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Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant
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Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant E. Piccinini a , V. Ferrari a,∗ , G. Campanelli a , F. Fusari a , L. Righetti b , R. Matteo c , L. Lazzeri b a Council for Agricultural Research and Economics – Research Unit for Vegetable Crops in Central Areas (CRA-ORA), Via Salaria, 63077 Monsampolo del Tronto (AP), Italy b Council for Agricultural Research and Economics – Research Centre for Industrial Crops (CRA-CIN), Via di Corticella, 133, 40128 Bologna, Italy c External consultant, Italy
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Article history: Received 11 July 2014 Received in revised form 10 April 2015 Accepted 25 May 2015 Available online xxx Keywords: Allelopathic effect Biofumigation Eggplant Red spider mite
a b s t r a c t Two liquid formulations based on Brassica carinata oil and defatted seed meal were tested as treatments to control the red spider mite (Tetranychus urticae Koch) on eggplants in open field conditions. These products were compared to a commercial chemical acaricide (fenazaquin), which is very effective, but whose use was recently limited to ornamentals in greenhouse due to its environmental risks. The application in these trials was therapeutic, the first treatment was distributed at the time of appearance of at least five mobile mites (adults and/or larvae) per plant. The 2-year results indicated that the application of both formulations have a clear effect in containing mites, statistically different from the untreated control. Moreover, the ability of pest control of the formulation with the higher concentrations of oil and defatted seed meal was not different from the chemical treatment. Treatment efficacy clearly decreased after 21 days, but within a preventive treatment plan these formulations could represent an interesting additional bio-based option both in conventional and organic farming. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The red spider mite (RSM), Tetranychus urticae Koch (Acari: Tetranychidae), is a widespread pest all over the world, which attacks a large number of host plants (Tsagkarakou et al., 2002). It causes, in fact, considerable damage on eggplant (Solanum melongena L.), bean (Phaseolus vulgaris L.), melon (Cucumis melo L.), tomato (Solanum lycopersicum L.), strawberry (Fragaria vesca L.), pumpkin (Cucurbita sp.) and many other crops, both in greenhouse and in open field (Ahmadi et al., 2007; Chaudhri et al., 1985). Such a broad diffusion of RSM populations is partially due to the harmful effects that some widely applied pesticides have on the natural RSM enemies and to an increased resistance to these treatments. RSM, in fact, can develop resistance symptoms even after just few treatments, due to its high reproductive potential and the shortness of its life cycle (Cranham and Helle, 1985; Devine et al., 2001; Stumpf and Nauen, 2001), that in the region and the season in which the tests were carried out and on the basis of experience is estimated to be about 10 days in open field. In a study on the resis-
∗ Corresponding author. Tel.: +39 0735 703684; fax: +39 0735 703684. E-mail address: [email protected] (V. Ferrari).
tance of pests to active ingredients of insecticides and acaricides, RSM proved to be resistant to 92 pesticides, and resistance phenomena have been reported in 389 cases (Whalon et al., 2013). All these chemical control failures on RSM are reported for several pesticides such as organophosphates (Sato et al., 1994), dicofol (FergussonKolmes et al., 1991), organotin (Edge and James, 1986; Flexner et al., 1988), hexythiazox (Herron and Rophail, 1993), clofentezine (Herron et al., 1993), fenpyroximate (Sato et al., 2004; Stumpf and Nauen, 2001) and abamectin (Beers et al., 1998). Furthermore, the wide application of chemical pesticides could cause the presence of residues in foods with obvious negative consequences on safety and marketing, especially for fresh food such as vegetables and fruits (Baker et al., 2002). In this ambit, in fact, the European Community has presented the EC Regulation No. 1907/2006EC Regulation No. 1907/2006 that concerns the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH). In the same years, the Directive 2009/128/EC, 2009 clearly underlined the need of innovative nonchemical alternatives to conventional pesticides in the Integrated Crop Management, in order to produce safer food in a less polluted environment. The definition of new strategies, which could counteract resistance phenomena, is therefore mainly linked to non-chemical
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Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060
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options such as: (i) application of natural antagonists (predators, parasitoids and entomopathogens); (ii) new cultivation methods aimed at improving crop vigor by correct mineral fertilization management (Opit et al., 2005; Chow et al., 2009; Chen et al., 2007); (iii) new techniques that modify microclimate (mainly in greenhouse) for example fogging (Duso et al., 2004); (iv) use of bio-based plant extracts which contain allelopathic molecules or have physical effects on pests. In particular, the development of new bio-based materials with an allelopathic effect could be of great help both in conventional and in organic farming, for which the European Community Regulation no. 889/2008 (Official Journal, 2008) allows only the use of products based on sulphur and paraffinic oil. With regard to the specific issue of RSM on eggplant, a study conducted by El Kady et al. (2007) showed the effectiveness of the two biopesticides spinetoram 12% SC and abamectin (Vertimec 1,8EC) on this mite species. In addition, some interesting results in containing California red scale (Aonidiella aurantii Maskell), a small insect with low mobility, were observed using a patented formulation based on a vegetable oil–water emulsion after the addition of a suitable amount of B. carinata A. Braun defatted seed meals (DSM) (Rongai et al., 2008a). In this kind of formulations, the water activates the enzymatic hydrolysis of glucosinolates (GLs) via myrosinase with a controlled release of the corresponding degradation products, mainly allylisothiocyanate (AITC) (Fahey et al., 2001). This glucosinolate-myrosinase system, a typical defensive tool of Brassicaceae, releases allelopathic compounds able to influence the growth, survival and reproduction of some living organisms (Lazzeri et al., 2011). The first results on California red scale suggested that there may be an effect also in RSM containment and the aims of this paper were the definition and optimization of the above mentioned three-phase emulsions and a first validation in open field trials, in order to investigate their potential in containing RSM infestation on eggplant. 2. Materials and methods In 2012 and 2013, two trials were conducted on eggplants (Solanum melongena L.), applying the same protocol and experimental design in order to verify the efficacy on RSM containment of two formulations based on Brassicaceae materials. The formulations were compared to an untreated control and to the commercial acaricide Magister® (active ingredient fenazaquin) (DuPont). The fenazaquin was inserted for its well-known efficacy in mite control, even if its use was recently limited to ornamentals in greenhouse, due to its environmental risks.
2.1.2. Defatted seed meal The meal was characterized as follows: • Moisture content was determined by oven-drying DSM at 105◦ C for 12 h and evaluating the difference in weight before and after treatment. • Nitrogen content was determined by the Kjeldahl method (Standard UNI 22604, 1992), using a Tecator digestion system 20 and an automatic Büchi distillation unit (B-324). • GLs content was determined following the ISO 9167-1 method (ISO 9167), with some minor modifications in the extraction phase, as described in Lazzeri et al. (2011). All data are reported as mean ± SD of four determinations. 2.1.3. The formulations Two types of formulations were defined and prepared by the authors and applied in the trials. They were compared to an untreated control and to a chemical acaricide. Summarizing, the tested treatments were: (a) an untreated control, sprayed with water. (b) a basic bio-based experimental formulation (Formulation 1) that in 100 liters of water contained: (i) 1.5% of B. carinata oil supplemented with a natural emulsifier, (ii) 300 g of DSM formulated by the authors according to a patented procedure (Lazzeri et al., 2010) aimed at optimizing the release of allylisothiocyanate. This formulation was essentially based on the commercial fertilizer Duolif (Agrium Italia SpA, Italy), changing doses. (c) a second bio-based experimental formulation (Formulation 2) that in 100 liters of water contained: (i) 1.5% of B. carinata oil with a natural emulsifier and (ii) 450 g of the same formulated DSM as in (b). This formulation was essentially based on the commercial fertilizer Duofruit (Agrium Italia SpA, Italy), changing doses. (d) Magister® 100EC (fenazaquin) (DuPont), applied at a dose of 100 mL per 100 L of water. A contact acaricide was chosen for a proper comparison with the new formulations (acting on contact). Both B. carinata formulations were totally based on natural compounds. In fact, due to the complete absence of chemicals and the high volatility of isothiocyanates that leave no residues on the plant, these two formulations have a shorter withholding period (WHP) than fenazaquin (WHP of 7 days). Formulations 1 and 2 were prepared mixing the meals to the oil before water addition. To favor the enzymatic reaction, the suspensions were mixed every 5 min, and after 20 min, the time required for maximizing glucosinolate hydrolysis, they were considered ready for application. The emulsion amount that permitted a good plant wetting ranged between 300 and 500 L ha−1 .
