The relationship between insecticidal effects and chitinase activities of Coleopteran-originated entomopathogens and their chitinolytic profile

Abstract

Peritrophic membrane (PM) is present in most insects’ midgut and acts as a mechanical barrier to protect the epithelium from various harmful factors such as pathogens or toxins. Chitinase is a virulence factor due to its ability to degrade the chitin content of PM. Therefore, chitinase is a mediator for easier binding of toxins to gut epithelium and intercepting nutritional absorption in the midgut. One hundred and eight bacterial isolates derived from microbial flora of coleopteran pests were screened to determine chitin-producing entomopathogenic bacteria. The M9 chitin–-agar method and polymerase chain reaction with specific primers for a conserved domain of chitinase genes indicated that 23 of the 108 isolates have chitinase activity. The chitinase activities of the chitinase-positive bacteria were measured. We compared these results with the insecticidal activities results to determine, statistically, the potential relationship between the chitinase activities and the insecticidal activity. Consequently, 21.3% of bacterial isolates showed chitinolytic ability and among these the chitinase-positive bacteria, Serratia marcescens, was found the most active one in the M9-CAD method. More importantly, our study indicated a very strong positive correlation between the insecticidal activities of isolates and the chitinase activities with the M9-CAD method (r ² = 0.96, p ≤ 0.01), but not with the DNS method (r ² = −0.279, p ≤ 0.01). This strong relationship of entomopathogens has a high potential for biocontrol of Coleopteran pests.

Introduction

The Coleopterans are the largest order of insects, representing about 40% of the known insect species (Liebherr and McHugh 2003). Certain Coleopterans are serious pests of forest or agricultural crops throughout the world. Chemical insecticides which have a variety of side effects on the environment have been used for several years against these pests. Because of their hazardous effects, these chemicals are no longer recommended for agricultural pest management. Fortunately, entomopathogens do not harm other animals or plants. Serratia marcescens is an efficient biological degrader of chitin and also a potential biological agent for fungi and other organisms (Tu et al. 2010).

Chitin, homopolymer of β 1–4 linked N-acetylglucosamine, is the second most abundant polysaccharide and renewable source in nature after cellulose (Duo-Chuan 2006). Insects’ peritrophic membranes (PM) are film-like structures including important amounts of chitin embedded in a protein–carbohydrate matrix. PM separates food from midgut tissues and protects the epithelium against food abrasion and microorganisms (Granados et al. 2001). Consequently, any defect in PM reduces feeding and protection against microbial attack.

Chitinase, an extra-cellular enzyme, is able to effectively break down glycosidic bonds of chitin polymer in the content of PM and allow the passage of all kinds of components found in the midgut through this barrier. In this way, some bacteria and their toxic components reach the endothelial cells of the midgut and become a threat to insect life (Huber et al. 1991; Langer et al. 2000).

Degradation of the PM by chitinases has long been a very attractive biological control method against insect pests of crop plants (Regev et al. 1996; El–Tarabilya et al. 2000). Up to now, some chitinolytic bacteria have been shown to be potential biological control agents of insect pests (Khmel et al. 1998). Furthermore, chitinolytic bacteria can be pathogens of fungi, such as Ewingella americana, the causative agent of internal stipe necrosis of the commercial mushroom Agaricus bisporus (Jolles and Muzzarelli 1999). Therefore, chitinases are of great biotechnological interest and all chitinolytic bacteria have the potential to be used as biological control agents (Yanhua et al. 2007).

At the present time, there is an increasing interest in using biological control agents as alternatives for chemical insecticides. Therefore, chitinases have been used directly or as improvers of virulence with many pathogenic agents (Otsu et al. 2003).

So far, there has been no study on the chitinolytic profile of enthomopathogenic bacteria from the microbial flora of Coleopteran pests. In this article, we determined the chitinolytic bacteria profile in a bacterial library of 108 Coleopteran pests, tested their chitinase activities and compared these values with their insecticidal activities obtained from our previous studies.

