Predictive Factors for the Suppression of Fusarium Wilt of Tomato in Plant Growth Media

Borrero, C., 1, Phytopathology Predictive Factors for the Suppression of Fusarium Wilt of Tomato in Plant Growth Media Celia Borrero, Mª Isabel Trillas, José Ordovás, Julio C. Tello, Manuel Avilés First, third and fifth authors: Dept. Ciencias Agroforestales, Universidad de Sevilla, Ctra. Utrera, Km. 1. 41013 Sevilla, Spain. Second author: Dept. Biología Vegetal, Universitat de Barcelona, Avda Diagonal, 645, 08028 Barcelona, Spain. Fourth author: Dept. Producción Vegetal, Universidad de Almería, Ctra. Sacramento, s/n, 04120 La Cañada de San Urbano, Almería, Spain. Corresponding author: [email protected] Borrero, C., 2, Phytopathology ABSTRACT Fusarium wilts are economically important diseases for which there are no effective chemical control measures. However, biological control and fertility management are becoming efficient alternatives for controlling this disease. Growth media formulated with composts that are able to suppress Fusarium wilt of tomato provide a control system that integrates both strategies. The aim of this study was to predict Fusarium wilt suppression of growth media using abiotic and biotic variables. Grape marc compost was the most effective medium used to suppress Fusarium wilt. Cork compost was intermediate, and light peat and expanded vermiculite were the most conducive growth media. The growth media evaluated were in a pH range of 6.26 to 7.97. Both composts had high β-glucosidase activity. When pH and β-glucosidase activity were taken into account as predictive variables, more than 91% of the variation in severity of Fusarium wilt was explained. This relationship illustrates the effect of nutrient availability and the degree of microbiostasis, two key factors in this pathosystem. Microbial populations involved in suppressiveness were cellulolytic and oligotrophic actinomycetes, fungi, and the ratios cellulolytic actinomycetes/cellulolytic bacteria, oligotrophic bacteria/copiotrophic bacteria, and oligotrophic actinomycetes/oligotrophic bacteria. Based on community level physiological profiles, different community structures were evident among growth media evaluated. Keywords: Fusarium oxysporum f. sp. lycopersici, Lycopersicon esculentum, microbial activity, pH, community level physiological profiles, Biolog, biological control Borrero, C., 3, Phytopathology Fusarium wilt diseases, caused by pathogenic formae speciales of the soil-inhabiting fungus Fusarium oxysporum (Sacc.) Snd. & Hans., cause severe losses in a wide variety of crop plants including tomato (Lycopersicon esculentum Mill.). This pathogen occurs throughout most tomato growing areas and devastates the crop. Fusarium-resistant cultivars have been available for decades and provide some degree of control; however, the occurrence and development of new pathogenic races is a continuing problem. There are no commercially acceptable tomato cultivars with adequate resistance (28). In addition, the resurgent interest in planting heirloom and other susceptible tomato cultivars has increased the incidence of Fusarium wilt. Effective soil fungicide treatments for this disease are also unavailable. Difficulties in controlling Fusarium wilt have stimulated the search for biocontrol systems. The nature of soils and composts that are suppressive to Fusarium wilt is the result of complex interactions between the abiotic characteristics of the media and microbial populations (1, 2, 21, 60). Numerous biocontrol agents have been identified in these soils and composts (21, 60). Unfortunately, the introduction of biocontrol agents has provided inconsistent results (59). Growth media formulated with composts such as hardwood bark (9, 55), pine bark (8, 42, 44), poplar bark (16), wood shavings (8), cork (54), grape marc (54), olive pumice (44), cattle manure (45), sewage sludges (11), and vermicompost (16, 51, 52) can suppress Fusarium wilt. Several of these have been used successfully for consistent biological control, particularly for containerized crops (21, 22). The microbiological nature of Fusarium wilt-suppressiveness for most of these compostamended growth media has been demonstrated (8, 9, 11, 16, 22, 42, 43, 45, 47, 51, 54, 55). The role of nutritional factors in wilt-suppressiveness has also been suggested (11, 22, 42, 55). For some composts, it has been demonstrated that fungitoxic substances are not involved (43, 45, 47, 51). Borrero, C., 4, Phytopathology The main purpose of this study was to determine the relationship between biotic and abiotic properties of growth media and the severity of Fusarium wilt in order to predict disease suppressiveness of growth media. In addition, nutrient availability, nutrient status of plants and microbial diversity were evaluated at the conclusion of the experiments in order to understand possible mechanisms involved in the suppressive phenomena. For this