Biological control of soft rot and biofilm by microbial predators encapsulated in carrier

Abstract

The present invention provides a system for differential delivery of live BALO. The system comprises: a carrier comprising a polysaccharide hydrocolloid; and BALO immobilized within the carrier wherein the BALO is present in a physiological state selected from the group consisting of leech plastid, leech cyst and AP, which is present at a cell density of at least about 1.0 * 108 PFU / g carrier wherein the polysaccharide hydrocolloid is in the form of a wet or dry gel, and wherein the carrier preserves the viability and lysolytic efficacy of the immobilized BALO. Further provided are methods for making the systems for differential delivery of live BALO, as well as methods for using the same, e.g., for treating phytopathogenic bacterial diseases in plants or crops.

Description

Field of the Invention

[0001] The present invention relates to hydrocolloid-based compositions for encapsulation and delivery of live Bdellovibrio-and-like organisms (BALOs) and their uses. Background of the Invention

[0003] Biofilm is an aggregate of microorganisms surrounded by a matrix attached to a biological or abiotic surface. The complex microbial community of biofilm is highly resistant to antibiotics and sanitizers and confers long-lasting survival, which is a challenge to overcome. Biofilm formation is an important problem in various industries and can lead to major economic and health problems. Many pathogen outbreaks have been attributed to biofilms. In water and sewage treatment facilities and water distribution systems, biofilms (biofouling) cause metal corrosion, increase pollution risks, reduce water quality and reduce heat exchange efficiency.

[0004] Pectobacterium and Dickeya pectinolytic bacteria belonging to the Pectobacteriaceae are plant pathogens responsible for several maceration diseases (soft rot, blackleg and aboveground stem rot) on a wide range of crops and ornamental plants under a wide variety of climates. These diseases are responsible for huge economic damage, with estimated global crop losses of at least 30%. As of today, there is no effective physical and chemical control for this group of bacteria, and most practices for protecting crops are based on hygiene, nutrition and growth management.

[0005] However, recent findings show that Bdellovibrio and similar organisms (BALOs) are promising biocontrol agents for soft rot Pectobacteriaceae (SRP).BALOs are a group of small, highly motile Gram-negative bacteria that prey on other Gram-negative bacteria and are found in many terrestrial and aquatic environments (Rotem et al., 2014). These bacteria interact with their prey as highly motile attack phase (AP) cells, by attaching to the outer membrane and consuming prey (surface parasitic predation) or by penetrating their periplasm (periplasmic predation) outside the cell. However, most BALO isolates, including bacteriovorous Bdellovibrio (Bdellovibrio bacteriovorus) (most studied in BALO), are periplasmic predators. During prey invasion, predators change the prey cell wall, thereby preventing repeated infection, and convert it into a shielded feeding niche known as bdelloid plastid. Inside it, BALO cells grow to eventually produce daughter AP cells that escape from prey debris (Rotem et al., 2014). The prey range of BALOs varies from strain to strain, with some showing high specificity for a certain prey, while others are generalists, using a wide range of prey. Colony and single-cell analyses reveal complex prey interactions. Genetic resistance to BALOs is not easily acquired, but prey populations can exhibit plastic phenotypic resistance (Shemesh et al., 2004; Duncan et al., 2018; Aharon et al., 2021).

[0006] These properties give BALO the potential to be an alternative source of antibiotics (Sockett, 2009). However, the use of AP cells or bdelloid plasmids is not common, although showing great potential for protection against pathogens of humans and other animals as well as against plant pathogens (Duncan et al., 2018). It requires freshly grown BALO cultures, which imposes several limitations in terms of shelf life, transportation and handling (Mansfield et al., 2012). Therefore, practical implementation requires a suitable delivery vehicle.

[0007] BALO encapsulation in gelatin microparticles has been reported for use in aquaculture systems (Mansfield et al., 2012). Encapsulated Bdellovibrio powder has been suggested as a potential biodisinfectant against pathogenic Vibrio species of whiteleg shrimp (Cao et al., 2019). Polyhydroxyalkanoate carriers have been developed for BALO delivery in the industrial production of bioplastics, such as Bdellovibrio HD100 encapsulation (González et al., 2020).

[0008] Since biocontrol involves the use of sensitive live organisms, suitable encapsulation systems offer several advantages over free cell inoculation. Encapsulation is the process of forming a continuous layer of encapsulated cells and / or compounds within a matrix core. Encapsulation within the matrix protects living cells from biotic and abiotic stress factors by providing a beneficial microenvironment. Appropriate matrix formulations have the potential to improve the application of microbial biocontrol agents by extending shelf life, reducing the number of applications and reducing dosage, because it can provide sufficient cell density while maintaining cell performance and simplifying handling (Vemmer, 2013). For such preparations, water-soluble polymeric materials (hydrocolloids) extracted from natural sources have been widely used to produce gel-based carriers for encapsulation of microorganisms in the food, biotechnology and agricultural industries.

[0009] Hydrocolloid carriers for encapsulated microorganisms can be customized for specific needs. For example, pectin has been widely used as a delivery vehicle for colon-specific oral drugs and, to a lesser extent, probiotics (low-methoxyl pectin and rice bran) (Chotiko et al., 2016; Jung et al., 2013). Few reports have investigated the controlled release of kasugamycin (antibiotic) conjugated to pectin against plant pathogens with pectinase activity (Fan et al., 2017; Liu et al., 2015).

[0010] Recently, Sason et al. disclosed biological control of soft rot in potatoes by BALOs encapsulated in κ-carrageenan carriers (Applied Microbiology and Biotechnology (2023) 107:81-96) and self-elimination of soft rot bacteria by activating BALOs via pectin-based carriers (Microbial Biotechnology (2023) 16:1561-1576).

[0011] There remains an unmet need for encapsulation and delivery systems for live BALOs, particularly to be used in agricultural and industrial applications to prevent bacterial soft rot infection and reduce biofilm formation in plant tissues. The encapsulation and delivery systems should provide long shelf life, as well as controllable release time and properties of the encapsulated live BALOs that can be customized for specific applications and bacteria types. SUMMARY OF THE INVENTION

[0013] The present invention provides a system for differential delivery of live BALOs, a method for preparing the same, and a method for treating plants and crops by using the system. The system for differential delivery of live BALOs is based on encapsulation in a polysaccharide hydrocolloid. The polysaccharide hydrocolloid with encapsulated BALOs can be in a dried gel form, thereby providing a long shelf life and high stability of the live BALOs.

[0014] The present invention is based in part on the surprising discovery that polysaccharide hydrocolloids can provide an effective immobilization and delivery system for BALOs, whereby carriers based on the polysaccharide hydrocolloids can be delivered at a rate of at least about 1.0×10 8 The BALOs are encapsulated at a cell density of PFU / g carrier. The encapsulated BALOs in the delivery system of the present invention exist in the physiological state of broomraplasts, broomraplasts and / or attack phase (AP) cells. According to some embodiments, the BALOs exist in the physiological state of broomraplasts and AP cells.

[0015] Surprisingly, it has been found that after drying the hydrocolloid-based delivery system, the survival rate of BALO is higher for leech plastids than for attack-phase BALO cells. In addition, different polysaccharide hydrocolloids are used to provide time-dependent controlled release of encapsulated BALO. As exemplified below, carrageenan was found to enable long-term entrapment of BALO and rapid delivery thereof after swelling and dissolution of the dried hydrocolloid beads after contact with water. Thus, carrageenan-based carriers provide an effective BALO rapid delivery system for water-related applications, such as crop protection and biofilm disruption against soft rot diseases. In contrast, pectin-based carriers ensure long-term entrapment of BALO within the carrier and allow targeted delivery at a certain location and in a sustained release manner. Without wishing to be bound by any theory or mechanism of action, it is believed that the degradation of the pectin-based delivery system is caused by pectolytic enzymes secreted from the prey itself (i.e., Enterobacter spp. (SRE)), which are triggered by the pectin of the delivery system, and as a result of the degradation of the delivery system, the predator is released in an "attack and kill" state. In this way, the use of BALOs can be localized, thereby minimizing the potential damage caused by biocontrol agents to beneficial microorganisms. Therefore, the present invention provides a universal system for differential delivery of live lytic BALOs that can be customized to provide the desired delivery characteristics of the live BALOs.

[0016] It is now further disclosed that the viability of BALO in liquid suspension at 25°C drops dramatically, resulting in no viable cells after two months of storage, whereas the dry delivery system of the present invention comprising BALO immobilized in a carrageenan-based carrier has a viability of at least 1.0×1010 It is also disclosed that a liquid suspension of BALO loses viability within six months of storage at 4°C, whereas a dry delivery system of the present invention comprising BALO immobilized in a carrageenan-based carrier maintains viable BALO at a cell density of at least 1.0×10 PFU / g carrier after storage at 4°C for one year. 10 The cell density of 1.0×10 PFU / g carrier preserves viable BALOs, and the dry delivery system of the present invention comprising BALOs immobilized in a pectin-based carrier maintains viable BALOs at a cell density of at least 1.0×10 8 The cell density of PFU / g carrier preserved the viable BALOs. Therefore, the delivery system of the present invention is highly useful for the long-term storage of viable lytic BALOs.

[0017] It is further disclosed that the dry delivery system of the present invention comprising BALO immobilized within a carrageenan-based carrier was found to be highly effective in reducing bacterial soft rot infection in potato tubers under controlled conditions as well as under natural environmental conditions.

[0018] Thus, the delivery systems of the present invention comprising BALOs immobilized / entrapped within polysaccharide hydrocolloid-based carriers, particularly the dry forms of these systems, are highly effective, non-toxic, biodegradable biocontrol agents that enable the achievement of at least 1.0×10 8 , or at least 1.0×10 9 , or even at least 1.0×10 10 The invention provides a cell density of live bacterial predators of 100 PFU / g carrier; provides a controlled and localized release of BALOs; and preserves BALO viability and lytic potency during storage for several months. Thus, the delivery system of the invention is useful in a variety of applications, including in agriculture, such as reducing maceration diseases on crops.

[0019] According to one aspect, the present invention provides a system for differential delivery of live Bdellovibrio and similar organisms (BALOs), comprising:

[0020] a carrier comprising a polysaccharide hydrocolloid; and

[0021] BALOs immobilized in the carrier, wherein the BALOs are in a physiological state selected from the group consisting of bdelloid plasmids, bdelloid cysts, and attack phase cells (AP), which are present at a rate of at least about 1.0×10 8 The cell density of PFU / g vector exists,

[0022] wherein the polysaccharide hydrocolloid is in the form of a wet or dry gel, and wherein the carrier preserves the viability and bacteriolytic potency of the immobilized BALO.

[0023] According to some embodiments, the BALO comprises or consists of Bdellovibrio bacteriovorus. According to further embodiments, Bdellovibrio bacteriovorus is selected from the group consisting of Bdellovibrio bacteriovorus HD100, Bdellovibrio bacteriovorus 109J and Bdellovibrio bacteriovorus ΔmerRNA. Each possibility represents a separate embodiment of the present invention. According to an exemplary embodiment, Bdellovibrio bacteriovorus comprises or consists of Bdellovibrio bacteriovorus HD100. According to a certain embodiment, the BALO comprises or consists of a bdellovibrio and an AP. According to another embodiment, the BALO comprises or consists of a bdellovibrio.

[0024] According to further embodiments, the carrier is present as granules, beads, capsules, granules, powders and any combination thereof. Each possibility represents a separate embodiment of the present invention. According to still further embodiments, the granules, beads, capsules or granules have an average Feret diameter of about 0.01 mm to about 10 mm. According to further embodiments, the granules, beads, capsules or granules have an average Feret diameter of about 0.1 mm to about 5 mm, or about 2 mm to about 4 mm.

[0025] According to yet further embodiments, the polysaccharide hydrocolloid is in the form of a dried gel and the system is a dry system. According to an exemplary embodiment, the dry system is present as a powder.

[0026] According to some embodiments, the polysaccharide hydrocolloid is selected from the group consisting of: kappa-carrageenan, pectin, alginate, agar, gellan gum, carboxymethyl cellulose (CMC), furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan and derivatives and combinations thereof. Each possibility represents a separate embodiment of the present invention. According to an exemplary embodiment, the polysaccharide hydrocolloid is kappa-carrageenan. According to other exemplary embodiments, pectin is low methoxyl pectin (LMP).

[0027] According to further embodiments, the polysaccharide hydrocolloid is present in the system in a weight percentage of about 2% to about 25% based on the total weight of the dry system.

[0028] According to some embodiments, the polysaccharide hydrocolloid is crosslinked by a crosslinking agent and / or a gelling inducing agent. According to other embodiments, the crosslinking agent comprises a metal cation selected from the group consisting of potassium ions, calcium ions, barium ions, magnesium ions, aluminum ions, zinc ions, lead ions, iron ions, strontium ions, copper ions, cadmium ions, nickel ions, cobalt ions and combinations thereof. Each possibility represents a separate embodiment of the present invention. According to further embodiments, the metal cation is present in the system in a weight percentage of about 0.1% to about 10% of the total weight of the dry system.

[0029] According to some embodiments, the carrier further comprises a protein hydrocolloid and / or a protein. According to further embodiments, the protein hydrocolloid or protein is selected from the group consisting of gelatin, collagen, collagen hydrolysate, albumin and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0030] According to yet further embodiments, the protein hydrocolloid is gelatin, which is present in the system in a weight percentage of about 20% to about 85% based on the total weight of the dry system.

[0031] According to some embodiments, the carrier further comprises a water absorbing agent. According to a further embodiment, the water absorbing agent is selected from the group consisting of starch, polyvinylpyrrolidone (PVP) and derivatives and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0032] According to further embodiments, the water absorbent is present in the system in a weight percentage of about 25% to about 85% based on the total weight of the dry system.

[0033] According to some embodiments, the carrier further comprises at least one osmoprotectant.

[0034] According to a further embodiment, the at least one osmoprotectant is selected from the group consisting of trehalose, inositol, glycerol, arabinitol, betaine, amino acids and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0035] According to yet further embodiments, the at least one osmoprotectant is present in the system in a weight percentage of about 20% to about 85% based on the total weight of the dry system.

[0036] According to some embodiments, the system comprises about 0.1% (w / w) to about 10% (w / w) water based on the total weight of the dry system.

[0037] According to some embodiments, the differential delivery of the BALO comprises a rapid release or a delayed release of the BALO from the carrier.

[0038] According to some embodiments, a carrier suitable for rapid release of BALOs comprises kappa-carrageenan and potassium cations. According to further embodiments, the carrier further comprises trehalose. According to exemplary embodiments, kappa-carrageenan is present in the system at a weight percentage of about 5% to about 25% of the total weight of the dry system, potassium cations are present in the system at a weight percentage of about 0.5% to about 10% of the total weight of the dry system, and trehalose is present in the system at a weight percentage of about 25% to about 85% of the total weight of the dry system. According to a certain embodiment, kappa-carrageenan is present in the system at a weight percentage of about 15% to about 25% of the total weight of the dry system, potassium cations are present in the system at a weight percentage of about 0.5% to about 10% of the total weight of the dry system, and trehalose is present in the system at a weight percentage of about 70% to about 80% of the total weight of the dry system.

[0039] According to some embodiments, a carrier suitable for delayed release of BALO comprises low methoxyl pectin (LMP) and calcium ions. According to further embodiments, the LMP has a degree of esterification (DE) of about 5% to about 30%, alternatively about 5% to about 10%, and preferably about 7%; and a degree of amidation (DA) of about 0% to about 25%, alternatively about 0% to about 5%, and preferably the LMP is non-amidated. Each possibility represents a separate embodiment of the present invention.

[0040] According to further embodiments, the carrier suitable for delayed release of BALO further comprises gelatin or a water absorbent. According to certain embodiments, LMP is present in the system at a weight percentage of about 2% to about 15% of the total weight of the dry system, and gelatin is present in the system at a weight percentage of from about 20% to about 85% of the total weight of the dry system.

[0041] According to further embodiments, the carrier suitable for delayed release of BALO further comprises trehalose. According to still further embodiments, LMP is present in the system at a weight percentage of about 2% to about 15% of the total weight of the dry system, calcium cations are present in the system at a weight percentage of about 0.1% to about 5% of the total weight of the dry system, gelatin is present in the system at a weight percentage of about 20% to about 50% of the total weight of the dry system, and trehalose is present in the system at a weight percentage of about 20% to about 50% of the total weight of the dry system. According to a certain embodiment, LMP having a DE of about 7% and a DA of zero is present in the system at a weight percentage of about 5% to about 10% of the total weight of the dry system, calcium cations are present in the system at a weight percentage of about 0.1% to about 5% of the total weight of the dry system, gelatin is present in the system at a weight percentage of about 40% to about 50% of the total weight of the dry system, and trehalose is present in the system at a weight percentage of about 40% to about 50% of the total weight of the dry system.

[0042] According to some embodiments, LMP, gelatin and trehalose for delayed release of BALO are present in the dry system in a weight percent ratio of 1:1:1 to 1:10:10. According to certain embodiments, LMP, gelatin and trehalose are present in the dry system in a ratio of about 1:5:5. Preferably, the LMP has a DE of about 7% and a DA of zero.

[0043] According to some embodiments, the drying system is at a temperature of up to about 25°C at a temperature of at least about 1.0×10 8 The density of PFU / g carrier preserves the viability of the immobilized BALO for at least 3 months, at least 6 months, or at least 12 months. According to further embodiments, the dry system is at a temperature of about -20°C to about 25°C at a density of at least about 1.0×10 8 The density of PFU / g carrier preserves the viability of the immobilized BALO for at least 3 months, at least 6 months, or at least 12 months. Each possibility represents a separate embodiment of the present invention. According to an exemplary embodiment, the dry system comprising kappa-carrageenan and trehalose is heated at a temperature of about 25°C at a temperature of at least about 1.0×10 10 The density of PFU / g carrier preserves the viability of the immobilized BALO for at least 12 months. According to another exemplary embodiment, the dry system comprising LMP, gelatin and trehalose is maintained at a temperature of about 4°C at a density of at least about 1.0×10 8 The density of PFU / g carrier preserved the viability of the immobilized BALOs for at least 12 months.

[0044] According to another aspect, the present invention provides a method for preventing, reducing or eliminating a phytopathogenic bacterial disease in a plant, plant part or crop before or after harvest, comprising contacting the plant, plant part or crop with an effective amount of a delivery system according to the principles of the present invention before or after harvest, thereby preventing, reducing or eliminating the phytopathogenic bacterial disease in the plant, plant part or crop.

[0045] According to some embodiments, the phytopathogenic bacterial disease is caused by a phytopathogenic bacterium,The phytopathogenic bacteria are selected from the group consisting of Pectobacterium carotovorum, Pectobacterium chrysanthemi, Pectobacterium aroidearum, Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium cacticida, Pectobacterium colocasium, Pectobacterium cypripedii, Pectobacterium melonis, Pectobacterium parmentieri, Pectobacterium Polaris, Pectobacterium punjabense, Pectobacterium rhapontici), Pectobacterium versatile, Pectobacterium wasabiae, Pectobacterium zantedeschiae, Xanthomonas campestris, Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas tolaasii, Pseudomonas glycinea, Erwinia amylovora, Ralstonia solanacearum, Acidovorax citrulli, Acidovorax oryzae, Acidovoraxavenae, Dickeya solani), Dickeya chrysanthemi, Dickeya dadantii, Xylella fastidiosa and Pantoea sp. Each possibility represents a separate embodiment of the present invention.

[0046] According to some embodiments, the phytopathogenic bacterial disease is a maceration disease. According to further embodiments, the maceration disease is selected from the group consisting of soft rot, blackleg, and aboveground stem rot. According to an exemplary embodiment, the phytopathogenic bacterial disease is a soft rot caused by Pectobacterium carotovora.

[0047] According to yet further embodiments, the plant or plant part is selected from the group consisting of seeds, seedlings, plantlets, bulbs, rhizomes, trees, grasses, shrubs, creepers, vegetables, fruits, legumes, mushrooms, flowers and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0048] According to certain embodiments, the plant, plant part or crop is selected from the group consisting of potato (Solanum tuberosum), onion, carrot, soybean, mushroom and flower. According to an exemplary embodiment, the crop is potato and the phytopathogenic bacterial disease is soft rot.

[0049] According to some embodiments, contacting the plant, plant part or crop with the system is carried out by a process selected from the group consisting of: foliar application, soil application, application through an irrigation system, starter solution, injection into the soil, patch application, aerial application and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0050] According to another aspect, the present invention provides a method for reducing the bacterial load of an article in at least one of the pharmaceutical industry, microbiological industry, medical industry, food industry, marine industry, paper industry, textile industry, plant nursery, aquaculture, soil treatment, ship maintenance, water treatment facilities, sewage treatment facilities and water distribution systems, the method comprising contacting the article with an effective amount of a delivery system according to the principles of the present invention, thereby reducing the bacterial load of the article.