Materials and methods

Bacterial isolates and growth conditions

Bacterial isolates were provided from the Bacterial Library of the Karadeniz Technical University Microbiology laboratory (BLCP) (www.microlabktu.com) (Table 1). All bacterial strains have been isolated from Coleoptera. All isolates were incubated in Luria-Bertani broth (LB) medium at 30°C for 16 h. After incubation cultures were centrifuged at 2,300 g for 5 min to pellet the cells. The pellets were washed two times to remove any nutrient substances from the LB medium and were re-suspended in sterilized phosphate-buffered saline (PBS). The cell densities were adjusted to one at OD₆₀₀ (Sambrook et al. 1989). These suspensions were used for inoculations in chitinolytic ability and chitinase activity tests.

Table 1 The source and chitinolytic activities of bacterial isolates

Chemicals

Colloidal chitin (Sigma, C7170 USA) was prepared as described by Hsu and Lockwood 1975. Five grams of powdered chitin were dissolved in 100 ml concentrated hydrochloric acid and stirred overnight. This mixture was dissolved in 2 l ethanol with vigorously stirring for 2 days. The colloids were collected and concentrated by centrifugation for 10 min at 15,000 g. The chitin pellet was re-suspended and re-centrifuged in distilled water until the pH reached 5.5–6 several times. The final chitin cake was re-suspended in distilled water (1%), autoclaved and stored at 4°C. M9 medium was prepared according to the protocol described by Sambrook et al. (1989).

Screening the bacterial isolates for their chitinolytic ability

Two different approaches, molecular detection with specific degenerate primers and M9-Chitin Agar (M9-CA) methods were used to determine if the bacterial isolates have conserved regions of chitinase coding genes and chitinolytic ability on agar plates, respectively.

Molecular detection of chitinolytic ability with specific degenerate primers

The degenerate chitinase primers GA1F (5′-CGTCGACATCGACTGGGARTDBCC-3′) and GA1R (5′-ACGCCGGTCCAGC CNCKNCCRTA-3′) were used to amplify part of chitinase coding gene. PCR was performed in a final volume of 50 μl containing 400 nM of each primer, 0.2 mM of each dNTP in 1.5 mM MgCl₂, 10 mM Tris–HCl (pH 9.0 at 25°C), 50 mM KCl, 0.1% Triton X-100, and 0.5 units of Taq DNA polymerase (Promega). PCR reaction conditions was adjusted as: denaturation for 5 min at 94°C, one cycle, and then 35 cycles of 94°C for 1 min, 55°C for 10 s and 72°C for 1 min followed by one cycle of 72°C for 10 min. PCR products were analyzed in a 1.5% agarose gel stained with EtBr. The size of the amplified products were 450 bp long. This region codes conserved blocks of amino acids within the catalytic domain of bacterial chitinase proteins (Williamson et al. 2000).

M9-chitin agar (M9-CA) method

M9 minimal agar plates were incorporated with 0.5% (v/v) of colloidal chitin to prepare M9-CA plates. Ashless Whatman filter paper disks, 5 mm in diameter, were located on M9-CA plates and used for bacterial inoculation. The paper disks were saturated with 5 μl of the PBS suspensions in triplicate for each bacterium. These plates were incubated at 30°C. After 10 days of incubation, bacterial isolates which had clear zones surrounding the colony were evaluated as chitinase-positive.

Chitinase activity assays of chitinase positive entomopathogenic bacteria

The chitinase activities of chitinase positive isolates were evaluated by two different methods. These methods were the M9-Chitin Agar Diffusion (M9-CAD) method and the colorimetric 3.5-Dinitrosalicylic acid (DNS) method.

M9-chitin agar diffusion (M9-CAD) method

M9 Chitin Broth (M9-CB) medium that includes 0.5% (v/v) of colloidal chitin was inoculated with 10% (v/v) of the PBS suspensions of each bacterial strain and incubated at 30°C for 18 h. After incubation, cultures were centrifuged at 1,000 g for 10 min. The bacterial pellets were re-suspended at 1.89 at OD₆₀₀ in sterilized PBS (Moar et al. 1995) and 5 μl of these was used to saturate the paper disks. These disks were inoculated into separate M9-CA plates in triplicates and incubated at 30°C. The chitin-free halos which occurred by diffusion of secreted chitinases around the colonies were measured 1 week after incubation. The milimetric values were evaluated as chitinase activity.

