ORIGINAL PAPER

Impact of UV-B radiation on Clonostachys roseagermination and growth

Lucio B. Costa • Drauzio E. N. Rangel •

Marcelo A. B. Morandi • Wagner Bettiol

Received: 13 October 2011 / Accepted: 9 April 2012 / Published online: 22 April 2012

� Springer Science+Business Media B.V. 2012

Abstract Sensitivity to UV-B radiation is one of the main

limitations of biological control of plant pathogens in the

field. The effect of UV-B radiation on germination and leaf

tissue colonization by the biological control agent Clono-

stachys rosea was evaluated. There were variations among

C. rosea strains in sensitivity to UV-B radiation. The most

tolerant strain (LQC62) had relative germination of about

60 % after irradiation of 4.2 kJ m-2. The deleterious

effects of UV-B radiation on C. rosea colonization were

overcome by higher conidial concentration. In addition, the

tolerance of C. rosea conidia was higher when irradiated

over leaf disks compared to agar media, and this is very

important information to determine the dose and spray

strategies for applying C. rosea in the field.

Keywords Gliocladium roseum � Ultraviolet radiation �Biocontrol � Conidial UV-B tolerance � Conidial viability �Climate change

Introduction

Clonostachys rosea (sin. Gliocladium roseum: teleomorfo

Bionectria ochroleuca; Schroers et al. 1999) is commonly

found as a saprophyte fungus in soil with cosmopolitan

distribution (Schroers 2001; Sutton and Peng 1993; Toledo

et al. 2006). This fungus is capable of suppressing the

sporulation of several plant pathogens by competing for

saprophytic growth, which limits the colonization of

senescent tissues by the pathogen. Because Clonostachys

rosea can colonize endophytically green tissues, the results

are better when the biocontrol agent is applied before or at

the same time as the pathogen appears (Morandi et al.

2003, 2006; Sutton and Peng 1993). In the greenhouse, this

bioagent suppressed Botrytis cinerea sporulation and

infection by competing for nutrients and colonizing

wounds (Peng and Sutton 1991; Sutton and Peng 1993).

C. rosea was more efficient than fungicides in controlling

Botrytis blight in strawberries cultivated in field conditions

in Brazil (Cota et al. 2008a, b).

Clonostachys rosea, as a biological control agent of plant

pathogens, however, may be less efficient in the field due to

solar UV radiation, which is harmful to most microorgan-

isms. Ultraviolet radiation can be conventionally classified

by the wavelengths. UV-C radiation (100–280 nm) is com-

pletely filtered by the ozone layer and absorbed by other

atmospheric gases (Kuluncsics et al. 1999). UV-B radiation

(280–315 nm) is only partially filtered by the ozone layer and

has significant biological effects when compared to the other

ultraviolet radiations (Madronich et al. 1998). UV-A radia-

tion (315–400 nm) is not absorbed by the ozone layer and

directly reaches the Earth (Paul 2000). UV-B and UV-A

radiations cause cellular membrane disorganization, protein

denaturation, oxidative stress, and damage to DNA, RNA,

and ribosome (Griffiths et al. 1998). DNA damage is detected

as strand breaks or as base lesions. The most common lesions

in DNA are 8-hydroxydeoxyguanosine (8OHdG) from UV-

A exposure and cyclobutane pyrimidine dimers from UV-B

exposure, which impair the duplication of genetic material

L. B. Costa

Departamento de Fitopatologia, Universidade Federal de Lavras,

CP 3027, Lavras, MG 37200-000, Brazil

D. E. N. Rangel

Instituto de Pesquisa e Desenvolvimento, Universidade do Vale

do Paraıba, Sao Jose dos Campos, SP 12244-000, Brazil

M. A. B. Morandi � W. Bettiol (&)

Embrapa Environment, CP 69, Jaguariuna, SP 13820-000, Brazil

e-mail: bettiol@cnpma.embrapa.br

123

World J Microbiol Biotechnol (2012) 28:2497–2504

DOI 10.1007/s11274-012-1057-7

(Griffiths et al. 1998). In this way, UV-A and UV-B radiation

can inactivate conidia of biocontrol agents in a few hours due

to the genetic and morphologic changes, resulting in lost

efficacy of the biocontrol agent (Braga et al. 2001c).

Therefore, sensitivity to solar radiation is one of the

main limitations for applying biocontrol agents in the field

(Braga et al. 2001b; Li and Feng 2009; Morandi et al.

2006). The effects of UV-B radiation on biocontrol agents

have been well studied for Metarhizium spp., which is used

to control insect pests in agriculture (Braga et al. 2001b, d;

Rangel et al. 2005). Isolates of Metarhizium spp. from

tropical areas are more tolerant to the UV-B radiation than

isolates from temperate areas (Braga et al. 2001d). In

addition, the growth substrate and nutritional and physical

environment in which conidia are produced influences

increased M. anisopliae conidial UV-B tolerance and

higher speed of germination; and manipulation of these

variables could be used to obtain conidia with increased

tolerance to UV-B radiation and reduced germination rates

(Rangel 2011; Rangel et al. 2004, 2008, 2011).

