Introduction and establishment ofTrioxys pallidus [Hym.: Aphidiidae] in Oregon,U.S.A. for control of filbert aphidMyzocallis coryli [Hom.: Aphididae]

Hazelnut orchards in Spain, France, and Italy were searched for parasitoids of the filbert aphid,Myzocallis coryli (Goetze). A biotype ofTrioxys pallidus Haliday was found to parasitize the aphid throughout western Europe. Wasps were imported, quarantined, mass-reared, and released in Oregon orchards. The wasp successfully attacked and completed its development on Oregon populations of the filbert aphid. A greenhouse culture of the parasitoid was maintained continuously for over 50 generations, and approximately 30,000 adult wasps were released in western Oregon. Overwintering survival has been documented in at least 12 different locations. In 3 commercial hazelnut orchards, the parasitoids proved capable of reducing aphid population peaks by 33–48%.      

Genetic improvement of Amblyseius finlandicus (Acari: Phytoseiidae): laboratory selection for resistance to azinphosmethyl and dimethoate

Amblyseius finlandicus (Oudemans) was selected in the laboratory for resistance to azinphosmethyl and dimethoate by subjecting adult females to increasing concentrations of dried residues of dimethoate and azinphosmethyl on detached bean leaves. The first eight selections were done with dimethoate. Slide-dip bioassays indicated selection with dimethoate increased dimethoate resistance 1.8-fold and azinphosmethyl resistance 2.6-fold. These resistances appeared to be quite stable: a 1.2 to 1.3-fold decrease in resistance ratios was observed in a subculture after 10 months without selections. No decrease was observed after 9 months without selections in a pooled colony that consisted of both resistant and susceptible mites. The dimethoate-selected colony was subsequently selected eight times with azinphosmethyl. About 15 % of the mites survived the last selection round with 2,500 ppm, which is 2.5 times the highest recommended field rate in Finnish apple orchards. At the end of the selection program, based on slide-dip bioassays, the total increase in resistance to dimethoate was about two-fold and to azinphosmethyl about 5.4-fold compared to the unselected base colony from which the selected colony was derived. The LC₅₀ value for azinphosmethyl was 14 times higher in the selected colony (451.3 ppm a.i.) compared to the most susceptible colony tested. A similar level of resistance to both pesticides was achieved after six azinphosmethyl selections on a mixed colony that was initiated by pooling mites from five field-collected colonies and the dimethoate-selected lines. Year-to-year variation in azinphosmethyl LC₅₀ values of the unselected base colony was high, with values varying from 83.8 to 348.7 ppm a.i., demonstrating the need to test a reference strain in each bioassay. Results of the azinphosmethyl selections and the subsequent slide-dip bioassays suggest that the resistant strain could tolerate field rates of azinphosmethyl (300–950 ppm a.i.) used in Finnish apple orchards.     

Genetic analysis of resistance to azinphosmethyl in the pear psylla Cacopsylla pyri

Laboratory selection with azinphosmethyl had little effect on the chemical resistance in Cacopsylla pyri strains, in comparison to the original wild populations. The resistance ratio relative to a susceptible strain varied from 10- to 40-fold depending on the generation studied. Crosses between two resistant strains and the susceptible strain show resistance to be autosomally inherited and semi-dominant in expression. Backcrosses between F1 and the susceptible strain were unable to distinguish unabigously between monogenic and polygenic inheritance. In the majority of experiments, however, the overall dose-response relationship for backcross progeny was consistent with a single gene hypothesis. Additional bioassays showed azinphosmethyl-resistant strain to cross-resistant monocrotophos and phosmet, but not carbamates, pyrethroids, amitraz or the organophosphates chlorpyrifos and mevinphos.     

