Seasonal Population Dynamics of Megacopta cribraria (Hemiptera: Plataspidae) in Kudzu and Soybean, and Implication for Insecticidal Management in Soybean.

Megacopta cribraria (F.), an invasive species introduced from Asia in 2009, is now prolific in the southeastern United States. Megacopta cribraria develops primarily on kudzu and soybean completing two generations. It is not well understood how this economic pest is affected by changes in geographic distribution in the United States or how population levels have changed since its establishment. The effect of insecticide application timing on field populations of M. cribraria is not well documented. These studies seek to understand how population dynamics of M. cribraria vary with geographic regions in Georgia. Effect of application timing on populations throughout the growing season was also examined. Weekly from 2012 to 2013, all life stages were enumerated from kudzu and soybean environments at several locations throughout Georgia from sweeps samples and flight intercept captures. Coordinates were recorded for locations, and classified as belonging to the Piedmont or Coastal Plain region of the state. Single spray trials were conducted from 2011-2014, and applications were made to soybean at intervals throughout the season. From 2012 to 2015, two kudzu patches near Griffin, GA, were monitored to detect population changes. Differences in population dynamics from locations around the state were found, but no clear effect of latitude, longitude, or region was observed. Insecticide applications applied in July suppressed nymph populations significantly better than treatments made earlier or later. Megacopta cribraria populations declined in 2014 and 2015 compared with 2012 and 2013. These studies provide the critical information for M. cribraria management in soybean in the southeastern United States.

Lepidoptera (Crambidae, Noctuidae, and Pyralidae) Injury to Corn Containing Single and Pyramided Bt Traits, and Blended or Block Refuge, in the Southern United States.

Fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae); corn earworm, Helicoverpa zea Boddie (Lepidoptera: Noctuidae); southwestern corn borer, Diatraea grandiosella Dyar (Lepidoptera: Crambidae); sugarcane borer, Diatraea saccharalis F. (Lepidoptera: Crambidae); and lesser cornstalk borer, Elasmopalpus lignosellus Zeller (Lepidoptera: Pyralidae), are lepidopteran pests of corn, Zea mays L., in the southern United States. Blended refuge for transgenic plants expressing the insecticidal protein derivative from Bacillus thuringiensis (Bt) has recently been approved as an alternative resistance management strategy in the northern United States. We conducted a two-year study with 39 experiments across 12 states in the southern United States to evaluate plant injury from these five species of Lepidoptera to corn expressing Cry1F and Cry1Ab, as both single and pyramided traits, a pyramid of Cry1Ab×Vip3Aa20, and a pyramid of Cry1F×Cry1Ab plus non-Bt in a blended refuge. Leaf injury and kernel damage from corn earworm and fall armyworm, and stalking tunneling by southwestern corn borer, were similar in Cry1F×Cry1Ab plants compared with the Cry1F×Cry1Ab plus non-Bt blended refuge averaged across five-plant clusters. When measured on an individual plant basis, leaf injury, kernel damage, stalk tunneling (southwestern corn borer), and dead or injured plants (lesser cornstalk borer) were greater in the blended non-Bt refuge plants compared to Cry1F×Cry1Ab plants in the non-Bt and pyramided Cry1F×Cry1Ab blended refuge treatment. When non-Bt blended refuge plants were compared to a structured refuge of non-Bt plants, no significant difference was detected in leaf injury, kernel damage, or stalk tunneling (southwestern corn borer). Plant stands in the non-Bt and pyramided Cry1F×Cry1Ab blended refuge treatment had more stalk tunneling from sugarcane borer and plant death from lesser cornstalk borer compared to a pyramided Cry1F×Cry1Ab structured refuge treatment. Hybrid plants containing Cry1F×Cry1Ab within the pyramided Cry1F×Cry1Ab blended refuge treatment had significantly less kernel damage than non-Bt structured refuge treatments. Both single and pyramided Bt traits were effective against southwestern corn borer, sugarcane borer, and lesser cornstalk borer.

Seed yield of near-isogenic soybean lines with introgressed quantitative trait loci conditioning resistance to corn earworm (Lepidoptera: Noctuidae) and soybean looper (Lepidoptera: Noctuidae) from PI 229358.

