Effectiveness reduction of nucleopolyhedrovirus against Chrysodeixis includens days after application in soybean plants

The use of nucleopolyhedroviruses (NPVs) can provide an effective and environmentally benign alternative to synthetic chemicals. The efficacy and persistence of their occlusion bodies may be affected by several factors. The present study investigated the efficacy of isolate Chin-IA (I-A) (ChinSNPV) on third-instar Chrysodeixis includens larvae after 10 days of its application in soybean plants under semi-field conditions. Two concentrations of virus (2x10 11 and 10x10 11 PIB ha -1 ) and water (control treatment) were applied in soybean plants. Leaves were collected 0, 2, 4, 6, 8 and 10 days after application of treatments, and then used as disc format to feed the soybean looper larvae in the lab. It was considered a split plot scheme with a randomized complete block design (three treatments and eight repetitions) and the mortality data adjusted to a regression equation using the exponential decay model. In the day of application, the lowest and highest virus concentration caused 87.5 and 100% of mortality of larvae, respectively. However, their efficiency was reduced, on average, 70% after four days of the application. Among the factors may have compromised the persistence and activity of the isolate studied, the time after its application and ultraviolet radiation may have had a strong influence on this results. Its notable the potential use of ChinSNPV in programs for the management of this pest in soybean. Similarly, the properly application of this virus at sundown will ensure its efficiency for an extended period.

Brazilian grain production was estimated in 229.5 million ton to the 2017/2018 harvest with soybean crops contributing with around 50% (114.9 million ton) to this scenario (CONAB, 2018).Simultaneous with this large production, soybean producers have had significative economic losses due to the damages caused mainly by defoliating pests such as Chrysodeixis (= Pseudoplusia) includens (Walker, [1858]) (Lepidoptera: Noctuidae).The soybean looper, as it is known, was considered as a secondary pest.However, has become the primary soybean leaf eater in the past years possibly due to the increasing number of pesticide applications, especially those fungicides used to control Asian soybean rust, Phakopsora pachyrhizi Sydow & P. Sydow, which has action against natural biological control agents, particularly the entomopathogen fungus Nomuraea rileyi (Farlow) (Bernardi et al., 2012).
The development of management strategies that integrate the potential of microorganisms may lead to a more effective control of this important defoliating pest.Among the microbial control agents, the virus of genus Nucleopolyhedrovirus (NPVs) from Baculoviridae family is a promisor candidate being highly virulent in laboratory and field conditions, persisting during years in the soil renewing the infecting cycles.NPVs represents a sustainable alternative to the environment, humans, pollinators and vertebrates (Moscardi et al., 2011).The C. includens Single Nucleopolyhedrovirus (ChinSNPV) was morphological and molecularly characterized by Alexandre et al., (2010).The authors have shown that, the virions present a single nucleocapsid per envelope occluded in a protein matrix, forming the occlusion bodies (OB).Furthermore, ChinSNPV's genetic diversity, phylogeny and the complete genome was revealed recently and compared with other Alphabaculoviruses (Craveiro et al., 2013;2015).
The maintenance of OB integrity is crucial to viral persistence outside the host and for initiating infections in new host insects, thus some climatic factors may be limiting for its appropriate performance in the different agroecosystems (Stevenson et al., 2010).Solar radiation is the major factor affecting field persistence of baculoviruses with mean half-life generally ranging from 2 to 5 days (Moscardi et al., 2011;Jeyarani et al., 2013).Several studies reported the use of microencapsulation (Gifani et al., 2015) and UV protectants to prevent baculovirus inactivation such as reflectants (Wu et al., 2015), optical brighteners (Bernal et al., 2014), natural additives (Sutanto et al., 2017), and other general and selective absorbents, and chromophores (Beas-Catena et al., 2014).Temperature is another factor that can also affect the success of applied viruses by increasing the lethal time in regions with low mean temperatures, and by inhibiting the infection at low or high temperatures (Moscardi et al., 2011).Hence, timing of applications associated with an appropriate host age structure and density is a key feature for successful use of baculoviruses (Silva and Moscardi, 2002).
