Pakistan Journal of Phytopathology

Published by: Pakistan Phytopathological Society www.pakps.com www.pjp.pakps.com Pak. J. Phytopathol., [email protected] Vol 23(2):125-130, 2011.

INVESTIGATIONS ON SUDDEN DEATH SYNDROME AFFECTED PLANT PARTS IN DISTRICT MUZAFFARGARH

Abdul Rehman, Tasawar Abbas, Nasir Ahmad Khan and *Saira Mehboob Department of Plant Pathology, University of Agriculture Faisalabad. *Plant Pathology Institute, Ayyub Agriculture Research Institute Faisalabad.

ABSTRACT

Mango (Magifera indica L.) is an important fruit crop. A survey for disease incidence was recorded in four tehsils of district Muzaffargarh which showed that Kot Addu was the most affected tehsil followed by Ali pur and Jatoi, while least affected was Muzaffargarh. Maximum incidence was recorded on variety , followed by , Anwar Ratol and Malda. Chaunsa proved to be the most susceptible amongst four observed commercial varieties, while Malda showed resistance in field observation. The infected plants showed abundant gum secretion, bark splitting, rotting and twig blight. The wide spread fungus Ceratocystis manginecans was isolated in maximum percentage, followed by Lasiodiplodia theobromae. The maximum percentage of these two fungi from all diseased samples suggests that either both of them are responsible for Mango Sudden Death Syndrome. Maximum temperature for colony growth of Ceratocystis manginecans was 25°C and best pH was 7. Carbendazim fungicide was found most effective against C. manginecans while Agrofit was the least effective at all concentrations. Key words: , Ceratocystis manginecans, Mango Sudden Deaths Syndrome, Management, Carbendazim

