Use of Biological and Chemical Pesticides in Agricultural Production: What Fate for Entomopathogenic Fungi?

Abstract

In the context of integrated pest management, the compatibility between the products used and even with the natural enemies of crop pests should still be elucidated. In this study, interviews were done with about 60 coffee growers to assess the use of pesticides in the protection of coffee berries. Then, in vitro tests were used to evaluate the effects of extracts of the seed powders of Thevetia peruviana, Azadirachta indica, the chlorpyriphos-ethyl insecticide and the chlorothalonil + dimethomorph fungicide, on the development parameters of the entomopathogenic fungus Beauveria bassiana, the natural enemy of the coffee berry borer, Hypothenemus hampei. The said tests consisted of the method of poisoning the culture medium with pesticides, observation and counting of spores under the optical microscope. The analysis of the collected data showed that depending on the type and severity of the pest pressure, growers apply several types of mainly chemical pesticides. Among the pesticides tested, extracts of T. peruviana, A. indica and chlorpyriphos-ethyl considerably reduced the development of B. bassiana. These results show that in crop protection, the use of biological or chemical substances should be done in a judicious way, to ensure the conservation and the valorization of natural enemies of phytosanitary pressures.

1. Introduction

Some studies point out that today’s, food and agricultural systems have succeeded in supplying large quantities of food to global markets with high use of chemical inputs, but are degrading land, water, ecosystems, biodiversity and human health [1]. To address the increasing use of chemical pesticides in agricultural production, a fundamentally different model of agriculture is now required, one that reduces or rejects chemical inputs, optimizes biodiversity, and stimulates interactions between different species [2]. Pesticidal plants and entomopathogenic fungi, both gifts of nature, are effective alternatives to chemical pesticides in the integrated management of crop pests and diseases [3, 4, 5, 6]. In this sense, several authors have already proven the effectiveness of plants of the genera Azadirachta (Neem) and Thevetia (Yellow Laurel) [7, 8], as well as biological control agents of the genera Beauveria and Metarhizium [9, 10, 11, 12]. However, the introduction of these exotic pests is the most widely used approach in agriculture, at the expense of conserving and valorizing existing biological control agents in the agroecosystem [13, 14, 15, 16], and without verification of their compatibility.

Given the effectiveness of pesticidal plant extracts and biological pest control agents in pest management, could they be used synergistically? This probably requires compatibility [17] which has been assessed so far by a few disparate studies [18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37]. These cited studies focus on the compatibility or synergy between chemical pesticides and entomopathogenic fungi. In view of the rareness of studies on the compatibility between biological pesticides (pesticidal plants and entomopathogenic fungi), and our previous research on the use of B. bassiana and extracts of Thevetia peruviana and Azadirachta indica in the control of the coffee berry borer (Hypothenemus hampei Fer.), this study was initiated to provide some information. Specifically, the aim is to evaluate the compatibility between extracts of T. peruviana and A. indica seed powders, the chlorpyrifos-ethyl insecticide and the chlorothalonil + dimethomorph fungicide, on the development parameters of the entomopathogenic fungus B. bassiana, in the control of phytosanitary pressures on coffee.

2. Methodology

The biological material consisted of Thevetia peruviana seed powder, Azadirachta indica seed powder and oil, and two isolates of Beauveria bassiana. Both strains were isolated from composite soil samples of Arabica (Bb-IRAD.Fbt) and Robusta (Bb-IRAD.Nkoe) coffee plantations [12], and stored at the Central Laboratory of Phytopathology (CLP) of the Mbalmayo Agricultural Research Centre (MARC) of the Institute of Agricultural Research for Development (IRAD), Cameroon. Compatibility tests were carried out in the same laboratory. The effects of the plant extracts on B. bassiana were compared to those caused by synthetic pesticides. These synthetic pesticides consisted of the chemical insecticide, Pyriforce composed of chlorpyriphos-ethyl 600 g/l as active substance, and the chemical fungicide, Sphinx composed of chlorothalonil 400 g/kg + dimethomorph 80 kg as active substances.

