Chethankumar K.1, Kirankumar K.C.1, H.B. Patil2,
Sadanand K. Mushrif3 and Babu A.G.4

1Department of Plant Pathology (University of Horticultural Sciences), Bagalkot, Karnataka, India.
2Department of Vegetable Science (University of Horticultural Sciences), Bagalkot, India.
3Department of Plant Pathology (University of Horticultural Sciences), Kolar, India.
4Department of Crop physiology (University of Horticultural Sciences), Bagalkot, Karnataka, India.

ABSTRACT

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-bromo- 2-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.

Keywords : Chemicals, Bioagents, Citrus canker, Botanical, Xantomonas citri.

Introduction

Citrus canker caused by Xanthomonascitrisubspcitri is a serious disease reducing the external quality of citrus fruits. It affects all types of citrus and severely infects on Citrusaurantifolia (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 bioagentsfor 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 andGrainge1987). Plant extracts and bioagents are of interest as an alternative source of natural pesticide for controlling of plant pests.

Material methods:-

The antibiotic compounds (2-Bromo – 2 nitropropane-1,3-diol, 2-Bromo – 2 nitropropane-1,3-diol (Immuno modulator), Kasugamycin, Plantamycin, Streptocycline and Validamycin) and fungicides(Carbendazim, Copper hydroxide, Copper oxychloride, Copper sulphate and Mancozeb) were evaluated in three different concentrations for their efficacy against Xanthomonascitrisubspcitriby inhibition zone assay method.

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±1oC 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 4oC for 30 minutes to allow the diffusion of chemicals into the medium.Then plates were incubated at 28±1oC 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, Bagalkotviz.,were used to evaluate the efficacy of bioagents against Xanthomonascitrisubspcitri.

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-13Awere tested using the dual culture technique, the 48 hr old pure culture of 13 bacterial bio-agents and Xanthomonascitrisubspcitriwerestreakedin 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 ofinhibition of Xanthomonascitrisubspcitri by bio-agent was recorded.

To identify the effective bioagents against Xanthomonascitrisubspcitri, the scale developed by Ramesh 2015, which ranged from I to VI was used.

Sl.No
Per cent inhibition of growth of X.citrisubspcitriby bio-agent
Grade
1
>90
I
2
76-90
II
3
51-75
III
4
26-50
IV
5
1-25
V
6
0
VI

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 fiftygram of sample and macerated in the surface sterilized pestle and mortar under aseptic conditionby adding fifty ml of sterile water (1:1 w/v) and kept overnight at 40 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 Xanthomonascitrisubspcitri 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.

Results

The in-vitro evaluation of different antibiotics in inhibiting the growth of Xanthomonascitrisubspcitri wereassessed by paper disc method and results are presented in Table1. Among the different antibiotics, 2-Bromo-2 nitropropane-1,3-diol (14.67, 16.33 and 18.17mm) and Streptocycline(13.50, 15.00 and 16.33mm) showed highest inhibition at 300, 400 and 500ppm. Among the fungicides copper hydroxide (14.33, 15.67 and 17.33mm) and copper oxy chloride (11.00, 13.33 and

14.83mm) recorded highest inhibition at 1500, 2000 and 2500ppm respectively. Whereas plantamycin and copper sulphate recorded average values of 12.50 mm and 3.88mm respectively. However 2-Bromo – 2 nitropropane-1,3-diol (Immuno-modulator),kasugamycin, validamycin, carbendazim,mancozeb and untreated control recorded no inhibition zone in all the concentrations tested.

The results of the dual culture technique indicated that all 13 isolates inhibited growth of Xanthomonascitrisubspcitri. A maximum inhibition of >90% was recorded by two isolates VK-6B(Lysinibacillusxylanilyticus) and KK-3A of bacterial bio-agents. Eight isolates vizPM-1A, KK-9A, VK-10C, BK-5, BK-3, CK-13A, PM-2A and BK-1L recorded 76-90% inhibition whereas three isolates BK-6, BK-8 and BK-7 recorded 26.-50% inhibition. The results are presented in Table 2.

The data on in-vitro evaluation of botanicals on the growth of Xanthomonascitrisubsp. citriis 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 thehighest inhibition of 14.00, 10.00 and 8.50 mm respectively followed by Kokum (13.50, 8.50 and 6.50) and Soapnut (11.00, 7.50 and 5.50 mm) respectively. There was no inhibition observed in remaining treatments. However positive check Streptocycline @ 500ppm + Copper oxy chloride @ 3000ppm showed maximum inhibition of 21.50 mm and found significantly different from all other botanicals tested.

