EMERGING RESISTANCE AGAINST DIFFERENT FUNGICIDES IN LASIODIPLODIA THEOBROMAE AS THE CAUSE OF MANGO DIEBACK IN PAKISTAN

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1 Arch. Biol. Sci., Belgrade, 67 (1), , 2015 DOI: /ABS R EMERGING RESISTANCE AGAINST DIFFERENT FUNGICIDES IN LASIODIPLODIA THEOBROMAE AS THE CAUSE OF MANGO DIEBACK IN PAKISTAN Ateeq ur Rehman 1, Ummad ud Din Umar 1, Syed Atif Hasan Naqvi 1,*, Munaza Rana Latif 1, Sajid Aleem Khan 2, Muhammad Tariq Malik 3 and Shoaib Freed 4 1 Department of Plant Pathology, Bahauddin Zakariya University, Multan, Pakistan 2 Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan 3 Mango Research Institute, Multan, Pakistan 4 Department of Entomology, Bahauddin Zakariya University, Multan, Pakistan * Corresponding author: atifhasanshah@hotmail.com Abstract Dieback of mango caused by Lasiodiplodia theobromae is among several diseases responsible for low crop production in Pakistan. To further complicate the issue, resistance in L. theobromae is emerging against different fungicides. L. theobromae was isolated from diseased samples of mango plants collected from various orchards in the Multan District. The efficacy of different fungicides viz. copper oxychloride, diethofencarb, pyrachlostrobin, carbendazim, difenoconazole, mancozeb, and thiophanate-methyl was evaluated in vitro using a poison food technique. Thiophanate-methyl at all concentrations was found to be the most effective among five systemic fungicides against L. theobromae, followed by carbendazim, difenoconazole and diethofencarb. The fungicides, i.e., thiophanate-methyl, difenoconazole, carbendazim and diethofencarb showed maximum efficacy with increasing concentration. The isolates of L. theobromae showed some resistance development against the tested fungicides when compared with previous work. These investigations provide new information about chemical selection for the control of holistic disease in mango growing zones of Pakistan. Key words: Mangifera indica; Lasiodiplodia theobromae; fungicides; resistance Received September 4, 2014; Revised October 13, 2014; Accepted October 20, 2014 INTRODUCTION Mango (Mangifera indica L.) is one of the most important fruit in Punjab and Sindh Provinces of Pakistan with an average yield of tons hectare (Anonymous, 2011). Various factors including diseases and insects are responsible for its low productivity (Shahbaz et al., 2009). Malik et al. (2005) described the enigma of dieback as a serious disease for mango growers. The onset of dieback becomes clear by discoloration and darkening of twigs, oozing of gum, wilting of leaves, dieback, browning of vascular bundles and death of the entire plant (Narasimhudu and Reddy, 1992; Khanzada et al., 2004a). Previous research has proved Lasiodiplodia theobromae (Pat.) Griff. and Maubl. (syn. Botryodiplodia theobromae Pat.) as the cause of dieback of mango (Sutton, 1980; Khanzada et al., 2004b). 241

2 242 Ateeq ur Rehman et al. Similar associations between L. theobromae and mango decline have been observed by quite a number of researchers (Das Gupta and Zachariah, 1945; McSorely et al., 1981; Narasimhudu and Reddy, 1992; Sharma, 1993; Schaffer, 1994; Simone, 1999; Gonztilez et al., 1999). The fungus causes similar symptoms in other economic plants like leaf necrosis and stem canker on Protea cynaroides (Deman, 2002; Denman et al., 2003), root rot of Brachychiton populneus seedlings (Sandlin and Ferrin, 1992), Anacardium occidentale gummosis in Brazil (Cedeno, 1992; Cardoso et al., 1998; Cardoso et al., 2004), gummosis of Prunus armeniaca and Prunus persica trees (Li et al., 1995), collar rot of Arachis hypogaea (Phipps and Porter, 1998), canker in Thuja occidentalis (Sandrock et al., 1999), dieback of Citrus japonica (Ko et al., 2004), storage rot of Colocasia esculenta, crown rot of Musa acuminata and dieback in Citrus limon (Alam and Nahar, 1990; Mortuza and Ilag, 1999; Anthony et al., 2004; Alam et al., 2001), while research has reported that in Pakistan, L. theobromae is associated with more than 50 plant