). This has negative consequences for both the economy and the environment. Eutrophication is the main environmental concern related to the application of excess phosphorus and subsequent run-off into waterways, an example being the algal blooms, which are increasingly common in Australian inland rivers. The spiralling costs of
ARTICLE IN PRESS 1522
J.N. Harris et al. / Soil Biology & Biochemistry 38 (2006) 1521–1526
conventional fertilisers have left many searching for alternatives in order to maintain profitability. One area of increasing interest is the use of microorganisms with the ability to solubilise mineral and organic phosphates (Goldstein, 1986). Phosphate solubilisation has been attributed to a number of processes including acidification, chelation and exchange reactions (Beckie et al., 1998). As phosphate-solubilising microbes are common in soil (Chabot et al., 1996), they have been easily isolated in the laboratory since the 1950s using methods such as those described by Sperber (1958). Though the ability to solubilise phosphate is common in soil isolates and a few are commercially produced as biofertilisers (Beckie et al., 1998), the application of these organisms as plant inoculants has varied in its effectiveness (Kucey et al., 1989). There are a number of possible reasons for this, including the inappropriate nature of the screening media utilised (Gyaneshwar et al., 2002). The aim of this study was to selectively isolate rhizosphere-competent phosphate solubilisers and to assess their potential as plant inoculants. The study focussed on organisms capable of solubilising dicalcium phosphate, for the following reasons: (1) the precipitation of soil phosphorus by calcium is common particularly under neutral to alkaline conditions (Kumar and Narula, 1999); (2) superphosphate, which is commonly applied to improve yield, contains enough calcium to precipitate half of its own phosphorus thereby increasing the amount of insoluble calcium phosphate in the soil (Chabot et al., 1996); (3) dicalcium phosphate is cheaper than superphosphate per unit of phosphorus but requires much larger additions to be effective and is reported to be beneficial only in acid soils (Kucey, 1987). Therefore, it was hoped that the isolates would improve the efficiency of calcium phosphate utilisation.
0.1% sodium deoxycholate and 2.5% polyethylene glycol 6000 (Sigma, St Louis). The bottle was sealed and shaken at 4 1C for 1 h on an orbital shaker at 100 rpm, interspersed with intense agitation by hand at intervals of approximately 7 min. The soil particles were then pelleted by centrifuging (960g, 15 min) and the Chelex 100 was removed from the supernatant by aseptically filtering through sterile gauze. The bacteria were harvested by centrifuging at 22,100g for 20 min.
2. Materials and methods
2.2.4. Maintenance Cultures were maintained on modified nutrient (MN) agar, containing (per litre): lab-lemco (Oxoid, London, England) 3 g, bacteriological peptone 5 g, yeast extract 5 g, glucose 0.5 g, sucrose 0.5 g, agar 15 g; pH 7.0. For MN broth the agar was omitted.
2.1. Plants Wheat (Triticum aestivum L. cultivar Bowerbird) was used. Non-sterile seed was planted in the pot trial, and sterile seedlings were prepared for other experiments by germinating surface-sterilised seeds (70% EtOH 1 min then 0.5% NaOCl 20 min, followed by six rinses in sterile distilled water (SDW)) in the dark on 1% water agar in inverted plates for 2 d at 25 1C. 2.2. Bacterial isolation 2.2.1. Separation from soil particles A cation-exchange resin was employed to separate the bacteria from the soil particles (Jacobsen and Rasmussen, 1992). A 100 g soil sample (Vertisol A horizon, pH 6.4 (1:5, v/v, 0.01 M CaCl2), Narrabri, NSW) was placed in a 250 ml centrifuge bottle containing 10 g Chelex 100 (Bio-Rad, Copenhagen, Denmark) in 100 ml of solution containing
2.2.2. Enrichment Flasks containing 100 ml of Modified Illmer and Schinner (MIS) medium were inoculated with the harvested bacteria and incubated at 25 1C with shaking (200 rpm) for 1 week. MIS was modified from Illmer and Schinner (1992) by reducing the amount of added sugars to 0.5 g per litre each of sucrose and glucose and replacing soil extract with root exudates. The root exudates were prepared by growing sterile wheat seedlings in SDW for 2 weeks. Each seedling was placed on a filter paper (Whatman 3 mm chromatography paper) support in a test tube (18 150 mm) containing 10 ml SDW, and incubated for 2 weeks in a light chamber that simulated a day/night cycle (12 h/12 h) at 25 1C. The plants were removed and the root exudate solution was filtered through a 0.2 mm membrane filter (Gelman Sciences, Ann Arbor Michigan). 2.2.3. Selection Samples of 0.1 ml of enrichment were spread over the surface of plates of MIS agar medium, poured in two layers, with phosphate precipitated in the top layer as described by Sperber (1958). The plates were incubated at 25 1C for 1 week and colonies of phosphate solubilisers were selected, based on their ability to form clear halos in the cloudy layer of precipitated phosphate.
