Biological control of crown gall disease on grapevines

Abstract

Derivatives of Agrobacterium vitis strain F2/5 are disclosed. These derivatives were generated following homologous recombination with an internal fragment of targeted genes resulting in gene disruption by insertion of a copy of suicide vector pVIK165. The genes disrupted were F-avi5813 encoding a phosphopantetheinyltransferase, F-avi4329 encoding an aminotransferase and F-avi0838 (rirA) encoding an iron responsive transcriptional regulator. Such derivatives control crown gall on grapevines. In addition, these derivatives did not induce roots necrosis but enhanced root development and callus formation. On young stem explants, it was shown as well that the F2/5 derivatives are necrosis-negative.

Claims

1. An isolated, necrosis-minus, Agrobacterium vitis derivative of strain F2/5, said derivative comprising a nucleic acid molecule encoding an aminotransferase comprising 95% sequence identity to SEQ ID NO:4, wherein expression of said aminotransferase is diminished or abrogated.

2. The A. vitis of claim 1, wherein said nucleic acid molecule is inactivated.

3. The A. vitis of claim 2, wherein said aminotransferase is a homolog of A. vitis S4 (avi4329).

4. A culture comprising the A. vitis of claim 1.

5. A composition comprising the A. vitis of claim 1.

6. A method for reducing crown gall disease on a grapevine or a grapevine component, said method comprising administering to said grapevine or grapevine component an effective amount of the A. vitis of claim 1, wherein said method reduces crown gall disease on said grapevine or grapevine component.

7. The method of claim 6, wherein said A. vitis, culture, or composition is administered at grafting time; is administered on graft unions or the base of the grapevine; is administered during field grafting of grapevine; is administered to a dormant cane cutting; composition is administered to a green shoot cutting; or is administered to a grapevine plant part above the ground.

8. A method for reducing necrosis on a grapevine or a grapevine component, said method comprising administering to said grapevine or grapevine component an effective amount of the A. vitis of claim 1, wherein said method reduces necrosis on said grapevine or grapevine component.

9. A method for controlling crown gall disease, said method comprising administering to a locus for planting a grapevine or grapevine component an effective amount of the A. vitis of claim 1, wherein said method controls crown gall disease.

10. The method of claim 9, wherein said locus is a furrow or soil.

11. A method for promoting callus development, said method comprising administering to a grapevine or grapevine component an effective amount of the A. vitis of claim 1, wherein said method promotes callus development on said grapevine or grapevine component.

12. A method for promoting root development, said method comprising administering to a grapevine or grapevine component an effective amount of the A. vitis of claim 1, wherein said method promotes root development on said grapevine or grapevine component.

13. A method for reducing necrosis, said method comprising administering to a grapevine or grapevine component an effective amount of the A. vitis of claim 1, wherein said method reduces necrosis of grapevine or grapevine component tissue below ground.

14. The A. vitis of claim 1, wherein said aminotransferase comprises 96% sequence identity to SEQ ID NO:4.

15. The A. vitis of claim 1, wherein said aminotransferase comprises 97% sequence identity to SEQ ID NO:4.

16. The A. vitis of claim 1, wherein said aminotransferase comprises 98% sequence identity to SEQ ID NO:4.

17. The A. vitis of claim 1, wherein said aminotransferase comprises 99% sequence identity to SEQ ID NO:4.

18. The A. vitis of claim 1, wherein said aminotransferase comprises SEQ ID NO:4.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows crown gall at wounds on woody grape tissues inoculated with CG49 or CG49 mixed with F2/5 or with F2/5 mutants. Arrows point to inoculated wound sites.

(2) FIG. 2 shows dormant grape cuttings were soaked in suspensions of F2/5, F2/5 mutants or water and then rooted. Arrows point to necrosis on roots.

(3) FIG. 3 shows effects of F2/5 and three necrosis-minus derivatives of F2/5 on grape shoot necrosis.

