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Endophytic fungal entomopathogens with activity against
plant pathogens: ecology and evolution
Bonnie H. Ownley •Kimberly D. Gwinn •
Fernando E. Vega
Received: 17 September 2009 / Accepted: 12 October 2009 / Published online: 28 October 2009
ÓInternational Organization for Biological Control (IOBC) 2009
Abstract Dual biological control, of both insect
pests and plant pathogens, has been reported for the
fungal entomopathogens, Beauveria bassiana (Bals.-
Criv.) Vuill. (Ascomycota: Hypocreales) and Lecan-
icillium spp. (Ascomycota: Hypocreales). However,
the primary mechanisms of plant disease suppression
are different for these fungi. Beauveria spp. produce an
array of bioactive metabolites, and have been reported
to limit growth of fungal plant pathogens in vitro. In
plant assays, B. bassiana has been reported to reduce
diseases caused by soilborne plant pathogens, such as
Pythium, Rhizoctonia, and Fusarium. Evidence has
accumulated that B. bassiana can endophytically
colonize a wide array of plant species, both monocots
and dicots. B. bassiana also induced systemic
resistance when endophytically colonized cotton seed-
lings were challenged with a bacterial plant pathogen
on foliage. Species of Lecanicillium are known to
reduce disease caused by powdery mildew as well as
various rust fungi. Endophytic colonization has been
reported for Lecanicillium spp., and it has been
suggested that induced systemic resistance may be
active against powdery mildew. However, mycopara-
sitism is the primary mechanism employed by Lecan-
icillium spp. against plant pathogens. Comparisons of
Beauveria and Lecanicillium are made with Tricho-
derma, a fungus used for biological control of plant
pathogens and insects. For T. harzianum Rifai (Asco-
mycota: Hypocreales), it has been shown that some
fungal traits that are important for insect pathogenicity
are also involved in biocontrol of phytopathogens.
Keywords Beauveria bassiana Fungal
endophyte Hypocreales Induced systemic
resistance Lecanicillium Mycoparasite
Trichoderma
Introduction
Resource availability can trigger shifts in functional-
ity within a fungal species, thereby changing the
ecological role of the organism (Termorshuizen and
Jeger 2009). Shifts from one resource to another may
necessitate significant adaptations in metabolism,
particularly if the resources are dissimilar (Leger
Handling Editor: Helen Roy.
B. H. Ownley (&)K. D. Gwinn
Department of Entomology and Plant Pathology,
The University of Tennessee, 2431 Joe Johnson Drive,
205 Ellington Plant Sciences Bldg, Knoxville,
TN 37996-4560, USA
e-mail: bownley@utk.edu
K. D. Gwinn
e-mail: kgwinn@utk.edu
F. E. Vega
Sustainable Perennial Crops Laboratory,
United States Department of Agriculture,
Agricultural Research Service, Building 001,
BARC-West, Beltsville, MD 20705, USA
e-mail: fernando.vega@ars.usda.gov
123
BioControl (2010) 55:113–128
DOI 10.1007/s10526-009-9241-x
et al. 1997). Among members of the Hypocreales,
animal, fungal, and plant resources are exploited.
These fungi gain nutrition in a variety of ways,
including: saprotrophs that colonize the rhizosphere
and phyllosphere, endophytic saprotrophs, hemibio-
trophs and necrotrophs of plants, entomopathogens,
and mycoparasites. Some of these fungi function in
more than one econutritional mode. Fungi tradition-
ally known for their entomopathogenic characteris-
tics, such as Beauveria bassiana (Bals.-Criv.) Vuill.
(Ascomycota: Hypocreales) and Lecanicillium spp.
(Ascomycota: Hypocreales), have recently been
shown to engage in plant-fungus interactions (Vega
2008; Vega et al. 2008), and both have been reported
to effectively suppress plant disease (Goettel et al.
2008; Ownley et al. 2008).
Mechanisms of plant disease suppression
by biocontrol fungi
Biological control of plant pathogens usually refers to
the use of microorganisms that reduce the disease-
causing activity or survival of plant pathogens.
Several different biological control mechanisms
against plant pathogens have been identified. With
some mechanisms, such as antibiosis, competition,
and parasitism, the biocontrol organism is directly
involved. With other modes of biological control,
such as induced systemic resistance and increased
growth response, endophytic colonization by the
biocontrol organism triggers responses in the plant
that reduce or alleviate plant disease.
Antibiosis, competition, and mycoparasitism
The mechanism of antibiosis includes production of
antibiotics, bioactive volatile organic compounds
(VOCs), and enzymes. Volatile bioactive compounds
include acids, alcohols, alkyl pyrones, ammonia,
esters, hydrogen cyanide, ketones, and lipids (Ownley
and Windham 2007). The fungal endophyte Muscodor
albus Worapong, Strobel & W.M. Hess (Ascomycota:
Xylariales) produces a mixture of VOCs that are lethal
to a variety of microorganisms (Strobel et al. 2001;
Mercier and Jime
´nez 2004; Mercier and Smilanick
2005; Strobel 2006), as well as to insects (Riga et al.
2008; Lacey et al. 2009). In the first report of VOCs
released by a fungal entomopathogen, carbon source
played a major role in VOC production by B.
bassiana. When cultured on glucose-based media,
the VOCs identified were diisopropyl naphthalenes
(\50%), ethanol (ca. 10%) and sesquiterpenes (6%),
but in media with n-octacosane (an insect-like
alkane), the primary VOCs were n-decane (84%)
and sesquiterpenes (15%) (Crespo et al. 2008).
Enzymes involved in antibiosis are distinctly
different from those involved in mycoparasitism of
plant pathogens. For example, the biocontrol fungus
Talaromyces flavus Tf1 (Klo
¨cker) Stolk & Samson
(Ascomycota: Eurotiales) produces the enzyme glu-
cose oxidase, whose reaction product, hydrogen
peroxide, kills microsclerotia of phytopathogenic
Verticillium (Fravel 1988).
Fungal biocontrol organisms actively compete
against plant pathogens for niche or infection site,
carbon, nitrogen, and various microelements. The site
of competition is often the rhizosphere, phyllosphere,
or intercellularly within the plant. Successful com-
petition is often a matter of timing as resources are
likely to go to the initial colonizer.
Mycoparasitism is the parasitism of one fungus by
another. Varying degrees of host specificity are
displayed by mycoparasites. Within a given species
of mycoparasite, some isolates may infect a large
number of taxonomically diverse fungi, while others
demonstrate a high level of specificity (Askary et al.
