Background

Sitophilus weevils, including Sitophilus oryzae (rice weevil), S. zeamais (maize weevil), and S. granarius (granary weevil) (Coleoptera: Curculionidae), are well-known stored-grain insect pests in Turkey and many other countries in the world (Bağcı et al. 2014). These weevils have a nearly cosmopolitan distribution, occurring throughout all warm and tropical parts of the world (Hong et al. 2018). Generally, because of the storage-grain pests infestation, it has been estimated that during storage, 10–25% of the grain crops are damaged yearly worldwide. Damages caused by the insects not only contain the direct feeding harm resulting in loss of weight, but they also seriously decrease nutrients, lowering seeds germination rate, reducing quality, and lowering their marketing value due to the mass of waste, webbing, and insect cadavers (Abdel-Raheem et al. 2015).

Stored-grain protection against the pests is currently based on the use of synthetic insecticides and fumigants (Arthur 1996). As a result, these have caused problems including insecticide resistance along with contamination of many food products with chemical residues and consumer demand for pesticide-free grain. Thus, there is a growing interest in using biological control agents against the pests as an alternative (Wakil et al. 2015).

Entomopathogenic fungi (EPFs) are common natural enemies of arthropods worldwide, attracting attention as a potential biological control agent. There are more than 700 species of EPFs (Sandhu et al. 2012; Erper et al. 2016). Fungal entomopathogens such as Beauveria bassiana, B. brongniartii, Isaria farinosa, I. fumosorosea, Lecanicillium spp., and Metarhizium anisopliae play an important role in the regulation of insect populations (Zimmermann 2008; Gurulingappa et al. 2011). Also, since they exist in nature, EPFs have low environmental impact and are generally considered environmentally safe agents with low mammalian toxicity (Rumbos and Athanassiou 2017).

The using of EPFs for the control of the insect pests in stored-grain products is one of the most promising alternative control methods (Moore et al. 2000). Especially, the species B. bassiana and M. anisopliae have a wide host range and have been tested against most of the major stored-grain pests (Batta 2018; Rumbos and Athanassiou 2017).

Temperature plays a significant role on the effectiveness of EFPs, especially high temperatures affect negatively conidial viability and germination (Rumbos and Athanassiou 2017). For example, B. bassiana was found to be more effective against R. dominica, S. oryzae at 26 °C than at 30 °C (Vassilakos et al., 2006), and S. granarius (Athanassiou and Steenberg 2007) in stored wheat. Similarly, Michalaki et al. (2007) found that Isaria fumosorosea was more effective at 20 °C than at 25 °C. In another study, I. fumosorosea was effective against Tribolium confusum and Ephestia kuehniella, but its effectiveness was highly dependent on the target species and life stage, exposure interval, and temperature (Michalaki et al. 2007).

The aim of this study was to evaluate the efficacy of five EPFs isolates, belonging to I. fumosorosea, Simplicillium lamellicola, B. bassiana, M. anisopliae, and L. muscarium, against the storage-grain pests, S. granarius and S. oryzae, at two different temperatures under laboratory conditions.

Materials and methods

Fungi cultures

Five respect isolates (TR-01, TR-07, TR-78-3, TR-106, and TR-217) of the entomopathogenic fungi (EPFs), I. fumosorosea, Simplicillium lamellicola, B. bassiana, M. anisopliae, and L. muscarium, were used in the present study. They were isolated from different infected hosts in hazelnuts orchards in the Black Sea region of Turkey (Erper et al. 2016; Kushiyev et al. 2018). The single-spore cultures of B. bassiana (TR-217 isolate), I. fumosorosea (TR-78-3 isolate), L. muscarium (TR-07 isolate), M. anisopliae (TR-106 isolate), and S. lamellicola (TR-01 isolate) were stored at 4 °C on Sabouraud dextrose agar (SDA; Merck Ltd., Darmstadt, Germany) slants and deposited in the fungal culture collection of the Mycology Laboratory at the Ondokuz Mayis University, Faculty of Agriculture’s Department of Plant Protection in Samsun, Turkey.

