Somatic
structures. The majority of fungi are filamentous, their
somatic thalli consisting of hyphae, that may or may not
be septate.
Taxonomic
Status
Ainsworth
(1973) separated fungi into two divisions, Myxomycota
(plasmodial forms) and Eumycota (true fungi).
Entomopathogenic fungi are placed in the following
subdivisions of Eumoycota:
- Mastigomycotina
(zygosporic fungi)
Zygomycotina
Ascomycotina (sac fungi)
Basidiomycotina (club fungi)
Deuteromycotina (imperfect fungi)
Reproduction
Reproduction
is the formation of new individuals having all the
characteristics typical of the species. Fungi reproduce
asexually and sexually. Asexual reproduction does not
involve the union of sex nuclei, cells, or organs. Sexual
reproduction is characterized by the union of two nuclei
(Alexopoulos, 1952).
Asexual
The
most common method of asexual reproduction in fungi is by
means of spores such as conidia, blastospores,
chlamydospores, sproangiospores, and
azygospores.
Sexual
Sexual
reproduction is widespread in the fungi and typically
consists of three distinct phases.
- Plasmogamy
- union of two protoplasts brings the nuclei close
together within the same cell
- Karyogamy
- cell conjugation with the union of
nuclei
- Meiosis
- reduces the number of chromosomes to
haploid
- Zygospores
occur in subdivision Mastigo mycotina or aquatic
phycomycetes. The genera Coleomomyces and
Lagenidium comprise aquatic fungi, which are
obligate parasites of mosquitoes.
Zygomycotina
-- Entomophthorales
Entomogenous
zygomycetes are located principally in the order
Entomophthorales. For this publication we selected insect
pathogens from the order Entomophthorales with potential
for use in biological control of aphids and bollworms in
Mississippi. The genera Entomophthora, Neozygites,
and Pandora all have species recognized as being
able to cause epizootics that decimate arthropod
populations. All three genera are well-represented in
Mississippi. The insect hosts of these fungi occur in
more than 32 families in the orders Hemiptera, Homoptera,
Diptera, Lepidoptera, Coleoptera, Orthoptera, and
Hymenoptera (MacLeod and Muller-Kogler, 1973). In our
laboratory, we frequently find heliothine larvae
overgrown with sporulated Entomophthora spp. on
heads of field-collected sorghum. Smith et al. (1976)
also found H. zea and H. virescens infected
with Entomophthora sp. on cotton and H. zea
on corn.
Life
Cycle
The
conidiophore forcibly discharges the conidium, which is
covered with a mucilaginous sticky substance (Eilenberg
et al., 1986). When the conidium lands on a moist
substrate other than the host, it may produce a secondary
conidiophore and secondary conidium, which may produce
tertiary conidiophores and conidia. This process may
continue until the protoplasm is depleted or a suitable
host is found (Tanada and Kaya, 1993). Secondary and
tertiary conidia may become resting spores. Secondary
conidia (capilliconidia) may be borne on narrow capillary
conidiophores. The surface of the capilliconidia has
sticky substances, which adhere the spores to objects
with which they come in contact. Upon germination, the
conidium produces a germ tube that penetrates the insect
cuticle and enters the hemocoel. Inside the hemocoel, the
fungus forms protoplasts, hyphal bodies, and hyphae. The
protoplasts appear early in infection and have amoeboid
movement. The reason for the production of protoplasts by
certain species of Entomophthorales is not well
understood. The time required for entomophthorous species
to kill an insect is dependent upon many factors such as
the host it infects, temperature, moisture, and
developmental stage of the insect. At the time of death,
nearly all of the internal organs of the insect are
utilized by the fungus. The period from infection to
death of the insect may be from 3 days to 12 days, with
most deaths occurring at 5 to 8 days (MacLeod, 1963).
Shortly after the death of the insect, conidiophores grow
out from the hyphal bodies and emerge through the less
resistant portions of the exoskeleton. Terminally-borne
conidia are produced via septum formation.
Entomophthorous fungi produce various types of resistant
or resting structures: chlamydospores, zygospores,
thick-walled hyphae, and azygospores. Zygospores may be
produced when two hyphal bodies of opposite mating types
fuse. Thick-walled zygospores are important for survival
in temperate climatic zones. Germinating resting
zygospores produce a germ tube, which may produce
infectious primary-discharge conidia or secondary
adhesive spores.
Geographical
Distribution and Host Range
Species
of Entomophthora have worldwide distribution and
play an effective role in destroying many insects of
economic importance (MacLeod, 1963).
Microbial
Control
The
use of the Entomophthoraceae as microbial control agents
has been hindered by the poor survival of conidia (McCoy
et al., 1988). This group of fungi has been tested for
microbial control of aphids with some degree of success
in the former U.S.S.R. and Australia (McCoy et al.,
1988). In the United States, Hajek et al. (1996)
successfully introduced Entomophthora maimaiga
into areas more recently colonized by the gypsy moth
(Lymantria dispar).
