Cross-Reference to Related Applications

This application claims benefit of U.S. Provisional Application No. 61/635,160, filed April 18, 2012, which is hereby incorporated by reference in its entirety.

Statement as to Federally Funded Research

This invention was made with government support under Hatch Grant No. 2009-10-381 awarded by the U.S. Department of Agriculture. The government has certain rights in the invention.

Background of the Invention

This invention relates to biological control of crown gall disease on grapevines as well as enhancing grapevine development.

Crown gall disease of grapevine caused by Agrobacterium vitis is a limiting factor to grape production in several regions of the world (Burr et al. (1999) Annu. Rev. Phytopathol. 37:53-80). A. vitis survives systemically in grapevines and therefore is disseminated in propagation material (Burr et al. (1984) Plant Dis. 68:976-978; Burr and Katz (1983)

Phytopathology 73: 163-165; Goodman et al. (1987) Am. J. Enol. Vitic 38:189-193). Like crown gall on other plant species, tumorigenesis results following the transfer of a set of oncogenes called the T-DNA that are carried on a bacterial Ti plasmid; the T-DNA becomes integrated and expressed in the plant host (Chilton et al., (1977) Cell 11 : 263-271) leading to crown gall. A. vitis strains can be grouped based on the type of Ti plasmid they carry and may also be designated by the opine catabolic gene(s) that resides on their Ti plasmid (Otten et al. (1996) Mol. Plant-Microbe Interact. 9:782-786).

There are no effective chemical controls to combat crown gall and therefore the interest in developing a biological control. Biological control of crown gall on specific crops, such as stone fruits has been successfully implemented in commercial agriculture using A. radiobacter strain K84 (Farrand, (1990) In R. R. Baker, and P. E. Dunn (Eds.) pp. 679-691). K84 is not effective against A. vitis strains on grapevine and therefore several research groups have investigated other candidate biological control strains for crown gall control on grape. Some of the more recent research on biological control of grape crown gall included nontumorigenic A. vitis strain E26 (Liang et al. (1990) Acta Microbio Sin 30: 165-171) and strain VAR03-1 (Kawaguchi et al. (2008) Phytopathology 98: 1218-1225). Other strains including Rahnella aquatilis strain HX2 (Chen et al. (2007) Plant Disease 91 : 957-963), Serratia plymuthica strain IC1270, Pseudomonas fluorescens strain Q8rl-96, P. fluorescens strain B-4117 (Dandurishvili et al. (2010) Journal of Applied Microbiology 110: 341-352) and others were shown to provide various levels of grape crown gall inhibition under experimental conditions (Eastwell et al. (2006) Crop Protection 25: 1191-1200).

Accordingly, there is a need in the art for effective controls of crown gall disease on grapevines. The present invention provides such a need.

Summary of the Invention

The invention described herein relates to genetically engineered Agrobacterium strains (for example, Agrobacterium vitis F2/5) useful as a biological control agent for the prevention of crown gall disease caused by the infection of nursery stock and vineyards by many virulent strains of Agrobacterium such as A. vitis.

Accordingly, in general, the invention features genetic derivatives of a non-tumorigenic Agrobacterium vitis strain F2/5. In particular, such derivatives have a reduced ability to cause a necrosis of grape tissue while retaining biological control of crown gall disease. Identification of such derivatives is accomplished through standard methods such as those described herein.

In one aspect, the invention features a necrosis-minus, Agrobacterium vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control. Exemplary derivatives are typically provided in an isolated or pure form. In one embodiment, the necrosis-minus A. vitis derivative of F2/5 has an inactivated gene encoding phosphopantetheinyl transferase (PPTase) (e.g., an inactivated homolog of A. vitis S4 (avi5813)), an inactivated gene encoding aminotransferase (e.g., an inactivated homolog of A. vitis S4 ( avi4329)), an inactivated gene encoding iron response regulator (e.g., an inactivated rirA homolog of A. vitis S4 (avi0838)); or a combination thereof. Other genes of necrosis which may be inactivated to engineer a necrosis- minus A. vitis derivative of strain F2/5 include a member of transcriptional regulator LuxR family aviR (F-avi4374), a proteolytic subunit of ATP-dependent Clp protease clpPl(F- avil696), nonribosomal peptide synthetase (F-avi3342) and polyketide synthase (F-avi4330). Methods for inactivating such genes in bacteria are well known in the art and include, without limitation, standard methods such as gene disruption by insertion of a DNA fragment and gene replacement in which the target gene is replaced by a select marker such as antibiotic resistance gene via double recombination at flanking regions of the target gene as well as the methods and biological control assays for analyzing such derivatives as is disclosed herein.

