Patent Application: US-51135900-A

Abstract:
the invention accelerates the growth of all types of plants , i . e . tomato , cucumber , bell pepper , tobacco , horticultural crops , and others , and increases plant health . the invention is designed to increase the rate of seedling growth in the soil or in the greenhouse ; and produce seeds having a coating imparting resistance to disease , thereby decreasing the time required to produce transplants in seedling greenhouses prior to transplanting into agricultural fields ; and develop “ disease - suppressive ” transplant plugs , which are protected for a time from multiple diseases through combined mechanisms of stimulated plant defense and increased activity of indigenous antagonistic microorganisms on plant roots . compositions and methods of use are provided .

Description:
the invention relates to various combinations of biocontrol tactics applied together into soil or plant growth media used for production of plants or transplant plugs . the “ tactics ” may include mixtures of two or more pgpr strains where one or more can induce plant resistance . these “ tactics ” may further include organic amendments , which may have nematode - control activity , and “ selector compounds ”, such as botanical aromatic compounds ( including thymol , benzaldehyde , citral , furfural , menthol and alpha - terpineol ), which alter soil microflora to enhance activity of indigenous antagonistic microorganisms . the invention may also consist of using two of these three tactics , i . e . pgpr mixtures together with organic amendments , but without addition of selector compounds . the invention provides other embodiments further discussed hereinafter . in accordance with the invention , a novel synergy of biocontrol tactics of a heretofore unidentified composition has been discovered . the composition , in accordance with the invention , initiates and promotes plant growth and synergistically induces systemic disease resistance in plants . the characteristics of the composition are both novel and synergistic . the invention provides a composition comprised of chitinolytic and nonchitinolytic constituents for plant growth and disease resistance . the composition results in a synergy of constituent characteristics including initiation and promotion of plant growth and the inducing of systemic plant disease resistance . the composition is comprised of at least one pgpr strain and an organic amendment . the biological pgpr mixture preferably comprises spore preparations of the bacteria . additionally , in the pgpr mixture , at least one pgpr strain can induce systemic plant resistance to disease . the presence of the bacteria in the mixture is generally on the order of 10 3 to 10 10 bacteria per seed or per liter of soil - less mix . a preferred embodiment of the invention is where the bacteria presence is approximately 1 × 10 10 bacteria per liter of soil - less mix and 1 × 10 8 bacteria per seed . examples of two formulated pgpr strains include but are not limited to bacillus sublilis strain gb03 ( available from gustafson llc ., plano , tex . as kodiak ™) and bacillus amyloliquefaciens strain in937a . other types of nonchitinolytic bacteria suitable for the composition include : root - colonizing bacteria including the family bacilliaceae which is spore - forming and comprises the genera bacillus , paenibacillus , brevibacillus , virgibacillus , alicyclobacillus and aneurinibacillus ; fluorescent pseudomonads ; isolates of pseudomonas spp ., serratia spp ., cornynebacterium spp ., enterobacterspp ., arthrobacter spp ., and burkholderia spp . ; and benefical fungi such as trichoderma spp ., gliocladium spp ., and others ; yeasts , and actinomycetes . additional examples of the group of bacilli and their spore - forming genera , including bacillus , are referenced in the “ atcc catalogue of bacteria ”, published by the american type culture collection , and are incorporated herein by reference . the organic amendment is also known as a chitinolytic component as it exhibits nematode - control activity . the chitinolytic component in the composition is in an amount sufficient to cause a chitinolytic effect . the amount of the chitinolytic component present may range from 0 . 1 % to 10 . 0 %. a preferred embodiment of the invention is where the composition is comprised of approximately 2 . 5 % of the chitinolytic component . examples of the chitinolytic component include but are not limited to chitin , flaked chitin , and chitosan . the chitinolytic component can also be derived from its precursors which upon hydrolysis or other chemical or biochemical breakdown will yield the chitinolytic component . preferably , the organic amendment is a glucose polysaccharide . such precursors are organic natural compounds like pine bark , crab or shrimp shells , soybean meal , cotton seed meal and casein . a further embodiment of the invention is a composition comprising at least one of the pgpr strains , an organic amendment , and an optional botanical aromatic compound . the optional addition of a botanical aromatic compound , also known as a selector compound , to the primary composition further