[0051] According to some embodiments, the method of reducing the bacterial load of an article comprises preventing or stopping biofilm formation and / or reducing or eradicating mature biofilm. According to further embodiments, the bacterial load is caused by bacteria selected from the group consisting of: Gram-negative bacteria, Gram-positive bacteria, antibiotic-resistant bacteria, and any combination thereof. According to a further embodiment, the Gram-negative bacteria are pathogenic Gram-negative bacteria, including but not limited to Escherichia coli, Pseudomonas, Klebsiella, Plesiomonas shigelloides, Salmonella, Shigella, Vibrio spp., such as Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. Each possibility represents a separate embodiment of the present invention.

[0052] According to another aspect, a method for preparing a delivery system for differential delivery of live Bdellovibrio and similar organisms (BALOs) is provided, wherein the delivery system comprises: a carrier comprising a polysaccharide hydrocolloid; and the BALOs immobilized within the carrier, wherein the BALOs are in a physiological state of a leech plasmid, a leech cyst, and an AP, which are at a rate of at least about 1.0×10 8 PFU / g carrier cell density, the method comprising:

[0053] (i) mixing a suspension comprising BALOs with a hydrocolloid solution comprising a polysaccharide hydrocolloid to form a hydrocolloid composition comprising the BALOs; and

[0054] (ii) mixing the hydrocolloid composition comprising the BALO of step (i) with a cross-linking solution to form a polysaccharide hydrocolloid in a wet gel form, thereby forming the delivery system in a wet form.

[0055] According to some embodiments, the method for preparing the delivery system further comprises the step of drying the delivery system of step (ii), thereby forming a dry delivery system.

[0056] According to a further embodiment, drying the delivery system in gel form is performed by at least one of: air drying, desiccant drying, vacuum drying, fluidized bed drying, spray drying, sunlight drying and freeze drying. According to a certain embodiment, drying the delivery system is performed by vacuum drying.

[0057] According to yet further embodiments, the method further comprises the steps of grinding, crushing, pulverizing and / or any process known in the art to be useful for reducing the Feret diameter of granules, beads, capsules or granules.

[0058] According to further embodiments, mixing the hydrocolloid composition comprising the BALO with the cross-linking solution is performed by at least one of: dripping, spraying, emulsifying, and dispersing in oil with subsequent cooling. According to an exemplary embodiment, mixing the hydrocolloid composition comprising the BALO with the cross-linking solution is performed by dripping.

[0059] According to further embodiments, the polysaccharide hydrocolloid used to prepare the delivery system is selected from the group consisting of: kappa-carrageenan, pectin, alginate, agar, gellan gum, carboxymethylcellulose (CMC), furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan and derivatives and combinations thereof. According to an exemplary embodiment, the polysaccharide hydrocolloid is kappa-carrageenan. According to another exemplary embodiment, the pectin is low methoxyl pectin (LMP).

[0060] According to yet further embodiments, the polysaccharide hydrocolloid used to prepare the delivery system is present in the hydrocolloid solution at a weight percentage of about 0.25% to about 10% of the total weight of the hydrocolloid solution. According to yet further embodiments, the polysaccharide hydrocolloid is present in the hydrocolloid solution at a weight percentage of about 1% to about 5% of the total weight of the hydrocolloid solution. According to one embodiment, the polysaccharide hydrocolloid is present in the hydrocolloid solution at a weight percentage of about 1% to about 3% of the total weight of the hydrocolloid solution.

[0061] According to still further embodiments, the volume ratio between the BALO suspension and the hydrocolloid solution is about 1:3 to about 1:20, preferably about 1:4 to about 1:9.

[0062] According to some embodiments, the hydrocolloid composition for preparing the delivery system further comprises a protein hydrocolloid and / or a protein. According to further embodiments, the protein hydrocolloid or protein is selected from the group consisting of gelatin, collagen, collagen hydrolysate, albumin, and any combination thereof. According to still further embodiments, gelatin is present in the hydrocolloid solution at a weight percentage of about 0.25% to about 15% of the total weight of the hydrocolloid solution. According to still further embodiments, gelatin is present at a weight percentage of about 1% to about 10% of the total weight of the hydrocolloid solution. According to a certain embodiment, gelatin is present at a weight percentage of about 5% of the total weight of the hydrocolloid solution.

[0063] According to some embodiments, the hydrocolloid composition for preparing the delivery system further comprises a water absorbing agent. According to further embodiments, the water absorbing agent is selected from the group consisting of starch, polyvinylpyrrolidone (PVP) and derivatives and combinations thereof. According to still further embodiments, the water absorbing agent is present in the hydrocolloid solution in an amount of about 0.25% to about 15% by weight of the total weight of the hydrocolloid solution.

[0064] According to some embodiments, the hydrocolloid composition used to prepare the delivery system further comprises at least one osmoprotectant.

[0065] According to further embodiments, the at least one osmoprotectant is selected from the group consisting of trehalose, inositol, glycerol, arabinitol, betaine, amino acids, and combinations thereof. According to still further embodiments, the osmoprotectant is present in the hydrocolloid solution in an amount of about 0.25% to about 15% by weight, alternatively about 1% to about 10% by weight, based on the total weight of the hydrocolloid solution. According to one embodiment, the osmoprotectant is present in the hydrocolloid composition in an amount of about 5% by weight, based on the total weight of the hydrocolloid solution.

[0066] According to some embodiments, the cross-linking solution comprises metal cations and / or a gelation inducing agent.

[0067] According to further embodiments, the cross-linking solution and the hydrocolloid composition are mixed in a volume ratio of about 10:1 to about 500:1. According to a certain embodiment, the cross-linking solution and the hydrocolloid composition are mixed in a volume ratio of about 100:1.

[0068] According to yet further embodiments, the metal cations present in the cross-linking solution useful for preparing the system are selected from the group consisting of: potassium (K + ), calcium (Ca 2+ ), barium (Ba2+ ), magnesium (Mg 2+ ), aluminum (Al 3+ )、Zn 2 + ), lead (Pb 2+ ), iron (Fe 2+ ), Sr 2+ ), copper (Cu 2+ ), cadmium (Cd 2+ ), nickel (Ni 2+ ), cobalt (Co 2+ ) and combinations thereof. According to an exemplary embodiment, when the polysaccharide hydrocolloid is κ-carrageenan, the crosslinking solution comprises potassium ions as metal cations. According to another exemplary embodiment, when the polysaccharide hydrocolloid is LMP, the crosslinking solution comprises calcium ions as metal cations.

[0069] According to still further embodiments, the cross-linking solution comprises the metal cation in an amount of about 0.1% to about 5% by weight based on the total weight of the cross-linking solution.

[0070] According to some embodiments, the drying is performed at a temperature of about 10°C to 60°C and / or at a pressure of 10 mbar to 1000 mbar. According to an exemplary embodiment, the drying is performed at a temperature of about 40°C at a pressure of about 400 mbar.

[0071] According to some embodiments, after the drying step, the system comprises at least about 1.0×10 8 PFU / g carrier of BALO.

[0072] Further embodiments and the full scope of applicability of the present invention will become clear from the detailed description given below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, they are given by way of illustration only, because various changes and modifications within the spirit and scope of the present invention will become clear to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figures 1A-1F Before immersion in excess DW ( Figure 1A ) and after immersion in excess DW for 2.5 min, 5 min, 7.5 min, 10 min and 12.5 min (respectively Figures 1B-1F) Digital image of a dried κ-carrageenan carrier (350 μm in diameter) acquired under a binocular (model 5ZX16, Olympus America Inc., Center Valley, PA, USA). Scale bar = 200 μm.

[0075] Figure 2 A histogram showing the concentration of potassium released over time from dried kappa-carrageenan carriers immersed in DW is presented. n = 6. (Student's t-test, P < 0.05).

[0076] Figure 3A-3B The effects of predator cell type and added osmoprotectant on encapsulation efficiency are shown ( Figure 3A ) and the survival of predators after vacuum drying ( Figure 3B ). The results represent the mean of two independent experiments with three technical replicates for each treatment. n=5. (Student's t-test, P<0.05).

[0077] Figures 4A1-4D4 SEM micrographs are shown. Figures 4A1-4A4 : κ-carrageenan carrier. Figure 4B1-4B4 : κ-carrageenan carrier + bee plastid. Figure 4C1-4C4 : Kappa-carrageenan carrier with trehalose + bee plasmids. Figure 4D1-4D4 : Kappa-carrageenan carrier + bedding plasmids with glycerol. In each column: Figures 4A1-4D1 : Complete vector; Figure 4A2-4D2 : Close-up of the outside of the carrier; Figure 4A3-4D3 : A cut-away carrier showing the inner part; 4A4-4D4: Close-ups at the inner side of the carrier.

[0078] Figure 5A-5B Growth and predation kinetics of Bdellovibrio virulentus HD100-Td-tomato encapsulated in released dry κ-carrageenan carriers on E. coli ML35 are depicted. Prey populations were tracked by optical density (OD) ( Figure 5A ), and predatory populations were tracked by fluorescence (relative fluorescence units, RFU) ( Figure 5B ). Two independent experiments with three technical replicates were performed for each treatment.

[0079] Figure 6A-6BGrowth and predation dynamics of Bdellovibrio spp. HD100-Td-tomato encapsulated in dry κ-carrageenan carriers released on P. carotovorum subsp. carotovorum WPP14 are depicted. Prey populations were tracked by optical density (OD) ( Fig. 6A ), and predatory populations were tracked by fluorescence (relative fluorescence units, RFU) ( Figure 6B ). Two independent experiments with three technical replicates were performed for each treatment.

[0080] Figure 7A-7B Growth and predation dynamics of released Bdellovibrio spp. encapsulated in dry κ-carrageenan carriers on Pectobacterium carotovorum subsp. brasiliense are depicted. Prey populations were tracked by optical density (OD) ( Fig. 7A ), and predatory populations were tracked by fluorescence (relative fluorescence units, RFU) ( Figure 7B ). Two independent experiments with three technical replicates were performed for each treatment.

[0081] Figures 8A-8B Growth and predation kinetics of released Bdellovibrio spp. encapsulated in dry κ-carrageenan carriers on B. solanacearum are depicted. Prey populations were tracked by optical density (OD) ( Fig. 8A ), and predatory populations were tracked by fluorescence (relative fluorescence units, RFU) ( Figure 8B ). Two independent experiments with three technical replicates were performed for each treatment.

[0082] Fig. 9 The effect of carrier preparation conditions on the survival of encapsulated bdelloids with or without trehalose is shown. Results represent the mean of two independent experiments with three technical replicates for each treatment. n=5. (Student's t-test, P<0.05).

[0083] Figures 10A-10F SEM (AD) and high-resolution SEM (EF) micrographs of κ-carrageenan-trehalose carriers encapsulating predators prepared with 0.5% KCl. Fig. 10A ), a close-up of the outside of the carrier ( Fig. 10B ), the inner part of the carrier ( Fig. 10C ), a close-up of the inside of the carrier ( Fig. 10D), bungoplasts and AP cells encapsulated in the vector (marked by black and white arrows, respectively) ( Fig.10E ), and AP cells encapsulated in the vector ( Fig.10F ).

[0084] Figures 11A-11C The effect of encapsulated Bdellovibrio bacteriovorus HD100 in a κ-carrageenan-trehalose carrier applied 60 minutes before the application of Pectobacterium carotovora subsp. brasiliensis (Pcb) on the development of soft rot in potato slices is depicted: Maceration area (cm 2 )( Fig.11A ), the number of diseased sections ( Fig. 11B ), Photograph of potato slice with macerated tissue revealed as dark patches ( Fig. 11C ). Error bars represent the standard error of 18 replicates. (Student's t-test, P < 0.05).

[0085] Figures 12A-12C The effect of the κ-carrageenan-trehalose carrier (without predators) applied 60 minutes before the application of Pectobacterium carotovora subsp. brasiliensis (Pcb) on the development of soft rot in potato slices is depicted: Maceration area (cm 2 )( Fig. 12A ), the number of diseased sections ( Fig. 12B ), Photograph of potato slice with macerated tissue revealed as dark patches ( Fig. 12C ). Error bars represent the standard error of 18 replicates. (Student's t-test, P < 0.05).

[0086] Figures 13A-13B Pseudomonas fluorescens ( Fig.13A ) and Escherichia coli ZK2686 ( Fig. 13B ) of biofilm disruption. The results are the mean ± SD of 6 replicates from two different batches. (Student's t-test, P < 0.05).

[0087] Figures 14A-14C The results of the biosynthesis of Pectobacterium carotovora subsp. brasiliensis (Pc1692, Pc3; respectively) on three different LMP solutions (106S-YA, 104AS, and 5CS) as the sole carbon source are presented. Fig.14A and 14B ) and Pectobacterium carotovora subsp. carotovora (WPP14; Fig. 14C ) were used to analyze the growth curves of the cells. The results are the mean ± SD of 10 replicates from two different batches.

[0088] Figures 15A-15C The results of the biosynthesis of Pectobacterium carotovora subsp. brasiliensis (Pc1692, Pc3; respectively) on three different LMP carriers (106S-YA, 104AS, and 5CS) as the sole carbon source are presented. Fig.15A and 15B ) and Pectobacterium carotovora subsp. carotovora (WPP14; Fig. 15C ) were used to analyze the growth curves of the cells. The results are the mean ± SD of 10 replicates from two different batches.

[0089] Figures 16A-16C Depicted is the growth of Pectobacterium carotovora subsp. brasiliensis (Pc1692, Pc3; respectively) after exposure to LMP solutions (106S-YA, 104AS, and 5CS) as a single carbon source. Fig.16A and 16B ) and Pectobacterium carotovora subsp. carotovora (WPP14; Fig. 16C ) were used to measure the activity of extracellular PCWDE (Pel, Peh and Prt) secreted by 10 different batches of cells. The results are the mean ± SD of 10 replicates from two different batches. (Student's t-test, P < 0.05).

[0090] Figures 17A-17D The attraction of GFP-tagged Pcb to different LMP 5CS, 104AS and 106S-YA vectors is depicted. Fig.17A ). GFP-tagged Pcb is directed toward LMP 5CS ( Fig. 17B )、104AS( Fig. 17C ) or 106S-YA( Fig.17D ) Fluorescence binocular image after 48 hours. Scale bar = 1 cm.

[0091] Figures 18A-18F Degradation of different LMP vectors by Pectobacterium carotovora subsp. brasiliensis (Pcb) is depicted. Representative digital images (x2 magnification) of the size and appearance of the vectors were obtained under a binocular (model 5ZX16, Olympus America Inc., Center Valley, PA, USA): 5CS ( Fig.18A , 18D )、104AS( Fig.18B , 18E )、106AS-YA( Fig.18C , 18F ); incubated in DDNB for 72 h without Pcb ( Figures 18A-18C ) or with Pcb( Figures 18D-18F ). Changes in weight percentage of carrier in DDNB without or with Pcb ( Figure 18G ). The results are the mean ± SD of 6 replicates from two different batches. (Student's t-test, P < 0.05).

[0092] Figures 19A-19E Degradation of 5CS vectors by Pectobacterium carotovora subsp. brasiliensis (Pcb) is depicted. Representative digital images (x2 magnification) of vectors of different sizes in DDNB were acquired under a binocular (model 5ZX16, Olympus America Inc., Center Valley, PA, USA) for 72 hours: 3 mm ( Figures 19A-19B ), 2mm( Figures 19C-19D ), no PCB( Fig.19A , Fig.19C ) or with Pcb( Fig.19B , Fig.19D ). Changes in weight percentage of carrier in DDNB without or with Pcb ( Fig.19E ). The results are the mean ± SD of 6 replicates from two different batches. (Student's t-test, P < 0.05).

[0093] Figures 20A-20G Degradation of 5CS-gelatin carriers by Pectobacterium carotovora subsp. brasiliensis (Pcb) is depicted. The images of 5CS-gelatin carriers were obtained under a binocular (model 5ZX16, Olympus America Inc., Center Valley, PA, USA) in DDNB for 72 h with 0% ( Fig. 20A , 20B )、2%( Fig. 20C , 20D ) and 5%( Fig.20E , 20F Representative digital images (x2 magnification) of carriers with added gelatin and without PCB ( Fig. 20A , 20C , 20E) or with Pcb( Fig. 20B , 20D , 20F). Changes in weight percentage of carrier in DDNB without or with Pcb ( Figure 20G ). Results are mean ± SD of 6 replicates from two different batches. (Student's t-test, P < 0.05). ND, not determined.

[0094] Figures 21A-21DDegradation of dried 5CS-gelatin carriers by Pectobacterium carotovora subsp. brasiliensis (Pcb) is depicted. The dried carriers were acquired under a binocular (model 5ZX16, Olympus America Inc., Center Valley, PA, USA) Fig.21A ), in the absence of PCB( Fig.21B ) or with Pcb( Fig. 21C Representative digital images of dry carriers in DDNB for 72 hours without or with Pcb (x2 magnification). Fig.21D ). Results are mean ± SD of 6 replicates from two different batches. (Student's t-test, P < 0.05). ND, not determined.

[0095] Figures 22A-22F SEM micrograph of an optimized dried carrier comprising LMP and gelatin for encapsulating Bdellovibrio bacteriovorus predators ( Fig.22A -D) and high-resolution SEM ( Fig.22E , 22F ): Complete vector ( Fig.22A ); the outer surface of the carrier ( Fig. 22B ); a vector cut in half, showing the thin shell wall and empty core ( Fig. 22C ); the inner surface of the carrier ( Fig.22D ); AP cells and bead plasmids embedded on the surface of the carrier ( Fig.22E , 22F ). The black and white arrows point to bdelloids and AP cells, respectively.

[0096] Figures 23A-23B The growth and predation efficiency of encapsulated Bdellovibrio bacteriovorus HD100-Td-tomato on Pectobacterium carotovora subsp. brasiliensis (Pcb) prey in optimized dry carriers containing LMP and gelatin are depicted, with 1, 2 or 3 carriers / well. The optical density (OD, 600nm) of the prey population ( Fig.23A ). Td-tomato fluorescence of predator populations (Relative fluorescence units, RFU) ( Fig. 23B ). Two independent experiments were performed for each treatment, each with three technical replicates.

[0097] Fig.24 Depicted are long-term survival of Bdellovibrio bacteriovorus HD100 in carrageenan-trehalose and pectin-gelatin based carriers and in liquid suspension at different temperatures. Results represent the mean of two independent experiments with three technical replicates for each treatment.

[0098] Figures 25A-25DThe protection of potato tubers against Pectobacterium brasiliense by a hydrated kappa-carrageenan-trehalose-based Bdellovibrio formulation under controlled conditions is depicted. Tubers were soaked in a suspension of Pectobacterium brasiliense and treated with predators as follows: control (water only); Pb1692 inoculation followed by sterile tap water (Pecto only); pathogen inoculation and application of predators (either as freshly grown AP cells or as encapsulated predators released from carriers). Fig.25A The percentage of tuber decay (by weight) is depicted. Fig.25B Disease index is depicted. Fig.25C Photographs of potato tubers in planting trays after Pbl692 inoculation, predator treatment and before covering with planting soil. Fig.25D The number of infected potatoes is depicted. Results represent the mean ± SD of eight independent experiments with 15 replicates for each treatment [[IS - treatments not associated by the same letter are significantly different - DELETE]] (Student's t-test, P < 0.05).

[0099] Figures 26A-26E The protection of potato tubers against Pectobacterium brasiliensis by a hydrated kappa-carrageenan-trehalose based Bdellovibrio formulation was shown for potato tubers in pots under natural net-house conditions. Fig.26A The photographs are of potted plants after inoculation with Pb1692 in a net house. Fig.26B Depicted are the number of infected potatoes at the end of a 4-week period with the following treatments: control (water only); Pb1692 inoculation followed by sterile tap water (Pecto only); pathogen inoculation and application of predators (either as freshly grown AP cells or as predators released from carriers). Fig.26C Photographs of potato tubers cut open at the end of the experiment. Fig.26D The percentage of tuber decay (by weight) is depicted. Fig.26E Disease index is depicted. Results represent mean±SD of two independent experiments with 32 replicates for each treatment [[IS—treatments not associated by the same letter are significantly different—DELETE]] (Student's t-test, P<0.05). DETAILED DESCRIPTION OF THE INVENTION

[0101] The present invention provides a system for differential delivery of live Bdellovibrio and similar organisms (BALOs), comprising: a carrier comprising a polysaccharide hydrocolloid; and BALOs immobilized in the carrier, wherein the BALOs are in the physiological state of AP cells, leech cysts, and leech plastids, which are at least 1.0×108 The invention relates to a method for preparing a delivery system comprising: providing a polysaccharide hydrocolloid having a cell density of 100 PFU / g carrier, wherein the polysaccharide hydrocolloid is in the form of a wet or dry gel, and wherein the carrier preserves the viability and lytic potency of the immobilized BALO. The invention further provides uses of the delivery system and methods for preparing the same.

