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Efficacy of some local isolates of the fungus Beauveria bassiana (Balsamo) Vuillemin on the alfalfa weevil Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) larvae, under laboratory conditions

  • Emine Baysal
  • Turgut Atay
  • Yusuf Yanar
Open Access
Research

Abstract

Control potential of 10 Beauveria bassiana isolates, isolated from Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) and Gonioctena fornicata (Brüggemann) (Coleoptera: Chrysomelidae), collected from alfalfa fields in Tokat province, Turkey, was evaluated against H. postica larvae under laboratory conditions. Concentration-response tests were carried out using the concentrations (1 × 103, 1 × 105, 1 × 107, and 1 × 109 conidia/ml) of isolates GN-23, GN-4, HP-30, and HP-6, which performed more than 95% efficacy in screening tests (1 × 107 conidia/ml) 5 days post treatments. LT50 and LT90 values at 1 × 109 conidia/ml were determined. According to the obtained results, H. postica larvae were susceptible to all the tested B. bassiana isolates.

Keywords

Entomopathogenic fungi Beauveria bassiana Efficacy Alfalfa weevil Larvae Hypera postica 

Background

Alfalfa (Medicago sativa L.) is one of the well-known and widely used forage crops in the world. Its high yield and quality allows it to be used in feeding programs of different types of livestock (Bates 1998). Alfalfa is attacked by a numerous number of insect species that cause considerable damage and reduce forage yield. Alfalfa weevil Hypera postica (Gyllenhal) (Coleoptera: Curculionidae) is one of the most important pests that attack this crop. Both adults and larvae feed on alfalfa foliage, but the larvae cause the majority of the damage. Larvae feed initially on the inside of terminal leaves and later move to the lower portions of the plant, while adults generally feed on the leaf margins. Injured leaves dry very quickly giving the field a grayish to whitish cast. This pest can be found in all alfalfa production areas of Turkey and worldwide (Cook et al. 2004 and Atanasova 2012).

Frequent cuttings are the most effective cultural method of the pest in the alfalfa fields. Chemical control can be recommended, if there are 25 adults in 1-m2 area before the first mowing (Erdoğan 2008). However, the chemical insecticides used to manage this pest are extremely hazardous to bees and other beneficial insects (Reddy et al. 2016). Some efficient biological control agents, particularly entomopathogenic fungi (EPF), and some hymenopteran parasitoids have been recorded (Atay et al. 2015; Reddy et al. 2016; Efil 2018 and Yücel et al. 2018).

The application of EPF in bio-control of insects is of immense significance because of their environmental and food safety concerns (Reddy et al. 2016). Species of the genera Beauveria, Metarhizium, Lecanicillium and Isaria are commercially produced (Vega et al. 2009). B. bassiana was reported to infect 707 species of insect hosts, including 521 genera and 149 families of 15 orders (Imoulan et al. 2017). Some studies have been conducted to determine the potential of B. bassiana as a bio-agent against various insect pests in Turkey. These studies have mostly focused on lepidopteran and coleopteran pests (Güven et al. 2015; Yanar et al. 2017).

There are several studies concerning biological control of H. postica by EPF in Turkey (Atay et al. 2015 and Yücel et al. 2018) and in the world (Hedlund and Pass 1968; Roberts et al. 1994; Mustafa et al. 2014 and Reddy et al. 2016).

The objective of this study was to determine the efficacy of 10 local B. bassiana isolates, isolated from H. postica and Gonioctena fornicata, collected from alfalfa fields in Tokat province, Turkey, against larvae of the alfalfa weevil under laboratory conditions.

