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Grishechkina S.D., Kovalenko T.K., Kirpicheva T.V., Antonets K.S., Nizhnikov A.A. Modified semisynthetic medium MMBt for production of preparations based on Bacillus thurin-giensis Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2023, Vol. 58, № 3, p. 416-428.
(how to cite this article)

doi: 10.15389/agrobiology.2023.3.416eng

UDC: 632.7:579.64

Acknowledgements:
Supported by the Ministry of Science and Higher Education of the Russian Federation (agreement № 075-15-2021-1055 dated September 28, 2021 on providing a grant in the form of subsidies from the Federal budget of the Russian Federation). The grant was provided for the implementation of the project: “Mobilization of the genetic resources of microorganisms on the basis of the Russian Collection of Agricultural Microorganisms (RCAM) at the All-Russia Research Institute for Agricultural Microbiology (ARRIAM) according to the network principle of organization”.
The authors declare no conflict of interests

 

MODIFIED SEMISYNTHETIC MEDIUM MMBt FOR PRODUCTION OF PREPARATIONS BASED ON Bacillus thuringiensis

S.D. Grishechkina1 ✉, T.K. Kovalenko2, T.V. Kirpicheva3,
K.S. Antonets1, A.A. Nizhnikov1

1All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail svetagrishechkina@mail.ru (✉ corresponding author), k.antonets@arriam.ru, a.nizhnikov@arriam.ru;
2Far Eastern Research Institute of Plant Protection — Branch of the Chaika Federal Research Center of Agricultural Biotechnology of the Far East, 42-a, ul. Mira, s. Kamen’-Rybolov, Primorsky Krai, 692682 Russia, e-mail
biometod@rambler.ru;
3Ekaterininskaya Experimental Station — Branch of the Federal Research Center Vavilov All-Russian Institute of Plant Genetic Resources, Ekaterininsky experimental station, s. Ekaterino, Nikiforovsky District, Tambov Province, 393023 Russia, e-mail ecosvir@yandex.ru

ORCID:
Grishechkina S.D. orcid.org/0000-0002-4877-705X
Antonets K.S. orcid.org/0000-0002-8575-2601
Kovalenko T.K. orcid.org/0000-0003-1432-4500
Nizhnikov A.A. orcid.org/0000-0002-8338-3494
Kirpicheva T.V. orcid.org/0000-0002-9459-507
Tikhomirova N.Yu. orcid.org/0000-0001-8530-6698

