Physiological response of Metarhizium rileyi with linoleic acid supplementation.

Metarhizium rileyi has a broad biocontrol spectrum but is highly sensitive to abiotic factors. A Colombian isolate M. rileyi Nm017 has shown notorious potential against Helicoverpa zea. However, it has a loss of up to 22 % of its conidial germination after drying, which limits its potential as a biocontrol agent and further commercialization. Conidial desiccation resistance can be enhanced by nutritional supplements, which promotes field adaptability and facilitates technological development as a biopesticide. In this study, the effect of culture medium supplemented with linoleic acid on desiccation tolerance in Nm017 conidia was evaluated. Results showed that using a 2 % linoleic acid-supplemented medium increased the relative germination after drying by 41 % compared to the control treatment, without affecting insecticidal activity on H. zea. Also, the fungus increased the synthesis of trehalose, glucose, and erythritol during drying, independently of linoleic acid use. Ultrastructural analyses of the cell wall-membrane showed a loss of thickness by 22 % and 25 %, in samples obtained from 2 % linoleic acid supplementation and the control, respectively. Regarding its morphological characteristics, conidia inner area from both treatments did not change after drying. However, conidia from the control had a 24 % decrease in length/width ratio, whereas there was no alteration in conidia from acid linoleic. The average value of dry conidia elasticity coefficient from linoleic acid treatment was 200 % above the control. Medium supplementation with linoleic acid is a promising fermentation strategy for obtaining more tolerant conidia without affecting production and biocontrol parameters, compatible solutes synthesis, or modifying its cell configuration.

Quantitative microbial spoilage risk assessment (QMSRA) of pasteurized strawberry purees by Aspergillus fischeri (teleomorph Neosartorya fischeri).

Aspergillus fischeri ascospores are known as potential spoilage microorganisms of pasteurized fruit products due to their high incidence in fruits, the ability to survive pasteurization and to grow in acidic conditions. This study aimed to develop a quantitative microbial spoilage risk assessment (QMSRA) model approach to estimate the spoilage risk of packaged strawberry purees due to A. fischeri under various scenarios regarding product formulation, processing and storage conditions. The development of the risk assessment comprised three steps: (1) initial contamination level of raw material by ascospores (N0), (2) inactivation of ascospores during thermal processing (Np) and (3) determination of the number of ascospores which are able to survive thermal processing and develop visible mycelia (D = 2 mm) during storage (Nf). Data of visible growth (tv, days) comprised distributions previously obtained as function of water activity (aw) (0.860-0.985), oxygen (0-21%), temperature (8-30 °C) and pasteurization (95-105 °C/15 s). The simulations were performed in triplicate with 100,000 iterations using the software R. The outcome "spoilage risk" was defined as the probability of having at least one ascospore (Nf) capable of forming visible colonies in 100 g-pack strawberry puree within the typical use-by dates. Overall, high probabilities of spoilage were estimated for purees pasteurized at milder treatments at 85 °C/15-60 s (67%) and 90 °C/15-60 s (≥40%) stored at ambient temperature (22 °C). The spoilage risk was only effectively reduced (0.02%) by increasing pasteurization conditions to 95 °C for at least 45 s. Moreover, the microbial stability of such purees, i.e., spoilage risk <0.001% (=less than 1 spoilage pack out of 105 produced units) was predicted to occur for purees treated at 100 °C/15 s or stored at chilled conditions (≤8 °C) or at strict anaerobic conditions or produced as concentrates (aw ≤ 0.860). Based on the outcomes obtained, a set of specifications for Heat-Resistant Moulds (HRMs) in raw material and pasteurized purees aimed to be used as an ingredient was suggested. Furthermore, the results can be used to support risk management decisions in identifying and quantifying the impact of possible interventions during formulation, processing and storage conditions of fruit purees to effectively reduce this risk.

Assessment of minimum oxygen concentrations for the growth of heat-resistant moulds.

