Patent Publication Number: US-2022232834-A1

Title: Pseudomonas chlororaphis species and its use in the control of diseases caused by bacteria and fungi

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims priority to U.S. Patent Application Ser. No. 63/142,691, filed Jan. 28, 2021, entitled “ PSEUDOMONAS CHLORORAPHIS  SPECIES AND ITS USE IN THE CONTROL OF DISEASES CAUSED BY BACTERIA AND FUNGI,” the contents of which are herein incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     This present invention relates to a novel strain of the bacterial species  P. chlororaphis  subsp.  aurantiaca  1214-CHY4 (1214-CHY4) and the application of the metabolites produced by this strain to protect against pathogenic bacteria and fungi of plants. 
     BACKGROUND OF THE INVENTION 
     The control of many bacterial and fungal pathogens is challenging due to the resistance to the existing treatments.  Clavibacter michiganensis  subsp.  michiganensis  is a gram-positive bacterium that causes bacterial canker of solanaceous crops such as tomato, pepper, and eggplant (Sen et al., 2015).  C. michiganensis  subsp.  michiganensis  causes plant wilting, stunting, reduced yields, and eventually plant death. Yield losses vary with years, locations, cultivars, and phenological stages of host infection. For example, in Ontario, Canada,  C. michiganensis  subsp.  michiganensis  accounted for yield losses of up to 84% in commercial fields, whereas in artificially inoculated crops, yield losses varied from 46 to 93% (Poysa, 1993). The economic losses are also high: in Michigan, the bacterial canker has cost individual processing tomato growers as much as $300,000 in a single year (Hausbeck et al. 2000). As  C. michiganensis  subsp.  michiganensis  causes severe yield and economic losses; it is a quarantine microorganism in the European Union and many other countries (de Leon, et al. 2008).  C. michiganensis  subsp.  michiganensis  was first discovered in Michigan but is found globally wherever tomatoes are grown. It is the most important bacterial disease of tomatoes; yield losses can be severe. China, US (East of Mississippi), Brazil, and some parts of EU are significant areas for the disease. It was reported that the continuous use of streptomycin leads to emergence of resistant  C. michiganensis  subsp.  michiganensis  strains, challenging researchers to look for novel alternatives to control this plant pathogenic bacterium (Valenzuela, et al. 2019). 
     Apple scab caused by  Venturia inaequalis  is one of the most challenging diseases for growers to control. It requires the right timing of protectant sprays early in the season. Most growers need to spray 3 to 6 times, depending upon the weather conditions such as rainfall triggers release of ascospores for primary infection and the amount of inoculum in the orchard from the previous season. The disease is most prevalent in Eastern and upper Midwest growing areas in the US but is also found in Pacific Northwest and California. Two types of resistant genes have been reported for  Venturia inaequalis  (Schouten et al. 2014) Apple scab is a major problem in most apple-producing regions, including EU, Canada, and China. 
     Late blight caused by  Phytophthora infestans  is the most important globally disease of potatoes and also causes substantial losses in tomatoes. It can also infect other Solanaceous species and a range of ornamentals. The disease is found globally wherever potatoes are grown. It overwinters in crop residue and cull piles from the previous season. Late blight can be a devastating disease in potatoes, causing total loss of crop if uncontrolled. Under cool and wet conditions, the disease can cause complete plant/crop collapse in 7 to 10 days. As the disease tends to strike late in the growing season, it is especially damaging as the grower has already invested a significant amount of money and resources into the crop. Chemical fungicides are used extensively for late blight control with 6 to 10 fungicide treatments, including both older protectant active ingredients like mancozeb, copper, and chlorothalonil as well as newer actives, including phenylamides, carbamates, triazoles, strobilurines, and others. Resistance development can be rapid as multiple sprays are required during the growing season, and if growers rely on a single mode of action for repeated sprays, the microorganism quickly develops resistance. During the 1990s, several new strains of  P. infestans  were introduced into the US from Mexico, which have led to increased severities of the disease as well as higher degrees of resistance to fungicides due to sexual reproduction among the different strains (Inglis et al, 1996). 
       Botrytis cinerea  attacks an extremely broad and diverse range of crops, with more than 200 crops documented as host species. Infection is favored by cool and wet weather in spring and summer. Some of the most important host species include vines, strawberries, tomatoes, cucurbits, beans, tree nuts, and tree fruits. A broad range of chemical fungicides is used against  Botrytis  as nearly all manufacturers target this important disease. Current microbial biofungicides approved for use against  Botrytis  include  Trichoderma harzianum, Bacillus amyloliquefaciens, B. subtilis, Streptomyces griseoviridis , and  Streptomyces lydicus . There are also plant extract-based biofungicides that actively against  B. cinerea , such as Regalia from Marrone and Timorex from the Stockton Group (STK). 
     Bacterial spot, caused by  Xanthomonas euvesicatoria, X. gardneri, X. perforans , and  X. vesicatoria , is the most common disease seen in tomatoes (Potnis et al. 2015). Bacterial speck, caused by  Pseudomonas syringae  pv. tomato, is an increasing problem as well and it is a bigger problem when temperatures are cooler (Basim et al. 2004). In both cases, copper-based products or copper combined with mancozeb have been used as the primary control methods, but pest resistance to copper has been increasing. Some growers have resorted to using Actigard (acibenzolar-S-methyl) in rotation with copper, but this is a costly treatment for growers. Biological treatments like bacteriophages have not gained much market share in Florida due to cost and lack of performance. 
     Bacterial wilt, caused by  Ralstonia solanacearum , is one of the major diseases of tomato and other solanaceous plants. The disease is known to occur in the wet tropics, subtropics, and some temperate regions of the world. It is one of the most damaging plant pathogens. This pathogen affects more than 300 plant species in 44 families globally, including a wide range of crop plants, ornamentals, and weeds (Li et al, 2006). Even though the biological control agents such as  Pseudomonas  and  Bacillus  have been tested for the bacterial wilt, they have no effects after 40 days of field planting (Li et al. 2006). 
     Walnut blight is caused by  Xanthomonas arboricola  pv.  juglandis  (Xaj). It is a major problem in California (Buchner et al. 2001). The application of copper for the treatment of walnut blight has resulted in the resistance of Xaj and side effects on the soil. Biological control of walnut blight pathogen by using kasugamycin is an alternative to chemical control. However, kasugamycin alone has more moderate efficacy and is at an elevated risk for Xaj resistance development. Current management of walnut blight in California includes the Cu-mancozeb, kasugamycin-mancozeb, kasugamycin-Cu rotation. 
     There is an imperative need for new multi-functional biopesticides derived from novel strains, cell broths and novel metabolites produced from such strains that can inhibit the growth of a variety of crop disease-causing pathogens. 
     BRIEF SUMMARY OF THE INVENTION 
     In a first aspect, a method of growing bacteria to enhance production of protective metabolites is provided. The method includes a step of growing  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (1214-CHY4) (Accession No. PTA-126941) bacteria in liquid media in a vessel to produce a bacterial fermentate, wherein the vessel is shaken at a rate between about 150 and 250 RPM. 
     In a second aspect, an agricultural composition comprising the bacterial fermentate or the protective supernatant produced by any of the foregoing methods. 
     In a third aspect, a method of controlling bacterial crop diseases is provided. The method includes several steps. A first step includes producing an agricultural composition comprising the bacterial fermentate or the protective supernatant produced by any one of foregoing methods or any of the foregoing agriculture compositions. A second step includes applying said agricultural composition to crops to inhibit the growth of pathogenic microorganisms. 
     In a fourth aspect, a method of purifying at least one protective metabolite from  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (Accession No. PTA-126941) bacteria is provided. The method includes several steps. A first step includes producing a bacterial fermentate or protective supernatant or using their formulations. A second step includes extracting the bacterial fermentate or protective supernatant by ethyl acetate extraction. A third step includes producing an eluate containing at least one protective metabolite by eluting the bacterial fermentate or protective supernatant using 50% hexane and 50% ethyl acetate or by eluting the bacterial fermentate or protective supernatant using 25% hexane and 75% ethyl acetate ( FIG. 2 ). 
     In a fifth aspect, a method of controlling bacterial crop diseases is provided. The method includes several steps. A first step includes producing an agricultural composition comprising at least one protective metabolite from  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (Accession No. PTA-126941) bacteria purified by one of the foregoing methods of the fourth aspect. A second step is applying said agricultural composition to crops to inhibit the growth of a pathogenic microorganism. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates an exemplary scheme for isolation and purification of Peaks 1-3. 
         FIG. 2  illustrates an exemplary scheme for isolation and purification of Peaks 4-5. 
         FIG. 3  illustrates bioactive metabolites produced by  P. chlororaphis  subsp  aurantiaca  1214-CHY4. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention relates to a novel metabolite produced by  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (1214-CHY4) that exhibits antimicrobial activity against pathogenic microorganisms, including bacteria and fungi. The cell broth of this bacterial strain contains a novel natural products. These compounds, their method of production, and applications for inhibiting plant microbial pathogens are disclosed in greater detail herein. 
     Definitions 
     When introducing elements of aspects of the disclosure or particular embodiments, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. The term “or” means any one member of a particular list and also includes any combination of members of that list, unless otherwise specified. 
