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Aerobic gut bacterial flora of Cydia pomonella (L.) (Lepidoptera: Tortricidae) and their virulence to the host

Egyptian Journal of Biological Pest Control201828:30

https://doi.org/10.1186/s41938-018-0036-1

  • Received: 4 January 2018
  • Accepted: 21 February 2018
  • Published:

Abstract

This study aimed to isolate and characterize bacteria from the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), and determine their virulence to its larvae. A total of 16 bacteria were isolated from larvae belonging to different instars. Based on morphological, biochemical, physiological, and molecular studies, the bacterial isolates were identified as Pseudomonas sp. (Cp1, 3, 5, and 13), Corynebacterium sp. (Cp2), Bacillus sp. (Cp4, 7, 9, 10, 12, and 15), Pectobacterium carotovorum (Cp6), Paenibacillus sp. (Cp8), Bacillus megaterium (Cp11), Bacillus pumilus (Cp14), and Terribacillus saccharophilus (Cp16). It was also determined the virulence of these isolates, where the highest potential activity was obtained by Bacillus sp. Cp9, with (76%) mortality. These results could be beneficial for future biocontrol programs of C. pomonella.

Keywords

  • Bacteria
  • Codling moth
  • Cydia pomonella
  • Virulence
  • Microbial control

Background

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is one of the most important pests in many orchards worldwide, mainly apples, pears, quince, peach, plum, apricot, and walnut. It causes economic losses in fruit production (Pajac et al. 2012 and Alford 2014). The larvae of this pest which overwinter in the cracked bark of tree trunks and in cocoons at packaging and storage places develop to pupae in late April to early May. The mated female moths emerging from pupae lay eggs at appropriate temperatures. The hatched larvae burrow into the fruit within 4–8 h and render them unsalable (Beers et al. 2003). In order to control this pest and to obtain undamaged fruits, traditional insecticides such as organophosphorus compounds and synthetic pyrethroids have been used. However, these insecticides cause unfavorable environmental impacts. In addition, some strains of this pest have acquired resistance to several insecticides (Lacey and Unruh 2005).

Entomopathogenic microorganisms such as bacteria, viruses, fungi, nematodes, and protists are able to infect different insect species, and they can be used as biological control agents against insect pests (Khetan 2001). Among entomopathogens, the entomopathogenic bacteria (EPB) play a key role in the commercial control of insect pests and Bacillus thuringiensis (Bt) is the species on which most of the scientific community and industry efforts have been focused (Owuama 2001 and Ruiu et al. 2013). Apart from Bt, many different EPB belonging to different species of Bacillus and other genera, such as Bacillus sphaericus, Paenibacillus papillae, and Serratia entomophila, are available as insecticides (Federici 2007).

Many insect life cycles are associated with symbiotic microorganisms, and there is increasing evidence that symbiotic microorganisms influence many insect features such as sex determination, nutrient exchange, nutrition, and digestion processes (Rajagopal 2009; Douglas 2014; and Brune 2014). However, some insect groups are not obligatory dependent on their microbiota (Douglas 2014). Symbiotic microorganisms, especially bacteria, can be used in the biological control of insect pests through the use of different methods, e.g., they can be used to express insecticidal toxins or proteins by using genetic engineering techniques (Beard et al. 1998). In addition, changing the dynamics among bacterial microbes in the insect gut could be used for controlling insect pests. For different purposes, the microbiota of many insect species has been determined (Sevim et al. 2012; Demirci et al. 2013; and Roopa et al. 2014).

This study aimed to isolate and characterize bacterial species from C. pomonella. Additionally, these bacterial isolates were tested against the larvae of the codling moth.

Methods

Collection of larvae

Different larval instars of C. pomonella were collected from infested walnut fruits at the vicinity of Kırşehir, Turkey, in the summer of 2015. The obtained larvae were separated according to the developmental stage (instars) and used in the process of bacterial isolation.

