Characterization of acetylcholinesterases of acaricide resistant and susceptible strains of the cattle tick Boophilus microplus (Can.): II. The substrate specificity and catalytic efficiency of the critical enzyme component

Concentrations and turnover numbers have been determined for the critical acetylcholinesterase component of the organophosphorus susceptible Yeerongpilly and resistant Biarra and Ridgelands strains. The lower specific activity for the substrate acetylthiocholine in the resistant strains was shown to be due to decreased catalytic efficiency and not to any change in the titer of the enzyme. Thioester analogs of acetylthiocholine, with additional methylene groups between the carbonyl carbon and the quaternary nitrogen of the molecule and in the alkyl moiety, have been used to derive K_m and V_max values for the enzymes from each strain. An hypothesis is proposed concerning the alteration of the active site that has occurred to produce the dissimilar aberrant enzymes of the resistant strains and the implications of this hypothesis for inhibitor design are discussed.     

Toxicology and metabolism of some synthetic pyrethroids in larvae of susceptible and resistant strains of the cattle tick boophilus microplus (Can.)

Larvae of the Malchi and DDT-R pyrethroid-resistant strains of ticks, compared with larvae of the Yeerongpilly (susceptible) strain, exhibited knock-down resistance equally to (1 RS)-cis- and (1 RS)-trans-permethrin but little resistance to either (1 RS)-cis- or (1 RS)-trans-cypermethrin. Kill resistance was higher in Malchi than in DDT-R ticks and more pronounced against the permethrin isomers. The cis-isomers were more potent overall than the trans-isomers. Cypermethrin isomers penetrated more slowly than permethrin isomers, especially into Malchi larvae. Metabolic detoxication, mainly esteratic, was fastest in Malchi larvae, trans-isomers consistently being attacked faster than cis-isomers. The greatei resistance of Malchi ticks is therefore probably due to a combination of these two differences. Dual mechanisms of the toxic action of pyrethroids are discussed.     

Acaricide resistance and synergism between permethrin and amitraz against susceptible and resistant strains of Boophilus microplus (Acari: Ixodidae)

The control of the southern cattle tick, Boophilus microplus (Canestrini), in Mexico and many other countries relies on chemical acaricides. Boophilus microplus has developed resistance to all major classes of acaricides in recent years. To gain a better understanding of the resistance and to develop resistance management strategies that benefit both Mexican ranchers and USDA's cattle fever tick eradication program (CFTEP), the authors used larval bioassay techniques to determine levels of resistance to permethrin and amitraz and then evaluated synergism between these two acaricides in one susceptible laboratory tick strain and four resistant strains originating from Mexico and Brazil. To examine mechanisms of resistance to permethrin in these strains, the frequency of a mutated sodium channel gene was determined using a PCR assay. The tick strains from Mexico and Brazil demonstrated 49.4- to over 672.2-fold resistance to permethrin, and up to 94.5-fold resistance to amitraz. While the San Roman strain from Mexico was the most permethrin-resistant strain, the Santa Luiza strain from Brazil was the most amitraz-resistant strain. A significant correlation was found between the permethrin resistance ratio and the allelic frequency of the sodium channel mutation. Significant synergism between permethrin and amitraz was found when one acaricide was tested in the presence of another. Synergism ratios ranged from 1.5 to 54.9 when amitraz was tested as a synergist for permethrin. Similar synergism ratios were obtained when permethrin was tested as a synergist for amitraz. Permethrin caused virtually no mortality in the San Roman strain, even at the highest concentration (3294 microg cm(-2)). Adding amitraz (11.0 microg cm(-2)) to permethrin led to a dramatic increase in larval mortality, even at very low concentrations of permethrin.     

