An endogenous rhythm of trap formation in the nematophagous fungus Arthrobotrys oligospora

In the predacious fungus Arthrobotrys oligospora Fres., the number and distribution of traps formed after the addition of living nematodes to the colonies were determined. At 21°C the traps were formed periodically; the mean period was 42.3±0.8 h. The periodicity was independent of light-dark (LD) cycles of 24 h (10:14). Temperature influenced the hyphal elongation but did not affect the periodic trap formation; at lower temperatures the peaks of trap formation were close together, showing partial overlapping. Induction of rhythmic mycelial growth and conidiation by chemical means was effective only in LD-cycles. The latter diurnal rhythm was weakly correlated with the trap formation and did not affect the endogenous period of approximately 42 h.Abbreviations LD light-dark - DD continuous darkness - LNM low-nutrient medium - CMA corn meal agar     

Carbon sources,growth, sclerotium formation and carbohydrate composition of Sclerotinia sclerotiorum

Summary Sclerotinia sclerotiorum (Lib.) D By. was grown in stationary liquid mineral-salts medium, pH 4.3, containing various carbon sources and the weight of mycelia and sclerotia was determined at regular intervals. When grown on various glucose concentrations (0–24 g of C/l), more sclerotia were produced at 8–12 g of C/l. Sclerotia were not usually formed in shake cultures. The ability of the fungus to use other carbon sources for growth and sclerotium formation was tested at 12 g of C/l in the stationary mineral-salts medium. The highest weights of mycelia and sclerotia occurred with raffinose, sucrose, maltose, lactose, d-mannose, d-glucose, d-fructose or l-arabinose. Good growth but decreased sclerotium production were found on cellobiose and d-xylose. Reduced or poor growth, a long lag period and few or no sclerotia occurred on trehalose, melibiose, l-sorbose, l-rhamnose, d-ribose, d-arabinose, l-xylose or 8 polyols. No growth was observed with erythritol or i-inositol. A combination of glucose plus trehalose or polyols resulted in increased growth and the formation of sclerotia. Organic acids supported little or no growth and no sclerotia were produced. Generally culture filtrates which supported growth and formation of sclerotia became acid (about pH 3.5). The pH of the culture filtrate usually increased slowly during the growth period when the fungus grew poorly and no sclerotia were formed. The alcoholsoluble sugars and polyols present in culture filtrates, mycelia and sclerotia were determined by paper and thin-layer chromatography. Regardless of the carbon source, mannitol was usually present in culture filtrates. The occurrence of other compounds in the filtrates depended on the carbon source. Trehalose, mannitol and usually small quantities of glucose or fructose were present in mycelia and sclerotia from all carbon sources. Galactitol or pentitols occurred in mycelia and sclerotia when the fungus grew on galactose and oligosaccharides containing galactose or the corresponding pentose, sugars. Acid hydrolyzates of the alcohol-insoluble fraction of mycelia or sclerotia contained glucose, smaller amounts of galactose and mannose and traces of ribose and rhamnose.     

Biocompatibility of Steinernema glaseri with a nematode trapping fungus Arthrobotrys superba on different nutrient media

Steinernema glaseri is known to be the most efficacious biocontrol agent against many soil insects belonging to various orders and of diverse habitats. In nature the efficacy of such entomogenous nematodes may be challenged by many micro pathogens. Keeping this in view, the biocompatibility of Steinernema glaseri with a nematophagous fungus Arthrobotrys superba was assessed on eight different nutrient media. The predatory activity of A. superba was evaluated in terms of trap formation, conidiophore formation, and number of adhesive cells formed in the presence and absence of nematodes. The fungus failed to form any trap on any of the culture media in the absence of nematodes. However, in the presence of nematodes, the trap formation by the A. superba was increased and found to be maximum on LNM (low nutrient mineral salt) medium and minimum at WA (water agar) of all the culture media tested. In the present investigation, the effect of CMA (2% and 5%) was not considerable; however, gradually higher dilutions of CMA induced greater number of traps. This less pronounced effect might be due to nutrient richness because low nutrient levels were found to be necessary for the formation of conidial traps. The number of conidiophores decreased with increase in dilution of Corn Meal Agar from 5% to 2%. Higher number of chlamydospores was observed in phenylalanine treated medium which indicates the inhibiting effect of phenylalanine on the growth of A. superba. Our results suggest that richness of soil has pronounced effect on the predatory activity of nematophagous fungus, Arthrobotrys superba which may further affect the biopotential of entomogenous nematode Steinernema glaseri, therefore, care should be taken while releasing entomogenous nematodes in an agroecosystem for managing various insect pests in a more efficient manner.     

Isolation,identification and characterisation of the nematophagous fungus Arthrobotrys thaumasia (Monacrosporium thaumasium) from China

The objective of this study is to select a native isolate of Arthrobotrys thaumasia (Monacrosporium thaumasium), a nematophagous fungus that shows potential for use in biocontrol programmes for ruminants. First, we looked for native isolates of A. thaumasia and characterised them using light microscopy and molecular markers. Then, we determined the effect of temperature, pH and nutrition on the growth rate and trap formation of a representative isolate. Of the 1532 samples of different types related to sheep and cattle, 11 isolates of A. thaumasia were isolated, and their occurrence frequency in the samples was 0.71% (11/1532). We sequenced the rDNA internal transcribed spacer of isolate NBS005, submitted it to GenBank (ID: KX640093) and then sequenced it using BLAST. The NBS005 could not grow at 37.5°C but could grow from 11°C to 35°C, and it exhibited its optimum growth at 30°C on 1% corn meal agar (CMA). Over 4 days, the fungus did not grow in the pH interval from 1 to 3 or from 13 to 14 but did grow in the pH interval from 4 to 12 and exhibited its optimum growth between pH 9 and 10 on 2% CMA. The factors responsible for the trap formation of NBS005 in liquid culture were identified. Trap formation was induced only by contact with L3 lysate. The concentrations of sucrose, ammonium chloride and tryptone of 0.4, 0.2 and 0.2%, respectively, promoted trap formation, and there were higher numbers of trapping nets in 2% wheat bran liquid medium containing L3 lysate.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Oxalic Acid Production and Mycelial Biomass Yield of Sclerotinia minor for the Formulation Enhancement of a Granular Turf Bioherbicide

The fungus Sclerotinia minor is presently under development in this laboratory as a granular bioherbicide for broadleaf weed species. With a view to enhancing the virulence of the fungus, the effect of increasing endogenous oxalic acid concentration through modification of the growth conditions was investigated. S. minor was grown in 125 ml of eight different liquid culture media in shake flasks incubated at 20°C for 7 days. The final pH, mycelial dry weight, and oxalic acid content of the spent growth media were determined and the virulence of S. minor grown on each solid culture medium was screened on detached dandelion leaves. A 330% increase in oxalic acid was obtained plus 56 mM of sodium succinate to Modified Richard's solution (MRS) as compared to MRS alone. A concomitant increase in virulence of 218% was expressed as increased lesion diameter.     

Almond seed wasp (Hymenoptera: Eurytomidae) sex pheromone: effect of trap type, trap position, blend ratio and time of the day on male attraction

Field trials were carried out in order to evaluate some of the factors affecting male response of the almond seed wasp, Eurytoma amygdali Enderlein, to traps baited with the alkadienes (Z,Z)-6,9-tricosadiene and (Z,Z)-6,9-pentacosadiene, the male attractant of this species. The trials were conducted in Latomia, Microthivae, Kanalia and Sykourion (Thessaly, Central Greece) during the 2004 growing season. In all regions, captures in traps baited with a 7:3 ratio of the above blend significantly and positively correlated with male emergence from infested almonds that had been placed in cages in the experimental fields. Traps baited with the 7:3 pheromone ratio were significantly more effective in capturing males than traps baited with the 1:1 ratio, supporting the hypothesis that (Z,Z)-6,9-tricosadiene is the main component of this attractant. Of four trap types tested, Rebel and Lasiotraps attracted significantly more males than Delta and Funnel traps. Height had no effect on trap efficiency; however, males were captured earlier in the day in traps placed 2.3 m above grown than in traps placed 1.3 m above grown. Most male activity was recorded between 10 and 12 h, but captures were continuously recorded during the entire monitoring interval from 6 to 17 h.     

