Effect of Bacillus amyloliquefaciens A1, Paenibacillus ...

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Bacterial isolates, Bacillus amyloliquefaciens A1, Paenibacillus polymyxa AB3 and Providencia rettgeri K10, originating from soil samples were screened for ...
Egyptian Journal of Biological Pest Control, 27(1), 2017, 41-47

Effect of Bacillus amyloliquefaciens A1, Paenibacillus polymyxa AB3 and Providencia rettgeri K10 on the Citrus Mealybug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae) Mohamedova1, M. S.; I. S. Valcheva2; D. G. Draganova2; M. K. Naydenov2 and Y.B. Borisov2 1 2

Department of Entomology, Agricultural University, Plovdiv, [email protected]. Department of Microbiology and Ecological Biotechnologies, Agricultural University, Plovdiv. (Received: November 30, 2016 and Accepted: February 2, 2017)

ABSTRACT Bacterial isolates, Bacillus amyloliquefaciens A1, Paenibacillus polymyxa AB3 and Providencia rettgeri K10, originating from soil samples were screened for their capability and efficacy to control the citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudocoсcidae). All of the tested bacterial strains indicated approximately the same effectiveness. The mortality rate of the1st instar larvae caused by B. amyloliquefaciens A1, P. rettgeri K10 and P. polymyxa AB3 reached 84.29, 82.62 and 90.37%, respectively. The bacterial isolates were tested for production of serinproteases (EC 3.4.21) and metalloproteases (EC 3.4.24). Specific proteolytic enzymes were investigated in crude enzyme filtrate by means of specific protease inhibitors: phenylmethylsulfonyl fluoride (PMSF), which successfully inhibited serinproteases and ethylenediamine tetraacetic acid (EDTA) for the inhibition of metalloproteases. By addition of PMSF, the remaining activity decreased by 25.73 and 69.46% in B. amyloliquefaciens A1 and P. polymyxa AB3, respectively. The remaining enzyme activity after adding EDTA was 87% in B. amyloliquefaciens A1 and 1.4% in P. polymyxa AB3.

Key words: Biocontrol, Bacillus amyloliquefaciens A1, Paenibacillus polymyxa AB3, Planococcus citri, Providencia rettgeri K10, Protease inhibitors. INTRODUCTION The citrus mealybug, Planococcus citri (Risso) (Hemiptera: Pseudocoсcidae) is a cosmopolitan species occurs in all subtropical and tropical areas of the world (Williams and Watson, 1988). Various crops of economic importance severely attack by this pest (Ahmed and Abd-Rabou, 2010). P. citri sucks plants sap from all parts of the infested plants. The damage caused by this species includes: stunted growth; wilted, chlorotic, and distorted leaves; hyperpigmentation; premature leaf and flower drop (Hattingh and Moore, 2003). P. citri is also a vector of several plant viruses on citrus, ornamentals and grape (Charles et al., 2006). Pseudococcus citri is difficult to control with insecticides as it is able to rapidly develop resistance and exhibit cryptic behavior. Further, it is covered by waxes that protect its body from insecticide penetration (Franco et al., 2004). The low efficacy of insecticide use has encouraged biocontrol strategies as the mealybugs can be controlled by coccinellid species, parasitoids (Daane et al., 2004 and Fallahzadeh et al., 2011) and entomopathogenic nematodes (EPN) of the genera Heterorhabditis and Steinernema spp. as the nymphs and adults of P. citri are greatly affected by Steinernema yirgalemense and Heterorhabditis zealandica (van Niekerk and Malan, 2012), as well as S. feltiae (Negrisoli et al., 2013). The application of enthomopathogenic fungi (EPF) is a valuable method of biological control of

mealybugs. According to Muştu et al. (2015), the application of Isaria farinosa resulted in significant mortality of the vine mealy bug P. ficus, even when using fungicides. The EPF Beauveria bassiana and Verticillium lecanii produce enzymes, destroying body cuticle (Kulkarni and Patil, 2013). To date, a few observations have been done on the effect of entomopathogenic bacteria on Psedococcidae species, as a result of extra cellular enzyme production or lipopolysaccharide(s) that destroys the hemocytes and internal organs of the insects, once the bacteria have penetrated the hemocoele (Pseudomonas aeruginosa, Serratia marcescens and Providencia rettgeri) (Lysenko, 1985) or because of the ability to produce toxins that destroy the epithelial cells lining the insect gut (Bacillus thuringiensis) (Van-Rie et al., 1990). Salunkhe et al. (2013) reported that the wax degrading bacteria S. marcescens, P. aeruginosa and Bacillus subtilis affected female longevity, fecundity and offspring weight of the pink hibiscus mealy bug, Maconellicoccus hirsutus. B. amyloliquefaciens A1 and P. polymyxa AB3 are root-colonizing microorganisms that have shown anti-microbial activity against a wide variety of fungi and the ability to improve root tolerance of abiotic stress. Studies on B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10 against different hemipteran species, and significant mortality of the rose aphid Macrosiphum rosae and the greenhouse whitefly Trialeurodes vaporariorum were carried out (unpublished data).

