Patent Application
20160227789
1. Field of the Invention
The present invention relates to bacteria and bacterial combinations which can be used in methods to improve the health and vigor, including enhancement of the growth of plants, including important crop plants, while improving the sustainability of the agro-ecosystem. The bacterial strains herein include Paenibacillus sp. (ATY16); Bacillus megaterium (PT6); Bacillus subtilis (PT26A); and combinations thereof, and can be useful for treatment of healthy plants and plants which are susceptible to plant disease or which have been infected with plant disease. Although the methods and compositions are useful for administration to any plant or seed, preferred plants are those which are commercial crops, for example citrus, corn, soybean, and tomato. The methods and compositions of embodiments of the invention can ameliorate the effects of plant diseases, including microbial diseases such as huanglongbing (HLB) disease (also known as citrus greening disease).
2. Description of the Related Art
Conventional pest control technologies based on the use of agricultural chemicals have contributed to efficient agricultural productivity. However, their use also has led to increasing public concerns regarding their negative impacts on the environment. Environmentally-beneficial agriculture using no or reduced amounts of agricultural chemicals and satisfying cultivation efficiency, while assuring human safety is desired and necessary. Therefore, pest and disease control technology fulfilling such demand is needed in the art.
Crops in different ecosystems around the world may suffer less than ideal conditions due to soil or weather conditions, or various stresses, as well as diseases that can negatively affect the health and vigor of the crop plants. Such factors can reduce productivity of the crops to a greater or lesser degree, even under good growing conditions. Thus, crop plants can benefit from treatment that will increase the health and vigor of the plants, whether the plants are stressed by poor conditions, by disease, or even when the plants are healthy or grown under favorable conditions.
A number of plant diseases have negative effects on crop plants worldwide. Microbial plant pathogens can lead to losses in yield, and can even kill crop plants. Therefore, strategies to improve plant defenses against pathogens are needed to improve cultivation, crop yield, and crop quality, while avoiding environmental pollution of the plants and the soil in which they are grown. Biological approaches, such as the use of beneficial bacteria as described herein, therefore are helpful to improve crop plant health generally, and to reduce the effects of plant pathogens.
An example of a harmful plant disease is HLB or citrus greening disease also sometimes referred to as yellow shoot or yellow dragon. This is a major bacterial disease of citrus crops and can be found in Asia, in the Americas and in Africa. It has been spreading worldwide, resulting in economic loss. Huanglongbing (HLB) is currently the most economically devastating disease of citrus worldwide and no established cure is available. All commercial citrus varieties currently available are susceptible to HLB and the citrus industries in affected areas have suffered a decline in both production and profit (Bové, 2006; Gottwald et al., 2007; Wang and Trivedi, 2013). In Florida, HLB is now present in all commercial citrus-producing counties and is destroying the $9 billion citrus industry at a rapid pace. It was estimated that HLB has played a key role in the loss of about 100,000 citrus acres since 2007 in Florida and has cost Florida's economy approximately $3.6 billion in lost revenues since 2006 (Gottwald, 2010; Wang and Trivedi, 2013).
Citrus HLB is associated with a phloem-limited fastidious α-proteobacterium belonging to the ‘Candidatus’ genus Liberibacter, formerly known as Liberobacter (Jagoueix et al., 1994). Currently, three species of ‘Ca. Liberibacter ’ have been identified to cause HLB disease: ‘Ca. L. asiaticus’ (Las), ‘Ca. L. africanus ’, and ‘Ca. L. americanus’ (Gottwald, 2010). These bacteria have not been cultivated in pure culture. HLB pathogen is mainly spread by the insect (psyllid) vector Diaphorina citri in the field. There are two psyllid species transmitting Liberibacters: Asian citrus psyllid (Diaphorina citri) in Asia and the Americas (Bové, 2006; Halbert, 2005; Teixeira et al., 2005) and African citrus psyllid (Trioza erytreae) in Africa (Bové, 2006). Las and Asian citrus psyllid are the most prevalent and important throughout HLB-affected citrus-growing areas worldwide (Bové , 2006). Las propagates in the phloem of the host plants, resulting in die-back, small leaves, yellow shoots, blotchy mottles on leaves, corky veins, malformed and discolored fruit, aborted seed, premature fruit drop, root loss, and eventually tree death (Bové, 2006; Gottwald et al., 2007; Wang and Trivedi, 2013). The life span for the profitable productivity of infected citrus trees is dramatically shortened as the disease severity increases and the yield is significantly reduced while the tree is still alive (Gottwald et al., 2007). The understanding of virulence mechanism of the bacterial pathogen is limited, due to the difficulty in culturing Las. So far, most molecular insights of the HLB biology and Las pathogenicity are derived from the genome sequences of Las and other related Liberibacters (Duan et al., 2009; Lin et al., 2011; Leonard et al., 2012; Wulff et al., 2014).
Particularly sensitive citrus includes Citrus halimii, ‘Nules’ clementine mandarin, Valencia sweet orange, ‘Madam Vinous’ sweet orange, ‘Duncan’ grapefruit, ‘Ruby’ red grapefruit, and ‘Minneola’ tangelo, however, any Citrus species is vulnerable to HLB. In addition, some related plants in the genus Rutaceae, and other plants may become infected with Ca. Liberibacter species. Those of skill in the art are able to test for infection by Ca. Liberibacter, and therefore are able to determine which plants suffer from HLB or Ca. Liberibacter infection. Treatment of such plants is considered part of this invention.
Current methods in use for HLB control include the use of HLB-free citrus seedlings, destruction of infected trees, and application of insecticides such as aldicarb (Temik®) or imidacloprid (Admire®). These insecticides are aimed at controlling psyllids, a possible insect vector for the disease, although it is not known if insecticides have a direct effect on the spread of HLB. These insecticide treatments do not reduce disease in trees already infected, in any case. An integrated control program has been recommended for HLB in commercial orchards by the United Nations Development Program, Food and Agriculture Organization (UNDP, FAO) Southeastern Asian citrus rehabilitation project (Aubert, 1990). The program highlights controlling psyllid vectors with insecticides, reducing inoculum through removal of HLB-symptomatic trees, propagating and using pathogen-free budwood and nursery trees. In Florida, foliar nutrition programs coupled with vector control are often used to slow down the spread of HLB and reduce devastating effects of the disease (Gottwald, 2010). These control practices have shown limited effect for preventing the further spread of HLB. Other than destruction and removal of diseased trees, there is no effective control for HLB in infected trees, and there is no known cure for HLB. New and improved treatments for citrus (and other) HLB disease therefore are needed in the art.
Other plant pathogens of the greatest interest include the bacterium Xanthomonas citri causing citrus canker, Xanthomonas axonopodis pv. citrumelo causing citrus bacterial spot disease, and Xylella fastidiosa causing citrus variegated chlorosis; the pathogenic fungus Alternaria citri causing leaf and stem rot and spot, Phytophthora spp. causing foot and root rot, and Guignardia citricarpa causing citrus black spot, all of which can result in crop loss.
Induced resistance can confer long-lasting protection against a broad spectrum of plant diseases either locally or systemically (Durrant and Dong, 2004; Walters et al., 2013). Plant defense mechanisms can be activated by pathogens (Durrant and Dong, 2004), beneficial microorganisms (Weller et al., 2012; Zamioudis and Pieterse, 2012), or by chemical inducers (Walters et al., 2013). Overall, maximizing crop plant health and vigor has been a difficult problem with no comprehensive solution. Therefore, the embodiments of the invention described herein are provided for the control of crop pathogens such as HLB, Xanthomonas citri causing citrus canker, Xanthomonas axonopodis pv. citrumelo causing citrus bacterial spot disease, and Xylella fastidiosa causing citrus variegated chlorosis; the pathogenic fungus Alternaria citri causing leaf and stem rot and spot, Phytophthora spp. causing foot and root rot, and Guignardia citricarpa causing citrus black spot and to improve plant health and vigor, including germination, growth, disease resistance, and improvement of crop quality and quantity.
Techniques are provided for improving the health and disease resistance of plants, including important crop plants such as citrus, corn, soybeans, tomatoes and others. The bacterial strains according to embodiments of the invention described herein can be applied to plants to improve health and vigor, increase seed germination, increase growth, enhance crop or fruit production, and increase plant defense mechanisms. Therefore, the bacterial strains can be used to benefit any plant, including healthy plants and diseased plants. The methods described here involve application of the bacterial strains to the plant, including application to the soil around the plant by soil injection or soil drench methods, application to the surface of the plant, such as by spraying onto the plant or parts of the plant, such as by foliar spraying, or injection into the plant such as by trunk injection. Plants for which the invention is contemplated include any plant, particularly crop plants, but including ornamental plants as well.
Still other aspects, features, and advantages of embodiments of the invention are readily apparent from the following detailed description, simply by illustrating a number of particular embodiments and implementations, including the best mode contemplated for carrying out the invention. Other embodiments also are capable of other and different features and advantages, and its several details can be modified in various obvious respects, all without departing from the spirit and scope of the invention. Accordingly, the drawings and descriptions are to be regarded as illustrative in nature, not as restrictive.
It has now been found that certain bacterial strains have beneficial effects on the growth, general health and vigor, pathogenic defenses, fruit and crop productivity, fruit quality and crop quality of plants. The bacteria have benefits to the cellular health of plants and stimulate growth of the plants, even when the plants are infected with a disease, such as HLB disease. These bacterial strains can be used individually or as mixtures with other beneficial bacteria. The consortia of various compatible bacteria possessing multiple plant beneficial traits and antagonistic ability against plant pathogens may improve disease control, with broad spectrum of action and enhanced reliability and efficacy (Lugtenberg and Kamilova 2009).
Therefore, embodiments of the invention include a bacterial composition for administration to plants, which comprises a botanically compatible vehicle and at least one isolated bacterial strain selected from the group consisting of Bacillus megaterium PT6 having accession number PTA-122799, Bacillus subtilis PT26A having accession number PTA-122797, Paenibacillus sp. ATY16 having accession number PTA-122798. Preferably, the compositions comprise 103-1011 cfu/mL of the at least one bacterial strain or at least 103 cfu/mL of the at least one bacterial strain, at least 105 cfu/mL of the at least one bacterial strain, or at least 107 cfu/mL of the at least one bacterial strain.
Additional embodiments of the invention include compositions as described herein wherein the at least one bacterial strain is Bacillus megaterium PT6 having accession number PTA-122799, wherein the at least one bacterial strain is Bacillus subtilis PT26A having accession number PTA-122797, or wherein the at least one bacterial strain is Paenibacillus sp. ATY16 having accession number PTA-122798.
Further embodiments of the invention include a composition which comprises seeds treated or coated with the composition described above in the summary of invention, and also include compositions which further comprise a plant defense inducer compound selected from the group consisting of β-aminobutyric acid (BABA) or a salt thereof, and salicylic acid (SA) or a salt thereof.
A preferred embodiment of the invention is a method of improving the health and vigor of a plant, comprising administering to the plant an effective amount of the bacterial composition described throughout the application and in paragraph 16 herein, wherein the improvement in health and vigor is one or more of: a) improved resistance to disease; b) improved ability to defend against disease; c) reduction of disease symptoms; d) faster growth; e) improved crop productivity; f) improved crop quality; g) improved seed germination; and h) improved seedling emergence. Preferably, the at least one bacterial strain is Bacillus subtilis PT26A having accession number PTA-122797.
