EP1765061A2

EP1765061A2 - Citrus sudden death

Info

Publication number: EP1765061A2
Application number: EP05763870A
Authority: EP
Inventors: Todd W. Gusek; Richard F. Lee; Manjunath L. Keremane; Eyal Aharon
Original assignee: Cargill Inc
Current assignee: Cargill Inc
Priority date: 2004-06-29
Filing date: 2005-06-28
Publication date: 2007-03-28
Also published as: IL180320A0; WO2006004634A2; BRPI0512586A; WO2006004634A3

Abstract

A pathogenic bacterium has been identified which is associated with Sudden Death disease. The bacterium can be used to generate antibodies for immunological tests. Antibacterial agents can be used to treat infected trees or to keep uninfected trees from becoming infected. The environment of an infected tree, including soil and air, can also be treated to prevent or cure infections and/or spread of infections. Antibodies can be raised agains the pathogenic bacterium and can be used for identifying the pathogenic bacterium in soil and tree samples.

Description

CITRUS SUDDEN DEATH

[0001] This application claims the benefit of U.S. provisional applications Serial Number 60/600,128 filed August 10, 2004, and Serial Number 60/583,589 filed June 29, 2004.

FIELD OF THE INVENTION

[0002] The present invention generally relates to Citrus Sudden Death (CSD) and the causative organism responsible for it. The present invention relates to methods for treating CSD infected trees and the reagents for doing so. The present invention also relates to diagnosis of CSD in infected trees. In addition, it relates to prophylactic treatment to protect trees from CSD. Moreover, it relates to treatments for blocking the spread of CSD. The present invention also relates to CSD-infected or CSD- susceptible trees or rootstocks, or inarch or a combination of both of CSD-infected or CSD-susceptible citrus tree with an agent, which is a bactericide or a bacterial control agent or an agent for enhancing resistance to CSD infection or symptoms.

BACKGROUND OF THE INVENTION

[0003] A disease that causes rapid decline in citrus trees on Rangpur lime and Volkamer lemon (Citrus volkameriana) rootstock was first observed in the state of Minas Gerais, Brazil in or about 1999. The disease was named Citrus Sudden Death (CSD). The symptoms of CSD are typically characterized according to the following progression: (a) the loss of small tertiary roots (before most other symptoms become visible); (b) black spots on secondary roots (see FIG. 1); (c) the infected tree is "stunted" or shorter compared to uninfected trees; (d) the interior portion of the bark of infected trees is yellow (CSD was originally called "yellow bark disease") (see FIG. 2); (e) reduced new growth or very little new flushes are seen; (f) the infected tree defoliates (see FIG. 2); and (g) complete defoliation and death of the tree, possibly with fruit left hanging on the branches.

[0004] CSD has already spread south into Sao Paulo state since the first confirmed finding in Minas Gerais state. Approximately 3 million trees may have been killed by CSD, and CSD has the potential to inflict great economic hardship on the citrus industry. As many as about 85% of the approximately 150 to 200 million trees planted in the state of Sao Paulo may be at risk.

[0005] According to one theory, CSD is caused by a mutant of the Citrus Tristeza Virus ("CTV") and is spread by an aphid. According to another theory, CSD is caused by a CSD associated virus (CSDaV) belonging to the Tymoviridae family. No cure has been found for CSD up until now.

[0006] Thus there is a need in the art for methods to prophylactically and therapeutically treat trees at risk of CSD infection and CSD-infected citrus trees, respectively. There also is a need for a method to identify and diagnose citrus trees infected by CSD causative organisms.

SUMMARY OF THE INVENTION

[0007] A method is provided for treating a CSD-infected citrus tree with an agent. The method comprises administering a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection. An additional but optional step of the method involves the harvesting of fruit from the tree. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree.

[0008] A CSD-infected citrus tree is provided which comprises an agent. The agent is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection.

[0009] A rootstock or an inarch of a CSD-infected citrus tree or a combination of both are provided. They comprise an agent, which is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection. [0010] Also provided by the present invention are a Rangpur lime rootstock or a Volkamer lemon (Citrus volkameriana) rootstock of CSD-infected citrus tree. The rootstock comprises an agent which is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection.

[0011] A method is provided for protecting a citrus tree against CSD infection. An agent is administered to the citrus tree. The agent is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection. An additional but optional step of the method involves the harvesting of fruit from the tree. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree.

[0012] Also provided is a citrus tree which comprises an agent. The agent is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection

[0013] A rootstock or inarch of a citrus tree or a combination of both is provided. The rootstock or inarch comprises an agent which is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection.

