PROCEDE DE PREPARATION DE SUSPENSION DE SPORE DE H. PULVINATA

Abstract

The present invention relates to a new method for preparing spores of Hansfordia pulvinata (H. pulvinata), enabling the production of a culture medium for these spores which can be stored, transported and used for the preparation of a suspension of said spores, a suspension which can be applied to an agricultural crop to protect it against parasitic fungi, in particular against Passolora fulva

Description

Title of the invention: PROCESS FOR PREPARING H. PULVINATA SPORE SUSPENSION

[0001] The present invention relates to a new method for preparing spores of Hansfordia pulvinata (H. pulvinata), allowing the production of a culture medium for these spores which can be stored, transported and used for the preparation of a suspension of said spores, a suspension which can be applied to an agricultural crop in order to protect it against parasitic fungi, in particular against Passolorafulva.

[0002] Passalora fulva, formerly Fulvia fulva (P. fulva) or Cladosporium fulvum, is a foliar fungus of tomato responsible for cladosporium leaf spot. It is frequently found on greenhouse tomato crops worldwide. When crops are heavily affected, it impacts yield and leads to significant economic losses. Furthermore, allergic asthma has been reported by workers in heavily contaminated greenhouses with high spore concentrations in the ambient air. Since the 1970s, numerous varieties resistant to cladosporium leaf spot have been commercialized. However, since the late 1990s and early 2000s, resistance gene breakdown has been observed in all greenhouse tomato production areas.

[0003] The parasitic fungus develops in the leaf parenchyma. Diffuse chlorotic spots, light green to pale yellow in color, are observed on the upper surface of the leaves, and an olive-brown mycelium sporulates on the lower surface. Infection generally begins on the lower leaves before spreading to the upper parts of the plants. The incubation period is 10 to 15 days. P. fulva is then disseminated from plant to plant within shelters via conidia (spores) carried by air currents, work clothes, insects, water droplets, etc.

[0004] The pathogen can survive as a saprophyte on culture debris, tools, and shelter walls. Thus, P. fulva is capable of contaminating a new culture if the disinfection of shelters and tools is not carried out properly.

[0005] P. fulva secretes virulence factors in the form of small cysteine-rich proteins, called effectors, within the apoplast of plant cells during infection. These proteins interfere with the plant defense system, are recognized by the products of the (dominant) Cf resistance genes, and induce a hypersensitivity (HR) response. Various resistance genes have been progressively introduced into many cultivated varieties since the 1970s and confer varying levels of resistance against different races of P. P. fulva. The intensive use of these resistant varieties has generated significant selection pressure on P. fulva, to the point of giving rise to new strains capable of overcoming all the resistance genes introduced into current commercial varieties. In addition to overcoming resistance, phenomena of tolerance / resistance to fungicides have also been described in P. fulva, further complicating the control of this pathogen.

[0006] In 1978, a study highlighted the presence of a hyperparasitic fungus of P. fulva in market garden greenhouses in northern Finistère. This hyperparasite, belonging to the family Deuteromycetes or Fungi imperfecti, was identified as Hansfordia pulvinata, a species first described in the literature in the 1950s and reported in Italy under the name Botrytis yuae.

[0007] The potential of H. pulvinata as a biological control agent against cladosporium has been highlighted on numerous occasions over the past few decades.

[0008] However, despite these encouraging prospects, no development of a biocontrol product from H. pulvinata has yet emerged.

[0009] Indeed, the spores (or conidia) of this fungus do not preserve well in water, and inoculation must be carried out immediately after preparation of a spore suspension at the required concentration for application to a crop. Long-distance transport of a suspension of H. pulvinata spores is therefore not possible due to the significant risk of spore mortality and subsequent treatment ineffectiveness.

[0010] There is therefore a need for a process for producing H. pulvinata spores which allows on the one hand the optimization of the production of spores in sufficient quantity and to allow their transport while guaranteeing their viability and effectiveness when a suspension is prepared and applied to a culture to be treated.

