Effect of olive mill wastewater on growth and bulb production of tulip plants infected by bulb diseases

The effect of olive mill wastewater (OMW) on growth of tulip plants infected by common diseases as well as on their new bulbs production is analyzed in this work. Filtered and sterilized OMW was tested as growth inhibitor of Botrytis tulipae, Fusarium oxysporum, Aspergillus niger and Penicillium spp. mycelium. The effect of filtered OMW on uninfected tulip bulbs was also tested as well as on the growth of bulbs infected with the fungus B. tulipae and A. niger in vivo. The mycelium length, severity of scab-like lesions, plant height (PH), fresh mass (FM) and dry mass (DM) of plants and production of new bulbs were recorded. Only the filtered OMW inhibited the in vitro mycelium growth of all tested fungi. However filtered OMW caused infections when it sprayed on uninfected bulbs, malformations on 30% of the plants grown from these bulbs and decrease PH, FM and DM as well as new bulbs production at 75%, 72.4%, 79.1% and 50% respectively. The treatment of B. tulipae infected bulbs with filtered OMW reduced further the PH, FM, DM and the production of new bulbs in 92.1%, 81.4%, 78.7% and 97% respectively. In contrast the treatment of infected bulbs by B. tulipae + A. niger with filtered OMW did not affect PH, FM and the number of new bulbs produced and significantly improved plants DM and the mass of new bulbs. Additional key words: OMW; Tulipa spp.; scab-like lesions; malformation; dry mass; biofungicides. * Corresponding author: chlikas@uth.gr Received: 25-06-13. Accepted: 20-02-14 Abbreviations used: AFP (antifungal protein); CMI (Commonwealth Mycological Institute); DBU (dibutylurea); DM (dry mass); FM (fresh mass); GAE (gallic acid equivalents); OMW (olive mill wastewater); PDA (potato dextrose agar); PH (plant height); SDW (sterilized distilled water); TDS (total dissolved solids); TP (total polyphenols); WP (wettable powder); Un (untreated bulbs). Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Spanish Journal of Agricultural Research 2014 12(1): 233-243 Available online at www.inia.es/sjar ISSN: 1695-971-X http://dx.doi.org/10.5424/sjar/2014121-4662 eISSN: 2171-9292 234 C. Lykas et al. / Span J Agric Res (2014) 12(1): 233-243 plication could inhibit seed germination (Casa et al., 2003; D’Annibale et al., 2004), shoot and root elongation and biomass production (Asfi et al., 2012), plant growth (Ouzounidou et al., 2010), uptake and translocation of nutrients (Ouzounidou et al., 2008) and cause various physiological changes (Mekki et al., 2006; Ouzounidou et al., 2008; El Hassani et al., 2010) including decreased water use efficiency mainly due to decreased CO2 assimilation rate (Asfi et al., 2012). The presence of phenols as well as short and long-chain fatty acids is believed to contribute to the phytotoxic nature of OMW (Casa et al., 2003; Kistner et al., 2004; Isidori et al., 2005). Accordingly, the current scientific knowledge seems to be insufficient to support the safe agricultural use of untreated OMW (Azbar et al., 2004), since the existing results are sometimes contradictory and not always useful to draw practical conclusions. The main objective of this study was to investigate the effect of OMW on growth and new bulb production of tulip plants, when these wastes were used for biological control of common tulip bulb diseases (Botrytis tulipae and Fusarium oxysporum) and tulip bulb moulds, caused by Aspergillus niger and Penicillium spp. as an alternative to disinfection of the bulbs with chemical fungicides. Material and methods


