Fluorescein-labeled wheat germ agglutinin stains the pine wood nematode, <Emphasis Type="Italic">Bursaphelenchus </Emphasis><Emphasis Type="Italic">xylophilus</Emphasis>

Pine wilt disease, caused by the pine wood nematode (PWN, Bursaphelenchus xylophilus), is a major threat to pine forests throughout East Asia. Nonetheless, its mechanism of invasion has not yet been described in detail. To better understand the pathology of this disease, it is important to examine the distribution of PWNs within pine tissue during the course of disease development. We attempted to stain nematodes with fluorescein-conjugated wheat germ agglutinin (F-WGA) as a means to locate and track the spread of PWNs. Although PWNs proliferated on Botrytis cinerea fungus were successfully stained only on their vulvas and spicule holes, PWNs extracted from inoculated Pinus thunbergii seedlings were stained on their surface. Stainability, or the percentage of PWNs stained with F-WGA over more than half of their surface, was about 20% by 1 day after inoculation, but increased to 80% at 10 days. The stainability of PWNs extracted from a 5-cm main stem segment that included the inoculation site was less than that of PWNs extracted from other parts of the main stem farther away (i.e., those that had dispersed). These results suggest that stainability is related to dispersal activity in time. Thus, to raise the stainability of PWNs at shorter timeframes after inoculation, PWNs with more than 80% stainability were re-inoculated into pine seedlings. This resulted in more than 70% stainability from 1 to 6 days after inoculation. In F-WGA stained thin paraffin sections of pine tissue of re-inoculated seedlings, PWNs brightly fluoresced under epifluorescence and were easily detected against the dark background of pine tissue. This staining technique with F-WGA is an excellent tool for detecting PWNs in pine tissue.     

Control of a new turf pest, <Emphasis Type="Italic">Dorcadion pseudopreissi</Emphasis> (Coleoptera: Cerambycidae), with the entomopathogenic nematode <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis>

The use of biological control in turf has increased to avoid possible negative effects on humans. Entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae have control potential against many economically important insect pests. In the present study, the efficacy of Heterorhabditis bacteriophora against a new pest on turf, Dorcadion pseudopreissi, was examined in the field. Cages (1 × 1 × 1 m) with female and male D. pseudopreissi were placed on two kinds of turf: Lolium perenne and Festuca arundinacea. After beetles had deposited eggs into the moist soil surface, the cages were removed and H. bacteriophora was applied at 0.5 million infective juveniles/m² to half the plots. Application of H. bacteriophora caused a statistically significant reduction in numbers of beetle larvae in L. perenne plots and a non-significant reduction in F. arundinacea plots. The area damaged by D. pseudopreissi was significantly reduced by nematode application in both turf species. The number of nematodes declined after application, but small numbers could still be detected after 6 months.     

Migration of pine wood nematodes in the tissues of <Emphasis Type="Italic">Pinus thunbergii</Emphasis>

The distribution of pine wood nematodes (Bursaphelenchus xylophilus, PWNs) in Japanese black pine (Pinus thunbergii) tissues was investigated by staining with fluorescein isothiocyanate-conjugated wheat germ agglutinin. After PWNs were inoculated to current-year stems of pine seedlings, their distribution at about 5 cm below the inoculation site was confined only to cortical resin canals 1 day after inoculation, and then spread to other tissues, including resin canals of short branches. When PWNs were inoculated onto cross or tangentially cut surfaces of stem segments, maximal PWN migration speed was estimated to be faster through cortical resin canals and xylem axial resin canals vertically (>6.7 and <2.3 mm/h, respectively) than through cortical tissues both vertically and horizontally (<1.2 and <0.2 mm/h). To examine whether PWNs in cortical resin canals could invade surrounding tissues, segments in which PWNs resided only in cortical resin canals were prepared by removing the top portion 6 h after inoculation. Additional incubation of such segments caused extended PWN distribution to xylem axial resin canals and then to other tissues. A similar experiment with top portions of girdled segments removed 12 h after inoculation also showed extended PWN distribution from xylem axial resin canals and pith to cortical resin canals and then to other tissues. These results provided direct evidence that PWNs have the ability to migrate from cortical resin canals and xylem axial resin canals to other tissues.     