2.3. Bacterial isolates 2.3.1. Ability to solubilise phosphate using nutrients in the rhizosphere Illmer and Schinner medium (1992) was further modified by removing all carbon sources (C-free MIS medium), and double-layered plates were prepared in large (145 mm diameter) acid-washed sterile glass Petri dishes. Bacteria were grown in 10 ml MN broth at 25 1C for 24 h with shaking (200 rpm), then centrifuged and resuspended in the same volume of peptone diluent (0.1% peptone in water). The roots of sterile wheat seedlings were soaked in the bacterial suspensions for approximately 1 h before being placed on the surface of the agar and the plates were
ARTICLE IN PRESS J.N. Harris et al. / Soil Biology & Biochemistry 38 (2006) 1521–1526
incubated on their edges, allowing the roots to grow down the surface of the medium. Effective strains were recognised by zones of clearing of insoluble phosphate around the plant roots. 2.3.2. Colonisation of wheat roots Sterile wheat seedlings were sown in large test tubes (30 200 mm) into sterile perlite wetted to field capacity with Fa˚hraeus (1957) solution modified by the addition of NH4NO3 (0.355 g per l) and precipitated calcium phosphate (per 100 ml: 0.5% CaCl2, 1.62 ml; 0.5% K2HPO4, 1.08 ml). Bacteria were grown in 10 ml MN broth at 25 1C for 24 h with shaking (200 rpm), then centrifuged and resuspended in P-free Fa˚hraeus solution (as in Fa˚hraeus (1957) plus 0.355 g/l NH4NO3). Cell concentrations were determined using a Petroff–Hausser counter, and appropriate dilutions in P-free Fa˚hraeus solution were added to the test tubes to give a final concentration of 103 cells/g wet perlite. Plants were incubated for 2 weeks in a light chamber at 25 1C (day night cycle 12 h/12 h). The roots were removed aseptically and suspended in peptone diluent. Dilutions in peptone diluent were incorporated into molten MN agar for enumeration by the plate count method. The plates were incubated at 25 1C for 48 h. 2.3.3. Peat cultures Bacterial isolates were inoculated into sterile peat for use in pot trials. Bacteria were grown in 10 ml MN broth overnight with shaking (200 rpm) at 30 1C and the culture was aseptically added to 50 ml SDW. A 38 ml aliquot of diluted culture was injected into a surface-sterilised (70% ethanol) sealed bag of 50 g sterile peat (Bio-Care Technology, Somersby, NSW) using a 50 ml syringe with a 19gauge needle and the injection hole sealed with a sticky label. After kneading to evenly mix the culture through the peat, the bag was incubated at 30 1C for 1 week. The peat was aseptically transferred to sterile McCartney bottles, loosely capped to allow oxygen transfer, and stored in the dark at room temperature for up to 1 week. 2.4. Validation of laboratory evaluation method 2.4.1. Pot trial Each pot (125 mm diameter 140 mm height) contained a 1:1 (v/v) mixture of sand and a phosphorus-deficient soil (sandy loam A horizon with 3–4 ppm Bray No. 2 available phosphorus from Camden, NSW). A complete chemical fertiliser lacking phosphorus (15N:0P:21.6K:0.2Fe, Sierraform, Scotts Australia, Baulkham Hills NSW) was mixed evenly through the surface soil of the pots at a rate equivalent to 25 g/m2 of the pot surface. For each bacterial strain and the uninoculated control, two sets of pots were prepared, one containing insoluble phosphorus in the form of dicalcium phosphate (Rindies Fertilisers, Silverdale, NSW) evenly worked into the soil surface and the other without added phosphorus. An additional set of pots contained single superphosphate (INCITEC, Elders,
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NSW). Both dicalcium phosphate and single superphosphate were applied to a final rate of 10 kg P/ha. Wheat seeds (cv. Bowerbird) were coated with a sticky solution of seed pelleting adhesive (Bio-Care Technology, Somersby, NSW), then dipped in peat containing one of the strains or in sterile peat in the case of the controls. Three peat-coated seeds were sown to a depth of 15 mm in each pot, which was then watered to field capacity with distilled water. The plants were grown in a temperature-light-controlled plant growth cabinet (Thermoline Scientific Equipment, Smithfield, NSW) with a day/night cycle of 14 h day/10 h night and light intensity of 550–600 mmol/m2/s. The temperature cycle began with 18/12 1C (day/night), and at late anthesis, it was increased to 25/22 1C (day/night). The seedlings were thinned to one per pot, selected on the basis of uniformity over all pots, after the first leaf developed. Plant development was measured weekly and soil water content was replenished to constant weight. After maturation, the above-ground parts were harvested and air-dried before plant and grain weights were measured. 2.4.2. Plant and grain phosphorus levels Plant material was digested by the method of Hutton and Nye (1958). A 1 g sample of material was placed in a block digester tube (35 300 mm). Selenium powder (0.05 g) and concentrated H2SO4 (20 ml) were added before the tube was placed in a block digester in a fume hood, heated to 350 1C and digested for 2.5 h with occasional swirling to mix. The tube was removed from heat and cooled before 50 ml of deionised water was added and the mixture was again allowed to cool. Then 3 ml 7.9% Na2SO3 solution was added to precipitate selenium remaining in the digest and the tube returned to the digester and boiled for a minimum of 15 min. After cooling, the solution was filtered (Whatman No. 42 filter paper) and the volume was made up to 100 ml with distilled water. Phosphorus levels were determined by the method of Kitson and Mellon (1944). A 5 ml sample was placed in an acid-washed test tube to which 5 ml (NH4)6Mo7O24 solution (5 g per 100 ml) and 5 ml NH4VO3 solution (2.5 g dissolved in 20 ml concentrated HNO3/l) were added sequentially. The mixture was thoroughly mixed with a Vortex mixer and placed in a water bath at 80 1C for 10 min. After cooling, absorbance was measured at 660 nm and the phosphorus level calculated from a standard curve. 2.5. Statistical analysis Data from phosphate solubilisation and root colonisation experiments were statistically analysed using Minitab 11.21 software (Minitab Inc., State College, PA, USA). One-way analysis of variance was performed and significance of differences between the means was determined using Tukey’s wholly significant difference method (Tukey, 1977). Two-way analysis of variance of data from the pot trial was performed using GenStat (Release 7.1, Lawes
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3. Results 3.1. Characterisation of strains In all, 48 strains were isolated and showed wide variation in their ability to colonise wheat roots and to solubilise phosphate. Seven strains were selected to validate the laboratory methods in a pot trial. They were identified as Flavobacterium sp. (strains 3, 5, 12, 17), Microbacterium sp. (strain 4), Arthrobacter sp. (strain 16), and Enterobacter sp. (strain 22) through standard biochemical tests and sequencing of 16S rDNA. These strains varied from poor to good in their abilities to solubilise phosphate and colonise wheat roots. On the basis of relative size of the zones of clearing around the roots in the cloudy phosphate precipitate (Fig. 1a), strain 3 was found to be an excellent phosphate solubiliser using root exudates as sole carbon source. It was significantly better than the moderately solubilising strains 4, 5 and 12. Strains 16 and 22 had large zones of clearing on some plates but showed a high degree of variation. Strain 17 was a very poor solubiliser. The ability to colonise wheat roots expressed as colony forming units (cfu) per gram root, (Fig. 1b) was independent of the ability to solubilise phosphate. Strain 3 was a poor coloniser but all other strains achieved counts in excess of 5 107 cfu/g root.
