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EVALUATION OF THE PARASITIC RELATIONSHIP OF MEZANOSPORA AND
OTHER ALLIED GENERA WITH FUSARIUMOXYSPORUM
ROBERT MARTIN HARVESON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
I would first like to extend my thanks to Dr. G. N. Agrios and the committee in
charge of awarding plant pathology departmental assistantships. Without this possibility, I
would not have been able to come to Florida to complete my education. Most
importantly, I want to thank my major professor, Dr. Jim Kimbrough for listening to my
unorthodox research ideas and being willing to support them at a very difficult time in my
career. His constant sense of humor, enthusiasm, and guidance will always be appreciated
and never forgotten. I would also like to thank my other committee members-Dr. Don
Hopkins, Dr. Gary Simone, Dr. Andy Ogram, and Dr. Bill Zettler-for agreeing to serve on
my committee at the last minute and their input throughout the course of this project. Dr.
Hopkins also agreed to support peripheral areas related to this work by furnishing his
research plots and laboratory in Leesburg. Unfortunately, none of this research will
appear in this dissertation. Dr. Simone was instrumental in also sharing his lab facilities
and being there to listen to me vent and advise me when my course of study changed. Dr.
Ogram has been helpful in finding flaws in this work, which subsequently led to an
improved final product. Dr. Zettler's contribution is particularly important. He served as
proxy for both the oral exam and the final defense, hence he has become a formal
committee member in literally the twelfth hour. Without the encouragement and
assistance from Dr. Gerry Benny and Ulla Benny over the last three years, this project and
my sanity would probably not be where they are now. I want to also thank Drs. Dave
Mitchell, Dan Purcifill, and Tom Kucharek for advice and academic discussions over the
years. The influence and mentoring ability of Dr. Charlie Rush on my career is
incalculable. I owe him a debt of gratitude that can never fully be expressed or repaid. I
am also grateful to Dr. Corby Kistler for so succinctly illustrating to me that my ultimate
future led down a different path than I first began when starting my doctoral work. I want
to also express my appreciation to Galen Jones and Jack Bishko for their consultation and
recommendations for the various statistical analyses used in this study. I need to also
acknowledge my parents, Wayne and Maralyn Harveson, and my grandmother, Marietta
Martin, for always believing in me and giving unconditional support and encouragement
for any endeavor that I have chosen to undertake. Finally, I wish to thank my wife,
Tammy, for understanding my sometimes unreasonable drive to complete this work and
for reluctantly tolerating the 12-14 hour work days spent over the last year and a half.
Her willingness to consider moving to a new and totally foreign location has been a
tremendous comfort to me while finishing my work. The completion of this project would
not have been possible without her.
TABLE OF CONTENTS
ACK NOW LEDGM ENTS........................................................................... ii
A B ST R A C T ..................................................................................... vi
1 IN TR O D U C TIO N ............................................... ........................... 1
2 A TAXONOMIC STUDY OF MELANOSPORA AND ITS ALLIES..... 17
Introduction........................... ....................... 17
Materials and Methods............................ ............... 22
Results................ ........... ...... ............. ........ 25
D iscussion................................... ............................ ..... 38
3 ENHANCEMENT OF GROWTH AND SPORULATION OF
MELANOSPORA AND ITS ALLIES BY FUSARIUM 40
M materials and M ethods................................................ ......... 43
R esults.............................................. ................. ......... 51
D iscussion.................................. ....................... ......... 63
4 PARASITISM AND MEASUREMENT OF DAMAGE TO FUSARIUM
OXYSPORUMBY SPECIES OF MELANSOPORA,
SPHAERODES AND PERSICIOSPORA................... .......... 70
Introduction........................... ...................................... 70
M materials and M ethods....................................................... 72
Results..................................................... .... ................. ....... 79
D iscu ssio n.............................................................. ............ 90
5 ECOLOGY AND USE OF SPHAERODES RETISPORA VAR.
RETISPORA FOR THE CONTROL OF FUSARIUM WILT
OF WATERMELON ............................................................ 93
Introduction.............................. ..... 93
Materials and Methods............................. .......... ...... 95
R esults.................................................................. ............. 100
D iscussion.................................................................. ............ 110
6 SUMMARY AND CONCLUSIONS..................................... 116
REFERENCES ................................................ 120
BIOGRAPHICAL SKETCH.......................................... 129
Abstract of Dissertation Presented to the Graduate School of the University of Florida
in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
EVALUATION OF THE PARASITIC RELATIONSHIP OF MELANOSPORA AND
OTHER ALLIED GENERA WITH FUSARIUMOXYSPORUM
Robert Martin Harveson
Chairman: J. W. Kimbrough
Major Department: Plant Pathology
Fusarium wilt, caused by Fusarium oxysporum, has been a problematic disease for
many different crops for over 100 years. The discovery of a group ofpyrenomycetous
ascomycetes found naturally in association with severalformae specials ofF. oxysporum
from field samples, has generated the interest to investigate the use of these fungi as
biocontrol agents for watermelon wilt caused by F. oxysporum f sp. niveum (FON). The
first of these fungi was isolated from the sugar beet pathogen, F. oxysporum f. sp. betae,
and was identified asMelanospora zamiae. Later findings from other wilt-infected crops
yielded another five isolates that very closely resembled the original M zamiae. However,
based upon current taxonomic criteria, these five isolates were determined to consist of
two distinctly different genera, Sphaerodes retispora var. retispora, and Persiciospora
All six isolates were found to be strongly dependent upon F. oxysporum for
growth and sporulation. Linear growth and production ofperithecia were significantly
improved for all isolates in dual culture with F. oxysporum or growing in filtrate from a
liquid culture ofFusarium. Significant differences in growth were observed among and
between the different pyrenomycetes. Without the presence ofF. oxysporum or its
metabolic growth products, growth and reproduction were generally very poor for all
isolates. The better media without Fusarium tended to be those composed of simple
sugars like dextrose or maltose, while those producing the worst results contained starch
or cellulose. All isolates were determined to be parasitic on F. oxysporum through the use
of several cultural assays, but not all were pathogenic to FON. Persiciospora caused no
detectable reduction in hyphal growth, whereas the Sphaerodes isolates were the most
Isolate LE (S. retispora) was selected for disease reduction evaluations in the
greenhouse. It was incorporated into sodium alginate pellets and delivered to the infection
court by either root treatment or amendment to infested soils before transplantation. Yield
data were collected after eight weeks, and consisted of mortality counts and total plant dry
weights. This technique did significantly improve plant dry weights of treated plants
compared to the FON-inoculated controls. These are promising results for an alternative
method for managing Fusarium wilt of watermelon. However, it is not known whether
this procedure is efficacious or even feasible under field conditions. If proven to be
successful in the field, this procedure could also presumably be utilized for disease control
with other vegetable crops in Florida that are similarly grown as transplants. However,
care must be taken for the selection of the potential biocontrol isolates, because not all
included in this study were found to equally colonize or induce deleterious effects upon all
formae specials ofF. oxysporum.
The genus Fusarium was first erected in 1809 by Link when he described F.
roseum as the first species (Booth 1984). Fusarium species are ubiquitous soil fungi
found in many climatic extremes such as deserts and the arctic, in addition to temperate
and tropical regions of the world. They can be saprophytes and pathogens infecting both
animals and plants (Nelson 1981). Because of variation in culture, it has been difficult for
workers to correctly classify this genus into species. The basis for Fusarium classification
was established by Appel and Wollenwebber (1910). They placed species studied into
sections based on various morphological characteristics (Coons 1928). These sections
were later named and expanded to include a type species for each section (Wollenwebber
Of the 43 species of Fusarium listed by Booth (1971), 27 are pathogenic to plants
and cause some of the most important diseases in agriculture. The pathogens may be
divided into three main categories: the wilts, the root rots caused primarily by F. solani,
and those fungi attacking graminaceous plants, such as F. moniliforme, F graminearum,
F. culmorum, and F. avenaceum (Price 1984). The vascular wilts, caused byformae
specials ofF oxysporum, are considered to be the most economically important due to
the diversity of plants attacked and the destructiveness of the diseases (Nelson 1981).
Fusarium oxysporum was the first pathogenic species described, and was included
in the section Elegans by Wollenwebber and Reinking (1935). They divided this section
into three subsections and further subdivided these subsections into 10 species, 18
varieties, and 12 forms (Nelson 1981). Their taxonomic system was difficult to use by
others because they placed such great emphasis on spore measurements and septations,
which are highly variable.
Snyder and Hansen (1940) simplified Wollenwebber and Reinking's system by
combining all members of the section Elegans into one species, F. oxysporum, by the
utilization of standard culture media and incubation temperatures. The different parasitic
forms were recognized by their selective pathogenicity to specific crops and were
designated asformae specials. Formae specials are also further subdivided into
biological races based on pathogenicity to differential cultivars containing different
Recognition of the limiting effect of the Fusarium wilts on crop production goes
back to the 1890's. Atkinson (1892) described the first Fusarium wilt disease, and
referred to it as "Frenching." It had been causing serious losses to Alabama cotton
growers. He demonstrated the presence of gummy substances blocking the vascular
elements and illustrated the phialids producing the microconidia, which has proven to be
the main diagnostic criterion for identification ofF. oxysporum. He also named the causal
agent as F. vasinfectum (Atkinson 1892), which is now known to be the form name for
Fusarium wilt of cotton. There are many additional early examples of crops threatened by
Fusarium wilts including potatoes, celery, tomatoes, flax, bananas, cabbage, cowpea, and
The cultivated watermelon, Citrullus lanatus (Thunb.) Matsum and Nakai, is the
most economically important member of the genus Citrullus, and is grown for fresh
consumption as a desert vegetable. The citron, C. lanatus var. citroides (L. H. Bailey)
Mansf, is not eaten fresh, but is candied. It has also been important in breeding for
disease resistance because it readily crosses with the watermelon, and is resistant to
Fusarium wilt. Watermelons are an important truck crop in the South. Commercial
production is approximately 184,000 acres nationally, concentrated in Florida, Texas,
Georgia, and California (Peirce 1987). It has an estimated gross market value of $150
million (Martyn 1986).
The watermelon is considered to be native to the southern deserts of Africa.
David Livingstone reported that wild populations covered vast areas of the Kalahari
Desert after unusually heavy rains. Both natives and animals were observed consuming
the fruit (Livingstone, 1859). Carrier, however, concluded wrongly that it was indigenous
to North America based on descriptions by French explorers of Indians growing melons in
the Mississippi Valley (Carrier 1924).
The actual time and place of domestication of the watermelon is unknown, but by
2000 B.C. it was being cultivated along the Nile by Egyptians and widely grown by
prehistoric agricultural peoples of sub-Sahara Africa (Peirce 1987, Sauer 1994).
Watermelons were introduced to India and China by A.D. 1100, and brought to Europe by
the Moors. Popularity in Europe spread slowly, presumably because of the long, hot
growing season required for good yields (Sauer 1994). In North America, Spanish
colonists were growing them in Florida during the 1500's, and by 1650, Dutch and British
colonies in the Northeast were cultivating them abundantly (Peirce 1987). Watermelons
were enthusiastically adopted by the North American Indians. By 1700, they were being
grown from the Great Lakes region to the American southwest and lower Rio Grande
Valley of Texas (Sauer 1994). This probably explains Carrier's mistaken conclusion that
they originated in North America. Hawaiians also began growing watermelons after being
given seeds by Captain Cook on one of his voyages (Sauer 1994).
Fusarium wilt of watermelon was first described in 1894 from Georgia and South
Carolina by E. F. Smith (Smith 1894). By the time of his second report (Smith 1899), it
had been identified throughout the melon-growing areas of the Southeast ranging from
Texas to Maryland. Taubenhaus began investigations on this disease in Texas in the late
teens because it was considered to be the most important and destructive disease affecting
watermelons (Taubenhaus 1920). He showed that the organism was widely distributed at
different soil depths, that disease became more severe and spread more rapidly after using
manure than with commercial fertilizers, and that activity of the pathogen was greater
under warm conditions than cool. He further concluded that after a three year absence of
melons in fields, pathogen levels were reduced, but not completely eradicated
(Taubenhaus 1920). Elsewhere throughout the South, large tracts of land, previously
cropped profitably to watermelons, were rendered unfit and abandoned because of this
disease. It is now known to be widespread throughout the U.S. and wherever else
watermelons are grown, and is considered to be the primary limiting factor to production
in many of these areas.
Smith's (1899) research on the watermelon wilt was combined with a wilt of
cowpea and extended work on the wilt disease of cotton originally described by Atkinson.
He gave the fungi causing these diseases different names: F. niveum, F. tracheiphila, and
F. vasinfectum for watermelon, cowpea, and cotton, respectively. This was done because
they could not be distinguished in pure culture, but he determined that they differed in
pathogenicity after cross inoculation studies on each crop host. He further observed that
land infested with the melon fungus should not be planted with watermelons, but could be
grown with cotton or cowpea. He also demonstrated that the pathogen could be spread to
new land on seeds, in run-off water, barnyard manure, and in soil clinging to farm
equipment (Smith 1899).
These outstanding researches were marred however, by Smith's erroneous
conclusion that Neocosmospora, found on dead stems, was the teleomorph of the
Fusarium wilt pathogen (Smith 1899). This conclusion caused some confusion until it was
first challenged by Higgins in 1909 (Higgins). He proved that Neocosmospora was a
saprophyte and distinct from the organism causing the wilt disease of watermelon. This
was subsequently confirmed by others (Taubenhaus 1920, Wollenwebber, 1913).
The pathogen survives in soil primarily as chlamydospores and may remain
dormant for many years. This stage serves as the predominant source of inoculum
although infection may also occur via macro- and microconidia (Martyn 1996).
Chlamydospores may germinate in response to host or nonhost roots, or by contact with
fresh noncolonized plant debris (Nelson 1981). After chlamydospore germination, conidia
and new chlamydopsores may be formed, in addition to hyphae. Penetration then occurs
on the host plant either directly or through wounds.
Once the pathogen enters a susceptible host, it invades the vascular system where
it becomes systemic and moves throughout the plant by means of hyphal growth or
movement of microconidia in the vascular stream (Martyn 1996, Nelson 1981). It is
generally confined to the xylem vessels and tracheids initially, but may also enter the
parenchyma later. As disease development progresses, the fungus may also move into
adjacent tissues such as the pith, cambium, phloem, or cortex (Nelson 1981). At this point
wilt symptoms are severe and often result in plant mortality. After the plant dies,
chlamydospores are reformed from mycelium or macroconidia and serve as the
overwintering propagule. In resistant plants, colonization is generally limited to the cortex
and several vessel elements (Martyn 1996).