[0014] A Rangpur lime rootstock or a Volkamer lemon (Citrus volkameriana) rootstock is provided in combination with an agent which is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection.

[0015] Moreover, with regard to the method described in paragraph 11 the citrus tree may be located within 200 kilometers from a CSD-infected citrus tree.

[0016] Also provided by the present invention is a method for blocking the spread of CSD to other non-infected trees. At least one CSD-infected tree is treated with an agent, which is an insecticide for control of an insect vector that spreads CSD {e.g., insect, beetle, weevil, aphid, etc.). An additional but optional step of the method involves the harvesting of fruit from the tree. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree.

[0017] Another method for blocking the spread of CSD from one tree to another is provided by the present invention. The soil within at least 100 meters of a CSD- infected tree is treated with an agent. The agent is an insecticide for control of an insect vector that spreads CSD. An additional but optional step of the method involves the harvesting of fruit from the tree(s) growing in the treated soil. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree(s) growing in the treated soil.

[0018] Another method for blocking the spread of CSD is provided by the present invention. The air within at least 10 kilometers of a CSD-infected tree is treated with an agent. The agent is an insecticide for control of an insect vector that spreads CSD. An additional but optional step of the method involves the harvesting of fruit from the tree(s) growing in the treated region. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree(s) growing in the treated region.

[0019] The method described in paragraphs 7 and 11 may be practiced using an agent useful to control bacteria selected from a group consisting of Pseudomonadaceae, Xanthomonadaceae, Enterobacteriaceae, and Microbacteriaceae pathogens.

[0020] The method described in paragraphs 7 and 11 may be practiced using an agent useful to control bacteria selected from a group consisting of Pseudomonas, Xanthomonas, Erwinia, Pantoea and Clavibacter pathogens.

[0021] The method described in paragraphs 7 and 11 may be practiced using an agent useful to control bacteria selected from a group consisting of Burkholderia, Acidovorax and Ralstonia pathogens. [0022] The method described in paragraphs 7 and 11 may be practiced using one or more of: an antibiotic, a metallic element with an atomic number of 21 through and including 82, salts thereof, and organic or inorganic complexes thereof.

[0023] The method described in paragraph 22 may employ a metallic element that is selected from the group consisting of copper, zinc, and combinations thereof.

[0024] The method described in paragraphs 7 and 11 may be practiced using as the agent a protein which enhances a tree's resistance to bacterial infection.

[0025] The method described in paragraphs 7 and 11 may be practiced using as the agent an antibiotic, an antibiotic-producing microorganism, a metabolite of such microorganism, or an extract of such microorganism.

[0026] The method described in paragraph 25 may be practiced using an antibiotic selected from the group consisting of oxytetracycline, rifampicin, cefoxitin, and cefotaxime.

[0027] The method described in paragraph 25 may be practiced by administering the agent at least once during the crop cycle.

[0028] The method described in paragraphs 7 and 11 may be practiced by administering the agent by a means selected from a group consisting of spraying, foliar spraying, injection, trunk injection, root injection, root soaking, and combinations thereof.

[0029] The method described in paragraph 22 may be practiced using a slow-release form of the metallic element with an atomic number of 21 through and including 82, salts thereof, and organic or inorganic complexes thereof.

[0030] The method described in paragraph 22 may be practiced by administering the metallic element with an atomic number of 21 through and including 82, salts thereof, and organic or inorganic complexes thereof in a nutrient solution or suspension. [0031] The method described in paragraph 30 may be practiced by applying the nutrient solution or suspension to a foliar region of the citrus tree.

[0032] The method described in paragraph 30 may be practiced by applying the nutrient solution or suspension to the rootstock region of the citrus tree.

[0033] The method described in paragraph 22 may be practiced by applying the metallic element with an atomic number of 21 through and including 82, salts thereof, or organic or inorganic complexes thereof, in an about 1-30% solution or suspension by weight.

[0034] The method described in paragraphs 7 and 11 may comprise further steps. For example, before or after administering an agent to the tree "inarching" of the tree can be performed. (Inarching, commonly referred to as "grafting by approach," is grafting by uniting, as a scion to a stock, without separating either from its root before the union is complete).

[0035] The method described in paragraph 22 may comprise further steps. Before or after application of the antibiotic, metallic element, its organic or inorganic complex, or salts, or combinations thereof, the citrus tree can be pruned.

[0036] The method described in paragraphs 7 and 11 may be performed on a citrus tree comprising a Rangpur lime or a Volkamer lemon (Citrus volkameriana) rootstock.

[0037] A method for detection of CSD-infected citrus trees is provided. A method is employed selected from a group consisting of immunoblot, ELISA, PCR, nucleic acid hybridization or other means of pathogen detection. An antibody specific for a bacterium denominated RT 120, which has been deposited in a public depository, or a nucleic acid primer or probe specific for RT120 is used.

