Characterization of fungi with molecular methods

Characterization of fungi with molecular methods

by

Katharina Pelant

This work was carried out as diploma thesis at

Karl-Franzens-Universität Graz,

Institut für Botanik.

Graz, 2002

Supervisors:

Univ.-Ass. Mag. Dr. Martin Grube

Ao. Univ.-Prof. Mag. Dr. Helmut Mayrhofer

רחשל

Acknowledgement

I would like to express my gratitude to all those who gave me the possibility to complete this thesis.

Prof. Dr. Helmut Mayrhofer for giving me the opportunity to start the diploma thesis at the Institute of Botany, Graz, for providing the working place, and for his support at every step of my thesis.

Dr. Martin Grube for introducing me to the world of molecular biology and genetics and improving my knowledge in long discussions, his advice during the practical work in the laboratory and his continuous discussion of the thesis. Furthermore I want to thank him for the recruitment of the project that became the main part of this work.

Dr. Michael Stelzl, director of Hygienicum AG, for giving me the project and financial support. Here I also want to acknowledge the employees of Hygienicum AG, who showed me the microbiological techniques required in the project.

Dr. Herbert Huss for information regarding Ramularia collo-cygni and for organising samples from all over Europe.

D.I. Herbert Bistrich and Dr. Edith Sachs for the provision of Ramularia collo-cygni samples.

Ing. Sigrun Kraker and Mag. Elisabeth Baloch for their practical introduction to the laboratory at the beginning of my work and their never-ending help and support.

My colleagues for all their help, support, interest and valuable hints.

My parents for financing my studies, but much more for their encouragement and their love during this time.

My sisters Lena and Johanna and my brother Thomas for their friendship and motivation.

My fiance Shahar for spending weekends together with me at the university, and being always there for technical and mental support.

IndexABBREVIATIONS......................................................................................................................2APPROACH...............................................................................................................................3

I MOLECULAR CHARACTERIZATION OF THE MYCOFLORA OF HAZELNUTS.....................................................................................................................................................4

1. INTRODUCTION...................................................................................................................51.1 Mycotoxins...................................................................................................................51.2 Hazelnuts (Corylus sp.) as food crop...........................................................................91.3 Introduction to food borne fungi................................................................................111.4 Fungal species on hazelnuts......................................................................................13

2. MATERIALS AND METHODS..............................................................................................212.1 Materials....................................................................................................................212.2 Cultivation of the moulds...........................................................................................232.3 Determination of germ numbers................................................................................242.4 Identification of fungi using morphological criteria.................................................242.5 Identification of fungi using molecular methods.......................................................25

3. RESULTS...........................................................................................................................323.1 Germ numbers............................................................................................................323.2 Hazelnut contaminating fungal species.....................................................................333.3 PKS fragment patterns of hazelnut contaminating fungi...........................................38

4. DISCUSSION......................................................................................................................434.1 Quantitative analysis of the mycoflora......................................................................434.2 The source of mycotoxins in hazelnut paste...............................................................454.3 Comparison of the suppliers of the hazelnuts............................................................464.5 Mycotoxic fungi found in raw, roasted hazelnuts and hazelnut paste.......................50

II MOLECULAR CHARACTERIZATION OF THE PHYTOPATHOGENIC FUNGUS RAMULARIA COLLO-CYGNI.............................................................................52

1. INTRODUCTION.................................................................................................................532. MATERIALS AND METHODS..............................................................................................54

2.1 Materials....................................................................................................................542.2 Cultivation of Ramularia collo-cygni........................................................................552.3 Sequencing of ITS, IGS, mtSSU and chitin synthase genes.......................................552.4 Molecular Markers....................................................................................................56

3. RESULTS...........................................................................................................................593.1 Sequences of ITS, IGS, mtSSU and chitin synthase genes.........................................593.2 Molecular Markers....................................................................................................60

4. DISCUSSION......................................................................................................................62SUMMARY..............................................................................................................................63APPENDIX 1...........................................................................................................................66APPENDIX 2...........................................................................................................................90References.............................................................................................................................91

Abbreviations

AFLP Amplified fragment length polymorphismaw Water activityBBA Federal Biological Research Center for Agriculture and ForestryBLAST Basic Local Alignment Search Toolbp Base pairdATP Desoxyadenine triphosphatedCTP Desoxycytidine triphosphateddNTP Dideoxyribonucleotide triphosphateDGGE Denaturing gradient gel electrophoresisdGTP Desoxyguanidine triphosphateDNA Desoxyribonucleic aciddTTP Desoxytyridine triphosphatedNTP Deoxyribonucleotide triphosphateELISA Enzyme linked immuno sorbent assayETS External transcribed spacerEU European UnionEDTA Ethylenediamine tetra-acetic acidFAO Food and Agriculture Organization of the United NationsHPLC High-performance liquid chromatographyIGS Intergenic SpacerITS Internal transcribed SpacerJECFA Joint FAO/WHO Expert Committee on Food Additiveskb Kilo base pairsME Malt extractMgCl2 Magnesium chloridemtSSU Mitochondrial small subunitNCBI National Center for Biotechnology Informationnt Nucleotide(s)OTA Ochratoxin APCR Polymerase chain reactionPKS Polyketide synthasePTDI Provisional Tolerably Daily IntakeRFLP Restriction fragment length polymorphismRAPD Randomly amplified polymorphic DNARNA Ribonucleic acidRPM Rotations per minuteRT Room temperatureTaq Thermus aquaticusTLC Thin-layer chromatographytRNA Transfer ribonucleic acidU UnitsUV Ultra violetWHO World Health OrganizationYGC Yeast glucose chloramphenicol

Approach

Identification

Identification of micro fungal species has relied mainly on morphological characters and taxonomic criteria. However, morphological features are often insufficient for identification of microfungi due to morphological divergence among isolates of the same species. Molecular methods are therefore used in addition for identification and taxonomic classification of microfungi. Discrepancies in morphological and molecular typification will be presented in Part I of this investigation. Most studies that are based on DNA sequences emphasize ribosomal DNA genes (e.g. KURTZMAN & ROBNETT 1997, PEDERSEN et al. 1997, HENRY et al. 2000, CAPPA & COCCONCELLI 2001), only recently started to use protein-encoding loci (e.g. GEISER et al. 1998). Other approaches use fragment patterns (RFLP, RAPD, AFLP), or detection of particular biosynthesis genes of secondary metabolites (e.g. FÄRBER et al. 1997). Allele specific typification of strains like heteroduplex analysis (e.g. KUMEDA & ASAO 2001), DGGE (e.g. VAINIO & HANTULA 2000) and microsatellites (e.g. MOON et al. 1999) is only rarely used. In this study identification of hazelnut contaminating fungi was carried out using the ubiquitous nuclear internal transcribed spacer (ITS) regions 1 and 2, which separate the coding rDNA genes. The ITS 1 and ITS 2 regions were chosen due to their high abundance in public databases (GenBank), which were queried for comparisons with the sequences produced in this work. Another reason to choose these loci is their high number in the genome. They are arranged as tandem repeats, which makes it easy to amplify fragments even from very low concentrations of template DNA. This might prove useful for rapid detection of fungi, and without the necessity to culture them.

Characterization

Several molecular tools can be utilized for characterization, differentiation and strain typing of fungi. Multilocus sequence typing requires a series of simple, well-characterized, independent and stable polymorphic loci (TAYLOR et al. 1999). One of the options to gather this kind of data is sequencing of known genes as it was applied to the phytopathogenic fungus Ramularia collo-cygni. Fingerprinting methods with molecular markers are useful for distinguishing clones. This is why restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD) analysis were implemented on R. collo-cygni.Secondary metabolite encoding genes can be used for identification as well as for characterization of fungi. As for the hazelnut contaminating fungi, the genera Aspergillus and Penicillium were characterized further by fragment patterns of biosynthetic genes for polyketides (PKS).

I Molecular characterization of the mycoflora of hazelnuts

I Introduction

1. Introduction

1.1 Mycotoxins

1.1.1 General information about mycotoxins

The definition of mycotoxins is somehow vague. According to WEIDENBÖRNER (2000) mycotoxins are low-molecular, aromatic, sometimes aliphatic compounds of microfungi that are produced in the steady state of growth when secondary metabolism is dominant. Beside mycotoxins not being of low molecular weight, this definition is inconsistent as there is a wide range of secondary metabolites (e.g. antibiotics, alkaloids and gibberellins) that are produced by microfungi and are not considered as mycotoxins. The definition of BENNET (1987) adds one neglected aspect: Mycotoxins are toxic in low concentrations to higher vertebrates and other animals when introduced via a natural route. The “Dictionary of the Fungi” gives the widest definition, stating that a toxin is a non enzymatic metabolite of one organism which is injurious to another, and a mycotoxin is a toxin produced by a fungus, especially one affecting humans or animals (KIRK et al. 2001).

Diseased or mouldy food and feed can cause mycotoxicosis (see 1.1.2), which includes the induction of cancer and immune deficiency. Therefore, mycotoxin contamination of food and feed is a worldwide problem with great relevance to human and animal health. Presumably 25 % of worldwide produced foods are contaminated by mycotoxins (WEIDENBÖRNER 2000). By now almost 400 mycotoxins formed by 350 species are known. Some fungi produce a single toxin only, while others may produce many toxic compounds, which may be shared across fungal genera. Nevertheless, there are mycotoxins related to a specific genus, whereby the emphasis is placed on the genera Aspergillus, Penicillium, Fusarium, Alternaria and Claviceps. These fungi produce mycotoxins belonging to eight groups that are of relevance in food industry: aflatoxins, citrinin, fumonisins, ochratoxins, patulin and other small lactones, trichothecenes, zearalenone and ergot alkaloids (Table 1).

Table 1. Mycotoxins of relevance to human health (GEISEN 1998)

Genus Mycotoxins

Aspergillus Aflatoxins, sterigmatocystin, cyclopiazonic acid

Penicillium Patulin, ochratoxin A, citrinin, penitrems, cyclopiazonic acid, PR toxin

Fusarium Trichothecenes (T2 Toxin, deoxynivalenol, nivalenol, diacetoxyscirpenol), zearalenone (F2 toxin), fumonisins

Alternaria Tenuazonic acid, alternariol, alternariol methylether, altertoxins

Claviceps Ergot alkaloids

I Introduction

1.1.2 Mycotoxicosis and food safety

The relevance of mycotoxins to human health received little attention until the early 1960s when the Turkey X disease killed thousands of turkey poults in Great Britain and the aflatoxins where discovered (see 1.1.3). Since then, many diseases of man and animals could be associated with mycotoxins spoiling food and feed. Ergotism (Saint Anthony’s fire) is the oldest known mycotoxicosis caused by the toxic sclerotia of Claviceps sp. that contaminate rye flour. Like ergotism, other diseases claimed also thousands of deaths. Some examples are the yellow rice disease, initiated by the consumption of citreoviridin contaminated rice in Japan, the alimentary toxic aleukia in various parts of Russia, caused by the consumption of Fusarium-contaminated grain, and human primary liver cancer, predominantly found in Africa and South East Asia and correlated with the ingestion of aflatoxins. An example for an epidemic mycotoxicosis of animals is the mycotoxic porcine nephropathy of pigs, which is caused by the ingestion of ochratoxin A. In addition to these complex diseases, mycotoxins induce other severe biological effects at low levels of exposure. STEYN (1995) summarized these effects as carcinogenic (aflatoxins, ochratoxins and fumonisins), mutagenic (aflatoxins and sterigmatocystin), teratogenic (ochratoxins), estrogenic (zearalenone), hemorrhagic (trichothecenes), immunotoxic (aflatoxins and ochratoxins), nephrotoxic (ochratoxins), hepatotoxic (aflatoxins), dermotoxic (trichothecenes) and neurotoxic (ergotoxins and others). In recent years, the general concern about the potential effects of mycotoxins on the health of man and animals is increasing. For this reason, many countries have regulations governing the maximum concentrations of mycotoxins in food and feed. Food standards like the legislation of maximum limits for mycotoxins are formulated and harmonized by the Codex Alimentarius, which was created in 1963 by FAO and WHO. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) is the body responsible for the risk assessment and provides Codex Alimentarius with scientifically based evaluation of toxicity of food additives and contaminants. The EU regulations and proposals are roughly similar to the worldwide Codex legislation, but contain more detail. The basic principles of EU legislation on contaminants in food are presented in Council Regulation 315/93/EEC (1993). Maximum levels are set for certain contaminants in foodstuffs in Commission Regulation 466/2001.Via risk assessment maximum levels of food contaminants that are unlikely to be of health concern are set. For the risk assessment, the results of the exposure assessment (estimated probable daily intake) are compared with the hazard assessment (estimated tolerable daily intake). The hazard assessment is usually based on the determination of a no-observed-effect-level in long-term toxicological studies, and the application of a safety factor. The exposure assessment is evaluated with data on the occurrence of mycotoxins in various commodities and food intake data. Both the hazard assessment and the exposure assessment contain many uncertainties, and thus the actual health risks are suggested to be somewhat less critical than estimated (KUIPER-GOODMAN 1995).Determination of mycotoxin occurrence and concentration in food is necessary to receive data for the exposure assessment. Analytical methods routinely used nowadays are mainly based on thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and enzyme linked immuno sorbent assay (ELISA). BOENKE (1998) gives a summary of

I Introduction

mycotoxin determination in food and feed and a validation for standard methods of the European Commission.Commission Directive 1998/53/EC defines acceptable sampling and analysis methods for aflatoxins, and Commission Directive 2002/26/EC for ochratoxin A (European Commission).In Austria, the federal institutes for food inspection (Bundesanstalten für Lebensmittel-untersuchung), the Q-lab of Agrarmarkt Austria and the Institute for Agrobiotechnology (IFA) Tulln execute mycotoxin analysis.

Table 2. Maximum aflatoxin limits in various nuts, groundnuts, dried fruits and products thereof, according to the Annex to Commission Regulation (EC) No 466/2001.

ProductMaximum aflatoxin limit (µg/kg)

B1 B1+B2+G1+G2

Groundnuts, nuts and dried fruit and processed products thereof, intended for human consumption or as an ingredient in foodstuffs

2 (6) 4(6)

Nuts and dried fruit to be subjected to sorting, or other physical treatment, before human consumption or use as an ingredient in foodstuffs

5 (6) (8) 10 (6) (8)

Table 3. Maximum limits for several mycotoxins in foods in Austria (CREPPY 2002)

Mycotoxin Maximum limit (µg/kg or µg/l)

Foods

Aflatoxin B1 1 All

Aflatoxin B2+G1+G2 5 All

Aflatoxin M1+B1+B2+G1+G2

0.02 Children’s food

Aflatoxin M1 0.050 Milk

Desoxynivalenol 750 Wheat

Ochratoxin A 5 Cereals

Zearalenone 60 Cereals

In Austria, the regulations of aflatoxin levels in hazelnuts are set according to the Annex to Commission Regulation (EC) No 466/2001 (Table 2) with 2 µg/kg for aflatoxin B1 and 4 µg/kg for total aflatoxins. In contrast, there are no regulations for ochratoxin A in hazelnuts, although this mycotoxin has been found in hazelnuts (WEIDENBÖRNER 2001a, ELMADFA & BURGER 1999). However, according to ELMADFA & BURGER (1999) this fact should not be a matter of concern, as the by the JECFA postulated Provisional Tolerably Daily Intake (PTDI) of 14 ng/kg body weight is achieved only by an average of 10 % of Austrians. Cereals,

I Introduction

beverages and meat products contribute 93 % to dietary intake of ochratoxin A, dried fruits though high contents only 0.3 %. Therefore hazelnuts can be considered as safe concerning dietary intake of ochratoxin A.

1.1.3 Aflatoxins

As aflatoxins and ochratoxin A have been reported on hazelnuts, these two groups of mycotoxins shall be described in more detail.The aflatoxins were discovered in 1960, when the Turkey X disease caused the deaths of more than 100 000 of turkey poults in Great Britain. It turned out, that Brazilian peanut meal in the feed was contaminated by four highly toxic compounds, the aflatoxins B1, B2, G1 and G2. Since then the aflatoxins have been the most widely studied mycotoxins. Aflatoxins are polycyclic, unsaturated and highly substituted coumarins. There are approximately 20 aflatoxins identified with the aflatoxins B1, B2, G1, G2, M1, M2 as most common. The B aflatoxins fluoresce blue under UV light, whereas the G aflatoxins show green fluorescence under UV light. From these, the M aflatoxins are distinguished as results of metabolic processes in the digestive tract of mammals and were found in milk the first time. However, they were also isolated from maize and peanuts. Aflatoxins with the index number 1 show greater toxic property compared to aflatoxins with the index number 2. For aflatoxins with the index 1, there is no threshold dose below that no tumor formation would occur. Only a zero level of exposure will result in no risk. Therefore Aflatoxin B1 is considered as the strongest natural genotoxic carcinogen, causing hepatic cancer. Besides their carcinogenic effect aflatoxins are mutagenic, teratogenic and hepatotoxic.Aflatoxins are as far as known produced by Aspergillus flavus, A. nomius and A. parasiticus. However only approx. 50 % of A. flavus strains are aflatoxin producers and also only a certain part of A. nomius and A. parasiticus strains. Foods most commonly contaminated by aflatoxins include maize, peanuts, pecans, almonds, hazelnuts, Brazil nuts, pistachio nuts, and walnuts (BETINA 1989; MILLER 1995; WEIDENBÖRNER 1998, 2000, 2002; CREPPY 2002).

1.1.4 Ochratoxin A

Ochratoxin A (OTA) is the major toxic compound out of a group of nine or more orchratoxins, which are composed of a 3,4-dihydroxy-3-methylisocoumarin linked via the 7-carboxy group to L--phenylalanine by an amide bond (WEIDENBÖRNER 2001a).OTA is strong nephrotoxic, hepatotoxic, immunosuppressive, teratogenic, mutagenic and cancerogenic. Recent data from in vitro and in vivo tests have also provided evidence of the genotoxic potential of OTA (Scientific Committee on Food, 1998). Furthermore it is suspected as the partial cause of kidney damage in areas of chronic exposure in parts of Eastern Europe, e.g. of the Balkan endemic nephropathy. For the first time OTA was isolated from cultures of Aspergillus ochraceus in 1965, but several other fungi also produce this mycotoxin often together with citrinin. The main producers are members of the Aspergillus ochraceus group. Further producers are Aspergillus

I Introduction

melleus, A. sclerotiorum, A. sulphureus, A. niger, Eurotium herbariorum, Penicillium ssp. and Petromyces alliaceus. Penicillium verrucosum predominates in the temperate climate of Europe or North America infecting stored cereals and cereal products. These foods seem to be the main contributor to the dietary intake of OTA in the EU (Scientific Committee on Food 1998).

1.2 Hazelnuts (Corylus sp.) as food crop

1.2.1 Cultivation of Corylus sp.

The genus Corylus belongs to the Corylaceae, a family of deciduous, monoecious trees and shrubs. However, several authors include Corylus in the Betulaceae. All species of Corylus produce edible nuts. C. colurna, C. avellana var. pontica and C. maxima are cultivated in Turkey for production of hazelnuts (MANSFELD 1986, ÖZDEMIR 2001).C. avellana (hazelnut, cobnut, filbert, Haselnuss) is growing as shrubs up to 6 m with smooth brown bark. The nuts grow in infructescences of 1-4 and each nut is surrounded by the involucre, which is about as large as the nut. The pericarp is hard, loosely covering the smooth to shriveled kernel. Time of pollination is mid-January to mid-February. Fertilization then takes place in July, and the nut rapidly develops, maturing by late August.C. avellana is native to the Temperate Zone and the Subtropics and distributed worldwide. The cultivated hazelnut prefers regions with mild, moist winters and cool summers. For this reason, most production is located near large bodies of water at mid latitudes in the Northern Hemisphere (along the Black Sea in Turkey, the Atlantic coast in France, the Willamette Valley in Oregon) (RIEGER 2002).C. colurna (Turkish hazel, Turkish filbert, Baumhasel, Türkische Hasel) is a tree, sometimes up to 22 m. It has a characteristic involucre, which is much longer than the nut. C. colurna is native to the subtropics and locally distributed in the Balkan Peninsula, Romania and Turkey (MANSFELD 1986).C. maxima (giant filbert, Lambertsnuss) is growing as a shrub or small tree like C. avellana, but the involucre is tubular, contracted above the nut and dentate at the apex. It is native to the temperate zone and the subtropics and distributed regionally in southeast Europe (MANSFELD 1986).

I Introduction

1.2.2 Hazelnut industry

In 2001 the worldwide production of shelled hazelnuts amounted 875,375 t (FAO). Turkey is with 75-80 % the most important producer followed by Italy with 15 % and finally the USA with 2 %. In 2001 Turkey produced 630,000 t hazelnuts, Italy 120,000 t and the USA 43,540 t. However, the yield per area unit was two times higher in the USA than in Turkey and Italy, due to different production methods.In Turkey, hazelnuts are cultivated in an area of about 550,000-600,000 ha. The production area is spread along the hilly Black Sea coast and on shallow land outside the Istanbul area. Turkish hazelnuts usually ripen between early and late August depending on the altitude of the orchard and climatic conditions. The hazelnuts are harvested by hand from the trees and traditionally sun dried (Istanbul Hazelnut Exporters Union 2002).Economically important varieties of Corylus in Turkey are selected from C. avellana var. pontica, C. maxima and C. colurna var. glandulifera (ÖZDEMIR 2001). There are three main varieties of hazelnuts: Ordu, Akcacoga and Giresun. These varieties are further classified as either Levant quality (which includes Ordu and Akcacoga) or Giresun quality (which is named after the region in which it is grown). Giresun quality hazelnuts have an oil content of 62 %, which make them very flavorful and ideal for blanching. Giresun nuts are preferred for confectionery goods like chocolate bars, truffles and chocolate-covered hazelnuts. But the largest percentage of Turkish hazelnuts is Levant quality with an oil-content of 55 %. Levant nuts are widely used as an ingredient in confectioneries, bakery goods, ice cream and mixed nuts (SAMON 1999).

Table 4. Examples for the usage of hazelnuts (Istanbul Hazelnut Exporters Union 2002)

Usage Types of processed hazelnuts

Chocolate Whole kernel, diced, paste, meal

Bakery Diced, meal, paste

Mixed nuts Roasted, blanched, natural whole kernels

Ice cream Diced

Salads, coffee Hazelnut oil

Home cooking Hazelnut oil

Hazelnuts are among the nuts (almonds, walnuts, pistachios, groundnuts) the most important export goods in Turkish agriculture. Hazelnut export of Turkey comprises shelled hazelnut kernels by 70 % and processed hazelnuts by 30 %. In 2001 258,124 t of shelled hazelnuts with a value of 739,970 130 US$ were exported from Turkey (FAO). Shelled and peeled hazelnuts are exported in 50 kg or 80 kg jute bags. Processed hazelnuts are consigned in polypropylene vacuum packed range from 1-25 kg or in covered carton boxes. Hazelnut paste is exported in barrels (Black Sea Hazelnuts and Product Exporter’s Union 2002).

I Introduction

1.3 Introduction to food borne fungi

1.3.1 Definition of moulds

Fungal organisms can contaminate nearly every food. Most food borne fungi are commonly summarized in the ecological term “moulds”, although they belong to different systematic taxa (WEIDENBÖRNER 1998). Moulds are growing on organic material as fuzzy, cottony, woolly, or powdery textured colonies (SCHMIDT-LORENZ 1977). According to WEIDENBÖRNER (2000) a scientific definition of moulds includes following characteristics: Moulds appear with a filamentous growth, high growth rates, high sporulation, mainly vegetative propagation and a parasexual cycle. Furthermore they are ubiquitous in nature, show a cosmopolitan distribution and produce secondary metabolites (mycotoxins). KIRK et al. (2001) defines moulds as microfungi that have a well-marked mycelium or spore mass and are economically important saprobes.When speaking about food borne fungi, the term “moulds” will also be used further on.

1.3.2 Environmental factors affecting mycotoxin production

Both fungal growth and mycotoxin production are dependent on environmental factors, with the limits for mycotoxin production usually being narrower than those for growth only (FRISVAD & SAMSON 1991). The relevant factors for mycotoxin production are summarized in Table 5 and their interaction is shown in Figure 1. The factors influence the fungus interacting with each other, either increasing or decreasing growth and mycotoxin production.

Fig. 1. Interaction of environmental factors influencing the mycotoxin production

I Introduction

Table 5. The relevant factors for mycotoxin production

Physical factors Chemical factors Biological factors

Temperature Atmosphere Fungal plant pathogens

Water content Substrate composition Microbial Competition

Mechanical damage pH

Time/season Fungicides

Temperature. Mycotoxin production is greatly influenced by temperature and water activity. Usually mycotoxin production occurs at the same temperatures as the optimal growth. Penicillium grows well and produces mycotoxins at lower temperatures than Aspergillus. Aflatoxin synthesis can happen from 12-42 °C and is optimal from 24-28 °C (REISS 1998). Both, A. flavus and A. niger are able to grow between 8 and 45 °C (PITT & HOCKING 1997). At 5 °C Aspergillus cannot produce aflatoxins and ochratoxin anymore, whereas Penicillium and Fusarium are able to produce mycotoxins (BULLERMAN 1984, WEIDENBÖRNER 1998).

Water content. The water content of a substrate is given as water activity (aw) or as water content in percent (%). But using the second is problematic, as it includes also the “bound” water, which is unavailable for fungi. For this reason aw is the most commonly used value. aw

is defined as the ratio of the vapor pressure of water in a material (p) to the vapor pressure of pure water (p0) at the same temperature.

aw = p/p0

There are several factors, which control aw in a system. These factors are osmotic and matrix effects, that reduce the relative humidity as compared to pure water. aw is also temperature dependent. Most of food borne fungi grow at a minimal aw of 0.8, which is lower than the aw

needed for bacterial growth (0.9). Xerophilic moulds grow at minimal aw from 0.75-0.65 and can spoil low water activity products, for example grain, nuts, herbs, jam, dried fish and fruits. Foodstuffs with aw ≤ 0.6 are protected from microbial spoilage. The optimal aw for moulds are usually close to 1, for xerophilic fungi the values range from 0.96-0.90 (WEIDENBÖRNER 1998). Mycotoxin production occurs at higher water contents than needed for growth.

pH. Most food borne fungi can grow from pH 2.5 to pH 9.5 with an optimal pH from 4.5 to 6.5. Mycotoxin production usually takes place at a different pH optimum than fungal growth (WEIDENBÖRNER 1998).

