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Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae
FEMS Microbiology Ecology 51 (2004) 79–86 www.fems-microbiology.org
Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae Chaorong Tang, Fuzai Sun *, Xinjian Zhang, Tingchang Zhao, Jiyan Qi State Key Lab for the Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China Received 13 January 2004; received in revised form 26 June 2004 First published online 20 August 2004
Abstract The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at 2.5 C, significantly higher than their parent strains (active at approx 10 C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were 3 to 4 C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of 7 C, and more than 95% died after 12 h at 5 C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Transgenic INA En. cloacae; Biological control; SCP; Gut colonization; Corn borer and cotton bollworm; Epiphytic fitness
1. Introduction The severity of insect damage to crops often depends on the size of the surviving fraction of the overwintering population [1,2]. Increasing winter mortality of insect pests is therefore an effective way to reduce crop loss in agricultural fields. Since the vast majority of insects is freeze-intolerant, i.e., unable to tolerate internal ice formation, a key factor in their overwintering survival is the regulation of the temperature at which they spontaneously freeze, termed the supercooling point (SCP) or * Corresponding author. Tel.: +86-10-62815933; fax: +86-1062896114. E-mail addresses: [email protected], [email protected] (C. Tang), [email protected] (F. Sun).
the temperature of crystallization [3,4]. Overwintering insects enhance the supercooling of body fluids by several means. First, due to their small size and therefore small water volume, insects have the inherent ability to extensively supercool [4]. Second, they can enhance their supercooling capacity by evacuating or inactivating heterogeneous ice-nucleating agents and amassing lowmolecular-mass polyols and sugars, sometimes in high concentrations [5–7]. In addition, some insects can accumulate anti-freeze proteins in haemolymph tissues or seek overwintering shelters [1,8,9]. Combining some or all of these measures, insect pests can decrease their SCPs to remain unfrozen to 20 C or lower, and thus readily survive harsh winters to reduce crop yields in the following growing season.
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.08.001
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Ice nucleation-active (INA) bacteria, the most potent ice nuclei known in nature, were reported to raise the SCPs and reduce the cold hardiness of insect pests such as Tribolium castaneum (red flour beetle), Helicoverpa armigera (cotton bollworm), Plodia interpunctella (Indian mealmoth), Anoplophora glabripennis and Rhyzopertha dominica, when these bacteria were sprayed on insect surfaces or ingested by the insects [6,10–13]. These interesting results prompted a number of investigators to explore the possibility of exploiting INA bacteria to control overwintering insect pests. In practice, problems emerge when INA bacteria are released in agricultural fields to control insect pests. First, after several days, the SCPs of insects treated with INA bacteria often decrease to levels similar to that of untreated controls [13–15]. These results have been attributed to unstable colonization of INA bacteria [15]. Due to the time interval between the spread of INA bacteria and the drop of the ambient temperature to levels low enough for them to function, it is essential for these bacteria to stably colonize in the guts or on the surfaces of the insects, so that they can maintain their ability of reducing insect cold hardiness over a long time. Second, frost damage to frost-sensitive plant species has been related to the population size of INA bacteria on the plants [16,17]. Therefore, the most serious problem regarding the future release of INA bacteria in agricultural fields as biological insecticides may be the occurrence of incited frost injury. One feasible approach of dealing with these problems is to isolate new kinds of INA microorganisms, which would be stably retained by insect pests, and simultaneously have weaker epiphytic fitness (the ability of bacteria to grow and maintain a population on leaves) [18] than frequently isolated epiphytic INA bacteria such as Erwinia ananas and Pseudomonas syringae, the predominant species in China [19]. So far, such ideal strains have not been obtained. We were therefore interested in the application of other strains. For example, both insect-origin and plant-origin Enterobacter cloacae strains grow well and stably in the guts of silkworm [15]. Furthermore, En. cloacae is not commonly isolated from plant leaves, except for some opportunistic phytopathogenic strains [20–23]. It was thus reasonable to expect some En. cloacae strains to stably colonize in insect guts, but not to persist long on plants and therefore, INA En. cloacae should be an ideal candidate for controlling insect pests in agricultural fields. However, attempts to isolate wild-type INA En. cloacae have failed [24,25]. Ice nucleation (IN) genes responsible for bacterial ice nucleation have been cloned from several INA bacteria, including E. ananas [26,27]. Watanabe et al. [25] first reported that transgenic INA En. cloacae could be useful in controlling mulberry pyralid larvae, G. duplicalis (Lepidoptera), in agricultural fields. They
transformed a recombinant pUC18 vector containing the IN gene inaA into an En. cloacae strain. The resulting transgenic En. cloacae, unlike the parent strain, showed high INA, and after ingestion by mulberry pyralid larvae, it exhibited marked efficacy in reducing their supercooling capacity and cold hardiness. The efficacy maintenance was found to result from the efficient gut colonization of this transgenic En. cloacae. On the other hand, the growth ability of this strain on mulberry leaves tended to be lower than that of epiphytic INA E. ananas, reducing the incidence of frost injury after its release in agricultural fields. In their study, the IN gene existed in the form of plasmids. In our previous study [24], however, we demonstrated that in the absence of antibiotic pressure, En. cloacae strains harboring IN gene-containing plasmids were likely to lose their plasmids and INA, and may therefore not retain their activity in agricultural fields. In this study, the IN gene iceA we previously cloned from E. ananas 110 [24,26] was integrated into the chromosomes of two En. clocae strains, and their efficacy was tested in reducing the cold hardiness of two of the most hazardous insect pests in China, corn borer and cotton bollworm. Their epiphytic fitness on corn leaves was also compared to that of epiphytic E. ananas by pot tests, conducted in both greenhouse and outdoor conditions.
2. Materials and methods 2.1. Bacteria, plasmids and insects Escherichia coli S17-1 bears a chromosomally integrated RP4 that provided broad host-range conjugal transfer functions [28]. INA bacterium E. ananas 110, the parent strain for the IN gene iceA, was previously isolated from the leaves of Zea mays in Shanxi, China [29]. Enc1.181 (ATCC7256) and Enc1.2022 (ATCC13047) are two biosafety level 1, non-ice-nucleation-active and ampicillin (Amp) resistant En. cloacae strains isolated from well water and spinal fluid, respectively, initially acquired from the American Type Culture Collection and now available in the China General Microbiological Culture Collection Center. Recombinant plasmid pINA105, carrying the cloned iceA gene, was constructed by Tang et al [26]. The Tn5-mob fragment endows the plasmid pSZ21 [28,30] with the functions of Tn5 transposition, kanamycin (Km) resistance and conjugal mobilization in gram-negative bacteria. Two kinds of Lepidoptera insect pests, cotton bollworm (H. armigera) and corn borer (Pyrausta nubialis) larvae, were used as test insects for the measurement of SCP and death by freezing.
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2.2. Construction of transgenic INA En. cloacae
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Biotechnology (Dalian) Co., Ltd., Dalian, China) and with the iceA full-length coding region as template.
The IN gene iceA was excised as a 4-kb SalI-BamHI fragment from pINA105, and cloned into pSZ21 by inserting it between the SalI and BamHI sites within Tn5. The resulting construct, named Tn5-mob-iceA, was transformed into E. coli S17-1 to obtain the Tn5mob-iceA -containing transformant (designated E. coli S17-1 (Tn5-mob-iceA)). The Tn5-mob-iceA construct was then transferred from donor strain S17-1 (Tn5mob-iceA) into the two target En. cloacae strains (Enc1.181 and Enc1.2022), using a filter mating technique. Filters (Nitrocellulose filter, U 50 mm, 0.2 lm, Cole-parmar, etc.) with a mixture of donor and recipient cells at logarithmic phase in a 1:5 ratio were incubated for 4 h at 30 C and spread on LB agar plates supplemented with ampicillin (Amp) and chloramphenicol (Cm), incubated at 30 C for 24 h, and then for 2h at 4 C for the enhancement of INA. The resulting En. cloacae transconjugants originating from Enc1.181 and Enc1.2022 were named Enc181(Tn5-mob-iceA) and Enc2022(Tn5-mob-iceA), respectively. These transconjugants were then isolated and further identified with the INA assays, as described by Lindow [31], and plasmid restriction analysis. Bacterial suspensions for INA assays were prepared as follows. One single colony, from a newly cultured bacterial plate, was inoculated into 5 ml of KingÕs medium B (KB), and grown for 12 h with shaking of 200 rpm at 30 C. After a 2-h treatment at 4 C, bacterial cells were pelleted and suspended in sterile distilled water (DW) to a concentration of 108 cells per ml. Plasmids were eliminated from En. cloacae transconjugants by growing in the absence of antibiotic pressure and in the presence of acridine orange as previously described [32]. En. cloacae colonies with plasmids eliminated and iceA integrated into their chromosomes through Tn5 transposition should be resistant to kanamycin (Kmr) and sensitive to chloramphenicol (Cms). INA assays, plasmid isolation, PCR and Southern blot analysis (see below) were performed to further verify chromosomal integration of iceA. The resulting transgenic INA En. cloacae strains, originating from Enc181(Tn5-mob-iceA) and Enc2022(Tn5-mob-iceA), were designated En. cloacae 181-I (abbr. Enc181-I) and En. cloacae 2022-I (abbr. Enc2022-I), respectively.
