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Ann Microbiol (2012) 62:647–653 DOI 10.1007/s13213-011-0301-y ORIGINAL ARTICLE The relationship between insecticidal effects and chitinase activities of Coleopteran-originated entomopathogens and their chitinolytic profile Ali Adem Bahar & Kazım Sezen & Zihni Demirbağ & Remziye Nalçacioğlu Received: 4 January 2011 /Accepted: 14 June 2011 /Published online: 13 July 2011 Springer-Verlag and the University of Milan 2011 . . Abstract Peritrophic membrane (PM) is present in most Keywords Chitinase Chitinolytic bacteria . . insects’ midgut and acts as a mechanical barrier to Enthomopathogen Insecticidal activity protect the epithelium from various harmful factors such Serratia marcescens 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 Introduction toxins to gut epithelium and intercepting nutritional absorption in the midgut. One hundred and eight The Coleopterans are the largest order of insects, represent- bacterial isolates derived from microbial flora of coleop- ing about 40% of the known insect species (Liebherr and teran pests were screened to determine chitin-producing McHugh 2003). Certain Coleopterans are serious pests of entomopathogenic bacteria. The M9 chitin–-agar method forest or agricultural crops throughout the world. Chemical and polymerase chain reaction with specific primers for a insecticides which have a variety of side effects on the conserved domain of chitinase genes indicated that 23 of environment have been used for several years against these the 108 isolates have chitinase activity. The chitinase pests. Because of their hazardous effects, these chemicals activities of the chitinase-positive bacteria were mea- are no longer recommended for agricultural pest manage- sured. We compared these results with the insecticidal ment. Fortunately, entomopathogens do not harm other activities results to determine, statistically, the potential animals or plants. Serratia marcescens is an efficient relationship between the chitinase activities and the biological degrader of chitin and also a potential biological insecticidal activity. Consequently, 21.3% of bacterial agent for fungi and other organisms (Tu et al. 2010). isolates showed chitinolytic ability and among these the Chitin, homopolymer of β 1–4 linked N-acetylglucosamine, chitinase-positive bacteria, Serratia marcescens,was is the second most abundant polysaccharide and renewable found the most active one in the M9-CAD method. More source in nature after cellulose (Duo-Chuan 2006). Insects’ importantly, our study indicated a very strong positive peritrophic membranes (PM) are film-like structures including correlation between the insecticidal activities of isolates important amounts of chitin embedded in a protein–carbohy- and the chitinase activities with the M9-CAD method (r = drate matrix. PM separates food from midgut tissues and 0.96, p≤0.01), but not with the DNS method (r =−0.279, protects the epithelium against food abrasion and micro- p≤0.01). This strong relationship of entomopathogens has organisms (Granados et al. 2001). Consequently, any defect in a high potential for biocontrol of Coleopteran pests. 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 : : : A. A. Bahar K. Sezen Z. Demirbağ R. Nalçacioğlu (*) components found in the midgut through this barrier. In this Faculty of Science, Department of Biology, way, some bacteria and their toxic components reach the Karadeniz Technical University, endothelial cells of the midgut and become a threat to insect Trabzon, Turkey life (Huber et al. 1991; Langer et al. 2000). e-mail: remziye@ktu.edu.tr 648 Ann Microbiol (2012) 62:647–653 Degradation of the PM by chitinases has long been a bacterial strains have been isolated from Coleoptera. All very attractive biological control method against insect isolates were incubated in Luria-Bertani broth (LB) pests of crop plants (Regev et al. 1996;El–Tarabilya et al. medium at 30°C for 16 h. After incubation cultures 2000). Up to now, some chitinolytic bacteria have been were centrifuged at 2,300 g for 