purpose, two Fusarium suppressive composts, which can be used as plant growth media, were compared with two standard growth media. MATERIALS AND METHODS Plant growth media. Two residues from agricultural and industrial wastes were composted and evaluated for Fusarium wilt suppression: cork compost (CC) from cork (Quercus suber L.) production and grape marc compost (GMC) from the alcohol-producing industry (grape skins, seeds and stems). Both wastes were composted as described elsewhere (54). Composts were compared to Sphagnum light peat (P) (Klasmann, Valinex, Palleter, Spain) and expanded vermiculite (V) (Vermiculita y derivadas, Gijón). Peat was neutralized with 4 g per liter CaCO3. To determine the role of microflora in suppression, the two composts also were heated to 60ºC for 6 days, (CC-60, GMC-60). Assessment of disease severity. Disease suppressive properties of plant growth media were measured by a Fusarium wilt bioassay described elsewhere (11). To standardize initial conditions, the growth media were incubated at a water tension of 1000 Pa (adjusted for weight) for 14 days at 25ºC. Liquid culture, 500 ml 1% malt extract (Sigma Chemical Company, St Louis, MO), of Fusarium oxysporum f. sp. lycopersici race 2, isolate RAF 70 obtained from an infected tomato plants (FOL-2) was prepared and grown with continuous Borrero, C., 5, Phytopathology agitation (130 rpm) for 10 days at 25ºC. Conidia were recovered by centrifugation at 5,000 rpm, for 15 min, (Selecta Medifriger BL-S, Abrera, Spain) and rinsed twice with sterile distilled water. The concentration of conidia was determined with a hemocytometer. The six plant growth media were infested with FOL-2 (105 microconidia/cm3 plant growth medium), mixed vigorously and poured into 9-cm-diam. plastic pots (330-cm3 volume). Pots of growth media without FOL-2 were prepared as controls. Four tomato seedlings (1 to 2 true leaf stage) grown in sterile vermiculite were transplanted into each pot. Plants were irrigated as needed and fertilized with a nutrient solution containing: 0.5 g/liter Peter’s foliar feed 27-15-12 (Scotts, Heerlen, The Netherlands), 0.6 g/liter CaCl2 and 0.696 g/liter MgSO4.7H2O (pH 5.68). Plants were grown in a growth chamber (25ºC, 280 μE/m2/s PAR intensity, 16:8 h light: dark photoperiod and 60% RH). Bioassays were repeated at least three times with four plants per pot and five pots per treatment. Treatments were arranged in a randomized block design. Disease severity was monitored at 2-day intervals for 25 days after planting, and was scored based on a symptom severity scale where: 0 = asymptomatic plants; 1 = weakly infected plants (≤ 50% of leaves chlorotic or wilted); 2 = highly infected plants (> 50% of leaves wilted but plants not dead) and 3 = dead plants (11). Disease severity was expressed as proportion of the maximum possible disease severity. At each disease assessment, the mean of disease severity per pot was calculated. The area-under-the-disease-progress-curve-standardized (AUDPCs) per pot was calculated by disease severity integrated between symptoms onset and bioassay final time and dividing by the total duration (days) of the epidemic in each bioassay, in order to compare the various bioassays, which had a variety of epidemic durations. At the end of the bioassays, mean values per pot (four plants) of the relative-length-of-the-stem-with-brown- Borrero, C., 6, Phytopathology xylem (RLSBX), and the fresh and dry weight of leaves and stems means per pot were recorded. These values per pot were considered as replicates for each observed variable. Physical and physico-chemical properties of growth media. The bulk density (BD) for the plant growth media was determined according to De Boot et al. (13). Electrical conductivity (EC) and pH of media were measured in a water extract (2:1; vol /vol), as described by Bunt (6) and Gabriëls et al. (15), respectively. Physical and chemical variables of the growth media were determined at the beginning of the bioassays. The pH also was measured at the end of each bioassay. Three samples were analyzed for each growth medium. Nutrient availability and nutrient status of plants. Plant growth media samples (50 g) were collected from the pots and extracted with ammonium acetate 0.5 M (pH 4.65). Plant samples (0.25 g dry weight) from all treatments were incinerated at 550ºC for 8 h and dissolved in 20 ml 1N HCl, filtered as necessary and stored at 4ºC until analysis. Calcium, magnesium, iron, copper, manganese and zinc were determined by atomic absorption spectroscopy (63) and sodium and potassium were analyzed by atomic emission spectrometry (63). Phosphorous was measured with colorimetry (37), nitrate (plant growth media) with reflectometry (RQ flex Merck, Darmstadt, Germany) (50) and boron (plants) with colorimetry (31). Total nitrogen in plants was measured with Kjeldahl digestion, modified to include nitrates, and ammonium was subsequently