3.2. Validation of laboratory evaluation 3.2.1. Pot trial There was no significant difference between the bacterial strains and the uninoculated control in the growth parameters of height, tillering (rate of tiller development and total number of tillers) and ear development over the growth period (data not shown). Average weight per grain and weight of tops minus grain were also not statistically different between treatments (data not shown). However, there were statistically significant differences between treatments in grain yield and the number of grains per pot (Fig. 2a and b). Two-way analysis of variance revealed significant effects of fertilisation with dicalcium phosphate (increase of yield from 1.20 to 1.60 g and of P content from 2.08 to 2.84 mg per pot) and of microbial inoculation. Strains 4, 5, 12 and 16 to a lesser extent, stimulated grain yield and grain number per pot compared with uninoculated controls (Fig. 2a, b). For these strains, yields in the dicalciumphosphate-treated pots approached the levels attained by fertilisation with superphosphate at the normal field application rate.
4 a
Phosphate Solubilisation
Agricultural Trust, Rothamsted, UK) and the significance of differences between the pooled means was determined using Fisher’s protected LSD.
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b
b
ab
c
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Fig. 1. Properties of selected bacterial strains. (a) Phosphate-solubilising ability of selected bacterial strains growing in the rhizosphere of wheat. Phosphate solubilisation was scored on a scale of 0–3: 0, no solubilisation; 1, poor solubilisation (clearing barely visible); 2, moderate solubilisation (clearing up to 10 mm from roots); 3, excellent solubilisation (clearing 410 mm from roots). (b) Root colonisation of sterile wheat roots by selected strains after 2 weeks, expressed as log10 colony-forming units (cfu)/g root. The error bars are standard error, and strains with the same letter are not significantly different (p ¼ 0.05). Solubilisation was based on three replicates, and colonisation was based on four replicates.
3.2.2. Plant and grain phosphorus levels The level of phosphorus passed to the seed is seen in Fig. 2c. The same four strains (4, 5, 12 and 16) also caused an increased content of grain phosphorus per pot compared with the uninoculated controls. There was no significant difference between treatments in the amount of phosphorus in the shoots and leaves (data not shown). 4. Discussion Many other studies have used media rich in added carbon sources, such as the medium of Sperber (1958) to isolate phosphate-solubilising organisms, but we have found that the great majority of strains isolated by this method colonised wheat roots poorly (data not shown). Sperber medium provides organisms with a relatively high concentration (1%) of a single carbon source, glucose, which is only found in very low concentrations in root
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40.5
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31.9
31.0
27.4
50
ab
c
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c
bc
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a
40 30 20 10 0 3 4 LSD 6.41
Grain yield (g/pot)
(a)
2
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12
16
17
22
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1.65
1.56
1.61
1.34
1.23
1.37
1.02
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e
cde
de
bc
ab
bcd
a
S
1
0 3 4 LSD 0.25
Grain P (mg/pot)
(b) 4
5
12
16
17
22
C
2.33
3.05
2.91
2.88
2.41
1.94
2.34
1.80
bc
d
d
d
c
ab
bc
a
S
3 2 1 0
(c)
3 4 LSD 0.43
5
12
16 17 Treatment
22
C
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ability to solubilise phosphate under these more natural conditions, although all had produced obvious zones of clearing of precipitated phosphate on the medium containing added carbon. The results of the pot trial demonstrate that these screening methods are an effective tool for evaluating potential phosphate solubilisers. Strains identified as both good root colonisers and phosphate solubilisers in laboratory tests (strains 4, 5, 12 and 16) were shown to improve grain phosphorus content, which was accompanied by a corresponding increase in grain yield. Another strain (3) was found to be a poor root coloniser but a good solubiliser in the wheat rhizosphere. It is interesting that the pot trial showed that this strain was still able to benefit plant growth through solubilisation. The other strains were predicted to be either poor phosphate solubilisers (strain 17) or extremely variable in this ability (strain 22), and these performed worst in the pot trial. The laboratory tests described here have been shown to be good tools for the quick evaluation of rhizosphere and solubilisation competence. However, they do not answer all relevant questions about a given organism. Properties such as the ability to produce phytohormones, to compete for rhizosphere sites with indigenous microflora, and the extent of adaptation to different soil conditions will also affect the effectiveness of each strain as a soil inoculant. These tests are simply an effective first step to understanding the overall rhizosphere situation.
