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Survival of phytopathogenic bacteria during waste composting
Agriculture, Ecosystems and Environment 96 (2003) 141–146
Survival of phytopathogenic bacteria during waste composting M.A. Elorrieta∗ , F. Suárez-Estrella, M.J. López, M.C. Vargas-Garc´ıa, J. Moreno Unidad de Microbiolog´ıa, Departamento de Biolog´ıa Aplicada, Escuela Politécnica Superior, Universidad de Almer´ıa, 04120 Almer´ıa, Spain Received 6 November 2001; received in revised form 1 August 2002; accepted 19 August 2002
Abstract The survival of phytopathogenic bacteria, Erwinia carotovora subsp. carotovora, Xanthomonas campestris pv. vesicatoria, and Pseudomonas syringae pv. syringae was investigated during a horticultural waste composting process to determine to what extent they were eliminated during the process. The relative influence of temperature, antagonistic compost microorganisms, and phenolic compounds was assessed and compared. Results showed that all phytopathogenic bacteria disappeared in less than 60 h of composting, these bacteria showing low resistance to high temperatures (50, 60 and 70 ◦ C). The highest survival was 1 h at 60 ◦ C for P. s. syringae and 15 min at 70 ◦ C for E. c. carotovora. None survived >15 h at 50 ◦ C. Some culture filtrates of other compost microorganisms and some phenolic compounds also had an inhibitory effect which differed according to the phytopathogenic bacterial species. Composting is therefore considered a useful method for recycling horticulture wastes and eliminating phytopathogenic bacteria. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Composting; Horticultural waste; Temperature; Antagonism; Phenolic compounds; Erwinia carotovora subsp. carotovora; Xanthomonas campestris pv. vesicatoria; Pseudomonas syringae pv. syringae
1. Introduction Composting is a microbial biotransformation process which offers viable solutions for recycling plant waste. The use of the compost material obtained, however, requires an adequate microbiological and physico-chemical quality. The persistence of phytopathogenic microorganisms in crop residues is well known (Conway, 1996). Any composting therefore requires the confirmed capacity of the process to eliminate phytopathogens that might be present in the raw material (Hoitink et al., 1976; Ylimäki et al., 1983; Bollen, 1985). ∗ Corresponding author. Tel.: +34-9500-15892; fax: +34-9500-15476. E-mail address: [email protected] (M.A. Elorrieta).
Many scientists noted that the elimination was mainly due to the high temperatures reached during the process (Ylimäki et al., 1983; Yuen and Raabe, 1984; Bollen, 1993), though additional effects of other microorganisms may occur during the curing phase (Hoitink and Fahy, 1986; Bollen, 1993). Secondary vegetable metabolites like phenolic compounds produced during composting (Trankner, 1992) may also have an effect on phytopathogens (Hoitink and Fahy, 1986; Kai et al., 1990). Horticulture has become one of the principal sources of income in Almer´ıa Province (Spain). This intensive production system has been developed on a relatively small area (about 23,000 ha). The main problems of this system are the generation of an enormous quantity of horticultural waste (around 900,000 t per year cf. Cara and Rivera, 1998), and land exhaustion.
0167-8809/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 8 8 0 9 ( 0 2 ) 0 0 1 7 0 - 6
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M.A. Elorrieta et al. / Agriculture, Ecosystems and Environment 96 (2003) 141–146
Waste recycling through composting maybe a viable alternative if the destruction of any vegetable phytopathogens present in the raw material is guaranteed. The present study was intended to (1) determine the capacity of the composting process to eliminate the phytopathogenic bacteria Erwinia carotovora ssp. carotovora (Skerman et al., 1980), Xanthomonas campestris pv. vesicatoria (Vauterin et al., 1995), and Pseudomonas syringae pv. syringae (Skerman et al., 1980); (2) measure the survival of these bacteria in vegetable residues exposed to constant temperatures of 50, 60 and 70 ◦ C; (3) detect any antagonistic microorganism, likely to biologically control phytopathogenic bacteria in the compost; (4) study phenolic compounds able to affect the viability of the phytopathogenic bacteria.