Introduction
Damage during mechanical harvesting and planting of tulip bulbs as well as handling during storage may result in significantly infected bulbs by fungi, bulb rot, reduction of root growth and increasing floral abortion (Saaltink, 1971;Piwoni, 2007).A common practice to reduce the rotting of bulbs is to disinfect them with chemical fungicides.However, several benzimidazolresistant fungi strains of Penicillium spp., Botrytis elliptica and Fusarium oxysporum have been already isolated on many bulb ornamental plants like tulips (Tulipa gesneriana L.), lilies (Lilium sp.) and gladiolus (Gladiolus communis L.) (Migheli et al., 1990;Bus et al., 1991;Chung et al., 2009).To overcome this problem and reduce the pesticide use, alternative methods based on the use of organic amendments for the biological control of soilborne plant pathogenic fungi has been reported (Hoitink et al., 1997;Abawi & Widmer, 2000).One alternative and economical solution is the use of olive mill wastewater (OMW), to reduce soil-borne plant pathogens in many crops and postharvest rot diseases of flower bulbs (Smid et al., 1995;Kotsou et al., 2004).In addition, aerobic biological of OMW and their use in agriculture to protect plant material from storage fungi (Bonanomi et al., 2006) will contribute to the reduction of the environmental pollution, since OMW constitutes a major environmental problem due to its high content in phenolic compounds as tannins and flavonoids (Hamdi, 1992;Gonzales et al., 1999).
However, apart from its antimicrobial properties, OMW may become phytotoxic (Bonari et al., 1993;Capasso et al., 1995), since uncontrolled OMW ap-plication could inhibit seed germination (Casa et al., 2003;D'Annibale et al., 2004), shoot and root elongation and biomass production (Asfi et al., 2012), plant growth (Ouzounidou et al., 2010), uptake and translocation of nutrients (Ouzounidou et al., 2008) and cause various physiological changes (Mekki et al., 2006;Ouzounidou et al., 2008;El Hassani et al., 2010) including decreased water use efficiency mainly due to decreased CO 2 assimilation rate (Asfi et al., 2012).The presence of phenols as well as short and long-chain fatty acids is believed to contribute to the phytotoxic nature of OMW (Casa et al., 2003;Kistner et al., 2004;Isidori et al., 2005).Accordingly, the current scientific knowledge seems to be insufficient to support the safe agricultural use of untreated OMW (Azbar et al., 2004), since the existing results are sometimes contradictory and not always useful to draw practical conclusions.
The main objective of this study was to investigate the effect of OMW on growth and new bulb production of tulip plants, when these wastes were used for biological control of common tulip bulb diseases (Botrytis tulipae and Fusarium oxysporum) and tulip bulb moulds, caused by Aspergillus niger and Penicillium spp. as an alternative to disinfection of the bulbs with chemical fungicides.

The study site
The experiments were carried out at the Plant Pathology Laboratory of the Technological Education Institute of Larissa, Greece and in an experimental polyethylene covered greenhouse, N-S oriented, located at the University of Thessaly near Volos, (Velestino: Latitude 39 o 22', longitude 22 o 44', altitude 85 m) on the continental area of Eastern Greece.

Olive mill wastewater characterization
Olive mill wastewater was obtained from a 3-phase olive mill plant, located near the city of Volos, central eastern Greece.The OMW was stored in PVC vessels tightly closed at 18°C until use.
Total polyphenols (TP) were determined with Folin-Ciocalteau reagent (Singleton & Rossi, 1965) and were expressed as gallic acid equivalents (GAE).Organic carbon was analysed by chemical oxidation with 1 mol L -1 K 2 Cr 2 O 7 and titration of the remaining reagent with 0.5 mol L -1 FeSO 4 .The organic carbon content was transformed in organic matter content by multiplying by 1.724, which is an experimental factor, reported by Hesse (1972).Total N, K and P content were determined by using Kjeldhal device, flame emission spectroscopy and molecular absorption spectroscopy, respectively, after sulphuric digestion.The concentration of Cu and Zn were measured by using atomic absorption spectroscopy, after the digestion of OMW with HNO 3 and HClO 4 (in a 2:1 portion).The characteristics of OMW used in this experiment are summarized in Table 1.

Pathogen preparation used for infections
Botrytis tulipae, Fusarium oxysporum, Aspergillus niger and Penicillium spp.isolated from infected tulip bulbs were used for this experiment.Potato dextrose agar (PDA) and malt extract medium was used for fungi isolation.The identification of fungi was made based on Hopkins (1921) description for B. tulipae and CMI descriptions of Pathogenic Fungi and Bacteria according to Brayford (1987) for F. oxysporum, Onions (1966) for A. niger and Kozakiewicz (1992) for Penicillium spp.(Sheets No. 921,94,111 respectively).
tested in vitro by using two procedures.In the first one, 25 mL of OMW were added into 1 L of PDA and further sterilized by autoclaving (121°C for 20 min).These substrates were placed in 9 cm Petri dishes.In the second procedure, Petri dishes containing PDA were sterilized by autoclaving (121 o C for 20 min) and afterwards 50 μL of filtered OMW (using syringe filler 0.2 μm) was added onto the centre of each Petri dish.Fifteen Petri dishes per treatment were inoculated with a mycelium plug (5 mm in diameter) colonized by the fungus taken from the periphery of 7 days old fungal colonies.Mycelia plugs were placed onto the centre of each Petri dish or next to the OMW drop.Equal Petri dishes numbers were used as control (without OMW).All cultures were incubated in darkness at 21°C for 6 days.At the end of this period, fungus mycelium length (mm) was measured.