determined with colorimetry (36). All analyses were performed with three samples for plants and two samples of plant growth media from one of the three bioassays, at the end of the experiment. Borrero, C., 7, Phytopathology Biological characteristics of growth media. All determinations were performed in incubated growth media at the beginning of bioassays, except for microbial diversity. Microbial biomass. Three techniques were used to assess microbial biomass: i) phosphates from phospholipids, (10) with digestion as described by Van Veldhoven and Mannaerts (57) and phosphate determination with colorimetry (37); ii) fumigation-extraction, based on the estimation of cell carbon by extraction with saline solution of fumigated and nonfumigated samples and their conversion to biomass (46); and iii) acridine orange direct counting (AODC) technique which was performed by counting cells from an aqueous extraction with an epifluorescence microscope (30). The aqueous extraction was filtered through 0.2 μm pore-size black polycarbonate membranes (Isopore, Millipore Iberica S.A., Madrid, Spain). Counting was performed at 1,250 x with a minimum of 400 cells per filter. Controls were counted also in sterile water. Three samples were analyzed for each plant growth medium except for those that were heated. Microbial activity. Microbial activity was estimated by measuring β-glucosidase activity. This method is based on colorimetric determination of the p-nitrophenol released by βglucosidase when the plant growth medium was incubated with p-nitrophenil-β-D- glucoside (pH 6.0). The p-nitrophenol released was extracted by filtration and determined colorimetrically. β-glucosidase activity was measured according to Bandick and Dick (4). Two samples were analyzed for each plant growth medium. Microbial diversity. The density of cultivable groups of bacteria and fungi associated with biocontrol phenomena was determined by dilution plating on semi-selective media according to Tuitert et al. (56) with modifications. Samples were taken from the rhizosphere at the end of bioassays. Plant growth media (5 to10 g) were suspended in 250 Borrero, C., 8, Phytopathology ml of 0.1% sodium pyrophosphate. The suspension was shaken and tenfold dilution series were prepared with 0.1% water agar. Suspensions were pipetted onto three plates per culture medium and dilution. Four or five dilutions per series were placed on plates. For isolation of copiotrophic bacteria, Bacillus spp., fluorescent Pseudomonas spp., oligotrophic and cellulolytic bacteria and actinomycetes, 100 μg of cycloheximide was substituted for 10 μg of benomyl per ml (Energía e Industrias Aragonesas, S.A., Madrid) and 0.3 μl of Previcur (Propamocarb, 72.2%, Schering, Alcácer, Spain) per ml. Fungal counts were made on potato dextrose agar amended with 1,000 ppm of Tergitol-7 (Fluka Chemie AGB, Buchs, Switzerland) and 50 μg of oxytetracycline hydrochloride (Sigma Chemical Company, St Louis, MO) (10). Data for population density was normalized by natural log (Ln) transformations. Analyses were performed twice with one sample per each control and each infested plant growth medium from each of the two bioassays. Community level physiological profiles (CLPPs). Eco-microplates (Biolog Inc., Hayward, CA) were used to determine differences in physiological abilities of the microbial communities from different plant growth media and the similarities between these communities and the pathogen. The microplates contained 96 wells with 31 carbon sources plus control, with three replicates each, as has been proposed for community characterization in environmental samples (25). The growth media extraction method was performed according to Insam et al. (26). Population cell density was determined by AODC to standardize the inoculum density. The density of FOL-2 conidia in suspension was determined by hemocytometer. Five eco-microplates were inoculated for plant growth medium suspensions and three for FOL-2 conidial suspension. The microplates were incubated in darkness at 25ºC for 5 days. Color formation was measured with a microplate reader (Labsystem Multiskan MS, Helsinki, Finland) at 590 nm and readings were made at Borrero, C., 9, Phytopathology 12-h intervals. For the analysis, the average absorbance of the control wells in each plate was subtracted from that of wells containing the same C-source. Negative values were considered as zero. Microbial community diversity was calculated with these data using Shannon (48) and Gini (19) indices to compare functional diversity of microbial communities. For further analysis of Biolog data, a Riemann’s sum for each C-source was calculated from all readings taken during the 5 days of incubation (49). The transformed data were subjected to principal component analysis (PCA) (17), and distances in Euclidean space from the first five principal components were calculated (24). Statistical analysis. Data collected