In field control of Botrytis cinerea by synergistic action of a fungicide and organic sanitizer

Fatima Ayoub, Najwa Ben oujji, Mohamed Ayoub, Athman Hafidi, Rachid Salghi, Shehdeh Jodeh

1 Laboratory of Applied Chemistry and Environment, National School of Applied Science, Ibn Zohr University, P.O Box 1136,Agadir 80000, Morocco

2 Ecolink International, Zone Industrielle, Ait Melloul, Agadir 80000, Morocco

3 Department of Chemistry, An-Najah National University, P.O. Box 7, Nablus, Palestine

1. Introduction

Agricultural crops are exposed to approximately 70 000 species of pests, including insects, mites, plant pathogens,and weeds that potentially cause the reduction of world food production by more than 40% if pesticides are not applied (Suprapta 2016). These phytosanitary products play a vital role in the economic production of wide ranges of vegetable, fruit, cereal, forage, fibre and oil crops which now constitute a large part of successful agricultural industry in many countries (Al Hattab and Ghaly 2012).Although tremendous benefits have been derived from the use of pesticides in agriculture, increasing awareness on their negative effects on human health and biodiversity urges to find more eco-friendly and healthier alternatives(Anderssonet al. 2014; Bernardeset al. 2015). In the last few years, different alternatives to chemical products have been reported in the literature, including: i) biocontrol agents(Heydari and Pessarakli 2010; Liuet al. 2010); ii) biologically active natural products (Ben-Shalomaet al. 2003; Dafereraet al. 2003; Nigroet al. 2006; Vitoratoset al. 2013; Aminiet al. 2016; Suprapta 2016; Todorovi?et al. 2016); (iii) GRAS(generally recognized as safe)-classified sanitizers (Vendittiet al. 2008; Elbouchtaouiet al. 2015); and iv) Integrated Pest Management (IPM) methods (Romanazziet al. 2006; Xuet al. 2007; Camiliet al. 2010; Solimanet al. 2015). In that regard, we describe in this paper a new IPM method based on combination of an organic sanitizer and fungicide for the control ofB.cinereain tomato fields in the attempt to reduce the negative impacts of chemical pesticides on environment and consumer’s health. IPM is increasingly perceived as a workable solution to chemical pesticides problems. It can be defined as an ecosystem approach to crop production and protection that combines different management strategies and practices to grow healthy crops and minimize the use of pesticides (FAO 2012). Therefore, IPM utilizes the best mix of control tactics for a given pest problem when compared with the crop yield, profit and safety of other alternatives(Ehi-Eromoseleet al. 2013).

Gray mold caused byBotrytis cinerea(Botrytis cinereaPersoon: Fries (teleomorphBotryotinia fuckeliana) is one of the most economically important diseases and the most redoubtable threatof commercially produced tomatoes(McDougall 2016). This fungus can affect yield in different ways. The pathogen may cause blight on leaf or petal tissues, crown rot, stem cankers, cutting rot, and dampingoff (Liet al. 2014). The fungus produces germ tubes from conidia that can infect through natural openings or wounds.It is a cool-season disease and infection is favored under wet conditions with temperatures below 22°C. In addition to actively causing disease during the growing season,the fungus is also able to cause latent infections leading to disease after harvest, either during storage or transit,in the store, or after purchase by the consumer (Liet al.2014). In the absence of resistant tomato varieties to this pathogen, the control of this disease is still based upon multiple applications of fungicides, mainly benzimidazoles and dicarboximides, which can leads to the development of pathogen resistance, chemical residues in fruits,phytotoxicity to other organisms or serious environmental and public health problems as described above.

In a previous work (Ayoubet al. 2017), we have described a new treatment approach based on the combination of a commercially available peroxyacetic acid mixture(PERACLEAN?5) with two commonly used fungicides (each alone) to control grey mold. Thein vitrotests described in Ayoubet al. (2017) allowed us to suppress the pathogen while minimizing the amounts of applied fungicides by more than 95%. In this paper, thein fieldtrials of this new developed approach is presented. This new practice presents several advantages over the conventional methods to controlB.cinerea: i) It will minimize the 100% effective concentration of the two products, enhance fungicidal action and consequently reduce the well known negative impacts of pesticide; ii) lengthen the period of effectiveness;iii) peroxyacetic acid being rapidly active will speed up the biocide action against the pathogen; iv) being highly acid, Peroxyacetic acid will induce the acidification of the fungicide, necessary before application on field, so no additional acidifying product is needed; v) the use of this mixture will delay the selection of resistant strains; and vi)it’s a cost effective way to limit the grey mold incidence.To the best of our knowledge, this new practice was not previously described elsewhere.