Atmosphere. According to FRISVAD & SAMSON (1991) the concentrations of oxygen and carbon dioxide in the atmosphere and especially of dissolved oxygen in the substrate strongly influence growth and mycotoxin production by various moulds. The required amounts differ from species to species. Generally a combination of low oxygen content and high carbon dioxide inhibits growth and mycotoxin production of Aspergillus and Penicillium species.

I Introduction

TANIWAKI et al. (2001) showed that in 40 % CO2 and 1 % O2 the growth of A. flavus in cheese is reduced by 65 %, and the level of aflatoxin B1 production is insignificant. Also in the most favorable atmosphere studied (20 % CO2 and 5 % O2) the aflatoxin B1 production is reduced by a factor of 1000 compared to production in air. The level of cyclopiazonic acid production by P. commune in 20 % CO2 and 5 % O2 decreased to 8 % of that in air.

Substrate composition. Mould fungi are heterotroph organisms and therefore need organic compounds as glucose, maltose, saccharose and other water-soluble carbohydrates. Moulds cause mainly spoilage of carbohydrate-rich substrates, sometimes very specific to a certain composition. For example Penicillium crustosum, P. commune and P. echinulatum are common only on nuts and other lipid- and protein-rich substrates like meat and cheese (FRISVAD & SAMSON 1991).

Microbial Competition. The presence of competing microorganisms can restrict fungal growth and mycotoxin production. For example Aspergillus niger, Rhizopus stolonifer or lactic bacteria decrease or inhibit aflatoxin production (WEIDENBÖRNER 2001a).

1.4 Fungal species on hazelnuts

1.4.1 Phylogenetic classification of fungal genera found on hazelnuts

The fungi of the hazelnut mycoflora belong either to the Zygomycetes, the Ascomycetes or represent the anamorphic state of an Ascomycete. The Zygomycetes are characterized by the production of zygospores, nonseptate hyphae and asexual reproduction by sporangia or conidia. Particularly the Zygomycetes order Mucorales is related to food and beverage spoilage (BEUCHAT 1987). Ascomycetes have septate hyphae and reproduce sexual by ascospores borne inside asci and asexual by conidia.Anamorphic fungi (Fungi Imperfecti) are fungi that are disseminated by propagules not formed from cells where meiosis has occurred. Most of these propagules can be referred to as conidia (KIRK et al. 2001). Aspergillus and Penicillium are anamorphic genera that can be related to Ascomycetes teleomorphs. However, many anamorphic fungi stay unresolved and cannot be connected to a teleomorphic state. Mycologists have long used a system of classification that allows anamorphs to be named separately from the holomorph of which they form a part. As a consequence, many fungi can have two different names. For example, the name Eurotium chevaleri pertains to a holomorph with both ascospores and conidia, whereas Aspergillus chevaleri pertains only to the anamorph of the same fungus.

I Introduction

According to PITT & HOCKING (1997) and DE HOOG et al. (2000) the fungal genera found on hazelnuts are classified as follows (abbreviations of the authors according to KIRK & ANSELL 1992):

Division: ZygomycotaOrder: Mucorales

Family: MucoraceaeMucor P. Micheli: Fr.Rhizopus Ehrenb.

Family: SyncephalastraceaeSyncephalastrum J. Schröt.

Division: AscomycotaOrder: Eurotiales

Family: TrichocomaceaeAspergillus Fr.: Fr.Penicillium Link

Order: DothidealesFamily: Mycosphaerellaceae

Cladosporium LinkFamily: Dothioraceae

Aureobasidium Viala & G. BoyerOrder: Pleosporales

Family: PleosporaceaeAlternaria Nees: Fr.

Order: HypocrealesFamily: Hypocreaceae

Trichoderma Pers.Order: Moniliales

Trichothecium Link

1.4.2 Propagation of fungi on hazelnuts

Mould contamination of hazelnut is widespread, and is an important risk for human health (SANCHIS et al. 1988).Turkish hazelnuts are mainly contaminated by Aspergillus fumigatus, A. flavus, A. versicolor, and Penicillium chrysogenum (SHAHIN et al. 1994). In hazelnuts of Egyptian provenience Aspergillus spp., Penicillium spp., Eurotium spp. and Cladosporium spp. are dominant (ABDEL-HAFEZ & SABER 1993). SENSER (1979) found the genera Aspergillus, Penicillium, Fusarium and Rhizopus stolonifer dominant on hazelnut samples from German wholesale and retail industry. Saudia Arabian Hazelnuts are particularly spoiled by Aspergillus spp., Penicillium spp., Eurotium spp., Rhizopus stolonifer and Trichoderma hamatum (ABDEL-GAWAD & ZOHRI 1993).

I Introduction

Particularly fungi of the genera Penicillium, Aspergillus and Eurotium cause spoilage of hazelnuts (ÖZDEMIR 1997).

The mycoflora of hazelnuts has been investigated by SANCHIS et al. (1988), ABDEL-GAWAD & ZOHRI (1993), ABDEL-HAFEZ & SABER (1993), SAHIN & KALYONCUOGLU (1994), SENSER (1979), REISS (1998), WEIDENBÖRNER (1998) and ÖZDEMIR & ÖZILGEN (2001). On average 19 fungal taxa have been found. ABDEL-GAWAD & ZOHRI (1993) reported the highest number of 38 taxa. A total count of 77 fungal species has been reported on hazelnuts (Table 6).

Table 6. Fungi that have been reported on hazelnut kernels and their potential mycotoxins

Fungal speciesPotential Mycotoxins (not verified on hazelnut) according to FRISVAD & SAMSON (1991), SAMSON et al. (1995), PITT & HOCKING (1997)

Absidia corymbifera 3, 5 Not reported

Acremonium strictum 8 Not reported

Acremonium sp.6 (Cephalosporium sp.) Not reported

Alternaria sp. 6 Alternariols and others

A. alternata 1, 8 (A. tenuis 2) Alternariols, altertoxins, tenuazonic acid

A. humicola 2 Alternariols

A. tenuissima 1 Alternariols, tenuazonic acid

Aspergillus sp. 6 Several

Aspergillus candidus 2, 8 Candidulin, terphenyllin, xanthoascin

Aspergillus flavus 1, 2, 3, 4, 5, 7, 8 Aflatoxin B1, cyclopiazonic acid

A. fumigatus 1, 2, 5, 8 Fumagilin, gliotoxin

A. glaucus 7 Physcion

A. granulosus 2 Not reported

A. japonicus 1 Not reported

A. niger 1, 4, 5, 6, 8 Ochratoxin A

A. ochraceus 1 Ochratoxins A, B, C; penicillic acid

A. oryzae 1, 2 Cyclopiazonic acid

A. parasiticus 1, 2, 3, 8 Aflatoxin B1

A. sydowii8 Not reported

A. proliferans 1 -

A. tamarii 1, 2, 8 Cyclopiazonic acid, fumiclavine, kojic acid

A. terreus 1, 2, 8 Territrems

A. versicolor 1, 2, 5, 8 Sterigmatocystin

A. wentii 1 Emodin

Botryotrichum piluliferum 1 -

Chaetomium globosum 1, 8 Not reported

Cladosporium sp. 6 Cladosporic acid

I Introduction

Table 6. Fungi that have been reported on hazelnut kernels and their potential mycotoxins (continued)

Fungal species Potential Mycotoxins

Cladosporium cladosporioides 1, 8 Not reported

C. herbarum 2, 8 Not reported

C. macrocarpum8 Not reported

C. epiphyllum 2 Not reported

Cochliobolus lunatus 1, 8 Not reported

Cochliobolus spiciferus 1 Not reported

Emericella nidulans 1, 8 Sterigmatocystin, nidulotoxin

Epicoccum nigrum 1 Not reported

Eurotium amstelodami 1, 2, 8 (A. amstelodami) Physicon, sterigmatocystin ?

E. chevalieri 1, 8 Emodin, physicon, gliotoxin ?, xanthocillin X ?, echinulin

E. cornoyi 1 -

E. herbariorum 3 Ochratoxin A, physicon‚ echinulin, xanthocillin

E. repens8 Not reported

E. montevidensis 1 -

E. rubrum 1, 8 Unknown toxins

Fusarium sp. 7 Fusarium mycotoxins incl. trichothecenes

F. graminearum 2 Deoxynivalenol, zearalenone and almost 50 other toxins

F. moniliforme 2 Fumonisins, moniliform, zearalenone, deoxynivalenol and others

F. oxysporum 2, 3, 4 Moniliform, zearalenone and others

Humicola grisea8 -

Mucor racemosus 2 Not reported

M. circinelloides8 Not reported

M. hiemalis8 Not reported

Paecilomyces sp. 3, 4 P. fulvus - patulin

Paecilomyces variotii 1 Not reported

Penicillium sp. 7 Several

Penicillium aurantiogriseum 1, 3 Penicillinic acid, ochratoxin A, patulin and many others

P. cyclopium 2, 4, 8 Penicillinic acid, ochratoxin A, patulin and many others

P. puberulum 1 Penicillinic acid, ochratoxin A, patulin and many others

P. verrucosum var. cyclopium 5 Penicillinic acid, ochratoxin A, patulin and many others

P. brevicompactum 3 Mycophenolic acid

P. chrysogenum 1, 2, 3, 4, 5, 8 Patulin, roquefortine C, cyclopiazonic acid

P. citrinum 1, 8 Citrinin

P. corylophilum8 -

P. crustosum 2, 3, 4 Penitrem A, chloroanisols

P. decumbens 2, 3, 4 Not reported

I Introduction

Table 6. Fungi that have been reported on hazelnut kernels and their potential mycotoxins (continued)

Fungal species Potential Mycotoxins

P. digitatum 2, 5 Unknown toxins

P. echinulatum 5 Not reported

P. funiculosum 1 Patulin

P. granulatum 1 (P. glandicola) Penitrem A

P. nalgiovense 5 Penicillin

P. oxalicum 1, 3, 8 Secalonic acid D

P. simplicissimum8 Janthitrems

P. viridicatum 3 Citrinin, ochratoxin A, penicillinic acid and others

Pestalotia sp. 2 *

Pestilazza sp. 6 -

Phoma sp. 6 P. sorghina – tenuazonic acid, P. lingam – sirodesmin H

Pleospora herbarum 1 -

R. nigricans 2 Rhizonine

Rhizopus sp. 3, 4, 6 Rhizonine

Rhizopus stolonifer 1, 5 Rhizonine

R. oryzae 5 Isofumigaclavine A

Scopulariopsis brevicaulis8 Not reported

Syncephalastrum racemosum 1, 3, 5 Not reported

Trichoderma hamatum 3, 1 Not reported

Trichoderma sp. 2 Several

Trichothecium roseum 2, 5, 6, 8 Trichothecenes

Ulocladium atrum8 Not reported

Verticillium sp. 6 -1 ABDEL-GAWAD & ZOHRI (1993) – 38 taxa2 SENSER (1979) – 26 taxa3 WEIDENBÖRNER (1998) – 16 taxa4 REISS (1998) – 13 taxa5 SAHIN & KALYONCUOGLU (1994) – 13 taxa6 ÖZDEMIR & ÖZILGEN (2001) – 10 taxa7 SANCHIS et al. (1988) – 5 taxa (and a number of Mucorales)8 ABDEL-HAFEZ & SABER (1993) – 33 taxa- No reference found* According to PITT & HOCKING (1997) Pestalotia sp. has frequently been incorrectly applied to food borne fungi, which should have been identified as Pestalotiopsis or, less frequently, Truncatella. In food the non-toxic species Pestalotiopsis guepinii can be found.

I Introduction

1.4.3 Moulds and mycotoxins in hazelnuts and the prevention of their formation

1.4.3.1 Prevention of the formation of moulds and mycotoxins in hazelnuts

Fungi that produce mycotoxins in crops have been divided into two distinct groups. The first includes those, which invade and produce their toxins before harvest, the so-called “field fungi”. The second group, which primarily grow on the crop after harvest, are known as “storage fungi” (WEIDENBÖRNER 2000). Alternaria, Cladosporium and Fusarium are examples for field fungi. They require high water contents and therefore they do not compete well with the storage fungi. Xerophilic moulds like Penicillium, Aspergillus and Eurotium are typical storage fungi.Hazelnuts pass a number of steps from harvesting to the final product, which involve harvesting, drying, storage, and processing (roasting, grounding, baking). Hazelnuts are contaminated during all steps from harvesting to the final product, whereby major mould growth occurs after harvest by storage fungi. The susceptibility to mould and mycotoxin contamination during these steps depends on a variety of factors. As mentioned above (Table 5 and Figure 1) both fungal growth and mycotoxin production are dependent on environmental factors (FRISVAD & SAMSON 1991), which are physical (temperature, aw, mechanical damage, time), chemical (atmosphere, substrate composition, pH, fungicides) and biological factors (fungal plant pathogens, microbial competition). Whenever these required parameters are fulfilled, contamination of hazelnuts may occur. By denying only one of the parameters to the moulds, crop protection can succeed.

Harvest. Aspergillus flavus invades the hazelnuts on the tree, but unless a minor crack on the shell of the hazelnuts occurred during harvest and post-harvest treatment, A. flavus was not isolated from the hazelnut kernel. Aflatoxin could be isolated of sun-dried hazelnuts with a crack in their shells (ÖZDEMIR 1998). This implies that safe post-harvest handling is essential for prevention of both mould growth and mycotoxin contamination.

Drying. Immediate, proper drying is the most important means to avoid fungal growth and mycotoxin production in crop after harvest. Sun drying by spreading on a paved floor with intermitted stirring is the most commonly used method especially in developing countries. Usually sun drying requires 6 - 10 days to reduce the moisture content to aw of 0.38 - 0.24, at which hazelnut kernels can be stored safely. But in rainy weather it is not possible to dry the crop in a reasonable time. Re-wetting due to insufficient protection from rain or due to vapor condensation at night is a further problem (ÖZDEMIR & ÖZILGEN 2001). The result is increased mould growth, because at water activities between 0.78 and 0.81 hazelnuts become a good substrate for aflatoxin contaminating fungi (SANCHIS et al. 1988, ÖZDEMIR 1998). Fast mechanical drying at 40 °C may decrease the risk of mycotoxin contamination.

I Introduction

Storage. Crops to be stored must be – whenever possible – of high quality: free from moulds and insects and dried to safe moisture level. Traditionally, moisture control has been the method of choice for prevention of mould growth in stored crop. Also the constancy of temperature and relative humidity is of great significance. Constant temperature and relative humidity is important since moisture migration and condensation resulting from thermal gradients within stored crop masses can cause an accumulation of moisture and enhance mould growth in certain areas (ÖZDEMIR & ÖZILGEN 2001). The rapidity of the moisture transfer depends on the moisture content of the stored material and on the magnitude of temperature differential. Also respiration by insects, mites and fungi produces water, so that once spoilage gets under way it is self-perpetuating, and usually self-accelerating. Moisture transfer and deterioration can be avoided by maintaining of a uniform temperature throughout the bulk (BEUCHAT 1978).Low temperature storage must be preferred, as mycotoxin contamination is correlated directly with temperature except for some species of Fusarium and Penicillium that can produce mycotoxins (e.g. trichothecenes and penicillinic acid) at low temperature (5 °C) (BULLERMAN 1984, WEIDENBÖRNER 1998). The optimum of Aspergillus parasiticus for aflatoxin production is 30 °C and even at 20 °C aflatoxin production is possible (SANCHIS et al. 1988). Most storage fungi have a maximum temperature for growth of 40 ° to 45 °C, but A. flavus can grow vigorously at 50 ° to 55 °C, and can raise the temperature of the materials in which it is growing to that figure (BEUCHAT 1978).Control of the atmosphere in storage is of importance, as oxygen is one of the critical components of mould growth. Mould growth and mycotoxin production is depressed by low oxygen and high concentration of other gases. According to WEIDENBÖRNER (2001a) aflatoxin production is inhibited at 1 % oxygen.

Processing. Varying processed hazelnuts provide different media for fungal growth and mycotoxin production. SANCHIS et al. (1988) showed that ground raw hazelnuts are most susceptible to aflatoxin contamination. Ungrounded roasted hazelnuts are least susceptible, however at a water activity of 0.78 aflatoxin contamination can occur.

1.4.3.1 Mycotoxic fungi on hazelnuts

According to ÖZDEMIR (1998) and SANCHIS et al. (1988) Aspergillus flavus, A. parasiticus, A. tamarii, A. ochraceus, A. terreus and A. wentii can produce aflatoxins on hazelnuts. This conclusion can be corrected according to later publications (PITT & HOCKING 1997, WEIDENBÖRNER 2001a among others), which summarize, that only A. flavus, A. parasiticus and A. nomius are aflatoxin producers. As A. nomius has not been found on hazelnuts, only the first two Aspergillus species are a threat of aflatoxin production on hazelnuts.SENSER (1979) tested all isolated mould genera from hazelnuts (Table 6) for their aflatoxin production ability on hazelnut substrate. Except for 6 of 17 A. flavus isolates and 1 of 3 A. parasiticus isolates from hazelnut, all were found to be non-producing strains. Hazelnut spoiling fungi like A. ochraceus, A. niger, Eurotium herbariorum, and Penicillium spp. are ochratoxin A producers (FRISVAD & SAMSON 1991, PITT & HOCKING 1997), but it is not

I Introduction

clear, if all of them synthesize ochratoxin A on hazelnut substrate. It is not known as well, whether other fungal genera reported on hazelnuts produce their potential mycotoxins on hazelnut substrate.

1.4.3.1 Mycotoxins in hazelnuts

WEIDENBÖRNER (2001a) summarizes, that hazelnuts may contain the following mycotoxins: aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 and ochratoxin A. Aflatoxins occur at concentration ranges from 0.5-50,000 µg/kg. Ochratoxin A was found at concentrations of 4.7 µg/kg and 1.49 µg/kg, respectively. SENSER (1979) surveyed mould suspected nuts and found the aflatoxins B1, B2, G1 and G2 on an average of 30 ppb (~30 µg/kg). ABDEL-HAFEZ & SABER (1993) found the aflatoxins B1, B2, G1 and G2 at concentration ranges of 25-175 µg/kg in mouldy hazelnut samples. ELMADFA & BURGER (1999) published the occurrence of ochratoxin A in an average concentration of 0.02 µg/kg with a maximum concentration of 0.08 µg/kg in hazelnuts.

I Materials and methods

2. Materials and methods

Fig. 2. Flowchart illustrating the methods for identification and characterization of fungi contaminating hazelnuts.

2.1 Materials

For investigation on the mycoflora of hazelnut based food, a company provided 39 samples of the three main steps of food processing. The steps are raw hazelnuts, roasted hazelnuts and hazelnut paste (Figure 3). All samples of raw and roasted hazelnuts were obtained from Turkish hazelnut producers, but the sampling method was not conveyed to the company. Hazelnut paste samples were taken according to following method: The paste was delivered in tanks subdivided into sections. The paste in each section was stirred well. Then an incremental sample of approx. 10 kg was taken out of each section. Three incremental samples were combined to an aggregate sample of approx. 30 kg and mixed well. 1 kg of each aggregate sample was sent to an external laboratory for aflatoxin analysis and to Hygienicum AG for investigation of the mycoflora (TANJA MEINDL pers. comm.). All the samples of raw


and roasted hazelnuts and hazelnut paste delivered to Hygienicum AG were stored at RT until initial sample preparation.

A B C

Fig. 3. The three main steps of hazelnut processing. A: Raw hazelnuts. B: Roasted hazelnuts. C: hazelnut paste.

Table 7. Covering letter of the samples provided by the company

Sample No. Product Date Supplier No. Comments

835 Nuts raw 24-03-2002 7667

836 Nuts roasted 06-04-2002 7667

837 Hazelnut paste 11-04-2002 7667

838 Nuts raw 16-04-2002 7667 Sample 4

Sample 4

Sample 4

839 Nuts roasted 16-04-2002 7667

840 Hazelnut paste 16-04-2002 7667

841 Nuts raw 16-04-2002 7667 Sample 3

Sample 3

Sample 3

842 Nuts roasted 16-04-2002 7667

843 Hazelnut paste 16-04-2002 7667

844 Hazelnut paste - 7542

857 Nuts raw 1 29-04-2002 7661

858 Nuts raw 2 29-04-2002 7661

859 Nuts roasted 29-04-2002 7661

860 Nuts roasted 25-04-2002 7667 Lief. f. Wolkersdorf

861 Hazelnut paste 29-04-2002 7661

989 Nuts raw 24-04-2002 7667 Charge no. 3-141

990 Nuts roasted 24-04-2002 7667 Charge no. 3-141

991 Hazelnut paste 26-04-2002 7667 Charge no. 3-141

992 Nuts raw 08-05-2002 7667 Charge no. 3-142



995 Hazelnut paste 13-05-2002 7667 Kammer 1

996 Hazelnut paste 13-05-2002 7667 Kammer 2

1059 Nuts raw 22-05-2002 7661


Table 7. Covering letter of the samples provided by the company (continued)

Sample No. Product Date Supplier No. Comments

1060 Nuts roasted 22-05-2002 7661

1061 Hazelnut paste 22-05-2002 7661

1062 Nuts raw 18-05-2002 7667



1065 Hazelnut paste 27-05-2002 7667 Lieferung Fässer

1066 Hazelnut paste 21-05-2002 7596

1699 Nuts raw 21-05-2002 7596

1700 Nuts roasted 21-05-2002 7596

1701 Hazelnut paste 21-05-2002 7596 Charge no. 077

1702 Nuts raw 12-06-2002 7596

1703 Nuts roasted 12-06-2002 7596

1704 Hazelnut paste 12-06-2002 7596 Charge no. 095

1705 Nuts roasted (air) 01-07-2002 7596

1706 Hazelnut paste 01-07-2002 7596

2.2 Cultivation of the moulds

Isolation and cultivation of the moulds were carried out following the subsequently described procedure.

2.2.1 Production of the nutrient agar

The solid substances were weighed into an Erlenmeyer flask, the H2O was added and boiled until the liquid was lucent. Then the agar was autoclaved for 20 min, and chilled to about 50 °C. It was poured until about one third of the volume of a Petri dish was filled. The plates were solidified at room temperature over night.

Composition of the yeast glucose chloramphenicol (YGC) agarYeast extract 5.0 gD(+)-Glucose 20.0 gChloramphenicol 0.1gAgar-Agar 14.9 gH2O 1000 ml


2.2.2 Dilution plating

The fungi were isolated from the nuts using direct plating and dilution plating methods. The hazelnut paste was applied on the dishes only by dilution plating methods. For a dilution scheme of 1:10, 10 g of nuts or paste, respectively, were combined with 90 ml of Maximum Recovery Diluent, ground in a homogenisator and then 1000 µl of the dilution were added to 15 ml pre-cooled YGC medium and poured into Petri dishes. A 1:100 dilution scheme was done by surface plating of 100 µl of the dilution on agar plates.

Composition of the Maximum Recovery Diluent (Merck)Peptone 1.0 gNaCl 8.5 gH20 1000 mlThe solid substances were dissolved in 1000 ml H20 and the diluent was autoclaved.

2.2.3 Direct plating

5-6 hazelnut kernels were placed on solidified agar, rolled on the surface and then taken out of the dish again.

2.3 Determination of germ numbers

All plates were incubated 2-4 days at RT and daylight. Every day the colonies were observed. The germ numbers were counted in 26 out of 39 samples each day of incubation. Yeasts were included in the germ numbers, but were not subcultured and investigated further. Pure cultures were received by transferring single colonies to new Petri dishes.

2.4 Identification of fungi using morphological criteria

Optical evaluation of the species was based on the color and surface characteristics of the cultures and microscopic morphological criteria like size, color and shape of reproductive and vegetative organs, spores and hyphae (SAMSON et al. 1995, PITT & HOCKING 1997, DE HOOG et al. 2000). A Leica M3Z stereoscopic microscope with a continuous zoom up to 40x was used to distinguish Aspergillus and Penicillium species. For further identification of all the species a Zeiss AXIOSKOP 20 microscope with Achroplan objectives of magnifying power 4x, 10x, 20x, 40x and 100x (oil immersion) was used. Pictures were taken with a Canon Digital Camera PowerShot S10 for documentation of the cultures (see Appendix 2).


2.5 Identification of fungi using molecular methods

2.5.1 DNA isolation

DNA isolation was mostly carried out with the DNeasy Plant Mini Kit of QUIAGEN. The precipitation isolation protocol of CUBERO et al. (1990), which takes more time, was applied only on a minor amount of samples. Around 0.1 g of the culture-grown fungi was taken for DNA extraction with the kit. The mycelium was ground with a plastic pistil in a 1.5 ml Eppendorf tube until a fine powder was obtained. Extraction was performed according to the manufacturer’s protocol. The purified DNA was diluted in 200 µl H2O.Extraction according to CUBERO et al. (1990) was performed as described below. 500 µl lysis buffer were added to the ground mycelium and the mixture was incubated at 65 °C for 1 hour. The tubes were vortexed every several minutes. Protein extraction was carried out twice with 500 µl chloroform/isoamylalcohol 24:1. After vortexing the tubes were centrifuged for 5 minutes at 12,000 rpm. The upper phase was transferred into a new tube and 1 ml precipitation buffer was added. The tubes were incubated for 1 hour at RT and then centrifuged for 15 minutes at 12,000 rpm. The supernatant was discarded and the pellet was resuspended in 350 µl 1.2 M NaCl. 500 µl chloroform were added. The tubes were centrifuged again for 5 minutes at 12,000 rpm. The upper phase was taken and 210 µl isopropanol with a temperature of –20 °C were added to precipitate the DNA. After incubation at –20 °C for 15 minutes to overnight the tubes were centrifuged for 20 minutes at 12,000 rpm and 4 °C. The supernatant was discarded and the pellet was washed with 200 µl ethanol. The pellet was dried for 10 minutes at 45 °C in a drying closet and resuspended in 50 µl H2O. The purified DNA can be stored for several years at –20 °C.