42 The objective of this study was to determine the potential of the three bacteria; B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10 against the 1st instar larvae of P. citri and to examine the ability of these microorganisms to produce enzymes responsible for suppressing larval development. MATERIALS AND METHODS Maintenance of Pseudococcus citri Planococcus citri initial population was collected from begonia (Begonia sp.), gerbera (Gerbera sp.), rose (Rosa sp.) and cucumber (Cucumis sp.) plants infested in greenhouses of the Plovdiv region in the autumn of 2014. Infested plants parts were transferred to the laboratory and placed on tobacco plants (Nicotianatabacum L., cv. Krumovgrad 58). The 1st instar larvae moved from desiccated leaves to the tobacco plants. This procedure provides a minimal risk of mealy bugs’ body injury (Hogendrop et al., 2006). To produce a homogenous nymphal population of P. citri for virulence experiments, the procedure was repeated several times and 3 mealybug generations were completed. The plants were kept in a growth room at the following conditions: 28±1°C, 70±5% RH and 16:8 (L: D) photoperiod. Microorganisms and culture condition The bacterial isolates B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10 were obtained from the Culture Collection of the LMBT, Agricultural University. They were previously isolated from soil. The bacterial strains were cultured in 500 ml Erlenmeyer flasks with 200 ml of specific medium broth containing (g/l): casein hydrolysate, 5.0; yeast extract, 2.5; glucose, 1.0; skim milk powder, 28.0 and pH 8.1. The flasks were cultivated on a rotary shaker with agitation (185 rpm) at 28°C for 48 h. The obtained cultural fluid was used for treatment of P. citri 1st instar nymphs. Qualitative estimation of enzymes Cellulolytic activity (Congo red test) Test medium containing (g/l): 10.0 carboxymethyl cellulose sodium salt (CMC-Na); 10.0 Peptone; 2.0 K2HPO4; 0.3 MgSO4; 2.5 (NH4)2SO4; 10.0 agar-agar pH 7.0. Single colonies were transferred to the Petri dish. After 48 h of incubation at 30°C, all the plates were stained with 1% (w/v) Congo red solution for 15 min and discolored with 1 M NaCl for 15 min (Ponnambalam et al., 2011). Wax degradation Screening of wax degradation was carried out using the modified Davis minimal agar medium containing (g/l): 8.0 beeswax; 1.0 (NH4)2SO4; 7.0 K2HPO4; 2.0 KH2PO4; 0.1 MgSO4; 18.0 Agar-agar. The halo zone diameters were measured after 48 h of incubation at 37°C (Salunkhe et al., 2013).