     As intended herein, the terms “substantially,” “approximately,” and “about” and similar terms are intended to have a broad meaning in harmony with the common and accepted usage in the art to which the subject matter of this disclosure pertains. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to precise numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the invention as recited in the appended claims. 
     “Biological control agents (or BCAs)” are a way of managing pests, such as pathogens, weeds, and insects, safely, sustainably, and cost-effectively. These agents are introduced into the environment to target a pest species, with the aim of reducing the pest&#39;s population or abundance in the environment. 
     “Biologicals” are preparations of living microorganisms (bacteria and yeasts) that produce colonies on the hosts. These microorganisms are applied mainly to slow the pathogen buildup during the epiphytic phase (Tianna et al. (2018)). 
     “Biorational” is a term applied to microbe-based biopesticides. These biopesticides are often made by fermenting microbial strains. Most of these products have both anti-bacterial and anti-fungal activity (Tianna et al. (2018)). 
     “Biopesticides” is defined by The US Environmental Protection Agency (EPA) to be pesticides derived from natural materials and categorizes them as either biochemical pesticides, containing substances that control pests by nontoxic mechanisms, microbial pesticides, consisting of microorganisms that typically produce bioactive natural products (BNPs), or plant-incorporated-protectants with activity produced by plants because of added genetic materials (Gwinn K. D. (2018)). 
     In the present invention, the term “strain of the invention” refers to the strain  P. chlororaphis  subsp.  aurantiaca  1214-CHY4. 
     In a first aspect, a method of growing bacteria to enhance production of protective metabolites is provided. The method includes a step of growing  Pseudomonas  bacteria in liquid media in a vessel to produce a bacterial fermentate, wherein the vessel is shaken at a rate between about 150 and 250 RPM. In a first respect, the method further includes a step separating the liquid media from the bacteria after a period of time to produce a protective supernatant comprising the protective metabolites. In a second respect, the bacterial strain is  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (Accession No. PTA-126941). In a third respect, the growing temperature is between about 10 degrees C. and 35 degrees C. In a fourth respect, the liquid media for the production of cells is selected from glucose media, malt extract media, and yeast extract media. In a fifth respect, the growing temperature is between about 16 degrees C. and 28 degrees C. In a sixth respect, the bacteria are grown for a period of at 18 h to 2 days. In a seventh respect, the bacteria are grown for a period of at least 2 days. 
     In a second aspect, an agricultural composition comprising the bacterial fermentate or the protective supernatant produced by any of the foregoing methods. In a first respect, the formulation of the protective supernatant or its metabolites is selected from a solution (SL), a soluble powder (SP), a soluble granule (SG), a suspension concentrate (SC), a wettable powder (WP), a water dispersible granule (WG), a suspoemulsion (SE), a granule (GR) and an encapsulated formulation. In a second respect, the formulation of bacterial fermentate and cells is selected from a suspension concentrate (SC), a wettable powder (WP), and a water dispersible granule (WG). 
     In a third aspect a method of controlling bacterial crop diseases is provided. The method includes several steps. A first step includes producing an agricultural composition comprising the bacterial fermentate or the protective supernatant produced by any one of foregoing methods or any of the foregoing agriculture compositions. A second step includes applying said agricultural composition to crops to inhibit the growth of pathogenic microorganisms. In a first respect, the crop diseases are selected from the group consisting of grey mold, fire blight, citrus canker, soft rot, olive knot, tomato bacterial speck, bacterial canker or blast (stone and pome fruits), angular leaf spot of cucurbits, bacterial spot of peach, tomato bacterial spot, walnut blight, bacterial wilt, tomato canker, potato late blight, apple scab, bacterial leaf blight, and bacterial leaf streak. In a second respect, the pathogenic microorganism is selected from the group consisting of  Mycosphaerella fijiensis, Botrytis cinerea, Erwinia amylovora  (Ea) (especially the streptomycin-resistant  E. amylovora  strains),  Xanthomonas axonopodis  pv.  citri  (Xac),  Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum  subsp.  brasiliensis, Pectobacterium carotovorum  subsp.  carotovorum, Dickeya dadantii, Pseudomonas savastanoi  pv.  savastanoi  (Psv),  Pseudomonas syringae  pv.  tomato, Pseudomonas syringae  pv.  syringae, Pseudomonas syringae  pv.  lachrymans, Xanthomonas campestris  pv.  pruni, Xanthomonas campestris  pv.  vesicatoria, Xanthomonas arboricola  pv. juglandis,  Ralstonia solanacearum, Clavibacter michiganensis  subsp.  michiganensis, Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae  pv.  oryzae, Xanthomonas oryzae  pv.  oryzicola  and  Xanthomonas citri  pv.  citri . In a third respect, the crop is selected from the group consisting of bananas, apples, pears, crabapples, citrus, potatoes, pumpkins, onions, rice, African violets, plant species of Cruciferae, Solanaceae, Cucurbitaceae including carrots, potatoes, tomatoes, eggplants, leafy greens, squashes and cucurbits, peppers and green peppers, olive, stone and pome fruit plants including olives, peaches, walnuts. 
     In a fourth aspect, a method of purifying at least one protective metabolite from  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (Accession No. PTA-126941) bacteria is provided. The method includes several steps. A first step includes producing a bacterial fermentate or protective supernatant or using their formulations. A second step includes extracting the bacterial fermentate or protective supernatant by ethyl acetate extraction. A third step includes apply the extract into a silica gel column (as in  FIG. 1 ) and producing an eluate containing at least one protective metabolite by eluting the bacterial fermentate or protective supernatant using 50% hexane and 50% ethyl acetate or by eluting the bacterial fermentate or protective supernatant using 25% hexane and 75% ethyl acetate. In a first respect, the at least one protective metabolite is selected from peaks 1-5 or structural compounds derived therefrom, as depicted in  FIG. 3 . 
     In a fifth aspect, a method of controlling bacterial crop diseases is provided. The method includes several steps. A first step includes producing an agricultural composition comprising at least one protective metabolite from  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 (Accession No. PTA-126941) bacteria purified by one of the foregoing methods of the fourth aspect. A second step is applying said agricultural composition to crops to inhibit the growth of a pathogenic microorganism. In a first respect, the crop disease is selected from the group consisting of grey mold, fire blight, citrus canker, soft rot, olive knot, tomato bacterial speck, bacterial canker or blast (stone and pome fruits), angular leaf spot of cucurbits, bacterial spot of peach, tomato bacterial spot, walnut blight, bacterial wilt, tomato canker, potato late blight, apple scab, bacterial leaf blight, and bacterial leaf streak. In a second respect, the pathogenic microorganism is selected from the group consisting of  Mycosphaerella fijiensis, Botrytis cinerea, Envinia amylovora  (Ea) (especially the streptomycin-resistant  E. amylovora  strains),  Xanthomonas axonopodis  pv.  citri  (Xac),  Pectobacterium parmentieri, Pectobacterium atrosepticum, Pectobacterium carotovorum  subsp.  brasiliensis, Pectobacterium carotovorum  subsp.  carotovorum, Dickeya dadantii, Pseudomonas savastanoi  pv.  savastanoi  (Psv),  Pseudomonas syringae  pv. tomato,  Pseudomonas syringae  pv.  syringae, Pseudomonas syringae  pv.  lachrymans, Xanthomonas campestris  pv.  pruni, Xanthomonas campestris  pv.  vesicatoria, Xanthomonas arboricola  pv. juglandis,  Ralstonia solanacearum, Clavibacter michiganensis  subsp.  michiganensis, Phytophthora infestans, Venturia inaequalis, Xanthomonas oryzae  pv.  oryzae, Xanthomonas oryzae  pv. oryzicola and  Xanthomonas citri  pv.  citri . In a third respect, the crop is selected from the group consisting of bananas, apples, pears, crabapples, citrus, potatoes, tomatoes, eggplants, leafy greens, squashes and cucurbits, peppers and green peppers, olive, stone and pome fruit plants including olives, peaches, walnuts. 
     Biological Deposit Information 
     The bacterial strain  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 was submitted to the American Type Culture Collection (ATCC®), P.O. Box 1549, Manassas, Va. 20110 USA (“ATCC Patent Depository”) on Dec. 22, 2020. Following viability testing, the ATCC Patent Depository accorded this deposited bacterial strain the following Accession number, effective Dec. 22, 2020:  Pseudomonas chlororaphis  subsp.  aureofaciens  (now  aurantiaca )1214-CHY4 (Accession No. PTA-126941). 
     One of the inventors, Dr. Ching-Hong Yang (residing at 10120 N. Sheridan Dr., Mequon, Wis. 53902, US), acting on behalf of Applicants, submitted the bacterial strain  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4 to the American Type Culture Collection (ATCC®), P.O. Box 1549, Manassas, Va. 20110 USA (“ATCC Patent Depository”) on Dec. 22, 2020, as evidenced by the Form PCT/RO/134, “Indications Relating to Deposited Microorganism,” pursuant to PCT Rule 13 bis (filed with this application). Following viability testing, the ATCC Patent Depository accorded this deposited bacterial strain the following Accession number, effective Dec. 22, 2020:  Pseudomonas chlororaphis  subsp.  aureofaciens  (now  aurantiaca )1214-CHY4 (Accession No. PTA-126941). Dr. Yang grants permission to Applicants to include this biological deposit disclosure in the present application and gives his unreserved and irrevocable consent to it being made available to the public as of the date of filing. 
     EXAMPLES 
     Example 1. Isolation and Characterization of  P. chlororaphis  Subsp.  Aurantiaca  1214-CHY4 
     1214-CHY4 is generally classified to the species  Pseudomonas chlororaphis  based on the search of the full length sequences of 16S rRNA, gyrB, rpoB and rpoD against the NCBI BLAST database. All four sequences of 1214-CHY4 show 99% identities to  Pseudomonas chlororaphis  strains (Table 1). 1214-CHY4 produces orange pigments that lead to the classification of subspecies either  aureofaciens  or  aurantiaca  and the strain cannot utilize 5-ketogluconate as the sole carbon source that further classifies the strain to subspecies  aurantiaca  (Peix et al, 2007). Therefore, the strain is classified as  Pseudomonas chlororaphis  subsp.  aurantiaca  1214-CHY4. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Search results of 1214-CHY4 16S sRNA/gyrB/rpoB/rpoD 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                 Length of 
                   
