International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(6), 39-45, June (2013) Int. Res. J. Biological Sci. International Science Congress Association 39 Characterization of Leaf Blight Pathogen, Pseudomonassyringae pv. syringaeof mango in BangladeshIslam S.I.A., Islam M.R., Dastogeer K.M.G. and Hossain I.Department of Plant Pathology, Bangladesh Agricultural University, Mymensingh-2202, BANGLADESHAvailable online at: www.isca.in Received 5th April 2013, revised 24th April 2013, accepted 1st June 2013Abstract Bacterial leaf blight of mango caused by Pseudomonas syringae pv. syringae is an emerging disease and great threat for production of healthy mango saplings in different nurseries of Bangladesh. A survey was conducted in nurseries of some selected areas viz. Dinajpur, Rajshahi, Khulna, Mymensingh and Madhupur of Bangladesh to know the status of bacterial leaf blight of mango in terms of its incidence and severity and to characterize the pathogen causing the disease. The results showed that the highest (10.42%) bacterial leaf blight incidence was recorded in Rajshahi followed by Mymensingh, Madhupur and Dinajpur with 9.73, 9.12, 8.64 % disease incidence respectively and the lowest (8.58%) incidence was recorded in Khulna. The maximum (7.34%) severity of bacterial leaf blight was recorded in Rajshahi followed by Dinajpur, Mymensingh and Madhupur with 7.17, 7.13, 7.13% disease severity and the minimum (7.03%) severity was recorded in Khulna. Twenty four bacterial isolates were obtained from the infected leaves by tissue planting method from different locations surveyed. Pathogenicity test on detached mongo leaves confirmed the bacterium was Pseudomonas syringae pv. syringae. The hypersensitivity test in tobacco leaves with P. syringae pv. syringae isolates did not show rapid death leaf of tissues where infiltrated. The causal bacterium, P. syringae pv. syringae was also characterized by a series of biochemical tests viz. KOH solubility test, Gram staining test, Kovac’s oxidase test, Temperature sensitivity test, Levan test, Sugar utilization test, Arginine dihydrolase activity, Catalase test and Pectolytic test. The results of the all the tests revealed that the causal organism of leaf blight of mango was Pseudomonas syringae pv. syringae. Keywords: Characterization, leaf blight, Pathogen, Mango Introduction Mango (Mangifera indica L.) belonging to the family Anacardiaceae is said to have originated in the region of eastern Indo-Bangladesh, Burma, Malaysia or Thailand. It is also grown all over the tropics and subtropics and one of the most important fruit crops in the world. The leading mango producing countries are India, Pakistan, Mexico, Brazil, Haiti and Philipines. Mango is also grown in Indonesia, Java, Thailand, Bangladesh, Srilanka and Northwest Australia. India is the largest producers with approximately 66% of the total mango production in the world. In Europe, mango orchards are not widespread, but mango plantings in southern Spain have escalated over the last 10 years, presently covering about 800 ha. It is widely grown all over Bangladesh with the quality mangoes solely concentrated in the north-western areas especially greater Rajshahi, Dinajpur and Rangpur. Mango ranks third among the tropical fruits grown in the world. Bangladesh produced 82816 metric ton of mango in 31028 ha of mango orchard during the period of 2008-09. Mango is now recognized as one of the desirable fruit in the world market for its excellent flavor, pleasant aroma, attractive color and taste. Mango has got a unique position in respect of nutritional quality, taste, consumer’s preference etc. among the 50 kinds of fruits grown in Bangladesh. Mango is being the most demanding fruit. So the demand for fruit mango is increasing day by day with growing population but declining in production which results in scarcity every year. Disease is one of the predominating causes for lower production of mango in Bangladesh. Mango is reported to be attacked with as many as 18 different diseases in Bangladesh . The major diseases occur in mango are scab, anthracnose, stem end rot, bacterial leaf spot, bacterial leaf blight, powdery mildew, die-back, rust, malformation etc. Bacterial leaf blight has been observed in mango in southern Spain in 1991 . The symptoms of the disease are characterized bya rapid enlargement of necrotic lesions in buds and leaves. Mango buds, leaves, and stems are all predisposed to infection, but fruit lesions have not been detected. Disease symptoms comprise necrosis of vegetative and flower buds and bud failure before bud break. Necrotic lesions in buds occasionally outspread to the leaf petiole through the stem. Leaf blight of mango in Bangladesh has been reported by Hossain. They also identify the causal agent to be the bacteria Pseudomonas syringae pv. syringae but the pathogen was not characterized in details. Hence, a comprehensive survey was made in some nurseries of the selected mango growing areas to know the status of bacterial leaf blight of mango in terms of its incidence and severity in some selected nurseries and to characterize different isolates of its causal organism in Bangladesh. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(6), 39-45, June (2013) Int. Res. J. Biological Sci. International Science Congress Association 40 Material and Methods Survey and collection of diseased plant samples: A survey was carried out to know the status of bacterial leaf blight of mango in nurseries of Rajshahi, Dinajpur, Mymensingh, Madhupur and Khulna during July to September 2011. At least ten nurseries from each growing areas were surveyed to record the bacterial leaf blight incidence and severity. The infected leaf samples from different nurseries were brought into the laboratory for the isolation of the pathogen. Isolation, purification, identification and preservation: The infected leaves were surface disinfected by immersion in 10 % Clorox solution and then rinsed in sterile distilled water. Leaf pieces after drying on blotting paper were transferred to nutrient agar (NA) medium and incubated at 25°C for 2 to 3 days. Bacterialisolates were purified by streaking a single colony of each isolate on NA medium as described by Kelman.The isolates of bacteria were preserved in 10% skim milk kept at -20C refrigerator for subsequent biochemical studies. Characterization of Pseudomonas syringae pv. Syringae: Pathogenicity Test: To confirm the isolates of . syringae pv. syringae, the pathogenicity test was performed on the detached leaves of mango. A single colony of syringae pv. syringaeshowing virulent, fluidal, irregular and creamy white color was selected for each group of isolates and multiplied in a NA medium. After 24 hours bacterial isolates were suspended in sterile water. The tip of the leaves was cut about 2.5cm. Then the cut leaves were dipped into bacterial suspension for 24hr. The inoculated leaves were then incubated in a Petridish at room temperature for at least 5-7days for the appearance of symptom. Host range test: The host range of syringae pv. syringaeisolates were tested in some other fruit species such as Guava, Jujube and Litchi by pathogenicity test on detached leaves (described earlier) of those fruit species. This test was performed to observe the reaction or infection ability of . syringae pv. syringae, on fruits other than mango. Hypersensitivity response (HR) test: To determine the pathogenic nature of the isolates, hypersensitive reaction was studied on tobacco (Nicotiana rustica) plants by infiltration of bacterial suspension into the intrveinal areas of the tobacco leaves. Biochemical studies for of P. syringae pv. Syringae: Gram staining reaction: 24 hrs old fresh bacterial cultures for each group of isolates were taken to perform the gram reaction test. Bacterial mass were taken out from culture, spread on clean glass slide followed by drying in air without heat and was flamed it lightly and gently to settle the bacteria. Crystal violet was then poured over the bacterial smear on the slide for 2-3 minutes and washed in tap water for 1 minute and lightly blot dry on a paper towel to remove crystal violet. The smear was flooded with iodine solution for 1 minute and washed in tap water for few seconds and then blot dried. It was decolorized with solvent e.g. alcohol, until the solvent flowed colorlessly from the slide (about 2-4 seconds) and rinse in tap water for 5 seconds. Then the smear was counterstained for about 1 minute with safranin solution and washed briefly in tap water followed by blot dry. One drop emulsion oil was then added on the slide and placed it on compound microscope with 100 x magnification for observation. Potassium hydroxide test: Gram staining results were confirmed by potassium hydroxide test (KOH) 3% as described by Suslow et al.10. The bacteria were aseptically removed from Petriplates with tooth pick, placed on glass slide in a drop of 3% KOH solution and stirred for 10 second using a quick circular motion of hand. Oxidase test: A 24h old bacterial colony on nutrient agar, augmented with 1% glucose was used in this test. A loopful of the inoculum was rubbed on filter paper pervaded with 15%(w/v) freshly prepared aqueous solution of Tetramethyl-p-phenylene diamine dihydrochloride11. Temperature sensitivity test: The ability of bacterial isolates to grow at different selected temperature at 27°C, 37°C and 41°C were tested by initially growing isolate on NA for 24h. The bacterial suspension of isolate was prepared from 24 hrs NA culture to 5 ml of nutrient broth. 2-3 drops of bacterial suspension was taken in nutrient broth contained in test tubes which were placed at temperature 27°C, 37°C and 41°C. Levan test: Sucrose peptone agar or a nutrient agar medium with 5% sucrose is suitable substrate for Levan test. For the test a single colony for each isolate was stabbed with a sterilized tooth pick on NA medium containing 5% sucrose. Then the plates were incubated at 28C for 2 to 4 days to have distinctive dome shaped colonies. Sugar Utilization test: Preparation of medium:The medium consists of peptone water to which fermentable sugar was added in the proportion of 1%. Peptone water was prepared by adding 1g of Bacto peptone (Difco, USA) and 0.5 g of sodium chloride in 100 ml distilled water. The medium was boiled for 5 minutes, adjusted to pH at 7.0, cooled and then filtered through filter paper. Phenol red, an indicator at the strength of 0.2% solution was added to peptone