Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2018, Vol. 53, ¹ 5, p. 1045-1053.
(how to cite this article)

doi: 10.15389/agrobiology.2018.5.1054eng

UDC 632.51:582.28:57.083.1

Acknowledgements:
Supported financially by Russian Science Foundation (grant ¹ 16-16-00085 «Technologies for production and use of mycobiocides against difficult-to-eradicate weeds»)

 

LIQUID FERMENTATION OF Stagonospora cirsii C-163,
A POTENTIAL MYCOHERBICIDE FOR Cirsium arvense (L.) Scop.

S.V. Sokornova, A.O. Berestetskiy

All-Russian Research Institute of Plant Protection, Federal Agency for Scientific Organizations, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail toxbiotech@vizr.spb.ru (✉ corresponding author), s.sokornova@spbu.ru

ORCID:
Sokornova S.V. orcid.org/0000-0001-6718-4818
Berestetskiy A.O. orcid.org/0000-0002-0612-6996
The authors declare no conflict of interests

Received May 20, 2017

 

The bioherbicides should exhibit stable effectiveness in the field, be specific and quick in action, compatible with other preparations and meet market demand. In many ways, the cost and quality of product is determined by the technology of obtaining infectious material. An infectious material is used as a mycelium and its modifications and as conidia as well. The extreme sensitive of the mycelium to drying is often referred as main disadvantage of using it as the basis of a formulation. At the same time, the technological process of obtaining conidia is often more complicated, and the efficiency in the field is less than the mycelium has. The phytopathogenic fungus Stagonospora cirsii J.J. Davis, which is causative agent of a leaf spot disease of creeping perennial weeds in the family Asteraceae, is considered a potential mycoherbicide of Canadian thistle Cirsium arvense (L.) Scop. However, the yield of C. cirsii C-163 mycelium on standard nutrient media is significantly lower than that used in biotechnology (3 g/l). Our paper is the first to report that manipulation with liquid nutrient medium allows a significant increase in S. cirsii mycelium pathogenicity and tolerance to exsiccation. The study is devoted to the optimization of liquid-phase deep fermentation parameters, as well as the duration of cultivation and composition of a nutrient medium, in order to obtain the C. cirsii C-163 mycelium with improved mycoherbicidal properties. This infection material is a good basis for development formulations that can be used both individually and jointly with other protective agents for perennial weed control. The advantage of the approach used in the work is that the resistance to drying, an important technological parameter which largely determines the success of the herbicides, was additionally considered, along with virulence and mycelial yield, during the optimization of fermentation parameters. The strain C. cirsii C-163 was used. The 10-day inoculum was obtained in Petri plates on potato dextrose agar medium. The mycelium was incubated in 250-ml Erlenmeyer flasks containing 50 ml of the medium at 130 rpm and 24±2 °C for 2-7 days. The base liquid nutrient media contained carbon source (20 g/l), organic (10 g/l) or inorganic (3.5 g/l) nitrogen source, yeast auto lysate (1 g/l), KH2PO4 (1 g/l), MgSO4 (0.5 g/l). Dulcite, rhamnose, L-inositol, L-arabinose, D-sorbitol, glucose, trehalose, and sucrose were a source of carbon. Casein, soy peptone, enzyme peptone, soy flour, gelatin, lecithin, ammonium dihydrogen phosphate, ammonium chloride, ammonium sulfate, and sodium nitrate were a source of nitrogen was. The pH of all liquid nutrient media was adjusted to 6.0. To establish the optimum concentrations of sucrose and soy flour in a nutrient medium with yeast auto lysate (1 g/l), KH2PO4 (1 g/l), MgSO4 (0.5 g/l), the amount of sucrose was changed from 10 to 70 g/l with a step of 10 g/l and the concentration of soy flour was changed from 5 to 20 g/l with a step of 2.5 g/l. The degree of leaf damage caused by disease was estimated by the necrosis area of leave disks or whole plants (5-6 true leaves). Drying of harvest mycelium, humidity 85-87 %, was carried out in a thin layer (1-2 mm) with flowing air at 30 °C without protectors for 3 hours. The highest yield of mycelium is when the carbon source in the nutrient medium is L-inositol. When inositol is substituted with sucrose or D-sorbitol, the biomass yield reduces by 25 %. At the same time, these nutrient media gave the most aggressive mycelium. Among nitrogen sources, the maximum yield of mycelium is in the case of casein, soy flour and enzymatic peptone. In the process of drying mycelium, loss of viability of propagules turned out to be significant. The mycelium obtained on sucrose-soy nutrient medium is the most resistant to drying. The most viable and aggressive mycelium was formed in the middle of the exponential growth phase which occurred on day 3 when cultivated in flasks on a soya-sucrose nutrient medium. Optimization of the concentration of soybean flour (15 g/l) and sucrose (60 g/l) makes it possible to increase the yield and aggressiveness of mycelium 12 and 4 times, respectively, as compared to  Czapek medium. Thus, the present study provides a method for the preparation of a mycelium having a high aggressiveness to the host-plant and a capability to remain viable during drying. The prospects of such a method of obtaining an infectious material are proved.

Keywords: phytopathogenic fungi, Stagonospora cirsii J.J. Davis, Cirsium arvense (L.) Scop., Canada thistle, submerged liquid cultivation, carbon source, nitrogen source, mycelium, mycoherbicide.

 

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