Copper resistance in Desulfovibrio strain R2

A sulfate-reducing bacterium, designated as strain R2, was isolated from wastewater of a ball-bearing manufacturing facility in Tomsk, Western Siberia. This isolate was resistant up to 800 mg Cu/l in the growth medium. By comparison, Cu-resistance of reference cultures of sulfate-reducing bacteria ranged from 50 to 75 mg Cu/l. Growth experiments with strain R2 showed that Cu was an essential trace element and, on one hand, enhanced growth at concentrations up to 10 mg/l but, on the other hand, the growth rate decreased and lag-period extended at copper concentrations of >50 mg/l. Phenotypic characteristics and a 1078 bp nucleotide sequence of the 16S rDNA placed strain R2 within the genus Desulfovibrio. Desulfovibrio R2 carried at least one plasmid of approximately of 23.1 kbp. A 636 bp fragment ot the pcoR gene of the pco operon that encodes Cu resistance was amplified by PCR from plasmid DNA of strain R2. The pco genes are involved in Cu-resistance in some enteric and aerobic soil bacteria. Desulfovibrio R2 is a prospective strain for bioremediation purposes and for developing a homologous system for transformation of Cu-resistance in sulfate-reducing bacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biological control of strawberry tarsonemid mite Phytonemus pallidus and two-spotted spider mite Tetranychus urticae on strawberry in the UK using species of Neoseiulus (Amblyseius) (Acari: Phytoseiidae)

Two species of Neoseiulus, N. californicus and N. cucumeris, showed potential for biocontrol of phytophagous mites on strawberry. N. californicus controlled Tetranychus urticae on potted strawberry plants in a gauze-sided glasshouse at temperatures comparableto early summer in the UK (8–20°C). Both species of phytoseiid reducednumbers of the tarsonemid Phytonemus pallidus on potted strawberry plants under glasshouse conditions (15–23°C). In several experiments reductions in the range of 71–81% in numbers of tarsonemid active stages and eggs, compared to non-release plants, were obtained. The importance of establishing a suitable predator: prey ratio at an earlystage was demonstrated in an experiment where an initial ratio of 1 N. cucumeris: 10 P. pallidus gave a greater degree of controlthan 1:20 or 1:40.     

Sources and inheritance of resistance to leaf curl virus in Lycopersicon

Summary One hundred and twenty-two varieties, lines and wild accessions of Lycopersicon were screened under three different regimes during the autumn/winter season of 1982–83 and 1983–84 for resistance to tomato leaf curl virus (TLCV). L. hirsutum f. glabratum (B6013) and L. hirsutum f. typicum (A1904) proved to be highly resistant to TLCV in all three environments. Various accessions of L. peruvianum were also highly resistant. L. pimpinellifolium (A1921) exhibited no TLCV symptoms within 90 days. Of the cultivated varieties, Acc 99 exhibited the minimim score for susceptibility; AC 142, Collection No. 2, Kalyanpur Angurlata and HS 101 had a low rating for virus incidence. The inheritance of resistance was studied in the interspecific crosses between a TLCV resistant line of L. pimpinellifolium (A1921) and five (HS 101, HS 102, HS 110, Pusa Ruby and Punjab Chhuhara) susceptible cultivars of L. esculentum. Parents, F₁, F₂ and backcross progenies were artificially inoculated with local strains of TLCV using vector the viruliferious whitefly, Bemisia tabaci (Genn.). Data indicated that the resistance of L. pimpinellifolium (A 1921) is monogenic and incompletely dominant over susceptibility.     

Selection for kanamycin resistance in transformed petunia cells leads to the co-amplification of a linked gene

A cell suspension culture was established from a transgenic petunia (Petunia hybrida L.) plant which carried genes encoding neomycin phosphotransferase II (nptII) and -glucuronidase (uidA, GUS). Two selection experiments were performed to obtain cell lines with increased resistance to kanamycin. In the first, two independently selected cell lines grown in the presence of 350 g/ml kanamycin were eight to ten-fold more resistant to kanamycin than unselected cells. Increased resistance was correlated with amplification of the nptII gene and an increase in nptII mRNA levels. Selection for kanamycin resistance also produced amplification of the linked GUS gene, resulting in increased GUS mRNA levels and enzyme activity. Selected cells grown in the absence of kanamycin for twelve growth cycles maintained increased copy numbers of both genes, and GUS enzyme activity was also stably overexpressed. In a second selection experiment, a cell line grown continuously in medium containing 100 g/ml kanamycin exhibited higher nptII and GUS gene copy numbers and an increase in GUS enzyme activity after eleven growth cycles. In this cell line, amplification of the two genes was accompanied by DNA rearrangement.     

