TY - JOUR
AU - Babcock,, Jonathan
AB - Abstract Maize (Zea mays L.) is one of the most important and widely cultivated crops in Argentina. Fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith), is a common maize pest capable of causing significant yield losses and is most destructive in late-planted maize in subtropical regions, going through five to six generations per growing season. The Bacillus thuringiensis (Bt) trait Herculex I Insect Protection technology by Dow AgroSciences and Pioneer Hi-Bred (HX I, event DAS-Ø15Ø7-1), expressing Cry1F protein, was launched in the 2005–2006 season in Argentina and was widely adopted because of the high level of efficacy against S. frugiperda, as well as other pests such as Diatraea saccharalis (J.C. Fabricius). However, increased late-season plantings, limited adoption of refuge, and properties of S. frugiperda biology (high number of generations and migratory behavior) have led to high S. frugiperda exposure to Cry1F and resistance selection pressure. Field efficacy monitoring has been conducted throughout the main maize production areas in Argentina from 2009 to 2016. Laboratory monitoring has been conducted throughout the same areas from 2010 to 2015. Here, we describe changes in field efficacy of HX I and the results of laboratory-based susceptibility monitoring conducted using purified Cry1F protein. Increases in larval survival and crop damage were evident throughout the 2012–2016 period and spanned the majority of maize production areas in Argentina. Over the same period, random larval collections showed increasing survivorship on diet containing purified Cry1F protein. These field and laboratory studies confirmed that resistance to Cry1F has developed and is now widely distributed in S. frugiperda populations in Argentina. Bacillus thuringiensis, maize, Argentina, Spodoptera frugiperda, resistance Maize is one of the three most important crops grown in Argentina, with 5.1 MM hectares planted in the 2016–2017 season (Bolsa de Cereales de Buenos Aires 2017). One of the main pests for this crop is fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), which prefers maize as host crop (Perdiguero et al. 1967; Willink et al. 1993a,b; Murúa et al. 2008). This lepidopteran pest causes significant yield losses, especially in tropical and subtropical regions of Latin America (Sifuentes et al. 1971, Sparks 1979, Artigas 1994, Clavijo and Pérez Greiner 2000, Molina-Ochoa et al. 2003). In Argentina, S. frugiperda is distributed in most maize production areas; however, the northern maize production region (Tucumán, Chaco, and Misiones) is estimated to be the southern limit where S. frugiperda can overwinter (Murúa and Virla 2004a). In northern Argentina, S. frugiperda is considered the most important pest in maize, but it is of lower pest importance in Cordoba province and to the south (Willink et al. 1991a,b, 1993a; Virla et al. 1999). Spodoptera frugiperda has a range of feeding behaviors in maize, which includes cutting of young plants, leaf and ear feeding, and whorl damage (Artigas 1994, Bentancourt and Scatoni 1995, Clavijo and Pérez Greiner 2000). Damage to maize at early vegetative stages can produce severe losses; however, at advanced vegetative stages, plants can recover and reach a normal reproductive stage (Casmuz et al. 2010). Yield losses from S. frugiperda infestations can range from 9 to 72% depending on the stage of development at which the infestation occurred (Perdiguero et al. 1967; Willink et al. 1991a,b; Sosa 2002). Considering an average yield of 8,000 kg/ha (Bolsa de Cereales de Buenos Aires 2017), this represents a total yield loss of between 720 and 5,760 kg/ha. Historically, the most widespread method of control for S. frugiperda in maize was chemical insecticide sprays; however, the efficacy of this method is typically low because of the tendency of the pest to feed inside the whorl, which makes it difficult for the chemical application to come in contact with the larvae. Additionally, the timing of applications is often not optimal (García Roa et al. 1999, Berta et al. 2000, García Degano et al. 2009). The introduction of maize plants transformed to express Bt proteins (derived from Bacillus thuringiensis (Berliner) (Bacillales: Bacillaceae), a soil bacteria) in Argentina started with events 176 (SYN-EV176-9), MON 810 (MON-ØØ81Ø-6), and BT11 (SYN-BTØ11-1) in 1998. These Bt maize events express the Cry1Ab protein and provided high levels of protection against some lepidopteran maize pests, particularly sugarcane borer, Diatraea saccharalis (J.C. Fabricius) (Lepidoptera: Crambidae) (Trigo and Cap 2003). HX Ian event co-developed by Dow AgroSciences LLC and Pioneer Hi-Bred International, expressing Cry1F protein, was approved in 