Identifying characteristics of Verticillium wilt suppressiveness in olive mill composts

Avilés, Plant Disease 1 TITLE 1 Identifying characteristics of Verticillium wilt suppressiveness in olive mill composts 2 AUTHORS 3 Manuel Avilés and Celia Borrero 4 Dept. Ciencias Agroforestales, Escuela Técnica Superior de Ingeniería Agronómica, Universidad 5 de Sevilla, Ctra. Utrera km 1, 41013 Sevilla, Spain. e-mail: [email protected] and cbor[email protected] 6 Corresponding author: Celia Borrero. Tel: +34 954486457. Fax: +34 954486436. e-mail: 7 [email protected] 8 ABSTRACT 9 The aims of this study were to assess the potential suppressive effects of different olive mill 10 composts on Verticillium wilt and to elucidate the suppressive mechanisms. To this end, four olive 11 mill composts from different crop areas with two maturation levels were selected. After conducting 12 the Verticillium wilt bioassays in cotton, the suppressive effect was observed in only one compost. 13 Compost maturation level did not affect disease development. The standardized area-under-the-14 disease-progress-curveand microsclerotia concentration were associated with low API-ZYM 15 enzymatic diversity, β-glucosidase activity, pH and high electrical conductivity (EC). To assess 16 the nature of suppressiveness in the supressive compost, additional bioassays were performed 17 with three treated compost-amended growing media (N-supplemented, autoclaved and heat 18 treated at 60ºC for 6 days). Suppressiveness was partially reduced with heat treatments, where 19 N-acetyl-β-glucosaminidase activity disappeared. In this compost high oligotrophic actinomycete 20 populations were associated with disease reduction. Therefore, plant growth media amended with 21 different olive mill composts do not always show suppressiveness against Verticillium wilt. 22 Enzymatic diversity, β-glucosidase activity, pH and EC may be sufficient to predict where olive 23 Avilés, Plant Disease 2 mill compost plant growth media will be effective in reducing Verticillium wilt and microsclerotia 24 concentration. General and specific suppressiveness are involved in the mechanism of compost 25 suppression. 26 Keywords: Plant growth media, olive mill compost, Verticillium dahliae, suppressiveness 27 28 Verticillium wilt is currently considered a major soilborne disease threatening many crops 29 worldwide and unfortunately, there are limited chemical control options (López-Escudero and 30 Mercado-Blanco, 2011). In Andalusia, southern Spain, this disease causes severe problems in 31 both young and old olive orchards and has spread to all the other major olive-growing areas in 32 Spain (Jiménez-Díaz et al., 2012). Verticillium wilt of olive and cotton are very similar 33 pathosystems. Verticillium dahliae isolates infecting both crops are traditionally classified into 34 defoliating and non-defoliating pathotypes. Thus, an isolate recovered from cotton or olive 35 displays cross-virulence in both crops (López-Escudero and Mercado-Blanco, 2011). The primary 36 forms of V. dahliae dispersion are the establishment of new olive orchards on former cotton fields 37 and pathogen infection of susceptible crops nearby (Jiménez-Díaz et al., 2012; López-Escudero 38 and Mercado-Blanco, 2011). Preventive and/or biological control strategies must be developed to 39 reduce this disease. 40 Spain is the main olive fruit and olive oil producer in the world, accounting for nearly 40% of 41 production (FAO, 2015). Andalusia is the largest olive cultivation area (61%) and accounts for 42 83% of the total national production of olives for oil production (Ministerio de Agricultura, 43 Alimentación y Medio Ambiente, 2015). Nowadays, a two-phase centrifugation system is the most 44 commonly used olive oil extraction method in Spain. This system generates olive oil plus a semi-45 solid waste, known as olive mill waste or alperujo (Morillo et al., 2009), which represents 90% of 46 olive weight (Ministerio de Medio Ambiente, 2007). 47 Avilés, Plant Disease 3 Olive mill waste is not recommended for use as an organic soil amendment in olive orchards due 48 to its acidic pH, potential phytotoxicity and the possibility of soil contamination with V. dahliae 49 (Jiménez-Díaz et al., 2012; Morillo et al., 2009). There is increasing interest in composting as a 50 sustainable strategy to recycle this waste for agricultural purposes. To achieve this, due to its 51 semi-solid consistency and low porosity, the residue should be mixed with bulking agents 52 (Chowdhury et al., 2013; Morillo et al., 2009). The type of bulking agent, the ratio of olive mill 53 waste to bulking agent and the composting method used may vary. Many types of bulking agents 54 exist, including olive leaves, cotton gin trash, rice hulls, sheep manure and grape stalks (Alfano et 55 al., 2011, 2009; Borrero et al., 2009; Cayuela et al., 2008; Moreno et al., 2016). After composting, 56 non-phytotoxic organic matter-rich materials free of pathogens are obtained (Morillo et al., 2009; 57 Principi et al., 2001), and these materials are suitable for use as organic soil amendments and 58 plant growing media (Caballero et al., 2009). It is particularly significant that composting can be 59 an efficient way of reducing V. dahliae propagules (Noble and Roberts, 2004). 60 Some composts have been shown to have suppressive effects on different phytopathogens, 61 including V. dahliae, when added to plant growth media or soils (Bonanomi et al., 2010; Litterick 62 et al., 2004; Noble and Coventry, 2005). In particular, the suppressive effects of some olive mill 63 composts on Verticillium wilt disease have been observed in tomato (Alfano et al., 2011; 64 Arriagada et al., 2012; Vitullo et al., 2013), eggplant (Malandraki et al., 2008; Markakis et al., 65 2008; Papasotiriou et al., 2013), and cotton (Castaño and Avilés, 2013). Also, olive mill extracts 66 can inhibit V. dahliae growth in agar culture media (Alfano et al., 2011; Arriagada et al., 2012; 67 Lima et al., 2008) and reduce microsclerotia concentration in soils or plant growth media 68 (Castaño and Avilés, 2013; Lima et al., 2008; Vitullo et al., 2013). 