1998). As reviewed in Harmon et al. (2004), parasit-
ism by the biocontrol fungus Trichoderma (Ascomy-
cota: Hypocreales) begins with detection of the
fungal host before contact is made. Trichoderma
produces low levels of an extracellular exochitinase,
which diffuse and catalyze the release of cell-wall
oligomers from the target host fungus. This activity
induces Trichoderma to release fungitoxic endoch-
itinases, which also degrade the fungal host cell wall.
Attachment of the mycoparasite to the host fungus is
mediated by binding of carbohydrates in the Trich-
oderma cell wall to lectins in the cell wall of the
fungal host. Upon contact, hyphae of Trichoderma
coil around the host fungus and form appressoria.
Several lytic enzymes are involved in degradation of
the cell walls of fungal and oomycetous plant
pathogens, including chitinases, ß-1,3 gluconases,
proteases, and lipases.
In many cases, mechanisms of biocontrol are not
mutually exclusive, i.e. multiple mechanisms may be
114 B. H. Ownley et al.
123
operating against a specific plant pathogen, or a given
biocontrol fungus may employ different mechanisms
against different phytopathogens. For example, con-
trol of Botrytis cinerea Pers. (Ascomycota: Heloti-
ales) on grapes (Vitis) with Trichoderma involves
competition for nutrients and mycoparasitism of
sclerotia, the overwintering, long-term survival struc-
ture of Botrytis. Both mechanisms contribute to
suppression of the pathogen’s capability to cause
and perpetuate disease (Dubos 1987). Following
application to leaves as a preventative, Trichoderma
induced resistance to downy mildew, Plasmopara
viticola (Berk. & M.A. Curtis) Berl. & De Toni
(Oomycota: Peronosporales), in grape (Perazzolli
et al. 2008). Therefore, it is possible that induced
systemic resistance may also play a role in biocontrol
of Botrytis. Induced resistance to Botrytis, following
application of T. harzianum T39 Rifai (Ascomycota:
Hypocreales) to roots and leaves of several ecotypes
of Arabidopsis thaliana (L.) Heynh. has been
reported (Korolev et al. 2008).
Induced systemic resistance
Plants are sessile organisms that must develop a
complex chemical arsenal in order to withstand biotic
and abiotic attack. Colonization of plants with
nonpathogenic fungi and bacteria can lead to induced
systemic resistance (ISR) in the host plant. Induced
resistance is a plant-mediated biocontrol mechanism
whereby the biocontrol agent and the phytopathogen
do not make physical contact with one another. Plants
react to the presence of a pathogen with a rapid
expression of defense-related genes. For example,
dramatic cellular changes, characterized by rapid
necrotization of lemon (Citrus 9limon (L.) Burm. f.)
fruit exocarp cells were observed in fruit treated with
Lecanicillium muscarium DAOM 198499 (Petch)
Zare & W. Gams (formerly Cephalosporium musca-
rium Petch). Phenolic compounds and phenol oxidase
were both present in reactive cells (Benhamou 2004).
In contrast, gene expression changes in plants
infected with beneficial fungi tend to be mild, and the
relationship is allowed to develop resulting in an
infected or colonized plant. The signaling mecha-
nisms for this induced resistance are based on
jasmonic acid (JA) and ethylene (Van Loon et al.
1998; Van Wees et al. 2008; Gutjahr and Paszkowski
2009). Induction of systemic resistance via the JA/
ethylene signaling pathway has been reported pri-
marily for plant growth-promoting bacteria, however,
it is also operative for many mycorrhizal fungi
(Gutjahr and Paszkowski 2009) and biocontrol fungi
(Harmon et al. 2004; Vinale et al. 2008).
Endophytism by fungal entomopathogens
Even though the term ‘‘endophyte’’ has several
definitions (Hyde and Soytong 2008), it is widely
accepted that endophytes are microorganisms present
in plant tissues without causing any apparent symp-
toms. Fungal endophytes are widespread and quite
diverse in nature (Arnold et al. 2000; Arnold 2007).
For example, Vega et al. (2009b) reported 257 unique
ITS genotypes for fungal endophytes isolated from
coffee plants in Hawaii, Mexico, Colombia, and
Puerto Rico. Infection by fungal endophytes can be
localized (i.e., not systemic; see Saikkonen et al.
1998 and references therein), and establishing a long-
term systemic infection with endophytic fungal
entomopathogens that can act against plant pathogens
will remain a challenge, and should be the focus of
intensive study.
Isolation of B. bassiana as a fungal endophyte has
been reported for many plants under natural condi-
tions, as well as in plants inoculated using various
methods (Vega 2008; Vega et al 2008). In contrast to
the several studies dealing with endophytic Beauveria
spp., only a handful of studies have been conducted
on endophytic Lecanicillium spp. For example,
Lecanicillium dimorphum (J.D. Chen) Zare & W.
Gams and L cf. psalliotae (Treschew) Zare & W.
Gams have been introduced as endophytes in date
palms (Phoenix dactylifera L.) (Go
´mez-Vidal et al.
2006), and L. muscarium strain DAOM 198499
(=Verticillium lecanii (Zimm.) Vie
´gas) and L. mus-
carium strain B-2 have been introduced as endo-
phytes in cucumber (Cucumis sativus L.) roots
(Benhamou and Brodeur 2001; Hirano et al. 2008).
In cytological investigations of cucumber roots, the
entomopathogen grew actively at the root surface and
colonized a small number of epidermal and cortical
cells, without inducing extensive host cell damage.
Ingress into the root tissue was primarily intercellular
and cell wall penetration was seldom observed
(Benhamou and Brodeur 2001). Verticillium
Endophytic fungal entomopathogens 115
123
(=Lecanicillium)lecanii has been reported as a
natural endophyte in an Araceae (Petrini 1981), in
Arctostaphylos uva-ursi (L.) (Widler and Mu
¨ller
1984), and in Carpinus caroliniana Walter (Bills
and Polishook 1991).
Although traditionally categorized as a soil sapro-
phyte, Beauveria spp. are considered to be poor
competitors for organic resources against other
ubiquitous saprophytic soil fungi (Keller and Zim-
mermann 1989; Hajek 1997). The endophytic habit of
B. bassiana may provide benefits to both plant and
fungus. It is well known that plant species has a
significant impact on shaping plant-associated micro-
bial communities (Berg et al. 2005; reviewed in Berg
and Smalla 2009). As suggested by the bodyguard
hypothesis, the plant gains through reduction of
damage against herbivorous insects (Elliot et al.
