The Journal of Plant Pathology. Photon 114 (2014) 177-187 https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-plant-pathology Original Research Article. ISJN: 3752-1938: Impact Index: 3.76

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The Journal of Plant Pathology

Prevalence of canker on seedlings of citrus (Citrus spp.) in selected areas of Bangladesh and its management Rashid M.*, Chowdhury M.S.M., Sultana N. Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh Article history: Received: 13 April, 2014 Accepted: 19 April, 2014 Available online: 18 August, 2014 Keywords: Canker, citrus seedling, prevalence, management Corresponding Author: Rashid M.* Ph.D Student Email: [email protected] Chowdhury M.S.M. Professor Sultana N. Professor

Abstract A survey conducted in eight nurseries of Bangladesh during January, 2012 to December, 2013 revealed that the highest incidence (72.22%) and severity (27.55%) of canker of citrus was recorded in the month of July at Khagrachari and the lowest incidence (28.33%) and severity (5.55%) was recorded in the month of January at Dhaka. An important bacteria Xanthomonus axonopodis

pv. citri was isolated from the canker infected part of citrus seedlings and identified by studies on morphological, biochemical and cultural features of the bacteria. In vitro evaluation of different antibacterial chemicals indicated that Mancozeb (Indofil M-45 showing 37.67 mm inhibition zone) and Copper compounds (Champion and Cupravit showing 20.67 mm and 20.33 mm inhibition zone respectively) were highly effective against Xanthomonas axonopodis pv. citri. In vivo evaluation of different antibacterial chemicals indicated that the highest reduction of incidence (44.92%) and severity (72.24%) was recorded when Indofil M-45 was applied as foliar spray @ 0.3%. In vitro evaluation revealed that Bacillus subtilis has the maximum zone of inhibition (19.00 mm) against Xanthomonas axonopodis pv. citri compared to other biocontrol agents. Citation: Rashid M., Chowdhury M.S.M., Sultana N., 2014. Prevalence of canker on seedlings of citrus (Citrus spp.) in selected areas of Bangladesh and its management. The Journal of Plant Pathology. Photon 114, 177-187. All Rights Reserved with Photon. Photon Ignitor: ISJN37521938D689418082014

1. Introduction Citrus (Citrus spp.) is one of the most important, popular and nutritious fruit crop in the world as well as in Bangladesh. Annual citrus production in the world is 115 million tons (FAO, 2012). Bangladesh produces only 21 thousand metric tons citrus fruits every year according to a study of 2009-2010 (BBS, 2010). Among the various factors, plant diseases play an important role in lowering the yield. Different species of citrus grown in the world suffers from more than 100 diseases (Klotz, 1973). In Bangladesh, twelve diseases are known to occur in different species of citrus seedlings. Most of the commercial citrus species grown in nursery around the world including Bangladesh are suffering from a bacterial disease, citrus canker caused by Xanthomonus axonopodis pv. citri (Graham et al., 2004). The disease is endemic in many tropical and subtropical citrus growing areas (Goto, 1992). Temperatures Ph ton

between 15 to 20° C and 35 to 40° C are conducive for infection and development of citrus canker disease (Pria et al., 2006). The disease is mostly prevalent in area with more than 1000 mm rainfall per year (Verniere et al., 2003). The pathogen is dispersed by splashing rain and winds in excess of 8.0 ms-1 (Gottwald et al., 1989). Prophylactic sprays of copper oxychloride or other coppercontaining compounds provide protection against initial infection of citrus canker during growth flushes and fruit development (Graham and Leite, 2004; Das, 2003; Leite and Mohan, 1990). But the indiscriminate use of agrochemicals leads to degradation of the ecosystem, may induce pathogen resistance to the pesticide, and may cause human and animal health problems (Huang, 1997). Therefore, public concern is focused on alternative methods of pest control (Sutton, 1996). Bacillus subtilis and Pseudomonas fluorescens are most

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effective antagonist against Xanthomonas axonopodis pv. citri (Kalita et al., 1996). Bacillus subtilis shows biological activity against phytopatogenic bacteria by producing peptide antibiotics (Backman et al., 1997). Biochemical studies by Valasubramanian et al. (1994) showed that efficient strains of Pseudomonas fluorescens produce an antibiotic phenazine-1-carboxylic acid (PCA), which hinders the growth of plant pathogenic bacteria. Despite of economic importance, sufficient information has not been generated so far about this worldwide disease and its management. Therefore, the current study was conducted to survey on the prevalence of the disease, identify the causal agent and evaluate the effective management strategies against it.

(Schaad, 1992), motility indole urease agar (MIU) test (Schaad, 1992), gelatine liquefaction test (Salle, 1961), tobacco hypersensitivity test (Klement and Goodman, 1967) and study on growth and colony morphology of the bacterium on NA, YDCA and SX media as per method described by Schaad (1992). 3.2 Pathogenicity test A bacterial suspension (5 µl) containing 108 colony forming units per ml (CFU/ml) was inoculated into the lower surface of citrus leaf with a sterile syringe and observed for 15 days. Visual symptoms were recorded and examined. To confirm Koch’s postulates, bacteria reisolated from diseased leaves were streaked on NA plate and reidentified using the methods outlined by Lin et al. (2008).

2. Objective of Research The main objective of this study is to know the epidemiology of canker on seedlings of citrus and find out suitable management strategies for controlling it. 3. Materials and Methods Prevalence of canker on seedlings of citrus was surveyed in eight nurseries of Dhaka, Gazipur, Barisal and Khagrachari during January, 2012 to December, 2013; lab experiments were conducted in Disease diagnostic laboratory, Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka and field experiments were conducted in Nursery, Department of Plant Pathology, Sher-eBangla Agricultural University, Dhaka. 3.1 Isolation and identification of the causal organism Leaves collected from different survey areas were surface sterilized with 5% sodium hypochlorite solution and Xanthomonas axonopodis pv. citri was isolated from it using the techniques described by Goszczynska and Serfontein (1998). Colonies of bacteria were purified on NA plate. Identification of the pathogen involved in causing of citrus canker was done by gram’s staining reaction (Gerhardt, 1981), potassium hydroxide (3% KOH) test (Suslow et al., 1982), starch hydrolysis test (Cowan, 1974), catalase test (Schaad, 1992), oxidase test (Kovacs, 1956), citrate utilization test

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3.3 Isolation and identification of biocontrol agents Trichoderma harzianum and Aspergillus flavus were isolated and purified on PDA plates following the method described by Ashrafuzzaman (1976). Antagonistic bacteria were isolated from rhizosphere soil by dilution plate technique as described by Goszczynska and Serfontein (1998) on NA plate, King's B (KB) plate and YMA plate. For isolation of spore forming Bacillus subtilis, stock solution was heated at 850C for 10 minutes (Nemeckova et al., 2011). Colonies of bacteria were purified on NA plates. Identification of antagonistic bacteria was done by gram’s staining reaction (Gerhardt, 1981), potassium hydroxide (3% KOH) test (Suslow et al., 1982), starch hydrolysis test (Cowan, 1974), catalase test (Schaad, 1992), oxidase test (Kovacs, 1956), citrate utilization test (Schaad, 1992), motility indole urease agar (MIU) test (Schaad, 1992) and gelatine liquefaction test (Salle, 1961). 3.4 Epidemiology of disease incidence and severity During the survey, every seedling along with canker infected was counted in thenursery for calculating the incidence of the disease. Thirty seedlings were randomly selected for counting diseased leaves and disease free leaves. Disease incidence of canker on seedlings of citrus was determined by the following formula:

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-Percent disease incidence of foliar diseases was determined by the following formula (Rai and Mamatha, 2005) The disease severity was recorded by using the following scale developed by Anon. (2006). Grade 0 1 2 3 4 5

Percent leaf infection 0.00 Up to 1 >1-10 >10-20 >20-40 >40-100

3.5 In vitro evaluation of antibacterial chemicals against Xanthomonas axonopodis pv. citri A supply of cotton swabs on wooden applicator sticks was prepared following the method described by Vandipitte et al. (1991). Six selected antibacterial chemicals viz. Cupravit 50 WP (Copper oxychloride), Sulcox 50 WP (Copper oxychloride), Champion 77 WP (Copper hydroxide), Indofil M-45 (Mancozeb), Dithane M45 (Mancozeb) and Bavistin 50 WP (Carbendazim) were screened against the bacterium by well diffusion method measuring the inhibition zone (Anon., 1996). Four holes of 5 mm in diameter were punched into the same NA plate maintaining equal distance and the broth culture of Xanthomonas axonopodis pv. citri was spreaded

uniformly on it with sterile cotton swabs. Chemical suspension at definite concentration with different volume was added into the hole each at three replications. In case of control only sterile water was used instead of chemical. The plates were incubated at 30±1ºC. Zone of inhibition around the holes were measured and recorded after every 24 hours for 5 days. 3.6 In vivo evaluation of antibacterial chemicals against Xanthomonas axonopodis pv. citri For In vivo evaluation of antibacterial chemicals against Xanthomonas axonopodis pv. citri, four selected antibacterial chemicals viz. Cupravit 50 WP (Copper oxychloride), Champion 77 WP (Copper hydroxide), Indofil M-45 (Mancozeb), and Bavistin 50 WP (Carbendazim) were sprayed at 30 days interval by a hand sprayer. One seedling per pot and 5 seedlings per treatment were used. The experiment was laid out in Randomized Complete Block Design (RCBD) with five replications. The data on incidence, severity and plant height were collected at one month interval before each spray schedule. Assessment of disease incidence and severity was calculated using the formula described in previous section. Percent disease reduction (PDR) was calculated using the formula of Rai and Mamatha (2005).

Percent height increase/decrease over first count was calculated using the formula:

Percent height increase/decrease over control was calculated using the formula:

3.7 In vitro evaluation of biocontrol agents against Xanthomonas axonopodis pv. citri Five biocontrol agents viz. Bacillus subtilis, Pseudomonas fluorescens, Rhizobium leguminosarum, Trichoderma harzianum and Aspergillus flavus were screened against the growth of Xanthomonas axonopodis pv. citri by well diffusion method measuring the inhibition zone (Yenjerappa, 2009). Supernatants of biocontrol agents were dropped on the hole of previously swabbed plate with the pathogenic bacteria each at three replications. In case of control, only sterile water was used instead of supernatant. The plates were then incubated at 30±1º C in incubation chamber. Zone of inhibition

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around the holes were measured and recorded after every 24 hours for 5 days. 3.8 Meteorological data collection Day to day meteorological data on temperature, relative humidity and rainfall were collected from Meterological Department, Agargaon, Dhaka-1207. 3.9 Statistical analysis Data were analyzed through computer software MSTAT-C (Anon., 1989). To determine the level of significant differences and to separate the means within the parameters, Duncan’s Multiple Range Test (DMRT) and Least Significant Difference (LSD) test were performed.

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Table 1: Average temperature, relative humidity 2012 to December, 2013 Average Mean average Month temp. (ºC) temp. (ºC ) of 2 years Jan, 2012 16.88 17.89 Jan, 2013 18.90 Apr, 2012 20.50 24.25 Apr, 2013 28.00 Jul, 2012 30.50 29.57 Jul, 2013 28.63 Oct, 2012 29.50 28.40 Oct, 2013 27.45

and rainfall of Dhaka, Gazipur, Barisal and Khagrachari from January, Average relative humidity(%) 73.80 72.00 70.43 74.75 83.50 83.50 81.50 78.00

Mean average relative humidity (%) of 2 years 72.90 72.59 83.50 79.75

Average rainfall (cm) 0.52 0.46 3.85 2.48 5.50 3.47 5.95 1.48

Mean average rainfall (cm) of 2 years 0.49 3.17 4.49 3.72

4. Results and Discussion 4.1 Isolation and identification of the causal organism Repeated isolation from the infected leaves of citrus yielded well separated, typical, yellow, convex, mucoid, colonies of bacterium Xanthomonas axonopodis pv. citri on NA plates after 48 hours of incubation at 30±1º C. Colonies were purified by restreaking the isolated colony on NA plates. Chand and Kishun (1991) reported that Xanthomonas produce mucoid, circular, convex, yellow, round, glistening and raised colonies on nutrient agar medium. The isolated bacteria identified as Xanthomonas axonopodis pv. citri according to morphological, biochemical and cultural characters of the bacterium as per standard microbiological procedures. Table 2. Reaction of isolated Xanthomonas axonopodis pv. citri to different tests Name of tests Reaction Gram Staining + KOH solubility test + Starch hydrolysis test + Catalase test + Oxidase test Citrate utilization test + Motility indole urease agar (MIU) + test Gelatine liquefaction test + Tobacco hypersensitivity test +

Data in Table (2) summaries the important characters of Xanthomonas axonopodis pv. citri. The bacterium was rod shaped with rounded ends, cells appeared singly and also in pairs, gram negative (red colour) and capsulated under the compound microscope at 100x magnification with oil immersion. It produced a mucoid thread when lifted with the loop, showed amylase activity, no bubbles were formed after adding 3% H2O2, formed dark purple colour on oxidase disk, used citrate as a carbon source for their energy, migrated away from the original line of inoculation, gelatin was liquefied and infiltrated area of tobacco leaf became dry and necrotized within 48 hours. This is Ph ton

also in agreement with the findings of Yenjerappa (2009) and Chand and Pal (1982), where they found Xanthomonas axonopodis liquefied the gelatin, hydrolysed the starch, positive for catalase and oxidase, utilized various carbon sources. Colonies of Xanthomonas axonopodis pv. citri on NA medium appeared as circular, mucoid, convex, yellow to orange colour (Figure 1 a). Circular, flattened or slightly raised, yellow to bright yellow colour, mucoid colonies were found on YDCA medium (Figure 1 b). Bacterium exhibited very poor growth with light yellow to slightly blue, mostly circular, small, flattened, mucoid colonies on SX medium (Figure 1 c). Jabeen et al. (2012) observed that after 48-72 h of incubation at 28oC, Xanthomonas gave yellow, circular, smooth, convex and viscous bacterial colonies on YDCA medium and light yellow, mucoid, round colonies on XS medium. Figure 1: Starch hydrolysis test for Xanthomonas axonopodis pv. Citri

Figure 2: Tobacco hypersensitivity test for Xanthomonas axonopodis pv. Citri

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Figure 3: Colonies of Xanthomonas axonopodis pv. citri on different growth media (a) On NA media (b) On YDCA media and (c) On SX media

a

b

4.2 Pathogenicity test The characteristic symptoms were observed on citrus leaf after ten days of inoculation as small, blister-like lesions, which later on turned gray to tan brown surrounded by a yellow halo. Reisolations were carried out from these lesions and comparisons were made with the original culture to confirm the identity of the pathogen. Artificially inoculated plants yielded the bacterial colonies similar to the original ones. 4.3Incidence and severity of citrus canker during different growing seasons at different experimental location Incidence of canker of citrus varied significantly from season to season as well as location to location that ranged from 28.33 to 72.22% (Table 3). The highest (72.22%) incidence was recorded in the month of July (2012 & 2013) at Khagrachari and the lowest (28.33%) incidence was recorded in the month of January (2012 & 2013) at Dhaka. Statistically similar incidence (28.89%) was recorded in the month of January (2012 & 2013) at Gazipur. The severity of canker of citrus also varied significantly from season to season as well as location to location that ranged from 5.55 to 27.55% (Table 3). The highest (27.55%) severity was recorded in the month of July (2012 & 2013) at Khagrachari and the lowest (5.55%) severity was recorded in the month of January (2012 & 2013) at Dhaka. 4.4 Effect of weather components on the incidence and severity of canker on seedlings of citrus during January, 2012 to October, 2013 The incidence of canker of citrus was influenced by average temperature, relative humidity and rainfall (Figure 4). The highest incidence (63.89%) was recorded in the month of July (2012 & 2013) when the average temperature, relative humidity and rainfall were 29.57°C, 83.50% and 4.49cm, respectively. On the other hand, lowest incidence (30.97%) was recorded in the month of January (2012 & 2013) having average temperature, relative humidity and rainfall 17.89°C, 72.90% and 0.49 cm, respectively. The severity of canker of Ph ton

c citrus was also influenced by average temperature, relative humidity and rainfall. The highest severity (24.26%) was recorded in the month of July (2012 & 2013) when the average temperature, relative humidity and rainfall were 29.57°C, 83.50% and 4.49cm, respectively. On the other hand, lowest severity (6.86%) was recorded in the month of January (2012 & 2013) having average temperature, relative humidity and rainfall 17.89°C, 72.90% and 0.49 cm, respectively. Khan and Abid (2007) observed that environmental conditions (relative humidity, rainfall, maximum temperature, minimum temperature and wind speed) were correlated with citrus canker development and the disease increased with the increase in rain fall and relative humidity and decreased with the increase in maximum temperature. 4.5 In vitro evaluation of antibacterial chemicals against Xanthomonas axonopodis pv. citri Among the six antibacterial chemicals, Indofil M45 (Mancozeb) showed the highest amount of inhibition zone after 24 hours (34.33 mm), 48 hours (37.67 mm), 72 hours (35.00 mm), 96 hours (31.00 mm) and 120 hours (29.33 mm) of incubation at 0.3% concentration when 80 µl/well was used where Bavistin 50 WP (Carbendazim) showed the lowest amount of inhibition zone (Figure 5, Table 4). Meneguim et al. (2007) reported that copper as well as mixture of copper with mancozeb were effective in controlling citrus canker. 4.6 In vivo evaluation of antibacterial chemicals against Xanthomonas axonopodis pv. citri 4.6.1 Effect of different antibacterial chemicals on the incidence of canker on seedlings of citrus All the treatments significantly reduced the incidence of canker on seedlings of citrus over control during the period of October, 2012 to September, 2013 (Table 5). Considering the mean incidence, out of all the treatments applied the highest incidence (74.83%) was recorded in T5 (untreated control), which was statistically different from all other treatments and the lowest disease

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Table 3: Incidence and severity of canker of citrus during January, 2012 to October, 2013 at different experimental locations of Bangladesh Time of data Canker of citrus Location collection % Disease Incidence % Disease Severity (Jan, 2012-Oct, 2013) (Jan, 2012-Oct, 2013) January 28.33 h 5.55 j Dhaka April 47.22 de 17.33 f

Gazipur

Barisal

Khagrachari

July October January

55.00 c 33.33 gh 33.89 g

21.50 de 10.77 h 7.33 i

April

49.44 de

20.00 e

July October January April July October January

64.44 b 40.00 f 28.89 gh 51.11 cd 63.89 b 51.11 cd 32.78 gh

24.66 b 11.11 h 6.55 ij 18.22 f 23.33 bc 10.66 h 8.00 i

April July October

47.77 de 21.77 cd 72.22 a 27.55 a 44.44 ef 12.77 g LSD(p≤ 0.05) 4.90 1.63 CV(%) 6.25 6.25 Each data represents the mean value of two nurseries at each location of two years. Values followed by the same letter within a column are not significantly different (p≤ 0.05 according to Duncan’s multiple range test). Figure 4: Effect of weather components on the incidence and severity of canker on seedling of citrus during January, 2012 to October, 2013

