Characterisation of <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. and <Emphasis Type="Italic">Ochrobactrum</Emphasis> sp. isolated from volcanic soil

Soil bacteria may have properties of plant growth promotion but not be sufficiently beneficial for plants under stress conditions. This challenge has led researchers to extend their searches into extreme environments for potential soil bacteria with multiple plant beneficial traits as well as abiotic stress tolerance abilities. In the current study, an attempt was made to evaluate soil bacteria from an extreme environment, volcano soils, based on plant growth promoting and abiotic stress mitigating characteristics. The screening led to the isolation of eight (NBRISH4, NBRISH6, NBRISH10, NBRISH11, NBRISH13, NBRISH14, NBRISH16 and NBRISH26) bacterial isolates capable of withstanding stresses, namely temperature (up to 45 °C), salt (up to 2 M NaCl) and drought (up to 60% Poly Ethylene Glycol 6000) in vitro. Further, the selected isolates were notable for their in vitro temporal performance with regards to survival (in terms of colony count), phosphate solubilisation, biofilm formation, auxin, alginate and exo-polysaccharide production abilities under abiotic stresses i.e. 40 °C temperature; 500 mM NaCl salt and drought (PEG) conditions. In vivo seed treatments of individual selected bacteria to maize plants resulted into significant enhancement in root and shoot length, root and shoot fresh and dry weight and number of leaves per plant. Overall, the plant growth promoting and abiotic stress tolerance ability was most evident for bacterial isolate NBRISH6 which was identified as an Ochrobactrum sp. using 16S rRNA based phylogenetic analysis.     

Dearomatization of diesel oil using <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Objectives

To improve the quality of diesel fuel via removal of aromatic compounds using Pseudomonas sp.

Results

In the present study Pseudomonas sp. was able to remove 94% of fluorene, 59% of phenanthrene, 49% of anthracene, 52% of fluoranthene, 45% of pyrene and 75% carbazole present in diesel oil. Additionally, it also does not affect the aliphatic content of fuel thus maintaining the carbon backbone of the fuel.

Conclusions

Pseudomonas sp. is a potential biocatalyst that can be used in the refining industry.

     

Expression and Characterization of the TNT Nitroreductase of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V _max and K _m values of the enzyme for TNT were 12.6 μmol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain.     

Salicylate degradation by a cold-adapted <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Pseudomonas sp. strain MC1 was characterized as a cold-adapted, naphthalene-degrading bacterium that is able to grow in a broad temperature range of 5–30°C. MC1 harbors a catabolic plasmid, designated pYIC1, which is almost identical to the archetypal NAH7 plasmid from the mesophilic bacterium Pseudomonas putida G7. On pYIC1, the catabolic genes for naphthalene degradation are clustered in two operons: nahAa-Ab-Ac-Ad-B-F-C-Q-E-D encoding the conversion of naphthalene to salicylate, and nahG-T-H-I-N-L-O-M-K-J encoding the conversion of salicylate through meta-cleavage pathway to pyruvate and acetyl CoA. NahH, the bona fide extradiol dioxygenase in MC1 salicylate metabolism, is thermolabile and is a cold-adapted enzyme. The thermal profiles of the NahH enzyme activities expressed in different hosts indicate the presence of a factor(s) or mechanism(s) to protect the thermolabile NahH enzyme (100% aa identity with MC1 counterpart) in G7. Overall, the results reported in the present work suggest that the thermolabile NahH might be a product of the cold-adaptation process of MC1 and thus contribute to the survival and growth ability of MC1 on salicylate and naphthalene in cold environments.     

Characterization of pNI10 plasmid in <Emphasis Type="Italic">Pseudomonas</Emphasis>, and the construction of an improved <Emphasis Type="Italic">Escherichia</Emphasis> and <Emphasis Type="Italic">Pseudomonas</Emphasis> shuttle vector,pNUK73

The complete nucleotide sequence of pNI10 (3.75 kb), from which pNI105 and pNI107 were constructed as medium-host-range vectors for Gram-negative bacteria, was determined. A fragment of about 2.1 kb of pNI10 was essential for replication in Escherichia coli and Pseudomonas fluorescens. This fragment encodes a putative origin of replication ( ori) and one putative replication-controlling protein (Rep). An improved version of the medium-host-range plasmid vector pNUK73 (5.13 kb) was constructed with the basic-replicon of pNI10 and pHSG298 (2.68 kb). We show that expression in pseudomonads of the bromoperoxidase gene ( bpo) of Pseudomonas putida, inserted downstream of the lac promoter in pNUK73, resulted in about 30% (13.6 U/l culture) of the enzyme level obtained in E. coli.     