5 min to pellet the cells. shown to be potential biological control agents of insect The pellets were washed two times to remove any pests (Khmel et al. 1998). Furthermore, chitinolytic bacteria nutrient substances from the LB medium and were re- can be pathogens of fungi, such as Ewingella americana, suspended in sterilized phosphate-buffered saline (PBS). the causative agent of internal stipe necrosis of the The cell densities were adjusted to one at OD commercial mushroom Agaricus bisporus (Jolles and (Sambrook et al. 1989). These suspensions were used Muzzarelli 1999). Therefore, chitinases are of great for inoculations in chitinolytic ability and chitinase biotechnological interest and all chitinolytic bacteria have activity tests. the potential to be used as biological control agents (Yanhua et al. 2007). Chemicals At the present time, there is an increasing interest in using biological control agents as alternatives for chemical Colloidal chitin (Sigma, C7170 USA) was prepared as insecticides. Therefore, chitinases have been used directly described by Hsu and Lockwood 1975.Fivegrams of or as improvers of virulence with many pathogenic agents powdered chitin were dissolved in 100 ml concentrated (Otsu et al. 2003). hydrochloric acid and stirred overnight. This mixture was So far, there has been no study on the chitinolytic profile dissolved in 2 l ethanol with vigorously stirring for of enthomopathogenic bacteria from the microbial flora of 2 days. The colloids were collected and concentrated by Coleopteran pests. In this article, we determined the centrifugation for 10 min at 15,000 g. The chitin pellet chitinolytic bacteria profile in a bacterial library of 108 was re-suspended and re-centrifuged in distilled water Coleopteran pests, tested their chitinase activities and until the pH reached 5.5–6 several times. The final chitin compared these values with their insecticidal activities cake was re-suspended in distilled water (1%), autoclaved obtained from our previous studies. and stored at 4°C. M9 medium was prepared according to the protocol described by Sambrook et al. (1989). Materials and methods Screening the bacterial isolates for their chitinolytic ability Bacterial isolates and growth conditions Two different approaches, molecular detection with specific degenerate primers and M9-Chitin Agar (M9-CA) methods Bacterial isolates were provided from the Bacterial Library were used to determine if the bacterial isolates have con- of the Karadeniz Technical University Microbiology labo- served regions of chitinase coding genes and chitinolytic ratory (BLCP) (www.microlabktu.com)(Table 1). All ability on agar plates, respectively. Table 1 The source and chiti- Hosts of bacterial isolates Total (108) Chitinolytic (23) References nolytic activities of bacterial isolates Agelastica alni (Chrysomelidae) 4 3 Sezen et al. 2001 Amphimallon solstitiale (Scarabaeidae) 4 0 Sezen et al. 2005 Anoplus roboris (Curculionidae) 4 0 Demir et al. 2002 Balaninus nucum (Curculionidae) 5 1 Sezen and Demirbağ 1999 Curculio elephas (Curculionidae) 7 2 Unpublished data Dendroctonus micans (Scolytidae) 7 2 Yılmaz et al. 2006 Ips sexdentatus (Scolytidae) 10 4 Unpublished data Ips typographus (Curculionidae) 8 0 Muratoğlu et al. 2011b Leptinotarsa decemlineata 6 0 Muratoğlu et al. 2011a (Chrysomelidae) Agriotes sp. (Elateridae) 28 5 Unpublished data Bacterial Library Pests in Melolontha melolontha (Scarabaeidae) 7 2 Sezen et al. 2007 Karadeniz Technical University Oberea linearis (Cerambycidae) 13 3 Bahar and Demirbag 2007 Microbiology Laboratory (BLCP) originated from Xyleborus dispar (Scolytidae) 5 1 Sezen et al. 2008 Coleopterans Ann Microbiol (2012) 62:647–653 649 Molecular detection of chitinolytic ability with specific around the colonies were measured 1 week after incubation. degenerate primers The milimetric values were evaluated as chitinase activity. The degenerate chitinase primers GA1F (5′-CGTCGA 3,5-Dinitrosalicylic acid (DNS) method CATCGACTGGGARTDBCC-3 ′) and GA1R (5 ′- ACGCCGGTCCAGC CNCKNCCRTA-3′) were used to The PBS suspensions