(4) FIG. 4 shows effects of treatments with A. vitis F2/5 and its mutants on grapevine grafts

(5) FIGS. 5A and 5B show the gene organization and annotation of Avs locus (A) and the effects of knock-down mutant ΔP1391 and ΔP1396 on biological control (B).

(6) FIGS. 6A through 6P show the sequences listed in Table 1.

DETAILED DESCRIPTION

(7) Crown gall of grapevines is caused by the bacterium, Agrobacterium vitis. The disease is initiated at wound sites, such as graft unions and freeze injuries and limits grape production worldwide. There are no chemical or biological controls that have been successful to control this disease to date. A non-tumorigenic strain of A. vitis, F2/5, prevents crown gall on grapevines at wounds but also causes a necrosis that can be deleterious to graft union development and to root formation. A. vitis strain F2/5 is publically available from a number of laboratories throughout the world including from the laboratory of the Applicant's inventor, Dr. Thomas J. Burr (Department of Plant Pathology & Plant-Microbe Biology, New York State Agricultural Experiment Station, Cornell University, A104 Barton Hall 630 W. North Street, Geneva, N.Y. 14456-0462). As is disclosed herein, specific necrosis-minus derivatives of F2/5 that still retain biological control activities have been engineered. One mutant was generated by knocking out of a gene encoding phosphopantetheinyl transferase (PPTase), which is required for post-translational modification of polyketide synthase and non-ribosomal peptide synthase genes; another mutant was generated through the disruption of an aminotransferase; yet another mutant was targeted at an iron-response regulator. These mutations result in abolished necrosis but did not affect the ability of F2/5 to function as a biological control agent.

(8) Such strains may be used in nurseries at grafting time (on graft unions and base of plants) as well as during field grafting of vines to prevent crown gall. Other uses are described herein.

(9) The following examples are intended only to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.

Example 1

(10) Development of Agrobacterium vitis F2/5 Mutants:

(11) Mutants of specific genes were generated through disruption with suicide vector pVIK165 (Kalogeraki et al. (1997) Gene 188: 69-75). Internal fragments of specific target genes were amplified from F2/5 genomic DNA with corresponding primer pairs. The specific genes that were mutated and primers used to amplify internal gene fragments are listed in the Table 2.

(12) TABLE-US-00002 TABLE 2 Genes mutated and primers used in generation of the mutants size of Gene primer fragment targeted name primer sequence* amplified F-avi5813 avi5813-F: 5′-AAGAATTCTTGATCTCGCATCGCGCTG-3′ 336 bp (PPTase) avi5813-R: 5′-AATCTAGATGCGCTGTCATGACCTGTTCTG-3′ F-avi4329 avi4329-F: 5′-ACGAGCTCAGCTCGTCAGCGAGCAGCTG-3′ 382 bp (amino- avi4329-R: 5′-AGTCTAGATGAACTCGTCATGGATCACCAG-3′ transferase) F-avi0838 avi0838-F 5′-ACGAGCTCTGCCAAGGCCTGTGGTGTCTC-3′ 224 bp (rirA) avi0838-R 5′-AGTCTAGATGTCGATCAACGGGCAATCGAC-3′ *Restriction sites are underlined

(13) XbaI and SacI restriction sites were introduced into forward and reverse primers respectively. PCR products were purified, digested and then ligated into suicide vector pVIK165 at XbaI and SacI sites. The constructs were transformed into E. coli strain S17-1/λpir by electroporation and transformants were selected on Luria-Bertani agar amended with kanamycin (50 μg/ml). After verification by sequencing, the constructs were transferred to F2/5 via conjugal mating and gene disruptions occurred following single homologous recombination. Derivatives of F2/5 were selected on AB minimal medium amended with 10% mannitol and kanamycin (50 μg/ml). Mutations were verified by PCR using primers derived from sequences of the F2/5 chromosome that flank the insertion site and sequence from the pVIK165 vector. Derivatives were then tested for their ability to prevent crown gall formation as described below.