Bulbs inoculation
Spore suspension was prepared from 7 days old cultures of Botrytis and Aspergillus species.Six Petri dishes with Botrytis and Aspergillus species cultures on PDA media (three dishes per fungus species) were used as spores' sources.Spores were collected by washing the agar surface with 3 mL distilled water.The produced solution was filtered through sterilized muslin in 250 mL conical flask that contained distilled water.In each flask spores, suspension was adjusted for a final concentration 10 6 spores mL -1 .A total of eighty healthy precooled tulip bulbs cv.Kees Nelis (with circumference of 11-12 cm) were divided in two groups (40 bulbs each) and sprayed with 50 mL of the above mentioned solution, containing spores of B. tulipae or spores of B. tulipae + A. niger.

Treatments of plant material
One hour after inoculation, ten bulbs from each group (infected with B. tulipae or B. tulipae + A. niger) as well as 10 healthy uninfected bulbs, were sprayed with: a) 20 mL f iltered OMW, b) 20 mL sterilized distilled water (SDW) or c) 20 mL Carbendazim (methyl 1H-benzimidazol-2-ylcarbamate) 50 WP (wettable powder) solution 120% w/v, while 10 bulbs from the infected groups and 10 healthy uninfected bulbs remained without any farther treatment.All bulbs were placed in plastic box chambers and kept for 12 days at 21°C.The plastic box chambers were spayed every two days with SDW to achieve high humidity (>85% RH).After the incubation period, the scab-like lesions on each tulip bulb were recorded and classified in six damage rating scales (0-5), where 0 is equal to healthy tulip bulb, 1 = brown halos without scab lesion, 2 = slightly visible scab lesion, 3 = scab lesions up to 1 mm, 4 = scab lesions up to 2 mm, and 5 = heavily infected tulip bulb with large scab-like lesions (>4 mm).
All the above mentioned tulip bulbs, were planted individually in 0.7 L plastic pots.A mixture of sterilized black pit and perlite (3:1 v/v) was used as substrate.The top 1-2 cm of substrate was removed to facilitate the exposures of bulb tip and stem measurements.
Pots were placed on benches inside the greenhouse with natural daylight and without any other OMW and Carbendazim application.Minimum day and night temperature inside the greenhouse was 18°C, with automatic ventilation at 22°C.The plants were irrigated every two days with distilled water, while, once a week, 2 g L -1 of Ca(NO 3 ) 2 was added with the irrigation water (Hanks, 1984).The pH of the irrigation solution was adjusted to 6.5.Pots were set out in dense rows.A plot was a pot and all experiments were complete randomised blocks with 10 replications.
At the end of the experiment (50 days after planting): a) the number of malformed plants, b) the plant height (PH) from the bulb tip, c) the fresh mass (FM) and dry mass (DM) of shoot, leaves and flowers and e) the number, FM and diameter of new bulbs produced from each plant were measured.

Statistical analysis
Data were analyzed using the Minitab statistical package (Ryan et al., 2005).One-way analysis of variance (ANOVA) was applied to test the effect of OMW on: a) mycelium length of B. tulipae, F. oxysporum, A. niger and Penicillium spp., grown in vitro, b) diameter of scab-like lesions on tulip bulbs caused from B. tulipae or with B. tulipae + A. niger infections and c) growth characteristics and new bulb production of plants grown in pots.Significance was evaluated at p < 0.05 for FM, DM and new bulbs production and b) p < 0.001 for all other parameters measured.

In vitro assessment of antimicrobial activity of OMW
The mycelia growth of B. tulipae, F. oxysporum, A. niger and Penicillium spp., decreased (p < 0.001) when filtered OMW was added on the agar surface where fungi were growing (Fig. 1).Mycelia growing on untreated PDA and on substrate with autoclave sterilized OMW had similar growth or a slight growth reduction.In contrast, filtered OMW strongly affected the fungi growth (Fig. 1).