species (Ahmed et al., 1997). Chemical fungicides have turned out to be a very significant source for controlling fungal pathogens (Da Silva et al., 2012), restricting or preventing their growth (Malik et al., 1997, 2003; Karaoglanidids et al., 2003). Keeping in mind the above-mentioned status of mango parasites and taking into consideration the magnitude of dieback of mango and its drastic losses, the present study was conducted with the aim to evaluate the most efficient fungicide against L. theobromae. MATERIALS AND METHODS Sample collection: Three hundred diseased samples from different orchards in the Multan region were collected to determine the infestation of various fungi associated with dieback of mango. The samples were brought in plastic bags to the Plant Pathology Laboratory of the Department of Plant Pathology, Bahauddin Zakariya University, Multan, within 4-7 h after collection for further processing. Isolation of pathogen parts (3-4 mm) of diseased tissues from the collected samples were excised, disinfected in 1% sodium hypochlorite solution and washed twice in sterilized distilled water. They were then dried on blotting paper and placed on sterilized Petri dishes lined with potato dextrose agar (PDA) (Bio Basic Inc.) at 25 C with illumination of 600 Lux (Ploetz and Gregory, 1993). After 7-8 days of incubation, the Petri dishes were observed for the identification of different fungi based on the specific characteristics of the particular fungus (Nelson et al., 1983). Poison food technique Using the poison food technique, (Borum and Sinclair, 1968), fungicides at different concentrations of 25, 50, 75 and 100 µg ml 1 were mixed in PDA and poured onto Petri dishes; the inoculation was performed using small pieces of 72-h-old culture of L. theobromae. The inoculated Petri dishes were randomized in four replicates, incubated at 25 C until the fungus acquired a 9-cm radial mycelial growth in control treatment. Petri dishes with only PDA medium were reserved as control. The efficacy of the various fungicides was examined against the radial mycelial growth of the pathogen, and percent decrease over control of fungicides was calculated using the following formula (Sahi et al., 2012): PD = (X - Y):X 100, where, PD is the percent decrease; X is growth of L. theobromae in the control plate; Y is growth of L. theobromae in the fungicide-treated plate. Statistical analysis The collected data of radial mycelial growth of fungus was subjected to analysis of variance (ANOVA) using (SAS 2002). Treatment means were compared using Fisher s least significant differences (LSD) at (P = 0.05). Inhibition zone of mycelial growth for each concentration was calculated as a percent decrease with respect to control treatment.

3 FUNGICIDAL RESISTANCE IN LASIODIPLODIA THEOBROMAE 243 Table 1. List of fungicides tested against in vitro mycelial growth of Lasiodiplodia theobromae. Trade Name Active ingredient Action a.i* (%) Formulation Toxicity class Manufacturer Cobox Copper oxychloride C* 35 WP* III Syngenta Precure combi Diethofencarb + Thiophanate-methyl S* 65 WP III FMC Cabrio Top Pyrachlostrobin+ Metiram S 60 WP III Arysta Life Sciences Score Difenoconazole S 250 EC* III Syngenta Bavistin Carbendazim S 50 WP III Syngenta Dithane M-45 Mancozeb C 80 WP IV Arysta Life Sciences Topsin-M Thiophanate-methyl S 70 WP III/IV Arysta Life Sciences *a.i: active ingredient, *WP: wettable powder, *EC: emulsifiable concentrate,* C: contact, *S: systemic, Table 2. Fungi associated with different parts of diseased mango plants. S. No. Nature of sample Fungi associated Infection frequency % 1. Roots 2. Leaves/ flowers 3. Twigs/ branches Fusarium sp. L. theobromae Rhizoctonia sp. Alternaria sp. Fusarium sp. L. theobromae Fusarium sp RESULTS Association of fungi with diseased mango plants A number of plant pathogenic fungi including Alternaria sp., Fusarium sp., L. theobromae and Rhizoctonia sp. were isolated from different parts of mango plant. It was observed that the most universally prevailing Alternaria sp. was isolated from