CA2053295A1 - Method of endophyte-enhanced protection of plants - Google Patents

Abstract

2053295 9013224 PCTABS00002 A method of endophyte-enhanced protection in commercially-valuable plants is described which comprises providing an endophytic bacterium that can be harbored within the plant but creates no visible manifestation of disease and introducing that organism to the plant to enhance protection against a broad spectrum of diseases.

Description

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METHOD OF ENDOPHYTE-ENHANCED PROTECTION OF PLANTS
FIELD OF THE INVENTION
The present invention relates to a method of providing enhanced protection agai.nst disease in commercially-valuable plants. More particularly, the present invention relates to such a method employing endophytic microorganism~.
BAC~GROUND OF TXE INVENTION
Since the first development of agriculture, man has battled to protect ~aluable plants from attack by bacteria, viruses, fungi, and insect pests that can rob him of the product of his labor and, on occasion, even thxeaten his existence. The focus in the past has been on chemical means of protection. Recently, the increased awareness of ~he effects of chemicals on the environment has led to the search for other, less toxic means of protecting plants.
Mechanisms for biological control may provide a solution to this problem; however, to date they have proven to be largely ineffective.
Biological control of plant pathogens can be defined as "the decrease of inoculum or the disease-producing activity of a pathogen accomplishe~ through one or more organisms, including the host plant but excluding man.~' See, K.F. Baker, Annual Review of Phytopathology 28:
67-8S (1987). The term was first used in relation to plant pathogens in 1914 and to insects in 1919.
One form of biological control is the phenomenon of induced resistance, that is, an increase in a plant's ability to resist disease after prior exposure to a pathogen. Although the mechanism of action of induced resistance (also called cross protection, acquired resistance or acquired immunity) has never been fully understood, it has been reported in the scientific literature since the 1950's. C.W. Bennett described virus infection to protect plants by induced resistance in Advance~ in Virus Research 1:39 ~1953). Other early SUBS rITUT@: SHEET

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reports on induced resistance include T.O. Biener (Annual Review of Phytopathology 1:197 (1963)) and B. Kassanis (Advances in Virus Research 10:219 (1963)).
These early reports of induced resistance described resistance that was conferred to plants by the introduction of biotic inducers, i.e., pathogenic inducers that were either live or attenuated (i.e., unable to live and increase within the plant). Although these inducers may have created the desired response in the plant, target crops and non-target species were subjected to pathogens that could be potentially harmful to them. In addition, these pathogens were often applied topically thus enhancing the opportunities for environmentally mediated inactivation of the organism (e.g. W degradation). Moreoverj topical application required relatively large amounts of the pathogen, enhancing the opportunity for unwanted exposure of non-target species. In addition when attenuated pathogens were us~d, multiple applications were often required.
In addition to the biotic inducers described above, abiotic (i.e., biochemical) inducers have also been reported in the scientific literature, (~odderman, P.W., et al., Phytopath. Zeit. 113:165-170 (198S); Albersheim, P.A., et al., Structure and Function_of Plant Genomes, NATO
Adv. Study Inst. Series. Plenum Publ. Corp. N.Y. pp. 293-312 (O. Ciferri (ed.) 1982); Graham, T.L., et al., Applied & Environmental ~icrobiology 34:424-432 (1977); ~an Loon, L.C., Netherlands Journal of Plant Pathology 89:265-273 :
(1983); Soliman, H.N., Egyptian Journal of Phytopathology 18(1):35-45 (1986); Salt, S.D., et al., Physiological &
Molecular Plant Pathology 28(2):287-297 (1986); White, R.F., Virology 99:410-412 (1979); Gianinazzi, S. & B.
Kassanis, Journal of General Virolo~y 23:1-9 (1974); and van Loon, L.C., Virology 80:417-420 (1977)). These ~5~ E 5~:.EF I

