Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Repellency of methyl jasmonate to <Emphasis Type="Italic">Ixodes ricinus</Emphasis> nymphs (Acari: Ixodidae)

In our search for tick repellents of plant origin, to be used as alternatives to commercial arthropod repellents, we investigated the effect of the well known plant signaling compound methyl jasmonate (MJ) using nymphs of the tick Ixodes ricinus. In laboratory tests, pieces of cloth with MJ at 0.075, 0.15, 0.30 and 0.75 mg/cm² yielded increasing repellencies against the nymphs: 57%, 71%, 92% and 99%, respectively, of the nymphs did not cling to the cloth. Repellency of MJ was also investigated in a tick-infested woodland area in central Sweden. Cotton flannel cloths sprayed with 0.05, 0.1 or 0.2 mg/cm² MJ dissolved in acetone were dragged over the ground vegetation. The numbers of nymphs on the treated cloths were significantly lower than those on the untreated cloth. Thus, MJ has, at the concentrations tested, significant repellent activity against I. ricinus nymphs.     

Combined field efficacy of <Emphasis Type="Italic">Paranosema locustae</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">acridum</Emphasis> for the control of sahelian grasshoppers

Field trials were conducted to evaluate the efficacy of wheat bran bait formulations of Paranosema locustae and Metarhizium anisopliae for controlling grasshoppers in southeast Niger. Treatments consisted of wheat bran baits mixed with M. anisopliae, P. locustae + M. anisopliae or with P. locustae spores and P. locustae + sugar. Oedaleus senegalensis, Pyrgomorpha cognata and Acrotylus blondeli were the predominant species at the time of application representing ca. 94% of the total population. Bran application was done when O. senegalensis (ca. 75% of the population) was at its early developmental stages, with first, second and third instars accounting for 64–85%. Grasshopper population reduction, P. locustae prevalence and level of infections in the predominant species were monitored. Manual application of P. locustae and M. anisopliae formulated in wheat bran has proven to induce consistent pathogen infection in grasshopper populations. Population density over the three weeks monitoring, typically decreased by 44.7 ± 6.9%, 52.8 ± 8.4%, 73.7 ± 5.5% and 89.1 ± 1.8% in P. locustae, P. locustae + sugar, M. anisopliae and P. locustae + M. anisopliae treated plots respectively. Paranosema locustae prevalence in surviving adult grasshoppers at 28 after application was 48.1 ± 2.3%, 28.9 ± 4.8% and 27.4 ± 3.7%, with infection level of 6.2 ± 0.8 × 10⁶, 2.3 ± 0.3 × 10⁴ and 2.1 ± 0.3 × 10³ spores mg⁻¹ host weight in O. senegalensis, A blondeli and P. cognate respectively. Other species that each accounted for <2% of the community, namely Aiolopus thalassinus, A. simulatrix, Acorypha glaucopsis, Acrotylus patruelis, Anacridium melanorhodon, Diabolocatantops axillaris, Kraussaria angulifera and Schistocerca gregaria were found to show sign of infection. The results from this study suggest that wheat bran application of M. anisopliae and P. locustae alone or in combination, targeting early instars grasshopper could be a valuable option in grasshopper control programs.     

Influence of Temperature on Virulence of Fungal Isolates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and <Emphasis Type="Italic">Beauveria bassiana</Emphasis> to the Two-Spotted Spider Mite <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Twenty-three isolates of Metarhizium anisopliae (Metschnikoff) Sokorin and three isolates of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales: Clavicipitaceae) were assessed for their virulence against the two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Based on the screening results, nine isolates of M. anisopliae and two isolates of B. bassiana were tested for their virulence against young adult (1- to 2-day-old) female T. urticae at constant temperatures of 20, 25, 30 and 35°C. At all temperatures tested, all the fungal isolates were pathogenic to T. urticae but mortality varied with isolates and temperatures. Fungal isolates were more virulent at 25, 30 and 35°C than at 20°C. The lethal time to 50% mortality (LT₅₀) and lethal time to 90% mortality (LT₉₀) values decreased with increased temperature. There were no significant differences in virulence between fungal isolates at 30 and 35°C; however, significant differences were observed at 20 and 25°C.     

