Ecofriendly management of bacterial leaf rot of onion caused by Enterobacter cloacae under in vitro

Introduction

Onion (Allium cepa L.) belongs to the family Alliaceae and having chromosome number 2n=16. It is the most widely cultivated species of the genus Allium. It is commonly known as "ullagaddi or irulli" in Kannada and "pyaaz" in Hindi. The onion is called as "queen of kitchen" (Priya et al., 2015) [4] . The south-west Asia is considered as the primary centre of domestication. Onion is one of the most important commercial vegetable crops in India and throughout the world. Onion has manifold use such as spice, vegetable, salad-dressing etc.. India (288.77 lakh tons) is the largest producer of onion. In India Maharashtra is the leading producing state. In Karnataka the area under onion is 2.32 lakh hectares, with production of 38.91 lakh tons, (Anon., 2024) [1] . Onion has high value both in domestic use and export purpose, but growing of onion has some constraints such as insect pests and diseases. The pests are thrips, whiteflies, beetles etc. Now a day's bacterial leaf rot is becoming an important disease in northern part of Karnataka from past 8-10 years. There is a need to develop ecofriendly management practice for mitigating the plant diseases. Hence, commercially available bio-agents and botanicals were evaluated against the disease under in vitro.

Materials and method Evaluation of different bio agents

About 10 isolates of bacterial bio agents which were isolated and maintained in laboratory in Department of Plant Pathology of College of Horticulture, Bagalkot were used to evaluate their efficacy against Enterobacter cloacae by inhibition zone assay method. The botanicals and bio agents were evaluated under in vitro against the growth of bacteria causing leaf rot of onion at different concentrations by zone of inhibition method. A loop full of 24h old E. cloacae culture was inoculated into nutrient broth and incubated for 48h to allow bacterial growth. This broth was seeded into nutrient agar media and then poured into Petri plates and allowed for solidification. Media was bored with cork borer to get 9 mm well, into which different concentrations of botanicals were poured, as mentioned in Table 1. The bio agents were multiplied by following the procedure given by Vijayan et al., 2013 [9] . The bio agents were poured into the well in three replications @ 10 8 cfu/ml concentration. The list of botanicals and bio agents evaluated were given in table 1.

The inoculated plates were incubated at 28±2 °C for 24 h. Each concentration was repeated thrice and observations were recorded by measuring the diameter of the inhibition zone for each concentration and analyzed statistically.

Results

In vitro evaluation of botanicals against the growth of Enterobacter cloacae

Aqueous extraction method

The aqueous extracts of 15 botanicals were evaluated against Enterobacter cloacae under in vitro. Among the different botanicals tested, Prosopis juliflora has shown the highest inhibition of 23.33, 19.16 and 15.16 mm, this was followed by Garcinia indica has given 22.83, 15.50 and 12.83 mm at different contrations viz., 1:1, 1:5 and 1:10 w/v respectively. The least inhibition was expressed in citronella (12.00, 10.67 and 0.00 mm). While Pongemia pinnata and Amrithpani were unable to inhibit the growth of pathogen. The standard check streptocycline (K-cyclin) 500 ppm + copper oxychloride 3000 ppm recorded maximum inhibition of 38.33 mm which found superior over all the botanicals tested, whereas no inhibition zone was recorded in water control (Table 2 andPlate 1).

Alcoholic extraction method

The results on effect of botanicals with alcoholic extraction are presented in Table 15. Among the tested botanicals Prosopis juliflora has shown the highest inhibition of 32.16, 23.33 and 18.17 mm and this was followed by Garcinia indica, recorded inhibition zone of 26.50, 20.17 and 17.50 mm at 1:1, 1:5 and 1:10 w/v concentrations, respectively. The least inhibition was seen in case of amrithpani 11.33, 10.66 and 0.00 mm. Pongemia pinnata (0.00 mm) failed to show inhibition of bacterial growth at all three concentrations. The standard check (streptocycline 500 ppm + copper oxychloride 3000 ppm) was recorded maximum inhibition of 38.33 mm, which found superior to all the botanicals tested. While the water control was shown no inhibition zone (Table 3 andPlate 1).