ReagentsChloroform/Isoamylalcohol 24:1Lysis buffer 1.4 % N-Cetyl-N,N,N-trimethyl-ammoniumbromid (CTAB),

1 M NaCl, 7 mM Tris, 30 mM EDTAPrecipitation buffer 0.5 % CTAB, 40 mM NaCl

2.5.2 PCR

2.5.2.1 DNA template

In eucaryotic cells, 50-5000 identical copies of the rDNA genes specify the 18S (small sub unit), 5.8S and 28S (large sub unit) in the ribosomes. These genes are tandem-wise arranged in large clusters. The non-coding external transcribed spacers (ETS), and internal transcribed spacers (ITS) 1 and 2 separate the coding genes.


rDNA sequences like the ITS regions, 18S rDNA and 28S rDNA have been used for taxonomy, phylogeny and identification in a number of fungi. Due to the wide use of the rDNA sequences in molecular biology, a high abundance of these genes is revealed in gene banks. According to BRIDGE & ARORA (1998) the variation among the non-coding ITS regions is useful for differentiation of species or populations.

The fungal specific primer ITS1F and the universal primer ITS4 that anneal to the conserved 18S and 28S rDNA genes were used to amplify the ITS 1 and ITS 2 regions.

Table 8. Primers used for amplification of the ITS 1 and ITS 2 regions

Primer Sequence 5’-3’ Reference

ITS1F CTTGGTCATTTAGAGGAAGTAA Gardes & Bruns 1993

ITS4 TCCTCCGCTTATTGATATGC White et al. 1990

2.5.2.2 PCR reaction

ReagentsDeoxyribonucleotide triphosphates (dNTPs) dATP, dGTP, dCTP, dTTP; each 10 mM;

Amersham BiosciencesPrimer 10 pM/µl each; GenXpressDNA polymerase BioTherm DNA polymerase, 5 U/µl; GenXpressReaction buffer (10x) 160 mM (NH4)2SO4, 670 mM Tris-HCl (pH 8.8

at 25 °C), 15 mM MgCl2, 0.1 % Tween 20; GenXpress

DNA polymerase Taq DNA polymerase, 5 U/µl; Amersham Biosciences

Reaction buffer (10x) 500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl (pH 9 at RT); Amersham Biosciences

MgCl2 25 mM, Perkin ElmerMineral oil

First, a master mix with all components excluding the water and template DNA was prepared (Table 9). Aliquots of the master mix were transferred into 0.5 ml Eppendorf tubes. Then water and template DNA were added. The reaction mixtures were overlaid with a drop of mineral oil to avoid evaporation. In each PCR experiment a negative control with water instead of template DNA was included.

PCR amplification was carried out using a Perkin Elmer DNA thermal cycler 480 and a Perkin Elmer Cetus DNA Thermal Cycler. The temperature profile for the PCR is shown in Table 10 and Figure 4.


Table 9. The components of the 30 µl PCR mix

Component Volume (µl)

Template DNA 11.00

H2O 8.00

Reaction buffer 3.00

Primer 3.00

MgCl2 1.80

DNA polymerase 0.15

Table 10. Temperature profile for ITS PCR

Step Time/Temperature

Initial denaturation 5 min at 94 °C

30 to 35 cycles

Denaturation 1 min at 94 °C

Primer annealing 1 min at 52 °C

Extension 2 min at 72 °C

Final extension 10 min at 72 °C

Storage Unlimited at 4 °C

Fig. 4. Scheme illustrating the temperature profile for the PCR.


2.5.2.3 Visualization of PCR products on an agarose gel

ReagentsTBE buffer 0.82 M Tris, 0.67 M Boric acid, 22 mM Na2EDTA; pH 8.3;Loading buffer 30 % glycerin, 0.25 % bromophenol blue, 0.25 % xylene cyanol

in H2ODNA standard 100 bp ladder; New England BiolabsEthidiume Bromide (EtBr) 10 mg/ml H2O

The amplification of products of the correct size was verified on 1 % agarose gels. 3-5 µl of the PCR products and 0.8 µl of size marker, respectively, were mixed with a drop of loading buffer and pipetted into the slots. The electrophoresis was carried out at 100 V for 20 minutes. The EtBr-stained DNA fragments were visualized under a UV-transilluminator.

2.5.2.4 Purification of the PCR products

The PCR products were purified with the QIA quick PCR Purification Kit of QIA according to the manufacturer’s protocol. The DNA was dissolved in 50 µl H2O. Then an aliquot of 2 µl of the purified DNA was run on an agarose gel to estimate the final DNA concentration. The brightness of the band provides a reference on the amount of DNA, which is utilized for the cycle sequencing.

2.5.3 Cycle Sequencing

The method used was the dideoxynucleotide sequencing method, also called chain termination or Sanger method (SANGER et al. 1977). This technique utilizes 2’,3’-dideoxyribonucleotide triphosphates (ddNTPs), which differ from deoxyribonucleotides (dNTPs) by lacking the OH group at the 3’ carbon. ddNTPs terminate DNA chain elongation because they cannot form a phosphodiester bond with the next deoxynucleotide. The cycle sequencing yields DNA fragments ending with a particular ddNTP, varying in one base pair steps. The DNA sequence can be analyzed in an automated sequencer, as the four ddNTPs are labeled with different fluorescence dyes.

The primers ITS1F, ITS2, ITS3 and ITS4 were used for cycle sequencing.

Table 11. The primers additionally used for cycle sequencing


ITS2 GCTGCGTTCTTCATCGATGC WHITE et al. 1990

ITS3 GCATCGATGAAGAACGCAGC WHITE et al. 1990


Cycle Sequencing was carried out using a Perkin Elmer Gene Amp PCR System 2400 with heated lid and an Applied Biosystems Gene Amp PCR System 2700 with heated lid. MicroAmp Reaction Tubes 0.2 ml with cap, N801-5040 from Perkin Elmer were used to hold the reagents. The temperature profile is shown in Table 13 and Figure 5.

ReagentsPrimer 1.6 pM/µl eachSequencing mix BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (dNTPs,

fluorescent labeled ddNTPs, DNA polymerase, MgCl2, pyrophosphatase, buffer), ABI Prism; Perkin Elmer

Table 12. The components of a 10 µl cycle sequencing mix (half size as given by the manufacturer)


Primer 1

Sequencing mix 3

Template DNA + H2O 6

Table 13. Temperature profile for cycle sequencing



25 cycles

Denaturation 20 sec at 96 °C

Primer annealing 5 sec at 50 °C



94°C96°C

50°C

60°C

4°C

25 cyclesInitialdenaturation

Storage5:00

0:20

0:05

4:00

Fig. 5. Scheme illustrating the temperature profile for the cycle sequencing.


The cycle sequencing products were purified by ethanol precipitation. 25 µl 96 % ethanol and 1 µl 3 M sodium acetate (pH 4.4) were added to the products. The DNA was precipitated for 10 minutes at RT and then pelleted by centrifugation for 15 minutes at 12,000 rpm. The supernatant was discarded and the pellet was washed carefully with 150 µl 70 % ethanol. The pellet was dried for 10 minutes at 45 °C in a drying closet and resuspended in 30 µl H 2O. The tubes were placed for 2 minutes in boiling water to denature the DNA and then chilled on ice.

2.5.4 Sequence analysis

The sequence analysis was carried out using an automated genetic analyzer, ABI Prism 310, Applied Biosystems, Perkin Elmer. The samples were hold in Genetic Analyzer 0.5 ml Sample Tubes with Genetic Analyzer Septa, Applied Biosystems.

ReagentsPolymer Performance Optimized Polymer (POP6), Applied BiosystemsBuffer 310 Genetic Analyzer Buffer with EDTA, Applied Biosystems

The ABI Prism 310 detects and analyses the DNA fragments automatically. The fragments are injected in a capillary, which is filled with the polymer, and separated electrophoretically by length. The detection is performed on the dNTPs, which are labeled with 4 different fluorescent dyes. The GeneScanTM-Software collects the data and compiles the chromatogram and related sequence. From each investigated culture 4 sequences of the ITS regions were analyzed with the ABI Prism 310. The sequences were assembled using the ABI Prism Auto Assembler software (version 1.4.0, 1995; Applied Biosystems Division, Perkin Elmer Biosystems). The consensus sequences were aligned using the freeware-program ClustalX.

2.5.5 Identification of the species

The species were identified using BLAST (Basic Local Alignment Search Tool) at the NCBI website (http://www.ncbi.nlm.nih.gov/). The BLAST package provides programs, which establish an alignment with a high score between a query sequence and sequences of a database (nucleotide or protein sequences). For alignments of nucleotide sequences the program BLASTN is used. The BLAST concept assumes that a significant alignment of two sequences includes with high probability short sections of identical fragments with very high score (HSPs, high-scoring segment pairs). The result of a BLAST search is a list of HSPs, which are evaluated with a score (RAUHUT 2001). The ITS 1 and the ITS 2 regions were used whenever complete as query sequences. The species showing the highest score was taken as result. When more than one species got the same score, all optional species names are mentioned in the results. The results received by

http://www.ncbi.nlm.nih.gov/


BLAST were supplemented by PKS fragment patterns, the optical evaluation of the color and surface characteristics of the cultures and microscopic morphological criteria.

2.5.6 Fragment patterns using primers for the PKS (polyketide synthase) genes

The biosynthetic genes of secondary metabolites (e.g. mycotoxins) of fungi are typically clustered (MOORE & PIEL 2000). The clusters include genes for the polyketide synthases (PKS) and genes for other enzymes involved in the biosynthesis of secondary metabolites. The fungal PKS genes are usually 6-8 kb and encode multifunctional proteins with iteratively used active domains (MIAO et al. 2001). The primers FKS1 and FKS2 are specific to the conserved ketosynthase domain of polyketide synthases and were applied on Aspergillus and Penicillium samples to provide information on inter- and intraspecific differences. The primers not only amplify one fragment of approx. 500 bp expected size, but a pattern of several fragments of various sizes. The PKS-PCR was carried out using Biotherm polymerase from GenXpress.

Table 14. Sequences of oligonucleotide primers for the PKS genes


FKS1 GCNBHNCARATGGAYCCNGCNCA LEE et al. 2001

FKS2 GCNBHNCARATGGAYCCNCARCA LEE et al. 2001

Table 15. Temperature profile for the PKS-PCR

Steps Time/Temperature


35 cycles






2.5.7 Testing the reproducibility of PKS patterns

PCR were carried out according to the temperature profile in Table 15 varying the annealing temperatures (53 °C, 55 °C, 60 °C) to find the temperature, which reveals the most intense and distinct fragments. DNA polymerases from GenXpress and Amersham Biosciences were compared by preparing the PCR twice, adding either the Taq or the Biotherm polymerase.


I Results

3. Results

3.1 Germ numbers

The germs were counted in 26 of 39 agar plates to receive information about the intensity of fungal contamination of the hazelnut samples in three stages of processing. The direct plating method revealed the highest germ numbers except for once (no. 1699). Roasted hazelnuts and hazelnut paste reveal in average 0-3 germs/plate depending on the plating method compared to raw hazelnuts, which reveal in average 12-163 germs/plate (Table 16).

Table 16. Germ numbers obtained from hazelnuts (roasted, raw, paste), which were applied on Petri dishes by different plating methods (direct, dilution 1:10, dilution 1:100)

No. 989 (nuts raw) 990 (roasted) 991 (paste) 992 (nuts raw)Days directa 1:10 1:100 direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:1001st 0 0 0 0 0 0 - 0 0 0 0 02nd 189 55 15 0 0 0 - 0 0 113 14 73rd 0 0 0 - 0 04th 0 0 0 - 0 05th 1 0 0 - 0 0No. 993 (roasted ) 994 (paste) 995 (paste) 996 (paste)Days direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:1001st 0 0 0 - 0 0 - 0 0 - 0 02nd 0 0 0 - 0 0 - 1 1 - 0 13rd 0 0 1 - 0 1 - 1 1 - 0 14th 0 0 1 - 0 1 - 1 1 - 1 15th 0 0 1 - 0 1 - 1 1 - 1 2No. 1059 (nuts raw) 1060 (roasted ) 1061 (paste) 1062 (nuts raw)Days direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:1001st -2nd 72 13 2 3 0 0 - 0 0 ∞ 123 123rd 3 0 0 - 0 04th 3 0 0 - 0 0No. 1063 (roasted) 1064 (paste) 1065 (paste) 1066 (paste)Days direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:1001st - - -2nd 0 0 0 - 0 1 - 0 0 - 0 03rd 0 0 0 - 0 1 - 0 0 - 0 04th 0 0 0 - 0 1 - 0 0 - 0 0No. 1699 (nuts raw) 1700 (roasted ) 1701 (paste) 1702 (nuts raw)Days direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:1001st 0 0 0 0 0 0 - 0 0 0 0 02nd 41 0 6 0 0 0 - 0 0 162 0 13rd 69 103 27 4 0 0 - 1 0 370 100 84th 0 0 - 0

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Table 16. Germ numbers obtained from hazelnuts (roasted, raw, paste), which were applied on Petri dishes by different plating methods (direct, dilution 1:10, dilution 1:100) (continued)

No. 1703 (roasted ) 1704 (paste) 1705 (roasted) 1706 (paste)Days direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:100 direct 1:10 1:1001st 0 0 0 - 0 0 0 0 0 - 0 02nd 7 0 0 - 0 0 0 0 0 - 0 03rd 10 0 1 - 0 0 4 0 1 - 1 04th 0 - 0 0 0 - 0a Application of the nuts on the Petri dishes by the direct plating method.- Hazelnut paste samples were not applied on the Petri dishes by the direct plating method.Empty box: The germ number was equal to the previous day, or it was uncountable.

Table 17. Average of germ numbers obtained from hazelnuts (roasted, raw, paste), which were applied on Petri dishes by different plating methods (direct, dilution 1:10, dilution 1:100).

Product Germ number (average)

Direct 1:10 1:100

Raw 162.6 (5)a 68 (6) 11.8 (6)

Roasted 3.1 (7) 0 (7) 0.4 (7)

Paste - 0.4 (11) 0.4 (11)aFigures in parentheses are the numbers of samples for calculation of the average (n)- Hazelnut paste samples were not applied on the Petri dishes by the direct plating method.

3.2 Hazelnut contaminating fungal species

Each sample of raw hazelnuts, roasted hazelnuts and hazelnut paste contributed 1-8 single colonies that were studied further. Two samples of roasted hazelnuts (no. 836 and 1063) and five samples of hazelnut paste (no. 991,1061, 1065, 1066 and 1704) did not reveal any fungi when applied to the agar plates. Therefore the sample numbers are not mentioned in Table 19, 20, 21 and 22. The remaining hazelnut samples provided a total number of 113 fungal colonies. Out of this, 39 samples of Penicillium and 29 samples of Aspergillus were identified. The residual is made up of 26 Zygomycetes samples and 19 various Ascomycetes, which were grouped to the so-named miscellaneous fungi (Table 21). In total 10 fungal genera were identified (Figure 6, Table 18). In some cases the same fungus was subcultured twice from one hazelnut sample. Penicillium cf. commune was taken twice from the samples no. 858, 992 and 1059, and Rhizopus stolonifer was taken twice from the samples no. 835 and 838.

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Table 18. Fungal genera identified among the mycoflora of hazelnuts

Order Genus Incidence

Eurotiales Aspergillus 29Penicillium 39

Mucorales Rhizopus 9Mucor 8Syncephalastrum 9

Dothideales Cladosporium 11Aureobasidium 1

Pleosporales Alternaria 2

Hypocreales Trichoderma 3Trichothecium 1

Unknown Ascomycetes 1

Total 113

Fig. 6. Fungal genera identified among the mycoflora of hazelnuts

Penicillium and Aspergillus samples were combined in groups according to the criteria ITS sequence (see Appendix 1) and PKS pattern (Figures 7-11). The PKS types were named A a, Ba, Ca, Da, Ea and Ap, Bp, Cp, Dp, Ep, Fp with the indices indicating the genus (a for Aspergillus and p for Penicillium). Few samples could not be identified as member of one of these groups, because of insufficient PCR results, and were summarized in Xa and Xp, respectively.According to the ITS sequences, four species groups of Aspergillus were identified. The groups are A. niger agg., A. fumigatus, A. flavus group and A. parasiticus. Two species could not be identified (no. 837.1 and 859.6). According to the scores as results of the BLASTN search on Genbank, A. niger and A. tubingensis could not be distinguished. Thus, the term A. niger agg. is used for these species, as both have been associated with the same group. Recently, it has been suggested, that the Aspergillus niger aggregate should be divided into four taxa: A. niger, A. tubingensis, A. brasiliensis and A. foetidus (PAŘENICOVÁ et al. 2001). These black Aspergilli cannot be identified reliably on the basis of morphological features,

I Results

but require molecular and biochemical identification methods. A. oryzae and A. flavus could not be distinguished as well and are therefore summarized as A. flavus group. A. oryzae belongs to the A. flavus group and shows a high similarity with A. flavus. However, A. oryzae does not produce aflatoxins and is used for fermentation of different kinds of foodstuff in Asian countries (WEIDENBÖRNER 2001a). According to a multi-gene study by GEISER et al. (1998), A. oryzae is a clonal lineage within a subgroup of A. flavus.Penicillium spp. were identified according to ITS sequences and PKS pattern. P. cf. commune was the most common Penicillium. However, BLAST search with ITS sequences provides the same scores for P. commune as well as for P. crustosum, P. italicum and in few cases P. camemberti and P. echinulatum. As a consequence, P. cf. commune was subgrouped using the PKS patterns to P. cf. commune A, B and X. Less abundant Penicillium spp. were P. brevicompactum, P. geastrivorus, P. glabrum, P. citrinum, P. nalgiovense, P. chrysogenum, P. cyclopium, and P. expansum.The main part of Zygomycetes was identified by morphological criteria and not by the ITS sequence due to sequencing difficulties. The species identified were Rhizopus stolonifer, R. oryzae, Syncephalastrum racemosum, Mucor circinelloides and M. plumbeus. Five Mucor spp. could not be identified to the species level.The miscellaneous fungi were identified by ITS sequencing and/or morphological criteria. Alternaria alternata, A. infectoria, Aureobasidium pullulans, Trichoderma longibrachiatum, Trichothecium roseum and Cladosporium sp. were identified. Concerning Cladosporium, BLAST search provided results that did not point on a single species alone. Two groups were distinguished and named Cladosporium sp.1 and Cladosporium sp.2. The species no. 1705.4 could not be resolved.

Table 19. Aspergillus spp. identified among the mycoflora of hazelnuts

Product Sample No. PKS pattern ITS sequence SpeciesNuts raw 835.5 (1) Xa + A. parasiticusHazelnut paste 837.1 (2) Xa A. sp.Nuts raw 838.3 (3) Aa + A. niger agg.

838.4 (4) Ba + A. flavus groupHazelnut paste 840.2 (5) Da + A. fumigatusNuts raw 841.2 (6) Xa + A. niger agg.

841.4 (24) Ba A. flavus groupNuts roasted 842.2 (8) Da + A. fumigatusHazelnut paste 843.2 (9) Xa + A. flavus group

843.3 (10) Xa A. niger agg.Hazelnut paste 844.1 (11) Ba + A. flavus group

844.2 (12) Ba + A. flavus group844.3 (13) Ca + A. flavus group844.4 (14) Da A. niger agg.

Nuts raw 857.2 (15) Aa + A. niger agg.857.3 (16) Ca A. flavus group

Nuts raw 858.2 (17) Aa A. niger agg.858.3 (18) Xa + A. flavus group

Nuts roasted 859.4 (19) Xa + A. flavus group859.6 (20) Xa A. sp.

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Table 19. Aspergillus spp. identified among the mycoflora of hazelnuts (continued)

Product Sample No. PKS pattern ITS sequence SpeciesNuts roasted 860.1 (21) Aa + A. niger agg.Nuts raw 989.1 (22) Ba + A. flavus group

989.3 (23) Aa + A. niger agg.Nuts roasted 990.1 (24) Da + A. fumigatusNuts raw 1059.2 (25) Aa + A. niger agg.Nuts raw 1699.1 (26) Aa + A. niger agg.Nuts roasted 1700.1 (27) Aa + A. niger agg.Nuts raw 1702.3 (28) Ba + A. flavus groupNuts roasted 1703.4 (29) Aa A. niger agg.aFigures in parentheses are the numbers used for the PKS gels.+: ITS sequencing was used to identify the species.

Table 20. Penicillium spp. identified among the mycoflora of hazelnuts

Product Sample No. PKS pattern ITS sequence SpeciesNuts raw 835.4 (1) Xp + P. cf. commune XNuts raw 838.5 (2) Cp + P. brevicompactum

838.7 (3) Fp + P. geastrivorusNuts roasted 839.1 (4) Cp P. sp.Nuts raw 841.3 (5) Cp P. sp.

841.5 (6) Xp + P. glabrumNuts roasted 842.3 (7) Xp P. sp.Nuts raw 857.4 (8) Ap + P. cf. commune A

857.5 (9) Xp + P. glabrumNuts raw 858.6 (10) Ab + P. cf. commune A

858.7 (11) Xp + P. brevicompactum858.8 (12) Ap + P. cf. commune A

Nuts roasted 859.3 (13) Cp P. sp.859.7 (14) Cp + P. brevicompactum

Nuts roasted 860.3 (15) Cp P. sp.Hazelnut paste 861.1 (16) Bp + P. cf. commune BNuts raw 989.2 (17) Ap + P. cf. commune A

989.4 (18) Ep + P. citrinum989.6 (19) Xp + P. commune

Nuts raw 992.2 (20) Bp + P. commune992.3 (21) Ap + P. cf. commune A992.5 (22) Ap + P. cf. commune A

Hazelnut paste 995.2 (23) Ep + P. citrinumHazelnut paste 996.3 (24) Xp + P. nalgiovenseNuts raw 1059.1 (25) Ap + P. cf. commune A

1059.3 (26) Ap + P. cf. commune A1059.5 (27) Cp + P. cyclopium

Nuts raw 1062.1 (28) Bp + P. cf. commune B1062.3 (29) Ap + P. expansum1062.5 (30) Ap P. sp.

Nuts raw 1699.2 (31) Ap + P. cf. commune A1699.3 (32) Cp + P. polonicum

Nuts roasted 1700.2 (33) Xp + P. cf. commune XNuts raw 1702.1 (34) Bp + P. cf. commune B

1702.2 (35) Cp P. sp.Nuts roasted 1703.1 (36) Ap + P. cf. commune A

1703.2 (37) Cp P. sp.Nuts roasted 1705.1 (38) Dp + P. chrysogenumHazelnut paste 1706.1 (39) Xp + P. chrysogenumaFigures in parentheses are the numbers used for the PKS gels.+: ITS sequencing was used to identify the species.Table 21. Miscellaneous fungi identified among the mycoflora of hazelnuts

I Results

Product Sample No. ITS sequence SpeciesHazelnut paste 840.1 + Alternaria alternataNuts roasted 842.1 + Aureobasidium pullulansHazelnut paste 843.1 + Trichoderma longibrachiatumNuts raw 858.4 Trichoderma longibrachiatum

858.5 + Trichothecium roseumNuts roasted 860.4 + Cladosporium sp.1Nuts raw 992.4 + Cladosporium sp.1Nuts roasted 993.1 + Cladosporium sp.1Hazelnut paste 994.1 + Cladosporium sp.1Hazelnut paste 995.1 + Trichoderma longibrachiatumHazelnut paste 996.1 + Cladosporium sp.1Nuts raw 1062.2 + Cladosporium sp.1Hazelnut paste 1064.1 + Cladosporium sp.2Hazelnut paste 1701.1 + Cladosporium sp.2Nuts roasted 1703.3 + Cladosporium sp.2

1703.6 Cladosporium sp.1Nuts roasted 1705.2 Cladosporium sp.1

1705.3 + Alternaria infectoria1705.4 X

+: ITS sequencing was used to identify the species.X: This species was not identified.

Table 22. Zygomycetes identified among the mycoflora of hazelnuts

Product Sample No. ITS sequence SpeciesNuts raw 835.1 Rhizopus stolonifer

835.2 Mucor sp.835.3 Rhizopus stolonifer

Hazelnut paste 837.2 Mucor sp.837.3 Rhizopus stolonifer

Nuts raw 838.1 Mucor sp.838.2 Syncephalastrum racemosum838.6 Rhizopus stolonifer838.8 Syncephalastrum racemosum

Nuts roasted 839.2 Rhizopus stoloniferNuts raw 841.1 + Rhizopus oryzaeNuts raw 857.1 Rhizopus stoloniferNuts raw 858.1 Rhizopus stoloniferNuts roasted 859.1 + Mucor circinelloides

859.2 Mucor sp.859.5 + Rhizopus oryzae

Nuts roasted 860.2 Mucor sp.Nuts raw 989.5 + Mucor plumbeusNuts raw 992.1 Syncephalastrum racemosumHazelnut paste 996.2 Syncephalastrum racemosumNuts raw 1059.4 Syncephalastrum racemosumNuts roasted 1060.1 + Mucor plumbeusNuts raw 1062.4 Syncephalastrum racemosumNuts raw 1699.4 Syncephalastrum racemosumNuts raw 1702.4 Syncephalastrum racemosumNuts roasted 1703.5 Syncephalastrum racemosum+: ITS sequencing was used to identify the species.

I Results

3.3 PKS fragment patterns of hazelnut contaminating fungi

A PCR using primers that are specific for the ketosynthase domain of polyketide synthase genes was carried out with isolates assigned to Aspergillus and Penicillium to obtain fragment patterns for further molecular characterization of the strains. Aspergillus could be associated with 5 general PKS pattern types and Penicillium with 6 general PKS pattern types. Within these types variation of intensity, lack of single fragments or lack of fragment groups occurred.

3.3.1 PKS patterns of Aspergillus

S 26 29 25 17 4 11 12 22 18 8 24 5 16 13 7 28 14 X 1 27 0 S

- 1 kb

- 500 bp

Fig. 7. Agarose gel electrophoresis of PCR products from Aspergillus spp. with the PKS primers FKS1 und FKS2. Lane S: 100 bp ladder. Lane X: Unknown sample no.

S 23 27 1 18 6 10 9 19 2 0 S S 3 15 17 20 21 2 0 S

- 1 kb

- 500 bp

- 1 kb

- 500 bp

Fig. 8. Agarose gel electrophoresis of PCR products from Aspergillus spp. with the PKS primers FKS1 und FKS2. Lane S: 100 bp ladder.