2. Materials and methods

2.1. Chemical products

A commercially available 5% peracetic acid (PAA) solution PERACLEAN?5 was kindly provided by Green Solutions Company, Morocco. It’s a fungicidal, bactericidal and yeast-active disinfectant with a broad spectrum of action,consisting of 5% of peroxyacetic acid, 26.4% of hydrogen peroxide and 6.8% of acetic acid.

Two commonly used fungicides to control grey mold disease caused byB. cinereawere selected so as to study their antifungal efficacy, separately and in combination with PERACLEAN?5: (1) SIGNUM?WG by BASF, Germany;active ingredients: 26.7% (w/w) boscalid and 6.7% (w/w)pyraclostrobin; chemical groups: pyridinecarboximide and methoxy-carbamate, respectively and (2) SWITCH?62.5 WG by Syngenta Crop Protection Pty Ltd., Switzerland;active ingredients: 375 g kg–1cyprodinil and 250 g kg–1fludioxonil; chemical groups: anilinopyramidine and phenylpyrrole, respectively.

2.2. Antifungal activities

The antifungal activities of the above cited chemicals were testedin fieldagainst growth ofB.cinereaon stems, leaf and fruits. PERACLEAN?5, SWITCH and SIGNUM were tested either separately or as combinations as described in Table 1. The choice of the concentrations was made according to the manufacturers recommended field doses for both fungicides: The highest concentrations of SWITCH and SIGNUM reported in Table 1 (60 and 125 g L–1,respectively) represent the advised concentrations used by the Moroccan farmer for the control ofB.cinereain tomatofields.Since our goal is to decrease the used concentrations of pesticide, 50 and 25% of these concentrations were tested either alone or combined with the organic sanitizer(PERACLEAN?5) to test their efficiency. All the solutions were prepared immediately before their application in the greenhouse where a randomized complete block design was adopted.

Table 1 Tested treatments

2.3. Greenhouse experiments

The current study was conducted in a commercial tomato production site located in Ben Guemoud region (Latitude:30°14′4.3′′ (30.2345°) North; Longitude: 9°34′28.6′′(9.5746°) West), Souss-Massa, Agadir-Morocco, where a Canarian greenhouse with soil culture system was adopted. Tomato plants var. Pristyla (from Gautier)susceptible toB.cinerea, were planted at the beginning of August in sandy loam soil that had been disinfected with 1,3-dichloropropene+chloropicrine (55.4%+32.7%)(Ajaanid 2016). Plants were spaced 0.4 m apart and 1.25 m between rows. Natural infestation withB.cinereaoccurred in the greenhouses during winter seasons, so the tests were conducted during the winter in January 2017.Irrigation, fertilization and other agricultural procedures were carried out according to common practices in Moroccan farming.

Naturally infected tomato plants were used. Each of the 27 treatments was applied to a separated tomato row containing 10 to 15 tomato plants with one row treated with distilled water to serve as the control. Buffer rows were used between treated lines and during all the treatment process, care was taken to avoid drift to adjacent plots.The treatments were applied on the entire plant using a backpack-type sprayer. The efficiency of the tested treatments was evaluated by the decrease of the infection rate after 24, 48 and 120 h after treatment application.Before treatment, plants infected withB.cinerea, showing brown or spotted plant material with masses of silver-gray spores on the dead or dying tissue, were counted and considered as initial rate of infection with report to the total number of plants in the row. After treatments, the number of healed plant showing dry spot without spore in surface was counted. The infection rate was calculated as follow:

Where,X0andXare the number of infected plants before and after treatments, respectively.

