Tolerance of an entomophagous fungus (Lecanicillium muscarium, Beijing-P strain) against pesticides

Abstract

The tolerance of the entomophagous fungus Lecanicillium muscarium to eight pesticides using a poisoned medium method in the laboratory was evaluated. The purpose was to identify compatible combinations between biopesticides based on pathogenic fungi and conventional pesticides. Pyridalyl slightly inhibited the germination of L. muscarium spores and very slightly inhibited hyphal growth at low concentrations. The maximum inhibition rate of pyridalyl was <30%. Difenoconazole very slightly inhibited the germination of L. muscarium of conidia, and thiamethoxam and dimethomorph both very slightly inhibited hyphal growth. In most treatments, the maximum inhibition rates of difenoconazole, dimethomorph, or thiamethoxam were <20%. Phoxim at a low concentration slightly inhibited both L. muscarium conidial germination and hyphal growth (maximum inhibition rate, <25%). The minimum inhibition rate of most chlorothalonil, triadimefon, or tetraconazole treatments exceeded 60%. At low concentrations, pyridalyl and phoxim showed weak inhibitory effects, and therefore, may be compatible with L. muscarium. These data will support recommendations for the use of pesticides in integrated pest management programmes that involve L. muscarium as the biocontrol agent.

Keywords:

• Entomophagous fungus
• Lecanicillium muscarium
• conidia germination
• hyphal growth
• compatible pesticides
• inhibition

1. Introduction

Biopesticides based on fungi as biological control agents are important pest management tools because most of them can be used to avoid the negative effects of synthetic pesticides (e.g. environmental pollution and killing the natural enemies of pests) (Marrone 2019). However, most biopesticides based on fungi have limited shelf-lives, act slowly, and have narrow target ranges (Faria and Wraight 2001; Jackson, Dunlap, and Jaronski 2010). Combining an effective biopesticide based on fungi and compatible pesticides in an integrated pest management (IPM) programme offers promise (Sain et al. 2019). It is necessary to investigate the effects of synthetic pesticides on various entomopathogenic fungi to determine which fungi and pesticides can be combined successfully in such programmes.

The entomopathogenic fungus Lecanicillium muscarium Zare and Gams (previously called Verticillium lecanii [Zimmerman]) are widely distributed ascomycete fungi of the order Hypocreales. Colonies of L. muscarium on PDA agar media grow white or pale yellow, cottony or velvety, turning cream in colour with mealy appearance (Upadhyay et al. 2014). The Lecanicillium genus is notable in that conidia are borne in slime balls and rarely in dry chains, unlike the other Hyprocreales fungi, which produce hydrophobic conidia (Jaronski 2014). L. muscarium can naturally infects various sucking pests (e.g. aphids, mites, thrips, and whiteflies) and plant-pathogenic nematodes that are economically important pests of horticultural crops (Gillespie 1986; Hazarika et al. 2009; Jackson, Heale, and Hall 2008; Meyer and Huettel 1993; Wang et al. 2004; Hamdi et al. 2011). L. muscarium could also be used as an effective hyperparasite of several rust and powdery mildew fungi (Allen 1982; Verhaar, Hijwegen, and Zadoks 1996; Askary et al. 1998). Some L. muscarium strains are very virulent and have high epizootic efficiencies against certain insects. Such strains have been used as commercial mycoinsecticides against aphids and whitefly (Hall 1981a, 1981b; Quinlan 1988).

Some studies on the compatibility of L. muscarium with pesticides have been published (Cuthbertson et al. 2010; Cuthbertson, Walters, and Deppe 2005; Hall 1981a, 1981b). However, the effects of some currently used pesticides on L. muscarium have not been evaluated, and different pesticides inhibit the germination and mycelial growth of entomopathogenic fungi species and strains to varying degrees (Anderson et al. 1989; Poprawski and Majchrowicz 1995; Todorova et al. 1998). In addition, the formulation of a pesticide will markedly affect its compatibility with fungi (Inglis et al. 2016; Morjan, Pedigo, and Lewis 2002; Celar and Kos 2020). In this study, the tolerance of L. muscarium Beijing-P strain to each of eight chemical pesticides that are currently used to control insect pests and diseases were examined to address whether these chemicals inhibit the conidial germination and hyphal growth of L. muscarium. The results will provide data for further studies on interaction between the pathogen and chemical pesticides, as well as their applications in integrated pests management programs.