Isolation of bacteria

The collected larvae were divided into three groups based on their instars. The first group consisted of the first and second larval instars, the second group consisted of the third instar, and the third group consisted of the fourth and fifth larval instars. The bacterial isolation was separately performed from these groups. A total of ten larvae were used for each group for the bacterial isolation. The larvae were surface-sterilized with 70% ethanol for 2–3 min and washed three times with sterile distilled water (Lipa and Wiland 1972). Thereafter, the larvae belonging to the different groups were separately placed into glass test tubes (10 ml) including 3 ml nutrient broth (Difco, NJ, USA) with sterile forceps and completely homogenized, using a sterile glass tissue grinder. The homogenates were filtered through two layers of sterile cheesecloth to remove insect debris. A series of dilutions from 10−1 to 10−8 were prepared from the insect homogenates, and 10−1, 10−3, 10−5, and 10−8 dilutions from each homogenate were plated on nutrient agar and then incubated at 30 °C for 3 days. In addition, these dilutions were heated at 80 °C for 10 min to eliminate non-spore-forming bacteria. Then, they were plated on nutrient agar and incubated at 30 °C for 3 days; then, the bacterial colonies were counted, and the total number of bacteria per larvae was calculated as 2 × 106 cfu (colony forming units). Moreover, the different bacterial colonies were streaked on nutrient agar and incubated at 30 °C for 18 and 48 h for slow-growing isolates to obtain pure cultures. The obtained pure cultures were stored in 20% glycerol at − 20 °C. The bacterial isolates were identified based on various tests. All isolates from this study are publicly accessible and were deposited at Microbiology Laboratory, Genetic and Bioengineering, Ahi Evran University, Kırşehir, Turkey.

Morphological characterization of the bacterial isolates

The bacterial isolates were morphologically characterized on the basis of their colony, cell, and spore features. Colony morphologies of the bacterial isolates were evaluated on nutrient agar plates by using a stereomicroscope (Demirci et al. 2013). Cell properties of the isolates were evaluated by the gram and endospore staining. The capsule layer of the bacterial isolates was determined by negative staining. The motility of the isolates was determined according to the method of Soutourina et al. (2001).

Physiological characterization of the bacterial isolates

The bacterial isolates were also physiologically characterized on the basis of their growth at different temperatures, NaCl concentrations, and pH. All isolates were inoculated into nutrient broth (3 ml) and incubated at different temperatures ranging from 4 to 55 °C. Also, all isolates were incubated into nutrient broth (3 ml) with different concentrations of NaCl, ranging from 3 to 15%. Finally, all isolates were incubated into nutrient broth (3 ml) with different pH values ranging from 3 to 12. Evaluations were visually made.

VITEK 2 microbial identification system

The bacterial isolates were also identified using the VITEK 2 microbial identification system. Firstly, the bacterial isolates were streaked on nutrient agar plates to obtain single colonies. The bacterial suspensions were prepared from a single colony, using 2 ml of 0.45% sterile saline solution to the equivalent of a 0.5 McFarland turbidity standard. Concentrations were checked with the VITEK colorimeter for each isolate. Additionally, the oxidase and catalase production of the isolates were manually determined. A total of two cards were used to identify the isolates. The GN ID card was used for gram-negative bacterial identification and the GP ID card was used for gram-positive bacterial identification. The bacterial suspensions prepared as above were inoculated onto these cards and incubated at 30 °C for 18 h. The time between preparation of the suspension and card filling was less than half an hour. The results were automatically evaluated with the VITEK 2 device (Ligozzi et al. 2002).

16S rRNA gene sequencing

The bacterial isolates were further characterized, using the partial sequencing of 16S rRNA gene. Genomic DNAs were extracted by the Genomic DNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNAs were stored at − 20 °C until PCR was done.

