Number Of Viable Spores Research Articles

Effects of cold plasma on the growth and aflatoxin production of Aspergillus flavus

Cold plasma (CP) has an inactivating effect on bacteria, but little research has been done on fungi, especially on aflatoxin (AF)-producing strains of Aspergillus flavus (A. flavus). In this study, a CP device was utilized to investigate the destructive effects and mechanisms of different voltage treatments on AF-producing A. flavus strains. The inactivation curve was fitted using Weibull and Logistics kinetic models (R2 > 0.930). The results of viable spore count and AF detection showed that after 6 min of 60 V treatment, the total number of viable spores decreased by 4.47 log10 CFU/mL, and the AF level was found to be significantly lower after treatment (p < 0.05). The microstructure of A. flavus spores was severely damaged after DBDP treatment. The leakage of nucleic acids and proteins, lipid oxidation level, reactive oxygen level, and Fourier transform infrared spectroscopy analysis of spores all confirmed that the cell membrane of A. flavus was damaged after DBDP treatment. This study could provide reference methods for CP technology in the control of AF-producing A. flavus.

Microencapsulation of Bacillus thuringiensis strains for the control of Aedes aegypti

In this study, we investigated the microencapsulation of two strains of the entomopathogenic bacteria Bacillus thuringiensis (B. thuringiensis) (BtMA-750 and BtMA-1114), which are biopesticides of high toxicity for the mosquito vector Aedes aegypti. The encapsulation of different concentrations of microorganisms in starch microparticles was evaluated, and the inverse suspension polymerization technique was explored. It was possible to observe that the higher amounts of the biopesticide caused a slight decrease in the diameter of the particles; however, even when encapsulated, the biopesticide still presents an average diameter that is able to be consumed by the larvae of Aedes aegypti. Furthermore, it was noticed that the presence of both of the B. thuringiensis strains did not affect the thermal stability of the particles. The microencapsulated bacterial strains presented a high number of viable spores and preserved the expression of proteins with molecular masses corresponding to the insecticidal toxins Cry and Cyt, indicating that the encapsulation process was conducted satisfactorily. Finally, the encapsulated strains were tested against Ae. aegypti larvae and maintained 100% larval mortality even after 35 days. Therefore, microencapsulation of B. thuringiensis not only guarantees the bacterial activity, but also prolongs the action of the biopesticide. Collectively, such findings highlight the great potential of the new biopesticides, which may help to reduce the population indices of the mosquito vector Ae. aegypti via a sustainable and environment-friendly route.

A novel ultrasonic crushing method for accurate detection of viable spore counts in Paenibacillus polymyxa-based microbial pesticides is superior to the conventional method of Shaker shaking

ABSTRACT The number of viable spores is the most important quality index of Bacillus-based microbial pesticides. This study aimed to establish a novel pre-treatment method of samples for improving the traditional plate counting method thereby detecting the number of viable spores accurately and reproducibly in Paenibacillus polymyxa-based microbial pesticides. Firstly, three pre-treatment methods of Shaker shaking (SSP), ultrasonic oscillation (UOP), and ultrasonic crushing (UCP) were optimised and compared. The results showed that only the UCP-based plate counting method could detect accurately the viable spore counts (5.2 × 109 CFU/g) in the 5 × 109 CFU/g P. polymyxa original powder, and were repeated well with a coefficient of variation (CV) of 5.0%, compared to SSP (3.46 × 109 CFU/g, CV was 23.4%) and UOP (3.5 × 109 CFU/g, CV was 26.0%). The optimal conditions of UCP were ultrasonic power of 200 W, ultrasonic/intermittent time of 3 s/3 s, and total time of 3 mins (30 cycles). Furthermore, the scanning electron microscope observed that spores were completely desorbed from the carrier by the UCP method. and the average particle size of the carrier pellet was about 2–5 μm, which is significantly smaller than the average particle size of the carrier pellet without pre-treatment (about 15–20 μm) and by the SSP (about 7–12 μm). The plate counting method based on UCP could desorb all spores from the carrier, and the detection results of viable spore counts in P. polymyxa microbial pesticides were accurate and reproducible.