2. Materials and methods

2.1. Fungus and pesticides

The L. muscarium strain Beijing-P was originally isolated from Trialeurodes vaporariorum on radish planted in a greenhouse of Beijing, China in 1986. The fungus was cultured on potato dextrose agar (PDA), which contained 200 g L⁻¹ potato infusion, 20 g L⁻¹ dextrose, and 20 g L⁻¹ agar. The fungus was incubated at 23 °C in the dark. Eight synthetic pesticides were acquired from a local market for use in these experiments. The compatibility of each pesticide with the fungus was tested by performing in vitro bioassays for each pesticide at five concentrations. We used the recommended concentration for use in the field (R), a quarter of the recommended concentration (R/4), half of the recommended concentration (R/2), double the recommended concentration (2 R), and four times the recommended concentration (4 R). The active ingredients, formulations, suppliers, and chemical classifications of the pesticides used are shown in Table 1.

Table 1. Pesticides tested against Lecanicillium muscarium in this study.

Active ingredients	Formulation type	Chemical type	Recommend dilution	Company
10.5%Pyridalyl	Emulsifiable concentrate	Propylene ether	1000×	Weierda Chemical Co. Zhejiang China
10%Difenoconazole	Water dispersible granule	Heterocycle	1000×	Syngenta Plant Protection Co. Nantong China
25%Thiamethoxam	Water dispersible granule	Neonicotinoid	2000×	Guanlong Agro-chemical Co. Hebei China
50%Dimethomorph	Wettable powder	Morpholines	2000×	BASF Co. Europe
40%Phoxim	Emulsifiable concentrate	Organophosphorus	1000×	Baoling Chemical Co. Jiangsu China
75%Chlorothalonil	Wettable powder	Organochlorine	1000×	Suli Chemical Co. Jiangyin China
20%Triadimefon	Emulsifiable concentrate	Triazoles	1500×	Jiannong Plant Protection Co. Jiangsu China
4%Tetraconazole	Emulsion in water	Triazoles	800×	Isagro Co. Italy

2.2. Hyphal growth inhibition

A solution of the pesticide at the desired concentration was added to melted PDA medium (100 mL) in a flask and mixed thoroughly at 48 °C before solidification. About 20 mL of the medium was then added to each of a series of sterile Petri dishes (9 cm diameter). Each Petri dish was inoculated with a 7.5 mm diameter disc of a L. muscarium culture, which was placed in the centre using a sterile toothpick. The culture disc was removed 7 d later old L. muscarium culture on PDA. Three to six replicate Petri dishes were prepared for each pesticide concentration, and PDA medium containing no pesticides was used as the control. The Petri dishes were incubated at 23 °C for 5 d, then the diameter of the colony on each dish was determined using the cross method, using the mean of three measurements.

2.3. Inhibition of conidial germination

The fungus was cultured on PDA at 23 °C in the dark for 7 d. Fungi were harvested by rinsing the culture plate with sterile distilled water containing 0.1% v/v Tween 80, vortexing the conidial suspension for 2 min, and then passing the mixture through a single layer of gauze to collect the mycelia. The conidial concentration in the suspension was estimated using a microscope with a blood cell counting chamber, and then the concentration was adjusted to the desired concentration (2 × 10⁶ conidia mL⁻¹) by adding sterile distilled water. Each pesticide was dissolved in sterile distilled water containing 0.1% v/v Tween 80, and then the solution volume was adjusted to give the required pesticide concentration. For each test, an aliquot of the conidial suspension was mixed with a pesticide solution and 4% w/v glucose medium (1 + 1 + 1 by volume), and then the mixture was spread on a sterile glass slide with grooves. The slide was placed on sterile wet filter paper in a sterile Petri dish (9 cm diameter) and incubated at 23 °C in the dark. The conidial suspension with no pesticides was used as a control. Three or four replicate samples at each concentration were prepared. The percentage of conidia that germinated was determined after 24 h. Between 100 and 200 conidia (randomly selected) in each Petri dish were counted. Conidia were considered to have germinated if the germ tube was longer than the conidial diameter.

2.4. Statistical analyses

Conidial germination inhibition and hyphae growth inhibition (each as a percentage) in each treatment relative to the control were determined as described by Hokkanen and Kotiluoto (1992), using the following equation: $$\text{I}\hspace{0.17em}\left(\text{\%}\right)=\frac{C-P}{C}\times 100,$$ where I, C, and P are percentage conidial germination inhibition or hyphae growth inhibition, conidial germination or hyphal growth of the fungus in the controls, and conidial germination or hyphae growth of the fungus in the medium containing pesticide, respectively. An arcsine square-root transformation of the percentage germination inhibition or hyphal growth inhibition data was performed before the data were subjected to one-way analysis of variance. Means were compared using Turkey’s multiple range tests (p = 0.05). The statistical analyses were performed using IBM SPSS statistics 20.0 software (IBM, Armonk, NY, USA).