The development of superior soybean, Glycine max (L.) Merr., cultivars exhibiting resistance to insects has been hindered due to linkage drag, a common phenomenon when introgressing alleles from exotic germplasm. Simple-sequence repeat (SSR) markers were used previously to map soybean insect resistance (SIR) quantitative trait loci (QTLs) in a'Cobb' X PI 229358 population, and subsequently used to create near-isogenic lines (NILs) with SIR QTL i n a 'Benning' genetic background. SIR QTLs were mapped on linkage groups (LGs) M (SIRQTL-M), G (SIRQTL-G), and H (SIRQTL-H). The objectives of this study were to 1) evaluate linkage drag for seed yield by using Benning-derived NILs selected for SIRQTL-M, SIRQTL-H, and SIRQTL-G; 2) assess the amount of PI 229358 genome surrounding the SIR QTL in each Benning NIL; and 3) evaluate the individual effects these three QTLs on antibiosis and antixenosis to corn earworm, Helicoverpa zea (Boddie), and soybean looper, Pseudoplusia includens (Walker). Yield data collected in five environments indicated that a significant yield reduction is associated with SIRQTL-G compared with NILs without SIR QTL. Overall, there was no yield reduction associated with SIRQTL-M or SIRQTL-H. A significant antixenosis and antibiosis effect was detected for SIRQTL-M in insect feeding assays, with no effect detected in antixenosis or antibiosis assays for SIRQTL-G or SIRQTL-H without the presence of PI 229358 alleles at SIRQTL-M. These results support recent findings concerning these loci.

Effects of defoliating insect resistance QTLs and a cry1Ac transgene in soybean near-isogenic lines.

The crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) have shown considerable value in providing effective insect resistance in a number of crop species, including soybean, Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance/resistance to crystal proteins by defoliating insects and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three insect resistance quantitative trait loci (QTLs; QTL-M, QTL-H, and QTL-G) originating from Japanese soybean PI 229358 and a cry1Ac gene in a "Benning" genetic background. A set of 16 BC(6)F(2)-derived near isogenic lines (NILs) was developed using marker-assisted backcrosses and evaluated for resistance to soybean looper [SBL, Pseudoplusia includens (Walker)] and corn earworm [CEW, Helicoverpa zea (Boddie)] in field cage, greenhouse, and detached leaf assays. Both Bt and QTL-M had significantly reduced defoliation by both SBL and CEW and reduced larval weight of CEW. The antibiosis QTL-G had a significant effect on reducing CEW larval weight and also a significant effect on reducing defoliation by SBL and CEW in some assays. The antixenosis QTL-H had no main effect, but it appeared to function through interaction with QTL-M and QTL-G. Adding QTL-H and QTL-G further enhanced the resistance of the Bt and QTL-M combination to CEW in the field cage assay. These results should help guide the development of strategies for effective management of insect pests and for sustainable deployment of Bt genes.

Efficacy of transgenic cotton expressing Cry1Ac and Cry1F insecticidal protein against heliothines (Lepidoptera: Noctuidae).

Cotton, Cossypium hirsutum L, plants expressing Cry1Ac and Cry1F (Phytogen 440W) insecticidal crystal proteins of Bacillus thuringiensis (Bt) Berliner, were evaluated against natural populations of tobacco budworm, Heliothis virescens (F.), and bollworm, Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), across 13 southern U.S. locations that sustained low, moderate, and high infestations. The intrinsic activity of Phytogen 440W was compared with nontreated non-Bt cotton (PSC355) and with management strategies in which supplemental insecticides targeting heliothines were applied to Phytogen 440W and to PSC355 cotton. Infestations were composed primarily of bollworm, which is the least sensitive of the heliothine complex to Cry toxins. Therefore, damage recorded in these studies was primarily due to bollworm. Greater than 75% of all test sites sustained heliothine infestations categorized as moderate to high (10.6-64.0% peak damaged bolls in nontreated PSC355). Phytogen 440W, alone or managed with supplemental insecticide applications, reduced heliothine-damaged plant terminals, squares (flower buds), flowers, and bolls equal to or better (1.0-79.0-fold) than managing a non-Bt cotton variety with foliar insecticides across all infestation environments. Rarely (frequency of < or = 11% averaged across structures), sprayed Phytogen 440W reduced damaged structures compared with nontreated Phytogen 440W. Protection against heliothine-induced plant damage was similar across the three levels of infestation for each viable management strategy, with exception to damaged squares for nontreated Phytogen 440W. In situations of moderate to high heliothine infestations, cotton plants expressing Cry1Ac and Cry1F may sustain higher levels of damage compared with that same variety in low infestations. No significant difference in yield was observed among heliothine management strategies within each infestation level, indicating cotton plants may compensate for those levels of plant damage. These findings indicate Phytogen 440W containing Cry1Ac and Cry1F provided consistent control of heliothines across a range of environments and infestation levels.

Functional response of the tiger beetle Megacephala carolina carolina (Coleoptera: Carabidae) on twolined spittlebug (Hemiptera: Cercopidae) and fall armyworm (Lepidoptera: Noctuidae).