of all chitinolytic isolates were amplify part of chitinase coding gene. PCR was performed inoculated into M9-CB with 1% (v/v) and incubated for in a final volume of 50 μl containing 400 nM of each 72 h at 30°C. Cultures w ere centrifuged at 25,000 g (Sigma primer, 0.2 mM of each dNTP in 1.5 mM MgCl , 3 K18) at 4°C for 10 min to pellet the bacterial cells and 10 mM Tris–HCl (pH 9.0 at 25°C), 50 mM KCl, 0.1% chitin content. The crude cell-free culture supernatants were Triton X-100, and 0.5 units of Taq DNA polymerase taken and used to determine the chitinase activities and (Promega). PCR reaction conditions was adjusted as: existing glucose and protein levels. These crude super- denaturation for 5 min at 94°C, one cycle, and then 35 natants contain extra-cellular proteins, especially chitinases, cycles of 94°C for 1 min, 55°C for 10 s and 72°C for and a small amount of glucose from chitin degradation by 1 min followed by one cycle of 72°C for 10 min. PCR existing chitinases. products were analyzed in a 1.5% agarose gel stained The chitinase activities were assayed by the method of with EtBr. The size of the amplified products were 450 bp Monreal and Reese (1969)with some modifications. An long. This region codes conserved blocks of amino acids amount of 300 μl of crude cell-free culture supernatant of within the catalytic domain of bacterial chitinase proteins each isolates were reacted with 150 μl colloidal chitin (Williamson et al. 2000). (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 M9-chitin agar (M9-CA) method 7 min to pellet the colloidal chitin. Next, 300 μlof supernatants containing the resultant glucose monomers M9 minimal agar plates were incorporated with 0.5% (v/v) degraded from chitin were treated with 300 μl3.5- of colloidal chitin to prepare M9-CA plates. Ashless Dinitrosalicylic acid reagent for 5 min in vigorously boiling Whatman filter paper disks, 5 mm in diameter, were located water to estimate the concentration of these reducing sugars on M9-CA plates and used for bacterial inoculation. The according to the specific activities of chitinases. The crude paper disks were saturated with 5 μlofthe PBS cell-free culture supernatants boiled alone for 5 min was used suspensions in triplicate for each bacterium. These plates in controls. The reaction absorbances were measured at were incubated at 30°C. After 10 days of incubation, 540 nm and were run with a glucose standard. One unit of the chitinase activity was defined as the amount of enzyme bacterial isolates which had clear zones surrounding the colony were evaluated as chitinase-positive. that liberates 1 μM of N-acetylglucosamine; the resultant glucose monomers, per 1 min at 30°C (pH 5.5 or 9.5). Chitinase activity assays of chitinase positive Spectra Max M2 microplate reader with 96-well standard flat entomopathogenic bacteria bottom plates were used for all these measurements. To determine existed glucose contents of the crude cell- The chitinase activities of chitinase positive isolates were free culture supernatants, 300 μl of each of them were evaluated by two different methods. These methods were reacted with 300 μl DNS reagent in the same way and the M9-Chitin Agar Diffusion (M9-CAD) method and the conditions. These values were excluded from previous colorimetric 3.5-Dinitrosalicylic acid (DNS) method. crude supernatant–colloidal chitin reaction results to pre- vent any mistake because of existing glucose content of M9-chitin agar diffusion (M9-CAD) method supernatants. Total protein quantities of the crude cell-free culture M9 Chitin Broth (M9-CB) medium that includes 0.5% (v/v) supernatants were determined by Biorad bradford reagent of colloidal chitin was inoculated with 10% (v/v) of the according to the manufacturer’s instructions (Bio-Rad PBS suspensions of each bacterial strain and incubated at Protein Assay Kit, 0006). Existing protein contents of 30°C for 18 h. After incubation, cultures were centrifuged supernatants were used to calculate chitinase activity with at 1,000 g for 10 min. The bacterial pellets were re- crude