infected flowers and leaves of diseased plants. The infestation of L. theobromae twigs/branches and roots was observed as maximum colonization. Fusarium spp. were isolated on leaves, twigs and roots, whereas Rhizoctonia sp. was isolated only from root samples. Maximum infection frequencies and 75% percent were observed on roots and twigs/branches, respectively, by L. theobromae. On the other hand, the infection frequency of Fusarium sp. was 41.66% from roots and 25% from leaves and twigs samples, while Rhizoctonia sp. was only isolated from roots samples with infection of 16.66% (Table 2). Efficacy of fungicides on Lasiodiplodia theobromae The results of current study suggested that among the five systemic fungicides evaluated against L. theobromae, thiophanate-methyl was significantly superior to all the other fungicides with inhibition zones of 0.15, 0.30, 0.43 and 1.16 cm at 100, 75, 50 and 25 µg ml 1 concentrations, respectively. The results of difenoconazole were at par with carbendazim with the inhibition zone of mycelial growth of L. theobromae i.e., 0.15, 0.33, 0.42 and 1.65 cm at different concentrations, while in the case of carbendazim these were 0.33, 0.47, 0.58 and 1.53 cm at similar concentrations. In the case of diethofencarb, there was a significant decrease in the colony diameter of L. theobromae as compared to control with the inhibition zone of mycelial growth 0.15, 0.36, 1.28, and 1.63 cm at the tested doses. Pyrachlostrobin was found to be the least effective among the systemic fungicides while between the two contact fungicides, copper oxychloride showed inhibition to some extent at 100 and 75

4 244 Ateeq ur Rehman et al. Table 3. Effect of different fungicides on mycelial growth of Lasiodiplodia theobromae Mycelial growth (cm ± S.E*) Concentration (µg ml 1 *) Copper oxychloride Diethofencarb + Thiophanate methyl Pyrachlostrobin + Metiram Carbendazim Difenoconazole Mancozeb Thiophanate-methyl 25 µg ml ± 0.09 b 1.28 ± 0.19 b 8.85 ± 0.09 a 1.53 ± 0.41 b 1.65 ± 0.43 b 7.13 ± 0.24 b 1.16 ± 0.23 b 50 µg ml ± 0.12 c 0.36 ± 0.07 c 8.25 ± 0.00 b 0.58 ± 0.05 c 0.42 ± 0.12 c 7.13 ± 0.38 b 0.43 ± 0.09 c 75 µg ml ± 0.10 d 1.63 ± 0.55 b 8.44 ± 0.19 b 0.33 ± 0.08 c 0.33 ± 0.06 c 7.90 ± 0.50 b 0.30 ± 0.07 c 100 µg ml ± 0.06 e 0.15 ± 0.02 c 9.00 ± 0.00 a 0.47 ± 0.34 c 0.15 ± 0.02 c 8.95 ± 0.03 a 0.15 ± 0.02 c Control 8.95 ± 0.02 a 9.00 ± 0.00 a 9.00 ± 0.0 a 8.57 ± 0.25 a 8.85 ± 0.12 a 9.23 ± 0.30 a 8.88 ± 0.13 a LSD* Means followed by the same letters in each column are not statistically different (*P < 0.05); LSD*= Least significant difference; S.E*= Standard error; µg ml 1 * Table 4. Average decrease in colony growth of Lasiodiplodia theobromae on PDA mixed with different fungicides. Average decrease in colony growth (%) (cm ± S.E*) Concentration (µg ml 1 *) Copper oxychloride Diethofencarb + Thiophanate methyl Pyrachlostrobin + Metiram Carbendazim Difenoconazole Mancozeb Thiophanate-methyl 25 µg ml ± 1.05 d ± 3.20 bc 1.72 ± 1.03 b ± 4.54 b ± 4.83 b ± 2.67 a ± 2.52 b 50 µg ml ± 1.37 c ± 0.80 ab 6.94 ± 0.80 a ± 0.56 a ± 1.33 a ± 4.17 a ± 0.97 a 75 µg ml ± 1.14 b ± 6.16 c 6.25 ± 2.08 a ± 0.86 a ± 0.73 a ± 5.61 ab ± 0.76 a 100 µg ml ± 0.70 a ± 0.23 a 0.00 ± 0.00 b ± 3.80 a ± 0.23 a 1.67 ± 1.32 bc ± 0.23 a Control 0.53 ± 0.20 e 0.00 ± 0.00 d 0.00 ± 0.00 b 1.67 ± 1.32 c 1.67 ± 1.32 c 1.39 ± 1.39 c 1.39 ± 1.39 c LSD* Means followed by the same letter in each column are not statistically different (*P < 0.05); LSD*= Least significant difference; S.E*= Standard error ; µg ml 1 *

