Fungal Spore Suspension Research Articles

Potential bioremediation of mercury-contaminated substrate using filamentous fungi isolated from forest soil

The use of filamentous fungi in bioremediation of heavy metal contamination has been developed recently. This research aims to observe the capability of filamentous fungi isolated from forest soil for bioremediation of mercury contamination in a substrate. Six fungal strains were selected based on their capability to grow in 25 mg/L Hg2+-contaminated potato dextrose agar plates. Fungal strain KRP1 showed the highest ratio of growth diameter, 0.831, thus was chosen for further observation. Identification based on colony and cell morphology carried out by 18S rRNA analysis gave a 98% match to Aspergillus flavus strain KRP1. The fungal characteristics in mercury(II) contamination such as range of optimum pH, optimum temperature and tolerance level were 5.5–7 and 25–35°C and 100 mg/L respectively. The concentration of mercury in the media affected fungal growth during lag phases. The capability of the fungal strain to remove the mercury(II) contaminant was evaluated in 100 mL sterile 10 mg/L Hg2+-contaminated potato dextrose broth media in 250 mL Erlenmeyer flasks inoculated with 108 spore/mL fungal spore suspension and incubation at 30°C for 7 days. The mercury(II) utilization was observed for flasks shaken in a 130 r/min orbital shaker (shaken) and non-shaken flasks (static) treatments. Flasks containing contaminated media with no fungal spores were also provided as control. All treatments were done in triplicate. The strain was able to remove 97.50% and 98.73% mercury from shaken and static systems respectively. A. flavus strain KRP1 seems to have potential use in bioremediation of aqueous substrates containing mercury(II) through a biosorption mechanism.

Development of high throughput screening protocol and identification of novel sources of resistance against bakanae disease in rice (Oryza sativaL.)

Bakanae or foot rot disease caused by Fusarium fujikuroi (teleomorph: Gibberella fujikuroi, Sawada, Wollenweber) is emerging as a serious disease of rice. A simple, reliable and high-throughput method for screening the disease would enable rapid screening of germplasm aimed at identifying resistance sources, mapping QTLs/genes and developing resistant rice cultivars. In the present study, a highthroughput, reliable bioassay to screen rice germplasm for resistance to bakanae disease was developed and compared with the conventional screening technique. This technique involves soaking of rice seeds in fungal spore suspension (1.0x106 spores ml–1) for 24 hours at room temperature. Seedling growth at 30°/25° (±3)°C day/night temperature and 60/80(±10)% day/night relative humidity in glasshouse gave the best results. The new protocol described here produces consistent and reproducible bakanae disease symptoms and enables screening of hundreds of rice germplasm within 15 days without any loss of precision in screening of rice genotypes against bakanae disease. The resistant and susceptible genotypes can be used for developing mapping population and identification of QTLs/genes conferring resistance to bakanae disease.

Decolorization applicability of sol–gel matrix immobilized manganese peroxidase produced from an indigenous white rot fungal strain Ganoderma lucidum

BackgroundAn eco-friendly treatment of industrial effluents is a major environmental concern of the modern world in the face of stringent environmental legislations. By keeping in mind the extensive industrial applications of ligninolytic enzymes, this study was performed to purify, and immobilize the manganese peroxidase (MnP) produced from an indigenous strain of Ganoderma lucidum. The present study was also focused on investigating the capability of immobilized MnP for decolorization of dye containing textile effluents.ResultsA large magnitude of an indigenous MnP (882±13.3 U/mL) was obtained from white rot fungal strain G. lucidum in solid state bio-processing of wheat straw under optimized fermentation conditions (moisture, 50%; substrate, 5 g; pH, 5.5; temperature, 30°C; carbon source, 2% fructose; nitrogen source, 0.02% yeast extract; C: N ratio, 25:1; fungal spore suspension, 5 mL and fermentation time period, 4 days). After ammonium sulfate fractionation and Sephadex-G-100 gel filtration chromatography, MnP was 4.7-fold purified with specific activity of 892.9 U/mg. G. lucidum MnP was monomeric protein as evident by single band corresponding to 48 kDa on native and denaturing SDS-PAGE. The purified MnP (2 mg/mL) was immobilized using a sol–gel matrix of tetramethoxysilane (TMOS) and proplytrimethoxysilane (PTMS). The oxidation of MnSO4 for up to 10 uninterrupted cycles demonstrated the stability and reusability of the immobilized MnP. Shelf life profile revealed that enzyme may be stored for up to 60 days at 25°C without losing much of its activity. To explore the industrial applicability of MnP produced by G. lucidum, the immobilized MnP was tested against different textile effluents. After 4 h reaction time, the industrial effluents were decolorized to different extents (with a maximum of 99.2%). The maximally decolorized effluent was analyzed for formaldehyde and nitroamines and results showed that the toxicity parameters were below the permissible limits.ConclusionsIn conclusion, G. lucidum MnP was immobilized by sol–gel matrix entrapment with an objective to enhance its practical efficiencies. The MnP was successfully entrapped into a sol- gel matrix of TMOS and PTMS with an overall immobilization efficiency of 93.7%. The sol- gel entrapped MnP seems to have prospective capabilities which can be useful for industrial purposes, especially for bioremediation of industrial effluents.

Bacteria-induced systemic resistance and growth promotion in Glycine max L. Merrill upon challenge inoculation with Fusarium oxysporum

In the present study, two bacterial strains along with Pseudomonas sp. strain VS1 were employed and screened on the basis of plant growth-promotion characteristics. Strains showed in vitro antibiosis against the test fungal pathogen Fusariumoxysporum together with plant growth-promotion properties that included indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid and inorganic phosphorus solubilization production. The activities of stress responsive enzymes that included lipoxygenase, phenylalanine ammonia-lyase, catalase, peroxidase and superoxide dismutase were reported in bacteria and control-treated soybean plants. Bacteria treated seeds showed higher enzymatic activities in leaves in comparison with fungal spore treated seeds and were considered as positive control. Treatment with bacteria resulted in remarkable increase in the chlorophyll content in leaves as compared to water (negative control) and fungal spore treated seeds (P < 0.05). Soybean seeds treated with bacteria were subjected to fungal spore stress at 0 day after seeding, strain SJ5 significantly enhanced emergence at 28 days after seeding than other treated seeds and positive control. The fresh weight of both shoot and root of plant was significantly increased when soybean seeds were treated with bacteria in the presence of fungal spores as compared to positive control. The dry weights of both roots and shoots were significantly increased by the bacterial treatments as compared to positive control (P < 0.05). Strain treated seeds significantly enhanced root and shoot length together with a number of lateral roots when spore suspension was applied at 0 day after seeding. Moreover, soybean plants treated with strain SJ5 exhibited significantly greater root and shoot weights as compared to the other treated plants when fungal spore suspension was applied at 0 day after seeding.

COLLETOTRICHUM COCCODES: A SOIL-BORNE PATHOGEN DANGEROUS TO A PEPPER ROOTSTOCK IN ITALY

During a survey carried out in July 2011 in Piedmont (north- ern Italy), root rots were repeatedly observed on grafted and non- grafted sweet pepper (Capsicum annuum) plants. Affected plants were stunted, had yellow leaves and a heavily damaged root sys- tem, exhibiting extensive necrosis of old and young roots, which were blackish and cracked. The cortex of necrotic roots could readily be removed, showing the heavily darkened internal tis- sues. Symptoms were particularly evident on older rootstock roots, whose tissues were grey to black. Disease incidence in the pepper rootstock cv. Rocal grown under plastic tunnel was about 35%. Colletotrichum coccodes (Bailey and Jeger, 1992) was re- peatedly and consistently isolated from infected tissues in potato dextrose agar. Hyaline, aseptate and thin- walled conidia (7.7- 18.9×3.2-5.3, average 11.4×4.3 μm size) were abundantly pro- duced in acervuli, and black round sclerotia (diameter 90×68 μm) were also observed. DNA was extracted from a pathogen isolate denoted CC1, using the Nucleospin Plant kit (Macherey Nagel, USA). The nuclear rDNA region (ITS) was amplified using the universal primers ITS1/ITS4 (White et al., 1990), the amplicon (451 bp) was custom-sequenced by LMU (Germany) and the se- quence deposited in GenBank (accession No. JQ290351). This sequence showed 99% homology with that of C. coccodes (AB233340.1). For pathogenicity tests (repeated twice) 15 healthy 20-day-old cv. Rocal plants were inoculated by dipping the roots in a fungal spore suspension (106 CFU/ml), whereas control plants were dipped in water. All plants were grown in a greenhouse at 22-25°C. Thirty days after transplanting, root rot symptoms were observed on all inoculated plants. C. coccodes was consistently reisolated from inoculated plants but not from the controls.

