Histopathological studies of sclerotia of ...

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Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain Sabrina SARROCCOa, Lisbeth MIKKELSENb, Mariarosaria VERGARAc, Dan Funck JENSENb, Mette LU¨BECKb, Giovanni VANNACCIa,* a

Department of Tree Science, Entomology and Plant Pathology ‘‘G. Scaramuzzi’’, Plant Pathology Section, Faculty of Agriculture, University of Pisa, Via del Borghetto 80, I-56124 Pisa, Italy b Department of Plant Biology, Section for Plant Pathology, the Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Denmark c Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56100 Pisa, Italy

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abstract

Article history:

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein

Received 29 December 2004

(GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, character-

Received in revised form

ized as having a promising biocontrol activity against a large number of phytopathogenic

2 August 2005

fungi. On the basis of molecular and biological results, a stable GFP transformant was se-

Accepted 25 August 2005

lected for further experiments. In order to evaluate the effects of GFP transformation on

Corresponding Editor:

mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum

Derek T. Mitchell

and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the trans-

Keywords:

formation of the antagonistic isolate with the GFP reporter gene did not modify mycopar-

Biocontrol

asitic activity against sclerotia. Sclerotium colonization was followed by fluorescent

Crop protection

microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and in-

Mycoparasites

ter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed

Sclerotinia

mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum

Sclerotium

suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry. ª 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction Fungi belonging to the genus Trichoderma have received considerable attention as potential biocontrol agents against a wide range of soilborne plant-pathogenic fungi. Strains of Trichoderma spp. have proven to be effective mycoparasites of several economically important plant-pathogenic fungi. Parasitism by Trichoderma species has been reported against

pathogens, such as Rhizoctonia solani (Benhamou & Chet 1993; Elad et al. 1983), Fusarium spp. (Chet 1990; Cherif & Benhamou 1990), as well as Sclerotinia spp. (Knudsen & Eschen 1991; Gracia-Garza et al. 1997) and Sclerotium spp. (Vannacci et al. 1989; Tsahouridou & Thanassoulopoulus 2001). Sclerotinia sclerotiorum, Sclerotium rolfsii, and Sclerotinia minor, represent three of the most destructive pathogens of

* Corresponding author. E-mail address: [email protected] 0953-7562/$ – see front matter ª 2005 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2005.08.005

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many economically important crops (Purdy 1979; Punja 1985; Abawi & Grogan 1979). These pathogens produce resting structures known as sclerotia, which have a strong resistance both to chemical and biological degradation (Punja 1985) and permit the fungi to survive in the absence of a host (Abawi & Grogan 1979). Sclerotia are composed of an outer rind layer of melanized cells that are thought to be responsible for resistance to microbial degradation in soil (Jones 1970). The mechanisms, whereby Trichoderma spp. antagonistic strains control diseases caused by sclerotial fungi, may involve interference with sclerotial germination that may or may not be accompanied by sclerotial degradation, growth inhibition of the pathogen in soil and prevention of host penetration by the pathogen. Some species of Trichoderma can penetrate the rind and colonize the inner cell layers of sclerotia, often completely destroying them or rendering them not viable. The incidence of diseases incited by sclerotia-producing pathogens is frequently proportional to the inoculum density of these structures in soil (Benhamou & Chet 1996; Tu 1980). Genetic engineering of biocontrol agents with reporter or marker genes has provided useful tools for detecting and monitoring introduced biocontrol agents in confined natural environments (Green & Jensen 1995; Lo et al. 1998). The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been developed as a reporter for gene expression (Chalfie & Kain 1998) and has received considerable attention as a vital marker in many prokaryotes and eukaryotes, including filamentous fungi. The fluorescence of GFP requires only UV, or blue light and oxygen, unlike the case of other reporters (bglucuronidase, b-galacturonidase, chloramphenicol acetyltransferase and firefly luciferase), which rely on cofactors or substrate activity. Therefore, in vivo observation of the gfp gene expression is possible (Lorang et al. 2001). Many studies have shown GFP to be an useful tool for studying plant-fungus interactions in vivo (Mikkelsen et al. 2001; Rohel et al. 2001; Jensen & Schulz 2003). The aim of this work was to produce a stable transformant of T. virens I10, expressing the GFP marker gene, in order to determine whether it can provide an useful tool for histological studies concerning plant pathogen/antagonist interactions, exemplified by colonization of sclerotia of Sclerotium rolfsii and Sclerotinia sclerotiorum.

