High somatic instability of a microsatellite locus in a clonal tree,<Emphasis Type="Italic"> Robinia pseudoacacia</Emphasis>

Robinia pseudoacacia L. is a clonal tree species. To investigate a mutation within eight microsatellite loci of R. pseudoacacia, we analyzed DNA samples obtained from different leaf samples within each ramet, leaves from ramets within the genet, and seeds. Of the eight loci, locus Rops15 (AG motif) displayed hypermutability. The mutation rates of Rops15 within each ramet, among ramets within the genet, and offspring were 6.27% (ranging from 0 to 31.1%), 6.11% (from 0 to 25.0%) and 3.78% (from 0 to 10.9%), respectively. The mutation rate increased with allele size (13–71 repeat units). The mutation patterns observed in Rops15 were distinctive in two ways. First, there was a significant bias toward additions over deletions, and both addition and deletion of single repeats were dominant at alleles with lengths less than 232 bp (63 repeats). Second, for the longest allele of 248 bp (71 repeats), the number of losses was higher than the number of gains. These observations suggest that the mutation patterns of microsatellites in R. pseudoacacia may follow a generalized stepwise mutation model, and that the tendency of long alleles to mutate to shorter lengths acts to prevent infinite growth. Finally, the observation of somatic hypermutability at locus Rops15 highlights the need for caution when using highly polymorphic microsatellites for population genetic structure and paternity analysis in tree species.Communicated by H.F. Linskens     

<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> in Poland and Japan is nodulated by <Emphasis Type="Italic">Mesorhizobium amorphae</Emphasis> strains

Robinia pseudoacacia microsymbionts from plants growing in Poland and Japan were evaluated for phylogeny and taxonomic position by genomic approach. Based on the comparative analyses of atpD (368 bp) and dnaK (573 bp) gene sequences as well as 16S rDNA restriction analysis (RFLP-16S rDNA), R. pseudoacacia microsymbionts were identified as Mesorhizobium strains. In dnaK and atpD gene phylograms R. pseudoacacia nodulators formed robust, monophyletic clusters with Mesorhizobium species with the nucleotide sequence similarity of 91–98% and 90–98%, respectively. The classification of R. pseudoacacia rhizobia to the genus Mesorhizobium was also supported by amplified 16S rDNA restriction analysis. The studied bacteria formed common clusters with Mesorhizobium species, and their DNA patterns were identical or nearly identical to Mesorhizobium genus strains. When DNA-DNA hybridization was performed, the total DNA of the representative R. pseudoacacia rhizobia exhibited 51–75% relatedness to DNA of Mesorhizobium amorphae ICMP15022 strain and below 41% to DNA of other Mesorhizobium species. These results showed that R. pseudoacacia and M. amorphae belong to the same genomospecies. The G+C content of DNA of R. pseudoacacia two microsymbionts was 59.7 and 60.6 mol% compared to 61–64 mol% across M. amorphae strains.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

Natural hybridisation between <Emphasis Type="Italic">Populus nigra</Emphasis> L. and <Emphasis Type="Italic">P</Emphasis>. <Emphasis Type="Italic">x canadensis</Emphasis> Moench. Hybrid offspring competes for niches along the Rhine river in the Netherlands

Black poplar (Populus nigra L.) is a major species for European riparian forests but its abundance has decreased over the decades due to human influences. For restoration of floodplain woodlands, the remaining black poplar stands may act as source population. A potential problem is that P. nigra and Populus deltoides have contributed to many interspecific hybrids, which have been planted in large numbers. As these Populus x canadensis clones have the possibility to intercross with wild P. nigra trees, their offspring could establish themselves along European rivers. In this study, we have sampled 44 poplar seedlings and young trees that occurred spontaneously along the Rhine river and its tributaries in the Netherlands. Along these rivers, only a few native P. nigra L. populations exist in combination with many planted cultivated P. x canadensis trees. By comparison to reference material from P. nigra, P. deltoides and P. x canadensis, species-specific AFLP bands and microsatellite alleles indicated that nearly half of the sampled trees were not pure P. nigra but progeny of natural hybridisation that had colonised the Rhine river banks. The posterior probability method as implemented in NewHybrids using microsatellite data was the superior method in establishing the most likely parentage. The results of this study indicate that offspring of hybrid cultivated poplars compete for the same ecological niche as native black poplars. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Codominance of <Emphasis Type="Italic">Acer saccharum</Emphasis> and <Emphasis Type="Italic">Fagus grandifolia</Emphasis>: the role of <Emphasis Type="Italic">Fagus</Emphasis> root sprouts along a slope gradient in an old-growth forest

