Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 917
Approaches to Species Delineation in Anamorphic (mitosporic) Fungi: A Study on Two Extreme Cases
BY OLGA VINNERE
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Preface
This work consists of results of two loosely connected studies I conducted during my PhD. The truth is that the original topic of my dissertation was supposed to be “The Fungal Group Mycelia Sterilia – Its Interactions With Crop Plants and Their Pathogens”, implying research on taxonomy and biological activity of sterile fungal strains. While studying this subject we (my supervisors and myself), however, decided to include a project on Colletotrichum that was a part of research done for my Master of Science degree and which turned to be much more interesting that we could possibly expect. Since I have used similar methods and addressed similar questions in studies of these two unrelated groups of fungi, I will defend my work under the title: Approaches to Species Delineation in Anamorphic (mitosporic) Fungi: A Study on Two Extreme Cases
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Contents
Introduction...... 11 Species concepts in fungi ...... 11 Fungal holomorph ...... 12 Combination of morphological and molecular approaches...... 14 Outline of this study ...... 15 The studied cases ...... 16 Case No 1: Colletotrichum acutatum ...... 16 Importance of C. acutatum as plant pathogen ...... 16 History of the C. acutatum taxon...... 18 Morphological studies ...... 20 Biochemical and molecular studies ...... 24 Sexuality of C. gloeosporioides and C. acutatum ...... 26 Current state of C. acutatum...... 27 Is C. acutatum sensu lato a single species? ...... 32 Case No 2: Mycelia Sterilia...... 36 What are Mycelia Sterilia? ...... 36 Distribution in nature...... 36 Importance of plant-associated Mycelia Sterilia ...... 37 Our isolation and screening strategy...... 38 Taxonomical studies of Mycelia Sterilia ...... 39 New biologically active Mycelia Sterilia found in this study and remarks on their taxonomic position ...... 42 General remarks, or what did I learn?...... 50 Conclusions...... 52 Acknowledgements...... 53 References...... 55
Introduction
In this work, I will be discussing two rather extreme cases in fungal taxonomy. One of the case studies was carried out on a plant pathogen, Colletotrichum acutatum – an anamorphic (mitosporic) fungus that has too many variable morphological features, usually causing overlapping descriptions with other species of the same genus. Another studied case is Mycelia Sterilia, an artificial taxonomic group of fungi deficient in production of any kind of spores, therefore lacking the main morphological features that could have helped in identifying these fungi to the species and sometimes even genus or family level. On one hand, due to its high variability, the genus Colletotrichum has drawn major attention from mycologists and plant pathologists. There are dozens of papers published on this plant pathogen, and many of them are quite controversial. This fungus has been extensively studied from taxonomical, enzymatic, plant pathological, genetic, bio- and chemical control points of view. On the other hand, Mycelia Sterilia are not well studied, mainly because of the difficulties in their identification. Therefore, many sterile fungi may have been neglected, although they could be extremely interesting objects of studies and research programs on, for instance, discovery of novel drugs or the development of biological control agents against important plant pathogens.
Species concepts in fungi There are many traits that are used in the traditional and modern mycology to contribute to taxonomical studies of fungi. These include morphology, anatomy, biochemistry, nucleic acid sequences, and many others. However, starting with works by de Bary and Fuckel from the early beginning of mycology as a science and up to the last decades of the 20th century, taxonomic species in fungi continued to be almost entirely derived from morphology (Braiser 1997), therefore meeting the criteria of the Morphological Species Concept (MSC). MSC, proposed by Linnaeus (1758), recognizes species as groups of individuals sharing similar morphological (sometimes also anatomical) traits that are distinctly different from other groups of organisms. This concept proved to be handy for many plants and animals, where morphological and anatomical traits are abundant. However in fungi, especially microscopic ones, morphological traits are 11 considerably fewer and use of MSC is therefore severely biased. Despite all limitations of the MSC, many fungal species described more than 100 years ago by Saccardo, Fuckel, Petrak and others and based on morphology alone, are still regarded as valid. In 1942, Ernst Mayr proposed a Biological Species Concept (BSC), defining species as “groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups” (Mayr, 1942). Ability to mate resulting in a fertile progeny is one of the main points of the BSC. It works well for many fungi, where sexual reproduction exists. But there are also fungal groups, where sexual reproduction has never been discovered. Let us get some insight into the fungal life cycle by looking at the perception of the fungal holomorph.
Fungal holomorph Historically, as was pointed out by several researchers, fungal taxonomy was quite anthropocentric and too much emphasis was placed on classification and too little on understanding the concept of species as it is present in nature (Brasier, 1997, Taylor et al., 2000). Therefore, modern fungal systematics is quite artificial, especially when it concerns different stages of the fungal life cycle (fungal holomorph). Namely, sexual (teleomorph) and asexual (anamorph) stages of the life cycle of a single species until recently were classified under two different phyla of the Fungal Kingdom: anamorphic (mitosporic) fungi under the Fungi Imperfecti (subdivision Deuteromycotina), whereas their teleomorphs were placed in Ascomycetes or Basidiomycetes, depending on the type of the reproductive structures. According to the last Dictionary of Fungi (Kirk et al., 2001), this classification is quite convenient for practical reasons, but it is also “meaningless in terms of natural or phylogenetic classification”. According to the latest opinion in fungal taxonomy, anamorphic fungi are not forming a separate taxonomic unit anymore, but instead are assigned to appropriate existing levels of teleomorphic hierarchy (Kirk et al., 2001). Although the Fungal Kingdom has been separated from plants and animals, its nomenclature and taxonomy are still governed by the International Code of Botanical Nomenclature (Greuter et al., 2000), due to its history. The Article 59 of the Code states that in fungal systematics priority should be given to the teleomorphic taxon, so for instance anamorphic fungus Colletotrichum gloeosporioides should instead be called by the name of its teleomorph, Glomerella cingulata. Currently, there are different opinions on the Article 59 and on the last edition of the Dictionary of Fungi, and both anamorphic and teleomorphic names are still widely used both by mycologists and plant pathologists.
12 First of all, the old system of classification of the anamorphic fungi based on morphology, such as peculiarities of conidiomata and conidia, as well as the conidiogenesis, is very handy for practical identification reasons. Another important point why we cannot completely discard the Fungi Imperfecti (anamorphic or mitosporic fungi) is that many of them are lacking teleomorphs – they have lost it during the process of evolution, it has never been observed in nature or in the laboratory, or the connection of anamorph with teleomorph has never been proven. There are also several teleomorphs, for which anamorphs have never been found, e.g. Myrangium, Geoglossum, etc. (Hanlin, 1990). Another point is that the same teleomorph can have different anamorphic stages (synanamorphs), for example ascomycete Mycoshpaerella has at least 18 different anamorphs, including such important plant pathogens as Cercospora and Ramularia (Kirk et al., 2001).
BSC defines species based on isolating mechanisms, of which the most important one is reproductive isolation. Therefore it cannot be applied to anamorphic fungi in many of which sexual recombination is unknown (Harrington & Rizzo, 1999). There are some opinions, even among mycologists that asexual organisms cannot form species (Perkins, 1991). However, asexually reproducing (anamorphic) fungi are extremely common in nature, therefore Harrington & Rizzo (1999) believe they still should be included in a “workable species concept for fungi”, even though the level of variation within these taxa is supposed to be lower than in sexually recombining ones. Despite reproducing asexually, many anamorphic fungi are known to possess a surprisingly high level of genetic variation (Kohn, 1995; Tahlinhas et al., 2002). Since BSC is not fully satisfactory for fungal taxa, a new approach, based on modelling evolutionary relationships and on analysis of genotypic traits has been developed. The phylogenetic approach to fungal classification became standard during the past decades due to the rapid development of DNA based techniques (e.g. Mayden, 1997; Taylor et al., 2000). Along with development of molecular markers, came also changes in understanding the species concept in different groups of fungi. The Phylogenetic species concept (PSC) recognizes species as “… the smallest aggregation of populations with a common lineage that share unique, diagnosable phenotypic characters” (Harrington & Rizzo, 1999). As it has been pointed out in an excellent review by Taylor et al. (2000), this concept seems to be “well suited for fungi and likely to become very popular with mycologists”, first of all because it applies equally to both sexual and asexual organisms, including fungi.
13 Combination of morphological and molecular approaches As science developed, it became obvious that many morphological features can be quite plastic and easily change depending on the environmental conditions and not necessarily because of variation inside the genomes. This would lead to overlapping descriptions of several species, and, therefore to creation of numerous synonyms, like in Trichoderma (Gams & Bissett, 1998), Colletotrichum (von Arx, 1957) and several other fungal genera. It became clear that the MSC applied to fungi does not always work. One of the main limitations of the MSC is that differences in morphological features might not necessarily represent different species, but indeed an extensive phenotypic variation within the same species. Besides, organisms that are morphologically similar might indeed represent different species (especially in cases when similar phenotypic trait evolved several times in several distinct groups). It is also very difficult to set boundaries on phenotypic variation, especially in cases when organisms exhibiting intermediate morphology are involved. In order to assist identification of species based on morphology and to overcome limitations of MSC, several other traits were employed. Various biochemical markers have been used along with morphological traits, including isozymes (Micales, Bonde & Peterson, 1986), ability to utilize different carbon sources (Buyer et al., 2001; Kubicek et al., 2003), production of secondary metabolites (Talbot, Vincent & Wildman, 1996), etc. Some of those are still successfully used for identification of the species boundaries (Levenfors et al., 2003; Kubicek et al., 2003) The golden age of molecular fungal systematics began with the development of DNA sequencing techniques and computer programs using the nucleotide sequence data for inferring phylogenetic relationships between organisms. One of the most extensively studied parts of the fungal genomes is the ribosomal DNA (rDNA) array that has been proven to be of great value for resolving taxonomic positions in many living organisms, (including fungi) at different levels (Woese, 1987; Hillis & Dixon, 1991). Since all the genes and spacers constituting the rDNA array evolve at different rates, but the speeds of evolution in all the arrays in eukaryotic organisms are roughly the same, some of the rDNA fragments are useful for delineation of higher taxonomic ranks, such as classes and phyla (5.8S rRNA gene, parts of the Large Subunit rRNA [LSU] and Small Subunit rRNA [SSU] genes) (Bruns et al., 1992), others are used for separation of genera and species (Internally Transcribed Spacer [ITS] 1 and 2 regions, Domain 2 of the LSU) (Hillis & Dixon, 1991; Bruns, White & Taylor, 1991; White et al., 1990; Donaldson et al., 1995; James et al., 2001). Sequences of ITS1 and Intergenic Spacer (IGS) regions can be used in some organisms to resolve relationships even between populations within the same species 14 (Hillis & Dixon, 1991; James et al., 2001). Besides rDNA, several other genes are widely used for phylogenetic studies: i.g. nuclear (E-tubulin gene, Elongation Factor [EF] - D, actin gene, etc.) and mitochondrial (mitochondrial rRNA genes, Cytochrome Oxydase [CO] gene, etc.) (Li, Rouse & German, 1994; Glass & Donalsdon, 1995; Donaldson et al., 1995; O’Donnel, Cigelnik & Nirenberg, 1998; Baayen et al., 2000; Schoch et al., 2001; Zhou & Stanosz, 2001; Baayen et al., 2001). As sequencing information accumulated, it turned out that in many cases phylogenetic trees inferred from the DNA sequencing data were not concordant with traditional morphology-based grouping of fungal taxa (Waalwijk, de Koning & Baayen, 1996; Yang & Sweetengham, 1998; Nirenberg, Feiler & Hagedorn, 2002). Moreover, phylogenetic trees of different genes sometimes are discordant. However, there are also many cases when morphological groupings are highly supported by the sequencing data (Sherif et al., 1994; Abeln, de Pagter, & Verkley, 2000; James et al., 2000). Obviously, it is not clear which region should or should not be used, and if morphological features or nucleotide sequences alone should be used in studies of fungal taxonomy (Seifert, Wingfield & Wingfield, 1995). After being in the field for couple of years I dare to say that in my opinion, molecular data should be used just as one component supplementing the taxonomical and phylogenetic studies, and the value of classical morphological methods should not be neglected or underestimated.
Outline of this study In this thesis, I would like to address the question of identifying species units in anamorphic fungi, in two cases when morphological traits are too abundant and variable or too few or even lacking. Using DNA sequence data, my co-workers and myself made an attempt to resolve phylogenetic relationships among several morphologically similar species in Colletotrichum on one hand, and tried to clarify taxonomic positions of several sterile strains on the other hand. During selection of plant-associated sterile strains I came across three interesting (supposedly novel) fungi, one of which had pronounced deleterious effect on the plants, and another two possessed biocontrol properties. Reports on the biological activities of these isolates are also included in this thesis.
15 The studied cases
Case No 1: Colletotrichum acutatum Importance of C. acutatum as plant pathogen Distribution and host range C. acutatum is an economically important plant pathogenic fungus with a wide host range and worldwide distribution. Originally, it was described as a causal agent of ripe fruit rot of strawberry (Fragaria ananassa) in Queensland, Australia (Simmonds, 1965). In his paper, Simmonds also mentions papaw (Carica papaya), walnut (Juglans regia), avocado (Persea americana), slash pine (Pinus elliotii), apple (Malus sylvestris), tomato (Lycopersicon esculentum), and several other plants as hosts of this species. Later, C.acutatum designated as forma specialis pinea was shown to be associated with terminal crook disease of pine (Pinus spp.) in New Zealand. The same fungus was also recognized as a pathogen of several pasture legumes, namely sweet pea (Lathyrus odoratus), lupine (Lupinus spp.) and vetch (Vicia spp.) (Dingley & Gilmour, 1972). Hindorf in 1973 (a, b & c) mentioned C. acutatum among other species of Colletotrichum pathogenic to coffee (Coffea arabica) in Kenya. In later years, there were several reports about occurrences of C. acutatum in different countries and on various crops (Baxter, Eiker & van der Westhuizen, 1982; Smith & Black, 1986; de Clauser et al., 1990; Bernstein et al., 1995; Martín & García-Figueres, 1999, Saha et al., 2002, I & II). Until recently, it was believed to be present in all the continents, excluding South America (Walker, Nikandrow & Millar, 1991). The latest data show the presence of this pathogen even on this continent (Henz, Boiteux & Lopez, 1992; Kuramae-Izioka et al., 1997; Afanador-Kafuri et al., 2003), therefore this species is now considered cosmopolitan. Paper I reports, probably, the first case of C. acutatum on plants of the genus Rhododendron. Besides, to the best of our knowledge, paper I contains the first report of occurrence of this species in Sweden and Latvia, and II reports the first case of C. acutatum-caused anthracnose of azalea in Italy. Additionally, this species to our knowledge was detected for the first time in Vietnam, on leaves of mango (Mangifera ingica), during the survey done by a Master student under my co-supervision (Nguyen, 2002). If we combine the information contained in the available literature sources, it turns out that C. acutatum infects plants of at least 24 families worldwide
16 (Farr et al., 1989; Walker, Nikandrow, Millar 1990; Sutton, 1998; Lardner et al., 1999; I). Due to the high aggressiveness and tremendous yield losses in many crops, C. acutatum was put on the lists of quarantine objects in many countries, including Latvia and Sweden.
Disease symptoms and course of infection C. acutatum causes a range of different symptoms on all possible plant parts, most commonly on crowns, petioles, leaves, flowers and fruit (e.g. Simmonds, 1965; Legard, 2000; Timmer & Brown, 2000; Adaskaveg & Förster, 2000). The most common disease caused by this fungus is anthracnose – appearance of dark, sunken lesions on the infected organs, which is common on strawberries, rhododendrons, mango, and many other plant species (Simmonds, 1965; Legard, 2000; I). It also causes crown rot of strawberry (Smith & Black, 1990; Freeman, Katan & Shabi, 1998), fruit rot in many tropical plants (Lardner et al., 1999; Nguyen, 2002), terminal crook disease in pine (Dingley & Gilmoure, 1972), stem and twig cankers of lilac and willows (IV), etc. Another common problem caused by this pathogen is flower anthracnose, post-harvest diseases and prevention of fruit set of various fruit crops (e.g. oranges, lime, avocado), resulting in severe yield losses in fruit industry worldwide (Timmer & Brown, 2000). Fungi of the genus Colletotrichum have adapted to saprophytic existence on decaying plant parts, where they usually sporulate abundantly, and which serve as an efficient source for new infections (Simmonds 1965, Dyko & Mordue, 1979; Zulfiqar, Brlansky & Timmer, 1996). In many cases, especially in the case of fruit rot, C. acutatum causes latent infection that does not show until the fruit ripens (Timmer & Brown, 2000).
Measures of control Mostly chemicals are used for controlling spread of C. acutatum. This species has a lower sensitivity to chemicals than other species of Colletotrichum (Adaskaveg & Förster, 2000). In comparison with C. gloeosporioides, C. acutatum is less sensitive to benomyl, captan, and propiconazole, but more sensitive to myclobutanil and tebuconazole (Bernstein, Zehr & Dean, 1995; Adaskaveg & Förster, 2000). Based on that, benomyl sensitivity test is frequently applied to separate C. acutatum from C. gloeosporioides (Bernstein, Zehr & Dean, 1995; Brown, Sreenivasaprasad & Timmer, 1996; Freeman, Katan & Shabi, 1998; Tahlinhas et al., 2002). Still, there is a report of successfully controlling C. acutatum infection by benomyl, which is called “unique” by its authors (Peres et al. 2002). These authors state that the fungus might not be highly sensitive to benomyl in cultures, but this fungicide still gives good results when applied in the field as preventive for early disease development. However, it has a very little effect on further disease development and 17 spread (Peres et al. 2002). There also were reports of lower doses of ineffective chemicals being conductive for disease spread and development (Peterson, 1973). Recently, the possibilities of biological control of this pathogen by using plant beneficial microorganisms were discussed. However, there are no registered biocontrol agents against C. acutatum specifically (Korsten & Jeffries, 2000; Jeffries & Koomen, 1992).
Pathogenicity to animals Very recently, there was a single report of C. acutatum causing disseminated mycosis in hypothermic immuno-compromised Kemp’s riddle sea turtle (Lepidochelys kempi) in Florida, U.S. (Manire et al. 2002). Any anti-fungal drug treatment proved to be ineffective. The fungal hyphae were post mortem detected in kidneys by silver staining and the fungus was isolated in a pure culture. Its morphology fully fitted the original description of C. acutatum. The fungus did not grow at temperatures above 30 °C, and is thus assumed not to be pathogenic to humans. How the turtle acquired the fungus still reminds a mystery (Manire et al. 2002). This particular clinical isolate was kindly supplied to us by Dr. Deanna Sutton. We have included it in our study and the original identification of this strain done by Manire et al. (2002) based on morphology alone was further confirmed by the sequencing of the rRNA genes (Vinnere, unpublished). So far, to the best of our knowledge, there are no records of C. acutatum causing infections in any other animals or human.
