Phytogeny of the Quambalariaceae fam. nov., including important Eucalyptus pathogens in South Africa and Australia

The genus Quambalaria consists of plant-pathogenic fungi causing disease on leaves and shoots of species of Eucalyptus and its close relative, Corymbia. The phylogenetic relationship of Quambalaria spp., previously classified in genera such as Sporothrix and Ramularia, has never been addressed. It has, however, been suggested that they belong to the basidiomycete orders Exobasidiales or Ustilaginales. The aim of this study was thus to consider the ordinal relationships of Q. eucalypti and Q. pitereka using ribosomal LSU sequences. Sequence data from the ITS nrDNA were used to determine the phylogenetic relationship of the two Quambalaria species together with Fugomyces (= Cerinosterus) cyanescens. In addition to sequence data, the ultrastructure of the septal pores of the species in question was compared. From the LSU sequence data it was concluded that Quambalaria spp. and F. cyanescens form a monophyletic clade in the Microstromatales, an order of the Ustilaginomycetes. Sequences from the ITS region confirmed that Q. pitereka and Q. eucalypti are distinct species. The ex-type isolate of F. cyanescens, together with another isolate from Eucalyptus in Australia, constitute a third species of Quambalaria, Q. cyanescens (de Hoog & G.A. de Vries) Z.W. de Beer, Begerow & R. Bauer comb. nov. Transmission electron-microscopic studies of the septal pores confirm that all three Quambalaria spp. have dolipores with swollen lips, which differ from other members of the Microstromatales (i.e. the Microstromataceae and Volvocisporiaceae) that have simple pores with more or less rounded pore lips. Based on their unique ultrastructural features and the monophyly of the three Quambalaria spp. in the Microstromatales, a new family, Quambalariaceae Z.W. de Beer, Begerow & R. Bauer fam. nov., is described.

Soral synapomorphies are significant for the systematics of the Ustilago-Sporisorium-Macalpinomyces complex (Ustilaginaceae)

The genera Ustilago, Sporisorium and Macalpinomyces are a polyphyletic complex of plant pathogenic fungi. The four main morphological characters used to define these genera have been considered homoplasious and not useful for resolving the complex. This study re-evaluates character homology and discusses the use of these characters for defining monophyletic groups recovered from a reconstructed phylogeny using four nuclear loci. Generic delimitation of smut fungi based on their hosts is also discussed as a means for identifying genera within this group. Morphological characters and host specificity can be used to circumscribe genera within the Ustilago-Sporisorium-Macalpinomyces complex.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Rhizoctonia -Scots pine interactions: detection, impact on seedling performance and host defence gene response

Rhizoctonia spp. are ubiquitous soil inhabiting fungi that enter into pathogenic or symbiotic associations with plants. In general Rhizoctonia spp. are regarded as plant pathogenic fungi and many cause root rot and other plant diseases which results in considerable economic losses both in agriculture and forestry. Many Rhizoctonia strains enter into symbiotic mycorrhizal associations with orchids and some hypovirulent strains are promising biocontrol candidates in preventing host plant infection by pathogenic Rhizoctonia strains. This work focuses on uni- and binucleate Rhizoctonia (respectively UNR and BNR) strains belonging to the teleomorphic genus Ceratobasidium, but multinucleate Rhizoctonia (MNR) belonging to teleomorphic genus Thanatephorus and ectomycorrhizal fungal species, such as Suillus bovinus, were also included in DNA probe development work. Strain specific probes were developed to target rDNA ITS (internal transcribed spacer) sequences (ITS1, 5.8S and ITS2) and applied in Southern dot blot and liquid hybridization assays. Liquid hybridization was more sensitive and the size of the hybridized PCR products could be detected simultaneously, but the advantage in Southern hybridization was that sample DNA could be used without additional PCR amplification. The impacts of four Finnish BNR Ceratorhiza sp. strains 251, 266, 268 and 269 were investigated on Scot pine (Pinus sylvestris) seedling growth, and the infection biology and infection levels were microscopically examined following tryphan blue staining of infected roots. All BNR strains enhanced early seedling growth and affected the root architecture, while the infection levels remained low. The fungal infection was restricted to the outer cortical regions of long roots and typical monilioid cells detected with strain 268. The interactions of pathogenic UNR Ceratobasidium bicorne strain 1983-111/1N, and endophytic BNR Ceratorhiza sp. strain 268 were studied in single or dual inoculated Scots pine roots. The fungal infection levels and host defence-gene activity of nine transcripts [phenylalanine ammonia lyase (pal1), silbene synthase (STS), chalcone synthase (CHS), short-root specific peroxidase (Psyp1), antimicrobial peptide gene (Sp-AMP), rapidly elicited defence-related gene (PsACRE), germin-like protein (PsGER1), CuZn- superoxide dismutase (SOD), and dehydrin-like protein (dhy-like)] were measured from differentially treated and un-treated control roots by quantitative real time PCR (qRT-PCR). The infection level of pathogenic UNR was restricted in BNR- pre-inoculated Scots pine roots, while UNR was more competitive in simultaneous dual infection. The STS transcript was highly up-regulated in all treated roots, while CHS, pal1, and Psyp1 transcripts were more moderately activated. No significant activity of Sp-AMP, PsACRE, PsGER1, SOD, or dhy-like transcripts were detected compared to control roots. The integrated experiments presented, provide tools to assist in the future detection of these fungi in the environment and to understand the host infection biology and defence, and relationships between these interacting fungi in roots and soils. This study further confirms the complexity of the Rhizoctonia group both phylogenetically and in their infection biology and plant host specificity. The knowledge obtained could be applied in integrated forestry nursery management programmes.

