JP2005102510A - Plant insect pest controlling agent and method for controlling plant insect pest utilizing insect pathogenic bacterium pseudomonas fluorescens kpm-018p - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for controlling plant insect pests by which the plant insect pests are suppressed. <P>SOLUTION: The plant insect pets are effectively suppressed by spraying a controlling agent using a bacterium Pseudomonas fluorescens KPM-018P isolated from a leaf of a tomato. The Pseudomonas fluorescens KPM-018P secrets chitinase and has lethal effects on ingested insects. Furthermore, the KPM-018P produces a biosurfactant and forms a stable colony on a plant body. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a plant pest control agent and control method using a novel entomopathogenic bacterium Pseudomonas fluorescens KPM-018P.

  Microorganisms that stably form colonies on the leaves of plants have the potential to be used as biological agents that suppress foliar pathogens or plant pests. The present inventors succeeded in suppressing leaf feeding and egg laying by phytophagous ladybirds by using alginate gel beads encapsulating chitinolytic foliar bacteria isolated from greenhouse tomatoes in the previous study. (Non-Patent Documents 1 and 2). Such suppression is mainly due to enzymatic degradation of the chitinous envelope in the midgut of pests that have eaten tomato leaves treated with bacteria. Although this treatment was simple and effective, only adult ladybirds could be suppressed. In the tomato cultivation system of the present inventors, the leaves of seedlings before transplanting are remarkably fed by both larvae and adults. Therefore, the present inventors further needed to screen effective biological materials by focusing on bacteria that settled on the leaves of tomato seedlings at the stage before planting. The present specification discloses a phytobacteria Pseudomonas fluorescens isolated and identified from tomato leaves, and further discloses its effectiveness as an entomopathogenic bacterium against phytophagous pests.

  Some Pseudomonas fluorescens strains have been extensively investigated as endoparasite biomaterials that exhibit antibacterial activity by inducing host resistance against phytopathogenic bacteria and fungi Some also affect the growth and development of insects at various stages of development (Non-patent Document 3). For example, certain Pseudomonas fluorescens can be used to control pests such as Leptinotarsa decemlineata and rice leaf holders (Cnaphalocrocis medinalis) by feeding on tubers and shoots of bacterially treated plants. in use. Beattie and Lindow (1995) have shown that bacteria are localized at specific parts of the leaf surface, ie, junctions of trichomes, stomata, and epidermal cell walls. In an attempt to biologically control plant leaf pathogens using antagonistic strains of Pseudomonas fluorescens, powdered bacterial cell suspensions were sprayed onto the leaves to sustain the formation of bacterial colonies. (Non-Patent Documents 4 to 6).

Our approach using bacteria is also based on the ability of the bacteria to survive stably on the surface of the tomato leaves that the pest eats. Recent molecular techniques, in particular the use of green fluorescent protein gene (gfp) as a reporter to visualize colonization of target bacteria on plants, allows bacteria to be labeled with a reporter gene and observed. . This bacterial labeling technique with gfp was also used in our system to follow the persistent colonization of bacteria in the treated leaves. With these approaches, we evaluated the feasibility of using the entomopathogenic bacterium Pseudomonas fluorescens against phytophagous pests.
Otsu. Y., Matsuda, Y., Shimizu, H., Ueki, H., Mori, H., Fujiwara, K., Nakajima, T., Miwa, A., Nonomura, T., Sakuratani, S., Tosa, Y, Mayama, S. & Toyoda, H. (2003a) Biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Coleoptera: Coccinellidae) by chitinolytic phylloplane bacteria Alcaligenes paradoxus entrapped in alginate beads.Journal of Applied Entomology (press) Otsu, Y., Mori, H., Komuta, K., Shimizu, H., Nogawa, S., Matsuda, Y., Nonomura, T., Sakuratani, S., Tosa, Y., Mayama, S. & Toyoda, H. (2003b) Suppression of leaf feeding and oviposition of phytophagous ladybird beetles (Coleoptera: Coccinellidae) by chitinase gene-transformed phylloplane bacteria and their specific bacteriophages entrapped in alginate gel beads. Journal of Economic Entomology 96, 555-563 Ramamoorthy, V., Viswanathan, R., Raguchander, T., Prakasam, V. & Samiyappan, R. (2001) Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pests and diseases.Crop Protection 20, 1- 11. Gnanamanickam, SS & Mew, TW (1992) Biological control of host disease of rice (Oryza sativa L.) with antagonistic bacteria and its mediation by a Pseudonzonas antibiotic. Annals of the Phytopathological Society of Japan 58, 380-385. Chatterjee, A., Valasubramanian, R., Ma, WL., Vachhani, AK, Gnanamanickam, S. & Chatterjee, AK (1996) Isolation of ant mutants of Pseudomonas fluorescens strain Pf7-14 altered in antibiotic production, cloning of ant + DNA, and evaluation of the role of antibiotic production in the control of blast and sheath blight of rice.Biological Control 7, 185-195. Vidhyasekaran, P., Rabindran R., Muthamilan, M., Nayar, K., Rajappan, K., Subramanian, N. & Vasumathi, K (1997) Development of a powder formulation of Pseudomonas fluorescens for control of rice blast. Pathology 46, 291-297.

