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Antifungal Activity of Bacillus Subtilis Subsp. Spizizenii (MM19) for the Management of Alternaria Leaf Blight of Marigold

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Antifungal Activity of Bacillus Subtilis Subsp. Spizizenii (MM19) for the Management of Alternaria Leaf Blight of Marigold Journal of Biological Control, 32(2): 95-102, 2018, DOI: 10.18311/jbc/2018/21134 Volume: 32 No. 2 (June 2018) Research Article Antifungal activity of Bacillus subtilis subsp. spizizenii (MM19) for the management of Alternaria leaf blight of marigold R. PRIYANKA1*, S. NAKKEERAN1, I. ARUMUKA PRAVIN1, A. S. KRISHNA MOORTHY1 and U. SIVAKUMAR2 1Department of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore – 641003, Tamil Nadu, India 2Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India *Corresponding author E-mail: [email protected] ABSTRACT: Biological control with bioagents is a cost effective alternate method for the management of crop diseases. The antagonistic bacterial strains were explored for the management of leaf blight of marigold which is caused by Alternaria alternata. The present study clearly proved that the mycelial growth of A. alternata was inhibited up to 83% by Bacillus subtilis subsp. spizizenii (MM19) in vitro. GC/MS analysis of partially purified extracts of B. subtilis subsp. spizizenii (MM19) revealed the presence of antifungal Phthalic acid esters which might be responsible for the inhibition of the pathogen. Foliar application of B. subtilis subsp. spizizenii (MM19) under field conditions suppressed leaf blight by 77%. This study highlighted the potential ofB . subtilis subsp. spizizenii (MM19) for the management of Alternaria leaf blight. KEY WORDS: Alternaria, Bacillus subtilis subsp. spizizenii, GCMS, PCR (Article chronicle: Received: 15-03-2018; Revised: 22-05-2018; Accepted: 30-05-2018) INTRODUCTION cide leads to the resistance in pathogen and also it affects the human and environment directly or indirectly resulting Marigold is a seasonal flower and can be grown round in ecological imbalance (Waghmare et al., 2011). Exploita- the year. Marigold flowers gained popularity amongst gar- tion of biocontrol agents for the management of this disease deners and dealers on its easy cultivation and wide adapt- is an alternative. Effective inhibition of mycelial growth of ability. Marigold as a cut flower and loose flower is exten- A. solani causing leaf blight of tomato by Bacillus subtilis sively used in the social and religious function for internal has been reported (Babu et al., 2000; Mishra et al., 2013). decoration and garlands. The leaves and flowers are known It was also found that Bacillus have strong antifungal ac- to possess high phenolic and antioxidant properties can be tivity both in vitro as well as in vivo conditions against A. exploited in pharmaceutical industry (Khalil et al., 2007). solani isolated from tomato (Zhao et al., 2008). Bacillus Marigold is used as the trap crop in the borders to attract the species are capable of producing a wide variety of second- insects attacking the main crop (Kolambkar et al., 2013). ary metabolites that are diverse in structure and function. Marigold is infected with fungal, viral and bacterial dis- The production of antimicrobial metabolites determines eases. Among the diseases, inflorescence blight caused by their ability to control plant diseases (Silo-suh et al., 1994). Alternaria zinnia Ellis is the most common disease in mari- Hence, the present investigation was carried out to find al- gold, the infection causes considerable damage to the orna- ternate methods for controlling the leaf blight of marigold mental crops (Karlatti and Hiremath, 1989). In 2014, Aktar by evaluating the bioefficacy of bacterial bioagents for the and Shamsi reported the leaf, bud and flower blight of Ta- management of marigold leaf blight. getes erecta and T. patula. was caused by A. alternata. In 1966, Shome and Mustafee reported that the leaf spot and MATERIALS AND METHODS flower blight was a serious disease of marigold in northern Madhya Pradesh. The leaf spot in marigold initially appear Survey and collection of samples as necrotic spot, latter it progresses as leaf blight and com- Survey for the occurrence of leaf blight in marigold pletely coalesce leads to drying of entire leaf. This disease was conducted in Krishnagiri, Tharmapuri, Madurai and emerged as major constraint in exploitation of high yielding Sakthiyamangalam district of Tamil Nadu during 2015- marigold varieties in the country. Excessive use of fungi- 2016. The fields were having marigold hybrids like US45, 95 Bacillus subtilis subsp. spizizenii for the management of Alternaria leaf blight of marigold Benz tall, Gold colour, Yellow. Plants showing characteris- three plants per replication. The fungus was reisolated from tic blighting symptom were collected. The per cent disease the plants expressing the typical symptoms to confirm index for each hybrids/variety was observed and they were Koch’s postulates. classified using the disease score as described by Manojku- mar et al., (2013). In vitro screening of antagonistic bacterial strains of Bacillus spp. against Alternaria alternata Sumofindividualrating Antagonistic activity of twenty antagonistic bacterial of infected leaves Per centdiseaseindex = ×100 strains of Bacillus spp (B. cereus -BSC5; B. megaterium Nuumberofleavesobserved -BmTNAU5; B. subtilis subsp. spizizenii -MM19; B. am- ×Maximumdiseasescore yloliquefaciens- MM 12; B. subtilis - VB3 and B. licheni- Isolation and identification of pathogen formis -B1TNAU2) against the pathogen Alternaria alter- nata was evaluated in vitro by standard protocol given by Small segments of diseased tissue along with some Matar et al. (2009) with slight modifications. Agar wells 2 healthy portion (5 × 5 mm ) from the infected leaf sam- were created in four corners of the Petri plates at equal ples were cut by sterile razor and surface sterilized in 0.5% distance from the periphery using sterile cork borer. Later, sodium hypochlorite (NaOCl) for 1 minute. Surface steri- 10 µl of 48 h old, bacterial suspension (OD Value @ 600 lized plant tissue were rinsed twice by sterilized distilled nm) was transferred into the wells. Then, mycelia disc (9 water for removing the traces of Sodium hypochlorite, mm dia) was placed at the centre of the plate under sterile dried on filter paper and plated on Petri plates containing condition. Simultaneously, control plate without antago- Potato Dextrose Agar medium (PDA) amended with 100µg/ nists was also maintained. The cultures were incubated at ml of streptomycin sulphate. The plates were incubated at room 150 ± 20C for 3–5 days and diameter of the mycelial growth temperature for 48 to 72 hrs. Later the plates were observed for was measured. Per cent Inhibition (PI) was calculated using growth of fungus. The fungal pathogen was purified by single the formula spore isolation method on plain agar medium (Tutte, 1969). Morphological characters of all the strains were studied on CT− PI = ×100 PDA as described by Simmons (2007). C Where, Molecular characterization The fungus was cultured on potato dextrose broth C is the growth of test pathogen (mm) in the absence at room temperature for two weeks. Then the myce- of the antagonist. lium was collected, dried and powdered by freezing in T is the growth of test pathogen (mm) in the presence liquid nitrogen. The genomic DNA was extracted by Ce- of the antagonist. tyl Trimethyl Ammonium Bromide (CTAB) method as described by Chakraborty et al. (2010). The extract- Extraction and detection of nonvolatile metabolites ed genomic DNA was subjected to PCR with ITS 1 The effective bacterial strain (Bacillus subtilis subsp. (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS 4 (5’-TC- spizizenii -MM19) was cultured in Nutrient Broth (NB) and CTCCGCTTATTGATATGC-3’) primer pair (White et al., incubated at 28°C for 3 days. The culture filtrates along 1990). The PCR product was analyzed on 1.2 % agarose with the bacterial cells were centrifuged at 5000 rpm for gel, stained with ethidium bromide and viewed under tran- 15 minutes at 4°C. The bacterial cells were discarded. Then silluminator. The amplified fragments were sequenced and supernatant was adjusted to acidic pH 2.0 with 1N HCl. confirmed using NCBI database. Antifungal compounds in culture broth were extracted by adding equal volume of ethyl acetate and shaked for 2 h in Pathogenicity an orbital shaker at 200 rpm. Culture broth was extracted 5 Spore suspension was prepared with 10 conidia twice with ethyl acetate for complete extraction. The sol- per ml. Tween 20 was added to the suspension at a final vent fraction with antifungal compounds were combined concentration of 0.05%. The spore suspension containing and concentrated by evaporation in the rotary flask evapo- 10000 conidia/ml in phosphate buffer (pH 7) was sprayed rator maintained at 60°C at 80 rpm. The concentrated crude over healthy plants using atomiser. Inoculated plants were metabolites of the extracellular antifungal compounds ob- covered in polythene bags and incubated at room tempera- tained from the culture broth were dissolved in 1 ml HPLC ture for one week. Simultaneously, a healthy control was grade methanol and used for GC/MS analysis (Dheepa et also maintained. The experiment was replicated thrice with al., 2016). 96 PRIYANKA et al. Evaluating the bio efficacy of Bacillus spp. against Al- rial suspensions (1% FS at 108CFU/ml) after disease ternaria Blight under open field conditions initiation at weekly intervals for four weeks. An un- Field experiment was conducted using Completely treated control was also maintained. All the treatments Randomized Block Design during 2018 at Paruvachi, were replicated thrice. Fifty plants in each treatment Erode to assess the efficacy of Bacillus spp. for the were selected at random and tagged to record obser- management of leaf blight of marigold (variety-Benz vations on various traits and assessed for leaf blight tall). The plants were sprayed with respective bacte- incidence. Treatment details 8 T1 Foliar spray with Bacillus cereus (BSC5) (1%) @ 10 CFU/ml 8 T2 Foliar spray with B. megaterium (BmTNAU5) - (1%) @ 10 CFU/ml 8 T3 Foliar spray with B. subtilis subsp. spizizenii(MM19) -(1%) @ 10 CFU/ml 8 T4 Foliar spray with B. amyloliquefaciens (MM 12) - (1%) @ 10 CFU/ml 8 T5 Foliar spray with B.
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