Influence of the ectomycorrhizal fungus Laccaria laccata on pre-emergence, post-emergence an late damping-off by Fusarium oxysporum and F. verticillioides on Stone pine seedlings

1 1 Influence of the ectomycorrhizal fungus Laccaria laccata on pre-emergence, post-2 emergence and late damping-off by Fusarium oxysporum and F. verticillioides on 3 Stone pine seedlings. 4 5 P. Machón, J. A. Pajares, J. J. Diez and F. M. Alves-Santos* 6 7 Departamento de Producción Vegetal y Recursos Forestales. ETSIIAA de Palencia. 8 Universidad de Valladolid. Avda. Madrid 57. 34071 Palencia. Spain 9 10 Phone number: ++34 979 10 84 31 11 Fax number: ++34 979 10 84 40 12 E-mail: 13 pamadi[email protected] 14 [email protected].es 15 [email protected] 16 [email protected] 17 18 19 *Corresponding author This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s13199-009-0015-0 Machon, P., Pajares, J. A., Diez, J. J., & Alves-Santos, F. M. (2009). Influence of the ectomycorrhizal fungus Laccaria laccata on preemergence, post-emergence and late damping-off by Fusarium oxysporum and F. verticillioides on stone pine seedlings. Symbiosis, 49(2), 101-109. https://doi.org/10.1007/s13199-009-0015-0 2 Influence of the ectomycorrhizal fungus Laccaria laccata on pre-emergence, post-1 emergence and late damping-off by Fusarium oxysporum and F. verticillioides on 2 Stone pine seedlings. 3 4 5 ABSTRACT 6 In greenhouse experiments, the ectomycorrhizal fungus Laccaria laccata was evaluated 7 for biological control of pre-emergence, post-emergence and late damping-off of Pinus 8 pinea caused by Fusarium verticillioides and F. oxysporum. In pre-emergence damping-9 off assays, preinoculation with Laccaria laccata did not significantly improve 10 germination of seeds and no statistical significant differences were found in Fusarium 11 treatments when compared with controls. At 18 weeks after sowing, inoculation with L. 12 laccata reduced the incidence of post-emergence damping-off but differences were 13 significant only in F. oxysporum treatments. Pinus pinea transplanted plants were used 14 in late damping off assays, and only F. oxysporum produced significant damage. 15 Inoculation with L. laccata did not attenuate significantly the virulence of F. 16 oxysporum. However, mycorrhization percentage did not reached significant level, so 17 the mycorrhizal fungus level could be not enough for an effective protection. Although 18 very low percentages of mycorrhization were recorded in all mycorrhized treatments, 19 and Fusarium occurrence significantly reduced mycorrhization, those levels have been 20 efficient to reduce damage in F. oxysporum post-emergence damping-off assays. 21 In short, pre-emergence damping-off was not found; only F. oxysporum produced 22 significant damage on P. pinea seedlings and L. laccata reduced damage when the 23 percentage of mycorrhization reached significant levels. These results have been 24 compared with previous work on P. sylvestris inoculated with the same mycorrhizae 25 isolate and Fusarium pathogens. 26 3 Keywords: forest nursery, Fusarium damping-off, Laccaria laccata, mycorrhization, 1 Pinus pinea, plant protection 2 3 4 4 INTRODUCTION 1 2 Forest nurseries are known to be affected by damping-off (Nef and Perrin, 1999). 3 Damping-off is caused by different fungal and oomycetes species like Pythium spp., 4 Fusarium spp. and Rhizoctonia solani, which occur very frequently. Another species as 5 Botrytis cinerea, Phytophthora spp., Alternaria spp., Phoma spp. and Phomopsis spp. 6 can also be present. Fusarium and Pythium are the most important genera that cause 7 damage to containerized plants in nurseries (Jones and Benson, 2000) and Fusarium 8 verticillioides Sheld. (teleomorph Gibberella fujikuroi) and Fusarium oxysporum 9 Schltdl. are the most aggressive species within Fusarium pathogens involved in 10 damping-off (Bloomberg, 1985; Chakravarty et al., 1999; Dick and Dobbie, 2002). 11 Those pathogens have already been detected in Spanish nurseries and Fusarium 12 oxysporum, F. verticillioides and the saprophytic fungus Trichoderma viride, have been 13 very often isolated from rhizosphere of Pinus and Quercus in forest nurseries of Castilla 14 y León region (West-central Spain) where those pathogens are responsible for 15 considerable losses (Martin-Pinto et al. 2006a). 16 Primary inoculum of the pathogenic fungi can be present on seed, contaminated 17 substrate or in water. Several fungicides are used to control this disease. However, 18 many of them are not effective and do not protect the seedling (Williams, 1989; 19 Dumroese et al., 1996) and most fungicides have only a temporary effect and therefore 20 require repeated applications that increase their negative impact, since surface 21 recolonization by Fusarium via blowing soil, surface water flow, and infested seed is 22 observed (Bloomberg, 1985). 23 In recent decades, holistic integrated strategies for nurseries protection have been 24 considered as the best approach to mitigate losses from this disease (Dumroese and 25 5 James, 2005). Those strategies have take into account the fungal resistance to chemicals 1 and its residual toxicity and have considered the biological control as an important tool 2 available (Dumroese et al., 1996; 1998). In order to develop integrated control 3 procedures according to environmental principles, several microorganisms have been 4 tested (Le Tacon and Bouchard, 1986; Pedersen et al., 1999; Mandeel, 2006 and 2007). 5 Mycorrhizal symbiosis is an important factor in the establishment of seedlings in 6 semiarid or degraded areas (Helm and Carling, 1993a and 1993b) and the effectiveness 7 of mycorrhizal inoculation in producing an increase in the growth of Pinus halepensis 8 Mill. has been demonstrated (Roldan and Albadalejo, 1994). Mycorrhizae have a 9 positive influence on the performance of seedlings planted in reforestation (Krop and 10 langlois, 1990), owing to the mutual beneficial relationship between plants and 11 mycorrhizal fungi. Besides other beneficial effects, mycorrhizal fungi increase nutrient 12 uptake, facilitate the transport of water to plant roots (Parke et al. 1983) and act as a 13 defense mechanism against pathogenic organisms. 14 In 1942 Davis et al. suggested that ectomycorrhizae could protect feeder roots of 15 nursery seedling against pathogens and since then several studies have noted this 16 protective capability of mycorrhizae (Davis et al., 1942; Chakravarty and Hwang, 1991; 17 Chakravarty et al., 1991; Duchesne, 1994; Hwang et al. 1995; Morin et al. 1999; ). 18 Several mechanisms may be involved in plant protection. Mycorrhizal fungi can create 19 physical barrier between roots and pathogens, exude antimicrobial metabolites, and use 20 surplus carbohydrates, thereby reducing the attractiveness of roots to pathogenic 21 organisms (Duchesne et al., 1987). Several studies of conifer seedling protection by 22 mycorrhizal fungi against fungal pathogens such as Phytophthora (Marx and Davie, 23 1969), Pythium (Perrin and Garbaye, 1983) (both them now considered as fungal-like 24 organisms), Fusarium (Machon et al., 2006), Cylindrocladium, and Cylindrocarpon 25 6 (Buscot et al, 1992; Chakravarty et al., 1999; Morin et al., 1999) have been recorded. 1 Protective effect of mycorrhizae could be extended to other pathogens such as 2 nematodes (Diedhiou, 2003). The effectiveness of root protection varies with the 3 species of mycorrhizal fungi, host species, and soil conditions (Chakravarty and 4 Unestam, 1987a and 1987b). 5 Laccaria laccata (Scop.:Fr) Berk. & Broome can be easily isolated from fruiting bodies 6 and grown in laboratory conditions. The ability of this fungus to form mycorrhizae and 7 its wide host range makes it very interesting organism for artificial inoculation of 8 nursery plants (Molina, 1983; Molina and Chamard, 1983; Hung and Molina, 1986; 9 Perrin and Soulas, 1996). This fungus has been recorded to give protection against 10 several pathogenic fungi when associated with different plant species as Pseudotsuga 11 menziesi (Mirb.) Franco, Picea abies (L.) Karst, Pinus banksiana Lamb, Pinus nigra 12 Arnold or Pinus sylvestris L. (Chakravarty and Hwang, 1991; Chakravarty and 13 Unestam, 1987a and 1987b; Sinclair et al. 1982; Sylvia and Sinclair, 1983a and 1983b, 14 Martin-Pinto et al. 2006b). In our lab the effective protection of P. sylvestris seedlings 15 by L. laccata have been recorded (Machon et al. 2006). 16 The aim of this study was to evaluate the protective effect of L. laccata against the 17 damage caused by Fusarium spp in Pinus pinea seedlings. In this study we have take 18 into account the three different stages on damping-off disease: pre-emergence damping-19 off that reduces the germination percentage; post-emergence damping-off affecting the 20 early stages of seedlings and late damping-off when plants are older than four-months-21 old. 22 23 MATERIALS AND METHODS 24 Organisms 25 7 Pinus pinea seeds used in this study were provided by the forest nursery “Viveros 1 Fuenteamarga” in Cabezón de Pisuerga (Castilla y Leon region in west central Spain). 2 Seeds were disinfected by using 30% H2O2 for 30 min, and then washed 10 times with 3 sterile distilled water to eliminate disinfectant before sowing 4 The root pathogens Fusarium verticillioides (Fm6) and F. oxysporum (Fo4), isolated 5 from diseased seedlings in a greenhouse at the Imave Nursery (León) were used through 6 the experiment. They were previously tested against Pinus spp (Martín-Pinto et al., 7 2006b) and both of them behaved as pathogens and produced damping-off symptoms. 8 The monosporic cultures of Fusarium spp. were maintained on solid Komada medium 9 (K) and inoculum of Fusarium spp was produced by culturing the fungus in liquid PDB 10 (Potato dextrose broth) medium for 7 days in the dark. Spores were separated from the 11 medium and resuspended at a concentration of 106 spores/ml. 12 The ectomycorrhizal fungus, Laccaria laccata (isolated from fruiting bodies) was 13 provided by Dr. M. Fernandez from the Valonsadero Forestry Centre (Soria) on 14 modified Melin Norkrans´(MMN) medium (Marx and Davie 1969). Inoculum of L. 15 laccata consisted of mycelium growing at 25ºC for two months in 2-l Flasks containing 16 1000 ml vermiculite, 100 ml peat and 500 ml MMN liquid medium (pH adjusted to 17 5.0). The vermiculite and peat were previously sterilised twice at 120ºC during 60 min. 18 The resulting mixture was autoclaved for 90 min at 121ºC prior to inoculation with the 19 mycorrhizal fungus. Uninoculated flasks were prepared for control treatments. 20 21 Pre-emergence and post-emergence Damping-off 22 The experiment consisted of 6 treatments: 1 control (Not inoculated), 2 Laccaria 23 laccata (Myc), 3 Fusarium verticillioides (Fm), 4 Fusarium oxysporum (Fo), 5 24 8 Fm+Myc, and 6 Fo+Myc. Each treatment consisted of 3 replicates of 48 seeds. The 1 experiment was carried out in a completely randomised design. 2 Pine seeds were sown in multipots (250 ml) in a greenhouse (ETSIIA-Palencia) in 3 early February 2004. All seedlings were grown in a mixture of Sphagnum (Finn peat) 4 and vermiculite (1:1). The pot substrate was autoclaved twice at 121ºC for 90 min 5 before sowing. The Myc treatment was inoculated with 50 ml of media containing L. 6 laccata inoculum. A 5 ml spore suspension (106 spores ml-1) of Fusarium 7 verticillioides or Fusarium oxysporum was added to each pot of Fm, Fm+Myc and Fo, 8 Fo+Myc treatments. Control seedlings were inoculated with 5 ml of MMN medium. 9 Seedlings were grown in a greenhouse until mid-July. Watering and other 10 procedures were common nursery practice; no fungicides applied. Fifteen seedlings of 11 each treatment were randomly taken at the end of July. 12 Thirteen weeks after sowing, Pre-emergence damping-off was estimated by 13 counting the number of germinated seeds in each treatment. 