US-12616207-B2 - Boxwood endophyte Burkholderia sp SSG as potential biocontrol agent against a wide range of pathogens

Abstract

The disclosure, in one aspect, relates to compositions containing non-pathogenic Burkholderia species SSG and/or extracts from SSG cultures and methods of making and using the same. Also disclosed are methods for treating and preventing plant diseases caused by pathogens, the methods including applying the disclosed compositions to plants and/or soil, diseased leaf debris, or other plant growth media surrounding the plants. Finally, disclosed are methods for increasing plant growth using the disclosed compositions. The compositions are effective as well as environmentally benign and are not detrimental to human or animal health This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present disclosure.

Inventors

• Ping Kong

Assignees

• VIRGINIA TECH INTELLECTUAL PROPERTIES, INC.

Dates

Publication Date

20260505

Application Date

20210303

Claims (18)

1. 1 . A composition comprising a cell-free supernatant produced by culturing a microbial strain comprising a DNA sequence exhibiting at least 99.5% sequence identity to SEQ ID NO. 1 and at least one of SEQ ID NOs. 2-6; and at least one excipient, diluent, or carrier.
2. 2 . The composition of claim 1 , wherein the at least one excipient, diluent, or carrier comprises a surfactant, a solvent, an emulsifier, a buffer, a cryoprotectant, a salt, a microbial culture medium, a wetting agent, a bulking agent, an anti-caking agent, a thickener, a clay, a mineral, a lipid, a gum, a dye or colorant, a biological waste material, or any combination thereof.
3. 3 . The composition of claim 1 , wherein the composition further comprises an effective amount of at least one additional active ingredient, wherein the additional active ingredient comprises a fertilizer, a pesticide, an herbicide, or any combination thereof.
4. 4 . A method for treating or preventing a plant disease caused by a pathogen, the method comprising applying the composition of claim 1 to a plant or a material in a bed surrounding a plant.
5. 5 . The method of claim 4 , wherein the composition is applied from about 2 to about 30 days prior to exposure to the pathogen.
6. 6 . The method of claim 4 , wherein the composition is applied from about 3 hours to about 48 hours after exposure to the pathogen.
7. 7 . A method for treating or preventing a plant disease caused by a pathogen, the method comprising applying the composition of claim 1 to at least one environmental component of a plant, wherein the at least one environmental component comprises leaf debris, soil, the plant's rhizosphere, or any combination thereof.
8. 8 . The method of claim 4 , wherein applying the composition reduces primary inocula by at least 30% after 5 days.
9. 9 . The method of claim 4 , wherein the pathogen comprises a bacterium, a fungus, an oomycete, or a virus.
10. 10 . The method of claim 9 , wherein the fungus comprises Calonectria pseudonaviculata, Alternaria tenuissium, Botrytis cinerea, Collectortrichum acutatum , Collectortrichum frutticola, Collectortrichum gleosporiodes, Collectortrichum viniferum, Fusarium proliferaturm, Fusarium solani , or Volutella buxi.
11. 11 . The method of claim 4 , wherein the composition is applied to roots, leaves, fruits, flowers, stems, or seeds of the plant, soil, compost, mulch, leaf litter, sawdust, straw, pine straw, wood chips, gravel, plant growing medium, other material in a bed surrounding the plant, or any combination thereof.
12. 12 . The method of claim 4 , wherein the plant comprises Fraser fir, petunia, rhododendron , annual vinca , bell pepper, cucumber, tomato, hydrangea , pansy, boxwood, geranium, or impatiens.
13. 13 . The method of claim 12 , wherein the plant pathogen is a fungus and wherein the fungus comprises Calonectria pseudonaviculata and the plant comprises boxwood.
14. 14 . The method of claim 4 , wherein performing the method reduces at least one symptom of the plant disease by at least 50%.
15. 15 . The method of claim 14 , wherein the at least one symptom comprises leaf yellowing, leaf loss, wilting, dwarfing or hypoplasia, gall formation, mycelium or mold growth, smuts, rusts, sclerotia, tissue necrosis, cankers, blight, rot, hypertrophy, or any combination thereof.
16. 16 . The method of claim 4 , wherein performing the method reduces transmission of the plant disease by at least 50%.
17. 17 . The method of claim 4 , wherein the plant pathogen is a fungus and wherein performing the method lyses at least a portion of fungal conidia, causes defects in formed fungal conidia, suppresses mycelial growth, reduces survival of fungal microsclerotia, reduces sporulation, or any combination thereof.
