versión impresa ISSN 1517-8382
Braz. J. Microbiol. v.37 n.2 São Paulo abr./jun. 2006
ENVIRONMENTAL AND SOIL MICROBIOLOGY
Purificação parcial de elicitores a partir de basidiocarpos de Lentinula edodes protegendo plântulas de pepino contra Colletotrichum lagenarium
Robson M. Di PieroI; Nelson A. WulffII; Sérgio F. PascholatiII,*
IDepartamento de Fitotecnia, Centro de Ciências Agrárias, Universidade Federal de Santa Catarina, Florianópolis, SC, Brasil
IIDepartamento de Entomologia, Fitopatologia e Zoologia Agrícola, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Piracicaba, SP, Brasil
The shiitake mushroom (Lentinula edodes) has been used in research involving the prevention and the control of human and plant diseases. In cucumber plants, treated with aqueous extracts from mushroom basidiocarps, there was a reduction in anthracnose severity caused by Colletotrichum lagenarium, and an increase in peroxidase activity in the leaves. With the aim of obtaining molecules of agronomic interest, the crude aqueous extract from L. edodes basidiocarp was fractioned with ammonium sulfate. The fraction corresponding to 40-80% of saturation (p40-80), the most effective in reducing anthracnose on cucumber cotyledons, was submitted to anion exchange chromatography (AEC). After AEC, six protein peaks were obtained and the peak V, containing 34% of the proteins present in p40-80, induced peroxidase increase in the cucumber cotyledons besides reducing anthracnose severity. Separation of peak V proteins by SDS-gel electrophoresis revealed the presence of more than one band in the gel. Thus, a partial purification of elicitors present in the L. edodes basidiocarp was achieved.
Key words: shiitake, anthracnose, peroxidase
O cogumelo shiitake (Lentinula edodes) vem sendo utilizado com objetivos de prevenir e controlar doenças em humanos e em vegetais. O tratamento com o extrato aquoso de basidiocarpos do cogumelo propiciou uma redução significativa na severidade da antracnose em plantas de pepino, provocada por Colletotrichum lagenarium, e um aumento na atividade de peroxidases nos tecidos foliares. Desse modo, com o objetivo de obter moléculas de interesse agronômico destinadas à proteção de plantas, o extrato aquoso bruto de basidiocarpos de L. edodes foi fracionado inicialmente com sulfato de amônio. A fração correspondente a 40-80% de saturação (p40-80) foi a mais efetiva em reduzir a antracnose em cotilédones de pepino, sendo então submetida à cromatografia de troca aniônica (CTA). Após a CTA, seis picos protéicos foram obtidos, e o pico V, contendo em média 34% das proteínas presentes em p40-80, provocou o acúmulo de peroxidases nos cotilédones de pepino, bem como reduziu a antracnose. A separação eletroforética das proteínas do pico V revelou a presença de mais de uma banda no gel de poliacrilamida. Portanto, estes procedimentos resultaram na purificação parcial de elicitores presentes no basidiocarpo de L. edodes.
Palavras-chave: shiitake, antracnose, peroxidases
Elicitors are molecules that stimulate any of a number of defense responses in plants, such as synthesis of phytoalexins and pathogenesis-related proteins (PR-proteins). Such responses occurs after the binding of elicitor molecules to receptors normally located on the plant cell surface, promoting a signal transduction pathway that will lead to the activation of one or more defense mechanisms. The first characterized elicitors were oligosaccharide fragments from fungal cell walls, including hepta-b-glucoside, oligochitin and oligochitosan (10).
Glucans, with the ability to induce the phytoalexin accumulation (glyceollin) in soybean tissues, were detected in the culture filtrates and on the mycelial walls of Phytophthora sojae, a phytopathogenic oomycete (2). Another glyceollin elicitor was isolated from autoclaved urediniospores of Hemileia vastatrix. This elicitor was water-soluble, heat-stable, resistant to the pronase treatment and sensitive to sodium metaperiodate. Besides, it had no affinity neither for Concanavalin-A lectin nor for the anion exchanger diethylaminoethyl-celulose (DEAE-cellulose), suggesting to be a polysaccharide (9).
Microbial elicitors can also be glycoproteins, such as the ones synthesized by the yeast Saccharomyces cerevisae, that induce phenylalanine ammonia-lyase synthesis in tomato cell suspension (7) or phytoalexin accumulation in sorghum mesocotyls (25).
Recently, biologically active peptides have been purified and characterized. A protein-elicitor isolated from P. sojae induced phytoalexin synthesis in parsley plants (18). Several species of Phytophthora also produce other small proteins (around 10 kDa), known as elicitins. When applied in tobacco plants, elicitins increased PR-protein activity on the leaves and induced resistance against the plant pathogen P. parasitic var. nicotianae (19).
