- Open Access
Efficacy of some entomopathogenic fungi against Aphis fabae Scopoli (Hemiptera: Aphididae)
© The Author(s) 2018
- Received: 9 July 2018
- Accepted: 31 October 2018
- Published: 20 November 2018
The present study was carried out to evaluate the efficacy of three different conidial concentrations (1 × 104, 1 × 105, and 1 × 106 conidia/ml) of five isolates (TR-04, TR-05, TR-07, TR-08, and TR-10) of Lecanicillium muscarium, one isolate (TR-01) of Simplicillium lamellicola, a commercial bioinsecticide Verticillium lecanii, and a synthetic insecticide (Imidacloprid) against the black bean aphid, Aphis fabae Scopoli (Hemiptera: Aphididae) under laboratory conditions. Bioassays were conducted in Petri dishes, and insect mortality rate was recorded daily. The LT50 values (days) of the isolates at 1 × 106 conidia/ml were 2.96 (TR-05), 3.08 (TR-10), 3.21 (TR-07), 3.45 (TR-01), 3.73 (TR-04), and 3.83 (TR-08), while they were 4.37 and 0.73 for the commercial bioinsecticide and insecticide, respectively. The LT90 values (days) of the same conidial concentrations of the isolates attained 4.30 (TR-07), 4.35 (TR-05), 4.80 (TR-10), 5.15 (TR-04), 5.25 (TR-01), 6.06 (TR-08), 6.72 (commercial bioinsecticide), and 2.36 (insecticide). The 1 × 105 and 106 conidia/ml concentrations of all the entomopathogenic fungal isolates tested against A. fabae caused > 90% mortality by the end of the seventh day. It is concluded that both conidial concentrations of these isolates had significant potential to control black bean aphid.
- Lecanicillium muscarium
- Simplicillium lamellicola
- Entomopathogenic fungi
- Aphis fabae
Aphids (Hemiptera: Aphididae) are one of the most significant threats to agriculture and forests (Blackman and Eastop 2007). Aphis fabae Scopoli (Hemiptera: Aphididae), known as the black bean aphid, is one of the most important species causing yield losses in several cultivated crops, including broad bean and sugar beet (Volkl and Stechnann 1998). A. fabae is known to have > 200 host plants globally, and 50 plant species in Iran are vulnerable to this aphid species (Adabi et al. 2010).
The aphids are predominantly controlled by synthetic insecticides; however, their use has raised serious environmental problems (Scorsetti et al. 2007). The overuse of pesticides not only resulted in insect resistance but also forced many countries to reduce pesticide use through alternative methods, including biological control in wake of the increasing consumer tend to prefer pesticide-free food as well as environmental concerns (Kim et al. 2001). The biological control agents used against harmful insects have gained increased importance in recent years. A large number of entomopathogenic fungi (EPF) have been identified and used against aphids (Vu et al. 2007; Scorsetti et al. 2007 and Saruhan et al. 2014, 2015).
EPF such as Beauveria bassiana, Isaria farinosa, I. fumosorosea, Lecanicillium lecanii, L. muscarium, and Metarhizium anisopliae play an important role in the control of insect populations (Zimmermann 2008; Gurulingappa et al. 2011). B. bassiana, L. muscarium, L. lecanii, and S. lamellicola are known to be substantial entomopathogens of A. gossypii (Saruhan et al. 2015). L. lecanii recorded the highest virulent pathogenicity rate against Myzus persicae and A. gossypii, and their control values reached approximately 100% after 5 and 2 days, respectively, following the treatment (Vu et al. 2007). Yeo et al. (2003) investigated the implications of temperature on growth and germination of prospective bioinsecticides (B. bassiana and V. lecanii) and various isolates of B. bassiana, V. lecanii, M. anisopliae, and I. fumosorosea, formerly known as Paecilomyces fumosoroseus and their pathogenicity against aphids. Three isolates were tested against A. fabae and M. persicae at 10, 18, and 23 °C. A. fabae was generally found more vulnerable than M. persicae to infection by the tested fungal isolates. A meaningful interaction between aphid types and temperature signified that the pathogenic nature of an isolate was susceptible to both target aphid types as well as the temperature of the bioassay (Yeo et al. 2003). Saruhan et al. (2015) evaluated two isolates of L. muscarium and S. lamellicola, commercial bioinsecticide V. lecanii, and two different insecticides against A. fabae at 20 and 25 °C. At the end of the seventh day, mortality rates were approximately 100% at all treatments at both temperatures.