(14) Biological Control Assays:

(15) F2/5 derivatives, Δ F-avi5813, Δ F-avi4329 and Δ F-avi0838, which are all grape necrosis-minus, were tested for their ability to prevent crown gall on potted 1- to 2-month-old grapevines (V. vinifera cv. Chardonnay) in the greenhouse. Avi numbers correspond to gene homologs in the A. vitis S4 genome as described in the literature (see, for example, http://agro.vbi.vt.edu/public/index.html) (Slater et al., J. Bacteriol. 191:2501-11 (2009).). F2/5, derivatives of F2/5, and tumorigenic A. vitis strain CG49 were grown overnight on Potato Dextrose Agar (PDA) or PDA plus kanamycin and suspended in sterile distilled water and adjusted to OD.sub.600=0.1 (corresponding to about 10.sup.8 cells/ml). F2/5 and derivatives of F2/5 were mixed 1:1 with CG49, and 50 μl of the mixed suspensions were inoculated on slice wounds made with a scalpel on woody trunks and/or green shoot tissues of the potted grapevines. Wounds were made just below a side shoot on woody tissue and below a bud on green shoots. After inoculum suspensions dried, the wound sites were wrapped with Parafilm. In general eight plants were inoculated for each treatment and experiments were repeated. The number of inoculation sites at which galls developed was recorded at least 6 weeks after inoculation for green shoots and 8 weeks for woody cutting tissue (Table 3 and FIG. 1). The necrosis-minus derivatives of F2/5 maintained their biological control activity.

(16) TABLE-US-00003 TABLE 3 Biological control of crown gall in grapevine with F2/5 and F2/5 mutants Average Gall.sup.c/ gall size.sup.d Treatment Experiment.sup.b Inoculations (mm.sup.2) CG49 T-1 8/8 217.45 T-2 8/8 141.78 T-3 8/8 175.21 T-4 6/6 203.75 CG49 + F2/5 T-1 0/8 T-2 0/8 T-3 0/8 T-4 0/6 CG49 + Δavi0838 (rirA).sup.a T-1 0/8 T-2 2/8 >10 T-3 1/9 170.10 T-4  3/12 28.98 CG49 + Δavi4329 (amino- T-1 2/8 92.45 transferase) T-2 1/8 33.50 T-3 1/8 145.92 T-4  4/10 27.04 CG49 + Δavi5813 (PPTase) T-1 0/8 T-2 0/8 T-3 0/8 T-4  4/12 21.84 .sup.aavi numbers correspond to genes annotated in the A. vitis strain S4 genome sequence .sup.bT-1, experiment initiated on Dec. 6, 2011; T-2. initiated on Dec. 13, 2011; T-3, initiated on Dec. 19, 2011; T-4 initiated on Jan. 28, 2012. .sup.c“Gall” on the avi0838, avi4329 and avi5813 mutants treated vines may be callus development and not crown gall. .sup.dThe average gall size is calculated as total gall size/gall number instead of inoculation number.
Effect of F2/5 and Derivatives of F2/5 on Necrosis, Root and Callus Development:

(17) Dormant grapevine cuttings were inoculated with strain F2/5 and derivatives of F2/5, ΔF-avi5813), ΔF-avi4329 and ΔF-avi0838 by submersing basal ends of cuttings in water suspensions of the bacteria (about 10.sup.8 cfu/ml) for 4 hours prior to planting them in perlite to initiate callus and rooting. Data on necrosis, callus and root development and incidence of crown gall infection were collected 8 weeks after inoculation (Table 4 and FIG. 2). Cuttings treated with necrosis-minus derivatives produced enhanced roots and callus and lacked the significant necrosis seen on F2/5 treated cuttings.

(18) TABLE-US-00004 TABLE 4 Effect of F2/5 and derivatives on root and callus development and on the level of necrosis on roots. Phenotypes were rates as 0-2 for necrosis (2 being more than 90% of roots having necrosis, 0-3 for root development with a 3 having the largest root mass and 0-3 for callus development with 3 equalling the largest amount of callus development at the cutting base. ΔF- ΔF-avi4329 Treatment/ avi0838 (amino- ΔF-avi 5813 Response F2/5 Water (rirA) transferase) (PPTase) Necrosis (0-2) 2 0.1 0.3 0.2 0.3 Root 1.5 1.5 1.9 1.9 1.7 development (0-3) Callus 0.3 1.5 2.3 2.2 1.7 development (0-3)
On young stem explants, it was also shown that derivatives of F2/5 are necrosis-negative (FIG. 3).