INTRODUCTION Mango is known to suffer from a number of diseases caused by different pathogens, which bring Mango (Mangifera indica L.) is the most important significant losses in the form of reduced yield and fruit grown in many tropical and sub tropical regions low quality of produce. Over 140 diseases are known of the world. It is the favorite fruit of the to cause damage to the mango crop in different subcontinent and is called as the king of fruits countries of the world (Parkash, 1998). Mango is (Purseglove, 1972). Being useful and delicious, it has internationally favorite fruit with multiple uses. It is been a part of global cultural heritage for long time. used to fulfill the nutritional needs of people and also The mango production has increased in non to earn foreign exchange through its export. MSDS is traditional mango producing areas which include causing major loss to growers and to the country. All parts of Asia, West Africa, Australia, South America, commercial varieties are susceptible to MSDS. It is and Mexico. It is the second major fruit crop in the disease with multiple symptoms that’s why it is Pakistan after citrus. The climate and soil conditions called syndrome. of Pakistan are highly suitable for mango cultivation. Almost all the commercial mango verities are In Pakistan, mango is cultivated on an area of 167.5 susceptible to this disease (Mahmood and Gill, 2002). thousand ha with annual production of 1732 thousand Al-Adawi et al. (2003) reported MSDS as a new tones (Anonymous, 2010). Pakistan produces 5.99% disease of mango affecting 60% of the trees in Al of world mango (Anonymous, 2006) being the fifth Batinah region of Oman. Al-Adawi et al. (2006) largest producer after India, China, Thailand and revealed Ceratocystis manginecans as primary factor Mexico producing 43.51, 13.03, 6.68 and 5.73 associated with MSDS in Oman. In Pakistan C. percent, respectively (FAO, 2004) manginecans has been recorded first time by Malik et According to taxonomy, mango is member of class al. (2005). Recently C. manginecans was isolated Dicotyledonae and its subclass is Arhichlamydae. It from vascular bundles of declining mango trees in belongs to order Sapindales and family is , Pakistan (Fateh et al., 2006). Yadahalli et al. Anacardiaceae (Fernald, 1950). In Pakistan, 250 (2007) reported 250C to 300C is best temperature and varieties are under cultivation. Most widely grown pH is 7 for Colony growth of Ceratocystis paradoxa. varieties are Chaunsa, , Sindhri, Anwar Vijaya et al. (2007) evaluated five systemic and five Ratole, Fajri, Samar Bahisht, Shah Pan, and non-systemic fungicides in two concentrations in (Anonymous, 2004). vitro against Ceratocystis paradoxa causing sett rot 125 of sugar cane. Among the systemic fungicides transferred to sterilized filter paper in Petri plates for carbendazim and propiconazole were most effective drying. Then sterilized pieces were placed on petri in complete inhibition of the pathogen at both the plates containing PDA medium using sterilized concentrations (0.05 and 0.1%), while benomyl and forceps. All petri plates were incubated at 25oC with hexaconazole only at 0.1 per cent concentration. 12 hour alternate periods of light and darkness for 7 Among the non-systemic fungicides tested thiram days and observed on daily bases. The colony of was found best followed by captan at both the fungus was purified and identified with the help of concentrations (0.1 and 0.2%), whereas mancozeb literature, Ellis (1971) and Sutton (1980). The and copper oxychloride were least effective. frequencies of each isolated fungus from each plant Systemic fungicides were found more effective in part were calculated using the following formula: controlling the pathogen growth than that of non- systemic fungicides. Number of pieces colonized by a pathogen MSDS is a complex disease and different fungi are Colonization %= ------x 100 reported as causal organism. It is important to study Total number of pieces etiology of this disease, Physiological studies of isolated fungi and their in vitro management. Physiological studies of causal organism: In Etiology and in vitro management will provide basis physiological studies the effect of different of a plan of management strategies to control this temperatures and pH on the colony growth of isolated disease. fungi was observed. Effect of Temperature: Fungus was inoculated in