14 Eighteen weeks after sowing, Post-emergence damping-off was analysed and 15 classified into four damage classes: (0) no damage; (1) slight damage, (2) moderate 16 damage; (3) dead seedling (Halldorsson et al. 2000). 17 The shoot dry weight, diameter, root length, root dry weight and number of 18 mycorrhizal short roots were measured 18 weeks after planting. Soil was washed off the 19 root, which was subsequently cut off the seedlings and the mycorrhizae were examined 20 by a binocular magnifier (Nikon SMZ2T). The intensity of root colonization was 21 expressed as percentage of mycorrhized apexes within 250 observed apexed plants. 22 23 Late damping-off 24 9 Simultaneously, in order to analyze late damping-off, Stone pine seeds were sown in 1 multipots (50 ml) as previously described. Two-month-old seedlings were transferred to 2 multipots (250 ml) for assay and six treatments were applied: 1 control (Not 3 inoculated), 2 Laccaria laccata (Myc), 3 Fusarium verticillioides (Fm), 4 Fusarium 4 oxysporum (Fo), 5 Fm+Myc, and 6 Fo+Myc. Each treatment consisted of 3 replicates of 5 36 seeds. The experiment was carried out in a completely randomised design. 6 Inoculum of Fusarium spp. and L. laccata was prepared as described previously. 7 The Myc treatment was inoculated with 50 ml of media contained L. laccata inoculum. 8 Fifteen days after sowing, a 5-ml spore suspension (106 spores ml-1) of Fusarium 9 verticillioides and Fusarium oxysporum was added to each pot of Fm, Fm+Myc and Fo, 10 Fo+Myc treatments respectively. 11 Eighteen weeks after inoculation, Late damping-off was estimated by recording 12 seedling damage. All sample seedlings were evaluated into four damage classes: (0) no 13 damage; (1) slight damage, (2) moderate damage; (3) dead seedling (Halldorsson et al. 14 2000). 15 Pine seedling measures and mycorrhizal colonization were evaluated as previously 16 described in post-emergence assays. 17 18 Statistical analysis 19 All the data were processed by one-way analysis of variance (ANOVA), and 20 repeated measures test ANOVA (p<0.05) using STATISTICA Software. The 21 differences between means were considered significant (p<0.05) according to a least 22 significant difference (LSD) multiple range test. For each treatment (Control, Myc, Fm, 23 Fo, Fm+Myc, Fo+Myc), data were obtained by calculating the mean of three different 24 16 competition for nutrients and space, and the low level of mycorrhizae could reflect the 1 fast colonization by Fusarium (even when the mycorrhizal fungus was ground earlier). 2 In nurseries, microbiological activity in the soil is the principal factor affecting 3 mycorrhizae formation (Kropp and Langlois, 1990). For bare-root production, soil 4 fumigation should be carried out to reduce pathogen populations before sowing and 5 introducing mycorrhizal fungi (Marx and Cordell, 1987). To eliminate chemical 6 products and inoculate seedlings with ectomycorrhizal fungi before exposing them to 7 pathogens, containerized inoculated seedlings should be produced and transplanted in 8 infested soil, since the inhibition of mycorrhizal formation by substrate microbial 9 activity is rarely a problem in containers where excellent results have been obtained 10 (Morin et al., 1999). 11 The present study suggests that inoculation of P. pinea seedlings by ectomycorrhizal 12 fungi in nurseries reduces both damage intensity and seedlings mortality caused by 13 Fusarium damping-off. However several points should be taken into account: (a) 14 Fusarium verticillioides isolate Fm6 that has been reported as pathogen against Pinus 15 nigra and P. sylvestris (Martin-Pinto et al., 2006a; Machon et al., 2006], did not 16 produce severe damage in Pinus pinea seedlings; (b) statistically, the pre-emergence 17 damping-off damage was no significant; (c) the percentage of mycorrhization was low 18 in all cases and it did not reached significant level in F. oxysporum late damping-off 19 assays. 