The present study was undertaken to investigate the efficacy of isolate Chin-IA (I-A) (ChinSNPV) on thirdinstar C. includens larvae days after its application in soybean plants under semi-field conditions.We made an association with the climatic conditions of the region with a view to provide better recommendations to use of this virus on soybean crops.
The experiment was carried out in semi-field conditions, during soybean crop 2016/2017 season, at Embrapa Western Agriculture located in Dourados city (22º16'30"S, 54º49'00"W, 408m), Mato Grosso do Sul State.Soybean plants from BRS 255 RR cultivar, adapted to edaphoclimatic conditions of region, were maintained in plastic vases (8L of capacity) containing a mixture (1:1:1) of land, sand and cattle manure under greenhouse conditions until to achieve the flowering stage (R2).The vases were then exposed to natural conditions being randomly placed under wooden structures.Two concentrations of isolate Chin-IA (I-A) (ChinSNPV) (2x10 11 and 10x10 11 PIB ha -1 ) and water (control treatment) were applied in soybean plants at 8 a.m. using a constant pressure bar (CO 2 ) sprayer equipped with empty cone nozzles and water volume equivalent to 100 L ha -1 .The isolate Chin-IA (I-A) was provided by Embrapa Soybean (Londrina/PR) and is considered one of the most pathogenic isolates to C. includens (Alexandre et al., 2010).
Leaves from the middle third of the plants, portion where soybean loopers remains mainly in field conditions (Czepak and Albernaz, 2014), were obtained in the day of application and two, four, six, eight and ten days after application of treatments (DAA).Soybean leaf discs were prepared (area of 12.56 cm²) using a metal hole-puncher with approximately 4 cm of diameter in the Entomology's laboratory.Third-instar C. includens larvae from an existing colony were placed in sterile plastic Petri dishes (1 larvae/Petri dish), fed with one soybean leaf disc and incubated in an environmental chamber at 25±2 ºC, relative humidity of 70 ± 10% and photoperiod of 12L:12D.The leaf discs (not treated) were replaced every 48 hours until the death of larvae.Mortality was checked daily, and dead larvae were storage in eppendorf vials at -20 ºC.
A split plot scheme with a randomized complete block design was considered in this study comprising three treatments (two virus concentration and control) with a total of 10 soybean plants per treatment and eight replications (1 larvae/Petri dish).The evaluation times (0, 2, 4, 6, 8 and 10 days after application of treatments) corresponded to the subplots.The mortality results were submitted to variance analysis using the ASSISTAT program (Silva and Azevedo, 2002) and therefore, adjusted to a regression equation using the Exponential decay model with two parameters given by: y = a.℮ -b.x where, y = Average percentage of mortality (%); x = Days after application of treatments (DAA); a = Reflects the percentage of mortality in the day of isolate Chin-IA (I-A) and b = Reflects the speed rate in which mortality decreases.
The meteorological data (temperature, relative humidity, rainfall and solar radiation) during the experimental period were collected at the Embrapa Meteorological Station.A significant interaction between treatments and days in which the soybean leaves were collected after their application was observed, F (3, 6) = 8.56, P<0.01 (Table 1).In addition, there was significative effect of treatments applied into soybean plants against soybean lopper larvae (P< 0.05).The mortality of C. includens larvae at 0 DAA, was and 100% for the lowest and highest virus concentration used in this study, respectively, whereas all larvae of control treatments remained alive (Table 2).At 2 DAA, only the concentration 10x10 11 PIB ha -1 was able to cause a significative mortality to the larvae when compared to the control, without differing from that mortality found for the concentration 2x10 11 PIB ha -1 .After larvae ingest the OBs present on plants, they are solubilized in the midgut, releasing the virions who initiate infection in the midgut cells and then, spread through tracheal epidermal cells to other tissues (Harrison and Hoover, 2012).A series of behavioral and morphological changes may be observed between 3 rd and 4 th days after infection in the insect, such as the reduction in their feeding, retardation of their growth, discoloration and rupture of the integument, culminating in their death (Katsuma, 2015).