3. Results and discussion

3.1. L. muscarium tolerates pyridalyl

Pyridalyl at the recommended concentration (R) and the two low concentrations (R/4 and R/2) very weakly inhibited L. muscarium hyphal growth, but at high concentrations (4 R and 2 R) it strongly inhibited hyphae growth (Figure 1A, B). The hyphal growth inhibition rates differed significantly among the five pyridalyl treatments (F = 83.28, df = 4, p = 0.002 < 0.05) (Figure 1B). All five pyridalyl treatments very weakly inhibited L. muscarium conidial germination, and significant inhibition was found at higher pyridalyl concentrations (F = 8.91, df = 4, p < 0.0001) (Figure 1B). The main targets of pyridalyl are lepidopteran pests, such as Plutella xylostella, an important pest of cruciferous vegetables, whereas the hosts of L. muscarium, such as aphid, thrip, and whitefly are also important pests of cruciferous vegetables. Therefore, the combination of these two agents could complementally control more insect pests and reduce application costs.

Figure 1. Inhibition of Lecanicillium muscarium conidial germination and hyphal growth by pyridalyl at different concentrations. R, R/4, R/2, 2R, and 4R indicate recommended concentration for use, a quarter of the recommended concentration, half of the recommended concentration, double the recommended concentration, and four times the recommended concentration, respectively. Colony morphology of L. muscarium grown on potato dextrose agar (PDA) containing pyridalyl at different concentrations at 5 d after inoculation (A). The plates were 9 cm in diameter. Control (PDA medium) did not contain any pesticides (A). Conidial germination inhibition rate and hyphal growth inhibition rate were determined relative to that in the control (see Materials and Methods) (B). In the box chart, uppermost point, lowest point, upper box edge, lower box edge, and line within the box represent the maximum, minimum, upper quartile, lower quartile, and median of three or four replicates, respectively. A single line in the box chart indicates 100% inhibition rate in all replicates. Data were arcsine square-root transformed before one-way analysis of variance. Means were compared using Turkey’s multiple range test. Different letters indicate significant differences among treatments (p < 0.05).

3.2. L. muscarium spores or hyphae tolerate difenoconazole, thiamethoxam, and dimethomorph

Difenoconazole completely inhibited L. muscarium hyphal growth, only very weakly inhibited conidial germination (Figure 2A, C). Neither hyphae growth nor conidial germination differed significantly among the five difenoconazole test concentrations (Figure 2C). Thiamethoxam and dimethomorph both strongly inhibited L. muscarium conidial germination, but only very weakly inhibited hyphal growth (Figure 2B, C). Notably, hyphal growth was enhanced in some of the thiamethoxam and dimethomorph treatments (Figure 2C). The conidial germination inhibition rates differed significantly among the five thiamethoxam treatments (F = 21.31, df = 4, p < 0.0001) (Figure 2C), but the hyphal growth inhibition rates did not. The hyphal growth inhibition rates differed significantly among the five dimethomorph treatments (F = 4.95, df = 4, p = 0.0053 < 0.05), so did the conidial germination inhibition rates (F = 186.9, df = 4, p < 0.0001) (Fig. 2C). Previous studies have reported similar results for the effect of thiamethoxam on L. muscarium hyphal growth but contrasting results for its effect on conidial germination (Gurulingappa, Gee, and Sword 2011). To our knowledge, no other studies have explored the compatibility of L. muscarium with difenoconazole and dimethomorph. In the present study, we investigated the inhibitory effects on L. muscarium by direct exposure to pesticides. However, different inhibitory effects may occur when L. muscarium is exposed to pesticides sequentially. It was reported that copper oxychloride stimulated the insecticidal properties of Beauveria bassiana when it was used 2–4 days after B. bassiana application. However, when copper oxychloride was applied 2–4 days before, or in combination with B. bassiana, it showed inhibitory effects (Kouassi, Coderre, and Todorova 2003). Some IPM schemes require pesticides and entomopathogenic fungi to be applied sequentially, rather than simultaneously, under field conditions. In another study, exposure of L. muscarium to spiromesifen for 24 h resulted in very low conidial germination, at levels unsuitable for commercial application, but infectivity (hyphal growth) was not negatively affected when L. muscarium was applied to plants that had been sprayed with spiromesifen 24 h previously (Cuthbertson et al. 2008). In the present study, thiamethoxam and dimethomorph both strongly inhibited L. muscarium conidial germination, but only weakly inhibited hyphal growth. Based on this, these two pesticides are probably compatible with L. muscarium if applied sequentially in the field. The insecticidal spectra of thiamethoxam and L. muscarium are similar, so this study will lay the foundation for further research on whether the combination of this two pesticides can have a synergistic effect. Furthermore, whether the combination of thiamethoxam and L. muscarium can accelerate pests to produce resistance because of dosage reduction in the mixture need to be researched further. The main targets of dimethomorph are oomycete fungi that cause various plant diseases such as downy mildew, and late blight in cucumber, tomato, and grape. The hosts of L. muscarium, such as aphid, thrip, and whitefly are also important pests of these economic plants. Therefore, the combination of these two agents could control of plant diseases and insect pests simultaneously. In addition, the phenomenon that pesticides can strongly inhibited L. muscarium conidial germination, but only weakly inhibited hyphal growth is interesting and the mechanisms needed to be solved in future.