This study evaluated the effect of both gaseous and dissolved oxygen (O2) concentration (0 - 21%) on the growth of six heat-resistant moulds (HRMs) (Neosartorya and Byssochlamys spp.) previously isolated from high-acid fruit products. The study was performed in acidified potato dextrose agar (aPDA) with all six HRMs and with B. fulva and N. fischeri in strawberry, apple and orange juice-based media. At ≥ 0.15% O2, visible growth of the HRMs occurred within 3-6 days. Complete inhibition on aPDA did not occur even at very low levels of dissolved O2 (ca. 0.01% O2). With the exception of B. fulva, decrease of the O2 concentration to ≤0.03% resulted in significantly (p < 0.05) longer times to visible growth. The growth of N. laciniosa, N. fischeri, B. nivea and B. fulva was inhibited for 30 days when they were incubated under strict anaerobic conditions. As in aPDA, B. fulva and N. fischeri grew in the three fruit-based media at O2 concentrations ≥0.15%. Significantly slower (p < 0.05) growth was observed for N. fischeri in orange juice medium. Strategies to inhibit the growth of HRMs should therefore not be based entirely on establishing low headspace O2 levels. With this in mind, the effect of low O2 concentrations (<1%) should be studied in combination with other factors (hurdles) such as antioxidants, organic acids, sugars (aw), storage temperature and pasteurization intensity, in order to predict the growth inhibition of the HRMs.

Synthesis of chitosan biocomposites loaded with pyrrole-2-carboxylic acid and assessment of their antifungal activity against Aspergillus niger.

A wide variety of chitosan (CS) biomaterials have been loaded with different antimicrobial agents to improve the activity of CS against phytopathogenic fungi. Recently, the antimicrobial activity of 1H-pyrrole-2-carboxylic acid (PCA) has been reported as a secondary metabolite of Streptomyces griseus, which was identified as the main bioactive compound in the biological control. However, it is sensitive to light and its activity against filamentous fungi has not yet been reported. The aim of the present research work was to evaluate the biological activity of CS-PCA biocomposites for the control of Aspergillus niger. CS-PCA biocomposites were obtained through nanoprecipitation. In vitro antifungal activity was determined by viability assay, spore germination, morphometric analysis of spores and hyphae, and the analysis of cellular components by fluorescence microscopy. CS-PCA showed an average size and Z potential of 502 ± 72 nm and + 54.7 ± 15 mV, respectively. Micrographs demonstrated well-distributed biocomposites with an apparently spherical shape. A new signal at 1473 cm-1 in the FT-IR spectrum of the CS-PCA biocomposite was observed, confirming the presence of PCA in the composition of the CS-PCA nanosystem. CS-PCA biocomposites reduced the spores' viability by up to 58%. Effects on fungi morphometry, observed as an increase in the spores' average diameter, swelling, distortion, and an increase in the branching of hyphae, were observed. Fluorescence analysis showed oxidative stress and membrane and cell wall damage, mainly at early growth stages. The inhibitory effect against CS-resistant fungi, such as A. niger, opens a door for the control of CS-sensitive fungi.

Assessment of intraspecies variability in fungal growth initiation of Aspergillus flavus and aflatoxin B1 production under static and changing temperature levels using different initial conidial inoculum levels.

Intraspecies variability in fungal growth and mycotoxin production has important implications for food safety. Using the Bioscreen C we have examined spectrophotometrically intraspecies variability of A. flavus using 10 isolates under different environments, including temperature shifts, in terms of growth and aflatoxin B1 (AFB1) production. Five high and five low AFB1 producers were examined. The study was conducted at 5 isothermal conditions (from 15 to 37 °C) and 4 dynamic scenarios (between 15 and 30 °C). The experiments were carried out in a semisolid YES medium at 0.92 aw and two inoculum levels, 102 and 103 spores/mL. The Time to Detection (TTD) of growth initiation was determined and modelled as a function of temperature through a polynomial equation and the model was used to predict TTD under temperature upshifts conditions using a novel approach. The results obtained in this study have shown that a model can be developed to describe the effect of temperature upshifts on the TTD for all the studied isolates and inoculum levels. Isolate variability increased as the growth conditions became more stressful and with a lower inoculum level. Inoculum level affected the intraspecies variability but not the repeatability of the experiments. In dynamic conditions, isolate responses depended both on the temperature shift and, predominantly, the final temperature level. AFB1 production was highly variable among the isolates and greatly depended on temperature (optimum temperature at 30-35 °C) and inoculum levels, with often higher production with lower inoculum. This suggests that, from an ecological point of view, the potential isolate variability and interaction with dynamic conditions should be taken into account in developing strategies to control growth and predicting mycotoxin risks by mycotoxigenic fungi.

Assessment of carvacrol for control of avian aspergillosis in intratracheally challenged chickens in comparison to voriconazole with a reference on economic impact.