From the fourth day after treatments' application, there is no significative effect of mortality to the C. includens larvae.The isolate Chin-IA (I-A) reduced its efficiency against C. includens larvae in 75 and 62.5% to the lowest and highest concentrations, respectively, between 0 and 4 days after application.These findings are similar to that found by Young and Yearian (1974) when the virus B. heliothis lost around 75% of its original activity into control Heliothis virescens (Fabricius, 1781) (Lepidoptera: Noctuidae) after 48 hours exposure on the soybean leaves in natural conditions.The purified B. anticarsia also had 50% of reduction in the control of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Noctuidae) in the second day after to be applied in soybean plants (Batista Filho et al., 1992).Revista de Agricultura Neotropical, Cassilândia-MS, v. 5, n. 4, p. 94-99, out./dez.2018.
The exponential decay model provided for the lowest Chin-IA (I-A) concentration the values of 87.53 ± 0.86 and 0.99 ± 0.03 for a and b parameters, respectively, and a value of coefficient of determination (R 2 ) equal 0.99 (Figure 1).Regarding the highest Chin-IA (I-A) concentration, the values for a and b parameters were 99.73 ± 5.74 and 0.45 ± 0.05, respectively, with a R² of 0.98.Thus, it was observed low standard error values to the both parameters evaluated and R², indicating that the model regression chosen have fit very well.The persistence and activity of the virus studied here may be compromised by several factors.Host plants plays an important role in the efficiency of ChinNPV against C. includens producing some chemical substances responsible for the loss of viral activity (Stevenson et al., 2010).Climatic conditions are also recurring concern when microorganisms are exposure to natural conditions affecting their performance, persistence, virulence and dispersion (Jaronski, 2010).During the experimental period, the isolate Chin-IA (I-A) was exposure to mean temperatures ranging from 25.8 to 29.1 ºC (Table 3) falling into favorable limits for this pathogen according to Alves and Lecuona (1998).Similar conditions were not harmful to the activity of virus B. anticarsia (Johnson et al., 1982).For this reason, we believe that the temperature did not influence in the loss of activity of isolate used.In addition, theses temperatures are pretty convenient since the C. includens outbreaks are related with warmer and wetter periods of the year as well as higher availability of host plants (Santos et al., 2017).Virus has shown to be the less affected by the relative humidity of the air among the microorganisms control agents (Alves and Lecuona, 1998).During the experiment, the air relative humidity remained above of 50% achieving a peak of 73% in the 5 th day due to rain.Considering that around 70% of efficiency of our nucleopolyhedrovirus was reduced during the four first days, this factor does not seem to have had expressive influence over time.
The most likely reason for the loss of activity of ChinNPV (I-A) against soybean lopper larvae may be attributed to the solar radiation, in which it ranged from 8.5 to 15.7 MJm² day -1 during the period that the virus was exposure on the plants.Ultraviolet radiation has been extensively reported as the major cause of activity loss in field-applied baculoviruses.The UV acts directly on the nucleic acids causing alterations or destroying them (Grzywacz, 2016), being necessary the reapplication of virus in the field at 7-10 days interval (Jeyarani et al., 2013).In this scenario, it is important that virus be applied at sundown to reduce the effects of UV radiation on the activity of them, ensuring its efficiency during the first night after application on the crop (Silva and Moscardi, 2002).The time of application used in this study may have been  We have shown in the present study that two concentrations of isolate Chin-IA (I-A) (ChinSNPV) lost, on average, 70% of their efficiency into control the third-instar C. includens larvae after 4 days of application in soybean plants.Solar radiation and time of application may have had strong influence in the results obtained here.The implementation of ChinSNPV in soybean integrated pest management (IPM) programs is a sustainable alternative where its correct use, whether associated with other agricultural practices may bring successful results and hence, facilitated farmer acceptance.

*
Means followed by the same letter in the column are not statistically different by the Tukey's test at the 0.05 level.
responsible for the fast effectiveness reduction of ChinNPV (I-A) in control C. includens larvae.AccordingSilva and Moscardi (2002), the highest efficacy of the AgMNPV (nucleopolyhedrovirus of A. gemmatalis) was observed for applications made at 2:00 a.m. or 8:00 p.m. compared to those made at 8:00 a.m. and 2:00 p.m.Despite the loss of the activity of the baculovirus applied, its polyhedral bodies might be continuously renewed through dead caterpillars in natural conditions(Moscardi et al., 2011).

Table 3 .
Meteorological data collected at the Embrapa Meteorological Station during the experimental period.