Figure 2. Inhibition of Lecanicillium muscarium conidial germination and hyphal growth by difenoconazole, thiamethoxam, and dimethomorph at different concentrations. Colony morphology of L. muscarium grown on potato dextrose agar (PDA) containing difenoconazole (A) and thiamethoxam (B) at different concentrations at 5 d after inoculation.The plates were 9 cm in diameter. Different letters indicate significant differences among treatments (p < 0.05) (C). The meanings of R, R/4, R/2, 2R, 4R and box chart, as well as data processing methods are all the same as Figure 1.

3.3. L. muscarium tolerates phoxim at low concentrations

Phoxim at low concentrations (R/4 and R/2) weakly inhibited L. muscarium conidial germination and hyphal growth (Figure 3A,B). The hyphal growth inhibition rates increased at higher concentrations (F = 165.70, df = 4, p < 0.0001), as did the conidial germination inhibition rates (F = 143.90, df = 4, p < 0.0001) (Figure 3B). The main targets of phoxim are lepidopteran insects pests, whereas L. muscarium are against homopetran insect pests. Therefore, the combination of these two agents could control of this two orders of insect pests simultaneously.

Figure 3. Inhibition of L. muscarium conidial germination and hyphal growth by phoxim at different concentrations. Colony morphology of L. muscarium grown on potato dextrose agar (PDA) containing phoxim at different concentrations at 5 d after inoculation (A). The plates were 9 cm in diameter. Different letters indicate significant differences among treatments (p < 0.05) (B). The meanings of R, R/4, R/2, 2 R, 4 R and box chart, as well as data processing methods are all the same as Figure 1.

3.4. L. muscarium does not tolerate chlorothalonil, triadimefon, or tetraconazole

Chlorothalonil strongly inhibited both conidial germination and hyphal growth. The conidial germination inhibition rates differed significantly among the five chlorothalonil treatments (F = 146.10, df = 4, p < 0.0001), as did the hyphal growth inhibition rates (F = 5.2, df = 4, p < 0.0001) (Figure 4). Similar results were found in a study performed by Ali et al. (2012), but in another study, chlorothalonil was found to be very toxic and completely inhibited L. muscarium conidial germination and hyphal growth (Krishnamoorthy et al. 2007). Triadimefon and tetraconazole both extremely strongly inhibited L. muscarium conidial germination and hyphal growth, particularly hyphal growth, for which the inhibition rate was 100% (Figure 4). The conidial germination inhibition rates differed significantly among the five triadimefon treatments (F = 4.06, df = 4, p = 0.0035 < 0.05), and among the five tetraconazole treatments (F = 3.43, df = 4, p = 0.0457 < 0.05). The hyphal growth inhibition rates did not differ significantly among the five triadimefon treatments or among the five tetraconazole treatments (Figure 4). The results of a study conducted by Ali et al. showed that the triazole fungicides myclobutanil and propiconazole strongly decreased L.muscarium hyphal growth, but did not completely inhibit it (Ali et al. 2012).

Figure 4. Inhibition of Lecanicillium muscarium conidial germination and hyphal growth by chlorothalonil, triadimefon, and tetraconazole at different concentrations. The meanings of R, R/4, R/2, 2 R, 4 R and box chart, as well as data processing methods are all the same as Figure 1. Different letters indicate significant differences among treatments (p < 0.05).

4. Conclusion

In summary, L. muscarium tolerated pyridalyl and phoxim (at low concentrations), both in terms of its conidial germination and hyphal growth. The conidial germination of L. muscarium, but not its hyphal growth, was inhibited by thiamethoxam and dimethomorph. On the basis of our results, we conclude that three insecticides, pyridalyl, phoxim, thiamethoxam, and one fungicide, dimethomorph, could be compatible with L. muscarium. These data will support recommendations for the use of pesticides in IPM programmes involving L. muscarium as the biocontrol agent.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This research was supported by grants from Shandong Academy of Agricultural Sciences (CXGC2021B12), the Shijiazhuang City Key Research & Development Program (221490404A), and Beijing Science and Technology Association (ZZ22013).