69 Historically, compost suppressiveness has been divided into two major categories: general and 70 specific. General suppression is related to the sum of the activities of the overall microbial 71 biomass, and specific suppression is a consequence of the activities of a reduced number of 72 Avilés, Plant Disease 4 microorganism populations (Alfano et al., 2011; Avilés et al., 2011; Hoitink and Boehm, 1999; 73 Stone et al., 2004). The abiotic environment (aeration, pH, mineral composition, etc) is also 74 relevant in explaining suppressiveness (Avilés et al., 2011; Termorshuizen and Jeger, 2008). If 75 suppressiveness is eliminated by pasteurization, biocides or harsher treatments like autoclaving, 76 then the suppressiveness is due to biological factors (Weller et al., 2002). In contrast to specific 77 suppression, general suppression is usually recovered after sterilization due to the rapid 78 colonization of microorganisms (Termorshuizen and Jeger, 2008). Another characteristic of 79 general suppression is that the number of pathogen propagules does not decline rapidly in 80 suppressive media (Hoitink et al., 1993). 81 Verticillium dahliae can be transported long distances in infected planting stock and/or infested 82 potting soil, and thus can be introduced in pathogen-free olive-growing areas. The spread of the 83 pathogen can be further enhanced if olive nurseries are established in V. dahliae-infested areas 84 and the asymptomatic host plants distributed across the olive production region (Jiménez-Díaz et 85 al., 2012). Compost application in the field can be expensive due to the large quantities required; 86 however, its application in nurseries is easier and more practical than in large-scale field 87 production (Malandraki et al., 2008). Thus, olive mill compost obtained after a strong thermophilic 88 phase followed by extensive recolonization of mesophilic microorganisms would be free of 89 pathogens and could confer Verticillium wilt suppressiveness to the plant growth media used in 90 olive nurseries, even after transplanting in open fields (Hoitink et al., 1999; Noble and Roberts, 91 2004). Additionally, the incorporation of olive mill compost in plant growth media would give value 92 to this residue. 93 The first objective of this work was to study the potential suppressive effects of different olive mill 94 composts on Verticillium wilt. For this purpose, commercial olive mill composts obtained from 95 different sources and having different maturation levels were studied. The second objective was 96 to elucidate the primary mechanisms explaining these suppressive effects. 97 Avilés, Plant Disease 5 98 MATERIALS AND METHODS 99 Olive mill waste composts and plant growth media. Four olive mill composts were obtained 100 from four companies in different olive production areas in Andalusia. These composts 101 encompassed the usual variability found in these types of products (i.e. bulk density, pH, organic 102 matter, etc.), and were named OC1, OC2, OC3 and OC4 (Supplementary Table S1). The 103 composts represented two maturation levels, with curing phases of either four months or one 104 year, and each contained source materials specific to each company (Supplementary Table S1). 105 For each type of compost and maturation level, two subsamples were collected and assessed for 106 a number of properties including: bulk density (BD) according to de Boodt et al. (1974), organic 107 matter following the European Standard (EN 13039, 2011), total C and N by the Dumas method 108 described in UNE-EN 13039 (AENOR, 2002), electrical conductivity (EC) and pH. The latter two 109 properties were determined in 1:5 v/v compost:water extracts. 110 To reduce salinity, the composts were flushed with three volumes of water per volume of compost 111 (Sullivan and Miller, 2001) and then mixed at a ratio of one part compost to two parts peat 112 substrate. The commercial peat substrate used (Kekkilä Iberia S.L., Valencia, Spain) was 113 composed of a mixture of light and brown peat (50:50 v/v) slightly amended with a NPK fertilizer 114 and pH corrected. These plant growth media formulated with the different composts were 115 designated OC1-GM, OC2-GM, OC3-GM and OC4-GM and compared with peat substrate as the 116 standard plant growth medium (control). The peat used as the control was enriched with 0.17 g / 117 liter of NH4H2PO4 (12% N and 61% P2O5, Fertiberia, Madrid, Spain), 0.52 g / liter of NH4NO3 118 (33.5% N, Fertiberia, Madrid, Spain), and 6.15 g / liter of K2SO4 (50% K2O, Compo Agricultura 119 S.L., Barcelona, Spain) in order to achieve nutrient availability comparable to the other compost 120 Avilés, Plant Disease 6 amended plant growth media. To standardize the initial conditions, all plant growth media were 121 incubated in 7-liter bags at 25ºC for 14 days (Inbar et al., 1991) with 40% water by volume. 122 Olive mill compost bioassays. Four bioassays were performed to study the potential 123 suppressive effects of the different compost amended plant growth media on Verticillium wilt. Two 124 bioassays were performed with short-matured composts (composts matured for four months) and 125 two with long-matured composts (composts matured for one year). Each bioassay was arranged 126 in a randomized block design with 4 compost types x 3 blocks x 5 inoculated pots (replications) 127 for a total of 60 pots. In addition, in each bioassay three pots of each plant growth medium were 128 not inoculated and served as the negative check in order to evaluate compost effects on plant 129 growth in the absence of disease, resulting in an additional 36 control pots for a total of 96 pots 130 per bioassay. 131 The suppressive capacity of olive mill compost against Verticillium wilt was evaluated in cotton 132 plants cv. Carlota (Eurosemillas, S.A., Córdoba, Spain). Cotton bioassays are faster than those 133 for olive, and facilitate standardized plant development. The similarity of both cotton and olive 134 pathosystems makes this host substitution possible (López-Escudero and Mercado-Blanco, 135 2011). 