This article was downloaded by: [Umeå University Library]

On: 26 September 2013, At: 07:02

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK

Canadian Journal of Plant Pathology

Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tcjp20

Antagonism of the endophytic insect pathogenic fungus

Metarhizium robertsii against the bean plant pathogen
Fusarium solani f. sp. phaseoli
a a
Ramanpreet K. Sasan & Michael J. Bidochka
a
Department of Biological Sciences , Brock University , St. Catharines , ON , L2S 3A1 ,
Canada
Accepted author version posted online: 24 Jul 2013.Published online: 29 Jul 2013.

To cite this article: Ramanpreet K. Sasan & Michael J. Bidochka (2013) Antagonism of the endophytic insect pathogenic
fungus Metarhizium robertsii against the bean plant pathogen Fusarium solani f. sp. phaseoli , Canadian Journal of Plant
Pathology, 35:3, 288-293, DOI: 10.1080/07060661.2013.823114

To link to this article: http://dx.doi.org/10.1080/07060661.2013.823114

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions
 Can. J. Plant Pathol., 2013
Vol. 35, No. 3, 288–293, http://dx.doi.org/10.1080/07060661.2013.823114

Disease control/Moyens de lutte

Antagonism of the endophytic insect pathogenic fungus

Metarhizium robertsii against the bean plant pathogen Fusarium
solani f. sp. phaseoli

RAMANPREET K. SASAN AND MICHAEL J. BIDOCHKA

Downloaded by [Umeå University Library] at 07:02 26 September 2013

Department of Biological Sciences, Brock University, St. Catharines, ON L2S 3A1, Canada

(Accepted 25 June 2013)

Abstract: The antagonism of bean (Phaseolus vulgaris) root rot fungus Fusarium solani f. sp. phaseoli by the endophytic insect pathogenic
fungus Metarhizium robertsii was investigated in vitro and in vivo. Dual cultures on Petri dishes showed antagonism of M. robertsii against F.
solani. A relative inhibition of c. 60% of F. solani growth was observed in these assays. Cell-free culture filtrates of M. robertsii inhibited the
germination of F. solani conidia by 83% and the inhibitory metabolite was heat stable. Beans plants colonized by M. robertsii, then exposed to
F. solani, showed healthier plant growth and lower disease indices compared with plants not colonized by M. robertsii. These results suggest
that the endophytic insect pathogenic fungus M. robertsii could also be utilized as a biocontrol agent against certain plant root pathogens.

Keywords: antagonism, biocontrol, rhizosphere, root rot

Résumé: L’antagonisme du champignon entomopathogène Metarhizium robertsii à l’endroit du pourridié Fusarium solani f. sp. phaseoli du
haricot commun (Phaseolus vugaris) a été étudié in vitro et in vivo. Des cultures en duel sur boîtes de Petri ont démontré l’antagonisme de M.
robertsii à l’égard de F. solani. Au cours de ces tests, une inhibition relative d’environ 60 % de la croissance de F. solani a été observée. Des
filtrats de cultures acellulaires de M. robertsii ont réduit de 83 % la germination des conidies de F. solani, et le métabolite inhibiteur était
thermostable. Les plants de haricot colonisés par M. robertsii, puis exposés à F. solani, ont affiché une meilleure croissance et de plus faibles
indices de la maladie que les plants qui n’avaient pas été colonisés par M. robertsii. Ces résultats suggèrent que le champignon
entomopathogène M. robertsii pourrait également être utilisé comme agent de lutte biologique contre certains organismes pathogènes
s’attaquant aux racines des plantes.

Mots clés: Antagonisme, lutte biologique, pourridié, rhizosphère

Introduction die. In some cases, the plant hypocotyl is also affected

along with the development of lateral or adventitious roots
Haricot beans (Phaseolus vulgaris L.) are highly sus-
above the damaged taproot. Infected plants show stunted
ceptible to root rot disease which is caused by the soil-
growth when compared with healthy plants (Burke &
borne fungal pathogen Fusarium solani f. sp. phaseoli
Barker, 1966; Campbell, 1989; Schneider & Kelly, 2000;
(Steadman et al., 1975; Graham, 1978; Tan & Tu, 1995;
Cichy et al., 2007).
xCichy et al., 2007). Disease symptoms are initially char-
Generally, fungicides are used to control fusarium root
acterized by narrow, long, red to brown lesions on stems
rot (Campbell, 1989). Application of fungicides is not
extending down to the main taproot, which may decay and

