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The influence of potato endophytes on Leptinotarsa decemlineata endosymbionts promotes mortality of the pest
Journal of Invertebrate Pathology 136 (2016) 65–67
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
The influence of potato endophytes on Leptinotarsa decemlineata endosymbionts promotes mortality of the pest Antonina V. Sorokan ⇑, Galina V. Ben’kovskaya, Igor’ V. Maksimov Institute of Biochemistry and Genetics, Ufa Science Center, Russian Academy of Science, Russia
a r t i c l e
i n f o
Article history: Received 28 December 2015 Revised 24 February 2016 Accepted 8 March 2016 Available online 9 March 2016 Keywords: Bacillus subtilis 26D Endophytes Colorado potato beetle Symbionts
a b s t r a c t Plants are exposed to pervasive attack by diverse attackers, such as pathogens and pests. But plants have their own endophytic microflora as well as the attacking insects. These microbiomes contact face to face in the nature. It has been found that the endophytic strain Bacillus subtilis 26D increases mortality of Colorado potato beetles, disturbing the development of insect microsymbionts Enterobacter ssp. and Acinetobacter ssp. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Relationships in ecosystems are mainly based on trophic chains. Host-parasite interactions can be considered as the most diverse and multilevel among them. So, parasites in an animal or plant organism can become useful to their owner, i.e. can affiliate to the host microbiome in the process of co-adaptation. Of course, natural plant-phytophage systems include microbiomes of the host plant and the pest alike. The role of the interaction between macroorganisms’ microbiomes, related to each other by a trophic chain, is one of the most intriguing areas of modern synecology. Apart from the fundamental aspects of research, this area has obvious practical importance for the creation of selective agents, controlling the number of phytophagous pests and causing minimal damage to agricultural plants and ecosystems. Given the diversity of habitats for Bacillus subtilis, it is not surprising that B. subtilis not only has antibiotic properties, but also can be an endophytic colonist from several plant species and protect plants against plant pathogens. Many strains of these bacteria are used already in protection and growth-promoting schemes (Kloepper and Tuzun, 1996; Pieterse et al., 2014). Strain B. subtilis 26D was used for the development of Fytosporine-M that is considered as environmentally desirable alternatives to synthetic chemicals for the control of agricultural plant diseases (Smirnov et al., 1987). It has been shown that this strain has the ability to penetrate into the plant organism and exists inside of tissues of ⇑ Corresponding author. E-mail address: [email protected] (A.V. Sorokan). http://dx.doi.org/10.1016/j.jip.2016.03.006 0022-2011/Ó 2016 Elsevier Inc. All rights reserved.
different plant families, including Solanaceae (Maksimov et al., 2014). It should be noted, the majority of applied research in this area is evaluated without taking into account the fact that these microorganisms interact not only with plants, but also with plant microbiome, insect pest and insect microbiome. So, the role of endophytes in plant protection against pathogenic organisms has been established repeatedly. At the same time the role of phytophagous insect endosymbionts, which are potentially able to prevent the heterogeneous microflora invasion and also to inhibit potential protective reaction of the plant, has been investigated to a less degree (Muratoglu et al., 2011; Chung et al., 2013). So, what happens when two armies of endosymbionts face each other? 2. Materials and methods Leptinotarsa decemlineata were collected from potato fields in the vicinity of Bashkortostan, Russia and were used for two variants of the experiments: 2.1. Variant 1. Potato plants, infected by B. subtilis 26D Sterile potato plants (Solanum tuberosum L.), cultivar Early Rose, were cultured in a test tube for 30 days at 16 h illumination of 12,000–16,000 lx (Lamp LD-4, LB-40, Russia) in a KC-200 growth chamber (SKTB SPU, Russia). 7-days-old plants were inoculated with 5 lL of B. subtilis 26D strain cell suspension on the stem zone adjacent to the zone of adventitious roots formation. Concentration of B. subtilis cells was 1 ⁄ 108 cells/mL. Plants were grown in a
66
A.V. Sorokan et al. / Journal of Invertebrate Pathology 136 (2016) 65–67
Fig. 1. Influence of B. subtilis 26D on Acinetobacter ssp. and Enterobacter ssp. colonies growth.