3,5-Dinitrosalicylic acid (DNS) method

The PBS suspensions of all chitinolytic isolates were inoculated into M9-CB with 1% (v/v) and incubated for 72 h at 30°C. Cultures w ere centrifuged at 25,000 g (Sigma 3 K18) at 4°C for 10 min to pellet the bacterial cells and chitin content. The crude cell-free culture supernatants were taken and used to determine the chitinase activities and existing glucose and protein levels. These crude supernatants contain extra-cellular proteins, especially chitinases,and a small amount of glucose from chitin degradation by existing chitinases.

The chitinase activities were assayed by the method of Monreal and Reese (1969) with some modifications. An amount of 300 μl of crude cell-free culture supernatant of each isolates were reacted with 150 μl colloidal chitin (12.5 mg/ml chitin) as a substrate and incubated at 30°C for 3 h. Then, the reactions were centrifuged at 25,000 g for 7 min to pellet the colloidal chitin. Next, 300 μl of supernatants containing the resultant glucose monomers degraded from chitin were treated with 300 μl 3.5-Dinitrosalicylic acid reagent for 5 min in vigorously boiling water to estimate the concentration of these reducing sugars according to the specific activities of chitinases. The crude cell-free culture supernatants boiled alone for 5 min was used in controls. The reaction absorbances were measured at 540 nm and were run with a glucose standard. One unit of the chitinase activity was defined as the amount of enzyme that liberates 1 μM of N-acetylglucosamine; the resultant glucose monomers, per 1 min at 30°C (pH 5.5 or 9.5). Spectra Max M2 microplate reader with 96-well standard flat bottom plates were used for all these measurements.

To determine existed glucose contents of the crude cell-free culture supernatants, 300 μl of each of them were reacted with 300 μl DNS reagent in the same way and conditions. These values were excluded from previous crude supernatant–colloidal chitin reaction results to prevent any mistake because of existing glucose content of supernatants.

Total protein quantities of the crude cell-free culture supernatants were determined by Biorad bradford reagent according to the manufacturer’s instructions (Bio-Rad Protein Assay Kit, 0006). Existing protein contents of supernatants were used to calculate chitinase activity with crude supernatant–colloidal chitin reaction results.

Statistical analysis

The statistical analysis was done using SPSS 13.0 (SPSS, 1989–2004). Linear regression analysis was used to compare chitinase activity results and insecticidal effects of enthomopathogenic isolates. Values for insecticidal activities (%), DNS activities (U/mg), and M9-CAD activities (mm) were Log10-transformed, to ensure linearity of the data prior to statistical analysis. Statistical significance was judged at the P ≤ 0.01 level.

Results and discussion

Finding more effective and safer biological control agents against hazardous insects has been very important in biological control. After the discovery of the Bacillus thuringiensis toxins, there has been a growing interest to increase the effect of existing biological control agents as well as finding new ones. So researchers have been focused on Bt toxins and chitinase enzymes, the two most important biocontrol materials, produced by pathogenic and sometimes nonpathogenic bacteria. The importance of chitinases in biological control of fungi, nematodes, and insect pests has become an emerging field of research (Ajit et al. 2006). Chitinases of some insect pathogens have also been utilized for enhancing their pathogenity (Kramer and Muthukrishnanm 1997; Merzendorfer and Zimoch 2003).

