Abstract

Key Words

Soil biology, microbial biomass, substrate induced respiration, fungal bacterial ratio.

Introduction

Fungi and bacteria are drivers of major soil processes such as carbon and nutrient cycling (Gregorich et al. 1997; Milne and Haynes 2004). Changes in their abundance and functioning may be linked to soil sustainability, fertility and crop productivity (Bardgett et al. 1999; Beare 1997; Feng et al. 2004). The high functional and species diversity of soil fungi and bacteria make quantifying their relative contribution to soil biomass challenging.

Several approaches are in use for determining the fungal and bacterial biomass in soil, and each one has its own merits and disadvantages. The direct counting method (Bloem et al. 1995) requires higher manipulative skills and a longer time commitment. The indirect methods such as phospho-lipid fatty acid, ergosterol and DNA-based fingerprinting (Grant and West 1986; van Elsas et al. 1998; Zelles et al. 1992) may overestimate the microbial biomass as there are no ways of discriminating dead and living components during analysis. The rRNA-amplification methods that assess metabolically active microbial fractions require expensive instrumentation (Pennanen et al. 2004). The SIR method, first developed by Anderson and Domsch (1975) which has since been substantially improved, is relatively quick and inexpensive. It is an accurate measure of the biomass of the active soil microbial communities as it measures their respiration directly. The method uses a microbial ‘booster’, usually glucose, and a fungal and bacterial inhibitor separately and in combination to suppress the activity of those microbial fractions.

Even though it is simpler, the SIR method has to be locally calibrated because the interaction between microorganisms and inhibitors may dictate the experimental outcome. Different microorganisms respond differently to various biocides (Ingham and Colman 1984); non-target inhibition is characteristic with certain biocides (Tremaine and Mills 1987). Moreover, Ingham et al. (1986) and Stamatiadis et al. (1990) have proposed that some soil microorganisms may use biocides directly as nutrient sources.

Previous research has shown Captan (N-[Trichloromethylthio]-4-cyclohexene-1,2-dicarboximide), a broad spectrum fungicide, and the prokaryotic inhibitor Oxytetracycline hydrochloride, to be effective biocides to use in fungal bacterial ratio assessments in agricultural soils (Bailey et al. 2002; 2003). We tested the efficacy of these compounds in inhibiting microbial activity of common Western Australian agricultural soils. Our aim was to determine the precise inhibitor concentrations to use in fungal bacterial ratio assessments in these soils.

Materials and methods

Sampling sites

Soil samples collected from 3 paddocks were used for calibration. Sample 1 originated from Boyanup, 2 from Pingaring and 3 from Dunsborough. Boyanup and Dunsborough are situated approximately 225 km south, and Pingaring about 300 km southeast of Perth, WA. Some properties of these soils are given in Table 1. Each soil was a composite sample of 10 random soil cores collected to a depth of 10 cm by crossing the entire paddock diagonally.

Table 1. Properties of experimental soils.

	Sample 1	Sample 2	Sample 3
Soil texture Current crop Soil pH1 Electrical conductivity (mS/cm)1 Bulk density (g/cm3) Total organic C (%)2 C/N ratio2	Sandy loam Pasture 6.92 514.0 0.92 5.18 10.22	Loamy sand Wheat 6.99 546.0 1.4 1.22 17.68	Sandy loam Vineyard 7.13 401.0 0.91 3.18 14.04

¹Soil pH and EC were measured in 1:1 soil/water solution; ²TOC and C/N were measured by dry combustion in a Leco CHN analyzer

Soil treatments

a. The field moist soils were sieved upon arrival in the laboratory and stored at 4° C in aerated bags until further processing. They were pre-incubated at 25° C for 36 hrs prior to SIR treatments. Fresh soil equivalents to 2 g dry weight were weighed into 30 ml McCartney bottles and treated with:
Control (20 mg talc/g dry soil)
Fungal inhibitor Captan (rates 2, 4 & 6 mg/g soil)
Bacterial inhibitor Oxytetracycline hydrochloride (rates 1, 2, 4, 6 & 8 mg/g soil)
Mixture of fungal and bacterial inhibitors at rates described above

Inhibitors were mixed with talc for better mixing, and made to final concentrations of 20 mg dry material/g soil (Bailey et al. 2002). The control treatments received only 20 mg talc/g soil. The treatment mixtures were thoroughly mixed with soil. Glucose (1.0 ml of 4 g/L) was added into each vial after 1 hr of incubation at 25° C, mixed thoroughly, stoppered with rubber bungs and re-incubated. Each treatment included 3 replicates.