However, there are few reports on the effect of UV-B

radiation on other biocontrol agents, especially those used

against plant pathogens, like C. rosea (Morandi et al.

2008). Considering the growing market of this biocontrol

agent in Brazil and other countries, the objectives of this

work was to evaluate the effects of UV-B on several iso-

lates of C. rosea.

Materials and methods

Isolates and inocula preparation

Eight isolates of C. rosea obtained from different regions in

Brazil and deposited in the Embrapa Environment Col-

lection of Microorganisms were used in these studies

(Table 1). The isolates were grown on 20 ml of Potato-

Dextrose-Agar (PDA) (Acumedia Manufacturers, Michi-

gan) in plates (polystyrene, 90 9 10 mm, Pleion) and

incubated at 25 ± 2 �C and 12 h light/12 h dark for

21 days. The strain LQC62 was used for preliminary tests

to establish the appropriate irradiance and incubation per-

iod for germination evaluation.

The conidia were suspended in Tween 80 solution with

distilled water (0.01 % v/v) and the suspensions were

vigorously shaken using a vortex and filtered through a

polycarbonate membrane (80 mm diameter, 8 lm pore

size, Whatman Nucleopore, Clifton, NJ, USA) to remove

spore aggregates. Conidial concentrations were estimated

by hemocytometer counts and dilutions made with sterile

Tween 80 solution (0.01 % v/v) for immediate use in the

irradiation and germination studies.

Irradiation chambers, lamps, and filters

Irradiation experiments were conducted in a temperature-

controlled room with four UV-B 313EL lamps (Q-lab

Cleveland, OH, USA). The lamps were aged prior to the start

of the experiments, resulting in a stable level of irradiation.

The temperature inside the chambers was adjusted to

25 ± 2 �C and was verified periodically throughout the

sequence of experiments. Controlled room temperature was

allowed to equilibrate for 1 h prior to all experiments. Every

lamp was covered with a 0.13 mm-thick cellulose diacetate

film (Malaga Ltda), which had a cutoff point at 290 nm.

This permitted the passage of most UV-B and UV-A

(290–400 nm), but prevented exposure to UV-C (\280 nm)

and short-wavelength UV-B (\290 nm). Control-plates

inside each of the chambers were wrapped with aluminum

foil and thus physically protected from radiation. The Petri

plates were randomized at intervals of 30 min to homogenize

the received doses of UV-B radiation.

The DNA damage action spectrum developed by Quaite

et al. (1992) and normalized to unity at 300 nm was used to

calculate the weighted UV irradiances (mW m-2). This

spectral weighting was selected based on the spectral

characteristics of nine fungal responses reviewed by Paul

et al. (1997), who concluded that this DNA damage spec-

trum is closely approximated to the fungal responses. All

the light measurements were made with a spectroradiom-

eter (Ocean Optics model USB2000 ? rad) connected to a

portable computer.

Table 1 Geographic origin of

C. rosea strainsStrain Host Geographic origin Date of isolation

LQC 59 Strawberry Serra Negra, Sao Paulo State, Brazil 14/03/2003

LQC 60 Rose Holambra, Sao Paulo State, Brazil 07/04/2005

LQC 62 Rose Vicosa, Minas Gerais State, Brazil 18/07/2002

LQC 73 Violet flower Jaguariuna, Sao Paulo State, Brazil 01/05/1993

LQC 87 Lettuce Jaguariuna, Sao Paulo State, Brazil 25/05/2005

LQC 111 Cacao tree Belem, Para State, Brazil 08/11/2005

LQC 112 Cacao tree Belem, Para State, Brazil 08/11/2005

LQC 114 Cacao tree Belem, Para State, Brazil 08/11/2005

2498 World J Microbiol Biotechnol (2012) 28:2497–2504

123

Evaluation of conidial germination

Each conidial suspension (20 ll, 105 conidia ml-1) was

placed on 7 ml-agar medium (PDA ? 0.002 % benomyl

with 25 % of active ingredient; Hi-yield Chemical, Bon-

ham, TX, USA) in plates (polystyrene, 50 9 10 mm, Ple-

ion) for evaluation of germination. The benomyl has little

effect on the germination of the fungus and was used to

reduce the speed of growth of the germinative tube, pre-

venting hypha superposing to allow monitoring of the

germination for a longer time (Milner et al. 1991). The

plates were immediately exposed to the UV-B radiation.

The germination was interrupted with lactofenol ?

0.05 % tripan blue. Germination was observed with 4009

magnification. Conidia with a germ tube longer than the

diameter of the conidia were considered germinated. A

total of 300 conidia per treatment were evaluated. Relative

percent germination after each period of incubation was

calculated by the following equation: Relative germination

(%) = (Wt/Wt) 9 100, where Wt is the number of germ-

lings at exposure time t and Wc is the number of germlings

of the control plate (Braga et al. 2001b).

