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
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2004
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“Ordo fungorum chaos est”
Carl von Linné
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
53
BAA67875
BAA70884
99
397
99
BAA67886
94
C.
TUT 5954
BAA70351
C. acutatum
sensu
Simmonds
Clemson SF21
100
Glomerella acutata
60
56
Mya663
Mya662
BAA65797
100
BAA70991
UPSC2066
UPSC926
86
1117 4
G. miyabeana
1115 1
1160 1
a
c
u
t
a
t
u
m
s
e
n
s
u
BAA67435
96
L3
66
S2
NI90
64
UPSC1045
67
S17
C. acutatum
with
intermediate
morphology
l
a
t
o
S4
80
S7
L1
100
64
231
TN3
100
C. gloeosporioides
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 wellknown 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 anticancer 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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
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.
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.
Aberra, M.B., Seah, S. & Sivasithamparam, K. (1998) Suppression of the takeall 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.
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.
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.
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.
Agrios, G.N. (1997) Plant Pathology. Academic Press, USA. Pp. 410-418.
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.
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.
Alexopulos, C.J., Mims, C.W. & Blackwell, M. (1996) Introductory
Mycology. 4th edition. John Wiley & Sons, Inc., New York-…-Singapore.
Ando, N. & Takatori, K. (1982) Fungal flora of the conjuctival sac. American
Journal of Ophtalmology 94: 67-74.
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.
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.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
von Arx, J. A. (1957) Die Arten der Gattung Colletotrichum Cda.
Phytopathologische Zeitschrift 29: 413-468.
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.
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.
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.
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.
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
Barrow, M.O. (1995) Comparisons between different isolation procedures for
isolation of sterile mycelia from cruciferous and monocotyledonous plants.
M.Sc. diploma thesis, SLU.
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
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
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.
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.
Berkeley, M.J. (1954) Septoria rufo-maculans. Gardeners’ Chronicle: p. 676.
Berkeley, M.J. (1956) Glœosporium fructigenum. Gardeners’ Chronicle: p.
245.
Berkeley, M.J. (1860) Outlines of British Fungology, p. 320.
Berkeley, M.J. & Curtis, M.A. (1874). Glœosporium versicolor. Grevillea 3:
13.
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.
Boerema, G.H. & Bolle, G.L. (1975) Conidiogenesis and conidial septation as
differentiating criteria between Phoma and Ascochyta. Persoonia 8: 111-144.
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.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
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.
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.
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.
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.
Bonde, M.R., Peterson, G.L. & Maas J.L. (1991) Isozyme comparisons for
identification of Colletotrichum species pathogenic to strawberry.
Phytopathology 81: 1523-1528.
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.
Bridge, P.D., Roberts, P.J., Spooner, B.M. & Panchal, G. (2003) On the
unreliability of published DNA sequences. New Phytologist 160: 43-48.
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.
Brown, A. & Soepena, H. (1994) Pathogenicity of Colletotrichum acutatum
and C. gloeosporioides on leaves of Hevea spp. Mycological Research 98(3):
264-266.
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.
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.
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.
Bruns, T.D., White, T.J. & Taylor, J.W. (1991) Fungal molecular systematics.
Annual Review of Ecology and Systematics 22: 525-564
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.
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.
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.
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: 399402.
57
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
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.
Cisar, C.R. & TeBeest, D.O. (1999) Mating system of the filamentous
ascomycete, Glomerella cingulata. Current Genetics 35: 127-133.
de Clauser, R., Magnano, P., Corazza, L. & Chilosi, G. (1990). Colletotrichum
acutatum: agente causale di antracnose della fragola. L’Informatore Agrario
46: 71-72.
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.
Cove, D.J. (1966) The induction and repression of nitrate reductase in the
fungus Aspergillus nidulans. Biochimica et Biophysica Acta 113: 51-56
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.
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.
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.
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.
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.
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.
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.
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.
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.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
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.
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.
Desjardin, D.E. (1990) Culture morphology of Marasmius species. Sydowia
42: 17-87
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.
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: 156160.
Dewan, M.M. & Sivasithamparam, K. (1989b) Behavior of a plant growthpromoting sterile fungus on agar and roots of rye-grass and wheat.
Mycological Research 93 (2): 161-166.
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.
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.
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.
Dhingra, O.D. & Sinclair, J.B. (1986) Basic plant pathology methods. CRS
Press, U.S.
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.
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.
Domsch, K.H., Gams, W. & Anderson, T.H. (1980) Compendium of Soil
Fungi. IHW-Verlag: London - … - Toronto.
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.
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.
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.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
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.
Edgerton, C.W. (1915) Effect of temperature on Glomerella. Phytopathology
5: 247-259.
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.
Ferraz, J.F.P. (1977) Morfologia, comportamento cultural e patologenicidade
de espécies de Colletotrichum e Gloeosporium. Agronomia Lusitana 38(2):
163-179.
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): 10141020.
Freeman, S., Katan, T. & Shabi, E. (1998) Characterization of Colletotrichum
species responsible for anthracnose diseases of various fruits. Plant Disease
82(6): 596-605.
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.
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.
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.
Frost, R.R. (1964) Setae formation in Colletotrichum spp. Nature 201: 730731.
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.
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.
Förster, H. & Adaskaveg, J.E. (1999) Identification of subpopulations of
Colletotrichum acutatum and epidemiology of almond anthracnose in
California. Phytopathology 89: 1056-1065.
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.
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.
