Quorum sensing activity in Ophiostoma ulmi: effects of fusel oils and branched chain amino acids on yeast-mycelial dimorphism

Aims: For Ophiostoma (Ceratocystis) ulmi, the ability to undergo morphological change is a crucial factor for its virulence. To gain an understanding of quorum‐sensing activity in O. ulmi as it relates to yeast‐mycelium dimorphism control, this study examines the effects of branched‐chain amino acids as well as their fusel alcohols and fusel acids as quorum sensing molecules. Methods and Results: In a defined medium containing glucose, proline and salts, O. ulmi grew as yeasts when the culture was inoculated with a high density of spores (2 × 10⁷ CFU ml^?1) and as mycelia when inoculated with a low spore density (4 × 10⁵ CFU ml^?1). The cultures displaying yeast morphology secreted a quorum‐sensing factor that shifted the morphology from mycelia to yeast. This quorum‐sensing molecule was lipophilic and extractable by organic solvents from the spent medium. Using GC/MS analysis, it was determined that the major compound in the extract was 2‐methyl‐1‐butanol. A similar effect was observed when the branched‐chain amino acids (fusel alcohol precursors) were used as the nitrogen source. E, E‐farnesol had no effect on the morphology of O. ulmi. Conclusions: Addition of the branched‐chain amino acids or one of the compounds detected in the spent medium, 2‐methyl‐1‐butanol or 4‐hydroxyphenylacetic acid, or methylvaleric acid, decreased germ tube formation by more than 50%, thus demonstrating a quorum sensing molecule behaviour in O. ulmi cultures. Significance and impact of the study: This study presents advances in the investigation of dimorphism in O. ulmi, complementing the existing scientific basis, for studying, understanding and controlling this phenomenon.     

Some insectary experiments with predacious mites to determine their effect on the development of Metatetranychus ulmi (Koch) populations

Summary As field spraying experiments give results which are difficult to resolve in terms of individual predator species, a series of experiments has been carried out in an insectary to find out what effect predacious mites of various species have upon populations of Metatetranychus ulmi (Koch), in the absence of other prey. Known numbers of each mite species were placed on seedling plants, and the development of the resulting populations followed.In one experiment, where initial numbers of 5, 25 or 50 M. ulmi females were placed on a plant, together with five females of Typblodromus tiliae Oudms., the M. ulmi population in every case remained throughout the following three months at a density of less than one mite per leaf, which was less than a total of 50 mites per plant. Where the same numbers of M. ulmi started in the absence of T. tiliae, a rapid increase of M. ulmi resulted in populations of over 3000 mites per plant in up to eleven weeks.It was also found that both the size of the host plant, and the initial ratio of M. ulmi and T. tiliae, had an effect on the development of the T. tiliae populations.Of other predacious species, T. finlandicus was similar to T. tiliae in its effect on M. ulmi. Although T. umbraticus itself increased steadily, it exerted little effect on the M. ulmi population, nor did T. tiliarum which, however, did not develop well under these conditions. Phytoseius macropilis (Banks) was also slow to increase and had little effect on the M. ulmi population.