2.4. Chemicals interaction trials

For simplicity, most interaction experiments are performed under laboratory or greenhouse conditions (in vivoandin vitro)with a single isolate (Gisi 1996). In this work, the results obtained from a greenhouse trial were used to determine the interaction nature between tested chemicals:antagonistic, additive or synergistic interaction. Fungicide and organic sanitizer interactions were assessed with the widely used model Abotts’ formula (Gisi 1996). The model compares expected and observed suppressions where expected suppressions expressed as percent Cexp(%) can be predicted as follows:

Where, A and B are the inhibitions caused when the fungicide and sanitizer act alone respectively. The ratio of inhibition (RI) was then calculated as follows for each mixture:

Interactive effects were evaluated by comparing RI with 1. RI values＞1 indicated synergism; RI values=1 indicated additivity; and RI values＜1 indicated antagonism (Chesworthet al. 2004).

2.5. Statistical analysis

Data were analyzed using MINITAB statistical software version 18. Treatment means were separated by Tukey’s test atP≤0.05.

3. Results

3.1. Antifungal activity of PERACLEAN?5

The antifungal activity of PERACLEAN?5, againstB.cinerea, in tomato greenhouse was assessed using three concentrations: 0.5, 1 and 1.5%. The infection rate by grey mould was counted before treatment (0 h), and 24, 48 and 120 h after treatment. The obtained results are presented in Table 2.

As reported in Table 2, after 24 h of treatment application,all concentrations of peroxyacetic acid preparation allowed the suppression of the pathogen by 40, 50 and 62.5%using 0.5, 1 and 1.5% of PERACLEAN?5, respectively,which demonstrate that increasing the concentration of PERACLEAN?5 increase to efficiency of the product.After 48 h, the decrease observed in the infection rate has remained constant for the treatments T1 and T3 (0.5 and 1.5% of PERACLEAN?5, respectively) while using treatment T2, new infections withB. cinereaappeared,which may be due to the biodegradation of the product.After 5 days of treatment (120 h), new infections by the fungus were observed in the rows treated with 0.5 and 1% of PERACLEAN?5 while in the row treated with 1.5%the disease incidence remained stable. Even if thisconcentration of PERACLEAN?5 permits a good control ofB.cinereawith a persistent action, it may cause severe damages to the plant by provoking burns on leaves.

Table 2 Disease incidence of Botrytis cinerea after 0, 24, 48 and 120 h of PERACLEAN?5 treatment application

3.2. Antifungal activity of SWITCH

Three concentrations of SWITCH (37.5% cyprodinil+25%fludioxonil) were tested either alone or combined with 0.5,1 and 1.5% of PERACLEAN?5 to test their efficiency in the control ofB.cinerea. The obtained results are shown in Table 3.

In general, all the treatments significantly reduce (P≤0.05)the incidence of fruit and stem infections except 15 g L–1of SWITCH alone. When the fungicide was used alone, it was observed that increasing the pesticide concentration increases the inhibition ofB.cinereawith 62.5% of infection suppressed by the highest concentration: 60 g L–1of SWITCH. The ×1/2 of this concentration (30 g L–1)only suppresses 25% of the pathogen infection while 15 g L–1of SWITCH alone doesn’t exhibit any inhibition of the fungus. When combined with PERACLEAN?5, an increase of the efficiency of the pesticide was observed.The results in Table 3 show that all mixtures of SWITCH with PERACLEAN?5 were more effectives in suppressingB.cinereathan the recommended dose of the fungicide and with a persisting action. The best results were obtained when combining 1/4 of the recommended concentration of SWITCH (15 g L–1) with 0.5% of PERACLEAN?5, a result that may be due to the interactions between the fungicide and the sanitizer which were assessed with the widely used model Abotts’ formula (1925). The obtained results are presented in Table 4.

When PERACLEAN?5 was combined with the recommended dose of SWITCH (60 g L–1), a slight antagonistic interaction was observed (Table 4). Using 30 g L–1of the fungicide, a synergistic interaction between the twoproducts was observed and with 15 g L–1, a synergistic to highly synergistic interaction was expressed. The highly synergistic effect was obtained when combining 15 g L–1of SWITCH with 0.5% of PERACLEAN?5. The obtained results are in concordance with those reported in Gisi (1996)where the synergistic interactions between biocides always decrease rapidly with increasing control levels of the single component and maybe almost nil at high control levels.

