Method for infecting plants with Liberibacter sp.

Abstract

To provide a method that allows Liberibacter bacteria to stably infect even non-cultivars and wild relatives in a fixed bacterial density condition.SOLUTION: The present invention discloses a method for allowing Liberibacter bacteria to infect a plant, the method including bringing the bacteria-containing liquid that contains Liberibacter bacteria into contact with plant roots.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for infecting plants with bacteria of the genus Liberibacter, and more particularly to a method for infecting host and non-host plants with difficult-to-culture bacteria of the genus Liberibacter, including citrus greening pathogenic bacteria. [Background technology]

[0002] Many Liberibacter bacteria are known to infect various plants. For example, citrus greening (huanglongbing) is known to be a disease that causes devastating damage to citrus. Citrus greening is caused by several phloem-localized bacteria, 'Candidatus Liberibacter spp.' (hereinafter referred to as 'Liberibacter bacteria'). Among them, 'Candidatus Liberibacter asiaticus (Las)' is the main pathogenic bacterium, mainly in Asia (Non-Patent Document 3). This pathogen is transmitted via the Asian citrus psyllid (Diaphorina citri). This disease causes great damage to citrus production around the world (Non-Patent Document 4). In Japan, cases of citrus greening disease have been confirmed on Tokunoshima, Okinoerabujima, and Yoronjima in Kagoshima Prefecture, as well as throughout Okinawa Prefecture, and there are concerns that the disease may invade major citrus producing areas.

[0003] Candidatus Liberibacter solanacearum (Lso) is known to cause zebra chip disease, which shows striped symptoms on potato tubers, and yellowing and shrinkage symptoms in plants of the Apiaceae family. Lso is transmitted by vectors such as the potato psyllid.

[0004] However, little is known about the biology and pathogenicity of these pathogenic bacteria, and there is also limited information about the biochemical responses and resistance of host plants to the disease. As a result, little progress has been made in breeding resistant varieties or developing control techniques such as therapeutic drugs.

[0005] Patent Document 1 discloses a medium capable of stably culturing difficult-to-culture Liberibacter bacteria. Patent Document 2 discloses a method for diagnosing infection of a plant by a pathogen, which includes a rooting step of rooting a part of a plant by inserting the cutting into a cutting bed and performing a cutting, and a first detection step of detecting the presence or absence of the pathogen using the rooted root, as a method for enabling early diagnosis of asymptomatic infected trees of citrus greening disease.

[0006] Citrus species that are hosts for Liberibacter bacteria are broadly divided into three types: cultivated varieties such as Unshu mandarin and Yuzu, non-cultivated varieties such as Acanthus nigra used as hedges, and wild species closely related to citrus. Infection of citrus with Liberibacter bacteria has been reported not only in cultivated varieties but also in Acanthus nigra plants of the Rutaceae family. On the other hand, it is empirically known that Liberibacter bacteria only infect Acanthus nigra only transiently and cannot survive in the Acanthus nigra for long periods of time. Furthermore, Acanthus nigra does not develop citrus greening disease. There have been no reports of Liberibacter bacteria in wild species closely related to citrus. Therefore, it is believed that non-cultivated varieties such as Acanthus nigra and wild species closely related to citrus have a high immune response to Liberibacter bacteria. Therefore, these species are considered to be highly useful as new materials for elucidating the physiological mechanism of infection of Liberibacter bacteria, breeding resistant varieties, and developing control techniques such as therapeutic drugs.

[0007] Infection of plants with Liberibacter bacteria can be achieved by artificial inoculation using the graft inoculation method and the inoculation method via the parasitic plant Cuscuta campestris, in addition to insect vector transmission by the Asian citrus psyllid. For example, Non-Patent Document 5 discloses a method for producing infected citrus by rearing Asian citrus psyllids carrying Liberibacter bacteria on healthy citrus. Non-Patent Document 6 discloses a method for producing infected citrus by grafting plant tissue infected with Liberibacter bacteria onto healthy citrus. Non-Patent Document 7 discloses a method for producing infected citrus by inoculating a citrus infected with Liberibacter bacteria and a healthy citrus via a dodder.

