NL2024726B1 - Enzymatic crop protection and process for preparing biological crop protection composition - Google Patents

Abstract

The invention relates to a method for preparing a chitinase comprising composition by fermenting a bacteria strain which is capable of producing chitinase, which bacteria 5 strain is immobilized on a carrier, wherein the carrier comprises a porous polymeric material, wherein chitin is present during fermentation, and separating the chitinase comprising composition from the immobilized bacteria. Preferably, the bacterial strain is Pseudomonas gessardii, deposited under CBS 146242. The chitinase comprising composition can effectively be used as a plant protection product, such as a fungicide, lO insecticide or the like, by applying the composition to a plant or seed.

Description

ENZYMATIC CROP PROTECTION AND PROCESS FOR PREPARING

BIOLOGICAL CROP PROTECTION COMPOSITION Field of the invention This application concerns enzymatic crop protection, and a method for preparing a biological crop protection composition. More in particular, the invention relates to a biological crop protection composition comprising chitinase prepared by immobilized bacteria capable of producing chitinase on a porous polymer carrier comprising chitin. Further provided are embodiments of a novel Pseudomonas gessardii species that can effectively produce compositions comprising effective amounts of chitinase.

Background of the invention Chitin is a biopolymer, which is found as the structural component of cell walls of many fungi and in the shells, exoskeletons and gut linings of arthropods.

Chitinase is any type of enzyme that can cleave chitin. Chitinase compositions can thus degrade the cell walls of fungi and/or shells, exoskeletons and/or gut linings of arthropods, and thereby exhibit antifungal and/or insecticidal properties.

Many chitinases exists, including endo- and exochitinases which cleave the esterbond between sugar moieties, as well as chitinases that specifically hydrolyse the acylamide bond.

Pesticides are necessary to provide high yields and output of crop, however current pesticides generally are considered also harmful to the environment. Further, insects and/or fungi can and have adapted to develop resistance against known pesticides.

Fungi and arthropods themselves also synthesize chitinase, for reasons, including nutrient recycling and morphogenesis. Additionally, chitin comprises the main part of their outer defense layer. As such, widespread resistance to chitinase is highly unlikely.

As chitinases are enzymes, they are themselves easily degraded in the environment to small peptides that are used by other organisms for growth. These enzymes therefore are short-lived after application and are as such not harmful to the environment.

Chitinolytic bacteria decompose chitin in aerobic and anaerobic environments. These bacteria use chitin as a source of carbon and nitrogen and therefore produce chitinases. Chitinase containing compositions are described as fungicidal preparation.

For example, US4534965 discloses growing Streptomyces on a suspension of dried shrimp waste, separating a supernatant from the culture via centrifugation and/or decanting, and contacting seeds with the supernatant. Also, chitinolytic bacteria can beused as a form of pest control, in particular Streptomyces, Bacillus and Pseudomonas. EP0353689 discloses application of Pseudomonas putida directly to plants for fungicidal control.

However, storage of live bacteria is not easy, and shelf life of many enzymatic compositions is short. Further, separating the chitinase from the bacteria can be difficult, and yields generally are low, which may be a reason that such compositions are not used in practice yet.

It is desirable to provide a method for preparing a composition comprising chitinase which solves one or more of these issues. It is furthermore desirable to provide chitinase comprising compositions as effective fungal and/or arthropods.

Summary of the invention In one aspect, the invention is directed to a method for preparing a chitinase comprising composition by fermenting a bacteria strain which is capable of producing chitinase, which bacteria strain is immobilized on a carrier, wherein the carrier comprises a porous polymer material, wherein chitin is present during fermentation, and separating the chitinase comprising composition from the immobilized bacteria.

Preferably, the carrier comprises chitin. Such chitin may be present during preparation of the carrier in any suitable form, such as for example as colloidal particles or as powdery particles.

The carrier may also after preparation be provided with chitin, in a suitable form, such as for example colloidal or dissolved chitin. Providing chitin can be attained by impregnating the carrier material, and/or by applying the chitin together with growth medium.

The invention is further directed to the bacteria strain Pseudomonas Gessardii, deposited at the International Depositary Authority of the Budapest treaty under CBS 146242, and the use thereof in the preparation of a composition comprising chitinase.

The invention is further directed to a method of preparing a chitinase comprising composition by immobilizing a bacteria strain, which is capable of producing chitinase, on a carrier, wherein the carrier comprises a porous polymer and chitin, and wherein the bacteria strain comprises the Pseudomonas Gessardii deposited under CBS 146242.

The invention is further directed to the use of the composition comprising chitinase obtainable by any of the methods of the invention, as crop or seed protection agent or biostimulant.

