Comparative study on the Efficacy of Agrotis segetum Granulosis virus ( Agse GVEG ) against Agrotis ipsilon under semi-field conditions

The present work was carried out to evaluate the efficacy of Agrotis segetum granulosis virus (AgseGV) against Agrotis ipsilon larvae in semi-field experiments using spray and wheat bran bait method. A Fluorescent brightener (Tinopal UNPAGX) was added for enhancement of Agrotis segetum granulosis virus (AgseGV). Results showed that, addition of F. brightener 1% to AgseGV suspension using surface contamination technique of semi synthetic diet increased the susceptibility of 3 and 4 instars of A. ipsilon larvae; the LC50 was 2.08×10 OB / ml and 6.10×10 OB/ml, respectively. The LT50 values of AgseGV (9.07×10 OB/ml) +F. brightener 1% was7.67 and 7.85 days for 3 and 4 instars. In addition, semi-field conditions, F. brightener 1% as sun screen enhanced AgseGV persistence on Cotton foliage after spray, the calculated lethal inactivation median time for 50 % reduction in virulence (lethal median inactivation time, LIT50) for 2 instar larvae was0.97 or 2.27 days for virus alone-treatment and AgseGV+ F. brightener additive, respectively. The calculated potency value for F. brightener was 2.335 Fold. Mixture of AgseGV+ F. brightener 1% in bran bait formulation increased mortality% of A. ipsilon in two concentrations of AgseGV (9.07×10 and 9.07×10 OB/ ml) + F. brightener 1% and decreased the number of cut cotton plants infested with 3 instar larvae of A. ipsilon compared to untreated control. The damage reduction to Cotton seedlings with A. ipsilon, was 83.33% and 70% for 3 and 4 instars larvae, respectively, in the AgseGV (9.07×10 OB/ml) + F. brightener 1% compared to control. In conclusion in both spray and bran tested treatments larvae infected with AgseGV+ F. brightener 1% died more rapidly than those infected with AgseGV alone ; indicating that AgseGV appears to have better potential as a control agent for A. ipsilon by adding enhancement additive.


INTRODUCTION
The term cutworms is used to describe the caterpillars of a range of noctuid moths that spend a large portion of their time in the soil and have a tendency to feed wastefully on a wide range of crops by severing plants near the soil surface.They have been recorded as pests on the seedlings of many vegetable crops.In Egypt, most of the recorded damages are produced by the larvae of Agrotis ipsilon and the closelyrelated A. segetum.Cutworms' attacks about 50 plant species, e.g., maize, faba bean, wheat, cotton, berseem, soybean, tomatoes, potato, cantaloupe, cucumber and many other vegetable plant species and can be very wasteful feeders, destroying far more plant seedlings than they consume, (Bourner et al., 1992).There are numerous problems associated with treating cutworms; in particular they are hard to locate until the larvae are large and the damage has been done, since at this stage the larvae spend most of their time below ground where they feed on untreated parts of the crop (Bowden et al., 1983).
Control of cutworms depends exclusively on various kinds of insecticides that have been used with varying success for many decades at least.Baits based on wheat bran, finely spread, have met with some success.
Baculoviruses have high potential to be used as microbial control agent against lepidopteron pests due to their high pathogenicity, environmental safety, and low stability, i.e., restricted host range.Two baculovirus species have been isolated from A. segetum larvae, the granulovirus AgseGV and the nucleopolyedrovirus AgseMNPV (Allaway and Payne, 1983;Lipa and Ziemnicka, 1971;Lipa et al., 1971 andKhattab, 2012.Both types of viruses showed bio control potential in the field (Caballero et al., 1991;Bourner et al., 1992).
A granulovirus of A. segetum (AgseGV) is effective in spray formulation against both A. segetum and A. ipsilon in field trial (e.g.Shah et al., 1979;Zethner et al., 1987), and has also been used in a wheat bran bait trial in Spain (Caballero et al.,1991;Khattab, 2007).
One way to increase the activity of the pathogen is to incorporate it with small quantities of synergistic substances such as F.brighteners.The enhancers decrease, slightly, the time to mortality and decrease the amount of virus necessary to produce mortality (Shapiro, 1992&Shaprio andDougherty, 1994).
The present work describes a semi -field trial to compare the efficacy of granulovirus A. segetum (AgseGV EG ) Egyptian isolate in both spray and wheat bran bait formulations with and without enhancement additive Florescent brightener to control 3 rd and 4 th instar A. ipsilon larvae on cotton seedlings.