supernatant–colloidal chitin reaction results. suspended at 1.89 at OD in sterilized PBS (Moar et al. 1995) and 5 μl of these was used to saturate the paper Statistical analysis disks. These disks were inoculated into separate M9-CA plates in triplicates and incubated at 30°C. The chitin-free The statistical analysis was done using SPSS 13.0 (SPSS, 1989–2004). Linear regression analysis was used to halos which occurred by diffusion of secreted chitinases 650 Ann Microbiol (2012) 62:647–653 compare chitinase activity results and insecticidal effects of been utilized for enhancing their pathogenity (Kramer and enthomopathogenic isolates. Values for insecticidal activi- Muthukrishnanm 1997; Merzendorfer and Zimoch 2003). ties (%), DNS activities (U/mg), and M9-CAD activities Epithelium of the insect midgut, which is very important in (mm) were Log10-transformed, to ensure linearity of the insect feeding, is protected by the peritrophic membrane. This data prior to statistical analysis. Statistical significance was barrier can be easily degraded by chitinases. Therefore, insect judged at the P≤0.01 level. 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 Results and discussion the Bt toxins to the gut epithelium receptors enhanced by adding chitinases resulted in higher insecticidal activity (Sneh Finding more effective and safer biological control et al. 1983; Regev et al. 1996; Wiwat et al. 1996;Sampson agents against hazardous insects has been very important and Gooday 1998). Downing et al. (2000)used two different in biological control. After the discovery of the Bacillus recombinant bio-control agents: Pseudomonas fluorescens- thuringiensis toxins, there has been a growing interest to secreted cry toxins and chitinase, against the sugarcane borer. increase the effect of existing biological control agents as In their study, an increased toxic effect, fivefold more than well as finding new ones. So researchers have been individual applications, was observed when recombinant cry focused on Bt toxins and chitinase enzymes, the two most producer P. fluorescens combined with P. fluorescens carrying important biocontrol materials, produced by pathogenic the Serratia marcescens chitinase gene. Thus, chitinases are and sometimes nonpathogenic bacteria. The importance of the most important enzymes through biological control chitinases in biological control of fungi, nematodes, and materials by means of these versatile effects. Furthermore, insect pests has become an emerging field of research (Ajit all chitinolytic bacteria have a great potential to use as et al. 2006). Chitinases of some insect pathogens have also biological control agents. Table 2 Chitinase activities determined bythe M9-CAD and DNS methods and the insecticidal activities of bacterial isolates Isolate codes Bacteria Pests Insecticidal activity (%) DNS activity M9-CAD activity (crude extract, U/mg) (zone distance, mm) Aa3 Listeria sp. Agelastica alni 48 0.401639 7.2 Aa4 Pseudomonas chlororaphis Agelastica alni 37 0.284803 6.4 Aa5 Serratia marcescens Agelastica alni 70 0.138339 10.38 Ag01 UI Agriotes sp. ND 0.041105 5.825 Ag02 UI Agriotes sp. ND 0.557054 3.59 Ag14 UI Agriotes sp. ND 0.151668 5.085 Ag16 UI Agriotes sp. ND 0.16975 3.52 Ag21 UI Agriotes sp. ND 0.337794 3.55 BnSm Serratia marcescens Balaninus nucum 100 0.251294 12.03 Ce4 UI Curculio elephas ND 0.45039 8.595 Ce7 UI Curculio elephas ND 0.441952 8.02 Dm3 Serratia grimesii Dendroctonus micans ND 0.518604 10.815 Dm5 Enterobacter intermedius Dendroctonus micans ND 0.501664 3.5 Is10 UI Ips sexdentatus ND 0.367967 8.38 Is3.1 UI Ips sexdentatus ND 0.333498 8.185 Is3.2 UI Ips sexdentatus ND 0.86506 12.355 Is6 UI Ips sexdentatus ND 0.599349 10.58 Mm3 Pseudomonas sp. Melolontha melolontha 50 0.36964 9.54 Mm4 Enterobacter sp. Melolontha melolontha 20 0.123859 2.45 Ol10 Xanthomonas sp. Oberea linearis 5 0.72304 2.14 Ol12 Xanthomonas maltophilia Oberea linearis 5 0.290336 2.07 Ol13 Serratia marcescens Oberea linearis 65 0.16446 10.835 Xd1 Serratia marcescens Xyleborus dispar 79 0.501167 10.785 UI Unidentified, ND not determined