from all trials were analyzed with Statgraphics Plus, Version 6 (SGS, 1999). The effect of growth medium on AUDPCs, pH, EC, bulk density, nutrient availability, nutrient status in plants and biological characteristics of container medium were analyzed with ANOVA. Significant means were compared by Tukey’s whole significant difference test (P = 0.05). Overall relationships between AUDPCs and continuous measured variables were analyzed with regression analysis. RESULTS Suppressiveness of plant growth media. Fusarium wilt was suppressed most effectively with GMC, with more than a 92% reduction in AUDPCs and RLSBX than in peat or vermiculite. Media CC, GMC-60 and CC-60 were intermediate and P and V were the most conducive growth media (Fig. 1A). The heated composts had reduced suppressive ability; however, GMC-60 maintained moderate levels of suppressiveness compared with P and V (Fig. 1A, B). Medium CC-60 was not different from P for disease severity measured by RLSBX (Fig 1B). At an inoculum concentration of 1x104 microconidia / cm3 plant growth Borrero, C., 16, Phytopathology Pyrenochaeta lycopersici. Other strong negative correlations related to suppressive phenomena found in this study involved oligotrophic actinomycetes, oligotrophic actinomycetes / oligotrophic bacteria and oligotrophic bacteria / copiotrophic bacteria, Tuitert et al. (56) found higher oligotrophic populations in growth media that were suppressive of, rather than conducive to, R. solani. Ohta and Hatory (41) suggested that oligotroph life is associated with cellulose decomposers on organic debris. Our results regarding the negative correlation of fungi populations and AUDPCs are also in agreement with those for organic household waste composts suppressive of R. solani (56). On the other hand, Kuter et al. (32) found no differences in total numbers of fungi for hardwood bark composts that were suppressive and non-suppressive of Rhizoctonia damping-off; however, there were quantitative differences in the relative abundance of certain taxa which separated suppressive from conducive media amended with hardwood bark compost. The high population densities of Bacillus spp. associated with the two composts, either natural or heated (Table 7), may account for the suppressiveness in our composts. Similarly in a study on potting mix amended with chicken manure, high populations densities of Bacillus spp. were associated with the survival of seedlings infested with Phytophthora cinnamomi (3). The high population densities of fluorescent Pseudomonas spp. in CC (controls) may be involved in the suppressiveness of this material (2, 14). The community structures of the plant growth media studied were different at the beginning of the bioassays, based on community level physiological profiles, the similar catabolic potential of CC microbial communities and FOL-2, together with the lower diversity index and the higher β-glucosidase activity and AOCD microbial biomass, suggest a high competition for C-sources. These results, along with the low level of disease suppression of heated CC, suggest that control is biological, and basically concerns competition between Borrero, C., 17, Phytopathology F. oxysporum and endogenous microbial communities. Although the high biological diversity found in vermiculite is noteworthy, its very low microbial activity and biomass should not be overlooked. The high diversity found for this inorganic material can be explained by the fact that vermiculite is almost totally sterile prior to use. The primary source of microorganisms in this medium is probably handling processes, although aerial transmission may also play a role (7). Higher diversity cannot explain disease control in this study, as evidenced by the Shannon diversity index between the conducive peat and the suppressive grape marc compost and the lower 1-G and Shannon index of the suppressive cork compost with respect to peat. Thus, our results are in agreement with those of Boehm et al. (5) for control of Pythium root rot, but not with those of Nitta (39) or Toyota et al. (53), where lower diversity was associated with conduciveness to brown stem rot of bean and Fusarium wilt of radish, respectively. In conclusion, the strong relationship found at the beginning of the bioassays for AUDPCs with pH of growth media and β-glucosidase activity of microbial populations supports its predictive value. The pH reflected the availability of inorganic nutrients, in this multiple correlation. For Fusarium wilt control, the importance of availability of specific nutrients is demonstrated by fertility management (27) and by the biocontrol induced for Fe competition by fluorescent Pseudomonas (2, 14). Based on our results, microbial competition (measured as β-glucosidase activity), especially for the C-source present in the media and root exudates (33, 38, 60), plays a significant role in disease suppression. The microbial community