gnotobiotic system for 14 days. Then, the leaves of infested and non-infested plants with petioles covered by paper-gauze and inserted into tubes with water were placed in glass vessels and beetles were fed on potato leaves of these plants (1 beetle on 1 plant). 2.2. Variant 2. Potato plants with surface contaminated by B. subtilis 26D Sterile potato plants were dipped into a suspension of B. subtilis 26D (109 cells/ml) for 1 min and were exposed to beetle attack at once. Subsequently beetles in all vessels ate non-infested plants to remove B. subtilis 26D containing intestinal contents. The control groups of both the variants were fed with potato leaves of plants treated with sterile water. Activity of feeding (mg/day/beetle) and % of surviving beetles were assessed. After 24 h remaining plants were weighed. The survived beetles were selected into groups for identification endosymbiotic microorganisms and analyses of their quantity in their guts. The data were analyzed statistically using a one-way ANOVA test. Identification of bacterial endosymbionts of L. decemlineata was carried out through DNA sequencing of 16S RNA gene fragments on the device ABI PRISM 310 (Applied Biosystems, USA), by using sequencing kits Big Dye Terminator v3.1 (Life technologies, USA). Nucleotide sequence analysis was carried out by using international database GenBank. The method of perpendicular strokes was used for antagonistic activity tests. Individual guts were homogenized with 100 lM of 0.9% saline for count of colony forming units (CFU). 3. Results and discussion In our laboratory B. subtilis 26D was isolated from a section of surface-sterilized tissues of the tube-growth potato with traditional planting methods onto a potato-glucose agar medium. Surface-sterilization kills all microorganisms except those living in internal tissues. This property of B. subtilis 26D strain is very important for the development of growth- and resistance-
stimulating agricultural agents, because internal plants microorganisms are protected from unfavourable environmental conditions (Maksimov et al., 2015). So, this strain is able to colonize stem and leaf tissues and it’s clear that L. decemlineata eating potato leaves also swallow bacterial cells too. Table 1 shows the L. decemlineata imagos feeding rate (plant mass) and their survival in 7 days after feeding. A significant change in the rate wasn’t observed. Treatment of cut potato leaves with B. subtilis 26D in the laboratory also resulted in a decrease of the survival among affected L. decemlineata imagos. It should be noted that on the 7th day of the experiment the natural death rate of insects wasn’t more than 15%, but amount of beetles, which were fed on both the B. subtilis 26D infected potato plants, was halved. Thus, our findings suggest that L. decemlineata do not have certain ‘‘feeding habits”, but eating of plants, which contain B. subtilis 26D (Maksimov et al., 2015) or directly B. subtilis 26D suspension, lead to the dramatic reduction of survival. So, we supposed that this effect was caused by B. subtilis 26D. L. decemlineata microsymbionts isolated from anterior and posterior parts of beetle gut were defined as Enterobacter ssp. and Acinetobacter ssp. An insect unit in the normal conditions has about 20.6 ⁄ 102 CFU of Enterobacter ssp. and 133 ⁄ 102 CFU of Acinetobacter ssp. Eating of B. subtilis 26D treated plants or B. subtilis 26D suspension led to the significant decrease of their amount. So, in both variants amount of Enterobacter ssp. was decreased more than two-times. The quantity of Acinetobacter ssp. in the guts of beetles, eating infected plants, almost vanished and was more than 50-times less in comparison with the results of control group (beetles eating plants with suspension on its surface). Besides, it was found that B. subtilis 26D inhibited growth of endosymbiotic microflora of the Colorado potato beetle, especially isolates of Acinetobacter ssp. on basal medium (Fig. 1). It should be noted that neither isolate inhibits the growth of the each other and has no influence on B. subtilis 26D growth. B. subtilis 26D disturbs L. decemlineata microbiome stability. It is possible that this ability is the reason for the subsequent low survival rate of the insects. Thus, we confirmed the active influence of B. subtilis 26D on endosymbionts of L. decemlineata and its role in the development
Table 1 Effect of eating of plants inoculated with B. subtilis 26D on the beetle mortality and L. decemlineata gut endosymbionts amount on the 7th day after feeding.
Control B. subtilis 26D (inoculated plants) B. subtilis 26D (surface contaminated plants) *
Mortality, %
Mg/day/beetle
Enterobacter ssp., cells/beetle 102
Acinetobacter ssp., cells/beetle 102
15.5 ± 4.1 50* ± 7.3 46.3* ± 7.5
83.75 ± 2.4 86.67 ± 14.6 –
20.6 ± 1.2 9* ± 0.4 8* ± 0.3
133 ± 15.8 0 3* ± 0.1
Effect of B. subtilis 26D was significant at P 6 0.001.
A.V. Sorokan et al. / Journal of Invertebrate Pathology 136 (2016) 65–67
of a pathological process in the pest organism. This property, high antagonistic potential against phytopathogenic fungi and ability to induce systemic resistance of potato to late blight (Maksimov et al., 2014) allows us to consider this strain as an efficacious resource for the development of multipurpose and environmentally friendly pesticides. Additional research is needed to solve the question about antagonistic and synergistic relationships between plants and pest’s endosymbionts and the possibility of changing these relations during infection and protective reactions.