Table 3 Disease incidence of Botrytis cinerea on tomatoes plants after 0, 24, 48 and 120 h of treatment with SWITCH alone or mixed with PERACLEAN?5

Table 4 Synergistic interactions between SWITCH and PERACLEAN?5 in the greenhouse conditions

3.3. Antifungal activity of SIGNUM

As for SWITCH, three concentrations of SIGNUM were tested either alone or combined with 0.5, 1 and 1.5% of PERACLEAN?5 to test their efficiency in the control ofB.cinereaunder the greenhouse conditions. The reported concentrations are the recommended dose of the fungicide(125 g L–1), ×1/2 (62.5 g L–1) and ×1/4 (31.25 g L–1) of this concentration. The obtained results are presented in Table 5.

Used alone, the recommended dose of SIGNUM (125 g L–1) resulted in suppression of 66.7% of grey mould infection,while ×1/2 and ×1/4 of this concentration suppressed 59.5 and 54.6%, respectively. When combined with PERACLEAN?5, all mixtures gave better results. The treatment providing the maximum control ofB.cinereawas the result mixture of 62.5 g L–1of SIGNUM with 1.5%of PERACLEAN?5. Even if this combination permits a good control of the fungus, its application maybe limited by the negative impact that a concentration of 1.5% of PERACLEAN?5 may induce to the plant. Therefore, in this study, the recommended mixture will be the combination of 31.25 g L–1of SIGNUM with 0.5% of PERACLEAN?5,which suppress 66.7% of the disease. This mixture resulted in similar results to these obtained with the recommended concentration of SIGNUM while minimizing the applied amount of pesticide by 75%.

In order to better understand the nature of interactions between SIGNUM and PERACLEAN, interactive effects were calculated according to the Abotts’ formula and the results are presented in Table 6.

In general, a slight antagonistic effect was observed between SIGNUM and PERACLEAN?5, which may be due to the chemical composition of both products. This later interpretation is based on the common definition of the synergism where it’s considered as a joint action of mixture components in which the total effect is greater than the sum of the effects of the individual components(Gisi 1996). However, in our case, this definition may be an underestimation of the obtained results since our goal is to enhance the efficiency of the synthetic pesticide while minimizing the applicable amount without taking into consideration the effect of the organic sanitizer.

4. Discussion

Peroxyacetic acid (PAA) is a biodegradable product and a highly effective biocide, used in a wide range of applications and demonstrates excellent bactericidal and fungicidal activity against a wide range of microorganisms (Baldry 1983; Ossia-Ongagna and Sabatier 1993; Kitis 2004; Bernetet al. 2005). It had been used for purposes ranging from disinfestations of bulbs to prevention of other horticultural diseases through disinfecting potting soil and cleaning irrigation equipment (Alvaro 2009). There are several registered products on the market, containing peroxyacetic acid, hydrogen peroxide and acetic acid in different proportions as stabilized mixtures. These mixtures are environmentally-friendly products as their manufacture does not involve polluting processes; they have the same function as other chemicals, and after use, there is no toxic residue left in the environment (Carrasco and Urrestarazu 2010).They can be used in the application of green chemistry in agriculture and none of the products formed during the degradation process are harmful. Acetic acid, hydrogen peroxide, water, and oxygen, are biodegradable, not harmful or even secondarily useful (Carrasco and Urrestarazu 2010). Several studies have reported the antifungal activity of these mixtures againstPenicillium digitatumandB. cinerea(Elbouchtaouiet al. 2015),Monilinia laxaandRhizopus stolonifer(Mariet al. 2004) and many other pathogens. However, to have a fungicide effect, 1.5 to 2%of a mixture based on 5% of PAA is needed. Even if this concentration may suppress the pathogen, it may cause severe damages when applied to the plant. In addition,the rapid decay of those products limits their application as a biofungicide (Pedersen 2015). To cover these products limitations, we described in this study, a new Integrated Pest Management program (IPM) based on the combination of a commercially available peroxyacetic acid mixture(PERACLEAN?5) with two commonly used fungicides to control grey mold disease in tomato greenhouse.