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3.3.2 PKS patterns of Penicillium

S 21 26 36 16 8 31 12 25 10 22 7 20 28 34 35 27 5 13 S

- 1 kb

- 500 bp

Fig. 9. Agarose gel electrophoresis of PCR products from Penicillium spp. with the PKS primers FKS1 und FKS2. Lane S: 100 bp ladder.

S X 32 38 39 21 23 31 15 2 37 4 0 S

- 1 kb

- 500 bp

Fig. 10. Agarose gel electrophoresis of PCR products from Penicillium spp. with the PKS primers FKS1 und FKS2. Lane S: 100 bp ladder. Lane X: Unknown sample no.

S 30 17 29 3 14 37 4 7 6 9 19 1 24 33 11 0 0 S

- 1 kb

- 500 bp

Fig. 11. Agarose gel electrophoresis of PCR products from Penicillium spp. with the PKS primers FKS1 und FKS2. Lane S: 100 bp ladder.

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3.3.3 Reproducibility of PKS patterns

PKS-PCR was carried out with three different annealing temperatures: 53 °C, 55 °C and 60 °C (Figure 12, 13 and 15). At 53 °C and 55 °C the PKS pattern is almost the same, sometimes varying in intensity. At 60 °C much less fragments were received: 4 samples provided less than 10 fragments, 4 samples provided less than 3 fragments, and 4 samples failed. At 60 °C less random amplicons may be produced due to the increased specificity of the primer at higher annealing temperature.Two different DNA polymerases (BioTherm from GenXpress and Taq from Amersham) were used for PKS-PCR (Figure 13 and Figure 14). It turned out, that both polymerases produce almost the same pattern. Taq polymerase has a tendency to produce more of the longer fragments, which is clearly shown by the samples no. 19 and 27 in Figure 14.Concluding, PKS-PCR can be considered to be of high reproducibility. Changing the annealing temperature by 2 °C does not influence the result seriously. The same is true for the use of different DNA polymerases.

Penicillium Aspergillus S 25 26 27 28 29 17 18 19 20 21 23 X 24 S 25 22 31a 24 0 S

- 1 kb

- 500 bp

Fig. 12. Agarose gel electrophoresis of PCR products from Aspergillus spp. and Penicillium spp. with the PKS primers FKS1 und FKS2. PCR carried out with BioTherm DNA polymerase (GenXpress) at 53 °C annealing temperature. Lane S: 100 bp ladder. Lane X: Purified DNA from ITS PCR.aSample no. 31 is Penicillium.

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Penicillium Aspergillus S 25 26 27 28 29 17 18 19 20 21 23 22 X S 25 22 31a 24 0 S

- 1 kb

- 500 bp

Fig. 13. Agarose gel electrophoresis of PCR products from Aspergillus spp. and Penicillium spp. with the PKS primers FKS1 und FKS2. PCR carried out with BioTherm DNA polymerase (GenXpress) at 55 °C annealing temperature. Lane S: 100 bp ladder. Lane X: Purified DNA from ITS PCR.aSample no. 31 is Penicillium.

Penicillium Aspergillus S 25 26 27 28 29 17 18 19 20 21 23 22 24 S 25 22 31a 24 0 S

- 1 kb

- 500 bp

Fig. 14. Agarose gel electrophoresis of PCR products from Aspergillus spp. and Penicillium spp. with the PKS primers FKS1 und FKS2. PCR carried out with Taq DNA polymerase (Amersham) at 55 °C annealing temperature. Lane S: 100 bp ladder.aSample no. 31 is Penicillium.

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Penicillium Aspergillus S 25 27 28 29 17 18 19 24 25 22 23 24 0 S

17.7.02

PKS vg l 16.7.

100b p 1 2 3 4 5 6 7 8 1 2 3 4 0 100b p Pe nic illium Asp e rg illus

Pe nic illium1 1059.12 1059.53 1062.14 1062.35 989.36 989.47 989.68 996.3

Asp e rg illus1 1059.22 989.13 989.24 990.1

- 1 kb

- 500 bp

Fig. 15. Agarose gel electrophoresis of PCR products from Aspergillus spp. and Penicillium spp. with the PKS primers FKS1 und FKS2. PCR carried out with BioTherm DNA polymerase (GenXpress) at 60 °C annealing temperature. Lane S: 100 bp ladder.

I Discussion

4. Discussion

4.1 Quantitative analysis of the mycoflora

4.1.1 Isolation and culturing of moulds

4.1.1.1 Omitting of surface disinfection

In many studies of fungi in dried particular food, such as cereals and nuts, a surface disinfection procedure with a sodium hypochlorite solution is carried out before the seeds are plated on agar plates. Surface disinfection inactivates the surface mycoflora and enables the assessment of the internal mycoflora (ANDREWS 1996). Usually germ numbers of disinfected kernels are much lower than those of non-disinfected. For example WEIDENBÖRNER (2001b) found significant lower fungal counts in disinfected pumpkin seeds compared to non-disinfected seeds. Surface disinfected hazelnuts were investigated by SAMSON et al. (1995), whereas non-disinfected hazelnuts were investigated by ABDEL-GAWAD & ZOHRI (1993), ABDEL-HAFEZ & SABER (1993) and SAHIN & KALYONCUOGLU (1994). Surface disinfection was not carried out in this study. Like fungi of the internal mycoflora, also fungi of the surface mycoflora can contribute to deterioration and mycotoxin contamination. They are therefore of interest to food safety. Another reason for not applying surface disinfection is, that hazelnut paste cannot be surface disinfected, because it is a viscous liquid. For an equal comparison of the hazelnut samples in three stages of processing (raw, roasted, paste), the same preparation procedure was used, which omitted surface disinfection.

4.1.1.2 Evaluation of the plating methods

Three different plating methods were used to isolate fungi from hazelnuts. For direct plating, 5-6 hazelnut kernels were placed on solidified agar, rolled on the surface and then taken out of the dish again. For dilution plating, the hazelnuts were combined with an isotonic diluent and ground in a homogenisator. The liquid was plated in 1:10 and 1:100 dilutions. The number of fungi and yeasts (germ number) obtained from each plate varied depending on the plating method used. Bacteria were eliminated by the antibiotic chloramphenicol, and were therefore not included in the germ number.The direct plating method (raw and roasted hazelnuts) revealed the highest germ numbers compared to the dilution plating methods, except for one sample. The same resulted in a study of RABIE et al. (1997), who compared direct plating and dilution methods. They enumerated a much larger number and variety of fungi on barley when using the direct plating method. High germ numbers of approx. 100 were obtained from raw hazelnuts. These are sometimes disadvantageous when single colonies are overgrowing each other. Otherwise more germs may include more species. Therefore direct plating as well as 1:10 and 1:100 dilution plating

I Discussion

methods should be applied to raw hazelnuts, to get one plate usable for germ counting and subculturing. Contrary a 1:100 dilution scheme is not necessary for roasted hazelnuts and hazelnut paste, because the highest germ number was 6 at a 1:10 dilution scheme, which was easily counted. In 5 samples 1 colony grew on the 1:100 plates and none on the 1:10 plates, but this random effect can be obviously ignored due to the 0 cultures on the 1:10 plates.

4.1.1.3 Contaminations

In some samples ITS sequences and morphological criteria provided different results. Usually the reason was a contamination of the culture, often by Rhizopus stolonifer, which overgrows e.g. Penicillium and Aspergillus very fast. If the culture was not contaminated, the reason was probably a contamination during DNA isolation, PCR preparation or cycle sequencing preparation. Contaminated cultures were cleaned mechanically by transferring a piece of the primary fungus on a new agar plate, repeating this procedure until the culture was clean.Contamination is a known problem in microbiology. Some species are considered as very dangerous to contaminate other cultures, due to their high number of conidia or sporangiospores. According to PITT & HOCKING (1997) Trichoderma isolates should be handled carefully, because of its small conidia and its ability to produce chitinases and cellulases. Also Rhizopus is a known contaminator. Cladosporium and Rhizopus were the main contaminators of the hazelnut mould cultures in this study. Contamination can be avoided by cleaning the clean bench when working with mould cultures. Petri dishes should be closed with laboratory film (parafilm) and opened as little as possible. Dishes stored in the refrigerator should not be turned upside down, because dropping condense water might cause contaminations. Cultures should be observed from time to time to verify their cleanness.

4.1.2 Evaluation of the substrate: roasted, raw hazelnuts and hazelnut paste

Raw hazelnuts revealed 3 to 50 times more germs than roasted hazelnuts and hazelnut paste. This tendency could be observed among all samples and is supported by SANCHIS et al. (1988). According to their article raw hazelnuts presented the highest fungal contamination with a total infection of 85.8 % compared to 56.2 % in the roasted hazelnut samples. As shown in the results, roasting hazelnuts has a negative influence on the mycoflora. The purpose of roasting nuts is to promote flavor and texture changes involving a number of physio-chemical processes. A complex mixture of more than 20 different substances is formed, which contribute to the total flavor impression. The water content is lowered from approx. 5 % to 1 % (SAKLAR et al. 2001). The heating during the roasting process to 165 °C for 25 minutes kills fungal hyphae and spores of primary contaminators. It is not clear, whether the changes in the chemical composition of roasted hazelnuts or their dryness are a reason for decreased secondary fungal contamination. However, the decrease of the water content is an important factor in suppression of mould growth and may be an explanation why roasted hazelnuts are less contaminated.

I Discussion

Another reason for the increased mould contamination of raw hazelnut might be storage conditions and duration. The mature hazelnuts are harvested by hand from August to September and dried in the sun or in dryers until the water content drops below 6 %. Hazelnuts that fall from the trees and are collected from the floor supposed to be more contaminated than hazelnuts picked straight from the trees depending on the conditions (moisture) on the floor. The raw hazelnuts are stored for at least one year in shell under uncontrolled storage conditions regarding atmosphere and moisture. Only in few cases temperature control is provided. All hazelnuts are used for production of hazelnut paste in Turkey, which is accomplished by cracking, roasting and grinding twice (TANJA MEINDL pers. com.). If roasted hazelnuts are processed quickly to paste, moulds do not grow intensively. In contrast, raw hazelnuts stored for one year under uncontrolled conditions provide a high risk for contamination through storage fungi.Germ numbers obtained from hazelnut paste were around the same range like the germ numbers obtained from roasted hazelnuts. This is reasonable, as the production of paste from roasted hazelnuts includes only mechanical treatments (grinding twice, TANJA MEINDL pers. com.) and no heat or chemical treatment, which might decrease the mycoflora. Contrary, a higher contamination can be expected due to the fact that paste has no surface protection like a closed nut. Also storage conditions under uncontrolled temperatures in a range of 8-45 °C for several weeks may lead to increased mould growth, whereas storage temperatures above 45 °C prohibit mould growth. The paste is stored in Turkey 3-6 weeks in tanks, whereby the uncontrolled temperature sometimes reaches to 80 °C. Then the paste is transported in heated tanks for approx. 1 week and stored in Austria for another 4 weeks at 40 °C until processing (TANJA MEINDL pers. comm.). In this context it is worth to mention, that according to REISS (1998), aflatoxin production can occur until 42 °C. A. flavus and A. niger are able to grow until 45 °C (PITT & HOCKING 1997). Thermophilic fungi with an optimum between 40-50 °C (WEIDENBÖRNER 2000) are usually found in pasteurized fruit juices and jams, and are not relevant in hazelnuts. Concluding, storage above 45 °C will avoid both mould growth and consolidation with certainty.

4.2 The source of mycotoxins in hazelnut paste

Our supplier of material states that the mycotoxin contents of approximately 20 % of hazelnut paste deliveries are above the limits (TANJA MEINDL pers.com.). The source of mycotoxins in hazelnut paste, produced in Turkey and then delivered to the company, can be either a primary contamination of raw hazelnuts or a secondary contamination of the roasted hazelnuts or the paste itself. Determination of the contaminating mycoflora in the first place might provide useful information for risk assessment of mycotoxin contents, as lack of fungal contamination is equivalent with lack of mycotoxin contamination. According to the germ numbers obtained from several samples (Table 16), main fungal contamination occurs in raw hazelnuts. Roasted hazelnuts and hazelnut paste are negligible contaminated compared to raw hazelnuts (Table 17). As a consequence, major mycotoxin production can be related to fungi on raw hazelnuts.

I Discussion

4.3 Comparison of the suppliers of the hazelnuts

Four suppliers named A, B, C and D delivered hazelnut samples to Hygienicum AG.

Table 23. Suppliers of hazelnut samples (numbers according to the covering letter in Table 7)

Supplier Number

A 7542

B 7596

C 7661

D 7667

Only one hazelnut paste sample was obtained from supplier A. Therefore this supplier is not included in the comparison. Comparison of the suppliers B, C and D regarding germ numbers of hazelnuts shows, that hazelnuts of supplier B present the highest germ numbers, followed by D and C. It should be pointed out that hazelnuts of supplier C show a considerable lower germ number than those of the other suppliers. It would be of interest, if this pattern is also reflected by possible aflatoxin contents. Figure 16 shows the varying germ numbers of raw hazelnuts within the suppliers and within the plating methods.

Table 24. Average germ numbers obtained from hazelnuts (roasted, raw, paste) provided by the suppliers B, C and D, applied with different plating methods (direct, dilution 1:10 and 1:100).

NutsDirect 1:10 1:100

B C D B C D B C D

Raw 439 (2)a 72 151(2)b 102 (2) 13 64 (3) 17.5 (2) 2 11.3 (3)

Roasted 6 (3) 3 0.3 (3) 0 (3) 0 0 (3) 0.7 (3) 0 0.3 (3)

Paste - - - 0.5 (3) 0 0.3 (6) 0 (3) 0 0.8 (6)aFigures in parentheses are the numbers of samples for calculation of the average (n).b Sample no. 1062 with an infinite germ number was not included.

Fig. 16. Germ number obtained from raw hazelnuts provided by the suppliers B, C and D, applied with different plating methods (direct, dilution 1:10 and 1:100).

I Discussion

4.4 Identification of fungi

4.4.1 Morphological features

The results obtained from the ITS sequences were verified and improved using morphogical features according to SAMSON et al. (1995), PITT & HOCKING (1997) and DE HOOG et al. (2000). In doubtful cases the genus only was identified. Species of Aspergillus and Penicillium were difficult to distinguish by morphological criteria and therefore were not identified in some cases. Usually physiological features like growth temperatures, growth rates on certain media and colony diameters are also applied to identify fungal species (SAMSON et al. 1995, PITT & HOCKING 1997). However, the required methods are most time consuming among all.

4.4.2 Molecular methods

4.4.2.1 Precautions with molecular methods

A major problem in PCR can be cross-contamination of reagents with DNA, which results in the amplification of both the template DNA and the contaminating DNA. In order to detect false-positive amplifications due to contamination, in each PCR experiment a negative control without template DNA was included. Whenever the negative control was positive, the PCR experiment was repeated while trying to find the source of contamination. Precautions to minimize the risk of contaminations were taken. Sterile pipette tips and tubes were used, aliquots of NTPs, sterile water and primers were prepared, and furthermore one specific pipette set for the preparation of PCR reactions and one for the preparation of cycle sequencing reactions were used. Sequence data are obtained using an automatic sequencer ABI 310 (Applied Biosystems). Buffer and water was changed once a week, and the capillary was changed after approx. 300 runs. The polymer was refilled, whenever needed, and the syringe block was cleaned from urea crystals. The template DNA can sometimes cause complications. For example, the Zygomycetes sequencing results showed characteristic problems throughout all genera. The sequences seemed, as if more than one cycle sequencing reaction was in the tube. Peaks were overlapping and built a background noise that made it impossible to analyze and assemble the sequences (Figure 17). Remarkable are homopolymeric sections in the ITS region of Rhizopus (Figure 18). After such a section, the sequence is not resolved anymore, either due to a loop in this part of the DNA strand, which disturbs the DNA polymerase during cycle sequencing, or due to erroneous shifting of the polymerase during extension. Changing the temperature profile did not result in better sequences.

I Discussion

Fig. 17. Part of the ITS sequence of Syncephalastrum racemosum (no. 1703.5) with overlapping peaks

Fig. 18. Homopolymeric sections in the ITS sequence of Rhizopus stolonifer (no. 835.1)

4.4.2.2 ITS sequences for identification of fungi

The ITS sequences of 72 samples were used to query the Genbank using BLASTN program and resolved the species or the genus. Yet, ITS sequencing of the Zygomycetes did not provide reliable results. Characteristic for the Zygomycetes genus Rhizopus are few homopolymeric sections that probably build loops and cause detachment of the DNA polymerase. Detachment and attachment of the polymerase can result in incorrect positioning of 1-4 nt. Therefore the sequences are usually bad after the first 100-150 nt and hardly usable for running a query on nucleotide databases. Only few sequences were used for identification of Zygomycete species. The main part of Zygomycetes including the genus Rhizopus was identified by morphological criteria.A disadvantage of using the method of running a sequence query with BLASTN is that the result does not point on a single species alone. Furthermore there is no reliability, whether the sequences in the database represent the species, they are referred to. This dilemma can be resolved by establishing a database with more variable sequences than ITS or fragment pattern types of all fungi, which are mentioned in the literature as contaminators of hazelnuts. Another alternative is to use morphological features to filter one species out of all the options.

4.4.3 PKS patterns for characterization of Aspergillus and Penicillium isolates

Aspergillus and Penicillium were investigated in more detail because they show clear predominance in the fungal spectrum. Second they are very efficient mycotoxin producers. And third their species are difficult to distinguish by morphological criteria. The polyketide synthases (PKS) are an important enzyme class involved in the production of secondary metabolites of fungi. These enzymes synthesize the basic structure of a compound, whereas the final secondary product is then produced after a number of post-PKS enzyme-

I Discussion

mediated transformations. The PKSs are relatives of fatty acid synthetases, and likewise they sequentially condensate C2-units. They are responsible for the assembly of potent carcinogens such as aflatoxins (a precursor of this carcinogen, norsolorinic acid, is named after its occurrence in the lichen Solorina) and for metabolic regulators such as the cholesterol biosynthesis inhibitor lovastatin. Data from fungi suggest that PKS genes encode multifunctional proteins containing only a single, reiteratively used ketide synthesis domain. The gene fragment encoding this domain can be targeted by PCR primers (LEE et al. 2001, SAUER et al. 2002). These acquired ‘tag’ sequences indicate for the genetic potential for the synthesis of PKSs (MIAO et al. 2001).PKS pattern seem to be a good tool to group Aspergillus spp. and Penicillium spp., since both genera produce several mycotoxins in polyketide pathways (SWEENEY & DOBSON 1998). The advantage of PKS pattern is, that certain synthase genes of secondary metabolites (polyketides) are amplified instead of amplifying an unknown part of the genome by a random short primer (RAPD). Therefore a PKS pattern includes also information about presence or absence of PKS genes. Not necessarily all of the numerous fragments obtained from Aspergillus and Penicillium are representing PKS genes, although work by MIAO et al. (2001) shows that most of the fragments actually are. The ones that are not PKS genes could be distant paralogs from the ketosynthase domain superfamily (possibly mitochondrial oxo-acyl synthetase and similar).As shown in the results, each Aspergillus and Penicillium sample has a characteristic PKS pattern, which is good reproducible. Lack of PKS pattern does not seem to be characteristic for any of the Aspergillus and Penicillium isolates investigated. Hence, all species seem to possess PKS genes. PCR, that did not provide a pattern, were repeated again. Nevertheless some samples did not provide a pattern even after few trials and were excluded from the results.

4.4.4 Are PKS patterns characteristic for species?

The PKS types of Aspergillus and Penicillium were named Aa, Ba, Ca, Da, Ea and Ap, Bp, Cp, Dp, Ep, Fp with the indices indicating the genus (a for Aspergillus and p for Penicillium). A few samples could not be identified as member of one of these groups (Xa and Xp,) and are not included in the discussion. Aspergillus fumigatus and A. niger agg. could be precisely associated with one PKS type (Da

and Aa, respectively). The A. flavus group showed in 6 cases PKS pattern Ba and in 2 cases PKS pattern Ca. Recent studies of this species complex by GEISER et al. (1998) revealed two reproductively isolated groups in this complex. One of the subgroups includes the non-toxic clonal derivative Aspergillus oryzae. However, it still needs to be tested whether the two different PKS patterns found in the present study correlate with these subgroups or are restricted to one of them. Penicillium citrinum showed one characteristic PKS type (Ep). Also P. brevicompactum provided a distinct pattern (Fp), however only one sample was analyzed, and it cannot be excluded, that additional patterns exist for P. brevicompactum. One sample of P.

I Discussion

chrysogenum showed PKS pattern Dp. P. cf. commune was subgrouped according to the PKS pattern (10 samples were of type Ap, and 3 of type Bp). Also P. expansum provided pattern type Ap. P. brevicompactum, P. cyclopium and P. polonicum provided all PKS pattern Cp.A number of species show only one PKS pattern, whereas others are shared by two patterns, and one PKS type can also occur in various species. Further analyzes must show, whether PKS patterns might be a useful tool for fast identification of fungi. If mycotoxin production ability is correlated with certain PKS types this could also support quick risk assessments. Because the existence of PKS genes does not necessarily imply the capacity to produce mycotoxins, strains with certain pattern could be sorted out in further experiments and tested for their mycotoxin production.

4.5 Mycotoxic fungi found in raw, roasted hazelnuts and hazelnut paste

By now, 77 fungal species have been reported on hazelnuts. In this study 28 taxa were identified, whereby 9 taxa have not been reported (Table 25). Hazelnuts may contain the mycotoxins aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2 and ochratoxin A (SENSER 1979, ABDEL-HAFEZ & SABER 1993, ELMADFA & BURGER 1999, WEIDENBÖRNER 2001a). Several strains of Aspergillus flavus, A. parasiticus and A. nomius can produce aflatoxins. Potential ochratoxin A producers are A. ochraceus, A. niger, Eurotium herbariorum, and Penicillium spp. (FRISVAD & SAMSON 1991; PITT & HOCKING 1997; WEIDENBÖRNER 1998, 2001a). By now only A. flavus and A. parasiticus are known to produce mycotoxins on hazelnut substrate (SENSER 1979). It is not clear, which fungi are responsible for ochratoxin A contamination, and whether other fungal genera reported on hazelnuts produce their potential mycotoxins on hazelnut substrate. In this study the potential aflatoxin producers Aspergillus flavus and A. parasiticus, and the potential ochratoxin A producers A. niger and Penicillium cyclopium were identified. Table 25 shows that most of the fungi found on the hazelnut samples are potential mycotoxin producers. However, the risk of these toxins to human health is not clear, because both the hazard assessment and the exposure assessment include many uncertainties. Investigation of fungal strains to verify their mycotoxin production ability on hazelnut substrate could enable a first assessment of their hazard as hazelnut contaminators. Mycotoxin production can be expected, if the required environmental factors are optimal. It would be of interest to analyze potential mycotoxins (Table 25) in hazelnuts, whether they occur in reasonable amounts or not, although there are no regulations of limits for most of these mycotoxins.

I Discussion

Table 25. Fungi that were found in raw, roasted hazelnuts and hazelnut paste, their potential mycotoxins and their report as hazelnut contaminating fungi (FRISVAD & SAMSON 1991, SAMSON et al. 1995, PITT & HOCKING 1997, WEIDENBÖRNER 1998).

Fungus Potential mycotoxins Report

Aspergillus flavus Aflatoxin B1, cyclopiazonic acid yes

A. fumigatus Fumagilin, gliotoxin yes

A. parasiticus Aflatoxin B1 yes

A. oryzae Cyclopiazonic acid yes

A. tubingensis - no

A. niger Ochratoxin A yes

Penicillium expansum Patulin, citrinin, roquefortine C and others no

P. cf. commune Cyclopiazonic acid and others no

P. geastrivorus - no

P. glabrum Citromycetin no

P. brevicompactum Mycophenolic acid yes

P. citrinum Citrinin yes

P. chrysogenum Patulin, roquefortine C, cyclopiazonic acid yes

P. cyclopium Penicillinic acid, ochratoxin A, patulin and many others

yes

P. nalgiovense Penicillin yes

P. polonicum Penicillinic acid, verrucosidin, nephrotoxic glycopeptides

no

Rhizopus stolonifer Rhizonine yes

R. oryzae Isofumigaclavine A yes

Mucor circinelloides Not reported yes

M. plumbeus Not reported no

Syncephalastrum racemosum Not reported yes

Cladosporium sp. Cladosporic acid yes

Aureobasidium pullulans Not reported no

Alternaria alternata Alternariols, altertoxins, tenuazonic acid yes

Alternaria infectoria Alternariols, altertoxins, tenuazonic acid no

Trichoderma longibrachiatum Trichoderma sp. produces several yes

Trichothecium roseum Trichothecenes yes- No reference found.

II Molecular characterization of the phytopathogenic fungus Ramularia collo-cygni

II Introduction

1. Introduction

Ramularia collo-cygni B. Sutton & J. Waller is a parasitic hyphomycete on barley and other Poaceae that causes leaf spots and threatens both yield and quality of the crop. In the last few years R. collo-cygni emerged to be a problem in European agriculture causing crop loss of 10-15 % (NEUHOLD 1995).The biology, morphology and epidemiology of R. collo-cygni have been investigated among others by HUSS et al. 1992, HUSS & NEUHOLD 1995, NEUHOLD 1995. The species was included in phylogenetic studies, which clearly show a position in Mycosphaerella (CROUS et al. 2001), and close to Mycosphaerella fragariae (GOODWIN et al. 2001).However, molecular approaches for strain typing and characterization of the fungus have never been applied. In this study first steps in this new field using molecular methods as described above should suggest future research options.The aim was to apply multilocus sequence typing with the sequences of 3-5 genes to gather data for the identification of strains within populations and to assess intraspecific variation. However, the investigated genes revealed no interspecies differences and therefore other molecular markers (RFLP, RAPD) were investigated. 11 samples were analyzed. ITS, IGS, mtSSU and chitin synthase genes were partly sequenced and the samples were tested for several RFLP- (in the case of IGS) and RAPD-markers.