               
               
                   
                   
                 GenBank 
                 full gene 
                   
               
               
                 Gene 
                 Top matched species 
                 Sequence ID 
                 sequence 
                 Identities 
               
               
                   
               
               
                 16S rRNA 
                 
                   Pseudomonas chlororaphis 
                 
                 CP045221.1 
                 1532bp 
                 1531/1532 
               
               
                   
                 subsp.  aurantiaca  strain ARS 38 
                   
                   
                 (99%) 
               
               
                 gyrB 
                 
                   Pseudomonas chlororaphis 
                 
                 KX696668.1 
                 2418bp 
                 2398/2418 
               
               
                   
                 strain 48B8 
                   
                   
                 (99%) 
               
               
                 rpoB 
                 
                   Pseudomonas chlororaphis 
                 
                 CP014867.1 
                 4074bp 
                 4063/4074 
               
               
                   
                 isolate 189 
                   
                   
                 (99%) 
               
               
                 rpoD 
                 
                   Pseudomonas chlororaphis 
                 
                 CP027712.1 
                 1848bp 
                 1838/1848 
               
               
                   
                 subsp.  chlororaphis  strain DSM 
                   
                   
                 (99%) 
               
               
                   
                 50083 
               
               
                   
               
            
           
         
       
     
     Example 2. Culture, Crude Extract Preparation and Identification of Bioactive Compounds 
     The strain  P. chlororaphis  subsp.  aurantiaca  1214-CHY4 was streaked onto LB plate and cultivated in 28° C. incubator for one day. Several colonies were picked up and inoculated into YME medium in a fermenter for 3 days at 28° C. with an agitation speed at 200 rpm. The bacterial culture was extracted with an equal volume of ethyl acetate. The concentrated ethyl acetate extract solution was applied onto a Yamazen flash system (AI-580) equipped with a silica gel column (I.D. 3.0×20.0 cm, 55 g, 30μ, 60 Å) and separated by different concentrations of ethyl acetate/hexane (See  FIG. 2 ). 
     The flash fractions were subjected to the antimicrobial test against  Staphylococcus aureus, Venturia inaequalis, Phytophthora infestans , and  Botrytis cinerea . The most active fractions are from Tube 21-30, Tube 37-62 (Table 2.1). 
     