[0102] Delivery System

[0103] The terms "system for differential delivery of live Bdellovibrio and similar organisms (BALOs)", "delivery system", "system" and "delivery system for BALOs" are used interchangeably herein and refer to the system of the present invention.

[0104] As used herein, the term "Bdellovibrio and similar organisms (BALOs)" are Gram-negative, small, obligate predators of other Gram-negative bacteria. These bacteria interact with their prey as highly motile attack phase (AP) cells, where they attach to the outer membrane and consume the prey extracellularly (epizootic predation) or penetrate their periplasm (periplasmic predation). The former divide in a binary fashion, while the latter grow as polynucleotide filaments to eventually divide into daughter attack cells. Most BALO isolates, including Bdellovibrio bacterivorans, are periplasmic predators. According to certain embodiments, BALOs include or consist of Bdellovibrio bacterivorans, Bdellovibrio stolpii, and Bdellovibrio starrii.

[0105] As used herein, the term "hydrocolloid" refers to a water-soluble polymer of plant, animal, microbial or synthetic origin, which typically contains a plurality of hydroxyl, carboxyl, amine and / or amide groups.

[0106] The terms "polysaccharide hydrocolloid" and "protein hydrocolloid" used in this article refer to polysaccharides or proteins that can thicken aqueous solutions by increasing viscosity values ​​or forming gels in aqueous media. Gel formation involves the association or crosslinking of polymer chains to form a three-dimensional network, which captures or immobilizes water within it to form a rigid structure that resists flow. Gel formation can be spontaneous, or is induced by using a suitable crosslinking agent or when combined with other hydrocolloids (under determined conditions and concentrations). For example, the combination of xanthan gum and locust bean gum (LBG) (all non-gelling hydrocolloids) causes the formation of a firm gel.

[0107] The term "bdelloplast" as used herein refers to a modified Gram-negative cell formed in a host of parasitic bacteriovorous Bdellovibrio bacteria. Bdelloplasts are generated by penetrating the periplasm of Gram-negative bacteria (which constitute the main prey for BALO). After prey invasion, the prey peptidoglycan is modified, thereby producing bdelloplasts. Acylation of the prey peptidoglycan by long-chain fatty acids and solubilization of 25% of lipopolysaccharide (LPS) glucosamine by lipopolysaccharidase activity increase the hydrophobicity of the bdelloplasts and stabilize the outer membrane, which now serves as a permeation barrier. Therefore, bdelloplasts can provide enhanced protection compared to naked AP cells during dehydration of the carrier (wherein the osmotic pressure is expected to increase).

[0108] As used herein, the term "Bdellovibrio sp." refers to bacterial cells of the genus Bdellovibrio sp. that infect prey and produce resting cells therein called Bdellovibrio cysts. Bdellovibrio cysts typically contain more deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins, and carbohydrates than attack-stage cells. Bdellovibrio cysts are more resistant to the effects of elevated temperatures, sonication, and drying than attack-stage cells.

[0109] As used herein, the term "immobilized" refers to bacterial cells that are entrapped, embedded and / or encapsulated within a polysaccharide hydrocolloid and released therefrom by disintegration, dissolution and / or degradation of the hydrocolloid.

[0110] According to some embodiments, the immobilized BALO, such as bdelloid, is present at a density of at least 1.0×10 8 According to a further embodiment, the immobilized BALO, such as bdelloid, is present at a cell density of at least 1.0×10 9 According to a further embodiment, the immobilized BALO, such as bdelloid, is present at a cell density of at least 1.0×10 10 The cell density was present at PFU / g vector.

[0111] As used herein, the term "wet form" refers to a gel formed from a polysaccharide hydrocolloid comprising at least 75 wt% water based on the total weight of the hydrocolloid or gel. According to a further embodiment, the term "wet form" refers to a carrier prepared from an aqueous solution that has not been subjected to drying.

[0112] As used herein, the term "dried gel form" refers to a gel that has been formed from a polysaccharide hydrocolloid and then dried such that it contains less than about 10 wt % water based on the total weight of the dried gel.

[0113] As used herein, the term "preservation viability" means a retention of at least about 1.0×10 8PFU / g carrier of live BALO immobilized within the carrier. In some embodiments, the term "preserving viability" refers to bdelloid plastids and / or AP cells that remain viable after release from the carrier.

[0114] Quantitative measurement of cell viability can be performed by dissolving the gel carrier or dry carrier in sterile sodium hexametaphosphate (SHMP) or sterile DW to release the encapsulated Bdellovibrio cells. PFU (which represents viable cells) can be counted by dilution plating in double agar.

[0115] The percentage of live BALO cells immobilized in the carrier was expressed as the encapsulation efficiency (EE) index and calculated according to the following equation:

[0116] EE(%)=(N / N0)×100Equation I

[0117] where EE is the encapsulation efficiency expressed as a percentage, N is the number of cells released from the carrier (CFU / g carrier), and N0 is the number of cells in the hydrocolloid solution (CFU / g suspension).

[0118] As used herein, the term "preserved lytic potency" refers to a BALO that is immobilized in a carrier, released from the carrier, and retains at least 10%, or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%, or 100% of the lytic activity of the BALO prior to immobilization. In further embodiments, the term "preserved lytic potency" refers to a BALO that retains lytic potency after release from a dried carrier. As long as the viability of the BALO is preserved, the lytic potency of the BALO is preserved.

[0119] The bacteriolytic efficacy of BALOs after encapsulation in carriers was examined by using a predation kinetics assay. Predation of soft rot bacteria by encapsulated BALOs in dry carriers was followed by fluorescence (predator growth) and OD (prey decay) in a plate reader incubated at 28°C.

[0120] According to some embodiments, the carrier is present as granules, beads, capsules, granules, powder and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0121] The Feret diameter of the carrier is measured along its longest chord. According to some embodiments, the carrier has an average Feret diameter of about 0.01 mm to about 10 mm. In further embodiments, the carrier has an average Feret diameter of about 0.1 mm to about 5 mm. In further embodiments, the carrier has an average Feret diameter of about 2 mm to about 4 mm.

[0122] According to some currently preferred embodiments, the polysaccharide hydrocolloid is in a dried gel form. In such embodiments, the system is referred to as a "dry system" or "dry system". Without wishing to be bound by any theory or mechanism of action, it is considered that the dried gel form of the hydrocolloid-based carrier provides an extended shelf life and stability of the encapsulated BALO, compared to the wet gel form. Further, it has been surprisingly found that the dried gel carrier provides a faster release of the BALO after contact with the aqueous phase compared to the wet gel carrier. The inventors of the present invention have further shown that the dried hydrocolloid-based carrier is highly effective for the encapsulation and rapid release of the bacterial predator Bdellovibrio bacteriovorus, thereby keeping the cells alive and preventing bacterial infection in plant tissues. It has been further unexpectedly found that the bacteriolytic efficiency of the bletillar plasmids released from the dried gel carrier is higher than the bacteriolytic efficiency of the released AP cells. Therefore, the combination of the dried gel carrier and the bletillar plasmid form of Bdellovibrio bacteriovorus is the most effective delivery system for BALO.

[0123] The polysaccharide hydrocolloids may be naturally occurring or semi-synthetic polysaccharide hydrocolloids (e.g., chemically modified). The naturally occurring polysaccharide hydrocolloids may be obtained or extracted from various natural sources, such as plant, microbial or animal sources. Polysaccharide hydrocolloids obtained / extracted from natural sources include, but are not limited to, kappa-carrageenan, pectin, alginates, agar, gellan gum, furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan, and derivatives and combinations thereof. Semi-synthetic polysaccharide hydrocolloids include, but are not limited to, carboxymethyl cellulose and other cellulose ethers, hydroxyethyl and propyl cellulose.

[0124] The type of hydrocolloid can be selected based on the intended use of the delivery system of the BALO. For example, if the delivery system is to be used in the food industry, naturally occurring hydrocolloids should be used, which are food grade materials. Further, the type of hydrocolloid can be selected based on the desired release profile of the immobilized BALO, as described in detail below.

[0125] Non-limiting examples of suitable polysaccharide hydrocolloids that can be used in the delivery system include: kappa-carrageenan, pectin, alginate, agar, gellan gum, carboxymethylcellulose (CMC), furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan, and derivatives and combinations thereof.

[0126] As used herein, the term "derivative" includes a salt, amide, ester, enol ether, enol ester, acetal, ketal, orthoester, hemiacetal, hemiketal, acid, base, solvate, hydrate or prodrug of a hydrocolloid.

[0127] In some exemplary embodiments, the carrier comprises kappa-carrageenan.

[0128] Carrageenan is a component of the intercellular matrix in many species of red seaweed. Chemically, the polymer is a linear, anionic, sulfated galactan composed of repeating galactose units and 3,6-anhydrogalactose (both sulfated and non-sulfated) linked by alternating α-(1-3)- and β-(1-4)-glycosidic bonds (Nussinovitch & Hirashima, 2013). Three major types of carrageenan have been identified: iota, kappa, and lambda, depending on the number and position of sulfate groups. Both kappa- and iota-carrageenan dispersions were stabilized upon cooling and addition of counterions such as K. + and Ca 2+ After undergoing a sol-gel transition (Thrimawithana, Young, Dunstan, & Alany, 2010). Carrageenan is used in a variety of commercial applications, especially in foods and sauces, for its gelling, thickening and stabilizing abilities (Necas & Bartosikova, 2013).

[0129] In some exemplary embodiments, the carrier comprises pectin.

[0130] Pectin is an anionically charged structural plant polysaccharide consisting of a linear backbone of α-(1-4)-D-galacturonic acid residues that may be partially methyl-esterified or amidated. Pectin is therefore characterized by its degree of esterification (DE) as well as its degree of amidation (DA). Low methoxylated pectin (also known as low methoxy pectin, LMP) (DE < 50%) (whether amidated or not) can form gels in the presence of divalent cations. Pectin has been widely used as a delivery vehicle for colon-specific oral drugs and, to a lesser extent, probiotics (low methoxy pectin and rice bran).

[0131] According to some embodiments, the pectin is low methoxyl pectin (LMP).

[0132] According to some embodiments, the carrier comprises LMP having a degree of esterification of about 5% to 30% and a degree of amidation of about 0% to 25%. According to further embodiments, the carrier comprises LMP having a degree of esterification of about 5% to 10% and a degree of amidation of about 0% to 5%. In certain embodiments, the carrier comprises non-amidated LMP.

[0133] According to some embodiments, the polysaccharide hydrocolloid is present in the system in a weight percentage of about 2% to about 25% based on the total weight of the dry system.

[0134] According to some embodiments, the polysaccharide hydrocolloid is present in the system at a weight percentage of about 0.5% to about 25% of the total weight of the wet system. In further embodiments, the hydrocolloid is present in the system at a weight percentage of about 0.5% to about 5% of the total weight of the wet system. In still further embodiments, the hydrocolloid is present in the system at a weight percentage of about 1% to about 3% of the total weight of the wet system.

[0135] According to some embodiments, the polysaccharide hydrocolloid is crosslinked by a crosslinking agent and / or in combination with a gelling inducing agent. The most common type of hydrocolloid crosslinking agent is a metal ion, particularly a monovalent or divalent metal ion. In some embodiments, the crosslinking agent comprises a metal cation selected from the group consisting of: calcium (Ca 2+ ), barium (Ba 2 + ), magnesium (Mg 2+ ), aluminum (Al 3+ ), potassium (K + )、Zn 2+ ), lead (Pb 2+ ), ferrous iron (Fe 2+ ), Sr 2+ ), copper (Cu 2+ ), cadmium (Cd 2 + ), nickel (Ni 2+ ), cobalt (Co 2+ ) and combinations thereof. Each possibility represents a separate embodiment of the present invention. In some exemplary embodiments, the carrier comprising kappa-carrageenan is loaded with potassium ions (K + In a further exemplary embodiment, the carrier comprising LMP is cross-linked by calcium ions (Ca 2+ ) for cross-linking.

[0136] In some embodiments, gelation inducing agents include polycationic hydrocolloids and polycationic agents such as LBG, genipin, chitosan, polylysine, polyarginine, polyethyleneimine (PEI), mannosylated PEI, and derivatives and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0137] According to some embodiments, the carrier further comprises a water absorbing agent. The term "water absorbing agent" as used herein refers to an agent configured to enhance the absorption of water by the carrier.

[0138] In some embodiments, the carrier comprises pectin as a polysaccharide hydrocolloid. In some embodiments, the carrier further comprises gelatin as a protein hydrocolloid. Without wishing to be bound by any theory or mechanism of action, it is considered that depending on pH, ionic strength and pectin type, gelatin forms ionic interactions with pectin, thus helping to overcome the stability of the reduction when using low concentrations of pectin in the carrier.

[0139] According to some embodiments, gelatin is present in the system in a weight percentage of about 20% to about 85% of the total weight of the dry system.

[0140] In further embodiments, gelatin is present in the system at a weight percentage of about 20% to about 50% of the total weight of the dry system. In still further embodiments, gelatin is present in the system at a weight percentage of about 40% to about 50% of the total weight of the dry system.

[0141] According to some embodiments, gelatin is present in the system in a weight percentage of about 0.25% to about 15% based on the total weight of the wet system.

[0142] According to some embodiments, the system and / or the carrier does not comprise gelatin as the sole hydrocolloid component. In further embodiments, the system and / or the carrier does not comprise gelatin.

[0143] The dry pectin carrier has a compact structure that hinders the activity of soluble pectin, which can be overcome by adding gelatin or water absorbing agent to increase swelling. In some embodiments, the water absorbing agent is the hydrocolloid selected from the group consisting of the following: starch, polyvinyl pyrrolidone (PVP), polyacrylic acid and polymethacrylic acid compounds, vinyl polymers, polycarboxylic acids, polyethers, polyimines, polyamides and derivatives and combinations thereof. Every kind of possibility represents a separate embodiment of the present invention.

[0144] According to some embodiments, the water absorbent is present in the system in a weight percentage of about 25% to about 85% based on the total weight of the dry system.

[0145] In further embodiments, the water absorbent is present in the system at a weight percentage of about 35% to about 80% of the total weight of the dry system. In still further embodiments, the water absorbent is present in the system at a weight percentage of about 45% to about 75% of the total weight of the dry system.

[0146] According to some embodiments, the moisture absorbent is present in the system in a weight percentage of about 0.25% to about 15% based on the total weight of the wet system.

[0147] According to some embodiments, the carrier further comprises at least one osmotic protectant. The term "osmotic protectant" as used herein refers to a compound that acts as an osmotic agent and helps the predator bacteria described in this article survive under osmotic stress. The osmotic protectant can be added to the carrier composition before drying to reduce / eliminate the stress induced by salt and improve the survival of encapsulated bacteria during dehydration. Osmotic protectants include various compound classes, such as sugars, amino acids, polyols and isoglycosides.

[0148] The inventors of the present invention have surprisingly found that the higher the molecular weight of the added osmoprotectant, the higher the entrapment efficiency of BALO within the carrier.

[0149] According to some embodiments, the molecular weight of the osmoprotectant is at least 50 g / mole. In further embodiments, the molecular weight of the osmoprotectant is at least 200 g / mole. In still further embodiments, the molecular weight of the osmoprotectant is at least 300 g / mole.

[0150] According to some embodiments, the at least one osmotic protectant is selected from the group consisting of trehalose, inositol, glycerol, arabinitol, betaine, mannitol, sucrose, amino acids and combinations thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the at least one osmotic protectant is trehalose and / or glycerol. In certain embodiments, the at least one osmotic protectant is trehalose.

[0151] According to some embodiments, the at least one osmotic protectant is present in the system at a weight percentage of about 20% to about 85% of the total weight of the dry system. According to alternative embodiments, the at least one osmotic protectant is present in the system at a weight percentage of about 10% to about 90% of the total weight of the dry system.

[0152] According to some embodiments, the at least one osmoprotectant is present in the system in a weight percentage of about 0.25% to about 15% based on the total weight of the wet system.

[0153] According to some embodiments, the system comprises up to about 0.1% (w / w) water, based on the total weight of the dry system. According to further embodiments, the system comprises up to about 0.5% (w / w) water, based on the total weight of the dry system. According to still further embodiments, the system comprises up to about 2.5% (w / w) water, based on the total weight of the dry system.

[0154] According to some embodiments, the system comprises about 0.1% (w / w) to about 10% (w / w) water, based on the total weight of the dry system. In further embodiments, the system comprises about 0.2% (w / w) to about 5% (w / w) water, based on the total weight of the dry system. In still further embodiments, the system comprises about 0.5% (w / w) to about 2.5% (w / w) water, based on the total weight of the dry system.

[0155] According to some embodiments, the system comprises about 75% (w / w) to about 99% (w / w) water based on the total weight of the wet system.

[0156] As mentioned above, dry hydrocolloid-based carriers are highly effective in BALO encapsulation, thereby preserving the viability and lytic potency of BALO. BALO is a bacterial predator that has a mandatory requirement for Gram-negative prey for its growth and replication. The release of BALO (e.g., bdelloid plasmid) from the carrier activates this mandatory requirement for prey in order for it to survive outside the carrier. Controlling the predatory ability of BALO by differential release of immobilized BALO (e.g., bdelloid plasmid) from the carrier allows for efficient predation.

[0157] The system of the present invention is configured to release the immobilized live BALOs (eg, bead plasmids) upon disintegration of the carrier.

[0158] According to some embodiments, the differential delivery of BALOs (e.g., benguelosomes) includes rapid release or delayed release of the live BALOs (e.g., benguelosomes) from the carrier. Differential delivery systems with rapid predator release profiles are suitable for preventive antimicrobial treatments, while systems with delayed predator release can be beneficially used in targeted treatments. According to some embodiments, the carrier composition is customized according to the desired release profile of the BALOs (e.g., benguelosomes) from the carrier.

[0159] According to some embodiments, a carrier composition suitable for rapid release of BALOs comprises kappa-carrageenan and potassium cations.

[0160] According to some embodiments, the carrier comprising kappa-carrageenan is configured to undergo swelling and dissolution / disintegration upon contact with an aqueous solution, thereby releasing the BALO (e.g., leech plastids). According to certain embodiments, the aqueous solution is water / DW / DDW. Without wishing to be bound by any theory or mechanism of action, it is considered that the swelling and disintegration behavior of the dried kappa-carrageenan-based carrier is due to the loss of potassium ions (which are the main cross-linking agent), and / or due to the exchange of these cations with other monovalent cations present when the delivery system is applied. Without further wishing to be bound by any theory or mechanism of action, the swelling and disintegration behavior depends on the external volume of the aqueous solution, which affects the potassium concentration gradient during diffusion, and on the carrier size, which determines the surface area from which potassium ions can escape.

[0161] According to some embodiments, disintegration of the carrier comprising kappa-carrageenan occurs from about 1 minute to about 48 hours after contact with an aqueous solution. This rapid disintegration may be particularly useful in aquaculture. In further embodiments, disintegration of the carrier comprising kappa-carrageenan occurs from about 1 minute to about 12 hours after contact with an aqueous solution. In still further embodiments, disintegration of the carrier comprising kappa-carrageenan occurs from about 1 minute to about 3 hours after contact with an aqueous solution.

[0162] According to some embodiments, the carrier comprises kappa-carrageenan and trehalose. According to some embodiments, kappa-carrageenan is present in the system at a weight percentage of about 5% to about 25% of the total weight of the dry system, and trehalose is present in the system at a weight percentage of about 25% to about 85% of the total weight of the dry system.

[0163] In further embodiments, kappa-carrageenan is present in the system at a weight percent of about 5% to about 25% based on the total weight of the dry system, and trehalose is present in the system at a weight percent of about 25% to about 80% based on the total weight of the dry system. In still further embodiments, kappa-carrageenan is present in the system at a weight percent of about 15% to about 25% based on the total weight of the dry system, and trehalose is present in the system at a weight percent of about 70% to about 80% based on the total weight of the dry system.

[0164] According to some embodiments, kappa-carrageenan is present in the system at a weight percent of about 0.25% to about 10% based on the total weight of the wet system, and trehalose is present in the system at a weight percent of about 0.25% to about 15% based on the total weight of the wet system.

[0165] According to some embodiments, the carrier comprises kappa-carrageenan and glycerol. According to some embodiments, kappa-carrageenan is present in the system at a weight percentage of about 5% to about 25% of the total weight of the dry system, and glycerol is present in the system at a weight percentage of about 25% to about 85% of the total weight of the dry system.

[0166] In further embodiments, kappa-carrageenan is present in the system at a weight percent of about 15% to about 25% based on the total weight of the dry system, and glycerin is present in the system at a weight percent of about 45% to about 80% based on the total weight of the dry system. In still further embodiments, kappa-carrageenan is present in the system at a weight percent of about 15% to about 25% based on the total weight of the dry system, and glycerin is present in the system at a weight percent of about 50% to about 80% based on the total weight of the dry system.

