Screening of Fungicides, Botanicals and Bioagents against Colletotrichum dematium In Vitro

Screening of Fungicides, Botanicals and Bioagents against Colletotrichum dematium In Vitro *Rajkumar Kothikar and Mina Koche Department of Plant Pathology, Post Graduate Institute, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra, India *rajkumarkothikar@gmail.com Abstract- Turmeric is an important and economical medicinal and aromatic crop. It is subjected to variety of diseases like leaf spot, leaf blotch, leaf blight, rhizome rot etc. which are threat in turmeric production. Among these diseases, leaf spot caused by Colletotrichum dematium is important and major limiting factor for yield loss. Realizing the potentiality of the disease in causing economic losses, the different fungicides (propiconazole, mancozeb, carbendazim, hexaconazole, penconazole), botanicals (Azadirachta indica, Agael marmalos, Eucalyptus sp.) and bio-agents (Trichoderma viride, Trichoderma harzianum) were screened against Colletotricum dematium in vitro. In vitro studies by poisoned food technique revealed that, fungicide propiconazole 0.1% found to be most effective among all the chemicals, botanicals and bioagents and gave 91.98% mycelia inhibition of C. dematium. Among the botanicals, Azadirachta indica seed extract 5% (74.69%) found to be superior. Dual culture studies of antagonism reported that, the bio-agent Trichoderma viride (80.11%) was found most effective against C. dematium. KEYWORDS: Turmeric, Colletotrichum dematium, Fungicides, Botanicals, Bio-agents I. INTRODUCTION Turmeric is one of the most commercially important crop is suffering from foliage and rhizome rot diseases. The most important of them are leaf spot (Colletotrichum dematium), leaf blotch (Taphrina maculans), leaf blight (Alternaria alternata) and rhizome rot (Pythium aphanidermatum). Colletotrichum dematium causing leaf spot is the most important among foliar diseases of turmeric cause severe reduction in yield due to loss of photosynthetic area. [1] Reported 50 per cent yield loss due to Colletotrichum leaf spot. [2] While working on the chemical control of leaf spot caused about 35 per cent yield loss. [3] Reported a loss of 62 per cent of yield in pot culture studies due to Colletotrichum leaf spot. [4] Also observed turmeric leaf spot caused by Colletrotrichum capsici as a serious disease in Karnataka. Realizing the potentiality of this foliar disease in causing economic losses, it was found necessary to initiate systemic studies on foliar diseases. Taking into consideration the importance of crop and seriousness of disease prevailing on turmeric, it is thought worthwhile to undertake the study on screening of fungicides, botanicals and bio-agents against C. dematium causes leaf spot in turmeric for the economical management of the disease. 1

II. MATERIAL AND METHODS The present investigation on in vitro screening of fungicides, botanicals and bio-agents against Colletotrichum dematium was conducted at the Department of Plant Pathology, Post Graduate Institute, Dr. Panjabrao Deshmukh Krishi Vidyapeeth (Dr. PDKV), Akola. The culture of C. dematium used in this study was isolated from infected leaves of turmeric plant collected from the fields of Department of Horticulture, Dr. PDKV, Akola. In order to isolate pathogen from the leaf spot, infected parts were cut in to 3 mm small bits and surface sterilized with 0.1 per cent HgCl2 solution for one minute and three subsequent washing with sterilized distilled water were given. The bits were placed in previously poured solidified PDA medium in Pertiplates. The plates were incubated at 27± 2 0 C for 7 days. The isolated fungi were identified as Colletotrichum dematium on the basis of morphological characters and compared with published literature. The inoculum was prepared and sprayed on plants of turmeric which were planted in pots. The infected leaf spot plants of turmeric were used for re-isolation as per the standard procedure. The culture obtained was confirmed for growth with original culture from which isolations were made to prove pathogenicity. PREPARATION OF AQUEOUS EXTRACTS OF BOTANICALS Sufficient quantity of fresh green leaves of different test plants were collected from campus of Dr. PDKV, Akola. The following method was used for preparation of aqueous extracts as followed by [5]. Sufficient quantity of selected plant leaves were soaked in mercuric chloride solution (1:1000) for 5 minutes and then thoroughly washed with sterilized distilled water by three washings. One ml of sterilized distilled water was used for each gram of fresh material for maceration. In this way required quantity of water and leaves were macerated in mortar and pestle for 5 minutes and the extract was collected. The extract thus obtained was first filtered through three folds of muslin cloth so as to remove fibrous and suspended material. This filtrate was taken as stock material of crude aqueous extract and stored at 4 O C. Streptocycline (200 ppm) was mixed as per need for controlling growth of bacteria. This crude aqueous extract was used in the study. POISONED FOOD TECHNIQUE AGAINST C. DEMATIUM The principle involved in this technique was to make the nutrient medium toxic with a fungitoxicant and allow the test fungi to grow on medium and study the mycelial inhibition in laboratory. Hundred ml of liquefied potato dextrose agar medium (PDA) was taken in 250 ml flask plugged cotton and sterilized. To this medium requisite quantities of botanicals/ fungicides were added as per their concentrations mentioned. About 20 ml of melted toxic PDA was poured in sterilized Petriplates and allowed to solidify. These Petriplates were then inoculated by test organisms separately. Two mm disc of 10 days old test fungal cultures were cut with sterilized cork borer and transferred aseptically in the centre of the Petriplates containing the poisoned medium. The surface of inoculum disc was kept in inverted position with agar surface in plates. The control plates were also grown under same condition on PDA without fungicides/ aqueous extracts of botanicals. All these operations were carried out under aseptic condition in sterilized isolation chamber. Three plates were inoculated for each fungus for every fungicides/ aqueous plant leaf extract. Plates were incubated at room temperature of 27 C ± 2 C and observation recorded. 2