introduces a fumigant for altering soil microflora . the presence of the fumigant reduces parasitic nematodes and increases antagonists . an example of the botanical aromatic compound includes but is not limited to benzadehyde . other types of botanical aromatic compounds suitable for the composition include citral . further optional components include components which do not negatively affect the function of the two principal components of the composition , referred to as non - essential ingredients . a further embodiment of the invention is a composition comprising the two pgpr strains , an organic amendment , and an optional botanical aromatic compound . an embodiment of the invention is a method for promoting plant growth and synergistically inducing disease resistance in plants . the method comprises exposing seed to the novel biological composition or growing seedlings in a soil - less potting media containing the novel biological composition , for a period of time sufficient to initiate improved growth and disease - suppressive characteristics . a seed is planted in the soil or in the soil - less media and grown in a greenhouse under conditions previously discussed . by way of example , the biological composition may be incorporated into a soil - less media such as in a styrofoam transplant flat and then seeded . during this growth period , if desired , additional treatments of the composition are provided to the plant in predetermined amounts at predetermined times . then , if desired , when the seedlings have advanced to an age of approximately four to six weeks , or where the plants are ready for transplant , the plant is transplanted to field conditions or greenhouse conditions where the plant has been observed to continue to grow normally . further , the plants are then optionally treated with the composition , in which the composition is in either a liquid or solid formn . preferably , this treatment is in the form of a foliar spray , drench application , drip application or through irrigation where it has been observed that the plants achieve a greater resistance to disease . this optional treatment may also be performed at any time during the growth period . further , untreated seeds are coated by exposing the seed with the composition , either in a liquid or solid form , preferably in a foliar spray , drench application , drip application or through irrigation . this treatment may be optional , and may additionally be performed at any time to untreated or treated seeds . particularly , this method stimulates and promotes plant growth in the early seedling stages which is known to be a difficult stage of plant growth to stimulate with pgpr alone . the method also induces disease resistance systemically in plants to a greater extent than previously achieved in the prior art . the invention also synergistically enables both the stimulation and promotion of plant growth in combination with systemically protecting a plant from disease by exposure to the biological composition . further embodiments of the invention provide methods for inhibiting the growth of disease agent phytophtora infestans ( a causal agent of late blight disease ), xanthomonas axonopodis pv . vesicatoria ( a causal agent of bacteria spot disease ), pseudomonas syringae pv . lachrymans ( a causal agent of angular leaf spot disease ), and fusarium spp . by using the method discussed above and challenging the plant with a treatment of the disease agent . the challenged seedling is then evaluated and the incidence and severity of disease are measured . it has been observed that plants grown under the present invention and challenged with the disease exhibited a significantly greater ability to resist the disease . an additional embodiment of the invention is a plant which having been exposed to the composition exhibits the synergistic effects of improved growth in combination with the systemic protection from disease . seeds , seedling and plants which may be treated and obtained in accordance with the invention include both monocotyledonous and dicotyledonous plant species including barley , oats , rice , wheat , soybean , corn ; melons including cucumber , muskmelon , cantaloupe and watermelon ; vegetables including beans , pea , peanut ; oil crops including canola and soybean ; solanaceous plants including tobacco ; tuber crops including potato ; vegetables including tomato , pepper , cucumber , broccoli , cabbage , cauliflower , lettuce and radish ; fruits including strawberry ; fiber crops including cotton ; other plants including coffee , bedding plants , perennials , woody ornamentals , turf and cut flowers including carnation and roses ; sugar cane ; containerized tree crops ; evergreen trees including fir and pine ; deciduous trees including maple and oak ; and fruit trees including cherry , apple , pear and orange . in general any seed , seedling or plant that is susceptible to plant disease and does respond to the composition of the invention may be treated in accordance with the invention . the resulting plant of the present invention exhibits improved plant growth in the early seedling stages , which is known to be a difficult