2.1. Biomass description The two three-phase patented formulations (Rongai et al., 2008b) used in the trials were based on B. carinata oil and DSM purchased from Agrium Italia SpA (Livorno, Italy). DSM was obtained from B. carinata seeds defatted by an endless screw press in which temperature was kept lower than 75◦ C (Lazzeri et al., 2010). 2.1.1. The oil After extraction, the oil was filtered, partially refined (degummed) and characterized for its fatty acid composition according to the UNI EN ISO 5508 method. Briefly, the oil was transformed into the corresponding methyl-ester via rapid transmethylation with basic catalysis (Christopherson and Glass, 1969) and fatty acid composition was determined by a Carlo Erba mod. HRGC 5300 mega series gas-chromatograph with a flame-ionisation detector (FID), using a capillary column from Restek (Rtx 2330, 30 m length, 0.32 mm Id and 0.2 m df).
2.1.4. Determination of AITC release from the formulations over time A kinetic curve of the AITC released over time by the two formulations was estimated by using headspace gas chromatography as described in De Nicola et al. (2013). Samples were collected every 5 min up to 40 min after the preparation of the formulations. An external calibration curve was calculated using dilutions of pure AITC standard (Fluka, Steinheim, Germany) in 1.5% of oil–water emulsion. Results were expressed as mol of AITC and the percentage of the theoretical value achievable from a complete hydrolysis of 2-propenyl glucosinolate (trivial name: Sinigrin; SIN) contained in B. carinata DSM was calculated. 2.1.5. Determination capacity of retention of pure AITC of the formulations used in the trials AITC was dissolved in 50 mL of water, or in the same volume of 1.5% B. carinata oil–water emulsion. AITC concentrations were
Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060
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Fig. 1. Mean temperatures and rainfall recorded at CRA-ORA from May to August 2012 and 2013 and their respective thirty-year average.
equivalent to total theoretical release of AITC from formulations at 0.15%, 0.3%, 0.45% and 0.6% (w/v) of DSM. Tubes were immediately closed with gas-tight Teflon caps and placed in agitation for 30 min at room temperature. A volume of 200 L was collected for AITC basal determination and tubes were left open in agitation for other 30 min, simulating the preparation of the products for spraying in field. AITC determination was measured 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 hours at 65◦ C, cooled at temperatures ranging from 21 ± 1◦ C, and their absorbance at 365 nm was determined against a paired blank containing all the ingredients except AITC. Every analysis was carried out in triplicate.
2.2. Trial protocol Trials were conducted in 2012 and 2013 on eggplants of outdoor cultivation at the experimental farm of CRA-ORA at Monsampolo del Tronto (Ascoli Piceno, Italy) (42◦ 53 N; 13◦ 47 E; 184 m a.s.l.) on a medium textured soil. Fig. 1 shows mean temperatures and rainfall recorded at CRA-ORA during the growing cycle of eggplant in 2012 and 2013 and their respective thirty-year average; particularly in the month of August, in which treatments were performed, temperatures were above normal in both years but with a higher deviation in 2012, whose rainfall in the same month was much lower than normal, unlike August 2013 for which a regular amount of rainfall was recorded. In both years, the 4 experimental treatments were arranged in a randomized complete block design with 3 replications. Each treatment covered a surface area of 5.5 m2 ; the elementary plot comprised 12 plants, arranged in twin rows with a distance of 1.6 m between their centers, a distance of 0.5 m between the 2 rows of a twin row and a distance of 0.7 m between plants along a row, which results in a density of 1.8 plants m−2 . Each year, the soil was ploughed to a depth of 20 cm prior of harrowing. Black plastic mulch (50 m thick) was laid down by covering 80 cm-wide bands of the soil. Eggplant seedlings of the cultivar Violetta allungata (Arcoiris, organic seeds) were transplanted at the 3rd–4th true leaf stage on the first decade of May. Throughout the crop cycle, irrigation was conducted by a drip irrigation system placed under the plastic mulch with drip holes (water flow rate 2.5 L h−1 ) spaced 20 cm from each other. The trials were planned to verify for each product the pest containment capacity and not its prevention; with this aim, the treatments started at the appearance of at least 5 mobile mites (adults and larvae) on a plant and, in order to assess persistence, a single treatment was carried out at Time 0 (T0 ). Single
plots were screened with protective plastic partitions to avoid any drifting effect. Treatments were applied by a hand-sprayer, as normally performed in open field ; insecticides were sprayed at 9 a.m. uniformly on the two leaf blades (both from the top downwards and from below upwards) up to dripping. At the end of the trials, before eggplant harvesting time, all the plants were removed and in no case any of their parts were used for food or feeding.
2.3. Sampling methods During the 2-year trials sampling of mobile stages of RSM present on the middle leaves was conducted using a binocular microscope (Bausch and Lomb); one person was holding the microscope and another one the leaf while counting the mites. Four leaves from each plant were sampled, so the samples were 384 (4 leaves/plant × 8 plants/treatment × 4 treatments × 3 replications). The first measurement (T0 ) coincided with the application time; samplings were carried out on the middle leaves because at T0 the degree of infestation on them was rather uniform and thus comparable in subsequent surveys. Following samplings were conducted weekly at 7, 14 and 21 days after treatment (T0 ) aiming at defining the allelopathic effect and the effectiveness of plant protection products tested. Mean numbers of mobile mites per leaf were calculated for each treatment and day of sampling.
2.4. Statistical analysis AITC trapping ability of water and B. carinata oil–water emulsions was measured spectrophotometrically at 365 nm. Data are expressed as means ± SE from at least three experiments. Statistical analysis was performed with Student’s t-test (Sigma Plot 10.0SPSS, Chicago, IL, USA). Differences between groups were considered statistical significant at P < 0.05. The effectiveness of the treatments was calculated using Abbott’s formula (Abbott, 1925), after angular transformation of the percentage values, and results were analyzed with Duncan’s multiple range test.
3. Results and discussion 3.1. Biomass characterization 3.1.1. Oil fatty acid composition The analysis on oil confirmed the high level of long chain fatty acids with a % of erucic acid (C22:1) higher than 40%.
Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060
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corresponding to Formulation 2, instead, the difference with water was significant and showed an increase in the retention of AITC of about 35%. 3.2. Experimental trials
Fig. 2. Allylisothiocyanate (AITC) retention in water and Brassica carinata oil–water emulsion. Data are expressed as relative % retention after 30 min of shaking in open vials compared to AITC basal concentration in closed vials. F1 and F2 indicate retention at Formulation 1 and 2 composition, respectively. Statistical analysis on the absorbance at 365 nm was performed by Student’s t-test; P < 0.05 was considered significant.