[0008] However, in all of the above-mentioned transmission methods, since live insects or plant tissues containing Liberibacter bacteria are used as inoculation sources, the density of Liberibacter bacteria varies for each carrier Asian citrus psyllid or infected citrus tested. The density of Liberibacter bacteria carried by Asian citrus psyllids varies from individual to individual. In infected citrus, Liberibacter bacteria are localized, so the density of bacteria varies from plant part to plant part. Therefore, it is impossible to reproduce infection with Liberibacter bacteria under conditions of a constant density of bacteria using conventional methods.

[0009] In citrus, the difference in the palatability of the Asian citrus psyllid makes it extremely difficult to inoculate wild relatives of citrus.Furthermore, due to differences in compatibility between citrus species, there are some combinations that cannot be inoculated by grafting, so at present it is technically not possible to artificially inoculate non-cultivated varieties and wild relatives of citrus with Liberibacter bacteria.

[0010] Even when Liberibacter bacteria are inoculated via dodder, the density of Liberibacter bacteria varies depending on the part of the infected citrus plant, making it impossible to reproduce infection with Liberibacter bacteria under conditions of a constant bacterial density. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2018-130058 [Patent Document 2] Japanese Patent Application Laid-Open No. 2017-163968 [Non-Patent Document]

[0012] [Non-Patent Document 1] Fujiwara et al., Front Microbiol, 2018, Vol.9, Article 3089 [Non-Patent Document 2] Li et al., J Microbial Methods, 2006, Vol.66, p.104-115 [Non-Patent Document 3] Bove JM, Journal of Plant Pathology, 2006, Vol.88, p.7-37 [Non-Patent Document 4] United States Department of Agriculture, (US), Citrus: World Markets and trade, July 2015 [Non-Patent Document 5] Ammar et al., PLoS ONE, 2016, Vol.11, e0159594 [Non-Patent Document 6] Lopes SA and Frare GF., Plant Disease, 2008, Vol.92, p.21-24 [Non-Patent Document 7] Garnier G and Dove JM., Phytopathology, 1983, Vol.73, p.1358-1363 [Non-Patent Document 8] Fujikawa et al., PlosOne, 2013, Vol.8, e57011 [Non-Patent Document 9] Wells et al., Applied and Environmental Microbiology, 1981, Vol.42, 357-363 Summary of the Invention [Problem to be solved by the invention]

[0013] As described above, in the conventional method, live insects or plant tissues containing Liberibacter bacteria are used as inoculum sources, and therefore the bacterial density varies, and it is not possible to reproduce the infection of plants with Liberibacter bacteria under a constant bacterial density condition. In addition, due to the differences in the palatability of the Asian citrus psyllid and the affinity between citrus species, it is difficult to artificially inoculate non-cultivated varieties and wild species closely related to citrus with Liberibacter bacteria in the conventional method.

[0014] Therefore, an object of the present invention is to provide a method by which non-cultivated varieties and closely related wild species can be stably infected with Liberibacter bacteria under certain bacterial density conditions. [Means for solving the problem]

[0015] As a result of intensive research to solve the above problems, the present inventors have focused on a method in which cultured cells of Liberibacter bacteria and crude sap from infected plants are used as inoculum sources. The present inventors have also focused on the property of Liberibacter bacteria preferentially accumulating in the roots at the early stage of infection. Thus, when Liberibacter bacteria were inoculated into the roots of plant seedlings, it was found that the Liberibacter bacteria could be stably infected into plants. Furthermore, it was found that this method also makes it possible to infect non-cultivated varieties such as Citrus orientalis and wild species closely related to Citrus with Liberibacter bacteria. Based on these findings, the present inventors have completed the present invention.

[0016] The present invention provides a method for infecting a plant with a bacterium of the genus Liberibacter, the method comprising contacting a bacteria-containing liquid containing a bacterium of the genus Liberibacter with a root part of the plant.

[0017] The present invention also provides the above-mentioned method, wherein the bacteria-containing liquid is a culture liquid of Liberibacter bacteria, a crude sap of a plant infected with Liberibacter bacteria, or a crude sap of a vector insect carrying Liberibacter bacteria.

[0018] The present invention also relates to the above-mentioned method, wherein the bacterial density of the bacteria-containing liquid is 1×10 0 ~1×10 8 cells / ml.