Correspondence to: M. J. Bidochka. E-mail: bidochka@brocku.ca

© 2013 The Canadian Phytopathological Society

 Antagonism of Metarhizium against Fusarium 289

always successful and may enhance fungicide resistance, Antagonism of M. robertsii to F. solani on Petri dishes
environmental contamination, harm to human health and
The antagonism of M. robertsii against F. solani was
production costs. In this context, biological control can
screened using dual culture Petri dish assays (Sobowale
be an effective alternative and a number of biological
et al., 2010). Metarhizium robertsii and F. solani were
antagonists are currently available. For example, several
inoculated 3 cm apart from the edge of the Petri dish
species of Trichoderma (Grosch et al., 2006; Rojo et al.,
(9 cm diameter) containing PDA. Metarhizium robert-
2006; El-Kassas & Khairy, 2009), Pythium oligandrum
sii was inoculated first, allowed to grow for two days,
Dreschler (Floch et al., 2005), and other fungal species
before the introduction of F. solani. Controls contained M.
are reportedly used as biocontrol agents of plant root dis-
robertsii or F. solani alone, in order to observe the growth
eases. In particular, several endophytic fungi have been
in the absence of interactions. Additionally, controls with
shown to be antagonists of F. solani (Hassan Dar et al.,
F. solani and F. oxysporum inoculated 3 cm apart, each
1997).
1.5 cm from the centre of the Petri dish, were utilized.
Metarhizium robertsii J.F. Bisch., Rehner & Humber
Fungal growth was observed for 14 days at 25 ◦ C. Growth
sp. nov., a well-known insect pathogenic fungus, has
of F. solani was measured as the average diameter of the
recently been reported to be a plant endophyte and pro-
colony size in four different directions. Fusarium solani
Downloaded by [Umeå University Library] at 07:02 26 September 2013

motes plant root growth (Sasan & Bidochka, 2012).

