Identification of entomopathogenic fungus Metarhizium rileyi infested in fall armyworm in the cornfield of Korea, and evaluation of its virulence.

The fall armyworm (FAW) Spodoptera frugiperda is an important invasive pest in Africa and Asia. It is a polyphagous pest with at least 353 recorded host plant species, including corn. Chemical control of this pest is unsuccessful because of a developed resistance and harmful effects on the environment. Entomopathogenic fungi are potential biological control agents for FAW. In this study, the native strain of Metarhizium rileyi (KNU-Ye-1), collected from a cornfield at Yeongcheon, Korea, was identified by morphological and molecular characterization. The susceptibility of the fourth-instar larvae of FAW to the native strain M. rileyi was examined in the laboratory. The results showed that the Korean strain of M. rileyi (KNU-Ye-1) was highly virulent to FAW larvae, causing 89% mortality 7 days posttreatment. Therefore, M. rileyi (KNU-Ye-1) identified in this study is highly valuable for the biological control of FAW in the field.

Integrated Identification and Genetic Diversity of Potentially Invasive Clearwing Moths (Lepidoptera: Cossoidea: Sesiidae) in Korea.

The populations of clearwing moth borers in Korea have recently caused extensive and severe damage to pin oaks (Quercus palustris Munchh.). We conducted field monitoring and molecular analyses to identify them in an integrated manner. Morphological examination and molecular analyses of the COI gene, based on intra- and interspecific genetic divergences (GDs), revealed that the borers were identified as two invasive species, Sphecodoptera sheni and Paranthrenella pinoakula sp. nov. The maximum intraspecific GD was found to be 1.9%, whereas the minimum interspecific GD was confirmed as 8.1%, indicating a distinct barcoding gap. Both the MJ network and NJ tree also showed that 18 haplotypes (Hs) were detected from the 52 COI sequences. The borers revealed a total of 17 Hs: (i) H1-H7 were detected in all seven regions with S. sheni; (ii) Wonju and Goyang populations of S. sheni revealed more than three Hs; (iii) H7 was closely connected with H8 of the Chinese population of S. sheni; (iv) H9-H10 were detected in other samples from the Wonju population with P. pinoakula sp. n. and were closely located with congeneric species. A maximum likelihood tree also revealed that P. pinoacula sp. n. nested within the congeneric species, genetically separating from S. sheni.

Non-destructive environmental safety assessment of threatened and endangered plants in weed biological control.

Assessing the risk of nontarget attack (NTA) for federally listed threatened and endangered (T&E) plant species confamilial to invasive plants targeted for classical biological control, is one of the most important objectives of pre-release environmental safety assessments in the United States. However, evaluating potential NTA on T&E species is often complicated by restrictive agency requirements for obtaining propagules, or the ability to propagate plants and rear agents to the appropriate phenostages synchronously for testing, or both. Here, we assessed whether plant cues associated with a host recognition can be used for testing the attractiveness of four T&E and one rare single population plant species non-destructively for a candidate biocontrol agent. We used the seed-feeding weevil, Mogulones borraginis, a candidate for the biological control of the invasive plant, Cynoglossum officinale (Boraginaceae) as the study system. We collected olfactory and visual cues in the form of flowering sprigs from T&E plant species confamilial to the invasive plant in a non-destructive manner and used them to measure behavioral responses and searching time of weevils. Female weevils preferred C. officinale to all tested plant species in dual-choice bioassays using either olfactory or visual cues in a modified y-tube device. Furthermore, female weevils were repelled by the combined olfactory and visual cues from all tested T&E plant species in a dual-choice test against controls (e.g., purified air in an empty arm), indicating that it would be extremely unlikely for the weevil to attack any of these species upon release in the United States. Principal component analysis based on 61 volatile organic compounds effectively separated the five confamilial plant species and C. officinale, corroborating the results of behavioral bioassays. We conclude that studies on pre-alighting host selection behavior and the underlying physiological mechanisms of how organisms select host plants they exploit can aid in environmental safety testing of weed biological control agents.