Measurement of larval SCP was performed at 0, 3, 5, 7 and 9 days after bacterial ingestion. At each time point, 15 larvae were collected, starved for 1 day at 22 C and kept 12 h at 4 C for the enhancement of INA. Then, their SCPs were measured based on the method of Watanabe and Tanaka [35]. After measurement, 5 larvae for each treatment were surface-sterilized for 1 min in 70% alcohol, then rinsed three times with sterile DW and macerated with sterile DW in a mortar. The macerated suspensions were diluted and spread on KB plates with amphotericin B (1 lg ml1) to inhibit fungi and Km (20 lg ml1) to estimate the population size of transgenic bacteria. To determine which colonies originated from the inoculated bacteria, 20 colonies were randomly picked and INA was assayed according to the method of Lindow [31].
2.3. Southern blot analysis of transgenic En. cloacae
2.6. Survival estimation
Genomic DNA from non-transgenic En. cloacae strains (Enc1.181 and Enc1.2022) and their corresponding transgenic strains (Enc181-I and Enc2022-I) was isolated, digested with BamHI or SalI, fractionated by agarose electrophoresis, transferred to nylon membranes and hybridized to the iceA probe as previously described [33]. The iceA probe was prepared using the ‘‘Random Prime DNA Labeling Kit ver. 2.0’’ (TaKaRa
From each treatment at 6 days after ingestion, 14–28 larvae of corn borer and cotton bollworm were brought to a frost chamber, where the temperature was lowered from room temperature to 5 or 7 C at a rate of 10 C h1.The larvae were kept at 5 or 7 C and checked after 3, 6 and 12 h to see if they were frozen before being returned to the chamber for continued incubation. Freezing status was determined by slightly
2.4. Feeding insects and bacterial ingestion Newly hatched larvae of corn borer and cotton bollworm were transferred into sterile 1-l beakers and separately fed in an incubator at 22 C and 70% relative humidity on healthy fresh corn stems and cotton leaves, which were collected from agricultural fields, washed with tap water, rinsed with DW and wiped with absorbent paper. Three days later, these larvae were allowed to ingest the bacterial strains Enc181-I and Enc2022-I, using an approach similar to that described by Watanabe and Sato [34], as follows. Corn stems and cotton leaves were thoroughly immersed in bacterial suspensions (108 cells ml1 in DW, prepared from cells cultured in LB liquid with shaking to stationary phase) or just sterile DW as a control treatment for 20 min, and airdried at room temperature. Then, 250 larvae of each kind of insect were fed on them. After 3 days feeding (the so-called bacterial ingestion), the insects were shifted to sterile glass tubes, one larva per tube, and fed fresh untreated plant tissues. Every 2 days, old plant tissues were discarded and fresh tissues were administered. 2.5. Measuring insect SCP and bacterial population size
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prodding the larva with a matchstick to check for softness. After incubation at 5 or 7 C for 12 h, all larvae were transferred to an incubator at 22 C for 1 h, and then examined for survival or death.
mine which colonies on the KB plates originated from the inoculated bacteria, 20 colonies were randomly selected and their colony characteristics were compared with those of the inoculated bacteria and examined for INA using the freezing droplet assay [31].
2.7. Bacterial epiphytic fitness 2.8. Statistical methods The epiphytic fitness of transgenic En. cloacae 181-I and 2022-I on corn leaves was investigated based, with some modifications, on the method of Lindow [18], and compared with that of wild-type INA bacterium E. ananas 110 (Ea110). Experiments were conducted with potted corn seedlings using the same procedure both outdoors and in a greenhouse. The outdoor assay was performed between late May and early June of 2001, in the open ground behind the experimental building of the Institute of Plant Protection of CAAS (Beijing, China). Outdoor air temperatures ranged from 14 to 30 C,with daily mean air temperatures of 20–23 C. Greenhouse conditions were maintained at 20 C and 60% relative humidity. Bacteria (Enc181-I, Enc2022-I or Ea110) were cultured to the stationary phase in LB medium, pelleted by centrifugation and suspended in sterile DW to suspensions of 108 cells per ml. Leaves of potted corn seedlings at the 5-leaf stage (with five primary leaves) were evenly sprayed with a suspension of a given bacterial strain, and these seedlings were then covered with a polyethylene bag dipped in water for 1 day. After removing the bag, five samplings were sequentially made at 1, 5, 10, 15 and 20 days after bacterial inoculation, and the population size of each respective inoculated bacterium was quantified as described below. Two days before each sampling, the seedlings were sprayed with water. On the day of sampling, leaves were collected, cut into small pieces and about 0.5 g (fresh weight) of leaves were ground in 5 ml sterile DW in a sterile mortar. The resulting suspension was incubated for 2 h at 4 C, vortexed for 30 s, and appropriate 10-fold serial dilutions were plated onto KB plates containing either amphotericin (1 lg ml1) and Km (20 lg ml1) to isolate transgenic En. cloacae or just amphotericin (1 lg ml1) to isolate E. ananas 110. To deter-
Statistical analyses were done using the software SAS 6.11 (SAS Institute, Cary, NC). The comparison of insect SCPs, gut-colonizing ability and epiphytic fitness of bacteria were performed by ANOVA, Scheffe test and t-test. The v2 test and FisherÕs Exact test were used to compare freezing percentages and mortality rates of larvae in different treatments.
3. Results 3.1. Expression of iceA in transgenic En. cloacae The filter mating produced 2 and 3 Kmr Cms colonies, respectively, from En. cloacae transconjugants of Enc181(Tn5-mob-iceA) and Enc2022(Tn5-mob-iceA). One such colony from each strain was randomly selected for subsequent assays. No plasmid could be isolated from these En. cloacae colonies, indicating that plasmid vectors of the Tn5-mob-iceA constructs were eliminated. Both PCR and Southern blot analyses demonstrated that the IN gene (iceA) was integrated into their chromosomal DNAs. Also, Southern blot analysis showed that the transconjugants had only one copy of iceA (data not shown). These transgenic En. cloacae, designated En. cloacae 181-I (abbr. Enc181-I) and En. cloacae 2022-I (abbr. Enc2022-I), displayed ice nucleation activity (INA) at 2.5 C, similar to that of E. ananas 110 (active at 2.3 C), the parent strain for iceA (Table 1). In contrast, their parent strains Enc1.181 and Enc1.2022 barely showed INA, even at 10 C. These results indicated that the high INA of transgenic En. cloacae was an acquired trait and iceA was fully expressed in En. cloacae.
Table 1 Ice nucleation activity (INA) of different bacterial strains Percentages of freezing drops (%)a
Strain
Enterobacter cloacae Enterobacter cloacae Erwinia ananas 110 Enterobacter cloacae Enterobacter cloacae Distilled water a b
181-I 2022-I 1.181 1.2022
10.5 C
10 C
5 C
4 C
3 C
2.5 C
2.3 C
2.1 C
NDb ND ND 28.7 ± 3.5 4.0 ± 1.0 0
ND ND ND 3.0 ± 1.0 0 0
100 100 100 0 0 0
100 100 100 0 0 0
100 100 100 ND ND ND
7.0 ± 2.0 3.7 ± 0.6 37.0 ± 3.1 ND ND ND
0 0 5.7 ± 1.5 ND ND ND
0 0 0 ND ND ND
INA assays were conducted according to the droplet-freezing method of Lindow [31], and were repeated three times. ND, not detected.
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Corn borer larvae
Cotton bollworm larvae
-12
-16
Supercooling point(˚C)
Supercooling point(˚C)
-20
-12 -8 -4 0 0
3
5
7
83
-8
-4
0
9
0
Days after treatment
3
5
7
9
Days after treatment
Fig. 1. Supercooling point changes in corn borer larvae and cotton bollworm larvae ingesting ice nucleation-active transgenic En. cloacae over the 9day period after treatment. Each larva was treated with: n, distilled water; }, Enc181-I; h, Enc2022-I. Vertical bars represent standard errors for means.