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Zusammenfassung Man hat aus Freilandversuchen gefolgert, daß gewisse Typhlodromus-Arten eine wichtige Rolle bei der Bekämpfung von Metatetranychus ulmi (Koch) spielen können, obwohl die Beurteilung der. Ergebnisse nach der Anwendung von Chemikalien und in Gegenwart räuberischer Insekten oft erschwert ist. Um diese Komplikationen auszuschalten, wurden Versuche in einem Insektarium durchgeführt, wo die Wirkung von Typhlodromus auf M. ulmi in Abwesenheit anderer Beute geschätzt werden kann.Wenn Ausgangszahlen von 5, 25 oder 50 M. ulmi-Weibchen zusammen mit 5 Weibchen von Typhlodromus tiliae Oudms. an kleine Pflanzen gesetzt wurden, blieb die M. ulmi Population während der folgenden drei Monate in jedem Fall niedrig und überstieg die durchschnittliche Dichte von einer Milbe pro Blatt oder eine Summe von 50 Milben pro Pflanze nicht. Wenn die gleichen Anzahlen von M. ulmi-Weibchen in Abwesenheit von T. tiliae angesetzt wurden, so ergab sich ein jäher Anstieg zu Populationen von über 3000 Milben je Pflanze im Zeitraum bis zu 11 Wochen und bald folgte Bronzefärbung der Blätter.Die Größe der Wirtspflanzen beeinflußte die Entwicklung der T. tiliae-Populationen ebenso wie das Ausgangsverhältnis von M. ulmi zu T. tiliae. Sowohl größere Wirtspflanzen wie höhere M. ulmi-Werte führen zu größeren T. tiliae-Zahlen.Von anderen räuberischen Arten hat Typhlodromus finlandicus (Oudms.) eine ähnliche Wirkung auf M. ulmi wie T. tiliae. Obwohl Typhlodromus umbraticus Chant sich selbst stetig vermehrte, übte er einen geringen Einfluß auf die M. ulmi-Populationen aus; ebenso wenig Typhlodromus tiliarum Oudms., welcher sich jedoch unter der gegebene Bedingungen nicht gut entwickelte. Auch Phytoseius macropilis (Banks) zeigte nur langsames Wachstum und hatte wenig Wirkung auf die M. ulmi-Population.So wurde nachgewiesen, daß T. tiliae unter etwas begrenzten Bedingungen und in Abwesenheit irgendeiner anderen Beute fähig ist, Populationen von M. ulmi auf einer relativ niedrigen und gleichbleibenden Dichte zu halten, woraus sich eine Stütze für die Theorie ergibt, daß das auch unter Obstgartenverhältnissen zutrifft.

     

Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in Ceratocystis ulmi

We studied the inoculum size effect in Ceratocystis ulmi, the dimorphic fungus that causes Dutch elm disease. In a defined glucose-proline-salts medium, cells develop as budding yeasts when inoculated at ≥10⁶ spores per ml and as mycelia when inoculated at <10⁶ spores per ml. The inoculum size effect was not influenced by inoculum spore type, age of the spores, temperature, pH, oxygen availability, trace metals, sulfur source, phosphorous source, or the concentration of glucose or proline. Similarly, it was not influenced by added adenosine, reducing agents, methyl donors, amino sugars, fatty acids, or carbon dioxide. Instead, growing cells excreted an unknown quorum-sensing factor that caused a morphological shift from mycelia to budding yeasts. This yeast-promoting effect is abolished if it is extracted with an organic solvent such as ethyl acetate. The quorum-sensing activity acquired by the organic solvent could be added back to fresh medium in a dose-dependent fashion. The quorum-sensing activity in C. ulmi spent medium was specific for C. ulmi and had no effect on the dimorphic fungus Candida albicans or the photomorphogenic fungus Penicillium isariaeforme. In addition, farnesol, the quorum-sensing molecule produced by C. albicans, did not inhibit mycelial development of C. ulmi when present at concentrations of up to 100 μM. We conclude that the inoculum size effect is a manifestation of a quorum-sensing system that is mediated by an excreted extracellular molecule, and we suggest that quorum sensing is a general phenomenon in dimorphic fungi.     

