WO2009083819A1

WO2009083819A1 - Trichoderma granule production

Info

Publication number: WO2009083819A1
Application number: PCT/IB2008/054757
Authority: WO
Inventors: Yahya Sems Yonsel; Mehmet Sevki Batum
Original assignee: Simbiyotek Biyolojik Urunler Sanayi Ve Ticaret Anonim Sirketi
Priority date: 2007-12-31
Filing date: 2008-11-13
Publication date: 2009-07-09
Also published as: TR200709242A2; EP2227538A1

Abstract

This invention is about producing and applying Trichoderma granules to agricultural plants and especially olive trees and using Trichoderma harzianum spores as a microbial fertilizer and microbial bio-control agent to protect against the wilt (Verticillium dahliae) observed in trees.

Description

Description TRICHODERMA GRANULE PRODUCTION

[ 1 ] TECHNICAL FIELD

[2] This invention relates to the application of the Trichoderma harzianum spores as microbial fertilizer and microbial biocontrol agent against the Verticillium dahliae wilt in trees and to the production process of the Trichoderma granules.

[3] PRIOR ART

[4] It is known that Trichoderma rapidly multiplies and covers the roots. This symbiosis lasts throughout the life span of annual plants whereas in perennials an application is recommended each year.

[5] Trichoderma benefits the formation of roots; they reach greater depths. It can be observed that roots of maize covered with trichoderma grow deeper than 1 m. This development of the roots results in a more developed shoot and enhances the maturation of the plant. The plant is also more resistant by drought due to deeper reaching roots.

[6] Trichoderma triggers the immune system and growth hormones of plants; many plant diseases are prevented this way: for example root rot in Citrus trees, sunflower, sugar cane, soy bean, pea, groundnut, tea, coffee and rubber tree, wilt in cotton, tomatos and peppers, capsule rotting in cardamom is prevented [1, 2].

[7] Trichoderma prevents attacks of soil borne fungi against the plant. The prevention of such diseases caused by fungi is due to the antagonist character of Trichoderma. The effectiveness of Trichoderma against pathogenic fungi in roots and vineyards has been proven [1,2,3].

[8] Trichoderma makes nutrients like phosphorus, manganese, copper and iron soluble, therefore easily digestable for the plant; this enables the plant to grow faster. Also, substances such as hydrocyanic acid (HCN) that inhibit the development of the roots are turned into a harmless form by Trichoderma. These activities of Trichoderma make it possible to reduce chemical fertilizer usage; Trichoderma treated maize requires 40% less nitrogen [I].

[9] Once Trichoderma has established itself on the roots, it is not affected by chemicals.

Hence, Trichoderma' s beneficial effects continue even by applications of fungicides, pesticides or insecticides on the field. Trichoderma can be applied on a wide field ranging from greenhouse plants to field crops. In greenhouses improvements on measurable parameters such as leaf development and chlorophyll amount have been observed.

[10] Trichoderma harzianum spores, registered as Trichoderma harzianum KUEN 1585, sprout on the roots, establish themselves on the roots with one application, multiply rapidly and cover the roots, form a symbiosis with the plant, increase the yield throughout the life span of the plant and decrease the need for fertilizer. Once Tri- choderma harzianum covers the roots, it inhibits pathogens from colonizing on the roots; it can be used as a precautionary measurement against root diseases.

[11] A state of art application, the international patent document WO2005068609 is concerned with clones of Trichoderma harzianum. This document relates to a method used to separate, select and improve fungi causing tree diseases and biocontrolling thereof.

[12] A state of art application, the Japanese patent document JP2002153129 is concerned with a method that reactivates fruit and other trees. The method is characterized through an application of preparing a clamydospore or conidia from Trichoderma harzianum SK-5-5 to be used in reactivating old or non-active fruit and other trees.

[13] A state of art application, the English patent GB 1274291A is concerned with a method related to harmful microorganisms that live in trees, various plants and wood products. Other methods like benefits of microrganisms on woody plants like young trees or saplings or methods to prevent microbial parasites are described in this document.