[0038] The method described in paragraph 30 can also be performed on non-CSD- infected citrus trees to provide a prophylactic effect. [0039] A method for treating a CSD-infected citrus tree is provided. A copper containing compound is administered to the tree to control growth or kill pathogenic bacteria. An additional but optional step of the method involves the harvesting of fruit from the tree. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree.

[0040] A method for treating a CSD-uninfected citrus tree is provided. A copper containing compound is administered to the tree to prevent infection by bacteria. An additional but optional step of the method involves the harvesting of fruit from the tree. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree.

[0041] A method of inhibiting CSD in a tree in need thereof is provided by the invention. An inhibitory amount of a bacterial protein that enhances resistance to CSD is administered to the tree. An additional but optional step of the method involves the harvesting of fruit from the tree. Yet another additional but optional step of the method involves obtaining juice from the fruit from the tree.

[0042] A microbiologically pure culture is provided of bacterial strain RT 120, which has been deposited at the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209 USA on July 19, 2004, and assigned the deposit designation PTA-6121.

[0043] An isolated antibody is provided. The antibody is useful for identifying CSD in infected citrus trees. The antibody specifically binds to one or more antigens on the surface of RT120.

[0044] A method is provided for screening for substances useful for treating or preventing CSD. A test substance is contacted with RT120 bacteria. Growth rate or viability of the bacteria is determined after the contacting. A test substance which diminishes the growth rate or decreases the viability is identified as a candidate agent for treating or preventing CSD. [0045] A method is also provided for identifying a CSD-infected tree or plant. A sample of bacteria is isolated from a citrus tree. The sample is applied to leaf tissue of a plant such as geranium, tobacco and certain resistant citrus varieties, such as Cleopatra mandarin, Swingle, and Sunki. If necrotic tissue develops within 1-4 days, the test tree or plant is identified as potentially CSD-infected.

[0046] Another method provides a means of identifying a CSD-resistant tree or plant. A sample of RT120 bacteria is applied to leaf tissue of the tree or plant being tested. If necrotic tissue develops within 1-4 days, the tree or plant is identified as CSD-resistant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Fig. 1 shows CSD affected roots, including black spots and loss of small, tertiary roots.

[0048] Figs. 2A-2C compare a healthy part to a CSD-affected part of a tree. The mterior portion of the bark of infected trees is yellow (Fig. 2C) whereas no yellow bark is observed on the trunk of the healthy part of the tree (Fig. 2B) and the infected portion of the tree defoliates (Fig.2A).

[0049] Fig. 3 shows that RT 120 causes hypersensitivity reaction (HR) on tobacco and geranium.

[0050] Fig. 4A-4D show that RT120 causes hypersensitivity reaction (HR) on citron (Fig. 4A) and Cleopatra mandarin (Fig. 4C), while RTl 10 does not cause HR on either citron (Fig. 4B) or Cleopatra (Fig. 4D).

[0051] Fig. 5A-5C compare the HR reaction on Rangpur lime (Fig. 5A), citrumelo (Fig. 5B), and sweet orange (Fig. 5C). No HR reaction was observable at this time point. [0052] Fig. 6A-6D show antibody blots using antibodies to DRIZl (Erwinia caratovora; Fig. 6B), DRIZ3 {Enterobacter hormaechei; Fig. 6C), and RT120 (Pseudomonas cepacia; Fig. 6D). Top row of each blot contains the reference bacteria, with other rows containing different test bacteria. The layout of the antibodies is shown in Fig. 6A.

[0053] Fig. 7A-7D show inoculation of plants in greenhouse by injuring the stem (Fig. 7A) and roots (Fig. 7D) and adding bacterial culture (Fig. 7B-D).

DETAILED DESCRIPTION OF THE INVENTION

[0054] Without intending to be limited to any particularly theory, it is presently believed that Citrus Sudden Death (CSD) disease is caused at least in part by a plant pathogenic bacterium. Without intending to be limited to any particularly theory, it is also presently believed that the bacterium (referred to in this disclosure as "RT120") is Pseudomonas cepacia (e.g., by fatty acid analysis) or Burfcholderia/ Acidovorax/ Ralstonia (e.g., by biochemical tests such as the diagnostic assays included in the API 2OE kit available from BioMerieux). Its 16S rRNA most closely resembles Burkholderia cepacia. RT120 is resistant to antibiotics such as carbenicillin, penicillin and streptomycin. RT120 is sensitive to antibiotics such as oxytetracycline, rifampicin, cefoxitin and cefotaxime.