We studied how the unusual capacity of mature Fagus grandifolia to form clumps of clonal stems from root sprouts can contribute to its frequent codominance with Acer saccharum in southern Quebec, Canada. In an old-growth forest, the degree of dominance by the two species shifted along topographic gradients spanning a few hundreds of meters, with Fagus more frequent on lower slopes and Acer on upper slopes. The frequency distribution of Fagus stem diameter had an inverse J distribution at all slope positions, which is indicative of continuous recruitment. Acer stem diameter also had an inverse J pattern, except at lower slope positions where size structure was discontinuous. For stems <2 m tall, Fagus regenerated mainly by sprouts at the upper and mid-slopes, while regeneration from seed was more pronounced on the lower slope. This change of regeneration mode affected the spatial pattern of Fagus stems. Understory trees of Fagus were positively correlated with conspecific canopy trees on upper and mid-slopes, but not on lower slopes where Fagus regenerated mainly by seedlings. Understory trees of Acer were positively correlated with conspecific canopy trees only on the mid-slope. There were many Fagus seedlings around Acer canopy trees at the lower slope, suggesting the potential replacement of Acer canopy trees by Fagus. This study suggests that the regeneration traits of the two species changed with slope position and that Fagus patches originating from root sprouts can contribute to the maintenance of Acer–Fagus codominance at the scale of local landscapes.     

Host physiological condition regulates parasitic plant performance: <Emphasis Type="Italic">Arceuthobium vaginatum</Emphasis> subsp. <Emphasis Type="Italic">cryptopodum</Emphasis> on <Emphasis Type="Italic">Pinus ponderosa</Emphasis>

Much research has focused on effects of plant parasites on host-plant physiology and growth, but little is known about effects of host physiological condition on parasite growth. Using the parasitic dwarf mistletoe Arceuthobium vaginatum subsp. cryptopodum (Viscaceae) and its host Pinus ponderosa, we investigated whether changes in host physiological condition influenced mistletoe shoot development in northern Arizona forests. We conducted two studies in two consecutive years and used forest thinning (i.e., competitive release) to manipulate host physiological condition. We removed dwarf mistletoe shoots in April, before the onset of the growing season, and measured the amount of regrowth in the first season after forest thinning (Study I: n=38 trees; Study II: n=35 trees). Thinning increased tree uptake of water and carbon in both studies, but had no effect on leaf N concentration or δ¹³C. Mistletoe shoot growth was greater on trees with high uptake of water and carbon in thinned stands than trees with low uptake in unthinned stands. These findings show that increased resource uptake by host trees increases resources to these heterotrophic dwarf mistletoes, and links mistletoe performance to changes in host physiological condition.     

Cryopreservation of <Emphasis Type="Italic">Robinia</Emphasis><Emphasis Type="Italic">pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Nutrient partitioning and storage in arid-zone forests of the mangroves Rhizophora <Emphasis Type="Italic">stylosa</Emphasis> and <Emphasis Type="Italic">Avicennia</Emphasis><Emphasis Type="Italic">marina</Emphasis>

