Colonization of geranium foliage by Clonostachysrosea f. catenulata, a biological control agent ofbotrytis grey mould

S. Chatterton and Z.K. Punja

Abstract: The ecological requirements for the colonization of geranium leaves by the biocontrol agent Clonostachys roseaf. catenulata strain J1446 were investigated. Although this biocontrol agent is a soil-inhabiting fungus, treatment of gera-nium foliage with the agent can reduce grey mould caused by Botrytis cinerea in the greenhouse. To characterize the extentof foliar colonization, a GUS-transformed isolate of C. rosea f. catenulata was applied to foliage of two geranium cultivars,Pelargonium × hortorum and Pelargonium × domesticum. Population levels of C. rosea f. catenulata were found to behighest on senescent leaves and stems, followed by fully expanded leaves, and lowest on newly emerged leaves of both culti-vars. Optimum temperature for leaf and petiole colonization was 20–25 °C for both cultivars. The biocontrol agent requiredat least 12 h of continuous leaf wetness to achieve maximum population densities on the leaves and stems of both cultivars.On whole plants, colonization was significantly higher on wounded leaves, stems, and senescing leaves compared with thaton nonwounded leaves, stems, and mature leaves, respectively. GUS staining indicated that the fungus preferentially colon-ized the wound sites of leaves and the cut portions of stems. Results indicate that this biocontrol agent can successfully col-onize the foliage of geraniums, thus demonstrating the endophytic ability of C. rosea f. catenulata in both root and foliartissues.

Key words: biological control, geranium, leaf colonization, Gliocladium catenulatum.

Résumé : L’auteur a examiné les besoins écologiques pour la colonisation des feuilles du géranium par l’agent de maitrisebiologique, Clonostachys rosea f. catenulata, souche J1446. Bien que cet agent de lutte biologique soit un champignon dusol, le traitement des feuilles de géranium avec cet agent peut réduire la moisissure grise causée par le Botrytis cinerea enserre. Pour caractériser l’étendue de la colonisation foliaire, on a appliqué un isolat du C. rosea gus-tranformé sur le feuil-lage de deux cultivars de géranium, le Pelargonium × hortorum et le Pelargonium × domesticum. On constate une impor-tance décroissante des populations du C . rosea sur les feuilles sénescentes et sur la tige, suivie par les feuilles pleinementdéveloppées, et enfin les feuilles les plus basses nouvellement émergées, chez les deux cultivars. Les températures optimalespour la colonisation des feuilles et des pétioles voisinent les 20–25 °C, chez les deux cultivars. L’agent de maitrise biolo-gique nécessite au moins 12 heures d’humectation continue des feuilles pour l’obtention de densités de population maxima-les sur les feuilles et les tiges, chez les deux cultivars. Sur la plante entière, on observe une colonisation significativementplus importante sur les feuilles blessées, les tiges et les feuilles sénescentes, comparativement aux feuilles non blessées, auxtiges et aux feuilles matures respectivement. La coloration GUS indique que le champignon colonise préférentiellement lessites de blessures des feuilles et les parties coupées des tiges. Les résultats indiquent que cet agent de maitrise biologiquepeut coloniser avec succès le feuillage des géraniums, démontrant ainsi la capacité endophytique du C. rosea f. catenulata,dans les racines aussi bien que dans les tissus foliaires.

Mots‐clés : lutte biologique, géranium, colonisation foliaire, Gliocladium catenulatum.

[Traduit par la Rédaction]

Introduction

The fungus Clonostachys rosea f. catenulata Schroers, Sa-muels, Seifert & Gams (= Gliocladium catenulatum), com-mercially formulated as Prestop (Verdera Oy, Finland), is aneffective biocontrol agent for management of root diseases inthe greenhouse (Punja and Yip 2003; Rose et al. 2003) and isregistered for use in Canada on greenhouse vegetables, herbs,

and ornamentals. Although this biocontrol agent is princi-pally associated with the rhizosphere and is an efficient rootcolonizer (Lahdenperä and Korteniemi 2005; Chatterton et al.2008), it also shows efficacy against foliar pathogens. For ex-ample, the agent reduced anthracnose symptoms on blueberryfruits caused by Colletotrichum acutatum Simmonds whenapplied to blossoms and developing fruit (Verma et al.2006). Applications of C. rosea f. catenulata reduced the in-

Received 11 July 2011. Accepted 15 September 2011. Published at www.nrcresearchpress.com/cjb on 9 December 2011.

S. Chatterton. Department of Biological Sciences, 8888 University Drive, Simon Fraser University, Burnaby, BC V5A 1S6, Canada;Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, AB T1J 4B1, Canada.Z.K. Punja. Department of Biological Sciences, 8888 University Drive, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.

Corresponding author: S. Chatterton (e-mail: Syama.Chatterton@agr.gc.ca).

