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Centro de Investigação das Ferrugens do Cafeeiro, Quinta do Marquês, 2784-505, Oeiras, Portugal
Maria A. Nunes
Centro de Produção e Tecnologia Agrícolas, Tapada da Ajuda, 1304 Lisboa, Portugal
Maria C. Silva
Carlos J. Rodrigues, Jr. 1
Centro de Investigação das Ferrugens do Cafeeiro, Quinta do Marquês, 2784-505, Oeiras, Portugal
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The method of penetration of fungi through the host cuticle by means of cutinase versus mechanical pressure exerted by melanized appresoria has been the subject of debate. Colletotrichum kahawae Bridge & Waller infects green coffee berries in Africa, inducing 70–80% losses. Turgor pressure (TP) of the appresoria was estimated in vitro to be 2.6 MPa, about twice the osmotic pressure (OP) of the green berries. Appresoria exposed in vitro to polyethylene glycol (PEG) solution with OPs of 7.0 MPa and above immediately collapsed. However, collapsed appresoria subjected to OP as high as 46.5 MPa could recover. Green berries inoculated with conidial suspensions, if subjected to OP of 28.5 MPa, showed 7% of them with necrotic lesions. Total inhibition of infection was achieved at 46.5 MPa. The OP of PEG solutions applied to inoculated green fruit decreased to the OP of the green berries in 48 h. The resistance of appresoria to osmotic stress, combined with the rapid dilution of PEG by solutes (water) from the fruit might explain the rate of infection even at very high OP. Unmelanized appresoria induced by tricyclazole showed TPs as low as one-quarter of melanized ones and, as a consequence, the percentage of infection on leaves and green berries was much lower. Cutinase was present in conidial mucilage and in extracellular fluids of germinated conidia in vitro and in planta. Cutinase was induced by growing the fungus in Czapek-Dox medium if cutin was used as the sole carbon source. Diisopropyl fluorophosphate, a cutinase inhibitor, totally inhibited cutinase activity of culture filtrates and extracellular fluids but did not prevent infection. It is suggested that the TP of C. kahawae appresoria might play a major role in coffee cuticle penetration, according to our results.
Key words: Coffea arabica, coffee berry disease, cutinase, fungal penetration, osmotic pressure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The enzymatic hydrolysis of the polyester cutin by cutinase has been identified as an essential factor of fungal pathogenicity, and the involvement of cutinase has been established by different methods for many host-pathogen systems such as rape-Pyrenopeziza brassicae (Li et al 2003), pea-Fusarium solani f. sp. pisi (Nectria haematococca) (Dickman et al 1989), maize-Colletotrichum graminicola (Pascholati et al 1993) and papaya-Colletotrichum gloeosporioides (Dickman et al 1982, Dickman and Patil 1986). However, the validity of this concept remains uncertain because some fungi can deform synthetic surfaces and even pierce them, consistent with a primary role for mechanical force during penetration, as it has been proposed for the rice pathogen Pyricularia oryzae (Magnaporthe grisea) (Howard et al 1991, Sweigard et al 1992). Lack of evidence for an important role of cutinolytic enzymes also has been reported for fungal pathogens of several plant species: Colletotrichum lagenarium in cucumber by Bonnen and Hammerschmidt (1989), Pyricularia oryzae (Magnaporthe grisea) in rice by Howard et al (1991) and Sweigard et al (1992), and N. haematococca in pea by Stahl and Schäfer (1992).
To check whether mechanical forces are involved in leaf penetration, measurements of the turgor pressure (TP) of fungal penetration structures are critical. Such measurements have been made by (i) inducing collapse of cells by solutions of various osmolytes (Howard et al 1991), (ii) determining the melting point of ice crystals in individual cells (Money and Howard 1996) and (iii) using an optical technique to measure the indentation of a waveguide (Bechinger et al 1999). Few attempts have been made to measure the turgor of these fungal cells on plants.
Some fungi produce unicellular infection structures, called appresoria, which adhere tightly to the host surface and produce slender infection pegs that pierce the underlying cell wall. The cell walls of appresoria contain a dense layer of melanin, the presence of which correlates with a build-up of high TP essential in penetration (Kubo et al 1982, Kubo and Furusawa 1991, Howard and Ferrari 1989, Howard and Valent 1996). Indirect measurement of TP obtained through osmotically induced collapse of Magnaporthe grisea appresoria indicated that the infection apparatus of this fungus can generate TP in excess of 8.0 MPa (40 times that of a typical automobile tire) (Howard et al 1991, Talbot 1999, Money 1999, Tucker and Talbot 2001).
Colletotrichum kahawae Waller & Bridge is the causal agent of coffee berry disease (CBD) inducing 70–80% losses if no control measures are applied. This fungus, like many other Colletotrichum species, produces appresoria and penetrates the cuticle usually after 19 h of conidia germination (Garcia 1999). The main objectives in our investigation were to assess TP of C. kahawae appresoria in vitro and in planta, as well as the OP of coffee leaves and fruit. We also tried to elucidate whether TP, fungal cutinase, or both pressure and enzymes are involved on cuticle penetration.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant tissues. – Leaves (full size but still tender) of Coffea arabica var. Caturra as well as green (4–5 mo old) and ripe fruit (8–9 mo old) were used for the determination of OP of the plant tissues. Inoculated green and ripe fruit of the same coffee variety were used for the in planta estimation of TP of the fungal appresoria.
Turgor pressure and osmotic pressure evaluations. – PEGs with molecular weight of 600 (Fluka) and 8000 (Sigma) were chosen to obtain the desired OP solutions. The determination of OP was made with a thermocouple psychrometer SC-10A from Decagon Devices, Washington City (USA).
The cytological counting of 50% cytorrhyzed cells as induced by PEG solutions of known OP was used as indication of conidial and appresorial TP. The observation of cytorrhysis (i.e. the collapse of the cell wall) was used (Davis et al 2000, Money et al 1998).
The OP of leaves and green or ripe berries was determined in 0.5 mL sap obtained from one fully turgid leaf or from one fruit pericarp smashed with a hand mortar inside a wet chamber (to avoid evaporation).
Measurement of appresorial turgor pressure and survival in vitro. – Conidial suspensions of 30 µL were placed on glass slides and allowed to germinate at 22 C for 19, 24 or 47 h. The water was removed with Whatman No.1 filter paper and immediately replaced by drops of PEG-8000 solutions of various OPs (0.5–4.4 MPa). After 10 min incubation, the cytorrhyzed appresoria were counted under the microscope and expressed as percentage of the total. Individual experiments were repeated at least three times, and counts of the appresoria were made on a minimum of six microscope fields of 100 each per experiment.
To test the viability of appresoria obtained after conidial germination on sterile cover slips during 22–24 h incubation, appresoria were subjected to PEG solutions with OP ranging from 7.3 to 46.5 MPa for 20 min. After aseptic removal of the PEG, the cover slips were placed upside down in contact with PDA (Holmstrom-Ruddick and Mortensen 1995).
To ensure that surviving ungerminated conidia or mycelia would not reflect appresoria survival, conidial suspensions were prepared in PEG-8000 solutions with OP of 0–3.3 MPa and incubated on glass slides at 22 C for 24 h and the percentage of germination was counted. For further confirmation of the survival ability of conidia and mycelia, they were subjected to PEG-600 solutions with OP of 3.3–46.5 MPa for 20 min, washed three times with sterilized distilled water by centrifugation (10 000 g, 10 min) and plated on PDA at 22 C.
Conidial, mycelial and appresorial survival was evaluated by measuring the growth at 8 d after inoculation at 22 C and compared with the controls.
Assessement of turgor pressure in planta as measured by symptom development and appresorial penetrations. – Five µL of conidial suspensions were deposited carefully on the abaxial side of detached leaves and on the fruit surface premarked with an ink circle. The inoculated leaves or fruit were put in small trays at 22 C, lined on the bottom with a wet sponge and tightly covered with plastic bags. The plant materials in some experiments were placed in contact with the water-saturated sponge, whereas in others they were separated from the sponge by four layers of a plastic net (partially hydrated). At 16–19 h incubation at 22 C, water droplets were removed carefully from plant tissues with a piece of filter paper and 15 µL of PEG solutions of various OPs were placed immediately on the marked circle. Controls were identical, except for the use of distilled water instead of PEG. The expression of symptoms was evaluated after 2 wk incubation at 22 C. Three leaves (each leaf with 10 inoculated sites) and 12 fruit were replicated three times for each OP, and individual experiments were repeated 4–5 times.
To evaluate the PEG influence on fungus appresorial penetration, cross sections of preinoculated fruit fragments (0.3 x 0.3 cm), subjected to different OPs 16 h after inoculation, were made with a freezing microtome (Leica, CM 1850, Germany) at 48, 72 and 96 h after inoculation. Sections were stained with cotton blue plus lactophenol and mounted on glass slides for observation. The percentage of appresorial penetrations was counted in a minimum of 600 appresoria, and the hyphal lengths were measured in 60 infected sites per experiment. All observations were made with a Leitz Dialux 20 microscope with the aid of a micrometer eyepiece.
Turgor pressure of nonmelanized appresoria and symptoms expression on leaves and on green and ripe berries. –
The fungicide tricyclazole (5-methyl-1, 2, 4-triazole (3,4-b) benzothiazole) acts as an antipenetrant, blocking melanin biosynthesis in appresoria and reducing their TP (Kubo et al 1982, Kubo and Furusawa 1991, Woloshuk et al 1983). Conidial suspensions were prepared in 10, 50, 100, 150 and 200 µg/mL of tricyclazole (75% w/w, from Dow AgroSciences Ltd, Spain), and the TP of the unmelanized appresoria were checked on glass slides by the method described above. Symptom expression on leaves and on green and ripe berries inoculated with tricyclazole-treated conidia was evaluated by counting the percentage of lesions with symptoms.
Cutinase production and assays. –
Cutinolytic enzyme production in vitro followed the method described by Dickman et al (1982) by growing the fungus in Czapek-Dox mineral medium at pH 7.5 using apple cutin as sole carbon source. Erlenmeyer flasks (500 mL) containing 100 ml medium and 0.5 g of 60 mesh cutin were inoculated with 5 mL of 2 x 106 conidia/mL. The flasks were incubated in the dark at 22 C without shaking. Sucrose was used instead of cutin in controls. After 25 d of growth, culture filtrates were harvested by filtration through two layers of muslin to remove mycelium and remaining cutin, centrifuged at 20 000 g for 20 min to remove conidia; supernatant was used for protein and cutinase activity determinations (Chen 2002). Individual experiments were repeated twice.
The enzymatic activity of extracellular fluid during conidial germination in vitro and in planta also was determined. To obtain the extracellular fluid in vitro, 1.5 mL of a conidial suspension (washed free of mucilage) was germinated 3, 6 and 24 h at 22 C on Petri dishes, recovered with a pipette and centrifuged at 11 000 g for 15 min at 4 C. The supernatant then was assayed for enzyme activity. The procedure to obtain extracellular fluid of conidial germination in planta was similar but started with 15 µL of spore suspensions, which were deposited carefully on each of 180–240 green and ripe berries at 22 C. After 3, 6 and 24 h of incubation, the droplets on all fruit were recovered with a pipette and centrifuged at 11 000 g for 5 min at 4 C.
Cutinase activity was measured with p-nitrophenyl butyrate (PNB) as a substrate, and the release of p-nitrophenol was determined by measurement of the absorbance at 405 nm, according to the published methods (Bostok et al 1999, Kolattukudy et al 1981). It had been confirmed that PNB esterase activity provides an accurate and simple measurement of cutinase activity that correlates with hydrolysis of 3H-cutin (Bonnen and Hammerschmidt 1989, Bostok et al 1999).
Effect of diisopropyl fluorophosphate on conidial germination, appressorium formation, appresorial penetration, enzyme activity and symptom development. –
The cutinase inhibitor diisopropyl fluorophosphate (DFP, Aldrich D12 600-4) is a potent serine inhibitor of fungal cutinases (Dickman et al 1982). The influence of DFP on fungal morphogenesis was evaluated by measuring the percentage of conidial germination and appressorium formation on glass slides or on berries. Conidial suspensions were prepared in 10, 100 and 1000 µM solutions of DFP and incubated at room temperature 15 min. Fifteen µL droplets of these mixtures were applied to slides or to berries. The slides or berries were incubated in closed moist chambers 24 h at 22 C. The slides then were studied microscopically. The berries were painted with nail polish on the inoculated sites and left to dry. The film of polish then was removed and stained with lacto phenol/cotton blue for microscopic observation.
To assess the influence of DFP on fungus penetration, cross sections were made on the green berries at 36 and 48 h after inoculation with conidial suspensions containing 1000 µM of DFP, following the method described above. To determine the effect of DFP on cutinase activity, aliquots from both culture filtrate and extracellular fluid surrounding germinated conidia on green berries were mixed with the same volume of 50, 500, 1000 and 2000 µM DFP and incubated at room temperature 15 min, then submitted to spectrophotometry using PNB as substrate. Controls used distilled water instead of DFP.
The influence of DFP on disease development was assessed in fruit of the susceptible coffee variety Caturra. Fifteen µL of conidial suspensions in different concentrations of DFP (1, 10, 100, 1000 and 10 000 µM) were inoculated on green and ripe berries. After 7–10 d in a 22 C growth chamber, the percentage of symptoms was evaluated. Three replicates of 36 berries were used for each DFP concentration.
Statistical analysis. –
Data concerning TP of appresoria and fungal growth were presented as the combined values of at least two experiments. Arcsine-transformed percentages and Tukey’s test for multiple range analysis were used with 99% confidence. For comparison between means using Student’s t-test, statistical significance was indicated as follows: NS = not significant, * = significant (P 
5%), ** = highly significant (P 1%), *** = very highly significant (P 0.1%).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Appresoria subjected to a PEG 600 solution with OP of 7.3 MPa and above collapsed almost instantly. However, the survival capacity of collapsed appresoria was very high based upon their ability to grow in PDA plates after exposure to PEG solutions with OPs as high as 46.5 MPa, although with slower growth (about 50–60% of the control, data not shown).
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Osmotic pressure of coffee tissues. –
Leaves and green berries had a similar OP value (approximately 1.1 MPa), whereas ripe berries had about double this value (TABLE I).
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). Complete inhibition of symptoms was achieved with 46.5 MPa. However, even with this high OP, 11% of the ripe fruit showed lesions.
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).
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), although the general results were the same. In fact, 87% of green berries submitted to an OP of 17.6 MPa in contact with the wet sponge (TABLE II
) showed necrotic symptoms, versus 39% when the berries were separated from the wet sponge (TABLE III). These results indicate that the dilution of PEG by the wet sponge played an important role in the recovery of the appresoria.
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). In the inoculated berries subjected to an OP higher than 10.3 MPa no infection hyphae could be detected at 72 h after inoculation (data not shown).
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). At about 48 h of contact OP fell to 5 and 8% of the initial value (i.e., it approached the OP value of the green fruit—1.0 MPa). When the fruit were separated from the wet sponge, the decline in OP of the PEG was slower because 14–24% of the initial OP values were reached at the same time (48 h). These results indicated that the concentration of PEG rapidly fell (i.e., the initial very high value was not maintained).
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). However, even at the lowest concentration (10 µg/mL) the appresoria were not melanized.
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). Necrotic lesions developed on about 50% of the green berries 7 d after inoculation with conidia that had been incubated in 10 µg/mL tricyclazole. No lesions appeared when the green berries were inoculated with conidial suspensions in tricyclazole concentration in excess of 100 µg/mL. Delay in the appearance of normal lesions might be due to the lower number of penetrating hyphae resulting from the small number of appresoria that maintained viability or could recover slowly from the treatment. In ripe berries, no total inhibition was achieved even with the highest concentration of tricyclazole (200 µg/mL) tested (TABLE V). Controls showed normal expanding necrotic lesions 4–5 d after inoculation.
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).
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). This activity was very high in green berries, much lower in ripe ones and very low in vitro.
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Cutinase activities of the culture filtrates and of the recovered extracellular fluids during conidia germination on green berries were inhibited by the cutinase inhibitor DFP (TABLE VII). The inhibition by the lowest concentration assayed (25 µM) was 84% (for the culture filtrate) and 92% (for the extracellular fluid in planta) relative to the control.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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by submitting the appresoria in vitro and in planta to various concentrations of the inert PEG with various OPs. With this technique we concluded that the appressorium TP of the C. kahawae at 19 h after conidia germination was about 2.6 MPa (50% of appresoria collapsed). Maximum appresorial collapse (about 90%) was obtained at an OP of 4.4 MPa. These values suggest a possible role for the TP of C. kahawae appresoria in the mechanical penetration of the coffee leaves and green berries whose tissues have TPs about one-third of those of the fungus. TPs in fungal infection structures, if elevated above those of host cells, might play a significant role during penetration (Aist 1976, Harold 2002, Davis et al 2000, Bastmeyer et al 2002). The obtained value of 4.4 MPa to induce 90% collapsed appresoria in vitro is in the same range of those obtained by Howard et al (1991) whose work demonstrated that 100% of appresoria of Magnaporthe grisea at 18 h of conidial germination collapsed at an extracellular OP of 5.0 MPa. Our value is also similar to estimates for Colletotrichum gramminicola of 5.4 MPa (Bechinger et al 1999) and for Erysiphe graminis f. sp. hordei (2.0–4.0 MPa), although in the latter case the appresoria are hyaline and not melanized (Pryce-Jones et al 1999). It, however, is considerably higher than the maximum TP recorded for rust Uromyces appendiculatus where 0.35 MPa is sufficient to cause deformation of the guard cell lips (Terhune et al 1993, Dean 1997).
Tricyclazole applied at 10 µg/mL in vitro to conidia of C. kahawae inhibited the synthesis of melanin and subsequent formation of melanized appresoria. Hence, TP of these unmelanized appresoria decreased dramatically to about one-quarter of the melanized ones. Only 7% of the leaf spots inoculated with tricyclazole-treated conidia showed symptoms that expressed themselves very slowly. On the inoculated sites with symptoms, pin-point lesions were observed only at 10 d after inoculation. Green coffee berries inoculated with tricyclazole treated conidia (10 µg/mL) showed only 50% with necrotic lesions about 7 d after inoculation, whereas the controls already were destroyed. Therefore these results indicated that the inhibition of melanin synthesis in the appresoria of C. kahawae, reducing their TP to about 0.7 MPa, had a strong negative effect on infection. This is in agreement with the fact that induced melanin-deficient mutants of Colletotrichum spp. and Magnaphorthe grisea, as well as wild-type isolates treated with inhibitors of melanin biosynthesis, are unable to penetrate their host (Chumley and Valent 1990, Kubo and Furusawa 1991, Howard and Valent 1996, Deising et al 2000).
We demonstrated that C. kahawae presents cutinase activity in conidial mucilage and in extracellular fluids of germinated conidia in vitro and in planta. Culture filtrates of the fungus grown in Czapek’s medium displayed cutinase activity only if cutin was used as the sole carbon source (Chen 2002). The serine cutinase inhibitor DFP did not affect either conidial germination or appressorium formation in vitro or in planta. At the lowest concentration tested (25 µM), however, the DFP greatly inhibited the cutinase activity of the culture filtrates and extracellular fluid but had no effect upon infection. The observed inhibition of enzyme activity (measured spectrophotometrically) with continued infection is consistent with the hypothesis that cuticle can be penetrated mechanically. This suggestion also is supported by the weak performance of nonmelanized appresoria that induced only a small percentage of infections with a lengthy delay in the expression of symptoms. TP of C. kahawae appresoria then might play a major role in the coffee cuticle penetration. The present evidence that cell-wall degrading enzymes other than cutinase might have a role in the penetration and virulence cannot be disregarded, as shown by Tonukari et al (2000) for Cochliobolus carbonum on maize. This finding, however, does not exclude the mechanical action of the melanized appresoria because it applies to nonmelanized appresoria (Bastmeyer et al 2002).
Conidia and mycelium of C. kahawae subjected to an OP of 3.3 MPa did not survive. Appresoria of C. kahawae submitted to OPs of 7.3 MPa and higher collapsed immediately. However, the survival capacity of appresoria was very high, as demonstrated by further growth on PDA after exposure to an OP as high as 46.5 MPa.
Pathogenicity tests allowed a complementary insight on appresoria behavior regarding cuticle penetration. Assessment of symptoms on green and ripe coffee berries, inoculated with C. kahawae submitted to different OPs, incubated in contact with a wet sponge, indicated that the fungus still produced 7% infected green berries with necrotic lesions at an OP as high as 28.5 MPa. Complete inhibition of symptoms was achieved by exposure to an OP of 46.5 MPa. However, even with this high OP, 11% of the ripe fruit still showed necrotic lesions, although the development was much slower than controls. The manifestation of symptoms at such high OP raised obvious doubts about the validity of our results. We decided to check the OP variation of the PEG applied to the green fruit, either leaving them in contact with the wet sponge or separating them from the wet sponge by four layers of an alveolated plastic net. It was observed in the former experiment that OP decreased to about half of the initial value after 2 h of PEG solution in contact with the coffee berries. At 48 h of contact, the OP decreased to the turgor of the green fruit. For the fruit not in contact with the wet sponge, the OP of PEG also decreased to a large extent, although slowly.
The high resistance of melanized appresoria to external OP, and their functional recovery, combined with the rapid dilution of PEG fed by the moisture of the fruit might explain the development of disease lesions even at very high OPs. Higher values of infections in ripe fruit as compared with green ones subjected to the same high value of OP very likely are related to the highest available content of water and solutes of the ripe fruit, therefore leading to rapid and higher dilution of PEG.
Our work provides plausible data to support the TP role of C. kahawae appresoria in coffee cuticle penetration. Because resistance to penetration is one key mechanism of plant defense against pathogens, this mechanism should be explored to help its use in plant breeding for resistance to pathogens.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: cifc{at}clix.pt
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Bastmeyer M, Deising HB, Bechinger C. 2002. Force exertion in fungal infection. Annu Rev Biophys Biomol Struct 31:321–341.[Medline]
Bechinger C, Giebel KF, Schnell M, Leiderer P, Deising HB, Bastmeyer M. 1999. Optical measurements of invasive forces exerted by appresoria of a plant pathogenic fungus. Science 285:1896–1899.
Bonnen AM, Hammerschmidt R. 1989. Role of cutinolytic enzymes in infection of cucumber by Colletotrichum lagenarium. Physiol Molec Plant Pathol 35:475–481.
Bostock RM, Wilcox SM, Wang G, Adaskaveg JE. 1999. Suppression of Monilinia fructicola cutinase production by peach fruit surface phenolic acids. Physiol Molec Plant Pathol 54:37–50.
Chen ZJ. 2002. Morphocultural and pathogenic comparisons between Colletotrichum kahawae and C. gloeosporioides isolated from coffee berries [Doctoral dissertation]. Lisbon, Portugal: Univ Techn de Lisboa. 163 p.
Chumley FG, Valent B. 1990. Genetic analysis of melanin-deficient, non-pathogenic mutants of Magnaporthe grisea. Molec Plant-Microb Interact 3:135–143.
Davis DJ, Burlak C, Money NP. 2000. Biochemical and biomechanical aspect of appresorial development in Magnaporthe grisea. In: Tharreau D, eds. Advances in rice blast research. The Netherlands: Kluwer Academic Publisher. p 248–256.
Dean RA. 1997. Signal pathways and appressorium morphogenesis. Annu Rev Phytopathol 35:211–234.[Medline]
Deising HB, Werner S, Wernitz M. 2000. The role of fungal appresoria in plant infection. Microbe Infect 2:1631–1641.[Medline]
Dickman MB, Patil SS. 1986. Cutinase deficient mutants of Colletotrichum gloeosporioides are non-pathogenic to papaya fruit. Physiol Molec Plant Pathol 28:235–242.
———, Patil SS, Kolattukudy PE. 1982. Purification, characterization and role in infection of an extracellular cutinolytic enzyme from Colletotrichum gloeosporioides Penz. on Carica papaya L. Physiol Molec Plant Pathol 20:333–347.
———, ———, ——— 1989. Insertion of cutinase gene into a wound pathogen enables it to infect intact host. Nature 342:446–448.
Garcia IVAN. 1999. Histologia e ultra-estrutura do processo de infecção de Colletotrichum kahawae e C. Gloeosporioides em Coffea arabica. Mestrado em Protecção Intergrada [Doctoral dissertation]. Lisbon, Portugal: Instit Superior de Agro, Univ Techn de Lisboa. 96 p.
Harold FM. 2002. Force and compliance: rethinking morphogenesis in walled cells. Fungal Genet Biol 37:271–282.[Medline]
Holmstrom-Ruddick B, Mortensen K. 1995. In vitro formation and survival of appresoria of a mycoherbicide agent, Colletotrichum gloeosporioides f. sp. malvae and a benomyl-resistant strain, HP4.5RR. Mycol Res 99:1103–1107.
Howard RJ, Ferrari MA. 1989. Role of melanin in appressorium function. Exp Mycol 13:403–418.
———, ———, Roach DH, Money NP. 1991. Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci USA 88:11281–11284.
———, Valent B. 1996. Breaking and entering: host penetration by fungal rice blast pathogen, Magnaporthe grisea. Annu Rev Microbiol 50:403–512.
Kolattukudy PE, Purdy RE, Maiti IB. 1981. Cutinase from fungi and pollen. Methods Enzymol 71:652–664.
Kubo Y, Furusawa I. 1991. Melanin biosynthesis. Prerequisite for successful invasion of the host by appresoria of Colletotrichum and Pyricularia. In: Cole GT, Hoch HC, eds. The fungal spore and disease initiation in plants and animals. New York: Plenum. p 205–218.
———, Suzuki K, Furusawa I, Yamamoto M. 1982. Effect of tricyclazole on appresorial pigmentation and penetration from appresoria of Colletotrichum lagenarium. Phytopathol 72:1198–1200.
Li D, Ashby AM, Johnstone K. 2003. Molecular evidence that the extracellular cutinase Pbc1 is required for pathogenicity of Pyrenopeziza brassicae on oilseed rape. Molec Plant Microbe Interact 16:545–552.
Martin JT. 1964. Role of cuticle in the defense against plant disease. Annu Rev Phytopathol 2:81–100.
Money NP. 1999. Fungus punches its way in. Nature 401: 332–333.[Medline]
———, Caesar-TonThat TC, Freserick B, Henson JM. 1998. Melanin systhesis is associated with changes in hyphopodial turgor, permeability, and wall rigidity in Gaeumannomyces graminis var. graminis. Fungal Genet Biol 24:240–251.[Medline]
———, Howard RJ. 1996. Confirmation of a link between fungal pigmentation, turgor pressure, and pathogenicity using a new method of turgor measurement. Fungal Genet Biol 20:217–227.
Pascholati SF, Deising H, Leite B, Anderson D, Nicholson RL. 1993. Cutinase and non-specific esterase activities in the conidial mucilage of Colletotrichum graminicola. Physiol Molec Plant Pathol 42:37–51.
Pryce-Jones E, Crver T, Gurr SJ. 1999. The roles of cellulase enzymes and mechanical force in host penetration by Erysiphe graminis f. sp. Hordei. Physiol Molec Plant Pathol 55:175–182.
Stahl DJ, Schäfer W. 1992. Cutinase is not required for fungal pathogenicity on pea. Plant Cell 4:621–629.
Sweigard JA, Chumley FG, Valent B. 1992. Disruption of a Magnaporthe grisea cutinase gene. Molec Gener Genet 232:183–190.
Talbot NJ. 1999. Forcible entry. Science 285:1860–1861.
Terhune BT, Bojko RJ, Hoch HC. 1993. Deformation of stomatal guard cell lips and microfabricated artificial topographies during appressorium formation by Uromyces. Exp Mycol 17:70–78.
Tonukari NJ, Scott-Craig JS, Walton JD. 2000. The Cochlibolus carbonum SNF1 gene is required for cell wall-degrading enzyme expression and virulence on Maize. Plant Cell 12:237–248.
Tucker S, Talbot NJ. 2001. Surface attachment and pre-penetration stage development by plant pathogenic fungi. Annu Rev Phytopathol 39:385–417.[Medline]
Woloshuk CP, Sisler HD, Vigil EL. 1983. Action of the antipenetrant, tricyclazole, on appresoria of Pyricularia oryzae. Physiol Plant Pathol 22:245–259.
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OD/min/mL) of the extracellular fluids after different conidia germination periods