  The present invention has been made in view of the above circumstances, and an object thereof is to control plant pests using a novel entomopathogenic bacterium.

  That is, the present invention provides Pseudomonas fluorescens KPM-018P, which is a bacterium having a deposit number of FERM P-19525, which has the property of producing chitinase and biosurfactant.

The present invention also provides a plant pest control agent comprising the bacterium, wherein the plant pest is killed by a chitinase secreted by the bacterium.
According to one aspect of the present invention, the plant pest is a Coleoptera insect, in particular an insect selected from the group consisting of ladybirds, scallops and kissabill beetles.
According to another aspect of the present invention, the plant pest is a lepidopteran insect, and in particular, an insect selected from the group consisting of Spodoptera litura, Tobacco moth, Pterodum, and Urinoma.
According to another aspect of the present invention, the plant pests are hemiptera, in particular insects selected from the group consisting of whitefly whitefly, silverleaf whitefly, cotton aphid, and peach aphid.
According to another aspect of the present invention, the plant pest is a thrips insect, and more particularly, a citrus thrips.

  Furthermore, the present invention provides a plant pest control method using the plant pest control agent, the method comprising spraying a liquid plant pest control agent containing the live bacteria on a plant body. provide. Preferably, the plant is tomato. The liquid plant pest control agent is preferably a culture solution of the bacteria.

  ADVANTAGE OF THE INVENTION According to this invention, the plant pest control agent and plant pest control method which suppress a plant pest effectively can be provided by using Pseudomonas fluorescens (Pseudomonas fluorescens) KPM-018P.

  The present inventors screened bacteria from tomato leaves and succeeded in isolating a novel bacterium Pseudomonas fluorescens KPM-018P having chitinolytic properties. The isolated Pseudomonas fluorescens KPM-018P has the property of producing chitinase secretion and biosurfactant. The colonies formed by KPM-018P are smooth and circular, have fluorescence and gloss, and change to light yellow at 37 ° C and red at 25 ° C. In a solid medium containing chitin, chitin is hydrolyzed by chitinase secreted by bacteria, and a transparent zone is formed around the colony. It is resistant to ampicillin, tetracycline, and chloramphenicol, and sensitive to kanamycin and streptomycin. As detailed later, this KPM-018P was shown to have entomopathogenicity against plant pests.

  In the present invention, a plant pest means an insect that eats leaves or other parts of a plant body or sucks a juice to cause damage to the plant. This insect is, for example, an insect of the order Coleoptera, and in particular, ladybirds, scallops, and kissing beetles. Other examples include lepidopteran insects, and in particular, Spodoptera litura, Tobacco moth, moth, and urinous moth. In addition, for example, insects of the order Hemiptera, particularly, white fly whitefly, silver leaf whitefly, cotton aphid and peach aphid. Furthermore, it is an insect of the order Thrips, for example, Citrus thrips.

  These plant pests have been shown to be lethal by ingestion of KPM-018P. This is thought to be because chitinase secreted by KPM-018P degrades the chitinous envelope in the midgut of the pest. In this invention, the plant pest control agent containing the living microbe of KPM-018P can be provided using such an entomopathogenicity of KPM-018P.

  The method of controlling plant pests using this control agent is by spraying this control agent on plants and ingesting them with plants.

  In order to maintain the effectiveness of this pest control agent, it is important that the spread KPM-018P settles on the plant body. At this time, the biosurfactant produced by KPM-018P works advantageously, imparts stability to the colony formation of KPM-018P on the plant body, and enables stable and continuous colonization of KPM-018P.