Tinopal:
Fluorescent brightener (Tinopal UNPA-GX) was obtained from (Sigma  ).It was obtained as a powder to be used as a sun screen for increasing AgseGV EG stability and persistence under field conditions.It was prepared as water suspension and tested at the concentration 0.1 %.

Tests insect:
Laboratory strain of A. ipsilon was provided by Cutworms Department at Plant Protection Research Institute, ARC. A. ipsilon larvae were mass reared on synthetic diet described by Shorey and Hale (1965), but formaldehyde was removed from diet component when it is used in bio-assay tests.

Laboratory Bioassay tests:
Bioassay tests were performed using surface contamination of synthetic diet with AgseGV EG concentrations (Khattab, 2007).A bioassay plates (L1CEFA, Bad-Salzuflen Germany) were used.Triton X -100 at concentration 2.5% was mixed with virus suspension to reduce AgseGV EG OB clumping.AgseGV EG was tested at concentrations 9.07×10 10 , 9.07×10 9 and 9.07×10 8 OB/ ml against 3 rd and 4 th instar larvae of A. ipsilon.For each concentration, five replicates were used (20 larvae each) after mixing virus suspension with brightener1%.In addition, five more replicates were used as control by adding distilled water only.Mortality % was recorded after 2 and 4 days post-feeding on virus contaminated diet.Abbot formula (1925) was used to calculate corrected mortality %.The LC 50 and LT 50 values were calculated.

Semi -field Treatments:
Seeds of the cotton variety (Giza 86) were cultivated in pots (12.5 cm in diameter × 12.5 cm in length) on May 2013.The efficacy of the Egyptian isolate granulovirus of A. segetum (AgseGV EG ) with or without F. brightener 1% additive was evaluated against of A. epsilon 2 nd , 3 rd and 4 th instar larvae on cotton seedlings.The virus was applied by two application methods, spray and bait formulations.

The spray treatments:
Spray of (AgseGV EG ) was applied to the cotton seedlings using a hand sprayer at concentration of (9.07×10 9 OB/ ml) in addition to 2.5 % (Triton X -100) as a wetting agent.The nozzle was directed towards the plants and spraying took place during sunset.After drying, sprayed leaf from each treatment was collected (as a zero time) then a daily collection was made through 1, 2 and 4 days.2 nd instar larvae were fed on the collected treated leaves for 24hrs.Mortality% of larvae or pupae due to viral infection was done at two days intervals (Elnagar et al. 2003).

Wheat bran bait treatments:
Infestation levels of 3 rd and /or 4 th instars larvae of A. ipsilon was calculated in 20 plants per concentration/ treatment on the soil into the pots near the stem of the cultivated cotton plants when the seedlings had 3-4 leaves.
Wheat bran bait was used as bait at a rate of 50 gm.+1mlAgseGV EG / concentration (two concentrations of AgseGV EG 9.07×10 9 and 9.07×10 8 OB / ml were tested) alone or mixed with F. brightener 1% additive.The wheat bran bait formulations were applied manually in the infested pots on soil surface around the cotton seedling after 2hrs.Control pots were treated with wheat bran bait mixed water only.To compare level of virus infection, samples of A. ipsilon were examined from the treated pots at times, 24 hr., 48 hr. and 72 hr. and 7 days after treatment (Khattab, 2007).

Larval diagnosis:
After 7 days of treatment the soil surface pots, soil samples were inspected to determine the number of dead and alive larvae by shaking them onto a cloth and then alive larvae were transferred individually to semi-synthetic diet described by Shorey and Hale (1965) until death or pupation.Diagnosis of viral deaths was performed visually since the symptoms of viral infection are easily observed.

Damage evaluation:
(AgseGV EG ) treated plants with or without F. brightener 1% additive applied to the cotton seedlings as well as control plants were examined for A. ipsilon damage in the feature of plants or leaves cut after the release of larvae.

