Abstract:
Biofungicidal composition from a biologically pure culture of a Chilean bacterial isolate obtained from soils of the seventh region of Maule, Chile, corresponding to  Paenibacillus polymyxa  SCHC33, strain with the deposit number RGM2141 granted by the depository authority of the Chilean Collection of Microbial Genetic Resources (CChRGM) to be used as an environmentally friendly, biological control agent against fungal plant diseases, particularly fruits susceptible to infection by  Botrytis cinerea , efficiently inhibiting conidial germination and mycelium proliferation of said phytopathogenic fungus, furthermore protects plant leaves and fruits from infection by the same fungus, and has the potential to be used in biological control of other fungi and in general of phytopathogenic microorganisms.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention discloses a bacterial strain,  Paenibacillus polymyxa  SCHC33, as a biofungicide against phytopathogenic filamentous fungi, particularly against gray mold,  Botrytis cinerea . This bacterial strain  P. polymyxa  SCHC33 possesses important qualities that will allow it to compete with commercial biofungicides of current use. Its fungicidal activity is very potent and independent of the medium in which the bacterium is cultivated, it is not toxic to humans or plants, it grows rapidly in simple culture media with a Monod kinetic behavior without substrate inhibition, so that it can be produced easily on a large scale and at a low cost. In addition to the above, its ability to sporulate will allow commercial formulations of high stability and long service life. 
         [0002]    The field of application of this invention comprises all fruits, vegetables and ornamental plants which are susceptible to infection by phytopathogenic fungi under pre- and post-harvest conditions. 
       SUMMARY OF THE INVENTION 
       [0003]    This invention provides an isolated wild type bacterial strain, characterized as  Paenibacillus polymyxa  SCHC33, and its use as biofungicide. This bacterial strain was deposited in the Chilean Collection of Microbial Genetic Resources (CChRGM), according to the Budapest treaty for patenting purposes. 
         [0004]    The deposit number granted on Jul. 23, 2014, by the depository authority is RGM2141, being the depository authority the Chilean Collection of Microbial Genetic Resources (CChRGM). This wild type Chilean native bacterium was isolated from soils in the seventh region of Maule, Chile and has the capacity to kill different isolated and wild type strains of the phytopathogenic fungus  B. cinerea . The fungicidal capacity of the bacterium is given by the secretion of fungitoxic molecules that interact with the fungus destroying it and as a consequence, inhibiting the germination of conidia and the proliferation of fungal vegetative mycelium. 
         [0005]    Many of the biofungicides currently in use could be potentially pathogenic to humans and plants. In contrast,  Paenibacillus polymyxa  SCHC33 has not been reported to be pathogenic or toxic to plants or fruits and has not been reported to be pathogenic to animals or humans. Experiments carried out in the laboratory, reveal that their inoculation in leaves of vines plants does not produce visible alteration in this host after 7 days of incubation at 20° C. in plates containing only 1.5% agar-agar (w/v) to maintain humidity at adequate levels. Likewise, when large inocula of the strain [2×10 10  cfu (colony-forming unit)] are added to intact or wounded grapes, no effect is seen after 7 days of incubation under the same conditions described above. 
         [0006]    The biological agent of the invention corresponds to a wild type bacterium, isolated from soils, and has been individualized by biochemical, microbiological, electron microscopy and 16S rDNA sequencing techniques.  P. polymyxa  SCHC33 is a Chilean autochthonous strain and corresponds to a saprophyte organism, non-pathogenic to plants or animals, and therefore the use of its live cells as biofungicide against  B. cinerea  is adequate. 
         [0007]    In addition, the present invention relates to compositions containing said bacterial strain or extracts containing the same, or to solutions or mixtures containing compounds derived therefrom (for example, fungitoxic molecules secreted by said bacterial strain), all of which are capable to protect plants and their fruits during the pre- and post-harvest periods, from the attack of phytopathogenic microorganisms. 
         [0008]    The present invention also includes any mutant derived from the wild type strain having essentially the same or better properties. 
         [0009]    Also, the invention relates to the process for the preparation of said bacterial strain or its derivatives and to the applications thereof in the protection of plants, and in particular the fruits. 
         [0010]    In addition, the present invention provides a formulation for controlling fungal plant diseases using the pure strain  P. polymyxa  SCHC33. The use of this bacteria constitutes a natural alternative for synthetic chemical fungicides, ensuring a safer environment to achieve the control or elimination of diseases caused by phytopathogenic fungi. 
         [0011]    Finally, the present invention provides a composition containing said strain in an adequate form and amount for its biological activity. The mixture contains non-toxic agents that allow the adherence of the bacterium to the vegetable on which it is inoculated, vegetable nutrients and preservatives in innocuous quantities, in the form of liquid suspension or powder obtained by freeze-drying. 
       BACKGROUND OF THE INVENTION 
       [0012]    Infections caused by phytopathogenic fungi in any type of plant tissue are extremely devastating, rapidly spreading and occur virtually anywhere on planet earth. The methods currently used to control these diseases are mainly based on the use of chemical fungicides which, despite their potential toxicity, since most of them are recalcitrant molecules, are still applied massively because they allow a relatively efficient control of fungal diseases and because there are still few effective and environmentally safe alternatives. 