Conflict of interest We declare that there are no conflicts of interest.
Acknowledgments Russian Federation Ministry of Education and Science is gratefully acknowledged for project No. 14.604.21.0016 (unique identifier RFMEFI57614X0039).
67
References Chung, S.H., Rosa, C., Hoover, K., Luthe, D.S., Felton, G.W., 2013. L. decemlineata manipulates plant defenses in local and systemic leaves. Plant Signaling Behav. 8, e27592, PMID: 24390091; http://dx.doi.org/10.4161/psb.27592. Kloepper, J.W., Tuzun, S., 1996. Induced systemic to diseases and increased plant growth by growth-promoting rhizobacteria under field conditions. Phytopathology 81, 1508–1516. http://dx.doi.org/10.1094/Phyto-86-221. Maksimov, I.V., Abizgildina, R.R., Sorokan’, A.V., Burkhanova, G.F., 2014. Regulation of peroxidase activity under the influence of signaling molecules and Bacillus subtilis 26D in potato plants infected with Phytophthora infestans. Appl. Biochem. Microbiol. 50 (2), 173–178, PMID: 25272739. Maksimov, I.V., Sorokan’, A.V., Nafikova, A.R., Benkovskaya, G.V., 2015. On principal ability and action mechanisms of joint use of Bacillus subtilis 26D and Beauveria bassiana Ufa-2 preparation for potato protection against Phytophthora infestans and Leptinotarsa decemlineata. Micologia I fitopatologia 49 (5), 317–324. Muratoglu, H., Demirbag, Z., Sezen, K., 2011. The first investigation of the diversity of bacteria associated with Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Biologia 66 (2), 288–293. http://dx.doi.org/10.2478/s11756011-0021-6. Pieterse, C.M., Zamioudis, C., Berendsen, R.L., Weller, D.M., Van Wees, S.C., Bakker, P. A., 2014. Induced systemic resistance by beneficial microbes. Annu. Rev. Phytopathol. 52, 347–375. http://dx.doi.org/10.1146/annurev-phyto-082712102340. Smirnov, V.V., Sorokulova, I.B., Bereznitskaya, T.G., 1987. Russian Federation Patent No. RU 2099947.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
The influence of potato endophytes on Leptinotarsa decemlineata endosymbionts promotes mortality of the pest Antonina V. Sorokan ⇑, Galina V. Ben’kovskaya, Igor’ V. Maksimov Institute of Biochemistry and Genetics, Ufa Science Center, Russian Academy of Science, Russia
a r t i c l e
i n f o
Article history: Received 28 December 2015 Revised 24 February 2016 Accepted 8 March 2016 Available online 9 March 2016 Keywords: Bacillus subtilis 26D Endophytes Colorado potato beetle Symbionts
a b s t r a c t Plants are exposed to pervasive attack by diverse attackers, such as pathogens and pests. But plants have their own endophytic microflora as well as the attacking insects. These microbiomes contact face to face in the nature. It has been found that the endophytic strain Bacillus subtilis 26D increases mortality of Colorado potato beetles, disturbing the development of insect microsymbionts Enterobacter ssp. and Acinetobacter ssp. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Relationships in ecosystems are mainly based on trophic chains. Host-parasite interactions can be considered as the most diverse and multilevel among them. So, parasites in an animal or plant organism can become useful to their owner, i.e. can affiliate to the host microbiome in the process of co-adaptation. Of course, natural plant-phytophage systems include microbiomes of the host plant and the pest alike. The role of the interaction between macroorganisms’ microbiomes, related to each other by a trophic chain, is one of the most intriguing areas of modern synecology. Apart from the fundamental aspects of research, this area has obvious practical importance for the creation of selective agents, controlling the number of phytophagous pests and causing minimal damage to agricultural plants and ecosystems. Given the diversity of habitats for Bacillus subtilis, it is not surprising that B. subtilis not only has antibiotic properties, but also can be an endophytic colonist from several plant species and protect plants against plant pathogens. Many strains of these bacteria are used already in protection and growth-promoting schemes (Kloepper and Tuzun, 1996; Pieterse et al., 2014). Strain B. subtilis 26D was used for the development of Fytosporine-M that is considered as environmentally desirable alternatives to synthetic chemicals for the control of agricultural plant diseases (Smirnov et al., 1987). It has been shown that this strain has the ability to penetrate into the plant organism and exists inside of tissues of ⇑ Corresponding author. E-mail address: [email protected] (A.V. Sorokan). http://dx.doi.org/10.1016/j.jip.2016.03.006 0022-2011/Ó 2016 Elsevier Inc. All rights reserved.