Ann Microbiol (2012) 62:647–653 651 Fig. 1 The logaritmic compari- 2.5 son of chitinase activities with insecticidal effects of isolates. All parameters were 1.5 compared via taking their log to the base 10 Log (%) Insecticidal activity 0.5 Log (mm) M9-CAD chitinase activity -0.5 Log (U/mg) DNS specific chitinase activity -1 -1.5 Ol12 Ol10 Mm4 Aa4 Aa3 Mm3 Aa5 Xd1 Ol13 BnSm In this study, we tested all bacterial flora members of 13 tiale, Anoplus roboris, Ips typographus or Leptinotarsa Coleopteran pests in BLCP to determine the potential of decemlineata. chitinase production (Table 1). The M9-CA method and We evaluated chitinase activities of these 23 chitinase PCR with specific degenerate primers were used to test the positive isolates using two methods. In the M9-CAD chitinolytic abilities of the bacteria. We tested not only method, the diameters of the colonies (mm) around the pathogenic bacteria but also nonpathogenic ones because of colonies on agar plates were measured. According to the their possible chitinase production capabilities and syner- M9-CAD method, five isolates of the highest seven (BnSm, gistic actions to pathogenic ones. Ol13, Dm3, Xd1, Aa5) are Serratia species and the other In M9-CA chitinolytic ability studies, the bacteria have isolates (Is3.2 and Is6) are as yet unidentified (Fig. 2; been forced to produce chitinase, to be able to degrade Table 2). These isolates produced higher chitinolytic zones complex chitin polymer, and to produce metabolites to than all other isolates on the diffusion agar plates. In the support their growth in the media incorporated with chitin DNS method, specific chitinase activities (U/mg) from as the only carbon and energy sources without any crude extract of cell-free culture supernatants were deter- nutrients. Twenty-three of 108 (21.3%) Coleopteran- mined as colorimetric in a spectrometer. Results of both originated bacteria were found to be chitinolytic using this experiments are shown in Table 2. method (Table 1). In PCR reactions, conserved domains of We compared chitinase activity results and insecticidal chitinase coding genes of all isolates, which showed clear effects (Table 2) of enthomopathogenic isolates statistically zone surrounding the colony on M9-CA plates, were by taking their log to the base 10 to be able to see the successfully amplified. However, among these pests, we possible relationship clearly in the graph (Fig. 1). Statistical could not get positive isolates from Amphimallon solsti- analyses revealed a very strong positive linear correlation Fig. 2 Chitinase activities of 14.0 bacterial isolates from M9- CAD and DNS results. DNS 12.0 results were multiplied by 10 within all values to make the results comparable with the 10.0 other activity results. All tests were repeated three times and 8.0 ranking was made according to M9-CAD activities to see the difference more easily 6.0 4.0 2.0 0.0 DNS activity (U/mg)x10* M9-CAD activity (mm) 652 Ann Microbiol (2012) 62:647–653 between the M9-CAD results and the insecticidal activities of between the M9-CAD and insecticidal activities. However bacterial isolates (F=75.415; df=1; P≤0.0001). The pre- this absence does not mean that the chitinase activity results of dicted regression equation between insecticidal efficiency and the DNS method are inconsiderable. On the contrary, it helps the M9-CAD activity result of the bacteria is shown below. 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 y ¼ b þ b ðÞ x 0 1 supernatants. Thus, the DNS method still keeps its importance y ¼ Insecticidal effect of entomopathogenic bacteriaðÞ % in developing new recombinant chitinases. Thus, researchers b and b ¼ standardized constants 0 1 x ¼ M9 CAD activity result of bacteriaðmmÞ can use DNS results as a helpful parameter to select the y¼ðÞ 9; 089þ 7; 719 ðÞ x convenient isolates to develop their chitinase mechanism or to use them as recombinant bio-control agents. The Pearson correlation coefficient r values between In Fig. 2, isolate Ol10 has the second lowest chitinase insecticidal activities of isolates and the