Carl T. Redmond

Recent Advances in Soybean Transformation

Soybean (Glycine max (L.) Merrill) transformation continues to be problematic even with the efforts of numerous researchers in the field. In spite of this low efficiency, soybean transformation is now routine utilizing the somatic embryogenie and meristematic tissue culture methods. A comparison, including the advantages and disadvantages, of each method are presented in this review. Soybean transformation is currently done using both Agrobacterium tumefaciens and particle bombardment. We review some of the current selectable marker genes in use, and we also describe additional non-standard transformation techniques that have been, or are being attempted, to produce transgenic soybeans.

Assessing Insecticide Hazard to Bumble Bees Foraging on Flowering Weeds in Treated Lawns

Maintaining bee-friendly habitats in cities and suburbs can help conserve the vital pollination services of declining bee populations. Despite label precautions not to apply them to blooming plants, neonicotinoids and other residual systemic insecticides may be applied for preventive control of lawn insect pests when spring-flowering weeds are present. Dietary exposure to neonicotinoids adversely affects bees, but the extent of hazard from field usage is controversial. We exposed colonies of the bumble bee Bombus impatiens to turf with blooming white clover that had been treated with clothianidin, a neonicotinoid, or with chlorantraniliprole, the first anthranilic diamide labeled for use on lawns. The sprays were applied at label rate and lightly irrigated. After residues had dried, colonies were confined to forage for six days, and then moved to a non-treated rural site to openly forage and develop. Colonies exposed to clothianidin-treated weedy turf had delayed weight gain and produced no new queens whereas those exposed to chlorantraniliprole-treated plots developed normally compared with controls. Neither bumble bees nor honey bees avoided foraging on treated white clover in open plots. Nectar from clover blooms directly contaminated by spray residues contained 171±44 ppb clothianidin. Notably, neither insecticide adversely impacted bee colonies confined on the treated turf after it had been mown to remove clover blooms present at the time of treatment, and new blooms had formed. Our results validate EPA label precautionary statements not to apply neonicotinoids to blooming nectar-producing plants if bees may visit the treatment area. Whatever systemic hazard through lawn weeds they may pose appears transitory, however, and direct hazard can be mitigated by adhering to label precautions, or if blooms inadvertently are contaminated, by mowing to remove them. Chlorantraniliprole usage on lawns appears non-hazardous to bumble bees.

Resistance to Bean pod mottle virus in Transgenic Soybean Lines Expressing the Capsid Polyprotein

ABSTRACT Transgenic fertile soybean plants were generated from somatic embryos of soybean (Glycine max) cv. Jack transformed via particle bombardment with the capsid polyprotein (pCP) gene of Bean pod mottle virus(BPMV). The plant transformation vector (pHIG/BPMV-pCP) utilized in these experiments contained the BPMV-pCP coding sequence, an intron-containing GUS gene, and the hygromycin phosphotransferase gene. Southern blot hybridization analysis showed that 19 transgenic soybean plants selected for resistance to hygromycin contained the genes for GUS and BPMV-pCP. The progeny of five of these transgenic soybean plants (plants 137, 139, 157, 183, and 186) were characterized in detail. An additional transgenic plant (plant 200) contained the intron-GUS and hygromycin resistance genes, but lacked the BPMV-pCP gene and was used as a negative control. Southern blot hybridization analysis of the five transgenic plants showed the presence of three copies of the T-DNA in a similar banding pattern suggesting that they were derived from a single transformation event. Western and northern blot analyses showed that the expression levels of BPMV-pCP and pCP transcript were high in these five pCP plants. Infectivity assays with detached leaves demonstrated that all five pCP plants exhibited resistance to virus infection because they accumulated lower levels of BPMV compared with plant 200 and nontransformed controls. Unlike the T(2) progeny of line 183-1 that segregated with respect to the pCP gene and, consequently, to BPMV resistance, the T(2) progeny of the homozygous line 183-2 showed little or no symptoms in response to rub-inoculation with virions of a severe strain of BPMV. Although BPMV accumulation was evident in leaves on which viruliferous beetles were allowed a 72-h inoculation access period, the upper noninoculated leaves of the T(2) progeny of line 183-2 plants were symptomless and accumulated little or no virus. Because the progeny of this homozygous transgenic line exhibited systemic resistance, they could potentially be useful in generating commercial cultivars resistant to BPMV.

Contribution of ergot alkaloids to suppression of a grass‐feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.

