Comparison of indigenous and exotic entomopathogenic nematode strains for control of the cedar web-spinning sawfly,Cephalcia tannourinensis in vitro

Cedars of Lebanon Cedrus libani have been attacked since 1990s by a hymenopteran sawfly, Cephalcia tannourinensis (Chevin), causing considerable damage to the foliar buds. A biological control programme using entomopathogenic nematodes (EPNs) was conducted in vitro against C. tannourinensis prenymphs. Two EPNs strains belonging to the same species Heterorhabditis bacteriophora were used; one indigenous to the country (LIB04) and the other a commercial strain (HbCom). Four EPNs concentration rates (40, 100, 250, 480 infective juveniles) were tested against C. tannourinensis prenymphs. Higher mortality was noted for the indigenous strain, LIB04, independently from all concentrations (100%). Emergence of EPNs was observed following 12–17 days from the first contact between EPNs and C. tannourinensis prenymphs. This parameter indicated that EPNs succeeded to complete all their reproductive cycle inside the C. tannourinensis cadavers before emerging. Bioassays were realised in jars containing soil extracted from the forest in order to validate the scenario suggested for application in situ. Most C. tannourinensis prenymphs were attacked by the EPNs on the surface.     

Maternal care in the red-headed spruce web-spinning sawfly,Cephalcia isshikii (Hymenoptera: Pamphiliidae)

We describe oviposition and maternal behavior in the sawfly Cephalcia isshikiiand examine the adaptive significance of this behavior. Females deposited eggs in a single but loose cluster on needles of terminal twigs of spruces, Piceaspp., and remained with the eggs usually on the underside of the twig facing toward the tip. The female attended her eggs until death without taking food but did not follow the first-instar larvae that moved from natal needles even if she survived until then. When the female was disturbed, she usually moved toward the source and attempted to bite it. Though at much lower frequencies, this aggressive behavior was also observed in gravid females and even in males. Field observations and female removal experiments indicated that the female enhanced the survival of the eggs through the reduction of arthropod prédation.     

Phylogeography of the Northern Atlantic species Chondrus crispus (Gigartinales, Rhodophyta) inferred from nuclear rDNA internal transcribed spacer sequences

Eighteen isolates of the red algae Chondrus crispus were collected from Northern Atlantic sites, together with C. ocellatus, C. yendoi and C. pinnulatus from the North Pacific. The nuclear rDNA internal transcribed spacer (ITS) was sequenced and compared, spanning both the ITS regions and the 5.8S rRNA gene. Percentage of nucleotide variation for C. crispus ranged from 0.3% to 4.0%. Phylogenetic analyses were performed using maximum parsimony (MP), neighbor-joining (NJ) and minimum evolution methods. They showed that two main clades existed within the C. crispus samples examined and that suggested C. crispus had a single Atlantic origin. The clustering however did not follow the geographic origin. We hypothesized that the current distribution of C. crispus populations might be a result of three main factors: temperature boundaries, paleoclimate and paleoceanography. ITS data exhibited abundant molecular information not only for phylogeographical investigation but also for systematics studies. Handling editor: C. Sturmbauer     

Interactions between the fungal pathogen Beauveria bassiana and three species of coccinellid: Harmonia axyridis, Coccinella septempunctata and Adalia bipunctata

Harmonia axyridis is a predatory coccinellid, native to central and eastern Asia. It has been available in many countries for use as a biological control agent of pest aphids and scale insects. In many of these countries, including the USA, H. axyridis has established. It is now considered an invasive alien species for a number of reasons, including its impact on functional biodiversity. Beauveria bassiana is known to be a natural mortality agent of overwintering coccinellids and is a potential candidate for the biological control of H. axyridis. In this paper we compare the susceptibility of three species of coccinellid, H. axyridis (cultures derived from Japan and UK), Coccinella septempunctata and Adalia bipunctata to infection by B. bassiana (commercial strain GHA) after exposure at three doses (10⁵, 10⁷, 10⁹ conidia ml⁻¹). The two subpopulations of H. axyridis (Japan and UK) were more resistant to B. bassiana infection than either A. bipunctata or C. septempunctata. This is exemplified by the median lethal doses at 10 days post-inoculation (LD₅₀) of 10^6.2, 10^6.0, 10^8.3, 10^9.6 conidia ml⁻¹ for A. bipunctata, C. septempunctata, H. axyridis (Japan) and H. axyridis (UK), respectively. Only doses of 10⁹ conidia ml⁻¹ resulted in mortality of H. axyridis, in contrast, 80% of C. septempunctata and 70% of A. bipunctata exposed to 10⁷ conidia ml⁻¹ of B. bassiana succumbed to infection. The fecundity (cumulative mean egg production over 10 days) of A. bipunctata and H. axyridis (UK) was also assessed. The fecundity of C. septempunctata could not be assessed because this species requires diapause prior to the onset of reproduction and these studies were on beetles that had recently eclosed (2–8 weeks). Harmonia axyridis (Japan) produced no eggs in most treatments including the control and so was excluded from analysis. High dose (10⁹ conidia ml⁻¹) inoculation reduced the fecundity of A. bipunctata to zero but egg production was similar for individuals inoculated with doses of 10⁵, 10⁷ conidia ml⁻¹ and control individuals. In contrast, all doses of B. basssiana reduced H. axyridis (UK) egg production dramatically. We discuss these results in relation to the potential for control of H. axyridis using B. bassiana.     