Materials and methods Fungal strains Trichoderma virens (syn. Gliocladium virens) strain I10 (CBS 116947) was isolated from a sandy soil collected in a pine wood within the Regional park of San Rossore, Pisa, Italy (Scaramuzzi et al. 1986, Vannacci & Pecchia 2000), and is also deposited in the fungal collection of the Department of Tree Science, Entomology and Plant Pathology ‘‘Giovanni Scaramuzzi’’, University of Pisa. Sclerotinia sclerotiorum strain 2048 was isolated in 1992 from the aerial part of tomato plants, cultivated in an experimental greenhouse in Pisa, Italy; S. minor strain 595 was isolated from a naturally infected lettuce plant (Vannacci et al. 1988); Sclerotium rolfsii strain 398 was isolated in 1987 from soil cultivated with

S. Sarrocco et al.

sugar beets. All fungi were maintained on Potato Dextrose Agar (PDA; Difco Laboratories, Detroit) under mineral oil at 4 C and grown on PDA at 24 C. Sclerotia of S. rolfsii, S. minor, and S. sclerotiorum were produced by inoculating 8 mm diam disks of PDA with fungal mycelium in flasks containing 100 g of barley seeds sterilized (116 C for 1 h) in 100 ml tap water. Flasks were incubated for one month at 24 C, in 16 h-8 h of light-darkness. After incubation sclerotia were collected by sieving and were dried under air flow at room temperature for 24 h.

Media and plasmids for transformation For optimization of transformation, three media, viz. PDA, modified TSM (Trichoderma Selective Medium; Elad et al. 1981) without chloramphenicol and brassicol and GYE (Glucose Yeast Extract) (Seh & Kenerley 1988) were tested with different hygromycin B (Calbiochem-Novabiochem, Darmstadt) concentrations. 150 ppm in PDA was the most efficient dose for the selection of transformants. The plasmid gGFP (Maor et al. 1998), containing the Escherichia coli hygB resistance gene and a synthetic version of the gfp gene, sGFP(ser65T), in which threonine replaced Ser65, both under the control of the constitutive Aspergillus nidulans glyceraldehyde-3-phosphate (gpd) promoter, was kindly donated by Amir Sharon (Tel Aviv University). Plasmid propagation was performed according to Sambrook et al. (1987), by employing the E. coli competent strain MC1061. Purification of plasmid was carried out using the Qiagen plasmid kit purification procedure (Quiagen, Chatsworth, CA).

Preparation of protoplasts and transformation Spore suspensions of Trichoderma virens I10 were inoculated in 100 ml of PDB (Potato Dextrose Broth) at a final concentration of 2.0 ! 106 cfu mlÿ1 and incubated at 28 C for 16 h with shaking at 120 rpm. The mycelium was collected on a nylon membrane (42 mm) and washed three times with 1.2 M MgSO4. The mycelium was weighed and 1.0 g was transferred to a Petri dish, in which protoplasts were prepared according to Lu¨beck et al. (2002), modified from Thrane et al. (1995), using 5 ml of an enzyme mixture containing lysing enzyme from T. harzianum (10 mg mlÿ1), driselase (20 mg mlÿ1) and cellulase (10 mg mlÿ1; Sigma Chemicals, St Louis, MO). The solution was made hypertonic by the addition of 1.2 M MgSO4, 10 mM sodium-phosphate buffer, adjusted to pH 6.0 and filtered through a 0.45 mm membrane filter. Release of protoplasts was monitored by microscopic observation at regular intervals and after 3 h they were collected by filtering through a nylon membrane, washed three times with 1.2 M MgSO4, pelleted by centrifugation at 750 g for 5 min, resuspended in 1.2 M MgSO4 and counted using a haemocytometer. Concentration was adjusted to 0.5 ! 106 cfu mlÿ1 in 1.2 M MgSO4 and the suspension was kept on ice until use. An aliquot of 200 ml protoplasts suspension was gently mixed with 5 mg of plasmid DNA and 50 ml of PEG-solution (25 % polyethylene glycol 6000, 50 mM CaCl2, 1 M sorbitol, 50 mM - pH 8.0 - tris-HCl), and incubated on ice for 20 min. Then 2 ml of PEG-solution were added and the suspension was incubated at room temperature for 5 min. Aliquots of 100 and 500 ml were added to 5 ml 0.8 % molten agar (50 C) in PDB supplemented by 1 M sorbitol and immediately

Colonization of sclerotia by GFP transformed Trichoderma virens

poured onto Petri dishes containing 20 ml of regenerating medium (PDA with addition of 1 M sorbitol). Plates were incubated at 28 C and overlaid after 16 h with 5 ml of selective agar (PDA, 0.8 % agar, 150 ppm hygromycin B). Regenerating protoplasts were observed, 24 h after antibiotic addition, in plates using a fluorescent stereomicroscope. Putative transformants grew through the selective agar layer and were transferred to fresh selective agar plates. Hygromycin B-resistant colonies were tested for GFP expression by examination under fluorescence stereomicroscope. Total DNA from nine T. virens GFP putative transformants and from the wild type was extracted according to Bulat et al. (1998) and 1 mg of genomic DNA was digested with either BglII or KpnI restriction enzymes to determine the copy number and sites of insertion of the gGFP plasmid (Lu¨beck et al. 2002; Mikkelsen et al. 2001). Digested DNA was size-fractionated by gel electrophoresis, blotted and hybridised using standard procedures (Sambrook et al. 1987). The 700 bp NcoI/PstI gfp fragment of the gGFP plasmid labelled with [32P]dCTP using the Megaprime DNA labeling system (Amersham Pharmacia Biotech, Little Chalfont) was used as probe. Hybridization signals were revealed after over-night exposure of membranes to autoradiographic films.

