Efficacy of Isolated Nomuraea Rileyi and Spinosad Against Corn Pests Under Laboratory and Field Conditions in Egypt

Annual Review & Research in Biology 3(4): 903-912, 2013

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Efficacy of Isolated Nomuraea rileyi and Spinosad against Corn Pests under Laboratory and Field Conditions in Egypt

M. M. Sabbour 1* and A. Abdel-Rahman 1

1Department of Pests and Plant Protection, National Research Centre, Dokki, Cairo, Egypt.

Authors’ contributions

This work was carried out in collaborations between both two authors. Author MMS designed the experiment structures of this study, performed the statistical analysis, wrote the protocol and first draft of the manuscript. Author AAR managed the literature and field studied. All authors read and approved the final manuscript.

Received 20 th March 2013 th Research Article Accepted 19 July 2013 Published 9th August 2013

ABSTRACT

Isolates of the entomopathogenic fungus, Nomuraea rileyi and spinosad were tested against the corn insect pests: Ostrinia nubilalis, Chilo agamemnon and Sesamia cretica under laboratory and field conditions . Results obtained showed that the LC 50 of N. rileyi for O. nubilalis, C. agamemnon and S. cretica were 124, 146 and 159 conidia/ml under laboratory conditions. The corresponding figure for Spinosad was 166, 179 and 185 microgram/ ml (µg/ml. Under field conditions, results showed that during season 2011, the percentage of O. nubilalis, S. cretica and C. agamemnon infestations were significantly decreased in plots treated with N. rileyi to 25±3.2, 21±2.4, and 24±2.3 individuals after 120 days after the first application as compared to 97±3.3 in the control. When spinosad was applied in the field, the percentage of infestations was significantly decreased to 26±3.3 as compared to 99±1.3 individuals in the control during both seasons. During the harvest season, the corn weight was significantly increased to 3822±45.6 and 3940±60.4 kg/Feddan (Feddan=2400m 2) in the plots treated with the fungus N. rileyi as compared to 2810±40.9 and 2710 ±73.2 Kg/Feddan in the control plots. Among plots treated with spinosad the yield significantly increased to 3000 ±60.7 and 3129 ±84.1 kg/Feddan during season 2011 and 2012, respectively.

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*Corresponding author: Email: [email protected];

Annual Review & Research in Biology, 3(4): 903-912, 2013

Keywords: Nomuraea rileyi; spinosad; Ostrinia nubilalis; Chilo agamemnon; Sesamia cretica.

1. INTRODUCTION

Maize ( Zea mays ) is an important crop all over the world and also in Egypt. Its demand continuously increases. Corn is subjected to attack by many insect pests that affect the yield quality and quantity. Among the most common pest species surveyed in Egypt are: Ostrinia nubilalis , Sesamia cretica and Chilo agamemnon . O. nubilalis is the one of key pest damaging corn in Egypt [1]. O. nubilalis is native to Mediterranean countries which has 98% of the world’s cultivated corn [2]. C. agamemnon is also one of the most important insect pests of corns in Egypt and many Mediterranean countries. The corn borers moth develops three generations per year [2]. Nomuraea rileyi and Paecilomyces fumosoroseus proved highly pathogenic to aphids and whiteflies [3]. The fungus ( N. rileyi ) exhibits host preferential infections in lepidopterous larvae, the fungi spores come in contact with the cuticle (skin) of susceptible insects, they germinate and grow into their bodies then penetrate the integuments; spread rapidly blocked the respiratory pores lead to pest death [4]. The fungi emerge from the infected insect for sporulation and these spores are disseminated in the air and spread as an inoculum spores for infection of other insects. Infected insects will stop feeding, thereby reducing further crop damage. Regular spraying of fungi ensures a protective coating of these beneficial fungi on the surface of leaves and stems and the invasion of the pests is effectively arrested. The infected insect dead due to tissue destruction and by secretion of toxins inside the body of the insect [4]. Spinosad is a broad- spectrum, organic insecticide. It is, however, relatively non-toxic to mammals and beneficial insects [5]. It is necessary to find alternative safe insecticides to reduce the heavy doses of chemical insecticides which is using for the control of corn pests [5,6]. The mode of action of spinosad is that spinosad is highly active, by both contact and ingestion, to numerous pests. Spinosad overall protective effect varies with pest species and life stage. Spinosad affects certain insect pests only in the adult stage, but can affect other pests at more than one life stage. The pests that are subject to very high rates of mortality as larvae, but not as adults, may gradually [3] be controlled through sustained larval mortality [7].

