Composts from agricultural waste and the Trichoderma asperellum1 strain T-34 suppress Rhizoctonia solani in cucumber seedlings

Composts from agricultural waste and the Trichoderma asperellum 1 strain T-34 suppress Rhizoctonia solani in cucumber seedlings 2 3 M. Isabel Trillasa, Eva Casanovaa , Lurdes Cotxarreraa, José Ordovásb, Celia Borrerob, 4 Manuel Avilésb 5 6 a Universitat de Barcelona, Departament de Biologia Vegetal, Facultat de Biologia, 7 Avgda. Diagonal 645, 08028 Barcelona, Spain 8 b Universidad de Sevilla, Departamento de Ciencias Agroforestales, EUITA, Ctra. 9 Utrera, Km. 1, 41013 Sevilla, Spain 10 11 Corresponding author. M.I. Trillas, [email protected] 12 13 Abstract 14 15 Using composts in agriculture to minimize organic wastes and to reduce the addition of 16 fertilizers and fungicides in crop production is highly effective. Our results show that 17 among those tested composts aged 0.5–1 year, cork compost reduced diseases caused by 18 Rhizoctonia solani in cucumber plants (53% of diseased plants) in comparison to peat (up 19 to 89%). However, all composts aged 1.5–3 years (comprised of cork, grape marc, olive 20 marc and spent mushroom) highly suppressed Rhizoctonia disease, measuring 3, 11, 27 21 and 29% of diseased plants, respectively. Plant growth media enriched with the biological 22 control agent Trichoderma asperellum (strain T-34) reduced the incidence of R. solani 23 disease when amended at 103 cfu ml¡1. In composts aged 0.5–1 year, T-34 was only 24 efficient when added to spent mushroom and cork compost, although it remained well 25 established in all of them. The fact that T-34 rendered all composts aged 1.5–3 years 26 highly suppressive is attributed to the low levels of easily biodegradable substances. 27 Rhizoctonia damping-off in cucumber plants can be reduced by using composts and/or 28 the biological control agent T. asperellum strain T-34. In addition, the extent to which 29 composts suppress this disease depends on the nature of the composted materials, 30 increasing with the composts’ maturity level. 31 32 Keywords: Biological control agents; Cork compost; Cucumis sativus; Grape marc 33 compost; Olive marc compost; Peat; Rhizoctonia solani; Spent mushroom 34 compost; Trichoderma asperellum. 35 36 1. Introduction 37 38 Increasing concern regarding food safety and environmental pollution, as well as 39 legislative pressures in European countries to reduce the number of approved active 40 pesticide ingredients, has generated an interest in compost and other biological control 41 agents to prevent and control plant diseases (http://europa.eu.int/comm/food/plant/ 42 protection/evaluation/framework_en.htm; UNEP, 2002). 43 The use of compost as a peat substitute to control root pathogens was first suggested by 44 Hoitink et al. (1975). Since then, several soil-borne plant pathogens have been reduced 45 by using composts made of different raw materials (Borrero et al., 2004; Cotxarrera et al., 46 2002; Hoitink and Boehm, 1999; Hoitink and Fahy, 1986; Litterick et al., 2004). 47 However, the capacity of composts to suppress Rhizoctonia solani, a pathogen that affects 48 both seedlings and adult plants of many species, remains limited (Hoitink and Boehm, 49 1999; Scheuerell et al., 2005). Composts that reportedly reduce Rhizoctonia damping-off 50 are detailed in Table 1. The capacity of certain composts to repress R. solani may be due 51 to the presence and activity of specific antagonists (Kuter et al., 1983; Scheuerell et al., 52 2005; Tuitert et al., 1998) and depends on the degree of compost decomposition (Hoitink 53 and Boehm, 1999). Moreover, matured composts can sustain biological control agents, 54 whereas immature composts do not support them, negatively affecting the growth of crop 55 plants and possibly containing pathogen populations (De Ceuster and Hoitink, 1999; 56 Litterick et al., 2004). 