The study was carried out in the localities of Melong, Bamendjou and Doumé where the producers surveyed were selected. These localities were chosen because they are among the major Arabica (Bamendjou) and Robusta (Melong and Doumé) coffee production basins in Cameroon, are accessible by national roads and are located in three different agroecological zones. At the farm level, farmers were surveyed to assess pest pressures and control strategies.

In the CLP and the Central Laboratory of Entomology (CLE) of the IRAD, insect rearing, fungus isolation and efficacy tests on the bark beetle and compatibility tests between plant extracts and B. bassiana were performed.

2.1 Evaluation of pest and disease control strategies in coffee farms

The different strategies for regulating phytosanitary constraints and improving yields were evaluated through surveys. The experimental unit consisted of sixty-three farmers surveyed, divided into twenty-two farmers in Bamendjou, twenty-one farmers in Melong and twenty farmers in Doumé. The selection of farmers was done using the referral sampling technique (non-probability sampling method), where a farmer refers to an individual with a farm. Thus, farmers with at least one dependent coffee farm were chosen as the basic sampling unit. Semi-structured comprehensive interviews were conducted using a pre-established open-ended questionnaire. The questionnaire included semi-structured questions that allowed for the collection of information on diseases and pests affecting coffee farms, as well as strategies and techniques to control these pests.

2.2 Obtaining the different concentrations of biological and chemical pesticides

The plant extracts were applied to each strain on the basis of four concentrations: C₁ (12.5), C₂ (25), C₃ (50) and C₄ (100) in mg/ml for the aqueous extracts and in μl/ml for the oil. The plant extracts were prepared and the tested doses were obtained according to the method used in previous studies [8, 38, 39]. The choice of used doses was based on the proof of their efficacy tested in some studies on Phytophtora megakarya, Sahbergella singularis, Lasiodiplodia theobromae and Fusarium sp. [7, 40]. Each treatment in the trial was repeated five times. The chemical fungicide and insecticide were used as positive controls (C₀₊) at the manufacturers’ recommended doses. The negative/absolute control (C₀₋) was simply the PDA culture medium.

The recommended doses for the chemical insecticide and fungicide are 1 l/ha (50 ml for a 16-liter sprayer) and 3.33 g/l water, respectively. Petri dishes were prepared by taking 7 ml of the stock solution of each pesticide, and mixing it with 93 ml of PDA medium for a final volume of 100 ml of each product. This final volume was poured into five Petri dishes serving as replicates, 20 ml per dish. The different Petri dishes were placed in an incubation room under conditioned air at a temperature of T = 25°C and a humidity of ψ = 60%.

2.3 Effect of treatments on germination of Beauveria bassiana

To assess spore germination, 10 ml of spore solution was prepared by mixing spores from pure cultures of B. bassiana (twenty-one days old) with sterile distilled water and 1% tween 80. After homogenization with a magnetic stirrer, the solution was calibrated using the Malassez cell at the concentration of 1 × 10⁶ spores/ml [41]. Then, three drops of each solution were individually placed in three different locations of five Petri dishes containing PDA medium, and covered with a coverslip. The plant extracts were calibrated at four concentrations (C₁, C₂, C₃ and C₄), the positive controls at one concentration each (C_0 + 1, C_0 + 2) and the absolute/negative control (C₀₋). For each strain (Bb-IRAD.Nkoe and Bb-IRAD.Fbt), the five prepared Petri dishes were incubated in the dark at room temperature for 16 hours. After incubation, the different plates were observed under a light microscope for the enumeration of germinated and ungerminated spores; this was repeated three times. Any spore with an elongated germ tube was considered as germinated and viable spore. The germination rate of the spores was calculated using the following formula from [42]:

$$\mathit{GR}\left(\%\right)=\frac{A}{A+B}\times 100$$E1

where: GR = spore germination rate; A = number of germinated spores; B = number of ungerminated spores.$\frac{}{}\mathrm{d}1+\mathrm{d}22$.