Isolation and characterization of 10 new microsatellite loci in the ambrosia beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis>

The ambrosia beetle, Platypus quercivorus, is an insect vector for the causal agent of Japanese oak wilt disease, Raffaelea quercivora. We have developed 10 new polymorphic microsatellite markers from Type 1, one of the genetic types of the beetle, using a biotin-enrichment procedure. Fifty-three adult beetles were used for characterization of the markers. The number of alleles per locus ranged from two to nine (average 4.9). The observed and expected heterozygosity ranged from 0.49 to 0.77 and from 0.44 to 0.80, respectively. None of the loci deviated significantly from Hardy–Weinberg equilibrium. No linkage disequilibrium was detected in any pairwise comparisons of loci. Most of the markers successfully amplified in other genetic types of the beetle. These markers could become useful tools for population genetic studies and for parentage and sibship analysis for this beetle.     

Population dynamics of <Emphasis Type="Italic">Monochamus galloprovincialis</Emphasis> Olivier (Coleoptera: Cerambycidae) in two pine species under laboratory conditions

The study of the life history dynamics of the pine sawyer beetle Monochamus galloprovincialis (Olivier) is important because the beetles vector the pinewood nematode. The objectives of this study were to investigate differences in beetle life history parameters between the two common host tree species in Turkey, Pinus sylvestris and Pinus nigra. Relationships between the number of oviposition sites, holding time (time between log cut and oviposition), log moisture content, log volume and area, and adult survivorship were evaluated. No significant differences were found between the two tree species except adult density per m². The holding time did not influence the number of oviposition sites, larval entry holes or adults for P. sylvestris. There was a negative correlation between the holding time and the number of oviposition sites for P. nigra. The number of adults emerged per log was correlated with the number of larval entry holes only for P. sylvestris. Only 12 and 15% of the initial cohort completed development and emerged as adults for P. sylvestris and P. nigra, respectively. Both tree species are equally suitable hosts for M. galloprovincialis development. We conclude that the high within-log mortality of beetles is a combination of resource quality and quantity factors and intraspecific competition and cannibalism during larval development.     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

Enzymatic saccharification and ethanol production of <Emphasis Type="Italic">Acacia mangium</Emphasis> and <Emphasis Type="Italic">Paraserianthes falcataria</Emphasis> wood,and <Emphasis Type="Italic">Elaeis guineensis</Emphasis> trunk

We examined the saccharification and fermentation of meals from Acacia mangium wood, Paraserianthes falcataria wood, and Elaeis guineensis trunk. The levels of enzymatic hydrolysis of cellulose and ethanol production were highest for P. falcataria wood and lowest for A. mangium wood. Ultrasonication pretreatment of meal further increased the rates of hydrolysis and ethanol production in meal from P. falcataria wood. Through this pretreatment, hemicelluloses (xylan and xyloglucan) and cellulose were released in the meal from P. falcataria wood. Loosening of hemicellulose associations can be expected to make P. falcataria wood more useful for bioethanol production.     

<Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda: Aphelenchoididae) vectored by <Emphasis Type="Italic">Monochamus </Emphasis><Emphasis Type="Italic">urussovi</Emphasis> (Coleoptera: Cerambycidae) in Hokkaido,Japan

Bursaphelenchus mucronatus Mamiya et Enda has been recovered for the first time from adults of the cerambycid beetle, Monochamus urussovi (Fischer), in Hokkaido, Japan. The nematode was also recovered from the inner bark of Picea jezoensis (Siebold et Zuccarini) Carrière and Abies sachalinensis (Fr Schmidt) Masters infested with M. urussovi larvae. PCR–RFLP analysis indicated that B. mucronatus in Hokkaido is the European type.     