57 The antagonistic activity (parasitism) of the genus Trichoderma and Gliocladium to R. 58 solani has been widely demonstrated (Elad et al., 1980; Harman et al., 2004; Koch, 1999; 59 Krause et al., 2001; Lewis et al., 1998; Nelson et al., 1983). Other antagonists to this 60 pathogen are also well documented: Bacillus spp. (Pleban et al., 1995; Yu et al., 2002), 61 Pseudomonas spp. (Kwok et al., 1987; Pal et al., 2000; Thrane et al., 2001), Streptomyces 62 spp. (Sabaratnam and Traquair, 2002), as well as binucleate Rhizoctonia strains (Harris, 63 2000; Hwang and Benson, 2003). 64 The purpose of this study was to examine the degree of suppression of R. solani in 65 cucumber seedlings by using several composts (cork, grape marc, olive marc and spent 66 mushroom), obtained from agricultural waste, as plant growth media. Trichoderma 67 asperellum (strain T-34), an efficient biological control agent against Fusarium 68 oxysporum (Cotxarrera et al., 2002), was tested against R. solani when added to these 69 composts at different stages of maturity. 70 71 2. Materials and methods 72 73 2.1. Composts 74 The suppressiveness of composts to damping-off, as generated by R. solani, was 75 evaluated using Sphagnum light peat (Klasmann or Floratorf, Germany) as a conducive 76 plant growth medium. The composts used were derived from turned piles as previously 77 described (Trillas et al., 2002). Cork compost (CC) was made from cork industry residues 78 (Carmona et al., 2003). Coarse CC, with 48% of particles>1mm, had the appropriate 79 physical properties (water and air availability) for use as a stand-alone plant growth 80 medium (Table 2). Fine CC, with 68% of particles <1mm, had low air capacity. 81 Consequently, after the composting process, the Wne CC was mixed with rice hulls 82 (CC:rice hulls, 2:1, v/v), yielding a compound referred to as CCr (Table 2). The physical 83 properties of cork compost including water release curve were assessed by the standard 84 method described in De Boodt et al. (1974). The grape marc compost (GMC) consisted 85 of grape skins, seeds and stalks from the alcohol industry. Oil residues, which consisted 86 of olive cake (marc) and olive mill wastewaters from the oil industry, were always 87 composted with a bulk agent (olive marc and cotton gin trash, 2:3, v/v) and mixed with 88 rice hulls (1:1, v/v), referred to as OMC. We also evaluated composts obtained 89 commercially (RECOMSA, Cuenca, Spain) made from spent mushrooms composted 90 alone and mixed with peat (1:1, v/v) (SMC). 91 Composts were chemically characterized 4–5.5 months after the start of the composting 92 process. At that time, they were all stable and none proved phytotoxic when used as the 93 plant growth medium. Peat and rice hulls were also characterized (Table 3). Chemical 94 analyses were carried out using aqueous extracts (1:2, v/v). Ca, Mg, Fe, Cu, Mn and Zn 95 were determined by atomic absorption spectroscopy and Na and K were analyzed by 96 atomic emission spectrometry (Wright and Stuczynski, 1996). P was measured with 97 colorimetry (Murphy and Riley, 1962). 98 Composts were tested for disease suppressiveness 0.5–1 year after the start of the 99 composting process (composts aged 0.5–1 year), then again after 1.5–2 years, except for 100 old CC, which was evaluated after 2.5–3 years (composts aged 1.5–3 years). 101 102 2.2. Fungal strains and inoculum preparations 103 R. solani (isolate AG-4) soil inoculum was prepared as a potato-soil mixture (Ko and 104 Hora, 1971). After 14 days, cultures were sieved, with those pieces remaining on the 1mm 105 sieve used to infest the container media. The quantity of inoculum used for the different 106 bioassays varied between 1.0–2.0 g l-1 of container medium. The soil inoculum 107 concentration of R. solani was adjusted to induce around 80– 100% damping-oV when 108 the cucumber was grown in peat. 109 The T. asperellum [Spanish collection of type culture, C.E.C.T. 20417, European patent 110 application EP 1 400 586 A1 (Trillas and Cotxarrera, 2002)] strain T-34 was derived from 111 a suppressive compost. The T-34 strain consistently reduced