Establishment of the appropriate irradiance

and incubation period to evaluate spore germination

Cclonostachys rosea conidia (strain LQC 62) were prepared

and placed on PDA ? benomyl in plates as described and

exposed to irradiances of 222, 600, and 823 mW m-2 of

UV-B radiation (Fig. 1). For this, the plates were positioned

at 18, 28, and 48 cm from the lamps, respectively. At the

end of the 2 h exposure, the doses corresponded to 1.6; 4.2,

and 5.9 kJ m-2, respectively. After irradiated, the Petri

dishes were incubated at 25 ± 2 �C, in the dark and the

germination of the conidia were evaluated at 12, 24, and 36 h

after treatment.

To establish the best incubation period after the expo-

sure to UV-B radiation and controls, a previous trial

was done. For this experiment, only the irradiance of

600 mW m-2 was used and the energy inside the chamber

was 2.1 kJ m-2 per hour. A conidial suspension of

C. rosea were placed on PDA ? benomyl and exposed to

4.2 kJ m-2 of UV-B radiation and then incubated at

25 ± 2 �C in the dark. The determination of germination

was started 8 h after inoculation and repeated at 4 h

intervals up to 24 h. Once the appropriate period of incu-

bation for germination evaluation was established, the eight

C. rosea isolates were compared for their tolerance to UV-

B radiation, following the same methodology.

Survival curve of C. rosea conidia exposed to UV-B

radiation on agar medium and on beans leaf disks

To establish the survival curve, a conidial suspension of

C. rosea (strain LQC 62) was placed in plates containing

PDA ? benomyl and was exposed to UV-B radiation

(irradiance 600 mW m-2) for 0–6 h, corresponding to 0

(control), 2.1, 4.2, 6.3, 8.4, 10.5, and 12.6 kJ m-2. After

irradiation, the plates were kept at 25 ± 2 �C in the dark

until the evaluation of germination.

To estimate the conidial germination incidence and germ

tube length of C. rosea on bean leaf disks, 1-cm-diameter leaf

disks of bean plants (cv. Talisman) between 30 and 60 days

after emergence were obtained and surface sterilized in 70 %

ethanol (1 min) and in 2 % sodium hypochlorite (1 min).

Then the disks were rinsed three times in sterile distilled

water and placed in disposable plates (10 9 90 mm, Pleion)

on humidified absorbent paper (5 ml of sterilized water;

Morandi et al. 2000a). In each plate, 19 disks were placed and

organized into five rows. After that, each disk received an

aliquot of 20 ll of C. rosea conidia suspension at 107 conidia

ml-1. The disks were exposed to UV-B radiation (irradiance

600 mW m-2) for 0–5 h, corresponding to 0 (control), 2.1,

4.2, 6.3, 8.4, and 10.5 kJ m-2. The disks were mounted with

lactophenol containing 0.05 % Trypan blue on microscope

slides, gently heated over a flame for 2 min to clear the tis-

sues, and examined on a compound microscope (Saha et al.

1988). The germination was evaluated by counting 100

conidia per disk at 24 h (control) or 36 h (irradiated with

UV-B) after inoculation.

Clonostachys rosea colonization on bean leaf disks was

evaluated indirectly by quantifying the potential of the

fungus to cover the surface of leaf disks and its sporulation

(Morandi et al. 2000b). The leaf disks were prepared and

treated as describe earlier. After irradiation, the disks were

transferred to paraquat-chloramphenicol-agar medium

Wavelength (nm)280 300 320 340 360 380 400

Spe

ctra

l irr

adia

nce

(µm

cm

-2 nm

-1)

0

2

4

6

8823 mW m-2

600 mW m-2

222 mW m-2

Fig. 1 Spectral irradiances of the lamp setups used for the different

UV treatments. Chamber providing UV-B irradiance of 222, 600, and

823 mW m-2 respectively, for the lamp heights of 48, 28, and 18 cm

from the sample. The lamps was covered with a 0.1-mm thick

cellulose acetate, which blocked radiation below 290 nm

World J Microbiol Biotechnol (2012) 28:2497–2504 2499

123

(PCA) in Petri dishes (Peng and Sutton 1991). Sporulation

was estimated after the tissues were incubated at

25 ± 2 �C (12 h light/12 h dark) for 3, 7, and 10 days. The

evaluation was accomplished following a scale of notes for

the area of the disks covered with conidiophores of the

fungus, as follow: 0 = 0 % (0 %); 1 = 2 % (1–3 %);

2 = 5 % (4–6 %); 3 = 10 % (7–13 %); 4 = 20 %

(14–27 %); 5 = 40 % (28–52 %); 6 = 70 % (53–87 %);

and 7 = 94 % (88–100 %; Morandi et al. 2000b).