different plant families, including Solanaceae (Maksimov et al., 2014). It should be noted, the majority of applied research in this area is evaluated without taking into account the fact that these microorganisms interact not only with plants, but also with plant microbiome, insect pest and insect microbiome. So, the role of endophytes in plant protection against pathogenic organisms has been established repeatedly. At the same time the role of phytophagous insect endosymbionts, which are potentially able to prevent the heterogeneous microflora invasion and also to inhibit potential protective reaction of the plant, has been investigated to a less degree (Muratoglu et al., 2011; Chung et al., 2013). So, what happens when two armies of endosymbionts face each other? 2. Materials and methods Leptinotarsa decemlineata were collected from potato fields in the vicinity of Bashkortostan, Russia and were used for two variants of the experiments: 2.1. Variant 1. Potato plants, infected by B. subtilis 26D Sterile potato plants (Solanum tuberosum L.), cultivar Early Rose, were cultured in a test tube for 30 days at 16 h illumination of 12,000–16,000 lx (Lamp LD-4, LB-40, Russia) in a KC-200 growth chamber (SKTB SPU, Russia). 7-days-old plants were inoculated with 5 lL of B. subtilis 26D strain cell suspension on the stem zone adjacent to the zone of adventitious roots formation. Concentration of B. subtilis cells was 1 ⁄ 108 cells/mL. Plants were grown in a
66
A.V. Sorokan et al. / Journal of Invertebrate Pathology 136 (2016) 65–67
Fig. 1. Influence of B. subtilis 26D on Acinetobacter ssp. and Enterobacter ssp. colonies growth.
gnotobiotic system for 14 days. Then, the leaves of infested and non-infested plants with petioles covered by paper-gauze and inserted into tubes with water were placed in glass vessels and beetles were fed on potato leaves of these plants (1 beetle on 1 plant). 2.2. Variant 2. Potato plants with surface contaminated by B. subtilis 26D Sterile potato plants were dipped into a suspension of B. subtilis 26D (109 cells/ml) for 1 min and were exposed to beetle attack at once. Subsequently beetles in all vessels ate non-infested plants to remove B. subtilis 26D containing intestinal contents. The control groups of both the variants were fed with potato leaves of plants treated with sterile water. Activity of feeding (mg/day/beetle) and % of surviving beetles were assessed. After 24 h remaining plants were weighed. The survived beetles were selected into groups for identification endosymbiotic microorganisms and analyses of their quantity in their guts. The data were analyzed statistically using a one-way ANOVA test. Identification of bacterial endosymbionts of L. decemlineata was carried out through DNA sequencing of 16S RNA gene fragments on the device ABI PRISM 310 (Applied Biosystems, USA), by using sequencing kits Big Dye Terminator v3.1 (Life technologies, USA). Nucleotide sequence analysis was carried out by using international database GenBank. The method of perpendicular strokes was used for antagonistic activity tests. Individual guts were homogenized with 100 lM of 0.9% saline for count of colony forming units (CFU). 3. Results and discussion In our laboratory B. subtilis 26D was isolated from a section of surface-sterilized tissues of the tube-growth potato with traditional planting methods onto a potato-glucose agar medium. Surface-sterilization kills all microorganisms except those living in internal tissues. This property of B. subtilis 26D strain is very important for the development of growth- and resistance-
stimulating agricultural agents, because internal plants microorganisms are protected from unfavourable environmental conditions (Maksimov et al., 2015). So, this strain is able to colonize stem and leaf tissues and it’s clear that L. decemlineata eating potato leaves also swallow bacterial cells too. Table 1 shows the L. decemlineata imagos feeding rate (plant mass) and their survival in 7 days after feeding. A significant change in the rate wasn’t observed. Treatment of cut potato leaves with B. subtilis 26D in the laboratory also resulted in a decrease of the survival among affected L. decemlineata imagos. It should be noted that on the 7th day of the experiment the natural death rate of insects wasn’t more than 15%, but amount of beetles, which were fed on both the B. subtilis 26D infected potato plants, was halved. Thus, our findings suggest that L. decemlineata do not have certain ‘‘feeding habits”, but eating of plants, which contain B. subtilis 26D (Maksimov et al., 2015) or directly B. subtilis 26D suspension, lead to the dramatic reduction of survival. So, we supposed that this effect was caused by B. subtilis 26D. L. decemlineata microsymbionts isolated from anterior and posterior parts of beetle gut were defined as Enterobacter ssp. and Acinetobacter ssp. An insect unit in the normal conditions has about 20.6 ⁄ 102 CFU of Enterobacter ssp. and 133 ⁄ 102 CFU of Acinetobacter ssp. Eating of B. subtilis 26D treated plants or B. subtilis 26D suspension led to the significant decrease of their amount. So, in both variants amount of Enterobacter ssp. was decreased more than two-times. The quantity of Acinetobacter ssp. in the guts of beetles, eating infected plants, almost vanished and was more than 50-times less in comparison with the results of control group (beetles eating plants with suspension on its surface). Besides, it was found that B. subtilis 26D inhibited growth of endosymbiotic microflora of the Colorado potato beetle, especially isolates of Acinetobacter ssp. on basal medium (Fig. 1). It should be noted that neither isolate inhibits the growth of the each other and has no influence on B. subtilis 26D growth. B. subtilis 26D disturbs L. decemlineata microbiome stability. It is possible that this ability is the reason for the subsequent low survival rate of the insects. Thus, we confirmed the active influence of B. subtilis 26D on endosymbionts of L. decemlineata and its role in the development
Table 1 Effect of eating of plants inoculated with B. subtilis 26D on the beetle mortality and L. decemlineata gut endosymbionts amount on the 7th day after feeding.
