In-vitro Evaluation of Different Chemicals, Bioagents and Botanicals against Xanthomonas citri subsp citri

Citrus canker caused by Xanthomonas citri subsp citri is one of the important disease in kagzi lime affecting the quality and market value of the fruits. The inhibitory activity of chemicals, bioagents and botanicals were evaluated against Xanthomonas citri subsp citri under in-vitro condition. Among the bactericides and fungicides tested 2-bromo2-nitropropane-1, 3-diol and copper hydroxide showed maximum inhibition of 16.39mm and 15.77mm respectively. However, the bioagents VK-6B and KK-3A recorded >90% inhibition among the thirteen bioagents evaluated. Similarly 21 different botanical were evaluated for their inhibition efficacy which revealed that Prosopis juliflora extract showed highest average inhibition of 10.83mm and 15.21mm in aqueous and alcoholic extracts respectively.

Citrus canker caused by Xanthomonas citri subsp citri is a serious disease reducing the external quality of citrus fruits.It affects all types of citrus and severely infects on Citrus aurantifolia (lime).Canker occurs in all areas where lime grows in Karnataka state.Control of the disease requires integrated cultural practices and chemical sprays.Copper compound products are recommended for canker control.One major limitation of using chemical control agents is that phytopathogenic bacteria frequently develop a resistance to these compounds (Sigee,1993).In recent year much interest has been developed in the antimicrobial effects of medicinal plants and bacterial bioagents for plant disease control.Some plant extracts and bioagents were reported as effective inhibitors of phytopathogenic bacterial growth and was also suppressed by plant extracts and bioagents (Leksomboon et al,1998;Leksomboon et al,2000;Khodakaramian 2008; Garden et al,1978 andGrainge 1987).Plant extracts and bioagents are of interest as an alternative source of natural pesticide for controlling of plant pests.
The bacterium was multiplied by inoculating the culture into 250 ml of nutrient broth taken in 'Erleyenmayers' flask.The inoculated flasks were incubated at 28±1 o C for 48hr.The bacterial suspension was then seeded to the lukewarm nutrient agar medium.The seeded medium was poured into the sterilized petri plates and plates were allowed to solidify.
The chemicals and antibiotics were prepared at different concentrations as mentioned.The filter paper discs (Whatman No. 42) measuring 5mm in diameter were soaked in the respective chemical solution for 5-10 minutes and transferred onto the surface of the seeded medium in petriplates.The inoculated plates were kept in the refrigerator at 4 o C for 30 minutes to allow the diffusion of chemicals into the medium.Then plates were incubated at 28±1 o C for 24 hr and observed for the production of inhibition zone around the filter paper discs.The results obtained were analysed statistically.
Similarly the bacterial antagonistic isolates maintained in the Department of Plant Pathology, College of Horticulture, Bagalkot viz., were used to evaluate the efficacy of bioagents against Xanthomonas citri subsp citri.
The 13 isolates viz., PM-1A, VK-6B, BK-6, KK-9A, VK-10C, BK-3, BK-5,BK-7, PM-2A, BK-8, BK-1L, KK-3 and CK-13A were tested using the dual culture technique, the 48 hr old pure culture of 13 bacterial bio-agents and Xanthomonas citri subsp citri were streaked in a single Plate containing NA medium with the help of inoculation loop.Two streaks of bio-agent were streaked first and the pathogen streaked at the middle of both streaks of bio agent.Later plates were kept for incubation at 30°C for 48 hours and finally the visual observation of inhibition of Xanthomonas citri subsp citri by bio-agent was recorded.
To identify the effective bioagents against Xanthomonas citri subsp citri, the scale developed by Ramesh 2015, which ranged from I to VI was used.

S.
Per cent inhibition of Grade No. growth of X. citri subsp citri by bio-agent

Aqueous extraction method
The plant extracts used in the study as mentioned in table 3 were collected and washed in the tap water and rinsed in the distilled water.The plant parts chopped in to small parts and taken fifty gram of sample and macerated in the surface sterilized pestle and mortar under aseptic condition by adding fifty ml of sterile water (1:1 w/v) and kept overnight at 4 0 C in a refrigerator for complete release of active component.After that the extract was filtered through two layered muslin cloth.The extracted solution was considered as stock solution of aqueous extract.