Statistical analysis:
Bioassay data were subjected to probit analysis using the method described by Ehab Mostafa Bakr (1998).The relative potencies of the treatments were calculated according to the changes in % mortality.Percent reduction of population was calculated according to the following equation (Mohamed et al., 2000).
Where C= the estimated parameter in check T= the same parameter in treatment

Laboratory bioassay:
Preliminary bioassay was conducted to estimate the relative virulence based on LC 50 values of A. segetum AgseGV EG alone or mixed with F.brightener1%.
Data in Table (1) show that mortality percentage of A. ipsilon 3 rd instar larvae was (14.00, 4.08 and 0.00 %) after treatment with different concentrations of AgseGV EG , while it was 0% at the same concentrations for the 4 th instar larvae, respectively.
Table 1: Mortality response of Agrotis ipsilon 3 rd and 4 th instar larvae fed on diet contaminated with the virulence (AgseGV EG ) alone and with the additive F.brightener1%: Mixing AgseGV EG with F. brightener at concentration 1% increased mortality percentage of 3 rd and 4 th instar larvae of A. ipsilon.Mortality percentage was 93.75, 85.41 and 64.00 for the 3 rd instar larvae of A. ipsilon at AgseGV EG concentrations (9.0 ×10 10 , 9.07×10 9 and 9.07×10 8 OB/ ml) mixed with 1% of F. brightener, respectively.While mortality percentage was 90.00, 82.00 and 52.00% for the 4 th instar larvae of A. ipsilon at the same concentrations of AgseGV EG mixed with F. brightener, respectively.
Taking into account the LT 50 values, it is clear that LT 50 values for 3 rd and 4 th instar larvae of A. ipsilon decreased with increasing viral tested concentration mixed The LT 50 values for the 3 rd instar larvae were 7.67, 8.48 and10.03days at 1%Fbrightener mixed with virus concentrations at 9.07×10 10 , 9.07×10 9 and 9.07×10 8 OB, s/ml, respectively.The LT 50 values for the 4 th instar larvae were 7.85, 7.94 and 10.33 days at the same concentration of virus, respectively (Table 2).Otherwise, no mortality occurred to A. ipsilon 3 rd and 4 th instar larvae treated with F. brightener1% alone.
It is clearly obvious that, the addition of F. brightener-28 additive to the AgseGV EG has increased their virulence against A. ipsilon larvae by reducing both the LC 50 and LT 50 values as reported previously by (Khattab et al., 2004).

Semi-field test: Spray test:
Effect of solar radiation on AgseGV EG efficacy against the 2 nd instar larvae of A. ipsilon after feeding on treated Cotton plants are shown in (Table 3).Data in Table (3) show that increasing time exposure of treated cotton plants by virus separately or mixed with F. brightener reduced mortality percentage of A. ipsilon 2 nd instar larvae.Mortality % after larval feeding on treated Cotton with virus was 73.46, 48.00, 34.00 and 16.00 % at exposure times 0, 1, 2 and 4 days, respectively.Mixing AgseGV EG with F. brightener at 1% increased mortality percentage while mortality % was 81.63, 78.00, 52.00 and 32.00 % at different time exposure of treated Cotton to solar radiation, respectively (Table 3).The calculated lethal median inactivation time for 50 % reduction (LIT 50 ) was 0.97 &2.27days for virus alone-treatment and AgseGV EG + F. brightener additive, respectively.The calculated potency value was 2.335 Fold for F. brightener (Table 3).
The present results are in agreement with those previously reported on the effect of F. brightener-28 as an enhancing additive to NPV virulence (Hamm and Shapiro, 1992;Shapiro and Robertson 1992;Shapiro and Dougherty, 1994;Adams et al. 1994;Shapiro and Vaughan, 1995;Zou and Young, 1996;Vail et al., 1996;and El-Salamouny et al.( 2001) who reported that susceptibility of A. ipsilon neonate larvae was increased by addition of F. brightener-28 (Tinopal LPW) 0.1% to the tested viruses concentration of baculoviruses, Agse MNPV, Auca MNPV and Mamestra brassicae Mabr MNPV by the rate of 1806, 1040 and 336 fold, respectively.Also, Boughton et al., (2001) reported a potential effect of F. brightener on the newly isolated Agrotis ipsilon MNPV.
From the obtained results, it is worth to conclude that, F. brightener additive enhanced virus persistence on cotton foliage and showed the best UV protection effect to AgseGV EG sprayed against Agrotis ipsilon under natural sunlight.