Deuteromycotina
(Fungi Imperfecti) - Hyphomycetes
Most
of the entomopathogenic fungi belong to Deuteromycetes.
About 30 genera have been reported to contain one or more
species that infect insects. Imperfect fungi are mycelial
fungi that reproduce by means of conidia that are
generally produced on free or aggregated conidiophores on
the substrate surface. Since these fungi apparently lack
a sexual or perfect stage, they are known as imperfect
fungi. Mycologists believe that many of these fungi have
lost the ability to reproduce sexually. They have
developed parasexual reproduction in which nuclear fusion
occurs but not meiosis proper. The parasexual process
provides a mechanism for genetic exchange among imperfect
fungi. Fungi that produce conidia on more or less loose,
cottony hyphae are often termed Hyphomycetes.
A
number of fungi in this class cause muscardine diseases
in insects, including heliothine larvae. The term was
first applied to the white muscardine of the silkworm
caused by Beauveria bassiana. Other muscardine
diseases associated with heliothine larvae are the green
muscardine caused by Metarhizium anisopliae, the
red muscardine by Sorosporella uvella, and yellow
muscardine by Aspergillus flavus. As a group, the
hyphomycetous fungi are facultative pathogens. Almost all
can be cultured on artificial media.
Beauveria
bassiana
Figure
1. Beauveria bassiana infected H. virescens
larvae, pupa, and adult.
Larvae
and pupa completely covered by white conidiophores and
conidia (asexual
spores).
Adult
overgrown by the fungus.
Invasion
of cuticle and hemoceol of H. virescens larva by
hyphae of B.
bassiana.
Conidiophores
with conidia of B.
bassiana
B
bassiana colony originating from infected larva on
agar plate.
The
first microorganism to be recognized as a disease agent
was the fungus B. bassiana (Bassi, 1835). The
genus Beauveria has been monographed by MacLeod
(1954), who recognized two species, B. bassiana
and B. Brongniartii, that attack all stages of
insects of all groups. They have also occasionally been
found in the lungs of wild rodents and nasal passages of
horses, man, and giant tortoises. Beauveria
bassiana occurs worldwide; it has one of the largest
host lists among the imperfect fungi and occurs in soil
as a ubiquitous saprophyte (McCoy et al., 1988; Tanada
and Kaya, 1993).
Pathogenicity
Beauveria
bassiana generally infects through the integument.
The fungus also invades the larval alimentary canal of
H. zea to cause starvation and nutrient depletion
that may lead to larval death (Cheung and Grula, 1982).
Beauveria bassiana produces mycotoxins, such as
beauvericin, in culture media. Toxic compounds rapidly
debilitate the insect after invasion of the hemolymph
(Roberts, 1981). However, Champlin and Grula (1979)
reported that beauvericin is not produced in sufficient
quantities to be involved in the pathogenesis of B.
bassiana on H. zea larvae. Dead larvae, pupae,
and adults originating from Beauveria-infected
insects are overgrown with external mycelium and white
conidia within one or two days after death. Conidia are
produced on conidiophores growing from the surface of an
insect.
Geographical
Distribution and Host Range
Beauveria
bassiana occurs worldwide. The hosts are mainly
Lepidoptera, Coleoptera, and Homoptera. Some of the major
economic insect pests that are susceptible to this fungus
are European corn borer, Ostrinia nubilalis;
codling moth, Laspeyresia pomonella; Japanese
beetle, Popillia japonica; Colorado potato beetle,
Leptinotarsa decemlineata chinch bug, Blissus
leucopterus; and the European cabbageworm, Pieries
brassicae (Tanada and Kaya, 1993). Beauveria
bassiana is also a frequent pathogen of pecan weevils
(Curculio caryae) in the southern United
States.
Nomuraea
rileyi
Figure
2. Nomuraea rileyi infected H.
virescens.
Larva
completely covered by conidiophores and
conidia.
Cross
section of N. rileyi infected larva showing
colonization of the larval tissues by the
fungus.
Conidia
(spores formed asexually) with conidiophores of N.
rileyi (arrows).
The
fungus Nomuraea rileyi (= Spicaria prasina
= Spicaria rileyi) is pathogenic to a number of
economically important lepidopterous pests, including
H. zea and H. virescens (Ignoffo et al.,
1976). Infected hosts are covered with a dense white mat
of hyphae that, upon conidia formation, turn pale
green.