Table 5 Disease incidence of Botrytis cinerea on tomatoes plants after 0, 24, 48 and 120 h of treatment with SIGNUM alone or mixed with PERACLEAN?5

Table 6 Synergistic interactions between SIGNUM and PERACLEAN?5 under greenhouse conditions

The obtained data showed that treatment with PERACLEAN?5 resulted in interested results in suppression grey mold disease, however, its fast biodegradability limits its use as biocide: When used alone, the PERACLEAN?5 efficiency decreases rapidly with time. After 5 days (120 h)of its application, new infections were observed in the rows treated with 0.5 and 1% of PERACLEAN?5, while in the row treated with 1.5% the infection rate remained stable at 37.5%. Even if this concentration of PERACLEAN?5 permits a good control ofB.cinereawith a persistent action, it may cause severe damages to the plant by provoking burns on leaves. When combined with the fungicides (SWITCH or SIGNUM), the effectiveness period was longer with no loss of activity observed even after 5 days of the application.

On the other hand, SWITCH fungicide permits a better control of grey mold disease than SIGNUM followed by PERACLEAN?5. When combined with the latter, the efficiencies of both fungicides were enhanced. The treatment providing the maximum control ofB.cinereawas the result mixture of ×1/4 of the recommended concentration of SWITCH (15 g L–1) with 0.5% of PERACLEAN?5 followed by ×1/2 of the advised dose of SIGNUM with 1.5% of PERACLEAN?5. These two mixtures suppressed respectively 85 and 78% of gray mould infection.

In order to better understand the mode of action of these mixtures, interaction trials were conducted. The results showed that the synergy levels are strongly dependent on the ration of components in the mixture and that minimum amounts of the components give a maximum synergy levels,which is in concordance with the results reported in Gisi(1996) that the synergistic interactions always decrease rapidly with increasing control levels of the single component and maybe almost nil at high control levels. When PERACLEAN?5 was combined with the recommended dose of SWITCH (60 g L–1), a slight antagonistic interaction was observed. Using 30 g L–1of the fungicide, a synergistic interaction between the two products was observed and with 15 g L–1, a synergistic to highly synergistic interaction was expressed. The highly synergistic effect was obtained when combining 15 g L–1of SWITCH with 0.5% of PERACLEAN?5.Concerning SIGNUM and PERACLEAN mixtures, a slight antagonistic effect was observed between both products.This interpretation of the antagonistic and synergistic interaction between PERACLEAN?5 and SWITCH or SIGNUM was based upon the common definition of the synergism where it’s considered as a joint action of mixture components in which the total effect is greater than the sum of the effects of the individual components (Gisi 1996). In our study, this definition may be an underestimation of the obtained results since our goal is to enhance the efficiency of the synthetic pesticide while minimizing the applicable amount without taking into consideration the effect of the organic sanitizer. Based on the terminology of Gaddum(1959), where a synergism is declared when the effect of the mixtures exceeds the effect of its more potent component,our results reveal remarkable synergistic effects when combining PERACLEAN?5 with both synthetic fungicides which allowed suppressing grey mould disease with higher efficiency.

5. Conclusion

In this work, the efficiency of a fungicide/organic sanitizer mixture to control grey mold disease caused byB.cinereawas described in order to exploit their additive and synergistic interaction, by which the overall activity is increased and the concentration of the pesticide can be reduced without loss of activity. As a conclusion, the combination of an organic peroxyacetic acid preparation (PERACLEAN?5) with two synthetic fungicides (SWITCH or SIGNUM separately)enhances the efficiency of both products while minimizing the amount of pesticides needed to controlB.cinereain the greenhouse conditions. This study allowed us to define the optimum ration of the components (PERACLEAN?5 and SWICH or SIGNUM) in the mixture to achieve the highest control levels of grey mould. This optimum was obtained by mixing 15 g L–1of SWITCH with 0.5% of PERACLEAN?5.The later combination resulted in suppression of 85% ofB.cinereainfections while decreasing the recommended dose of this fungicide by 75% reducing then the well known negative impacts of chemical pesticides on environment and consumer’s health. In SIGNUM case, the recommended mixture will be the combination of 31.25 g L–1of the fungicide with 0.5% of PERACLEAN?5, which suppress 66.7% of the disease. This mixture resulted in similar results than these obtained with the recommended concentration of SIGNUM while minimizing the applicable amount of pesticide by 75%.

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