The Efficacy and Persistence of Spodoptera littoralis Nucleopolyhedrovirus ( Spli MNPV ) Applied in UV Protectants against the Beet Armyworm , Spodoptera exigua ( Hübner ) ( Lepidoptera : Noctuidae ) under Saudi Field Conditions

Sukirno Sukirno1,2, Muhammad Tufail1,3,*, Khawaja Ghulam Rasool1, Said El Salamouny1, 4, Koko Dwi Sutanto1 and Abdulrahman Saad Aldawood1 1Economic Entomology Research Unit (EERU), Plant Protection Department, College of Food and Agriculture Sciences, King Saud University, Riyadh 11450, Kingdom of Saudi Arabia 2Entomology Laboratory, Faculty of Biology, Gadjah Mada University, Yogyakarta 55281, Indonesia 3Organization of Advanced Science and Technology, Kobe University, Japan 4Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, Giza, Egypt. Article Information Received 13 December 2016 Revised 27 August 2017 Accepted 22 October 2017 Available online 17 August 2018


INTRODUCTION
T he beet armyworm, Spodoptera exigua (Hübner)   (Lepidoptera: Noctuidae) is one of the most serious pests of agricultural crops including vegetables and flowers worldwide (Pogue, 2002;Saeed et al., 2010).This have been used to control S. exigua.Some synthetic insecticides have been studied in the laboratory conditions for the IPM of S. exigua in Pakistan (Saeed et al., 2012).However, recent reports have shown that use of these chemical pesticides lead to the resistance of the insect pests after five generations.Wang et al. (2006) have reported a 345.4 fold increase in resistance of S. exigua to spinosad.
The increasing awareness of the environmental pollution and the growing demand for safe food products lead to find the biopesticides as an alternative to chemicals.Baculoviruses are promising candidates due to their safety and target-specificity (Burges et al., 1980).
A NPV was isolated from the larvae of cotton leafworm, S. littoralis in Egypt (Abul-Nasr, 1956) and its characterization has been done (Seufi, 2008).The virus was tested under the field conditions and it is available in the market under the commercial name of Spodopterin ® and Littovir®.Under the sunny conditions, baculoviruses are affected by ultraviolet (UV).It has been proved that UV in sunlight has negative impact on persistence of microbial control agents (Jaques, 1977;Behle et al., 1997;Jones et al., 1993;Ignoffo et al., 1989).There is a considerable evidence that the UV-B portion of sunlight between 280-320 nm is responsible for baculoviruses inactivation (Ignoffo and Batzer, 1971).In order to overcome the inactivation problem and improve the persistence of baculoviruses under the field conditions, several additives like fluorescent brightener (Dougherty et al., 1996), Congo red, indigo carmine (Shapiro and Robertson, 1990) and carbon (Ignoffo et al., 1991) have been used.
Recently, the use of natural UV protectants in virus formulations has received attention of the researchers involved in biocontrol studies.Natural UV protectants like lignin, a side product of paper industry, has shown a potential as a UV protectant of S. littoralis MNPV (Elnagar et al., 2003;El Salamouny and Huber, 2004).Magnesium lignosulfonate also has been reported to improve the persistence of Helicoverpa armigera MNPV (El Salamouny et al., 2003).Potassium lignite in the presence of pre-gelatinized corn flour has the ability to improve the activity of Anagrafa falcifera MNPV.Moreover, beverages such as black tea, green tea, cocoa and coffee are shown to act as UV protectants for S. exigua MNPV (Shapiro et al., 2008;El Salamouny et al., 2009a, b).Extracts of spices, medicinal herbs and weeds showed a promise as UV protectants (Shapiro et al., 2009b;Shepard et al., 2010).Plant extracts also improve the persistence of biopesticides under the field conditions (Shapiro et al., 2008).
Saudi Arabia has arid weather in which the habitat is dominated with less humidity and rich sunny conditions.The activity of baculovirus, as a biocontrol agent, is rapidly declined when exposed to these harsh conditions.Finding an effective natural UV protectant will lead to resolve the issue of rapid inactivation of baculoviruses under the harsh sunny field conditions of Saudi Arabia.