The crop or seed can be protected against a fungus or arthropod, preferably a fungus. Detailed description of the invention Preparation of chitinase comprising composition The invention is directed to a method for preparing a chitinase comprising composition by immobilizing bacteria capable of producing chitinase, on a carrier, wherein the carrier comprises a porous polymer.

The bacteria can be any known in the art and should be capable of producing chitin.

Suitable bacteria are known as such and include those that are for example described in E. A. Velizl, P. Martinez-Hidalgo, A. M. Hirsch, Chitinase-producing bacteria and their role in biocontrol, Microbiology, 3(3) 2017.

The carrier is a solid carrier and comprises a porous polymeric material. Solid means that the carrier can be held in a fixed place, but the carrier may be flexible or gel like. The porous carrier material allows the bacteria to growth on the surface. Either the medium, and/or the carrier comprises chitin, as the chitin will induce chitinase production in the bacteria.

The presence of chitin not only induces the production of chitinase by the bacteria, but it will serve as a nitrogen and carbon source for the bacteria.

In a preferred embodiment, the carrier comprises chitin In one preferred embodiment of the invention, chitin is incorporated into the polymer framework of the carrier material. This can be achieved by having chitin present during preparation of the carrier material.

Chitin may be present during polymerization preferably as powder and/or as colloidal chitin. Thus, the chitin may be chemically bound into the polymer backbone of the carrier material, but may be just incorporated via e.g. van der Waals forces or both.

In the presence of chitinase, the chitin comprising carrier will break down, and thereby allow proper stress conditions allowing optimized chitinase production.

In another preferred embodiment, chitin is present in the growth medium. This allows easy optimization of amounts of chitin in (semi-)continuous processing.

The chitin may be present in the both the polymer backbone and the growth medium.

In a preferred embodiment, chitin is at least present in the polymer backbone, to improve immobilization of the bacteria on the carrier material.

The bacteria are immobilized onto the carrier. Bacteria are seeded onto the backbone material and allowed to grow for an appropriate time. Generally, excess is washed off after immobilizing the bacteria, yielding a carrier with immobilized bacteria thereon. The immobilized bacteria can be used to efficiently produce chitinase, while separating the supernatant comprising the chitinase is relatively easy and cost effective.

The carrier before or after immobilizing the bacterial strain is generally placed into a vessel. The vessel is generally aerated for the duration of the fermentation, including the growth phase and the cultivation phase. However, this depends on the type of strain (aerobic or anaerobic). Most bacterial strains producing chitinase are aerobic, therefore, aerobic cultivation generally is preferred.

It is preferred to use a bacterial strain that produces chitinase both during the growth phase as well as the cultivation phase.

Fermentation (comprising the growth and cultivation phase) will generally involve providing a growth medium to the immobilized bacteria and washing after a growth/cultivation cycle.

The liquid obtained from growth/cultivation generally comprises chitinase that can be harvested.

After the growth phase, a cultivation phase may take place. During this cultivation phase (also production phase), the bacteria generally produce chitinase, and the liquid obtained from this cultivation phase will be a chitinase comprising composition. As explained above, during the growth phase, substantial amount of chitinases will be produced as well, and the chitinase comprising composition equally will be suitable for use as fungicide.

During the growth and the cultivation phase, a growth medium will be present. Preferably, this growth medium provides part of the carbon and nitrogen requirements, in addition to required metallic and other elements like sulfur, phosphorous and the like.

Often, bacteria produce chitinases on stress conditions, if limited amount of at least part of necessary nutrients are available, including ‘easy’ energy sources. Hence, the growth medium for such natural bacteria generally is generally relatively poor in at least one of carbon or nitrogen.

5 In a preferable embodiment, the carrier acts as a growth medium and provides for at least part of the C- and/or N-source for preferably about 2 % or more, more preferably about 3% or more, and most preferably about 5%. The amount may be 20% or less, preferably about 10 wt% or less. By requiring the bacteria to use the carrier as carbon and/or nitrogen source, the production of chitinase is enhanced.

The chitinase comprising composition is generally separated from the bacteria immobilized on the carrier, for example using filtration or decanting. As the bacteria are immobilized on a solid carrier, the separation is easily performed.

The carrier In a preferable embodiment, the porous polymer is formed by an isocyanate and a polyol, wherein the polyol is degradable by the bacteria, in particular by one or more of the enzymes produced by the bacteria.

This carrier comprises a porous polymer preferably formed by reacting an isocyanate and a polyol. Such polymers are also known as polyurethanes.