Mangrove partitioning and storage of macronutrients and trace metals were examined in different arid coastal settings of Western Australia. Total living biomass in three Rhizophora stylosa forests, which ranged from 233 to 289 t DW ha^-1, was significantly greater than biomass in three Avicennia marina forests (range: 79-155 t DW ha^-1). Although prop roots and stems were the largest single tree components for R. stylosa and A. marina, respectively, most nutrients were stored in leaves and living roots of both species. However, only a small fraction of the total nutrient pool was vested in tree biomass; the vast bulk was in soils. A large below-ground pool of dead fine roots was identified at all stands, equivalent to 36-88% DW of total living tree biomass. The amount of Ca, S, Cl, Na, Si, Fe, Mn, Zn, B, Mo and Cu vested in dead roots of both species was greater than in the total living tree biomass. The proportion of Fe and S vested in live and dead roots was exceptionally large, consistent with previous evidence of metal plaques on mangrove roots. Sulphur, iron and zinc in dead roots of both species constituted the bulk of these metals. R. stylosa trees preferentially accumulated more Mg, S, Cl, Na, Si, Fe, Mn, B and Mo than A. marina trees. Proportionally greater storage of P, N, Ca, K, Cu and Zn occurred in two of the three A. marina forests. Foliar concentrations of Mg, S, Mn, B and Mo in mangrove leaves were at the high end of the range reported for other tropical trees, but other elemental concentrations were at the low or mid-range. Nitrogen limitation in these forests is implied by a positive correlation between total tree N and net canopy production and by a lower percentage of ecosystem N in tree biomass as compared with other forests. Unlike terrestrial forests where a large proportion of nutrient capital is vested in floor litter, most elements in these mangrove forests are stored in dead roots. A large reservoir of dead roots below the forest floor may serve as a conservation mechanism, particularly in such arid oligotrophic environments.     

Evaluation of a field experiment for the conservation of a <Emphasis Type="Italic">Magnolia stellata</Emphasis> stand using clear-cutting

Magnolia stellata is a rare subcanopy tree species that grows in secondary forests in warm temperate zones. It is now endangered due to habitat degradation by vegetation succession. In an attempt to improve the habitat, a 30 m?×?10 m plot (0.03 ha) was set up with all vegetation including M. stellata being clear-cut in January 2012. The number of sprouts increased for 1–2 years after clear-cutting and then gradually decreased or remained constant. Five years after clear-cutting, the numbers of individuals and stems, and the total basal area (BA), were 87.0, 165.5 and 3.2%, respectively, of the values before clear-cutting. BA was highest for Ilex pedunculosa, followed by M. stellata and Hydrangea paniculata. Some sprouted individuals of M. stellata produced flower buds in the second year after clear-cutting, and flowered and fruited in the spring and summer of the third year, respectively. The densities of potential canopy species were 18,533 ha^?1 (height >?0.5 m) and 7,267 ha^?1 (height >?1.2 m), vastly exceeding the value of the criterion for successful natural regeneration after clear-cutting of warm temperate forests in the region (3,000 ha^?1). Based on this criterion, it is thus considered that the natural regeneration has reached completion. However, 45.1% (height >?0.5 m) and 95.5% (height >?1.2 m) of M. stellata individuals were regenerated by sprouting. Further research is needed into how individuals, regenerated from seedlings, develop and reach sexual maturity, and how successive generations change.     

Higher phenotypic plasticity does not confer higher salt resistance to <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> than <Emphasis Type="Italic">Amorpha fruticosa</Emphasis>

A greenhouse experiment was conducted in which two leguminous species commonly used in the Yellow River Delta for vegetation restoration, Robinia pseudoacacia and Amorpha fruticosa, were subjected to five salt treatments: 0, 50, 100, 150, and 200 mmol L^?1. We aimed to determine which of the two species would be better suited for growth in a saline environment, and whether the acclimation capacity to salinity resulted from an inherently higher phenotypic plasticity. The results showed that salinity affected most growth and biomass parameters but had no effects on most leaf traits and physiological parameters of the two species. Height, relative growth rate of crown area, root biomass, and leaf mass ratio of R. pseudoacacia were reduced by higher salinity, while A. fruticosa was not affected. Chlorophyll a-to-chlorophyll b ratio and total antioxidative capacity of A. fruticosa increased with higher salinity, whereas those of R. pseudoacacia remained unchanged. Root mass ratio and vitamin C concentration of both species were not affected by salinity, whereas vitamin C concentration of A. fruticosa was higher than that of R. pseudoacacia. The root-to-shoot ratio of A. fruticosa was higher than that of R. pseudoacacia in most salt treatments. Of all leaf traits, only leaf area differed between treatments. R. pseudoacacia generally exhibited a greater plasticity than A. fruticosa in response to salinity, but A. fruticosa was more resistant to the higher salinities than R. pseudoacacia, and was thus a better candidate for vegetation restoration in saline areas.     