Control B. subtilis 26D (inoculated plants) B. subtilis 26D (surface contaminated plants) *
Mortality, %
Mg/day/beetle
Enterobacter ssp., cells/beetle 102
Acinetobacter ssp., cells/beetle 102
15.5 ± 4.1 50* ± 7.3 46.3* ± 7.5
83.75 ± 2.4 86.67 ± 14.6 –
20.6 ± 1.2 9* ± 0.4 8* ± 0.3
133 ± 15.8 0 3* ± 0.1
Effect of B. subtilis 26D was significant at P 6 0.001.
A.V. Sorokan et al. / Journal of Invertebrate Pathology 136 (2016) 65–67
of a pathological process in the pest organism. This property, high antagonistic potential against phytopathogenic fungi and ability to induce systemic resistance of potato to late blight (Maksimov et al., 2014) allows us to consider this strain as an efficacious resource for the development of multipurpose and environmentally friendly pesticides. Additional research is needed to solve the question about antagonistic and synergistic relationships between plants and pest’s endosymbionts and the possibility of changing these relations during infection and protective reactions.
Conflict of interest We declare that there are no conflicts of interest.
Acknowledgments Russian Federation Ministry of Education and Science is gratefully acknowledged for project No. 14.604.21.0016 (unique identifier RFMEFI57614X0039).
67
References Chung, S.H., Rosa, C., Hoover, K., Luthe, D.S., Felton, G.W., 2013. L. decemlineata manipulates plant defenses in local and systemic leaves. Plant Signaling Behav. 8, e27592, PMID: 24390091; http://dx.doi.org/10.4161/psb.27592. Kloepper, J.W., Tuzun, S., 1996. Induced systemic to diseases and increased plant growth by growth-promoting rhizobacteria under field conditions. Phytopathology 81, 1508–1516. http://dx.doi.org/10.1094/Phyto-86-221. Maksimov, I.V., Abizgildina, R.R., Sorokan’, A.V., Burkhanova, G.F., 2014. Regulation of peroxidase activity under the influence of signaling molecules and Bacillus subtilis 26D in potato plants infected with Phytophthora infestans. Appl. Biochem. Microbiol. 50 (2), 173–178, PMID: 25272739. Maksimov, I.V., Sorokan’, A.V., Nafikova, A.R., Benkovskaya, G.V., 2015. On principal ability and action mechanisms of joint use of Bacillus subtilis 26D and Beauveria bassiana Ufa-2 preparation for potato protection against Phytophthora infestans and Leptinotarsa decemlineata. Micologia I fitopatologia 49 (5), 317–324. Muratoglu, H., Demirbag, Z., Sezen, K., 2011. The first investigation of the diversity of bacteria associated with Leptinotarsa decemlineata (Coleoptera: Chrysomelidae). Biologia 66 (2), 288–293. http://dx.doi.org/10.2478/s11756011-0021-6. Pieterse, C.M., Zamioudis, C., Berendsen, R.L., Weller, D.M., Van Wees, S.C., Bakker, P. A., 2014. Induced systemic resistance by beneficial microbes. Annu. Rev. Phytopathol. 52, 347–375. http://dx.doi.org/10.1146/annurev-phyto-082712102340. Smirnov, V.V., Sorokulova, I.B., Bereznitskaya, T.G., 1987. Russian Federation Patent No. RU 2099947.