Comparative impact of an anthranilic diamide and other insecticidal chemistries on beneficial invertebrates and ecosystem services in turfgrass.

BACKGROUND Chlorantraniliprole, the first anthranilic diamide insecticide labeled for turf, combines strong selective activity against key pests with low vertebrate toxicity. The hypothesis that it is less disruptive to beneficial invertebrates and their ecosystem services than are other prevailing insecticide classes was tested. Plots in golf course settings were treated with chlorantraniliprole, or with a representative nicotinoid (clothianidin), pyethroid (bifenthrin) or a combination (clothianidin-bifenthrin) formulation. Non-target effects were assessed via pitfall traps (epigeal predators), Tullgren funnel extraction (soil microarthropods), hand sorting (earthworms), counting ant mounds and earthworm casts on tees and putting greens, assessing predation on sentinel pest eggs and comparing grass clipping decomposition in treated versus untreated turf. RESULTS Chlorantraniliprole had little or, in most cases, no impact on predatory or soil invertebrates, predation or decomposition. Each of the other insecticides temporarily reduced abundance and activity of one or more predator groups. Clothianidin and the clothianidin-bifenthrin combination retarded grass clipping decomposition, and the combination suppressed earthworms and casts more than did carbaryl, a toxic standard. CONCLUSION Chlorantraniliprole is compatible with conservation biocontrol and a good fit for industry initiatives to use relatively less toxic pesticides. One caveat is that its use on golf courses may require targeted management of ant mounds and earthworm casts that are suppressed as a side effect by some less selective insecticides.

Microbial Control of Black Cutworm (Lepidoptera: Noctuidae) in Turfgrass Using Agrotis ipsilon Multiple Nucleopolyhedrovirus

Abstract Agrotis ipsilon multiple nucleopolyhedrovirus (family Baculoviridae, genus Nucleopolyhedrovirus, AgipMNPV), a naturally occurring baculovirus, was found infecting black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), on central Kentucky golf courses. Laboratory, greenhouse, and field studies investigated the potential of AgipMNPV for managing black cutworms in turfgrass. The virus was highly active against first instars (LC50 = 73 occlusion bodies [OBs] per μl with 2-μl dose; 95% confidence intervals, 55–98). First instars that ingested a high lethal dose stopped feeding and died in 3–6 d as early second instars, whereas lethally infected fourth instars continued to feed and grow for 4–9 d until death. Sublethal doses consumed by third or fifth instars had little or no effect on subsequent developmental rate or pupal weight. Horizontal transmission of AgipMNPV in turfgrass plots was shown. Sprayed suspensions of AgipMNPV (5 × 108–6 × 109 OBs/m2) resulted in 75 to >93% lethal infection of third or fourth instars in field plots of fairway-height creeping bentgrass, Agrostis stolonifera (Huds.), and on a golf course putting green collar. Virus spray residues (7 × 109 OBs/m2) allowed to weather on mowed and irrigated creeping bentgrass field plots significantly increased lethal infection of implanted larvae for at least 4 wk. This study, the first to evaluate a virus against a pest in turfgrass, suggests that AgipMNPV has potential as a preventive bioinsecticide targeting early instar black cutworms. Establishing a virus reservoir in the thatch and soil could suppress successive generations of that key pest on golf courses and sport fields.

Managing earthworm casts (Oligochaeta: Lumbricidae) in turfgrass using a natural byproduct of tea oil (Camellia sp.) manufacture

BACKGROUND Earthworm casts are a worldwide problem on golf courses and sports fields when they disrupt the playability, aesthetics and maintenance of closely mowed playing surfaces. Currently, no pesticides are labeled for earthworms in the United States. Tea seed pellets (TSPs), a saponin-rich byproduct of Camellia oleifera Abel oil manufacture, were tested for expelling earthworms and reducing casts on creeping bentgrass turf. The fate of expelled worms, methods for removing them and impacts on pest and beneficial arthropods were also evaluated. RESULTS Application of TSPs at 2.93 kg 100 m(-2), followed by irrigation, quickly expelled earthworms from the soil. A single application reduced casts by 80-95% for at least 5 weeks. Mowing or sweeping removed expelled earthworms from putting green surfaces. Most expelled earthworms burrowed down when transferred to untreated turf, but few survived. Bioassay-guided fractionation confirmed the vermicidal activity results from a mix of saponins. TSPs did not reduce the abundance of beneficial soil arthropods, nor did they control black cutworms or white grubs in treated turf. CONCLUSION TSPs are an effective botanical vermicide that could be useful for selectively managing earthworm casts on closely mowed turfgrass. They might also be used to suppress earthworms in grassy strips alongside runways to reduce bird strike hazard at airports.