History of the C. acutatum taxon The taxonomy of Colletotrichum, as well as its teleomorph, Glomerella, is controversial and has been the object of many studies, especially during the last couple of decades. There are 18 generic synonyms known for Colletotrichum, and many species of this genus have several synonyms (Sutton, 1992). The champion in this sense is C. gloeosporioides, which alone has as many as 600 specific synonymous epithets (von Arx, 1957). This species is known since 1882, it is very heterogeneous in culture, its morphological features are extremely plastid and its host range is vast – that was the main reason for confusion that yielded such a tremendous amount of described synonymous species based on morphology and host specificity. At the present time, C. gloeosporioides is considered a so-called “group species”, containing several species that are genetically distinct and sharing similar morphology: gray, fast growing mycelium and cylindrical conidia with both ends obtuse (Sutton, 1992; 1998). C. acutatum has a somewhat similar story. First it was described in Australia in 1965, as a causal agent of fruit rot of strawberry, papaw, and tomato (Simmonds, 1965). At that time, Simmonds did not deposit any type 18 material and the name stayed invalid for three years, until he designated the holotype, thereby validating the name (Simmonds, 1968). According to the original definition of the species, the isolates have gray or pink mycelium; conidia are fusiform with both ends acute. The colony color is very variable. Some of the isolates initially are pink and later develop wine red color, others are from light- to dark-gray and never produce pink pigment (Simmonds, 1965; Hindorf 1970, 1973a & b). According to the production of the pigment, the pink form of C. acutatum has been separated into C. acutatum f.sp. chromogenum (Baxter, van der Westhuizen & Eicker, 1983). The main difference from C. gloeosporioides is the slow growth rate (Simmonds,1965), smaller and slenderer conidia and the shape of the conidial endings (Dyko & Mordue 1979). Several other characters, for example colony appearance, production of setae, etc. seem to be not informative, since they are dependant mostly on the culturing conditions (Buddie et al., 1999). During the past decades, there were several reports about finding intermediate isolates of Colletotrichum, which had morphological features fitting to descriptions of two species: C. acutatum and C. gloeosporioides (e.g. van der Aa, Nooderloos & de Gruyter, 1990; Sreenivasaprasad et al., 1996; I). In practice, it seemed that the morphology of C. acutatum appeared more variable than in the original description done by Simmonds (1965). And since C. acutatum is a quarantine object, there was a need for developing reliable methods that are able to make a clear identification of the strains with intermediate morphology. Since the discovery of C. acutatum in 1965, there clearly have been two periods of studies done on this pathogen. Relatively few articles were published in the time period between 1965 and 1980s. Most of them were reports of the presence of this taxon on new hosts and geographic locations (e.g. Hindorf, 1973 a, b & c; McGechan, 1977; Peredo, Osario & Santamaria, 1979); single papers contained descriptions of new formae speciales (Dingley & Gilmour 1972), or concentrated on aspects of chemical control (Peterson, 1973), etc. However, since the beginning of 1990s, there has been a burst of papers dealing with characterization of C. acutatum and differentiating isolates of this species from C. gloeosporioides. This remarkable increment was mostly due to the application of molecular methods to characterization of fungal isolates, which became popular in mycology exactly around that time. At present, despite of all those studies, the question about the species boundaries in C. acutatum (both morphological and molecular) still remains open.
19 Morphological studies C. acutatum versus C. gloeosporioides Sutton (1998) separated these species based on the conidial shape: C. gloeosporioides has cylindrical conidia, but C. acutatum is supposed to have fusiform ones. Many authors have pointed out a high variation in spore shape within both taxa (Smith & Black, 1990; Walker, Nikandrow & Millar, 1991; Lardner et al., 1999), but the problem of identification of the isolates with intermediate morphology, to the best of our knowledge, for the first time was discussed in the publication of van der Aa, Noordeloos and de Gruyter (1990). These authors suggested that C. acutatum was brought to Europe in the late 1970’s from Australia and started to cause leaf curl or leaf crinkle disease of the florist’s anemone, Anemone coronaria. They also proposed that pathogenicity to Anemone could be used as a characteristic feature for separating C. acutatum from C. gloeosporioides, since the isolates non-pathogenic to this plant fit the broader concept of C. gloeosporioides. In their opinion, Sutton (1980) narrowed the original description of Simmonds, especially regarding the conidial width. Besides the conidial characteristics, they also mention the in vitro absence of teleomorph and setae for C. acutatum, color of conidial masses, as well as the differences in the host range. They also pointed out the necessity of using molecular methods for species identification and suggested that the host specificity (biological identification tests) should remain in use as an additional character. Let us look closer and discuss the morphological features of these two species.
Conidial characteristics Shape: C. acutatum conidia should be fusiform or acuminate at least on one end (Fig. 1 A &B) (Simmonds, 1965; Dyko & Mordue, 1971; Sutton, 1998). This has been confirmed in several studies (i.e. Hindorf, 1973 a, b & c; Kuleshrestha, Mathur & Neergaard, 1976; Denoyes & Bauldry, 1995). In our work (I), we have included reference isolates of C. gloeosporioides and C. acutatum in the analysis, as well as the holotype strain (IMI 117617) and one of the paratype strains (IMI 117619) of C. acutatum. The reference isolates were used in different studies by several authors and their identification has been confirmed by various molecular methods (Smith & Black, 1990; Sreenivasaprasad et al., 1996; Freeman et al., 2000). Pictures obtained from scanning electron microscopy (SEM) clearly show that even C. gloeosporioides reference isolates can have conidia that acuminate towards one end (Fig. 1D). Isolates of C. acutatum, including the holotype isolate, show a high level of variation in the conidial shape, and cylindrical conidia are sometimes present in the microscopic slides, as is shown both on pictures from SEM and light microscopy (Fig 1 C, E &F). 20 B
C D
E F Fig. 1. Shape of conidia in C. acutatum and C. gloeosporioides.
A: C. acutatum* B: C. gloeosporioides* C: C. acutatum holotype** D: reference isolate of C. gloeosporioides** E. C. acutatum paratype** F. C. acutatum with intermediate morphology, isolate from Rhododendron sp.**
* Sutton, 1998 **Paper I
21 This phenomenon was already mentioned in the literature on C. gloeosporioides (Davids, Boland & Howitt, 1992; Liyanage, McMillan & Kistler, 1992; Agostini, Timmer & Mitchel, 1992). However, after a closer look at the full set of characters used in those studies (especially the colony color and the growth rate), one would suspect that those particular isolates with intermediate morphology might have belonged to C. acutatum instead. This was later proved by the work of Brown, Sreenivasaprasad and Timmer (1996) by PCR using C. acutatum specific primers.
Dimensions: Following the original descriptions of both species, C. acutatum generally have shorter and more slender conidia, than C. gloeosporioides. The original measurements of conidial length and width, as well as some extreme cases are gathered in Table 1. In some papers there are data about Colletotrichum (C. acutatum in particular) producing so-called secondary conidia - phialoconidia formed in artificial cultures shortly after spore germination (Stoneman, 1898; Baxter, van der Westhuizen & Eicker 1983, Buddie et al., 1999). This type of conidia is generally smaller and more variable in shape (Buddie et al., 1999). Besides, C. acutatum is known for producing conidia directly on the mycelium and not necessarily in the acervuli both in pure cultures and in artificial inoculations (Buddie et al., 1999; IV). Also, these conidia are more variable, than ones produced in the fruiting bodies. And, certainly, the dimensions of the spores produced on the natural substratum might differ from ones present in artificial cultures. This peculiarity is, unfortunately, usually neglected (especially by plant pathologists). The confusion in setting the limits between the species might well be caused by these facts.
Color of conidial masses: Simmonds (1965) pointed out that there are slight differences in the color of spores in mass between C. gloeosporioides var. minor and C. acutatum. He refers to the color of C. acutatum as capucine orange (salmon) and C. gloeosporioides as bitter sweet pink. This is true in most cases, however conidial masses of two C. acutatum isolates used in our study in I were light yellow. Besides, in several studies the color of C. acutatum conidial masses is reported to be “bright orange”, “orange” or “orange-pink” (Smith & Black, 1990; Walker, Nikandrow & Millar, 1991; Lardner et al., 1999; Talhinhas et al., 2002, I). It should be pointed out, that very often no conidial slime could be observed in the cultures due to the production of spores directly on the mycelium.
Appressorial characteristics Size and shape of appressoria until recently was widely used in taxonomic studies of whole genus Colletotrichum. According to Sutton (1998), C. gloeosporioides has slightly larger appressoria than C. acutatum. However, in practice it is very difficult to draw a clear destinction between these 22 species based only on this character (Smith & Black, 1990). Also, Simmonds (1965) mentions that the size and shape of appressoria formed by the several species of Colletotrichum that he used in his study, did not have so many differences that could be “sufficient to warrant a more detailed examination”. He also observed that from all the species he studied, appressoria of C. acutatum were the most regular.
Table 1. Spore dimensions in two species of Colletotrichum
Spore dimensions, Pm Reference
C. acutatum:
8.3-14.4 x 2.5-4 Simmonds, 1965
8-16 x 2.5-4 Dyko & Mordue, 1979
12.3-14.7 x 4.4–5.3 Smith & Black, 1990
10-18 x 2.5– 4.5* Walker, Nikandrow & Millar, 1991
12.5-20(-22.5) x 3-5 Gunnel & Gubler, 1992
10 x 3.5** I
11-15.5 x 3.5-4*** I
10-22.5 x 3-5.5 I, IV
C. gloeosporioides
16-18 x 4-6 Saccardo, 1884
12-22 x 4-6 von Arx, 1957
5.0-47.5 x 2.5-7.5 Mordue, 1971
* living subculture of the holotype isolate, ** dried holotype isolate, *** dried paratype isolate
Production of setae Production of setae was regarded as a diagnostic feature for separation of two genera: Colletotrichum and Gloeosporium before the revision by von Arx (1957). In his work, von Arx regarded the genus Gloeosporium as polymorphic and incorporated several of its species into Colletotrichum. 23 According to von Arx, fungi of the genus Colletotrichum may or may not produce setae. Also, production of setae was mentioned in his description of C. gloeosporioides. While defining a C. acutatum as a new species, Simmonds (1965) did not observe routine production of setae in the acervuli of C. acutatum isolates that he examined (he mentioned, however, scattered poorly developed setae occasionally appearing in culture), and did not mention setae in the description of this taxon. Therefore, absence of setae was used by some authors as one of the features to assist separation of the latter species from C. gloeosporioides for a long time (van der Aa, Nooredloos & de Gruyter, 1990; Smith & Black, 1990). A separate study was done to investigate the factors affecting setae production, and its results were published in Nature (Frost, 1964). The author has clearly demonstrated the effect of air humidity upon the production of setae, namely: more setae were produced at lower relative air humidity. This evidence positively discharges production of setae as a diagnostic feature in separating different taxa. In our work, we have observed production of setae by both species in pure cultures (I).
Temperature response and growth rate C. acutatum generally grows slower, than C. gloeosporioides. It seems to be the only character that is stable and can give reliable results for separation of these species. Many papers, including I, support this observation done by Simmonds (1965) (i.e. Smith & Black, 1990; Bernstein, Zehr & Dean, 1995; Denoyes & Baudry, 1995; Shi et al., 1996; Lardner et al., 1999; Talhinhas et al., 2002; I). The temperature optimum for C. acutatum is 25.0 – 26.5 oC (max. 33.0 oC), and 26.0 – 28.5 oC (max. 35.5 oC) for C. gloeosporioides.
This is just a short overview of the development of morphological studies done on separation of C. gloeosporioides from C. acutatum. As can be seen, many of the characters overlap between these two taxa, creating confusion and making morphological identification of some of the strains, especially ones with intermediate morphology, almost impossible. Therefore, other techniques were developed to aid in correct identification.
Biochemical and molecular studies In 1991, use of isozymes was demonstrated for differentiation of Colletotrichum species pathogenic to strawberry (Bonde, Peterson & Maas 1991). In this work, authors used isozymes for 12 enzymes and 14 putative isozyme loci. The intraspecific variation obtained in this study has demonstrated the importance of isozyme analysis in identification of Colletotrichum species. Later, this technique was used also for species delineation in Colletotrichum, including separation of C. acutatum from C. gloeosporioides (Buddie et al., 1999). 24 Isozyme analysis provides good information about the variation on the protein level, but it does not give any direct information about how variable the genomes are. With the advent of molecular techniques, numerous studies of nucleic acids of different Colletotrichum species have followed. Application of different DNA-based techniques for characterization of different species and populations of C. gloeosporioides and C. acutatum began in the early 1990s. A vast range of different techniques were used by different authors, including: RFLP and DNA-DNA hybridization (Liyanage, McMillan & Kistler, 1992; Bernstein et al., 1995; Brown, Sreenivasaprasad & Timmer, 1996), rDNA and mtDNA RFLP (Sreenivasaprasad, Brown & Mills, 1992; Alahakoon et al., 1994 a & b; Hodson, Mills & Brown, 1993; Buddie et al., 1999; Tahlinhas et al., 2002), RAPD (Alahakoon et al., 1994a, Mills, Sreenivasaprasad & Brown, 1992; Kuramae-Izioka et al., 19997; Lardner et al., 1999), PCR with species-specific primers (Mills, Sreenivasaprasad & Brown, 1992; Martínez-Culebras et al., 2003), arbitrarily-primed PCR and analysis of A+T rich fragments (Freeman, Pham & Rodriguez, 1993; Freeman & Rodriguez, 1995; Freeman & Katan, 1997), sequencing of Domain 2 of 28S rRNA gene (Sheriff et al., 1994; Bailey et al., 1996; Johnston & Jones, 1997), sequencing the ITS1 region of rDNA (Sreenivasaprasad, Brown & Mills, 1992; Sreenivasaprasad et al., 1996 a & b; Saha et al., 2002), AFLP (O’Neil et al., 1997) etc. Among all these molecular tools, the sequencing of the rDNA array seems to be the handiest, and most of the later published papers were based on this region. The ITS1 sequences were found to be informative to separate species within Colletotrichum and were used for inferring phylogenetic relationships between these species (Sreenivasaprasad, Mills & Brown, 1994; Sreenivasaprasad et al., 1996). In the case of some species, like C. coccodes, the phylogenetic groupings based on ITS 1 sequences were in concordance with morphological characteristics (Sreenivasaprasad et al., 1996 a & b), however in the case of C. acutatum and C. gloeosporioides morphology sometimes contradicted the molecular data (Sreenivasaprasad et al., 1996; Yang & Sweetengham, 1998; Nirenberg, Feiler & Hagedorn, 2002). Therefore, other molecular methods, for instance RFLP of A+T – rich fragments, were employed to complement the ITS sequencing data (Martín & García-Figueres, 1999; Talhinhas et al., 2002). To our knowledge, our group was the first to use sequencing of multiple loci for characterization of the isolates of C. acutatum and C. gloeosporioides (I, IV). In our study, we have used nucleotide sequencing data obtained from a portion of the mitochondrial small subunit rDNA (mtSSU) and a fragment of the E-tubulin gene in combination with the widely used ITS sequencing. These genes, as far as we are aware of, were never used for phylogenetic studies in Colletotrichum. The obtained results have shown that sequencing of additional regions strongly supported identification of Colletotrichum isolates as C. acutatum or C. gloeosporioides based of the ITS1 region. The 25 mtSSU gives the best resolution due to the species-specific length of the amplified fragments, therefore the differences between the strains can be picked up already while running the detection gel for the PCR products, thus no additional sequencing might be required. Thus, using this region can assist screening of a large amount of strains and assure reliable identification on the species level (Nguyen, 2002; I, IV).
Sexuality of C. gloeosporioides and C. acutatum Similarly to the anamorph, the taxonomy of the genus Glomerella (the teleomorph of Colletotrichum) is highly confused. Currently, only five species of this taxon are accepted (Sutton, 1992). As in the case of Colletotrichum, several species of Glomerella are often confused with each other due to high similarities in morphology. One of the best-studied species of Glomerella is G. cingulata, the teleomorph of C. gloeosporioides. This taxon is known since 1898, when it was described by Stoneman as Gnomoniopsis cingulata. Later, in 1903, Spaulding and von Schrenk transferred it into the genus Glomerella, adopting the currently used name, G. cingulata (Stonem.) Spauld et v. Schrenk. G. cingulata is known to have both homothallic and heterothallic strains (Wheeler, 1954). Homothallic isolates of C. gloeosporioides quite easily produce the Glomerella state in cultures on artificial substrates (Smith & Black, 1990; Agostini, Timmer & Mitchel, 1992; Cisar et al., 1994; Shabi et al., 1994). Mating type systems in Glomerella are studied quite intensively, although most of the studies were using not only G. cingulata, but also G. graminicola (teleomorph of C. graminicola), as the model (Wheeler, 1954, 1956; Cisar et al., 1994; Cisar & TeBeest, 1999; Vaillancourt & Hanau, 1991, 1999; Vaillancourt et al., 2000). It turns out that Glomerella, to some extent, is also an extreme case regarding fungal mating systems and does not fit any of the common fertility patterns by possessing a phenomenon called “unbalanced heterothallism” (Wheeler 1954, Vaillancourt et al. 2000). In this case, we assume that heterothallic strains might have evolved from homothallic ones through mutations in the genes controlling self-fertility (Vaillancourt & Hanau, 1999, Vaillancourt et al., 2000). This means that if two individuals have mutations in different genes in the homothallic pathway, they are sexually compatible. The difference from true heterothallism is that strains displaying unbalanced heterothallism by crossing can produce a recombinant homothallic progeny, whereas truly heterothallic individuals can only generate heterothallic progeny. If this assumption is correct, both homothallic and heterothallic strains can be present in the same species, as demonstrated in studies on G. cingulata (Wheeler, 1954) and G. graminicola (Vaillancourt et al., 2000). In contradiction, many other fungal taxa are strictly homothallic (Emericella
26 nidulans = anamorph Aspergillus nidulans) or heterothallic (Didymella spp. = anamorph Ascocshyta spp.). Sexual reproduction in fungi is controlled by mating type (MAT) genes. These are uniquely studied, in order to clarify the evolutionary processes driving fungal reproductive strategy as well as fungal sexuality itself (Arie et al., 1997). Studies of these genes are restricted so far to some genera of fungi (including Glomerella), mostly due to the difficulties in cloning of MAT genes from some fungal groups (Arie et al., 1997; Cozijnsen et al., 2000; Turgeon & Yoder, 2000; Yun et al., 2000; Dyer et al., 2001; Zhong & Steffenson, 2001). The sexual state of C. acutatum was not discovered for a very long time. In 1997, the first report about successful experimental mating of C. acutatum and production of the teleomorph appeared (Guerber & Correll, 1997) and in 2001 Glomerella acutata species nova was described (Guerber &Correll, 2001). In both cases, G. acutata was obtained in laboratory crossings of different C. acutatum isolates, meaning that the species is heterothallic. To our knowledge, there still are no records about the presence of G. acutata in nature. In 1999, the first report of single-spore C. acutatum isolates producing sexual state on culture media followed (Lardner et al., 1999), thus suggesting the existence of homothallic strains within this species. One explanation is that G. acutata could also be subjected to unbalanced heterothallism.