Preferably, the carrier is prepared in the presence of water, forming a water- blown polyurethane foam. In addition to water, other physical foaming agents commonly used in polyurethane chemistry may be used, like carbonate comprising compounds combined with some acid.

The foam generally will be an open-cell foam with a density of 10-200 kg/m}, preferably about 20-100 kg/m’, and preferably will have an open cell content in the range from 70-100%, more preferably higher than 80%.

The polymer preferably has cell sizes from about 0.1 — 9 mm, preferably from about 0.2-3 mm. Such cell size means that about 90% of the volume of the foam has such cell size, preferably about 95% or more of the volume of the foam has such cell size. The cell size can be assessed by cutting a foam, and measuring the cell size (diameter) of 5 cm x 5 cm foam. Such measurements can be automated.

The isocyanate can be a di-isocyanate, tri-isocyanate or higher functional isocyanates. The isocyanate preferably is a di- or tri-isocyanate, and most preferably a tri-isocyanate. The isocyanate preferably is an aliphatic isocyanate having 4-40 carbon atoms, preferably 5-20 carbon atoms.

Suitable isocyanates include hexamethyl-diisocyanate, (HDI), methylene dicyclohexyl diisocyanate, pentamethylene diisocyanate, or isophorone diisocyanate (IPDI), and trimerized compounds thereof, such as for example trimerized- hexamethylene-diisocyanate. The tri-isocyanate of castor oil is also suitable.

The polyol of this porous polymer carrier comprises a degradable polyol, in preferably at least about 50 wt% of the polyol is biodegradable. Preferably, a substantial amount of the polyol is degradable by the bacteria used in the method of the invention. Such substantial amount is about 80 wt% or more. This allows the bacteria to disperse/populate throughout the carrier. It further can allow the carrier to act as the source of carbon and/or nitrogen.

The polyol preferably has a relatively low glass transition temperature, to allow flexibility of the foam when swelling with water and allowing the bacteria to grow.

The polyol furthermore preferably is hydrophilic, which allows swelling in a water based medium.

The polyol is preferably a polyether-ester polyol having a Tg of about 0 °C or lower, preferably of about -20 °C or lower.

Preferably the polyols are largely aliphatic, both for providing a low glass transition temperature and/or for environmental purposes.

Preferred polyols are diethyleneglycol, tri-ethylene glycol, or tetra- ethyleneglycol, and more preferable tri- or tetra-ethyleneglycols.

Other polyols include hexanediol, trimethylolpropane, glycerol, pentaerythritol, polyetherified polyols and the like.

The acid component of the polyester preferably is a di-acid, such as for example adipic acid, succinic acid and the like. The acid components are preferably aliphatic, like for example more than 50 mol%. And more preferably more than 80% aliphatic. Non aromatic diacids may include hexahydro-phthalic acid, hexahydro iso- phthalic acid or hexahydro terephthalic acid Other suitable components include hydroxy-acids such as lactic acid, tartaric acid and lactons.

The polyether-esterpolyol may comprise diols such as polyethyleneglycol with a molecular weight of about 3000 Dalton or lower, preferably about 2000 or lower, such as for example about 400, about 500 or about 700.

The polyurethane foam may next to a polyether-ester-polyol comprise polyetherdiols such as polyethyleneglycol with a molecular weight of about 3000 Dalton or lower, preferably about 2000 or lower, such as for example about 400, about 500 or about 700.

The polymeric material preferably is biodegradable by the bacteria, and in order to achieve that goal, it preferred to include biologically common materials in the polymer.

The polymeric material comprises preferably about 5 wt% or more of ethyleneglycol units, more preferably about 10 wt% or more.

In a preferable embodiment, chitin is incorporated into the polymer structure. In one embodiment, the chitin may replace of part of the polyol. Chitin has a glass transition temperature far above 0 °C, and preferably the amount of chitin is such that the Tg of the polyol and chitin on average is below 0 °C.

Chitin can also be incorporated by having for example chitin powder or colloidal chitin present during polymerization in such a way that the chitin is homogeneously dispersed through the polymeric carrier material.

The amount of chitin in the polymeric material preferably is about 20 wt% or less (wt% relative to dry materials), preferably about 10 wt% or less. Generally, the amount of chitin will be about 0.1 wt% or higher, preferably about 0.5 wt% or more and even more preferably about 2 wt% or more. Suitable amounts and for example about 3 wt%, about 5 wt% or about 7 wt%.

The chitin does not have to be part of the polymeric material, and part or all of the chitin can be provided as dissolved/dispersed (colloidal) chitin in the fermentation medium. However, in a preferred embodiment of the invention, the chitin is part of the polymer structure. Chitin serves in that preferred embodiment as biodegradable polyol in the polymeric structure.

