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
Full size table

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
Full size table
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)
Full size table

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
Full size table
Fig. 1
figure 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

Full size image

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
figure 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

Full size image

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.