Effect of two liquid formulations based on Brassica carinata co-products in containing powdery mildew on melon

Industrial Crops and Products 75 (2015) 48–53

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Effect of two liquid formulations based on Brassica carinata co-products in containing powdery mildew on melon E. Piccinini a , V. Ferrari a,∗ , G. Campanelli a , F. Fusari a , L. Righetti b , E. Pagnotta b , L. Lazzeri b a Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria – Unità di Ricerca per l’Orticoltura di Monsampolo del Tronto (AP) (CRA-ORA), Via Salaria 1, Monsampolo del Tronto, AP 63077, Italy b Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria – Centro di ricerca per le colture industriali, Via di Corticella 133, Bologna 40129, Italy

a r t i c l e

i n f o

Article history: Received 3 July 2014 Received in revised form 4 May 2015 Accepted 14 May 2015 Available online 15 June 2015 Keywords: Allelopathic effect Co-products Biodiesel Biofumigation Bio-based Brassica carinata

a b s t r a c t Two liquid bio-based formulations were characterized and evaluated for powdery mildew (Podosphaera xanthii) control on melon. The new products were based on oil and defatted seed meals of Brassica carinata at different concentrations, formulated according to the patented procedure PCT WO 2006/136933. The abilities of innovative emulsions to release the bioactive molecule allyl-isothiocyanate and to contain its dispersal in the air were evaluated in lab conditions. In the years 2012 and 2013, trials on plants naturally infected with P. xanthii were performed at Monsampolo del Tronto (Ascoli Piceno, Italy) in open field. Both formulations showed disease control properties. In particular, the formulation with the higher concentration of oil and defatted seed meal gave results statistically not different from Topas 10 EC, a chemical pesticide widely applied for melon defence both in greenhouse and open field, that was inserted as control. Two weeks after the first treatment no leaves were presenting lesions covering more than 60% of their surface and the attack index, which takes into account both the area covered by lesions and the number of infected leaves, was halved if compared to the untreated control. The new bio-based formulations could thus be considered of great interest both for conventional and organic farming in the control of powdery mildew on melon. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The main spring–summer horticultural crops are extremely susceptible to attack of many insects and fungi, that can cause serious damages to the vegetative-productive structures of plants. Among these, powdery mildew is one of the most common fungal diseases in the world, attacking nearly ten thousand species of Angiosperms (Glawe, 2008). This disease frequently affects Cucurbitaceae (Braun, 1995) and, when not kept under control, it can cause considerable damages to melon (Cucumis melo L.) both in open field and greenhouse cultivation (Alvarez et al., 2000; Brunelli, 2007), where it strongly reduces qualitative and quantitative crop yields. Powdery mildew represents a considerable problem in Italy where melon is cultivated on more than 20,000 ha in open field and on 3,000 ha in protected cultivation farming, with a total yield of

∗ Corresponding author. Tel.: +39 0735 703684; fax: +39 0735 703684. E-mail address: [email protected] (V. Ferrari). http://dx.doi.org/10.1016/j.indcrop.2015.05.024 0926-6690/© 2015 Elsevier B.V. All rights reserved.

almost 700,000 ton year−1 (Istat, 2010), data that make Italy the second largest producer in Europe after Spain. It can be caused by several etiologic agents, and the most frequent on Cucurbitaceae are Podosphaera xanthii (Castag.) U. Braun & N. Shish. [sin. Sphaerotheca fuliginea Schlect. ex Fr. (Poll.)] and Golovinomyces cichoracearum (D.C.) Huleta (sin. Erysiphe cichoracearum D.C.) (Kˇrísková et al., 2009). P. xanthii is the most widespread in the Mediterranean basin (Torés Montosa, 1987; Branzanti and Brunelli, 1992; Pérez-Garcia et al., 2009), while G. cichoracearum is more common in Central Europe (Kˇrísková et al., 2009). In melon, buds and growing fruits are the most frequently affected structures, showing firstly a discoloration, followed by a progressive tissue necrosis and subsequent desiccation. The infection spread takes place through the air via chlamydospores that are favored in the hot and dry Mediterranean climatic conditions. In conventional farming, the control methods are essentially based on treatments with sulfur-containing products or with a range of pesticides. The use of sulfur, even if effective in pathogen control, can cause phytotoxicity symptoms on some melon cultivars, especially at the high temperatures typical of the Italian

