Phytotoxic action of biologically active substances BACTERIA of GENUS BACILLUS, extracted from tomato seed

Abstract

The aim of the research is to define antagonistic and phytotoxic activity of bacteria exometabolites of Bacillus genus, extracted from tomato seeds.

Materials and methods of research. The object of the research was untreated with disinfectants seeds of 5 determinant tomato varieties of Ukrainian selection, including Chaika, Klondaik, Oberih, Kimmeriiets and San’ka. For bacteria isolation, seeds, after 15 minutes douching with running tap water and sterile ground water, were rubbed in a mortar with 0,5 ml of saline after which a small amount of material was deposited on the surface of potato agar applying thick strokes from one end of petri dish to another. The bacteria were cultured in an incubator for 24 hours at a temperature of 28° C. The morphological properties of the bacteria were studied by classical methods. Antagonistic activity of isolates was determined by the deferred antagonism method. Test cultures were 
C. michiganensis subsp. michiganensis (Smith 1910) Davis et al. 1984 strains P8, P12, P73, P110, P115 and CFBP 4999, P. syringae pv. tomato (Okabe 1933) Young et al. 1978 strains Dappg-4213, Pst-2, Psr-120 and PstBB-9 (obtained from the collection of the Institute of pesticides and plant protection, Serbia), 
X. vesicatoria (Doidge 1920) Vauterin et al. 1995 strain 9098 (from the collection of the department of pathogenic bacteria of the Institute of Microbiology and Virology named after D. K. Zabolotny of NAS of Ukraine) and obtained on the farms of Dnipropetrovsk region isolates P. syringae pv. tomato IS-28, IS-46 and 
X. vesicatoria IS-30, IS-31. The level of antagonistic activity of isolates of tomato seeds was evaluated by zones of growth inhibition in test cultures, expressed in millimeters. To identify the influence of isolates on seed vigor and germination, tomato seeds were soaked for 2 hours in a solution of cell suspension. The number of seeds germinated in 3 – 5 days (depending on the variety of plants, the first record), was considered the laboratory vigor, and of those germinated in10 – 14 days (second record) – germination. During the second calculation they were counted sprouted seeds, it was measured the length of shoots and roots. Phytotoxic activity of isolates was tested by submerging of cut under water young tomato seedlings into cell suspension titre 20•10⁹ cells/ml. Within two days they were watched the signs of metabolites toxicity.  

Results and discussion. The obtained isolates from the surface of seeds of tomato varieties had colonies of different shapes, of yellow, white, beige, pink, brown and orange colors, were transparent and translucent, with a matte and glossy surface, smooth and rough edges. For each tomato variety it was characteristic different quantitative and qualitative composition of seed microflora. Most of the isolates from untreated with disinfectants tomato varieties suggest greater diversity of microbiota. From untreated tomato seeds, they were selected 6 isolates that showed antagonistic activity to test cultures. OB2 isolate showed high antagonistic activity to bacterial spotting, diameter of suppression of growing strains of P. syringae pv. tomato Dappg-4213, Pst-2, Psr-120, 9-PstBB and IS-28, IS-46 was 74 – 80 mm. Less sensitivity to the antagonist showed strains of C. michiganensis subsp. michiganensis P8, P12, P73, P110, P115 and CFBP 4999 (bacterial cancer agent), areas of growth inhibition ranged from 44 to 70 mm. The diameter of the growth inhibition of strains C. michiganensis subsp. michiganensis P8, P12, P73, P110, P115 and CFBP 4999 by isolate OB1 was 40 – 80 mm. In relation to the strains 
P. syringae pv. tomato Dappg-4213, Pst-2, Psr-120, PstBB-9 and IS-28, IS-46, the isolate OB1 predetermined formation of zones of inhibition of growth within 30 – 60 mm. Isolate CHA3 also had high antagonistic activity to bacterial cancer and bacterial spotting agents. Isolates KM2, FL1 and SA1 demonstrated somewhat less antagonistic activity to the test cultures. The level of antagonistic activity of FL1 isolate to strains C. michiganensis subsp. michiganensis P8, P12, P73, P110, P115 and CFBP 4999 was in the range of 30 to 70 mm, and P. syringae pv. tomato Dappg-4213, Pst-2, Psr-120, PstBB-9 and IS-28, IS-46 – 20 – 60 mm. Isolates also showed antagonistic activity to bacterial pathogen of black spot, the diameter of growth inhibition of strains X. vesicatoria 9098 and IS-30, IS-31 was equal to 38 – 74 mm. The selected isolates were identified in the study of their morphological, cultural, physiological and biochemical properties. It was established that bacterial isolates CHA3, OB1, OB2, KM2, FL1 and SA1 are aerobic spore-forming Gram-positive moving straight or slightly curved sticks; the location of cells is different - from single to filamentary chains. According to these characteristics the bacteria was pre-attributed to the Bacillus genus. To determine potential producer with a view to its use in agriculture, it was conducted a comparative assessment of phytotoxicity of the selected active isolates. It was found that treatment with aqueous suspension of antagonistic isolates does not have a negative effect on seed germination, but rather it marked an increase of 5 – 12 % when compared to the control. Selected strains significantly stimulated the growth of roots and shoots of tomatoes when compared to the control. In terms of studying of phytotoxicity of promising strains antagonistic to tomato seedlings it took place an active development of shoots and previously damaged roots in variants and the control. Clipped lateral roots remain healthy. Within 10 days it was seen not a single seedling death. In addition, the process of determining of phytotoxicity of the isolates it was revealed that some strains have a stimulating effect. When compared to the control, in the variants with isolates FL1, KM2 and SA1 shoots were longer and more developed, stronger and thicker at the base.

Conclusions. Isolates OB1, OB2, CHA3, KM2, FL1 and SA1 of epiphytic microflora of untreated with fungicide tomato seeds are promising to limit bacterial pathogens of cancer, bacterial spotting and bacterial black spot. Zones of growth inhibition provided by the isolates to C. michiganensis subsp. michiganensis are 
40 – 80 mm, P. syringae pv. tomato – 30 – 80 and X. vesicatoria – 40 – 74 mm. Isolates OB1, OB2, CHA3, KM2, FL1 and SA1 showed no phytotoxic effect on the seed and sprouts of tomatoes. Treating of seeds with isolates OB1, OB2, CHA3, KM2, FL1 and SA1 provided growth stimulation of tomato seedlings from 3 to 25% in the early stages of development, indicating growth stimulating properties. The highest increase in the length and weight of vegetative mass of shoots was typical for variants with isolates FL1, KM2 and SA1.