[0011] The applicator would then only have to prepare a suspension at a given spore concentration and apply this spore suspension extemporaneously to the plants to be treated.

[0012] Currently, the only study developing a culture medium suitable for the production of large quantities of H. pulvinata conidia (spores) is described by a Brazilian team in 2009 (Melo and Mello 2009, Melo, Débora Ferreira, and Sueli Corrêa Marques de Mello. 2009. “Ideal culture conditions for Dicyma pulvinata conidia mass production.” Pesquisa Agropecuâria Brasileira 44 (10): 1232-38). This study describes various culture media based on rice, maize, and cooked wheat, inoculated with H. pulvinata in several types of containers such as aluminum trays, polypropylene bags, and Erlenmeyer flasks. Several incubation conditions for the fungus were tested in parallel with the different media and containers: Continuous light, darkness, alternating light / dark cycles, and a temperature range of 19 to 31°C were used. The study shows that the best substrate appears to be cooked rice, placed in polypropylene bags and kept in a culture chamber set between 19 and 25°C with continuous lighting to promote sporulation. The team's objective is to produce large quantities of conidia (spores) for use as a biocontrol agent against the pathogenic fungus Microcyclus ulei, also known as Fusicladium macrosporum, which causes South American rubber tree leaf disease. Another study from 2015 confirms that the optimal incubation condition for conidia production is a constant temperature of 21°C combined with a photoperiod of 12h (JMS Vivas, Vivas, and Silveira 2015. Vivas, Janieli Maganha Silva, Marcelo Vivas, and Silvaldo Felipe da Silveira. 2015.“Effect of temperature on in vitro growth and sporulation of hyperparasites fungi of Asperisporium caricae”. Pesquisa Agropecuâria Tropical 45 (1):73-81). .

[0013] The invention thus aims at developing a culture medium suitable for the production of H. pulvinata conidia (spores) for large-scale professional use to cover thousands of square meters of tomato production greenhouses. The technique proposed by the inventors is adapted to the production of a culture medium conducive to the development and sporulation of H. pulvinata, allowing for simplified harvesting of the spores (or conidia) and the preparation of an inoculum for immediate use by spraying onto plants infected with P. fulva.

[0014] Thus, according to a first embodiment, the invention aims to:

[0015] A method for preparing a culture medium for H. pulvinata spores including the following steps: - Provide cooked millet seeds, - Fill a container equipped with a gas exchange device with the cooked millet seeds, - Close and sterilize the container once it is filled. - Inoculate the sterile container filled with cooked millet seeds with a suspension of H. pulvinata containing a mixture of H. pulvinata spores and mycelium, - Incubate the inoculated container at a temperature, lighting conditions and for a sufficient time to allow homogeneous colonization of the seeds by H. pulvinata and the appearance of a grey down on the surface of all the seeds containing H. pulvinata spores.

[0016] The term "container" here refers to a resealable package, preferably flexible, such as a bag, pouch, pocket, or jerrycan. Preferably, the container is a bag made of flexible plastic material, such as a thermoplastic polymer, for example polypropylene, or polyethylene, particularly polypropylene.

[0017] The volume of the container is not critical and it can be chosen according to the chosen destination in order to facilitate transport, storage and subsequent implementation.

[0018] Typically, the container may have a volume ranging from 0.2 liters to 20 liters, for example, more particularly between 0.2 liters and 10 liters, more particularly between 0.2 liters and 5 liters, or even between 0.2 liters and 2 liters, and more particularly about 1 liter. Advantageously, the containers are filled to about two-thirds full to ensure proper sealing / closing and stirring, as well as to allow for the respiration of the future mycelium.

[0019] The term "gas exchange device" here refers to a device that allows gases to enter and / or exit the container to enable the proper growth of H. pulvinata. Typically, such a device could be a 0.5 µm filter, which allows for greater gas exchange but also increases the potential risk of contamination due to its relatively large pores. More advantageous are 0.2 µm filters, which are well-suited for mycelium cultivation. Slide-on filters offer the advantage of even better gas exchange and are even more suitable.

