Control of seedling damping off caused by Rhizoctonia solani and Sclerotium rolfsii using onion broths

Resumen. El mal de los almácigos causa muerte de plántulas. Los controles cultural y biológico constituyen las únicas herramientas orgánicas para su manejo. Acerca de la eficiencia de preparaciones vegetales, sólo se encuentra información de divulgación. Este trabajo evalúa el efecto de caldos fermentados de cebolla sobre el crecimiento de Rhizoctonia solani y Sclerotium rolfsii y la incidencia de la enfermedad. Se obtuvieron caldo (B) y caldo esterilizado (SB), respectivamente, mediante hervido de trozos de cebollas y fermentación a temperatura ambiente, con o sin esterilización posterior. Se cultivaron los patógenos en APG suplementado con B y SB diluidos 1,7; 3,3; 8,3; 16,7 y 25%. Su crecimiento decreció con B al 8,3, 16,7 y 25%. La producción de esclerocios por S. rolfsii disminuyó con B, pero aumentó con SB. Penicillium purpurogenum, P. simplicissimum y Aspergillus niger aislados de B evidenciaron antibiosis, competencia e hiperparasitismo respecto de ambos patógenos. B y SB diluidos 10 y 50% fueron aplicados en almácigos de acelga (Beta vulgaris), tomate (Solanum lycopersicum), pimiento (Capsicum annuum) y berenjena (Solanum melongena) infestados. La pérdida de plántulas fue menor en el tratamiento con B al 50%, en relación al tratamiento con SB. Se concluye que la actividad antifúngica de B depende de su dilución y de su micota. La utilización de caldos de cebolla debería ser contemplada como alternativa para el manejo fitosanitario de bajo impacto ambiental. Se requiere continuar los estudios para completar el conocimiento acerca de los componentes químicos y microbiológicos de los caldos, y sus posibles cambios durante la fermentación.

] are ubiquitous soilborne pathogens with a broad host range (Sneh et al., 1995;Sarma & Singh, 2002). They survive forming resting structures that remain free or embedded in plant debris (Shlevin et al., 2003). Mycelia developed from sclerotia initiate infection causing pre or post emergence damping off (Bedendo, 1995).
Plant preparations are frequently used for disease protection in organic crops. Onion broth is applied by spraying the base of the plants or by soil drenches (Stoll, 1986). Although empirical information is available, it still has to be elucidated whether broth effectiveness depends on chemical or microbiological activity. Our hypotheses were that onion broths control the damping off pathogens R. solani and S. rolfsii through microbiological activity, and that their efficiency is dilutiondependent. The aims of this study were to evaluate the effect of fermented onion broth and sterilized broth on the development of R. solani and S. rolfsii and damping off incidence, and to determine optimal dilutions. In addition, broth mycobiota was explored regarding biological control of these pathogens.

MATERIALS AND METHODS
Onion broth and fungal cultures. Chopped yellow onions cv. Valcatorce INTA (Valenciana group) were boiled in water (1 kg/L) for 20 minutes and filtered through surgical gauze. Volume was adjusted to 1 L by addition of tap water. Half volume of the broth (herein called B) was transferred to an Erlenmeyer flask, and incubated for 14 days at room temperature covered with a piece of surgical gauze. The other half (herein called SB) was incubated as described above, and then sterilized by autoclaving at 121 °C for 90 minutes. New broths were prepared for each repetition of the experiments. A sample of B and SB was chemically analyzed (Table 1). Rhizoctonia solani O-IM-1 and S. rolfsii O-DI-1 were obtained respectively from Impatiens balsamina L. and Dichondra microcalyx (Hallier f.) Fabris. Their pathogenicity on the hosts selected for this work was confirmed by inoculation (results not shown).
Agar plate tests. Broth and SB were diluted in sterilized water at 5, 10, 25, 50 and 75% (v/v). Petri dishes (110-mmdiameter) were poured with 11 mL of potato dextrose agar Oxoid (PDA), cooled to 45-50 °C, and 5.5 mL of the dilutions of B, SB or water (control) were added so that the final dilutions of B and SB in the agar medium were 1.7, 3.3, 8.3, 16.7 and 25%, respectively, with five replicates for each treatment. An 8-mm diameter disk with fungal mycelium grown on PDA was placed in the centre of each plate and incubated at 20 °C, until the controls covered the whole surface (4-6 days). Sclerotia of S. rolfsii were counted after 14 days of incubation, while those of R. solani could not, as they were inconspicuous. Colony boundaries were daily reproduced on a sheet of paper and measured using a Leaf Area Scanner. The test was performed three times.
A sample of B prepared for the second test was used to explore its microbiological components. Fungal strains from B showing any kind of interactions with the pathogens on PDA were isolated and identified to species. Dual cultures of each pathogen and each B isolate were prepared by placing two 8-mm diameter PDA disks with mycelia on the centre of PDA plates separated by 4 cm and incubated for 21 days at 20 °C. Interactions between each pathogen and each B isolate were observed under the microscope (Zeiss Axioscop, Germany) at 100x magnification. The test was performed three times for each isolate.