Biology and molecular phylogenetics of Nematoceras sulcatum, a second endemic orchid species from subantarctic Macquarie Island

A second orchid species, Nematoceras sulcatum M.A.Clem. et D.L.Jones, has been found on subantarctic Macquarie Island. A history of its discovery and recognition is provided. The morphology, biology and ecology of the new species are compared with N. dienemum (D.L.Jones) D.L.Jones, M.A.Clem. et Molloy, the other species of orchid on Macquarie Island. Molecular studies based on the internal transcribed spacer (ITS) region of nuclear ribosomal DNA reveal the phylogenetic relationship of the two Macquarie Island species compared to others in the genus from New Zealand and its Southern Ocean islands.     

On-farm evaluation of Beauveria bassiana for control of Ostrinia nubilalis in Iowa, USA

Ostrinia nubilalis is (Lepidoptera: Crambidae) a severe pest ofcorn in the major corn growing areas of theUnited States. The efficacy of a Beauveria bassiana application, for season-long suppression of O. nubilalis was evaluated in 1996 and 1997 at locationsacross Iowa. Beauveria bassiana,Mycotech 726 (Mycotech Corporation, Butte, MT)formulated on corn grit granules (14–20 mesh)at 2.2 × 10⁹ conidia/g and applied with ahand-held applicator at the rate of 0.4 g/plant(8.8 × 10⁸ conidia/plant). Applicationswere made when plants were in the V7 or R1growth stage. The length of larval tunneling,percentage of plants not infested with O.nubilalis, percentage of plants with anendophyte, and yield from treated and controlplots were determined. Whorl-stage applicationof B. bassiana in 1996 resulted in asignificant reduction in centimeters oftunneling (46–55%) and the percentageplants not infested by O. nubilalis. In1997, B. bassiana caused significantreductions in larval tunneling at all locations(20–53%); however, a significant increase inthe percentage of plants not infested with O. nubilalis occurred at only one location. Treatment of plants with B. bassiana in1997 did not significantly increase thepercentage of plants with an endophyte;however, the trend, with the exception of onesite, was for a greater percentage ofendophytic plants in treated versus untreatedplots. A whorl-stage application of a granularformulation of B. bassiana was mostefficacious in reducing O. nubilalis larval damage.     

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State,Brazil

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.     

Lead,mercury, and certain nutrient elements in ulva lactuca (linnaeus) from Ras Beirut,Lebanon

Collections of Ulva lactuca (Linnaeus) at nine locations along the coast of Ras Beirut, Lebanon were analysed for lead, mercury, phosphate, calcium, magnesium, iron, copper, and zinc. Low lead concentrations, the same in all samples, could possibly indicate that U. lactuca has some means by which lead uptake and toxicity can be controlled. Phosphate levels may be a contributing factor to this process.Concentrations of all other elements seemed relatively uniform with few exceptions. This suggests that U. lactuca is subject to similar environmental conditions and element exposure at each of the collecting sites and might be capable of maintaining biochemical stability under high levels of stress.Further studies of toxic and nutrient elements as to their individual and combined effects in U. lactuca are needed.c/o Sturdy Memorial Hospital, Attleboro, Massachusetts U.S.A.     

