Carnation Fusarium wilt suppression in four composts

1 1 CARNATION FUSARIUM WILT SUPPRESSION IN FOUR COMPOSTS 2 3 Celia Borrero, Mª Isabel Trillas and Manuel Avilés 4 5 First, author: Dept. Ciencias Agroforestales, Escuela de Ingeniería Técnica 6 Agrícola, Universidad de Sevilla, Ctra Utrera km 1, E-41013 Sevilla, Spain. Fax: 34 7 954486436, e-mail: [email protected] 8 Second author: Dept. Biologia Vegetal, Universitat de Barcelona, Facultat de Biologia, 9 Avda Diagonal, 645, E-08028 Barcelona, Spain. Fax: 34 954486436, e-mail: 10 [email protected]. 11 Third author: Dept. Ciencias Agroforestales, Escuela de Ingeniería Técnica Agrícola, 12 Universidad de Sevilla, Ctra Utrera km 1, E-41013 Sevilla, Spain. Fax: 34 954486436, 13 e-mail: avile[email protected] 14 15 Corresponding author: Manuel Avilés. 16 17 2 Abstract 18 Fusarium wilt is now a major disease of carnation crops worldwide. Methyl 19 bromide, which is used to remedy it, is environmentally unsafe. An alternative approach 20 integrated in biological control is to grow crops in suppressive media. Suppressiveness 21 of seven plant growth media to carnation Fusarium wilt was evaluated in bioassays with 22 carnation cv. `Medea´. These media were: (i) grape marc compost, (ii) cork compost, 23 (iii) olive oil husk + cotton gin trash composted and mixed with rice husk, (iv) spent 24 mushroom composted and mixed with peat, (v) coir fibre, (vi) light peat and (vii) 25 vermiculite. In order to look for carnation Fusarium wilt suppressiveness indicators, 26 growth media pH and β-glucosidase activity were evaluated. Furthermore, Fusarium 27 oxysporum populations were measured in plant growth media at the beginning and at 28 the end of bioassays. All the compost media showed a range of suppressiveness. Grape 29 marc compost was the most effective plant growth media to suppress carnation 30 Fusarium wilt. On the other hand coir fibre, peat and vermiculite were conducive for 31 this disease. β-glucosidase activity and pH were correlated with severity like other 32 report with tomato. Therefore these two parameters are good indicators for carnation 33 Fusarium wilt suppressiveness, and possibly for other Fusarium oxysporum 34 pathosystems. All composts showed similar Fusarium oxysporum populations at the end 35 of bioassays than peat and vermiculite. 36 Keywords: biological control, compost, Dianthus cariophyllus, Fusarium 37 oxysporum f. sp. dianthi, soilless. 38 39 40 3 Introduction 41 The most important phytopathologycal problem affecting carnation (Dianthus 42 cariophyllus L.) in most areas of the world where the crop is grown is Fusarium wilt, 43 incited by Fusarium oxysporum Shlecht. f. sp. dianthi (Prill. & Del.) Snyd. & Hans. 44 (Fod). Fusarium wilt is prevalent in SW Spain, the site with 79% of the national 45 production area of carnation and 45% of total production (Anonymous, 2006). 46 Susceptible cultivars suffer severe yield losses and considerable yield losses are also 47 common in carnation cultivars previously described as resistant (Prados-Ligero, 48 Basallote-Ureba, López-Herrera & Melero-Vara, 2007). The crop is mainly produced as 49 a monoculture in plastic house; rooted cuttings are planted in late spring (May to June) 50 and removed after 22 to 23 months before preparation of the soil for new plantings. 51 Consequently, populations of soilborne plant pathogens often increase to unacceptable 52 levels (Prados-Ligero, Basallote-Ureba, López-Herrera & Melero-Vara, 2007). 53 Due to the great crop losses, growers relied on soil fumigation with chemicals 54 like methyl bromide. The use of methyl bromide is forbidden except for critical uses, 55 because of its environmental risks. Therefore, other culture alternatives may be tested. 