In certain embodiments of the invention, the plant is a crop plant, preferably a citrus plant. The citrus advantageously can be selected from the group consisting of Citrus maxima (Pomelo), Citrus medica (Citron), Citrus micrantha (Papeda), Citrus reticulata (Mandarin orange), Citrus paradisi (grapefruit), Citrus trifolata (trifoliate orange), Citrus japonica (kumquat), Citrus australasica (Australian Finger Lime), Citrus australis (Australian Round lime), Citrus glauca (Australian Desert Lime), Citrus garrawayae (Mount White Lime), Citrus gracilis (Kakadu Lime or Humpty Doo Lime), Citrus inodora (Russel River Lime), Citrus warburgiana (New Guinea Wild Lime), Citrus wintersii (Brown River Finger Lime), Citrus halimii (limau kadangsa, limau kedut kera) Citrus indica (Indian wild orange), Citrus macroptera, and Citrus latipes, Citrus×aurantiifolia (Key lime), Citrus×aurantium (Bitter orange), Citrus×latifolia (Persian lime), Citrus×limon (Lemon), Citrus×limonia (Rangpur), Citrus×sinensis (Sweet orange), Citrus×tangerina (Tangerine), Imperial lemon, tangelo, orangelo, tangor, kinnow, kiyomi, Minneola tangelo, oroblanco, sweet orange, ugli, Buddha's hand, citron, lemon, orange, bergamot orange, bitter orange, blood orange, calamondin, clementine, grapefruit, Meyer lemon, Rangpur, tangerine, and yuzu.
In the method embodiments for citrus, preferably the at least one isolated bacterial strain is selected from the group consisting of Bacillus megaterium PT6 having accession number PTA-122799, Bacillus subtilis PT26A having accession number Pta-122797, Paenibacillus sp. ATY16 having accession number PTA-122798, or any combination thereof.
In certain embodiments of the invention, the crop plant is selected from the group consisting of almond, apple, banana, cacao, carrot, cassava, chili, citrus, coconut, coffee, corn, cotton, cucumber, grape, legume, lettuce, mango, olive, onion, palm, peach, peanut, potato, rapeseed, rice, rubber, soybean, strawberry, sugar beet, sugar cane, sunflower, sweet potato, tea, tomato, walnut, wheat, and yam. Preferably, the crop plant is selected from the group consisting of corn, soybean, and tomato. In another embodiment, preferably the at least one isolated bacterial strain is selected from the group consisting of Bacillus subtilis PT26A having accession number PTA-122797.
In certain method embodiments of the invention, the plant is healthy. In other method embodiments, the plant is affected by a plant disease or plant disease symptoms. The disease can be a bacterial disease or a fungal disease, and can be selected from the group consisting of huanglongbing (HLB) disease, Fusarium, Phytophthora, citrus canker disease, citrus bacterial spot disease, citrus variegated chlorosis, citrus food and root rot, citrus and black spot disease.
In some embodiments of the invention, the administering to the plant is by a method selected from the group consisting of soil injection, soil drenching, application to seed, and foliar spraying. These methods of administering to the plant preferably provide at least 102 cfu or at least 103 cfu of the isolated bacterial strain per gram of plant root thirty days after administration.
Additional embodiments of the invention include a method of improving seed germination in a plant, the method comprising administering to the seed of the plant a composition as described above and a method of enhancing growth of a plant, the method comprising administering to the plant a composition as described above. A highly preferred method is a method of treating a plant disease in a plant in need thereof, which comprises administering to the soil within a ten foot radius surrounding the plant a composition as described above. Preferably, the plant is a Citrus plant and the disease is huanglongbing (HLB) disease.
Additionally, the method can further comprise administering to the plant a plant defense inducer compound selected from the group consisting of β-aminobutyric acid (BABA) or a salt thereof, and salicylic acid (SA) or a salt thereof.
In another embodiment, provided is a container that includes a housing with a composition including a botanically compatible vehicle and at least one isolated bacterial strain selected from the group consisting of Bacillus megaterium PT6 having accession number PTA-122799, Bacillus subtilis PT26A having accession number PTA-122797, Paenibacillus sp. ATY16 having accession number PTA-122798 disposed within the housing. The container may further include a mechanism of administration associated with the housing. The mechanism of administration may include, for example, a conduit in fluid communication with the housing and a spray nozzle in fluid communication with the conduit. The container may further include an access port for accessing the composition. Examples of suitable containers include, but are not limited to, a bin, a bucket, a barrel, a box, and the like.
Certain embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings.
The invention is described herein with reference to specific embodiments. However, various modifications and changes can be made to the invention without departing from its broader spirit and scope. The specification and drawings therefore are to be regarded as illustrative rather than restrictive. Throughout this specification and the claims, unless the context requires otherwise, the word “comprise” and its variations, such as “comprises” and “comprising,” are used to imply the inclusion of a stated item, element or step or group of items, elements or steps but not the exclusion of any other item, element or step or group of items, elements or steps. Furthermore, the indefinite article “a” or “an” is meant to indicate one or more of the item, element or step modified by the article.
In this study, the effects of various bacterial strains and combinations of bacterial strains, were evaluated for activation of natural plant defense mechanisms and for improvements in health and growth of the plants. The bacterial strains improve plant defenses against disease, with the effect of increasing the health and growth of plants. Therefore, this approach can be used to treat, for example, plants that have been infected with, plants that are susceptible to infection with, or plants that exhibit symptoms of HLB disease or infection with a Candidatus liberibacter species. Examples of such species include Candidatus liberibacter asiaticus, Candidatus liberibacter arnericanus, Candidatus liberibucter africanus, and any combination thereof.
While a number of embodiments of the present invention have been shown and described herein in the present context, such embodiments are provided by way of example only, and not of limitation. Numerous variations, changes and substitutions will occur to those of skill in the art without materially departing from the invention herein. Any means-plus-function and step-plus-function clauses are intended to cover the structures and acts, respectively, described herein as performing the recited function and not only structural equivalents or act equivalents, but also equivalent structures or equivalent acts, respectively. Accordingly, all such modifications are intended to be included within the scope of this invention as defined in the following claims, in accordance with relevant law as to their interpretation.
All technical and scientific terms used herein, unless defined herein, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise.
The term “applying,” “application,” “administering,” “administration,” and all their cognates, as used herein, refers to any method for contacting the plant with the bacteria and bacterial compositions discussed herein. Administration generally is achieved by application of the bacteria, in a vehicle compatible with the plant to be treated (i.e., a botanically compatible vehicle or carrier), such as an aqueous vehicle, to the plant or to the soil surrounding the plant. Any application means can be used, however preferred application is to the soil surrounding the plant, by injection, soaking or spraying, so that the applied bacteria preferably come into contact with the plant roots and can colonize the roots.
The term “bacteria,” as used herein, refers to any prokaryotic microorganism, and is intended to include both Gram positive and Gram negative bacteria, and unclassified bacteria. The term “beneficial bacteria,” as used herein, refers to the bacteria of strains PT6, PT26A and ATY16, described herein and deposited in the ATCC under accession numbers PTA-122799, PTA-122797 and PTA-122798, respectively, in accordance with the requirements of the Budapest Treaty. Further, strains that have at least 99% identity to the 16s rRNA of these deposited strains are considered “genetic equivalents” of the specific deposited strains. In addition, or alternatively, strains possessing at least 99% identity to at least 2, at least 3, at least 4, at least 5, or all of the uvrD, secA, carA, recA, groEL, dnaK, atpD, gyrB and infB genes of the deposited strains are considered “genetic equivalents” of the deposited strains. See the sequence information for these genes, provided below. in embodiments described and/or claimed herein, genetic equivalents may be used as an alternative in place of beneficial bacteria.
The bacterial strains PT6, PT26A and ATY 16 initially were isolated from plants in the St. Lucie County School District, Fla. The genome size of ATY16 is 6,788,192 by with 50.79% GC. The genome size of PT6 is 5,485,792 by with 37.76% GC. The genome size of PT26A is 4,360,593 by with 43.23% GC. At a 16s level, the rRNA of the strains is 99% identical to the deposit of Bacillus megaterium PT6 having accession number PTA-122799, Bacillus subtilis PT26A having accession number PTA-122797, Paenibacillus sp. ATY16 having accession number PTA-122798.
The term “botanically acceptable carrier/vehicle” or “botanically compatible carrier/vehicle,” as used herein, refers to any non-naturally occurring vehicle, in liquid, solid or gaseous form which is compatible with use on a living plant and is convenient to contain a substance or substances for application of the substance or substances to the plant, its leaves or root system, its seeds, the soil surrounding the plant, or for injection into the trunk, or any known method of application of a compound to a living plant, preferably a crop plant, for example a citrus tree, or corn, soybean or tomato plant. Useful vehicles can include any known in the art, for example liquid vehicles, including aqueous vehicles, such as water, solid vehicles such as powders, granules or dusts, or gaseous vehicles such as air or vapor. Any vehicle which can be used with known devices for soaking, drenching, injecting into the soil or the plant, spraying, dusting, or any known method for applying a compound to a plant, is contemplated for use with embodiments of the invention. Typical carriers and vehicles contain inert ingredients such as fillers, bulking agents, buffers, preservatives, anti-caking agents, pH modifiers, surfactants, soil wetting agents, adjuvants, and the like. Suitable carriers and vehicles within this definition also can contain additional active ingredients such as plant defense inducer compounds, nutritional elements, fertilizers, pesticides, and the like. In a particular embodiment, the botanically acceptable vehicle pertains to a vehicle component, or vehicle formulation, that is not found in nature. In another embodiment, the botanically acceptable vehicle may pertain to a vehicle found in nature, but where the vehicle and the bacteria strain(s) are not mixed or combined together in nature.
The term “Citrus” or “citrus,” as used herein, refers to any plant of the genus Citrus, family Rutaceae, and includes Citrus maxima (Pomelo), Citrus medica (Citron), Citrus micrantha (Papeda), Citrus reticulata (Mandarin orange), Citrus trifolata (trifoliate orange), Citrus japonica (kumquat), Citrus australasica (Australian Finger Lime), Citrus australis (Australian Round lime), Citrus glauca (Australian Desert Lime), Citrus garrawayae (Mount White Lime), Citrus gracilis (Kakadu Lime or Humpty Doo Lime), Citrus inodora (Russel River Lime), Citrus warburgiana (New Guinea Wild Lime), Citrus wintersii (Brown River Finger Lime), Citrus halimii (limau kadangsa, limau kedut kera) Citrus indica (Indian wild orange), Citrus macroptera, and Citrus latipes. Hybrids also are included in this definition, for example Citrus×aurantiifolia (Key lime), Citrus×aurantium (Bitter orange), Citrus×latifolia (Persian lime), Citrus×limon (Lemon), Citrus×limonia (Rangpur), Citrus×paradisi (Grapefruit), Citrus×sinensis (Sweet orange), Citrus×tangerina (Tangerine), Poncirus trifoliata×C. sinensis (Carrizo citrange), C. paradisi “Duncan” grapefruit×Pondirus trifoliate (Swingle citrumelo), and any other known species or hybrid of genus Citrus. Citrus known by their common names include, Imperial lemon, tangelo, orangelo, tangor, kinnow, kiyomi, Minneola tangelo, oroblanco, sweet orange, ugli, Buddha's hand, citron, lemon, orange, bergamot orange, bitter orange, blood orange, calamondin, clementine, grapefruit, Meyer lemon, Rangpur, tangerine, and yuzu, and these also are included in the definition of citrus or Citrus.