136 Each replication consisted of one 0.8 liter pot with one cotton plant derived from three seeds that 137 were pre-germinated, sown in individual pots and thinned after one week. The plants were grown 138 in a growth chamber set at 25ºC (day) and 23ºC (night), with 14 h light, 10 h dark, and drip-139 irrigated. The plants were fertilized with 1 g / liter of Peters foliar feed 27-15-12 (N-P-K, Scotts, 140 Heerlen, The Netherlands) on a weekly basis. 141 An isolate of Verticillium dahliae defoliant pathotype (isolate V25) obtained from a diseased olive 142 tree was used as the pathogen. The isolate was grown on PDA with streptomycin sulphate (0.05 143 g / liter). The plates were scraped with water, filtered through cheesecloth, and the conidia were 144 Avilés, Plant Disease 7 counted with a hemocytometer. Twelve days after sowing, each pot was placed in a plastic bag 145 which, in turn, was inserted into an empty pot. The inoculum was adjusted to 5x106 conidia per ml 146 of solution based on previous preliminary assays. A 400 ml aliquot of inoculum solution was 147 added to each pot as a drench, and allowed to infuse through the growth media for one hour. The 148 pots were then drained by removing the plastic bag from around each pot and incubated at 90% 149 relative humidity for 3 days in the dark. The controls were treated in the same way using water. 150 Disease severity was scored twice a week based on a symptom severity scale where: 0 = 151 asymptomatic plants; 1 = slightly symptomatic plants (1 to 33% of leaves affected); 2 = 152 moderately symptomatic plants (34 to 66% of leaves affected); 3 =severely symptomatic plants 153 (67 to 99% of leaves affected but plants not dead); and 4 = dead plants (Bejarano-Alcázar et al., 154 1995). Each score was converted to the midpoint of the corresponding disease severity range 155 prior to using parametric analyses. The standardized area-under-the-disease-progress-curve 156 (AUDPCs) per pot was calculated from the disease severity values by the trapezoidal integration 157 method between the onset of symptoms and the ending time of the bioassay divided by the total 158 duration (days) of the epidemic in each bioassay, in order to compare the various bioassays, in 159 which the duration of the epidemic varied (Campbell and Madden, 1990). The disease severity at 160 the end of bioassays and the AUDPCs data were used for further analyses. 161 The bioassays ended when the majority of the inoculated plants grown in the unamended peat 162 media were dead (about two months). At the end of the bioassays, fresh shoot weight was 163 recorded, as well as dry weight after drying in a forced-air oven until a constant weight was 164 obtained. 165 In order to quantify the concentration of V. dahliae microsclerotia in the rhizosphere at the end of 166 the bioassays, rhizospheric and non-rhizospheric growth media were separated following Dhingra 167 and Sinclair (1995). Rhizosphere samples from five pots for each compost amendment, block and 168 Avilés, Plant Disease 8 bioassay were collected, combined, crushed, mixed and air-dried for 4 weeks. For wet sieving 169 analysis (Harris et al., 1993), 25 g of each plant growth medium was suspended in 250 ml of 170 distilled water and shaken for 1 h at 270 rpm in an orbital shaker. The suspension was then 171 washed through nested 150 and 36 μm sieves with tap water, and the material on the 36 μm 172 sieve was recovered and made up to 100 ml with distilled water. Aliquots of 1-ml suspension 173 were plated on modified soil extract agar medium (Harris et al., 1993) with 10 plates per sample. 174 The plates were incubated at 22 °C in the dark for 2 weeks and the plant growth medium 175 residues were washed away with distilled water. The plates were then dried and incubated for an 176 additional 2 to 3 weeks, after which the V. dahliae colonies were counted. 177 Physical and chemical properties of the plant growth media. Bulk density, pH and EC of the 178 growth media were measured on sub-samples collected from each of the formulated and 179 incubated plant growth media used in each of the bioassays. Electrical conductivity and pH were 180 measured in 1:5 v/v water extracts, according to Handreck and Black (2002), in two samples for 181 each plant growth medium, and block, bioassay and mean values per block were used for 182 statistical analysis. Bulk density was also determined for each of these samples, following De 183 Boodt et al. (1974). 184 To assess phytotoxicity, a seedling growth test was performed directly in the plant growth media. 185 Prior to conducting the bioassays, three bags (replications) per plant growth medium were 186 prepared and incubated for 11 days, under the conditions previously described, and three 187 subsamples collected from each bag were tested for phytotoxicity following the modified method 188 of Ortega et al. (1996). For this test, 20 ml volume sample of the plant growth media was placed 189 in 9 cm diameter Petri dishes, wetted with distilled water, and ten lettuce (Lactuca sativa) seeds 190 were sown in each dish (3 Petri dishes per plant growth medium and bag). After sowing, the 191 dishes were placed in an incubator at 20ºC in the dark for one week. Subsequently, the 192 germinated seeds were counted (G) and radicle growth (L) measured. The germination index (GI) 193 Avilés, Plant Disease 9 was calculated according to the formula GI = G/Go x L/Lo x 100, where Go and Lo are the 194 germination percentage and radicle growth of the control, respectively (Zucconi et al., 1985). The 195 germination index can assess both low toxicity, which mainly affects root growth, and high 196 toxicity, which affects seed germination (Selim et al., 2012). 