Fusarium oxysporum f. sp. niveum has been shown to be seedborne in low levels,
however the major mechanisms of spread are via movement of infested soil or plant debris.
The disease is usually more severe on light, sandy soils with a low pH, and has an
optimum soil temperature requirement between 20-27 C (Martyn 1996).
Control of this disease is very difficult to achieve. It was noted long ago that
spraying plants with Bordeaux mixture (CaSO4 and lime) or a similar fungicide had little
or no effect because of the location of the fungus inside the vascular system (Taubenhaus
1920). Control with broad spectrum soil fumigants is erratic and expensive (Martyn
1986). However, it has been shown to be effective under certain conditions. Hopkins and
Elmstrom (1979) successfully obtained commercial yields on severely infested lands in
Florida utilizing sodium azide and chlorinated hydrocarbons combined with methyl
isothiocyanate (DD-MENCS). This was only possible with moderate or highly resistant
cultivars, but not with susceptible ones. They further demonstrated that control was more
consistent and effective when applications were made prior to bed formation due to
deeper penetration of the chemicals (Hopkins and Elmstrom 1979). However, some
studies since this time have provided evidence that recolonization ofFusarium after
fumigation still can occur in some cropping systems (Marois and Mitchell 1981).
Biological control is another form of disease management that is becoming
increasingly popular and important for Fusarium wilt control. Cook and Baker (1983)
define biological control as "the reduction of the amount of inoculum or disease-producing
activity of a pathogen accomplished by or through one or more organisms other than
man". Their very broad-based definition includes cultural practices, breeding for
resistance, or the use of antagonists, nonpathogenic, or other beneficial microorganisms
either introduced or resident (Cook and Baker 1983).
Numerous cultural methods that attempt to modify the environment to the
advantage of the host have proven to be of some value for Fusarium wilt management.
Avoiding monoculture of watermelons is one of the simplest and most important of these
methods. In most cases, continuous culture tends to increase inoculum potential of soils.
An exception to this was noted with the use of the moderately resistant cultivar Crimson
Sweet. After four-five years, it induced a wilt suppressive soil (Hopkins and Elmstrom
1984, 1987). Because of the capacity of Fusarium to survive as chlamydospores or on
roots of plants as symtomless carriers, control through crop rotation alone appears to be
unlikely. Rotation also should be combined with long periods (five-seven years) without
growing watermelons. Planting melons on new land is another excellent strategy to avoid
wilt, however, this is also very impractical for many producers. Soil solarization is
another cultural method that has shown promise by delaying onset of wilt and reducing
overall disease incidence. It reduced initial inoculum and overall population levels in soil,
but was only effective in fields with moderate to low inoculum levels (Martyn 1986,
The management of crop nutrition has also resulted in varying levels of success in
controlling Fusarium wilt diseases. These diseases have been demonstrated to be less
severe in soils with a high pH. Since the addition of many elements profoundly influences
soil pH, it is not known whether the pH itself or the increased plant nutrition is responsible
for wilt control. One possible explanation for this phenomenon has been hypothesized that
at high pH, many micronutrients tend to become unavailable. Fusarium oxysporum has a
high requirement for these elements, and its inability to grow and sporulate under these
conditions may explain reduced disease at high pH (Jones et al 1989). Evidence for this
idea was obtained when tomato wilt still occurred in limed soils in Florida after certain
combinations of trace elements (iron, manganese, and zinc) were applied (Jones and Woltz
1981). Regardless of the mechanism responsible for reducing disease severity, Jones et al.
(1989) demonstrated that the use of lime, N03-N instead of NH4-N, and low phosphorus
was highly effective for Fusarium wilt of tomato. However, Hopkins and Elmstrom
(1976) found no significant differences in watermelon wilt control comparing the nitrogen
fertilizer treatments of NO3 and NH4.
The most effective control measure for any Fusarium wilt disease, other than long
rotations and planting on new land, is generally considered to be the use of resistant
cultivars. The first known instance of developing a plant variety with resistance to a
Fusarium disease by hybridization was for watermelon wilt. Orton (1907, 1909) was able
to create the wilt resistant cultivar Conquerer by crossing the inedible but resistant citron
melon with the susceptible cultivar Eden. Conquerer was not widely used commercially
due to certain horticultural limitations, but it was employed as a parent for early resistance
breeding (Porter and Melhus 1932). Since that time, extensive breeding programs have
successfully produced a number of wilt resistant cultivars, which had their beginning with
this original cultivar (Walker 1957). The major disadvantage of using genetic resistance is
that performance of individual cultivars may vary among different locales because of
different disease reactions of pathogen races. Currently there are 3 known races ofF.
oxysporum f. sp. niveum (races 0,1,and 2). Race 0 is pathogenic only to cultivars with no
resistance genes. Since most modern cultivars have some Fusarium wilt resistance, this
race of the pathogen is no longer important. High levels of race I resistance are available
for many good commercial cultivars. Race 2 isolates have only been identified, to date,
from Israel, Texas, Oklahoma, and Florida (Martyn, 1996), but will undoubtedly be
problematic for watermelon growers from other locations within the near future.
Induced resistance and suppressive soils are two of the best known and successful
biological control methods that utilize microorganisms. Induced resistance, also known as
cross protection and immunization, has been extensively documented against all classes of
pathogens in many different plant pathosystems, however, it has been best exemplified for
disease control of foliar pathogens of cucurbits, beans, and tobacco (Kuc 1982, 1990).
Induced resistance has also been shown to be effective for a number of vascular
wilt diseases. Reduced Fusarium wilt has been reported by inoculation of susceptible
hosts with nonpathogenic races or different wilt pathogens prior to challenge with the
target pathogen. The selection and subsequent inoculation of nonpathogenic strains ofF.
oxysporum have been shown to successfully reduce wilt by pathogenic strains in celery
(Schneider 1984), cucumber (Paulitz et al. 1987), sweet potato (Ogawa and Komada
1985), and watermelon (Biles and Martyn 1989, Larkin et al. 1996).
The inoculation of non-host pathogens has additionally been documented in
reducing disease severity. Phillips et al. (1967) observed reduced wilt of tomato by
inoculations of Cephalosporium spp. Another interesting example by Gessler and Kuc
(1982) demonstrated that root protection from F oxysporum f. sp. cucumerina on
cucumber could be induced sytemically with otherformae specials (melonis and
conglutinans), or by leaf inoculation with Colletotrichum lagenarium or tobacco necrosis
virus. Specifically, watermelon wilt has similarly been proven to be effectively reduced by
preinoculation with the non-watermelon pathogens F oxysporum f.sp. cucumerina (Biles
and Martyn 1989) and F oxysporum f. sp. lycopersici, Verticillium alboatrum, and
Helminthosporium (Cochliobolus) carbonum (Shimotsuma et al. 1972).
Conversely, Wymore and Baker (1982), concluded that using this approach to wilt
control of tomatoes under greenhouse conditions was not practical. The induced
resistance initially obtained required inoculum densities of the cross protection agents to
be of equal or greater than that of the challenge pathogen. Additionally, the protection
that did occur was only temporary by delaying symptoms and eventually produced disease
equal to that of the inoculated controls (Wymore and Baker 1982).
Over the last 25-30 years, the greatest progress achieved in biological control
employing soil microorganisms has been investigating and understanding the principles
responsible for pathogen suppressive soils (Cook 1990). Most soils are suppressive some
degree, but those that are pathogen suppressive have a unique ability to reduce the impact
of soilborne pathogens following certain agronomic management systems. Suppressive
soils have been defined by Cook and Baker (1983) as those soils where disease never
becomes established or persists, becomes established but causes little damage, or is
established and causes disease for a short time but afterward becomes less problematic
even though the pathogen is still detectable in these soils.
The occurrence of pathogen suppressive soils over the years has been recognized
primarily because of observed disease severity in crops being lower than was anticipated
(Baker and Cook 1974). In general, suppressiveness may act directly on the pathogen by
affecting propagule growth and survival, or it conceivably can work indirectly through the
host in the form of cross protection or induced resistance (Cook 1990, Cook and Baker,
1983). This example of induced resistance is different from the one previously described
by being resident or naturally-occurring in the soil rather than being introduced.
Pathogen suppressive soils are now known for 10-15 plant diseases (Cook 1990,
Cook and Baker 1983). With the possible exception of take-all suppressive wheat soils
Gaeumannomyces graminis var tritici), the most extensively studied suppressive soils
worldwide are those of Fusarium wilt. The existence of wilt suppressive soils has been
recognized for over 100 years, and research on them has been conducted for greater than
70 years. Atkinson (1892) first noticed that cotton wilt was more severe on sandy soils in
Alabama. Later work on wilt suppressive soils from Central America involving banana
wilt (F. oxysporum f sp. cubense) and the pea wilt caused by F.oxysporum f. sp. pisi in
Wisconsin similarly noted that disease severity was less devastating on heavier clay soils
than on lighter, sandy ones (Toussoun 1975, Reinking and Mann 1933, Walker and
A number of other agricultural soils throughout the world exhibit a similar
phenomenon of disease reduction, including several areas of the San Joaquin and Salinas
valleys of California (Scher and Baker 1980, Smith and Snyder 1971), the Chateaurenard
region of the Rhone Valley in France (Alabouvette 1986, 1990), and on an experimental
farm in Central Florida (Hopkins and Elmstrom 1984, 1987). Most of these soils have a
number of characteristics in common. They tend be alkaline and heavy-textured.
Suppressiveness is destroyed by heat treatments, but can be restored by the addition of
small quantities of suppressive soil. This suggests a biological origin for induction and
maintenance of suppression. Suppression to a particularforma specialist is also
suppressive to other pathotypes ofF. oxysporum, but not to nonpathogenic F. oxysporum
or other soil pathogens. This also holds true for other species ofFusarium. Pathogen
suppressive soils also tend to have higher concentrations of overall soil microflora than do
wilt conducive soils (Cook and Baker 1983 ) Competition for iron or carbon between the
pathogens and resident bacteria and/or nonpathogenic fusaria is believed to be responsible
for suppression in many of these soils. The mechanism of suppression in other soils seems
to be a general resistance in plants induced by nonpathogenic F oxysporum or growth-
promoting rhizobacteria (GPR). They have the ability to infect plants before the
pathogens due to a higher degree of aggressiveness.
The watermelon wilt suppressive soil from Florida is the curious exception to
several of these traits characteristic of most wilt suppressive soils. It has similarly been
shown to harbor higher populations of soil microorganisms than wilt conducive soils, but
is different by its sandy, acidic nature and low organic matter content. This soil is also
unique by suppression being induced by monoculture of the moderately resistant cultivar
Crimson Sweet (Hopkins and Elmstrom 1984, 1987). The suppressive effect of the other
soils is believed to be constituitive. The primary mechanism of suppression in the Florida
soil is not competition for micronutrients among native soil organisms, but an induced
resistance caused by the high populations of nonpathogenic F. oxysporum isolates.
Mycoparasitism is another method that has been utilized in controlling soilborne
pathogens, and this term is defined as the parasitism of one fungus by another
(Hawksworth et al. 1995). Barnett and Binder (1973) divided mycoparasites into two
major categories based upon their mode of obtaining nutrition. The highly aggressive
necrotrophic parasites make contact with hosts, secrete hyphal wall-degrading enzymes
causing death of the host cell, and finally utilize nutrients released through this process
(Barnett and Binder 1973, Jeffries and Young 1994). Necrotrophic parasites tend to have
large host ranges and are largely unspecialized as to their mechanism of parasitism. Most
members of this group are opportunisitc and capable of living saprophytically or
competing with other organisms for space and nutrients (Jeffries and Young 1994).
The biotrophic parasites, conversely, obtain their nutrition from living host cells.
The living host supports the parasite and may appear unaffected at first. Throughout the
years of close association, some biotrophic parasites have lost the ability the synthethize
one or more required nutrients, and must rely upon the host to supply them. Not only
must the host supply these food stuffs, but the parasite must also be capable of
successfully retrieving them from the host. Thus a physiological balance is established in
this relationship and the parasite appears to be highly adapted (Jeffries and Young 1994).
This is probably why biotrophic parasites tend to be more restrictive in host range than do
necrotrophs. They also form specialized infection structures, or what Jeffries and Young
(1994) call "host-parasite interfaces." Additionally no biotrophic parasite has ever been
demonstrated to produce cell-degrading enzymes or exotoxins (Jeffries and Young 1994).
There are 3 separate types of biotrophic relationships that can be distinguished
based upon the morphology of this interaction. There are those in which the whole thallus
of the parasite enters the hyphae of the host, and are called intracellular biotrophs. The
haustorial biotrophs send a lobed haustorium into host cells via invagination of the
plasmalemma. Nutrients are absorbed from host cytoplasm back to the parasite through
this haustorium. The third type is apparently very unusual and the most specialized of the
three. It involves physical contact between host and parasite, but no penetration occurs.
Until the ultrastructural studies of Hoch (1977), it was not understood specifically how
this relationship functioned. His work showed that nutrients are obtained by the parasite
through the formation of channels of contact between closely appressed host-parasite
hyphae. Specialized contact or buffer cells form on the parasite and contact the host
hyphum without penetration. These cells serve as a stable interface for the transfer of the
nutrients through channels similar to plasmodesmata (Hoch 1977). The plasma membrane
of both organisms come into contact and fuse, therefore cytoplasmic continuity is achieved
(Hoch 1977, Jeffries and Young 1994). Since this is such an important feature of this type
of parasitism, it has been renamed fusion biotrophism, to replace the older term, contact
biotrophism (Barnett and Binder 1973, Jeffries and Young 1994).
Fusion biotrophs consist primarily ofanamorphic hyphomycetes, although several
teleomorphic states of the Ascomata have also been reported (Jeffries and Young 1994).
These include several closely related genera like Gonatobotrys, Gonatobotryum, and
Nematogonum (Whaley and Barnett 1963, and Barnett and Binder 1973). The
pyrenomycete Melanospora zamiae has also been reported to parasitize a large number of
fungi, (Hanlin et al 1993, Jordan and Barnett 1978), which is considered to be unusual for
a fusion biotrophic mycoparasite.
Interestingly, one of the first biotrophic parasites discovered was that of
Gonatobotrys simplex (Whaley and Barnett 1963). It also happened to serve as the model
for Hoch's ultrastructural work with fusion biotrophs (Hoch 1977). The teleomorph of G.
simplex has further been identified asMelanospora damnosa, which is itself a fusion
biotrophic parasite ofFusarium sacchari, causal agent of corn stalk rot (Vakili, 1989).
Because of the unique ability ofF. oxyporum to inhabit and colonize the
specialized niche inside a plant's vascular system, no studies have been published
attempting to control wilt diseases with mycoparasites that act directly on the pathogen.