Inducible nickel resistance in a river isolate of India phylogenetically ascertained as a novel strain of Acinetobacter junii

Summary A gram negative, motile, short rod-shaped, and nickel resistant (tolerating 6.5 mM Ni²⁺) bacterium, strain BB1A, was isolated from the waters of the River Torsa in Hashimara, Jalpaiguri district, West Bengal, India. The isolate BB1A was identified as a strain of Acinetobacter junii following detailed analysis of morphological, physio-biochemical and 16S rRNA gene sequence. The expression of nickel resistance in BB1A was inducible by exposure to nickel chloride at a concentration as low as 50 μM Ni²⁺. The other metal ions, Cu²⁺, Zn²⁺, or Pb²⁺ at a concentration range of 20–30 μM, also induced the nickel resistance system in this bacterium. Southern hybridizations of BB1A genomic DNA with digoxigenin-dUTP labeled DNA probes specific for well known nickel resistance determinants, cnr, ncc or nre, resulted in no detectable signal, but nir specific probe yielded weak hybridization signal with restricted genomic DNA of BB1A. The isolate BB1A, therefore, carries out a novel induction phenomenon of nickel resistance and presumably with a nickel resistance genetic system different from that previously characterized in other bacteria.     

Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase gene

Summary We have selected a tobacco cell line, SU-27D5, that is highly resistant to sulfonylurea and imidazolinone herbicides. This line was developed by selection first on a lethal concentration of cinosulfuron and then on increasing concentrations of primisulfuron, both sulfonylurea herbicides. SU-27D5 was tested against five sulfonylureas and one imidazolinone herbicide and was shown, in every case, to be two to three orders of magnitude more resistant than wild-type cells. The acetohydroxyacid synthase (AHAS) of SU-27D5 was 50- to 780-fold less sensitive than that of wild-type cells to herbicide inhibition. The specific activity of AHAS in the SU-27D5 cell lysate was 6 to 7 times greater than that in wild-type cells. Using Southern analysis, we showed that cell line SU-27D5 had amplified its SuRB AHAS gene about 20-fold while maintaining a normal diploid complement of the SuRA AHAS gene. Genomic clones of both AHAS genes were isolated and used to transform wild-type tobacco protoplasts. SuRB clones gave rise to herbicide-resistant transformants, whereas SuRA clones did not. DNA sequencing showed that all SuRB clones contained a point mutation at nucleotide 588 that converted amino acid 196 of AHAS from proline to serine. In contrast, no mutations were found in the SuRA clones. The stability of SuRB gene amplification was variable in the absence of selection. In one experiment, the withdrawal of selection reduced the copy number of the amplified SuRB gene to the normal level within 30 days. In another experiment, amplification remained stable after extended cultivation on herbicide-free medium. This is the first report of amplification of a mutant herbicide target gene that resulted in broad and strong herbicide resistance.     

Genetic analysis and cross-resistance spectrum of a laboratory-selected chlorfenapyr resistant strain of two-spotted spider mite (Acari: Tetranychidae)

A laboratory susceptible strain of Tetranychus urticae was selected with chlorfenapyr resulting in a resistant strain. After 12 cycles of exposure, the resistance ratio (RR) calculated from the LC50s of susceptible and selected strain was 580. The resistant strain was screened with 16 currently used acaricides for cross-resistance. Cross-resistance was detected with amitraz (RR = 19.1), bifenthrin (RR = 1.3), bromopropylate (RR = 7.5), clofentezine (RR = 29.6) and dimethoate (RR = 17.6). No cross-resistance was detected with the new molecules acequinocyl, bifenazate and spirodiclofen. Mortality caused by chlorfenapyr in the F1 progeny from reciprocal crosses between both strains indicated that the mode of inheritance was incomplete recessive. Mortality in F2 progeny indicated that the resistance was under the control of more than one gene. Synergist experiments with S,S,S-tributylphosphorotrithioate (DEF), piperonylbutoxide (PBO) and diethylmaleate (DEM), which are inhibitors of esterases, monooxygenases and glutathion-S-transferases respectively, suggested a major role of esterases in the resistance to chlorfenapyr.     