[0019] The present invention also provides the above method, wherein the plant is a plant of the Rutaceae, Solanaceae, or Apiaceae family.

[0020] The present invention also provides the above method, wherein the root portion is a root portion rooted by a cutting or a root portion of a seedling.

[0021] The present invention also provides the above method, wherein the bacteria-containing liquid is injected into the root portion using a syringe. Effect of the Invention

[0022] By using the method of the present invention, it is possible to stably infect non-cultivated varieties and wild species closely related to citrus with Liberibacter bacteria under certain bacterial density conditions. Therefore, although there has been no knowledge about the immune response of Citrus or wild species to Liberibacter bacteria, the use of the method of the present invention makes it possible to, for example, elucidate the infection physiological mechanism of Liberibacter bacteria, select resistant varieties, and develop varieties resistant to citrus greening disease. [Brief description of the drawings]

[0023] [Figure 1] FIG. 1 is a diagram for explaining the preparation of cuttings. [Diagram 2]Figure 2A shows the bacterial density of Liberibacter (greening disease bacteria) in leaves of inoculated rough lemons inoculated with a bacterial-containing liquid derived from diseased leaves with a bacterial density of 1 x 107 cells / ml, and Figure 2B shows the number of citrus greening disease strains among 20 inoculated rough lemon strains. [Diagram 3] Figure 3A shows the bacterial density of Liberibacter (greening disease bacteria) in leaves of inoculated rough lemons inoculated with a bacterial-containing liquid derived from diseased leaves at a bacterial density of 5 x 105 cells / ml, and Figure 3B shows the number of citrus greening diseased strains in five inoculated rough lemon strains. [Figure 4] Figure 4A shows the bacterial density of Liberibacter (greening disease bacteria) in leaves of inoculated rough lemons inoculated with a bacterial-containing liquid derived from diseased leaves at a bacterial density of 5 x 100 cells / ml, and Figure 4B shows the number of citrus greening diseased strains in five inoculated rough lemon strains. [Diagram 5] FIG. 5A shows the bacterial density of Liberibacter bacteria (greening disease bacteria) in leaves of inoculated rough lemons inoculated with a bacteria-containing liquid derived from cultured bacterial cells, and FIG. 5B shows the number of citrus greening diseased strains among 14 inoculated rough lemon strains. [Figure 6] FIG. 6A shows tomato seedlings, and FIG. 6B shows the presence or absence of detection of Liberibacter bacteria in four sown tomato plants. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present invention provides a method for infecting a plant with a bacterium of the genus Liberibacter. The method of the present invention comprises contacting a bacteria-containing liquid containing a bacterium of the genus Liberibacter with a root part of a plant.

[0025] In the present invention, examples of Liberibacter bacteria include Candidatus Liberibacter asiaticus (Las), Candidatus Liberibacter africanus (Candidatus Liberibacter americanus), Candidatus Liberibacter solanacearum (Lso), Candidatus Liberibacter europaeus, and Liberibacter crescens.

[0026] Candidatus Liberibacter asiaticus (Las), Candidatus Liberibacter africanus and Candidatus Liberibacter americanus are known as citrus greening pathogens. Candidatus Liberibacter solanacearum (Lso) is known as potato zebra chip pathogen. The method of the present invention can be used for the diagnosis and research of these plant diseases caused by Liberibacter bacteria.