The WNT Signaling Pathway Contributes to Dectin-1-Dependent Inhibition of Toll-Like Receptor-Induced Inflammatory Signature

Macrophages regulate cell fate decisions during microbial challenges by carefully titrating signaling events activated by innate receptors such as dectin-1 or Toll-like receptors (TLRs). Here, we demonstrate that dectin-1 activation robustly dampens TLR-induced proinflammatory signature in macrophages. Dectin-1 induced the stabilization of beta-catenin via spleen tyrosine kinase (Syk)-reactive oxygen species (ROS) signals, contributing to the expression of WNT5A. Subsequently, WNT5A-responsive protein inhibitors of activated STAT (PIAS-1) and suppressor of cytokine signaling 1 (SOCS-1) mediate the downregulation of IRAK-1, IRAK-4, and MyD88, resulting in decreased expression of interleukin 12 (IL-12), IL-1 beta, and tumor necrosis factor alpha (TNF-alpha). In vivo activation of dectin-1 with pathogenic fungi or ligand resulted in an increased bacterial burden of Mycobacteria, Klebsiella, Staphylococcus, or Escherichia, with a concomitant decrease in TLR-triggered proinflammatory cytokines. All together, our study establishes a new role for dectin-1-responsive inhibitory mechanisms employed by virulent fungi to limit the proinflammatory environment of the host.

Mapeamento de proteínas alvo para novos antifúngicos na fração microssomal e citosólica do patógeno humano Aspergillus fumigatus

A incidência de infecções fúngicas invasivas vem aumentando nos últimos anos. Estas infecções, em geral, apresentam altas taxas de mortalidade. A profilaxia com antifúngicos ainda é a estratégia mais comum na contenção da mortalidade e prevenção contra infecções fúngicas invasivas, porém, apresenta baixa eficiência, e relatos de resistência às drogas. Além disso, a terapia antifúngica é limitada a um pequeno grupo de drogas, como os polienos, azóis e equinocandinas. Desta forma, a busca de novos alvos de drogas é fundamental para o desenvolvimento de novos antifúngicos. Estudos in silico indicaram quatro genes como potenciais alvo de drogas em fungos patogênicos. Neste contexto, o objetivo deste trabalho foi verificar a expressão das proteínas codificadas por dois destes possíveis genes alvo, a proteína erg6, na fração microssomal, e trr1, na fração citosólica, em hifas de A. fumigatus. Visando alcançar este objetivo, foram primeiramente padronizadas todas as etapas de fracionamento celular visando isolar estas duas subfrações celulares de A. fumigatus. Posteriormente, foi otimizado o protocolo de extração e reidratação de proteínas microssomais bem como reidratação de proteínas citosólicas. Estes extratos foram submetidos a diferentes protocolos de fracionamento proteico em um sistema de eletroforese OFFGEL (OGE). Os resultados de Western immunoblot mostraram que estas duas proteínas, erg6 e trr1, são de fato expressas na fase filamentosa de A. fumigatus. O extrato proteico da fração microssomal submetido ao OGE em doze subfrações apresentou três subunidades da proteína erg6, reconhecidas pelo anticorpo monoclonal, com massas moleculares e pI distintos: uma subunidade de aproximadamente 79 kDa com pI entre 5,91 e 6,49, e outras duas subunidades de aproximadamente 35 kDa e 32 kDa, ambas com pI entre 6,49 e 7,08. A enzima erg6 foi descrita como um homotetrâmero em outros fungos. Porém, nossos resultados sugerem que, em A. fumigatus, a erg6 possui uma estrutura heterotetramérica. Quanto à proteína trr1, tanto no extrato total quanto nas frações resultantes do fracionamento em OGE, uma banda única de aproximadamente 40 kDa, com pI na faixa de 4,79 e 5,33, foi reconhecida pelo anticorpo policlonal. Desta forma, esta proteína parece ter uma estrutura homodimérica, assim como descrito em outros micro-organismos.