Methods

Isolation of fungi

Overwintered adults of H. postica and G. fornicata emerged from the soil were collected from alfalfa fields of Tokat, Turkey, during the period, April–May 2015 (Table 1). The collections were brought to the laboratory with fresh alfalfa plants, transferred to separate cages and checked daily. The dead adults were subjected to surface sterilization with 1% sodium hypochlorite solution for 1 min, washed twice with distilled water, placed in a sterile Petri-dish containing moistened filter paper and kept at 25 ± 2 °C and 16L: 8D h photoperiod (Ali-Shtayeh et al. 2002). By the end of incubation period, the fungus was isolated from the adults, with external fungal growth, with special care (Sevim 2010). PDAY (PDA + 1% yeast extract) media were used for isolation. To prevent bacterial contamination, 50 μg/ml ampicillin, 20 μg/ml tetracycline, and 200 μg/ml streptomycin were added to the medium (Ihara et al. 2001). Single-spore isolates of all the isolates were obtained by serial dilution (Dhingra and Sinclair 1995) and were identified as B. bassiana. Totally, ten B. bassiana isolates were isolated from the field-collections of H. postica and G. fornicata adults (Table 1), and they were deposited in the fungal culture collection of the Mycology Laboratory at the Gaziosmanpasa University, Faculty of Agriculture, Department of Plant Protection in Tokat, Turkey. In order to obtain sufficient amounts of spore suspensions, the fungi were sub-cultured in PDA (Potato Dextrose Agar) medium. The fungi cultures were incubated at 25 ± 2 °C for 17 days. Ten milliliters of sterilized water with 0.02% Tween 80 was added to each plate, and spore harvesting was done by gently rubbing the culture surface, using a sterilized glass hokey. Spore suspension from each isolate was adjusted to 1 × 103, 1 × 105, 1 × 107, and 1 × 109 conidia/ml (Şahin 2006).
Table 1

Hosts and locations of the tested enthomopathogenic Beauveria bassiana isolates

Isolates

Information of Locations

Hosts

Location

Coordinates

Altitude (m)

N

E

GN-23

Gümenek, Tokat, Turkey

40° 21′ 56″

36° 38′ 39″

637

G. fornicata

GN-20-2

Yağmurlu, Tokat, Turkey

40° 30′ 51″

36° 49′ 17″

829

G. fornicata

GN-12-3

Emirseyit, Tokat, Turkey

40° 20′ 16″

36° 24′ 21″

572

G. fornicata

GN-1

Ulaş, Tokat, Turkey

40° 19′ 18″

36° 26′ 12″

600

G. fornicata

GN-4

Güryıldız, Tokat, Turkey

40° 19′ 58″

36° 22′ 35″

582

G. fornicata

GN5–2

Güryıldız, Tokat, Turkey

40° 19′ 49″

36° 22′ 04″

525

G. fornicata

GN8–2

Büyükyıldız, Tokat, Turkey

40° 20′ 12″

36° 23′ 37″

567

G. fornicata

GN-8-1(2)

Büyükyıldız, Tokat, Turkey

40° 20′ 12″

36° 23′ 37″

567

G. fornicata

HP-30

Bedirkale, Tokat, Turkey

40° 03′ 56″

36° 26′ 48 ″

1133

H. postica

HP-6

Güryıldız, Tokat, Turkey

40° 19′ 45″

36° 21′ 40″

585

H. postica

Bioassays

Primarily, screening tests were conducted to determine the efficacy of the isolates against H. postica larvae at 1 × 107 conidia/ml. To test the effect of each isolate, H. postica larvae were dipped into conidial suspension of 1 × 107 conidia/ml of each isolate for 4–5 s and placed in a Petri-dish (10 larvae per dish) containing fresh alfalfa leaves. Mortality rates were recorded on the 1st, 3rd, 5th, and 7th days post treatment. In addition, concentration-response tests were carried out with isolates proved to have a high effect, using such tested concentrations. The experiments were carried out in completely randomized block design, with three replications and replicated two times.

Statistical analysis

Test results were converted into percentages and arcsine transformed. The transformed data was analyzed by analysis of variance (ANOVA) and the means compared by Tukey’s multiple comparison tests. All statistical analyses were carried out using the MINITAB Release 16 packet program. LT50 and LT90 values of the concentration causing the fastest effect were determined, using the probit analysis.

Results and discussion

The ten EPF isolates tested against H. postica at 1 × 107conidia/ml caused 100% mortality 7 days post treatment in almost all isolates (Table 2). Concentration-response tests, carried out using concentrations of 1 × 103, 1 × 105, 1 × 107, and 1 × 109 conidia/ml of the isolates (GN-23, GN-4, HP-30, HP-6), caused more than 95% mortality rate on the 5th day.
Table 2

Mortality of Hypera postica exposed to the ten isolates at 1x107conidia/ml

Mortality±SEM*(%)