Table 1. Effect of antibacterial chemicals against Xanthomonascitrisubspcitriunder in vitro condition

Treatments Antibacterial chemicals Mean diameter zone of Average
inhibition (mm)  
Bactericides 300 ppm 400 ppm 500 ppm
T1 2-Bromo – 2 nitropropane-1,3-diol 14.67 16.33 18.17 16.39
(3.89) (4.10) (4.32)  
T2 2-Bromo – 2 nitropropane-1,3-diol (Immuno modulator) 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T3 Kasugamycin 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T4 Plantamycin 10.50 12.67  14.33 12.50
(3.31) (3.62) (3.85)  
T5 Streptocycline 13.50 15.00 16.33 14.27
(3.74) (3.93) (4.10)  
T6 Validamycin 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T7 Control 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
SEm± 0.04 0.04 0.05  
CD (0.01) 0.09 0.14 0.18  
  Fungicides 1500 2000 2500  
ppm ppm ppm  
T1 Carbendazim 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T2 Copper hydroxide 14.33 15.67 17.33 15.77
(3.85) (4.02) (4.22)  
T3 Copper oxychloride 11.00 13.33 14.83 13.05
(3.38) (3.71) (3.91)  
T4 Copper sulphate 0.00 4.33 7.33 3.88
(0.70) (2.19) (2.79)  
T5 Mancozeb 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T6 Control 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
SEm± 0.04 0.05 0.02  
CD (0.01) 0.16 0.21 0.15  

 

Note: Figures in parentheses indicate Square root transformed values

 

Table 2. In-vitro evaluation of antagonistic bacterial bio-agents against Xanthomonascitrisubspcitri

Sl. No.
Code of the bio agents
Grade

(based on % inhibition by

bio agent)

1
PM-1A
II
2
VK-6B(Lysinibacillusxylanilyticus)
I
3
BK-6
IV
4
KK-9A
II
5
VK-10C
II
6
KK-3A
I
7
BK-5
II
8
BK-3
II
9
CK-13A
II
10
BK-8
IV
11
PM-2A
II
12
BK-7
IV
13
BK-1L
II

The alcoholic extracts of 21 botanicals were evaluated against Xanthomonascitrisubspcitriunder in-vitro condition and results are presented in Table 4. The similar trends of results were obtained as in case of aqueous extraction. 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 soapnutrecorded 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 checkStreptocycline 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 Xanthomonascitrisubspcitriby 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.

Table 3. Effect of aqueous extracts of selected botanicals against Xanthomonascitrisubspcitriunder in-vitro condition

 

 

Treatments Plant extracts Mean diameter zone of inhibition (mm) Average
1:01 1:05 1:10
T1 Adathodavasica 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T2 Calotropisgigantean 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T3 Cinnamomumverum 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T4 Cymbogonwinteranius 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T5 Eugenia caryophyllata 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T6 Eugenia caryophyllata 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T7 Eucalyptus citriodora 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T8 Allium sativum 12.00 0.00 0.00 4.00
 

(3.54)

(0.70) (0.70)  
T9 Kaliabith 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T10 Garciniaindica 13.50 8.50 6.50 9.50
 

(3.74)

(3.68) (2.64)  
T11 Lantena camera 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T12 Cymbopogoncitrates 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T13 Salvadorapersica 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T14 Azadiractaindica 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T15 Nitro sulph 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T16 Phosphorika 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T17 Pongamiapinnanta 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T18 Prosopisjuliflora 14.00 10.00 8.50 10.83
 

(3.81)

(4.00) (3.00)  
T19 Sapindussaponaria 11.00

(3.39)

7.50

(3.61)

5.50

(2.44)

8.00
T20 Ocimumbasilium 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T21 Control 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T22 Streptocycline + copper oxychloride 21.5(4.69)  
  SEm± 0.05 0.05 0.04  
  CD (0.01) 0.15 0.12 0.11  

Note: Figures in parentheses indicate Square 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 Xanthomonascitrisubspcitriunder in-vitro condition

Treatments Plant extracts Mean diameter zone of inhibition (mm) Average
1:01 1:05 1:10
T1 Adathodavasica 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T2 Calotropisgigantean 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T3 Cinnamomumverum 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T4 Cymbogonwinteranius 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T5 Eugenia caryophyllata 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T6 Eugenia caryophyllata 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T7 Eucalyptus citriodora 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T8 Allium sativum 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T9 Kaliabith 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T10 Garciniaindica 18.50 13.00 10.50 14.00
(4.30) (3.68) (3.32)  
T11 Lantena camera 16.50 11.00 0.00 9.16
(3.61) (3.39) (0.70)  
T12 Cymbopogoncitrates 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T13 Salvadorapersica 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T14 Azadiractaindica 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T15 Nitro sulph 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T16 Phosphorika 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T17 Pongamiapinnanta 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T18 Prosopisjuliflora 18.50 15.50 11.63 15.21
(4.36) (4.00) (3.48)  
T19 Sapindussaponaria 16.50 12.50 8.50 12.50
(4.13) (3.61) (3.00)  
T20 Ocimumbasilium 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T21 Control 0.00 0.00 0.00 0.00
(0.70) (0.70) (0.70)  
T22 Streptocycline + copper oxychloride 21.5(4.69)  
  SEm± 0.04 0.06 0.04  
  CD (0.01) 0.11 0.20 0.11  

Note: Figures in parentheses indicate Square root transformed values 1:1, 1:5 and 1:10 = Concentrations (1 part of crude extract and 1, 5 and 10 part of water)

 

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 shahidet al. (2005) who evaluated the efficacy of different chemicals against Xanthomonascampestrispv. citri with varied concentration of 0.01, 0.1 and 1% to check the multiplication of bacteria by paper disc method and found that Agrimycin –100, Streptomycin sulphate, Vitavax and DithaneM–45had maximum inhibiting capacity with 2.47, 2.28, 2.38 and 2.32 cm respectively.

Shahbazet al. (2007) studied the efficacy of Agrimycin–100, Cupravit, Bavistin, Dithane M-45, Vitavax, Daconil, Antracol, Benlate and Nimrod at 1% concentration in in-vitro condition againstXanthomonascampestrispv. citri. Among which Agrimycin–100 was found to be the most effective chemical with 2.89cm of inhibition.

Similar studies were also reported by Rashid et al., 2014 on in-vitro evaluation of six selected antibacterial chemicals viz.Cupravit 50 WP (Copper oxy chloride),Sulcox 50 WP (Copper oxy chloride), Champion 77 WP (Copper hydroxide),Indofil M-45 (Mancozeb),Dithane M- 45 (Mancozeb) and Bavistin 50 WP (Carbendazim) where Indofil-M45 recorded maximum inhibition of Xanthomonasaxonopodispv. citri

Thirteen isolates were examined against Xanthomonascitrisubspcitrifor their efficacy in inhibiting the growth of the pathogen, in which two isolates VK-6B(Lysinibacillusxylanilyticus) and KK-3A showed maximum inhibition (>90%) and were scaled as grade I. Eight isolates viz(PM-1A, KK-9A, VK-10C, BK-5, BK-3, CK-13A, PM-2A and BK-1L) recorded 76-90% inhibition with II grade. The last three isolates BK-6, BK-8 and BK-7 recorded 26.-50% inhibition with IV grade.

The antagonistic activity of ten strains of Pseudomonas fluorescensand five strains ofPseudomonas putidawere evaluated against Xanthomonasaxonopodispv. 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 putidastrain8, 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 (Khodakaramianet 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 fluorescenson 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 Xanthomonasaxanopodispv. punicae by Yenjerappa (2009), where antagonists like Bacillus subtilisand Pseudomonas fluorescenswere 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 bio-agent 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 Prosopisjuliflora 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 Sutaret 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 atthe 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 belericmyrobalan fruit, nut gall fruit and pomegranate fruit peel could inhibit the growth of bacteria at all concentration.

Srinivasachary (1995) investigated that, Ocimumplant extract was more effective in inhibiting the growth of Xanthomonascampestrispv. mori. Similar studies were also reported on in vitro efficacy of different botanicals in inhibiting growth of Xanthomonasaxonopodispv. punicaeby Manjula (2002) who found thatkolangi 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 Xanthomonasaxonopodispv. punicaeby 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.

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