SUMMARY

(19) Crown gall is a significant disease of grapevines that often is initiated at grafts. Grafting may be done in the nursery (scion grafted onto rootstocks) or in the field when changing varieties of an established vineyard. Strain F2/5 is able to prevent the development of crown gall at wound sites on grapevines however is detrimental to graft take. As shown in FIG. 2, F2/5 is also detrimental to root formation. This is likely due to the fact that F2/5, like other A. vitis strains, initiates a necrosis of certain grape tissues.

(20) Mutations in specific genes of strain F2/5 to generate F2/5 derivatives that no longer cause necrosis but have maintained biological control function were generated. These results have been repeatedly confirmed in the greenhouse as presented herein. In some cases small galls develop when the derivatives are applied together with a tumorigenic A. vitis strain (CG49) however the degree of galling is greatly reduced and, in most cases, no gall forms.

(21) It was also observed that when dormant cuttings were treated with the mutants compared to the wildtype F2/5 strain, necrosis on roots was absent, an increased amount of roots were formed and increased callus at the base of the cuttings was noted. This is significant because the treatment is therefore beneficial to use even on grape cuttings that are not grafted. For example certain American and hybrid varieties are not grafted on rootstocks but rather grown on their own roots. Therefore treatment of such varieties with the F2/5 mutants may be advantageous in stimulating callusing and root development.

Example 2

(22) Mutants that Display a Necrosis-Minus Phenotype and Remain Biological Control Positive:

(23) Effects of F2/5 Necrosis-Minus Mutants on Grapevine Graft Take

(24) Effects of F2/5 and the mutant ΔF-avi5813 (PPTase), ΔF-avi4329 (aminotransferase) and ΔF-avi0838 (rirA) on graft take and root or shoot development were determined by treatment of grapevine cuttings (Cabernet Franc) with the bacterial suspensions. Bacterial strains were grown on PDA or PDA amended with kanamycin (50 μg/ml) for 48 hr. The bacterial cells were suspended in water and adjusted to be OD=0.1. Dormant woody cuttings were cut with an omega graft tool. Following cutting the cut surfaces were dipped in bacterial suspensions and then fitted together and wrapped with Parafilm. The cuttings were planted in greenhouse potting mixture and kept moist until analyzed.

(25) TABLE-US-00005 TABLE 5 Effects of A. vitis F2/5 and mutants on grapevine grafts Vines that formed Graft take Root Treatment shoots* Root formation on plants (%) necrosis Water 8/11 (72.7%) 7 with roots, all from rootstock 87% + 1 without roots F2/5 9/12 (75.0%) 8 with roots, all from graft sites 0 ++ 1 without roots CG49 5/12 (41.7%) 4 with roots, 3 from graft sites 20% ++ and 1 from rootstock, 1 without roots ΔF-avi5813 10/14 (71.4)  10 with roots, 8 from rootstock 80% − (PPTase) and 2 from graft sites ΔF-avi0838 10/19 (52.6%)  9 with roots, 6 from rootstock 66% − (rirA) and 3 from graft sites, 1 without root ΔF-avi4329 9/14 (64.3%) 9 with roots, 7 from rootstock 77% − (aminotransferase) and 2 from graft sites F2/5 + CG49 7/18 (38.9%) 5 with roots, all from graft sites 0 ++ 2 without root ΔF-avi5813 + 15/18 (83.3%)  14 with roots,12 from rootstock 85% − CG49 and 2 from graft sites, 1 without root ΔF-avi0838 + 8/13 (61.5%) 8 with roots, 4 from rootstock 50% − CG49 and 4 from graft sites ΔF-avi4329 + 8/13 (61.5%) 8 with roots, 4 from rootstock 50% − CG49 and 4 from graft sites −, no root necrosis; ++, heavy roots necrosis *Cuttings had single buds. Some buds did not grow and therefore the plants did not develop as shown in results.