Metarhizium spp. have previously been shown to pos-
sess antifungal properties against Fusarium oxysporum
Schlecht. emend. Snyder & Hansen, Botrytis cinerea
(De Bary) Whetzel, and Alternaria solani Sorauer (Kang
et al., 1996) as well as Ceratocystis ulmi (Buisman) Melin
& Nannf. (Gemma et al., 1984). In this study, we explore
the possibility that M. robertsii could be antagonistic to
the bean root rot pathogen F. solani f. sp. phaseoli under
in vitro and in vivo conditions. To our knowledge, this is
the first report of biocontrol of a bean root pathogen by an
endophytic, insect pathogenic fungus.
Materials and methods
Fungal isolates
Metarhizium robertsii (ARSEF 2575) was obtained from
the United States Department of Agricultural Research
Service Collection of Entomopathogenic Fungal Cultures,
Ithaca, New York. Cultures were grown on potato
dextrose agar (PDA; Difco Laboratories, Sparks, MD)
at 27 ◦ C at a 16/8 h day/night cycle for 10 days
to obtain conidia. Fusarium solani f. sp. phaseoli
(DAOM 170970) was obtained from the Canadian Culture
Collections, Agriculture Canada, Ottawa. This strain was
originally isolated from infected bean roots. Fusarium
oxysporum Schlecht. emend. Snyder & Hansen, origi-
nally isolated from infected wheat roots, was obtained
from Wards Natural Science Limited (St. Catharines,
ON). Fusarium solani and F. oxysporum were grown on
PDA for 14 days at 25 ◦ C to obtain macroconidia. The
conidial suspensions were filtered through glass wool to
remove mycelia, and adjusted, using sterile distilled water
containing 0.01% (v/v) Triton X-100, to 106 conidia
mL−1 measured using a haemocytometer on a compound
microscope.
growth on day 14, measured as the average diameter of
the colony size in four different directions, was used for
data analysis. The per cent inhibition of the radial colony
growth was calculated as [(C-T)/C] ×100 (Mishra, 2010),
where C is the radial growth in the control set and T is the
radial growth in the treated set. There were 10 replicates
per experiment and the experiment was repeated thrice.
The formation of clearing zones by M. robertsii in
the presence of F. solani was investigated by introducing
M. robertsii on a F. solani lawn culture that was grown
for 5 days. These cultures were initiated by spreading a
macoconidial suspension onto the surface of PDA plates.
Metarhizium robertsii was inoculated (10 µL of 108 coni-
dia mL−1 ) at four equidistant points on the F. solani lawn.
Plates were incubated at 25 ◦ C for 14 days at a 16/8 h
day/night cycle and clearing zones due to M. robertsii
were measured on days 7 and 14. There were 10 replicate
dishes per experiment and the experiment was repeated
thrice.
Antagonism of Metarhizium robertsii cell-free culture
filtrate
The effect of cell-free culture filtrates of M. robertsii on
spore germination and growth in liquid culture of F. solani
was assessed. Metarhizium robertsii was inoculated into
100 mL of yeast extract broth (0.1% yeast extract in dis-
tilled water; YEB) and incubated at 25 ◦ C. Culture filtrates
were obtained on days 4 and 10 by vacuum filtration
through Whatman filter paper no. 44 followed by filtra-
tion through 0.22 µm Millipore membrane. The culture
filtrates were checked for the presence of M. robertsii by
plating a sample on PDA. The cell-free culture filtrates
were diluted in YEB, and 3 mL of 100%, 90%, 50% and
10% cell-free culture filtrate were inoculated with 300 µL
of a conidial suspension of F. solani (108 conidia mL−1 )
 R. K. Sasan and M. J. Bidochka
in glass tubes. YEB was used as the control. The coni-
dial suspensions were incubated at 25 ◦ C. The number
of F. solani conidia germinated per hundred cells was
counted at 24 and 48 h. Three counts of 100 were taken
from each treatment and the experiment was repeated
thrice.
The effect of M. robertsii cell-free culture filtrates on
F. solani growth was also observed for a 4-day period.
Autoclaved, as well as non-autoclaved, M. robertsii cell-
free culture filtrate was obtained after 4 days and 10 days
of M. robertsii growth as described above. Fusarium
solani conidial suspension (1.25 mL of 108 conidia mL−1 )
was added to 125 mL of M. robertsii 100% cell-free cul-
ture filtrate in glass flasks. For controls, YEB was used.
Flasks were placed on a rotary shaker at 150 rpm at 25 ◦ C
Downloaded by [Umeå University Library] at 07:02 26 September 2013
for four days. The cultures were harvested by vacuum fil-