2. Materials and methods Strains of E. c. carotovora (CECT 225), P. s. syringae (CECT 127), and X. c. campestris (CECT 97) were supplied by the Spanish Culture Type Collection (CECT, Valencia, Spain). Both E. c. carotovora and P. s. syringae were cultured on nutrient agar medium (NA) (Oxoid, Hampshire), X. c. vesicatoria on malt and yeast extract medium (YM: glucose 10 g/l, peptone 5 g/l, malt extract 3 g/l, yeast extract 2 g/l, and agar 20 g/l). The survival of phytopathogenic bacteria during composting process was studied using artificially infected plant wastes introduced into compost piles and regularly analysed. Two composting processes were used, i.e. (1) three composting piles formed by a mixture of pepper, bean, and cucumber wastes; (2) four piles of pepper wastes. In both cases, sawdust was added (1:4 ratio v/v), as conditioner of the relation of C/N. Each pile of 2 m3 was turned over and aerated periodically after the first 14 days of composting. Sterile plant material was inoculated with a suspension of each bacterial strain. The cultures were incubated at 120 rpm for 48 h at 30 ◦ C, then centrifuged at 5000 rpm for 5 min and the sediment was resuspended in Ringer solution 1/4 strength to inoculate flasks with melon seedling stems previously sterilised at 120 ◦ C for 21 min. The inoculated flasks were then incubated for 72 h at 30 ◦ C.
At the beginning of the composting process, all plant material infected was split in equal portions of 20 g placed in muslin bags which were introduced 60 cm deep in the compost piles. Samples of 1 g were taken from the first composting process, every 14 days, during the first 70 days, after 0, 12, 36, 60 and 132 h in the second assay. Three replicates of each pathogen were sampled on every occasion. To detect E. c. carotovora on NA and potato slices the standard plate dilution method was used (Schaad, 1988), YM and the selective medium of McGuire et al. (1986) being used to detect X. c. vesicatoria, NA and King B (Cultimed, Barcelona, Spain) media for P. s. syringae. The incubation time was 2–3 days at 30 ◦ C in all cases, except for X. c. vesicatoria which was maintained for 5–7 days. Identification was made using the biochemical, microscopic and colony characteristics described by Schaad (1988), with the initial CECT strains as control. Results are shown as the number of colony-forming units per 1 gram of compost (CFU/g). Test tubes with plant residues infected as mentioned above were placed into incubators at 50, 60 and 70 ◦ C. Both inoculated and non-inoculated controls were maintained at room temperature. Sampling occurred each 15 h at 50 ◦ C, each 1 h at 60 ◦ C and each 15 min at 70 ◦ C, three replicates being made for each bacteria. The standard dilution plate method was used to isolate bacteria and fungi from mature compost on NA and Rose Bengal (RB) (Oxoid) mediums, respectively. After 2 days of incubation at 30 ◦ C, 20 bacteria colonies were isolated onto fresh NA, 18 fungus colonies on potato dextrose agar (Oxoid) medium after 7 days of incubation. The bacteria were incubated for 2 days on 100 ml of nutrient broth (NB) (Oxoid) at 30 ◦ C, the fungi for 7 days on 100 ml of potato dextrose broth medium at 30 ◦ C. The cultures were filtered through sterile 0.45 mm pore size millipore filters following Landa et al. (1997) method and filtrates were frozen and dried. These filtrates were later put in suspension at 1, 2, 4, 8, and 16 concentrations. These were used to determine the growth inhibition of the phytopathogenic bacteria using the Kirby et al. (1966) technique. Forty millilitres of these concentrated filtrates were added to 0.2 Whattman paper discs placed on NA plates previously sown with 0.1 ml bacterial
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Fig. 1. Time course of temperatures during active composting of (i) first, and (ii) second assay.
culture on NB for 2 days at 30 ◦ C in E. c. carotovora and P. s. syringae case, on YM for X. c. vesicatoria. Inhibition was expressed as the diameter of the inhibition halo divided by the diameter of the control disc. For the control treatment, 40 l of sterile culture broth medium was added to a disc on each plate. Each test was made in three replicates. To detect any inhibitory effect of phenolic compounds, a gentisic, p-coumaric, vanillic, protocatechuic, caffeic, veratric, syringic, 3,4,5-trimethoxybenzoic and ferulic acid dilutions, at 0.1 and 0.01% was prepared using Kirby et al. (1966) technique. The effect was determined by measuring the inhibition halo. Discs inoculated with sterile water were used as controls, and each test was made in triplicate. Statistical analysis used Statgraphics Plus 4.0 software (Manugistics, Rockville, MD). One-way analysis of variance (ANOVA) was performed to compare the pile CFU/g mean values for the different sampling times for each phytopathogenic bacteria, and to test whether there was any significant difference among the means at the 0.95% level. Multiple comparison
Fig. 2. Evolution of colony former units per gram of (i) E. c. carotovora and (ii) X. c. vesicatoria during the second composting assay.