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_ 3 _ 2~53 inducers included such materialsi as chitin, oligosaccharides and polysaccharides, or other components of cell walls, in addition to chemicals such as salicylic acid.
Although those abiotic inducers were advantageous in that they were not likely to cause disease, there were numerous disadvantages inherent in the use of abiotic inducers including the ability to induce resistance only against a very limited spectrum of pathogens and the need for multiple applications. For example, application of oligosaccharides would induce resistance only against organisms with cell walls that were structurally similar to the inducing compound used. In addition, certain abiotics (e.g. HgCl2) were potentially hazardous to work with and the "cure~' caused damage to the plant which was worse than the disease. Other abiotics (e.g. W light) were impractical to apply.
Accordingly, there remains a need for biological control of plant pathogens. Specifically, there is a need for a method for enhancing the natural mechanisms for resisting diseases present in commercially-valuable plants using an organism that will not harm the environment, will not harm the plant, is capable of living within the plant, requires only a single application, and will enhance protection against a broad spectrum of disease organisms.
SUMMARY OF THEI NVENTION
The present invention overcomes the problems and disadvantages of the prior art by pro~iding of method of enhancing disease resistance in commercially-valuable plants, comprising providing an endophytic organism which is capable of being harbored wi~hin the plant and which creates no visible manifestations of disease and t in one embodiment, crea~es no ill effects on the host plant. This organism is introduced into the plants to enhance protection against a wide spectrum of diseases. In addition, the present invention provides a method of ~;UE~STll'UTE 5HE~T
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W090/l3224 PCT/US9a'02~0 ~'' % ~ ~3~ J _ 4 -enhancing protection in commercially-valuable plants using a vascular-inhabiting endophyte, i.e., one that lives in the vascular tissues of the plant. In another em~odiment, the invention relates to a method of enhancing protection using a vascular-inhabiting endophyte that is a gram positive bacterium. In still another embodiment, the present invention relates to a method of enhancing protection in commercially-valuable plants using an endophytic organism that lives in the vascular-inhabiting system of the plant, is gram positive, and is fastidious.
The invention also provides for a method of enhancing protection in commercially-valuable plants using an endophytic organism known as Clavibacter xYli subsp.
cynodontis ~Cxc).
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be clear from the description, or may be learned by practice of the invention. These objects and advantages of the invention will be realiæed and obtained by means of the methods particularly pointed out in the appended claims.
It is to be understood ~hat the general description above and the following detailed description and drawings are exemplary and explanatory only and do not limi~ the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustra~e several exemplary embodimen~s of the invention and, together with ~he description, serve to explain the principles of the invention.
Fig. 1 is a graph that depicts the effect of Cxc inoculation on leaf area of tobacco (varie~y C319) challenged fourteen days post-inoculation with tobacco mosaic virus (TMV).

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WO90/13224 PCr/US90/02240 .
_ 5 _ ~ ~ ~3~5 FigO 2 is a graph that depicts the effect of Cxc inoculation of leaf area of tobacco (variety C319) challenged twenty days post-inoculation with TMV.
Fig. 3 is a graph that depicts the effect of Cxc inoculation on leaf weight of tobac:co (variety C319) challenged fourteen days post-inoculation with TMV.
Fig. 4 is a graph that depicts the effect of Cxc inoculation on leaf weight of tobacco (variety C319) challenged twenty days post-inoculation with TMV.
Fig. 5 is a graph that depicts the titer of Pseudomonas syrinqae pv. tabaci titer in Cxc-inoculated tobacco (~ariety Ky-14) leaves.
DESCRIPTION OF THE PREFERRED EMBODI~MENTS
Reference will not be made in detail to the currently preferred em~odiments of the invention, examples of which are illustrated below and in the accompanying drawings.
As used herein, "endophyte-enhanced protection~' is defined as the reduction of disease in plants resulting from the introduction of an endophyte into plants. The present invention is not limited by the manner in which the endophyte enhances protection of the plant against disease, nor, as discussed more fully below, by the method of its introduction into plants.
Unlike the induced resistance previously described by the prior art, ~he endophytes of the present invention do not act as pathogens in the host plant. The endophytes are organisms that are capable of being harbored within the plant but create no visible manifestations of disease and, in one embodiment, have no ill effects on the host plan~.
The endophytic organisms of the present invention may also be referred to as organisms which are capable of entering into an endosymbiotic relationship wi~h a plant host. The endosymbiotic relationship is one in which the organism actually exists wIthin and may spread throughout all or a por~ion of the host plant, without causing any Sl 1BSTlTUl-E SHET
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The endophytes used in the method of the present invention are contained within the plant body. In a preferred embodiment, the endophytes are contained within the vascular system of the plant or, in an alternative embodiment, within the intercellular spaces of the plant.
In another embodiment, the vascular-inhabiting or intercellular-space-inhabiting endophytes are gram-positive. "Gram-positive~' refers to a classification of microorganisms based on the components of the cell wall as that term is described by Davis et al. in Microbiolo~Y, 3rd ed., (1980), specifically incorporated herein by reference. -In still another embodiment of the invention, the gram-positive vascular-inhibiting endophytes are fastidlous in nature. As used herein, the term "fastidious" refers to organisms having complicated nutritional requirements, as that term is defined by McCoy, R.E., in ~Chronic and insidious disease: The fastidious vascular pathogens,~
PhYtopathoqenic ProkarYotes (Mount M.S. and Lacy, G.H., eds. 1982), specifically incorporated herein by reference.
In still another embodiment, the present invention relates to endophytes of ~he Coryneform family as that term is defined by M.J. Davis in Annual Review Phytopathology 24: 115-40 (1986), specifically incorporated herein by reference. In another embodiment the present invention relates to the genus Clavibacter. In a particularly preferred embodiment, the invention relates to the endophyte known as Clavibacter xyli subsp. cynodontis (hereinafter "Cxc''), as that term is defined by ~.J. Davis et al. in International Journal of Systematic Bacteriology 34(2):107-117 (April 1984), specifically incorporated herein by reference.

