Questions & Answers for NOGALL

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1 Questions & Answers for NOGALL 1. What is crown gall? A) Crown gall is a cancerous disease of many dicotyledonous plants that is caused by four species of the soil and plant inhabiting ubiquitous bacterium Agrobacterium: Agrobacterium tumefaciens, A. rhizogenes, A. rubi and A. vitis. Bacteria of these species are thought to be present in most agricultural soils and cause disease that affects plants in nurseries, landscapes, vineyards, and orchards worldwide. Shrubs, ornamental flowering trees, fruit trees in temperate to tropical areas, tubers, grapevines, tobacco, annuals and members of Coniferae are just some of the plants affected by crown gall. Galls are typically located on the crown of the plant at or below the soil surface. Fruit trees infected with crown gall produce lower yields, fruit quality and variable fruit set and size. In severe conditions crown gall can weaken or kill the host plant. Losses to the nursery industry can be particularly severe because plants with crown gall must be culled. (Ref. 2, 7, 20, 31) 2. What is the NOGALL product used for? A) NOGALL is a wettable powder inoculant containing a pure culture of the bacterium Agrobacterium radiobacter, strain K1026. It is used as a treatment for the biological control of the pathogenic bacterium Agrobacterium tumefaciens that causes crown gall tumours on some horticultural and ornamental plants. NOGALL is a preventative and not a curative treatment for crown gall diseased plants. 3. NOGALL is recommended for use against crown gall in what plants? A) NOGALL is effective against crown gall pathogens in stone fruit trees, (cherries, apricots, peaches, nectarines, plums, etc) nut trees (almonds, walnuts, pecans, etc) roses, cranberries (boysenberry, raspberry, etc), clematis, hops, kiwifruit, persimmons; and although lab studies show K1026 not directly lethal to pathogens that infect apple and pear trees, field evidence shows some efficacy of K1026 probably due to its strong competitive and colonisation ability, thus, excluding pathogens from infection sites. (Numbers in brackets at the end of answers refer to published papers for further information on this question see references list at the back of the questions.) 1

2 4. How does NOGALL work? A) NOGALL works against many crown gall agrobacteria strains by various mechanisms such as: 1. colonisation of plant wound sites; 2. competition for plant foods and attachment sites; 3. antibiotic production by K1026 including agrocin 84, agrocin 434 and ALS84. (Ref. 6, 9, 10, 12, 14, 15, 19, 20) 5. How should NOGALL be applied? A) NOGALL should be applied as an aqueous dip solution made up by adding NOGALL peat culture to cool clean drinking quality water, free of chemical residues including chlorine. Plant stock should be dipped into the solution and all roots, lower stems and any other parts where wounds may have occurred should be well saturated with the dip solution. NOGALL solution can also be sprayed on wounded tissue both on roots and above ground plant stems. Survival of K1026 on aerial parts will be poor due to detrimental affects of desiccation and sunlight. Separate spraying of aerial plant stems and leaves will not protect roots from infection by crown gall agrobacteria. Make sure the plant stock is thoroughly wetted with the NOGALL solution. (Ref. 5, 19, 20, 22) 6. Can NOGALL be diluted and still work OK? A) No, dilution reduces the bacterial cell count in the dip solution and could reduce efficacy. Less bacterial cells can reduce the colonisation of plant wounds and lead to insufficient production of agrocin 84, 434 and ALS84, thus reducing protection against pathogenic bacteria. (Ref. 6, 9, 17, 18) 7. Is over inoculation harmful? A) No, over inoculation is not harmful to plants and does not impose a safety risk to humans, insects, birds, fish or other animals. 8. How often should the grower change the dip solution, especially if stock has lots of soil adhering to roots? A) If dip solution becomes very dirty change to fresh solution. This will depend on how much soil is attached to propogation stock. 2