The objective of the present study was to evaluate the pathogenicity of five different EPF isolates of L. muscarium and one isolate of S. lamellicola against A. fabae and compare the results with one commercially bioinsecticide and a synthetic insecticide under laboratory conditions.
A stock culture of A. fabae, originated from field collection of infesting broad bean (Vicia faba L.) plants in the experimental area of Ondokuz Mayis University, Turkey, during 2017 was established. The pest was reared in 30 × 20 × 40 cm cages at 22 ± 1 °C and 16 h photoperiod for several generations (Mohammed 2018).
The fungal isolates were obtained from the stock cultures at the Mycology Laboratory, Department of Plant Protection, Faculty of Agriculture, Ondokuz Mayis University, Samsun, Turkey. A total of six isolates of two EPF, i.e., L. muscarium (five isolates) and S. lamellicola (one isolate) were used for bioassays. The fungal isolates used in the present study were identified by Dr. Richard A. Humber, an insect mycologist (USDA-ARS). The isolates were cultured on potato dextrose agar (PDA; Merck, Darmstadt, Germany) for 5–7 days at 25 ± 1 °C. The fungi were stored at 4 °C on PDA dishes until the start of the bioassay experiments.
Conidial germination assessment
The conidium viability of the fungal isolates (TR-01, TR-04, TR-05, TR-07, TR-08, and TR-10) was determined following Saruhan et al. (2015). The conidial suspension was adjusted to 104 conidia/ml, and 100 μl was sprayed on Petri dishes (6 cm diameter) containing PDA. The dishes were then sealed by a parafilm (American National CanTM) and incubated at 25 ± 1 °C. The presence of germinating and non-germinating conidia was counted using an Olympus CX-31 compound microscope (Olympus America Inc., Lake Success, NY) at × 400 magnification after 24 h of incubation. Conidia were regarded as germinated when they produced a germ tube having at least half of the conidial length. The germination ratios of the fungi were determined by examining a minimum of 400 conidia from each of the replicate dishes.
One commercial bioinsecticide, V. lecanii (Nibortem SL, 250 ml/100 l of water), and a synthetic insecticide, Imidacloprid (Conmirid SC 350, 20 ml/100 l of water), were used in the study as a positive control.
Inoculum of isolates of EPF
The six fungal isolates belonging to L. muscarium and S. lamellicola were cultured on PDA at 25 ± 1 °C for 14 days before the initiation of the experiment. Conidial suspensions of the isolates were initially prepared in Tween 20 (0.02% in sterile distilled water) and then filtered through four layers of sterile cheesecloth to remove mycelium and agar pieces. The conidial suspensions were then vortexed for 3 min for homogenization. The concentration of conidial suspension was determined by a Neubauer hemocytometer and adjusted at 1 × 104, 1 × 105, and 1 × 106 conidia/ml.
Conidial suspensions of L. muscarium (five isolates: TR-04, TR-05, TR-07, TR-08, and TR-10) and S. lamellicola (TR-01), V. lecanii, and Imidacloprid were applied on fresh broad bean leaves, obtained from 3-week-old plants grown in pots, containing ten A. fabae (third nymphal instar) placed in Petri dishes (9 cm diameter) with sterile distilled water-soaked blotters. For eight treatments, a 2-ml solution was sprayed by a Potter spray tower (Burkard, Rickmansworth, Hertz, UK) on the nymphs of A. fabae. The Petri dishes were loosely capped to prevent the escape of insects. The same number of nymphs was used for control, where only sterile distilled water containing 0.02% Tween 20 was sprayed. All dishes were incubated at 25 ± 1 °C in 16 h light/8 h dark cycle and in 70 ± 5% RH for 7 days and inspected daily. Dead nymphs were counted, using a Leica EZ4 stereo dissecting scope at × 40–70 magnification, and the mortality rate was calculated per Petri dish. The experiment was repeated twice, with four replicates per treatment.