S
Fig. 2. Response of wheat to bacterial inoculation. (a) Number of grains per pot, (b) grain yield per pot and (c) grain phosphorus content per pot. Plants were inoculated with strains 3, 4, 5, 12, 16, 17 or 22, or were uninoculated controls (C). Pots were fertilised with single superphosphate ( ), dicalcium phosphate ( ) or had no added phosphate ( ). The error bars are standard error. There were four replicates for each treatment. Results were analysed by factorial analysis of variance omitting the superphosphate treatment (8 bacterial treatments 2 fertilisation regimes) and numbers above the bars are pooled means of all dicalciumphosphate-treated and-untreated pots for each bacterial treatment. Means with the same letter are not significantly different based on Fisher’s protected LSD (p ¼ 0.05).
exudates. Thus, organisms capable of solubilising phosphate on Sperber medium may not be typical rhizosphere inhabitants or capable of solubilising phosphate under rhizosphere conditions. Therefore, a medium that more realistically reflects nutrient levels in the rhizosphere was designed (modified MIS medium), using low amounts of glucose and sucrose (0.05%) and exudates from wheat roots. To establish that the isolates could solubilise phosphate in the rhizosphere, a simple laboratory test was developed in which inoculated wheat seedlings were placed on the surface of MIS agar lacking any added carbon sources. Zones of clearing around the roots identified bacterial strains able to solubilise phosphate when supplied only with root exudates. The tested strains varied greatly in their
Acknowledgements We are grateful to Jim Hull of the Plant Breeding Institute at the University of Sydney for assistance with the phosphorus assays. References Beckie, H.J., Schlechte, D., Moulin, A.P., Gleddie, S.C., Pulkinen, D.A., 1998. Response of alfalfa to inoculation with Penicillium bilaii (Provide). Canadian Journal of Plant Science 78, 91–102. Chabot, R., Antoun, H., Cescas, M.P., 1996. Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar phaseoli. Plant and Soil 184, 311–321. Fa˚hraeus, G., 1957. The infection of clover root hairs by nodule bacteria, studied by a simple glass slide technique. Journal of General Microbiology 16, 374–381. Goldstein, A.H., 1986. Bacterial solubilization of mineral phosphates: historical perspective and future prospects. American Journal of Alternative Agriculture 1, 51–57. Gyaneshwar, P., Naresh Kumar, G., Parekh, L.J., Poole, P.S., 2002. Role of soil microorganisms in improving P nutrition of plants. Plant and Soil 245, 83–93. Hutton, R.G., Nye, P.H., 1958. The rapid determination of the major nutrient elements in plants. Journal of the Science of Food and Agriculture 9, 7–14. Illmer, P., Schinner, F., 1992. Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biology & Biochemistry 24, 389–395. Jacobsen, C.S., Rasmussen, O.F., 1992. Development and application o f a new method to extract bacterial DNA from soil based
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on separation of bacteria from soil with cation-exchange resin. Applied and Environmental Microbiology 58, 2458–2462. Kitson, R.E., Mellon, M.G., 1944. Colorimetric determination of phosphorus as molybdivanadophosphoric acid. Industrial and Engineering Chemistry 16, 379–383. Kucey, R.M.N., 1987. Increased phosphorus uptake by wheat and field beans inoculated with a phosphorus-solubilizing Penicillium bilaji strain and with vesicular–arbuscular mycorrhizal fungi. Applied and Environmental Microbiology 53, 2699–2703. Kucey, R.M.N., Janzen, H.H., Leggett, M.E., 1989. Microbially mediated increases in plant-available phosphorus. Advances in Agronomy 42, 199–228.
Kumar, V., Narula, N., 1999. Solubilization of inorganic phosphates and growth emergence of wheat as affected by Azotobacter chroococcum mutants. Biology and Fertility of Soils 38, 301–305. Sperber, J.I., 1958. The incidence of apatite-solubilizing organisms in the rhizosphere and soil. Australian Journal of Agricultural Research 9, 778–781. Tukey, J.W., 1977. Exploratory Data Analysis. Addison-Wesley, Reading. Wild, A. (Ed.), 1988. Russell’s Soil Conditions and Plant Growth, 11th ed. Longman, Harlow (chapter 21).
Soil Biology & Biochemistry 38 (2006) 1521–1526 www.elsevier.com/locate/soilbio
Laboratory tests can predict beneficial effects of phosphate-solubilising bacteria on plants Jodie N. Harris, Peter B. New, Peter M. Martin Received 16 April 2004; received in revised form 15 November 2005; accepted 22 November 2005 Available online 7 April 2006
Abstract Phosphorus is important in plant growth but in the soil it reacts readily to form insoluble compounds, which are not readily available for plant utilisation. Although the presence of soil microorganisms capable of solubilising phosphate has been known for many years, their isolation and use as crop inoculants have met with only limited success. This project aimed to develop a more robust method of selecting strains, which would give a more reliable indication of their usefulness for Australian crops, with a particular focus on wheat. Bacteria were isolated from a typical black wheat growing soil from the North-Western wheat belt of New South Wales using a differential medium containing insoluble phosphate. In all, 48 isolates were tested in the laboratory for their ability to colonise the roots of wheat seedlings in sterile perlite and to solubilise phosphate in the rhizosphere of plants grown on agar plates. To establish whether strains selected for their laboratory performance could benefit wheat grown in unsterilised field soil, a pot trial was carried out in a highillumination growth chamber. Seven strains of varying capabilities were used to determine the effects of inoculation on releasing unavailable phosphorus and improving growth and yield, in comparison with conventional phosphorus fertiliser. Strains that were good phosphorus solubilisers in laboratory tests resulted in increased grain yield and grain phosphorus content in the presence or absence of applied dicalcium phosphate. It was also seen that the ability to colonise plant roots in high numbers was not always necessary for a positive effect. Strains that were predicted in the laboratory to be either poor or highly variable phosphorus solubilisers performed worst in the pot trial. The laboratory tests were found to be useful tools for quickly ranking isolates in terms of their ability to supply the phosphorus requirements of wheat grown in pots of non-sterile soil. r 2006 Elsevier Ltd. All rights reserved. Keywords: Phosphate solubilisation; Rhizosphere; Laboratory screening; Wheat
1. Introduction Phosphorus is an essential part of the energy transfer system in plants. The element is generally abundant in soil but, due to its highly reactive nature, the amount of phosphorus in a form available to plants is a limiting factor; deficiencies lead to slower and often stunted growth and decreased yields. Phosphorus is sequestered mainly through the mechanisms of precipitation and adsorption. It is capable of adsorption to the surface of soil particles and also reacts readily with soil cations, particularly iron, aluminium and calcium, to form insoluble compounds, which plants are unable to utilise (Wild, 1988). Corresponding author. Tel.: 61 2 9351 2540; fax: 61 2 9351 4571.