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Botany 90: 1–10 (2012) doi:10.1139/B11-076 Published by NRC Research Press

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cidence of gummy stem blight, caused by Didymella bryo-niae, on cucumbers by approximately 67% compared withthat on untreated plants (Utkhede and Koch 2004). Greymould, caused by Botrytis cinerea on tomato stems, wasalso suppressed under semicommercial conditions (Utkhedeand Mathur 2002), and this was the most effective microbialagent against grey mould on lettuce seedlings (Lahdenperäand Korteniemi 2005). Prestop also controlled Botrytis onstrawberries under field conditions in Finland, and market-able yield was increased (Lahdenperä and Korteniemi 2005).Geranium foliage treated with Prestop WP showed reduceddisease symptoms caused by B. cinerea in the greenhouse,and Prestop provided the best disease protection comparedwith that provided by other commercially available biocontrolagents and fungicides (Elmhirst et al. 2011).To ensure success, a biocontrol agent should be able to

survive under different environmental conditions (Fravel2005). Environment affects not only the survival of biocon-trol agents, but also their efficacy against pathogens, and amajor impediment to successful biocontrol is the extent towhich disease control may differ under different environmen-tal parameters (Fravel 2005). The identification and quantifi-cation of variables that affect the efficacy of an antagonistshould make biocontrol efforts more predictable and effective(Longa et al. 2008). Basic environmental conditions, such astemperature, moisture, and plant age, can not only affect thephysiology of the host plant, but can also alter the interac-tions among the plant, pathogen, and biocontrol agent (Lar-kin and Fravel 2002). Therefore, environmental parametersand conditions that optimize the survival of biocontrol agentsshould be determined. The effectiveness of an introduced bi-ocontrol agent in controlling disease is also influenced by itscolonization ability, which determines the population size ofthe agent at the time of pathogen infection and the degree towhich the pathogen population is potentially affected by thebiocontrol agent (Larkin and Fravel 1999; Paulitz 2000).Consequently, inconsistent colonization has often been attrib-uted to the variable success of biocontrol of foliar diseases(Daughtrey and Benson 2005). Variability in disease resist-ance and susceptibility among host cultivars can also affectcolonization and may influence the degree of biological con-trol (Hoitink and Boehm 1999). To date, experimental dataon the ability of C. rosea f. catenulata to survive on foliagehas not been reported, despite its efficacy against some foliarpathogens. Therefore, the objectives of this research were to(i) visualize the extent of foliar colonization by C. rosea f.catenulata, through the use of a GUS-marked strain (Chatter-ton et al. 2008), on two geranium hybrids; (ii) identify envi-ronmental conditions required for successful colonization bythe biocontrol agent; and (iii) monitor long-term survival ofC. rosea f. catenulata on whole geranium plants.

Materials and methods

Geranium growing conditionsPlants of two different geranium species, Pelargonium ×

hortorum (‘Zonal’) and Pelargonium × domesticum (‘MarthaWashington’ or ‘Regal’), were grown in 20 cm diameter potscontaining a soilless mix (Sunshine Mix 4, Sun Gro Horticul-ture Canada Ltd., Vancouver, B.C., Canada). These two hy-brid species were chosen because they were available

commercially at the time and are popular ornamental plantsgrown in Canadian nurseries. Both species are susceptible toinfection by B. cinerea (Elad and Volpin 1988; Daughtreyand Benson 2005), although there is no current literature de-scribing the availability of resistant or susceptible varieties.However, they differ substantially in growth habit, especiallyfoliage type and flowering time, and require different envi-ronmental conditions for optimal growth (Lis-Balchin 2002;Norman et al. 2009). Therefore, these two species werechosen to determine whether these differences would resultin variable responses to treatment with C. rosea f. catenulata.Plants were supplied with soluble N–P2O5–K2O (20:20:20;Plant Products Co., Brantford, Ont., Canada) fertilizer once aweek. For detached leaf assay studies, plants were maintainedin a growth room under ambient temperature (22–24 °C)with a 12 h photoperiod provided by sodium vapour lights(light intensity of 100 mmol·m–2·s–1). For studies on whole-plant colonization, plants were grown in growth cabinets(Conviron, Winnipeg, Man., Canada) with day and nighttemperatures of 20 and 16 °C, respectively, and relative hu-midity ranging from 55% to 70%.

Inoculum production and measurement of colonizationConidia of a GUS-transformed isolate of C. rosea f. cate-

nulata strain J1446 were produced as described previously(Chatterton and Punja 2010). The conidia were suspended insterile distilled water plus Tween 20 (0.05% v/v), filteredthrough two layers of cheesecloth, and adjusted to a concen-tration of 5 × 106 conidia/mL. Conidial suspensions weresprayed on detached leaves or whole plants with a standardgarden spray bottle until run-off. A control treatment of steriledistilled water plus Tween 20 was included in all experiments.Colonization of C. rosea f. catenulata on leaves was deter-mined by collecting 15 leaf discs (6 mm diameter) from eachinoculated leaf in triplicate, while petioles were cut into 1 cmpieces. Five leaf discs and two stem pieces from each leafwere assessed visually for colonization by staining for GUSactivity according to the method described by Chatterton etal. (2008). The remaining 10 leaf discs and stem pieces werehomogenized in GUS buffer, and the homogenate was used todetermine colony forming units (CFU) by dilution plating andGUS activity (expressed as nmol MU·(mg fresh tissue)–1·h–1,where MU is 4-methyl-umbelliferyl-b-D-glucuronide) as de-scribed previously (Chatterton et al. 2008).

Colonization and survival of C. rosea f. catenulata ongeranium plantsConidial suspensions of C. rosea f. catenulata were

sprayed onto ‘Zonal’ and ‘Martha Washington’ plants thathad been rooted for approximately 4 weeks. Plants weremaintained in a plant growth chamber. Two leaflets fromeach of three plants were removed at 3, 10, 17, 24, 31, and45 days after application. Leaf discs were removed, and colo-nization was assessed as described above. In a similar experi-ment, to assess colonization of different plant tissues and todetermine the effect of wounding on colonization, plants of‘Zonal’ geraniums were treated with C. rosea f. catenulata.Immediately prior to inoculation, two leaves from each ofthree plants were wounded with a fine needle (insect mount-ing pin), and two leaves from each plant were removed tocreate deleafing wounds on the petiole. At 10 and 24 days

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after application, the entire shoot system was harvested, andpopulation levels of C. rosea f. catenulata on the followingplant parts were assessed: meristem, petioles, stalk, woundedpetiole (deleafing wound), new leaves, mature leaves,wounded mature leaves, and senescent leaves. There werethree replicated plants in each trial, and the experiment wasrepeated twice.