2005 in Argentina. Unlike the earlier Bt events, HX I provided excellent control of S. frugiperda (Murúa et al. 2013), as well as other pests such as black cutworm (Agrotis ipsilon) (Hufnagel) (Lepidoptera: Noctuidae) and D. saccharalis, and also provided tolerance to the broad-spectrum herbicide glufosinate ammonium. Prior to registration for use in Argentina, HX I was registered in the United States and researchers showed high levels of Cry1F efficacy against S. frugiperda (Buntin 2008; Siebert et al. 2008a,b). At the time of launch in Argentina, HX I hybrids provided excellent control of S. frugiperda ((Murúa et al. 2013). For commercial cultivation of HX I in Argentina, technology developers established a locally adapted insect resistance management (IRM) program to delay and manage resistance development. This plan included recommendations for growers (refuge planting, scouting, and insecticide application when damage thresholds were reached) as well as field and laboratory monitoring program to detect variations in the susceptibility of S. frugiperda field populations to Cry1F. For the laboratory monitoring that was conducted using purified Cry1F protein, a susceptibility baseline was established in 2006; the diagnostic concentration was estimated as 10 μg Cry1F/ml and used in the annual monitoring efforts. Spodoptera frugiperda resistance to HX I maize was first reported in Puerto Rico in 2006 (Blanco et al. 2010, Storer et al. 2010), Florida between 2011 and 2013 (Huang et al. 2014), North Carolina in 2014 (Li et al. 2016), and Brazil in 2011 (Farias et al. 2016). In Puerto Rico, resistance was characterized as autosomal and recessive (Storer et al. 2010), and this resistance is believed to be the source of resistance in Florida (Huang et al. 2014) and North Carolina (Li et al. 2016), due to the ability of this species to migrate long distances and its polyphagous behavior. Reports from resistant populations in Brazil that were tested in laboratory bioassays with artificial diet also characterize this resistance as autosomal and incompletely recessive (functional dominance values between 0.0 and 0.14; Farias et al. 2014). High pest pressure, optimal weather conditions for the pest, maize production for many years, geographic isolation (resulting in reduced gene flow), insufficient refuge planted, and adoption of late planting all may have been causes for evolution of resistant populations in S. frugiperda (Storer et al. 2010) and D. saccharalis (Malacarne et al. 2017) in Argentina. In this work, we report the results from a systematic monitoring program launched in Argentina in 2005 and conducted until resistance confirmation in 2015. The field and laboratory monitoring program targeted maize production areas with medium to high pressure of S. frugiperda and, therefore, high rates of technology adoption. With these results, we identified the evolution of resistance in S. frugiperda to Cry1F in Argentina. The resistance was subsequently characterized by Chandrasena et al. (2018). Materials and Methods Laboratory Monitoring for Cry1F Susceptibility in S. frugiperda Spodoptera frugiperda susceptibility to Cry1F was monitored annually by evaluating the number of survivors reaching third instar or greater after 7 d, using the diagnostic concentration, previously established, of 10 µg Cry1F/ml diet (Chandrasena et al. 2018). Insect Source and Rearing Conditions From 2010 through 2015, 15–25 S. frugiperda populations per year were collected at random from non-Bt fields (maize or sorghum) in Argentina, including the provinces of Buenos Aires, Chaco, Córdoba, Corrientes, Entre Ríos, Misiones, Salta, Santa Fe, Santiago del Estero, and Tucumán. These provinces are within the four primary maize-growing regions of Argentina: northwest (NOA), northeast (NEA), Corn Belt, and Central. Insect collections were made with the greatest intensity in the NOA and NEA regions, medium intensity in the Central region, and lowest intensity in the Corn Belt. This variation in insect collection was done to account for the differences in survival of S. frugiperda due to temperature variations (i.e., there is no overwintering in the Central and Corn Belt regions). For each location, approximately 300 viable larvae (which is considered enough to ensure genetic diversity), of mixed instar, were collected to start a population for laboratory assessment (Chandrasena et al. 2018). During collection, each larva was placed in a plastic test tube (10 cm long) with a fresh piece of non-Bt maize leaf tissue. The larvae were reared under controlled conditions (27 ± 2°C, 14L: 10D photoperiod and 70–75 % RH) and were carefully monitored to remove sick or parasitized individuals. Prior to pupation, larvae were fed artificial diet (Southland