Epithelium of the insect midgut, which is very important in insect feeding, is protected by the peritrophic membrane. This barrier can be easily degraded by chitinases. Therefore, insect feeding can be stopped by this degradation and the epithelium becomes indefensible. As a consequence of this circumstance, insects undergo a lot of suffering or death. Moreover, access of the Bt toxins to the gut epithelium receptors enhanced by adding chitinases resulted in higher insecticidal activity (Sneh et al. 1983; Regev et al. 1996; Wiwat et al. 1996; Sampson and Gooday 1998). Downing et al. (2000) used two different recombinant bio-control agents: Pseudomonas fluorescens-secreted cry toxins and chitinase, against the sugarcane borer. In their study, an increased toxic effect, fivefold more than individual applications, was observed when recombinant cry producer P. fluorescens combined with P. fluorescens carrying the Serratia marcescens chitinase gene. Thus, chitinases are the most important enzymes through biological control materials by means of these versatile effects. Furthermore, all chitinolytic bacteria have a great potential to use as biological control agents.

In this study, we tested all bacterial flora members of 13 Coleopteran pests in BLCP to determine the potential of chitinase production (Table 1). The M9-CA method and PCR with specific degenerate primers were used to test the chitinolytic abilities of the bacteria. We tested not only pathogenic bacteria but also nonpathogenic ones because of their possible chitinase production capabilities and synergistic actions to pathogenic ones.

In M9-CA chitinolytic ability studies, the bacteria have been forced to produce chitinase, to be able to degrade complex chitin polymer, and to produce metabolites to support their growth in the media incorporated with chitin as the only carbon and energy sources without any nutrients. Twenty-three of 108 (21.3%) Coleopteran-originated bacteria were found to be chitinolytic using this method (Table 1). In PCR reactions, conserved domains of chitinase coding genes of all isolates, which showed clear zone surrounding the colony on M9-CA plates, were successfully amplified. However, among these pests, we could not get positive isolates from Amphimallon solstitiale, Anoplus roboris, Ips typographus or Leptinotarsa decemlineata.

We evaluated chitinase activities of these 23 chitinase positive isolates using two methods. In the M9-CAD method, the diameters of the colonies (mm) around the colonies on agar plates were measured. According to the M9-CAD method, five isolates of the highest seven (BnSm, Ol13, Dm3, Xd1, Aa5) are Serratia species and the other isolates (Is3.2 and Is6) are as yet unidentified (Fig. 2; Table 2). These isolates produced higher chitinolytic zones than all other isolates on the diffusion agar plates. In the DNS method, specific chitinase activities (U/mg) from crude extract of cell-free culture supernatants were determined as colorimetric in a spectrometer. Results of both experiments are shown in Table 2.

Table 2 Chitinase activities determined bythe M9-CAD and DNS methods and the insecticidal activities of bacterial isolates

We compared chitinase activity results and insecticidal effects (Table 2) of enthomopathogenic isolates statistically by taking their log to the base 10 to be able to see the possible relationship clearly in the graph (Fig. 1). Statistical analyses revealed a very strong positive linear correlation between the M9-CAD results and the insecticidal activities of bacterial isolates (F = 75.415; df = 1; P ≤ 0.0001). The predicted regression equation between insecticidal efficiency and the M9-CAD activity result of the bacteria is shown below.

$$ \begin{array}{*{20}{c}} {{\text{y}} = {{\text{b}}_0} + {{\text{b}}_{{1}}}*\left( {\text{x}} \right)} \hfill \\ {{\text{y}} = {\text{Insecticidal}}\,{\text{effect \, of}}\,{\text{entomopathogenic}}\,{\text{bacteria}}\,\left( {\text{\% }} \right)} \hfill \\ {{{\text{b}}_0}\,{\text{and}}\,{{\text{b}}_1} = {\text{standardized}}\,{\text{constants}}} \hfill \\ {{\text{x}} = {\text{M9 - CAD activity result of bacteria (mm)}}} \hfill \\ {{\text{y}} = \left( { - {9},0{89}} \right) + {7},{719}*\left( {\text{x}} \right)} \hfill \\ \end{array} $$

Fig. 1

The logaritmic comparison of chitinase activities with insecticidal effects of isolates. All parameters were compared via taking their log to the base 10

The Pearson correlation coefficient r ² values between insecticidal activities of isolates and the chitinase activities of M9-CAD method, insecticidal activities and DNS activity results, and M9-CAD and DNS activity results were 0.968, −0.279, and 0.180, respectively.