197 A fungitoxicity assay was performed to detect inhibition of V. dahliae mycelial growth in each 198 plant growth medium amended with either shortand long-matured compost. Sub-samples 199 collected from the same bags used for phytotoxicity measurement were dried at room 200 temperature, milled and sieved at 250 μm. As described by Kokalis-Burelle and Rodríguez-201 Kábana (1994), the culture media were prepared with each milled plant growth medium by 202 suspending 100 g of this material and 15 g of agar in one liter of deionized water. Control plates 203 without the plant growth medium (water agar) were also prepared. The media were autoclaved at 204 105ºC for 30 min. The plates were then inoculated with a 5-mm-diameter plug of V. dahliae taken 205 from the leading edge of a 6-day-old culture. Twenty replicate plates per treatment and replication 206 (n=60) were incubated at 25ºC in the dark. After 20 days, radial fungal growth was evaluated and 207 the percentage of growth inhibition (PGI) calculated as follows: 208 PGI = (Growth in control – Growth in treatment) x 100 / Growth in control 209 210 Biological characteristics of the plant growth media. Biological analyses were performed on 211 sub-samples collected from incubated growth media at the beginning of each bioassay prior to 212 being divided into the pots (three bags (replications) per plant growth medium). The enzymatic 213 activities in the plant growth media were determined using the API-ZYM system (BioMereux SA, 214 Marcy-l’Etoile, France). With the API-ZYM system, semi-quantitative evaluations were performed 215 on the activities of 19 hydrolyticenzymes [alkaline phosphatase, esterase (C4), esterase–lipase 216 (C8), lipase (C14), leucine arylamidase, valine arylamidase, cistine arylamidase, trypsin, α-217 Avilés, Plant Disease 16 Of the cultural microorganisms, the population of Bacillus spp. and the ratio of cellulolytic 364 actinomycetes / cellulolytic bacteria were the highest in growth medium heat treated at 60ºC. 365 Heating at 60ºC seemed to favor copiotrophic bacteria, but the concentration of these populations 366 did not differ significantly from the untreated OC3s-GM. Nevertheless, copiotrophic bacteria 367 developed higher populations in OC3s-GM-H than in OC3s-GM-A (Table 8). 368 For OC3s-GM treatments, the best MLR model was found when oligotrophic actinomycetes and 369 Bacillus spp. populations were taken into account as predictive variables. This model explained 370 86% of the variation in Verticillium wilt disease severity (r2 =0.86, P < 0.001; Supplementary Fig. 371 2A). These variables were also included in the best model for estimating microsclerotia 372 concentration in the rhizosphere at the end of bioassays. This model explained 74% of the 373 variation in a Napierian logarithm of the microsclerotia concentration (r2 = 0.74, P < 0.01; 374 Supplementary Fig. 2B) (both dependent variables are correlated). Final Verticillium wilt severity 375 explained 84.5% of the variation in a Napierian logarithm of the microsclerotia concentration 376 (Severity = 18.17 + 15.04 x ln microsclerotia concentration; r2 = 0.85, P < 0.001) meaning that as 377 disease severity increased so did the concentration of microsclerotia enumerated from the 378 rhizosphere. 379 DISCUSSION 380 There is evidence that certain olive mill composts are suppressive to Verticillium wilt, reduce 381 microsclerotia concentration or inhibit mycelial growth (Alfano et al., 2011; Arriagada et al., 2012; 382 Castaño and Avilés, 2013; Lima et al., 2008; Malandraki et al., 2008; Markakis et al., 2008; Vitullo 383 et al., 2013; Yildiz and Benlioglu, 2010). However, our results demonstrate that not all olive mill 384 composts are suppressive to Verticillium wilt when used as an amendment in plant growth media; 385 they can even exacerbate disease compared to peat alone. In these bioassay trials, only the peat 386 formulated with OC3 compost, regardless of maturation level, was suppressive to Verticillium wilt 387 Avilés, Plant Disease 17 when compared to peat, an effect which was related to a lower concentration of microsclerotia at 388 the end of bioassays. On the other hand, compost maturation did not affect disease 389 suppressiveness, despite the fact that short matured composts were shown to have a lower 390 microsclerotia concentration at the end of bioassays than long matured composts. The 391 relationship between the compost curing duration and disease suppression was not always clear. 392 There is evidence for decreased suppressiveness in compost with increased age for 393 Phytophthora spp. (Danon et al., 2007), Pythium spp. (Darby et al., 2006; Stone et al., 2001), and 394 Sclerotium rolfsii (Danon et al., 2007). However, evidence also exists for an increased 395 suppressive effect in more matured compost, in the case of Fusarium wilt on melon (Saadi et al., 396 2010), and Rhizoctonia solani on cucumber (Trillas et al., 2006; Tuitert et al., 1998). Thus, the 397 effects of maturation on compost suppressiveness seem to depend on the nature of the 398 composted materials and the pathosystem (Avilés et al., 2011; Hoitink and Boehm, 1999). 399 For the four olive mill composts studied in these bioassays, the explicative factors for Verticillium 400 wilt disease severity and the final rhizosphere microsclerotia concentration, were determined to 401 be API-ZYM Shannon’s diversity index, β-glucosidase activity and pH (negatively related) and EC 402 (positively related) based on the best MRL models. In the present experimental design, disease 403 severity and microsclerotia concentration were considered dependent variables, because V. 404 dahliae inoculation was carried out in plant growth media free from this pathogen. Papasotiriou et 405 al. (2013) also proposed that the suppressive effect of olive mill compost on V. dahliae on 406 eggplant was due to abiotic and biotic factors. In order to explain the possible biological meaning 407 of these predictive factors and the complex mechanism involved, it is important to discuss the 408 biological effect of each factor. Castaño and Avilés (2013), also working with