Products® Inc., Lake Village, AR). Emerging adults from each population were placed in cylindrical polyethylene terephthalate cages (30 cm long, 10 cm diameter), which were covered at both ends with muslin tissue for proper aeration and included a folded piece of paper as an oviposition substrate. Adults were fed using a piece of cotton wool soaked in a honey solution (1:1; honey: water). Daily egg masses were taken from the cages and placed in glass tubes (10 cm long, 1.5 cm diameter) with artificial diet (Southland Products® Inc.) until larvae reached third instar. Third-instar larvae were isolated into individual tubes to prevent cannibalization. Population Sensitivity to Cry1F Assays were conducted when suitable numbers of larvae were available, which corresponded to either the F1, F2, or F3 generation. Each colony established from field collections was bioassayed separately and neonate larvae were exposed to Cry1F truncated purified protein at the diagnostic concentration. From 2009 to 2014, monitoring was performed using the diet-incorporation technique with the diagnostic concentration of 10 µg/ml of purified Cry1F protein. For the 2015 season, monitoring was performed using a diet-overlay method with the diagnostic concentration of 2,000 ng/cm2, which was based on studies conducted for evaluating susceptibility of S. frugiperda in Brazil (Farias et al. 2014). Use of the overlay technique allowed comparison with monitoring data from other countries such as Brazil and Colombia and made more efficient use of the available Cry1F protein. To prepare the solution used for the diet incorporation, lyophilized purified Cry1F protein powder (TSN303216 toxicological lot prepared by Dow AgroSciences LLC, Indianapolis, IN) was diluted in 10 mM potassium phosphate buffer (pH 10.5) and carefully mixed with warm (approximately 50°C) artificial diet (Southland Products® Inc.) at the desired concentration of 10 µg Cry1F/ml diet. Bioassays were conducted in 128-well bioassay trays from CD International (Pitman, NJ). Each well of the trays was filled with 0.5 ml of warm diet mixed with Cry1F protein. In the diet-overlay bioassays, 0.5 ml of warm diet that had not been mixed with Cry1F protein was added to each well of the bioassay plates. After the diet had cooled and solidified, 50 µl of a concentrated Cry1F solution at the desired concentration was carefully pipetted onto the top of the artificial diet in each well. In both cases, the negative control consisted of the artificial diet mixed with or overlaid with potassium phosphate buffer without Cry1F. Trays containing diet incorporated with the Cry1F protein were left to cool, solidify, and dry for 30 min, were then covered with plastic bags to maintain dark conditions, and were stored at 4°C for 24 h. After this time, trays were removed from the fridge and left 1 h at 25°C. Each well was infested with neonate (unfed larvae of less than 24 h of age) using a soft paint brush. Wells were sealed with adhesive covers (CD International) to prevent escape of larvae and allow proper aeration. Inoculated trays were placed in a chamber maintained at 27 ± 2°C, 14L: 10D photoperiod and 70–75 % RH. Six or seven replications of 16 larvae per population were evaluated in each treatment. Seven days after inoculation, bioassays were assessed under a binocular magnifying glass. A criterion of ≤20% dead larvae in control replicates was established for validity of the assay. In each assay, the number of live and dead larvae and their larval instar were recorded. Percentages were calculated on the following criteria: mortality (number of dead and live larvae in the first instar); growth inhibition (indicated by visually assessing larval survival as well as failure to develop out of the first and second instar); and survival (number of live larvae that reached the third or higher instar). For each population, the corrected mortality (CM%) was calculated (Abbott 1925) and corrected survival (CS%) was calculated as 100 − CM. Field Monitoring Field Monitoring Locations Efficacy of the Cry1F protein in commercial hybrids was evaluated in the field using experimental trials, which were run every summer season (November to March) from 2009 to 2016. Commercial hybrids expressing the Cry1F protein (HX I) were compared to non-Bt hybrids. The hybrids used in the field trials were chosen according to their adaptability to each geographical area. A total of 42 trials were strategically located throughout the main maize production areas in Argentina. These areas (Central region, Corn Belt, NEA, and NOA) reflected a wide range of environmental conditions and S. frugiperda pressure. All studies were maintained using agronomic practices for optimal productivity. Seed used for these trials