Effect of development stage of geranium leaves oncolonization by C. rosea f. catenulataSix each of newly formed mature or senescent leaves with

petioles attached were removed from ‘Zonal’ and ‘MarthaWashington’ geranium plants. New leaves were light greenand just emerging from actively growing tips, mature leaveswere green and fully expanded, and senescent leaves wereyellowing but still attached to the plant when removed foruse. Leaves were surface-sterilized for 1 min in 1% Javexbleach and rinsed three times in distilled water before beingtreated with C. rosea f. catenulata. Leaves were placed in hu-mid chambers (12 cm × 20 cm Rubbermaid containers) on a2 mm nylon screen placed on four paper towels moistenedwith sterile distilled water and were maintained at room tem-perature (22–24 °C). At 3 and 6 days after spore application,three leaves and three petiole segments were sampled and as-sessed for colonization as described above.

Effect of temperature on colonization of geranium leavesby C. rosea f. catenulataMature leaves were removed from ‘Zonal’ and ‘Martha

Washington’ plants, surface-sterilized, and treated with C. ro-sea f. catenulata. Leaves were placed in plastic boxes, as de-scribed above, and boxes were placed in incubators at 16, 20,24, 30, and 35 °C. At 3 and 6 days after treatment, threeleaves and three petiole segments from each temperaturetreatment were sampled and assessed for colonization as de-scribed above.

Effect of leaf wetness duration on colonization ofgeranium leaves by C. rosea f. catenulataMature leaves from ‘Zonal’ and ‘Martha Washington’

geranium plants were removed, surface-sterilized, treatedwith C. rosea f. catenulata, and placed in plastic boxes withmoistened paper towels. After 0, 6, 12, 24, or 48 h of incu-bation at room temperature, the lids of the boxes were re-moved, and leaves were air-dried in a laminar flow hood for30 min. The lids were replaced, and the boxes were placed atroom temperature for 3 and 6 days after initial application ofC. rosea f. catenulata, after which time leaves and petioleswere assessed for colonization.

Statistical analysisA completely randomized design was used in each experi-

ment, and each geranium experiment was conducted twice.Data from individual trials were combined for analysis whenF tests indicated that variances of the data did not differ sig-nificantly; if variances differed, the results from each trialwere analyzed separately. Density estimates of C. rosea f. cat-enulata associated with geranium leaves were log-transformed(y+1) prior to analysis. Analysis of variance was performedusing the proc mixed statement to determine significance ofmain treatment effects and the interaction of treatment with

other experimental effects (day, variety) using SAS version9.1 (SAS Institute Inc. 2008). Data sets from each samplingday and for the two geranium species were analyzed sepa-rately when analysis of variance indicated that day or varietywas a significant source of variation. For all experiments, ex-cept for colonization of C. rosea f. catenulata on whole gera-nium plants, significant differences between treatment meanswere separated using Fisher’s protected least significant differ-ence test. The type 1 error rate (a) was set at 0.05 for all stat-istical tests.

Results

Colonization and survival of C. rosea on geranium plantsFor both geranium species, in two independent trials, colo-

nization of geranium leaves and petioles was greatest 3 daysafter application and decreased steadily throughout the exper-imental period (Figs. 1A, 1B). By 17 days after application,colonization levels on the leaves and petioles were reducedby approximately 50%, and by 31 days, population levelswere approaching undetectable levels in many samples(Figs. 1A, 1B); by day 45, C. rosea f. catenulata was not de-tected (data not shown). Both geranium species displayedsimilar results, and there was no difference in their responsefor either leaves or stems (trial 1: P = 0.8081 and 0.3651,respectively; trial 2: P = 0.4681 and 0.4281, respectively).Colonization levels on leaves were slightly higher in the sec-ond trial compared with those in the first trial, and thereforethe two trials were not combined for data analysis. GUS ac-tivity of colonized leaves and petioles displayed similartrends as those observed for CFU values, except that GUSactivity was not detected in some samples at 24 days afterapplication (Figs. 1C, 1D).Leaf colonization was also visualized by staining leaf

pieces from whole plants for the presence of blue hyphae ofthe GUS-marked strain. On mature leaves of ‘Zonal’ gera-niums, small colonies of C. rosea f. catenulata were distrib-uted over the upper leaf surface (Fig. 2A). Hyphae wereoften associated with trichomes (Fig. 2D) and were also ob-served emerging from stomata (Fig. 2E) on the lower surface.Senescing leaves supported denser colonies of C. rosea f.catenulata, with the most intense staining observed at pointsof natural decay and spreading outwards (Fig. 2B). On artifi-cially wounded mature leaves, colonization was seen in thearea immediately surrounding the wound site but without fur-ther spread to unwounded tissues (Fig. 2C). On mature leavesof ‘Martha Washington’ geraniums, colonization was sparseand primarily concentrated along the main leaf vein (Fig. 2H).Similar to results for ‘Zonal’ geraniums, senescing leavesfrom ‘Martha Washington’ geraniums were colonized at sitesof natural decay with spread outwards from dying tissues(Fig. 2I). In intact tissues, colonization appeared to be con-fined to intercellular spaces around leaf cells (Fig. 2J).Population density of C. rosea f. catenulata on whole ‘Zo-

nal’ geranium plants was significantly affected by samplingday and tissue type. Mean separations of all the different tis-sue types was not performed owing to the large number oftreatments. However, contrast partitioning was performed tocompare wounded treatments with the corresponding un-wounded controls. Colonization numbers and GUS activitywere the lowest in new leaves and the shoot tip area at both

