Modification of Medium Composition for Enhancing the Production of Antifungal Activity from Xenorhabdus stockiae PB09 by Using Response Surface Methodology.

Xenorhabdus stockiae PB09 bacterium has been shown to exhibit antifungal activity against several plant pathogens. To improve its efficacy, the optimization of the nutritional components in culture media was performed. The medium components that have significant effects on antifungal activity of X. stockiae PB09 were initially identified using a fractional factorial design. Response surface methodology and central composite design were then used to create a model for optimizing the levels of carbon, nitrogen, and mineral sources that maximize antifungal activity of X. stockiae PB09. After that, the suitable carbon, nitrogen, and mineral sources were selected and adjusted by the second-order polynomial regression model, which predicted that 98.62% of antifungal activity could be obtained when the medium contained sucrose, yeast extract, NaCl, and K2HPO4 at 3.24, 23.71, 5.46, and 2.73 g/L, respectively. Laboratory verification of this recipe resulted in the antifungal activity at 97.95% in the shake flask experiment after 48-hour cultivation, which was significantly 27.22% higher than that obtained by using the TSB medium. In addition, X. stockiae PB09 cultured in the verified recipe by using 5 L fermenter could effectively inhibit the mycelial growth of Phytophthora sp., Rhizoctonia solani, Pythium sp., and Fusarium oxysporum. This study demonstrated that the RSM and CCD were shown to be valuable tools for optimizing the culture medium that maximize the antifungal activity of X. stockiae PB09.

Fast Population Growth in Physogastry Reproduction of Luciaphorus perniciosus (Acari: Pygmephoridae) at Different Temperatures.

Luciaphorus perniciosus Rack is one of the most serious pests of several cultivated mushroom species including Ganoderma lucidum (Fr.), Flammulina velutipes Karst., Auricularia polytricha (Mont.) Saac., Lentinus polychrous Lev., and Lentinus squarrosulus (Mont.) Singer in Thailand. Adult female Lu. perniciosus produce offspring inside their physogastric hysterosomas, with all embryos developing through to the adult stage while remaining in the abdomen. Once the abdomen ruptures, the female parent dies and the offspring consisting of mostly fertilized female adults along with a few male adults continue to emerge from the cadaver of the mother for a period of several days. This peculiar type of reproduction after the death of the mother is a special case for life table analysis and has not been discussed previously in demographic analyses. In this study, the life table data of this mite fed on Le. squarrosulus were collected at 25, 30, and 35 °C and analyzed by using the age-stage, two-sex life table. The standard errors of population parameters were estimated by using the bootstrap technique (200,000 bootstraps). At 25, 30, and 35 °C, females started reproduction at ages 9, 5, and 3 d, respectively; the net reproductive rates (R0) were 192.27, 253.81, and 234.11 offspring. Due to their rapid development and high fecundity, the r values were as high as 0.4189, 0.8653, and 1.0892 d-1 at 25, 30, and 35 °C, respectively. Computer projection indicated that the mushroom mites Lu. perniciosus is capable of a threefold daily increase at 35 °C.

A new species of Blattisocius (Acari: Mesostigmata: Blattisociidae), with a new characterisation of the genus.

A new species of Blattisocius Keegan, Blattisocius thaicocofloris n. sp., is described from Thailand from adult females and males, raising to 18 the number of species known in this genus. This is the first species of this genus described from flowers (of Cocos nucifera L.; Arecaceae), although two species, Blattisocius apis Basha & Yousef and Blattisocius apisassociae Chinniah & Mohanasundaram, were reported in association with honeybees, which could suggest an association with flowers. The new species requires updates to the diagnostic characteristics of the genus and recently published keys for generic identification of blattisociid subfamilies, blattisociine genera and Blattisocius species.

Synergistic repellent and irritant effect of combined essential oils on Aedes aegypti (L.) mosquitoes.

This study was designed to compare the behavioral responses of Aedes aegypti to a single essential oil and to a mixture of two or three essential oils using an excito-repellency test chamber. Mixtures were prepared from essential oils extracted from Litsea cubeba (LC), Litsea salicifolia (LS), and Melaleuca leucadendron (ML). In general, the mixture of essential oils produced a much stronger escape response by Ae. aegypti, regardless of the test conditions. No significant difference in escape responses was seen when the mixture of oils was compared with a standard commercial product containing DEET. Greater contact irritancy was seen from mixed oils of LC and LS than with other mixed oils. Mixtures of LC and LS at 0.075% showed the highest synergistic action (65.5% escaped) compared to that with unmixed oil alone at the same concentration (LC/20% and LS=32.2%). In addition, mixtures of LC and LS at 0.075% demonstrated the highest non-contact repellency (62.7%) and showed a greater effect than the use of LC (20%) or LS (20.3%) alone. We conclude that mixtures of two essential oils show potential as active ingredients for mosquito repellents.