Efficacy of culture filtrates of <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> against larvae of <Emphasis Type="Italic">Anopheles stephensi</Emphasis> and <Emphasis Type="Italic">Culex quinquefasciatus</Emphasis>

Efficacy of culture filtrates of five strains of Metarhizium anisopliae isolated from insects were evaluated against Anopheles stephensi and Culex quinquefasciatus. The culture filtrates released from the strains of M. anisopliae in the YpSs and chitin broths were filtered and used for the bioassays after a growth of 7 days. Among the culture filtrates of five strains, M. anisopliae 892 was found to be more effective against both the mosquitoes. The LC(50) values of culture filtrates of M. anisopliae 892 in chitin broth was lower than the LC(50) of culture filtrates in YpSs broth against first and fourth instars of both the mosquitoes. The LC(50) values of culture filtrates were significantly different between first and fourth instars of A. stephensi (t test; P = 0.0001) and C. quinquefasciatus (t test; P = 0.02). The larvae of A. stephensi were more susceptible than C. quinquefasciatus except in two cases. This is the first report of efficacy of culture filtrates produced by M. anisopliae in chitin broth against mosquitoes and have potential as a biological control agent of mosquitoes.     

Resistance of the termite, <Emphasis Type="Italic">Coptotermes formosanus</Emphasis> Shiraki to <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> due to grooming

Termites, Coptotermes formosanus, reared individually, were highly susceptible to the entomopathogenic fungus, Metarhizium anisopliae, while termites reared in␣groups were highly resistant. When reared in groups, the termites treated with M.␣anisopliae conidia on the body surface were groomed by their nestmates and more than 80% of the conidia were removed from the cuticle within 3 h. However, there was not a significant reduction in the numbers of conidia on the body surfaces of termites reared individually. For the termites maintained in groups, conidia were found in foreguts, midguts and hindguts, but very few conidia were detected in the guts of termites reared individually. Conidia in the alimentary tracts did not germinate, but some of were alive. As a result, it seems that the removal of foreign bodies, such as fungal conidia, from the␣cuticle is one function of termite mutual grooming behavior and that conidia removed from the cuticle are eliminated through alimentary tracts. This study indicates that mutual grooming behavior is very effective in protecting these termites from M.␣anisopliae infection.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Influence of growth medium on antifungal activity of neem oil (<Emphasis Type="Italic">Azadirachta indica</Emphasis>) against <Emphasis Type="Italic">Lagenidium giganteum</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The inhibition of mycelial growth of Lagenidium giganteum by neem oil was lower than that of Metarhizium anisopliae in PYG and Emerson’s YpSs agar media. However, neem oil did not inhibit the mycelial growth of L. giganteum in sunflower seed extract agar medium, but did it inhibit the mycelial growth of M. anisopliae. The minimum inhibitory concentration of neem oil for L. giganteum was higher than that for M. anisopliae. The minimum fungicidal concentration of neem oil in PYG medium was lower than in YpSs for both fungi. The spores of L. giganteum grown in SFE medium could be used with neem oil for vector control.     

Pathogenicity of hyphomycetous fungi against <Emphasis Type="Italic">Cyclocephala signaticollis</Emphasis>

Susceptibility of the white grub Cyclocephala signaticollis Burmeister (Coleoptera: Scarabaeidae: Dynastinae) larvae to seven isolates of Beauveria bassiana (Balsamo) Vuillemin, five of Metarhizium anisopliae (Metschnikoff) Sorokin and two of Paecilomyces lilacinus (Thom) Samson (Deuteromycotina: Hyphomycetes) was investigated. Among 14 fungal isolates screened the most virulent was a B. bassiana isolate (Bb 53) that caused 70% mortality of third instar larvae in 40 days after inoculation at 1 × 10⁸ conida/ml. Strains of M. anisopliae and P. lilacinus showed low efficacy or no virulence to the target host.     