Effect of imidacloprid on the reproduction of acaricide-resistant and susceptible strains of Tetranychus urticae Koch (Acari: Tetranychidae)

Occasional reports linking neonicotinoid insecticide applications to field population outbreaks of the two-spotted spider mite, Tetranychus urticae Koch, have been a topic of concern for integrated pest management (IPM) programmes, particularly in apples. In order to shed light on the factors which may contribute to the occasional field population increase of T. urticae following the application of neonicotinoid insecticides, greenhouse experiments have been set up. Four different T. urticae strains, namely GSS (acaricide-susceptible), WI (organophosphate-selected), USA (a largely uncharacterised strain) and Akita (METI (mitochondrial electron transport inhibitor) acaricide-resistant and cross-resistant to dicofol), were compared for their fecundity without insecticide treatment and for their ovipositional response to foliar and drench applications of the field-relevant dose of imidacloprid (100 mg litre(-1)). Without insecticide treatment, strain GSS laid significantly more eggs (162.50 (+/- 5.43)) than the multiple resistant strain Akita (139.90 (+/- 5.54)) during a 16 day oviposition period. With imidacloprid treatment the highest effect was observed with GSS, with a significantly reduced number of eggs in drench (143.40 (+/- 4.22)) and foliar (144.60 (+/- 5.85)) applications. For strains Akita and USA, no significant differences were observed in oviposition between imidacloprid treatments and controls. The proportion of F1 female offspring decreased significantly with drench application for GSS and WI, while no differences were observed among strains in the survival of F1 immature stages, except for strain USA. The viability of eggs was relatively high (from 82.9 (+/- 4.5)% for USA to 95.2 (+/- 1.2)% for GSS) and not affected by imidacloprid treatments.     

Characterization and Inhibitor Studies of Chitinases from a Parasitic Blowfly (Lucilia cuprina), a Tick (Boophilus microplus), an Intestinal Nematode (Haemonchus contortus) and a Bean (Phaseolus vulgaris)

The molecular weight pattern and the stage-specific activities of chitinases from the blowfly Lucilia cuprina, the tick Boophilus microplus and the intestinal nematode Haemonchus contortus were examined. Chitinolytic enzymes could be detected in all parasite species tested, but the activity was different between the stages. Highest chitinolytic titers were found in blowfly pupae (83 kDa, 118 kDa), hatching larvae of ticks (58 kDa, 94 kDa) and nematode eggs (43 kDa). Leaves from ethylene-treated bean plants Phaseolus vulgaris expressed two basic Class I chitinases (Ia, Ib) of 34 kDa, differing in their amino acid sequences at residue 33 and 34 (Ia: glycine, proline; Ib: lysine, aspartic acid). Inhibitor studies with blowfly pupae revealed that allosamidin (IC₅₀=0·32 (±0·02) μM ) was by far the best inhibitor when compared with various amino sugar derivatives. This compound also inhibited chitinases from tick larvae (IC₅₀=0·69(±0·10) μM ) and nematode eggs (IC₅₀=0·048(±0·0045) μM ) specifically. Whereas Class Ia chitinase from bean leaves was inhibited only up to 18% by 10 μM allosamidin, it had an IC₅₀ of 1(±0·14) μM for the Ib type, which is the first plant chitinase described to be highly sensitive to allosamidin.     

New compounds active against resistant cattle ticks (Boophilus spp.): Relationship between structure and activity within the group of cyclic amidines

A series of cyclic amidines is described, many of which are highly active against resistant cattle ticks, Boophilus spp. The preferred compound is clenpyrin, 1-butyl-2-(3,4-dichlorphenylimino)pyrrolidine which paralyses the cattle ticks and inhibits their egg-production.     

Metabolism of methyl bromide by susceptible and resistant strains of the granary weevil, Sitophilus granarius (L.)