chitinase activities activity with the M9-CAD method while this isolate has the of M9-CAD method, insecticidal activities and DNS second highest specific chitinase activity with the DNS activity results, and M9-CAD and DNS activity results method. In contrast, isolate Is3.2 has the highest activities were 0.968, −0.279, and 0.180, respectively. in both methods. Therefore, the absence of any relationship Insecticidal activity results of bioassay studies were between the DNS and M9-CAD chitin-agar methods is an tested by using the whole bacterial cells in previous admissible result. enthomopathogenic agent finding and developing studies Using bacteria in the environment at a particular level at our laboratory (Sezen and Demirbağ 1999; Sezen et al. will be advantageous to protect that area for decades, 2001, 2007, 2008; Bahar and Demirbag 2007). In this because of the permanent regulation of bio-control agents study, bacterial cells were also used in the M9-CAD method (Nowierski 1984). In some critical situations, bio-control as in bioassays. Therefore, these two parameters appear to agents must be used promptly to protect the agricultural be more related. In the M9-CAD method, measured activity area or forestry region against Coleopteran pests. In such a is not based only on specific enzyme activity but also case, the M9-CAD method is very important thanks to its comes from the enzyme-producing and secretion capability strong relationship (r 0.96, p≤0.01) with the insecticidal of the bacteria to the extracellular media. In contrast, the activities of the bacteria. crude extracts taken from the cell-free culture medium of The insecticidal efficiencies can be rapidly predicted bacterial isolates were used to determine chitinase-specific over the M9-CAD method for each bacterial isolate. So, we activity with the DNS method. Therefore, we made an can make very important decisions about the insecticidal assumption that there must be a stronger relationship activity of potential biocontrol agents and discover new between the variables of M9-CAD and insecticidal activity ones. Thanks to this, researchers can save their time and results than any other parameter. To prove this idea, some money prior to long-term, comprehensive and very sensi- statistical analyses were carried out. tive bioassay studies. Three different variables were compared in statistical This is the first detailed statistical investigation on the studies to assess possible correlations. These parameters are determination of the chitinase-producing ability and the chitinase activity results of the M9-CAD and DNS methods chitinase activity of enthomopathogens from Coleopteran in the current study and insecticidal activities of enthomo- pests which mainly damage agriculture and forestry. In pathogens from our previous studies. addition, some of them appear to be promising for use Figure 1 clearly indicates that the insecticidal activities against these pests. Moreover, the M9-CAD method proves and M9-CAD activity results of Coleopteran-originated itself as a powerful tool for biological control agent-finding bacterial isolates were strongly correlated with each other, studies thanks to its fast and high capacity to estimate the but we could not get any significant correlation between the insecticidal efficiency of bacterial isolates. specific activity from the DNS method and the other two Acknowledgment This work was supported by The Scientific and parameters. The first five of the most active isolates in M9- Technological Research Council of Turkey (107 T926). We thank Dr. CAD are Serratia species (Table 2;Fig. 2). Serratia Cemal Sandalli for his support during this investigation. marcescens is an important microorganism with its strong chitinase mechanism, after Bacillus thuringiensis that has been widely used in biological control of hazardous insects References and fungi species (Brurberg et al. 2001). 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Annals of Microbiology – Springer Journals
Published: Jul 13, 2011
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