Selection of a stable transformant and detection of gfp gene insertion by PCR The nine transformants were submitted to ten successive subcultures on a non-selective medium (PDA without antibiotic), checking every time for emission of fluorescence from the mycelium by microscopic observation, in order to evaluate the stability of gfp gene insertion in the genome. To compare all nine transformants with the wild type, in terms of mycelial growth and antagonistic activity, in vitro tests were performed. Mycelial growth test was made placing one PDA disc of 8 mm diam, collected from the edge of actively growing colonies of each transformant and the wild type, in the centre of PDA and WA (Water Agar) plates. Plates were incubated in darkness at 24 C (three replicates for each strain) and measures of radial growth were made every 8 h. Radial growth rates were expressed as mm hÿ1. To evaluate antagonistic activity of the nine transformants against a Rhizoctonia solani strain (AG4 isolated from tobacco), PDA disks of 8 mm diam of both the antagonist and the pathogen, were placed on opposite sides of a sterilized cellophane membrane laid down on PDA or WA. Plates were incubated in darkness at 24 C and were observed at regular intervals up to 22 d. Overgrowth and sporulation over the pathogen colony were considered as signs of activity. During incubation, interaction zones or overlap regions were examined microscopically: coilings of the antagonist around hyphae of the pathogen were considered as sign of mycoparasitism. The growth rate of each transformant on different media and the ability to overgrow, to sporulate on Rhizoctonia solani colony and to coil around pathogen hyphae were compared with those of the wild type. Insertion of the gfp gene in the fungal genome was checked by PCR. Specific primers, designed on the gfp gene sequence, GFPF (5#-ATGGTGAGCAAGGGCGAGG-3#) and GFPR (5#TTCTGCTGGTAGTGGTCGGC-3#), amplifying a 557 bp fragment, were used. PCR amplification was performed on

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100 ng DNA with the cycling parameters: 3 min at 94 C (denaturation); 35 cycles at 94 C for 1 min, 65 C for 1 min, 72 C for 1 min (amplification) and finally elongation at 72 C for 10 min.

Histopathological studies of mycoparasitic activity of GFP transformants Further experiments involved the Trichoderma virens I10 3.4.10 GFP transformant, on the basis of the stable insertion of the gfp gene and the fluorescence emission from mycelium. In order to evaluate the effects of GFP transformation on the mycoparasitic ability of T. virens I10 against sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor, a 24 well microplate (‘Beal’, Pbi International, Milano, Italy) test was arranged. Each well was half-filled with PDA and was inoculated with 3.4.10 GFP or the wild type. After 7 d of incubation, four sclerotia of each pathogen were sown in each well, considering a row of six wells as a replicate. Each microplate contained four replicates. 4 ! 24 sclerotia were tested for each isolate. After further 7 (Sclerotinia minor), 11 (Sclerotium rolfsii) and 13 (Sclerotinia sclerotiorum) days of incubation, firmness of sclerotia was evaluated by pressure (Artigues & Davet 1982) and soft sclerotia were considered as decayed. Decay percentages were submitted to analysis of variance (ANOVA) after angular transformation, using the SYSTAT 10 package; P % 0.05 was considered as a significant level of difference. Colonization of Sclerotium rolfsii and Sclerotinia sclerotiorum sclerotia by T. virens I10 3.4.10 GFP was histologically assessed by microscopy at regular intervals. Sclerotia of S. sclerotiorum colonized by T. virens I10 wild type were examined under UV or white light to verify the autofluorescence of mycoparasite structures. Presence of the transformant in sclerotia sections was detected by using a Leica MZ FLIII stereomicroscope, equipped with a mercury lamp and filters GFP2 (excitation filter at 480/40 and a barrier filter at 510 nm LP) and a Leitz dialux 22 epifluorescence microscope with a filter having excitation at 470/20 nm and barrier filter at 515 LP. Both instruments were equipped with a Leica DC 300F digital camera. Images were handled with the software Leica DC Twain version 5.1.1.