Methyl bromide was metabolized by susceptible and resistant strains of adult granary weevil, Sitophilus granarius (L.), mainly by conjugation with glutathione. S-Methyl glutathione and S-methyl cysteine were produced by both strains and S-methyl glutathione sulfoxide was identified as a metabolite in the resistant strain. In the untreated insects, no significant difference was observed in glutathione S-transferase activity but the resistant contained approximately twice as much glutathione per insect as the susceptible strain. When the insects were treated with methyl bromide, the glutathione content of both strains was lowered; proportionally, however, the decrease was considerably higher in the susceptible than in the resistant strain. These results indicate that conjugation of methyl bromide with glutathione is a major detoxication pathway and tolerance to this fumigant is related, in part at least, to the level of glutathione in the granary weevil.     

Identification of the anthracnose fungus of walnut (Juglans spp.) and resistance evaluation through physiological responses of resistant vs. susceptible hosts

Walnut anthracnose is one of the most serious diseases of walnut globally. In this study, the pathogen was identified as Colletotrichum gloeosporioides by morphological and molecular approaches. Then, the resistance of 18 walnut genotypes was evaluated by field investigation as well as field and detached leaflet inoculation. A field inoculation assay was used as a benchmark for comparing field evaluation and the detached leaflet inoculation assay. Seven genotypes, Panhe1 and clones 71 and 200 of Juglans sigillata, Yanyuanzao and Qingxiang of J. regia, and Shujiang1 and Chuanzao1 of J. regia × J. sigillata, displayed high resistance in all assays, suggesting that they may be good candidates for further evaluation. Subsequently, two genotypes (Panhe1 with high resistance, and clone 199 with high susceptibility) were selected to analyse the different physiological responses between highly resistant and susceptible genotypes after inoculation. C. gloeosporioides infection induced the activities of superoxide dismutase (SOD), peroxidase (POD), polyphenol oxidase (PPO), and chitinase; they were all significantly increased in both Panhe1 and clone 199. However, the POD activity and its change rate in Panhe1 were significantly higher than those in clone 199. The change rates of PPO activity, and total phenol and proline content in Panhe1 were significantly higher than those in clone 199. Moreover, the change rates of SOD activity, malondialdehyde, and soluble sugar content in clone 199 were significantly higher than those in Panhe1. These findings not only enhance our understanding of the resistance of walnut to anthracnose, but also lay the foundation for breeding anthracnose-resistant walnuts in the future.     

Development and physiology of infection by the downy mildew fungus Peronospora parasitica (Pers. ex Fr.) Fr. in susceptible and resistant Brassica species

The growth of two isolates of the downy mildew fungus Peronospora parasitica , one obtained from cauliflower ( Brassica oleracea ) and the other from oilseed rape ( B. napus ) was assessed in their respective hosts of origin, and also in the alternative combination. Both isolates were capable of infecting either host, but there were marked contrasts in the time course and extent of mycelial development, the amounts of associated host-cell necrosis, and eventual intensity of sporulation. Oilseed rape, which was partially resistant to the isolate from cauliflower, exhibited extensive necrosis of mesophyll cells, in conjunction with reduced mycelial development, and delayed and reduced sporulation by the pathogen. The isolate from oilseed rape was virulent on both host species. Pathogenesis in the susceptible combinations was accompanied by large increases in electrolyte leakage, and increased activity of the enzymes β-glucosidase, ribonuclease, and peroxidase. Effects on chlorophyll content were variable and activities of acid phosphatase and acid phosphodiesterase were unaffected. Electrophoretic analyses of extracts from fungal sporangia and infected seedlings indicated that the large increases in β-glucosidase were of pathogen origin, while evidence from inhibitor studies suggested that enhanced ribonuclease activity was due to a new post-infectional form of the enzyme. The significance of these findings is discussed in relation to pathogenesis and host resistance mechanisms.     