2.4 Effect of treatments on radial growth of Beauveria bassiana

To assess the radial growth of B. bassiana isolates, a 6 mm diameter mycelial disc was taken (from 21-day-old pure cultures of Bb-IRAD.Nkoe and Bb-IRAD.Fbt) and placed in the center of each Petri dish containing media supplemented with the different plant extracts and chemical pesticides. A negative control not supplemented with extracts and chemical pesticides was also prepared. Each treatment was repeated 5 times, each repetition corresponding to one Petri dish. Petri dishes were incubated at a temperature of T = 25°C, a humidity of ψ = 60% under a photoperiod of 12/12 and for 21 days. Using the perpendicular line method, each diameter or line was measured daily. The average of the two perpendicular measurements, subtracted from the diameter of the starting explant, was the measure of radial growth of the fungus. It was obtained using the following formula [43]:

$$\mathit{RG}=\frac{d1+d2}{2}-d0$$E2

where: RG = Radial Growth; d₁ = first growth diameter (cm); d₂ = second growth diameter (cm); d₀ = diameter of the deposited explant (cm).

2.5 Effect of treatments on Beauveria bassiana spore production

The Petri dishes used in the evaluation of radial growth of B. bassiana, were used to evaluate the effects of treatments on spore production. Thus, a quantity of 10 ml of spore suspension of each isolate and each concentration of the different treatments was prepared as in the evaluation of the germination of isolates. In order to quantify the number of conidia produced by the fungus, five 1 ml samples of the suspension were successively taken from each dish and placed on the Malassez cell. The conidia were counted under a light microscope and the average number of conidia per observation was recorded.

2.6 Correlative and comparative assessment of compatibility between biological pesticides, chemical pesticides and Beauveria bassiana

The correlative and comparative assessment of compatibility was done using the percentages of inhibition or reduction of mycelial growth, germination and spore production of B. bassiana [44]. These percentages were calculated according to the following formula [45]:

$$\mathit{IP}\mathit{\text{or}}\mathit{RR}\left(\%\right)=\frac{\mathit{Vt}-\mathit{Vx}}{\mathit{Vt}}\times 100$$E3

where: IP or RR = inhibition percentage or reduction rate; Vt = value of growth diameter, germination rate or number of conidia produced estimated on control medium; Vx = value of growth diameter, germination rate or number of conidia produced estimated in the presence of the extract or fungicide tested.

2.7 Statistical analysis

The experimental set-up adopted for the incubation of the Petri dishes was a completely randomized set-up with: two strains of B. bassiana; two aqueous extracts (aqueous extract of A. indica and T. peruviana seed powder) and an oil cold extract of A. indica seeds; and two chemical pesticides.

Microsoft Excel spreadsheet software was used to create the databases and XLSTAT 2014 software was used for statistical analysis. The experimental test data were first subjected to a Shapiro–Wilk normality test [46], followed by a square root transformation (radial growth and number of spores germinated or produced) or an ArcSin angular transformation (inhibition rate) [47, 48]. The sampled and transformed data underwent descriptive analysis, a general linear regression model with analysis of variance (ANOVA), followed by the multiple comparison test of means at 5% risk (α).

After these calculations, the inhibitory effect of the tested products was classified firstly by means of hierarchical ascending classification (HAC). This allowed treatments with the same effect to be grouped together. Secondly, the different groups were categorized on the basis of a list of inhibition levels [49], where:

• very toxic = >80% inhibition;

• toxic = 10–79% inhibition;

• low toxic or compatible = <10% inhibition.

At the end of each analysis, all interpretations and conclusions were drawn at the transformed scale, but the results presented were converted back to the original units [48, 50].

3. Results

3.1 Weed, disease and pest control strategies

Clearing of coffee fields and structural/sanitary pruning of coffee trees were carried out by 100% of farmers in each locality. Insecticides were applied by 100%, 80% and 59.1% of farmers in Melong, Doumé and Bamendjou, respectively. Fungicides were applied in Bamendjou by a significantly higher proportion of farmers (86.36%) than in other localities such as Doumé (5% of farmers). Fertilizers were widely applied in Melong and Bamendjou by 85.71 and 54.55% of farmers, respectively. Herbicides were applied in all localities by more than half of the farmers, but this percentage was significantly lower (59.1% of farmers) in Bamendjou (Fisher’s exact test at 0.05 significance level) (Figure 1).

Figure 1.

Practices applied to manage pests and diseases in coffee farms. Values shown with the symbol < or > in bold are significant according to Fisher’s exact test at the 0.05 significance level.