Results Transformation of T. virens I10 with the gfp gene and selection of a stable transformant For stabilization of putative transformants, agar plugs from each protoplast regeneration plate were transferred onto new selective medium and incubated until sporulation at 24 C. Spore suspensions from each putative transformant culture of the antagonist were plated on the selective medium and colonies arisen from single conidia of each transformant were tested for GFP expression by microscopic observation. Since the gGFP plasmid contains both the hygromycin B resistance and the gfp gene, all antibiotic-resistant colonies were expected to be fluorescent. Nine transformants from singlespore colonies of Trichoderma virens I10, expressing both hygromycin B resistance and GFP after several single spore subcultures, had inserted the gfp gene into the genome in

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1-2 copies at a single site as shown by DNA hybridisation (data not shown). There were no background signals of the wildtype genomic DNA. In order to compare all nine T. virens I10 GFP transformants with the wild type in terms of mycelial growth and antagonistic activity against a Rhizoctonia solani strain (AG4), in vitro tests were performed. Growth rate of the wild type was 0.584 (G0.022 S.D.) mm hÿ1 on PDA and 0.558 (G0.004 S.D.) mm hÿ1 on WA. Growth rates of transformants vary from 0.523 (G0.101 S.D.) mm hÿ1 to 0.648 (G0.039 S.D.) mm hÿ1 on PDA, and from 0.530 (G0.011 S.D.) mm hÿ1 to 0.577 (G0.013 S.D.) mm hÿ1 on WA. Both the wild type and the nine transformants were able to grow and sporulate profusely over the R. solani colonies on both media. Microscopic observations demonstrated a coiling formation on WA, but not on PDA, by the wild type and the transformants around R. solani hyphae. Based on results obtained in these tests, no differences were detectable between the nine stable transformants and the wild type. Stereomicroscopic observation showed that PDA cultures of all nine T. virens I10 GFP transformants had the typical green fluorescent colour under epifluorescence. GFP clearly accumulated in the cytoplasm. All nine transformants had strong GFP expression, but the transformant I10 3.4.10 GFP was slightly brighter than the others. DNA hybridization confirmed that it contained two gfp gene insertions. On the basis of this observation and considering that it showed no differences in growth rate, or in antagonistic and mycoparasitic activity against a R. solani strain, when compared with the wild type, T. virens I10 3.4.10 GFP was selected for further experiments. To evaluate the stability of GFP expression, a conidial suspension of T. virens I10 3.4.10 GFP was plated on PDA and fluorescence of single colonies was checked. 94.4 % out of 261 colonies checked resulted as fluorescent. Results confirmed that this was a stable transformant. PCR amplification of DNA from T. virens I10 wild type and 3.4.10 GFP strains with GFPF and GFPR primers produced a single band of approximately 600 bp, corresponding to the size of the expected gfp gene fragment, only in the transformed isolate (data not shown).

Histopathological studies of mycoparasitic activity of GFP transformants In order to evaluate the effects of GFP transformation on the mycoparasitic ability of Trichoderma virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were plated in 24 wells microplates, inoculated with the transformant I10 3.4.10 GFP or the wild-type strain. After incubation, a number of sclerotia of all three pathogenic fungi became soft in the presence of T. virens I10 (both wild type and 3.4.10 GFP transformant) (Table 1). Statistical analysis of decay percentages showed that the transformation of the antagonistic isolate with the GFP reporter gene had not modified its mycoparasitic activity against sclerotia. T. virens I10 3.4.10 GFP was therefore used to follow the colonization of sclerotia of Sclerotinia sclerotiorum and Sclerotium rolfsii. Microscopic observation of parasitized sclerotia of S. rolfsii showed that the fungus grew profusely on the sclerotial surface, producing mycelial tufts (Fig 1A) that, under UV light at low magnification, appeared bright green where the mycelium

S. Sarrocco et al.

Table 1 – Proportion (%) of decaying sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor as a result of colonization by Trichoderma virens (either wild type or strain I10 3.4.10 GFP) after 11, 13 and 7 d incubation, respectively

Wild type I10 3.4.10 GFP

S. rolfsii

S. sclerotiorum

S. minor

54.8 a 49.1 a

20.8 a 20.2 a

38.0 a 43.1 a

Average of four replicates of 24 sclerotia each. Within each column, figures followed by the same letter are not significantly different at 5 % level.

was more dense (Fig 1B). Examination of sclerotial sections showed that, after 9 d, T. virens I10 had penetrated uniformly through the rind cells and grew in the cortex (Fig 1C). After 13 d, the antagonist’s growth into the sclerotium was clearly evident at low magnification, appearing as a green layer just behind the rind without any evident preferential entry point (Fig 1D). At higher magnification, fungal growth in the cortex was seem to be mainly intracellular, producing hyphal mats within host cells. Adjacent cells were colonized through small lesions in the cell wall, but later large portions of walls were destroyed (Fig 1E). Colonization of the medulla by T. virens hyphae (15 d) was characterized by a shift in the mode of antagonist’s growth from intra- to intercellular (Fig 1F) . T. virens I10 3.4.10 GFP grew and sporulated profusely on S. sclerotiorum sclerotia (Fig 2A). Microscopic examination of sections of sclerotia 9 and 14 d after inoculation revealed the presence of antagonist’s mycelium in the medulla (Fig 2B) and colonization was characterized by intercellular growth (Fig 2C). Under the stereomicroscope, sclerotial tissues colonized by transformed T. virens appeared as a green layer along the rind without any evident preferential entry point (Fig 2D). After 20 d, the medulla was completely colonized by the antagonist and most sclerotial tissues were replaced by Trichoderma hyphae (Fig 2E) forming chlamydospores (Fig 2F). Approaching maturity, chlamydospore walls were characterized by autofluorescence (Fig 2G). Visualization of wild type hyphae within tissues of S. sclerotiorum sclerotia has been quite difficult but its behaviour is similar to that of transformed strain. After 20 d intercellular hyphae forming chlamydospores are present, and chlamydospores, but not hyphae, autofluoresce under UV (Fig 2H and 2I).