Effect of olive OMW on scab-like lesions of tulip bulbs
Untreated bulbs as well as uninfected bulbs sprayed with SDW remained without scab-like lesions 12 days after inoculation (Fig. 2).However, uninfected bulbs sprayed with OMW, showed scab-like lesions probably caused from microbial flora present in OMW (Amaral et al., 2008).Infected with B. tulipae or with B. tulipae + A. niger bulbs showed a great number of scab-like lesions with large diameter, which decreased by about 50% after the treatment with filter sterilized OMW.Bulbs treated with Carbendazim shown only few scablike lesions with shorter diameter (Fig. 2).

Effect of olive OMW on plant growth
Treatment with OMW did not affect shoot emergence from the bulbs unless they were infected with B. tulipae, when shoot emergence decreased by 10%.In addition, 33.3% of uninfected bulbs sprayed with Carbendazim as well as 30.1% and 70% of bulbs infected with B. tulipae or B. tulipae + A. niger, respectively, and treated with Carbendazim, failed to emerge.The bulbs from the other treatments showed 100% emergence.
A great percentage of malformations occurred in plants grown from control bulbs sprayed with OMW (30%), control bulbs infected only with B. tulipae (20%) and untreated bulbs (16%) (Fig. 3).In contrast, plants from control bulbs infected with B. tulipae + A. niger, sprayed with SDW or Carbendazim did not show any abnormality.Bulbs infected with B. tulipae and treated with OMW resulted in 90% of plants with   malformed shoot, leaves and flowers, while bulbs infected with B. tulipae + A. niger and treated with OMW showed very few malformations (10%), mainly on leaves (Fig. 3).The results also indicate that treatment of infected bulbs with Carbendazim failed to reduce plants organ malformations, which were 90% and 100% for bulbs infected with B. tulipae or with B. tulipae + A. niger, respectively.
The effect of OMW application on PH, FM and DM production was also significant.Plants from control bulbs sprayed with OMW were shorter by 75% (Fig. 4), and had reduced FM and DM production by 72.4% and 79.1% respectively, compared to plants grown from bulbs sprayed with SDW with maximum height, FM and DM (Table 2).Bulbs infected with B. tulipae + A. niger gave plants of similar height (p < 0.001) to plants grown from SDW treated bulbs, and reached almost the maximum height plants of this variety can reach (26 to 35 cm).In contrast, plants grown from untreated bulbs (Un) and control bulbs sprayed with Carbendazim or infected with B. tulipae were shorter, having around half the height of SDW plants (Fig. 4).However (except of plants grown from control bulbs sprayed with OMW), only plants grown from B. tulipae infected bulbs suffered signif icant FM and DM reduction (Table 2).
Nevertheless, OMW treatment of bulbs did not limit final plant height, when bulbs were previously infected with B. tulipae + A. niger.OMW application resulted in plants with homogeneous height (Fig. 4) and improved their DM by about 18.2% compared to those grown from infected with B. tulipae + A. niger bulbs without any further treatment (Table 2).In contrast, bulbs infected with B. tulipae produced plants with 46.7%, 56.4% and 59.6% height, FM and DM reduction, respectively, compared to plants grown from SDW bulbs.These reductions became greater (92.1%, 81.4% and 78.7%, respectively) after their treatment with OMW (Fig. 4

Effect of olive OMW on new bulb production
New bulb production was strongly influenced by OMW or Carbendazim spraying, since plants grown from control bulbs sprayed with these solutions produced almost half of the number of bulbs in the next generation, compared with untreated and SDW treated bulbs (Table 3).In addition, almost 40% of plants from the above mentioned treatments did not produce any new bulb.OMW or Carbendazim spraying had slightly different influence on the FM of bulbs of the next generation.The production of new bulbs remained almost unaffected when control bulbs were sprayed with Carmendazim, but reduced by 37.5% when  control bulbs sprayed with OMW, compared to SDW treated bulbs.Infection of control bulbs with B. tulipae did not affect new bulb production as well as their FM and diameter, whereas 28.6% of the plants grown from these bulbs did not produce new bulbs.However, new bulbs FM decreased by 45.5%, when control bulbs were infected with B. tulipae + A. niger.During this treatment 40% of the growing plants did not produce new bulbs.
The application of OMW on infected with B. tulipae bulbs signif icantly reduced (97%) the new bulb production, as well as 82.5% the FM of new bulbs and increased 62.7% the number of plants without any new bulb compared to SDW treated bulbs (Table 3).In contrast, B. tulipae infected bulbs treated with Carbendazim left unaffected the mentioned parameters.In the other hand, plants from bulbs infected with B. tulipae + A. niger had similar new bulb production, new bulbs FM and their maximum diameter to plants from control treatments, when bulbs sprayed with OMW; but there was no new bulb production, when these bulbs were treated with Carbendazim (Table 3).

