Organic and Conventional Bean Pesticides in Development of Autochthonous Trichoderma Strains

Pesticides of chemical synthesis have mainly been used to control pests, diseases and adventitious plants up until now. However, it has been shown that some pesticides can remain in the soil for long periods of time, thus affecting the development of organisms in the rhizosphere as well as human health, which are two of the most noteworthy side effects. The aim of this research was to analyze the compatibility of autochthonous Trichoderma strains with different synthetic fungicides, acaricides, insecticides (including an entomopathogenic fungus) and herbicides. Sulfur encouraged the growth of all autochthonous strains assayed, and the combination Trichoderma-B. bassiana did not disturb their growth. So, the combination of the autochthonous Trichoderma strains with these organic pesticides will be a positive strategy to apply in the field to control pests and some diseases. Conventional pesticides modified the development of all autochthonous Trichoderma strains, demonstrating that not only do they affect weeds, fungus or pests but also rhizosphere microorganisms. In conclusion, conventional pesticides indiscriminately used to control pests, diseases and weeds could reduce the development of autochthonous Trichoderma strains, especially fungicides and herbicides.


Introduction
Food consumption must not only be guided by aspects related to the safety of the product but must also be healthy, nutritious and environmentally friendly. Thus, it is necessary to look for production techniques that guarantee consumer safety and environmental sustainability. In this sense, the reduction in chemical measures by using natural production techniques is more highly appreciated by consumers. Until now, pesticides of chemical synthesis have been used mainly to control pests, diseases and adventitious plants. A total of 160,660 tons of fungicides and bactericides were used in the European Union (EU) in 2019 (Spain, France and Italy being the highest consumers, representing 51.56% of the EU); 56,669 tons of insecticides and acaricides (Germany, Turkey and Spain, with 67.74% of the EU); and 128,121 tons of herbicides (France, Spain and Germany, representing 41.72% of the EU) [1]. However, it has been proven that some pesticides can remain in the soil for long periods of time, altering the development of organisms in the rhizosphere and affecting plants, human and animal health. For example, since 2019, the use of the herbicide Diquat is no longer approved by the EU due to its high persistence in soil under aerobic conditions. Moreover, it presented a high risk for aquatic organisms Table 1. Autochthonous Trichoderma strains used in this study.
Isolate (1) Culture Collection ( (1) [8,17]. (2) All "PAULE" strains are in "Pathogens and Antagonists" collection of the Laboratory Diagnosis of Pests and Diseases (PALDPD), University of León, León, Spain; All IASULE strains are in the "Pathogens and Antagonists" collection of the Research Group of Engineering and Sustainable Agriculture Collection, University of León, León, Spain.

Pesticides
Three acaricides, four insecticides (including an insect entomopathogenic fungus), seven fungicides and three herbicides were tested in this study to evaluate their suitability for application in bean crops ( Table 2).

In Vitro Evaluation
Pesticides (Table 2) were incorporated into a potato-dextrose-agar medium (PDA, Merck KGaA, Darmstadt, Germany). Media were sterilized (121 • C, 20 min), the pesticides were added at the recommended field doses and distributed in 90 mm diameter Petri plates (18 mL/plate). Six mm diameter plugs collected from the edge of growing fungal colonies of each isolate were used to inoculate the plates, which were then incubated in the dark at 25 • C for seven days. Control PDA plates without any pesticide were prepared in the same conditions as above ( Figure 1).

In Vitro Evaluation
Pesticides (Table 2) were incorporated into a potato-dextrose-agar medium (PDA, Merck KGaA, Darmstadt, Germany). Media were sterilized (121 °C, 20 min), the pesticides were added at the recommended field doses and distributed in 90 mm diameter Petri plates (18 mL/plate). Six mm diameter plugs collected from the edge of growing fungal colonies of each isolate were used to inoculate the plates, which were then incubated in the dark at 25 °C for seven days. Control PDA plates without any pesticide were prepared in the same conditions as above ( Figure 1).
The development of each Trichoderma strain was measured after two, five and seven days after sowing. Experiments were performed with four replicates.

Statistical Analysis
Kolmogorov-Smirnov's and Shapiro-Wilk's tests were used to check the normality of the data for ANOVA and variance homogeneity among the treatments.
The results were compared by two analyses of variance. Firstly, a two-way ANOVA The development of each Trichoderma strain was measured after two, five and seven days after sowing. Experiments were performed with four replicates.