[14] A state of art application, the international patent document WO0064837 is concerned with an inorganic fertilizer that nourishes and protects plants. The mentioned fertilizer for protecting is prepared biologically and contains at least one of the nitrogen, phosphorus, potassium, magnesium, sulfur or calcium containing groups. Biological preparation protects the plants against diseases; Trichoderma consists of an inert substrate with a mix of conidia and at least one type of a filamentus fungus chosen from a group containing verticillium wilt (Verticillium dahliae) genes. The inorganic fertilizer s produced by using at least one of the a.m. nutrients and generally by spraying on the surface of granules.

[15] A state of art application, the US patent document US19767A1 is concerned with controlling and defining the genes of plants containing disease causing genes.

[16] Brief description of the invention

[17] Purpose of this invention is to produce Trichoderma granules from Trichoderma harzianum spores to be used as a microbial fertilizer and a microbial biocontrol agent in trees suffering from verticillium wilt (Verticillium dahliae) and the application of this product on olive trees and other agricultural crops.

[18] The invention in details

[19] Olive trees have various diseases, one of them being a wilt disease: Verticillium dahliae. This wilt (Verticillium dahliae) has established itself on many various hosts and in many regions of the world. Verticillium dahliae multiplies on dying host tissue and lives in microsclerotia form in soil. This situation changes accordingly as the soil humidity and temperature change. It can live on for 10 or more years in soil; the formation of microsclerotia plays an important role in this longevity.

[20] It has been established that in woody plants the cause of wilt is more often due to

Verticillium dahliae. Three types of effects have been observed: Leaf symptoms (wilting, deformation), vascular system symptoms (discoloration, water-conducting tissue blockage) and branch dieback. In many types of woody plants after wilting there is a strong branch dieback and in some woody plants new leaf formation has been observed after defoliation. Especially olive trees improve in this manner. Although the wilt disease (Verticillium dahliae) can affect old trees, it usually harms 5-6 year old trees more. Symptoms of the wilt observed in olive trees are apoplexy, dieback (acute form) and slow death (chronic form).

[21] Apoplexy develops from early spring to winter. First the leaves start to wilt, and then they turn light brown and start turning backwards. Apoplexy is seen as a rapid death of twigs and branches and later death of the tree. The branches become a slight purple color and in young trees partial defoliation can be observed. In some cases the dried leaves of the trees stay attached on the twigs and branches and the vascular system becomes brown in color.

[22] Necrosis of the flowers is a sign of the chronic form. The flowers become mummified and remain attached to the twigs. The leaves of the twigs that are affected turn into a stale green color and fall without wilting. Generally symptoms are seen first in flowers and then in leaves, afterwards signs of necrosis are seen on the twigs. The barks of twigs become a reddish brown while the vascular system becomes dark brown in color. Signs of slow death are seen in spring and continue towards the summer. Olive trees showing these signs can improve and the signs of disease lessen in the following years.

[23] Usually there is a cross-pathogenic relationship between olive trees and other hosts.

It is known that a reason behind the common occurrence of the wilt (Verticillium dahliae) in olive plantations is the prior cultivation of tomatos, peppers, cotton and such susceptible plants on the same soil. Also disease carrying leaves of olive trees have a considerable affect on the increase of the microsclerotia level in the soil. The pathogen propagules are spread in the immediate area by ploughing and irrigation. Long distances are infested through transportation of infected plant material. Ploughing practices and frequencies are also thought to severe the impact of the disease. Irrigated vs. non-irrigated land has shown 21% and 9% of disease occurrence respectively. Soil and air temperature has a considerable effect on the disease; observations show a good growth of the disease at 20-25⁰C in spring with a peak of 30-35⁰C in the summer.