Insect cultures

Adults of S. granarius and S. oryzae were used. Adult insects were obtained from stock cultures in the Black Sea Agricultural Research Institute (Samsun-Turkey). Insects in cultures were grown at 25 ± 2 °C, 65 ± 3% RH in 16-h light/8-h dark conditions and fed on sterile wheat grains. Adults from cultures were collected by an oral aspirator and 1-day-old adults were used in the study.

Inoculum of EPF

The five isolates of EPFs were incubated on potato dextrose agar (PDA; Merck Ltd., Darmstadt, Germany) at 25 ± 1 °C for 10–14 days. Conidia were harvested by sterile distilled water, containing 0.02% Tween 20. Then, conidia suspensions were filtered through four layers of sterile cheesecloth to remove mycelium, and conidia were counted under an Olympus CX-31 compound microscope (Olympus America Inc., Lake Success, NY), using a Neubauer hemocytometer to calibrate a suspension of 1 × 108 conidia ml−1 of each isolate (Erper et al. 2016).

Conidial germination assessment

The viability of conidia of the five isolates belonging to B. bassiana, I. fumosorosea, L. muscarium, M. anisopliae, and S. lamellicola was determined. A conidial suspension (200 μl) of each isolate at (1 × 104 conidia ml−1) obtained by dilution was sprayed onto Petri plates (9-cm dia.), containing PDA (Merck Ltd., Darmstadt, Germany). These plates were incubated at 25 ± 1 °C. After 24 h of incubation, the percentage of germinated conidia was counted, using an Olympus CX-31 compound microscope at × 400 magnification. Conidia were regarded as germinated, when they produced a germ tube, at least half of the conidial length. The germination ratios for each isolate were calculated after examining a minimum of 200 conidia from each of the three replicate plates (Saruhan et al. 2015).

Experimental design

Ten S. granarius and S. oryzae adults were released in each Petri plate (9-cm dia.), containing 10 pieces of sterilized wheat grain. Bottoms of plate cups were covered by a filter paper moisturized with sufficiently sterile distilled water. Conidial suspension (1 × 108 conidia ml−1) of each EPF (TR-217, TR-78-3, TR-07, TR-106, and TR-01) was applied to the S. granarius and S. oryzae adults (2 ml per plate), using a Potter spray tower (Burkard, Rickmansworth, Hertz UK). Control Petri plates were treated by sterile distilled water (2 ml), containing 0.02% Tween 20. All the plates were loosely covered by a Parafilm to prevent their escape and incubated both at 20 ± 1 and at 25 ± 1 °C in 16 h light/8 h dark and in 75 ± 5% RH, using the Memmert incubator (Model ICP 110; Germany). The spray tower was cleaned by 70% ethanol and sterile distilled water after each application of the fungus suspension.

Dead adults were counted, using a Leica EZ4 stereo dissecting scope at × 40–70 magnification. They were removed daily from the plates and immediately surface disinfected by dipping it in 1% sodium hypochlorite (NaOCl) for 3 min and in 70% ethanol for 3 min. Then, the dead insects (belonging to S. granarius and S. oryzae) were washed three times in sterile distilled water and placed in 75 ± 5% RH, using the Memmert incubator. Mortality rates were confirmed by examining of hyphae on the cadavers under Leica EZ4 stereomicroscope, 10 days after placing the dead insects (Kocaçevik et al. 2016). The bioassay was performed by using a completely randomized experimental design with five replicates. Each replicate consisted of 10 1-day-old adults of the pests and placed in a Petri plate (9-cm dia.), and the experiment was conducted once (Saruhan et al. 2015).

Statistical analysis

The mortality rate was observed at 7 days, following each application. Dead individuals were counted under a stereoscopic microscope and the mortality rate was calculated. Data was corrected by Abbott’s formula (Abbott 1925). Fifty percent lethal time (LT50) and 90% lethal time (LT90) were determined, using the probit analysis by SPSS (ver. 21) program. The effects on mortality rates of the S. granarius and S. oryzae were analyzed, using the two-way analysis of variance (ANOVA), followed by a comparison of means, using the Tukey HSD test (SPSS) (P < 0.05).