3 9. How long can the dip solution be held for use? A) Use dip solution within 24 hours. Contaminant microorganisms can multiply in the dip solution from nutrients introduced with NOGALL. 10. Is there any benefit leaving stock soaking in NOGALL solution? A) There is scientific evidence in recent years showing that the attachment of bacteria to plant wounds happens within 30 minutes of application. Three distinct stages take place before polar cell attachment to wound sites and a suite of genes are involved. Hence, it is particularly important to keep plant stock moist for at least 30 minutes to help strain K1026 attach to wound infection sites and increase chance of biological control of crown gall. (Ref. 13, 21, 27, 31) 11. How much product (units) do nurseries need for treating propogation stock? A) A 12 litre dip solution made up from a 250g pack of NOGALL will treat up to 500 bare rooted trees with stem diameters of about 8-10mm or up to 1000 bare rooted seedlings with 4-6mm stems, or up to 5000 cuttings and up to 10,000 seeds (depending on seed size). Hence, a gallon of dip solution should treat about (with soft ball size root system) bare rooted trees or up to 500 small seedlings with about ¼ caliper stems, depending on how much soil is attached to the root system. 12. Are there any ways to improve use and survival of NOGALL bacteria? A) Any method that protects NOGALL dip solution on plant material will increase colonisation and attachment of K1026 cells to plant wounds. For example, plastic or hessian (burlap?) bags can be used to protect treated stock against sunlight, wind and desiccation following application of the inoculant. 13. Why is the NOGALL peat formulation better than other inoculant products? A) NOGALL is formulated to deliver high numbers of viable cells of the active ingredient Agrobacterium radiobacter strain K1026. It is formulated in a neutralised microfine peat medium with suspending and adhesion agents to aid in plant attachment and to protect the cells after application to plant wounds. K1026 cells are better able to survive desiccation conditions on plant material because they have been grown-up in peat and formulated with additives. The peat formulation also protects the inoculant bacteria during transport and storage of the product. 3

4 14. How long does NOGALL survive on plant material and in soil after planting? A) Inoculation of plant rootstock with NOGALL can add between 1-10 million bacteria per gram of fresh weight of root. This population can reduce to 10,000 to 100,000 after weeks. NOGALL bacteria can survive for at least 2 years in a field environment as a rhizosphere inhabitant on treated stock or on weeds or grass roots, in soil or in association with crown gall tissue. Plant galls are genetically programmed to produce special food (opines) for agrobacteria; some non-disease agrobacteria can also utilise the secreted opines and other food compounds produced by the plant cells in the gall tissue. (Ref. 29) 15. When should nurseries treat with NOGALL? A) Any nursery that buys in propagation stock from a source that has not used NOGALL risks purchasing diseased stock. Disease development usually takes two to four weeks after infection at soil temperatures of 68ºF or above. Development stops altogether at soil temperatures below 50ºF. Always specifiy treatment with NOGALL at each propagation stage. NOGALL treatment should be done immediately or within 2 hours after transferring or lifting plant stock, wounding or removal from cold storage. This minimises risk of disease contact and infection. Value adding by use of NOGALL should be promoted by nurseries. Treating plant wounds with NOGALL at each transfer stage is essential to protect against crown gall infection. If plant material is in contact with crown gall causing agrobacteria prior to treatment with NOGALL, infections can occur and disease can develop. I often explain that a plant wound has infection sites and by saturating these infection sites with K1026 cells, it excludes and protects the plant from disease infection. I often correlate the infection sites to the dimples in a golf ball, fill each one with a K1026 cell and the ball is protected. Crown gall infections have been mostly associated with plant material where active plant cell division is occurring, such as wound areas, next to grafts, emerging lateral roots and lenticels that are accessible by pathogenic bacteria. 16. What should customers do if stock was dipped 3 months ago and they want to plant out? A) Even though plant stock was dipped 3 months prior to planting out NOGALL should be used again at the transfer stage from its growing medium to field planting as new wounds can occur during this transfer stage and offer new infection sites for pathogens in the field. 4