         [0013]      Botrytis cinerea  is a phytopathogenic fungus, polyphagous and necrotrophic, infecting plant species of great economic importance, including fruit trees, ornamental plants and vegetables. It produces a disease known as gray rot causing a serious problem pre- and post-harvest in strawberries, raspberries, peaches, apples, pears, chestnuts, kiwi and grapes, among other fruits. In the vine this fungus produces the rot of the bunch of grapes, a disease that is considered at the moment as one of the most serious in the production of export fruits in Chile, since it is capable of causing large losses not only at field level but also during its storage and transportation (Williamson B, Tudzynski B, Tudzynski P, van Kan J A 2007.  Botrytis cinerea : the cause of gray mould disease Mol Plant Pathol 8: 561-80; Elad, Y., Williamson B Tudzynski, P. and Delen, N. eds. 2007.  Botrytis: Biology, Pathology and Control . The Netherlands: Kluwer Academic Publishers). 
         [0014]    At present, the control of this important phytopathogen is mainly carried out with chemical fungicides, however, in recent years some microorganisms with antifungal activity against  B. cinerea  have been described. One of the most promising examples is the Serenade®, whose active ingredient is a bacteria of the genus  Bacillus , specifically  Bacillus subtilis  QST 713, discovered in soil samples from a vegetable garden by AgraQuest Inc. in Davis, Calif. This biofungicide is registered in several countries, including Chile, has low toxicity and is being used commercially in Chile and the United States for the control of diseases such as powdery mildew ( Uncinula necator ) and acid rot in vines. 
         [0015]    In patent KR20100024454,  Paenibacillus lentimorbus  CMC3723 (deposit number KACC91379) is disclosed, with the ability to suppress pathogens in plants, in particular the pear tree scab (pear scab),  Botrytis  and  Sclerotinum cepivorum , in addition to preventing the growth of plant pathogens.  Paenibacillus lentimorbus  CMC3723 was isolated from the soil and has the effect of suppressing  Venturia nashicola, Botrytis cinerea, Sclerotium cepivorum  and  Colletotrichum acutatum . The agent for preventing the diseases caused by these pathogens in plants contains a culture of  Paenibacillus lentimorbus  CMC3723. It is also disclosed a method for isolating, culturing, determining activity against phytopathogenic fungi and bacteria, identifying  Paenibacillus lentimorbus  CMC3723 and preparing a culture medium containing it. In this case, the bacterial strains  Paenibacillus lentimorbus  CMC3723 and  Paenibacillus polymyxa  SCHC33 belong to the same genus, but to totally different species. 
         [0016]    Publication KR20090105149 discloses  Paenibacillus polymyxa  NB1 with antibacterial activity and a composition containing it, useful for preventing diseases caused by plant pathogens.  Paenibacillus polymyxa  NB1 acts as an antagonistic agent against plant pathogens and its deposit number is KFCC 11413P. The plant pathogens described are  Colletotrichum acutatum, Pythium ultimum, Phytophthora capsici, Rhizoctonia solani  AG4,  Botrytis cinerea  and  Fusarium oxysporum.    
         [0017]    Patent KR20030075092 teaches a microorganism  Paenibacillus  sp. SD17 that produces antifungal agents for the biological control of plant diseases and a composition for the biological control of plant diseases containing the microorganism for the effective control of diseases in plants. The microorganism  Paenibacillus  sp. SD17 (KCTC10016BP) produces antifungal agents for the biological control of plant diseases including diseases caused by  Pythium  spp.,  Rhizoctonia solani  AG1-1,  Rhizoctonia solani  AG 2-2,  Phytophthora infestans  or  Botrytis cinerea . A composition for the biological control of plant diseases containing 5 to 30% by weight of the culture medium of  Paenibacillus  sp. SD17 (KCTC-10016BP); 0.2 to 3.0% by weight of an agent which activates spore germination, corresponding to yeast extract; 0.02 to 0.5% by weight of a water-soluble pigment; 1 to 5% by weight of a surfactant and an additive or resin. 
         [0018]    Publication EP1241247 discloses antagonistic bacteria for the protection of plants against phytopathogenic bacteria and fungi. The isolated bacteria are of the species  Paenibacillus polymyxa, Pseudomonas chlororaphis, Pseudomonas putida, Serratia plymuthica  and  Bacillus subtilis . They may be applied as a mixture or separately for the control of, for example, the following plant diseases: crown gill caused by  Agrobacterium tumefaciens  in grapes; diseases caused by  Corynebacterium  spp.,  Pseudomonas syringae  and  Xanthomonas campestris , in tomato and cabbage. Diseases caused by  Botrytis cinerea  in cucumber and tomato; powdery mildew caused by  Erysiphales  spp. in cucumber and tomato; damping-off caused by  Fusarium oxysporum  in cucumber, melon and tomato; diseases caused by  Helminthosporum sativum  in cereals; the root rot disease caused by  Rhizoctonia solani  in cotton and beans; diseases caused by  Sclerotium rolfsii  in beans;  Dactylium dendroides  in mushrooms and others. 
         [0019]    If we compare the antifungal activity, which is obtained in plaque confrontation bioassays against  Botrytis cinerea , of  Paenibacillus polymyxa  from publication EP1241247 with that of the bacterial strain  Paenibacillus polymyxa  SCHC33 under identical assay conditions, strain SCHC33 produces an inhibition halo of greater diameter and therefore, its fungicidal activity is greater. 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0020]      FIG. 1  corresponds to scanning electron micrographs of  Paenibacillus polymyxa  SCHC33. In (A) is clearly observed material secreted by the bacteria, corresponding to a biofilm formed by 15 exopolysaccharides. In (B) it is observed grouping of bacilli surrounded by ellipsoidal spores obtained from a liquid culture. 
           [0021]      FIG. 2  is a transmission electron micrograph of  Paenibacillus polymyxa  SCHC33. There are dividing cells and their corresponding peritrichous flagella. 
           [0022]      FIG. 3  shows the partial nucleotide sequence of the 16S rDNA of  Paenibacillus polymyxa  SCHC33. 
           [0023]      FIG. 4  corresponds to confrontation bioassays of  Paenibacillus polymyxa  SCHC33 against  Botrytis cinerea . In (A), the result of the bioassay performed in a glucose-free minimal medium is shown. In (B), the result of the bioassay performed in a culture medium containing glucose as a carbon source. 
       