       
         
           
               
             
               
                 TABLE 2.1 
               
             
            
               
                   
               
               
                 Summary of the assay results for the 57 flash fractions 
               
            
           
           
               
               
               
            
               
                 Pathogen 
                 Positive Flash Fractions 
                   
               
               
                   
               
            
           
           
               
               
               
            
               
                 
                   Staphylococcus aureus 
                 
                 T22-T30 (T2230) a   
                 T40-T62 (T4062) 
               
               
                 
                   Venturia inaequalis 
                 
                 T21-T29 (T2129) 
                 T37-T45 (T3745) 
               
               
                 
                   Phytophthora infestans 
                 
                 T23 
                 T37-T59 (T3759) 
               
               
                 
                   Botrytis cinerea 
                 
                 T27-T29 (T2729) 
                 T39-T43 (T3943) 
               
               
                   
               
               
                   a ‘T’ represents ‘Tube’. The samples from the odd number of flash fraction/Tubes were picked up to dry and dissolved in DMSO/MeOH, and then was used for the assay. 
               
            
           
         
       
     
     After HPLC purification of the active flash fractions, five bioactive compounds named Peak 1 (7.1 mg), Peak 2 (142 mg), Peak 3 (115.3 mg), Peak 4 (11.0 mg), and Peak 5 (3.0 mg) were obtained from 15 L of the 1214-CHY4 culture using preparative HPLC ( FIG. 1 ,  FIG. 2 ,  FIG. 3 ). The structures of the 5 compounds were further determined by Liquid chromatographic-mass spectrometer (LCMS), High resolution-mass spectrometer (HR-MS), and Nuclear magnetic resonance (NMR) spectroscopy. LCMS and NMR data suggest that Peak 2 is 2-hexyl-5-pentylbenzene-1,3-diol which is also known as resorstatin. Resorstatin was reported as a free radical scavenging substance and has antimicrobial for various microbes (Table 2.2). The structure of Peak 3 is 2-hexyl-5-heptylbenzene-1,3-diol that was confirmed by X-ray crystallography analysis of the crystal structure in this disclosure. It has the same structure as compound 27 in the reported literature, but no crystal structure was available to confirm the structure of Peak 3. (Table 2.2). The backbones of Peak 2 and Peak 3 are related to the family of 2,5-dialkylresorcinols (DARs) including DB-2073 (Table 2.2). In 2021, a paper has reported that a  Pseudomonas aurantiaca  strain produced resorstatin (Compound 2 in the report) and Peak 3 (Compound 1 in the report) that both compounds have certain antimicrobial activity (Table 2.2). SciFinder search showed three journal articles (Table 2.2, Ref. [2,6,7]) mentioned the structure of Peak 3, but no other prior disclosures are reported for Peak 2 and Peak 3. 
     
       
         
           
               
             
               
                 TABLE 2.2 
               
             
            
               
                   
               
               
                 Summary of the producers and activities of Peak 2, Peak 3 and DB2073 
               
            
           
           
               
               
               
               
            
               
                 Compounds 
                 Producers 
                 Activity (MIC values) 
                 Ref 1   
               
               
                   
               
               
                 Resorstatin 
                 
                   P. chlororaphis 
                 
                 See this disclosure for further information 
                 Present 
               
               
                 (2-hexyl-5- 
                 subsp.  aurantiaca   
                   
                 disclosure 
               
               
                 pentylbenzene- 
                 1214-CHY4 
                   
                   
               
               
                 1,3-diol) 
                   Pseudomonas  sp. 
                   Aspergillus fumigatus  (50 μg/mL) 
                 1 
               
               
                 (Peak 2) 
                 Ki19 
                   Candida albicans  (50 μg/mL) 
                   
               
               
                   
                   
                   Drechslera sorokiniana  (50 μg/mL) 
                   
               
               
                   
                   
                   Fusarium culmorum  (50 μg/mL) 
                   
               
               
                   
                   
                   Pseudomonas sevastanoi  (50 μg/mL) 
                   
               
               
                   
                   
                   Staphyllococcus aureus  (7 μg/mL) 
                   
               
               
                   
                   Pseudomonas  sp. 
                 Free radical scavenging substances 
                 5 
               
               
                   
                 DC165 
                   
                   
               
               
                   
                 
                   Pseudomonas 
                 
                 
                   Staphylococcus aureus 
                 
                 6 
               
               
                   
                 
                   aurantiaca 
                 
                 ATCC 12600 (2.1 μg/mL) 
                   
               
               
                   
                   
                 
                   Staphylococcus epidermidis 
                 
                   
               
               
                   
                   
                 ATCC 14990 (4.2 μg/mL) 
                   
               
               
                   
                   
                 
                   Enterococcus hirae 
                 
                   
               
               
                   
                   
                 ATCC 8043 (4.2 μg/mL) 
                   
               
               
                   
                   
                 
                   Streptococcus mutans 
                 
                   
               
               
                   
                   
                 ATCC 25175(4.2 μg/mL) 
                   
               
               
                   
                   
                   Bacillus subtilis  subsp.  spizizenii   
                   
               
               
                   
                   
                 ATCC 6633(4.2 μg/mL) 
                   
               
               
                 2-hexyl-5- 
                 
                   P. chlororaphis 
                 
                 See this disclosure for further information 
                 Present 
               
               
                 heptylbenzene- 
                 subsp.  aurantiaca   
                   
                 disclosure 
               
               
                 1,3-diol 
                 1214-CHY4 
                   
                   
               
               
                 (Peak 3) 
                   Azoarcus  BH72 
                 Compound 27 of FIG. S3, antimicrobial 
                 2 
               
               
                   
                   
                 activity not provided 
                   
               
               
                   
                 
                   Pseudomonas 
                 
                 
                   Staphylococcus aureus 
                 
                 6 
               
               
                   
                 
                   aurantiaca 
                 
                 ATCC 12600 (4.2 μg/mL) 
                   
               
               
                   
                   
                 
                   Staphylococcus epidermidis 
                 
                   
               
               
                   
                   
                 ATCC 14990 (4.2 μg/mL) 
                   