<Emphasis Type="Italic">Hymeniacidon perleve</Emphasis> associated bioactive bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.From Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 35–39.Original English Text Copyright © 2005 by Li Zheng, Xiaojun Yan, Jilin Xu, Haimin Chen, Wei Lin.This article was submitted by the authors in English.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

Kinetics of styrene biodegradation by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. E-93486

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value Y_\textxs^\textM = 0.913 Y_{\text{xs}}^{\text{M}} = 0.913 . Chemostat experiments confirmed the high value of yield coefficient Y_\textxs^\textapp Y_{\text{xs}}^{\text{app}} observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Quinua</Emphasis> and Relatives (<Emphasis Type="Italic">Chenopodium</Emphasis> sect.<Emphasis Type="Italic">Chenopodium</Emphasis> subsect.<Emphasis Type="Italic">Celluloid</Emphasis>)

Traditionally viewed as an Andean grain crop,Chenopodium quinoa Willd. includes domesticated populations that are not Andean, and Andean populations that are not domesticated. Comparative analysis of leaf morphology and allozyme frequencies have demonstrated that Andean populations, both domesticated(quinua) and free-living(ajara), represent an exceptionally homogeneous unit that is well differentiated from allied domesticates of coastal Chile(quingua) and freeliving populations of the Argentine lowlands(C. hircinum). This pattern of relationships indicates that Andean populations represent a monophyletic crop/weed system that has possibly developed through cyclic differentiation (natural vs. human selection) and introgressive hybridization. Relative levels of variation suggest that this complex originated in the southern Andes, possibly from wild types allied withC. hircinum, with subsequent dispersal north to Colombia and south to the Chilean coast. Coastal populations were apparently isolated from post-dispersal differentiation and homogenization that occurred in the Andes. Other data point toward a center of origin in the northern Andes with secondary centers of genetic diversity subsequently developing in the southern Andes and the plains of Argentina. Comparative linkage of South American taxa, all tetraploid, with North American tetraploids of the subsection will eventually clarify this problem. While the possibility of a direct phyletic connection betweenC. quinoa and the Mexican domesticate(C. berlandieri subsp. nuttalliae,) cannot be excluded, available evidence indicates that the latter represents an autonomous lineage that is associated with the basal tetraploid, C. b. subsp.berlandieri, through var.sinuatum, whereas South American taxa show possible affinities to either var. zschackei or var.berlandieri. An extinct domesticate of eastern North America,C. b. subsp.jonesianum, represents either another instance of independent domestication, possibly from subsp. b. var.zschackei, or a northeastern outlier of subsp.nuttalliae.     

Degradation of ethylbenzene by free and immobilized <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">fluorescens-</Emphasis>CS2

Pseudomonas fluorescens-CS2 metabolized ethylbenzene as the sole source of carbon and energy. The involvement of catechol as the hydroxylated intermediate during the biodegradation of ethylbenzene was established by TLC, HPLC and enzyme analysis. The specific activity of Catechol 2,3-dioxygenase in the cell free extracts of P. fluorescens-CS2 was determined to be 0.428 μmoles min⁻¹ mg⁻¹ protein. An aqueous-organic, Two-Phase Batch Culture System (TPBCS) was developed to overcome inhibition due to higher substrate concentrations. In TPBCS, P. fluorescens-CS2 demonstrated ethylbenzene utilization up to 50 mM without substrate inhibition on inclusion of n-decanol as the second phase. The rate of ethylbenzene metabolism in TPBCS was found enhance by fivefold in comparison with single phase system. Alternatively the alginate, agar and polyacrylamide matrix immobilized P. fluorescens-CS2 cells efficiently degraded ethylebenzene with enhanced efficiency compared to free cell cultures in single and two-phase systems. The cells entrapped in ployacrylamide and alginate were found to be stable and degradation efficient for a period of 42 days where as agar-entrapped P. fluorescens was stable and efficient a period of 36 days. This demonstrates that alginate and polyacrylamide matrices are more promising as compared to agar for cell immobilization.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Effect of Drought on the Growth and Survival of the Stress-Tolerant Bacterium <Emphasis Type="Italic">Rhizobium</Emphasis> sp. NBRI2505 <Emphasis Type="Italic">sesbania</Emphasis> and Its Drought-Sensitive Transposon Tn<Emphasis Type="Italic">5</Emphasis> Mutant