An ustilaginomycetous yeast,Pseudozyma graminicola sp. nov., isolated from the leaves of pasture plants

Two strains belonging to a novel anamorphic species, Pseudozyma graminicola, were isolated from the leaves of herbaceous plants in the Moscow region (Russia). This species was genetically distinct from all known Pseudozyma species, based on sequence divergence in the D1/D2 domains of the large subunit rDNA and the ITS region. It is related phylogenetically to species of the genus Sporisorium (Ustilaginaceae, Ustilaginales). Physiological characteristics distinguishing this novel species from the other species of the genus Pseudozyma are presented.     

ITS region of the rDNA of Pythium longandrum, a new species; its taxonomy and its comparison with related species

Pythium longandrum (F-73.0) was isolated, from soil samples taken in Lille in northern France. Morphologically the fungus resembles closely Pythium rostratum, however its antheridial characters are unique. The oogonia of this species are provided with hypogynous and monoclinous antheridia. The antheridial cells are inflated and are probably the largest and longest for the genus. The internal transcribed spacer region of its nuclear ribosomal DNA indicates that it is entirely different from all other species of Pythium. This new species is characterized by its spherical to elongated sporangia, smooth-walled oogonia and hypogynous to monoclinous antheridia bearing long antheridial cells closely applied to the oogonia. Morphological features of this new species, together with the sequences of the ITS region of its nuclear ribosomal DNA and comparison with related species are discussed here.     

Use of a novel multiplex probe array for rapid identification of <Emphasis Type="Italic">Mycobacterium</Emphasis> species from clinical isolates

Conventional identification of mycobacteria is based on the analysis of their phenotypic and biochemical characteristics after culture; thus this method is time-consuming, laborious, and is not always conclusive. Developing a fast and accurate method for rapid identification of Mycobacterium species is in urgent need for early diagnosis of mycobacteriosis and effective patient management. In this study, an efficient and affordable novel multiplex probe array which allows simultaneous identification of 15 medically important mycobacterial species was developed. A pair of genus-specific primers and a set of genus- and species-specific probes were designed according to the conserved and polymorphic regions of the 16S rRNA gene, internal transcribed spacer (ITS) sequence, and 23S rRNA gene of mycobacteria. This probe array was applied for the identification of 78 clinical mycobacterial isolates recovered from Henan, China. The results showed that the specificity and sensitivity of the probe array were 100% for both genus-specific probe and Mycobacterium tuberculosis complex-specific probe. Among 52 isolates of nontuberculous mycobacteria, 43 isolates (82.7%) can be rapidly identified to the species level. Genetic variability of 16S-23S rRNA gene ITS region in M. avium, M. intracellulare, M. chelonae, M. abscessus and M. fortuitum were analyzed. With the accumulation of the sequences of ITS identified and further optimization of probes, the multiplex probe array has the potential to be developed into a practical tool for rapid and accurate identification of mycobacterial species in clinical laboratory.     

Molecular phylogeny of ectomycorrhizal <Emphasis Type="Italic">Pisolithus</Emphasis> fungi associated with pine,dipterocarp, and eucalyptus trees in Thailand

The phylogenetic relationships among 135 Pisolithus basidiomes and two isolates collected from three pine forests, a pine-dipterocarp forest, two dipterocarp forests, and 29 eucalyptus plantations in Thailand were investigated. Internal transcribed spacer (ITS) polymorphism analyses, including terminal RFLP, divided them into 26 groups. The ITS in a representative basidiome of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggested that at least three Pisolithus species are present in Thailand. Pisolithus basidiomes collected in the pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5 as previously described by Martin et al. in 2002. Those collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from the eucalyptus plantations were all identified as Pisolithus albus.     

ITS1 region of the rDNA of Pythium proliferatum, a new species: its taxonomy and its comparison with related species

A new species, Pythium proliferatum (F-382), was isolated from soil samples taken in Genlis in the burgundian region of France. The fungus is unique because of the character combinations of its large, spherical to elongated, proliferating sporangia, and its smooth walled oogonia supplied with different types of antheridia like hypogynous, monoclinous sessile, monoclinous stalked, diclinous and wrapping around the oogonia. Almost all types of antheridia that are found in the genus are present in this new species. Morphological features of this new species, together with the sequences of the ITS1 region of its nuclear ribosomal DNA and its comparison with related species are discussed in this article.     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