56 Carnations can be cultured in containers or bags using plant growth media where 57 fumigation is not needed (Pizano, 2001). A rapid technique to identify suppressive plant 58 growth media before use could facilitate the choice of such media. Two of the growth 59 media most widely used in Spain are peat and coir fibre. However, media formulated 60 with composts can suppress different formae speciales of Fusarium wilt in comparison 61 to peat. These formae speciales are Fusarium wilt of carnation (Cebolla & Pera, 1983; 62 Orlikowski, 1983; Pera & Calvet, 1989), Fusarium wilt of chrysanthemum (Chef, 63 Hoitink, & Madden, 1983), Fusarium wilt of cyclamen (Garibaldi, 1988), Fusarium wilt 64 4 of flax (Chef, Hoitink, & Madden, 1983), Fusarium wilt of iris (Garibaldi, 1988), 65 Fusarium wilt of radish (Trillas-Gay, Hoitink, & Madden, 1986), Fusarium wilt of 66 sweet basil (Reuveni, Raviv, Krasnovsky, Freiman, Medina, Bar & Orion, 2002); and 67 Fusarium wilt of tomato (Szczech, Rondomanski, Brzeski, Smolinska, & Kotowski, 68 1993; Szczech, 1999; Cotxarrera, Trillas-Gay, Steinberg, & Alabouvette, 2002). 69 Furthermore, we have evaluated the suppressiveness of four composts plant growth 70 media in comparison to peat and vermiculite to tomato Fusarium wilt (Trillas, Avilés, 71 Ordovás, Bello & Tello, 2002; Borrero, Trillas, Ordovás, Tello & Avilés, 2004; 72 Borrero, Infantes, González, Tello & Avilés, 2005). In these assays pH and microbial 73 activity were studied as predictive factors of tomato Fusarium wilt (Borrero, Trillas, 74 Ordovás, Tello & Avilés 2004). 75 Our study objectives were to determine: (i) the capacity of four compost plant 76 growth media as well as peat, coir fibre and vermiculite to suppress carnation Fusarium 77 wilt, (ii) if plant growth media pH and microbial activity indicate carnation disease 78 suppressiveness. 79 Materials and methods 80 1. Plant growth media. Four different composted residues from the agricultural 81 industry wastes were evaluated for carnation Fusarium wilt suppression: (i) Cork 82 compost (CC) from cork (Quercus suber L.) transformation, (ii) grape marc compost 83 (GMC) from the alcohol distilling industry (grape skins, seeds and stems) and (iii) olive 84 oil husk + cotton gin trash, 1:1 v/v, composted and mixed with rice husk (1:1 v/v) 85 (OC+R) were composted according to procedures previously described (Trillas, Avilés, 86 Ordovás, Bello, & Tello, 2002). The fourth plant growth medium (iv) was spent 87 mushroom composted (Recomsa, Quintanar del Rey, Spain) mixed with amended light 88 5 peat (1:1 v/v) (SM+P). Composts were evaluated with three plant growth media that are 89 widely used in Spain: fertilized and amended Sphagnum light peat (Klasmann, Valimex, 90 Palleter, Spain), coir fibre (Cocopeat, Projar, Valencia, Spain) and expanded vermiculite 91 (Vermiculita y derivadas, Gijón, Spain). Peat was amended with 4g l-1 CaCO3 and 92 fertilized with 0.33 g l-1 of K2O (50% high solubility granulated; Compo Agricultura 93 S.L., Barcelona, Spain) and 4.15 g l-1 of P2O5 (18% granulated; Fertiberia, Madrid, 94 Spain). To standardize the initial condition, the different plant growth media were 95 incubated at a water tension of 1000 Pa (adjusted for weight) for 14 days at 25ºC. 96 2. Assessment of disease severity. Disease suppressive properties of plant 97 growth media were measured by a Fusarium wilt bioassay. The bioassays were 98 developed with the susceptible carnation cultivar ‘Medea’ and a monosporic isolate of 99 Fod. This isolate was obtained from an infected carnation plant and stored in silica gel. 100 The pathogenicity of this isolate was confirmed in a previous assay. The Fod was grown 101 for 7 days in AMAP culture media: agar 10g l-1, malt extract (Difco, Le Pont de Claix, 102 France) 10 g l-1, asparagine (Difco, Le Pont de Claix, France) 2 g l-1 and Peter’s foliar 103 feed 27-15-12 and micronutrients (Scotts, Heerlen, The Netherlands) 0.5 g l-1. Five ml 104 of sterile water were added to each culture plate. The surface of the culture was scraped 105 with a sterile spreader. The suspension of Fod was transferred to MAP liquid culture (as 106 described above, but without agar) and grown with continuous agitation (130 rev min-1) 107 for 10 days at 25ºC. Conidia were recovered after filtration and centrifugation at 5000 108 rev min-1, 15 min, (Eppendorf 5810 R, Hamburg, Germany) and rinsed twice in sterile 109 distilled water. The concentration of conidia was determined with a hemocytometer. 