Current state of C. acutatum Genetic diversity within C. acutatum sensu lato In 1997-1999, a group of New Zealand researchers did two extensive studies on different morphological groups recognized within C. acutatum and for the first time used the terms “C. acutatum sensu lato” and “C. acutatum sensu Simmonds”, thereby recognizing C. acutatum as “group species” (Johnston & Jones, 1997; Lardner et al., 1999). These studies employed both morphological and molecular analyses, including sequence data of the rDNA D2 region and RAPD data. They were able to divide C. acutatum sensu lato into seven distinct biological groups and for the first time showed close relationship between C. acutatum and the ascomycete Glomerella miyabeana (Fig. 2). Authors also made a comment that at least some of those biological groups within C. acutatum could be regarded as separate taxa, but it is yet to be decided if they should be separate species or subspecies (Lardner et al., 1999; Johnston, 2000). Later works on C. acutatum were concentrated mostly on molecular characterization of the strains originating from different hosts in different countries (Förster & Adaskaveg, 1999; Saha et al., 2002; Tahlinhas et al., 2002; Nguyen, 2002; I; Afanador-Kafuri et al., 2003; II). If combined together, some common trends can be observed. Disregarding the geographic 27 origin C. acutatum isolates are quite divergent, which partially could be explained by the fact that the fungus had a man-driven dispersal as many other plant pathogens. No clear phylogeographic pattern could be inferred from any of the phylogenetic trees based on any of the currently studied DNA regions in this species (I, IV). However, populations of C. acutatum on the same host are usually uniform (Lardner et al., 1999; Talhinhas et al., 2002; Nirenberg, Feiler & Hagedorn, 2002; I) and sometimes the switch to a new host can be traced back in time by using DNA sequencing data (Talhinhas et al., 2002). Reasons for the lack of a general pattern are several, including presence of both asexually and sexually recombining populations in different parts of the world. Besides sexual recombination, in asexual populations certain levels of variation can be achieved through anastomosis and heterocariosis that occur if the strains are vegetatively compatible, in other words, that belong to the same vegetative compatibility groups (VCGs). Studies on VCGs in C. acutatum are quite scarce and mostly concentrated on strains pathogenic to strawberry, anemone and lupine, proving existence of several VCGs with different host and geographic preferences in populations where sexual recombination was not observed (Lardner et al., 1999; Katan, 2000). This question definitely requires more thorough investigation. Strains with intermediate morphology that cannot be reliably identified either as C. acutatum, or as C. gloeosporioides and showing higher genetic affinity to C. acutatum still remain a problem. As it was pointed out by the New Zealand group, they could represent different biological forms within C. acutatum if we accept a wider species concept, that is: C. acutatum sensu lato. But how wide can those species boundaries be? While designing species-specific primers based on the ITS1 sequences, Sreenivasaprasad with colleagues (1992) accepted 6% variation in sequences as a threshold for species in Colletotrichum. Our study on Rhododendron isolates have shown that variation among isolates that are grouping in the C. acutatum sensu lato cluster on the ITS1 tree can reach up to 9.6% (I). Is it still valid to group all those isolates into the same species?
28 Fig. 2. Neighbour-joining tree of C. acutatum sensu lato constructed based on RAPD data (from Lardner et al., 1999)
29 C. acutatum versus C. fructigenum While describing a new species, Simmonds (1965) noticed that the growth rates of C. acutatum at different temperatures resembles those of another fruit-rot causing fungus – Gloeosporium fructigenum, which was used in a similar comparative study on growth rates of different anthracnose-causing fungi by Edgerton (1915). He writes: “In fact, there is nothing in Edgerton’s description to indicate they are not the same organism. However, the spore dimensions usually attributed to G. fructigenum set it apart from C. acutatum and the former perhaps more correctly considered a synonym or form of C. gloeosporioides” (Simmonds, 1965). The history of C. fructigenum goes back to 1856, when this fungus was described by Berkeley as Gloesporium fructigenum (Berkeley, 1856). He also described a very similar fungus two years before that, and called it Septoria rufo-maculans (Berkeley, 1854). Later Berkeley himself renamed it into Ascochyta rufo-maculans, and in 1879 von Tümen placed this species into the genus Gloeosporium (Berkeley, 1860; von Tümen, 1879). Later, this species, G. rufo-maculans, and several other morphologically similar species described by the same Berkeley were combined in one and, despite the priority of G. rufo-maculans, were called G. fructigenum since it was the most widely used name at that time (Berkeley & Curtis, 1874; Saccardo, 1884; Southworth, 1891; Vassiljevsky & Karakulin, 1950). The sexual state of this fungus was described in the U.S. under the name Gnomoniopsis fructigenum and was then renamed into Glomerella rufo-maculans by Spaulding and von Shrenk, which was later considered synonymous to G. cingulata (Saccardo, 1902, 1903, 1904; Shear & Wood, 1913; Vassiljevsky & Karakulin, 1950). This particular sexual stage of G. fructigenum, to our knowledge, has never been found in Europe. In 1950, Vassiljevsky and Karakulin made a new combination and placed G. fructigenum into Colletotrichum, adopting the name C. fructigenum. The description of the species given in the revision by Vassiljevsky and Karakulin (1950) is very similar to C. acutatum, however presence of slightly curved conidia is mentioned – a trait not typical for the latter taxon. In his dissertation, von Arx (1957) put C. fructigenum as a synonym of C. gloeosporioides. But he did not examine the type material, or any pure culture of this fungus. Neither did Shear and Wood (1913) in their monograph on Glomerella, which he refers to. Baxter, van der Westhuizen & Eicker (1983) considered that in several descriptions of C. fructigenum (Vasiljevsky & Karakulin, 1950; Gorter, 1962; Simmonds, 1965; Hindorf, 1973b; Ferraz, 1977), its spore shape and size as well as cultural morphology is very similar to descriptions provided for C. acutatum. Consequently, for the first time, they proposed that these two species were conspecific. A single strain of C. fructigenum was used in the study concerning identification C. acutatum strains based on the ITS1 sequences 30 (Sreenivasaprasad, Mills & Brown, 1994). The particular strain obtained from DSIR culture collection in Auckland, New Zealand, had 97% homology to C. acutatum. Therefore, it was assigned to the latter taxon. The correctness of the original identification of that strain remains doubtful, however. Examination of Berkeley’s type material could help answer the question if C. acutatum and C. fructigenum are conspecific. This research is hampered by the age of the herbarium material and the quality of the specimens (III). To clarify this question, we have examined the authentic material (syntypes) of C. fructigenum identified and deposited by Berkeley himself. Simple morphological analysis revealed that C. fructigenum in no sense can be considered conspecific with C. acutatum nor with C. gloeosporioides. Conidia coming from acervuli in Berkeley’s material are not just clearly curved, but according to the mycological nomenclature should rather be considered falcate. Several less curved and straight conidia were also observed, but the fusiform shape was clearly prevailing. Detailed results of this study can be found in III. This is additional demonstration of usefulness of examining the type material for taxonomic study in fungi, especially fungi of such confusing genera as Colletotrichum.
C. acutatum and the taxonomic position of Glomerella miyabeana As mentioned before, Johnston and his colleagues were the first to make a comment on relatedness of these two taxa, mainly due to similarities in host range and colony appearance, as well as based on molecular evidence (Johnston & Jones, 1997; Lardner et al., 1999). Originally, G. miyabeana was described by Fukushi in 1921 in Sapporo, Japan, under the name Physalospora miyabeana, as the causal agent of willow canker disease and was proven to have a connection with a Gloesoporium anamorph. The disease and the pathogen were further studied by Nattrass (1928), who also was the first to find it in the UK. Based on his observations, Nattrass proposed that the fungus could be closer Fig. 3. Conidia of G. miyabeana, related to Glomerella, than to isolate UPSC 886 (Courtesy Dr. Physalospora. This was later confirmed by Ovidiu Constantinescu) von Arx in his dissertation (1957), where he not only validated the G. miyabeana name, but also mentioned it within aberrant forms of G. cingulata, while still regarding it as a separate species. 31 G. miyabeana was observed on willows in USA (Hepting, 1971), Georgia (former USSR) (Shishkina & Mamukashvili, 1976), later also in New Zealand (Spiers & Hopcroft, 1993) and in Sweden (Astrom & Ramstedt, 1994). For a long time it was believed to be restricted solely to willows, but Johnston & Jones (1997) demonstrated that G. miyabeana can be an opportunistic pathogen also on fruits of other crops, including strawberry, apple, nashi and tomato in cases when its main host, willow, was grown in close contact with orchards. The description of the anamorph, including conidial size and shape done by Nattrass, in our opinion is similar to what is typical for currently recognized C. acutatum sensu lato (Fig. 3). In the publication of Spiers and Hopcroft (1993), production of the pink pigment by anamorphic isolates of G. miyabeana is mentioned – a trait that is typical for C. acutatum, but not for C. gloeosporioides. Besides, spore dimensions of the anamorphic state in both descriptions of Nattrass (1929) and Spiers and Hopcroft (1993) are similar to those observed by isolates of C. acutatum with intermediate morphology. Due to morphological resemblance of G. miyabeana and C. acutatum, mainly because of production of the pink pigment and conidia of variable shape, molecular markers were employed in order to clarify the relationship between these two fungi (Johnston, personal communication). Use of RAPD markers and D2 rDNA sequences gave concordant results and on the constructed phylogenetic trees these taxa appear closely related (Fig. 2), and it became evident that G. miyabeana has much closer genetic affinity to C. acutatum sensu lato, than to G. cingulata (Johnston & Jones, 1997; Lardner et al., 1999, Johnston, 2000; IV).
Is C. acutatum sensu lato a single species? The notion that C. acutatum should possibly be divided into two different species, due to the exceptionally high inter-specific sequence variation within the ITS region, to the best of my knowledge was proposed for the first time by Sreenivasaprasad et al. in 1996. Johnston & Jones (1997) and later also Lardner et al. (1999) have suggested that the biological groups currently recognized within C. acutatum sensu lato should be considered as separate taxa, however they did not propose segregation of any new species. During analysis of the phylogenetic trees generated from the ITS, mtSSU and E-tubulin sequence data, we have observed that C. acutatum sensu lato isolates formed two distinct internal clades with a relatively high bootstrap support, therefore confirming the proposal of above-mentioned authors (Fig. 4) (I, IV). Our observations were later supported also by a study of Martinez-Culebras et al. (2003). Recent studies on sexuality of C. acutatum (Guerber & Correll, 2001) and our sequencing of multiple loci of several strains of G. miyabeana (IV) 32 provided a basis for speculation about the reason for existence of those two groups. G. acutata and the presumably heterothallic isolates producing teleomorph in the laboratory crossings are present in the upper clade of the phylogenetic tree together with the holotype of C. acutatum, but G. miyabeana is grouping together with homothallic isolates from Latvia and New Zealand and also with many other strains where production of the teleomorph was never observed. This remains true for phylogenetic trees obtained from all three loci separately as well as in combination (IV).
Bootstrap BAA67875 53 BAA70884 99 397 C. TUT 5954 99 C. acutatum 94 BAA67886 sensu a BAA70351 Simmonds c Clemson SF21 u 100 Glomerella acutata t 60 Mya663 56 a Mya662 t BAA65797 u 100 m BAA70991 UPSC2066 s UPSC926 86 G. miyabeana e 1117 4 n 1115 1 s 1160 1 u 96 BAA67435
L3 l 66 S2 a t NI90 C. acutatum o 64 UPSC1045 with 67 intermediate S17 morphology S4 80 S7
L1 231 100 64 TN3 C. gloeosporioides 100 FLBB
UPSC2866
S 15
Fig. 4. Phylogenetic tree inferred from sequences of a joint alignment of three loci, numbers above lines correspond to bootstrap values higher than 50% (IV).
33 Available isolates of the G. acutata clade have morphology similar to C. acutatum sensu Simmonds, whereas isolates that comprise the G. miyabeana clade are the ones that are called “the isolates with intermediate morphology”, meeting the criteria of C. acutatum sensu lato. As we conclude in IV, it is likely that G. acutata is a teleomorph of presumably heterothallic isolates of the upper clade of C. acutatum, and G. miyabeana is a teleomorph of the isolates comprising the second, lower clade. This could be a scientific ground for separation of C. acutatum sensu lato into (at least) two separate species. Evidence presented in IV suggest that G. miyabeana indeed is the teleomorph of one of the biological groups recognized within C. acutatum sensu lato. I see two possible solutions for the current confusion concerning the species definition in C. acutatum. As a first option, we may accept the current level of genetic and morphological variation existing within C. acutatum sensu lato. In this case, considering name priority, G. miyabeana should be recognized as the sole teleomorph of C. acutatum sensu lato, G. acutata should be considered synonymous to it, and all biological groups currently recognized within C. acutatum sensu lato should be treated at the sub-species level. Despite the fact that G. acutata is strictly heterothallic (Cuerber & Correl, 2001), G. miyabeana appears to be homothallic. However, as we have discussed above, two other well-studied species of Glomerella, namely G. cingulata & G. graminicola possess the phenomenon of unbalanced heterothallism due to peculiar genetic organization of the mating system, and both homothallic and heterothallic populations are present within both species. In our opinion, this solution requires a very careful consideration, because, as was previously demonstrated, genetic and biological variation within C. acutatum sensu lato is much greater than usually accepted for species in Colletotrichum. Another solution, which I consider much more appealing, is separation of C. acutatum sensu lato into at least two biological species: first – C. acutatum sensu Simmonds, with G. acutata as a teleomorph and the second – strains currently recognized as “C. acutatum with intermediate conidial morphology” with G. miyabeana as the sexual state. With this approach, the taxonomic position of homothallic isolates of C. acutatum sensu lato remains unresolved, because the teleomorph of those isolates is genetically different from both G. miyabeana and G. acutata (IV). Possibly, these isolates should also be described as a separate species. In the second solution, C. acutatum sensu Simmonds would regain its original meaning and status, which, due to all above-mentioned confusion, has been lost. In both cases, the results presented in I and IV, as well as studies by Sreenivasaprasad, et al. (1996), Johnston & Jones (1997), Lardner et al. (1999) and Guerber and Correll (2001) strongly suggest re-defining C. acutatum.
34 Such a revision of C. acutatum sensu lato cannot be the effort of our single scientific group alone, since we can only justify our own point of view and methods of choice. Studies conducted in order to eliminate the existing level of taxonomic confusion should employ as many methods of analysis as possible and as many opinions as available. I am certain that gradual accumulation of information about Colletotrichum genome, and future comparison of complete genomes of different species could help us to understand what is a species definition applicable to this genus.
35 Case No 2: Mycelia Sterilia What are Mycelia Sterilia? By the definition given in a previous edition of the Dictionary of Fungi, Mycelia Sterilia is “an artificial taxonomic group including fungi deficient in production of spores of any kind” (Hawksworth, Sutton & Aisworth, 1983). As we have discussed before, modern fungal systematics is based on morphology of spores and spore bearing structures, so the absence of any of these makes Myceila Sterilia isolates virtually unidentifiable. As a consequence, no scientific name can be given to such isolates, which can result in difficulties with publication. Furthermore, if such isolates possess any sort of biological activities that can appear useful for use by human, registration of the isolates themselves as well as their produced active substances becomes very difficult. Besides registration and nomenclature problems, propagation of sterile isolates, their maintainance and preservation is much more troublesome than for sporulating fungi (Parmeter, 1964; Currah & Tsuneda, 1993). This is a very short list of the reasons why the fungi of this group are usually avoided, and sometimes even neglected, by both mycologists and plant pathologists. As pointed out in the beginning of this chapter, Mycelia Sterilia is an artificial taxonomic unit. Many fungi belonging to different classes of the Fungal Kingdom are lacking spores at certain stages of their development. Therefore, the lack of sporulation is the only unique trait shared by all sterile mycelia. Historically, this group was considered a part of Fungi Imperfecti – another questionable higher taxon of the Kingdom. To simplify the matter, later in the text I will refer to Mycelia Sterilia as “a group”, however this can hardly be justified from the scientific point of view. During the standard isolation procedures of fungi from diverse ecological niches, including various plant parts, the isolates producing no spores during a certain period of time, which varies among different scientific groups, are usually discarded (Parmeter, 1964). However, if we look at the lists of fungal species originating from different plants, miscellaneous Mycelia Sterilia sometimes comprise quite a large portion of the recovered strains. Thus far, we have a very scarce knowledge about the ecological role of sterile isolates, mainly due to the problems with their classification (Waid, 1974; Hall, 1987).
Distribution in nature Fungi of the Mycelia Sterilia group are ubiquitous. They are common inhabitants of soil, plants and plant debris, but most of them prefer decaying
36 wood (Parmeter, 1964; Taylor & Parkinson, 1965; Thornton, 1965; Pugh, 1967; Hall, 1987; Vrålstad, Myhre & Schumacher, 2002). Many sterile fungi are common inhabitants of plant roots, and several of them are endophytes (Hall, 1987; Jumpponen & Trappe, 1998; Hashimoto & Hayakumachi, 2001; Schadt, Mullen & Schmidt, 2000; Mucciarelli et al., 2002; Girlanda, Ghihnone & Luppi, 2002). The term “endophyte” in the thesis is used according to the definition by Petrini (1991), who considered a fungus being an endophyte if it is able “asymptomatically colonize the living, internal tissues of their hosts”. Certain Mycelia Sterilia are routinely isolated from household environments (Ebner, Haselwandter & Frank, 1992; Strachan et al., 1990). Several fungi of this group are known to be associated with eye and skin infections in humans (Ando & Takatori, 1982; Khairallah, Byrne & Tabbara, 1992), human allergies (Ebner, Haselwandter & Frank, 1992; Kwaasi et al., 1998) and animal mycoses (Singh & Singh, 1969; Villa, 1979; Duarte et al., 2001).