Effects of conidial concentration and irradiation doses

on C. rosea survival on bean leaf disks

Bean leaf disks, prepared as previously described, were

inoculated with C. rosea suspensions at 103, 104, 105, and

106 conidia ml-1 and exposed to UV-B (irradiance

600 mW m-2) for 0, 1, 2, and 3 h, corresponding to 0

(control), 2.1, 4.2, and 6.3 kJ m-2. The colonization of leaf

disks was evaluated as described before.

Experimental design and data analysis

Each experiment was conducted with a completely ran-

domized design and repeated three times. For experiments

on agar media, there were two plates as replicates for each

treatment. For evaluations on leaf disks, three replication

plates each contained 10 disks. The data from the three

experimental repetitions resulted in treatment effects in the

same significance classes. Therefore, the mean of trials for

each experiment was used in the analyses. Statistical

computations were performed using the Statistical Analysis

Systems (SAS Institute Inc., Cary, NC, USA). Data for

conidial germination and fungal sporulation were exam-

ined using analysis of variance (ANOVA), the area under

growth curve (AUGC) was calculated and in some

instances and treatment means were compared by Tukey

test.

Regression models were used to examine quantitative

relationships between germination of C. rosea conidia and

hours of exposure after inoculation. A factorial arrange-

ment was used to evaluate the interactions between length

of exposure and inoculum concentration of C. rosea.

Results

Establishment of the appropriate incubation period

for conidial germination evaluation

The germination of C. rosea conidia was inversely pro-

portional to the irradiance, i.e. higher conidial germination

was found in the lower dose and low conidial germination

was found in the higher dose (Fig. 2). The medium

irradiance (600 mW m-2, which corresponds to a final

dose of 4.2 kJ m-2) provided the medium lethal dose and

this dose was considered for the next experiments. The

most appropriate incubation period for conidia germination

evaluation was between 12 and 24 h for the control and the

irradiated treatments, respectively. After 36 h of incuba-

tion, it was impossible to count germination because the

germinative tubes were long, intermingled, and not easily

distinguishable.

Once the appropriated irradiances were selected, a new

trial was conducted to confirm the most appropriate period

of incubation using incubation periods of 8, 12, 16, 20,

24 h. The germination incidence of C. rosea conidia in the

control treatment was less than 40 % after 8 h and reached

98 % after 12 h of incubation. After 12 h, it was impos-

sible to count germination for the control treatment. For the

conidia submitted to UV-B radiation, the germination was

observed only after 16 h and reached the maximum of

60 % after 24 h (Table 2). There were variations among

C. rosea strains in sensitivity to UV-B radiation. After

irradiation of 4.2 kJ m-2, two strains germinated approxi-

mately 40 %; and five strains had viability below 10 %.

The LQC 62 strain was the most tolerant, showing relative

germination above 60 %, statistically superior to the other

isolates. Therefore, the LQC 62 strain was selected to be

used in the subsequent experiments (Fig. 2).

Survival curve of C. rosea conidia exposed to UV-B

radiation on agar medium and on bean leaf disks

There was a negative exponential reduction of germination

of C. rosea LQC 62 conidia on agar medium when

IsolatesLQC62 LQC112 LQC114 LQC87 LQC 60 LQC111 LQC59 LQC73

Rel

ativ

e ge

rmin

atio

n (%

)

0

20

40

60

80

100

A

B

C

D D DDD

Fig. 2 Relative germination of C. rosea conidia of several strains

after the exposure for 2 h to UV-B irradiation (Irradiance of

600 mW m-2 at a weighted dose of 4.2 kJ m-2). Relative germina-

tion was calculated in relation to control plates. Graph bars with the

same letters are not significantly different by the Tukey test at 5 % of

significance (p \ 0.001). Errors bar are standard deviation of three

independent experiments

2500 World J Microbiol Biotechnol (2012) 28:2497–2504

123

submitted to irradiation between 0 and 12.6 kJ m-2. The

medium lethal dose (LD50) was 4.3 kJ m-2 and the lethal

dose of 100 % (LD100) was 7.1 kJ m-2 (Fig. 3).

On the surface of the leaf disks, the germination of

C. rosea LQC 62 conidia had a linear reduction when

submitted to irradiation from 0 to 10.5 kJ m-2. The med-

ium lethal dose (LD50) was 6.6 kJ m-2 and the lethal dose

of 100 % (LD100) was not reached (Fig. 4).

The growth of C. rosea on leaf disks after 3 days was

initially reduced by increasing the irradiation dose, but it

reached 100 % with no significant differences after 7 and

10 days (Fig. 5a). The colonization of C. rosea on leaf

disks (measured indirectly by surface growth and sporu-

lation) followed a similar pattern; however, after 3 days,

the control treatment differed significantly from the irra-

diated treatments, but there were no differences among

doses. After 10 days, the colonization varied from 80 to

94 % and the control treatment differed only from the

irradiated treatment at 6.3 kJ m-2 (Fig. 5b).

Effects of conidial concentration and irradiation doses

on C. rosea survival on bean leaf disks

There was a significant interaction between irradiation

doses and conidial concentrations for C. rosea growth

(p = 0.019) and colonization (p = 0.0349) on leaf disks

(Fig. 6a, b).

