INTRODUCTION

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In Japan, the typical method of controlling turf grass disease has been the direct application of fungicides. This management strategy is increasingly viewed as being ecologically undesirable and has led to an increased interest in the development and use of more ecologically sound integrated pest management practices such as biological control. This study describes a reduction in the use of chemical pesticide on a golf course through the use of disease-suppressive compost. Currently, grass clippings from Japanese golf courses are disposed of either by incineration or by landfilling and so are readily available as raw material for compost.

The turf grass disease in this study was Rhizoctonia large patch, caused by Rhizoctonia solani AG2-2 on mascarene grass (Zoysia tenuifola Willd.). This soilborne disease first develops on turf grass near the soil surface and then infects from the hypocotyl to the leaves before appearing as leaf blight.

Numerous studies have been published on the biological control of plant diseases (9, 13, 15, 34) and on the use of composts to reduce disease in agricultural crops (1, 3, 6, 13, 14, 18, 24, 27, 29, 32, 33). Recently, experiments have succeeded in suppressing major soilborne plant pathogens with compost-amended container media (1, 3, 27, 29). Various trials have also reported biological control of turf grass diseases with suppressive soil (35), inoculants (2, 11, 17, 22, 35), and composts containing suppressive microorganisms (4, 13, 23). Studies of disease-suppressive composts have focused mainly on the disease control and on the mechanisms underlying suppressiveness. With the exception of the studies by Phae et al. (25, 26) and Hoitink (12), there have been no reports on the deliberate growth during composting of the necessary suppressive microbes. Phae et al. did not succeed in growing suppressive bacteria inoculated in the course of composting, probably because composting under nonisothermal conditions with high temperatures was unsuitable for bacterial growth or because the lower growth rate of the bacterium used made it unable to effectively compete for nutrients required for growth and proliferation. Hoitink has succeeded in manufacturing suppressive compost from bark with the addition of one or more microorganisms antagonistic to the plant pathogen. This was based on his knowledge that the inoculation of antagonists would be most favorable at 44 weeks after composting (12). However, as he himself points out, this method cannot be applied as it stands, since even after the prescribed 44 weeks, the temperature and degree of organic matter decomposition will differ according to environmental factors, the raw material being composted, and the composting system used. The purpose of this study was to produce a compost capable of consistently suppressing Rhizoctonia large patch on mascarene grass by inoculating the compost with suppressive bacteria and controlling the composting temperatures.

	MATERIALS AND METHODS

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Pathogen. R. solani AG2-2 was isolated from turf grass exhibiting symptoms of Rhizoctonia large-patch disease at a golf course in Shizuoka Prefecture, Japan. The upper parts of the hypocotyl of diseased turf grass were surface sterilized in 1% (vol/vol) sodium hypochlorite for 30 s, rinsed twice in sterile water, and placed on a water agar plate. Mycelium of R. solani AG2-2 was produced on potato dextrose agar (PDA; Eiken) at 25°C for 5 days. The mycelium was scraped from the agar plate and suspended in sterile water (6 log₁₀ CFU/ml) before being used as an inoculum for disease suppression assays.

Determination of disease-suppressive composts and the isolation of bacterial antagonists from the composts. Seven different compost products developed from various raw materials (two from chicken manure, two from cow dung, one from biosolids, one from brewery sludge, and one from food waste, each mixed with a bulking agent such as bark, wood chips, and rice hulls) were screened for their disease-suppressive capabilities. PDA plates for in vitro inhibition of R. solani AG2-2 were inoculated with 100 µl of the mycelial fragment suspension, which was spread over the entire surface of the plate. Then, 100 µl of a compost suspension made by homogenizing 10 g (wet weight) of compost into 90 ml of sterile water was placed on sterile circular filter paper (20-mm diameter) in the center of the PDA plate. The disease suppression assay was established with three replicates and was repeated twice for each compost sample. If, after incubation at 25°C for 5 days, the compost had made a clear inhibitory zone on the PDA plate, it was judged to be suppressive.