Entomopathogenic fungi are found worldwide associated to insects and phytophagous mite populations, contributing to biological control of these arthropods on several economically important crops [8]. Commercial products have been developed with entomopathogenic fungi [9]. [10] reported that fungal concentrations of 10 6 and 10 7 conidia/ml of B. bassiana and N. rileyi affected the larval development, movement and mobility of corn borers larvae during the seedlings and vegetative stages of corn plant under laboratory; greenhouse and field conditions. Success of a pest control program using B. bassiana however depends on conidia survival in the field environment [11]. Conidia survival may be affected either by environmental factors [12] or chemical products used to protect plants [13]. [14] controlled the cereal aphids with the fungus B. bassiana and found that the infestation was reduced after fungal applications under laboratory and field conditions [4]. Spinosad is a broad- spectrum, organic insecticide (where spinosad is a commercial Bt.). It is, however, relatively non-toxic to mammals and beneficial insects. If used carefully only against insects that actually eat something that has been treated, such as a leaf, are affected. This is different than a lot of other broad-spectrum insecticides that are toxic if the insect merely comes in contact with dry insecticide residues [5]. The entomopathogenic fungus, Beauveria bassiana is one of several fungi that are of particular research interest because of its potential as commercial bioinsecticides. Some studies had focused on identifying nutrient substrates that B. bassiana can utilize with application to industrial production, while others focused on the

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pathogenic processes of B. bassiana and interactions with insect cuticle [15]. The present study aims to evaluate the pathogenicity of the isolates of entomopathogenic fungus, Nomuraea rileyi and spinosad (bio-insecticide) against corn insect pests under laboratory and field conditions. It is necessary to find alternative safe insecticides to reduce the heavy doses of chemical insecticides which is using for the control of corn pests [5,6].

The present study aims to estimate the effect of Nomuraea rileyi, and spinosad against Sesamia cretica ; Ostrinia nubilalis; Chilo agamemnon .

2. MATERIALS AND MEHODS

2.1 Tested Insects

Sesamia cretica ; Ostrinia nubilalis ; Chilo agamemnon reared on corn leaves under laboratory conditions 26 ± 2ºC and 60± 5 RH. Leaves changed every two days.

2.2 Tested Spinosad

Spinosad-based, GF-120 (Conserve 0.24% CB) as a microbial insecticide, prepared 6 concentrations (prepared according [16]. Percentages of mortality were calculated according to [17] , while LC 50 was calculated throughout probit analysis [18]. The experiment was carried out under laboratory conditions at 26ºC± 2 and 60-70 % RH.

2.3 Entomopathogenic Fungus

The fungus, Nomuraea rileyi was obtained from Florida University USA. They were reproduced on potato dextrose agar (PDA) plus 0.4% yeast extracts (PDAY) and poured onto sterilized Petri-dishes [9]. Plating was performed according to the full dish method. The conidia were transferred from the vial to dish containing medium by platinum loop and then streaked. Plates were incubated at 25ºC with 12 hours photo phase for fungus growth and sporulation. After ten days, conidia were scraped and transferred to conical flasks (200 ml) containing 200 ml sterilized distilled water with 0.02% the speeder sticker (tween, 80). Conidial concentrations in the suspensions were quantified directly under the optical microscope with a haemacytometer. Then the suspensions were standardized until the direct concentration 1x10 7 conidia/ ml was obtained.

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2.4 Laboratory Tests

2.4.1 Treatment of pests

concentrations of N. rileyi ranged from 1x10 2 to 1x10 8 spores/ml were prepared by 1-10 fold dilution from the main stock culture and tested under controlled conditions (26 ± 2ºC and 65± 5 %RH.) against O. nubilalis , S. cretica and C. agamemnon third instar nymphs. Fresh corn leaves were sprayed with the desired fungus concentration ( 3 shots as spurts / leaf ) (Matter et al 1993), left to dry and placed in 1 L plastic containers (one/each). Then, twenty nymphs of either species were placed on each leaf. five containers (replicates) were used / cocentration / microbial pathogen / species. Each container was covered with muslin and incubated at 25 ºC, thereafter, untreated leaves were introduced in the plastic containers to allow gentle transfer of survivors to them and the previous treated leaves were discarded. Untreated leaves were placed in plastic containers sprayed with water only and used as the control treatment. The experiment was replicated 4 times.