[0020] By way of example, suitable containers for culturing mycelium according to the invention include polyethylene bags equipped with filters having a pore size of 0.2 µm, in particular slide-on filters with a pore size of 0.2 µm. Such types of bags are available from UNICORNBAGS (Texas, USA).

[0021] The inventors chose and selected millet seeds as a particular culture medium suitable for the purpose.

[0022] The expression "cooked millet seeds" means millet seeds which have undergone heat treatment in an aqueous medium, water or steam, in a conventional manner, in order to make them digestible, in particular to achieve hydration of the starch contained, softening of the grain's outer layer and enabling the mycelium to develop.

[0023] Millet, or Panicum miliaceum, commonly known as "common millet," "white millet," or "spike millet," is a plant approximately 1.3 m tall, with loose, branched, drooping panicles of inflorescences. This cereal is cultivated in temperate regions, in Eastern Europe, the USA, Argentina, and Australia.

[0024] Panicum miliaceum, common millet, is a species of monocotyledonous plant in the family Poaceae, subfamily Panicoideae, native to temperate Asia.

[0025] This annual herbaceous plant, cultivated for its edible seeds, is a secondary cereal, well adapted to semi-arid areas, which still constitutes a food crop in some regions of Asia.

[0026] Common millet is the true millet (milium) of the Romans. Cultivated since prehistoric times, and probably before the cultivation of wheat in Europe. Millet Common corn is an annual herbaceous plant, growing to a height of approximately 30 cm to 1 m, but can exceed 1.5 m. The stems are rough, woody, and hairy. It is a C4 photosynthetic plant, like maize. The compound inflorescence is a fairly dense, highly branched panicle that droops at maturity. Depending on the variety, the panicle can be nodding (closed, like a broom) or erect (widely open). The fruit is an ovoid caryopsis, 3 mm long and 2 mm wide, enclosed by two robust glumes: the lemma (lower lemma) and the palea (upper lemma). The enclosed seed is highly variable in color, ranging from very light to very dark: white, cream, yellow, orange-red, olive-brown, gray, and brownish-black; when naked, it is creamy white. An average of 175 seeds are needed to make one gram. Proso millet includes the subspecies Panicum miliaceum subsp. agricola H. Scholz & Mikolâs, Panicum miliaceum subsp. Miliaceum, Panicum miliaceum subsp. ruderale (Kitagawa) Tzvelev, Panicum miliaceum var. badium Kôm, Panicum miliaceum 'Album' as well as Panicum miliaceum 'Violaceum'.

[0028] Thus, the expression "millet seeds" in the context of the present invention means common millet seeds, P. miliaceum and its subspecies.

[0029] The millet seeds are thus cooked conventionally in hot water or steamed until softened and swollen seeds are obtained.

[0030] The seeds can be spread out, for example on drying racks, or left to drain in order to remove excess water. This drying or draining step can last a few hours at room temperature or under refrigeration and allows the cooking water to be removed; typically for 12 to 24 hours at room temperature, for example, i.e., around 20°C.

[0031] The inventors have indeed noted that the use of millet seeds, compared to other growing media such as rice, wheat, or rye, gives a particularly advantageous and also surprising result in terms of production and especially in the recovery of active spores after removal of the millet seed as a growing medium. The inventors cannot explain this phenomenon and, without being bound by the theory, suppose that the size and spherical geometry of the millet grain allows for the harmonious development of the mycelium and spores, as well as their recovery by solid / liquid separation.

[0032] The container is then filled with the cooked millet seeds, typically half or 2 / 3 full.

[0033] The container is hermetically sealed, for example by a cap if it is a can or bottle, and preferably by heat sealing if it is a bag, in particular a polypropylene bag.

[0034] The container filled with cooked millet seeds and hermetically sealed can then be sterilized.