Pathogenicity
(based
mainly on the works of Mohamed et al., 1978)
Conidia of N. rileyi germinated in 2 days post
treatment of 7-day-old H. zea larvae at 25 °C
and high humidity. The germ tube passed directly into the
epicuticle. Evidence of direct penetration of the cuticle
was observed one day later. They reported that the points
of entry were darkened, indicating lysis, presumably due
to enzymatic action. Lysis was observed in the epicuticle
and exocuticle but not in the endocuticle. By the fourth
day, laterally branched hyphae penetrated the
endocuticle. These hyphae grew parallel to the
endocuticular laminae. They observed penetration into the
hemocoel five and one-half days after application of
conidia. Hyphal bodies were formed by budding from
pre-existing hyphae and by abstriction of terminal pegs.
The hyphal bodies were short, thick, mostly 1-, 2-, or
3-celled filaments and distinctly nucleated. The blood
cells were the first to be invaded, followed by fat
lobes, Malpighian tubules, muscles, and mesentron. At
death of the host, hyphae began to grow outward. Mohamed
et al. (1978) also reported that in vitro
enzymatic tests showed that N. rileyi secretes
chitinase, protease, and lipase.
Geographical
Distribution and Host Range
The
results of two surveys by Sikorowski (unpublished) and
Smith et al. (1976) of the pathogens of heliothine larvae
in Mississippi showed that N. rileyi was one of
the major pathogens of Heliothis/Helicoverpa spp.
Sprenkel and Brooks (1975) reported that N. rileyi
infects all of the major lepidopterous pests of soybean
in North Carolina. Mohamed et al. (1977) reported that
the third to fifth instars were more susceptible to
infection than were those in the first and second
instars. They also reported that the fungus was most
effective at 20° and 25 °C, with a mortality of
80% and 71%, respectively. Epizootics severe enough to
eliminate a population of heliothines in localized areas
are common in Mississippi. Such epizootic outbreaks are
usually associated with a humid, cool environment and a
dense host population, frequently present during autumn
in this state.
Metarhizium
anisopliae
The
green-muscardine fungus is rarely found associated with
heliothine larvae in the cotton and soybean producing
areas in Mississippi, although we isolated it from
southern pine beetle, Dendroctonus frontalis (SPB)
and pecan larvae. We also found that SPB larvae were very
susceptible to this fungus. Because we failed to detect
the association of the heliothine with this fungus, we
decided to present only a short description of M.
anisopliae in this publication. Metarhizium
anisopliae was isolated from the beetle Anisoplia
austriaca by Metchnikoff in 1879. He suggested it be
used as a microbial agent against insect pests. Cultures
of M. anisopliae produce destruxins A, B, C, D,
and E and desmethyldestruxin B, substances toxic to
insects (Suzuki et al., 1966, 1970, and 1971). The rapid
production of destruxins in the larvae causes death.
Metarhizium anisopliae also produces toxic
proteolytic enzymes (Kucera, 1980). Metarhizium
anisopliae has two types of conidia, the short-spored
(3.9-9.0 µm) and long-spored (9.0-18.0
µm).
On
SPB larvae, M. anisopliae forms a white mat that
upon formation of conidia turns green. The conidiophores
are branched, and a chain of conidia is formed on each
conidiophore. The mass of spore chains becomes so dense
and coheres with others to produce prismatic masses of
columns of spore chains (Tanada and Kaya,
1993).
Pathogenicity
- Infection
generally takes place through the integument. However,
the exact site of infection is dependent on the stage
of the insect, environmental conditions, and the
opportunity. The cuticle is penetrated with the aid of
enzymes secreted at the apex of the penetrant
hypha.
- Penetrant
hyphae give rise to hyphal bodies before death of the
host.
- Hyphal
bodies become distributed throughout the body cavity
and give rise to secondary hyphae.
- In
moist, warm environments, hyphae emerge a few days
after the insect's death, usually through weaker parts
of the integument, and conidia are produced borne on
conidiophores by the millions. This fungus also
produces several toxic compounds that may kill the
host.
- The
infection cycle of hyphomycetes is as follows: conidia
attachment, germination, germ tube penetration,
vegetative growth, and conidia formation.
Geographical
Distribution and Host Range
Metarhizium
anisolpliae is reported to be capable of infecting
more than 100 different insect species belonging to a
variety of insect orders (McCoy et al., 1988).
Aspergillus
spp.
Figure
3. H. virescens infected with Aspergillus
niger.
A.
niger infected larva (arrow) on a grossly
contaminated artificial rearing
diet.
A.
niger colonies originating from infected larva on
agar plate.