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sampling times (Table 1). Population densities and GUS ac-tivities were similar on mature leaves, petioles, and the mainstem of the plant, while senescent leaves displayed the high-est levels on the unwounded tissues (Table 1). Contrast parti-tioning indicated that colonization of wounded leaves andstems was significantly higher than that of unwounded leavesand stems on both day 10 (P = 0.0280 and 0.0351, respec-tively) and day 24 (P = 0.0037 and 0.0098, respectively).Similar results were found for GUS activity, where activitylevels on wounded leaves and stems were significantly higherthan those on unwounded leaves and stems on day 10(P = 0.0211 and 0.0151). However, by day 24, there was nosignificant difference in GUS activity between unwoundedand wounded leaves (P = 0.1373), whereas levels were sig-nificantly higher on wounded stems than on unwoundedstems at this time (P < 0.0001).

Effect of developmental stage of geranium leaves oncolonization by C. rosea f. catenulataThe interaction between Pelargonium species and treatments

and between sampling day and treatments were significant for

CFU values associated with detached leaves (P = 0.0352 and0.0001, respectively); therefore, main treatment effects wereanalyzed separately for the two varieties. At 3 and 6 days afterinoculation, population densities were highest on senescentleaves and petioles of both ‘Zonal’ and ‘Martha Washington’plants (Figs. 3A, 3B, 3C, 3D). These levels were significantlyhigher than those observed on young and mature tissue typesat both sampling times. Colonization levels on mature leaves,from both varieties, were significantly higher than those onyoung leaves at day 3 only; on day 6, colonization levels weresimilar to each other. Colonization was significantly lower onmature petioles compared with that on young tissues on day 3from ‘Zonal’ geraniums, whereas colonization was lowest onmature petioles on day 6 from ‘Martha Washington’ gera-niums. GUS activity was also significantly higher on senescentleaves compared with that on mature and young leaves at allsampling times in both species (Figs. 3E, 3F). On young andmature leaves from both species, GUS activity levels in leafand petiole extracts did not differ significantly from each otheron day 3 or 6 (Figs. 3G, 3H). GUS activity was highest onpetioles from senescent leaves at both sampling times.

Fig. 1. Population levels of Clonostachys rosea f. catenulata on leaves (A, C) or petioles (B, D) of ‘Zonal’ or ‘Martha Washington’ geraniumplants as determined by colony plate counts (top) or fungal biomass of C. rosea f. catenulata expressing the GUS gene (bottom). Means andstandard errors were obtained from three replicates per treatment from two repeated trials (Tr1 and Tr2). Vertical bars indicate the standarderrors of the mean. CFU, colony-forming units; MU, 4-methyl-umbelliferyl-b-D-glucuronide.

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Effect of temperature on colonization of geranium leavesby C. rosea f. catenulataThe interaction between Pelargonium species and treat-

ments and between sampling day and treatments was signifi-cant for CFU values associated with detached leaves (P <0.0001); therefore, main treatment effects were analyzed sep-arately for the two varieties. On leaves from both species ofgeraniums, population densities were all significantly differ-ent from each other after 3 days of incubation (Figs. 4A,4B). The highest population densities were observed at 24

and 20 °C for ‘Zonal’ and ‘Martha Washington’ plants, re-spectively. After 6 days, population levels on leaves from‘Zonal’ geraniums were not significantly different from eachother when incubated at 16, 20, 24, or 30 °C, but levels fromthe 35 °C treatment were significantly lower. Population lev-els on ‘Martha Washington’ geraniums at day 6 were highestat 20 and 24 °C and were significantly higher than coloniza-tion densities from all other temperature treatments. On pe-tioles of both ‘Zonal’ and ‘Martha Washington’ geraniums,population levels of C. rosea f. catenulata were significantly

Fig. 2. Colonization pattern of leaves and petioles from ‘Zonal’ (A–G) or ‘Martha Washington’ (H–J) geranium plants 10 days after treatmentwith conidia of a GUS-marked strain of Clonostachys rosea f. catenulata. (A) Mature leaf; (B) naturally senescing leaf; (C) wounded leaf; (D)fungal hyphae associated with trichomes and (E) stomata; (F) mature and (G) cut petiole from ‘Zonal’ geraniums; (H) mature leaf and (I, J)senescing leaves from ‘Martha Washington’ geraniums. Arrows indicate areas of plant tissues colonized by the blue GUS-stained hyphae ofC. rosea f. catenulata.

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higher at 20 and 24 °C than those at all other incubation tem-peratures on days 3 and 6 (Figs. 4C, 4D). GUS activity washighest in leaves of both geranium species at incubation tem-peratures of 20 and 24 °C after 3 days (Figs. 4E, 4F). By thesixth day of incubation, GUS activity was highest in leavesof ‘Zonal’ geraniums incubated at 24, 30, or 35 °C, whileGUS activity levels were highest at 20 and 24 °C in ‘Martha

Washington’ geraniums at this sampling time. In the petioles,GUS activity levels were highest at 24 °C in ‘Zonal gera-niums’ at both days 3 and 6 (Fig. 4G). ‘Martha Washington’geraniums had the highest GUS activity levels in the petiolesafter 6 days of incubation at 20, 24, or 30 °C, whereas at3 days of incubation the highest GUS activity levels werefound at temperatures of 16, 20, and 24 °C (Fig. 4H).

