- Email: [email protected]
An ice-nucleating active fungus isolated from the gut of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)
J. lnsecfPhysiol.Vol. 38, No. 2, pp. 119425, 1992 Printedin Great Britain.All rightsreserved
0022-1910/92 $5.00+ 0.00 Copyright0 1992PergamonPressplc
AN ICE-NUCLEATING ACTIVE FUNGUS ISOLATED FROM THE GUT OF THE RICE STEM BORER, CHILO SUPPRESSALIS WALKER (LEPIDOPTERA: PYRALIDAE) HISAAKI TSUMUKI,‘,* HARUYOSHI KONNO,’ TAKANORI MAEDA’
and YASUHIRO OKAMOTO’ ‘ResearchInstitute for Bioresources, Okayama University, Kurashiki 710 and 20kayama Prefectural Agricultural Experiment Station, Sanyo-cho, Okayama 709-08, Japan (Received 21 May 1991; revised 30 August 1991)
Abstract-The existence of a fungus with the ability to nucleate ice formation in supercooled water was revealed. The fungus was isolated from the gut of larvae of the rice stem borer, Chifo suppressalis Walker, and from rice seedlings which were host plants of this insect. The fungus was identified as a Fusarium sp. on the basis of its morphology. Ice-nucleating activity, at around - YC, was detected in the mycelial suspension of the fungus and also in the culture filtrate. The presence of the exogenous ice-nucleating active fungus in the gut and on the body surface caused an elevation in crystallization temperature of the larvae. Ke\j Word Index: Ice-nucleating lization temperature
active fungus; Fusarium sp.; Chilo suppressalis; gut; crystal-
INTRODUCTION
The ice-nucleating active agents contained in insects can be classified into two types: exogenous and endogenous. The endogenous types occur in the haemolymph (Zachariassen and Hammel, 1976; Zachariassen, 1982; Duman and Horwarth, 1983) and muscle (Tsumuki and Konno, 1991) and are constructed from protein (Duman and Horwarth, 1983; Tsumuki and Konno, 1991). In contrast to the endogenous types, the exogenous agents are distributed in the gut or its contents (Salt, 1968; Sdmme, 1982) and on the body surface (Bale et al., 1989). Salt (1968) and Sijmme (1982) have proposed that food particles in the gut act as exogenous ice nuclei, but the characteristics are obscure. It is well known that several epiphytic bacteria serve as ice nuclei (Lindow, 1983). Insects reported to contain ice-nucleating active bacteria in their gut include Plutella xylostella (Kaneko et al., 1989, 1991), and Ceratoma trlfurcata and Hippodamia convergens (Lee and Strong-Gunderson, 1991). Some workers have also reported ice-nucleating ability from lichen fungi (Kieft, 1988; Kieft and Ruscetti, 1990). In this study, we identify an ice-nucleating active fungus isolated from the gut of larvae of the rice stem borer (Chile suppress&s Walker), and measure its ice-nucleating ability. We further examine the effect *To whom all correspondence
should be addressed.
of the exogenous fungus on the crystallization perature of the iarvae. MATERIALS
tem-
AND METHODS
Isolation of ice-nucleating active microorganisms
Mature larvae of the rice stem borer, reared on rice seedlings at 25°C under long-day conditions (16 h light-8 h dark), were used (Tsumuki and Kanehisa, 1978). They were dipped in 70% (v/v) ethanol for 1 min and 0.1% (w/v) mercuric chloride solution for 3 min, and the isolated guts, including the contents, were suspended in sterile distilled water under sterile conditions. Serial dilutions of these suspensions were plated onto potato-sucrose-agar (made from 200 g of potato, 20 g of sucrose and 10 g of agar in 1 litre of distilled water): (i) adjusted to pH 7.0 for bacteria, and (ii) supplemented with 100ppm streptomycin and adjusted to pH 5.6 for the fungi. After incubation for 2-3 days at 25”C, discrete colonies were subcultured in test tubes (180 x 16 mm i.d.) containing 4 ml of liquid potato sucrose medium at 25°C with shaking (140 strokes/min). After incubation for 3-6 days, the crystallization temperatures of the resulting microorganism suspensions were measured. Culture of ice-nucleating active fungus
An ice-nucleating active fungus colonies on the potato-sucrose-agar 119
found from medium was
120
HISAAKITSUMUKIet al.