Approximately 1.450 bp of the 16S rRNA gene region was targeted and amplified. The primer pairs of 27F (5′-AGAGTTTGATCMTGGCTCAG-3′ as forward) and 1492R (5′-GGYTACCTTGTTACGACTT-3′ as reverse) were purchased from MACROGEN and used for amplification. The total volume of PCR reactions was 50 μl to which 50–100 ng genomic DNA was added. The PCR mix of 50 μl per sample contained 25 pmol of each primer, 200 mM each of the dNTPs, 1×PCR buffer, 3 mM MgCl2 and 1.5 U Taq DNA polymerase. After adding all the components, the final volume was adjusted to 50 μl with sterile distilled water. The PCR program consisted of 95 °C (60 s) for the initial denaturation, followed by 35 cycles of 94 °C (45 s) for denaturation, 55 °C (30 s) for annealing, 72 °C (1.5 min) for extension, and a final extension of 72 °C (5 min). After performing PCR, 5 ml of the products was analyzed by electrophoresis on 1.0% agarose gel containing ethidium bromide to check the sizes and amounts of the amplicons. After checking PCR products, the accurate products were sent to MACROGEN (the Netherlands) for sequencing. The PCR products were sequenced with the primer pairs 518F (5′-CCAGCAGCCGCGGTAATACG-3′) and 800R (5′-TACCAGGGTATCTAATCC-3′). The obtained sequences were subjected to the nucleotide BLAST searches in the NCBI GenBank database to get the percentage similarity of the bacterial isolates to the most related bacterial species (Altschul et al. 1990).

Phylogenetic analysis

Phylogenetic analysis of the bacterial isolates and their closely related species was performed for molecular characterization of the bacterial isolates. The sequences were edited using Bioedit, and multiple sequence alignments were created by using 16S rRNA sequences belonging to our strains and different bacterial species from the NCBI GenBank database for the purpose of developing a phylogenetic tree. The multiple sequence alignment was performed with ClustalW in Bioedit (Hall 1999). Finally, the sequences were subjected to neighbor-joining analysis with p-distance correction, gap omission, and 1.000 bootstrap pseudoreplicates using MEGA 6.0 (Tamura et al. 2013).

Nucleotide sequence accession numbers

The GenBank accession numbers of the 16S rRNA gene sequences belonging to the bacterial isolates from this study are listed in Table 4.

Bioassay

Each bacterium isolated from C. pomonella in the stock culture was streaked on nutrient agar to obtain a single colony and to check the purity of the cultures. After that, 3 ml of nutrient broth was inoculated from each single colony of 16 bacteria and incubated at 30 °C overnight. At the end of the incubation period, the bacterial density was measured at 600 nm absorbance and adjusted to 1.8 × 109 cfu/ml by centrifugation (4.000 rpm for 15 min) and using sterile phosphate buffer solution (PBS) (Moar et al. 1995). The bacterial solutions were freshly prepared and used for bioassay.

For the bioassay experiments, healthy C. pomonella larvae were obtained from the laboratory culture at Ahi Evran University, Genetic Bioengineering and Microbiology Laboratory. Healthy larvae were randomly selected and used for the bioassay. Twenty-five grams of freshly prepared artificial diet (for diet ingredients and rearing conditions (Fukova et al. 2005)) was inoculated with 1 ml of the bacterial suspension prepared as described above for each isolate. For the control group, 25 g of the artificial diet had 1 ml of the sterile PBS added. The contaminated artificial diets were separately placed into plastic boxes (20 × 10 × 8 mm) with ventilated lids to permit airflow. After that, ten third instar C. pomonella larvae were placed into the box for each replicate and allowed to feed on the contaminated diets. A total of ten larvae were used for each replicate, and all experiments were repeated three times. Finally, the plastic boxes were incubated at 25 °C under 16:8 (day:night) light regime. After 10 days, the boxes were checked for larval mortality, and the number of dead larvae was recorded. Mortality data were corrected based on Abbott’s formula (Abbott 1925). To determine the differences among the isolates and the control group, the data were subjected to ANOVA and subsequently to the LSD multiple comparison test. Before performing ANOVA, all data were tested for homogeneity of variance using Levene’s statistic. All tests were performed with SPSS 16.0 statistical software.

Results and discussion

A total of 16 isolates of bacteria were obtained from the treated C. pomonella larvae. Among these isolates, five were from the first and second larval instars, eight from the third larval instar, and three from the fourth and fifth larval instars. The isolates were characterized on the basis of their morphological, physiological, and molecular characteristics. Colonies of all isolates were smooth, except for Cp9 and Cp11, which were rough. Only one isolate (Cp12) had a mucoid colony. Three isolates (Cp3, Cp5, and Cp13) had yellow colonies, and one isolate (Cp4) had a pink colony. The other isolates produced creamy-colored colonies. Five isolates (Cp1, Cp3, Cp5, Cp6, and Cp13) were gram-negative, and the others were gram-positive. All isolates were bacilli-shaped, except for Cp2 and Cp3 which were coccus shaped. It was found that six isolates (Cp7, Cp8, Cp9, Cp10, Cp14, and Cp16) formed spores. Only two isolates (Cp1 and Cp13) had capsules. All morphological characteristics of the bacterial isolates are given in Table 1.
Table 1