EVALUATION OF FUNGICIDES AND BOTANICALS Following fungicides were evaluated for their relative efficacy against the pathogen in vitro. Sr. No. Common name Trade name Conc. (%) 1. Propiconazole Tilt 0.1 2. Dithane M-45 Mancozeb 0.25 3. Carbendazim Bavistin 0.1 4. Hexaconazole Contaf 0.1 5. Penconazole Topas 0.1 Following plant extracts were evaluated for their relative efficacy against the pathogen in vitro. Sr. No. Botanicals Plant part used Conc.(%) 1. Azadirachta indica Seed 5.0 2. Agael marmalos Leaf 5.0 3. Eucalyptus spp. Leaf 5.0 The colony diameter were recorded and compared with the control treatment after 7 days incubation. [6] formula was used to calculate the percentage inhibition of growth. C - T Per cent Inhibition = x 100 Where, PI = Percentage of inhibition of growth C = Growth of fungus in control T = Growth of fungus in treatment C EVALUATION OF TRICHODERMA SPP. AGAINST COLLETOTRICHUM DEMATIUM BY DUAL CULTURE METHOD The culture of T. harzianum and T. viride obtained from Plant Pathology section, Department of Plant Pathology, College of Agriculture, Akola were grown separately on PDA in plates of 8 days. Each culture was used for evaluation of antagonistic effect against Colletotrichum dematium. A well sterilized melted PDA was poured in sterilized Petriplates and after solidification 5 mm diameter circular bits were prepared from Trichoderma culture with sterilized cork borer. In each plate, 4 bits of antagonists were inoculated at four peripheral points and at the centre 5 mm disc of C. dematium isolate was inoculated per plate and incubated at room temperature (27 C ± 2 C). For each isolate three replications were maintained. In control, only 5 mm disc of C. dematium was placed per plate at centre. Antagonistic effects of both the Trichoderma spp. were tested separately for the fungus of C. dematium. Inhibition of C. dematium was calculated by deducting growth of C. dematium growth in association and Trichoderma spp. from that of individually grown fungal colony. III. RESULT AND DISCUSSION Four systemic fungicides viz.,propiconazole (0.1%), carbendazim (0.1%), hexaconazole (0.1%), penconazole (0.1%); one non-systemic fungicide viz.,dithane M-45 (0.25%); three botanicals viz.,azadirchta indica seed extract (5%), Agael marmalos leaf extract (5%), Eucalyptus leaf extract (5%) and two bioagents viz.,trichoderma viride and T. harzianum were selected to study the effectiveness against the foliar pathogen of turmeric in vitro. 3

SCREENING OF FUNGICIDES AND BOTANICALS AGAINST TEST FUNGUS C. DEMATIUM IN VITRO BY POISONED FOOD TECHNIQUE The data on evaluation of fungicides and botanicals against Colletotrichum dematiumin vitro is presented in Table 1. It was evident from Table 1 that test fungus C. dematium produced minimum (6.44 mm) growth in propiconazole (0.1%). This treatment was significantly superior over all other treatments. Hexaconazole (0.1%), penconazole (0.1%), carbendazim (0.1%) and dithane M-45 (0.25%) were at par with each other and produced colony diameter of 10.00, 11.55, 12.83 and 13.33 mm, respectively. Amongst the botanicals, A. indica seed extract (5%) was the efficient treatment which produced 20.33 mm growth as against 80.33 mm in control. In vitro evaluation of fungicides and botanicals were made by poisoned food technique against C. dematium. Among the treatments propiconazole (0.1%), hexaconazole (0.1%), penconazole (0.1%), carbendazim (0.1%) and dithane M-45 (0.25%) were effective to inhibit the growth of C. dematium in the range of (92.98-82.58%). [7] [8] [9] and [4] reported that propiconazole, hexconazole, penconazole, carbendazim at various concentration (0.025, 0.05, and 0.1 %) completely inhibited the growth of C. capsici causing leaf spot in turmeric. Among botanicals, Azadiracta indica seed extract (5%) was effective treatment to inhibit the growth of C. dematium (74.69%). [10] and [11] reported that among the botanicals Azadiracta indica effective against C. capsici. [12] and [13] reported propiconazole (0.1%) was significantly superior while [14] found carbendazim @ 0.5 g lit -1 was the most efficient fungicide to control Colletotrichum leaf spot. SCREENING OF TRICHODERMA SPP. AGAINST C. DEMATIUM BY DUAL CULTURE METHOD AFTER 7 DAYS The results of evaluation of Trichoderma spp. against Colletotrichum dematium by dual culture method is presented in Table 2. The data from the Table 2 revealed that Trichoderma spp. inhibited the growth of C. dematium. Maximum inhibition was noticed by Trichoderma viride (80.11%) while minimum in T. harzianum (71.51%). Both the species of Trichoderma effectively reduced the mycelial growth of test fungus C. dematium causing leaf spot disease in turmeric. But T. viride was superior over T. harzianum in inhibiting growth of C. dematium. Besides chemical control, biological control is an effective, eco-friendly and alternative approach for any disease management strategy. Two Trichoderma spp. viz., T. viride and T. harzianum were tested against, C. dematium by dual culture method. Both the spp. of Trichoderma found effective in restricting the growth of C. dematium. The highest percent inhibition was recorded by T. viride followed by T. harzianum. The similar results were recorded by [15] and [16]. [17] and [18] screened five Trichoderma spp. against C. capsici causing leaf spot in turmeric in vitro by dual culture method and reported that T. koningii and T. harzianum were best in inhibiting the growth of C. capsici. [19] in efficacy of bioagent test, T. viride was observed most effective antagonist and [20] reported T. harzianum and T. viride were most effective against Colletotrichum. [21] reported highest percentage of inhibition of mycelia germination of Colletotrichum was obtained from T. viride followed by T. harzianum and Psuedomonas flueorescens. Present results confirms that T. viride inhibit the growth of C. dematium causing leaf spot in turmeric and thus can be used for management of leaf spot disease. Conclusion Efficacy of five fungicides and three botanicals was studied in vitro against C. dematium by poisoned food technique. Propiconazole (0.1%) was significantly superior over all other treatments for inhibiting the growth of C. dematium followed by hexaconazole, penconazole, carbendazim and dithane M-45. Azadiracta indica seed extract (5%) inhibited the growth of test fungus and was found most effective among the botanicals. Trichoderma viride found to be the most effective antagonist of the fungus Colletotrichum dematium followed by T. harzianum. 4

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TABLE 1.Screening of fungicides and botanicals against C. dematium in vitro (poisoned food technique) Sr. Conc. Mean colony diameter Per cent growth in Treatments No. (%) after 10 days (mm) inhibition (%) T 1 Propiconazole 0.1 6.44 91.98 T 2 Dithane M-45 0.25 13.33 82.58 T 3 Carbendazim 0.1 12.83 84.02 T 4 Hexaconazole 0.1 10.00 87.55 T 5 Penconazole 0.1 11.55 85.62 T 6 Azadirachta. indica seed extract 5.0 20.33 74.69 T 7 Agaelmarmalosleaf extract 5.0 33.66 58.09 T 8 Eucalyptus leaf extract 5.0 35.88 55.33 T 9 Control -- 80.33 0.00 F test Sig. SE (M) ± 1.86 CD (P = 0.05) 5.50 TABLE 2.Evaluation of Trichoderma spp. against C. dematiumin vitro(dual culture studies) Mean colony diameter of C. dematium(mm) Per cent growth inhibition (%) Untreated T. viride T. harzianum T. viride T. harzianum 62.00 12.33 17.66 80.11 71.51 6