Antimicrobial activity of hydroxylbenzenesulfonailides derivatives of chitosan, chitosan sulfates and carboxymethyl chitosan

Chitosan, carboxymethyl chitosan (CIVICS) and chitosan sulfates (CSS) with different molecular weight were modified by reacting with 4-hydroxyl-5-chloride-1,3-benzene-disulfo-chloride or 2-hydroxyl-5-chloride-1,3 -benzene-disulfo-chloride to give 12 kinds of new hydroxylbenzenesulfonailides derivatives of them. The preparation conditions of the derivatives were discussed in this paper, and their structures were characterized by FT-IR and C-13 NMR spectroscopy. The solubility of the derivatives was measured in the experiment. In addition, their antimicrobial activities against four bacteria and five crop-threatening pathogenic fungi were tested in the experiment. Besides, the rule and mechanism of their antibacterial activities were discussed in this paper. (C) 2009 Published by Elsevier B.V.

Synthesis of acyl thiourea derivatives of chitosan and their antimicrobial activities in vitro

Three different acyl thiourea derivatives of chitosan (CS) were synthesized and their structures were characterized by FT-IR spectroscopy and elemental analysis. The antimicrobial behaviors of CS and its derivatives against four species of bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Sarcina) and four crop-threatening pathogenic fungi (Alternaria solani, Fusarium oxysporum f. sp. vasinfectum, Colletotrichum gloeosporioides (Penz.) Saec, and Phyllisticta zingiberi) were investigated. The results indicated that the antimicrobial activities of the acyl thiourea derivatives are much better than that of the parent CS. The minimum value of MIC and MBC of the derivatives against E coli was 15.62 and 62.49 mu g/mL, respectively. All of the acyl thiourea derivatives had a significant inhibitory effect on the fungi in concentrations of 50 - 500 mu g/mL; the maximum inhibitory index was 66.67%. The antifungal activities of the chloracetyl thiourea derivatives of CS are noticeably higher than the acetyl and benzoyl thiourea derivatives. The degree of grafting of the acyl thiourea group in the derivatives was related to antifungal activity; higher substitution resulted in stronger antifungal activity. (c) 2007 Elsevier Ltd. All rights reserved.

Phenotypical characterization in "Plasmopara halstedii" (sunflower downy mildew) isolates of several races

Phenotypic variation (morphological and pathogenic characters), and genetic variability were studied in 50 isolates of seven Plasmopara halstedii (sunflower downy mildew) races 100, 300, 304, 314, 710, 704 and 714. There were significant morphological, aggressiveness, and genetic differences for pathogen isolates. However, there was no relationship between morphology of zoosporangia and sporangiophores and pathogenic and genetic characteristics for the races used in our study. Also, our results provided evidence that no relation between pathogenic traits and multilocus haplotypes may be established in P. halstedii. The hypothesis explaining the absence of relationships among phenotypic and genetic characteristics is discussed.

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

Characterisation of ZG16p, a unique mammalian lectin from pancreatic zymogen granules

The mechanisms of secretory granule biogenesis and regulated secretion of digestive enzymes in pancreatic acinar cells are still not well understood. To shed light on these processes, which are of biological and clinical importance (e.g., pancreatitis), a better molecular understanding of the components of the granule membrane, their functions and interactions is required. The application of proteomics has largely contributed to the identification of novel zymogen granule (ZG) proteins but was not yet accompanied by a better characterization of their functions. In this study we aimed at a) isolation and identification of novel membrane-associated ZG proteins; b) characterization of the biochemical properties and function of the secretory lectin ZG16p, a membrane-associated protein; c) exploring the potential of ZG16p as a new tool to label the endolysosomal compartment. First, we have performed a suborganellar proteomics approach by combining protein analysis by 2D-PAGE and identification by mass spectrometry, which has led to the identification of novel peripheral ZGM proteins with proteoglycan-binding properties (e.g., chymase, PpiB). Then, we have unveiled new molecular properties and (multiple) functions of the secretory lectin ZG16p. ZG16p is a unique mammalian lectin with glycan and proteoglycan binding properties. Here, I revealed for the first time that ZG16p is highly protease resistant by developing an enterokinase-digestion assay. In addition I revealed that ZG16p binds to a high molecular weight complex at the ZGM (which is also protease resistant) and forms highly stable dimers. In light of these findings I suggest that ZG16p is a key component of a predicted submembranous granule matrix attached to the luminal side of the ZGM that fulfils important functions during sorting and packaging of zymogens. ZG16p, may act as a linker between the matrix and aggregated zymogens due to dimer formation. Furthermore, ZG16p protease resistance might be of higher importance after secretion since it is known that ZG16p binds to pathogenic fungi in the gut. I have further investigated the role of ZG16p binding motifs in its targeting to ZG in AR42J cells, a pancreatic model system. Point mutations of the glycan and the proteoglycan binding motifs did not inhibit the targeting of ZG16p to ZG in AR42J cells. I have also demonstrated that when ZG16p is present in the cytoplasm it interacts with and modulates the endo-lysosomal compartment. Since it is known that impaired autophagy due to lysosomal malfunction is involved in the course of pancreatitis, a potential role of ZG16p in pancreatitis is discussed.