The morphological properties of the bacterial isolates

Isolate

Colony shape

Colony color

Gram staining

Cell shape

Spore staining

Motility

Capsule

Growth in NBa

Instar

Cp1

Smooth

Cream

Bacil

+

Turbid

1–2

Cp2

Smooth

Cream

+

Coccus

Turbid

1–2

Cp3

Smooth

Yellow

Coccus

Turbid

1–2

Cp4

Smooth

Pink

+

Bacil

Turbid

1–2

Cp5

Smooth

Yellow

Bacil

Turbid

1–2

Cp6

Smooth

Cream

Bacil

+

Turbid

3

Cp7

Smooth

Cream

+

Bacil

+

+

Turbid

3

Cp8

Smooth

Cream

+

Bacil

+

+

Turbid

3

Cp9

Rough

Cream

+

Bacil

+

+

Turbid

3

Cp10

Smooth

Cream

+

Bacil

+

+

Turbid

3

Cp11

Rough

Cream

+

Bacil

Turbid

3

Cp12

Mucoid

Cream

+

Bacil

+

Precipitated

3

Cp13

Smooth

Yellow

Bacil

+

+

Turbid

3

Cp14

Smooth

Cream

+

Bacil

+

Turbid

4–5

Cp15

Smooth

Cream

+

Bacil

+

Turbid

4–5

Cp16

Smooth

Cream

+

Bacil

+

+

Turbid

4–5

aNutrient broth

All isolates were able to grow in 3% NaCl, and only one isolate (Cp3) could not grow in 4% NaCl. Growth characteristics of the isolates in other NaCl concentrations were variable, depending on the isolate. All isolates were able to grow in the pH range of 3, 4, and 5, except for Cp12. All isolates were able to grow at pH 6 and 7. Growth characteristics of the isolates at other pHs were variable, depending on the isolate. None of the isolates grew at pH 4 and 55 °C, and growth properties of the isolates at 30, 37, 45, and 50 °C were variable, depending on the isolate. Physiological properties of the isolates are given in Table 2. The VITEK 2 microbial characterization system for biochemical characterization of the isolates and gram-negative and gram-positive cards was used to identify them. Five isolates (Cp6, Cp12, Cp14, Cp15, and Cp16) were not able to be characterized by VITEK 2. Other identifications are given in Table 3.
Table 2

The physiological properties of the bacterial isolates. Luria-Bertani broth was used as growth medium

Isolate

Growth

NaCl (%)

pH

Temperature (°C)

3

5

7

10

12

15

3

4

5

6

7

8

9

10

12

4

30

37

45

50

55

Cp1

+

+

+

+

+

+

+

+

+

+

+

Cp2

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp3

+

+

+

+

+

+

+

+

+

+

+

Cp4

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp5

+

+

+

+

+

+

+

+

+

+

+

+

Cp6

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp7

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp8

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp9

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp10

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp11

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp12

+

+

+

+

+

+

+

+

+

+

+

+

Cp13

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp14

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp15

+

+

+

+

+

+

+

+

+

+

+

+

+

Cp16

+

+

+

+

+

+

+

+

+

+

+

+

+

+

+

Table 3

Percent similarity of the bacterial isolates with their closely related species based on the BLAST searches in NCBI GanBank database (Altschul et al. 1990)

Isolate

Bacterial species

GenBank accession number

Query cover (%)

Similarity (%)

VITEK2 (%)

Cp1

Pseudomonas sp. PDD-59b-7

Pseudomonas sp. R3ScM3P1C11

Pseudomonas syringae strain PDD-48b-5

KR922145

KF147001

KR922059

96

96

96

99

99

99

Lysinibacillus sphaericus/Lysinibacillus fusiformis (91)

Cp2

Corynebacterium variabile strain C3-13

Corynebacterium sp. ZT10-3

Corynebacteriumvariabile DSM 44702

KP114214

KT597082

NR102874

96

96

96

96

96

96

Gardnerella vaginalis (93)