was treated only with a commercial fungicide and no preventive treatments of soil or foliar insecticides were applied to avoid any interference with the trait efficacy. Field Data Collection HX Imaize expressing the Cry1F protein and non-Bt near-isoline hybrids were planted in four identical blocks arranged in a randomized block design. Each plot area was four rows by 5 m in length, and row spacing varied from 35 to 70 cm, according to commercial practices in each region. Assessment of S. frugiperda foliar feeding damage was carried out using the visual scale developed by Frank M. Davis and W. P. Williams (Davis et al. 1992) which assigns a score to each plant according to the severity of feeding damage. This scale ranges from 0 to 9 with 0 representing no damage and 9 representing almost complete destruction of the plant. An average of two visual evaluations were performed for each trial at plant development growth stages ranging from V2 to V10 (Ritchie et al. 1993). At each evaluation date, 10–30 continuous plants were scored for feeding damage from the central two rows of each plot. Only the evaluation achieving the highest score in damage was included in statistical analysis in order to assess the efficacy of HX I maize versus non-Bt maize under the worst-case scenario. Statistical Analysis Field monitoring data of plant damage using the Davis scale (0–9) was analyzed with the linear mixed model: ηijkl=η+Treatmenti+Yearj+Trialk+Treatment×Yearij+Treatment×Trialik+Blockl(k) with observations normally distributed, yijkl ∼ N (μijkl,σ2) ⁠, and identity link function ηijkl = μijkl. In order to improve the normality and homogeneity of variance of the dataset, both requirements for linear model application, values were transformed using x+0.5transformation. Least square means values presented in tables are back-transformed values and their SEs were estimated with the delta method (Stroup 2012). Survivorship in laboratory monitoring was analyzed with the generalized linear mixed model: ηij=η+Yeari+Locationj+Year×Locationij with observations binomially distributed, yij∼Binomial (Nij,πij) ⁠. The link function for the binomial distribution is the logit function ηij=log[πij╱1−πij] ⁠. In both models above, treatment, year, and interaction treatment × year were modeled as fixed effects. Trial, location, block, and interactions containing any of these factors were modeled as random effects. Significance of treatment and year effects were evaluated with the F-approximate test (α = 0.05) and least square means were compared with Tukey’s test. In the case of linear mixed model, the estimation method was restricted maximum likelihood and Kenward–Roger for df. For the generalized linear mixed model, the estimation method was maximum likelihood with Laplace approximation. Linear mixed models were estimated with Proc MIXED and generalized linear mixed models were estimated with Proc GLIMMIX (SAS Institute Inc. 2011). Results Laboratory Monitoring Routine monitoring bioassays on field populations of S. frugiperda collected from maize and sorghum fields in four regions in Argentina indicated that the population susceptibility gradually decreased over time as measured by insect survivorship when exposed to the Cry1F diagnostic concentration (Table 1). Spodoptera frugiperda collected from maize and sorghum from 2010 to 2013 were defined as susceptible to Cry1F, based on <1% survival to third-instar stage when larvae were exposed to the diagnostic concentration. However, monitoring from the 2013 season onwards showed a significant increase in survival of S. frugiperda populations to the Cry1F protein (F5, 41 = 105.07, P < 0.01), which represents the first documented shift in susceptibility of this pest to Cry1F purified protein in Argentina. In the 2013 season, the average survival was 2.06 %, with survival greater than 2% (proportion of larvae reaching third instar) in 8 out of 15 collections from the Central, NOA, and NEA regions (no collection occurred in the Corn Belt region). During the 2014 monitoring season, all tested populations (from three regions, no collections on Corn Belt region), showed increased survival ranging from 5.2 to 91%, with an average of 38.57%. In the 2015 season, 26 populations were monitored, and survival on the diagnostic concentration of 2,000 ng Cry1F/cm2 ranged from 16.6% (Los Altos, Catamarca) to 97.90% (Avia Terai, Chaco) with an average of 67.77% (Table 1). Susceptibility of S. frugiperda to Cry1F protein across consecutive years and regions was mapped. Ranges of CS were established according to potential expected risk of damage in the field and are represented by bubbles of increasing size (Fig. 1). Table 1. Laboratory monitoring of changes in survival of Spodoptera frugiperda populations