Cook and Baker (1983) point out that in general, the more internal a pathogen is during
the host-pathogen interaction, the less vulnerable it is to antagonists because of the degree
of protection afforded by the plant. The vascular wilt fungi, and F. oxysporum in
particular, have only a brief epiphytic stage and exist almost entirely as endophytes within
the root cortex and vascular tissue. However, these same authors also point out that the
specialization required for this type of life strategy can also lead to an increased
vulnerability to the physical environment and antagonists when not residing within the host
(Cook and Baker 1983, Baker and Cook 1974).
Owing to the difficulties associated with direct observation of living fungi in the
soil, most information concerning mycoparasites and their hosts have been obtained, by
necessity, from dual cultures in vitro, or by inference from field observations (Jeffries and
Young 1994). Mycoparasites are also notoriously difficult to observe in the lab because
of slow or poor growth in axenic culture. Very little is known about the natural
occurrence of fusion biotrophic mycoparasitism, however because of these reasons, this
field is wide open for exploration.
The impetus behind this research began while collecting isolates ofF. oxysporum f
sp. betae from sugar beet in Texas. After several weeks, approximately 10-15% of the
isolates from the field became contaminated with an unknown ascomycete that seriously
affected growth and vigor ofFusarium in culture. This unknown fungus was identified as
Melanospora zamiae, and was determined to be occurring naturally with F. oxysporum f.
sp. betae. Over the last year, five additional isolates ofPyrenomycecious ascomycetes
have been found in Florida associated with field populations of three additional plant
pathogenicformae specials ofF. oxysporum. These organisms are closely related to, but
distinct from, M. zamiae.
This study will attempt to address some of these aspects involving the
mycoparasitism ofF. oxysporum byM. zamiae and other allied genera. None of these
additional genera have been conclusively shown to be parasitic. Chapter 2 will cover the
identification and taxonomy of this group of Pyrenomycetes. Chapter 3 will address the
stimulation of growth and reproduction of the parasites by the F. oxysporum hosts.
Chapter 4 will determine host range and measurable damage caused by the parasites to F.
oxysporum in culture. Chapter 5 will delve into some ecological aspects of the parasite
and its ability to colonize both plant hosts and F. oxysporum, and to survive in soil.
Chapter 5 will also attempt to combine knowledge from other aspects of this study and
attempt to utilize one or more of these parasites as a biological control agent for
watermelon wilt at the greenhouse level.
A TAXONOMIC STUDY OF MELANOSPORA AND ITS ALLIES
During the course of studying soilborne isolates ofFusarium causing root diseases
of a number of crops, species resembling Melanospora were found in constant association
with these organisms. These Melanospora-like strains appeared to be mycoparasitic and
causing damage to the fusaria. Because there has been confusion as to the limits of
Melanospora, a study of this genus and its closely related allies appeared warranted.
The genus Melanospora was first erected in 1837 by Corda (1837). He described
three species in the following order, M. zamiae, M. chionea, and M. leuchotricha (Shear
and Dodge, 1927). Because he did not designate a type species, most authors have
considered M zamiae to be the type (Von Arx 1981, Cannon and Hawksworth, 1982),
although Clements and Shear described M chionea as the type (1931).
Melanospora is currently classified as belonging to the Ceratostomataceae in the
Sordariales (Hawksworth et al, 1995, Barr, 1990). However, this genus has long been a
taxonomic problem for mycologists and has resided in at least five orders and six families
since its first description. Gilmer (1957), and Gwynne-Vaughn and Barnes (1927) both
included it in the Nectriaceae of the Hypocreales while Alexopoulos (1962) and Stevens
(1974) placed it in the Melanosporaceae of the same order.
Numerous authors have placed Melanospora in the now invalid order Sphaeriales.
Within this order, Melanospora has been included in the Melanosporaceae (Dennis 1978,
Bessey 1935,1961, Munk 1957), Hypocreaceae (Clements and Shear 1931),
Ceratostomataceae (Fitzpatrick 1934), and the Xylariaceae (Nannfeldt 1932).
Based on centrum development, Luttrell (1951) included Melanospora in the
Melanosporaceae of the Diaporthales. This placement was established to include genera
from the Hypocreales that had a Diaporthe-like centrum, but resembled members of the
Diaporthaceae in centrum-type only.
Weymeyer (1975) recognized three families in the Melanosporales,
Chaetomiaceae, Melanosporaceae, and Ceratocystaceae, and placed Melanospora in the
Melanosporaceae. He regarded the order as intermediate between the Plectascales and the
Hypocreales or Sphaeriales. Alexopoulos et al. (1996), listed the genus in the
Melanosporales, but designated it as a monotypic order with no familial rank.
Over one hundred species have been named Melanospora (Goh and Hanlin 1994),
but Doguet (1955) considered most to be synonyms. Doguet introduced a broad concept
of the genus and arranged species into groups based on shape and ornamentation of the
ascospores. Recently this concept of Melanospora has been narrowed, and many genera
have now been separated from Doguet's broad treatment of the genus (Udagawa and Cain
1969, Hawksworth and Udagawa 1977, Cannon and Hawksworth 1982). Based on
Cannon and Hawksworth's (1982) study utilizing scanning electron microscopy,
Melanospora is now restricted to those fungi with smooth ascospore walls and depressed
Of all the many synonyms used for species ofMelanospora, the two most
commonly accepted have been Sphaeroderma Fuckel, and Microthecium Corda.
Sphaeroderma was distinguished from Melanospora by the absence of an ostiolar neck,
but this trait is considered to be highly variable (Cannon and Hawksworth 1982).
Microthecium was retained for cleistothecial species, but otherwise identical to
Melanospora (Udagawa and Cain, 1969, Hawksworth and Udagawa 1977). Scanning
electron microscopy studies have now confirmed that both cleistothecial and perithecial
species with reticulately ornamented ascospores constitute a distinct genus for which the
name Sphaerodes Clem. was available (Von Arx 1981). Because species ofMicrothecium
are characterized by smooth-spored ascospores with depressed germ pores, they are now
synonomized with Melanospora, while those with reticulated spores formally placed there
have been transferred to Sphaerodes (Cannon and Hawksworth 1982).
Sphaerodes was erected by Clements in 1909 to include S. episphaeria, and was
distinguished from Sphaeroderma by the lack of a subiculum (Clements and Shear 1931).
Besides the reticulated spores, Sphaerodes differs from Melanospora in the structure of
the germ pores. It has protruding, tuberculate pores, while Melanospora has pores either
level with spore surface or slightly sunken or depressed (Cannon and Hawksworth 1982).
Persiciospora is a newly created genus to retain Melanospora-like pyrenomycetes
with yet another type of ascospore morphology. It is distinguished from Melanospora's
smooth spores and Sphaerodes' reticulated ones by producing spores with delicately
pitted walls similar to a peach seed (Cannon and Hawksworth 1982).
Apart from ascospore structure and morphology, Melanospora and its allies,
Sphaerodes and Persiciospora, are similar in numerous respects. They are characterized
by superficial, nonstromatic perithecia composed of angular pseudoparechymatous cells.
Perithecia are spherical, amber to pale yellow in color and are solitary to gregarious.
Four-to eight-spored, clavate asci are soon deliquescent with spores filling the perithecial
cavity and exuding out of the ostiolar neck in a slimy cirrhus. Ascospores are dark, and
ellipsoid or oval-shaped. The yellowish, translucent perithecia appear black when filled
with mature ascospores. Ostiolate species have long or short beaks, often ringed with
hyaline setae of varying lengths. Centra are composed of thin-walled psuedoparenchyma
and lack paraphyses at maturity. Many species have additionally been suspected of being
parasitic on other fungi.
The primary reason for the extensive taxonomic changes for Melanospora is that
its characteristics are not consistently applicable to all the families and orders within which
it has been classified. It does not fit well within any of the ranks. It was placed in the
Hypocreales because of its fleshy, unpigmented perithecium, and phialidic anamorphs
(Rehner and Samuels 1995) Many species of this order, such asMelanospora, also have
clavate asci and a presumed ecological role as mycoparasites. However, Melanospora
differs from other members of the Hypocreales in its aseptate, dark ascospores with germ
pores at each end, and deliquescent asci. Melanospora also lacks the apical paraphyses or
pseudoparaphyses of the Nectria-type centrum development characteristic of this order
(Rehner and Samuels 1995).
There has never been a standard, universally recognized classification for the
Sphaeriales. It once included all pyrenomycetes, but the Hypocreales, Clavicepitales, and
Laboulbeniales were removed based on centrum development and ascal characteristics
(Luttrell 1951). The remainder constituted the single, heterogeneous order Sphaeriales,
which did not fit well within any other order of the Euascomycetes (Wehmeyer 1975).
Therefore, it is no longer recognized as an acceptable taxonomic rank. Members of this
old order were characterized by clavate or cylindric asci, that were borne in a hymenial
layer. Asci opened by a round, apical pore, and were forcefully ejected. They additionally
contained haemathecia consisting of varying numbers of paraphyses (Alexopoulos 1962).
Melanospora and its allies were considered members of this catch-all group probably
because of their varied, hybrid-like characteristics. Since they had to be placed
somewhere, this was considered to be the best location. They were similar in the
possession of clavate asci borne in a true perithecium, but were different by lacking the
paraphyses or poroid asci.
Luttrell (1951) placed Melanospora in the Diaporthales based on similar early
perithecial development. However, this group is different from Melanospora in almost
every other respect, including persistent, poroid asci with a refractive ring at the apex, and
ascal development at several levels within the perithecium (Alexopoulos et al. 1996).
The current placement ofMelanospora in the Sordariales is not without
inconsistencies either. This order is characterized by dark perithecia or cleistothecia with
tough, leathery peridia. Asci are produced in persistent, basal fascicles and usually have a
thin refractive apical ring, although ascus walls may be evanescent in some species. The
Sordariales also have both deliquescent paraphyses and psuedoparenchyma. Spores are
variable in color, shape, and ornamentation, and many species have germ pores.
Melanospora and its allies have a number of like characteristics, such as evanescent asci
and variation in spore morphology and ornamentation, but still defy placement
comfortably within this order.
Very little information exists concerning the biology and natural occurrences of
this group of fungi. In addition, extensive confusion has prevailed over the years
regarding the taxonomic placement of various genera within this ascomycete complex.
Therefore, this study was conducted in order to correctly identify and characterize a
number ofMelanospora-like pyrenomycetes isolated from production agriculture soils in
Texas and Florida. A secondary goal was to identify and reclassify isolates of similar fungi
that have been deposited in the University of Florida fungal herbarium.
Materials and Methods
During the fall of 1994, while surveying Texas sugar beet-producing counties for
isolates of the fusarial root rot pathogen, Fusarium oxysporum f. sp. betae,, approximately
10-15% of cultures became contaminated after two-three weeks with an unknown
pyrenomycetous ascomycete. Growth and vigor of the Fusarium isolates was severely
affected in vitro with the dual cultures. The consistent association with and observed
damage to the pathogen generated the concept of mycoparasitism and the utilization of
this ascomycete as a potential biological control agent for fusarial wilt. Several cultures
containing both organisms were dried and saved on sterile filter paper as previously
described, and stored at 40C (Harveson and Rush 1997).
Over the last 14 months, an additional five isolates closely resembling the Texas
sugar beetMelanospora were obtained from agricultural fields in Florida. All were
observed growing in dual culture with variousformae specials of Fusarium oxysporum
recovered from diseased plant roots. These isolates reacted like the one from Texas by
not becoming evident until after a period of several week's incubation. The recovered
isolates were given a two letter acronym and are illustrated in Table 2-1 along with their
provenance. The Texas sugar beet isolate was designated as TX, while the Florida
isolates were named FL, FP, LE, GI, and GI-2. FL was found in association with the
causal agent of Fusarium wilt of Canary Island date palm, F. oxysporum f. sp. canariensis.
Isolate FP was recovered from the tomato wilt pathogen, F. oxysporum f. sp. lycopersici,
while the remaining three were isolated from the watermelon wilt pathogen, F. oxysporum
f. sp. niveum (Table 2-1).
All isolates were separated from their respective hosts by one of two methods.
Individual ascospore cirrhi were picked off culture plates and streaked on potato dextrose
agar (PDA) or Mycological agar (MA). However, because of the difficulty of avoiding
the numerous Fusarium microconidia, this was not always successfully accomplished.
The other method entailed removing mature perithecia and shaking in two ml water blanks
amended with 50% lactic acid. After several minutes, the water was spread on PDA and
left at room temperature overnight. The following day, individual ascospores were
removed with the aid of a dissecting microscope and transferred to PDA or MA amended
with 100 m/1 of a filtrate from a three to four day old liquid culture of Fusarium. This
greatly enhanced the germination and growth of the ascomycetes (Chapter 3).
Morphological Characterization and Identification
Cultures produced from germinated ascospores were transferred to and observed
on 12 different growth media. Results of growth characteristics on these various media
and subsequent inferred nutritional requirements are included in Chapter 3. The
identification and correct taxonomic placement of these fungi was based upon current
protocol for the Ceratostomataceae by Cannon and Hawksworth (1982), and Horie et al.
(1986). This was accomplished by viewing and measuring the fungal morphological
Table 2-1. Isolates collected and used for taxonomic identification
Isolate Host* Geographic Taxonomic
Designation Origin Identity
FL FOCA Brevard Co., FL Sphaerodes retispora var. retispora
FP FOL Indian River Co., FL Sphaerodes retispora var retispora
GI FON Gilchrist Co., FL Persiciospora moreaui
GI-2 FON Gilchrist Co., FL Sphaerodes retispora var. retispora
LE FON Lake Co., FL Sphaerodes retispora var. retispora
TX FOB Randall Co., TX Melanospora zamiae
SFOCA = F. oxysporum f. sp. canariensis; FOL = F. oxysporum f. sp. lycopersici; FON =
F. oxysporum f. sp. niveum; and FOB = F. oxysporum f sp. betae
structures with both light and scanning electron microscopy. The shape, ornamentation,
and germ pore structure were central criteria for establishing the identity of these fungi.
In addition to the six isolates found in association with F. oxysporum, another
collection of isolates identified as Melanospora and deposited in the University of Florida
fungal herbarium were examined and reclassified according to modem taxonomic criteria.
These fungi included three isolates originally named as Melanospora sphaerophila, M.
ornata, and M. cameronse. TheM. sphaerophila isolate was collected originally in
Kittery Point, ME in 1906 in association with Plowrightia morbosa on branches of Prunus
virginiana (chokecherry). TheM. ornala isolate was found in deer dung from Cheboygan
Co., Michigan in 1969, while that ofM. cameronse was found in Gainesville, Florida
colonizing dead asparagus fern stems (Asparagus plumosus) in 1927.