[0027] In the present invention, the plant to be infected may be a plant of the genus Citrus (Citrus spp.) of the family Rutaceae that can be infected by bacteria of the genus Liberibacter. In the present invention, the plants to be infected include, for example, the genus Boronia (Boronia heterophylla, Boronia megastigma, Boronia pinnata Sm.), the genus Fortunella japonica (Citrus japonica), the genus Coleonema (Coleonema pulchrum), the genus Crowea (Crowea saligna Andrews, Crowea exalata F. Muell., Crowea angustifolia Sm.), the genus Melicope grisea var. crassifolia, the genus Murraya (Murraya paniculata Sm.), the genus Prunus persica (Prunus persica ... Plants of the Rutaceae family, including the genera Phellodendron amurense, Philotheca myoporoides, Poncirus trifoliata, Ruta graveolens, Skimmia japonica, Tetradium glabrifolium var. glaucum, and Zanthoxylum piperitum; plants of the Solanum lycopersicum L., Capsicum annuum L., Nicotiana benthamiana Domin), Datura metel L., Brugmansia suaveolens L.), Physalis alkekengi L. var. franchetii, Physaliastrum echinatum (Yatabe) Makino, Tubocapsicum anomalum (Franch. et Sav.) Makino, Petunia x hybrida, Scopolia japonica Maxim., Hyoscyamus niger L., Atoropa belladonna L., Mandrake Mandrake Mandrake officinarun L., Lycium plants of the Solanaceae family, such as Angelica pubescens Maxim. var. pubescens, Anthriscus sylvestris (L.) Hoffm., Bupleurum longiradiatum Turcz., Centella asiatica (L.) Urb., Chamaele decumbens (Thunb.) Makino, Cicuta virosa L., Cnidium japonicum L., and others; Miq.), Mitsuba (Cryptotaenia canadensis DC. subsp. japonica (Hassk.) Hand.-Mazz.), Cyclospermum leptophyllum (Pers.) Sprague), Daucus carota L.), Dystaenia ibukiensis (Y.Yabe) Kitag.)), Fennel (Foeniculum vulgare Mill.), Glehnia littoralis F.Schmidt ex Miq.), Heracleum sphondylium L. subsp. sphondylium var. nipponicum (Kitag.) H.Ohba), Libanotis coreana (H.Wolff) Kitag.), Oenanthe javanica (Blume) DC subsp. Javanica), Osmorhiza aristata (Thunb.) Rydb. var. aristata), Ostericum sieboldii (Miq.) The plant may be a plant of the Umbelliferae family, such as Peucedanum japonicum Thunb., Pleurospermum uralense Hoffm., Sanicula chinensis Bunge, Sium ninsi L., Spuriopimpinella calycina (Maxim.) Kitag., and Torilis japonica (Houtt.) DC. .

[0028] The bacteria-containing liquid can be, for example, a culture liquid of Liberibacter bacteria. Liberibacter bacteria are difficult to culture, but a culture liquid cultured in a medium described in Patent Document 1 can be used as the bacterial culture liquid. For example, Liberibacter bacteria can be cultured in a medium containing glucose and / or an oligosaccharide containing glucose, and ketoglutaric acid. The medium for culturing Liberibacter bacteria may further contain the following components: carbohydrates other than glucose and oligosaccharides containing glucose; at least one selected from the group consisting of CoA, CoA derivatives, glycerophosphate, and glycerol; at least one selected from the group consisting of valine, isoleucine, proline, ornithine, tyrosine, tryptophan, histidine, and cysteine; and antibiotics.

[0029] Examples of media for culturing Liberibacter bacteria include the medium described in Non-Patent Document 8 (Fujikawa et al., PlosOne, 2013, Vol. 8, e57011) and the BYCE medium described in Non-Patent Document 9 (Wells et al., Applied and Environmental Microbiology, 1981, Vol. 42, 357-363).

[0030] The bacteria-containing liquid may be, for example, crude sap from a plant infected with Liberibacter bacteria or crude sap from a vector insect carrying Liberibacter bacteria. The crude sap can be prepared, for example, by grinding leaves of a plant infected with Liberibacter bacteria or parts of a vector insect carrying Liberibacter bacteria in sterilized water using a homogenizer or the like, filtering the mixture through a filter to obtain a precipitate, and then adding sterilized water to resuspend the mixture.

[0031] The density of bacteria in the bacteria-containing liquid is not particularly limited, but may be, for example, 1×10 0 ~1×10 8The bacterial density can be measured in units of cells / ml. The bacterial density can be quantified by performing real-time PCR using Las931 / Lss primers according to the method described in Non-Patent Document 1 (Fujiwara et al., 2018. Front Microbiol 9:3089).

[0032] Insect vectors of Liberibacter bacteria include, for example, Diaphorina citri Kuwayama, Bactericera cockerelli, Trioza apicalis Forster, Bactericera trigonica Hodkinson, and Bactericera nigricornis.

[0033] The method of contacting the bacteria-containing liquid with the roots of a plant is not particularly limited, and for example, a method of injecting bacteria into the phloem of a plant can be used. For example, the bacteria-containing liquid may be injected into the roots of a plant using a syringe. In the method of injecting into the roots with a syringe, for example, the bacteria-containing liquid in the syringe can be injected into the phloem of the roots through a needle attached to the syringe.