Predicting the Recovery of <Emphasis Type="Italic">Pinus halepensis</Emphasis> and <Emphasis Type="Italic">Quercus ilex</Emphasis> Forests after a Large Wildfire in Northeastern Spain

Quercus ilex and Pinus halepensis are two of the most common tree species of the western Mediterranean basin. Both species regenerate reliably after fire: P. halepensis colonizes recently disturbed areas by effective seedling recruitment, while Q. ilex resprouts vigorously after disturbances. For this reason, the natural regeneration of these species after fire should ensure the re-establishment of a forest similar to that which existed before the fire. This study analyzes with a simple simulation model whether or not the relative abundance of monospecific and mixed forests of these species in the landscape is altered by fire. We also analyze the topographic factors and the forest structure before the fire that determine the changes in forest composition after fire. This study has been carried out in a large fire that occurred in NE Spain. Overall, 33% of plots changed to another community type, but this probability of change varied considerably among community types before the fire. Monospecific forests of P. halepensis or Q. ilex had a high probability of remaining in their original composition after the fire, whereas the resilience of mixed forests of these two species was quite low. Mixed forests changed for the most part to monospecific P. halepensis or Q. ilex forests. Analysis of several factors determining these changes indicated that only elevation as a significant topographical variable. The effect of fire was to increase the altitudinal differentiation between the two species. P. halepensis forests that changed to mixed or Q. ilex forests were those of highest elevation, while the mixed and Q. ilex plots that changed to P. halepensis forests were those located at the lowest elevations. Concerning structural variables before fire, density of Q. ilex trees before the fire showed a much greater effect than P. halepensis density in determining the post-fire community. Finally, burn severity also influenced the changes observed. For both P. halepensis and Q. ilex forests, plots that changed to another forest type were mainly those that burned more severely. In the case of mixed forests, even low fire severities involved high probabilities of change to monospecific forests.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Variations in <Emphasis Type="Italic">BM224</Emphasis> microsatellite in green frogs of genus <Emphasis Type="Italic">Rana</Emphasis>

The variable microsatellite repeat BM224 has been revealed in the genomes of eight species of green frogs (Rana ridibunda, R. cf. bedriagae, R. cretensis, R. esculenta, R. lessonae, R. shquiperica, R. saharica, R. nigromaculata). Previously, this repeat was observed in members of the genus Bufo. In this paper, the possibility of using this genetic marker for species identification is discussed.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Analysis of interactions between the invasive tree-of-heaven (<Emphasis Type="Italic">Ailanthus altissima</Emphasis>) and the native black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis>)

Invasive exotic plants can persist and successfully spread within ecosystems and negatively affect the recruitment of native species. The exotic invasive Ailanthus altissima and the native Robinia pseudoacacia are frequently found in disturbed sites and exhibit similar growth and reproductive characteristics, yet each has distinct functional roles such as allelopathy and nitrogen fixation, respectively. A four-month full additive series in the greenhouse was used to analyze the intraspecific and interspecific interference between these two species. In the greenhouse experiment, the inverse of the mean total biomass (g) response per plant for each species was regressed on the density of each species and revealed that the performance of the plants was significantly reduced by interspecific interference and not by intraspecific interference (p < 0.05). Other biomass traits such as root dry weight, shoot dry weight, stem dry weight, and leaf dry weight were also negatively affected by interspecific interference. Competition indices such as Relative Yield Total and Relative Crowding Coefficient suggested that A. altissima was the better competitor in mixed plantings. Ailanthus altissima consistently produced a larger above ground and below ground relative yield while R. pseudoacacia generated a larger aboveground relative yield in high density mixed species pots.     

Species differentiation and gene flow in the Blackbutts (genus <Emphasis Type="Italic">Eucalyptus</Emphasis> subgenus <Emphasis Type="Italic">Eucalyptus</Emphasis> section <Emphasis Type="Italic">Pseudophloius</Emphasis>)