tration through Whatman No. 44 filter paper and dried at
40 ◦ C for 48 h and weighed. There were four replicate
flasks per experiment and the experiment was repeated
thrice.
Antagonism in host plants
Haricot bean seeds were obtained from OSC seeds
(Waterloo, ON) and surface disinfected prior to use by
a modification of the method of Miche & Balandreau
(2001). Seeds were soaked in sterile distilled water for
30 min and then immersed in 4% sodium hypochlorite
thrice (10 min each) followed by a single 30 min wash
with 30% hydrogen peroxide. Seeds were then washed
with sterile distilled water and kept overnight at 4 ◦ C to
allow for synchronization of growth. Seeds were plated
on PDA as well as nutrient agar to test for fungal and
bacterial contamination.
The effect of pretreating the potting mixture (Schultz
Potting Mixture, Brantford, ON) with M. robertsii as an
antagonist of F. solani infection in bean plants was inves-
tigated in growth chamber experiments. Potting mixture
was moistened with sterile distilled water and autoclaved
in trays (3 cm × 19 cm × 28 cm) twice before use.
Eight plugs (1 cm2 each) from an actively growing
fungal colony were mixed in each tray and incubated for
10 days at 25 ◦ C. The medium was mixed every alter-
nate day to ensure uniform inoculation. Treatments for
biocontrol assays were: (1) no treatment; (2) M. robert-
sii; (3) F. solani; and (4) M. robertsii and F. solani
(1 : 1 ratio). Inoculum of F. solani consisted of 8 plugs
(1 cm2 ) taken from a PDA colony. Bean seeds were
individually placed at a 0.5 cm depth in each tray.
The potting mixture was moistened with 10 mL half-
strength MMN solution (0.05 g CaCl2 , 0.025 g NaCl,
0.5 g KH2 PO4 , 0.5 g (NH4 )2 HPO4 , 0.15 g MgSO4 .7H2 O,
290
1 mg FeCl3 .6H2 O, 100 µg thiamine hydrochloride,
5 g glucose monohydrate), 10 mL L−1 stock solution
of trace elements (containing KCl 3.728 g, H3 BO3
1.546 g, MnSO4 .H2 O 0.845 g, ZnSO4 .7H2 O 0.575 g,
CuSO4 .5H2 O 0.125 g, (NH4 )6 Mo7 O24 .4H2 O 0.018 g, per
litre) (Kottke et al., 1987) once a week to promote plant
growth and sterile distilled water was added daily to main-
tain soil moisture. Plants were maintained in a growth
chamber (25/20 ◦ C, 60/80% relative humidity and 16/8 h
day/night cycle (Grosch et al., 2006) for four weeks. Plant
growth parameters such as seedling emergence, shoot
height, number of leaves, and any visible disease symp-
toms were assessed daily. After four weeks, whole plants
were removed from the potting mixture and roots were
rinsed with sterile water. Plants were individually rated for
disease severity based on a root rot index and colour inten-
sity according to Dar et al. (1997). Individual plants were
rated on a scale of 0–1, where 0 = no root rot symptoms;
0.10 = less than 10% root area rotted; 0.25 = 11–25%
root area rotted; 0.50 = 26–50% root area rotted; 0.75
= 51–75% root area rotted; and 1.0 = more than 75%
root area rotted. The individual ratings were converted to
a mean disease index by taking the quotient of the sum
of the individual plant rating values and the number of
plants assessed. Plants were also rated for disease severity
based on necrotic lesions on the roots and hypocotyl as
described by Filion et al. (2003). The ratings were based
on a scale of 0–5, where 0 = no disease symptoms, 1
= slightly brown or <50% surface discolouration of the
hypocotyl, firm upon pressure from thumb and forefin-
ger, and slight root pruning; 2 = as 1 but >50% surface
discolouration; 3 = discoloured hypocotyl and roots col-
lapsing under pressure and extensive root pruning, 4 =
darkly discoloured hypocotyl and roots completely col-
lapsed or collapsing easily under pressure and severe root
pruning; and 5 = dead or dying plant. Root and shoot
fresh weights, height, number of leaves, and dry weights
of entire plants were measured for all plants in each
treatment. Each treatment included five plants and the
experiment was repeated four times.
Results and discussion
Antagonism of M. robertsii to F. solani on Petri dishes
When F. solani and M. robertsii were co-cultured on
Petri dishes, an inhibition clearing zone was observed
on day 4 and this effect was observed up to day 14
(Fig. 1a). Clearing zones were absent when F. oxyspo-
rum and F. solani were co-cultured (data not shown).
Inhibition of F. solani by M. robertsii was also observed as
a decrease in radial growth. A relative inhibition of 59.4%
 Antagonism of Metarhizium against Fusarium 291