The insect mass rearing
The beet armyworm, Spodoptera exigua (Hübner) adults were collected by light trap from Al-Amariyah, Riyadh, Saudi Arabia.The captured moths were kept in plastic jars (20 cm x 35 cm) provided with sugar soakedcotton as a food source and opaque paper as an egg laying substrate.The eggs were surface sterilized by dipping the egg mass in 1% chlorox solution (v/v) for 10 seconds and then washed in the running tap water.After hatching, the neonate larvae were transferred into white bean artificial diets (Shorey and Hale, 1965) placed in plastic cups (50 mm x 80 mm).The 4 th instar larvae were then transferred and maintained individually on the artificial diet until the pupal stage.The pupae were surface sterilized in chlorox solution (1%) and then washed in the running tap water.The pupae, 20-25 individuals were then placed in the plastic jars until adults emerge.The colony was maintained under controlled conditions at 25°C and 60-70% RH.

Virus source and natural additives preparations
The virus used in the present study was Spodoptera littoralis multiplenucleopolyhedrovirus (SpliMNPV) available as commercial product Littovir® (Biocontrol, Switzerland).The concentration of the product SpliMNPV was 2x10 12 PIB/L.Dilutions of virus suspensions were prepared as required using autoclaved distilled water.The serial solutions were kept in glass tubes (Lab.Glass, India) at 4°C until further use.
Three plant extracts used as natural UV protectants were: henna, Lawsonia inermis L. (Myrtales: Lythraceae), Green tea leave, Camellia sinensis (L.) Kuntze (Ericales: Theaceae) and clove flower, Syzygium aromaticum (L.) Merrill and Perry (Myrtales: Myrtaceae).The plant materials were air dried for at least 72 h, then ground to make powder.100 gram of each powder was soaked in a 1000 mL (10% w/v) distilled water for 24 h at the room temperature.The mixture then blended and filtered using two layers of muslin cloth.The filtrates were kept at 4°C until use.
The prepared filtrates were added to virus solution to get the final concentration of SpliMNPV as 2.23x10 7 PIBs/mL according to the method described by Shapiro et al. (2008).This preparation was done prior to the field experiment.

Preliminary bioassay
Preliminary laboratory tests were performed to determine the lethal concentration of SpliMNPV against the beet army worm by using diet surface contamination bioassays.For this bioassay, six concentrations of 2x10 1 , 2x10 4 , 2x10 5 , 2x10 6 , 2x10 7 and 2x10 8 PIB/mL of the virus were tested against first and second instar larvae of the beet army worm.While distilled water was used as the control treatment.For bioassay, one mL of each concentration was evenly poured onto artificial diet that was prepared in bioassay plates (5 x 10x 1.5 cm) (LICEFA, Bad Salzuflen (DE), Germany).Then a 50 bioassay cells plate (1cm x 1cm) was fixed into the bioassay plate.The diet was then air died at room temperature for 2 h and 1 larva was introduced in each cell.Each bioassay plate was then covered with two layers of tissue paper and a glass plate that was fixed with rubber band to prevent escaping of the larvae.The preliminary experiments were done against 1 st and 2 nd instar larvae.Each treatment was replicated 3 times using 50 larvae in each.Larval mortality was observed daily for 10 days post-application.

The UV absorption spectra of the plant extracts
To study the UV absorption capacity of the plant additives, the absorption spectra of the green tea, olive, henna, beetroot, lemon, grapes, dates, kiwi, clove and pomegranate were measured by spectrophotometer (JENWAY, 6705 UV/Vis.England) at the range of 190-500 nm.One mL of each sample at 0.5% concentration was used for the UV absorption measurement.

Field trials of natural UV
Field trials were conducted to evaluate the UV protectants for their effectiveness to enhance the persistence of the virus by using 6 week old cabbage plants cultivated at the Educational Farm, King Saud University, Dierab, Saudi Arabia.The cabbage plants were planted at 25 cm by 50 cm distance between plants.
The virus formulations with the best three natural additives (clove, henna and green tea) were prepared six hours before the application.The virus concentration of 100 X LC 95 was diluted with 10% plant extracts and 1% surfactant (final concentration) (Triton X-100, Bio Basic Inc., CA) to obtain a final volume of 500 mL with 10 folds of LC 95 concentration.The formulated virus solution was then placed into a 1000 mL hand sprayer for application.
The treatment was done by evenly spraying the upper surface of eight cabbage leaves per plant in the field.Each treatment was using three replicates (as mentioned above) while six different exposure timings of 0, 1, 3, 5 and 7 days post-application were utilized to test efficacy under field-sunlight conditions.After desired exposure timing, one treated-leave was taken from the field, kept in zip plastic bag and stored at -20°C until used for the bioassay.The field trials were conducted during beginning of the summer in 2015 when the average temperature recorded was 36°C.
For bioassays, a frozen leaf sample (kept at -20°C) for each treatment was taken (30 mm diameter) and placed onto the surface of 10 mm thick bacteriological agar provided in the plastic cup (50 mm diameter 80 mm height) almost six hours before the analysis.The leave discs were then thawed at room temperature for about 1 h.After thawing, 20 1-day-old larvae were released on each leaf disc.The cabbage leaves treated with H 2 O and exposed for 7 days under the sunlight were used as the control treatment.The larvae were allowed to feed on the leaf disc for 24 h and then were transferred to an artificial diet in 50 cells bioassay plate.The effectiveness of the treatment in terms of larval mortality was recorded daily for 10-d post-application.A further bioassay following the same protocol was done by using only clove, the best natural additive against the 2 nd instar larvae of S. exigua.