               
               
                   
                   
                 
                   Enterococcus hirae 
                 
                   
               
               
                   
                   
                 ATCC 8043 (8.4 μg/mL) 
                   
               
               
                   
                   
                 
                   Streptococcus mutans 
                 
                   
               
               
                   
                   
                 ATCC 25175(8.4 μg/mL) 
                   
               
               
                   
                   
                   Bacillus subtilis  subsp.  spizizenii   
                   
               
               
                   
                   
                 ATCC 6633(33.4 μg/mL) 
                   
               
               
                 DB2073 
                   Pseudomonas  sp. B- 
                 Active against 22 bacteria and fungi, 
                 3-4 
               
               
                   
                 9004 
                 including Gram-positive bacteria 
                   
               
               
                   
                   
                 mycobacteria and  Staphylococcus aureus   
                   
               
               
                   
                   
                 (25 μg/mL) 
               
               
                   
               
               
                   1 References for this Table 
               
               
                 1 Pohanka, A., Levenfors, J., &amp; Broberg, A. (2006). Antimicrobial dialkylresorcinols from  Pseudomonas  sp. Ki19.  Journal of Natural Products , 69(4), 654-657. 
               
               
                 2 Fuchs, S. W., Bozhüyük, K. A. J., Kresovic, D., Grundmann, F., Dill, V., Brachmann, A. O., Waterfield, N. R., &amp; Bode, H. B. (2013). Formation of 1,3-cyclohexanediones and resorcinols catalyzed by a widely occurring ketosynthase.  Angewandte Chemie  -  International Edition , 52(15), 4108-4112. 
               
               
                 3 Kanda, N., Ishizaki, N., Inoue, N., Oshima, M., Handa, A., &amp; Kitahara, T. (1975). Db-2073, a new alkylresorcinol antibiotic. I. Taxonomy, isolation and characterization.  The Journal of Antibiotics , 28(12), 935-942. 
               
               
                 4 Kitahara, T., &amp; Kanda, N. (1975). Db-2073, a new alkylresorcinol antibiotic. II. the chemical structure of Db-2073.  The Journal of Antibiotics , 28(12), 943-946. 
               
               
                 5 Kato, S., Shindo, K., Kawai, H., Matsuoka, M., &amp; Mochizuki, J. (1993). Studies on free radical scavenging substances from microorganisms III. Isolation and structural elucidation of a novel free radical scavenger, resorstatin.  Journal of Antibiotics , 46(6), 1024-1026. 
               
               
                 6 Li, J., Shi, Y. &amp; Clark, B. R. (2021). Semi-synthesis of antibacterial dialkylresorcinol derivatives.  Journal of Antibiotics  74, 70-75. 
               
               
                 [7] Budzikiewicz, H., Scholl, H., Neuenhaus, W., Pulverer, G. and Korth, H. (1980). “Dialkylresorcine aus  Pseudomonas aureofaciens /Dialkyl Resorcinols from  Pseudomonas aureofaciens ” Zeitschrift für Naturforschung B, 35 (7), 909-910. (In German) 
               
            
           
         
       
     
     Peak 4 and Peak 5 are phenazine natural products. Peak 4 was analyzed by HR-MS and further confirmed by parallel analysis of the NMR spectra with the authentic compound phenazine-1-carboxylic acid (PCA). The PCA producer  Pseudomonas aeruginosa  M18 is an effective biocontrol agent that was isolated from the rhizosphere of sweet melon. The PCA yield of the genetically modified strain M18UMS/Phz reached approximately 4.7 g/L. PCA was commercially named shenqinmycin and a 1% shenqinmycin suspension was registered in China as an environmentally friendly fungicide (Product no. PD20110315) in 2011. This product was marketed in China to control rice and vegetable diseases caused by  Rhizoctonia solani  and  Fusarium oxysporum  (Table 2.3 and Table 2.4). 
     
       
         
           
               
             
               
                 TABLE 2.3 
               
             
            
               
                   
               
               
                 Summary of the published producer and effects of Phenazine- 
               
               
                 1-carboxylic acid on the pathogenic bacteria and fungi 
               
            
           
           
               
               
               
            
               
                 Producers 
                 Active Against/Yield 
                 Ref 1   
               
               
                   
               
            
           
           
               
               
               
            
               
                   P. aeruginosa  LV Strain 
                 
                   Botrytis cinerea 
                 
                 1 
               
               
                   P. fluorescens  LBUM636 
                 
                   Phytophthora infestans 
                 
                 2 
               
               
                   P. fluorescens  LBUM223 
                 
                   Phytophthora infestans 
                 
                 3 
               
               
                   P. chlororaphis  subsp.  aureofaciens  M71 
                 
                   Seiridium cardinale 
                 
                 4 
               
               
                   P. aeruginosa  PA31x 
                   Vibrio anguillarum  C312 
                 5 
               
               
                   P. fluorescens  2-79 
                   Gaeumannomyces graminis  var.  tritici.   
                 6 
               
               
                   P. fluorescens  strain Psd 
                 
                   Fusarium oxysporum 
                 
                 7 
               
               
                   P. aeruginosa  MSH ΔlasR 
                 ~250 mg/L 
                 8 
               
               
                   Pseudomonas  sp. M18G 
                 1987 mg/L 
                 9 
               
               
                   Pseudomonas  sp. M18UMS 
                 4.7 g/L 
                 10 
               
               
                   P. syringae  pv. tomato (Pst) DC3000 
                 13.4 μg/L 
                 11 
               
               
                   P. chlororaphis  MCC2693 
                   Alternaria alternata  &gt;  Phytophthora  sp. &gt; 
                 12 
               
               
                   
                   Fusarium solani  &gt;  F. oxysporum   
               
               
                   
               
               
                   1 References for this Table 
               
               
                 1 Simionato, A. S., Navarro, M. O. P., de Jesus, M. L. A., Barazetti, A. R., da Silva, C. S., Simões, G. C., Balbi-Peña, M. I., de Mello, J. C. P., Panagio, L. A., de Almeida, R. S. C., Andrade, G., &amp; de Oliveira, A. G. (2017). The effect of phenazine-1-carboxylic acid on mycelial growth of  Botrytis cinerea  produced by  Pseudomonas aeruginosa  LV strain.  Frontiers in Microbiology , 8(JUN), 1-9. 
               
               
                 2 Morrison, C. K., Arseneault, T., Novinscak, A., &amp; Filion, M. (2017). Phenazine-1-carboxylic acid production by  Pseudomonas fluorescens  LBUM636 alters  Phytophthora infestans  growth and late blight development.  Phytopathology , 107(3), 273-279. 
               
               
                 3 Roquigny, R., Novinscak, A., Arseneault, T., Joly, D. L., &amp; Filion, M. (n.d.). Transcriptome alteration in  Phytophthora infestans  in response to phenazine-1-carboxylic acid production by  Pseudomonas fluorescens  strain LBUM223.  BMC Genomics , 19, 474. 
               