MATERIALS AND METHODS Petri plates containing PDA and incubated at 5oC, 10oC, 15oC, 20oC, 25oC and 30oC with 12 hours Survey: A survey of MSDS worst hit district of alternate periods of light and darkness for 7 days in Punjab i.e. Muzzafargarh was conducted in spring an incubator SANYO MIR 254. Colony diameter was (March-April) 2009. Randomly, 5 orchards were noted on daily basis to establish the best temperature selected from each tehsil in spring and a total of 20 for the growth of causal organism. orchards were visited in spring 2009 in spring. Four Effect of pH: Fungus was inoculated in Petri plates mango commercial cultivars, Chaunsa, Malda, containing PDA and incubated at optimum Anwar Ratol, and Sindhri were observed to know the temperature 25+ 1oC.. The pH of PDA medium was resistant and susceptible towards MSDS during the adjusted 3, 4, 5, 6, 7 and 8 with 0.1 N NaOH/Hcl survey and disease incidence measured by the with pH meter. Optimum pH was determined by following formula. calculating colony diameter on daily bases for 7 days. Number of plants died Chemical control: Different doses of fungicides Disease incidence% = ------x 100 were used for in vitro evaluation by poison food Total number of plants technique (Dhingra & Sinclair, 1985). Five fungicides Carbendazim, Dithane M-45, Daconil, The infected plants were observed on the base of Agrofit and Alliete were used. These fungicides with following symptoms. Twig Blight: Drying of the twig three concentrations 20, 60 and 100 ppm of each (Ragab et al., 1971).Gummosis: Produce oozing of fungicide were added to PDA medium at the time of the cell sap of honey color on the surface of the bark pouring into 9 cm glass Petri plates. After of the trunk (Parkash,1996). Wilting: Drying of solidification, 5 mm discs of seven days old culture whole plant with defoliation (Malilk et al., 2004). of test fungus were placed in the center of test plates Samples were collected from four tehsils, under and incubated at 250C temperature. Data on colony survey in Muzaffargarh district. These samples growth of appearing colonies were recorded for one including bark of stem at collar region, roots and week on daily basis. branches were collected and brought to laboratory for Data analysis: The recorded data were subjected to isolation of associated fungi. analysis of variance (ANOVA) under CRD and LSD- Isolation, purification and identification of test at 5% level of significance (Steel et al., 1996). associated fungi: Potato dextrose agar (PDA), a general purpose media was used for isolation of RESULTS AND DISCUSSION fungal pathogens. The infected specimens comprising roots, twigs and bark were cut into small pieces of 4- The result regarding a survey of disease incidence in mm length and were surface sterilized with 70% Muzzaffargarh district in Table 1, shows that ethyl alcohol for 1-2 minutes. The sterilized pieces maximum disease incidence was in Kot Addu were rinsed twice with sterilized water and (2.50%) followed by Ali pur ( 1.50% ), Jatoi (1.18%) 126 and Muzzaffargarh (1.32 %). A statistically, orchards were found in Kot Addu which may have significant difference was found between Kot Addu affected the probability of high disease incidence in and other tehsils but Ali pur, Jatoi ,and Kot Addu was more as compared to incidence in Ali Muzzaffargarh were not significantly difference with pur, Jatoi and Muzzaffargarh. to each other. It was revealed by the study of disease incidence in These results are contradictory to Mehmood et al. different varieties that maximum disease incidence (2007) who reported a higher disease incidence was in Chaunsa (2.53%), Sindhri (1.99), Anwar Ratol (13%) but in agreement with Kazmi et al. (2005) who (1.19%) and Malda (0.79%). According to statistical reported (3.69%) disease incidence in Muzzaffargarh analysis the disease incidence was significantly district. greater in Chaunsa and lowest in Malda. Chaunsa and During 2009 disease incidence was lower than Sindhri have non significant disease incidence with expected due to unfavorable environmental each other. Anwar Ratol and Malda have non conditions i.e. maximum temperature in early month significant disease incidence of 2009 reached 43°C, which is lethal for pathogen. with each other. So Malda was significantly different In these visited tehsils a maximum number of (resistant) to all varieties as mentioned in Table 1,