Statistical analysis
Probit analysis was used to calculate LC 50 and LC 95 values (Finney, 1977).A complete randomized design and ultimately with two factorial analyses of variance (ANOVA) was used to determine the effectiveness of treatments against the S. exigua.The means were separated using LSD test.All analyses were done using SPSS 13.0 (SPSS, 2005).

RESULTS AND DISCUSSION
Preliminary bioassay of SpliMNPV efficacy against 1 st and 2 nd instar larvae of S. exigua The preliminary bioassay of SpliMNPV effectiveness was conducted against the first and second instar larvae of S. exigua to determine LC 50 and LC 95 values in the laboratory.The effectivity of virus against S. exigua larvae was presented in Table I.SpliMNPV showed 76%, 70.67%, 77.33%, 96.67 and 100% mortality at the concentrations of 2x10 4 , 2x10 5 , 2x10 6 , 2x10 7 and 2x10 8 PIB/mL, respectively against the 1 st larval instar.Whereas, mortality in 2 nd instar larvae was 64%, 64.67%, 64%, 84.67% and 98.67%, at the above concentrations.The LC 50 and LC 95 values obtained against the 1st instar larvae were 1.59 x 10 3 and 4.91 x 10 7 PIB/mL, respectively.Whereas in second instar larvae the LC 50 and LC 95 values were 4.99 x 10 6 , 1.06 x 10 8 PIB/mL, respectively.
These data suggest that the baculovirus SpliMNPV is pathogenic to both the larval instars of the beet armyworm although this insect is a non-homologous host of the virus SpliMNPV, a virus strain isolated from the other lepidoperan pest Spodoptera littoralis, as it has been reported to be effective for S. exigua (Pudjianto et al., 2016).It was reported that NPV application might also have some impacts on non-target Lepidoptera (Takatsuka et al., 2007).SpliMNPV was reported to be highly virulent to the 2 nd larval instar of Spodoptera littoralis with the lethal time 50% of 7.32 days (Masetti et al., 2008).SpliMNPV is also reported to be pathogenic to S. frugiperda, whereas the baculoviruses isolated from S. exigua (SeNPV) could not cause infection to S. frugiperda or S. littoralis (Murillo et al., 2003) and termite (Reticulitermes speratus) (Takatsuka et al., 2007).Similar to that of SpliMNPV, a non-homologous pathogenicity has also been observed in cabbage moth (Mamestra brassicae) NPV when applied to non-lepidopteran species (Doyle et al., 1990).The most recent study of the local SeNPV has been reported from Egypt which causes 91.02% and 6.66% mortality against 5 th instar larvae and pupal stage of the beet army worm, respectively (Khattab, 2013).Means in the same column followed by the same letterwere not significantly different at α: 0.05.PIB indicates concentration of polyhedral inclusion bodies of the virus.Means in the same column followed by the same letter (small letter) are not significantly different at α: 0.05.Means in the same row followed by the same capital letters are not significantly different at α: 0.05.V, HN, GT and CV are the virus, henna, green tea and clove.

The UV absorption spectra of the plant extracts
The absorption spectra of the tested natural additives are presented in Figure 1.Among the 10 natural additives, clove, henna and green tea have shown the highest rate of UV absorption.The UV absorption ability of these additives reveals that these natural additives might have the potential to be used as UV protectants for virus when applied under field sunlight conditions.Based on our absorption spectra experiments, clove, henna and green tea extracts showed a good potential as UV protectants even against a wide range of UV light starting from UV-B (280-320 nm) to UV-C (<280 nm).Moreover, our previous findings indicated that black tea is also a good UV absorber of UV-B (El Salamouny et al., 2009b) and provides the UV protection to the S. exiqua MNPV (SeMNPV) when used against beet armyworm.