Exemplary sequences for A. vitis strain F2/5 related to necrosis and crown gall biological control are listed in Table 1. Table 1

P1397 Nucleic acid (717 nucleotides) SEQ ID NO: 34

A. vitis strain Protein (238 amino acids) - transposase SEQ ID NO: 35 F2/5

F-avi5730 Nucleic acid (3879 nucleotides) SEQ ID NO: 36

F-avi5730 Protein (1292 amino acids) SEQ ID NO: 37

In another aspect, the invention features a biologically-pure culture including a necrosis- minus, A. vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control. Exemplary derivatives are described herein.

In another aspect, the invention features a composition including a necrosis-minus, A. vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control.

Exemplary derivatives are described herein.

In still other aspects, the invention features a grapevine or grapevine component including a necrosis-minus, A. vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control. Exemplary derivatives are described herein. In one embodiment, the 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 another embodiment, the grapevine or grapevine component includes one or more of the F2/5 derivatives, described herein.

Exemplary grape plants for use in the practice of the invention include, but are not limited to, grapevines (for example, Vitis spp., Vitis spp. hybrids, and all members of the subgenera Euvitis and Muscadinia), including scion or rootstock cultivars. Exemplary scion cultivars include, without limitation, those which are referred to as table or raisin grapes and those used in wine production such as Cabernet Franc, Cabernet Sauvignon, Chardonnay (for example, CH 01, CH 02, CH Dijon), Merlot, Pinot Noir (PN, PN Dijon), Semillon, White Riesling, Lambrusco, Thompson Seedless, Autumn Seedless, Niagrara Seedless, and Seval Blanc. Other scion cultivars which can be used include those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson seedless, and Thomuscat. They also include those used in wine production, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Caberet, Sauvignon, Calzin, Carignan, Charbono, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green

Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint- Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Lampia, Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitivo di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tarmat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling, and Zinfandel.

Rootstock cultivars that are useful in the invention include, without limitation, Vitis rupestris Constantia, Vitis rupestris St. George, Vitis California, Vitis girdiana, Vitis rotundifolia, Vitis rotundifolia Carlos, Richter 110 (Vitis berlandieri x rupestris), 101-14 Millarder et de Grasset (Vitis riparia x rupestris), Teleki 5C (Vitis berlandieri x riparia), 3309 Courderc (Vitis riparia x rupestris), Riparia Gloire de Montpellier (Vitis riparia), 5BB Teleki (selection Kober, Vitis berlandieri x riparia), S0 ₄ (Vitis berlandieri x rupestris), 41B Millardet (Vitis vinifera x berlandieri), and 039-16 (Vitis vinifera x Muscadinia). Additional rootstock cultivars which can be used include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33EM, Freedom, Ganzin 1 (A x R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (S0 ₄ ), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint- George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis California, and Vitis girdiana.

In still another aspect, the invention features a method for reducing crown gall disease on a grapevine or grapevine component, the method including administering to a grapevine or grapevine component an effective amount of a necrosis-minus, A. vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control; a culture including a necrosis- minus, A. vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control or a composition including a necrosis-minus, A. vitis derivative of strain F2/5, wherein the derivative retains crown gall biological control. Exemplary derivatives are described herein. In one embodiment, one or more of the bacterial derivatives, culture, or composition is administered at grafting time. In another embodiment, one or more of the bacterial derivatives, culture, or composition is administered on graft unions or the base of grapevine. In still another embodiment, one or more of the bacterial derivatives, culture, or composition is administered during field grafting of grapevine. In this case, the biocontrol agent is applied when vinyardists are top-grafting vines; a process that is used to change varieties in vineyards without removing current vines. In other embodiments, one or more of the bacterial derivatives, culture, or composition is administered to a dormant grapevine cutting. The biocontrol agent may also be applied to the basal end of each grafted or nongrafted vine to control crown gall and to stimulate callus and root formation. In certain embodiments, one or more of the bacterial derivatives, culture, or composition is administered to a dormant cane cutting or to a green shoot cutting. And in yet other embodiments, one or more of the bacterial derivatives, culture, or composition is administered to grapevine plant part above the ground (e.g., a trunk or cane). If desired, one or more of the necrosis-minus F2/5 derivatives is administered in any of the aforementioned embodiments.