Controlling the sugar beet beetle <Emphasis Type="Italic">Cassida vittata</Emphasis> with entomopathogenic nematodes

Sugar beet, Beta vulgaris L. is a strategic crop of sugar industry in Egypt. It is threatened by several insect pests among most important of them is the beet beetle Cassida vittata. This work deals with the biological control of this insect using four Egyptian and imported entomopathogenic nematodes (EPNs). The nematodes included Steinernema carpocapsae S2, Heterorhabditis bacteriophora S1, S. feltiae and H. bacteriophora (1-3). Daily mortality of larvae, pupae and adults of C. vittata were recorded after treatment with serial concentrations (from 500 to 4,000 infective juveniles/ml) of each of four studied EPNs. Development of nematodes in insect bodies was followed up. S. carpocapsae S2 was chosen for the application against different stages of the pest in a sugar beet field. In the field, single application of S. carpocapsae S2 killed 65% of the larvae, 92% of the pupae and 57.3% of the adults of C. vittata within a week. This work is the first report on using the EPNs to control sugar beet beetle.     

Bioremediation of CCA-treated wood by brown-rot fungi <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>, <Emphasis Type="Italic">Coniophora puteana</Emphasis>, and <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis>

This study evaluated oxalic acid accumulation and bioremediation of chromated copper arsenate (CCA)-treated wood by three brown-rot fungi Fomitopsis palustris, Coniophora puteana, and Laetiporus sulphureus. The fungi were first cultivated in a fermentation broth to accumulate oxalic acid. Bioremediation of CCA-treated wood was then carried out by leaching of heavy metals with oxalic acid over a 10-day fermentation period. Higher amounts of oxalic acid were produced by F. palustris and L. sulphureus compared with C. puteana. After 10-day fermentation, oxalic acid accumulation reached 4.2 g/l and 3.2 g/l for these fungi, respectively. Fomitopsis palustris and L. sulphureus exposed to CCA-treated sawdust for 10 days showed a decrease in arsenic of 100% and 85%, respectively; however, C. puteana remediation removed only 18% arsenic from CCA-treated sawdust. Likewise, chromium removal in F. palustris and L. sulphureus remediation processes was higher than those for C. puteana. This was attributed to low oxalic acid accumulation. These results suggest that F. palustris and L. sulphureus remediation processes can remove inorganic metal compounds via oxalic acid production by increasing the acidity of the substrate and increasing the solubility of the metals.An erratum to this article can be found at     

Nutritional requirements for larval development of the dry wood borer <Emphasis Type="Italic">Trichoferus holosericeus</Emphasis> (Rossi) in laboratory cultures

Despite the dangerousness of the dry wood-boring beetle Trichoferus holosericeus (Rossi) (=Hesperophanes cinereus (Villiers)) (Coleoptera: Cerambycidae), little is known about its biological and nutritional requirements. The aim of this research was therefore to investigate into the dietary preferences of this insect, in order to determine if carbohydrates can be considered as its main nutritional source, to clarify whether this cerambycid is specialised on hardwoods and finally to select a suitable diet for laboratory cultures. Different substrates were tested and the decay grade, as well as the survival of larvae, was recorded after 3 and 6 months. Chemical analyses were also carried out on excrements, residues of wood after larvae excavation and sound wood. Results indicated that T. holosericeus fed mainly on glucose and peptone added to an artificial diet, while on natural wood it preferred structural carbohydrates constituting the hemicelluloses and the non-crystalline cellulose. Furthermore, Scots pine sapwood had no toxic effect to the beetle. In terms of number and dimensions of emerged larvae, the artificial diet resulted to be the most suitable substrate for laboratory cultures of T. holosericeus among those tested.     