a) 80
a)
70

F. solani % conidial germination

Day 7

60

50

40

30
Day 14

20

10

0
F.s/ F.s F.s/ M.r 100 90 50 10 Con 100 90 50 10
b) 4d 10 d
0.7
b)
0.6
Downloaded by [Umeå University Library] at 07:02 26 September 2013

F. solani dry wt (g)

0.5

0.4

0.3
Control+ F.s
0.2

0.1

0
100 90 50 Con 100 90 50
4d 10 d
+ M.r + F.s M. robertsii (4 and 10 d) cell free extract [%]

Fig. 1. Images of dual plate cultures of Metarhizium robertsii (M.r)
and Fusarium solani (F.s) and biocontrol of F. solani by M. robertsii
on bean root. (a) Dual plate assays showing antagonistic activity of
M. robertsii on F. solani on day 7 and 14. In each photo, a zone of F.
solani clearing is evident at the colony interface. (b) Images of bean
root rot caused by F. solani after four weeks. Roots were obtained
from control plants, F. solani treatment and M. robertsii + F. solani
treatments.
of F. solani growth occurred when co-cultured with M.
robertsii. A zone of F. solani inhibition of 3.7 ± 0.5 mm
and 5.9 ±1.1 mm on a lawn culture was observed at days
7 and 14, respectively, by M. robertsii. Furthermore, M.
robertsii could be observed overgrowing F. solani in dual
culture plate assays at day 14 and white mycelial growth
of M. robertsii could be seen at the interface with F. solani.
The darker zone of confluent growth could be indicative of
melanization or necrosis of F. solani hyphae.
Antagonism of Metarhizium robertsii cell-free culture
filtrate
Fusarium solani conidia incubated in cell-free culture
filtrates of M. robertsii showed delayed germination
(Fig. 2a). Fusarium solani conidia incubated in 100%
M. robertsii cell-free culture extract collected on day
10 showed 83% and 66% germination inhibition, com-
pared with the control, at 24 and 48 h, respectively
Fig. 2. Inhibition of Fusarium solani in Metarhizium robertsii cell-
free culture extracts. (a) Germination of F. solani conidia in M.
robertsii cell-free culture extract. Fusarium solani conidia were
suspended in different concentrations (100%, 90%, 50% and 10%)
of 4-day-old and 10-day-old M. robertsii cell-free culture filtrates.
Con = control, no cell-free extract. Germination of 100 F. solani
conidia was counted (N = 4) after 24 h (light bars) and 48 h
(dark bars). Each bar represents mean % germination ± S. E. (b)
Fusarium solani biomass yield in M. robertsii cell-free culture fil-
trate (autoclaved – light bars and non-autoclaved – dark bars) after
four days of growth. Data represents the mean ± S.E. of dry weights
of F. solani grown under different concentrations (100%, 90% and
50%) of M. robertsii autoclaved and not autoclaved cell-free extract
and the control (Con = control, no cell-free extract).
(P<0.0001, t = 26.85, df = 6). At day 4, M. robertsii
cell-free extract germination inhibition was 65% and 39%
at 24 and 48 h, respectively (P<0.0001, t = 16.29, df = 6).
These results suggest that M. robertsii secretes a com-
pound that inhibits F. solani germination. Additionally,
the amount of this compound in M. robertsii cultures
increased with culture age.
Metarhizium robertsii cell-free culture filtrates
also decreased F. solani vegetative growth (Fig. 2b).
Autoclaving the cell-free culture filtrate did not reduce
the inhibitory effect on F. solani. No significant dif-
ferences were observed in F. solani biomass when
grown in autoclaved compared with non-autoclaved M.
robertsii cell-free culture filtrates (P>0.05, df = 8). This
 R. K. Sasan and M. J. Bidochka 292

suggests that the inhibitory compound(s) are heat stable. Table 1. The effect of M. robertsii (M.r) as an antagonist of
Fusarium solani biomass obtained in control cultures was Fusarium solani (F.s) infection of bean plants.
significantly higher (P<0.0001, df = 8) than without M. Root rot and colour Disease index based on necrotic
robertsii cell-free culture filtrate. intensityb lesions on roots and hypocoty
Many species of endophytic fungi show antagonism
Controla 0.25 ± 0.03 1.73 ± 0.28
against plant pathogenic fungi. Furthermore, cell-free F.s 0.69 ± 0.03 3.93 ± 0.07
extracts from endophyte fungal cultures inhibited growth F.s + M.r 0.35 ± 0.03 2.31 ± 0.11
of various plant pathogenic fungi (Liu et al., 2001; Inácio
a Controlsrepresent plants grown without F. solani or M. robertsii. Values
et al., 2006; Kim et al., 2007). For example, treatment
are mean (±S.E.).
of wheat with the endophytic fungi Chaetomium sp. and b The indices are described in the Materials and methods. The root rot
Phoma sp. resulted in reduced severity of foliar disease indices (±S.E.) and colour intensities are according to Dar et al. (1997)
caused by Puccinia and Pyrenophora spp. The same pro- and disease indices based on necrotic lesions on the roots and hypocotyl are
tective effect was observed when the endophytic fungal as described by Filion et al. (2003).
culture filtrates were applied to the plants (Dingle &
presence of the pathogen alone. After four weeks, roots
McGee, 2003; Istifadah & McGee, 2006). Metarhizium
were collected from all treatments and rated for growth
Downloaded by [Umeå University Library] at 07:02 26 September 2013