Statistical Analysis
Kolmogorov-Smirnov's and Shapiro-Wilk's tests were used to check the normality of the data for ANOVA and variance homogeneity among the treatments.
The results were compared by two analyses of variance. Firstly, a two-way ANOVA for a completely randomized design was carried out, including the main effects of autochthonous Trichoderma strains (T008, T019, T028 and T032) and the groups of pesticides (acaricides, insecticides, fungicides and herbicides). The other two-way ANOVA was performed, including the effects of autochthonous Trichoderma strains and all pesticides ( Table 2). Analysis of Fisher's least significant difference (LSD) was performed using IBM SPSS Statistics (IBM SPSS Statistics for Windows, Version 26.0. IBM Corp. Armonk, NY, USA).

Results
As a result of the Kolmogorov-Smirnov and the Shapiro-Wilk tests, together with the two-way analyses of variance of Trichoderma strains and pesticide group on Trichoderma development, significant differences were observed among autochthonous Trichoderma strains and among the groups of pesticides analyzed but no significant interaction between Trichoderma strains and pesticide group ( Table 3). The two-way analysis of variance of Trichoderma strains and pesticides on Trichoderma development is significant for Trichoderma strains, pesticides and the interaction of Trichoderma strains x pesticides (Table 4). Therefore, one-way analyses of variance were independently performed for each Trichoderma strain and for each group of products (acaricide, insecticides, fungicides and herbicides). The results are presented in Figures 2, 4, 6 and 8.

Acaricides
Regarding the development of Trichoderma strains in contact with chemically synthesized acaricides, it was observed that Sulfur did not prevent the development of any tested strain, highlighting T. virens where it stimulated the growth compared to the control. In the case of Abamectin, the growth of the different Trichoderma strains was lower compared to all the other treatments. However, in the medium with Deltamethrin, all strains grew at a lower level on the second day after inoculation, but this lower development was only observed for T. virens and T. citrinoviride after 5 and 7 days following inoculation (Figures 2 and 3).

Insecticides
Among the analyzed insecticides was Chlorpyrifos, whose application has been banned by the European Food Safety Authority (EFSA), as it causes problems in children and unborn neurological development. In the current study, we observed that this was the cause of significant reduction in the development of all Trichoderma strains, followed

Insecticides
Among the analyzed insecticides was Chlorpyrifos, whose application has been banned by the European Food Safety Authority (EFSA), as it causes problems in children and unborn neurological development. In the current study, we observed that this was the cause of significant reduction in the development of all Trichoderma strains, followed by Pirimicarb. B. bassiana, while, only significantly modifying the development of T. citrinoviride and T. velutinum in the first two days after inoculation. Imidacloprid significantly reduced the growth of all Trichoderma strains, except T. velutinum, when compared to the control (Figures 4 and 5).

Fungicides
Mancozeb, Thiophanate-methyl and Thiram are also unauthorized in the EU because they presented a high risk to birds, mammals, non-target arthropods and soil macroorganisms. All autochthonous Trichoderma strains assayed were affected by the presence of fungicides in the growth medium (Figures 6 and 7). In the case of Azoxystrobin + Difenoconazole, Trichoderma development was affected on the second and fifth day, but on the seventh day, they did not show any differences compared to the control. As for Thiram, its action affected the development of T. citrinoviride, which exhibited a lower growth than the control for the evaluation period, but the other strains became similar to the control from the fifth day. Chlorothalonil, Methyl thiophanate, Tebuconazole and Copper drastically reduced the growth of all Trichoderma strains analyzed.

Fungicides
Mancozeb, Thiophanate-methyl and Thiram are also unauthorized in the EU because they presented a high risk to birds, mammals, non-target arthropods and soil macroorganisms. All autochthonous Trichoderma strains assayed were affected by the presence of fungicides in the growth medium (Figures 6 and 7). In the case of Azoxystrobin + Difenoconazole, Trichoderma development was affected on the second and fifth day, but on the seventh day, they did not show any differences compared to the control. As for Thiram, its action affected the development of T. citrinoviride, which exhibited a lower growth than the control for the evaluation period, but the other strains became similar to the control from the fifth day. Chlorothalonil, Methyl thiophanate, Tebuconazole and Copper drastically reduced the growth of all Trichoderma strains analyzed.  Table 2. Upper and lower error bars are represented and indicate standard error of the mean showing the accuracy of the calculations. Different letters indicate significant differences between synthetic fungicides (ANOVA, LSD, p < 0.05).

Herbicides
All herbicides inhibited the development of the autochthonous Trichoderma strains but at different levels depending on each strain (Figures 8 and 9). T. citrinoviride did not grow or had a weak growth in the presence of all the herbicides analyzed, with Diquat showing the highest level of inhibition. T. harzianum and T. virens exhibited a lower development than the control, but their growth was not totally inhibited, with Pendimethalin causing the highest inhibition. In fact, this was the only herbicide able to reduce growth of T. velutinum, among those tested in the present work.