In another embodiment, the porous polymeric material consists largely of a polyvinyl alcohol matrix. Generally, to immobilize the bacteria onto the carrier, a dispersion or solution of polymer and a dispersion comprising the bacteria are mixed.

The resulting mixture can then be dried, yielding an article of polyvinyl alcohol matrix encapsulating the bacteria. Chitin can be mixed with the polymer as powder or colloidal component as well.

Generally, the resulting polymeric material with immobilized bacteria is washed to remove excess bacteria on the outside.

Preferably, chitin is provided as colloidal chitin and mixed into either the dispersion comprising the bacteria or in the solution or dispersion employed during gel production. After mixing and drying, the resulting article thus also comprises colloidal chitin.

Processing Fermentation media comprise generally used media, preferably relatively lean media, as the bacteria need to be sufficiently stressed to start producing chitinase. A suitable medium is BSM (Basal Salt Medium) supplemented with some C and/or N source.

An exemplary BSM medium comprises the following components.

Chemical Concentration Trace elements Concentration Ee i K2HPO4 ZnSO4-7H20 60 KH2PO4 MnSO4-H20 (NH4)2S04 CuSO4:5H20 eee oo eeen Additionally, a carbon and/or nitrogen source is used during the growth phase and the cultivation phase. The medium may be richer during the growth phase. Suitable carbon and nitrogen sources include peptone and yeast extract.

Suitable carbon sources include and for example acetic acid in a concentration of 0.24-1.0 mL Lt, glucose up to 10 g Lt or ethanol. For example, peptone or yeast extract can be used during the growth phase in a concentration of 10.0 g Lt. During cultivation, the amounts may be for example 0.2 g/L to 1 g/L.

Processing conditions generally include room temperature, or a temperature of about 20 °C or higher. Temperatures above room temperature may be preferred to increase bacterial growth. Suitable temperatures are e.g. 25 °C, 30 °C or 35 °C. Generally, the temperature will be about 45 °C or lower, preferably about 40 °C or lower, unless thermophilic bacterial strains are used.

Processing condition generally include largely neutral to slightly acidic or basic pH. However, depending on the bacterial strain, it is easy to optimize such conditions. Suitable pH can be e.g. about 6, about 7 or about 8. Generally, the pH will be about 9 or lower, and of about 4 or higher. Generally, cultivation will be performed for some days to achieve suitable amounts of enzymes.

Cultivation can be done batch wise, semi continuous or continuous. Generally, the bacterial strain requires a first growth phase, after which cultivation is performed. Growth and cultivation can be done continuously. In another embodiment, growing and thereafter cultivation can be done in a number of production runs, like 1, 2, 3, 4, or 5 runs.

At a certain amount of time, e.g. because the carrier material is broken down to a certain extent, production and/or separation will be less efficient, and it is economically more appropriate to start production with a new batch.

Continuous production may take place in a column. Suitable flow rates may be 5-20 mL/min, depending on the reactor design.

Generally, an hydrolic retention time of between 3 and 13 hours will be adequate. Pseudomonas Gessardii The invention is further directed to the bacteria strain Pseudomonas Gessardii deposited under CBS 146242. Pseudomonas are a genus of bacteria known in the art. These are generally found not to have adverse effects on humans, animals or plants. Pseudomonas strains are commonly found in soil and water.

Pseudomonas are typically aerobic and commonly produce chitinase. Applicant has found that a strain of Pseudomonas, P. gessardii deposited under CBS 146242, shows very desirable chitinase production characteristics.

The composition comprising chitinases obtainable with this strain comprises relatively high activity of chitinase, low activity of proteases (even if no protease inhibitor is added), and this is achieved without artificial genetic modification.

In a preferred method for obtaining a chitinase comprising composition, the method disclosed above is used with the immobilized bacteria comprising 7.

gessardii, more preferably the P. gessardii strain deposited under CBS 146242.

The proportion of active compounds depends on the technological process of down-stream processing and the composition of the culture medium.

The chitinase comprising composition obtained by any of the methods of the invention may comprise proteolytic enzymes. It may be desirable to remove these,

optimize fermentation conditions to lower the amount and number of proteases produced to as low as possible, and/or inhibit these proteases with commonly known protease inhibitors. Any of these measures can be done via methods known in the art.

The composition obtained from the fermentation comprising the chitinase can be concentrated to lower transport costs. Concentration can be achieved for example by membrane filtering or freeze drying. Preferably, in view of costs, simple concentration is preferred. Care should be taken not to deactivate chitinases during such treatments. Use of composition as a biological pesticide The chitinase comprising composition obtained by any of the methods of the invention may be used as a biological pesticide. As the composition comprises chitinase, the composition can be effective against insects and fungi, and is preferably used against fungi.