110 The seven plant growth media were infested with Fod (5·104 conidia / cm3 plant growth 111 medium), mixed vigorously and poured into plastic pots (1 l volume). Pots of growth 112 6 media without Fod were prepared as controls. Four carnation cuttings (8 to 12 true leaf 113 stage) grown in perlite were bare-root planted into each pot. Plants were irrigated as 114 needed and fertilized with a nutrient solution containing: 0.5 g l-1 Peter’s foliar feed, 0.6 115 g l-1 CaCl2, 0.7 g l-1 MgSO4.7H2O and 0.3 g l-1 urea (pH 6.1). Plants grew in a growth 116 chamber (27ºC, photosynthetically active radiation intensity 280 μE·m-2·s and 16:8 h 117 light: dark photoperiod). Bioassays were performed three times with five pots per 118 treatment. Treatments were arranged in a randomized block design. Disease severity 119 was monitored at 2-day intervals for 55 days after planting, and was scored based on a 120 symptom severity scale described by Baayen & Van der Plas (1992), where: 0 = 121 asymptomatic plant (0% disease); 1 = weakly infected plant (5% disease); 2 = local base 122 stem symptoms (20%); 3 = unilateral and well developed symptoms (50%); 4 = strong 123 disease symptoms all along the plant (80%); 5 = dead plant (100%). The symptoms 124 evaluated were wilted leaves and stems. At each rating time, the mean of the disease 125 severity per pot were calculated. Disease severity was expressed as the proportion of the 126 maximum possible disease severity. The area-under-the-disease-progress-curve-127 standardized (AUDPCs) per pot was calculated by disease severity integrated between 128 symptoms onset and bioassay final time and dividing by the total duration (days) of the 129 epidemic in each bioassay, in order to compare the various bioassays, which had a 130 variety of epidemic durations. At the end of the bioassays, mean values per pot (four 131 plants) of the relative-length-of-the-stem-with-brown-xylem (RLSBX), and the fresh 132 and dry weight of leaves and stems means per pot were recorded. These values per pot 133 were considered as replicates for each observed variable. 134 3. Fusarium oxysporum populations. The density of Fusarium oxysporum was 135 determined by dilution plating on a semi-selective media (Komada’s medium, Dhingra, 136 7 & Sinclair, 1995.). Samples were taken from incubated plant growth media at the 137 beginning of the bioassays and from the rizosphere at the end of the three bioassays. 138 Plant growth media (0.5-1g) were suspended in 10 ml of 0.2% water agar. The 139 suspension was shaken and tenfold dilution series were prepared with 0.2% water agar. 140 Suspensions were pipetted onto three plates per dilution. Four dilutions per series were 141 placed on plates. Colony forming units (CFU) were counted four days after plating and 142 expresed as CFU/ ml of plant growth media. Analyses were performed three times with 143 one sample per each plant growth medium from each of the three bioassays. Means 144 groups with homogeneous variance were studied separately for comparison of means. 145 4. Plant growth media characteristics. β-glucosidase activity and pH measures 146 were performed in incubated growth media at the beginning of the three bioassays. 147 Plant growth media pH were measured in a water extract (2:1; vol /vol), as 148 described elsewhere (Gabriëls, Van Keirsbulck, & Verdonck, 1991). Three samples 149 were analyzed for each growth medium. 