Analysis of the Italian Dutch Elm Disease Fungal Population

Sixty‐two Ophiostoma ulmi sensu lato strains have been collected from symptomatic trees in seven areas of Central Italy. Isolates were compared with 10 reference strains, belonging to the species O. ulmi and to the two subspecies of O. novo‐ulmi, in order to establish the genetic variability within the Italian population of this fungal pathogen. The structure of the population has been analysed by means of morpho‐physiological features and of the direct amplification of minisatellite‐region DNA polymerase chain reaction (DAMD‐PCR) by using the M13 core sequence. The DNA profiles have been compared with taxonomic parameters (growth rate, culture aspect and fertility barriers). Taxa could thus be well separated. None of the isolates collected was recognized as O. ulmi. Isolates assigned to the two subspecies of O. novo‐ulmi (novo ulmi and americana) by means of the fertility test, showed short genetic distances with the respective reference strains and they constituted subgroups according to their geographical origin. The high level of variation detected indicates a postepidemic situation in Italy. Some inconsistency was found within the subspecies clusters. Several isolates, assigned to subspecies americana using fertility test, were in the novo‐ulmi cluster and vice versa. A possible explanation is that these isolates are americana–novo‐ulmi hybrids.     

CONIDIOGENESIS IN CERATOCYSTIS ULMI,CERATOCYSTIS PICEAE,AND GRAPHIUM PENICILLIOIDES

Conidiogenous cells of both the synnematous and mononematous conidiophores in Ceratocystis ulmi and C. piceae develop by sympodial proliferation. Holoblastic conidia are produced on nodules or short denticles from the synnematous conidiogenous cells and on well-defined denticles from the mononematous conidiogenous cells. Graphium penicillioides is characterized by percurrent proliferation of the conidiogenous cells and the conidia are holoblastic and annellidic. A comparison of the type material of G. penicillioides with the lectotype specimen of C. piceae indicates that G. penicillioides is not the conidial state of C. piceae. The method of conidial development in C. ulmi and C. piceae is distinct from that of G. penicillioides, the lectotype species of Graphium; these conidial states are, therefore, placed in a new hyphomycete genus, Pesotum, as the Pesotum state of Ceratocystis ulmi and C. piceae, respectively.     

Biological control of Dutch elm disease by Pseudomonas species

Antagonism of a selected group of bacteria against Ophiostoma (=Ceratocystis) ulmi, the Dutch elm disease pathogen, was determined on agar media. Four promising bacterial isolates, all fluorescent Pseudomonads, were used for field experiments and for further tests on in vitro antagonism against several fungi. It was shown that only slight differences existed in antagonism against different O. ulmi isolates. Also Ceratocystis fagacearum and C. fimbriata were inhibited similarly to O. ulmi. In field experiments bacteria were applied to elm trees by low pressure injection or by injection with a specially developed ‘gouge-pistol’. Elms treated only with bacteria remained healthy throughout two growing seasons. Elms inoculated with O. ulmi developed severe Dutch elm disease symptoms. Trees, inoculated first with O. ulmi and treated subsequently with bacteria also developed severe Dutch elm disease symptoms. Trees treated with bacteria first and inoculated subsequently with O. ulmi showed significantly fewer symptoms, especially those where treatments were carried out with the gouge-pistol.     

DEVELOPMENT OF THE ASCOCARP OF CERATOCYSTIS ULMI

Rosinski , Martin A. (U. Maine, Orono.) Development of the ascocarp of Ceratocystis ulmi. Amer. Jour. Bot. 48(4): 285–293. Illus. 1961.—A study of the development of the perithecium of Ceratocystis ulmi was conducted using classic histological techniques. This study revealed the presence of a singular combination of primitive and advanced characteristics. The perithecium possesses a simple centrum made up only of ascogenous hyphae and small, spherical asci, but croziers are formed prior to ascus formation, and the ascogenous hyphae are arranged in a hymenium. Since development of C. ulmi compares closely with most other accounts of development in other members of the genus Ceratocystis, it appears that Ceratocystis is a good taxon. In addition, because of its intermediate nature and because Ceratocystis is the type genus of the family Ophiostomataceae, this family should be placed in a separate order, the Ophiostomatales.     