Cp3

Pseudomonas sp. MN11-3

Pseudomonas matsuisoli strain CC-MHH0089

Pseudomonas matsuisoli strain CC-MHH0089

JQ396614

NR134793

KJ720680

97

97

97

96

96

96

Dermacoccus nishinomiyaensis/Kytococcus sedentarius (96)

Cp4

Bacterium BEL C12

Bacillus sp. 13K7a2

Bacillus sp. 7Kp1a

Bacillus pumilus strain OU101

KT382407

KT825840

KT825839

KR140377

96

96

96

96

99

99

99

99

Lysinibacillus sphaericus/Lysinibacillus fusiformis (87)

Cp5

Pseudomonas matsuisoli strain CC-MHH0089

Pseudomonas matsuisoli strain CC-MHH0089

Pseudomonas sp. RBSB9_C3

NR134793

KJ720680

KT390731

97

97

96

96

96

97

Aeromonas salmonicida (98)

Cp6

Pectobacterium carotovorum subsp. brasiliense strain Y45 16S

Pectobacterium carotovorum subsp. brasiliense strain Y34 16S

Pectobacterium carotovorum subsp. brasiliense strain Y33 16S

KP187510

KP187504

KP187503

98

98

98

97

97

97

Unidentified organism

Cp7

Bacillus sp. BG2-9

Bacillus pumilus strain T246

Bacillus pumilus strain ML353

KP992115

KC764989

KC692160

98

99

99

97

97

97

Bacillus pumilus (85)

Cp8

Paenibacillus sp. MOLA 507

Bacterium UKR A17

Paenibacillus sp. S8

AM990732

KT382376

KR051059

96

96

96

97

97

97

Paenibacillus polymyxa (90)

Cp9

Bacillus sp. 210_50

Bacillus sp. strain RHH15

Bacillus sp. L11(2010)

GQ199752

HQ143613

HQ222333

99

99

99

96

96

96

Bacillus pumilus (86)

Cp10

Bacillus pumilus strain X22

Bacillus pumilus strain HN-30

Bacillus pumilus strain HN-10

FJ763645

KT003271

KT003256

99

99

99

98

98

98

Bacillus pumilus (88)

Cp11

Bacillus megaterium strain D5

Bacillus megaterium strain BCRh8

Bacillus megaterium strain BS9

KC441754

KT153604

KR063189

99

99

99

98

98

98

Bacillus megaterium (87)

Cp12

Bacillus sp. C26(2014)

Bacillussubtilis strain L-13

Bacillustequilensis strain YJ-S4

KM117217

HQ232422

KF876849

99

99

99

99

99

99

Unidentified organism

Cp13

Pseudomonas sp. BE07

Uncultured bacterium isolate 1112863845131

Pseudomonas sp. DR11(2011)

AY456700

HQ121073

JN210571

97

97

97

96

96

96

Aeromonas salmonicida (97)

Cp14

Bacillus pumilus strain ZA13

Bacillus pumilus strain LX11

Bacillus pumilus strain Y13

FJ263042

KP192031

KF641806

98

98

98

99

99

99

Unidentified organism

Cp15

Bacillus subtilis strain L-13

Bacillus subtilis strain YA4BZ

Bacillus sp. RKZ11262

HQ232422

JQ346075

EU835569

98

98

98

99

99

99

Unidentified organism

Cp16

Terribacillus saccharophilus strain MER_108

Terribacillus saccharophilus strain JP44SK46

Terribacillus saccharophilus strain WA2-4

KT719683

JX155763

JF496471

96

96

96

96

96

96

Unidentified organism

The bacterial isolates were also characterized on the basis of 16S rRNA gene sequencing to verify the recorded conventional characterizations of the isolates. Based on molecular characterization, the bacterial isolates were identified as Pseudomonas sp. (Cp1, 3, 5, and 13), Corynebacterium sp. (Cp2), Bacillus sp. (Cp4, 7, 9, 10, 12, and 15), Pectobacterium carotovorum (Cp6), Paenibacillus sp. (Cp8), Bacillus megaterium (Cp11), Bacillus pumilus (Cp14), and Terribacillus saccharophilus (Cp16) (Table 4). This identification was also supported by phylogenetic analysis (Fig. 1).
Table 4

The proposed identification results of the bacterial isolates and their GenBank accession numbers for 16S rRNA gene sequences

Isolate

Species

GenBank accession number

Cp1

Pseudomonas sp.