Results and discussion

The efficacy of the five different EPFs, I. fumosorosea, Simplicillium lamellicola, B. bassiana, M. anisopliae, and L. muscarium, against adults of storage-grain pests S. granarius and S. oryzae at two different temperatures (20–25 °C) under laboratory conditions was evaluated.

Among the EFPs, I. fumosorosea (92.69%) and M. anisopliae (90.35%) recorded the highest effects on S. granarius at 20 °C at the end of day 7, followed by B. bassiana (72.91%), S. lamellicola (62.02%), and L. muscarium (33.91%). The same isolates were tested at 25 °C, where the highest effect, recorded at this temperature, was by M. anisopliae (90.48%), followed by I. fumosorosea (84.21%), S. lamellicola (59.26%), B. bassiana (56.14%), and L. muscarium (22.81%). The effects of these isolates on S. granarius at different temperatures were similar but slightly low at 25 °C (Figs. 1 and 2). Sheeba et al. (2001) applied B. bassiana against S. oryzae and recorded (86.2%) the mortality rate in adults after day 25. In another study, Khashaveh et al. (2011) tested the commercial product of B. bassiana against S. granarius, Oryzaephilus surinamensis, and Tribolium castaneum at 24 ± 2 °C recording 88.33, 78.31, and 64.99% mortality, respectively. Among these three pests, S. granarius was reported to be the most sensitive.

Fig. 1
figure 1

Mortality rate of Sitophilus oryzae treated with entomopathogenic fungi at 20 °C

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Fig. 2
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Mortality rate of Sitophilus oryzae treated with entomopathogenic fungi at 25 °C

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Among the isolates applied on S. oryzae at 20 °C, M. anisopliae showed the highest effect (85.68%), followed by I. fumosorosea (63.32%), S. lamellicola (48.06%), L. muscarium (45.10%), and B. bassiana (40.74%). For the same pest at 25 °C, B. bassiana had the highest effect (93.66%) at this temperature, followed by M. anisopliae (90.40%), I. fumosorosea (58.02%), L. muscarium (56.86%), and S. lamellicola (54.74%). With the rise of temperature, the effect of isolates against S. oryzae was increased (Figs. 3 and 4).

Fig. 3
figure 3

Mortality rate of Sitophilus granarius treated with entomopathogenic fungi at 20 °C

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Fig. 4
figure 4

Mortality rate of Sitophilus granarius treated with entomopathogenic fungi at 25 °C

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Temperature plays a significant role for the effectiveness of EPFs. It is widely accepted that high temperatures affect negatively the conidial viability and germination (Rumbos and Athanassiou 2017). Generally, different fungal species have different temperature requirements. For instance, regarding several strains of B. bassiana, the optimum temperature for conidial germination and vegetative growth is around 25 °C (Rumbos and Athanassiou 2017), while I. fumosorosea was more effective at 20 °C than at 25 °C (Michalaki et al. 2007). In the present study, the efficacy of the isolate of I. fumosorosea was the highest (92.69%) on S. granarius at 20 °C, while it showed a lower effect (84.21%) on the pest at 25 °C. Similarly, the isolate of B. bassiana had the highest effect (93.66%) at 25 °C, while it showed the lowest effect (40.74%) on the S. oryzae at 20 °C. In contrast, B. bassiana was found to be more effective (72.91%) at 20 °C than (56.14%) at 25 °C against S. granarius. Similarly, Tefera and Pringle (2003) found that among different isolates of B. bassiana, germination, radial growth, and sporulation of all isolates were retarded at 15 and 35 °C, while the optimum temperature of different isolates of B. bassiana was between 20 and 30 °C (Tefera and Pringle 2003). Also, the pathogenicity and virulence of B. bassiana isolates vary remarkably among the host species and the life stage of the target pest. In the present study, the isolate of B. bassiana was more effective on S. granarius than on S. oryzae. This finding is also in line with Kassa et al. (2002) who tested 11 isolates of B. bassiana against adults of S. zeamais and Prostephanus truncatus (larger grain borer) (Coleoptera: Bostrychidae), and as a result, determined that P. truncatus was more susceptible to the B. bassiana than S. zeamais.