different composts 409 formulated as plant growth media, found negative correlations between olive and cotton 410 Verticillium wilt disease severity, β-glucosidase activity and pH. For green manure amendments, 411 Ochiai et al. (2008) included microbial activity, pH and inoculum density in their best MLR model 412 Avilés, Plant Disease 18 for Verticillium wilt disease in potato. Nevertheless, in this regression model, pH had a positive 413 relation to disease severity. The difference in the direction of these relationships with the pH 414 could be due to the range of pH studied. In the present work, the pH of plant growth medium 415 amended with olive mill composts ranged from 7.64 to 8.09, while in Ochiai et al. (2008) the pH 416 range was from 5.2 to 7.5. Therefore, in acid or neutral media, pH increase seems to favor 417 Verticillium wilt while in alkaline media, such as plant growth media with olive mill composts, 418 decreasing pH seemed to favor this disease and is consistent with other research where 419 Verticillium wilt of tomato and cotton increased in acidic soils limed to increase pH (Jones et al., 420 1971; Shao and Foy, 1982). Furthermore, certain actinomycetes, which are V. dahliae 421 antagonists, are more antagonistic in alkaline pHs (Abd-Allah, 2001; Bonjar and Aghighi, 2005). 422 As for EC, some authors have observed that irrigating with saline water boosted V. dahliae 423 infections in potato, pistachio and cotton (Besri, 1981; Kaufman et al., 1990; Mohammadi et al., 424 2007). Other works have demonstrated that salinity increases root and shoot colonization by V. 425 dahliae and, and consequently, the disease severity (Levin et al., 2007; Saadatmand et al., 2008; 426 Pascual et al., 2009). These authors also suggested salinity as a possible cause for the 427 enhancement of Verticillium wilt in pepper further confirming the important role of EC in 428 increasing Verticillium wilt development similar to the MLR models proposed here. 429 Compost amendments have been shown to affect microbial community structure and composition 430 and can enhance microbial diversity (Bonilla et al., 2012; D’Hose et al., 2014; Saison et al., 431 2006). Disease suppressiveness is often related to overall increases microbial biomass and 432 activity in plant growth media or soils, which create a competitive environment which is 433 deleterious to the pathogen (Bonanomi et al., 2010; Bonilla et al., 2012; Hadar and 434 Papadopoulou, 2012; Larkin, 2015). Larkin et al. (2011) working with green manures, found high 435 diversity (Shannon´s index) and the highest microbial activity in the treatment with the lowest 436 severity in Verticillium wilt on potato. In the same sense, other authors working with organic soil 437 Avilés, Plant Disease 19 amendments have found negative correlations between microbial activity and the disease 438 severity of Verticillium wilt on potato (Conn and Lazarovits, 1999; Davis et al., 1994, 1996). 439 Conversely, with composts formulated as plant growth media, Termorshuizen et al. (2006) found 440 a positive correlation between respiration and disease severity of Verticillium wilt on eggplant, but 441 were unable to clearly explain this relationship. 442 Regarding other properties evaluated in this study, the low fungitoxicity observed in the 443 suppressive medium OC3-GM compared to the other media did not seem to be an important 444 factor for suppressiveness, given that the medium with the highest fungitoxicity, OC2-GM, also 445 showed the highest AUDPCs value. On the other hand, the high level of lysogenic enzymatic 446 activity found in OC3-GM could be associated with fungal cell wall degradation. Cell wall-447 degrading enzymes are involved in the antagonistic activity of biocontrol agents against 448 phytopathogenic fungi (Elad, 1985; Gajera, 2012; Madi et al., 1997). 449 To elucidate the main mechanisms explaining the suppressive effects of OC3s-GM, it was altered 450 with treatments designed to disrupt the microbial community and assess the role of nitrogen on 451 disease suppression. The predictive factors for suppressiveness obtained from the initial 452 bioassays conducted using the growth media amended with four olive composts do not 453 necessary correspond to the explicative suppressive properties observed from the bioassays 454 conducted with OC3s-GM. The microbial communities of the different OC3s-GM treatments were 455 triggered by a response to disturbances created through the heat treatment or nitrogen 456 application. The same organic matter composition can show different functional abilities 457 depending on the microbial community it harbours (van Bruggen and Semenov, 2000). The 458 varying Verticillium wilt severity between the heat treatments could be due to their different effects 459 on the microbial community, without discarding possible minor changes in the organic matrix. 460 Avilés, Plant Disease 20 Nitrogen supplementation did not affect suppressiveness, rather, it contributed slightly to 461 increased microsclerotia concentration. An increment in nitrogen availability can help 462 microsclerotia to overcome fungistasis (Green and Papavizas, 1968). Therefore, this loss of 463 fungistasis could allow microsclerotia to thrive in the presence of the host plant, which would 464 explain the increase in concentration observed. In any case, a possible low availability of nitrogen 465 in untreated OC3s-GM as a cause of suppressiveness is ruled out, given that N-supplemented 466 OC3s-GM-N maintains the same level of suppressiveness. 