E-mail address: [email protected] (P.B. New). 0038-0717/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.11.016
The conventional approach to improving phosphorus nutrition for optimum crop yields is to apply a chemical fertiliser containing soluble phosphate. However, the applied phosphate is also subject to the same fixation processes, resulting in a large fraction of the fertiliser becoming unavailable for plant use. It has been estimated that the proportion of phosphorus fertiliser used by plants is in the order of only 5–25% (Wild, 1988). In order to maintain high yields, farmers need to continually apply more fertiliser than is used by crops (Goldstein, 1986). This has negative consequences for both the economy and the environment. Eutrophication is the main environmental concern related to the application of excess phosphorus and subsequent run-off into waterways, an example being the algal blooms, which are increasingly common in Australian inland rivers. The spiralling costs of
ARTICLE IN PRESS 1522
J.N. Harris et al. / Soil Biology & Biochemistry 38 (2006) 1521–1526
conventional fertilisers have left many searching for alternatives in order to maintain profitability. One area of increasing interest is the use of microorganisms with the ability to solubilise mineral and organic phosphates (Goldstein, 1986). Phosphate solubilisation has been attributed to a number of processes including acidification, chelation and exchange reactions (Beckie et al., 1998). As phosphate-solubilising microbes are common in soil (Chabot et al., 1996), they have been easily isolated in the laboratory since the 1950s using methods such as those described by Sperber (1958). Though the ability to solubilise phosphate is common in soil isolates and a few are commercially produced as biofertilisers (Beckie et al., 1998), the application of these organisms as plant inoculants has varied in its effectiveness (Kucey et al., 1989). There are a number of possible reasons for this, including the inappropriate nature of the screening media utilised (Gyaneshwar et al., 2002). The aim of this study was to selectively isolate rhizosphere-competent phosphate solubilisers and to assess their potential as plant inoculants. The study focussed on organisms capable of solubilising dicalcium phosphate, for the following reasons: (1) the precipitation of soil phosphorus by calcium is common particularly under neutral to alkaline conditions (Kumar and Narula, 1999); (2) superphosphate, which is commonly applied to improve yield, contains enough calcium to precipitate half of its own phosphorus thereby increasing the amount of insoluble calcium phosphate in the soil (Chabot et al., 1996); (3) dicalcium phosphate is cheaper than superphosphate per unit of phosphorus but requires much larger additions to be effective and is reported to be beneficial only in acid soils (Kucey, 1987). Therefore, it was hoped that the isolates would improve the efficiency of calcium phosphate utilisation.
0.1% sodium deoxycholate and 2.5% polyethylene glycol 6000 (Sigma, St Louis). The bottle was sealed and shaken at 4 1C for 1 h on an orbital shaker at 100 rpm, interspersed with intense agitation by hand at intervals of approximately 7 min. The soil particles were then pelleted by centrifuging (960g, 15 min) and the Chelex 100 was removed from the supernatant by aseptically filtering through sterile gauze. The bacteria were harvested by centrifuging at 22,100g for 20 min.
2. Materials and methods
2.2.4. Maintenance Cultures were maintained on modified nutrient (MN) agar, containing (per litre): lab-lemco (Oxoid, London, England) 3 g, bacteriological peptone 5 g, yeast extract 5 g, glucose 0.5 g, sucrose 0.5 g, agar 15 g; pH 7.0. For MN broth the agar was omitted.