Table 1. Population density of Clonostachys rosea f. catenulata, as determined by plate count or GUS activity, associated with differenttissue types of ‘Zonal’ geraniums at 10 and 24 days after application of the biocontrol fungus.

Plate count (log10 CFU·(mg fresh tissue)–1)a GUS activity (nmol MU·(mg fresh tissue)–1·h–1

Tissue type Day 10b Day 24 Day 10 Day 24Meristem 0.7 (0.18) 0.1 (0.21) 13.4 (4.19) 0New leaf 0.6 (0.14) 0.3 (0.16) 12.5 (2.81) 3.3 (3.13)Mature leaf 1.2 (0.12) 0.6 (0.14) 17.7 (3.20) 7.1 (2.81)Wounded leaf 1.5 (0.12) 1.2 (0.15) 30.4 (2.96) 12.2 (2.96)Senescent leaf 1.5 (0.19) 0.8 (0.15) 46.2 (4.19) 22.7 (2.96)Petiole 1.2 (0.15) 0.8 (0.16) 13.9 (3.20) 3.7 (3.20)Stem 1.3 (0.19) 0.8 (0.21) 17.0 (4.19) 4.1 (4.19)Wounded stem 1.6 (0.13) 1.4 (0.15) 30.4 (2.96) 25.2 (2.96)

Note: Standard error of the mean is in parentheses. CFU, colony-forming units; MU, 4-methyl-umbelliferyl-b-D-glucuronide.aData were log-transformed (y+1) for analysis.bTime of sampling in days after application of C. rosea f. catenulata.

Fig. 3. Population level of Clonostachys rosea f. catenulata on detached young, mature, or senescent leaves (A, B, E, F) or petioles (C, D, G,H) of ‘Zonal’ (A, C, E, G) or ‘Martha Washington’ (B, D, F, H) geranium plants as determined by colony plate counts (top) or fungal bio-mass of C. rosea f. catenulata expressing the GUS gene (bottom). Means and standard errors were obtained from six replicates per treatment.Values represent the combined means from two independent trials. Vertical bars indicate the standard errors of the mean. CFU, colony-forming units; MU, 4-methyl-umbelliferyl-b-D-glucuronide.

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Effect of leaf wetness duration on colonization ofgeranium leaves by C. rosea f. catenulataThe interaction between Pelargonium species and treat-

ments and between sampling day and treatments was signifi-cant for CFU values associated with detached leaves (P <0.0001); therefore, main treatment effects were analyzed sep-arately for the two varieties. Colonization levels in leaves andpetioles from ‘Zonal’ and ‘Martha Washington’ geraniumsdisplayed similar trends over both sampling days. Signifi-cantly higher population densities were observed on leavesthat experienced leaf wetness periods of 12, 24, and 48 hcompared with those that received 0 and 6 h of wetness,with values being highest in leaves that received 48 h of wet-ness (Figs. 5A–5D). GUS activity levels displayed similartrends for all treatments from both geranium species on bothsampling days, with the highest activity values occurringafter 48 h of leaf wetness (Figs. 5E–5H). However, in leaves,there was no significant difference between GUS activityfrom the 24 and 48 h treatments.

DiscussionThe efficacy of a biocontrol agent is based not only on its

antagonistic properties, but also on the extent to which it per-sists and grows in the environment where it is applied (Elad

and Kirshner 1993). Although the strain of C. rosea f. cate-nulata used in this study (strain J1446) was isolated fromfield soil and is found associated with roots (Teperi et al.1998), results from this study demonstrate that this funguscan also survive and proliferate on foliar tissues of two differ-ent geranium hybrids. When compared with the extent of cu-cumber root colonization, the population density of C. roseaf. catenulata was lower on geranium foliage than on rootsand was likely affected by fluctuations in environmental con-ditions (Chatterton et al. 2008; Chatterton and Punja 2010).In general, the rhizosphere environment can support higherpopulations of microorganisms owing to large amounts ofroot exudates that provide an energy-rich environment and toless fluctuating moisture conditions. Microbial populationson the leaf surface (phyllosphere), in contrast, are often nu-trient-limited owing to low carbon exudation rates and waterscarcity (Andrews and Harris 2000). Therefore, variable abio-tic conditions on the leaf surface make biocontrol of foliarpathogens more difficult to predict (Kessel et al. 2005).Understanding the requirements for an introduced biocontrolagent to colonize the leaf surface will aid in the selection ofecologically competent strains and more accurately predictunder which conditions the biocontrol agent will establish it-self (Hjeljord et al. 2000; Jeger et al. 2009).Expression of the GUS gene in the marked strain of C. rosea

Fig. 4. The effect of incubation temperature on population levels of Clonostachys rosea f. catenulata on detached leaves (A, B, E, F) orpetioles (C, D, G, H) of ‘Zonal’ (A, C, E, G) or ‘Martha Washington’ (B, D, F, H) geranium plants as determined by colony plate counts(top) or fungal biomass of C. rosea f. catenulata expressing the GUS gene (bottom). Means and standard errors were obtained from sixreplicates per treatment. Values represent the combined means from two independent trials. Vertical bars indicate the standard errors of themean. CFU, colony-forming units; MU, 4-methyl-umbelliferyl-b-D-glucuronide.

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f. catenulata used in this study was under the control of theconstitutive promoter from the glyceraldehyde-3-phosphate de-hydrogenase gene (Chatterton et al. 2008). Therefore, the pro-duction of the enzyme correlates with the physiologicalcondition of the fungus and is linked to the metabolic activityof the fungus in planta (Green and Jensen 1995; Bao et al.2000). On the other hand, the dilution-plating technique doesnot distinguish between propagules such as hyphal fragmentsor conidia and thus is not an accurate estimation of active fun-gal biomass (Park et al. 1992; Bae and Knudsen 2000). Fur-thermore, spore production is a measurement of the finalprocess of colonization, whereas measurement of active myce-lial biomass represents the nutrient acquisition phase duringwhich competition with pathogenic fungi is likely to take place(Kessel et al. 2002). Therefore, in these experiments, GUS ac-tivity measurements may correlate to the fungal biomass ofC. rosea f. catenulata, whereas CFU likely reflects sporulationon the foliage.Clonostachys rosea f. catenulata became established on

geranium plants, but leaf and petiole colonization decreasedover time, with naturally senescing leaves supporting thehighest density of fungal hyphae as visualized with GUSstaining. Colonization was concentrated at sites of senescenceand appeared to spread outwards from these points. Colonycounts were also highest on senescing tissue, indicating that

these tissues supported active sporulation. Botrytis cinerea isa necrotrophic pathogen that colonizes senescing tissue orplant tissues that has been killed by the pathogen, and sporu-lation occurs exclusively on necrotic tissues (Gerlagh et al.2001; Kessel et al. 2001). Colonization of necrotic onionleaves by G. catenulatum reduced sporulation and spread ofBotrytis aclada in senescing tissues (Köhl et al. 1997). Simi-larly, colonization of senescing leaves by C. rosea f. catenu-lata should result in the preemptive exclusion of saprophyticcolonization by B. cinerea, thus reducing pathogen spread.Furthermore, once B. cinerea infects necrotic leaf tissues, itoften spreads to the petioles that are least resistant to infec-tion, resulting in spread throughout the plant (Kessel et al.2001). Our results show that C. rosea can also colonize pe-tioles and would thus likely prevent Botrytis spread throughpetioles.There was no significant difference in colonization levels

by C. rosea f. catenulata on mature green leaves or petioleson whole plants of ‘Zonal’ or ‘Martha Washington’ gera-niums, but there was a difference on detached leaves. ‘MarthaWashington’ geraniums prefer cooler temperatures than ‘Zo-nal’ geraniums (Lis-Balchin 2002); therefore, detached leavesfrom ‘Martha Washington’ geraniums decayed faster thanleaves from ‘Zonal’ geraniums in the humid chambers attemperatures >20 °C. Principal nutrient sources in naturally

Fig. 5. The effect of leaf wetness duration on population levels of Clonostachys rosea f. catenulata on detached leaves (A, B, E, F) or pe-tioles (C, D, G, H) of ‘Zonal’ (A, C, E, G) or ‘Martha Washington’ (B, D, F, H) geranium plants as determined by colony plate counts (top)or fungal biomass of C. rosea f. catenulata expressing the GUS gene (bottom). Means and standard errors were obtained from six replicatesper treatment. Values represent the combined means from two independent trials. Vertical bars indicate standard errors of the mean. CFU,colony-forming units; MU, 4-methyl-umbelliferyl-b-D-glucuronide.

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senescent leaves are cell wall components such as cellulose,hemicelluloses, and lignin; however, under conditions of arti-ficial senescence, large amounts of soluble sugars and aminoacids are available owing to the absence of relocation (Kesselet al. 2002). As a result, it is likely that increased nutrientleakage under artificial senescence supported the higher pop-ulations of C. rosea f. catenulata observed in detached leafassays on both geranium species.In addition to the age of the leaves, preinoculation wound-

ing greatly increased sporulation and biomass of C. rosea f.catenulata on geranium plants compared with that in non-wounded mature leaves. Wounding results in nutrient leakagefrom the wounded cells onto the surface of the phylloplane(Morandi et al. 2000). Deleafing wounds on stems also sup-ported significantly higher levels of C. rosea f. catenulatapopulations compared with those on nonwounded stems of in-tact geranium plants. The wound sites left after removing sen-escent lower leaves of geraniums serve as entry points forinvasion by B. cinerea (Yohalem et al. 2007). Therefore, suc-cessful competitive colonization of these tissues by C. rosea f.catenulata should result in exclusion of B. cinerea fromwound sites. Clonostachys rosea successfully established ongeranium leaves and petioles at all temperatures, but popula-tion levels were highest at 20–24 °C. Similar results werefound for colonization of cucumber roots by C. rosea f. cate-nulata (Chatterton and Punja 2010), demonstrating that thisbiocontrol fungus has an optimal growing temperature thatcoincides with recommended temperatures for plant growth.For maximum colonization potential, C. rosea f. catenulatarequired at least 12 h of leaf wetness, after which time coloni-zation levels were stable at increasing hours of leaf wetness.Efficacy of C. rosea f. catenulata in suppressing root-

infecting pathogens has been attributed to both its root coloni-zation ability and production of cell wall degrading enzymesin situ, resulting in reduced sporulation and establishment offungal propagules (Chatterton et al. 2008; Chatterton andPunja 2009). The ability of C. rosea f. catenulata to producecell wall degrading enzymes on foliar tissues and how thisimpacts foliar pathogens remains to be determined. Our re-sults show that this biocontrol fungus can establish and colo-nize foliar tissues of an ornamental plant and thus mayprovide broad-spectrum protection against both root- and fo-liar-infecting pathogens on different host plants.

AcknowledgementsThis research was supported by the Natural Sciences and

Engineering Research Council of Canada (NSERC) throughthe Discovery Grants program. We thank Verdera Oy for pro-viding samples of Prestop and Leslie Dodd (Simon FraserUniversity) for providing cuttings of geraniums.

ReferencesAndrews, J.H., and Harris, R.F. 2000. The ecology and biogeography

of microorganisms on plant surfaces. Annu. Rev. Phytopathol.38(1): 145–180. doi:10.1146/annurev.phyto.38.1.145. PMID:11701840.

Bae, Y.S., and Knudsen, G.R. 2000. Cotransformation of Tricho-derma harzianum with b-glucuronidase and green fluorescentprotein genes provides a useful tool for monitoring fungal growthand activity in natural soils. Appl. Environ. Microbiol. 66(2): 810–815. doi:10.1128/AEM.66.2.810-815.2000. PMID:10653755.

Bao, J.R., Velema, J., Dobinson, K.F., and Lazarovits, G. 2000. UsingGUS expression in a nonpathogenic Fusarium oxysporum strain tomeasure fungal biomass. Can. J. Plant Pathol. 22(1): 70–78.doi:10.1080/07060660009501163.

Chatterton, S., and Punja, Z.K. 2009. Chitinase and b-1,3-glucanaseenzyme production by the mycoparasite Clonostachys rosea f.catenulata against fungal plant pathogens. Can. J. Microbiol.55(4): 356–367. doi:10.1139/W08-156. PMID:19396235.

Chatterton, S., and Punja, Z.K. 2010. Factors influencing coloniza-tion of cucumber roots by Clonostachys rosea f. catenulata, abiological disease control agent. Biocontrol Sci. Technol. 20(1):37–55. doi:10.1080/09583150903350253.

Chatterton, S., Jayaraman, J., and Punja, Z.K. 2008. Colonization ofcucumber plants by the biocontrol fungus Clonostachys rosea f.catenulata. Biol. Control, 46(2): 267–278. doi:10.1016/j.biocontrol.2008.02.007.

Daughtrey, M.L., and Benson, D.M. 2005. Principles of plant healthmanagement for ornamental plants. Annu. Rev. Phytopathol.43(1): 141–169. doi:10.1146/annurev.phyto.43.040204.140007.PMID:16078880.

Elad, Y., and Kirshner, B. 1993. Survival in the phylloplane of anintroduced biocontrol agent (Trichoderma harzianum) and popula-tions of the plant pathogen Botrytis cinerea as modified by abioticconditions. Phytoparasitica, 21(4): 303–313. doi:10.1007/BF02981048.

Elad, Y., and Volpin, H. 1988. The involvement of ethylene andcalcium in gray mold of Pelargonium, Ruscus and rose plants.Phytoparasistica, 16(2): 119–131. doi:10.1007/BF02980466.

Elmhirst, J.F., Haselhan, C., and Punja, Z.K. 2011. Evaluation ofbiological control agents against powdery mildew on rose andbotrytis blight on geranium. Can. J. Plant Pathol. doi:10.1080/07060661.2011.630758. In press.

Fravel, D.R. 2005. Commercialization and implementation ofbiocontrol. Annu. Rev. Phytopathol. 43(1): 337–359. doi:10.1146/annurev.phyto.43.032904.092924. PMID:16078888.

Gerlagh, M., Amsing, J.J., Molhoek, W.M.L., Bosker-van Zessen,A.I., Lombaers-van Der Plas, C.H., and Köhl, J. 2001. The effectof treatment with Ulocladium atrum on Botrytis cinerea — attackof geranium (Pelargonium zonale) stock plants and cuttings. Eur.J. Plant Pathol. 107(4): 377–386. doi:10.1023/A:1011248318499.

Green, H., and Jensen, D.F. 1995. A tool for monitoring Trichodermaharzianum. 2. The use of a Gus transformant for ecological studiesin the rhizosphere. Phytopathology, 85(11): 1436–1440. doi:10.1094/Phyto-85-1436.

Hjeljord, L.G., Stensvand, A., and Tronsmo, A. 2000. Effect oftemperature and nutrient stress on the capacity of commercialTrichoderma products to control Botrytis cinerea and Mucorpiriformis in greenhouse strawberries. Biol. Control, 19(2): 149–160. doi:10.1006/bcon.2000.0859.

Hoitink, H.A.J., and Boehm, M.J. 1999. Biocontrol within the contextof soil microbial communities: a substrate-dependent phenom-enon. Annu. Rev. Phytopathol. 37(1): 427–446. doi:10.1146/annurev.phyto.37.1.427. PMID:11701830.

Jeger, M.J., Jeffries, R., Elad, Y., and Xu, X.-M. 2009. A generictheoretical model for biological control of foliar plant diseases.J. Theor. Biol. 256(2): 201–214. doi:10.1016/j.jtbi.2008.09.036.PMID:18983855.

Kessel, G.J.T., De Haas, B.H., Lombaers-van Der Plas, C.H., van denEnde, J.E., Pennock-Vos, M.G., van der Werf, W., and Köhl, J.2001. Comparative analysis of the role of substrate specificity inbiological control of Botrytis elliptica in lily and B. cinerea incyclamen with Ulocladium atrum. Eur. J. Plant Pathol. 107(3):273–284. doi:10.1023/A:1011273613406.

Kessel, G.J.T., De Haas, B.H., van der Werf, W., and Köhl, J. 2002.

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Competitive substrate colonisation by Botrytis cinerea andUlocladium atrum in relation to biological control of B. cinereain cyclamen. Mycol. Res. 106(6): 716–728. doi:10.1017/S0953756202005956.

Kessel, G.J.T., Köhl, J., Powell, J.A., Rabbinge, R., and van der Werf,W. 2005. Modeling spatial characteristics in the biological controlof fungi at leaf scale: competitive substrate colonization byBotrytis cinerea and the saprophytic antagonist Ulocladium atrum.Phytopathology, 95(4): 439–448. doi:10.1094/PHYTO-95-0439.PMID:18943048.

Köhl, J., Belanger, R.R., and Fokkema, N.J. 1997. Interaction of fourantagonistic fungi with Botrytis aclada in dead onion leaves: acomparative microscopic and ultrastructural study. Phytopathol-ogy, 87(6): 634–642. doi:10.1094/PHYTO.1997.87.6.634. PMID:18945082.

Lahdenperä, M.A.L., and Korteniemi, M. 2005. Application ofmicrobial fungicides on horticultural crops: a case study withGliocladium catenulatum J1446. In Proceedings of the 1stInternational Conference on Plant–Microbe Interactions: Endo-phytes and Biocontrol Agents, Saariselkä, Lapland, Finland, 18–22 April 2005. Edited by S. Sorvari and O. Toldi. pp. 80–90.

Larkin, R.P., and Fravel, D.R. 1999. Mechanisms of action and dose–response relationships governing biological control of fusariumwilt of tomato by nonpathogenic Fusarium spp. Phytopathology,89(12): 1152–1161. doi:10.1094/PHYTO.1999.89.12.1152.PMID:18944639.

Larkin, R.P., and Fravel, D.R. 2002. Effects of varying environmentalconditions on biological control of fusarium wilt of tomato bynonpathogenic Fusarium spp. Phytopathology, 92(11): 1160–1166. doi:10.1094/PHYTO.2002.92.11.1160. PMID:18944240.

Lis-Balchin, M. (Editor). 2002. Growing pelargoniums in the garden,conservatory and for shows. In Geranium and Pelargonium:history of nomenclature, usage, and cultivation. Taylor & Francis,New York.

Longa, C.M.O., Pertot, I., and Tosi, S. 2008. Ecophysiologicalrequirements and survival of a Trichoderma atroviride isolate withbiocontrol potential. J. Basic Microbiol. 48(4): 269–277. doi:10.1002/jobm.200700396. PMID:18720503.

Morandi, M.A.B., Sutton, J.C., and Maffia, L.A. 2000. Effects of hostand microbial factors on development of Clonostachys rosea andcontrol of Botrytis cinerea in rose. Eur. J. Plant Pathol. 106(5):439–448. doi:10.1023/A:1008738513748.

Norman, D.J., Huang, Q., Yuen, J.M.F., Mangravita-Novo, A., andByrne, D. 2009. Susceptibility of Geranium cultivars to Ralstoniasolanacearum. HortScience, 44(5): 1504–1508.

Park, Y.H., Kenerley, C.M., and Stack, J.P. 1992. Inoculum dynamicsof Gliocladium virens associated with roots of cotton seedlings.Microb. Ecol. 23(2): 169–179. doi:10.1007/BF00172638.

Paulitz, T.C. 2000. Population dynamics of biocontrol agents andpathogens in soils and rhizospheres. Eur. J. Plant Pathol. 106(5):401–413. doi:10.1023/A:1008733927515.

Punja, Z.K., and Yip, R. 2003. Biological control of damping-off androot rot caused by Pythium aphanidermatum on greenhousecucumbers. Can. J. Plant Pathol. 25(4): 411–417. doi:10.1080/07060660309507098.

Rose, S., Parker, M., and Punja, Z.K. 2003. Efficacy of biological andchemical treatments for control of fusarium root and stem rot ongreenhouse cucumber. Plant Dis. 87(12): 1462–1470. doi:10.1094/PDIS.2003.87.12.1462.

SAS Institute Inc. 2008. SAS version 9.1 [computer program]. SASInstitute Inc., Cary, N.C.

Teperi, E., Keskinen, M., Ketoja, E., and Tahvonen, R. 1998.Screening for fungal antagonists of seedborne Fusarium culmorumon wheat using in vivo tests. Eur. J. Plant Pathol. 104(3): 243–251.doi:10.1023/A:1008647607310.

Utkhede, R.S., and Koch, C.A. 2004. Evaluation of biological andchemical treatments for control of gummy stem blight oncucumber plants grown hydroponically in greenhouses. BioCon-trol, 49(1): 109–117. doi:10.1023/B:BICO.0000009394.75429.a7.

Utkhede, R.S., and Mathur, S. 2002. Biological control of stemcanker of greenhouse tomatoes caused by Botrytis cinerea. Can. J.Microbiol. 48(6): 550–554. doi:10.1139/w02-043. PMID:12166682.

Verma, N., MacDonald, L., and Punja, Z.K. 2006. Inoculumprevalence, host infection and biological control of Colletotrichumacutatum: causal agent of blueberry anthracnose in BritishColumbia. Plant Pathol. 55(3): 442–450. doi:10.1111/j.1365-3059.2006.01401.x.

Yohalem, D.S., Paaske, K., Kristensen, K., and Larsen, J. 2007.Single application prophylaxis against grey mold in pot rose andpelargonium with Ulocladium atrum. Biol. Control, 41(1): 94–98.doi:10.1016/j.biocontrol.2007.01.007.

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