The growth of C. rosea on leaf disk surfaces at 3 and

7 days after irradiation was delayed as a function of

increased doses of irradiation and reduction in conidial

concentration (Fig. 6a). After 10 days, however, there was

no difference in C. rosea incidence among treatments

(p = 0.1676). On the other hand, the leaf disk area colo-

nized by the fungus was significantly reduced by increasing

the irradiation and reducing conidial concentration

(Fig. 6b). The deleterious effects of UV-B radiation over

C. rosea colonization were overcome by higher conidial

concentration. At 105 and 106 conidia ml-1, there were no

significantly effects of irradiation doses (105 p = 0.1008

and 106 p = 0.0232).

Discussion

The effects of increased UV-B radiation have been studied in

several biological systems, such as bacteria (Flores et al.

2009; Peak and Peak 1983), filamentous fungi (Braga

et al. 2001a; Duguay and Klironomos 2000), plants (Barnes

et al. 2009; Caldwell et al. 1995); and animals (Corsini et al.

1997; Fahlman and Krol 2009). In general, the results indi-

cate deleterious effects over all organisms.

Table 2 Germination of C. rosea spores (strain LQC 62) to different

doses of UV-B radiation

UV-B radiation dose (KJ m-2)

Control 1.6 4.3 5.9

Germination (%)

12 h 98 ± 0.31 70 ± 7 10 ± 3 1.4 ± 0.6

24 h a 88 ± 2.9 42 ± 6.1 4 ± 3

36 h a a a 5 ± 2.1

Spores were exposed for 2 h to UV-B irradiation. Irradiances of 222,

600, and 823 mW m-2 at a weighted doses of 0 (Control), 1.6, 4.3

and 5.9 kJ m-2. ± are standard deviation of three independent

experimentsa Impossible to count due to excessive germination and germ tube

length

f= 96,4/(1+exp(-(x-4,5)/(-0,8)))R2=0,99

Dose (kJ m-2)0.0 2.1 4.2 6.3 8.4 10.5 12.6

Rel

ativ

e ge

rmin

atio

n (%

)

0

20

40

60

80

100

RegressionObservated date95% Confidence Band 95% Prediction BandLethal dose50

Fig. 3 Survival curve of C. rosea conidia exposed to UV-B radiation

on agar medium (strain LQC 62) to different weighted doses of UV-B

radiation (Irradiance of 600 mW m-2)

f = 100,6+(-7,6)*xR2=0,94

Dose (kJ m-2)0.0 2.1 4.2 6.3 8.4 10.5

Rel

ativ

e ge

rmin

atio

n (%

)

0

20

40

60

80

RegressionObservate date95% Confidence Band 95% Prediction BandLethal dose50

Fig. 4 Survival curve of C. rosea conidia exposed to UV-B radiation

on bean leaf disks (strain LQC 62) to different weighted doses of UV-

B radiation (Irradiance of 600 mW m-2)

World J Microbiol Biotechnol (2012) 28:2497–2504 2501

123

For filamentous fungi, most of the studies found a

reduction in germination and/or growth when UV-B irra-

diation increased. We observed the same tendency for

C. rosea. Conidial germination was reduced and, in some

cases, total inactivation of the conidia after a few hours of

exposure to UV-B radiation with doses usually found in

natural conditions. Morandi et al. (2008) also observed that

the conidial viability of C. rosea was significantly reduced

with increasing length of exposure to sunlight during the

day. These authors reported reduction up to 30 % after 2 h

of exposure to sunlight.

We found some variability in tolerance to UV-B radia-

tion in eight isolates of C. rosea. Other authors have found

that similar UV-B irradiances are able to greatly reduce

germination of different entomopathogenic fungi (Fargues

et al. 1996; Fernandes et al. 2007). For example, the rela-

tive germination of sixth isolates of Beauveria spp. was

found to range from 0 to 100 % at an irradiance of

7.04 kJ m-2 (Fernandes et al. 2007).

Another important observation was the reduction of

germination speed of C. rosea conidia (Table 2). This

effect has been reported by several authors working with

0 2,1 4,2 6,3 8,4 10,5

Pre

sens

e of

Clo

nost

achy

s (%

)

0

20

40

60

80

100

3 Day7 Day10 Day

Dose (kJ m-2)Dose (kJ m-2)0 2,1 4,2 6,3 8,4 10,5

Leaf

are

a w

ith c

onid

ioph

ores

of

C. r

osea

(%

)

0

20

40

60

80

100

3 Day7 Day10 Day

A

AB

AB

B B

B

A AA A A

AB B BB B

A

A

AA

A A

A

B

AB

ABAB AB

(A) (B)