               
                 4 Raio, A., Reveglia, P., Puopolo, G., Cimmino, A., Danti, R., &amp; Evidente, A. (2017). Involvement of phenazine-1-carboxylic acid in the interaction between  Pseudomonas chlororaphis  subsp.  aureofaciens  strain M71 and  Seiridium cardinale  in vivo.  Microbiological Research , 199, 49-56. 
               
               
                 5 Zhang, L., Tian, X., Kuang, S., Liu, G., Zhang, C., &amp; Sun, C. (2017). Antagonistic activity and mode of action of phenazine-1-carboxylic acid, produced by marine bacterium  Pseudomonas aeruginosa  PA31x, against  Vibrio anguillarum  In vitro and in a zebrafish in vivo model.  Frontiers in Microbiology , 8(FEB), 1-11. 
               
               
                 6 Thomashow, L. S., Weller, D. M., Bonsall, R. F., &amp; Pierson, L. S. (1990). Production of the antibiotic phenazine-1-carboxylic acid by fluorescent  Pseudomonas  species in the rhizosphere of wheat.  Applied and Environmental Microbiology , 56(4), 908-912. 
               
               
                 7 Upadhyay, A., &amp; Srivastava, S. (2011). Phenazine-1-carboxylic acid is a more important contributor to biocontrol Fusarium oxysporum than pyrrolnitrin in  Pseudomonas fluorescens  strain Psd.  Microbiological Research , 166(4), 323-335. 
               
               
                 8 Sun, S., Zhou, L., Jin, K., Jiang, H., &amp; He, Y. W. (2016). Quorum sensing systems differentially regulate the production of phenazine-1-carboxylic acid in the rhizobacterium  Pseudomonas aeruginosa  PA1201.  Scientific Reports , 6(February), 16-18. 
               
               
                 9 Li, Y., Jiang, H., Du, X., Huang, X., Zhang, X., Xu, Y., &amp; Xu, Y. (2010). Enhancement of phenazine-1-carboxylic acid production using batch and fed-batch culture of gacA inactivated  Pseudomonas  sp. M18G.  Bioresource Technology , 101(10), 3649-3656. 
               
               
                 10 Du, X., Li, Y., Zhou, W., Zhou, Q., Liu, H., &amp; Xu, Y. (2013). Phenazine-1-carboxylic acid production in a chromosomally non-scar triple-deleted mutant  Pseudomonas aeruginosa  using statistical experimental designs to optimize yield.  Applied Microbiology and Biotechnology , 97(17), 7767-7778. 
               
               
                 11 Li, W., Xu, Y. ping, Jean-Pierre, M., Xu, X., Qi, X. F., Gu, Y., &amp; Cai, X. Z. (2016). Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria  Pseudomonas syringae  pv. tomato and  Xanthomonas oryzae  pv.  oryzae .  Journal of Integrative Agriculture , 15(4), 812-821. 
               
               
                 12 Jain, R., &amp; Pandey, A. (2016). A phenazine-1-carboxylic acid producing polyextremophilic  Pseudomonas chlororaphis  (MCC2693) strain, isolated from mountain ecosystem, possesses biocontrol and plant growth promotion abilities.  Microbiological Research , 190, 63-71. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2.4 
               
             
            
               
                   
               
               
                 Summary of SciFinder search for Peak 5 
               
            
           
           
               
               
               
            
               
                 Name of the compound 
                 Notes 
                 Ref 1   
               
               
                   
               
               
                 4-hydroxy-5,10-dioxo-phenazine-1- 
                 Intermediate of 
                 1 
               
               
                 carboxylic acid. 
                 chemical synthesis 
                   
               
               
                 4-hydroxyphenazine-1-carboxylic acid 
                 natural product from 
                 2 
               
               
                   
                 
                   Pseudomonas 
                 
                   
               
               
                 4-hydroxyphenazine-1-carboxylic acid 
                 Intermediate of 
                 3 
               
               
                   
                 chemical synthesis 
                   
               
               
                 4-hydroxyphenazine-1-carboxylic acid 
                 Intermediate of 
                 4 
               
               
                   
                 chemical synthesis 
               
               
                   
               
               
                   1 References for this Table 
               
               
                 1 WO 2020035548. 2020, Redox-active compounds and uses thereof. 
               
               
                 2 Roemer. (1981). Bacterial constituents. Part II. Phenazines from pseudomonads.  Zeitschrift F ü r Naturforschung. , 8. 
               
               
                 3 Checchi, S. (1958). 5-Aminopyrazole derivatives. V. Synthesis of new tri- and tetraheterocyclic pyrazopyrimidopyridines and pyrazopyrimidopyridopyridazones.  Gazzetta Chimica Italiana. , 88. 
               
               
                 4 Nakamura, S. (1958). Structure of griseolutein-B, a  Streptomyces  antibiotic. II. Decarboxylation and periodic acid oxidation.  Chemical &amp; Pharmaceutical Bulletin. , 6. 
               
            
           
         
       
     
     Example 3. Test for the Antimicrobial Activity of 1214-CRY4 and its Metabolites Against Plant Disease-Causing Bacteria and Fungi 
     We investigated the biological activity of the 1214-CHY4 metabolites against plant-associated pathogenic bacteria, including the Gram-positive bacterium  Clavibacter michiganensis  subsp.  michiganensis  (Cmm), which causes tomato canker. The biological activities of the crude extract of 1214-CHY4 were also tested against plant-associated gram-negative pathogenic bacteria,  Erwinia amylovora  (Ea),  Ralstonia solanacearum  (Rs), and  Xanthomonas arboricola  pv.  juglandis  (Xaj), etc. 
     The crude extract from 1214-CHY4 showed inhibition zones against a broad range of different types of phytopathogenic bacteria, including  E. amylovora, R. solanacearum, C. michiganensis  subsp.  michiganensis , and  X. arboricola  pv.  juglandis  at 25 mg/mL and 5 mg/mL according to the plate diffusion assay results (Table 3.1). 
     The MIC values were studied for Peak 2, Peak 3 and Peak 4 against three fungal and four bacterial species. MIC values of 1214-CHY4 crude extract against  Venturia inaequalis VI 19-032,  Phytophthora infestans  88069, and  Botrytis cinerea  CA17 are 31.3 μg/mL, 1.56 μg/mL, and 62.5 μg/mL, respectively (Table 3.2). 
     In terms of antibacterial activity, 1214-CHY4 crude extract, Peak 2, and Peak 3 are the most active metabolites against  Clavibacter michiganensis  subsp.  michiganensis  (Cmm), with a MIC value of 1.95 μg/mL, 1.56 μg/mL and 0.78 μg/mL, respectively. Additionally, Peak 4 showed the strongest activity against  Ralstonia solanacearum  K60 (tomato wilt), with a MIC value of 6.25 μg/mL (Table 3.2). 
     Due to the limited amount, Peak 1 was only tested against  Venturia inaequalis , which shows 100% inhibition of  Venturia inaequalis  at 80 μg/mL (Table 3.3). 
     The overnight living bacterial culture of 1214-CHY4 at 1:500 dilution in PDA plate showed 100% antifungal activity against  Venturia inaequalis, Phytophthora infestans  Pi 1306,  Botrytis cinerea  CA17,  Botrytis cinerea  CA177,  Botrytis cinerea  CA31 (Table 3.3). 
     Peak 2 and Peak 3 have notable activity (1.56 μg/mL and 0.78 μg/mL respectively) against Cmm and have not been reported. Peak 1 is a novel structure with antifungal activity against  Venturia inaequalis . Phenazine-1-carboxylic acid, is a potential antifungal compound, which has been reported to be effective against  Botrytis cinerea  and  Fusarium oxysporum  (Upadhyay and Srivastava, 2011); however, there is no report on the efficacy of this compound against  Venturia inaequalis , the fungal pathogen causing apple scab. Our discovery demonstrates that the 1214-CHY4 metabolites are the biocontrol agents against tomato diseases and apple scab since they have nice activity against Cmm,  R. solanacearum , and  V. inaequalis  (Table 3.2 and Table 3.3). 
     
