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was called fertilizer un-amended control. Fivereplicates were prepared for each treatment. Theabove-ground dry biomass was measured at the end ofthe experiment.

2-2 Filter paper cultures

Seeds of weeping love grass were placed onpairs of filter paper (Advantec) in sterile plastic dishesand let to germinate for 3 days in a growth chamber atthe same temperature and illumination conditions as inexperiment 1-2. Evenly seedlings were selected andfurther grown for 7 days with or without Nostocapplication of 2 g (fresh weight, equivalent to 20 mgas dry weight) in N-free Knop’s culture solution(Namiki, 1990). Sodium chloride solution (0.1 or0.2 M) or distilled water was then added to the Knop’sculture solution as treatments. Four replications, eachwith 6 seeds, were prepared for the individualtreatments. Plant growth was evaluated by measuringthe height of the shoot at the end of the experiment.

2-3 Brown Forest soil

Sixty-five grams of the Brown Forest soil (thesame soil as in experiment 1-1) were placed in plasticPetri dishes, and autoclaved after they were amendedwith a quantity of Na2CO3 equivalent to 15 cmol kg-1

dry soil. The pH and EC of the Na2CO3-amendedsoil were 10.0 and 0.8 dS m-1, while those of theNa2CO3-un-amended soil were 6.6 and 0.07 dS m-1,respectively. The Na2CO3-amended soil was thendivided into four parts and further treated as follows:(i) no fertilizer amendment (referred to as salt-amended[-] fertilizer), (ii) amendment with a chemical fertilizer(N:P:K 8:8:8) at a rate equivalent to 200 kg N ha-1

(referred to as salt-amended[+]fertilizer), (iii) Nostoc(3 g fresh weight) amendment mixed with thesoil (referred to as salt-amended[+]Nos mixed) and(iv) Nostoc (3 g fresh weight) amendment applied onthe soil surface (referred to as salt-amended[+]Nossurface). Half of the Na2CO3-un-amended soil sampleswere also further amended with a chemical fertilizer atthe same rate as for the Na2CO3-amended soils(referred to as salt un-amended[+]fertilizer), the otherhalf did not receive any fertilizer (referred to as salt un-amended[-]fertilizer). Seeds of weeping love grass(0.05 g) were planted in each Petri dish and grown ina growth chamber for 7 days at the same temperatureand illumination conditions as in experiment 1-2. Threereplications were prepared for each treatment. Plantgrowth was evaluated by measuring the height of theshoot and the fresh weight of the above-groundbiomass at the end of experiment.

Experiment 3: Comparison of growth and salt-tolerance between isolates from Khon Kaen,Thailand and from Japan

The growth of four strains of Nostoc was com-pared after a stay of 168-192 h in a growth chamber.Salinity tolerance was also examined by growing thestrains for 72 h in a 0.1-0.6 M NaCl media. Totalpolysaccharide production was also compared after a72 h incubation in BG 11 media and filtrated (0.47 µm)then determined by the phenol sulphate method.

Results and discussion

Effect of Nostoc on soil characteristics

When Nostoc was applied on the surface ofsoils in plastic containers kept outdoor and cultivatedfor 90 days in experiment 1-1, soil moisture andtemperature were more steady when compared with theun-amended soils. After heavy rain, the amount ofdrained water was the same in the Nostoc amended andun-amended treatments. Without rainfall or irrigation,soil pF in the topsoil remained low, indicating moistconditions, for a longer period in the soil where Nostochad been applied compared with Nostoc un-amendedsoils (Figure 2). In addition, the pF in the deeper soillayers remained unchanged for about one week in theNostoc treatment whilst it started to rise after four days[RP3] in the Nostoc un-amended soils. The maximumdaily soil temperature with Nostoc application wasabout 4 to 6oC lower than without Nostoc application(Figure 3). Thus Nostoc application to the soil surfaceled to a decrease in the maximum soil temperature andthe retention of soil moisture under the dry conditionsof mid summer. These two effects can be attributed tothe crust mat of Nostoc developed on the soil surface.Crust mat development resulted in a reducedevaporation from the soil surface, and therefore helpedretain the water, as indicated by the low water potential(pF), for longer period than without Nostoc application.The crust mat also reduced the incident solar radiationon the soil surface, and this led to the observed

Figure 2. Effect of Nostoc application on soil waterpotential

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decrease in maximum soil temperature. The decreasedmaximum soil temperature and the retention of soilmoisture by Nostoc may influence other soil biologicaland chemical properties. Indeed soil moisture is animportant factor in determining the quantity andactivity of soil biota. Soil borne organisms, includingmicroorganisms, are very active at soil moisturecontent of about 60% of the field capacity, but theiractivity becomes restricted at a lower moisture content.The optimum soil temperature for soil organisms isbetween 20-30oC. Some soil organisms may endure upto 35oC, but they become severely restricted above40oC.

Soluble organic C and N contents at soil surface(0 to 2.5 cm) increased significantly (p <0.01; t-test)with Nostoc application after 90 days of cultivation(Table 1). Nostoc application also significantlyincreased the amount of soluble N at depths from 2.5to 5.0 cm. The increase in soluble C and N wasattributed to the secretion of polysaccharides fromNostoc. Polysaccharides are known to contribute to thestructural stability of the soil, to increase soil C and N,and to promote plant growth (Foth 1990). Someorganic compounds were applied when Nostoc was

inoculated, but this initial amount of polysaccharideswas much less than the quantity observed after 90 daysof cultivation.

There were no significant effects of Nostocapplication on the other soil chemical and biologicalcharacteristics such as soil pH, electrical conductivity,number of fungi and bacteria, CEC and exchangeablecations (Table 1). However, significant differences insome of these soil properties between the different soildepths were observed. For example, the electricalconductivity, the amount of soluble N and the numberof microorganisms decreased with depth in bothNostoc amended and un-amended soils. Interestingly,while the amount of soluble C increased with depth inthe control, it decreased with Nostoc application. Thesoil used in this experiment was neutral, and rather richin soil organic matter, as indicated by its biochemicalproperties listed under experiment 1-1. The effects ofNostoc application on the soil chemical and biologicalproperties might have been limited in such an unfertilesoil.

In experiment 1-2, when Nostoc was applied onthe surface of pots of Brown Forest and sandy soilseither in the dry ground or freshly minced forms,soluble organic C and N were significantly increased,even in a period shorter (30 days) than in the outdoorexperiment (90 days) (Table 2). Like in experiment1-1, the biochemical properties were not modified byNostoc application. The similarities in the effects of dryor fresh minced Nostoc on soluble C and N may be dueto the fact that the photosynthetic and nitrogen fixingactivities of the dried Nostoc can recover within a fewhours after re-wetting to become comparable tofresh samples (Apte and Thomas 1997). From this

Figure 3. Effect of Nostoc application on soil temperature(1 cm depth)

Table 1. Effect of Nostoc application on soil properties

Soil depth (cm) Treatment pHEC (mS/m)

Number of microorganisms(CFU/kg.d.s.)