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M.A. Elorrieta et al. / Agriculture, Ecosystems and Environment 96 (2003) 141–146
tests (Fisher’s least significant difference) were used to separate means at P < 0.05. Similar analysis were made to compare the inhibition mean values for the different microbial culture filtrate or phenolic compound concentrations.
3. Results The temperature curves obtained during both composting processes are shown in Fig. 1. The first composting assay showed a total absence of pathogen bacteria in all samples taken. The time to eliminate these bacteria was therefore less than 14 days, during which the piles were neither aerated nor turned over. The results obtained during the second composting
assay are shown in Fig. 2. Only E. c. carotovora was able to maintain itself up to 36 h (Fig. 2(i)), with some differences between piles. In pile D, the bacterium was not detected after 36 h already. In the case of X. c. vesicatoria (Fig. 2(ii)) a significant reduction was detected after 12 h in the four piles, whereas P. s. syringae did not survive at all. No phytopathogenic bacteria was found after 15 h of exposure at 50 ◦ C, and 1 h exposure at 60 ◦ C eliminated E. c. carotovora and X. c. vesicatoria. P. s. syringae maintained about 11 CFU/g after 1 h but no presence was detected after 2 h. At 70 ◦ C, only 3 CFU/g of E. c. carotovora were detected after 15 min of exposure, and none survived 30 min, the other two species being eliminated during the first 15 min. At room temperature, controls maintained the
Fig. 3. Effect of 0.1 and 0.01% concentrations of the phenolic compounds inhibiting (i) E. c. carotovora, (ii) X. c. vesicatoria and (iii) P. s. syringae growth. Relative inhibition expressed as the ratio of the diameter of inhibition halo to the diameter of halo on a control disc (0 = control).
M.A. Elorrieta et al. / Agriculture, Ecosystems and Environment 96 (2003) 141–146
initial levels, in some cases with a slight increase. Negative controls did not show any contamination. The filtrates of six bacterial and nine fungal cultures showed some antibiosis on at least one of the pathogenic bacteria. Only one bacterial filtrate significantly inhibited growth of E. c. carotovora, the effect of different concentrations being significant. The growth of X. c. vesicatoria was inhibited by five filtrates, and that of P. s. syringae by four filtrates. The concentration effect was significant in the majority of them. Six fungi filtrates inhibited E. c. carotovora development. The inhibition produced by one of them was outstanding, which also inhibited X. c. vesicatoria, whereas P. s. syringae was significantly affected by six filtrates. A significant concentration effect was observed for several filtrates. All phenolic compounds tested inhibited E. c. carotovora (Fig. 3(i)), with a significant effect of the concentration in most of them. X. c. vesicatoria was not affected by any phenolic compound except to some extent by trimethoxybenzoic and ferulic acids (Fig. 3(ii)). Most compounds except veratric and protocatechuic acids significantly inhibited P. s. syringae development. There were differences in the degree of inhibition according to the concentration of some of these compounds (Fig. 3(iii)).
4. Discussion The three phytopathogenic bacteria were rapidly eliminated during composting, probably because of the high temperature generated in the piles (Ylimäki et al., 1983; Yuen and Raabe, 1984; Bollen, 1993). This effect was less pronounced in E. c. carotovora, which maintained its initial levels 36 h in piles A and B with the slowest increase in temperature (Fig. 1(ii)). Results obtained “in vitro” also indicated highest survival times of <30 min at 70 ◦ C, <2 h at 60 ◦ C and <15 h at 50 ◦ C. The results corroborated observations made with other pathogens and viruses (Hoitink et al., 1976; Ylimäki et al., 1983; Bollen, 1985; López-Real and Foster, 1985; Avgelis and Manios, 1992). Pseudomonas phaseolicola was inactivated in 4 days at 35 ◦ C (López-Real and Foster, 1985). The recommendations issued by the USEPA (1994) to maintain
145
the residual piles at above 55 ◦ C for at least 5 days, to eliminate these pathogens are therefore perfectly accurate. In addition, the presence of a variety of antagonist microorganisms in the compost piles may add to the physical action on the pathogens (Hoitink et al., 1997). Several phenolic compounds may also contribute to controlling phytopathogenic bacteria. Composting is therefore a useful method for recycling horticultural wastes, making sure that any pathogenic bacteria are eliminated in the process.