Discussion The antagonistic abilities of the Trichoderma species make them potential candidates for biocontrol against phytopathogenic fungi in integrated pest management. Trichoderma virens I10 has been tested in numerous experiments in order to control different phytopathogenic fungi, such as Rhizoctonia solani, Bipolaris sorokiniana, Sclerotinia spp. and Sclerotium spp., showing promising results (Scaramuzzi et al. 1986; Vannacci & Pecchia 1986; Cortellini et al. 1989; Vannacci & Pecchia 2000; Sarrocco 2004). As a tool for monitoring and visualising this antagonistic strain in ecological studies, T. virens I10, belonging to sect.


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Fig 1 – Fluorescent micrographs of colonization of Sclerotium rolfsii sclerotia by Trichoderma virens I10 3.4.10 GFP. (A) Sclerotial surface producing mycelial tufts. (B) Mycelial tufts appear bright green under UV light. (C) Penetration of T. virens I10 through the rind cells and growth in the cortex after 9 d incubation. (D) A green layer behind the rind without any clear preferential entry point after 13 d incubation. (E) Intracellular fungal growth, producing hyphal mats within host cells. (F) Intercellular growth characterizes colonisation of medulla by T. virens after 15 d incubation.

Fig 2 – Fluorescent micrographs of colonization of Sclerotinia sclerotiorum sclerotia by Trichoderma virens I10 3.4.10 GFP. (A) Sclerotial surface producing T. virens I10 mycelial tufts that, under UV light appear bright green. (B) After 9 d. (C) After 14 d. (D) Presence of T. virens I10 in the medulla showing intercellular growth; penetration through the rind cells and, evidence of a green layer just behind the rind without any evident preferential entry point. (E) After 20 d Trichoderma hyphae forming chlamydospores. (F) Chlamydospores forming in the medulla. (G) UV light autofluorescence of walls of mature chlamydospores. (H) After 20 d T. virens wild-type chlamydospores in the medulla shows autofluorescence under UV (I).


Pachybasium, was transformed with the Green Fluorescent Protein (GFP) marker gene. Other Trichoderma species, belonging to the sect. Trichoderma, have previously been GFP tagged, and have been used to study the induction of different chitinolytic enzymes-encoding genes: T. atroviride (Zeilinger et al. 1999; Kullnig et al. 2000), T. asperellum, (Viterbo et al. 2002), to monitor T. harzianum (Bae & Knudsen 2000) and to quantify hyphal growth in soil (Orr & Knudsen 2004), and to visualize mycoparasitic interactions against Pythium ultimum or Rhizoctonia solani hyphae (T. atroviride; Lu et al. 2004). T. virens I10 isolate had the ability to integrate the gfp plasmid into its genome, and to express the fluorescent protein. In general, GFP transformants of T. virens I10 seemed to be stable. A high mitotic stability of the selected 3.4.10 GFP transformant was demonstrated, and this transgenic strain was successfully subcultured, expressing GFP in the absence of selective pressure. In a previous study, T. virens I10 was successfully transformed by using the DsRed-Express fluorescent protein, isolated from the reef coral Discosoma sp. (Matz et al. 1999), showing a good expression of the inserted gene (Mikkelsen et al. 2003). In order to be useful tools for inferring biological information on the wild-type isolate, reporter genes must not modify important phenotypic traits (Thrane et al. 1995; Lu¨beck et al. 2002). In this work, GFP transformants were compared with the wild-type in order to examine growth rate, the ability to grow on both rich and poor substrates (PDA and Water Agar, respectively), to overgrow colonies of fungal pathogens, and to parasitize structures of pathogens such as sclerotia, and to make sure that the inserted genes did not interfere with any of these important traits. T. virens I10 3.4.10 GFP was morphologically similar to the wild type, showing no differences in growth rate and antagonistic activity against Rhizoctonia solani. The presence of the gfp gene in the fungal genome did not compromise the ability of T. virens I10 to decay sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor. Bae & Knudsen (2000) reported that their co-transformed strain T. harzianum thzID1-M3 showed a lower growth rate on agar plates and soil and a similar ability, compared to the wildtype, to colonize S. sclerotiorum sclerotia. The different species and the presence of three foreign genes could account for the somewhat different results reported by cited authors if compared with data reported here. A microscopic study allowed the direct observation of T. virens I10 colonizing the sclerotia of both Sclerotium rolfsii and Sclerotinia sclerotiorum. The uniformly distributed mycelium of I10 3.4.10 GFP just beneath the rind of sclerotia of both pathogens suggests that the sclerotia became infected at numerous, randomly distributed locations without any preferential entry point, even if we cannot exclude that cracks in the rind can favour penetration (Merriman 1976). The rind of sclerotia of Sclerotinia sclerotiorum and Sclerotium rolfsii is composed of cells whose walls are heavily melanized (Jones 1970; Punja 1985). The uniform penetration of T. virens I10 through the rind of sclerotia of both pathogens suggests its ability to induce enzymatic degradation of rind walls and, probably, to degrade melanin. Melanin protects fungi against lysis in natural soils (Butler & Day 1998), or against antifungal drugs in human pathogens (Gomez & Nosanchuk 2003). Degradation of melanin, as