Some diagnostic, biologic and morphologic characteristics of Francisella tularensis strains isolated from the ticks Ixodes ricinus (L.) in the Prague agglomeration

Four infectious agents were isolated from the ticks Ixodes ricinus (L.) collected in the recreational area and park-forest of Prague. On the basis of cultivation, staining, biochemical, serologic properties, pathogenicity for animals and histological tests they were identified as Francisella tularensis with the following features: they are short, gram-negative rods of approximate dimensions of 0.3 X 0.8 micron, growing in enriched media after 3-4 day incubation at 37 degrees C. They form small circular, at first transparent, later greyish turbid colonies with regular rims. They are little active biochemically. They are susceptible to streptomycin and some broad spectrum antibiotics. They react positively with tularemic serum, but in lower titres than those in which this serum reacts with standard antigen. The microbes are highly pathogenic for mice, guinea-pigs, young rats, in which a massive bacteriemia occurs before death, but they do not kill rabbits. They multiply well in chick embryo, but do not grow in cell or tissue cultures. The most important histologic changes were observed in liver and spleen of mice. No pathologic changes were found in brain, lungs, heart, kidneys. Necroses were found in liver and in their marginal zones the microbes were present. Conspicuous were changes in numerous hepatocytes which became enlarged due to microbial multiplication and finally transformed into "sacs" packed with microbes. Histological and electronoptical examination showed that these are intracellular parasites fringed with a light lytic zone. Discussed is the problem to what extent the properties of the isolated strains are typical of F. tularensis as well as the importance of their detection from the aspect of epidemiology and differential diagnostics.     

Effect of multiple infestations from insect pests and other stresses on irrigated rice in the Philippines: I. Damage functions

Artificial infestation methods were employed to generate damage function graphs for guilds of three rice insect pests. In the vegetative stage, infestation of whorl maggot (Hydrellia philippina Ferino) and defoliators (a mixed population of Naranga aenescens Moore and Rivula atimeta [Swinhoe]) was applied as a combined or single pest attack. Infestation of yellow stemborer (Scirpophaga incertulas [Walker]) and leaffolder (Cnaphalocrocis medinalis [Guenée]) each were infested in one or two crop growth stages. Combinations of three stresses (four N application rates, solar radiation from the wet or dry season, and/or the presence or absence of weeds) were applied to each guild to develop more holistic economic injury levels (EILs). Slopes on the linear portion of damage curves of each pest guild became steeper with each additional stress applied, whether from multiple insect pest attack, low N, low solar radiation, or weeds. EILs can be developed by pest managers from the graphs of the damage functions for the variables quantified in this study.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Studies on the biology of black mould (Aspergillus niger) on temperate and tropical onions. 1. A comparison of sources of the disease in temperate and tropical field crops

Aspergillus niger colonies were present on 60–90% of the leaves sampled from onion crops grown under hot, tropical conditions (Shambat, Sudan), but only on 21% of leaves in crops grown under temperate conditions (Wellesbourne, UK). The seed stocks used in the Sudan were naturally contaminated with A. niger , whereas the fungus was absent from seed used in the UK. Inoculation of seed with A. niger resulted in an increase in the incidence of the fungus on onion plants (mean 83% of plants with affected leaves) in the field and on bulbs in store in crops grown in the UK. In the UK, A. niger is infrequent in the soil and air, but it is a major component of the soil and air mycoflora in the Sudan. Most of the bulbs grown in Sudan were contaminated with A. niger at harvest because of the high incidence of the fungus in the field.     

Potential of zimtaldehyde, 4-allyl-anisol, linalool, terpineol and other phytochemicals for the control of the confused flour beetle (Tribolium confusum J. d. V.) (Col., Tenebrionidae)

Toxicity and repellency effects of several pure compounds (of plant origin) against the confused flour beetle (Tribolium confusum) were investigated in a filter paper test. The compounds were α-pinen, β-pinen, 4-allyl-anisol, camphor, 1,8-cineol, eugenol, linalool, menthol, piperin, terpineol, thymol and zimtaldehyde. Five compounds (4-allyl-anisol, linalood, terpineol, thymol and zimtaldehyde) caused enough mortality of this insect to allow the determination of LD₅₀ and LD₉₅ values. Zimtaldehyde and 4-allyl-anisol showed the strongest toxicity effects with LD₅₀ values of 0.04–0.05 μl/cm² within an exposure period of 24 h. When the isolated fumigant effects were tested by separating the insects from the filter paper, the toxicity of zimtaldehyde (LD₅₀=0.29 μl/cm²) was much lower than that of 4-allyl-anisol (LD₅₀=0.10μl/cm²).     