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WO90/13224 PCT/US90/02~0 2~S3235 The present invention contemplates the introduction of live endophytes capable of being harbored within the plant host. The endophytes of the present invention may multiply within the plant host but the present invention is not limited to endophytes that multiply within the host.
The endophytes of the present invention may be unmodified or modified or formulated with other components to provide beneficial properties in addition to enhanced protection. ~odification of endophytes is accomplished by techniques that are known to those of ordinary skill in the art. Any means of modification and any modification of - endophyte~ are specifically contemplated by the present invention.
The endophytes used in the method of the present invention may be modified, for example, by mutagenesis or recombinant techniques known to those of ordinary skill in the microbiology and molecular biology art in light of the teachings contained herein. The endophyte may be modified by the induction and isolation of mutant strains effective in protecting plants against disease. The DNA of the endophytes may be modified by the addition of DNA that codes for the production of particular compounds, including but not limited to proteins, antibiotics, and other biochemical compaunds. Thus, the endophyte could, in addition to enhancing protection, provide agricultural chemicals that might benefit the plant. On such method for ~he production of such endophytes is pro~ided copending in United States Patent Application No. 166,819 (filed ~arch

3, 1988), No. 266,232 ~filed October 10, 1988), and No.
266,221 filed October 10, 1988), all of which are commonly assigned to the assignee of the present invention and are incorporated specifically herein by reference.
Alternati~ely, endophytes may be modified by mutagenesis or recombinant techniques to produce inducer compounds, such as, for example, dihydroxy benzoic acid or beta-ionone. The techniques for these modifications are 5U BSTITlJTE SH EET

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similarly within the routine skill of one ordinary skill in the art in light of the teachings contained herein.
In addition, plant protection provided by the present invention may be enhanced by formulating the endophyte with one or more abiotic inducers. The techniques to select abiotic inducexs and de~elop formulations including them are within the routine skill of those of ordinary skill in the art in light of the teachings contained herein.
The modified, unmodified or formulated endophytes may be introduced to the plants by any technique known to those ordinary skill in the art. The method of endophyte introduction does not in any way limit the present invention. Introduction techniques, which vary with the plant host, include, bu~ are not limited to, latex plugs, slow releases, root drips for transplanted plants, abrasive sprays, needle or needless injection, pressure injection and the like.
In a preferred embodiment, the endophytes are introduced by stem stabbing. "Stem stabbing" refers to the introduction of endophytes by wounding the plant and ; -delivering the endophytes to that wound. A preferred method of stem stabbing involves a scalpel or other sharp instrument that is first coated wi~h an endophyte and then used to simultaneously wound and deliver the organism.
In another embodiment, the endophytes are introduced to plants by stem injection. "Stem injection"
refers to the introduction of organisms into the stem of the plant via a puncture created by a needle of, for example, a tuberculin intradermal syringe. In one preferred method, the needle of the syringe, containing the endophytic organisms to be introduced, i~ gently pushed ~ -into the stem and the contents of the syringe gently and slowly injected into the stem.
In another embodiment, the endophytes are introduced to plants either by injection into the petiole - - ~iU~S~,TUTF SffEET

4 PCT/US90/02240 21:95~%~5 .
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g by needle or by deposition onto a previously broken petiole.
In still another embodiment, the endophytes can be introduced by intercellular infiltration, where a suspension of endophytes is injected into the intercellular spaces of a leaf.
In still another embodiment of the invention, the endophytes may be introduced by inoculating the seeds of the plant with the endophytes. The method of seed inoculation is provided in co-pending United States Patent Application No. 194,247, filed May 16, 1988, to Jed W.
Fahey, incorporated specifically herein in its entirety by reference.
The invention relates to enhanced protection in all commercially-valuable plants. Persons of ordinaxy skill in the art are generally familiar with agriculturally-valuable plants. These include the horticultural plants, such as those producing fruits, vegetables, flowers and ornamental trees and plants. In addition, commercially-valuable plants include agricultural trees and plants such as field and row plants. Field and row plants include, but are not limited to, corn, sorghum, wheat, barley, oats, rice, tomato, potato, cabbage, broccoli, melons, cucumbers and related plants. In another embodiment, commercially-valuable plants encompass plants of forestry. This list is exemplary only and does not in any way limit the application of the present invention.
In accordance with the present invention, the protected plants become resistant to one or more of a broad spectrum of diseases including, but not limited to, mildews, rusts, smuts, rots, scabs, spots, blights, blasts, decay, damping-off, leaf rolls, vascular wilts, warts, galls, yellows, cankers, mosaics, ring spots and other stunting, dwarfing or disfiguring plant diseases. These diseases include those caused by bacteria, viruses, and fungi and othex biotic pathogens. In a preferred SU13STITUTE~ SHEET
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, ~ ~S3~43'3 embodiment~ the plants are resistant to tobacco mosaic virus, potato viruses X and Y, Pseudomonas svrin~a_ pv.
tabaci, Clavibacter michiqanense subsp. michiqanense and Fusarium oxys orum f.sp. melonisO
The invention will be further illustrated by the following examples, which are intended to be purely exemplary of the invention.

