Strain of fungus stagonospora cirsii davis having herbicidal activity against canada thistle

FIELD: biotechnology.

SUBSTANCE: strain of the fungus Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 has herbicidal activity against canada thistle and species closely related to it - S. Cirsii. It was deposited in the State Collection of Microorganisms of All-Union Research Institute of Plant Protection under the collection number of Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 and can be used for biological control of canada thistle.

EFFECT: invention enables to increase the herbicidal activity against canada thistle.

4 tbl, 3 ex

The invention relates to agricultural Microbiology, in particular, to the field of plant protection, in particular of controlling undesirable vegetation. Can be used to obtain mycoherbicide to fight Cirsium arvense L.

It is known that the Thistle field (Cirsium arvense) and related species Thistle bristly (.setosum) and creeping Thistle gray (SEM) is a perennial soboliferous weed. In the world agriculture yield losses with an average contamination of crops (about 15 plants/m²these species Cirsium row crops make up about 10%. The use of chemical herbicides justified when the density of their growth 2-3 PCs/sqm (Donald, 1994; Ulyanova, 1997 [8,4]).

As producers of mycoherbicides to fight Thistle field have been previously proposed various kinds of phytopathogenic fungi, for example: Septoria cirsii (Hershenhorn et al., 1993 [11]), Sclerotinia sclerotiorum (Bourdot et al., 2006, [7]), Alternaria cirsinoxia (Green&Bailey, 2000, [9]), Phoma destructiva (Guske et al., 2004, [10]) and P. macrostoma (Bailey&Derby, 2006, [5]). While infecting the Thistle field crushed mycelium Septoria cirsii infectious load of 80 g/l and the 48-hour period, increased leaf wetness defeat in controlled conditions causes the death of plants (Leth, 1988 [12]). However, such a long period of moisture necessary for the effective destruction of plants of the Thistle field Septoria cirsii, difficult to reproduce field conditions is. Alternaria cirsinoxiae and Phoma destructiva affect only the lower leaves of the weed. Sclerotinia sclerotiorum and Phoma macrostoma broad specialization, affecting a wide range of dicotyledonous plants.

As a producer of mycoherbicide to fight Thistle field known strain of the fungus Stagonospora cirsii Davis 1.41 (VIZR), adopted for the prototype. Grain substrates intensity of sporulation of strain 1.41 (VIZR) S.cirsii was 5.1×10⁸conidia/g of substrate. Pathogenicity on the leaf disks strain 1.41 (VIZR) S.cirsii in infectious load 5×10⁶conidia/ml was about 50%. The same pathogenicity showed a water suspension on the basis of the mycelium of this strain at a concentration of 100 mg/ml. Output virulent mycelium on sacharose-soy environment was 36 g/L. the Advantage of strain 1.41 (VIZR) compared with the prototype strain of the fungus Phoma macrostoma was the high specificity of the fungus S.cirsii dedicated to the family Asteraceae (Sochorova, 2011, [3]), the disadvantages of this strain include low efficiency.

The task of the invention to provide strain, superior strain 1.41 (VIZR) S.cirsii on herbicide activity in respect of the Thistle field and closely related species - .setosum, SEM, biomass productivity, and not inferior to him specificity of action.

The task was solved by obtaining a strain of the fungus Stagonospora cirsii Davis room 1.42 (VIZR). He was kind of the flax from the leaves of Canada Thistle (Cirsium arvense L. Scop.). The strain deposited in the Public collections of microorganisms wildebeest VIZR RAAS. His collection number 1.42 (VIZR).

The strain is designed for biological control of creeping Thistle field. Strain 1.42 (VIZR) was selected by screening the collection of strains of Stagonospora cirsii Davis, selected from the Thistle field, different geographical origin. Screening of strains was carried out according to the results of a rapid method of determining the pathogenicity area of necrosis produced on the third day, cut from the leaves of creeping Thistle (Berestetskiy et al., 2007, [6]) and growth rate on potato-glucose agar (CCA). The pathogenicity of selected strain was confirmed in experiments with whole plants of the Thistle field. Specialization strain was studied in whole plants 6 families in controlled conditions.

The strain was isolated Ahoerstemeier from the affected leaf spot Cirsium arvense L. from the sample collected in the Penza region p/o Ilia in 2003.

Identification was carried out on the determinant Saccardo (Saccardo, 1921, [13]).

The strain deposited in the Public collections of microorganisms wildebeest VIZR RAAS, where he was awarded the collection number 1.42 (VIZR). The strain is designed to develop bioherbicide against Thistle field and closely related species.

Cultural and morphological characteristics of strain S.cirsii 142 (VIZR).