acts as a nutrient sink that is independent of the microbial community structure in this study. Future research may provide additional information that would allow Borrero, C., 18, Phytopathology us to generalize the relationships between pH and β-glucosidase activity as predictive variables for the suppressiveness of other growth media and other Fusarium wilt diseases. ACKNOWLEDGEMENTS This research was supported by grant IFD97-1322-C04 from the Comisión Interministerial de Ciencia y Tecnología (Proyectos I+D del Programa FEDER) and by grant AGL2002- 04313-C03 from the Ministerio de Ciencia y Tecnología (Proyectos I+D del Plant Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica) of Spain. We thank S. Castillo, M. C. Mauri and M .J. Infantes for excellent technical assistance and Dr. H. A. J. Hoitink for kindly reviewing the manuscript. LITERATURE CITED 1. Alabouvette, C. 1986. Fusarium-wilt suppressive soils from the Châteaurenard region: review of a 10-year study. Agronomie 6:273-284. 2. Alabouvette, C., Hoeper, H., Lemanceau, P., and Steinberg, C. 1996. Soil suppressiveness to diseases induced by soilborne plant pathogens. Pages 371-413 in: Soil Biochemistry, Vol 9. G. Stotzky, and J. M. Bollag, eds. Marcel Dekker, Inc, New York. 3. Aryantha, I. P., Cross, R., and Guest, D. I. 2000. Suppression of Phytophthora cinnamomi in potting mixes amended with uncomposted and composted animal manures. Phytopathology 90:775-782. 4. Bandick, A. K., and Dick, R. P. 1999. Field management effects on soil enzyme activities. Soil Biol. Biochem. 31:1471-1479. 5. Boehm, M. J., Wu, T., Stone, A. G., Kraakman, B., Iannotti, D. A., Wilson, G. E., Madden, L. V., and Hoitink, H. A. J. 1996. Cross-Polarized magic-angle spinning 13C nuclear magnetic resonance spectroscopic characterization of soil organic matter relative to culturable bacterial species composition and sustained biological control of Pythium root rot. Appl. Environ. Microbiol. 63:162-168. Borrero, C., 19, Phytopathology 6. Bunt, A. C. 1988. Media and Mixes for Container-Grown Plants: a Manual on the Preparation and Use of Growing Media for Pot Plants. 2nd ed. Academic Division of Unwin Hyman Ltd. London. 7. Carlile, W. R., and Wilson, D. P. 1991. Microbial activity in growing media - A brief review. Acta Horticult. 294:197-206. 8. Cebolla, V., and Pera, J. 1983. Suppressive effects of certain soils and substrates against Fusarium wilt of carnation. Acta Horticult. 150:113-119. 9. Chef, D. G., Hoitink, H. A. J., and Madden, L. V. 1983. Effects of organic components in container media on suppression of Fusariurn wilt of chrysanthemum and flax. Phytopathology 73:279-281. 10. Chen, W., Hoitink, H. A. J, Schmitthenner, A. F., and Tuovinen, O. H. 1988. The role of microbial activity in suppression of damping-off caused by Pythium ultimum. Phytopathology 78:314-322. 11. Cotxarrera, L., Trillas-Gay, M. I., Steinberg, C., and Alabouvette, C. 2002. Use of sewage sludge compost and Trichoderma asperellum isolates to suppress Fusarium wilt of tomato. Soil Biol. Biochem. 34:467-476. 12. Davet, P., Messiaen, C. M., and Rieuf, P. 1996. Interpretation des manifestations hivernales de la fusariose de la tomate en Afrique du nord, favorisées par la presence de sels dan les eaux d’irrigation. Proc. First Cong. Mediterr. Phytopathol. Union: 407-416. 13. De Boodt, M., Verdonk, O., and Cappaert, I. 1974. Method for measuring the water release curve of organic substrates. Acta Horticult. 37:2054-2062. 14. Duijff, B. J., Meijer, J. W., Bakker, P. A. H. M., and Schippers, B.1993 Siderophore-mediated competition for iron and induced resistance in the suppression of Fusarium wilt of carnation by fluorescent Pseudomonas spp. Neth. J. Pl. Path. 99:277-289. 15. Gabriëls, R., Van Keirsbulck, W., and Verdonck, O. 1991. Reference method for physical and chemical characterization of growing media: an international comparative study. Acta Horticult. 294:147-160. 16. Garibaldi, A. 1988. Research on substrates suppressive to Fusarium oxysporum and Rhizoctonia solani. Acta Horticult. 221:271-277. 17. Glimm, E., Heuer, H., Engelen, B., Smalla, K., and Backhaus, H. 1997. Statistical comparisons of community catabolic profiles. J. Microbiol. Meth. 30:71-80. 18. Handreck, K. and Black, N. 1991. Growing Media for Ornamental Plants and Turf. New South Wales University Press, Kensington, Australia. Borrero, C., 20, Phytopathology 19. Harch, B. D., Correll, R. L., Meech, W., Kirkby, C. A., and Pankhurst, C. E. 1997. Using the Gini coefficient with BIOLOG substrate utilisation data to provide an alternative quantitative measure for comparing bacterial soil communities. J. Microbiol. Meth. 30:91-101. 20. Hoitink, H. A. J., and Boehm, M. J. 1999. Biocontrol within the context of soil microbial communities: a substrate-dependent phenomenon. Annu. Rev. Phytopathol. 37:427-446. 