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summer time. In addition, it is characterized by a withholding period of 5 days, an aspect that at harvest time may determine serious economic damages for the farmer. Pesticides, instead, are generally cheap and practical, but are often characterized by important negative drawbacks, i.e., a withholding period varying from 3 to 14 days and the reduction of fungicidal efficacy due to the occurrence of pathogen resistance, as reported in the case of benzimidazoles, sterol biosynthesis inhibitors (SBI), and strobil˜ urin analogues (McGrath and Shishkoff, 2003; Fernandez-Ortuno et al., 2006). In addition, they are often characterized by a high environmental impact and by risks of toxicity for operators and consumers. In this ambit, the European Community has defined several procedures such as the EC Regulation No. 1907/2006 concerning the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) aimed at a more regulated application of chemistry. The Directive 2009/128/EC comprises several actions targeted at a sustainable use of pesticides including the need of defining new non-chemical control systems in crop management and defence, as a partial or total alternative to conventional pesticides. This concept is also reported in the CAP 2014 (“The CAP towards 2020”) in order to promote and encourage competitive and sustainable agriculture and forestry. For these reasons, there is a growing interest in innovative bio-based techniques of cultivation, with a low impact on the agro-ecosystem (Gilardi et al., 2011), and short or no withholding periods (Liu et al., 2010). In organic farming, the admitted options for powdery mildew control are limited to microorganisms, such as the hyperparasite Ampelomyces quisqualis (Legler et al., 2009), and to products based on sulfur (EC Regulation No. 889/2008) with the above mentioned problems of phytotoxicity. In recent years, several studies have been conducted on powdery mildew control with natural products such as chabazite (Passaglia, 2006; Romanazzi, 2008), Stifenia, a natural compound extracted from Trigonella foenum-graecum L. seeds (Camele et al., 2009), and more recently, water/oil emulsions containing defatted seed meals of the Brassicaceae (Rongai et al., 2009; Candido et al., 2014). This last option is based on the well known defensive tool of Brassicaceae, the glucosinolate-myrosinase system (Agerbirk and Olsen, 2012), and its capability of releasing allelopathic compounds, so called for their ability to influence the growth, survival, and reproduction of several organisms. The agricultural technique exploiting this system is called biofumigation and it is of particular interest, considering also the potential cultivation of endemic oleaginous Brassicaceae species in a full biorefinery approach (Lazzeri et al., 2011). According to this strategy, oil and defatted seed meal can also be used to produce bio-based formulations for plant management and disease control in agriculture (Lazzeri et al., 2013). The aims of this work were to compare two formulations based on Brassica carinata oil and defatted seed meal, characterizing their ability to release and retain the bioactive molecule allylisothiocyanate and evaluate their efficacy in controlling P. xanthii on melon cultivated in open field, in order to provide a new suitable alternative to systemic chemical pesticides.

2. Materials and methods 2.1. Biomass description Two patented formulations based on B. carinata A. Braun (common name Ethiopian mustard) oil and defatted seed meals (DSM) (Rongai et al., 2006) were used. B. carinata oil and DSM, defatted by an endless screw press in which temperature and pressure were continuously monitored, were purchased from Agrium Italia S.p.A. (Livorno, Italy).