Antifeedant activities of secondary metabolites from <Emphasis Type="Italic">Ajuga nipponensis</Emphasis> against adult of striped flea beetles, <Emphasis Type="Italic">Phyllotreta striolata</Emphasis>

Antifeedant activities of the isolated chemical compounds from Ajuga nipponensis, were studied against adult of striped leaf beetles. The methanol, petroleum ether, ethyl acetate and n-butanol extracts at 1.0 mg/ml, were used in this study. All four extracts exhibited more than 65 antifeedant index at 24 h and ethyl acetate extract showed significant activity against striped leaf beetles with 83.12 antifeedant index. Six compounds and one fraction were isolated by chromatography and their structures were identified by NMR, MS and FTIR spectra. At 2.0 mg/ml for 24 h the three compounds 20-hydroxyecdysone, acacetin and apigenin showed considerable activities with antifeedant indexes 59.29, 51.22 and 61.55, respectively. In contrast to this the antifeedant indexes of acacetin and apigenin, were sharply reduced as the time extended and that of 20-hydroxyecdysone remained unchanged. In addition, the synergistic effects of two mixtures of secondary metabolites, were studied and no sharp difference was observed.     

Insecticidal properties of <Emphasis Type="Italic">Thymus persicus</Emphasis> essential oil against <Emphasis Type="Italic">Tribolium castaneum</Emphasis> and <Emphasis Type="Italic">Sitophilus oryzae</Emphasis>

Red flour beetle Tribolium castaneum (Herbst) and rice weevil Sitophilus oryzae (L.) are considered to be the major insect pests in storage. Essential oils from aromatic plants are recognized as proper alternatives to fumigants. Thymus persicus (Ronniger ex Rech. f.) is one of these plants that have medicinal properties and is indigenous to Iran. The essential oil was obtained from aerial parts of the plant and analyzed by GC and GC–MS. Carvacrol (44.69%) and thymol (11.05%) were the major constituents of the oil extracted. In this experiment, fumigant toxicity of the essential oil was studied against T. castaneum, S. oryzae at 27 ± 1°C and 60 ± 5% RH in dark condition. The adult insects were exposed to the concentrations of 51.9, 111.1, 207.4 and 370.4 μl/l air to estimate median lethal time (LT₅₀) values. The fumigant toxicity was increased in response to increased essential oil concentrations. The LT₅₀ values at the lowest and the highest concentrations tested were ranged from 28.09 to 13.47 h for T. castaneum, and 3.86 to 2.30 h for S. oryzae. It was found that S. oryzae adults were much more susceptible to the oil than T. castaneum. After 24 h of exposure, the LC₅₀ values (95% fiducial limit) for T. castaneum and S. oryzae were estimated to be 236.9 (186.27–292.81) and 3.34 (2.62–4.28) μl/l air, respectively. These results suggest that T. persicus essential oil merits further study as potential fumigant for the management of these stored-product insects.     

Effects of organic acid treatments on small hive beetles, <Emphasis Type="Italic">Aethina tumida</Emphasis>, and the associated yeast <Emphasis Type="Italic">Kodamaea ohmeri</Emphasis>

In honeybee, Apis mellifera, colonies infested with larval and adult small hive beetles (SHB), hive material, and in particular honey, tends to ferment, probably due to SHB-associated yeasts such as the predominant Kodamaea ohmeri. Here, we test the effects of organic acids on K. ohmeri and on SHB-infested honey/pollen combs. Organic acids were applied at standard concentrations used by beekeepers to control other pests. In laboratory tests, the growth of K. ohmeri was significantly inhibited by lactic, formic and acetic acids. Treatments of SHB-infested honey/pollen combs (N = 18 colonies) with acetic acid significantly increased mortality of adult SHB and treatments with formic acid significantly reduced larval infestation. Our data suggest that treatment of honeybee colonies and storage rooms with organic acids could also help in reducing SHB damage.     