The LT50 values of the I. fumosorosea and M. anisopliae isolates (2.75 and 2.88 days, respectively), used against S. oryzae at 20 °C at a concentration of 1 × 108 conidia/ml, showed that they were the most effective ones and they were statistically different from the other used isolates. Correspondent LT50 values of L. muscarium, S. lamellicola, and B. bassiana at the same concentration and at 20 °C were 4.78, 4.87, and 5.14 days, respectively. The LT90 values of the EPFs used against S. oryzae at 20 °C had a similar trend to those of LT50. Considering the LT90 values, the most effective EPFs were I. fumosorosea and M. anisopliae (4.17 and 4.47 days), respectively, although the mortality period in adults was lengthen out. These EPFs were determined as L. muscarium, S. lamellicola, B. bassiana (7.67, 7.84, 8.26 days), respectively. The most effective isolate at LT50 at 25 °C was M. anisopliae (2.20 days), followed by B. bassiana (3.17 days), I fumosorosea (3.34 days), L. muscarium (3.73 days) and S. lamellicola (4.57 days). Similarly, LT90 values of M. anisopliae and B. bassiana were 3.82, 3.94 days, respectively, followed by I. fumosorosea with 5.62 days. The lowest effect was recorded for L. muscarium and S. lamellicola (6.42 and 5.92 days, respectively) (Table 1). When the temperature sensitiveness of LT50 values of the used isolates was analyzed, L. muscarium and S. lamellicola isolates were found statistically in the same group (P < 0.05). M. anisopliae, I. fumosorosea, and B. bassiana were different from these two isolates. In a study, the LT50 value was determined as 3.52 days after AAU D (Metarhizium) application against S. oryzae at a dose of 1 × 108 conidia ml−1, and (96.6%) the mortality rate was determined at the end of day 10. In the same study, the LT50 value of DLCO 141 (Beauveria) was reported as 6.53 days and the mortality rate was 70.0%, and the LT50 value of DLCO 26 (Metarhizium) was 6.21 days and the mortality rate was 60.0% (Kassaye 2011). In this study, the effect of isolates used against S. oryzae was similar to those of the isolates that were used against the same pest species.

Table 1 Lethal time (LT50 and LT90) for Sitophilus oryzae treated with the tested entomopathogenic fungal isolates at 20 and 25 °C
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According to the results of the isolates of EPFs against S. granarius, there was insignificant difference between the results obtained at 20 and 25 °C (P < 0.05) (Table 2). In the present study, LT50 values of the five isolates of EPFs, used against S. granarius at 20 °C, were evaluated; S. lamellicola, B. bassiana, I. fumosorosea, and M. anisopliae were found in the same group, while L. muscarium was in a different group. LT50 values of the isolates, used against S. granarius, were 3.21 days (S. lamellicola), 2.95 days (B. bassiana), 2.43 days (I. fumosorosea), and 2.71 days (M. anisopliae), while L. muscarium was determined as (6.03 days). LT90 values of EPF applied against S. granarius at 20 °C were found to be at the same group statistically (Table 2). The LT50 results of the isolates used against S. granarius at 25 °C showed a similar trend to those obtained at 20 °C. In terms of LT50 values, I. fumosorosea (2.78 days), M. anisopliae (2.82 days), B. bassiana (3.91 days), and S. lamellicola (4.01 days) were found to be at the same group statistically, whereas L. muscarium (7.26 days) isolate was found to be low in effect and in a different group statistically. Additionally, in terms of LT90, it was determined that L. muscarium had a low effect (11.39 days) and it was in a different group than other isolates statistically, and LT90 values of the other four isolates ranged 4.52 to 6.11 days and were at the same group statistically (Table 2) (P < 0.05).

Table 2 Lethal time (LT50 and LT90) for Sitophilus granarius treated with the tested entomopathogenic fungal isolates at 20 and 25 °C
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Conclusion

In conclusion, the five different EPFs evaluated in this study showed that they were effective against S. oryzae and S. granarius, and may be considered as alternatives to chemical control. In addition, M. anisopliae and I. fumosorosea showed about 90% efficacy against both pests at the end of the day 7. Thus, they are promising biocontrol agents in terms of practical application according to the results obtained from similar studies. Further studies are necessary to evaluate the efficacy of the isolate on the pests under storage conditions.