[0055] The presence of RT 120 in a citrus tree may be identified by several methods. Such methods include: (a) isolation on media with certain antibiotics, such as carbenicillin, penicillin, or streptomycin; (b) hypersensitivity reaction ("HR") on geranium, tobacco and citrus varieties; and (c) antibody tests. The presence of RT120 in a citrus tree may be identified by isolation on media with antibiotics according to the following method: a tree root is cut and the exterior of the root is sterilized to remove extraneous microorganisms. The root is chopped to expose its interior and the pieces suspended in sterile water. Bacteria that are present in the root are thereby suspended in the water. The bacterial suspension is plated onto microbiological media that allows for the growth of bacteria (such as nutrient agar or orange serum agar) into discrete colonies. Inclusion of any of the antibiotics to which RT 120 is resistant will allow it to outgrow other bacteria that are sensitive to the same antibiotic or combination of antibiotics.

[0056] Table 1 shows examples of antibiotics that can be used to help in the isolation and identification of the bacterial pathogen that causes CSD.

Table 1.

[0057] The presence of RT 120 in a citrus tree may alternatively be identified by the hypersensitivity reaction ("HR") on geranium, tobacco and citrus varieties according to the following method. (See FIGS. 3 through 5.) Less than about 1 ml (e.g., about 0.2 ml) of live RT120 culture suspended in water at a concentration of approximately 0.1 absorbance units (turbidometric units) at 660 nm is injected with a blunted syringe through the epidermis or skin and into the middle lamellae of a leaf of a geranium, tobacco or citrus plant (e.g., Cleopatra mandarin, Citron, Swingle, Rangpur lime, Sunki, Volkamer lemon). Multiple injections may be made between the veins of the leaf and spaced about 1 cm apart from an adjacent injection. An HR response is evident by the formation of necrotic tissue in approximately 1 to 4 days following the injection of the geranium and tobacco and any citrus plant which is able to recognize RT120 as a pathogen.

[0058] According to another alternative embodiment, the presence of RT120 in a citrus tree may be identified by antibody tests according to the following method (see FIG. 6): Live culture of RT120 is washed two times in physiological buffer saline (PBS), re-suspended in PBS, boiled for 5 minutes, diluted to a concentration of 10⁷ cells per ml, and mixed with an equal volume of complete adjuvant. A rabbit is injected with the resulting mixture at 10-12 intradermal sites, using approximately 10 microliters at each injection site. The same rabbit is also injected at a single intramuscular site using a larger dosage (e.g., 800 microliters of the same mixture). The preceding protocol of injections is repeated at weekly intervals for a total of four times. The rabbit is also bled at weekly intervals to recover approximately 20 ml serum from each bleeding. The serum contains antibodies to RT120. The antibodies can be diluted approximately 50,000-fold and used to analyze plant tissue (e.g., citrus) infected with RT 120. Detection of RT 120 can employ an immunoblot procedure in which live twigs or roots are collected and the sap is deposited onto a membrane (e.g., nitrocellulose). Antibodies to RT120 are applied and excess antibody washed away. The presence of RT 120 in the sap is determined by application of a second antibody conjugated to alkaline phosphatase. The second antibody is reactive with the first antibodies. A chemical reaction catalyzed by alkaline phosphatase using two substrates BCIP and NTB yields a detectable product.

[0059] Bacteria which cause CSD may also be identified by comparison of 16S rRNA to that of RT120. The sequence of RT120's 16S rRNA gene has been determined and is provided as SEQ ID NO: 1. Bacteria whose 16S rRNA gene is highly similar to that of SEQ ID NO: 1 can be identified as potential disease causative organisms. Highly similar sequences are those which are at least about 95 % identical. Percent sequence identity between the nucleotide sequence of SEQ ID NO:1 and a putative related 16S rRNA nucleotide sequence is determined using computer programs which employ the Smith- Waterman algorithm, as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with the following parameters: a gap open penalty of 12, and a gap extension penalty of 1.

[0060] A CSD-infected tree may be treated to "cure" or inhibit the disease. Alternatively, a CSD-uninfected tree (or a tree within a predetermined proximity to a CSD-infected tree) may be treated to "prevent" or resist the disease.

[0061] The CSD-infected tree (or CSD-uninfected tree) is treated with an amount of agent effective for inhibiting (or preventing) CSD. If the intended treatment is to stimulate resistance, the effective amount of bacterial protein that should be administered to inhibit or prevent CSD is about 0.1 to 2 grams per tree, more suitably about 0.5 to 1 grams per tree. If the treatment comprises administering a metallic element, for example copper or zinc, alone or in the form of a salt or slow-release complex, the effective amount of metal to inhibit or prevent or recover CSD is about 4 to 20 grams per tree, more suitably about 8-14 grams per tree. If the treatment is an antibiotic, the effective amount of antibiotic to inhibit or prevent or recover from CSD is about 0.5 to 4 grams per tree, more suitably about 1 to 2 grams per tree. Preferably, the above agents are first dissolved or suspended in water and then applied to the tree.

