INTEGRATED CONTROL OF ALTERNARIA SOLANI WITH TRICHODERMA SPP. AND FUNGICIDES UNDER IN VITRO CONDITIONS

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1 Sarhad J. Agric. Vol. 26, No. 4, INTEGRATED CONTROL OF ALTERNARIA SOLANI WITH TRICHODERMA SPP. AND FUNGICIDES UNDER IN VITRO CONDITIONS FAZLI RAZIQ and SANA ISHTIAQ Department of Plant Pathology, KP, Agricultural University, Peshawar-Pakistan ABSTRACT The antagonistic efficiency of Trichoderma spp in integration with three fungicides (Captan, Cobox and Dithane M-45) against Alternaria solani was studied in an in vitro experiment conducted at the Department of Plant Pathology, KP Agricultural University, Peshawar, during Six isolates of Trichoderma were used individually and in combination with the fungicides by inoculating each of the antagonist at four sites around the pathogen on potato dextrose agar medium amended with the of 200 mg l -1. In the control treatment, neither an antagonist nor a fungicide was applied. Each treatment was replicated five times in a completely randomized factorial design. After 10 and 20 days of incubation, data were taken by measuring the mean colony diameters of A. solani. The results showed that main effects of antagonists and fungicides and the interaction effect between antagonists and fungicides were significant (P<0.05). The greatest reduction in the colony diameter of A. solani was caused when Trichoderma harzianum (Th12) was integrated with Dithane M-45. These results show that integration of Trichoderma with the lower doses of the fungicides may offer a promising control of A. solani. This will reduce reliance on the indiscriminate use of fungicides, leading to more cost-effective and environmentfriendly control of the disease. Key Words: Alternaria solani, integrated control, Trichoderma spp. Citation: Raziq., F and S. Ishtiaq Integrated control of alternaria solani with trichoderma spp and fungicides under in vitro conditions. Sarhad J. Agric 26(4): INTRODUCTION The early blight disease is caused by the fungus Alternaria solani (Chester, 1950) and it is of common occurrence wherever potatoes and tomatoes are grown in the world (Singh, 1983). The fungus is pathogenic on solanaceous crops and has also been reported on other hosts such as Brassica sp. (Hooker, 1986). A. solani is an imperfect fungus with no sexual stage known so far. The mycelium consists of septate, branched, light brown hyphae which become darker with age. The intercalary cells contain 0-14 nuclei while the terminal cells have nuclei. The hyphae in the host are at first intercalary, later penetrating into the cells of the invaded tissues. Conidiophores emerge through the stomata from the centers of the spots. They are relatively shorter, x 9 µm and dark colored. Conidia are x µm in size, beaked, muriform, dark-colored and borne singly. However, in culture they form short chains. They develop from a bud formed by the apical cell of the conidiophore. Five to ten transverse septa and few longitudinal septa are present. In moist weather, these conidia germinate readily and 5-10 germ tubes arise from a single conidium (Singh, 1983). Both on potato and tomato, early blight takes the form of brown leaf spots marked with concentric rings to give a target effect. These spots enlarge slowly and may eventually destroy the leaves. The fungus causes stem canker or collar rot of young seedlings, sunken spots or cankers on older stems, blossom drop and loss of young fruits and dark leathery fruit spots, usually about the point of attachment of the stem on tomato and circular, decayed lesions may form on the tubers, permitting the entrance of decay organisms on potato. In severe attacks, lesions appear on upper stems and petioles (Chester, 1950). To minimize crop losses of potato, tomato and other economically important crops from A. solani and improve the quality, it is important to develop management strategies to reduce fungicide use in these crops and adopt integrated disease management. Ideally, resistant cultivars should be planted, but currently there are no commercial cultivars of both the vegetables resistant to the disease. Physical methods are not highly effective in controlling leaf diseases under field conditions, as they require large investments and are difficult to use in large acreages of tomatoes and potatoes (Batista et al. 2006).

2 Fazli Raziq and Sana Ishtiaq et al. Integrated control of alternaria solani with trichoderma spp 614 The best method to control diseases is through integrated pest management wherein biological control component would be significant. Trichoderma, which is known to play an important role in the biological control of soil-borne diseases, has been recorded to inhibit the leaf pathogens also. Competition for nutrients and space, the production of antibiotics and hyperparasitism all play important roles in the antagonism of pathogens by Trichoderma (Mukerji and Garg, 1988). The present investigation was carried out to evaluate a number of Trichoderma species and isolates, alone and in integration with different fungicides, viz captan, mancozeb (Dithane M- 45) and copper oxychloride (Cobox), for suppressing growth of A. solani. MATERIALS AND METHODS Isolation of Alternaria solani Early blight infected potato leaves were collected from Pir Piai, District Nowshera, in April The infected leaves were cut into small pieces (1cm 2 ), surface sterilized with mercuric chloride (0.1%) for seconds, rinsed with three changes of sterile distilled water to remove the disinfectant and blotted dry. The sterilized pieces were plated (4 pieces/dish) on potato dextrose agar (PDA) medium in Petri dishes under aseptic conditions and incubated at 25 o C for 2 weeks. For obtaining sufficient quantity of inoculum, pure cultures were obtained by subculturing. For this purpose, small bits of the fungus were taken at the tip of a sterilized needle and transferred aseptically to the centre of fresh PDA medium in Petri dishes. The dishes were incubated for 2 weeks at 25 o C in the dark. Isolation of local Trichoderma strains Two isolates of Trichoderma, T. harzianum from Swat and one unidentified Trichoderma isolate from Thana were obtained from the culture collection of Department of Plant Pathology, KP Agricultural University, Peshawar. Originally, these were isolated by sprinkling the soil ( g) on the PDA medium in Petri dishes under aseptic conditions and incubated at 25 o C for 2 weeks. Pure cultures were obtained by subculturing and used for the studies. Revival of exotic isolates of Trichoderma Four isolates of Trichoderma, viz T. harzianum isolate Th12, T. hamatum isolate Tham and Trichoderma isolates TRC14 and TRN3, acquired from the University of Reading (U.K.), were revived from silica gel on PDA medium in Petri dishes under aseptic conditions and incubated at 25 o C for 2 weeks, during which they grew fully on the medium. Treatments The above six isolates were tested in vitro against A. solani at the laboratory of Department of Plant Pathology, KP Agricultural University Peshawar, in A. solani was inoculated at the centre of the PDA medium in Petri dishes as 1cm diameter inoculum plug was cut out from 2 weeks old culture with sterile scalpel. Inoculum plugs of the same size were then cut out from 2 weeks old cultures of Trichoderma isolates and applied at four sites around A. solani. In the control treatment, A. solani was allowed to grow alone. Each of the Trichoderma isolates and control were tested on the PDA medium to which Dithane M-45, Captan or Cobox were added at the fixed concentration of 200 mg l -1 just before pouring in Petri dishes. Each of the above treatments was replicated five times in a factorial arrangement using the completely randomized (CR) design. The Petri dishes were sealed with parafilm and incubated at 25 o C in the dark for an extended period of time. The whole work was performed aseptically in the laminar flow unit to avoid contamination. Measurements and observations The colony diameters of A. solani were measured after 10 and 20 days of incubation to evaluate the antagonistic efficiency of Trichoderma isolates and effect of the fungicides on the growth of the pathogen. To minimize variability in the growth diameters of A. solani, measurements were taken along two perpendicular lines on the dishes. The growth of the antagonists on or around the pathogen was also recorded.

3 Sarhad J. Agric. Vol. 26, No. 4, Statistical Analyses The data were subjected to ANOVA test, using MSTATC. Means for the two factors (antagonists and fungicides) as well as the interaction were separated by LSD test when ANOVA revealed P<0.05. RESULTS AND DISCUSSION All the six isolates of Trichoderma significantly (P<0.05) reduced the colony diameters of A. solani as compared to the control (Tables I & II). T. harzianum isolate Th12 was found to be the most effective in reducing the colony diameters of A. solani followed by TRN3 but these initially did not differ significantly from the other antagonists. These antagonists overgrew A. solani, resulting in greatly restricted growth of the latter (Fig. 1-4). Similarly, the fungicides (Captan, Cobox and Dithane 200 mg l -1 significantly (P<0.05) reduced the colony diameters of A. solani compared with the control (no fungicide) treatment. Dithane M- 45 was found to be more effective than Captan and Cobox. The interaction between the Trichoderma isolates and the fungicides was also found to be significant (P<0.05). The fungicides alone were effective in reducing the growth of A. solani. Th12 showed greater inhibitory effect with Dithane M-45. T. harzianum (Swat) was more effective with Captan against the pathogen than with Cobox and Dithane M-45. TRC14 in integration with Captan reduced the colony diameter of the pathogen more effectively. Tham showed more effectiveness when integrated with Captan and Dithane M-45. The Trichoderma sp isolated from Thana was as effective alone as with all the three fungicides used to reduce the colony diameter of the pathogen. A successful disease-control program could involve just a single practice, but the long-term reduction of disease losses generally requires the application of several control measures. The best way to ensure success of a disease-management program is to use integrated disease-control measures. Generally, IPM is regarded as the use of environmentally safe practices to reduce the disease incidence and development or use of multiple control tactics integrated into a single pest control strategy (Nofal and Haggag, 2006). Trichoderma species are used as potential biocontrol agents in the integrated biological control of plant pathogens, along with the other pest management practices, Trichoderma is exceptionally good model of biocontrol because it is ubiquitous, easy to isolate and culture, grows rapidly on many substrates, affects a wide range of plant pathogens, acts as mycoparasite, competes well for food and site, produce antibiotics and has an enzyme system capable of attacking a wide range of plant pathogens (Mukerji and Garg, 1988). Harman et al. (2004) reported that the rhizosphere competent strain of T. harzianum, Th-22, provided control that was both spatially and temporally distant from the point of application. In field trials with tomato, disease caused by natural infection with A. solani was substantially reduced on the foliage by root application of Th-22 more than 100 days earlier. Leaves were protected from infection when Trichoderma was present only on the roots. Thus there was 80% reduction in early blight symptoms from natural field infection. Wilson (1997) reported that chemical fungicides typically have provided adequate control of most foliar fungal pathogens. However, fungicide resistance problems, concerns regarding pesticide residues and revocation of registration of certain widely used fungicides have led to increased activity in the development of biocontrol agents of foliar fungal pathogens. Much of this activity has centered around the use of Trichoderma spp. and Gliocladium spp. to control Botrytis cinerea on grape and strawberry. The biocontrol agent T. harzianum T39 is commercially available in Israel as Trichodex for control of grey mold in grapes. Choulwar and Datar (1989) assessed the efficacy of eight fungicides (copper oxychloride, Zineb, mancozeb, carbendazim, dithianon, iprodione, thiophanate-methyl and captafol) to reduce mycelial growth of A. solani in vitro. Mancozeb (1000 ppm) was the most effective (77% growth inhibited) followed by captafol. Sinha and Prasad (1991) tested seven fungicides in the field over three seasons against A. solani. Dithane M % was the best and cost-effective treatment with the highest yield. In the present study too, Dithane M-45 controlled A. solani more effectively. Elad et al. (1995) tested several fungicides, fungicide mixtures and spraying programs. Some trials also included a biological preparation based on T. harzianum isolates T39 and TF. Dicarboximide fungicides (iprodione or procymidone) applied alone suppressed the disease by 40 88%, as effectively as

4 Fazli Raziq and Sana Ishtiaq et al. Integrated control of alternaria solani with trichoderma spp 616 its mixture with thiram, dichlofluanid or tebuconazole. Similar disease suppression was achieved by mixtures of tebuconazole + dichlofluanid and carbendazim + diethofencarb. T. harzianum T39 alone reduced disease by 31 82% but in more than half of the cases the reduction was non-significant. Disease control achieved by the biocontrol preparations did not differ significantly (P = 0.05) from that achieved by the chemical fungicides. Adequate control was achieved when the biocontrol and the chemical products were applied alternately although the quantity of chemical sprays was reduced by one half. The consistency among treatments in various trials with respect to percent disease control was greater in the T. harzianumfungicide alternation treatments, than that in the T. harzianum or the fungicide treatments applied alone. Similar results were obtained in this study. Latorre et al. (1997) tested T. harzianum Rifai, found to be antagonistic to B. cinerea Pers. ex Fr. on apple fruits. Isolate S10B from soil in Chile provided similar control of Botrytis bunch rot under field conditions to reference isolate P1 (ATCC 74058) and T39 (Trichodex 25 WP). Elad (1994) tested the biological and chemical control of grey mold in vineyards of table and wine grapes. Treatments with T. harzianum ( g l -1 ), dicarboximide fungicides (vinclozolin or iprodione) (0.5 g l -1 ) or diethofencarb plus carbendazim (0.25 g l -1 ) resulted in up to 78% disease reduction. A tank mix of the biocontrol agent with a dicarboximide fungicide was not superior to either treatment alone. It was suggested that alternate sprays of the biocontrol preparation with a fungicide should be employed in vineyards in order to reduce the use of chemicals. Populations of Trichoderma on grapes treated with the biocontrol agent were per berry compared with per berry on untreated bunches. T. harzianum and iprodione applied alone in the vineyard reduced the postharvest rot of grapes in one of two experiments. Alternation of T. harzianum with diethofencarb plus carbendazim, or its mixture with iprodione in the vineyard, resulted in a 64 68% reduction in postharvest rot caused by Botrytis cinerea. In the present study, fungicides (Dithane M-45, Captan and Cobox ) and Trichoderma spp. controlled A. solani. It can be concluded that A. solani can be controlled more effectively when Trichoderma spp. are integrated with the fungicides (Dithane M-45, Captan and Cobox). However, Trichoderma spp. should be applied in formulations containing a suitable food base to ensure their survival and proliferation under in planta conditions. CONCLUSION AND RECOMMMENDATIONS These results show that integration of Trichoderma with the fungicides (Captan, Cobox and Dithane M-45) may offer a new and promising control of A. solani. However, further in vitro and in vivo studies are needed to evaluate the integrated use of these biocontrol agents with higher doses of these and other fungicides. Table-I Effect of Trichoderma isolates and fungicides (at 200 mg l -1 ) in PDA medium on the colony diameters (cm) of Alternaria solani 10 days after inoculation. Antagonist Fungicide No fungicide Captan Cobox DithaneM-45 Mean Control 6.88 A 5.60 B 4.96 C 3.44 D 5.22 A T. harzianum (Th12) 1.38 EF 1.32 EFG 1.42 EF 1.04 G 1.27 B T. harzianum (Swat) 1.48 E 1.30 EFG 1.46 EF 1.34 EF 1.39 B Trichoderma sp. (TRC14) 1.52 E 1.24 EFG 1.38 EF 1.32 EFG 1.36 B Trichoderma sp. (TRN3) 1.40 EF 1.34 EF 1.32 EFG 1.18 FG 1.31 B T. hamatum (Tham) 1.50 E 1.18 FG 1.46 EF 1.30 EFG 1.36 B Trichoderma sp. (Thana) 1.50 E 1.34 EF 1.42 EF 1.38 EF 1.41 B Mean A B B 1.57 C Category P-Value LSD (0.05) Fungicides (F) Antagonist (A) Interaction (F x A) Means for each category followed by different letters are significantly different from one another at 5% level of probability.