3.1.2. Defatted seed meals. B. carinata DSM, derived from a pressure industrial extraction plant, was characterized by a GL content of around 90 mol g−1 . Sinigrin corresponded to 97% of all glucosinolates. The presence of more than 5.5% of nitrogen gives the emulsions fertilizing and biostimulating activity. 3.1.3. Determination of AITC release in time of the formulations Water addition to the oil and DSM mixture permitted a defined released in the emulsion of GL degradation products, mainly AITC. The estimation of the kinetic of hydrolysis over time and the concentration of AITC in the two formulations, simulated in laboratory condition, could provide useful information about the best time of application and about the actual presence of this compound well known for its allelopathic activity on several pathogens and pests (Fahey et al., 2001). The hydrolysis reaction reached its maximum around 20 min after watering. Formulation 2 showed a conversion yield of sinigrin of 77%, slightly lower than Formulation 1 (79%). Nevertheless, considering the different concentrations of the DSM used, the concentration of AITC in Formulation 1 after 20 min was around 200 M, while for Formulation 2 was 300 M. 3.1.4. Determination capacity of retention of pure AITC of the formulations used in the trials The AITC retention in pure water was almost constant regardless of the basal concentration of AITC, while the presence of a low concentration of oil led to an increase of the retention of AITC that was highest (about 75%) at the lowest AITC basal concentration and that was around 40–50% at the other AITC concentrations tested (Fig. 2). The difference between the solutions simulating Formulation 1 and 2 was not significant, nor was that between the AITC retention in water and B. carinata oil emulsion, at a concentration of AITC corresponding to 0.3% of DSM (w/v). At the concentration of AITC
In both trial years an attack of mites on eggplant occurred, which appeared stronger in the second one. In 2013, the exacerbation of assay conditions permitted a clearer evaluation of the acaricidal performance of the tested products. The preparation of both the experimental formulations could be conveniently performed directly on the site of the trials; the application proved to be practical and their distribution easy by the mechanization normally used on a horticultural farm. No phytotoxic effects of both bio-based formulations were observed on plants during the trials. 3.2.1. First trial year In 2012, the trial started on August 16th , when all the plots showed a sufficiently uniform RSM natural infestation: from 6 to 13 mobile mites per leaf. Table 1 reports the mean number of mobile mites found in each treatment at different sampling times. These data make it possible to show the clear knock-down effect of both formulations at 7 (T7 ) and 14 (T14 ) days after the treatments and their persistence during the first 14 days. The fenazaquin showed a highly lethal effect, determining the almost complete death of mites until the end of the trial: the mortality rate was always higher than 99% even after 21 days (Table 1). Both experimental formulations are characterized by a mechanism of action based on the oil film that covers the insect isolating the respiratory system from the air, an action improved by the allelopathic effect of GL degradation products (essentially AITC). It was therefore not surprising that these products, and in particular Formulation 2, determined a stronger acaricide action, reducing the infestation to only one mobile mite at T7 with a corresponding degree of effectiveness of 99%, statistically not different from the effect of the chemical pesticide. Formulation 1 was instead rather less effective, and 7 days after the treatment an average of 7 mobile forms were found, compared to 13 recorded at T0 . The degree of effectiveness, calculated on the evolution of populations in control plants, amounted therefore to 61.2%, a percentage significantly lower than fenazaquin and Formulation 2. This may be in part explained by the data in Fig. 2 from which it is evident that the lower concentration of oil in Formulation 1 is not able to trap a quantity of AITC significantly different from water. The persistence of the acaricide action of Formulation 2 showed a good ability of counteracting repopulation of the pest for 14 days. In fact, the degree of effectiveness, still high (93.9%) at T14 , was not statistically different from those found in fenazaquin plots. At T21 , a mean number of 20 mobile mites per leaf was found on plots treated with Formulation 2, showing that the product ended its acaricide action, reducing the degree of effectiveness to 69.2%. Fenazaquin, instead, maintained the same efficacy even 21 days after the treatment.
Table 1 Year 2012: mean number of mobile mites (N) per leaf found on eggplants, standard error (SE) and effectiveness of the products expressed as % (Abbott’s formula). Treatment
Untreated control Fenazaquin Formulation 1 Formulation 2
T0
T7
T14
T21
N
SE
N
SE
%
N
SE
%
N
SE
%
6.0 g 11.0 eg 13.0 ef 10.0 eg
3.2 4.1 4.0 3.8
18.0 de 0.1 h 7.0 fg 1.0 h
5.1 1.2 3.7 1.4
– 99.4 a 61.2 bc 94.4 a
33.0 bc 0.1 h 15.0 e 2.0 h
7.4 1.4 6.8 1.6
– 99.6 a 54.6 c 93.9 a
65.0 a 1.0 h 40.0 b 20.0 d
10.4 1.4 7.6 2.0
– 99.8 a 38.5 d 69.2 b
Different letters correspond to significantly different values for p < 0.05 (Duncan test).
Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060
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Table 2 Year 2013: mean number of mobile mites (N) per leaf found on eggplants, standard error (SE) and effectiveness of the products expressed as % (Abbott’s formula). Treatment
Untreated control Fenazaquin Formulation 1 Formulation 2
T0
T7
T14
T21
N
SE
N
SE
%
N
SE
%
N
SE
%
10.0 ce 15.0 cd 10.0 ce 14.0 cd
4.0 5.1 3.9 6.1
24.0 c 0.1 e 4.0 e 1.0 e
6.2 1.3 3.0 1.2
– 99.6 a 83.3 bc 95.8 a
80.0 b 0.1 e 7.0 de 3.0 e
9.1 1.4 4.6 1.9
– 99.8 a 91.2 a 96.2 a
150.0 a 2.0 e 35.0 c 13.0 cd
14.5 1.8 6.7 3.4
– 98.7 a 76.7 c 91.3 a
Different letters correspond to significantly different values for p < 0.05 (Duncan test).
4. Conclusions
Fig. 3. Comparison of number of mobile forms of Tetranychus urticae per leaf on eggplant under different treatments in the two trial years.
3.2.2. Second trial year In 2013, the trial started on August 7th , when all the plots showed a sufficiently uniform natural infestation: from 10 to 15 mobile mites per leaf. Table 2 reports the mean number of mobile mites from the beginning until the end of the trial after 21 days. In the second year too, the knock-down effect and persistence of fenazaquin was confirmed till 21 days after treatment, with 98.7% (Table 2) of degree of effectiveness, even in the presence of environmental conditions particularly favorable to RSM development. Formulation 2 also gave results similar to the first year, showing a good knock-down effect of the first treatment, but with a higher persistence till 21 days after the treatment with a degree of effectiveness of 91.3%, a value statistically not different from that of fenazaquin. In the second year of trial, Formulation 1 confirmed an acaricide action milder than Formulation 2 in terms of knock-down effect and persistence of the action, but in both formulations the related number of mobile mites was statistically lower than for the untreated plots. The average values of the two years of trial, regarding the number of mobile mites detected at T0, T7 , T14 and T21 , showed a knock-down effect for Formulation 2 comparable to fenazaquin even if with a lower persistence (Fig. 3). The natural patented Formulations 1 and 2 had different but significant effects on RSM. The oil in the emulsion suffocates the spiders, creating a microfilm that remains on the leaf apparatus and on the insects that even alone showed a clear containment of pests that was observed as not statistically different from paraffinic oils on red scale (Rongai et al., 2008). This effect was summed to the allelopathic effect of DSM that, after water addition, released AITC which had an additional control effect on the disease and a fertilizing one. This system confirmed the capability to release AITC at a rate of conversion of sinigrin of about 80%, after water addition, and of limiting, if compared to water, the dispersion in the air of AITC released from the DSM from B. carinata of around 35% in Formulation 2.