stage of plant growth to stimulate with pgpr alone . the plant further exhibits measurably improved plant physical characteristics such as greater height , weight , vigor , leaflets and leaflet surface area , at growth stages earlier than non - treated , control plants . similarly , the plant displays less disease than untreated , control plants . further embodiments of the invention include various media for culturing the plants using the composition , in particular tomato , cucumber , and classes previously discussed , and parts thereof which have been developed in accordance with the invention . other embodiments of the invention will become apparent in the further detailed description of preferred and other embodiments of the invention . the term “ transplant ” as used herein is a term of art used to designate a plant of any age of any variety that is moved from one growing location to another . the term “ transplant plug ” as used herein is a plant of any age situated in a contained growth medium in which the plant is prepared to be transplanted or transported from one location to another . the term “ soil - less medium ” as used herein is a growth medium comprising peat - based products which may contain perlite , vermiculite , a fertilizer component and other ingredients . examples of a soil - less medium include readily available products such as “ pro - mix ”, “ redi - gro ” and “ speedling mix ”. the term “ seedling ” as used herein is a term of art used to designate a plant of an age ranging from the day of emergence to one year after planting or an age where transplant of the plant occurs and is not limited to or by any plant class . the term also includes “ forest seedlings ” which can be one year old . the term “ synergy ” as used herein is used to designate the resulting action of two or more substances to achieve an effect of which each is individually incapable of achieving . as the first example of the invention , a biological preparation was made containing two pgpr strains together with chitin . specifically , the biological preparation ( termed ls213 ), which is publicly available , contained bacillus subtilis strain gb03 , has been shown to control some diseases by production of iturin antibiotics , and bacillus amyloliquefaciens strain in937a which was selected for activation of induced resistance . with both of these bacteria , industrial formulations of spores were prepared and were mixed with flaked chitin . the biological preparation was added to soil - less potting media which was used to prepare tomato transplant plugs . additional treatments consisted of each bacterial strain alone with and without chitin , chitin alone , and a nontreated control . transplant plugs were grown in transplant trays in the greenhouse and effects on seedling growth were noted . results are presented in table 1 . across all three measurements ( vigor , shoot fresh weight , and leaflet surface area ), significantly greater seedling growth occurred with ls213 than with the various individual components . this indicates a clear synergy in plant growth promotion by the combination of chitin and the two pgpr strains . 1 biological treatments were incorporated into soil - less mix at 1 : 40 ( v / v ), placed into styrofoam transplant flats , and then seeded with tomato cv . solar set . there were four replicated flats per treatment . gb03 and in937a are pgpr strains . 2 seedling vigor was rated at 3 weeks after seeding on a scale of 1 - 5 ; 1 = poor , 2 = average , 3 = good , 4 = very good , 5 = excellent . mean of four replications . 3 seedling shoot fresh weight . mean of 4 replications , 10 seedlings per replication . 4 largest leaflet surface are ( usually from the 4rth or 5 th true leaf ). mean of 4 replications , 10 leaflets per replication . means followed by different letters are significantly different according to the protected least significance difference ( lsd ) test at p = 0 . 05 . certain of the transplant plugs of the above experiment , those being treated with the control , the chitin alone and the ls213 , were transplanted to larger pots immediately after making the measurements of growth shown in table 1 . ten plants of each treatment type were then challenged with phytophthora infestans ( causal agent of late blight disease ) or xanthomonas axonopodis pv . vesicatoria ( causal agent of bacterial spot disease ). after a predetermined time , the plants were measured for disease resistance . results of disease development are shown in table 2 . the highest degree of disease protection against both pathogens occurred with ls213 . with bacterial spot , the protection afforded by ls213 was significantly greater than both the nontreated control and the chitin alone treatment . this indicates that the synergy in plant growth promotion noted in table 1 translates into synergy in disease protection . an experiment similar to that described in example 1 was conducted . the components of chitin , gb03 and in937a were tested in soil - less plant growth medium for effects on seedling growth using tomato and cucumber . additional measurements of plant growth were conducted as shown in tables 3 and 4 . results