spp. produce a number of metabolites, such as destruxins,

and disease indices (Table 1). Roots obtained from the
swainsonine, serinocyclins and cytochalasins (Roberts
F. solani treatment were necrotic when compared with
& St Leger, 2004; Krasnoff et al., 2007). These com-
the control roots (without F. solani or M. robertsii) or
pounds have insecticidal and phytotoxic activities; how-
the F. solani + M. robertsii treatment plants (Fig. 1b).
ever, their antagonistic effects on plant pathogens have yet
Roots infected with F. solani showed severe disease symp-
to be elucidated. Metarhizium spp. have previously been
toms when compared with the F. solani + M. robertsii
shown to have antifungal activity against F. oxysporum,
treatment (Fig. 1b). Per cent root rot area rating for F.
Botrytis cinerea (De Bary) Whetzel, and Alternaria solani
solani treatment was greater (0.69±0.03) than the control
Sorauer (1896) (Kang et al., 1996). Other endophytic
plants (0.25±0.03) (P<0.0001, t = 8.86, df = 14) and F.
insect pathogenic fungi, including Beauveria bassiana
solani + M. robertsii treatments (0.34±0.03) (P<0.0001,
(Bals.-Criv.) Vuill. and Lecanicillium spp., provide dual
t = 8.37, df = 14). There were no statistical differ-
biological control properties (i.e. against insect pests and
ences in root rot rating between the control plants and
plant pathogens) (Ownley et al., 2010). Metarhizium
F. solani + M. robertsii treatment (P > 0.05, t = 1.97,
anisopliae and B. bassiana were reported to show in-
df = 14). Additionally, the disease index based on inten-
vitro inhibition of Ceratocystis ulmi (Buisman) Melin &
sity of hypocotyl and root rot was also higher in the F.
Nannf. (1934) (Gemma et al., 1984). Beauveria bassiana,
solani treatment (3.93±0.07) when compared with control
which is also an endophyte, provided plant protection
plants (1.73±0.28) (P<0.0001, t = 9.89, df = 14) or F.
against Rhizoctonia solani J.G. Kühn 1858 and Pythium
solani + M. robertsii treatments (2.31±0.11) (P<0.0001,
myriotylum Drechsler, (1930) (Ownley et al., 2008). The
t = 12.22, df = 14).
mechanism by which Metarhizium provides plant pro-
We have previously shown that M. robertsii is a plant
tection against plant fungal pathogens is not known, but
endophyte (Sasan & Bidochka, 2012), and the present
our results suggest that an extracellular, water-soluble
study describes antagonistic activity against a fungal
metabolite is implicated. Earlier, it was suggested that
plant root pathogen. A more complete understanding of
M. anisopliae inhibition of Fusarium oxysporum f. sp.
the ecology of these endophytes is likely to aid in the
vasinfectum could also be due to competition for nutri-
development of more efficient insect and phytopathogen
tion and space and antibiosis (Qi et al., 2010). However,
biocontrol programmes, and also provide beneficial traits
multiple mechanisms may be involved. For example, it
such as increased yields (Kabaluk & Ericsson, 2007)
has been suggested that B. bassiana, Lecanicillium spp.
through plant growth and root system promotion, and
and Trichoderma spp. induce systemic resistance in plants
improved compatibility with other beneficial microorgan-
(Harman et al., 2004; Ownley et al., 2010).
isms (Sasan & Bidochka, 2012).