2.5 Preparation of Entomopathogenic Fungus N. rileyi against Pests Larvae

Spores of the entomopathogenic fungi; N. rileyi , collected from the surface of mycelium growth and spore suspensions with 2 drops of Tween 80 were prepared and adjusted at 1x10 7 conidia/ ml. Conidial viability was determined by counting germ tubes produced on potato dextrose agar (PDA) plus 0.4% yeast extracts (PDAY) medium after 18 hrs, using light microscope at 400 x. Conidial viability was 95-100%. The surface of cultures was gently brushed in the presence of 20ml of sterilized water in order to free the spores and the suspension was filtered through muslin. Six concentrations of spore suspensions were prepared i.e., 10 7, 10 6, 10 5, 10 4, 10 3, and 10 2 conidia/ml. Piece of corn leaves were dipped in the prepared suspensions and left for drying under laboratory conditions then placed in Petri-dishes (one/dish). For each concentration (4 replicates/ each), ten L3 larvae of each of the tested insects were transferred into each Petri-dish. Control larvae were fed on untreated castor leaves. Percentages of mortality were calculated according to [17], while LC 50 was calculated throughout probit analysis. The experiment was carried out under laboratory conditions at 26°C± 2 and 60-70 % RH. Physiological and metabolic characteristics of N. rileyi.

2.6 Field Trials

Field trials were carried out at Nobaria region (Behera Governorate), Egypt during the two successive corn seasons 2011 and 2012 to study the effectiveness of the tested fungus and spinosad on corn borers. Corn (variety Giza 2) was cultivated by end of May during the two seasons in an area of about 1200 m 2, divided into 12 plots of 100 m 2each. Four plots were assigned for each pathogen, while 4 plots were treated with water and used as controls and fungi were applied at 1x10 8 conidia/ml concentration and 5L / plot. Spinosad was applied at 500 µg/ml. Treatments were performed in a randomized plot design at the sunset with a five liter sprayer. Three applications were made at one week interval at the commencement of the experiment, then 20 samples of plants were randomly collected at certain time intervals from each plot and transferred to laboratory for examination. Average number of each of the tested pests / sample / plot / treatment was calculated, 20, 50, 90 and 120 days post 1st application. The infestation of corn borers were then estimated in each case. After harvest, yield of each treatment was weighed as kgs/Feddan.

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2.7 Yield Assessment

Yield data in treated and untreated plots in the corn harvest seasons (2011and 2012), represented by weight in kgs were determined. Yield loss was estimated according to the following equation:

Yield loss = Potential yield – Actual yield X 100 Potential yield

Potential yield is N. rileyi treatment.

Potential yield was that of (N.r) which gave the best results among the tested pathogens, and was taken as a base for comparing with the other treatments.

3. RESULTS AND DISCUSSION

3.1 In-vitro Effect of Entomopathogenic Fungi on the Target Insects

4 Data in Table 1, show that under laboratory conditions the LC 50 obtained was 142X10 , 146 x10 4, 166 x10 4 conidia/ml after O. nubilalis , S . cretica and C. agamemnon treated with different concentrations of Nomuraea rileyi respectively. When the corresponding pests were treated with spinosad the corresponding LC 50 obtained 166, 179 and 185 µg/ml; respectively (Table 2). Results show that S. cretica and C. agamemnon are less susceptible to spinosad than O. nubilalis Table 2. At the same time, results show that O. nubilalis was susceptible to N. rileyi more than spinosad but the 2 other pests susceptible to both at the same degree.

Similar Results Obtained by [19]) reported that when Phyllotreta cruciferaem, Pegomyia hyoscami and Cassida vittata treated with the fungi Verticillium lecanii, N. rileyii and 6 7 Paecilomyces fumosoroseus the LC 50 ranged between 5.4x10 and 1.43x10 conidia/ml. These results agree with [20, 21]. [22] as they found that the fungi; B. bassiana and M. anisopliae reduced the LC 50 of S. littoralis under laboratory conditions.

Data in Table 3 show that the application of the bioinsecticides which affected on decreasing the infestation under field conditions. The number of infestations of O. nubilalis significantly decreased to 21±2.2 and 24±2.1 after treatment with N. rileyi after 50 day as compared to 59±4.3 and 70±3.1 individual in the control during both two seasons 2011 and 2012. In all treatments the numbers of corn pests were significantly decreased. C. agamemnon infestation decreased to 24±2.3and 21±4.2 individuals after 90 days as compared to97±3.3and 99±1.3 in the control plots in both two seasons. I all treatments Nomuraea rileyi gave the pest results for controlling the target pests.

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The obtained results are similar to other studies carried out by [23] and [24] on their work on C. capitata who reported that after fungi applications during field experiments the yield increased . These results agree with [22, 25, 26], who proved that the application with bioinsecticides increased the yield and decreased the infestation with insect pests in the field. Also, results were in accordance with [23] reported that the virulence of B. bassiana against C. capitata ranged between 8 to 30% and decrease the infestation among the olive fruits. [24] recorded that C. capitata mortality ranged between 69 and 78% after bioinsecticides treatments and the infestation were moderator decreased. At the harvest time the corn weight obtained 3822±54.6 and 3941±60.4 kg/Feddan among the harvested plots treated with N. rileyi as compared to 2810±40.9 and 1710±73.2 kg/Feddan during seasons 2011 and 2012, respectively Table 4. The same results [27,28] controlled cereal aphids with entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae . They found that the infestation was reduced after fungi applications under laboratory and field conditions [29,30,31,32,33] found that the fungi B. bassiana, M. anisopliae, Paecilomyes fumosoroseus Verticillium lecanii ; reduced insect infestations of cabbage and tomato pests under laboratory and field conditions [33,28].

Table 1. Effect of some entomopathogenic fungi N. rileyi against the target insect pests larvae under laboratory conditions

Insects LC 50 slope variance 95%confidence limits Ostrinia nubilalis 142X10 4 0.1 1.01 99-166 Sesamia cretica 146X10 4 0.2 1.00 110-189 Chilo agamemnon 166X10 4 0.1 1.03 135-199

Table 2. Effect of the spinosad against target insect pests under laboratory conditions

Insects LC 50 slope variance 95%confidence limits Ostrinia nubilalis 166 1.01 0.02 111-176 Sesamia cretica 179 0.1 1.01 134-187 Chilo agamemnon 185 0.1 1.01 145-199

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Table 3. Effect of different treatments on the target insect pests under field conditions

Post 1 st Treatments Number of infestation (means)±s.e during both two seasons application Ostrinia nubilalis Sesamia cretica Chilo agamemnon date 2011 2012 2011 2012 2011 2012 20 Control 40 ±3.2 48±3.1 61±2.5 70±2.3 67±3.4 73±2.2 50 59±4.3 70±3.1 79±3.4 83±2.5 84±3.4 91±1.3 90 78±2.3 86±2.1 88±5.1 92±2.1 97±3.3 99±1.3 20 N. rileyi 16±3.1 20±2.1 21±4.4 24±5.1 21±4.3 20±1.2 50 21±2.2 24±2.1 22±4.7 24±3.2 22±3.4 24±4.4 90 25±3.2 19±1.2 21±2.4 19±2.3 24±2.3 21±4.2 20 Spinosad 45±3.3 39±2.1 41±2.6 38±2.4 41±3.4 37±2.2 50 45±4.2 35±1.2 42±3.5 39±3.2 34±2.3 31±4.2 90 39±4.2 30±1.3 35±4.1 29±2.5 28±1.3 26±3.3 F value = 29.4 13.1 33.2 23.1 30.1 29.4 Lsd5%= 15.4 17.4 14.8 16.2 15.3 16.5

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Table 4. Assessments of damage caused in corn field after the fungi treatment

Treatments Season 2011 Season 2012 Wt of corn crop Wt of corn crop yield loss % (kg/ Feddan) yield loss% (kg/Feddan) N. rileyi 3822 ± 54.6 - 3941 ±60.4 - Spinosad 3000 ±60.7 21.5 3129 ±84.1 20.6 control 2810 ±40.9 26.4 2710 ±73.2 31.2 F value 35.6 31.9 Lsd5%= 121.7 125.5

4. CONCLUSION

The fungus, Nomuraea rileyi and natural insecticide spinosad were evaluated against the corn insect pests: Ostrinia nubilalis, Chilo agamemnon and Sesamia cretica under laboratory and field conditions . Results obtained showed that the target insect pests were susceptible to treatments with different concentrations of the isolated entomopathogenic fungus N. rileyi. Under field conditions the percentages of infestation were significantly decreased. The yield loss were significantly decreased among the plots treated with N. rileyi during season 2011 and 2012. When the target insect pests were treated with the natural product spinosad the infestation number of pests were significantly decreased under laboratory and field conditions during season 2011 and 2012.

ACKNOWLEDGEMENTS

This research was supported by Agric. Department, National Research Centre, Cairo, Egypt. Project No ( 9050309 ).

COMPETING INTERESTS

Authors have declared that no competing interests exist.

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Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history.php?iid=239&id=9&aid=1831

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