are shown in table 3 and table 4 . the results with both tomato ( table 3 ) and cucumber ( table 4 ) demonstrate again that there is a clear synergy of tactics for growth promotion . for example , with tomato ( table 3 ), chitin alone had some growth promotion , causing significant increases , relative to the control , in height , number of leaflets , and leaflet surface area ; but not causing significant increases in vigor , weight , or chlorophyll content . in contrast , ls213 treatment resulted in significant increases , relative to the control , for all of these parameters . similarly on cucumber ( table 4 ), ls213 caused significant increases in all parameters , compared both to the nontreated control and chitin alone . after making the growth measurements described above , plants from three treatments of tomato and cucumber were transplanted to pots . treatments tested were the nontreated control , chitin alone , and ls213 . tomato plants were inoculated with the tomato spot pathogen ( as described in example 1b ), and cucumber plants were inoculated with pseudomonas syringae pv . lachrymans , causal agent of angular leaf spot disease . the results are shown in table 5 . the results shown in table 5 indicate that seedlings prepared from ls213 treatments had induced disease resistance . interestingly , the seedlings prepared from treatment with chitin alone , which caused some significant plant growth increases , were not resistant . the lack of induced resistance with chitin when significant growth promotion occurred further demonstrates that the benefits of ls213 are synergistic . 1 biological treatments were incorporated into soil - less mix at 1 : 40 ( v / v ) and placed into styrofoam transplant flats and then seeded with tomato cv . solar set . there were four replicated flats per treatment . 2 seedling vigor was rated at 3 weeks after seeding on a scale of 1 - 5 ; 1 = poor , 2 = average , 3 = good , 4 = very good and 5 = excellent . mean of four replication . 3 seedling height from the soil level to the tip . mean of 4 replications , 5 seedlings per replication . 4 seedling shoot fresh weight . mean of 4 replications , 5 seedlings per replication . 5 number of leaflets per plant . mean of 4 replications , 5 plants per replication . 6 largest leaflet surface area ( usually from the 4rth or 5 th true leaf ). mean of 4 replications , 5 plants per replication . 7 chlorophyll content from the largest leaflet either from 4rth or 5 th true leaf . mean of 4 replications , 5 plants per replication . means followed by different letters are significantly different according to the protected least significance difference ( lsd ) test at p = 0 . 05 . 1 biological treatments were incorporated into soil - less mix at 1 : 40 ( v / v ) and placed into styrofoam transplant flats and then seeded with tomato cv . solar set . there were four replicated flats per treatment . 2 seedling vigor was rated at 3 weeks after seeding on a scale of 1 - 5 ; 1 = poor , 2 = average , 3 = good , 4 = very good and 5 = excellent . mean of four replication . 3 seedling height from the soil level to the tip . mean of 4 replications , 5 seedlings per replication . 4 seedling shoot fresh weight . mean of 4 replications , 5 seedlings per replication . 5 number of leaflets per plant . mean of 4 replications , 5 plants per replication . 6 largest leaf surface area ( usually from the 4rth or 5 th true leaf ). mean of 4 replications , 5 plants per replication . 7 chlorophyll content from the largest leaf either from 4rth or 5 th true leaf . mean of 4 replications , 5 plants per replication . means followed by different letters are significantly different according to the protected least significance difference ( lsd ) test at p = 0 . 05 . an example was conduced to determine if the synergistic effects on seeding plant growth noted in examples 1 and 2 occurred with additional crops . in this experiment , a non - treated control , chitin alone , ls213 treatments were preparedas described in example 1 and used to grow seedlings of tomato ( cv . solar set ), cucumber ( cv . smr 48 ), bell pepper ( cv . california wonder ), and tobacco ( cv . tn90 ). seedling growth was monitored by measuring the parameters listed in table 6 . with tomato and cucumber , the results previously seen repeated in which ls213 treatment generally resulted in significant growth promotion compared both to the nontreated control and the treatment with chitin alone ( table 6 ). this synergistic effect on growth was seen with pepper and and tobacco . an experiment was conducted to determine if one of the pgpr strains used in ls213 could be replaced with another strain . more specifically , pgpr strain in937a from ls213 was replaced with 8 differnt bacilli , each of which had induced resistance activity by itself . the bacteria were mixed with pgpr strain gb03 and chitin prior to incorporating into soil - less plant growth medium . effects on seedling growth of cherrry tomato ( cv . rx335 ) were tested along with effects on induced resistance against bacterial spot . results shown in table 7 show that ls213 caused significant promotion of plant growth and induction of resistance , compared to the control with this cultivar of tomato similar to what was seen previously on ‘ solar set ’ tomato . other combinations of bacteria and chitin generally caused significant enhancements in growth compared to the control ; however , not all of these caused significant protection against bacterial spot . overall the results show that the beneficial effects of the invention are bacterial - strain dependent , but that differnt mixtures of bacteria may be used to achieve the same results . 