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2.1.1. The oil After extraction, the oil was filtered, partially refined (degummed) and characterized for its fatty acid composition by the UNI EN ISO 5508:1998 method. 2.1.2. Defatted seed meal characterization Moisture content in DSM was determined by evaluating the difference between its weight before and after oven-drying at 105 ◦ C for 12 h. Nitrogen content was determined by the Kjeldhal method as reported in the UNI 22604:1992 method, using a Tecator digestion system 20 and an automatic Büchi distillation unit (B 324). Glucosinolates (GLs) were determined following the ISO 91671:1992 method, with some minor modifications reported in Lazzeri et al. (2011). All data are reported as mean ± SD of four determinations. 2.1.3. The formulations DSMs were formulated according to patented procedures (Lazzeri et al., 2010) aimed at optimising the enzymatic system that catalyzes GLs hydrolysis. The details must be considered as confidential information and will not be extensively reported. Four treatments were defined and compared through in vivo trials: (a) Untreated control, sprayed with a volume of water equivalent to that of insecticide treatments. (b) Topas 10EC (active ingredient: Penconazole, Syngenta) at a dose of 25 mL/100 L of water. It is a systemic triazolic fungicide characterized by a curative and preventive activity, widely applied in conventional agriculture for powdery mildew control. (c) Bio-based experimental formulation (Formulation 1) containing 1.5% of B. carinata oil supplemented with a natural emulsifier, 3 g L−1 of formulated DSM and other minor natural components for a total amount ≤5%. The composition was essentially based on commercial fertilizer Duolif (Agrium Italia, Livorno, Italy), changing proportions. (d) Bio-based experimental formulation (Formulation 2) containing 2% of B. carinata oil with a natural emulsifier and 4.5 g L−1 of formulated DSM. The composition was essentially based on commercial fertilizer Duofruit (Agrium Italia, Livorno, Italy), changing proportions. Formulations 1 and 2 were prepared mixing the meals with the oil and then adding water, which activated the enzymatic hydrolysis of GLs via Myrosinase with the controlled release of the corresponding degradation products, mainly allyl-isothiocyanate (AITC) (Fahey et al., 2001). The suspensions were mixed every 5 min. 20 min was the time required after water addition for maximizing GLs hydrolysis, therefore it was set as the time needed before distribution. 2.1.4. Determination of allyl-isothiocyanate release from the formulations The kinetic of AITC release was estimated by gas headspace chromatography as in De Nicola et al. (2013). Formulations 1 and 2 were tested and a separate calibration curve for the two concentrations of oil was calculated using a pure AITC standard (Fluka, Steinheim, Germany). Results are expressed as micromoles of AITC and referred to the sinigrin (2-propenyl glucosinolate) content in B. carinata DSM to calculate the hydrolysis rate. The data derived from three replicates of the formulations. 2.1.5. Determination of the formulations capability of allyl-isothiocyanate retention AITC (Fluka, Steinheim,Germany) was dissolved in 50 mL of water, or in the same volume of 1.5% and 2% B. carinata oil/water emulsion. AITC concentrations were equivalent to total theoretical

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release of AITC from formulations at 3 g L−1 and 4.5 g L−1 of seed meal. Tubes were immediately closed with gas-tight Teflon caps and placed in agitation for 30 min at room temperature (21 ± 1 ◦ C). A volume of 200 ␮L of solution was collected for AITC basal determination and tubes were left open in agitation for another 30 min before a second sampling. AITC was determined according to Zhang et al. (1992). Briefly 200 ␮L of AITC solution were added to 800 ␮L of 100 mM potassium phosphate buffer (pH 8.5), 900 ␮L of methanol and 100 ␮L of 80 mM 1,2 benzenedithiol (Sigma–Aldrich, Milan, Italy) in isopropanol. The vials were heated for 2 h at 65 ◦ C and cooled to room temperature, after which their absorbance at 365 nm was determined against a paired blank containing all ingredients except AITC. Every solution was tested in triplicate. 2.2. Trial protocol The research was conducted in open field in the years 2012 and 2013 at the experimental farm of CRA-ORA at Monsampolo del Tronto (Ascoli Piceno, Italy) (42◦ 53 N; 13◦ 47 E; 60 m above sea level) on a medium textured soil. Each year, the soil was ploughed to a depth of 20 cm before harrowing, and fertilized with 80, 80 and 150 kg ha−1 of N, P2 O5 and K2 O, respectively. Plastic mulching was laid down, covering 80 cm wide strips of soil with black plastic (50 ␮m thick). Melon seedlings of the Supermarket cultivar (FourBlumen), chosen for its high susceptibility to powdery mildew, were transplanted at the 3rd–4th leaf stage in the first week of June. The seedlings were spaced at 1 m on rows that were 2 m apart, in order to obtain a density of 0.5 plants m−2 . Throughout the crop cycle, the field was irrigated by a drip irrigation system placed under the plastic mulch with drip holes spaced 20 cm from each other (water flow rate at 2.5 L h−1 ). Post-transplanting, fertilization provided 40 kg ha−1 of N, 70 kg ha−1 of P2 O5 and 50 kg ha−1 of K2 O. The trials were focused on verifying, for each treatment, the capacity of pathogen containment and not on disease prevention. Thus, the treatments were carried out after the symptoms of powdery mildew started to appear on every plot. The treatments were applied with a sprayer powered by a 2-cycle engine. A spray pressure of 5 bar was used, and the volume of solutions was about 500 L ha−1 . Plots were sprayed 3 times during the crop cycle, starting at the beginning of the flowering stage and repeating the treatment twice, with a seven day interval. At the end of the trials, before melon harvesting time, all the plants were removed, and soil incorporated; in no case the plants were used for food or feeding.