Evaluation of SpliMNPV persistence with natural UV protectants against the 1 st and 2 nd larval instars of S. exigua under field conditions
The persistence of SpliMNPV after blended with natural plant extracts (clove, henna and green tea) against S. exigua was tested under field-sunlight conditions in Riyadh, Saudi Arabia.The data presented in Table II revealed the effectiveness of virus formulations on the mortality (%) of S. exiqua.The highest mortality was recorded on day 0 of sunlight exposure and was declined in parallel to increase of the sunlight exposure timings.It is worth mentioning that in the virus alone treatment, the mortality was decreased up to 10% on day 7 of sunlight exposure as compared to those of un-exposed treatment.Meanwhile in henna, green tea and clove treated virus applications, the decrease in mortality was up to 23, 16 and 4%, respectively.The results indicated that the virus (SpliMNPV) treated with henna and green tea has presented less protection as compared to clove treatedvirus where the persistence remains high on day 7 of sunlight exposure.Means in the same column followed by the same letters (small letters) are not significantly different (in the exposure timings effect on the insect mortality) at α: 0.05.Means in the same row followed by the same capital letters are not significantly different (in the formulation effect on the insect mortality) at α: 0.05.
A further experiment on effectiveness of virus formulated with clove, the best UV protectant in this report, against second larval instar of S. exigua was also performed under field conditions (Table III).The results indicated that the mortality (%) of S. exiqua in virus alone treatment was decreased from 88% at day 0 to 5.37% (F= 29.71; P< 0.001) at day 7 of sunlight exposure.On the other hand, the mortality in clove-treated virus applications was 78% on day 0 and 32% on day 7 of sunlight exposure.The low mortality (%) in 2 nd larval instar as compared to that of the 1 st larval instar suggests that the 2 nd larval instar is probably less susceptible than that first larval instar.The addition of clove provides at least some preliminary UV protection, because the mortality of tested larvae was higher than virus alone treatments.
Studies have documented that persistence of baculoviruses is affected by the ultraviolet sunlight.Ultraviolet portion in sunlight is deleterious to the virus activity as it causes DNA and protein matrix degradations (Bandyopadhyay et al., 1999) by producing two types of pyrimidine dimers; cyclobutane pyrimidine dimers and pyrimidine-pyrimidine 6-4 photoproducts (Friedberg et al., 1995).Based on our finding, the virus alone (without additives) lost its 83% pathogenicity after 7 days of sunlight exposure when applied against the 2 nd larval instar of beet army worm.These results are in parallel to those reported by Shapiro et al. (2008) where 98.7% decrease in pathogenicity was observed.
The finding of clove as a good UV protectant agrees with the results reported by Shapiro et al. (2008) since it contains high concentration of antioxidant thus able to absorb the ultraviolet (Shapiro et al., 2009b;El Salamouny et al., 2009a, b).It is known that clove extract contains tannins and flavonoid that play an important role in absorbing the UV light (Shapiro et al., 2009a).Clove gave more improvement in the persistence of SpliMNPV in the field as compared henna and green tea.
The addition of clove extract improved the persistence of SpliMNPV under field conditions.The present results are in agreement with the previous findings on green tea, black tea and kudzu as SeMNPV additives against larvae of the S. exigua (Shapiro et al., 2009a;Shepard et al., 2010).The most recent research on additives to increase the virus persistence under ultraviolet A (UV-A) has been done by using microencapsulation polymer methods of starch (3%), gelatine (3-5%), sodium alginate (3%) against another lepidopteran pest, the cotton bollworm (Helicoverpa armigera).It was reported that gelatine (5%) provides high protection up to 93.3% after being exposed under UV-A for 72 h, while starch (3%) has shown only a protection of 3.7% (Gifani et al., 2015).

CONCLUSIONS
The present findings conclusively demonstrate that SpliMNPV that was originally isolated from Spodoptera littoralis has high potential to be used as a biopesticide to control another lepidopteran pest, the beet army worm (Spodoptera exigua).Although, the effectiveness of commercial formulation remains high until 7 days of field application, however, the addition of clove extract as a natural UV protectant provides more persistence than that of virus alone applications for the later instar infestations.This extract will inhibit the fast inactivation of baculoviruses as a potent UV protectant under the harsh Saudi sunny field conditions.Further screening of natural UV protectants, especially from the local resources to enhance the effectiveness/persistence of the virus is recommended.