Integrated control of apple pests in New Zealand 6. Incidence of European red mite,Panonychus ulmi (Koch), and its predators

Mite relationships were investigated in an orchard near Nelson from 1962 to 1974 in conjunction with a long‐term experiment into integrated control of apple pests. From 42% to 57% of the total winter eggs of Panonychus ulmi hatched over a period of 48–53 days from the last week of September until mid November. The main hatch—from 10% to 90%—took 19–20 days. Five generations occurred in each season, from October to March, although some females laid winter eggs from mid January if leaf condition became poor. Insect predators found were Stethorus bifidus, Sejanus albisignata, Arthrocnodax sp., and Xylapothrips sp. The mite Typhlodromus pyri (Phytoseiidae), the most important predator, had up to 5 generations from October to March; some females ceased to feed or oviposit from mid February onwards, and took on the characteristics of the overwintering diapause phase. With the integrated programme based on ryania, a favourable balance of P. ulmi and T. pyri was established after an initial outbreak of P. ulmi in the 1st or 2nd season, and was maintained until 1970–71, when P. ulmi outbreaks occurred on several cultivars in a season marked by hot, dry conditions in November and December. Natural control was restored the next year except on cy. Delicious, where P. ulmi remained in greater numbers for several years. With a complete commercial spray programme from 1962 to 1967 P. ulmi became resistant to all acaricides used; with the regular azinphos‐methyl programme and no acaricides in 1967–68 a population of T. pyri resistant to this insecticide emerged. The approximately × 10 resistance level did not increase appreciably, so the azinphos‐methyl dosage was reduced from 0.05% to 0.025% to allow greater survival. Several hot, dry summers followed, and commercial control of P. ulmi by T. pyri did not occur until 1973–74. A programme of low‐dosage carbaryl allowed T. pyri and S. albisignata to establish in fair numbers, but control of P. ulmi was not achieved. Experiments with mineral oils and cyhexatin have shown them to have some potential for inclusion against P. ulmi in future integrated programmes.     

Feeding by Scolytus bark beetles to test for differently susceptible elm varieties

Dutch elm disease (DED), caused by the fungi Ophiostoma ulmi and O. novo‐ulmi, has reduced elm populations severely in Europe and North America. Breeding programmes are in action to find less susceptible elm varieties suitable for re‐establishing elm stands. Bark beetles, mainly Scolytus spp., are the only known natural vectors of DED. During twig feeding, beetles transfer Ophiostoma spores to healthy elms. Thus, less palatable elms should run a lower risk of DED infections. In feeding preference bioassays, we offered twigs from elms exhibiting different degree of susceptibility to O. novo‐ulmi, together with non‐host trees to Scolytus beetles. Scolytus multistriatus preferred wych elm, Ulmus glabra, to 100% in two‐choice tests, whereas S. laevis did not discriminate between a tolerant and a susceptible variety of field elm, U. minor. We suggest that the feeding assay is useful as a low‐tech method in breeding programmes for evaluating the suitability of promising elm genotypes to vector insects.     

Functional response of Euseius finlandicus and Amblyseius andersoni to Panonychus ulmi on apple and peach leaves in the laboratory

The functional response of adult females of the predatory mites Euseius (Amblyseius) finlandicus and Amblyseius andersoni to larvae and adult females of the fruit tree red spider mite Panonychus ulmi was determined on apple and peach leaf disks in the laboratory at 25°C and 16:8 (L:D). For adult females of P. ulmi the predation efficiency of E. finlandicus was higher on peach than on apple, whereas that of A. andersoni was higher on apple than on peach. Efficiency of predation on larvae of P. ulmi by either predator did not differ significantly between apple and peach. On both plants, A. andersoni had a higher predation rate than E. finlandicus on larvae of P. ulmi. It is concluded that in the laboratory the host plant has a substantial effect on predation efficiency of A. andersoni and E. finlandicus when they preyed on adults but not when they preyed on larvae of P. ulmi.     

