Introduction

Potato (Solanum tuberosum L.) belongs to the Solanaceae family and is one of the world’s major food crops. The potato tuber moth (PTM), Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae), is an important and prevalent pest of potato in 90 countries in Africa, Asia, Central and South America with tropical and subtropical climates (Sporleder et al. 2004; Jensen et al. 2005; Lacey et al. 2010). It causes direct damage by feeding on leaves, stems and tubers, and indirect damage by promoting growth of pathogens. Hence, it causes a severe reduction in quality and quantity of tubers in most potato cultivation areas (Valencia-Jimenez et al. 2008). Damage by the pest in appropriate conditions in storage is more than in the field (Das 1995).

Control of P. operculella is primarily accomplished through using conventional insecticides. Chemical control has resulted in the development of resistance in P. operculella, and its negative environmental impact has encouraged development of alternative pest management strategies, in which microbial control may play a fundamental role (Collantes et al. 1986; Llanderal-Cazares et al. 1996). The present contribution is an attempt to assess different components of IPM (integrated pest management) in controlling the potato tuber moth. Entomopathogenic fungi are not only safe alternatives to synthetic insecticides but also compatible with synthetic and botanical pesticides. Additionally, they are insect-specific pathogens (Neves et al. 2001; Patil1 et al. 2014).

Some laboratory and field studies have demonstrated the insecticidal activity of three entomopathogenic fungi, Metarhizium anisopliae (Metchnikoff) Sorokin, Nomuraea rileyi (Farlow) Samson and Paecilomyces tenuipes Samson (Santi et al. 2010), in which N. rileyi was a good candidate for potato tuber moth management (Sabbour and Singer 2014; Sabbour and Abdel-Raheem 2015). Ignoffo (1981) reviewed insecticidal activity of N. rileyi to lepidopteran pests and concluded that the fungus was a potential agent for further development as a bio-insecticide. N. rileyi secretes a proteinaceous substance inhibiting larval moult and metamorphosis (Kiuchi et al. 2003). The entomopathogenic filamentous fungus M. anisopliae is popular as a pathogen of arthropods applied in biological control strategies against numerous serious insect pests (Santi et al. 2010). Some Paecilomyces isolates produce several metabolites of the antibiotic group cephalosporins (Sabbour and Singer 2014).

The aim of our study was to determine ovicidal and larvaecidal activity of three native entomopathogenic fungi, M. anisopliae, N.rileyi and P. tenuipes, against P. operculella to discover the most effective one to include in IPM of PTM.

Materials and Methods

Insect

The colony of potato tuber moth was obtained from the University of Mohaghegh Ardabili, Ardabil, Iran, and reared on potato cultivar Agria. Experiments were conducted under laboratory (26 ± 1 °C, 60 ± 5% relative humidity (RH) and a photoperiod of 16-h light:8-h dark) and greenhouse (26 ± 2 °C, 55 ± 5% RH and a photoperiod of 14-h light:10-h dark) conditions.

Culture of Fungal Species

Three entomopathogenic fungal species (M. anisopliae IRAN 2252, N. rileyi IRAN 1020C and P. tenuipes IRAN 1026C) were purchased from the Iranian Research Institute of Plant Protection, Tehran, Iran. Selection of the entomopathogenic fungi was based on their importance against lepidopteran species. Each fungal species was cultured in potato dextrose agar (PDA) at 25 ± 1 °C. After 2 weeks (enough growth and sporulation of fungi), conidia were scrapped to make an aqueous suspension with 0.02% Tween-80. The conidia suspension was filtered through three layers of sterile cheesecloth to remove mycelium and was enumerated with an improved Neubauer haemacytometer (Weber Scientific International Ltd., United Kingdom). Before the bioassay tests, spore viability percentage was determined by inoculating plates of PDA with the suspension. After 24 h, the germination rate was observed under a light microscope. Germination was considered positive when the length of germ tube was as long as the width of the conidia (Hall 1981). The germination of M. anisopliae, N. rileyi and P. tenuipes conidia was 97, 96 and 97%, respectively.

Bioassays

Larval Penetration

Potato-Dipping

To investigate larval penetration, each potato was dipped in spore suspensions of M. anisopliae, N. rileyi and P. tenuipes determined by preliminary dose setting experiments. The ranges for M. anisopliae, N. rileyi and P. tenuipes were 104–109, 102–106 and 105–109 conidia/ml, respectively. For controls, the tubers were immersed in 0.02% Tween-80 aqueous solution. When the water had evaporated and the tubers were dry, they were transferred into plastic containers with ventilated lids kept at 26 ± 1 °C, 60 ± 5% RH and a photoperiod of 16-h L: 8-h D. Then 20 new larvae (< 5-h old) were placed on each tuber with a soft hair brush. The criterion of larval penetration was the number of pupae and adult emergence in all experiments. There were three replicates of 20 larvae per fungal isolate and control.