II Materials and methods

2. Materials and methods

Ramularia collo-cygni sample

DNA-Isolation

Characterisation with molecular methods

DNA Sequencing RAPDs RFLPs

Fig. 1. Flowchart illustrating the methods for characterization of Ramularia collo-cygni

2.1 Materials

Isolated cultures from Ramularia collo-cygni were kindly provided by D.I. Herbert Bistrich from the IFA Tulln and by Dr. Edith Sachs from the BBA, Kleinmachnow Branch.

Table 1. Investigated isolates of Ramularia collo-cygni

Synonym No. Origin Host

Kru 2 1 Krumpendorf, Carinthia, Austria Barley

Malh 2A 2 Markt Allhau, Burgenland, Austria Barley

Lam 8/6/1A 3 Lambach, Upper Austria, Austria Barley

Lam 2 4 Lambach, Upper Austria, Austria Barley

Aum 1B 5 Aumühle, Lower Austria, Austria Barley

Dlbg 1A 6 Deutschlandsberg, Styria, Austria Barley

Gle 1 7 Gleisdorf, Styria, Austria Barley

Ilz 2A 8 Ilz, Styria, Austria Barley

Fek 3 9 Feldkirchen, Carinthia, Austria Barley

No. 139 10 Reutlingen, Baden-Württemberg, Germany Winter barley

No. 140 11 Argentina Summer barley


2.2 Cultivation of Ramularia collo-cygni

R. collo-cygni cultures were grown on ME agar. Usually three days after subculturing, incubated at RT, first hyphae were visible with the naked eye. After 10 days the plates were half covered by the mycelia and then stored in the refrigerator. Differences in growth rates within the strains could not be observed.

Composition of the malt extract (ME) agarMalt extract 30 gPeptone 5 gAgar 15 gH2O 1000 ml

2.3 Sequencing of ITS, IGS, mtSSU and chitin synthase genes

The PCR were carried out as described in part I (2.5.2) using the primers listed in Table I.8. The temperature profile for the PCR of ITS, IGS and mtSSU was programmed according to table I.10 with higher annealing temperatures of 53 °C for mtSSU and 60 °C for IGS. The PCR with chitin synthase primers required a major deviation from the standard PCR concerning denaturation, annealing and extension time (30 sec each) and annealing temperature (Table 3).

Table 2. Sequences of oligonucleotide primers for the ITS, IGS and mtSSU region and the chitin synthase gene


ITS1F CTTGGTCATTTAGAGGAAGTAA GARDENS & BRUNS 1993

ITS4 TCCTCCGCTTATTGATATGC WHITE et al. 1990

LR12R GAACGCCTCTAAGTCAGAATCC Vilgalys laba

InvSR1R ACTGGCAGAATCAACCAGGTA Vilgalys laba

mtSSU1 AGCAGTGAGGAATATTGGTC ZOLLER et al. 1999

mtSSU2 CTGACGTTGAAGGACGAAGG ZOLLER et al. 1999

Chitin synthase1 TGGGGCAAGGATGCTTGGAAGAAG CARBONE & KOHN 1999

Chitin synthase2 TGGAAGAACCATCTGTGAGAGTTG CARBONE & KOHN 1999a http://www.biology.duke.edu/fungi/mycolab/


Table 3. Temperature profile for the chitin synthase PCR



30 cycles



Extension 30 sec at 72 °C



Table 4. Sequences of oligonucleotide primers additionally used for cycle sequencing of the ITS, IGS and the mtSSU region


ITS1R TCCGTAGGTGAACCTGCGG WHITE et al. 1990

ITS2 GCTGCGTTCTTCATCGATGC WHITE et al. 1990

ITS3 GCATCGATGAAGAACGCAGC WHITE et al. 1990

5SRNA (IGS primer) ATCAGACGGGATGCGGT Vilgalys laba

5SRNAr (IGS primer) ACCGCATCCCGTCTGAT Vilgalys laba

mtSSU2 CCTTCGTCCTTCAACGTCAG ZOLLER et al. 1999

mtSSU3r ATGTGGCACGTCTATAGCCC ZOLLER et al. 1999a http://www.biology.duke.edu/fungi/mycolab/

2.4 Molecular Markers

2.4.1 RFLP analysis

Restriction enzymes cut the DNA in or near the recognition sequence and produce a collection of DNA fragments. The presence or absence of a restriction sequence is a useful genetic marker and is revealed by the presence or absence of a restriction fragment. A disadvantage of RFLP analysis is the high amount of DNA that is required.In Ramularia collo-cygni, RFLP analysis was applied on the IGS region, as it is too long for direct sequencing (see Results). The IGS region was first amplified with the primers LR12R and invSR1R and then assayed for polymorphism by RFLP analysis. Three different restriction enzymes were investigated: AvaI, HinfI and MspI.


Table 5. Restriction enzymes and recognition sequences

Enzyme Buffer Recognition sequence

AvaI B: 100 mM Tris-HCl, pH 8.0, 50 mM MgCl2, 1000 mM NaCl, 10 mM 2-ME

C/(T,C)CG(A,G)G

HinfI H: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTE, 1000 mM NaCl

G/ANTC

MspI L: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl2, 10 mM DTE

C/CGG

Table 6. The components of the RFLP mix


Template DNA 10

H2O 7

10x restriction buffer 2

Enzyme 1

The samples were incubated for 4 hours at 37 °C and then analyzed on a 2 % agarose gel.

2.4.2 RAPD analysis

RAPD analysis is a popular tool for genotyping the diversity among and within species. Usually, a short primer of about 8-10 nucleotides is applied at low annealing temperature. It typically reveals a characteristic pattern of differently sized PCR amplification products. Alternatively, it is possible to use longer primers as long as the annealing temperature is low. The reproducibility of RAPD analysis can be a problem, due to mispriming at the low annealing temperature. Furthermore RAPD analysis can be impaired by biogenic contaminations leading to undesired amplifications. Thus, the samples were handled with great care and amplification was always carried out with the same sets of chemicals and in the same PCR machine.To identify primers, which can detect polymorphisms, six different 8-10-mer oligonucleotides (Table 7) were screened with several samples of R. collo-cygni.


Table 7. Oligonucleotide primers that were used for RAPD analysis

Primer Sequence 5’-3’

Afu1 GCTGGTGG

Afu2 TCACCCTGA

Derm1 ACTACTCGCA

OPAA11 ACCCGACCTG

OPD18 GAGAGCCAAC

R108 GTATTGGCCT

Table 8. Temperature profiles for the RAPD-PCR



30 to 35 cycles

Denaturation 1 min at 92 °C

Primer annealing 1 min at 33 °C

Extension 10 sec at 72 °C



2.4.3 tRNA primer

The intention of using the tRNA (Table 9) primer was to reveal a fragment pattern of the tRNA cluster. CARTER et al. (2000) stated that the primers would be complementary to a consensus sequence of tRNAs, but such could not be found in comparisons of tRNAs retrieved from GenBank. It is here concluded that the tRNA primer behaves similar to a RAPD primer, and reveals PCR-fragment patterns by binding to anonymous loci. The temperature profile used for all RAPD-PCR was then also used for the tRNA primer (Table 8).

Table 9. Oligonucleotide primer that was used for tRNA-RAPD analysis


tRNA TCCAGCCGGGAATCGAAC CARTER et al. 2000

II Results

3. Results

3.1 Sequences of ITS, IGS, mtSSU and chitin synthase genes

The sequences of the ITS, IGS and mtSSU region and the chitin synthase gene are shown below. The alignments of the received sequences in ClustalX revealed no significant differences in any of the investigated genes and are therefore not shown. The IGS sequences obtained have a size of approx. 300 bp starting from the primers on both ends of the fragment. For the sequencing of the entire IGS region, it would be necessary to develop reverse primers, as the PCR fragment is too long (3 kb) for a complete complementary sequencing with 2 primers.

ITS (no. 10)

1 ATCATTACTG AGTGAGGGAG CAATCCCGAC CTCCAACCCT TTGTGAACGC ATCATGTTGC TTCGGGGGCG ACCCTGCCGC 81 GCAAGCGGCA TTCCCCCCGG AGGTCATTCA AACACTGCAT TCTTACGTCG GAGTAAAAAG TTAATTTAAT AAAACTTTCA 161 ACAACGGATC TCTTGGTTCT GGCATCGATG AAGAACGCAG CGAAATGCGA TAAGTAATGT GAATTGCAGA ATTCAGTGAA 241 TCATCGAATC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT GCCTGTTCGA GCGTCATTTC ACCACTCAAG 321 CCTCGCTTGG TATTGGGCGT CGCGAGTCTC TCGCGCGCCT CAAAGTCTCC GGCTGAGCGG TTCGTCTCCC AGCGTTGTGG 401 CAACTATTCG CAGAGGAGTT CGAGTCGTCG CGGCCGTTAA ATCTTTCAAA GGTTGACCTC GGATCAGGTA GGGAT

IGS 1 (no. 5)

1 TCCCCCGCAA GCACACGCGG GGGCGACGGG CGTATCGNAA TTTTATCGCG CGCTGGGATG AAACCCTTGC ATACGACTTG 81 GACGTCTGAG CGGGTCGTGT AAGCAGTCGA GTAGCCTTGT TGTTACGAGC TGCTGAGCGT AAGCCCGATC TCAGCTAGTT 161 TTGTTTAATA CCTCCCCATC GTTTCGTGGT ATCTCACCAC GGGCCGGAGG GGACTCTGGT GGCTTCCCAT CACACCTTGA

IGS 2 (no. 5)

1 TACTTCACCA GTCTCACCAT CCAATTGCCA CAACCGAATT CCCCAAGGCG ACTTCCGCCG AAGAATTGAC TAGCGAGCTA 81 ATCAAACTTT GACATGGCCG TCTCAAGTGG AGATCTCTCG TCGCCGGGTC ACATTCCGAG GAACGGCTAC CGGTACACGA 161 GTATATCCCC GAGTGTCTTT CGATAATGAT AGCCAACGGC CAATTATCCA CAGACTCCTT CGGCGCCGCC TCATCAGTAA 241 TTGAAGAGAC GGGCTTCGGG CCGCCACAAT GTACGCCCGA TCCTTGCGAA TCAAAGTGCA ACCGAGGCGG TGCGATCAAC 321 CAATAGAACC CAGGATGGCT G

mtSSU (no. 1)

1 TATATGCAAA TCGGCTATGC CGAATAAAGT TCTAAATAGA TAATTTATTA TGAAAAACTT CTATTTATAT GTCTCGACCA 81 ATTCTTGTGC CAGCAGTCGC GGCAACACAA GTGAGACTAG TGTTATTCAT CTTTATTAGG TTTAAAGGGT ACCTAGACAG 161 TATTTCCAGC CCCCAAAGGG TACAGATTTA CTAGAGTTTT ATACGGGAGG TAGATATTAG GACCATTGGT GTAGAGATGA 241 AATTCTTTGA TACTAATGGG ATGTGTAACG GCGAAGGC-A CCCTT--ATG TAAAA-CTGA CGTT-AGGAC GAA-GCTTGG

II Results

321 GGAGCGAATA GGA-TTAGAT ACCCTAGTAG TCCAGGCAGA AAATTATGAG TGTTATAGAC TAAATTAAAT TGGTGTATAA 401 ATGAAAGTGT AAGCATTCCA CCTCAAGAGT AATGTGGCAA CACAGGAACT GAAATCATTA GACCGTTTCT GAAACCAGTA 481 GTGAAGTATG TTATTTAATT CGATGATCCT CGAAAAACCT TACCACAATT TGAATGTTTT CTAAAACGGG ATATACATTT 561 ATACCGCATA CATTATTTTT TTATTTATGT ATAATTAAAC ACAAGCGTTG CACGGCTGTC TTCAGTTAAT GTCGTGAGAT 641 TTTGGTTAGA TCCATTAAAT TAACGTAAAC CCTTGCTTTT TTTATGTATT TTTCCTTTTA TTTTGTTTTA TAATTTAATT 721 TAAGAAGAGG AAAATAATTA ATGAAGTAGT TCGCCGCTAG ATTGGCTTAT GATAACAGG

Chitin synthase (no. 11)

1 TTTGCATTGT CTCCGATGGG CGTGCCAAGA TCAACCCGAG GACACGATCT GTGCTCGCTG CCATGGGTAT CTACCAGGAC 81 GGCATTGCGA AGCAGCAAGT GAATGGAGAA GATGTCACGG CTCACATCTA CGAATACACC ACGCAGATGA CCTTGGAAAT 161 CAAGAAGGGC ATTGTGCAGG TCAAGAAAGA TCTTGTTCTG TCTCAAGGAG AAGAACC

II Results

3.2 Molecular Markers

As the sequencing data did not provide any variation useful for multilocus sequence typing, molecular markers based on anonymous loci, either serving as priming regions (RAPD) or as restriction enzyme recognition regions (RFLP), were investigated.

3.2.1 RFLP analysis

RFLP analysis revealed more or less banding variation among the investigated isolates in the IGS region, depending on the enzyme used. AvaI provided 3 different fragment types combining each, sample no. 1 and 2, no. 3, 4 and 5 and no. 10 and 11. According to the pattern provided by HinfI, sample no. 1, 2, 3, 4 and 5 can be grouped and distinguished from no. 10 and 11. Interestingly, both AvaI and HinfI produce a similar pattern for no. 10 and 11 and no. 1, 2, 3, 4 and 5, respectively. Among the bands produced by MspI not much variation can be observed. The main fragments range from approx. 100 to 500 bp, whereby sample no. 2 and 3 reveal several longer fragments. Samples no. 6-9 were not tested.

AvaI HinfI MspI S 1 2 3 4 5 10 11 1 2 3 4 5 10 11 1 2 3 4 5 10 11 S

- 1 kb

- 500 bp

Fig. 2. Agarose gel electrophoresis of RFLP products from the IGS region of Ramularia collo-cygni with the enzymes AvaI, HinfI and MspI. Lane S: 100 bp ladder. Sample numbers are according to Table 1.

3.2.2 RAPD analysis

As already mentioned in 2.4.3 (Materials and methods) the tRNA primer is not specific for tRNA. It was therefore treated as a RAPD primer and included in this part of the results. Afu1 failed to amplify fragments, OPD18 and Derm1 did not show any variation in patterns among the investigated isolates and thus, the results are not shown. tRNA, Afu2, OPAA11 and R108 provided differences in the fragment pattern (Figures 3 and 4).

II Results

Sample no. 1, 2, 3, 4, 5 and 9 show the same tRNA pattern with 2 characteristic fragments and a smear. Sample no. 8 shares the fragments of approx. 450 bp and 1500 bp with no. 6 and 7, but lacks the fragment of approx. 600 bp. The OPAA11 pattern of sample no. 1, 2, 5, 9 and 11 shows a cluster of 4 strong fragments from 1000 to 1600 bp. No. 6, 7 and 8 show only 2 close fragments around 1200 bp and 1 more characteristic fragment of 700 bp, which no. 8 is lacking.

II Results

The Afu2 pattern reveals variability among all samples, whereby also differences in intensity are observed. Sample no. 6, 7 and 8 share 3 fragments of approx. 120, 600 and more than 1700 bp, which the residual samples are missing. The R108 pattern is characterized by varying intensity of the fragments. Similar patterns are observed within no. 1 and 4, no. 2 and 3, and no. 5 and 10. Sample no. 9 and 11 are different in intensity and number of fragments.

A B S 0 1 2 3 4 5 6 7 8 9 S 11 0 9 8 7 6 5 2 1

- 1kb

- 500 bp

1 Kru22 M alh 2A5 M alh2A6 D lbg1A7 G le18 Ilz2A9 Fek3S1 139S2 140

RAP D w ith O PAA11 prim er

11.3.02

Fig. 3. Agarose gel electrophoresis of PCR products from Ramularia collo-cygni with the primers (A) tRNA and (B) OPAA11. Lane S: 100 bp ladder. Sample numbers are according to Table 1.

A B 1 2 5 6 7 8 9 10 11 0 S S 1 2 3 4 5 9 10 11 S

- 1kb

- 500 bp

- 1kb

- 500 bp

Fig. 4. Agarose gel electrophoresis of PCR products from Ramularia collo-cygni with the primers (A) Afu2 and (B) R108. Lane S: 100 bp ladder. Sample numbers are according to Table 1.

II Discussion

4. Discussion

Multilocus sequence typing of Ramularia collo-cygni using ITS, IGS, mtSSU and chitin synthase primers revealed no variability. Major molecular methods, applicable on species level, have been developed from the rDNA cluster. Part of this cluster are the ITS regions, which are a particularly useful area for molecular characterization of fungi (BRIDGE & ARORA 1998). The variation among the ITS regions may be useful in resolving relationships between close taxonomic relatives including strains of one species. For example CHARCOSSET & GARDES (1999) revealed intra-specific genetic diversity in an aquatic hyphomycete. However, in R. collo-cygni the ITS regions do not provide enough variability for strain typing within the species. Applied to an inter-specific level the ITS regions were used among the genus Mycosphaerella for phylogenetic investigations on its anamorph genera including R. collo-cygni (CROUS et al. 2001).In contrast to the ITS region, fewer studies have considered the IGS region, located between the rDNA regions (BRIDGE & ARORA 1998). ARORA et al. (1996) used RFLP derived from the IGS to determine variability within the species Verticillium chlamydosporium and others, and found a low level of variability within species, but distinct IGS types characteristic for single species. In R. collo-cygni RFLP patterns showed variability, in contrast to the sequences of the first and last approx. 300 nt. That means, that variable parts must be located within the sequenced strands. Sequencing of the entire IGS region could reveal more information. The mtSSU genes are the more conserved parts of the rDNA gene cluster useful in phylogenetics for investigating relationships among distantly related fungi (BRIDGE & ARORA 1998). Nevertheless, part of the mtSSU was sequenced in R. collo-cygni, but did not reveal intra-specific variation as expected.Chitin synthase genes neither showed intra-specific variation, but this was not surprising as this gene is generally more conserved than the ITS. The molecular markers RFLP and RAPD contributed more results, but due to the low number of samples definite conclusions cannot be done. Summarizing, the RAPD primers tRNA, Afu2, OPAA11 and R108 reveal intraspecific differences in R. collo-cygni. The samples no. 6, 7 and 8 are similar and clearly distinguishable from the other samples in the tRNA, Afu2 and OPAA11 patterns. The Afu2 and R108 patterns show the highest variability, but also strong differences in band intensity, which may lead to misinterpretation. A correlation between origin and RAPD pattern can be found in the samples no. 6, 7 and 8, which all were collected in Styria (Austria). However, the wider distributed samples from Austria, Germany and Argentina show variability, which cannot be related to their origin. Therefore it can be assumed, that the origin may play a minor role in the occurrence of fragment variability. An investigation of a correlation between host and PCR fragment types may contribute more results.Further researches should include more samples of a wider geographic distribution and from different hosts, so strains could actually be determined and related to origin and/or host. Furthermore investigation on better reproducible methods, e.g. AFLP (amplified fragment length polymorphism) genotyping, should be considered.

II Discussion

Summary

Summary

This study comprises two parts, which both deal with molecular methods for identification and characterization of microfungi. The investigated fungi have an economical impact, either as a pathogen on crops or as food contaminators. In the latter case, this involves fungi, which may produce toxic metabolites. Principal questions in applied mycological studies can be addressed by molecular methods. For example fungi that are difficult to determine phenotypically can be identified using molecular tools.

Part I of this study was carried out as a cooperation with Hygienicum AG, a company for microbiology and hygiene consulting in Graz, Austria (URL: www.hygienicum.at). The aim of the project was to identify and characterize the mycoflora of raw hazelnuts and processed hazelnuts (roasted, paste) provided by a costumer company. Molecular methods were applied to achieve this target. Identification of hazelnut contaminating fungi was carried out using the ubiquitous nuclear internal transcribed spacer (ITS) regions 1 and 2, which separate the coding rDNA genes. The ITS 1 and ITS 2 regions were chosen due to their high abundance in public databases (GenBank), which were queried for comparisons with the sequences produced in this work. Another reason to choose these loci is their high number in the genome. The species were identified using BLAST (Basic Local Alignment Search Tool) at the NCBI website (http://www.ncbi.nlm.nih.gov/), and the results received were supplemented by PKS fragment patterns, the optical evaluation of the color and surface characteristics of the cultures and microscopic morphological criteria. 28 taxa could be identified, whereby 9 have not been reported before on hazelnuts (Alternaria infectoria, Aspergillus tubingensis, Aureobasidium pullulans, Mucor plumbeus, Penicillium cf. commune, P. expansum, P. geastrivorus, P. glabrum, P. polonicum). Aspergillus spp. and Penicillium spp. were characterized further by fragment patterns of biosynthetic genes for polyketides (PKS), because several species of these two genera are important mycotoxin producers on hazelnut substrate. Additionally, a risk assessment of the mycoflora based on the mycotoxic properties of the fungi identified, the environmental factors relevant during hazelnut processing, and the quantity of contaminating fungi during the processing steps was carried out. Until now, only aflatoxins and ochratoxin A have been investigated in hazelnuts. Regulation limits have been set only for aflatoxins. Taxa identified on hazelnuts which produce mycotoxins include Aspergillus flavus and A. parasiticus as potential aflatoxin producers and A. niger and Penicillium cyclopium as potential ochratoxin A producers. Significant mycotoxin production can be related to high fungal contamination in raw hazelnuts.

In Part II the phytopathogenic fungus Ramularia collo-cygni, which causes worldwide crop loss of barley, was characterized using a number of PCR based methods. ITS, intergenic spacer (IGS), mitochondrial small subunit (mtSSU) and chitin synthase genes were sequenced and aligned using the free-ware program ClustalX. The investigated genes revealed no intraspecific differences and therefore fingerprinting methods with molecular markers were


Summary

tested. Restriction fragment length polymorphism (RFLP) of the nuclear IGS region and randomly amplified polymorphic DNA (RAPD) analysis were applied to R. collo-cygni and revealed variety among some isolates.

Zusammenfassung

Diese Studie ist aus zwei Teilen zusammengesetzt, die sich beide mit molekularen Methoden zur Identifizierung und näheren Charakterisierung von Mikropilzen auseinandersetzen. Die untersuchten Pilze haben eine ökonomische Bedeutung als Pathogen auf Getreide bzw. als Lebensmittelverderber. Letzteres schließt Pilze ein, die Mykotoxine produzieren können. In angewandten mykologischen Studien können viele Fragestellungen mit molekular-biologischen Methoden gelöst werden. Beispielsweise können phänotypisch schwer bestimmbare Pilze mit molekularen Methoden identifiziert werden.

Teil I dieser Studie wurde in einer Kooperation mit Hygienicum AG, einer Firma für Mikrobiologie und Hygiene Consulting in Graz, Österreich (URL: www.hygienicum.at) ausgeführt. Das Ziel des Projektes war die Identifizierung und Charakterisierung der Mykoflora von rohen Haselnüssen und verarbeiteten Haselnüssen (geröstet, Paste), die von einer Kundenfirma zur Verfügung gestellt wurden. Molekulare Methoden wurden angewandt um dieses Ziel zu erreichen. Die auf Haselnüssen gefundenen Pilze wurden mit den ubiquitären nukleären Internal Transcribed Spacer (ITS) Regionen, die die kodierenden rDNA Gene trennen, identifiziert. Die ITS 1 und ITS 2 Regionen wurden aufgrund ihrer Häufigkeit in öffentlichen Datenbanken (GenBank) und ihrer hohen Zahl im Genom gewählt. Die Arten wurden mit BLAST (Basic Local Alignment Search Tool) auf der NCBI Website (http://www.ncbi.nlm.nih.gov/) verglichen und bestimmt. Die Ergebnisse wurden mit PKS Fragmentmustern, optischer Beurteilung der Farbe und Oberflächenbeschaffenheit der Kulturen sowie mikroskopischen morphologischen Kriterien unterstützt. Es konnten 28 Taxa bestimmt werden, wobei 9 bisher noch nicht auf Haselnüssen nachgewiesen worden waren (Alternaria infectoria, Aspergillus tubingensis, Aureobasidium pullulans, Mucor plumbeus, Penicillium cf. commune, P. expansum, P. geastrivorus, P. glabrum, P. polonicum). Vertreter der Gattungen Aspergillus und Penicillium wurden mit Fragmentmustern von Genen für Polyketidsynthase (PKS) näher charakterisiert, weil einige Arten dieser Gattungen wichtige Mykotoxinproduzenten auf Haselnuss-Substrat sind. Zusätzlich wurde eine Risiko-abschätzung der Mykoflora basierend auf den mykotoxischen Eigenschaften der identifizierten Pilze, den während der Haselnussverarbeitung relevanten Umweltfaktoren und der Quantität der kontaminierenden Pilze während der Verarbeitungsschritte durchgeführt.Bisher sind nur Aflatoxine und Ochratoxin A in Haselnüssen untersucht worden, und Grenzwerte sind nur für Aflatoxine festgelegt. Aspergillus flavus und A. parasiticus als potentielle Aflatoxinproduzenten und A. niger and Penicillium cyclopium als potentielle Ochratoxin A Produzenten zählen zu den auf Haselnüssen identifizierten Taxa, die


Summary

Mykotoxine produzieren. Signifikante Mykotoxinproduktion kann mit starker Pilzkontamination roher Haselnüsse korreliert werden.

Im Teil II wurde der phytopathogene Pilz Ramularia collo-cygni, Verursacher weltweiter Ernteschäden bei Gerste, mit einer Reihe von PCR-basierten Methoden charakterisiert. ITS, Intergenic Spacer (IGS), Mitochondrial Small Subunit (mtSSU) und Chitinsynthase Gene wurden sequenziert und ein Alignment mit dem Freeware Programm Clustal X erstellt. Die untersuchten Gene zeigten keine intraspezifischen Unterschiede. Aus diesem Grund wurden Fingerprinting-Methoden mit molekularen Markern getestet. Restriktionsfragment- Längen-Polymorphismus (RFLP) der nuklearen IGS Region und Randomly-Amplified-Polymorphic-DNA (RAPD) Analyse wurden auf R. collo-cygni angewandt und zeigten Variabilität innerhalb einiger Isolate.

Appendix 1

Appendix 1

Sequences of Aspergillus samples

Aspergillus niger agg.