[0034] The roots of the plant to be contacted with the bacteria-containing liquid may be roots that have been rooted by cuttings. For example, the roots may be roots that have been rooted by inserting a part of the plant into a cutting bed. The "part of the plant" refers to a so-called cutting for cutting, and may be any part that will take root when cutting is taken, such as the stem, branch, leaf, and root of the plant.

[0035] The method for rooting by cuttings is not particularly limited, and any conventionally known cutting method can be suitably used. For example, a part of a plant (a cutting) is first taken and then inserted into a cutting bed for rooting, so that the part of the plant can be rooted. A drug for promoting rooting (rooting promoter) may be used, and any conventionally known rooting promoter can be used as the rooting promoter.

[0036] The cutting bed may be any material that allows cuttings to root, such as potting soil, culture medium, mat material, water-retaining material, and water. Potting soil may be, for example, Akadama soil, Kanuma soil, peat moss, vermiculite, perlite, coco peat, etc. The cutting bed may be a sterile cutting bed. The sterile cutting bed may be, for example, sterilized soil (potting soil).

[0037] The roots of the plant to be contacted with the bacteria-containing liquid may be the roots of a plant layer or a seedling. A "layer" is a plant in which new roots are generated by artificially damaging a part of a branch or stem or burying it in soil. A "seedling" is a plant seedling grown from seeds. When inoculating the roots of a seedling, the bacterial liquid can be inoculated into the inner cells 0.5 mm to 5 mm deep inside the epidermis of the roots (ground edge) of a seedling 1 to 1.5 months after sowing.

[0038] Since the method of the present invention can perform artificial inoculation of Liberibacter bacteria, it can be used in both academic research activities such as elucidating the mechanism of plant disease caused by Liberibacter bacteria, and in the application field of developing new resistant varieties. The method of the present invention can be used not only in Japan, but also in countries that are seriously damaged by citrus greening disease, potato zebra chip disease, and carrot Lso disease caused by closely related Liberibacter bacteria. If resistant varieties are developed by the method of the present invention and it becomes possible to introduce resistant varieties in areas where citrus greening disease occurs, it is expected that it will be possible to prevent new outbreaks of infected trees and minimize the risk of infection spreading. EXAMPLES

[0039] Example 1 (Preparing cuttings) Healthy young branches and stems of rough lemons were collected to prepare cuttings including leaves. The cuttings were soaked in a diluted solution of Menedair (Menedair Co., Ltd.), a commercially available germination and rooting promoter, to prevent the cut surfaces from drying out.

[0040] Sandy soil (Kanuma soil) that had been preliminarily sterilized at high pressure (121°C, 15 minutes) was placed into a plastic pot.

[0041] The cuttings were coated with Luton (Sumitomo Horticultural Chemicals), a commercially available rooting agent, and then inserted into sandy soil (Kanuma soil). Aqueous Menedil solution was added to the Kanuma soil, and the underground parts of the cuttings were thoroughly soaked. Care was taken to prevent the Kanuma soil from losing moisture by adjusting the moisture level frequently and to avoid overhydration. The cuttings were also covered with a bag or similar to keep them moist (Figure 1A).

[0042] After one month, symptom-free cuttings (rooted seedlings) were gently removed from the Kanuma soil, and those that had rooted were used (Figure 1B).

[0043] (Preparation of bacteria-containing solution) An inoculum (bacteria-containing liquid) was prepared from infected leaves (disease-affected leaves) and cultured bacteria of Liberibacter genus bacteria. The method for preparing the inoculum was according to the method described in Non-Patent Document 1 (Fujiwara et al., 2018. Front Microbiol 9:3089).