Importance of plant-associated Mycelia Sterilia From the very few available studies performed by different scientific groups, we know that among the fungi of this group there are both plant deleterious and plant growth promoting ones (Jacobs, 1994). Among sterile fungi, there are several well-known plant pathogens of high economical importance (Agrios, 1997). One of them is Rhizoctonia solani (teleomorph Thanatephorus cucumeris), a difficult-to-control fungus, responsible for severe yield losses in potatoes, cereals, oil-seed rape and other agricultural crops worldwide. Another example of deleterious Mycelia Sterilia is Sclerotium rolfsii (teleomorph Athelia rolfsii), the causal agent of southern blight on a great range of agricultural crops. There are also reports of less common plant pathogenic sterile fungi (e.g. Howard, Conway & Albregts, 1977; Kaiser, et. al. 1987; Jumpponen & Trappe, 1998; Harveson, 2002; V). On the other hand, several sterile isolates have shown good biocontrol abilities against root pathogens of agricultural crops (De La Cruz & Hubbel, 1975; Martin, Abawi, & Hoch, 1984; Narita & Suzui, 1991; Prashar, Singhan & Hooda, 1992; Garsoni, Stegman-DeGurfunkel & Fortugno, 1993; Shivanna, Meere & Hyakumachi, 1994; Vinnere et al., unpublished). One of the best-know examples is Sterile Red Fungus isolated from Australia, which has potential for biological control of Gaeumannomyces graminis var. tritici – a pathogen of worldwide importance, the causal agent of take-all disease (Dewan & Sivasithamparam, 1988, 1989a, b, c, 1990, 1991; DeJong et al., 1993; Rowland et al., 1994; Shankar, Kurtboke & Sivasithamparam, 1994; Aberra, Seah & Sivasithamparam, 1998; Sivasithamparam, 1998). Besides studies on pronounced plant pathogens and plant growth promoters, sterile fungi are becoming more and more popular subject of ecological studies 37 (Shivanna et al., 1995; Hashimoto & Hyakumachi, 2001; Yanna & Hyde, 2001). Search for novel drugs to cure untreatable diseases is another recent opening in science, and there is evidence that some Mycelia Sterilia could contribute to this research (e.g. Huang et al., 1995; Fujita et al., 1996, Vinnere et al., unpublished). The ecological role of the Mycelia Sterilia definitely cannot be generalized and it should be studied separately in each individual case. There are studies, showing that some of the sterile fungi may play a role as mycorrhiza in plant families that usually lack it (e.g. Brassicaceae) (Barrow, 1995; Horton, Cázares & Bruns, 1998). Such isolates are able to cause asymptomatic infections of roots and to colonize root cortex, and in some cases also the stele (Sivasithamparam, 1998). The beneficial effect exhibited by such isolates was the subject of several studies (Deacon, 1981; Wong, 1981; Newsham, 1999; Worth, 2002). It was speculated that, among other cortical fungi, Mycelia Sterilia could contribute to plant fitness, including its defense against pathogens (Marx, 1969; Richard, Fortin & Fortin, 1971; Deacon, 1980; Wong, 1981), improving nutrition conditions (Newsham, Fitter & Watkinson, 1995; Worth, 2002), fecundity (Newsham, Fitter & Watkinson, 1994), etc.
Our isolation and screening strategy Papers concentrating on studies of Mycelia Sterilia exclusively are few. Thus, I would like to describe our approach in detail. The original aim of the study was to discover sterile fungi that could be used as agents of biocontrol against soil-borne diseases of agricultural plants with later attempt to clarify their taxonomic position. In our opinion, our approach had to be restricted and target-oriented, thus we performed isolations only from plant roots, believing that we have the best chance to isolate potential biocontrol agents from the site of their saprophytic competence. First, we performed a large-scale isolation aiming for strains that live inside the cortical tissues as well as beyond the Casparian belt, and therefore, presumably, are endophytic. Collections of isolates were performed in Australia, Latvia and Sweden, from both agricultural crops and wild plant species. The collected roots were surface-sterilized with 1.25% NaOCl (sodium hypochlorite) for 1-3 minutes in order to ensure elimination of the fungi on the root surface. Small pieces of root tissue were transferred onto water agar amended with lactic acid (in order to minimize bacterial contamination) and the advancing fungal mycelia were isolated in pure cultures. Two runs of screening for sporulation were done: first of all, the fungal cultures were grown on PDA (Potato Dextrose Agar), a medium routinely used both by plant pathologists and mycologists. Cultures starting to sporulate after one month of incubation under laboratory conditions were discarded. Isolates failing to produce any kind of sporulation were taken into 38 the second round of the screening. These fungi were grown on a set of media, routinely used for such purpose: Malt Extract Agar, Corn Meal Agar, Oat Meal Agar, Water Agar and Potato-Carrot Agar (Smith & Onions, 1994). Besides standard media, fungal isolates were also grown on Water Agar and Minimal Medium (Cove, 1966) plates containing birch toothpicks and sterilized lupin stems (Dhingra & Sinclair, 1986). All cultures were grown in duplicates: one of each was grown under standard laboratory conditions, at approx. 12 hours of day light and 12 hours of darkness, second replica were placed under constant near-UV light (Phillips, TL20W/08). Isolates that remained sterile after two months of treatment were considered sterile. Some of the isolates produced very scarce sporulation or begun to sporulate vary late during the experiment. Such strains were considered slowly-sporulating, and we still decided to include them in further biotests, believing that without a special treatment they could have been considered sterile. Out of approximately 3000 initially isolated fungal cultures, 1220 were slowly sporulating, and only 53 so far are considered truly sterile. After discarding fast-sporulating isolates, sterile ones were used in greenhouse experiments, as described in V and only isolates having a pronounced effect on plants were selected for further study. During those screenings, we found several strains which had a clear deleterious effect on the test plants (V, VI), as well as strains that were obviously plant growth promoting (Vinnere et al., unpublished). In further studies on characterization of biologically active Mycelia Sterilia we decided to concentrate on three isolates only, which in our opinion were the most interesting among the fungi we studied. One of them, No 3034 in our collection, was a strong pathogen on all crops tested (V, VI), and two others, No 3035 and 3041, promoted root development in wheat and oats (Vinnere et al., unpublished).
Taxonomical studies of Mycelia Sterilia As mentioned before, the classification of fungi is based manly on the morphology of reproductive structures, which Mycelia Sterilia lack. Interested readers can be referred to an excellent review by Parmeter (1964), which describes the problems of taxonomy of sterile fungi. Although Mycelia Sterilia have been and remain an enormously difficult group for taxonomists, there have been many attempts to utilize the classic mycological approaches, which, in combination, can bring a better understanding of possibile of classification of sterile mycelia. Synthetic use of morphological, biochemical and molecular traits seems to be the only opportunity to discriminate among sterile isolates at our current state of knowledge.
39 Morphology When dealing with sterile fungi, a researcher can confidently assign the isolates only to one of the classes of the Fungal Kingdom - Ascomycetes or Basidiomycetes, using the very few mycelial features that are available. Two of the principal differences between mycelia of fungi belonging to these classes are: i) the structure of hyphal septa (simple septum in Ascomycetes and dolipore septum in Basidiomycetes), and ii) presence or absence of clamp connections, which are a characteristic feature of Basidiomycetes (Alexopulos, Mims & Blackwell, 1996). The fact that the nature of septation can be diagnostic and that presence of a septal pore apparatus indicates Basidiomycete mycelium, was proposed in 1962 by Moore and McAlear. The techniques for studies of hyphal septation are rather easy, and can be performed by using simple light microscopy and/or conventional stains (tripan blue), if hyphal diameter is large enough, or otherwise with a bit more sophisticated in vivo staining with fluorescent dyes (Hoescht Dye 33258 pH 10.5) with subsequent fluorescent microscopy (Yang, Sivasithamparam & O’Brien, 1991). The only constraint in studies of septa in fungi is the age of the mycelium, since the dolipore septum can be visualized only in young living hyphae. The second character, presence of clamp connections, can be easily observed under a simple light microscope. Clamps appear in Basidiomycetes after the nuclear division, and with some exceptions are usually present in most fungi of this class. However, clamp connections are never observed in one of the most studied sterile fungi, Rhizoctonia solani (Domsch, Gams & Anderson, 1980). Combination of presence of dolipore septum and presence or absence of clamp connections can be used as important features for separation of the sterile basidiomycetous isolates. Besides septation and clamps, there is a range of hyphal characters that can be informative for characterization of sterile strains. These include presence of so called moniloid cells (enlarged hyphae of irregular shape), formation of sclerotia, differences in hyphal diameter, length, character of hyphal branching, overall colony appearance, margins, texture, color, growth rate, temperature response, etc. (Nobles, 1965, 1971; Stalpers, 1978; Desjardin, 1990). Most of these features, however, are dependent on growth conditions, such as media composition, light intensity, etc. Although quite variable, these characters were informative enough to tell apart isolates of Sterile White Basidiomycete in V. It is common knowledge that many Basidiomycetes are unable to produce spores under laboratory conditions. Several diagnostic keys were developed, based on the previously mentioned morphological characters, aiming to assist identification of the sterile mycelia of some Basidiomycetes, and several of them are still in use. The best examples of such keys are the ones by Nobles (1965) and Stalpers (1978). Moreover, Warcup alone and with his colleagues (e.g. Warcup, 1959; Warcup & Talbot, 1962), produced several 40 comprehensive descriptions of the most abundant sterile fungi that can be used quite successfully.
Biochemical studies Simple chemical tests for many of the non-sporulating isolates especially the ones belonging to the Basidiomycetes are widely used. These include color reactions in response to adding certain chemicals (peroxidase, KOH, dyes, etc.: Stalpers, 1978; Desjardin, 1990). Isozyme analysis is also frequently employed for characterization of sterile strains especially ones belonging to the genus Rhizoctonia (Sweetingham, Cruickshank & Wong, 1986; Yang et al., 1994; Worth, 2002).
Molecular analyses Similarly to the case of Colletotrichum, various analyses of the DNA are used as a powerful tool for separation of sterile strains and their tentative identification, especially in such economically important sterile fungi as Rhizoctonia (e.g. Cubeta et al., 1991; Liu, Domier & Sinclair, 1993, 1995; Bryan, Daniels & Osbourn, 1995; Mazzola, Wong & Cook, 1996). It is no surprise that rDNA sequencing data are widely used for this purpose. But what is the best region for identification of Mycelia Sterilia? The answer is, in my opinion, the most abundant sequenced fungal regions contained in the Gen Bank. ITS regions in most of the known fungi are too variable and can help us only to group closely related isolates; 5.8S, on the other hand, is too conserved, as we realized during our work on VI. The two remaining genes of the array are 18S and 25-28S. On which of these is better, the opinions of scientists split and which region is used seems to be dependent on traditions followed in different scientific groups. Overall, it looks like most work on sequencing of the 25S-28S is done on Basidiomycetes (Vilgalys & Sun, 1994; Hopple & Vilgalys, 1997; Moncalvo et al., 2000; 2002). Sequences of the small subunit rRNA gene have been conducted on both Basidiomycetes and Ascomycetes (Bruns & Szaro, 1992; Berbee, 1996; Berbee, Carmean & Winka, 2000). Despite all difficulties in identification of sterile strains, there has been several works attempting to resolve taxonomic position of biologically active (deleterious or benefitial) Mycelia Sterilia, and as it was expected, all of them employ rDNA analysis for this particular purpose. Several sterile fungi have been tentatively identified based on the ITS region (Schadt, Mullen & Schmidt, 2001; Girlanda, Ghignone & Luppi, 2002; Vrålstad, Myhre & Schumacher, 2002; Vinnere et al., unpublished), small subunit rRNA gene (Jumpponen & Trappe, 1998, Mucciarelli et al., 2001; Girlanda, Ghignone & Luppi, 2002; VI) and large subunit rRNA gene (Klonowska et al., 2003; VI)
41 Species concept in Mycelia Sterilia There are currently at least 28 genera and around 200 species of Mycelia Sterilia known to science according to the current edition of the Dictionary of Fungi (Kirk et al., 2001). However, it is very difficult to justify these numbers. Some of these sterile mycelia were found to be just a mycelial stage of already known fungi with mainly basidiomycetous teleomorphs (including such well-known examples as Rhizoctonia and Sclerotium), others fail to sporulate on artificial substrata, etc. The structures observed in vegetative fungal thallus are usually too few to create a reliable key system for separation of different mycelia into species. Besides, we do not know the level of variation exhibited by the isolates belonging to the same species of Mycelia Sterilia, we even do not know if this variation level is the same among different species of sterile fungi. Also, we do not know how unique certain traits are (Parmeter, 1964). Historically, the tendency very often was to name sterile isolates by the closest (from researcher’s subjective point of view) species of sterile fungi (i.e. Howard, Conway & Albregts, 1977). As was already mentioned several times, accumulation of DNA sequencing data in the future can shed some light upon problems of systematics of Mycelia Sterilia and will definitely lead to revision of several existing taxa. Based on experience gained over several years of work with sterile isolates, with a certain degree of confidence I can say that induction of spore production in sterile isolates can be achieved in most cases (Vinnere et al., unpublished). The only serious restriction is our lack of knowledge of fungal biological requirements for the process of sporulation; therefore, the hunt for fungal spores can take quite a long time and substantial resources, spent in search for the optimal combination of various methods and conditions. As was pointed out by Parmeter (1964), growing interest in studies of fungi from unique environments, as well as perfection of isolation and handling techniques are likely to increase the overall number of Mycelia Sterilia taxa, and, hopefully, our knowledge of these fungi.
New biologically active Mycelia Sterilia found in this study and remarks on their taxonomic position Novel plant pathogens Several fungi within Mycelia Sterilia, which are pathogenic to agricultural crop plants and most of those belong to Basidiomycetes, including such well- known genera as Rhizoctonia and Sclerotium. Since these two are the most wide-spread plant pathogenic sterile fungi, many diseases caused by mycelia with similar morphology are probably wrongfully assigned to these two genera (Parmeter, 1964). A sterile white basidiomycete isolated during our study from roots of buffalo grass (Stenotaphrum secundatum) in Perth, Western Australia, was
42 found to be pathogenic to 12 different plant genera (V). While making a literature search on the subject, we have found that there were several reports of incidence of a similar plant pathogen in North America. A fungus originally called SWB (Sterile White Basidiomycete) was identified as the causal agent of root rot of snap bean (Phaseolus vulgaris) in central Florida. Later, a similar fungus was found also in coastal areas of Georgia (US) (Sumner, Bell & Huber 1979; Bell & Sumner 1984), and then in Puerto Rico (Kaiser, et. al. 1987), and finally in Western Nebrasca (Harveson, 2002). The host range of that pathogen includes plants of at least 16 different genera, including several economically important legumes and cereals. Comparisons of the isolates done in previous studies have shown that the isolates have very similar morphology and several independent researchers have concluded that those fungi are probably identical. Initially, the SWB fungus was compared to both Rhizoctonia and Sclerotium. It is even still kept in the American Type Culture Collection (ATCC) under the name Rhizoctonia sp. However, cultural and enzymatic studies have shown that the SWB isolate is quite different from either of those well-known sterile plant pathogens, i.e. it differed from Rhizoctonia by having clamp connections, and from Sclerotium by being unable to produce sclerotia and by its aminopeptidase profile (Howard, Conway & Albregts, 1987; Sumner, Bell & Huber, 1979). Later studies aiming at inducing sporulation in several SWB isolates have revealed that the fungus belongs to the genus Marasmius (Baird, Wilson & Sumner, 1992). However, the fruiting bodies (teleomorph) were produced only by one of the studied isolates and interestingly, they belonged to two different species of Marasmius – M. graminum and M. rotula. Therefore, authors suggested that SWB possibly represents a complex of several fungal species, and maybe even fungal genera not yet known (Baird, Wilson & Sumner, 1992). A study of unusual cases of maize stalk rot in Queensland has demonstrated that the disease was caused by two species of Marasmius, namely M. sacchari var. hawaiiensis and M. graminum var. brevispora. Symptoms reported in that particular paper were very similar to ones caused by both American and Australian strains of SWB (Pont, 1973). A similar fungus was also reported to cause root-rot disease in maize in Egypt (Sabet, Samra & Abdel-Azim, 1968). The fungus produced symptoms very similar to what was observed for the American SWB strain and for our Australian isolate, but the Egyptian fungus had several distinctive features: i) it did not grow well on artificial substrates, unless yeast extract was added; ii) it was producing chlamidospores (which were never observed neither for the American, nor for the Australian strains); iii) attempts to induce sporulation were successful, but the spores could belong to either Omphalina, Gerronema, Mycena or Clitocybe (from which fungi of the genus Omphalina are known to cause root rots in tropical areas) (Sabet, Samra & Abdel-Azim, 1968). 43 Being unable to induce sporulation in our SWB isolate (code 3034 in our collection), we acquired the original SWB strain originally isolated from snap been in Florida (ATCC 28344), used in studies by Howard, Conway & Albregts (1977) and Sumner, Bell & Huber (1979). Both isolates had slightly different colony morphology (V), therefore we suspected from the very beginning that they might not be the same. Since the American isolate was kept on artificial media for almost 30 years, we made a passage through a susceptible host (maize) and were using a freshly re-isolated pathogenic strain for comparison with the Australian isolate 3034. Strains were compared morphologically, using overall colony appearance, hyphal diameter, growth rate, presence/absence of clamp connections and moniloid cells, amount of nuclei in the cells, temperature response and ability to anastomose. Both isolates had clamp connections, binucleate mycelia, moniloid cells, rhizomorph-like structures, but other characteristics were clearly different. The American isolate had thicker hyphae than the Australian one, it grew slightly slower and had different temperature maximum. Isolates also failed to anastomose (V). Both isolates were also compared by host range, ability to cause infection at different levels of inoculum, and by the way they infected the root tissues. Together, these studies have shown that the fungi are clearly different (V). Finally, when comparison of most of phenotypic traits suggested that the fungi were not the same, molecular analysis was used in order to confirm our previous observations and to make an attempt to identify the sterile isolate originating from Australia. For this reason, we have used sequencing of both small and large subunit rRNA genes, as well as of the ITS region (VI). As expected, the American and Australian isolates were not the same. But we encountered some problems while trying to make a more precise identification of both strains. It was clear from all the studied regions that the American isolate does belong to the genus Marasmius, as proposed by Baird, Wilson & Sumner, 1992. However, comparison of our data with the sequences of the 25S available in the GenBank, concluded that the ATCC 28344 strain is closely related or identical to M. graminum, but not M. rotula (VI). Several species of Marasmius are known to be plant pathogenic (Baird, Wilson & Sumner, 1992). One of the best-known examples is M. oreades, the causal agent of “fairy rings” in turf grasses (Singer, 1975). Another example is M. sacchari, which is an important pathogen of sugar cane. Other species of Marasmius (e.g. M. tritici) are usually associated with roots of different grasses, but have never been proven to be pathogenic (Sabet, Samra and Abdel-Azim, 1968; Pont, 1973; Singer, 1975). As seen from the phylogenetic trees (VI), the genus Marasmius is not well resolved, and is polyphyletic. While studying the literature on this genus, one can notice that there still is some confusion about taxonomy of Marasmius and other closely related genera. The generic concept of
44 Marasmius as well as relationships between the species of this genus is the object of several independent studies (Gilliam, 1976; Desjardin, 1990). But what about the 3034 strain? Overall, the 25S rRNA gene seems to be the most popular region for taxonomic studies in Basidiomycetes, therefore also the Australian isolate could be identified, at least to a generic level, based on analysis of sequences of fungi representing 154 species of Agaricales. The closest match was with two different species of Campanella: C. subdendrophora and Campanella sp. (VI). However, no sequences for ITS or 18S rDNA of these fungi were available in GenBank. Therefore, to check if our assumption is correct, we have purchased a strain of C. subdendrophora from ATCC (ATCC 42449). Similarly to the data obtained from 25S rRNA gene, ITS and 18S sequences showed high level of similarity to our isolate and the reference strain of Campanella. However, sequences of the 5.8S rRNA gene of these two fungi were different at 4 bp (VI). Based on these data, we suggested that our isolate 3034 might belong to this genus or be closely related to it. Usually, fungi of the genus Campanella are saprophytic inhabitants of decaying wood in tropical areas, but there are some exceptions (Singer, 1986). Recently, several species of Campanella were reported in temperate regions, and some species are found associated with grasses (Redhead, 1974; Singer, 1986). However, to our knowledge there are no reports about plant pathogenic species of Campanella. C. subdendrophora, which was used as a reference isolate for our morphological study originates from stalks of grass from Canada, so it belongs to one of several temperate species of this genus (Redhead, 1974). The choice of this particular isolate was done based only on 25S rDNA sequence similarities. During our study, we did not encounter any pure cultures of tropical and/or Australian species of Campanella, despite contacts with several culture collections. The sterile white basidiomycete strain 3034 with high affiliation to the genus Campanella, seems to be a novel plant pathogen. Although it was isolated from symptomless roots of buffalo grass, it causes severe damage to every crop that it infects (V). This far, we have observed this disease only in the greenhouse. It could possibly be that buffalo grass is immune to this particular fungus and just harbors it in its roots. The possibility remains that the fungus is able to cause disease in the field, but it might be routinely mistaken for Rhizoctonia due to high resemblance of the induced symptoms (V). As follows from several reports of Marasmius-caused infections, those are considered unusual, and local outbreaks sometimes are caused by specific weather conditions, e.g. draught (Pont, 1973). Therefore it may happen that the disease caused by the fungus that we describe in V & VI is overlooked or potentially dangerous under specific climatic conditions, especially if the host range and caused mortality rate is taken into account.