water and then dispensed 5 ml in Durham's fermentation tubes, placed inversely. These were then sterilized in autoclave at 121C maintaining at 15 PSI pressure for 15 minutes. The sugars used for fermentation test were prepared separately as 10% solution in distilled water (10 g sugar was dissolved in 100 ml of distilled water). A little heat was given to dissolve the sugar completely. The sugar solutions were sterilized using filters. An amount of 0.5 ml of sterile sugar solution was added aseptically in each culture tubes containing sterile peptone water and the indicator. Before use, the sugar media was sterilized and International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(6), 39-45, June (2013) Int. Res. J. Biological Sci. International Science Congress Association 41 incubated it for 24 hours at 37C. The carbohydrate fermentation test was performed by inoculating a loop full of nutrient broth culture of the organisms into the tubes containing different sugar media (four basic sugars such as dextrose, sucrose, lactose, and manitol) and incubated for 24 hours at 37C. Acid production was indicated by the color change from reddish to yellow in the medium and the gas production was noted by the appearance of gas bubbles in the inverted Durham's tubes. Arginine dihydrolase activity test: The test was performed to observe the presence of two enzymes that permits certain Pseudomonads to grow under anaerobic conditions. 0.2g peptone, 1g NaCl, 0.06g KHPO4, 0.6g Agar, 0.02mg Phenol red and 2g Arginine HCl was taken in a conical flask and mixed with 200mL water. The conical flask was shaken gently for mixing the chemicals properly. The pH was adjusted at 7.2. These were then sterilized in autoclave at 121C maintaining at 15PSI pressure for 15 minutes. A fresh culture was stabbed into a soft agar tube of Thornley’s medium, sealed with sterilized mineral oil or melted agar and incubated at 28C. A color change from faint pink to red within four days is positive reaction. Catalase Test: This test was performed to isolates to check their liveliness. Standard cultures are used for control purpose. One milliliter of 3% hydrogen peroxide (H) was placed on the microscope slides. The bacterial isolates collected from the leaf samples were tested for catalase activity. Bacterial isolates were grown in NA medium for 24 hrs and a loopful of bacterial cells was added in the drop of H. Bubbles’ arising from the solution was recorded as positive reaction. This enzyme is responsible for protecting bacteria from hydrogen peroxide (H) buildup, which can arise during aerobic metabolism. Catalase breakdowns H into water and O2. Pectolytic activity: Well washed, healthy looking, firm potatoes were surface sterilized and were peeled aseptically. Three standard slices were placed in sterile petridishes and the petridishes were incubated in a moist chamber. Two slices were then inoculated with loopful of bacteria previously grown on Nutrient Agar culture of each isolate and incubated at room temperature for 24-48 hrs for the detection of soft rot symptoms. A control for each isolate was maintained using loopful of sterile water. Pectolytic activity was performed to observe the condition of the sliced potato. Results and Discussion Leaf blight of mango a new disease was identified in ten nurseries of each of five areas of Bangladesh viz. Rajshahi, Dinajpur, Mymensingh, Madhupur and Khulna (figure-1 and table-1). The symptoms appear on buds, leaves, and stems of mango but fruit lesions have not been observed. Disease symptoms include necrosis of vegetative and flower buds and bud failure before bud break. Necrotic lesions in buds sometimes spread to the leaf petiole through the stem. Generally, a white creamy gum exudes from necrotic lesions on buds, stems, and less frequently on petioles. Necrotic symptoms also affect flower panicles, causing the most severe economic losses because of the decrease in fruit set. Lesions on leaves start as interveinal, angular, water-soaked spots (1 to 3 mm in diameter) that coalesced, becoming dark brown to black (figure-2). The survey results indicated a regional variation in bacterial leaf blight incidence and severity. The results showed that the highest (10.42%) incidence was recorded in Rajshahi and the lowest (8.58%) incidence was in Khulna (figure-3). At the same time the highest (7.34%) severity was recorded in Rajshahi and the lowest (7.03%) severity was recorded in Khulna (figure-4). Morphological appearance preliminary showed that mango with bacterial leaf blight have shown the presence of bacterial strains resembling Pseudomonas syringae pv. syringae. Table-1 Isolates of Pseudomonas syringae pv. syringae obtained from the infected mango leaf samples collected from some selected areas of BangladeshAreas surveyed Group of Isolates Number of isolates obtained in each area Dinajpur Group 1 (A) 6 Rajshahi Group 2 (B) 6 Khulna Group 3 (C) 4 Mymensingh Group 4 (D) 6 Madhupur (Tangail) Group 5 (E) 2 Isolation and Identification of the Pseudomonas syringae pv.syringae isolates: A total of twenty four P. syringae pv.syringae isolates were obtained from the infected mango leaves collected from different locations surveyed. All of the syringae pv. syringae isolates