The functional response of the tiger beetle Megacephala carolina carolina L. (Coleoptera: Carabidae) was determined on adult twolined spittlebug, Prosapia bicincta (Say) (Hemiptera: Cercopidae), and fourth instars of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), in single-prey and two-prey systems. In the laboratory, M. carolina carolina demonstrated a type II functional response for P. bicincta and S. frugiperda in both single- and two-prey systems. Search efficiency of M. carolina declined for both prey as the initial number of prey increased. Of the total prey consumed, M. carolina carolina killed significantly more S. frugiperda than P. bicincta in the single-prey system (8.0 and 4.5, respectively) and the two-prey system (5.0 and 2.0, respectively). Estimates of attack coefficient, a, were not significantly different for P. bicincta and S. frugiperda in the single-prey (0.07 and 0.02) and two-prey systems (0.04 and 0.06), respectively. The handling time, T(h), was significantly greater for P. bicincta (5.02 and 10.64 h) than for S. frugiperda (2.66 and 4.41 h) in single- and two-prey systems, respectively. Estimations of attack coefficient and handling time in the single-prey system were used to predict prey preference of M. carolina carolina. No strong prey switching response was observed. M. carolina carolina showed no preference for either prey. However, in the presence of S. frugiperda, the functional response of the predator for P. bicincta was reduced. M. carolina carolina is a potential predator of one or more turfgrass pests and should be considered in conservation efforts.

Susceptibility of twolined spittlebug (Hemiptera: Cercopidae) life stages to entomophagous arthropods in turfgrass.

Prosapia bicincta (Say) (Hemiptera: Cercopidae), the twolined spittlebug, is an economic pest of turfgrass in the southeastern United States. No data concerning natural enemies of P. bicincta in turfgrass have been reported previously. We compared predation of spittlebug eggs, nymphs, and adults in the laboratory by potential generalist predators commonly found in turfgrass: bigeyed bugs Geocoris uliginosus Say and Geocoris punctipes Say; red imported fire ant, Solenopsis invicta Buren; wolf spiders (Lycosa sp. Walckenaer); carabid beetles Harpalaus pensylvanicus DeGeer and Calosoma sayi Dejean; and tiger beetles Megacephala carolina carolina L. Eggs were readily consumed by generalist predators. S. invicta consumed 100% of the eggs offered. H. pensylvanicus and C. sayi were also significant predators of P. bicincta eggs. Nymphs live in spittlemasses that protect them from attack by predators, but exposed nymphs were susceptible to attack when mechanically removed from their spittlemasses. S. invicta and M. carolina carolina caused significant mortality of exposed nymphs. P. bicincta adults are aposematic and have the ability to reflex bleed; however, reflex bleeding did not prevent attack by predators. S. invicta and M. carolina carolina killed 100% of the adult spittlebugs offered in laboratory bioassays. Lycosa sp. are less voracious predators of adults. Sound background knowledge about P. bicincta and its potential natural enemy complex is important for the development and implementation of a detailed, site-specific, biologically based pest management program in turfgrass.

Precision application of aldicarb to enhance efficiency of thrips (Thysanoptera: Thripidae) management in cotton.

Field studies were conducted during 1999-2001 in two climatic/edaphic areas of Georgia (Southern Piedmont and East Gulf Coastal Plain) to test the hypothesis that precision placement of aldicarb with cotton seed in hill planting at spatially specific intervals could decrease insecticide use for management of tobacco thrips, Frankliniella fusca (Hinds). Precision-placed aldicarb controlled thrips during cotton seedling stages using per ha amounts of one-half or less than standard in-furrow application rates with no significant differences in yield. Residual analysis of cotton plants showed that plants in precision placement plots had as much or more aldicarb and aldicarb metabolites present as compared with cotton treated with conventional in-furrow treatments. Higher rates of precision-placed aldicarb did cause phytotoxic burning early in the growing season, but no significant impact on yield was observed.

Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer.

Laboratory, greenhouse and field studies were conducted to characterize the insecticidal properties of genetically altered forms of Autographa californica (Speyer) nucleopolyhedrovirus (AcNPV) and Helicoverpa zea (Boddie) NPV (HzNPV) against selected heliothine species. The altered viruses each contained a chimeric 0.8-kb fragment encoding the insect-specific, sodium channel neurotoxin from the Algerian scorpion Androctonus australis Hector (AaIT, hence recombinant viruses designated Ac-AaIT and Hz-AaIT). Based on LD50 values, results from diet-overlay bioassays showed Ac-AaIT and Hz-AaIT to be equally virulent against larval tobacco budworm, Heliothis virescens (F.), but Hz-AaIT averaged 1,335-fold greater bioactivity than Ac-AaIT against larval cotton bollworm, Helicoverpa zea (Boddie). Hz-AaIT killed larvae of both heliothine species at rates significantly faster than those imparted by HzNPV (viral LT50 values averaged 2.5 and 5.6 d, respectively). In greenhouse studies, foliar sprays of Ac-AaIT and Hz-AaIT were equally effective in controlling H. virescens on cotton; however, Hz-AaIT provided control of H. zea on cotton at a level superior to that of Ac-AaIT. For example, after three weekly sessions of foliar application and H. zea artificial infestation, cotton treated with Ac-AaIT or Hz-AaIT at 10 x 10(11) occulsion bodies (OB)/ha averaged 2.5 and 16.2 nondamaged flower buds per plant, respectively. Another greenhouse study conducted against heliothine species on cotton showed that the quicker killing speed exhibited by Hz-AaIT led to improved plant protection versus HzNPV. Finally, results from three field trials demonstrated that Hz-AaIT at 5-12 x 10(11) OB/ha provided control of the heliothine complex in cotton at levels slightly better than Bacillus thuringiensis, equal to the macrolide, spinosad, and only slightly less than that of selected pyrethroid and carbamate insecticides. Overall, results from these studies indicate that, because of host range differences between the two wild-type viruses, HzNPV is the better vectoring agent (versus AcNPV) for designing recombinant clones as insecticides targeted at the multi-species heliothine complex. Further, these studies suggest that if appropriately tailored for the pest complex, recombinant NPVs may be very effective, insect-specific approaches to managing pests in many cropping scenarios. Possible Hz-AaIT deployment strategies for control of heliothine species on conventional and transgenic cotton varieties are discussed.

Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae).

A transgenic line of the soybean 'Jack', Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field. Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hübner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996. Susceptible controls included Jack (1996-1998), 'Cobb' (1996), and Jack-HPH (1996). GatIR 81-296 was used as the resistant control in all 3 yr. Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar. Defoliation of GatIR 81-296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar. Jack-Bt exhibited significant, but lower resistance to soybean looper. Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998. Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.

Effect of ecdysteroid UDP-glucosyltransferase gene deletion on efficacy of a baculovirus against Heliothis virescens and Trichoplusia ni (Lepidoptera: Noctuidae).

Laboratory, greenhouse, and field studies were conducted to characterize the biological activity of a genetically altered form of Autographa californica (Speyer) nucleopolyhedrosis virus (AcNPV). The altered baculovirus (vEGTDEL) had a deletion in the ecdysteroid UDP-glucosyltransferase gene. Results from bioassays conducted with neonate and 3rd-instar tobacco budworm, Heliothis virescens (F.), as well as with 3rd-instar cabbage looper, Trichoplusia ni (Hübner), showed vEGTDEL caused larval death slightly, but significantly, quicker than AcNPV. Based on supposition (LT50 values were not calculated), it appeared that larval mortality occurred 0.5-1.0 d faster following exposure to vEGTDEL versus AcNPV. Greenhouse studies conducted against H. virescens on cotton showed that hastened virulence exhibited by vEGTDEL led to improved plant protection versus AcNPV. For example, following 5 weekly sessions of foliar application and H. virescens artificial infestation, cotton treated with wettable powder formulations of vEGTDEL or AcNPV at 2.5 x 10(12) OB/ha averaged 25.7 and 61.8% damaged flower buds, respectively. Although vEGTDEL tended to provide more consistent control of T. ni than AcNPV in greenhouse and field trials conducted on leafy vegetables, differences in efficacy between the 2 baculoviruses were marginal and usually not statistically significant. Generally, results from these studies suggest that genetic modification of NPVs to hasten their lethal effect may be a promising strategy for improving the insecticidal properties of the insect-specific pathogens.

Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cryIAc gene.

Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene. Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants. The recovered lines contained the HPH gene, but the Bt gene was lost in one line. The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged. The CryIAc protein accumulated up to 46 ng mg-1 extractable protein. In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis). Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars. Unlike previous reports of soybean transformation using this technique, all plants were fertile. To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene.

Insect Control and Dosage Effects in Transgenic Canola Containing a Synthetic Bacillus thuringiensis cryIAc Gene.

Zygotic hypocotyls of canola (Brassica napus L.) cv Oscar, cv Westar, and the breeding line UGA188-20B were transformed with a truncated synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) under the control of the cauliflower mosaic virus 35S promoter using Agrobacterium tumefaciens-mediated transformation. Fifty-seven independently transformed lines were produced, containing 1 to 12 copies of the transgenes. A range of cry expressors was produced from 0 to 0.4% Cry as a percentage of total extractable protein. The Brassica specialists, the diamondback month (Plutella xylostella L.) and the cabbage looper (Trichoplusia ni Hubner), were completely controlled by low-, medium-, and high-expressing lines. Whereas control of the generalist lepidopteran, the corn earworm (Helicoverpa zea Boddie), was nearly complete, the other generalist caterpillar tested, the beet armyworm (Spodoptera exigua Hubner), showed a dose response that had a negative association between defoliation and cry expression. These plants were produced as models for an ecological research assessment of the risk involved in the field release of naturalized transgenic plants harboring a gene (Bt) that confers higher relative fitness under herbivore-feeding pressure.