Gilliam, M.S. (1976) The genus Marasmius in the Northeastern United States
and adjascent Canada. Mycotaxon 4: 1-144
60
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
Girlanda, M., Ghihnone, S. & Luppi, A.M. (2002) Diversity of sterile rootassociated fungi of two Mediterranean plots. New Phytologist 155: 481-498.
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.
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.
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.
Guerber, J.C. & Correll, J.C. (2001) Characterization of Glomerella acutata,
the teleomorph of Colletotrichum acutatum. Mycologia 93(1): 216-229.
Gunnel, P.S. & Gubler, W.D. (1992) Taxonomy and morphology of
Colletotrichum species pathogenic to strawberry. Mycologia 84(2)157-165.
Hall, G. (1987) Sterile fungi from roots of winter wheat. Transactions of
British Mycological Society 89: 447-456.
Hanlin, R.T. (1990) Illustrated genera of Ascomycetes. APS Press MN, U.S.A.
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.
Harveson, R.M. (2002) A new soilborne disease of dry, edible beans in
Western Nebrasca. Plant Disease 86:1051.
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.
Hawksworth, D,L., Sutton, B.C. & Aisnworth, G.C. (1983) Ainsworth and
Bisby’s Dictionary of the Fungi. CMI, Kew, UK.
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.
Hepting, G.H. (1971) Disearses of forest and shade trees of the United States.
USDA Forest Service Handbook 386: 658 pp.
Hillis, D.M. (1987) Molecular versus morphological approaches to
systematics. Annual Review of Ecology and Systematics 18: 23-42.
Hillis, D.M. & Dixon, M.T. (1991) Ribosomal DNA: Molecular evolution and
phylogenetic inference. Quarterly Review of Biology 66(4): 411-453.
Hindorf, H. (1970) Colletotrichum spp. isolated from Coffea arabica L. in
Kenya. Zeitschrift für Pflanzkrankheiten und Pflanzschutz 77(6): 328-331.
Hindorf, H. (1973a). Colletotrichum – Population auf Coffea arabica L. in
Kenia. I. Eine Methode zur systematischen Trennung von Pilzpopupationen.
Phytopathologische Zeitschrift 77: 97-116.
Hindorf, H. (1973b). Colletotrichum – Population auf Coffea arabica L. in
Kenia. II. Qualitative und quantitative Unterschiede in der ColletotrichumPopulation. 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, vesiculararbuscular 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: 420430.
128. Johnston, P.R. (2000) The importance of phylogeny in understanding host
relationships within Colletotrichum. In: Colletotrichum. Host specificity,
62
129.
130.
131.
132.
133.
134.
135.
136.
137.
138.
139.
140.
141.
142.
143.
Pathology, and Host-Pathogen Interaction, D. Prusky, S. Freeman, & M.
Dickman eds. APS Press, St. Paul, MN, USA.
Jumpponen, A. & Trappe, J.M. (1998) Dark septate endophytes: a review of
facultative biotrophic root-solonizing fungi. New Phytologist 140: 295-310.
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
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: 10361041.
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.
Khairallah, S.H., Byrne, K.A. & Tabbara, K.F. (1992) Fungal keratitis in Saudi
Arabia. Documenta Ophtalmologica 79: 269-276.
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.
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.
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
Kohn, L.M. (1995) The clonal dynamic in wild and agricultural plant-pathogen
populations. Canadian Journal of Botany 73 (Suppl. 1): S1231-1240.
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.
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.
Kubicek, C.P., Bissett, J., Druzhinina, I., Kulling-Gradinger, C. & Szakacz, G.
(2002) Genetic and metabolic diversity of Trichoderma: a case study on SouthEast Asian isolates. Fungal Genetics and Biology 38: 310-319.
Kulshrestha, D.D., Mathur, S.B. & Neergaard, P. (1976) Identification of seedborne species of Colletotrichum. Friesia 11: 116-125.
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.
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: 260265.
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
159.
160.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
171.
172.
173.
174.
M.F., Dawah, H.A. & Wilson, M.R. eds.), Chapman and Hall, London,
pp.381-424.
Mayr, E. (1942) Systematics and the Origin of Species From the Viewpoint of
a Zoologist. New York: Columbia University Press.
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.
McGechan, J.K. (1977). Black spot of strawberry. Agricultural Gazette of New
South Wales 88: 26-27.
Micales, J.A., Bonde, M.R. & Peterson, G.L. (1986) The use of isozyme
analysis in fungal taxonomy and genetics. Mycotaxon 27: 405-449.
Mills, P.R., Sreenivasaprasad, S. & Brown, A.E. (1992) Detection and
differentiation of Colletotrichum gloeosporioides isolates using PCR. FEMS
Microbiology Letters 98: 137-144.
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
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.
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.
Mordue, J.E.M. (1971) Colletotrichum gloeosporioides. CMI Descriptions of
Pathogenic Fungi and Bacteria 315. Commonwealth Mycological Institute:
Kew, U.K.
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.
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.
Nattrass, R.M. (1928) The Physalospora disease of the Basket Willow.
Transactions of British Mycological Society 13: 286-304.
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.
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.
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.
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. 226.
187. Peterson, R.A. (1973) Fungicidal control of black spot (Colletotrichum
acutatum) and grey mould (Botrytis cinerea) on strawberry fruit in SouthEsatern 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): 14531457.
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): S760767.
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) PCRbased 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. 211216.
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 HostPathogen 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: 14581471.
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.
72
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