Acknowledgements This study was supported by the Comisión Interministerial de Ciencia y Tecnolog´ıa (CICYT), Ministerio de Educación y Ciencia, Project AMB96-1171. References Avgelis, A.D., Manios, V.I., 1992. Elimination of cucumber mottle mosaic tobamovirus by composting infected cucumber residues. Acta Hort. 302, 311–314. Bollen, G.J., 1985. The fate of plant pathogens during composting of crop residues. In: Gasser, J.K.R. (Ed.), Composting of Agricultural and Other Wastes. Elsevier, London, pp. 282–290. Bollen, G.J., 1993. Factors involved in inactivation of plant pathogens during composting of crop residues. In: Hoitink, H.A.J., Keener, H.M. (Eds.), Science and Engineering of Composting: Design, Environmental, Microbiology and Utilization Aspects. Renaissance Publications, Worthington, OH, pp. 301–318. Cara, G., Rivera, J., 1998. Residuos en la Agricultura Intensiva. El caso de Almer´ıa. In: Encuentro Medioambiental Almeriense: En busca de soluciones. Gestión de Residuos, Universidad de Almer´ıa, Almer´ıa, Spain, pp. 128–132. Conway, K.E., 1996. An overview of the influence of sustainable agricultural systems on plant diseases. Crop Prot. 15, 223–228. Hoitink, H.A.J., Fahy, P.C., 1986. Basis for the control of soil-borne plant pathogens with composts. Annu. Rev. Phytopathol. 24, 93–114. Hoitink, H.A.J., Herr, L.J., Schmitthenner, A.F., 1976. Survival of some plant pathogens during composting of hardwood tree bark. Phytopathology 66, 1369–1372. Hoitink, H.A.J., Stone, A.G., Han, D.Y., 1997. Suppression of plant diseases by compost. HortScience 32 (2), 184–187. Kai, H., Ueda, T., Sakaguchi, M., 1990. Antimicrobial activity of bark-compost extracts. Soil Biochem. 22 (7), 983–986. Kirby, W.M.M., Bauer, A.W., Sherrits, J.C., Turck, M., 1966. Antibiotic susceptibility testing by standarized single disc method. Am. J. Clin. Pathol. 45, 493–496.
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Landa, B.B., Hervás, A., Bettiol, W., Jiménez-D´ıaz, R.M., 1997. Antagonistic activity of bacteria from the chickpea rhizosphere against Fusarium oxysporum f.sp. ciceris. Phytoparasitica 25 (4), 305–318. López-Real, J., Foster, M., 1985. Plant pathogen survival during composting of agricultural wastes. In: Gasser, J.K.R. (Ed.), Composting of Agricultural and Other Wastes. Elsevier, London, pp. 291–300. McGuire, R.G., Jones, J.B., Sasser, M., 1986. Tween medium for semiselective isolation of Xanthomonas campestris pv. vesicatoria from soil and plant material. Plant Dis. 70, 887–891. Schaad, N.W. (Ed.), 1988. Laboratory Guide for Identification of Plant Pathogenic Bacteria. APS Press, St. Paul, MN, pp. 81–94. Skerman, V.B.D., McGowan, V., Sneath, P.H.A. (Eds.), 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30, 225–420. Trankner, A., 1992. Use of agricultural and municipal organic wastes to develop suppressiveness to plant pathogens. In:
Tjamos, E.C., Papavizas, G.C., Cook, R.J. (Eds.), Biological Control of Plant Diseases: Progress and Challenges for the Future. NATO ASI Series No. 230. Plenum Press, New York, NY, pp. 35–42. United States Environmental Protection Agency, 1994. A Plain English Guide to the EPA Part 503 Biosolids Rule EPA/832/R-93/003. Office of Wastewater Management, Washington, DC. Vauterin, L., Hoste, B., Kersters, K., Swings, J., 1995. Reclassification of Xanthomonas. Int. J. Syst. Bacteriol. 45, 472–489. Ylimäki, A., Toiviainen, A., Kallio, H., Tikanmäki, E., 1983. Survival of some plant pathogens during industrial-scale composting of wastes from a food processing plant. Ann. Agric. Fenniae 22, 77–85. Yuen, G.Y., Raabe, R.D., 1984. Effects of small-scale aerobic composting on survival of some fungal plant pathogens. Plant Dis. 68, 134–136.