LOCAL LESION (HYPERSENSITIVE) RESPONSE IN ENDOPHYTE~
INOCULATED TOBACCO PLANTS FOLLOWING CHALLENGE
WITH TOBACCO MOSAIC VIRUS
Plants of Nicotiana tabacum L. cv. 'Ky 14', a variety hypersensitive to tobacco mosaic virus (hereinafter "TMV"), were planted in one gallon pots in the greenhouse.
Thirty days after sowing, the plants were selected for uniformity. Plants were randomly assigned as either `
control plants or treatment plants.
A. Pre~aration of the Endophyte ~Cxc !:
For six days at 28C + 3C, Cxc was grown on SC
Media, consisting of 1000 ml distilled water; 17 g cornmeal -agar; 8 g papaic digest of soy meal; 1 g K2HPO4; 1 g KH2P04; 0.2 g ~gS04 7H20; lS mg (15 ml of a 0.1 percent solution in 0.05N NaOH) bovine hemin chloride; 2 g (10 ml of a 20 percent aqueous solution) bovine serum albumin fraction 5; 0.5 g (1.0 ml of a 50 percent aqueous solution) glucose; and 1 g (free basef 10 ml of a 10 percent aqueous solution) cysteine. After incubation, the cells were washed and suspended in 10 ml sterilized tap water.
Suspensions of Cxc cells were centrifuged at 6000 rpm for 15 minutes and resuspended in sterile water or phosphate buffered saline (PBS). Bacterial concentration was determined spectrophotometrically at 600 nm and adjusted to ca. 10 bacteria/ml.

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B. Inoculation with the EndoPhvte tCxc):
The treatment plants were inoculated with solutions containing Cxc as follows:
1. - Stem Inoculations a) Iniection of Cxc cells into the stem 4 cm above the soil surface by hypodermic syringe.
b) Stabbinq of Cxc into the stem by sterile scalpel blades containing Cxc scraped from streaked plates where the scalpel tip was inserted completely through the stem.
2. - Petiole Injections Injection of Cxc into the petiole by hypodermic syringe.
3. - Intercellular Infiltration.
Injection of ca. 10 ul of bacterial suspension into the intercellular space of the leaf anima to create water soaking; eight injections per leaf, with hypodexmic syringe.
In addition, control plants were inoculated with control solutions, i.e., the s~me solutions as above except that Cxc was absent.
C. Inoculation with the Challenae Or~anism:
Two weeks later, partially purified suspensions of strain U-1 of TMV in phosphate buffered saline were used for all challenge inoculations, as in R.W. Fulton, ~Nicotiana As Experimental Virus Hosts, "Nicotiana Procedures for Experimental Use - Technical_Bulletin No.
1586 (U.S.D.A. P.D. Durkin, ed., 1979), specifically incorporated herein by reference. A gauze pad was soaked in the ~NV inoculum and rubbed onto all expanded leaves following a light dusting with 600 mesh carborundum, an abrasive powderl to facilitate viral infection.
As set forth in Table 1, prior inoculation of tobacco variety RY-14 with Cxc resulted in a consistent ,.. .. .. . . . . . . . . .. .. . . . ....

WO90/t3224 PCT/US90/02240 "' :' reduction of TMV lesion numbers. Accordingly, prior inoculation with Cxc resulted in a dramatic reduction of the hypersensitive response compared to controls.

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%~3~`~ - 14 -LOCAL LESION (HYPERSENSITIVE) RESPONSE IN
ENDOPHYTE-INOCULATED TOBACCO PLANTS FOLLOWING
C~L~LLENGE WITH TOBACCO MOSAIC VIRUS

In the same manner as Ex~mple 1, in a field experiment, Cxc-inoculated tobacco plants t~ariety KY 14), planted in a randomized complete ~lock design with five replications, exhibited a reduction in lesion number over controls when plants were challenged at fourteen days and twenty days (two separate readings) post-inoculation (see Table 2 below). All plants (both control and experimental) challenged at thirty-one days showed such low numbers of lesions that comparisons between treatments is not valid, likely due to environmental conditions in the field. :~

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' A '' . ''' Table 2 Effect of Cxc Inoculation on TMV Lesion Number in Field-Planted Tobacco Variety KY 14 . . . ~
Mean ~ of lesions/100 sq cm leaf Method of Inoculation Days After Cxc Inoculation ExperimentalStem Stab 60 49 20 8.4 with Cxc Stem Inject 60 41 14 6.9 with Cxc _______________________________________________ ______ ___ ControlS~em Stab 60 57 24 8.1 with Water Stem Inject 63 52 23 8.7 with Water Unioculated 85 58 29 6.7 Control LSD = 32.4 13.6 9.85 4.5 N = 100 68 80 100 *L$D refers to Least Significant Difference ... .
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TOBACCO PhANTS FOLLOWING CHALLENGE WITH TMV
Using the protocol of Example 1, sixty plants of tobacco variety Coker 319 (C319) were planted in the greenhouse. Af~er five weeks of growth, uniform plants were selected and subjected to the following treatments:
1) untreated control 2) stem stab with water (control) 3) stem stab with Cxc 4) stem injection with Cxc.
The entire experiment was repeated three separate times.
Plan~s were inoculate~ with Cxc at the stage of growth when two true leaves had formed. Fourteen days later, the plants were challenged with TMV by inoculation of true leaves four and five (counted from the soil line).
Twenty days later, leaves numbered 5, 6, 7, and 8, counted from the challenged leaf, were removed and leaf area and leaf fresh weight were assessed. Leaf fresh weight was determined by removing any adherent water or debris and weighing the entire leaf and subtending petiole. Nine days later, plants were re-assessed by measuring leaf area and fresh weight for leaves numbered 9, 10, 11, and 12. In addition, plant height was determined by measuring total height of the plant from the 50il line to the uppermost leaves and plant weight was scored as the weight of the above-ground portion of the plant.
In two of the three replications, Cxc-inoculated plants exhibited greater leaf area and leaf weight than their respective controls. Table 3 sets forth results from one of those replications in which the leaf areas of leaves numbered 7, 8, 9, 10, 11, and 12, from Cxc inoculated plants, were significantly greater than their respective controls.