(26) The results indicate that the mutants improve graft take and root growth as compared to strain F2/5, tumorigenic strain CG49, or CG49 combined with F2/5 (FIG. 4 and Table 5). The roots from cuttings treated with the mutants grow with no or less necrosis and produce more roots (FIG. 4). Graft take was increased in mutant treatments as compared to F2/5, CG49 or CG49 and F2/5 (Table 5).

(27) A. vitis F2/5 Genes Required for Biological Control but not for Necrosis: Avs Locus and F-avi5730

(28) Avs Locus

(29) The Avs locus as being unique to A. vitis strain F2/5 as no homologous loci present have been identified in other A. vitis strains using PCR screens. The Avs locus is approximately 12 kb in length and contains 10 ORFs that are flanked by two genes putatively encoding transposases (FIG. 5A) thereby suggesting horizontal gene transfer as a potential means of inquiry by F2/5. The DNA sequences and deduced amino acid sequences of the genes within Avs locus are shown in FIG. 5. Mutation of either gene P1391 encoding a siderophore synthase or gene P1396 encoding a siderophore ferrichrome-iron receptor resulted in loss of biological control activity by F2/5 (FIG. 5B). All of the genes listed in FIG. 5A are common in A. vitis strains except for those that reside in the Avs locus. Accordingly, one or more genes of the Avs locus is useful for generating new biological control strains by transferring it and expressing in non-tumorigenic A. vitis strains. Exemplary non-tumorigenic A. vitis strains are described in Burr et al. (1999) Plant Disease 83:102-107. Examples of such non-tumorigenic strains are nontumorigenic A. vitis from Vitis riparia (CG511, CG515, CG517, CG518, CG523, CG526, CG529, CG531, CG535, CG537, CG538, CG542, and CG544), nontumorigenic A. vitis from V. riparia (CG546, CG548, CG550, CG553, CG555, CG556, CG559, CG561, and CG565), nontumorigenic A. vitis from V. riparia (CG567, CG569, CG571, and CG572), nontumorigenic A. vitis, biocontrol for grape crown gall (F2/5), and nontumorigenic A. rhizogenes, biocontrol strain (K-84).

(30) NRPS (F-avi5730)

(31) A non-ribosomal peptide synthase (NRPS) gene (F-avi5730) in F2/5 as being required for biological control but not for necrosis was also identified. Avi5730 in F2/5 was mutagenized by gene disruption. The F-avi5730 (residing on chromosome II) mutant did not affect grape necrosis. Gene disruption also resulted in loss of biological control similar to results described above for disruption of the P1391 encoding the siderophore synthase and disruption of P1396 encoding the siderophore ferrichrome-iron receptor. Like Avs and its genes, the NRPS is useful for generating new biological control strains by transferring it and expressing in non-tumorigenic A. vitis strains.

(32) Use

(33) Exemplary uses of any of the engineered strains of Agrobacterium described herein are as follows.

(34) Methods and rates of application of a necrosis-minus, non-tumorigenic Agrobacterium sp. such as a mutant derivative of A. vitis F2/5 which retains biological control of crown gall disease (for example, the PPTase, rirA, and aminotransferase gene mutants described herein) is accomplished according to standard practices known in the art. Typically, bacteria are applied as a dip solution (such as an inoculant) to any grapevine or grapevine component (grapevine component is a somatic embryo, a seed, a seedling, a scion, a rootstock, a cane, a cutting (e.g., a green cutting or a dormant cutting), a leaf, a stem, or a root in, for example, laboratories, nurseries, greenhouses, or in vineyards.

