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Evaluation of Secondary Metabolites of Hirsutella Citriformis Against Udaspes Folus Infecting Curcuma Longa L

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Evaluation of Secondary Metabolites of Hirsutella Citriformis Against Udaspes Folus Infecting Curcuma Longa L journal of pharmacy research 7 (2013) 7e14 Available online at www.sciencedirect.com journal homepage: www.elsevier.com/locate/jopr Original Article Evaluation of secondary metabolites of Hirsutella citriformis against Udaspes folus infecting Curcuma longa L. Arutselvi Ramachandran a,b,*, Bala Saravanan Thangappan c, Ponmurugan Ponnusamy b a Research and Development Centre, Bharathiar University, Coimbatore 646 041, India b Department of Biotechnology, K.S. Rangasamy College of Technology, K.S.R Kalvi Nagar, Tiruchengode 637 215, Tamil Nadu, India c Department of Biotechnology, Nehru Arts and Science College, Coimbatore 641 105, India article info abstract Article history: Objective: A field experiment was conducted in a turmeric field during 2010e11 to evaluate Received 9 November 2012 the efficiency of suitable biopesticides for the management of Udaspes folus larvae on Accepted 22 January 2013 turmeric leaves. Available online 18 February 2013 Methods: The treatments contained 11 treatments with three replicates covering isolated and indigenous fungal strains along with neem based formulations. Moreover, a commercial Keywords: biopesticide and a chemical pesticide were also tested to find out the efficiency of the product. Biochemical parameters Results: Pooled data on mortality of the larvae revealed significant superiority of Hirsutella GCeMS analysis citriformis and Metarhizium anisopliae. Among the four larval stages tested the second, third Leaf roller and fourth instar larvae showed higher percent of mortality rate than the fifth instar. Leaf damage Between the two isolates of H. citriformis, the isolate HC 28 revealed significant mortality of Turmeric U. folus in field conditions. Similarly, the physiological and biochemical parameters were also increased in plants greatly after imposing various treatments. GCeMS analysis of H. citriformis showed the presence of Phthalic acid to be the major constituent. Natural en- emies were recorded in all the treatment plots. Conclusion: The present study provides foremost solid proof for the use of entomopatho- genic fungi H. citriformis as an important component for integrated pest management strategy against U. folus infecting leaves of turmeric plants. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. 1. Introduction seasonally affected by few major and minor pests which includes shoot borer, Conogethes punctiferalis and leaf roller, Curcuma longa L. (turmeric) is an important spice valued for Udaspes folus3,4 which leads to major crop loss5 observed U. its export earnings and culinary uses in India. India is the folus harboring Elettaria cardomum, Aframomum melegueta and largest producer (80%) and exporter (60%) of turmeric in the Curcuma amada too. The larvae of this lepidopteron pest world.1 Turmeric plants are propagated by vegetative cause destruction in the plant leaf and cause considerable method using mother and finger rhizomes.2 The plant is yield loss by 20e34%. * Corresponding author. Department of Biotechnology, K.S. Rangasamy College of Technology, K.S.R Kalvi Nagar, Tiruchengode 637 215, Tamil Nadu, India. Tel.: þ91 9790346831 (mobile), þ91 04288 274741; fax: þ91 04288 274745. E-mail address: [email protected] (A. Ramachandran). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.01.019 8 journal of pharmacy research 7 (2013) 7e14 Entomopathogenic fungi like Beauveria bassiana (Bals.) 2.3. Larval bioassays Vuillemin and Metarhizium anisopliae (Metsch.) Sorokin has been used successfully for managing insect pests in tem- The 2nd, 3rd, 4th and 5th instar larvae were grown in plastic perate regions.6 The present study was aimed in developing containers covered by a muslin cloth for aeration. Each con- a biopesticide against U. folus with rapid growth rate and tainer consists of 10 larvae and three replicates were main- high pathogenicity. The study was conducted in PTS tur- tained. Ten milliliters of spore suspension of the fungi were meric variety which is a famous cultivar of India now pre- taken in which each larva was dipped thoroughly for 10 s. The ferred by most farmers for its high yield and its high control larvae were dipped in 0.02% Tween 80 alone. The tolerance to disease and pest attack. Neem products are also containers with larvae were maintained at 26 Æ 1 C temper- used selectively in controlling pests of various economically ature; relative humidity 70 Æ 10% and photoperiod of 16:8 L:D. useful plants.7 The seeds contain a complex secondary Larval mortality was recorded at every 24 h interval for seven metabolite azadirachtin which imparts a bitter taste. It acts days after treatment and the data was analyzed statistically. as an anti-feedant, repellent and egg-laying deterrent, pro- The cadavers were used for re-isolating the pathogen in pure tecting the crop from damage. Similarly the leaves of Vitex culture for confirming the pathogenicity of fungi. negundo are also capable of causing mortality of lepidopteron pests.8 So these two plant products were also used in the 2.4. Semi-synthetic diet current study for comparison purpose. To keep in mind on all these parameters, studies were conducted to evaluate The larvae were fed twice a day with a specially formulated indigenous biocontrol agents to control U. folus under field diet (slightly modified diet of6) which consists of caesin-10 g, conditions. sucrose-20 g, ascorbic acid-2 g, Brewer’s yeast-2 g, sorbic acid- 0.65 g, formaldehyde-1 ml, agar-6 g, turmeric leaves-50 g and water-275 ml. The unfed feed and leaves were removed 2. Materials and methods periodically. 2.1. Fungal isolates 2.5. Field experiments of fungal isolates against U. folus Surveys were conducted in naturally infected turmeric farms Field trials were conducted for two years at one of the tur- to isolate and identify virulent entomopathogenic fungi meric farms in Karungalpalayam, Erode, Tamil Nadu, India infecting U. folus of PTS turmeric plants in Erode region, during 2010e2011 in randomized complete block design hav- [1120N77431 E], Tamil Nadu, India. The collections were ing 11 treatments which includes an untreated control plot made during SeptembereNovember in 2010. The cadavers with three replicates for each treatment. Each treatment plot were collected in sterile glass vials separately from which size was 10 m2 with 50 plants in each plot. Treatments were the pathogens were isolated using Potato Dextrose Agar applied as foliar sprays and comprised as follows: (PDA) medium following standard mycological techniques.9 Two fungi were subjected to 18S rDNA sequencing and 2.5.1. Treatments BLAST and identified as Hirsutella citriformis and Nomuraea T1 e M. anisopliae;T2e B. bassiana;T3e Standard N. rileyi (MTCC rileyi. The fungal sequences were deposited in NCBI (JQ 4175); T4 e Standard H. citriformis (MTCC 6800); T5 e H. cit- 675289 and JQ 686668; respectively). Along with M. anisopliae riformis HC28; T6 e N. rileyi NR07; T7 e Neem leaf extract; T8 e and B. bassiana, which are commonly used entomopatho- Neem seed kernel þ V. negundo leaf extract; T9 e Commercial Ò genic fungi; Standard H. citriformis (MTCC 6800) and N. rileyi Biopesticide (Biopower ); T10 e Acephate; T11 e Untreated (MTCC 4171) cultures were obtained from Microbial Type control. Culture Collection, Chandigarh, India and used for compar- The spraying of bioformulations was done using a Knap- À ison studies. sack sprayer with a spray volume of 300 L ha 1. The treatment sprays were applied twice at two days interval. Soap powder 2.2. Mass multiplication of fungal isolates (2 g/L) and/or starch powder was added to enhance the adhesiveness of the sprays as the whole experiments were For B. bassiana, H. citriformis and M. anisopliae PDA medium conducted during rainy season.10 The observations were and N. rileyi, Sabarouds Yeast Maltose Peptone (SYMP) me- recorded on ten randomly selected plants in each plot. Data on dium was used for multiplication. Spore suspensions of each the death of larval population after 3, 5 and 7 days after pathogenic fungus were prepared by using 80e100 ml of spraying were calculated. sterile distilled water containing 0.05% Tween 80 solution. The conidial count of cultures were then determined by using 2.6. Leaf damage due to pest infestation a Neubauer hemocytometer and adjusted to 1 Â 107/ml and stored in 4 C until use. The neem leaf extract was prepared To assess the damage caused to turmeric leaves by the four by crushing 100 g of neem leaves in water and soaking in larval stages of the pest, around ten plants from each exper- water overnight; the neem seed kernel e V. negundo leaf imental plot were selected randomly in each replicate in extract was prepared by taking 100 g each neem seed kernel which the plant height, number of leaves and disease index powder and V. negundo leaves. They are then crushed and were calculated by measuring the damaged leaf with the un- soaked in water overnight and filtered before use for field affected leaf. The range of the disease index was grouped into trials. four types as 25%, 50%, 75% and 100% depending upon the journal of pharmacy research 7 (2013) 7e14 9 damage caused to the leaves. The disease index was calcu- error (SE) was segregated by critical difference (CD) at various lated to evaluate the damage caused to the leaves and know levels of significance (CV) was calculated for the assessment of the severity of the problem caused by the larvae. disease incidence.20 2.6.1. Biochemical changes in turmeric leaves Turmeric leaves (5 g) were collected from all experimental 3. Results plots and ground separately with 80% aqueous acetone using a chilled pestle and mortar. The aqueous layer was transferred 3.1. In vitro effect of fungal application on U.
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