3.2 Effects of treatments on Beauveria bassiana spore germination

The results showed that spore germination of both B. bassiana isolates was reduced by the different treatments and concentrations used. Compared to the absolute control, the percentage of germination was higher in Bb-IRAD.Nkoe than in Bb-IRAD.Fbt at concentrations C₁, C₂ and C₃, respectively for reduction rates ranging from 54 to 86% for AEAI on Bb-IRAD.Nkoe against 46 to 84% for AETP on Bb-IRAD.Fbt. Thus, at C₄ concentrations of these extracts, reduction rates of 100% were observed. This rate is identical to that obtained with chlorpyrifos-ethyl, hence no significant difference (Figure 2B).

Figure 2.

Germination and germination inhibition rates of conidia under the influence of plant extract, chemical fungicide and insecticide treatments. AEAI = Azadirachta indica aqueous extract; AETP = Thevetia peruviana aqueous extract; OEAI = Azadirachta indica oil extract; pyriforce = chemical insecticide composed of chlorpyrifos-ethyl; sphinx = chemical fungicide composed of chlorothalonil + dimethomorph; 1, 2, 3 and 4 correspond to C₁ (12.5), C₂ (25), C₃ (50) and C₄ (100) in mg/ml for the aqueous extracts and in μl/ml for the oil extract. *Means with the same letter are not significantly different at the 0.05 level according to Fisher’s test.

With the A. indica oil extract (OEAI), the germination rate was lower for Bb-IRAD.Nkoe than for Bb-IRAD.Fbt at concentrations C₁ and C₂ (86 and 77% versus 89 and 85% respectively). Furthermore, this extract totally inhibited germination (0% germination rate) of spores at concentrations C₃ and C₄. Nevertheless, Fisher’s test showed no significant difference between germination rates at these concentrations and that of chlorpyrifos-ethyl (Figure 2A).

Tests with the chlorothalonil + dimethomorph fungicide showed little or no reduction in spore germination of both isolates. The lowest germination inhibition rates (7 and 6%) were recorded for Bb-IRAD.Nkoe and Bb-IRAD.Fbt, respectively. Nevertheless, Fisher’s test showed significant differences between the germination inhibition rates recorded with this fungicide and those with the negative control (60.33 and 56.33% for Bb-IRAD.Nkoe and Bb-IRAD.Fbt, respectively) (Figure 2A).

Finally, with chlorpyrifos-ethyl, AETP₄, AEAI₄ and OEAI₃, a germination inhibition rate of 100% was recorded in both B. bassiana isolates. However, for the same concentrations, AETP had more effect on spore germination of both isolates than AEAI (Figure 2B).

3.3 Effects of treatments on radial growth of Beauveria bassiana isolates

The aqueous extract of A. indica seed powder inhibited the growth of both isolates at all concentrations. This inhibition of radial growth was proportional and significant at the concentrations tested in both isolates (Figure 3). Thus, a total inhibition (100%) of radial growth was observed with AEAI₄ in both isolates and only in Bb-IRAD.Fbt with AEAI₃ (Table 1).

Figure 3.

Inhibition of mycelial growth of bb-IRAD.Nkoe by aqueous extract of Azadirachta indica on day 21 of growth. N = neem or Azadirachta indica; _N = Nkoemvone isolate; 1, 2, 3 and 4 correspond to C₁ (12.5), C₂ (25), C₃ (50) and C₄ (100) in mg/ml for the aqueous extracts and in μl/ml for the oil extract; C₀ = sterile distilled water + tween 80.