H2O KCl Fungi Bacteria0~2.5 Cont 6.11 ± 0.4 4.53 ± 0.03 43.4 ± 15.4 (5.7 ± 1.8) × 104 (2.9 ± 1.3) × 106

+Nos 5.95 ± 0.1 4.66 ± 0.1 41.9 ± 10.8 (4.3 ± 1.2) × 104 (3.7 ± 1.0) × 106

2.5~5.0 Cont 6.18 ± 0.1 4.18 ± 0.1 12.1 ± 2.65 (2.4 ± 0.6) × 104 (2.2 ± 0.5) × 106

+Nos 6.10 ± 0.1 4.33 ± 0.02 16.9 ± 1.71 (2.0 ± 0.5) × 104 (1.8 ± 0.6) × 106

Soil depth (cm) TreatmentSoluble C Soluble N CEC Exchange cation (cmol/kg d.s.)

(mg/kg d.s.) (cmol/kg d.s.) Na Mg K Ca Mn0~2.5 Cont 177 ± 34 12.1 ± 1 25.1 ± 1 0.78 3.65 1.24 7.44 0.03

+Nos 242 ± 14* 26.3 ± 4** 28.6 ± 3 0.76 3.66 1.14 7.25 0.06

2.5~5.0 Cont 232 ± 64 07.7 ± 1 26.7 ± 2 1.18 4.25 1.12 8.14 0.07+Nos 197 ± 27 16.6 ± 1 30.6 ± 5 1.22 4.00 1.17 7.79 0.06

5.0~7.5 Cont 230 ± 74 17.6 ± 1 30.6 ± 3 0.99 3.74 1.16 7.82 0.07+Nos 160 ± 18 12.7 ± 4 29.8 ± 2 1.03 3.54 1.18 7.25 0.04

* and **: Significantly different from control at p <0.05 and p <0.01, respectively (t – text). (n = 3)

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observation, it can be anticipated that it is possible touse dry Nostoc instead of fresh material.

Nitrogen fixation is an important N source inmarginal soils like sandy or coastal saline soils. In salt-affected areas in India, application of cyanobacteriacombined with gypsum or sulphur changed the soil pHfrom alkaline to neutral, reduced exchangeable Na andEC, and led to the development of soil aggregates inthe long term (Kaushik and Mutri 1981; Kaushik 1989,Subhashini and Kaushik 1984). It is necessary,therefore, to conduct long-term experiments to see allthe effects of Nostoc application.

Effect of Nostoc on plant growth

In experiment 2-1, when Nostoc was appliedeither on the surface or mixed homogeneously with thesoil, and the soils were sown to weeping love grass, themixing of Nostoc enhanced plant growth, comparedwith Nostoc un-amended soils, even though the bestplant growth was obtained with fertilizer application(Figure 4). The plant growth in the control (no fertilizerapplication) and surface-applied Nostoc were similar,and these were significantly lower than the plantgrowth obtained with the mixing of Nostoc with thesoil. Due to the absence of light in the topsoil, Nostoc

mixed with the soil did not grow, but was probablymineralized by heterotrophic soil microorganisms. Thismineralization provided probably plant-availablenutrients, especially N, that was taken up by plants toimprove their growth. This growth was better than inthe samples where Nostoc was applied to the surface.The soil microbial biomass may have also immobilizedpart of the C and N mineralized from the Nostoc cells,and a great portion of this C and N may have ended upbeing incorporated into the soil organic matter.

When the seedlings of weeping love grass weretransplanted in a N-free growth medium with orwithout addition of NaCl in experiment 2-2, plantgrowth was significantly decreased with NaCl addition(Figure 5). However, at the NaCl concentration of 0.1and 0.2 M, the growth of the seedlings in Nostocamended dishes was significantly higher than that inthe control. In experiment 2-3, the addition of Na2CO3

also reduced plant growth by decreasing both the shootheight and fresh weight, but this decrease wasmitigated by the application of Nostoc, especially whenit was applied at the soil surface (Figure 6). The resultspresented in the last two figures (5 and 6) indicate thatNostoc can partly compensate the negative effects ofsalts on plant growth in sandy or saline soils. There hasbeen no report so far to describe such an effect ofcyanobacteria on the growth of higher plants, so furtherstudy is important.

Table 2. Effect of Nostoc application on soil properties

Soil type TreatmentSoluble C Soluble N CEC Exchange cation (cmol/kg d.s.)

(mg/kg d.s.) (cmol/kg d.s.) Na Mg K Ca MnRiver sand Cont 13 ± 2 N.D. 2.0 ± 0.1 0.46 N.D. 0.90 0.92 0.02

+Nos (dry) *48 ± 10 *18 ± 3 2.2 ± 0.1 0.52 N.D. 0.87 1.05 0.02+Nos (wet) *47 ± 11 *15 ± 3 2.0 ± 0.1 0.40 N.D. 0.77 1.00 0.03

Brown Cont 151 ± 11 15.2 ± 2 20.8 ± 2.0 0.62 2.29 2.13 7.59 0.34forest soil +Nos (dry) *209 ± 10 *25.0 ± 2 18.6 ± 2.1 0.65 2.63 2.35 8.29 0.57

+Nos (wet) *227 ± 33 *31.5 ± 3 18.6 ± 0.9 0.66 2.32 1.53 7.63 0.22

*: Significant difference at p <0.01 (t – test) n = 3

Figure 4. Effect of Nostoc application on plant growth ina sandy soil

Vertical bars represent S.D. ( n = 5)Bars with the same letter are not different (Fisher’s LSP: p <0.05)

Figure 5. Effect of Nostoc application on plant growthunder sodium stress in growth medium

Vertical bars represent S.D. (n = 24)*: Significant difference at p <0.01 (t – test)

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Figure 6. Effect of Nostoc application on plants shootheight (i) and wet weight (ii) in soils

Vertical bars represent S.D. (i) shoot height (n = 60), (ii) wet weight(n = 3)Bars with the same letter are not different (Fisher’s LSD: p <0.05)

Figure 7. Effects of Nostoc application on plants shootwet weight in soils

Vertical bars represent S.D. (n = 3)Bars with the same letters are not different (Fisher’s LSD: p <0.05)

Comparison of growth and salt-tolerancebetween tropical and temperate isolates

Growth was faster in the Khon Kaen isolatewhen compare to temperate (Japanese) isolates, exceptHK which had most rapid growth (Figure 8). Thesalinity tolerance of Nostoc isolated from Khon Kaenwas comparable to other isolates (Figure 9). Nostocisolated from Khon Kaen produced the largest amountof polysaccharides followed by HK, Tateyama andMorioka (Figure 10). HK and Khon Kaen Nostocisolates produced substantial amounts of poly-saccharides in saline condition, which may haveinteresting implications to remediate saline soils.

