Functional Comparison of Three Chitinases from Symbiotic Bacteria of Entomopathogenic Nematodes.

Xenorhabdus and Photorhabdus, bacterial symbionts of entomopathogenic nematodes Steinernema and Heterorhabditis, respectively, have several biological activities including insecticidal and antimicrobial activities. Thus, XnChi, XhChi, and PtChi, chitinases of X. nematophila, X. hominickii, and P. temperata isolated from Korean indigenous EPNs S. carpocapsae GJ1-2, S. monticolum GJ11-1, and H. megidis GJ1-2 were cloned and expressed in Escherichia coli BL21 to compare their biological activities. Chitinase proteins of these bacterial symbionts purified using the Ni-NTA system showed different chitobiosidase and endochitinase activities, but N-acetylglucosamidinase activities were not shown in the measuring of chitinolytic activity through N-acetyl-D-glucosarmine oligomers. In addition, the proteins showed different insecticidal and antifungal activities. XnChi showed the highest insecticidal activity against Galleria mellonella, followed by PtChi and XhChi. In antifungal activity, XhChi showed the highest half-maximal inhibitory concentration (IC50) against Fusarium oxysporum with 0.031 mg/mL, followed by PtChi with 0.046 mg/mL, and XnChi with 0.072 mg/mL. XhChi also showed the highest IC50 against F. graminearum with 0.040 mg/mL, but XnChi was more toxic than PtChi with 0.055 mg/mL and 0.133 mg/mL, respectively. This study provides an innovative approach to the biological control of insect pests and fungal diseases of plants with the biological activity of symbiotic bacterial chitinases of entomopathogenic nematodes.

High level of genetic diversity among Steinernema monticolum in Korea revealed by single-enzyme amplified fragment length polymorphism.

Steinernema monticolum was first described from a mountainous forest at sites of Sancheong, Gyeongnam province in Korea. Since S. monticolum is one of the most commonly isolated entomopathogenic nematodes from Korea, it is desirable to investigate the diversity of this species. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analyses were used to differentiate 32 S. monticolum populations. Our results revealed a high level of genetic diversity within S. monticolum at the population level. On the geographic scale, SE-AFLP analysis revealed that there was no correlation between the genetic similarity of populations of this species and their geographical proximity.

Temperature effects on Korean entomopathogenic nematodes, Steinernema glaseri and S. longicaudum, and their symbiotic bacteria.

We investigated the temperature effects on the virulence, development, reproduction, and motility of two Korean isolates of entomopathogenic nematodes, Steinernema glaseri Dongrae strain and S. longicaudum Nonsan strain. In addition, we studied the growth and virulence of their respective symbiotic bacterium, Xenorhabdus poinarii for S. glaseri and Xenorhabdus sp. for S. longicaudum, in an insect host at different temperatures. Insects infected with the nematode-bacterium complex or the symbiotic bacterium was placed at 13 degrees C, 18 degrees C, 24 degrees C, 30 degrees C, or 35 degrees C in the dark and the various parameters were monitored. Both nematode species caused mortality at all temperatures tested, with higher mortalities occurring at temperatures between 24 degrees C and 30 degrees C. However, S. longicaudum was better adapted to cold temperatures and caused higher mortality at 18 degrees C than S. glaseri. Both nematode species developed to adult at all temperatures, but no progeny production occurred at 13 degrees C or 35 degrees C. For S. glaseri, nematode progeny production was best at inocula levels above 20 infective juveniles/host at 24 degrees C and 30 degrees C, but for S. longicaudum, progeny production was generally better at 24 degrees C. Steinernema glaseri showed the greatest motility at 30 degrees C, whereas S. longicaudum showed good motility at 24 degrees C and 30 degrees C. Both bacterial species grew at all tested temperatures, but Xenorhabdus sp. was more virulent at low temperatures (13 degrees C and 18 degrees C) than X poinarii.

Distribution and adult activity of Popillia quadriguttata (Coleoptera: Scarabaeidae) on golf courses in Korea.