Lipolytic activity assay Lipase activity assay was evaluated in an Agar Tween medium, (g/l): 10.0 peptone; 0.1 CaCl2.2 H2O; 5 NaCl; 10 ml Tween 80; 15 agar-agar at pH 7-7.4. The formation of a white precipitate around the colony indicates positive lipolytic activity (Harrigan, 1998 andMazzucotelli et al., 2013). Chitinase activity assay The three bacterial strains were tested in a chitinase-inducing medium containing (g/l): 50.0 colloidal chitin; 1.0 (NH4)2SO4; 0.2 KH2PO4; 1.6 K2HPO4; 0.2 MgSO4; 0.01 FeSO4; 20.0 agar-agar. The pH was adjusted to 7.0 prior to autoclaving. Test strains were transferred to Petri dishes by spot inoculation and cultivated for 21 days at 28°C. The presence of a clear zone around the bacterial colony indicated chitinolytic activity (Cattelan et al., 1999). Nutrient Gelatin stab method The ability of microorganisms to produce gelatinases was tested using a gelatin specific medium containing (g/l): 4.0 peptone; 3.0 proteoso peptone; 120.0 gelatin. Twenty-four-hour-old test bacteria were stab-inoculated into tubes with gelatin media and were cultivated at 28°C for 5 days. After an incubation time, the test tubes were transferred to a refrigerator for 30 minutes. The low temperature solidified the unhydrolyzed gelatin. The hydrolysis of gelatin indicate the secretion of gelatinases by the test organism into the medium (Harley, 2005). Protease activity assay The isolates were tested for protease production using a Skim milk Agar medium containing (g/l): 5.0 casein hydrolysate; 2.5 yeast extract; 1.0 dextrose; 28.0 skim milk powder; 20.0 agar-agar. The test was conducted at three pH 10.0, 6.75 and 5.0 adjusted with 1N NaOH and 1N HCl. The strains were incubated for 48 h at 28°C. Quantitative estimation of protease Enzyme production The bacterial strains were cultured in 500 ml Erlenmeyer flasks with 100 ml of specific medium broth containing (g/l): 5.0 casein hydrolysate; 2.5 yeast extract; 1.0 dextrose; 28.0 skim milk powder at pH 8.1. The flasks were cultivated on a rotary shaker with agitation (185 rpm/min) at 28°C for 120 h. Protease activity was determined every 24 h of cultivation. Enzyme assays Protease activity in the culture supernatant was assessed by the modified procedure of Tsuchida et al. (1986) using 2% casein (Hammerstan casein, Merck, Germany) in 50 mMTris – HCl buffer, pH 8.1 (Jooet al., 2002). The assay mixture consisting of 2 ml of substrate and 2 ml of enzyme solution was incubated at 35ºC for 20 min and the reaction was terminated by

43 the addition of 4ml of 5% trichloroacetic acid (TCA) and kept at room temperature for 15-20 min. The reaction mixture was centrifuged at 15000 rpm for 10 min to remove the resulting precipitate. The supernatant (1 ml) was mixed with 5 ml Na2CO3 (0.5 M) and 1 ml Folin-Ciocalteu (33%). The solution was incubated at room temperature in the dark for 30 min and then measured at 670 nm. Protease activity was determined as released tyrosine from the supernatants, according to a modified Lowry method of (Lowry et al., 1951). One unit of enzyme activity was defined as the amount of the enzyme resulting in the release of 1 µg of tyrosine per min at 35°C under the reaction conditions. Effect of protease inhibitors on enzyme activity The supernatants were used to investigate the type of protease produced by the bacteria. Phenylmethylsulfonyl fluoride (PMSF, stock solution of 10 mM in isopropanol) and ethylenediaminetetraacetic acid (EDTA, stock solution of 100 mM in H2O) in a final concentration of 0.9 mM and 4.76 mM were added to a cell-free supernatant and pre-incubated at 35°C for 30 min before the addition of substrate. PMSF was used as a serine inhibitor and EDTA as a metalloprotease inhibitor. The remaining protease activity was measured as described previously. The protease activity without inhibitor was considered as 100% activity (Wilson and Remigio, 2012). Bioassay protocol Tobacco seedlings were planted in small pots (12 cm diameter) with soil and grown for two weeks at 28±1°C, 70±5% RH and 16:8 (L:D). Twenty-four well-developed plants were chosen and placed individually in screened cages (30×40×30 cm). At the beginning of the experiment, 10 newly hatched nymphs were transferred to each tobacco plant using a fine brush. The cages were covered with voile to prevent the larvae migration and allow efficient ventilation. For better adaptation of the mealybug larvae the plants were kept for 24 h at the same growth room conditions. At 24 h after larvae transfer the plants were treated with 20 ml cultural fluid (109 CFU) of B. amyloliquefaciens, P. polymyxa and P. rettgeri. Four replications were used for each bacterial strain (isolate): 4 untreated plants received 20 ml distilled water and served as controls. At 24, 48 and 72 h after incubation at 28±1°C, 70±5% RH and 16:8 (L:D) dead individuals were counted under a stereomicroscope and percent mortality was calculated per plant. The experiment was repeated 3 times in 2015. Statistical analysis The results obtained in the present study were analyzed by two-way ANOVA, using statistical software SPSS for Windows version 19.0. Treatment means were compared using Duncan’s test at P≤0.05.