Applied Treatment	Average growth diameter (cm)		Inhibition rate (%)
	Beauveria bassiana isolates
	Foumbot	Nkoémvone	Foumbot	Nkoémvone
AEAI1	0.47 ± 0.25^h	2.14 ± 0.37^k	94.36 ± 2.94^fg	47.09 ± 1.49^d
AEAI2	0.33 ± 0.20^ef	1.52 ± 0.33^j	96.48 ± 2197^ghij	64.10 ± 3.67^e
AEAI3	0 ± 0^a	0.39 ± 0.19^fg	100 ± 0^m	94.18 ± 1.86^fg
AEAI4	0 ± 0^a	0 ± 0^a	100 ± 0^m	100 ± 0^m
AETP1	0.32 ± 0.17^ef	0.57 ± 0.26^i	96.31 ± 2.01^ghij	92.79 ± 3.35^f
AETP2	0.20 ± 0.13^cd	0.36 ± 0.21^fg	98.08 ± 1.48^ijkl	96.39 ± 2.81^ghi
AETP3	0.14 ± 0.1^bc	0.21 ± 0.13^cd	98.86 ± 1.08^klm	97.98 ± 1.57^ijkl
AETP4	0 ± 0^a	0.08 ± 0.09^ab	100 ± 0^m	99.66 ± 0.86^lm
OEAI1	0.12 ± 0.14^bc	0.41 ± 0.28^gh	99.46 ± 1.41^klm	96.20 ± 3.69^gh
OEAI2	0.08 ± 0.09^ab	0.27 ± 0.19^de	99.66 ± 0.88^lm	97.73 ± 2.37^hijk
OEAI3	0.05 ± 0.07^ab	0.18 ± 0.12^c	99.82 ± 0.59^lm	98.58 ± 1.60^jkl
OEAI4	0 ± 0^a	0 ± 0^a	100 ± 0^m	100 ± 0^m
Control	4.22 ± 0.59^n	4.21 ± 0.60^n	0 ± 0^a	0 ± 0^a
Pyriforce	0 ± 0^a	0 ± 0^a	100 ± 0^m	100 ± 0^m
Sphinx	3.40 ± 0.53^m	3.31 ± 0.52^l	16.27 ± 3.50^b	21.03 ± 1.32^c
Analysis of Variance	R^2 = 0.993; F = 295.936; Pr < 0.0001		R^2 = 0.985; F = 133.447; Pr < 0.0001

Table 1.

Radial growth and inhibition rate of Beauveria bassiana isolates according to treatments after 21 days of incubation.

AEAI = Aqueous extract of Azadirachta indica; AETP = Aqueous extract of Thevetia peruviana; OEAI = Oil extract of Azadirachta indica; Pyriforce = Insecticide composed of Chlorpyriphos-ethyl; Sphinx = Fungicide composed of Chlorothalonil + Dimethomorph; 1, 2, 3 and 4 correspond to C₁ (12.5), C₂ (25), C₃ (50) and C₄ (100) in mg/ml for the aqueous extracts and in μl/ml for the oil extract. * Means with the same letter are not significantly different at the 0.05 level according to Fisher’s test.

The inhibition of mycelial growth of B. bassiana isolates in the presence of AEAI and AETP was significant and proportional to the concentrations tested compared to the negative control (0 ± 0). This inhibition was relatively stronger in Bb-IRAD.Fbt with both types of aqueous extracts than in Bb-IRAD.Nkoe with the aqueous extract of A. indica. In contrast to the negative control, Fisher’s test showed no significant difference between the two aqueous extracts and chlorpyrifos-ethyl at the AETP₄ concentration (Table 1).

With OEAI the inhibition of mycelial growth of isolates was more pronounced at all concentrations. No significant difference was observed between the oil extract of A. indica and chlorpyrifos-ethyl in the two B. bassiana isolates (Table 1).

With the Chlorothalonil + Dimethomorph complex, a very small reduction in the growth of both B. bassiana isolates was observed (Figure 4). Thus, low percentages of inhibition were observed (16.27 ± 3.50 and 21.03 ± 1.32%) in Bb-IRAD.Fbt and Bb-IRAD.Nkoe, respectively. However, Fisher’s test showed a significant difference between these percentages of inhibition and that caused by the negative control (Table 1).

Figure 4.

Mycelial growth of the two isolates of Beauveria bassiana under the effect of sphinx and pyriforce. A and a’ = control; b and b’ = sphinx (chlorothalonil + dimethomorph); c and c’ = pyriforce (chlorpyriphos-ethyl).