Japanese beetle traps baited with the Popillia japonica Newman (Coleoptera: Scarabaeidae) pheromone lure and a eugenol feeding attractant were placed at five golf courses in Korea to determine how well they work for detecting activity of a closely related species, Popillia quadriguttata (F.) (Coleoptera: Scarabaeidae), a turf pest in Korea. The traps also were used to determine the time of day and time of year that P. quadriguttata is most active. Nineteen scarab species of 13 genera were attracted to the Japanese beetle traps with P. quadriguttata clearly being the most abundant (383 beetles per trap), followed by Adoretus tenuimaculatus Waterhouse (10 per trap), Popilliaflavosellata Fairmaire (seven per trap), Exomala orientalis Waterhouse (four per trap), and Maladera japonica (two per trap). Other scarab species were trapped at a rate of <1.0 per trap. Popillia quadriguttata adults were active over a 5-wk period in late June and early July. At Yongwon Golf Club in 2002, peak adult activity was during the last week of June in visual counts and approximately 1 wk later in the Japanese beetle traps. In Korea, P. quadriguttata adults are most active between 12:00 p.m. and 4:00 p.m. This information should be helpful to golf course superintendents in Korea and to entomologists interested in finding natural enemies of P. quadriguttata to evaluate as potential biocontrol organisms for the very closely related species, the Japanese beetle.

Attraction of Piezodorus hybneri to the aggregation pheromone components of Riptortus clavatus.

The aggregation pheromone of Riptortus clavatus (Heteroptera: Alydidae) consists of (E)-2-hexenyl (Z)-3-hexenoate (E2HZ3H), (E)-2-hexenyl (E)-2-hexenoate (E2HE2H), and myristyl isobutyrate (MI). Experiments were conducted to examine the attraction of Piezodorus hybneri (Heteroptera: Pentatomidae) to the pheromone components of R. clavatus, individually and in combination, and to determine if P. hybneri produces these compounds. Electroantennogram (EAG) responses of P. hybneri to E2HE2H were significant in a dose dependent manner, while those to E2HZ3H were not significantly different from those to hexane and air. Field trapping tests using E2HZ3H, E2HE2H, and MI, individually or in combination, showed that only E2HE2H was attractive to P. hybneri. E2HE2H was not detected in whole-body extracts or volatile collections from either sex of P. hybneri adults. We propose that the response of P. hybneri adults to the R. clavatus aggregation pheromone may be related to finding suitable host plants.

Effects of purified persimmon tannin and tannic acid on survival and reproduction of bean bug, Riptortus clavatus.

We evaluated the effects of tannic acid and purified perrsimmon tannin on survival and reproduction of bean bugs, Riptortus clavatus.Feeding behavior of R. clavatus was also examined on sweet (cv. Fuyu) and astringent (cv. Chongdosi) persimmon fruits. Soluble tannin in sweet persimmon fruits decreased from 3% in early June to 0.5% in late September, but it increased from 2 to 8% during the same period in astringent persimmon fruits. More bugs visited sweet than astringent persimmon. Numbers of piercing/sucking spots were higher on sweet than on astringent persimmon. When fed 1 and 3% solutions of persimmon tannin, adult bugs ingested only 64.1 and 9.5% of the amount of water ingested by those offered the control (distilled water). Amounts of persimmon tannin ingested by the adult bugs were 6.5 and 2.8 times higher at 1 and 3% tannin solutions compared to a 0.1% solution. Persimmon tannin exerted negative effects on survival and reproduction of R. clavatus at higher concentrations (1 and 3% solutions). Feeding of R. clavatus adults decreased with increasing tannin concentrations. When results from both sexes were pooled, 50% mortality was achieved at 11 and 4 days after treatment with the 1 and 3% tannin solutions, respectively. Reproduction decreased with 1% tannin, and no eggs were produced with 3% tannin solution. Tannic acid was similar in its effects on R. clavatus. All nymphs died 14, 12, and 7 days after feeding on 0.1, 1, and 3% tannic acid, respectively. Adults were less sensitive than nymphs, and their survival was not affected by 0.1% tannic acid. However. 1 and 3% tannic acid solutions were fatal. Survivorship decreased to 50% at 11 and 6 days after supplying tannin solutions of 1 and 3% concentrations. Higher concentrations (1 and 3%) resulted in reduced reproduction, as was seen with persimmon tannin. Our data may explain why R. clavatus does not invade sweet persimmon orchards until late July, when concentrations of soluble tannin are low enough to allow them to feed.