Seasonal population trends of<Emphasis Type="Italic"> Liriomyza huidobrensis</Emphasis> (Blanchard, 1926) (Diptera: Agromyzidae) on cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) in Western Turkey

Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae) is an important pest in cucumber greenhouses in Izmir, Western Turkey. This study was carried out during the spring and autumn seasons of 1999 and 2000 in two adjacent cucumber greenhouses, each 1,000 m² in size. Population densities of adult L. huidobrensis in insecticide-treated and non-treated greenhouses were compared by using yellow sticky traps. Ten traps were placed in the non-treated greenhouse and ten traps in the insecticide-treated greenhouse. Yellow sticky traps were changed weekly and the number of flies counted in order to compare the two greenhouses. In addition to monitoring the leaf miner population, in each greenhouse 100 plants were checked and the ratio of infested plants, the ratio of infested leaves per plant and the number of mines per leaf were recorded weekly. The population density of the leaf miner was almost the same in both insecticide-treated and non-treated greenhouses. Thus, it was concluded that using yellow sticky traps will be healthier and more cost effective than using pesticides. Furthermore using pesticides is risky for the environment and the use of yellow sticky traps can easily reduce the number of pesticide applications. If yellow sticky traps are used in the greenhouse in a completely isolated environment and the appropriate insecticide is used when it is necessary, this will provide successful and integrated management against L. huidobrensis.     

Spectrophotometric assay of a wood preservative, <Emphasis Type="Italic">N,N</Emphasis>-didecyl-<Emphasis Type="Italic">N</Emphasis>-methylpoly(oxyethyl) ammonium propionate (DMPAP), in aqueous solution

A spectrophotometric assay based on the color reaction between N,N-didecyl-N-methyl-poly(oxyethyl) ammonium propionate (DMPAP) and 4-[4-(dipropylamino) phenylazo]-benzenesulfonic acid (propyl orange) was used to determine DMPAP concentrations as a wood preservative. The assay was carried out using a propyl orange solution at pH 2.9. The visible absorption spectrum of propyl orange showed an absorption maximum at 510 nm, which decreased linearly with increasing DMPAP concentration from 0 to 10 ppm. To apply this assay method to determine DMPAP retention in treated wood, the influence of wood extractives on the assay was investigated. Extractives from Japanese cedar, hinoki cypress, and Japanese larch were found to increase apparent DMPAP concentration. However, it was also found that measuring visible absorption at 477 nm prevented overestimation and gave precise values. This assay can be a viable alternative to the current methods for the determination of DMPAP concentrations.     

Relative dominance of the mold fungus,<Emphasis Type="Italic">Raffaelea</Emphasis> sp., in the mycangium and proventriculus in relation to adult stages of the oak platypodid beetle,<Emphasis Type="Italic">Platypus quercivorus</Emphasis> (Coleoptera; Platypodidae)

Microorganisms associated with the oak platypodid beetle,Platypus quercivorus (Murayama), were isolated from the mycangium of adult females, from the proventriculus of adults of both sexes, and from galleries in the period before dispersal. Fungi isolated from female mycangia were separated into three groups:Raffaelea sp., yeasts (mainlyCandida spp.), and other species.Raffaelea sp. was isolated predominantly from the teneral stage to the dispersal stage, but was not isolated from the mycangia of mother beetles in new galleries. Yeasts were isolated in every beetle stage tested and their isolation rate was over 80% from the teneral stage to the dispersal stage. All three fungal groups were found in the female proventriculus.Raffaelea sp. was isolated only in the dispersal stage at a rate of 40%, whereas the isolation rate of yeasts gradually increased beginning in the mature stage and reached 100% in the rearing stage. In contrast, in the male proventriculus, onlyRaffaelea sp. and yeasts were isolated in the dispersal and rearing stages. The isolation rate in the rearing stage ofRaffaelea sp. was less than 20%, but that of yeasts was 100%, although these rates were almost the same in the dispersal stage.Raffaelea sp. and yeasts were also isolated from cradles containing eclosing pupae. These results suggest thatP. quercivorus acquiresRaffaelea sp. and yeasts in their mycangia from the cradles immediately after eclosion, and maintain them to the dispersal stage. Then, they transmit these fungi from old to new galleries by way of mycangia, and possibly the digestive system.     