Evaluation of various substrates for mass production and shelf life of Trichoderma asperellum

Trichoderma species, which belong to the genus Trichoderma and the order Hypocreales within the Ascomycota phylum, are highly promising bio-control agents. They are commonly present in natural soil, decaying organic plant matter, and wood, demonstrating remarkable efficacy in combating various plant diseases, including those caused by parasitic nematodes. Being imperfect fungi, the formulation and shelf-life of Trichoderma species play vital roles in ensuring their successful utilization in commercial applications. The present study aimed to assess different substrates for mass production and shelf-life of two Trichoderma asperellum isolates, FbMi4 and FbMi6. The initial spore count of T. asperellum FbMi4 at three concentrations (30, 40, and 50 ml/100 g carrier) on the first day were 226×106, 255×106, and 291×106 spores/ml, respectively. Over a storage period of 60 days, the spore populations gradually decreased to 149×106, 169×106, and 192×106 spores/ml, with reductions in viability of 14.9%, 14.2%, and 14.6% for the respective concentrations. The study also found similar trends in T. asperellum FbMi6 and sorghum grains talc-based formulations. Neem cake, an organic amendment, outperformed potato dextrose broth in producing spores, even after 45 days of storage. Remarkably, the formulated solutions maintained a substantial number of viable spores for up to 60 days, indicating their potential for extended shelf-life and storage. These findings emphasize the significance of selecting appropriate substrates to achieve mass production and long-term viability of Trichoderma as an effective bio-control agent against various plant diseases, including those caused by parasitic nematodes.

Ability of Essential Oil Vapours to Reduce Numbers of Culturable Aerosolised Coronavirus, Bacteria and Fungi.

Transmission of pathogens present in the indoor air can occur through aerosols. This study evaluated the efficacy of an evaporated mix of essential oils to reduce the numbers of culturable aerosolized coronavirus, bacterium and fungus. The essential oil-containing gel was allowed to vaporize inside a glass chamber for 10 or 20 min. Aerosols of a surrogate of SARS-CoV-2, murine hepatitis coronavirus MHV-1, Escherichia coli or Aspergillus flavus spores were produced using a collision nebuliser and passed through the essential oil vapours, then collected on a six-stage Andersen sampler. The six-stages of the impact sampler capture aerosols in sizes ranging from 7 to 0.65 µm. The number of culturable microbes present in the aerosols collected in the different stages were enumerated and compared to the number of culturable microbes in control microbial aerosols that were not exposed to the evaporated essential oils. After 10 and 20 min evaporation, the essential oils reduced the numbers of culturable aerosolized coronavirus by 48% (log10 reduction = 0.3; p = 0.002 vs. control) and 53% (log10 reduction = 0.3; p = 0.001 vs. control), respectively. The essential oils vaporised for 10 min, reduced the number of viable E. coli by 51% (log10 reduction = 0.3; p = 0.032 vs. control). The Aspergillus flavus spores were mostly observed in the larger aerosols (7.00 µm to 2.10 µm) and the essential oils vaporised for 10 min reduced the number of viable spores by 72% (log10 reduction = 0.6; p = 0.008 vs. control). The vapours produced by a gel containing naturally occurring essential oils were able to significantly reduce the viable numbers of aerosolized coronavirus, bacteria and fungal spores. The antimicrobial gel containing the essential oils may be able to reduce aerosol transmission of microbes when used in domestic and workplace settings.

Plasmodiophora brassicae in Its Environment: Effects of Temperature and Light on Resting Spore Survival in Soil.

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot is challenging, largely because of the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 × 107 spores/g soil) were stored at various temperatures for 2 years. Additionally, other samples were buried in soil or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative PCR (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C, and being buried in the soil were more conductive conditions for spore survival than -20°C, 30°C, and at the soil surface. Most (99.99%) of the spores kept on the soil surface were nonviable, suggesting a negative effect of light on spore viability. Additional experiments confirmed the negative effect of ultraviolet light on spore viability because spores receiving 2 and 3 h ultraviolet light exhibited lower disease potential and contained less DNA content than the nontreated control. Finally, this work confirmed that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential because of the presence and persistence of DNA from dead spores.

Cycling versus Continuous High Pressure treatments at moderate temperatures: Effect on bacterial spores?