AIM: This study was designed to investigate the efficacy of essential oils as an alternative prophylaxis and treatment for avian aspergillosis. METHODS AND RESULTS: The in vitro susceptibility of Aspergillus fumigatus strains to antifungal drugs and carvacrol, thymol, eugenol, thymoquinone and cinnamon was determined using the macrodiffusion and microdilution methods. Carvacrol has antifungal activity in comparison to voriconazole (VCZ) (MIC 0·5, 0·25 µg ml-1 respectively). While cinnamon, euganol, thymol and thymoquinone displayed moderate to weak inhibitory activity. For the efficacy study, five groups of 10-day-old chicks (n = 48) were infected intratracheally either with A. fumigatus conidia or saline (negative control). Chicks in carvacrol prophylactic and treatment (CRPT) group were fed for 10 days beginning from hatch with carvacrol (200 mg kg-1 per diet) supplemented diets. VCZ (VCZT:20 mg kg-1 body weight (BW)), carvacrol treatment (CRT, CRPT) was started upon appearance of the first clinical signs and continued for 10 days. Birds were monitored for an additional 15 days following treatment. Fungal burden and therapeutic efficacy were assessed by survival, BW, quantitative (q) culture (CFU), quantitative real-time PCR (qPCR) and histopathological changes at several time points. Serum biochemical changes were also assessed. VCZT, CRPT, CRT in comparison to the sham-treated (SHAM) group have prolonged survival (87·5, 83·4, 79·2, 41·7% respectively). In VCZT and CRPT, a significant reduction in clinical signs, lesions, CFU and qPCR counts to the limit of detection were observed. CRPT has the lowest BW reduction, economic losses and significant low total cholesterol levels. CONCLUSIONS: Carvacrol has a promising potential to be used as a prophylactic and treatment against A. fumigatus. SIGNIFICANCE AND IMPACT OF THE STUDY: Prognosis of avian aspergillosis is often poor due to delayed diagnosis and treatment failure. However, the widespread uses of azole prophylaxis in birds are thought to be the major driver of azole resistance. These findings create a possibility to develop an effective drug-free alternative strategy for control of avian aspergillosis.

Flow Cytometry Is a Powerful Tool for Assessment of the Viability of Fungal Conidia in Metalworking Fluids.

Fungal contamination of metalworking fluids (MWF) is a dual problem in automated processing plants because resulting fungal biofilms obstruct cutting, drilling, and polishing machines. Moreover, some fungal species of MWF comprise pathogens such as Fusarium solani Therefore, the development of an accurate analytical tool to evaluate conidial viability in MWF is important. We developed a flow cytometric method to measure fungal viability in MWF using F. solani as the model organism. To validate this method, viable and dead conidia were mixed in several proportions and flow was cytometrically analyzed. Subsequently, we assessed the fungicidal activity of two commercial MWF using flow cytometry (FCM) and compared it with microscopic analyses and plating experiments. We evaluated the fungal growth in both MWF after 7 days using quantitative PCR (qPCR) to assess the predictive value of FCM. Our results showed that FCM distinguishes live from dead conidia as early as 5 h after exposure to MWF, whereas the microscopic germination approach detected conidial viability much later and less accurately. At 24 h, microscopic analyses of germinating conidia and live/dead analyses by FCM correlated well, although the former consistently underestimated the proportion of viable conidia. In addition, the reproducibility and sensitivity of the flow cytometric method were high and allowed assessment of the fungicidal properties of two commercial MWF. Importantly, the obtained flow cytometric results on viability of F. solani conidia at both early time points (5 h and 24 h) correlated well with fungal biomass measurements assessed via a qPCR methodology 7 days after the start of the experiment.IMPORTANCE This result shows the predictive power of flow cytometry (FCM) in assessing the fungicidal capacity of MWF formulations. It also implies that FCM can be implemented as a rapid detection tool to estimate the viable fungal load in an industrial processing matrix (MWF).

Risk assessment of fungal spoilage: A case study of Aspergillus niger on yogurt.