838.3 Species Accession No. ScoreAspergillus tubingensis AJ 280008 983A. tubingensis AJ 280007 983A. tubingensis AJ 223853 983A. phoenicis U65307 983Gliocladium cibotii AF021264 967 1 ?TATTGATA TGCTTAAGT TCAGCGGGT ATCCCTACC TGATCCGAG GTCAACCTG GAAAAAATG GTTGGAAAA CGTCGGCAG 81 GCGCCGGCC AATCCTACA GAGCATGTG ACAAAGCCC CATACGCTC GAGGATCGG ACGCGGTGC CGCCGCTGC CTTTCGGGC 161 CCGTCCCCC CGGAGAGGG GGACGGCGA CCCAACACA CAAGCCGGG CTTGAGGGC AGCAATGAC GCTCGGACA GGCATGCCC 241 CCCGGAATA CCAGGGGGC GCAATGTGC GTTCAAAGA CTCGATGAT TCACTGAAT TCTGCAATT CACATTAGT TATCGCATT 321 TCGCTGCGT TCTTCATCG ATGCCGGAA CCAAGAGAT CCATTGTTG AAAGTTTTA ACTGATTGC ATTCAATCA ACTCAGACT 401 GCACGCTTT CAGACAGTG TTCGTGTTG GGGTCTCCG GCGGGCACG GGCCCGGGG GGCAAAGGC GCCCCCCCG GCGGCCGAC 481 AAGCGGCGG GCCCGCCGA AGCAACAGG GTATA

841.2 Species Accession No. ScoreAspergillus tubingensis AJ 280008 924A. tubingensis AJ 280007 924A. tubingensis AJ 223853 924A. phoenicis U65307 924Gliocladium cibotii AF021264 916

1 ATCATTACCG AGTGCGGGTC CTTTGGGCCC AACCTCCCAT CCGTGTCTAT TATACCCTGT TGCTTCGGCG GGCCCGCCGC 81 TTGTCGGCCG CCGGGGGGGC GCCTTTGCCC CCCGGGCCCG TGCCCGCCGG AGACCCCAAC ACGAACACTG TCTGAAAGCG 161 TGCAGTCTGA GTTGATTGAA TGCAATCAGT TAAAACTTTC AACAATGGAT CTTCTTTGGT TCCCGCATTC GATGAAAGAA 241 CGCACCCAAT GCCGATTACT AATGTGAATT GCAGAATTCA GTGAATCATC GAGTCTTTGA ACGCACATTG CGCCCCCTGG 321 TATTCCGGGG GGCATGCCTG TCCGAGCGTC ATTGCTGCCC TCAAGCCCGG CTTGTGTGTT GGGTCGCCGT CCCCCTCTCC 401 GGGGGGACGG GCCCGAAAGG CAGCGGCGGC ACCGCGTCCG ATCCTCGAGC GTATGGGGCT TTGTCACATG CTCTGTAGGA 481 TTGGCCGGCG CCTGCCGACG TTTTCCAACC ATTTTTTCCA GGTTGACCTC GGATCAGGTA GGGATA

857.2

Species Accession No. ScoreAspergillus tubingensis AJ 280008 993A. tubingensis AJ 280007 993A. tubingensis AJ 223853 993A. phoenicis U65307 993Gliocladium cibotii AF021264 993

1 ATCATTACCG AGTGCGGGTC CTTTGGGCCC AACCTCCCAT CCGTGTCTAT TATACCCTGT TGCTTCGGCG GGCCCGCCGC 81 TTGTCGGCCG CCGGGGGGGC GCCTTTGCCC CCCGGGCCCG TGCCCGCCGG AGACCCCAAC ACGAACACTG TCTGAAAGCG 161 TGCAGTCTGA GTTGATTGAA TGCAATCAGT TAAAACTTTC AACAATGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA 241 GCGAAATGCG ATAACTAATG TGAATTGCAG AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT 321 CCGGGGGGCA TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGT CGCCGTCCCC CTCTCCGGGG 401 GGACGGGCCC GAAAGGCAGC GGCGGCACCG CGTCCGATCC TCGAGCGTAT GGGGCTTTGT CACATGCTCT GTAGGATTGG 481 CCGGCGCCTG CCGACGTTTT CCAACCATTT TTTCCAGGTG ACCTCGGATA AGGAGGGATA

Appendix 1

858.2


1 ATCATTACCG AGTGCGGGTC CTTTGGGCCC AACCTCCCAT CCGTGTCTAT TATACCCTGT TGCTTCGGCG GGCCCGCCGC 81 TTGTCGGCCG CCGGGGGGGC GCCTTTGCCC CCCGGGCCCG TGCCCGCCGG AGACCCCAAC ACGAACACTG TCTGAAAGCG 161 TGCAGTCTGA GTTGATTGAA TGCAATCAGT TAAAACTTTC AACAATGGGA TCTTTTTGGG TTCCGGCATT CGAATAAAGA 241 ACGCAACGAA ATTGCCAATT ACCTTAATGG TGGAATTGGA CAATTGCAGA ATTCAGTGAA TCATCGAGTC TTTGAACGCA 321 CATTGCGCCC CCTGGTATTC CGGGGGGCAT GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGTC 401 GCCGTCCCCC TCTCCGGGGG GACGGGCCCG AAAGGCAGCG GCGGCACCGC GTCCGATCCT CGAGCGTATG GGGCTTTGTC 481 ACATGCTCTG TAGGATTGGC CGGCGCCTGC CGACGTTTTC CAACCATTTT TTCCAGGTTG?

860.1


1 ATCATTACCG AGTGCGGGTC CTTTGGGCCC AACCTCCCAT CCGTGTCTAT TATACCCTGT TGCTTCGGCG GGCCCGCCGC 81 TTGTCGGCCG CCGGGGGGGC GCCTTTGCCC CCCGGGCCCG TGCCCGCCGG AGACCCCAAC ACGAACACTG TCTGAAAGCG 161 TGCAGTCTGA GTTGATTGAA TGCAATCAGT TAAAACTTTC AACAATGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA 241 GCGAAATGCG ATAACTAATG TGAATTGCAG AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT 321 CCGGGGGGCA TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGT CGCCGTCCCC CTCTCCGGGG 401 GGACGGGCCC GAAAGGCAGC GGCGGCACCG CGTCCGATCC TCGAGCGTAT GGGGCTTTGT CACATGCTCT GTAGGATTGG 481 CCGGCGCCTG CCGACGTTTT CCAACCATTT TTTCCAGGTT GACCTC?

989.3


1 ATCATTACCG AGTGCGGGTC CTTTGGGCCC AACCTCCCAT CCGTGTCTAT TATACCCTGT TGCTTCGGCG GGCCCGCCGC 81 TTGTCGGCCG CCGGGGGGGC GCCTTTGCCC CCCGGGCCCG TGCCCGCCGG AGACCCCAAC ACGAACACTG TCTGAAAGCG 161 TGCAGTCTGA GTTGATTGAA TGCAATCAGT TAAAACTTTC AACAATGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA 241 GCGAAATGCG ATAACTAATG TGAATTGCAG AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT 321 CCGGGGGGCA TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGT CGCCGTCCCC CTCTCCGGGG 401 GGACGGGCCC GAAAGGCAGC GGCGGCACCG CGTCCGATCC TCGAGCGTAT GGGGCTTTGT CACATGCTCT GTAGGATTGG 481 CCGGCGCCTG CCGACGTTTT CCAACCATTT TTTCCAGGTT GACCTCGGAT CAGGTAGGGA TA

1059.2


Appendix 1


1700.1

Species Accession No. ScoreAspergillus niger AF455522 1025A. niger AF138904 1025A. niger AJ280006 1025A. niger U65306 1025A. tubingensis AJ280008 1001

1 ATCATTACCG AGTGCGGGTC CTTTGGGCCC AACCTCCCAT CCGTGTCTAT TGTACCCTGT TGCTTCGGCG GGCCCGCCGC 81 TTGTCGGCCG CCGGGGGGGC GCCTCTGCCC CCCGGGCCCG TGCCCGCCGG AGACCCCAAC ACGAACACTG TCTGAAAGCG 161 TGCAGTCTGA GTTGATTGAA TGCAATCAGT TAAAACTTTC AACAATGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA 241 GCGAAATGCG ATAACTAATG TGAATTGCAG AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT 321 CCGGGGGGCA TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGT CGCCGTCCCC CTCTCCGGGG 401 GGACGGGCCC GAAAGGCAGC GGCGGCACCG CGTCCGATCC TCGAGCGTAT GGGGCTTTGT CACATGCTCT GTAGGATTGG 481 CCGGCGCCTG CCGACGTTTT CCAACCATTC TTTTCAGGTT GACCTCGGAT CAGGTAGGGA TA

1699.1

Species Accession No. ScorePenicillium brevicompactum AF521657 1039Penicillium sp. AF125943 1031Penicillium sp. AF177735 1023P. brevicompactum AJ270769 1017A. niger AF108474 1011

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG AGCCTGCCTT 81 TTGGCTGCCG GGGGACGTCT GTCCCCGGGT CCGCGCTCGC CGAAGACACC TTAGAACTCT GTCTGAAGAT TGTAGTCTGA 161 GATTAAATAT AAATTATTTA AAACTTTCAA CAACGGATCT CTTGGTTCCG GCATCGATGA AGAACGCAGC GAAATGCGAT 241 ACGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCT CTGGTATTCC GGAGGGCATG 321 CCTGTCCGAG CGTCATTGCT GCCCTCAAGC ACGGCTTGTG TGTTGGGCTC CGTCCTCCTT CCGGGGGACG GGCCCGAAAG 401 GCAGCGGCGG CACCGCGTCC GGTCCTCAAG CGTATGGGGC TTTGTCACCC GCTTTGTAGG ACTGGCCGGC GCCTGCCGAT 481 CAACCAAACT TTTTTCCAGG TTGACCTCGG ATCAGGTAGG GATA

Aspergillus fumigatus

840.2

Species Accession No. ScoreAspergillus fumigatus AB055971 1070A. fumigatus AF455542 1057A. fumigatus AF455534 1057A. fumigatus AF455475 1057A. fumigatus AF455474 1057

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTCTAT CGTACCTTGT TGCTTCGGCG GGCCCGCCGT 81 TTCGACGGCC GCCGGGGAGG CCTTGCGCCC CCGGGCCCGC GCCCGCCGAA GACCCCAACA TGAACGCTGT TCTGAAAGTA 161 TGCAGTCTGA GTTGATTATC GTAATCAGTT AAAACTTTCA ACAACGGATC TCTTGGTTCC GGCATCGATG AAGAACGCAG 241 CGAAATGCGA TAAGTAATGT GAATTGCAGA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC 321 CGGGGGGCAT GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CACGGCTTGT GTGTTGGGCC CCCGTCCCCC TCTCCCGGGG 401 GACGGGCCCG AAAGGCAGCG

Appendix 1

GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCTGCTCTG TAGGCCCGGC 481 CGGCGCCAGC CGACACCCAA CTTTATTTTT CTAAGGTTGA CCTCGGATCA GGTAGGGATA

Appendix 1

842.2

Species Accession No. ScoreAspergillus fumigatus AF348420 908A. fumigatus AB055971 908A. fumigatus AF455542 906A. fumigatus AF455534 906A. fumigatus AF455475 906

1 ?TGTCCTATT CGTACCTTGT TGCTTCGGCG GGCCCGCCGT TTCGACGGCC GCGGGGAGGC CTTGCGCCCC CGGGCCCGCG 81 CCCGCCGAAG ACCCCAACAT GAACGCTGTT CTGAAAGTAT GCAGTCTGAG TTGATTATCG TAATCAGTTA AAACTTTCAA 161 CAACGGATCT CTTGGTTCCG GCATCGATGA AGAACGCAGC GAAATGCGAT AAGTAATGTG AATTGCAGAA TTCAGTGAAT 241 CATCGAGTCT TTGAACGCAC ATTGCGCCCC CTGGTATTCC GGGGGGCATG CCTGTCCGAG CGTCATTGCT GCCCTCAAGC 321 ACGGCTTGTG TGTTGGGCCC CCGTCCCCCT CTCCCGGGGG ACGGGCCCGA AAGGCAGCGG CGGCACCGCG TCCGGTCCTC 401 GAGCGTATGG GGCTTTGTCA CCTGCTCTGT AGGCCCGGCC GGCGCAGCCG ACACCCAACT TTATTTTCTA AGGTTGACCT 481 CGGATCAAGT AAGGGTACCC CGTTGACTTA AGCATATAAA G?

990.1

Species Accession No. ScoreAspergillus fumigatus AB055971 1070A. fumigatus AF455542 1057A. fumigatus AF455534 1057A. fumigatus AF455475 1057A. fumigatus AF455474 1057

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTCTAT CGTACCTTGT TGCTTCGGCG GGCCCGCCGT 81 TTCGACGGCC GCCGGGGAGG CCTTGCGCCC CCGGGCCCGC GCCCGCCGAA GACCCCAACA TGAACGCTGT TCTGAAAGTA 161 TGCAGTCTGA GTTGATTATC GTAATCAGTT AAAACTTTCA ACAACGGATC TCTTGGTTCC GGCATCGATG AAGAACGCAG 241 CGAAATGCGA TAAGTAATGT GAATTGCAGA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC 321 CGGGGGGCAT GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CACGGCTTGT GTGTTGGGCC CCCGTCCCCC TCTCCCGGGG 401 GACGGGCCCG AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCTGCTCTG TAGGCCCGGC 481 CGGCGCCAGC CGACACCCAA CTTTATTTTT CTAAGGTTGA CCTCGGATCA GGTAGGGATA

Aspergillus flavus group

838.4

Species Accession No. ScoreAspergillus flavus AF027863 997A. flavus D84353 997A. flavus AB008416 997A. oryzae AF459735 997A. oryzae D84355 997

1 ATCATTACCG AGTGTAGGGT TCCTAGCGAG CCCAACCTCC CACCCGTGTT TACTGTACCT TAGTTGCTTC GGCGGGCCCG 81 CCATTCATGG CCGCCGGGGG CTCTCAGCCC CGGGCCCGCG CCCGCCGGAG ACGACCGACG AACTCTGTCT GATCTAGTGA 161 AGTCTGAGTT GATTGTATCG CAATCAGTTA AAACTTTCAA CAATGGATCT CTTGGTTCCG GCATCGATGA AGAACGCAGC 241 GAAATGCGAT AACTAGTGTG AATTGCAGAA TTCCGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCC CTGGTATTCC 321 GGGGGGCATG CCTGTCCGAG CGTCATTGCT GCCCATCAAG CACGGCTTGT GTGTTGGGTC GTCGTCCCCT CTCCGGGGGG 401 GACGGGCCCC AAAGGCAGCG GCGGCACCGC GTCCGATCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC 481 CGGCGCTTGC CGAACGCAAA TCAATCTTTT TCCAGGTTGA CCTCGGATCA GGTAGGGATA

Appendix 1

843.2


1 ATCATTACCG AGTGTAGGGT TCCTAGCGAG CCCAACCTCC CACCCGTGTT TACTGTACCT TAGTTGCTTC GGCGGGCCCG 81 CCATTCATGG CCGCCGGGGG CTCTCAGCCC CGGGCCCGCG CCCGCCGGAG ACACCACGAA CTCTGTCTGA TCTAGTGAAG 161 TCTGAGTTGA TTGTATCGCA ATCAGTTAAA ACTTTCAACA ATGGGATCTC TTGGTTTCCG GCATCGATGA AAGAACGCAA 241 CCGAAAATGG CGATAAACTA GTGTGAATTG CAGAATTCCG TGAATCATCG AGTCTTTGAA CGCACATTGC GCCCCCTGGT 321 ATTCCGGGGG GCATGCCTGT CCGAGCGTCA TTGCTGCCCA TCAAGCACGG CTTGTGTGTT GGGTCGTCGT CCCCTCTCCG 401 GGGGGGACGG GCCCCAAAGG CAGCGGCGGC ACCGCGTCCG ATCCTCGAGC GTATGGGGCT TTGTCACCCG CTCTGTAGCC 481 CGGCCGGCGC TTGCCCGAAC GCAAATCAAT CTTTTTTCAA GGTTGACCTT GGATTAAAGT AAGGGATA

844.1


1 ATCATTACCG AGTGTAGGGT TCCTAGCGAG CCCAACCTCC CACCCGTGTT TACTGTACCT TAGTTGCTTC GGCGGGCCCG 81 CCATTCATGG CCGCCGGGGG CTCTCAGCCC CGGGCCCGCG CCCGCCGGAG ACACCACGAA CTCTGTCTGA TCTAGTGAAG 161 TCTGAGTTGA TTGTATCGCA ATCAGTTAAA ACTTTCAACA ATGGATCTCT TGGTTCCGGC ATCGATGAAG AACGCAGCGA 241 AATGCGATAA CTAGTGTGAA TTGCAGAATT CCGTGAATCA TCGAGTCTTT GAACGCACAT TGCGCCCCCT GGTATTCCGG 321 GGGGCATGCC TGTCCGAGCG TCATTGCTGC CCATCAAGCA CGGCTTGTGT GTTGGGTCGT CGTCCCCTCT CCGGGGGGGA 401 CGGGCCCCAA AGGCAGCGGC GGCACCGCGT CCGATCCTCG AGCGTATGGG GCTTTGTCAC CCGCTCTGTA GGCCCGGCCG 481 GCGCTTGCCG AACGCAAATC AATCTTTTTC CAGGTTGACC TCGGATCAGG TAGGGATA

844.2

Species Accession No. ScoreAspergillus flavus AF027863 910A. flavus AF078894 910A. flavus AB078893 910A. oryzae AF459735 910A. oryzae D84355 910

1 ?AAGTGTAGG GTTCCTAGCG AGCCAACCTC CCACCCGTGT TTACTGTACC TTAGTTGCTT CGGCGGGCCC GCCATTCATG 81 GCCGCCGGGG GCTCTCAGCC CCGGGCCCGC GCCCGCCGGA GACACCACGA ACTCTGTCTG ATCTAGTGAA GTCTGAGTTG 161 ATTGTATCGC AATCAGTTAA AACTTTCAAC AATGGATCTC TTGGTTCCGG CATCGATGAA GAACGCAGCG AAATGCGATA 241 ACTAGTGTGA ATTGCAGAAT TCCGTGAATC ATCGAGTCTT TGAACGCACA TTGCGCCCCC TGGTATTCCG GGGGGCATGC 321 CTGTCCGAGC GTCATTGCTG CCCATCAAGC ACGGCTTGTG TGTTGGGTCG TCGTCCCCTC TCCGGGGGGG ACGGGCCCCA 401 AAGGCAGCGG CGGCACCGCG TCCGATCCTC GAGCGTATGG GGCTTTGTCA CCCGCTCTGT AGGCCCGGCC GGCGCTTGCC 481 GAACGCAAA?

859.4

Species Accession No. ScoreAspergillus flavus AF027863 690A. flavus AF078894 690A. flavus D84353 690A. oryzae AF459735 690A. oryzae D84355 690

Appendix 1

1 ?ACCTGGAAA AANATGANTT GCGTTCGGCA AGCGCCGGCC GGGCCTACAG AGCGGGTGAC AAAGCCCCAT ACGCTCGAGG 81 ATCGGACGCG GTGCCGCCGC TGCCTTTGGG GCCCGTCCCC CCCGGAGAGG GGACGACGAC CCAACACACA AGCCGTGCTT 161 GATGGGCAGC AATGACGCTC GGACAGGCAT GCCCCCCGGA ATACCAGGGG GCGCAATGTG CGTTCAAAGA CTCGATGATT 241 CACGGAATTC TGCAATTCAC ACTAGTTATC GCATTTCGCT GCGNTCTTCA ACGATGCCGG AACCAAGAGA ACCATTGGTG 321 AAAGNTTTAA CTGATTGCGA TCAATCAACT TAAACTTTAC TAGATCAGAC AGAGTTCGTG GNGTCTNCGG CNGGCGCGGG 401 CCCCGGGCTG ANANCCCCCG GCGGGCATGA ATGGCGGGCC CGCNAACAACT?

844.3



858.3



989.1



Appendix 1

842.3


1 ATCATTACCG AGTGTAGGGT TCCTAGCGAG CCCAACCTCC CACCCGTGTT TACTGTACCT TAGTTGCTTC GGCGGGCCCG 81 CCATTCATGG CCGCCGGGGG CTCTCAGCCC CGGGCCCGCG CCCGCCGGAG ACACCACGAA CTCTGTCTGA TCTAGTGAAG 161 TCTGAGTTGA TTGTATCGCA ATCAGTTAAA ACTTTCAACA ATGGATCTCT TGGTTCCGGC ATCGATGAAG AACGCAGCGA 241 AATGCGATAA CTAGTGTGAA TTGCAGAATT CCGTGAATCA TCGAGTCTTT GAACGCACAT TGCGCCCCCT GGTATTCCGG 321 GGGGCATGCC TGTCCGAGCG TCATTGCTGC CCATCAAGCA CGGCTTGTGT GTTGGGTCGT CGTCCCCTCT CCGGGGGGGA 401 CGGGCCCCAA AGGCAGCGAC GGCACCGCGT CCGATCCTCG AGCGTATGGG GCTTTGTCAC CCGCTCTGTA GGCCCGGCCG 481 GCGCTTGCCG AACGCAAATC AATCTTTTTC CAGGTTGACC TCGGATCAGG TAGGGATA

1702.3

Species Accession No. ScoreAspergillus oryzae AF459735 1063A. oryzae AB008417 1045A. flavus AB008416 1045A. flavus AB008414 1029A. flavus AB008414 1021

1 ATCATTACCG AGTGTAGGGT TCCTAGCGAG CCCAACCTCC CACCCGTGTT TACTGTACCT TAGTTGCTTC GGCGGGCCCG 81 CCATTCATGG CCGCCGGGGG CTCTCAGCCC CGGGCCCGCG CCCGCCGGAG ACACCACGAA CTCTGTCTGA TCTAGTGAAG 161 TCTGAGTTGA TTGTATCGCA ATCAGTTAAA ACTTTCAACA ATGGATCTCT TGGTTCCGGC ATCGATGAAG AACGCAGCGA 241 AATGCGATAA CTAGTGTGAA TTGCAGAATT CCGTGAATCA TCGAGTCTTT GAACGCACAT TGCGCCCCCT GGTATTCCGG 321 GGGGCATGCC TGTCCGAGCG TCATTGCTGC CCATCAAGCA CGGCTTGTGT GTTGGGTCGT CGTCCCCTCT CCGGGGGGGA 401 CGGGCCCCAA AGGCAGCGGC GGCACCGCGT CCGATCCTCG AGCGTATGGG GCTTTGTCAC CCGCTCTGTA GGCCCGGCCG 481 GCGCTTGCCG AACGCAAATC AATCTTTTTC CAGGTTGACC TCGGATCAGG ?

Aspergillus parasiticus

835.5

Species Accession No. ScoreAspergillus parasiticus AF027862 1025A. parasiticus D84356 1025A. sojae D84357 1025A. sojae AB008419 1025A. toxicarius AB008421 1025

1 ATCATTACCG AGTGTAGGGT TCCTAGCGAG CCCAACCTCC CACCCGTGTT TACTGTACCT TAGTTGCTTC GGCGGGCCCG 81 CCGTCATGGC CGCCGGGGGC GTCAGCCCCG GGCCCGCGCC CGCCGGAGAC ACCACGAACT CTGTCTGATC TAGTGAAGTC 161 TGAGTTGATT GTATCGCAAT CAGTTAAAAC TTTCAACAAT GGATCTCTTG GTTCCGGCAT CGATGAAGAA CGCAGCGAAA 241 TGCGATAACT AGTGTGAATT GCAGAATTCC GTGAATCATC GAGTCTTTGA ACGCACATTG CGCCCCCTGG TATTCCGGGG 321 GGCATGCCTG TCCGAGCGTC ATTGCTGCCC ATCAAGCACG GCTTGTGTGT TGGGTCGTCG TCCCCTCTCC GGGGGGGACG 401 GGCCCCAAAG GCAGCGGCGG CACCGCGTCC GATCCTCGAG CGTATGGGGC TTTGTCACCC GCTCTGTAGG CCCGGCCGGC 481 GCTTGCCGAA CGCAAAACAA CCATTTTTTC CAGGTTGACC TCGGATCAGG TAGGGATA

Appendix 1

Sequences of Penicillium samples

Penicillium commune

835.4 Species Accession No. ScorePenicillium commune AF455527 1049P. commune AF455418 1049P. crustosum AF033472 1049P. italicum AJ250548 1049P. commune AF455471 1041

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCCCCG ATCTCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC CGGCGCTTGC 481 CGATCAACCC AAATTTTTAT CCAGGTTGAC CTCGGATCAG GTAGGGATA

857.4

Species Accession No. ScorePenicillium commune AF455527 798P. commune AF455418 798P. crustosum AF033472 798P. italicum AJ250548 798P. commune AF455471 790

1 ATCATTACCG ATGAGGGCCC TCTGGGTCCA ACCTCCCACC CGTGTTTATT TTACCTTGTT GCTTCGGCGG GCCCGCCTTA 81 ACTGGCCGCC GGGGGGCTTA CGCCCCCGGG CCCGCGCCCG CCGAAAAGAC ACCCTCGAAC TCTGTCTGAA GATTGAAGTC 161 TGAGTGAAAA TATTAAATTA TTTAAAACTT TTCAACAACG GTCTTCTTGG TTCCGGCATC GATGAAAGAA CGCAGCGAAA 241 TGCGATACGT AATGTGAATT GCAAATTCAG TGAATCATCG AGTCTTTGAA CGCACATTGC GCCCCCTGGT ATTCCGGGGG 321 GCATGCCTGT CCAACGTNTC ATTGCTGCCC TTCAAGCCCG GCTTGTGTGT TGGGCCCCGT CCCCCGATCT TCCCGGGGAC 401 GGGCCCCGAA AGGCAGCGGC GGCACCGCGT CCGGTCYTCG AGCGTATGGG GCTTTGTCAC CCGCTCTGTA GGCCCGGCCG 481 GCGCTTGCCG ATCAACCCAA ATTTT?