5 FUNGICIDAL RESISTANCE IN LASIODIPLODIA THEOBROMAE 245 µg ml 1 with the inhibition of mycelial growth of L. theobromae to 1.29 and 3.29 cm, but statistically no significant difference was found. Mancozeb was the least effective among all the fungicides tested on L. theobromae (Table 3). The percent decrease in colony growth of L. theobromae over control was calculated at all concentrations of the fungicides. Thiophanate-methyl was found to be the best fungicide, showing significant decreases of 98.30, 96.69, and 87.08% as compared to the control, at 100, 75, 50 and 25 µg ml 1, respectively, followed by 98.33, 96.52, and 81.66% decreases as compared to the control. This was statistically at par with carbendazim with 96.33, 4.72, and 83.05% decreases at similar concentrations (Table 4). DISCUSSION Dieback is an important disease of mango fruit crop. The infectious fungus L. theobromae was isolated from leaves and twigs/branches, and different fungicides were evaluated for its control. In the current research, primarily we found the association of four fungi, i.e. Alternaria sp., Fusarium sp., L. theobromae and Rhizoctonia sp. with the dieback-affected plants. Among the isolated fungi, L. theobromae is the cause of mango decline. A high percentage of infestation of L. theobromae viz and 51% from roots and twigs was also reported by Banik et al. (1998). The infestation of L. theobromae in roots and twigs/branches of affected mango plants was observed, which might affect the xylem tissues of the stem and disturb the translocation process in the plant (Mahmood et al., 2002, 2007). In our experiments, fungicides viz. diethofencarb in combination with thiophanate-methyl, carbendazim, difenoconazole and thiophanate-methyl showed significantly promising results with 94-98% reduction in the colony growth of L. theobromae with respect to control. Our findings coincide with the results of Sahi et al. (2012), in which thiophanate-methyl was proved the best chemical fungicide in controlling the growth of L. theobromae. The outcome of current research also supports the statement of Rehman et al. (2011) that difenoconazole showed to be an excellent fungicide in controlling the growth of L. theobromae. Difenoconazole and thiophanate-methyl belong to the triazole (demethylation-inhibiting DMI) and benzimidazole (methyl benzimidazole carbamates MBC) group of fungicides, respectively, which are being used against L. theobromae. Shelar et al. (1997) observed the in vitro efficacy of several fungicides viz. carbendazim, benomyl, mancozeb, copper oxychloride and thiophanate-methyl against L. theobromae and concluded that 100 µg ml 1 carbendazim, 25 µg ml 1 mancozeb and 100 µg ml 1 thiophanate-methyl showed the best results against L. theobromae. Carbendazim at the concentration of 40 µg ml 1 totally suppressed the growth of L. theobromae followed by thiophanate-methyl at 45 µg ml 1 (Banik et al., 1998). Similarly, Khanzada et al. (2005) and Jamadar and Lingaraju (2011) efficiently controlled mycelial growth of L. theobromae by carbendazim and thiophanate-methyl at 50 µg ml 1 concentration. Mahmood et al. (2002) also reported the suppression of colony growth of L. theobromae by thiophanate-methyl at 50 µg ml 1, while copper oxychloride failed to inhibit growth. The evaluation of various fungicides against L. theobromae proved carbendazim to be the most effective at 100 µg ml 1, while thiophanate-methyl completely inhibited growth of the fungus at 100 µg ml 1 concentration (Li et al., 1995). Mahmood et al. (2007) appraised diethofencarb to completely inhibit the mycelial growth of L. theobromae at 100 µg ml 1, whereas thiophanate-methyl produced the same result at 25 µg ml 1. Shahbaz et al. (2009) assessed some fungicides viz. thiophanate-methyl, carbendazim, diethofencarb and copper oxychloride at 50 and 100 µg ml 1 and concluded that thiophanate-methyl, carbendazim and diethofencarb showed 100% inhibition over control, while copper oxychloride revealed only 20.20% inhibition. Similar findings were recorded by Sharma (1995) and Johnson et al. (1990) where carbendazim and thiophanate-methyl completely suppressed the growth of L. theobromae at 50 µg ml 1. In our exper-