Discussion
The antimicrobial activity of OMW against B. tulipae, F. oxysporum, A. niger and Penicillium spp.
indentified in vitro experiments can be attributed to the phenol content (0.43%) and antioxidants remaining in the f iltered OMW (Fodale et al., 1999;Paredes et al., 1999;Kotsou et al., 2004).A solution with similar phenol concentration can kill 75% of Botrytis sp.spores in almost 120 min (Henderson, 1982).In contrast, many Aspergillus, Penicillium and Fusarium species are resistant to phenol since characterized from their ability to degrade aromatic compounds (García et al., 2000;Millan et al., 2000;Mendonça et al., 2004).The decreased mycelia growth observed in vitro, when filtered OMW was added on the agar surface where the fungi were grown, could be also attributed to Cu 2+ concentration (12.7 mg kg -1 ) measured in OMW, since minimum Cu 2+ inhibitory concentrations for fungi colony growth is 10 mg L -1 for Fusarium spp., Penicillium spp.and Aspergillus spp.(Iqbal et al., 2010;Rathod et al., 2010;Askarne et al., 2011).Olive mill wastewater seems to lose its properties when is sterilized at 121°C for 20 min, since normal mycelia growth was observed on substrate containing autoclave sterilized OMW.This is due to the decreased antioxidant activity of phenolic compounds with increasing temperature (Réblová, 2012), but this is not similar for all phenolic compounds, which can be more than 30 in olive mill wastewater, as reported by several authors (Greco et al., 2006;De Marco et al., 2007).However, use of OMW on uninfected bulbs may cause primary infections, probably due to the presence of moulds like Penicillium sp. and Aspergillus sp.detected and isolated from the olive mill waste water (Mouncif et al., 1993).Hamdi et al. (1991a,b), Hamdi & Ellouz (1992a,b) and Martínez-Nieto et al. (1992), studied the ability of A. niger to grow in the presence of OMW.Scab-like lesions observed on tulip bulbs caused from these infections were limited, without any further evolution compared to those caused from B. tulipae or B. tulipae + A. niger infections, probably because of the phenols and antioxidants content of OMW that hinder their further evolution.It is noteworthy that these scab-like lesions had similar evolution to those developed on tulip bulbs infected with the above mentioned fungus and treated with filter-sterilized OMW.Shoot emergence was the only parameter that remained unaffected after OMW application, probably because shoot emergence depends mainly from internal physiological factors, such as dormancy and maturity.However, shoot emergence was slightly negatively affected (10% decrease) only when bulbs were previously infected with B. tulipae.This must probably be attributed to the action of B. tulipae, which attacks mainly to the emerged shoot, rather to the OMW toxicity.In contrast, a great percentage (33.3%) of uninfected bulbs sprayed with Carbendazim failed to emerge shoots.Benomyl (methyl (1-(butylaminocarbonyl)-1H-benzimidazol-2-ylcarbamate) and its main metabolite Carbendazim have been shown to negatively affect root growth and shoot development of several ornamental plants (Klingensmith, 1961;Hocking & Thomas, 1979;Lee et al., 1983).In addition, increased fungicide concentration may cause abnormalities in mitotic cell divisions and mitosis decreases (Dane & Dalgiç, 2005) as well as significant changes in morphology of bulb plants (Reyes, 1975).An additional reduction (about 36.7%) of shoot emergence observed in Carbendazim-treated bulbs previously infected with B. tulipae + A. niger, whereas there was no significant further reduction when bulbs were previously infected only with B. tulipae.It seems that the net effect of Carbendazim on shoot emergence and growth was the result of interplay between its disease controlling and phytotoxic effects.
Spraying uninfected (control) bulbs with OMW signif icantly increased shoot, leaf and flower malformations and reduced plants height (by about 75%) and FM and DM compared to SDW control bulbs.A common adverse effect of OMW treatment is the reduction in fresh and dry biomass production in different crops and other plants such as tomato, spearmint, peppermint, and wheat (Mekki et al., 2006;Ouzounidou et al., 2008;El Hassani et al., 2009a,b).This can be attributed to a number of factors such as: a) decrease of leaf chlorophyll and carotenoids concentrations (Asfi et al., 2012), which can affect plant photosynthesis, b) root growth reduction, since phytotoxic substances existing in OMW such as phenols, organic acids, and fats affect root membrane structure and modify its functions including metabolic efficiency and stability (El Hadrami et al., 2004) , c) nutrients depletion which severely affects metabolism and resulting in growth inhibition (Wulff-Zotelle et al., 2010) and d) low water use efficiency mainly due to decreased CO 2 assimilation (Asf i et al., 2012).However, the intensity of this phytotoxicity is related to the concentration of OMW and the sensitivity of plant species to OMW.Increased phytotoxicity after OMW treatment can be attributed also to the increased microbial activity that can lead to phytotoxic effects, through the release of phytotoxins from the microbial breakdown of the organic residues (Stroo et al., 1988;Tiquia et al., 1996).Spraying Carbendazim on control bulbs did not result to the production of malformed plants however may cause plants height, FM and DM reduction compared to OMW application.The negative effect of Carbendazim on plant growth can be probably attributed to their metabolites like dibutylurea (DBU), which can decrease the net photosynthesis of the plant.Although there have been no published reports of the effects of DBU on the gas exchange of higher plants, Van Lersel & Bugbee (1996) reported that DBU decreased the dry matter production and shoot height of cucumber.