Conclusion

Nostoc has some potential to help re-vegetatearid, saline or alkaline soils. The Khon Kaen isolatemay be an option for some tropical sandy soils. Indeed,the Khon Kaen isolate produced the largest amounts ofpolysaccharides and exhibited an average tolerance tosalinity. However, further studies are needed beforeNostoc can be introduced to farmers. In particular,long-term field experiments are needed to ascertainmedium-to long-term effects.

Acknowledgment

Part of this research was supported by theBRAIN project (leader Dr. Masanori Saito, NIAES).We are grateful to Prof. Masayuki Ohmori (SaitamaUniversity) and Dr. Solomon Acquaye (ChibaUniversity) for their valuable comments.

References

Apte, S.K. and Thomas, J. 1997. Possible amelioration ofcoastal soil salinity using halotolerant nitrogen-fixingcyanobacteria. Plant Soil, 189, 205-211.

Eldridge, D.J., and Greene, R.S.B. 1994. Microbiotic crusts:a review of their roles in soil and ecological processesFigure 8. Growth rate of Nostoc isolates from Khon

Kaen and from Japan

Figure 9. Salinity tolerance of Nostoc isolates from KhonKaen and from Japan

Figure 10. Total polysaccharide production of Nostoc insaline conditions

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in rangelands of Australia. Australian Journal of SoilResearch, 32, 389-415.

Foth, H.D. 1990. Fundamentals of Soil Science. 8th ed. JohnWiley, New York, 136 p.

Johansen, J.R. 1993. Cryptogamic crust of semiarid and aridlands of North America. Journal of Phycology, 29,140-147.

Kaushik, B.D., and Mutri, G.S.R.K. 1981. Effect of bluegreen algae and gypsum application on physico-chemical properties of alkali soil. Phykos, 20, 91-94.

Kaushik, B.D. 1989. Reclamative potential of cyanobacteriain salt-affected soils. Phykos, 28, 101-109.

Muramoto, J., Goto, I., and Ninaki, J. 1992. Rapid analysisof exchangeable cation and cation exchange capacity(CEC) of soils by a shaking extraction method.Japanese Journal of Soil Science and Plant Nutrition,63, 210-215.

Namiki, Y. 1990. Hydroponic culture of vegetables.Yokendo, Tokyo, 50-51 (in Japanese).

Okitsu, S., Imura, S., and Ayukawa, E. 2003. Structure anddynamics of the Ceratodon purpureus-Bryumpseudotriquetrum community in the Yukidori Valley,Langhovde, continental Antarctica. Polar Bioscience,16, 49-60.

Sakamoto, K., and Hayashi, A. 1999. A rapid method fordetermining the microbial biomass-N in soil –Determination of total-N in soil extracted by peroxy-disulfate oxidation method. Soil Microorganisms,53, 57-62 (in Japanese).

Soil Microbial Society 1992: Methods in Soil Microbiology– New Edition, ed. Soil Microbiological Society ofJapan, Youken-dou, Tokyo,41 1 p. (in Japanese).

Subhashini, D., and Kaushik, B.D. 1984. Amelioration ofsalt affected soils with blue green algae, I. Influenceof algalization on the properties of saline-alkali soil.Phykos, 23, 273-277.

Zaddy, E. 1997. Nitrogen fixation in macro and micro-phytic patches in Negev desert. Soil Biology andBiochemistry, 30, 449-454.

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Dry matter production and digestibility of Centrosema pubescens andPueraria phaseoloides with rock phosphate fertilization and mycorrhizae

inoculation in Latosolic soil

Lukiwati, D.R.1

Keywords: Centrosema pubescens, Pueraria phaseoloides, rock phosphate, mycorrhizae

Abstract

Centrosema pubescens (centro) and Pueraria phaseoloides (puero) are important forage legumes asprotein and mineral sources for ruminant livestock in the tropics. However, most of the land that is used forforage production is characterized by a low phosphorus content. As the high cost of superphosphate is a majorlimiting factor, a combination of rock phosphate (RP) fertilization and vesicular-arbuscular mycorrhizae(VAM) inoculation maybe a promising approach to increasing available P. A field experiment was conductedon a latosolic soil (low pH and low available Bray II extractable P) to evaluate the effects of RP fertilizationand VAM inoculation and their interaction on dry matter (DM) production and in vitro dry matter digestibility.A completely randomized block design with 3 replicates was used. The main experiment consisted therein thecombination of three factors as follows 1) legume species (centro, puero), 2) VAM inoculation (with, andwithout VAM inoculation), and 3) rock phosphate fertilization (0, 44, 87, 131, and 175 kg P ha-1). The periodof defoliation was used as sub factor (defoliation I, II, and III). Results showed that DM production and DMdigestibility of puero was higher compared to centro after defoliation. Dry matter digestibility of VAMinoculated puero was higher compared to uninoculated one. Rock phosphate fertilization increased DMproduction of VAM inoculated legume. Dry matter production was not significantly different with or withoutVAM inoculation. When inoculated, rock phosphate fertilization increased DM production. Success of VAMinoculation in the field is affected by the effectiveness of indigenous-VAM fungi or is dependent upon VAMinoculum potential.

1 Faculty of Animal Agriculture, Diponegoro University,Campus UNDIP Tembalang, Semarang-Indonesia

Introduction

Centro (Centrosema pubescens) and puero(Pueraria phaseoloides) are important forage legumesas protein and mineral sources for ruminant livestockin the tropics. Centro and puero have also been used ascover crops in forest plantations or in agroforestrysystems. However, most land used for forageproduction in Indonesia is characterized by a lowphosphorus (P) content and a low soil pH (latosolicsoil). The application of P fertilizer during the periodsof active growth increases forage legumes productionand quality (Coates et al.,1990).

Superphosphate (SP) fertilizer has been widelyused to improve agricultural production. However,its high cost makes the use of rock phosphate (RP)attractive. Rock phosphate is a slow release source ofphosphorus, thus the inoculation with vesicular-arbuscular mycorrhizae (VAM) fungus is a promising

technique to increase P bio-availability. According toJones (1990), the response obtained to applications ofP fertilizers is a function of many factors i.e. the initialavailability of soil P, the form of fertilizer applied, andthe presence or absence of effective mycorrhizae in thesoil.

Most research on VAM inoculation has beendone on forest-trees and agricultural crops, but rarelyon forage crops. The VAM fungus of Glomus mosseae,for instance, is the most common species associatedwith agricultural crops or forests (Chen et al., 1998).Centro and puero are suitable host plants for VAMfungi culture (Lukiwati & Supriyanto, 1995). Twospecies of VAM (Glomus fasciculatum, Entrophosphoracolumbiana) proved to increase dry matter productionand nutrient uptake of Pueraria phaseoloides similarly(Lukiwati & Simanungkalit, 2004). The results showedthat SP can be replaced by RP when combined withVAM fungi inoculation (Lukiwati & Simanungkalit,2001). Success of VAM inoculation in the fieldaffected by effectiveness of indigenous-VAM fungi ordepending upon VAM inoculum potential (Mitiku-

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Habte & Fox, 1993). The symbiosis between VAMfungi and legumes has been less studied in unsterilizedsoils than in sterilized soils. The objective of this workwas to investigate under field conditions the effect ofRP fertilization, VAM (Glomus sp) inoculation, andtheir combination on dry matter production anddigestibility of centro and puero in a latosolic soil lowin available P.