Influence of biofilm contamination on electrical performance of silicone rubber based composite materials

In this research, three kinds of silicone rubber based composite materials, SIR, NSIR and DSIR, were prepared. SIR is the reference silicone rubber without addition of antimicrobial biocide. Two different antimicrobial agents at a concentration of 3 wt% were added to the reference silicone rubber to form the other two types of materials. The materials were inoculated with a fungal spore suspension containing nutrients. Fungal growth could be observed visually in form of spots by naked eye, and biofilm was formed and detected by SEM observation on the surfaces of SIR and NSIR samples, whereas DSIR samples were completely free of the fungal growth. Then the electrical performance of both clean and biofilm contaminated samples, including the surface and volume conductivity, surface flashover voltage and leakage current before flashover, were measured. The obtained results indicate that as compared with SIR base material, the biocides have not changed the surface conductivity and surface flashover voltage of NSIR and DSIR materials. At the same time, the volume conductivity of NSIR remains almost the same as SIR, while that of DSIR increases by about two orders of magnitude. Biofilm could increase the surface conductivity and decrease the surface flashover voltage significantly. The time domain and frequency domain analyses of the leakage currents flowing before flashover event provide useful information on the severity of contamination by biofilm and the eventual risk for surface flashover.

&lt;i&gt;In Vitro&lt;/i&gt; Mutation Induction and Selection of Chrysanthemum (&lt;i&gt;Dendranthema Grandiflora&lt;/i&gt; Tzelev) Lines with Improved Resistance to &lt;i&gt;Septoria Obesa&lt;/i&gt; Syd

This paper describes in vitro mutation selection technique for improved resistance in chrysanthemum (Den- dranthema grandiflora Tzelev) cv. Snow Ball against culture filtrate of Septoria obesa, a leaf spot pathogen. The callus was initiated fro m leaf exp lant on MS med iu m supplemented with 10 mg/ l kinetin and 1 mg/l NAA. Opt imal doses of gamma radiation and culture filtrate on the per cent survival of calli were standardized. The optimal dose of gamma radiation was 20 Gy and that of culture filtrate was 15%. The selection of the resistant calli was made at 15% cu lture filtrate concentration and the calli were subjected to two more cycles of selection (30 day cycle) to obtain resistant cell lines. 100% of the plants raised fro m cuttings had acquired resistance against the pathogen in the pot in greenhouse. The plants were maintained for four years without any symptoms with regular inoculation of fungal spore suspension. No phenotypic variation was observed in the resistant plants.

Inhibition of biofilm formation on silicone rubber samples using various antimicrobial agents

High-temperature-cured silicone rubber samples (silicone rubber (SIR) based on polydimethylsiloxane (PDMS)) and SIR samples containing three different antimicrobial agents, sodium benzoate (NaB), DCOIT (4,5 Dichloro-2-octyl-2H-isothiazolone-one) and p-aminobenzoic acid (PABA) were inoculated with fungal spore suspensions and incubated for 28 days at 29 ± 1 °C and ≥90% humidity, according to the ISO 846:1997(E) protocol. Prior to the biodegradation test, a powder test was conducted to study the efficacy of the chosen antimicrobial compounds and to determine the correct concentration of the compounds for sample preparation. The extent of the microbial growth was studied visually and by Scanning Electron Microscopy (SEM). Changes in surface hydrophobicity and surface chemical composition were studied by contact angle measurements and Fourier Transform Infrared (FTIR) spectroscopy, respectively. Microbial growth and biofilm formation were observed on the surface of reference samples. DCOIT was the most effective antimicrobial agent, as demonstrated by the lack of microbial growth and unaltered surface hydrophobicity. On the surface of samples containing NaB, an initiation of microbial growth and therefore a slight change in surface hydrophobicity was observed. PABA did not inhibit the fungal growth.

Highly accurate deterministic lateral displacement device and its application to purification of fungal spores.