45 Biocontrol agents During the initial screening of the isolates on plants of different species, we came across several sterile and slowly sporulating isolates with similar morphology. Several isolates of this group seemed to cause syptomless infections on roots of wheat, and some of them, like the isolates 3035 and 3041, had a slight plant growth promoting effect. Later greenhouse tests aiming for detection of antagonistic activity against Gaeumannomyces graminis f. sp. avenae have revealed that the isolate 3035 was also able to protect cereals from this dangerous pathogen (Vinnere et al., unpublished). Therefore, we decided to also investigate the mode of action that could be involved in this process. First, the isolates 3035 and 3041 were re-isolated from the roots showing no signs of any infection (Vinnere et al., unpublished). This could mean that they are able to colonize root tissue and possibly compete with the pathogen for infection sites. Also, we decided to check if any secondary metabolites excreted by these fungi can have antifungal effects. A range of plant and human pathogenic fungi, one species of yeast, one gram-negative and one gram-positive bacterium were selected for biotests (Vinnere et al., unpublished). Several HPLC fractions of the fungal supernatant have shown strong activity against all of the studied microorganisms. Work on purification of the active substances as well as structure elucidation is currently under way in collaboration with the Dept. of Chemistry, SLU. There are early indications of presence of antifungal compounds of terpenoid nature produced by the 3035 isolate. Since we knew that these slowly sporulating fungi possess interesting antimicrobial features, we also needed to make a tentative identification of the most interesting isolates. For this reason, both morphological and molecular studies were performed. A vast range of media and growth conditions were used to induce sporulation of 3035 and 3041. The 3041 isolate readily produced fertile pycnidia under direct sunlight or near-UV light. The type of pycnidia, as well as the structure of conidiogenous cells strongly suggested that this isolate belongs to the genus Phoma (Vinnere et al., unpublished). The isolate 3035 was much more reluctant to form any kind of spores. We have observed a single pycnidium-like structure once on Potato-Carrot Agar plates, but spores were never produced. However, when it was placed on low-nutrient agar containing sterile birch toothpicks, it finally started to sporulate and we were able to examine pycnidia, conidia and conidiogenous cells. Morphologically these were very similar to the 3041 isolate. However, overall colony appearance was quite different. The isolate 3035 had intense red pigment diffusing into the agar; it also produced droplets of exudates of the same color on top of the cultures. Sequencing of the ITS region and first 500 bp of the LSU rDNA have shown that the isolates in fact belong to the same species, since there were different only at 6 bp. A BLAST search in the GenBank database gave closest hits to species 46 of Ampelomyces, Epicoccum, Phoma and Leptosphaeria. Leptosphaeria is known as the teleomorph of several species of Phoma and Ampelomyces (Fig. 5). This is not suprpising, since Arenal et al. (2000) have recently demonstrated that E. nigrum and P. epicoccina indeed belong to the same biological species based on ITS sequencing data and therefore should be considered syanamorphic. Besides, recent studies on Ampelomyces have shown high genetic similarity of two currently recognized species of this anamorphic genus to other species of Phoma, as well as pointed out possible confusion in distinguishing among these two genera (Kiss & Nakasone, 1998; Sullivan & White, 2000). Although being morphologically similar to P. epicoccina, our slowly sporulating Phoma-like isolates 3035 and 3041 did not produce an Epicoccum state in culture, even under conditions favourable for its development, as described by Domsch, Gams & Anderson (1980). Besides, after inducing sporulation in the isolates 3035 and 3041, their pycnidia were clearly sessile (Vinnere et al., unpublished), which is typical for the genus Phoma in contrary to fungi of the genus Ampelomyces, which are known to have stipitate pycnidia (Sutton, 1980). Moreover, Ampelomyces occupies a very restricted ecological niche being inhabitant of plant phylloplane and mycoparasite of powdery mildews (Sutton, 1980), whereas the slowly sporulating isolates 3035 and 3041 were isolated from plant roots. All the above-mentioned pieces of evidence suggest that the latter isolates indeed belong to the genus Phoma and are different from Ampelomyces and P. epicoccina/E.nigrum. Further identification of those isolates to the species level, however, has not been achieved, this matter requires further investigations.
The taxonomy of Phoma is puzzling and difficult. The phenotypical features that are used to separate species within this genus usually are: production and type of chlamydospores, sclerotia, Epicoccum state, color of cultures and zonation, colour reaction with NaOH and KOH, size and presence/absence of septation in conidia. Currently, there are more than 2000 species described within this genus (Sutton, 1998). As in the case of Colletotrichum, most of them were described based on minor morphological differences and host affinity. There are several fundamental works by Boerema and his co-workers, who have been trying to create a reliable identification system for Phoma isolates grown in culture (Boerema, Dorenbosch & van Kestern, 1965, 1968, 1971, 1973, 1977; Boerema & Bollen, 1975). His keys currently are the most widely used for differentiation the Phoma species.
47 Fig. 5. Phylogram of the ITS region of isolates representing relationships among biologically active Phoma-like isolates 3041 and 3035 and several species of Phoma and other closely related genera. Numbers above branches correspond to bootstrap values higher than 50%.
The ecological niche occupied by our biologically active Phoma sp. isolates is quite unusual for fungi of this genus. Phoma are usually saprotrophs or plant parasites, which prefer to occupy above-ground plant parts or plant debris in the soil, but they can be rarely found in plant roots (Domsch, Gams & Anderson, 1980). There is a species of Phoma isolated from rizosphere of wheat in Western Australia, namely P. chryzanthemicola, but its morphological features differed from what we observed in our slowly 48 sporulating isolates. There have been reports on Phoma-like sterile isolates being inhabitants of diverse environments including soil, plants as well as tropical waters (Hyde, 1986; Sugano et al., 1991, Howlett et al., 2001; Pelaez et al., 1998). Several strains of Phoma-like Mycelia Sterilia have been demonstrated to possess plant growth-promoting properties, as well as the ability to control fungal and bacterial infections and certain weeds (Pelaez et al., 1998; Koike et al., 2001; Neumann & Boland, 2002). Different modes of action such as mycoparasitism (Sullivan & White, 2000), induced systemic resistance (Koike et al., 2001) and antagonism including production of secondary metabolites are usually implicated in their biological activity and/or antagonistic properties (Brown et al., 1987; Pelaez et al., 1998). The mode of action of biologically active Phoma and Phoma-like sterile strains has been a subject of several studies. These fungi are common producers of various important secondary metabolites (Liu et al., 2001). Non-pathogenic isolates of this group were recently demonstrated to produce divergent plant antimicrobial metabolites (e.g. Sugano et al., 1991, 1996; Dawson et al., 1992; Che, Gloer & Wicklow, 2002). Besides secondary metabolites that can be used in agriculture for controlling plant diseases and weeds, or for promoting plant growth, several Phoma-like sterile strains have been found to produce compounds of interest in human medicine. Such metabolites include antifungal compounds (Kamigiri et al., 2002; Koji et al., 2003; Osterhage et al., 2003; Vinnere et al., unpublished), as well as anti- cancer and anti-viral agents (Singh et al., 1998; Takafumi et al., 2000), etc. Moreover, from a taxonomic point of view, secondary metabolite profiles are used both to distinguish terrestrial from marine Phoma species (Osterhage et al., 2000) and to differentiate among phytopathogenic terrestrial species (Soledage et al, 2000).
The problem of correct identification of biologically active Phoma-like sterile or slowly sporulating strains remains unsolved. Even if production of spores has been achieved, it is very difficult to assign the isolate to any certain species, mainly due to overlapping descriptions of species in Phoma. Many biologically active Phoma-like fungi have been isolated from rather exotic environments or locations, therefore they might represent novel, yet undescribed species (e.g. Koji et al., 2002; Yamaguchi et al., 2002; Vinnere et al., unpublished). Sequences of rDNA of various species of Phoma available in the GenBank for comparison are often unreliable (Bridge et al., 2003), which makes correct identification of important strains extremely difficult. Comparison of the biologically active Phoma-like isolates 3041 and 3035 with several type materials of various Phoma species should be performed for further identification.
49 General remarks, or what did I learn?
No secret, that the best person to do any job is the person who is educated and experienced for exactly this particular kind of job. If a tailor will start making jewelry and a painter will suddenly decide to make shoes, what result can one expect? But strangely enough, there are many examples of non-mycologists trying to do the job that only a qualified mycologist can succeed with. As the result of this, we have plenty of fungal species described based only on some minor morphological differences without taking into account all the peculiarities of fungal lifestyle, biology, phylogeny, etc. Other important points, like using holotypes or other type specimens for taxonomic studies, establishing the anamorph-teleomorph connection, collecting representative samples, etc. are very frequently neglected. Loosing mycological traditions is one point I observed while compiling the literature for this thesis. Another, not less important, is misusing DNA sequencing analysis, a sin performed also by some mycologists. Any person who used GenBank at least once knows how many sequences of wrongly identified taxa are in there, due to lack of knowledge, ignorance, or just old data that need to be revised. Besides problems with correct identification, there are also many bad sequences out there, e.g. chimeric sequences, sequences containing vectors, unresolved base pairs, as well as wrongly identified region boundaries or even wrongly identified regions. According to one of the recent reviews, around 20% of GenBank sequences contain errors (Bridge et al., 2003).
After several years of being subjected to the problem of identifying fungal species, I learnt that there are plenty of questions that cannot be answered at our current state of knowledge, no matter which system I was dealing with: over-studied Colletotrichum or understudied Mycelia Sterilia. What is the true fungal species in nature? What is the correct definition that should be given to it? Does it differ from our anthropomorphic species concept? Is there such a thing as the “fungal species” and do we really need this concept or do we just make our life more difficult by trying to classify things that have no real meaning or reason? Being honest, I do not know and am not sure if anyone can give a proper answer. How many fungal species are there? Presuming that only around 1/5 of all fungal species are known to mankind (estimation by Hawksworth, 1991), 50 how can we be sure that our systematic models built up on the present data can be extrapolated to all fungal species existing in nature? While using molecular techniques, everyone faces the reality that a vast amount of information is still lacking, therefore comparison only with existing data cannot be complete and, even, might be completely misleading or false. Accumulating knowledge is an extremely important thing, which can help us to answer at least part of the above-mentioned questions. Educating specialists in mycology, who can combine knowledge of several aspects of fungal systematics, and not just simple molecular techniques, is an important issue. Broadening our horizons, being able to accept our mistakes and also other people’s opinion also counts. This work that I have performed and presented in this thesis is just an attempt to understand how the things work or might work in the real life. Although it consists of sometimes very loosely connected bits and pieces of information, it is still a part of a search for the answer…
51 Conclusions
x The most correct approach to delineation of species of anamorphic fungi is one that combines as many aspects of knowledge as possible, including studies of morphological, molecular, biochemical, and other traits. x Morphological features cannot be underestimated and should be used along with molecular data for complete species identification in fungi. x Use of the type and/or authentic material has a great value in taxonomic studies of any groups of fungi.
x G. miyabeana is the sexual state of one of the biological groups of C. acutatum. x C. acutatum sensu lato should possibly be divided into two species. x C. fructigenum cannot be synonymous to C. gloeosporioides or C. acutatum and should still be considered a valid taxon until proven otherwise.
x There are many understudied (and possibly even novel for science) fungal organisms, which belong to the Mycelia Sterilia group; among them there are both plant deleterious and plant growth promoting strains. x The plant pathogenic Sterile White Basidiomycete isolate originating from Australia is closely related to the teleomorphic genus Campanella, where, to our knowledge, no plant pathogenic species were previously reported. x The plant growth promoting isolate 3035 belongs to the anamorphic genus Phoma and has a strong antimicrobial activity.
52 Acknowledgements
There are many people without whom this thesis would never be written. Be prepared, because this is going to be a long one (besides, this is usually the only chapter everyone reads anyway)… First of all, my deepest thanks to my supervisors: Prof. Berndt Gerhardson and Dr. Jamshid Fatehi. Thank you, Berndt, for believing in me and never restricting my interests! I will never forget your saying that the most important thing in research is having fun (I really did!). Thank you Jamshid, not just for being the World’s Best Supervisor, but also the person I could always rely on, even when it did not concern scientific matters. My deepest respect to both of you!… My family – thank you for what I am, allowing me to be free in my choices in life and all the moral support at all times! Being far away from you was not easy… Deepest thanks to my grant providers: Royal Swedish Academy of Agriculture and Forestry (KSLA), MISTRA and the Swedish Institute for opportunity to study, work and live in Sweden. My first teacher in Science, Galina Svedberga (Sultanova) – thank you for introducing me into the world of research and spiritual guiding through all these years. Dr. Edgars Vimba (my first mycology teacher) and Prof. Rihards Kondratovics, my Latvian supervisors, for their endless support and encouragement. Prof. Kondratovics, thank you for all your rhododendrons, I still have a passion for them. Prof. Yurijs Hrols – sorry I left you group. It was fun and pleasure being at KKI and being a part of The Serpentarium! Thank you Sandra for supervising me in the very beginning of my stay in Sweden. Sorry it did not work out, but I do appreciate the time we spent together. Prof. Krishnapillai Sivasithamparam – for teaching me philosophy of science and giving an example of being The Scientist. Ja-On, Nura, Titik, Nicolin, Brett and PJ, thank you for a wonderful time in Perth! I will never forget my stays in Western Australia! Dr. Ovidiu Constantinescu – thank you for all the valuable discussions about fungi (and not only), patience and constant consultations in any aspect of mycology, identification of my strains, supplying UPSC isolates, and, of course, Jazz!
53 My Latvian friends, Oksana, Sveta and Olga for being there for me and reminding that there are other things in life besides science. BioFak gang: Dima, Kristina, Karina and Zhanna, thank you for your Friendship! Latvian Mafia in Sweden: dear soul-sisters Inga and Daiga, and “brothers” Maris and Agnis – for being around when I needed it the most, sharing problems and happiness, and having a great fun together! Jolanta and Jens – thank you for being great friends and perfect work colleagues! Thank you for all your kind help both outside the department and during writing the thesis! My newly gained friends in Sweden: Dmitrijs, Aneta, Andrzej, Galia, Martin, Iveta, Vitek and Jola – thanks for the wild parties, “intellectual discussions” and just being great! Brother Idress – thank you for your support, wise life advices, being a great office-mate and Marlboro when I needed it the most! Maria – thank you for all your help, holding up and your thunderstorm temper and believing in yourself and me as well. Valentin – thank you for friendship; for a while you were the only person I could practice my Russian with. Sture, Lennart and Tahsein – thank you for being always kind and correct, please accept my deepest respect. Sture – thank you for bringing me to Sweden for the second time and your endless help with immigration authorities! Dear Plant Pathology gang (Ibrahim, Moje, Sergio, Janne, Fredrik, Paul, Janna, Britt-Marie, Titti, Zahra, Ola, Anna, Ragnar, Pablo, Jeshetla, Riccardo, Phuong, …) – was nice working together with you! Pity that the old group is gone… Dear Ewon, Catarina and Karin – life without a good secretary is a disaster! (Especially Ewon, the Guardian Angel of the old Department – thank you for everything!) Dear Molecular Evolution group – I have found many friends here. Everyone – it was nice meeting you! Thank you Siv for accepting the strange greenish-white plant pathologists among your virgin-white group. I had a great time here, honest. Dear Wagied, thank you very much for proofreading one of the manuscripts and the final version of the thesis! No PhD can be done without a computer (or Mac, which I learnt is not the same). Therefore, thank you Håkan, Ola & Ola for constant fixing my Foxy (may it rest in peace)! Did I forget anyone? Oups… MATS – thank you. For critical revision of my manuscripts, including this one. For Tomta, Bob Dylan, golf and crayfish. And for Life. And the Universe. And EVERYTHING.
54 References
1. van der Aa, H.A., Noordeloos, M.E. & de Gruyter, J. (1990) Species concept in some larger genera of the Coelomycetes. Studies in Mycology 32:3-19. 2. Abeln, E.C.A., de Pagter, M.A. & Verkley, G.J.M. (2000) Phylogeny of Pezicula, Dermea and Neofabrea inferred from partial sequences of the nuclear ribosomal RNA gene cluster. Mycologia 92: 685-693. 3. Aberra, M.B., Seah, S. & Sivasithamparam, K. (1998) Suppression of the take- all fungus (Gaeumannomyces graminis var. tritici) by a sterile red fungus through induced systemic resistance in wheat (Triticum aestivum) seedling roots. Soil Biology and Biochemistry 30(10-11): 1457-1461. 4. Adaskaveg, J.E. & Förster, H. (2000) Occurrence and management of anthracnose epidemics caused by Colletotrichum species on tree fruit crops in California. In: Colletotrichum. Host specificity, Pathology, and Host-Pathogen Interaction, D. Prusky, S. Freeman, & M. Dickman eds. APS Press, St. Paul, MN, USA. 5. Afanador-Kafuri, L., Minz, D., Maymon, M. & Freeman, S. (2003) Characterization of Colletotrichum isolates from tamarillo, passiflora, and mango in Colombia and identification of a unique species from this genus. Phytopathology 93: 579-587. 6. Agostini, J.P., Timmer, L.W. & Mitchel, D.J. (1992) Morphological and pathological characteristics of strains of Colletotrichum gloeosporioides from citrus. Phytopathology 82(11): 1377-1382. 7. Agrios, G.N. (1997) Plant Pathology. Academic Press, USA. Pp. 410-418. 8. Alahakoon, P.W., Brown A.E. & Sreenivasaprasad, S. (1994a) Genetic characterization of Colletotrichum gloeosporioides isolates obtained from mango. International Journal of Pest Management 40(2): 225-229. 9. Alahakoon, P.W., Brown A.E. & Sreenivasaprasad, S. (1994b) Cross-infection potential of genetic groups of Colletotrichum gloeosporioides on tropical fruits. Physiological and Molecular Plant Pathology 44: 93-103. 10. Alexopulos, C.J., Mims, C.W. & Blackwell, M. (1996) Introductory Mycology. 4th edition. John Wiley & Sons, Inc., New York-…-Singapore. 11. Ando, N. & Takatori, K. (1982) Fungal flora of the conjuctival sac. American Journal of Ophtalmology 94: 67-74. 12. Arenal, F., Platas, G., Monte & Peláez, F. (2000) ITS sequencing support for Epicoccum nigrum and Phoma epicoccina being the same biological species. Mycological Research 104: 301-303. 13. Arie, T., Christiansen, S.K., Yoder, O.C. & Turgeon, B.G. (1997) Efficient cloning of ascomycete mating type genes by PCR amplification of the conserved MAT HMG box. Fungal Genetics and Biology 21: 118-130.