[0035] The sterilization step can be carried out using any technique suitable for the container material to ensure sterilization of the contents. For example, it could involve sterilization in an autoclave at 120°C for 15 minutes to 1 hour, depending on the size of the container.

[0036] Autoclaving may be carried out twice in order to ensure complete sterilization of the millet seeds.

[0037] The seeding / inoculation step of the container containing the cooked millet seeds with H. pulvinata is then carried out with a suspension comprising mycelium and spores.

[0038] H. pulvinata is cultured either on solid medium (such as, for example, PDA, potato dextrose agar), or in a stirred liquid culture medium based on potato extract, for example.

[0039] A suspension containing a mixture of H. pulvinata spores and mycelium is prepared from a fresh culture (less than 10 days old) from either a stirred solid or liquid culture medium.

[0040] This suspension of H. pulvinata is used to inoculate containers containing cooked and sterilized millet grains.

[0041] Advantageously, H. pulvinata is strain HP1-OBS-2021 filed with the NCIMB (Aberdeen, Scotland) under NCIMB Number 43776 on May 26, 2021.

[0042] This strain has proven to be particularly advantageous both in terms of spore productivity and in terms of the activity of the spores thus produced with regard to the protection of an agricultural crop against parasitic fungi, in particular against Passolorafulva.

[0043] After inoculation the containers can be incubated in a culture chamber at a temperature between 18 and 30°C, in particular between 20 and 25°C, more particularly around 22°C.

[0044] The photoperiod of the culture advantageously corresponds to a natural photoperiod, lasting approximately 7 to 21 days, in particular between 10 and 15 days, in order to obtain a homogeneous colonization of the seeds by the white mycelium of H. pulvinata and a development of spores which is observed by the appearance of a light grey down on the surface of all the seeds.

[0045] Containers containing seeds covered with H. pulvinata spores can then be stored for several days and transported to the treatment sites to be used for the preparation of spore suspensions intended to be applied to the crops to be treated.

[0046] Thus, another object of the invention is to provide a method for preparing a suspension of H. pulvinata spores comprising the following steps: - Mix the culture medium obtained previously, comprising millet seeds with a grey down on their surface containing H. pulvinata spores, with an aqueous phase in a closed container, - Shake the mixture, - Perform a liquid / solid separation using a suitable device that allows the spores to pass through the liquid phase and the millet seeds to remain in the solid phase - Recover the liquid phase containing the spores of H. pulvinata.

[0047] The v / v ratio of culture support (i.e. millet seeds coated with spores) / aqueous phase in the mixture to be stirred may be between L? and 1 / 10 or even between L? and 1 / 3.

[0048] This ratio will depend on the desired spore concentration in the resulting liquid phase. This concentration will advantageously be between 10³ and 10⁷, in particular between 10⁴ and 10⁶, or even between 10⁵ and 10⁶ spores / ml.

[0049] The aqueous phase may be water, in particular demineralized water, or any suitable aqueous phase, i.e. water supplemented with various excipients suitable for use in the subsequent solid phase.

[0050] The aqueous phase / culture medium mixing step consists of separating the spores and suspending them in said aqueous phase. The method of carrying out this agitation is not critical in itself and can be performed by any means known to a person skilled in the art, for example, stirring tables or magnetic stirrers.

[0051] The liquid / solid separation step aims to recover a liquid phase containing the spores and free of millet seeds and any plant debris contained.

[0052] This liquid / solid separation can be achieved by a suitable technique, such as centrifugation or filtration.

[0053] In particular, it will involve filtration using a filter that retains seeds, particles and plant debris while allowing H. pulvinata spores to pass through.

[0054] Typically, filtration using a 200 µm filter, or even a 100 µm filter, will allow spores to pass through while retaining plant debris and seeds.

[0055] The advantage of this liquid / solid separation step is to obtain a liquid phase containing the spores in suspension, usable in conventional spraying equipment used in agriculture. In particular, the method allows for the control of Passolorafulva. Even more advantageously, it is suitable for agricultural plots and tomato plots, especially tomato greenhouses.