Strain 1.42 (VIZR) in herbarium material gives sporulation in pycnidia. Conidia cylindrical, with rounded tops, straight or slightly curved, hyaline, 1-3, rarely with 4 partitions or without walls, without constrictions, (16) 21-23 (28)×(4) 5-6 (7) mm.

In pure culture strain grows well on potato-glucose medium, forming a white, velvety fluffy colonies, darkening with age. Pycnidia are formed only when the lighting colonies erythema lamps (for example, PE-30 with a wavelength of 290-400 nm). Pycnidia 120-150 µm diam., rounded, hemispherical, single, 1 (4) hole, brown, later becoming black. Conidial slime cream, light orange to pinkish color. Conidia obtained in pure culture, mostly without partitions (>70%) with numerous fatty inclusions (4.5) 7-14×2-3 .5 μm. Conidia with 1 to 3 partitions (7.5) 9-21×3-4 (5) mm. Chlamydospores are absent.

Physiological and biochemical characteristics S.cirsii 1.42 (VIZR).

The strain grows well on a standard semi-synthetic agar media. When the cultivation temperature 24°C, the diameter of colonies of strains of S. cirsii on potato-glucose agar is 70,8±2.8 mm, ovsanna agar 57,8±2.5 mm for maltose agar 60,0±2,0 mm On a standard agar medium of čapek mushroom grows poorly.

In pure culture strain 1.42 (VIZR) S. cirsii not sporanos the t in the dark. The fungus produces pycnidia on solid nutrient substrates (agarized medium or grain substrates) at a constant irradiation growing crops near ultraviolet light in the range of 290-400 nm. The radiation source 2 lamp LE-30 (Russia) at a distance of about 50 cm from the Petri dishes or flasks with culture. The output of conidia on the CCA is about 1×10⁶conidia/cm²and 1.2×10⁸conidia/g of grain substrate (pearl barley).

Storage of strains of S. cirsii is carried out in test tubes beveled on potato-glucose agar in refrigerator at 5°C for 1 year without replanting, and inoculated with mushroom pieces of pearl barley in 10%glycerol at -80°C in a freezer (Methods..., 1982; [1]).

Methods using strain described in the examples, and the results are presented in tables.

Example 1. Getting infectious material and the assessment of its quality.

In a 100-ml conical flask were introduced 10 g of barley. The substrate was moistened 8 ml of tap water. The substrate was sterilized by autoclaving for 30 min at 121°C, 2 times with a daily interval. Sterile substrate was inoculable agar blocks of 5 mm in diameter, cut from the edge of the 2-week-old colonies of the fungus. Flasks were incubated at 24°C in the dark for 2 days and then 8 days under irradiation lamps LAYE-30. Flasks were shaken every 2 days for PR is preventing clumping of the substrate. After 10 days of fermentation of the biomaterial were dried with sterile air laminar Boxing 2 days. Seroprotective of the fungus was determined from a sample of 1 g in 10 ml of 0.01% solution of Tween-80, the concentration of conidia in the suspension was counted using a camera Goryaeva.

To determine the viability of conidia on a glass slide was applied 3 drops of conidial suspension volume of 50 ál and put it in a humid chamber (Petri dish with two layers of filter paper moistened with 2 ml of distilled water). Glass incubated for 6 h at 24°C in the dark. Then a drop of the suspension was dried by a current of air in a laminar box. To account for the germination of conidia on the dried drops added drop lactophenol (lactic acid, glycerol, phenol and water in equal proportions). The resulting smears were at 100-fold magnification, viewing 50 conidia per drop, 6 drops/option. Conidia considered germinated if the length of germ tubes exceeded 2 times the width of conidia (Naumov, 1937, [2]).

To assess the pathogenicity concentration of conidia in the suspension was brought to 5×10⁶germinating conidia/ml by diluting the calculated volume of a 0.01% solution of Tween-80. A drop of suspension volume of 10 μl were applied to leaf disks cut from leaves of the Thistle field. One option used on 6 CDs. Accounting for the development of the disease (the area of necrosis of the total of Plamadeala, %) was performed after 3 days of incubation at 24°C and 12-hour photoperiod (Berestetskiy et al., 2007, [5]).

The good development of the mycelium of the fungus Stagonospora cirsii strain 1.42 (VIZR) at all studied grain substrate except wheat. Maximum spore productivity identified in the cultivation of the fungus in barley grain (5,1×10⁸conidia/g of substrate). Mushroom satisfactory to spironol also the grains of rye and barley (table 2). Much worse sporulation S. cirsii formed on rice, millet and wheat. The viability of conidia of the fungus obtained in barley grits, accounted for more than 90%, the development of necrotic spots on the leaf disks Thistle was about 5 mm in diameter after 72 h after inoculation.

