CC BY
27FUNGICIDE EFFECTIVENESS OF BACILLUS SUBTILIS HB-21 STRAIN IN STORED POTATO
AND CARROT
Zhappar N.,
Junior researcher, «EcoSave» LLP Shaikhutdinov V., Junior researcher, «EcoSave» LLP Myrzabayev B., Researcher, «EcoSave» LLP Shibayeva A., Junior researcher, «EcoSave» LLP Kaliaskar D.
Junior researcher at the laboratory of applied biotechnology The branch of National Center for Biotechnology
Abstract
The loss of harvested vegetables and fruits during long storage varies from 30 to 75% annually. Chemical pesticides cannot use often due to their harmful effect on public health and developed pathogens resistance. An alternative to chemical pesticides is microorganisms and their metabolites as a biocontrol against common plant diseases. Bacillus subtilis is one of the well-studied bacteria that can be used to inhibit the growth of common vegetable and fruits diseases. This paper aims to test the antagonistic effect of B. subtilis strain HB-21 that was extracted in Akmola region against common pathogens such as Sclerotinia sclerotiorum, Botrytis cinerea, and Fusarium solani.
Keywords: Bacillus subtilis, Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium solani, carrot, potato.
Introduction
Pathogens of plant products cause significant worldwide economic losses, amounting to 30-75% of the crop that stays for long-term storage [3]. The use of chemical pesticides for the safety of products is restricted by sanitary standards and is harmful to public health [2,5,10]. Another problem is developed resistance of pathogens against chemical pesticides [2,5]. A potential solution for long-term saving harvested vegetables and fruits is using microorganisms and their metabolites as a biocontrol against common plant diseases [4]. The use of biological products immediately after harvesting prevents the active development of pathogenic microflora of potatoes and vegetables, prevents their penetration into the tissues, contributing to the preservation of agricultural products and their consumer properties [4,9]. Besides, the advantage of bio-logics over chemical pesticides is the possibility of multiple treatments of storage facilities during the storage period of products.
Bacillus subtilis (B. subtilis) is one of the well-studied genera that can be broadly used in the agriculture industry as a biocontrol in the field and food storage places [4,9]. This genus produces a variety of metabolites such as enzymes and antibiotics that can control the growth and degrade pathogens cell-membrane [6]. Moreover, B. subtilis is fast replicating and resistant to changes in environmental conditions (temperature, moisture, pH) [8]. According to the US Food and Drug Administration, B. subtilis is generally recognised as a safe (GRAS) organism that can be used in the food industry [4,9].
This paper aims to test antagonistic effect B. sub-tilis strain HB-21 against common pathogens such as Sclerotinia sclerotiorum, Botrytis cinerea, and Fusarium solani. Due to the high interest of vegetable growers to extend the shelf life of carrots and potato, these products were chosen for this experiment.
Materials and methods
Growth inhibition of pathogens in dual culture Vegetable and fruit common pathogens, Sclero-tinia sclerotiorum (white mould) and Botrytis cinerea (grey mould) were obtained from infected parts of carrots with an inoculation loop. Then these pathogens were cultivated on Potato Dextrose Agar (PDA) in Petri plates to obtain pure cultures. Antagonist, B. subtilis HB-21 strain was extracted from the soil layer of carrot rootzone in Akmola region, Kazakhstan. The strain is gram-positive, aerobic, spore-forming rods that produce catalase, glucose, arabinose, xylose, maltose, lactose, mannitol, and sucrose with the formation of acid. The strain hydrolyzes starch, gelatin, recycles citrate, and does not hydrolyze urea.
Two biological treatments, biopreparation-1 (based on B. subtilis, Russia) and biopreparation-2 (based on Pseudomonas fluorescens, Russia), and one chemical (1% solution of copper sulfate) were included in this experiment to evaluate B. subtilis HB-21 strain effect on the pathogens growth inhibition on a carrot. Pathogens were inoculated to Petri plates on top of PDA. Then, one of the treatments was inoculated in four sides around pathogen at 2.5 cm distance from plate's borders (Fig.1). Each plate had one pathogen and one treatment. As a control, two pathogens were inoculated alone with no treatments. All treatments and control were triplicated. The plates were incubated at 25°C for five days. The inhibition of fungi growth was measured as the diameter of inhibition zone and evaluated by following categories: highly sensitive (inhibition zone 3-5 mm), sensitive (2-3 mm), low sensitive (1-2 mm), and stable (no effect of antagonist).