According to the present results the extent of shoot and leaf malformation as well as plants height, FM and DM reduction in plants grown from infected bulbs, depend on infection by one or two fungi as well as on the treatment (OMW or Carbendazim).Infection of bulbs with B. tulipae + A. niger, did not cause plant malformations or PH and FM reduction, probably because of the production of antifungal substances by A. niger (Moreno et al., 2003;Brzezinska & Jankiewicz, 2012) and/or antagonism between the two fungi.The mold Aspergillus giganteus is known to produce and secrete a basic low molecular weight protein, the antifungal protein (AFP).This protein strongly inhibits mycelial growth as well as conidial germination of B. cinerea.The antagonism between B. tulipae and A. niger may strongly be influenced by pH, diameter of colonies and soil environment conditions (Sahile et al., 2011) (Millán et al., 2000), reducing the total phenol content in OMW and decreasing their phytotoxicity.Nevertheless, we observed malformation in 10% of plants, probably as an adverse effect of OMW treatment.In contrast, when bulbs were previously infected with B. tulipae, OMW treatment increased shoot, leaf and flower malformations, FM and DM reduction.These results indicate that OMW treatment of bulbs infected with B. tulipae + A. niger was more effective than OMW treatment of bulbs infected with B. tulipae alone.Inoculation of bulbs with B. tulipae resulted in the formation of primary infections but this happened only where the shoot tip came into contact with the fungi (Coley & Javed, 1972), preventing shoot and leaf emergence and causing leaf malformations and blemishes flowers.The treatment with OMW did not reduce malformations caused from B. tulipae infections, but it seemed to act synergistically increasing the percentage of abnormalities in plants.The same negative effect was observed with Carbendazim treatments, which not only failed to improve the height, FM and DM of plants produced from B. tulipae or B. tulipae + A. niger infected bulbs, but it seemed to further decrease them.
The reduced new bulb production and new bulb weight produced from plants grown from control bulbs sprayed with OMW can be attributed probably to the decreased plant DM and increased leaf malformation due to OMW (Timmer & Van Der Valk, 1973;Benschop & Van Der Valk, 1984).In contrast, limited plant FM and DM reduction, as well as the absence of malformations in plants grown from control bulbs sprayed with Carbendazim, resulted in a higher number of new bulbs with higher FM.According to the same pattern, the inability of OMW and Carbendazim treatment to limit DM reduction of plants grown from infected by B. tulipae or B. tulipae + A. niger bulbs, respectively, resulted in a proportional reduction of the number and the FM of the new bulb produced from the plants of these treatments.In general, new bulbs production followed a logarithmic correlation with plant DM reduction (R 2 = 0.8), regardless of the cause of this reduction (fungal infection, type of treatment solution used or both), whereas plants with DM lower than 0.6 g did not produce any new bulb (Fig. 5).
Olive mill wastewater can reduce the growth of Botrytis tulipae, Fusarium oxysporum, Aspergillus niger and Penicillium spp., when it is used with in vitro cultured fungi, probably because of its phenolic and antioxidant components, but this ability will be lost if OMW is sterilized at 121 o C for 20 min.Consequently, treatment of infected bulbs with OMW may decrease significantly the scab-like lesions resulting from these infections on bulb surface.The treatment of B. tulipae infected bulbs with OMW seems to be unable to reduce malformations caused from infections.The OMW seems to act synergistically to fungi infection, increasing the malformations of plants and decreasing further plant height and dry mass as well as new bulb production (their number and weight).In contrast, treatment of B. tulipae + A. niger infected bulbs with OMW, does not affect plants height, their fresh mass and the number of new bulbs produced from these plants and improve significantly plant dry mass and daughter bulb weight.However, spraying of uninfected bulbs with OMW may cause primary infections because of the microbial flora (like Penicillium sp. and Aspergillus sp.) that had been yet detected from other researchers in OMW.The scab-like lesions from these infections will probably remain limited, without any further evolution.OMW application does not affect shoot emergence but will cause shoot, leaves and flowers malformations and will decrease significantly plant height as well as fresh and dry mass production.Due to leaf malformations and plant dry mass reduction caused from bulbs spraying with OMW, the number of new bulbs will be reduced.