We also examined adult emergence, and their progeny (F1), from neonate larvae of PTM exposed to a lethal concentration of each fungus resulting in 25% mortality (LC25) and non-treated potato tubers. The procedure of this experiment was the same as the previous one. After neonate larval penetration and adult emergence, their oviposition rates were recorded up to 24, 48 and 72 h in laboratory conditions. Egg hatch was also recorded.

Leaf-Dipping in the Laboratory and Greenhouse

The fresh leaves used in leaf-dipping under laboratory conditions came from potatoes grown in the research field of Urmia University, Urmia, Iran. For greenhouse trials, potatoes were grown in pots under greenhouse conditions and fresh leaves, attached to the plants, were used for the experiments.

M. anisopliae, N. rileyi and P. tenuipes were applied to fresh leaves of potato in laboratory and greenhouse conditions at 102–105, 4 × 101–4 × 104 and 7 × 104–7 × 108 and 103–106, 103–105 and 7 × 105–7 × 109 conidia/ml, respectively. For each trial, the fresh leaves were dipped in the spore suspensions. In the laboratory, the leaves were then dried and placed in Petri dishes. To supply humidity, wet filter papers were placed on the bottom of the Petri dishes. Then 20 new larvae (< 5-h old) were placed on each leaf. Mortality was recorded after pupae and adult emergence. The leaves were replaced every 3 days. Each treatment and the untreated control had three replications per experiment.

Egg-Dipping

M. anisopliae, N. rileyi and P. tenuipes were tested at 107–109, 4 × 101–4 × 104 and 106–1010 conidia/ml, respectively. For treatments, each filter paper contained 20 1-day-old eggs dipped in the spore suspensions of the fungi. When the water had evaporated and the filter paper was dry, they were transferred into plastic containers with ventilated lids which contained potato tubers to stimulate egg hatching and penetration into the potatoes. The plastic containers were kept under laboratory conditions. Egg hatch was observed under a light microscope after 8 days. Each experiment was replicated three times.

Data Analysis

To determine LC50 (lethal concentration resulting in 50% mortality) and LC25 values, a probit analysis of the data was done using the Statistical Package for the Social Sciences (SPSS) for Windows® release 16. Percentages were transformed to arcsin√x before analysis of variance (ANOVA). Significant differences between means were identified using Tukey’s test.

Results

Bioassays of toxicity of M. anisopliae, N. rileyi and P. tenuipes to neonate larval penetration of potato tubers by potato tuber moth gave LC50 values equal to 1.9 × 105, 1 × 103 and 2.4 × 106 conidia/ml, respectively (Table 1). N. rileyi exhibited the lowest LC50 value, and hence the highest preventive effect on PTM neonate larval penetration into potatoes and development to pupal and adult stages. Results presented in Table 2 show that egg number and hatching was significantly reduced (F3,32 = 16.26, P < 0.05) in F1 progenies of neonate larvae of PTM exposed to potato tubers treated (at LC25 value) with the three entomopathogenic fungi. The results in Tables 3 and 4 show the toxicity of leaves dipped in the three fungi to neonate larvae of PTM. Under laboratory conditions (Table 3), the most effective entomopathogenic fungus was N. rileyi (LC50 = 2.6 × 102 conidia/ml). Under greenhouse conditions (Table 4), the LC50 values for M. anisopliae, N. rileyi and P. tenuipes were 1.4 × 104, 4.2 × 103 and 3.4 × 107 conidia/ml, respectively. Hence, N. rileyi also demonstrated a high efficacy against neonate larvae of P. operculella in greenhouse conditions. One-day-old eggs of PTM were sensitive to N. rileyi (LC50 = 4.8 × 104 conidia/ml), whereas M. anisopliae and P. tenuipes had low activity (LC50 = 5.8 × 107 and 3.2 × 107 conidia/ml, respectively) (Table 5). Mean percentage mortalities in pupae from neonate larvae exposed to LC25 value of fungal suspensions of M. anisopliae, N. rileyi and P. tenuipes were 30, 58 and 28%, respectively (Fig. 1). Hence, neonate larvae of PTM were susceptible to the entomopathogenic fungi. In general, N. rileyi caused higher mortality rate than M. anisopliae and P. tenuipes (% adult emergence = 43, 62 and 69%, respectively) (Fig. 1).