collected from different locations throughout Argentina from 2010 to 2015 Year No. of populations CS (%) SE 2010 20 0.23 0.08d 2011 19 0.14 0.06d 2012 19 0.44 0.14d 2013 15 2.06 0.67c 2014 17 38.57 7.02b 2015 26 67.77 5.24a Year No. of populations CS (%) SE 2010 20 0.23 0.08d 2011 19 0.14 0.06d 2012 19 0.44 0.14d 2013 15 2.06 0.67c 2014 17 38.57 7.02b 2015 26 67.77 5.24a Spodoptera frugiperda populations were exposed to the diagnostic concentration of Cry1F in laboratory monitoring bioassays for 7 d (n = 96–112 larvae/population) and the number of live and dead larvae and their larval instar were used to calculate corrected survival (CS%) and standard error (SE). Different letters indicate significant difference at α = 0.05 (Tukey’s HSD test). Open in new tab Table 1. Laboratory monitoring of changes in survival of Spodoptera frugiperda populations collected from different locations throughout Argentina from 2010 to 2015 Year No. of populations CS (%) SE 2010 20 0.23 0.08d 2011 19 0.14 0.06d 2012 19 0.44 0.14d 2013 15 2.06 0.67c 2014 17 38.57 7.02b 2015 26 67.77 5.24a Year No. of populations CS (%) SE 2010 20 0.23 0.08d 2011 19 0.14 0.06d 2012 19 0.44 0.14d 2013 15 2.06 0.67c 2014 17 38.57 7.02b 2015 26 67.77 5.24a Spodoptera frugiperda populations were exposed to the diagnostic concentration of Cry1F in laboratory monitoring bioassays for 7 d (n = 96–112 larvae/population) and the number of live and dead larvae and their larval instar were used to calculate corrected survival (CS%) and standard error (SE). Different letters indicate significant difference at α = 0.05 (Tukey’s HSD test). Open in new tab Fig. 1. Open in new tabDownload slide Spatial and temporal changes in survival of Spodoptera frugiperda populations collected from different locations throughout Argentina from 2010 to 2015. Spodoptera frugiperda populations were exposed to the diagnostic concentration of Cry1F in laboratory monitoring bioassays for 7 d and the number of live and dead larvae and their larval instar were used to calculate CS%. Ranges of %CS were established according to potential expected risk of damage in the field and are represented by bubbles of increasing size. The size gradient from smaller to bigger bubbles represents the %CS (percentage of individuals that survived exposure to the diagnostic concentration of Cry1F protein) (Table 1). Fig. 1. Open in new tabDownload slide Spatial and temporal changes in survival of Spodoptera frugiperda populations collected from different locations throughout Argentina from 2010 to 2015. Spodoptera frugiperda populations were exposed to the diagnostic concentration of Cry1F in laboratory monitoring bioassays for 7 d and the number of live and dead larvae and their larval instar were used to calculate CS%. Ranges of %CS were established according to potential expected risk of damage in the field and are represented by bubbles of increasing size. The size gradient from smaller to bigger bubbles represents the %CS (percentage of individuals that survived exposure to the diagnostic concentration of Cry1F protein) (Table 1). Field Monitoring Data from field evaluations of the damage of fall armyworm on maize showed a significant effect of treatment (Cry1F, non-Cry1F) (F1, 34.7 = 215.76, P < 0.01), year (F1, 34.1 = 3.78, P = 0.0038), and interaction treatment × year (F1, 34.7 = 11.47, P < 0.01). During the first 4 y of evaluation (2009–2012), levels of damage in Cry1F-expressing maize remained low under natural pest pressure (average values below 1 on the Davis scale); damage on non-Cry1F-maize isoline hybrids was significantly higher with averages ranged from 3.23 to 5.37 on the Davis scale (Table 2). However, average damage in Cry1F-expressing hybrids significantly increased from the 2013 season on (F7, 46.2 = 8.44, P < 0.01), whereas there was no significant variation of damage on the isoline over time (F7, 46.2 = 1.49, P = 0.1936). In the last two seasons of evaluation (2015 and 2016), field data showed no statistical difference (P > 0.05) between Cry1F and conventional maize in terms of foliar damage. Furthermore, most of the damage scores in Cry1F-expressing hybrids are above 3 in 2015 and almost all in 2016 (Fig. 2). Table 2. Field monitoring of foliar feeding damage on HX I maize expressing the Cry1F protein and non-Bt near-isoline hybrids by Spodoptera frugiperda from 2009 to 2016 was assessed using the 0–9 Davis scale (Davis et al. 1992) where 0 representing no damage and 9 representing almost complete destruction of the plant (n = 40–120 plants/trial) Year No. of trials Treatment Avg. damage SE 2009 3 HERCULEX 0.02 0.34b Non-bt 3.23 0.89a 2010 4 HERCULEX 0.62 0.42b Non-bt 5.37 0.97a 2011 5 HERCULEX 0.60 0.38b Non-bt 4.52 0.80a 2012 2 HERCULEX 0.15 0.45b Non-bt 3.57 1.14a 