RESULTS AND DISCUSSION The 3 tested bacteria; B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10 showed a high pathogenicity on the 1st P. citri instar larvae. The biochemical profiles of the microorganisms, particularly their ability to produce specific enzymes defined the potential of the bacteria as biocntrol agents. Production of specific enzymes The ability of the 3 tested bacteria to produce specific enzymes is indicated in the table (1).They were unable to produce lipolytic and chitinolytic enzymes and could not degrade the wax. B. amyloliquefaciens A1 and P. polymyxa AB3 were able to produce cellulolytic enzymes and alkaline, neutral and acid proteases. Only B. amyloliquefaciens A1 was able to produce a proteolytic enzyme that hydrolyzed gelatin. P. rettgeri K10 produced only acid protease. Protease activity assay Eight hours after incubation, the first zone of hydrolysis was observed at B. amyloliquefaciens A1 at pH 10, whereas 24 h after incubation B. amyloliquefaciens A1 successfully hydrolyzed the substrate either at the pHs 10.0, 6.75 or 5.0 (Fig. 1). P. polymyxa AB3 formed a clear zone after 24 h at pH 10.0. At the end of the test period (48 h), both strains; B. amyloliquefaciens A1 and P. polymyxa AB3 grew at all the three pHs and completely hydrolyzed the substrate. P. rettgeri K10 showed active growth, without a clear halo zone at pH 10 and pH 6.75 and hydrolyzed the substrate at pH 5.0. Enzyme production The maximum protease activity was attained at 120 h by isolates of B. amyloliquefaciens A1 (35.01 U/ml) and P. polymyxa AB3 (17.2 U/ml). The lowest extracellular enzyme activity was measured in the isolate P. rettgeri K10 (1 U/ml) at 24 h (Fig. 2). Effect of protease inhibitors For classifying these proteases, their activities were measured in the presence of specific protease inhibitors (Table 2). After the addition of PMSF, the remaining activity decreased by 25.73 and 69.46% in B. amyloliquefaciens A1 and P. polymyxaAB3, respectively. The remaining enzyme activity after adding EDTA was 87% for B. amyloliquefaciens A1 and 1.4% for P. polymyxaAB3. This result indicated that the cell-free supernatant contained serine proteases and metalloenzymes. Proteases have a potential for use as insecticidal agents. They can destroy proteins and tissues of the insect body. Cysteine proteases, as well as metalloproteases and serine proteases showed toxic activity to insect

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Fig.(1): Skim-milk agar plates with halo zones after the degradation of substrate at three pH values (A – 8 h after incubation; B – 24 h after incubation; C – 48 h after incubation).

Table (1): Lytic enzymes activity of the bacterial strains Bacillus Paenibacillus Providenc amyloliquefaciens polymyxa iarettgeri A1 AB3 K10 Lipase Cellulase + + Wax degradation Gelatinase + Chitinase Alkaline protease + + Neutral protease + + Acid protease + + + Test

Table(2): Effect of protease inhibitors (PMSF and EDTA) Strains (remaining activity %)

Fig.(2): Protease activity of B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10.

B. amyloliquefaciens A1 P. polymyxa AB3

Inhibitor concentration PMSF, EDTA, 0.9 mM 4.76 mM 25.73 87 69.46 1.4

Table(3): Effects of bacterial treatments on first instar larvae of Pseudococcus citri. Means (SD) after different bacterial exposition times Exposition (hours) Mean 24 48 72 B. amyloliquefaciensA1 87.63 (14.43)a 85.23 (17.41)ab 80.00 (14.14)ab 84.29 (14.32)a a ab b P. rettgeriK10 97.92 (4.17) 80.68 (14.38) 69.26 (24.75) 82.62 (19.48)a ab ab a P. polymyxaAB3 90,92 (12.42) 85.46 (9.73) 94.72 (6.11) 90,37 (9.69)a c c c Control 22.88 (7.02) 24.22 (21.74) 31.59 (16.01) 26.23 (15.11)b Means within each column followed by the same letter are not significantly different, Duncan’s test (P < 0.05). Treatment