5 17. If my plant stock has crown gall can it be removed then treated with NOGALL to save the plant? A) No, once a plant cell is infected with DNA from the crown gall disease agrobacteria, galling will occur and is then uncontrolled by strain K1026. In some cases the disease-causing agrobacteria can colonise the vascular tissue and cause galling in other parts of the plant where wounds are caused by wind damage, insects such as borers, nematodes, grafting, cracks formed by developing lateral roots or root lenticels, frost injury and other causes. In these cases removing galled tissue would be ineffective against prevention of further galling. A list of plant stock that can have systemic infection is not available but walnuts, almonds, roses, cherries, pears, apples and grapevines are known to have pathogen present in vascular tissue. (per. comm. L. Moore). 18. Can cuttings be put in sawdust for 24 hrs then dipped with NOGALL, then 3 days later planted out? A) Cuttings must be dipped before placing in a sawdust medium. When planting out they should be dipped again as new wounds could be caused by the transfer. Pathogens can be found in soil, water and utensils that have come into contact with them, hence, the sawdust could contain disease agrobacteria. 19. What else can growers do to reduce crown gall? A) The risk of crown gall infection can be reduced by: Using disease free plant stock, Choosing rootstock that is known to be less susceptible to crown gall, Removing and destroying galled plants (e.g. burning) as soon as possible, Using aseptic techniques such as disinfecting pruning and propagation equipment frequently, for example, implements used when grafting, taking cuttings or root prunings, sterilising soil potting mixes, avoiding soil or water that contains crown gall pathogens, Not planting seedlings too deeply, Avoiding soils which are heavily infested with nematodes or root attacking insects, Avoiding clay soils which are subject to water-logging or poor drainage, Avoiding unnecessary damage to roots and cuttings during planting or cultivation, Keeping grafts and buds above soil line, Solarise lighter soils where infestations are known, Irrigate with well water or sanitised pond water, Avoiding fields with recent history of high crown gall infestations. (Ref. 3, 17, 18) 5

6 20. Can NOGALL be mixed with other chemicals, for example, fungicides, insecticides, hormone treatments? A) Some yes, some no, for example: Yes No Benomyl (Benlate) Captan Iprodione (Rovral) Chlorinated water Metalaxyl (Apron) Dazomet Methyl thiophanate Dichlofluanid Proconazole Dodine (Melprex) Tridimifon Guazatine (Panoctine) Bayleton Mancozeb (Dithane&Manzate) Root hormones TMDT when applied Zineb (Curit) properly (see Truban Q. 21) Zyban Previcur Agristrep (streptomycin) (Ref. 12, 23) 21. What method of treatment should be implemented when using growth hormones? A) Treat NOGALL first, then dip in non-alcohol powder based hormones and then plant. For alcohol or water based hormone solutions treat cuttings first with hormone, allow alcohol and/or water to evaporate, then treat with NOGALL and then plant. (per. comm. A. Kerr) 22. How does the disease spread? A) Crown gall disease caused by agrobacteria can spread by splashing rain, cultivation, contaminated utensils, soil, water. Bacteria can be spread in soil by water transport or along developing roots such as on weeds and grasses. Galls can contain 1-10 million disease bacteria per gram of tissue and root rhizosphere counts have been recorded between 10,000 to 1 million per gram of root tissue of cherry, ryegrass and 11 other herbaceous plants. Soil can frequently contain 500 cells per gram, however, most agrobacteria are found in the rhizosphere along roots and in crown gall tissue. Damage to roots of field grown nursery stock from cultivation should be avoided as this creates new infection sites and leads to a build up of galls and disease agrobacteria in the field. (Ref. 29) 6

7 23. How long does the disease survive in soil? A) Populations of pathogenic agrobacteria can survive for years as soil inhabiting bacteria, rhizosphere inhabitant or with crown gall tissue. Generally, populations slowly decline in fallow soil and in water. In one study, agrobacteria soil populations of 100,000 per gram of dry soil declined to about 100 per gram after 8 weeks and less than 100 after 16 weeks. Higher populations are generally more prevalent in heavy clay soils or in soils where water can stand for several days. (Ref. 29) 24. Can I drench the soil around a tree? A) Drenching the soil around a tree with NOGALL solution will increase populations of K1026 temporarily and may lead to increased colonisation of the root rhizophere. This would aid in the protection against crown gall infection but no studies have been done on its field effectiveness against crown gall disease. Drenching is appropriate for potted plants and for seedlings or cuttings in cell trays. 25. If I discover a plant with crown gall can I plant a NOGALL dipped plant there and be safe? A) It is recommended to avoid soil or fields with a history of crown gall. The risk of future infections will be increased if frequent root damage occurs due to cultivation, nemotodes, soil larvae, wind damage or other means. If field planting stock that is susceptible to crown gall ensure all trees are treated with NOGALL just before planting. 26. If K84 has worked for me in the past why do I need to switch to NOGALL? A) Due to: 1. high risk of conjugation by K84 with disease bacteria and subsequent loss of efficacy, 2. NOGALL is an easy to use formulation, 3. NOGALL is a robust product and can be out of refrigeration for several weeks before use without loss of quality, 4. NOGALL has a long shelf life of 6 months. 7