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Leaf fresh weight exhibited a similar pattern, as depicted in Table 4. Leaves 7, 8, 9, 10, 11 and 12 from Cxc-inoculated plants exhibited significantly greater fresh lea weight than control plants.

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Similarly, as depicted in Table 5, inoculated plants exhibited increased plant height and weight compared to controls.
Table 5 Effect of Cxc Inoculation on Plant Height and Weight of Tobacco Variety C-319 Challenged with TMV (REP#1) Method of Plant HeightPlant Weight Inoculation (cm) (g) Experimental Stem Stab 46 211 with Cxc S~em Inject 43 189 with Cxc ____________________________________ .______________ _______ Control 5tem Stab 38 161 with Water Uninoculated 42 166 Control .
LSD = 5 . 5 25 SYSTEMIC RESPONSE IN ENDOPHYTE- INOGUI~ATED :
TOBACCO PLANTS FO~LOWING CHALLENGE WITH TMV
In the same manner as Example 3, in three field -experiments, Cxc-inoculated plants exhibited significantly greater leaf area and leaf weight when challenged fourteen and twenty days post inoculation. As shown in Figures 1 and 2, the a~erage leaf area of Cxc-inoculated plants was greater than control plants. This pattern was exhibited when challenge occurred at either fourteen days (Figure 1) or twenty days (Pigure 2) after inoculation. Similarly, leaf weight of Cxc-inoculated plants challenged fourteen (Figure 3) or twenty days post inoculation (Figure 4) was significantly greater than that of control plants.

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SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED
TOMATO PLANTS FOLLOWING CHALLENGE WITH TMV
Using the protocol of Example 1, tomato plants (Lyco~ersicon esculentum cv. Marglobe), susceptible to TMV, were planted in a greenhouse, tran.splanted into one gallon pots approximately six days later, and allowed to continue to grow in the greenhouse. This experiment was replicated twice. In both replications, plants were inoculated with Cxc one week after transplanting. All plants were challenged with TMV approximately three weeks after Cxc inoculation.
Approximately 25 days after challenge, the plants were assessed for the number of flowers produced, the number of fr-liting bodies produced, and plant height.
Table 6 sets forth the effect of Cxc inoculation on flowering and fruiting. Flowering scores were higher for inoculated plants than controls. Similarly, inoculated plants exhibited greater fruiting than did controls.
Table 6 Effect of Cxc on the Flowering and Fruiting of TMV Challenged Tomato ~ .
FLOWERING FRUITING
TREATMENT SCORE SCORE
... ._ _ Experimental Stem stab 2.3a 1.4a with Cxc Stem inject 1.9 1.2 with Cxc ___________________________________________________________ Control Stem stab 1.5 1.0 with Water Stem in~ect 1.8 1.0 with Water a = Scores:
3=100% of plants flowering or fruiting 2=50 % of plants flowering or fruiting 1=0 ~ of plants flowering or fruiting SU85TlTUTE~ SHET

WO?0/13224 PCT/US90/02~0 2~2~ - 22 -When TMV challenge of tomato plants was performed in the field, Cxc-inoculated plants exhibited an increase in yield over control plants, as set forth in Table 7.
Table 7 Effect of Cxc Inoculation on Yield of Tomato Plants Following Challenge with TMV
Method of Tomato Yield Inoculation (Kg) . . .
Experimental Stem Stab 2.8 with Cxc Stem Inject 3.3 with Cxc _________________________ _____________________________ __ Control Stem Stab 2.3 with Water Stem Inject 2.7 with Water Unioculated 2.7 Control --:
LSD= .6 Similarly, when the quality of tomatoes at harvest time was assessed, as set forth in Table 8, fruit from ~he Cxc-inoculated plants was of superior quality. In contrast, ratings for controls were predominantly in th~
lowest quality categories (three and four).

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WO90/13224 PCT~U~90/02240 - ~3 -Table 8 Effect of Cxc Inoculation on Tomato Fruit Quality Following TMV Challenge with TMV- -Quality Categorya Method of ----~
Inoculation lb 2 3 4 Average Experimental Stem Stab 0% 12~i 80% 8~ 3.0 with Cxc Stem Inject 0% 27% 68% 5% 2.7 with Cxc ________________________~__________________________________ Control Stem Stab 0% 0% 62% 38% 3.4 .:
with Water Stem Inject 0% O~i 59% 41~i 3.4 with Water Uninoculated 0% 4% 48% 48% 3.4 Control .
a = % of total plants in each quality cate~ory b = Quality categories:
1=mostly red :
2=half red and half green : :
3=mostly green and large 4=mostly green but small ~ .