Table.1. Diseases incidence in four tehsils of districts Muzffar garh in different varieties. Varieties (V) Locations (L) Means Kot Addu Ali Pur Jatoi M.Garh Chunsa 2.620 2.600 2.060 2.860 2.53 a Sindhri 3.860 1.640 1.340 1.120 1.99 ab Anwar ratol 2.180 1.080 0.680 0.840 1.19 bc Malda 1.360 0.700 0.640 0.460 0.79 c

Means 2.50 a 1.50 b 1.18 b 1.32 b Alpha=0.05. LSD value (V) = 0.120, LSD value (L) = 0.0797

Table-2: Infection % of Different Fungi colonized on Different Plant Parts Sr.No Name of fungus. Name of Sample Infection % age 1 Ceratocystis manginecans Stem, Root 41.50 2 Lasiodiplodia theobromae Twigs, Stem, Root 33.33 3 Colletotrichum gloeosporioides Twigs 11.10 regarding MSDS disease incidence. In Chaunsa majority of the selected samples yielded the fungus variety maximum disease incidence 2.53% was noted Ceratocystis manginecans, which was isolated from and minimum 0.79% in Malda. One possible reason all locations with a maximum frequency 41.50%. for the susceptibility of Chaunsa is that it is heavy Lasiodiplodia theobromae (33.33%) was the second bearer of fruit. Its canopy is large in size and fruit frequently isolated fungus which was isolated from bearing capacity is excellent. Due to large number of root, stem and twigs. Colletotrichum gloeosporioides fruit, plant uses its nutrition for fruit development and was the third and least frequent fungus as mentioned it’s physiological processes change during fruit in Table 2. Ceratocyctis spp has been identified as the bearing, which might be leading to susceptibility to cause of MSDS, and it is known to be soil borne pathogen. However in comparison, Malda is dwarf fungus (Rossetto and Rinero, 1990). As the fungus variety and produces fewer fruits as compared to spreads through soil and enters plants through roots, Chaunsa. Due fewer fruits,being produced with less the effect of root ball size and root area to soil born effect on physiological processes, Malda may not fungi. Malik et al. (2005) isolated Ceratocystis change and it remains more resistant to pathogen than manginecans from the mango plants infected by other varieties. The examination of infected parts of MSDS. Fateh et al. (2006) isolated Ceratocystis mango plants revealed the association of few fungi fimbriata from mango plants in Sindh infected by viz., Ceratocystis manginecans, Lasiodiplodia MSDS. theobromae and Colletotrichum gloeosporioides. The 127