Antagonism in host plants

Bean plant bioassays were performed in the presence of
F. solani and M. robertsii. While all seeds germinated into
healthy seedlings by day 11 in the control and F. solani
+ M. robertsii treatment, 60% of seeds emerged in the
Acknowledgements
This research was conducted with funds provided by a
Natural Sciences and Engineering Research Council of
Canada Discovery Grant to M.J.B.
 Antagonism of Metarhizium against Fusarium
References
B URKE, D.W., & BARKER, A.W. (1966). Importance of lateral roots in
Fusarium root rot of beans. Phytopathology, 56, 292–294.
C AMPBELL, R.E. (1989). Biological Control of Microbial Plant Pathogens.
Cambridge: Cambridge University Press.
C ICHY, K.A., S NAPP, S.S., & K IRK, W.W. (2007). Fusarium root rot inci-
dence and root system architecture in grafted common bean lines. Plant
Soil, 300, 233–244.
DAR, G.H.H., Z ARGAR, M.Y., & B EIGH, G.M. (1997). Biocontrol of
Fusarium root rot in the common bean (Phaseolus vulgaris L.) by
using symbiotic Glomus mosseae and Rhizobium leguminosarum. Microb.
Ecol., 34, 74–80.
D INGLE, J., & M CGEE, P.A. (2003). Some endophytic fungi reduce the den-
sity of pustules of Puccinia recondita f.sp. tritici in wheat. Mycol. Res.,
107, 310–316.
E L -K ASSAS, H.Y., & K HAIRY, H.M. (2009). A trial for biological control of
a pathogenic fungus (Fusarium solani) by some marine microorganisms.
Amer.-Euras. J. Agric. Environ. Sci., 5, 434–440.
Downloaded by [Umeå University Library] at 07:02 26 September 2013
F ILION, M., S T-A RNAUD, M., & JABAJI -H ARE, S.H. (2003).
Quantification of Fusarium solani f. sp. phaseoli in mycorrhizal
bean plants and surrounding mycorrhizosphere soil using real-time
polymerase chain reaction and direct isolations on selective media.
Phytopathology, 93, 229–235.
F LOCH, G.L., B ENHAMOU, N., M AMACA, E., S ALERNO, M-I., T IRILLY,
Y., & R EY, P. (2005). Characterisation of the early events in atypical
tomato root colonization by a biocontrol agent, Pythium oligandrum.
Plant Physiol. Biochem., 43, 1–11.
G EMMA, J.N., H ARTMANN, G.C., & WASTI, S.S. (1984). Inhibitory
interactions between Ceratocystis ulmi and several species of ento-
mopathogenous fungi. Mycologia, 76, 256–260.
G RAHAM, P.H. (1978). Some problems and potentials of field beans
(Phaseolus vulgaris L.) in Latin America. Field Crops Res., 1,
295–317.
G ROSCH, R., S CHERWINSKI, K., L OTTMANN, J., & B ERG, G. (2006).
Fungal antagonists of the plant pathogen Rhizoctonia solani: selection,
control efficacy and influence on the indigenous microbial community.
Mycol. Res., 110, 1464–1474.
H ARMAN, G.E., H OWELL, C.R., V ITERBO, A., C HET, I., & L ORITO, M.
(2004). Trichoderma species – opportunistic, avirulent plant symbionts.
Nat. Rev. Microbiol., 2, 43–56.
H ASSAN DAR, G.H., Z ARGAR, M.Y., & B EIGH, G.M. (1997). Biocontrol
of Fusarium root rot in the common bean (Phaseolus vulgaris L.) by
using symbiotic Glomus mosseae and Rhizobium leguminosarum. Microb.
Ecol., 34, 74–80.
I NÁ C IO, M.L., S ILVA, G.H., T ELES, H.L., T REVISAN, H.C., C AVALHEIRO,
A.J., B OLZANI, V.S., YOUNG, M.C.M., P FENNING, L.H., & A RAUJO,
A.R. (2006). Antifungal metabolites from Colletotrichum gloeospo-
rioides, an endophytic fungus in Cryptocarya mandioccana Nees
(Lauraceae). Bioch. Syst. Ecol., 34, 822–824.
I STIFADAH, N., & M CGEE, P.A. (2006). Endophytic Chaetomium globosum
reduces development of tan spot in wheat caused by Pyrenophora tritici-
repentis. Austral. Plant Pathol., 35, 411–418.