The advantage of using high pressure (HP) cycling treatment compared with continuous HP treatment was investigated for the inactivation of bacterial spores. The effects of parameters such as pulse number, pressure level, treatment temperature, compression and decompression rates, and time between pulses were evaluated. For this purpose, Bacillus subtilis and B. cereus spores (108 and 106 CFU/mL respectively) were suspended in 2-(N-morpholino) ethanesulfonic acid (MES) buffer solution, tryptone salt (TS) buffer solution, or infant milk and treated by HP cycling at 300–400 MPa, at 38–60 °C, for 1–5 pulses. Pressure cycling reduced the number of viable spores by 1.8 and 5.9 log respectively for B. subtilis and B. cereus species. Continuous HP treatments were performed at the same pressure and temperature for similar treatment durations. Our results showed that the spore inactivation ratio was correlated with the cumulative exposure time to pressure rather than to effects of the cycling process. Greater spore inactivation caused by HP cycling was observed only when faster compression and decompression rates were applied, probably due to adiabatic heating. A three-step kinetic model was developed, which seemed to support our hypothesis regarding the mechanisms of inactivation by pressure cycling and continuous HP treatments. Industrial relevanceThe resistance of bacterial spores to HP limits the industrial applications to refrigerated food products. In this study, we investigated the use of pressure cycling as a means to improve spore baroinactivation at moderate temperatures (T < 60 °C). We showed that cycling pressure does not significantly increase bacterial spore inactivation in comparable treatment duration, but certainly increases material fatigue in HP vessels. Thus, under moderate temperature, cycling pressure treatment is not industrially relevant.

INFLUENCE OF CHITOSAN SOLUTION ON THE VIABILITY OF ARBUSCULAR MYCORRHIZAL SPORES

The study was aimed to fortify the Arbuscular Mycorrhizal Fungal (AMF) spores, widely used in Bio-fertilizers with chitosan and check the inuence of chitosan on the viability of AMF spores. Chitosan was prepared from shrimp shells using chemical method involving demineralization, deproteinization and deacetylation.AMF spores were kept in three different concentrations of chitosan (0.1%, 0.5 %and 1%) which was prepared in 0.1% acetic acid and 0.1% ascorbic acid. Spore viability was checked by the MTT 3-(4, 5 th th th dimethylthiazol-yl-2, 5-diphenyl-2H-tetrazolium bromide) after a day, 10 day, 20 day and 30 day. Highest number of viable spores was observed in chitosan dissolved in 0.1% ascorbic acid as compared to chitosan in acetic acid.

Assessment of temperature and acid tolerance of Bacillus subtilis isolated from a Brazilian fruit juice-added soy beverage

Bacillus subtilis is a spore-forming bacterium and an important food contaminant. The aim of this study was to analyze the ability of B. subtilis spores to survive under conditions of low pH and high temperature. The package was purchased at a local supermarket, in Uberaba, Minas Gerais. A sample was collected, diluted and plated on Brain-Heart-Infusion agar (BHI). After incubation, suspected colonies of B. subtilis were transferred to BHI agar. Cell morphology, the presence of spores and Gram stain were examined, and the isolate was identified by 16S rRNA gene sequencing . The microscope evaluation indicated the presence of spores. The thermal tolerance of the spores was evaluated by the addition of 3x109spores/mL in test tubes containing peptone water. Heat treatments were carried out at 80 and 90°C at different incubation times (0, 10, 20, 30, 40, 50 and 60 min). After heating, the tubes were cooled and the number of viable spores was determined in BHI Agar. For the analysis of spore survival, D and Z values were calculated. Tolerance to acid conditions was evaluated using BHI broth with different pH values. After incubation, the bacterial concentration was determined by determining viable cell count on BHI Agar medium. The vegetative cells were transferred to the BHI broth and the pH was adjusted to different values (3, 4 or 5). Sampling were taken 8, 12 and 24 h after incubation. The samples were serially diluted in peptone water and spread in BHI Agar to determine the viable cell count . The 16S rRNA gene sequencing indicated high similarity (99.99%) with B. subtilis. D values were 17.01 min at 80°C and 13.42 min at 90°C. The Z-value was 97.13°C. B. subtilis was not able to grow at pH 3 and pH 4, but its survival was confirmed after the growth of colonies on BHI agar. At pH 5, B. subtilis grew after 24 h and the final pH changed to 7. Our results suggest that the spores of B. subtilis isolated from fruit juice-added soy beverage are tolerant to low pH and high temperature.