In vivo antagonists test on carrot and potato pathogens in storage
Carrot and potato samples were taken from vegetable storages in Akmola and North Kazakhstan regions. Carrot samples were treated with B. subtilis HB-
21 strain, biopreparation-1, and potassium permanganate solution. Each treatment was sprayed on carrots. Three replicates of nine carrot taproots per treatment were used. The control was untreated taproots. Carrots were stored at 0.5-1°C for 63 days in the laboratory refrigerators.
Potato tubers were treated with B. subtilis (108 cells/mL), biopreparation-1 (108 cells/mL), and chemical fungicide (fludioxonil 25g/L). Wounds were made on each tuber (5mm in diameter and 1-2mm deep) with a sterile punch, 50 ^L of B. subtilis and biopreparation-1 were inoculated in the wounds. The chemical fungicide was sprayed on tubers at 10mg/kg rate. Fusarium solani (F. solani) was chosen as a pathogen model. After the wounds were dried, 50^L of F. solani were inoculated into the wounds. The control was untreated potato tubers with inoculated F. solani in the wounds. Potato tubers were stored at 3-4°C for 72 days in the laboratory refrigerators.
Variants with S. sclerotiorum
Results and Discussion
The dual culture experiment showed a strong inhibitory effect of B. subtilis for both pathogens (Fig. 1). The results revealed that on the fifth day of incubation S. sclerotiorum and B. cinerea were highly sensitive to the presence of B. subtilis in the plate (Table 1). Biopreparation-1 had the same level of pathogen growth inhibition as B. subtilis HB-21 strain while in the presence of P. fluorescens (biopreparation-2) pathogens had a low level of sensitivity. Copper sulfate and control had no inhibitory effect on the pathogens in this experiment. The inhibitory effect of biological treatments based on the substances produced by microorganisms and competition during growth [10]. B. subtilis produces enzymes (chitinases and glucanases) and antibiotics (surfactin, iturin, and gramicidin) that have an an-tifungal effect [12]. The result of this experiment correlates with other findings which confirm the antifungal effect of B. subtilis [1,11].
Variants with B. cinerea
B. subtilis HB-21 top B. subtilis HB-21 bottom B. subtilis HB-21 top
B. subtilis HB-21 bottom
Biopreparation -1 top
Biopreparation -1 bottom
Biopreparation -1 top
Biopreparation -1 bottom
Biopreparation-2 top
Biopreparation-2 bottom
/
Biopreparation-2 top
Biopreparation-2 bottom
1% solution of copper sulfate
Control
1% solution of copper sulfate
Control
Figure 1 - Antagonistic activity of B. subtilis strain HB-21, biopreparation-1 and 2, and 1% solution copper sulfate against Sclerotinia sclerotiorum and Botrytis cinerea, top and bottom views of treated plates.
Table 1
Antagonistic activity of biopreparations against pathogens, S. sclerotiorum and B. cinerea
Treatments Diameter of inhibition zone, mm Pathogens sensitivity
Botrytis cinerea (Grey mould) Sclerotinia sclerotiorum (Wight rot)
Bacillus subtilis HB-21 3-4 3-5 highly sensitive
Biopreparation -1 2-3 2-3 sensitive
Biopreparation-2 1-2 1-2 low sensitive
1% of copper sulfate - - no effect
Control - - -
White mould was observed on the control carrot taproots on the day 16 of storage. Chemical treatment prohibited the growth of white mould on two weeks compared with the control. Meanwhile, the taproots treated with B. subtilis and biopreparation were not affected by pathogens during the experiment. The experiment with potato tubers showed a strong effect of B. subtilis and biopreparation-1 compare with control
(Fig. 2). The pathogen spread on a quarter of inoculated tubers in the control on the day 72 of the experiment. Chemical fungicide prohibited the growth of pathogens on all treated tubers (Fig. 3). It is possible that B. subtilis of strain-21 and biopreparation-1 outgrow pathogen due to competition for space and by releasing fungi membrane degrading enzymes and antibiotics they terminate the spread of pathogens on tubers' surface [7].