Survival of phytopathogenic bacteria during waste composting M.A. Elorrieta∗ , F. Suárez-Estrella, M.J. López, M.C. Vargas-Garc´ıa, J. Moreno Unidad de Microbiolog´ıa, Departamento de Biolog´ıa Aplicada, Escuela Politécnica Superior, Universidad de Almer´ıa, 04120 Almer´ıa, Spain Received 6 November 2001; received in revised form 1 August 2002; accepted 19 August 2002
Abstract The survival of phytopathogenic bacteria, Erwinia carotovora subsp. carotovora, Xanthomonas campestris pv. vesicatoria, and Pseudomonas syringae pv. syringae was investigated during a horticultural waste composting process to determine to what extent they were eliminated during the process. The relative influence of temperature, antagonistic compost microorganisms, and phenolic compounds was assessed and compared. Results showed that all phytopathogenic bacteria disappeared in less than 60 h of composting, these bacteria showing low resistance to high temperatures (50, 60 and 70 ◦ C). The highest survival was 1 h at 60 ◦ C for P. s. syringae and 15 min at 70 ◦ C for E. c. carotovora. None survived >15 h at 50 ◦ C. Some culture filtrates of other compost microorganisms and some phenolic compounds also had an inhibitory effect which differed according to the phytopathogenic bacterial species. Composting is therefore considered a useful method for recycling horticulture wastes and eliminating phytopathogenic bacteria. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Composting; Horticultural waste; Temperature; Antagonism; Phenolic compounds; Erwinia carotovora subsp. carotovora; Xanthomonas campestris pv. vesicatoria; Pseudomonas syringae pv. syringae
1. Introduction Composting is a microbial biotransformation process which offers viable solutions for recycling plant waste. The use of the compost material obtained, however, requires an adequate microbiological and physico-chemical quality. The persistence of phytopathogenic microorganisms in crop residues is well known (Conway, 1996). Any composting therefore requires the confirmed capacity of the process to eliminate phytopathogens that might be present in the raw material (Hoitink et al., 1976; Ylimäki et al., 1983; Bollen, 1985). ∗ Corresponding author. Tel.: +34-9500-15892; fax: +34-9500-15476. E-mail address: [email protected] (M.A. Elorrieta).
Many scientists noted that the elimination was mainly due to the high temperatures reached during the process (Ylimäki et al., 1983; Yuen and Raabe, 1984; Bollen, 1993), though additional effects of other microorganisms may occur during the curing phase (Hoitink and Fahy, 1986; Bollen, 1993). Secondary vegetable metabolites like phenolic compounds produced during composting (Trankner, 1992) may also have an effect on phytopathogens (Hoitink and Fahy, 1986; Kai et al., 1990). Horticulture has become one of the principal sources of income in Almer´ıa Province (Spain). This intensive production system has been developed on a relatively small area (about 23,000 ha). The main problems of this system are the generation of an enormous quantity of horticultural waste (around 900,000 t per year cf. Cara and Rivera, 1998), and land exhaustion.
0167-8809/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 8 8 0 9 ( 0 2 ) 0 0 1 7 0 - 6
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M.A. Elorrieta et al. / Agriculture, Ecosystems and Environment 96 (2003) 141–146
Waste recycling through composting maybe a viable alternative if the destruction of any vegetable phytopathogens present in the raw material is guaranteed. The present study was intended to (1) determine the capacity of the composting process to eliminate the phytopathogenic bacteria Erwinia carotovora ssp. carotovora (Skerman et al., 1980), Xanthomonas campestris pv. vesicatoria (Vauterin et al., 1995), and Pseudomonas syringae pv. syringae (Skerman et al., 1980); (2) measure the survival of these bacteria in vegetable residues exposed to constant temperatures of 50, 60 and 70 ◦ C; (3) detect any antagonistic microorganism, likely to biologically control phytopathogenic bacteria in the compost; (4) study phenolic compounds able to affect the viability of the phytopathogenic bacteria.