The pressure procedure made it possible to classify the oil and DSM used in these trials as not chemically modified materials and, in this way, both the oil and the bio-products are considered in the European Regulament REACH (EC Regulation No. 1907/2006) as exempt from registration (Annex IV). The experiments carried out in 2012–2013 showed that both experimental liquid formulations were characterized by an allelopathic action, expressed in particular by Formulation 2. Its higher DSM amount should be the reason for its stronger effectiveness, generally statistically not different from the chemical fenazaquin, widely applied in the past on eggplant both at open field and greenhouse level. It can thus be speculated that Formulation 1 could be used as a preventive treatment, while Formulation 2 could be applied in the presence of stronger attacks. Summarizing, the average values of the two years of trial, regarding the number of mobile mites detected during the periods of trials, showed a knock-down effect for Formulation 2 comparable to chemical even if with a lower persistence. Considering that only sulfur and paraffinic oil are currently allowed as acaricides in organic farming, the definition of new low environmental impact products with a shorter withholding period could represent considerable technical progress for conventional and organic farmers in the control of spider mites. The interest for these partial or total innovative alternatives to chemicals can be confirmed if we point out that, since 2011 (Commission Implementing Directive 2011/39/EU, 2011), all the pesticides based on fenazaquin (including Magister® ) are not allowed for application as acaricides on food crops and are authorized only for applications on ornamental plants. Acknowledgement The trials were performed as a 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. 17,533/7303/10 del 29/04/2010) and coordinated by CRA-CIN of Bologna. We thank Lorena Malaguti (CRA-CIN Bologna) for her help in chemical characterization of the biomasses used in the trials. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 163, 265–267. Ahmadi, M., Fathipour, Y., Kamali, K., 2007. Population growth parameters of Tetranychus urticae (Acari: Tetranychidae) on different bean varietiesm. J. Entomol. Soc. Iran 26, 1–10. Baker, B.P., Benbrook, C.M., Groth, E., Lutz Benbrook III, K., 2002. Pesticide residues in conventional, integrated pest management (IPM)-grown and organic foods: insights from three US data sets. Food Addit. Contam. 19, 427–446, C32]. Beers, E.H., Riedl, H., Dunley, J.E., 1998. Resistance to abamectin and reversion to susceptibility to fenbutatin oxide in spider mite (Acari: Tetranychidae) populations in the Pacific Northwest. J. Econ. Entomol. 91, 352–360. Chaudhri, W.M., Akbar, S., Rasool, A., 1985. Studies on biosystematics and control of mites of field crops, vegetable and fruit plants in Pakistan UAF. Tech. Bull. 3, 314.
Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060
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Chen, Y., Opit, G.P., Jonas, V.M., Williams, K.A., Nechols, J.R., Margolies, D.C., 2007. Two spotted spider mite population level, distribution, and damage on ivy geranium in response to different nitrogen and phosphorus fertilization regimes. J. Econ. Entomol. 100, 1821–1830. Chow, A., Chau, A., Heinz, K.M., 2009. Reducing fertilization for cut roses: effect on crop productivity and twospotted spider mite abundance, distribution, and management. J. Econ. Entomol. 102, 1896–1907. Christopherson, S.W., Glass, R.L., 1969. Preparation of milk fat methylesthers by alcoholysis in essentially non alcoholic solutions. J. Dairy Sci. 52, 1289–1290 [L33]. Commission Implementing Directive 2011/39/EU, 2011. April 11th. Cranham, J.E., Helle, W.,1985. Pesticide resistance in Tetranychidae. In: Helle, W., Sabelis, M.W. (Eds.). Elsevier, Amsterdam, pp. 405–421. 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. Devine, G.J., Barber, M., Denholm, P.S., 2001. Incidence and inheritance of resistance to METI acaricides in European strains of the two-spotted spider mite (Tetranychus urticae), (Acari: Tetranychidae). Pest Manag. Sci. 57, 443–448. Directive 2009/128/EC of the European Parliament and of the Council, 2009. October 21th. Duso, C., Chiarini, F., Conte, L., Bonora, V., Dalla Montà, L., Otto, S., 2004. Fogging can control Tetranychus urticae on greenhouse cucumbers. J. Pest Sci. 77, 105–111. EC, Regulation No. 1907/2006 on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) Annex IV. Edge, V.E., James, D.G., 1986. Organotin resistance in Tetranychus urticae Acari: Tetranychidae in Australia. J. Econ. Entomol. 79, 1477–1483. El Kady, G.A., Sharabasy, H.M., Bahgat, I.M., 2007. Toxicity of Two Potential Bio-insecticides Against Moveable Stages of Tetranychus urticae Koch. J. Appl. Sci. Res. 3, 1315–1319. Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5–51. Fergusson-Kolmes, L.A., Scott, J.G., Dennehy, T.J., 1991. Dicofol resistance in Tetranychus urticae (Acari: Tetranychidae): Cross-resistance and pharmacokinetics. J. Econ. Entomol. 84, 41–48. Flexner, J.L., Westigard, P.H., Croft, B.A., 1988. Field reversion of organotin resistance in the twospotted spider mite (Acari: Tetranychidae) following relaxation of selection pressure. J. Econ. Entomol. 81, 1516–1520. Herron, G.A., Rophail, J., 1993. Genetics of hexythiazox resistance in two spotted spider mite, Tetranychus urticae Koch. Exp. Appl. Acarol. 17, 423–431.
Herron, G.A., Edge, V., Rophail, J., 1993. Clofentezine and hexythiazox resistance in Tetranychus urticae Koch in Australia. Exp. Appl. Acarol. 17, 433–440. Lazzeri, L., Leoni, O., Manici, L.M., Palmieri, S., Patalano, G., 2010. Use of Seed Flour as Soil Pesticide. Patent no US 7749,549, July 6th. Lazzeri, L., D’Avino, L., Ugolini, L., De Nicola, G.R., Cinti, S., Malaguti, L., Bagatta, M., Patalano, G., Leoni, O., 2011. Bio-based products from Brassica carinata A. Braun oils and defatted meals by a second generation biorefinery approach. EU BC&E Conference Proceedings, 1080–1092. Official Journal of the European Union, 2008. L 250, September 18th, pp. 1–84. Opit, G.P., Chen, Y., Williams, K.A., Nechols, J.R., Margolies, D.C., 2005. Plant age, fertilization, and biological control affect damage caused by twospotted spider mites on ivy geranium: development of an action threshold. J. Am. Soc. Hortic. Sci. 130, 159–166. Regulation (CE) N. 889/2008 of the European Commission, 2008. September 5th. Rongai, D., Cerato, C., Lazzeri, L., Palmieri, S., Patalano, G., 2008a. Vegetable oil formulation as biopesticide to control California red scale (Aonidiella aurantii Maskell). J. Pest Sci. 81, 179–185. Rongai, D., Cerato, C., Lazzeri, L., Palmieri, S., 2008. Composition for the treatment and/or prevention of attacks by biological agents. Patent no (US 2008/0199451A1), August 21st. Sato, M.E., Suplicy Filho, N., De Souza Filho, M.F., Takematsu, A.P., 1994. Resistencia do acaro rajado Tetranychus urticae (Koch,1836) (Acari: Tetranychidae) a diversos acaricidas emmorangueiro (Fragaria sp) nos municipios de Atibaia- Sp e Piedade-Sp. Ecossistema 19, 40–46. Sato, M.E., Miyata, T., Da Silva, M., Raga, A., De Souza Filho, M.F., 2004. Selections for fenpyroximate resistance and susceptibility, and inheritance, cross-resistance and stability of fenpyroximate resistance of Tetranychus urticae Koch (Acari: Tetranychidae). Appl. Entomol. Zool. 39, 293–302. Standard UNI 22,604. 1992. Seed and vegetable oils and derived products. Vegetable proteins. Determination of crude protein. Stumpf, N., Nauen, R., 2001. Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor - acaricide resistance in Tetranychus urticae (Acari: Tetranychidae). J. Econ. Entomol. 94, 1577–1583. Tsagkarakou, A., Pasteur, N., Cuany, A., Chevillon, C., Navajas, N., 2002. Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece. Insect Biochem. Mol. Biol. 32, 417–424. Whalon, M.E., Mota-Sanchez, D., Hollingworth, R.M., Gutierrez, R., 2013. Arthropod Pesticide Resistance Database 2012. www.pesticideresistance.org, consulted on 15.04.2014. Zhang, Y., Cho Posner Talalay, C.G.G.H.P., 1992. Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols. Anal. Biochem. 205, 100–107.