Table 1 Probit analysis of three entomopathogenic fungi toxicity against neonate larval penetration of Phthorimaea operculella into potato tubers
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Table 2 F1 progeny from neonate larvae of PTM exposed to the treated (LC25 value of each fungus) and non-treated potato tubers after 24, 48 and 72 h
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Table 3 Probit analysis of three entomopathogenic fungi toxicity against neonate larval penetration of Phthorimaea operculella into potato leaves in the laboratory condition
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Table 4 Probit analysis of three entomopathogenic fungi toxicity against neonate larval penetration of Phthorimaea operculella into potato leaves in the greenhouse condition
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Table 5 Probit analysis of three entomopathogenic fungi toxicity against 1-day-old eggs of Phthorimaea operculella
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Fig. 1
figure 1

Efficacy of three entomopathogenic fungal isolates on % pupae and adult emergence from neonate larvae of PTM exposed to the treated (with LC25 value of each fungus) and non-treated potato tubers

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Discussion

To our knowledge, this is the first investigation showing the susceptibility of P. operculella eggs and neonate larvae to local entomopathogenic fungi in Iran. Based on our findings, neonate larvae of PTM are susceptible to infection by entomopathogenic fungi. While 1st instar larvae of the pest are the only free-living ones, it could be a potential practical target to apply entomopathogenic fungi as bio-control measures in potato storage and field conditions.

In the present study, N. rileyi displayed high insecticidal activity against neonate larvae of potato tuber moth and their development to pupal and adult stages, whereas M. anisopliae and P. tenuipes exhibited lower efficacy. The entomopathogenic fungi show various degrees of pathogenicity, which is correlated with the physiology of the immune response of their hosts (Lee et al. 2005). The high virulence of N. rileyi may be due to a low rate of proteolytic activity, activating glucose dehydrogenase. Additionally, the number of nodules formed is significantly smaller in larvae inoculated with N. rileyi. Sabbour and Singer (2014) evaluated the insecticidal activity of two Paecilomcyes isolates in laboratory and semi-field conditions. They reported that P. lilaceous was more effective than P. fumosoroseus and that the LC50 values ranged from 1.06 × 106 conidia/ml to 1.22 × 106 for P. fumosoroseus and P. lilaceous, respectively, in laboratory condition. This is higher than our results for P. tenuipes.

Based on our results, adult emergence and F1 progenies from neonate larvae of PTM exposed to treated potato tubers (with LC25 value of each fungus) were significantly affected by the three species of entomopathogenic fungi. Treatment of potato tubers with the three fungi significantly reduced the numbers of eggs of P. operculella laid and hatched. Lacy and Neven (2006) assayed the potential of the fungus Muscodor albus as a microbial control agent of P. operculella in stored potatoes. They showed that neonate larvae were significantly affected by exposure to volatiles produced by M. albus but adults were more sensitive than neonate larvae. Moreover, when 15 and 30 g of formulated M. albus mycelia were used against the neonate larvae of P. operculella, their development to the pupal stage was reduced by 61.8 and 72.8%, respectively, in comparison with controls. Lacey et al. (2010) examined microbial control of PTM exposed as neonate larvae to M. anisopliae and showed that the fungus decreased development to pre-pupal, pupal and adult stages.

In our study, N. rileyi presented the highest toxicity against neonate larval penetration of PTM and pupae and adult emergence in laboratory and greenhouse conditions with LC50 values equivalent to 2.6 × 102 and 4.2 × 103 conidia/ml, respectively. Sabbour and Abdel-Raheem (2015) noted that B. brongniartii and N. rileyi reduced the number of eggs laid/female on potato leaves relative to the control under laboratory and semi-field conditions. In addition, the yield of potatoes increased in plots treated with B. brongniartii and N. rileyi. Pathogenicity of M. anisopliae and Baeuveria bassiana was assessed against early stages of Capnodis tenebrionis by Marannino et al. (2006), who demonstrated that neonate larvae of C. tenebrionis were affected by B. bassiana and M. anisopliae isolates. However, B. bassiana isolates caused lower mortality than M. anisopliae isolates. Based on our probit analysis for 1-day-old eggs of P. operculella, the most effective entomopathogenic fungus was N. rileyi (LC50 = 4.8 × 104 conidia/ml). Marannino et al. (2006) studied susceptibility of 7-day-old eggs of C. tenebrionis to entomopathogenic fungal isolates by egg-dipping masses individually in a spore suspension. They showed that egg hatch was significantly reduced by entomopathogenic fungi and that B. bassiana isolates caused significant reduction of egg hatching relative to M. anisopliae isolates.

In general, our results indicated that these entomopathogenic fungi had ovicidal and larvicidal acivities against P. operculella and can be used in IPM programmes. However, N. rileyi demonstrated the highest activity in all experiments. Despite the potential of entomopathogenic fungi in pest control, these bio-control agents have some disadvantages (sensitivity to environmental factors such as moisture, light and temperature), limiting their applications in storage and field conditions. However, new formulation technologies such as nano formulation strategies can improve their efficacy and pathogenicity.