3.4 Effects of treatments on Beauveria bassiana spore production

The test results revealed that there was very little influence of Sphinx on spore production of both B. bassiana isolates. Consequently, sporulation reduction rates were higher with Bb-IRAD.Fbt than with Bb-IRAD.Nkoe regardless of the treatment. The aqueous extracts (AEAI and AETP) of both plants caused 100% reduction rates in both isolates only with C₄, while 100% reduction was observed with OEAI₃ and OEAI₄. However, all treatments except the negative control caused more than 50% reduction in sporulation in both isolates (Figure 5B).

Figure 5.

Average number of conidia produced and sporulation inhibition rate in each treatment. AEAI = aqueous extract of Azadirachta indica; AETP = aqueous extract of Thevetia peruviana; OEAI = oil extract of Azadirachta indica; pyriforce = insecticide composed of chlorpyriphos-ethyl; sphinx = fungicide composed of chlorothalonil + dimethomorph; 1, 2, 3 and 4 correspond to C₁ (12.5), C₂ (25), C₃ (50) and C₄ (100) in mg/ml for the aqueous extracts and in μl/ml for the oil extract. *means with the same letter are not significantly different at the 0.05 level according to Fisher’s test.

Finally, spore production in both B. bassiana isolates was relatively low with all OEAI concentrations. The absence of spore production was noted with OEAI₃ and OEAI₄ in both isolates. Bb-IRAD.Fbt showed spore production with OEAI₁ and OEAI₂, but this was not significant compared to that with OEAI₃, OEAI₄ and pyriforce (Figure 5A).

3.5 Relationship between treatments and inhibition of Beauveria bassiana development

The hierarchical ascending classification (HAC) of the inhibition rates of B. bassiana by the different treatments shows that all the treatments, AETP, OEAI, pyriforce and AEAI were very toxic (inhibition between 98.31 or 92.79%) for the radial growth of both isolates. Only AEAI₁ and AEAI₂ were just toxic (inhibition between 64.10 and 47.09%) to Bb-IRAD.Nkoe. Water and sphinx were compatible with both isolates (Table 2).

Classification variable	Classes	Barycentre (%); [Interval]	Treatment-Isolate	Classification status
Inhibition rate of radial growth (%)	1	98,190; [0,117—5398]	AEAI1-Fbt; AEAI2-Fbt; AEAI3-Fbt; AEAI4-Fbt; AETP1-Fbt; AETP2-Fbt; AETP3-Fbt; AETP4-Fbt; OEAI1-Fbt; OEAI2-Fbt; OEAI3-Fbt; OEAI4-Fbt; Pyriforce-Fbt; AEAI3-Nk; AEAI4-Nk; AETP1-Nk; AETP2-Nk; AETP3-Nk; AETP4-Nk; OEAI1-Nk; OEAI2-Nk; OEAI3-Nk; OEAI4-Nk; Pyriforce-Nk	Very Toxic
	2	9325; [6942—11,709]	Control-Fbt; Sphinx-Fbt; Control-Nk; Sphinx-Nk	Compatible
	3	55,597; [8507—8507]	AEAI1-Nk; AEAI2-Nk	Toxic
Inhibition rate of spore germination (%)	1	53,301; [0,249—7445]	AEAI1-Fbt; AEAI1-Nk; AEAI2-Nk	Toxic
	2	90,484; [1726—19,478]	AEAI2-Fbt; AEAI3-Fbt; AEAI4-Fbt; AETP1-Fbt; AETP2-Fbt; AETP3-Fbt; AETP4-Fbt; OEAI1-Fbt; OEAI2-Fbt; OEAI3-Fbt; OEAI4-Fbt; Pyriforce-Fbt; AEAI3-Nk; AEAI4-Nk; AETP1-Nk; AETP2-Nk; AETP3-Nk; AETP4-Nk; OEAI1-Nk; OEAI2-Nk; OEAI3-Nk; OEAI4-Nk; Pyriforce-Nk	Very Toxic
	3	3008; [2516—3500]	Control-Fbt; Sphinx-Fbt; Control-Nk; Sphinx-Nk	Compatible
Inhibition rate of Sporulation (%)	1	76,130; [1143—23,189]	AEAI1-Fbt; AEAI2-Fbt; AETP1-Fbt; AETP2-Fbt; AETP3-Fbt; OEAI1-Fbt; OEAI2-Fbt; AEAI1-Nk; AEAI2-Nk; AEAI3-Nk; AETP1-Nk; AETP2-Nk; AETP3-Nk; OEAI1-Nk; OEAI2-Nk	Toxic
	2	100,000; [0–0]	AEAI3-Fbt; AEAI4-Fbt; AETP4-Fbt; OEAI3-Fbt; OEAI4-Fbt; Pyriforce-Fbt; AEAI4-Nk; AETP4-Nk; OEAI3-Nk; OEAI4-Nk; Pyriforce-Nk	Very Toxic
	3	2737; [1185—4288]	Control-Fbt; Sphinx-Fbt; Control-Nk; Sphinx-Nk	Compatible