467 For the different treatments of suppressive OC3s-GM, both heat treatments (60ºC heat treated 468 and autoclaved) caused an important drop in suppressiveness, but heating at 60ºC most 469 significantly increased disease severity. Nevertheless, the heat treatments were still suppressive 470 in comparison to unamended peat, despite showing a similar microsclerotia concentration at the 471 end of bioassays. In contrast, the untreated OC3s-GM medium showed very low microsclerotia 472 concentration. Therefore, the suppressiveness and the absence of a reduction in microsclerotia in 473 heat treatments compared to peat could indicate that untreated OC3s-GM suppressiveness is 474 due to the presence of both general and specific suppressiveness. Alfano et al. (2011) also 475 suggested general and specific suppressiveness in this kind of compost against Pythium ultimum 476 and Fusarium oxysporum f.sp. lycopersici in tomato. The differential suppressive effect of 60º C 477 heat treatment compared to untreated OC3s-GM seems to indicate that mesophilic thermo-478 tolerant taxa are not involved in specific suppression in this compost. Therefore, the relatively 479 higher Bacillus spp. concentration observed in OC3s-GM-H compared to the other treatments 480 proves that these microorganisms, which are usually described as biocontrol agents, do not play 481 a major role in the specific suppressiveness of this plant growth medium. On the other hand 482 increased cellulolytic actinomycetes compared to cellulolytic bacteria in OC3s-GM-H seem to be 483 related to decreased suppressiveness, which contradicts observations by Tuitert et al. (1998) for 484 Rhizoctonia solani in household waste composts and Borrero et al. (2004) for tomato Fusarium 485 Avilés, Plant Disease 21 wilt in other agro-industrial waste composts. The higher mean level of API-ZYM activity and 486 diversity, alkaline phosphatase, esterase lipase and trypsin activities in OC3s-GM-H than in 487 OC3s-GM-A must be due to these thermo-tolerant taxa. 488 After both heat treatments, N-acetyl-β-glucosaminidase activity was not detected and acid 489 phosphatase activity decreased. Therefore, microorganisms contributing to these activities 490 (especially N-acetyl-β-glucosaminidase) could be involved in the specific suppressiveness 491 observed in this plant growth medium. N-acetyl-β-glucosaminidase activity is used as an index of 492 chitinolytic activity in environmental samples (O’Brien and Colwell, 1987). Chitinolytic enzymes 493 have been considered important in the biocontrol of soilborne pathogens because of their ability 494 to degrade fungal cell walls (Haran et al., 1996; Harman et al., 1993; Inbar and Chet, 1995; 495 Nguyen et al., 2008). Several chitinolytic enzymes have been identified in different kinds of 496 microorganisms, including various species of Streptomyces (Kinkel et al., 2012; Xue et al., 2013). 497 In the same way, acid phosphatase has been suggested to play a role in cell wall hydrolysis of 498 phytopathogens (Monteiro et al., 2010) and microbial phosphatase activity in the rhizosphere has 499 been demonstrated to enhance plant nutrition and consequently plant development and health 500 (Recena et al., 2015). The best LMR model found to explain disease severity and microsclerotia 501 concentration in the different treatments of the suppressive compost OC3s-GM showed the 502 importance of populations of oligotrophic actinomycetes and Bacillus spp. In this compost, 503 oligotrophic actinomycetes seem to favour disease suppression, while Bacillus spp. populations 504 did not. Many actinomycete taxa, such as Streptomyces, are associated with plant disease 505 suppression in many soils (Kinkel et al., 2012; Xue et al., 2013) and are soil saprophytes with a 506 crucial role in nutrient cycling (Kennedy, 1999). On the other hand, oligotrophic populations are 507 related to soils with high microbial diversity and suppressiveness (van Bruggen and Semenov, 508 2000). In the same way, in other plant growth media formulated with composts, higher 509 oligotrophic actinomycetes populations were found that were suppressive to Rhizoctonia solani 510 Avilés, Plant Disease 22 on cucumber (Tuitert et al., 1998) and Fusarium wilt on tomato (Borrero et al., 2004). In plant 511 growth media amended with olive mill compost which was suppressive to Fusarium wilt on tomato 512 and Pythium ultimum, high actinomycete and microbial quitinolytic aerobic populations were 513 found (Alfano et al., 2011). 514 These results suggest that olive mill composts demonstrated to be suppressive to Verticillium wilt, 515 such as OC3, and used to amend nursery plant growth media, can protect plantlets from this 516 disease at least during the transplant phase. The lack of disease symptoms that developed in 517 nonV. dahliae inoculated control treatments containing olive mill composts indicated that these 518 composts are also generally free of this pathogen. We can conclude that olive mill composts 519 amended plant growth media are not always suppressive against Verticillium wilt. However, for 520 these four compost sources, maturation level did not affect disease development. API-ZYM 521 Shannon’s diversity index, β-glucosidase activity and pH and EC in olive mill compost plant 522 growth media were the factors that best described Verticillium wilt disease severity and 523 rhizosphere microsclerotia concentration. In the olive mill compost that was characterized as 524 suppressive (OC3s-GM) general and specific suppressiveness were both involved. In this 525 compost, N-acetyl-β-glucosaminidase activitiy and the oligotrophic actinomycete populations 526 seemed to be involved in suppressiveness. 527 Acknowledgements. This research was supported by grants from Spain’s Ministerio de Ciencia 528 e Innovación (AGL2010-21982-C02-01). We would like to thank A. Gata, S. Castillo, S. 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For each column and plant growth medium or level of maturation factor, values followed by different letters are 790 significantly different according to ANOVA and Tukey’s Test (P<0.05). Data for analysis were transformed with x2 for Severity, x0.67 for AUDPCs and x0.17 for 791 Avilés, Plant Disease 35 microsclerotia concentration in growth medium. Severity and AUDPCs were n=60 for growth medium and n=120 for level of maturation. Microsclerotia 792 concentration was n=12 for growth medium and n=24 for level of maturation. 