20 It could be possible that Pinus pinea was more resistant to Fusarium damping-off than 21 other Pinus species; despite the Fm6 isolate was recovered from Pinus pinea diseased 22 plants [10]. Our results showed that percentage of mycorrhization as low as 2.6 percent 23 or 2.06 percent in P. sylvestris (Machon et al., 2006) could be enough to provide 24 effective protection, thus indicating that the principal method of protection could be not 25 17 the barrier effect, nor the metabolites produced by ectomycorrhizal fungi. According to 1 our results, the hypotheses of plant-produced antifungal products could be the most 2 suitable explanation. Nevertheless, more detailed biochemical examination should be 3 carried out in order to confirm this hypothesis. 4 The protective effect of mycorrhizae in F. oxysporum post-emergence damping-off 5 assays was not complete. Previous studies (Morin et al., 1999) suggested a correlation 6 between mycorrhizal colonization rate and infection rate, therefore higher level of 7 mycorrhization should be attempted in order to achieve mycorrhization percentage that 8 should completely restore the seedlings health as found in F. oxysporum post emergence 9 damping-off on P. sylvestris seedlings (Machon et al., 2006). 10 Our results suggest that mycorrhization can protect pine seedling and could be used as 11 biological control of damping-off in forest nurseries, but further studies are required to 12 establish the effectiveness of L. laccata against other pathogens, the level of 13 mycorrhization required and the mechanisms involved in mycorrhizal protection in 14 Pinus species 15 16 17 18 Acknowledgements 1 This research was supported by grant AGL2001-1771 (Ministerio de Ciencia y 2 Tecnología). We thank M. Fernández for providing L. laccata isolate. 3 4 5 REFERENCES 6 Bloomberg W.J. 1998. 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Phytopathology 73: 384-389. 4 Sylvia D.M. and Sinclair W.A. 1983b. Phenolic compounds and resistance to fungal pathogens induced in 5 primary roots of Douglas-fir seedlings by the ectomycorrhizal fungus Laccaria laccata. 6 Phytopathology 73: 390-397. 7 Williams F. 1989. Benomyl drenches do not control Fusarium or Cylindrocarpon caused root rots. 8 Victoria, British Columbia: BC Ministry of Forests, Seed and Seedling extension Topics 9 1(1):11–12 10 Table 1. Pre-emergence damping-off of Pinus pinea (expressed as mean percentage of seed germinating 13 weeks after sowing in three inoculation experiments) in media with and without Laccaria laccata inoculum and with Fusarium verticillioides, F. oxysporum or no Fusarium. Means are followed by the standard deviation. No significant differences were found. No Fusarium F. verticillioides F. oxysporum No Myc 40.28±7.3 32.64±6.7 40.97±2.4 Myc 45.14±2.4 37.50±5.5 47.22±3.2 Table 2. Post-emergence damping-off of Pinus pinea. Measures were taken 18 weeks after sowing. Diameter in mm was measured at the shoot base and dry weight was used. Within columns values sharing the same letter did not show significant differences (LSD test p<0.05) Treatment Shoot height (cm) Shoot weight (g) Diameter (mm) Root length (cm) Root weight (g) Control 11.14 a 0.300 a 1.593 b 13.31 b 0.107 b Fm6 10.95 a 0.348 ab 1.553 ab 12.93 ab 0.110 b Fo4 10.89 a 0.328 ab 1.463 ab 12.30 a 0.098 ab Myc 10.94 a 0.347 ab 1.573 ab 12.95 ab 0.105 b Fm6 + Myc 11.33 a 0.372 b 1.597 b 12.25 a 0.101 ab Fo4 + Myc 10.57 a 0.296 a 1.410 a 12.80 ab 0.078 a Table 3. Late damping-off of Pinus pinea. Measures were taken 18 weeks after planting. Diameter in mm was measured at the shoot base and dry weight was used. Within columns values sharing the same letter did not show significant differences (LSD test p<0.05) Treatment Shoot height (cm) Shoot weight (g) Diameter (mm) Root length (cm) Root weight (g) Control 15.52 c 0.448 b 1.543 b 13.19 ab 0.145 b Fm6 12.43 a 0.387 ab 1.503 b 13.20 ab 0.141 b Fo4 11.84 a 0.332 a 1.427 ab 14.65 b 0.119 ab Myc 13.61 b 0.343 a 1.450 ab 12.25 a 0.103 a Fm6 + Myc 11.69 a 0.325 a 1.440 ab 12.98 a 0.101 a Fo4 + Myc 11.55 a 0.318 a 1.333 a 13.18 ab 0.095 a