Suitable fungi that can be combatted with this chitinase comprising composition include the generally present and harmful fungi such as for example Alternaria solamum, Botrytis cinereal, Dydimella bryoniae, Fusarium oxysporum, Phytophthora cactorum, Phytophthora infestans, Pythium ultima, Rhizoctonia solani, Sclerotinia sclerotium, Pestalotiopsis.

Suitable insects that can be combatted for example larva’s of Spodoptera litovalis, Culex quinduefasciatus and aphids and Rhinoceros. Mortality of aphids was observed to amount to 48% with application of suspension of a composition according to the invention. Mortality of rhineceros beetle was observed to an extent of 68% with application of suspension of the bacterial strain according the present invention.

The chitinase comprising composition preferably is applied to plants, seeds or other places of interest.

The chitinase comprising composition preferably is dissolved or diluted before application.

Application may be done by any method known in the art such as foliar spray, spraying and the like.

The present invention also relates to a method for treating plants with at least a biological fungicidal composition comprising chitinase, wherein the above described water based spraying liquid 1s sprayed on plants in an amount such that the effective amount of fungicidal composition is about 1 L or more, preferably about 2 L or more up to about 100 L or less, preferably SOL or less of said fungicidal composition per hectare.

The spray solution made by dilution of the concentrate will generally be sprayed at a volume of about 20 L/ha to about 2500 L/ha, preferably about 30 L/ha to about 2000 L/ha and even more preferably between 500 L/ha to 1500 L/ha.

A suitable dilution comprises about 95 wt% water or more and about 5 wt% of said ani fungicidal composition or less. Preferably, the concentrate is diluted with water in a range of about 1:30 to 1:100 (in volume/volume). Suitable amounts of concentrate in water include 1 wt%, 2 wt%, 3 wt%, 4 wt% or the like. Generally, for foliar spray higher dilutions are used than with in furrow application.

The water based spraying liquid preferably comprises the concentrate in an amount of about 1 L up to about 50 L of said concentrate per hectare for foliar spray, preferably between about 3 L to 20 L/ha.

The composition or spraying liquid can contain other actives, such as for example a herbicide, a fungicide, a bactericide, an insecticide, a nematicide, a miticide, a plant growth regulator, a plant growth stimulant, and a fertilizer.

The method according the invention is preferably applied to field crops, vegetable crop or fruit crops.

Suitable field crops comprise broad acre crops which include tuber or root crops, cereal crops, oil crops and other crops. Suitable tuber or root crops include potatoes or sugar beet. Suitable cereal crops include maize or corn, rice, wheat, barley, rye, sorghum, flax, oat and grain. Suitable oil crops include soy bean, sunflower, rape-seed (canola) or peanut. Other crops include clover, alfalfa, cotton, mustard or tobacco.

Suitable vegetable crops include asparagus, beans broccoli, Brussels sprouts, cabbage, cantaloupe, carrots, celery, cauliflower, sweet corn, cucumbers, eggplant, lettuce, melons, okra, onions, parsley, peas, peppers, potatoes, radishes, spinach, squash, tomatoes, turnips, water melon and the like.

Suitable fruit crops include small fruits, vines and tree fruits, like apples, bananas, grapes, olives, citrus (oranges, lemon, lime, grapefruit), pineapples, pomegranates, strawberries, papayas, stone fruit (apricot, cherry, nectarine, peach), and the like.

Further, the composition according the invention can suitably be applied on young trees, ornamental flowering plants (like roses, azaleas and the like) and on sod and turf.

Examples Example 1: Chitinase production by Pseudomonas sp (qualitative).

Bacteria strains of Pseudomonas (P. fluorescens I., P. fluoerescens I1., P. veronii, P. stutzeri, P. fragii and P. gessardii) were tested for chitinase production by cultivating a strain for 48 hrs in Erlenmeyer flasks with BSM (Bacterial Standard Medium) medium with peptone addition.

An agar medium comprising colloidal chitin (1% w/v, see Table X) was prepared, and the cultivated colonies applied thereon. Production of chitinase was detected by observing a clear zone around colonies. All Pseudomonas sp. showed chitinase production. Table 1: composition of agar medium ao: Example 2: Chitinase production by Pseudomonas sp. (quantitative) Cultivation of Pseudomonas sp. was done at 35°C and pH 7 in Basal Salt Medium 1 (BSM medium 1), as detailed in tables 2-3, and with colloidal chitin addition (1 g/L). The source of carbon added was either acetic acid (0.3 ml/l) or glucose (500 mg/l); during fermentation the mixture was stirred at 90 rpm.