Final revision received October 16, 2022
Accepted February 28, 2023

One of the trends in the biological control of pests is the use of bacteria belonging to the genus Bacillus and, first of all, entomopathogenic strains of Bacillus thuringiensis. Of great interest to industrial biotechnology are studies related to the search for optimal cultivation conditions that can improve the manufacturability of the production of microbiological preparations and their effectiveness. Previously, the nutrient media for the production of microbiological preparations based on B. thuringiensis which include natural organic components have been developed. Nevertheless, during the production of biopreparations based on this bacterium, the foaming of the culture frequently occurs and expensive filters of bioreactors have to be replaced. Also, during the treatment of plants, working solutions containing organic components of the liquid medium can clog the nozzles. This effect complicates the treatment process. In addition, organic cultural media components are not standard and depend on the quality and source origin. In this regard, it is important to carry out the screening of optimal synthetic media that could eliminate these shortcomings. Our study was aimed at selecting the optimal synthetic media and evaluating the effectiveness of the obtained preparation samples in laboratory and field conditions. The objects of study were the cultures of B. thuringiensis var. thuringiensis 800/15 (BtH1 800/15) and B. thuringiensis var. darmstadiensis 25 (BtH10 25). The composition of the culture media was as follows: CCY medium — 0.5 mM MgCl2·6H2O, 0.01 mM MnCl2·4H2O, 0.05mM FeCl3·6H2O, 0.05 mM ZnCl2, 0.2 mM CaCl2·6H2O, 13 mM KH2PO4, 26 mM K2HPO4, 20 mg /l glutamine, 1 g/l casein hydrolysate, 0.4 g/l yeast extract, 0.6 g/l glycerol; MBt medium: 7 g/l casein hydrolyzate, 6.8 g/l KH2PO4, 0.12 g/l MgSO4·7H2O, 0.0022 g/l MnSO4·4H2O, 0.014 g/l ZnSO4·7H2O, 0.02 g/l Fe2(SO4)3, 0.18 g/l CaCl2·4H2O; LB medium: 10 g/l trypton, 5 g/l yeast extract, 10 g/l NaCl; modified semi-synthetic medium MMBt (modified MBt): 7 g/l casein hydrolyzate, 6.8 g/l KH2PO4, 0.12 g/l MgSO4·7H2O, 0.0022 g/l MnSO4·4H2O, 0.014 g/l ZnSO4·7H2O, 0.02 g/l Fe2(SO4)3, 0.18 g/l CaCl2·4H2O (25), glucose (1.0 %), Na citrate (2 g/l). Yeast polysaccharide media (YPM) for BtH1 and BtH10 served as a reference. Bt strains were cultivated in 750 ml Erlenmeyer flasks filled with 40-50 ml of medium on a shaker at 220 rpm and 29 °C for 48-72 h until the maturation of culture, accompanied by the formation of spores and crystalline endotoxin. On the basis of BtH1 800/15 and BtH10 25 strains, batches of liquid preparations were obtained, the effectiveness of which was evaluated in 2020 and 2021 on potatoes (Solanum tuberosum L.) of the Yantar variety in the Far East (Ussuri district of Primorsky Krai) against Henoseplachna vigintioctomaculata Motsch and on potatoes of the Emelya variety in the Tambov region against Leptinotarsa decemleniata Say. In the experiments, liquid preparations obtained on YPM and MMBt were used, which were used at consumption rates of 15 and 20 l/ha. The biological effectiveness of the preparations was calculated according to the formula W.S. Abbot. The antifungal activity of the preparation BtH10 25, obtained on MMBt and YPM, was determined by the method of agar blocks in vitro in Petri dishes. The control medium was used without the addition of drugs. Fungi Botrytis cinerea Pers (strain C-5) and Bipolaris sorokiniana (Sacc.) Shoemaker (strain C-20) served as test cultures. The inhibitory activity was calculated according to the W.S. Abbot. Cultivation of BtH1 800/15 and BtH10 25 strains on different nutrient media showed that on semi-synthetic media MBt and LB CFU titers were 2 times lower than on YPM, while on CCY medium they were 10 times lower. Their activity, determined by the content of exotoxin, was also lower, but on the MBt medium it was slightly inferior to YPM for BtH1 800/15. Therefore, MBt medium was chosen for further studies, and the composition of this medium was modified by adding glucose (1.0 %) and Na citrate (2 g/l). The resulting MMBt medium made it possible to achieve a significant increase in titers, activity, and the rate of culture development compared to the initial MBt. In 2020 in the Tambov region, the effectiveness of the preparation based on BtH1 800/15 obtained on DPS was high against the Colorado potato beetle and on the 5th day was 95.3 %, slightly inferior to the chemical standard. In the case of preparation obtained on MMBt, it was slightly lower (83.3 %), but the protective effect lasted longer, and on day 15 the efficiency was 73.7 %. In 2021, the efficacy of BtH1 800/15 was lower than in 2020. In the preparation obtained on MMBt, it was slightly inferior to the effectiveness of the preparation obtained on YPM, amounting to 75.3 and 67.7 %, respectively, on the 5th day after treatment. The effect of the BtH10 25 preparation obtained on MMBt was weaker than in the variant with BtH1 800/15 (47.7 % on day 5). In Primorsky Krai, the high efficacy of liquid preparations against H. vigintioctomaculata was also noted. In 2020, at a rate of application of the BtH1 800/15 preparation of 15 l/ha, the effectiveness in the YPM and MMBt variants was 60.5 and 63.9 %, respectively, on day 5. Similar data was obtained in 2021. The inhibitory activity of the BtH10 25 preparation obtained on MMBt was 12 % higher on day 5 than that of the preparation obtained on YPM, and was 72.3 and 60.8 % for B. sorokiniana and 78.9 and 67.4% for B. cinerea. On day 10, this trend persisted, but for the preparation produced on YPM, a decrease in the inhibition of the growth of B. sorokiniana and B. cinerea colonies, respectively, to 57.3 and 44.3% was noted. Thus, preparations based on Bacillus thuringiensis obtained on the MMBt medium were only slightly inferior in terms of effectiveness against pests to preparations obtained on YPM, while their effectiveness against phytopathogens was higher than that of preparations with YPM. The MMBtmedium is promising for agricultural biotechnology, since its use reduces the time required for the formation of spores and crystalline protein endotoxin by increasing the growth rate of the B.thuringiensis culture. Thus, on the MMBt medium, this process ends after 48 h, and on the YPM medium, after 72 h, which makes it possible to reduce energy consumption.

Keywords: biopreparation, Bacillus thuringiensis var. thuringiensis, Bacillus thuringiensis var. darmstadiensis, Bipolaris sorokiniana, Botrytis cinerea, Colorado potato beetle, potato ladybug, inhibitory activity, cultural medium.

 

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