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WO90/13224 PcT/Us9o/o224o ~ ;3Z~ ` f SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED POTATO
PLANTS FOLLOWING CHALLENGE WITH POTATO VIRUSES
In a field experiment, one hundred and eight potato plants (Solanum tuberosum cv. Kennebec) were planted by hand in a randomized complete block design with 3 replications.
After appxoximately seventeen days of growth, plants were treated with Cxc, introduced by either stem injection or stem stabbing, as described in Example 1 above. Con~rol plants were stem injected with water, stem stabbed with water, or left un~reated.
Twenty days after inoculation with Cxc, the potato plants were challenged with potato virus X (PVX) using the same procedure used for challenging tobacco plants with TMV
as described in Example 1.
Plants were scored for flowering and for disease severity. As set forth in Table 9, potato plants inoculated by either injection or stabbing with Cxc exhibited a significantly higher percentage of flowering than did any of the three sets of control p-lants.
Similarly, inoculated plants exhibited reduced disease severity (as evidenced by the number of discolored and wilted leaves) than controls. Disease severity in each plant was rated on a scale of 1-4 based on quality of leaves, such that:
1 - less than 10% of plants' leaves exhibited disease symptoms 2 = greater than 10% to less than 30%
of the leaves exhibited disease symptoms 3 = greater than 30~ but less than 60% of the leaves exhibited disease symptoms 4 = greater than 60% of the leaves exhibited disease symptoms .. . , . . , _ . , BS ~ JT~-; StlEE~

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Table 9 Effect of Cxc Inoculation on Potato ~lowering and Disease Severity Following Challenge with PVX
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DISEASE RATINGa FLOWERIIIG~
___________________.____________.____________ AVERAGE
METHOD OF DISEASE

Experi-Stem stab 4% 87% 9% 0% 1.9 52%
mentalwith Cxc Stem inject 5% 76% 19% 0% 2.2 71%
with Cxc ____________ _______ ______________________________________ ControlSte~ stab 0% 64% 32% 4% 2.3 20% . .
with Water Stem inject 0% 52% 40% 8% 2.6 24%
with Water Uninoculated 0% 77~ 14~ g% 2.3 9%
Control , a = % of total plants in each disease rating b = % of total plants flowering in each category Example 7.
SYSTEMIC RESPONSE IN ENDOPXYTE-INOCULATED
POTATO PLANTS FOLLONING CH~LLEN&E WITH POTATO VIRUSES .
In the same manner as Example 6, 180 potato plants (S. tuberosum. cv. Kennebec) were planted by hand in the field. Approximately two weeks later, the plants were randomly assigned to fi~e groups and subjected to ~ive treatments. As above, the five treatments were: stem stab with Cxc, stem inject with Cxc, uninoculated c~ntrol, stem ~ :
stab wi~h water and s~em inject with water. Twenty d ys after inoculation with Cxc, all plants were challenged with potato ~irus Y (PVY) Thirty days later, the plants were scored for flowering and for di ease severity. As set forth in Table 10, although there was no difference in flowering between ~ ;, Cxc-inoculated and oontrol plants, Cxc-inoculated plants did exhibit.a reduction in disease severity over control SV~35TITlJTE~: SHE:E:T - - ~

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W~90/1322~ PCT/US90/02240 3~5 plants. Specifically, as set forth in Table 10, Cxc-inoculated plants predominantly ranked in the lowest damage category.
Table 10 Effect of Cxc Inoculation on Potat:o Flowering and Disease Severity Following Challenge with PVY
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DISEASE RATINGa FLOWERINGD
___________.________________________ _________ METHOD OF AVERAGE

RATING
-Experi-Stem stab 22% 78% 0% 0% 1.8 65%
mentalwith Cxc Stem inject 5% 73% 23% 0% 2.2 55%
with Cxc _________________________________________._________________ Control Stem stab 0% 80% 20% 0% 2.2 48%
with Water Stem inject 0% 50~ 45% 5~ 2.6 65%
with Water Uninoculated 0% 67% 29% 4~ 2.4 46%
Control ~-r a = % of total plants in each disease rating b = ~ of total plants flowering in each category SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED POTATO
PLANTS FOLLOWING CHALLENGE WITH POTATO VIRUSES
In the same manner as Example 6, 180 potato plarts were planted in the ield and allowed to grow for approximately two weeks. The plants were then inoculated with Cxc, using the same five treatments as set forth abo~e. Eighteen days after inoculation with Cxc, the plan~s were challenged with a mixture of PVY and PVX.
Seventeen days later the plants were evaluated. As set forth in Table 11, the Cxc inoculated plants exhibited a greater percentage of flowering than did the control ,. . . ~ . . . i . :

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-. , WO90/13224 ~53% ~ PCT/US90/02~0 ~ ' '.,`,' '' ,, plants. Similarly, Table 11 depicts that the Cxc-inoculated plants exhibited less damage due ~o viral infection than did the control plants.