[24] The technique developed in the invention of Trichoderma granule production goes through the following steps. [25] 10. Preparation of the culture medium by mixing the raw materials with water

[26] 20. Culture medium is introduced to the fermentation tank and feed tank

[27] 30. The cooled inoculation tank is inoculated with a pure microorganism culture

[28] 40. The inoculant is transferred to the main fermentor by means of air pressure

[29] 50. Temperature and pH are monitored throughout the fermentation

[30] 60. Samples are taken everyday under asceptic conditions and analyzed

[31] 70. Water is separated through a centrifuge

[32] 80. The resulting biomass is washed with water to rid of culture medium rests

[33] 90. Water is removed through ultra-filtration; a filter cake is formed

[34] 100. Dough is won

[35] 110. The dough is put through an extruder that froms the granules

[36] 120. The granules are dried

[37] Corn starch, soy flour, sugar and wheat flour or molasses are used as raw materials and mixed with water in preparing the culture medium (10) that goes into the fermentors and feed tanks (20) at 120 OC for two hours. The cooled fermentation tank is inoculated with a pure microorganism culture and production starts. The inoculant that matures after a period of 36-48 hrs is transferred to the main fermentation tank by means of sterile air pressure (40). Depending on the properties of the microorganism to be produced the temperature and pH are monitored throughout the fermentation process (50) and sterile air is given. Samples are taken everyday under asceptic conditions and analyzed (60). After 3-7 days the maximum microorganism count is reached and the separation process begins. By means of the centrifuge water is removed (70) and a biomass is won. This mass is washed with water to get rid of culture medium rests (80) and if needed a further step of ultrafiltration through 0.2 micrometer membranes is done to reach the desired microorganism count (90). At this stage the unfinished product is a liquid organic fertilizer. [38] To increase the spore count in the fermentation liquid the solid part has to be separated from the liquid part prior to the drying process. This separation process demands the use of filters (e.g. pressurized, cloth, vacuum), centrifuges (e.g. high rotation, continuous, dscontinuous centrifuges), micro and ultra filters. The solid part or filter cake contains Trichoderma harzianum spores and water. The filter cake is enriched with various supportive ingredients prior to drying in order to increase the dry matter percentage. These supportive ingredients in powder form can be inorganic and include sepiolite, vermiculite, leonardite, coal, aerosil, silica, montmorillonite or such organic substances as cellulose, yeast, starch, sugar, flour and sawdust.

[39] The filter cake that reaches a higher percentage of dry matter with the addition of the supportive ingredients is formed into a dough with the help of a mixer (100). The percentage of dry matter in this dough is about 25-27 '. The dough is trasferred into granules by putting it through an extruder (110) and the desired shape is given. The granules are dried in tray, drum or fluidized bed type of driers (120). The temperature of the air going into the drier should be about 40-125 oC, preferably 70-90 ⁰C. Drying continues until the temperature of the drier cabin is 35-60 oC, preferably 40-50 oC. The ingoing air temperature is than controlled to 35-60 oC, preferably 40-50 oC. Drying continues until the level of humidity reaches 1-15%, preferably 3-7%. The size of the granules are 0.05-5 mm, preferably 0.5-3 mm. In order to get a powder product granules are grinded in ball or rod mills or attritor types of grinding mills. Powder size should be 100-1000 micrometers, preferably 300-500 micrometers.

[40] Solid microbial fertilizer can be in granule or powder form. Granule formulations are produced by means of fixing Trichoderma harzianum spores on to a material that does not dissolve in water. This formulation increases the spore's ability to cope with environmental stress factors and is designed to be used in applications involving seedlings, saplings, trees, and soil.

[41] Another method of producing Trichoderma granules is solid fermentation. The solid fermentation environment is prepared by mixing various nutritious elements at a percentage of 30-70, preferably 40-50% into minerals such as vermiculite, zeolite, bentonite, calcite, organic materials such as wood chips, saw dust, hackled corncob or crops such as bran, bulgur, semolina, wheat, barley, rice and adding water (10). Pure Trichoderma culture in form of spores or mycelia is inoculated to the environment (30). The inoculated environment is taken into a solid phase tray fermentor or a mixing deep bed fermentor (40).