2013 5 HERCULEX 1.32 0.48b Non-bt 2.97 0.67a 2014 11 HERCULEX 2.54 0.42b Non-bt 4.67 0.55a 2015 7 HERCULEX 2.85 0.55a Non-bt 3.65 0.61a 2016 5 HERCULEX 4.87 0.83a Non-bt 5.63 0.88a Year No. of trials Treatment Avg. damage SE 2009 3 HERCULEX 0.02 0.34b Non-bt 3.23 0.89a 2010 4 HERCULEX 0.62 0.42b Non-bt 5.37 0.97a 2011 5 HERCULEX 0.60 0.38b Non-bt 4.52 0.80a 2012 2 HERCULEX 0.15 0.45b Non-bt 3.57 1.14a 2013 5 HERCULEX 1.32 0.48b Non-bt 2.97 0.67a 2014 11 HERCULEX 2.54 0.42b Non-bt 4.67 0.55a 2015 7 HERCULEX 2.85 0.55a Non-bt 3.65 0.61a 2016 5 HERCULEX 4.87 0.83a Non-bt 5.63 0.88a Mean damage ratings are reported with SEs. Significant differences between treatments within a year were assessed (Tukey’s HSD test), where a common letter indicates that mean values were not significantly different at α = 0.05. Open in new tab Table 2. Field monitoring of foliar feeding damage on HX I maize expressing the Cry1F protein and non-Bt near-isoline hybrids by Spodoptera frugiperda from 2009 to 2016 was assessed using the 0–9 Davis scale (Davis et al. 1992) where 0 representing no damage and 9 representing almost complete destruction of the plant (n = 40–120 plants/trial) Year No. of trials Treatment Avg. damage SE 2009 3 HERCULEX 0.02 0.34b Non-bt 3.23 0.89a 2010 4 HERCULEX 0.62 0.42b Non-bt 5.37 0.97a 2011 5 HERCULEX 0.60 0.38b Non-bt 4.52 0.80a 2012 2 HERCULEX 0.15 0.45b Non-bt 3.57 1.14a 2013 5 HERCULEX 1.32 0.48b Non-bt 2.97 0.67a 2014 11 HERCULEX 2.54 0.42b Non-bt 4.67 0.55a 2015 7 HERCULEX 2.85 0.55a Non-bt 3.65 0.61a 2016 5 HERCULEX 4.87 0.83a Non-bt 5.63 0.88a Year No. of trials Treatment Avg. damage SE 2009 3 HERCULEX 0.02 0.34b Non-bt 3.23 0.89a 2010 4 HERCULEX 0.62 0.42b Non-bt 5.37 0.97a 2011 5 HERCULEX 0.60 0.38b Non-bt 4.52 0.80a 2012 2 HERCULEX 0.15 0.45b Non-bt 3.57 1.14a 2013 5 HERCULEX 1.32 0.48b Non-bt 2.97 0.67a 2014 11 HERCULEX 2.54 0.42b Non-bt 4.67 0.55a 2015 7 HERCULEX 2.85 0.55a Non-bt 3.65 0.61a 2016 5 HERCULEX 4.87 0.83a Non-bt 5.63 0.88a Mean damage ratings are reported with SEs. Significant differences between treatments within a year were assessed (Tukey’s HSD test), where a common letter indicates that mean values were not significantly different at α = 0.05. Open in new tab Fig. 2. Open in new tabDownload slide Average of foliar feeding damage (Davis scale) (Davis et al. 1992) on HX I and non-Bt maize hybrids from 2009 to 2016, where 0 representing no damage and 9 representing almost complete destruction of the plant. Median indicated by a solid line. Mean indicated by spot in the box plot. Twenty-five percentage of the higher and lower values are indicated by the whiskers. Fig. 2. Open in new tabDownload slide Average of foliar feeding damage (Davis scale) (Davis et al. 1992) on HX I and non-Bt maize hybrids from 2009 to 2016, where 0 representing no damage and 9 representing almost complete destruction of the plant. Median indicated by a solid line. Mean indicated by spot in the box plot. Twenty-five percentage of the higher and lower values are indicated by the whiskers. When the field dataset was evaluated by agroecological region (Central, Corn Belt, NEA, and NOA), levels of damage showed a similar trend, with a more significant increase in the damage on Cry1F-expressing hybrids from 2013 onwards the NOA and Corn Belt regions (F5, 62 = 60.23, P < 0.01 and F5, 28 = 23.22, P < 0.01 respectively). Due to fewer trials and years of field monitoring in the other two regions, Central and NEA, the increase in damage for Cry1F over time was also significant but more variable (F3, 19 = 10.15, P = 0.0003 and F4, 25 = 7.80, P = 0.0003, respectively) although during the most recent season (2016), the average damage was higher than 4 on the Davis scale in both regions. There was significant linear correlation between the annual average values of survival from lab monitoring and damage on Cry1F-expressing maize from field monitoring (years 2010–2015), with Pearson correlation = 0.922, P = 0.0088. Discussion Field monitoring was conducted from 2009 to 2016 to determine the performance of HX I technology in the field under natural S. frugiperda pressure across Argentinean maize-growing regions. Increasing crop damage was observed in the field trials from 2013 onwards and in a routine laboratory monitoring program run in parallel from 2010 to 2015. Collectively, the laboratory monitoring data clearly show an increase in the survival of S. frugiperda in Argentina and are indicative of resistance evolution to the Cry1F protein. These results show that the development of resistance was rapid after an initial lag phase (consistent with the classical pattern of recessive resistance evolution), and was broadly established in seven crop seasons (from product launch in 2006 through 2013). These results