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Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant E. Piccinini a , V. Ferrari a,∗ , G. Campanelli a , F. Fusari a , L. Righetti b , R. Matteo c , L. Lazzeri b a Council for Agricultural Research and Economics – Research Unit for Vegetable Crops in Central Areas (CRA-ORA), Via Salaria, 63077 Monsampolo del Tronto (AP), Italy b Council for Agricultural Research and Economics – Research Centre for Industrial Crops (CRA-CIN), Via di Corticella, 133, 40128 Bologna, Italy c External consultant, Italy
a r t i c l e
i n f o
Article history: Received 11 July 2014 Received in revised form 10 April 2015 Accepted 25 May 2015 Available online xxx Keywords: Allelopathic effect Biofumigation Eggplant Red spider mite
a b s t r a c t Two liquid formulations based on Brassica carinata oil and defatted seed meal were tested as treatments to control the red spider mite (Tetranychus urticae Koch) on eggplants in open field conditions. These products were compared to a commercial chemical acaricide (fenazaquin), which is very effective, but whose use was recently limited to ornamentals in greenhouse due to its environmental risks. The application in these trials was therapeutic, the first treatment was distributed at the time of appearance of at least five mobile mites (adults and/or larvae) per plant. The 2-year results indicated that the application of both formulations have a clear effect in containing mites, statistically different from the untreated control. Moreover, the ability of pest control of the formulation with the higher concentrations of oil and defatted seed meal was not different from the chemical treatment. Treatment efficacy clearly decreased after 21 days, but within a preventive treatment plan these formulations could represent an interesting additional bio-based option both in conventional and organic farming. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The red spider mite (RSM), Tetranychus urticae Koch (Acari: Tetranychidae), is a widespread pest all over the world, which attacks a large number of host plants (Tsagkarakou et al., 2002). It causes, in fact, considerable damage on eggplant (Solanum melongena L.), bean (Phaseolus vulgaris L.), melon (Cucumis melo L.), tomato (Solanum lycopersicum L.), strawberry (Fragaria vesca L.), pumpkin (Cucurbita sp.) and many other crops, both in greenhouse and in open field (Ahmadi et al., 2007; Chaudhri et al., 1985). Such a broad diffusion of RSM populations is partially due to the harmful effects that some widely applied pesticides have on the natural RSM enemies and to an increased resistance to these treatments. RSM, in fact, can develop resistance symptoms even after just few treatments, due to its high reproductive potential and the shortness of its life cycle (Cranham and Helle, 1985; Devine et al., 2001; Stumpf and Nauen, 2001), that in the region and the season in which the tests were carried out and on the basis of experience is estimated to be about 10 days in open field. In a study on the resis-
∗ Corresponding author. Tel.: +39 0735 703684; fax: +39 0735 703684. E-mail address: [email protected] (V. Ferrari).
tance of pests to active ingredients of insecticides and acaricides, RSM proved to be resistant to 92 pesticides, and resistance phenomena have been reported in 389 cases (Whalon et al., 2013). All these chemical control failures on RSM are reported for several pesticides such as organophosphates (Sato et al., 1994), dicofol (FergussonKolmes et al., 1991), organotin (Edge and James, 1986; Flexner et al., 1988), hexythiazox (Herron and Rophail, 1993), clofentezine (Herron et al., 1993), fenpyroximate (Sato et al., 2004; Stumpf and Nauen, 2001) and abamectin (Beers et al., 1998). Furthermore, the wide application of chemical pesticides could cause the presence of residues in foods with obvious negative consequences on safety and marketing, especially for fresh food such as vegetables and fruits (Baker et al., 2002). In this ambit, in fact, the European Community has presented the EC Regulation No. 1907/2006EC Regulation No. 1907/2006 that concerns the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH). In the same years, the Directive 2009/128/EC, 2009 clearly underlined the need of innovative nonchemical alternatives to conventional pesticides in the Integrated Crop Management, in order to produce safer food in a less polluted environment. The definition of new strategies, which could counteract resistance phenomena, is therefore mainly linked to non-chemical
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options such as: (i) application of natural antagonists (predators, parasitoids and entomopathogens); (ii) new cultivation methods aimed at improving crop vigor by correct mineral fertilization management (Opit et al., 2005; Chow et al., 2009; Chen et al., 2007); (iii) new techniques that modify microclimate (mainly in greenhouse) for example fogging (Duso et al., 2004); (iv) use of bio-based plant extracts which contain allelopathic molecules or have physical effects on pests. In particular, the development of new bio-based materials with an allelopathic effect could be of great help both in conventional and in organic farming, for which the European Community Regulation no. 889/2008 (Official Journal, 2008) allows only the use of products based on sulphur and paraffinic oil. With regard to the specific issue of RSM on eggplant, a study conducted by El Kady et al. (2007) showed the effectiveness of the two biopesticides spinetoram 12% SC and abamectin (Vertimec 1,8EC) on this mite species. In addition, some interesting results in containing California red scale (Aonidiella aurantii Maskell), a small insect with low mobility, were observed using a patented formulation based on a vegetable oil–water emulsion after the addition of a suitable amount of B. carinata A. Braun defatted seed meals (DSM) (Rongai et al., 2008a). In this kind of formulations, the water activates the enzymatic hydrolysis of glucosinolates (GLs) via myrosinase with a controlled release of the corresponding degradation products, mainly allylisothiocyanate (AITC) (Fahey et al., 2001). This glucosinolate-myrosinase system, a typical defensive tool of Brassicaceae, releases allelopathic compounds able to influence the growth, survival and reproduction of some living organisms (Lazzeri et al., 2011). The first results on California red scale suggested that there may be an effect also in RSM containment and the aims of this paper were the definition and optimization of the above mentioned three-phase emulsions and a first validation in open field trials, in order to investigate their potential in containing RSM infestation on eggplant. 2. Materials and methods In 2012 and 2013, two trials were conducted on eggplants (Solanum melongena L.), applying the same protocol and experimental design in order to verify the efficacy on RSM containment of two formulations based on Brassicaceae materials. The formulations were compared to an untreated control and to the commercial acaricide Magister® (active ingredient fenazaquin) (DuPont). The fenazaquin was inserted for its well-known efficacy in mite control, even if its use was recently limited to ornamentals in greenhouse, due to its environmental risks.
2.1.2. Defatted seed meal The meal was characterized as follows: • Moisture content was determined by oven-drying DSM at 105◦ C for 12 h and evaluating the difference in weight before and after treatment. • Nitrogen content was determined by the Kjeldahl method (Standard UNI 22604, 1992), using a Tecator digestion system 20 and an automatic Büchi distillation unit (B-324). • GLs content was determined following the ISO 9167-1 method (ISO 9167), with some minor modifications in the extraction phase, as described in Lazzeri et al. (2011). All data are reported as mean ± SD of four determinations. 2.1.3. The formulations Two types of formulations were defined and prepared by the authors and applied in the trials. They were compared to an untreated control and to a chemical acaricide. Summarizing, the tested treatments were: (a) an untreated control, sprayed with water. (b) a basic bio-based experimental formulation (Formulation 1) that in 100 liters of water contained: (i) 1.5% of B. carinata oil supplemented with a natural emulsifier, (ii) 300 g of DSM formulated by the authors according to a patented procedure (Lazzeri et al., 2010) aimed at optimizing the release of allylisothiocyanate. This formulation was essentially based on the commercial fertilizer Duolif (Agrium Italia SpA, Italy), changing doses. (c) a second bio-based experimental formulation (Formulation 2) that in 100 liters of water contained: (i) 1.5% of B. carinata oil with a natural emulsifier and (ii) 450 g of the same formulated DSM as in (b). This formulation was essentially based on the commercial fertilizer Duofruit (Agrium Italia SpA, Italy), changing doses. (d) Magister® 100EC (fenazaquin) (DuPont), applied at a dose of 100 mL per 100 L of water. A contact acaricide was chosen for a proper comparison with the new formulations (acting on contact). Both B. carinata formulations were totally based on natural compounds. In fact, due to the complete absence of chemicals and the high volatility of isothiocyanates that leave no residues on the plant, these two formulations have a shorter withholding period (WHP) than fenazaquin (WHP of 7 days). Formulations 1 and 2 were prepared mixing the meals to the oil before water addition. To favor the enzymatic reaction, the suspensions were mixed every 5 min, and after 20 min, the time required for maximizing glucosinolate hydrolysis, they were considered ready for application. The emulsion amount that permitted a good plant wetting ranged between 300 and 500 L ha−1 .