[0062] According to an exemplary embodiment, CSD-infected citrus tree is treated with at least one agent which is a bactericide or a bacterial growth control agent (bacteriostatic) or an agent which enhances a tree's resistance to bacterial infection.

[0063] According to an exemplary embodiment, a citrus tree is protected against CSD infection by applying an agent which is a bactericide or a bacterial growth control agent or an agent which enhances a tree's resistance to bacterial infection.

[0064] According to an embodiment, the protected citrus tree is located within about 200 kilometers from a CSD-infected citrus tree.

[0065] According to another embodiment, the protected tree is located within about 100 kilometers from a CSD-infected citrus tree.

[0066] According to an exemplary embodiment, the spread of CSD is blocked by treating at least one infected tree or its adjacent soil with an agent, which is an insecticide for control of a vector that spreads CSD. The vector may be airborne, water borne, soil borne (Le., carried by or through the soil), ground borne, etc. The vector may be carried by equipment used in maintaining citrus trees such as tractors, sprayers, harvesting and pruning equipment. The agent can be, for example, administered by fumigation or into the soil as pellets.

[0067] According to a preferred embodiment, the agent is selected from a group consisting of agents found useful to control bacteria selected from the group consisting of Pseudomonadaceae, Xanthomonadaceae, Enterobacteriaceae, and Microbacteriaceae pathogens.

[0068] According to another preferred embodiment, the agent is selected from the group consisting of agents found useful to control bacteria selected from a group consisting of Pseudomonas, Burkholderia, Acidovorax, Ralstonia, Xanthomonas, Erwinia, Pantoea and Clavibacter pathogens.

[0069] According to a preferred embodiment, the agent is selected from the group consisting of metallic elements with atomic number 21 through and including 82, and their organic and inorganic complexes, salts and combinations thereof.

[0070] According to a particularly preferred embodiment, the agent is selected from a group consisting copper, zinc, organic and inorganic compounds of copper and/or zinc, salts, complexes, and combinations thereof.

[0071] According to a preferred embodiment, metallic elements with atomic number 21 through and including 82, their organic and inorganic compounds, salts, complexes or combinations of those is applied in a slow-release form. An example of a slow- release form is Tech-Flo Nutricop-20, which can be used as such or after dilution.

[0072] According to a preferred embodiment, nutrient is also applied, preferably in a solubilized or suspended form.

[0073] According to a preferred embodiment, the agent is applied in a nutrient solution or suspension. Examples are Tech-Flo Copocal products containing phosphorous and sulfur compounds in addition to copper such as those commercially available from Nutrient Technologies, Dinuba CA: Alpha and Beta containing phosphorous compounds in addition to zinc, MN-15 containing phosphorous compounds in addition to manganese, PHI containing phosphorous and nitrogen compounds in addition to iron, ZMC containing phosphorous and potassium compounds in addition to copper and manganese and zinc, and Sigma containing phosphorous, potassium and nitrogen compounds in addition to zinc. [0074] According to a preferred embodiment, the nutrient solution or suspension is applied to the foliar region of the citrus tree.

[0075] According to another preferred embodiment, the nutrient solution or suspension is applied to the rootstock region of the citrus tree.

[0076] According to a preferred embodiment, the metallic element, its organic and inorganic complexes, salts, or a combination of those is applied in a solution or suspension containing it at a concentration of between about 1% and 30% by weight. Examples are Tech-Flo Copocal products Alpha and Sigma containing 5% zinc each, Zeta containing 22% zinc, Beta containing 6.5% zinc and 6.5% manganese, ZM 13.5- 7 containing 13% zinc and 5% manganese, Copocal containing 5% copper, MN- 15 containing 15% manganese, Nutricop containing 20% copper, PHI containing 5% iron, ZMC containing 5% zinc and 5% manganese and 2.5% copper, their diluted solutions and their mixtures.

[0077] According to another preferred embodiment, the agent which enhances resistance to infection is a protein or a polypeptide. Preferably, the protein or polypeptide elicits a hypersensitivity response in plants as described, for example, in U.S. Patents 5,849,868, 6,174,717 and 6,228,644. The protein or a polypeptide, e.g., HARPIN and MESSENGER™ products of Eden Bioscience, initiates a set of metabolic responses in the treated citrus tree, causing gene expression and eliciting the tree's natural defense against bacteria.