KMnO.
Control
Figure2 - Comparison of the antagonistic effectiveness of Bacillus subtilis HB-21with analogues at storage
temperature of 0.5-1°C
Chemical fungicide Control
Figure3 - Comparison of the antagonistic effectiveness of Bacillus subtilis HB-21 with analogues against
Fusarium solani at storage temperature of 3-4 °C
In conclusion, the B. subtilis strain HB-21 showed a strong antagonistic effect on common vegetable pathogens. The treatment of carrot taproots and potato tubers with strain HB-21 immediately prior storage inhibits the growth of pathogens and saves harvested vegetables for long period. The results of testing showed a high growth delay of pathogenic microorganisms when using B. subtilis strain HB-21 and it is not inferior in efficiency to its foreign counterpart.
This publication was funded under the project "Stimulating productive innovation", supported by the world Bank and the Government of the Republic of Kazakhstan. Statements may not reflect the official position of the world Bank and the Government of the Republic of Kazakhstan
References
1. Arroyave-Toro, J.J., Mosquera, S. and Villegas-Escobar, V., 2017. Biocontrol activity of Bacillus subtilis EA-CB0015 cells and lipopeptides against posthar-vest fungal pathogens. Biological control, 114, pp.195200.
2. Benítez, T., Rincón, A.M., Limón, M.C. and Codon, A.C., 2004. Biocontrol mechanisms of Tricho-derma strains. International microbiology, 7(4), pp.249-260.
3. Buchholz, F., Kostic, T., Sessitsch, A. and Mitter, B., 2018. The potential of plant microbiota in reducing postharvest food loss. Microbial biotechnology, 11(6), pp.971-975.
4. Denner W, Gillanders T. The legislative aspects of the use of industrial enzymes in the manufacture of food and food ingredients. In: Godfrey T, Reichelt J, editors. Industrial enzymology. New York: Stockton Press; 1996. p. 397-412.
5. Kim, B.S. and Hwang, B.K., 2007. Microbial fungicides in the control of plant diseases. Journal of Phytopathology, 155(11-12), pp.641-653.
6. Kim, H.M., Lee, K.J. and Chae, J.C., 2015. Postharvest biological control of Colletotrichum acuta-tum on apple by Bacillus subtilis HM1 and the structural identification of antagonists. J. Microbiol. Bio-technol, 25(11), pp.1954-1959.
7. Kim, Y.S., Balaraiu, K. and Jeon, Y., 2016. Effects of rhizobacteria Paenibacillus polymyxa APEC136 and Bacillus subtilis APEC170 on biocontrol of postharvest pathogens of apple fruits. Journal of Zhejiang University-SCIENCE B, 17(12), pp.931-940.
8. Lastochkina, O., Baymiev, A., Shayahmetova, A., Garshina, D., Koryakov, I., Shpirnaya, I., Pusen-kova, L., Mardanshin, I.D., Kasnak, C. and Pal-amutoglu, R., 2020. Effects of Endophytic Bacillus Subtilis and Salicylic Acid on Postharvest Diseases (Phytophthora infestans, Fusarium oxysporum) Development in Stored Potato Tubers. Plants, 9(1), p.76.
9. Shafi, J., Tian, H. and Ji, M., 2017. Bacillus species as versatile weapons for plant pathogens: a review. Biotechnology & Biotechnological Equipment, 31(3), pp.446-459.
10. Tapwal, A., Thakur, G., Chandra, S. and Tyagi, A., 2015. In-vitro evaluation of Trichoderma species against seed borne pathogens. Int J Biol Chem Sci, 1(10), pp.14-19.
11. Vijayalakshmi, S.K., Abha, S. and Chander, P., 2012. Isolation and characterization of Bacillus subtilis KC3 for amylolytic activity. International journal of bioscience, biochemistry and bioinformatics, 2(5), pp.336-341.
12. Yánez-Mendizábal, V., Usall, J., Viñas, I., Casals, C., Marín, S., Solsona, C. and Teixidó, N., 2011. Potential of a new strain of Bacillus subtilis CPA-8 to control the major postharvest diseases of fruit. Biocontrol Science and Technology, 21(4), pp.409-426.