Table 1 Moisture, nitrogen and GLs content in B. carinata defatted seed meal (DM means dry matter). Mean ± S.D Moisture Nitrogen GLs

4.3 ± 0.3% 5.70 ± 0.01% DM 90.3 ± 2.0 ␮mol g−1

2.4. Statistical analyses The comparison between the AITC trapping ability of water and B. carinata oil/water emulsions was performed in triplicate and statistical analysis was performed on the absorbance at 365 nm data by Student’s test (Sigma Plot 10.0-SPSS, Chicago, IL, USA). P < 0.05 was considered as statistically significant. In both years, a total of 4 treatments was performed in a randomized block design with 3 replications each on a surface area of 50 m2 . For statistical analysis, the percentage data were transformed into their respective angular values and subjected to analysis of variance and to the Duncan test. Each sampling datum was analyzed separately. 3. Results 3.1. Biomass characterization 3.1.1. Oil fatty acid composition The oil, obtained by a pressure extraction procedure, confirmed the high level of long chain fatty acids with an erucic acid (C22:1) content higher than 40%. The extraction method made it possible to classify the oil as produced without any chemical treatment. 3.1.2. Defatted seed meals B. carinata DSM showed a GLs content of about 90 ␮mol g−1 (Table 1), 97% of which was identified as 2-propenyl glucosinolate (trivial name: sinigrin). The presence of more than 5.5% of nitrogen (Table 1) may grant the emulsion a secondary fertilizing or biostimulating activity. 3.1.3. Determination of allyl-isothiocyanate release from the formulations Water addition to the oil/DSM mixture activated the GLsmyrosinase hydrolysis reaction, allowing AITC release in the emulsion. After 20 min, the hydrolysis reaction reached its plateau and the amount of released AITC was 79 and 68% of the potential amount calculated on the GLs content in DSM, for Formulations 1 and 2, respectively.

2.3. Disease assessment The powdery mildew agent was identified at CRA-PAV (Rome, Italy). The disease assessment was carried out on 10 leaves per plant (7 plants) in three successive surveys at intervals of 7 days (T0, T7 and T14), a period coinciding with the interval fixed for the treatments. Leaves, depending on the level of infection, were grouped into classes and the representativeness of each class was expressed as a percentage. Class 0 referred to leaves without any symptoms (healthy leaves), class 1 to leaves with 1–10% of lesioned area, class 2 to leaves with 11–30% of lesioned area, class 3 to leaves with 31–60% of lesioned area and class 4 to leaves with more than 60% of lesioned area, including senesced leaves. For a concise description of plant health, an Attack Index (A.I. = % leaf area covered by lesions /Log10 % infected leaves) was used, derived from the ratio between the sum of the weighting averages of each class of attack and the base−10 logarithm of the percentage of leaves variously attacked by the fungus (Ferrari and Piccirillo, 1995).

3.1.4. Determination of the formulation capacity of allyl-isothiocyanate retention As shown in Fig. 1, emulsion with 1.5% of B. carinata oil (the concentration of Formulation 1) retained about 40% of pure AITC. This effect was not significantly different from that observed in water where, for different AITC concentrations, the retention was always by 25–30%. Instead, a slight increase of B. carinata oil concentration in emulsions till 2%, as in Formulation 2, caused a 60% retention of AITC. This result was approximately double if compared to AITC retention in water. 3.2. Experimental trials In both years a powdery mildew attack caused by the agent Podosphaera xanthii was recorded, with a higher A.I. at T0 in the first year. The preparation of the formulations proved to be practical and their distribution easy, for an average mechanization level

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B. carinata oil

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Table 3 Evaluation of P. xanthii attack in 2013. Different letters correspond to significantly different values for p ≤ 0.05 (Duncan test) (A.I. means attack index) Second trial year (2013)

B. carinata oil

L-1 Fig. 1. AITC retention in water and B. carinata oil/water emulsions. Data are expressed as relative retention % after 30 min of stirring in open vials and compared to AITC basal concentration in closed vials. AITC concentrations of 270 and 405 ␮mol L−1 correspond to those of 3 g L−1 and 4.5 g L−1 DSM, respectively. F1 and F2 indicate the AITC retentions at starting AITC concentrations corresponding to Formulations 1 and 2, respectively. Statistical analysis on data relating to absorbance at 365 nm was performed by Student’s test; F1 vs F2 gives P = 0.03.