Discovering the Role of Patagonian Birds in the Dispersal of Truffles and Other Mycorrhizal Fungi

Dispersal is a key process that impacts population dynamics and structures biotic communities. Dispersal limitation influences the assembly of plant and microbial communities, including mycorrhizal fungi and their plant hosts. Mycorrhizal fungi play key ecological roles in forests by feeding nutrients to plants in exchange for sugars, so the dispersal of mycorrhizal fungi spores actively shapes plant communities. Although many fungi rely on wind for spore dispersal, long-distance dispersal is uncommon and most spores land close to fungal fruiting bodies. Some fungi have lost the ability to shoot their spores into the air and instead produce enclosed belowground fruiting bodies (truffles) that rely on animals for dispersal. Animal-facilitated spore dispersal increases the chances that spores reach suitable habitats and colonize plant roots. The role of mammals in fungal spore dispersal is well documented but the relevance of birds as dispersal agents of fungal spores has been understudied, despite the prominence of birds as seed dispersal vectors. Here we use metagenomics and epifluorescence microscopy to demonstrate that two common, widespread and endemic birds from Patagonia, Chucao Tapaculos (Scelorchilus rubecula) and Black-throated Huet-huets (Pteroptochos tarnii), regularly consume mycorrhizal fungi and disperse viable spores via mycophagy. We sampled more than 100 fecal samples that were collected across a 700 kilometer area in Patagonia. Our metagenomic analysis indicates that these two species of generalist, ground-dwelling birds routinely consume diverse mycorrhizal fungi, including many truffles, that are symbiotically associated with Nothofagaceae trees that dominate Patagonian forests. We used soil sample controls to ensure that fecal samples were not contaminated from nearby soil as well as extraction, PCR and sequencing controls to account for lab contamination. Epifluorescence microscopy of fecal samples confirmed that the birds dispersed large numbers of viable spores from truffles and other mycorrhizal fungi, including Cystangium nothofagi and species of Cortinarius, Descolea, and Inocybe, which are important root colonizers of seedling and saplings of several Nothofagaceae species. Our results show that fungi are a common food for both bird species and that this cryptic animal-fungi symbiosis is widespread and ecologically important in Patagonia. Our findings suggest that birds may also act as cryptic but critical agents of fungal spore dispersal in other ecosystems.

Sporicidal evaluation of different substances against Nosema ceranae for surface disinfection

Nosema ceranae is the microsporidium causing a disease highly prevalent in honey bees. Since few treatments are available to control this disease, cleaning and disinfection of beekeeping equipment could be a useful tool for decreasing the number of viable spores and therefore the risk of infection. Up to date, the information about the efficacy of substances suitable for beekeeping use is scarce. In this work, the sporicidal activity of different products against N. ceranae spores has been tested using a new method here developed based on International harmonisation norms. Stainless steel surfaces (5 cm2) were contaminated with 100,000 fresh percoll® purified N. ceranae spores. The surfaces were exposed for 5 and 15 minutes with different products selected either because they are common use disinfectants (sodium hypochlorite, acetic acid, ethanol 96°, ammonia solution, or povidone-iodine), or because they are commercial products marketed for surface disinfection (Inokuo®, Mycoplasma OFFTM, Biocidal ZFTM) or for Varroa control (Ecoxal). After exposure, the viability of spores was analysed by flow cytometry after staining with propidium iodide solution and compared to spores exposed to water (negative control) or autoclaved (positive control). Among tested products, ammonia solution, sodium hypochlorite and Mycoplasma OFFTM, showed sufficient effect to be used in equipment disinfection. Other products as acetic acid causing an increase of cell debris could be also useful although they require further investigations.