We have designed, built, and evaluated a microfluidic device that uses deterministic lateral displacement for size-based separation. The device achieves almost 100% purity and recovery in continuously sorting two, four, and six micrometer microspheres. We have applied this highly efficient device to the purification of fungal (Aspergillus) spores that are spherical ( approximately 4 mum diameter) with a narrow size distribution. Such separation directly from culture using unfiltered A. niger suspensions is difficult due to a high level of debris. The device produces a two to three increase in the ratio of spores to debris as measured by light scatter in a flow cytometer. The procedure is feasible at densities up to 4.4x10(6) sporesml. This is one of the first studies to apply microfluidic techniques to spore separations and has demonstrated that a passive separation system could significantly reduce the amount of debris in a suspension of fungal spores with virtually no loss of spore material.

A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays

BackgroundInterest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared.MethodsWired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes.ResultsK-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time.ConclusionsK-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.

Mortality of the Nut-Leaf Weevil Strophosoma Melanogrammum (Forster) and Damage Rate of Needles After Treatment with Entomopathogenic Fungi

The susceptibility of the nut-leaf weevil, Strophosoma melanogrammum (Forster) (Coleoptera; Curculionidae) to the select- ed strains of Beauveria bassiana (Bals.-Criv.) Vuillemin, Isaria farinosa (Holm) Brown & Smith, I. fumosorosea (Wize) Brown & Smith, Metarhizium anisopliae (Metschnikoff) Sorokin and M. flavoviride Gams & Rozsypal, was tested under laboratory conditions. Two ways of insect exposure to fungus were studied; direct inoculation of weevils and indirect by contact with inoculated A. procera twigs that served as food for S. melanogrammum. All of the tested isolates were pathogenic to S. melanogrammum. Direct inoculation of adult weevils in fungal spore suspensions resulted in the greatest mortality compared to indirect inoculation where A. procera twigs were immersed in spore suspensions. Direct inoculation with B. bassiana and Metarhizium spp. isolates caused over 80% mortality after 28 days at 20°C, while LT 50 values ranged from 15.0 to 21.6 days. Indirect inoculation with the most effective isolate M. anisopliae (275) resulted in 72% mortality after 28 days with a LT 50 value of 22.1 days. Both direct and indirect spore application failed to reduce needle damage compared with the control. However, significant differences between particular isolates have appeared.

Characterization and Pathogenicity of Fusicladium eriobotryae, the Fungal Pathogen Responsible for Loquat Scab.

To characterize the fungal pathogen responsible for loquat scab and establish differences in pathogenicity in loquat, eight strains identified as Fusicladium eriobotryae were isolated from either loquat leaves or fruit showing scab symptoms in Spain. Loquat plants belonging to the cv. Peluche were infected via a newly developed infection system that was based on spraying susceptible loquat plants with fungal spore suspensions, keeping the plants for 1 week in 100% humidity, and then transferring the plants to ambient relative humidity in greenhouses. Scab symptoms were analyzed and pathogenic characterization of all F. eriobotryae strains revealed different degrees of aggressiveness. Based on infection progression and severity of scab symptoms, strain ST1 was confirmed as the most aggressive in cultivars in the Mediterranean region. Strain ST1, which even caused chlorotic spots in loquat stems, is so aggressive it can be used to identify highly resistant cultivars using this in vivo system. Molecular characterization of internal transcribed spacer ribosomal DNA and, particularly, the glyceraldehyde 3-phosphate dehydrogenase gene, clearly distinguished loquat strains from Venturia inaequalis. Moreover, random amplified polymorphic DNA (RAPD) and microsatellite-primed polymerase chain reaction techniques were used to qualitatively discriminate between species and report the variations within fungal populations. Molecular variability was checked by comparing all the different strains and enabled the specific identification of F. eriobotryae. Although no association was observed between any pattern and phenotypic traits, such as aggressiveness, RAPD provided a specific profile that allowed fungal identification.

Exo‐β‐1,3‐Glucanase from Yeast Inhibits Colletotrichum lupini and Botrytis cinerea Spore Germination

AbstractYeast exo‐β‐1,3‐glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 103 to 80 × 103 spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 103 to 80 × 103, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5‐day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β‐glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea, respectively, could be considered.

A Simple Method for the Assessment of Crown Rot Disease Severity in Wheat Seedlings Inoculated with Fusarium pseudograminearum

AbstractCrown rot caused by Fusarium pseudograminearum (teleomorph: Gibberella coronicola) is a serious disease of wheat. A simple and reliable method for assessing this disease would enhance our capacity in identifying resistance sources and developing resistant wheat cultivars. In searching for such a method, several inoculation techniques were tested. One technique, soaking 1‐ to 4‐day old seedlings in fungal spore suspension, produced consistent and severe crown rot symptoms. Glasshouse testing of several wheat genotypes using this method produced results that agreed well with previously established field ratings for crown rot disease, suggesting that this assay can be conveniently employed for screening of wheat genotypes for crown rot resistance.

Pathogenicity of Two Fungi; Trichoderma harzianum and Cladosporium herbarium on The Two-spotted SpiderMite; Tetranychus urticae Koch

spider mite Tetranychus urticae Koch. Pathogenicity of the two fungi against T. urticae were studied and evaluated underlaboratory and semi-field conditions. Three fungal spore suspensions were prepared; T. harzianum (2x106 spores/ml),C. herbarium (1x106 spores/ml) and a mixture of both fungi (50%: 50%). The effect of fungal spore suspensions on thedeferent stages of T. urticae was studied at 30 & 35oC and 90 % R.H. The mortality percentages of all mite stagesincreased with prolonging the period after spraying. The mixture of the two fungi gave higher mortality than a single oneafter 3 and 7 days for all mite stages reaching 46.8% and 59.8% after 3 and 7 days respectively at 30oC, while at 35oC, itincreased to 65.1% and 83.0%after the two periods respectively. Under semi-field conditions, the mean reduction in mitepopulation resulted from using suspensions of T. harzianum and C. herbarium recorded 42.4 and 43.9% respectivelyafter 10 days of spraying, while it increased to 58.2% when using the mixture of the two fungi. Both fungi T. harzianumand C. herbarium exhibited a positive effect in the pathogenicity of the two-spotted spider mite, T. urticae, but it isrecommended to use a mixture of both fungi to obtain more efficient control.

Biocontrol and Plant Pathogenic Fusarium oxysporum‐Induced Changes in Phenolic Compounds in Tomato Leaves and Roots

AbstractThe biocontrol fungus Fusarium oxysporum strain CS‐20 was previously shown to reduce the incidence of Fusarium wilt of tomato through an uncharacterized host‐mediated response. As phenolic compounds are involved in the defence response of tomato to pathogens and other stressors, this work was undertaken to determine whether biocontrol strains induced changes in phenolic compounds in leaves and roots of tomato seedlings in the presence and absence of pathogenic F. oxysporum f. sp. lycopersici. Roots of intact tomato seedlings were placed in water or aqueous fungal spore suspensions. Two biocontrol F. oxysporum strains [CS‐20 (host‐mediated mechanism) and 85SK‐1 (control mechanism unknown)] and two plant pathogenic strains of F. oxysporum f. sp. lycopersici Race 1 were used. After 24 or 72 h exposure, phenolic compounds were extracted from leaves and roots before identification by HPLC. There were significant qualitative and quantitative differences between the two sampling times. Compared with the control treatment, strain CS‐20 significantly altered (usually increasing) the ferulic, caffeic and vanillic acid contents, and concentrations once unidentified phenolic compounds recovered from leaves and roots. In another experiment, tomato seedlings growing in sterile sand were drenched with spores of strain CS‐20 the day before treating them with varying concentrations of spores of the pathogen for 24 or 72 h. The amount of pathogen present did not significantly affect the plant phenolic response to the presence of strain CS‐20. This work demonstrates that tomato responds within 24 h to the presence of the biocontrol strain CS‐20 by alterations in secondary metabolism that are typical of resistance responses in tomato.

Inoculation of coffee plants with the fungal entomopathogen Beauveria bassiana ( Ascomycota: Hypocreales)

The entomopathogenic fungus Beauveria bassiana was established in coffee seedlings after fungal spore suspensions were applied as foliar sprays, stem injections, or soil drenches. Direct injection yielded the highest post-inoculation recovery of endophytic B. bassiana. Establishment, based on percent recovery of B. bassiana, decreased as time post-inoculation increased in all treatments. Several other endophytes were isolated from the seedlings and could have negatively influenced establishment of B. bassiana. The recovery of B. bassiana from sites distant from the point of inoculation indicates that the fungus has the potential to move throughout the plant.