The term “crop plant,” as used herein, includes any cultivated plant grown for food, feed, fiber, biofuel, medicine, or other uses. Such plants include, but are not limited to, citrus, corn, soybean, tomato, sugar cane, strawberry, wheat, rice, cassava, potato, cotton, and the like. The term “crop,” as used herein, refers to any of the food (including fruits or juice), feed, fiber, biofuel, or medicine derived from a crop plant. All crop plants are contemplated for use with the invention, including monocots and dicots.
The term “effective amount” or “therapeutically effective amount,” as used herein, means any amount of the bacterial strain, combination of bacterial strains or composition containing the bacterial strains, which improves health, growth or productivity of the plant, or which reduces the effects, titer or symptoms of the plant disease, or prevents worsening of the plant disease, symptoms or infection of the plant. This term includes an amount effective to increase seed germination of a plant or a plant population, to increase the speed of seed germination of a plant or a plant population, to increase growth rates of a plant or a plant population, to increase crop yield of a plant or plant population, increase crop quality in a plant or plant population, reduce the plant pathogen titer, to inhibit plant pathogen growth, to reduce the percent of infected plants in a plant population, to reduce the percent of plants showing disease symptoms in a plant or plant population, to reduce the disease symptom severity rating or damage rating of a plant or plant population, to reduce average pathogen population or titer in a plant or plant population by about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, or more, compared to plants or a plant population not treated with the active ingredient.
The term “faster growth,” as used herein, refers to a measurable increase in the rate of growth of a plant, including seedlings, stems, roots, seeds, flowers, fruits, leaves and shoots thereof.
The term “health,” as used herein, refers to the absence of illness and a state of well-being and fitness, and refers to the level of functional or metabolic efficiency of the plant, including the ability to adapt to conditions and to combat disease, while maintaining growth and development. The term “vigor,” as used herein, refers to the health, vitality and hardiness of a plant, and its capacity for natural growth and survival. Therefore, the phrase “health and vigor of a plant,” as used herein, means the absence of illness, a high level of functional or metabolic efficiency, the ability to combat disease, and the maintenance of good growth and development, and the efficient production of crops.
The term “healthy,” as used herein, refers to a plant or plant population which is not known currently to be affected by a plant disease.
The term “Huanglongbing disease,” as used herein, is a disease of plants caused by microorganisms of the Candidatus genus Liberibacter, such as L. asiaticus, L. africanus, and L. americanus. This disease, for example, can be found in citrus plants, or other plants in the genus Rutaceae. Symptoms of Huanglongbing disease include one or more of yellow shoots and mottling of the plant leaves, occasionally with thickening of the leaves, reduced fruit size, fruit greening, premature dropping of fruit from the plant, low fruit soluble acid content, fruit with a bitter or salty taste, or death of the plant. The term “treating” or “treatment,” or its cognates, as used herein indicates any process or method which cures, diminishes, ameliorates, or slows the progress of the disease or disease symptoms. Thus, treatment includes reducing bacterial titer in plant tissues or appearance of disease symptoms relative to controls which have not undergone treatment.
The term “improved ability to defend against disease,” as used herein, refers to a measurable increase in plant defense against a disease. This can be measured in terms of a measurable decrease in disease symptoms, pathogen titer, or loss of crop yield and/or quality, or a measurable increase in growth, crop quantity or quality.
The term “improved crop productivity,” as used herein, refers to a measurable increase in the quantity of a crop in a plant or a population of plants, in terms of numbers, size, or weight of crop seeds, fruits, vegetable matter, fiber, grain, and the like.
The term “improved crop quality,” as used herein, refers to a measurable increase in the quality of a crop, in terms of numbers, size, or weight of crop seeds, fruits, vegetable matter, fiber, grain, and the like, or in terms of sugar content, juice content, unblemished appearance, color, and/or taste.
The term “improved resistance to disease,” as used herein, refers to an increase of plant defense in a healthy plant or a decrease in disease severity in a plant or in a population of plants, or in the number of diseased plants in a plant population.
The term “improved seed germination,” as used herein, means a measurable increase of the chance of successful germination of an individual seed, a measurable increase in the percentage of seeds successfully germinating, and/or a measurable increase in the speed of germination.
The term “improved seedling emergence,” as used herein means a measurable increase in the speed of growth and/or development of successfully germinated individual seeds or population of seeds.
The term “measurable increase” (or “measurable decrease”), as used herein, means an increase (or decrease) that can be detected by assays known in the art as greater (or less) than control. For example, a measurable increase (or decrease) is an increase (or decrease) of about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, or more, compared to plants or a plant population not treated with the active ingredient.
The term “plant in need thereof,” as used herein, means any plant which is healthy or which has been diagnosed with a plant disease or symptoms thereof, or which is susceptible to a plant disease, or may be exposed to a plant disease or carrier thereof.
The term “plant disease,” as used herein, refers to any disease of a crop plant, caused by any plant pathogen, including but not limited to, bacterial, viral, fungal nematode, phytomyxean, protozoan, algal and parasite plant pathogens.
The term “plant disease symptoms,” as used herein, refers to any symptom of disease, including the detectable presence of a known plant pathogen, or the presence of rot, mottling, galls, discoloration such as yellowing or browning, fruit greening, stunted growth, plant death, cellular death, cell wall breakdown, and/or the presence of spots, lesions, dieback, wilting, dwarfing, Witch's broom and/or knots.
The term “population of plants,” as used herein, refers to a group of plants, all of the same species, that inhabit a particular area at the same time. Therefore, the plants in a nursery, a grove, a farm, and the like are considered a population.
The term “reduction of disease symptoms,” as used herein, refers to a measurable decrease in the number or severity of disease symptoms.
The term “treating,” treatment,” and all its cognates, as used herein, refers to any application or administration to a plant, the soil surrounding the plant, the water applied to the plant, or the hydroponic system in which the plant is grown, which is intended to improve the health, growth or productivity of a plant, particularly a crop plant. For example, a treatment intended to increase the health or growth or a crop plant, increase crop yield of a plant or population of plants is contemplated as part of this definition, as well as treatment intended to improve disease symptoms or pathogen titer in the plant.
The invention relates to bacterial strains and combinations of bacterial strains that assist in inducing plant defenses against diseases, which are able to induce plant resistance effective against pathogens involved in HLB disease, and other diseases, in plants, as well as increase the health and vigor of any plants, including healthy plants, and increase seed germination and crop quantity and quality. These bacteria can be incorporated into a botanically acceptable vehicle and administered or applied to a plant of interest by any convenient known method. Preferable methods include injection to the soil surrounding the plant, application to the soil by spraying, soaking/drenching, seed treatment, or any other means conventionally used in agriculture, to induce uptake by the roots, or by injection into the plant itself, such as injection into the trunk, or by application to the leaves or any part of the plant. Application or administration methods that provide contact of the bacteria to the roots of the plant, such as soil applications within about a 10-foot radius of the trunk of the plant are preferred.
Introduction
Methods and compounds for use in enhancing the health and vigor of plants are needed in the art, both to improve the productivity of healthy plants, and to increase the plant's natural ability to combat disease. Embodiments of this invention provide methods and compositions to assist in these goals, and to treat plants that are affected by disease as well. For example, plants treated according to embodiments of the invention can exhibit improved growth, seed germination, and defense against disease progression and disease symptoms, for example against HLB or Ca. Liberibacter infection. In the following description, for the purposes of explanation, certain specific details are set forth in order to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without these specific details.
Some bacteria have the ability to induce systemic resistance in plants, providing them with an advantage against plant pathogens and generally increasing health and vigor in the plants. The resulting induced systemic resistance (ISR) allows the plant to evoke a stronger and faster defense responses against a broad spectrum of pathogens in a systemic way. Physiologically, the ISR response is similar to the systemic acquired resistance (or SAR), triggered after an encounter with some plant pathogens. Both ISR and SAR are similar in the resulting defense against a broad number of pathogens. However, some differences exist between the responses. While SAR is thought to be activated through the salicylic acid pathway and accumulate PRs as a consequence, ISR is believed to be activated through the ethylene/jasmonate (ET/JA) pathways, and whether PRs accumulate is less understood. Plant defense responses were monitored using qRT-PCR by studying expression of selected defense genes on the leaf tissue. Genes involved in the ethylene/jasmonate (I/J) and salicylic acid (SA) pathways, as well as pathogensis-related (PR) protein-encoding genes were used as markers. Identities of three bacteria were determined by genomic DNA fingerprinting using 16SrRNA gene sequencing based analysis.
Beneficial bacteria have been isolated from the rhizosphere of healthy citrus trees in severely HLB-diseased groves in Florida. The phytobiome associated with such trees were found to harbor microbes able to improve plant defenses against citrus pathogens. It now has been discovered that the bacterial strains described here, alone and in combination, are able to induce systemic resistance and benefits to health and vigor, in a wide variety of plants, including any crop plants, and most preferably citrus, corn, soybean, and tomato crop plants.
Methods of Bacterial Isolation
Molecular identification of beneficial strains was achieved by amplification and sequencing of 16s rRNA. 16s rRNA was amplified from seven antibiotic-producing bacterial strains using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′; SEQ ID NO:1) and 1492R (5′-GGTTACCTTGTTACGACTT-3′; SEQ ID NO:2). The corresponding amplification products then were sequenced with both primers and the universal primer 519F (5′- CAGCMGCCGCGGTAATAC-3′; SEQ ID NO:3) to obtain a fragment of 1350 by or more. Each strain was assigned to its closest microorganism based on BLAST analysis, and a phylogenetic tree was constructed with Neighbor Joining method with a bootstrap of 1000 using MEGA 6.0.
A phylogenetic tree was constructed to show the relationship of strains ATY16, PT6, and PT26A to other known strains. See
Bacterial Strains
The phylogenetic tree indicates that the strain ATY16 groups most closely with Paenibacillus sp. JDR-2 and Paenibacillus mucilaginosus K02, forming a distinct Glade from other Bacillus and Paenibacillus species. The closest relative of the strain PT6 is Bacillus megaterium QMB 1551. The closest relative of the strain PT26A is Bacillus subtilis.
The strain ATY16 shared 99% homology with the Paenibacillus sp. strain JDR-2 (Accession No. CP001656), Paenibacillus glycanilyticus strain NBRC 16618 (Accession No. NR_113853) and Paenibacillus sp. strain A12 (Accession No. KF479531). The strain PT6 shared 99% homology with the Bacillus megaterium strain GMA327 (Accession No. AB738784), Bacillus aryabhattai strain IHB B 7024 (Accession No. KJ721203), and Bacillus sp. strain NH1 (Accession No. JN208177). The strain PT26A shared 99% homology with several strains of Bacillus subtilis (Accession No. KJ767313, CP010052, and CP010053, etc.), and Bacillus tequilensis strain IHB B 6839 (Accession No. KF668464).