2000; White et al. 2002) or plant diseases; the fungus
benefits through protection from environmental
stress, acquisition of limited nutrients from endo-
phytic colonization as well as exudates on the plant
surface, and use of the plant surface as a staging
platform for insect parasitism. On tomato (Solanum
lycopersicum L.) and other dicots, as well as mono-
cots, colonization by B. bassiana is not restricted to
growth as an endophyte (Ownley et al. 2008; Powell
et al. 2009; authors, unpublished data). From initial
establishment as a seed treatment, the fungus can be
found on the outer surfaces as the plant ages,
particularly in areas where new leaves or shoots have
emerged. The fungus also gains from nutrients
acquired during saprophytic colonization of the plant
when it, or parts of it senesce. Similar epiphytic
growth was observed by Posada and Vega (2005)
with cocoa (Theobroma cacao L.) seedlings.
Beauveria bassiana: Potential for biological
control of plant pathogens
Beauveria bassiana is known to occur naturally in
more than 700 species of insect hosts (Inglis et al.
2001). Infection of host insects results in the
production of large numbers of conidia, thereby
serving to increase the population size of the fungus
(Meyling and Eilenberg 2007). There is now
substantial evidence that B. bassiana can provide
protection against some soilborne plant pathogens
(Ownley et al. 2004; Ownley et al. 2008; Vega et al.
2009a,b). It is likely that more than one mode of
action is operative in suppression of plant disease by
B. bassiana. Isolates of the fungus are known to
produce numerous secondary metabolites (e.g. beau-
vericin, beauverolides, bassianolides, oosporein,
cyclosporin A, and oxalic acid) with antibacterial,
antifungal, cytotoxic, and insecticidal activities
(Grove and Pople 1980; Genthner et al. 1994; Gupta
et al. 1995; Boucias and Pendland 1998; Copping and
Menn 2000). Effects of these compounds on micro-
organisms and insects have been reported (Kanaoka
et al 1978; Taniguchi et al. 1984; Eyal et al. 1994;
Boucias et al. 1995). Recently, another antimicrobial
compound, bassianolone, from B. bassiana fermen-
tation culture under low nitrogen conditions, was
characterized (Oller-Lo
´pez et al. 2005). Bassianolone
has activity against fungi and Gram-positive cocci.
Antibiosis assays with B. bassiana against various
plant pathogens in vitro have been reported (Table 1).
However, the antimicrobial compounds were not
identified.
Beauveria bassiana strain 11-98 suppresses plant
disease caused by the soilborne plant pathogens
Rhizoctonia solani Ku
¨hn (Basidiomycota: Cantharell-
ales) (Ownley et al. 2004) and Pythium myriotylum
Drechsler (Oomycota: Pythiales) (Clark et al. 2006).
This isolate produces beauvericin (Leckie et al. 2008)
and oosporein (authors, unpublished data), but it is not
known if these compounds play a role in suppression
of plant disease. Biological control of plant pathogens
with B. bassiana 11-98 is likely to involve competi-
tion for resources (Ownley et al. 2004), since the
fungus is a plant colonist. Application of B. bassiana
11-98 to tomato seed resulted in endophytic and
epiphytic colonization of seedlings and subsequent
protection against damping-off. Similarly, seed treat-
ment of cotton (Gossypium hirsutum L.) reduced
severity of R. solani damping-off in seedlings (Griffin
2007; Ownley et al. 2008). In both tomato and cotton,
the degree of disease control achieved with Beauveria
bassiana was correlated with the population density of
conidia established on seed (Ownley et al. 2008;
authors, unpublished data). Smaller seeds, such as
tomato were protected more effectively with rates of
1910
6
–10
7
CFU/seed, while higher rates (1 910
7
–
10
9
CFU/seed) gave the greatest protection against
seedling disease in cotton.
Parasitism of Pythium myriotylum by B. bassiana
may be involved in suppression of Pythium damping-
116 B. H. Ownley et al.
123
Table 1 Studies reporting activity of Beauveria spp. against plant pathogens
Strain or species of Beauveria Type of study Plant pathogen Activity against plant pathogen Reference
Beauveria bassiana, isolated from
wheat rhizosphere
In vitro bioassay
In planta (wheat), pot
assays
Gaeumannomyces graminis var. tritici J. Walker
(Ascomycota: Sordariomycetidae)
Inhibited growth; produced chitinase
and b-gluconases
Suppressed take-all disease
Renwick et al.
(1991)
Beauveria bassiana (Bals.-Criv.)
Vuill. (Ascomycota:
Hypocreales), five different
isolates
In vitro bioassay Fusarium oxysporum E.F. Smith & Swingle
(Ascomycota: Hypocreales)
Armillaria mellea (Vahl) P. Kumm
(Basidiomycota: Agaricales)
Rosellinia necatrix Berl. ex Prill. (Ascomycota:
Xylariales)
All Beauveria isolates inhibited
mycelial growth of the pathogens
tested
Reisenzein and
Tiefenbrunner
(1997)
Culture filtrate of B. bassiana In vitro bioassay Fusarium oxysporum f. sp. lycopersici (Sacc.) W.C.
Snyder & H.N. Hansen (Ascomycota Hypocreales)
Botrytis cinerea Pers. (Ascomycota: Helotiales)
Inhibited mycelial growth;
Inhibited and delayed conidial
germination
Bark et al.
(1996)
B. bassiana
B. brongniartii (Sacc.) Petch
(Ascomycota: Hypocreales)
In vitro bioassay Pythium ultimum Trow (Oomycota: Pythiales),
Pythium debaryanum R. Hesse (Oomycota:
Pythiales), Septoria nodorum (=Phaeosphaeria
nodorum (E. Mu
¨ll.) Hedjar. (Ascomycota:
Pleosporales)
Rhizoctonia solani Ku
¨hn (Basidiomycota:
Cantharellales), Pythium irregular Buisman
(Oomycota: Pythiales), Phoma betae
(=Pleospora betae Bjo
¨rl. (Ascomycota:
Pleosporales)), Phoma exigua var. foveata
Malc. & E.G. Gray (Ascomycota: Pleosporales)
Caused cell lysis; inhibited mycelial growth
Did not inhibit mycelial growth of these
pathogens
Vesely and
Koubova
(1994)
Culture filtrates of Beauveria sp. In vitro bioassay Rhizoctonia solani Inhibited mycelial growth; stimulated
growth of cucumber
Lee et al. (1999)
B. bassiana 142, applied to onion
bulbs
In planta (onion), field
and greenhouse
Fusarium oxysporum f. sp. cepae (Hanzawa)
W.C. Snyder & H.N. Hansen (Ascomycota:
Hypocreales)
Increased bulb germination; reduced
plant infection
Flori and
Roberti (1993)
B. bassiana 11-98, applied
as a seed treatment
In planta (tomato),
greenhouse
Rhizoctonia solani Reduced damping off of seedlings;
increased plant growth
Ownley et al.
(2000) and
Ownley et al.
(2004)
B. bassiana 11-98, applied
as a seed treatment
In planta (tomato),
growth chamber
Pythium myriotylum Drechsler (Oomycota:
Pythiales)
Reduced damping off of seedlings Clark et al.
(2006)
B. bassiana 11-98
B. bassiana 11-98, applied
as a seed treatment
In vitro bioassay
In planta (cotton),
growth chamber
Rhizoctonia solani
Pythium myriotylum
Did not inhibit mycelial growth of R.
solani; but hyphae of 11-98 coiled around
hyphae of P. myriotylum, which
suggested parasitism
Reduced damping-off of seedlings
Griffin (2007)
and Ownley
et al. (2008)
Endophytic fungal entomopathogens 117
123
off in tomato seedlings. In dual culture, hyphae of
isolate 11-98 were observed coiling around the larger
coenocytic hyphae of P. myriotylum (Griffin 2007).
The extent of endophytic colonization of tomato
by B. bassiana 11-98 was also correlated with the rate
of conidia applied to seed. Rates that were most
effective in disease control also resulted in the
greatest degree of plant colonization. Beauveria
bassiana was detected in root, stem, and leaf sections
of surface-sterilized tomato seedlings with standard
dilution plating procedures onto semi-selective med-
ium (Ownley et al. 2008). In addition to seedlings,
B. bassiana 11-98 has been recovered from foliage,
stem, and root tissues of surface-sterilized 18-week-
old tomato plants produced from treated seed (Powell
et al. 2009). Beauveria bassiana has also been
recovered as an endophyte of eastern purple cone-
flower (Echinacea purpurea L. Moench), cotton, snap
bean (Phaseolus vulgaris L.), soybean (Glycines max
L.), and switchgrass (Panicum virgatum L.) following
application of conidia to seed (Griffin 2007; Ownley
et al. 2008; authors, unpublished data).
Endophytic B. bassiana 11-98 has been observed
with scanning electron microscopy (SEM), and
detected with polymerase chain reaction (PCR) in
cotton seedlings (Griffin 2007). Using SEM on
seedlings maintained in a sterile system, conidial
germination and hyphal growth were observed in
association with areas of leaf exudation. Penetration
points through epithelial cells were observed, without
formation of a specialized structure. Hyphae ramified
through the palisade parenchyma and mesophyll
layers of leaf tissues. Beauveria bassiana 11-98 was
also detected with PCR in a mixed DNA sample of 1
part B. bassiana DNA to 1,000 parts cotton DNA, and
from surface-sterilized tissues of cotton seedlings
grown from B. bassiana-treated seed (Griffin 2007;
Ownley et al 2008; authors, unpublished data).
The results of a study with cotton seedlings
suggested that induced systemic resistance is also a
probable mechanism of biological control for
B. bassiana 11-98 (Griffin 2007; Ownley et al.
2008; authors, unpublished data). Isolate 11-98 was
evaluated for its ability to induce systemic resistance
in cotton against Xanthomonas axonopodis pathovar
malvacearum (causes bacterial blight). Conidia of
B. bassiana were applied as a root drench to 5-day
old seedlings, 13 days prior to pathogen challenge.
Treatment with B. bassiana (at 10
7
CFU/seedling
root) resulted in significantly lower foliar disease
ratings for bacterial blight than the untreated control
and was as effective as 2,6-dichloro-isonicotinic acid,
which has been shown to induce systemic resistance
against plant pathogens.
Lecanicillium spp. and biological control of plant
pathogens
Lecanicillium spp. (formerly classified in the single
species Verticillium lecanii) are well known as
entomopathogens of aphids and scale insects (Hall
1981; Goettel et al. 2008). These fungi are also
known as mycoparasites of species of plant patho-
genic, biotrophic powdery mildew (Hall 1980; Ver-
haar et al. 1996) and rust fungi (Spencer and Atkey
1981; Allen 1982; Whipps 1993) on various vegeta-
ble, fruit, and ornamental crops, and as pathogens of
plant parasitic nematodes (Meyer et al. 1990; Shinya
et al. 2008). Activity of Lecanicillium spp. against
both plant pathogens and insects has been demon-
strated in bioassays (Askary et al. 1998; Askary and
Yarmand 2007; Kim et al. 2007) and greenhouse
studies (Kim et al. 2008) (Table 2).
Commercial products containing Lecanicillium
spp. have not been developed for plant disease
control. However, a formulation of L. longisporum
(Petch) Zare & W. Gams, known as Vertalec
Ò
,is
available for control of insect pests. Lecanicillium
longisporum (applied as Vertalec
Ò
), Lecanicillium
attenuatum Zare & W. Gams CS625, and Lecanicil-
lium sp. DAOM 198499 suppressed development of
powdery mildew, Podosphaera fuliginea (Schltdl.) U.
Braun & S. Takam. (Ascomycota: Erysiphales)
(=synonym Sphaerotheca fuliginea) on cucumber
leaf discs when applied one or eight days after
powdery mildew inoculation. When applied to highly
infected leaf discs 11–15 days after pathogen inoc-
ulation, Lecanicillium treatments significantly sup-
pressed subsequent production of powdery mildew
spores, compared to controls (Kim et al. 2007). In
greenhouse experiments, L. longisporum (applied as
Vertalec
Ò
) suppressed spore production of powdery
mildew on potted cucumber plants under conditions
of low and high infection levels (Kim et al. 2008).
Askary et al. (1997) provided ultrastructural and
cytochemical evidence for the process of parasitism
of P. fuliginea by Lecanicillium sp. DAOM 198499
118 B. H. Ownley et al.
123
(formerly V. lecanii DAOM 198499), including
production of cell-wall degrading enzymes such as
chitinases. They suggested that prior to invasion of P.
fuliginea, the powdery mildew fungus was weakened
by antibiotics produced by Lecanicillium (Askary
et al. 1997). Subsequently, Benhamou and Brodeur
(2000) showed that this strain does produce anti-
fungal compounds in culture that are effective against
Penicillium digitatum (Pers.) Sacc. (Ascomycota:
Eurotiales), which causes postharvest green mold of
citrus. It has been suggested that production of
antimicrobial compounds that weaken or kill the
target host cells prior to parasitism is a form of
specialized saprophytism, rather than parasitism
(Be
´langer and Labbe
´2002).
In most of the studies with Lecanicillium as a
biological control against plant pathogens, activity
has been attributed to parasitism. Indeed, an array of
extracellular lytic enzymes have been reported for
isolates of Lecanicillium, including cellulases, prote-
ases, b-1,3-glucanases, chitinases (Bidochka et al.
1999; Saksirirat and Hoppe 1991) and more recently,
pectinases (Benhamou and Brodeur 2001). However,
induction of plant host defense reactions against P.
digitatum (Benhamou and Brodeur 2000; Benhamou
2004), Pythium ultimum Trow (Oomycota: Pythiales)
(Benhamou and Brodeur 2001), and powdery mildew
(Hirano et al. 2008) have been reported. In studies on
biological control of P. ultimum,Lecanicillium sp.
DAOM 198499 grew intercellularly among epider-
mal and cortical cells on cucumber roots treated with
the fungus (Benhamou and Brodeur 2001). Endo-
phytic colonization of cucumber roots was also
observed when blastospores of L. muscarium B-2
Table 2 Studies on Lecanicillium spp. as dual biological controls for plant pathogens and insect pests
Species or strain
of Lecanicillium
a
Type
of study
Plant pathogen Mode of action
against plant
pathogen
Insect Reference
V. lecanii
Vertalec
DAOM 216596
(see below)
DAOM 198499
(see below)
Laboratory
bioassay
Podosphaera fuliginea (Schltdl.)
U. Braun & S. Takam.
(Ascomycota: Erysiphales)
(syn. Sphaerotheca
fuliginea) Powdery mildew
Parasitism/
antibiosis
Macrosiphum
euphorbiae
(Hemiptera:
Aphididae)
Askary et al.
(1998)
L. muscarium (Petch)
Zare & W. Gams
(Ascomycota:
Hypocreales) strain
DAOM 198499
Laboratory
bioassay
P. fuliginea (syn. S. fuliginea) Parasitism M. euphorbiae
Aphidius nigripes
(Hymenoptera:
Braconidae)
Askary and
Yarmand
(2007)
L. longisporum (Petch)
Zare & W. Gams
(Ascomycota:
Hypocreales)
(Vertalec)
L. attenuatum Zare
& W. Gams
(Ascomycota:
Hypocreales)
strain CS625
Lecanicillium sp. strain
DAOM 198499
Laboratory
bioassay
P. fuliginea (syn. S. fuliginea) Not reported Myzus persicae
(Hemiptera:
Aphididae)
M. euphorbiae
Aulacorthum solani
(Hemiptera:
Aphididae)
Kim et al.
(2007)
L. longisporum
(Vertalec)
Greenhouse P. fuliginea (syn. S. fuliginea) Not reported Aphis gossypii
(Hemiptera:
Aphididae)
Kim et al.
(2008)
L. lecanii (Zimm.) Zare
& W. Gams (Ascomycota:
Hypocreales)
Field (survey) Hemileia vastatrix Berk.
& Broome (Basidiomycota:
Pucciniales) Coffee leaf rust
Parasitism Coccus viridis
(Hemiptera:
Coccidae)
Vandermeer
et al. (2009)
a
Name listed is the same as was given in the reference
Endophytic fungal entomopathogens 119
123
were applied to roots. Subsequently induced resis-
tance to powdery mildew on the cucumber leaf
surface was reported (Hirano et al. 2008). Koike et al.
(2004) demonstrated that L. muscarium B-2 is also a
very successful epiphytic colonist of cucumber leaf
surfaces, suggesting that competition for nutrients
and space may also be operative against powdery
mildew.
Fungal endophytism and induced systemic
resistance
Recently, proteomic analysis of P. dactylifera
infected with endophytic B. bassiana or two Lecan-
icillium spp. was reported by Go
´mez-Vidal et al.
(2009). Colonization by B. bassiana,L. dimorphum,
or L. cf. psalliotae resulted in induction of proteins
related to plant defense or stress response, and
proteins involved in energy metabolism and photo-
synthesis were also affected. As additional studies on
molecular analysis of plants infected with endophytic
fungal entomopathogens are conducted, it will
become evident that endophytism is inducing impor-
tant changes in plant metabolism, even though the
plant does not present any symptoms of endophyte
infection. It will be important to take into consider-
ation that endophytes may cause plants to enter a
‘‘primed state’’ (sensu Conrath et al. 2006; see also
Schulz and Boyle 2005), which could be contributing
to the antagonistic effects of B. bassiana and
Lecanicillium on plant pathogenic fungi. It is also
possible that endophyte infection might result in
positive effects such as enhanced plant growth (Ernst
et al. 2003; Schulz and Boyle 2005). Plant growth-
related variables should be measured in all studies
dealing with the introduction of fungal entomopath-
ogens as possible endophytes, as was recently done
by Tefera and Vidal (2009) for sorghum plants
inoculated with B. bassiana, although it will be
difficult to elucidate the role of a specific endophyte
if others are already present in the plant.
When endophytism results in ‘‘primed’’ plants,
subsequent biotic challenge leads to a transitory
period of strongly potentiated gene expression that is
associated with accelerated defense responses. These
responses confer broad-spectrum resistance to patho-
gens and insects (Van Wees et al. 2008). In this
respect, plants colonized by fungal entomopathogens
resemble plants colonized with plant growth-promot-
ing rhizobacteria (Harmon et al. 2004). Much of the
research on systemic resistance of plants infected
with endophytic beneficial fungi has focused on
mycorrhizal fungi (reviewed in Gutjahr and Pasz-
kowski 2009). These obligate fungi live on plant
roots and stimulate plant growth and development by
increasing nutrient uptake and decreasing disease and
insect problems. While plants infected with hypo-
crealean fungi do not have the complex structures
associated with mycorrhizal infection, they can
occupy a nutritional niche in or on the plant and
develop an active cross talk with their plant hosts that
results in induced resistance (Vinale et al. 2008).
Induction of plant resistance has been reported for
several species of Trichoderma (Harmon et al. 2004;
Jeger et al. 2009), and mechanisms for induced
resistance are beginning to emerge (Segarra et al.
2007; Vinale et al 2008). Mechanisms for induced
resistance by other hypocrealean fungi are scant, but
much information on mechanisms of induced resis-
tance obtained from studies with Trichoderma can be
applied to other fungal entomopathogens.
Many species of Trichoderma have been commer-
cially developed for biological control of plant
diseases and insects (Harmon et al. 2004; Shakeri
and Foster 2007). Some of these isolates induce
resistance to plant pathogens (Table 3). Typically,
Trichoderma is applied to soil or to plant roots grown
in co-culture with the fungus. However, some species
induce systemic resistance when leaves are treated
with Trichoderma conidia (Perazzolli et al. 2008;
Korolev et al. 2008). Plant hosts in which resistance
is induced are taxonomically diverse and include both
monocots and dicots. Several recent studies support
jasmonate/ethylene signaling as the mechanism for
induced systemic resistance (Table 3), further sug-
gesting that the response is similar to that induced by
rhizobacteria (reviewed in Harmon et al. 2004).
Induced resistance is broad spectrum, and subsequent
challenges of the primed plant by taxonomically
diverse pathogens (e.g., bacteria, necrotrophic fungi,
biotrophic fungi) induce a rapid and intense activa-
tion of cellular defense mechanisms somewhat rem-
iniscent of hypersensitive responses.
Species in the genus Trichoderma (Ascomycota:
Hypocreales) are well known for the production of
bioactive metabolites that play a role in the myco-
parasitic or entomopathogenic lifestyles of the
120 B. H. Ownley et al.
123
fungus, as well as in the induction of resistance in
plant hosts. Elicitors or resistance inducers can be
divided into three broad categories: proteins with
enzymatic activity, avirulence-like gene products,
and low molecular weight compounds released from
cell walls (either fungal or plant) as a result of
hydrolytic enzymes (e.g., chitinase, glucanase) (Vi-
nale et al. 2008). In several recent studies, various
proteins and peptides from Trichoderma have been
shown to induce host defense responses (Table 4).
Volatiles released after treatment with alamethicin, a
20-amino acid polypeptide isolated from T. viride
Pers., affect the behavior of the parasitoid Cotesia
glomerata (L.) (Hymenoptera: Braconidae) (Bru-
insma et al. 2009). Wasps chose alamethicin-treated
plants over nontreated plants, but chose plants on
which Pieris brassicae (L.) (Lepidoptera: Pieridae)
had fed over alamethicin-treated plants.
Sm1, a hydrophobin-like small protein secreted by
Trichoderma virens (J.H. Mill., Giddens & A.A.
Foster) Arx, was the first non-enzymatic
proteinaceous elicitor determined to be involved in
induced resistance responses in rice (Oryza sativa L.),
cotton, and maize (Zea mays L.) (Djonovic
´et al.
2006,2007). Recently a second small hydrophobin-
like protein (Epl1) was isolated from Hypocrea
atroviride (=Hypocrea atroviridis Dodd, Lieckf. &
Samuels (Ascomycota: Hypocreales)) (teleomorph of
T. atroviride P. Karst.) (Vargas et al. 2008). Epl1 was
produced as a dimer. Sm1 can also be a dimer, but
upon dimerization, the glycosyl moiety and activity
are lost. Both hydrophobins are active as resistance
inducers when configured as a monomer. Vargas
et al. (2008) have proposed that aggregation of the
elicitor disrupts the molecular cross-talk between the
beneficial fungal colonizer and plant.
Recent proteomic studies provide a glimpse into
the complexity of the Trichoderma-plant interaction.
In cucumber, 51 proteins were different in treatments
with T. asperellum Samuels, Lieckf. & Nirenberg and
untreated controls; 17 proteins were up-regulated,
and 11 were down-regulated. Proteins were divided
Table 3 Recent evidence for involvement of the jasmonate/ethylene pathway in systemic resistance induced by Trichoderma species
Species and strain
or extract
Plant Pathogen Evidence of effects Efficacy References
T. asperellum
Samuels,
Lieckf. & Nirenberg
(Ascomycota:
Hypocreales) strain
T34, (10
7
spores)
Cucumis sativus
L. (cucumber)
Pseudomonas
syringae pv
lachrymans
Significant increase of
jasmonic acid (JA),
but not salicylic acid (SA)
at 1 h, both peaked at 3 h;
JA levels not above untreated
control after 6 h, SA
decreased until 24 h;
Significant increase
of peroxidase by 6 h
Reduced bacterial
colony forming
units by ca. 50%
Segarra
et al.
(2007)
T. harzianum Rifai
(Ascomycota:
Hypocreales)
strain T39
Arabidopsis
thaliana (L.)
Heynh.
Botrytis cinerea
Pers. (Ascomycota:
Helotiales)
Col-0 ecotype, and auxin-
resistant and SA acid
mutants were ISR-inducible;
Mutants impaired in ABA,
gibberillic acid, or ethylene/
JA were not ISR-inducible
Disease severity
reduced in Col-0
following either
root or leaf
application
Korolev
et al.
(2008)
T. harzianum
strain T39
Vitis vinifera
L. cv. Pinot
Noir (grape)
Plasmopara viticola
(Berk. & M.A. Curtis)
Berl. & De Toni
(Oomycota:
Peronosporales)
Timing and persistence
differed from BTH
which is SA-dependent
Leaf treatment
decreased
disease
severity; Root
treatment did not
Perazzolli
et al.
(2008)
T. virens (J.H. Mill.,
Giddens & A.A.
Foster) Arx
(Ascomycota:
Hypocreales)
strain Gv29-8
Zea mays
L. (corn)
Colletotrichum
graminicola
(= Glomerella
graminicola
D.J. Politis
(Ascomycota:
Sordariomycetidae)
Induction of JA and green
leaf volatile biosynthetic
genes
Reduced lesion
area in leaves
from endophytic
plants
Djonovic
´
et al.
(2007)
Endophytic fungal entomopathogens 121
123
Table 4 Effects of selected Trichoderma-derived peptides and proteins on host defense responses
Peptide/protein Plant Effects and efficacy Reference Similar compounds described for Beauveria
or Lecanicillium spp.
Alamethicin: Ion channel-
forming peptide mixture
Brassica oleracea L. var.
gemmifera DC. ‘Cyrus’ (brussel
sprouts)
20-fold more potent inducer of ISR than JA;
volatile emissions; increased preference for
parasitoid wasps (Cotesia glomerata (L.)
(Hymenoptera: Braconidae))
Bruinsma et al.
(2009)
Suspension cells of Arabidopsis
thaliana (L.) Heynh. (Col-1) and
Nicotiana tabacum L. ‘BY-2’
(tobacco)
Activation of callose synthase; callose
deposition
Aidemark et al.
(2009)
Mitogen-activated protein kinase
TMK1: Serine-threonine
kinases
Phaseolus vulgaris L. (var. nanus
L.) (bean)
Deletion tmk1 mutants had reduced
mycoparasitism and host-specific regulation
of ech42 gene transcription; deletion mutants
had an increased ability to protect plants against
Rhizoctonia solani Ku
¨hn (Basidiomycota:
Cantharellales)
Reithner et al.
(2007)
Zhang et al. (2009)—Beauveria—regulation
of environmental stress
and virulence to insects
Sm1: Cerato-platanin
protein that is hydrophobin-
like
Zea mays L. (corn) Deletion or over-expression of Sm1 in mutants
did not affect normal growth and development
of Trichoderma virens (J.H. Mill., Giddens
& A.A. Foster) Arx (Ascomycota: Hypocreales);
Root colonization was not affected in mutants, but
ability to induce resistance to a foliar pathogen was
reduced in deletion mutants and increased
in some over-expression mutants
Djonovic
´et al.
(2007)
Ying and Feng (2004)Beauveria—
relationship between hydrophobins
and thermotolerance Kamp (2002)
Lecanicillium—Hydrophobins abundant in
sporulating cultures, but not in mycelial
cultures
Oryza sativa L. ‘M-202’ (rice);
Gossypium hirsutum L.
‘Paymaster 2326BG/RR’
and ‘DeltaPine 50’ (cotton)
Induced expression of defense genes
(glucanase, chitinase) locally and
systemically; H
2
O
2
produced in Sm1-
treated levels, but no resulting necrosis
Djonovic
´et al.
(2006)
Ethylene-inducing xylanase: 18
Kd protein similar to serine
protease
Gossypium hirsutum ‘DeltaPine
50’ (cotton)
The 18 Kd protein increased terpenoid production
and peroxidase activity
Hanson and
Howell (2004)
ThPG1 endopolygalacturonase:
Cell-wall degrading enzyme
associated with pectin
degradation
Lycopersicon esculentum
(=Solanum lycopersicon L. var.
lycopersicon) ‘Marmande’
(tomato)
ThPG1-silenced mutants had lower
polygalacturonase activity and less growth
on pectin medium; protection against Botrytis
cinerea Pers. (Ascomycota: Helotiales) was
the same for ThPG1-silenced mutants and
wild type, even though root colonization
by mutants was lower
Mora
´n-Diez et al.
(2009)
Fenice et al. (1997)
Lecanicillium—Antarctic strains of
V.(=Lecanicillium)lecanii had wide
enzymatic competence, including
polygalacturonase activity
ABC transporter membrane
pump: ATP-binding cassette
with transmembrane domain
L. esculentum Gene up-regulated in fungus by pathogen-secreted
metabolites and some fungicides; deletion mutants
were sensitive to fungicides and lost ability to
protect against Pythium ultimum Trow (Oomycota:
Pythiales) and R. solani
Ruocco et al.
(2009)
122 B. H. Ownley et al.
123
into four categories: stress and defense, energy and
metabolism, secondary metabolism, and protein syn-
thesis/folding (Segarra et al. 2007). In maize, 114
proteins were up-regulated and 50 were down-
regulated in response to treatment with T. harzianum.
Most of the upregulated genes were for proteins
involved in carbohydrate metabolism, defense, and
photosynthesis (Shoresh and Harman 2008).
There are several parallels between Trichoderma
and Beauveria and/or Lecanicillium spp. that suggest
similar mechanisms of induced resistance:
1. These fungi can live endophytically between
plant cells without causing negative effects on
plant growth and development. Genes with sim-
ilar function (e.g., plant defense/stress response,
energy metabolism, and photosynthesis) are up-
regulated in plants colonized by Beauveria and
Lecanicillium (Go
´mez-Vidal et al. 2009) and
those colonized by Trichoderma spp. (Segarra
et al. 2007; Shoresh and Harman 2008).
2. Plant colonization can be established horizon-
tally by application of spores to seed, roots, or
leaves. Even though the relationship between the
fungi and their hosts is intimate, plants can easily
be infected. This is similar to mycorrhizae but
contrasts markedly with the grass endophytes in
the genus Neotyphodium (Ascomycota: Hypo-
creales), which are transmitted vertically via seed
(Gime
´nez et al. 2007; Hartley and Gange 2009).
3. Beauveria and Trichoderma spp. are natural and
introduced colonists of a wide variety of plants
that include both dicots and monocots. Although
there is less information available on the plant
host range of Lecanicillium spp., it has also been
recovered as a natural and introduced endophyte
of monocots and dicots.
4. All three fungi produce a wide array of enzymes
and avirulence-like products. Hydrolytic
enzymes that can attack substrates as diverse as
plant cell walls, insect cuticle, and oomycetous
and fungal plant pathogens are important for the
varied nutritional niches occupied by these fungi.
5. Beauveria bassiana and many species of Trich-
oderma produce hydrophobins or hydrophobin-
like molecules. It has been suggested that the
functions of hydrophobins in the life cycle of
fungi include: formation of protective layers,
attachment, structural components of cell walls,
and reduction of surface tension to allow aerial
growth (Linder 2009). Hydrophobins produced
by B. bassiana have been shown to be important
in conidial thermotolerance (Ying and Feng
2004) and attachment to substrates (Holder and
Keyhani 2005). Hydrophobins of T. asperellum
were proposed to protect hyphae from defense
compounds during the early stages of infection
(Viterbo and Chet 2006). Therefore, it is possible
that they play a similar role in B. bassiana.
Hydrophobins have been detected in Lecanicil-
lium (Kamp 2002), but little is known on their
role in the fungal life cycle.
6. Mitogen-associated protein kinases (MAP
kinases) in the subfamily HOG-1 (High osmo-
larity glycerol (1) are associated with host
infection and with protection from osmotic stress
in Beauveria and Trichoderma spp. The MAP
kinases interfere with the ability of T. atroviride
to induce resistance to the soilborne plant
pathogen, R. solani, in bean plants. Deletion
mutants had a greater ability than wild type to
protect the plants. In B. bassiana, MAP kinases
regulated response of the fungus to stress.
Deletion mutants were more sensitive to hyper-
osmotic stress, high temperature, and oxidative
stress than the wild type (Zhang et al. 2009).
When transcript levels of hydrophobin-encoding
genes in the deletion mutants were low, conidial
attachment to cicada hind wings was severely
impaired (Zhang et al. 2009).
7. Both Beauveria and Trichoderma spp. can
induce systemic resistance to bacterial patho-
gens. In cucumber, plants infected by T. asper-
ellum (10
7
conidia ml
-1
) supported less than
50% the number of colony-forming units (CFU)
after challenge with Pseudomonas syringae
pathovar lachrymans (Segarra et al. 2007).
Treatment of cotton with 1 910
7
CFU B.
bassiana 11-98 per root induced systemic resis-
tance against bacterial blight (Xanthomonas
axonopodis pathovar malvacearum) on cotton
foliage. Although bacterial populations were not
assessed, foliar disease ratings were significantly
lower for Beauveria-treated plants than the
untreated control (Griffin 2007).
8. Both Lecanicillium and Trichoderma spp. can
induce systemic resistance to oomycetous plant
pathogens. Host plant signaling and subsequent
Endophytic fungal entomopathogens 123
123
intense defense responses have been proposed for
Lecanicillium-treated cucumber. Ingress of P.
ultimum into roots resulted in the deposition of
an electron-opaque material that frequently encir-
cled pathogen hyphae and accumulated in unin-
fected xylem vessels (Benhamou and Brodeur
2001). Inoculation of roots with L. muscarium
resulted in root colonization and endophytic
growth. Plant leaves were protected from powdery
mildew, but defense enzymes were not different in
colonized and non-colonized plants (Hirano et al.
2008). Trichoderma harzianum induced systemic
resistance in pepper plants grown from seed
treated with T. harzianum spores (Ahmed et al.
2000). Stem lesions, caused by inoculation with
Phytophthora capsici Leonian (Oomycota: Per-
onosporales), were 40% shorter than lesions in
inoculated plants grown from non-treated seed. P.
capsici was isolated from zones immediately
contiguous with the necrotic tissue, but T. harzia-
num was not, suggesting that there was no direct
contact between them. The percentage of P.
capsici isolated nine days after inoculation was
greater in non-treated inoculated plants than in
Trichoderma-treated plants inoculated with P.
capsici. In addition to induced resistance against
P. capsici in the upper part of the plant, concen-
tration of the phytoalexin capsidiol was more than
7-fold greater than in non-treated plants inocu-
lated with P. capsici, six days after inoculation
(Ahmed et al. 2000).
Conclusions
The ability of many hypocrealean entomopathogens
to occupy nutritional niches as diverse as insects,
fungi, and plants provides unique opportunities for
biological control of multiple plant pathogens and
insect pests. Use of these fungi may overcome some
of the challenges faced in plant disease control. For
example, many foliar phytopathogens have a very
high sporulation rate and are well-suited for wide-
spread dissemination as air-borne propagules. If
genetic resistance is not available in the crop,
fungicide applications are often the primary means
of disease control. The rapid reproduction rate of
foliar pathogens coupled with frequent applications
of systemic fungicides, many of which are narrow
spectrum, increases the chances of developing
fungicide resistance in these pathogens (Fry 1982).
The ability of the hypocrealean fungi to use several
strategies reduces the probability of development of
resistance. For example, treatment of roots or seeds
with Beauveria or Lecanicillium spp. conidia poten-
tially produces endophyte-infected plants that reduce
initial establishment of the disease through induced
resistance. Studies have shown that both Beauveria
and Lecanicillium spp. can become established as
epiphytes, which provides opportunities for plant
disease suppression through antibiosis, competition,
or mycoparasitism. Endophytic and epiphytic popu-
lations of these fungi could also reduce insect damage
to the plant.
Plant diseases caused by soilborne fungi are
notoriously difficult to control since these fungi
generally have wide host ranges and can survive in
soil for long periods of time as saprophytes or as
specialized survival structures (e.g., sclerotia, chlam-
ydospores). Resistant cultivars are available for a
limited number of host-pathogen combinations. Soil-
borne pathogens often cause disease at multiple life
stages of the plant (i.e., seed rot, damping-off of
seedlings, and root rots), but typically, the greatest
impact is on the seed or newly emerged seedling. Use
of hypocrealean fungi as plant, seed, or soil treat-
ments facilitates rapid colonization of plant hosts and
creates potential for subsequent induced resistance.
Older plants may be protected from root rots by
induced systemic resistance, although this has not
been documented. Seed treatment may also create a
potential ‘antibiotic’ spermosphere that inhibits pop-
ulations of seed rot pathogens. Mycoparasitism by
hypocrealean fungi can be directed against survival
structures of soilborne plant pathogens, thus reducing
their inoculum potential.
Although much has been accomplished in the
commercial development of Beauveria and Lecani-
cillium spp. as fungal entomopathogens in plant
production, more work is needed to understand the
roles of these fungi as epiphytes and endophytes
involved in suppression of plant diseases. Some
strains of these fungi have been approved for use as
bioinsecticides. Use in plant disease control extends
development of these products. Future studies should
focus on the ecology of these fungi (Vega et al.
2009a,b), their role in plant-microbe interactions,
and their antagonism against pathogenic and nontar-
get microorganisms.
124 B. H. Ownley et al.
123
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Author Biographies
Dr. Bonnie H. Ownley conducts research on the biology,
biological and cultural control, and epidemiology of soilborne
plant pathogens, and on the ecology and population dynamics
of beneficial plant-associated microorganisms.
Dr. Kimberly D. Gwinn focuses her research on natural
products for control of plant disease, the roles of natural
products in host-symbiont relationships, and secondary metab-
olism of fungi.
Dr. Fernando E. Vega conducts research on biological
methods to control the coffee berry borer, the most important
insect pest of coffee throughout the world.
128 B. H. Ownley et al.
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