2. Materials and methods Strains of E. c. carotovora (CECT 225), P. s. syringae (CECT 127), and X. c. campestris (CECT 97) were supplied by the Spanish Culture Type Collection (CECT, Valencia, Spain). Both E. c. carotovora and P. s. syringae were cultured on nutrient agar medium (NA) (Oxoid, Hampshire), X. c. vesicatoria on malt and yeast extract medium (YM: glucose 10 g/l, peptone 5 g/l, malt extract 3 g/l, yeast extract 2 g/l, and agar 20 g/l). The survival of phytopathogenic bacteria during composting process was studied using artificially infected plant wastes introduced into compost piles and regularly analysed. Two composting processes were used, i.e. (1) three composting piles formed by a mixture of pepper, bean, and cucumber wastes; (2) four piles of pepper wastes. In both cases, sawdust was added (1:4 ratio v/v), as conditioner of the relation of C/N. Each pile of 2 m3 was turned over and aerated periodically after the first 14 days of composting. Sterile plant material was inoculated with a suspension of each bacterial strain. The cultures were incubated at 120 rpm for 48 h at 30 ◦ C, then centrifuged at 5000 rpm for 5 min and the sediment was resuspended in Ringer solution 1/4 strength to inoculate flasks with melon seedling stems previously sterilised at 120 ◦ C for 21 min. The inoculated flasks were then incubated for 72 h at 30 ◦ C.
At the beginning of the composting process, all plant material infected was split in equal portions of 20 g placed in muslin bags which were introduced 60 cm deep in the compost piles. Samples of 1 g were taken from the first composting process, every 14 days, during the first 70 days, after 0, 12, 36, 60 and 132 h in the second assay. Three replicates of each pathogen were sampled on every occasion. To detect E. c. carotovora on NA and potato slices the standard plate dilution method was used (Schaad, 1988), YM and the selective medium of McGuire et al. (1986) being used to detect X. c. vesicatoria, NA and King B (Cultimed, Barcelona, Spain) media for P. s. syringae. The incubation time was 2–3 days at 30 ◦ C in all cases, except for X. c. vesicatoria which was maintained for 5–7 days. Identification was made using the biochemical, microscopic and colony characteristics described by Schaad (1988), with the initial CECT strains as control. Results are shown as the number of colony-forming units per 1 gram of compost (CFU/g). Test tubes with plant residues infected as mentioned above were placed into incubators at 50, 60 and 70 ◦ C. Both inoculated and non-inoculated controls were maintained at room temperature. Sampling occurred each 15 h at 50 ◦ C, each 1 h at 60 ◦ C and each 15 min at 70 ◦ C, three replicates being made for each bacteria. The standard dilution plate method was used to isolate bacteria and fungi from mature compost on NA and Rose Bengal (RB) (Oxoid) mediums, respectively. After 2 days of incubation at 30 ◦ C, 20 bacteria colonies were isolated onto fresh NA, 18 fungus colonies on potato dextrose agar (Oxoid) medium after 7 days of incubation. The bacteria were incubated for 2 days on 100 ml of nutrient broth (NB) (Oxoid) at 30 ◦ C, the fungi for 7 days on 100 ml of potato dextrose broth medium at 30 ◦ C. The cultures were filtered through sterile 0.45 mm pore size millipore filters following Landa et al. (1997) method and filtrates were frozen and dried. These filtrates were later put in suspension at 1, 2, 4, 8, and 16 concentrations. These were used to determine the growth inhibition of the phytopathogenic bacteria using the Kirby et al. (1966) technique. Forty millilitres of these concentrated filtrates were added to 0.2 Whattman paper discs placed on NA plates previously sown with 0.1 ml bacterial
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Fig. 1. Time course of temperatures during active composting of (i) first, and (ii) second assay.
culture on NB for 2 days at 30 ◦ C in E. c. carotovora and P. s. syringae case, on YM for X. c. vesicatoria. Inhibition was expressed as the diameter of the inhibition halo divided by the diameter of the control disc. For the control treatment, 40 l of sterile culture broth medium was added to a disc on each plate. Each test was made in three replicates. To detect any inhibitory effect of phenolic compounds, a gentisic, p-coumaric, vanillic, protocatechuic, caffeic, veratric, syringic, 3,4,5-trimethoxybenzoic and ferulic acid dilutions, at 0.1 and 0.01% was prepared using Kirby et al. (1966) technique. The effect was determined by measuring the inhibition halo. Discs inoculated with sterile water were used as controls, and each test was made in triplicate. Statistical analysis used Statgraphics Plus 4.0 software (Manugistics, Rockville, MD). One-way analysis of variance (ANOVA) was performed to compare the pile CFU/g mean values for the different sampling times for each phytopathogenic bacteria, and to test whether there was any significant difference among the means at the 0.95% level. Multiple comparison
Fig. 2. Evolution of colony former units per gram of (i) E. c. carotovora and (ii) X. c. vesicatoria during the second composting assay.