8 27. What evidence is there for the breakdown of biological control of crown gall disease from use of K84? A) This phenomena has been observed in field and laboratory trials in Greece, USA, Spain and Italy. The breakdown of biological control is caused by K84 mating (conjugation) with disease agrobacteria which results in a build-up of resistant pathogens. K1026 protects against this breakdown possibility and ensures the long-term use of K1026 for the biological control of crown gall disease. (Ref. 1, 16, 24, 25, 28, 30, 32, 33) 28. What tests have been done between Norbac and NOGALL? A) One experiment showed Norbac equal to NOGALL for delivery of numbers and their survival on rose cuttings. The survival of NORBAC K84 cells was attributed to its formulation in carboxymethyl cellulose. Both strains produced equal amounts of agrocin What comments do we have from growers going from K84 to K1026? A) See attached letters of support in Australia. (Ref. 6) 30. There is one month left to go on the NOGALL label, what should NBI do with the stock? A) Only sell stock to growers if they will use it within the expiry period. Sales of NOGALL outside the labelled expiry date may lead to reduced efficacy due to lower numbers of viable cells of K1026 in the product and then legal issues may arise if efficacy is in doubt. Outdated stock can be watered into plant stock or disposed of in an authorised landfill. 31. Is the NOGALL label as serious as it insinuates on safety issues? A) NOGALL is very safe for humans, insects, birds, fish and plants. Label precautions use generic statements for use with any bacterial inoculant product and are intended to safeguard misuse and unnecessary exposure to foreign bacteria to eyes, skin and ingestion. Contact with eyes could cause minor irritation. (Ref. 4, 17, 19) 8

9 32. Why doesn t NOGALL or K84 control crown gall in grapevines? A) Grapevines can have infected root or stems and/or vascular system colonisation by pathogens that are not sensitive to the antibiotics produced by K1026 and are genetically quite different to the controllable disease agrobacteria. Hence, root and stem applications of K1026 on grapevine stock will not control crown gall disease. 33. Why is NOGALL not recommended for control of crown gall in propagation stock of apples and pears trees? A) Crown gall disease agrobacteria that infect apples and pears are typically not sensitive to the antibiotics produced by K1026 but have been controlled in many cases in field planting, probably due to the strong colonising and competitive ability of K1026 resulting from their high populations applied from the dip solution. Successful control of crown gall in apples and pears has been generally less successful than in other hosts. The higher risk of infections and generally reduced efficacy precludes it from inclusion in our recommended host range. REFERENCES 1. Alconera, R.A Crown Gall of Peaches from Maryland, South Carolina, and Tennessee and problems with biological control. Plant Disease 64: DeCleene, M., and DeLey, J The host range of crown gall. Botanical Review 42: Fawzi, El-Fiki and Giles, K.L Agrobacterium tumefaciens in Agriculture and Research. Internation Review of Cytology 13: Glenister, C.S The story of a successful biological control. American Nurseryman. April 15, 1987: Htay, K. and Kerr, A Biological Control of Crown Gall: Seed and Root Inoculation. J. appl. Bact. 37: Jones, D.A. and Kerr, A Agrobacterium radiobacter strain K1026, a Genetically Engineered Derivative of Strain K84, for Biological Control of Crown Gall. Plant Disease. 73: Kennedy, B.W., and Alcorn, S.M Estimate of U.S. crop losses to prokaryote plant pathogens. Plant Disease 64: Kerr, A Biological control of crown gall: Seed inoculation. J. Appl. Bacteriol. 35: Kerr, A Soil Microbiological Studies on Agrobacterium radiobacter and Biological Control of Crown Gall. Soil Science. 118: Kerr, A Biological Control of Crown Gall Through Production of Agrocin 84. Plant Disease. 64:

10 11. Kerr, A., Beer, S.V., Schroth, M.N. and Bahme, J.B Biological Control Chapter In: Methods in Phytobacteriology Eds. Klement, Rudolph and Sands. Hungarian Acad. Of Science, Budapest 12. Kerr, A Commercial Release of a Genetically Engineered Bacterium for the Control of Crown Gall. Agricultural Science, Nov Lippincott, B.B. and Lippincott, J.A Bacterial Attachment to a Specific Wound Site as an Essential Stage in Tumor Initiation by Agrobacterium tumefaciens. Journal of Bacteriology. 97: Lopez, M.M., Gorris, M.T., Temprano, F.J. and Orive, R.J Results of seven years of biological control of Agrobacterium tumefaciens in Spain. EPPO Bulletin. 17: Lopez, M.M., Gorris, M.T., Salcedo, C.I. Montojo, A.M. and Miro, M Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees. Appl. Environ. Microbiol. 55: Lu, S. F Isolation of putative pagk84 transconjugants from commercial cherry and raspberry plants treated with Agrobacterium radiobacter strain K84. M.S. Thesis. Oregon State University, Corvallis. (Thesis available on request). 17. Moore, L.W Biological Control of Crown Gall. Proc. 2 nd Woody Ornamental Disease Workshop Moore, L.W Practical use and success of Agrobacterium radiobacter strain 84 for crown gall control. Biology and Control of Soil Borne Pathogens, New York: Academic Press, pp Moore, L.W. and Warren G Agrobacterium radiobacter strain 84 and biological control of crown gall. Ann. Rev. Phytopathol. 17: Moore, L.W Controlling crown gall. American Nurseryman. May 15, 1997: Nester, E.W., Gordon, M.P., Amasino, R.M. and Yanofsky M.F Crown gall: A molecular and physiological analysis. Ann. Rev. Plant Physical 35: New, P.B., and Kerr, A Biological control of crown gall: field measurements and glasshouse experiments. J. of Appl. Bacteriol. 35: Oros, G The influence of pesticides on the antagonistic effect of Agrobacterium radiobacter to A. tumefaciens. P. Int. Conf. Integr. Plant Prot. 4: Panagopoulos, C.G., Psallidas, P.G., and Alivizatos, A.S Evidence of a breakdown in the effectiveness of biological control of crown gall. In Schippers, B., Gams, W. (Eds.): Biology and Control of Soil-Borne Plant Pathogens, New York: Academic Press, pp Peluso, R., Zoina, A. Morra, F., Sigillo, L., and Raio, A Outbreak of dangerous transconjugant Agrobacterium strains in a stone-fruit nursery field. Journal of Plant Pathology. 83:

11 26. Peňalver, R., Vicedo, B., and Lopez, M.M Use of genetically engineered Agrobacterium strain K1026 for biological control of crown gall. European Journal of Plant Pathology. 106: Reuhs B.L., Kim J.S. and Matthysse A.G Attachment of Agrobacterium tumefaciens to carrot cells and arabidopsis wound sites in correlated with the presence of a cell-associated, acidic polysaccharide. Journal of Bacteriology 179: Stockwell, V.O., Kawalek, M.D., Moore, L.W., and Loper, J.E Plasmid transfer between Agrobacterium radiobacter strain K84 and A. tumefaciens in crown gall tissue. Phytopathol. 80:1001 (abstr). 29. Stockwell, V.O., Moore, L.W. and Loper, J.E. (1)1993. Fate of Agrobacterium radiobacter K84 in the environment. Applied and Environmental Microbiology 59: Stockwell, V.O., Kawalek, M.D., Moore, L.W., and Loper, J.E Transfer of pagk84 from the biocontrol agent Agrobacterium radiobacter K84 to A. tumefaciens under field conditions. Biological Control 86: Sykes, L.C. and Matthysse, A.G Time required for tumor induction by Agrobacterium tumefaciens. Applied and Environmental Microbiology. 52: Sule, S. and Kado, C.I Agrocin resistance in virulent derivatives of Agrobacterium tumefaciens harbouring the pti plasmid. Physiological Plant Pathology 17: Vicedo, B., Peňalver, R., Asina, M.J. and Lopez, M.M Biological control of Agrobacterium tumefaciens, colonization, and pagk84 transfer with Agrobacterium radiobacter strain K84 and the tra - mutant strain K1026. Appl. Environ. Microbiol. 59: