Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent under Field-Sunlight Conditions in Egypt

Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent under Field-Sunlight Conditions in Egypt

Alexandra El-Helaly 1 , Magda Khattab 2 , Said El-Salamouny 1 , Mohammed El-Sheikh 1 and Salah Elnagar 1 1. Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, Cairo, Egypt 2. Cutworm and Mole Cricket Plant Protection Research Institute, Agriculture Research Center, Giza, Egypt

Received: December 24, 2012 / Accepted: March 11, 2013 / Published: May 30, 2013.

Abstract : Baculoviruses are effective biocontrol agents except of their short persistence under sunlight conditions. Four promising additives containing different groups of antioxidants were tested on cotton plant foliage. Spodoptera littoralis test insect and its

nuclepolyhedrovirus (SpliNPV) are the standard material used in the investigation. Results are based on leaf-bioassays, to test Original Activity Remaining (OAR) and Lethal Infectivity Time to 50% (LIT 50 ) of tested population of the virus after exposure to natural sunlight. The results showed that cacao additive at 10%, sustained 50% of virus activity for five days post application (113.11 hours) and three days and a half (83.33 hours) at 5% concentration. The virus alone treatment sustained 50% of its activity for only

24.07 hours. The obtained results suggested the possibility of prolonging the virus activity on plant foliage under field applications.

Key words : Antioxidants, baculovirus activity, field-biocontrol application, Spodoptera littoralis NPV, virus protection.

1. Introduction  provide a degree of protection from UV irradiation. They are safe and selective bioinsecticids used world

Spodoptera littoralis (Bosid.) is a major pest of wide against many insect pests, especially cotton and several field and vegetables in Egypt and

Lepidopterous. Their use in insect pest management is many countries in Middle East, during the last two limited because of their sensitivity to limitations can decades research has been made for new and

be overcame by chemical or biological substances non-traditional control agents effective against such as ultraviolet. Baculoviruses are very effective Lepidopterous pest. Control of this pest has depended biological control agents towards several important primary and heavily on insecticides, and this excessive lepidopterous pests [1, 2]. However, baculoviruses are use has led to occurrence of resistant problem. That is rapidly inactivated by the UV spectrum of sunlight why the insect viruses have been used recently as this with most having a half life from two to several hours microbial agent which may play an important role in [3-5]. Thus, field stability is one of the most important reducing pest insect populations steps to judge a protective material. Recently natural Baculoviruses are insect pathogens that are usually materials containing antioxidants such as green tea found when their hosts reach outbreaks densities and a and black tea [6], eucalyptus and mango [7] cacao, disease epizootic occurs. The long term persistence of green coffee, red cabbage, green cabbage and other 16 baculovirus relies on their survival in the external natural materials [8] have been examined. The field environment in the form of occlusion bodies which application of virus-biocontrol agent is facing

Corresponding author: Alexandra Magdalena Ahmed inactivation by UV in the sunlight. Therefore, the El-Helaly, Ph.D., lecture, research field: virology. E-mail:

virus application in the field has a short duration of Alex.ahmad@yahoo.com.

Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent

under Field -Sunlight Conditions, in Egypt

activity against the targeted pests. Means and ways of two layers of clean muslin cloth with a thin layer of enhancing and/or prolonging the persistence of virus

cotton wool in-between them. The mixture of virus activity are needed in order to maximize the benefit of

and tested additive was prepared at a final such a promising bio-control agent. The main

concentration of 5% and 10% additive in the tested objective of this work is to develop a standard additive

mixture [11].

(s) suitable to be used at a large scale to sustain the

2.4 Field Experiment

effectiveness of baculovirus in bio-controlling agricultural pests. This can be obtained through

An experimental area under cotton plants (Variety Giza 83) about 1/4 feddan (1 feddan = 4200 m prolonging virus stability and persistence under the 2 ),

natural sunlight. located at the Agriculture Experimental Station, Accordingly, the present investigation tests 4

Faculty of Agriculture, Cairo University, Giza superior protective additives containing natural Governorate was selected and one small scale open antioxidants [8] under laboratory conditions using a

field test was set up. Two different concentrations of baculovirus (SpliMNPV) and the test insect tested additives were prepared (5% and 10% w/v) and (Spodoptera littoralis), this work was done under two

kept in the fridge 12 hr before spraying. The virus different concentrations and evaluating their role in

inocula SpliNPV was diluted in distilled de-ionized protecting the virus product exposed to sunlight under 8 water and the suspension was adjusted to contain 10

field conditions. OBs/mL (= LC 90%-95%). At the time of field application, the virus and tested additive were

2. Material and Methods

thoroughly mixed together and the measured volume

2.1 Insect Colony was transferred into a hand sprayer. The sticker arabic gum (0.25%) was added to each NPV dilute as a

A laboratory colony of the cotton leaf worm, sticking agent. Virus suspension treatments were Spodoptera littoralis (Boisd.), was established as the

applied separately to cotton foliage using one liter test insect species on a semi synthetic diet of Shorey

hand sprayer. Leaves were randomly collected from and Hale [9].

treated / untreated pants at 0, 0.41, 1, 2, 4, and 7 days

2.2 Virus Inoculum post application and kept individually. Each leaf was placed into a glass bottle, on which 10 neonate larvae

A Local isolate of Spodoptera littoralis multiple were allowed to feed for 48 hr. before transferred embedded nucleopolyhedrovirus (SpliMNPV) was

daily to fresh leaves from the same treatment. Larval originally isolated in Egypt by Abul Nasr [10].

mortality was recorded until day 14. Virus persistence

2.3 UV Protective Additives in the Virus Inoculum was calculated as OAR% (percentage of original

activity remaining) based upon 100% mortality at “0” Four natural materials originated from plants, cacao,

day post treatment. Bioassay tests were repeated in green coffee, green cabbage and red cabbage were

four replicates with 10 larvae per treatment [11]. obtained either fresh (green cabbage and red cabbage),

2.5 Statistical Analysis

where they were washed with distilled water, and dried under cooling or were already in a dry form

Slope and LC 50s values were calculated according (cacao and green coffee). All products were ground to

to the method described by Finney [12]. Original obtain powder, and the required concentrations were

activity remaining percentages were determined for prepared using distilled water, and kept for 24 hours,

each tested treatment using both parameters as then centrifuged. Suspensions were filtered through

described by Muro and Paul [13] to insure the

Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent

under Field -Sunlight Conditions, in Egypt

potential of the tested material to prolong the virus increased the persistence of 50% of its activity for persistence.

almost five days (113.11 hours = 4.712 days), compared to 58.40 h (2.433 days), 59.22 h (2.467 days)

3. Results and Discussion

and 51.91 h (2.162 days) for red cabbage, green coffee In the case of using SpliNPV alone treatment, the

and green cabbage, respectively. recorded rates of mortality among S. littoralis neonate

At the lower concentration 5% (Table 3) also cacao larvae after exposure periods of 10, 24, 48, 96 and 168

gave the best protection of 50% of virus activity for hours to natural conditions were 96.00%, 48.78%,

almost three days and half (83.34 h = 3.47 days) 6.97%, 0%, and 0%, respectively, compared to

compared with (49.78 h = 2.074 days), (44.96 h = 100.00% in case of un-irradiated virus (the calculated

1.873 days) and (53.57 h = 2.232 days) for red LIT 50 was only 24.07 h) (Table 1).

cabbage, green coffee and green cabbage, respectively, The data shows that cacao 10% additive (Table 2)

while virus treatment without additive gave only

Table 1 Effect of cacao, red cabbage, green coffee or green cabbage additive at 5% concentration on the persistence of Spodoptera littoralis NPV (SpliNPV) sprayed on cotton foliage and bioassayed against S. littoralis neonate larvae.

Mortality % among larvae tested with NPV + the indicated additives

Spli NPV alone

Green cabbage M% R% M% R% M% R% M% R% M% R% Zero time 100.00 -

Cacao

Red cabbage

Green coffee

0.00 - LIT 50 hr 24.07

83.34 49.78 44.96 53.57 Slope -48656E-4

-32681E-4 Potency -

-33818E-4 -25225E-4

-40493E-4

3.40 2.00 1.88 2.22 * Refers to either distilled water or additives alone at 10%. M% = Mortality percent. R%= Reduction percentage of virus activity.

Table 2 Effect of cacao, red cabbage, green coffee or green cabbage additive at 10% concentration on the persistence of Spodoptera littoralis NPV (SpliNPV) sprayed on cotton foliage and bioassayed against S. littoralis neonate larvae.

Mortality % among larvae tested with NPV + the indicated additives

Spli NPV alone

Green cabbage M%

Cacao

Red cabbage

Green coffee

M% R% Zero time

97.50 - 10 96.00 4.00 90.00 7.50 86.48 6.37 100.00 - 91.66 5.84

0.00 - LIT 50 hr

-27109E-4 Potency -

4.69 2.43 2.47 2.16 * Refers to either distilled water or additives alone at 10%. M% = Mortality percent. R%= Reduction percentage of virus activity.

Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent

under Field -Sunlight Conditions, in Egypt

Table 3 Effect of cacao, red cabbage, green coffee or green cabbage additive at 5% concentration on the OAR (original activity remaining) of Spodoptera littoralis NPV (SpliNPV) bioassayed against S. littoralis neonate larvae.