The mushroom Lentinula edodes, known as shiitake, produces polysaccharides and glycoproteins that activate the animal immune system and some of them show antibiotic activity too (16,21,22). From shiitake basidiocarps, Kobayashi et al. (13) purified a protein that inhibited the infectivity of the Tobacco mosaic virus (TMV) on tobacco plants. That protein did not cause TMV aggregation and it did not exhibit a RNA hydrolyzing activity (11). The action of this L. edodes protein could have been on the plant by activating defense mechanisms, however, there are no reports about that.
In cucumber plants with three true leaves kept under greenhouse conditions, the basidiocarp aqueous extract from several isolates of L. edodes significantly reduced the disease caused by Colletotrichum lagenarium, and increased the peroxidase activity on the leaves (5). Thus, the aim of the present work was to obtain fractions from L. edodes fruiting bodies, showing biological activity towards disease reduction and peroxidase increase, by using the interaction cucumber-C. lagenarium.
MATERIALS AND METHODS
The crude aqueous extract from L. edodes
L. edodes basidiocarps (isolate LE 96/22) were obtained from the mushroom grown in Eucaliptus grandis and E. saligna trunks. To obtain the aqueous extract, dehydrated powder from basidiocarps was suspended into distilled water (14 mL.g-1) and, after 24 h incubation at 4ºC, the suspension was filtered through a common filter paper (8g.cm-2) and centrifuged at 20,000 g for 25 min. The supernatant obtained, after this procedure, was considered as the crude aqueous extract.
Fractionation of crude aqueous extract from L. edodes basidiocarps
The proteins present in 100 mL of crude aqueous extract were precipitated with ammonium sulfate in saturations from 0 to 20%, 20 to 40%, 40 to 60%, 60 to 80% and 80 to 100%. The precipitates were dissolved in 2 mL distilled water. After dialysis against distilled water (4ºC, 48 h, three changes), accomplished in membrane with a cut off of 6 - 8 kDa, the volume of each precipitate was adjusted to 4 mL with distilled water. The protein content in the samples was quantified according to the Bradford procedure (3). The samples were used in a bioassay to check the protection of cucumber seedlings against C. lagenarium.
Bioassay with cucumber seedlings
Seeds of cucumber (Cucumis sativus cv. Caipira Verde) were placed in trays containing Plantimax® substrate and kept inside a greenhouse. When the cotyledons were 3 cm in length, the seedlings were removed from the substrate, washed, drought in the air and immersed into the fractions during 5 s, in a way where just the cotyledons were reached (14). The treated seedlings were placed inside glass tubes (9.0 cm height x 1.4 cm diameter) containing distilled water and kept in a growth chamber (25ºC; 12 h light period) until the end of the experiment. Three days later, the cotyledons were inoculated with C. lagenarium (1 x 105 spores.mL-1), by spraying. Each treatment had four replicates and a replicate was represented by four seedlings. The bioassay was evaluated 5 days after the inoculation, by estimating the disease severity visually, and expressed as percentage.
After the evaluation, the protein fractionation was made from a 275 mL sample of crude aqueous extract of L. edodes (isolate LE 96/22), by using ammonium sulfate in saturations from 0 to 40%, 40 to 80% and 80 to 100%. Each precipitate was dissolved in 6 mL of distilled water, dialyzed, and the final volume adjusted to 14 mL with distilled water. The suspended materials were then evaluated for cucumber protection based upon the seedling bioassay.
Chromatography of the precipitate corresponding to 40-80% of saturation (p40-80)
A sample of the precipitate p40-80, containing 18 mg protein, was submitted to cation exchange chromatography (CEC) in a column (2.5 x 10 cm) filled with carboxy-methyl cellulose (CM-Cellulose) and equilibrated with 10 mM acetate buffer (pH 5.0). The unbound material was eluted in equilibration buffer at a flow rate of 3 mL.min-1 and the bound material was displaced by increasing NaCl concentration (0 to 1.0 M) in the same buffer.
Another sample of p40-80, containing 24 mg protein, was submitted to anion exchange chromatography (AEC) in a column (2.5 x 15 cm) filled with DEAE-Cellulose and equilibrated with 25 mM sodium phosphate buffer (pH 6.0). The unbound material was eluted in equilibration buffer at a flow rate of 2.5 mL.min-1 and the bound material was displaced by NaCl in the same buffer, by increasing the salt concentration (0, 0.2, 0.4, 0.6, 0.8 and 1.0 M), through a step-wise procedure. The collected fractions were pooled according to their profiles at 280 nm, dialyzed three times against water (12 h intervals with 7 L of distilled water at 4ºC), concentrated with polyethylene-glicol 20,000 to a final volume of 5 mL and tested in the bioassay with cucumber seedlings.
Peroxidase activity in cucumber cotyledons
Cucumber seedlings, obtained as mentioned above, had their cotyledons immersed into the fractions of interest (peaks obtained from AEC). The seedlings were kept inside glass tubes containing distilled water during 3 days, under growth-chamber conditions (25ºC; 12 h light period), when the cotyledons were harvested. Four replicates were undertaken by treatment, and a replicate was represented by three cotyledons.