[42] Spore formation is obtained after a period of keeping the temperature with the help of humid air at 25-30 oC, preferably at 25-28 oC (50), for 72-96 hours.

[43] The humid mixture containing spores of Trichoderma is put through the extruder, made into granules (110) and dried in tray, drum or fluid bed types of driers (120).

[44] The spores of the Trichoderma harzianum formulation developed for olive trees are fixed on a carier material. The supportive ingredients on these granules that do not dissolve in water enable the spores to sprout and colonize on the roots of the olive tree. The following materials are used: nutrients: sugar, starch, soy protein, wheat flour, citric acid; natural gums as adhesives; aerosol, silica, sepiolite, vermiculite, bentonite, talc, coal dust, leonardite, bran, semolina, soy pulp, wood chips, cellulose and tree bark as carriers. The size of the carriers should be 0.01-10 mm, preferably 0.1-2 mm.

[45] The following trial has been made with the above mentioned invention.

[46] TRIAL:

[47] A reactor with a capacity of 300 litres has been used for a production of a 240 litre working volume.

[48] The spore count in the liquid has been noted as 1*108 /ml. [49] The resulting liquid was put through a centrifuge, the filter cake was dried and a spore count of 2.5*109/g was obtained.

[50] The spore count has been adjusted to read 2* 107/g for the powder formulation.

[51] After a 6 month period the spore count was found 2* 107/g, after a year it was 1*107

/g- [52] Industrial applicability

[53] The wilt disease (Verticillium dahliae) can be summarized as a clogging of the vascular system, which inhibits water and nutrient supply to the branches, by entering of the pathogen into the plant through the roots. Hence, the symptom of drying out in trees can be observed. An intervention of the wilt disease (Verticillium dahliae) is not possible once the pathogen is in the vascular system. That is why it is important to inhibit this pathogen from colonizing the roots. The invention inhibits this pathogen from colonizing the roots and protects olive trees from the wilt disease (Verticillium dahliae).

[54] Due to the above mentioned reasons in areas where the wilt (Verticillium dahliae) is seen treatment of healthy saplings and trees should have priority. The best treatment is to apply the granule to saplings before they come in contact with infested soil. Applying the granule to saplings guarantees; colonization of roots with Trichoderma harzianum, highest protection and an easy application.

[55] The most important part is to reach all of the roots growing radially from the stem and pouring the Trichoderma granule solution or dry granules directly into the holes opened up to the roots. Coverage of all the roots with Trichoderma harzianum provides the best protection. Hence, tree applications have to be carried out with great care.

[56] The subject of the invention is a microbial fertilizer containing spores of Trichoderma harzianum that rapidly colonizes the roots and forms a symbiosis with the plant. This fertilizer enhances rooting of the plant and protects it from different causes of disease. After it colonizes the roots it makes them reach greater depths, solubilizes nutrients such as phosphorus, manganese, copper and iron found in soil, turns root growth inhibiting HCN (hydrocyanic acid) into a harmless form, is not affected by chemical fungicides once it is established on the roots and is resistant to pesticides or insecticides. One application is enough for it to establish itself on the roots, increase the output of the plant through out its life span and lessen the need for fertilizer.

[57] Trichoderma harzianum products can also be applied to olive trees. The recommended application method and dosages for olive trees are explained below.

[58] The formulation developed for olive tree applications contains the spores fixed on a carrier material. These granules that do not dissolve in water enable the spores to sprout and establish themselves on the roots with the help of supportive ingredients. 200 g of the product is mixed with 20 lires of chlorine-free water and laid to rest to wait for the granules to soak up the water. During the application the mixture is blended to maintain the granules suspended. The liquid containing spores and supportive granules has to come in contact with the roots of the olive tree.

[59] Another preferred application method is to use dry granules: pour them into the holes opened up to the roots of the olive tree and pour 50-200 mililiters of water afterwards and cover the holes with soil.

[60] A preferred field of application for the invention is olive cuttings ; 20Og of the product is mixed into 20 litres of chlorine free water set at room temperature and filled into a vessel suitable for the height of the olive cuttings. The olive cuttings are laid to rest for at least 15-20 min. and then transferred to plastic tubes or pots. The granules have to reach the soil together with the roots.