Wheat bran baits tests:
First observation of A. ipsilon larvae used in the present experiment demonstrated that the 3 rd & 4 th instars were often feed directly on the prepared bait .The role of F. brightener in enhancing AgseGV EG persistence was also observed in semi-field tests using wheat bran bait.The reduction damage of A. ipsilon 3 rd instar larvae after 7days of AgseGV EG + F. brightener 1%treatment was 83.33 % and 75.00 % ( in two concentrations 9.07×10 9 and 9.07×10 8 OB/ ml ), respectively.While % of cut plants by A. ipsilon larvae was 60 % after 72 hrs.and reached 100% after 7 days in control treatments (Table 4).On the other hand, the reduction of damage reached 70% in both above-mentioned tested concentrations of AgseGV EG + F. brightener 1% in wheat bran bait applied against A. ipsilon 4 th instar larvae.In control treatment 100% damage was obtained after 72 hrs. of treatment (Table 4).Results indicated that the mixture of AgseGV EG + F. brightener 1% in wheat bran bait decreased the number of cut Cotton plants infested with 3 rd instar larvae of A. ipsilon compared to untreated control (Table 4).Data presented in Table (5) show the mortality % among A. ipsilon larvae collected from Cotton plants 7 days after wheat bran bait application and fed on semisynthetic diet.Mortality% in two concentrations of AgseGV EG (9.07x10 9 and 9.07x10 8 OB/ ml) + F. brightener 1% was( 100 or 90%) and ( 60 or 50%) for3 rd and 4 th instar larvae, respectively.
The present results are in agreement with several authors who reported the successful use of A. segetum GV (AgseGV) for control A. segetum and the closely related A. ipsilon and reducing the cutworm damages in tobacco plots in Pakistan by72 and 100% when AgseGV was used against 2 nd instar larvae of A. ipsilon (Shah et al., 1979), Denmark (Zethner, 1980) Also, AgseGV reduced the natural occurrence of cutworms (A.ipsilon and A. segetum) damage by 64.82% in tobacco, 85% in Okra, 77% in potato and 78% in sugar beet (Zethner et al., 1987) and Spain (Caballero et al., 1990(Caballero et al., , 1991) ) in tobacco, root crops and maize, respectively.Boruner et al. (1992) found that, infection rates in A. segetum 2 nd instar larvae after treatment with AgseNPV and AgseGV were 87.5 or 91% for NPV and 12.5 or 55% for GV in spray or bait treatment, respectively.Mixed inocula of AgseNPV and AgseGV gave intermediate results on maize and beet root (Bourner et al., 1994).Consequently, results obtained by the author, and those presented in the present work show that F. brightener additive 1%, may be used in viral formulations to increase their virulence and speed of kill and protects the virus from the adverse effects of sunlight (UV light) for several days.
Previous reports demonstrated that, baculoviruses were rapidly inactivated after exposure to natural sunlight under natural field conditions.Also, purified virus was more affected than the crude extract (Elnagar and Abul-Nasr, 1980;Tamez-Guerra et al., 2000;El-Salamouny et al., 2000 andElnagar et al., 2003).
From all the previous results such strategies could be stated by early application in field trials using AgseGV EG + F. brightener 1% during cutworm season, to enhance viral persistence and increase speed of kill as well as reduce larval feeding activity of A. ipsilon.Death of those larvae will in turn create a reservoir of virus in the soil and thatch that perpetuates control of A. ipsilon thought the growing season.Thus the soil acts as a reservoir of virus and provides inoculum to initiate new infections each year as reported by Fuxa and Richter (1994).

Comparative study on the Efficacy of A. segetum Granulosis virus against A. ipsilon 25
with F. brightener 1%.

Table 3 :
Effect of the virulence of Agrotis segetum GV (AgseGV EG ) alone and with F. brightener 1% additive sprayed against Agrotis ipsilon 2 nd instar larvae on Cotton foliage exposed to natural sunlight during May, 2013:

Table 4 :
Percentage damage cotton seedling treated with virus-wheat bran bait formulation + F. brightener 1% for controlling 3 rd and 4 th instar of Agrotis ipsilon on Semi-field test, during May, 2013: