Biological Control 65 (2013) 95–100

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Biological Control

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Effects of UV-B radiation on the antagonistic ability of Clonostachys roseato Botrytis cinerea on strawberry leaves

Lúcio B. Costa a, Drauzio E.N. Rangel b, Marcelo A.B. Morandi c, Wagner Bettiol c,⇑a Departamento de Fitopatologia, Universidade Federal de Lavras, CP 3027, 37200-000 Lavras, MG, Brazilb Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, 12244-000 São José dos Campos, SP, Brazilc Embrapa Environment, CP 69, 13820-000 Jaguariúna, SP, Brazil

h i g h l i g h t s

" The UV-B radiation caused a strongdeleterious effect to Clonostachysrosea conidia.

" UV-B radiation significantly reducedthe growth of Clonostachys rosea onleaf discs.

" The UV-B radiation caused reductionon antagonized ability ofClonostachys rosea to control Botrytiscinerea.

1049-9644/$ - see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.biocontrol.2012.12.007

⇑ Corresponding author. Fax: +55 19 3311 2740.E-mail address: wagner.bettiol@embrapa.br (W. B

g r a p h i c a l a b s t r a c t

Clonostachys rosea on strawberry leaf discs exposed to doses of UV-B radiation and challenged by Botrytiscinerea, 3, 7, and 10 days after inoculation (dai). A. Incidence of C. rosea with different inoculum concen-trations at several doses of UV-B radiation; B. Incidence of B. cinerea on leaf discs challenged by C. rosea; C.Leaf disc area with C. rosea conidiophores; D. Leaf disc area with B. cinerea conidiophores.

a r t i c l e i n f o

Article history:Received 1 June 2012Accepted 18 December 2012Available online 27 December 2012

Keywords:Climate changeGliocladium roseumUltraviolet - BGray moldClonostachys rosea

a b s t r a c t

Clonostachys rosea is effective to control of Botrytis cinerea on strawberry, although is highly susceptible toultraviolet radiation and has reduced ability to antagonize a pathogen in solar radiation conditions. Theobjective of this work was to evaluate the ability of an isolate of C. rosea, previously selected for its tolerantto UV-B radiation, to control B. cinerea on strawberry leaves in controlled experiments. Leaf discs of 1 cmdiameter were placed on Petri dishes and each received 20 lL of a C. rosea LQC 62 concentrations (104, 105,and 106 conidia mL�1). They were then exposed to UV-B irradiance 600 mW m�2 (0, 2.1, 4.2, and6.3 kJ m�2), and after radiation, half of the discs were inoculated with an aliquot of 10 lL B. cinerea (105 -conidia mL�1). The colonization of fungi on the leaf disc was measured with diagrammatic scale formationof conidiophores. The presence and sporulation of C. rosea on leaf disc was influenced by the dose of UV-Bradiation and the conidial concentration of antagonist. The incidence and severity of B. cinerea on leaf discswere inversely correlated to presence and sporulation of C. rosea. The growth of the pathogen was higherin the lower C. rosea concentration. The highest concentration of C. rosea (106 conidia mL�1) reduced theincidence and severity by 91% and 98% of B. cinerea on strawberry leaf discs. The UV-B radiation reducedthe ability of C. rosea to control B. cinerea. The higher dose of UV-B reduced the presence and sporulation ofC. rosea by 20% and 42%, respectively. Consequently, the incidence of B. cinerea increased twice and theseverity was three-folder higher. Taken together this data means that, for the development of biologicalcontrol agents based products, the effect of UV-B should be considered on the efficacy studies.

� 2013 Elsevier Inc. All rights reserved.

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ettiol).

96 L.B. Costa et al. / Biological Control 65 (2013) 95–100

1. Introduction

The gray mold fungus Botrytis cinerea Pers. ex Fr., is a cosmo-politan necrotrophic pathogen, able to attack more than 200 gen-era of plants (Jarvis, 1989), and is severely destructive inprotected cultivation systems. Crops such as strawberry are highlyaffected when produced in large scale. The fungus can causelosses of up to 50% in a strawberry field (Blanco et al., 2006),and is capable of attacking the crop in the field, during harvest,transportation, and commercialization (Card et al., 2009;Timudo-Torrevilla et al., 2005). All parts of strawberry plants,including stems, leaves, flowers, and fruits, are susceptible tothe fungus (Sutton, 1990).

The chief inoculum sources of B. cinerea are the conidia pro-duced in senescent foliage, petals and fruit, which accumulate onthe soil surface (Guetsky et al., 2001). The abundant B. cinerea spor-ulation on plant tissues contributes to the development and main-tenance of an epidemic within a crop (Blanco et al., 2006). Thesuppression of pathogen sporulation on crop debris was proposedas a potential strategy of biological control (Morandi et al., 2003;Cota et al., 2009).

For many years, the control of gray mold was successfully per-formed exclusively by fungicides during the flowering of the cul-tures (Mertely et al., 2002). However, there have been manyreports of populations resistant to the main fungicides used againstB. cinerea (Bardas et al., 2008; Timudo-Torrevilla et al., 2005). Thishas led to an increasingly smaller application intervals, with higherconcentrations of active ingredients, and the mix of active princi-ples (Card et al., 2009), which increases pesticide residues on fruitsfor human consumption (Rabolle et al., 2006). Resistant cultivarshave not been effective, due to the great genetic variability andthe necrotrophic habit of the pathogen (Williamson et al., 2007).Biological control has been shown to be a promising strategyagainst the pathogen, achieving good results in a variety of crops(Boff et al., 2002; Card et al., 2009; Cota et al., 2009; Suttonet al., 1997; Swadling and Jeffries, 1996).

Clonostachys rosea (Link: Fr.) Schroers, Samuels, Seifert & W.Gams [(sin. Gliocladium roseum: teleomorph Bionectria ochroleuca)(Schroers et al., 1999)] is found commonly as saprophyte fungusin soil and crop residues, with cosmopolitan distribution (Schroers,2001). The competition for nutrients and substrate are the majormechanism that C. rosea uses against pathogens such as B. cinerea,which requires an external source of sugars to cause infection(Sutton et al., 1997).

Although effective against gray mold, C. rosea is highly suscep-tible to ultraviolet radiation and has reduced ability to antagonizea pathogen in solar radiation conditions (Costa et al., 2012; Moran-di et al., 2008). Arguably, solar radiation is an important factor thatinterferes with application of biological control agents in the field(Braga et al., 2001; Li and Feng, 2009; Morandi et al., 2006). UV-Aand UV-B can inactivate propagules of the agents in a few hours,due to genetic and morphological changes (Rangel et al., 2006b;Rotem et al., 1985; Santos et al., 2011). UV-B and UV-A causeprotein denaturation, inactivation of respiratory metabolism,oxidative stress, and damage DNA, RNA, and ribosomes. The accu-mulation of cyclobutane pyrimidine dimer and pyrimidine basesinside the cells prevent the duplication of genetic material(Griffiths et al., 1998).

Few works have studied the effect of the UV-B radiation on bio-control agents of plant pathogens, like C. rosea (Costa et al., 2012;Morandi et al., 2008). Considering the growing market of this bio-pesticides in Brazil and other countries, the objective of this workwas to evaluate the ability of an isolate of C. rosea, previously se-lected for its tolerant to UV-B radiation, to control B. cinerea onstrawberry leaves in controlled experiments.

2. Materials and methods

2.1. Isolates and inocula preparation

C. rosea LQC 62, previously selected as a tolerant isolate to UV-Bradiation (Costa et al., 2012), and B. cinerea LQC 126 isolated fromstrawberry crops at São Paulo state, Brazil (altitude: 925 m; lati-tude: 22�360 South; longitude: 46�420 West) were used in the stud-ies. The isolates are deposited in Embrapa Environment’s collectionof microorganisms.

The isolates were grown in 20 mL of Potato-Dextrose-Agar(PDA) (Acumedia Manufacturers, Michigan) in plates (polystyrene,90 � 10 mm, Pleion) and incubated at 25 ± 2 �C and 12 h light/12 hdark for 21 days. The conidia were suspended in a Tween 80 solu-tion with distilled water (0.01% v/v), vigorously shaken using a vor-tex and filtered through a polycarbonate membrane (80 mmdiameter, 8 lm pore size, Whatman Nucleopore, Clifton, NJ, USA)to remove spore aggregates. Conidial concentrations were esti-mated by hemocytometer counts and dilutions were made withsterile Tween 80 solution (0.01% v/v) for immediate use in the irra-diation studies.

2.2. Irradiation chambers, lamps, and filters

Irradiation experiments were conducted in a temperature-con-trolled chamber with four UV-B 313EL lamps (Q-lab Cleveland,OH). The lamps were aged prior to the start the experiments,resulting in a stable level of irradiation. Each lamp was coveredwith a 0.13 mm–thick cellulose diacetate film (Málaga Ltda), whichhad a cutoff point at 290 nm. This permitted the passage of mostUV-B and UV-A (290–400 nm), but prevented exposure to UV-C(>280 nm) and short-wavelength UV-B (>290 nm). The tempera-ture inside the controlled room was adjusted to 25 ± 2 �C andwas verified periodically throughout the sequence of experiments.

The DNA damage action spectrum developed by Quaite et al.(1992) and normalized to unity at 300 nm was used to calculatethe weighted UV irradiances (mW m�2). This spectral weightingwas selected based on the spectral characteristics of nine fungal re-sponses reviewed by Paul et al. (1997), which concluded that thisDNA damage spectrum is closely approximated to the fungal re-sponses. All the light measurements were made with a spectrora-diometer (Ocean Optics model USB2000 + rad) connected to aportable computer.

2.3. Effects UV-B radiation on C. rosea applied on strawberry leaf discsat different conidial concentrations

C. rosea growth on strawberry leaf discs was evaluatedindirectly by quantifying the potential of the fungus to cover thesurface of leaf discs and sporulate (Morandi et al., 2000). One-cm-diameter leaf discs of 30–60 day old strawberry plants (cv.Camarosa) were surface sterilized in 70% ethanol (1 min) followedby 2% sodium hypochlorite (1 min) and rinsed three times in steriledistilled water. After that, the leaf discs were placed in disposableplates (90 � 10 mm, Pleion) on humidified sterile absorbent paper(5 mL of sterilized water). Subsequently, each disc received an ali-quot of 20 lL of C. rosea conidial suspension at 104, 105, or 106 -conidia mL�1 and the material was exposed to UV-B radiation(irradiance 600 mW m�2) (Costa et al., 2012) for 0, 1, 2, or 3 h, cor-responding to doses 0 (control), 2.1, 4.2, and 6.3 kJ m�2. Control-plates were placed inside each of the chambers and wrapped withaluminum foil to physically protect them from radiation. The Petriplates were randomized in intervals of 30 min to homogenize thereceived doses of UV-B radiation. The discs were then transferredto paraquat chloramphenicol agar medium (PCA) in Petri dishes

L.B. Costa et al. / Biological Control 65 (2013) 95–100 97

(Peng and Sutton, 1991), at a number of 10 discs per plate. Sporu-lation of C. rosea was estimated after the tissues were incubated at25 ± 2 �C (12 h light/12 h dark) for 3, 7, and 10 days. The evaluationwas accomplished following a scale of notes for the area of thediscs covered with conidiophores of the C. rosea, 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., 2000). All the evaluations were madewith a stereomicroscope.

2.4. Reduction of B. cinerea sporulation on leaf discs treated withdifferent concentrations of C. rosea conidia, irradiated with UV-B andchallenged by the pathogen

Half of the discs treated with C. rosea and irradiated with UV-Bradiation received an aliquot of B. cinerea (10 lL–105 -conidia mL�1). The growth and sporulation of the pathogen wereestimated after the tissues had been incubated at 25 ± 2 �C (12 hlight/12 h dark) for 3, 7, and 10 days. The control treatment wasB. cinerea inoculated on leaf discs without C. rosea and not treatedwith UV-B radiation. The evaluation was accomplished following ascale of notes for the area of the discs covered with conidiophoresof B. cinerea, as follow: 0 = 0% (0%), 1 = 2% (1–3%), 2 = 5% (4–6%),3 = 10% (7–12%), 4 = 20% (13–26%), 5 = 40% (27–53%), 6 = 65%(54–76%), and 7 = 90% (77–100%) (Peng and Sutton, 1991).

2.5. Experimental design and data analysis

Each experiment was conducted with a completely randomizeddesign and repeated three times. For evaluations on leaf discs therewere three replication plates each containing 10 discs. The datafrom the three experimental repetitions invariably resulted intreatment effects in the same significance classes; therefore, thedata were grouped for analyses. Statistical computations were per-formed using the Statistical Analysis Systems (SAS Institute Inc.,Cary, NC). Data for fungal sporulation were transformed when nec-essary and examined using analysis of variance (ANOVA). The AreaUnder Presence Curve (AUPC) and Area Under Sporulation Curve(AUSC) were calculated for C. rosea and B. cinerea growth on leafdiscs and treatment means were compared by Tukey test. A facto-rial arrangement was used to evaluate the interactions betweenlength of exposure to UV-B radiation and inoculums concentrationof C. rosea. When there were no significant interactions among fac-tors, the data were grouped for analysis.

3. Results

3.1. Effects UV-B radiation on C. rosea applied on strawberry leaf discsat different conidial concentrations

The presence and sporulation of C. rosea on leaf disc surface wasinfluenced by the dose of UV-B radiation and the conidial concen-tration of antagonist, but there were no significant interactions be-tween the factors (P > 0.05; Table 1). The higher conidialconcentration greater the presence and sporulation of the antago-nist, regardless of the dose of UV-B radiation (Fig. 1 A and B). Sim-ilarly, increases in the UV-B dose reduced the presence andsporulation of the antagonist, regardless of conidial concentration(Fig. 1 C and D).

The deleterious effects of UV-B radiation on C. rosea growthwere more pronounced at lower spore concentrations. At 104 -conidia mL�1 the AUPC and AUSC of C. rosea was reduced by 40%and 60%, respectively, when irradiated at 0 (control) and 6.3(kJ m�2). At 106 conidia mL�1, the reductions were 3% and 40%

for AUPC and AUSC C. rosea, respectively, at the same doses ofUV-B radiation.

3.2. Reduction of B. cinerea sporulation on leaf discs treated withdifferent concentrations of C. rosea conidia, irradiated with UV-B andchallenged by the pathogen

The incidence and severity of B. cinerea on leaf discs were inver-sely correlated to presence and sporulation of C. rosea (Pearson cor-relation coefficients of p = �0.8184 and p = �0.5962, respectively,for B. cinerea incidence and severity).

The growth of the pathogen was higher in the lower C. roseaconcentration. The application of 104 conidia mL�1 of the biocon-trol agent regardless of the dose of UV-B reduced the average inci-dence and severity of the pathogen by 40% and 80%, respectively.The highest concentration of C. rosea (106 conidia mL�1) reducedthe incidence and severity by 91% and 98% of B. cinerea on straw-berry leaf discs after 10 days after inoculation (Fig. 2).

The UV-B radiation reduced the ability of C. rosea to control B.cinerea. The higher dose of UV-B reduced the presence and sporu-lation of C. rosea by 20% and 42%, respectively. Consequently, theincidence of B. cinerea increased twice and the severity wasthree-folder higher after 10 days after inoculation (Fig. 3).

4. Discussion

The ability of C. rosea to establish on leaf tissues is a critical fac-tor for the effectiveness of biocontrol against B. cinerea. In studiesinvolving dose-response to C. rosea–B. cinerea interactions per-formed on leaves and flowers of begonia, cyclamen, and raspberryunder controlled conditions (Li et al., 1996), significant control wasachieved only when the inoculum of C. rosea was 10–100 timesthat of B. cinerea (Yu and Sutton, 1998). Variables such as pheno-logical stage of the host or organ to be colonized, concentrationof bioagent inoculum, and environmental factors (temperature,humidity and UV-B radiation) can affect the viability of the conidiaand the establishment of C. rosea and, consequently, the level of B.cinerea control (Sutton et al., 1997).

UV-B and UV-A, naturally present in solar radiation, reduces thelongevity of fungal spores and their establishment on the host tis-sue (Ragaei, 1999; Rotem et al., 1985). However, few studies on theeffects of radiation on C. rosea are available (Morandi et al., 2008).In a previous study (Costa et al., 2012), comparing the effects ofUV-B radiation on C. rosea conidia on agar or bean leaves surfaces,found that in lower concentrations of conidia, C. rosea were moresensitive to UV-B radiation and that the effects were more pro-nounced in agar medium compared to leaf surface. The pigmenta-tion on leaf tissues and the physical protection to the conidia dueto the presence of trichomes, grooves, and bumps were suggestedas hypothesis to explain that results.

The present work found that UV-B radiation can affect the inoc-ulum viability of C. rosea and reduce its ability to control B. cinereaon strawberry leaves. Increases in UV-B radiation dose reduced theviability of the antagonist, and the effects were most pronouncedat lower spore concentrations. At 106 conidia mL�1 the viabilityof the conidia was reduced to 40%, while at 104 conidia mL�1, thereduction was up to 60%. Consequently, the incidence of B. cinereaincreased up to twice and the severity was up to three-folder high-er. Although not evaluated in this study, it is possible that the in-creased UV-B radiation may not be as detrimental to thepathogen as it is for C. rosea.

Our results indicate that the applied inoculum of the biocontrolagent must be higher than the effective dose to control the patho-gen. Part of this problem could be overcome by more effectivemethods of multiplication (Rangel et al., 2006a, 2004) and use of

Fig. 1. Effect of concentrations of Clonostachys rosea conidia and doses of UV-B radiation on leaf disc of strawberry and challenged to Botrytis cinerea. Area Under PresenceCurve – AUPC (A) and Area Under Sporulation Curve – AUSC (B) of C. rosea (__) and challenged of B. cinerea (–) on leaf discs in different concentrations of C. rosea 0–106 conidia mL�1 and B. cinerea; Area Under Presence Curve – AUPC (C) and Area Under Sporulation Curve – AUSC (D) of C. rosea (__) and challenged of B. cinerea (–) on leafdiscs with only C. rosea were irradiated with UV-B radiation doses 0–6.3 kJ m�2 (Quaite weighted irradiance of 600 mW m�2 at a different weighted dose). Curves show meanvalues plus standard error bars of three independent experiments. P = values from the analyses of variance and respective level of significance.

Table 1C. rosea development to differences conidia concentrations (104, 105 and 106 conidia mL�1) and irradiated with 0, 2.1; 4.2 and 6.3 kJ m�2 of UV-B radiation doses (Quaite weightedirradiance of 600 mW m�2 at a different weighted dose) at evaluation.

Area under presence curve Area under growth curve

UV-B dose (kJ m�2) UV-B dose (kJ m�2)

0 2.1 4.2 6.3 0 2.1 4.2 6.3

104 393 305 265 235 104 37.9 30.3 21.4 15.4105 438 426 390 371 105 54.5 52.9 47.5 38.9106 433 465 417 423 106 55.0 50.1 33.4 31.3P = 0.12 P = 0.93

P > 0.05 there were significantly different according analysis of variance.

98 L.B. Costa et al. / Biological Control 65 (2013) 95–100

additives in the formulation to increase resistance and/or reduceexposure of the conidia to UV-B radiation (Moore et al., 1993;Reddy et al., 2008).

In greenhouses, this problem can be partially solved by the plas-tic covering materials such as polyethylene (Costa et al., 2001).Most greenhouse polyethylene plastic films contain UV light-blocking components to prolong the life of the plastic coveringwhile maintaining transmission of photosynthetically active radia-tion. The majority of the greenhouse plastics on the market block

transmission of UV light with wavelengths of 360 nm or less. An-other well-known effect of polyethylene films that trap the radia-tion in the wavelength 300–400 nm is the decrease on B. cinereasporulation (Elad, 1997). Therefore, the effectiveness of biologicalcontrol in greenhouses is expected to be higher not only by the in-crease in the viability of conidia of C. rosea but also due to the re-duced sporulation of the pathogen.

The knowledge of the ecological behavior of the pathogens andthe antagonists is essential for the development of successful

Fig. 2. Incidence and severity of Botrytis cinerea in strawberry leaf disc at 10 days after inoculation. (A) Effect of concentrations of Clonostachys rosea on incidence of B. cinerea(%); (B) effect of concentrations of C. rosea on severity of B. cinerea (%).

Fig. 3. Effect of UV-B radiation (0–6.3 kJ m�2 mL�1) at 10 days after inoculation on incidence (A) and leaf disc area with conidiophores (B) of Clonostachys rosea and Botrytiscinerea.

L.B. Costa et al. / Biological Control 65 (2013) 95–100 99

biopesticides (Köhl et al., 2011). In addition to studies on toleranceto UV radiation, further studies are needed involving other envi-ronmental factors in the development of C. rosea in the future be-cause the scenarios of global climate change project not only analteration in background radiation, but also in global temperatureand CO2 concentration.

According to our results, in addition to showing less growth un-der UV-B, conidia of C. rosea had lower antagonistic ability. Furtherstudies are needed to observe the tolerance of B. cinerea conidia toUV-B radiation and thereby prove that an environment with in-creased UV-B radiation may be favoring the pathogen due to a low-er ability of C. rosea to control the pathogen B. cinerea in conditionsof increased UV-B.

Acknowledgments

This article was part of a M.Sc. dissertation of the first author.He was supported by a scholarship provided by the Fundação deAmparo à Pesquisa do Estado de Minas Gerais (FAPEMIG). We sin-cerely thank the project ‘‘Impacts of climate change on plant dis-eases, pests, and weeds’’ (Embrapa 010706002-04-Climapest).We also thank Alene Alder-Rangel at UNIVAP for reviewing the

English, and CNPq for a scholarship granted to Wagner Bettiol(CNPq 302724/2008-7).

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