858.6


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TGTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCCCCG ATCTCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC CGGCGCTTGC 481 CGATCAACCC AAATTTTTAT CCAGGTTGAC CTCGGATCAG TAGGGATA

Appendix 1

858.8



861.1

Species Accession No. ScorePenicillium commune AF236103 1049P. commune AJ004813 1049P. camemberti AJ004814 1049P. commune AF455471 1041P. camemberti AF033474 1041

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTC ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCTCCG ATCTCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC CGGCGCTTGC 481 CGATCAACCC AAATTTTTAT CCAGGTTGAC CTCGGATCAG GTAGGGATA

989.2


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCCCCG ATCTCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC CGGCGCTTGC 481 CGATCAACCC AAATTTTTAT CCAGGTTGAC CTCGG?

989.6


Appendix 1

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACCGTAAT GTGAATTGCA AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA 321 TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGC CCCGTCCCCC GATCTCCGGG GGACGGGCCC 401 GAAAGGCAGC GGCGGCACCG CGTCCGGTCC TCGAGCGTAT GGGGCTTTGT CACCCGCTCT GTAGGCCCGG CCGGCGCTTG 481 CCGATCAACC CAAATTTTTA TCCAGGTTGA CCTCGGATCA GGTAGGGATA

992.2

Species Accession No. ScorePenicillium commune AF348419 1035P. echinulatum AF033473 1035P. commune AF455477 1027P. commune AF236103 1027P. camemberti AJ004814 1027

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTC ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA AGCGAAATGC 241 GATACGTAAT GTGAATTGCA AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA 321 TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGC CCCGTCCTCC GATTTCCGGG GGACGGGCCC 401 GAAAGGCAGC GGCGGCACCG CGTCCGGTCC TCGAGCGTAT GGGGCTTTGT CACCCGCTCT GTAGGCCCGG CCGGCGCTTG 481 CCGATCAACC CAAATTTTTA TCCAGGTTGA CCTCGGATCA GGTAGGGATA

992.3

Species Accession No. ScorePenicillium commune AF455527 1059P. commune AF455418 1059P. commune AF455471 1051P. crustosum AF033472 1045P. italicum AJ250548 1045

1 ATCATTACCT GATCCGAGGT CAACCTGGAT AAAAATTTGG GTTGATCGGC AAGCGCCGGC CGGGCCTACA GAGCGGGTGA 81 CAAAGCCCCA TACGCTCGAG GACCGGACGC GGTGCCGCCG CTGCCTTTCG GGCCCGTCCC CCGGAGATCG GGGGACGGGG 161 CCCAACACAC AAGCCGGGCT TGAGGGCAGC AATGACGCTC GGACAGGCAT GCCCCCCGGA ATACCAGGGG GCGCAATGTG 241 CGTTCAAAGA CTCGATGATT CACTGAATTT GCAATTCACA TTACGTATCG CATTTCGCTG CGTTCTTCAT CGATGCCGGA 321 ACCAAGAGAT CCGTTGTTGA AAGTTTTAAA TAATTTATAT TTTCACTCAG ACTTCAATCT TCAGACAGAG TTCGAGGGTG 401 TCTTCGGCGG GCGCGGGCCC GGGGGCGTAA GCCCCCCGGC GGCCAGTTAA GGCGGGCCCG CCGAAGCAAC AAGGTAAAAT 481 AAACACGGGT GGGAGGTTGG ACCCAGAGGG CCCTCACTCG GTAATGATCC TTCCGCAGG?

992.5


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTTGGTT CCGGCATCGA TGAAGAACGC AGCGAAATGC 241 GATACGTAAT GTGAATTGCA AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA 321 TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGC CCCGTCCCCC GATCTCCGGG GGACGGGCCC 401 GAAAGGCAGC GGCGGCACCG CGTCCGGTCC TCGAGCGTAT GGGGCTTTGT CACCCGCTCT GTAGGCCCGG CCGGCGCTTG 481 CCGATCAACC CAAATTTTTA TCCAGGTTGA CCTCGGATCA GGTAGGGATA

Appendix 1

1059.1



1059.3


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCCCCG ATCTCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC CGGCGCTTGC 481 CGATCAACCC AAATTTTTAT CCAGGTGACC TCGGACAGGT AGGGATA

1062.1


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTC ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATTCGA TGAAGAACGC AGCGAAATGC 241 GATACGTAAT GTGAATTGCA AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA 321 TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCCCGGCTTG TGTGTTGGGC CCCGTCCTCC GATTTCCGGG GGACGGGCCC 401 GAAAGGCAGC GGCGGCACCG CGTCCGGTCC TCGAGCGTAT GGGGCTTTGT CACCCGCTCT GTAGGCCCGG CCGGCGCTTG 481 CCGATCAACC CAAATTTTTA TCCAGGTTGA CCTCGGA?

1699.2


Appendix 1


1700.2


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 AACTGGCCGC CGGGGGGCTT ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCTCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCCCCG ATCTCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCTC GAGCGTCATG GGGCTTTGTC ACCC?

1702.1


1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC CAACCTTCCC ACCCGTGTTT ATTTTACCTT GTTGCTTCGG CGGGCCCGCC 81 TTAACTGGCC GCCGGGGGGC TCACGCCCCC GGGCCCGCGC CCGCCGAAGA CACCCTCGAA CTCTGTCTGA AGATTGAAGT 161 CTGAGTGAAA ATATAAATTA TTTAAAACTT TCAACAACGG ATCTCTTGGT TCCGGCATCG ATGAAAGAAC GCAGCCGAAA 241 TGCGATACCG TAATGTGAAT TGCAAATTCA GTGAATCATC GAGTCTTTGA ACGCACATTG CGCCCCCTGG TATTCCGGGG 321 GGCATGCCTG TCCGAGCGTC ATTGCTTGCC CTCAAGCCCG GCTTGTGTGT TGGGCCCCGT CCTCCGATTT CCGGGGGACG 401 GGCCCGAAAG GCAGCGGCGG CACCGCGTCC GGTCCTCGAG CGTATGGGGC TTTGTCACCC GCTCTGTAGG CCCGGCCGGC 481 GCTTGCCGAT CAACCCAAAT TTTTATCCAG GTTGACCTCG GATCA?

1703.1


1 ?GCCCTCTCT GGGTCCAACC TCCCACCCGT GTTTATTTTA CCTTGTTGCT TCGGCGGGCC CGCCTTAACT GGCCGCCGGG 81 GGGCTTACGC CCCCGGGCCC GCGCCCGCCG AAGACACCCT CGAACTCTGT CTGAAGATTG AAGTCTGAGT GAAAATATAA 161 ATTATTTAAA ACTTTCAACA ACGGATCTCT TGGTTCCGGC AATTCGATGA AAGAACGCAC CGAAATGCGA ATTACGTAAT 241 GTGAATTGCA AATTCCAGTG AATCATCGAG TCTTTGAACG CACATTGCGC CCCCTGGTAT TCCGGGGGGC ATGCCTGTCC 321 GAGCGTCATT GCTGCCCTCA AGCCCGGCTT GTGTGTTGGG CCCCGTCCCC CGATCTCCGG GGGACGGGCC CGAAAGGCAG 401 CGGCGGCACC GCGTCCGGTC CTCGAGCGTA TGGGGCTTTG TCACCCGCTC TGTAGGCCCG GCCGGCGCTT GCCGATCAAC 481 CCAAATTTTT ATCCAGGTTG ACCTCGGATC AGGTA?

Appendix 1

Penicillium brevicompactum

838.5

Species Accession No. ScorePenicillium brevicompactum AF521657 1039Penicillium sp. AF125943 1031Penicillium sp. AF177735 1023P. brevicompactum AJ270769 1017Aspergillus niger AF108474 1011

1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG AGCCTGCCTT 81 TTGGCTGCCG GGGGACGTCT GTCCCCGGGT CCGCGCTCGC CGAAGACACC TTAGAACTCT GTCTGAAGAT TGTAGTCTGA 161 GATTAAATAT AAATTATTTA AAACTTTCAA CAACGGATCT CTTGGTTCCG GCATCGATGA AGAACGCAGC GAAATGCGAT 241 ACGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCT CTGGTATTCC GGAGGGCATG 321 CCTGTCCGAG CGTCATTGCT GCCCTCAAGC ACGGCTTGTG TGTTGGGCTC CGTCCTCCTT CCGGGGGACG GGCCCGAAAG 401 GCAGCGGCGG CACCGCGTCC GGTCCTCAAG CGTATGGGGC TTTGTCACCC GCTTTGTAGG ACTGGCCGGC GCCTGCCGAT 481 CAACCAAACT TTTTTCCAGG TTGACCTCGG ATCAGGTAGG GATA

858.7

Species Accession No. ScorePenicillium brevicompactum AF521657 987P. brevicompactum AJ270769 981Penicillium sp. AF125943 979Penicillium sp. AF177735 971Aspergillus niger AF108474 971

1 ATCATTACCG AAGTAAGGGC CCTCTGGGTC CAACCTCCCA CCCGTGTTTA TTTTACCTTG TTGCTTCGGC GAGCCTGCCT 81 TTTGGCTGCC GGGGGACGTC TGTCCCCGGG TCCGCGCTCG CCGAAGACAC CTTAGAACTC TGTCTGAAGA TTGTAGTCTG 161 AGATTAAATA TAAATTATTT AAAACTTTCA ACAACGGATC TCTTGGTTCC GGCATCGATG AAGAACGCAA GCGAAATGCG 241 ATACGTAATG TGAATTGCAG AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CTCTGGTATT CCGGAGGGCA 321 TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCACGGCTTG TGTGTTGGGC TCCGTCCTCC TTCCGGGGGA CGGGCCCGAA 401 AGGCAGCGGC GGCACCGCGT CCGGTCCTCA AGCGTATGGG GCTTTGTCAC CCGCTTTGTA GGACTGGCCG GCGCCTGCCG 481 ATCAACCAAA CTTTTTTCCA GGTTGACCTC GGATCAGG?

859.7

Species Accession No. ScorePenicillium brevicompactum AF521657 894P. brevicompactum AJ270769 894Aspergillus niger AF108474 894Penicillium sp. AF125943 886Penicillium sp. AF177735 878

1 ?GTCAACCTG GAAAAAGTTT GGTTGATCGG CAGGCGCCGG CCAGTCCTAC AAAGCGGGTG ACAAAGCCCC ATACGCTTGA 81 GGACCGGACG CGGTGCCGCC GCTGCCTTTC GGGCCCGTCC CCCGGAAGGA GGACGGAGCC CAACACACAA GCCGTGCTTG 161 AGGGCAGCAA TGACGCTCGG ACAGGCATGC CCTCCGGAAT ACCAGAGGGC GCAATGTGCG TTCAAAGACT CGATGATTCA 241 CTTGAATTCT GCAATTCACA TTACGTATCG CATTTCGCTG CGTTCTTCAT CGATGCCGGA ACCAAGAGAT CCGTTGTTGA 321 AAGTTTTAAA TAATTTATAT TTAATCTCAG ACTACAATCT TCAGACAGAG TTCTAAGGTG TCTTCGGCGA GCGCGGACCC 401 GGGGACAGAC GTCCCCCGGC AGCCAAAAGG CAGGCTCGCC GAAGCAACAA GGTAAATAAA CACGGTGGGA GGTTGGACCC 481 AAGGGCTCAC TCGGATGA?

Appendix 1

Penicillium geastrivorus

838.7

Species Accession No. ScorePenicillium geastrivorus AF125941 957P. paraherquei AF178511 938P. brasilianum AF178523 932P. simplicissimum AF203084 866P. simplicissimum AF033440 866 1 ATCATTACTT GAGTGAGGGC CCTCTGGGTC CAACCTCCCA CCCGTGTTTA TTGTACCTTG TTGCTTCGGC GAGCCCGCCT 81 CACGGCCGCC GGGGGGCACT TGCCCCCGGG CCCGCGCCCG CCGAAGACAC CATTGAACTC TGTCTGAAGA TTGCAGTCTG 161 AGTAGATTAG CTAAATCAGT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA AGCGAAATGC 241 GATACGTAAT GTGAATTGCA GAATTCAGTG AATCATCGAG TCTTTGAACG CACATTGCGC CCCCTGGTAT TCCGGGGGGC 321 ATGCCTGTCC GAGCGTCATT GCTGCCCTCA AGCACGGCTT GTGTGTTGGG CTTCGCCCCC CGTTCTTCGG GGGGCGGGCC 401 CGAAAGGCAG CGGCGGCACC GCGTCCGGTC CTCGAGCGTA TGGGGCTTTG TCACCCGCTC TGTAGGCCCG GCCGGCGCCC 481 GCCGGCGACA CCCAAATCAA TCTATCCAGG TTGACCTCGG ATCAGG?

Penicillium glabrum

841.5

Species Accession No. ScorePenicillium glabrum AF455487 1033P. glabrum AF033407 1033P. thomii AF034448 1025Eupenicillium lapidosum AF033409 1025P. purpurescens AF033408 1025 1 ATCATTACTG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TGTACCTTGT TGCTTCGGTG CGCCCGCCTC 81 ACGGCCGCCG GGGGGCTTCT GCCCCCGGGT CCGCGCGCAC CGGAGACACT ATTGAACTCT GTCTGAAGAT TGCAGTCTGA 161 GCATAAACTA AATAAGTTAA AACTTTCAAC AACGGATCTC TTGGTTCCGG CATCGATGAA GAACGCAGCG AAATGCGATA 241 ACTAATGTGA ATTGCAGAAT TCAGTGAATC ATCGAGTCTT TGAACGCACA TTGCGCCCCC TGGTATTCCG GGGGGCATGC 321 CTGTCCGAGC GTCATTGCTG CCCTCAAGCA CGGCTTGTGT GTTGGGCTCC GTCCCCCCGG GGACGGGTCC GAAAGGCAGC 401 GGCGGCACCG AGTCCGGTCC TCGAGCGTAT GGGGCTTTGT CACCCGCTCT GTAGGCCCGG CCGGCGCCAG CCGACAACCA 481 ATCATCCTTT TTTCAGGTTG ACCTCGGATC AGGTAGGGAT A

857.5

Species Accession No. ScorePenicillium glabrum AF455487 991P. glabrum AF033407 991P. thomii AF034448 983Eupenicillium lapidosum AF033409 983P. purpurescens AF033408 983 1 ATCATTACTG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTAT TGTACCTTGT TGCTTCGGTG CGCCCGCCTC 81 ACGGCCGCCG GGGGGCTTCT GCCCCCGGGT CCGCGCGCAC CGGAGACACT ATTGAACTCT GTCTGAAGAT TGCAGTCTGA 116 GCATAAACTA AATAAGTTAA AACTTTCAAC AACGGGATCT CTTGGTTCCG GCATCGATGA AAGAACGCAG CGAAATGCGA 241 TAACTAATGT GAATTGCAGA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TCGCCCTCAA GCACGGCTTG TGTGTTGGGC TCCGTCCCCC CGGGGACGGG TCCGAAAGGC 401 AGCGGCGGCA CCGAGTCCGG TCCTCGAGCG TATGGGGCTT TGTCACCCGC TCTGTAGGCC CGGCCGGCGC CAGCCGACAA 481 CCAATCATCC TTTTTTCAGG TTGACCTCGG ATCAGGTAGG GATA

Appendix 1

Penicillium citrinum

989.4

Species Accession No. ScorePenicillium citrinum AF033422 942P. westlingii AF033423 942P. sartoryi AF033421 926Eupenicillium anatolicum AF033425 567P. roseopurpureum AF033415 565 1 ATCATTACCG AGTGCGGGCC CCTCGGGGCC CAACCTCCCA CCCGTGTTGC CCGAACCTAT GTTGCCTCGG CGGGCCCCGC 81 GCCCGCCGAC GGCCCCCCTG AACGCTGTCT GAAGTTGCAG TCTGAGACCT ATAACGAAAT TAGTTAAAAC TTTCAACAAC 161 GGATCTCTTG GTTCCGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAACT AATGTGAATT GCAGAATTCA GTGAATCATC 241 GAGTCTTTGA ACGCACATTG CGCCCTCTGG TATTCCGGAG GGCATGCCTG TCCGAGCGTC ATTGCTGCCC TCAAGCCCGG 321 CTTGTGTGTT GGGCCCCGTC CCCCCCGCCG GGGGGACGGG CCCGAAAGGC AGCGGCGGCA CCGCGTCCGG TCCTCGAGCG 401 TATGGGGCTT CGTCACCCGC TCTAGTAGGC CCGGCCGGCG CCAGCCGACC CCCAACCTTT AATTATCTCA GGTTGACCTC 481 GGATCAGGTA GGGATA

995.2

Species Accession No. ScorePenicillium citrinum AF033422 942P. westlingii AF033423 942P. sartoryi AF033421 926Eupenicillium anatolicum AF033425 567P. roseopurpureum AF033415 565

1 ATCATTACCG AGTGCGGGCC CCTCGGGGCC CAACCTCCCA CCCGTGTTGC CCGAACCTAT GTTGCCTCGG CGGGCCCCGC 81 GCCCGCCGAC GGCCCCCCTG AACGCTGTCT GAAGTTGCAG TCTGAGACCT ATAACGAAAT TAGTTAAAAC TTTCAACAAC 161 GGATCTCTTG GTTCCGGCAT CGATGAAGAA CGCAGCGAAA TGCGATAACT AATGTGAATT GCAGAATTCA GTGAATCATC 241 GAGTCTTTGA ACGCACATTG CGCCCTCTGG TATTCCGGAG GGCATGCCTG TCCGAGCGTC ATTGCTGCCC TCAAGCCCGG 321 CTTGTGTGTT GGGCCCCGTC CCCCCCGCCG GGGGGACGGG CCCGAAAGGC AGCGGCGGCA CCGCGTCCGG TCCTCGAGCG 401 TATGGGGCTT CGTCACCCGC TCTAGTAGGC CCGGCCGGCG CCAGCCGACC CCCAACCTTT AATTATCTCA GGTTGACCTC 481 GGATCAGGTA GGGATA

Penicillium nalgiovense

996.3

Species Accession No. ScorePenicillium nalgiovense AJ004895 914P. nalgiovense AJ004894 906P. dipodomyis AJ004896 906P. chrysogenum AF034857 898P. viridicatum AF033477 898 1 ?TCCAACCTC CCACCGTGTT TATTTTACCT TGTTGCTTCG GCGGGCCCGC CTTAACTGGC CGCCGGGGGG CTCACGCCCC 81 CGGGCCCGCG CCCGCCGAAG ACACCCTCGA ACTCTGTCTG AAGATTGTAG TCTGAGTGAA AATATAAATT ATTTAAAACT 161 TTCAACAACG GATCTCTTGG TTCCGGCATC GATGAAAGAA CGCAGCGAAA TGCGATACGT AATGGTGAAT TGCAAAATTC 241 AAGTGAATCA TCGAGTCTTT GAACGCACAT TGCGCCCCCT GGTATTCCGG GGGGCATGCC TGTCCGAGCG TCATTGCTGC 321 CCTCAAGCCC GGCTTGTGTG TTGGGCCCCG TCCTCCGATC CCGGGGGACG GGCCCGAAAG GCAGCGGCGG CACCGCGTCC 401 GGTCCTCGAG CGTATGGGGC TTTGTCACCC GCTCTGTAGG CCCGGCCGGC GCTTGCCGAT CAACCCAAAT TTTTATCCAG 481 GTTGACCTCG GATCAGGTAG G?

Appendix 1

1705.1

Species Accession No. ScorePenicillium chrysogenum AF034857 898P. chrysogenum AF034451 898P. chrysogenum AF033465 898P. commune AF455544 890P. chrysogenum AF455544 890

1 ?AGTGAGGGC CCTCTGGGTC CAACCTCCCA CCCGTGTTTA TTTTACCTTG TTGCTTCGGC GGGCCCGCCT TAACTGGCCG 81 CCGGGGGGCT TACGCCCCCG GGCCCGCGCC CGCCGAAGAC ACCCTCGAAC TCTGTCTGAA GATTGTAGTC TGAGTGAAAA 161 TATAAATTAT TTAAAACTTT CAACAACGGA TCTCTTGGTT CCGGCATCGA TGAAGAACGC AGCGAAATGC GATACGTAAA 241 TGTGAATTGC AAATTCAAGT GAAATCAGTC GAAGTCTTTG AACGCACATT GCGCCCCCTG GTATTCCGGG GGGCATGCCT 321 GTCCGAGCGT CATTGCTGCC CTCAAGCACG GCTTGTGTGT TGGGCCCCGT CCTCCGATCC CGGGGGACGG GCCCGAAAGG 401 CAGCGGCGGC ACCGCGTCCG GTCCTCGAGC GTATGGGGCT TTGTCACCCG CTCTGTAGGC CCGGCCGGCG CTTGCCGATC 481 AAACCCAAAT TTTTATCCAG GT?

1706.1

Species Accession No. ScorePenicillium chrysogenum AF034857 1033P. commune AF455544 1025P. nalgiovense Aj004894 1011P. dipodomyis AJ004896 1011P. expansum AF033479 1009 1 ATCATTACCG AGTGAGGGCC CTCTGGGTCC AACCTCCCAC CCGTGTTTGA TTTTACCTTG TTGCTTCGGC GGGCCCGCCT 81 TAACTGGCCG CCGGGGGGCT TACGCCCCCG GGCCCGCGCC CGCCGAAGAC ACCCTCGAAC TCTGTCTGAA GATTGTAGTC 161 TGAGTGAAAA TATAAATTAT TTAAAACTTT CAACAACGGA TCTCTTGGTT CCGGCATCGA TGAAGAACGC AGCGAAATGC 241 GATACGTAAT GTGAATTGCA AATTCAGTGA ATCATCGAGT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA 321 TGCCTGTCCG AGCGTCATTG CTGCCCTCAA GCACGGCTTG TGTGTTGGGC CCCGTCCTCC GATCCCGGGG GACGGGCCCG 401 AAAGGCAGCG GCGGCACCGC GTCCGGTCCT CGAGCGTATG GGGCTTTGTC ACCCGCTCTG TAGGCCCGGC CGGCGCTTGC 481 CGATCAACCC AAATTTTTAT CCAGGTTGAC CTCGGATCAG GTAGGGATA

838.3

Species Accession No. ScorePenicillium chrysogenum AF034857 1033P. commune AF455544 1025P. nalgiovense Aj004894 1011P. dipodomyis AJ004896 1011P. expansum AF033479 1009


Appendix 1

Penicillium cyclopium

1059.5

Species Accession No. ScorePenicillium cyclopium AJ005491 1023P. aurantioviride AJ005490 1023P. tricolor AJ005489 1015P. polonicum AF033475 1007P. expansum AJ270767 1007

1 ATCATTACCG AGTGAGGGCC CTTTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 TACTGGCCGC CGGGGGGCTC ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCTCCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGCGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGACGCAG CGAAATGCGA 241 TACGTAATGT GAATTGCAAA TTCAGTGAAT CATCGAGTCT TTGAACGCAC ATTGCGCCCC CTGGTATTCC GGGGGGCATG 321 CCTGTCCGAG CGTCATTGCT GCCCTCAAGC ACGGCTTGTG TGTTGGGCCC CGTCCTCCGA TTCCGGGGGA CGGGCCCGAA 401 AGGCAGCGGC GGCACCGCGT CCGGTCCTCG AGCGTATGGG GCTTTGTCAC CCGCTCTGTA GGCCCGGCCG GCGCTTGCCG 481 ATCAACCCAA ATTTTTATCC AGGTTGACCT CGGATCAGGT AGGGATA

Penicillium expansum

1062.3

Species Accession No. ScorePenicillium expansum AF218786 1013P. expansum AJ005676 1013P. italicum AJ250549 997P. expansum AF455466 991P. polonicum AF033475 959

1 ATCATTACCG AGTAGGGCCC TTTGGGTCCA CCTCCCACCC GTGTTTATTT ACCTCGTTGC TTCGGCGGGC CCGCCTTAAC 81 TGGCCGCCGG GGGGCTCACG CCCCCGGGCC CGCGCCCGCC GAAGACACCC CCGAACTCTG CCTGAAGATT GTCGTCTGAG 161 TGAAAATATA AATTATTTAA AACTTTCAAC AACGGATCTC TTGGTTCCGG CATCGATGAA GAACGCAGCG AAATGCGATA 241 CGTAATGTGA ATTGCAAATT CAGTGAATCA TCGAGTCTTT GAACGCACAT TGCGCCCCCT GGTATTCCGG GGGGCATGCC 321 TGTCCGAGCG TCATTGCTGC CCTCAAGCCC GGCTTGTGTG TTGGGCCCCG TCCTCCGATT CCGGGGGACG GGCCCGAAAG 401 GCAGCGGCGG CACCGCGTCC GGTCCTCGAG CGTATGGGGC TTTGTCACCC GCTCTGTAGG CCCGGCCGGC GCTTGCCGAT 481 CAACCCAAAT TTTTATCCAG GTTGACCTCG GATCAGGTAG GGATA

Penicillium polonicum

1699.3

Species Accession No. ScorePenicillium polonicum AF033475 1047P. polonicum AJ005493 1047P. viridicatum AF033478 1039P. viridicatum AJ005482 1039P. tricolor AJ005489 1039

1 ATCATTACCG AGTGAGGGCC CTTTGGGTCC AACCTCCCAC CCGTGTTTAT TTTACCTTGT TGCTTCGGCG GGCCCGCCTT 81 TACTGGCCGC CGGGGGGCTC ACGCCCCCGG GCCCGCGCCC GCCGAAGACA CCCCCGAACT CTGTCTGAAG ATTGAAGTCT 161 GAGTGAAAAT ATAAATTATT TAAAACTTTC AACAACGGAT CTCTTGGTTC CGGCATCGAT GAAGAACGCA GCGAAATGCG 241 ATACGTAATG TGAATTGCAA ATTCAGTGAA TCATCGAGTC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT 321 GCCTGTCCGA GCGTCATTGC TGCCCTCAAG CCCGGCTTGT GTGTTGGGCC CCGTCCTCCG ATTCCGGGGG ACGGGCCCGA 401 AAGGCAGCGG CGGCACCGCG TCCGGTCCTC GAGCGTATGG GGCTTTGTCA CCCGCTCTGT AGGCCCGGCC GGCGCTTGCC 481 GATCAACCCA AATTTTTATC CAGGTTGACC TCGGATCAGG TAGGGATA

Appendix 1

Sequences of miscellaneous fungi

Cladosporium sp.1

860.4

Species Accession No. ScoreCladosporium cladosporioides AF393691 908C. cladosporioides AJ300334 908C. tenuissimum AF393724 908C. cucumerinum AF393696 908C. oyxysporium AJ300332 908

1 ATCATTACAA GTGACCCCGG TCTAACCACC GGGATGTTCA TAACCCTTTG TTGTCCGACT CTGTTGCCTC CGGGGCGACC 81 CTGCCTTCGG GCGGGGGCTC CGGGTGGACA CTTCAAACTC TTGCGTAACT TTGCAGTCTG AGTAAACTTA ATTAATAAAT 161 TAAAACTTTT AACAACGGAT CTCTTGGTTC TGGCATCGAT GAAGAACGCA GCGAAATGCG ATAAGTAATG TGAATTGCAG 241 AATTCAGTGA ATCATCGAAT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA TGCCTGTTCG AGCGTCATTT 321 CACCACTCAA GCCTCGCTTG GTATTGGGCA ACGCGGTCCG CCGCGTGCCT CAAATCGACC GGCTGGGTCT TCTGTCCCCT 401 AAGCGTTGTG GAAACTATTC GCTAAAGGGT GTTCGGGAGG CTACGCCGTA AAACAACC?