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a mechanism of action in biocontrol, has been discussed elsewhere (Butler et al. 2004). Results reported here along with the evidence that sclerotia-degrading ability is isolate-specific (Sarrocco et al. 2004), and the observation that Lentinula edodes produces melanin when attacked by Trichoderma (Savoia et al. 1998) suggest that melanin degradation as a mechanism for sclerotia infection by Trichoderma needs a more detailed study. Growth of I10 3.4.10 GFP into the cortex region of S. rolfsii sclerotia is clearly intracellular and it is accompanied by destruction of the cell walls. This is a behaviour quite different from that described by Henis et al. (1983), where the mycelium of the Trichoderma species (T. harzianum, T. viride, and T. hamatum) seemed to grow through the rind and cortex without affecting the host cells. This behaviour, conversely, confirms the findings of Benhamou & Chet (1996) for their isolate of T. asperellum (Kullnig et al. 2001). Intercellular growth of T. virens in medullary tissue of S. rolfsii is well established (Henis et al. 1983; Benhamou & Chet 1996), and has been confirmed by our experiments. Intracellular parasitism of T. virens in S. sclerotiorum sclerotia seems dominant according to some previous electron microscopic observations (Tu 1980), but not according to our results. The inter- or intracellular growth into medullary tissues could be time-dependent, since after extensive colonization, medullary tissue begins to disintegrate. At this time, it is reasonable to imagine that transmission electron microscope sections would show hyphae of Trichoderma inside cells of S. sclerotiorum. Discrepancies between the findings reported by different authors can have several explanations. Mycoparasitism of sclerotia of S. rolfsii and S. sclerotiorum by Trichoderma is widespread, but seems isolate-specific (Sarrocco et al. 2004). Therefore, that different species or isolates are being studied may account for the different reported behaviours. GFP transformation enables a simple fluorescent microscope to be used to trace the growth of labelled Trichoderma strains inside host structures, and it is a prerequisite to set up protocols for exploiting more sophisticated equipment, such as the confocal microscope, for an even more detailed study of the interaction.

Acknowledgements We acknowledge the skilful technical assistance of Maurizio Forti, and the support of Nicola Falaschi for microscopic investigations. S.S. performed fungal transformation during a stay at KVL (Frederiksberg) funded by the University of Pisa, during her PhD studies.

references

Abawi GS, Grogan RG, 1979. Epidemiology of disease caused by Sclerotinia species. Phytopathology 69: 899–904. Artigues M, Davet P, 1982. Recherche de crite`res de se´lection de clones de Trichoderma actifs contre des champignons a` scle´rotes. In: La Se´lection des Plantes, Bordeaux (France) 21–26 March 1982 [Les Colloques de l’INRA No. 11.]: 181–193.

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Bae YS, Knudsen GR, 2000. Cotransformation of Trichoderma harzianum b-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. Applied Environmental Microbiology 66: 810–815. Benhamou N, Chet I, 1993. Hyphal interactions between Trichoderma harzianum and Rhizoctonia solani: ultrastructure and gold cytochemistry of the mycoparasitic process. Phytopathology 83: 1062–1071. Benhamou N, Chet I, 1996. Parasitism of sclerotia of Sclerotium rolfsii by Trichoderma harzianum: ultrastructural and cytochemical aspects of the interaction. Phytopathology 86: 405–416. Bulat SA, Lu¨beck M, Mironenko NV, Jensen DF, Lu¨beck PS, 1998. UP-PCR analysis and ITS1 ribotyping of Trichoderma and Gliocladium fungi. Mycological Research 102: 933–943. Butler MJ, Day AW, 1998. Fungal melanins: a review. Canadian Journal of Microbiology 44: 1115–1136. Butler MJ, Gardiner RB, Day AW, 2004. Degradation of melanin or inhibition of its synthesis: are these a significant approach as a biological control of phytopathogenic fungi? Biological Control 32: 326–336. Chalfie M, Kain S, 1998. Green Fluorescent Protein: properties, applications and protocols. In: Chalfie M, Kain S (eds). WileyLiss, New York. Cherif M, Benhamou N, 1990. Cytochemical aspects of chitin breakdown during the parasitic action of Trichoderma spp. on Fusarium oxysporum f. sp. radicis-lycopersici. Phytopathology 80: 1406–1414. Chet I, 1990. Mycoparasitism: recognition, physiology and ecology. In: Baker RR, Dunn PE (eds), New Directions in Biological Control: alternatives for suppressing agricultural pests and diseases. Alan Liss, New York, pp. 725–733. Cortellini W, Vannacci G, Bergonzoni P, Ravagli S, 1989. Impiego di funghi antagonisti in programmi di difesa biologica in colture orticole. Notiziario sulle Malattie delle piante 110: 252–264. Elad Y, Chet I, Henis Y, 1981. A selective medium for improving quantitative isolation of Trichoderma spp. from soil. Phytoparasitica 9: 59–67. Elad Y, Chet I, Boyle P, Henis Y, 1983. Parasitism of Trichoderma spp. on Rhizoctonia solani and Sclerotium rolfsii. Scanning electron microscopy and fluorescence microscopy. Phytopathology 73: 85–88. Gomez BL, Nosanchuk JD, 2003. Melanin and fungi. Current Opinion in Infectious Diseases 16: 91–96. Gracia-Garza JA, Bailey BA, Paulitz TC, Lumdsen RD, Releder RD, Roberts DP, 1997. Effect of sclerotial damage on Sclerotinia sclerotiorum on the mycoparasitic activity of Trichoderma hamatum. Biocontrol Science and Technology 7: 401–413. Green H, Jensen DF, 1995. A tool for monitoring Trichoderma harzianum. II. The use of a GUS transformant for ecological studies in the rhizosphere. Phytopathology 74: 1436–1440. Henis Y, Adams PB, Lewis JA, Papavizas GC, 1983. Penetration of sclerotia of Sclerotium rolfsii by Trichoderma spp. Phytopathology 73: 1043–1046. Jensen DF, Schulz A, 2003. Exploitation of GFP-technology with filamentous fungi. In: Arora DK (ed), Handbook of Fungal Biotechnology. Marcel Dekker, New York, pp. 441–451. Jones D, 1970. Ultrastrucure and composition of the cell walls of Sclerotinia sclerotiorum. Transactions of British Mycological Society 54: 351–360. Knudsen GR, Eschen DJ, 1991. Potential for biocontrol of Sclerotinia sclerotiorum through colonization of sclerotia by Trichoderma harzianum. Plant Disease 75: 466–470. Kullnig C, Mach LM, Lorito M, Kubicek CP, 2000. Enzyme diffusion from Trichoderma atroviride (Z T. harzianum P1) to Rhizoctonia solani is a prerequisite for taggering of Trichoderma ech42 gene