Chitinase production by Pseudomonas sp. was measured after 4, 8 and 24 hrs.

See table 4.

Optical density of the bacterial cultivations was measured over time to provide insight in growth stages (exponential, stationary phase of growth). Table 2: Composition of Basal Salt Medium 1 Substance Amount (g/l) Table 3: Composition of trace elements of BSM 1 Table 4: Chitinase activity (nmol h™ 1) in cultures with carbon sources being either acetic acid or glucose CC Acetic Acid Glucose 4h 8 h 24h 4h 18h 24h

9.8 00 0.0 0.0 00 0.0 EC Cu LL J LS LL en Li LCR LN CC LL re [o0 [10 [wT Jo Jor Jou

It was noted that P. gessardii shows chitinase production at all stages of growth. P. fragii showed high chitinase activity, but only in the exponential growth phase. Example 3: Effect of chitin concentration on chitinase production Chitinase activity was measured after cultivation for 3 and 4 days for 7. gessardii, P. fluorescence II, P. veronii and P fragii. Batch cultivations were realized in Erlenmeyer flasks with 100 ml of BSM 1, temperature 35°C and orbital stirring 90 rpm.

Table 5: Chitinase activity (nmol h™ 1?) in cultures with chitin concentration of 1 g/l or 5 g/l. ee Team Geward [90 162 [11 [1064 | Sorecawl! [00 [Bs 17 Je0 oo Jee joo Je Sa ee In the cultivation with P. gessardii, a positive effect was shown of higher colloidal chitin concentration on chitinase production. This was not observed for any of the other strains. Example 4: Chitinase production by bacteria immobilized on a porous carrier Ps. fragii and Ps. gesardii were immobilised into a polyvinyl alcohol matrix, prepared according to EP-B 1996156 and EP-B-1091996 at a biomass concentration of

1.1 gow/kgear (dry weight of bacteria in grams / kg carrier) and 1.6 gpw/kgcar respectively. After immobilization, the polyvinyl alcohol matrix was washed three times with 0.85% NaCl. The immobilized bacteria were cultivated on the cultivation medium as detailed in Table 6, in five repeated batch cycles. After each cycle, the carrier comprising bacteria was washed with physiologic saline solution. The cultivated carrier was used for enzyme-chitinase production. Acetic acid was added during production as a carbon source.

Table 6: Cultivation and production media composition Compound Concentration (g | Trace Element Concentration (mg ae a CH3COOH 0.24-20mlLt | onw | Chitinase activity in pmol/h/l was determined during each of the cultivation cycles with P. gessardii and P. fragii.

Cultivation was performed in opened polypropylene beaker (2L). The carriers comprising immobilized bacteria (150 g) were cultivated in 1 L of cultivation medium with peptone in a water bath at 35°C with vigorous mixing (500-600 rpm). Each cultivation batch took 20-24 hours.

Table 7 Chitinase activity in umol/h/l for P. gessardii and P. fragii during cultivation batch cycles Enzyme activity in umol/h/l was determined during production cycles using HPLC-RI, leupeptin (1 pM) was added to samples as a stabiliser.

The production was done in 4 repeated batches of 23.5 hours.

Production was performed in open polypropylene beaker (21). 1 L of production medium (with acetic acid) and 150 g of cultivated carrier were used for chitinase production.

Table 8 Enzyme activity in pmol/h/l for P. gessardii (G) and P. fragii (F) during production batch cycles.

Proteolytic stabilizer was added to some batches, this is indicated with STAB.

4F-6h-STAB 0.008 4F-16.5h-STAB 0.056 Chitinase activity with peptone was 3.1-3.7 umol/h/l for P. gessardii and about

0.18 pmol/h/1 for P. fragii. Chitinase activity during production with acetone was

0.118-0.154 pmol/h/1 for P. gessardii and 0.022-0.056 umol/b/1 for P. fragii.

Example 5: Chitinase production by bacteria immobilized on polyurethane carrier A porous polymer carrier was made according to Table 9. Table 10 details the properties of the polymer. Table 9: Composition of polyurethane carrier PDEGAD poly(diethyleneglycol 92 pbw ae Table 10: Properties of polyurethane carrier of Table 9

Apparent density Polyurethane carriers according to table 9 were prepared further comprising 5 and 10 % chitin by performing the foam production reaction in the presence of colloidal chitin. However, comparable results were obtained when chitin was incorporated as dispersed very fine powder.