incidence (41.22%) was observed in T3 (Indofil M45 was applied as foliar spray @ 0.3%). In case of percent reduction of disease incidence over control, the highest reduction (44.92%) was observed in T3 (Indofil M-45 was applied as foliar spray @ 0.3%) and the lowest reduction (6.98%) was observed in T4 (Bavistin was applied as foliar spray @ 0.3%). Behlau et al. (2007) observed copper sprays significantly reduced citrus canker on leaves. He observed that plants subjected to frequent copper sprays showed 43.5% lower canker incidence than those not protected with chemical sprays. Ph ton

4.6.2 Effect of different antibacterial chemicals on the severity of canker on seedlings of citrus For the application of different management practices, significant variations in the severity of canker on seedlings of citrus were observed during the period of October, 2012 to September, 2013 (Table 6). Considering the mean severity, out of all the treatments applied the highest severity (3.89%) was recorded in T5 (untreated control), which was statistically different from all other treatments and the lowest severity (1.08%) was recorded in T3 (Indofil M-45 was applied as foliar spray @ 0.3%).

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Figure 5: Evaluation of antibacterial chemicals against Xanthomonas axonopodis pv. citri (a) Cupravit 50 WP (b) Sulcox 50 WP (c) Champion 77 WP (d) Indofil M-45 (e) Dithane M-45 and (f) Bavistin 50 WP

C C

a

C

c

b

C C C

e

d

f

Table 4. Comparative efficacy of different antibacterial chemicals against Xanthomonas axonopodis pv. citri Antibacterial Conc. Volume Inhibition zone (mm) chemicals (%) (µl) 24h 48h 72h 96h 120h Cupravit 0.3 80 18.33 c 19.67 c 19.67 c 19.67 c 17.00 cd 50 WP Sulcox 0.3 100 19.33 c 20.33 c 19.33 c 18.00 c 15.67 d 50 WP Champion 0.2 100 20.33 c 20.67 c 20.00 c 19.67 c 18.00 c 77 WP Indofil M-45 0.3 80 34.33 a 37.67 a 35.00 a 31.00 a 29.33 a Dithane M-45

0.3

80

26.00 b

29.00 b

25.33 b

23.67 b

Bavistin 0.3 100 9.67 d 0.00 d 0.00 d 0.00 d 50 WP LSD(p≤0.05) 2.51 2.78 1.44 1.91 CV % 6.46 7.18 3.97 5.62 Each data represents the mean value of three replications. Values followed by the same letter within a column are not significantly different (p≤ 0.05 according to range test).

In case of percent reduction of disease severity over control, the highest reduction (72.24%) was observed in T3 (Indofil M-45 was applied as foliar spray @ 0.3%) and lowest reduction (18.51%) was observed in T4 (Bavistin was applied as foliar spray @ 0.3%). Behlau et al. (2007) also observed that plants subjected to frequent copper sprays showed 37.1% lower canker severity than those not protected with chemical sprays. 4.7 In vitro evaluation of biocontrol agents against Xanthomonas axonopodis pv. citri The study conducted revealed that among the five biocontrol agents, Bacillus subtilis and Pseudomonas fluorescens were significantly superior in inhibiting the growth of Xanthomonas axonopodis pv. citri. Bacillus subtilis showed Ph ton

21.33 b 0.00 e 1.36 4.41 Duncan’s multiple

highest amount of inhibition zone (19.00 mm) after 72 hours of incubation followed by Pseudomonas fluorescens with inhibition zone (14.33 mm) after 48 hours of incubation. Other biocontrol agents viz. Rhizobium leguminosarum, Trichoderma harzianum and Aspergillus flavus were ineffective as they failed to inhibit the growth of X. axonopodis pv. citri (Figure 6, Table 7). Kalita et al. (1996) also reported Bacillus subtilis was most effective antagonist producing largest inhibition zone followed by Pseudomonas fluorescens against Xanthomonas axonopodis pv. citri.

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Table 5. Effect of different antibacterial chemicals on the incidence of canker of citrus during the growing period of October, 2012 to September, 2013 % Disease Incidence Treatments Oct, Nov, Dec, Jan, Feb, Mar, Apr, May, Jun, Jul, 2013 Aug, 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 T1 66.00 c 52.67 c 36.00 b 0.00 c 0.00 c 11.33 c 34.00 c 39.33 c 96.00 b 97.33 a 94.00 b

Sep, 2013 89.33 b

51.34 c

% reduction over control 31.39

T2

66.00 c

50.67 c

35.33 b

0.00 c

0.00 c

0.00 d

33.33 c

38.00 c

94.00 b

94.00 b

92.67 b

89.33 b

49.45 d

33.92

T3

60.67 d

36.67 d

22.67 c

0.00 c

0.00 c

0.00 d

0.00 d

34.00 d

89.33 c

88.67 d

86.67 c

76.00 c

41.22 e

44.92

T4 90.67 b 69.33 b 60.67 a 39.30 b 38.67 b 41.33 b 47.33 b 48.00 b 100.0 a 100.0 a T5 100.0 a 100.0 a 63.33 a 42.67 a 43.33 a 44.00 a 50.67 a 53.98 a 100.0 a 100.0 a LSD(p≤0.05) 2.74 3.21 3.03 2.17 2.17 2.00 2.95 3.38 2.97 2.45 CV(%) 2.66 3.87 5.19 9.85 9.85 7.71 6.65 5.91 2.31 1.90 Data represent the mean value of 5 (five) replications. Values followed by the same letter within a column are not significantly different (p≤ 0.05 according to Duncan’s multiple range test). T1= Cupravit was applied as foliar spray @ 0.3% T2= Champion was applied as foliar spray @ 0.2% T3= Indofil M-45 was applied as foliar spray @ 0.3% T4= Bavistin was applied as foliar spray @ 0.3% T5= Untreated control

100.0 a 100.0 a 2.83 2.23

100.0 a 100.0 a 2.49 2.04

69.61 b 74.83 a 0.90 1.18

6.98

Sep, 2013 3.44 c 1.82 d 1.84 d

Mean 1.57 c 1.40 d 1.08 e

% reduction over control 59.64 64.01 72.24

6.61 b 8.44 a 0.41 6.97

3.17 b 3.89 a 0.04 1.61

Table 6. Effect of different antibacterial chemicals on the severity of canker of citrus during the growing period of October, 2012 to September, 2013 % Disease severity Treatments Oct, Nov, Dec, Jan, Feb, Mar, Apr, May, Jun, Jul, 2013 Aug, 2012 2012 2012 2013 2013 2013 2013 2013 2013 2013 T1 1.01 d 0.11 c 0.24 c 0.00 b 0.00 c 0.06 c 0.43 c 0.87 b 3.88 c 4.98 c 3.77 c T2 1.42 c 0.17 c 0.22 cd 0.00 b 0.00 c 0.00 d 0.41 c 0.83 b 3.74 c 4.76 c 3.45 d T3 0.73 e 0.08 c 0.20 d 0.00 b 0.00 c 0.00 d 0.00 d 0.06 c 3.28 d 3.91 d 2.87 e T4 2.69 b 1.84 b 0.39 b 0.19 a 0.54 b 0.73 b 0.81 b 0.87 b 4.96 b 9.56 b T5 3.57 a 3.30 a 0.44 a 0.21 a 0.80 a 0.81 a 1.02 a 1.02 a 7.09 a 10.32 a LSD(p≤0.05) 0.25 0.14 0.04 0.04 0.04 0.04 0.07 0.10 0.15 0.23 CV(%) 9.79 9.52 9.44 9.14 5.00 6.30 9.89 9.36 2.34 2.53 Data represent the mean value of 5 (five) replications. Values followed by the same letter within a column are not significantly different (p≤ 0.05 according to Duncan’s multiple range test). T1= Cupravit was applied as foliar spray @ 0.3% T2= Champion was applied as foliar spray @ 0.2% T3= Indofil M-45 was applied as foliar spray @ 0.3% T4= Bavistin was applied as foliar spray @ 0.3% T5= Untreated control

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8.83 b 9.63 a 0.28 3.66

Mean

18.51

Figure 6: Evaluation of biocontrol agents against Xanthomonas axonopodis pv. citri (a) Bacillus subtilis (b) Pseudomonas fluorescens

a

b

Table 7. Efficacy of different biocontrol agents against the growth of Xanthomonas axonopodis pv. citri Bioagents Inhibition zone (mm) 24h 48h 72h 96h 120h Bacillus subtilis 13.67 a 16.33 a 19.00 a 16.67 a 13.33 a Pseudomonas fluorescens 11.33 b 14.33 b 12.67 b 10.67 b 8.667 b Rhizobium leguminosarum 0.00 c 0.00 c 0.00 c 0.00 c 0.00 c Trichoderma harzianum 0.00 c 0.00 c 0.00 c 0.00 c 0.00 c Aspergillus flavus 0.00 c 0.00 c 0.00 c 0.00 c 0.00 c Control 0.00 c 0.00 c 0.00 c 0.00 c 0.00 c LSD(p≤0.05) 0.66 0.64 0.79 0.64 0.58 CV % 8.76 6.84 8.23 7.67 8.62 Each data represents the mean value of three replications. Values followed by the same letter within a column are not significantly different (p≤ 0.05 according to Duncan’s multiple range test).

Conclusion Xanthomonas axonopodis pv. citri showed positive result in KOH solubility test, starch hydrolysis test, catalase test, citrate utilization test, motility indole urease agar (MIU) test, gelatine liquefaction test, salt tolerant test, tobacco hypersensitivity reaction and negative result in oxidase test. Highest incidence (72.22%) and severity (27.55%) of canker of citrus was recorded in the month of July at Khagrachari and the lowest incidence (28.33%) and severity (5.55%) was recorded in the month of January at Dhaka. A positive correlation was observed between the incidence and severity of cankerof citrus with temperature, relative humidity and rainfall. In vitro as well as field evaluation of antibacterial chemicals indicated that Mancozeb and Copper compounds were highly effective against Xanthomonas axonopodis pv. citri. Among different biocontrol agents, in vitro evaluation revealed that Bacillus subtilis was highly effective against the bacterium. Further study should be conducted to apply these biocontrol agents in field condition.

are immense possibilities to work on the molecular basis of citrus canker. Justification of Research The results showed that the highest incidence and severity of canker of citrus is higher in seedlings when temperature, relative humidity and rainfall are higher. We have also observed Mancozeb and Copper compounds are highly effective against Xanthomonas axonopodis pv. citri. Bacillus subtilis a biocontrol agent is also effective against the bacterium. Authors’ Contributions Rashid M. has carried out the laboratory and field experiments, analysed the data, interpreted the results and wrote the paper. Chowdhury M.S.M. and Sultana N. defined the research him, participated in the analysis and interpretation of results and helped in drafting the manuscript. All authors read and approved the final manuscript.

Recommendations Acknowledgment Our study showed that the highest incidence and severity of canker of citrus was in the month of July at Khagrachari and the lowest incidence and severity in the month of January at Dhaka. There

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We especially thankful to PIU-BARC (NATP Phase-1), Bangladesh Agricultural Research Council for financial help for conducting the research under the project “Surveillance of seedling

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