Combined high pressure and heat treatment effectively disintegrates spore membranes and inactivates Alicyclobacillus acidoterrestris spores in acidic fruit juice beverage

The commercial potential of high pressure and thermal processing (HPTP) was investigated against Alicyclobacillus acidoterrestris spores in commercial acidic apple juice beverage and in acidified and neutral potassium buffers. With starting spore counts prior to treatments being 6.5 and 7.2 log10 respectively for strains AJA 66 (D90°C 15.4 min) and ATCC 49025 (D90°C 8.5 min), HPTP at 600 MPa at 80 °C for 3 min provided an optimal treatment with spore viability reduced below the detection limit for both strains. HPTP at 80 °C for 1 min and HPTP at 70 °C for 3 min achieved 4.1–4.5 log10 CFU/mL reduction. HPTP at 70 °C for 1 min reduced the number of viable spores by 2.0–2.5-log10 CFU/mL. Flow cytometry revealed the presence of membrane-compromised spores among culturable spores following HPTP and heat alone treatments at different temperatures. Electron microscopy clearly showed the efficiency of HPTP with crushed or hollow spores predominating after treatments. No correlation between HPTP susceptibility and genetic diversity was observed for two genotypes of A. acidoterrestris spores. The treatment combination provides a promising option for industrial utility since it requires lower heat and processing time.

Inactivation of Bacillus anthracis and Bacillus atrophaeus spores on different surfaces with ultraviolet light produced with a low‐pressure mercury vapor lamp or light emitting diodes

To obtain quantitative efficacy data of two ultraviolet light (UVC) technologies for surface inactivation of Bacillus anthracis Ames and Bacillus atrophaeus spores. Spores were deposited onto test coupons and controls of four different materials, via liquid suspension or aerosol deposition. The test coupons were then exposed to UVC light from either a low-pressure mercury vapor lamp or a system comprised of light emitting diodes, with a range of dosages. Positive controls were held at ambient conditions and not exposed to UVC light. Following exposure to UVC, spores were recovered from the coupons and efficacy was quantified in terms of log10 reduction (LR) in the number of viable spores compared to that from positive controls. Decontamination efficacy varied by material and UVC dosage (efficacy up to 5·7 LR was demonstrated). There was no statistical difference in efficacy between the two species or between inoculation methods. Efficacy improved for the LED lamp at lower relative humidity, but this effect was not observed with the mercury vapor lamp. This study will be useful in determining whether UVC could be used for the inactivation of B. anthracis spores on different surface types.

Spore Production of Toxigenic and Non-toxigenic Clostridium difficile Isolates in Sub-MIC of Vancomycin, Clindamycin, and Ceftazidime

Background: The non-toxigenic variant of Clostridium difficile is prevalent in clinical samples. The reason for the high prevalence of these strains in the clinical diarrhea specimens has not yet been studied. Objectives: Evaluation of spore production in non-toxigenic C. difficile isolates (A-/B-/CDT-) compared to toxigenic isolates (A+/B+/CDT-) in the absence and presence of antibiotics. Methods: Minimum inhibitory concentration (MIC) for bacteria was performed by the microdilution technique. About ~106 bacteria from 18-hour culture were inoculated to pre-reduced media containing ½ × MIC of vancomycin (VAN), clindamycin (CLI) and ceftazidime (CAZ). After 24, 48, and 96 hours, one milliliter of broth culture was added and heated for killing vegetative forms. One hundred microliters of appropriate dilution were cultured on Columbia blood agar in triplicate. After 72 hours, the number of viable spores was counted based on the colony forming unit. Results: The result showed the spore production of non-toxigenic C. difficile isolates in free antibiotic media and ½ × MIC of antibiotics was similar to toxigenic isolates. The VAN, CLI, and CAZ inhibited spore production in non-toxigenic isolates as much as toxigenic isolates of C. difficile. Conclusions: It seems the non-toxigenic C. difficile isolates are able to produce spores in the absence and presence of antibiotic in a similar manner to toxigenic isolates. Generally, their ability to produce toxin is lost, but they are able to remain, sporulate, and survive in hospitalized patients who receive antibiotics.