The general features of the three genomes are summarized in Table 1, below. The genome size of ATY16 is 6,788,192-bp, with 50.79% GC, 6,129 predicted CDSs (coding sequences), 85 types of tRNA and rRNA, and 39 estimated missing genes. The genome size of PT6 is 5,485,792-bp, with 37.76% GC, 5,666 predicted CDSs, 124 types of tRNA and rRNA, and 16 estimated missing genes. The genome size of PT26A is 4,360,593-bp, with 43.23% GC, 4,578 predicted CDSs, 84 types of tRNA and rRNA, and 38 estimated missing genes.
Analysis also indicated the presence of putative plant beneficial gene clusters in the three genomes. In B. subtilis PT26A, there are nonribosomal peptide synthetase (NRPS) gene clusters encoding surfactin production (comS, srfA, and rapA), genes for bacilysin (bacA, bacB, and bacE) and a bacillibactin siderophore, and several polyketide synthase (PKS) genes. The bacterium was equipped with genes for resistance to drugs and heavy metals, aromatic compound degradation, motility, and chemotaxis and for the synthesis of auxin precursors probably useful in the beneficial relationship with plants. It also harbors genes for synthesis of exopolysaccharides and biofilm, capsule and endospore proteins, and genes for assimilation of nitrogen (moaCDE, mobAB, mobAB; and nar/nas) and minerals, including Phosphate (phoPR), and Magnesium (mgtE, and conA). It also contains genes for the synthesis of 2,3-butandiol that can elicit a plant defense response. In B. megaterium PT6, there are genes encoding probable nikkomycin biosynthesis proteins, siderophore biosynthesis proteins, and genes for resistance to drugs and heavy metals, for motility and chemotaxis, for the synthesis of auxin precursors, for endospore proteins, and for assimilation of nitrogen and minerals. In Paenibacillus sp. ATY16, there are NRPS genes encoding antimicrobial(s) to be determined, Phenazine biosynthesis protein encoding genes, and genes for resistance to drugs and heavy metals, for motility and chemotaxis, for the synthesis of auxin precursors, for exopolysaccharides biosynthesis, for endospore protein biosynthesis, for assimilation of nitrogen and minerals, for xylan, chitin and N-cetylglucosamine utilization, and genes for putative insecticidal toxin complex.
B. megaterium
B. subtilis
Paenibacillus sp.
Deposit Information
The bacterial strains Bacillus megaterium PT6, Bacillus subtilis PT26A, and Paenibacillus sp. ATY16 have been deposited in an international depository under conditions that assure that access to the culture will be available during the pendency of this patent application and any patent(s) issuing therefrom to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. 1.14 and 35 U.S.C. 122. These strains have been deposited in the American Type Culture Collection (ATCC), at 10801 University Boulevard, Manassas, Va., 20110-2209 United States of America, under the following accession numbers: Bacillus megaterium PT6 (accession number PTA-122799), Bacillus subtilis PT26A (accession number PTA-122797), and Paenibacillus sp. ATY16 (accession number PTA-122798).
Plants
The studies described herein have shown that the bacterial cultures according to embodiments of the invention have desirable effects on the growth of plants, their productivity and their ability to combat disease. The description herein provides data showing effects on a variety of crop plants, including both monocots and dicots, indicating that the effects are generalized and widespread, and can provide benefits to the health and vigor of all plants, and improvements in fighting a wide variety of diseases. Any plant is contemplated for use with the invention, both healthy plants and those which have been exposed to or may be exposed to a plant pathogen or a carrier of a plant pathogen.
Preferred plants for use with the invention include citrus, corn, soybean, potato, tomato, sugar cane, and strawberry, which are major fruit or food crops, however any plant, including both crop and ornamental plants can be treated with the methods and compositions described herein.
The invention is contemplated for use on plants at all stages of development, including seeds, seedlings and mature plants, which are cultivated by any method known in the art which is convenient for the plant in question. Plants in the field, on farms or in a natural environment are included as useful for practicing the invention, as well as plants in a nursery or greenhouse, or a raised bed, home garden, or hydroponics facility, on a large or small scale.
Bacterial Effects
Without wishing to be bound by theory, the bacterial strains described herein beneficially affect plants to which they are exposed, by increasing the expression of certain genes in the plant, at least some of which are related to the natural plant defense mechanisms of the plant. The bacteria improve the metabolism of the plant, thereby enhancing growth, productivity and disease resistance and the ability to combat disease.
The data provided herein show both general effects on growth and productivity in plants, which demonstrate an effect on the health and vigor of the plants, as well as effects on plant pathogens that beneficially affect infected plants. PT26A inoculation promoted germination of seeds in all three crops and also improved the vigor of germinating seeds. See Examples.
Methods for study of the effects of the beneficial bacterial strains include root inoculation with the bacteria (or control), monitoring expression of defense genes in the inoculated plants, a pathogen challenge on leaves, for example Xanthomonas citri subsp. citri, and monitoring the development of disease in the plants.
Plant Diseases
Diseases for which embodiments of the invention are contemplated for use include diseases of citrus, including, but not limited to:
Bacterial diseases (bacterial spot, black pit (fruit), blast, citrus canker, citrus variegated chlorosis, huanglongbing (citrus greening);
Viral diseases (citrus mosaic, bud union crease, citrus leaf rugose, citrus yellow mosaic, crinkly leaf, infectious variegation, navel infectious mottling, psoriasis, satsuma dwarf, tatter leaf, tristeza, citrus leprosis),
Fungal diseases (albinism, alternaria brown spot, anthracnose, areolate leaf spot, black mold rot, black root rot, black rot, blue mold, botrytis, branch knot, brown rot (fruit), charcoal root rot, citrus black spot, dumping off, dry rood complex, dry rot, fly speck, fusarium, green mold, heart rot, leaf spot, mucor fruit rot, phymatotrichum root rot, phomopsis stem-end rot, phytophthora, pink disease, pink mold, pleospora rot, poria root rot, post bloom fruit drop, powdery mildew, rootlet rot, rosellinia root rot, scab, sclerotinia twig blight, septoria spot, sooty blotch, sour rot, sweet orange scab, thread blight, Trichoderma rot, twig blight, ustulina root rot, whisker mold and white root rot);
and diseases of corn, including, but not limited to:
Bacterial diseases (bacterial leaf blight and stalk rot, bacterial leaf spot, bacterial stalk rot, bacterial stripe, chocolate spot, Goss's bacterial wilt and blight, holcus spot, purple leaf sheath, seed rot-seedling blight, Stewart's disease, and corn stunt);
Fungal diseases (phytophthora, anthracnose leaf blight, anthracnose stalk rot, aspergillus ear and kernel rot, banded leaf and sheath spot, black bundle disease, black kernel rot, borde blanco, brown spot, black spot, stalk rot, cephalosporium kernel rot, charcoal rot, corticium ear rot, diplodia, didymella, downy mildews, dry ear rot, ergot, eyespot, fusarium, gibberella, grey leaf spot, cercospora, helminthosporium, hormodendrum, cladosporium, hyalothyridium, late wilt, white blast, stripe, northern corn leaf spot, penicillium ear rot, blue eye, blue mold, phaeocytostroma stalk rot, phaeosphaeria leaf spot, botrysphaeria ear rot, pythium root rot, red kernel disease, sclerotial rot, rhuzoctonia root rot, rust, southern blight, and smut);
Viral diseases (maize bushy stunt, maize chlorotic dwarf, maize chlorotic mottle, maize leaf fleck, maize mosaic, maize pellucid ringspot, maize red leaf, maize ring mottle, maize streak, maize stripe, maize tassel abortion, maize white leaf, and northern cereal mosaic);
and diseases of soybean, including, but not limited to:
Bacterial diseases (bacterial blight, bacterial pustules, bacterial tan spot, bacterial wilt, and wildfire);
Fungal diseases (alternaria leaf spot, anthracnose, black leaf blight, black root rot, brown spot, charcoal rot, choanephora leaf blight, downy mildew, fusarium root rot, fusarium sudden death syndrome, leptosphaerulina leaf spot, mycoleptodiscus root rot, phomopsis seed decay, phyllosticta leaf spot, powdery mildew, Pythium rot, phytophthora, red crown rot, rhizoctonia, rust, scab, southern blight, target spot, and yeast spot);
Viral diseases (bean pod mosaic, bean yellow mosaic, Brazilian bud blight, peanut mottle, soybean chlorotic mottle, soybean crinkle leaf, soybean mosaic, soybean severe stunt, and bud blight);
and diseases of tomato, including, but not limited to:
Bacterial diseases (bacterial canker, bacterial speck, bacterial wilt, pith necrosis, and syringae leaf spot);
Fungal diseases (alternaria stem canker, anthracnose, black mold rot, black root rot, cercospora leaf mold, charcoal rot, didymella stem rot, early blight, fusarium, grey leaf spot, gray mold, late blight, leaf mold phoma rot, phytophthora, powdery mildew, rhizoctonia, rhizopus rot, septoria leaf spot, sour rot, southern blight, target spot, verticillium wilt, and white mold);
Viral diseases (curly top, tomato bushy stunt, tomato etch, tomato mosaic, tomato mottle, tomato necrosis, tomato spotted wilt, tomato yellow top, tomato bunchy top, common mosaic of tomato, and tomato big bud);
and diseases of sugar cane, including, but not limited to:
Bacterial diseases (gumming disease, leaf scald, mottled stripe, ratoon stunting disease, and red stripe);
Fungal diseases (banded sclerotial disease, black rot, black stripe, brown spot, brown stripe, downy mildew, eye spot, fusarium, iliau, leaf blast, leaf blight, leaf scorch, marasmius sheath and shoot blight, phyllosticta leaf spot, phytophthora, phytophthora rot of cuttings, pineapple disease, red leaf spot, rhizoctonia sheath and shoot rot, rind disease, ring spot, common rust, orange rust, schizophyllum rot, sclerophthora disease, seedling blight, smut, wilt, yellow spot, and zonate leaf spot);
Viral diseases (chlorotic streak, Fiji disease, mosaic, streak disease, and yellow leaf);
and diseases of strawberry, including, but not limited to:
Bacterial diseases (angular leaf spot, bacterial wilt and cauliflower disease);
Fungal diseases (powdery mildew, alternaria fruit rot, anthracnose, armillaria crown and root rot, black leaf spot, black root rot, cercospora leaf spot, charcoal rot, common leaf spot, coniothyrium diseases, diplodina rot, downy mildew, brown cap, byssochlamys rot, gray mold leaf blight, hainesia leaf spot, leaf blotch, leaf rust, leaf scorch, powdery mildew, botrytis crown rot, idriella root rot, olpidium root infection, phytophthora, synchytrium root gall, rhizoctonia bud and crown rot, rhizopus rot, southern blight, and verticillium wilt);
Viral diseases (strawberry chlorotic fleck, strawberry crinkle, strawberry mottle, strawberry vein banding, strawberry green petal, strawberry lethal decline, strawberry latent ringspot, strawberry mycoplasma yellows disease, arabis mosaic virus, and strawberry pallidosis).