Soil sampling for <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> spores in Queensland sugarcane fields

Three sugarcane fields in Bundaberg and four fields in each of the Burdekin, Tully and Innisfail (Queensland, Australia) were sampled for spores of Metarhizium anisopliae (Metchnikoff) (Deuteromycotina: Hyphomycetes). This entomopathogenic fungus is the active ingredient in the biocide “BioCane^TM”, which was developed for the management of the greyback canegrub Dermolepida albohirtum (Waterhosue) (Coleoptera: Scarabaeidae) and other scarabs in cane fields. Fields sampled were of different crop ages and all had a history of BioCane^TM treatment in Plant Cane in past years. Soil samples were taken in each field from four depths (0–10, 10–20, 20–30 and 30–40 cm below soil level) with the use of an auger. Spore levels were highest at the depths of 10–20 and 20–30 cm. Spore levels differed between locations with Innisfail and Tully recording the highest spore counts. Spores were also found in the inter-row space in plots sampled in Tully. Sampling statistics were determined for M. anisopliae spores at the four soil depths with 0.1 and 0.25 precision levels. Three sampling methods were compared (use of marker beads; use of 100 mm auger and 150 mm auger). Samples that relied on marker beads resulted in higher spore counts, however, an auger can still be used since BioCane^TM does not normally contain coloured markers. Results obtained demonstrate the ability of the pathogen to translocate in soil profile and across rows, most likely due to grub movements and other soil fauna. Sampling for M. anisopliae spores provides good monitoring of their levels in soil. The implications of this on grub management decisions are discussed.     

Selection of entomopathogenic fungi for use in combination with sub-lethal doses of imidacloprid: perspectives for the control of the leaf-cutting ant <Emphasis Type="Italic">Atta sexdens</Emphasis><Emphasis Type="Italic">rubropilosa</Emphasis> Forel (Hymenoptera: Formicidae)

In the first part of this study, four isolates of the fungus Beauveria bassiana (Bals.) Vuillemin (LPP1, LPP2, CG05 and CG24) and one isolate of Metarhizium anisopliae (Metsch.) Sorokin (CG46) were tested against adult foragers of Atta sexdens rubropilosa. Ants were allowed to walk on filter paper discs, inside Petri dishes, previously impregnated with 1 ml of a conidia suspension (2 × 10⁷ conidia ml⁻¹), maintained at 80% RH and 26°C for 24 h and subsequently, transferred to sterile Petri dishes, maintained at 23°C, 80% RH, 24 h dark. The mean values of LT₅₀ for LPP2, LPP1, CG46, CG24 and CG05 were 3.5, 3.7, 3.8, 4.2 and 4.4 days, respectively. Control insects for all tests in this study showed less than 10% mortality. Experiments were carried out to test the toxicity of imidacloprid (IMI) to A. sexdens rubropilosa. Mortality was evaluated 10 days following a 24 h exposure to the insecticide. Percent mortality caused by 500, 200, 100 and 10 ppm IMI was 77.8, 56.7, 45.5 and 5.5 respectively. Insects treated with 10 ppm IMI were observed to have reduced locomotor activity 24 h after exposure to the insecticide. The LC₅₀ of IMI was 154.3 ppm. Subsequent tests were carried out to evaluate the combination of a sub-lethal dose of IMI (10 ppm) and infection by CG24 (1 × 10⁷ conidia ml⁻¹). Mortality due to fungal infection alone was 43.3%. Mortality of insects treated with IMI followed by exposure to the fungus was 64.3%. These results indicate that IMI significantly increases the susceptibility of ants to infection by B. bassiana CG24.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Molecular analysis of <Emphasis Type="Italic">Ixodes granulatus</Emphasis>, a possible vector tick for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in Taiwan

The genetic identity of Ixodes granulatus ticks was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2–99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.     

The parasitism of immature stages of <Emphasis Type="Italic">Ixodes loricatus</Emphasis> (Acari: Ixodidae) on wild rodents in Argentina

Ixodes loricatus has long been considered with strict-total specificity to New World Marsupials. However, frequent findings of its immature stages on rodents suggest that these vertebrates play an important role in the tick’s life cycle. Aspects dealing with the ecology of Sigmodontinae rodents infestation by I. loricatus are unknown. To contribute to the knowledge of the ecology of this tick species, environmental factors, as well as host species, sex and age, were evaluated to find associations of immature I. loricatus infestation of the most abundant wild rodent species from riparian locations of Buenos Aires province (Argentina). A total of 290 hosts belonging to the subfamily Sigmodontinae were captured and examined for ticks at six locations. The data analysis showed that there was a preference of I. loricatus towards Akodon azarae, but only for those hosts captured in lands not prone to flooding. The results suggest that the host preference of I. loricatus is mainly related to factors such as habitat type and host density.     