2.1. Plants Wheat (Triticum aestivum L. cultivar Bowerbird) was used. Non-sterile seed was planted in the pot trial, and sterile seedlings were prepared for other experiments by germinating surface-sterilised seeds (70% EtOH 1 min then 0.5% NaOCl 20 min, followed by six rinses in sterile distilled water (SDW)) in the dark on 1% water agar in inverted plates for 2 d at 25 1C. 2.2. Bacterial isolation 2.2.1. Separation from soil particles A cation-exchange resin was employed to separate the bacteria from the soil particles (Jacobsen and Rasmussen, 1992). A 100 g soil sample (Vertisol A horizon, pH 6.4 (1:5, v/v, 0.01 M CaCl2), Narrabri, NSW) was placed in a 250 ml centrifuge bottle containing 10 g Chelex 100 (Bio-Rad, Copenhagen, Denmark) in 100 ml of solution containing
2.2.2. Enrichment Flasks containing 100 ml of Modified Illmer and Schinner (MIS) medium were inoculated with the harvested bacteria and incubated at 25 1C with shaking (200 rpm) for 1 week. MIS was modified from Illmer and Schinner (1992) by reducing the amount of added sugars to 0.5 g per litre each of sucrose and glucose and replacing soil extract with root exudates. The root exudates were prepared by growing sterile wheat seedlings in SDW for 2 weeks. Each seedling was placed on a filter paper (Whatman 3 mm chromatography paper) support in a test tube (18 150 mm) containing 10 ml SDW, and incubated for 2 weeks in a light chamber that simulated a day/night cycle (12 h/12 h) at 25 1C. The plants were removed and the root exudate solution was filtered through a 0.2 mm membrane filter (Gelman Sciences, Ann Arbor Michigan). 2.2.3. Selection Samples of 0.1 ml of enrichment were spread over the surface of plates of MIS agar medium, poured in two layers, with phosphate precipitated in the top layer as described by Sperber (1958). The plates were incubated at 25 1C for 1 week and colonies of phosphate solubilisers were selected, based on their ability to form clear halos in the cloudy layer of precipitated phosphate.
2.3. Bacterial isolates 2.3.1. Ability to solubilise phosphate using nutrients in the rhizosphere Illmer and Schinner medium (1992) was further modified by removing all carbon sources (C-free MIS medium), and double-layered plates were prepared in large (145 mm diameter) acid-washed sterile glass Petri dishes. Bacteria were grown in 10 ml MN broth at 25 1C for 24 h with shaking (200 rpm), then centrifuged and resuspended in the same volume of peptone diluent (0.1% peptone in water). The roots of sterile wheat seedlings were soaked in the bacterial suspensions for approximately 1 h before being placed on the surface of the agar and the plates were
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incubated on their edges, allowing the roots to grow down the surface of the medium. Effective strains were recognised by zones of clearing of insoluble phosphate around the plant roots. 2.3.2. Colonisation of wheat roots Sterile wheat seedlings were sown in large test tubes (30 200 mm) into sterile perlite wetted to field capacity with Fa˚hraeus (1957) solution modified by the addition of NH4NO3 (0.355 g per l) and precipitated calcium phosphate (per 100 ml: 0.5% CaCl2, 1.62 ml; 0.5% K2HPO4, 1.08 ml). Bacteria were grown in 10 ml MN broth at 25 1C for 24 h with shaking (200 rpm), then centrifuged and resuspended in P-free Fa˚hraeus solution (as in Fa˚hraeus (1957) plus 0.355 g/l NH4NO3). Cell concentrations were determined using a Petroff–Hausser counter, and appropriate dilutions in P-free Fa˚hraeus solution were added to the test tubes to give a final concentration of 103 cells/g wet perlite. Plants were incubated for 2 weeks in a light chamber at 25 1C (day night cycle 12 h/12 h). The roots were removed aseptically and suspended in peptone diluent. Dilutions in peptone diluent were incorporated into molten MN agar for enumeration by the plate count method. The plates were incubated at 25 1C for 48 h. 2.3.3. Peat cultures Bacterial isolates were inoculated into sterile peat for use in pot trials. Bacteria were grown in 10 ml MN broth overnight with shaking (200 rpm) at 30 1C and the culture was aseptically added to 50 ml SDW. A 38 ml aliquot of diluted culture was injected into a surface-sterilised (70% ethanol) sealed bag of 50 g sterile peat (Bio-Care Technology, Somersby, NSW) using a 50 ml syringe with a 19gauge needle and the injection hole sealed with a sticky label. After kneading to evenly mix the culture through the peat, the bag was incubated at 30 1C for 1 week. The peat was aseptically transferred to sterile McCartney bottles, loosely capped to allow oxygen transfer, and stored in the dark at room temperature for up to 1 week. 2.4. Validation of laboratory evaluation method 2.4.1. Pot trial Each pot (125 mm diameter 140 mm height) contained a 1:1 (v/v) mixture of sand and a phosphorus-deficient soil (sandy loam A horizon with 3–4 ppm Bray No. 2 available phosphorus from Camden, NSW). A complete chemical fertiliser lacking phosphorus (15N:0P:21.6K:0.2Fe, Sierraform, Scotts Australia, Baulkham Hills NSW) was mixed evenly through the surface soil of the pots at a rate equivalent to 25 g/m2 of the pot surface. For each bacterial strain and the uninoculated control, two sets of pots were prepared, one containing insoluble phosphorus in the form of dicalcium phosphate (Rindies Fertilisers, Silverdale, NSW) evenly worked into the soil surface and the other without added phosphorus. An additional set of pots contained single superphosphate (INCITEC, Elders,