[0044] Specifically, the midrib of a rough lemon leaf infected with Liberibacter asiaticus (Las) was cut out and ground in sterile water using a commercially available disposable homogenizer (Biomasher III (Nippon Gene)). The ground sample was placed in a filter with a column (Biomasher III accessory (Nippon Gene)) and centrifuged at 7000 rpm for 2 minutes. The supernatant was discarded, and the precipitate was resuspended in sterile water. Centrifugation and resuspension were repeated three times to obtain a bacteria-containing liquid. A portion of the bacteria-containing liquid was quantified by real-time PCR to examine the bacterial density. Specifically, real-time PCR was performed using the intercalator method using Las931 / Lss primers according to the method described in Non-Patent Document 1 (Fujiwara et al., 2018. Front Microbiol 9:3089). This bacteria-containing liquid was subjected to a centrifugation experiment using a Liberibacter genus bacteria density of 1 × 10 7 cells / ml, 5×10 5 cells / ml or 5 × 10 0 The concentration was adjusted to cells / ml and used as an inoculum source derived from diseased leaves.

[0045] In addition, the bacteria-containing liquid obtained from the diseased leaves was added to a liquid medium and cultured. The liquid medium was prepared according to the method described in Non-Patent Document 1 (Fujiwara et al., 2018. Front Microbiol 9:3089) and contained 0.5 g of peptone, 0.2 g of yeast extract, 0.1 g of glucose, 0.1 g of fructose, 0.1 g of sucrose, 0.1 g of soluble starch, 0.1 g of α-ketoglutaric acid, 0.5 ml of MEM essential amino acid solution (×50), 1 ml of MEM non-essential amino acid solution (×100), 0.1 g of glutamine, 0.1 g of methionine, 0.1 g of cysteine, 0.1 g of cystine, 0.5 g of sodium glycerophosphate, 0.1 g of coenzyme A, 1 ml of glycerol, and 1 ml of glycerol. 0.001 g of bromomethylene blue was dissolved in 100 mL of water, and the pH was adjusted to 7 with 10N aqueous sodium hydroxide solution. The solution was then sterilized in an autoclave (121°C, 15 minutes), and oxytetracycline was added to a final concentration of 1000 ppm. After one month, the culture solution was incubated at 37°C until the bacterial density of Liberibacter asiaticus (Las) reached 1×10 5 The cultured bacterial inoculum was diluted to 1000 cells / ml and used as an inoculum. The bacterial density was determined by quantifying a portion of the inoculum by real-time PCR.

[0046] (Plant inoculation) The above inoculum was filled into a syringe. 100 μl of the inoculum was inoculated into the rooting site of the prepared cutting seedling. The inoculated seedling was transplanted into high-pressure sterilized (121°C, 15 minutes) seedling soil (Kennae (Yae Nogei Co., Ltd.)) and cultivated.

[0047] (Inoculation using inoculum derived from diseased leaves) Bacterial density 1×10 7In the inoculation using the inoculum derived from diseased leaves adjusted to 1.5 cells / ml, 20 inoculated rough lemons were used as the target, and leaves of the inoculated rough lemons were collected every two months from 2 months after cultivation to 12 months after cultivation, and the bacterial density of Liberibacter bacteria was examined. The bacterial density of Liberibacter bacteria was measured by real-time PCR using the method described in Non-Patent Document 2 (Li et al., 2006. J Microbial Methods 66:104-115). Specifically, real-time PCR was performed by the Taqman probe method using HLBas / HLBr primers and HLBp probe.

[0048] Bacterial density 1×10 7 The bacterial density of Liberibacter bacteria (greening disease bacteria) in rough lemon leaves inoculated with bacteria-containing liquid derived from diseased leaves at 1000 cells / ml is shown in Figure 2A. The number of citrus greening disease strains in 20 inoculated rough lemon strains is shown in Figure 2B. As shown in Figure 2A, Liberibacter bacteria were detected in leaves two months after inoculation. Furthermore, as shown in Figure 2B, symptoms of citrus greening disease were observed in the inoculated rough lemons from 10 months after inoculation.

[0049] Bacterial density 5×10 5 cells / ml or 5×10 0 In the inoculation using the inoculum adjusted to each cell / ml, five strains of inoculated rough lemon were inoculated under each inoculation condition, and leaves of the inoculated rough lemon were collected every two months from two months to 16 months after cultivation, and the bacterial density of Liberibacter bacteria was examined. The bacterial density of Liberibacter bacteria was measured by real-time PCR using the method described in Non-Patent Document 2 (Li et al., 2006. J Microbial Methods 66:104-115). Specifically, real-time PCR was performed by the Taqman probe method using HLBas / HLBr primers and HLBp probe.