KX094470

Cp2

Corynebacterium sp.

KX094471

Cp3

Pseudomonas sp.

KX094472

Cp4

Bacillus sp.

KX094473

Cp5

Pseudomonas sp.

KX094474

Cp6

Pectobacterium carotovorum

KX094475

Cp7

Bacillus sp.

KX094476

Cp8

Paenibacillus sp.

KX094477

Cp9

Bacillus sp.

KX094478

Cp10

Bacillus sp.

KX094479

Cp11

B. megaterium

KX094480

Cp12

Bacillus sp.

KX094481

Cp13

Pseudomonas sp.

KX094482

Cp14

B. pumilus

KX094483

Cp15

Bacillus sp.

KX094484

Cp16

Terribacillus saccharophilus

KX094485

Fig. 1
Fig. 1

Phylogenetic tree derived from neighbor-joining analysis of 16S rRNA sequences (1400 bp) from the flora members of C. pomonella and their closely related species. Bootstrap values based on 1000 replicates were indicated above nodes. Bootstrap values C ≥ 70 are labeled. C. pomonella isolates were indicated with black circle. The scale on the bottom of the dendrogram indicates the degree of dissimilarity

All isolates caused different mortality values in comparison to each other (F = 15.43, df = 16, p < 0.05). The highest mortality values were obtained from Bacillus sp. Cp4, Cp9, and Cp10 with 70, 76, and 63%, respectively (F = 15.43, df = 16, p < 0.05). Other mortalities ranged from 3 to 56% (Fig. 2). Ertürk and Demirbağ (2006) studied the ability of culturing a bacterial flora of C. pomonella. They obtained eight bacterial isolates from the larvae of this pest, collected from apple fruits. Also, the bacterial flora were Proteus rettgeri (Cp1), Eschericia coli (Cp2), Pseudomonas stutzeri (Cp3), Pseudomonas aeroginosa (Cp4), Bacillus laterosporus (Cp5), Micrococcus sp. (Cp6), Proteus vulgaris (Cp7), and Deinococcus sp. (Cp8). However, in this study, 16 bacterial isolates from the same insect collected from walnut fruits were obtained. The microbiota of insects was affected by many factors such as diet, development stage, habitat, and phylogeny of the host (Yun et al. 2014). The difference between these studies with respect to the bacterial diversity might be due to the use of different diets of C. pomonella larvae.
Fig. 2
Fig. 2

Virulence of the bacterial isolates using the bacterial concentration of 1.89 × 109 cfu/ml against C. pomonella larvae within 10 days after application. Mortality data were corrected according to Abbott’s formula (Abbott 1925). Bars indicate standard deviation. Different lowercase letters represent statistically significant differences among larval mortalities. Cp1 Pseudomonas sp., Cp2 Corynebacterium sp., Cp3 Pseudomonas sp., Cp4 Bacillus sp., Cp5 Pseudomonas sp., Cp6 Pectobacterium carotovorum, Cp7 Bacillus sp., Cp8 Paenibacillus, Cp9 Bacillus sp., Cp10 Bacillus sp., Cp11 B. megaterium, Cp12 Bacillus sp., Cp13 Pseudomonas sp., Cp14 B. pumilus, Cp15 Bacillus sp., and Cp16 Terribacillus saccharophilus

Among the EPB, spore-forming bacilli are the major group of species of bacteria that infect and kill insects (Aronson et al. 1986). Many different Bacillus species have been isolated from different insects which are harmful in both agriculture and forestry. In the present study, eight different Bacillus species were obtained and characterized, and some of them, namely Cp4, Cp9, and Cp10, showed a high virulence against larvae of C. pomonella.

The genus Pseudomonas contains 191 currently described species (Euzeby 1997). Some of them are entomopathogenic such as Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas entomophila, and Pseudomonas taiwanensis (Khetan 2001; Mahar et al. 2005; Chen et al. 2014; and Dieppois et al. 2014). In the present study, four isolated Pseudomonas species were not characterized at the species level. Among them, Pseudomonas sp. Cp1 showed insecticidal activity against the larvae of the codling moth. This may suggest that probably a new Pseudomonas species might be isolated from the codling moth. However, more detailed identification studies should be conducted to verify this probability.