Dynamics ofPanonychus ulmi andTyphlodromus pyri: factors contributing to persistence

We addressed the question of persistence of predator and prey in a biological control system by examining temporal patterns ofPanonychus ulmi (Koch) and its predator,Typhlodromus pyri Scheuten at two geographic locations and at two spatial scales. At the scale of an orchard, bothP. ulmi andT. pyri were persistent over the time frame of 6 years. At the scale of an individual tree,T. pyri appeared to be more persistent than its prey,P. ulmi. We used a simulation model of single populations ofP. ulmi andT. pyri to determine which of several aspects of the biology of the two species could contribute to such a pattern. Spatial incongruity between predator and prey was essential for persistence of both species. The generalist food habit ofT. pyri probably contributes to the persistence ofT. pyri on individual trees, and may cause occasional extinction ofP. ulmi at this spatial scale. The presence of alternate food is likely an essential element for successful biological control in this system. Cannibalism byT. pyri results in higher prey densities, that is, it is detrimental to the biological control ofP. ulmi, but has no effect on the relative persistence of the two species.     

The Transfer of Typhlodromus pyri on Grape Leaves for Biological Control of Panonychus ulmi (Acari: Phytoseiidae, Tetranychidae) in Vineyards in Ontario, Canada

The phytophagous mite Panonychus ulmi Koch has become a significant problem in Ontario vineyards. We attempted to introduce and establish populations of the predatory mite Typhlodromus pyri Scheuten for P. ulmi biological control. Grape leaves were transferred from a vineyard containing T. pyri in early summer 1998, by picking leaves from a donor vineyard and attaching them onto leaves in the release vineyard where T. pyri were extremely rare. Two release treatments were used with rates of 8.5 (1×) and 25.5 (3×) mobiles per vine. In the first season, T. pyri established in similar densities in both release treatments, which were significantly higher than controls. However, there were no differences among treatments in P. ulmi densities in 1998 as a result of predator release. During summer 1999, significantly fewer P. ulmi mite-days were observed in release plots compared to the control. Amblyseius fallacis (Garman) was common throughout the release vineyard in 1998 and in 1999, but appeared on the vines too late in the season to maintain low P. ulmi densities. T. pyri appeared to out-compete A. fallacis in 1999 because A. fallacis densities were significantly lower in plots where T. pyri had been released than in control plots. We conclude that T. pyri can be effective for P. ulmi biological control in Ontario vineyards and may be introduced by transferring leaves. In Europe, transferring prunings has been the standard method of inoculating T. pyri into new vineyards. Here we show that transferring leaves is another practical method.     

Integrated control of apple pests in New Zealand 16. Progress with integrated control of European red mite

Abstract

Studies were made in an orchard near Nelson from 1974 to 1978 on integrated control of Panonychus ulmi (Koch) using the organophosphate-resistant predator Typhlodromus pyri Scheuten. Correct timing of selective acaricides during the October-January period is essential, and can be achieved by close observation of P. ulmi development. When the majority of winter eggs have hatched (late October to early November), or the majority of the summer eggs laid by the first generation (very late November to early or mid December), there is a short period when a maximum of immature mites occurs. This is before many adult females have matured, and is the most effective time for application of an acaricide such as cyhexatin. The development of P. ulmi, in particular the incubation period of the summer eggs, is controlled by temperature, and in seasons when mean temperatures are well below average in November and December the delayed mite development makes accurate timing of the December spray more difficult. Other factors which also affect mite development are the use of dormant oil, cultivar, and orchard aspect and shelter.

On the block of trees where no acaricides were used since 1968, although regular applications of azinphos-methyl were made, T. pyri maintained P. ulmi at acceptable levels over the 5 years 1973-78, a maximum of P. ulmi occurring each season in January with the third generation. In three seasons the peak was well below the threshold for commercial spraying; in two seasons this threshold was exceeded, but despite the absence of acaricides P. ulmi numbers soon fell.

A further experiment demonstrated the utility of mineral oil and cyhexatin as selective acaricides in integrated programmes. Cycloprate, tricyclotin, and fenbutatin oxide were shown to be as effective and selective as cyhexatin, a single application in early December giving good survival of T. pyri and season-long control of P. ulmi. In another experiment propargite was as effective and selective as cyhexatin; amitraz was more toxic to T. pyri and less persistent against P. ulmi, and resulted in a poor P. ulmi : T. pyri ratio; and chlorpyrifos, though not more toxic than cyhexatin to T. pyri, was less effective against P. ulmi.

Mancozeb, and dinocap + mancozeb, used in a summer-long programme of 12 treatments, were more toxic to T. pyri than were binapacryl or dinocap alone.     

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

The effect of pyrethroid lambdacyhalothrin applications on the spatial distribution of phytophagous and predatory mites in apple orchards

The spatial distribution of three phytophagous mites,Panonychus ulmi (Koch),Tetranychus urticae Koch andAculus schlechtendali (Nalepa), and two predacious mites,Zetzellia mali (Ewing) andAmblyseius fallacis (Garman), and the effect of pyrethroid lambdacyhalothrin applications on mite spatial dispersion were investigated over a 3-year period in an apple orchard in Ontario. The index of dispersion and the slope of Taylor's power law were used to evaluate dispersion patterns of mites. Panonychus ulmi showed that between-tree spatial variation decreased with an increase of population densities, whereas between-leaf variation increased with population densities. With all other four species it appeared that between-tree variation is much greater than between-leaf variation at all field population density levels. The values ofb by Taylor's power law suggested that all five species of mites are aggregated, but that in generalP. ulmi andT. urticae (b=1.427–1.872) are more aggregated than their predators (b=1.254–1.393). Taylor's regression technique suggests that pyrethroid applications causedP. ulmi, T. urticae, Z. mali andA. fallacis to be less aggregated whileA. schlechtendali was more aggregated. The impact of changes in mite spatial distribution following pyrethroid applications on sampling plans is discussed.     

Loop-mediated isothermal amplification for rapid detection of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> spores

A loop-mediated isothermal amplification (LAMP) assay system was employed for detecting Bacillus anthracis spores in pure cultures as well as in various simulated powder samples. The specificity of the designed LAMP primer sets was validated by assaying 13 B. anthracis strains and 33 non-B. anthracis species. The detection limits of the LAMP assay were 10 spores/tube for pure cultures and 100 spores/2 mg powder for simulated powder samples. The results show that the LAMP protocol is a promising method for detecting B. anthracis.     

The effect of an alternative food supply on the relationship between two Typhlodromus species and Panonychus ulmi (Koch) (Acarina)

Using seedling plants in an insectary, populations were recorded of two species of Typhlodromus (Phytoseiidae) when provided with known numbers of Panonychus ulmi (Koch) (Tetranychidae) and/or large numbers of Aculus fockeui (Nal.) (Eriophyidae). T. pyri Scheut. and T. finlandicus (Oudms.) both maintained P. ulmi at a low level compared with populations where Typhlodromus was not present, and whether or not A. fockeui was also available. Both species of Typhlodromus increased more rapidly when provided with A. fockeui as well as P. ulmi, and under these conditions T. pyri reduced P. ulmi to a lower level than where A. fockeui was not provided.

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Zusammenfassung Der Versuch sollte zeigen, ob die Raubmilbe Typhlodromus spp. (Phytoseiidae) die Spinnmilbe Panonychus ulmi (Koch) (Tetranychidae) weiter niederhalten könnte, wenn gleichzeitig eine andere günstige Beute angeboten würde. Es wurden getopfte Prunus-Sämlinge in einem Gewächshaus benutzt, die zur Hälfte mit einer großen Anzahl von Aculus fockeui (Nal.) (Eriophyidae) infiziert wurden, bevor bekannte Anzahlen von P. ulmi und Typhlodromus pyri Scheut. oder T. finlandicus (Oudms.) auf ihnen angesetzt wurden. Während eines Zeitraumes von 8 Wochen wurden in annähernd wöchentlichen Abständen einzelne, als Wiederholungen angesetzte Pflanzen jeder Versuchsvariante entnommen und ihre Milbenpopulationen ausgezählt. Bei Abwesenheit der Raubmilben stieg die P. ulmi-Population schneller auf Pflanzen an, die von A. fockeui frei, als auf solchen, auf denen sie vorhanden waren. Sowohl T. pyri wie T. finlandicus hielten P. ulmi auf einem vergleichsweise niedrigen Niveau, gleichgültig ob A. fockeui gleichfalls vorhanden war oder nicht. Beide Typhlodromus-Arten vermehrten sich rascher, wenn ihnen neben P. ulmi zusätzlich A. fockeui zur Verfügung stand, und unter diesen Bedingungen reduzierte T. pyri die P. ulmi-Population auf ein niedrigeres Niveau als bei Abwesenheit von A. fockeui. Diese Ergebnisse zeigen, daß die Gegenwart von A. fockeui die Bekämpfung von P. ulmi durch Typhlodromus spp. nicht beeinträchtigt, daß sie aber eine günstige Beute darstellt und eine erhöhte Vermehrungsrate der Raubmilben bewirkt.

     

Resistance to clofentezine and hexythiazox inPanonychus ulmi from apples in Australia

The ovicides clofentezine and hexythiazox have been used in Australia againstPanonychus ulmi (Koch) since 1984 and 1987, respectively. Failure of clofentezine in the field againstP. ulmi was first reported in early 1988. In 1988–89, laboratory bioassays were carried out against susceptible reference strains, and discriminating doses for clofentezine and hexythiazox established. Suspected resistant strains were tested, as were randomly collected strains.Panonychus ulmi from 23 apple orchards in eastern Australia were found to be resistant to clofentezine, and 19 of these strains were also resistant to hexythiazox. In all cases, a total of four or more ovicide sprays had been used. Of 15 samples ofP. ulmi from blocks where less than four ovicides had been used, none were resistant to either clofentezine or hexythiazox. A survey of orchards in the Orange district of New South Wales found that 30% had received four or more ovicide sprays and were likely to experience resistance problems. The control problems with clofentezine and hexythiazox means the few remaining acaricides will be subject to extreme selection pressure.     

Decontamination of a hard surface contaminated with Bacillus anthracisΔSterne and B. anthracis Ames spores using electrochemically generated liquid-phase chlorine dioxide (eClO2)

Aims: To evaluate the inactivation of Bacillus anthracisΔSterne and Ames spores using electrochemically generated liquid‐phase chlorine dioxide (eClO₂) and compare two sporulation and decontamination methods with regard to cost, safety and technical constraints. Methods and Results: Spores were prepared via agar and broth methods and subsequently inoculated and dried onto clean, autoclave‐sterilized glass coupons. Bacillus anthracis spore inactivation efficacy was evaluated using the modified three‐step method (AOAC 2008.05) and a single‐tube extraction method. Spores (7·0 ± 0·5 logs) were inactivated within 1 min at room temperature using freshly prepared eClO₂. Bacillus anthracisΔSterne spores decreased in size after eClO₂ treatment as measured using a Beckman Coulter Multisizer. Conclusions: eClO₂ saturation of a hard surface was an effective B. anthracis sporicide. Broth sporulation and the single‐tube extraction method required less time and fewer steps, yielded a higher percentage of phase‐bright spores and showed higher spore recovery efficiency compared with AOAC 2008.05, making it more amenable to biosafety level 3 (BSL3) testing of virulent spores. Significance and Impact of the Study: Two test methods demonstrated the sporicidal efficacy of eClO₂. A new single‐tube extraction test protocol for decontaminants was introduced.