WO90/13224 PCT/US90/~2240 2~53~

Table 11 Effect of Endophyte-Inoculation on Potato Flowering and Disease Severit~ Following Challenge with PVX + PVY
DISEASE RATINGa FLO~ERINGD
_________ .________________________ __________ METHOD OF AVERAGE

- RATING
Experi-Stem stab 77% 23% 0% 0% 1.2 88 mentalwith Cxc Stem inject 77% 23% 0% 0% 1.2 . 95%
with Cxc ___________________________________ _ _____________________ . .
Control Stem stab 4% 84%12% 0% 2.1 68%
with Water Stem inject 12% 72~16% 0% 2.0 80%
with Water Uninoculated 7% 86%7% 0% 2.0 42%
Control -a = % of total plants in each disease rating b = % of total plants flowering in each category SYSTEMIC RESPONSE OF ENDOPHYTE-INOCULA~ED TOMATO CHALLENGED
WI'rH CLAVIBA~CTER MICHIGANENSE SUBSP. MICHIGANENSE
Using the protocol of Example, tomato plants (Lvco~ersicon esculentum cv. Marglobe) were planted in a greenhouse and allowed to grow for twelve days.
Thereafter, plants were divided into five groups as ~ollows:
1) untreated control 2) stem stab with water 3) stem inject with water 4) stem stab with Cxc

5) stem inject with Cxc.
Eleven days later, ~he tomato plantæ were challenged by introduction of the bacteria Clavibacter michi~anense subsp. michiganense ("Cmm") (syn.
C rvnebacterium mLchiqanense subsp. michiqanese) grown on ~UBSTlTU~E SHEET

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WO90/13224 PCr/US90/0~240 nutrient broth yeast extract agar (NBY) and incubated at 26C for approximately four days. Cell suspensions of Cmm in distilled water were used for challenge inoculation.
Challenge inoculation was performed by breaking the petiole and applying a suspension containing approximately 108 cells/ml to the broken area.
Eighteen days after challenge, the plants were scored for disease severity. The severity of disease in each plant was rated on a scale of 0 to 4 as follows:
0 = no evidence of disease 1 = 0-10% of plant wilted a = 10%-40% of plant wilted 3 = 40-75% of plant wilted 4 = greater than 60% of plant wilted As set forth in Table 12, the inoculated plants exhi~ited a dramatic reduction in disease compared to control plants.
Table 12 Effect of Endophyte-Inoculation on Tomato Disease Severity Following Challenge with C. michi~anense subsp.
michiq~ense DISEASE RATING , -______________________________________________ , . .
METHOD OF
INOCULATION la 2 3 4 AVG.
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Experimental Stem stab 33% 56% 11% 0% 1.8 with Cxc Stem inject 22% 56% 22% 0% 1.9 with Cxc ___________________________________________________________ Control Stem stab 0% 20% 60% 20~ 3.0 with Water Stem inject 0% 20% 80% 0~ 2.8 wi~h Water Uninoculated 0% 0% 100%0~ 3.0 Control .._ _ _ . . _ . .. .
a - ~ of total plants in each disease categcry _ . _ _ . .

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WO90/1322~ PCTtUS90/022~

2~353'~5 EXAMPLE l0 SYSTEMIC RESPONSE IN ENDOPHYTE-INOCULATED TOBACCO PLANTS
FOLLOWING CHALLENGE WITH PSEU _MONAS SYRINGAE
pv. TABACI

Using the protocol of Example 1, tobacco plants were planted in the greenhouse. Af~er an initial growth period of about 1 month, in each of three randomly assigned replications, ten plants were stem injec~ed with Cxc, ten were stem stabbed with Cxc, five were stem stabbed with sterilized tap water, and five were untreated.
Fourteen days after inoculation with Cxc, the plants were challenged with Pseudomonas s~rinqae pv. tabaci (~'Ps tabaci ). At 1, 2, 3 and 4 days after inoculation with this bacterium, a disc was punched from the leaf tissue between lesions. The discs were homogenized and plated onto Kings B Agar to calculate the number of Ps.
tabaci cells per gram of leaf tissue. These numbers provided an index of bacterial infestion.
As set forth in Figure 5, inoculated plants exhibited a reduction in the number of bacterial cells per gram of leaf tissue each day after inoculation.
Accordingly, it appeared that the Cxc-inoculated plants permitted less multiplication of Ps. tabaci in leaf tissue.
This reduction of pathogen/titer in the plants has a direct impact on the rate of spread of ~he resultant disease (wildfire disease) in the field.