  The form of the plant control agent may be any form capable of spraying live KPM-018P bacteria on plants. For example, it may be a powder obtained by freeze-drying bacteria, or may be a suspension of a carrier such as alginate gel beads enclosing bacteria. However, in order to effectively use the biosurfactant produced by KPM-018P, a liquid form is preferable, and an aqueous suspension containing viable KPM-018P bacteria is preferable. In particular, since a culture solution in which KPM-018P is cultured is considered to have the highest biosurfactant concentration, a control agent using such a culture solution containing viable KPM-018P bacteria is most preferable.

As a method of spraying the plant pest control agent on a plant body, a liquid control agent can be sprayed using a spray. A suspension of KPM-018P cultured for 2 days (10 ⁹ cells / ml) was sprayed on tomato leaves at a bacterial density of 10 ⁴ to 10 ⁵ cells / 1 cm ² , and the number of bacteria was measured over 7 days. It was suggested that the bacterial population on the leaves did not decrease significantly within 7 days after inoculation. Therefore, by treating the control agent at least once a week, KPM-018P can be fixed on the plant body, and a pest control effect can be obtained. The KPM-018P bacterial density in the control agent shown here is a relatively high density, but is not limited thereto, and a suspension or culture solution having an arbitrary density can also be used.

  A typical plant that can control pests with the plant pest control agent of the present invention is tomato, and it was shown that the ladybug of the pest is effectively suppressed in the leaves of tomato.

[Example]
Examples of the present invention will be described below.

1. Materials and methods <Plants and insects>
The germinated tomato was transferred to a 12 cm pot containing vermiculite and left in a growth box covered with a transparent vinyl film blocking the UV spectrum of 390 nm or less and on the top and sides. This growth box was left in a greenhouse controlled at 26 ± 4 ° C. The variation in relative humidity in the growth box was 50 to 90%.

  Adult adults of Epilachna vigintioctopunctata (Coleoptera, Ladybirdidae) were collected from field tomatoes and raised in tomato seedlings 1 month after germination in a growth chamber (26 ± 1 ° C, light period 16 hours) .

<Isolation and identification of entomopathogenic bacteria>
Fully grown leaves were randomly collected from tomatoes grown in a greenhouse under humid conditions (26 ± 6 ° C., relative humidity 70 ± 20%) for 2 months. The top surface of the picked leaves was placed on M9 minimal agar medium (12.8 g Na ₂ HPO ₄ 7H ₂ O, 3 g KH ₂ PO ₄ , 0.5 g NaCl, 1 g NH ₄ Cl, and 4 g in 1000 ml water). And was allowed to stand for 2 minutes. The bacterial colonies that grew on the medium were transferred to an M9 medium (Chi-M9 medium) containing colloidal chitin. Colloidal chitin was prepared according to the method of Hirano and Nagao (1988). After incubation at 26 ° C. for 3 days, bacterial colonies that formed halos (clear zones formed around the colonies in the medium by chitin being hydrolyzed by the chitinase secreted by the bacteria) were treated with standard concentrations of antibiotics. Transferred to Chi-M9 medium containing (tetracycline 20 μg / ml, ampicillin 50 μg / ml, streptomycin 50 μg / ml, kanamycin 50 μg / ml, and chloramphenicol 100 μg / ml). Isolated fungi were classified into 18 types based on colony color and antibiotic susceptibility. Each of the classified bacteria was suspended in distilled water and tested for pathogenicity against ladybird larvae in the preliminary screening described below. As a result, KPM-018P isolated and identified had entomopathogenic activity, and its microbiological characteristics were determined by Bergey's Manual of Determinative Bacteriology (9th Ed.) As described below.

<Isolation of KPM-018P and its entomopathogenic activity test>
For preliminary screening of the entomopathogenic bacterium KPM-018P, the 4th instar larvae were isolated in a desiccator kept at 14-15% relative humidity and starved for 12 hours, and the bacterial suspension (10 ⁹ cells in sterile water) / Ml was suspended, and placed on paper immersed in a hemocytometer and a phase contrast microscope for 30 minutes. After confirming the inoculation of bacteria due to the expansion of the abdomen of the larvae, the larvae were left in a petri dish containing fresh leaves and incubated until the next wing stage was completed (10 days). Based on this preliminary screening, KPM-018P was isolated.

  The isolated entomopathogenic activity test of KPM-018P was tested by the direct injection method. That is, starved larvae were placed on their back on 1.5% agar (weight / volume) on a glass slide and 2 μl suspended in sterile water using a micropipette (tip diameter 10 μm) under a stereo microscope. The bacterial suspension was injected directly into the larval mouth. The larvae inoculated with the bacteria were left for 10 days in a petri dish containing leaves as described above. E. coli HB101 / ampicillin resistance (HUC101 transformed with pUC119) and Alcaligenes paradoxus KPM-012A were used as negative control bacteria and treated in the same manner. KPM-012A was previously obtained from the surface of tomato leaves (Otsu et al., 2003a).