K ABALUK, J.T., & E RICSSON, J.D. (2007). Metarhizium anisopliae seed
treatment increases yield of field corn when applied for wireworm control.
Agron. J., 99, 1377–1381.
293
K ANG, C.S., G OO, B.Y., G YU, L.D., & H EON, K.Y. (1996). Antifungal
activities of Metarhizium anisopliae against Fusarium oxysporum,
Botrytis cinerea and Alternaria solani. Kor. J. Mycol., 24, 49–55.
K IM, H.Y., C HOI, G.J., L EE, H.B., L EE, S.W., L IM, H.K., JANG, K.S., S ON,
S.W., L EE, S.O., S UNG, N.D., & K IM, J.C. (2007). Some fungal endo-
phytes from vegetable crops and their anti-oomycete activities against
tomato late blight. Lett. Appl. Microbiol., 44, 332–337.
KOTTKE, I., G UTTENBERGER, M., H AMPP, R., & O BERWINKLER F
(1987). An in vitro method for establishing mycorrhizae on coniferous
tree seedlings. Trees, 1, 191–194.
K RASNOFF, S.B., K ERESZTES, I., G ILLILAN, R.E., S ZEBENYI, D.M.E.,
D ONZELLI, B.G.G., C HURCHILL, A.C.L., & G IBSON, D.M. (2007).
Serinocyclins A and B, cyclic heptapeptides from Metarhizium aniso-
pliae. J. Nat. Prod., 70, 1919–1924.
L IU, C.H., Z OU, W.X., L U, H., & TAN, R.X. (2001). Antifungal activity
of Artemisia annua endophyte cultures against phytopathogenic fungi. J.
Biotech., 88, 277–282.
M ICHE, L., & BALANDREAU, J. (2001). Effects of rice seed steriliza-
tion with hypochlorite on inoculated Burkholderia vietnamiensis. Appl.
Environ. Microbiol., 67, 3046–3952.
M ISHRA, V.K. (2010). In vitro antagonism of Trichoderma species against
Pythium aphanidermatum. J. Phytol., 2, 28–35.
OWNLEY, B.H., G RIFFIN, M.R., K LINGEMAN, W.E., G WINN, K.D.,
M OULTON, J.K., & P EREIRA, R.M. (2008). Beauveria bassiana:
endophytic colonization and plant disease control. J. Invert. Pathol., 3,
267–270.
OWNLEY, B.H., G WINN, K.D., & V EGA, F.E. (2010). Endophytic fungal
entomopathogens with activity against plant pathogens: ecology and
evolution. BioControl, 55, 113–128.
Q I, Y-X., C HEN, F-X., & L I, Z-Z. (2010). Inhibitory mechanisms of
Metarhizium anisopliae against the pathogens of Fusarium wilt of cotton.
Cotton Sci., 2, 591–596.
ROBERTS, D., & S T. L EGER, R.J. (2004) Metarhizium spp., cosmopolitan
insect-pathogenic fungi: mycological aspects. In A.I. Laskin, J.W. Bennet
and G.M. Gadd (Eds.). Advances in Applied Microbiology (pp. 1–70).
Amsterdam: Elsevier Academic Press.
ROJO, F.G., R EYNOSO, M.M., F EREZ, M., C HULZE, S.N., & T ORRES,
A.M. (2006). Biological control by Trichoderma species of Fusarium
solani causing peanut brown root rot under field conditions. Crop Prot.,
26, 549–555.
S ASAN, R.K. & B IDOCHKA, M.J. (2012). The insect-pathogenic fun-
gus Metarhizium robertsii (Clavicipitaceae) is also an endophyte that
stimulates plant root development. Am. J. Bot., 99, 101–107.
S CHNEIDER, K.A., & K ELLY, J.D. (2000). A greenhouse screening protocol
for Fusarium root rot in bean. HortScience, 35, 1095–1098.
S OBOWALE, A.A., O DEBODE, A.C., C ARDWELL, K.F.,
BANDYOPADHYAY, R., & J ONATHAN, S.G. (2010). Antagonistic
potential of Trichoderma longibrachiatum and T. hamatum resident on
maize (Zea mays) plant against Fusarium verticillioides (Nirenberg)
isolated from rotting maize stem. Arch. Phytopathol. Plant Prot., 43,
744–753.
S TEADMAN, J.R., K ERR, E.D., & M UMM, R.F. (1975). Root rot of bean in
Nebraska: primary pathogen and yield loss appraisal. Plant Dis. Rep., 59,
305–308.
TAN, C.S., & T U, J.C. (1995). Tillage effect on root rot severity, growth and
yield of beans. Can J. Plant Sci., 75, 183–186.