Herbicides
All herbicides inhibited the development of the autochthonous Trichoderma strains but at different levels depending on each strain (Figures 8 and 9). T. citrinoviride did not grow or had a weak growth in the presence of all the herbicides analyzed, with Diquat showing the highest level of inhibition. T. harzianum and T. virens exhibited a lower development than the control, but their growth was not totally inhibited, with Pendimethalin causing the highest inhibition. In fact, this was the only herbicide able to reduce growth of T. velutinum, among those tested in the present work.

Trichoderma Development and Groups of Pesticides
Analyzing the effect of the pesticide groups on the development of the autochthonous Trichoderma strains, it was observed that acaricides and insecticides reduced the development of the Trichoderma strains to a lesser extent than the other pesticides. However, herbicides significantly reduced the growth of these BCAs, as well as fungicides (Figure 10a).
Regarding the evaluation of the development of the autochthonous Trichoderma strains in the presence of pesticides, T. harzianum T019 and T. velutinum T028 showed greater development than T. virens T032 and T. citrinoviride T008, with less growth ( Figure  10b).

Trichoderma Development and Groups of Pesticides
Analyzing the effect of the pesticide groups on the development of the autochthonous Trichoderma strains, it was observed that acaricides and insecticides reduced the devel-opment of the Trichoderma strains to a lesser extent than the other pesticides. However, herbicides significantly reduced the growth of these BCAs, as well as fungicides (Figure 10a).

Discussion
At present, agriculture is imposing an integrated model of production. The use of chemical compounds is alternated with cultural measures, the application of resistant varieties and the use of agents of biocontrol in order to guarantee environmental sustainability. In a combined treatment of a chemical product and a biological agent, it is suitable to know how the former will affect the development of the latter. Therefore, in this research, the effect of different phytosanitary products on the development of several autochthonous Trichoderma strains was studied. There are different research works where the combined use of phytosanitary products and biological control agents produced different responses. This partnership can be positive-by improving the action of both-or negative, by inhibiting growth.
In the case of acaricides, their influence caused different responses in the Trichoderma strains. Abamectine, produced by Streptomyces avermitilis (ex Burg et al.) Kim and Goodfellow, is firmly fixed to the soil, and it is rapidly degraded by microorganisms [22,23]. The action of this compound was different on each Trichoderma strain. Thus, T. citrinoviride T008 and T. virens T032 had a lower level of growth than the control, which would presumably indicate that they are not able to degrade this compound, but T. harzianum T019 and T. velutinum T028 growth was not affected.
Deltamethrin is another insecticide-acaricide that eliminates Euseius spp., Amblyseius spp., Typhlodromus spp. and other phytoseids. Its affinity for soils is relatively high, with a half-life of 11-72 days [24][25][26]. This pyrethroid is readily degraded by microorganisms Regarding the evaluation of the development of the autochthonous Trichoderma strains in the presence of pesticides, T. harzianum T019 and T. velutinum T028 showed greater development than T. virens T032 and T. citrinoviride T008, with less growth (Figure 10b).