Discovery of a North American genetic variant of the entomopathogenic fungus <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var. <Emphasis Type="Italic">anisopliae</Emphasis> pathogenic to grasshoppers

A genetic variant of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, isolated from a soil in Alberta, Canada, from a location with a history of severe grasshopper infestations, was evaluated for pathogenicity in bioassays of living grasshoppers. Mortality in treated individuals drawn from a laboratory colony was 99% (LT₅₀ = 6.7 days, LT₉₀ = 9.6 days) at 12 days post-inoculation compared to 100% (LT₅₀ = 4.1 days, LT₉₀ = 5.8 days) mortality at 8 days in insects exposed to a commercial isolate of M. anisopliae var. acridum (IMI 330189). Experimental infection of field-collected grasshoppers under laboratory conditions with the native isolate of M. anisopliae var. anisopliae resulted in 100% (LT₅₀ = 4.4 days, LT₉₀ = 5.4 days) mortality attained within 7 days compared to 100% (LT₅₀ = 4.7 days, LT₉₀ = 6.3 days) mortality in 9 days in insects treated with M. anisopliae var. acridum. Amplification of fungal genomic DNA from the indigenous isolate with primers for the specific detection of M. anisopliae var. anisopliae produced a product almost 300 bp larger than expected based on previously known isolates. This is the first demonstration of a highly virulent, indigenous non-chemical control agent of grasshoppers in North America. GenBank Accession Nos. DQ342236, DQ342237.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

The ontogenetic basis for floral diversity in <Emphasis Type="Italic">Agonis</Emphasis>, <Emphasis Type="Italic">Leptospermum</Emphasis> and <Emphasis Type="Italic">Kunzea</Emphasis> (Myrtaceae)

Floral development in three species each of Leptospermum and Kunzea, and one species of Agonis, is described and compared. Differences in the number of stamens and their arrangement in the flower at anthesis are determined by the growth dynamics of the bud. In Leptospermum, early expansion of the bud is predominantly in the axial direction and causes the stamen primordia to be initiated in antepetalous chevrons. In Kunzea, early expansion occurs predominantly in the lateral direction and successive iterations of stamen primordia are inserted alternately at more or less the same level. In both genera, further expansion in the lateral plane spreads the stamens into a ring around the hypanthium. Agonis flexuosa is similar to Leptospermum. Other variable factors include the timing at which stamen initiation commences (earlier in Leptospermum than Kunzea), the duration of stamen initiation (hence the total number of stamens produced – varies within genera), and very late differential expansion that forces stamens into secondary antesepalous groups in A. flexuosa and L. myrsinoides.The authors thank Dr H. Toelken for kindly providing some material and the impetus for this project. This research was supported by Australian Research Council grant AS19131815.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Evidence of <Emphasis Type="Italic">Babesia microti</Emphasis> infection in multi-infected <Emphasis Type="Italic">Ixodes persulcatus</Emphasis> ticks in Russia

To detect Babesia-infected Ixodes persulcatus Shulze in a suburb of St. Petersburg, Russia, 738 adult ticks were studied using Babesia specific primers and PCR techniques. The entire sample (more than 1,200 individuals) was screened for the presence of Borrelia spp., Ehrlichia spp. and tick-borne encephalitis virus (TBEV). All 7 ticks infected with Babesia microti, were also infected with other pathogens (all 7 among 417 infected ticks, zero amongst the remaining 321 naive ones (χ² = 5.25, p < 0.05). Babesia microti occurred twice with Borrelia afzelii, 3 times with Borrelia garinii, once with both, and once with both B. garinii and TBEV. The prevalence of infection with Borrelia spp. was 34.0%, with Ehrlichia spp. 6.2%, with TBEV 1.5%, and with Ba microti 0.9%. Babesia microti infection was not found in combination with Ehrlichia sp. or Borrelia burgdorferi sensu stricto. The latter pathogen (prevalence 2.6%), just like Ba. microti, was not encountered as a monoinfection. The data suggest that Ba. microti infection can only survive in I. persulcatus in combination with Borrelia spp. (7 of 7 infections). The disease in humans is more severe and longer-lasting when more than one pathogen is involved. Our observations show that the well known St. Petersburg focus of tick-borne encephalitis and Lyme disease is also a focus of ehrlichiosis and babesiosis. This revised version was published online in July 2006 with corrections to the Cover Date.