3.2. Supercooling point of corn borer and cotton bollworm larvae
Table 2 Colonization of transgenic Enterobacter cloacae in the guts of larvae of corn borer and cotton bollworm
In pilot experiments, it was demonstrated that the SCPs of corn borer and cotton bollworm larvae ingesting wild-type En. cloacae (Enc1.2022 and Enc1.181) were similar to those of DW (distilled water)-treated larvae (data not shown). Therefore, we simply used DW instead of non-transgenic En. cloacae as controls for the remainder of the experiments. The progressive changes in the SCPs of Enc181-I, Enc2022-I and DW treated larvae were monitored. Samples were collected at 0, 3, 5, 7 and 9 days after ingestion and their SCPs were measured. The SCPs of corn borer and cotton bollworm larvae remained constant for 7 days after ingesting transgenic En. cloacae (Fig. 1), and displayed a significant increase against those of untreated larvae, from 11 to 17 C and 10 C, respectively, to 3 to 4 C. Although the SCPs of these Enc181-I and Enc2022-I treated larvae markedly declined at 9 days after ingestion, they were still over 5 C and significantly higher than those of the DW controls. Some larvae of corn borer and cotton bollworm began to pupate at 9 days after ingestion, and no transgenic En. cloacae were isolated from these pupae, suggesting that these bacteria were unable to stably colonize in the pupal stage. In addition, individuals of transgenic bacteriatreated larvae had a narrow range of SCP distribution, the vast majority with SCPs of 3 to 5 C. In contrast, the SCPs of DW-treated insects were distributed over a much broader range, from 6 to 22 C for corn borer and 5 to 14 C for cotton bollworm.
Days after ingestion (days)
3.3. Colonization of transgenic En. cloacae in larval guts Both transgenic strains were able to grow in the guts of corn borer and cotton bollworm larvae (Table 2). Over 7 days after bacterial ingestion, a large population of target bacteria (Kmr, INA+) (104–105/larva) was detected in the guts of these larvae. The population size of target bacteria declined to 103 per larva 9 days after
0 3 5 7 9
Number of bacteria (log CFU/larva)a Corn borer
Cotton bollworm
Enc181-I
Enc2022-I
Enc181-I
Enc2022-I
4.8 ± 0.1a 5.0 ± 0.3a 5.3 ± 0.2a 4.7 ± 0.2a 3.5 ± 0.3b
4.6 ± 0.2a 4.9 ± 0.1a 5.2 ± 0.3a 5.0 ± 0.3a 3.8 ± 0.2b
4.7 ± 0.2a 5.1 ± 0.3a 5.2 ± 0.2a 4.8 ± 0.3a 3.4 ± 0.3b
4.4 ± 0.2a 4.9 ± 0.2a 5.4 ± 0.3a 4.6 ± 0.3a 3.6 ± 0.3b
a Means ± standard error. Within a column, means followed by different letters are significantly different (P 6 0.05; ANOVA, Scheffe test). Means are not significantly different (P > 0.05; t-test) between the two transgenic En. cloacae strains.
bacterial ingestion. The colonizing ability was not significantly different between these two transgenic bacteria. 3.4. Mortality of corn borer and cotton bollworm larvae by freezing The freezing percentages and mortality of larvae were significantly increased after ingesting transgenic INA En. cloacae (Table 3). More than 80% of bacteria-treated larvae froze after 3 h exposure to 5 C and over 90% after 12 h exposure. In contrast, even after 12 h exposure to 5 C, none or only a minority of untreated corn borer and cotton bollworm larvae froze. After 6 h exposure to 7 C, all treated larvae froze, compared to only a small proportion of untreated larvae (7.4% of corn borer and 14.3% of cotton bollworm). More than 90% of the treated larvae died after 12 h exposure to 5 C, and 100% died after 12 h exposure to 7 C. On the other hand, all or most untreated larvae survived under the same conditions. 3.5. Epiphytic fitness of transgenic En. cloacae The epiphytic fitness of transgenic bacteria on corn leaves was tested on potted corn seedlings (5-leaf stage), grown both outdoors and in a greenhouse, and
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C. Tang et al. / FEMS Microbiology Ecology 51 (2004) 79–86
Table 3 Percentages of freezing and mortality of corn borer and cotton bollworm larvae after exposure to low temperaturea Insect
Treatment
Freezing percentages of larvae (%) 5 C
Mortality rate of larvae (%)b
7 C
3h
6h
12 h
3h
6h
12 h
5 C
7 C
Corn borer
Enc181-I Enc2022-I DWc
84.6a 87.5a 0.0b
96.1a 91.7a 0.0b
96.1a 91.7a 0.0b
92.3a 95.8a 3.7b
100a 100a 7.4b
100a 100a 7.4b
96.1a 95.8a 0.0b
100a 100a 7.4b
Cotton bollworm
Enc181-I Enc2022-I DW
76.9a 87.5a 0.0b
92.3a 93.7a 7.1b
92.3a 93.7a 7.1b
100a 93.7a 7.1b
100a 100a 14.3b
100a 100a 14.3b
92.3a 93.7a 7.1b
100a 100a 14.3b
a b c
Means followed by different letters are significantly different (P 6 0.05; the v2 test and FisherÕs exact test). Larvae treated for 12 h were transferred to a 22 C incubator and their mortality examined. DW, distilled water.
been investigated by several research groups, and some inspiring results have been obtained [6,10–13]. In those studies, P. syringae strains were mainly used as INA bacteria. Being common epiphytic residents of plants, however, these strains incite frost injury by limiting the supercooling ability of water in plant tissues. Moreover, the effectiveness of the treatment with these bacteria rapidly decreased after application because of their inability to colonize insect guts [13–15]. Therefore, these strains are not the best suited for release in agricultural fields to control insect pests. In the present study, two transgenic INA En. cloacae strains (Enc181-I and Enc2022-I) were constructed by integrating the IN gene iceA into the chromosomes of two En. cloacae strains (Enc1.181 and Enc1.2022). These resulting transgenic bacteria displayed ice nucleation activity at 2.5 C, only slightly weaker than that of the parent INA strain E. ananas 110 (active at 2.3 C) for iceA (Table 1), indicating that the introduced iceA was actively expressed in En. cloacae. Expression of iceA was also identified in transgenic En. cloacae colonizing in insect guts. The mean SCPs of Enc181-I and Enc2022-I treated larvae were 3 to 4 C throughout the 7-day period after ingestion, significantly higher than those of untreated
compared to that of the bacterium E. ananas 110 (Ea110) (a corn epiphytic strain). A large difference existed in the epiphytic fitness among different bacterial strains (Fig. 2). In both experiments, transgenic En. cloacae (especially Enc2022-I) had a weaker fitness than Ea110 (P 6 0.05; ANOVA, Scheffe test). Strain Ea110 retained a population size of over 106 cells per gram of fresh leaf weight (cells/gFLW) 20 days after spraying bacteria, while Enc2022-I was unable to be recovered at this time period and Enc181-I had a population size of 103–104 cells/gFLW. Significant differences in epiphytic fitness between these two transgenic En. cloacae strains also existed (P 6 0.05; ANOVA, Scheffe test). After spraying, the population size of Enc2022-I decreased rapidly, and this strain could not be re-isolated 15 or 20 days later. In contrast, Enc181-I maintained, in both experiments, a population size of over 103 cells/gFLW during the 20 day post-inoculation period.
4. Discussion In recent years, decrease of cold hardiness and biological control of insect pests using INA bacteria have
B
LOG(Cells/gFLW)
LOG(Cells/gFLW)
10
A
8 6 4 2
8 6 4 2 0
0 1
5
10
15
Days after inoculation
20
1
5
10
15
20
Days after inoculation
Fig. 2. Population sizes of Erwinia ananas 110 and transgenic En. cloacae on corn leaves in a field study (A), and in a greenhouse study (B). n, E. ananas 110; }, Enc181-I; h, Enc2022-I; ·, no bacteria detected; vertical bars represent standard errors for means calculated from five independent measurements; cells/gFLW, cells per gram of fresh leaf weight.