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NSW). Both dicalcium phosphate and single superphosphate were applied to a final rate of 10 kg P/ha. Wheat seeds (cv. Bowerbird) were coated with a sticky solution of seed pelleting adhesive (Bio-Care Technology, Somersby, NSW), then dipped in peat containing one of the strains or in sterile peat in the case of the controls. Three peat-coated seeds were sown to a depth of 15 mm in each pot, which was then watered to field capacity with distilled water. The plants were grown in a temperature-light-controlled plant growth cabinet (Thermoline Scientific Equipment, Smithfield, NSW) with a day/night cycle of 14 h day/10 h night and light intensity of 550–600 mmol/m2/s. The temperature cycle began with 18/12 1C (day/night), and at late anthesis, it was increased to 25/22 1C (day/night). The seedlings were thinned to one per pot, selected on the basis of uniformity over all pots, after the first leaf developed. Plant development was measured weekly and soil water content was replenished to constant weight. After maturation, the above-ground parts were harvested and air-dried before plant and grain weights were measured. 2.4.2. Plant and grain phosphorus levels Plant material was digested by the method of Hutton and Nye (1958). A 1 g sample of material was placed in a block digester tube (35 300 mm). Selenium powder (0.05 g) and concentrated H2SO4 (20 ml) were added before the tube was placed in a block digester in a fume hood, heated to 350 1C and digested for 2.5 h with occasional swirling to mix. The tube was removed from heat and cooled before 50 ml of deionised water was added and the mixture was again allowed to cool. Then 3 ml 7.9% Na2SO3 solution was added to precipitate selenium remaining in the digest and the tube returned to the digester and boiled for a minimum of 15 min. After cooling, the solution was filtered (Whatman No. 42 filter paper) and the volume was made up to 100 ml with distilled water. Phosphorus levels were determined by the method of Kitson and Mellon (1944). A 5 ml sample was placed in an acid-washed test tube to which 5 ml (NH4)6Mo7O24 solution (5 g per 100 ml) and 5 ml NH4VO3 solution (2.5 g dissolved in 20 ml concentrated HNO3/l) were added sequentially. The mixture was thoroughly mixed with a Vortex mixer and placed in a water bath at 80 1C for 10 min. After cooling, absorbance was measured at 660 nm and the phosphorus level calculated from a standard curve. 2.5. Statistical analysis Data from phosphate solubilisation and root colonisation experiments were statistically analysed using Minitab 11.21 software (Minitab Inc., State College, PA, USA). One-way analysis of variance was performed and significance of differences between the means was determined using Tukey’s wholly significant difference method (Tukey, 1977). Two-way analysis of variance of data from the pot trial was performed using GenStat (Release 7.1, Lawes
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3. Results 3.1. Characterisation of strains In all, 48 strains were isolated and showed wide variation in their ability to colonise wheat roots and to solubilise phosphate. Seven strains were selected to validate the laboratory methods in a pot trial. They were identified as Flavobacterium sp. (strains 3, 5, 12, 17), Microbacterium sp. (strain 4), Arthrobacter sp. (strain 16), and Enterobacter sp. (strain 22) through standard biochemical tests and sequencing of 16S rDNA. These strains varied from poor to good in their abilities to solubilise phosphate and colonise wheat roots. On the basis of relative size of the zones of clearing around the roots in the cloudy phosphate precipitate (Fig. 1a), strain 3 was found to be an excellent phosphate solubiliser using root exudates as sole carbon source. It was significantly better than the moderately solubilising strains 4, 5 and 12. Strains 16 and 22 had large zones of clearing on some plates but showed a high degree of variation. Strain 17 was a very poor solubiliser. The ability to colonise wheat roots expressed as colony forming units (cfu) per gram root, (Fig. 1b) was independent of the ability to solubilise phosphate. Strain 3 was a poor coloniser but all other strains achieved counts in excess of 5 107 cfu/g root.
3.2. Validation of laboratory evaluation 3.2.1. Pot trial There was no significant difference between the bacterial strains and the uninoculated control in the growth parameters of height, tillering (rate of tiller development and total number of tillers) and ear development over the growth period (data not shown). Average weight per grain and weight of tops minus grain were also not statistically different between treatments (data not shown). However, there were statistically significant differences between treatments in grain yield and the number of grains per pot (Fig. 2a and b). Two-way analysis of variance revealed significant effects of fertilisation with dicalcium phosphate (increase of yield from 1.20 to 1.60 g and of P content from 2.08 to 2.84 mg per pot) and of microbial inoculation. Strains 4, 5, 12 and 16 to a lesser extent, stimulated grain yield and grain number per pot compared with uninoculated controls (Fig. 2a, b). For these strains, yields in the dicalciumphosphate-treated pots approached the levels attained by fertilisation with superphosphate at the normal field application rate.
4 a
Phosphate Solubilisation
Agricultural Trust, Rothamsted, UK) and the significance of differences between the pooled means was determined using Fisher’s protected LSD.
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b
b
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Fig. 1. Properties of selected bacterial strains. (a) Phosphate-solubilising ability of selected bacterial strains growing in the rhizosphere of wheat. Phosphate solubilisation was scored on a scale of 0–3: 0, no solubilisation; 1, poor solubilisation (clearing barely visible); 2, moderate solubilisation (clearing up to 10 mm from roots); 3, excellent solubilisation (clearing 410 mm from roots). (b) Root colonisation of sterile wheat roots by selected strains after 2 weeks, expressed as log10 colony-forming units (cfu)/g root. The error bars are standard error, and strains with the same letter are not significantly different (p ¼ 0.05). Solubilisation was based on three replicates, and colonisation was based on four replicates.