To obtain mycelium strain was cultured on sacharose-soy medium of the following composition: sucrose, 60 g soy flour 15 gr, KN₂RHO₄1 g, MgSO₄0.5 g, yeast extract 1 g, water to 1 l, 250 ml flasks with 50 ml medium on a shaker at 180 rpm for 3 days (Sochorova, 2011, [1]). Wednesday was inoculable 2 mycelial agar blocks (diameter 0.5 cm), cut using a drill of the 14-day culture of the fungus on potato-sucrose agar medium. The mycelium was filtered through the mill gas and dried to constant weight to determine productivity, or to obtain mycelial suspensions were crushed p and using blender. On sacharose-soy environment strain forms numerous light yellow pellets of medium size, the bezel is well developed, the culture fluid of a pale brown colour, the yield of dry biomass on the 3rd day of cultivation is 38 g/l (0,12×10⁶CFU/ml), which is comparable with the output of the prototype 36 g/L.

Example 2. Evaluation of herbicide activity of the strain.

The AMF inoculum was obtained in a 300-ml conical flasks on barley grain (20 g pearl barley, 16 ml of water). After 14 days incubation in the dark substrate is formed pycnidia were dried for 2 days. Prepared conidial suspension concentration of 0.6×106 to 2×10⁷conidia/ml (viability of conidia was about 90%) in 0.01% solution of tween-80. The Thistle plants grown from seeds in the rosette (the age of about 4 weeks) were sprayed with a suspension (2 ml/plant). Inoculated plants were kept in plastic bags to maintain high humidity at 20°C for 24 or 36 hours Then the packages were removed, the plants were incubated at 24°C and artificial lighting (16 h) and at 20°C in the dark (8 h). In 7 days after infection carried out the assessment of disease development on leaves of creeping Thistle (area of necrosis in relation to the total area of leaves, %), and determined the fresh biomass of aboveground parts of plants (Methods..., 1982; [2]).

When the concentration of inoculum 2×10 conidia/ml and 36-h hydration is istlef area of necrosis on the leaves of the Thistle in 7 days after inoculation was approximately 100%. The loss of fresh above-ground biomass was about 85%, and dry weight of underground shoots about 50%. When the concentration of inoculum 5×10⁶conidia/ml, the area of necrosis on the leaves of plants of the Thistle field after treatment was about 50%loss of biomass of the aerial part of this plant was approximately 35% (table. 3), which is comparable with the prototype.

Assessment of the pathogenicity of mycelium obtained in liquid culture was carried out for whole plants under controlled conditions. For inoculation used a 3-day culture of strain on sacharose-soy medium obtained in submerged cultivation on the rocking chair. The crude mycelium (relative humidity 80%) in suspension were tested at the following concentrations: 25, 50, 75 and 100 mg/ml Vessels with plants after inoculation were placed for 24 hours in a humid chamber.

The strain shows high pathogenicity in respect of the Thistle field. On day 7 after treatment of plants under controlled conditions mycelial suspension of strain at 24 h duration Rosanova period and concentration of raw inoculum 50 mg/ml (5×10⁴CFU/ml) resulted in complete destruction of plants (table 3), the loss of biomass of the aerial part of this plant was approximately 65%, which exceeds mycoherbicide activity of the prototype.

Example 3. Example evaluate the specificity of the strain.

The plants were grown from the mJy in greenhouses up to rosette stage (5-6 leaves). Inoculation was performed as in example 2. At 14 days after infection assessed the development of the disease on the leaves of plants (area of necrosis in relation to the total area of leaves, %).

Estimated 14 plant species from 7 families (Asteraceae, Brassicaceae, Cucurbitaceae, Fabaceae, Malvaceae, Poaceae, Solanaceae).

Phytopathogen Stagonospora cirsii is characterized by narrow specialization and dedicated to the family Asteraceae. Of representatives of the Asteraceae family strain was pathogenic for Cirshim arvense, Chrysanthemum segetum and Cynara scolymus, to a lesser extent for Zinnia elegans. Among the species of other families marked the defeat of Althaea rosea and Cucumis sativus. On the plants of other families lesions conidia S.cirsii not detected (table 4).

Thus, the data for the strain S. cirsii 1.42 (VIZR) indicate higher aggressiveness in relation to the target plants (table 2, 3)and comparable with the prototype on biomass yield and specialization. It can be used to combat the Thistle field in crops of most cultivated plants and natural protected areas.

Bibliography

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The strain of the fungus Stagonospora cirsii Davis VIZR 1.42 with herbicide activity against Canada Thistle (Cirsium arvense L. Scop.).