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controls (Fig. 1). The maintenance of the high SCPs was apparently due to the stable gut colonization of the transgenic bacteria (Table 2). These transgenic bacteria markedly reduced the cold hardiness of corn borer and cotton bollworm larvae (Table 3), leading to a 100% death rate after exposure for 6 h to 7 C, while, under the same conditions, most DW-treated larvae did not freeze or die. In addition, the transgenic En. cloacae strains (especially Enc2022-I), unlike epiphytic E. ananas 110, did not persist longterm on plants (Fig. 2). These findings suggested that the transgenic En. cloacae were potentially useful as microbial control measures in order to reduce insect survival over winter in agricultural fields. In a different study, Watanabe et al. [25] reported the use of INA En. cloacae for the control of mulberry pyralid larvae. Our studies were different from theirs in several aspects. First, the IN gene iceA (GenBank Accession No. AF387802) cloned and used here was different from the IN gene inaA used in the previous study, sharing 94% amino acid identity [26,27]. Second, in their study, the IN gene inaA was transformed on plasmids, while in the present study, iceA was integrated into the chromosomes of En. cloacae, and these transgenic bacteria were more ice nucleation-active (2.5 C) (Table 1) than the previously reported transgenic strain (active at 2.9 C) [25]. Third, the present study focused on controlling two of the predominant insect pests in China, corn borer and cotton bollworm, undoubtedly expanding the application potential of this novel kind of biological control. Several problems should be pointed out regarding the field application of transgenic INA En. cloacae. In this study, these bacteria were unable to colonize the pupal stage of tested insects, suggesting that they would not be effective in controlling those insect pests overwintering in the form of pupae. For best efficiency, the transgenic bacteria should be extensively examined for their effects on the cold hardiness of different insect species and at different stages. In addition, the effects of INA bacteria in reducing crop loss caused by insect pests will not be seen until the year following bacterial release, so farmers tend to take quicker control means (such as chemical spraying) instead. Therefore, we suggest that this approach could be used concomitantly with other control means for integrated pest management of a given species. Also, since En. cloacae strains have been detected in the guts of a variety of insects [15,21,36–39], including the beneficial Collembolan Folsomia candida, and INA bacteria seem to be effective against a diverse range of insects [6,24], consideration must be given to avoid possible detrimental effects on the overwintering survival of beneficial insects. It has been suggested that applications of INA bacteria should be targeted to specific areas, where few nontarget insects would be exposed, such as in
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the trap crops for Colorado potato beetles or storage product sites [1,11]. We suggest here that, prior to the release of INA bacteria into agricultural fields, intensive surveys should be made to determine the species composition, the distributions of insects in selected target fields and the effects of these bacteria on overwintering species. Lastly, field conditions are very complex, and many factors may affect the effects of INA bacteria, so the results of laboratory studies must be confirmed with field tests. Using transgenic INA bacteria for biological control of insect pests is a recent area of scientific investigation. As it often occurs, results from initial studies give rise to many new questions, such as the following: are these transgenic INA bacteria safe to humans, animals and the environment? How do we develop application methods for better use of these bacteria in agricultural fields? Could insects develop resistance to this type of control and if so, how rapidly would this occur? In agricultural fields, to what extent will the microbial habitat in insect guts affect the function of these INA bacteria? Is it possible to actively and stably express the IN gene in those bacteria colonizing stably in overwintering insect pupae, eggs or adults? The answers to these questions will help us to evaluate the potential for environmental application of these bacteria.
Acknowledgements We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 30170624). We thank Prof. Stephen B. Goodwin (Dept. of Botony and Plant Pathology, Purdue University) and Prof. Yingchuan Tian (Ins. of Microbiology, CAS) for critically reading the manuscript, and Dr. Zhiwen Li and Dr. Jianghui Zhu (Ins. of Birth Health, Peking University) for valuable assistance in statistical analyses. We also thank Prof. Bingfu Shen (Ins. of Plant Physiology, CAS) for granting pSZ21, and Yongjun Li (Ins. of Atomic Energy, CAAS) and Hong Zhu (Ins. of Plant Protection, CAAS) for providing the test insects.
References [1] Lee, R.E., Costanzo, J.P., Kaufman, P.E., Lee, M.R. and Wyman, J.A. (1994) Ice nucleating active bacteria reduce the cold-hardiness of the freeze-intolerant Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 87, 377–381. [2] Zhou, Z.L. (1980) Insect Ecology, pp. 42–49. Shanghai Scientech Press, Shanghai (in Chinese). [3] Lee, R.E. (1989) Insect cold-hardiness: to freeze or not to freeze. BioScience 39, 308–313.
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[4] Lee, R.E. (1991) Principles of insect low temperature tolerance In: Insects at Low Temperature (Lee, R.E. and Denlinger, D.L., Eds.), pp. 17–46. Chapman and Hall, New York. [5] Cannon, R.J.C. and Block, W. (1988) Cold tolerance of microarthropods. Biol. Rev. 62, 23–77. [6] Lee, R.E., Lee, M.R. and Strong-Gunderson, J.M. (1993) Review: insect cold-hardiness and ice nucleating active microorganisms including their potential use for biological control. J. Insect Physiol. 39, 1–12. [7] Zachariassen, K.E. (1985) Physiology of cold tolerance in insects. Physiol. Rev. 65, 799–832. [8] Doucet, D., Tyshenko, M.G., Davies, P.L. and Walker, V.K. (2002) A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferan. Eur. J. Biochem. 269, 38–46. [9] Duman, J.G. (2002) The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. J. Comp. Physiol. [B.] 172, 163–168. [10] Feng, Y.X. and He, W.X. (1996) Studies on elevating the supercooling point of Indian meal moth larvae with bacterial ice nucleators. Acta Entomol. Sin. 39, 53–57. [11] Lee, R.E., Strong-Gunderson, J.M., Lee, M.R. and Davidson, E.C. (1992) Ice nucleating active bacteria decrease the coldhardiness of stored grain insects. J. Econ. Entomol. 85, 71–374. [12] Sun, F.Z., Xing, W., Zhang, Y.X. and Gao, R.T. (1997) Preliminary study on effect of INA bacteria to kill larva of Anoplophora glabripennis through freezing induction. Chinese J. For. Res. 10, 96–99. [13] Zhu, H., Sun, F.Z., Zhang, Y.X. and Wang, W.L. (1994) The effects of INA bacteria on the freezing temperature of cotton bollworm. Sci. Agr. Sin. 27, 23–27. [14] Strong-Gunderson, J.M., Lee, R.E. and Lee, M.R. (1990) Ingestion of ice nucleating active bacteria increases the supercooling point of the lady beetle Hippodamia convergens. J. Insect Physiol. 36, 53–157. [15] Watanabe, K. and Sato, M. (1998) Plasmid-mediated gene transfer between insect-resident bacteria, Enterobacter cloacae and plant-epiphytic bacteria, Erwinia herbicola in guts of silkworm larvae. Curr. Microbiol. 37, 352–355. [16] Lindow, S.E. (1983) The role of bacterial ice nucleation in frost injury to plants. Annu. Rev. Phytopathol. 21, 263–384. [17] Sun, F.Z. and Zhao, T.C. (2003) Ice nucleation active (INA) bacteriaÕs biological characteristics and their role in frost incitement and frost injury prevention. Acta Ecol. Sin. 23, 121–131. [18] Lindow, S.E. (1993) Novel method for identifying bacterial mutants with reduced epiphytic fitness. Appl. Environ. Microbiol. 59, 1586–1592. [19] Sun, F.Z. and Wei, J.F. (1996) Investigation on the dominant species of ice nucleation active bacteria in China. Acta Ecol. Sin. 16, 618–623. [20] Bishop, A.L. and Davis, R.M. (1990) Internal decay of onions caused by Enterobacter cloacae. Plant Disease 74, 692–694. [21] Richard, C. (1984) Genus VI. Enterobacter Hormaeche and Edwards 1960, 72AL (Krieg, N.R. and Holt, J.G., Eds.), BergeyÕs Manual of Systematic Bacteriology, Vol. 1, pp. 465–469. Williams & Wilkins, Baltimore, Md. [22] Sato, M. and Watanabe, K. (1988) A conjugative plasmid from epiphytic Enterobacter cloacae that confers multi-drug resistance. Ann. Phytopathol. Soc. Jpn. 64, 390 (Abstract).