1

Raw materials and compost production. Grass clippings collected from golf courses were used as raw material for production of the compost. Compost raw materials were either mixed with a bulking agent or prepared without one. For compost with a bulking agent, grass clippings, matured compost, and sawdust as a bulking agent were mixed at the ratio of 10:1:10 on a dry weight basis. For compost without a bulking agent, grass clippings and matured compost were mixed at the ratio of 10:1. We initially thought it necessary to mix a bulking agent in order to maintain favorable aerobic conditions, but later we ascertained that the aerobic condition could be maintained even without it. Eliminating the bulking agent allowed us to obtain matured compost more easily; thus, we did not include it at the later stage of this study. The grass clippings procured on different days possessed similar moisture contents (ca. 58%) and C/N ratios (ca. 14:1), whereas the population densities of mesophilic bacteria in them varied from 7.2 to 8.2 log₁₀ CFU/g (dry weight). The C/N ratios of the raw materials with and without the bulking agent were 24.9:1 and 12.2:1, respectively.

View larger version (28K): [in this window] [in a new window]   FIG. 1.   Schematic diagram of bench-scale composting system. Components are numbered as follows: 1, flow meter; 2, temperature controller; 3, gas meter; 4, perforated plate; 5, reactor; 6, water bath; 7, thermocouple; 8, ammonia trap; 9, silica gel adsorber; 10, CO2 analyzer; 11, recorder; 12, microcomputer.

Effect of temperature and mesophilic populations on growth of N4-1 in the composting material. B. subtilis N4-1 was added to the raw material containing sawdust to achieve a population density of approximately 8.2 log₁₀ CFU/g (dry weight) of composting material in the minireactor. In three separate experiments, the incubator temperature was set to set points of 60°C (run A-1), 50°C (run A-2), and 40°C (run A-3). These and all subsequent experimental runs were carried out twice to ascertain the reproducibility of the obtained data.

Change in spore density of strain N4-1 during composting. To confirm N4-1's time of sporulation during composting at 40°C, we carried out an additional run at 40°C in the bench-scale reactor. The raw material was free of sawdust, and B. subtilis N4-1 was incorporated at approximately 6 log₁₀ CFU/g (dry weight). Changes in the spore and total population densities were determined over time (total population density was taken as the sum of the vegetative cell and spore densities). In a preliminary experiment, the vegetative cells of N4-1 lacked heat resistance, and only spores survived in compost treated at 60°C for 24 h. The total population density of B. subtilis N4-1 was measured for each compost sample collected in situ, and the spore density was measured after the sample was treated at 60°C for 24 h.

Compostings with constant temperature and incremental temperature change. The sawdust-free raw material and the bench-scale reactor were used. Temperature was controlled in two different ways. One was to maintain a constant temperature of 40°C, the optimal temperature for the growth of N4-1. The raw material was sterilized, the N4-1 was inoculated at around 5 log₁₀ CFU/g (dry weight), and the mixture was heated to 40°C and maintained for 12 days (run C-1). The other method was to change the temperature incrementally from 40 to 60°C. We expected that an incremental change in the compost temperature might not only allow the survival of suppressive bacteria at high densities in the products but also accelerate organic decomposition. The raw material was sterilized and inoculated with N4-1 (ca. 5 log₁₀ CFU/g [dry weight]), and the resulting mixture was heated to 40°C and maintained for 3 days of composting. Next, the temperature was raised rapidly to 60°C within 5 h, where it was maintained for 9 additional days. This composting was designated as run C-2.

Self-heating of compost to reduce microbial population. The reduction of the microbial population in compost raw material was attempted by maintaining a temperature of 80°C, thereby utilizing the self-heating reaction and avoiding the previous sterilization procedure. The sawdust-free raw material and the bench-scale reactor were used. The raw material, not previously sterilized, was heated and maintained at 80°C for 1 day to reduce the density of microorganisms. Then, it was cooled to 23°C and inoculated with 6 log₁₀ CFU/g (dry weight) of B. subtilis N4-1. After being heated to 40°C and maintained at this temperature for 3 days, the mixture was heated to 60°C and maintained for an additional 7 days. This composting was designated as run C-3.