       
         
           
               
             
               
                 TABLE 3.1 
               
             
            
               
                   
               
               
                 Summary of the antimicrobial effect of 1214-CHY4 
               
               
                 crude extract against phytopathogenic bacteria. 
               
            
           
           
               
               
            
               
                   
                 Diameter of Growth Inhibition Zone (cm) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 CHY4 crude 
                 CHY4 crude 
                   
                   
               
               
                   
                   
                 extract 
                 extract 
                 Streptomycin 
                 Vancomycin 
               
               
                 Bacteria 
                 Media 
                 (25 mg/mL) 
                 (5 mg/mL) 
                 (200 μg/mL) 
                 (200 μg/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   Erwinia amylovora  1189 (Fire 
                 LB 
                 0.4 
                 0.4 
                 0.5 
                 NA 
               
               
                 blight on apples/pears) 
                   
                   
                   
                   
                   
               
               
                   Erwinia amylovora  110 a  (Fire 
                 LB 
                 0.5 
                 0.3 
                 0.5 
                 NA 
               
               
                 blight on apples/pears) 
                   
                   
                   
                   
                   
               
               
                   Erwinia amylovora  DM1 b   
                 LB 
                 0.5 
                 0.4 
                 0.0 
                 NA 
               
               
                 (Fire blight on apples/pears) 
                   
                   
                   
                   
                   
               
               
                   Erwinia amylovora  CA11 b   
                 LB 
                 0.5 
                 0.4 
                 0.0 
                 NA 
               
               
                 (Fire blight on apples/pears) 
                   
                   
                   
                   
                   
               
               
                   Erwinia amylovora  88 c  (Fire 
                 LB 
                 0.4 
                 0.3 
                 0.0 
                 NA 
               
               
                 blight on apples/pears) 
                   
                   
                   
                   
                   
               
               
                 
                   Pectobacterium parmentieri 
                 
                 LB 
                 0.8 
                 0.4 
                 0.5 
                 NA 
               
               
                 UPP163 936 (Causer of soft 
                   
                   
                   
                   
                   
               
               
                 rot in multiple crops) 
                   
                   
                   
                   
                   
               
               
                 
                   Pectobacterium atrosepticum 
                 
                 LB 
                 0.3 
                 0.4 
                 0.0 
                 NA 
               
               
                 942 (Causer of soft rot in 
                   
                   
                   
                   
                   
               
               
                 multiple crops) 
                   
                   
                   
                   
                   
               
               
                 
                   Pectobacterium carotovorum 
                 
                 LB 
                 0.7 
                 0.3 
                 0.0 
                 NA 
               
               
                 subsp  brasillensis  944 (Causer 
                   
                   
                   
                   
                   
               
               
                 of soft rot in multiple crops) 
                   
                   
                   
                   
                   
               
               
                 
                   Pectobacterium carotovorum 
                 
                 LB 
                 0.5 
                 0.3 
                 0.0 
                 NA 
               
               
                 subsp.  carotovorum  wpp14 
                   
                   
                   
                   
                   
               
               
                 945 (Causer of soft rot in 
                   
                   
                   
                   
                   
               
               
                 multiple crops) 
                   
                   
                   
                   
                   
               
               
                   Dickeya dadantii  3937 
                 LB 
                 0.5 
                 0.3 
                 0.5 
                 NA 
               
               
                 (Causer of soft rot in multiple 
                   
                   
                   
                   
                   
               
               
                 crops) 
                   
                   
                   
                   
                   
               
               
                   Pseudomonas savastanoi  pv. 
                 LB 
                 0.5 
                 0.5 
                 0.6 
                 NA 
               
               
                   savastanoi  (Olive knot) 
                   
                   
                   
                   
                   
               
               
                   Pseudomonas syringae.  pv 
                 LB 
                 0.5 
                 0.4 
                 0.5 
                 NA 
               
               
                   syringae  7046 (Bacterial 
                   
                   
                   
                   
                   
               
               
                 canker or blast (stone and 
                   
                   
                   
                   
                   
               
               
                 pome fruits)) 
                   
                   
                   
                   
                   
               
               
                   Pseudomonas syringae.  pv. 
                 LB 
                 0.5 
                 0.4 
                 0.6 
                 NA 
               
               
                   lachrymans  1188-1 (Angular 
                   
                   
                   
                   
                   
               
               
                 leaf spot of cucurbits) 
                   
                   
                   
                   
                   
               
               
                   Pseudomonas syringae.  pv. 
                 LB 
                 0.5 
                 0.5 
                 0.6 
                 NA 
               
               
                 tomato PT30 (Tomato 
                   
                   
                   
                   
                   
               
               
                 bacterial speck) 
                   
                   
                   
                   
                   
               
               
                   Ralstonia solanacearum  K60 
                 LB 
                 0.5 
                 0.4 
                 0.3 
                 NA 
               
               
                 (Tomato wilt) 
                   
                   
                   
                   
                   
               
               
                   Ralstonia solanacearum  Pss4 
                 LB 
                 0.3 
                 0.0 
                 0.3 
                 NA 
               
               
                 (Tomato wilt) 
                   
                   
                   
                   
                   
               
               
                 
                   Clavibacter michiganensis 
                 
                 LB 
                 1.1 
                 1.0 
                 NA 
                 1.1 
               
               
                 subsp.  michiganensis  Cmm 
                   
                   
                   
                   
                   
               
               
                 0317 (Tomato canker) 
                   
                   
                   
                   
                   
               
               
                 
                   Clavibacter michiganensis 
                 
                 LB 
                 1.0 
                 1.0 
                 NA 
                 1.2 
               
               
                 subsp.  michiganensis  Cmm 
                   
                   
                   
                   
                   
               
               
                 0690 (Tomato canker) 
                   
                   
                   
                   
                   
               
               
                 
                   Clavibacter michiganensis 
                 
                 LB 
                 1.3 
                 1.1 
                 NA 
                 1.3 
               
               
                 subsp.  michiganensis   
                   
                   
                   