[23] Wick, R.L., Rane, K.K. and Sutton, D.K. (1987) Mung bean sprout disease caused by Enterobacter cloacae. Phytopathology 77, 123 (Abstract). [24] Tang, C.R. (2001) Ph.D. thesis: cloning of a new ice nucleation active gene and construction of ice nucleation active Enterobacter cloacae for insect pest control. The Graduate School, Chinese Academy of Agricultural Sciences, Beijing. [25] Watanabe, K., Abe, K. and Sato, M. (2000) Biological control of an insect pest by gut-colonizing Enterobacter cloacae transformed with ice nucleation gene. J. Appl. Microbiol. 88, 90–97. [26] Tang, C.R., Sun, F.Z., Zhao, T.C. and Li, R.F. (2002) Cloning and characterization of a new ice nucleation active gene from ice nucleation active bacterium Erwinia ananas 110. Chinese J. Microbiol. 29, 45–51. [27] Warren, G.J. (1995) Identification and analysis of INA genes and proteins In: Biological Ice Nucleation and its Applications (Lee, G.J., Warren, G.J. Jr. and Gusta, L.V., Eds.), pp. 85–91. APS Press, St. Paul, Minnesota. [28] Simon, R. (1984) High frequency mobilization of gram-negative bacterial replicons by the in vivo constructed Tn5-Mob transposon. Mol. Gen. Genet. 196, 413–420. [29] Sun, F.Z., Zhu, H., He, L.Y. and Zhang, Y.X. (1994) Identification of ice nucleation active bacteria on plants in China. Prog. Nat. Sci. 4, 449–456. [30] Jiang, Q.Y., Zhou, Z.H., Shen, S.E., Jin, R.Z., Huang, Y.D. and Shen, S.J. (1990) Construction of recombinant plasmid of Tn5nifA having conjugal mobilization of broad host range. Chinese Sci. Bull. 35, 302–305. [31] Lindow, S.E. (1990) Bacterial ice nucleation activity In: Methods in Phytobacteriology (Klement, S., Rudolf, K. and Sands, D.C., Eds.), pp. 185–198. Akademiai Kiado, Budapest, Hungary. [32] Qiu, D.Y. and Shen, B.F. (1999) Construction of genetically engineered strains of Enterobacter cloacae (nlfLAc) by gene targeting mutation. Acta Phytophysiol. Sin. 25, 269–273. [33] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [34] Watanabe, K. and Sato, M. (1999) Gut colonization by an ice nucleation active bacterium, Erwinia (Pantoea) ananas reduces the cold-hardiness of mulberry pyralid larvae. Cryobiology 38, 281– 289. [35] Watanabe, M. and Tanaka, K. (1997) Photoperiodic control of adult diapause, cold hardiness, and inositol accumulation in a beetle, Aulacophora nigripennis (Coleoptera, Chrysomelidae). Zool. Sci. 14, 233–237. [36] Brooks, M.A. (1963) The microorganisms of healthy insects (Steinhaus, E.A., Ed.), Insect Pathology, Vol. 1, pp. 215–250. Academic Press Inc., New York. [37] Yamvrias, C., Panagopoulos, C.G. and Psallidas, P.G. (1970) Preliminary study of the internal bacterial flora of the olive fruit fly Dacus oleae (Gmelin). Ann. Inst. Phytopathol. Benakai. 9, 201–206. [38] Armstrong, J.L., Wood, N.D. and Porteous, L.A. (1990) Transconjugation between bacteria in digestive tract of the cutworm Peridroma saucia. Appl. Environ. Microbiol. 56, 1492– 1493. [39] Dilloa, R.J. and Dillon, V.M. (2004) The gut bacteria of insects: nonpathogenic interactions. Ann. Rev. Entomol. 49, 71–92.
Transgenic ice nucleation-active Enterobacter cloacae reduces cold hardiness of corn borer and cotton bollworm larvae Chaorong Tang, Fuzai Sun *, Xinjian Zhang, Tingchang Zhao, Jiyan Qi State Key Lab for the Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China Received 13 January 2004; received in revised form 26 June 2004 First published online 20 August 2004
Abstract The ice nucleation (IN) gene iceA of Erwinia ananas 110 was integrated into the chromosomes of two Enterobacter cloacae strains (Enc1.2022 and Enc1.181). These two newly derived transgenic strains, designated Enc2022-I and Enc181-I, respectively, possessed ice nucleation activity at 2.5 C, significantly higher than their parent strains (active at approx 10 C or lower). After ingesting these transgenic bacteria, the mean supercooling points (SCPs) of corn borer and cotton bollworm larvae were 3 to 4 C, significantly higher than those of untreated controls. The SCPs remained significantly elevated over the 9-day period after ingestion, which matched well with the efficient gut colonization of the bacteria during this period. All treated larvae froze and eventually died after exposure for 6 h to a temperature of 7 C, and more than 95% died after 12 h at 5 C. In contrast, few or none of the untreated control larvae froze and died under the same conditions. Furthermore, the growth ability of these transgenic ice nucleation-active (INA) En. cloacae strains on corn leaves was reduced, compared to that of wild-type epiphytic E. ananas, as revealed by pot tests conducted in both greenhouse and outdoor conditions. The stable colonization in insect guts and their lower affinity to plants would make these transgenic INA bacteria useful as a novel tool for biological control of insect pests in agricultural fields. 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Transgenic INA En. cloacae; Biological control; SCP; Gut colonization; Corn borer and cotton bollworm; Epiphytic fitness
1. Introduction The severity of insect damage to crops often depends on the size of the surviving fraction of the overwintering population [1,2]. Increasing winter mortality of insect pests is therefore an effective way to reduce crop loss in agricultural fields. Since the vast majority of insects is freeze-intolerant, i.e., unable to tolerate internal ice formation, a key factor in their overwintering survival is the regulation of the temperature at which they spontaneously freeze, termed the supercooling point (SCP) or * Corresponding author. Tel.: +86-10-62815933; fax: +86-1062896114. E-mail addresses: [email protected], [email protected] (C. Tang), [email protected] (F. Sun).
the temperature of crystallization [3,4]. Overwintering insects enhance the supercooling of body fluids by several means. First, due to their small size and therefore small water volume, insects have the inherent ability to extensively supercool [4]. Second, they can enhance their supercooling capacity by evacuating or inactivating heterogeneous ice-nucleating agents and amassing lowmolecular-mass polyols and sugars, sometimes in high concentrations [5–7]. In addition, some insects can accumulate anti-freeze proteins in haemolymph tissues or seek overwintering shelters [1,8,9]. Combining some or all of these measures, insect pests can decrease their SCPs to remain unfrozen to 20 C or lower, and thus readily survive harsh winters to reduce crop yields in the following growing season.
0168-6496/$22.00 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsec.2004.08.001
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Ice nucleation-active (INA) bacteria, the most potent ice nuclei known in nature, were reported to raise the SCPs and reduce the cold hardiness of insect pests such as Tribolium castaneum (red flour beetle), Helicoverpa armigera (cotton bollworm), Plodia interpunctella (Indian mealmoth), Anoplophora glabripennis and Rhyzopertha dominica, when these bacteria were sprayed on insect surfaces or ingested by the insects [6,10–13]. These interesting results prompted a number of investigators to explore the possibility of exploiting INA bacteria to control overwintering insect pests. In practice, problems emerge when INA bacteria are released in agricultural fields to control insect pests. First, after several days, the SCPs of insects treated with INA bacteria often decrease to levels similar to that of untreated controls [13–15]. These results have been attributed to unstable colonization of INA bacteria [15]. Due to the time interval between the spread of INA bacteria and the drop of the ambient temperature to levels low enough for them to function, it is essential for these bacteria to stably colonize in the guts or on the surfaces of the insects, so that they can maintain their ability of reducing insect cold hardiness over a long time. Second, frost damage to frost-sensitive plant species has been related to the population size of INA bacteria on the plants [16,17]. Therefore, the most serious problem regarding the future release of INA bacteria in agricultural fields as biological insecticides may be the occurrence of incited frost injury. One feasible approach of dealing with these problems is to isolate new kinds of INA microorganisms, which would be stably retained by insect pests, and simultaneously have weaker epiphytic fitness (the ability of bacteria to grow and maintain a population on leaves) [18] than frequently isolated epiphytic INA bacteria such as Erwinia ananas and Pseudomonas syringae, the predominant species in China [19]. So far, such ideal strains have not been obtained. We were therefore interested in the application of other strains. For example, both insect-origin and plant-origin Enterobacter cloacae strains grow well and stably in the guts of silkworm [15]. Furthermore, En. cloacae is not commonly isolated from plant leaves, except for some opportunistic phytopathogenic strains [20–23]. It was thus reasonable to expect some En. cloacae strains to stably colonize in insect guts, but not to persist long on plants and therefore, INA En. cloacae should be an ideal candidate for controlling insect pests in agricultural fields. However, attempts to isolate wild-type INA En. cloacae have failed [24,25]. Ice nucleation (IN) genes responsible for bacterial ice nucleation have been cloned from several INA bacteria, including E. ananas [26,27]. Watanabe et al. [25] first reported that transgenic INA En. cloacae could be useful in controlling mulberry pyralid larvae, G. duplicalis (Lepidoptera), in agricultural fields. They
transformed a recombinant pUC18 vector containing the IN gene inaA into an En. cloacae strain. The resulting transgenic En. cloacae, unlike the parent strain, showed high INA, and after ingestion by mulberry pyralid larvae, it exhibited marked efficacy in reducing their supercooling capacity and cold hardiness. The efficacy maintenance was found to result from the efficient gut colonization of this transgenic En. cloacae. On the other hand, the growth ability of this strain on mulberry leaves tended to be lower than that of epiphytic INA E. ananas, reducing the incidence of frost injury after its release in agricultural fields. In their study, the IN gene existed in the form of plasmids. In our previous study [24], however, we demonstrated that in the absence of antibiotic pressure, En. cloacae strains harboring IN gene-containing plasmids were likely to lose their plasmids and INA, and may therefore not retain their activity in agricultural fields. In this study, the IN gene iceA we previously cloned from E. ananas 110 [24,26] was integrated into the chromosomes of two En. clocae strains, and their efficacy was tested in reducing the cold hardiness of two of the most hazardous insect pests in China, corn borer and cotton bollworm. Their epiphytic fitness on corn leaves was also compared to that of epiphytic E. ananas by pot tests, conducted in both greenhouse and outdoor conditions.