2.1. Biomass description The two three-phase patented formulations (Rongai et al., 2008b) used in the trials were based on B. carinata oil and DSM purchased from Agrium Italia SpA (Livorno, Italy). DSM was obtained from B. carinata seeds defatted by an endless screw press in which temperature was kept lower than 75◦ C (Lazzeri et al., 2010). 2.1.1. The oil After extraction, the oil was filtered, partially refined (degummed) and characterized for its fatty acid composition according to the UNI EN ISO 5508 method. Briefly, the oil was transformed into the corresponding methyl-ester via rapid transmethylation with basic catalysis (Christopherson and Glass, 1969) and fatty acid composition was determined by a Carlo Erba mod. HRGC 5300 mega series gas-chromatograph with a flame-ionisation detector (FID), using a capillary column from Restek (Rtx 2330, 30 m length, 0.32 mm Id and 0.2 m df).
2.1.4. Determination of AITC release from the formulations over time A kinetic curve of the AITC released over time by the two formulations was estimated by using headspace gas chromatography as described in De Nicola et al. (2013). Samples were collected every 5 min up to 40 min after the preparation of the formulations. An external calibration curve was calculated using dilutions of pure AITC standard (Fluka, Steinheim, Germany) in 1.5% of oil–water emulsion. Results were expressed as mol of AITC and the percentage of the theoretical value achievable from a complete hydrolysis of 2-propenyl glucosinolate (trivial name: Sinigrin; SIN) contained in B. carinata DSM was calculated. 2.1.5. Determination capacity of retention of pure AITC of the formulations used in the trials AITC was dissolved in 50 mL of water, or in the same volume of 1.5% B. carinata oil–water emulsion. AITC concentrations were
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Fig. 1. Mean temperatures and rainfall recorded at CRA-ORA from May to August 2012 and 2013 and their respective thirty-year average.
equivalent to total theoretical release of AITC from formulations at 0.15%, 0.3%, 0.45% and 0.6% (w/v) of DSM. Tubes were immediately closed with gas-tight Teflon caps and placed in agitation for 30 min at room temperature. A volume of 200 L was collected for AITC basal determination and tubes were left open in agitation for other 30 min, simulating the preparation of the products for spraying in field. AITC determination was measured 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 hours at 65◦ C, cooled at temperatures ranging from 21 ± 1◦ C, and their absorbance at 365 nm was determined against a paired blank containing all the ingredients except AITC. Every analysis was carried out in triplicate.
2.2. Trial protocol Trials were conducted in 2012 and 2013 on eggplants of outdoor cultivation at the experimental farm of CRA-ORA at Monsampolo del Tronto (Ascoli Piceno, Italy) (42◦ 53 N; 13◦ 47 E; 184 m a.s.l.) on a medium textured soil. Fig. 1 shows mean temperatures and rainfall recorded at CRA-ORA during the growing cycle of eggplant in 2012 and 2013 and their respective thirty-year average; particularly in the month of August, in which treatments were performed, temperatures were above normal in both years but with a higher deviation in 2012, whose rainfall in the same month was much lower than normal, unlike August 2013 for which a regular amount of rainfall was recorded. In both years, the 4 experimental treatments were arranged in a randomized complete block design with 3 replications. Each treatment covered a surface area of 5.5 m2 ; the elementary plot comprised 12 plants, arranged in twin rows with a distance of 1.6 m between their centers, a distance of 0.5 m between the 2 rows of a twin row and a distance of 0.7 m between plants along a row, which results in a density of 1.8 plants m−2 . Each year, the soil was ploughed to a depth of 20 cm prior of harrowing. Black plastic mulch (50 m thick) was laid down by covering 80 cm-wide bands of the soil. Eggplant seedlings of the cultivar Violetta allungata (Arcoiris, organic seeds) were transplanted at the 3rd–4th true leaf stage on the first decade of May. Throughout the crop cycle, irrigation was conducted by a drip irrigation system placed under the plastic mulch with drip holes (water flow rate 2.5 L h−1 ) spaced 20 cm from each other. The trials were planned to verify for each product the pest containment capacity and not its prevention; with this aim, the treatments started at the appearance of at least 5 mobile mites (adults and larvae) on a plant and, in order to assess persistence, a single treatment was carried out at Time 0 (T0 ). Single
plots were screened with protective plastic partitions to avoid any drifting effect. Treatments were applied by a hand-sprayer, as normally performed in open field ; insecticides were sprayed at 9 a.m. uniformly on the two leaf blades (both from the top downwards and from below upwards) up to dripping. At the end of the trials, before eggplant harvesting time, all the plants were removed and in no case any of their parts were used for food or feeding.
2.3. Sampling methods During the 2-year trials sampling of mobile stages of RSM present on the middle leaves was conducted using a binocular microscope (Bausch and Lomb); one person was holding the microscope and another one the leaf while counting the mites. Four leaves from each plant were sampled, so the samples were 384 (4 leaves/plant × 8 plants/treatment × 4 treatments × 3 replications). The first measurement (T0 ) coincided with the application time; samplings were carried out on the middle leaves because at T0 the degree of infestation on them was rather uniform and thus comparable in subsequent surveys. Following samplings were conducted weekly at 7, 14 and 21 days after treatment (T0 ) aiming at defining the allelopathic effect and the effectiveness of plant protection products tested. Mean numbers of mobile mites per leaf were calculated for each treatment and day of sampling.
2.4. Statistical analysis AITC trapping ability of water and B. carinata oil–water emulsions was measured spectrophotometrically at 365 nm. Data are expressed as means ± SE from at least three experiments. Statistical analysis was performed with Student’s t-test (Sigma Plot 10.0SPSS, Chicago, IL, USA). Differences between groups were considered statistical significant at P < 0.05. The effectiveness of the treatments was calculated using Abbott’s formula (Abbott, 1925), after angular transformation of the percentage values, and results were analyzed with Duncan’s multiple range test.
3. Results and discussion 3.1. Biomass characterization 3.1.1. Oil fatty acid composition The analysis on oil confirmed the high level of long chain fatty acids with a % of erucic acid (C22:1) higher than 40%.
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corresponding to Formulation 2, instead, the difference with water was significant and showed an increase in the retention of AITC of about 35%. 3.2. Experimental trials
Fig. 2. Allylisothiocyanate (AITC) retention in water and Brassica carinata oil–water emulsion. Data are expressed as relative % retention after 30 min of shaking in open vials compared to AITC basal concentration in closed vials. F1 and F2 indicate retention at Formulation 1 and 2 composition, respectively. Statistical analysis on the absorbance at 365 nm was performed by Student’s t-test; P < 0.05 was considered significant.
3.1.2. Defatted seed meals. B. carinata DSM, derived from a pressure industrial extraction plant, was characterized by a GL content of around 90 mol g−1 . Sinigrin corresponded to 97% of all glucosinolates. The presence of more than 5.5% of nitrogen gives the emulsions fertilizing and biostimulating activity. 3.1.3. Determination of AITC release in time of the formulations Water addition to the oil and DSM mixture permitted a defined released in the emulsion of GL degradation products, mainly AITC. The estimation of the kinetic of hydrolysis over time and the concentration of AITC in the two formulations, simulated in laboratory condition, could provide useful information about the best time of application and about the actual presence of this compound well known for its allelopathic activity on several pathogens and pests (Fahey et al., 2001). The hydrolysis reaction reached its maximum around 20 min after watering. Formulation 2 showed a conversion yield of sinigrin of 77%, slightly lower than Formulation 1 (79%). Nevertheless, considering the different concentrations of the DSM used, the concentration of AITC in Formulation 1 after 20 min was around 200 M, while for Formulation 2 was 300 M. 3.1.4. Determination capacity of retention of pure AITC of the formulations used in the trials The AITC retention in pure water was almost constant regardless of the basal concentration of AITC, while the presence of a low concentration of oil led to an increase of the retention of AITC that was highest (about 75%) at the lowest AITC basal concentration and that was around 40–50% at the other AITC concentrations tested (Fig. 2). The difference between the solutions simulating Formulation 1 and 2 was not significant, nor was that between the AITC retention in water and B. carinata oil emulsion, at a concentration of AITC corresponding to 0.3% of DSM (w/v). At the concentration of AITC
In both trial years an attack of mites on eggplant occurred, which appeared stronger in the second one. In 2013, the exacerbation of assay conditions permitted a clearer evaluation of the acaricidal performance of the tested products. The preparation of both the experimental formulations could be conveniently performed directly on the site of the trials; the application proved to be practical and their distribution easy by the mechanization normally used on a horticultural farm. No phytotoxic effects of both bio-based formulations were observed on plants during the trials. 3.2.1. First trial year In 2012, the trial started on August 16th , when all the plots showed a sufficiently uniform RSM natural infestation: from 6 to 13 mobile mites per leaf. Table 1 reports the mean number of mobile mites found in each treatment at different sampling times. These data make it possible to show the clear knock-down effect of both formulations at 7 (T7 ) and 14 (T14 ) days after the treatments and their persistence during the first 14 days. The fenazaquin showed a highly lethal effect, determining the almost complete death of mites until the end of the trial: the mortality rate was always higher than 99% even after 21 days (Table 1). Both experimental formulations are characterized by a mechanism of action based on the oil film that covers the insect isolating the respiratory system from the air, an action improved by the allelopathic effect of GL degradation products (essentially AITC). It was therefore not surprising that these products, and in particular Formulation 2, determined a stronger acaricide action, reducing the infestation to only one mobile mite at T7 with a corresponding degree of effectiveness of 99%, statistically not different from the effect of the chemical pesticide. Formulation 1 was instead rather less effective, and 7 days after the treatment an average of 7 mobile forms were found, compared to 13 recorded at T0 . The degree of effectiveness, calculated on the evolution of populations in control plants, amounted therefore to 61.2%, a percentage significantly lower than fenazaquin and Formulation 2. This may be in part explained by the data in Fig. 2 from which it is evident that the lower concentration of oil in Formulation 1 is not able to trap a quantity of AITC significantly different from water. The persistence of the acaricide action of Formulation 2 showed a good ability of counteracting repopulation of the pest for 14 days. In fact, the degree of effectiveness, still high (93.9%) at T14 , was not statistically different from those found in fenazaquin plots. At T21 , a mean number of 20 mobile mites per leaf was found on plots treated with Formulation 2, showing that the product ended its acaricide action, reducing the degree of effectiveness to 69.2%. Fenazaquin, instead, maintained the same efficacy even 21 days after the treatment.
Table 1 Year 2012: mean number of mobile mites (N) per leaf found on eggplants, standard error (SE) and effectiveness of the products expressed as % (Abbott’s formula). Treatment
Untreated control Fenazaquin Formulation 1 Formulation 2
T0
T7
T14
T21
N
SE
N
SE
%
N
SE
%
N
SE
%
6.0 g 11.0 eg 13.0 ef 10.0 eg
3.2 4.1 4.0 3.8
18.0 de 0.1 h 7.0 fg 1.0 h
5.1 1.2 3.7 1.4
– 99.4 a 61.2 bc 94.4 a
33.0 bc 0.1 h 15.0 e 2.0 h
7.4 1.4 6.8 1.6
– 99.6 a 54.6 c 93.9 a
65.0 a 1.0 h 40.0 b 20.0 d
10.4 1.4 7.6 2.0
– 99.8 a 38.5 d 69.2 b
Different letters correspond to significantly different values for p < 0.05 (Duncan test).
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Table 2 Year 2013: mean number of mobile mites (N) per leaf found on eggplants, standard error (SE) and effectiveness of the products expressed as % (Abbott’s formula). Treatment
Untreated control Fenazaquin Formulation 1 Formulation 2
T0
T7
T14
T21
N
SE
N
SE
%
N
SE
%
N
SE
%
10.0 ce 15.0 cd 10.0 ce 14.0 cd
4.0 5.1 3.9 6.1
24.0 c 0.1 e 4.0 e 1.0 e
6.2 1.3 3.0 1.2
– 99.6 a 83.3 bc 95.8 a
80.0 b 0.1 e 7.0 de 3.0 e
9.1 1.4 4.6 1.9
– 99.8 a 91.2 a 96.2 a
150.0 a 2.0 e 35.0 c 13.0 cd
14.5 1.8 6.7 3.4
– 98.7 a 76.7 c 91.3 a
Different letters correspond to significantly different values for p < 0.05 (Duncan test).
4. Conclusions
Fig. 3. Comparison of number of mobile forms of Tetranychus urticae per leaf on eggplant under different treatments in the two trial years.
3.2.2. Second trial year In 2013, the trial started on August 7th , when all the plots showed a sufficiently uniform natural infestation: from 10 to 15 mobile mites per leaf. Table 2 reports the mean number of mobile mites from the beginning until the end of the trial after 21 days. In the second year too, the knock-down effect and persistence of fenazaquin was confirmed till 21 days after treatment, with 98.7% (Table 2) of degree of effectiveness, even in the presence of environmental conditions particularly favorable to RSM development. Formulation 2 also gave results similar to the first year, showing a good knock-down effect of the first treatment, but with a higher persistence till 21 days after the treatment with a degree of effectiveness of 91.3%, a value statistically not different from that of fenazaquin. In the second year of trial, Formulation 1 confirmed an acaricide action milder than Formulation 2 in terms of knock-down effect and persistence of the action, but in both formulations the related number of mobile mites was statistically lower than for the untreated plots. The average values of the two years of trial, regarding the number of mobile mites detected at T0, T7 , T14 and T21 , showed a knock-down effect for Formulation 2 comparable to fenazaquin even if with a lower persistence (Fig. 3). The natural patented Formulations 1 and 2 had different but significant effects on RSM. The oil in the emulsion suffocates the spiders, creating a microfilm that remains on the leaf apparatus and on the insects that even alone showed a clear containment of pests that was observed as not statistically different from paraffinic oils on red scale (Rongai et al., 2008). This effect was summed to the allelopathic effect of DSM that, after water addition, released AITC which had an additional control effect on the disease and a fertilizing one. This system confirmed the capability to release AITC at a rate of conversion of sinigrin of about 80%, after water addition, and of limiting, if compared to water, the dispersion in the air of AITC released from the DSM from B. carinata of around 35% in Formulation 2.