SYSTEMIC RESPONSE IN ENDOPHYTE-INOC~LATED MUSRMELON
CHALLENGE WITH FUSARIUM OXYSPQRUM f.sp. MELONIS
Cucumis melo Muskmelon (variety Honey Rock) plants were grown in a greenhouse. At approximately one week pos~-emergence, plants were subjected to one of two treatments: hypodermal inoculation with ~ashed Cxc cells resuspended in phosphate buffered saline ("PBS") at approximately 108 CFU/ml; or a control inoculation using PBS alone.- After approximately 26 days of growth, all 51JE~15TITUTE SHEET

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WO90/13~24 PCT/US90/02~0 2~5 plants were challenged with Fusari~ oxysPorum f.sp.
melonis by transplanting into pots containing Fusarium infested soil. All plants were allowed to grow with a photoperiod of about 14:10 (L:D), at 95~ relative humidity and 23C until symptoms appeared. Two replications were conducted.
Plants were assessed for disease se~erity and plant dry weight. Plant weight was determined by weighing the harvested, above-ground portions of the plant. The severity of each disease was assessed using standard phytopathological methods and plants were rated on a scale of 0-5:
0 = no evidence of disease 1 = 0 - 10% of plant wilted 2 = 10% - 30% of plant wilted 3 = 30~ - 60% of plant wilted 4 = greater than 60% of plant wilted 5 = plant death.
As set forth in Table 13, Cxc-inoculated plants exhibited greater dry weight and reduced disease severity compared to controls.

Table 13 Effect of Cxc Inoculation on Vascular Wilt of Muskmelon Following Challenge with Fusarium oxYsporum f.sp. melonis.

_ Mean Disease Mean Treatment Severity Score Dry wt. (g) _____________________ _. ___~_______________________________ .
Replication 1 Cxc-inoculated 1.7 1 85 PBS-inoculated control 3.7 1 58 Replication 2 Cxc-inoculated 2.58 4 01 PBS-inoculated control 3.75 2 15 35 ~ ~TU~ 5~.EET

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WO90/13224 PCT/~S90/02~0 . .
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%~5~ 5 .i Other embodiments of the invention will be apparent to those skilled in the axt from a consideration of the specification and practice of the inventicn disclosed herein. It is intended that the specification and the examples be considered as exemplary only, with the true scope of the spirit of the invention being indicated by the following claims and their equivalents.

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Claims (33)

WHAT IS CLAIMED IS:

1. A method of endophyte-enhanced protection in commercially-valuable plants comprising:
a. providing an endophytic organism that can be harbored within the plant and which creates no visible manifestations of disease, and b. introducing the organisms to the plants such that the organisms enhance protection against disease.

2. The method of claim 1 wherein the endophytic organism is a vascular-inhabiting endophyte.

3. The method of claim 1 wherein the endophytic organism is a vascular-inhabiting bacterium.

4. The method of claim 2, wherein the vascular-inhabiting endophytic organism is a gram positive organism.

5. The method of claim 4 wherein the vascular-inhabiting gram positive endophytic organism is fastidious.

6. The method of claim 1 wherein the endophytic organism is a Coryneform bacterium.

7. The method of claim 1 wherein the endophytic organism is a Clavibacter spp.

8. The method of claim 1 wherein the endophytic organism is a Clavibacter xyli subsp. cynodontis.

9. The method of claim 1 wherein Clavibacter xyli subsp. cynodontis is introduced to tobacco plants to enhance protection against disease.

10. The method of claim 9 wherein the disease is caused by a virus.

11. The method of claim 10 wherein the virus is tobacco mosaic virus.

12. The method of claim 9 wherein the disease is caused by bacteria.

13. The method of claim 12 wherein the bacterium is Pseudomonas syringae pv. tabaci.

14. The method of claim 1 wherein Clavibacter xyli subsp. cynodontis is introduced to tomato plants to enhance protection against disease.

15. The method of claim 14 wherein the disease is caused by a virus.

16. The method of claim 15 wherein the virus is a tobacco mosaic virus.

17. The method of claim 14 wherein the disease is caused by a bacterium.

18. The method of claim 17 wherein the bacterium is Clavibacter michiganese subsp. michiganese.

19. The method of claim 1 wherein Clavibacter xyli subsp. cynodontis is introduced to potato plants to enhance protection against disease.

20. The method of claim 19 wherein the disease is caused by virus.

21. The method of claim 20 wherein the disease is caused by potato virus X.

22. The method of claim 20 wherein the disease is caused by potato virus Y.

23. The method of claim 20 wherein the disease is caused by a combination of potato virus X and potato virus Y.

24. The method of claim 1 wherein Clavibacter xyli subsp. cynodontis is introduced to muskmelon to enhance protection against disease.

25. The method of claim 24 wherein the disease is caused by a fungus.

26. The method of claim 24 wherein the disease is caused by vascular wilt fungus.

27. The method of claim 26 wherein the fungus is a Fusarium sp.

28. The method of claim 1 wherein the endophytic organism is genetically unmodified.

29. The method of claim 1 wherein the endophytic organism is genetically modified.

30. The method of claim 28 wherein the endophytic organism is modified by recombinant DNA techniques.

31. The method of claim 28 wherein the endophytic organism is modified by mutagenesis techniques.

32. The method of claim 1 wherein the endophytic organism is formulated with one or more abiotic inducers.

33. The method of claim 1 wherein the endophytic organism creates no visible manifestation of disease.