150 Microbial activity was estimated by measuring β-glucosidase activity. This 151 method is based on colorimetric determination of the p-nitrophenol released by β-152 glucosidase when the plant growth medium was incubated with 4-nitrophenyl-β-D-153 glucopyranoside (pH 6.0). The p-nitrophenol released was extracted by filtration and 154 determined colorimetrically. β-Glucosidase activity was measured according to Bandick 155 & Dick, (1999). Four samples were analyzed for each plant growth medium and 156 bioassay. 157 5. Statistical analysis. Data collected from three bioassays were analyzed with 158 Statgraphics Plus, (version 5.1; Statistical Graphics Corp., Rockville, MD, 2002). The 159 8 effect of growth medium on AUDPCs, RLSBX, height, fresh and dry weight, pH, and 160 microbial activity were analyzed with ANOVA. Significant means were compared by 161 appropriated tests (P ≤ 0.05). Overall relationships between AUDPCs or RLSBX and 162 continuous measured variables were analyzed with regression analysis. 163 164 Results 165 1. Suppressiveness of plant growth media. Both severity indexes (AUDPCs 166 and RLSBX) indicated that the four composts had suppressive characteristics to 167 Fusarium wilt in comparison to peat, vermiculite, and the coir fibre, which were 168 conducive (Fig. 1). This disease was suppressed most effectively with GMC and the 169 most conducive plant growth media were coir fibre and peat, according to AUDPCs 170 measures, GMC reduced 99% of disease in comparison to these conducive media (Fig. 171 1A). Medium OC + R was not different from GMC for disease severity measured by 172 RLSBX, OC + R reduced 80% of disease respect to peat and coir fibre (Fig. 1B). 173 Height, fresh and dry weight of carnation plants (Fig. 2) showed inverse patterns to the 174 severity indexes (Fig. 1). The GMC medium had the tallest plants, followed by OC + R 175 plants (Fig. 2A). However, fresh and dry weights were no significantly different in these 176 two composts (Fig. 2B, 2C). 177 The best negative correlations between measured disease severity variables 178 (AUDPCs and RLSBX) and the height, fresh and dry weight of leaves and stems (data 179 not shown) were obtained for RLSBX. These correlations were found between RLSBX 180 and the square root of height (R2 = 0.79, P = 0.000, n=105), RLSBX and the square root 181 of fresh weight (R2 = 0.91, P = 0.000, n=105) and RLSBX and the square root of dry 182 9 weight (R2 = 0.84, P = 0.000, n=105). Based on the correlation between RLSBX and 183 plant fresh weight, dry weight and height Fusarium wilt was the main growth-limiting 184 factor in these infested growth media. Consequently, RLSBX was selected for further 185 correlation analysis with the different variables measured. None of the plants grown in 186 the seven non-infested control growth media developed symptoms of Fusarium wilt. 187 2. Fusarium oxysporum population densities. Composts showed significantly 188 lower Fusarium oxysporum densities at the end of bioassays than at the beginning. 189 Nevertheless, the population density was no different at the beginning and at the end of 190 bioassays in coir fibre, peat and vermiculite (Fig. 3). Vermiculite had a low population 191 density of F. oxysporum at the beginning of bioassay while SM + P had the highest 192 population (Fig. 3). At the end of bioassay coir fibre had the highest F. oxysporum 193 population than the rest of media, except peat (Fig. 3). There was found a positive 194 correlation between AUDPCs and F. oxysporum population density at the end of 195 bioassays (R2 = 0.68, P = 0.0000, n=21, AUDPC = 1.35436 – 9336.48/ F. oxysporum 196 density). 197 3. Plant growth media properties before the bioassays. Composts had higher 198 pH and β-glucosidase activity than the other plant growth media (Table 1). Coir fibre 199 and peat had the lowest pH and β-glucosidase activity (Table 1). The OC + R medium 200 had the highest and SM + P the lowest β-glucosidase activity of the four composts 201 tested (Table 1). 