Research Article - (2013) Volume 3, Issue 1
1Department of Plant Pathology, University of Agriculture, Faisalabad
2Department of Plant Breeding & Genetics, University of Agriculture, Faisalabad
3Institute of Soil & Environmental Sciences, University of Agriculture, Faisalabad
4Directorate of Pest Warning & Quality Control of Pesticides, Gujranwala
5Directorate of Pest Warning & Quality Control of Pesticides, Sheikhupura
6Directorate of Pest Warning & Quality Control of Pesticides, Sialkot
7Directorate of Pest Warning & Quality Control of Pesticides, Toba Tek Singh
8Directorate of Pest Warning & Quality Control of Pesticides, Nankana Sahib
Cotton (Gossypium hirsutum L.) occupies a significant place in cash crops of Pakistan. Bacterial blight is the most destructive disease of cotton. Three chemicals (Plant Protector, Agrimycine and Copper oxy Chloride) and plant extracts (Citrullus colocynthis, Nicotiana tobaccum and Curcuma Lunga) were evaluated in vitro by using inhibition zone technique against colony growth of Xanthomonas axonopodis pv. malvacearum. Plant Protector was found most effective at 600ppm after 72 hours of treatment. In chemicals inhibition zone area increased with increasing dose and time. No relation was seen among plant extracts in colony growth inhibition of bacteria with respect to time and concentrations. Tobacco’s extract (Nicotiana tobaccum) was most significant at 10% concentration. Treatments means were compared by Tukey’s HSD test.
Bacterial Blight of Cotton, Chemical Control, Inhibition zones, Plant extracts, Xanthomonas axonopodis pv. malvacearum.
Cotton (Gossypium hirsutum) is an important cash crop. It is a backbone of Pakistan’s economy. It is grown over 12% of the total cultivated area in the country [1] . It accounts for 8.6% of the value added in agriculture and about 1.9% to GDP. In Pakistan, Bacterial blight of cotton was first observed near Multan in 1965 reduces the yield of the crop up to 50% under favorable environmental conditions [2]. Bacterial blight (BL) of cotton caused by Xanthomonas axonopodis pv. malvacearum (Smith) Dye affects the entire aerial parts of cotton plant i.e. necrosis of parenchymatous tissue in the local phase and blockage of xylem vessels in its systemic phase [3]. In Faisalabad district (Pakistan), Disease incidence was recorded 20-37% [4]. The use of resistant varieties is an economical option for disease management but currently none of the high yielding commercial varieties has a durable resistance against the disease [5, 6]. In the absence of durable resistance in varieties, control of the disease through chemicals, seed treatment or acid delinting is recommended but bactericide alone or in combination with fungicides dose not eradicate the pathogen completely [7, 8]. The use of chemicals for disease management is recommended as an alternative method but has very limited success [7, 9] due to systemic nature of the bacterium. Disease management through plant extracts has been reported by different workers in different crops [10-12] but very little is known about the antibacterial affects of plant extracts against bacterial blight of cotton.
The objective of these studies was to evaluate chemicals and plant extracts at different concentration against Xanthomonas axonopodis pv. malvacearum In vitro . Study will be an effective tool for choosing the most effective plant extracts and chemicals at best concentration for disease management in field conditions.
Isolation Purification & Multiplication of bacterium from infected leaves
Cotton leaves showing typical symptoms of bacterial blight were collected from the field and the bacterium was isolated by dilution plate technique [13]. Isolated bacterium produced yellow and round colonies on nutrient agar. It was rod shaped and motile with single polar flagellum. The bacterium was purified by streaking method on Nutrient Glucose Agar (NGA) medium (Nutrient agar 28g L-1and glucose 2 g L-1). Stock culture of bacterium was maintained on nutrient glucose broth in culture tubes at 4°C.
Inhibition zone Technique
For In vitro evaluation of plant extracts against colony growth of Xanthomonas axonopodis pv. malvacearum, inhibition zone technique was used. Bacterial culture was multiplied by adding the freshly growing aqueous bacterial suspension to the Luke warm NGA media in a flask. After solidification of media, using inhibition zone technique, wells (I cm diameter) were made in the centre of each Petri plate. Plant extracts and chemicals were poured in the well with the help of a sterilized syringe.
Preparation of Plant Extract:-
Three plant extracts (Nicotiana tobaccum, Colocunthus citrulus and Curcuma longa) were used. For tobacco (Nicotiana tobaccum) and colocynth (Colocunthus citrulus), 25g leaves of each were thoroughly washed, chopped and 25g were macerated separately in an electric grinder with 100 ml of distilled water. The mixtures were taken in a pellet and centrifuged at 9000 rpm for 5 minutes.
100g piece of turmeric (Curcuma longa) bulb was thoroughly washed in water, macerated well in pestle and mortar in 100ml of distilled water. Mixture was centrifuged at 9000 rpm and extract was separated.
All extracts were filtered separately through filter paper and were arbitrarily considered as standard. Each plant extract was used at 5%, 10% and 15%. Each treatment was replicated thrice.
Data Recording:-
The experiment was conducted in completely randomized design with three replications and having nine Petri dishes in each treatment. All the petri dishes were placed in a refrigerator at 4°C for 24 hr and then incubated at 30°C. The data was recorded by measuring the inhibition zones after 24, 36 and 48 hrs were subjected to analysis of variance and treatment means were compared by Tuckey’s HSD test.