A bacteria strain (P. gessardii) was immobilized onto the carriers. The carriers were then rinsed to remove excess bacteria. The bacteria immobilized onto the carriers were cultivated in BSM media. To this was optionally added glucose (1% solution) or ethanol (1% solution). The biomass growth was observed, and it was shown that even in a low carbon environment (i.e. only BSM), growth occurred. Significant growth was obtained using added glucose. Example 6: Chitinase production by bacteria immobilized on another polyurethane foam carrier A mixture of 7.3 g of chitin (Sigma-Aldrich) and 75 g of Desmodur TM N 3300 aliphatic polyisocyanate was homogenized in a plastic crucible for 10 min using a high speed 20 stirrer (2000 rpm). 60 g of poly(tetraethylene glycol adipate) triol having a hydroxyl value of 49 mgKOH / g, 3.1 g of water, 3.1 g of ammonium bicarbonate, 1.4 g of Niax TM Silicone L-6900 (Momentive Performance Materials) silicone surfactant, 1.1 g of dibutyltin dilaurate (DBTL, Aldrich, Germany) and 1.1 g of N,N,N',N”,N"-pentamethyldiethylenetriamine (Polycat TM 9, Air Products) were then added to the mixture, and the mixture was homogenized for a further 60 s. The reaction mixture was then poured into an open mold and left freely foamed at 55° C and then post-cured at room temperature for 48 h. The foam had a density of 68 kg/m3, an % open cells of 94% and an average pore diameter of 0.5 mm. The prepared block of foam was cut to cubes of 10x10x10 mm, which were used as porous carriers of microbial biomass.

Analogously, biodegradable polyurethane foams with 2.5 and 7.5 % (w/w) of chitin were prepared, using 3.6 or 15 g of chitin, respectively, for the synthesis.

Three experiments were done with 2.5%, 5% and 7.5% chitin. Results of chitinase production are shown in table 11.

In a further set of experiments, the foam with 5% chitin was used for measuring the area covered by the bacteria after 24 hr at 20, 25 and 30 °C. Coverage was good for all experiments (>70%), but highest for the reaction performed at 25 °C (92%). This value was somewhat influenced by the amount of inoculum used. Although all amounts between 0.5 and 2 % (percent by volume; inoculum contained 10? bacteria) were good (>70%), best results were obtained with 1 vol% of inoculum (92%). Chitinase production of the three foams was measured over 72 hr at 25°, as shown in table 12. olin G0 20h : Hence, all three inoculation amounts gave good results, with those of 1 vol% most consistent. In a further set of experiments, three sources of carbon were tested at a concentration of 200 mg/L. Fermentation at 25 °C and pH 7. Results are given in table

13. Gamow [Eh [En EE

All three sources can be used with good results.

Further tests were done at a pH of 6-8, buffered by using appropriate amounts of phosphate acid salts. Results are given in table 14.

Covering of the foam by the bacteria was >90% at both pH 7 and pH 8. Production was measured over 72 hr at 25 °C, results are shown in table 14. Ew fw hw EL ea a All results are judged as good. This bacterial strain performs best at pH 8.

Examples 7: Use of composition comprising chitinase as a biopesticide A composition obtained from example 4, from P. gessardii was tested against the growth of 10 fungi: Alternaria solamum, Botrytis cinereal, Dvdimella bryoniae, Fusarium oxysporum, Phytophthora cactorum, Phytophthora infestans, Pythium wltimum, Rhizoctonia solani, Sclerotinia sclerotium, Pestalotiopsis.

Petri dishes with PDA (Potato Dextrose Agar) were prepared. After preparation, 4 holes were made into the PDA layer. Onto the PDA, spores of fungi were applied. Directly after application of the fungi on the PDA, application of water / product was performed by dripping the products into the holes in the PDA.

Comparative examples were water as negative control and Serenade (Com. Ex. 1) (known biofungicide) as positive control.

After the application, the development of the fungi was observed by taking pictures at days 3, 7, 10 and 14. At the end of the trial period at day 14 an assessment on efficacy was performed.

In table 15, the efficacy of the product against fungicidal growth is presented on a scale of 0 — 10. Table 15 Efficacy of applied product on fungi Fungus Water Serenade Example 6 ee [TT gen | Bw [0p Jes Dydimella 8.5 7 me Fusarium 3.2 6.67

EE Phytophthora 5.33 7.83 ame Phytophthora 8.33 6.0 [Ee Oe | Remane OE Sclerotinia 0 7 ee Tanne | A further range of tests has been performed with the chitinase containing mixture of example 6 (the foam comprised 5% chitin). The efficacy of the chitinase containing mixture (simply denoted as ‘chitinase’ below) was assessed compared to a water sprayed check, a chemical reference and a biological reference (generally Serenade). Assessments were performed on 3, 7, 10 and 14 days after application of the products. Fungal development was assessed by means of percentage coverage of the petri dish by fungus. Per treatment, 6 petri dishes were assessed with the following results. Alternaria solani Fungal development of Alternaria solani for the chitinase was lower than the water sprayed check and lower than the chemical reference

Ranman Top.