A person of skill in the art is aware of methods for determining whether a plant is in need of treatment for a plant disease (for example HLB or Ca. Liberibacter infection), and which plants may be or may become susceptible to a plant disease. Therefore, the invention described and claimed herein is contemplated for use in any plant which is or which may become infected with a plant disease, as determined by a person of skill. Due to its nature in inducing plant defenses and in improving health and vigor, it is recommended to use it when the trees are firstly planted in the field to prevent disease development, or to treat seeds prior to planting. Methods according to embodiments of the invention preferably are used when the person of skill in the art becomes aware of a plant with early symptoms of HLB disease on leaves, which may be small and upright, with vein yellowing and an asymmetrical chlorosis referred to as “blotchy mottle.” Methods according to embodiments of the invention also advantageously can be used when a person of skill in the art becomes aware that a plant is becoming infected by HLB as determined using PCR methods known in the art, for example quantitative real time PCR (qPCR) tests. The inventive methods can also be used prophylactically or in a more severely infected plant with disease of longer standing.
Methods of Administration
Persons of skill are aware of various methods to apply compounds, including live bacteria, to plants for surface application or for uptake, and any of these methods are contemplated for use in this invention. Methods of administration to plants include, by way of non-limiting example, application to any part of the plant, by inclusion in irrigation water, by injection to the plant or to the soil surrounding the plant, or by exposure of the root system to aqueous solutions containing the compounds, by use in hydroponic or aeroponic systems, by seed treatment, by exposure of cuttings of citrus plants used for grafting to aqueous solutions containing the compounds, by application to the roots, stems or leaves, by application to the plant interior, or any part of the plant to be treated. Any means known to those of skill in the art is contemplated.
Application of the bacteria can be performed in a nursery setting, a greenhouse, hydroponics facility, or in the field, or any setting where it is desirable to treat plants which have been or can become exposed to a plant disease, such as HLB or Ca. Liberibacter infection, or which can benefit from an enhancement of health and vigor. The methods and bacteria of this invention can be used to treat infection with a plant pathogen and can be used to improve plant defenses or health, growth and productivity in plants which are not infected. Thus, any plant in need, in the context of this invention, includes any plant susceptible to a lack of optimum health and vigor, or susceptible to a plant disease, whether currently infected or in potential danger of infection, in the judgement of the person of skill in this and related arts.
Application to seeds is preferably accomplished as follows, however any method known in the art can be used. Seeds may be treated or dressed prior to planting, by soaking the seeds in a solution containing the bacteria at a concentration of 103 to 1011 cells/mL over a period of minutes or hours, applying the bacteria at a concentration of 103 to 1011 cells/mL to seeds during planting, or by coating the seeds with a carrier containing the compounds at a concentration of 103 to 1011 cells/mL. The concentrations, volumes, and duration may change depending on the plant.
Application to soil is preferably performed by soil injection or soil drenching, however any method known in the art can be used. These methods of administration are accomplished as follows. Soil drenching may be performed by pouring a solution or vehicle containing the bacteria at a concentration of 103 to 1011 cells/mL at 0.5 to 1 gallon/tree to the soil surface in a crescent within 10 to 100 cm of the trunk on the top side of the bed to minimize runoff, and /or by using the irrigation system. Soil injection may be performed by directly injecting a solution or vehicle containing the bacteria at a concentration of 103 to 1011 cells/mL into the soil within 10 to 100 cm of the trunk using a soil injector. The concentrations, volumes, and duration may change depending on the plant and can be determined by one of skill in the art, however preferred methods are those wherein the administering to the plant provides at least 102 cfu of the isolated bacterial strain per gram of plant root thirty days after administration or at least 103 cfu of the isolated bacterial strain per gram of plant root thirty days after administration.
Application to hydroponic or culture media preferably is performed as follows, however any method known in the art can be used. A solution or vehicle containing the bacteria at a concentration of 103 to 1011 cells/mL may be added into the hydroponic or culture media at final concentrations suitable for plant growth and development. The concentrations, and volumes may change depending on the plant, and can be determined by one of skill in the art.
Application to the roots preferably is performed by immersing the root structure in a solution or vehicle in a laboratory, nursery or hydroponics environment, or by soil injection or soil drenching to the soil surrounding the roots, as described above. Emersion of the root structure preferably is performed as follows, however any method known in the art can be used. A solution or vehicle containing the bacteria at a concentration of 103 to 1011 cells/mL may be applied to the roots by using a root feeder at 0.5 to 1 gallon/tree. The concentrations, volumes, and duration may change depending on the plant and can be determined by one of skill in the art, however preferred methods are those wherein the administering to the plant provides at least 102 cfu of the isolated bacterial strain per gram of plant root thirty days after administration or at least 103 cfu of the isolated bacterial strain per gram of plant root thirty days after administration.
Application to the stems or leaves of the plant preferably is performed by spraying or other direct application to the desired area of the plant, however any method known in the art can be used. A solution or vehicle containing the bacteria at a concentration of 103 to 1011 cells/mL may be applied with a sprayer to the stems or leaves until runoff to ensure complete coverage, and repeat three or four times in a growing season. The concentrations, volumes and repeat treatments may change depending on the plant and can be determined by one of skill in the art.
Application to the plant interior preferably is performed by injection directly into the plant, for example by trunk injection or injection into an affected limb, however any method known in the art can be used. A solution or vehicle containing the bacteria at a concentration of 103 to 1011 cells/mL may be applied with an injector into the plant interior, and repeat three or four times in a growing season. The concentrations, volumes and repeat treatments may change depending on the plant and can be determined by one of skill in the art.
Preferred methods of administration are soil application methods, including soil injection, soil soaking or soil spraying. A highly preferred method according to the invention for treatment of trees is application to the soil by soil injection within a 10-foot radius of a plant to be treated, for example a plant exhibiting infection with or symptoms of infection with a plant pathogen. Any method of administering the bacteria which contacts the bacteria with the roots of the plant is preferred. The concentrations, volumes, and duration may change depending on the plant and can be determined by one of skill in the art, however preferred methods are those wherein the administering to the plant provides at least 102 cfu of the isolated bacterial strain per gram of plant root thirty days after administration or at least 103 cfu of the isolated bacterial strain per gram of plant root thirty days after administration.
Typically, the bacterial strain or strains are administered so as to achieve at least 102-403 cfu, preferably at least about 102 cfu, of each bacterial strain administered per gram of root after a thirty day period, or preferably more than 102-103 cfu of each bacterial strain per gram of root, or more than 103 cfu or 109 cfu of each bacterial strain per gram of root. in a specific embodiment, administering involves administering bacterial strains according to an amount and frequency to achieve at least 102 or 103 cfu/g of root for each bacterial strain after a thirty day period. The composition administered preferably contains more than 103 cfu/ mL of each bacterial strain or 103 to 1012 cfu/mL, or 104-1010 cfu/mL, or 105-109 cfu/mL or 107 cfu/mL of each bacterial strain. A preferred goal of the administration of the bacteria according to embodiments of the invention is to increase the colony-forming units of the bacterial strains at the roots of the plants, and particularly to increase those levels above any natural levels, if any. Therefore compositions are administered to deliver an amount of bacteria to achieve this goal.
A particularly preferred embodiment involves providing the at least one bacterial strain in an amount and with a frequency to achieve at least 102 cfu, 103 cfu, 104 cfu, 105 cfu or more of each bacteria per gram of root after a thirty day period. In one embodiment, the composition administered contains a botanically acceptable vehicle and at least 103 cfu/mL of each bacterial sample. The method can include one bacterial strain, two bacterial strains, three bacterial strains, four bacterial strains, or more, provided in separate carriers or provided together as a mixture.
Compositions
Compositions according to embodiments of the invention preferably include a botanically acceptable vehicle or carrier, preferably a liquid, aqueous vehicle or carrier such as water, and at least one bacterial strain. Preferably, the composition contains 103 cfu/mL to 1010 cfu/mL of each bacterial strain, most preferably' about 107 cfu/mL to about 109 cfu/mL of each bacterial strain. The composition may be formulated as an emulsifiable concentrate(s), suspension concentrate(s), directly sprayable or dilutable solution(s), coatable paste(s), dilute emulsion(s), wettable powder(s), soluble powder(s), dispersible powder(s), dust(s), granule(s) or capsule(s).
The composition may optionally include a botanically acceptable carrier that contains or is blended with additional active ingredients and/or additional inert ingredients. Active ingredients which can be included in the carrier formulation can be selected from any combination of pesticides, herbicides, plant nutritional compositions such as fertilizers, and the like. Plant inducer compounds such as salicylic acid or β-aminobutyric acid (BABA) also can be included in the compositions. Additional active ingredients can be administered simultaneously with the bacterial strains described here, in the same composition, or in separate compositions, or can be administered sequentially.
Inert ingredients which can be included in the carrier formulation can be selected from any compounds to aid in the physical or chemical properties of the composition. Such inert ingredients can be selected from buffers, salts, ions bulking agents, colorants, pigments, dyes, fillers, wetting agents, dispersants, emulsifiers, penetrants, preservatives, antifreezes, evaporation inhibitors, bacterial nutrient compounds, anti-caking agents, defoamers, antioxidants, and the like.
Endophytic bacterial strains were isolated from healthy citrus rhizosphere of asymptomatic Valencia orange (Citrus sinensis) trees in a heavily Huanglongbing (BEM diseased grove of citrus in Fort Pierce, Fla., and were isolated and morphologically characterized as described by Trivedi et al., 2011. The bacteria were isolated using nutrient agar (NA) (BD, Sparks, Md., USA) or tryptone yeast (TY) extract agar medium (Sigma, St. Louis, Mo., USA). The bacterial isolates were also evaluated in vitro for plant growth promoting activity and biocontrol ability, using the methods described by Trivedi et al., 2011. Then the bacterial isolates were subjected to molecular identification using 16S rDNA analysis. DNA extraction, 16S rRNA gene amplification, and sequencing was performed as previously described (Trivedi et al. 2011). Homology was determined using the Blastn programme within the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/). Bacterial strains also were screened for the ability to elicit induced systemic resistance (ISR) as described in the art.
Genomic DNA was extracted from bacterial culture grown overnight at 28° C. in Luria-Bertani (LB) broth medium (Bertani, et al., 1951) using the Wizard genomic DNA purification Kit (Promega, Madison, Wis.) according to the manufacturer's instructions. Quantity and quality of the DNA samples were determined spectrophotometrically (Nanodrop ND-1000; NanoDrop Tech. Inc., Wilmington, Del.). Whole-genome sequencing was performed using an Illumina HiSeq 2000 system at the Beijing Genomic Institute (BGI, Shenzhen, China). All generated paired-end reads were qualitatively assessed, trimmed to remove the vector sequences, and assembled de novo using the Short Oligonucleotides Alignment Program (SOAP) according to the instructions (http://soap.genomics.org.cn/index.html#intro2). The draft genome sequence was annotated using both RAST server (Aziz et al., 2008) and NCBI Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). The annotations were manually refined by direct comparison to closely related completed genomes.