Conditions to Terminate Reproductive Diapause of a Univoltine Insect: Ceratapion basicorne (Coleoptera: Apionidae), a Biological Control Agent of Yellow Starthistle.

Ceratapion basicorne (Illiger) is a recently approved univoltine biological control agent that develops inside the rosette of yellow starthistle (Centaurea solstitialis L.), an invasive annual plant. Adult weevils normally emerge in early summer, and females are thought to be in reproductive diapause until the following spring, when they oviposit in rosettes. The long period of reproductive diapause constrains mass-rearing this weevil because only one generation per year can be produced. Determining the environmental conditions that regulate diapause termination may enable shortening diapause under laboratory conditions to increase production of adults to release. We tested three hibernating conditions (greenhouse [ambient temperature and photoperiod], glass door refrigerator [5°C and ambient photoperiod], and growth chamber [5°C and 24 h dark]) for three durations (4, 8, 11 wk). The highest proportion of females laying eggs came from the growth chamber, with 40% terminating diapause after 4 wk, 80% after 8 wk, and 95% after 11 wk of exposure. Our study demonstrates that duration of cold temperature is an important stimulus to terminate reproductive diapause of C. basicorne, and that exposure to ambient light had no effect at 5°C. However, 47% of females held at ambient greenhouse conditions, without any chilling period, completed diapause within 11 wk. Thus, a cold period can accelerate diapause development, but it is not necessary for its completion. Reducing the winter diapause period from about 6 mo to 11 wk should enable the production of multiple generations per year to increase the number of insects available to release.

Topical Application of Synthetic Hormones Terminated Reproductive Diapause of a Univoltine Weed Biological Control Agent.

Classical biological control is an important method for controlling invasive alien weeds. Univoltine insects can be highly effective biological control agents of annual weeds because they are well synchronized with their host plant. However, having only one generation per year makes it difficult and slow to multiply them in the laboratory for initial field releases. If it were possible to terminate reproductive diapause early, then we could rear multiple generations per year, which would greatly increase annual production. We used a recently approved biocontrol agent, Ceratapion basicorne (a univoltine weevil), for yellow starthistle (Centaurea solstitialis) as a model system to study the use of two insect hormones, 20-hydroxyecdysone (20E) and methoprene, to terminate reproductive diapause. Methoprene (1 µg applied topically) terminated reproductive diapause of female weevils, whereas doses of 0.0, 0.01 and 0.1 µg did not. The combination of methoprene and 20E had a stronger effect and induced an increase in eggs (1.51 ± 0.16 eggs/day, mean ± SE) compared with a methoprene only group (1.00 ± 0.13 eggs/day), and a control group (0.21 ± 0.04 eggs/day). Thus, topical application of these hormones should enable us to rear the weevil out of its normal season and produce more than one generation per year, which will increase productivity of mass-rearing it for field release. Once released in the field, the insect would continue as a univoltine agent that is well-synchronized with its host plant.

Delineating the Asphondylia prosopidis Complex (Diptera: Cecidomyiidae): Possible Biological Control Agents of Honey Mesquite.

The Asphondylia prosopidis Cockrell complex with its fungal associate produces four distinct bud galls on honey mesquite (Prosopis glandulosa). They are considered as potential biological control agents to reduce seed production of invasive mesquites. Previous studies suggest that the complex may consist of more than one gall midge species or biotype. Therefore, before conducting host specificity tests, it is essential to understand the relationships among the gall midges in the complex. Each gall type was collected from sympatric regions in Arizona, New Mexico, and Texas for four years. Here we show that midges producing each gall type were clearly separated based on phylogenetic analysis using DNA sequences in the cytochrome oxidase subunit I region. Furthermore, we confirmed that morphological differences between pupae from each gall type were discernible, although variable, using a scanning electron microscope. Based on these differences, we suggest that the A. prosopidis complex consists of four different gall midge species, three of which are cryptic species. Among them, Asphondylia species producing a barrel gall type and A. prosopidis producing the original gall type are potential biological control agents of P. glandulosa because they are multivoltine species with four to five generations per year that complete their annual life cycle exclusively on flower buds of mesquite.