2. Materials and methods 2.1. Bacteria, plasmids and insects Escherichia coli S17-1 bears a chromosomally integrated RP4 that provided broad host-range conjugal transfer functions [28]. INA bacterium E. ananas 110, the parent strain for the IN gene iceA, was previously isolated from the leaves of Zea mays in Shanxi, China [29]. Enc1.181 (ATCC7256) and Enc1.2022 (ATCC13047) are two biosafety level 1, non-ice-nucleation-active and ampicillin (Amp) resistant En. cloacae strains isolated from well water and spinal fluid, respectively, initially acquired from the American Type Culture Collection and now available in the China General Microbiological Culture Collection Center. Recombinant plasmid pINA105, carrying the cloned iceA gene, was constructed by Tang et al [26]. The Tn5-mob fragment endows the plasmid pSZ21 [28,30] with the functions of Tn5 transposition, kanamycin (Km) resistance and conjugal mobilization in gram-negative bacteria. Two kinds of Lepidoptera insect pests, cotton bollworm (H. armigera) and corn borer (Pyrausta nubialis) larvae, were used as test insects for the measurement of SCP and death by freezing.
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2.2. Construction of transgenic INA En. cloacae
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Biotechnology (Dalian) Co., Ltd., Dalian, China) and with the iceA full-length coding region as template.
The IN gene iceA was excised as a 4-kb SalI-BamHI fragment from pINA105, and cloned into pSZ21 by inserting it between the SalI and BamHI sites within Tn5. The resulting construct, named Tn5-mob-iceA, was transformed into E. coli S17-1 to obtain the Tn5mob-iceA -containing transformant (designated E. coli S17-1 (Tn5-mob-iceA)). The Tn5-mob-iceA construct was then transferred from donor strain S17-1 (Tn5mob-iceA) into the two target En. cloacae strains (Enc1.181 and Enc1.2022), using a filter mating technique. Filters (Nitrocellulose filter, U 50 mm, 0.2 lm, Cole-parmar, etc.) with a mixture of donor and recipient cells at logarithmic phase in a 1:5 ratio were incubated for 4 h at 30 C and spread on LB agar plates supplemented with ampicillin (Amp) and chloramphenicol (Cm), incubated at 30 C for 24 h, and then for 2h at 4 C for the enhancement of INA. The resulting En. cloacae transconjugants originating from Enc1.181 and Enc1.2022 were named Enc181(Tn5-mob-iceA) and Enc2022(Tn5-mob-iceA), respectively. These transconjugants were then isolated and further identified with the INA assays, as described by Lindow [31], and plasmid restriction analysis. Bacterial suspensions for INA assays were prepared as follows. One single colony, from a newly cultured bacterial plate, was inoculated into 5 ml of KingÕs medium B (KB), and grown for 12 h with shaking of 200 rpm at 30 C. After a 2-h treatment at 4 C, bacterial cells were pelleted and suspended in sterile distilled water (DW) to a concentration of 108 cells per ml. Plasmids were eliminated from En. cloacae transconjugants by growing in the absence of antibiotic pressure and in the presence of acridine orange as previously described [32]. En. cloacae colonies with plasmids eliminated and iceA integrated into their chromosomes through Tn5 transposition should be resistant to kanamycin (Kmr) and sensitive to chloramphenicol (Cms). INA assays, plasmid isolation, PCR and Southern blot analysis (see below) were performed to further verify chromosomal integration of iceA. The resulting transgenic INA En. cloacae strains, originating from Enc181(Tn5-mob-iceA) and Enc2022(Tn5-mob-iceA), were designated En. cloacae 181-I (abbr. Enc181-I) and En. cloacae 2022-I (abbr. Enc2022-I), respectively.
Measurement of larval SCP was performed at 0, 3, 5, 7 and 9 days after bacterial ingestion. At each time point, 15 larvae were collected, starved for 1 day at 22 C and kept 12 h at 4 C for the enhancement of INA. Then, their SCPs were measured based on the method of Watanabe and Tanaka [35]. After measurement, 5 larvae for each treatment were surface-sterilized for 1 min in 70% alcohol, then rinsed three times with sterile DW and macerated with sterile DW in a mortar. The macerated suspensions were diluted and spread on KB plates with amphotericin B (1 lg ml1) to inhibit fungi and Km (20 lg ml1) to estimate the population size of transgenic bacteria. To determine which colonies originated from the inoculated bacteria, 20 colonies were randomly picked and INA was assayed according to the method of Lindow [31].
2.3. Southern blot analysis of transgenic En. cloacae
2.6. Survival estimation
Genomic DNA from non-transgenic En. cloacae strains (Enc1.181 and Enc1.2022) and their corresponding transgenic strains (Enc181-I and Enc2022-I) was isolated, digested with BamHI or SalI, fractionated by agarose electrophoresis, transferred to nylon membranes and hybridized to the iceA probe as previously described [33]. The iceA probe was prepared using the ‘‘Random Prime DNA Labeling Kit ver. 2.0’’ (TaKaRa
From each treatment at 6 days after ingestion, 14–28 larvae of corn borer and cotton bollworm were brought to a frost chamber, where the temperature was lowered from room temperature to 5 or 7 C at a rate of 10 C h1.The larvae were kept at 5 or 7 C and checked after 3, 6 and 12 h to see if they were frozen before being returned to the chamber for continued incubation. Freezing status was determined by slightly
2.4. Feeding insects and bacterial ingestion Newly hatched larvae of corn borer and cotton bollworm were transferred into sterile 1-l beakers and separately fed in an incubator at 22 C and 70% relative humidity on healthy fresh corn stems and cotton leaves, which were collected from agricultural fields, washed with tap water, rinsed with DW and wiped with absorbent paper. Three days later, these larvae were allowed to ingest the bacterial strains Enc181-I and Enc2022-I, using an approach similar to that described by Watanabe and Sato [34], as follows. Corn stems and cotton leaves were thoroughly immersed in bacterial suspensions (108 cells ml1 in DW, prepared from cells cultured in LB liquid with shaking to stationary phase) or just sterile DW as a control treatment for 20 min, and airdried at room temperature. Then, 250 larvae of each kind of insect were fed on them. After 3 days feeding (the so-called bacterial ingestion), the insects were shifted to sterile glass tubes, one larva per tube, and fed fresh untreated plant tissues. Every 2 days, old plant tissues were discarded and fresh tissues were administered. 2.5. Measuring insect SCP and bacterial population size
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prodding the larva with a matchstick to check for softness. After incubation at 5 or 7 C for 12 h, all larvae were transferred to an incubator at 22 C for 1 h, and then examined for survival or death.
mine which colonies on the KB plates originated from the inoculated bacteria, 20 colonies were randomly selected and their colony characteristics were compared with those of the inoculated bacteria and examined for INA using the freezing droplet assay [31].