[0078] According to a preferred embodiment, the agent is an antibiotic. A method of treating a tree may involve applying an effective amount of antibiotic, a combination of antibiotics, an antibiotic-producing strain, a metabolite of such strain or an extract thereof. The effective amount of antibiotic may be about 0.5 to 4 grams per tree according to any preferred or alternative embodiments.

[0079] According to a particularly preferred embodiment, the antibiotic is selected from a group consisting of oxytetracycline, rifampicin, cefoxitin and cefotaxime. [0080] According to a preferred embodiment, the agent is applied at least once, twice, or three times during the crop cycle.

[0081] According to a preferred embodiment, the treatment is by a means selected from a group consisting of spraying, foliar spraying, injection, trunk injection, root injection, root soaking and other means of root uptake and combinations thereof.

[0082] According to a preferred embodiment, the tree may be pruned or cut either before or after application of the agent.

[0083] According to a particularly preferred embodiment, the method comprises both pruning and inarching.

[0084] According to an exemplary embodiment, CSD infected citrus trees are detected or identified by means such as immunoblot, ELISA, PCR, nucleic acid hybridization or other means of pathogen detection.

[0085] According to an exemplary embodiment, a CSD-infected citrus tree is treated with a metallic element with an atomic number 21 through and including 82, or its organic and inorganic salts and complexes.

[0086] According to another exemplary embodiment, a CSD infected citrus tree is treated with a copper compound.

EXAMPLES

[0087] While the invention will now be described in connection with certain embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, the examples are not intended to limit the invention to these particular examples.

Example 1

[0088] Citrus plants in a greenhouse were inoculated by injuring the stem (i.e. pricking) and adding a live culture of RT 120. The stem of the plant was also cut. Flexible tubing was placed around the cut stem. The culture in an aqueous suspension was provided into the tubing and onto the freshly cut stem with a pipette for introduction into the open wound. The concentration of the culture was approximately 0.1 absorbance units at 660 nm (e.g., about 1 X 10⁸ cells of RT120/ml). It is expected that root decline will be observed and that symptoms of CSD will be observed. (See FIG. 7.)

Example 2

[0089] Citrus plants in a greenhouse were inoculated by injuring the stem and adding a live culture of RT120. The stem of the plant was pricked or pierced with a pin in the vicinity of a droplet of live suspended culture, to provide a point of entry for RT120. A root of the plant was cut and flexible tubing was placed around the cut root. The culture in an aqueous suspension was provided into the tubing and onto the root with a pipette for introduction into the open wound. The concentration of the culture was approximately 0.1 absorbance units at 660 nm (e.g. about I X lO⁸ cells of RT120/ml). It is expected that root decline will be observed and that symptoms of CSD will be observed. (See FIG. 7.)

Example 3

[0090] We inoculated citrus trees that are at risk of Citrus Sudden Death disease with RT 120 under controlled (greenhouse) conditions. Our RT 120 microorganism survived and effectively colonized the roots of the trees. Survival and colonization of roots are requirements for pathogenesis. So far, we have observed root decline as a symptom of disease in these trees. Other field symptoms of CSD have not yet been observed, although we believe that this is because they are not yet of fruit-bearing age. Flowering and fruiting pose huge physiological^" stresses^' on a weakened tree and accelerate its death.

[0091] We performed the same inoculation using CSD-resistant rootstocks. The RT 120 bacteria did not survive. We infer that the rootstocks of the resistant plants have a mechanism to recognize and contain/eradicate the RT120 infection. [0092] While the preferred and other exemplary embodiments described in this disclosure are presently preferred, it should be understood that these embodiments are offered by way of example only. The invention is not limited to a particular embodiment, but extends to various modifications, combinations, and permutations.

Claims

We claim:

1. A method for treating a Citrus Sudden Death (CSD)-infected citrus tree, comprising:

administering an agent to a CSD-infected citrus tree, wherein the agent is selected from the group consisting of bactericidal, bacteriostatic, or bacterial infection resistance enhancing.

2. The method of claim 1 further comprising:

harvesting fruit from the tree.

3. The method of claim 2 further comprising:

extracting juice from the fruit from the tree.

4. The method of claim 1 wherein the tree comprises Rangpur lime rootstock.

5. The method of claim 1 wherein the tree comprises Volkamer lemon rootstock.

6. The method of claim 1 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonadaceae, Xanthomonadaceae, Enterobacteriaceae, and Microbacteriaceae pathogens.

7. The method of claim 1 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonas, Xanthomonas, Erwinia, Pantoea and Clavibacter pathogens.