cultured. To prevent the contamination of adjacent species on the medium, single conidia were isolated by the standard dilution plate method, and germinating conidia were taken from the plates under a dissecting microscope (Booth, 1971). The resulting fungal cultures were used in the subsequent tests. A growth medium containing 20 g of galactose, 2 g of asparagine, 1 g of K2HP0.,, 0.5 g of KCI, 0.5 g of MgS04:7H,0 and 10 mg of Fe-Na-EDTA in 1 litre of distilled water (Komada, 1976), adjusted to pH 5.6, was used to culture the ice-nucleating active fungus. The fungus was inoculated into 250 ml of the liquid medium in a 500ml shaking flask, and cultivation was carried at 25°C with reciprocal shaking at 140 strokes/min. After 12-14 days growth, the mycelial pellets were separated from the culture medium by filtration through a nitrocellulose filter (0.45 ,um pore size). Determination of ice-nucleus concentrations
Serial lo-fold dilutions of the culture filtrate in sterile distilled water were assayed. Ice-nucleating activity was defined as the reciprocal of the highest dilution factor of the dilutions that catalysed the freezing above - 10°C. The crystallization temperatures of the filtrates were estimated from an average of six measurements. Preparation of sterile rice seedlings and larvae
Unpolished rice grain surfaces were sterilized by several immersions in 1% sodium hypochloride solution for 20 min then the grains placed on 1% agar under sterile conditions. After growing for 10 days at 25”C, the rice seedlings were used for rearing sterile larvae. Rice stem borer eggs, sterilized by the same methods as described for larvae, were transferred to the sterile plants. The rice seedlings were exchanged every week and larvae of about 35 days old used for the following experiments. Inoculation of the fungus
The effect of ingesting ice-nucleating active fungus on the crystallization temperature of the insects was determined as follows. Sterile rice plants (10-12 days of growth) were dipped in the fungal suspension (12-14 days of growth) and incubated at 25°C for 3 days and then sterile larvae (35 day old) were placed on the treated plants at 25°C for 1 day. After feeding on the fungus, the larvae were ligated head and tail, and then immersed in 70% ethanol for 3 min. To estimate the activity of the ice-nucleating agents on the body surface, sterile larvae were tied head and tail to prevent the incorporation of ice-nucleating agents into the body and then brought into contact with the fungus by incubation on the treated plants
at 25°C for 1 day. The resulting larvae were immersed in 70% ethanol for 3 min or washed four times with sterile distilled water. The crystallization temperatures of the sterile and treated larvae and plants were measured. Measurement of crystallization temperature
The microorganism suspensions and/or culture filtrates were collected in glass capillary tubes (60 x 1.1 mm i.d.), sandwiched between two layers of paraffin oil. Rice shoot and root pieces in 0.9% NaCl solution were also placed between two layers of the oil in the capillary tubes (Zachariassen et al., 1982). The capillary tubes containing the samples and the larvae were attached to 30-gauge copper-constantan thermocouples connected to a multichannel temperature recorder (Thermodac 5OOlA, Eto-denki, Japan) and cooled at a rate of about l”C/min until frozen. The crystallization temperature was determined as the temperature at which a temporary rapid increase in temperature occurred. The temperatures of 29 samples could be measured simultaneously by the recorder. RESULTS
IdentiJication and activity of the ice-nucleating active fungus
About 600 colonies were obtained from the dilution plates inoculated with a suspension of the gut samples. Bacteria were the predominant microorganisms, accounting for about 75% of all colonies obtained. Fungi including yeasts were also common and accounted for about 25% of all colonies. All microorganisms were tested for their ice-nucleating activities and selected on the basis of their activities above - 10°C. One mycelial fungus showed nucleating activity, but no bacteria or yeasts having such activity were isolated. The fungus which produced long, crescent-shaped, multiseparate macroconidia and small oval-shaped microconidia (Fig. I), was identified as a Fusariam sp. The same fungus was also obtained from rice seedlings reared under natural conditions. Ice nucleation in a suspension of the fungus was detected at around -5°C (Table 1). The ice-nucleating activity was also detected in the mycelium-free culture filtrate (Fig. 2). The activity was detected from 4 days after initiating the culture at 25°C and increased with mycelia growth, reaching a maximum at 12 days. Effect of the fungus on the crystallization temperatures of sterile larvae and rice seedlings
Sterile larvae and those fed on Peniciflium sp., supercooled to below - 2O”C, while the larvae fed on
Fig. 1. Macroconidia and microco mnidiaproduced by the ice nucleating active fungus Ir. __.. (ruscrrium sp.) scale = 65 pm.
121
Ice-nucleating fungus isolated from the gut of C. suppress&
0
10
5
15
123
20
Cultivation time (days)
Fig. 2. Production of ice nuclei in culture filtrate by the ice nucleating active fungus (Fusurium sp.).
the Fusarium sp. isolated showed an elevation in the crystallization temperature to around - 5°C which was equivalent to that of the fungal suspension (Table 1). The crystallization temperature of sterile rice seedlings was - 13 to - 15°C while that of the plants inoculated with the fungus also to around -5°C (Table 1). Inoculation of the larval body surface with the Fusarium sp. also caused the crystallization temperature to increase (Table 2). However, this effect was greatly reduced by immersion in 70% ethanol or washing with water (Table 2). In order to exclude the influence of body surface ice nucleation, larvae fed on Fusarium sp. inoculated rice seedlings were then
Table 1. Effect of the fungus (Fusurium sp.) on the crystallization temperature of sterile larvae and rice seedlings Crystallization temperature (“C) Larvae Feeding Feeding Feeding Ligated
without Fusarium sp. Penicillium sp. Fusarium
sp.
larvae treated with ethanolt
Plants Control (sterilized) Shoot Root Inoculation with Fusnrium sp. Shoot Root Fusarium sp. suspension
ligated head and tail, and immersed in 70% ethanol prior to measurement of the crystallization temperature. The crystallization temperature of the treated larvae was maintained as high as that of the larvae fed on the Fusarium sp. (Table 1). DISCUSSION
The existence of ice-nucleating active bacteria have been reviewed in detail by Lindow (1983) and recently ice-nucleating lichen fungi have been reported (Kieft, 1988; Kieft and Ruscetti, 1990). We have isolated an ice-nucleating active fungus and identified it as Fusarium sp. on the basis of its morphology (Booth, 1971). The crystallization temperature of the fungal suspension was around - 5’C, which was similar to that of several ice-nucleating bacteria (Lindow, 1983) and lichen fungi (Kieft, 1988; Kieft and Ruscetti, 1990), showing that the ability of the fungus in ice nucleation may be comparable
- 20.1 + 0.9* (5) -21.0+0.5*(5)
- 5.7 f 0.6 (5) -5.8 k 0.5 (5)
Table 2. Effect of treatment of 70% ethanol and washing with water on the crystallization temperatures of ligated larvae kept on rice seedlings inoculated with the fungus (Fusarium
sp.) Crystallization temperature (“C)
- 13.6 + 0.7* (12) -15.2+ 1.0*(12) -5.1 kO.3 (14) -4.6*0.2(13) - 5.5 f 0.8 (10)
All values are mean f SE (N). *Significantly different (Student r-test) from the value of Fusarium sp. suspension, P < 0.0 1. tSterile larvae fed on rice seedlings inoculated with Fusurium sp. were ligated head and tail and then immersed in 70% ethanol.
Sterile larvae Larvae inoculated with Fusurium sp. Fusarium sp. inoculated larvae dipped in ethanol Fusarium sp. inoculated larvae washed with water
- 20.6 k 0.3* (5) -7.0 + 1.1 (5) - 14.5 * I.58 (5) - 14.2 + 0.9* (5)
All values are mean + SE (A’). *Significantly different (Student r-test) from the value of larvae inoculated with Fusarium sp., P < 0.01.
124
HISAAKITSUMUKIet al.
to that of these other ice-nucleating active microorganisms. Ice-nucleating active bacteria have been isolated from the guts of some insects and ingestion of the bacteria elevates ice crystallization temperature of the insects (Kaneko et al., 1989, 1991; Strong-Gunderson et al., 1990; Lee and Strong-Gunderson, 1991). In this experiment, an ice-nucleating active fungus (Fusarium sp.) which caused an elevation in crystallization temperature, was isolated from the gut of insect for the first time. The crystallization temperature of rice stem borer larvae reared in natural conditions has been shown to be around -8°C and is due to the gut containing its natural contents (Tsumuki and Konno, 1991). However, the crystallization temperature of sterile larvae was below -20°C as was that of the larvae fed the rice seedlings inoculated with icenucleating negative Penicillium sp. In contrast the presence of the Fusurium sp. isolated here limited the supercooling capacity. Consequently, it seems that the high crystallization temperature under natural conditions is due, at least in part, to the presence of the fungus in the gut. Under natural conditions, the fungus was also isolated from rice seedlings, showing that the fungus isolated from the gut may originate from the food. In diapausing larvae which excrete their gut contents, the crystallization temperature is lower than non-diapausing larvae (Tsumuki and Konno, 1991). Consequently the gut contents, possibly including the fungus, may play a less important role in the supercooling capacity in diapausing larvae. The differences in the crystallization temperatures between rice seedlings and sterile larvae fed on the seedlings (Table 1) show that the ice-nucleating agents in the plants are decomposed or masked in the guts of larvae (Baust and Rojas, 1985). Furthermore, these results indicate that ice nuclei may not be produced from the plant material in the gut of the larvae. The crystallization temperature of larvae exposed to the Fusurium sp. isolated showed a pronounced increase in the crystallization temperature from approx. -20 to -5°C. The ice-nucleating activity on the body surface was reduced by immersion in 70% ethanol and by washing the surface with water, but the crystallization temperatures did not drop as low as -2O”C, as was the case for sterile larvae. These results show that the ice nuclei on the body surface also affect the determination of the crystallization temperature of larvae as suggested by Bale er al. (1989). Preliminary investigations (unpublished data) indicate that the ice nuclei produced by the Fusurium sp. are composed of protein, as reported for icenucleating active bacteria (Corotto et al., 1986;
Warren and Corotto, 1989). Currently, we are attempting to purify the active protein from the culture filtrate and mycelia. Acknowledgemenrs-We
wish to sincerely thank Dr Michael N. Pearson of the Department of Botany, University of Auckland, for his critical reading of the manuscript. This study was supported in part by a Grant-in-Aid for Scientific Research (No. 01560053) and a Grant for Special Project Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES Bale J. S., Hansen T. N. and Baust J. G. (1989) Nucleators and sites of nucleation in the freeze tolerant larvae of the gallfly Eurosta soiidaginis (Fitch). J. Insect Physiol. 35, 291-298.