Mortality % among larvae tested with NPV+ the indicated additives

Spli NPV alone

Green cabbage M% OAR% M% OAR% M% OAR% M% OAR% M% OAR% Zero time 100.00 -

Cacao

Red cabbage

Green coffee

100.00 - 24 48.78 48.78 95.12 - 87.50 92.10 78.04 80.04 77.50 79.48 48 6.97 7.69 97.50 - 75.00 78.94 62.50 64.10 75.00 76.92

96 0.00 0.00 32.50 34.21 5.00 5.26 2.50 2.56 12.50 12.82 168 0.00 0.00 12.50 13.15 0.00 0.00 0.00 0.00 0.00 0.00 Control*

0.00 - Refers to either distilled water or additives alone at 5%.

Table 4 Effect of cacao, red cabbage, green coffee or green cabbage additive at 10% concentration on the OAR (original activity remaining) of Spodoptera littoralis NPV (SpliNPV) bioassayed against S. littoralis neonate larvae.

Mortality % among larvae tested with NPV+ the indicated additives

Spli NPV alone

Green cabbage M% OAR% M% OAR% M% OAR% M% OAR% M% OAR% Zero time 100.00 -

Cacao

Red cabbage

Green coffee

0.00 - * Refers to either distilled water or additives alone at 10%. **Between brackets are the No. of virus dead larvae / total No. tested.

(24.07 h = 1.00 day). There was not any OAR% [3, 4, 14]. Obtained results proved that the promising remained in examined additives expect of cacao after

additives in present investigation (cacao, green coffee, 168 hr which gave 13.15 OAR% for the concentration

green cabbage, and red cabbage) have pH range of of 5%, for 10% concentration only cacao and green

6.85, 6.67, 5.92 and 6.31, respectively, of which cacao cabbage have 17.94 and 2.5 OAR%, respectively,

was almost neutral which is considered as a safe after 168 hours (Table 4).

substance for NPV formulation, as any alkalinity or From the above mentioned result, it was evident

acidity may break up the polyhedra. The first record of that cacao is the best protective additive. Previous

the use of plant extracts to increase the persistence of studies [14-16] demonstrated that, baculoviruses were

insect viruses was by Shapiro et al. [17, 18]. Both of rapidly inactivated after exposure to SUV or natural

the green tea and black tea were reported to be UV sunlight, in the field. Elnagar and Abul-Nasr [3] found

protective additives to the beet armyworm that under UV in sunlight, purified virus suspension

nucleopolyhedrovirus [5, 6, 11]. This effect could be was less effective than the crude extract as the latter

due to the natural antioxidants present in tea. However, contains coloring material. In Egypt, the effect of

when green tea was included in the present sunlight on SpLiMNPV was thoroughly investigated

investigation, it did not standout as the best protective

Promising Additives to Protect the Activity of Baculovirus Biocontrol Agent

under Field -Sunlight Conditions, in Egypt

additive. The obtained results in the present work albistriga (Walker) (Lepidoptera: Arctiidae), Journal of Biological Control 21 (1) (2007) 169-174.

showed that cocoa and possibly coffee were the best [3] S. Elnagar, S. Abul-Nasr, Effect of direct sunlight on the

UV protective additives to SpliMNPV which also virulence of NPV (nuclear polyhedrosis virus) of the agree with that recently found by El Salamouny et al.

cotton leaf worm Spodoptera littoralis (Bosid.), Z. [6] for the beet armyworm nucleopolyhedrovirus.

Angew. Entomology 90 (1980) 75-80. [4] K.A. Jones, G. Moawad, D.J. McKinley, D. Grzywacz,

Thus, cacao additive showed the highest protection The effect of natural sunlight on Spodoptera littoralis

rate followed by green coffee. In conclusion, cacao nuclear polyhedrosis virus, Biocontrol Science and was the best additive at both tested concentrations (5%

Technology 3 (2) (1993) 189-197.

and 10%). It is worth mentioning that Theobroma [5] L. Villamizar, C. Espinel, A.M. Cotes, Effect of ultraviolet radiation on the insecticidal activity of

cacao L. (Sterculiaceae) and cocoa-derived products Spodoptera frugiperda (Lepidoptera: Noctuidae)

are phenolics-rich food; these products are largely nucleopolyhedrovirus, Journal Revista Colombiana de studied because of the antioxidant and antiradical in

Entomologia 35 (2) (2009) 116-121.

vitro properties of phenolic constituents [19]. The [6] S. El Salamouny, M. Shapiro, K.S. Ling, B.M. Shepard, Black tea and lignin as ultraviolet protectants for the beet

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