8. The method of claim 1 wherein the agent is effective against bacteria selected from a group consisting of Burkholderia, Acidovorax and Ralstonia pathogens.

9. A citrus tree which comprises an agent wherein the agent is selected from the group consisting of bactericidal, bacteriostatic, or bacterial infection resistance enhancing.

10. The citrus tree of claim 9 which is CSD-infected.

11. The method of claim 9 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonadaceae, Xanthomonadaceae, Enterobacteriaceae, and Microbacteriaceae pathogens.

12. The method of claim 9 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonas, Xanthomonas, Erwinia, Pantoea and Clavibacter pathogens.

13. The method of claim 9 wherein the agent is effective against bacteria selected from a group consisting of Burkholderia, Acidovorax and Ralstonia pathogens.

14. A plant product which is a rootstock or an march of a citrus tree or a combination thereof which comprises an agent wherein the agent is selected from the group consisting of bactericidal, bacteriostatic, or bacterial infection resistance enhancing.

15. The plant product of claim 14 which is CSD-infected.

16. The plant product of claim 14 which is free of CSD-infection.

17. The plant product of claim 15 which is a rootstock.

18. The plant product of claim 16 which is a rootstock.

19. The plant product of claim 17 wherein the rootstock is a Rangpur lime rootstock.

20. The plant product of claim 18 wherein the rootstock is a Rangpur lime rootstock.

21. The plant product of claim 17 wherein the rootstock is a Volkamer lemon (Citrus volkameriana) rootstock.

22. The plant product of claim 18 wherein the rootstock is a Volkamer lemon (Citrus volkameriana) rootstock.

23. The plant of claim 14 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonadaceae, Xanthomonadaceae, Enterobacteriaceae, and Microbacteriaceae pathogens.

24. The method of claim 14 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonas, Xanthomonas, Erwinia, Pantoea and Clavibacter pathogens.

25. The method of claim 14 wherein the agent is effective against bacteria selected from a group consisting of Burkholderia, Acidovorax and Ralstonia pathogens.

26. A method for protecting a citrus tree from CSD infection, comprising:

administering an agent to the citrus tree, wherein the agent is selected from the group consisting of bactericidal, bacteriostatic, or bacterial infection resistance enhancing.

27. The method of claim 26 further comprising:

harvesting fruit from the tree.

28. The method of claim 27 further comprising:

extracting juice from the fruit from the tree.

29. The method of claim 26 wherein the citrus tree is located within 200 kilometers from a CSD-infected citrus tree.

30. The method of claim 26 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonadaceae, Xanthomonadaceae, Enterobacteriaceae, and Microbacteriaceae pathogens.

31. The method of claim 26 wherein the agent is effective against bacteria selected from a group consisting of Pseudomonas, Xanthomonas, Erwinia, Pantoea and Clavibacter pathogens.

32. The method of claim 26 wherein the agent is effective against bacteria selected from a group consisting of Burkholderia, Acidovorax and Ralstonia pathogens.

33. A method for blocking the spread of CSD from an infected tree to one or more non-infected trees, comprising;

treating at least one CSD-infected tree with an insecticide, wherein the insecticide inhibits growth and/or reproduction of an insect that spreads CSD.

34. The method of claim 33 further comprising:

harvesting fruit from the tree.

35. The method of claim 34 further comprising:

extracting juice from the fruit from the tree.

36. A method for blocking the spread of CSD from an infected tree to one or more non-infected trees, comprising;

treating soil within 100 meters of a CSD-infected tree with an insecticide, wherein the insecticide inhibits growth and/or reproduction of an insect that spreads CSD.

37. The method of claim 36 further comprising:

harvesting fruit from trees in the treated soil.

38. The method of claim 37 further comprising:

extracting juice from the fruit from the trees.

39. A method for blocking the spread of CSD from an infected tree to one or more non-infected trees, comprising;

aerially delivering an insecticide to a region within 10 kilometers of a CSD-infected tree, wherein the insecticide inhibits growth and/or reproduction of an insect that spreads CSD.

40. The method of claim 39 further comprising:

harvesting fruit from trees within the region.

41. The method of claim 40 further comprising:

extracting juice from the fruit from the trees.

42. The method of claim 1 or 26 wherein the agent is selected from the group consisting of: an antibiotic, a metallic element with an atomic number of 21 through and including 82, salts thereof, organic or inorganic complexes thereof, and combinations thereof.

43. The method of claim 1 or 26 wherein the agent comprises a metallic element selected from the group consisting of copper, zinc, organic and inorganic complexes, salts, and combinations thereof.

44. The method of claim 1 or 26 wherein the agent is a protein which enhances a tree's resistance to bacterial infection.