Alcoholic extraction method
Fifty gram of the of the respective plant parts was mixed with a fifty ml of ethyl alcohol and macerated in a pestle and mortar under aseptic condition.The sample was transferred to a beaker and kept overnight under refrigerated condition for evaporation of alcohol.The fifty ml of distilled water was added to make the 1:1 w/v.Finally the Alcohol extract was squeezed through double layered muslin cloth.The filtrate was collected used as stock solution of alcoholic extract.
The inhibition zone technique as followed in chemical evaluation were done to know the effect of botanicals on Xanthomonas citri subsp citri at different concentration of 1:1, 1:5 and 1:10.The Streptocycline 0.5g (500ppm) + Copper oxychloride 3g (0.3%) per liter of water treated as positive control and untreated paper discs considered as negative control.
The data on in-vitro evaluation of botanicals on the growth of Xanthomonas citri subsp.citri is presented in Table 3.In aqueous extraction method the results showed difference among the treatments.Among the botanicals tested prosopis with concentration of 1:1, 1:5 and 1:10 w/v was found to be most effective with the highest inhibition of 14.00, 10.00 and 8.50 mm The results revealed that among all the botanicals, prosopis with concentration of 1:1, 1:5 and 1:10 w/ v was found to be most effective with the maximum inhibition of 18.50, 15.50 and 11.63mm respectively.Prosopis treatment was followed by Kokum at 1:1, 1:5 and 1:10 w/v recorded inhibitory zone of 18.50, 13.00 and 10.50 mm respectively.However soapnut recorded inhibitory zone of 16.50, 12.50 and 8.50 mm at 1:1, 1:5 and 1:10 w/v concentrations respectively.There was no inhibition of bacterial growth in the remaining treatments at all the three concentrations.The standard check Streptocycline 500ppm + Copper oxychloride 3000ppm recorded maximum inhibition of 21.50 mm which found superior to all the botanicals tested.Whereas water control recorded no inhibition zone DISCUSSION A total of eleven chemicals were evaluated for their efficacy against Xanthomonas citri subsp citri by paper disc method and the mean inhibition diameter was recorded.Highest inhibition was noticed in 2-Bromo-2 nitropropane-1,3-diol (14.67, 16.33 and 18.17mm) and Streptocycline (13.50, 15.00 and 16.33mm) at 300, 400 and 500ppm respectively, followed by copper hydroxide (14.33, 15.67 and 17.33mm) and copper oxy chloride (11.00, 13.33 and 14.83) which recorded highest inhibition at 1500, 2000 and 2500ppm respectively.
Streptocycline is a protein inhibitor whereas streptomycin sulphate play role in interfering with formyl-methionine tRNA and binds to 30s RNA whereas tetracyclin hydrochloride interferes with amino-acyl tRNA.However 2-Bromo -2 nitropropane-1,3-diol interacts with cysteine, cysteine methyl ester and glutathione in the presence of air and acts as catalyst for oxidation of thiol groups of disulfides (Julia 1988).
The present findings were supported by shahid et al. (2005)    The antagonistic activity of ten strains of Pseudomonas fluorescens and five strains of Pseudomonas putida were evaluated against Xanthomonas axonopodis pv.citri, under in-vitro condition.The strain Pseudomonas fluorescens -19 had recorded a maximum inhibition with 6.40cm and there was no inhibition by the Pseudomonas putida strain 8, 13 and 18.The variation in the antagonistic activity of bacterial isolates might be due to the difference in the mode of action and mechanism (Khodakaramian et al., 2008).
The study is supported by Mohammed et al. (2014) where a total of 22 potential bacterial antagonists isolated as epiphytes from the phylloplane of healthy citrus trees were screened for their in vitro efficacy against Xcc.These strains were identified as Pseudomonas fluorescens on the basis of biochemical and physiological tests and 16S rDNA.Out of these 22 potentially bacterial antagonists, five strains (KSA1, KSA9, KSA14, KSA17 and KSA20) showed high potential growth inhibition.