5 Sarhad J. Agric. Vol. 26, No. 4, Table-II The effect of Trichoderma isolates and fungicides (at 200 mg l -1 ) in PDA medium on the colony diameters (cm) of Alternaria solani 20 days after inoculation. Fungicide Antagonist No fungicide Captan Cobox DithaneM-45 Mean Control 8.26 A 6.62 B 6.42 C 5.96 D 6.76 A T. harzianum (Th12) 1.38 EFGH 1.32 FGHI 1.36 EFGHI 1.04 J 1.27 D T. harzianum (Swat) 1.50 EF 1.30 GHI 1.46 EFG 1.34 EFGHI 1.40 BC Trichoderma sp. (TRC14) 1.52 E 1.24 HI 1.38 EFGH 1.32 FGHI 1.36 BCD Trichoderma sp. (TRN3) 1.40 EFGH 1.34 EFGHI 1.32 FGHI 1.18 IJ 1.31 CD T. hamatum (Tham) 1.50 EF 1.18 IJ 1.46 EFG 1.30 GHI 1.36 BCD Trichoderma sp. (Thana) 1.50 EF 1.34 EFGHI 1.42 EFGH 1.38 EFGH 1.41 B Mean 2.43 A 2.04 B 2.11 B C Category P-Value LSD (0.05) Fungicides (F) Antagonist (A) Interaction (F x A) Means for each category followed by different letters are significantly different from one another at 5% level of probability. Fig.1 Growth of Alternaria solani alone (right) and in the presence of Trichoderma harzianum (Th12) (left) on unamended potato dextrose agar medium. Fig.2 Growth of Alternaria solani alone (right) and in the presence of Trichoderma harzianum (Th12) (left) on potato dextrose agar medium amended with Dithane M-45 (200 mg l -1 ).

6 Fazli Raziq and Sana Ishtiaq et al. Integrated control of alternaria solani with trichoderma spp 618 Fig.3 Growth of Alternaria solani alone (right) and in the presence of Trichoderma harzianum (Th12) (left) on potato dextrose agar medium amended with Cobox (200 mg l -1 ). Fig.4 Growth of Alternaria solani alone (right) and in the presence of Trichoderma harzianum (Th12) (left) on potato dextrose agar medium amended with Captan (200 mg l -1 ). REFERENCES Batista, C.D., M.A. Lima, F. Haddad, L.A. Maffia and E.S.G. Mizubuti Validation of decision support systems for tomato early blight and potato late blight under Brazilian conditions. Crop Prot. 25: Chester, K.S Nature and Prevention of Plant Diseases. 2 nd Edn. McGraw-Hill Book Comp. Inc., New York. Choulwar, A.B. and U.V. Datar Efficacy of fungi toxicants on the mycelial growth of Alternaria solani. Pestol. India. 13: Elad, Y Biological control of grape grey mould by Trichoderma harzianum. Crop Prot. 13: Elad, Y., M.L. Gullino, D. Shtienberg and C. Aloi Managing deitalicize cinerea on tomatoes in greenhouses in the Mediterranean. Crop Prot. 14: Harman, G.E., C.R. Howell, A. Viterbo, I. Chet. and M. Lorito Trichoderma species opportunistic, avirulent plant symbionts. Nature Rev. Microbiol.. 2:43-56.

7 Sarhad J. Agric. Vol. 26, No. 4, Hooker, W.J Compendium of Potato Diseases. American Phytopath. Soc., St. Paul, Minnesota, USA. pp Latorre, B. A., E. Agosín, R.S. Martín and G.S. Vásquez Effectiveness of conidia of Trichoderma harzianum produced by liquid fermentation against Botrytis bunch rot of table grape in Chile. Crop Prot. 16: Mukerji, K.G. and K.L. Garg Biocontrol of Plant Diseases, Vol. ll. CRC Press Inc., Boca Raton, Florida. Nofal, M.A. and W.M. Haggag Integrated management of powdery mildew of mango in Egypt. Crop Prot. 25: Singh, R.S Plant Diseses, 5 th Edn. Oxford and IBH Pub. Co., New Delhi. Sinha, P.P. and R.K. Prasad Evaluation of fungicides for the control of early blight of tomato. Madras Agric. J. 78: Wilson, M Biocontrol of aerial plant diseases in agriculture and horticulture: current approaches and future prospects. J. Ind. Microbiol. Biotechnol. 19:

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