Table-3: Effect of Temperature on Colony Growth (cm) of Ceratocystis manginecans Temperature Duration (Hours) Means (°C) Time in Hours 24 48 72 96 120 144 168 5 0.200 0.200 0.200 0.200 0.200 0.200 0.200 0.20 f u u u u u u u 10 0.400 0.433 0.633 0.800 0.966 1 .133 1.367 0.81 e t t s rs qr pq o 15 0.433 0.633 0.900 1.333 1.600 1.833 2.167 1.27 d t s r op n m l 20 0.9000 1.400 2.133 3.600 4.167 4.467 5.267 3.13 c r o l i h g de 25 1.333 2.500 3.200 4.033 5.100 6.300 b 7.300 a 4.25 a op k j h e 30 1.200 2.233 3.133 4.000 4.667 5.400 6.000 3.80 b op l j h f d c Means 0.74 g 1.23 f 1.70 e 2.32 d 2.78 c 3.22 b 3.71 a

Alpha= 0.05 LSD value (°C) = 0.06997; LSD value (H) = 0.07558; LSD value (°C x H) = 0.1851

Table-4: Effect of pH on Colony Growth (cm) of C. manginecans pH (P) Duration (Hours) Means 24 48 72 96 120 144 168 3 0.266 0.433 0.633 0.800 1 .000 1.200 1.367 0.81 e y xy vwx uv tu qrst opqr 4 0.400 0.466 0.733 1.100 1.333 1.567 2.067 1.09 d xy wxy v rst opqr nop kl 5 0.700 1.033 1.200 1.600 1.967 2.300 2.700 1.64 c vw stu qrst no lm jk hi 6 1.033 1.400 1.767 2.300 2.900 3.633 4.300 2.47 b stu opq mn jk gh f d 7 1.300 1.733 2.367 2.700 3.800 5.233 6.333 a 3.35 a pqrs mn j hi ef c 8 1.100 1.733 2.533 3.100 3.967 e 5.067 5.900 3.34 a rst mn ij g c b Means 0.80 g 1.13 f 1.54 e 1.9 d 2.49 c 3.16 b 3.77 a Alpha= 0.05; LSD value (H) = 0.09832; LSD value (P) = 0.09103; LSD value (P x H) = 0.2408 anatomy may be important in the development of growth to 7.30 cm was maximum at 25oC followed disease. Chaunsa is large plant with a big root zone by 30oC, 20oC, 15oC and 10oC.At 5oC C.manginecans which give more area to pathogen to attack. In did not show any growth. This indicated that comparison, Malda is a dwarf plant and has small temperature levels 25oC and 30oC were best for the root zone which gives less colony growth of C. manginecans but the Physiological Studies temperature levels 5oC, 10oC, 15oC reduced growth Effect of Temperature on colony growth of rate respectively. Statistically there was significant Ceratocystis manginecans: Colony growth of relationship of colony growth on 5oC, 10oC, 15oC, Ceratocystis manginecans was observed at different 20oC, 25oC and 30oC. temperature levels with respect to time. The results The effect of temperature on colony growth with showed in Table 3, demonstrate that temperature respect to time also showed statistically significant effect on colony growth was significant. After the relationship. Results indicate with in time colony statistical analysis, results revealed that colony growth also increased. Colony growth was maximum 128 after 168 hour, 144 hour, 120 hour, 96 hour, 72 hour, optimum pH of 6.5 while on pH 7 produced abundant 48 hour and 24 hour respectively. Statistically colony sporulation. growth 0.74 cm was significantly lower after 24 hour and maximum 3.71 cm after 168 hour. Chemical control: The results presented in Table 5, The optimum temperature for development is 25°C. clearly indicate that there is significant difference Baker et al. (2004) reported that Ceratocystis among the different fungicides in inhibiting the manginecans usually grows best at temperatures from mycelial growth of C. manginecans. Statistically 18oC to 28oC and is able to produce ascospores Carbendazim was found significantly to be the most within a week. These results are agreement to effective in inhibiting the growth of C. manginecans Yadahalli et al. (2007) they reported 25°C to 30°C is with the radial growth of 2.86, 2.10 and 1.13 cm, best temperature for Colony growth of Ceratocystis Agrofit was least effective with colony radii of 5.50, paradoxa. 4.73 and 3.80 cm at 20, 60 and 100 ppm against C. manginecans respectively. Daconil was second most Effect of pH on colony growth of Ceratocystis effective followed by Alliete and Dithane M-45 at all manginecans: Colony growth of C. manginecans concentrations. The results revealed that growth of C. was observed on different pH levels with respect to manginecans decreased as the concentrations of time. The results showed in Table 4, pH levels effect fungicides increased. All the concentrations reduced on colony growth significantly. After the statistical the growth of C. manginecans, but at 100 ppm of analysis, results revealed, colony growth of 6.33 cm Carbendazim gave the maximum control over was maximum at pH level 7 followed by pH level 8, mycelial growth of C. manginecans after 144 hours 6, 5, 4 and 3. This indicated that pH levels 7 and 8 of incubation at 25oC temperature. were best for the colony growth of C. manginecans These results are in agreement with the finding of but the pH levels 3, 4, 5 and 6 reduced their growth Vijaya et al. (2007). They evaluated five systemic rate respectively. Statistically there was non and five non-systemic fungicides at two significant relationship of colony growth with 7 and 8 concentrations in vitro against C. paradoxa causing pH levels. sett rot of sugar cane. Among the systemic Effect of pH on colony growth with respect to time fungicides, Carbendazim and Propiconazole were also showed statistically significant relationship. most effective in complete inhibition of the pathogen Results indicated with increase in time colony growth at both the concentrations (0.05 and 0.1%), while increased. Colony growth was maximum after 168 Benomyl and Hexaconazole only at 0.1 per cent hour, 144, 120, 96, 72, 48 and 24 hour respectively. concentration. Among the non-systemic fungicides Statistically, colony growth after 24 hour 0.80 cm tested, Thiram was found best followed by Captan at was significantly lower and 3.77 cm was the both the concentrations (0.1 and 0.2%), whereas maximum after 168 hour. Mancozeb and Copper Oxychloride were least The best suitable pH for the growth of C. effective. Systemic fungicides were found more manginecans is 7. These results are in agreement to effective in controlling the pathogen growth than that Yadahalli et al. (2007), as they determine C. of non-systemic fungicides. paradoxa was best good at pH of 6-7 with an