Insecticidal activity results of bioassay studies were tested by using the whole bacterial cells in previous enthomopathogenic agent finding and developing studies at our laboratory (Sezen and Demirbağ 1999; Sezen et al. 2001, 2007, 2008; Bahar and Demirbag 2007). In this study, bacterial cells were also used in the M9-CAD method as in bioassays. Therefore, these two parameters appear to be more related. In the M9-CAD method, measured activity is not based only on specific enzyme activity but also comes from the enzyme-producing and secretion capability of the bacteria to the extracellular media. In contrast, the crude extracts taken from the cell-free culture medium of bacterial isolates were used to determine chitinase-specific activity with the DNS method. Therefore, we made an assumption that there must be a stronger relationship between the variables of M9-CAD and insecticidal activity results than any other parameter. To prove this idea, some statistical analyses were carried out.

Three different variables were compared in statistical studies to assess possible correlations. These parameters are chitinase activity results of the M9-CAD and DNS methods in the current study and insecticidal activities of enthomopathogens from our previous studies.

Figure 1 clearly indicates that the insecticidal activities and M9-CAD activity results of Coleopteran-originated bacterial isolates were strongly correlated with each other, but we could not get any significant correlation between the specific activity from the DNS method and the other two parameters. The first five of the most active isolates in M9-CAD are Serratia species (Table 2; Fig. 2). Serratia marcescens is an important microorganism with its strong chitinase mechanism, after Bacillus thuringiensis that has been widely used in biological control of hazardous insects and fungi species (Brurberg et al. 2001).

Fig. 2

Chitinase activities of bacterial isolates from M9-CAD and DNS results. DNS results were multiplied by 10 within all values to make the results comparable with the other activity results. All tests were repeated three times and ranking was made according to M9-CAD activities to see the difference more easily

We could not get any significant correlation between the specific activity from the DNS method and the other two parameters, while there is a very strong positive correlation between the M9-CAD and insecticidal activities. However this absence does not mean that the chitinase activity results of the DNS method are inconsiderable. On the contrary, it helps to understand which bacterial crude extract contains the enzyme that has a high activity thanks to its power for determining specific chitinase activity in cell-free culture supernatants. Thus, the DNS method still keeps its importance in developing new recombinant chitinases. Thus, researchers can use DNS results as a helpful parameter to select the convenient isolates to develop their chitinase mechanism or to use them as recombinant bio-control agents.

In Fig. 2, isolate Ol10 has the second lowest chitinase activity with the M9-CAD method while this isolate has the second highest specific chitinase activity with the DNS method. In contrast, isolate Is3.2 has the highest activities in both methods. Therefore, the absence of any relationship between the DNS and M9-CAD chitin-agar methods is an admissible result.

Using bacteria in the environment at a particular level will be advantageous to protect that area for decades, because of the permanent regulation of bio-control agents (Nowierski 1984). In some critical situations, bio-control agents must be used promptly to protect the agricultural area or forestry region against Coleopteran pests. In such a case, the M9-CAD method is very important thanks to its strong relationship (r ² 0.96, p ≤ 0.01) with the insecticidal activities of the bacteria.

The insecticidal efficiencies can be rapidly predicted over the M9-CAD method for each bacterial isolate. So, we can make very important decisions about the insecticidal activity of potential biocontrol agents and discover new ones. Thanks to this, researchers can save their time and money prior to long-term, comprehensive and very sensitive bioassay studies.

This is the first detailed statistical investigation on the determination of the chitinase-producing ability and the chitinase activity of enthomopathogens from Coleopteran pests which mainly damage agriculture and forestry. In addition, some of them appear to be promising for use against these pests. Moreover, the M9-CAD method proves itself as a powerful tool for biological control agent-finding studies thanks to its fast and high capacity to estimate the insecticidal efficiency of bacterial isolates.