Fusarium wilt disease in 112 tomato plants (Cotxarrera et al., 2002). T-34 was grown in petri dishes on Malt Agar 113 (Scharlau, Barcelona, Spain) for 7 days. Sterile water was added to the culture and the 114 surface was scraped to obtain a conidial suspension. The concentration was determined 115 by measuring with a haemocytometer. T-34 was added to the peat, as well as to different 116 composts at 103, 104, or 105 cfu ml-1, following the experimental design. Populations of 117 T-34 were counted at 1, 2 and 3 weeks as total populations of Trichoderma spp. by serial 118 dilution on semi-selective Trichoderma medium (Chung and Hoitink, 1990). Peat and the 119 different composts amended with T-34 were incubated for 2 weeks at a water tension of 120 1KPa (adjusted on a weight basis) and an incubation temperature of 25±2 °C prior to use 121 in the bioassay described below. 122 123 2.3. Bioassays and assessment of disease incidence and severity 124 The suppression of Rhizoctonia damping-off in cucumber seedlings was assessed by 125 bioassays adapted from that described by Nelson et al. (1983). For each treatment, we 126 used five pots (330 ml) with 15 cucumber (Cucumis sativus) cv. Negrito seeds per pot. 127 Pots were placed in a growth chamber (25±2 °C, 16 h light and 150–210 µE m-2s-1). 128 Seedlings were fertirrigated twice a day with 50 ml of Peter’s foliar feed 27-15-12 at 0.5 129 g l-1 (Scotts, Heerlen, The Netherlands), complemented with CaCl2 at 0.6 g l-1 and MgSO4 130 7H2O (pH 5.68) at 0.7 g l-1. For each bioassay, and for peat and each of the composts, the 131 treatments were as follows: (1) control pots, with no R. solani or T-34, (2) pots infested 132 with R. solani, and (3) pots infested with R. solani and amended with T. asperellum strain 133 T-34 at one (103 cfu ml-1) or several concentrations (103, 104 and 105 cfu ml-1). 134 Disease incidence was the percentage of diseased plants over the total number of plants 135 and was evaluated after 7 days. Disease severity was evaluated as follows: 1, healthy 136 plants, 2, small lesions, 3, large lesions, 4, postemergence damping-off and 5, pre-137 emergence damping-off. At least three bioassays were performed for each experiment. 138 139 2.4. Statistical analysis 140 Differences in the disease incidence of R. solani were assessed with one-way ANOVA at 141 the end of each bioassay between treatments (plant growth media and/or T-34 142 concentration). Duncan’s multiple range test was applied when one-way ANOVA 143 revealed significant differences (P<0.05). All statistical analyses were performed with 144 SPSS 12.0 (SPSS Inc., Chicago, IL). 145 146 3. Results 147 148 3.1. Colonisation of T-34 in different plant growth media 149 When T-34 was added at 103 cfu ml-1 to peat or various composts, the total Trichoderma 150 spp. stabilised between 0.5 and 3.6x103 cfu ml-1 for composts and at higher levels for peat 151 (4.1 and 9.6x103 cfu ml-1) (Table 4). When T-34 was added at 105 cfu ml-1, the 152 Trichoderma spp. Populations stabilised between 2.1x104 and 1.3x105 cfu ml-1 both for 153 peat and composts (Table 4). Trichoderma spp. Populations were similar after 1, 2 and 154 even 3 weeks of incubation (Table 4). 155 156 3.2. Effects of composts aged 0.5–1 year and T-34 on suppressiveness to R. solani 157 Between 0.5–1 year after the start of the composting process, the only compost that 158 significantly reduced R. solani disease in cucumber seedlings was the CC (53% diseased 159 seedlings) (Table 5), which produced a disease severity of 1.4 compared with 2.8 obtained 160 in the peat. At the same level of maturity, OMC also significantly reduced this disease, 161 with diseased seedlings totalling 76% (Table 5) and a disease severity of 1.9. Cucumber 162 seedlings grown in GMC, SMC and CCr (with rice hulls) had the same disease incidence 163 (80–90%) and severity as that of Floratorf peat. The chemical composition of plant 164 growth media at that age of maturity (Table 3) did not correlate with their level of 165 suppressiveness to R. solani. 