55 14. von Arx, J. A. (1957) Die Arten der Gattung Colletotrichum Cda. Phytopathologische Zeitschrift 29: 413-468. 15. Astrom, B. & Ramstedt, M. (1994) Stem cankers on Swedish biomass willows caused by Cryptodiaporthe salicella and other fungi. European Journal of Forest Pathology 24(5): 264-276. 16. Baayen, R.P., O’Donnel, K., Breewsma, S., Geiser, D.M. & Waalwijk, C. (2001) Molecular relationships of fungi within the Fusarium redolens – F. hostae clade. Phytopathology 91:1 037-1044. 17. Baayen, R.P., O’Donnel, K., Bonants, P.J.M., Cigelnik, E., Kroon, L.P.N.M., Roebroeck, E.J. & Waalwijk, C. (2000) Gene genealogies and AFLP analysis in the Fusarium oxysporum complex identify monophyletic and nonmonophyletic formae speciales causing wilt and rot disease. Phytopathology 90: 891-900. 18. Bailey, J.A., Nash, C., Morgan, L.W., O’Connell, R.J.O. & TeBeest, D.O. (1996) Molecular taxonomy of Colletotrichum species causing anthracnose on the Malvaceae. Phytopathology 86: 1076-1083. 19. Baird, R.E, Wilson, J.P. & Sumner D.R. (1992) Identity and pathogenicity of two Marasmius species from the Sterile White Basidiomycete complex. Plant Disease 76:244-247 20. Barrow, M.O. (1995) Comparisons between different isolation procedures for isolation of sterile mycelia from cruciferous and monocotyledonous plants. M.Sc. diploma thesis, SLU. 21. Baxter, A., van der Westhuizen, G.C.A. & Eicker, A. (1983) Morphology and taxonomy of South African isolates of Colletotrichum. South African Journal of Botany 2(4): 259-289 22. Bell, D.K. & Sumner, D.R. (1984) Ecology of a sterile white pathogenic basidiomycete in corn, soybean, and snapbean field microplots. Plant Disease 68:18-22 23. Berbee, M.L. (1996) Loculoascomycete origins and evolution of filamentous ascomycete morphology based on 18S rRNA gene sequence data. Molecular Biology and Evolution 13: 462-470. 24. Berbee, M.L., Carmean, D.A. & Winka, K. (2000) Ribosomal DNA and resolution of branching order among the ascomycota: How many nucleotides are enough? Molecular Phylogenetics and Evolution 17:337-344. 25. Berkeley, M.J. (1954) Septoria rufo-maculans. Gardeners’ Chronicle: p. 676. 26. Berkeley, M.J. (1956) Glœosporium fructigenum. Gardeners’ Chronicle: p. 245. 27. Berkeley, M.J. (1860) Outlines of British Fungology, p. 320. 28. Berkeley, M.J. & Curtis, M.A. (1874). Glœosporium versicolor. Grevillea 3: 13. 29. Bernstein, B., Zehr, E.I., Dean, R.A. & Shabi, E. (1995) Characteristics of Colletotrichum from peach, apple, pecan, and other hosts. Plant Disease 79: 478-482. 30. Boerema, G.H. & Bolle, G.L. (1975) Conidiogenesis and conidial septation as differentiating criteria between Phoma and Ascochyta. Persoonia 8: 111-144. 31. Boerema, G.H., Dorenbosch, M.M.J. & van Kersteren, H.A. (1965) Remarks ob species of Phoma referred to Peyronellaea I. Perssoonia 4: 47-68. 56 32. Boerema, G.H., Dorenbosch, M.M.J. & van Kersteren, H.A. (1968) Remarks ob species of Phoma referred to Peyronellaea II. Perssoonia 5: 201-205. 33. Boerema, G.H., Dorenbosch, M.M.J. & van Kersteren, H.A. (1971) Remarks ob species of Phoma referred to Peyronellaea III. Perssoonia 6: 171-177. 34. Boerema, G.H., Dorenbosch, M.M.J. & van Kersteren, H.A. (1973) Remarks ob species of Phoma referred to Peyronellaea IV. Perssoonia 7: 131-139. 35. Boerema, G.H., Dorenbosch, M.M.J. & van Kersteren, H.A. (1977) Remarks ob species of Phoma referred to Peyronellaea V. Kew Bulletin 31: 533-544. 36. Bonde, M.R., Peterson, G.L. & Maas J.L. (1991) Isozyme comparisons for identification of Colletotrichum species pathogenic to strawberry. Phytopathology 81: 1523-1528. 37. Brasier, C.M. (1997) Fungal species in practice: identifying species units in fungi. In Species: The Unites of Biodiversity, Claridge, M.F., Dawah, H.A. & Wilson, M.R. eds., pp. 135-170. 38. Bridge, P.D., Roberts, P.J., Spooner, B.M. & Panchal, G. (2003) On the unreliability of published DNA sequences. New Phytologist 160: 43-48. 39. Brown, A.E., Finley, R. & Ward, J.S. (1987) Antifungal compounds produced by Epicoccum purpurascens against soil-borne plant pathogenic fungi. Soil Biology and Biochemistry 19: 657-664. 40. Brown, A. & Soepena, H. (1994) Pathogenicity of Colletotrichum acutatum and C. gloeosporioides on leaves of Hevea spp. Mycological Research 98(3): 264-266. 41. Brown, A., Sreenivasaprasad, S. & Timmer, L.W. (1996) Molecular characterization of slow-growing orange and key lime anthracnose strains of Colletotrichum as C. acutatum. Phytopathology 86: 523-527. 42. Bruns, T.D. & Szaro, T.M. (1992) Rate and mode differences between nuclear and mitochondrial small subunit rRNA genes in mushrooms. Molecular Biology and Evolution 95: 836-855. 43. Bruns, T.D., Vilgalys, R., Barnes, S.M., Gonzales, D., Hibbet, D.S., Lane, D.J., Simon, L., Stickel, S., Szaro, T.M., Weisburg, G.W. & Sogin, M.L. (1992) Evolutionary relationships within the fungi: Analyses of nuclear small subunit rRNA sequences. Molecular Phylogenetics and Evolution 1: 231-241. 44. Bruns, T.D., White, T.J. & Taylor, J.W. (1991) Fungal molecular systematics. Annual Review of Ecology and Systematics 22: 525-564 45. Bryan, G.T., Daniels, M.J. & Osbourn, A.E. (1995) Comparison of fungi within Gaeumannomyces-Phialophora complex by analysis of ribosomal DNA sequences. Applied and Environmental Microbiology 61: 681-689. 46. Buddie, A.G., Martinez-Cullebras, P., Bridge, P.D., Garcia, M.D., Querol, A., Cannon, P.F. & Monte, E. (1999) Molecular characterization of Colletotrichum strains derived from strawberry. Mycological Research 103: 385-394. 47. Buyer, J.S., Roberts, D.P., Millner, P. & Russek-Cohen, E. (2001) Analysis of fungal communities by sole carbon source utilization profiles. Journal of Microbiological Methods 45: 53-60. 48. Che, Y., Gloer, J.B., & Wicklow, D.T. (2002) Phomadecalins A-D and Phomapentenone A: new bioactive metabolites from Phoma sp. NRRL 25697, a fungal colonist of Hypoxylon stromata. Journal of Natural Products 65: 399- 402. 57 49. Cisar, C.R., Spiegel, F.W., TeBeest, D.O. & Trout, C. (1994) Evidence for mating between isolates of Colletotrichum gloeosporioides with different host specificities. Current Genetics 25: 330-335. 50. Cisar, C.R. & TeBeest, D.O. (1999) Mating system of the filamentous ascomycete, Glomerella cingulata. Current Genetics 35: 127-133. 51. de Clauser, R., Magnano, P., Corazza, L. & Chilosi, G. (1990). Colletotrichum acutatum: agente causale di antracnose della fragola. L’Informatore Agrario 46: 71-72. 52. Couch, B.C. & Kohn, L.M. (2002) A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycologia 94(4): 683-693. 53. Cove, D.J. (1966) The induction and repression of nitrate reductase in the fungus Aspergillus nidulans. Biochimica et Biophysica Acta 113: 51-56 54. Cozijnsen, A.J., Popa, K.M., Purwantara, A., Rolls, B.D. & Howlett B.J. (2001) Genome analysis of the plant pathogenic ascomycete Leptosphaeria maculans; maping mating type and host specificity loci. Molecular Plant Pathology 1(5): 293-302. 55. Cubero, O.F., Crespo, A., Fatehi, J. & Bridge, P.D. (1999) DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi. Plant Systematics and Evolution 216: 243-249. 56. Cubeta, M.A., Echandi, E., Abernethy, T. & Vilgalys, R. (1991) Characterization of anastomozis groups of binucleate Rhizoctonia species using restriction analysis of an amplified ribosomal RNA gene. Phytopathology 81: 1395-1400. 57. Currah, R.S. & Tsuneda, A. (1993) Vegetative and reproductive morphology of Phialocephala fortinii (Hyphomycetes, Mycelium radicis atrovirens) in culture. Transaction of Mycological Society of Japan 34: 345-356. 58. Davids, R.D., Boland, R.M. & Howitt, C.J. (1992) Colony descriptions, conidium morphology, and the effect of temperature on colony growth of Colletotrichum gloeosporioides isolated from Stylosanthes spp. growing in several countries. Mycological Research 96(2): 128-134. 59. Dawson, M.J., Farthing, J.E., Marshall, P.S., Middelton, R.F., O’Neill, M.J., Shuttleworth, A., Stylli, C., Tait, R.M., Taylor, P.M., Wildman, HG., Buss, A.D., Langley, D. & Hayes, M.V. (1992) The squalestatins novel inhibitors of squalene synthase produced by a species of Phoma. I. Taxonomy, fermentation, isolation physico-chemical properties and biological activities. Journal of Antibiotics 45: 639-647. 60. Deacon, J.W. (1980) Ectotrophic growth by Phialophora radicola var. graminicola and other parasites of cereal and grass roots. Transactions of the British Mycological Society 75: 158-160. 61. Deacon, J.W. (1981) Ecological relationships with other fungi: competition and hyperparasites. In: Biology and Control of Take-all,. Shipton, M.J.C., & Asher, P.J. eds. Academic Press, London. Pp 75-102. 62. DeJong, D., Kurtboke, D.I., Shankar, M. & Sivasithamparam, K. (1993) Effect of placement of inoculum in soil on infectivity and disease protection ability of a sterile red fungus. Soil Biology and Biochemistry 25(12): 1641-1647.
58 63. De La Cruz, R.E. & Hubbel, D.H. (1975) Biological control of the charcoal root rot fungus Macrophominia phaseolina on slash pine seedlings by a hyperparasite. Soil Biology and Biochemistry 7: 25-30. 64. Denoyes, B. & Baudry, A. (1995) Species identification and pathogenicity study of French Colletotrichum strains isolated from strawberry using morphological and cultural characteristics. Phytopathology 85(1): 53-57. 65. Desjardin, D.E. (1990) Culture morphology of Marasmius species. Sydowia 42: 17-87 66. Dewan, M.M. & Sivasithamparam, K. (1988) A plant-growth-promoting sterile fungus from wheat and ryegrass roots with a potential for suppressing take-all. Transactions of British Mycological Society 91: 687-692. 67. Dewan, M.M. & Sivasithamparam, K. (1989a) Growth promotion of rotation crop species by a sterile fungus from wheat and the effect of soil temperature and moisture on its suppression of take-all. Mycological Research 93: 156- 160. 68. Dewan, M.M. & Sivasithamparam, K. (1989b) Behavior of a plant growth- promoting sterile fungus on agar and roots of rye-grass and wheat. Mycological Research 93 (2): 161-166. 69. Dewan, M.M. & Sivasithamparam, K. (1989c) Efficacy of treatment with a sterile red fungus for control of take-all in wheat. New Zealand Journal of Crop and Horticultural Science 17:333-336. 70. Dewan, M.M. & Sivasithamparam, K. (1990) Effect of colonization by sterile red fungus on viability of seed and growth and anatomy of roots. Mycological Research 94: 553-557. 71. Dewan, M.M. & Sivasithamparam, K. (1991) Promotion of growth of wheat seedlings by a sterile red fungus in relation to plant density and soil fertility. Plant and Soil 135: 306-308. 72. Dhingra, O.D. & Sinclair, J.B. (1986) Basic plant pathology methods. CRS Press, U.S. 73. Dingley, J.M. & Gilmour, J.W. (1972) Colletotrichum acutatum Simmds. f.sp. pinea associated with “terminal crook” disease of Pinus spp. New Zealand Journal of Forestry Science 2(2): 192-201. 74. Donaldson, G.C., Ball, L.A., Axelrood, P.E. & Glass, N.L. (1995) Primer set to amplify conserved genes from filamentous Ascomycetes are useful in differentiating Fusarium species associated with conifers. Applied and Environmental Microbiology 61(4): 1331-1340. 75. Domsch, K.H., Gams, W. & Anderson, T.H. (1980) Compendium of Soil Fungi. IHW-Verlag: London - … - Toronto. 76. Duarte, E.R., Resende, J.C.P., Rosa, C.A. & Hamdan, J.A. (2001) Prevalence of yeasts and mycelial fungi in bovine parasitic otitis in the sate of Minas Gerais, Brazil. Journal of Veterinary Medicine, Series B 48: 631-635. 77. Dyer, P.S., Furneaux, P.A., Douhuan, G. & Murray, T.D. (2001) A multiple PCR test for determination of mating type applied to the plant pathogens on Tapesia yallundae and Tapesia acuformis. Fungal Genetics and Biology 33: 173-180. 78. Dyko, B.J. & Mordue, J.E.M. (1979) Colletotrichum acutatum. CMI Descriptions of Pathogenic Fungi and Bacteria 630. Commonwealth Mycological Institute: Kew, U.K. 59 79. Ebner, M.R., Haselwandter, K. & Frank, A. (1992) Indoor and outdor incidence of airborne fungal allergens at low and high altitude alpine environments. Mycological Research 96: 117-124. 80. Edgerton, C.W. (1915) Effect of temperature on Glomerella. Phytopathology 5: 247-259. 81. Farr, D.F., Bills, G.F., Chamuris, G.P. & Rossman, A.Y. (1989) Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, USA. 82. Ferraz, J.F.P. (1977) Morfologia, comportamento cultural e patologenicidade de espécies de Colletotrichum e Gloeosporium. Agronomia Lusitana 38(2): 163-179. 83. Freeman, S., Katan, T. & Shabi, E. (1996) Characterization of Colletotrichum gloeosporioides isolates from avocado and almond fruits with molecular and pathogenicity tests. Applied and Environmental Microbiology 62(3): 1014- 1020. 84. Freeman, S., Katan, T. & Shabi, E. (1998) Characterization of Colletotrichum species responsible for anthracnose diseases of various fruits. Plant Disease 82(6): 596-605. 85. Freeman, S., Minz, D., Jurkevitch, E., Maymon, M. & Shabi, E. (2000) Molecular analyses of Colletotrichum species from almond and other fruits. Phytopathology 90: 608-614. 86. Freeman, S., Pham, M. & Rodriguez, R.J. (1993) Molecular genotyping of Colletotrichum species based on arbitrarily primed PCR, A + T-rich DNA, and nuclear DNA analysis. Experimental Mycology 17: 309-322. 87. Freeman, S. & Rodriguez, R.J. (1995) Differentiation of Colletotrichum species responsible for anthracnose of strawberry by arbitrarily primed PCR. Mycological Research 99(4): 501-504. 88. Frost, R.R. (1964) Setae formation in Colletotrichum spp. Nature 201: 730- 731. 89. Fujita, T., Hamamichi, N., Kiuchi, M., Matsuzaki, T., Kitao, Y., Inoue, K., Hirose, R., Yoneta, M., Sasaki, S. & Chiba, K. (1996) Determination of absolute configuration and biological activity of new immunosuppressants, mycestericins D, E, F and G. Journal of Antibiotics Tokyo 49:846-853. 90. Fukushi, T. (1921) A willow-canker disease caused by Physalospora miyabeana and its conidial form Gloesoporium. Annals of Phytopathological Society of Japan 1: 1-12. 91. Förster, H. & Adaskaveg, J.E. (1999) Identification of subpopulations of Colletotrichum acutatum and epidemiology of almond anthracnose in California. Phytopathology 89: 1056-1065. 92. Gams, W. & Bissett, J. (1998) Morphology and identification of Trichoderma. In: Kubicek, C.P., Harman, G.E. (Eds.), Trichoderma and Gliocladium. Basic Biology, Taxonomy and Genetics. Taylor & Francis, London, UK, pp. 3-34. 93. Gasoni, L., Stegman – De Gurfinkel, B. & Fortugno, C. (1993) Supression of damping-off caused by Rhizoctonia solani through a nonpathogenic sterile septate fungus. Zeitschrift fuer Pflanzenkrankheiten und Pflanzenschutz 100: 467-473. 94. Gilliam, M.S. (1976) The genus Marasmius in the Northeastern United States and adjascent Canada. Mycotaxon 4: 1-144 60 95. Girlanda, M., Ghihnone, S. & Luppi, A.M. (2002) Diversity of sterile root- associated fungi of two Mediterranean plots. New Phytologist 155: 481-498. 96. Glass, N.L. & Donaldson, G.C. (1995) Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes. Applied and Environmental Microbiology 61(4): 1323-1330. 