Materials and Methods

A completely randomized field experimentwith three blocks was conducted for 7 months on an acid(pH (H2O) 5.1 to 5.3) latosolic soil low in available P(Bray II extractable between 4.0 and 5.7 g/kg-1). Theexperiment was conducted on 4 × 5 m plots.

The design consisted in three factors as follows1) legume species (centro, puero), 2) VAM inoculation(with, and without VAM inoculation), and 3) fivelevels of rock phosphate fertilization (0, 44, 87, 131,and 175 kg P ha-1). The periods of defoliation wereused as sub factors. The defoliation of the plants wasdone three times. Standard fertilizers, i.e. 50 kg N ha-1

as urea and 83 kg K ha-1 as KCl, were applied to eachplot. Legume seed of centro and puero were dibbledinto small holes made with a wooden stick at the rateof two seeds per hole, spaced 100 × 50 cm. Each plotwas inoculated with 100 gram of VAM inoculum/holeat sowing. The inoculum of VAM contained approxi-mately 820 spores/100 gram. The parameters measuredwere dry matter (DM) production at the three times ofdefoliation, and in vitro DM digestibility on the secondand third defoliation only, because of the limitedbiomass at the time of the first defoliation.

The first defoliation was conducted threemonths after sowing and subsequent defoliations wereconducted every two months. The plants were cut closeto the soil surface to determine dry matter production.Dry matter production of each replicate was calculatedfrom 1 m2 subplots. To measure DM production thedefoliated forage legumes was chopped, subsampled,and oven-dried to constant weight at 70ºC for 48 hours.The samples of the second and the third defoliationwere finely ground and analysed to determine in vitrodigestibility by the Terry and Tilley method (1963).

The analysis of variance on DM production andDM digestibility was done using the general linearmodel procedure of SAS. The significant differencesamong the treatments were tested using Duncan’sMultiple Range Test (DMRT).

Results and Discussion

Results

The effect of RP fertilization was not significanton the DM production of uninoculated legume (Table 1).Rock phosphate fertilization increased DM productionon inoculated legume. The DM production ofinoculated legume was not significantly higher than ofthe uninoculated one, at the same level of RP excepton the unfertilized treatment.

Table 1. Dry matter production (kg ha-1) of foragelegumes with rock phosphate fertilization andmycorrhiza inoculation

P levels (kg P ha-1) Uninoculated Inoculated

0 92.2 ab 67.0 c*44 92.7 ab 80.7 bc87 90.1 ab 88.4 ab

131 88.7 ab 93.9 ab175 97.4 ab 106.8 a

* Means followed by the same letters are not significantlydifferent at DMRT 5%

Dry matter production of centro and pueroincreased after the first and after the second defoliation(Table 2). Dry matter production was higher for puerothan for centro at the second period of defoliation(120.8 kg ha-1 against 90.6 kg ha-1).

For the same level of RP, dry matter productionof the second and third defoliation was significantlyhigher compared to the first defoliation. Dry matterproduction of the third defoliation was significantlyhigher compared to the second defoliation for 0, 131,and 175 kg P ha-1. RP fertilization did not significantlyincrease DM production at the first defoliation.However, RP fertilization increased DM production onthe second and the third defoliation (Table 2).

Table 2. Dry matter production (kg ha-1) of foragelegumes on three periods of defoliation

TreatmentPeriod of defoliation

I II IIISpecies of legume:

Puero 40.7 c 120.8 a 121.1 a*Centro 50.3 c 90.6 b 115.1 a

P levels (kg ha-1):0 39.6 e 89.5 d 109.8 bc

44 47.9 e 110.3 bc 102.0 c87 45.9 e 111.6 bc 110.4 bc

131 48.0 e 99.1 cd 126.7 ab175 46.3 e 118.2 bc 141.9 a

* Means followed by the same letters are not significantlydifferent at DMRT 5%

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Dry matter digestibility of puero was signi-ficantly higher compared to centro, with or withoutVAM inoculation on the second and third defoliation(Table 3). Dry matter digestibility of centro and pueroinoculated with VAM was not significantly differentcompared to uninoculated one on the seconddefoliation. However, DM digestibility of centro wassignificantly lower compared to uninoculated one onthe third defoliation. Contrasting this, DM digestibilityof puero inoculated by VAM was significantly highercompared to uninoculated one on the same defoliation.

Rock phosphate fertilization did not signi-ficantly increase DM production of forage legumes atthe first defoliation (Table 2). That was because theplants were still at the initial growth, while rockphosphate belongs to the group of slow release sourceof phosphorus (Jones, 1990).

The DM production of puero was highercompared to centro after first and second defoliation(Table 2). Performance of plant growth and rootgeometry (number of roots and distribution in the soil)of each plant species are different, as well as theirresponse to the treatments (Kerridge and Ratcliff,1982). Dry matter production stimulated after the firstand the second defoliation promoted new stolongrowth. Defoliation promoted vegetative regrowth oflegume as shown in the DM production. At the earliestperiod, the growth of puero was slower compared tocentro, therefore, DM production of puero was lowercompared to centro. However, the regrowth of puerowas faster than centro, therefore, the DM production ofpuero was higher than centro after each defoliationwhich in turn decreased the copper (Cu) content ofpuero (data not showed). This is so because of Cu asco-factor phenoloxidase enzyme which influenced thelignification process (Dell et al, 1995). Increase inphenoloxidase activity tend to increase the lignificationprocess. As a result, in vitro DM digestibility of centrowas lower than puero on the second and the thirddefoliation with or without VAM inoculation (Table 3).

Conclusion

Dry matter production on the second defoliationand DM digestibility of puero was higher than centro.Rock phosphate fertilization could increased the DMproduction of VAM inoculated legumes. Success ofVAM inoculation in fields is affected by theeffectiveness of indigenous-VAM fungi or dependingupon VAM inoculum potential.

References

Bagyaraj, D.J. 1988. Technology for inoculum production ofVAM fungi. In: Mahadevan, A., Raman, N., andNatarajan, K., eds., 1988. Proc. of the First AsianConference on Mycorrhizae. Madras, 29-31 January,p. 326-328.