18. 18 . The composition of claim 1 , wherein the at least one excipient, diluent, or carrier comprises 0.01% polysorbate 20.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a U.S. national phase application under 35 U.S.C. 371 of International Patent Application No. PCT/US2021/020606, filed Mar. 3, 2021, which claims priority upon U.S. Provisional Application No. 62/985,165, filed on Mar. 4, 2020, and U.S. Provisional Application No. 63/010,644, filed on Apr. 15, 2020. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with U.S. Government support under grant numbers 16-8130-0202-CA and 17-8130-0282-CA, and by Specialty Crop Block Grant for Virginia (FFY 2018-586, 301-190934), all awarded by the U.S. Department of Agriculture. The U.S. government has certain rights in the invention. CROSS REFERENCE TO SEQUENCE LISTING The genetic components described herein are referred to by sequence identifier numbers (SEQ ID NO). The SEQ ID NOs correspond numerically to the sequence identifiers <400>1, <400>2, etc. The sequence listing in written computer readable format (CRF) as a text file named “222204-1875_Replacement_Sequence_Listing_ST25.txt” created on Jan. 30, 2023 and updated on Mar. 6, 2023, and having a size of 11,144,438 bytes, is incorporated by reference in its entirety. BACKGROUND Boxwood (Buxus) is a genus of about 70 species in the family Buxaceae. As slow-growing evergreen shrubs and small trees, they are planted worldwide, especially in landscapes and gardens. Boxwood blight is a destructive disease caused by Calonectria pseudonaviculata (Cps). In the United States, the disease was first observed and confirmed in North Carolina and Connecticut in 2011 and it has been reported in 30 states as of February 2021. English boxwood (Buxus sempervirens ‘Suffruticosa’), an iconic plant in American and European landscapes including many historic gardens and plantings, is highly susceptible to this disease. Currently, boxwood protection relies largely on repeated fungicide applications because there are essentially no cultivars that are immune to Cps and eradication of seriously diseased plants results in significant economic loss to the growers and owners. Furthermore, replacing English boxwood in the gardens of royal and historical estates with less susceptible varieties or cultivars is affected by the plants' market and historic values. Cps also attacks several pachysandra, sweet box, and potentially some common groundcovers and boxwood companion plants outside of the Buxaceae family. While chemical control is effective, it is also expensive and potentially a threat to human health and to the environment. Specifically, chlorothalonil, one of the most effective compounds for controlling Cps, has recently been classified as a category one carcinogen and removed from the market in Europe. In addition, the use of fungicides in historic gardens and residential and commercial landscapes is particularly challenging. Economically viable and environmentally sound disease control methods are urgently needed. Recent studies on mulching and biological control alternatives provided safer and more sustainable management of boxwood blight. Mulching over the infested soil and diseased leaf litter protect plants by effectively preventing pathogen inoculum from splashing onto healthy boxwood foliage. However, this method is powerless to avert transmission above the ground. Two recently identified Cps antagonists, Trichoderma koningiopsis from wild mushrooms and Pseudomonas protegens from plant nursery recycled irrigation water, have shown potential to be used to reduce disease by about 60%. However, ornamental plants at such protection levels are not marketable. Plant diseases, including but not limited to boxwood blight, can wipe out entire crops and historical plantings in a short period of time. Globally, plant diseases pose an increasing risk to food security and plant biosecurity. The current approach to disease management depends mainly on chemical protection. This approach is not sustainable due to growing costs, fungicide resistance risk, and human and environmental safety concerns. Managing plant diseases in the environmental horticulture industry is particularly challenging as this industry produces a live commodity that requires the highest level of plant health for aesthetic value. More importantly, this commodity is expected to survive the stresses of shipment, retail, and being planted, then become established at its final destination. Contaminated plants at production facilities may appear healthy without any disease symptoms under intensive chemical protection programs. However, plants may become diseased when they reach their final destinations due to severe stress during shipment, sale, and field establishment, or from lack of continuing chemical protection. There is an urgent need to build a long-term disease-fighting mechanism into horticultural crop products to sustainably safeguard plants. Endophytes are the microorganisms that reside within the host plant's tissues in a commensal