97. Greuter, W., McNeill, J., Barrie, F.R., Burdet, H.-M., Demoulin, V., Filgueras, T.S., Nicolson, D.H., Silva, P.C., Skog, J.E., Trehane, P., Turland, N.J. & Hawksworth, D.L. eds. (2000) International Code of Botanical Nomenclature (St. Louis Code). Regnum Vegetabile 138. Koeltz Scientific Books, Königstein. 98. Guerber, J.C. & Correll, J.C. (1997) The first report of the teleomorph of Colletotrichum acutatum in the United States. Plant Disease 81(11): 1334. 99. Guerber, J.C. & Correll, J.C. (2001) Characterization of Glomerella acutata, the teleomorph of Colletotrichum acutatum. Mycologia 93(1): 216-229. 100. Gunnel, P.S. & Gubler, W.D. (1992) Taxonomy and morphology of Colletotrichum species pathogenic to strawberry. Mycologia 84(2)157-165. 101. Hall, G. (1987) Sterile fungi from roots of winter wheat. Transactions of British Mycological Society 89: 447-456. 102. Hanlin, R.T. (1990) Illustrated genera of Ascomycetes. APS Press MN, U.S.A. 103. Harrington, T.C. & Rizzo, D.M. (1999) Defining Species in the Fungi. In: Structure and Dynamics of Fungal Populations. Worrall, J.J ed.). Kluwer Press, Dordrecht, The Netherlands. 104. Harveson, R.M. (2002) A new soilborne disease of dry, edible beans in Western Nebrasca. Plant Disease 86:1051. 105. Hashimoto, Y. & Hyakumachi, M. (2001) Effects of isolates of ectomycorhizal fungi and endophytic Mycelium radicis atrovirens that were dominant in soil from disturbed sites on growth of Betula platyphylla var. japonica seedlings. Ecological Research 16: 117-125. 106. Hawksworth, D,L., Sutton, B.C. & Aisnworth, G.C. (1983) Ainsworth and Bisby’s Dictionary of the Fungi. CMI, Kew, UK. 107. Henz, G.P., Boiteux, L.S. & Lopes, C.A. (1992) Outbreak of strawberry anthracnose caused by Colletotrichum acutatum in Central Brazil. Plant Disease 76: 212. 108. Hepting, G.H. (1971) Disearses of forest and shade trees of the United States. USDA Forest Service Handbook 386: 658 pp. 109. Hillis, D.M. (1987) Molecular versus morphological approaches to systematics. Annual Review of Ecology and Systematics 18: 23-42. 110. Hillis, D.M. & Dixon, M.T. (1991) Ribosomal DNA: Molecular evolution and phylogenetic inference. Quarterly Review of Biology 66(4): 411-453. 111. Hindorf, H. (1970) Colletotrichum spp. isolated from Coffea arabica L. in Kenya. Zeitschrift für Pflanzkrankheiten und Pflanzschutz 77(6): 328-331. 112. Hindorf, H. (1973a). Colletotrichum – Population auf Coffea arabica L. in Kenia. I. Eine Methode zur systematischen Trennung von Pilzpopupationen. Phytopathologische Zeitschrift 77: 97-116. 113. Hindorf, H. (1973b). Colletotrichum – Population auf Coffea arabica L. in Kenia. II. Qualitative und quantitative Unterschiede in der Colletotrichum- Population. Phytopathologische Zeitschrift 77: 216-234. 61 114. Hindorf, H. (1973c). Colletotrichum – Population auf Coffea arabica L. in Kenia. III. Verbreitung von Colletotrichum-Arten auf den einzelnen Organen des Kaffeestrauches. Phytopathologische Zeitschrift 77: 324-338. 115. Hodson, A., Mills, P.R. & Brown, A.E. (1993). Ribosomal and mitochondrial DNA polymorphisms in Colletotrichum gloeosporioides isolated from tropical fruits. Mycological Research 97: 329-335. 116. Hopple, J. S., Jr. & Vilgalys, R. (1999) Phylogenetic relationships in the mushroom genus Coprinus and dark-spored allies based on sequence data from the nuclear gene coding for the Large Ribosomal Subunit RNA: divergent domains, outgroups, and monophyly. Molecular Phylogenetics and Evolution 13(1): 1-19. 117. Horton, T.R., Cázares, E. & Bruns, T.D. (1998) Ectomycorrhizal, vesicular- arbuscular and dark septate fungal colonization of bishop pine (Pinus muricata) seedlings in the first 5 months of growth after wildfire. Mycorrhiza 8: 11-18. 118. Howard, C.M., Conway, K.E. & Albregts, E.E. (1977) A stem rot of bean seedlings caused by a sterile fungus in Florida. Phytopathology 67:430-433 119. Howard, C.M., Maas, J.L., Chandler, C.K. & Albregts, E.E. (1992) Anthracnose of strawberry caused by the Colletotrichum complex in Florida. Plant Disease 76(10): 976-981. 120. Howlett, B.J., Idnurm, A. & Pedras, M.S. (2001) Leptosphaeria maculans, the causal agent of blackleg disease of Brassicas. Fungal genetics and Biology 33: 1-14. 121. Huang, L., Lingham, R.B., Harris, G.H., Singh, S.B., Dufresne, C., Nallin, O.M., Bills, G.F., Mojena, M., Sanchez, M., Karkas, J.D., Gibbs, J.B., Clapp, W.H., Meinz, M.S.; Silverman, K.C. & Bergstrom, J.D. (1995) New fungal metabolites as potential antihypercholesterolemics and anticancer agents. Canadian Journal of Botany 73 (Suppl. 1): S898-906. 122. Hyde, K.D. (1986) Frequency of occurrence of lignicolous fungi in the tropics. In: The biology of marine fungi, Moss, S.T. ed. Cambridge University Press, Cambridge, UK, pp. 311-322. 123. James, T.Y., Moncalvo, J.-M., Li, S. & Vilgalys, R. (2001) Polymorphism at the ribosomal DNA spacers and its relation to breeding structure of the widespread mushroom Schizophyllum commune. Genetics 157: 149-161. 124. James, T.Y., Porter, D., Leander, C.A., Vilgalys, R. & Longcore, J.E. (2000) Molecular phylogenetics of the Chytridiomycota supports the utility of ultrastructural data in chytrid systematics. Canadian Journal of Botany 78: 336-350. 125. Jacobs, A. (1994) Untersuchungen zu Eigenschaften und Vorkommen der aus Wurzeln isolierten Mycelia sterilia. M. Sc. Diploma theses, SLU. 126. Jeffries, P. & Koomen, I. (1992) Strategies and prospects for biological control of diseases caused by Colletotrichum. In Colletotrichum: Biology, Pathology & Control, Bailey, J.A. & Jeger, M.J. eds. CABI, UK, pp. 127. Johnston, P.R. & Jones, D. (1997) Relationships among Colletotrichum isolates from fruit-rots assessed using rDNA sequences. Mycologia 83: 420- 430. 128. Johnston, P.R. (2000) The importance of phylogeny in understanding host relationships within Colletotrichum. In: Colletotrichum. Host specificity, 62 Pathology, and Host-Pathogen Interaction, D. Prusky, S. Freeman, & M. Dickman eds. APS Press, St. Paul, MN, USA. 129. Jumpponen, A. & Trappe, J.M. (1998) Dark septate endophytes: a review of facultative biotrophic root-solonizing fungi. New Phytologist 140: 295-310. 130. Kaiser, W.J., Melendez, P.O. Hannan, R.M. & Zapata, M. (1987) Crown canker of pigeon pea (Cajanus cajan) caused by a sterile white basidiomycete in Puerto Rico. Plant Disease 71:1006-1009 131. Kamigiri, N.K., Matsumoto, H., Kawano, Y., Yamaoka, M., Shimo, S., Watanabe, M. & Suzuki, K. (2002) YM-202204, a new antifungal antibiotic produced by marine fungus Phoma sp. Journal of Antibiotics Tokyo 55: 1036- 1041. 132. Katan, T. (2000). Vegetative Compatibility in Colletotrichum. In: Colletotrichum. Host specificity, Pathology, and Host-Pathogen Interaction, D. Prusky, S. Freeman, & M. Dickman eds. APS Press, St. Paul, MN, USA. 133. Khairallah, S.H., Byrne, K.A. & Tabbara, K.F. (1992) Fungal keratitis in Saudi Arabia. Documenta Ophtalmologica 79: 269-276. 134. Kirk, P.M., Cannon, P.E., David, J.C. & Stalpers, J.A. (2001) Ainsworths & Bisby’s Dictionary of Fungi, 9th edition. CABI Bioscience, Egham, UK. 135. Kiss, L. & Nakasone, K.K. (1998) Ribosomal DNA internal transcribed spacer sequences do not support the species status of Ampelomyces quisqualis, a hyperparasite of powdery mildew fungi. Current Genetics 33: 362-367. 136. Klonowska, A., Gaudin, C., Ruzzi, M., Colao, M.C. & Tron, T. (2002) Ribosomal DNA sequence analysis shows that the basidiomycete C30 belongs to Trametes. Research in Microbiology 154: 25-28 137. Kohn, L.M. (1995) The clonal dynamic in wild and agricultural plant-pathogen populations. Canadian Journal of Botany 73 (Suppl. 1): S1231-1240. 138. Koike, N., Hyakumachi, M., Kageyama, K., Tsuyumu, S. & Doke, N. (2001) Induction of systemic resistance in cucumber against several diseases by plant growth-promoting fungi: Lignification and superoxide generation. European Journal of Plant Pathology 107: 523-533. 139. Korsten, L. & Jeffries, P. (2000) Potential for biological control of diseases caused by Colletotrichum. In Colletotrichum. Host Specificity, Pathology and Host-Pathogen Interactions, Prusky, D., Freeman, S., & Dickman, M.B., eds. APS Press, St. Paul, MN, USA, pp. 266-292. 140. Kubicek, C.P., Bissett, J., Druzhinina, I., Kulling-Gradinger, C. & Szakacz, G. (2002) Genetic and metabolic diversity of Trichoderma: a case study on South- East Asian isolates. Fungal Genetics and Biology 38: 310-319. 141. Kulshrestha, D.D., Mathur, S.B. & Neergaard, P. (1976) Identification of seed- borne species of Colletotrichum. Friesia 11: 116-125. 142. Kuramae-Izioka, E.E., Lopes, C.R., Souza, N.L. & Machado, M.A. (1997) Morphological and molecular characterization of Colletotrichum spp. from citrus orchards affected by postbloom fruit drop in Brazil. European Journal of Plant Pathology 103:323-329. 143. Kwaasi, A.A.A., Parhar, R.S., Al-Mohanna, F.A.A., Harfi, H.A., Collison, K.S. & Al-Sedairy, S.T. (1998) Aeroallergens and viable microbes in sandstorm dust: Potential triggers of allergic and nonallergic respiratory ailments. Allergy Copenhagen 53: 255-265.
63 144. Lardner, R., Johnston P.R., Plummer, K.M. & Pearson, M.N. (1999) Morphological and molecular analysis of Colletotrichum acutatum sensu lato. Mycological Research 103: 275-285. 145. Legard, D.E. (2000) Colletotrichum diseases of strawberries in Florida. In: Colletotrichum. Host specificity, Pathology, and Host-Pathogen Interaction, D. Prusky, S. Freeman, & M. Dickman eds. APS Press, St. Paul, MN, USA. 146. Levenfors, J.P., Wikström, M., Persson, L. & Gerhardson, B. (2003) Pathogenicity of Aphanomyces spp. From different leguminous crops in Sweden. European Journal of Plant Pathology 109: 535-543. 147. Li, K.-N., Rouse, D.I. & German, T.L. (1994) PCR primers that allow intergenic differentiation of Ascomycetes and their application to Verticillium spp. Applied and Environmental Microbiology 60(12): 4324-4331. 148. Linnaeus, C. (1758) Systema Naturae: facsimile of the first edition / Carolus Linnaeus; with an introduction and a first English translation of the "Observationes" by Engel-Ledeboer, M.S.J. & Engel, H. (1964). Nieuwkoop: B. de Graaf, The Netherlands. 149. Liu, Z.L., Domier, L.L. & Sinclair, J.B. (1993). ISG-specific ribosomal DNA polymorphism of the Rhizoctonia solani species complex. Mycologia 85: 260- 265. 150. Liu, Z.L., Domier, L.L. & Sinclair, J.B. (1995). Polymorphism of genes coding for nuclear 18S rRNA indicates genetic destinctiveness of anastomosis group 10 from other groups in the Rhizoctonia solani species complex. Applied and Environmental Microbiology 61: 2659-2664. 151. Liu, Z., Jensen, P.R. & Fenical, W. (2003) A cyclic carbonate and related polyketides from a marine-derived fungus of the genus Phoma. Phytochemistry 64: 571-574. 152. Liyanage, H.D., McMillan, R.T. & Kistler, H.C. (1992) Two genetically distinct populations of Colletotrichum gloeosporioides from citrus. Phytopathology 82(11): 1371-1376. 153. Manire, C.A., Rhinehart, H.L., Sutton, D.A., Thompson, E.H., Rinaldi, M.G., Buck, J.D. & Jacobson, E. (2002) Disseminated mycotic infection caused by Colletotrichum acutatum in a Kemp’s ridley sea turtle (Lepidochelys kempi). Journal of Clinical Microbiology 40(11): 4273-4280. 154. Martin, S.B., Abawi, G.S. & Hoch, H.C. (1984) Influence of the antagonist Laetisaria arvalis on infection of table beet by Phoma betae. Phytopathology 74:1092-1096. 155. Martín, M.P. & García-Figueres, E. (1999) Colletotrichum acutatum and C. gloeosporioides cause anthracnose on olives. European Journal of Plant Pathology 105: 733-741. 156. Martinez-Culebras, P.V., Querol, A., Suarez-Fernandez, M.B., Garcia-Lopez, M.D. & Barrio, E. (2003) Phylogenetic relationships among Colletotrichum pathogens of strawberry and design of PCR primers for their identification. Journal of Phytopathology 151: 135-143. 157. Marx, D.H. (1969) The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59: 153-163. 158. Mayden, R.L. (1997) A chierarchy of species concepts: the denouement in the saga of the species problem. In Species: The Units of Biodoversity (Claridge, 64 M.F., Dawah, H.A. & Wilson, M.R. eds.), Chapman and Hall, London, pp.381-424. 159. Mayr, E. (1942) Systematics and the Origin of Species From the Viewpoint of a Zoologist. New York: Columbia University Press. 160. Mazzola, M., Wong, O.T. & Cook, R.J. (1996) Virulence of Rhizoctonia oryzae and R. solani AG-8 on wheat and detection of R. oryzae in plant tissue by PCR. Phytopathology 86: 354-360. 161. McGechan, J.K. (1977). Black spot of strawberry. Agricultural Gazette of New South Wales 88: 26-27. 162. Micales, J.A., Bonde, M.R. & Peterson, G.L. (1986) The use of isozyme analysis in fungal taxonomy and genetics. Mycotaxon 27: 405-449. 163. Mills, P.R., Sreenivasaprasad, S. & Brown, A.E. (1992) Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR. FEMS Microbiology Letters 98: 137-144. 164. Moncalvo, J.-M., Lutzoni, F.M., Rehner, S.A., Johnson, J. & Vilgalys, R. (2000) Phylogenetic relationships of agaric fungi based on nuclear large subunit ribosomal DNA sequences. Systematic Biology 49: 278-305 165. Moncalvo, J.-M., Vilgalys, R., Redhead, S.A., Johnson, J.E., James, T.Y., Aime, M.C., Hofstetter, V., Verduin, S.J.W., Larsson, E., Baroni, T.J., Thorn, G.R., Jacobson, S., Clémençon, H. & Miller, O.K. Jr. (2002) One hundred and seventeen clades of euagarics. Molecular Phylogenetics and Evolution 23: 357-400. 166. Moore, R.T. & McAlear, J.H. (1962) Fine structures of Mycota. 7. Observations on septa of ascomycetes and basidiomycetes. American Journal of Botany 49:86-94. 167. Mordue, J.E.M. (1971) Colletotrichum gloeosporioides. CMI Descriptions of Pathogenic Fungi and Bacteria 315. Commonwealth Mycological Institute: Kew, U.K. 168. Mucciarelli, M., Scannerini, S., Bertea, C.M. & Maffei, M. (2002) An ascomycetous endophyte isolated from Mentha piperita L.: biological features and molecular studies. Mycologia 94: 28-39. 169. Narita, Y. & Suzu, T. (1991) Influence of a sterile dark mycelial fungus on take-all of wheat. Annales of Phytopathological Society of Japan 57: 301-305. 170. Nattrass, R.M. (1928) The Physalospora disease of the Basket Willow. Transactions of British Mycological Society 13: 286-304. 171. Neumann, S. & Boland, G.J. (2002) Influence of host and pathogen variables on the efficacy of Phoma herbarum, a potential biological control agent of Taraxacum officinale. Canadian Journal of Botany 80: 425-429. 172. Newsham, K.K., Fitter, A.H. & Watkinson, A.R. (1994) Root pathogenic and arbuscular mycorrhizal fungi determine fecundity of asymptomatic plants in the field. Journal of Ecology 82: 805-814. 173. Newsham, K.K., Fitter, A.H. & Watkinson, A.R. (1995) Arbuscular mycorrhiza protect an annual grass from root pathogenic fungi in the field. Journal of Ecology 83: 991-1000. 174. Newsham, K.K. (1999) Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata spp. ambigua. 144: 517-524.