<Evaluation of enzyme activity in bacterially infected larvae>
McCreath and Goody using 4-methylumbellifery-β-DN, N ′, N ″ -triacetylchitotrioside [4MU- (GlcNAc) 3] (Sigma) as substrate (1992) was used to measure chitinase activity. Bacteria-inoculated larvae and non-inoculated larvae were each homogenized in distilled water, 100 μl of the homogenate was centrifuged, and 900 μl of McIlvaine buffer (pH 7, 0.1 mol / L citric acid 17.8 ml, and 0.2 mol / L dibasic sodium phosphate (82.2 ml). Next, 50 μl of 0.134 mg / ml 4MU- (GluNAc) 3 was added to initiate the reaction. After incubation at 37 ° C. for 10 minutes, 1.2 ml of 1 mol / L glycine / NaOH buffer (pH 10.6) was added to stop the reaction, and the fluorescence due to the release of 4-methylumbelliferon (4MU) was spectroscopically analyzed. Observation was performed using a fluorometer (RF-5000; Shimazu, Kyoto, Japan). The amount of 4MU was evaluated according to a standard curve with standard 4MU in the same buffer (pH 7.0).

  Proteolytic activity was measured according to the method of Bowen et al. (2000) using azocasein (Sigma) as a substrate. The larvae were homogenized with 50 mM Tris buffer saline (pH 8.0), and the supernatant of the homogenate (20 μl) was added to 500 μl of the same buffer containing 10 mg / ml azocasein, and the orbital shaker at 30 ° C. for 4 hours. Incubated with. Non-hydrolyzed azocasein was precipitated by adding 500 μl of 10% trichloroacetic acid. The mixture was centrifuged and the absorbance (440 nm) of the resulting supernatant was measured. Increased absorbance indicated proteolytic activity.

<Evaluation of biosurfactant production by bacteria>
The production of biosurfactant by KPM-018P was evaluated by the rapid water droplet disintegration test (Jain et al., 1991). Bacteria are cultured in a liquid M9 medium (without agar) and shaken at 25 ° C., 10 μl of bacterial culture solution or supernatant obtained by centrifuging the culture solution is dropped onto a hydrophobic membrane (Parafilm) or tomato leaf surface, I examined the shape. As a control, a droplet containing a synthetic surfactant (10% (v / v) Tween 20) was used as a positive control. Moreover, the culture solution which culture | cultivated Escherichia coli HB101 / ampicillin resistance which does not produce | generate biosurfactant, and an alkali gene Paradoxas KPM-012A for 2 days was used for negative control.

<Observation by gene labeling of bacteria using gfp gene>
The original plasmid pUTgfp (Tombolini et al., 1997) was digested with Not I to excise the gfp gene. The excised gfp gene was inserted as a selectable marker into pBluescript (Stratagene, CA, USA) containing the ampicillin resistance gene. This new plasmid (pBlue / gfp) was introduced into KPM-018P according to the electroporation method disclosed previously (Toyoda et al., 1991). Colonies cultured in L broth containing 150 μg / ml ampicillin were irradiated with UV and tested for green fluorescence production. The GFP-labeled bacteria (KPM-018P / gfp) selected in this test were cultured for 2 days, and the culture solution (10 ⁹ cells / ml) was sprayed on the seedling leaves. After incubating the seedlings in the growth box for 7 days, the leaves sprayed with bacteria were collected and directly observed by Olympus fluorescence microscope BX-60 (IB excitation with BP460-490 excitation filter and BA510IF absorption filter).

<Recovering bacteria sprayed on leaves>
In order to confirm that KPM-018P was fixed on the plant body, the following test was conducted.
A 1-month-old tomato seedling (6 leaves with 20 leaflets) was suspended in KPM-018P suspension (2 days of culture, 10 ⁹ cells / ml) to a bacterial density of 10 ⁴ to 10 ⁵ cells per 1 cm ^{2 of} leaves. It sprayed so that it might become. Leaves are collected daily from a tomato seedling sprayed with this bacterium over a 7-day test period, homogenized and diluted in sterile water on Chi-M9 medium containing antibiotics to monitor the survival of the sprayed bacteria. It was applied to. After 2 days of incubation, the number of bacterial colonies formed was determined as colony forming units (cfu) per cm ² of leaf. The bacteria were distinguished on the basis of their colony appearance, ie chitin hydrolytic ability and antibiotic resistance. KPM-018P is ampicillin and tetracycline resistant, forms red colonies and is positive for chitin hydrolysis. KPM-012A is kanamycin resistant, pale yellow colony, chitin hydrolysis positive. And E. coli HB101 / ampicillin resistance has ampicillin resistance and is white colony and negative for chitin hydrolysis.

<Feeding by insects on leaves treated with bacteria>
Ten larvae of the 3rd instar ladybird were released per 10 seedlings on the leaves of 1 month old seedlings sprayed with the KPM-018P suspension by the above method. The seedlings with these ladybirds were placed separately in a transparent box in the growing cabinet. Every day during the 2-week experimental period, the seedlings were replaced with new seedlings that had been sprayed with bacteria, and larval survival and larval leaf feeding were recorded. A suspension of E. coli HB101 / ampicillin resistant and Alkaline Genes paradoxus KPM-012A was also used as a non-pathogenic control. To assess the extent of leaf feeding by ladybirds, the fed region of all leaves of each plant was scored on a scale of 0-4. 0 is no feeding, 1 is a fed leaf area of 25% or less, 2 is 50% or less, 3 is 75% or less, and 4 is 75 to 100%. The severity of feeding for each plant was determined using the following formula:

[(0A + 1B + 2C + 3D + 4E) / (A + B + C + D + E)]
Here, A, B, C, D, and E are the numbers of leaves corresponding to the scores (0, 1, 2, 3, and 4), respectively. All seedling leaves fed during the larval stage were scored until this stage was completed.

2. Results The phytophagous ladybirds were collected from our tomato field and successfully bred on tomato leaves under laboratory conditions. Adult beetles can be raised on tomato leaves and lay many eggs (Otsu et al., 2003a). Under these conditions (26 ± 1 ° C., 3000 lux, light irradiation for 16 hours), the duration of the egg stage was 2.8 ± 0.9 days, and the hatching rate was 77.5 ± 13.3%. . The larval growth period is 2.9 ± 0.4 days for 1 worm, 2.3 ± 0.5 days for 2 worms, 2.6 ± 0.5 days for 3 worms, and 4 worms Is 4.1 ± 0.4 days. The stage of pupae was 4.5 ± 0.7 days, and the incidence of adults from pupae was 98.5 ± 7.7%. Since 3 and / or 4 worms are active and feed on tomato leaves synchronously, they were used in the experiments.

  Tomato leaves (500) were randomly collected from 100 plant tomatoes and used as stamps to screen for bacteria. By gently pressing the leaves against the M9 medium, 820 bacteria were obtained from 98 leaves and 7110 yeast (sprouting cell form) colonies were obtained from 315 leaves. All bacterial colonies were transferred to Chi-M9 medium supplemented with antibiotics and chitinolytic bacteria were pre-classified into 18 types based on colony morphology and antibiotic sensitivity or resistance. From the obtained bacteria, KPM-018P having lethal activity against 4th instar larvae was isolated in a preliminary test. 70.5 ± 21.5% (average of 3 separate experiments) of the larvae that ate the bacteria died before becoming pupae. Other isolated bacteria did not have any lethal effect on the larvae that ate them. Therefore, KPM-018P was employed as an entomopathogenic bacterium and used in subsequent experiments.

  KPM-018P forms smooth, circular, fluorescent and glossy colonies that are pale yellow at 37 ° C and turn red when transferred to 25 ° C. This KPM-018P colony becomes liquid for the production of exopolysaccharide (EPS) when cultured in solid medium for 3 days (Allison et al., 1998). This isolate is resistant to ampicillin, tetracycline, and chloramphenicol and is sensitive to kanamycin and streptomycin. Chitinase secretion by KPM-018P is high, and colloidal chitin in the medium is decomposed to produce a clear transparent zone around the colony (FIG. 1). According to these properties (colony color change, halo formation, and antibiotic responsiveness), KPM-018P was first cultured at 37 ° C. for 1 day in Chi-M9 agar medium supplemented with antibiotics, then 25 It can be specifically detected by culturing at a temperature of 1 day.

  According to the criteria of Bergey's Manual of Determinative Bacteriology, KPM-018P was characterized as gonococci with gram negative and polar flagella (width 0.6-0.7 μm). The optimum temperature for growth is 25-30 ° C. The bacterium was aerobic and positive for catalase reaction, nitrate reduction, arginine dihydrolase reaction, and gelatin hydrolysis, negative for oxidase reaction, starch hydrolysis, and oxidation of ethanol to acetic acid. In addition, this bacterium produced aerobic acid from D-glucose and using D-glucose, D-mannitol, and L-histidine as carbon sources. Based on these data, it was proposed to assign the bacterial strain of the present invention to Pseudomonas fluorescens KPM-018P.

In order to further clarify the relationship between the bacterial concentration of KPM-018P and the lethality of bacteria feeding insects, the bacterial suspension was injected directly into the mouth of 4th instar larvae using a pipette under a microscope ( FIG. 1B). In this approach, the volume of suspension (2 μl) injected into each larva was quantified. Larvae viability decreased with increasing KPM-018P cells in the injected suspension (Table 1), but no lethal effect was detected in larvae injected similarly with non-pathogenic bacteria or distilled water, Therefore, it was shown that pipetting does not cause mechanical damage to the larvae.

The histopathological changes of larvae fed with bacteria were well synchronized. Larvae stopped working 36-40 hours after injection, and the body changed from yellow to dark pink or brown, especially when 10 ⁷ cells of bacteria were administered per larva (FIG. 1C). The input bacteria initially decreased but then gradually increased and increased vigorously as soon as the larvae lost color (FIG. 2). The first bacteria reduction was due to the bacteria being excreted as fecal pellets after injection. KPM-018P (10 ³ to 10 ⁶ bacteria / pellet) was recovered from a randomly collected pellet (30) 12 hours after injection. The bacterial population within the larvae reached 10 ¹¹ cells per larvae 4 days after injection. At this stage, the larvae became soft and bacteria spouted from the infected larvae (FIG. 1C). The enzymatic activity of chitinase and protease in infected larvae was markedly increased in equilibrium with the rapid increase in KPM-018P.

The production of biosurfactants by bacteria was thought to be an important factor for bacteria to obtain a water-insoluble substrate through the hydrophobic epidermis of leaves and form stable colonies on the leaf surface (Bunster et al. al., 1989). FIG. 3A (top and bottom) shows the change in droplet shape on the hydrophobic membrane. Water droplets containing more than 1% Tween 20 increased the surface activity and decreased the spherical tension. A similar effect was observed in the supernatant droplets of KPM-018P cultured for 48 hours, indicating that the bacteria secreted the surfactant into the culture. On the other hand, neither Escherichia coli nor Alkaligenes paradoxus produced surfactants that would disrupt the spherical shape of the droplets on the membrane. Similar results were obtained even when the culture solution was dropped onto tomato leaves (FIG. 3A; middle). From these results, it was shown that KPM-018P cultured for 48 hours is suitable for spraying leaves. In addition, colony formation on the leaf surface was investigated at various stages after spraying. FIG. 3B shows gfp-labeled bacteria (KPM-018P / gfp) that inhabit the tomato leaves 7 days after spraying. Many bacteria with fluorescence were detected at the junctions of the epidermal cells of the leaves sprayed with the bacteria. Furthermore, KPM-018P could be recovered by pressing the leaves against the medium (FIG. 3C). The numbers of bacterial colonies recovered from the leaf-pressed medium were 5.2 ± 1.1 × 10 ³ and 3.8 ± 2.6 × 10 ³ per leaf 30 minutes and 7 days after spraying, respectively. It was a colony. In addition, the number of bacteria recovered from the bacterially sprayed leaf homogenate ranged from 4.6 ± 1.8 × 10 ⁴ to 2.3 ± 1.2 × 10 ⁴ over the course of the experiment (FIG. 4). These results suggested that the bacterial population on the leaves was not significantly reduced within 7 days after inoculation.

In order to test the effectiveness of the control approach of the present invention, 3rd instar larvae were released on seedlings sprayed with bacteria or water. Table 2 shows the mortality, hatchability and adult incidence of larvae at the 3rd and 4th instar stages, and the severity of leaf feeding by the larvae during the test period. Almost all the larvae that ingested the leaves sprayed with KPM-018P were effectively killed and slightly hatched, but no adults were generated. At the same time, leaf intake by larvae was significantly reduced. On the other hand, neither Alkaligenes paradoxus nor Escherichia coli killed the larvae at all, and did not inhibit the hatching and adult development. This was similar to that sprayed with water.