Residual Effects of Imidacloprid on Japanese Beetle (Coleoptera: Scarabaeidae) Oviposition, Egg Hatch, and Larval Viability in Turfgrass

Abstract Preventive control of turf-infesting scarabaeid grubs by neonicotinoid insecticides is presumed to mainly result from residues killing first instars in the soil. The extent to which sublethal behavioral effects or intoxication of other life stages contribute to such control is poorly known. We tested whether Japanese beetle, Popillia japonica Newman, females lay fewer eggs in turf treated with imidacloprid (Merit 75 WP) or an imidacloprid–bifenthrin combination (Allectus GC SC), and whether exposure to those residues in thatch and soil reduces their survival and subsequent ability to feed or take flight. Effects of imidacloprid residues on egg hatch and viability of successive larval instars also were studied. In two sets of choice tests, 68 and 82% fewer eggs were laid in Kentucky bluegrass with Allectus residues than in controls. When females were confined in treated turf, however, neither insecticide consistently reduced their fecundity or affected depth at which eggs were laid, although exposure to fresh Allectus residues reduced the beetles’ subsequent viability. Imidacloprid residues up to 2 ppm in soil did not affect egg viability or days to hatch, but they killed neonates soon after eclosion. Imidacloprid curatively applied at label rate (0.34 kg active ingredient/ha) reduced weight gain, burrowing capability, frass production, and survival of second and third instars in turfgrass cores, with high mortality within 30 d. Intoxication and behavioral impairment of third instars also occurred in autumn field trials. Our data suggest that imidacloprid has greater activity against late instars than is generally appreciated.

Early spring defoliation, secondary leaf flush, and leafminer outbreaks on American holly

SummaryAdults of Phytomyza ilicicola (Diptera: Agromyzidae), a univoltine specialist leafminer, emerge in close synchrony with leaf flush of American holly and feed on and oviposit in soft, partially expanded leaves. Early spring defoliation, such as commonly results from freezing injury to young shoots, is followed several weeks later by a second flush of young leaves from lateral buds. We simulated this phenomenon by manually defoliating whole small trees and individual shoots of large trees to test the hypothesis that freezing injury can encourage leafminer outbreaks by inducing an abundance of soft, protein rich young leaves late in the adult activity period, when availability of vulnerable leaves becomes limited. Defoliation of small trees one or two weeks after bud break resulted in six- to 13-fold increases in the incidence of feeding punctures and larval mines on second flush leaves as compared with densities on original young leaves of control trees. Similarly, we induced significant increases in feeding punctures and larval mines on second flush leaves of individual defoliated shoots, although leaves that did not open until after the flight period escaped this injury. These observations underscore the capability of adult female P. ilicicola to locate and exploit a small number of phenologically available leaves among many hundreds of older leaves on the same tree. By altering the phenology of leaf flush, certain kinds of environmental stress may predispose perennial plants to outbreaks of early season folivores that restrict their feeding or oviposition to very young leaves.

Relative resistance or susceptibility of maple (Acer) species, hybrids and cultivars to six arthropod pests of production nurseries.

BACKGROUND Maples (Acer spp.) in production nurseries are vulnerable to numerous arthropod pests that can stunt or even kill the young trees. Seventeen cultivars representing various Acer species and hybrids were evaluated for extent of infestation or injury by shoot and trunk borers (Proteoteras aesculana, Chrysobothris femorata), potato leafhopper (Empoasca fabae), Japanese beetle (Popillia japonica), maple spider mite (Oligonychus aceris) and calico scale (Eulecanium cerasorum). Evaluations were done in replicated field plots in central and western Kentucky. RESULTS All of the maples were susceptible, to varying degrees, to one or more key pest(s). Red maples (A. rubrum) were relatively vulnerable to potato leafhopper injury and borers but nearly free of Japanese beetle feeding and spider mites. Sugar maples sustained conspicuous Japanese beetle damage but had very low mite populations, whereas the opposite was true for Freeman maples (A. × freemanii). A. campestre was heavily infested by calico scale. Within each species or hybrid there were cultivar differences in degree of infestation or damage by particular pests. CONCLUSION The results should help growers to focus pest management efforts on those plantings at greatest risk from particular pests, and to choose cultivars requiring fewer insecticide inputs to produce a quality tree.