S. Sarrocco et al.

expression before mycoparasitic contact. Applied and Environmental Microbiology 66: 2232–2234. Kullnig C, Krupica T, Woo SL, Mach RL, Rey M, Benitez T, Lorito M, Kubicek CP, 2001. Confusion abounds over identities of Trichoderma biocontrol isolates. Mycological Research 105: 770–772. Lo CT, Nelson EB, Hayes CK, Harman GE, 1998. Ecological studies of transformed Trichoderma harzianum 1295-22 in the rhizosphere and on the phylloplane of creeping bentgrass. Phytopathology 88: 129–136. Lorang JM, Tuori RP, Martinez JP, Sawyer TL, Redman RS, Rollins JA, Wolpert TJ, Johnson KB, Rodrigues RJ, Dickman MB, Ciuffetti LM, 2001. Green fluorescent protein is lighting up fungal biology. Applied and Environmental Microbiology 67: 1987– 1994. Lu ZX, Tombolini R, Woo S, Zeilinger S, Lorito M, Jansson JK, 2004. In vivo study of Trichoderma-pathogen–plant interactions, using constitutive and inducible green fluorescent protein reporter systems. Applied and Environmental Microbiology 70: 3073–3081. Lu¨beck M, Knudsen IMB, Jensen B, Thrane U, Janvier C, Jensen DF, 2002. GUS and GFP transformation of the biocontrol strain Clonostachys rosea IK726 and the use of these marker genes in ecological studies. Mycological Research 106: 815–826. Maor R, Puyesky M, Horwitz BA, Sharon A, 1998. Use of green fluorescent protein (GFP) for studying development and fungal–plant interaction in Cochliobolus heterostrophus. Mycological Research 102: 491–496. Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA, 1999. Fluorescent proteins from non bioluminescent Anthozoa species. Nature Biotechnology 17: 969–973. Merriman PR, 1976. Survival of sclerotia of Sclerotinia sclerotiorum in soil. Soil Biology and Biochemistry 8: 385–389. Mikkelsen L, Roulund N, Lu¨beck M, Jensen DF, 2001. The perennial ryegrass endophyte Neotyphodium lolii genetically transformed with the green fluorescent protein gene (gfp) and visualizing in the host plant. Mycological Research 105: 644–650. Mikkelsen L, Sarrocco S, Lu¨beck M, Jensen DF, 2003. Expression of the red fluorescent protein DsRed-Express in filamentous Ascomycete fungi. FEMS Microbiology Letter 223: 135–139. Orr KA, Knudsen GR, 2004. Use of green fluorescent protein and image analysis to quantify proliferation of Trichoderma harzianum in non sterile soil. Phytopathology 94: 1383–1389. Punja ZK, 1985. The biology, ecology and control of Sclerotium rolfsii. Annual Review of Phytopathology 23: 97–127. Purdy LH, 1979. Sclerotinia sclerotiorum: history, disease and symptomatology, host range, geographic distribution, and impact. Phytopathology 69: 875–880. Rohel EA, Payne AC, Fraaije BA, Hollomon DW, 2001. Exploring infection of wheat and carbohydrate metabolism in Mycosphaerella graminicola transformants with differentially regulated green fluorescent protein expression. Molecular Plant– Microbe Interaction 14: 156–163. Sambrook J, Fritsch EF, Maniatis T, 1987. Molecular Cloning: a laboratory manual, 2nd edn. Cold Springer Harbor Press, New York. Sarrocco S, Forti M, Vannacci G, 2004. Mycoparasitism against sclerotia of Sclerotium rolfsii and Sclerotinia sclerotiorum is widespread within the genus Trichoderma. IOBC/WPRS Bullettin 27: 375–379. Sarrocco S, 2004. Physiological characterization of the antagonists Trichoderma virens I10 and T. asperellum I252 and their transformation with genes encoding for fluorescent proteins. PhD thesis, University of Pisa. Savoia JM, Mata G, Billette C, 1998. Extracellular laccase production during hyphal interactions between Trichoderma spp. and Shiitake, Lentinula edodes. Applied Microbiology and Biotechnology 49: 589–593.


Scaramuzzi G, Vannacci G, Pecchia S, 1986. Valutazione dell’attivita` antagonistica di microrganismi applicati al seme nei confronti di Rhizoctonia solani Ku¨hn. Atti del Convegno Agrobiotec 1986, Bologna: 1–16. Seh ML, Kenerley CM, 1988. Protoplast isolation and regeneration and nuclear staining of mycoparasitic Gliocladium species. Journal of Microbiological Methods 8: 121–130. Thrane C, Lu¨beck M, Green H, Degefu Y, Allerup S, Thrane U, Jensen DF, 1995. A tool for monitoring Trichoderma harzianum I: transformation with the GUS gene by protoplast technology. Phytopathology 85: 1428–1435. Tsahouridou PC, Thanassoulopoulos CC, 2001. Trichoderma koningii as a potential parasite of sclerotia of Sclerotium rolfsii. Cryptogamie Mycologie 22: 289–295. Tu JC, 1980. Gliocladium virens, a destructive mycoparasite of Sclerotinia sclerotiorum. Phytopathology 70: 670–674. Vannacci G, Pecchia S, 1986. Evalution of biological seed treatment for controlling seed-borne inoculum of Drechslera sorokiniana on barley. Mededeling en van de Faculteit

187

Landbouwwetenschappen, Rijksuniversiteit Gent 51/2B: 741–750. Vannacci G, Pecchia S, 2000. Discovery of Trichoderma I252 and Gliocladium I10, components of the biofungicide GT10/252. European Cost 830 Workshop Selection Strategies for Plant Beneficial Microorganisms. Nancy, 3–5 April 2000. Vannacci G, Triolo E, Materazzi A, 1988. Survival of Sclerotinia minor Jagger sclerotia in solarized soil. Plant and Soil 109: 49–55. Vannacci G, Pecchia S, Resta E, 1989. Presence of Sclerotinia minor Jagger antagonists in soil from different Mediterranean areas. II. Sclerotia decaying fungi. Acta Horticulturae 225: 293–302. Viterbo A, Montero M, Ramot O, Friesem D, Monte E, Llobell A, Chet I, 2002. Expression regulation of endochitinase chit36 from Trichoderma asperellum (T. harzianum T-203). Current Genetics 42: 114–122. Zeilinger S, Galhaup C, Payer K, Woo SL, Mach RL, Fekete C, Lorito M, Kubicek CP, 1999. Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host. Fungal Genetics and Biology 26: 131–140.