Pheromone Traps for the Dissemination of an Entomopathogen by the Damson-hop Aphid Phorodon humuli

Two types of sex pheromone-emitting trap, a simple louvred trap and a commercially-produced wasp trap (Waspy®) designed or modified as delivery systems for entomopathogens, were tested using an isolate of the pathogen Verticillium lecanii . Their efficiencies for capturing winged autumn females (gynoparae) and males of Phorodon humuli in a hop garden and a plum orchard were compared with each other and with catches by yellow-painted water traps. Gynoparae rarely entered either type of pathogen dissemination trap. Males only entered traps that released (1 R , S ,4a R , 7 S , 7a S )-nepetalactol, the sex pheromone of P. humuli . In the hop garden, approximately five times as many males entered the modified Waspy® as entered the louvred traps, whereas in the plum orchard similar numbers of males were caught in both traps. On average, males visited the modified Waspy® trap for 34.1 ±7.9 (SE) min. Males spent about 50% of their time in the arena where the entomopathogen would normally be present and a further 20% of the time on the inside of the arena lid alongside the pheromone source. Out of a sample of 16 live aphids removed from a trap dispensing V. lecanii , 9 died from infection by the pathogen and 5 of the 16 initiated colonies of the fungus after they walked on a sterile agar plate for five minutes. None of the 15 aphids collected from traps without V. lecanii became infected or initiated colonies of the fungus on agar plates.     

Carbohydrates,invertase activity,growth and dimorphism inSporisorium reilianum

Sporisorium reilianum, the fungus that causes sorghum head smut, was grown with sucrose, lactose, trehalose or raffinose in liquid suspension or on a solid medium. Liquid culture media were analyzed for hydrolysis products of these carbohydrates to determine extracellular enzyme activity of the fungus. Increased amounts of glucose and fructose in the culture medium ofS. reilianum grown with sucrose or raffinose indicated that invertase (-fructofuranosidase, 3.2.1.26) activity was present. No evidence of extracellular galactosidase or trehalase activity was found. Enhanced sporidial colony formation on carbohydrates that can be hydrolyzed to hexoses, and specific forms of mycelial growth on lactose, trehalose or on a carbohydrate-deficient medium might suggest that mycelial growth is a way of foraging for food sources. However, the rapid and profuse mycelial growth on the host cell wall glycoprotein appears to be in response to abundant food supply (probably of a different type). Therefore availability of different kinds of carbon sources in the environment of the growing fungus might determine dimorphism and associated pathogenesis byS. reilianum.Technical Article No: 30699 from the Texas Agricultural Experiment Station.     

Micropropagation of the Thai orchid <Emphasis Type="Italic">Grammatophyllum speciosum</Emphasis> blume

Protocorm-like bodies (PLBs) were induced from shoot tips of Grammatophyllum speciosum, a Thai orchid. The highest frequency of PLBs (93%) were observed on explants incubated on 1/2-Murashige and Skoog (MS) liquid medium containing 2% (w/v) sucrose without any plant growth regulators (PGRs). Tests with different carbon sources compared to sucrose revealed that maltose promoted the highest relative growth of G. speciosum PLBs (7-fold increase), while trehalose and sucrose yielded 5-fold and 4-fold increases, respectively. In 1/2 MS liquid medium, addition of 15 mg/l of chitosan promoted a 7-fold increase in PLB growth while 25 mg/l promoted a 4-fold increase. However, the relative growth rate in solid culture was significantly lower than that in liquid culture. In addition, chitosan supplementation in solid medium promoted shoot formation but not rooting. Plantlet regeneration was induced using a combination of NAA and BA supplementation in 1/2 MS solid medium with optimum induction shoot and root formation at 2.0 mg/l NAA and 1.0 mg/l BA. Using this protocol, approximately 8 months was required to obtain a hundred plantlets from one shoot tip. The plantlets showed no changes in ploidy when tested by flow cytometry.     

Impact of trap design, windbreaks, and weather on captures of European corn borer (Lepidoptera: Crambidae) in pheromone-baited traps

Pheromone-baited traps are often used in ecological studies of the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae). However, differences in trap captures may be confounded by trap design, trap location relative to a windbreak, and changes in local weather. The objectives of this experiment were, first, to examine differences in 0. nubilalis adult (moth) captures among the Intercept wing trap, the Intercept bucket/funnel UNI trap, and the Hartstack wire-mesh, 75-cm-diameter cone trap (large metal cone trap) as well as among three cone trap designs. Second, we examined the influence of the location of the large metal cone trap relative to a windbreak on the number of moths captured. Third, we examined the relationship between nightly mean air temperature, relative humidity, wind speed, precipitation, and the number of moths captured in large metal cone traps. The number of moths captured was significantly influenced by trap design, with large metal cone traps capturing the most moths. Wing and bucket traps were ineffective. Differences among trap captures were significant among trap locations relative to a windbreak. Under strong (>14 kph) or moderate (7 <14 kph) wind speeds, traps located leeward of the windbreak captured the most moths, but when wind speeds were light (<7 kph), traps not associated with windbreaks captured the most moths. The multiple regression model fitted to the relationship between number of moths captured per Julian date and nightly weather patterns was significant. Nightly mean air temperature was the most influential parameter in the model, and its relationship with moth capture was positive.     

Evaluation of an oviposition‐stimulating kairomone for the yellow fever mosquito,Aedes aegypti,in Recife,Brazil

A synthetic mixture of an oviposition‐stimulating kairomone for the yellow fever mosquito, Aedes aegypti, comprising of 83% tetradecanoic acid, 16% nonanoic acid and 1% tetradecanoic acid methyl ester (NTT, in short) was tested in a dengue endemic area in Recife, Brazil. Gravid female mosquitoes confined to a cage under semi‐field conditions deposited significantly higher numbers of eggs in traps baited with NTT at doses ranging from 0.6 to 600 ng/μl than in control (water) traps. When tested in homes, egg‐laying in traps baited with 60 ng NTT/μl (final concentration in trap, ≈3.33 ng/ml) and in control traps was not significantly different, but egg deposited in traps with lower dosage (6 ng NTT/μl; final concentration in trap, ≈0.33 ng/ml) was significantly higher than in control traps. In subsequent trials, the numbers of eggs laid in traps baited with 0.6 ng NTT/μl (final concentration in trap, ≈0.033 ng/ml) were not significantly different from the numbers deposited in trap loaded with 6 ng NTT/μl. Egg‐laying was significantly higher in these treatments than in control traps.     

Effect of activated charcoal and pruning of the taproot on the in vitro mycorrhization of Hevea brasiliensis Müll. Arg.

In vitro mycorrhization of Hevea brasiliensis under autotrophic culture conditions is a promising methodology to produce plantlets adapted to overcome stresses during acclimatization. However, to succeed in the in vitro production of mycorrhizal plantlets, root production and subsequent colonization by the mycorrhizal fungus need to be optimized. Plantlets of H. brasiliensis clone PB 260 were grown in contact with the extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833. Addition of activated charcoal to the medium and pruning of the taproot were evaluated for their effects on root growth and colonization. None of the treatments stimulated the early formation of new roots. However, total root length, total root colonization, and production of arbuscules and intraradical spores/vesicles were significantly higher in plantlets grown in the presence of activated charcoal (especially after 13 wk of culture). In contrast, total root colonization was significantly lower in the pruned plantlets, while total root length and arbuscule formation were not affected. None of the treatments affected activities of succinate dehydrogenase and alkaline phosphatase measured in the extraradical mycelium of the fungus. It appeared that the addition of activated charcoal to the culture medium favored root growth and mycorrhization of rubber plantlets under in vitro culture conditions, while taproot pruning did not favor these parameters.     

Abscisic acid metabolism in Ceratocystis coerulescens

The fungus Ceratocystis coerulescens Bakshi (strain RWD 390) has been shown to produce the plant hormone, abscisic acid (ABA). The production of ABA in defined liquid medium during a culture period of 50 days was measured by gas-liquid chromatography. A considerable accumulation of ABA occurred in the stationary phase. Maximum ABA contents were 3.5 ng ml⁻¹ in culture media and 218 ng (g dry weight)⁻¹ in mycelial extracts.
The ABA-metabolizing capability of the fungus was investigated. Dihydrophaseic acid, and phaseic acid, ABA metabolites in higher plants, were not present in cultures of Ceratocystis coerulescens . When [2-¹⁴C]-ABA was fed to the fungus, the formation of [2-¹⁴C]- 2-trans , 4- trans -ABA and a second metabolite, less polar than ABA, was observed. This suggests a different metabolic pathway of ABA in the fungus.     

光周期对飞虱虫疠霉离体培养的生物量及其产孢节律的影响

飞虱虫疠霉（Ｐａｎｄｏｒａｄｅｌｐｈａｃｉｓ）是侵染飞虱和叶蝉等重要作物害虫的昆虫病原真菌。通过在液体及平板培养中研究光周期对菌丝及菌落生长量、产孢节律和产孢量的影响,作者发现光照对液培菌丝的营养生长无显著影响,但长光照液培菌丝的产孢延迟并且产孢量减少,而全黑暗液培菌丝产孢早且产孢量大。但是,光照明显促进全黑暗下液培菌丝的产孢。在平板培养中,长光照能促进菌落生长和产孢;半光照半黑暗虽有利菌落生长,但产孢量很低;光照９ｈ和１８ｈ产孢量较大,但菌落生长较小。无论液体还是平板培养,飞虱虫疠霉均生长良好,但建议在全黑暗条件下进行液体培养,在长光照下进行平板培养。     

Some responses of cabbage root fly (Delia brassicae) to ally I isothiocyanate and other volatile constituents of crucifers

Vapour components emanating from disrupted cauliflower, turnip, radish, wallflower and brown mustard tissue were assessed for their effects on the cabbage root fly (Delia brassicae). Of about 20 vapour components detected and separated by gas chromatography, six elicited sufficiently large electroantennal responses to warrant further testing, but of these only allyl isothiocyanate and hexyl acetate markedly affected the behaviour of gravid flies in an olfactometer. In wind-tunnel tests at a wind speed of 1–2-1-7 m/s, the numbers of females caught was increased when allyl isothiocyanate vapour was released at 32–130 mg/h but decreased at higher evaporation rates. The only effect of hexyl acetate vapour released at 40–140 mg/h was a reduction in the numbers caught at the highest concentration. In a cabbage crop, yellow water traps fitted with allyl isothiocyanate sources, each evaporating 2–3 g/day, caught 11 times as many female and seven times as many male flies as unmodified traps during the early period of the third generation but the improvement in trap efficiency later diminished. Trap efficiency was slightly reduced when the rate of evaporation of allyl isothiocyanate from a trap was decreased from 2–3 to 0–51 g/day. On fallow ground, allyl isothiocyanate improved trap performance in catching female flies by about seven-fold, but along a hedgerow adjacent to Brussels sprouts the improvement was barely two-fold. Hexyl acetate did not improve the performance of traps in a cabbage crop.     

Errors in determining the flow rate of Hirst-type pollen traps

Standardisation of methods of pollen monitoring networks is vital for data quality. In pollen monitoring networks in Europe, the Hirst-type trap is standard. Hirst traps are calibrated with handheld rotameters. We detected a systematic error in the flow rate calibrated by these standard handheld rotameters. We measured the flow rate of 19 Hirst traps from three commercial brands during calibration but also during monitoring. We used three different rotameters supplied by the manufacturers of the traps, respectively. The actual air flow rate was measured using an electronic heat anemometer with negligible air flow resistance. After calibration to 10 l/min, the rotameter was removed, which led to a significant increase in the flow rate in the range of 10.5–17.2 l/min, a systematic error between 5 and 72%. No significant difference was found between the different commercial trap brands. The analysis revealed that the error depended on the type of the rotameter and the individual trap. The error may be explained by the additional air flow resistance of each rotameter. The total resistance of the system—trap plus rotameter—is higher during calibration when the rotameter is held on the inlet compared to the routine monitoring without the rotameter. Depending on the characteristic curve of the suction pump in the trap (fan), the air flow rate increases to values considerably higher than 10 l/min. Thus, monitoring is done under a higher flow rate than that was calibrated. In order to obtain comparable data within a monitoring network, a solution for correction of this systematic error seems advisable, preferably in cooperation with the manufacturers.     

Decolourisation of synthetic textile dyes by Phlebia tremellosa

Phlebia tremellosa decolourised eight synthetic textile dyes (200 mg l(-1)) by greater than 96% within 14 days under stationary incubation conditions. High performance liquid chromatography analysis of culture supernatants indicated that Remazol Black B was degraded by the fungus, however, complete mineralisation did not occur as a colourless organic breakdown product accumulated. Laccase activity was detectable in culture supernatants after 5 days when the fungus was grown in the presence of an artificial textile effluent, with activity reaching a maximum of 15 U l(-1) on day 14.     

A gas chromatographic investigation of the influence of different carbon sources on the production of volatile compounds by Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was cultivated aerobically on different carbon sources. The growth was measured turbidimetrically and related to the simultaneus production of volatile compounds, which was determined by a gas chromatographic head-space technique. The same culture could be analyzed many times and up to 15 components were detected in a chromatogram recorded in 18 min. The main peaks were identified and the chromatograms quantitatively evaluated by peak height measurements.All the carbon sources tested were utilized for growth even if the lag phase was prolonged on xylose and ethanol. The production of volatile compounds from the different carbon sources decreased as follows; ethanol>glucose>fructose>glycerol >xylose and succinic acid. A good carbon source for growth could be unsuitable for the formation of volatile products and vice versa.The time course production of volatile components was recorded. On each separate carbon source the formation of volatiles was correlated to growth until the end of the exponential phase of growth.An extraction procedure including the addition of an internal standard was used to determine the exact concentrations of 9 components at near optimum production. The amounts varied between 0.1–32 mg/l medium when the fungus was grown on 5% glucose.