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M.A. Elorrieta et al. / Agriculture, Ecosystems and Environment 96 (2003) 141–146
tests (Fisher’s least significant difference) were used to separate means at P < 0.05. Similar analysis were made to compare the inhibition mean values for the different microbial culture filtrate or phenolic compound concentrations.
3. Results The temperature curves obtained during both composting processes are shown in Fig. 1. The first composting assay showed a total absence of pathogen bacteria in all samples taken. The time to eliminate these bacteria was therefore less than 14 days, during which the piles were neither aerated nor turned over. The results obtained during the second composting
assay are shown in Fig. 2. Only E. c. carotovora was able to maintain itself up to 36 h (Fig. 2(i)), with some differences between piles. In pile D, the bacterium was not detected after 36 h already. In the case of X. c. vesicatoria (Fig. 2(ii)) a significant reduction was detected after 12 h in the four piles, whereas P. s. syringae did not survive at all. No phytopathogenic bacteria was found after 15 h of exposure at 50 ◦ C, and 1 h exposure at 60 ◦ C eliminated E. c. carotovora and X. c. vesicatoria. P. s. syringae maintained about 11 CFU/g after 1 h but no presence was detected after 2 h. At 70 ◦ C, only 3 CFU/g of E. c. carotovora were detected after 15 min of exposure, and none survived 30 min, the other two species being eliminated during the first 15 min. At room temperature, controls maintained the
Fig. 3. Effect of 0.1 and 0.01% concentrations of the phenolic compounds inhibiting (i) E. c. carotovora, (ii) X. c. vesicatoria and (iii) P. s. syringae growth. Relative inhibition expressed as the ratio of the diameter of inhibition halo to the diameter of halo on a control disc (0 = control).
M.A. Elorrieta et al. / Agriculture, Ecosystems and Environment 96 (2003) 141–146
initial levels, in some cases with a slight increase. Negative controls did not show any contamination. The filtrates of six bacterial and nine fungal cultures showed some antibiosis on at least one of the pathogenic bacteria. Only one bacterial filtrate significantly inhibited growth of E. c. carotovora, the effect of different concentrations being significant. The growth of X. c. vesicatoria was inhibited by five filtrates, and that of P. s. syringae by four filtrates. The concentration effect was significant in the majority of them. Six fungi filtrates inhibited E. c. carotovora development. The inhibition produced by one of them was outstanding, which also inhibited X. c. vesicatoria, whereas P. s. syringae was significantly affected by six filtrates. A significant concentration effect was observed for several filtrates. All phenolic compounds tested inhibited E. c. carotovora (Fig. 3(i)), with a significant effect of the concentration in most of them. X. c. vesicatoria was not affected by any phenolic compound except to some extent by trimethoxybenzoic and ferulic acids (Fig. 3(ii)). Most compounds except veratric and protocatechuic acids significantly inhibited P. s. syringae development. There were differences in the degree of inhibition according to the concentration of some of these compounds (Fig. 3(iii)).
4. Discussion The three phytopathogenic bacteria were rapidly eliminated during composting, probably because of the high temperature generated in the piles (Ylimäki et al., 1983; Yuen and Raabe, 1984; Bollen, 1993). This effect was less pronounced in E. c. carotovora, which maintained its initial levels 36 h in piles A and B with the slowest increase in temperature (Fig. 1(ii)). Results obtained “in vitro” also indicated highest survival times of <30 min at 70 ◦ C, <2 h at 60 ◦ C and <15 h at 50 ◦ C. The results corroborated observations made with other pathogens and viruses (Hoitink et al., 1976; Ylimäki et al., 1983; Bollen, 1985; López-Real and Foster, 1985; Avgelis and Manios, 1992). Pseudomonas phaseolicola was inactivated in 4 days at 35 ◦ C (López-Real and Foster, 1985). The recommendations issued by the USEPA (1994) to maintain
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the residual piles at above 55 ◦ C for at least 5 days, to eliminate these pathogens are therefore perfectly accurate. In addition, the presence of a variety of antagonist microorganisms in the compost piles may add to the physical action on the pathogens (Hoitink et al., 1997). Several phenolic compounds may also contribute to controlling phytopathogenic bacteria. Composting is therefore a useful method for recycling horticultural wastes, making sure that any pathogenic bacteria are eliminated in the process.
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