6 246 Ateeq ur Rehman et al. iment, the fungicides diethofencarb, carbendazim, difenoconazole and thiophanate-methyl, although performing well could not give 100% inhibition of the pathogen, which shows some variation from the previous findings; mancozeb completely failed to inhibit fungal growth at all concentrations. With reference to the above review, it is clear that thiophanate-methyl, carbendazim and difenoconazole showed 100% inhibition of the mycelial growth of L. theobromae in vitro at different concentrations from 25 to 100 µg ml 1, whereas in our experiment all the fungicides failed to show 100% inhibition even at µg ml 1. Keeping in sight the previous research, our results are alarming, as it seems that the sensitivity of L. theobromae is decreasing against the most commonly used fungicides. Similarly, Banik et al. (1998) and Sales (2009) showed difenoconazole to provide 100% inhibition of the pathogen in vitro at 40 and 50 µg ml 1, while our results showed difenoconazole to inhibit 98% over control at 100 µg ml 1. As for as carbendazim and thiophanate-methyl are concerned, our results show some variation to the findings of Shelar et al. (1997), Banik et al. (1998), Khanzada et al. (2005), Shahbaz et al. (2009) and Sharma (1995), in which 100% inhibition of L. theobromae was recorded at 100, 40 and 50 µg ml 1, which is not in line with our result that no concentration showed 100% inhibition even at 100 and 50 µg ml 1. The reason might be that the L. theobromae isolates from the different mango orchards of the Multan region were previously exposed to many chemical fungicides that proved to be resistant to some extent due to the frequent use of fungicides in this particular region. Fungicidal resistance is a stable, inheritable adjustment by the fungus to fungicides that results in reduced sensitivity or increased resistance (Ma and Michailides, 2005). The prolonged use of DMIs + MBCs reduced performance due to high infection pressure, which is associated with the resistance of L. theobromae. This suggests that due to reduced fitness of the resistant isolates or mutants, the resistance-associated problems occur if prolonged and selective use of a fungicide continues under high disease inoculum pressure (Bever et al., 1982); resistance development is also due to the use of single mode of action fungicides (Bernardo and Rene, 2012). The effectiveness of carbendazim may be lost where heavy selection pressure is created by fungicides in favor of isolates that are resistant to many other chemicals (Pappas, 1997). The increased possible application of thiophanate-methyl (MBCs) in the mango groves has greatly increased the probability of selecting resistant isolates and strains of mango pathogens. Keeping in mind the facts, the resistance development in L. theobromae in mango groves of Pakistan is not surprising. In our study, multiple fungicidal resistances reflect that the intensive use of selective fungicides is a serious threat for the mango crop in Pakistan. Cross-resistance may occur and it is common in fungal species (Pereira et al., 2012). It is also possible that resistance development to one fungicide could accelerate the development of resistance to another unrelated fungicide (Bernardo and Rene, 2012). However the occurrence of point mutation cannot be ruled out, and more research is needed on this issue. The data indicate that fungicide chemistries are slowly but surely losing their effectiveness for controlling dieback disease of mango due to reduced sensitivity in L. theobromae. Donald and Spadling (1982) stated that the problem of resistance development to fungicides in L. theobromae could be solved by formulating more effective fungicide chemistries so that their use can be altered to avoid selection of resistant fungal isolates for a longer period. More work in the future will be required for sampling of dieback of affected mango plants at an increased number of sites in Pakistan, and a data set of many years is needed to estimate accurately the current frequency and potential of fungicide resistance in Pakistan. This study demonstrates that the mango growers must be conscious of the significance of rotation tactics to discourage the selection of single-chemistry fungicides, which are presently in development