Biological reference Serenade performed slightly better than the chitinase.

Efficacy: Moderate to good Botrytis cinerea Fungal development of Botrytis cinerea was quick, therefor only assessment A03 and A07 should be considered to base conclusions on.

On A03, the chitinase performed the best of all treatments.

On A07, biological reference Serenade performed better than the chitinase.

Chitinase was comparable to chemical reference Luna Privilege and slightly better than the water sprayed check.

Efficacy: Good (at early assessment stage) Fusarium oxysporum lactuca Fungal development of Fusarium oxysporum lactuca for the chitinase was slightly higher, though quite comparable to chemical reference Topsin and biological reference Trianum-P and lower than the water sprayed check.

Efficacy: Good Mycosphaerella / Didvmella bryoniae Fungal development of Mycosphaerella for the chitinase was slightly lower (though comparable to) biological reference Serenade and lower than chemical reference Fungaflash and the water sprayed check.

Efficacy: Good Phytophthora cactorum Fungal development of Phytophthora cactorum was comparable to chemical reference Fenomenal and biological reference Aliette during the trial.

At the end, fungal development was even lower for the chitinase than all other treatments.

Efficacy: Good Phytophthora nicotianae Fungal development of Phytophthora nicotianae was comparable to chemical reference Ridomil gold and slightly higher than biological reference Trianum-P.

Efficacy: Moderate to good Pythium ultimum Fungal development of Pythium ultimum while applying the chitinase was the lowest of all treatments at all assessment timings.

Efficacy: Good Veticillium dahliae Fungal development of Verficillium dahlige for the chitinase was comparable to biological reference Trianum-P and lower than for chemical reference Ridomil gold and the water sprayed check.

Efficacy: Moderate to good

It can be observed from table 15, that the product of the invention performed better than Com.

Ex. 1 (the commercially available gold standard) on 5 of the 10 fungi.

The product of the invention performed slightly lower against Alternaria and had lower efficacy against Dydimella brvoniae and Botrytis cinerea than comparative example 1. Nevertheless, the chitinase comprising composition according the invention showed a very broad-spectrum of activity against a wide variety of fungi.

This is confirmed with the second round of experiments.

Claims (15)

Conclusions 1, Method for preparing a chitinase-comprising composition | by fermenting a bacterial strain capable of producing chitinase | in which the bacterial strain is immobilized on a support, | wherein the carrier comprises a porous polymeric material, wherein chitin } is present during the fermentation and for separating the chitinase | comprising composition of the immobilized bacteria. î Process according to claim 1, wherein the porous polymer material is prepared | is made from an isocyanate and a polyol, the polyol being produced by the bacteria | can be broken down. The method of claim 2 wherein the polyol is a polyester polyol. | | A method according to any one of claims 1 to 3 wherein the polymer comprises about 5% by weight or more ethylene glycol units, preferably about 10% by weight or more. 9 The method of claim 1 wherein the porous polymeric material is a polyvinyl alcohol. A method according to any one of claims 1 to 5, wherein the polymer material | has cell sizes from about 0.1 to about 9 mm, preferably 0.2-3 mm, | a A method according to any one of claims 1 to 6, wherein the polymeric material | . has a Tg of about 0°C or less, preferably of about -20°C or less. | A method according to any one of claims 1 to 7, wherein the polymer material | a density between 10-200 kg/m? preferably about 20-100 | kg/m. | 9. Method for preparing a chitinase-comprising composition | according to any one of claims 1 to 8, wherein the bacterial strain is a Pseudomonas strain, preferably P. fluorescens I, P. fluorescens II, P. | veronii, P. stutzeri, P. fragii or P. gessardii. | The method of claim 9 wherein the bacterial strain is a Pseudomonas | ssardii is, preferably a species deposited under CBS 146242. 11. Chitinase-comprising composition obtainable by the | method according to any one of claims 1-10, preferably by the method according to claim 10. A chitinase-comprising composition obtainable by the method of claim 11 and further comprising a protease inhibitor, Use of the composition according to any one of claims 11 to 12 as a | crop protection agent, such as a fungicide, biostimulant or insecticide, by applying the composition to the plant or seed. } Use according to claim 13 as a fungicide or as a biostimulant, in | preferred as a fungicide. | 15. Bacterial strain of Pseudomonas gessardii deposited under CBS 146242. |