[0050] Bacterial density 5×10 5The bacterial density of Liberibacter (greening disease bacterium) in rough lemon leaves inoculated with a bacterial solution containing 5 × 10 cells / ml is shown in Figure 3A. 0 The bacterial density of Liberibacter (greening disease bacterium) in rough lemon leaves inoculated with a bacterial solution containing 5 × 10 cells / ml is shown in Figure 4A. The number of infected citrus greening strains in each of the five inoculated rough lemon strains is shown in Figures 3B and 4B. As shown in Figure 3A, the inoculation density was 5 × 10 5 When the inoculation density was 5 × 10 cells / ml, Liberibacter bacteria were detected in the leaves from 2 months after inoculation. As shown in Figure 3B, symptoms of citrus greening disease were observed in the inoculated rough lemon from 10 months after inoculation. As shown in Figure 4A, when the inoculation density was 5 × 10 0 When the number of cells / ml was measured, Liberibacter bacteria were detected in the leaves from 2 months after inoculation. In addition, as shown in Figure 4B, symptoms of citrus greening were observed in the inoculated rough lemon from 16 months after inoculation.

[0051] (Inoculation with inoculum derived from cultured bacteria) For inoculation using an inoculum derived from cultured bacteria, 14 strains of inoculated rough lemons were used. Leaves from the inoculated rough lemons were collected every two months from two months to 16 months after cultivation, and the density of Liberibacter bacteria was examined using the method described above.

[0052] The density of Liberibacter bacteria (greening disease bacteria) in the leaves of rough lemons inoculated with a bacteria-containing liquid derived from cultured bacteria is shown in Figure 5A. The number of diseased strains of citrus greening disease in 14 inoculated rough lemon strains is shown in Figure 5B. As shown in Figure 5A, Liberibacter bacteria were detected in the leaves 4 months after inoculation. As shown in Figure 5B, symptoms of citrus greening disease were observed in the inoculated rough lemons 14 months after inoculation.

[0053] These results demonstrated that Liberibacter bacteria can be infected into citrus plants by contacting a bacteria-containing liquid containing Liberibacter bacteria with the phloem of the roots of citrus plants.

[0054] Example 2 Tomatoes were inoculated using the inoculum derived from the diseased leaves in Example 1.

[0055] After sowing tomato seeds, four seedlings were obtained one month later (Figure 6A). The inoculum derived from the diseased leaves described above was inoculated into the soil edge of these seedlings in the same manner as in Example 1. Leaves were collected one month after inoculation, and Liberibacter asiaticus (Las) was detected. Specifically, PCR was performed using Las606 / LSS primers at an annealing temperature of 55°C according to the method described in Non-Patent Document 8 (Fujikawa et al., PlosOne, 2013, Vol.8, e57011), and agarose gel electrophoresis was performed. The results of the agarose gel electrophoresis are shown in Figure 6B. As shown in Figure 6B, Liberibacter bacteria were detected in the leaves of all four inoculated tomato plants.

[0056] These results demonstrated that Liberibacter bacteria can be infected into tomatoes by contacting a bacteria-containing liquid containing Liberibacter bacteria with the phloem of the roots of tomatoes. [Industrial Applicability]

[0057] The present invention can be utilized for elucidating the host and non-host recognition mechanism of Liberibacter bacteria, for selecting resistant varieties, and for developing varieties resistant to citrus greening disease.

Claims

1. A method for infecting a plant with a bacterium of the genus Liberibacter, comprising the steps of: contacting a bacteria-containing liquid containing the Liberibacter bacterium with a root part of the plant, The bacteria-containing liquid has a bacterial density of 1×10 0 to 1×10 8 cells / ml; The method, wherein the root portion is a root portion rooted by a cutting or a root portion of a seedling.

2. The method according to claim 1, wherein the bacteria-containing liquid is a culture liquid of the Liberibacter bacterium, a crude sap of a plant infected with the Liberibacter bacterium, or a crude sap of a vector insect carrying the Liberibacter bacterium.

3. The method according to claim 1 or 2, wherein the plant is a plant of the Rutaceae, Solanaceae or Apiaceae family.

4. The method according to any one of claims 1 to 3, wherein the bacteria-containing liquid is injected into the roots using a syringe.

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