21. Hoitink, H. A. J., Boehm, M. J., and Hadar, Y. 1993. Mechanisms of suppression of soilbome plant pathogens in compost-amended substrates. Pages 601-621 in: Science and Engineering of Composting: Design, Environmental, Microbiological and Utilization Aspects. H. A. J. Hoitink, and H. M. Keener, eds. Renaissance Publications, Worthington, OH. 22. Hoitink, H. A. J., Inbar, Y., and Boehm, M. J. 1991. Status of compost-amended potting mixes naturally suppressive to soilborne diseases of floricultural crops. Plant Dis. 75:869-873. 23. Hoitink, H. A. J., Madden, L. V., and Boehm, M. J. 1996. Relationships among organic matter decomposition level, microbial species diversity and soilborne disease severity. Pages 237-249 in: Principles and practice of managing soilborne plant pathogens. R. Hall, ed. A.P.S. Press, St. Paul, MN. 24. Howard, P. J. A. 1999. Analysis of inter-sample distances from BIOLOG plate data in euclidean and simplex spaces. Soil Biol. Biochem. 31:1323-1330. 25. Insam, H. 1997. A new set of substrates proposed for community characterization in environmental samples. Page 259 in: Microbial Communities. Functional Versus Structural Approaches. H. Insam, and A. Rangger, eds. Springer, Heidelberg Germany. 26. Insam, H., Amor, K., Renner, M., and Crepaz, C. 1996. Changes in functional abilities of the microbial community during composting of manure. Microbial Ecol. 31:77-87. 27. Jones J. P., Engelhard A. W., and Woltz S. S. 1993. Management of Fusarium wilt of vegetables and ornamentals by macro-and microelement nutrition. Pages 18-32 in: Soilborne Plant Pathogens: Management of diseases with macro-and microelements. W. A. Engelhard, ed. American Phytopathological Society, St Paul, MN. 28. Jones, J. B., Jones, J. P., Stall, R. E., and Zitter, T. A., eds. 1991. Compendium of tomato diseases. American Phytopathological Society, St. Paul, MN. 29. Jones, J. P., and Woltz, S. S. 1981. Fusarium-incited diseases of tomato and potato and their control. Pages 157-168 in: Fusarium: Diseases, Biology and Taxonomy. P. E. Nelson, T. A. Toussoun, and R. J. Cook, eds. Penn. State Univ. Press, University Park, PA. Borrero, C., 21, Phytopathology 30. Kepner Jr., R. L., and Pratt, J. R. 1994. Use of fluorochromes for direct enumeration of total bacterial in environmental samples: Past and present. Microbiol. Rev. 58:603-615. 31. Keren, R. 1996. Boron. Pages: 603-626 in: Methods of Soil Analysis. D. L. Sparks, ed. Soil Science Society of America, Inc., Madison, WI. 32. Kuter, G. A., Nelson, E. B., Hoitink, H. A. J., and Madden, L. V. 1983. Fungal populations in container media amended with composted hardwood bark suppressive and conducive to Rhizoctonia damping-off. Phytopathology 73:1450- 1456. 33. Lockwood, J. L. 1988. Evolution of concepts associated with soilborne plant pathogens. Annu. Rev. Phytopathol. 26:93-121. 34. Marshall, K. C., and Alexander, M. 1960. Competition between soil bacteria and Fusarium. Plant Soil 12:143-148. 35. Mondal, S. N., Kagejama, K., and Hyakumachi, M. 1995. Germinability, viability, and virulence of chlamydospores of Fusarium solani f. sp. phaseoli as affected by the loss of endogenous carbon. Phytopathology 85:1238-1244. 36. Mulvaney, R. L. 1996. Nitrogen-inorganic forms. Pages 123-1184 in: Methods of Soil Analysis. D. L. Sparks, ed. Soil Science Society of America, Inc., Madison, WI. 37. Murphy, J., and Riley, J. P. 1962. A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27:31-36. 38. Nelson, E. B. 1990. Exudate molecules initiating fungal responses to seeds and roots. Plant Soil 129:61-73. 39. Nitta, T. 1991. Diversity of root fungal floras: its implications for soil-borne diseases and crop growth. JARQ-Jpn. Agr. Res. Q. 25:6-11. 40. Nogués, S., Cotxarrera, L. Alegre, L., and Trillas, M. I. 2002. Limitations to photosynthesis in tomato leaves induced by Fusarium wilt. New Phytol. 154:461- 470. 41. Ohta, H., and Hattori, T. 1983. Oligotrophic bacteria in organic debris and plant roots in a paddy field soil. Soil Biol. Biochem. 15:1-8. 42. Orlikowski, L. B. 1983. Influence of substratum type and fungicidal treatment on the development of Fusarium wilt of carnation. Acta Horticult. 150:127-139. 43. Pera, A., and Filippi, C. 1987. Controlling of Fusarium wilt in carnation with bark compost. Biological Wastes 22:219-228. Borrero, C., 22, Phytopathology 44. Pera, J., and Calvet, C. 1989. Suppression of Fusarium wilt of carnation in a composted pine bark and composted olive pumice. Plant Dis. 73:699-700. 45. Reuveni, R., Raviv, M., Krasnovsky, A., Freiman, L., Medina, S., Bar, A., and Orion, D. 2002. Compost induces protection against Fusarium oxysporum in sweet basil. Crop Prot. 21:583-587. 46. Rice, W. C., Moorman, B. T., and Beare, M. 1996. Role of microbial biomass carbon and nitrogen and soil quality. Pages 203-215 in: Methods for Assessing Soil Quality. D. L. Sparks, ed. Soil Science Society of America, Inc., Madison,WI. 47. Serra-Wittling, C., Houot, S., and Alabouvette, C. 1996. Increased soil suppressiveness to Fusarium wilt of flax after addition of municipal solid waste compost. Soil Biol. Biochem. 