Chen Yinglong, Gong Mingqin, Wang Fengzhen, Chen Yu,Zhang Meiqing, Dell, B., and Malajczuk, N. 1998.Diversity putative ectomycorrhizal fungi andarbuscular mycorrhizal fungi in Eucalyptusplantations in Southern China. In: Gong Mingqin, XuDaping, Zhong Chonglu, Chen Yinglong, Dell, B.,and Brundrett, M., eds., 1998. Proc. of Guangzhou

Table 3. Dry matter digestibility (%) of forage legumeson the second and third period of defoliation withmycorrhiza inoculation

Uninoculated InoculatedPeriod of defoliation

II III II IIIPuero 47.3 cd 49.4 b 46.1 cd 50.8 a*Centro 43.4 e 47.3 c 43.5 e 45.3 d

* Means followed by the same letters are not significantlydifferent at DMRT 5%

Speciesof

legume

Discussion

Dry matter production of VAM inoculatedlegumes was increased by rock phosphate fertilizer.However, both VAM inoculated and uninoculatedlegumes gave a similar DM production at the samelevel of rock phosphate (Table 1). Previous fieldexperiments have shown that the response to VAMinoculation in the field greatly varies, and sometimesinoculation did not increase the production (Lin andHao, 1991). This is because most of agricultural soilsalready contain indigenous populations of VAM fungi.Mycorrhizal inoculation would be successful in thefield only if the native population was low and loweffectiveness of indigenous-VAM fungi or dependingupon VAM inoculum potential (Mitiku-Habte and Fox,1993). Field experiment was carried out on theunsterilized soils. A year before, cassava had beenharvested from the field, and since then the field wasunderfallow. Cassava is a VAM-obligate type. Cassavarhizosphere could increase the effectiveness ofindigenous-VAM fungi (Potty, 1988). Spore isolationin the beginning of the field experiment showed that,there was an indigenous population of VAM fungi witha density up to 496 spores/100 g. During an experimentaiming at isolating spores from the soil, some sporesinfected by pathogen fungi were found. Thesepathogens could have decreased the effectiveness ofVAM inoculum. Mycorrhizal spores could have beeninfected by pathogen fungi during the storage of thesoil inoculum (Bagyaraj, 1988).

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ACIAR International Workshop. Guangzhou,31 August – 6 September, p. 21-28.

Coates, D.B., Kerridge, P.C., Miller, C.P., and Winter, W.H.1990. Phosphorus and beef production in NorthernAustralia. 7. The effect of phosphorus on thecomposition, yield and quality of legume-basedpasture and their relation to animal production.Tropical Grasslands. 24: 209-220.

Dell, B., Malajczuk, N., and Grove, T.S. 1995. NutrientDisorders in Plantation Eucalyptus. ACIARMonograph 31: 110 p.

Jones, R.J. 1990. Phosphorus and beef production in NorthernAustralia. 1. Phosphorus and pasture productivity.Tropical Grasslands. 24: 131-139.

Kerridge, P.C., and Ratcliff, D. 1982. Comparative growth offour tropical pasture legumes and guinea grass withdifferent phosphorus sources. Tropical Grasslands.16: 33-40.

Lin Xian-Gui, and Hao Wen-Yin. 1991. Occurrence andefficiency of VA Mycorrhizaein Fluvo-Aquic soilsin North China. BIOTROP Special Publication.42: 111-121.

Lukiwati, D.R. and Simanungkalit, R.D.M. 2001.Improvement of maize productivity with combinationof phosphorus fertilizer from difference sources andvesicular-arbuscular mycorrhizae inoculation. In:Rajan, S.S.S, and Chien, S.H., eds. 2001. Proc. ofInternational Meeting “Direct Application of Phos-phate Rock and Related Appropriate Technology-

Latest Developments and Practical Experiences”.Kuala Lumpur, 16-20 July. p. 329-333.

Lukiwati, D.R., and Simanungkalit, R.D.M. 2004.Production and nutritive value of Puerariaphaseoloides with vesicular-arbuscular mycorrhizaeinoculation and hosphorus fertilization, abstr.p.90,Abstract of the 4th International Symposium of theWorking Group MO “Environmental Significanceof Mineral-Organic Component-MicroorganismInteractions in Terrestrial Systems”. Wuhan-China,20-23 September.

Lukiwati, D.R. and Supriyanto. 1995. Performance of threeVAM species from India for inoculum productionin centro and puero. In: Prana, S.S., ed. 1995.International Workshop on Biotechnology andDevelopment of Species for Industrial TimberEstates. Proc. LIPI Bogor. 27-29 June. Pp. 257-265.

Mitiku-Habte and Fox, R.L. 1993. Effectiveness of VAMfungi in non sterile soils before and after optimizationof P in soil solution. Plant and Soil. 151(2): 219-226.

Potty, V.P. 1988. Response of cassava (Manihot esculenta) toVAM inoculation in acid lateritic soil. In:Mahadevan, A., Raman, N, and Natarajan, K. 1988.Proc. of the First ACOM. Madras, 29-31 January.pp. 246-249.

Terry, R.A. and Tilley, J.M.A. 1963. A two state techniquefor the in vitro digestion of forage crops. JournalBritish Grassland. F.O.C. 18: 104-111.

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Short-term dynamics of soil organic matter and microbial biomass aftersimulated rainfall on tropical sandy soils

Sugihara, S.1; S. Funakawa1; H. Shinjo1 and T. Kosaki2

Keywords: Soil organic matter, microbial biomass, microbial activity, soil organic matter management

Abstract

Simulated-rainfall experiments were conducted during the dry season on sandy soils under two differentfarming systems in terms of the amounts of residue input to soils – that is, a sugarcane field in NortheastThailand (SJ) and a millet field in Niger (SD). The main objective of the experiment was to evaluate thepossible effects of rapid wetting/drying on soil microbial activity and the rate of soil organic matter (SOM)decomposition on tropical sandy soils in the two systems under field conditions. Three treatments wereimposed: (1) C plot, receiving no water; (2) W plot, treated with 10 mm of rainfall water; and (3) G plot,sprayed with glucose as a substrate, together with the 10 mm of rainfall treatment. The CO2 efflux rate andmicrobial biomass (MB) were measured at the field for about 2 weeks. After the rainfall treatment, a rapidCO2 flush was observed in the SJ-W, SJ-G and SD-G plots during the initial 4 days. At the same time, the MBincreased rapidly in these plots and resulted in a higher CO2 efflux. In the SJ-G plot, the cumulative CO2

efflux was twice that of the SD-G plot due to a higher growth rate of MB in the former. In contrast, thissimultaneous increase of MB and CO2 efflux rate was not observed in the SD-W plot or in the C plots.Therefore, the effects of rapid wetting/drying on SOM dynamics were considered to depend both on thedynamics of MB and on the microbial activity in tropical sandy soils. In particular, the multiplication of MBlargely contributed to a prolonged CO2 flush. The increase in CO2 flush after the addition of substratesand/or water was more pronounced in the SJ plots, which had been receiving higher amounts of residue inputin recent years. To conclude, it is necessary to take account of such historical factors of land management toappropriately simulate SOM dynamics in tropical sandy soils.