Complementary description of Colomerus novahebridensis Keifer (Acari, Eriophyidae), with a discussion about the constitution of the genus and its economic importance, and a tentative key to Colomerus Newkirk & Keifer species.

Colomerus Newkirk & Keifer, 1971 is an eriophyid genus described by Newkirk and Keifer about 43 years ago, that contains species from all continents, except Antarctica. They live mostly on dicotyledonous plants. Colomerus novahebridensis Keifer, 1977 was described from coconut (Cocos nucifera L., Arecaceae) fruits from Vanuatu. A description of a Thai population of this species is given in this paper. A revised characterization of Colomerus and a dichotomous key for the separation of the species presently considered to belong to this genus are provided, and a consideration about the importance of Colomerus species is presented.

New species and new records of Tetranychidae (Acarina, Prostigmata) from Thailand.

Sampling efforts conducted in several provinces from Thailand disclosed three new species of tetranychid mites. Two of them belong to the genus Tetranychus, namely Tetranychus occultaspina sp. nov. and Tetranychus truncatissimus sp. nov. and the third species belongs to the genus Schizotetranychus, Schizotetranychus krungthepensis sp. nov. They were collected on Ipomoea aquatica, Bambusa multiplex and Saccharum officinarum, respectively. New records and new hosts are also mentioned. 

Acaricidal activities of whole cell suspension, cell-free supernatant, and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.).

Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp., a mushroom mite endemic in Thailand. To develop an effective formulation of Xenorhabdus stokiae, treatments using different parts of X. stokiae isolate PB09 culture, including whole cell suspension, cell-free supernatant, and crude cell extract, were performed. The results show that different parts of X. stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity. Application with cell-free supernatant of X. stokiae culture resulted in both the highest mite mortality rate [(89.00±3.60)%] and the lowest mite fecundity [(41.33±23.69) eggs/gravid female]. Whole cell suspension of X. stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant, suggesting that X. stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media. Crude cell extract of X. stokiae was not effective against mites. Cell-free supernatant of X. stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

Contact toxicity, feeding reduction, and repellency of essential oils from three plants from the ginger family (Zingiberaceae) and their major components against Sitophilus zeamais and Tribolium castaneum.

The essential oils from rhizomes of Alpinia conchigera Griff, Zingiber zerumbet Smitt, Curcuma zedoaria (Berg.) Roscoe; their major compounds (camphene, camphor, 1,8-cineole, alpha-humulene, isoborneol, alpha-pinene, beta-pinene and terpinen-4-ol); and synthetic essential oils comprised of mixtures of major pure compounds in the same ratios as the extracted essential oils were tested for contact, feeding reduction, and repellency against Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) adults. Via topical applications, the three extracted oils had similar toxicity against S. zeamais (LD50 fiducial limits: 18-24 microg oil/mg insect). T. castaneum had similar sensitivity to all three oils (35-58 microg/mg), and it was less sensitive than S. zeamais. The LD50 values of synthetic A. conchigera and synthetic Z. zerumbet oils were similar to those of their corresponding extracted essential oils. The synthetic C. zedoaria oils showed lower contact toxicity than the extracted C. zedoaria oils to both insects. Sitophilus zeamais and T. castaneum were sensitive to terpinen-4-ol and isoborneol in contact toxicity tests. In antifeedant tests, the three extracted oils were able to decrease the consumption of flour disks, especially Z. zerumbet oils, whereas both insect species could feed on the flour disks treated with three synthetic essential oils. Only terpinen-4-ol deterred feeding in both insects. In repellency tests, A. conchigera oils at highest concentration repelled S. zeamais and T. castaneum. None of the synthetic essential oils repelled S. zeamais (315 microl/cm2) and T. castaneum (31 microl/cm2) Only terpinen-4-ol showed repellent activity against both insects.

Phylogenetic and cophylogenetic relationships of entomopathogenic nematodes (Heterorhabditis: Rhabditida) and their symbiotic bacteria (Photorhabdus: Enterobacteriaceae).