7 FUNGICIDAL RESISTANCE IN LASIODIPLODIA THEOBROMAE 247 to avoid the evolution of resistance in pathogens. To the best of our knowledge, this is the first report of L. theobromae isolates having instantaneous resistance to most commonly used fungicides from mango groves, reinforcing the knowledge that fungicide resistance is a serious problem of mango production in Pakistan. Our findings question the sustainability of the present management strategy against L. theobromae, which depends exclusively on fungicides and suggests the need to incorporate cultural and biological control strategies to achieve a satisfactory control. Acknowledgments - We were very fortunate to have the opportunity of working at the Mango Research Institute (MRI) Multan, in the Agriculture Sector Linkage Program (ASLP). Our thanks to Mr. Mwema Felix, Research Scientist, Tropical Pesticides Research Institute, Tanzania for critically reviewing the manuscript. Author s contribution The research work was conducted under the supervision of Dr. Ateeq ur Rehman and Dr. Ummad ud Din Umer by Syed Atif Hasan Naqvi and Munaza Rana Latif. Statistical analysis was performed by Syed Atif Hasan Naqvi. Technical assistance was provided by Shoaib Fareed and Sajid Aleem Khan during manuscript compilation. Conflict of interest disclosure There is no conflict of interest among the authors about this manuscript regarding any aspect of the research conducted and publishing. REFERENCES Akhar, K. P. (2000): Fresh potato extract the best source for the growth of Colletotrichum gloeosporioides causing anthracnose of mango and Fusarium subglutinans isolated from malformed inflorescence of mango. Pak J. Phytopathol. 12, Ahmed, S., Iqbal, S. H. and A. N. Khalid. (1997): Fungi of Pakistan. Sultan Ahmed Mycol. Soc. Pakistan, Deptt. Bot. Uni. Punjab, Lahore Pakistan. Alam, M. S., Begum, M. F., Sarkar, M. A and M. R. Islam. (2001): Effect of temperature, light and media on growth, sporulation, formation of pigments and pycnidia of Botryodiplodia theobromae. Pak. J. Bio. Sci. 10, Al Adawi, A.O., Deadman, M. L., Al Rawahi, A. K., Khan, A. J. and Y. M. Al Maqbali. (2003): Diplodia theobromae associated with sudden decline of mango in the Sultanate of Oman. Pl. Pathol. 52, Alam, M.S. and S. Nahar. (1990): Post fungal infection changes in ascorbic acid content of mango fruit. Bangla. J. Bot. 19, Alba, V. S. P., Ricardo, B. M., Sami, J. M., Marcelo, B. S. and P. S. C. Marcos. (2012): Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides. Eur. J. Pl. Pathol. 132, Anthony, S. K., Abeywickrama, R., Dayananda, S. and A. L. Wijeratnam. (2004): Fungal pathogens associated with Banana fruit in Sri Lanka and their treatment with essential oils. Mycopathol. 157, Anonymous. (2011): Agricultural statistics of Pakistan. Ministry of Food, Agric. & Crop. Govt. Pak, Food Agric. Div. Planning Units Islamabad. PP 89. Banik, A. K, Kaiser, S. A. K. and R. S. Dhua. (1998): Evaluation of some systemic and non-systemic fungicides against Botryodiplodia theobromae, the cause of dieback disease of mango (Mangifera indica L.). J. Soils Crops. 8, Bernardo, A. L. and T. Rene. (2012): Prevalence of isolates of Botrytis cinerea resistant to multiple fungicides in Chilean vineyard. Crop Proct. 40, Bever, R.E. and R. J. W. Byrde. (1982): Resistance to dicarboxymide fungicides. Page : Fungicide resistance in crop protection. Dekker, J., Georgoplous, S. G., (Eds.) Centre for agricultural publication and documentation. Weginengen Netherlands. PP 265. Borum, D. E. and J. B. Sinclair. (1968): Evidence for systemic fungicides protection against R. solani with vitavax in cotton seedlings. Phytopathol. 58, Cardoso, J. E. and F. C. O. Freire. (1998): Disseminação e controle da resinose em troncos de cajueiro decepados para substituição de copa. Fitopatologia Brasileira 23, Cardoso, J. E., Santos, A. A., Rossetti, A. G. and J. C. Vidal. (2004): Relationship between incidence and severity of cashew gummosis in semiarid north-eastern Brazil. Pl. Pathol. 53, Cedeno, L. and P. Palaces. (1992): Identification of Botryodiplodia theobromae as the cause of lesions and gummosis on citrus. Phytopathol Venz. 5,

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