    
    
       [0024]    In  FIG. 5 , the results of the germination inhibition bioassays of  Botrytis cinerea  conidia on vines leaves are presented. (A) and (B) correspond to the negative controls. (C) and (D) are the positive controls. In (E) and (G) is shown the Serenade® biocontrol effect on germination of conidia for a conidia:bacteria ratio of 1:10 and 1:100, respectively. In (F) and (H) is observed the biocontrol effect of  Paenibacillus  on germination of conidia for a conidia:bacteria ratio of 1:10 and 1:100, respectively. 
         [0025]      FIG. 6  shows the bacterial growth curves and glucose consumption for an initial glucose concentration of 2 [g/L]. In (A), the increase of biomass and glucose consumption over time is appreciated. (B) corresponds to the semi-logarithmic curve of biomass over time. 
         [0026]      FIG. 7  presents the curve of the initial glucose concentration effect on the specific growth rate of 20  Paenibacillus polymyxa  SCHC33. 
         [0027]      FIG. 8  shows the experimental growth curve and the fitting to the kinetic models evaluated (Monod, Moser and Tessier models). 
         [0028]    In  FIG. 9  was schematized the region of the 16S rDNA which was amplified by PCR and from which its sequence was obtained. In red primers used for PCR. In blue primers used for sequencing. 
         [0029]      FIG. 10  shows the 2-liter bioreactor with constant aeration that was used for the determination of the kinetic growth parameters of  Paenibacillus polymyxa  SCHC33. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0030]    The strain SCHC33 corresponds to the  polymyxa  species and to the  Paenibacillus  genus. Its characterization by microbiological and biochemical tests is presented in Table 1. It produces colorless/white colonies without pigmentation in potato-dextrose agar (PDA) and in MLG medium (malt extract 10 g/L, glucose 2 g/L, agar-agar 15 g/L). 
         [0000]                                  TABLE 1               Morphological and physiological characteristics of         Paenibacillus polymyxa  SCHC33.                                    Shape     Bacillus             Gram staining   Positive           Motility   (+)           Pigment production   (−)           Exopolysaccharides   (+)           production (EPS)           Growth at 4° C.   (+)                        
Ultra Structural Characterization of  Paenibacillus polymyxa  SCHC33 by Scanning Electron Microscopy and Transmission Electron Microscopy
 
         [0031]    At the level of optical microscopy, the cultures are constituted by Gram-positive mobile bacilli, whereas at the scanning electron microscopy (SEM) level, the bacilli morphology was clearly observed with a cell size in the range of 3 to 5 μm of length, and 0.5 to 0.8 μm in diameter (see  FIG. 1 ). Also, clusters of cells bound by a material of adhesive characteristics corresponding to exopolysaccharides (EPS), which allow the formation of biofilms (see  FIG. 1A ), very important for the adhesion of the bacteria to the surface of the plant to be protected against the attack of phytopathogenic organisms (bacteria or fungi). No bacterial appendages were observed by SEM, however, using the negative staining technique, it was possible to detect under Transmission Electron Microscopy (TEM), the presence of peritrichous flagella of approximately 3 to 5 μm in length (see  FIG. 2 ), which agrees perfectly with the background described for this type of bacteria (Lal S. and Tabacchioni S. 2009. Ecology and biotechnological potential of  Paenibacillus polymyxa : a minireview, Indian J. Microbiol 49: 2-10). 
         [0000]    Molecular Characterization of  Paenibacillus polymyxa  SCHC33 
         [0032]    In addition to the microbiological and biochemical characterization of the bacteria, the molecular characterization was carried out by obtaining the nucleotide sequence of a portion of the 16S rDNA and its subsequent bioinformatic analysis. In  FIG. 3  is shown the partial nucleotide sequence of the 16S rDNA of  Paenibacillus polymyxa  SCHC33 (1,255 nucleotides). The comparison of this sequence with existing sequences in databases using BlastN, generated the results shown in Table 2. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Results of the alignment of 16S rDNA of  Paenibacillus polymyxa   
               
               
                 SCHC33 on BlastN. The values of the parameters calculated 
               
               
                 by the computer program are indicated. 
               