Figure 3 .
Figure3.Malformed plants grown from untreated tulip bulbs (Un), control bulbs sprayed only with olive mill wastewater (OMW), control bulbs infected with B. tulipae (Bt) and bulbs treated with filtered OMW or Carbendazim after infection with Bt or Bt + Aspergillus niger (Bt + An).Treatments with the same letter were not significantly different at the 0.001 threshold.

Figure 2 .
Figure2.Scab-like lesions on tulip bulbs: A) untreated, B) sprayed with sterilized distilled water (SDW), C) uninfected sprayed with olive mill wastewater (OMW), D) infected with Botrytis tulipae (Bt), E) infected with Bt + Aspergillus niger (An), F) infected with Bt and treated with filtered OMW, (G) infected with Bt + An and treated with f iltered OMW, H) infected with Bt and treated with Carbendazim and I) infected with Bt + An and treated with Carbendazim.Treatments with the same letter were not significantly different at the 0.001 threshold.All treatments were significantly different (p < 0.001) from control (A and B treatments).

Figure 4 .
Figure 4. Height of control plants grown from untreated tulip bulbs (Un), sprayed only with sterilized distilled water (SDW), sprayed only with mill wastewater (OMW), sprayed only with Carbendazim (CA), infected with B. tulipae (Bt) or Bt + Aspergillus niger (Bt + An) and treated with filtered OMW or Carbendazim after infection.Vertical bars indicate standard deviation.

Figure 5 .
Figure 5. Correlation between the number of new bulbs and green plant dry matter (DM).

Table 1 .
Main physical and chemical characteristics of olive mill wastewater (OMW) and Table 2).Treatment with Carbendazim decreased PH and FM by about 73.3% and 31.1%,respectively, compared to plants grown from SDW bulbs, but increased DM by 42% compared to plants grown from B. tulipae infected bulbs and decreased plants height, FM and DM by 93.9%, 73.7% and 72.2%, respectively, compared to plants grown from B. tulipae + A. niger.

Table 2 .
Fresh mass (FM) and dry mass (DM) of tulip plants grown from treated bulbs e Means in each column followed by the same letter are not statistically significant (Duncan, p ≤ 0.05).

Table 3 .
Number of new bulbs, fresh mass (FM), maximum diameter and percentage of plants without new bulb production SDW: sterilized distilled water.OMW: olive mill wastewater.Means in each column followed by the same letters are not statistically significant (Duncan, p ≤ 0.05).
. In our experiments, infection of bulbs with B. tulipae + A. niger without any other treatment caused a slight plant FM and DM reduction, compared to plants grown from SDW sprayed bulbs.However, this FM and DM reduction was lower than the reduction caused when bulbs were infected only with B. tulipae.The above mentioned results confirm the inhibitory action of A. niger against B. tulipae.The presence of A. niger in bulbs microflora probably restrict B. tulipae action and consequently can reduce B. tulipae effect on plants FM and DM reduction.Plant FM and DM reduction caused from B. tulipae + A. niger infection was deterred after the treatment of the infected bulbs with OMW.The beneficial role of A. niger in this treatment seems to be twofold since it acts against B. tulipae and also it can biodegrade OMW