Mutualistic association between entomopathogenic Photorhabdus bacteria and Heterorhabditis nematodes represents one of the emerging model systems in symbiosis studies, yet little is known about this partnership from a coevolutionary perspective. Herein, we investigated phylogenetic and cophylogenetic relationships of Heterorhabditis and Photorhabdus strains using molecular markers Internal Transcribed Spacer and gyrase B gene sequences, respectively. The phylogenies presented consistent, well supported, monophyletic groups in the parsimonious and likelihood analyses for both the nematode and bacterial strains and supported the placement of currently recognized taxa, from which a potentially new Heterorhabditis species represented by a Thailand strain MP68 was identified. While the nematode strains with distant geographic distributions showed no detectable phylogenetic divergence within H. bacteriophora or H. georgiana monophyletic groups, their respective symbiotic bacteria speciated into two Photorhabdus species: P. luminescens and P. temperata, indicating the occurrence of duplication. Although such evolutionary process reduces the phylogenetic congruence between Heterorhabditis nematodes and Photorhabdus bacteria, global cophylogenetic tests using ParaFit detected a highly significant correlation between the two phylogenies (ParaFitGlobal = 0.001). Further, the associations between H. zealandica, H. indica and H. megidis strains and their symbiotic bacteria exhibited significant contribution to the overall cophylogenetic structure. Overall, this study reveals evidence of coevolution between Photorhabdus bacteria and Heterorhabditis nematodes and provides a framework for further examination of the evolution of these associations.

Characterization of New Entomopathogenic Nematodes from Thailand: Foraging Behavior and Virulence to the Greater Wax Moth, Galleria mellonella L. (Lepidoptera: Pyralidae).

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria (Xenorhabdus spp. and Photorhabdus spp., respectively) are lethal parasites of soil dwelling insects. We collected 168 soil samples from five provinces, all located in southern Thailand. Eight strains of EPNs were isolated and identified to species using restriction profiles and sequence analysis. Five of the isolates were identified as Heterorhabditis indica, and one as Heterorhabditis baujardi. Two undescribed Steinernema spp. were also discovered which matched no published sequences and grouped separately from the other DNA restriction profiles. Behavioral tests showed that all Heterorhabditis spp. were cruise foragers, based on their attraction to volatile cues and lack of body-waving and standing behaviors, while the Steinernema isolates were more intermediate in foraging behavior. The infectivity of Thai EPN strains against Galleria mellonella larvae was investigated using sand column bioassays and the LC(50) was calculated based on exposures to nematodes in 24-well plates. The LC(50) results ranged from 1.99-6.95 IJs/insect. Nine centimeter columns of either sandy loam or sandy clay loam were used to determine the nematodes' ability to locate and infect subterranean insects in different soil types. The undescribed Steinernema sp. had the greatest infection rate in both soil types compared to the other Thai isolates and three commercial EPNs (Heterorhabditis bacteriophora, Steinernema glaseri and Steinernema riobrave).

Excito-repellency properties of essential oils from Melaleuca leucadendron L., Litsea cubeba (Lour.) Persoon, and Litsea salicifolia (Nees) on Aedes aegypti (L.) mosquitoes.

We compared the behavioral responses of Aedes aegypti females to essential oils extracted from native plants Melaleuca leucadendron (ML), Litsea cubeba (LC), and Litsea salicifolia (LS) by using an excito-repellency test chamber. Mortality of Ae. aegypti females following 24 h holding period post-contact and non-contact trials were observed. No mortality was seen in escaped mosquitoes after direct contact with essential oils ML and LS, and low mortality (2.3-20.4%) with LC. Likewise, in all non-contact trials, no mortality was observed in escaped females from all three treatments, whereas low mortality was seen in non-escaped mosquitoes exposed to LC (0-14.3%) and LS (0-17.1%). Ae. aegypti showed significantly higher escape rates from contact chambers treated with ML and LC compared to LS, regardless of test concentrations (P<0.05). Non-contact repellent responses were significantly pronounced with LS, except at the highest (6.0%) concentration. We conclude that essential oils from all three botanicals exhibit significant irritant and repellent properties against Ae. aegypti and deserve further investigation for possible use as active ingredients in topical (skin) and indoor dispersed repellent systems.

Sensilla on the mouthparts and antennae of the elephant louse, Haematomyzus elephantis piaget (Phthiraptera: Haematomyzidae).

The labial palpus of the elephant louse Haematomyzus elephantis has six sensilla that represent three different types: trichoid, basiconic, and styloconic. Two rows of basiconic sensilla are situated on the dorsal and ventral surfaces of the rostrum, and each row consists of three sensilla. Male and female antennae have 15-17 trichoid sensilla situated on the scape, pedicel, and three antennal annuli. Both sexes have two sensilla basiconica on the dorsal surface of the pedicel near the junction of the scape and pedicel. Two coeloconic (tuft) sensilla are situated on the antennae of both sexes, one sensillum on each of the last two annuli. There are three plate organs, two on the last annulus and one on the penultimate annulus of the male and female antennae. Sexual dimorphism is exhibited in the male and female antennae, in that the male has about twice as many sensilla basiconica on the apex of the last annulus as does the female. The total number of sensilla basiconica on the apex of the male antennae is at least two times the number that is known to be present in any other species of lice. © 1992 Wiley-Liss, Inc.