Assessment of the suppressive effect of compost. Six potting mixes in total were prepared by blending various kinds and amounts of compost with the steam-pasteurized soil. One blend was made from a compost that received no inoculum and was produced by maintaining the material at 60°C for 12 days. Another blend was from an autoclaved compost product of run C-3. Both of these kinds of compost were blended with the soil at a 5% loading rate on a dry weight basis. The other mixes were loaded from the compost product of run C-3 at rates of 0, 2, 5, and 10% (dry weight basis). Water was added to all mixes during the hand blending in order to bring the moisture level to 50% (wt/wt). The potting mixes (250 ml) were then distributed into polycarbonate pots (70-mm diameter; 121 mm in height; AGRIPOT-1; Iuchi Co., Ltd.), and 25 surface-sterilized mascarene grass seeds (with a germination rate of ca. 70% on filter paper moistened with distilled water) were planted in each pot. In order to evaluate suppressiveness, eight pots were prepared for each mix. The pots were placed randomly in a growth chamber (BEC-II-350HUP; Shimadzu Rika Kikai Co., Ltd.) and incubated at 25°C for 10 days with continuous illumination (10,000 lx with ordinary fluorescent lamps). Inoculation of the pathogen was delayed for 10 days postseeding in order to avoid immediate infection and the death of the seeds. Ten days after the seedlings were sown, 1 ml of the mycelial fragment suspension adjusted to 6 log₁₀ CFU/ml was added to each seedling's base. After inoculation, the pots were returned to the incubator. The disease severity was rated on a scale from 1 to 5 by calculating a percentage of the number of diseased seedlings (those which showed reddish brown necrosis and rot) over the total number germinated 40 days after sowing, where 1 is asymptomatic turf, 2 is from 1 to 25% of the seedlings being symptomatic, 3 is from 26 to 50% of the seedlings being symptomatic, 4 is from 51 to 75% of the seedlings being symptomatic, and 5 is from 76 to 100% of the seedlings being symptomatic. The disease severity for one potting mix was obtained by averaging the values of disease severity for eight pots. The upper parts of the hypocotyl of diseased seedlings were surface sterilized in 1% (vol/vol) sodium hypochlorite for 30 s, rinsed twice in sterile water, and placed on a water agar plate in order to reisolate the pathogen.

Experimental design and statistical analysis. For all composting experiments, the reproducibility of data was checked by conducting duplicate runs. Microbial population data were analyzed by analysis of variance. Means were separated by a least significant difference (LSD) test. Disease prevention assay experiments were conducted three times, and disease ratings were subjected to the Kruskal-Wallis test, a nonparametric ranking procedure. When significant treatment effects were observed (P  0.05), rankings were subjected to analysis of variance, and mean ranking values were separated by the LSD test.

	RESULTS

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Effect of temperature on growth of strain N4-1 in the composting material. Figure 2 compares changes in the population density of N4-1 and other mesophilic bacteria during composting at different temperatures (runs A-1 to A-3).

View larger version (24K): [in this window] [in a new window]   FIG. 2.   Concentrations of B. subtilis N4-1 and other mesophilic bacteria during composting at 60°C (run A-1), 50°C (run A-2), and 40°C (run A-3). Closed and open circles indicate B. subtilis N4-1 and the other mesophilic bacteria, respectively (n = 3). The error bar (indicating the estimate interval) was too small to be seen in this and subsequent figures. wt, weight.

Effect of mesophilic bacteria on growth of N4-1. Population densities of N4-1 and of other mesophilic bacteria during runs B-1 through B-3 are shown in Fig. 3. Initial population densities of native mesophilic bacteria for runs B-1, B-2, and B-3 were 8.2, 5.5, and less than 2.0 log₁₀ CFU/g (dry weight), respectively. Strain N4-1 was able to grow rapidly and attain a high population density in run B-3, whereas it decreased to a very low level at the early stage of composting in run B-1. The highest population of N4-1 was found in the compost product of run B-3, where the lowest population of mesophilic bacteria other than N4-1 originally existed. Population densities of the other mesophilic bacteria for both runs B-1 and B-2 rose to around 10 log₁₀ CFU/g (dry weight) and were greater than those in run B-3. In run B-3, the population density of other mesophilic bacteria was hardly detectable in the initial stage of composting, since at that time N4-1 existed in much greater abundance than the other mesophilic bacteria.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Population densities of B. subtilis N4-1 and other mesophilic bacteria during composting with population densities of mesophilic bacteria in the raw material of 8.2 (run B-1), 5.5 (run B-2), and less than 2.0 (run B-3) log10 CFU/g (dry weight). Closed and open circles indicate B. subtilis N4-1 and the other mesophilic bacteria, respectively (n = 3). wt, weight.

Spore formation by strain N4-1 during composting at 40°C. Figure 4 shows changes in the total population density of N4-1, a sum of densities related to spores and vegetative cells, and the spore density of N4-1 in the course of composting at 40°C. The total population density of N4-1 increased rapidly in the first 2 days of composting and then remained almost constant. Spores were not observed in inoculum examined by microscopy. The spore density of 0 immediately after the start of composting increased gradually in the first day and then remained constant after 3 days, much like the total population density.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Total (sum of vegetative cells and spores) and spore population densities of B. subtilis N4-1 during composting at 40°C. Circles and squares indicate total and spore populations of N4-1, respectively (n = 3). wt, weight.

Compostings with constant temperature and incremental temperature change. Figure 5A shows changes over time in population densities of strain N4-1 and other mesophilic bacteria in runs C-1 and C-2. In both experimental runs, N4-1 grew to nearly 10 log₁₀ CFU/g (dry weight) after 3 days, and the population size remained almost constant thereafter. In run C-2, the temperature was increased to 60°C after 3 days, but the population density of strain N4-1 did not decrease. Since N4-1 had formed spores by this time (cf. Fig. 4), a high concentration was also maintained thereafter.

View larger version (20K): [in this window] [in a new window]   FIG. 5.   (A) Population densities of B. subtilis N4-1 and other mesophilic bacteria during composting at 40°C (constant) (run C-1) and incremental changes of 40 to 60°C (run C-2). Closed and open circles indicate B. subtilis N4-1 and the other mesophilic bacteria, respectively (n = 3). wt, weight. (B) Temperature and conversion of carbon during runs C-1 (dotted lines) and C-2 (solid lines).

Compost self-heating to reduce the microbial population. Figure 6 shows temperature changes over time in run C-3 and the population densities of strain N4-1 and other mesophilic bacteria. The other mesophilic bacteria first increased as the temperature rose but died at 80°C and were reduced by 2 orders of magnitude to around 5 log₁₀ CFU/g (dry weight). After that, when the temperature was maintained at 40°C for 3 days, the other mesophilic bacteria again increased to a population density of 9 log₁₀ CFU/g (dry weight) or higher. On the other hand, N4-1 inoculated at a population density of around 6 log₁₀ CFU/g (dry weight) after heating to 80°C increased to ca. 8 log₁₀ CFU/g (dry weight) and then remained constant.

View larger version (23K): [in this window] [in a new window]   FIG. 6.   Temperature and population densities of B. subtilis N4-1 and other mesophilic bacteria during run C-3 (reduction of microbial population in the compost raw material by self-heating). Closed and open circles indicate B. subtilis N4-1 and the other mesophilic bacteria, respectively (n = 3). wt, weight.

Assessment of the suppressive effect of the compost on turf grass pathogens. Figure 7 shows the results of the turf grass disease prevention tests. Adding compost from run C-3 to the soil resulted in the suppression of Rhizoctonia large patch in turf grass, and maximum disease suppression was obtained when compost comprised 5% of the total potting mixture. By contrast, the compost that received no inoculum, or the autoclaved compost of run C-3, showed no suppressive effect on Rhizoctonia disease. In addition, only R. solani AG2-2 was detected in the diseased seedlings, indicating that disease symptoms were due to the introduced pathogen. These results indicate that inoculated B. subtilis N4-1 was indeed a factor in the suppression of the disease.

View larger version (64K): [in this window] [in a new window]   FIG. 7.   Suppression of Rhizoctonia patch disease of mascarene grass with a soil-compost mixture. The abscissa represents six different potting mixes prepared by blending various kinds and amounts of compost: a compost that received no inoculum (N); an autoclaved compost of run C-3 (A); and the compost of run C-3 with four different loading rates, 0, 2, 5, and 10%. Disease severity was rated 40 days after sowing on a scale of 1 to 5, where 1 is asymptomatic turf and 5 is 100% necrotic and rotting seedlings. (Scale details are given in the text.) Ratings represent the means of three separate bioassays. Means with the same letter are not significantly different (P = 0.05), based on the analysis of variance and LSD test of mean Kruskal-Wallis ranking values (Kruskal-Wallis statistic H = 14.2; P < 0.05).

	DISCUSSION

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This study attempted production of a more consistently suppressive compost by inoculation with a suppressive bacterium. While many researchers have studied microbial inoculants for composting of wastes, especially for minimizing and eliminating the lag time typically observed early in the process, negative effects have frequently been observed (5, 7, 8, 10, 20) because the microorganisms added as inocula were not more effective than those indigenous to the refuse. Thus, seeding or inoculation was effective only in the early stages of the process for raw materials containing indigenous microorganisms at low population density (21) and for some special composting applications in which a raw material of relatively homogeneous composition, such as straw or wood, was used (19, 36).

The results of this study show that, in order to foster high concentrations of disease-suppressive bacteria in compost, the concentration of other bacteria originally present in the raw material should be reduced (Fig. 3). However, the higher cost of reducing the population density of other bacteria by prior treatment of the compost raw material could render the method impractical. Thus, we made use of the self-heating reaction in the initial stage of composting. As the reactor here was small, the 80°C level was achieved in a water bath, though in large-scale composting favorably decomposing organic matter will produce a high temperature of about 80°C spontaneously.

The method utilized in this study was to inoculate the suppressive bacterium in the initial stage of composting, immediately after heating to 80°C, when the concentration of the other mesophilic bacteria was at its lowest. After that, a low temperature of 40°C was maintained till the suppressive bacteria grew sufficiently to form spores. This method, which is characterized by microbe inoculation in combination with temperature control, may be applicable to a variety of composting systems. When the suppressive bacterium was inoculated in this study, residual organic matter was still abundant in the compost; it was therefore absolutely necessary to control the temperature, as it would otherwise have become too high. The method advocated by Suler and Finstein (30), by Kuter et al. (16), and by Stentiford (28) for regulating aeration rate can be used as a simple way to control composting temperatures in actual practice. This method has already been practically applied to high-rate composting for the purpose of controlling temperatures without exceeding the optimal level.

In another method for producing suppressive compost, suppressive microbes were inoculated not into the compost raw material but into the final product (26). Phae and Shoda have confirmed that a very high suppressive population can be maintained by inoculation at around 10 log₁₀ CFU/g (dry weight) or after sterilization of the product with gamma radiation, although they were not successful in growing the inoculated bacterium in the compost (26). The method of composting shown in this study is superior to that of inoculation into the product, because it allows growth of the suppressive bacterium to a concentration more than 2 orders of magnitude greater than that upon inoculation.

The reason why the disease-suppressing effect was higher for 5% compost-loaded soil than for 10% compost-loaded soil is not known. One possible explanation is that this compost product might not have been fully matured. Each of the tests revealed a germination rate of ca. 70%, equivalent to that on water-moistened filter paper, indicating that this compost product was not germination inhibitory. However, it might have had a negative influence on the growth of turf grass itself at a loading rate of 10%. Another possible explanation is that the pathogen was activated by the compost.

Ultimately, it will be necessary to test the compost product's effectiveness and optimum application rate in the field and on golf courses where the turf grass is grown and mown.