Table 2 Evaluation of P. xanthii attack in 2012. Different letters correspond to significantly different values for p ≤ 0.05 (Duncan test) (A.I. means attack index). First trial year (2012) Class

0

1

2

3

4

A.I.

T0 Test Formulation 1 Formulation 2 Topas 10 EC

0g 0g 0g 0g

40 cd 25 de 30 de 30 de

40 cd 55 ab 50 ac 60 a

20 ef 20 ef 20 ef 10 fg

0g 0g 0g 0g

14.0 a 14.5 a 13.9 ab 13.4 b

T7 Test Formulation 1 Formulation 2 Topas 10 EC

0d 0d 0d 5d

30 b 30 b 30 b 30 b

30 b 50 a 45 a 50 a

30 b 15 c 25 c 15 c

10 cd 5d 0d 0d

21.1 a 16.5 b 14.2 bc 13.7 c

T14 Test Formulation 1 Formulation 2 Topas 10 EC

0e 0e 0e 0e

10 d 13 d 20 c 25 c

25 c 37 b 50 a 50 a

35 b 40 a 30 bc 25 c

30 bc 10 de 0e 0e

33.4 a 18.7 b 15.8 c 15.5 c

of a horticultural farm. No phytotoxic effect was observed on plants for either formulation during the trials. 3.2.1. First trial year In 2012, the first significant and uniform presence of powdery mildew was recorded on August, 7 (T0). The A.I. were very similar in all the plots ranging from 13.4 to 14.5 (Table 2). The second sampling showed on the untreated plots an increase of 50.7% of the disease level compared to T0, reaching an A.I of 21.1. Differently, the treated plots were characterized by a slower disease development, representing an increase of 13.8% with Formulation 1 and only 2.2% in the plots treated with Formulation 2, whose performance did not significantly differ from Topas. The third rating (T14) showed an intense spread of the pathogen on plants of the untreated plots, reaching an A.I. of 33.4, 58.3% higher than 7 days before. Instead, after the second treatment with Formulation 1, the plants showed a significant containment of powdery mildew attack with an A.I. of 18.7, only 13.3% more than the infection rate of 7 days before. A greater effect on disease containment was observed using Formulation 2, with an A.I. of 15.8, a value not statistically different from plants treated with the chemical Topas (A.I. = 15.5).

Class

0

1

2

3

4

A.I.