28:1207-1214. 48. Shannon, C. E., and Weaver, W. 1949. The mathematical theory of communication. University of Illinois Press, Urbana, IL. 49. Sharma, S., Piccolo, A., and Insam, H. 1997. Different carbon source utilization profiles of four tropical soils from Ethiopia. Pages 132-140 in: Microbial Communities. Functional Versus Structural Approaches. H. Insam and A. Rangger, eds. Springer, Heidelberg Germany. 50. Sharman, K. V., and Whitehouse, M. 1993. Nitrogen drawdown index as a predictor of nitrogen requirements for Nephrolepis in sawdust media. Sci. Hortic. (Amsterdam) 54:32-33. 51. Szczech, M. M. 1999. Suppressiveness of vermicompost against Fusarium wilt of tomato. J. Phytopathol. 147:155-161. 52. Szczech, M., Rondomanski, W., Brzeski, M. W., Smolinska, U., and Kotowski, J. F. 1993. Suppressive effect of a commercial earthworm compost on some root infecting pathogens of cabbage and tomato. Biol. Agric. Hortic. 10:47-52 53. Toyota, K., Kimura, M., Takechi, S., and Kuninaga, S. 2000. Comparison of microbial community structure between soils suppressive and conducive to Fusarium-wilt of radish. Soil Microor. 54:69-79. 54. Trillas, I., Avilés, M., Ordovás, J., Bello, A., and Tello, J.C. 2002. Using compost as a methyl bromide alternative. Biocycle 43:64-68. 55. Trillas-Gay, M. I., Hoitink, H. A. J., and Madden, L. V. 1986. Nature of suppression of Fusarium wilt of radish in a container medium amended with composted hardwood bark. Plant Dis. 70:1023-1027. 56. Tuitert, G., Szczech, M., and Bollen, G. J. 1998. Suppression of Rhizoctonia solani in potting mixtures amended with compost made from organic household waste. Phytopathology 88:764-773. Borrero, C., 23, Phytopathology 57. Van Veldhoven, P. P., and Mannaerts, G. P. 1987. Inorganic and organic phosphate measurements in the nanomolar range. Anal. Biochem. 161:45-48. 58. Waksman, S. A. 1927. Principles of Soil Microbiology. The Williams and Wilkins Co., Baltimore, MD. 59. Warrior, P., Konduru, K., and Vasudevan, P. 2002. Formulation of biological control agents for pest and disease management. Pages 421-441 in: Biological Control of Crops Diseases. Gnanamanickam, S. S. ed. Marcel Decker, Inc. New York. 60. Weller, D. M., Raaijmakers, J. M., McSpadden Ggardener, B. B., and Thomashow, L. S. 2002. Microbial populations responsible for specific soil suppressiveness to plant pathogens. Annu. Rev. Phytopathol. 40:309-348. 61. Woltz, S. S., and Jones, J. P. 1981. Nutritional requirements of Fusarium oxysporum: Basis for a disease control system. Pages 340-349 in: Fusarium: Diseases, Biology, and Taxonomy. P. E. Nelson, T. A. Toussoun, and R .J. Cook, eds. Penn. State Univ. Press, University Park, PA. 62. Workneh, F., and van Bruggen, A. H. C. 1994. Microbial density, composition, and diversity in organically and conventionally managed rhizosphere soil in relation to suppression of corky root of tomatoes. Appl. Soil Ecol. 1:219-230. 63. Wright, R. J., and Stuczynski, T. 1996. Atomic absorption and Flame Emission Spectrometry. Pages: 65-90 in: Methods of Soil Analysis. D. L. Sparks, ed. Soil Science Society of America, Inc., Madison, WI. Borrero, C., 24, Phytopathology Fig 1. Standardized area under disease progress curve (AUDPCs) and relative length of stem with brown xylem (RLSBX) for tomato plants in six growth media. CC: composted cork; CC-60: heated composted cork; P: peat; V: vermiculite; GMC: grape marc compost; GMC-60; heated grape marc compost. Disease severity scale was 0: asymptomatic plants, 1: ≤ 50% affected leaves, 2: > 50% affected leaves, 3: dead plants. Plant growth media were infested with Fusarium oxysporum f. sp. lycopersici race 2 at the dose of 1 x 105 microconidia / cm3. Data for AUDPCs and RLSBX were transformed for analysis with the arcsine √x. Bars with the same letter are not significantly different according to Tukey's test at P < 0.05. Standard error of the mean is indicated by vertical line. Fig 2. Correlation (multiple) between standardized area under disease progress curve (AUDPCs), β-glucosidase activity and pH, recovered from six plant growth media at the beginning of bioassays. These growth media were infested with Fusarium oxysporum f. sp. lycopersici race 2 at 1 x 105 microconidia / cm3. CC: composted cork; CC-60: heated composted cork; P: peat; V: vermiculite; GMC: grape marc compost; GMC-60; heated grape marc compost. Disease severity scale was 0: asymptomatic plants, 1: ≤ 50% affected leaves, 2: > 50% affected leaves, 3: dead plants. Fig 3. Ordinate plot of principal components (PC1 and PC2) of microbial communities of six plant growth media at beginning of bioassay and Fusarium oxysporum f. sp. lycopersici (FOL-2) according to Biolog pattern. CC: composted cork; P: peat; V: vermiculite; GMC: grape marc compost. Borrero, C., 25, Phytopathology Plant growth medium CC CC-60 P V GMC GMC-60 