Investigations on the host-parasite interface of a mycoparasite system /

The cell wall composition of Choanephora cucur - bitarum and the host-parasite interface, after infection with Piptocephalis virginiana , were examined in detail. The cell walls of C_. cucurbitarum were determined to be composed of chitin (17%), chitosan (28.4%), neutral sugars (7.2%),uronic acid (2.4%), proteins (8.2%) and lipids (13.8%). The structure of hyphal walls investigated by electron microscopy of shadowed replicas before and after alkali-acid hydrolysis, showed two distinct regions: microfibrillar and amorphous. The microfibrils which were composed of mainly chitin, were organized into two distinct layers: an outer, thicker layer of randomly orientated microfibrils and an inner, thin layer of parallel microfibrils.Electronmicrographs of the host-parasite interface of C_. cucurbitarum and the mycoparasite , P_. virginiana , 30 h following inoculation, showed that the sheath zone has a similar electron density to that of the host cell wall. The sheath was not present around the young (18 h old) haustorium. High-resolution autoradiographs of infected host hyphae showed that radioactive N-acetyl-D-glucosamine , a precursor of chitin, was incorporated preferentially in the host cell wall and sheath zone. Cell fractionation of label fed hyphae showed that 84% of the label was present in the cell wall and specifically in the chitin portion of the wall. The antifungal antibiotic, Polyoxin D, a specific inhibitor of the enzyme, chitin synthetase, suppressed the incorporation of the label in the cell wall and sheath zone and resulted in a decrease in electron density of the developing sheath. The significance of these results is discussed in the light of host resistance.

Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.

Micoses sistémicas

Este trabalho teve como objetivo a análise das micoses sistémicas, no que respeita à sua identificação, morfologia dos agentes patogénicos que lhes dão origem, onde são endémicas, os sintomas que apresentam, os métodos de diagnóstico e as terapêuticas disponíveis para cada patologia. As micoses sistémicas são infeções causadas por fungos patogénicos primários que têm o trato respiratório como porta de entrada, e a partir daí podem disseminar-se por todo o organismo. Os agentes antifúngicos são usados no tratamento destas infeções, em que, no caso das infeções sistémicas, predominam o uso de cetoconazol (polieno), fluconazol e itraconazol (azóis). Existem ainda estudos que demonstram que as vacinas atenuadas podem ser usadas na profilaxia destas infeções. Com este trabalho conclui-se que as infeções fúngicas estão longe de ser extintas, uma vez que se verifica cada vez mais a existência de resistências por parte dos fungos aos fármacos, sendo o diagnóstico e a terapêutica usada dois parâmetros fundamentais para um tratamento eficaz.

Folsomia candida (Collembola): A "standard" soil arthropod

Folsomia candida Willem 1902, a member of the order Collembola (colloquially called springtails), is a common and widespread arthropod that occurs in soils throughout the world. The species is parthenogenetic and is easy to maintain in the laboratory on a diet of granulated dry yeast. F. candida has been used as a "standard" test organism for more than 40 years for estimating the effects of pesticides and environmental pollutants on nontarget soil arthropods. However. it has also been employed as a model for the investigation of numerous other phenomena such as cold tolerance, quality as a prey item, and effects of microarthropod grazing on pathogenic fungi and mycorrhizae of plant roots. In this comprehensive review. aspects of the life history, ecology, and ecotoxicology of F candida are covered. We focus on the recent literature, especially studies that have examined the effects of soil pollutants on reproduction in F candida using the protocol published by the International Standards Organization in 1999.