Studies were conducted to elucidate the nature of drought tolerance in the bacterium Rhizobium sp. NBRI2505 sesbania and its transposon Tn5 induced mutant to assess the role of salt, pH, and temperature stresses in contributing to drought tolerance, and to correlate drought tolerance and symbiotic effectiveness. Rhizobium sp. NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 28% salt (NaCl; wt/vol) for up to 18 h of incubation at 30°C, survived a 2-h incubation in YEB at 65°C, and when subjected to drought stress, tolerated YEB containing 45% polyethylene glycol 6000 (PEG; wt/vol) for up to 5 days of incubation at 30°C. One drought-sensitive mutant Rhizobium sp. NBRI2505 sesbania T112 (T112) containing a single Tn5 insertion was selected after screening about 10,000 clones. T112 was specifically defective in its tolerance for drought: when subjected to drought stress, it tolerated YEB containing 45% PEG for up to 2 days of incubation at 30°C. T122 mutant was also more sensitive to the heat and desiccation stresses, compared with Rhizobium sp. NBRI2505 sesbania in the presence of 45% PEG. Our results demonstrated a positive effect of calcium on the survival of Rhizobium sp. sesbania under acidic stress conditions. The observed enhanced survival at pH 3 of Rhizobium sp. NBRI2505 sesbania and T112 in the presence of 5% CaCO₃ suggests the requirement of calcium for growth and survival, which may have an ecological significance in acidic soils. Mutant strain T112 produced ineffective symbiosis with the plant host in the presence of 2.5 and 5% PEG, indicating that drought tolerance is required for effective symbiosis. Received: 11 January 2002 / Accepted: 18 February 2002     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

Biodegradation of malachite green by strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. K9 and cloning of the <Emphasis Type="Italic">tmr2</Emphasis> gene associated with an IS<Emphasis Type="Italic">Ppu12</Emphasis>

A bacterial strain K9 capable of degrading malachite green was isolated from the sludge of the wastewater treatment system of a chemical plant. It was identified preliminarily as Pseudomonas sp. Strain K9 was also able to degrade other triphenylmethane dyes, such as Crystal Violet and Basic Fuchsin. The gene tmr2, encoding the triphenylmethane reductase, was cloned from strain K9, and functionally expressed in E. coli. A 5946-bp DNA fragment including the tmr2 gene was cloned from the genomic DNA of strain K9 by chromosome walking. Its sequence analysis showed that tmr2 was associated with a typical mobile element ISPpu12 consisting of tnpA (encoding a transposase), lspA (encoding a lipoprotein signal peptidase) and orf1 (encoding a putative MerR family regulator), orf2 (encoding a CDF family heavy metal/H⁺ antiporter). This association was also found in another malachite green-degrading strain Pseudomonas sp. MDB-1, which indicated that the tmr2 gene might be a horizontally transferable gene.     

Specificity of <Emphasis Type="Italic">Anagyrus</Emphasis> sp. nov. nr. <Emphasis Type="Italic">sinope</Emphasis> and <Emphasis Type="Italic">Leptomastix dactylopii</Emphasis> for six mealybug species

In order to understand better non-target effect and potential uses, the host specificity of two parasitoid species (Anagyrus sp. nov. nr. sinope Noyes & Menezes and Leptomastix dactylopii Howard) (both Hymenoptera: Encyrtidae) for six mealybug species [Ferrisia virgata (Cockerell), Phenacoccus madeirensis Green, Phenacoccus solani Ferris, Planococcus citri (Risso),Pseudococcus longispinus (Targioni-Tozzetti) and Pseudococcus viburni (Signoret)] (all Hemiptera: Pseudococcidae) was studied through behavioral observations and laboratory rearing. The selected mealybug species represent major subfamilies and tribes of Pseudococcidae. Except for F. virgata, all mealybug species induced examinations by Anagyrus sp. nov. nr. sinope and L. dactylopii. Anagyrus sp. nov. nr. sinope was specific to P. madeirensis, which was the only mealybug species selected for oviposition and suitable for complete development of the parasitoid. No encapsulation of Anagyrus sp. nov. nr. sinope in P. madeirensis was observed. Leptomastix dactylopii accepted multiple species for oviposition, with the ranking of species preference as P. citri > P. viburni > P. longispinus > P. solani > P. madeirensis. Only P. citri, P. longispinus and P. viburni supported the development of L. dactylopii. Parasitoids developing in P. longispinus and P. viburni suffered from high encapsulation rates, while no encapsulation was observed when developing in P. citri. The results of this study suggest that Anagyrus sp. nov. nr. sinope is highly host specific. Leptomastix dactylopii, on the other hand, has a wider host range. The use of Anagyrus sp. nov. nr. sinope in a mealybug biological control program is limited to P. madeirensis and L. dactylopii to P. citri. The results presented in this study also lead us to question the accuracy of the reported host range of L. dactylopii, which include all six mealybug species tested.