992.4


1 ATCATTACAA GTGACCCCGG TCTAACCACC GGGATGTTCA TAACCCTTTG TTGTCCGACT CTGTTGCCTC CGGGGCGACC 81 CTGCCTTCGG GCGGGGGCTC CGGGTGGACA CTTCAAACTC TTGCGTAACT TTGCAGTCTG AGTAAACTTA ATTAATAAAT 161 TAAAACTTTT AACAACGGAT CTCTTGGTTC TGGCATCGAT GAAGAACGCA GCGAAATGCG ATAAGTAATG TGAATTGCAG 241 AATTCAGTGA ATCATCGAAT CTTTGAACGC ACATTGCGCC CCCTGGTATT CCGGGGGGCA TGCCTGTTCG AGCGTCATTT 321 CACCACTCAA GCCTCGCTTG GTATTGGGCA ACGCGGTCCG CCGCGTGCCT CAAATCGACC GGCTGGGTCT TCTGTCCCCT 401 AAGCGTTGTG GAAACTATTC GCTAAAGGGT GTTCGGGAGG CTACGCCGTA AAACAACCCC ATTTCTAAGG TTGACCTCGG 481 ATCAGGTAGG GATA

993.1


1 ?ATATGCTTA AGTTCAGCGG GTATCCCCTA CCCTGATCCG AGGTCAACCT TAGAAATGGG GTTGTTTTAC GGCGTAGCCT 81 CCCGAACACC CTTTAGCGAA TAGTTTCCAC AACGCTTAGG GGACAGAAGA CCCAGCCGGT CGATTTGAGG CACGCGGCGG 161 ACCGCGTTGC CCAATACCAA GCGAGGCTTG AGTGGTGAAA TGACGCTCGA ACAGGCATGC CCCCCGGAAT ACCAGGGGGC 241 GCAATGTGCG TTCAAAGATT CGATGATTCA CTGAATTCTG CAATTCACAT TACTTATCGC ATTTCGCTGC GTTCTTCATC 321 GATGCCAGAA CCAAGAGATC CGTTGTTAAA AGTTTTAATT TATTAATTAA GTTTACTCAG ACTGCAAAGT TACGCAAGAG 401 TTTGAAGTGT CCACCCGGAG CCCCCGCCCG AAGGCAGGGT CGCCCCGGAG GCAACAGAGT CGGACAACAA AGGGTTATGA 481 ACATCCCGGT GGTTAGACCG GGGTCAC?

Appendix 1

994.1



996.1



1062.2


1 ATCATTACAA GTGACCCCGG TCTAACCCAC CGGGATGTTC ATAACCCTTT GTTGTCCGAC TCTGTTGCCT CCGGGGCGAC 18 CCTGCCTTCG GGCGGGGGCT CCGGGTGGAC ACTTCAAACT CTTGCGTAAC TTTGCAGTCT GAGTAAACTT AATTAATAAA 161 TTAAAACTTT TAACAACGGA TCTCTTGGTT CTGGCATCGA TGAAGAACGC AGCGAAATGC GATAAGTAAT GTGAATTGCA 241 GAATTCAGTG AATCATCGAA TCTTTGAACG CACATTGCGC CCCCTGGTAT TCCGGGGGGC ATGCCTGTTC GAGCGTCATT 321 TCACCACTCA AGCCTCGCTT GGTATTGGGC AACGCGGTCC GCCGCGTGCC TCAAATCGAC CGGCTGGGTC TTCTGTCCCC 401 TAAGCGTTGT GGAAACTATT CGCTAAAGGG TGTTCGGGAG GCTACGCCGT AAAACAACCC CATTTCTAAG GTTGACCTCG 481 GATCAGGTAG GGATA

Appendix 1

Cladosporium sp.2

1064.1

Species Accession No. ScoreCladosporium herbarum AF455517 977C. herbarum AF393712 977C. herbarum AF222830 977C. magnusianum AF393712 977Mycosphaerella macrosporium AF362049 977

1 ATCATTACAA GAACGCCCGG GCTTCGGCCT GGTTATTCAT AACCCTTTGT TGTCCGACTC TGTTGCCTCC GGGGCGACCC 81 TGCCTTCGGG CGGGGGCTCC GGGTGGACAC TTCAAACTCT TGCGTAACTT TGCAGTCTGA GTAAACTTAA TTAATAAATT 161 AAAACTTTTA ACAACGGATC TCTTGGTTCT GGCATCGATG AAGAACGCAG CGAAATGCGA TAAGTAATGT GAATTGCAGA 241 ATTCAGTGAA TCATCGAATC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT GCCTGTTCGA GCGTCATTTC 321 ACCACTCAAG CCTCGCTTGG TATTGGGCAA CGCGGTCCGC CGCGTGCCTC AAATCGTCCG GCTGGGTCTT CTGTCCCCTA 401 AGCGTTGTGG AAACTATTCG CTAAAGGGTG TTCGGGAGGC TACGCCGTAA AACAACCCCA TTTCTAAGGT TGACCTCGGA 481 TCAGGTAGGG ATA

1701.1


1 ?TTGATATGC TAAGGTTAAG GGGGATCCCT TCCTTTATCC GAAGGGTCAC CCTTAAAAAA TGGGGTTGTT TTACGGCGGT 81 AGCTTCCGAA CACCCTTTAG CGAATAGTTC CACAACGCTT AGGGGACAGA AGACCCAGCC GGACGATTTG AGGCACGCGG 161 CGGACCGCGT TGCCCAATAC CAAGCGAGGC TTGAGTGGTG AAATGACGCT CGAACAGGCA TGCCCCCCGG AATACCAGGG 241 GGCGCAATGT GCGTTCAAAG ATTCGATGAT TCACTGAATT CTGCAATTCA CATTACTTAT CGCATTTCGC TGCGTTCTTC 321 ATCGATGCCA GAACCAAGAG ATCCGTTGTT AAAAGTTTTA ATTTATTAAT TAAGTTTACT CAGACTGCAA AGTTACGCAA 401 GAGTTTGAAG TGTCCACCCG GAGCCCCCGC CCGAAGGCAG GGTCGCCCCG GAGGCAACAG AGTCGGACAA CAAAGGGTTA 481 TGAATAACCA GGCCGAAGCC CGGGCGTTCT TGTAATGATC CCT?

1703.3


1 ATCATTACAA GAACGCCCGG GCTTCGGCCT GGTTATTCAT AACCCTTTGT TGTCCGACTC TGTTGCCTCC GGGGCGACCC 81 TGCCTTCGGG CGGGGGCTCC GGGTGGACAC TTCAAACTCT TGCGTAACTT TGCAGTCTGA GTAAACTTAA TTAATAAATT 161 AAAACTTTTA ACAACGGATC TCTTGGTTCT GGCATCGATG AAGAACGCAG CGAAATGCGA TAAGTAATGT GAATTGCAGA 241 ATTCAGTGAA TCATCGAATC TTTGAACGCA CATTGCGCCC CCTGGTATTC CGGGGGGCAT GCCTGTTCGA GCGTCATTTC 321 ACCACTCAAG CCTCGCTTGG TATTGGGCAA CGCGGTCCGC CGCGTGCCTC AAATCGTCCG GCTGGGTCTT CTGTCCCCTA 401 AGCGTTGTGG AAACTATTCG CTAAAGGGTG TTCGGGAGGC TACGCCGTAA AACAACCCCA TTTCTAAGGT GACCTCGGAT 481 CAGGT?

Appendix 1

Alternaria alternata

840.1

Species Accession No. ScoreAlternaria alternata AF455516 934A. arborescens AF404667 934A. tenuissima AF404666 934A. longipes AF314587 934A. pomicola AF314583 934

1 ATCATTACAC AAATATGAAG GCGGGCTGGA ACCTCTCGGG GTTACAGCCT TGCTGAATTA TTCACCCTTG TCTTTTGCGT 81 ACTTCTTGTT TCCTTGGTGG GTTCGCCCAC CACTAGGACA AACATAAACC TTTTGTAATT GCAATCAGCG TCAGTAACAA 161 ATTAATAATT ACAACTTTCA ACAACGGATT CTCTTGGGTT CTGGCATCGA TGAAAGAACG CAGCGAAATG CGATAAGTAG 241 TGTGAATTGC AGAATTCAGT GAATCATCGA ATCTTTGAAC GCACATTGCG CCCTTTGGTA TTCCAAAGGG CATGCCTGTT 321 CGAGCGTCAT TTGTACCCTC AAGCTTTGCT TGGTGTTGGG CGTCTTGTCT CTAGCTTTGC TGGAGACTCG CCTTAAAGTA 401 ATTGGCAGCC GGCCTACTGG TTTCGGAGCG CAGCACAAGT CGCACTCTCT ATCAGCAAAG GTCTAGCATC CATTAAGCCT 481 TTTTTTCAAC TTTTGACCTC GGATCAGGTA GGGATA

Alternaria infectoria

1705.3

Species Accession No. ScoreLewia infectoria AF229458 1033Alternaria conjuncta AF392988 1027Lewia infectoria AF397248 1017Alternaria infectoria Y17066 1011Lewia infectoria AF397239 1009 1 ATCATTACAC AATAACCAGG CGGGCTGGAC ACCCCCCGCT GGGCACTGCT TCACGGCGTG CGCGGCGGGG CCGGCCCTGC 81 TGAATTATTC ACCCGTGTCT TTTGCGTACT TCTTGTTTCC TGGGTGGGCT CGCCCGCCCT CAGGACCAAC CACAAACCTT 161 TTGCAATAGC AATCAGCGTC AGTAACAACG TAATTAATTA CAACTTTCAA CAACGGATCT CTTGGTTCTG GCATCGATGA 241 AGAACGCAGC GAAATGCGAT ACGTAGTGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT TTGAACGCAC ATTGCGCCCT 321 TTGGTATTCC AAAGGGCATG CCTGTTCGAG CGTCATTTGT ACCCTCAAGC TTTGCTTGGT GTTGGGCGTC TTTTGTCTCC 401 AGTTCGCTGG AGACTCGCCT TAAAGTCATT GGCAGCCGGC CTACTGGTTT CGGAGCGCAG CACAAGTCGC GCTCTTCGCC 481 AGCCAAGGTC AGCGTCCAGC AAGCCTTTTT TTCAACCTTT GACCTCGGAT CAGGTAGGGA TA

Aureobasidium pullulans

842.1

Species Accession No. ScoreAureobasidium pullulans AF455533 938A. pullulans AJ244232 902A. pullulans AF121285 890A. pullulans AF121281 890A. pullulans AJ244233 888

1 ATCATTAAAG AGTAAGGGTG CTTAGCGGCC GACCTTCCAA CCCTTTGTTG TTAAAACTAC CTTGTTGCTT TGGCGGGACC 81 GCTCGGTCTC GAGCCGCTGG GGATTCGTCC CAGGCGAGCG CCGCCAGAGT TAAACCAAAC TCTTGTTATT TAACCGGTCC 161 GTTCTGAGTT AAAAATTTTG AATAAATCAA AACTTTCAAC AACGGATCTC TTGGTTCTCG CATCGATGAA GAACGCAGCG 241 AAATGCGATA AGTAATGTGA ATTGCAGATT CAGTGAATCA TCGAATCTTT GAACGCACAT TGCGCCCCTT GGTATTCCGA 321 GGGGCATGCC TGTTCGAGCG TCATTACACC ACTCAAGCTA TGCTTGGTAT TGGGCGTCGT CCTTAGTTGG GCGCGCCTTA 401 AAGACCTCGG CGAGGCCACT CCGGCTTTAG GCGTAGTAGA ATTTATTCGA ACGTCTGTCA AAGGAGAGGA ACTCTGCCGA 481 CTGAAACCTT TATTTTTCTA GGTTGACCTC GGATCAGGTA GGGATA

Appendix 1

Trichoderma longibrachiatum

843.1

Species Accession No. ScoreTrichoderma longibrachiatum AF362105 1170Hypocrea schweinitzii X93966 1170T. longibrachiatum AF362099 1164T. longibrachiatum AF362104 1162T. longibrachiatum AF359258 1156

1 ATCATTACCG AGTTTACAAC TCCCCAAACC CCAATGTGAA CGTTACCCAA TCTGTTGCCT CGGCGGGATT CTCTTGCCCC 81 GGGCGCGTCG CAGCCCCGGA TCCCATGGCG CCCGCCGGAG GACCAACTCC AAACTCTTTT TTCTCTCCGT CGCGGCTCCC 161 GTCGCGGCTC TGTTTTATTT TTGCTCTGAG CCTTTCTCGG CGACCCTAGC GGGCGTCTCG AAAATGAATC AAAACTTTCA 241 ACAACGGATC TCTTGGTTCT GGCATCGATG AAGAACGCAG CGAAATGCGA TAAGTAATGT GAATTGCAGA ATTCAGTGAA 321 TCATCGAATC TTTGAACGCA CATTGCGCCC GCCAGTATTC TGGCGGGCAT GCCTGTCCGA GCGTCATTTC AACCCTCGAA 401 CCCCTCCGGG GGGTCGGCGT TGGGGATCGG CCCCTCACCG GGCCGCCCCC GAAATACAGT GGCGGTCTCG CCGCAGCCTC 481 TCCTGCGCAG TAGTTTGCAC ACTCGCACCG GGAGCGCGGC GCGGCCACAG CCGTAAAACA CCCCAAACTT CTGAAATGTT 561 GACCTCGGAT CAGGTAGGAA TACCCGCTGA ACTTAAGCAT ATCAAT?

858.4


1 ATCATTACCG AGTTTACAAC TCCCAAACCC CAATGTGAAC GTTACCAATC TGTTGCCTCG GCGGGATTCT CTTGCCCCGG 81 GCGCGTCGCA GCCCCGGATC CCATGGCGCC CGCCGGAGGA CCAACTCCAA ACTCTTTTTT CTCTCCGTCG CGGCTCCCGT 161 CGCGGCTCTG TTTTATTTTT GCTCTGAGCC TTTCTCGGCG ACCCTAGCGG GCGTCTCGAA AATGAATCAA AACTTTCAAC 241 AACGGATCTC TTGGTTCTGG CATCGATGAA GAACGCAGCG AAATGCGATA AGTAATGTGA ATTGCAGAAT TCAGTGAATC 321 ATCGAATCTT TGAACGCACA TTGCGCCCGC CAGTATTCTG GCGGGCATGC CTGTCCGAGC GTCATTTCAA CCCTCGAACC 401 CCTCCGGGGG GTCGGCGTTG GGGATCGGCC CCTCACCGGG CCGCCCCCGA AATACAGTGG CGGTCTCGCC GCAGCCTCTC 481 CTGCGCAGTA GTTTGCACAC TCGCACCGGG AGCGCGGCGC GGCCACAGCC GTA?

995.1


1 ATCATTACCG AGTTTACAAC TCCCAAACCC CAATGTGAAC GTTACCAATC TGTTGCCTCG GCGGGATTCT CTTGCCCCGG 81 GCGCGTCGCA GCCCCGGATC CCATGGCGCC CGCCGGAGGA CCAACTCCAA ACTCTTTTTT CTCTCCGTCG CGGCTCCCGT 161 CGCGGCTCTG TTTTATTTTT GCTCTGAGCC TTTCTCGGCG ACCCTAGCGG GCGTCTCGAA AATGAATCAA AACTTTCAAC 241 AACGGATCTC TTGGTTCTGG CATCGATGAA GAACGCAGCG AAATGCGATA AGTAATGTGA ATTGCAGAAT TCAGTGAATC 321 ATCGAATCTT TGAACGCACA TTGCGCCCGC CAGTATTCTG GCGGGCATGC CTGTCCGAGC GTCATTTCAA CCCTCGAACC 401 CCTCCGGGGG GTCGGCGTTG GGGATCGGCC CCTCACCGGG CCGCCCCCGA AATACAGTGG CGGTCTCGCC GCAGCCTCTC 481 CTGCGCAGTA GTTTGCACAC TCGCACCGGG AGCGCGGCGC GGCCACAGCC GTAAAACACC CCAAACTTCT GAAATGTTGA 561 CCTCGGATCA GGTAGGAATA CCCGCTGAAC TTAAGC?

Appendix 1

Trichothecium roseum

858.5

Species Accession No. ScoreTrichothecium roseum U51982 844

1 ATCATTATAG AGTTAACAAA ACAACTCCCA ACCCTTTGTG AACCTTACCT ACCGTTGCTT CGGCGGACCG CCCCGGGCGC 81 TGCGTGCCCC GGACCCAAGG CGCCCGCCGG GGACCACACG AACCCTGTTT AACAAACATG TGTATCCTCT GAGCGAGCCG 161 AAAGGCAACA AAACAAATCA AAACTTTCAA CAACGGATCT CTTGGTTCTG GCATCGATGA AGAACGCAGC GAAATGCGAT 241 AAGTAATGTG AATTGCAGAA TTCAGTGAAT CATCGAATCT TTGAACGCAC ATTGCGCCCG CCAGTATTCT GGCGGGCATG 321 CCTGTCCGAG CGTCATTTCA ACCCTCGGGC CCCCCCCTTT TCCCCTCGCG GGGGAGGGGG CGGGCCCGGC GTTGGGGCCC 401 AGGCGTCCTC CAAGGGCGCC TGTCCCCGAA ACCCAGTGGC GGCCTCGCCG CTGCCTCCTC CGCGTAGTAG CACAAACCTC 481 GCGGGCGGAA ACGGCGCGGC CACGCCGTAA AACCCCAAAC TTTTACCAAG GTTGACCTCG GATCAGGTAG GGATA

Sequences of Zygomycetes

Mucor circinelloides

859.1

Species Accession No. ScoreMucor circinelloides AF412286 824M. circinelloides AF412287 808M. circinelloides AF412288 792M. rouxii AF412295 792M. fragilis AF474242 699 1 ?GACGCTGGA GGGATGCTCC ACTGCTATAA GGATAGGCGG TGGGGATGCT AACCGAGTCA TAATCAAGCT TAGGCTTGGN 81 ATCCTATTAT TATTTACCAA AAGAATTCAG AATTAATATT GTAACATAGA CCTAAAAAAT CTATAAAACA ACTTTTAACA 161 ACGGATCTCT TGGTTCTCGC ATCGATGAAG AACGTAGCAA AGTGCGATAA CTARTGTGAA TTGCATATTC AGTGAATCAT 241 CGAGTCTTTG AACGCAACTT GCGCTCATTG GTATTCCAAT GAGCACGCCT GTTTCAGTAT CAAAACAAAC CCTCTATCCA 321 ACATTTTGTT GAATAGGAAT ACTGAGAGTC TCTTGATCTA TTCCTGATCT CGAACCTCTT GAAATGTACA AAGGCCTGAT 401 CTTGTTTGAA TGCCTGAACT TTTTTTTAAC ATAAAGANAA GCTCTTNCGG TANGCTGTGC TGGGGCCTCC CAAAA

Mucor plumbeus

989.5 Species Accession No. ScoreMucor plumbeus AF412293 1049M. plumbeus AF412292 1033M. plumbeus AF412291 1029M. fragilis AF474242 783M. racemosus AJ271061 733 1 ATCATTAAAT AATCAATAAT CTTGGCTTGT CCATTATTAT CTATTTACTG TGAACTGTAT TATTATTTGA CATTTGAGGG 81 ATGTTCCAAT GTTATAAGGA TAGACATTGG AAATGTTAAC CGAGTCATAA TCAGGTTTAG GCCTGGTATC CTATTATTAT 161 TTACCAAATG AATTCAGAAT TAATATTGTA ACATAGACCT AAAAAATCTA TAAAACAACT TTTAACAACG GATCTCTTGG 241 TTCTCGCATC GATGAAGAAC GTAGCAAAGT GCGATAACTA GTGTGAATTG CATATTCAGT GAATCATCGA GTCTTTGAAC 321 GCAACTTGCG CTCATTGGTA TTCCAATGAG CACGCCTGTT TCAGTATCAA AACAAACCCT CTATCCAACT TTTGTTGTAT 401 AGGATTATTG GGGGCCTCTC GATCTGTATA GATCTTGAAA TCCTTGAAAT TTACTAAGGC CTGAACTTGT TTAAATGCCT 481 GAACTTTTTT TTAATATAAA GGAAAGCTCT TGTAATTGAC TTTGATGGGG CCTCCCAAAT AAATCTTTTT TAAATTTGAT 561 CTGAAATCAG GCGGGATTAC CCGCTGAACT TAAGCATATC AA?

Appendix 1

1060.1

Species Accession No. ScoreMucor plumbeus AF412293 1080M. plumbeus AF412292 1057M. plumbeus AF412291 1037M. fragilis AF474242 771M. racemosus AJ271061 743 1 ATCATTAAAT AATCTAATAA TCTTGGCTTG TCCATTATTA TCTATTTACT GTGAACTGTA TTATTATTTG ACGTTTGAGG 81 GATGTTCCAA TGTTATAAGG ATAGACATTG GAGATGTTAA CCGAGTCATA ATCAGGTTTA GGCCTGGTAT CCTATTATTA 161 TTTACCAAAT GAATTCAGAA TTAATATTGT AACATAGACC TAAAAAATCT ATAAAACAAC TTTTAACAAC GGATCTCTTG 241 GTTCTCGCAT CGATGAAGAA CGTAGCAAAG TGCGATAACT AGTGTGAATT GCATATTCAG TGAATCATCG AGTCTTTGAA 321 CGCAACTTGC GCTCATTGGT ATTCCAATGA GCACGCCTGT TTCAGTATCA AAACAAACCC TCTATCCAAC TTTTGTTGTA 401 TAGGATTATT GGGGGCCTCT CGATCTGTAT AGATCTTGAA ACCCTTGAAA TTTACTAAGG CCTGAACTTG TTTAATGCCT 481 GAACTTTTTT TTAATATAAA GGAAAGCTCT TGTAATTGAC TTTGATGGGG CCTCCCAAAT AAATCTTTTT TAAATTTGAT 561 CTGAAATCAG GTGGGATTAC CCGCTGAACT TAAGCATAT?

Rhizopus oryzae

841.1

Species Accession No. ScoreAmylomyces rouxii AF115724 994Rhizopus arrhizus AF455429 978R. oryzae AF115722 971Amylomyces rouxii AF115723 957R. oryzae AF115725 953 1 ?TGACCCGCG GAGGGYCTTA TTATGTTAAA GCGCCTTACC TTAGGGTTTC CTCTGGGGTA AGTGATTGCT TCTACACTGT 81 GAAAATTTGG CTGAGAGACT CAGACTGGTC ATGGGTAGAC CTATCTGGGG TTTGATCGAT GCCACTCCTG GTTTCAGGAG 161 TACCCTTCAT AATAAACCTA GAAATTCAGT ATTATAAAGT TTAATAAAAA ACAACTTTTA ACAATGGATC TCTTGGTTCT 241 CGCATCGATG AAGAACGTAG CAAAGTGCGA TAACTAGTGT GAATTGCATA TTCAGTGAAT CATCGAGTCT TTGAACGCAG 321 CTTGCACTCT ATGGTTTTTC TATAGAGTAC GCCTGCTTCA GTATCATCAC AAACCCACAC ATAACATTTG TTTATGTGGT 401 GATGGGTCGC ATCGCTGTTT TATTACAGTG AGCACCTAAA ATGTGTGTGA TTTTCTGTCT GGCTTGCTAG GCAGGAATAT 481 TACGCTGGTC TCAGGATCTT TTTTTTTGGT TCGCCCAGGA AGTAAAGTAC AAGAGTATAA ACCAGTAACT TCAAACTAG?

859.5

Species Accession No. ScoreRhizopus oryzae AF115725 1059R. oryzae AF115722 1033R. arrhizus AF455429 1029Amylomyces rouxii AF115723 1023Amylomyces rouxii AF115724 1013 1 ATCATTAATT ATGTTAAAGC GCCTTACCTC TTAGGGTTTC CTCTGGGGTA AGTGATTGCT TCTACACTGT GAAAATTTGG 81 CTGAGAGACT CAGACTGGTC ATGGGTAGAC CTATCTGGGG TTTGATCGAT GCCACTCCTG GTTTCAGGAG CACCCTTCAT 161 AATAAACCTA GAAATTCAGT ATTATAAAGT TTAATAAAAA ACAACTTTTA ACAATGGATC TCTTGGTTCT CGCATCGATG 241 AAGAACGTAG CAAAGTGCGA TAACTAGTGT GAATTGCATA TTCAGTGAAT CATCGAGTCT TTGAACGCAG CTTGCACTCT 321 ATGGTTTTTC TATAGAGTAC GCCTGCTTCA GTATCATCAC AAACCCACAC ATAACATTTG TTTATGTGGT AATGGGTCGC 401 ATCGCTGTTT TATTACAGTG AGCACCTAAA ATGTGTGTGA TTTTCTGTCT GGCTTGCTAG GCAGGAATAT TACGCTGGTC 481 TCAGGATCTT TTTCTTTGGT TCGCCCAGGA AGTAAAGTAC AAGAGTATAA TCCA?

Appendix 2

Appendix 2

Pictures of hazelnut samples and fungal cultures obtained from these are attached on a CD-ROM and are available on the internet (URL: https://livne.co.il/thesis/picture_appendix.html).

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Characterization of fungi with molecular methods