ISOLATES

1 DAT**

3 DAT

5 DAT

7 DAT

GN-23

0.29 ± 0.70a***

24.83 ± 0.16c

98.85 ± 1.12abc

100.00 ± 0.00a

GN-20-2

0.29 ± 0.70a

60.22 ± 0.52ab

94.43 ± 1.49abcd

99.71 ± 0.70a

GN-12-3

0.00 ± 0.00a

51.82 ± 0.92ab

85.66 ± 0.49d

100.00 ± 0.00a

GN-1

0.60 ± 1.45a

39.79 ± 0.68bc

86.99 ± 0.22 cd

99.71 ± 0.70a

GN-4

2.57 ± 1.25a

56.84 ± 0.62ab

97.43 ± 1.25abcd

99.71 ± 0.70a

GN-5-2

0.29 ± 0.70a

53.50 ± 0.79ab

83.64 ± 0.22d

100.00 ± 0.00a

GN-8-2

2.57 ± 1.25a

60.22 ± 0.52ab

88.54 ± 0.14bcd

99.71 ± 0.70a

GN-8-1(2)

2.57 ± 1.25a

65.50 ± 0.72a

85.36 ± 0.25d

100.00 ± 0.00a

HP-30

1.70 ± 1.74a

60.22 ± 0.52ab

99.40 ± 1.45ab

100.00 ± 0.00a

HP-6

4.53 ± 1.12a

68.72 ± 0.48a

99.71 ± 0.70a

100.00 ± 0.00a

CONTROL

0.00 ± 0.00a

0.00 ± 0.00d

1.15 ± 1.12e

13.01 ± 0.22b

*SEM Standard error of the mean

**DAT Days after treatment

***Means in a column followed by the same letter are not statistical significantly different (ANOVA P < 0.05, Tukey’s test)

The mortality rates of H. postica larvae varied from 13.66 to 72.08% 3 days post treatment. The highest mortality rate (72.08%) was recorded for HP-6 at 1 × 109 conidia/ml (Table 6). HP-30 was the most effective isolate with mortality rate of 62.22% at 1 × 103 conidia/ml (Table 5). On the 5th day, all isolates and concentrations caused more than 70% mortality, while on the 7th day the mortality rate reached 100% by almost all isolates at 1 × 107 and 1 × 109 conidia/ml (Tables 3, 4, 5, and 6).
Table 3

Mortality of Hypera postica exposed to GN-23 isolate

Mortality±SEM* (%)

Doses (conidia/ml)

1 DAT**

3 DAT

5 DAT

7 DAT

1 × 103

1.65 ± 1.25a***

13.66 ± 0.25b

70.95 ± 1.10b

86.70 ± 0.54b

1 × 105

0.29 ± 0.70a

20.86 ± 0.65b

82.19 ± 0.40b

99.71 ± 0.70a

1 × 107

0.29 ± 0.70a

24.83 ± 0.16b

98.85 ± 1.12a

100.00 ± 0.00a

1 × 109

0.60 ± 1.45a

48.58 ± 1.49a

98.85 ± 1.12a

99.71 ± 0.70a

Control

0.00 ± 0.00a

0.00 ± 0.00c

1.15 ± 1.12c

13.01 ± 0.22c

*SEM Standard error of the mean

**DAT Days after treatment

***Means in a column followed by the same letter are not statistical significantly different (P < 0.05)

Table 4

Mortality of Hypera postica exposed to GN-4 isolate

Mortality±SEM* (%)

Doses(conidia/ml)

1 DAT**

3 DAT

5 DAT

7 DAT

1 × 103

1.15 ± 1.12a***

28.24 ± 0.09b

77.18 ± 0.43b

96.63 ± 1.75a

1 × 105

1.15 ± 1.12a

41.43 ± 0.75ab

80.69 ± 0.54b

99.71 ± 0.70a

1 × 107

2.57 ± 1.25a

56.84 ± 0.62a

97.43 ± 1.25a

99.71 ± 0.70a

1 × 109

0.00 ± 0.00a

50.00 ± 0.21a

98.35 ± 1.25a

100.00 ± 0.00a

Control

0.00 ± 0.00a

0.00 ± 0.00c

1.15 ± 1.12c

13.01 ± 0.22b

*SEM Standard error of the mean

**DAT Days after treatment

***Means in a column followed by the same letter are not statistical significantly different (P < 0.05)

Table 5

Mortality of Hypera postica exposed to Hp-30 isolate

Mortality±SEM* (%)

Doses(conidia/ml)

1 DAT**

3 DAT

5 DAT

7 DAT

1 × 103

0.41 ± 0.84a***

62.22 ± 0.52a

78.11 ± 0.11b

94.13 ± 2.44a

1 × 105

0.00 ± 0.00a

56.03 ± 0.12a

80.82 ± 0.68b

95.18 ± 1.78a

1 × 107

1.70 ± 1.74a

60.22 ± 0.52a

99.40 ± 1.45a

100.00 ± 0.00a

1 × 109

0.00 ± 0.00a

66.92 ± 0.33a

100.00 ± 0.00a

100.00 ± 0.00a

Control

0.00 ± 0.00a

0.00 ± 0.00b

1.15 ± 1.12c

13.01 ± 0.22c

*SEM Standard error of the mean

**DAT Days after treatment

***Means in a column followed by the same letter are not statistical significantly different (P < 0.05)

Table 6

Mortality of Hypera postica exposed to Hp-6 isolate

Mortality±SEM* (%)