The genus Paenibacillus includes bacteria which are facultative anaerobic and endospore-forming. This genus was previously included in the Bacillus genus but was reclassified as a separate genus (Ash et al. 1993). The members of this genus, which have been isolated from various environments such as soil, rhizosphere, water, clinical samples, and insects, are becoming important in agricultural and medical applications (McSpadden Gardener 2004; Lal and Tabacchioni 2009; and Danismazoglu et al. 2012). This genus includes some insect pathogenic bacteria such as Paenibacillus larvae, Paenibacillus popilliae, and Paenibacillus lentimorbus (Ruiu 2015). In the present study, one Paenibacillus sp. (Cp8) from live larva was isolated. However, it did not show any insecticidal activity against the larvae of the codling moth.

The genus Corynebacterium contains gram-positive, aerobic, and rod-shaped bacteria. The members of this genus are widespread in nature and have been isolated from different human and animal habitats (Collins et al. 2004). Some non-pathogenic members of this genus are also intensely used in industrial applications such as the production of amino acids, bioconversion of steroids, degradation of hydrocarbons, and cheese aging (Yamada et al. 1972 and Lee et al. 1985). Some members of this genus have been isolated from insects (Bucher 1981 and Hoeven et al. 2008). In this study, a Corynebacterium sp. (Cp2) was isolated from the codling moth, but it had no insecticidal activity against the larvae.

The genus Pectobacterium (formerly known as Erwinia) is a member of the family Enterobacteriaceae, and some species have been isolated from different environments such as soil, water, plants, and invertebrates (Ian et al. 2003 and Glasner et al. 2008). Some species within this genus, such as P. carotovorum, is an important plant pathogen of many vegetable plants such as tomato, potato, and carrot (Ma et al. 2007). Some studies showed an isolation of the members of this genus from insects (Gnanamanickam 2006). In the present study, the species (Cp6) was also isolated, but it had no any insecticidal activity.

Terribacillus is a genus of the family Bacillaceae that contains species that are aerobic, spore-forming, gram-positive, rod-shaped, and halophilic (An et al. 2007). The members of this genus have been isolated from various environments such as soil and saline lake sediments (An et al. 2007 and Liu et al. 2010). Some studies showed an association of some species of this genus with invertebrates (Menezes et al. 2010 and Vicente et al. 2013). In this study, also one Terribacillus species (T. saccharophilus Cp16) was isolated but it had no any insecticidal activity.

Conclusions

The aerobic gut bacteria of C. pomonella were isolated and characterized searching for bacterial control agents which may be used against it. Some of the flora members (Cp1, Cp4, Cp9, and Cp10) showed significant insecticidal activity under laboratory conditions, especially Bacillus sp. Cp9 that showed promising results against larvae of the pest. Further studies are still needed to determine the efficacy of this isolate under field conditions. Mass production and formulation studies are also warranted.

Declarations

Acknowledgements

We would like to thank Dr. Frantisek Marec for providing C. pomonella eggs.

Funding

This study was supported by Ahi Evran University Scientific Research Projects Coordination Unit, project number: PYO-MÜH.4001.15.008.

Availability of data and materials

Not applicable.

Authors’ contributions

ES carried out a large part of the whole experiments. MÇ collected the insect specimens in the field and participated in the bacterial isolation experiments. FMS carried out the VITEK-2 bacterial identification experiments. AS participated in the 16S rRNA gene sequencing, phylogenetic analysis, statistical analysis, and writing of the whole manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

None of the authors have any competing interests in the manuscript.

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Open Access This 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’ Affiliations

(1)
Faculty of Engineering and Architecture, Genetic and Bioengineering, Ahi Evran University, 40100 Kırşehir, Turkey
(2)
Faculty of Arts and Sciences, Department of Biology, Ahi Evran University, 40100 Kırşehir, Turkey
(3)
Department of Medical Microbiology, Ahi Evran University School of Medicine, 40100 Kırşehir, Turkey

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