2.7. Bacterial epiphytic fitness 2.8. Statistical methods The epiphytic fitness of transgenic En. cloacae 181-I and 2022-I on corn leaves was investigated based, with some modifications, on the method of Lindow [18], and compared with that of wild-type INA bacterium E. ananas 110 (Ea110). Experiments were conducted with potted corn seedlings using the same procedure both outdoors and in a greenhouse. The outdoor assay was performed between late May and early June of 2001, in the open ground behind the experimental building of the Institute of Plant Protection of CAAS (Beijing, China). Outdoor air temperatures ranged from 14 to 30 C,with daily mean air temperatures of 20–23 C. Greenhouse conditions were maintained at 20 C and 60% relative humidity. Bacteria (Enc181-I, Enc2022-I or Ea110) were cultured to the stationary phase in LB medium, pelleted by centrifugation and suspended in sterile DW to suspensions of 108 cells per ml. Leaves of potted corn seedlings at the 5-leaf stage (with five primary leaves) were evenly sprayed with a suspension of a given bacterial strain, and these seedlings were then covered with a polyethylene bag dipped in water for 1 day. After removing the bag, five samplings were sequentially made at 1, 5, 10, 15 and 20 days after bacterial inoculation, and the population size of each respective inoculated bacterium was quantified as described below. Two days before each sampling, the seedlings were sprayed with water. On the day of sampling, leaves were collected, cut into small pieces and about 0.5 g (fresh weight) of leaves were ground in 5 ml sterile DW in a sterile mortar. The resulting suspension was incubated for 2 h at 4 C, vortexed for 30 s, and appropriate 10-fold serial dilutions were plated onto KB plates containing either amphotericin (1 lg ml1) and Km (20 lg ml1) to isolate transgenic En. cloacae or just amphotericin (1 lg ml1) to isolate E. ananas 110. To deter-
Statistical analyses were done using the software SAS 6.11 (SAS Institute, Cary, NC). The comparison of insect SCPs, gut-colonizing ability and epiphytic fitness of bacteria were performed by ANOVA, Scheffe test and t-test. The v2 test and FisherÕs Exact test were used to compare freezing percentages and mortality rates of larvae in different treatments.
3. Results 3.1. Expression of iceA in transgenic En. cloacae The filter mating produced 2 and 3 Kmr Cms colonies, respectively, from En. cloacae transconjugants of Enc181(Tn5-mob-iceA) and Enc2022(Tn5-mob-iceA). One such colony from each strain was randomly selected for subsequent assays. No plasmid could be isolated from these En. cloacae colonies, indicating that plasmid vectors of the Tn5-mob-iceA constructs were eliminated. Both PCR and Southern blot analyses demonstrated that the IN gene (iceA) was integrated into their chromosomal DNAs. Also, Southern blot analysis showed that the transconjugants had only one copy of iceA (data not shown). These transgenic En. cloacae, designated En. cloacae 181-I (abbr. Enc181-I) and En. cloacae 2022-I (abbr. Enc2022-I), displayed ice nucleation activity (INA) at 2.5 C, similar to that of E. ananas 110 (active at 2.3 C), the parent strain for iceA (Table 1). In contrast, their parent strains Enc1.181 and Enc1.2022 barely showed INA, even at 10 C. These results indicated that the high INA of transgenic En. cloacae was an acquired trait and iceA was fully expressed in En. cloacae.
Table 1 Ice nucleation activity (INA) of different bacterial strains Percentages of freezing drops (%)a
Strain
Enterobacter cloacae Enterobacter cloacae Erwinia ananas 110 Enterobacter cloacae Enterobacter cloacae Distilled water a b
181-I 2022-I 1.181 1.2022
10.5 C
10 C
5 C
4 C
3 C
2.5 C
2.3 C
2.1 C
NDb ND ND 28.7 ± 3.5 4.0 ± 1.0 0
ND ND ND 3.0 ± 1.0 0 0
100 100 100 0 0 0
100 100 100 0 0 0
100 100 100 ND ND ND
7.0 ± 2.0 3.7 ± 0.6 37.0 ± 3.1 ND ND ND
0 0 5.7 ± 1.5 ND ND ND
0 0 0 ND ND ND
INA assays were conducted according to the droplet-freezing method of Lindow [31], and were repeated three times. ND, not detected.
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Corn borer larvae
Cotton bollworm larvae
-12
-16
Supercooling point(˚C)
Supercooling point(˚C)
-20
-12 -8 -4 0 0
3
5
7
83
-8
-4
0
9
0
Days after treatment
3
5
7
9
Days after treatment
Fig. 1. Supercooling point changes in corn borer larvae and cotton bollworm larvae ingesting ice nucleation-active transgenic En. cloacae over the 9day period after treatment. Each larva was treated with: n, distilled water; }, Enc181-I; h, Enc2022-I. Vertical bars represent standard errors for means.
3.2. Supercooling point of corn borer and cotton bollworm larvae
Table 2 Colonization of transgenic Enterobacter cloacae in the guts of larvae of corn borer and cotton bollworm
In pilot experiments, it was demonstrated that the SCPs of corn borer and cotton bollworm larvae ingesting wild-type En. cloacae (Enc1.2022 and Enc1.181) were similar to those of DW (distilled water)-treated larvae (data not shown). Therefore, we simply used DW instead of non-transgenic En. cloacae as controls for the remainder of the experiments. The progressive changes in the SCPs of Enc181-I, Enc2022-I and DW treated larvae were monitored. Samples were collected at 0, 3, 5, 7 and 9 days after ingestion and their SCPs were measured. The SCPs of corn borer and cotton bollworm larvae remained constant for 7 days after ingesting transgenic En. cloacae (Fig. 1), and displayed a significant increase against those of untreated larvae, from 11 to 17 C and 10 C, respectively, to 3 to 4 C. Although the SCPs of these Enc181-I and Enc2022-I treated larvae markedly declined at 9 days after ingestion, they were still over 5 C and significantly higher than those of the DW controls. Some larvae of corn borer and cotton bollworm began to pupate at 9 days after ingestion, and no transgenic En. cloacae were isolated from these pupae, suggesting that these bacteria were unable to stably colonize in the pupal stage. In addition, individuals of transgenic bacteriatreated larvae had a narrow range of SCP distribution, the vast majority with SCPs of 3 to 5 C. In contrast, the SCPs of DW-treated insects were distributed over a much broader range, from 6 to 22 C for corn borer and 5 to 14 C for cotton bollworm.
Days after ingestion (days)
3.3. Colonization of transgenic En. cloacae in larval guts Both transgenic strains were able to grow in the guts of corn borer and cotton bollworm larvae (Table 2). Over 7 days after bacterial ingestion, a large population of target bacteria (Kmr, INA+) (104–105/larva) was detected in the guts of these larvae. The population size of target bacteria declined to 103 per larva 9 days after
0 3 5 7 9
Number of bacteria (log CFU/larva)a Corn borer
Cotton bollworm
Enc181-I
Enc2022-I
Enc181-I
Enc2022-I
4.8 ± 0.1a 5.0 ± 0.3a 5.3 ± 0.2a 4.7 ± 0.2a 3.5 ± 0.3b
4.6 ± 0.2a 4.9 ± 0.1a 5.2 ± 0.3a 5.0 ± 0.3a 3.8 ± 0.2b
4.7 ± 0.2a 5.1 ± 0.3a 5.2 ± 0.2a 4.8 ± 0.3a 3.4 ± 0.3b
4.4 ± 0.2a 4.9 ± 0.2a 5.4 ± 0.3a 4.6 ± 0.3a 3.6 ± 0.3b
a Means ± standard error. Within a column, means followed by different letters are significantly different (P 6 0.05; ANOVA, Scheffe test). Means are not significantly different (P > 0.05; t-test) between the two transgenic En. cloacae strains.