[0056] The resulting liquid phase containing the spores may optionally be diluted with water or any other aqueous liquid in order to obtain the appropriate spore concentration.

[0057] The resulting suspension must be able to pass through the spray nozzles used in greenhouses without them becoming clogged.

[0058] It is advantageous to apply a suspension according to the invention at relatively low pressures in order to avoid alteration of the spores.

[0059] METHODS OF IMPLEMENTING THE INVENTION

[0060] Embodiment 1. According to a first embodiment, the invention relates to a method for preparing a culture medium for H. pulvinata spores comprising the following steps: - Provide cooked millet seeds, - Fill a container equipped with a gas exchange device with the cooked millet seeds, - Close and sterilize the container once it is filled. - Inoculate the sterile container filled with cooked millet seeds with a suspension of H. pulvinata containing a mixture of H. pulvinata spores and mycelium, - Incubate the inoculated container at a temperature, lighting conditions and for a sufficient time to allow homogeneous colonization of the seeds by H. pulvinata and the appearance of a grey down on the surface of all the seeds containing H. pulvinata spores.

[0061] Embodiment 2. In another embodiment, the invention relates to a method according to embodiment 1, characterized in that the container is a bag made of flexible plastic material, for example polypropylene.

[0062] Embodiment 3. In another embodiment, the invention relates to a method according to one of the preceding embodiments in which the strain of H. pulvinata is the strain HP1-OBS-2021 filed with the NCIMB (Aberdeen, Scotland) under NCIMB Number 43776.

[0063] Embodiment 5. In another embodiment, the invention relates to a method for preparing a suspension of H. pulvinata spores comprising the following steps: -Mix the culture medium obtained according to one of embodiments 1 to 4, comprising millet seeds with a grey down on their surface containing H. pulvinata spores, with an aqueous phase in a closed container, -Shake the mixture, -Perform a liquid / solid separation using a suitable device that allows the spores to pass through the liquid phase and the millet seeds to remain in the solid phase -Collect the liquid phase containing the spores of H. pulvinata.

[0064] Embodiment 6. In another embodiment, the invention relates to a process according to embodiment 5, characterized in that the v / v ratio of culture support / aqueous phase in the mixture to be stirred is between L2 and 1 / 10 or even between L2 and 1 / 3.

[0065] Embodiment 7. In another embodiment, the invention relates to a process according to one of the embodiments 5 or 6, characterized in that the concentration of spores in the liquid phase obtained is between 103 and 107 spores / ml.

[0066] Embodiment 8. In another embodiment, the invention relates to a process according to one of the embodiments 5 to 7, characterized in that the liquid / solid separation step is carried out by filtration, in particular with a 200pm size filter.

[0067] Embodiment 9. In another embodiment, the invention relates to a suspension of H. pulvinata spores obtained according to one of embodiments 5 to 8.

[0068] Embodiment 10. In another embodiment, the invention relates to a method for treating an agricultural plot against pests, comprising the application of a suspension of H. pulvinata spores, in particular a suspension according to embodiment 9. In particular, the method makes it possible to control Passolorafulva. Advantageously, the agricultural plot is a tomato plot, in particular a tomato greenhouse.

[0069] EXAMPLE

[0070] A strain of Hansfordia pulvinata was purified in 2020 from a tomato leaf sample infected with P. fulva from a greenhouse in Côtes-d'Armor. This strain was identified based on morphological criteria described by Peresse and Le Picard (Peresse and Le Picard 1980). This H. pulvinata strain is stored in the mycotheque of the pathology laboratory of OBS innovation (strain HP1-OBS-2021 deposited with the NCIMB (Aberdeen, Scotland) under NCIMB Number 43776 on May 26, 2021).