1 Graduate School of Agriculture, Kyoto University, Japan,Sohs@kais.kyoto-u.ac.jp

2 Graduate School of Global Environmental Studies,Kyoto University, Japan

Introduction

It is necessary both to understand the dynamicsof the soil organic matter (SOM) from anenvironmental perspective (for example, whether soilsare possible sinks or sources of atmospheric CO2), andalso from an agricultural perspective, as it is widelyrecognized that SOM is closely related to soil fertility.Microbial biomass (MB), which is defined as the livingmicrobial component of the soil, is the primary agentof the soil ecosystem that is responsible for thedecomposition of SOM, nutrient cycling and energyflow (Wardle, 1992). Above all, many studies havebeen carried out to evaluate the effects of drying/rewetting on the decomposition of SOM and microbialdynamics due to its importance in the overall dynamicsof SOM and atmospheric CO2 (Kieft et al., 1987; VanGestel et al., 1993a, b; Pulleman and Tietema, 1999;

Franzluebbers et al., 2000; Fierer and Schimel, 2002;Fierer and Schimel, 2003; Wu and Brooks, 2005;Mihka et al., 2005). However, most of theseexperiments were conducted under laboratoryconditions in which soil moisture contents were mostlyfixed at high values after rewetting, although soilsactually redry rapidly after rewetting under fieldconditions. There are few reports studying the effect ofthe dry-wet cycle on SOM and/or soil microbes underfield conditions (Murphy et al., 1998; McNeil et al.,1998). To evaluate the actual dry-rewetting effect,responses and sensitivity of soil microbes to dry-rewetting changes should be investigated in more detailunder field conditions. In addition, there is littleinformation in arid/semi-arid ecosystems (Schwinninget al., 2004; Saetre and Stark, 2005), as most studieswere carried out in temperate regions. Austin et al.(2004) report that pulsed-water events have a key rolein a number of below-ground processes in arid/semi-arid ecosystems, and that changes in the nature ofpulsed events due to human impact may be moreimportant than larger-scale changes in total rainfall or

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average temperature in affecting biogeochemicalcycling in water-limited ecosystems. As ourunderstanding of the MB in tropical climates remainspoor, it is important to identify whether general trendsgenerated for mostly temperate ecosystems can alsoapply to tropical ecosystems (Wardle, 1992).

In tropical sandy soil, it is expected that theshort-term dynamics of SOM and MB are crucial in theoverall dynamics of SOM, because sandy soils areinherently poor in SOM retention and high temperatureusually accelerates rapid SOM decomposition. It ispossible, therefore, that the effect of drying/rewettingon tropical sandy soil is different from the resultsobtained in temperate ecosystems. In this study,simulated rainfall experiments were conducted inThailand and Niger. Both soils are classified as tropicalsandy soil. In Thailand, relatively high amounts of cropresidues (sugarcane), composed of leaf and rootbiomass (1.1 and 1.0 Mg C ha-1 y-1, respectively), wereincorporated into the soils at harvest, in addition toa considerable amount of litter-fall occurring during thecropping period and, according to Funakawa et al.(2005), approximately 4 Mg C ha-1 y-1 of SOM wasannually decomposed. On the other hand, most of thecrop residues were removed from the cropland afterharvest either by humans or by cattle, so that onlybelow-ground crop residues were incorporated into thesoil in Niger. The Niger plots had been left fallow formore than 10 years and were converted to millet field2 years ago. Annual SOM decomposition rates wereabout 1.0 Mg C ha-1 y-1 (Shinjo 2005; data not shown).Therefore, the dynamics of SOM are very different inthe two regions.

The objectives of this study were (1) to evaluatethe possible effects of rapid wetting/drying on thedynamics of soil microbes and the rate of SOMdecomposition on tropical sandy soils by applyingsimulated-rainfall experiments under field conditionson the two tropical sandy soils, and (2) to compare theeffect of different farming systems on the short termdynamics of SOM and microbial biomass. Theinfluence of the residue management was also analysedin relation to microbial responses to water/substrateadditions.

Materials and methods

Description of the study sites

The experiments were conducted on twotropical sandy soils at the end of dry season; the soilshad different farming systems. One was Sam JanVillage near Khon Kaen City, Thailand (SJ) and the

other was ICRISAT (International Crops ResearchInstitute for the Semi-Arid Tropics) Sahelian Center(ISC) near Niamey City, Niger (SD). The soils of SJand SD are classified into Typic Ustipsamments andPsammentic Paleustalfs, respectively, according to SoilTaxonomy (Soil Survey Staff, 2003). The soilproperties of SJ and SD were, respectively: pHdetermined in water, 6.0 and 5.2; soil texture, sand with90.7 and 95.1%; and organic C, 1.2 and 1.5 g kg-1, andtotal N contents, 0.1 and 0.1 g kg-1. Mean annualprecipitation and average temperature in SJ and SDwere 1,189 and 560 mm, and 27.1 and 29.1ºC,respectively.

Experimental design

The experiments were carried out at the end ofthe dry season (13-28 March 2004 in SJ and 26 May –8 June 2004 in SD, respectively). We installed eachof the three experimental plots (C, W and G plots) inthe cropping field of SJ and SD (Figure 1-a). Eachcropping field was bared and there were just plantresidues.

1) The C plots (SJ-C and SD-C) were left withoutsimulated rainfall as a control.

2) The W plots (SJ-W and SD-W) were treatedwith 10 mm of simulated rainfall at the start ofthe experiments.

3) The G plots (SJ-G and SD-G) were sprayed witha glucose solution (at a rate of 75 g C m-2) as asubstrate, together with the 10 mm of rainfalltreatment. The G plots were designed tocompare the possible activities of soil microbeswith the effect of substrate addition at each site.

Each plot was further divided into subplots Aand B (Figure 1-b) to measure the CO2 efflux rate andMB, respectively, after removing visible plant residuesfrom the soil surface.

The rainfall treatment was then applied, and ittook 0.5 h for the irrigation procedure; following this,the start time of the experiment was set to 07:00 h toobserve the effect of the drastic evaporation in theinitial stage of the experiment. The W and G plots wereleft dry after the simulated rainfall.

Monitoring of air and soil temperature and moisturecontent

The air and soil temperature at a depth of 5 cmand the volumetric soil moisture content in the surface0-15 cm were continuously monitored at the C and W

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plots using a data logger system (107 thermistor probefor soil temperature and CS616 for soil volumetricmoisture connected to CR10X data logger; CampbellScientific, Inc., Logan, USA).