A quantitative risk assessment model of yogurt spoilage by Aspergillus niger was developed based on a stochastic modeling approach for mycelium growth by taking into account the important sources of variability such as time-temperature conditions during the different stages of chill chain and individual spore behavior. Input parameters were fitted to the appropriate distributions and A. niger colony's diameter at each stage of the chill chain was estimated using Monte Carlo simulation. By combining the output of the growth model with the fungus prevalence, that can be estimated by the industry using challenge tests, the risk of spoilage translated to number of yogurt cups in which a visible mycelium of A. niger is being formed at the time of consumption was assessed. The risk assessment output showed that for a batch of 100,000 cups in which the percentage of contaminated cups with A. niger was 1% the predicted numbers (median (5th, 95th percentiles)) of the cups with a visible mycelium at consumption time were 8 (5, 14). For higher percentages of 3, 5 and 10 the predicted numbers (median (5th, 95th percentiles)) of the spoiled cups at consumption time were estimated to be 24 (16, 35), 39 (29, 52) and 80 (64, 94), respectively. The developed model can lead to a more effective risk-based quality management of yogurt and support the decision making in yogurt production.

Banana peel culture as an indigenous medium for easy identification of late-sporulation human fungal pathogens.

AIM: Fungi are increasing in incidence as human pathogens and newer and rarer species are continuously being encountered. Identifying these species from growth on regular culture media may be challenging due to the absence of typical features. An indigenous and cheap medium, similar to the natural substrate of these fungi, was standardised in our laboratory as an aid to species identification in a conventional laboratory setting. MATERIALS AND METHODS: Ripe banana peel pieces, sterilised in an autoclave at 121°C temperature and 15 lbs pressure for 15 min promoted good growth of hyphae and pycnidia or acervuli in coelomycetes, flabelliform and medusoid fruiting bodies of basidiomycetes and fruit bodies such as cleistothecium in ascomycetes. The growth from the primary isolation medium was taken and inoculated onto the pieces of double-autoclaved ripe banana peel pieces in a sterile glass Petri dish with some moisture (sprinkles of sterile distilled water). A few sterile coverslips were placed randomly inside the Petri dish for the growing fungus to stick on to it. The plates were kept at room temperature and left undisturbed for 15-20 days. At a time, one coverslip was taken out and placed on a slide with lactophenol cotton blue and focused under the microscope to look for fruit bodies. RESULTS: Lasiodiplodia theobromae, Macrophomina phaseolina, Nigrospora sphaerica, Chaetomium murorum, Nattrassia mangiferae and Schizophyllum commune were identified by characteristic features from growth on banana peel culture. CONCLUSIONS: Banana peel culture is a cheap and effective medium resembling the natural substrate of fungi and is useful for promoting characteristic reproductive structures that aid identification.

Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum, and F. verticillioides.

Young maize plants were inoculated on unfolded mature leaves and on folded immature leaves with Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides suspensions. Infection and symptom development of disease on these asymptomatic mature leaves and immature leaves were then documented. Subcuticular infection was found by the three Fusarium species on both symptomatic and symptomless leaves. The three Fusarium species penetrated the stomata of immature leaves by the formation of appressoria-like structures, infection cushions or by direct penetration. Infection by the three species of Fusarium via stomata is reported here for the first time. The superficial hyphae and re-emerging hyphae of the three species produced conidia. The macroconidia of F. graminearum produced secondary macroconidia and F. proliferatum formed microconidia inside the leaf tissues that sporulated through stomata and trichomes. The infection of maize leaves by the three species of Fusarium and their sporulation may contribute inoculum to cob and kernel infection.

A phylogenetic assessment and taxonomic revision of the thermotolerant hyphomycete genera Acrophialophora and Taifanglania.

We assessed the phylogenetic relationships of 19 isolates belonging to Acrophialophora and Taifanglania based on internal transcribed spacer (ITS), nuclear 18S subunit (nuc 18S) rDNA and ß-tubulin sequences. Phylogenetic data showed that Acrophialophora and Taifanglania comprise a monophyletic clade, but did not support the distinction of two genera. Being the older and more frequently used name, Acrophialophora is adopted as the generic name and Taifanglania is treated as a synonym. The generic concept of Acrophialophora is emended to include the morphological characters formerly used to distinguish Taifanglania. Three new thermotolerant species isolated from soil samples in China are described and illustrated, (i) A. ellipsoidea, with solitary phialides tapering into thin necks and long chains of ellipsoidal to fusiform conidia, (ii) A. angustiphialis with single phialides terminal or lateral on hyphae, and long chains of ellipsoidal or fusiform conidia and, (iii) A. acuticonidiata with single phialides and fusiform conidia with acute ends. Phylogenetic analyses show that A. acuticonidiata, A. angustiphialis and A. ellipsoidea are most closely related to A. curticatenata, A. hechuanensis and A. major, respectively. Growth tests showed that the three new species are thermotolerant, with optimal growth temperatures of 37-40 C, and maximum growth temperatures near 50 C. A key to the accepted species of Acrophialophora is provided.