Doses(conidia/ml)

1 DAT**

3 DAT

5 DAT

7 DAT

1 × 103

0.41 ± 0.84ab***

52.01 ± 0.49b

86.34 ± 0.25b

99.59 ± 0.84a

1 × 105

0.00 ± 0.00b

59.68 ± 0.34ab

86.99 ± 0.22b

97.43 ± 1.25a

1 × 107

4.53 ± 1.12a

68.72 ± 0.48a

99.71 ± 0.70a

100.00 ± 0.00a

1 × 109

2.57 ± 1.25ab

72.08 ± 0.48a

100.00 ± 0.00a

100.00 ± 0.00a

Control

0.00 ± 0.00b

0.00 ± 0.00c

1.15 ± 1.12c

13.01 ± 0.22b

*SEM Standard error of the mean

**DAT Days after treatment

***Means in a column followed by the same letter are not statistical significantly different (P < 0.05)

LT50 for HP-30 was 2.401 days, followed by HP-6 (2.476 days), GN-23 (3.074 days), and GN-4 (3.110 days). LT90 values for the isolates of HP-6, HP-30, GN-4, and GN-23 were 3.608, 4.196, 4.343, and 4.376 days, respectively (Table 7).
Table 7

Lethal time (LT50 and LT90) values of the entomopathogenic fungi, Beauveria bassiana isolates (day)

Isolates

Slope ± SE

LT50 (95% fiducial limit)

LT90 (95% fiducial limit)

χ 2

GN-4

1.040 ± 0.148

3.110 (2.808–3.404)

4.343 (3.977–4.907)

7.4

GN-23

0.984 ± 0.122

3.074 (2.796–3.350)

4.376 (4.024–4.890)

15.7

HP-6

1.132 ± 0.151

2.476 (2.215–2.735)

3.608 (3.283–4.097)

5.9

HP-30

0.174 ± 0.167

2.401 (1.348–2.885)

4.196 (3.768–5.014)

5.8

Yücel et al. (2018) tested 7 isolates of B. bassiana and one isolate of B. pseudobassiana, isolated from H. postica, on larvae and adults of H. postica at 1 × 105, 1 × 106 1 × 107, and 1 × 108 conidia/ml. The results showed that the highest mortality rate in larvae was obtained by the isolate HpA-5 (B. bassiana) (100%) and HpI-4 (B. pseudobassiana) (97%), within 14 days at 1 × 108 conidia/ml. The highest mortality rate of adults were obtained by the isolates HpA-5 (B. bassiana) and HpI-4 (B. pseudobassiana) with 98 and 95% mortality rates within 14 days at 1 × 108 concentration, respectively.

Mustafa et al. (2014) reported that conidial suspension with 1 × 107 conidia/ml of two isolates of B. bassiana caused 100% mortality on adults of H. postica, 6 days post treatments. Also, Reddy et al. (2016) investigated the efficiency of six biorational-insecticides against H. postica larvae under laboratory conditions and found that Mycotrol® ESO (B. bassiana GHA) lasted 5–9 days to kill 100% of H. postica larvae in all the tested concentrations (0.072, 0.36, 0.72, and 1.44 ml/l). In addition, Atay et al. (2015) stated that 23.25% of H. postica adults, overwintered in alfalfa growing areas of Tokat province in Turkey, were found naturally infected with Beauveria spp. Harcourt et al. (1977) reported that larvae of this pest were found infected with Entomophthora phytonomi Arthur, which considerably reduced the weevil population in Canada.

Conclusions

The present study showed that the local isolates of B. bassiana could be suggested as bio-control agent against H. postica larvae; however, further studies should be conducted under field conditions.

Notes

Funding

The study was supported by Gaziosmanpaşa University Scientific Research Fund (project number: 2015/138).

Availability of data and materials

The dataset(s) supporting the conclusions of this article is(are) included within the article (and its additional file(s)).

Authors’ contributions

The whole team jointly planned the experiments. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of Plant Protection, Faculty of AgricultureGaziosmanpasa UniversityTokatTurkey

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