Fig. 5 Effect of UV-B radiation on C. rosea (107 conidia ml-1) in

bean leaf disk a Presence of C. rosea (%) on leaf disks after exposure

for 0 to 10.5 kJ m-2 to UV-B irradiation (Irradiance of 600 mW m-2

at a different weighted dose); b Leaf area with conidiophores of

C. rosea per disk after exposure for 0–10.5 kJ m-2 to UV-B

irradiation. Graph bars with the same letters are not significantly

different by the Tukey test at 5 % of significance (p \ 0.05). Errorsbar are standard deviation of three independent experiments

0.0

2.1

4.2

6.3

Concentration

105

0 20 40 60 80 100

3 Day

7 Day

10 Day

3 Day

7 Day

10 Day0

20

40

60

80

100

Pre

senc

e of

Clo

nost

achy

s (%

)

(A)

103

104

105

106

Dose (kJ m-2 )

0

20

40

60

80

100

Leaf

are

a w

ith c

onid

ioph

ores

(%

)(B)

103

104

106

Concentration

0.0

2.1

4.2

6.3 Dose (kJ m-2 )

Fig. 6 Effect of UV-B radiation on C. rosea (103–106 conidia ml-1)

in bean leaf disk. a Presence of C. rosea (%) on leaf disks after

exposure for 0–6.3 kJ m-2 to UV-B irradiation (Irradiance of

600 mW m-2 at a different weighted dose). C. rosea were sprayed

at four different concentrations, evaluated 3, 7 and 10 days; b Leaf

area with conidiophores of C. rosea per disk after exposure for

0–6.3 kJ m-2 to UV-B irradiation. C. rosea were sprayed at four

different concentrations, evaluated 3, 7 and 10 days

2502 World J Microbiol Biotechnol (2012) 28:2497–2504

123

Metarhizium spp. (Zimmermann 1982), Simplicillium lan-

osoniveum (formerly Verticillium lecanii), Lecanicillium

aphanocladii (formerly Aphanocladium album), and

Trichoderma sp. (Braga et al. 2002). Similar to our results,

Braga et al. (2006) observed that maximum germination of

Metarhizium robertsii conidia irradiated with UV-B was

reached after 24 h, and the extension of incubation period

until 36 h did not increased germination. These results

indicate that the conidia exposed to UV-B radiation need

some time to recover from the effects of UV-B radiation

and resume the germination process (Braga et al. 2001b).

The color of the conidia is involved in the tolerance to

UV-B radiation (Braga et al. 2006; Kawamura et al. 1999;

Rangel et al. 2006; Wang and Casadevall 1994). In general,

dark spores were significantly more stable than the lighter-

pigmented, such as C. rosea conidia. We observed that the

tolerance of C. rosea conidia was higher when irradiated

over leaf disks compared to agar media. One hypothesis to

explain this result is that the presence of pigmentation on

leaf tissues could absorb part and reflect another part of the

incident radiation mimicking the effects of conidia pig-

mentation. Another possibility is the presence of trichomes,

grooves, and bumps could provide physical protection to

the conidia. Because these variables were not evaluated, it

was not possible to determine the cause of the observed

difference in conidia tolerance on leaf disks and agar

media.

Our study found that UV-B radiation can affect the

inoculum viability of C. rosea. Increases on UV-B radiation

dose reduced the viability of the fungus, and the effects were

most pronounced at lower spore concentrations. In the

highest concentration (106 conidia ml-1) on the third day, the

non-irradiated disks had a difference of 30 % for the pres-

ence of the fungus and 50 % for sporulation on the leaf disks

with the highest irradiation doses (6.3 kJ m-2), but with

minimal difference at 7 and 10 days. However, the lowest

concentration (103 conidia ml-1) on third day had four times

as much presence and sporulation, and this continued

through all subsequent evaluations.

According to our results, C. rosea showed high sensi-

tivity to UV-B radiation. Therefore, the increase of envi-

ronmental UV-B radiation could reduce its survival. The

discover that the fungus was less susceptible to UV-B

radiation when conidia were inoculated on the leaves

compared to agar medium is very important to determine

dose and spray strategies to apply C. rosea in the field.

Acknowledgments This article was part of a M.Sc. dissertation of

the first author. He was supported by a scholarship provided by the

Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAP-

EMIG). We sincerely thank the project ‘‘Impacts of climate change

on plant diseases, pests, and weeds’’ (CLIMAPEST Embrapa) which

is connected to the Macroprogram 1—Great National Challenges, as

part of Embrapa Environment, and to Alene Alder-Rangel at

UNIVAP for reviewing the English. We also would like to thank

CNPq for the scholarship granted to Wagner Bettiol.