On PDA, all isolates except GI produced hyaline to white mycelium. Colony
diameters ofFL, FP, LE, and GI-2 reached 80 mm after six-seven days. GI grew at the
same rate, but its mycelium was a creamy tan to straw color. Isolate TX was much
slower-growing, attaining growth approximately one half that of the others in the same
period of time. Mycelium on the agar surface was thick (4-8 grm) and verrucolose,
containing numerous bulbils (Fig.2-1). These were particularly evident with increasing
age of cultures. Aerial mycelium was much finer in structure measuring 0.5-2.0 pm in
diameter with phialids scattered profusely along hyphal branches (Figs. 2-2 and 2-3).
Most of the perithecia appeared to form within the branches of the aerial mycelium and
not as readily on the agar surface.
Hyphal growth on hemp agar was quite different than that on PDA. Aerial
mycelium was sparse, and closely appressed to substrate surface. Mycelial growth
occurred in an irregular, non-radial pattern, and perithecia were not as abundantly found
as on PDA due to the lack of extensive aerial mycelium.
Although all isolates were approximately the same size and possessed the same
morphological structures, several differences were also noted. FP was much more prolific
in the production of this phialidic anamorph than the others. TX had the least amount of
phialids, while the remaining four fungi were intermediate. In general, the production of
perithecia likewise, differed greatly among isolates. Isolates LE and FL tended to form
greater numbers of perithecia, while TX and GI produced the fewest. FP and GI-2 were
intermediate between these two extremes. GI additionally formed its perithecia more
often gregariously while the remaining taxa produced perithecia scattered more or less
evenly over the substrate.
Perithecia were superficial, globose to ampullaceus, and amber to yellowish-brown
in color. They formed fleshy, membranous walls composed of angular
pseudoparenchymatous cells. All taxa had clavate, eight-spored asci that soon became
deliquescent (Fig.2-4). Ascospores filled the perithecial cavity before being exuded out in
a slimy cirrhus. Ascospores produced by all taxa were dark brown to olivaceous in color
and ellipsoid to citriform in shape with two apical germ pores (Fig. 2-5). The light,
translucent perithecia appeared dark to the naked eye due to the spore mass inside. All
isolates were ostiolate with perithecial necks varying from 80-150 pm (Figs.2-6-2-11).
The presence and lengths of coronal setae varied depending on growth substrate.
Morphological structures ofMelanospora and allies
2-1. Hyphae at agar surface showing numerous bulbils (x 350)
2-2. Aerial hyphae showing anamorphic conidia or spermatia (x 350)
2-3. Close-up view of anamorphic state (x 500)
2-4. Squash mount ofperithecium depicting asci in various states of
maturity (x 500)
2-5. Germinating ascospore showing two polar germ pores (x 350)
0 D% .
-* : 1
Figures 2-6-2-11. Perithecia ofMelanospora and allies. Note that all are ostiolate.
2-6. Sphaerodes retispora var. retispora (isolate FL) (x 138)
2-7. Sphaerodes retispora var. retispora (isolate FP) (x 123)
2-8. Sphaerodes retispora var. retispora (isolate GI-2) (x 127)
2-9. Sphaerodes retispora var. retispora (isolate LE) (x 126).
2-10. Melanospra zamiae (isolate TX) (x 150)
2-11. Persiciospora moreaui (isolate GI) (x 141)
However, despite numerous similarities among isolates, there were some major
differences that are commonly used now to separate members of this group of fungi.
These include the structure of the germ pore and the morphology and ornamentation of
the ascospore. Spores of isolates LE, FL, FP, and GI-2 were all coarsely reticulate with
strongly apiculate germ pores. They contained 8-10 deep lumina on each face of the
spore (Figs. 2-12-2-15). The spores of TX were smooth with sunken or depressed germ
pores (Fig. 2-16). GI produced spores that were also smooth in outline, but differed
from the others by having finely pitted spore walls reminiscent of a peach seed, with
slightly apiculate germ pores (Fig.2-17). A comparison of morphological structures and
sizes among isolates is presented in Table 2-2.
Based upon all discussed taxonomic criteria, isolates LE, FL, FP, and GI-2 were
concluded to be Sphaerodes retispora var. retispora. TX was classified as the type
species forMelanospora, M. zamiae. GI was determined to be the third reported finding
for the newly created taxon, Persiciospora moreaui (Cannon and Hawksworth 1982)
One of the three herbarium specimens was not able to be identified to species.
Melanospora cameronse was correctly named at the generic level due to its dark, smooth
ascospores with depressed germ pores (Fig. 2-18). However, the sample was in poor
shape and perithecia were seldom able to be observed. They disintegrated upon contact
with the cover slip and all that remained was a mass of spores in the place previously
occupied by the perithecium (Fig. 2-19). Since no immature perithecia could be located,
no asci could be examined. An extensive search of the literature did not reveal other
Table 2-2. A comparison of biological and morphological characteristics among Melanospora and its allies.
Specimen Genus Geographic Perithecium (pm) Ascus (pm) Ascospore (pm) Ostiole Substrate Spore
FL Sphaerodes Florida 240-320 35-50 x 18-22 18-22 x 8-12 Yes F oxysporum Reticulated
FP Sphaerodes Florida 280-320 35-55 x 18-22 20-22 x 8-12 Yes F oxysporum Reticulated
GI Persiciospora Florida 250-300 40-45 x 20-22 20-22 x 8-10 Yes F oxysporum Pitted
GI-2 Sphaerodes Florida 240-300 35-50 x 18-21 18-20 x 8-10 Yes F oxysporum Reticulated
LE Sphaerodes Florida 240-350 35-50 x 18-22 18-22 x 10-12 Yes F. oxysporum Reticulated
TX Melanospora Texas 230-300 40-65 x 20-25 16-18 x 12-14 Yes F. oxysporum Smooth
sphaeophila Scopinella Maine 250-375 Yes Prunus virginiania Smooth
ornata Sphaerodes Michigan 220-400 Yes Deer dung Reticulated
mer Mel a loid A s plu u Soo
camerense Melanospora Florida ? ? Asparagus plumosus Smooth
Figures 2-12-2-17. Mature ascospores for Melanospora and allies.
2-12. Sphaerodes retispora var. retispora (FL), note the reticulations
with 8-10 lumina on the face of each spore. (x 1500)
2-13. Sphaerodes retispora var. retispora (FP), note same structures.
2-14. Sphaerodes retispora var retispora (GI-2), note same structures
2-15. Sphaerodes retispora var. retispora (LE), note same structures.
2-16. Melanospora zamiae (TX), note smooth spores with no
additional ornamentation. Lighter spore is immature. (x 1470)
2-17. Persiciospora moreaui (GI), note pores or pitted surface of
spore characteristic of this genus. (x 1309)
reports of similar fungi containing the specific epithet, cameronse. Therefore, the name
used for this specimen, M. cameronse, was retained. Conversely, the remaining two
specimens were easily identified and redesignated with their currently accepted generic
names. The specimen labeled as Melanospora ornata needed to be renamed. This taxon
had been synonomized with Sphaerodesfimicola (Cannon and Hawksworth 1982). The
sample easily fit into this category because of its eight-spored asci (Fig. 2-20), globose
perithecium (220-400 pm), very short neck and poorly developed coronal setae, and dark,
citriform spores with strongly apiculate germ pores and coarse reticulations (Fig. 2-21).
The other specimen from the herbarium that needed renaming was Melaospora
sphaerophila. It had been synonomized with and described as Scopinella sphaerophila by
Malloch in 1976. The herbarium sample proved to be identical with Malloch's description
(1976) by possessing black, carbonaceous perithecia measuring 250-375 pm in diameter
with very long necks (450-700 pm) (Fig. 2-22). Its smooth, barrel-shaped ascospores
contained a thick walled, dark brown band in the middle of the spore with hyaline,
protruding walls at each end that collapsed bilaterally after drying (Malloch 1976) (Fig. 2-
23). This feature was thought to be a vertical germ slit by Cannon and Hawksworth
(1982), but Scopinella was later proven to germinate from both lateral, hyaline
protuberances (Tsuneda et al. 1985). Its identification was further confirmed by the
presence of the unique, 2-spored asci (Malloch 1976, Tsuneda et al. 1985) (Figs. 2-24 and
Figures 2-18-1-25. Morphological characteristics from specimens deposited in University
of Florida fungal herbarium.
2-18. Mature ascospore ofMelanospora cameronse. Note the
smooth spore with no ornamentation (x 1500)
2-19. Ascospores ofM. cameronse after mounting. Clump of spores
are the only structures remaining after degradation of
perithecium (x 500)
2-20. Eight-spored, clavate ascus ofSphaerodesfimicola (x 404)
2-21. Mature ascospores of S.fimicola. Note the fine reticulations on
spore surface (x 1300)
2-22. Perithcium of Scopinella sphaerophila, note the long neck
2-23. Mature ascospores of S. sphaerophila (x 1375)
2-24. Immature asci ofS. sphaerophila arising from an ascogenous
hyphal system (x 1100)
2-25. Nearly mature ascus of S. sphaerophila. Note characteristic
two-spored nature (x 1535)
The difficulty in classifying allied members of the Ceratostomataceae has been well
documented. Horie et al. (1986) state that the reason for this fact is that superficially
similar ascomycetes that are placed in this group have all been derived from other
unrelated sources through convergent evolution. For example, the presence of the
deliquescent ascus, long necks, and ostiolar setae are adaptations for passive dispersal of
spores by animal movement or rain splashing, rather than active dispersal by air currents
via forcibly discharged ascospores and poroid asci. Horie et al. (1986) further point out
that the characters now utilized for taxonomic separation such as ascospore morphology
and ornamentation, and germ tube structure, are useful as indicators of true evolutionary
relationships since they would not be subjected to strong selection pressures.
Recently, the tremendous insurgence of analyses using molecular techniques and
characters and the resulting inferred phylogenies among pyrenomycetes has not
extensively addressed this group of fungi. Very few members of this group consisting of
Melanospora and its closely related allied genera have been included in these types of
studies. However, two recent ones have been attempted utilizing M zmniae, and M
fallax (Rehner and Samuels 1995, Spatafora and Blackwell 1994). In both cases,
Melanospora was derived within or near the Hypocreales, specifically in clades containing
the genus Nectria. Although Melanospora and its allies were once included in this order
by some workers, they have many distinctly non-Hypocrealean features. They are similar
to the Hypocreales in the enteroblasitc, phialidic anamorphs, and the fleshy, light colored,
true perithecia. However they differ substantially from hypocrealean fungi by containing
deliquescent asci, and dark, aseptate ascospores with polar germ pores.
In a separate study, inference with another group of fungi has been found. These
same two Melanospora species, M. zamiae and M fallax have additionally been aligned
phylogenetically with the anamorphic, ambrosial species Raffaela hennebertii (Jones and
Blackwell 1998). Members of this genus have been difficult to derive because of the lack
of a known teleomorphic state. However, when R. hennebertii was excluded from the
analysis, seven different species from this genus formed a lineage that was resolved as a
sister group with the sexual genus Ophiostoma (Jones and Blackwell 1998). These
modern taxonomic studies have therefore proven to be equally as difficult to discern and
understand as have the morphological ones.
As pointed out earlier, the primary reasons for the problems of taxonomic
placement for this group ofpyrenomycetes is shared characteristics, both morphological
and molecular, with other fungal taxa. This can be further illustrated with the example of
Sordaria destruens. This taxon was first referred to Anthostomella before being
transferred toMelanospora by Shear (Shear and Dodge 1927). It was used for almost
thirty years as the model for studying fungal physiology and nutrition before being
transferred to Sordaria in 1951 (Asthana and Hawker 1936, Hawker 1936, 1951). The
inclusion of this taxon in the literature for many years as Melanospora has likely
contributed to the confusion between this genus and its allied genera in subsequent
studies, as well as contributing to the incorrect placement of these species.
ENHANCEMENT OF GROWTH AND SPORULATION OF MELANOSPORA AND
ITS ALLIES BY FUSARIUMOXYSPORUM
The phenomenon of enhanced sporulation of fungi by other organisms has been
observed for many years. The first account of increased reproduction of one organism by
another was recorded by Molliard in 1903. He found that species ofAscobolus growing
on carrots produced numerous apothecia in the presence of a contaminating bacterium,
but not when growing in pure culture (Molliard 1903). The first report of the
enhancement of sporulation by one fungus on another was made by Heald and Poole
(1909) in Nebraska. They found that Melanospora pampeana (syn. M zamiae) produced
perithecia abundantly in a mixed culture with Fusarium moniliforme, but only gave sparse
mycelial growth after being in pure culture (Heald and Poole 1909). A recent paper by
Watanabe (1997) has also demonstrated a similar process for Sordariafimicola and two
species ofArmillaria. In this case, A. mellea and A. tabescens were antagonistic to S.
fimicola by inhibiting its growth. However, at the same time, these Armillaria species
increased perithecium and ascospore production of Sordaria.
Many different fungi have also been demonstrated over the years to need certain
nutrients for adequate growth and sporulation in pure culture. A growth substance found
to be important for maximum culture of certain yeasts was identified in the late 1920's.
Eastcott (1928) was able to separate this substance into two parts, one of which was
identified as inositol. This compound is now known to be a hexahydroxycyclohexane that
occurs widely in the phospholipids of many microorganisms (Singleton and Sainsbury
1987). Robbins and Kavanagh (1938) showed that the addition of vitamin Bi (thiamin)
promoted the growth ofPhycomyces blakesleeans in synthetic culture. Using an isolate of
Melanospora destruens (syn. Sordaria destruens), Hawker (1938,1939) found that its
growth on a medium containing glucose and various inorganic salts was negligible. The
addition of thiamin had no beneficial growth effect on the fungus. Biotin (vitamin K)
added to the same medium, gave good vegetative growth, but no perithecial production.
However, when both biotin and thiamin were included, both reproduction and mycelial
growth were vastly improved.
Jordan and Barnett (1978) were similarly able to induce sporulation ofM. zamiae
better in dual culture with other fungi than by itself. They additionally attained greater
sporulation and hyphal growth with biotin and thiamin added to a glucose-based medium,
but discovered that the addition of zinc enhanced these properties to an even greater
extent. No growth of the fungus occurred without the inclusion of these nutrient
supplements. (Jordan and Barnett 1978). Conversely, Vakili (1989) found that the
majority ofMelanospora damnosa isolates produced from single ascospores would
germinate on PDA. His results determined that only 6% and 2% of the isolates required
thiamin and biotin respectively for germination and axenic growth.