             
          
           
               
                   
                   
                   
                   
                 Cover 
                   
                   
               
               
                   
                   
                 Max. 
                 Total 
                 of the 
                 E 
               
               
                 Accession 
                 Description 
                 Score 
                 Score 
                 request 
                 Value 
                 Identity 
               
               
                   
               
             
          
           
               
                 HE577054 
                 
                   Paenibacillus 
                 
                 1991 
                 27696 
                 97% 
                 0.0 
                 98% 
               
               
                   
                 
                   polymyxa 
                 
               
               
                   
                 M1 main 
               
               
                   
                 chromosome, 
               
               
                   
                 complete 
               
               
                   
                 genome 
               
               
                 CP002213 
                 
                   Paenibacillus 
                 
                 1991 
                 27658 
                 97% 
                 0.0 
                 98% 
               
               
                   
                 
                   polymyxa 
                 
               
               
                   
                 SC2, 
               
               
                   
                 complete 
               
               
                   
                 genome 
               
               
                 EF656457 
                 
                   Paenibacillus 
                 
                 1991 
                 1991 
                 97% 
                 0.0 
                 98% 
               
               
                   
                 
                   polymyxa 
                 
               
               
                   
                 M-1, partial 
               
               
                   
                 sequence of 
               
               
                   
                 the gene of 
               
               
                   
                 the 16S rRNA 
               
               
                 AY302439 
                 
                   Paenibacillus 
                 
                 1991 
                 1991 
                 97% 
                 0.0 
                 98% 
               
               
                   
                 
                   polymyxa 
                 
               
               
                   
                 WY110, 
               
               
                   
                 partial 
               
               
                   
                 sequence of 
               
               
                   
                 the gene of 
               
               
                   
                 the 16S rRNA 
               
               
                 JF683620 
                 
                   Paenibacillus 
                 
                 1989 
                 1989 
                 97% 
                 0.0 
                 98% 
               
               
                   
                 
                   polymyxa 
                 
               
               
                   
                 RS-10, 
               
               
                   
                 partial 
               
               
                   
                 sequence of 
               
               
                   
                 the gene of 
               
               
                   
                 the 16S rRNA 
               
               
                   
               
             
          
         
       
     
         [0033]    Therefore, microbiological tests, the electron microscopy, the 16S rDNA sequencing and bioinformatic analysis confirm that this is a new strain of  Paenibacillus polymyxa , which was isolated from soils of the Seventh Region of Maule, Chile and was named SCHC33. 
         [0000]    Determination of the Antifungal Activity of  Paenibacillus polymyxa  SCHC33 Against Phytopathogenic Fungus  Botrytis cinerea    
         [0034]    Plaque confronting bioassays were performed in which a fungal mycelium disc was placed in the center of the Petri dish and the bacteria were inoculated at the edges of the same (see  FIG. 4 ). The growth inhibition halos of the fungus were clearly observed, both in a glucose-free medium (see  FIG. 4A ) and in a medium containing this monosaccharide (see  FIG. 4B ). In addition if different culture media such as potato-dextrose agar (PDA) are used; Luria Bertani medium containing 0.5% yeast extract, 1% tryptone and 0.5% NaCl; ML medium containing 1.5% malt extract and 0.7% yeast extract, among others, the fungicidal activity remains intact. If in addition to the aforementioned culture media glucose is added, the fungitoxic activity against  B. cinerea  is not altered. This result is very relevant, since it has been observed in other Gram (−) bacteria like  Serratia plymuthica  that the secretion of fungitoxic molecules is repressed in culture media containing glucose. Therefore, for the production of bacterial biomass at industrial scale, any culture medium may be used. 
         [0000]    Determination of the Protective Capacity of  Paenibacillus Polymyxa  SCHC33 Against the Attack of  Botrytis cinerea  on Plant Tissue 
         [0035]    Two known amounts of bacteria were used 10 7  and 10 8  cfu/mL, in order to evaluate the efficacy of biocontrol compared to  Bacillus subtilis  QST 713, the active component of the commercial bio-fungicide Serenade®. 
         [0036]    The results at 7 days were satisfactory, obtaining a slightly superior level of protection of the vegetal tissue with the  Paenibacillus polymyxa  SCHC33 bacterium. The negative control showed small areas of necrosis attributed to the wounds caused in order to facilitate infection and the positive control, in which only conidia of the fungus inoculated, suffered an infection by  Botrytis cinerea  that covered practically 100% of the surface of the leaves. Both in the leaves protected by  Paenibacillus polymyxa  SCHC33 and in those protected by  Bacillus subtilis  QST 713, there were significant decreases in the degree of damage caused by the fungus. In the case of  Paenibacillus polymyxa  SCHC33, using a ratio of conidia:bacteria of 1:10, the damage produced by the fungus only covered 20.5% of the leaf surface, decreasing to 11.7% when the ratio was 1:100. In the trials with  Bacillus subtilis  QST 713, plant tissue necrosis produced by the fungus covered 36.9% of the leaf surface for a ratio of 1:10 and decreased to 15.4% when the ratio was Of 1:100 conidia:bacteria, respectively. These results are shown in Table 3 and  FIG. 5 , showing that the bacterium has a great potential to be used as biofungicide by direct inoculation in the field of Vegetables susceptible to be infected by  Botrytis cinerea . 
         [0000]                                                        TABLE 3                   Results of damaged surface in vine leaves.                Total Area   Damaged Area               [cm 2 ]   [cm 2 ]   Infection                        Control (−) 10 8  cfu/mL   28,208   2,561    9.1%       Control (+) 10 8     33,625   33,296     99%       conidia/mL         Bacillus subtilis  QST   42,195   15,575   36.9%       713 1:100         Bacillus subtilis  QST   40,595   6,258   15.4%       713 1:100         Paenibacillus     35,214   7,221   20.5%         polymyxa  SCHC33 1:10         Paenibacillus     45,596   5,340   11.7%         polymyxa  SCHC33 1:100                    
Determination of the Protective Capacity of  Paenibacillus polymyxa  SCHC33 Against  Botrytis cinerea  Attack on 5 Grape Clusters.
 