3. Discussion In Japan, a new powdery mildew was reported to occur in greenhouse-grown tomatoes (Matsuda et al., 2001; Ksashimoto et al., 2003). This pathogen also devastated young tomato seedlings in our greenhouse, especially with poor chemical control (Matsuda et al., 2001). Therefore, it was necessary to repeatedly use fungicides to completely prevent this disease. However, this is contrary to the policy of the university to which the present inventors try to reduce the use of chemical pesticides. Abikio (1978) tested optimal conditions for colonization and sporulation of tomato powdery mildew, and high humidity (100% relative humidity) suppresses mycelium growth and spore formation of pathogens Reported that. Based on these investigations, the seedlings were cultivated at high humidity until transplanted, and the seedlings were put in a growth box surrounded by a transparent vinyl film to maintain the humidity. This transparent vinyl film absorbs the UV spectrum below 300 nm, which is essential for gray mold (Botrytis cinerea) to form photospores on tomatoes and for rapid colonization (Honda & Yunoki, 1978). Aseptic seedlings of tomatoes were able to grow smoothly in a high-humidity growing box, but the box could not prevent the feeding of pests. In fact, it was rather suitable for insects that drop leaves, such as herbivorous ladybirds. Therefore, the inventors focused on the control of phytophagous pests, especially control using biocontrol agents.

  The phytophagous ladybird E.vigintioctopunctata is a pest of the solanaceous plant (Shirai & Katakura, 1999) and naturally occurs in our greenhouses and fields. These ladybirds often ruin tomatoes and eggplants, causing serious damage that compromises yield. In the laboratory, the inventors have developed a system for continuously raising and maintaining these pests on tomato leaves (Otsu et al., 2003a; 2003b). 3 and 4 instar larvae were particularly suitable for treatment in the current study due to their active and synchronous leaf feeding.

  Here, in the entomopathogenic strain Pseudomonas fluorescens (KPM-018P) obtained from the surface of tomato leaves, the color of the colony changes from pale yellow to red when the incubation temperature is lowered from 37 ° C. to 25 ° C. Thus, it was easily detected. Even after the bacteria were continuously subcultured for several months in minimal medium, their pathogenicity against ladybird larvae was stable. Although the pathogenic factor of KPM-018P has not been obtained, the stable pathogenicity even after spraying on leaves has the potential as a biocontrol material.

By using a micropipette, a known amount of bacterial suspension could be effectively injected into the larvae, which was useful for synchronizing disease progression. When a large amount of bacteria was injected, the bacteria were excreted in the fecal pellet in the early stage after injection. When a high-density (10 ⁷ cells / larvae) bacterial suspension was injected, 10 ⁴ to 10 ⁵ bacterial cells / larvae remained in the larvae, but these levels caused disease in the larvae. It was suggested that it is essential. After the infected larvae change color after incubation, the bacteria grow significantly and the insects do not move. Enzymatic activity (chitinase and protease) increases in parallel with bacterial growth and causes larval body softening. This activity causes the grown bacteria to erupt from the larvae.

  The present inventors previously controlled the insect pests by spraying bacteria (alkaligenes paradoxus) present in the leaves on arginic acid beads because of their low colony formation efficiency on the leaf surface (Otsu). et al., 2003a). In contrast, the novel strain of the present invention can form stable colonies on the surface of tomato leaves when used directly on the leaves as a cell suspension. In general, bacterial colonization on leaves depends on the ability of bacteria to survive under conditions of drought stress or UV irradiation stress (Andrews, 1992; Beattie & Lindow, 1995). KPM-018P was recovered from the leaves sprayed with bacteria even 7 days after spraying. This example provided valid data to confirm that bacteria form stable colonies on the leaf surface.

  In this example, the GFP fluorescence of bacteria on the leaf surface was directly observed using KPM-018P labeled with gfp, and the colonies were observed to be localized. Throughout the experimental period, GFP fluorescence was detected at the junctions of leaf epidermal cells sprayed with bacteria. It is considered that these junctions tend to become depressions for KPM-018P to survive and form colonies.

  Chemical properties of the strains of the present invention also contribute to stable settlement on the leaf surface. The production of biosurfactants by bacteria is useful for attaching to the leaf surface, and by controlling the balance of hydrophobic and hydrophilic substrates between the bacterial cells and the leaf surface, colonization in the chlorosphere is controlled. Helps (Bunster et al., 1989) and provides hydration for cells to attach to the hydrophobic epithelium (Bunster et al., 1989). In fact, KPM-018P mutants lacking biosurfactants by chemical treatment have a lower rate of recovery from leaves compared to bacteria that produce biosurfactants (data not shown), and these substrates are on the leaf surface. It was confirmed that it is involved in bacterial colonization in Desai and Banet (1997) reported strains with Pseudomonas fluorescens that produce lipopeptides or sugar-protein-lipid biosurfactants. Identification of the biosurfactant produced by KPM-018P is currently underway in our laboratory.