A viscometric study of the biodegradation of photographic gelatin by fungi isolated from cinematographic films

Abstract The biodegradation of photographic gelatin grade (Bloom 225) material was studied by viscometry in aqueous solution (at 37 °C, 6.67% w/w) using filamentous fungi isolated and identified from cinematographic film stored in different Spanish archives. From viscosity data, different variables such as molecular weight and chain scission were calculated. To ensure initial spore suspension concentration was standardized for all the biodegradation experiments, a correlation between transmittance at 530 nm of fungal spore suspensions and the corresponding cytometric determination of populations was established for all the fungal strains studied in this work. The bioassay experiments were carried out at 25 and 4 °C using an initial concentration of fungi of 4.5×105 conidia/mL except in the case of the genus Alternaria, where the concentration was 10 times lower. The fungal strains were three species of Aspergillus, i.e., A .ustus, A. nidulans var. nidulans, A. versicolor, seven Penicillium chrysogenum strains, and Cladosporium cladosporioides, Alternaria alternata, Mucor racemosus, Phoma glomerata, and Trichoderma longibrachiatum. All were gelatinase positive. Through the viscosity decay profiles with bioassay-time and the corresponding calculated chain scission, the relative quantitative gelatinase efficiency of these fungi has been evaluated.

Endophytic Colonisation of Opium Poppy, Papaver somniferum, by an Entomopathogenic Beauveria bassiana Strain

Beauveria bassiana strain EABb 04/01-Tip isolated from stem-borer larvae of Timaspis papaveris (Hymenoptera: Cynipidae), a serious pest of opium poppy in Spain, was shown to be able to become established endophytically in this pharmaceutical crop. Microbiological, molecular and light and electron microscopic methods were used to study fungal colonisation and to describe its mode of penetration. After inoculation with a foliar spray of conidia, microbiological methods showed 100% of plants examined 24, 48, 72 and 144 h after treatment to be colonised endophytically by the fungus, although the percentage of previously surface sterilised leaf pieces showing fungal growth was 100% at 24 and 48 h, and 80 and 75% at 72 and 144 h after treatment, respectively. The fungus was also observed in leaf pieces obtained from newly formed leaves, indicating that it could spread from treated leaves to leaves formed after fungal application. For molecular studies, a polymerase chain reaction (PCR) protocol was used to amplify the ITS1-5.8S-ITS2 regions of the rDNA of the plant and the fungus. This procedure allowed the detection of the fungus on the surface of the leaves and also endophytically, but only at 72 h after treatment. A nucleotide BLAST search revealed that the ITS1-5.8S-ITS2 sequence of strain EABb 04/01-Tip showed 100% homology with a similar sequence from Cordyceps bassiana. SEM images revealed that although numerous conidia were observed on the leaf surface, few germinated and penetrated. Intracellular colonisation by B. bassiana was not observed, but hyphae were detected growing into the xylem vessels. The fungus was found to colonise 40.5 +/- 4.3% of seedlings (with two cotyledons and the two first real leaves) from seeds dressed with a fungal spore suspension. These results may have implications in the biological control of T. papaveris, including the possible systemic protection of the plant against this cynipid.

Assessment of Fungal Growth on Sodium Polyborate-Treated Cellulose Insulation

Cellulose insulation has rapidly gained a large market share among general contractors and homeowners. Recent interest regarding health effects of high concentrations of fungi within indoor environments (building-related illnesses or sick building syndrome) has promoted concern about susceptibility of building materials, including wood products (in general) and cellulose insulation (specifically), to fungal attack. This study reports an assessment of fungal growth on cellulose insulation made from recycled paper and treated with varying concentrations of sodium polyborate within half-scale wall units exposed to variable and high ambient temperatures and relative humidities throughout the summer. Boron-treated and untreated (control) cellulose insulation within the wall units were challenged with a suspension containing high concentrations of spores of five fungal species commonly found in indoor environments. Our results suggest that cellulose insulation treated with sodium polyborate (a) precludes the growth of the five common fungal species; (b) harbors fewer fungal species before and after being challenged with the fungal spore suspension; and (c) is likely having a cytotoxic or sporocidal effect on many, if not all, fungal species. These results suggest that cellulose insulation treated with sodium polyborate, when properly applied and installed, precludes fungal growth for at least 124 days at high temperatures and relative humidities.