Baust J. G. and Rojas R. R. (1985) Review-insect cold hardiness: facts and fancy. J. Insect Physiol. 31,155~759. Booth C. (1971) The Genus Fusarium. Commonwealth Agricultural Bureaux, U.K. Corrotto L. V., Wolber P. K. and Warren G. J. (1986) Ice nucleation activity of Pseudomonas Jluorescens: mutagenesis, complementation analysis and identification of a gene product. EMBO JI 5, 231-236. Duman J. G. and Horwarth K. L. (1983) The role of hemolymph proteins in the cold tolerance of insects. A. Rev. Physiol, 45, 261-270.
Kaneko J., Kita K. and Tanno K. (1989) Bacteria in the gut determines the supercooling point of diamondback moth, Plutella xylostella, pupae reared on germinating radish seeds (Raphanus sativus L. var. accanthiformis Makino) (in Japanese with an English summary). Jap. J. appl. En!. Zool. 33, 82-91.
Kaneko J., Kita K. and Tanno K. (1991) INA bacteria isolated from diamondback moth, Plutella xylostella L. pupae (Lepidoptera: Yponomeutidae) (in Japanese with an English summary). JaD. J. Aool. Em. Zool. 35. 7-l I. Kieft T.-L. (1988) Ice &cation activity in lichens: Appl. Environ. Microbial. 54, 1678-168 1. Kieft T. L. and Ruscetti T. (1990) Characterization of biological ice nuclei from a lichen. J. Bacterial. 172, 3519-3523.
Komada H. (1976) Studies on the evaluation of activity of Fusarium oxysporum, Fusarium wilt pathogen of vegetable crops, in soil (in Japanese with an English summary). Bull. Tokai-Kinki Nat. Agric. Exp. Stn 29, 132-269. Lee R. E. and Strong-Gunderson
J. M. (1991) Isolation of ice nucleating active bacteria from insects. J. exp. Zoo/. 257, 124-127.
Lindow S. E. (1983) The role of bacterial ice nucleation in frost injury to plants. A. Rev. Phytopath. 21, 363-384. Salt R. W. (1968) Location and quantitative aspects of ice nucleators in insects. Can J. Zool. 46, 329-333. Siimme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73A, 519-543.
Strong-Gunderson J. M., Lee R. E., Lee M. R. and Riga T. J. (1990) Ingestion of ice-nucleating active bacteria increases the supercooling point of the lady beetle Hippodamia convergens. J. Insect Physiol. 36, 153-151.
Tsumuki H. and Kanehisa K. (1978) Carbohydrate content and oxygen uptake in larvae of the rice stem borer, Chilo suppressalis Walker. Ber. Ohara Inst. landwirt. Biol. Okayama Univ. 17, 95-1 IO. Tsumuki H. and Konno K. (1991) Tissue distribution of the ice-nucleating agents in larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae). Cryobiology 28, 37638 1.
Ice-nucleating fungus isolated from the gut of C. suppressulis Warren G. and Corotto L. (1989) The consensus sequence of ice nucleation proteins from Erwinia herbicola, Pseudomonas juorescens and Pseudomonas syringae. Gene 85, 239-242.
Zachariassen K. E. (1982) Nucleating agents in cold-hardy insects. Comp. Biochem. Physiol. 73A, 557-562.
125
Zachariassen K. E. and Hammel H. T. (1976) Nucleating agents in the haemolymph of insects tolerant to freezing. Nature 262, 285-287.
Zachariassen K. E., Baust J. G. and Lee R. E. (1982) A method for quantitative determination of ice nucleating agents in insect hemolymph. Cryobiology 19, 180-184.