Measurement of CO2 efflux rate from soil surface

The CO2 efflux rate from the soil surface wasmeasured using a closed-chamber system in the subplotA in triplicate. For each measurement, a polyvinylchloride (PVC) cylinder (13 cm in diameter × 15 cmheight) was inserted into the soil to a depth of 5 cmimmediately after the simulated rainfall. For eachmeasurement, after the top of the cylinder was coveredtightly with a plastic sheet and left for 40 min, a 50-mLgas sample was collected using a syringe, and then keptin a 30-mL glass vial until measurement, which waspreviously evacuated. At the same site, an air samplewas collected to determine a baseline atmospheric CO2

concentration. The concentration of CO2 was measuredusing an infrared CO2 controller (ZFP9AA11; FujiElectric, Tokyo, Japan) and the increase in CO2

concentration during the 40 min relative to the controlsample was assigned as the CO2 efflux from the soilsurface. Sample collection was carried out at 4, 6.5, 9,12, 14.5, 26, 32, 38, 50,5, 56, 61,5, 75,5, 86,5, 99.5,346.5 and 363.5 h in the SJ plots, and 3.5, 5.5, 8, 11,14, 26, 32, 37.5, 50, 55, 60.5, 99, 122, 317 and 320 hin the SD plots after the simulated rainfall in triplicate.

Measurement of MB and its activity

The microbial biomass was measured in thesubplot B every 4-6 h using an applied substrate-induced respiration (SIR) method (Anderson andDomsch 1978; Lin and Brooks 1999), as follows:

1) As the SIR is strongly influenced by soil watercontent, a sufficient amount of water was addedto the surface soil prior to each measurement toadjust water content to above 50% of the field-holding capacity. The amount of water addedwas determined based on the amount ofevaporation expected. After the upper 5-cmlayer of soil was well mixed, water was sprayedonto soil surface.

2) Glucose solution was sprinkled onto the soilsusing a syringe at a rate of 60 g C m-2. Then, theupper soil was mixed uniformly.

3) After 30 min, a cylinder (the same one as for theCO2 measurement described above) was placedinto the soil to a depth of 5 cm, and then the topwas covered tightly with a plastic sheet.

4) After 1 h, a 50-mL gas sample was collectedusing a syringe, and then kept in a 30-mL glassvial, which was previously evacuated. Theconcentration of CO2 was measured usingthe infrared CO2 controller (ZFP9AA11; FujiElectric).

5) At the same time, an air sample was alsocollected to determine the atmospheric CO2

concentration.

SIR method is commonly used for laboratoryexperiment, but we applied the method for fieldexperiments, in order to evaluate the short termdynamics (4-6 h) of microbial biomass.

Sample collection was carried out at 5.5, 9.5, 15,26.5, 38.5, 51, 62, 100, 124, 346.5 and 364 h in the SJplots, and 3.5, 8, 13, 25.5, 36.5, 49.5, 59.5, 98.5, 121.5and 316 h in the SD plots after the simulated rainfallin triplicate.

To calculate the microbial biomass based on theSIR measured, we used the following equation(Anderson and Domsch, 1978):

B = (40.04R + 0.37)/1000 (1)

where B is the microbial biomass C (g) and R is the soilrespiration rate (mL h-1). Since this equation isestablished on temperate soils and on laboratory

Figure 1. Design of experimental plots (a), and plotdescription (b)

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condition, we tentatively used this equation forcomparison.

It is widely recognized that temperature is oneof the controlling factors for soil CO2 efflux. Toeliminate the effect of temperature fluctuations over aday, we corrected the soil respiration rate based on theQ10 relationship, where the Q10 factor is the ratio ofrespiration rates observed at temperatures differing by10ºC (Fang and Moncrieff, 2001):

Q10 = (R2/R1) (10/(T2–T1)) (2)

where R2 and R1 are the respiration rates observed attemperatures T2 and T1, respectively. There are manyreports on the relationship between temperature andCO2 efflux from both a short-term and long-termperspective (Fang and Moncrieff, 2001; Parkin andKaspar, 2003). We used Q10 values of 2.2 and 1.7 forSJ and SD, respectively, which was preliminarycalculated by our laboratory incubations. Using theseparameters, we converted the respiration rate measured(R1) to that expected at 22ºC (R2) using MB determinedby SIR.

It has been suggested that microbial activityreflects the effect of environmental conditions(Anderson and Joergensen, 1997; Mamilov and Dilly,2002), and that the activity can be measured throughthe efficiency of substrate utilization (Dilly andMunch, 1998). In this study, we evaluated thecontribution of microbial activity to the decompositionof SOM after drying/rewetting, using the followingequation:

Microbial activity = CO2 efflux rate/microbialbiomass C (3)

In this equation, both the CO2 efflux rate and themicrobial biomass C were expressed on an area basis(g m-2 h-1 and g m-2, respectively). The CO2 efflux ratewas corrected to 22ºC based on the Q10 relationship, asin SIR methods, in order to eliminate the effect oftemperature fluctuation over a day.

Results

Fluctuation of air and soil temperature and soilmoisture during the experiments

The air temperature at the SJ and SD plotsfluctuated from 21ºC and 23ºC to 40ºC and 39ºC,respectively. Similarly the soil temperature at the SJand SD plots fluctuated from 23ºC and 29ºC to 46ºCand 49ºC, respectively (Figure 2).

The daily temperature fluctuation was thereforesubstantial. On the other hand, before the experimentthe soil was very dry – that is, the volumetric moisturecontent (VMC) in the SJ and SD plots was 1.5 and3.5%, respectively, which is equivalent to -4.3 and -1.9MPa, respectively. The simulated rainfall increasedVMC in the SJ and SD plots up to 9.2 and 9.7%,respectively, which is equivalent to -7.7 and -3.5 kPa,

Figure 3. Fluctuation of volumetric moisture contents ofsoil (0-15 cm) (%) at the SJ plot (a) and the SD plot (b)

Figure 2. Fluctuation of air and soil (5 cm) temperatureat the SJ plot (a) and the SD plot (b)

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respectively. Thereafter, the surface soil started to dryand VMC decreased continuously (Figure 3). Waterloss through evaporation was high, especially in thedaytime compared with the night-time. VMC in the SJand SD plots decreased to 5.7 and 7.0% after 1 day, 3.7and 3.9% after 3 days, and 2.0 and 2.9% after 2 weeks,respectively. The final VMC in the SJ and SD plotswas equivalent to -3.8 and -2.2 MPa, respectively.

CO2 efflux from the soil surface

The soil surface CO2 efflux, which was mostlydue to microbial respiration in our experiments, wassmall in the C plot compared with the W and G plots.After the simulated rainfall, the CO2 efflux rate in theSJ-W, SJ-G and SD-G plots increased immediately,whereas that in the SJ-C, SD-C and SD-W plots did notchange appreciably (Figure 4). In the SJ-W, SJ-G andSD-G plots, the CO2 efflux rate continued to increaseup to 6 h, and finally reached 2.6, 8.0 and 2.8 timeshigher than that of the control plots, respectively. Afterthe peak of CO2 efflux, it gradually decreased withfluctuation for fluctuated temperature. After correctionof CO2 data by soil temperature, however, there is no

fluctuation of the CO2 efflux (data not shown). TheCO2 efflux rate in the SJ-G plot was still 1.4 timeshigher than in the SJ-C plot at the end of theexperiment, whereas CO2 effluxes of the SJ-W andSD-G plots were almost equal to that of the controlplots after 100 and 122 h, respectively.