Discussion
At present, agriculture is imposing an integrated model of production. The use of chemical compounds is alternated with cultural measures, the application of resistant varieties and the use of agents of biocontrol in order to guarantee environmental sustainability. In a combined treatment of a chemical product and a biological agent, it is suitable to know how the former will affect the development of the latter. Therefore, in this research, the effect of different phytosanitary products on the development of several autochthonous Trichoderma strains was studied. There are different research works where the combined use of phytosanitary products and biological control agents produced different responses. This partnership can be positive-by improving the action of both-or negative, by inhibiting growth.
In the case of acaricides, their influence caused different responses in the Trichoderma strains. Abamectine, produced by Streptomyces avermitilis (ex Burg et al.) Kim and Good-fellow, is firmly fixed to the soil, and it is rapidly degraded by microorganisms [22,23]. The action of this compound was different on each Trichoderma strain. Thus, T. citrinoviride T008 and T. virens T032 had a lower level of growth than the control, which would presumably indicate that they are not able to degrade this compound, but T. harzianum T019 and T. velutinum T028 growth was not affected.
Deltamethrin is another insecticide-acaricide that eliminates Euseius spp., Amblyseius spp., Typhlodromus spp. and other phytoseids. Its affinity for soils is relatively high, with a half-life of 11-72 days [24][25][26]. This pyrethroid is readily degraded by microorganisms in soil [27,28]. In the present work, the growth of all Trichoderma strains was significantly reduced compared to the control.
Sulfur is widespread in agriculture as an acaricide but also as a fertilizer and fungicide. For example, its use is authorized to control mites, such as Tetranychus urticae Koch or eriophids, in addition to Powdery mildew. In our research, the growth of all Trichoderma strains analyzed was stimulated in presence of this compound. It should be pointed out that T. virens development was constantly greater in the presence of this compound than in the control and in all the points analyzed. Previous to this work, it has also been mentioned that Sulfur (2 g/L) significantly increased growth of T. virens and T. harzianum [29]. Similarly, it was also reported that the use of Sulfur at concentrations up to 500 µg/mL does not affect T. harzianum growth [30].
Pesticides are currently being taken off the market in the EU, but in other countries, their application is allowed. Jebakumar et al. [31] studied the compatibility of this pesticide with T. harzianum in vitro and in soil, and they observed that Chlorpyriphos could be safely applied with T. harzianum for the management of diseases, nematodes or insects. Suseela Bhai and Thomas [32] observed an inhibition of under 8% in T. harzianum with this compound in in vitro conditions. In our study, all the strains were inhibited by Chlorpyrifos, with growth diameters less than 30 mm in in vitro conditions.
Pirimicarb is a carbamate with specific activity for the control of aphids. It is systemic, slightly residual and penetrates through the leaves or is absorbed by the roots and translocated through the xylem. Widenfalk et al. [33] observed that pesticides, such as Deltamethrin, Isoproturon or Pirimicarb, induced toxic responses in microbial communities at concentrations that were predicted to be environmentally safe. In another study, Trichoderma strains were recorded to efficiently degrade Pirimicarb [34,35]. Unfortunately, in our research, this insecticide inhibited the development of all autochthonous Trichoderma strains, with T. citrinoviride T008 showing the lowest growth.
Another evaluated insecticide was Imidacloprid. It acts systemically as an antagonist of nicotinic acetylcholine receptors. Some studies focused on its application by foliar spraying, but they are highly toxic to honeybees [36], and residues of this compound were detected in samples from nectar, pollen, plant tissues and soils [37,38]. There are some studies about the combination of this pesticide with BCAs, e.g., combinations of Imidacloprid with entomopathogenic nematodes or with fungi, such as B. bassiana or Metarhizium anisopliae (Metschn). Sorokīn showed increased insect parasitism [39][40][41]. In the current study, this insecticide produced an inhibition in the development of T. citrinoviride T008 and T virens T032, on the seventh day. However, the growth of T. harzianum T019 and T. velutinum T029 was not affected, and they could be good candidates for combination with this insecticide for the treatment of insect pests. Nevertheless, concentrations of imidacloprid must be optimized to avoid its toxicity against honeybees.
B. bassiana is an entomopathogenic fungus, which infects the insect by adhering to its cuticle through fungal adhesion proteins [42]. It has been used to control some insects belonging, among others, to the Coleoptera and Lepidoptera orders. Daza et al. [43] observed that a combination of B. bassiana and Trichoderma lignorum spores was a viable alternative for the control of the leafcutter ant (Atta cephalotes L.). In our research, Trichoderma development was not inhibited after seven days of incubation, without significant differences with respect to the control plates.
Strobilurin-like fungicides, such as Azoxystrobin, are economically important fungicides that are used against a wide range of fungal-related plant diseases. These compounds can be used alone or in conjunction with BCAs. In a previous research work [44], Bacillus subtilis H158, in combination with fungicides of this family (Azoxystrobin, Trifloxystrobin, Pyraclostrobin, etc.), reduced the severity of rice sheath blight caused by Rhizoctonia solani J.G. Kühn. Difenoconazole is another triazole fungicide that acts by inhibiting the ergosterol biosynthesis through plant systemic response. A study carried out by Pinto et al. [45] showed that this compound was degraded to levels ranging from 51.3 to 72.1% by T. viride and Fusarium oxysporum Schltdl., respectively. In the present research, all Trichoderma species were affected by azoxystrobin and difenoconazole until the fifth day of growth. However, after the seventh day, their development was not inhibited and matched the control.
Other fungicides used in this research were Chlorothalonil, Mancozeb, Thiophanatemethyl and Thiram. They are fungicides with both preventive and curative activity for the control of a number of diseases in all types of crops. Malandrakis et al. [46] observed that Fusarium solani FsK-an isolate used as BCA-was highly insensitive to Thiophanatemethyl and Mancozeb, with an effective concentration as fungicide exceeding 100 mg/mL in in vitro conditions. In another study, some Trichoderma strains decreased the conidia production in presence of Mancozeb [47], but in the presence of this compound in the rhizosphere, the soil bacterial communities increased [48]. In the current research, these compounds reduced the growth in all strains. This was an expected result because these are broad-spectrum fungicides, which have been used for a long time to control fungal diseases. With regard to Thiram, only T. citrinoviride T008 was inhibited in its development. These results agree with previous data [49], indicating that some Trichoderma strains showed low sensitivity to Thiram.
Copper, just like Sulfur, is used as a fertilizer and fungicide. The presence of certain concentrations can cause different responses in the BCA. Banik and Pérez-de-Luque [50] observed that a T. harzianum isolate had more sporulation in the presence of Copper. In other research, Lal et al. [51] observed that eggplant seedlings immersed in a solution with oxychloride-dipped Copper and neem cake colonized with T. harzianum minimized the wilt incidence compared to some fungicides or BCA used separately. However, in our study, all autochthonous Trichoderma strains were affected in their growth by the presence of Copper oxide, which was a contrary result to that observed in other studies where sporulation of some Trichoderma strains was not affected by this compound [32,52]. In our research, all strains sporulated after seven days of growth.
Tebuconazole is a broad-spectrum triazole fungicide used worldwide in agriculture for disease control. It has a relatively high soil organic carbon-water binding coefficient and a half-life in soil ranging between 9 and 600 days under aerobic conditions [53,54]. In our study, the growth of autochthonous Trichoderma strains was inhibited compared to the control, which would represent a problem for their application as BCA in the presence of this compound. Interestingly, some studies indicate that, after several days following the application of this compound, the soil bacteria population increased significantly at all concentrations assayed compared to the control [55][56][57][58]. This stimulating effect of fungicides could be associated with an increased level of nutrients and energy sources released from the death of fungal hyphae or to a decrease in organisms competing for resources.
While discussing herbicides, we investigated Diquat, a desiccant-, Glyphosate, a systemic non-selective and a persistent, such as Pendimethalin. Unfortunately, T. citrinoviride T008 was inhibited by all these compounds. Regarding Diquat, Eberbach and Douglas [59] observed that Rhizobium sp. development was significantly retarded by all concentrations of this compound. In our work, T. harzianum T019, T. velutinum T028 and T. virens T032 were not inhibited by this herbicide. Meanwhile, Glyphosate-a non-specific organophosphate pesticide widely applied in weeds-binds to soil particles accumulating in the upper soil layer, often being detected in groundwater and surface water [60][61][62]. Glyphosate can also cause structural changes in local soil microbial communities by inhibiting the growth of soil microorganisms and facilitating the increase in phytopathogenic fungi in the soil [63][64][65]. In our study, T. velutinum T028 was not affected by this compound, while T. harzianum T019 and T. virens T032 were slightly inhibited. Regarding Pendimethalin, which is a dinitroaniline herbicide with residual activity that persists for 3-4 months, some previous research found that, when applied with Trichoderma, neither Pendimethalin nor any of the antagonists showed any mycelial radial growth inhibition in the presence of the herbicide at field doses [49,66]. However, in our study, all Trichoderma strains were inhibited on all analyzing days, except T. velutinum T028, which was only affected by this herbicide.