Biomass and other calculations

After 4 hrs of incubation, CO₂ gas accumulated in each vial was collected into a 1 ml syringe and injected to an infra-red gas analyzer. Prior to measurements, the analyzer was calibrated with 5% ultra pure standard CO₂ gas. The biomass was calculated by measuring peak heights and applying the following equation (Anderson and Domsch 1975):

μg CO₂-C g^-1 soil = ((A_sample × 10000/B_std)/10³ ×V X K))-((A_blank × 10000/B_std)/10³ × V × K))
where:
A_sample = Peak height (mm) of sample
A_blank = Peak height (mm) of blank bottle
B_std = Peak height (mm) of standard gas (1% CO₂)
V = Head space volume of bottle
K = Conversion constant for μl to μg of CO₂-C (assuming 1 atmosphere pressure: 1.7995 μg
CO₂ = 1 μl CO2 at 25° C and 1 atmosphere = 0.4908 μg CO₂-C)

For other calculations, treatments A, B, C and D were treated as follows:
(A-B), fungal biomass
(A-C), bacterial biomass
(A-B)/(A-C), fungal bacterial ratio
(A-B) + (A-C)/(A-D), inhibitor additivity ratio or IAR
(A-D/A) × 100, % inhibition

Results and Discussion

Relative to untreated controls, the SIR CO₂ production gradually decreased with increasing concentrations of the bacterial and fungal inhibitors (Figure 1 & 2), confirming their inhibiting potential in the soil types tested. The uninhibited portion might include biomass components other than bacteria and fungi (Vedder et al. 1996).

Figure 1. The effect of different concentrations of the fungal inhibitor on glucose mediated (SIR) CO2 production. N=3 at each data point.	Figure 2. The effect of different concentrations of the bacterial inhibitor on glucose mediated (SIR) CO2 production. N=3 at each data point.

In soil 1, inhibitor concentrations of 4 mg/g Captan and 8 mg/g Oxytetracycline hydrochloride and the combination thereof yielded the optimal fungal and bacterial inhibition that resulted in an IAR of 1.00 (Table 2). This means that these were the inhibitor concentrations that had acted only upon target organisms when treated alone and in combination. An IAR above 1.00 would have meant that the bacterial and/or fungal populations had been over-suppressed. Excessive concentrations of fungal inhibitors could act on bacteria, and vise-versa, and this is known as non-target inhibition. An IAR less than 1.00 would have meant incomplete inhibition.

IAR values were closer to 1.00 in soil 2 when 2 mg/g fungal and 1 mg/g bacterial inhibitor were combined, or when 4 & 6 mg Captan/g soil was combined with 2, 4, 6 and 8 mg Oxytetracycline hydrochloride/g soil (Table 3). IAR values ranged between 0.91 and 1.14 for soil 3, when either 2, 4 or 6 mg Captan and 1 mg Oxytetracycline hydrochloride, or 4 mg Captan and 8 mg Oxytetracycline hydrochloride or 6 mg Captan and 4 mg Oxytetracycline hydrochloride/g soil were used (Table 4). These results showed that an IAR of 1.00 ±0.15 was achievable in these soils with a wide range of inhibitor concentrations (highlighted in boldface in Tables 3 & 4). However, the FBR was higher with the lowest bacterial inhibitor concentration of 1 mg/g, indicating a higher representation of fungal biomass, and the degree of inhibition was the lowest, at least in soil 2 (Table 3). This might be because unrealistic bacterial biomasses were revealed by lower inhibitor concentrations. To be able to estimate fungal and bacterial components better, both the IAR and % inhibition should be given equal consideration (Bailey et al. 2002; Nakamoto and Wakahara 2004). According to our results, Captan (4 mg/g) and Oxytetracycline hydrochloride (8 mg/g) were shown to be the most appropriate biocide concentrations for the three WA soils tested.

Table 2. Rate of inhibition, fungal bacterial ratio and inhibitor additivity ratio with various inhibitor concentrations in soil 1. IAR values in the range 1.0 ± 0.15 are shown in bold type.