In still another aspect, the invention features a method for reducing necrosis on a grapevine or grapevine component, the method including administering to a grapevine or grapevine component an effective amount of one or more of bacterial derivatives, cultures, or compositions described herein, wherein the method reduces necrosis on the grapevine or grapevine component. Administration of the derivative of F2/5, culture, or composition is administered at grafting time; for example, on graft unions or the base of grapevine. In other embodiments, administration occurs during field grafting of grapevine or to a dormant cane cutting prior to rooting. In other embodiments administration is to a green shoot cutting.

Typically, the bacterial derivatives, culture, or composition, in this method, is administered to grapevine plant part which grows below the ground. If desired, one or more of the necrosis- minus F2/5 derivatives is administered in any of the aforementioned embodiments.

In still another aspect, the invention features a method for controlling crown gall disease, the method including administering to a locus for planting a grapevine or grapevine component an effective amount of the derivatives of F2/5, cultures, or compositions described herein, wherein the method controls crown gall disease. In one embodiment, the locus is a furrow or soil (e.g., the area where grape plants of a vineyard are to be planted). If desired, one or more of the necrosis-minus F2/5 derivatives is administered in any of the aforementioned embodiments.

In still another embodiment, the invention features a method for identifying an A. vitis F2/5 as a biocontrol agent for crown gall disease, the method including the steps of: a) administering the derivative of F2/5, culture, or composition to a grapevine or grapevine component; and b) assaying incidence of crown gall disease on the grapevine or grapevine component, wherein a reduction in crown gall disease compared to a control grapevine or grapevine component identifies the A. vitis strain F2/5 as a biocontrol agent for crown gall disease. In one embodiment, the A. vitis strain F2/5 is a biocontrol agent for crown gall disease on a grape plant part growing above the ground (e.g., where the grape plant part is a graft, trunk, or cane). Again, if desired, one or more of the necrosis-minus F2/5 derivatives is administered in any of the aforementioned embodiments.

In another aspect, the invention features a method for identifying an A. vitis F2/5 as a biocontrol agent for necrosis, the method including the steps of: a) administering any one of a derivative of F2/5, culture, or composition to a grapevine or grapevine component; and b) assaying incidence of necrosis on the grapevine or grapevine component, wherein a reduction in necrosis compared to a control grapevine or grapevine component identifies the A. vitis strain F2/5 as a biocontrol agent for necrosis. In one embodiment, the A. vitis strain F2/5 is a biocontrol agent for necrosis on a grape plant part growing below the ground (e.g., where the grape plant part is a root or callus). And still again, if desired, one or more of the necrosis-minus F2/5 derivatives is administered in any of the aforementioned embodiments.

In another aspect, the invention features a method for identifying an A. vitis F2/5 to promote callus development, the method including the steps of: a) administering any one of a derivative of F2/5, culture, or composition to a grapevine or grapevine component; to a grapevine or grapevine component; and b) assaying callus development on the grapevine or grapevine component, wherein an increase in callus development compared to a control grapevine or grapevine component identifies the A. vitis strain F2/5 promoting callus development.

In still another embodiment, the invention features a method for identifying an A. vitis F2/5 to promote root development, the method including the steps of: a) administering any one of a derivative of F2/5, culture, or composition to a grapevine or grapevine component; and b) assaying root development on the grapevine or grapevine component, wherein an increase in development compared to a control grapevine or grapevine component identifies the A. vitis strain F2/5 promoting root development.

In another aspect, the invention features a method of producing an A. vitis resistant vineyard, the method including the steps of: a) administering at least one necrosis-minus necrosis, A. vitis strain F2/5, wherein the A. vitis F2/5 retains crown gall biological control to a grapevine or grapevine component; b) planting the grapevine or grapevine component; c) growing the grapevine or grapevine component to establish the A. vitis resistant vineyard. In certain embodiments, administration of the derivative of F2/5 occurs in the vineyard. The invention accordingly features a vineyard produced according to this method. The invention further features a product derived from a grapevine produced in the vineyard. Such products include grapes, raisins, or other foodstuffs or beverages such as wine or juice.