Table 2.

Hierarchical ascending classification of treatments according to their inhibition of growth parameters of Beauveria bassiana isolates.

Fbt = Bb-IRAD.Fbt; Nk = Bb-IRAD.Nkoe; AEAI = Aqueous extract of Azadirachta indica; AETP = Aqueous extract of Thevetia peruviana; OEAI = Oil extract of Azadirachta indica; Pyriforce = Insecticide composed of Chlorpyriphos-ethyl; Sphinx = Fungicide composed of Chlorothalonil + Dimethomorph; 1, 2, 3 and 4 correspond to C₁ (12.5), C₂ (25), C₃ (50) and C₄ (100) in mg/ml for the aqueous extracts and in μl/ml for the oil extract.

Spores germination of the isolates was impacted by most of the treatments AETP, OEAI, pyriforce and AEAI which were highly toxic (90% inhibition on average) except AEAI₁ for Bb-IRAD.Fbt, AEAI₁ and AEAI₂ for Bb-IRAD.Nkoe which were toxic (50% inhibition on average) for these isolates. Water and sphinx were compatible with spore germination (Table 2).

As for sporulation, the high concentrations 3 and 4 of OEAI (for both isolates), 3 and 4 of AEAI (for Bb-IRAD.Fbt), 4 of AETP (for both isolates) and pyriforce were very toxic by totally (100%) inhibiting spore production. The low concentration C₁ of all treatments was found to be toxic for both isolates, however, concentrations 3 of AETP (for both isolates) and AEAI (for Bb-IRAD.Fbt) were also in this toxic class with inhibition ranging from 77.27 to 52.94%. Sterile distilled water and sphinx were found to be compatible with both isolates (Table 2).

4. Discussion

Coffee farmers use several strategies to cope with pest pressures. These practices are applied or not depending on the constraints encountered, their period of occurrence, incidence and severity [51]. Unfortunately, this study found that all the products used to control pests were synthetic products. In all study sites, there is a need to use organic products for quality coffee production and to ensure protection of human and environmental health. However, the unavailability of organic products in the market, low knowledge of their use and historical dependence on synthetic products complicate the adoption of organic products [52] and inexorably push producers towards synthetic products.

The products tested in vitro, with the exception of chlorothalonil + dimethomorph, significantly reduced germination, mycelial growth and spore production of the B. bassiana isolates used. Thus, the application of this fungicide to control fungal diseases such as anthracnose of coffee berries, allows the conservation of the natural inoculum of B. bassiana in the field and a synergistic control of phytosanitary pressures on berries. This finding corroborates with the results of some works [53, 54, 55] who respectively showed that sulfur, copper oxychloride and strobilurin fungicides are compatible with B. bassiana isolates although their primary faculty is antifungal.

All OEAI, AETP and AEAI treatments were found to be toxic to both isolates. This toxicity of the extracts of both plants to B. bassiana, is thought to be due to terpenes, phenols, alcohols, alkaloids, tannins and other secondary metabolites (capable of inducing toxicity of cell walls, membranes and organelles [56, 57]. A conservation of B. bassiana spores remains hypothetical in the presence of these pesticidal plant extracts (PPE) because they prevent conidial germination, a very important step in pest control with CEP. Indeed, the onset of the epizootic is conditioned by the ability of these conidia to germinate on the host [58]. Similarly, the success of CEP depends on the viability of its spores [44], which is therefore threatened by PPE in this study. These results are similar to other studies [22, 59, 60, 61, 62] which showed that A. indica oil extracts and azadirachtin 5EC (commercial biopesticide) at the recommended dose were incompatible with B. bassiana. Similarly, Margoside® (commercial formulation based on 0.3% neem oil) and neem extracts have been shown to delay in vitro spore germination of 23 isolates (out of 30 in total) of B. bassiana, but without significantly reducing it [23, 33].