Fungal inhibitor concentration (mg/g soil)		Bacterial inhibitor concentration (mg/g soil)
		1	2	4	6	8
	Inhibition (%)	34.4	31.9	45.4	40.8	52.8
2	FBR	11.0	2.2	1.3	1.4	1.0
	IAR	1.26	1.80	1.55	1.70	1.51
	Inhibition (%)	28.5	39.4	44.9	45.2	74.2
4	FBR	9.4	1.9	1.1	1.2	0.8
	IAR	1.32	1.31	1.43	1.40	1.00
	Inhibition (%)	43.5	47.3	45.4	47.8	52.3
6	FBR	14.7	3.0	1.8	1.8	1.3
	IAR	1.31	1.50	1.84	1.73	1.78

Fungal inhibitor, Captan; bacterial inhibitor, Oxytetracycline hydchloride

FBR, fungal bacterial ratio; IAR, inhibitor additivity ratio

Table 3. Rate of inhibition, fungal bacterial ratio and inhibitor additivity ratio with various inhibitor concentrations in soil 2. IAR values in the range 1.0 ± 0.15 are shown in bold type.

Fungal inhibitor concentration (mg/g soil)		Bacterial inhibitor concentration (mg/g soil)
		1	2	4	6	8
	Inhibition (%)	22.3	32.1	44.6	53.4	63.3
2	FBR	3.6	0.5	0.3	0.3	0.2
	IAR	0.91	1.43	1.37	1.38	1.28
	Inhibition (%)	30.8	42.0	54.2	60.1	69.4
4	FBR	3.2	0.5	0.3	0.2	0.2
	IAR	0.61	1.06	1.10	1.20	1.14
	Inhibition (%)	51.6	46.8	54.8	64.1	68.1
6	FBR	3.3	0.5	0.3	0.2	0.2
	IAR	0.37	0.95	1.10	1.13	1.17

Fungal inhibitor, Captan; bacterial inhibitor, Oxytetracycline hydchloride

FBR, fungal bacterial ratio; IAR, inhibitor additivity ratio

The incubation period is crucial because even with most suitable inhibitors, prolonged incubation may result in a flush of CO₂ production as those surviving microorganisms may use the soluble cell materials of the dead and degrading cells as energy and nutrient sources (Anderson and Domsch, 1975; Badalucco et al. 1994). However, we did not have to carry out tests for determining optimum period of incubation as research has shown CO₂ evolution to be the lowest at 2-4 hrs after biocide treatment (Bailey et al. 2002; 2003; Nakamoto and Wakahara, 2004). Therefore we measured the CO₂ production after 4 hrs of incubation following inhibitor application. The inhibitor concentrations that we worked out as the most appropriate for the soils tested appear to be higher in comparison with published reports (e.g. Bailey et al. 2002; 2003; Nakamoto and Wakahara 2004). This might be a consequence of the mode of biocide application (i.e. solid vs. liquid), differences in soil organic matter contents and microbial composition. Research is underway to further improve the technique using more effective, novel inhibitors to reduce their application rates.

Table 4. Rate of inhibition, fungal bacterial ratio and inhibitor additivity ratio with various inhibitor concentrations in soil sample 3. IAR values in the range 1.0 ± 0.15 are shown in bold type.

Fungal inhibitor concentration (mg/g soil)		Bacterial inhibitor concentration (mg/g soil)
		1	2	4	6	8
	Inhibition (%)	56.6	58.0	58.9	59.2	59.8
2	FBR	6.9	2.6	3.0	1.6	1.9
	IAR	1.05	1.23	1.17	1.41	1.33
	Inhibition (%)	67.6	58.6	60.9	66.2	72.1
4	FBR	7.1	2.7	3.1	1.7	1.9
	IAR	0.90	1.25	1.16	1.29	1.13
	Inhibition (%)	61.5	62.6	63.5	70.8	69.0
6	FBR	7.3	2.8	3.2	1.7	2.0
	IAR	1.03	1.20	1.14	1.23	1.21

Fungal inhibitor, Captan; Bacterial inhibitor, Oxytetracycline hydchloride
FBR, fungal bacterial ratio; IAR, inhibitor additivity ratio

Conclusion

When applied in combination, Captan (4 mg/g) and Oxytetracycline hydrochloride (8 mg/g) yielded maximum rates of respiration inhibition in all three soils (Table 2, 3 & 4). The IAR values were close to 1.00 with this treatment. These concentrations of the inhibitors were found to be most appropriate for determining FBR in the three WA agricultural soils tested.

Acknowledgements

We thank Phil Mulvey for valuable comments on the manuscript and the ERA Farming Company for technical and financial assistance.

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