793 x OC1-GM: growth medium formulated with OC1 compost and peat 1:2 v/v; OC2-GM: growth medium formulated with OC2 compost and peat 1:2 v/v; OC3-GM: 794 growth medium formulated with OC3 compost and peat 1:2 v/v; OC4-GM: growth medium formulated with OC4 compost and peat 1:2 v/v. 795 y Disease severity scale: 0 = asymptomatic plants; 1 = slightly symptomatic plants (1-33% of leaves affected); 2 = moderately symptomatic plants (34-66% of 796 leaves affected); 3 =severely symptomatic plants (67-99% of leaves affected but plants not dead); and 4 = dead plants. Each scale score was converted to the 797 midpoint of the corresponding disease severity range. 798 z Short: Compost with a curing phase of 4 months; Long: Compost with a curing phase of 1 year. 799 800 801 Avilés, Plant Disease 36 Table 2. The physical and chemical properties of the olive mill compost amended growth media prior to use in the bioassaysw. 802 Growth mediumy Propertiesx BD (g ml-1) pH EC (mS cm-1) GI (%) PGI (%) OC1-GM 0.24±0.01 a 7.81±0.12 b 0.82±0.06 bc 84.9±8.1 ab 21.2±0.8 c OC2-GM 0.32±0.05 a 7.64±0.07 b 0.88±0.10 bc 103.4±8.6 a 31.5±0.9 a OC3-GM 0.32±0.05 a 8.09±0.07 a 0.73±0.10 c 83.8±7.9 ab 3.9±1.0 d OC4-GM 0.24±0.02 a 7.87±0.08 ab 0.98±0.13 b 61.4±6.0 b 25.7±1.8 b peat 0.13±0.01 b 5.48±0.05 c 2.55±0.11 a 102.3±10.0 a 1.9±0.9 d Maturationz short 0.20±0.01 b 7.27±0.19 b 1.05±0.17 a 76.6±4.4 b 19.6±1.2 a long 0.29±0.03 a 7.49±0.18 a 1.33±0.10 a 97.6±6.2 a 14.5±0.7 b w Data represent mean ± standard error. For each column and plant growth medium or level of maturation factor, values followed by different letters are 803 significantly different according to ANOVA and Tukey’s Test (P<0.05). Data for analysis were transformed with x-1.5 for BD, x-0.2 for GI. BD: n=4 for growth 804 Avilés, Plant Disease 37 medium and n=8 for level of maturation. pH and EC: n=12 for growth medium and n=24 for level of maturation. GI: n=18 for growth medium and n=36 for level 805 of maturation. PICR: n=120 for growth medium and n=240 for level of maturation. 806 x BD: Bulk density; EC: Electrical conductivity; GI: Germination Index; PGI: Percentage of Verticillium dahliae micelial growth inhibition. 807 y OC1-GM: growth medium formulated with OC1 compost and peat 1:2 v/v; OC2-GM: growth medium formulated with OC2 compost and peat 1:2 v/v; OC3-GM: 808 growth medium formulated with OC3 compost and peat 1:2 v/v; OC4-GM: growth medium formulated with OC4 compost and peat 1:2 v/v. 809 z Short: Compost with a curing phase of 4 months; Long: Compost with a curing phase of 1 year. 810 811 812 Avilés, Plant Disease 38 Table 3. Oxygen uptake rate (OUR), β-glucosidase activity, API-ZYM mean activity and Shannon’s diversity index of the olive mill compost amended 813 growth media prior to use in the bioassaysx. 814 Growth mediumy OUR (mg O2 kg-1 day-1) β-glucosidase activity (μg ρnitrofenol ml-1h-1) API-ZYM mean activity API-ZYM Shannon’s diversity index OC1-GM 1880.1±177.3 a 177.1±12.6 ab 1.01±0.08 ab 2.09±0.08 ab OC2-GM 1189.7±150.6 bc 100.1±6.0 c 0.69±0.06 c 1.86±0.07 bc OC3-GM 1308.3±103.3 b 151.3±5.4 b 1.11±0.06 a 2.22±0.06 a OC4-GM 1439.1±160.8 b 201.6±19.8 a 0.90±0.10 b 2.06±0.07 ab peat 1037.7±130.9 c 62.4±9.5 c 0.56±0.06 c 1.68±0.12 c Maturationz short 1741.7±73.9 a 147.1±14.4 a 0.77±0.05 a 1.87±0.06 a long 1000.3±84.2 b 129.9±8.5 b 0.94±0.06 a 2.09±0.05 a x Data represent mean ± standard error. For each column and plant growth medium or level of maturation factor, values followed by different letters are 815 significantly different according to ANOVA and Tukey’s Test (P<0.05). n=12 for growth medium and n=24 for level of maturation. 816 Avilés, Plant Disease 39 y OC1-GM: growth medium formulated with OC1 compost and peat 1:2 v/v; OC2-GM: growth medium formulated with OC2 compost and peat 1:2 v/v; OC3-GM: 817 growth medium formulated with OC3 compost and peat 1:2 v/v; OC4-GM: growth medium formulated with OC4 compost and peat 1:2 v/v. 818 z Short: Compost with a curing phase of 4 months; Long: Compost with a curing phase of 1 year. 819 820 821 Avilés, Plant Disease 40 Table 4. The API-ZYM enzymatic activities of olive mill compost amended growth media prior to use in the bioassaysx. 822 Growth mediumy Alkaline phosphatase Esterase (C4) Esterase lipase (C8) Lipase (C14) Leucine arylamidase Valine arylamidase OC1-GM 2.2±0.3 b 2.8±0.1 ab 2.1±0.1 ab 0.1±0.1 b 2.7±0.2 a 0.4±0.1 ab OC2-GM 2.6±0.3 ab 2.3±0.2 bc 1.4±0.1 c 0.2±0.1 b 1.9±0.2 b 0.3±0.1 b OC3-GM 3.3±0.3 a 3.2±0.2 a 2.6±0.1 a 0.8±0.2 a 2.8±0.1 a 0.8±0.1 a OC4-GM 2.3±0.2 b 2.1±0.1 c 1.8±0.1 bc 0.1±0.1 b 2.4±0.2 ab 0.3±0.1 b peat 0.7±0.1c 2.0±0.1 c 1.3±0.1 c 0.0±0.0 b 0.8±0.2 c 0.1±0.1 b Maturationz short 2.0±0.2 a 2.6±0.1 a 1.8±0.1 a 0.3±0.1 a 2.1±0.2 a 0.3±0.1 a long 2.5±0.2 a 2.3±0.1 a 1.9±0.1 a 0.2±0.1 a 2.1±0.1 a 0.5±0.1 a x Data represent mean ± standard error. For each column and plant growth medium or level of maturation factor, values followed with different letters are 823 significantly different according to ANOVA and Tukey’s Test (P<0.05). n=12 for growth medium and n= 24 for level of maturation. 824 y OC1-GM: growth medium formulated with OC1 compost and peat 1:2 v/v; OC2-GM: growth medium formulated with OC2 compost and peat 1:2 v/v; OC3-GM: 825 growth medium formulated with OC3 compost and peat 1:2 v/v; OC4-GM: growth medium formulated with OC4 compost and peat 1:2 v/v. 826 z Short: Compost with a curing phase of 4 months; Long: Compost with a curing phase of 1 year. 827 Avilés, Plant Disease 41 828 Table 4 (Cont.). The API-ZYM enzymatic activities of olive mill compost amended growth media prior to use in the bioassaysx. 829 Growth mediumy Acid phosphatase Naphtol-AS-BIphosphohydrolase β-Galactosidase α-Glucosidase β-Glucosidase N-acetyl-β-glucosaminidase OC1-GM 4.0±0.3 a 1.2±0.1 ab 0.3±0.1 ab 0.2±0.1 b 1.1±0.1 a 1.3±0.3 a OC2-GM 2.8±0.3 b 0.8±0.1 b 0.1±0.1 b 0.1±0.1 b 0.1±0.1 b 0.2±0.1 b OC3-GM 3.1±0.2 b 0.8±0.1 b 0.3±0.1 ab 0.6±0.1 a 0.9±0.1 a 0.9±0.2 ab OC4-GM 2.9±0.4 b 1.7±0.2 a 0.4±0.1 ab 0.0±0.0 b 1.1±0.5 a 1.3±0.3 a peat 3.5±0.3 ab 0.8±0.2 b 0.6±0.1 a 0.0±0.0 b 0.4±0.1 ab 0.2±0.1 b Maturationz short 2.9±0.2 a 1.0±0.1 a 0.1±0.1 b 0.1±0.1 a 0.4±0.1a 0.7±0.2 a long 3.6±0.2 a 1.1±0.1 a 0.6±0.1 a 0.2±0.1 a 1.0±0.2 a 0.8±0.2 a x Data represent mean ± standard error. For each column and plant growth medium or level of maturation factor, values followed with different letters are 830 significantly different according to ANOVA and Tukey’s Test (P<0.05). Data for analysis were transformed with x0.59 for N-acetyl-β-glucosaminidase. n=12 for 831 growth medium and n= 24 for level of maturation. 832 Avilés, Plant Disease 48 871 Table 8 (Cont.). Microbial ecological-nutrient groups in treatments of identified suppressive olive mill compost (OC3s-GM) at the end of bioassaysv. 872 Treatmentw Bacillus spp. Talaromyces spp. Pseudomonas spp. Fungi OA/OBx CA/CBy OB/CoBz (CFU/ml growth medium x 105) OC3s-GM 16.43 b 0.00 a 0.01 a 4.11 a 0.12 a 0.11 b 1.17 a OC3s-GM-N 6.00 b 0.00 a 0.01 a 1.30 a 0.10 a 0.11 b 2.36 a OC3s-GM-A 24.64 b 0.00 a 0.06 a 3.30 a 0.01 a 0.03 b 8.82 a OC3s-GM-H 72.75 a 0.00 a 0.07 a 2.76 a 0.10 a 0.83 a 0.70 a Peat 1.48 b 0.00 a 0.00 a 1.31 a 0.10 a 0.06 b 1.55 a v Within each column, values followed by different letters are significantly different based on Tukey ’s test at P < 0.05. Analysis of variance was performed with 873 transformed data with log (x) for Fungi, n=3. 874 w OC3s-GM: growth medium formulated with short maturation OC3 compost and peat 1:2 v/v; OC3s-GM-N: OC3s-GM fertilized with 0.52 g/L of ammonium 875 nitrate; OC3s-GM-A: OC3s-GM autoclaved (two successive times within a 24 h interval for 1 h at 120 ºC, 1.2 atm.); OC3s-GM-H: OC3s-GM heated (to 60 ºC for 876 6 days). 877 Avilés, Plant Disease 49 x OA/OB: Oligotrophic Actinomycetes/ Oligotrophic Bacteria. 878 y CA/CB: Cellulolytic Actinomycetes / Cellulolytic Bacteria. 879 z OB/CoB: Oligotrophic Bacteria/ Copiotrophic Bacteria 880 Avilés, Plant Disease 50 881 Estimated microsclerotia concentration [ln (MS/g)] -1 0 1 2 3 4 Microsclerotia concentration observed [ln (MS/g)] -1 0 1 2 3 4 Line 1:1 B Estimated AUDPCs 0.0 0.2 0.4 0.6 0.8 1.0 AUDPCs observed 0.0 0.2 0.4 0.6 0.8 1.0 A Line 1:1 882 883 Supplementary Fig. S1. A) Area under the disease progress curve standardized (AUDPCs) 884 estimation in growth media formulated with olive mill composts as a function of: API-ZYM 885 Shannon’s diversity index, β-glucosidase activity, pH and electrical conductivity (EC); AUDPCs = 886 13.738 - 1.432 x API-ZYM Shannon’s diversity index - 0.005 x β-glucosidase activity - 1.239 x pH 887 + 0.002 x EC; R2 = 0.71, P < 0.0001, and coefficients for each explicative variable significant at P 888 < 0.01 in the least significant case; and mean absolute error = 0.42. Observed severity data were 889 mean values from each block and different olive mill composts in four bioassays, n=44. 890 Avilés, Plant Disease 51 B) Estimation of the napierian logarithm of microsclerotia concentration in growth media 891 formulated with olive mill composts as a function of: API-ZYM Shannon’s diversity index, β-892 glucosidase activity, pH and EC; ln (MS/g) = 14.543 - 1.063 x API-ZYM Shannon’s diversity index 893 - 0.001 x β-glucosidase activity - 1.626 x pH + 0.003 x EC; R2 = 0.68, P < 0.0001, and coefficients 894 for each explicative variable significant at P < 0.05 in the least significant case; and mean 895 absolute error = 0.64. Observed microsclerotia concentration data were mean values from each 896 block and different olive mill composts in four bioassays, n=44. 897 898 899 Avilés, Plant Disease 52 900 Estimated severity 020 40 60 80 Severity observed 0 20 40 60 80 A Line 1:1 02 4 0 2 4 Estimated microsclerotia concentration [ln (MS/g)] Microsclerotia concentration observed [ln (MS/g)] B Line 1:1 901 Supplementary Fig. S2. A) Verticillium wilt severity estimation in growth media with OC3s-GM 902 treatments as a function of: oligotrophic actinomycetes, and Bacillus spp. populations; Severity = 903 16.157 - 4.684 x 10-6 x oligotrophic actinomycetes + 1.235 x 10-5 x Bacillus spp.; R2 = 0.86, P < 904 0.001, and coefficients for each explicative variable significant at P < 0.01 in the least significant 905 case; and mean absolute error = 8.96. Observed severity data were mean values from each 906 assay and different treatments of suppressive OC3s-GM, n=12. 907 B) Estimation of the napierian logarithm of microsclerotia concentration in growth media with 908 OC3s-GM treatments as a function of: oligotrophic actinomycetes, and Bacillus spp. populations; 909 ln (MS/g) = -0.338 - 1.341 x 10-7 x oligotrophic actinomycetes + 6.456 x 10-7 x Bacillus spp.; R2 910 Avilés, Plant Disease 53 = 0.74, P < 0.01, and coefficients for each explicative variable significant at P < 0.05 in the least 911 significant case; and mean absolute error = 0.73. Observed microsclerotia concentration data 912 were mean values from each assay and different treatments of suppressive OC3s-GM, n=12 913 Avilés, Plant Disease 54 Supplementary Table S1. Characteristics of olive mill waste composts. 914 Compost Company Composted material (% w/w) Maturationa Propertiesb BD (g ml-1) OM (%) C/N EC (mS cm-1) pH OC1 Orobaena S.A.T., Baena, Córdoba, Spain wet olive husks (82%), olive leaves waste (15%), cereal straw (3%) short 0.29 66.3 15.3 5.1 9.1 long 0.39 68.8 20.5 1.9 9.0 OC2 La Reja S.L., Bobadilla, Málaga, Spain wet olive husks (50%), olive leaves waste (1%), manure and straw (49%) short 0.32 33.6 11.5 5.4 8.1 long 0.80 26.7 11.7 1.6 7.7 OC3 Vado Olivo S.A., Cazorla, Jaén, Spain wet olive husks (63%), olive leaves waste (30%), sheep manure (7%) short 0.55 55.0 21.4 4.3 8.5 long 0.65 39.8 19.6 2.2 7.5 OC4 Rafael Alonso S.L., Tabernas, Almería, Spain wet olive husks (94%), olive leaves waste (3%), chicken manure (3%) short 0.64 65.8 13.6 11.4 9.1 long 0.42 57.6 13.7 9.9 8.7 a Short: Compost with a curing phase of 4 months; Long: Compost with a curing phase of 1 year. 915 b BD: Bulk density; OM: Organic matter; C/N: Carbon/Nitrogen ratio; EC: Electrical conductivity. 916 917 918