65 175. Nguyen, T.H.P. (2002) Identification of Colletotrichum species in Vietnam and Thailand. M.Sc. diploma thesis, SLU, Sweden. 176. Nirenberg, H.I., Feiler, U. & Hagedorn, G. (2002) Description of Colletotrichum lupini comb. nov. in modern terms. Mycologia 94: 307-320. 177. Nobles, M.K. (1965) Identification of cultures of wood-enhabiting Hymenomycetes. Canadian Journal of Botany 43: 1097-1139. 178. Nobles, M.K. (1971) Cultural characters as a guide to the taxonomy of the Polyporaceae. In Evolution of the Higher Basidiomycetes, Petersen, R.H., ed. University of Tennessee Press, Knoxville, TN, pp 169-196. 179. O’Donnel, K., Cigelnik, E. & Nirenberg, H. I. (1998) Molecular systematics and phylogeography of the Giberella fujikuroi species complex. Mycologia 90(3): 465-493. 180. O’Neil, N.R., van Berkum, P., Lin J.-J., Kuo, J., Ude, G.N., Kenworthy, W. & Saunders, J.A. (1997) Application of Amplified Restriction Fragment Length Polymorphism for genetic characterization of Colletotrichum pathogens of alfalfa. Phytopathology 87(7): 745-750. 181. Osterhage, C., Schwibbe, M., Koening, G.M. & Wright, A.D. (2000) Differences between marine and terrestrial Phoma species as determined by HPLC-DAD and HPLC-MS. Phytochemical Analysis 11: 288-294. 182. Parmeter, J.R., Jr. (1964) The taxonomy of Sterile Fungi. Phytopathology 55: 826-828. 183. Peláez, F., Collado, J., Arenal, F., Basilio, A., Cabello, A., Díez Matas, M.T., García, J.B., González Del Val, A., Gorrochategui, J., Hernández, P., Martín, I., Platas, G. & Vincente, F. (1998) Endophytic fungi from plants living on gypsum soils as a source of secondary metabolites with antimicrobial activity. Mycological Research 102: 755-761. 184. Peredo, H., Osario, M. & Santamaria, A. (1979) Colletotrichum acutatum f.sp. pinea, a new pathogen of Pinus radiata in nurseries in Chile. Plant Disease Reporter 63: 121-122. 185. Peres, N.A.R. Souza, N.L., Zitko, S.E. & Timmer, L.W. (2002) Activity of benomyl for control of postbloom fruit drop of citrus caused by Colletotrichum acutatum. Plant Disease 86(6): 620-624. 186. Perkins, D.D. (1991) In prise of diversity. In More Gene Manipulations in Fungi (Benett, J.W. & Lasure, L.L. eds.), Academic Press, Ney York, pp. 2- 26. 187. Peterson, R.A. (1973) Fungicidal control of black spot (Colletotrichum acutatum) and grey mould (Botrytis cinerea) on strawberry fruit in South- Esatern Queensland. Queensland Journal of Agricultural and Animal Sciences 30: 327-329. 188. Petrini, O. (1991) Fungal endophytes on tree leaves. In: Microbial Ecology of Leaves, Andrews, J.H., & Hirano, S.S., eds. Springer Verlag, New York, US, pp. 179-187. 189. Pont, W. (1973) Studies on root rot and stalk rot of maize in north Queensland caused by Marasmius sacchari Wakker var. hawaiiensis Cobb and Marasmius graminum (Lib.) Berk. var. brevispora Dennis. Queensland Journal of Agriculture and Animal Sciences 30: 225 -237
66 190. Prashar, R.D., Sindhan, G.S. & Hooda, I. (1992) Biological control of bacterial blight (Xantomonas campestris pv. cyamopsidis) of clusterbean by epiphytes present on leaf surface. Crop Research Hisar 5: 551-558. 191. Pugh, G.J.F. (1967) Root colonization by fungi. In Progress in Soil Biology, Graff, O & Satchell, J.E. eds. Amsterdam, the Netherlands, pp. 21-26. 192. Redhead, S.A. (1974) A new species of Campanella from North America. Mycologia 66: 183-187 193. Richard, C., Fortin, J.-A. & Fortin, A. (1971) Protective effect of an ectomycorrhizal fungus against the root pathogen Mycelium radicis atrovirens. Canadian Journal of Forest Research 1: 246-251. 194. Rowland, C.Y., Kurtboke, D.I., Shankar, M. & Sivasithamparam, K. (1994) Nutritional and biological activities of a sterile fed fungus which promotes plant growth and suppresses take-all. Mycological Research 98(12): 1453- 1457. 195. Sabet, K.A., Samra, A.S. & Abdel-Azim, O.F.Z. (1968) Root-rot disease of Maize caused by non-sporing Basidiomycete fungus. Agricultural Research Review 46: 53-75 196. Saccardo, P.A. (1884) Sylloge Fungorum, vol. 3, Padova, pp. 718, 385. 197. Saccardo, P.A. (1902) Sylloge Fungorum, vol. 16, Padova, p. 999. 198. Saccardo, P.A. (1903) Sylloge Fungorum, vol. 17, Padova, p. 573. 199. Saccardo, P.A. (1904) Sylloge Fungorum, vol. 18, Padova, p. 450. 200. Saha, T., Kumar, A., Ravindran, M., Jacob C.K., Roy, B. & Nazeer, M.A. (2002) Identification of Colletotrichum acutatum from rubber using random amplified polymorphic DNAs and ribosomal DNA polymorphisms. Mycological Research 106(2): 215-221. 201. Schadt, C.W., Mullen, R.B. & Schmidt, S.K. (2001). Isolation and phylogenetic identification of a dark-septate fungus associated with the alpine plant Ranunculus adoneus. New Phytologist 150: 747-755. 202. Schoch, C.L., Crius, P.W., Wingfield, B.D. & Wingfield M.J. (2001) Phylogeny of Calonectria based on comparisons of E-tubulin DNA sequences. Mycological Research 105(9): 1045-1052. 203. von Schrenck, H. & Spaulding, P. (1903) Bitter rot of apples. Bulletin of U.S. Department of Agriculture, Bureau of Plant Industry 44:1-54. 204. Shabi, E., Katan, T., Gera, H. & Elisha, A. (1994) Taxonomic determination of pathogenic Colletotrichum gloeosporioides from almond, anemone and avocado according to fungicide sensitivity. Phytoparasitica 21: 130-131. 205. Shankar, M., Kurtboke, D.I. & Sivasithamparam, K. (1994) Nutritional and environmental factors affecting growth and antifungal activity of a sterile red fungus against Gaeumannomyces graminis var. tritici. Canadian Journal of Botany 72(2): 198-202. 206. Shear, C.L. & Wood, A.K. (1913) Studies of fungous parasites belonging to the genus Glomerella. U.S. Department of Agriculture, Bureau of Plant Industry, Bulletin 252: 5-129. 207. Sheriff, C., Whelan, M.J., Arnold, G.M., Lafay, J.-F., Brygoo, I. & Bailey, J.A. (1994) Ribosomal DNA sequence analysis reveals new species grouping in the genus Colletotrichum. Experimental Mycology 18: 121-138.
67 208. Shi, Y., Correll, J.C., Guerber, J.C. & Rom, C.R. (1996) Frequency of Colletotrichum species causing bitter rot of apple in the southeastern United States. Plant Disease 80: 692-696. 209. Shishkina A.K. & Mamukashvili C.I. (1976) Glomerella miyabeana (Fukushi) Arx na ive v Gruzii [Glomerella miyabeana (Fukushi) Arx on willows in Georgia]. Mikologia i fitopatologia 10(4): 334-335. [In Russian] 210. Shivanna, M.B., Meera, M.S. & Hyakumachi M. (1994) Sterile fungi from zoysiagrass rizosphere as plant promoters in spring wheat. Canadian Journal of Microbiology 40: 637-644. 211. Shivanna, M.B., Meera, M.S., Kageyama, K. & Mitsuro, H. (1995) Influence of zoysiagrass rhizosphere fungal isolates on growth and yield of soybean plants. Mycoscience 36: 25-30. 212. Siefert, K.A., Wingfield, B.D. & Wingfield, M.J. (1995) A critique of DNA sequence analysis in the taxonomy of filamentous Ascomycetes and ascomycetous anamorphs. Canadian Journal of Botany 73 (Suppl. 1): S760- 767. 213. Singer, R. (1975) The Agaricales in modern taxonomy, 3rd edition. J. Cramer, Vaduz, Liechtenstein. 214. Singer, R. (1986) The Agaricales in modern taxonomy, 4th edition. Koeltz, Koenigstein, Germany. 215. Singh, M.P. & Singh, C.M. (1969) Mycotic dermatitis in camels. Indian Veterinary Journal 46: 854-856. 216. Singh, S.B., Zink, D.L., Goetz, M.A. Dombrowski, A.W., Polishook, J.D. & Hasuda, D.J. (1998) Equisetin and a novel opposite stereochemical homolog Phomasetin, two fungal metabolites as inhibitors of HIV-1 Integrase. Tetrahedron Letters 39: 2243-2246. 217. Simmonds, J.H. (1965) A study of the species of Colletotrichum causing ripe fruit rots in Queensland. Queensland Journal of Agriculture and Animal Science 22: 437-459. 218. Simmonds, J.H. (1968) Type specimens of Colletotrichum gloeosporioides var. minor and Colletotrichum acutatum. Queensland Journal of Agriculture and Animal Science 25: 178A. 219. Sivasithamparam, K. (1998) Root cortex – the final frontier for the biocontrol of root-rot with fungal antagonists: a case study on a Sterile Red Fungus. Annual Review of Phytopathology 36: 439-452. 220. Smith, B.J. & Black, L.L. (1986) First report of Colletotrichum acutatum on strawberry in the United States. Plant Disease 70:1074. 221. Smith, B.J. & Black, L.L. (1990) Morphological, cultural, and pathogenic variation among Colletotrichum species isolated from strawberry. Plant Disease 74: 69-76. 222. Smith, D. & Onions, A.H.S. (1994) The Preservation and Maintenance of Living Fungi. CAB International, UK, p.89. 223. Soledage, M., Pedras, C. & Biesenthal, C.J. (2000) HPLC analyses of cultures of Phoma sp.: differentiation among groups and species through secondary metabolite profiles. Canadian Journal of Microbiology 46: 685-691. 224. Southworth, E.A. (1891) Ripe rot of grapes and apples. Mycological Journal 6: 164-173.
68 225. Spiers, A.G. & Hopcroft, D.H. (1993) Black canker and leaf spot of Salix in New Zealand caused by Glomerella miyabeana (Colletotrichum gloeosporioides). European Journal of Forest Pathology 23: 92-102. 226. Sreenivasaprasad, S., Brown, A.E. & Mills, P.R. (1992) DNA sequence variation and interrelationships among Colletotrichum isolates causing strawberry anthracnose. Physiological and Molecular Plant Pathology 41: 265-281. 227. Sreenivasaprasad, S., Mills, P. & Brown, A. (1994) Nucleotide sequence of the rDNA spacer 1 enables identification of isolates of Colletotrichum as C. acutatum. Mycological Research 98: 186-188. 228. Sreenivasaprasad, S., Mills, P. Meehan, B.M. & Brown, A. (1996) Phylogeny and systematics of 18 Colletotrichum species based on ribosomal DNA spacer sequences. Genome 39: 499-512. 229. Sreenivasaprasad, S., Sharada, K., Brown, A.E. & Mills, P.R. (1996a) PCR- based detection of Colletotrichum acutatum in strawberry. Plant Pathology 45: 650-655. 230. Sreenivasaprasad, S., Sharada, K., Brown, A.E. & Mills, P.R. (1996b) Molecular characterization and diagnosis of Colletotrichum acutatum. 1996 BCPC Symposium Proceedings 65: Diagnostics in Crop Production. P. 211- 216. 231. Stalpers, J.A. (1978) Identification of wood-ingabiting Aphyllophorales in pure culture. Studies of Mycology 16: 1-248. 232. Stoneman, B. (1898) The development of some anthracnoses. Botanical Gazette 26(2): 69-143. 233. Strachan, D.P., Flanningan, B., McCabe, E.M. & McGarry, F. (1990) Quantification of airborne molds in the homes of children with and without wheeze. Thorax 45: 382-387. 234. Sugano, M., Sato, A., Iijima, Y., Oshima, T., Furuya, K., Kuwano, H., Hata, T. & Hanzawa, H. (1991) Phomactin A: a novel PAF antagonist from a marine fungus Phoma sp. Journal of American Chemical Society 113, 5463-5464. 235. Sugano, M., Sato, A., Saito, K., Takashi, S., Matsushita, Y. & Ijima, Y. (1996) Structure – activity relationships of Phomadecin derivatives as platelet activating factor antagonists. Journal of Medical Chemistry 39: 5281-5284. 236. Sullivan, R.S. & White, J.F. Jr. (2000) Phoma glomerata as a mycoparasite of powderry mildew. Applied and Environmental Microbiology 66: 425-427. 237. Sumner, D.R., Bell, D.K. & Huber, D.M. (1979) Pathology, host range, and ecology of a sterile basidiomycete causing root disease on corn. Plant Disease Reporter 63:981-985 238. Sutton, B.C. (1980) The Coelomycetes. Commonwealth Mycological Institute: Kew, U.K. 239. Sutton, B.C. (1992) The genus Glomerella and its anamorph Colletotrichum. In Colletotrichum: Biology, Pathology & Control, Bailey, J.A. & Jeger, M.J. eds. CABI, UK, pp. 1-27. 240. Sutton, B.C. (1998) The Coelomycetes. Commonwealth Mycological Institute: Kew, U.K. 241. Sweetingham, M.W., Cruikshank, R.H. & Wong, D.H. (1986) Pectic zymograms and taxonomy and pathogenicity of the Ceratobasidiaceae. Thansactions of British Mycological Society 86: 305-311. 69 242. Takafumi, I., Kozo, H., Tsuneaki, H., Yasuharu, Y., & Yukimasa, N. (2000) TAN-1813, a novel Ras-farnesyltransferase inhibitor produced by Phoma sp.: Taxonomy, fermentation, isolation and biological activities in vitro and in vivo. Journal of Antibiotics Tokyo 53: 765-778. 243. Talbot, N.J., Vincent, P. & Wildman, H.G. (1996) The influence of genotype and environment on the physiological and metabolic diversity of Fusarium compactum. Fungal Genetics and Biology 20: 254-267. 244. Taylor, J.W., Jacobson, D.L., Kroken, S., Kasuga, T., Geiser, D.M., Hibbett, D.S. & Fisher, M.C. (2000) Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: 21-32. 245. Taylor, G.S. & Parkinson, D. (1965) Studies of fungi in the root region. IV. Fungi associated with roots of Phaseolus vulgaris L. Plant and Soil 2: 1-20. 246. Talhinhas, P., Sreenivasaprasad, S., Neves-Martins, J. & Oliveira, H. (2002). Genetic and morphological characterization of Colletotrichum acutatum causing anthracnose of lupins. Phytopathology 92: 986-996. 247. Timmer, L.W. & Brown, G.E. (2000) Biology and control of anthracnose diseases of Citrus. In: Colletotrichum. Host specificity, Pathology, and Host- Pathogen Interaction, Prusky, D., Freeman, S., & Dickman, M. eds. APS Press, St. Paul, MN, USA. 248. Thornton, R.H. (1965) Studies of fungi in pasture soils. I. Fungi associated with live roots. New Zealand Journal of Agricultural Research 8: 417-449. 249. Turgeon, B.G. & Yoder, O.C. (2000) Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genetics and Biology 31: 1-5. 250. von Tümen, F. (1879) Fungi Pomicoli, pp. 59-60. 251. Vassiljevsky, N.I. & Karakulin, B.P. (1950) >Pathogenic Imperfect Fungi@ Izdatelstvo Akademii Nauk SSSR, Moskva-Leningrad, USSR, pp. 296-300. >In Russian@ 252. Vaillancourt, L.J. & Hanau, R.M. (1991) A method for genetic analysis of Glomerella graminicola from maize. Phytopathology 81: 530-534. 253. Vaillancourt, L.J. & Hanau, R.M. (1999) Sexuality of self-sterile strains of Glomerella graminicola. Mycologia 91: 593-596. 254. Vaillancourt, L.J, Du, M., Wang, J., Rollins, J. & Hanau, R.M. (2000) Genetic analysis of cross fertility between two self-sterile strains of Glomerella graminicola. Mycologia 92: 430-435. 255. Vilgalys, R. & Sun, B.L. (1994) Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences. Proceedings of National Academy of Sciences, USA 91: 4599-4603. 256. Villa, J. (1979) Two fungi pathogenic to frog eggs in Central America. Copeia: 4. 257. Vrålstad, T., Myhre, E. & Schumacher, T. (2002) Molecular diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Heliotiales in burnt and metal polluted habitats. New Phytologist 155: 131-148. 258. Waalwijk, C., Mendes, O., Verstappen, E.C.P., de Waard, M.A. & Kema, G.H.J. (2002) Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola. Fungal Genetics and Biology 35: 277-286.
70 259. Waid, J.S. (1974) The decomposition of roots. In The Biology of Plant Litter Decomposition I, Dickinson, C.H., & Pught, G.J.F eds. Academic Press, London, UK, pp 175-210. 260. Walker, J., Nikandrow, A. & Millar, G.D. (1991) Species of Colletotrichum on Xanthium (Asteraceae) with comments on some taxonomic and nomenclatural problems in Colletotrichum. Mycological Research 95: 1175-1193. 261. Warcup, J.H. (1959) Studies on basidiomycetes in soil. Transactions of British Mycological Society 42: 45-52. 262. Warcup, J.H. & Talbot, P.H.B. (1962) Ecology and identity of mycelia isolated from soil. Transaction of Britich Mycological Society 45: 495-518. 263. Wheeler, H.E. (1954) Genetics and evolution of heterothallism in Glomerella. Phytopathology 44: 342-345 264. Wheeler, H.E. (1956) Sexual versus asexual reproduction in Glomerella. Mycologia 48:349-353. 265. White, T.J., Bruns, T., Lee, S. & Taylor, J.W. (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, In PCR protocols: a guide to methods and application, Innis, M.A., Gelfand, D.H., Sninsky, J.J. & White, Y.J. eds. Academic Press Inc.: San Diego, California, pp. 315-322 266. Woese, C.R. (1987) Bacterial Evolution. Microbiological reviews 51: 221-271. 267. Wong, P.T.W. (1981) Biological control by cross-protection. In Biology and Control of Take-All, Shipton, M.J.C., & Ascher, P.J. eds. Academic Press, London, pp. 417-432. 268. Worth, C.M (2002) Biological Activity of Fungi Isolated from the Roots of an Australian Grass (Neurachne alopecuroidea). PhD thesis, Soil Science and Plant Nutrition Group, Faculty of natural and Agricultural Sciences, University of Western Australia. 269. Yamaguchi, Y., Masuma, R., uchida, R., Arai, M., Tomoda, H. & Õmura, S. (2002) Phoma sp. FOM-8108, a producer of gentisylquinones, isolates from sea sand. Mycoscience 43: 127-133. 270. Yang, H.A., Sivasithamparam, K. & O’Brien, P.A. (1991) An improved technique for fluorescent staining of fungal nuclei and septa. Australasian Plant Pathology 20: 119-121. 271. Yang, H.A., Zhou, J., Sivasithamparam, K., Tommerup, I.C., Barton, J.E. & O’Brien, P.A. (1994) Genetic variability in pectic enzymes of Rhizoctonia solani isolates causing bare-patch disease of cereals. Journal of Phytopathology 141: 259-266. 272. Yang, H.A. & Sweetingham, M.W. (1998) The taxonomy of Colletotrichum isolates associated with lupine anthracnose. Australian Journal of Agricultural Research 49: 1213-1223. 273. Yanna, W.H.H. & Hyde, K.D. (2001) Fungal communities on decaying palm fronds in Australia, Brunei and Hong Kong. Mycological Research 105: 1458- 1471. 274. Yun, S.-H., Arie, T., Kaneko, I., Yoder, O.C. & Turgeon, B.G. (2000) Molecular organization of mating type loci in heterothallic, homothallic, and asexual Giberella/Fusarium species. Fungal genetics and Biology 31: 7-20. 275. Zhong, S. & Steffenson, B.J. (2001) Genetic and molecular characterization of mating type genes in Cochliobolus sativus. Mycologia 93(5): 852-863. 71 276. Zhou, S. & Stanosz, G.R. (2001) Primers for amplification of mtSSU rDNA, and a phylogenetic study of Botrysphaeria and associated anamorphic fungi. Mycological Research 105(9): 1033-1044. 277. Zulfiqar, M., Brlansky, R.H. & Timmer, L.W. (1996) Infection of flower and vegetative tissues of citrus by Colletotrichum acutatum and C. gloeosporioides. Mycologia 88: 121-128.
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