Laboratory and field evaluation of Korean entomopathogenic nematode isolates against the oriental beetle Exomala orientalis (Coleoptera: Scarabaeidae).

The oriental beetle Exomala (Anomala) orientalis (Waterhouse) is an important pest of turfgrass in Korean golf courses, and although a few chemical insecticides are registered for insect pest control, they are not very effective against scarab larvae. There is also a growing concern in Korea about the run-off of insecticides into sensitive habitats and the potential for groundwater contamination. A safe and environmentally sound alternative is needed to conventional insecticides. We therefore evaluated six Korean entomopathogenic nematode isolates: S. carpocapsae Pocheon, S. glaseri Dongrae, S. glaseri Mungyeong, S. longicaudum Gongju, S. longicaudum Nonsan, and Heterorhabditis sp. Gyeongsan for their potential as bioinsecticides for control of E. orientalis. In addition, we evaluated a reduced chemical insecticide approach that combined chlorpyrifos-methyl with nematodes. In laboratory tests Heterorhabditis sp. Gyeongsan was the most efficacious, causing 100% mortality of the second and 38% of the third instars. All other nematode isolates caused 50-80% mortality of the second and 15-30% of the third instars. E. orientalis pupae were highly susceptible to all the Korean entomopathogenic nematode isolates except S. carpocapsae. In artificially infested field plots, all Korean nematode isolates cause 50-70% mortality of the third instar. A combination of a one-half rate of Heterorhabditis sp. and a one-half rate chlorpyrifos-methyl was synergistic, causing 91% mortality compared with 69% for the full rate of Heterorhabditis sp. or 22% for the full rate of chlorpyrifos-methyl. In a second field trial, a natural infestation of preoverwintering third instar was treated. In this trial a one-half rate of S. longicaudum Nonsan plus a one-half rate of chlorpyrifos-methyl caused 96.8% mortality, much more than a full rate of S. longicaudum Nonsan (45.9% mortality) or a full rate of chlorpyrifos-methyl (28.7% mortality). The interactions of Heterorhabditis sp. and S. longicaudum Nonsan with chlorpyrifos-methyl in our field trials appear to be synergistic.

Life history and spatial distribution of Oriental beetle (Coleoptera: Scarabaeidae) in golf courses in Korea.

Larval and adult activity of the oriental beetle Exomala orientalis (Waterhouse), a pest of turfgrass in Korea, was investigated at four golf clubs in Pusan, Korea, from 1995 to 1999. Adult emergence was first observed on the greens in late May with peak activity occurring 2 wk later. During the day, E. orientalis adults were most active between 1800 and 2200 hours. First instars were found mostly in early July, second instars mostly in late July, and third instars from August to April. The density of larvae in fixed plots decreased steadily from the time of egg laying to pupation: 667/m3 on 26 July, 267/m3 on 29 August, and 122/m3 on 2 October 1997. All the observed E. orientalis completed one generation per year. Adult females were observed feeding on flowers of a late-blooming variety of Japanese chestnut (Castanea crenata Sieb & Zucc). E. orientalis larval densities were higher in greens with Japanese chestnut nearby, and where magpie, Pica pica sericea (Gould), feeding was observed. More E. orientalis adults emerged from the right, left, and back of greens than from the front or middle. The intensity of emergence was inversely proportional to the amount of golfer traffic on various parts of the green. Counting emergence holes may be a way that golf course superintendents can predict which greens and tees are most likely to be damaged from E. orientalis larvae without destructive sampling.