Table-5: In-vitro Effect of Fungicides against Ceratocystis manginecans after 168 hours Fungicides Concentration (ppm) Means 20 60 100 Carbendazim 3.38 h 2.46 j 1.40 k 2.41 f Daconil 4.86 e 3.90 g 2.93 i 3.90 e Alliete 5.43 d 4.45 f 3.55 h 4.47 d Dithane M-45 5.90 c 4.95 e 3.90 g 4.91 c Agrofit 6.23 b 5.25 d 4.23 f 5.23 b Control 7.40 a 7.40 a 7.40 a 7.40 a Means 5.53 a 4.73 b 3.90 c Alpha= 0.05, LSD value (F) = 0.1318, LSD value (C) = 0.08627, LSD value (F x C) = 0.2283

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Conclusively, all the four tehsils of district Muzaffar Kazmi, M. R., F. S. Fateh., K. Majeed., A. M. garh hit by MSDS, but the worst hit tehsil was Kot Kashkhely., I. Hussain., I. Ahmad and A. Addu. Among the varieties Chaunsa was severely Jabeen. 2005. Incidence and etiology of mango affected by the disease. Among the isolated fungi C. sudden death phenomenon. . Pak. J. Bot., 17(2): manginecans was isolated in abundant with the value 154-158. of 41.50% followed by B. theobromae. Cabendazim Mahmood, A. and M. A. Gill. 2002. Quick decline of was found most effective during in vitro management mango and in vitro response of fungicides of C. manginecans. against the disease. Int. J. Agri. Biol. 4(1): 39- 40. REFERENCES Mahmood. A., S. N. Khan., S. Ali and S. Alam. 2007. Survey for the prevalence of sudden death of Al-Adawi, A. O., M. L. Deadman., A. K. Al- mango in different districts of Punjab. Pak. J. Rawahi., Y. M. Al-Maqbali., A. A. Al-Jahwari., Phytopathol. 19 (1): 81-85. B. A. Al-Saadi., I. S. Al-Amri and M. J. Malik, M. T., S. M. Khan., A. A. Dasti and M. R. Wingsfield. 2006. Etiology and causal agents of Kazmi. 2005. First record of Ceratocystis mango sudden decline disease in the Sultanate manginecans causal organism of mango sudden of Oman. European J. Pl. Pathol. 116 (4): 247- death in Pakistan. Pak. J. Phytopathol. 17 (2): 254. 187-191. Al-Adawi, A. O., M. L. Deadman., A. K. Al Rawahi., Parkash, O. 1996. Principal diseases of mango causes A. J. Khan and Y. M. Al-Maqbali. 2003. and control. Advances in disease of fruit crops Diplodia theobromeae associated with sudden in India. Kalyani Publisheis. Ludhiana. : 19 1- death of mango in the “Sultanate of Oman.” Pl. 256. pathol. 52: 419-419. Parkash, O. 1998. Diseases of Mango. In: Sirvastava, Anonymous, 2006. Pakistan Statistical Year Book. R.P. ed. Mango cultivation. Int. Book Ministry of Food and Agriculture Economics Publishing Co., Plant diseases attributed to wing, Govt. of Pakistan, Islamabad. Botryodiplodia theobromeae. Cramer, Anonymous, 2010. Agriculture Statistics of Pakistan. Vaduz,Lichtenstein 123p. Government of Pakistan. Ministry of food, Ragab, M. M., K. A. Sabeth and N. A. Dawood. agriculture and livestock. Economic, trade and 1971. Botryodiplodia theobromae path. The investment wing. Islamabad, Pakistan. cause of the fruit rot and die hack of Mango in Anonymous, 2004, Database/production. crops. A.R.E. Agricultural Research Review. Cairo. Primary and Domain SUA. Room: Food and 49: 81-97. Agriculture Organization of United Nations. Rossetto, C. J. and I. J. A. Rinero. 1990. Mango wilt Dhingra, O. D. and J. B. Sinclair. 1985. Basic plant XII. Recommendation for control. Revista de pathology methods. Boca Raton, FL, USA, CRS Agriculture (Piracicaba), 65(2): 173-180. Press. Steel, R. G. D., J. H. Torrie and D. A. Dickey. 1996. Ellis, M. B. 1971. More Dematiaceous Principle and procedure of statistics. A Hypomycetes. CMI, Kew, Survey, England. Biometrical approach 3rd Ed McGraw Hill New 507pp. York. US.A. FAO, 2004 http:// apps.fao.org./lim Sutton, B. C. 1980. The Coelomycetes. Fungi 500/nphwrap.P/FAOSTAT Database/ imperfecti with pycnidia acervuli and stromata. Production. Crops. Primary & Domain= SUA. CMI. Kew. Surrey. England. 696 pp. Rome: Food and Agricultural organization of Vijaya, H. K; S. kulkarni and Y. Hedge.2007. the United Nations. Chemical control of sett rots of Sugarcane Fateh, F. S., M. R. Kazmi., I. Ahmad, and M. Ashraf. caused by Ceratocystis paradoxae. Karnatka J of 2006. Ceratocystis fimbriata isolated from Agri. Sci. 20 (1): 62-64. vascular bundles of declining mango trees in Yadahalli, KB; S.S. Adiver and S. Kulkarni. 2007. Sindh, Pakistan. Pak. J. Bot. 38(4): 1257-1259. Effect of pH, temperature and Relative Fernald, M. L. 1950. Grays. Manual Botany 8th Humuidity on growth and development of Edition book Co. New York. Ceratocystis paradoxae. A causal organism of pineapple disease of sugarcane. Karnatka J of Agri. Sci. 20 (1): 159-162.

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