T0 Test Formulation 1 Formulation 2 Topas 10 EC

25 c 15 d 0e 10 d

50 a 40 b 45 ab 50 a

25 c 45 ab 55 a 40 b

0e 0e 0e 0e

0e 0e 0e 0e

5.8 b 8.4 ab 10.2 ab 8.1 ab

T7 Test Formulation 1 Formulation 2 Topas 10 EC

0f 10 e 0f 10 e

25 cd 30 bc 35 bc 45 b

60 a 45 b 65 a 40 b

15 de 15 de 0f 5e

0f 0f 0f 0f

14.8 a 13.7 b 11.4 b 9.6 c

T14 Test Formulation 1 Formulation 2 Topas 10 EC

0f 5f 0f 0f

0f 30 de 25 e 45 bc

10 f 35 ce 70 a 45 bc

40 bd 25 e 5f 10 f

50 b 5f 0f 0f

44.6 a 17.3 b 13.3 b 11.8 b

3.2.2. Second trial year In order to confirm the results of the first year, the trial was repeated in 2013, a year in which the first significant and uniform presence of powdery mildew was recorded on August, 12 (T0) with an A.I. that ranged from 5.8 to 10.2 (Table 3). One week after the first treatment (T7), the disease spread was evident in the untreated plots (+155.2%), while the A.I. reached values of 13.7, 11.4, and 9.6 in the plots treated with Formulations 1, 2 and Topas, respectively. At T14, the untreated plants showed an A.I. increased of 201.4% compared to the second sampling, with all the leaves damaged. On plants treated with Formulation 1, the evolution rate of the disease was estimated at 26.3%, a value significantly lower than for untreated plants. On plants treated with Formulation 2, the increase of the disease level was limited to only 16.7%. The plots treated with Topas showed an A.I. only slightly higher (22.9%) than in the second sampling. 4. Discussion The pressure extraction procedure made it possible to classify oil and DSM as materials not chemically modified and, in this way, both are considered to be exempted from registration in the European REACH Regulation, EC Regulation No. 1907/2006, Annex IV. The components of the natural patented Formulations 1 and 2 showed different effects on disease control. In fact, paraffinic oils (“white oils”) are known to have an inhibitory effect on disease development, and Brassica oil/water emulsions alone were previously reported as not statistically different from paraffinic oils in red scale containment (Rongai et al., 2008). The paraffinic oils act essentially by asphyxiation, covering the body of the insect with a thin film, penetrating by capillarity in the tracheal channels and occluding them (Muccinelli, 2006). Considering the similar oily characteristics and the applied doses, even natural oils are expected to play a similar effect. Furthermore, as reported in Rongai et al. (2008), this effect was intensified by the supplement of DSM that, after water addition, activate the enzymatic hydrolysis of GLs via myrosinase, causing AITC release (Fahey et al., 2001). This molecule is well known for its allelopathic activity on several soil borne fungi (Manici et al., 1997). Lab trials demonstrated that the emulsions did not inhibit myrosinase activity and confirmed the capability of the formulations to release AITC from B. carinata DSM. Due to the higher oil concentration the conversion rate was lower in Formulation 2, that in any case, in absolute terms, produced a higher amount of

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50

Untreated control

Topas 10 EC

Formulation 1

Formulation 2

In addition, the nitrogen naturally present in the DSM could play a secondary fertilizing and/or biostimulating role, somehow enhancing plant vigor and natural defence. Finally, the interest of the EU for non-chemical systems in crop management and defence could make such formulations particularly appealing, especially if we consider that they are entirely biodegradable, renewable and based on biomasses that can be produced in Europe (Bezzi et al., 2007), in a full approach of a second generation biorefinery linked to the territory.

40

A.I. (%)

30 20 10

5. Conclusions

0 0

7 Days

14

Fig. 2. Mean Attack Index values (A.I.), defined in the two trial years (2012–2013).

AITC. In addition, another role of the emulsion was the containment of AITC dispersion in the air. Simulating in lab conditions the preparation of the formulations with 30 min of agitation in open vials, the difference in retention of AITC between Formulations 1 and 2 was significant, being of approximately 100 ␮mol L−1 and 250 ␮mol L−1 , respectively. This characteristic allows a slower release over time of the allelopathic molecule, reducing the risk of toxicity and enhancing the efficacy of treatments. Following these different properties, the Formulation 2 showed a greater effectiveness than Formulation 1, and it was in general not statistically different from Topas. This efficacy can be mainly ascribed to the higher amount of oil and DSMs in the emulsion. The tested doses did not cause any phytotoxic effect on plants. In Fig. 2, average A.I. values, obtained from 2012 to 2013 trials, show that Topas, currently one of the registered pesticides widely applied in powdery mildew control on melon, proved to be the most efficient treatment, but not significantly different from Formulation 2. The efficacy of these two products was significantly higher than Formulation 1, which anyway could still show an effect if compared to the untreated control. These results confirmed that both formulations, which are entirely based on vegetable materials, could be rightly considered an interesting additional option for limiting powdery mildew spread on melon. In particular, the application of Formulations 1 and 2 can be considered as a new bio-based physical method of pest control alternative to chemical pesticides (OJ, 2009). In conventional agriculture, they may be useful as a substitution product for limiting the onset of pathogen resistance and the problem of residues which are typical of chemical products, while in organic farming the two formulations could make it possible to reduce the application of sulfur-based products. Since the experiments were conducted on plants already infected, it is reasonable to hypothesize that efforts focused on disease prevention, consisting of regular treatments, may contribute to keep melon free from powdery mildew attacks even under climatic conditions favorable to disease development, although, further trials are necessary in order to confirm this aspect. The first impression is that for a prevention approach Formulation 1 could be useful due to the lower oil content that could permit more frequent treatments. Some other trials on this topic are planned in the next years. Another potentially interesting aspect is the effectiveness of the formulations against other pests and pathogens (Rongai et al., 2008; Piccinini et al., this issue; Benfatto et al., this issue). Therefore, exploiting the combination of the essentially physical action of oil and the allelopathic effect of DSM hydrolysis, various pests and diseases could be contained with the same treatment.