A number of fungi have also been observed occurring and growing in close
association with other fungi. These types of relationships have also been noticed to be
necessary for the growth and sporulation of some members. Those members of these
complexes presumed to be parasitic were often not successfully grown in pure culture
(Jordan and Barnett 1978, Whaley and Barnett 1963). Gonatobotrys simplex was found
in association with Alternaria tennis and attempts to grow it axenically failed unless an
extract of the Alternaria was added to the medium (Whaley and Barnett 1963). A growth
factor was isolated and partially purified from this extract and termed mycotrophein. It
was assumed to be vitamin-like because it was only needed in minute amounts (Whaley
and Barnett 1963), but its nature is still not fully understood.
Gonatobotrys and several species ofMelanospora have also been demonstrated to
be fusion biotrophs of other fungi (Jordan and Barnett 1978, Jeffries and Young 1994,
Vakili 1989). The biological behavior of these types of parasites appears to often be
associated with a nutritional dependency (Jeffries and Young 1994). It is assumed that
whatever growth factor or vitamin-like molecule that is responsible for enhanced growth
and sporulation of these organisms in culture can be obtained directly from the host
hyphae in nature.
The collection of pyrenomycetes found naturally occurring in association with F.
oxysporum-infected plant roots (Chapter 2) may also be reacting in a similar manner.
Species of Sphaerodes and Persiciospora, like Melanospora, have been suspected as
being parasitic on other fungi (Cannon and Hawksworth 1982). It is not known, however
if any of them exhibited a similar dependency upon their hosts or host products for
improved sporulation and growth. Therefore, this study was initiated to explore this
possibility and to better characterize the association and interrelationships between
members of this fungal group and the variousformae specials ofF. oxysporum from
which they were originally found.
Materials and Methods
Since all pyrenomycete isolates were found occurring in dual culture with F.
oxysporum, it was first necessary to separate the two organisms and isolate them in pure
culture. Once separation had been accomplished, it was quickly observed how slow the
pyrenomycetes germinated and grew on a selected group of standard synthetic media.
Preliminary attempts to grow the isolates using different concentrations of a
Fusarium culture filtrate provided promise for increased sporulation and growth. These
experiments consisted of filtrates from four-five day old F. oxysporum cultures grown in a
liquid medium composed of sucrose, yeast extract, and inorganic salts (Esposito and
Fletcher 1961). Cultures were vacuum-filtered through 150 ml bottle top flasks (0.45 um
capacity, Coming, Coming, NY) directly into media bottles using a hand-held Nalgene
(Cucamonga, CA) vacuum pump. Concentrations of the filtrate consisted of 25, 50, and
100 ml per one half liter of media. These experiments also included the addition of filtrate
before and after autoclaving. Filtrates produced by the still culture from the fourformae
specials ofF. oxysporum, niveum, lycopersici, canariensis, and betae, were all evaluated
on PDA and mycological agar with the corresponding pyrenomycetes in all possible
combinations. Once methods and techniques for optimum efficiency were established, all
subsequent tests routinely included filtrate from F oxysporum f. sp. niveum (FON) at a
rate of 50 ml per one half liter of media added prior to autoclaving.
Effects of Media with and without the Addition of Filtrate
Isolates used for this study included the four Sphaerodes strains and the one isolate
of Melanospora (Chapter 2). Persiciospora moreaui was include in the preliminary
studies, but was eventually lost in culture and was never able to be reconstituted. It has,
however, been deposited in the University of Florida fungal herbarium and is available for
There were 12 different media employed for this portion of the study. They and
their ingredients are illustrated in Table 3-1. These media were used in to determine the
most efficient source for growth and reproduction of these fungi for eventual mass
production of inoculum. It was also hoped that inferences could be made on the
nutritional requirements or preferences for this group of pyrenomycetes.
An eight mm diameter cork borer agar plug was placed in the center of each plate
utilizing six replications per medium. Each medium was evaluated both with the added
filtrate (50 ml/1/2 liter) and without. Plates were incubated in the dark at 250C. Colony
growth measurements were made after six days. After measurements were recorded,
plates were further incubated for another two weeks.
After the two week incubation period, growth morphology and characteristics
were recorded. Four cork borer plugs (eight mm) were also removed approximately one
inch away from the point of plate inoculation at the 12, 3, 6, and 9 o'clock positions Each
plug was viewed under a dissecting microscope and perithecia were counted. Four of the
24 plugs utilized for counting perithecia were randomly selected, prepared as squash
mounts, and observed microscopically. With the use of 10X magnification, 12 perithecia
from each plug were randomly measured for diameter at the widest point.
Table 3-1. Media used for evaluation of growth, perithecial development, and perithecial
size for isolates ofMelanospora zamiae, and Sphaerodes retispora var. retispora.
AK sporulating agar (AK)
Brain infusion agar (BIA)
Cornmeal agar (CMA)
Emerson's YPS (EYPS)
Hemp agar (HA)
Komada's agar (KA)
Malt extract agar (ME)
Mycological agar (MA)
Potato dextrose agar (PDA)
Starch agar (SA)
V82 (double strength)
Pancreatic digest of gelatin, pancreatic digest of
casein, yeast extract, beef extract, dexstrose,
Infusion of calf brains and beef hearts, proteose
peptone, dextrose, NaCI, Na2PO4
Infusion of cornmeal
Yeast extract, soluble starch, K2P04, MgSO4
Sterilized hemp seeds
glucose (dextrose), casein hydrolysate, MgSO4,
thiamin, biotin, microelement soln (Mn, Zn, Fe)
galactose, asparagine, K2PO4, KCL, MgSO4
Fe-Na EDTA, NaB407, PCNB, oxgall powder,
maltose, dextrin, glycerol, peptone
Infusion of potatoes, dextrose
Soluble starch, beef extract
V8 juice, CaCO
This study was initiated to more fully explore and understand possible
interrelationships between each of the four Fusariumformae specials and their
corresponding fungal partner with which they were recovered. There were two primary
questions which were being addressed. One was whether there was an added benefit with
the inclusion of a filtrate above that achieved with the host Fusarium alone. The second
was whether evidence could be gathered that suggested if a host specificity or preference
was involved with eachforma specialist and the specific fungus found with it. The major
assumption being made with this approach was that the ability of the each fungus to
reproduce in conjunction with the Fusarium was an important factor in host-parasite
evaluations. Therefore, perithecial production was the central criterion that was used for
evaluating this process. Peripheral goals that could be achieved at the same time included
evaluation of medium effect, relative efficiency of parasites to sporulate, and efficiency of
formae specials to induce formation of perithecia.
This study was designed as a four-level factorial using four media, four
formae specials, four suspected parasites, and the inclusion or exclusion of filtrate in
media (Table 3-2). Each host and parasite were paired on each medium one half inch
apart in all possible combinations using five replications for each combination. Each
medium was also evaluated with filtrate added or excluded. After two week's incubation
in the dark, plugs were removed along two transect lines on each plate, four plugs spaced
equidistantly from each transect (Fig. 3-1). Perithecia were counted on the eight plugs as
before with the aid of a dissecting microscope.
Table 3-2. Media, pyrenomycete isolates and Fusarium oxysporum isolates utilized in the
Medium F. oxysporum forma specialist Parasite
Potato dextrose agar betae TX
Double strength V8 agar canariensis FL
Emerson's YPS agar lycopersici FP
Starch agar niveum LE
TX = M. zamiae
FL, FP, and LE = S. retispora var. retispora
Each host-parasite combination was evaluated on each medium in all possible
combinations, and each medium was tested both with and without the addition of a filtrate.
Figure 3-1. Plate inoculated with both F. oxysporum and pyrenomycete for evaluation of
perithecial development in the factorial study. Note the plugs removed along
two transect lines. Perithecial counts were made from each plug. Tiny black
structures are mature perithecia (x 0.8)
Both studies were repeated once, and all data were analyzed by the general linear
model procedure. The means involving growth, sporulation, and perithecial size in the
presence or absence of filtrate on different media were separated by Duncan's mean
separation test. In the factorial study, all possible interactions and main effects were
tested before separating means by the least squares procedure (LS Means). The pairwise
probability values obtained were additionally adjusted by the Bonferroni correction.
Finally, two separate assays were attempted to establish some metabolic or
biological properties of the filtrate produced by the Fusarium isolates. Both involved the
use of dialysis tubing with a cut-off size of 14-16,00 MW (Spectra/Por, Spectrum Medical
Industries, Los Angeles, CA). Strips of tubing were taped to pieces of wire mesh and
autoclaved. These obstructions were placed in the center of Petri plates, dividing them in
half. Plugs of both parasite and Fusarium were inoculated on separate sides with the
parasite alone on separate plates as controls. Colony diameters of the parasite from both
treatments were measured after one week.
The other assay consisted of filling a six inch piece of tubing with 15 mls of PDA
broth and two plugs of the Fusarium. The ends were sealed and placed into sterile media
bottles containing 500 mis of PDA broth and two plugs of the parasite. This was
compared to bottles of liquid media containing only the parasite. After 10-14 days,
dialysis tubing was removed, contents were strained through cheesecloth and weighed.
After separation of isolates of the Melanospora and Sphaerodes from the F.
oxysporum, all isolates were evaluated after growing from a single ascospore. Because
they all grew and sporulated to some extent on the different media, they were concluded
to be homothallic and also not obligate parasites of the variousformae specials. From
the preliminary experiments, it was further determined that no differences existed in terms
of growth or perithecial production in response to the filtrates produced by the Fusarium
isolates. Because watermelon wilt was the pathosystem to be eventually evaluated for
disease control, filtrate from the Fusarium niveumforma specialist was routinely used for
all subsequent studies. It was also observed that few differences existed with the volume
of filtrate added to each medium. Therefore, a 1:10 ratio of filtrate to medium volume
was selected for all subsequent evaluations and general maintenance of isolates (50 ml/1/2
Linear growth after six days was significantly enhanced for all isolates, as was
perithecial production (Tables 3-3 and 3-4). However, the addition of filtrate had no
effect upon perithecial diameter (Tables 3-3 and 3-4). Significant differences were
observed among the three variables, and were influenced by choice of medium and
pyrenomycete isolate. These differences among growth, perithecial production and size
are illustrated in Tables 3-5 and 3-6 for combined media and isolates respectively.
With the exception of J&B, the better media for growth tended to be better for
sporulation as well. This medium consisted of glucose and inorganic salts amended with
biotin, thiamin, and a micronutrient solution (Mn, Zn, Fe), and was developed exclusively
for aM zamiae from West Virginia (Jordan and Barnett 1978). However, theM. zamiae
isolate used in this study from Texas did not respond well to this medium. In terms of
sporulation, none of the other Sphaerodes isolates in this study responded well either
Although among the leaders at producing linear growth, J&B medium was extremely poor
in inducing perithecial production (Table 3-5). The media that overall allowed the best
combination of growth and sporulation were PDA, MA, AK, and ME (Table 3-5). These
media also tended to produce the greatest amount of fluffy, aerial mycelium, which is
where the majority of the perithecia were formed. Those media that did not induce overall
good, vigorous growth and perithecial formation included Komada, starch, Emerson,
hemp, and cornmeal agars. Brain infusion agar was similar to J&B by producing extensive
mycelial growth, but the numerous perithecia produced after two weeks were primarily
immature. Interestingly, the Komada medium allowed absolutely no growth at all for any
of the pyrenomycete isolates. This is a F. oxysporum-selective medium that truly worked
correctly by inhibition of the pyrenomycetes completely. It was determined that a plug of
any of these isolates could be placed on this medium for at least a month without
producing any growth. However, the isolates were not killed, because the plugs could be
removed from the Komada, placed on a suitable growth medium and afterward function
Those media having high soluble starch (starch, Emerson) or cellulose
content (V82, hemp, cornmeal) produced very little aerial mycelium. Growth was often
extensive linearly but very thin, sparse and flat to the agar surface. Perithecial
development was additionally poor (Table 3-5). In general, perithecial diameter was
Table 3-3. General linear model data for growth, perithecial production, and perithecial
diameter of pyrenomycete isolates with and without an added filtrate from F. oxysporum f.
Source df growth (mm) perithecia perithecial size (mn)
Form 1 0.0001 0.0001 0.8743*
Medium 11 0.0001 0.0001 0.0001
Isolate 4 0.0001 0.0001 0.0001
Replication 5 0.9254* 0.9481* 0.3830*
* = not significant at P = 0.01
Table 3-4. The effect of an added fusarial filtrate to growth, perithecial production and
perithecial diameter of pyrenomycete isolates.
Growth (mm) Perithecia Perithecial size (unm)
Filtrate 68.3 a 26.4 a 245.6 a
No Filtrate 38.9 b 10.6 b 245.7 a
Means followed by the same letter are not significantly different according to Duncan's
multiple range test
Table 3-5. Growth, perithecial production and perithecial size of all pyrenomycete isolates
combined on different growth media.
Medium Growth (mm)" Perithecia' Perithecial size (unm)
AK 49.0d 29.9c 224.8ef
brain infusion 56.7c 4.9f 220.8f
cornmeal 30.5f 10.9e 256.8b
Emerson's YPS 50.2d 14.1e 277.6a
hemp 47.4d 12.2e 251.2bc
J&B 57.2c 3.7f 244.0cd
Komada's 0.Og 0.0h 0.0h
malt extract 58.9c 20.2d 199.2g
mycological 72.3b 38.4b 234.4de
potato dextrose 79.7a 42.2a 231.2ef
starch 47.5d 11.6e 277.6a
V82 38.9e 12.6e 273.6a
Means followed by the same letter are not significantly different according to Duncan's
multiple range test.
(P = 0.01)
Table 3-6. Growth, perithecial production and perithecial diameter for each pyrenomycete
isolate on all combined growth media.
Isolate' Growth (mm)b Peritheciab Perithecial size (nm)b
FL 54.3b 28.3a 261.6a
FP 59.7a 20.6a 237.6b
GI-2 54.2b 20.5a 232.0b
LE 59.6a 22.8a 259.2a
TX 39.9c 5.7b 233.6b
"FL, FP, GI-2, and LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
bMeans followed by the same letter are not significantly different according to Duncan's
multiple range test.
likewise influenced by choice of growth media and isolate. Those that produced the
poorest growth and sporulation also tended to induce significantly larger perithecia (Table
3-5). Isolates FL and LE were observed to form larger, but not more numerous perithecia
than the other pyrenomycetes with the exception of the M. zamiae isolate from Texas
Because of the significant differences detected among media and isolates, means of
the measured variables were sorted by fusarial filtrate effect and evaluated
individually. Tables 3-7 and 3-8 depict the effects of filtrate and medium upon the growth,
and sporulation, respectively, of the various isolates being evaluated in this study. Isolate
TX (M. zamiae) was consistently the slowest-growing and produced the fewest perithecia
among the other Sphaerodes isolates evaluated. This is particularly evident in Table 3-8
showing its production of perithecia without filtrate. In general, most media with the
added filtrate greatly improved both sporulation and growth, but ME was an exception.
With sporulation of isolates FL and LE, the difference between treatments was minimal.
Conversely, isolate FP actually produced more perithecia with the non-filtrate treatment
(Table 3-8). The linear growth produced by these isolates on PDA and mycological agars
could also be misleading. Although very little difference is noticed from looking at the
two treatments in Table 3-7, the filtrate was still better in inducing luxuriant, vigorous
aerial growth than was the same media without the filtrate. A more accurate picture of the
beneficial effect of filtrate is observed with the production ofperithecia (Table 3-8)
The factorial study provided further evidence of the importance or dependence of
these isolates upon F. oxysporum. After analyzing the perithecial production from the
different pyrenomycetes paired with the fourformae specials, it was determined that the
addition of filtrate to media is not a significant factor, like it had been with the
pyrenomycetes growing by themselves. The general linear model data obtained for main
effects and all possible interactions is presented in Table 3-9. Because all main effects
except for filtrate were significant, means from different treatments were separated and are
included in Table 3-10. After combining results for all fungal isolates, PDA was shown to
produce the greatest number ofperithecia followed by V82, starch and Emerson. Isolates
LE and FL formed greater numbers ofperithecia on all media andformae specials
followed by FP and TX. Finally, the sugar beet pathogen (FOB) was the poorest host for
all pyrenomycetes and media, whereas no differences were observed among the remaining
formae specials (Table 3-10).
The major questions being asked about the relationships between the
pyrenomycetes and the Fusariumformae specials were effectively answered by this
complicated factorial study. There were no significant differences observed with the
filtrate treatment, or with any interaction that included filtrate (Table 3-9). This essentially
answered the first question. Whatever advantage the pyrenomycetes received from the
filtrate was likewise obtained from the Fusarium isolates themselves, and there was no
added benefit to the filtrate in the media. However, there was a significant interaction
involving the pyrenomycete-F. oxysporum combination (Table 3-9), which alluded to the
second question being asked from these organismal relationships. Therefore, it was
important to investigate these relationships more completely with an additional analysis.
Table 3-11 shows the results of this additional analysis after subjecting data to the
least square means and pairwise comparison procedures. Isolate LE was the only one of
Table 3-7. Linear growth (mm) among pyrenomycete isolates after six day's incubation as
influenced by media and addition or exclusion offusarial filtrate.
Filtrate No Filtrate
FL FP GI-2 LE TX FL FP GI-2 LE TX
AK 49.3c 71.3a 60.3b 62.3b 47.3c 32.3c 45.0a 39.7b 46.7a 35.3c
Brain 63.7b 73.7a 54.0c 69.0ab 47.0d 50.3b 66.0a 43.7c 66.7a 33.0d
CM 48.3a 46.3a 46.3a 47.3a 47.0a 14.0a 13.7a 15.Oa 13.7a 13.0a
Emerson 79.3b 85.0a 84.3a 85.0a 53.7c 21.0b 32.7a 32.7a 17.3c 10.7d
Hemp 70.0a 72.0a 71.7a 69.0ab 64.3b 29.0a 24.7b 24.7b 28.3a 22.7b
J&B 85.0a 85.0a 80.0a 85.0a 64.7b 30.7d 43.3b 35.3c 49.0a 14.0e
Komada O.Oa O.Oa O.Oa O.Oa O.Oa O.Oa O.Oa O.Oa O.Oa O.Oa
ME 76.7a 75.7a 72.0a 76.0a 55.7b 37.3c 54.0a 54.0a 48.0b 40.0c
Mycol. 85.0a 85.0a 85.0a 85.0a 69.0b 73.7b 74.3b 53.3c 79.3a 33.3d
PDA 85.0a 85.0a 85.0a 85.0a 85.0a 83.3a 83.0a 80.0a 84.0a 41.7b
Starch 68.3b 80.7a 81.0a 81.0a 49.7b 25.3b 22.7b 28.7a 24.0b 14.0c
V82 58.3b 51.7c 34.0d 68.0a 26.0e 29.3d 45.0a 32.2c 42.3b 12.0e
'FL, FP, GI-2, and LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
bMeans followed by the same letter are not significantly different according to Duncan's
multiple range test.
Table 3-8. Perithecial production among pyrenomycete isolates after two week's
incubation as influenced by media and addition or exclusion of Fusarium filtrate.
Filtrate No Filtrate
FL FP GI-2 LE TX FL FP GI-2 LE TX
AK 44.7b 42.7b 37. lb
Brain 20.2a 2.7bc 5.0b
CM 21.2b 28.1a 29.1a
Emerson 20.5bc 29.3a 24.2ab
Hemp 21.lab 24.3ab 28.2a
J&B 7.3a 9.5a 0.0b
Komada 0.Oa 0.Oa 0.Oa
ME 40.2a 7.4c 24.7b
MA 66.6a 48.7b 28.2c
PDA 77.4a 67.la 84.la
Starch 22.4ab 23.5ab 27.9a
VS2 40.0a 27.7a 5.lb
11.4c 26.5a 22.0ab 13.0bc 7.3c
0.9ab 0.7b 0.Oc 1.3a O.Oc
0.Oa 0.Oa 0.Oa O.Oa O.Oa
6.7b 10.5a 7.06 4.6b .l1c
1.5b 0.Oc 0.Oc 2.0a 0.Oc
0.0b 3.2a 5.0a 3.2a 0.0b
0.Oa 0.Oa 0.Oa 0.Oa 0.Oa
33.6c 22.9b 0.0d 25.1b 1.2c
41.5a 24.0b 49.2c 46.7a 0.0d
42.2b 51.7a 31.8c 42.6b 0.0d
1.4a 2.3a 1.5a 1.3a 0.0b
37.3a 2.4b 3.4a 4.5a 0.7b 4.7a 0.Oc
"FL, FP, GI-2, and LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
bMeans followed by the same letter are not significantly different according to Duncan's
multiple range test. (P= 0.01)
Table 3-9. General linear model data describing sporulation of four pyrenomycetes paired
with their four corresponding F. oxysporumformae specials.
Source df Pr > F
Medium 3 0.0001
Pyrenomycete isolate 3 0.0001
Filtrate 1 0.8866*
Fusarium isolate 3 0.0001
Replication 4 0.8492*
Filtrate*Pyrenomycete isolate 3 0.2249*
Fusarium*Pyrenomycete isolate 9 0.0001
Filtrate*Fusarium isolate 3 0.2699*
* = not significant at P = 0.01
Table 3-10. Factorial study data illustrating perithecial development for pyrenomycete
isolates, Fusarium isolates, and growth media.
Mean' Medium Pyrenomycete isolated Fusarium isolate'
63.2 PDA a
'Means followed by the same letter are not significantly different according to Duncan's
Multiple Range Test (P = 0.01).
bLE, FL, FP = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
FOL = F. oxysporum f. sp. lycopersici; FOCA = F. oxysporum f sp. canariensis; FON =
F. oxysporum f. sp. niveum; and FOB = F. oxysporum f. sp. betae
the four pyrenomycetes that was capable of producing a significantly greater number of
perithecia on its original host from the field. In fact, no other isolate was able to match
LE's sporulation efficiency on any of theformae specials. This indicated that this isolate
was better than the others at reproducing on this group of Fusarium isolates. Although
numerous other examples of differences among and between isolates were detected, there
were no further incidences of significant perithecial production associated with each
pyrenomycete and theforma specialist from which it was obtained. So this effectively
addressed the second question and proved that only the combination including isolate LE
and F oxysporum f sp. niveum appeared to exhibit a significant host specificity or
preference in terms of reproductive ability.
The experiments with the dialysis tubing provided little information on the
chemical nature of the filtrate, however a great deal was learned about its biological
properties. Whatever metabolite or growth factor that was being produced in response to
the culture of Fusarium was heat and pressure stabile. It was able to retain its activity of
enhancing growth and sporulation regardless of whether it was added to media before or
after autoclaving. Secondly, it had a molecular weight smaller than 12-14,000, which was
the cut-off size for the pores in the dialysis tubing. Both assays demonstrated that the
active ingredient in the filtrate could pass through the tubing and still maintain its
beneficial effect upon growth and perithecial production for the pyrenomycetes.
The reported nutritional requirements for Melanospora growth and sporulation have
proven to be conflicting over the years. In many cases this study's results do not correlate
with those of others. For example, Hanlin et al. (1993) and Goh and Hanlin (1994),
successfully utilized V8 juice agar for both culture maintenance and developmental studies
because of superior perithecial production over that of ME agar. TheM. zamiae isolate
originally found in Texas was more effective in terms of growth and reproduction on ME
than on VS. Perithecial production on VS by isolate TX was among the poorest of all
media evaluated, and also resulted in very slow, limited growth (Tables 3-5, 3-9, and 3-
10). However, V8 was among the better media for growth and sporulation of several of
the Sphaerodes isolates (Tables 3-9, and 3-10).
Another major difference with the M zamiae isolate from Texas used in this study
concerns the increased growth and sporulation efficiency after the addition of a filtrate
from a liquid culture of the suspected host. Jordan and Barnett (1978) found that their
isolate ofM. zamiae responded positively to a mycelial extract from F. roseum and
Verticillium albo-atrum. They therefore concluded that all nutrients required for growth
and sporulation were contained within the living host and not from metabolites or
excretions from a liquid culture.
There are also discrepancies among different studies involving required growth
factors. Much of Hawker's (1938, 1939, 1950) work with fungal nutrition has found that
vitamins or vitamin-like substances are often needed for optimum growth and sporulation
of certain fungi. She determined that M destruens (syn. Sordaria destruens) needed the
combination of both thiamin and biotin. In addition, another vitamin-like growth factor
termed mycotrophein was found to be required for Gonatobotrys simplex (Whaley and
Barnett 1963). Jordan and Barnett (1978) determined that their isolates ofM. zamiae also
Table 3-11. Results of the factorial study showing least square means and pairwise
comparison probability values for perithecial production among pyrenomycetes' and F.
Perithecial Mean FOB-TX FOCA-FL FOL-FP FON-LE
Pr > mT| Pr > [T Pr > 1T Pr > ITl
FOB-FL 41.5 0.0001 0.0004 0.7676* 0.0001
FOB-FP 53.1 0.9427* 0.4890* 0.0001 0.0001
FOB-LE 42.9 0.0002 0.0022 0.4267* 0.0001
FOB-TX 52.9 0.5352* 0.0001 0.0001
FOCA-FL 51.2 0.5352* 0.0001 0.0001
FOCA-FP 39.0 0.0001 0.0001 0.5111* 0.0001
FOCA-LE 64.3 0.0001 0.0001 0.0001 0.2430*
FOCA-TX 52.3 0.8311* 0.6841* 0.0001 0.0001
FOL-FL 53.1 0.9252* 0.4753* 0.0001 0.0001
FOL-FP 40.8 0.0001 0.0001 0.0001
FOL-LE 67.1 0.0001 0.0001 0.0001 0.8757*
FOL-TX 46.3 0.0147* 0.0683* 0.0417* 0.0001
FON-FL 55.2 0.3855* 0.1370* 0.0001 0.0001
FON-FP 49.3 0.1890* 0.4876* 0.0016 0.0001
FON-LE 67.5 0.0001 0.0001 0.0001 -
FON-TX 29.4 0.0001 0.0001 0.0001 0.0001
FL, FP, LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
bFOB = F. oxysporum f. sp. betae; FOCA = F. oxysporum f sp. canariensis, FOL = F.
oxysporum f. sp. lycopersici; FON = F. oxysporum f sp. niveum.
*= not significant after use ofBonferroni correction (P = 0.0031)
needed biotin and thiamin, in addition to supplemental zinc, but were capable of
synthesizing their own mycotrophein. They developed a medium specific for M zamiae
that was glucose-based and supplemented with biotin and thiamin. It proved to be
excellent for inducing maximum growth and sporulation. The current study additionally
evaluated this same medium and referred to it as J&B (Table 3-1); however, it was still
very poor in production of perithecia for all isolates. It was also among the worst in
producing vigorous linear growth, unless supplemented with the fusarial filtrate.
Conversely, Hanlin et al. (1993) and Goh and Hanlin (1994) obtained adequate results
without the addition of these growth factors. Vakili (1994) found that most of his isolates
ofM damnosa could germinate without added vitamins, but single ascospores cultures
did not produce the phialidic, anamorphic state, Gonatobotrys simplex. However,
mycelium from compatible crosses ofM. damnosa did produce both microconidiophores
and perithecia. He concluded that the absence of the phialids in single-spored cultures and
their presence in crosses showed that they were spermatial in function and related to
sexual reproduction. The isolates used in this study still were capable of germinating and
growing without the extra nutrients although very poorly. They also formed both an
anamorphic stage and perithecia from single ascospores, proving that they were
Most physiological studies have found that nearly all fungi can utilize simple sugars
such as glucose or fructose equally as well as carbon sources (Cochrane 1958, Hawker,
1950, Griffin, 1981). These simple, monosaccharides appear to be more suitable for many
fungi than the more complex polysaccharides. In some instances the more complex sugars
seemed less able to support hyphal growth but may be more conducive to reproduction
(Hawker 1950). Galactose is another carbon source that is apparently difficult for a
number of fungi to utilize (Cochrane 1958, Hawker 1950, and Griffin 1981). Hawker
(1950) determined that M. destruens and several species of Chaetomium were unable to
grow vigorously with a galactose-based medium. However, the combination of good and
bad carbon sources had a overall beneficial effect. For example, the addition ofgalactose
to a glucose medium had no detrimental effect upon these fungi (Hawker, 1950). Perhaps
a similar phenomenon explains the effectiveness of PDA as a growth and perithecial
promoter for this group of pyrenomycetes. The combination of dextrose and starch would
not be as inhibitive as starch alone, and the beneficial effect of dextrose may override that
of the more complex sugar.
Several other previously published observations coincide well with the results from
this study. The best media overall for the current group of pyrenomycetes tended to
contain glucose or dextrose (PDA and MA), whereas the poorest contained more complex
carbohydrates such as cellulose or starch (cornmeal, starch, and Emerson). Griffin (1981),
points out that those fungi capable of utilizing complex carbohydrates such as starch or
cellulose were also able to synthesize exocellular, digestive or hydrolytic enzymes that
more readily broke the sugars down into glucose or maltose. Melanospora and the
closely related Sphaerodes and Persiciospora are suspected of being fusion, biotrophic
parasites. Organisms with this type of life strategy have additionally been determined to
obtain nutrition directly through contact with their hosts without producing any cellulitic
enzymes (Jeffries and Young 1994). Sordaria and Chaetomium, which were found to be
unable to use galactose, (Hawker 1950) are both closely related toMelanospora and
Sphaerodes. The current study concluded that Komada's agar would not allow growth of
any kind from these latter two genera. Komada's medium (Komada 1975) employs
galactose as the sole source of carbon (Table 3-1), and perhaps it is partially this
ingredient and not exclusively the antimicrobial supplements that are responsible for its
continued, successful use as a F. oxysporum selective medium.
Anamorphic fungi like Gonatobotrys, Gonatobotryum, and Phialophora have
often been linked with teleomorphic pyrenomycetes such as Melanospora. Melanospora
and related genera such as Sphaerodes and Persiciospora have also been found to be
ecologically associated with other fungi. Because of these close associations, many have
also been presumed to be contact parasites based upon the formation of hook cells or
contact branches on their hyphae. These types of fungi are also recognized to be slow-
growing or incapable of growing in axenic culture. They have also been demonstrated to
grow or sporulate better in mixed cultures with other organisms. Several other examples
have shown enhancement of these characteristics if extracts of suspected hosts are added
to media (Whaley and Barnett 1963). The current study discovered a very similar process
occurring with this group of pyrenomycetes and a filtrate produced by F. oxysporum. The
discovery of vitamin-like growth factors that improve growth and reproduction are
additionally assumed to be obtained from host fungi in nature or in synthetic media
insufficient for growth without added nutrients. This type of behavior has often been
associated with fusion biotrophs like Melanospora, but was completely unknown for allied
groups like Sphaerodes.
Although these nutritional relationships have been reported in the literature,
particularly with respect to Melanospora, they are very few in number. No other study
has attempted as extensively to address the aspect of enhancement of growth and
reproductive characteristics utilizing suspected hosts or host products. These
observations have been documented from several fungal complexes, but it is still not
known how common these types of interactions are outside the lab. Perhaps this type of
nutritional dependency between some fungi may explain why some can be easily cultured,
while others appear to be obligate.
This study has attempted to add new information that addressed Meanospora and
other related genera specifically with F. oxysporum. These particular organisms and their
interactions have been chosen for a number of reasons. First, these fungal complexes have
been consistently found occurring together in several geographically different locations
(Chapter 2). Secondly, because of the worldwide importance of Fusarium oxysporum and
the diseases it causes, it is desirable to find novel strategies for disease management.
Therefore the primary idea behind this chapter was to investigate these naturally-occurring
relationships and learn how to efficiently mass produce the pyrenomycetes in the attempt
to identify potential candidates for use as biological control agents for Fusarium wilt of
watermelon. Chapter 4 will focus on the establishment of host ranges and the evaluation
and measurement of damage to F. oxysporum at the bench level.
PARASITISM AND MEASUREMENT OF DAMAGE TO FUSARIUMOXYSPORUM
BY SPECIES OF MELANOSPORA, SPHAERODES, AND PERSICIOSPORA
The concept of parasitism can be defined or subdivided into a number of
categories. Parasitism usually refers to a relationship between two organisms where one
benefits at the expense of the other. However, it is not always correlated with damage or
deleterious effects upon one member. Parasitic relationships can also include mutualism,
in which both organisms receive equal benefit, or commensalism, where one organism
benefits from the relationship, leaving the other unaffected.
Melanospora, Sphaerodes, and Persiciospora have all been found in conjunction
with other fungi or from soil in field isolations (Cannon and Hawksworth 1982). All have
additionally been suspected of being parasitic based upon these intimate, natural
associations. Of these three genera, only M zamiae has been formally investigated as a
mycoparasite. Due to the formation of hook or contact cells and the ability to sporulate
on living hyphal mats ofFusarium roseum, Tritirachium sp., and Verticillium albo-atrum,
M. zamiae was concluded as being parasitic (Jordan and Barnett 1978). A collection of
11 other fungi, including F. oxysporum f sp. lycopersici, were also concluded to be
parasitic hosts for M zamiae based solely on the hook-shaped hyphal branches formed in
culture with this group of fungi. No other methods of evaluation were attempted for this
second group of fungi.
It has been fully established that all three genera being used in this study were
capable of more efficient growth and sporulation in dual culture with F. oxysporum or
with some of its metabolic products in the form of a liquid culture filtrate (Chapter 3)
however, this was not clear proof of parasitism. Therefore this study was initiated to
thoroughly establish in a more quantitative manner ifM. zamiae is a true mycoparasite.
Sphaerodes retispora var. reispora had previously only been isolated from soil or from
several different polypores (Cannon and Hawksworth 1982). Persiciospora moreaui has
been additionally isolated from soil, and in association with severalformae specials of F.
oxysporum (Cannon and Hawksworth 1982, Horie et al. 1986, Krug, 1988). However,
neither of the latter two pyrenomycetes have ever been tested as parasites, thus they have
been included along with M. zamiae in these evaluations. In addition, no other study has
ever attempted to show pathogenicity or damage caused by mycoparasites on their hosts
at the bench level.
The ultimate goal to this project was to find an alternative management option for
Fusarium wilt of watermelon. This chapter was another step in the process of selecting
potential candidates for use as biological control agents. Through the use of the
pyrenomycete isolates found to be naturally occurring in the field with F oxysporum, this
chapter has attempted to demonstrate parasitism at the cultural level and to measure the
amount of damage that these isolates cause to F. oxysporum. In addition to the
establishment of parasitism and pathogenicity, another purpose of this chapter was to
define a host range for these fungi among a collection ofF. oxysporumformae specials,
based upon the ability to sporulate in dual culture.
Materials and Methods
Fusarium oxysporum Host Range Determination
A collection ofF. oxysporum isolates were utilized to determine host range for the
Melanospora and Sphaerodes strains. This collection consisted of both pathogenic
isolates from various hosts, and non-pathogenic strains from a watermelon wilt-
suppressive soil (Table 4-1). For this portion of the study, only isolates TX, FL, FP, and
LE were included. Evaluations were made by monitoring perithecial production as
described in Chapter HI. Briefly, this entailed pairing eight mm agar plugs from actively
growing cultures of Fusarium with cultures ofMelanospora and Sphaerodes utilizing six
replications per treatment. After two weeks' incubation in the dark at 250C, four plugs
were removed from each replication from the 12, 3, 6, and 9 o'clock positions at a
distance of approximately two and one half cm from paired isolates. Perithecia were then
enumerated from each plug with the aid of a dissecting microscope.
Two separate analyses were performed for the pathogenic and non-pathogenic
isolates. Both data sets were sorted by Fusarium host, and analyzed by the general linear
model procedure. Tests were repeated once, and means were separated by Duncan's
multiple range test. The set consisting of the non-pathogenic isolates also included one
pathogenic isolate for comparison.
Establishment of Parasitism
Four separate methods were tested in order to provide proof of parasitism on F.
oxysporum. They included slide cultures, an assay using Komada's medium (KA)
Table 4-1. Fusarium oxysporum isolates collected from Florida and used to establish host
range among isolates ofMelanospora and Sphaerodes.
Isolate Isolation Host Plant Pathogen
Komada (1975) and PDA, an assay using Fusarium hyphal mats, and an assay using
watermelon seeds previously coated with both watermelon wilt pathogen and suspected
These studies consisted of Sphaerodes isolates FL, FP, and LE, Persiciospora
isolate GI, and Melanospora isolate TX. The host being evaluated was restricted to the
Fusarium wilt pathogen of watermelon (FON). All experiments were repeated at least
Slide cultures consisted of small (one mm2) agar pieces taken from actively
growing cultures of both FON and suspected mycoparasites. They were mounted in water
on sterile slides one half inch apart, and incubated in sterile, glass humidity chambers at
250 C. They were observed daily under a microscope for ascospore germination and
formation of the hook and contact cells attaching to hyphae of Fusarium, as described in
the literature (Jordan and Barnett 1978, Whaley and Barnett 1963).
Watermelon seeds (cultivar Charleston Gray), previously coated with a suspension
of mycoparasite spores and FON were allowed to air dry in petri plates at room
temperature. After 17 months, seeds were placed on moist paper towels in humidity
chambers and incubated at 250 to observe growth of fungi and to additionally establish
viability and survival over time of the mycoparasites.
For the hyphal mat assay, cultures of FON were grown in still liquid culture for 10-
14 days. After this time, hyphal mats were removed and washed thoroughly. The mats
were then placed on moist paper towels in humidity chambers and inoculated with agar
plugs from the different suspected parasites. These plates were incubated at 250 C and
observed periodically for growth and perithecial formation of parasites.
Lastly, a novel assay was developed that included the use of KA, one of the
standard, universally-employed F. oxysporum-selective media, and PDA. Previous work
had clearly demonstrated that Melanospora, Sphaerodes, and Persiciospora were all
completely inhibited on this medium, whereas PDA was one of the better media for
growth and sporulation (Chapter 3, Harveson and Kimbrough 1998). A sterile 40 mm
diameter beaker was placed in the center of a petri plate and molten PDA was poured
around the outside. After the agar solidified, the beaker was removed, leaving a 40 mm
diameter hole in the middle ringed by PDA. The hole was gently filled with KA and
allowed to harden. Agar plugs of FON and all suspected parasites were paired on the
center portion of plates containing the KA and incubated at 250 C (Fig. 4-1).
Measurement of Deleterious Effects of Parasites on F. oxysporum
Three separate techniques were developed to not only provide evidence of
deleterious effects on FON by parasites at the cultural level, but more importantly, to
measure the damage inflicted by Melanospora, Sphaerodes, or Persiciospora. The
measurement of damage in vitro to Fusarium was accomplished with several categorical
assays, including hyphal weight reduction, growth inhibition, and reduction of aerial
The hyphal weight reduction tests were performed in large petri plates (100 x 150
mm) using liquid culture (PD broth). A standard volume of 15 mis was put in each plate
along with eight mm agar plugs of various Fusarium and parasite treatments. The isolates
utilized for this study consisted of the four primaryformae specials evaluated in Chapter
Assays to determine parasitism of Fusarium oxysporum
4-1. Assay plates showing both parasite and Fusarium co-inoculated in
center portion of plates containing the Komada medium. Outside
ring contains PDA (x 0.6)
4-2. Germinating ascospores showing attachment of parasite germ
tubes on Fusarium hyphae (x 500)
4-3. Germinating ascospores showing attachment of parasite germ
tubes on Fusarium hyphae (x 500)
4-4. Hyphal strands of parasite attaching to Fusarium hyphae and
forming contact cells (arrows) (x 1100)
3, isolates FL, FP, LE, and TX and the corresponding pyrenomycete found in association
with eachforma specials, FOCA, FOL, FON, and FOB, respectively. Each Fusarium
forma specials was grown by itself which served as the control, and with each of the four
suspected parasites in all possible combinations. Each treatment consisted of five
replications. After five days' incubation at 250 resulting hyphal mats were filtered through
cheesecloth, squeezed free of liquid and weighed. Dry weights were additionally obtained
after air-drying hyphae for several days in petri plates. Data from hyphal weights were
analyzed by the general linear model procedure and means were separated by Duncan's
multiple range test.
Reduction of aerial hyphae was evaluated on F. oxysporum f sp. niveum only.
The various parasites were paired six cm apart on PDA and MA plates utilizing six
replications per assay. These plates were compared to FON growing singly on the same
media. Linear growth of FON for all medium treatments was measured daily for 12 days.
Collected data were analyzed as a repeated measures experiment and additionally
subjected to linear regression using the simple regression model: y = a(trt) + b(obs), where
the y equaled growth response and a and b were parameters to be estimated.
The growth inhibition assay was evaluated in a similar manner to that described
above for aerial hyphal reduction. This assay consisted of the same isolates, media, and
replications, only the mycoparasite was first put on plates to grow singly for three days.
After this time, FON was placed at the edge of the parasite colony. Linear growth of
FON was then measured as before on a daily basis. Data were similarly analyzed as a
repeated measures experiment before being subjected to linear regression. This assay
employed the quadratic model: y = a(trt) + b(obs) + c(obs2), where y = growth response
and a, b, and c were parameters to be estimated. Both of these two assays were repeated
once and results were separated by the least square means procedure. Pairwise probability
values obtained were additionally adjusted with the Bonferroni correction.
The results of the host range determinations are presented in Tables 4-2 and 4-3. Table 4-
2 shows the production of perithecia by Melanospora and Sphaerodes in conjunction with
the pathogenic isolates of F. oxysporum. There was no one parasite isolate that
statistically predominated over the others with respect to reproductive ability. Numerous
differences were observed between and among isolates with all four parasites being
represented at least once as being the best to sporulate on a fusarial isolate. Overall,
isolates LE and FL tended to produce higher numbers of perithecia than did FP or TX.
The data in Table 4-3 illustrate a similar story regarding differences among and between
parasite isolates and their abilities to efficiently produce perithecia on the non-pathogenic
isolates. However, large differences were observed in total perithecial production
between pathogenic and non-pathogenic isolates. There were no Fusarium isolates
among those tested that did not support at least some sporulation from the parasites.
With the exception of 96-14, sporulation for all parasites was much poorer on the non-
pathogenic isolates. This can be easily visualized by observing the one pathogenic isolate
analyzed with the rest in Table 4-3, and by also comparing overall values between Tables
4-2 and 4-3. The recognition that isolate 96-14 was capable of supporting good
perithecial production was an important observation and will be implemented and
discussed further in Chapter 5.
Table 4-2. Results of host range study for pathogenic isolates ofF. oxysporum using
isolates ofMelanospora and Sphaerodes
Fusarium isolate Parasite Isolate'
FLn' FP LE TX
1443 60.lb 59.7b 72.6a 43.2c
1453 70.2a 33.7c 46.9b 0.0d
1480 49.lc 34.4d 72.2b 82.3a
1481 43.2bc 36.6c 51.9b 68.3a
1742 59.9a 53.6a 60.2a 54.4a
1778 64.0a 51.6a 56.1a 51.7a
1779 63.1a 67.0a 66.3a 41.2b
2095 33.5a 32.3a 40.2a 4.3b
2099 49.1c 59.7bc 77.9a 70.lab
CL58 57.3b 41.1c 75.2a 78.1a
GD8 65.0a 58.7a 63.0a 37.9b
MN21 83.0a 66.5b 82.la 52.8b
MN27 78.9a 66.1a 79.1a 43.lb
NFL1 70.la 63.1a 60.9a 34.8b
*FL, FP, and LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
average number of perithecia produced by parasite isolates
'Means followed by the same letter are not significantly different according to Duncan's
multiple range test (P = 0.01)
Table 4-3. Results of the host range study for non-pathogenic isolates ofF. oxysporum
using isolates ofMelmospora and Sphaerodes.