1 biological treatments were incorporated into soil - less mix ) at 1 : 40 ( v / v ) and placed into styrofoam transplant flats and then seeded with tomato cv . cherry rx 335 . there were two replicated flats per treatment . 2 seedling vigor was rated at 4 weeks after seeding on a scale of 1 - 5 ; 1 = poor , 2 = average , 3 = good , 4 = very good and 5 = excellent . mean of 2 replications . 3 seedling height from the soil level to the tip . mean of 2 replications , 10 seedlings per replication . 4 number of leaflets per plant . mean of 2 replications , 10 leaflets per replication . 5 stem diameter is the mean of 2 replications , 10 seedlings per replication . 6 seedling shoot fresh weight . mean of 2 replications , 10 seedlings per replication . 7 largest leaflet surface area ( usually from the 4rth or 5 th true leaf ). mean of 2 replications , 10 leaflets per replication . means followed by different letters are significantly different according to the protected least significance difference ( lsd ) test at p = 0 . 05 . two of the biological preparations used in example 4 ( ls213 and ls254 ) were tested in a field trial . the tomato seedlings ( cv . ‘ solar set ’) were prepared as previously described by growing in soil - less mix trated with the biological preparations . a nontreated control group of plants was also prepared . the transplants were placed into a randomized complete block design , with 10 plants per replication and 5 replications of each treatment . the experiment was planted four times for assessment of 1 ) root - knot nematodes , 2 ) fusarium crown and root rot with methyl bromide fumigation of soil , 3 ) fusarium crown and root rot without soil fumigation , and 4 ) bacterial spot . results , shown in table 8 , demonstrate that the biological preparations led to reduced develpment of all the tested diseases under field conditions . two field trials were conducted on tomato to determine if different forms of the biological preparation ( differing in one of the bacterial components ) would protect gainst root - knot nematodes and bacterial spot as did tratment with ls213 in example 5 . results , shown in table 9 , indicaate that other forms of thee biological preparation did lead to protection of plants against the two tested pathogens . an objective was to determine if beneficial effects of the biological treatment could be enhanced by combining the standard treatment of the sil - less planting medium with a mid - season foliar spray of the bacterial components of the biological preparation . the results indicate that there was some additional protection seen by combining the standard treatment with a foliar spray . for example , without the foliar spray , ls254 caused significant protection against root - knot nematode but not against bacterial spot , while with the foliar spray , protection occurred against both pathogens . similar to example 6 , two field trials were conducted on cucumber to assess disease control activity of different forms of the biological preparation with and without foliar sprays . the diseases were both naturally occurring - root - knot nematodes and anthracnose , a foliar fungal disease . the results presentd in table 10 demonstrate that changing pgpr strain in937a with different pgpr can result in disease protection activtiy in the field . the results also demonstrate that the beneficial effects of the biological preparations are not limited to a single cultivar of cucumber , since some reductions in incidence of both root - knot nematodes and anthracnose occurred on each cultivar tested . a comparison of results in table 10 from the field with and without foliar spray demonstrate that this “ booster ” tratment of the same pgpr contained in the biological preparation used in the soil - less seeding mix provides additional efficacy for disease protection . this can be seen by comparisons of the frequency of significant disease reduction for one treatment with and without foliar spray . for example , with ls213 , with foliar spray , 3 of the 4 disease incidence means were significantly reduced compared to the control , while without foliar spray , 2 of the 4 means were significantly reduced . with ls260 , this “ frequency of significance ” increased from 2 of 4 without foliar spray to 4 of 4 with foliar spray . it will be understood that many variations can be made in the procedures described for the composition , the methods and the plant products of the present invention while still remaining within the bounds and the spirit of the invention . bauske , e . m ., backman , p . a ., harper , k . m ., brannen , p . m ., rodríguez - 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