[0167] According to some embodiments, kappa-carrageenan is present in the system at a weight percent of about 0.25% to about 10% based on the total weight of the wet system, and glycerol is present in the system at a weight percent of about 0.25% to about 15% based on the total weight of the wet system.

[0168] According to some embodiments, a carrier composition suitable for delayed release of BALOs (eg, leech plasmids) comprises LMP, gelatin or a water absorbent, and calcium cations.

[0169] According to some embodiments, LMP is present in the system at a weight percentage of about 2% to about 25% of the total weight of the dry system, and gelatin or a water absorbent is present in the system at a weight percentage of from about 20% to about 85% of the total weight of the dry system. According to further embodiments, LMP is present in the system at a weight percentage of about 2% to about 15% of the total weight of the dry system, and gelatin or a water absorbent is present in the system at a weight percentage of from about 25% to about 80% of the total weight of the dry system.

[0170] According to some embodiments, LMP is present in the system at a weight percent of about 0.25% to about 10% based on the total weight of the wet system, and gelatin or a water absorbent is present in the system at a weight percent of from about 0.25% to about 15% based on the total weight of the wet system.

[0171] According to some embodiments, a carrier composition suitable for delayed release of BALOs (eg, leech plasmids) comprises LMP, gelatin, trehalose, and calcium cations.

[0172] According to further embodiments, LMP is present in the system at a weight percent of about 2% to about 15% of the total weight of the dry system, gelatin is present in the system at a weight percent of about 20% to about 50% of the total weight of the dry system, and trehalose is present in the system at a weight percent of about 20% to about 50% of the total weight of the dry system. According to still further embodiments, LMP is present in the system at a weight percent of about 5% to about 10% of the total weight of the dry system, gelatin is present in the system at a weight percent of about 40% to about 50% of the total weight of the dry system, and trehalose is present in the system at a weight percent of about 40% to about 50% of the total weight of the dry system.

[0173] According to some embodiments, the carrier comprising the LMP is configured to undergo disintegration by plant cell wall degrading enzymes (PCWDE). PCWDE and pectinase are particularly produced by soft rot enterobacterium (SRE), a Gram-negative pathogenic bacteria preyed on by BALO. Without wishing to be bound by any theory or mechanism of action, it is contemplated that the LMP-based immobilization system for BALO in accordance with the principles of the present invention utilizes PCWDE secretion by the pathogen and chemotaxis induced by the pectin residues of the system, resulting in the disintegration of the immobilization system and the release of the encapsulated predator.

[0174] According to some embodiments, enzymatic degradation of the carrier comprising LMP occurs in soil or in any medium within a period of hours to days or weeks after contact with PCWDE. In further embodiments, disintegration of the carrier comprising LMP occurs from about 6 hours to about 84 hours after contact with PCWDE. In still further embodiments, disintegration of the carrier comprising LMP occurs from about 10 hours to about 72 hours, alternatively from about 12 hours to about 36 hours, or about 24 hours after contact with PCWDE.

[0175] According to some embodiments, the complete decomposition of the polymer under actual environmental conditions may take a period of several months. For example, the complete decomposition of the polymer in soil can take a period of 6 to 9 months.

[0176] The system may further comprise additives such as, but not limited to, anti-caking agents, anti-clamping agents, preservatives, antioxidants, binders, hydrophobic components, and resins.

[0177] According to some embodiments, the components of the delivery system, such as polysaccharide hydrocolloids, cross-linking agents, water absorbents and osmotic protectants, and other optional additives, such as anti-caking agents, anti-fastening agents, preservatives, antioxidants, adhesives, hydrophobic components and resins, are GRAS (generally recognized as safe), biodegradable and non-toxic. According to further embodiments, the components of the delivery system are approved by the FDA.

[0178] Uses of the delivery system

[0179] The system of the present invention can be used in agricultural applications to prevent bacterial soft rot infection in plant tissue or in crops. The inventors of the present invention have examined the efficacy of the system by using a potato slice model as well as potato tubers and showed reduced soft rot disease incidence, smaller maceration area and reduced disease index after treatment of SRE infected potato slices or potato tubers with carriers encapsulating BALOs.

[0180] Thus, the system of the invention can be used to treat phytopathogenic bacterial diseases in plants, plant parts or crops either pre-harvest or post-harvest.

[0181] In some embodiments, the plant pathogenic bacterial disease is caused by plant pathogenic bacteria, including but not limited to: Pectobacterium carotovora, Pectobacterium chrysanthemi, Pectobacterium alocasiae, Pectobacterium nigrorotum, Pectobacterium vascularis, Pectobacterium occidentalis, Pectobacterium taro, Pectobacterium cyperi, Pectobacterium melonis, Pectobacterium parmentieri, Pectobacterium polaris, Pectobacterium punjabi, Pectobacterium rhubarb, Pectobacterium polyhedralis, Pectobacterium wasabi, Pectobacterium calla, Xanthomonas campestris, Pseudomonas syringae, Pseudomonas fluorescens, Erwinia amylovora, Ralstonia solanacearum, Acidovorax citrullus, Acidovorax oryzae, Acidovorax avenae, Dickinia solanacearum, Dickinia chrysanthemi, Dickinia dadonii, Dickinia pectinolyticus, and Xylella fastidiosa. Each possibility represents a separate embodiment of the present invention.

[0182] In some embodiments, the crop is selected from the group consisting of: potato (Solanumtuberosum), onion (stem bulb and greens), shallot (Allium fistulosum), shallot, carrot, soybean, barley, buckwheat, millet, oats, rice, rye, quinoa, sugar beet, Ethiopian teff, Mexican maize, triticale, wheat, wild rice, corn, soybean, cotton, borage, buffalo gourd (Cucurbita foetidissima), canola, crambe, flax, jojoba, lesquerella, oilseed rape, safflower, sesame, sunflower, seed sorghum (milo), sugarcane, goat's horn palm, asparagus, bamboo shoots, artichoke, okra, peanut, pineapple, strawberry, multifruit, angelica, star anise, annatto (seed), balsam, basil, burnet, chamomile, caper bud, caraway seeds, black caraway seeds, cardamom, cassia bark, cinnamon buds, camphor grass, celery seeds, chervil (dried), chives, chives, cinnamon trees, salvia, clove buds, cilantro (coriander or coriander), coriander seeds (coriander), tansy, eryngium (leaf), eryngium (seed), cumin, curry (leaf), dill (dill leaves), dill (seed), nepeta, fennel seeds, fenugreek, white ginger flower, cardamom (Aframomum melegueta), Marrubium vulgare, hyssop, juniper berry, lavender, rue, lovage (leaves and seeds), mace, calendula, marjoram (including oregano), Mexican oregano, holly, mustard (seed), nasturtium, nutmeg, parsley, mint, pepper (black and white), pepper leaf, peppermint, basil, poppy (seed), rosemary, rue, saffron, sage, vanilla (summer and winter), spearmint, stevia, magnolia, tansy, tarragon, thyme, vanilla, wintergreen, woodruff, wormwood, broccoli, Chinese broccoli (kale), broccoli raab (rapini), Brussels sprouts, cabbage, Chinese cabbage (pek choy), Chinese cabbage (chinese cabbage), Chinese mustard (mustard), cauliflower, cavalo broccolo, kale, collard greens, kohlrabi, turnips, mustard greens, mustard spinach, rapeseed, garlic, garlic cloves, leeks, chayote, winter melon, citron, cucumber, West Indian cucumber, hyotan, cucuzza, loofah, Chinese okra, melon, balsam pear, balsam melon, bitter melon, Chinese cucumber, Roman melon, Casaba melon, Cranshaw melon, golden pershawmelon, honeydew melon, honeydew melon, citrus melon, Persian melon, pineapple melon, Santa Claus melon, snake melon, pumpkin, crookneck pumpkin, scalloped pumpkin, straightneck pumpkin, zucchini, squash, gourd, winter squash, acorn squash, spaghetti squash, watermelon, amaranth (Chinese spinach), rocket, beet greens, cardoon, celery, Chinese celery, celery lettuce (celtuce), worm amaranth, chervil, leaf daisy, chrysanthemum, watercress (garden and upland), dandelion, sorrel, houttuynia, endive (sonococcus), sweet fennel, gow kee, lettuce (head and leaf), saltbush, parsley, purslane (garden and winter), dadicchio (red endive), rhubarb, spinach, New Zealand spinach, vine spinach, chard, watercress (upland), water spinach, eggplant, Physalis species (Physalis spp), bell peppers, chili peppers, peppers, pimentos, sweet peppers, tomatillos, tomatoes, legumes, grain lupin, sweet lupin, white lupin, kidney beans, navy beans, pinto beans, runner beans, snap beans, broadleaf beans, wax beans, adzuki beans, cowpeas, black-eyed peas, pigeon peas, Chinese long beans, cowpeas, crowder beans pea), cowpea, mung bean, rice bean, southern pea, black mung bean, snap bean, broad bean, chickpea, guar, sword bean, lentil, lentil, dwarf bean, snap pea, English pea, field pea, garden pea, green pea, sugar pea, snap pea, pigeon pea, soybean (immature seed), sword bean, Peruvian carrot, arrowroot, manna, Jerusalem artichoke, beet (garden), burdock, canna, cassava (bitter and sweet), celeriac, chayote (root), chervil (turnip root), endive, Cyperus esculentus), taro, galangal, ginseng, horseradish, edible blue banana, kava (turnip type), parsley (turnip type), parsnip, radish, white radish, rutabaga, salsify, scorzonera, Spanish salsify, parsley, sweet potato, taro, turmeric, turnip, serrata, yacon, yam bean, ginseng potato, and any combination thereof. Each possibility represents a separate embodiment of the present invention.

[0183] In some embodiments, the treatment is for a plant or plant part selected from the group consisting of seeds, seedlings, plantlets, bulbs, rhizomes, trees, grasses, shrubs, creepers, vegetables, fruits, beans, mushrooms, flowers, and combinations thereof. As used herein, the term "plant" or "plant part" is intended to encompass any plant organ or tissue.

[0184] Treating a phytopathogenic bacterial disease in a plant, plant part or crop comprises contacting the plant, plant part or crop with an effective amount of a system according to the principles of the present invention.

[0185] As used herein, the term "effective amount" refers to an amount of the delivery system that can reduce the number of Gram-negative bacteria by at least 1-log, at least 2-log, at least 3-log, or at least 4-log, compared to the number of viable bacteria prior to treatment with / contact with the delivery system. Alternatively, the effective amount can reduce disease incidence and / or manifestations (e.g., maceration area) by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least about 90% or 100%, compared to disease incidence / manifestations prior to treatment with / contact with the delivery system.

[0186] The plants may be contacted with the system by at least one of foliar application, soil application, application through an irrigation system, initial fertilization, injection into the soil, patch application, and aerial application. Each possibility represents a separate embodiment of the present invention.

[0187] According to some embodiments, contacting the plant, plant part or crop with the system comprises direct contact or indirect contact.

[0188] In some embodiments, the direct contacting comprises foliar spraying with an aqueous composition comprising the system. In some embodiments, the carrier comprises LMP, gelatin, and trehalose. In an exemplary embodiment, the carrier comprises kappa-carrageenan and trehalose.

[0189] In some embodiments, the indirect contact of the plant with the system comprises applying the system to the soil in which the plant is planted. Ambient conditions such as ion content and moisture percentage can regulate the release pattern. In some related embodiments, the system is in the form of granules.

[0190] According to some embodiments, the delivery system can be applied directly or indirectly, once or as many times as desired, such as once a day, once every two days, once every three days, once a week, for a total of 1, 2, 3, 4, 5 or more times as desired, to prevent or treat the increase in the population of plant pathogenic bacterial disease pathogens to levels above those causing disease. The delivery system can be applied 2 times a day, 3 times a day, or as needed. According to an exemplary embodiment, the method comprises contacting potato tubers with a delivery system of the present invention (which comprises BALO immobilized in a carrier comprising kappa-carrageenan and trehalose) once a day for 3-4 days, wherein the delivery system is applied to the soil adjacent to the potato tubers.

[0191] According to further embodiments, the delivery system may be contacted with the plant, plant part or crop prior to infection by the plant pathogen. Alternatively or additionally, the delivery system may be contacted with the plant, plant part or crop after infection by the plant pathogen.

[0192] According to further embodiments, the delivery system can be contacted with the plant, plant part or crop before, simultaneously with or after treating the plant, plant part or crop with any known treatment, including but not limited to physical treatments, such as hot water at about 50°C to about 55°C or dry hot air at about 50°C, and chemical treatments, such as antibacterial agents such as antibiotic agents, copper-containing agents.

[0193] According to another aspect, there is provided a method for reducing the bacterial load of an article in at least one of the pharmaceutical industry, microbiological industry, medical industry, food industry, marine industry, paper industry, textile industry, plant nurseries, aquaculture, soil treatment, ship maintenance, water treatment facilities, sewage treatment facilities, drainage systems and water distribution systems, the method comprising contacting the article with an effective amount of a delivery system according to the principles of the present invention, thereby reducing the bacterial load of the article.

[0194] According to some embodiments, the bacterial load is caused by bacteria selected from the group consisting of, comprises bacteria selected from the group consisting of, or consists of bacteria selected from the group consisting of: Gram-negative bacteria, Gram-positive bacteria, antibiotic-resistant bacteria, and any combination thereof. Without wishing to be bound by any theory or mechanism of action, it is contemplated that BALO degrades the biofilm matrix of Gram-positive bacteria, however such degradation does not involve predation on Gram-positive bacteria.

[0195] According to some embodiments, the gram-negative bacteria are plant pathogenic bacteria. According to further embodiments, the gram-negative bacteria are pathogenic gram-negative bacteria. Pathogenic gram-negative bacteria include, but are not limited to, Escherichia coli, Vibrio species, such as white-leg shrimp pathogenic Vibrio, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Pseudomonas, Klebsiella, Shigella-like Pseudomonas, Salmonella, Shigella, Yersinia enterocolitica, and Yersinia pseudotuberculosis. Each possibility represents a separate embodiment of the present invention.

[0196] According to another embodiment, the gram-positive bacteria is a pathogenic gram-positive bacteria. Pathogenic gram-positive bacteria include but are not limited to: Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium and Clostridium. According to a further embodiment, the Staphylococcus or Streptococcus bacterium is selected from the group consisting of: Staphylococcus aureus, Staphylococcus simulans, Streptococcus suis, Staphylococcus epidermidis, Streptococcus equi, Streptococcus equi zoo, Streptococcus agalactiae (GBS), Streptococcus pyogenes (GAS), Streptococcus sanguinis, Streptococcus gordonii, Streptococcus dysgalactiae, Group G Streptococcus, Group E Streptococcus and Streptococcus pneumonia. Each possibility represents a separate embodiment of the present invention.

[0197] According to further embodiments, the antibiotic resistant bacteria include but are not limited to antibiotic resistant Staphylococcus bacteria and antibiotic resistant Streptococcus bacteria. According to further embodiments, the antibiotic resistant Staphylococcus or Streptococcus bacteria are selected from the group consisting of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), daptomycin-resistant Staphylococcus aureus (DRSA) and linezolid-resistant Staphylococcus aureus (LRSA). Each possibility represents a separate embodiment of the present invention.

[0198] According to some embodiments, the method of reducing the bacterial load of an article comprises reducing or eradicating biofilm.

[0199] The term "biofilm" refers to a population of microorganisms (e.g., bacteria) grown on a surface, wherein the bacteria are encapsulated in a matrix typically consisting of polysaccharides, proteins, and nucleic acids. In this state, the bacteria are less susceptible to phagocytic cells and antibiotics. The term "biofilm" is further intended to include biological films that develop and persist at interfaces in an aqueous environment.

[0200] The terms "biofilm reduction" and "biofilm eradication" are used interchangeably herein and are defined as the ability to degrade existing or mature biofilms and / or inhibit, prevent or reduce biofilm formation and / or kill bacteria embedded in or associated with biofilms in vitro and in vivo.

[0201] The terms "inhibit biofilm formation", "prevent biofilm formation" or "reduce biofilm formation" are used interchangeably herein and refer to the ability to avoid or reduce the formation of biofilm. According to some embodiments, preventing biofilm formation means inhibiting bacterial attachment to the outer surface of an article or the inner surface of an article. Additionally or alternatively, preventing biofilm formation means killing biofilm-forming bacteria.

[0202] Articles that may come into contact with the delivery system include, but are not limited to: machines and equipment for preparing or storing drugs; medical devices or implants; contact lenses; machines or equipment for preparing and storing food; tanks, pipes, filters, reservoirs; coolers; drainage systems, for example in medicine, such as infiltration pits, filters, pipes, etc.; water irrigation systems and any part thereof; boats and any part thereof.

[0203] As used herein, the term "reducing bacterial load" means that as a result of contacting an article with a delivery system of the present invention, the number of viable bacteria present on the outer surface of an article or on the inner surface of an article is reduced compared to the number of viable bacteria present on the surface of the article or in the article before contacting the delivery system. The number of viable bacteria can be reduced by at least 1-log, at least 2-log, at least 3-log, or at least 4-log compared to the number of viable bacteria before treatment with / contact with the delivery system. The number of viable bacteria can be determined by any method known in the art, such as PFU, which represents viable cells that can be counted by dilution plating in double agar.

[0204] The delivery system may be contacted with the article before bacterial contamination and / or biofilm formation occurs to prevent bacterial contamination and / or biofilm formation. Alternatively or additionally, the delivery system may be contacted with the article after bacterial contamination and / or biofilm formation.

[0205] Methods of preparing the delivery system

[0206] According to another aspect, a method for preparing a delivery system for differential delivery of live Bdellovibrio and similar organisms (BALOs) is provided, wherein the delivery system comprises: a carrier comprising a polysaccharide hydrocolloid; and the BALOs immobilized within the carrier, wherein the BALOs are in a physiological state of a leech plasmid, a leech cyst, and an AP, which are at a rate of at least about 1.0×10 8 PFU / g carrier cell density, the method comprises the following steps:

[0207] (i) mixing a suspension comprising BALOs with a hydrocolloid solution comprising a polysaccharide hydrocolloid to form a hydrocolloid composition comprising the BALOs; and

[0208] (ii) mixing the hydrocolloid composition comprising the BALO of step (i) with a cross-linking solution to form a polysaccharide hydrocolloid in a wet gel form, thereby forming the delivery system in a wet form.

[0209] According to some embodiments, the method further comprises the step of drying the delivery system of step (ii), thereby forming the system in dry form.

[0210] According to another embodiment, the system is dried by at least one of the following: air drying, desiccant drying, vacuum drying, fluidized bed drying, spray drying, sunlight drying and freeze drying. Each possibility represents a separate embodiment of the present invention. In some exemplary embodiments, the system is dried by vacuum drying. The advantage of vacuum drying over freeze drying is that the product is not frozen, thereby reducing energy consumption and related economic and environmental impacts. Further, potential freezing damage is avoided. However, the main problem with various drying techniques is the survival of microorganisms during dehydration. This may be particularly serious for bacteria that do not form spores (i.e., all Gram-negative taxa). After dehydration, the water availability in the carrier decreases until the immobilized cells reach a dormant state, during which metabolism slows down, sometimes to a complete standstill. The inventors of the present invention have surprisingly found that the survival rate of BALO in the carrier after drying is higher for bdelloid plastids than for AP cells. Survival was further improved in the presence of an osmoprotectant, with osmoprotectants having a high molecular weight increasing the survival of bdelloid plastids compared to osmoprotectants having a low molecular weight.

[0211] According to a further embodiment, the method further comprises the step of grinding, crushing, pulverizing and / or any process known in the art useful for reducing the Feret diameter of granules, beads, capsules or granules, thereby obtaining a dry system in powder form.

[0212] According to some embodiments, mixing the hydrocolloid composition with the cross-linking solution is performed by at least one of: dripping, spraying, emulsifying, and containing in oil and then cooling. In some exemplary embodiments, mixing the hydrocolloid composition with the cross-linking solution is performed by dripping the hydrocolloid composition into a cross-linking solution containing metal cations and / or gelation-inducing hydrocolloids.

[0213] According to some embodiments, the hydrocolloid composition is an aqueous composition. As used herein, the term "aqueous composition" refers to a solution or mixture wherein the solvent comprises about 75% (w / w) to about 99.8% (w / w) water.

[0214] According to some embodiments, the polysaccharide hydrocolloid is present in the hydrocolloid solution at a weight percentage of about 0.25% to about 10% of the total weight of the hydrocolloid solution. In further embodiments, the polysaccharide hydrocolloid is present in the hydrocolloid solution at a weight percentage of about 0.5% to about 5% of the total weight of the solution. In still further embodiments, the hydrocolloid is present in the hydrocolloid solution at a weight percentage of about 1% to about 3% of the total weight of the solution.