45. The method of claim 1 or 26 wherein the agent is selected from the group consisting of an antibiotic, an antibiotic-producing microorganism, a metabolite of such microorganism, and an extract of such microorganism.

46. The method of claim 1 or 26 wherein the agent is an antibiotic selected from the group consisting of oxytetracycline, rifampicin, cefoxitin, and cefotaxime.

47. The method of claim 33 wherein the treatment is administered by a means selected from a group consisting of spraying, foliar spraying, injection, trunk injection, root injection, root soaking, and combinations thereof.

48. The method of claim 43 wherein the agent is a slow-release form of the metallic element, its organic or inorganic complexes, salts, or combinations thereof.

49. The method of claim 43 wherein the metallic element, its organic or inorganic complexes, salts, or combinations thereof is administered in a nutrient solution or suspension.

50. The method of claim 49 wherein the nutrient solution or suspension is administered to a foliar region of the citrus tree.

51. The method 49 wherein the nutrient solution or suspension is administered to the rootstock region of the citrus tree.

52. The method of claim 42 wherein a metallic element, its organic or inorganic complexes, salts, or combinations thereof is administered and the metallic element is in an about 1-30 % solution or suspension by weight.

53. The method of claim 1 or 26 further comprising the step of inarching the citrus tree, either before or after the step of administering.

54. The method of claim 1 or 26 further comprising the step of pruning the citrus tree, either before or after the step of administering.

55. The method of claim 1 or 26 wherein the citrus tree comprises a Rangpur lime rootstock or a Volkamer lemon (Citrus volkameriana) rootstock.

56. A method for detecting a CSD-infected citrus tree, comprising;

detecting a bacterium in a sample from a citrus tree with antibodies which specifically bind to RT 120, but which do not specifically bind to Erwinia caratovora or Enterobacter hormaechei.

57. The method of claim 56 wherein a technique selected from the group consisting of immunoblot and ELISA is used.

58. A method for treating a CSD-infected citrus tree, comprising:

administering a copper-containing compound to a CSD-infected tree to control growth or kill pathogenic bacteria.

59. The method of claim 58 further comprising:

harvesting fruit from the tree.

60. The method of claim 59 further comprising:

extracting juice from the fruit from the tree.

61. A method for treating a tree which is not infected with CSD, comprising:

administering a copper-containing compound to a CSD-uninfected tree to control growth or kill pathogenic bacteria.

62. The method of claim 61 further comprising:

harvesting fruit from the tree.

63. The method of claim 62 further comprising:

extracting juice from the fruit from the tree.

64. The method of claim 1 wherein the agent is a bacterial protein that enhances resistance to CSD.

65. A microbiologically pure culture of bacterial strain RT120, which has been deposited at the American Type Culture Collection, Manassas, VA , USA on July 19, 2004, and assigned the deposit designation PTA-6121.

66. An isolated antibody which specifically binds to one or more antigens on the surface of RT 120 bacteria.

67. The antibody of claim 66 which does not specifically bind to antigens on the surface of Erwinia caratovora or Enterobacter hormaechei.

68. A method for screening for substances useful for treating or preventing CSD, comprising:

contacting a test substance with RT120 bacteria;

determining growth rate or viability of the bacteria after the step of contacting;

identifying as a candidate agent for treating or preventing CSD a test substance which diminishes the growth rate or decreases the viability of RT120.

69. A method for identifying a CSD-infected tree or plant, comprising:

isolating a sample of bacteria from a citrus tree;

applying the sample to a leaf tissue of a geranium or tobacco plant;

identifying the citrus tree as potentially CSD-infected if necrotic tissue develops in the leaf tissue within 1-4 days.

70. A method of identifying a CSD-resistant tree or plant, comprising:

applying a sample of RT 120 bacteria to leaf tissue of a citrus tree or plant;

observing whether necrotic tissue develops within 1-4 days;

identifying the tree or plant as CSD-resistant if necrotic tissue is observed.

71. A method for detecting a CSD-infected citrus tree, comprising;

comparing 16S ribosomal RNA or 16S ribosomal RNA gene from a sample of a citrus tree with a nucleic acid or sequence according to SEQ ID NO: 1, wherein a sample from a citrus tree is identified as CSD-infected if the 16S ribosomal RNA or 16S ribosomal RNA gene is determined to be at least 95 % identical to the nucleic acid or sequence according to SEQ ID NO: 1.

72. The method of claim 71 wherein the sequence of the 16S ribosomal RNA gene is determined.

73. The method of claim 71 wherein a hybridization technique is used to compare the 16S ribosomal RNA or 16S ribosomal RNA gene to a nucleic acid according to SEQ ID NO: 1.