Similar kinds of investigations were also reported on pomegranate bacterial blight caused by Xanthomonas axanopodis pv.punicae by Yenjerappa (2009), where antagonists like Bacillus subtilis and Pseudomonas fluorescens were found significantly superior in inhibiting the growth of pathogen.Ramesh (2015) isolated 170 isolates from different pomegranate orchards and found 57 isolates suppressing the growth of pathogen out of which 8 isolates viz, PM-1A, VK-6B, BK-6, KK-9A, VK-10C, KK-3A, BK-5 and BK-3 showed the maximum inhibition.
Biological control can achieve the objective of disease suppression through a number of ways such as antibiosis, competition, mycoparasitism, cell wall degradation, induced resistance, plant growth promotion and rhizosphere colonization capability.The most effective bioagent studied till date appears to antagonize pathogen using multiple mechanisms is Pseudomonas, which utilizes both antibiosis and induction of host resistance to suppress the disease causing microorganisms (Jan et al., 2013) Under biological control of plant diseases, various antagonistic organisms have been identified, which fight against the pathogens by different mechanisms viz., competition, lysis, antibiosis, siderophore production and hyper parasistism (Vidyasekaran, 1999).
The antagonism of Pseudomonas fluorescens against some Xanthomonas spp. was reported by Unnamalai and Gnanamanickam (1984).
In aqueous extraction of botanicals, the results showed significant difference among the treatments.Among the botanicals tested Prosopis juliflora was found to be most effective with maximum average inhibition of 10.83 mm which was significantly different from all other treatments.This was followed by kokum and soapnut which recorded average inhibitory zone of 9.50 and 8.00 mm respectively.Even in the alcoholic extraction method the same trend has been observed with the average inhibition of 15.21, 14.00 and 12.50 mm by the prosopis, kokum and soapnut respectively.
Kokum rind possesses an important phenolic compound called as garcinol identified by Sutar et al. (2012), beside garcinol the other compounds like furfural and its derivatives, cyanidin-3-glucose which are present as anthocyanin in the rind.This furfural and cyanidin -3-glucose is a potent antimicrobial.The extent to which these compounds get extracted in different solvents determine the degree of bactericidal action.Sasitorn (2003) reported the use of 23 different herbal extracts, extracted by 95% ethyl alcohol at the concentration of 100,000, 50,000, 10,000, 5,000 and 1,000 ppm respectively and found that guava leaf extract could inhibit the growth of bacteria at 50,000 ppm, the myrobalan wood fruit extract at 10,000 ppm, the extract of beleric myrobalan fruit, nut gall fruit and pomegranate fruit peel could inhibit the growth of bacteria at all concentration.
Srinivasachary (1995) investigated that, Ocimum plant extract was more effective in inhibiting the growth of Xanthomonas campestris pv.mori.Similar studies were also reported on in vitro efficacy of different botanicals in inhibiting growth of Xanthomonas axonopodis pv.punicae by Manjula (2002) who found that kolangi extract was superior followed by meswak, tulsi and patchouli.Yenjerappa (2009) reported that, garlic extract at 10 per cent concentration was significantly greater in efficacy than all other treatments followed by parthenium and lantana leaf extract and onion bulb.
The in-vitro efficacy of 15 different botanicals were tested against Xanthomonas axonopodis pv.punicae by Ramesh 2015 and revealed that garlic had recorded maximum average inhibition of 14.89 mm followed by kokum and prosopis with 13.20 and 11.44 mm respectively.

Table 1 .
Effect of antibacterial chemicals against Xanthomonas citri subsp citri under in vitro condition

Table 2 .
In-vitro evaluation of antagonistic bacterial bio-agents against Xanthomonas citri subsp citri Xanthomonas citri subsp citri under in-vitro condition and results are presented in Table4.The similar trends of results were obtained as in case of aqueous extraction.

Table 3 .
Effect of aqueous extracts of selected botanicals against Xanthomonas citri subsp citri under in-vitro condition Note: Figures in parentheses indicateSquare root transformed values 1:1, 1:5 and 1:10 = Concentrations (1 part of crude extract and 1, 5 and 10 part of water)

Table 4 .
Effect of alcohol extracts of selected botanicals against Xanthomonas citri subsp citri under in-vitro condition