45 mid-gut, hemocoel and cuticle (Harrison and Bonning, 2010). The mealybug cuticle, which completely surrounds the body, except small openings, is an exoskeleton with a very rigid, multilayered and multicomponent, but flexible structure and an effective barrier preventing the insect from being damaged. Many studies showed that extracellular hydrolytic enzymes involved in cuticle penetration and host-cell digestion was proteases, collagenase, lipase, chitinase, as well as wax degrading enzymes (Niuet al., 2006). Effect of the bacteria on first instar larvae of P.citri The bacteria of the genus Providencia have been isolated from the gut of insects from different taxon, including Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae) (Kuzina et al., 2001), peacock butterfly, Hylesia metabus (Lepidoptera: Saturniidae) (Osborn et al., 2002), mole cricket Gryllotalpa gryllotalpa (Orthoptera; Gryllotalpidae) (Sezen et al., 2013). Providencia rettgeri is usually engaged in a symbiotic relationship with EPNs from the genera Hetrorhabditis (Nematoda: Heterorhabditidae) (Jackson et al., 1995), Steinernema (Nematode: Steinernematidae) (Boemare et al., 1996) and Oscheius (Nematoda: Rhabditidae) (Torres-Baragan, 2011). EPNs showed high pathogenic effects on various insect and nematode hosts, but according to Jackson et al. (1995), the symbiotic nematode suppressed P. rettgeri pathogenicity. The authors suggest direct application of the bacterium may probably inhibit this suppression for P. rettgeri to fulfill its potential as a biological control agent. The genus Paenibacillus includes different species showing pathogenicity against insects. Ruiu (2015) reported that the spore-formers, P. popilliae and P. lentimorbus, are the agents of milky disease in coleopteran species. B. amyloliquefaciens strain AG1 produces a lipopeptide biosurfactant exhibiting serious injuries to the mid-gut tissues with rupture and disintegration of the epithelial layer and cellular vacuolization (Ben Khedher et al., 2015). However, to this date, there were no reports concerning the potential of these bacteria to control P. citri. The bacteria B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10 caused a significantly higher mortality of the 1st instar larvae (L1) of the citrus mealybug, compared to the control (Table 3, P < 0.05). P. rettgeri K10 demonstrated the highest initial efficacy among the bacterial strains leading to (97.92%) mortality of L1 after 24 h. P. polymyxa AB3 was found to be the most effective strain causing (94.72%) mortality of L1 after 72 h. At the same exposition, the highest percentages of L1 mortality, observed as a result of the

B. amyloliquefaciens A1 and P. polymyxa AB3 effect,were 80.00 and 69.26%, respectively. Larval mortality among the bacterial strains was insignificantly different in any of the observations (Table 3). The results indicated approximately the same effectiveness of all of the tested bacterial strains. The mortality rate in the 1st instar larvae was 84.29, 82.62 and 90.37%, caused by B. amyloliquefaciens A1, P. rettgeri K10 and P. polymyxa AB3, respectively. The high mortality of L1 of P. citri caused by P. polymyxa AB3 might be attributed to higher quantity of metalloproteases produced by this bacterium. Similar efficacy of the EPF, I. farinosa on the citrus mealybug life stages was reported by Demirici et al. (2011). The authors observed (89.4 and 78.7%) mortality of the eggs and the 1st instar larvae, respectively, at 1×108 conidia concentration (ml-1) and in 95% RH. In the present study, it was also found that P. polymyxa AB3 showed an inhibitory effect on the development of L1 larvae. At 48 and 72 h, the L1 treated with P. polymyxa AB3 was more indolent and colorless than those treated with B. amyloliquefaciens A1 and P. rettgeri AB3. This result suggests that P. polymyxa AB3 may be able to control the development of L2-L4 and the adult female of P. citri, as well. The results obtained suggest that the three bacteria, B. amyloliquefaciens A1, P. polymyxa AB3 and P. rettgeri K10, might be useful as biocontrol agents for mealybugs. However, further studies are needed to determine the mode of action of the tested bacteria and their influence on the development and reproduction of P. citri. REFERENCES Ahmed, N. and Abd-Rabou, S.M. 2010. Host plants, geographical distribution, natural enemies and biological studies of the citrus mealy bug, Planococcus citri (Risso) (Hemiptera: Pseudococcidae). Egypt. Acad. J. Biology. Sci., A, Entomology, 3 (1): 39- 47. Ben Khedher, S.B., Kilani-Feki, O.B., Chaib, I., Laarif, A., Abdelkefi-Mesrati, L. and Tounsi, S. 2015. Bacillus amyloliquefaciens AG1 biosurfactant: Putative receptor diversity and histopathological effects on Tuta absoluta midgut. J. Invertebr. Pathol. 132: 42-47. Boemare, E.N., Laumond, C. and Mauleon, H. 1996. The entomopathogenic nematode-bacterium complex: Biology, life cycle and vertebrate safety. Biocontrol Sci. Technol., 6: 333-345. Cattelan, M.E., Hartel, P.G. and Fuhrmann, J.J. 1999. Screening of plant growth-promoting rhizobacteria to promote early soybean growth. Soil Sci. Soc. Am. J., 63: 1670- 1680.

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