                   
                   
               
               
                 NCPPB382 (Tomato canker) 
                   
                   
                   
                   
                   
               
               
                   Xanthomonas axonopodis  pv. 
                 NB 
                 0.4 
                 0.4 
                 2.2 
                 NA 
               
               
                   citri  N40-SO5 (Citrus canker) 
                   
                   
                   
                   
                   
               
               
                   Xanthomonas axonopodis  pv. 
                 NB 
                 0.5 
                 0.4 
                 2.1 
                 NA 
               
               
                 citri-Miami XC2002-00010 
                   
                   
                   
                   
                   
               
               
                 (Citrus canker) 
                   
                   
                   
                   
                   
               
               
                   Xanthomonas campestris  pv. 
                 NB 
                 0.3 
                 0.0 
                 0.0 
                 NA 
               
               
                   pruni  (Bacterial spot of Peach) 
                   
                   
                   
                   
                   
               
               
                   Xanthomonas campestris  pv. 
                 NB 
                 0.4 
                 0.4 
                 1.0 
                 NA 
               
               
                   vesicatoria  XV-16 (Tomato 
                   
                   
                   
                   
                   
               
               
                 bacterial spot) 
                   
                   
                   
                   
                   
               
               
                   Xanthomonas arboricola  pv. 
                 YGC 
                 0.3 
                 0.3 
                 0.0 
                 NA 
               
               
                   juglandis  219 (Walnut blight) 
                   
                   
                   
                   
                   
               
               
                   Xanthomonas arboricola  pv. 
                 YGC 
                 0.4 
                 0.3 
                 1.0 
                 NA 
               
               
                   juglandis  417 d  (Walnut blight) 
               
               
                   
               
               
                   a Ea110 is the virulent strain used for the field trials in Michigan state; 
               
               
                   b Both CA11 and DM1 are streptomycin-resistant strains containing Tn5393 with the transposon on the acquired plasmid pEa34 and can grow in 100 μg/mL streptomycin containing media; 
               
               
                   c Ea88 is a spontaneous streptomycin-resistant strain with a mutation in the chromosomal rpsL gene and can grow in the media containing 2000 μg/mL streptomycin; 
               
               
                   d Copper resistant bacteria; 
               
               
                 NA: Not available. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3.2 
               
             
            
               
                   
               
               
                 Summary of MIC value study of 1214-CHY4 metabolites 
               
            
           
           
               
               
            
               
                   
                 MIC (μg/mL) 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Pathogen (Causing 
                 Crude 
                   
                   
                   
                   
                   
               
               
                 disease) 
                 extract 
                 Peak 2 
                 Peak 3 
                 Peak 4 
                 Vancomycin 
                 Streptomycin 
               
               
                   
               
               
                 
                   Venturia inaequalis 
                 
                   31.3 b   
                 &gt;100 b   
                 &gt;100 b   
                   31.3 b   
                 NA 
                 NA 
               
               
                 VI19-032 (Apple scab) 
               
               
                 
                   Phytophthora infestans 
                 
                    1.56 b   
                 &gt;100 b   
                 &gt;100 b   
                    1.56 b   
                 NA 
                 NA 
               
               
                 88069 (Potato late 
               
               
                 blight) 
               
               
                   Botrytis cinerea  CA17 
                   62.5 b   
                 &gt;100 b   
                 &gt;100 b   
                   62.5 b   
                 NA 
                 NA 
               
               
                 (Grey mold) 
               
               
                 
                   Ralstonia 
                 
                 &gt;25000 a    
                 &gt;25000 a   
                 &gt;25000 a   
                    6.25 b   
                 100 
                 25 c   
               
               
                   solanacearum  K60 
               
               
                 (Tomato wilt) 
               
               
                 
                   Pseudomonas syringae. 
                 
                 180 a   
                  312 a   
                  156 a   
                 100 b   
                 100 
                   3.13 c   
               
               
                 pv. tomato PT30 
               
               
                 (Tomato bacterial 
               
               
                 speck) 
               
               
                 
                   Xanthomonas 
                 
                 180 a   
                  78 a   
                  78 a   
                 100 b   
                 50 
                 50 c   
               
               
                   euvesicatoria  75-3 
               
               
                 (Tomato spot) 
               
               
                 
                   Clavibacter 
                 
                    1.95 c   
                     1.56 c   
                     0.78 c   
                 100 b   
                 0.39 
                  12.5 
               
               
                   michiganensis  subsp. 
               
               
                   michiganensis  382 
               
               
                 (Tomato canker) 
               
               
                   
               
               
                   a The values were obtained by a modified disk diffusion method; 
               
               
                   b The values were obtained by agar dilution method; 
               
               
                   c The values were obtained by a modified broth dilution (microdilution) method; 
               
               
                 NA: Not available. 
               
               
                 Peak 4: The standard compound Phenazine-1-carboxylic acid (PCA) was used for the assay since Peak 4 was proved to be PCA. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3.3 
               
             
            
               
                   
               
               
                 Summary of the antifungal study of 1214-CHY4 cells and peak 1. 
               
            
           
           
               
               
            
               
                   
                 Inhibitory % 
               
            
           
           
               
               
               
            
               
                   
                 1214-CHY4 
                 Peak 1 
               
               
                 Fungi 
                 (1:500 dilution) a   
                 (80 ug/mL) 
               
               
                   
               
               
                   Venturia inaequalis  19-032 
                 100 
                 100 
               
               
                   Phytophthora infestans  Pi1306 
                 100 
                 NA 
               
               
                   Botrytis cinerea  CA17 
                 100 
                 NA 
               
               
                   Botrytis cinerea  CA177 
                 100 
                 NA 
               
               
                   Botrytis cinerea  CA31 
                 100 
                 NA 
               
               
                   
               
               
                   a 1214-CHY4 cell culture grew at 28° C. overnight; then the cell culture was diluted to 1:500 in melted PDA media (40 μL cell broth in 20 mL PDA media in one petri dish plate). The results were checked on day 7 and day 14 for  Venturia inaequalis  and on day 3 for  Phytophthora infestans  and  Botrytis cinerea . 
               
               
                 NA: Not available. 
               
            
           
         
       
     
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         Anderson J. A., Staley J., Challender M., Heuton J. (2018) Safety of  Pseudomonas chlororaphis  as a gene source for genetically modified crops,  Transgenic Research  27:103-113. 
         Arrebola E., Tienda S., Vida C., de Vicente A., Cazorla F. M. (2019) Fitness features involved in the biocontrol interaction of  Pseudomonas chlororaphis  with host plants: The case study of PcPCL1606 , Frontiers in Microbiology  10:719. 
         Basim, H., Basim, E., Yilmaz, S., Dickstein, E. R., &amp; Jones, J. B. (2004). An outbreak of bacterial speck caused by  Pseudomonas syringae  pv. tomato on tomato transplants grown in commercial seedling companies located in the western Mediterranean region of turkey.  Plant Disease,  88(9), 1050. 
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