166 In one of two experiments, cucumber grown in Floratorf peat medium amended with T-167 34 at 103 cfu ml-1 exhibited significantly less disease (77%) than the unamended medium 168 (Table 5). When T-34 was added to CC and SMC, the disease incidence decreased to 26% 169 and 55%, respectively. When used to enrich CCr, GMC, and OMC (Table 5), T-34 at 103 170 cfu ml-1 had no effect on Rhizoctonia dampingoff. 171 172 3.3. Effects of composts aged 1.5–3 years and T-34 on suppressiveness to R. solani 173 Between 1.5–2 years after the start of the composting process, all studied composts 174 became suppressive to Rhizoctonia damping-off (3–29% diseased seedlings), with CC 175 (aged 2.5–3 years) proving to have the highest level of suppression (Table 6). It is worth 176 noting that 8% of seedlings grown in Klassman peat (95% of diseased seedlings) 177 presented pre-emergence damping-off symptoms, whereas none of those grown in 178 composts showed pre-emergence damping-off, only post-emergence damping-off. 179 Disease severity decreased from 3.1 in peat, to 1.0–1.5 in composts. Peat became 180 moderately suppressive when T-34 was added at 103 or 104 cfu ml-1. When T-34 was 181 added at 105 cfu ml-1, only 21% of seedlings showed disease symptoms (Table 6). T-34 182 added to OMC significantly reduced Rhizoctonia disease incidence in cucumber seedlings 183 from 27% to 5–9%. Analogously, SMC enriched with T-34 significantly reduced disease 184 incidence in cucumber seedlings from 29% to 3–15% (Table 6). The effects of CC (3% 185 of disease incidence) and GMC (11% of disease incidence) composts, as well as of T-34 186 enrichment, were not additive, since no improvement in disease suppression was found 187 at the various concentrations of T-34 tested. 188 189 4. Discussion 190 191 Among the composts aged 0.5–1 year, CC was the most highly suppressive plant growth 192 medium studied in controlling R. solani disease in cucumber seedlings, although there 193 was no suppression when rice hulls were needed to improve the air content of this plant 194 medium. This reduction in suppressiveness may be due not only to the dilution effect 195 caused by the addition of the rice hulls, but may also be considered that the composition 196 of fine cork particles can differ from that of coarse cork particles. GMC from the same 197 batches, which was conducive to disease as caused by R. solani, induced significant 198 disease reduction to Fusarium oxysporum in tomato plants (Borrero et al., 2004). This 199 GMC (aged 0.5–1 year), which proved conducive to disease produced by R. solani, 200 contains high populations of cellulolytic and oligotrophic actinomycetes and cellulolytic 201 bacteria (Borrero et al., 2004), despite the fact that these microorganisms have been 202 associated with Rhizoctonia suppression (Diab et al., 2003; Tuitert et al., 1998). Natural 203 compost suppressiveness to Rhizoctonia damping-off is not widespread, and only some 204 20% of composts are suppressive to this pathogen (Hoitink and Boehm, 1999). In 205 contrast, most composts naturally suppress diseases caused by Pythium spp. and 206 Phytophthora spp. (Hoitink and Boehm, 1999; Litterick et al., 2004; Noble and Coventry, 207 2005). 208 All studied composts aged 1.5–3 years showed a significant decrease in Rhizoctonia 209 damping-off in cucumber seedlings compared with composts aged 0.5–1 year. 210 Accordingly, it has been reported that the degree of maturity is especially important in 211 reducing disease caused by this pathogen (Diab et al., 2003; Hoitink and Boehm, 1999; 212 Kuter et al., 1988; Nelson et al., 1983). This phenomenon might be due to the low levels 213 of easily biodegradable substances that enhance the competitiveness of autochthonous 214 microflora in long-term matured composts (Hoitink and Boehm, 1999; Litterick et al., 215 2004). Thus, in our study with R. solani the most suppressive composts, cork composts, 216 liberate cellulose slowly, since the major compound in cork, suberine, is recalcitrant to 217 degradation. Consequently, natural microorganisms might remain in a competitive state 218 in both CC aged 0.5–1 year and 2.5–3 years. Natural Trichoderma spp. in cork composts 219 (unpublished data), the main mycoparasite described for Rhizoctonia (Harman et al., 220 2004), were particularly scarce. However, this compost contains high populations of 221 Fluorescent Pseudomonas (Borrero et al., 2004), which have also been associated with 222 suppression of Rhizoctonia damping-off (Kwok et al., 1987). 