The pressure procedure made it possible to classify the oil and DSM used in these trials as not chemically modified materials and, in this way, both the oil and the bio-products are considered in the European Regulament REACH (EC Regulation No. 1907/2006) as exempt from registration (Annex IV). The experiments carried out in 2012–2013 showed that both experimental liquid formulations were characterized by an allelopathic action, expressed in particular by Formulation 2. Its higher DSM amount should be the reason for its stronger effectiveness, generally statistically not different from the chemical fenazaquin, widely applied in the past on eggplant both at open field and greenhouse level. It can thus be speculated that Formulation 1 could be used as a preventive treatment, while Formulation 2 could be applied in the presence of stronger attacks. Summarizing, the average values of the two years of trial, regarding the number of mobile mites detected during the periods of trials, showed a knock-down effect for Formulation 2 comparable to chemical even if with a lower persistence. Considering that only sulfur and paraffinic oil are currently allowed as acaricides in organic farming, the definition of new low environmental impact products with a shorter withholding period could represent considerable technical progress for conventional and organic farmers in the control of spider mites. The interest for these partial or total innovative alternatives to chemicals can be confirmed if we point out that, since 2011 (Commission Implementing Directive 2011/39/EU, 2011), all the pesticides based on fenazaquin (including Magister® ) are not allowed for application as acaricides on food crops and are authorized only for applications on ornamental plants. Acknowledgement The trials were performed as a 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. 17,533/7303/10 del 29/04/2010) and coordinated by CRA-CIN of Bologna. We thank Lorena Malaguti (CRA-CIN Bologna) for her help in chemical characterization of the biomasses used in the trials. References Abbott, W.S., 1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 163, 265–267. Ahmadi, M., Fathipour, Y., Kamali, K., 2007. Population growth parameters of Tetranychus urticae (Acari: Tetranychidae) on different bean varietiesm. J. Entomol. Soc. Iran 26, 1–10. Baker, B.P., Benbrook, C.M., Groth, E., Lutz Benbrook III, K., 2002. Pesticide residues in conventional, integrated pest management (IPM)-grown and organic foods: insights from three US data sets. Food Addit. Contam. 19, 427–446, C32]. Beers, E.H., Riedl, H., Dunley, J.E., 1998. Resistance to abamectin and reversion to susceptibility to fenbutatin oxide in spider mite (Acari: Tetranychidae) populations in the Pacific Northwest. J. Econ. Entomol. 91, 352–360. Chaudhri, W.M., Akbar, S., Rasool, A., 1985. Studies on biosystematics and control of mites of field crops, vegetable and fruit plants in Pakistan UAF. Tech. Bull. 3, 314.
Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060
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Chen, Y., Opit, G.P., Jonas, V.M., Williams, K.A., Nechols, J.R., Margolies, D.C., 2007. Two spotted spider mite population level, distribution, and damage on ivy geranium in response to different nitrogen and phosphorus fertilization regimes. J. Econ. Entomol. 100, 1821–1830. Chow, A., Chau, A., Heinz, K.M., 2009. Reducing fertilization for cut roses: effect on crop productivity and twospotted spider mite abundance, distribution, and management. J. Econ. Entomol. 102, 1896–1907. Christopherson, S.W., Glass, R.L., 1969. Preparation of milk fat methylesthers by alcoholysis in essentially non alcoholic solutions. J. Dairy Sci. 52, 1289–1290 [L33]. Commission Implementing Directive 2011/39/EU, 2011. April 11th. Cranham, J.E., Helle, W.,1985. Pesticide resistance in Tetranychidae. In: Helle, W., Sabelis, M.W. (Eds.). Elsevier, Amsterdam, pp. 405–421. 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. Devine, G.J., Barber, M., Denholm, P.S., 2001. Incidence and inheritance of resistance to METI acaricides in European strains of the two-spotted spider mite (Tetranychus urticae), (Acari: Tetranychidae). Pest Manag. Sci. 57, 443–448. Directive 2009/128/EC of the European Parliament and of the Council, 2009. October 21th. Duso, C., Chiarini, F., Conte, L., Bonora, V., Dalla Montà, L., Otto, S., 2004. Fogging can control Tetranychus urticae on greenhouse cucumbers. J. Pest Sci. 77, 105–111. EC, Regulation No. 1907/2006 on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) Annex IV. Edge, V.E., James, D.G., 1986. Organotin resistance in Tetranychus urticae Acari: Tetranychidae in Australia. J. Econ. Entomol. 79, 1477–1483. El Kady, G.A., Sharabasy, H.M., Bahgat, I.M., 2007. Toxicity of Two Potential Bio-insecticides Against Moveable Stages of Tetranychus urticae Koch. J. Appl. Sci. Res. 3, 1315–1319. Fahey, J.W., Zalcmann, A.T., Talalay, P., 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56, 5–51. Fergusson-Kolmes, L.A., Scott, J.G., Dennehy, T.J., 1991. Dicofol resistance in Tetranychus urticae (Acari: Tetranychidae): Cross-resistance and pharmacokinetics. J. Econ. Entomol. 84, 41–48. Flexner, J.L., Westigard, P.H., Croft, B.A., 1988. Field reversion of organotin resistance in the twospotted spider mite (Acari: Tetranychidae) following relaxation of selection pressure. J. Econ. Entomol. 81, 1516–1520. Herron, G.A., Rophail, J., 1993. Genetics of hexythiazox resistance in two spotted spider mite, Tetranychus urticae Koch. Exp. Appl. Acarol. 17, 423–431.
Herron, G.A., Edge, V., Rophail, J., 1993. Clofentezine and hexythiazox resistance in Tetranychus urticae Koch in Australia. Exp. Appl. Acarol. 17, 433–440. Lazzeri, L., Leoni, O., Manici, L.M., Palmieri, S., Patalano, G., 2010. Use of Seed Flour as Soil Pesticide. Patent no US 7749,549, July 6th. Lazzeri, L., D’Avino, L., Ugolini, L., De Nicola, G.R., Cinti, S., Malaguti, L., Bagatta, M., Patalano, G., Leoni, O., 2011. Bio-based products from Brassica carinata A. Braun oils and defatted meals by a second generation biorefinery approach. EU BC&E Conference Proceedings, 1080–1092. Official Journal of the European Union, 2008. L 250, September 18th, pp. 1–84. Opit, G.P., Chen, Y., Williams, K.A., Nechols, J.R., Margolies, D.C., 2005. Plant age, fertilization, and biological control affect damage caused by twospotted spider mites on ivy geranium: development of an action threshold. J. Am. Soc. Hortic. Sci. 130, 159–166. Regulation (CE) N. 889/2008 of the European Commission, 2008. September 5th. Rongai, D., Cerato, C., Lazzeri, L., Palmieri, S., Patalano, G., 2008a. Vegetable oil formulation as biopesticide to control California red scale (Aonidiella aurantii Maskell). J. Pest Sci. 81, 179–185. Rongai, D., Cerato, C., Lazzeri, L., Palmieri, S., 2008. Composition for the treatment and/or prevention of attacks by biological agents. Patent no (US 2008/0199451A1), August 21st. Sato, M.E., Suplicy Filho, N., De Souza Filho, M.F., Takematsu, A.P., 1994. Resistencia do acaro rajado Tetranychus urticae (Koch,1836) (Acari: Tetranychidae) a diversos acaricidas emmorangueiro (Fragaria sp) nos municipios de Atibaia- Sp e Piedade-Sp. Ecossistema 19, 40–46. Sato, M.E., Miyata, T., Da Silva, M., Raga, A., De Souza Filho, M.F., 2004. Selections for fenpyroximate resistance and susceptibility, and inheritance, cross-resistance and stability of fenpyroximate resistance of Tetranychus urticae Koch (Acari: Tetranychidae). Appl. Entomol. Zool. 39, 293–302. Standard UNI 22,604. 1992. Seed and vegetable oils and derived products. Vegetable proteins. Determination of crude protein. Stumpf, N., Nauen, R., 2001. Cross-resistance, inheritance, and biochemistry of mitochondrial electron transport inhibitor - acaricide resistance in Tetranychus urticae (Acari: Tetranychidae). J. Econ. Entomol. 94, 1577–1583. Tsagkarakou, A., Pasteur, N., Cuany, A., Chevillon, C., Navajas, N., 2002. Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece. Insect Biochem. Mol. Biol. 32, 417–424. Whalon, M.E., Mota-Sanchez, D., Hollingworth, R.M., Gutierrez, R., 2013. Arthropod Pesticide Resistance Database 2012. www.pesticideresistance.org, consulted on 15.04.2014. Zhang, Y., Cho Posner Talalay, C.G.G.H.P., 1992. Spectroscopic quantitation of organic isothiocyanates by cyclocondensation with vicinal dithiols. Anal. Biochem. 205, 100–107.
Please cite this article in press as: Piccinini, E., et al., Effect of two bio-based liquid formulations from Brassica carinata in containing red spider mite (Tetranychus urticae) on eggplant. Ind. Crops Prod. (2015), http://dx.doi.org/10.1016/j.indcrop.2015.05.060