(35) For purposes of the present invention, the bacteria are used as formulations or compositions. Such formulations or compositions contain one or more bacterium described herein and optionally a carrier. The carrier component can be a liquid or a solid material for delivering the formulation or composition to a desired site on a grapevine or grapevine component. Liquids suitable as carriers include water, and any liquid which will not affect the viability of the bacteria. Similarly, solid carriers can be virtually anything that is non-toxic to the bacterium. Non-limiting examples of solid carriers include peat, vermiculite, perlite, and soil or any other material used in commercial propagation of grape. Bacteria may be lyophilized or in powder form prepared according to standard methods known in the art.

(36) Bacteria are administered according to standard practices for applying compositions to grapevine, grapevine component, or for application to a locus for planting a grapevine or a grapevine component such as soil. By the term “effective amount” or “amount effective for” is meant that minimum amount of a bacterial composition or bacterial formulation needed to at least reduce, or substantially eliminate crown gall disease on a grapevine or a grapevine component when compared to an untreated grapevine or grapevine component. Similarly, the terms refer to the minimum amount of bacteria needed to reduce necrosis or to promote callus or root development or both. The precise amount needed will vary in accordance with the particular bacterial composition used; the grapevine or grapevine component to be treated; and the environment in which the grapevine or grapevine component is located. The exact amount of bacteria in a composition needed can easily be determined by one having ordinary skill in the art given the teachings of the present specification. The examples herein show typical concentrations which will be needed to at least reduce crown gall disease as well as for promoting callus and root development.

(37) In some embodiments, the active ingredient of a solid formulation is a necrosis-minus, biological control positive derivative of A. vitis F2/5 (e.g., the PPTase, rirA and aminotransferase gene mutants described herein or any combination of these derivatives) that includes not less than 1,000 million colony forming units (cfu) per gram of moist peat medium. For preparation, 100 grams of solid formulation when mixed with 1 gallon of water typically provides a suspension of approximately 2.6×10.sup.7 (cfu) per milliliter in a dip solution.

(38) In other embodiments, the active ingredient of a liquid formulation is a necrosis-minus, biological control positive derivative of A. vitis F2/5 that includes not less than 1,000 million cfu per milliliter of water.

(39) Plants are typically dipped in a solution of a necrosis-minus, biological control positive derivative of Agrobacterium sp. such as any one of the mutant derivative of A. vitis F2/5 described herein. Alternatively, the formulation or composition is sprayed on a plant; for example, by spraying to graft surfaces during grafting. In other applications, the formulation or composition is applied to a locus where the plant is growing or to be planted.

(40) Compositions and formulations are applied to grapevine or grapevine components as needed according to standard viticultural methods. The compositions can be applied as needed to any plant wound or damaged plant tissue that allows entry of a tumorigenic A. vitis sp. Typically, such applications may be sprayed or painted on the plant. In other situations, applications of the compositions or formulations would be to grafts at time of grafting. This would be most typically done in a nursery as well as in field grafting. An exemplary grafting situation is where a grower changes varieties in the vineyard by grafting to a current vine, for example, changing from Riesling to Chardonnay by field grafting. For example, for grafting applications, grapevine or grapevine components should be treated with the bacteria after cutting or other handling operations that damage plant tissue and before and after cold storage. Bare rootstock or scion material should be dipped or sprayed with the formulation solution until all root surfaces or scion surfaces or the stem above the graft union are completely wet, or spray these tissues to runoff.

(41) Grapevines or grapevine components may also be treated with a formulation or composition before and after cold storage or at both times.

(42) Because the strains described herein also enhance callus and rooting of dormant cuttings, the bacteria useful in the nursery for rooting purposes. This is beneficial on grafted and non-grafted varieties. For example, many of grape hybrids and labrusca varieties are not grafted but callusing and rooting is important for regeneration. Another exemplary use involves a grower to dip rooted and grafted (or non-grafted) vines obtained from a nursery in a formulation or composition prior to planting. Such treatments would enhance growth in the vineyard.

(43) Any of the genes described herein are useful for engineering A. vitis strains (tumorigenic and non-tumorgenic alike) to necrosis minus phenotype and crown gall positive phenotype. Methods for engineering such strains are according to standard methods in the art including those described herein.

Other Embodiments

(44) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.

(45) All publications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.