         [0037]    Protection bioassays against  B. cinerea  were carried out on clusters of grapes of the Thompson seedless variety, using 2 known amounts of bacteria, 10 7  and 10 8  cfu/mL, to evaluate their efficiency as a biocontrol agent. The results at 30 days were of a protection comparable to that shown in vines leaves, since the clusters inoculated with the bacteria by spray did not present evident growth of mycelium of  B. cinerea , for both amounts of bacteria used. The negative controls did not show  B. cinerea  presence, either in clusters inoculated with only bacteria (10 8  cfu/mL) or in those inoculated with sterile water alone. In addition, these results are clear evidence of the innocuousness of the bacteria on the fruit used in the experimentation, since no morphological alteration was observed nor changes in the coloration and neither in the organoleptic characteristics of the grape used. Positive controls (bunches of grapes inoculated by spray only with fungal conidia) showed a clear infection by  B. cinerea , clearly observing the vegetative growth of the mycelium of the fungus in the berries of inoculated clusters. 
         [0000]    Determination of the Kinetic Parameters of the Growth of  Paenibacillus polymyxa  SCHC33 and Adjustment of the Results to One of the Pre-Established Models 
         [0038]    Using a bioreactor of 2 liters capacity, 5 curves of bacterial growth with different concentrations of glucose were obtained. In Table 4, the experimental data obtained are shown, and in  FIG. 6 , the corresponding growth curves for the culture in a medium with 2 g/L glucose are shown, in the first instance the curve that relates the formation of biomass over time and secondly the semi-logarithmic curve used to identify the phases of bacterial growth. Thus it can be seen that there is no latency phase and the exponential growth stage starts immediately at the beginning of the growth, this behavior was repeated in all the experimental runs performed. Glucose consumption occurred at a constant rate throughout the exponential growth stage, achieving in the experiments with a high glucose concentration (≧1 g/L) to be maintained during the period of growth slowdown. No inhibition was observed, either by substrate or product in the bacterial growth, so that the adjustment to kinetic models was restricted to those that do not present this type of situation, in this case Monod, Moser and Tessier (Shuler M., Kargi F. 2002. Stoichiometry of microbial growth and product formation In Bioprocess engineering: Basic concepts, Edited by Shuler M., Kargi F. Harlow: Pearson, 207-218). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Experimental data obtained from biomass increase 
               
               
                 and glucose consumption for a bacterial growth curve with 
               
               
                 an initial glucose concentration of 2 [g/L]. 
               
             
          
           
               
                   
                   
                 
                   Paenibacillus polymyxa 
                 
               
               
                   
                 Substrate 
                 SCHC33 
               
             
          
           
               
                   
                 Time 
                   
                 Glucose 
                   
                 Biomass 
                 ln 
               
               
                   
                 [h] 
                 Abs 500   
                 [g/l] 
                 DO 600   
                 [g/l] 
                 Biomass 
               
               
                   
                   
               
             
          
           
               
                 Expo- 
                 0 
                 0.647 
                 1.946 
                 0.122 
                 0.083 
                 −2.490 
               
               
                 nential 
                 0.5 
                 0.618 
                 1.860 
                 0.148 
                 0.088 
                 −2.425 
               
               
                 phase 
                 1 
                 0.576 
                 1.735 
                 0.198 
                 0.099 
                 −2.310 
               
               
                   
                 1.5 
                 0.570 
                 1.718 
                 0.225 
                 0.105 
                 −2.254 
               
               
                   
                 2 
                 0.562 
                 1.694 
                 0.283 
                 0.117 
                 −2.141 
               
               
                   
                 2.5 
                 0.513 
                 1.549 
                 0.323 
                 0.126 
                 −2.071 
               
               
                   
                 3 
                 0.500 
                 1.510 
                 0.430 
                 0.149 
                 −1.903 
               
               
                   
                 3.5 
                 0.480 
                 1.451 
                 0.493 
                 0.163 
                 −1.817 
               
               
                   
                 4 
                 0.443 
                 1.341 
                 0.621 
                 0.190 
                 −1.660 
               
               
                   
                 4.5 
                 0.402 
                 1.220 
                 0.665 
                 0.200 
                 −1.612 
               
               
                   
                 5 
                 0.353 
                 1.074 
                 0.788 
                 0.226 
                 −1.487 
               
               
                   
                 5.5 
                 0.311 
                 0.950 
                 0.866 
                 0.243 
                 −1.416 
               
               
                 Stationary 
                 6 
                 0.251 
                 0.772 
                 0.861 
                 0.242 
                 −1.420 
               
               
                 phase 
                 6.5 
                 0.241 
                 0.742 
                 0.880 
                 0.246 
                 −1.404 
               
               
                   
                 7 
                 0.189 
                 0.588 
                 0.943 
                 0.259 
                 −1.350 
               
               
                   
                 7.5 
                 0.150 
                 0.473 
                 0.964 
                 0.264 
                 −1.333 
               
               
                   
                 8 
                 0.106 
                 0.342 
                 0.988 
                 0.269 
                 −1.313 
               
               
                   
                 8.5 
                 0.060 
                 0.206 
                 0.992 
                 0.270 
                 −1.310 
               
               
                   
                 9 
                 0.038 
                 0.141 
                 0.995 
                 0.270 
                 −1.308 
               
               
                   
                 9.5 
                 0.033 
                 0.126 
                 0.994 
                 0.270 
                 −1.309 
               
               
                   
                 10 
                 0.016 
                 0.076 
                 1.011 
                 0.274 
                 −1.295 
               
               
                   
                 10.5 
                 0.000 
                 0.000 
                 1.030 
                 0.278 
                 −1.280 
               
               
                   
                 24 
                 0.000 
                 0.000 
                 0.998 
                 0.271 
                 −1.305 
               
               
                   
               
             
          
         
       
     
         [0039]    Obtaining the curve that correlates the specific growth rate with the initial concentration of glucose in the medium shown in  FIG. 7 , allowed to obtain the intrinsic kinetic parameters of this bacterium growing with glucose as the main substrate. However, the validation of these parameters first requires the statistical validation of the kinetic adjustment to one of the 3 aforementioned models shown in  FIG. 8 . In this case the curve obtained presented an expected behavior according to the literature, when increasing the substrate concentration the specific bacterial growth rate continuously increases, until reaching a maximum point from which the rate is constant (Acevedo F., Gentina J. 2004. Cinética de fermentación. In Fundamentos de ingeniería bioquímica. Edited by F. Acevedo, J. Gentina, A. Illanes. Valparaíso: Ediciones universitarias de Valparaíso, 151-168). 
         [0040]    The evaluation of the 3 kinetic models was based on the associated statistical parameters as such shown in Table 5, indicated that the growth of  Paenibacillus polymyxa  SCHC33 using glucose as the main substrate fits to a Monod type kinetic model since it is the model with a correlation coefficient closer to 1 and at the same time the model with a lower Chi square parameter, with a difference of one order of magnitude with respect to the other 2 models analyzed. Then the intrinsic kinetic parameters of  Paenibacillus polymyxa  SCHC33 are obtained by analyzing Monod, thus the maximum specific growth rate for this bacterium using Glucose as the main substrate is pmax=0.218 h −1 , its glucose affinity constant is Ks=0.087 g/L and the yield of biomass production from glucose is YX/S=0.159 [g biomass/g glucose]. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Statistical analysis of experimental data obtained and  
               
               
                 obtaining of the kinetic parameters of the 3 models evaluated. 
               
             
          
           
               
                 Kinetic 
                   
                   
                   
                   
                   
                   
               
               
                   
                 Equation 
                 μ max   
                 K S   
                 n 
                 R 2   
                 X 2   
               
               
                   
               
               
                   
               
             
          
           
               
                 Monod 
                 
                   
                     
                       
                         µ 
                         = 
                         
                           
                             µ 
                             max 
                           
                           · 
                           
                             S 
                             
                               
                                 K 
                                 S 
                               
                               + 
                               S 
                             
                           
                         
                       
                     
                   
                 
                 0.218 
                 0.087 
                 — 
                 0.99640 
                 0.00039 
               
               
                   
               
               
                 Moser 
                 
                   
                     
                       
                         µ 
                         = 
                         
                           
                             µ 
                             max 
                           
                           · 
                           
                             
                               S 
                               n 
                             
                             
                               
                                 K 
                                 S 
                               
                               + 
                               
                                 S 
                                 n 
                               
                             
                           
                         
                       
                     
                   
                 
                 0.216 
                 0.102 
                 0.879 
                 0.99500 
                 0.00116 
               
               
                   
               
               
                 Tessier 
                 μ = μ max  · (1-e -S/K     S   ) 
                 0.199 
                 0.112 
                 — 
                 0.98082 
                 0.00230 
               
               
                   
               
             
          
         
       
     