Systematics of the Trichoderma harzianum species complex and the re-identification of commercial biocontrol strains.

Trichoderma harzianum is known as a cosmopolitan, ubiquitous species associated with a wide variety of substrates. It is possibly the most commonly used name in agricultural applications involving Trichoderma, including biological control of plant diseases. While various studies have suggested that T. harzianum is a species complex, only a few cryptic species are named. In the present study the taxonomy of the T. harzianum species complex is revised to include at least 14 species. Previously named species included in the complex are T. guizhouense, T. harzianum, and T. inhamatum. Two new combinations are proposed, T. lentiforme and T. lixii. Nine species are described as new, T. afarasin, T. afroharzianum, T. atrobrunneum, T. camerunense, T. endophyticum, T. neotropicale, T. pyramidale, T. rifaii and T. simmonsii. We isolated Trichoderma cultures from four commercial biocontrol products reported to contain T. harzianum. None of the biocontrol strains were identified as T. harzianum s. str. In addition, the widely applied culture 'T. harzianum T22' was determined to be T. afroharzianum. Some species in the T. harzianum complex appear to be exclusively endophytic, while others were only isolated from soil. Sexual states are rare. Descriptions and illustrations are provided. A secondary barcode, nuc translation elongation factor 1-α (TEF1) is needed to identify species in this complex.

Antimicrobial properties of black grape (Vitis vinifera L.) peel extracts against antibiotic-resistant pathogenic bacteria and toxin producing molds.

AIM: Black grape peel possesses a substantial amount of polyphenolic antimicrobial compounds that can be used for controlling the growth of pathogenic microorganisms. The purpose of this study was to assess antibacterial and antifungal activity of black grape peel extracts against antibiotic-resistant pathogenic bacteria and toxin producing molds, respectively. MATERIALS AND METHODS: Peel of grape was subjected to polyphenolic extraction using different solvents viz., water, ethanol, acetone, and methanol. Antibiotic-resistant strains of Staphylococcus aureus, Enterococcus faecalis, Enterobacter aerogenes, Salmonella typhimurium, and Escherichia coli were screened for the antibacterial activity of different grape extracts. Antibacterial activity was analyzed using agar well diffusion method. Penicillium chrysogenum, Penicillium expansum, Aspergillus niger and Aspergillus versicolor were screened for the antifungal activity. Antifungal activity was determined by counting nongerminated spores in the presence of peel extracts. RESULTS: As compared to other solvent extracts, methanol extracts possessed high antibacterial and antifungal activity. S. typhimurium and E. coli showed complete resistance against antibacterial action at screened concentrations of grape peel extracts. Maximum zone of inhibition was found in case of S. aureus, i.e., 22 mm followed by E. faecalis and E. aerogenes, i.e., 18 and 21 mm, respectively, at 1080 mg tannic acid equivalent (TAE)/ml. The maximum and minimum percent of growth inhibition was shown by P. expansum and A. niger as 73% and 15% at 1080 TAE/ml concentration of grape peel extract, respectively. CONCLUSIONS: Except S. typhimurium and E. coli, growth of all bacterial and mold species were found to be significantly (P < 0.05) inhibited by all the solvent extracts.

Improvement of fruiting body production in Cordyceps militaris by molecular assessment.

Cordyceps militaris is a heterothallic ascomycetous fungus that has been cultivated as a medicinal mushroom. This study was conducted to improve fruiting body production by PCR assessment. Based on single-ascospore isolates selected from wild and cultivated populations, the conserved sequences of α-BOX in MAT1-1 and HMG-BOX in MAT1-2 were used as markers for the detection of mating types by PCR. PCR results indicated that the ratio of mating types is consistent with a theoretical ratio of 1:1 (MAT1-1:MAT1-2) in wild (66:70) and cultivated (71:60) populations. Cross-mating between the opposite mating types produced over fivefold more well-developed fruiting bodies than self- or cross-mating between strains within the same mating type. This study may serve as a valuable reference for artificial culturing of C. militaris and other edible and medicinal mushrooms and may be useful to develop an efficient process for the selection, domestication, and management of strains for industrial-scale production.

Influence of some parameters on the germination assessment of mycopesticides.

The substantial negative impact of some parameters on the germination of low-quality conidia (high proportion of slow-germinating propagules) was demonstrated, whereas for high-quality batches their effect was small or even absent. Germination was increased as the initial hydration status of conidia immediately prior to suspension preparation was increased, being ca. 33% and 80% for dehydrated Metarhizium anisopliae propagules (water activity ≤0.314) from low- or high-quality batches after an 18 h incubation period, respectively, and 63% and 95% for hydrated propagules (water activity = 0.933). Germination of low-quality propagules also increased as the time dry conidia were kept in aqueous suspension prior to inoculation onto culture media (15 min, 3 or 24 h) or the incubation time at 25°C before counts (18, 48 or 72 h) was increased. Depending on treatment conditions, average germination of low-quality conidia varied from 53% to 98%. On the other hand, germination for high-quality conidia was always ≥94%. Regarding the relative humidity (RH) of the incubation atmosphere, the average germination rates for low-quality conidia on Potato Dextrose Agar (PDA) in Petri plates was 49%, while germination of these conidia on PDA blocks kept under lower RH inside plastic boxes was ≤23%. Use of lactophenol-staining and/or use of coverslips had a negative effect when germination assessment was performed for low-quality conidia, resulting in distorted counts or increased standard deviations compared to high-quality conidial batches. The occurrence of dislodged conidia (ungerminated conidia outside the inoculation zone due to hydraulic pressure exercised by addition of stains and/or coverslips added to the substrate by the time germination is assessed) was common place, whereas dislodged conidia were not seen in treatments with high-quality batches. This work underscores the importance of a number of parameters that anyone working with low-quality fungi needs to be cognizant of in their research.

Nitric oxide generated by the rice blast fungus Magnaporthe oryzae drives plant infection.

Plant-derived nitric oxide (NO) triggers defence, priming the onset of the hypersensitive response and restricting pathogen ingress during incompatibility. However, little is known about the role of pathogen-produced NO during pre-infection development and infection. We sought evidence for NO production by the rice blast fungus during early infection. NO production was measured using fluorescence of DAR-4M and the role of NO assessed using NO scavengers. The synthesis of NO was investigated by targeted knockout of genes potentially involved in NO synthesis, including nitric oxide synthase-like genes (NOL2 and NOL3) and nitrate (NIA1) and nitrite reductase (NII1), generating single and double Δnia1Δnii1, Δnia1Δnol3, and Δnol2Δnol3 mutants. We demonstrate that Magnaporthe oryzae generates NO during germination and in early development. Removal of NO delays germling development and reduces disease lesion numbers. NO is not generated by the candidate proteins tested, nor by other arginine-dependent NO systems, by polyamine oxidase activity or non-enzymatically by low pH. Furthermore, we show that, while NIA1 and NII1 are essential for nitrate assimilation, NIA1, NII1, NOL2 and NOL3 are all dispensable for pathogenicity. Development of M. oryzae and initiation of infection are critically dependent on fungal NO synthesis, but its mode of generation remains obscure.

Monitoring and assessment of airborne fungi in Kolkata, India, by viable and non-viable air sampling methods.

The composition and variability of airborne fungal spores were studied using two complementary sampling methods in an outdoor environment in Kolkata suburb for 2 years, from November 2002 to October 2004. For monitoring the total fungal spore burden in the air, Burkard 7-day volumetric sampler was used, whereas Andersen two-sage viable sampler was used for isolating the cultivable airborne fungi. Among the 37 fungal spore types identified in the air samples, the predominant ones were Cladosporium, unidentified ascospores, unidentified basidiospores, Aspergilli/Penicilli, Nigrospora, Periconia, Chaetomium, Drechslera, Alternaria, Coprinus, Ganoderma, Pithomyces, and rust spores. Only six fungal spore types (Alternaria, Aspergilli/Penicilli, Cladosporium, Curvularia, Drechslera, and Nigrospora) were recovered in common by the two samplers. For Aspergilli/Penicilli, Drechslera, and Nigrospora, the spore concentration was underestimated in the non-viable sampling method (Burkard sampler). In general, higher spore count was recorded in winter. The highest fungal species variability was observed in early monsoon (June). Relative humidity could significantly predict the seasonal periodicity of the maximum number of airborne spores. The total airborne fungi concentration recorded in the study (15-16 × 10(3) spores m(-3) of air) was lower than the proposed threshold limit value for clinical significance, suggesting apparently no or less airborne-fungi-exposure-related health risk in the sampling area. Cladosporium cladosporioides was recorded beyond the proposed threshold limit value in January 2003 and March 2004; Aspergillus fumigatus and Aspergillus nidulans in winter that might have posed considerable health risk to sensitized individuals.

On-farm production of inoculum of indigenous arbuscular mycorrhizal fungi and assessment of diluents of compost for inoculum production.

On-farm production of arbuscular mycorrhizal [AM] fungus inoculum can be employed to make the benefits of the symbiosis more available to vegetable farmers. Experiments were conducted to modify an existing method for the production of inoculum in temperate climates to make it more readily adoptable by farmers. Perlite, vermiculite, and peat based potting media were tested as diluents of yard clippings compost for the media in which the inoculum was produced using bahiagrass (Paspalum notatum Flugge) as host plant. All produced satisfactory concentrations of AM fungus propagules, though vermiculite proved to be better than potting media (89 vs. 25 propagules cm(-3), respectively). Two methods were tested for the growth of AM fungi indigenous to the farm: (1) adding field soil into the vermiculite and compost mixture and (2) pre-colonizing the bahiagrass seedlings in media inoculated with field soil prior to transplant into that mixture. Adding 100 cm(3) of field soil to the compost and vermiculite produced 465 compared to 137 propagules cm(-3) for the pre-colonization method. The greater flexibility these modifications give will make it easier for farmers to produce inoculum of AM fungi on-the-farm.

Assessment of the suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic fungus Beauveria bassiana (Bals.) Vuillemin.

BACKGROUND: Biopesticides based on Beauveria bassiana (Bals.) Vuillemin hold great promise for the management of a wide range of insect pests. The conidia in the biopesticide formulation require an adjuvant to protect them from photoinactivation by sunlight. The suitability of Tinopal, an optical brightener used as sunscreen for baculovirus formulations, for use with B. bassiana was assessed. The aim was to study the effect of Tinopal on the growth and photoprotection of B. bassiana, and its effect on the susceptibility of insects to B. bassiana. RESULTS: Tinopal was found to have no adverse effect on the growth of B. bassiana. It was found to confer total protection (approximately 95% conidial germination at 10 g Tinopal L(-1)) from sunlight up to 3 h of exposure, and a better survival rate than controls even up to 4 h. Helicoverpa armigera Hübner larvae fed on diet with 5 g kg(-1) Tinopal were found to have reduced growth. The duration of the larval stage increased by 3-4 days in 1 and 5 g kg(-1) Tinopal treatments. Among the moths that emerged from larvae fed on diet with 5 g kg(-1) Tinopal, a significantly high number were malformed compared with controls. The larvae that were fed diet with Tinopal showed quicker and higher mortality and required a lower effective lethal dose (LC(50)) than the controls. Tinopal was found to have a synergistic effect with B. bassiana in causing insect mortality. CONCLUSIONS: Tinopal was found to be a suitable adjuvant for B. bassiana-based biopesticide formulations. It conferred tolerance to sunlight and caused stress in the insect, leading to a synergistic effect with B. bassiana.

Assessment of Mycosphaerella graminicola resistance to azoxystrobin.

Azoxystrobin resistance levels of twenty two strains sampled from ten French locations and two reference isolates (IPO323 and IPO94269) of the wheat pathogen Mycosphaerella graminicola were investigated in vitro. French strains assayed were selected from twenty two genetic groups determined from three hundred sixty three strains previously characterised using microsatellites, actine and beta-tubuline markers. For the first time, the evaluation was carried out using four distinct methods: spotting on PDA medium, spore germination on PDA medium and using microtitre plates with and without Alamar blue, a growth indicator. From dose-response curve, half maximal inhibitory concentration (IC50) was determined for each strain. The results obtained using microtitre plates with the addition of Alamar blue displayed high standard deviations from the growth averages observed. Therefore, we suggest that this method is inadequate to assess M. graminicolo resistance to fungicides. However, a good correlation was observed between the rankings of strains according to their IC50 values with the three other methods used. The two reference isolates, as expected, were inhibited by low azoxystrobin concentrations. On the other hand, the IC50 values obtained showed presence of a threshold between sensitive and resistant strains that corroborates the disruptive resistance of M. graminicola against strobilurin fungicides. In addition, the strains showing resistance were those sampled mainly from northern France, where a high frequency of strobilurin resistant isolates among M. graminicola populations was reported by several studies.