are consistent with the findings of inheritance assays that showed that resistance is due to a recessive and autosomal allele (Chandrasena et al. 2018) and consistent with the findings in other countries for the same pest (Storer et al. 2010, Vélez et al. 2013, Farias et al. 2014, Horikoshi et al. 2016). Susceptibility of S. frugiperda to Cry1F protein across consecutive years and regions was mapped in order to understand possible geographical incidence driven by temperatures, topography, crops, or even migration and the percentage of surviving individuals in each location was represented geographically by year (Fig. 1). The northeast region of Argentina represents the southern limit where S. frugiperda is able to over winter and maintain permanent populations (Murúa and Virla 2004a). Same authors suggest that this specie could migrate from the northeast and reach the province of Tucumán in the northwest of the country. However, it is important to consider that populations of this pest were found in different grasses during the spring in absence of maize, indicating that populations that affect this crop may not depend exclusively on migration of individuals from other regions (Murúa and Virla 2004b). Migration from southern Brazil to northern Argentina could be another source of resistant populations; however, this has not been characterized yet. This routine laboratory monitoring program confirmed that several populations of S. frugiperda, collected throughout maize-growing regions of Argentina, have reduced susceptibility to the Cry1F protein expressed in the event DAS-Ø15Ø7-1 (HX I). These findings confirmed that S. frugiperda resistance to Cry1F has become widespread in Argentina. Similar field-evolved resistance was reported in Brazil in 2012 (Farias et al. 2014) after 3 y of Bt maize expressing Cry1F and in Puerto Rico in 2006 following 3 y of commercial cultivation of Cry1F-expressing maize on the island (Storer et al. 2012b). In the case of Puerto Rico, the area of maize is surrounded by mountainous terrain, the lack of insect migration and the pressure of several generations of S. frugiperda per year (for an estimated 30–40 generations of S. frugiperda over the 4 y of commercial cultivation) contributed to a rapid field-evolved resistance. The same trend of field-evolved resistance was observed in Argentina as described previously in this paper. It is likely that the development of the first resistant populations started in the north of the country and moved progressively southwards. This assumption is based on the fact that S. frugiperda has more generations per year in the north and it is close to other maize-growing areas from Brazil, which also have a history of extensive Cry1F resistance. Late planting of maize is a practice widely adopted in Argentina due to increased rainfall and lower temperatures at flowering, which promote more stable grain production. However, this cultural practice increases the risk of higher insect pressure. Bt technologies helped to enable later planting dates by providing increased protection against S. frugiperda, which was very difficult in late-planted maize prior to Bt traits. These cultural practices also facilitated increased selection pressure. Results from Chandrasena et al. (2018) show that the resistance of S. frugiperda to Cry1F is incompletely recessive similarly to the results from Santos-Amaya et al. (2016) for some populations of Brazil. These results are consistent with the findings from Storer et al. (2010), which provide evidence that DAS- Ø15Ø7-1 does not meet the high-dose criteria for this pest (i.e., mortality of heterozygotes is not at least 95%, as recommended in the high dose + refuge strategy for single events). The impossibility of reaching 95% mortality on heterozygotes allows a small proportion of them to survive when exposed to HX I maize tissue and this could contribute to a more rapid evolution of resistance in S. frugiperda than in other species where this criteria is met. Also, other factors such as the migratory and multivoltine nature of this pest, as well as the high adoption rate of HX I technology without adherence to recommended IRM practices (like refuge planting), could also contribute to the quick evolution and the wide geographical spread of resistance. Despite the presence of Cry1F-resistant S. frugiperda field populations, DAS- Ø15Ø7-1 provides value to growers in Argentina as an effective control technology for other maize pests, such as D. saccharalis (with the exception of a small isolated area in NW San Luis province; Signorini et al. 2017). This represents the only case of resistance of D. saccharalis to HX I in Argentina that, unlike what happened with S. frugiperda, has been confined to this region. The agroecological characteristics makes of this place an excellent location for seed production. In fact, prior to D. saccharalis resistance discovery, 30–50% of the area was planted with maize for seed production (Malacarne et al. 2017). In this case, several factors contributed to high D. saccharalis population densities and high number of generations per year such as high temperatures during summer and mild winters with long frost-free periods (Trumper 2014, Signorini et al. 2017). The reason why the resistance has not spread to other regions is based on the fact that this is an area surrounded by mountains and isolated from other maize-producing areas. At the same time, a mitigation plan was put in place developed by the industry, farmers, and government upon discovery of the unexpected damage (Malacarne et al. 2017). Deployment of maize expressing multiple Bt proteins with different modes of actions (pyramided products) is key for resistance management. Additionally, it is known that the sustainability of Bt technologies requires the implementation of good agricultural and IRM practices. Trait providers have been working to align a unique message not only among seed companies but also with main influencers, universities, and government research institutions. These groups have reached agreement on best management practices which are being communicated through the Argentinean Seed Company Association (ASA) and IRAC Argentina. These practices include crop rotation, insect monitoring, application of insecticides, the use of seed treatments, management of weeds prior to and during cultivation, planting of structured refuge, and the stacking of Bt toxins (Box 1). The durability of technologies for insect control depends on multiple factors that involve various actors in the production chain. These include: 1) the companies that supply these technologies must guarantee a unified message of resistance management recommendations, and ensure the availability of refuge seed with similar agronomic characteristics to the Bt hybrids; 2) growers must follow the recommendations for the use of technologies, plant structured refuge, and utilize crop rotations that favor insect management and other best management practices; and 3) private consultants and research institutions, which are very important channels of influence and advocacy, should focus on communication strategies that increase the level of knowledge and commitment in the management of resistance. Box 1. Good agricultural and IRM practices recommended by ASA and IRAC Argentina Rotation of crops: This practice maintains the properties of the soil and reduces the populations of insect pests since generally, in Argentina, the insects that affect one crop are different from the ones that affect crops that follow in the rotation. Adequate control of weeds and insects before planting: Weeds act as reservoirs for pests; therefore, it is advisable to keep the field and surroundings clean 30 d before sowing. If desiccation is not possible and insects are found in the field, it is recommended to carry out an insecticide treatment prior to the emergence of the crop. The objective is to prevent the dispersal of larvae that will damage the crop during crop development. Use of seed treatment: To achieve correct implantation and a healthy and vigorous crop, it is necessary to protect the plants during the first stages. Insecticide applications: Use of an insecticide may be needed in Bt maize if infestation is high. Monitor the crop and spray an insecticide if damage achieves the level 3 in Davis scale and live larvae are found. Refuge: A portion of the field is planted with a non-Bt maize, with a similar characteristics and cycle to Bt maize, to provide susceptible adults to the Bt field (Bernardi et al. 2016). Stacking of Bt toxins: Pyramids producing two or more Bt toxins that target the same pest are becoming increasingly important for pest management (Storer et al. 2012a, Carrière et al. 2015). Seed companies are moving forward on stacking Bt toxins in order to delay resistance evolution. These recommendations are also available online at: http://asa.org.ar and http://irac-argentina.org. Acknowledgments The authors sincerely appreciate the support from Paula Bey (Dow AgroSciences Argentina SRL) who contributed developing the map across years. 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TI - Monitoring the Evolution of Resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) to the Cry1F Protein in Argentina
JF - Journal of Economic Entomology
DO - 10.1093/jee/toz076
DA - 2019-08-03
UR - https://www.deepdyve.com/lp/oxford-university-press/monitoring-the-evolution-of-resistance-in-spodoptera-frugiperda-6tm40XFbEd
SP - 1838
VL - 112
IS - 4
DP - DeepDyve
ER -