Greenhouse assays
Chard (Beta vulgaris L.). Broth and SB at dilutions of 10 and 50% v/v were tested. Soil was placed into 4.4-dm 3 metal containers, sterilized by tyndallization (Stanier et al., 1970), inoculated with 0.5-cm 3 plugs of R. solani on PDA (0.1% v/v), incubated at room temperature during 14 days (Rivera et al., 2004) and disposed in plastic trays (225 cm 2 ). Ten seeds of chard cv. Amarilla de Lyon were sown into plastic trays containing infested soil, for each of three replicates per treatment. The soil in each tray was daily sprayed with an average of 1.6 mL of water, B or SB at dilutions of 10% and 50%. Trays were kept in polyethylene bags, at 18 °C. Damping off incidence was evaluated 6 days after emergence. Samples of diseased seedlings were randomly taken, surface disinfected by immersion in 2% (v/v) of Cl as NaOCl, during 1 minute, plated on PDA, incubated at 22 °C and examined for pathogen development. A similar assay was conducted for S. rolfsii. The experiment was repeated twice.

Tomato (Solanum lycopersicum L.), pepper (Capsicum annuum L.) and eggplant (Solanum melongena L.).
Soil was prepared as previously described. Experimental units were plastic trays of 140 cm 2 filled with soil, either sterile or infested with R. solani, where tomato cv. Platense Redondo Grande, pepper cv. California Wonder or eggplant cv. Long Purple were sown (1,000 seeds/m 2 ). The soil in each tray was daily sprayed with an average of 1 mL of water, B or sterilized SB at dilutions of 10 and 50% during the experiment. The trays were individually kept in closed polyethylene bags. The experiment consisted of 10 treatments for each host, with 5 replicates, and was repeated twice. Average air temperature in the greenhouse was 16 °C. Disease incidence was evaluated as above, 25 days after emergence, and sampling for pathogen isolation was done as described. A similar assay was conducted for S. rolfsii.
Experimental design and data analysis. Lab data were analyzed by a two-factor study (broth-dilution) in a completely randomized design, and by Tukey multiple comparisons or DGC test (Di Rienzo et al., 2002). Shapiro-Wilks and Levene tests, respectively (Neter et al., 1996) verified normality of error terms and constancy of error variance. The number of sclerotia was analyzed by Kruskal Wallis nonparametric method (Conover, 1995). Greenhouse assays were designed as randomized complete blocks. A two-factor study was used for data analysis. Factors were analyzed separately when their interaction was non-significant. Friedman non-parametric tests were used when homogeneity of variance could not be verified (Conover, 1995). Analysis was done by InfoStat ® (FCA-UNC, Córdoba), with a significance level of 5%.

RESULTS AND DISCUSSION
Agar plate tests. Inhibition of R. solani by B and SB was dilution-dependent; the higher dilutions were less effective. The maximum growth of the pathogen was observed for the control, followed by SB treatments (Fig. 1). B diluted at 8.3, 16.7 and 25% was the most effective in inhibition of R. solani. SB was less effective, especially at 1.7%. There were no differences among SB treatments at 3.3-25%. On day two, there was neither interaction between infusions and dilutions (p=0.41) nor effect of dilutions on pathogen growth (p=0.21), which was reduced by both infusions (p=0.02). On the third and fourth days, interactions between infusions and dilutions were significant (p≤0.0001 and p=0.0003, respectively).
Broth reduced colony growth of S. rolfsii at all dilutions and SB only at 25% (Fig. 2). Control was not different between B dilutions from 8.3 to 25%. SB at 1.7 to 16.7% did not differ with the control. Infusion-dilution interaction was detected in the second day of incubation (p≤0.0001). Sclerotia development was markedly reduced by B at all dilutions (Table 2). On the contrary, SB diluted at 8.3-25% stimulated the production of sclerotia.  Fungal colonies from B cultivated in PDA, inhibited R. solani and S. rolfsii or overgrew their colonies. Isolations obtained from these interactions were identified as Penicillium simplicissimum (Oudem.) Thom (Raper & Tom, 1968), P. purpurogenum Stoll (Pitt, 2000) and Aspergillus niger Tiegh. (Singh et al., 1991). They were stored in BAFC Cult. (FCEN Culture Collection) and labeled as 3199, 3100 and 3198, respectively.
The growth of R. solani was reduced in the presence of each B isolate, but this was significant only in the case of P. simplicissimum (Table 3, Fig. 3a). Immature sclerotia of R. solani were evident in the interaction with P. simplicissimum. Pathogen growth was inhibited in the interaction with A. niger (Fig.  3b) and developing sclerotia appeared in the margins of the colonies On the reverse of the plates, P. purpurogenum showed an intense red pigmentation (Fig. 3c). The three B species, especially P. simplicissimum, grew and sporulated over the pathogen mycelium (Fig. 4a, 4b). After 14-days of incubation, R. solani formed brown sclerotia on the distal borders of the colony, against the three B fungi. Antagonist hyphal coiling (Fig.  4b) and pathogen necrosis (Fig. 4c) were observed for the in-  (Table 3). On the reverse, mycelium of P. simplicissimum appeared discolored in contact with the pathogen (Fig. 5d). P. purpurogenum developed a conspicuous zone of inhibition (Fig. 5b). A. niger showed variable behavior, from a thin band of inhibition ( Fig.  5c) to growth almost enclosing the pathogen colony. After 14 days, the production of sclerotia was similar to the control in confrontation with P. purpurogenum, reduced against A. niger and inhibited against P. simplicissimum (Table 3).

Greenhouse assays
Chard. Interaction between broths and dilutions was not significant for the assays with R. solani (p=0.22) and S. rolfsii (p=0.55), so the effects of both factors were analyzed separately. Rhizoctonia disease incidence was reduced by onion broth, diluted at 50%, (Table 5) either sterilized or not (Table  4). B was even more efficient in controlling Sclerotium disease (Table 4) at any dilution (Table 5). Control of damping off with onion broths

Tomato, pepper and eggplant.
Damping off caused by both pathogens was controlled by B at 50% for tomato, pepper and eggplant seedlings (p<0.0001) ( Table 6). Broth diluted at 10% was markedly less effective to control both pathogens on tomato, and R. solani on pepper, and behaved similar to dilutions at 50% for R. solani on pepper and S. rolfsii on pepper and eggplant. Sterilized broth at 10% and 50% stimulated disease on tomato and pepper plants, respectively.
Seedling loss was observed in tomato, pepper and eggplant sown in sterilized soil irrigated with SB (Table 6). Both pathogens were re-isolated from 100% of the samples taken from seedlings that died in inoculated soil, showing necrosis at the base that made them topple over at the soil surface. General chlorosis that turned into necrosis developed on the seedlings cultivated in sterilized soil, especially those irrigated with sterilized onion broth, and no pathogens developed from those samples. Severity of leaf symptoms was similar to that observed by Aktas et al. (2006). Seedling loss in plots with sterilized soil irrigated with B diluted at 10 or 50% was low, and similar to controls. Table 4. Diseased chard seedlings 6 days after emergence for the different sterilized (SB) and non-sterilized (B) onion broths.
Type and dilution of onion decoctions influenced pathogen growth as well as disease incidence. In general, treatments with B were more effective than those with SB. Dilution effectiveness to manage disease differed between pathogens. These broths constitute a source of beneficial fungi that develop during fermentation, and seem to be the basis of the effect of B on disease control. It is generally assumed that correlation between lab and field studies may be low (Knudsen et al., 1997). In our case, agar plate tests gave useful information to explain greenhouse results. Even for pepper seedlings, where control of R. solani was less effective with B at 50%, damping off incidence could be significantly reduced from 58% (control) to 35%. Antagonistic relationships observed among the pathogens and some fungal species developed in B are based on competition, antibiosis and/or hyperparasitism phenomena. Broth controlled the growth of R. solani better than the growth of S. rolfsii. However, B isolates controlled better the growth of S. rolfsii than that of R. solani. This behavior could evidence the contribution of some other not isolated antagonist/s to B total suppressiveness or any kind of synergism. Aspergillus and Penicillium species are frequent post harvest fungi (Fatima et al., 2009;Onilude et al., 2010). Aspergillus is commonly isolated from onions (Zohri et al., 1993) and A. niger has been reported as pathogenic on them (Summer, 1995). To our knowledge, P. simplicissimum, A. niger and P. purpurogenum are new records as antagonists of R. solani and S. rolfsii. Among them, the first appears to be the most efficient in reducing pathogen growth and sclerotia production. Table 6. Tomato, pepper and eggplant disease incidence 25 days after emergence in pathogen infested seedling plots.

Infusion Soil
Mean seedling loss (%) Even though this work evaluated preparations obtained from onion bulbs in relation to disease suppression, they may have nutritional value also. When compared with nutrient solutions, they are poor in Nitrogen and Calcium contents and balanced in relation to Potassium, Phosphorous and Magnesium (Hoagland & Syder, 1933); or poor in Nitrogen, rich in Potassium and balanced in other macronutrients (Hochmuth & Hochmuth, 2008). The electrical conductivity of standard solutions varies from 1.8 dS/m to 2.3 dS/m (Kayo et al., 2006;Rubio et al., 2011); and values obtained from B and SB range from 2.32 to 2.72 dS/m. Regarding pH, measured values were 5.3 and 6.4, similar to those suggested by Hochmuth & Hochmuth (2008). Broth and SB showed similar chemical composition, except for sodium content, which was markedly higher for the latter. Since salt sensitivity has been reported for Solanaceae crops, especially at the seedling stage (Aktas et al., 2006), ion toxicity may have been the cause of seedling loss in sterilized soil plots irrigated with SB.

Rhizoctonia solani Sclerotium rolfsii
The use of composted materials in the management of soilborne diseases has been deeply studied (Hoitink & Fahy, 1986). For instance, composted onion waste reduced viability of sclerotia of S. cepivorum Berk. in greenhouse pot tests (Coventry et al., 2002). In this work B behaviour was similar to those of compost teas, which are leachates obtained from composted materials (Özer & Köycü, 2006). Regarding likely mechanisms, Weltzien (1989) demonstrated that compost teas become rather ineffective after sterile filtration or heat treatments, thus attributing pathogen control to biological activ-ity. However, in our work, SB showed some activity suggesting that chemical components have their own antifungal role.
Onions are composed mainly of water (85-90 g/100 g fresh weight). Mature intact bulbs of Allium species contain cysteine sulphoxides (Block et al. 1992). As in other Allium species (Irkin & Korukluoglu, 2007) when onion tissues are chopped the enzyme alliinase is released, converting cysteine sulphoxides into thiosulfinates (Block et al., 1992), which are reactive, volatile, odorous and lachrymatory components (Benklebia, 2004). Soil microflora is also responsible for metabolising onion compounds (Coley-Smith & Parfitt, 1986). Alliin, allicin and ajoene, the most studied sulfur-containing compounds, are thermolabile (Block et al., 1992;Sinha et al., 1992;Naganawa et al., 1996). Although antifungal activity of onion extracts decreases with increasing temperatures, Yin & Tsao (1999) found that it is still evident over 100 °C suggesting that thermoresistant components may be responsible for it. Water soluble phenols and flavones are known as important antifungal compounds expressed in onion bulbs (Link & Walker, 1933) that prevent fungal spore germination and hyphal penetration in plant tissues (Özer & Köycü, 2006). Temitope et al. (2010) demonstrated that boiling during one or two hours does not affect onion total phenol content, and diminish total flavonoid content nearly 20%. Since B and SB used in this work were obtained by boiling during 20 minutes, heat-resistant onion components like phenols and, to a lesser extent, flavonoids may have been responsible for the antifungal performance observed.
Coley-Smith & Parfitt (1986) stated that onion volatile thiols and sulphides activate sclerotia germination and growth of the onion pathogen Sclerotium cepivorum (Assadi & Behroozin, 1987) in the soil, possibly due to chemotaxis. In this work, it was demonstrated that onion B decreased the number of sclerotia developed by S. rolfsii. On the contrary, SB stimulated it, which suggests any kind of unfavorable condition for pathogen's growth. It will be necessary to understand the molecular events involved in hyphal aggregation to form sclerotia (Gow & Gaad, 1995) and their requirements, to explain these effects.
Application of broth on seedling plots by irrigation, in a similar way as growers do in their fields or greenhouses, provided disease control in four different crops. As a result of this work, scientific evidence has arisen on the effectiveness of onion broth to control damping-off pathogens, and its effect was explained mainly by biological activity. As sterilized broth also showed efficiency, chemical components are supposed to have some influence on disease control. Despite the reported effectiveness against various food microorganisms, the strong odor of onions seems to have limited their use as additives or preservatives (Benklebia, 2004), but it does not seem to be an impediment for seedling protection. Further studies are needed for a complete understanding of chemical and microbiological components of the broths, as well as their changes during fermentation.