In another aspect, the invention features a method of engineering a necrosis-minus derivative of A. vitis F2/5, the method including the steps of: a) introducing into an A. vitis F2/5 bacterium a construct which inactivates a gene which induces grape necrosis; and b) determining whether the A. vitis F2/5 induces grape necrosis, wherein a reduction in grape necrosis compared to a control A. vitis F2/5 is taken as identifying a necrosis-minus derivative of A. vitis F2/5. In one embodiment, the gene encodes a PPTase, an aminotransferase or is a homolog of iron response regulator, rirA. Exemplary genes are described herein including homologs of A. vitis S4 avi5813, avi4329, avi0838, avi4374, avi3342, avil696, or avi3342. In certain embodiments, the gene includes a deletion of all or part of the gene with a suicide vector.

In another embodiment, the invention features a method for promoting callus development, the method including administering to a grapevine or grapevine component an effective amount of any one of a derivative of F2/5, culture, or composition, wherein the method promotes callus development on the grapevine or grapevine component.

In yet another aspect, the invention features a method for promoting root development, the method including administering to a grapevine or grapevine component an effective amount of any one of a derivative of F2/5, culture, or composition, wherein the method promotes root development on the grapevine or grapevine component.

In still another aspect, the invention features a method for reducing necrosis, the method includes administering to a grapevine or grapevine component an effective amount of any one of a derivative of F2/5, culture, or composition, wherein the method reduces necrosis of grapevine or grapevine component tissue growing below ground.

In still other aspects, the invention features an A. vitis bacterium attenuated by a non- reverting mutation in one or more of the PPTase gene (avi5813), the aminotransferase gene

(avi4329), and the iron-responsive regulator gene, rirA (avi0838) or a functional homolog of the PPTase gene, aminotransferase gene, or the iron-responsive regulator gene. Such a bacterium is a non-tumorigenic A. vitis, for example, the F2/5 strain. In some embodiments, the invention includes a method for reducing crown gall disease on a grapevine or grapevine component, the method including administering to the grapevine or grapevine component an effective amount of these described A. vitis strains.

In another aspect, the invention features an engineered Agrobacterium bacterium including (a) one or more recombinant genes encoding (i) a sequence including SEQ ID NO: 19 or a functional homolog, (ii) a multidrug resistance protein B (SEQ ID NO: 21) or a functional homolog, (iii) a siderophore synthetase (SEQ ID NO: 23) or a functional homolog, (iv) a tauropine dehydrogenase (SEQ ID NO: 25) or a functional homolog, (v) a cysteine synthetase (SEQ ID NO: 27) or a functional homolog, (vi) diaminopimelate decarboxylase (SEQ ID NO: 29) or a functional homolog, (vii) a sequence including SEQ ID NO: 31 or a functional homolog, or (viii) a ferrichrome-iron receptor (SEQ ID NO: 33) or a functional homolog; and optionally (b) one or more recombinant genes encoding a transposase or a functional homolog. In some embodiments of the engineered Agrobacterium bacterium, the one or more recombinant genes encoding one or more recombinant proteins encode at least one protein listed in Figure 5A or a functional homolog of at least one protein listed in Figure 5A. In some embodiments of the engineered Agrobacterium bacterium, expression of an operon including at least a siderophore synthetase or a ferrichrome-iron receptor is controlled by a recombinant promoter, and wherein the promoter is constitutive or inducible. In some other embodiments of the engineered

Agrobacterium bacterium, the Agrobacterium is a non-tumorgenic A. vitis, for example, A. vitis F2/5. Typically, the engineered Agrobacterium bacterium is necrosis minus. Such engineered bacteria are useful for controlling crown gall disease; in particular, biological control.

In other aspects, the invention features an engineered Agrobacterium bacterium including one or more recombinant gene encoding a sequence including SEQ ID NO: 37. In some embodiments, the engineered Agrobacterium bacterium is a non-tumorgenic A. vitis.

In other aspect, the invention features a method for producing an Agrobacterium species having biological control on grapevine, the method including the steps of (a) providing a non- tumorgenic Agrobacterium species having a control of crown gall disease negative phenotype; and (b) introducing into the Agrobacterium species a sequence including one or more recombinant genes encoding (i) a sequence including SEQ ID NO: 19 or a functional homolog, (ii) a multidrug resistance protein B (SEQ ID NO: 21) or a functional homolog, (iii) a siderophore synthetase (SEQ ID NO: 23) or a functional homolog, (iv) a tauropine

dehydrogenase (SEQ ID NO: 25) or a functional homolog, (v) a cysteine synthetase (SEQ ID NO: 27) or a functional homolog, (vi) diaminopimelate decarboxylase (SEQ ID NO: 29) or a functional homolog, (vii) a sequence including SEQ ID NO: 31 or a functional homolog, or (viii) a ferrichrome-iron receptor (SEQ ID NO: 33) or a functional homolog; and optionally, (viiii) a sequence including SEQ ID NO: 37 or a functional homolog thereof.

In other embodiments, the invention features an isolated and purified nucleic acid molecule, obtainable from A. vitis, which consists of at least one gene capable of conferring biological control of crown gall phenotype upon a genetically engineered microorganism, wherein the nucleic acid molecule is contained in the within a locus referred to herein as Avs (SEQ ID NO: 15). In some embodiments, the invention features a plasmid containing such a nucleic acid molecule. In other embodiments, the invention includes a genetically engineered Agrobacterium bacterium having stably and functionally incorporated therein the nucleic acid molecule. Typically, the Agrobacterium bacterium is A. vitis F2/5 such as a derivative that is necrosis minus.

In another aspect, the invention features an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule includes a nucleotide sequence substantially identical to any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 as described in Table 1 and herein. Typically, nucleic acid molecule includes a heterologous nucleotide sequence that inactivates, diminishes, or abrogates expression of the isolated nucleic acid molecule.

In another aspect, the invention features an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule includes a nucleotide sequence that encodes an amino acid sequence substantially identical to any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 as described in Table 1 and herein.

In another aspect, the invention features an engineered Agrobacterium bacterium, wherein the bacterium includes any one of the isolated nucleic acid molecules having a sequence according to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36 as described in Table 1 and herein.

By "substantially identical" is meant an amino acid sequence or nucleic acid sequence that exhibits at least 50% identity to a reference sequence. Such a sequence is generally at least, e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical at the amino acid level or nucleic acid level to a reference sequence. In general, for polypeptides, the length of comparison sequences can be at least five amino acids, e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, or more amino acids, up to the entire length of the polypeptide. For nucleic acids, the length of comparison sequences can generally be at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 900, or more nucleotides, up to the entire length of the nucleic acid molecule. It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to a uracil nucleotide.

As used herein, when a polypeptide or nucleic acid sequence is referred to as having "at least X% sequence identity" to a reference sequence, it is meant that at least X percent of the amino acids or nucleotides in the polypeptide or nucleic acid are identical to those of the reference sequence when the sequences are optimally aligned. An optimal alignment of sequences can be determined in various ways that are within the skill in the art, for instance, the Smith Waterman alignment algorithm (Smith et al., J. Mol. Biol. 147: 195-7, 1981) and BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215: 403-10, 1990). These and other alignment algorithms are accessible using publicly available computer software such as "Best Fit" (Smith and Waterman, Advances in Applied Mathematics, 482-489, 1981) as incorporated into GeneMatcher PlusTM (Schwarz and Dayhof, Atlas of Protein Sequence and Structure, Dayhoff, M.O., Ed pp 353-358, 1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR). In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve optimal alignment over the length of the sequences being compared.

The invention provides significant advantages for viticulturists, vineyardists, and grape nurserymen among others. Crown gall causes significant economic loss in nurseries worldwide. Application of derivatives of F2/5 offers great promise for controlling this disease and for improving survival and quality of grapevines in the nursery as well as in vineyards. The invention is especially advantageous because the disclosed necrosis-minus necrosis, A. vitis strain F2/5, which retains crown gall biological control minimizes necrosis. Furthermore, this biocontrol agent is not detrimental to grafting, callus formation and rooting of grapevines.

Indeed, such bacteria enhance callus and root formation on grapes.

Other features and advantages of the invention will be apparent from the following Drawings, Detailed Description, and Claims. Brief Description of Drawin2S

Figure 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.

Figure 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.

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

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

Figure 5 shows the gene organization and annotation of Avs locus (A) and the effects of knock-down mutant API 391 and API 396 on biological control (B).

Figure 6 shows the sequences listed in Table 1.

Detailed Description

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, NY 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.

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.

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

Development of Agrobacterium vitis F2/5 mutants:

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. Table 2. Genes mutated and primers used in generation of the mutants

Gene primer name primer sequence* size of fragment targeted amplified

F-avi5813 avi5813-F: 5 ' -AAGAATTCTTGATCTCGCATCGCGCTG-3 '

(PPTase) avi5813-R: 5 ' -AATCTAGATGCGCTGTCATGACCTGTTCTG-3 '

F-avi4329 avi4329-F: 5 ' -ACGAGCTCAGCTCGTCAGCGAGCAGCTG-3'

(aminoavi4329-R: 5 ' -AGTCTAGATGAACTCGTCATGGATCACCAG-3 ' 382bp transferase)

F-avi0838 avi0838-F 5 ' -ACGAGCTCTGCCAAGGCCTGTGGTGTCTC-3'

(rirA) avi0838-R 5 ' -AGTCTAGATGTCGATC AACGGGCAATCGAC-3 ' 224bp *: Restriction sites are underlined

Xbal and Sad restriction sites were introduced into forward and reverse primers respectively. PCR products were purified, digested and then ligated into suicide vector pVIKl 65 at Xbal and Sac I sites. The constructs were transformed into E. coli strain Sll-l/ pir by electroporation and transformants were selected on Luria-Bertani agar amended with kanamycin (5( g/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 (5( g/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 pVIKl 65 vector. Derivatives were then tested for their ability to prevent crown gall formation as described below.

Biological control assays:

F2/5 derivatives, Δ F-avi5813, A 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. Bacterid. 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 ₆ oo = 0.1 (corresponding to about 10 ⁸ cells/ml). F2/5 and derivatives of F2/5 were mixed 1 : 1 with CG49, and 50μ1 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 Figure 1). The necrosis-minus derivatives of F2/5 maintained their biological control activity. Table 3. Biological control of crown gall in grapevine with F2/5 and F2/5 mutants

a. avi numbers correspond to genes annotated in the A. vitis strain S4 genome sequence

b. T-l, experiment initiated on 12-06-2011; T-2.initiated on 12-13-2011; T-3, initiated on 12-19-2011; T-4 initiated on 01-28-2012. ^c . "Gall" on the avi0838, avi4329 and avi5813 mutants treated vines may be callus development and not crown gall.

d The 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:

Dormant grapevine cuttings were inoculated with strain F2/5 and derivatives of F2/5, AF -avi5813), AF- avi4329 and A.F-avi0838 by submersing basal ends of cuttings in water suspensions of the bacteria (about 10 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 Figure 2). Cuttings treated with necrosis-minus derivatives produced enhanced roots and callus and lacked the significant necrosis seen on F2/5 treated cuttings. Table 4. Effect of F2/5 and derivatives on root and callus development and on the level of necrosis on roots.

Phenotypes were rated 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.

On young stem explants, it was also shown that derivatives of F2/5 are necrosis-negative (Figure 3).

Summary

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 Figure 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.

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.

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

Mutants that display a necrosis-minus phenotype and remain biological control positive

Effects of F2/5 necrosis-minus mutants on grapevine graft take

Effects of F2/5 and the mutant AF-avi5813 (PPTase), AF-avi4329 (aminotransferase) and AF-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 (5( g/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.

Table 5. Effects of A. vitis F2/5 and mutants on grapevine grafts

-, no root necrosis; ++, heavy roots necrosis

*Cuttings had single buds. Some buds did not grow and therefore the p ants did not develop as shown in results.

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 (Figure 4 and Table 5). The roots from cuttings treated with the mutants grow with no or less necrosis and produce more roots (Figure 4). Graft take was increased in mutant treatments as compared to F2/5, CG49 or CG49 and F2/5 (Table 5). A. vitis F2/5 genes required for biological control but not for necrosis: Avs locus and F- avi5730

Avs locus

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 (Figure 5 A) 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 Figure 5. Mutation of either gene PI 391 encoding a siderophore synthase or gene PI 396 encoding a siderophore ferrichrome-iron receptor resulted in loss of biological control activity by F2/5 (Figure 5B). All of the genes listed in Figure 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).

NRPS (F-avi5730

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 PI 391 encoding the siderophore synthase and disruption of PI 396 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.

USE

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

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.

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.

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.

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 x 10 ⁷ (cfu) per millilter in a dip solution. 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 millilter of water.

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.

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.

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

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.

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

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.

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.

What is claimed is: