Survival of Rhizoctonia solani AG‐1 IA,the Causal Agent of Rice Sheath Blight,under Different Environmental Conditions

The ability of Rhizoctonia solani AG‐1 IA, the causal agent of rice sheath blight, to survive in diseased rice straw and as sclerotia and mycelia was investigated. After storage for 10 months at 4°C, 25°C and non‐air‐conditioned natural room temperature (NRT, temperature range from 6°C to 35°C), sclerotia placed inside a desiccator, soaked in sterile water or immersed in wet paddy soil were viable. In contrast, only 15% of sclerotia in dry paddy soil survived. Survival of mycelia was severely affected by temperature and humidity. After 10 months in a desiccator at 4°C, 55% of mycelia samples could survive, whereas at 25°C and NRT, mycelial samples survived for only 7 and 5 months, respectively. However, mycelia stored in sterile water at constant temperatures (4°C or 25°C) survived for 10 months. A certain amount of UV radiation had no obvious effect on the survival of sclerotia or mycelia. The survival rate of the fungus in diseased rice straw stored for 16 months could reach 100% at 4°C, 50% at 25°C and 35% at NRT. The survival rates of the pathogen in diseased rice straw buried in dry, wet and flooded paddy soils after 10‐month storage at NRT were 75, 100 and 100%, respectively, indicating that soil humidity is a crucial factor for the survival of this fungus.     

Fate of Clavibacter michiganensis ssp. sepedonicus, the Causal Organism of Bacterial Ring Rot of Potato, in Weeds and Field Crops

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inoculation. About 6–8 weeks after inoculation, Cms could be detected in most crops except onion and sugar beet, in larger numbers in stems (10⁵–10⁶ cells/g of tissue) than in roots (≤10³ cells/g of tissue) in immunofluorescence cell‐staining (IF). Cms was successfully re‐isolated only from IF‐positive stem samples of maize, bush bean, broad bean, rape and pea, but not from roots. Twelve solanaceous weeds and 13 other weeds, most commonly found in potato fields in Europe, were tested in IF as hosts of Cms by stem and root inoculations. Only in Solanum rostratum, a weed present in northern America, Cms persisted in high numbers (10⁸ cells/g tissue) in stems and leaves, where it caused symptoms. In the other solanaceous weeds, Cms persisted at low numbers (approximately 10⁵ cells/g of tissue) in stems but less so in roots. The bacteria could be frequently re‐isolated from stem but not from root tissues. In 2 consecutive years, plants from 14 different weed species were collected from Cms‐contaminated potato field plots and tested for the presence of Cms by dilution plating or immunofluorescence colony‐staining (IFC), and by AmpliDet RNA, a nucleic acid‐based amplification method. Cms was detected in roots but not in stems of Elymus repens plants growing through rotten potato tubers, and in some Viola arvensis and Stellaria media plants, where they were detected both in stems and roots, but more frequently by AmpliDet RNA than by IFC.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.     

A new formulation system for the application of biocontrol fungi to soil

A new biocontrol formulation system was devised that does not require sterile conditions during preparation. It involves mixing vermiculite and powdered wheat bran with wet or dry fermentor biomass of Trichoderma spp. or Gliocladium virens, moistening with 0.05 N HCl, and drying the mixture. Before application to soil, the preparation (VBA‐FB) is activated by re‐moistening with 0.05 N HCl and incubated at room temperature for 2–3 days to stimulate development of young hyphae of the biocontrol fungus. Populations of biocontrol fungi proliferated to greater than 10⁷ colony‐forming units (cfu) per g of soil when activated VBA‐FB was added to soil. In soil artificially infested with Rhizoctonia solani, seven isolates of the 14 studied added as VBA‐FB reduced survival and 12 reduced saprophytic growth of the pathogen. Of these, two isolates of T. hamatum (TRI‐4, Tm‐23) and one of T. harzianum (Th‐87) were the most effective. Preparations formulated with either wet or dry biomass effectively reduced pathogen survival, but activated VBA‐FB was more effective than non‐activated VBA‐FB. Storage of VBA‐FB at 25°C for 24 weeks before activation reduced viability of isolates considerably more than storage at 5°C for 24 weeks. In addition, VBA‐FB stored at 5°C before activation more effectively reduced survival of R. solani than VBA‐FB stored at 25° C. Survival of R. solani was reduced by activated VBA‐FB applied to several soil types (sandy loam, sandy clay loam, clay). Some nitrogen fertilizers increased the efficacy of VBA‐FB preparations of several isolates.     

Sporulation and regeneration efficiency of phosphobacteria (<Emphasis Type="Italic">Bacillus megaterium</Emphasis> var <Emphasis Type="Italic">phosphaticum</Emphasis>)

Sporulation in Bacillus megaterium var phosphaticum (PB — 1) was induced using modified nutrient media. This modified medium induced sporulation within 36 h. After spore induction the spores were kept under refrigerated (5°C) and room temperature (32°C) for five months and survival of spores was studied at 15 days intervals by plating them in nutrient agar medium. It was observed that there was not much variation in the storage temperature (5°C & 32°C). The spore cells of Bacillus megaterium var phosphaticum (PB — 1) were observed up to five months of storage under refrigerated (5°C) and room temperature (32°C). Regeneration of spore cells into vegetative cells was studied in tap water, rice gruel, nutrient broth, sterile lignite and sterile water at different concentrations of spore inoculum. The multiplication of sporulated Bacillus megaterium var phosphaticum culture was fast and reached its maximum (29.5 × 10⁸ cfu ml⁻¹) in nutrient broth containing 5 per cent inoculum level.     

Cold hardiness of diapausing and non-diapausing pupae of the European grapevine moth, Lobesia botrana

Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae) is a key pest of grapes in Europe. It overwinters as a pupa in the bark crevices of the plant. Supercooling point (SCP) and low temperature survival was investigated in the laboratory and was determined using a cool bath and a 1 °C min^?1 cooling rate. Freezing was fatal both to diapausing and non‐diapausing pupae. SCP was significantly lower in diapausing male (?24.8 °C) and female (?24.5 °C) pupae than in non‐diapausing ones (?22.7 and ?22.5 °C, respectively). Sex had no influence on SCP both for diapausing and non‐diapausing pupae. Supercooling was also not affected by acclimation. However, acclimation did improve survival of diapausing pupae at temperatures above the SCP. Survival increased as acclimation period increased and the influence was more profound at the lower temperatures examined. Diapausing pupae could withstand lower temperatures than non‐diapausing ones and lethal temperature was significantly lower than for non‐diapausing pupae. Freezing injury above the SCP has been well documented for both physiological stages of L. botrana pupae. Our findings suggest a diapause‐related cold hardiness for L. botrana and given its cold hardiness ability, winter mortality due to low temperatures is not expected to occur, especially in southern Europe.     

Survival and persistence of nonspore-forming biothreat agents in water

Aims: To determine whether nonspore‐forming biothreat agents can survive and persist in potable water that does not contain a disinfectant. Methods and Results: Autoclaved, de‐chlorinated Atlanta municipal water was inoculated with eight isolates of bacterial biothreat agents (10⁶ CFU ml^?1). The inoculated water samples were incubated at 5, 8 (Francisella tularensis only) or 25°C and assayed for viability by culture and by the presence of metabolic activity as measured by esterase activity (ScanRDI, AES Chemunex). Viability as determined by culture varied from 1 to 30 days, depending upon the organism and the temperature of the water. All organisms were determined viable as measured by esterase activity for the entire 30 days, regardless of the incubation temperature. Conclusion: Francisella tularensis was culturable for at least 21 days if held at 8°C. The remaining nonspore‐forming bacterial biothreat agents were found to be metabolically active for at least 30 days in water held at 5 or 25°C. Significance and Impact of the Study: The data can assist public health officials to determine the safety of drinking water after contamination with a biothreat agent.     

Susceptibility of the leatherjackets Tipula oleracea and T. paludosa to soil flooding

A relation was found between temperature and the survival of larvae of Tipula oleracea and T. paludosa when submerged in tap water alone or on turf growing on a sandy loam flooded with still or disturbed water (experiments with soil always starting with freshly flooded soil). Larvae under water alone usually survived until they starved, whereas larvae submerged on soil died in a relatively short time. The periods of survival at a depth of 25 mm were the same for both large and small larvae of either species at any one temperature. The regression of log.-log. survival time on temperature was a straight line, mean arithmetic values ranging from about 5 h at 20 °C to 122 h at 0 °C. Survival times at 25 mm were not increased by disturbing the water and the times at greater depths were no different, providing the water was still. If the water was regularly disturbed then the greater the depth the longer the survival time at any one temperature. Survival times in deoxygenated water indicated that lethal conditions may not be entirely due to deoxygenation of the water.     

Survival of Botrytis cinerea in Soil in South-Eastern Spain

The survival of Botrytis cinerea in sterile and unsterile soil at different temperatures and relative air humidities was investigated in south‐eastern Spain. Conidia survived only 7 days at 40^°C but, depending on relative humidity, for 30–90 days at 22^°C. High air humidity (95%) was needed to maintain soil humidity (8%) at a level that favoured conidial survival. Conidia survived better in sterile soil than in unsterile soil, probably because of the presence in the latter of soil microorganisms antagonistic to B. cinerea. Survival of conidia in environmental conditions simulating those in a greenhouse was less than 28 days. Results showed that B. cinerea conidia cannot survive over summer in south‐eastern Spain, and other primary sources of inocula are discussed.     

Effects of temperature and soil moisture on survival of eggs of the mosquito Psorophora columbiae (Diptera: Culicidae)

The results of laboratory tests indicated the average survival rates for Psorophora columbiae eggs remained quite high for all of the egg populations exposed to a temperature of 27°C (range 83.0–100.0% survival) after 96 days of exposure, except for the non‐diapausing eggs on dry soil (66.3%). In regard to the exposure of egg populations to moderately cold temperatures (i.e. 8°C, 4°C and ?2°C) for periods of up to 16 days, survival rates for egg populations exposed to 8°C continued to remain relatively high (average >85%) for the remainder of the experimental exposure period (i.e. 96 days). Diapausing Ps. columbiae eggs were more tolerant (82.0% survival) to low temperatures (?2°C) than non‐diapausing eggs (2.4% survival) for 64 days, particularly at temperatures of and below 4°C. Diapausing and non‐diapausing eggs were similar in their ability to survive under high temperatures (34°C and 38°C). High soil moisture (30–40%) or substrate moisture (95% relative humidity) content appeared to enhance the ability of the mosquito eggs to survive both low and high temperature extremes.     

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA,a new technology based on real time monitoring of NASBA amplicons with a molecular beacon

AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.     

Biotic and abiotic factors affecting stability of Beauveria bassiana conidia in soil

Beauveria bassiana conidia were stored in sterile and nonsterile soil under various temperature, relative humidity, soil water content, and pH regimes. Survival of the conidia was primarily dependent on temperature and soil water content. Conidia half-lives ranged from 14 days at 25°C and 75% water saturation to 276 days at 10°C and 25% water saturation. Conidia held at ?15°C exhibited little or no loss in viability regardless of water content, relative humidity, or pH. Conidia were not recoverable after 10 days from soils held at 55°C. Conidia survival in nonsterile soil that was amended with carbon sources, nitrogen sources, or combinations of carbon and nitrogen was greatly decreased and loss was often complete in less than 22 days whereas sterile soil treated in the same manner showed dramatic increases in number, demonstrating that B. bassiana is capable of growth in sterile soil. The obvious fungistatic effect in amended nonsterile soils was possibly related to Penicillium urticae which was routinely isolated from the soils and is shown to produce a water-soluble inhibitor of B. bassiana. The fungistatic effect was shown to be an active inhibition rather than due to competition.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

Modification of Macrosiphum euphorbiae colonisation behaviour and reproduction on potato plants treated by mineral oil

Although mineral oil spray is one of the most effective ways to control the transmission of non‐persistent aphid‐borne viruses in the field, its mode of action is poorly understood. In this study, the effects of mineral oil treatment of potato plants on host selection behaviour, growth, and reproduction of potato aphids, Macrosiphum euphorbiae (Thomas) (Hemiptera: Aphididae), were investigated. The effects were assessed 30 min, 1 day, and 7 days after treatment, (1) on aphid orientation behaviour by using a Y‐tube olfactometer, and (2) on aphid feeding behaviour by using the electrical penetration graph (EPG) technique. Olfactory experiments showed that the oil had a repulsive effect only 30 min after spraying. EPG experiments showed a slight modification of the aphid feeding behaviour mainly 7 days after treatment. The number of both salivation and sap ingestion events during the phloem phases were increased 7 days after treatment. In addition, irrespective of the time after treatment, xylem ingestion time was increased. Clip cage experiments were set up to assess potential effects of the oil treatment on aphid survival and population parameters. Nymphal mortality was increased on treated plants, whereas fecundity of surviving insects was enhanced. The antagonistic effects of oil treatment on aphids are discussed in a plant protection context.     

Effect of water storage and heat treatment on the cytotoxicity of soft liners

doi: 10.1111/j.1741‐2358.2011.00463.x Effect of water storage and heat treatment on the cytotoxicity of soft liners Objective: To evaluate the effect of water storage time on the cytotoxicity of soft liners. Methods: Sample discs of soft liners Dentusoft, Dentuflex, Trusoft, Ufi‐Gel‐P and denture base acrylic resin Lucitone‐550 were prepared and divided into four groups: GN: No treatment, G24: Stored in water at 37°C for 24 h; G48: Stored in water at 37°C for 48 h, GHW: Immersed in water at 55°C for 10 min. To analyse the cytotoxic effect, three samples of each group were placed in tubes with Dubelcco’s Modified Eagle Mediums and incubated at 37°C for 24 h. During this period, the toxic substances were leached to the culture medium. The cytotoxicity was analysed quantitatively by the incorporation of radioactivity ³H‐thymidine checking the number of viable cells (synthesis of DNA). The data were statistically analysed using two‐way anova and Tukey’s honestly significant difference tests (α = 0.05). Results: Treatments did not reduce the cytotoxicity effect of the soft liners (p > 0.05). It was found that Ufi‐Gel‐P had a non‐cytotoxic effect, Trusoft had a slightly cytotoxic effect, Dentuflex had a moderated cytotoxic effect, Dentusoft alternated between slightly and non‐cytotoxic effect, and Lucitone‐550 had non‐cytotoxic effect when stored in water for 48 h. Conclusion: The effect of water storage and the heat treatment did not reduce the cytotoxicity of the soft liners.     

Vulnerability to cavitation differs between current‐year and older xylem: non‐destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse‐porous species

Development of xylem embolism during water stress in two diffuse‐porous hardwoods, Katsura (Cercidiphyllum japonicum) and Japanese white birch (Betula platyphylla var. japonica), was observed non‐destructively under a compact magnetic resonance imaging (MRI) system in addition to conventional quantitation of hydraulic vulnerability to cavitation from excised stem segments. Distribution of white and dark areas in MR images corresponded well to the distribution of water‐filled/embolized vessels observed by cryo‐scanning electron microscopy in both species. Water‐filled vessels were observed in MR images as white areas in Katsura and as white dots in Japanese white birch, respectively, and embolisms could be detected as a change to dark areas. The increase in the relative embolized area (REA: %) in the cross‐sectional area of total xylem during water stress, which was estimated from the binarized MR images, was consistent with the hydraulic vulnerability curves of these species. From the non‐destructive MRI observations, cavitation induced by water stress was shown to develop earlier in 1‐ or 2‐year‐old xylem than in the current‐year xylem in both species; that is, the vulnerability to cavitation differs between vessels in the current‐year xylem and those in older annual rings.     