To determine the phylogenic relationship of the three bacterial isolates of ATY16, PT6, and PT26A to other Bacillus spp. and Paenibacillus spp., nine housekeeping genes, uvrD, secA, carA, recA, groEL, dnaK, atpD, gyrB, and infB, from 18 completely sequenced Bacillus spp. and Paenibacillus spp. were used to construct a phylogenic tree. Nucleotide sequences of the nine genes from the above genomes were aligned using DNAMAN and the resulting alignments were presented as a phylogenic tree with the maximum likelihood method.
Almost the full-length nucleotide sequence of the 16S rDNA region of the strain was determined and homology search was performed between the thus determined nucleotide sequence and the DDBJ/EMBL/GenBank international nucleotide sequence database using the Blastn homology search program. The ATY16, PT6, and PT26A strains were preliminarily identified by genomic DNA fingerprinting using 16S rRNA gene sequencing based analysis. The near full-length nucleotide sequence (about 1470 bp) of the 16S rDNA region of the strain was determined and homology search was performed using the GenBank /DDBJ/EMBL international nucleotide sequence database with the Blastn homology search program. The three bacterial strains ATY16, PT6, and PT26A were selected from 39 bacterial isolates for further characterization as they showed the potential to enhance plant growth and/or suppress plant diseases in preliminary tests for plant growth promoting (PGP) and biocontrol ability.
Genomic DNA fingerprinting was performed using housekeeping genes (uvrD, secA, carA, recA, groEL, dnaK, atpD, gyrB, and infB) which were determined by whole genomic sequencing according to methods well known in the art. For this analysis, bacteria with complete known genomes were used, and draft genomes were excluded due to their limitations. To further determine the position of the three bacterial strains in the groups of Paenibacillus spp. and Bacillus spp., a phylogenetic tree was constructed using the maximum-likelihood method for complete sequences of nine housekeeping genes (uvrD, secA, carA, recA, groEL, dnaK, atpD, gyrB, and infB) derived from sequenced genomes of Paenibacillus spp., along with the sequences of some members of the Bacillus spp. These genes have provided robust analysis and resolved evolutionary relationships reliably in other studies. For this analysis, we focused on bacteria with complete genomes and excluded draft genomes due to the limitations of draft genomes. An alignment of the nine genes were created and a phylogenetic tree constructed. See
The 16S rDNA sequence analysis showed that the strain PT6 shared 99% homology with the Bacillus aryabhattai strain IHB B 7024 (Accession No. KJ721203), Bacillus megaterium strain GMA327 (Accession No. AB738784), and Bacillus sp. strain NH1 (Accession No. JN208177). The strain PT26A shared 99% homology with several strains of Bacillus subtilis (Accession No. KJ767313, CP010052, and CP010053, etc.), and Bacillus tequilensis strain IHB B 6839 (Accession No. KF668464). The strain ATY16 shared 99% homology with Paenibacillus sp. strain JDR-2 (Accession No. CP001656), Paenibacillus glycanilyticus strain NBRC 16618 (Accession No. NR_113853) and Paenibacillus sp. strain Al2 (Accession No. KF479531).
The 16S rDNA sequences of these bacterial strains are available in the NCBI GenBank database with the accession number listed above. See also the sequence information below.
Based on the foregoing information, one skilled in the art would be able to identify the bacterial strains described above. Furthermore, the bacterial strains Bacillus megaterium PT6, Bacillus subtilis PT26A, and Paenibacillus sp. ATY16 have been deposited under the Budapest treaty in in the American Type Culture Collection (ATCC), under the following accession numbers: Bacillus megaterium PT6 (accession number PTA-122799), Bacillus subtilis PT26A (accession number PTA-122797), and Paenibacillus sp. ATY16 (accession number PTA-122798).
The deposit was received by the ATCC on Feb. 2, 2016 under the provisions of the Budapest Treaty, and all restrictions upon public access to the deposit will be irrevocably removed upon the grant of a patent on this application. The deposits will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. It should be understood, however, that the availability of the deposits does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Qualitative and quantitative assays were performed for trans related to mineral nutrition (phosphate (P) solubilization, siderophore production, nitrogen (N) fixation), plant development (indole acetic acid (IAA) synthesis), plant health (production of antibiotic and lytic enzymes (chitinase)), induction of systemic resistance (salicylic acid (SA) production), and stress relief (production of 1-aminocyclopropane-1-carboxylate (ACC) deaminase) using methods available in the art and described by Trivedi et al., 2011. All the experiments were done in triplicate and repeated three times.
Plant defense responses were studied by measuring the expression of genes involved in plant defense. A one-step qRT-PCR was performed with a 7500 fast real-time PCR system (Applied Biosystems, Foster City, Calif.) using a QuantiTect SYBR green RT-PCR kit (Qiagen, Valencia, Calif.) following the manufacturer's instructions. Total RNA was extracted from leaf samples by grinding two leaves per sample in liquid nitrogen and 200 mg of tissue was processed using the RNeasy® Mini kit for plant tissue (Qiagen, Md., USA), Contaminated genomic DNA was removed using a TURBO DNA-free kit (Ambion, Austin, Tex.), following the manufacturer's instructions. RNA purity and quality were assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del.). RNA concentration was adjusted to 50 ng/μL, and 2 μL of sample was used for quantitative reverse transcription-PCR (qRT-PCR) relative quantitation of gene expression. See Table 2, below for a list of genes of interest. The housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase-C (GAPDH-C) was used as the endogenous control. The relative fold change in target gene expression was calculated using the formula 2−ΔΔCT (Livak and Schmittgen, 2001), where ΔΔCT=(Cttarget−Ctreference)Treatment−(Cttarget−Ctreference)Control. qRT-PCR was repeated twice with three independent biological replicates each time.
Crop yield is calculated by harvesting the crop and comparing the weight of the crop obtained. For example, the yield of a citrus tree is estimated as the number of boxes of fruit per tree. One box is equivalent to approximately 90 lbs (40.8 kg) of fruit. A composite sample of the crop, randomly chosen from plants can be used for quality analysis. For example, fruit can be juiced and the percentage juice calculated according to Gottwald et al., 2012. Juice quality can be determined following standard methods as well (Gottwald et al., 2012). Fruit acidity can be expressed as percent citric acid. Total soluble solids are expressed as fruit brix (the measure of sugar content in fruit; i.e., 1 g of sugar/100 g of juice is equivalent to 1° of Brix). The fruit brix acidity ratio can be calculated, also according to known methods from the data collected.
D. Quantitative Real-time PCR (qPCR) to Estimate Las Titer in Leaf Samples
For the treatments which showed a suppressive effect against HLB disease development or progress after the initial application, the expression pattern of three plant defense-related genes in the treated citrus was determined by quantitative real time PCR. Samples were taken at four time points (1, 2, 3 or 4, and 6 days after a single application of the treatments). To estimate the Las bacterial titer in treated trees, eight leaves with mottling symptoms were collected from each tree and a combined sample of 100 mg of mid-rib was excised for DNA extraction. DNA from leaf samples was extracted using the Wizard Genomic DNA purification kit (Promega Corp., Madison, Wis., USA) following the protocol for isolating genomic DNA from the plant tissue. The extracted DNA was quantified using a nano-drop spectrophotometer (NanoDrop Technologies, Wilmington, Del.) and adjusted to 100 ng/μL.
qPCR assays were performed in a 96-well plate using an ABI 7500 fast real-time PCR system (Applied Biosystems, Foster City, Calif., USA). The primer/probe set CQULAO4F-CQULAP10P-CQULAO4R targeting the β-operon region of Las was used (Wang et al., 2006) and qPCR reactions were performed according to the conditions described by Trivedi et al. (2009). Each individual sample was replicated three times and the whole reaction was repeated twice. Raw data were analyzed using ABI SDS software with the default settings of the software except that the threshold was adjusted to 0.02 following the instruction of the QuantiTect Probe PCR Kits (Qiagen, Md., USA). The standard equation Y=11.607−0.288X, where Y is the estimated log concentration of templates and X is the qPCR Ct values, as described by Trivedi et al. (2009), was used to convert individual Ct values into bacterial population as genome equivalents or cells (1 cell=1 genome equivalent) per gram of samples.
The shelf life of three different formulations of the bacterial culture (AY16, PT6 and PT26A) were evaluated as follows. The bacterial strains were prepared in the following formulations: (1) solid alginate beads, and (2) broth-based preparations.
Solid alginate formulations were prepared following the method of Bashan (1986) with modifications under sterilized conditions. The bacterial strains were cultured in Luria-Bertani (LB) broth at 28° C. with shaking at 180 rpm for 24 hours to obtain a final concentration of 1010 CFU/mL. Bacterial suspension was aseptically mixed with autoclaved sodium alginate solution (2.0% wt/vol; Sigma, St. Louis, Mo., USA) and stirred gently for 1 hour. For preparing the beads, this mixture was added dropwise into sterilized 0.1 M CaCl2 (Sigma, St. Louis, Mo., USA). The resulting alginate beads were maintained in the solution at room temperature for 2 hours for further solidification. The beads were then washed twice with sterile distilled water and incubated in fresh LB broth for an additional 24 hours in a rotary shaker at 28° C. to allow bacteria to multiply inside the beads. Then the beads were washed twice with sterile distilled water and air dried overnight in a laminar flow hood.
Broth based formulations were prepared in LB and orange peer based broth (OPB) respectively. The broth was inoculated with a loop full of freshly grown bacterial culture and incubated at 28 at 28° C. with shaking at 180 rpm for 24 hours raising the final concentration of 108 cfu/mL. Fresh bacterial cultures were also diluted in sterilized tape water for viability determination.
Viable bacteria in the stored formulations were counted by dissolving 1 g in the case of the solid formulation or 1 mL of liquid formulation in 9 mL 0.85% NaCl in a test tube for 16-24 hours at 28° C. Further enumeration was performed using dilution-plate technique with NA medium. The plates were incubated at 28° C. and CFU were counted after 24 hours. The formulations were stored at two temperatures, 4±2° C. and room temperature (˜23° C.), and viability was checked at an interval of two months. The plate counts were conducted in triplicate and the final values (log10 cfu/g or /mL) of viable bacteria were the average of three readings. Under room temperature, after a 20-month storage, the bacterial populations in LB broth, OPB broth and sterilized tap water were about 102-103cfu/mL with initial populations of 108 cfu/mL. At 4±2° C., after 20-month storage, the bacterial populations in LB broth, OPB broth and sterilized tape water are around 105-106 cfu/mL with an initial population of 108 cfu/mL.
The bacterial viability of alginate bead formulations was also determined and the results were presented in Table 3, below. After a period of 9 months storage at 4±2° C., there was no loss in viability of AY16, PT6 and PT26A in alginate bead formulations; while at room temperature (˜23° C.), there was no loss in viability over a period of 4 months, and after 6 months, the loss in viability was observed readily.