Fusarium Isolate Parasite Isolate"
FL FP LE TX
101196 4.0bb.' 33.7a 29.6a 15.0b
3196-1 26.6a 34.9a 3.0b 8.0b
31196-1 26.2a 34.5a 25.9a 35.5a
96-10 22.0b 24.5b 47.6a 2.0c
96-14 72.5a 79.1a 88.6a 87.5a
96-17 26.9a 34.6a 36.1a 12.6b
96-2 23.7a 25.5a 15.4a 19.3a
96-5 24.9a 21.1a 18.1a 16.5a
96-6 18.7ab 5.0b 26.5a 7.0b
96-7 9.7b 29.9a 32.1a 24.2a
FO95-5* 76.0b 64.7b 96.5a 44.2c
'FL, FP, and LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
average number of perithecia produced by parasite isolates
*= pathogenic isolate ofF. oxysporum used for comparison
'Means followed by the same letter are not significantly different according to Duncan's
multiple range test (P = 0.01)
The slide cultures for all isolates produced similar results to those in the literature
describing mycoparasitism byM. zamiae. The isolates of Persiciospora and Sphaerodes
reacted in an identical manner. All three genera germinated and formed contact cells or
hooks on their hyphal strands that appeared to make contact with mycelial branches of the
Fusarium isolates. The ability to form these specialized branches was apparently not host
specific for each parasite and its corresponding field host. Eachforma specialist appeared
to be equally receptive to any of the parasites that were evaluated. These phenomena of
germination and attachment of ascospores and hyphae are demonstrated in Figures 4-2, 4-
3 and 4-4.
The watermelon seeds coated with both organisms simultaneously produced the
same results. After several weeks, perithecia were observed on the outer edges of the
paper towels in the humidity chambers, but no fusarial growth could be visualized.
Perithecia were removed from the periphery of the chambers and plated on media.
Resulting growth of both organisms indicated that the parasites were colonizing FON as it
grew saprophytically on the paper towel substrate. The hyphal mat assay produced the
same results. The isolates of Sphaerodes and Melanospora sporulated profusely on the
FON mats only, but not on the paper towels in the humidity chambers. The controls for
both assays consisted of parasite isolates placed singly on paper towels in chambers, and
did not result in perithecial formation. Therefore, it was concluded that the Sphaerodes
and Melanospora isolates were growing on and parasitizing FON due to the lack of any
additional nutritional source in the humidity chambers.
The assay consisting ofKA and PDA provided the final and perhaps most
convincing evidence for the parasitic abilities of these fungi with F. oxysporum. All three
genera were evaluated against FON for this test. Representatives of each (LE-
Sphaerodes, TX-Melanospora, and GI-Persiciospora) are illustrated in Figures 4-5-4-7.
In all cases, scattered perithecia were formed after several weeks on the central portion of
the plates containing the KA however there were differences in the ability to sporulate
with Sphaerodes forming the most perithecia followed by Melanospora and
Persiciospora. The outer ring of PDA in all examples consisted of a layer of perithecia
greater than that on the KA (Fig. 4-8). Komada agar was previously shown to inhibit
growth and sporulation for these isolates. However, perithecia formed on this normally
restrictive medium in conjunction with FON, and reproduction also was observed at points
removed from the initial inoculation site on the portion containing PDA. Therefore, it was
concluded that the most logical explanation for these events was the occurrence of
parasitism of Fusarium by the isolates of Sphaerodes, Melanospora, and Persiciospora as
it grew outward from the center.
The attempt to provide evidence for damage to F. oxysporum in culture was first
done by measuring the total hyphal weights of the variousformae specials alone and in
combination with the different parasites. Table 4-4 demonstrates the reductive effect on
Fusarium, but it includes only the fourformae specials and the corresponding parasites
originally found with them from the field. Although these evaluations were done with all
parasites and allformae specials in all possible combinations, there were not many
differences between the treatments containing both fungi. However, the important point
to consider is that both fungi together produced hyphal weights that were significantly
Figures 4-5-4-10. Results of Fusarium parasitism and pathogenicity assays.
4-5. Sphaerodes retispora var. retispora (LE) parasitizing FON.
Note heavier concentration of perithecia in outer ring of PDA.
4-6. Melanospora zamiae (TX) parasitizing FON. Note heavier
concentration of perithecia in outer ring of PDA, and the fewer
number of perithecia overall compared to that ofLE. (x 0.67)
4-7. Persiciospora moreaui (GI) parasitizing FON. Note the fewer
number of perithecia overall compared to both LE and TX.
4-8. Close-up view of parasitism of FON. Shows scattered perithecia
in center portion of plate with heavier concentration on outside
ring of PDA. Arrow depicts the line separating the two different
media. (x 0.58)
4-9. Plate showing the reduction of Fusarium aerial hyphae. Single
arrow depicts the final advance ofFON before being reduced.
Double arrows shows the extent of FON growth when picture
was taken (x 0.83)
4-10. Plate showing the inhibition ofFusarium hyphae. Note the clear
line of inhibition where the parasite stops FON growth (x 1.0)
Table 4-4. Reduction of total hyphal weights of oxysporumformae specials by each
parasite originally found with them.
Treatment" Wet weight (g) Dry weight (g)
FOCA 0.83 ab 0.145 ab
FOB 0.75 b 0.135 b
FON 0.73 bc 0.13 bc
FOL 0.69 c 0.12 c
FOCA+ FL 0.42 d 0.075 d
FON + LE 0.33 ef 0.057 ef
FOB + TX 0.32 ef 0.055 ef
FOL + FP 0.31 ef 0.055 ef
'FOCA = F. oxysporum f sp. canariensis; FOB = F oxysporum f sp. betae; FON = F.
oxysporum f. sp. niveum; FOL = F. oxysporum f sp. lycopersici
FL, FP, and LE = Sphaerodes retispora var. retispora; TX = Melanospora zamiae
bMeans followed by the same letter are not significantly different according to Duncan's
multiple range test (P = 0.01).
lower than any of theformae specials alone. This demonstrates that these four parasites
were pathogenic and caused measurable damage to the particularforma specialist with
which they were isolated. Each also induced highly significant weight reductions to the
otherformae specials as well (data not shown), but very few differences were observed
between these different parasite treatments.
The hyphal reduction test did not initially look very promising. Until about the
sixth day of taking measurements, the fusarial growth on all parasite treatments was equal
to that of the FON control. However, after this time, the hyphal growth of FON began to
recede and appeared to melt away. This occurred on the aerial growth only, one could
still see the mycelium on the agar surface. As the hyphae of Fusarium receded, the
parasite growth advanced in the same direction and perithecia were readily formed in the
process (Fig. 4-9). Table 4-5 exhibits the average growth of FON after 12 days for each
treatment. Isolate LE caused the most reduction to FON, but was no different statistically
than isolates FL or FP. Because these three treatments were statistically different than
that of FON alone, it was concluded that they significantly reduced the amount of aerial
mycelium compared to the control. This was interpreted to mean that they were
pathogenic to FON. The remaining two isolates, GI and TX, were not different
statistically than FON. Therefore, they were not causing distinguishable damage to FON
by the reducing aerial hyphae.
The knowledge gained from these prior observations led to the development of
another, slightly different assay. The growth reduction test entailed growing the parasites
on media for three days prior to the addition of FON. The final results were similar to
Table 4-5. Reduction of aerial hyphae ofF. oxysporum f. sp. niveum by Persiciopsora,
Melanospora, and Sphaerodes.
FON FL FP GI LE TX
Treatment" LSMean (mm) Pr >m Pr > I Pr > Pr > I Pr> IT Pr > I
FON 47.5 0.0001 0.0001 0.2868* 0.0001 0.2868*
FL 41.6 0.0001 0.7147* 0.0001 0.0063* 0.0001
FP 42.0 0.0001 0.7147* 0.0001 0.0025* 0.0001
GI 48.6 0.2868* 0.0001 0.0001 0.0001 0.0342*
LE 38.9 0.0001 0.0063* 0.0025* 0.0001 0.0001
TX 46.6 0.2868* 0.0001 0.0001 0.0342* 0.0001
'FON = F. oxysporum f sp. niveum; FL, FP, and LE = Sphaerodes retispora var.
retispora; GI = Persiciospora moreaui; TX = Melanospora zamiae
* = not significant after use of the Bonferroni correction (P = 0.0016)
Table 4-6. Growth inhibition ofF. oxysporum f. sp. niveum by isolates of Persiciospora,
Melanospora, and Sphaerodes.
FON FL FP GI LE TX
Treatment' LS Mean (mm) Pr > Trr Pr>m Pr > TIPr Pr>IT P > IT| Pr>
FON 50.8 0.0001 0.0001 0.0024* 0.0001 0.0001
FL 47.3 0.0001 0.0955* 0.0001 0.0031* 0.7202*
FP 46.4 0.0001 0.0955* 0.0001 0.1898* 0.1898*
GI 49.0 0.0024* 0.0045* 0.0001 0.0001 0.0014
LE 45.6 0.0001 0.0031* 0.1898* 0.0001 0.0091*
TX 47.1 0.0001 0.7202* 0.1898* 0.0014 0.0091* -
FON = F. oxysporum f. sp. niveum; FL, FP, and LE = Sphaerodes retispora var.
retispora; GI = Persiciospora moreaui; and TX = Melanospora zamiae
*= not significant after use of the Bonferroni correction (P = 0.0016)
the aerial hypahal reduction test, but were more visually spectacular (Fig. 4-10). The data
from this assay are presented in Table 4-6. Like the previous test, isolate LE was the most
aggressive and effective in halting fusarial growth. However, it was again no different
statistically from isolates FL or FP. The major difference between these two inhibition
assays was observed with isolate TX. Unlike its performance in the aerial reduction
TX was effective in inhibiting FON, and was statistically insignificant from the pathogenic
isolates LE, FL, and FP (Table 4-6).
Jordan and Barnett (1978) tested over 20 different fungi, and found that 11 were
judged to be hosts forM. zamiae by their evaluation method of observing contact cell
formation. This group included the tomato wilt pathogen, F. oxysporum f sp. lycopersici,
but no study has ever exclusively evaluated many different form species ofF. oxysporum.
There has also never been any type of parasitic host ranges established for Sphaerodes.
The results obtained in this study for host range determination were based upon the ability
to sporulate on the variousformae specials. They revealed a large number of differences
among and between the parasites and their reproductive abilities. This type of information
is not really very surprising, because variation in biological characteristics is a common
phenomenon in mycology.
Chapter 3 found that the parasites differed significantly in the ability to sporulate
on different media in the absence ofF. oxysporum. It further demonstrated that
production of perithecia was better for all parasites in the presence ofF. oxysporum. In
general, it was shown that M.zamiae tended to sporulate less than Sphaerodes, regardless
of whether F. oxysporum was present or not (Chapter 3). This is an interesting finding in
light of the fact that the species of Sphaerodes studied had never been found with F.
oxysporum (Cannon and Hawksworth 1982). However, after a larger number offormae
specials was tested, M. zamiae proved to have some clear host preferences in terms of
producing perithecia. These included isolates 1480 and 1481 (Table 4-2). It additionally
was among the poorest at reproducing on others like 1443, 1453, and most of the non-
pathogenic isolates (Tables 4-2 and 4-3). This suggests that ability to sporulate by these
groups of fungi is not simply a matter of some being able to and some not, but that other
factors like the presence of an adequate host also plays a role. It also suggests that ability
to sporulate may be an excellent criterion to initially judge parasitism and host-parasite
relationships. The host range study has also demonstrated that no one parasite is going to
be effective as a biological control agent for every F. oxysporum. There will be a need for
testing potential biological agents in a similar manner as this in order to determine the
most likely candidate.
This study has brought into question the validity or accuracy of establishing
parasitism by relying strictly on the formation of the specialized contact hyphal branches of
the suspected parasites. Although this is obviously an important finding, there should be
other methods tried in combination to completely address this question. This chapter has
combined the traditional technique with newly developed ones in the attempt to provide
more conclusive evidence for parasitism among this group ofpyrenomycetes on F.
oxysporum. Taken together, these methods have strongly indicated that the fungi found
occurring naturally with F. oxysporum in the field have the capacity to function as
Another important feature of parasitism has further been discovered. Just because
an organism may sporulate well on, or be able to parasitize its host, this is not always a
good indication that it will be an effective agent for biological control. All of the genera
from this study were demonstrated to be parasitic on the watermelon wilt pathogen but
there were different levels of damage caused by these organisms. Persiciospora moreaui
was isolated in conjunction with FON (Chapter 2), and was shown to be parasitic in all
tests in which it was evaluated. However, the pathogenicity assays (reduction of aerial
hyphae and growth inhibition) indicated that deleterious effects upon FON by P. moreaui
were negligible (Tables 4-5 and 4-6). This suggests that it functions as a mutualistic or
commensalistic parasite, but not as a pathogen.
This chapter has been the first attempt to demonstrate damage in culture by
mycoparasites on their hosts, and has shown that F. oxysporum is an excellent host for
Sphaerodes retispora var retispora. Sphaerodes is actually more aggressive and harmful
on Fusarium than the previously-studied parasite, M. zamiae. This also represents the
first definitive proof of parasitism for both Sphaerodes and Persiciospora, as well as the
first report ofP. moreaui in the western hemisphere.
The next chapter will focus on the selection of one of these parasites as a candidate
for biological control based upon all information gathered throughout the entire study. It
will then describe the methods and techniques used for evaluating the ability to reduce
disease severity on watermelon by the Fusarium wilt pathogen at the greenhouse level.
ECOLOGY AND USE OF SPHAERODES RETISPORA VAR. RETISPORA FOR THE
CONTROL OF FUSARIUM WILT OF WATERMELON
The Fusarium wilt and root rot diseases induced byformae specials ofFusarium
oxysporum are among the most severe plant diseases in the world. They cause significant
economic losses to a wide range of species and occur on plants belonging to all families
except the Graminaceae (Alabouvette et al. 1996).
These fungal diseases are some of the oldest recognized and among the most
difficult to manage as well. Most control methods available today are either inefficient or
difficult to apply. Chemical fungicides are ineffective because of the location of the
pathogen in the plant's vascular system. Fumigation can be successful, but is erratic and
often cost-prohibitive, depending upon the value of the crop. The beneficial effects it does
produce are only temporary and must be reapplied each season due to the pathogen's
propensity to rapidly recolonize soils (Marois and Mitchell 1981). Short term crop
rotation is of little value because F. oxysporum can colonize non-host plants and survive
as chlamydospores for long periods of time. Therefore, genetic resistance is generally
considered the be the most effective strategy for managing wilt diseases. However, this
can be a difficult procedure if no known resistance genes are available, or if many different
pathogen genotypes are present, necessitating the need for germplasm containing multiple
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