The significance of the taxonomic distinction of two species of Blackbutt was studied by analysing patterns of genetic (microsatellite markers; n = 13) and phenetic (capsule morphology) differentiation. Analysis of genetic structure using a Bayesian modelling approach on range-wide samples of both taxa (n = 457) showed the major division was within the more widely distributed species, Eucalyptus pilularis, and not aligned with taxonomy. Comparisons of intra- and inter-taxon genetic differentiation in paired-samples of taxa from each of four locations spanning the distribution of the more restricted E. pyrocarpa, showed that around twice as much variation was found among locations within taxa, than between taxa. Despite the lack of differentiation at effectively neutral microsatellite markers, significant phenetic differences (including capsule size) were evident between taxa at most sites. A landscape mosaic of taxa, coincident with changes in elevation, vegetation and soil types, suggested some phenetic differences were probably adaptive and spatial differentiation was stabilised by environmental factors. An absence of morphological intermediates and a lack of correlation in the rankings of locus inter-taxon differentiation (Phi_BT) across locations, was consistent with parapatric origins for E. pyrocarpa. We conclude the taxa are at the lower end of the speciation spectrum and might best be viewed as ecotypes, divergent in evolutionary potential, but with genomes broadly permeable to inter-taxa gene flow. Gene exchange between plantings of E. pilularis and nearby E. pyrocarpa forest is likely as the two taxa appear to have few barriers to reproduction.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

Impact of natural enemies on <Emphasis Type="Italic">Obolodiplosis robiniae</Emphasis> invasion

The gall midge Obolodiplosis robiniae is a pest of black locust Robinia pseudoacacia. The species was recently introduced into Europe and it is nowadays a usual insect in Slovakia, where two-year field surveys were undertaken during 2007–2008. The main objective of this study was to determine the natural enemies attacking O. robiniae and estimate their influence on midge population, less than 3 years after its introduction. Eight parasitoids and bushcricket Tettigonia viridissima were responsible for midge regulation. The parasitoid complex was not typically that of an introduced species as it was predicted. Firstly, the midge was heavily influenced by a specialized parasitoid Platygaster robiniae, which was a predominant parasitoid and accounted for 98% of all specimens reared. Secondly, annual larval parasitism varied from 5.4 to 10.8%, though the late season average parasitism achieved 26.0% and often exceeded 40%. P. robiniae was also very efficient in exploiting galls and was capable of consistently regulating midge population. The surveys demonstrated that the average cumulative impact of natural enemies on the midge was substantial. Stably around 20% of attacked galls were recorded during the late summer. Bushcrickets were as efficient as parasitoids and played an important role at control, especially during lack of parasitoids. Greater impact of parasitoids was found in the larger settlements than it was in the countryside, when countertendency was valid for bushcrickets. There is an indication that O. robiniae parasitized by specialized parasitoid, and attacked by bushcrickets represents only a moderate risk for R. pseudoacacia or forest stands.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from the immature seeds of <Emphasis Type="Italic">Cymbidium faberi</Emphasis>

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l^–1 -naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l^–1 NAA plus 2.0 or 5.0 mg l^–1N⁶-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l^–1 activated charcoal for 50 days and the plantlets grew normally after transplanting.     

Prevalence of <Emphasis Type="Italic">Wolbachia</Emphasis> Infection in <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Wolbachia are obligate intracellular bacteria present in reproductive tissues of many arthropod species. It has been reported that few silverleafing populations of Bemisia tabaci were positive for Wolbachia, whereas non-silverleafing populations were more likely infected with Wolbachia and all that infect B. tabaci are Wolbachia belonging to supergroup B. However, current detection methods were shown to be not sensitive enough to uncover all infections. Herein, a protocol based on polymerase chain reaction–restriction fragment length polymorphism analysis of Wolbachia 16S ribosomal DNA is presented. A systematic survey for the prevalence of Wolbachia infection in natural populations of B. tabaci using this method revealed that (1) all populations of B. tabaci tested positive for Wolbachia and the overall infection rate reached 80.5% (293 positives in 364 tests); (2) both single infection and superinfection existed within individual whiteflies tested; and (3) silverleafing populations of B. tabaci most likely harbored A Wolbachia as single infection, whereas non-silverleafing populations tend to carry B Wolbachia as superinfection. It is clear that the Wolbachia infection pattern is closely related to the genetic races of B. tabaci, and the infection frequencies are apparently much higher than those described previously. This study shows that detection methods can significantly influence estimation of Wolbachia infection. It is supposed that Wolbachia may be acting as a biotic agent promoting rapid differentiation and speciation of B. tabaci. This is the most systematic survey of Wolbachia infection within B. tabaci.