For assays with Arabidopsis, corn, soybean and tomato, sterile Petri dishes (150 mm diameter) containing 40 mL autoclaved water agar (0.65%) was used for seed germination. Petri dishes were partially sealed with parafilm to prevent water loss from evaporation. The experiment was repeated three times with 40 seeds per replicate and arranged in a completely randomized design with three replications. The experiments were conducted in the light and temperature-controlled growing cabinet with constant temperature of 28° C. and a daily cycle of 12 h light and darkness.
For assays with citrus (Citrus paradisi ‘Duncan’ Grapefruit), Deepot cell containers containing sterilized Metro-Mix professional growing soil was used for seed germination in a quarantine greenhouse at the Citrus Research and Education Center, Lake Alfred, Fla. The greenhouse was maintained at approximately 25-30° C. and 60% relative humidity. Arabidopsis, citrus, corn, soybean and tomato seeds were surface sterilized using 1% NaClO and 70% ethanol. Seeds were then washed with autoclaved Millipore water four times to remove the residual bleach and ethanol.
Inoculum of each strain of ATY16, PT6, and PT26A was taken from a pure culture stored with glycerol in −80° C. and streaked onto the nutrient agar plate and incubated at 28° C. for 2 days. A loop full of each culture was transferred separately into the nutrient broth (25 mL) (for ATY16, added 0.5% yeast extract and 0.5% xylan) and incubated for 16 h at 28° C. with constant agitation (180 rpm). Prior to inoculation, bacterial cultures were pelleted by centrifugation (4000×g, 15 min), washed with autoclaved 0.85% (w/v) NaCl, and resuspended in the same buffer solution. The number of colony forming unit (CFU) was determined after series of dilution and agar plating. Seeds were inoculated at population of 108-9/mL by soaking the surface sterilized seeds in bacterial suspension for 1 hour to allow bacteria bind to the seed coat and for seed imbibition. A similar procedure was used for control except the seeds were soaked in buffer solution. Inoculated Arabidopsis, corn, soybean and tomato seeds were spread out in sterile Petri dishes with autoclaved water agar (0.65%), and grapefruit seeds planted in the Deepot cells and maintained in the greenhouse.
For Arabidopsis, corn, soybean and tomato, daily germination was recorded and the germination rate (percent of germinated seeds) was calculated. Seedlings from germinated seeds were weighed at the final day of the experiment. For citrus, weekly germination was recorded and germination rate was calculated. Student's t-test (P<0.05) was used to test the significance of the difference between the means.
Bacteria were subjected to the following conditions. The compatibility of bacterial strains with ultraviolet (UV) protectant was determined by growing the tested bacterium in LB broth medium with different concentrations of a water soluble sodium salt of lignin (Sigma, St. Louis, Mo., USA), or without lignin, under short-wave UV radiation (254 nm in a biological safety cabinet) at a distance of 60 cm for 1 hour. The bacterial population was measured by counting the number of colonies using spread plate techniques on NA plates after incubating at 28° C. for 24 hours. The change in bacterial cell number in LB with or without UV protectant supplementation was determined The compatibility of bacterial strains with SAR inducers was determined by growing the tested bacterium in LB broth medium with or without different concentrations of salicylic acid (SA), acibenzolar-S-methyl (ASM), and 2,6-dichloroisonicotinic acid (INA) (Sigma, St. Louis, Mo., USA), at 28° C. for 20 hours. The bacterial population was measured, and the change in bacterial cell number in LB with or without SAR inducer supplementation was determined The tests were repeated three times with three replicates each time. Student's t-test was used to test the significance of the differences.
A water soluble sodium salt of lignin (Sigma, St. Louis, Mo., USA) at concentrations 0.2 and 0.3% (wt/vol) were able to enhance the survival of bacterial cells of ATY16, PT6 and PT26A versus controls when cells were exposed to UV for 1 hour. See Table 4, below. The assays for compatibility of ATY16, PT6 and PT26A with soil applied SAR inducers ASM and INA showed that these three bacteria could survive well at a 10-20 ppm concentration that is usually applied under field conditions. After 1 hour of exposure to UV radiation, there were less reduction in numbers of surviving cells of the bacterial strain incubated with 0.2% or 0.3% lignin, compared with the control. See Table 4. The results suggested that the (UV) protectant tested may be integrated to the bacterial formulations to enhance the resistance to UV. Lg=Log10.
The assays for compatibility of ATY16, PT6 and PT26A with SAR inducers SA, ASM and INA indicated that these three bacteria could survive well at a 10-20 ppm concentration that is usually applied under field conditions. Results obtained in the present study showed that bacterial cell numbers were almost the same when the cells were cultured in the presence of SA, ASM or INA (10 and 20 ppm) as the growth of the cells without SA, ASM or INA. See Table 5, below.
A series of experiments were conducted to test bacterial tolerance of ATY16, PT6, and PT26A to heat shock, saline stress, and heavy metal (copper) stress. The assays were performed as described previously in the art, with modifications. Bacterial cultures at mid-exponential stage in LB were used to test survival under the aforementioned stresses. In each stress treatment, cell viability was determined by plate-counting of CFU. The survival rate was defined as the percentage of viable cell counts from the culture with stress treatment compared with those from the non-treated culture. The stress treatments were applied as follows: for heat-shock stress, the culture was transferred to 50° C. for 15 mM (30° C. used as control); for sodium stress, NaCl was added to the bacterial culture at a final concentration of 5.0% (w/v) and incubated at 28° C. for 30 mM; and then survival was estimated respectively. Copper stress assays were conducted by growing the tested bacterium in NB broth medium with different concentrations of CuSO4. The growth of each strain was monitored by measuring the optical density at 600 nm after growth for 48 hours at 28° C. without shaking. Each stress test was repeated three times with three replicates each time. Student's t-test was used to test the significance of the difference. The copper resistant bacterium Xanthomonas citri subsp. citri strain A44 [XacA44(Cu—R) and copper sensitive bacterium X. citri subsp. citri strain 306 [Xac306 (Cu—S) were used as positive and negative control respectively.
The results showed that ATY16 can resist 1.0 mM of CuSO4, while PT6 and PT26A can resist 2.0 mM of CuSO4 in nutrient broth (NB) (BD, Sparks, Md., USA). After 48 hours growth in NB with 1.0 mM of CuSO4, the bacterial growth (optical density (OD) at 600 nm) of ATY16 was the same as the copper resistant bacterium Xanthomonas citri subsp. citri strain A44, and PT6 and PT26A was at a higher level than strain A44. See
These assays revealed that ATY16, PT6, and PT26A was of certain tolerance to heat shock. Following 15 minutes of exposure of bacteria to heat (50° C.), which was thought able to kill most bacterial cells, there were about 0.5% of ATY16, about 1.0% of PT6 and PT26A viable cells in the broth medium. See Table 6, below.
These assays also indicated that ATY16, PT6, and PT26A was of certain tolerance to salt stress. After 30 minutes of exposure of bacteria to 5.0% (wt/vol) NaCl, which was thought able to kill most bacterial cells, there were about 40-50% of ATY16, PT6 and PT26A viable cells in the broth medium. See Table 7, below.
Bacterial combinations were tested for survival as follows. The three strains of ATY16, PT6, and PT26A were tested to be compatible with each other. Bacterial growth compatibility test was performed in vitro in Nutrient agar (NA) plates by cross-streaking a fresh culture of the two bacterial strains to be tested. Plates were incubated at 28° C. and results were photo recorded after 24 hours. The antagonistic survival tests on nutrient agar (NA) plates showed that these three bacteria, ATY16, PT6, and PT26A, can survive well with each other under the experiment conditions. No growth inhibition was observed after 24 hours post co-streaking on NA plates. There is no antagonistic effect between the bacterial isolates. See
Grapefruit seedling plants were treated as follows.
Rifamycin resistance was used as the antibiotic selection marker to track the bacteria following application. The spontaneous rifamycin resistance of the bacterium was obtained using a gradient-inducing method as described elsewhere.
For greenhouse test, the spontaneous rifamycin resistant mutant of bacterial strains was grown under optimal conditions (28° C. and 180 rpm) for 24 hours in LB medium with rifamycin (50 μg/mL) and diluted with fresh sterile LB medium to OD (600nm)=0.3, equivalent to approximately 5×108 cfu/mL. Roots from 60 day-old citrus seedlings (Grapefruit) were carefully washed under a stream tap water to remove potting media, inoculated in bacterial suspensions for 60 minutes and transplanted into 0.5-liter pots containing sterile soil. Plants were transferred to a greenhouse as previously described. Once a week, the root systems of five independent plants initially inoculated with bacterial strains were collected and the roots and rhizospheric soil carefully separated, weighted and homogenized in sterile saline and platted on NA-agar plates with rifamycin (50 μg/mL) as described above. Finally, abundance of bacterial strains on root surfaces and in rhizospheric soil was expressed as log cfu/g fresh root weight and log cfu/g wet soil weight respectively.
For field experiments, the bacterial cultures (5×108 cfu/mL) were applied as soil drench around the trunk of 6-year old Valencia sweet orange trees in a grove at CREC, Florida. Once a week, the root and rhizospheric soil samples from three trees were collected and bacterial populations were determined as described above. The data showed that these bacteria can establish a population of 104-105 cfu/g root and 103-104 cfu/g soil at one month after inoculation with an initial population of 105-106 cfu/g root and 104-105 cfu/g soil respectively in greenhouse. Under field conditions, these bacteria can establish a population of about 103 cfu/ g root and about 102 cfu/g soil at one month after inoculation with an initial population of about 105 cfu/ g root and about 104 cfu/g soil respectively. See
The bacterial strains were cultured in Luria-Bertani (LB) broth at 28° C. with shaking at 180 rpm for 24 hours, then bacterial cells were collected by centrifugation (4000×g, 15 min). After subjecting the media to the conditions described above, they were tested for phosphate solubilization ability (P-sol) as an indicator of sufficient mineral nutrition; indole acetic acid production (IAA) as an indicator of plant development; siderophore production (Sid) as an indicator of sufficient mineral nutrition; antibiotic (antimicrobial) production (Anti-M) as an indicator of improved ability to defend against disease (antibiotic production was tested against several plant pathogenic fungi and bacteria including Alternaria, Aspergillus, Fusarium, Rhizoctonia, and the citrus canker bacterium Xanthomonas citri subsp. Citri); lytic enzyme production (Chitinase) as an indicator of production of antibiotic and lytic enzymes (plant health); salicylic acid production (SA) as an indication of systemic resistance induction and improved resistance to disease; nitrogen fixation (N-fix) as an indicator of nutritional health; and 1-aminocyclopropane-1-carboxylate deaminase production (ACCD) as an indicator of stress relief. The results showed that each of the three bacteria ATY16, PT6 and PT26A provided several effects that can be beneficial to plants, including phosphate solubilization ability, production of IAA, producing salicylic acid, nitrogen fixation ability, and producing ACC deaminase, and production of antibiotics against several pathogenic fungi and bacteria (including Alternaria, Aspergillus, Fusarium, Rhizoctonia, and the citrus canker bacterium Xanthomonas citri subsp. citri). See Table 8, below. “+” indicates the presence of trait; “−” indicates the absence of trait.