202 A multiple linear regression between RLSBX, pH and β-glucosidase activity in 203 the seven plant growth media was found. These two properties explained more than 204 67% of the variation in RLSBX (P < 0.000, n = 21). The resulting equation was RLSBX 205 = 2.457 – 0.000458 microbial activity – 0.267 pH. 206 16 9. Cebolla, V., and Pera, J. 1983. 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Plant growth media pH and β-glucosidase activity at the beginning of 417 bioassays. 418 Plant growth medium a pH b Microbial activity b (μg hydrolyzed p-nitrophenol / cm3 h) Coir fibre 5.66 ± 0.01 e 32.562 ± 2.184 d Peat 5.29 ± 0.19 e 30.252 ± 3.152 d Vermiculite 6.54 ± 0.09 d 1.567 ± 0.603 e CC 7.76 ± 0.01 ab 91.772 ± 10.268 b GMC 7.47 ± 0.00 bc 83.998 ± 3.113 b OC+ R 7.92 ± 0.01 a 252.201 ± 4.401 a SM+ P 7.14 ± 0.02 c 59.103 ± 8.655 c a CC: composted cork; GMC: grape marc compost; OC+R: olive oil husk + cotton gin 419 trash composted and mixed with rice husk; SM+P: spent mushroom composted and 420 mixed with peat. 421 b Mean values and standard errors followed by different letters are significantly different 422 based on Tukey's test at P < 0.05, n =3 for pH and n = 12 for β-glucosidase activity. 423 424 21 Figure 1 A-B. Standardized area-under–disease-progress curve (AUDPCs) and 425 relative length of stem with brown xylem (RLSBX) for carnation plants. CC: composted 426 cork; GMC: grape marc compost; OC+R: olive oil husk + cotton gin trash composted 427 and mixed with rice husk; SM+P: spent mushroom composted and mixed with peat. 428 Plant growth media were infested with Fusarium oxysporum f. sp. dianthi. Disease 429 severity scale was from 0: asymptomatic plants, to 5: dead plants. Data for AUDPCs 430 and RLSBX were transformed for analysis with the arcsine √x. For each square, bars 431 with the same letter were not significantly different according to Tukey's test at P < 432 0.05. Standard error of the mean (n=5) was indicated by vertical line. 433 Figure 2 A-C. Height, fresh and dry weight for carnation plants. CC: 434 composted cork; GMC: grape marc compost; OC+R: olive oil husk + cotton gin trash 435 composted and mixed with rice husk; SM+P: spent mushroom composted and mixed 436 with peat. Plant growth media were infested with Fusarium oxysporum f. sp. dianthi. 437 Data for fresh and dry weights were transformed for analysis with the √x. For each 438 square, bars with the same letter were not significantly different according to Tukey's 439 test at P < 0.05. Standard error of the mean (n=5) was indicated by vertical line. 440 Figure 3. Fusarium oxysporum populations at the beginning and at the end of 441 bioassays. CC: composted cork; GMC: grape marc compost; OC+R: olive oil husk + 442 cotton gin trash composted and mixed with rice husk; SM+P: spent mushroom 443 composted and mixed with peat. Plant growth media were infested with Fusarium 444 oxysporum f. sp. dianthi. Points with the same letter were not significantly different 445 according to LSD test at P < 0.05. Standard error of the mean (n=3) is indicated by 446 vertical line. 447 448 22 449 Fig.1 Plant growth medium Coir fibre Peat Vermiculite CC GMC OC+ R SM+ P RLSBX 0,0 0,2 0,4 0,6 0,8 1,0 AUDPCs 0,0 0,2 0,4 0,6 0,8 1,0 A B a a b cc c d a a a b bc d cd 450 451 452 23 453 454 Plant growth medium Coir fibre Peat Vermiculite CC GMC OC+ R SM+ P Dry weight (g) 0,0 0,5 1,0 1,5 2,0 Height (cm) 0 5 10 15 20 25 30 Fresh weight (g) 0 2 4 6 8 10 A C a a b b ccc b c c d dd a B a a bb c cd d 455 Fig. 2 456 457 458 459 24 Plant growth medium Coir fibre Peat Vermiculite CC GMC OC+ R SM+ P Fusarium population density (CFU / ml) 0 5 10 15 20 25 30 35 begining of bioassay end of bioassay cd c abcd abc bab d a c ab cd a e a (x 1000) 460 Fig. 3 461