223 The biological control agent T. asperellum strain T-34 was able not only to establish itself 224 in all composts aged 1.5–3 years, but also to reduce the disease caused by R. solani in 225 cucumber seedlings. However, in composts aged 0.5–1 year, even though T-34 was 226 established in all composts (data not shown), it improved suppressiveness only when 227 added to CC and SMC. The chitinase activity of T-34 might be enhanced in the low 228 cellulose environment of composts aged 1.5–3 years and of CC and SMC aged 0.5– 1 229 year (high in suberine and chitin, respectively), since cellulose-enriched composts repress 230 the chitin-degrading enzymes of Trichoderma spp. (Harman et al., 1993; Hoitink and 231 Boehm, 1999; Lorito et al., 1996). CC aged 2.5–3 years and GMC aged 1.5–2 years were 232 so suppressive to R. solani that the action of T-34 was underestimated vis-à-vis the other 233 suppression mechanisms involved in such composts. Compost manufacturers should 234 evaluate the economic viability of each compost in relation to longterm maturation and/or 235 the addition of a biological control agent. 236 The role of T-34 in disease suppression to Rhizoctonia was evident when it was added to 237 the conducive peat, since suppressiveness was clearly dose-dependent. The low 238 suppression observed, in some cases, in peat enriched with T-34 is consistent with results 239 obtained for peat enriched with another Trichoderma strain combined with 240 Chryseobacterium (Krause et al., 2001). 241 In conclusion, our results indicate that R. solani disease in cucumber seedlings can be 242 reduced by using composts and the biological control agent T. asperellum strain T-34. The 243 suppressiveness of composts to Rhizoctonia damping-off depends on the nature of the 244 composted materials, increasing with the maturity level of the composts. 245 246 Acknowledgments 247 248 This research was supported by grant AGL2002-04313 from the Ministerio de Ciencia y 249 Tecnologia (Proyectos I+D del Plan Nacional de Investigación Científica, Desarrollo e 250 Innovación Tecnológica) of Spain. We thank the Servei de Camps Experimentals of the 251 University of Barcelona for its technical support. 252 253 References 254 255 Borrero, C., Trillas, M.I., Ordovás, J., Tello, J., Avilés, M., 2004. Predictive factors for 256 the suppression of Fusarium wilt of tomato in plant growth media. Phytopathology 257 94, 1094–1101. 258 Carmona, E., Ordovás, J., Moreno, M.T., Avilés, M., 2003. Granulometric 259 characterization and alteration during composting of industrial cork residues for 260 use as a growing medium. 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Dwarf Baby Mix Stephens and Stebbins (1985). Cucumis sativus cv. Straight Eight Tunlid et al. (1989). Municipal sewage sludge Phaseolus vulgaris cv. Blue lake Lumsden et al. (1983). Gossypium hirsutum cv. Stoneville Raphanus sativus cv. Scarlet Globe Grape marc, cattle manure Epipremnum aureum Gorodecki and Hadar (1990). Raphanus sativus radicula alpha Organic household and garden waste Cucumis sativus cv. Lange Groene Tuitert et al. (1998). Spent forest mushroom, fish meal and blood waste Brassica oleracae cv. K-Y Cross Shiau et al. (1999). Cork Cucumis sativus cv. Negrito Trillas et al. (2002). Swine wastes and woodchips Impatiens balsamina cv. Super Elfin Diab et al. (2003). Bark, mushroom, nursery regrind Brassica oleracae cv. Cheers Scheuerell et al. (2005). 390