[0215] According to some embodiments, the polysaccharide hydrocolloid is selected from the group consisting of: kappa-carrageenan, pectin, alginate, agar, gellan gum, carboxymethylcellulose (CMC), furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan and derivatives and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0216] According to an exemplary embodiment, the polysaccharide hydrocolloid is kappa-carrageenan.

[0217] According to some embodiments, the volume ratio between the hydrocolloid solution and the BALO suspension (preferably, leech suspension) is about 1:3 to about 1:20, preferably about 1:4 to about 1:9. According to an exemplary embodiment, the volume ratio between the hydrocolloid solution and the BALO suspension is about 1:9.

[0218] According to some embodiments, the hydrocolloid composition further comprises a protein hydrocolloid and / or a protein. According to further embodiments, the protein hydrocolloid and protein include, but are not limited to, gelatin, collagen, collagen hydrolysate, albumin, casein, whey protein, zein, soy protein, and any derivatives or combinations thereof. Without wishing to be bound by any mechanism of action, the addition of a protein hydrocolloid such as gelatin may alter the degradation rate of the carrier via gelatinase secreted by the prey, thereby releasing the encapsulated BALO at a desired release rate.

[0219] According to some embodiments, the hydrocolloid composition further comprises a water absorbing agent. In some embodiments, the water absorbing agent is selected from the group consisting of starch, polyvinylpyrrolidone (PVP) and derivatives and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0220] In some embodiments, the polysaccharide hydrocolloid is LMP.

[0221] According to some embodiments, gelatin or a water absorbent is present in the hydrocolloid solution at a weight percentage of about 0.25% to about 25% of the total weight of the hydrocolloid solution. In further embodiments, gelatin or a water absorbent is present in the hydrocolloid solution at a weight percentage of about 0.5% to about 10% of the total weight of the solution. In further embodiments, gelatin or a water absorbent is present in the hydrocolloid solution at a weight percentage of about 1% to about 5% of the total weight of the hydrocolloid solution.

[0222] According to some embodiments, the hydrocolloid composition further comprises at least one osmotic protectant. According to further embodiments, the at least one osmotic protectant is selected from the group consisting of trehalose, inositol, glycerol, arabinitol, betaine, amino acid and combinations thereof. Each possibility represents a separate embodiment of the present invention. In some embodiments, the at least one osmotic protectant is trehalose and / or glycerol.

[0223] In certain embodiments, the hydrocolloid is kappa-carrageenan, and the at least one osmoprotectant is trehalose.

[0224] According to some embodiments, the osmotic protectant is present in the hydrocolloid solution at a weight percentage of about 0.25% to about 15% of the total weight of the hydrocolloid solution. In further embodiments, the osmotic protectant is present in the hydrocolloid solution at a weight percentage of about 0.5% to about 12.5% ​​of the total weight of the solution. In further embodiments, the osmotic protectant is present in the hydrocolloid solution at a weight percentage of about 1% to about 10% of the total weight of the hydrocolloid solution.

[0225] According to some embodiments, the cross-linking solution and the hydrocolloid composition are contacted at a volume ratio of about 10: 1 to about 500: 1. In further embodiments, the cross-linking solution and the hydrocolloid composition are contacted at a volume ratio of about 30: 1 to about 300: 1. In still further embodiments, the cross-linking solution and the hydrocolloid composition are contacted at a volume ratio of about 50: 1 to about 150: 1. According to a certain embodiment, the cross-linking solution and the hydrocolloid composition are contacted at a volume ratio of about 100: 1.

[0226] According to some embodiments, the cross-linking solution comprises metal cations and / or gelation inducing agents. According to other embodiments, the cross-linking solution comprises the metal cations and / or the gelation inducing agents in an amount of about 0.1% (w / w) to about 5% (w / w) by weight, based on the total weight of the cross-linking solution. In further embodiments, the cross-linking solution comprises the metal cations and / or the gelation inducing agents in an amount of about 0.2% (w / w) to about 4% (w / w) by weight, based on the total weight of the cross-linking solution. In still further embodiments, the cross-linking solution comprises the metal cations and / or the gelation inducing hydrocolloid in an amount of about 0.3% (w / w) to about 1% (w / w) by weight, based on the total weight of the cross-linking solution.

[0227] In some embodiments, the metal cation is selected from the group consisting of calcium (Ca 2+ ), potassium (K + ), barium (Ba 2+ ), magnesium (Mg 2+ ), aluminum (Al 3+ )、Zn 2+ ), lead (Pb 2+ ), iron (Fe 2+ ), Sr 2+ ), copper cation (Cu 2+ ), cadmium (Cd 2+ ), nickel (Ni 2+ ), cobalt (Co 2+ ) and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0228] In some exemplary embodiments, the metal cation is calcium (Ca 2+ ). In certain such embodiments, the hydrocolloid is LMP.

[0229] In another exemplary embodiment, the metal cation is potassium (K + ). In certain such embodiments, the hydrocolloid is kappa-carrageenan.

[0230] In some embodiments, the gelation inducing agent is a polycationic hydrocolloid and / or polycationic compound selected from the group consisting of LBG, genipin, chitosan, polylysine, polyarginine, and derivatives and combinations thereof. Each possibility represents a separate embodiment of the present invention.

[0231] According to an exemplary embodiment, the method for preparing the delivery system of the present invention comprises the following steps:

[0232] (i) mixing a suspension comprising BALOs with a hydrocolloid solution comprising a polysaccharide hydrocolloid to form a hydrocolloid composition comprising the BALOs,

[0233] wherein the polysaccharide hydrocolloid is kappa-carrageenan or LMP, which is present in the hydrocolloid solution in an amount of about 0.25% to about 10% by weight, preferably about 1% to about 3% by weight, based on the total weight of the hydrocolloid solution;

[0234] wherein the hydrocolloid solution further comprises trehalose in an amount of about 0.25% to about 15% by weight, preferably about 1% to about 10% by weight, based on the total weight of the hydrocolloid solution;

[0235] wherein the suspension of BALO is added to the hydrocolloid solution at a volume ratio of 1:100 to about 1:4, preferably at a volume ratio of 1:9; and

[0236] (ii) mixing the hydrocolloid composition comprising the BALO of step (i) with a cross-linking solution to form a polysaccharide hydrocolloid in the form of a wet gel,

[0237] wherein the cross-linking solution comprises metal cations;

[0238] wherein if the polysaccharide hydrocolloid is kappa-carrageenan, then the metal cation is a potassium ion, and if the polysaccharide hydrocolloid is LMP, then the metal cation is a calcium ion; and

[0239] The metal cation is present in the cross-linking solution in an amount of about 0.1% to about 5% by weight, preferably about 0.3% to about 1% by weight, based on the total weight of the cross-linking solution.

[0240] According to a further exemplary embodiment, if the hydrocolloid solution used in the method for preparing the delivery system comprises LMP, the hydrocolloid solution may further comprise gelatin in an amount of about 0.25% to about 25% by weight, preferably about 1% to about 5% by weight, based on the total weight of the hydrocolloid solution.

[0241] According to some embodiments, the drying is performed at a temperature of about 10° C. to 60° C. In further embodiments, the drying is performed at a temperature of about 20° C. to 50° C. In still further embodiments, the drying is performed at a temperature of about 30° C. to 40° C.

[0242] According to some embodiments, the vacuum drying is carried out at a pressure of 10 mbar to 1000 mbar. In further embodiments, the vacuum drying is carried out at a pressure of 50 mbar to 800 mbar. In further embodiments, the vacuum drying is carried out at a pressure of 100 mbar to 500 mbar.

[0243] According to some embodiments, after drying, the system comprises at least about 1.0×10 8 PFU / g carrier of BALO or preferably bdelloid plasmids. According to a further embodiment, after drying, the system comprises at least about 5.0×10 8 PFU / g carrier, about 1.0×10 9 PFU / g carrier or about 5.0×10 9 PFU / g carrier of BALO or preferably bdelloid plasmids.

[0244] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a vector" includes a plurality of such vectors and equivalents thereof (known to those skilled in the art), and so forth. It should be noted that the term "and" or the term "or" are generally used in their senses (including "and / or") unless the content clearly dictates otherwise.

[0245] As used herein, when referring to measurable values ​​such as amounts, durations, etc., the term "about" is meant to encompass variations of + / - 20%, preferably + / - 10%, more preferably + / - 5%, and even more preferably + / - 1% from the specified value, as such variations are appropriate for practicing the disclosed methods.

[0246] As used herein, the terms "plurality" or "multiplicity" mean two or more.

[0247] Numerical ranges disclosed throughout the specification and claims, such as amounts, ratios, and time periods, include any integer and numerical fraction therebetween.

[0248] It should be understood that each possibility disclosed throughout this specification represents a separate embodiment of the present invention.

[0249] Provide the following examples to fully illustrate some embodiments of the present invention. However, they should not be interpreted as limiting the wide scope of the present invention in any way. Those skilled in the art can easily design many changes and modifications of the principles disclosed in this article without departing from the scope of the present invention.

[0250] Example 1

[0251] Bacterial strains, media, and growth conditions

[0252] The Bdellovibrio and similar organisms (BALOs) used as predatory bacteria were Bdellovibrio bacteriovorus HD100 and HD100-Td-tomato, which contain the plasmid pMQ414 and constitutively express the fluorescent td-Tomato protein. Laboratory stocks of Bdellovibrio bacteriovorus were stored at -80°C.

[0253] Escherichia coli ML35, Pectobacterium carotovora subsp. carotovora WPP14, Pectobacterium carotovora subsp. brasiliensis (Pcb), and Diecilomyces solani were used as prey bacteria.

[0254] Prey bacteria P. carotovora and D. solanum were grown in LB medium at 28°C with shaking at 250 rpm. E. coli was grown at 37°C. All started from a single colony derived from a laboratory stock stored at -80°C. Overnight cultures were centrifuged at 4500 g for 10 minutes at 4°C, and the cell pellets were washed and resuspended in HEPES buffer (pH 7.8) (aHEPES) amended with 2 mM CaCl and 3 mM MgCl 6H O to a final optical density (OD) of 10 for P. carotovora, D. solanum and E. coli. 600 The E. coli suspension was further concentrated to produce ~10 11 The stock suspension was stored at 4°C until use, for up to two weeks.

[0255] To initiate the predation co-culture, transfer a bacteriological loopful of a frozen stock of the bacterial predator Bdellovibrio HD100 or HD100-Td-tomato into a 1.5 ml plate containing OD 20 diluted in 15 mL aHEPES. 600 = 10 bottles of E. coli suspension. The co-culture was incubated at 28°C with shaking at 250 rpm until the turbidity of the culture reached below OD 600=0.1. The suspension was then filtered through a 0.45 μm filter (Sartorius). Thereafter, 5 ml of the filtered predatory cells from the starter culture were inoculated into 40 ml of modified HEPES containing 5 ml of OD 600 = 10 E. coli suspension) and shaken at 250 rpm at 28°C until the turbidity of the suspension decreased. After most of the prey cells were lysed, the suspension was filtered through a 0.45 μm filter to produce ~10 9 Plaque forming units (PFU) / ml of predatory AP cells. The filtrate was stored at 4°C until use, up to one day. To further concentrate the filtered predatory cells, the suspension (50 ml) was centrifuged at 9000 rpm for 20 minutes at 10°C and the cell pellet was resuspended in 0.5 ml of aHEPES to ∼10 11 To generate large quantities of bdelloid plasmids, 500 μl of concentrated AP cell suspension (10 11 PFU / ml) was added with 250 μl of concentrated E. coli suspension (10 11 CFU / ml) and incubated at 28°C with shaking at 250 rpm for approximately 1 hour to synchronize the growth of the predators. The formation of bdelloids was confirmed by phase contrast microscopy.

[0256] Prey and predator cultures were routinely enumerated by dilution plating as CFU / ml and PFU / ml, respectively. The latter plating was performed in double-layer agar.

[0257] Example 2

[0258] Preparation and Characterization of κ-Carrageenan Dry Gel Carrier

[0259] Preparation of κ-carrageenan dry gel carrier

[0260] A 1.5% (w / w) κ-carrageenan dispersion was prepared by adding κ-carrageenan powder (Sigma, USA) to distilled water (DW). The mixture was then brought to a boil to complete the dissolution of the colloid. The solution was dripped into a 1% (w / w) solution of KCl (Biolab Chemicals, Israel) at a volume ratio of 1:100 (at 40°C) and stirred for 20 minutes. The carrier was then dried in a vacuum oven (Binder, Germany) at 37°C and 400 mbar.

[0261] Measurement of Swelling Ratio of κ-Carrageenan Dry Gel Support

[0262] The swelling measurement of the kappa-carrageenan dried gel carrier was performed by introducing the carrier into DW or 1% or 2% KCl solution. Periodically, the carrier was removed from the external solution, wiped with filter paper, and weighed (± 0.001 g) on ​​an analytical balance (Precisa, Switzerland). The test was terminated when the carrier began to disintegrate (in DW) or reached a constant weight (in 1 and 2% KCl solution).

[0263] The swelling ratio by weight is calculated as follows:

[0264]

[0265] where W(initial) is the initial sample weight (g), and W(final) is the final sample weight (g).

[0266] Potassium concentration measurement

[0267] To determine the potassium concentration (mg / L) released from the dried κ-carrageenan carrier, samples from the external solution (10 ml DW) were collected at different times (the samples at each time were taken from different test tubes) and tested by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Spectroflame ICP, Spectro Analytical Instruments, Germany). Negligible potassium concentration was found in DW (blank sample).

[0268] Preparation of κ-carrageenan carrier for encapsulating BALO

[0269] κ-carrageenan (1.67%; w / w) dispersion was prepared by adding κ-carrageenan powder (Sigma, USA) to DW. Then, different osmoprotectants were added to the κ-carrageenan dispersion to a final concentration (w / w) of 10% glycerol (Sigma, USA), 5% trehalose (Sigma, USA), 5% betaine (Sigma, USA) or 5% inositol (Sigma, USA). Then, the mixture was brought to boiling to complete the colloid dissolution. Then, the bungoid suspension was added to the κ-carrageenan-based solution at a volume ratio of 1:9 at 40°C. The final mixture was dripped into a 1% (w / w) (or other mentioned) sterile solution of KCl (Biolab Chemicals, Israel) at a volume ratio of 1:100 and stirred for 20 minutes. The crosslinked spherical supports obtained (having an average diameter of about 4 mm) were then dried in a vacuum oven (Binder, Germany) at 37° C. and 400 mbar to obtain supports having a diameter of about 1 mm.

[0270] Encapsulation efficiency

[0271] Encapsulation efficiency (EE) represents a comprehensive measure of the efficacy of entrapment and survival of living cells during the encapsulation procedure and is calculated according to the following equation (Gebara et al., 2013):

[0272] EE(%)=(N / N0)×100

[0273] where EE is the encapsulation efficiency expressed as a percentage, N is the number of cells released from the carrier (CFU / g carrier), and N0 is the number of cells in the hydrocolloid solution (CFU / g suspension).

[0274] Counting the number of bacteria in the vector

[0275] For quantitative measurement of cell viability, the carrier is dissolved to release the encapsulated bdelloids. For this purpose, the wet or dry gel carrier is immersed in sterile 2% sodium hexametaphosphate (SHMP) or sterile DW, respectively. Then, the mixture is shaken until all the carrier is dissolved. PFU (which represents viable cells) can be counted by dilution plating in double agar.

[0276] Scanning electron microscopy (SEM)

[0277] Different dried κ-carrageenan-based carriers were attached to aluminum stubs with double-sided carbon tape and coated with 3 nm Au-Pd in ​​a Q150TES coater (Quorum Technologies Ltd., Lewes, England). To study the characteristic carrier structures, a Jeol IT-100 SEM (Tokyo, Japan) was used at 20 kV. To visualize the bacteria residing in the carriers, a Jeol JSM-7800F high-resolution SEM (Tokyo, Japan) was used at 2 kV.

[0278] Predation dynamics by released encapsulated predators

[0279] Predation by encapsulated Bdellovibrio HD100-Td-tomato in κ-carrageenan carriers was tracked by monitoring predator growth using fluorescence and prey decline using OD in a plate reader (Tecan Spark 10M, Switzerland) incubated at 28°C. The detectable thresholds for low and high levels of fluorescence corresponded to ∼4 × 10 7 and ~10 9PFU / ml of Bdellovibrio spp. HD100. 500 μl of dissolved predator-encapsulated carriers at a ratio of 1 grain of dry carrier in 1 ml DW, 100 μl of prey (Escherichia coli and Pectobacterium carotovora, OD 600 =10) and 400 μl of modified HEPES buffer were added to an Eppendorf tube and mixed. For each treatment, 200 μl were pipetted onto the wells of a clear black flat-bottom 96-well plate (Greiner-Bio One, Germany) in three technical replicates in two independent experiments.

[0280] Potato slice assay

[0281] Red skin Desiree potato cultivar is sourced from a local supermarket and used in all experiments. Potatoes were surface sterilized by soaking in 1% HCl solution for 1 minute, washed twice with sterile DW, and dried in a laminar flow hood. After the potato surface was dried, the potato was washed with 70% ethanol and dried again on a sterile culture dish. Use a sterilized knife to cut out a slice of about 0.5 cm in width, and use sterile tweezers to transfer it to a culture dish containing a sterile filter paper (Whatman 1) of 70 mm diameter moistened with 330 μl of sterile DW.

[0282] Pectobacterium carotovora subsp. brasiliensis was grown as above and subcultured (~100 μl) in fresh LB to an OD of ~0.2. 600 , and washed twice in aHEPES buffer; OD 600 Adjust to 0.2 and then dilute to produce ~10 6 CFU / ml. Potato slices were inoculated with a 10 μl drop of the pathogen culture, and with a 10 μl or 20 μl drop of the different tested treatments; both were applied to the center of the slice. The tested treatments included two different concentrations (diluted or concentrated) of a dissolved κ-carrageenan-trehalose carrier cross-linked by 0.5% KCl and encapsulating bdelloid plasmids. The ratios were 1 grain of dry carrier in 0.5 ml DW and 1 grain of dry carrier in 0.15 ml DW for the diluted and concentrated liquids, respectively. The liquid from the dissolved carrier was applied 60 minutes before Bacillus carotovora subspecies brasiliensis. The potato slices were incubated at 28°C for 48 hours. All experiments were repeated three times with 6 replicates / treatment. After incubation, the slices were photographed using a ruler. The maceration area was calculated using the ImageJ image processing program.

[0283] Biofilm assay

[0284] Microtiter wells (200 μl / well) were inoculated from 18 h LB-grown cultures of Pseudomonas fluorescens and E. coli ZK2686 diluted 1:100 in LB (at 28°C and 37°C, respectively). Cells were grown at 28°C for 48 h (preformed biofilms). To evaluate the predation kinetics of encapsulated Bdellovibrio on these biofilms, preformed biofilms were grown as described above, washed three times with DDNB to remove any planktonic cells, and 200 μl of dissolved or ground carrier in DDNB was added to the wells (at a ratio of 1 carrier to 1 ml). As a control, 200 μl of DDNB was added to the wells. The microtiter plates were incubated at 28°C for 72 hours after which they were stained with crystal violet (CV) and quantified at 600 nm using a plate reader (Tecan Spark 10M, Switzerland) as described previously (O'Toole, GA and R. Kolter. 1998. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol. Microbiol. 30:295-304; Kadouri, D., & O'Toole, GA 2005. Susceptibility of biofilms to Bdellovibrio bacteriovorus attack. Applied and Environmental Microbiology, 71(7), 4044-4051).

[0285] Example 3

[0286] Characteristics of κ-carrageenan dry gel carrier

[0287] Several hydrocolloid gelling agents were tested with regard to fast predator release from the carrier. In preliminary experiments, a carrier based on kappa-carrageenan cross-linked by KCl was found to meet these requirements.

[0288] By comparing the disintegration rates of wet or dry kappa-carrageenan gel carriers in distilled water (DW), it was found that the dry carrier dissolved faster.

[0289] Figure 1 shows the characteristic swelling process and disintegration of a dry kappa-carrageenan carrier (350 μm diameter) when immersed in excess DW. The carrier swells gradually until it completely disintegrates after 12.5 minutes.

[0290] The effect of carrier size and water volume in the external solution on the swelling and disintegration of kappa-carrageenan dry carriers was examined. The weight ratio between carrier and water was maintained for each water volume by placing two half-grains of carrier together. Table 1 shows that the carrier swells until it reaches 30-34 times its initial weight, and the maximum swelling before disintegration occurs earlier for smaller carriers than for larger carriers. Further, the larger the water volume and the smaller the size of the carrier, the faster the disintegration occurs.