Effect Chemicals against colony growth of Xanthomonas axonopodis pv. malvacearum.
Initial reading was taken after 24 hours of treatment. Zones were measured by using an ordinary ruler in centimeter (cm) scale. After 24 hours Plant Protector was found most effect than other chemicals. Relation between chemical concentration and effectiveness was observed, results were more significant at 300ppm and at 500ppm concentrations as compared to 100ppm dose. No efficacy was seen between the 300ppm and 600ppm doses except for plant protector where at 600ppm the inhibition zone’s area significantly increased (see Table1).
Table 1:- Inhibition zones by different chemicals against bacterial colony measured after 24 hours.
After 48 hours of treatment, ascending trend was seen in Agrimycine, zone area was increased with respect to 100ppm, 300ppm, and 600ppm doses. In rest of treatments (Plant Protector and Copper oxy Chloride) no change was noticed at 100ppm and 300ppm concentrations. Plant protector at 600ppm was more effective. Agrimycine was more effective than copper oxy chloride but do not differ statistically (Table 2).
Table 2:- Inhibition zones by different chemicals measured after 48 hours.
No statistically difference was observed between plant protector and copper oxy chloride at 100 ppm and 300 ppm doses. Gradual increase in inhibition zone area was seen for Agrimycine at three doses were visualized after 72 hours of treatments (Table 3).
Table 3:- Inhibition zones by different chemicals measured after 72 hours.
All treatments were compared with control. Increased zone area was noticed when chemical concentration was increased. Similar effect was seen with respect to time. Zones area was increased gradually as time passed till after 72 hours ( see Table 4).
Table 4:- Inhibition zones formed by different chemicals at various concentrations after different time periods.
Jagtap, Jangam [14] conducted experiment to evaluate the efficiency of different chemicals and biocontrol agents against bacterial blight disease incidence (PI) and disease severity (PDI). Copper oxy chloride @ 0.25 % + streptocycline @ 100ppm significantly reduces the disease severity and disease incidence level (11.83% and 19.36%) respectively as compared to unsprayed control (27.56%).
Khan and Ilyas [15] evaluated antibacterial activity of various chemotherapuents i.e., Agromycine-100, Streptomycine sulphate and Carboxin were more effective in disease control while Pencozeb, Nemisopre, Cuperosan, Sandofan-M and Tricyclazole were comparatively less effective against Xanthomonas axonopodis pv. malvacearum.
Effect Plant extracts against colony growth of Xanthomonas axonopodis pv. malvacearum.
Three plant extracts (Citrullus colocynthis, Nicotiana tabacum and Curcuma longa) were used at 5%, 10% and 15%. After 24 hours, no significant difference was noted among treatments at 5% dose. Inhibition area increased twice at 10 % as compared to 5% concentration. 15% concentration results was not effective as was shown by 10% concentration. equal efficacy of Citrullus colocynthis was observed at 5% and 10% doses (Table 5).
Table 5:- Inhibition zones by different plant extracts measured after 24 hours.
Turmeric (Curcuma longa) was most effective relatively at 5% as compared to others. Negative trend was observed with the increase in dose concentration. Colocynth (Citrullus colocynthis) and tobacco (Nicotiana tabacum) were equally effective at 5%. Colocynth (Citrullus colocynthis) was effective at 10 % than all treatments of various concentrations. No effect of increased dose (10% to 15%) was observed in Colocynth (Citrullus colocynthis) and Tobaco (Nicotiana tabacum) extracts (Table 6).
Table 6:- Inhibition zones by different plant extracts measured a after 48 hours
Tobacco showed tremendous results after 72 hours of treatment. Significant effectiveness was noted at 10% . No effectiveness showed by Colocynth (Citrullus colocynthis) and Tobacco (Nicotiana tabacum) at 5%. Same results were seen by Tobacco (Nicotiana tabacum) and Colocynth (Citrullus colocynthis) at 10 and 15% doses respectively. Tobacco (Nicotiana tabacum) results were found most effective, though efficacy was equal with Colocynth at 15% but due to its low concentration it was preferred.
Change in zones with respects to time was unpredictable. Colocynth (Citrullus colocynthis) extract’s inhibition zone increased with increasing of time at 15%. No change was seen between Colocynth and Tobacco at different time periods (24 hours, 48 hours and 72 hours) at 5% concentration. Turmeric was unchanged at three time levels at 10% (see Table 8).
Table 7:- Inhibition zones by different plant extracts measured after 72 hours.
Table 8:- Inhibition zones formed by different plant extracts at various concentrations after different time periods.
Bambawale, Mohan [16], conducted research on 14 different medicinal plants against cotton pathogen In vitro study. Extracts were prepared in ethanol at 1:1 ratio. Extracts of Datura metel, Lawsonia alba (Lawsonia inermis) and D. stramonium inhibited colony growth of Xanthomonas campestris pv. malvacearum as well as spore germination and mycelial growth of Alternaria macrospora. In field condition, extract of Datura metel, Lawsonia alba and D. stramonium gave excellent control.
Khan, Rashid [17], used some plant extract as a biological control agent against bacterial blight of cotton. seed oil of neem (Azadirachta indica), neem seed bitter, leaf extract of Datura alba, and nimbokil 60 EC were evaluated at 1, 2 and 3% concentrations on the growth of Xanthomonas campestris pv. malvacearum. Datura alba significantly retarded the growth of bacterium at 3% concentration followed by nimbokil, neem seed bitter and neem seed oil respectively.