bacterial ingestion. The colonizing ability was not significantly different between these two transgenic bacteria. 3.4. Mortality of corn borer and cotton bollworm larvae by freezing The freezing percentages and mortality of larvae were significantly increased after ingesting transgenic INA En. cloacae (Table 3). More than 80% of bacteria-treated larvae froze after 3 h exposure to 5 C and over 90% after 12 h exposure. In contrast, even after 12 h exposure to 5 C, none or only a minority of untreated corn borer and cotton bollworm larvae froze. After 6 h exposure to 7 C, all treated larvae froze, compared to only a small proportion of untreated larvae (7.4% of corn borer and 14.3% of cotton bollworm). More than 90% of the treated larvae died after 12 h exposure to 5 C, and 100% died after 12 h exposure to 7 C. On the other hand, all or most untreated larvae survived under the same conditions. 3.5. Epiphytic fitness of transgenic En. cloacae The epiphytic fitness of transgenic bacteria on corn leaves was tested on potted corn seedlings (5-leaf stage), grown both outdoors and in a greenhouse, and
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Table 3 Percentages of freezing and mortality of corn borer and cotton bollworm larvae after exposure to low temperaturea Insect
Treatment
Freezing percentages of larvae (%) 5 C
Mortality rate of larvae (%)b
7 C
3h
6h
12 h
3h
6h
12 h
5 C
7 C
Corn borer
Enc181-I Enc2022-I DWc
84.6a 87.5a 0.0b
96.1a 91.7a 0.0b
96.1a 91.7a 0.0b
92.3a 95.8a 3.7b
100a 100a 7.4b
100a 100a 7.4b
96.1a 95.8a 0.0b
100a 100a 7.4b
Cotton bollworm
Enc181-I Enc2022-I DW
76.9a 87.5a 0.0b
92.3a 93.7a 7.1b
92.3a 93.7a 7.1b
100a 93.7a 7.1b
100a 100a 14.3b
100a 100a 14.3b
92.3a 93.7a 7.1b
100a 100a 14.3b
a b c
Means followed by different letters are significantly different (P 6 0.05; the v2 test and FisherÕs exact test). Larvae treated for 12 h were transferred to a 22 C incubator and their mortality examined. DW, distilled water.
been investigated by several research groups, and some inspiring results have been obtained [6,10–13]. In those studies, P. syringae strains were mainly used as INA bacteria. Being common epiphytic residents of plants, however, these strains incite frost injury by limiting the supercooling ability of water in plant tissues. Moreover, the effectiveness of the treatment with these bacteria rapidly decreased after application because of their inability to colonize insect guts [13–15]. Therefore, these strains are not the best suited for release in agricultural fields to control insect pests. In the present study, two transgenic INA En. cloacae strains (Enc181-I and Enc2022-I) were constructed by integrating the IN gene iceA into the chromosomes of two En. cloacae strains (Enc1.181 and Enc1.2022). These resulting transgenic bacteria displayed ice nucleation activity at 2.5 C, only slightly weaker than that of the parent INA strain E. ananas 110 (active at 2.3 C) for iceA (Table 1), indicating that the introduced iceA was actively expressed in En. cloacae. Expression of iceA was also identified in transgenic En. cloacae colonizing in insect guts. The mean SCPs of Enc181-I and Enc2022-I treated larvae were 3 to 4 C throughout the 7-day period after ingestion, significantly higher than those of untreated
compared to that of the bacterium E. ananas 110 (Ea110) (a corn epiphytic strain). A large difference existed in the epiphytic fitness among different bacterial strains (Fig. 2). In both experiments, transgenic En. cloacae (especially Enc2022-I) had a weaker fitness than Ea110 (P 6 0.05; ANOVA, Scheffe test). Strain Ea110 retained a population size of over 106 cells per gram of fresh leaf weight (cells/gFLW) 20 days after spraying bacteria, while Enc2022-I was unable to be recovered at this time period and Enc181-I had a population size of 103–104 cells/gFLW. Significant differences in epiphytic fitness between these two transgenic En. cloacae strains also existed (P 6 0.05; ANOVA, Scheffe test). After spraying, the population size of Enc2022-I decreased rapidly, and this strain could not be re-isolated 15 or 20 days later. In contrast, Enc181-I maintained, in both experiments, a population size of over 103 cells/gFLW during the 20 day post-inoculation period.
4. Discussion In recent years, decrease of cold hardiness and biological control of insect pests using INA bacteria have
B
LOG(Cells/gFLW)
LOG(Cells/gFLW)
10
A
8 6 4 2
8 6 4 2 0
0 1
5
10
15
Days after inoculation
20
1
5
10
15
20
Days after inoculation
Fig. 2. Population sizes of Erwinia ananas 110 and transgenic En. cloacae on corn leaves in a field study (A), and in a greenhouse study (B). n, E. ananas 110; }, Enc181-I; h, Enc2022-I; ·, no bacteria detected; vertical bars represent standard errors for means calculated from five independent measurements; cells/gFLW, cells per gram of fresh leaf weight.
C. Tang et al. / FEMS Microbiology Ecology 51 (2004) 79–86
controls (Fig. 1). The maintenance of the high SCPs was apparently due to the stable gut colonization of the transgenic bacteria (Table 2). These transgenic bacteria markedly reduced the cold hardiness of corn borer and cotton bollworm larvae (Table 3), leading to a 100% death rate after exposure for 6 h to 7 C, while, under the same conditions, most DW-treated larvae did not freeze or die. In addition, the transgenic En. cloacae strains (especially Enc2022-I), unlike epiphytic E. ananas 110, did not persist longterm on plants (Fig. 2). These findings suggested that the transgenic En. cloacae were potentially useful as microbial control measures in order to reduce insect survival over winter in agricultural fields. In a different study, Watanabe et al. [25] reported the use of INA En. cloacae for the control of mulberry pyralid larvae. Our studies were different from theirs in several aspects. First, the IN gene iceA (GenBank Accession No. AF387802) cloned and used here was different from the IN gene inaA used in the previous study, sharing 94% amino acid identity [26,27]. Second, in their study, the IN gene inaA was transformed on plasmids, while in the present study, iceA was integrated into the chromosomes of En. cloacae, and these transgenic bacteria were more ice nucleation-active (2.5 C) (Table 1) than the previously reported transgenic strain (active at 2.9 C) [25]. Third, the present study focused on controlling two of the predominant insect pests in China, corn borer and cotton bollworm, undoubtedly expanding the application potential of this novel kind of biological control. Several problems should be pointed out regarding the field application of transgenic INA En. cloacae. In this study, these bacteria were unable to colonize the pupal stage of tested insects, suggesting that they would not be effective in controlling those insect pests overwintering in the form of pupae. For best efficiency, the transgenic bacteria should be extensively examined for their effects on the cold hardiness of different insect species and at different stages. In addition, the effects of INA bacteria in reducing crop loss caused by insect pests will not be seen until the year following bacterial release, so farmers tend to take quicker control means (such as chemical spraying) instead. Therefore, we suggest that this approach could be used concomitantly with other control means for integrated pest management of a given species. Also, since En. cloacae strains have been detected in the guts of a variety of insects [15,21,36–39], including the beneficial Collembolan Folsomia candida, and INA bacteria seem to be effective against a diverse range of insects [6,24], consideration must be given to avoid possible detrimental effects on the overwintering survival of beneficial insects. It has been suggested that applications of INA bacteria should be targeted to specific areas, where few nontarget insects would be exposed, such as in
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the trap crops for Colorado potato beetles or storage product sites [1,11]. We suggest here that, prior to the release of INA bacteria into agricultural fields, intensive surveys should be made to determine the species composition, the distributions of insects in selected target fields and the effects of these bacteria on overwintering species. Lastly, field conditions are very complex, and many factors may affect the effects of INA bacteria, so the results of laboratory studies must be confirmed with field tests. Using transgenic INA bacteria for biological control of insect pests is a recent area of scientific investigation. As it often occurs, results from initial studies give rise to many new questions, such as the following: are these transgenic INA bacteria safe to humans, animals and the environment? How do we develop application methods for better use of these bacteria in agricultural fields? Could insects develop resistance to this type of control and if so, how rapidly would this occur? In agricultural fields, to what extent will the microbial habitat in insect guts affect the function of these INA bacteria? Is it possible to actively and stably express the IN gene in those bacteria colonizing stably in overwintering insect pupae, eggs or adults? The answers to these questions will help us to evaluate the potential for environmental application of these bacteria.
Acknowledgements We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grant No. 30170624). We thank Prof. Stephen B. Goodwin (Dept. of Botony and Plant Pathology, Purdue University) and Prof. Yingchuan Tian (Ins. of Microbiology, CAS) for critically reading the manuscript, and Dr. Zhiwen Li and Dr. Jianghui Zhu (Ins. of Birth Health, Peking University) for valuable assistance in statistical analyses. We also thank Prof. Bingfu Shen (Ins. of Plant Physiology, CAS) for granting pSZ21, and Yongjun Li (Ins. of Atomic Energy, CAAS) and Hong Zhu (Ins. of Plant Protection, CAAS) for providing the test insects.
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