[0291] Table 1: Parameters characterizing the disintegration of kappa-carrageenan dry carriers in DW at 25°C

[0292]

[0293] Superscript letters indicate significant differences (Student's t-test, P < 0.05).

[0294] Results presented are mean ± SD of 6 replicates from two different batches.

[0295] Without wishing to be bound by any theory or mechanism of action, it is believed that the kappa-carrageenan carrier disintegrates due to the loss of potassium ions, which are the primary cross-linking agent.

[0296] The swelling and disintegration process was further examined when the kappa-carrageenan dry carrier was immersed in KCl solution (main cross-linking agent) at different concentrations. As a result, the carrier did not disintegrate, but swelled. Table 2 shows that the higher the potassium concentration in the external solution, the less the carrier swells.

[0297] Table 2: Equilibrium swelling ratio values ​​of kappa-carrageenan dry supports immersed in 10 ml KCl solution at 25°C.

[0298]

[0299] Superscript letters indicate significant differences (Student's t-test, P<0.05). Results presented are mean±SD of 6 replicates from two different batches.

[0300] The concentration of potassium ions released from the kappa-carrageenan carriers before disintegration was measured as a function of time in an external solution of 10 ml DW. It was found that the potassium concentration in the external solution increased with time, and the smaller the carrier, the higher and faster the increase. The total surface area of ​​the 2 halves of the carrier was ~1.5 times the surface of 1 intact carrier (assuming a spherical shape), corresponding to the ratio of potassium concentration measured at each time point between carriers of different sizes. The results are presented in Figure 2 middle.

[0301] Example 4

[0302] Encapsulation of BALO in κ-carrageenan carriers and survival under vacuum drying

[0303] Encapsulation experiments were performed on Bdellovibrio virulens HD100 in two different physiological states (AP cells and bedelloid plasmids). Figure 3A and 3B The effects of different osmoprotectants (glycerol, trehalose, betaine, and inositol) on the encapsulation efficiency and survival of predator bacterial cells are shown in Figure 1. The encapsulation efficiency of AP cells and bdelloids ranged from 7.4% with glycerol to 41% with trehalose or without any addition, and appeared to decrease with decreasing molecular weight of the added osmoprotectant ( Figure 3A Compared with AP cells, the survival rate of bdelloid plastids after drying is higher ( Figure 3B AP cells without or with trehalose addition showed 0.05% and 2.8% survival rates, respectively, while betaine-added or trehalose-added bdelloid cells showed 0.3% and 6.1% survival rates, respectively ( Figure 3B ).

[0304] Table 3 shows that encapsulated bdelloids with trehalose produced the highest number of predators after drying (5.4×10 9 PFU / g carrier), while treatments without added osmoprotectant or with betaine performed the worst (Table 3). Regarding the survival percentage after desiccation, a clear advantage of bdelloids over AP cells was observed (Table 3).

[0305] Table 3: Effect of predator cell type and added osmoprotectant on the density of encapsulated Bdellovibrio bacteriovorus HD100 before and after vacuum drying.

[0306]

[0307] Results represent the mean of two independent experiments with three technical replicates for each treatment. In each test, five vectors were evaluated.

[0308] Example 5

[0309] Effect of κ-carrageenan carrier composition on its structure

[0310] Figures 4A1-4D4SEM images of typical structures observed in various dried kappa-carrageenan carrier samples are shown. KCl crystals were detected both on the surface of the carrier and on the inside of the carrier, with different crystallization patterns for each carrier sample depending on the added osmoprotectant. Dense areas (dark colors in the half-split carriers) and more porous areas (usually near the carrier walls) were observed. For the carrier with glycerol, a porous sponge-like structure was discerned on the outside.

[0311] Example 6

[0312] Predation dynamics of Bdellovibrio bacteriovorus encapsulated in kappa-carrageenan carriers on soft rot bacteria

[0313] To test the predation dynamics of predators encapsulated in κ-carrageenan carriers, the bacteriovorous Bdellovibrio HD100 expressing the Td-tomato fluorescent protein was used. Predation of Escherichia coli (“standard” laboratory prey) and Pectobacterium carotovora by encapsulated AP cell or bdelloplast predators when released from carriers with or without trehalose was examined. Figure 5A-5B and Figure 6A-6B ). Figure 5A and 6A The results showed a reduction in prey concentration in all treatments tested, with encapsulated bdelloids showing better performance compared to AP cells. The carrier with bdelloids and trehalose was better at preying on E. coli ( Figure 5A ), while the carrier with bdelloid plasmids without additives showed slightly faster predation of P. carotovora ( Figure 6B ). Predation by vehicle with AP cells without additives lagged behind the rest of the treatments, with a ~10 h delay ( Figure 5A and 6A ).

[0314] Figure 5B and 6B An increase in the predatory population was shown in all treatments exposed to prey. A higher increase in the concentration of bdelloid plasmids released from the carrier containing trehalose was obtained when E. coli constituted the prey population ( Figure 5B ), whereas for the P. carotovora prey population, a higher concentration increase was obtained for AP cells released from the carrier.

[0315] Encapsulated bdelloids and carriers including trehalose, glycerol, or no additives were tested against two additional soft rot bacteria: Pectobacterium brasiliensis ( Figure 7A-7B ) and D. solanacearum ( Figures 8A-8B The addition of glycerol resulted in much slower predation than the other treatments, exhibiting a ~25 h delay ( Fig. 7A and8A The predation kinetics on Pectobacterium did not differ between carriers with or without additives ( Fig. 7A ), and for Dickie'sella, a slight advantage was observed for the carrier with trehalose ( Fig. 8A Higher plateau RFU values ​​were obtained for the carrier with trehalose, as indicated by the increased number of predators after predation ( Figure 7B and 8B ).

[0316] Example 7

[0317] Effects of κ-carrageenan carrier preparation conditions on predator survival

[0318] To further improve predator survival, different drying conditions (vacuum and temperature) and cross-linker concentrations were examined. These experiments were performed with vectors of encapsulated bdelloid plastids with or without trehalose, as these previously produced the best results in terms of survival and predation. The results of these experiments are presented in Fig. 9 No differences in predator survival were observed between 37°C and 28°C. Increasing the vacuum from 400 mbar to 100 mbar resulted in a small decrease or no change in survival. Reducing the crosslinker concentration from 1% KCl to 0.5% KCl significantly increased survival (approximately 2-fold) to 3.2×10 9 PFU / g carrier when trehalose was not added. In its presence, survival increased to ~12% or 1.3×10 10 PFU / g carrier.

[0319] Figures 10A-10F The figure illustrates an SEM image of a κ-carrageenan-trehalose carrier encapsulating a predator prepared with 0.5% KCl. Salt-containing "flakes" ( Fig. 10B Crystals were also observed in the inner contents of the carrier, although they were smaller and had a different configuration ( Fig. 10D ). Overall, less salt was detected compared to FIG4 where a higher KCl concentration was used. High-resolution SEM micrographs show a mixed population of AP cells and bdelloid plastids embedded within the matrix ( Fig.10E and 10F HR-SEM micrographs showed that both Bdellovibrio as bedelloid plasmids and as AP cells maintained integrity after vacuum drying of the κ-carrageenan-trehalose carriers.

[0320] Example 8

[0321] Effectiveness of kappa-carrageenan carriers in preventing disease in potato slices

[0322] The ability of predators encapsulated in carrageenan-trehalose carriers to prevent in situ maceration of soft rot Enterobacteriaceae (SRE) was tested using a potato slice assay. The carriers were dissolved in water and the suspensions were applied 60 minutes prior to application of the highly virulent Pectobacterium brasiliensis (Pcb) pathogen. Addition of diluted or concentrated suspensions obtained from the dissolved carriers and application of two different volumes of each suspension significantly reduced the maceration area (by -87-99%) compared to the Pcb control ( Fig.11A ) and the number of diseased sections ( Fig. 11B The lowest disease incidence was obtained in the treatment using the largest volume of diluted suspension ( Fig. 11B ), as can also be seen in the photo of the potato slices ( Fig. 11C ).

[0323] To confirm that the observed effects were not the result of indirect interactions of vector components (eg, priming of plant defenses), vectors were tested without predators. Figures 12A-12C It was shown that the treatment was not significantly different from that with the pathogen-only control, indicating that the protective effect was due to the presence of high counts of live predators within the vector.

[0324] Example 9

[0325] Predation dynamics of Bdellovibrio bacteriovorus encapsulated in kappa-carrageenan carriers on biofilms

[0326] The biofilm assay was used to evaluate the predation kinetics of encapsulated Bdellovibrio bacteriovorus on Pseudomonas fluorescens and Escherichia coli ZK2686 biofilms. κ-carrageenan carriers (dissolved or ground, with or without trehalose) were added to the developed biofilms of both bacteria and the OD was 0.04 after 72 hours of incubation with the carriers. 600 Quantification of biofilm biomass was assessed by crystal violet (CV) staining measured at 4 °C.

[0327] As in Fig.13A As shown in , for Pseudomonas fluorescens biofilms, a ~65-75% reduction in crystal violet (CV) staining was observed after a 72-hour exposure period for the κ-carrageenan carriers. Although no differences in CV staining were observed between solubilized or ground carriers, carriers containing trehalose performed better than carriers without additives.

[0328] As in Fig. 13BAs shown in , for E. coli ZK2686 biofilms, a -70% reduction in CV staining was observed after a 72 hour exposure period for the kappa-carrageenan carriers. Further, no differences were observed in CV staining between solubilized or ground carriers, nor between carriers containing or not containing trehalose.

[0329] Example 10

[0330] Preparation and Characterization of Low Methoxy Pectin (LMP) Dry Gel Carrier

[0331] Purification of LMP materials

[0332] Three commercial LMP types (106S-YA, 104AS and 5CS) (CPKelco, Copenhagen, Denmark) with degrees of esterification (DE) and amidation (DA) of 7-27% and 0-24%, respectively, were used to prepare the carriers (Table 4). Of note, 5CS had the lowest DE and DA. All tested LMPs are used as food additives (under EU designation E440). Since these LMPs are routinely standardized with sucrose by the manufacturer, they were purified at 4°C in reverse double distilled water (DDW) in dialysis tubing with a molecular weight cutoff of 12,000-14,000 Da (Medicell Membranes Ltd, London, England) for 5 days, with water changed three times per day. The material remaining in the dialysis tubing was freeze-dried.

[0333] Table 4: Chemical characteristics of the LMPs used in this study

[0334]

[0335] pH adjustment and pasteurization of LMP solution and preparation of LMP gel carrier

[0336] The dialyzed purified LMP was oven heated at 90°C for 15 minutes and then dissolved in autoclaved HEPES to obtain a 3% w / w LMP solution. NaOH 3M was used to raise the acidic pH to 7.2. Alternatively, the dialyzed purified LMP was dissolved in distilled water (DW) to obtain a 3% w / w LMP solution. The solution was then pasteurized by heating on a hot plate at 100°C for 30 seconds and immediately cooling on ice. Pasteurization was selected relative to sterilization at 121°C because the extent of polymer hydrolysis was reduced at lower temperatures. The gel carrier was obtained by dripping the LMP solution into a sterile 1% (w / w) CaCl2 solution for 20 minutes under stirring. This procedure resulted in spontaneous cross-linking, resulting in spherical carriers with a diameter of ~4 mm.

[0337] To evaluate the inactivation of microorganisms in the samples, the carriers prepared from LMP dissolved in HEPES as described above were disintegrated in a 2% (w / w) sodium hexametaphosphate (SHMP) solution as described in detail below, and the solution was then plated on LB agar or potato dextrose agar (PDA) for total bacteria or yeast and mold, respectively. No colonies were detected.

[0338] Growth of Soft Rot Enterobacteriaceae (SRE) on LMP

[0339] To test the growth of SRE on LMPs, M9 minimal medium (400 μM MgSO4, 7.5 mM (NH4)2SO4, 20 mM K2HPO4, 15 mM KH2PO4, and 0.2% (w / v) sucrose) was supplemented with 0.1% (v / v) of each of liquid LMPs (106S-YA, 104AS, or 5CS), or alternatively one particle of carrier / well. Bacterial cultures were grown overnight in LB, washed in M9, and normalized to 1×10 7 CFU / ml. 20 μL of the standardized M9-treated bacterial culture was transferred to fresh 180 μL of M9 minimal medium supplemented with LMP in a 96-well microtiter plate to 1×10 6 Final bacterial concentration in CFU / ml. Bacterial growth was recorded for 48 h at 28°C. Each data point represents 8 replicates ± SD, and the experiment was repeated 3 times with similar results.

[0340] Secretion of plant cell wall degrading enzymes (PCWDEs) via SREs induced by LMP

[0341] SRE cells were grown overnight at 28°C with shaking at 150 rpm, washed twice in M9 medium, and diluted to 10 in 4 ml of M9 supplemented with liquid 0.1% LMP (106S-YA, 104AS, or 5CS). 6CFU / ml. The culture was incubated at 28°C with shaking at 150 rpm for 24 hours. 2 ml of the suspension was centrifuged at 13,000 rpm for 5 minutes, and 20 μL of the supernatant was used for semiquantitative plate assays of pectate lyase (Pel), polygalacturonase (Peh) and protease (Prt) activities as described by Chatterjee et al. (Chatterjee et al., 1995. Appl Environ Microbiol, 61, 1959-1967). Each plate contained a specific activity medium: for Pel, 1% polygalacturonic acid (PGA), 1% yeast extract, 100 mM Tris-HCl (pH=8.5) (1 M stock), 0.8% agar; for Peh, 1% polygalacturonic acid (PGA), 1% yeast extract, 2.2 mM ETDA (0.5 M stock), 0.8% agar; and for Prt, 3% gelatin, 0.4% nutrient broth and 0.8% agar. The plates were pierced to form 4 mm holes, sealed with melted agarose, and the supernatant containing the enzymes was added and incubated at 28°C for 18-24 hours, after which the Pel and Peh halos were visualized with 4N HCl, and for Prt activity, incubated for 24-36 hours. The activity was expressed as the size of the halo observed. Three independent experiments were performed, each with four replicates.

[0342] SRE chemotaxis towards LMP carriers

[0343] Chemotaxis was tested using culture dishes containing M9 minimal medium supplemented with 0.25% agar. Different LMPs (106S-YA, 104AS or 5CS) were applied at equal distances in the center as spherical carriers (4 on each plate). Pcb were grown in fresh LB medium containing 100 μg / ml ampicillin and incubated overnight at 28°C with continuous shaking at 150 rpm. Next, the bacterial culture was centrifuged at 13,000 rpm for 3 minutes and resuspended in DDW. 1×10 8 A 5 μL droplet of 100 CFU / ml of the bacterial strain (GFP-labeled Pcb) was applied 1.5 cm from each carrier. After 48 hours of incubation at 28°C, the bacteria had migrated towards the pectin carriers and GFP fluorescence was detected using a Leica MZFLIII stereomicroscope (Germany) with a Nikon DS-Fi1 digital camera.

[0344] Optimization of 5CS-based LMP vector

[0345] The support was prepared as described above except that the calcium chloride concentration was reduced to 0.35% (w / w) and the 5CSLMP concentration was reduced to 1% (w / w). The 5CSLMP was prepared using an electronic pipette ( Gilson, USA) prepared these carriers in smaller sizes by the following method: 20 μl and 5 μl droplets were injected into the cross-linking solution, thereby producing carriers with diameters of ~3-mm and 2-mm, respectively. Further optimization of the 5CS carrier was achieved by adding gelatin, which was prepared by the following method: gelatin powder (cow hide gelatin, Sigma, USA) was dissolved in HEPES and then autoclaved at 121°C for 20 minutes. The solution was mixed to a final concentration of 1% pectin + 2% gelatin or 1% pectin + 5% gelatin, and the carrier was obtained from a 5 μl droplet in a 0.35% CaCl2 solution. The carrier was then dried in a vacuum oven (Binder, Germany) at 37°C and 400 mbar.

[0346] Sphericity of pectin gel carrier

[0347] Digital photographs of LMP gel carriers were taken under binoculars (5ZX16 model, Olympus America Inc., Center Valley, PA, USA), and their sizes were measured using image analysis software Image J (version 1.48d, NIH, Bethesda, MD, USA). Sphericity was defined by calculating the ratio of the small diameter to the large diameter of a sphere:

[0348]

[0349] Values ​​closer to 1 indicate rounder beads (a value of 1 indicates a perfect sphere). Twelve replicates from two different batches were measured.

[0350] Mechanical properties of the carrier

[0351] The LMP gel carrier was compressed between parallel lubricated plates to failure at a constant deformation rate of 10 mm / min using an Instron Universal Testing Machine (UTM) (Model 5544, Instron Corporation, Canton, MA, USA). The software Bluehill 3 enables the conversion of the Instron voltage-time measurement data into digital force-deformation values. Stress (σ) and strain (ε E ) is defined as follows:

[0352] σ=F / A o

[0353] εE =ΔH / H o

[0354] Where F is the force at fracture (N), H o is the initial height of the carrier (m), ΔH is the total deformation (m), and A o is the initial cross-sectional area of ​​the carrier (m 2 ).

[0355] Swelling ratio measurement of LMP gel carrier

[0356] The LMP gel carrier was introduced into a plastic tube containing DW. To avoid the volume dilution effect, the volume ratio of liquid to sample exceeded 100:1. Periodically, the carrier was taken out of the tube, wiped with filter paper, and weighed (± 0.001 g) on ​​an analytical balance (Precisa, Dietikon, Switzerland). The test was terminated when the sample reached constant weight (balance, within 4-5 days).

[0357] The equilibrium swelling ratio by weight is calculated as follows:

[0358]

[0359] where W(initial) is the initial sample weight (g), and W(final) is the final sample weight (g) at equilibrium.

[0360] Reswelling capacity of dried LMP carriers

[0361] The dried support introduced into the DW at 25°C was weighed regularly (±0.001 g) on ​​an analytical balance (Precisa, Dietikon, Switzerland). The test was terminated when the sample reached constant weight (equilibrium), usually after less than ~24 hours. It is noteworthy that support swelling began within a few minutes.

[0362] The reswell capacity is calculated as follows:

[0363]

[0364] where W(initial wet state) is the weight of the sample as a gel carrier before drying (g), and W(final) is the weight of the re-swollen dry sample at equilibrium (g).

[0365] Preparation of LMP vector for BALO encapsulation

[0366] 5CS LMP vector compositions were prepared as above at a final concentration of 1% LMP + 5% gelatin. Concentrated bdelloids were then added to the LMP-gelatin solution at a 1:9 volume ratio and vectors were obtained from 20 μl or 5 μl drops in 0.35% CaCl2 solution followed by vacuum drying as above.

[0367] Encapsulation efficiency

[0368] To release encapsulated bdelloid plasmids from the LMP dry carrier, the carrier was mixed with a sterile 2% sodium hexametaphosphate (SHMP) solution and shaken until completely dissolved. Previous studies and preliminary experiments showed that SHMP had no harmful effects on predators (Sivakala et al., 2021. Journal of Applied Microbiology, 131, 2971-2980). The density of viable cells in the suspension was obtained by dilution plating and PFU counting in double-layer agar. EE was calculated as described above in Example 2 herein.

[0369] Scanning electron microscopy (SEM)

[0370] The dried LMP gel carriers with encapsulated predators and the bacteria residing within the carriers were visualized using SEM and high-resolution SEM (Tokyo, Japan), respectively, as previously described herein above in Example 2.

[0371] Vector degradation induced by SRE

[0372] Pcb were grown as above and subcultured in fresh LB to an OD of ~0.2. 600 , and washed twice in aHEPES buffer. 600 Adjust to 0.2 and then dilute to produce ~10 7CFU / ml. Pcb-induced degradation of LMP gel carriers prepared from different LMPs and different preparations of 5CS-based carriers was measured in double diluted NB medium (DDNB) amended with 2mM CaCl2 and 3mM MgCl2·6H2O in shake flasks at 28°C. One carrier was placed in 10 ml of DDNB with or without Pcb, in 6 replicates from two different batches. After 24, 48 and 72 hours, the carrier was removed from the tube, wiped with sterile filter paper, and weighed (±0.001 g) on ​​an analytical balance (Precisa, Dietikon, Switzerland) under sterile conditions. The weight change was calculated as the percentage difference between the initial and final weight of the carrier.

[0373] Predation dynamics of encapsulated predators released by SRE

[0374] Predation by encapsulated bdelloid plasmids of Bdellovibrio spp. HD100-Td-tomato in LMP dry carriers (obtained from 5 μl droplets) was followed by fluorescence (predator growth) and OD (prey decay) in a plate reader incubated at 28°C with shaking and measurements every 20 min (Tecan Spark 10M, Switzerland). The detectable thresholds for low and high levels of fluorescence corresponded to ∼4 × 10 7 and ~10 9 PFU / ml of Bdellovibrio spp. HD100. 1 ml of Pcb suspension (OD 600 =10) were added to 9 ml of amended DDNB and 200 μl aliquots were pipetted onto the wells of clear black flat-bottom 96-well plates (Greiner-Bio One, Germany) containing 1, 2 or 3 vectors in the wells. The experiments were performed twice independently and included three technical replicates / treatment.