Molecular phylogeny of species in the generaAmylostereum andEchinodontium

Analyses of DNA sequences from the internal transcribed spacer (ITS) region of the nuclear rDNA and from a portion of a manganese-dependent peroxidase gene were used to assess the species inAmylostereum, including isolates from the mycangia of horntails, decay, and basidiomes. Four species are recognized:A. areolatum, A. chailletii, A. laevigatum, andA. ferreum. An unidentifiedAmylosterum isolate from the mycangium ofXoanon matsumurae had an ITS sequence identical to that ofA. areolatum. Another unidentifiedAmylostereum isolate from the mycangium ofSirex areolatus was nearA. laevigatum, which appears to be the mycangial symbiont for those horntails attacking cedar-like trees. The other horntail isolates, primarily from Pinaceae, proved to be eitherA. areolatum orA. chailletii. The DNA sequences ofEchinodontium tinctorium, E. tsugicola andE. japonicum were similar to those of theAmylostereum species, andAmylostereum species are now recognized as members of the family Echinodontiaceae rather than the family Stereaceae.Echinodontium taxodii was found to be distinct from the Echinodontiacaea andStereum, andE. taxodii is recognized as aLaurilia species.     

<Emphasis Type="Italic">Beauveria bassiana</Emphasis> pathogenicity to the cherry slugworm, <Emphasis Type="Italic">Caliroa cerasi</Emphasis> (Hymenoptera: Tenthredinidae) larvae

The cherry slugworm Caliroa cerasi is a significant destructive pest of sour cherries (Prunus cerasus) in Turkey. The potential of entomopathogenic fungi for controlling C. cerasi was evaluated. The effects of exposure methods and conidial concentrations (1 × 10⁶, 1.5 × 10⁶, 1 × 10⁷ and 1.5 × 10⁷ conidia/ml) on mature larvae of C. cerasi infected by Beauveria bassiana were investigated under laboratory conditions. Larvae sprayed directly with B. bassiana conidial suspensions and larvae exposed to B. bassiana-treated leaves resulted in 100% mortality within 2.90 and 2.77 days, respectively. The median lethal time (LT₅₀) and days to mortality were highest in the 1.0 × 10⁷ concentrations of conidia for both direct spray and leaf exposure. The present study suggests that B. bassiana has good potential for control of the cherry slugworm, C. cerasi.     

Comparative study of wild and cultivated <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) based on molecular and morphological data

We compared the wild Porphyra strain OGATSU from northeastern Japan with cultivated Porphyra yezoensis f. narawaensis using the RuBisCO spacer, rbcL, and ITS-1 DNA sequences as well as early gametophyte development. Based on the molecular analyses and detailed morphological observations, OGATSU was identified as P. yezoensis, but also revealed important differences from the cultivated form. Under the same culture conditions, gametophytic blades of OGATSU produced more archeospores than P. yezoensis f. narawaensis strain HG-4. The length of blades and their length-to-width ratios were significantly lower in OGATSU than in HG-4, and the color of OGATSU blades was darker than that of HG-4. The first lateral cell division in conchospore germlings occurred significantly earlier in the OGATSU strain than in the HG-4 strain, resulting in the rounder shape of the OGATSU blade compared to that of P. yezoensis f. narawaensis. These results suggested that wild strains such as OGATSU can provide useful characters that could enhance cultivated varieties in a careful breeding program.     

Phylogenetic relationships ofPseudopanax species (Araliaceae) inferred from parsimony analysis of rDNA sequence data and morphology

Sequence data from internal transcribed spacer (ITS) regions of rDNA and data from morphology, cytology and wood anatomy are used to study phylogenetic relationships inPseudopanax. The molecular and non-molecular data are analysed as independent data sets and in combination using parsimony. Results supported the conclusion that the genusPseudopanax is polyphyletic.Pseudopanax species emerge in two major monophyletic groups. The Anomalus group containsPseudopanax anomalus, P. edgerleyi, andP. simplex; these species share a common ancestor withCheirodendron trigynum and more distantly withPseudopanax gunnii. The second major group consists of two smaller groups: the Arboreus group, includingPseudopanax arboreus, P. colensoi, P. kermadecensis, P. laetus, andP. macintyrei, and the Crassifolius/Discolor group, includingP. chathamicus, P. crassifolius, P. discolor, P. ferox, P. gilliesii, P. lessonii, andP. linearis. Meryta species are close relatives of thePseudopanax Arboreus and Crassifolius/Discolor groups.     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.