The cumulative CO2 efflux for the SJ-C, SJ-W,SJ-G, SD-C, SD-W and SD-G plots reached 4.25, 7.43,13.68, 2.46, 2.68 and 7.16 g C m-2, respectively, up tothe 13th day of the experiment. The difference in thecumulative CO2 efflux between each treated plot andthe control plots was 3.18, 9.43, 0.22 and 4.70 g C m-2

in the SJ-W, SJ-G, SD-W and SD-G plots, respectively.These differences were primarily contributed to by theCO2 flush in the initial 100 h.

Microbial biomass and activity

In the SJ-W, SJ-G and SD-G plots, MB wasincreased rapidly after the simulated rainfall (Figure 5).MB of the SJ-W plot was increased up to 10 h and thenstabilized without decreasing in spite of rapid drying.After 100 h, it started to decrease to the level of the

Figure 4. Fluctuation of CO2 efflux rates at the SJ plot(a) and the SD plot (b) Bar indicates standard deviation

Figure 5. Fluctuation of microbial biomass at the SJ plot(a) and the SD plot (b) Bar indicates standard deviation

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SJ-C plot. The MB of the SD-W plot fluctuatedsimilarly to that of the SD-C plot, keeping a low levelthroughout the experiment. The MB of the SJ-G andSD-G plots was 5 and 1.5 times, respectively, higherthan that of the control plots after 10 h and thengradually decreased with drying, but was twice that ofthe control plot by the end of the experiment (Figure 5).

Generally, the microbial activity reached thehighest a few hours after the simulated rainfall andthen decreased with water depletion (Figure 6). Theduration of high microbial activity of the G plots waslonger than that of the W plots.

Discussion

CO2 flush and multiplication of soil microbes

The simulated-rainfall treatment significantlyincreased the MB and the microbial activity in theSJ-W, SJ-G and SD-G plots, resulting in largedifferences in the cumulative CO2 efflux comparedwith the C plots. Many studies reported that the CO2

flush after drying/rewetting could be caused by theincrease of microbial activity and finished within a fewdays (Cui and Caldwell, 1997; Franzluebbers et al.,2000; Fierer and Schimel, 2003). Mihka et al. (2005)also reported that the flush of CO2 was mainly relatedto microbial activity and microbial turnover(or microbial origin), and it finished in a day underlaboratory conditions. In these studies, a rapidmultiplication of MB after the drying/rewetting wasnot postulated. In our field experiments, however, notonly microbial activity but also MB was rapidlyincreased in the SJ-W, SJ-G and SD-G plots after therainfall treatment and the CO2 flush lasted for 4 days,which were longer than the studies cited above. It ispossible to assume that the longer CO2 flush in ourexperiment was caused mainly by the multiplication

(and subsequent stabilization) of MB, in addition to theincreased microbial activity, although its reasons arestill unclear as there are few reports on the effect ofdrying/rewetting on tropical sandy soils (Murphy et al.,1998).

Response of soil microbes to rewetting under differentcropping systems in terms of residue management

The MB in the SD-W plot did not increaseappreciably, unlike that in the SJ-W plot. This is oneof the reasons why the CO2 efflux rate in the SD-Wplot did not increase significantly after water addition.As smaller amounts of visible plant residues wereobserved in the SD plots than in the SJ plots, readilydecomposable SOM may also be less in the former inspite of similar total carbon contents in the soils. Suchdifferences in the amount of available substrates, whichwere primarily caused by residue management in therespective farming systems, may have resulted in thedifferences in the response of soil microbes at the Wplots in the present study. The cumulative CO2 effluxin the SJ-G plot after simultaneous addition of waterand glucose reached twice that of the SD-G plot andthe difference was mainly caused by the CO2 effluxrates in the initial 100 h. To demonstrate themultiplication of MB at the W and G plots, the ratiosof MB of the W or G plot to the comparable C plotswere plotted in Figure 7. It is clearly shown that themultiplication of MB in the SJ-G plot was significantlylarger than the SD-G plot. There were small differencesin environmental conditions between plots – that is,the much carbon substrate, the soil temperature andmoisture contents condition, and soil texture. Therefore,the large differences observed for cumulative CO2

efflux between the SJ-G and SD-G plots might becaused by the responses of soil microbial communitiesto substrate addition – namely, some species couldincrease rapidly using substrates added in the SJ plots,unlike in the case of SD. It might be one of the possibleexplanations for this; namely, each farming system in

Figure 6. Fluctuation of microbial activity (CO2 effluxrate per microbial biomass)

Figure 7. The ratio of microbial biomass of the W or Gplot to the comparable C plot

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SJ and SD makes different amounts of crop residues,which were put into the soil, resulting in differentdecomposition dynamics. Another factor, which affectsthe microbial communities, still remains, for examples,climate and soil.

Generally, sandy soils cannot retain SOM insoils compared with clay soils, as sandy soils are moreaerated and SOM is scarcely protected fromdecomposition by being bound in clay-humuscomplexes or sequestered inside soil aggregates (Bradyand Weil, 2002). Actually, most plant residues in SJand SD would be decomposed within 1 or 2 yearsbecause of high temperature, so that there were fewvisible plant residues after 2 years from the lastaddition of plant residue in the SD plots (Funakawaand Shinjo 2005, data not shown). In addition, theturnover rates of MB and SOM in sandy soils and/ortropical regions are higher than in clay soils and/ortemperate regions (Gregorich et al., 1991; Sakamotoand Hodono, 2000; Wardle, 1992). Therefore, it ispossible to assume that the change of microbialcomposition is induced by rapid depletion of SOM(within 1 or 2 years) in the tropical sandy soils. Wetherefore suggest that in tropical sandy soils thefarming histories strongly affect the decompositiondynamics of SOM through inducing different microbialresponses, such as rapid multiplication on rainfallevents. In general, established models for simulatingSOM dynamics – for example, the Century model(Parton et al., 1987) and the Roth-C model (Jenkinsonet al., 1991) – do not take into account the directinfluence of the farming history on the composition ofthe soil microbial community. Further studies areneeded to elucidate the influence of land-use histories,such as past-residue incorporation, on the response ofsoil microbes under fluctuating environments ontropical sandy soils.

Conclusion

The effects of rapid wetting/drying on SOMdynamics lasted for a limited number of days anddepended on both the dynamics of the MB and themicrobial activity in the tropical sandy soils. Themultiplication of MB largely contributed to the initialCO2 flush.

The acceleration of the CO2 flush after additionof substrates and/or water was more pronounced in theSJ plots, which had been receiving higher amounts ofresidue input in recent years. Hence, it is necessary totake account of such historical factors of landmanagement to appropriately simulate SOM dynamicsin tropical sandy soils.

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