Potential impact of engineered silver nanoparticles in the control of aflatoxins, ochratoxin A and the main aflatoxigenic and ochratoxigenic species affecting foods

The potential use of nanotechnology in the control of toxigenic fungi and mycotoxin production has been little explored. In this report, engineered silver nanoparticles (AgNPs) were synthesized and characterized by single particle Inductively Coupled Plasma Mass Spectrometry. Then, their effectiveness in the control of the growth of the main aflatoxigenic and ochratoxigenic species affecting foods and aflatoxins (AFs) and ochratoxin A (OTA) production was studied. The target species and their associated mycotoxins were Aspergillus flavus (AFB1 and AFB2), A. parasiticus (AFB1, AFB2, AFG1 and AFG2), A. carbonarius, A. niger, A. ochraceus, A. steynii, A. westerdijkiae and Penicillium verrucosum (OTA). Spore suspensions supplemented with AgNPs (average diameter size 30 nm, range 14–100 nm) at doses 0–45 μg/mL were incubated for 2–30 h. At selected exposure times aliquots were removed and cultured on maize-based medium (MBM) for 10 days. In these cultures, percentage of viable spores, effective doses (EDs) to inhibit the number of viable spores to 50%, 90% and 100%, colony lag phases, colony growth rates (GR), EDs to inhibit the colony GR to 50%, 90% and 100% were estimated. AF and OTA levels were determined by UPLC-MS/MS. Under the assayed conditions, effective doses of the AgNPs against all the studied fungal species could be estimated. These doses generally decreased with increasing exposure time and were higher for A. flavus and A. parasiticus than for ochratoxigenic species. Colonies from spores treated at high exposure times (20–30 h) and variable AgNP doses showed long lag phases or even did not occur depending on the fungal species. Colony GR, as well as AF and OTA levels in MBM cultures decreased when AgNP dose and contact period increased. The factors species, AgNP dose, exposure time and their interactions significantly affect fungal growth and AF and OTA accumulation in MBM. The results suggest that AgNPs only or as active ingredient hosted in paints, films or other polymers could be a good strategy in the management of the main aflatoxigenic and ocratoxigenic species affecting food and AF and OTA contamination.

Microencapsulation of an indigenous isolate of Bacillus thuringiensis by spray drying

In this study, microencapsulation by spray drying was performed to protect spores and crystals of an indigenous isolate of Bacillus thuringiensis Se13 from environmental stress. The effects of wall material, inlet temperature, and outlet temperature on microencapsulation of Bt-Se13 were investigated using Taguchi’s orthogonal array. The most suitable wall material determined as maltodextrin DE10. The optimum inlet and outlet temperatures of spray drier were determined as 160 °C and 70 °C, respectively. The number of viable spores, mean particle size, wetting time, percentage of suspensibility and moisture content of the product produced under optimum conditions were determined as 8.1 × 1011 cfu g−1, 13.462 µm, 25.22 s, 77.66% and 7.29%, respectively. As a result of efficiency studies on Spodoptera exigua in the laboratory conditions, the LC50 was determined as 1.6 × 104 cfu mL−1. Microencapsulated Bt-Se13 based bio-pesticide may be registered for the control of S. exigua and can be tested against other lepidopterans which share the same environment.

Measurement of infection efficiency of a major wheat pathogen using time‐resolved imaging of disease progress

Infection efficiency is a key epidemiological parameter that determines the proportion of pathogen spores able to infect and cause lesions once they have landed on a susceptible plant tissue. In this study, an improved method to measure infection efficiency of Zymoseptoria tritici using a replicated greenhouse experiment is presented. Zymoseptoria tritici is a fungal pathogen that infects wheat leaves and causes septoria tritici blotch (STB), a major disease of wheat worldwide. A novel experimental setup was devised, where living wheat leaves were attached to metal plates, allowing for time‐resolved imaging of disease progress in planta. Because lesions were continuously appearing, expanding and merging during the period of up to 3 weeks, daily measurements were necessary for accurate counting of lesions. Reference membranes were also used to characterize the density and spatial distribution of spores inoculated onto leaf surfaces. In this way, the relationship between the number of lesions and the number of viable spores deposited on the leaves was captured and an infection efficiency of about 4% was estimated from the slope of this relationship. This study provides a proof of principle for accurate and reliable measurement of infection efficiency of Z. tritici. The method opens opportunities for determining the genetic basis of the component of quantitative resistance that suppresses infection efficiency. This knowledge would improve breeding for quantitative resistance against STB, a control measure considered more durable than deployment of major resistance genes.

Spore Acquisition and Survival of Ambrosia Beetles Associated with the Laurel Wilt Pathogen in Avocados after Exposure to Entomopathogenic Fungi.

Laurel wilt is a disease threatening the avocado industry in Florida. The causative agent of the disease is a fungus vectored by ambrosia beetles that bore into the trees. Until recently, management strategies for the vectors of the laurel wilt fungus relied solely on chemical control and sanitation practices. Beneficial entomopathogenic fungi (EPF) are the most common and prevalent natural enemies of pathogen vectors. Laboratory experiments demonstrated that commercial strains of EPF can increase the mortality of the primary vector, Xyleborus glabratus, and potential alternative vectors, Xylosandrus crassiusculus, Xyleborus volvulus and Xyleborus bispinatus (Coleoptera: Curculionidae: Scolytinae). Our study provides baseline data for three formulated commercially-available entomopathogenic fungi used as potential biocontrol agents against X. crassiusculus, X. volvulus and X. bispinatus. The specific objectives were to determine: (1) the mean number of viable spores acquired per beetle species adult after being exposed to formulated fungal products containing different strains of EPF (Isaria fumosorosea, Metarhizium brunneum and Beauveria bassiana); and (2) the median and mean survival times using paper disk bioassays. Prior to being used in experiments, all fungal suspensions were adjusted to 2.4 × 106 viable spores/mL. The number of spores acquired by X. crassiusculus was significantly higher after exposure to B. bassiana, compared to the other fungal treatments. For X. volvulus, the numbers of spores acquired per beetle were significantly different amongst the different fungal treatments, and the sequence of spore acquisition rates on X. volvulus from highest to lowest was I. fumosorosea > M. brunneum > B. bassiana. After X. bispinatus beetles were exposed to the different suspensions, the rates of acquisition of spores per beetle amongst the different fungal treatments were similar. Survival estimates (data pooled across two tests) indicated an impact for each entomopathogenic fungus per beetle species after exposure to a filter paper disk treated at the same fungal suspension concentration. Kaplan–Meier analysis (censored at day 7) revealed that each beetle species survived significantly shorter in bioassays containing disks treated with EPF compared to water only. This study demonstrated that ambrosia beetles associated with the laurel wilt pathogen in avocados are susceptible to infection by EPF under laboratory conditions. However, the EPF needs to be tested under field conditions to confirm their efficacy against the beetles.

PM 3/59 (3) Synchytrium endobioticum: descheduling of previously infested plots

Specific scopeThis Standard describes a procedure for descheduling plots previously infested by Synchytrium endobioticum.Specific approval and amendmentFirst approved in 1999‐09.Revisions approved in 2003‐09 and in 2017‐09.

Development of a Method to Determine the Effectiveness of Cleaning Agents in Removal of Biofilm Derived Spores in Milking System.

Microbial damages caused by biofilm forming bacteria in the dairy industry are a fundamental threat to safety and quality of dairy products. In order to ensure the optimal level of equipment hygiene in the dairy industry, it is necessary to determine the biofilm removal efficiency of cleaning agents used for cleaning-in-place (CIP) procedures. However, currently there is no standard method available for evaluating and comparing cleaning agents for use in CIP procedures in the dairy industry under realistic conditions. The present study aims to establish a CIP model system to evaluate the effectiveness of cleaning agents in removal of biofilm derived spores from the surfaces of stainless steel which is the predominant substrate in milking equipment on dairy farms. The system is based on Bacillus subtilis spores surrounded with exopolymeric substances produced by bacteria during biofilm formation. The spores applied on sampling plates were mounted on T-junctions protruding 1.5–11-times the milk pipe diameter from the main loop to resemble different levels of cleaning difficulty. The cleaning tests were conducted using commercial alkaline detergents and caustic soda at conditions which are relevant to actual farm environment. The spores removal effect was evaluated by comparing the number of viable spores (attached to sampling plates) before and after cleaning. Evaluation of the cleaning and disinfecting effect of cleaning agents toward biofilm derived spores was further performed, which indicates whether spores elimination effect of an agent is due to killing the spores or removing them from the surfaces of dairy equipment. Moreover, it was established that the presence of extracellular matrix is an important factor responsible for high level of cleaning difficulty characteristic for surface attached spores. In overall, the results of this study suggest that the developed model system simulates actual farm conditions for quantitative evaluation of the effectiveness of cleaning and disinfecting agents and their cleaning and disinfecting effect on removal of biofilm derived spores.