[0071] The millet seeds are first cooked until softened and swollen, then spread on drying racks to remove excess water for 24 hours at 20°C. The seeds are then bagged in flexible, 75 µm thick polypropylene bags measuring 18 cm x 32 cm (width x height) and equipped with filters to allow for gas exchange, at a rate of 11 seeds per bag. These bags are sealed by heat sealing and then autoclaved twice to ensure perfect sterility of the seeds.

[0072] H. pulvinata is cultured either on a solid medium (PDA, potato dextrose agar) or in a stirred liquid culture medium based on potato extract. A suspension containing a mixture of H. pulvinata spores and mycelium is prepared from from a fresh culture (less than 10 days old) obtained from either a stirred solid or liquid culture medium. This suspension of H. pulvinata is used to inoculate bags containing previously sterilized millet grains.

[0073] After inoculation, the bags are incubated in a culture chamber at 22°C, natural photoperiod, for approximately 10 to 15 days to obtain homogeneous colonization of the seeds by the white mycelium of H. pulvinata and spore development, which is observed by the appearance of a light gray down on the surface of all the seeds. The bags containing seeds covered with H. pulvinata spores can then be stored for several days and transported to the treatment site. The preparation of the spore suspensions is carried out extemporaneously at the treatment site, following the preparation instructions below.

[0074] To prepare 51 ml of spore suspension at a concentration between 10⁵ and 10⁶ spores / ml, the contents of a well-spore-coated bag should be placed in a closed container, agitating the grains in approximately 2 liters of water, preferably demineralized. After agitation, the liquid should be filtered through a suitable mesh filter (200 µm), allowing the H. pulvinata spores to pass through while retaining millet grains and other debris. The suspension should then be adjusted to obtain a final volume of 51 ml at the desired concentration. The resulting suspension must be able to pass through the spray nozzles of greenhouse growers without clogging them.

[0075] All the steps mentioned above were implemented and validated by application to a 2000m² area of ​​greenhouse tomato cultivation. The results observed are very promising for the prevention of parasitic fungi, particularly against Passolora fulva.

Claims

Demands

1. A process for preparing a spore suspension of H. pulvinata comprising the following steps: a) Preparation of a culture medium comprising the following steps: - Providing cooked millet seeds, - Filling a container equipped with a gas exchange device with the cooked millet seeds, - Closing and sterilizing the container thus filled, - Inoculating the sterile container filled with cooked millet seeds with a suspension of H. pulvinata containing a mixture of H. pulvinata spores and mycelium, - Incubating the inoculated container at a temperature between 18 and 30°C, at a natural photoperiod, for a time of approximately 7 to 21 days, to allow homogeneous colonization of the seeds by H. pulvinata and the appearance of a grey down on the surface of all the seeds containing H. pulvinata spores.pulvinata, b) Mix the culture medium obtained in the previous step with an aqueous phase in a closed container, c) Shake the mixture, d) Carry out a liquid / solid separation using a suitable device allowing the passage of spores into the liquid phase and the retention of millet seeds in the solid phase, e) Recover the liquid phase containing the H. pulvinata spores.

2. The method according to claim 1, characterized in that the container equipped with a device enabling gas exchange is a bag made of flexible plastic material, for example polypropylene.

3. A method according to any one of claims 1 or 2, characterized in that the incubation step is carried out in a culture chamber at a temperature between 20 and 25°C, more particularly around 22°C, for between 10 and 15 days.

4. A method according to any one of the preceding claims wherein the H. pulvinata strain is strain HP1-OBS-2021 filed with the NCIMB (Aberdeen, Scotland) under NCIMB Number 43776.

5. A method according to any one of claims 1 to 4, characterized in that the v / v ratio of culture support / aqueous phase in the mixture to be stirred is between F? and 1 / 10 or even between F? and 1 / 3.

6. A process according to any one of claims 1 to 5, characterized in that the concentration of spores in the liquid phase is between 103 and 107 spores s / ml.

7. A method according to any one of claims 1 to 6, characterized in that the liquid / solid separation step is carried out by filtration, in particular with a 200pm size filter.