References

Barnes PW, Flint SD, Ryel RJ (2009) Diurnal changes in UV-

shielding in plants. Comp Biochem Physiol Mol Integr Physiol

153A:S201–S201

Braga GUL, Flint SD, Messias CL, Anderson AJ, Roberts DW

(2001a) Effect of UV-B on conidia and germlings of the

entomopathogenic hyphomycete Metarhizium anisopliae. Mycol

Res 105:874–882

Braga GUL, Flint SD, Messias CL, Anderson AJ, Roberts DW

(2001b) Effects of UVB irradiance on conidia and germinants of

the entomopathogenic hyphomycete Metarhizium anisopliae: a

study of reciprocity and recovery. Photochem Photobiol

73:140–146

Braga GUL, Flint SD, Miller CD, Anderson AJ, Roberts DW (2001c)

Both solar UVA and UVB radiation impair conidial culturability

and delay germination in the entomopathogenic fungus Meta-rhizium anisopliae. Photochem Photobiol 74:734–739

Braga GUL, Flint SD, Miller CD, Anderson AJ, Roberts DW (2001d)

Variability in response to UV-B among species and strains of

Metarhizium isolated from sites at latitudes from 61� N to 54� S.

J Invertebr Pathol 78:98–108

Braga GUL, Rangel DEN, Flint SD, Miller CD, Anderson AJ, Roberts

DW (2002) Damage and recovery from UV-B exposure in

conidia of the entomopathogens Verticillium lecanii and Apha-nocladium album. Mycologia 94:912–920

Braga GUL, Rangel DEN, Flint SD, Anderson AJ, Roberts DW

(2006) Conidial pigmentation is important to tolerance against

solar-simulated radiation in the entomopathogenic fungus Meta-rhizium anisopliae. Photochem Photobiol 82:418–422

Caldwell M, Teramura AH, Tevini M, Bornman JF, Bjorn LO,

Kulandaivelu G (1995) Effects of increased solar ultraviolet-

radiation on terrestrial plants. Ambio 24:166–173

Corsini E, Sangha N, Feldman SR (1997) Epidermal stratification

reduces the effects of UVB (but not UVA) on keratinocyte

cytokine production and cytotoxicity. Photodermatol Photoim-

munol Photomed 13:147–152

Cota LV, Maffia LA, Mizubuti ESG (2008a) Brazilian isolates of

Clonostachys rosea: colonization under different temperature

and moisture conditions and temporal dynamics on strawberry

leaves. Lett Appl Microbiol 46:312–317

Cota LV, Maffia LA, Mizubuti ESG, Macedo PEF, Antunes RF

(2008b) Biological control of strawberry gray mold by Clono-stachys rosea under field conditions. Biol Control 46:515–522

Duguay KJ, Klironomos JN (2000) Direct and indirect effects of

enhanced UV-B radiation on the decomposing and competitive

abilities of saprobic fungi. Appl Soil Ecol 14:157–169

Fahlman BM, Krol ES (2009) UVA and UVB radiation-induced

oxidation products of quercetin. J Photochem Photobiol B Biol

97:123–131

Fargues J, Goettel MS, Smits N, Ouedraogo A, Vidal C, Lacey LA,

Lomer CJ, Rougier M (1996) Variability in susceptibility to

simulated sunlight of conidia among isolates of entomopatho-

genic Hyphomycetes. Mycopathologia 135:171–181

Fernandes EKK, Rangel DEN, Moraes AML, Bittencourt V, Roberts

DW (2007) Variability in tolerance to UV-B radiation among

Beauveria spp. isolates. J Invertebr Pathol 96:237–243

Flores MR, Ordonez OF, Maldonado MJ, Farias ME (2009) Isolation

of UV-B resistant bacteria from two high altitude Andean lakes

(4,400 m) with saline and non saline conditions. J Gen Appl

Microbiol 55:447–458

World J Microbiol Biotechnol (2012) 28:2497–2504 2503

123

Griffiths HR, Mistry P, Herbert KE, Lunec J (1998) Molecular and

cellular effects of ultraviolet light-induced genotoxicity. Crit Rev

Clin Lab Sci 35:189–237

Kawamura C, Tsujimoto T, Tsuge T (1999) Targeted disruption of a

melanin biosynthesis gene affects conidial development and UV

tolerance in the Japanese pear pathotype of Alternaria alternata.

Mol Plant Microbe Interact 12:59–63

Kuluncsics Z, Perdiz D, Brulay E, Muel B, Sage E (1999)

Wavelength dependence of ultraviolet-induced DNA damage

distribution: involvement of direct or indirect mechanisms and

possible artefacts. J Photochem Photobiol B Biol 49:71–80

Li J, Feng MG (2009) Intraspecific tolerance of Metarhiziumanisopliae conidia to the upper thermal limits of summer with

a description of a quantitative assay system. Mycol Res

113:93–99

Madronich S, McKenzie RL, Bjorn LO, Caldwell MM (1998)

Changes in biologically active ultraviolet radiation reaching the

earth’s surface. J Photochem Photobiol B Biol 46:5–19

Milner RJ, Huppatz RJ, Swaris SC (1991) A new method for

assessment of germination of Metarhizium conidia. J Invertebr

Pathol 57:121–123

Morandi MAB, Sutton JC, Maffia LA (2000a) Effects of host and

microbial factors on development of Clonostachys rosea and

control of Botrytis cinerea in rose. Eur J Plant Pathol

106:439–448

Morandi MAB, Sutton JC, Maffia LA (2000b) Relationships of aphid

and mite infestations to control of Botrytis cinerea by Clono-stachys rosea in rose (Rosa hybrida) leaves. Phytoparasitica