isolated from infected mango leaves produced cream color or off-white color colonies on NA media after 24 hours of inoculation. Confirmation test of P.syringae pv. Syringae: Pathogenicity test: The results of pathogenicity test revealed that all the isolate groups of P.syringae pv. syringae were able to cause blight symptoms on mango leaves identified by observing dark brown to black blight areas on the leaves (table-2). Host range test: The host range of P.syringae pv. syringae was determined by pathogenicity test on detached leaves of the three fruit species viz. guava, litchi and jujube. The result showed P.syringae pv. syringae causing blight symptoms on mango leaves but did not produce any symptom on the leaves of guava, litchi and jujube (table-2). Hypersensitivite response (HR) test: The result showed that none of the isolates were able to induce HR (rapid death of local cell of tissues) into the interveinal areas between of tobacco (cv. Banket –A1) and chili leaves. Biochemical tests: Potassium hydroxide solubility test: The result revealed that an elastic thread or viscious thread was 1 International Research Journal of Biological Sciences ________________________________________________ Vol. 2(6), 39-45, June (2013) International Science Congress Association observed when loop raised from the bacterial solution by toothpick a few centimeters from glass slides in case of all group of P. syringae pv. syringae isolates of groups of P. syringae pv. syringae isolates are gram negative (table-2). Gram staining test : The Gram’s staining reaction was performed using crystal violate. The microscopic results showed that all of the isolates of P. syringae pv. syringae violet color i.e. the isolates retained counter stain (pink color). Therefore, all isolates of P. syringae pv . syringae each group are gram negative and straight or curved rod shaped which is the characteristic feature of any plant pathogenic bacteria (table-2). Kovac’s oxidse test: In this study result showed that all group of P. syringae pv. syringae isolates were not able to develop deep blue color with oxidase reagent within few seconds which indicated that the resu lt of the test was positive for pv. syringae isolates (table-2). Temperature sensitivity test: Isolates of syringae grew at 27°C but failed to grow at 37°C and 41°C while conducting temperature sensitivity test ( Map showing the areas surveyed for the field based assessment of leaf blight of mango occurring in Bangladesh N W International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association observed when loop raised from the bacterial solution by toothpick a few centimeters from glass slides in case of all of indicating that all isolates are gram negative : The Gram’s staining reaction was performed using crystal violate. The microscopic results showed syringae did not retain violet color i.e. the isolates retained counter stain (pink color). . syringae representing are gram negative and straight or curved rod shaped of any plant pathogenic In this study result showed that all group isolates were not able to develop deep blue color with oxidase reagent within few seconds which lt of the test was positive for P. syringae Isolates of P. syringae pv. grew at 27°C but failed to grow at 37°C and 41°C while conducting temperature sensitivity test ( table-2). Levan test: The result showed that all group of syringae isolates were able to produce distinctive domed shaped or round colonies due to production of levan in sucrose containing NA medium (table-2). Sugar utilization test: The results of clearly showed that all group of isolates obtained from the diseased mango leaves samples were able to oxidize the four (4) basic sugars (Dextrose, sucrose, manitol and lactose) by producing acid and gas(red yellow) (table-2). Arginine dihydrolase activity : Results of this test showed that the isolates P. syringae pv . syringae pH and change the color (table-2). Catalase test: Results of this test showed that the isolates of syringae pv. syringae were able to raise bubbles on the slide and indicated as positive reaction (table - Pectolytic test : This results showed that pv. syringae does not respond against the pectinase enzyme as Erwinia sp. does (table-2). Figure-1 Map showing the areas surveyed for the field based assessment of leaf blight of mango occurring in Bangladesh International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 42 The result showed that all group of P. syringae pv. isolates were able to produce distinctive domed shaped or round colonies due to production of levan in sucrose The results of Sugar fermentation test clearly showed that all group of P. syringae pv. syringae isolates obtained from the diseased mango leaves samples were able to oxidize the four (4) basic sugars (Dextrose, sucrose, manitol and lactose) by producing acid and gas(red dish to : Results of this test showed that . syringae were not able to raise the Results of this test showed that the isolates of P. were able to raise bubbles on the slide and - 2) : This results showed that Pseudomonas syringae does not respond against the pectinase enzyme as Map showing the areas surveyed for the field based assessment of leaf blight of mango occurring in Bangladesh