The field trials carried out over two years with a curative approach for the control of powdery mildew (P. xanthii) on melon demonstrated the efficacy of two bio-based experimental formulations based on B. carinata biomasses. In particular, the formulation with a higher concentration of oil and DSM gave results statistically not different from those of the commonly used chemical pesticide Topas. The low environmental impact derived from this non-chemical approach and the clear advantages in term of CO2 saving and sequestration (Lazzeri et al., 2011) make the two biobased formulations of great interest both for conventional and organic farming. Acknowledgements The trials were performed as part of the activities of the Project “Sistema Integrato di Tecnologie per la valorizzazione dei sottoprodotti della filiera del Biodiesel” (VALSO) financed by MiPAAF (D.M. 17533/7303/10 del 29/04/2010) and coordinated by CRA-CIN of Bologna. The authors thank CRA-PAV (Rome, Italy) for identification of the powdery mildew agent in the trials, and Lorena Malaguti (CRA-CIN Bologna) for her help in chemical characterization of biomasses used in the trials. References Agerbirk, N., Olsen, C.E., 2012. Glucosinolates structure in evolution. Phytochemistry 77, 16–45. Alvarez, J.M., Gomez-Guillamon, M.L., Tores, J.A., Canovas, I., Floris, E., 2000. Virulence differences between two Spanish isolates of Sphaerotheca fuliginea race 2 on melon. Acta Hortic. 510, 67–70. Bezzi, G., Lazzeri, L., Monti, A., Venturi, G., 2007. Brassica carinata: un’interessante coltura oleaginosa per bioraffineria. Dal Seme 2, 56–63. Branzanti, B., Brunelli, A., 1992. Indagine eziologica sull’oidio delle Cucurbitacee in Emilia Romagna. Informatore Fitopatologico 11, 37–44. Braun, U., 1995. The Powdery Mildews (Erysiphales) of Europe. G. Fischer Verlag, Jena, Germany. Brunelli, A., 2007. Oidio delle cucurbitacee, quali strategie di difesa. L’Informatore Agrario 21, 57–59. Camele, I., Campanelli, G., Ferrari, V., Viggiani, G., Candido, V., 2009. Powdery mildew controland yield response of inodorus melon. Italian J. Agron. 2, 19–26. Candido, V., Campanelli, G., Viggiania, G., Lazzeri, L., Ferrari, V., Camele, I., 2014. Melon yield response to the control of powdery mildew by environmentally friendly substances. Sci. Hortic. 166, 70–77. De Nicola, G.R., D’Avino, L., Curto, G., Malaguti, L., Ugolini, L., Cinti, S., Patalano, G., Lazzeri, L., 2013. A new biobased liquid formulation with biofumigant and fertilising properties for drip irrigation distribution. Ind. Crops Prod. 42, 113–118. Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5–51. ˜ D., Perez-Garcia, A., Lopez-Ruiz, F., Romero, D., De Vicente, A., Fernandez-Ortuno, Torés Montosa, J.A., 2006. Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain. Eur. J. Plant Pathol. 115, 215–222. Ferrari, V., Piccirillo, F., 1995. Nuova difesa antioidica compatibile con i fitoseidi. L’Informatore Agrario 18, 3–6. Gilardi, G., Baudino, M., Garibaldi, A., Gullino, M.L., 2011. Efficacy of biocontrol agents and natural compounds against powdery mildew of zucchini. Phytoparasitica 40, 147–155, http://dx.doi.org/10.1007/s12600-011-0206-0 Glawe, D.A., 2008. The powdery mildews: a review of the world’s most familiar (yet poorly known) plant pathogens. Annu. Rev. Phytopathol. 46, 27–51.

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