In addition, extracellular polysaccharides produced by bacteria may form biofilms that protect bacteria from harmful environmental factors that interfere with colony formation (Allison et al., 1998; Morris et al., 1998). It is interesting from a practical point of view that KPM-018P forms a biofilm in sprayed leaves, as it assists the continuous colonization of bacteria when practicing the present invention in a field. KPM-018P strain continuously forms colonies on tomato leaves, and its pathogenicity is stably expressed. Therefore, it can be easily put into practical use, for example, by spraying a bacterial suspension on tomato seedlings every other week for two months. Application of the plant pest control agent using KPM-018P completely protects the seedlings by lethalizing the larvae and suppressing the occurrence of adults in the breeding box. After transplanting, the tomatoes with leafy leaves are not severely damaged when they are attacked by adults or larvae of ladybirds, but in order to protect the tomatoes at the seedling stage, measures against plant pests are necessary. .
[References]

A is a colony of the entomopathogenic bacterial strain Pseudomonas fluorescens KPM-018P, and has a transparent zone (arrow) formed by hydrolysis of chitin contained in the medium by chitinase secreted by the bacteria (in Chi-M9) 2 day incubation). B is a 4th instar larva orally injected with a bacterial suspension with a micropipette. C is KPM-018P-infected larvae, the left is before injection and the center is 24 hours after injection. The body color of infected larvae changed to red or light brown. On the right, larvae 72 hours after infection were placed in M9 medium, and KPM-018P was leached from the infected larvae (48 hours after transfer to the medium). Figure 5 shows the growth of Pseudomonas fluorescens KPM-018P injected with a micropipette into ladybird larvae, and kinase and protease activity. A, B, and C are each the period of pathological change of the infected larvae, respectively, and the period of bacterial leaching following cessation of movement, discoloration, and softening of the larval shells, respectively. The white squares and triangles indicate chitinase and protease activity of uninoculated larvae, respectively. Test of surfactant generation and bacterial colonization on tomato leaves by the entomopathogenic bacterial strain Pseudomonas fluorescens KPM-018P. A: Bacterial surfactant production test (top and bottom: droplets of bacterial suspension on hydrophobic membrane and middle: droplets of bacterial suspension on tomato leaves). B is a GFP fluorescent bacterium (KPM-018P / gfp) colonized on the leaf surface at the epidermal cell junction (7 days after spraying). C is a colony of KPM-018P recovered by pressing tomato leaves sprayed with bacteria against the medium. Bacteria recovered from tomato seedling leaves sprayed with bacterial suspension. Five leaves sprayed with bacteria were collected every day, homogenized with sterile water, and applied to Chi-M9 medium containing antibiotics. Data are the mean and standard deviation of three separate experiments.

Claims (13)

  Pseudomonas fluorescens KPM-018P, a bacterium with deposit number FERM P-19525.   A plant pest control agent comprising the bacterium according to claim 1, wherein the plant pest control agent kills the plant pest by a chitinase secreted by the bacterium.   The plant pest control agent according to claim 2, wherein the plant pest is a Coleoptera insect.   The plant pest control agent according to claim 3, wherein the insects of the order Coleoptera are selected from the group consisting of ladybirds, scallops, and kissabill beetles.   The plant pest control agent according to claim 2, wherein the plant pest is a lepidopteran insect.   6. The plant pest control agent according to claim 5, wherein the lepidopterous insect is selected from the group consisting of Spodoptera litura, Tobacco moth, Japanese moth, and Urino rice.   The plant pest control agent according to claim 2, wherein the plant pest is a hemiptera insect.   8. The plant pest control agent according to claim 7, wherein the hemiptera insects are selected from the group consisting of white fly whitefly, silver leaf whitefly, cotton aphid, and peach aphid.   The plant pest control agent according to claim 2, wherein the plant pest is a thrips insect.   The plant pest control agent according to claim 9, wherein the thrips insect is a citrus yellow thrips.   A plant pest control method using the plant pest control agent according to claim 2, wherein the plant is sprayed with a liquid plant pest control agent containing the live bacteria.   The plant pest control method according to claim 11, wherein the plant is a tomato.   13. The plant pest control method according to claim 11 or 12, wherein the liquid plant pest control agent is a culture solution of the bacterium.

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