[0375] Embodiment 11

[0376] Physical and mechanical properties of LMP carriers

[0377] pH adjustment and pasteurization significantly and differently affect the carriers, depending on the LMP material. Table 5 demonstrates that, while the diameters of the 106AS-YA and 5CS carriers decreased, the diameter of the 104AS did not change significantly. However, an increase in the sphericity of the carriers was consistently detected. Table 6 presents the results regarding load at break, stress, and strain at break measurements, which in all cases show increased values, but with large differences between the formulations tested. Table 7 presents the swelling ratios of the carriers after pH adjustment and pasteurization. The stability of the carriers was investigated by immersing them in DW; the carriers did not disintegrate, but swelled or shrunk by ~20% (Table 7). In summary, the enhanced features of the pH-adjusted, pasteurized carriers can contribute to their durability during application.

[0378] Table 5: Physical properties of LMP gel carriers without (-) and with (+) pH adjustment and pasteurization.

[0379]

[0380] Different superscript letters in the columns indicate significant differences (Student's t-test, P<0.05). Results are mean±SD of at least 10 replicates from two different batches.

[0381] Table 6: Mechanical properties of LMP gel carriers without (-) and with (+) pH adjustment and pasteurization.

[0382]

[0383] Different superscript letters in the columns indicate significant differences (Student's t-test, P<0.05). Results are mean±SD of at least 10 replicates from two different batches.

[0384] Table 7: Equilibrium swelling ratio values ​​of pectin gel carrier without (-) and with (+) pH adjustment and pasteurization in DW at 25°C.

[0385]

[0386] Different superscript letters in the columns indicate significant differences (Student's t-test, P<0.05). Results are mean±SD of at least 10 replicates from two different batches.

[0387] Example 12

[0388] Growth of Soft Rot Enterobacteriaceae (SRE) on LMP

[0389] SRE growth on LMPs 106S-YA, 104AS and 5CS (in solution or as a gel carrier) was tested in M9 minimal medium, where the material served as the sole carbon source. Initially, a calibration was performed to determine the inoculum size and LMP concentration. Therefore, 0.1% (w / w) LMP (or alternatively, 1 carrier / well in a 96-well plate) and 10 6 CFU / ml of inoculum. Solutions of all three materials were able to allow SRE growth, with 5CS producing significantly higher populations (Figure 14). Very poor growth was obtained with 106S-YA, 104AS vectors, in contrast to the 5CS vector, which produced populations at levels similar to those obtained in the 5CS solution, suggesting that the 5CS vector was dissolved and consumed (Figure 15).

[0390] Example 13

[0391] Secretion of plant cell wall degrading enzymes (PCWDEs) via SREs induced by LMP

[0392] LMP-dependent PCWDE secretion was tested by growing Enterobacterium rotum (SRE) for 8 h with LMP (as sole carbon source) and using culture supernatants in petri dishes containing each of the pectolytic enzymes pectate lyase (Pel) and polygalacturonase (Peh) and a protease-specific medium (Prt). All LMP solutions induced secretion of PCWDE. Among them, 5CS induced significantly higher Pel and Peh activities in the SRE strains tested. However, the different LMP solutions did not induce differential Prt secretion ( Figures 16A-16C These results are consistent with Figures 14A-14C and Figures 15A-15C and indicate that 5CS pectin is highly suitable as a material for inducing SRE pectinolytic activity to release BALOs in a pectin carrier.

[0393] Embodiment 14

[0394] SRE chemotaxis towards LMP carriers

[0395] The chemotaxis of green fluorescent protein (GFP)-tagged Pcb strains toward different LMP sources was tested in soft basic agar plates ( Fig.17A ). Figures 17B-17D It was shown that after 48 hours, the bacteria had migrated and taken up residence in the capsid of the 5CS LMP vector ( Fig. 17B ), and probably still multiplying within it, while much lower fluorescence was emitted from the 104AS and 106AS-YA vectors ( Fig. 17C and 17D), which suggests a lower associated bacterial population. Based on these results, it can be concluded that Pcb are attracted to the LMP 5CS carriers (in which the cells multiply).

[0396] Embodiment 15

[0397] Degradation kinetics of LMP carriers by SRE

[0398] The degradation of different LMP gel carriers by SRE was followed in DDNB with or without Pcb for 3 days. At the end of this period, the degradation of different LMP gel carriers by SRE was compared to the same carriers immersed in medium only ( Fig.18A ), the size of 5CS LMP carriers in the presence of Pcb was smaller and the surface was rougher ( Fig.18D ). In contrast, there were no noticeable changes in the appearance of the two other carriers ( Fig.18E and 18F ). Figure 18G It was shown that all carriers underwent weight changes in the culture medium alone, which may be due to gradual swelling (for 104AS and 106AS-YA) or shrinking (for 5CS) until equilibrium was reached. The tendency of each carrier type to swell or shrink in DDNB was consistent with the trends observed when those carriers were immersed in DW (Table 8). Figure 18G It was further shown that all LMP vectors lost more weight when incubated with Pcb compared to medium alone, with the differences increasing over time. The 5CS vector lost significantly more weight, losing approximately 40% of its initial weight within 72 hours of incubation with Pcb.

[0399] It is further observed in Table 8 that in the DDNB control treatment, the strength of the different carriers decreased compared to before treatment (Table 6). These results can be explained by the diffusion of cross-linking ions from the carriers to the medium, thereby weakening the structure of the carriers. Incubation with Pcb significantly weakened and embrittled all carriers (Table 8).

[0400] Table 8: Mechanical properties of LMP carriers incubated in DDNB without or with Pcb for 72 hours.

[0401]

[0402] Different superscript letters in the columns indicate significant differences (Student's t-test, P<0.05). Results are mean±SD of 6 replicates from two different batches.

[0403] Example 16

[0404] Optimization of pectin carrier based on 5CS

[0405] The 5CS LMP carrier was optimized to allow for faster SRE-induced degradation by examining the effects of reducing the CaCl2 crosslinker concentration and carrier size from 1% to 0.35% and from 4 mm to 2 or 3 mm in diameter, respectively. Figures 19A-19E It was shown that all carriers exposed to Pcb in DDNB lost significantly more weight than the DDNB control. Further, during the first 48 hours of incubation with Pcb, carriers of different sizes lost 50% of their weight. However, after 72 hours, the smaller carriers had lost approximately 80% of their initial weight, compared to 65% for the larger carriers ( Fig.19E This value is twice that obtained with the previous formulation ( Figures 18A-18G ).

[0406] Lowering the LMP concentration from 3% to 1% caused the support to become less stable, as revealed by the large weight loss that occurred even in the absence of Pcb ( Figure 20G Since gelatin can form ionic interactions with LMP and thereby improve gel properties, the effect of adding 2% and 5% gelatin to the matrix was examined. Figures 20A-20G It was demonstrated that gelatin addition stabilized the carriers, as they lost significantly less weight in the absence of Pcb. However, in the presence of Pcb, carriers with different gelatin concentrations lost approximately 80% of their initial weight within 48 hours. Before 72 hours, their weight could no longer be measured by existing means ( Figure 20G ).

[0407] The effect of vacuum drying of the LMP carriers was further examined. The reswelling capacity of the dried 5CS LMP carriers is an important parameter, as enzymatic degradation may be more effective in carriers that exhibit good reswelling (indicating a lower structural density). Table 9 demonstrates that the addition of gelatin to the LMP carriers resulted in improved reswelling capacity.

[0408] Table 9: Reswelling capacity of carriers comprising 5CS LMP and gelatin dried in DW at 25°C.

[0409]

[0410] Different superscript letters in the columns indicate significant differences (Student's t-test, P<0.05). Results are mean±SD of 10 replicates from two different batches.

[0411] Therefore, the dry carrier containing 1% LMP + 5% gelatin was first rewetted in DW for 24 hours and then incubated with Pcb in DDNB for 24 hours, resulting in a weight loss of approximately 88% ( Fig.21D), a value that can be reached in only 48 hours without a drying and rewetting step, as can be seen in Figure 20G After 48 hours, the weight was no longer measurable, and after 72 hours, the carrier had completely disintegrated ( Figures 21A-21D ).

[0412] Finally, the optimized carriers were examined for their ability to encapsulate a mixture of Bdellovibrio AP cells and bleedoplasts and preserve their viability. High numbers of live predatory bacteria (1.4 × 10 9 PFU / g carrier), although there was a 2-log decrease after the dehydration process. The initial entrapment of predators in the carrier before drying was approximately 57% (Table 10).

[0413] Table 10: Density and entrapment efficiency of Bdellovibrio bacteriovorus in optimized, dried pectin-gelatin carriers.

[0414]

[0415] Results represent the mean ± SD of two independent experiments with three technical replicates for each treatment. In each test, five vectors were evaluated.

[0416] Embodiment 17

[0417] Scanning electron microscopy of LMP and gelatin carriers

[0418] Observation under SEM revealed a thin shell wall and an empty core, which could explain the rapid degradation of the optimized carrier by Pcb. Figures 22A-22D A relatively smooth surface with few salt crystals was revealed. Further analysis with high-resolution SEM allowed visualization of Bdellovibrio bacteriovorus embedded within the matrix, either as AP cells or as elongated cells within the bdelloplasts ( Figures 22E-22F ). AP cells with their flagella can be seen, suggesting that the predator will be easily mobile after release. This mixed population results from the experimental protocol used to obtain bdelloids, in which AP cells were at twice the prey concentration.

[0419] Embodiment 18

[0420] Predation dynamics of encapsulated predators released by SRE

[0421] To test the predation efficiency of predators encapsulated in the optimized dry LMP carriers, Bdellovibrio spp. HD100 expressing Td-tomato fluorescent protein was immobilized in the carriers and vacuum dried. Pcb were inoculated in microwells with 1, 2, or 3 carriers ( Figures 23A-23BAll treatments with the vector resulted in a significant reduction in pathogen population size, as reflected by a decrease in OD values ​​( Fig.23A ), and lead to an increase in predator populations, as visualized by fluorescence ( Fig. 23B ). The final fluorescence level increased with the number of vectors, but the final prey OD values ​​were not consistent (i.e., lower OD values ​​were obtained with lower numbers of vectors). It should be noted that within this range, the RFU is linear and therefore reflects an approximately 1.5-fold difference in predator population density. Without wishing to be bound by any theory or mechanism of action, it is considered that the controlled release of the predator operates by gradually degrading the matrix of the vector via the enzymatic activity of the pathogen, resulting in predation on Pcb. Supporting this point, no plaques were observed from samples collected after 96 hours from the culture medium in the control wells containing the vector without Pcb. This suggests that the vector is not leaky and that degradation of the matrix is ​​required for predator release.

[0422] Embodiment 19

[0423] Protection of potato tubers against soft rot by encapsulated preparations of Bdellovibrio spp.

[0424] Bacterial strains, media, and growth conditions

[0425] The predatory bacteria used in this study was Bdellovibrio bacteriovorus HD100 (ATCC 15356). Escherichia coli ML35 (ATCC 43827) was used as prey bacteria.

[0426] Starting from a single colony derived from a laboratory stock stored at -80°C, E. coli was grown in LB medium at 37°C in a shake flask at 250 rpm. Overnight cultures were centrifuged at 3620 g for 10 min at 4°C, and the cell pellets were washed and resuspended in HEPES buffer (pH 7.8) amended with 2 mM CaCl2 and 3 mM MgCl2.6H2O (amended HEPES-aHEPES) to a final optical density (OD) of 10. 600 The E. coli suspension was further concentrated to produce ~10 11 The stock suspension was stored at 4°C until use, for up to two weeks.

[0427] To initiate the prey co-culture, transfer a bacteriological loopful from a frozen stock of the predator into a 1.5 ml plate containing OD 20 diluted in 15 mL aHEPES. 600 = 10 bottles of E. coli suspension. The co-culture was incubated at 28°C and 250 rpm until the turbidity of the culture reached below OD 600=0.1. The suspension was then filtered through a 0.45 μm filter (Sartorius). Thereafter, 5 ml of the filtered predatory cells from the starter culture were inoculated into 40 ml of aHEPES in a 250 mL bottle (which contained 5 ml of OD 600 =10 E. coli suspension). The bottle was shaken at 250 rpm at 28°C. The growth of the predator was monitored by the decrease in turbidity of the suspension. After most of the prey cells were lysed, the suspension was filtered through a 0.45 μm filter (Sartorius) to produce ~10 9 Plaque forming units (PFU) / ml of predatory cells. The filtrate was stored at 4°C until use, up to one day, without loss of viability. To further concentrate the filtered predatory cells, the suspension (50 ml) was centrifuged at 12,497 g for 20 minutes at 10°C, and the cell pellet was resuspended in 0.5 ml of aHEPES to ~10 11 The final concentration of predatory cells was 240 PFU / ml. To produce large quantities of bdelloids for encapsulation, synchronized cultures of predators were obtained by adding 250 μl of concentrated E. coli suspension (1:2 prey:predator) to 500 μl of concentrated AP cell suspension and incubating for about 1 hour at 28°C and 250 rpm. This experimental protocol resulted in a ratio of approximately 1:1 (AP cells:bdelloids). Bdelloid formation was verified by phase contrast microscopy. Prey and predator cultures were routinely counted by dilution plating as CFU / ml and PFU / ml, respectively. The latter plating was performed in double-layer agar (Jurkevitch 2012).

[0428] For the infection assay of potato tubers, Pectobacterium brasiliensis (Pcb 1692) was used. Bacterial cells were grown overnight in LB at 28°C, 150 rpm, washed twice in M9 medium, and normalized to 5×10 6 CFU / ml or 5×10 8 CFU / ml.

[0429] Preparation of kappa-carrageenan-based carriers for predator encapsulation

[0430] κ-carrageenan dispersion (1.67% (w / w)) was prepared by adding κ-carrageenan powder (Sigma, USA) to DW. Then, trehalose (Sigma, USA) was added to a final concentration of 5% (w / w). Then, the mixture was brought to boiling to complete the colloid dissolution. Then, the concentrated broomraplasts were added to a κ-carrageenan-based solution at 1:9 volume ratio at 40°C (yielding a final concentration of 1.5% carrageenan). The final mixture was dripped into a 0.5% (w / w) sterile KCl solution (Biolab Chemicals, Israel) at a volume ratio of 1:100 and stirred for 20 minutes. Spontaneous cross-linking reaction produced spherical gel / wet carrier. Then, the gel carrier was dried in a vacuum oven (Binder, Germany) at 37°C and 400mbar. To obtain a powder, the dry carrier was ground with an electric grinder (Graetz, Germany).

[0431] Preparation of pectin-based carriers for predator encapsulation

[0432] The carrier was prepared using 5CS pectin (CPKelco, Copenhagen, Denmark) with a degree of esterification (DE) of 7% and a degree of amidation (DA) of 0%. Pectin was purified and pasteurized before use. Gelatin solution was prepared in the following manner: gelatin powder (kraft gelatin, Sigma, USA) was dissolved in HEPES (or HEPES with trehalose), followed by autoclaving at 121°C for 20 minutes. The solution was mixed to a final concentration of 1% pectin + 5% gelatin or 1% pectin + 5% gelatin + 5% trehalose. Then, concentrated leech plasmids were added to the solution in a 1:9 volume ratio, and the suspension was then dripped into a sterile 0.35% CaCl2 solution, followed by vacuum drying at 37°C and 400mbar. In order to obtain powder, the dry carrier was ground with an electric grinder (Graetz, Germany).

[0433] Counting the number of bacteria in the vector

[0434] For quantitative measurement of cell viability, dry carrageenan-based or pectin-based carriers were immersed in sterile DW or sterile 2% sodium hexametaphosphate (SHMP), respectively. The mixture was then shaken until the carrier was completely dissolved. PFU (which represents viable cells) can be counted by dilution plating in double agar.

[0435] Stability of encapsulated predators under storage

[0436] The viability of encapsulated predators in carrageenan-based or pectin-based carriers (which were stored in sealed glass bottles containing silica gel) was measured over a period of one year or until no live bacteria were detected, which were stored at 3 different temperatures: 25°C, 4°C and -20°C. A liquid suspension of AP cells prepared as described above in this article and stored at the same temperature served as a control. Bacterial counts in the carrier / suspension were performed as described above, spanning intervals from weeks to months.

[0437] Infection assay and vector-based protection of potato tubers in small containers

[0438] White potato cv. 'Nicola' (with an average weight of ˜30 g) was sourced from a local supermarket. Potatoes were surface sterilized by soaking in a 0.5% sodium hypochlorite (NaOCl) solution for 20 min, washed twice with sterile DW (for 15 min), and allowed to dry in a laminar flow sterile fume hood. The Pectobacterium brasiliensis suspension was prepared as described above. Potato tubers were prepared by immersing them in 5×10 8 CFU / ml concentration of pathogen culture for 10 minutes. After inoculation, the tubers were planted in sterilized plant pots with a 6 cm 2 The cells were filled with autoclaved general purpose potting garden soil mix (ECOTERRA+ enriched with nutrients consisting of 70:20:10 coconut fiber, peat and compost). The treatments (2 ml of BALO suspension) were applied to the top of each potato tuber before covering with soil, or to the soil below the tuber, or near the corner of the pot. For the control treatment, 2 ml of DW was used. The tested treatments included dissolved carrageenan-based carrier (in tap water to produce ~10 8 PFU / ml) or predator suspension (~10 6 or ~10 8 PFU / ml). The pots were introduced into plastic boxes in a controlled environment room at 25°C / 28°C (±1°C), high relative humidity and a photoperiod of 16 hours fluorescent-8 hours dark. The humidity level was maintained by covering the plastic boxes / pots with plastic wrap. Seven or 14 days after infection, tubers were extracted and the average rot (%) and the number of infected potatoes were recorded. The disease index was calculated as follows: average rot (%) × number of infected potatoes. All experiments were repeated four times with 15 replicates / treatment.

[0439] Infection assay and vector-based protection of potato tubers in screenhouses

[0440] The experiment was carried out in a net house. Potatoes (average tuber weight of ~85 grams) were planted and irrigated twice a week by overhead spraying. The experiment was carried out for one month. Potatoes were prepared as above and treated with 2 ml of the dissolved carrier suspension, 4 times in total. Initial application was on the top of the potato tuber before covering with soil, and subsequent application was carried out directly on the soil. All experiments were repeated twice, with 32 repetitions / treatments.

[0441] The method described in this example is used in the examples described herein below.

[0442] Embodiment 20

[0443] Stability of encapsulated predators under storage

[0444] To examine the shelf life of dry formulations of encapsulated BALOs, the survival of Bdellovibrio spp. HD100 encapsulated in two different carriers (a carrageenan-based carrier and a pectin-gelatin-based carrier) was evaluated during one year of storage at three temperatures (25°C, 4°C, and -20°C) and compared with the survival of liquid suspensions of predator AP cells stored at the same temperature.

[0445] The carrageenan-trehalose carrier showed high stability at all temperatures ( Fig.24 ). In contrast, the cell density in liquid suspension at 25°C decreased dramatically: only 1.0 × 10 2 live bacteria, and no live bacteria were detected after two months (Table 11 and Fig.24 ). At 4°C, the decay rate of bacteria in liquid suspension was slower, but survival dropped dramatically after five months until no viable cells could be detected after seven months of storage. Although storage at -20°C improved survival, the concentration of predators in suspension was still reduced by approximately two orders of magnitude ( Fig.24 ).

[0446] Table 11: Survival of Bdellovibrio bacteriovorus HD100 in carrageenan-trehalose carrier and in liquid suspension at 25°C.

[0447]

[0448] The pectin-gelatin dry carriers showed improved survival at room temperature relative to the bacterial suspension, with a lower decay rate. However, after 3 months, no viable cells could be detected in the carriers at both 25°C and 4°C ( Fig.24The addition of trehalose significantly improved survival at all temperatures, with little loss and approximately an order of magnitude decrease in viability recorded for storage at -20°C and 4°C, respectively ( Fig.24 ). These results demonstrate the greater stability, and hence longer shelf life, of encapsulated Bdellovibrio preparations compared to liquid predator suspensions.

[0449] Embodiment 21

[0450] Protection of potato tubers against soft rot by predator formulations under controlled conditions

[0451] To evaluate the protective effect of potato tubers against P. brasiliensis by encapsulated Bdellovibrio spp. HD100, potato tubers were immersed in a suspension of P. brasiliensis and then treated with water as a control or with predators encapsulated in carrageenan-trehalose, the latter obtained after dissolving the dry carrier in water, which were applied to the top of the potato tubers. Incubation lasted for 7 days at 25°C under moist conditions. The effect of the encapsulated predators was compared with that of the predators at the same density (10 8 PFU / ml) which was also applied on the top of potato tubers.