However, results of some works have shown that neem oil (2.5%) and neem seed extracts, neem gold, Topneem, biospark and exodon (commercialized biological pesticides), show compatibility with all B. bassiana isolates obtained in these works [26, 32, 53, 63, 64]. Furthermore, some studies [65, 66] have shown that the combination of Azadirachtin (neem extract) with B. bassiana, has an additive effect. Similarly, a synergistic efficacy of A. indica leaf extracts and Azadirachtin (AzaMax; 200 ml × 100 l⁻¹) with B. bassiana has been proven respectively in the control of wheat aphids [67] and in the control of Plutella xylostella (L.) (Lepidoptera: Plutellidae) [68].

These contradictory results could be due to the qualitative and quantitative variability of the extracts used [69] by the different authors and to the genetic variability of the B. bassiana isolates used in this study [23]. It is therefore evident that the compatibility of plant protection products depends mainly on the nature of the compounds, the concentrations used and the nature of the isolates. Furthermore, studies have shown that commercial strains of B. bassiana are less resistant than wild strains possibly due to the effect of the products used for encapsulation/formulation [70, 71]. Further research on the effects of neem on the enzymatic activity of B. bassiana could be interesting to decide on these contradictions [72].

Apart from plant extracts, chlorpyrifos-ethyl at the recommended dose was also shown to be incompatible with B. bassiana by inhibiting all developmental stages of both isolates. This corroborates with some studies [53, 73, 74, 75] who reported that triazophos, chlorpyriphos and endosulfan formulations inhibited 100% of B. bassiana germination at all doses tested. This inhibition is due to the ability of the chemical insecticide to act as an acetylcholine esterase inhibiting neurotoxin [4].

Finally, a comparative look at the two isolates showed that Bb-IRAD.Fbt was more affected by EPP than Bb-IRAD.Nkoe. This further confirms that these two isolates are different hence their reactivity was variable to the applied pesticides. The different membrane and intracellular receptors of Bb-IRAD.Nkoe would be less specific to the toxic molecules AETP, AEAI and OEAI which act either at the membrane level (as a contact fungicide) or inside the cells (as a systemic fungicide) [76]. Therefore, these extracts can either inhibit metabolism by having a fungistatic effect or inhibit respiration by having a fungicidal effect on B. bassiana [7] resulting in incompatibility.

5. Conclusion

Cross-checking the results showed that all producers apply clearing and pruning, and disparately others apply insecticides, fungicides and herbicides. All the products used are chemical, with a large number for insecticides. AETP, OEAI, chlorpyrifos-ethyl and AEAI were found to be toxic to all developmental traits of B. bassiana isolates, with more than 50% inhibition at low and medium concentrations, and highly toxic at high concentrations, with 90% inhibition on average. However, the synthetic fungicide based on chlorothalonil + dimethomorph was found to be compatible with B. bassiana isolates as was the absolute control consisting of sterile distilled water.

This study shows that, although biological and with effects on Hypothenemus hampei or Colletotrichum kahawae, natural substances such as extracts of T. peruviana and A. indica, as well as chlorpyrifos-based products, do not allow either the preservation or the synergistic use of B. bassiana with extracts of these plants or chlorpyrifos-ethyl, especially at high concentrations of these substances. Therefore, these substances should be used with caution to ensure the sustainability and conservation of the diversity of natural enemies of coffee and other crop pests such as B. bassiana.

Acknowledgments

The authors are grateful to the International Foundation for Science (IFS) in Sweden [Research Grant Agreement No. I-1-C-6256-1] and the International Centre for Agricultural Research for Development (CIRAD) through the DP/Agroforestry in Cameroon, for their financial support. The authors are grateful to the Institute of Agricultural Research for Development (IRAD) for technical assistance.

Conflicts of interest

The authors have no conflict of interest to declare.