Effect of composting and pasteurisation on two important quarantine pests of potato

The effect of composting and pasteurization on the quarantine pests of potato Clavibacter michiganensis ssp. sepedonicus (Cms) and Synchytrium endobioticum (Se) were examined on an experimental scale. Composting was performed with 2-L pots and 60-L composters for two months at temperatures below 50 degrees C and for 12 and 21 days at temperatures above 65 degrees C. Pasteurization was performed via water bath at 70 degrees C for maximum 2 hours. Pathogens were introduced directly or via carriers into the processes. After composting for two months and for 12 and 21 days it was possible to isolate vital Cms cells from bioassay plants and vital resting spores of Se could be extracted from sample material. Likewise it was possible to isolate vital Cms cells and resting spores of Se after pasteurization for up to two hours. Both pests could not be killed completely during the performed processes. Further studies concerning sanitization of potato wastes are necessary.     

Growth,maturation and survival in a laboratory-reared Rhabdocoelid Turbellarian,Macrostomum orthostylum (BRAUN 1885)

Growth, maturation and survival of a free living turbellarian Macrostomum orthostylum (BRAUN), from a brackish water fish-farm, were studied in the laboratory under a constant temperature of 24 °C. The worms tolerated a wide range of salinity (1 to 30‰). Maximum growth (total length) of 1000 μm was attained in 56 days with a mean growth rate of 15.7 μm d^-1. Minimum maturation time (7 days) and highest longevity (112 days) were recorded in 9%. salinity. Survival period was considerably longer at lower salinities (1 to 10‰) and showed negative relationship with higher salinities (11 to 30‰).     

Fluorescent method for studying the morphogenesis and viability of dermatophyte cells

The dermatophyte Microsporum canis is commonly isolated from human and animal infection. The morphogenesis of this fungus was studied during its developmental stages through the fluorescent method Fluorescein Diacetate and Ethidium Bromide. To this end, 50 l dermatophyte suspension were transferred onto cellophane wrapping esterilized discs (2.5 cm of diameter) placed over the surface of Sabouraud dextrose agar on Petri dishes and incubated at 25 °C for 30 days. Every 60 minutes during the first 24 hours and every 12 hours for next 29 days, one disc was transferred onto glass slide, covered with equal volumes of freshly prepared fluorescein diacetate (FDA) and ethidium bromide (EB) solution, mounted with a coverslip and incubated in the dark for 30 minutes, at 25 °C. Each preparation was then examined on a fluorescent microscope. M. canis presented well defined growth stages: (1) tumescence of cells; (2) germination; (3) development of hyphae; (4) production of conidia and (5) tumescence and formation of arthroconidiae. Using the fluorescent method, non viable cells showed a light bright red coloration and viable cells presented green fluorescence. The principal morphological changes have occurred between the 3rd until the 18th day of culture. The method is very useful to demonstrate the dermatophyte growth stages as well as the perfect differentiation between viable and non viable cells.This revised version was published online in October 2005 with corrections to the Cover Date.     

Comparative study of two culture conservation methods in medical mycology

The efficiency of two fungal conservation methods was compared: Suspension in sterile distilled water and subcultures on potato dextrose agar (PDA) slants at 4 °C. One hundred and eleven strains corresponding to 84 different-species of microorganisms studied in medical mycology were evaluated. The efficiency of each method was estimated by the survival percentage and the preservation of the morphological features of each strain within a seven-year period. From the 111 strains, 79 (71.2%) were preserved viable in water, compared to 86 (77.5%) strains preserved by subculture on PDA slants. Concerning morphological features 75 of the 79 water viable strains (94.9%) conserved their morphology. In contrast, only 60 of the 86 strains (69.8%) conserved their typical morphology by the PDA subculture method. The water conservation method offers important benefits over serial subculture such as: Minimal pleomorphism, simple, rapid and requiring few materials. Thus, the water conservation method is recommended for laboratories where specialized conservation equipment is not available.