In vitro evaluation of antagonistic bacterial bio agents on the growth of Enterobacter cloacae

Ten antagonistic bio agents collected from the Dept. Plant Pathology were screened against growth of bacteria causing leaf rot of onion. The outcome of the in vitro evaluation showed that some bio agents did not able to inhibit the growth of the pathogenic bacterium viz., VK-6B (Lysinibacillus xylanilyticus), BK-6 (Brevibacilus borstelensis), KK-9A (Bacillus subtilis), KK-3A and BK-8, which showed 0.00 mm zone of inhibition. The highest mean zone of inhibition was observed in PM-2A (Brevibacilus sp.) (19.33 mm) which was followed by BK-5 (15.33 mm) and BK-3 (13.50 mm). The least mean zone of inhibition (11.50 mm) was observed in CK-13A. The results were depicted in table 4 and Plate 3.

Discussion

In vitro evaluation of botanicals agains the growth of Enterobacter cloacae of onion

Fifteen botanicals were screened against the growth of Enterobacter cloacae. Among the different tested botanicals Prosophis juliflora with concentration 1:1, 1:5 and 1:10 w/v was found to be most effective with the highest mean zone of inhibition of 21.88 mm, which is followed by Garcinia indica (19.22 mm) and Tamarindus indica (19.02mm) at different concentrations. The lowest inhibition was shown by Amrithpani (10.33 mm). Among the tested botanicals Prosopis juliflora has shown the highest inhibition of 24.56 mm and this was followed by Garcinia indica, which recorded inhibition zone of 21.39 mm at 1:1, 1:5 and 1:10 w/v concentrations respectively. The least inhibition was seen in case of amrithpani (10.67 mm). Pongamia pinnata (0.00 mm) failed to show inhibition of bacterial growth at all the three concentrations. The standard check (Streptocycline 500ppm + copper oxychloride 3000ppm) recorded maximum inhibition of 28.44 mm and was found superior to all the botanicals tested. Prosopis juliflora found to contain different group of alkaloids isolated from this plant are reported to possess significant antibacterial and antifungal activity which is comparable to the standard drugs (Ragavendra et al., 2010). Kokum rind possesses an important phenolic compound called as garcinol which possess antibacterial properties (Sutar et al., 2012) [8] .

In vitro evaluation of antagonistic bacterial bio agents on the growth of Enterobacter cloacae

Ten antagonistic bio agents collected from the Dept. Plant Pathology were screened against growth of bacteria causing leaf rot of onion. The outcome of the in vitro evaluation showed that some bio agents did not able to inhibit the growth of the pathogenic bacterium viz., VK-6B (Lysinibacillus xylanilyticus), BK-6 (Brevibacilus borstelensis), KK-9A (Bacillus subtilis), KK-3A and BK-8, which showed 0.00 mm zone of inhibition. The highest mean zone of inhibition was observed in PM-2A (Brevibacilus sp.) (19.33 mm) which was followed by BK-5 (15.33 mm) and BK-3 (13.50 mm). The least mean zone of inhibition (11.50 mm) was observed in CK-13A. While the water control was shown no inhibition zone (Table 4 and Plate 3). Under biological control of plants disease, various antagonistic organism has been identified, which fight against the pathogen by different mechanisms viz., competition, lysis, antibiosis, siderosphore production and hyper parasitism. The result obtained in the study showed similiarity with the earlier research works, by Xianling et al. (2008) [11] against bacterial wilt (Ralstonia solanacearum) by biocontrol management of mulberry by an endophytic Bacillus subtilis. In vitro, Ralstonia solanacearum by 4 isolates of bacillus in these 4 isolates L4144 was effectively reduced the disease incidence. This L4144 strain was identified as Bacillus subtilis. Khosro et al. (2012) [2] were evaluated antibacterial activity of different Bacillus spp. against X. campestris pv. campestris and Pectobacterium carotovorum subsp. carotovorum. They were used 8 epiphytic Bacilli viz., Bacillus pumilus (3 strains), Bacillus thuringiensis (2 strains), Bacillus megaterium, Bacillus subtilis, Bacillus cereus, which were isolated from plants such as cabbage, carrot, radish and kale. They used the paper disc method for screening of these bacteria against pathogen. Among the different species of bacillus spp., B. thuringiensis, B. cereus, B. subtilis, B. megaterium and Bacillus pumilus found effective against the pathogens (X. c. pv. campestris and P. c. subsp. carotovorum).

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