Conclusions
Fungicides, acaricides, insecticides (including the entomopathogenic fungus B. bassiana) and herbicides were checked in autochthonous Trichoderma strains. Even when all pesticides affected Trichoderma development, Sulfur encouraged the growth of all autochthonous strains assayed, and the combination Trichoderma-B. bassiana did not disturb their growth. So, the combination of the autochthonous Trichoderma strains with these compounds will be a positive strategy to apply in the field and for controlling pests and some diseases.
As expected, all the tested fungicides (except Azoxystrobin + Difenoconazole) inhibited the development of all autochthonous Trichoderma strains. However, all herbicides inhibited the development of all autochthonous Trichoderma strains, demonstrating that an excessive use of these pesticides not only affects weeds but also the microorganisms that live the rhizosphere.
In short, it would be necessary to control the use of conventional pesticides, considering that, in general, they could reduce the development of autochthonous Trichoderma strains, especially fungicides and herbicides. Funding: This research was funded by Junta de Castilla y León, Consejería de Educación for the project "Application of Trichoderma strains in sustainable quality bean production" (LE251P18), cofinanced by the European Social Fund, and the grants to Alejandra Porteous Álvarez (FPU19/03650) of Ministerio de Ciencia, Innovación y Universidades (Spain) and to Guzmán Carro Huerga (FPU 15/04681) of Ministerio de Educación, Cultura y Deporte (Spain).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.