[0452] Protection by the hydrated formulation of kappa-carrageenan was similar to that obtained by the predator suspension, with significantly lower mean values ​​of tissue decay (approximately 50%) and disease index ( Figures 25A-25B ).

[0453] Embodiment 22

[0454] Protection of potato tubers against soft rot by predator formulations under screenhouse conditions

[0455] Potato tuber experiments were conducted in pots in a screen house with controlled irrigation for four weeks until germination. Day / night conditions were natural conditions at ambient temperature, sometimes reaching 30°C.

[0456] Both the AP predator suspension and the dissolved predator-containing carrier increased the number of infected potatoes ( Fig.26B ),%rot( Fig.26D ) and disease index ( Fig.26E ) is reduced by at least ˜50%. Thus, not only does the cyst keep the predators alive for a long period of time, but it does not alter their potency in providing protection against soft rot disease, even under natural conditions.

[0457] Although the present invention has been specifically described, it will be appreciated by those skilled in the art that many changes and modifications may be made. Therefore, the present invention should not be interpreted as being limited to the specifically described embodiments, and the scope and concept of the present invention will be more easily understood by reference to the claims below.

[0458] References

[0459] Aharon,E.,Mookherjee,A.,Pérez- F.,da Silva,GM,Sathyamoorthy,R.,Burdman,S.,&Jurkevitch,E.(2021).Secretion systems play acritical role in resistance to predation by Bdellovibriobacteriovorus.Research in microbiology,172(7-8),103878.

[0460] Cao,H.,Wang,H.,Yu,J.,An,J.,&Chen,J.(2019).Encapsulated Bdellovibriopowder as a potential bio-disinfectant against whiteleg shrimp-pathogenicVibrios.Microorganisms,7(8),244.

[0461] Duncan, MC, Forbes, JC, Nguyen, Y., Shull, LM, Gillette, RK, Lazinski, DW, Ali, A., Shanks, RMQ, Kadouri, DE, & Camilli, A. (2018). Vibrio choleraemotility exerts drag force to impede attack by the bacterial predator Bdellovibrio bacteriovorus. Nature communications,9(1),1-9.

[0462] Fan,C.,Guo,M.,Liang,Y.,Dong,H.,Ding,G.,Zhang,W.,Tang,G.,Yang,J.,Kong,D.,&Cao,Y.(2017).Pectin-conjugated silica microcapsules as dual-responsivecarriers for increasing the stability and antimicrobial efficacy ofkasugamycin.Carbohydrate polymers,172,322-331.

[0463] González,E.,Herencias,C.,&Prieto,M.A.(2020).Apolyhydroxyalkanoate-based encapsulating strategy for‘bioplasticizing’microorganisms.MicrobialBiotechnology,13(1),185-198.

[0464] Jung,J.,Arnold,R.D.,&Wicker,L.(2013).Pectin and charge modifiedpectin hydrogel beads as a colon-targeted drug delivery carrier.Colloids andSurfaces B:Biointerfaces,104,116-121.

[0465] Liu,Y.,Sun,Y.,Ding,G.,Geng,Q.,Zhu,J.,Guo,M.,Duan,Y.,Wang,B.,&Cao,Y.(2015).Synthesis,characterization,and application of microbe-triggeredcontrolled-release kasugamycin-pectin conjugate.Journal of agricultural andfood chemistry,63(17),4263-4268.

[0466] Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, et al. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular plant pathology, 13(6), 614 - 629.

[0467] Necas, J., & Bartosikova, L. (2013). Carrageenan: a review. Veterinární medicina, 58(6).

[0468] Nussinovitch, A., & Hirashima, M. (2013). Cooking innovations. Boca Raton, FL: CRC Press (Chapter 4).

[0469] Rotem, O., Pasternak, Z., & Jurkevitch, E. (2014). Bdellovibrio and Like Organisms. In the Prokaryotes: Deltaproteobacteria and Epsilonproteobacteria. Rosenberg, E., DeLong, E.F., Lory, S., Stackebrandt, E., and Thompson, F. (eds). Berlin, Heidelberg: Springer Berlin Heidelberg, pp. 3 - 17.

[0470] Shemesh, Y., & Jurkevitch, E. (2004). Plastic phenotypic resistance to predation by Bdellovibrio and like organisms in bacterial prey. Environmental Microbiology, 6(1), 12 - 18.

[0471] Sockett,R.E.(2009).Predatory lifestyle of Bdellovibriobacteriovorus.Annual review of microbiology,63,523-539.

[0472] Thrimawithana,T.R.,Young,S.,Dunstan,D.E.,&Alany,R.G.(2010).Textureand rheological characterization of kappa and iota carrageenan in thepresenceof counter ions.Carbohydrate Polymers,82(1),69-77.

[0473] Vemmer,M.,&Patel,A.V.(2013).Review of encapsulation methodssuitablefor microbial biological control agents.Biological Control,67(3),380-389.

Claims

1. A system for differential delivery of live Bdellovibrio and similar organisms (BALOs), comprising: a carrier comprising a polysaccharide hydrocolloid; and BALOs immobilized in the carrier, wherein the BALOs are in a physiological state selected from the group consisting of bdelloid plasmids, bdelloid cysts, and attack phase cells (AP), which are present at a rate of at least about 1.0×10 8 The cell density of PFU / g vector exists, wherein the polysaccharide hydrocolloid is in the form of a wet or dry gel, and wherein the carrier preserves the viability and bacteriolytic efficacy of the immobilized BALO.

2. The system of claim 1, wherein the polysaccharide hydrocolloid is in the form of a dried gel and the system is a dry system.

3. The system of any one of claims 1 and 2, wherein the polysaccharide hydrocolloid is selected from the group consisting of: kappa-carrageenan, pectin, alginate, agar, gellan gum, carboxymethyl cellulose (CMC), furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan, and derivatives and combinations thereof.

4. The system of claim 3, wherein the polysaccharide hydrocolloid is kappa-carrageenan or low methoxyl pectin (LMP).

5. The system according to any one of claims 3 and 4, wherein the polysaccharide hydrocolloid is present in the system in a weight percentage of about 2% to about 25% based on the total weight of the dry system.

6. The system according to any one of claims 1 to 5, wherein the polysaccharide hydrocolloid is cross-linked by a cross-linking agent and / or a gelation inducing agent.

7. The system of claim 6, wherein the crosslinking agent comprises a metal cation selected from the group consisting of: potassium (K + ), calcium (Ca 2+ ), barium (Ba 2+ ), magnesium (Mg 2+ ), aluminum (Al 3+ )、Zn 2+ ), lead (Pb 2+ ), ferrous iron (Fe 2 + ), Sr 2+ ), copper (Cu 2+ ), cadmium (Cd 2+ ), nickel (Ni 2+ ), cobalt (Co 2+ ) and their combinations.

8. The system according to any one of claims 1 to 7, wherein the carrier further comprises a protein hydrocolloid and / or a protein.

9. The system of claim 8, wherein the protein hydrocolloid is gelatin and is present in the system in a weight percentage of about 20% to about 85% based on the total weight of the dry system.

10. The system according to any one of claims 1 to 9, wherein the carrier further comprises at least one osmoprotectant.

11. The system of claim 10, wherein the at least one osmoprotectant is selected from the group consisting of trehalose, inositol, glycerol, arabinitol, betaine, amino acids, and combinations thereof.

12. The system of any one of claims 10 and 11, wherein the at least one osmoprotectant is present in the system in a weight percentage of about 20% to about 85% based on the total weight of the dry system.

13. The system of any one of claims 2 to 12, wherein the system comprises about 0.1% (w / w) to about 10% (w / w) water based on the total dry system weight.

14. The system of any one of claims 1 to 13, wherein the differential delivery of the BALO comprises a rapid release or a delayed release of the BALO from the carrier.

15. The system of claim 14, wherein the carrier suitable for rapid release of BALO comprises kappa-carrageenan and potassium cations.

16. The system of claim 15, wherein the carrier further comprises trehalose.

17. The system of claim 16, wherein kappa-carrageenan is present in the system at a weight percent of about 5% to about 25% based on the total weight of the dry system, potassium cations are present in the system at a weight percent of about 0.5% to about 10% based on the total weight of the dry system, and trehalose is present in the system at a weight percent of about 25% to about 85% based on the total weight of the dry system.

18. The system of claim 17, wherein kappa-carrageenan is present in the system at a weight percent of about 15% to about 25% based on the total weight of the dry system, potassium cations are present in the system at a weight percent of about 0.5% to about 10% based on the total weight of the dry system, and trehalose is present in the system at a weight percent of about 70% to about 80% based on the total weight of the dry system.

19. The system of claim 14, wherein the carrier suitable for delayed release of BALO comprises low methoxyl pectin (LMP) and calcium cations.

20. The system of claim 19, wherein the carrier further comprises gelatin or a water absorbent.

21. The system of claim 20, wherein LMP is present in the system in a weight percentage of from about 2% to about 15% of the total weight of the dry system, and gelatin is present in the system in a weight percentage from about 20% to about 85% of the total weight of the dry system.

22. The system according to any one of claims 19 to 21, wherein the carrier further comprises trehalose.

23. The system of claim 22, wherein LMP is present in the system at a weight percent of about 2% to about 15% based on the total weight of the dry system, calcium cations are present in the system at a weight percent of about 0.1% to about 5% based on the total weight of the dry system, gelatin is present in the system at a weight percent of about 20% to about 50% based on the total weight of the dry system, and trehalose is present in the system at a weight percent of about 20% to about 50% based on the total weight of the dry system.

24. The system according to any one of claims 22 and 23, wherein LMP, gelatin and trehalose are present in the dry system in a weight percent ratio of 1:1:1 to 1:10:

10.

25. A method for preventing, reducing or eliminating a phytopathogenic bacterial disease in a plant, plant part or crop before or after harvest, comprising contacting the plant, plant part or crop with an effective amount of a system according to any one of claims 1 to 24 before or after harvest, thereby preventing, reducing or eliminating the phytopathogenic bacterial disease in the plant, plant part or crop.

26. The method of claim 25, wherein the phytopathogenic bacterial disease is caused by a phytopathogenic bacterium,The phytopathogenic bacteria are selected from the group consisting of Pectobacterium carotovorum, Pectobacterium chrysanthemi, Pectobacterium aroidearum, Pectobacterium atrosepticum, Pectobacterium betavasculorum, Pectobacterium cacticida, Pectobacterium colocasium, Pectobacterium cypripedii, Pectobacterium melonis, Pectobacterium parmentieri, Pectobacterium Polaris, Pectobacterium punjabense, Pectobacterium rhapontici), Pectobacterium versatile, Pectobacterium wasabiae, Pectobacterium zantedeschiae, Xanthomonas campestris, Pseudomonas syringae, Pseudomonas fluorescens, Pseudomonas tolaasii, Pseudomonas glycinea, Erwinia amylovora, Ralstonia solanacearum, Acidovorax citrulli, Acidovorax oryzae, Acidovorax avenae, Dickeya solani), Dickechachysanthemi, Dickeya dadantii, Xylella fastidiosa, and Pantoea sp.

27. The method according to any one of claims 25 and 26, wherein the crop is selected from the group consisting of: potato (Solanum tuberosum), onion (stem bulb and green), shallot (Allium fistulosum), shallot, carrot, soybean, barley, buckwheat, millet, oats, rice, rye, quinoa, sugar beet, Ethiopian teff, Mexican maize, triticale, wheat, wild rice, corn, soybean, cotton, borage, buffalo gourd (Cucurbitafoetidissima), canola, crambe, flax, jojoba, lesquerella, oilseed rape, safflower, sesame, sunflower, seed sorghum (milo), sugarcane, goosefoot, asparagus, bamboo shoots, artichoke, okra, peanut, pineapple, strawberry, polycarpon incense, angelica, star anise, annatto (seed), balm, basil, burnet, chamomile, caper bud, caraway, black caraway, cardamom, cassia bark, cassia bud, camphor, celery seed, chervil (dried), chives, chives, cinnamon, salvia, clove bud, cilantro (coriander or coriander), coriander seed (cilantro), tansy, eryngium (leaf), eryngium (seed), cumin, curry (leaf), dill (dill leaf), dill (seed), nepeta, fennel seed, fenugreek, white ginger flower, cardamom (Aframomummelegueta), Marrubium vulgare), hyssop, juniper berry, lavender, rue, lovage (leaves and seeds), mace, calendula, marjoram (including oregano), Mexican oregano, holly, mustard (seed), nasturtium, nutmeg, parsley, mint, pepper (black and white), pepper leaf, peppermint, basil, poppy (seed), rosemary, rue, saffron, sage, vanilla (summer and winter), spearmint, stevia, Virginia magnolia, tansy, tarragon, thyme, vanilla, wintergreen, woodruff, wormwood, broccoli, Chinese broccoli (kale), watercress moss, Brussels sprouts, cabbage, Chinese cabbage (pek choy), Chinese cabbage (Chinese cabbage), Chinese mustard (mustard), cauliflower, cavallo broccolo, kale, collard greens, kohlrabi, turnips, mustard greens, mustard spinach, rapeseed, garlic, garlic cloves, leeks, chayote, winter melon, citron, cucumber, West Indian cucumber, hyotan, cucuzza, loofah, Chinese okra, melon, balsam pear, balsam melon, bitter melon, Chinese cucumber, Roman melon, Casaba melon, Cranshaw melon, golden pershawmelon, honeydew melon, honeydew melon, citrus melon, Persian melon, pineapple melon, Santa Claus melon, snake melon, pumpkin, crookneck pumpkin, scalloped pumpkin, straightneck pumpkin, zucchini, courgette, squash, gourd, winter squash, acorn squash, spaghetti squash, watermelon, amaranth (Chinese spinach), rocket, beet greens, cardoon, celery, Chinese celery, celery lettuce, worm amaranth, chervil, leaf daisy, chrysanthemum, watercress (garden and upland), dandelion, sorrel, houttuynia, endive (sonococcus), sweet fennel, gow kee, lettuce (head and leaf), saltbush, parsley, purslane (garden and winter), dadicchio (red endive), rhubarb, spinach, New Zealand spinach, vine spinach, chard, watercress (upland), water spinach, eggplant, Physalis species (Physalis spp), bell peppers, chili peppers, peppers, Spanish pimentos, sweet peppers, tomatillos, tomatoes, legumes, lupine, sweet lupine, white lupine, kidney beans, kidney beans, lima beans, navy beans, pinto beans, runner beans, snap beans, broad beans, wax beans, adzuki beans, long beans, black-eyed peas, pigeon peas, Chinese long beans, cowpeas, cowpeas, aconite-leaved cowpeas, mung beans, rice beans, southern peas, black mung beans, beans, broad beans (broad beans), chickpeas (chicken beans), guar beans, sword beans, lentils, lentils, dwarf beans, snap peas, English peas, field peas, vegetable peas, green peas, sugar peas, snap peas, pigeon peas, large beans (immature seeds), sword beans, Peruvian carrots, arrowroots, honeydew seeds, Jerusalem artichokes, beets (garden), burdocks, cannas, cassava (bitter and sweet), celeriac, chayote (root), chervil (turnip type), chicory, Cyperus culentus, taro (taro), galangal, ginseng, horseradish, edible blue banana, kava (turnip type), parsley (turnip type), parsnip, radish, white radish, rutabaga, salsify, scorzonera, Spanish salsify, celery, sweet potatoes, taro, turmeric, turnips, serrata, yacon, yam, ginseng, and any combination thereof.

28. The method of any one of claims 25 to 27, wherein the plant or plant part is selected from the group consisting of seeds, seedlings, plantlets, tubers, bulbs, rhizomes, trees, grasses, shrubs, creepers, vegetables, fruits, legumes, mushrooms, flowers, and combinations thereof.

29. The method of any one of claims 25 to 28, wherein contacting the plant, plant part or crop with the system is performed by a process selected from the group consisting of foliar application, soil application, application through an irrigation system, initial fertilization, injection into the soil, patch application, aerial application, and any combination thereof.

30. A method for reducing the bacterial load of an article in at least one of the pharmaceutical industry, microbiological industry, medical industry, food industry, marine industry, paper industry, textile industry, plant nursery, aquaculture, soil treatment, ship maintenance, water treatment facilities, sewage treatment facilities, drainage systems and water distribution systems, the method comprising contacting the article with an effective amount of the delivery system according to any one of claims 1 to 24, thereby reducing the bacterial load of the article.

31. The method of claim 30, wherein reducing bacterial load comprises reducing or eradicating biofilm.

32. The method according to any one of claims 30 and 31, wherein the article is selected from the group consisting of: machines and equipment for preparing or storing medicines; medical devices or implants; machines or equipment for food preparation and storage; tanks, pipes, filters, reservoirs, infiltration pits, coolers; water irrigation systems and any parts thereof; and ships and any parts thereof.

33. A method for preparing a delivery system for differential delivery of live Bdellovibrio and similar organisms (BALOs), wherein the delivery system comprises: a carrier comprising a polysaccharide hydrocolloid; and the BALOs immobilized within the carrier, wherein the BALOs are in a physiological state selected from the group consisting of leech plasmids, leech cysts, and APs at a rate of at least about 1.0×10 8 PFU / g carrier cell density, the method comprising: (i) mixing a suspension comprising BALOs with a hydrocolloid solution comprising a polysaccharide hydrocolloid to form a hydrocolloid composition comprising the BALOs; and (ii) mixing the hydrocolloid composition comprising the BALO of step (i) with a cross-linking solution to form a polysaccharide hydrocolloid in a wet gel form, thereby forming the delivery system in a wet form.

34. The method of claim 33, further comprising the step of drying the delivery system of step (ii), thereby forming a dry delivery system.

35. The method of claim 34, wherein drying the delivery system is performed by at least one of vacuum drying, air drying, desiccant drying, fluidized bed drying, spray drying, sunlight drying, and freeze drying.

36. The method of any one of claims 33 to 35, wherein mixing the hydrocolloid composition with the cross-linking solution is performed by at least one of: dripping, spraying, emulsifying, or including in oil and subsequently cooling.

37. The method of any one of claims 33 to 36, wherein the polysaccharide hydrocolloid is selected from the group consisting of kappa-carrageenan, pectin, alginate, agar, gellan gum, carboxymethylcellulose (CMC), furcellaran, xanthan gum, locust bean gum (LBG), konjac mannan, chitosan, pullulan, curdlan, and derivatives and combinations thereof.

38. The method of claim 37, wherein the polysaccharide hydrocolloid is selected from the group consisting of kappa-carrageenan, low methoxyl pectin (LMP), and combinations thereof.

39. The method of any one of claims 33 to 38, wherein the polysaccharide hydrocolloid is present in the hydrocolloid solution in an amount of about 0.25% to about 10% by weight based on the total weight of the hydrocolloid solution.

40. The method of any one of claims 33 to 39, wherein the volume ratio between the BALO-containing suspension and the hydrocolloid solution is about 1:4 to about 1:

9.

41. The method of any one of claims 33 to 40, wherein the hydrocolloid solution further comprises a protein hydrocolloid and / or a protein.

42. The method of claim 41, wherein the protein hydrocolloid is gelatin and is present in the hydrocolloid solution in an amount of about 0.25% to about 15% by weight based on the total weight of the hydrocolloid solution.

43. The method of any one of claims 33 to 42, wherein the hydrocolloid solution further comprises at least one osmoprotectant.

44. The method of claim 43, wherein the at least one osmoprotectant is selected from the group consisting of trehalose, inositol, glycerol, arabinitol, betaine, amino acids, and combinations thereof.

45. The method of any one of claims 43 and 44, wherein the osmoprotectant is present in the hydrocolloid solution in an amount ranging from about 0.25% to about 15% by weight based on the total weight of the hydrocolloid solution.

46. ​​The method of any one of claims 33 to 45, wherein the cross-linking solution and the hydrocolloid composition are contacted in a volume ratio of about 10:1 to about 500:

1.

47. The method of any one of claims 33 to 46, wherein the cross-linking solution comprises metal cations and / or a gelation inducing agent.

48. The method of claim 47, wherein the metal cation and / or the gelation inducing agent is present in a weight percentage of about 0.1% to about 5% based on the total weight of the cross-linking solution.

49. The method according to any one of claims 47 and 48, wherein the metal cation is selected from the group consisting of: potassium (K + ), calcium (Ca 2+ ), barium (Ba 2+ ), magnesium (Mg 2+ ), aluminum (Al 3+ )、Zn 2+ ), lead (Pb 2+ ), iron (Fe 2+ ), Sr 2+ ), copper (Cu 2+ ), cadmium (Cd 2+ ), nickel (Ni 2+ ), cobalt (Co 2+ ) and their combinations.

50. The method of claim 34, wherein after the drying step, the system comprises at least about 1.0×10 8 PFU / g carrier of BALO.

Images