Measurement of mycelial growth and sporulation
Mycelial growth and sporulation of the isolates belonging to L. muscarium (TR-08) and S. lamellicola (TR-01) were assessed according to Cheng et al. (2016) with slight modifications. Mycelial disks (4 mm in diameter) from 10-day-old fungal cultures were placed in the centers of the Petri dishes (9 cm) containing PDA. Then, the dishes were sealed by a parafilm and incubated at 25 ± 1 °C. Mycelial growth was measured daily at two perpendicular colony diameters up to the point of nearly covering the Petri dishes, and their initial day of sporulation was recorded. At the end of the experiment, three agar pieces of 1 cm2/fungus were cut from the Petri dishes in which fungal growth occurred, using a sterile scalpel and put into 50-ml sterile polypropylene tubes. The conidia produced on each of the PDA pieces were shaken and dispersed in 20 ml of 0.02% Tween 20 solution. Then, the conidia were counted under Olympus CX-31 compound microscope using a Neubauer hemocytometer, and the spore amount per unit area was calculated. The experiment had three replicates/isolate repeated at different times.
Mortality data was corrected using Abbott’s formula (Abbott 1925). Serial time-mortality data from bioassays were analyzed by probit analysis using SPSS software (SPSS, version 21) to calculate the lethal times, 50% (LT50) and 90% (LT90). Mortality rates of A. fabae treated with EPF, mycelial growth rate and sporulation of six EPF isolates were compared by one-way analysis of variance (ANOVA), followed by Tukey student size post-hoc test where ANOVA indicated significance (P < 0.05).
The results of this study are in line with the results of previous studies. The cumulative mortality rates caused by the three isolates of EPF: Conidiobolus obscurus, C. thromboides, and Basidiobolus ranarum ranged from (51.2 to 91.7%) against A. fabae (Halımona and Jankevıca 2011). In another study, it was reported EPF, B. bassiana, caused 45% mortality of A. fabae at the end of the ninth day (Zamani et al. 2013). Guven et al. (2014) reported that at 1 × 108 conidia/ml of spore solution of B. bassiana isolates [BMAUM-A6-001 (90.78%), BMAUM-A6-002 (90.94%), BMAUM-005 (79.62%)], M. anisopliae (90.54%), and Paecilomyces lilacinus (84.15%) were the most effective against A. fabae on the third day based on the number of live individuals. In a study, to determine the biological effectiveness of the EPF, Fusarium subglutinans isolated from cotton aphid against A. fabae, applications of three different suspensions of two isolates of F. subglutinans resulted in meaningful differences in aphid mortality rate at 25 °C. Moreover, no differences in mortality rates were observed between 1 × 107 and 1 × 108 conidia/ml suspension (Arıcı et al. 2012).
Lethal times (LT50 and LT90) for Aphis fabae treated with six entomopathogenic fungi (1 × 106 conidia/ml), commercial bioinsecticide, and synthetic insecticide
LT50 (95% confidence limit)
LT90 (95% confidence limit)
3.45 (2.98–3.92) a*
5.25 (4.65–6.28) ab
3.73 (3.55–3.93) a
5.15 (4.86–5.55) ab
2.96 (2.78–3.14) a
4.35 (4.08–4.68) ab
3.21 (2.79–3.72) a
4.30 (3.79–5.28) ab
3.83 (2.97–5.37) a
6.06 (4.81–11.40) a
3.08 (2.25–3.88) a
4.80 (3.97–7.20) ab
4.37 (4.16–5.19) a
6.72 (6.09–7.01) a
0.73 (0.65–1.24) b
2.36 (2.01–2.98) b
Mycelial growth rate and sporulation of different entomopathogenic fungal isolates
Colony diameter (cm)
4.7 × 107 b
1.5 × 108 a
1.4 × 108 a
1.6 × 108 a
1.5 × 108 a
1.4 × 108 a
Different isolates belonging to the EPF: L. muscarium and S. lamelicolla, commercial bioinsecticide, and synthetic insecticide tested in this study showed similar pathogenicity against A. fabae. The isolates still need further evaluations under field conditions before recommending them in biological control of A. fabae.
I would like to thank Assoc. Prof. Dr. Ismail Erper for his help in collecting, reproducing, and applying the isolates.
Availability of data and materials
All data are available at the end of the article, and the materials used in this work are of high quality and grade.
IS designed the study, supervised the work, wrote the manuscript, carried out the experiments, and analyzed the data. The author read and approved the final manuscript.
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