[61] Another preferred application in olive trees is applied in three different methods

[62] In the first application 20Og of granules have been mixed into 20 litres of chlorine free water at room temperature and the saplings have been given 100 mililiters of this mixture, being constantly mixed to make sure that the granules also reach the roots, and then planted. Another application involved young saplings grown in plastic tubes where the mixture was poured in between the soil and the tube or directly on the roots after removing the plastic tube or by dipping the roots into the mixture. It was made sure that the granules reach the roots. In the last application some grams of dry granules were scattered on the soil of the planting hole, the roots of the olve sapling is set on the granules, the hole filled with soil and watered.

[63] All three application methods had the same results, there were no losses of saplings.

[64] Another preferred method of application was carried out on olive trees. Depending on the size of the tree 10-15 grams of granules are mixed with 1-1.5 litres of chlorine free water at room temperature. The mixture is diluted with more water to make a total of 10-15 litres. Then, a circle is drawn on the ground corresponding to the outer limits of the crown. A second circle on the ground is drawn about 75-100 cm away from the stem. Holes are opened so deep as to reach the roots in the area between the two circles and 1-2 litres of the mixture is poured into each hole. During the application the mixture has to be stirred or shaken to make sure that the granules reach the roots as well. Depending on the size of the tree 10-15 litres are needed.

[65] The Trichoderma harzianum spores that colonize the roots with application of the invention quickly sprout and cover the roots. The hormones produced by Trichoderma harzianum enhance root development, stimulate the green parts of the tree, new twigs increase, flowers increase. Trichoderma harzianum covers the roots like a glove and prevents pathogenic microorganisms from establishing on the roots.

Claims

[I] - Preparation of the culture medium by mixing corn starch or soy flour or sugar or wheat flour or molasses with water (10),

- Culture medium is introduced to the fermentation tank and feed tank (20),

- The cooled inoculation tank is inoculated with a pure microorganism culture (30),

- The inoculant is transferred to the main fermentor by means of air pressure (40),

- Temperature and pH are monitored throughout the fermentation (50),

- Samples are taken every day under asceptic conditions and analyzed (60),

- Water is separated through a centrifuge (70),

- The resulting biomass is washed with water to get rid of culture medium rests (80),

- Water is removed through ultra-filtration; a filter cake is formed (90) These steps characterize the production of Trichoderma fertilizer.

[2] A production characterized in requirement 1 ; Introduction of the culture medium into the culture tank after two hours of sterilization at 120 OC (20) [3] A production characterized in requirement 1-2; after a maturation period of

36-48 hours the inoculants is transferred to the fermentor by means of sterile air pressure. [4] A production characterized in requirement 1-3; separation of water with a centrifuge in 3-7 days. [5] A production characterized in requirement 1-4; the filter cake is ready after water separation utilizinf ultra filtration with a 0.2 micrometer membrane. [6] A production characterized in requirement 1-5; mixing of inorganic materials such as, sepiolite or vermiculite or leonardite or coal or aerosil or silica or mont- morillonite and organic materials such as, bran, or semolina, or soy pulp or wood chips or cellulose or tree bark with the filter cake to reach a dry mixture (100) [7] A production characterized in requirement 1-6; a dry mixture of 25-75 % (100)

[8] A production characterized in requirement 1-7; the dry mixture is made into granules by putting through the extruder (110) [9] A production characterized in requirement 1-8; granules are dried in a dryer

(120) [10] A production characterized in requirement 1-9; 70-90 ⁰C of air temperature in the dryer.

[I I] A production characterized in requirement 1-10; 40-50 oC of dryer cabin temperature. [12] A production characterized in requirement 1-11; drying up to a humidty of 3-7 %

[13] A production characterized in requirement 1-12; a granule size of 0.5-3 mm.

[14] A production characterized in requirement 1-12; a powder size of 300-500 micrometers.