Survival of a strain of Bacillus megaterium carrying a lepidopteran-specific gene of Bacillus thuringiensis in the phyllospheres of various economically important plants

The colonizing ability of a transcipient strain of Bacillus megaterium carrying a lepidopteran-specific cryIA (a) gene of Bacillus thuringiensis in the phyllospheres of various economically important plants was studied. Similar experiments were also carried out using the parental B. thuringiensis var. kurstaki strain HD1 for a comparison. While the transcipient remained on the leaves of cotton and okra for more than 28 days, its survival in phyllospheres of mulberry, peanut, chickpea, tomato and rice was rather limited to about 3 – 5 days. The persistence of B. thuringiensis, on the other hand, was extremely short (i.e. less than 4 days) on all the crop plants tested.     

Nickel resistance of Alcaligenes denitrificans strain 4a-2 is chromosomally coded

A newly isolated aerobic hydrogen-oxidizing bacterium, Alcaligenes denitrificans strain 4a-2, differs from related autotrophic bacteria by containing only a single cytoplasmic, NAD-reducing hydrogenase, and by its high resistance to nickel ions, i.e. tolerance to 20 mM NiCl₂. Strain 4a-2 harbors a single plasmid of about 250 kb. On helper-assisted mating of 4a-2 with Alcaligenes eutrophus strains H16,G29, and M85 nickelresistant transconjugants were selected; these did not contain the donor plasmid, however. All three transconjugants tolerated 3 to 10 mM NiCl₂. The resistance was constitutively expressed. DNA/DNA hybridization showed homology with EcoRI-digested DNA of the wild type 4a-2 and transconjugants using a DNA probe containing nickel resistance genes of pMOL28. This indicated that the 4a-2 nickel resistance genes are located on the chromosome.     

Bionomics ofTrioxys indicus [Hym.: Aphidiidae], a parasitoid ofAphis craccivora [Hem.: Aphididae]: 31. Effect of host haemolymph on the numerical response of the parasitoid

The influence of kairomones on the numerical response of the parasitoidTrioxys indicus against its hostAphis craccivora at its varying density was studied. The kairomones (applied as aqueous extract of the host) significantly enhanced the rate of parasitisation and multiplication and the area of discovery of the parasitoid and also the K-values of mortality of the host at all parasitoid densities introduced (1, 2, 4, 8, 12 and 16 parasitoids) into troughs having about 200 hosts. The sex-ratio of F₁ offspring decreased at lower parasitoid densities and remained more or less unchanged at higher parasitoid densities after the application of kairomones. The present findings indicate that if kairomones are applied properly, the number of hosts destroyed by a stimulated parasitoid will be about 200, twice the number reported earlier, thus fewer parasitoids will be needed to regulate an estimated population of the hosts.      

A test method for pesticide tolerance in minute parasiticHymenoptera

In contrast to many pest species, important biological control agents have only rarely been found to be pesticide resistant. Biochemical and ecological mechanisms have been implicated, but lack of toxicological techniques suitable for the minute and fragile insects concerned may limit screening and research projects. Standard techniques are criticized, and a method utilizing controlled access to pesticidecontaining sucrose solutions is described. The method was used to test tolerance ofAphytis holoxanthus to malathion, and is suggested for screening and selection experiments with this and similar species.      

Field Releases of the Predatory Mite Neoseiulus fallacis (Acari: Phytoseiidae) in Canada, Monitored by Pyrethroid Resistance and Allozyme Markers

The predacious phytoseiid mite Neoseiulus fallacis (Garman) is an important agent for the biological control of spider mites in deciduous fruit orchards in North America and Canada. It would be helpful to monitor the fate of released individuals to improve the results of introductions of the predators in biological control trials. We have used two types of genetic markers, pyrethroid resistance and allozymes, for indirect estimation of the survival of N. fallacis introduced in an apple orchard in Ontario, Canada. Mite samples were submitted to toxicological tests. The polymorphism of four enzymes was studied in individual females using an isoelectric focusing technique. A mite sample was taken from the field, mass-reared in the laboratory, and selected for permethrin resistance. This strain was released on several apple trees treated with permethrin, and mite samples were collected from the same trees 10 to 90 days later. The genetic composition and the insecticide resistance level of this sample were compared to those of two other samples from trees where mites had not previously been released, either in the same orchard or in a neighboring block. A control susceptible strain was compared using mites collected earlier from trees on the same site but outside the present experiment. The mites collected from the release trees and those from the strain used for the releases were found to be genetically closely related, as judged from a small genetic distance, and from similar levels of insecticide resistance in both samples. The control samples from the nonrelease trees were genetically distant from these and displayed low resistance levels. These results indicate that the released genotypes established and persisted in the release trees for the period of the experiment. The utility of the two approaches in assessing the fate of released natural enemies is discussed.     