Tanada
and Kaya (1993) reported a number of entomopathogenic
species of Aspergillus. These fungi are mainly
saprophytic but may infect a wide range of insect
species. Aspergillus and Penicillium
species together constitute the most omnipresent and
ubiquitously distributed molds in the world. There are
more than 300 species of Aspergillus (Rippon,
1973). In Mississippi, both A. flavus and A.
niger are frequent contaminants of heliothine diets
and are also pathogens of young larvae (Sikorowski and
Lawrence, 1994). The occurrence of Aspergilli in
commercially available insect diets is unavoidable
because key heliothine diet ingredients practically
always are contaminated with various species of
Aspergillus and Penicillium. In our
laboratory, heliothine rearing containers contaminated
with Aspergillus spp. are autoclaved before they
are opened or discarded for the following reasons: 1) to
avoid contamination of the laboratory with airborne
conidia; and 2) Aspergillus flavus and A.
niger are among species of this fungus that cause a
human disease known as aspergillosis. Aspergillosis
includes colonization of vulnerable areas within the
respiratory tract, infection of ear canal, burn eschar,
nail, and rarely other tissues (such as the central
nervous system) (Swatek et al., 1985; Rippon, 1973). In
addition, A. flavus and A. parasiticus (a
closely related species) are producers of the most potent
carcinogen yet discovered. The effects of this toxin on
heliothines is, at this time, unknown. Surveys of
pathogens of field-collected H. virescens and
H. zea in Mississippi showed a small percentage of
heliothines infected by A. flavus and an
unidentified Aspergillus sp. (Sikorowski,
unpublished).
Rearing
Fungus-free Heliothines
Aspergillus
niger and, to a lesser degree, A. flavus are
among the most frequent contaminants of heliothine diets
in rearing facilities in Mississippi. The combination of
heat (about 80 °C after incorporation of dry mix
with boiling water) and antimicrobials (sorbic acid and
methyl p-hydroxybenzoate) in heliothine diets is used
widely but produces diet only partially free of
contaminants (Sikorowski and Lawrence, 1994). The process
that uses a temperature below 100 °C is called
pasteurization, while above 100 °C is called
sterilization (Banwart, 1989).
Natural
diets of insects such as leaves, fruits, and flowers are
frequently contaminated with conidia of A. niger
and A. flavus. This type of food, if not
surface-disinfected and changed daily, may contaminate
the insectary with large numbers of undesirable
microorganisms, including various species of
Aspergillus and Penicillium.
Microbes
in the air, especially various spores including conidia,
are a part of an aerosol (suspension of solid or liquid
particles in a gas). Air in an insectary may contain
microbes from various sources (e.g. humans, diet, water,
floor, and walls), and the composition of microbes in
aerosols may vary from day to day (Sikorowski and
Lawrence, 1994).
Generally,
microbial control in insectaries requires personal
hygiene, maintenance of a clean and sanitary environment,
and various methods of sterilizing insectary equipment.
Sikorowski and Lawrence (1994) stated that current
technology allows us, with less effort, to obtain control
of microbial contamination with air filtration, flash
sterilization of diet, egg surface sterilization,
sterilization and/or sanitation of equipment, and
better-trained personnel. They also reported that air
filtration is one of the most important tools to remove
airborne microbial contaminants such as Aspergilli.
Laminar air systems equipped with high-efficiency
particulate air filters, known as HEPA filters, are
usually used for this purpose. In our insectary,
microbial contaminants are controlled by flash
sterilization of diet (141 °C for about 1 minute),
use of laminar air flow with high-efficiency particulate
air filters, and above all, training of employees in
contaminant control.
In
conclusion, Aspergilli are frequently a part of the wheat
germ diet typically used for rearing heliothines, produce
copious air borne conidia, can grow on most plant and
animal tissues, may be pathogenic to larvae, and may
overgrow and saturate the diet with various mycotoxins.
Some species are causative agents of human diseases known
as aspergillosis, and A. flavus and A.
parasiticus produce aflatoxin that is known to
produce acute necrosis, cirrhosis, and carcinoma of the
liver in a variety of animal species, including humans.
Effects of mycotoxins on heliothines are
unknown.
Microbial
Control with Fungi Imperfecti
Fungi
Imperfecti that show potential as microbial control
agents for commercial development include B. bassiana,
M. anisopliae, and N. rileyi. Beauveria
bassiana and M. anisopliae are used
commercially in countries outside the United States
(McCoy, 1990). All three can be produced on various
artificial and natural media in large enough quantities
for large-scale field tests. The characteristics that are
needed before an entomogenous fungus can be considered as
a potential microbial insecticide include 1) high
virulence, 2) rapid mode of action, 3) a broad host
range, 4) stability in culture and storage, 5)
amenability to submerged fermentation, 6) amenability to
quantitative bioassay, and 7) safety to workers (McCoy,
1990).
Several
companies in the United States and abroad are engaged in
commercial production of new mycoinsecticides, which will
be marketed for control of various insects in the near
future. Most formulations are based on recently isolated,
highly virulent strains of B. bassiana, M.
anisopilae, and Verticillium lecanii and are
formulated for greenhouse and field crop
pests.
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