RLSBX AUDPCs 0.0 0.1 0.2 0.3 0.4 0.5 0.6 aa b d bc c ab a c 0.0 bc d c A B 0.2 0.4 0.6 Fig 1. Borrero, C., 32, Phytopathology Table 5. Nutrient status of tomato leaves and stems grown in six plant growth media at the end of bioassay. Plant growth media were infested with Fusarium oxysporum f. sp. lycopersici race 2 at 1 x 105 microconidia / cm3 N P K Mg Ca Na Fe Cu (10-2) Zn Mn B (10-5) mg nutrient / g dry plant a Plant growth medium b CC 15.588 bc 21.106 bc 24.261 cd 7.964 ab 31.398 a 1.931 b 0.112 cd 1.173 ab 0.071 bc 0.125 bc 4.129 b CC-60 17.248 abc 24.588 a 27.893 bc 6.110 cd 31.237 a 1.930 b 0.157 bc 1.601 a 0.127 a 0.306 a 4.469 ab P 22.775 a 24.567 a 22.483 cd 4.590 d 26.552 ab 2.030 b 0.310 a 1.543 a 0.094 ab 0.120 bc 4.155 b V 21.616 ab 23.235 ab 20.025 d 8.953 a 22.076 b 3.276 a 0.240 ab 1.806 a 0.062 c 0.103 c 4.208 ab GMC 9.728 d 17.849 c 34.268 ab 7.176 bc 25.965 ab 1.166 c 0.093 d 0.897 b 0.072 bc 0.084 d 4.155 b GMC-60 14.357 c 20.603 bc 41.617 a 7.133 bc 27.149 ab 1.507 bc 0.125 cd 1.425 a 0.125 a 0.145 b 4.704 a a Analysis of variance was performed with transformed data: 1/x for Fe, Cu and Mn and Ln(x) for N to obtain a constant variance. Within each, values followed by different letters are significantly different based on Tukey's test at P < 0.05. b CC: composted cork; CC-60: heated composted cork; P: peat; V: vermiculite; GMC: grape marc compost; GMC-60; heated grape marc compost. Borrero, C., 33, Phytopathology Table 6. Correlation (simple) among AUDPCs and nutrient availability of plant growth media (mg of nutrients / cm3 plant growth medium) and tomato plants (mg of nutrients /g dry weight) at the end of the bioassay Element R2 r P b Equation Plant growth media AUDPCs a Na 81.36 -0.902 * AUDPCs = 0.54 – 2.04 * Na Cu 77.23 0.879 * AUDPCs = -0.006 + 12.96 * √Cu Ca 77.16 -0.878 * AUDPCs = 0.58 – 0.20 * Ln (Ca) Tomato plants AUDPCs a Fe 98.54 -0.993 *** AUDPCs = 0.65 - 0.06 / Fe N 97.24 -0.986 ** AUDPCs = e ( 1.29 - 45.14 / N ) Cu 93.01 -0.964 ** AUDPCs= e ( 1.85 - 0.05 / Cu ) P 87.69 -0.936 ** AUDPCs = e ( 5.56 - 155.53 / P ) Na 78.74 -0.887 * AUDPCs = e ( 1.02 – 4.68 / Na ) a The six plant growth media were inoculated with Fusarium oxysporum f. sp. lycopersici race 2 at 1 x 105 microconidia / cm3. b Significance levels: P < 0.05 *, P < 0.01 ** and P < 0.001 ***. Borrero, C., 34, Phytopathology Table 7. Microbiological properties of plant growth media at the end of the bioassay. Control: noninfested media and infested: Fusarium oxysporum f. sp. lycopersici race 2 at 1 x 105 microconidia / cm3 x 105 CFU / cm3 plant growth medium a Growth media b CONTROLS ca b c fp cb oa ob f CC 0.1120 ab 3.111 c 2863 ab 16.17 a 336.3 ab 2.596 b 1329 a 0.3523 bc CC-60 0 b 33.63 b 3686 ab 0.0075 c 1093 a 9.263 b 3519 a 1.993 ab P 0.0373 ab 0.122 d 1902 b 0.0886 bc 227.5 ab 0.8007 c 504.1 a 1.574 ab V 0 b 7.480 d 5956 ab 0.2689 bc 135.2 b 0.0787 d 2299 a 0.5135 bc GMC 4.824 a 19.99 b 3781 ab 0.3026 b 2127 a 44.21 a 2728 a 7.270 a GMC-60 ND c 137 a 9225 a 0.0238 c 402.7 ab 2.878 b 2687 a 0.0923 c Growth media INFESTED CC 0 b 8.766 ab 6646 1.109 ab 1083 2.651 b 3679 0.9636 b CC-60 0 b 32.07 ab 9311 0.7774 ab 688.0 1.354 b 6206 0.4951 b P 0 b 1.478 bc 4526 0.0290 b 671.4 1.082 b 2089 0.5765 b V 0 b 0.123 c 1799 0.0638 ab 227.0 0.0023 c 1377 0.2788 b GMC 1.599 a 25.31 a 3734 0.2308 ab 512.5 49.24 a 4454 3.706 a GMC-60 0 b 90.62 a 5494 2.5614 a 597.6 1.973 b 4201 2.434 b a ca: cellulolytic actinomycetes, b: Bacillus spp., c: copiotrophic bacteria, fp: fluorescent Pseudomonas spp., cb: cellulolytic bacteria, oa: oligotrophic actinomycetes, ob: oligotrophic bacteria, f: fungi. Within each, values followed by different letters are significantly different based on Tukey's test at P < 0.05. Analysis of variance was performed with transformed data in Ln(x). For cellulolytic actinomycetes (2m)-1, m = nº replicate was added to obtain normalized data. b CC: composted cork; CC-60: heated composted cork; P: peat; V: vermiculite; GMC: grape marc compost; GMC-60; heated grape marc compost. c ND: not determined. Borrero, C., 35, Phytopathology Table 8. Correlation (simple) among AUDPCs and different microorganisms recovered from growth media (CFU / cm3 plant growth medium) Microorganism group b R2 r P c Equation AUDPCs a oa 99.31 0.996 *** AUDPCs = 1/(2.8635+5.8 10-6·oa) oa/ob 99.12 0.996 *** AUDPCs = 1/(2.6899+2586.12·oa/ob) ca 98.50 0.992 *** AUDPCs = 1 / ( 3.694 + 0.172 10-3 ca) ca/cb 98.50 0.992 *** AUDPCs = 1/(3.6939+8829.72·ca/cb) f 80.68 -0.898 * AUDPCs = e(-0.6941-0.0000064· f) ob/c 68.90 -0.830 * AUDPCs = e(-0.7444-3.1961· ob/c) a The six plant growth media were infested with Fusarium oxysporum f. sp. lycopersici race 2 at 1 x 105 microconidia / cm3. b oa: oligotrophic actinomycetes, ob: oligotrophic bacteria, ca: cellulolytic actinomycetes, cb: cellulolytic bacteria, f: fungi, c: copiotrophic bacteria. c Significance levels: P < 0.05 *, P < 0.01 ** and P < 0.001 ***.