Additive inheritance of resistance to pod rot caused by Phytophthora palmivora in cocoa

Summary Quantitative inheritance of resistance to Phytophthora pod rot (Ppr) was studied in cocoa hybrid progeny from 12 Trinitario x Amazonian crosses and their reciprocal crosses. The crossing scheme was similar to a factorial design. Disease was assessed by the number and percentage of infected pods on each tree. Highly significant differences due to general combining abilities (GCA) were obtained for all characters, except for the GCA of Trinitario on total pod production. Differences for specific combining ability (SCA) were not significant for all characters. There were no significant differences between reciprocal crosses. The Trinitario clone K82 provided the only source for the hybrid progenies of strong Ppr resistance to the hybrid progenies, while K20 provided moderate resistance. Other parental clones — KA2-101, KA5-201, KEE 2, KEE 5, and KEE 52 — produced progenies which were susceptible to Ppr. It is evident that resistance to Ppr in cocoa is inherited additively. Maternal and cytoplasmic effects were assumed to have no influence on inheritance of resistance. It is also concluded that resistance to Ppr of the kind shown by K82 is likely to be horizontal resistance. Breeding for high-yielding cultivars combined with Ppr resistance is the most effective way of controlling Ppr of cocoa on the crops of growers with small holdings in Papua New Guinea.     

Ribosomal protein S12 as a site for streptomycin resistance in Nicotiana chloroplasts

Summary A streptomycin resistant Nicotiana plastome mutant, X/str ^R6, was subjected to molecular analysis. In this mutant, a single nucleotide transition, C » T, in the chloroplast gene for ribosomal protein S12 alters codon 90 from proline to serine while the nucleotide sequence of the chloroplast 16 S rRNA gene is identical to that of the wild type. Mutant X/str ^R6 thus differs from several previously reported streptomycin resistant chloroplast mutants which are altered in the gene for 16 S rRNA.     

Mechanism of zinc resistance in Pseudomonas putida strain S4

The mechanism of Zn resistance in multiple metal-resistant Pseudomonas putida strain S4 is based on inducible efflux. An ATPase in the strain S4 mediated active extrusion of Zn²⁺, which occurred during the exponential phase of growth. The ATPase activity was inhibited by micromolar concentrations (50 M) of vanadate, suggesting the involvement of a P-type ATPase. The effluxed Zn²⁺ were not ejected out of the cell but stored in the outer membrane and periplasm, which provided the required binding sites. The strain S4, thus, employs a dual strategy of efflux and binding to bring about a proper management of essential ions like Zn.     

Intra- and interspecific inheritance of some components of the resistance to leaf rust (Melampsora larici-populina Kleb.) in poplars

Clonal variation for rust resistance was studied in a factorial mating design of Populus trichocarpa and P. deltoides with 17 intraspecific and 15 interspecific progenies. Susceptibility to Melampsora larici-populina was assessed both under natural conditions in the nursery and after leaf-disk inoculation with two pathogen races in a controlled environment. Different components of the resistance were taken into account: immunity in the field or in the laboratory, latent period, number and size of uredia in the laboratory, and field resistance at the end of the growing season. Genes controlling immunity were contributed only by the P. deltoides parents. The distributions of clonal means within each family suggested a polygenic inheritance of field resistance in the P. trichocarpa x P. trichocarpa crosses, but major gene effects were suspected in the interspecific progenies. Inefficiency of the quantitative mechanisms of resistance in the interspecific F₁ hybrids might have important implications for future breeding strategies. Field and laboratory trials complemented each other well, and a combined selection approach is proposed.