       EXPERIMENTAL SECTION 
     Obtaining Soil Samples 
       [0041]    Sampling was carried out in situ from agricultural soils of the Seventh Region of Maule, About ten random samples, which were taken to the fungi Virology laboratory of the University of Santiago de Chile, where they were stored at room temperature until their posterior utilization. 
       Obtaining Bacterial Isolates 
       [0042]    The soil samples were submitted to a heat treatment at 67° C. for 48 hours and then 1 gram of each sample was suspended in 1 mL of sterile distilled water. Finally, 1 mL of this suspension for each Sample in triplicate, were inoculated on Petri dishes with MLG+C medium (10 g/L malt extract, 2 g/L glucose, 15 g/L agar-agar, cycloheximide 50 μg/mL), obtaining diverse microflora from which colonies were isolated and backed up for later analysis. 
       Determination of Antifungal Activity 
       [0043]    The various colonies obtained were individually backed up and plaque confrontation bioassays were performed on Petri dishes with MLG medium against  Botrytis cinerea  CCg149, a highly virulent virus-free strain from the fungal Virology laboratory and grown on potato-dextrose agar medium (PDA) until the completion of the tests. Variations in antifungal activity were also evaluated in media of different composition, mainly with and without glucose. 
         [0044]    Two types of bio-confrontations were carried out. The first consisted of planting a 5 mm diameter mycelial disk in the center of the Petri dish and at four equidistant points, the same amount of the different bacterial isolates were inoculated and the growth was observed for 7 days at 20° C. The second method consisted of planting 8 pieces of mycelium of 5 mm diameter at equidistant points from the center of the Petri dish, where the bacterial isolate was inoculated. Growth was again observed for 7 days at 20° C. As a control, the same tests were performed by replacing the bacterial isolates with sterile water. 
         [0045]    All those bacterial isolates that showed some degree of antifungal activity against the fungus, observable as a halo of inhibition in Petri dishes, were selected. 
         [0046]    Subsequently, similar assays were performed using suspension of conidia homogeneously distributed in Petri dishes, which were incubated for 24 hours at 20° C., to ensure the correct adsorption of the sample in the medium. Later, 10 μL of bacterial culture were inoculated, with an optical density of 0.9 at 600 nm in liquid medium, in the center of the plate and incubated during 7 days at 20° C. to observe the germination inhibition halo for  B. cinerea  conidia. 
         [0000]    Obtaining of Pure Bacterial Clones, DNA Isolation, Amplification of 16S rDNA by PCR and Sequencing 
         [0047]    From those bacterial isolates with increased antifungal activity (inhibition halos ≧1 cm in Petri dish), serial dilutions were performed to obtain isogenic clones which were considered pure bacterial strains. Genomic DNA of the obtained strains was extracted using the PureLink® Genomic DNA commercial kit and subsequently these samples were subjected to PCR amplification using the eubacterial universal primers designated 8F/1392R (see  FIG. 9 ) with the purpose of obtaining a specific 16S rDNA fragment of approximately 1400 base pairs. For PCR, 10 ng of genomic DNA, 0.5 μM of each primer, 200 μM dNTPs, 2.5 U of DNA polymerase, 1× reaction buffer and 1.5 mM MgCl 2  in a total volume of 50 μL were used. Cycles consisted of an initial denaturation step of 4.5 minutes at 95° C. and 40 cycles of 1 min at 95° C., 1 min at 60° C. and 2 min at 72° C., ending in a 5 min at 72° C. step. The PCR products were resolved in a 1% agarose (w/v) gel electrophoresis. For the sequencing the primer pair 27F/800R (see  FIG. 9 ) was used and the sequences obtained were analyzed by BlastN. 
       Ultra Structural Analysis by Electron Microscopy 
       [0048]    In order to obtain images that allowed the determination of morphological and structural aspects of the bacteria, samples of  Paenibacillus polymyxa  SCHC33 were prepared for visualization and analysis by scanning and transmission electron microscopy. For the scanning electron microscope (Jeol JSM-25-SII) samples of liquid bacterial cultures were used, which were prepared with a metallic shading technique using gold. In the case of transmission electron microscopy, negative staining with 1% (w/v) potassium phosphotungstate, pH 7.0, was performed on samples from liquid bacterial cultures and visualized on the Phillips Tecnai 12 Bio Twin microscope at 80 kV. 
       Obtaining Kinetic Parameters 
       [0049]    In order to obtain the kinetic parameters of growth using glucose as the main substrate, experimental runs of discontinuous growth of the bacterium with initial glucose concentrations of 0.1 g/L, 0.2 g/L, 0.5 g/L, 1 g/L, 2 g/L and 5 g/L in LG medium (yeast extract 5 g/L, glucose). Two-liter capacity bioreactors were constructed, as shown in  FIG. 10 , with an air feed sterilized with 2 μm filters from a 20 L/min capacity compressor and a sample-taker connected to a sterile syringe, with which bacterial culture samples were obtained. In all experimental runs, aeration (1 vvm), temperature (30° C.), pH (5) and agitation (200 rpm) were maintained constant at values recommended as optimal by bibliographic data. 
         [0050]    For each experimental run 200 mL of bacterial culture in exponential phase of growth were inoculated, in the bioreactor containing 2 liters of culture medium, considering as time 0 the moment when the bioreactor began the agitation and aeration of the sample. 
         [0051]    The increase in biomass as the optical density of the bacterial culture at a wavelength of 600 nm was recorded every 30 minutes, and the decrease of the dissolved glucose in the medium was measured using the commercial kit Liquicolor®. 
         [0052]    With these data were constructed graphs of bacterial growth and glucose uptake over time, and semi-logarithmic graphs to calculate the specific growth rate (p) of  Paenibacillus polymyxa  SCHC33, when glucose is used as the main substrate. In addition, the value of the substrate affinity constant (Ks) and the biomass yield per substrate (Y x/s ) were obtained from the same experimental data. 
       Adjustment to a Kinetic Model of Bacterial Growth 
       [0053]    To adjust the growth of  Paenibacillus polymyxa  SCHC33 to one of the bacterial growth kinetic models, the specific growth rate values obtained in each experimental run were used and a graph was constructed which relates the initial concentrations of glucose to the specific growth rates, obtained from the measurements. Three bacterial growth kinetics were evaluated where the different mathematical models adjusted to the experimental data varying one or more constants, depending on the model, which are estimated minimizing, by Newton&#39;s method, the residual sum of squares (RSS) between the experimental values and those calculated using the Microsoft Office add-on Excel Solver. To evaluate which model is the one that presented a better fit to the experimental data the following statistical parameters were used: 
         [0000]    
       
         
           
             
               
                 
                   
                     Correlation 
                      
                     
                         
                     
                      
                     coeficient 
                      
                     
                         
                     
                      
                     
                       R 
                       2 
                     
                   
                   = 
                   
                     
                       
                         ∑ 
                         
                           i 
                           = 
                           i 
                         
                         N 
                       
                        
                       
                         
                           ( 
                           
                             
                               V 
                               cal 
                             
                             - 
                             
                               V 
                               exp 
                             
                           
                           ) 
                         
                         2 
                       
                     
                     
                       
                         ∑ 
                         
                           i 
                           = 
                           1 
                         
                         N 
                       
                        
                       
                         
                           ( 
                           
                             
                               V 
                               exp 
                             
                             - 
                             
                               V 
                               cal 
                             
                           
                           ) 
                         
                         2 
                       
                     
                   
                 
               
               
                 
                   ( 
                   
                     4 
                     - 
                     3 
                   
                   ) 
                 
               
             
           
         
       
       
         
           
             where: 
             V c : Calculated value based on the model 
             V e : Experimental values 
             N: Data number 
           
         
       
     
         [0000]    
       
         
           
             
               
                 
                   
                     Chi 
                      
                     
                         
                     
                      
                     squared 
                      
                     
                         
                     
                      
                     
                       χ 
                       2 
                     
                   
                   = 
                   
                     RSS 
                     
                       N 
                       - 
                       n 
                     
                   
                 
               
               
                 
                   ( 
                   
                     4 
                     - 
                     2 
                   
                   ) 
                 
               
             
           
         
       
       
         
           
             where: 
             RSS: Residual sum of squares 
             N: Data number 
             n: Constant number 
           
         
       
     
       Bioassays in Plant Tissue 
       [0062]    Inhibition of conidia germination on plant tissue was observed, using leaves of vines harvested immediately before use. The leaves were washed with a solution of sodium hypochlorite 0.5% (v/v) and then with sterile distilled water. Subsequently, they were incubated in Petri dishes with 1.5% (w/v) agar-agar to maintain moisture during the 7-day duration of the assay. The leaves were wounded to facilitate infection and then inoculated with bacterial culture in liquid medium and suspension of conidia in proportions of 1:10 and 1:100, respectively. As a control, leaves inoculated only with conidia and leaves inoculated only with bacteria were prepared, in addition to a control consisting of leaves 10 inoculated only with sterile water and a control using the active principle of the commercial biofungicide named Serenade®,  Bacillus subtilis  QST 713 (Table 6). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Experimental treatments performed. 
               
             
          
           
               
                   
                   
                   
                   
                 
                   Bacillus 
                 
               
               
                   
                   
                 
                   Paenibacillus 
                 
                   
                 
                   subtilis 
                 
               
               
                   
                 Conidia 
                 
                   polymyxa 
                 
                 Distilled 
                 QST 713 
               
               
                 Treatment 
                 suspension 
                 SCHC33 culture 
                 H 2 O 
                 (Serenade ®) 
               
               
                   
               
               
                 1 
                 + 
                 − 
                 + 
                 − 
               
               
                 2 
                 + 
                 + 
                 − 
                 − 
               
               
                 3 
                 + 
                 − 
                 − 
                 + 
               
               
                 4 
                 − 
                 + 
                 − 
                 − 
               
               
                 5 
                 − 
                 − 
                 + 
                 − 
               
               
                 6 
                 − 
                 − 
                 − 
                 + 
               
               
                   
               
             
          
         
       
     
         [0063]    The results were quantified as percentage of leaf area damaged with respect to the total surface at 7 days of incubation at 20° C. For this purpose, ImageJ software (http://rsb.info.nih.gov/ij/index.html) was used to obtain the respective areas, all determined after 7 days of incubation at 20° C. 
         [0000]    Determination of the Protective Capacity of  Paenibacillus polymyxa  SCHC33 Against  Botrytis cinerea  Attack on Grape Clusters. 
         [0064]    The inhibition of the conidia germination on fruits was observed, using clusters of Thompson seedless grapes. The clusters were washed with a solution containing 0.5% (v/v) sodium hypochlorite and then with sterile distilled water, then incubated in disinfected closed containers for the 30 days of duration of the assay. The clusters were inoculated with suspensions of conidia and bacteria in proportions of 1:10 and 1:100 (conidia:bacteria). As controls, clusters were inoculated only with conidia, others only with bacteria and a control consisting of clusters inoculated only with sterile distilled water (Table 7). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Experimental treatments performed. 
               
             
          
           
               
                   
                   
                 Conidia 
                 
                   Paenibacillus 
                 
                 Distilled 
               
               
                   
                 Treatment 
                 suspension 
                 culture 
                 H 2 O 
               
               
                   
                   
               
               
                   
                 1 
                 + 
                 + 
                 − 
               
               
                   
                 2 
                 + 
                 + 
                 − 
               
               
                   
                 3 
                 + 
                 − 
                 − 
               
               
                   
                 4 
                 − 
                 + 
                 − 
               
               
                   
                 5 
                 − 
                 − 
                 + 
               
               
                   
                   
               
             
          
         
       
     
         [0065]    The results were analyzed qualitatively, determining the presence or absence of  Botrytis cinerea  mycelial growth on the surface of the clusters. Observations were made during the 30-day period of incubation at 20° C.