3.2.2. Plant and grain phosphorus levels The level of phosphorus passed to the seed is seen in Fig. 2c. The same four strains (4, 5, 12 and 16) also caused an increased content of grain phosphorus per pot compared with the uninoculated controls. There was no significant difference between treatments in the amount of phosphorus in the shoots and leaves (data not shown). 4. Discussion Many other studies have used media rich in added carbon sources, such as the medium of Sperber (1958) to isolate phosphate-solubilising organisms, but we have found that the great majority of strains isolated by this method colonised wheat roots poorly (data not shown). Sperber medium provides organisms with a relatively high concentration (1%) of a single carbon source, glucose, which is only found in very low concentrations in root
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27.4
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1.65
1.56
1.61
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1.37
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C
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3.05
2.91
2.88
2.41
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2.34
1.80
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d
d
c
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bc
a
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12
16 17 Treatment
22
C
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ability to solubilise phosphate under these more natural conditions, although all had produced obvious zones of clearing of precipitated phosphate on the medium containing added carbon. The results of the pot trial demonstrate that these screening methods are an effective tool for evaluating potential phosphate solubilisers. Strains identified as both good root colonisers and phosphate solubilisers in laboratory tests (strains 4, 5, 12 and 16) were shown to improve grain phosphorus content, which was accompanied by a corresponding increase in grain yield. Another strain (3) was found to be a poor root coloniser but a good solubiliser in the wheat rhizosphere. It is interesting that the pot trial showed that this strain was still able to benefit plant growth through solubilisation. The other strains were predicted to be either poor phosphate solubilisers (strain 17) or extremely variable in this ability (strain 22), and these performed worst in the pot trial. The laboratory tests described here have been shown to be good tools for the quick evaluation of rhizosphere and solubilisation competence. However, they do not answer all relevant questions about a given organism. Properties such as the ability to produce phytohormones, to compete for rhizosphere sites with indigenous microflora, and the extent of adaptation to different soil conditions will also affect the effectiveness of each strain as a soil inoculant. These tests are simply an effective first step to understanding the overall rhizosphere situation.
S
Fig. 2. Response of wheat to bacterial inoculation. (a) Number of grains per pot, (b) grain yield per pot and (c) grain phosphorus content per pot. Plants were inoculated with strains 3, 4, 5, 12, 16, 17 or 22, or were uninoculated controls (C). Pots were fertilised with single superphosphate ( ), dicalcium phosphate ( ) or had no added phosphate ( ). The error bars are standard error. There were four replicates for each treatment. Results were analysed by factorial analysis of variance omitting the superphosphate treatment (8 bacterial treatments 2 fertilisation regimes) and numbers above the bars are pooled means of all dicalciumphosphate-treated and-untreated pots for each bacterial treatment. Means with the same letter are not significantly different based on Fisher’s protected LSD (p ¼ 0.05).
exudates. Thus, organisms capable of solubilising phosphate on Sperber medium may not be typical rhizosphere inhabitants or capable of solubilising phosphate under rhizosphere conditions. Therefore, a medium that more realistically reflects nutrient levels in the rhizosphere was designed (modified MIS medium), using low amounts of glucose and sucrose (0.05%) and exudates from wheat roots. To establish that the isolates could solubilise phosphate in the rhizosphere, a simple laboratory test was developed in which inoculated wheat seedlings were placed on the surface of MIS agar lacking any added carbon sources. Zones of clearing around the roots identified bacterial strains able to solubilise phosphate when supplied only with root exudates. The tested strains varied greatly in their
Acknowledgements We are grateful to Jim Hull of the Plant Breeding Institute at the University of Sydney for assistance with the phosphorus assays. References Beckie, H.J., Schlechte, D., Moulin, A.P., Gleddie, S.C., Pulkinen, D.A., 1998. Response of alfalfa to inoculation with Penicillium bilaii (Provide). Canadian Journal of Plant Science 78, 91–102. Chabot, R., Antoun, H., Cescas, M.P., 1996. Growth promotion of maize and lettuce by phosphate-solubilizing Rhizobium leguminosarum biovar phaseoli. Plant and Soil 184, 311–321. Fa˚hraeus, G., 1957. The infection of clover root hairs by nodule bacteria, studied by a simple glass slide technique. Journal of General Microbiology 16, 374–381. Goldstein, A.H., 1986. Bacterial solubilization of mineral phosphates: historical perspective and future prospects. American Journal of Alternative Agriculture 1, 51–57. Gyaneshwar, P., Naresh Kumar, G., Parekh, L.J., Poole, P.S., 2002. Role of soil microorganisms in improving P nutrition of plants. Plant and Soil 245, 83–93. Hutton, R.G., Nye, P.H., 1958. The rapid determination of the major nutrient elements in plants. Journal of the Science of Food and Agriculture 9, 7–14. Illmer, P., Schinner, F., 1992. Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biology & Biochemistry 24, 389–395. Jacobsen, C.S., Rasmussen, O.F., 1992. Development and application o f a new method to extract bacterial DNA from soil based
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on separation of bacteria from soil with cation-exchange resin. Applied and Environmental Microbiology 58, 2458–2462. Kitson, R.E., Mellon, M.G., 1944. Colorimetric determination of phosphorus as molybdivanadophosphoric acid. Industrial and Engineering Chemistry 16, 379–383. Kucey, R.M.N., 1987. Increased phosphorus uptake by wheat and field beans inoculated with a phosphorus-solubilizing Penicillium bilaji strain and with vesicular–arbuscular mycorrhizal fungi. Applied and Environmental Microbiology 53, 2699–2703. Kucey, R.M.N., Janzen, H.H., Leggett, M.E., 1989. Microbially mediated increases in plant-available phosphorus. Advances in Agronomy 42, 199–228.
Kumar, V., Narula, N., 1999. Solubilization of inorganic phosphates and growth emergence of wheat as affected by Azotobacter chroococcum mutants. Biology and Fertility of Soils 38, 301–305. Sperber, J.I., 1958. The incidence of apatite-solubilizing organisms in the rhizosphere and soil. Australian Journal of Agricultural Research 9, 778–781. Tukey, J.W., 1977. Exploratory Data Analysis. Addison-Wesley, Reading. Wild, A. (Ed.), 1988. Russell’s Soil Conditions and Plant Growth, 11th ed. Longman, Harlow (chapter 21).