See
Two-year old potted Valencia sweet orange (Citrus sinensis) plants were trimmed about 3 weeks prior to bacterial inoculation to get new flushes. Once new flush emarginated, the plants were immediately covered with an insect proof cage, transferred to a secured greenhouse at CREC, FL, and exposed to HLB-infected psyllids for HLB inoculation. Then bacterial cultures (ATY16, PT6 and PT26A) were applied in a single application per month as a soil drench (500 mL of 5×108 cfu/mL solution per pot). The plants were checked monthly for HLB symptoms and Las quantification using qPCR analysis (Trivedi et al. 2011). The experiments were carried out in triplicate and tap water was used as non-treated control. The plants were analyzed for effects on growth of the HLB causative bacterium as determined by qPCR. See Example 1 for molecular biological methods. Ct values (cycle threshold values and Las titers are reported for each bacterial strain at 0, 90 and 120 days after treatment (DAT). See Table 9, below. The results showed that the three bacteria ATY16, PT6 and PT26A delayed the development of HLB symptoms and Las populations in new flushes of citrus plants exposed to HLB infected psyllids in greenhouse assays over a period of 4 months.
aqPCR and Las titer assays were performed as described by Trivedi et al (2009).
PT6, PT26A and ATY16 bacteria also delayed the development of HLB symptoms. Plants were treated as follows. Two-year old potted Valencia sweet orange (Citrus sinensis) plants were trimmed about 3 weeks prior to bacterial inoculation to get new flushes. Once new flush emarginated, the plants were immediately covered with an insect proof cage, transferred to a secured greenhouse at CREC, FL, and exposed to HLB-infected psyllids for HLB inoculation. Then bacterial cultures were applied in a single application per month as a soil drench (500 mL of 5×108 cfu/mL solution per pot). The plants were checked monthly for HLB symptoms and Las quantification using qPCR analysis (Trivedi et al. 2011). The experiments were carried out in triplicate and tap water was used as non-treated control. See
A series of experiments were conducted to determine the germination responses of Arabidopsis, citrus, corn, soybean and tomato seeds to plant beneficial bacteria inoculation. Seeds were inoculated with different bacterial strains of B. megaterium PT6, B. subtilis PT26A, and Paenibacillus sp. ATY16. Arabidopsis and Citrus seeds were treated as follows.
For assays with Arabidopsis, sterile Petri dish (150 mm diameter) containing 40 mL autoclaved water agar (0.65%) was used for seed germination. Petri dishes were partially sealed with parafilm to prevent water loss from evaporation. The set up was repeated three times with 40 seeds per replicate and arranged in a completely randomized design with three replications. The experiments were conducted in a light and temperature-controlled growing cabinet with a constant temperature of 28° C. and a daily cycle of 12 hours each light and darkness.
For assays with citrus (Citrus paradisi ‘Duncan’ Grapefruit), Deepot cells (containers) containing sterilized Metro-Mix professional growing soil was used for seed germination in a quarantine greenhouse at the Citrus Research and Education Center, Lake Alfred, Fla. The greenhouse was maintained at approximately 25-30° C. and 60% relative humidity. Arabidopsis and citrus seeds were surface sterilized using 1% NaClO and 70% ethanol. Seeds then were washed with autoclaved Millipore water four times to remove the residual bleach and ethanol.
Inoculum of each strain of ATY16, PT6, and PT26A was taken from a pure culture stored with glycerol at −80° C. and streaked onto the nutrient agar plate and incubated at 28° C. for 2 days. A loop full of each culture was transferred separately into the nutrient broth (25 mL) (for ATY16, with added 0.5% yeast extract and 0.5% xylan) and incubated for 16 hours at 28° C. with constant agitation (180 rpm). Prior to inoculation, bacterial cultures were pelleted by centrifugation (4000×g, 15 min), washed with autoclaved 0.85% (w/v) NaCl, and resuspended in the same buffer solution. The number of colony forming unit (cfu) was determined after series of dilution and agar plating. Seeds were inoculated at population log 8-9 cfu/mL by soaking the surface sterilized seeds in bacterial suspension for 1 hour to allow bacteria bind to the seed coat and for seed imbibition. A similar procedure was used for the control except seeds were soaked in buffer solution. Inoculated Arabidopsis seeds were spread out in a sterile Petri dish with autoclaved water agar (0.65%), and grapefruit seeds planted in the Deepot cells and maintained in the greenhouse.
The plant seeds were analyzed for seed germination and seedling growth (root length). See
The results showed that the three bacteria ATY16, PT6 and PT26A are able to promote seed germination and seedling growth of Arabidopsis in vitro; and increase seedling emergence and growth of citrus in greenhouse, with stronger root systems. The results also revealed that PT26A was able to promote seed germination and seedling growth of corn, soybean and tomato in vitro. Corn, soybean and tomato started to germinate on the following day post inoculation (DPI). Corn reached maximum germination 4 days from sowing while soybean and tomato reached peak germination after 3 days.
See
Seeds were germinated as follows: a sterile petri dish (150 mm diameter) containing 40 mL autoclaved water agar (0.65%) was used for seed germination. Petri dishes were partially sealed with parafilm to prevent water loss from evaporation. This set-up was repeated three times with 40 seeds per replicate, completely randomized with three replications. The experiments were conducted in a light and temperature-controlled growing cabinet with a constant temperature of 28° C. and a daily cycle of 12 hours each of light and darkness.
Corn, soybean and tomato seeds were surface sterilized using 1% NaClO and 70% ethanol. Seeds were then washed with autoclaved Millipore water four times to remove the residual bleach and ethanol.
An inoculum of each strain of Bacillus megaterium PT6, Bacillus subtilis PT26A, and Paenibacillus sp. ATY16 was taken from a pure culture stored with glycerol in −80° C. and streaked onto the nutrient agar and incubated at 28 C for 2 days. A loop full of each culture was transferred separately into the nutrient broth (25 mL), and incubated for 16 hours at 28° C. with constant agitation (180 rpm). Prior to inoculation, bacterial cultures were pelleted by centrifugation (4000×g, 15 min), washed with autoclaved 0.85% (w/v) NaCl, and resuspended in the same buffer solution. The number of colony forming unit (cfu) was determined after series of dilution and agar plating. Seeds were inoculated at population log 8-9 cfu/mL by soaking the surface sterilized seeds in bacterial suspension for 1 hour to allow bacteria bind to the seed coat and for seed imbibition. A similar procedure was used for control except seeds were soaked in buffer solution.
Daily germination was recorded and the germination rate (percent germinated seeds) was calculated. Seedlings from germinated seeds were weighed at the final day of the experiment. Student's t-test (P<0.05) was used to test the significance of the mean differences.
Among the three bacterial strains tested, PT26A was able to promote seed germination and seedling growth of corn, soybean and tomato in vitro. Corn, soybean and tomato started to germinate on the following day post inoculation (DPI). Corn reached maximum germination 4 days from sowing whiles soybean and tomato reached peak germination after 3 days. Regardless of the crops, seeds inoculated with the bacterial strain PT26A generally germinated earlier and faster than non-inoculated ones, with a higher total germination at 2 days post inoculation (DPI); whereas inoculation with ATY16 or PT6 did not affect corn, soybean or tomato seed germination (
PT26A inoculation also improved the vigor of germinating seeds of the three crops. PT26A-inoculated corn and tomato seeds, produced heavier seedlings compared to non-inoculated control (
The experiments of this study were performed in two citrus groves at Lake Wales, Florida. The first grove, subsequently noted as Mck block#15, was planted with Valencia sweet orange [Citrus sinensis (L.) Osbeck] Blanco] on Swingle citrumelo [Citrus paradisi Macf. “Duncan” grapefruit×Poncirus trifoliata (L.) in 2009. The second grove, subsequently noted as Hunt block#23, was planted with Murcott mandarin [Citrus reticulata (L.) Blanco] on Cleopatra mandarin [Citrus reticulata (L.) Blanco] rootstock in 2003. In each grove, the HLB disease severity was inspected and 20 HLB diseased trees were randomly selected for the study. A mixture of ATY16, PT6 and PT26A bacterial cultures (5×109 cfu/mL each) was applied monthly as a soil drench at 4.0 liters/tree to the soil surface in a crescent within 25 to 50 cm of the trunk on the top side of the bed to minimize runoff. The other 10 trees were provided water only as a non-treated control. Following the first application, the HLB disease severity and Las population was determined tri-monthly using visual inspection (Gottwald et al., 2007) and qPCR analysis (Trivedi et al., 2011) respectively.
The data collected showed that applications of a consortium of the three beneficial bacteria ATY16, PT6 and PT26A reduces the HLB disease progress and slows the pathogen population growth, compared with the non-treated control in the field trial with mild HLB affected citrus trees. See
All patents, patent applications, patent publications, technical publications, scientific publications, and other references referenced herein are hereby incorporated by reference in this application to the extent they are not inconsistent with the teachings herein. In particular, the following references are hereby incorporated by reference in their entirety.
ATP-dependent DNA helicase UvrD/PcrA, Paenibacillus sp. ATY16.
Protein export cytoplasm protein SecA ATPase RNA helicase (preprotein translocase subunit SecA), Paenibacillus sp. ATY16.
Carbamoyl-phosphate synthase small chain cara, Paenibacillus sp. ATY16.
RecA protein, Paenibacillus sp. ATY16.
Chaperone protein DnaK, Paenibacillus sp. ATY16.
Heat shock protein 60 family chaperone GroEL, Paenibacillus sp. ATY16.
ATP synthase beta chain atpD, Paenibacillus sp. ATY16.
gyrB (DNA gyrase subunit B), Paenibacillus sp. ATY16.
Translation initiation factor 2 (infB), Paenibacillus sp. ATY16.
ATP-dependent DNA helicase UvrD/PcrA, B. megaterium PT6.
Protein export cytoplasm protein SecA ATPase RNA helicase, B. megaterium PT6.
carbamoyl-phosphate synthase small chain (carA), B. megaterium PT6.
RecA, B. megaterium PT6.
DnaK, B. megaterium PT6.
Heat shock protein 60 family chaperone GroEL, B. megaterium PT6.
ATP synthase beta chain (atpD), B. megaterium PT6.
gyrB (DNA gyrase subunit B), B. megaterium PT6.
Translation initiation factor 2 (infB), B. megaterium PT6.
ATP-dependent DNA helicase Uvr/PcrA, B. subtilis PT26A.
Protein export cytoplasm SecA ATPase RNA helicase (TC3.A.5.1.1), B. subtilis PT26A.
Carbamoyl-phosphate synthase small chain (carA, macromolecular synthesis operon), B. subtilis PT26A.
RecA protein, B. subtilis PT26A.
Heat shock DnaK gene cluster chaperone protein, B. subtilis PT26A.
Heat shock protein 60 family chaperone GroEL, B. subtilis PT26A.
ATP synthase beta chain (atpD), B. subtilis PT26A.
gyrB (DNA gyrase subunit B), B. subtilis PT26A.
Translation initiation factor 2 (infB), B. subtilis PT26A.
| Number | Date | Country | |
|---|---|---|---|
| 62113048 | Feb 2015 | US | |
| 62199327 | Jul 2015 | US |
