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Departamento de Ciencias Biológicas, Facultad de Química y Biología, Universidad de Santiago de Chile
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Various stages of the infection process among B. cinerea strains isolated from tomatoes or grapes, belonging to different genetic groups, were compared. It was found that strains of B. cinerea isolated from either grapes or tomatoes showed differences in adhesion patterns and in the percentage of germination on tomato cutin. In strains isolated from tomato the first stage of adhesion occurred faster than in strains isolated from grape. At the same time strains isolated from tomato showed a higher percentage of germination on tomato cutin than the other strains after 9 h of incubation.
The production and isoenzymatic patterns of polygalacturonases, pectin methyl esterases, pectin lyases, p-nitrophenylbutyrate esterases and laccases by B. cinerea in solid-state fermentation also were analyzed. Correlation between the production of these enzymes and the origin of the strains was not found. On the other hand all strains produced different isoenzymes and a common pattern between the strains was not observed.
The ability of B. cinerea strains to colonize tomato leaves also differs between the isolated strains obtained from grapes and tomato. Strains isolated from tomato were more virulent on tomato leaves than strains isolated from grapes.
Key words: adhesion, B. cinerea, enzymatic production, germination, host specialization, isoenzymatic patterns
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The interaction of this fungus with plants begins with conidia attachment to the host surface and continues with conidia germination, formation of infection structures and penetration into the host (Mendgen and Deising 1993). It has been found that attachment of B. cinerea conidia occurs in two distinct stages (Doss et al 1993). The first stage or immediate adhesion occurs upon hydration and is characterized by relatively weak adhesive forces (Doss et al 1993). The second stage, or delayed adhesion, occurs after viable conidia have been incubated several hours under conditions that promote germination (Doss et al 1995). Delayed adhesion involves secretion of an extracellular matrix (Doss et al 1995, Doss 1999).
B. cinerea penetrates host tissue either via wounds or through natural openings. This fungus also can directly penetrate the cuticle of plants through enzymatic degradation (McKeen 1974, Rijkenberg et al 1980). It produces cutinases (Salinas et al 1986), pectin-degrading enzymes such as pectin methyl esterase, pectin lyase and a number of different polygalacturonases (Leone et al 1990, Movahedi and Heale 1990, Reignault et al 1994, Rha et al 2001), proteases (Movahedi and Heale 1990) and laccases (Slomczynski et al 1995).
Numerous studies have demonstrated that B. cinerea is a highly variable fungal species. These differences can be explained by genetic variation among B. cinerea isolates (van der Vlugt-Bergmans et al 1993). When Chilean B. cinerea strains, obtained from grape, tomato, apple and other fruit, were characterized by RAPD-PCR or PCR-RFLP, significant genetic variability was observed (Thompson and Latorre 1999, Muñoz et al 2002). Based on genetic similarities B. cinerea strains were divided into different groups. All strains isolated from grape were included in one group, whereas isolates from tomato belonged to another group (Thompson and Latorre 1999, Muñoz et al 2002). However, it has not been determined if B. cinerea isolates, belonging to different genetic groups, present phenotypic differences in the infection process.
The objective of this work was to compare genetically different strains of B. cinerea isolated from tomato or grape with regard to: (i) the first stages of the infection mechanism of B. cinerea (i.e., adhesion and germination into tomato cutin) and (ii) the enzymatic production and the isoenzymatic patterns of cutinases, pectinases and laccases produced by solid-stage fermentation on tomato peels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and were kindly provided by Dr Muñoz. Isolate TT was obtained in this study, and it was purified from an infected tomato. All isolates were maintained on potato-dextrose agar (Difco Laboratories, Detroit, Michigan) slants at 4 C. The fungi were cultivated in Petri dishes containing malt-yeast extract agar (malt extract 2% (w/v), yeast extract 0.2% (w/v) and 1.5% (w/v) agar). To induce sporulation, cultures were incubated 10–14 d at 22 C under continuous near-UV light (F15T8 BLB fluorescent tubes with an emission spectrum of 310–410 nm). Fluorescent tubes were suspended 10 cm above Petri dishes.
To obtain dry conidia, Petri dishes containing B. cinerea were inverted on their lids and tapped to dislodge conidia. Conidia deposited on lids were collected and used immediately.
Solid-state fermentation was carried out in glass (Raimbault) columns (10 cm x 1 cm) as described by Raimbault and Alazard (1980). These columns were packed with 7 g of vermiculite as inert support and 15 g of tomato peels as substrate. Vermiculite and substrate were pre-inoculated with 1 mL of a conidia suspension (5 x 105 conidia/mL) before package. Cultures were incubated 3 d at 22 C with a constant flow of wet air through the columns.
Purification.— –
Tomato cutin was purified as described by Baker and Bateman (1978) with these modifications: Tomato peels were autoclaved 20 min in 0.4% (w/v) oxalic acid and 1.4% (w/v) ammonium oxalate. Peels then were washed with distilled water and incubated 18 h at 30 C in 100 mM sodium acetate buffer pH 4.5 containing 0.5% (w/ v) cellulase (Sigma Chemical Co., St Louis, Missouri) and 0.1% (w/v) pectinase (Sigma Chemical Co., St Louis, Missouri). Cutin was extracted with methanol followed by 2:1 chloroform/methanol at room temperature. Finally, a soxhlet extraction with chloroform for 12 h (6 cycles/h) was carried out.
Adhesion assays.— –
Dry conidia were deposited on tomato cutin and incubated at 100% relative humidity and 22 C. The percentage of adhesion during incubation was determined as described (Deising et al 1992). Data were expressed as percentage of conidia originally present.
Assays.— – Germination assays were carried out on tomato cutin. Dry conidia were deposited on cutin. After 9 h at 100% relative humidity and 22 C, the percentage of germination was determined. Conidia were considered to have germinated when germ-tube length was equal to or greater than conidial diameter.
Determination of enzymatic activities.— – To quantify the activities produced by B. cinerea in solid-state fermentation, tomato peels and vermiculite were extracted from the Raimbault column after 3 d of incubation and were pressed and the enzymatic activities were quantified in the supernatants.
Esterase activity was measured using p-nitrophenylbutyrate as substrate, as described by Purdy and Kolattukudy (1973). One unit per mL of esterase (U/mL) corresponds to the amount of enzyme that releases 1 µmol of p-nitro-phenol in 1 min.
Polygalacturonase and pectin lyase activities were determined with 1% polygalacturonic acid or 1% pectin as substrate incorporated to a 1.5% agar solution (Leone and van der Heuvel 1987). Cups of 4 mm diam were cut into the agar layer and filled with 10 µL of culture supernatants. The plates were incubated 24 h at 22 C and developed with 0.5% (w/v) ruthenium red. One unit per mL of polygalacturonase and pectin lyase (U/mL) corresponds to the amount of enzyme that produces 1 cm of clear zone.
Pectin methyl esterase was evaluated by using absorbance changes at 620 nm of a solution of bromocresol green with pectin as substrate (Reignault et al 1994). One unit per mL of pectin methyl esterase (U/mL) corresponds to the amount of enzyme that decreases the absorbance in 0.1 in 1 min.
To evaluate laccase activity syringaldazine was used as substrate (Harkin et al 1974). One unit per mL of laccase (U/ mL) corresponds to the amount of enzyme that increases the absorbance in 0.1 in 1 min.
Protein concentration.— –
Protein concentration in the supernatants of the culture media was determined as described by Lowry et al (1951).
Isoelectric focusing (IEF).— – Isoenzyme identification by IEF was performed horizontally on a Multiphor II apparatus (Pharmacia) using 0.4 mm thick polyacrylamide gels containing 5% (v/v) ampholytes (Pharmacia) covering the pH range 3.5–10.0. Electrode solutions were 0.5 M NaOH for the cathode and 0.04 M aspartic acid for the anode. The gels were run at a constant power of 2 W for 1.5 h.
An ultrathin overlay gel for pectin lyase and pectin methyl esterase detection was prepared (Chilosi and Magro 1997). Ultrathin overlay gel contained 1% agarose and 1% pectin buffered at pH 4.5 with 37.4 mM sodium acetate. After 3 h at 37 C, pectin-containing gels were stained overnight in ruthenium red (0.5% w/v in water). Excess of ruthenium red was washed off with water. Detection of esterase activity was made directly on IEF gels with indoxyl acetate as had been reported by Deising et al (1992).
Laccase activity was detected on IEF gels. Gels were incubated in 50 mM sodium phosphate buffer (pH 6.0) for 20 min. Then 0.5 mM syringaldazine was added. Laccase activity was seen as a pink band.
To determinate the pI of different isoenzymes, broad pI kit from Amersham Pharmacia Biotech was used (Amylglucosidase pI 3.5; Methyl red pI 3.75; Trypsin inhibitor pI 4.44; ß-Lactoglobulin A pI 5.2; Carbonic anhydrase B (bovine) pI 5.85; Carbonic anhydrase B (human) pI 6.55; Myoglobin acidic band pI 6.85; Myoglobin basic band pI 7.35; Lentil lectin acidic pI 8.15; Lentil lectin middle pI 8.45; Lentil lectin basic pI 8.65; Trypsinogen pI 9.3).
Tomato leaves colonization assays.— –
The ability to colonize tomato leaves was assessed on detached tomato leaves as described by Benito et al (1998). B. cinerea conidia were harvested, washed and resuspended in Gamborg’s B5 medium (Benito et al 1998). The suspension was incubated 1 h at room temperature. Tomato leaves were sterilized with 10% sodium hypochloride and placed on Petri dishes containing water agar (1.5% w/v agar). Five µL of conidia suspension (105 conidia/mL) were inoculated on one-half of the upper side of detached tomato leaves. On the other half sterile Gamborg’s B5 medium was deposited as control. Petri dishes were incubated at 22 C.
Statistical analysis.— –
Germination and enzyme production were analyzed by using Mann-Whitney U-Test with GB-Stat V6.5 for MS Windows. Means were separated using the least significant difference test (P 
0.05).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Conidia adhesion occurred in two stages as described by Doss et al (1993). The first stage reached a maximum after 30 min. The second stage started after 120 min and coincided with the beginning of the germination.
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). Adhesion patterns of conidia obtained from isolates that belong to the same genetic group were similar. However, the adhesion patterns of conidia obtained from isolates of grape or tomato were different. In B. cinerea strains isolated from tomato, the first stage of adhesion occurred immediately after conidia interacted with tomato cutin. In contrast, in strains isolated from grapes, adhesion occurred after 30 min of incubation. In addition in strains isolated from tomato, the second adhesion stage started 30 min earlier than in strains isolated from grape.
Germination of conidia from B. cinerea.— –
A comparison of the germination on tomato cutin of conidia from B. cinerea strains obtained from grape or tomato is provided (FIG. 2). After 9 h of incubation, germination percentage of the strains TT, T6 and T50 was significantly higher (90%) than the germination of conidia from G1 and G5 (about 50–70%).
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). The secretion of p-nitrophenyl butyrate esterases, polygalacturonases and pectin lyases was similar in the strains isolated from tomato or grape. However, strain G5 produced a low level of polygalacturonase. Three different levels in the production of pectin methyl esterases were obtained. T6 produced a higher amount of enzyme, G1 and TT secreted an intermediate level of enzyme, and a lower level of production was observed in the isolates T50 and G5. Finally, the production of laccases showed a wider variation. The secretion of laccase by G5 was twice as high compared to other isolates.
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). In general all strains produced acidic p-nitrophenyl butyrate esterase isoenzymes with pI between 5.3 and 3.5. The same isoform pattern was shared by G5 and T50. G1 and T6 secreted two isoforms but with different pI, and TT produced three isoenzymes (FIG. 4A).
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). The patterns of pectin lyase isoenzymes obtained from these strains were different; G1 and T6 exhibited acidic and alkaline pectin lyase. Strain TT produced only one alkaline pectin lyase. The other strains produced acidic pectin lyases. With respect to pectin methyl esterase, G1 and T6 produced only alkaline isoenzymes. G5 exhibited one alkaline and one acidic pectin methyl esterase isoenzyme. TT and T50 secreted two and one acidic isoenzymes, respectively.
Patterns of laccase isoenzyme produced by B. cinerea strains isolated from tomato or grape are provided (FIG. 4C). All strains produced acidic isoenzymes. Strains G1 and G5 produced higher number of isoenzymes than strains isolated from tomato.
Colonization ability of tomato leaves by B. cinerea strains isolated from tomato or grape.— –
Conidia from strains isolated from grape or tomato were inoculated onto detached leaves. After 3 d of incubation the resulting lesions were measured (TABLE I). B. cinerea strains isolated from tomato were highly virulent on tomato leaves. In contrast, strains obtained from grape did not cause damage to the leaves after 3 d.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two distinct stages of adhesion of B. cinerea conidia were observed. Similar results have been reported for many plant pathogenic fungi (Doss et al 1993, Apoga et al 2001, Mercure et al 1994). The first stage of adhesion of B. cinerea conidia occurs by relatively weak forces that are disrupted easily, and it has been demonstrated that the surface of the attached conidia is altered by contact with the substrate (Doss et al 1993). These changes in the conidia surface could explain the decrease of the percentage of adhesion during the first stage of conidia adhesion.
We were interested by the fact that, in the strains isolated from tomato, the first stage of adhesion occurred more quickly than in the strains isolated from grape. It is possible that the conidia surface of strains isolated from tomato or grape are different. Conidia obtained from strains isolated from grape required an incubation period for the initial interaction. Doss et al (1995) have reported that during the first stage of adhesion of B. cinerea conidia, weak hydrophobic interactions are involved. This suggests that, in conidia obtained from strains isolated from grape, changes in the surface would occur promoting a more robust hydrophobic interaction.
On the other hand, in this work it was shown that conidia from B. cinerea strains isolated from tomato presented a higher germination rate on tomato cutin than those isolated from grape. Therefore the high germination rate could be due to the ability of the conidia to adhere earlier to tomato cutin because a correlation between early adhesion and germination was observed (FIGS. 1 and 2). Other authors have reported a correlation between the delayed adhesion and the germination in B. cinerea conidia (Doss et al 1995, Filonow 2001, Slawecki et al 2002). The results obtained in this study suggest that the early adhesion of the B. cinerea conidia would be an important factor in the virulence of this fungus. Other studies also suggest that the adhesion to the host surface is an important step during the infection process of plant pathogenic fungi ( Jones and Epstein 1990, Mercure et al 1994). Jones and Epstein (1990) reported that an adhesion-deficient mutant of the fungus Nectria haematococca was less virulent than the wild type strain.
It also was observed that B. cinerea strains exhibit different abilities to colonize tomato leaves. Strains isolated from tomato were more virulent on tomato leaves than strains isolated from grapes. These results suggest that a degree of host specialization exits between different isolates of B. cinerea. This observation supports previous results showing that B. cinerea isolates from grapevine were virulent against this species, while isolates from other plant species were avirulent (Derckel et al 1999). Similar results have been reported with B. cinerea isolated from roses, which were more virulent on rose petals than those obtained from gerbera flowers or pea leaves (Pie and Brower 1993). However, the mechanisms involved in the host specialization of B. cinerea have not been elucidated.
The production of hydrolytic and oxidative enzymes by the B. cinerea isolates in solid-state fermentation on tomato peels showed that there was no correlation between the enzymatic production levels and the origin of the strains. Similar levels of p-nitro-phenylbutyrate esterase, polygalacturonases and pectin lyase were produced by these isolates. Only variations in the secretion of pectin methyl esterases and laccases were observed. It can be assumed that strains obtained from grapes would produce more laccase because this enzyme has been associated with the detoxification of the phytoalexin resveratrol, a compound produced by grape (Sbagui et al 1996). However, under our culture conditions, only the isolate G5 secreted a high level of this enzyme. This suggests that laccase production is not an important factor in the mechanism of host specialization of B. cinerea.
Furthermore, strains belonging to the same genetic group did not secrete a common isoenzyme and all strains tested produced different isoenzymes. Enzymatic production by B. cinerea in solid-state fermentation had not been studied. This culture system was used because it mimics natural growth conditions of phytopathogenic fungi because water is scarcer than in liquid culture.
Strains G1 and T6 produced two groups of pectin lyases and pectin methyl esterases (acidic and alkaline) in solid-state fermentation. These results are in agreement with those reported by Chilosi and Magro (1997). These authors found that B. cinerea produced acidic and alkaline pectin lyase when the strain was cultivated on zucchini tissue. Therefore, solid-state fermentation may be a suitable replacement for cultures on plant tissues for certain investigations.
In conclusion, the first stages of infection process of B. cinerea (i.e., adhesion and germination of conidia) and the ability to colonize tomato leaves correlated with the origin of the isolates. Adhesion and germination, mainly the immediate adhesion, could be related with the degree of host specialization showed by the different isolates of B. cinerea.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. Departamento de Ciencias Biológicas, Facultad de Química y Biología, Universidad de Santiago de Chile. Casilla 40 Correo 33, Santiago, Chile. Fax: 56-2 681-2108. Voice: 56-2 681-1644. E-mail: mcotoras{at}lauca.usach.cl
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and germination (
) of the conidia from isolate G5. B. Early adhesion of the conidia from the isolates TT (
), T6 (•), T50 (
), G5 (
). Each point represents the mean of at least five independent experiments ± standard deviation.