Potential of zimtaldehyde, 4-allyl-anisol, linalool, terpineol and other phytochemicals for the control of the confused flour beetle (Tribolium confusum J. d. V.) (Col., Tenebrionidae)

Toxicity and repellency effects of several pure compounds (of plant origin) against the confused flour beetle (Tribolium confusum) were investigated in a filter paper test. The compounds were α-pinen, β-pinen, 4-allyl-anisol, camphor, 1,8-cineol, eugenol, linalool, menthol, piperin, terpineol, thymol and zimtaldehyde. Five compounds (4-allyl-anisol, linalood, terpineol, thymol and zimtaldehyde) caused enough mortality of this insect to allow the determination of LD₅₀ and LD₉₅ values. Zimtaldehyde and 4-allyl-anisol showed the strongest toxicity effects with LD₅₀ values of 0.04–0.05 μl/cm² within an exposure period of 24 h. When the isolated fumigant effects were tested by separating the insects from the filter paper, the toxicity of zimtaldehyde (LD₅₀=0.29 μl/cm²) was much lower than that of 4-allyl-anisol (LD₅₀=0.10μl/cm²).     

Molecular characterization,comparison of screening methods,and evaluation of cross‐pathogenicity of black rot (Xanthomonas campestris pv. campestris) strains from cabbage,choy sum,leafy mustard and pak choi from Taiwan

Choy sum (Brassica rapa var. parachinensis), leafy mustard (Brassica juncea) and pak choi (B. rapa var. chinensis) are highly nutritious components of diets in Taiwan and other Asian countries, and bacterial black rot caused by Xanthomonas campestris pv. campestris (Xcc) is a major biotic constraint in these crops. As very little was known about the Xcc strains from these crops in these regions, including their cross‐pathogenicity and aggressiveness on different hosts, Xcc strains were obtained from cabbage (Brassica oleracea var. capitata), choy sum, leafy mustard and pak choi crops in Taiwan. Two previously published PCR‐based assays reliably distinguished the Xcc strains from other Xanthomonas species and subspecies. Phylogenetic analysis based on repetitive sequence‐based PCR assays placed the Xcc strains in a clade distinct from other Xanthomonas species, and also showed host specificity. Although all of the Xcc strains from the different host species were pathogenic on all five Brassica test species in both a detached leaf assay and an intact plant assay, in the intact plant assay they showed differences in virulence or aggression on the different test hosts. The Xcc strains from leafy mustard and pak choi were consistently highly aggressive on all the test host genotypes, but the strains from choy sum and cabbage were less aggressive on leafy mustard and choy sum. The intact plant assay proved more discriminating and reliable than the detached leaf assay for comparing the aggressiveness of Xcc strains on different host genotypes, and so, with the new Xcc strains isolated in this study, will be useful for screening leafy brassica germplasm accessions for resistance to black rot.     

First report of the lacebug Corythauma ayyari (Drake) (Hemiptera: Tingidae) on Jasminum grandiflorum L. and Jasminum sambac (L.) from Syria

In Syria, defoliation was noted on several Jasminum grandiflorum and Jasminum sambac plants in various gardens in Latakia City and countryside in 2017. Subsequently, several male and female specimens of a lacebug insect were collected from the white jasmine, Jasminum grandiflorum, planted in the garden of the Latakia Center for Agricultural Research during 2018. Collected material was examined and identified as Corythauma ayyari (Drake). Infestation by this insect is considered by the authors to cause the defoliation of jasmine plants in Latakia City and it is reported to occur in Syria for the first time.