Field trap test for bioassay of synthetic (1<Emphasis Type="Italic">S</Emphasis>,4<Emphasis Type="Italic">R</Emphasis>)-4-isopropyl-1-methyl-2-cyclohexen-1-ol as an aggregation pheromone of <Emphasis Type="Italic">Platypus quercivorus</Emphasis> (Coleoptera: Platipodidae)

An ambrosia beetle, Platypus quercivorus, is a vector of Japanese oak wilt, which causes massive mortality of oak trees in Japan. Previous studies have identified (1S,4R)-4-isopropyl-1-methyl-2-cyclohexen-1-ol ((−)-IMCH) as the major component of the aggregation pheromone of P. quercivorus. We tested the ability of synthetic (−)-IMCH to attract P. quercivorus adults. The synthetic compound attracted 14.4 times as many P. quercivorus, of both sexes, as control traps. Control and (−)-IMCH-baited traps both captured 3.32 times as many males as females, probably because of the nature of Lindgren funnel traps and of male-biased emergence sex ratio early in the season of adult flights. (−)-IMCH was proved to be a major component of the aggregation pheromone of P. quercivorus.     

An inoculation experiment of Japanese <Emphasis Type="Italic">Bursaphelenchus</Emphasis> nematodes on Japanese black and red pine, <Emphasis Type="Italic">Pinus thunbergii</Emphasis> and <Emphasis Type="Italic">P. densiflora</Emphasis>

The pinewood nematode (PWN) Bursaphelenchus xylophilus is an invasive pathogen that was introduced from North America to Asian countries and Portugal and is devastating native pine forests. Some native European and Asian Bursaphelenchus nematodes also have weak to moderate pathogenicity to native pine species. To evaluate the potential risk of native Bursaphelenchus species, we inoculated ten Japanese Bursaphelenchus species into native pine species (the dominant forest species) in Japan, and evaluated their pathogenicity using mortality and tracheal tissue damage as indices. Inoculation was conducted on August 3, 2007, and the symptoms were observed every 2 weeks until February 1, 2008. None of the inoculated trees, excluding the pathogenic PWN inoculated control, showed external disease symptoms; however, four species [a less pathogenic PWN isolate, B. luxuriosae, Bursaphelenchus sp. NK215 (undescribed), and NK224 (undescribed)] caused tracheal tissue damage in inoculated seedlings and showed weak pathogenicity. Therefore, we conclude that there are some potentially pathogenic native species of nematodes distributed in Japan. Interestingly, two of these weakly pathogenic species, B. luxuriosae and NK215, are not associated with Pinaceae trees, suggesting that nematode pathogenicity may be a pre-adaptive character. More experimental studies under different conditions are necessary to accurately evaluate the potential risk of these pathogens.     

Pathogenic effects of <Emphasis Type="Italic">Xenorhabdus nematophilus</Emphasis> and <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> (Enterobacteriaceae) against pupae of the Diamondback Moth, <Emphasis Type="Italic">Plutella xylostella</Emphasis> (L.)

The relationship between Xenorhabdus nematophilus and Photorhabdus luminescens, the bacterial symbionts of the entomopathogenic nematodes Steinernema carpocapsae and Heterorhabditis bacteriophora, against the diamondback moth, P. xylostella (L.), pupae showed the pathogenic capability of P. luminescens to be over that of X. nematophilus. They gave 60 and 40?% mortality with LC₅₀ values of 5?×?10⁴ and 5.5?×?10⁵ cells/ml, respectively.The number of bacterial cells influences the rate of killing the pupae of P. xylostella and a direct correlation between mortality of the pupae, percentage of deformed adults and the spraying dose with significant differences were observed. An inverse correlation found between the no. of eggs laid/female, percentage of hatching, adult survival and the spraying dose. These bacterial effects have been attributed to different toxic bacterial enzymes which damage the haemocytes and inhibit activation of the humoral immune system of the insect.