28:55–64

Morandi MAB, Maffia LA, Mizubuti ESG, Alfenas AC, Barbosa JG

(2003) Suppression of Botrytis cinerea sporulation by Clono-stachys rosea on rose debris: a valuable component in Botrytis

blight management in commercial greenhouses. Biol Control

26:311–317

Morandi MAB, Maffia LA, Mizubuti ESG, Alfenas AC, Barbosa JG,

Cruz CD (2006) Relationships of microclimatic variables to

colonization of rose debris by Botrytis cinerea and the biocontrol

agent Clonostachys rosea. Biocontrol Sci Tech 16:619–630

Morandi MAB, Mattos LPV, Santos ER, Bonugli RC (2008)

Influence of application time on the establishment, survival,

and ability of Clonostachys rosea to suppress Botrytis cinereasporulation on rose debris. Crop Protect 27:77–83

Paul ND (2000) Stratospheric ozone depletion, UV-B radiation and

crop disease. Environ Pollut 108:343–355

Paul ND, Rasanayagam S, Moody SA, Hatcher PE, Ayres PG (1997)

The role of interactions between trophic levels in determining

the effects of UV-B on terrestrial ecosystems. Plant Ecol

128:296–308

Peak MJ, Peak JG (1983) Use of action spectra for identifying

molecular targets and mechanism of action of solar ultraviolet

light. Physiol Plant 58:367–372

Peng G, Sutton JC (1991) Evaluation of microorganisms for

biocontrol of Botrytis cinerea in strawberry. Can J Plant Pathol

13:247–257

Quaite FE, Sutherland BM, Sutherland JC (1992) Action spectrum for

DNA damage in alfalfa lowers predicted impact of ozone

depletion. Nature 358:576–578

Rangel DEN (2011) Stress induced cross-protection against environ-

mental challenges on prokaryotic and eukaryotic microbes.

World J Microbiol Biotechnol 27:1281–1296

Rangel DEN, Braga GUL, Flint SD, Anderson AJ, Roberts DW

(2004) Variations in UV-B tolerance and germination speed of

Metarhizium anisopliae conidia produced on artificial and

natural substrates. J Invertebr Pathol 87:77–83

Rangel DEN, Braga GUL, Anderson AJ, Roberts DW (2005)

Influence of growth environment on tolerance to UV-B radiation,

germination speed, and morphology of Metarhizium anisopliaevar. acridum conidia. J Invertebr Pathol 90:55–58

Rangel DEN, Butler MJ, Torabinejad J, Anderson AJ, Braga GUL,

Day AW, Roberts DW (2006) Mutants and isolates of Meta-rhizium anisopliae are diverse in their relationships between

conidial pigmentation and stress tolerance. J Invertebr Pathol

93:170–182

Rangel DEN, Alston DG, Roberts DW (2008) Effects of physical and

nutritional stress conditions during mycelial growth on conidial

germination speed, adhesion to host cuticle, and virulence of

Metarhizium anisopliae, an entomopathogenic fungus. Mycol

Res 112:1355–1361

Rangel DEN, Fernandes EKK, Braga GUL, Roberts DW (2011)

Visible light during mycelial growth and conidiation of Meta-rhizium robertsii produces conidia with increased stress toler-

ance. FEMS Microbiol Lett 315:81–86

Saha DC, Jackson MA, Johnsoncicalese JM (1988) A rapid staining

method for detection of endophytic fungi in turf and forage

grasses. Phytopathology 78:237–239

Schroers HJ (2001) A monograph of Bionectria (Ascomycota,

Hypocreales, Bionectriaceae) and its Clonostachys anamorphs.

Stud Mycol 46:1–214

Schroers HJ, Samuels GJ, Seifert KA, Gams W (1999) Classification

of the mycoparasite Gliocladium roseum in Clonostachys as

C. rosea, its relationship to Bionectria ochroleuca, and notes on

other Gliocladium-like fungi. Mycologia 91:365–385

Sutton JC, Peng G (1993) Biocontrol of Botrytis cinerea in strawberry

leaves. Phytopathology 83:615–621

Toledo AV, Virla E, Humber RA, Paradell SL, Lastra CCL (2006)

First record of Clonostachys rosea (Ascomycota: Hypocreales)

as an entomopathogenic fungus of Oncometopia tucumana and

Sonesimia grossa (Hemiptera: Cicadellidae) in Argentina. J In-

vertebr Pathol 92:7–10

Wang YL, Casadevall A (1994) Decreased susceptibility of melan-

ized Cryptococcus neoformans to UV light. Appl Environ

Microbiol 60:3864–3866

Zimmermann G (1982) Effect of high temperatures and artificial

sunlight on the viability of conidia Metarhizium anisopliae.

J Invertebr Pathol 40:36–40

2504 World J Microbiol Biotechnol (2012) 28:2497–2504

123