0022-1910/92 $5.00+ 0.00 Copyright0 1992PergamonPressplc
AN ICE-NUCLEATING ACTIVE FUNGUS ISOLATED FROM THE GUT OF THE RICE STEM BORER, CHILO SUPPRESSALIS WALKER (LEPIDOPTERA: PYRALIDAE) HISAAKI TSUMUKI,‘,* HARUYOSHI KONNO,’ TAKANORI MAEDA’
and YASUHIRO OKAMOTO’ ‘ResearchInstitute for Bioresources, Okayama University, Kurashiki 710 and 20kayama Prefectural Agricultural Experiment Station, Sanyo-cho, Okayama 709-08, Japan (Received 21 May 1991; revised 30 August 1991)
Abstract-The existence of a fungus with the ability to nucleate ice formation in supercooled water was revealed. The fungus was isolated from the gut of larvae of the rice stem borer, Chifo suppressalis Walker, and from rice seedlings which were host plants of this insect. The fungus was identified as a Fusarium sp. on the basis of its morphology. Ice-nucleating activity, at around - YC, was detected in the mycelial suspension of the fungus and also in the culture filtrate. The presence of the exogenous ice-nucleating active fungus in the gut and on the body surface caused an elevation in crystallization temperature of the larvae. Ke\j Word Index: Ice-nucleating lization temperature
active fungus; Fusarium sp.; Chilo suppressalis; gut; crystal-
INTRODUCTION
The ice-nucleating active agents contained in insects can be classified into two types: exogenous and endogenous. The endogenous types occur in the haemolymph (Zachariassen and Hammel, 1976; Zachariassen, 1982; Duman and Horwarth, 1983) and muscle (Tsumuki and Konno, 1991) and are constructed from protein (Duman and Horwarth, 1983; Tsumuki and Konno, 1991). In contrast to the endogenous types, the exogenous agents are distributed in the gut or its contents (Salt, 1968; Sdmme, 1982) and on the body surface (Bale et al., 1989). Salt (1968) and Sijmme (1982) have proposed that food particles in the gut act as exogenous ice nuclei, but the characteristics are obscure. It is well known that several epiphytic bacteria serve as ice nuclei (Lindow, 1983). Insects reported to contain ice-nucleating active bacteria in their gut include Plutella xylostella (Kaneko et al., 1989, 1991), and Ceratoma trlfurcata and Hippodamia convergens (Lee and Strong-Gunderson, 1991). Some workers have also reported ice-nucleating ability from lichen fungi (Kieft, 1988; Kieft and Ruscetti, 1990). In this study, we identify an ice-nucleating active fungus isolated from the gut of larvae of the rice stem borer (Chile suppress&s Walker), and measure its ice-nucleating ability. We further examine the effect *To whom all correspondence
should be addressed.
of the exogenous fungus on the crystallization perature of the iarvae. MATERIALS
tem-
AND METHODS
Isolation of ice-nucleating active microorganisms
Mature larvae of the rice stem borer, reared on rice seedlings at 25°C under long-day conditions (16 h light-8 h dark), were used (Tsumuki and Kanehisa, 1978). They were dipped in 70% (v/v) ethanol for 1 min and 0.1% (w/v) mercuric chloride solution for 3 min, and the isolated guts, including the contents, were suspended in sterile distilled water under sterile conditions. Serial dilutions of these suspensions were plated onto potato-sucrose-agar (made from 200 g of potato, 20 g of sucrose and 10 g of agar in 1 litre of distilled water): (i) adjusted to pH 7.0 for bacteria, and (ii) supplemented with 100ppm streptomycin and adjusted to pH 5.6 for the fungi. After incubation for 2-3 days at 25”C, discrete colonies were subcultured in test tubes (180 x 16 mm i.d.) containing 4 ml of liquid potato sucrose medium at 25°C with shaking (140 strokes/min). After incubation for 3-6 days, the crystallization temperatures of the resulting microorganism suspensions were measured. Culture of ice-nucleating active fungus
An ice-nucleating active fungus colonies on the potato-sucrose-agar 119
found from medium was
120
HISAAKITSUMUKIet al.
cultured. To prevent the contamination of adjacent species on the medium, single conidia were isolated by the standard dilution plate method, and germinating conidia were taken from the plates under a dissecting microscope (Booth, 1971). The resulting fungal cultures were used in the subsequent tests. A growth medium containing 20 g of galactose, 2 g of asparagine, 1 g of K2HP0.,, 0.5 g of KCI, 0.5 g of MgS04:7H,0 and 10 mg of Fe-Na-EDTA in 1 litre of distilled water (Komada, 1976), adjusted to pH 5.6, was used to culture the ice-nucleating active fungus. The fungus was inoculated into 250 ml of the liquid medium in a 500ml shaking flask, and cultivation was carried at 25°C with reciprocal shaking at 140 strokes/min. After 12-14 days growth, the mycelial pellets were separated from the culture medium by filtration through a nitrocellulose filter (0.45 ,um pore size). Determination of ice-nucleus concentrations
Serial lo-fold dilutions of the culture filtrate in sterile distilled water were assayed. Ice-nucleating activity was defined as the reciprocal of the highest dilution factor of the dilutions that catalysed the freezing above - 10°C. The crystallization temperatures of the filtrates were estimated from an average of six measurements. Preparation of sterile rice seedlings and larvae
Unpolished rice grain surfaces were sterilized by several immersions in 1% sodium hypochloride solution for 20 min then the grains placed on 1% agar under sterile conditions. After growing for 10 days at 25”C, the rice seedlings were used for rearing sterile larvae. Rice stem borer eggs, sterilized by the same methods as described for larvae, were transferred to the sterile plants. The rice seedlings were exchanged every week and larvae of about 35 days old used for the following experiments. Inoculation of the fungus
The effect of ingesting ice-nucleating active fungus on the crystallization temperature of the insects was determined as follows. Sterile rice plants (10-12 days of growth) were dipped in the fungal suspension (12-14 days of growth) and incubated at 25°C for 3 days and then sterile larvae (35 day old) were placed on the treated plants at 25°C for 1 day. After feeding on the fungus, the larvae were ligated head and tail, and then immersed in 70% ethanol for 3 min. To estimate the activity of the ice-nucleating agents on the body surface, sterile larvae were tied head and tail to prevent the incorporation of ice-nucleating agents into the body and then brought into contact with the fungus by incubation on the treated plants
at 25°C for 1 day. The resulting larvae were immersed in 70% ethanol for 3 min or washed four times with sterile distilled water. The crystallization temperatures of the sterile and treated larvae and plants were measured. Measurement of crystallization temperature
The microorganism suspensions and/or culture filtrates were collected in glass capillary tubes (60 x 1.1 mm i.d.), sandwiched between two layers of paraffin oil. Rice shoot and root pieces in 0.9% NaCl solution were also placed between two layers of the oil in the capillary tubes (Zachariassen et al., 1982). The capillary tubes containing the samples and the larvae were attached to 30-gauge copper-constantan thermocouples connected to a multichannel temperature recorder (Thermodac 5OOlA, Eto-denki, Japan) and cooled at a rate of about l”C/min until frozen. The crystallization temperature was determined as the temperature at which a temporary rapid increase in temperature occurred. The temperatures of 29 samples could be measured simultaneously by the recorder. RESULTS
IdentiJication and activity of the ice-nucleating active fungus
About 600 colonies were obtained from the dilution plates inoculated with a suspension of the gut samples. Bacteria were the predominant microorganisms, accounting for about 75% of all colonies obtained. Fungi including yeasts were also common and accounted for about 25% of all colonies. All microorganisms were tested for their ice-nucleating activities and selected on the basis of their activities above - 10°C. One mycelial fungus showed nucleating activity, but no bacteria or yeasts having such activity were isolated. The fungus which produced long, crescent-shaped, multiseparate macroconidia and small oval-shaped microconidia (Fig. I), was identified as a Fusariam sp. The same fungus was also obtained from rice seedlings reared under natural conditions. Ice nucleation in a suspension of the fungus was detected at around -5°C (Table 1). The ice-nucleating activity was also detected in the mycelium-free culture filtrate (Fig. 2). The activity was detected from 4 days after initiating the culture at 25°C and increased with mycelia growth, reaching a maximum at 12 days. Effect of the fungus on the crystallization temperatures of sterile larvae and rice seedlings
Sterile larvae and those fed on Peniciflium sp., supercooled to below - 2O”C, while the larvae fed on
Fig. 1. Macroconidia and microco mnidiaproduced by the ice nucleating active fungus Ir. __.. (ruscrrium sp.) scale = 65 pm.
121
Ice-nucleating fungus isolated from the gut of C. suppress&
0
10
5
15
123
20
Cultivation time (days)
Fig. 2. Production of ice nuclei in culture filtrate by the ice nucleating active fungus (Fusurium sp.).
the Fusarium sp. isolated showed an elevation in the crystallization temperature to around - 5°C which was equivalent to that of the fungal suspension (Table 1). The crystallization temperature of sterile rice seedlings was - 13 to - 15°C while that of the plants inoculated with the fungus also to around -5°C (Table 1). Inoculation of the larval body surface with the Fusarium sp. also caused the crystallization temperature to increase (Table 2). However, this effect was greatly reduced by immersion in 70% ethanol or washing with water (Table 2). In order to exclude the influence of body surface ice nucleation, larvae fed on Fusarium sp. inoculated rice seedlings were then
Table 1. Effect of the fungus (Fusurium sp.) on the crystallization temperature of sterile larvae and rice seedlings Crystallization temperature (“C) Larvae Feeding Feeding Feeding Ligated
without Fusarium sp. Penicillium sp. Fusarium
sp.
larvae treated with ethanolt
Plants Control (sterilized) Shoot Root Inoculation with Fusnrium sp. Shoot Root Fusarium sp. suspension
ligated head and tail, and immersed in 70% ethanol prior to measurement of the crystallization temperature. The crystallization temperature of the treated larvae was maintained as high as that of the larvae fed on the Fusarium sp. (Table 1). DISCUSSION
The existence of ice-nucleating active bacteria have been reviewed in detail by Lindow (1983) and recently ice-nucleating lichen fungi have been reported (Kieft, 1988; Kieft and Ruscetti, 1990). We have isolated an ice-nucleating active fungus and identified it as Fusarium sp. on the basis of its morphology (Booth, 1971). The crystallization temperature of the fungal suspension was around - 5’C, which was similar to that of several ice-nucleating bacteria (Lindow, 1983) and lichen fungi (Kieft, 1988; Kieft and Ruscetti, 1990), showing that the ability of the fungus in ice nucleation may be comparable
- 20.1 + 0.9* (5) -21.0+0.5*(5)
- 5.7 f 0.6 (5) -5.8 k 0.5 (5)
Table 2. Effect of treatment of 70% ethanol and washing with water on the crystallization temperatures of ligated larvae kept on rice seedlings inoculated with the fungus (Fusarium
sp.) Crystallization temperature (“C)
- 13.6 + 0.7* (12) -15.2+ 1.0*(12) -5.1 kO.3 (14) -4.6*0.2(13) - 5.5 f 0.8 (10)
All values are mean f SE (N). *Significantly different (Student r-test) from the value of Fusarium sp. suspension, P < 0.0 1. tSterile larvae fed on rice seedlings inoculated with Fusurium sp. were ligated head and tail and then immersed in 70% ethanol.
Sterile larvae Larvae inoculated with Fusurium sp. Fusarium sp. inoculated larvae dipped in ethanol Fusarium sp. inoculated larvae washed with water
- 20.6 k 0.3* (5) -7.0 + 1.1 (5) - 14.5 * I.58 (5) - 14.2 + 0.9* (5)
All values are mean + SE (A’). *Significantly different (Student r-test) from the value of larvae inoculated with Fusarium sp., P < 0.01.
124
HISAAKITSUMUKIet al.
to that of these other ice-nucleating active microorganisms. Ice-nucleating active bacteria have been isolated from the guts of some insects and ingestion of the bacteria elevates ice crystallization temperature of the insects (Kaneko et al., 1989, 1991; Strong-Gunderson et al., 1990; Lee and Strong-Gunderson, 1991). In this experiment, an ice-nucleating active fungus (Fusarium sp.) which caused an elevation in crystallization temperature, was isolated from the gut of insect for the first time. The crystallization temperature of rice stem borer larvae reared in natural conditions has been shown to be around -8°C and is due to the gut containing its natural contents (Tsumuki and Konno, 1991). However, the crystallization temperature of sterile larvae was below -20°C as was that of the larvae fed the rice seedlings inoculated with icenucleating negative Penicillium sp. In contrast the presence of the Fusurium sp. isolated here limited the supercooling capacity. Consequently, it seems that the high crystallization temperature under natural conditions is due, at least in part, to the presence of the fungus in the gut. Under natural conditions, the fungus was also isolated from rice seedlings, showing that the fungus isolated from the gut may originate from the food. In diapausing larvae which excrete their gut contents, the crystallization temperature is lower than non-diapausing larvae (Tsumuki and Konno, 1991). Consequently the gut contents, possibly including the fungus, may play a less important role in the supercooling capacity in diapausing larvae. The differences in the crystallization temperatures between rice seedlings and sterile larvae fed on the seedlings (Table 1) show that the ice-nucleating agents in the plants are decomposed or masked in the guts of larvae (Baust and Rojas, 1985). Furthermore, these results indicate that ice nuclei may not be produced from the plant material in the gut of the larvae. The crystallization temperature of larvae exposed to the Fusurium sp. isolated showed a pronounced increase in the crystallization temperature from approx. -20 to -5°C. The ice-nucleating activity on the body surface was reduced by immersion in 70% ethanol and by washing the surface with water, but the crystallization temperatures did not drop as low as -2O”C, as was the case for sterile larvae. These results show that the ice nuclei on the body surface also affect the determination of the crystallization temperature of larvae as suggested by Bale er al. (1989). Preliminary investigations (unpublished data) indicate that the ice nuclei produced by the Fusurium sp. are composed of protein, as reported for icenucleating active bacteria (Corotto et al., 1986;
Warren and Corotto, 1989). Currently, we are attempting to purify the active protein from the culture filtrate and mycelia. Acknowledgemenrs-We
wish to sincerely thank Dr Michael N. Pearson of the Department of Botany, University of Auckland, for his critical reading of the manuscript. This study was supported in part by a Grant-in-Aid for Scientific Research (No. 01560053) and a Grant for Special Project Research from the Ministry of Education, Science and Culture, Japan.
REFERENCES Bale J. S., Hansen T. N. and Baust J. G. (1989) Nucleators and sites of nucleation in the freeze tolerant larvae of the gallfly Eurosta soiidaginis (Fitch). J. Insect Physiol. 35, 291-298.
Baust J. G. and Rojas R. R. (1985) Review-insect cold hardiness: facts and fancy. J. Insect Physiol. 31,155~759. Booth C. (1971) The Genus Fusarium. Commonwealth Agricultural Bureaux, U.K. Corrotto L. V., Wolber P. K. and Warren G. J. (1986) Ice nucleation activity of Pseudomonas Jluorescens: mutagenesis, complementation analysis and identification of a gene product. EMBO JI 5, 231-236. Duman J. G. and Horwarth K. L. (1983) The role of hemolymph proteins in the cold tolerance of insects. A. Rev. Physiol, 45, 261-270.
Kaneko J., Kita K. and Tanno K. (1989) Bacteria in the gut determines the supercooling point of diamondback moth, Plutella xylostella, pupae reared on germinating radish seeds (Raphanus sativus L. var. accanthiformis Makino) (in Japanese with an English summary). Jap. J. appl. En!. Zool. 33, 82-91.
Kaneko J., Kita K. and Tanno K. (1991) INA bacteria isolated from diamondback moth, Plutella xylostella L. pupae (Lepidoptera: Yponomeutidae) (in Japanese with an English summary). JaD. J. Aool. Em. Zool. 35. 7-l I. Kieft T.-L. (1988) Ice &cation activity in lichens: Appl. Environ. Microbial. 54, 1678-168 1. Kieft T. L. and Ruscetti T. (1990) Characterization of biological ice nuclei from a lichen. J. Bacterial. 172, 3519-3523.
Komada H. (1976) Studies on the evaluation of activity of Fusarium oxysporum, Fusarium wilt pathogen of vegetable crops, in soil (in Japanese with an English summary). Bull. Tokai-Kinki Nat. Agric. Exp. Stn 29, 132-269. Lee R. E. and Strong-Gunderson
J. M. (1991) Isolation of ice nucleating active bacteria from insects. J. exp. Zoo/. 257, 124-127.
Lindow S. E. (1983) The role of bacterial ice nucleation in frost injury to plants. A. Rev. Phytopath. 21, 363-384. Salt R. W. (1968) Location and quantitative aspects of ice nucleators in insects. Can J. Zool. 46, 329-333. Siimme L. (1982) Supercooling and winter survival in terrestrial arthropods. Comp. Biochem. Physiol. 73A, 519-543.
Strong-Gunderson J. M., Lee R. E., Lee M. R. and Riga T. J. (1990) Ingestion of ice-nucleating active bacteria increases the supercooling point of the lady beetle Hippodamia convergens. J. Insect Physiol. 36, 153-151.
Tsumuki H. and Kanehisa K. (1978) Carbohydrate content and oxygen uptake in larvae of the rice stem borer, Chilo suppressalis Walker. Ber. Ohara Inst. landwirt. Biol. Okayama Univ. 17, 95-1 IO. Tsumuki H. and Konno K. (1991) Tissue distribution of the ice-nucleating agents in larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae). Cryobiology 28, 37638 1.
Ice-nucleating fungus isolated from the gut of C. suppressulis Warren G. and Corotto L. (1989) The consensus sequence of ice nucleation proteins from Erwinia herbicola, Pseudomonas juorescens and Pseudomonas syringae. Gene 85, 239-242.
Zachariassen K. E. (1982) Nucleating agents in cold-hardy insects. Comp. Biochem. Physiol. 73A, 557-562.
125
Zachariassen K. E. and Hammel H. T. (1976) Nucleating agents in the haemolymph of insects tolerant to freezing. Nature 262, 285-287.
Zachariassen K. E., Baust J. G. and Lee R. E. (1982) A method for quantitative determination of ice nucleating agents in insect hemolymph. Cryobiology 19, 180-184.