The samples were frozen in liquid nitrogen, homogenized in 4.0 mL of 100 mM phosphate buffer (pH 6.0) (extraction buffer) and centrifuged at 20,000 g by 30 min, at 4ºC. The supernatants were used to evaluate peroxidase activity as well as the total protein content. A 0.1 mL sample of the supernatant was added to 3 mL of the assay mixture, which consisted of extraction buffer, 0.1 M H2O2 and 0.25% (v/v) guaiacol. The reaction was carried out at 30ºC during 4 min, and the absorbance at 470 nm was recorded every 20 s, starting 1 min after the addition of the supernatant to the mixture. Protein content in the samples was evaluated by the Bradford procedure (3). Enzyme activity was expressed in units of absorbance at 470 nm / mg protein / min (Abs/mg prot/min).
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the biologically active fractions
Samples of the precipitate p40-80 and of the peak V, one of the peaks obtained after the p40-80 fractionation by AEC, were submitted to electrophoresis in polyacrylamide gel (8 x 8 cm) under dissociating conditions, using the procedure described by Laemmli (15). The running gel was prepared with 10% acrylamide, while the stacking gel with 4%. The electrophoresis was performed at 4ºC, under 100 V, during 2 h. Bands were visualized by gel staining with Coomassie Brilliant Blue R250 (Sigma). Apparent molecular weight was determined using a kit from Amersham Pharmacia Biotech (range between 29.0 and 116.0 kDa).
RESULTS AND DISCUSSION
L. edodes exhibits a protecting effect on cucumber plants against C. lagenarium under greenhouse conditions, depending upon the concentration of the basidiocarp aqueous extract used (5). The crude extract at 20% (v/v) also increased peroxidase activity systemically in cucumber leaves. Peroxidases are considered as resistance biochemical markers for this plant-pathogen interaction (4).
In the present investigation, a partial purification of peroxidase elicitors in cucumber seedlings from L. edodes basidiocarps was obtained, by using fractioned precipitation of molecules with ammonium sulfate and anion exchange chromatography.
In the first fractionation of the proteins present in the crude aqueous extract of L. edodes, utilizing ammonium sulfate, all the precipitates obtained reduced anthracnose severity in cucumber seedlings. However, the ones ranging from 20 to 80% saturation were the most effective (data not shown). Considering the relative efficiency and the protein concentration of the precipitates, a new separation of the proteins of L. edodes was made with ammonium sulfate, now in saturations from 0 to 40%, 40 to 80% and 80 to 100%. The treatment of cucumber seedlings with those materials revealed that the best protecting effect was obtained with the precipitate p40-80 (Table 1), which was then submitted to chromatography.
During the CEC, there was no binding of the molecules present in the precipitate p40-80 to the CM-Cellulose resin (data not shown). On the other hand, six peaks were obtained when a sample containing 24 mg of protein from p40-80 was submitted to AEC, as it is illustrated by Fig. 1. This indicates that according to our experimental conditions, active molecules present on p40-80 fraction have a negative net charge, showing affinity only to anionic resin. By incrementing a cationic chromatographic step did not result in further purification and higher specific activity.
In the first bioassay, when the material present in the six peaks was applied onto cucumber seedlings, only peak V promoted a significant control of anthracnose (Table 2). In a second one, the peaks were obtained from a new lot of L. edodes fruiting bodies, and each peak was used with the same quantities of proteins present in the first bioassay. In this experiment, the precipitate p40-80 was also evaluated to compare its activity directly to the activity of the peaks. None of the peaks, including peak V, were effective in reducing the anthracnose severity, while the precipitate p40-80 reduced disease in approximately 50% (data not shown).
In terms of elicitor activity, the peaks III and V as well as the precipitate p40-80 (Table 3) increased peroxidase activity in cucumber cotyledons in relation to the ones treated with distilled water (Table 3). The experiment with peroxidases was accomplished twice and they showed the same general results.
The ammonium sulfate precipitate p40-80 was fractionated into 6 bands after SDS-PAGE. In turn, Peak V, an inducer of peroxidase activity (Table 3), derived from p40-80 (Fig. 1), contained only two protein bands from fraction p40-80 (Fig. 2). Since one of these proteins (29 kDa) was not seen in peak V, derived from a second purification experiment from a different lot of basidiocarps (lot 2), it can be considered that the 29 kDa protein is the candidate to induce resistance in cucumber to anthracnose by the activation of peroxidases.
Protein or glycoprotein elicitors have been isolated from microorganisms, and some of those are able to induce resistance in plants against pathogens. It is the case of the glycoprotein purified from the mycelial walls of Pyricularia oryzae, causal agent of the rice blast (20). The previous treatment of rice leaves with the elicitor increased peroxidase, cinamil-alcohol desidrogenase and lipoxigenase activities and reduced disease symptoms in the treated leaves.
The commercial use of a protein-elicitor started with the discovery of harpin, a 44 kDa protein rich in glicin and heat-stable. The harpin is synthesized by Erwinia amylovora, Pseudomonas syringae and others plant-pathogenic bacteria, and represents a pathogenicity factor for those microorganisms during the interaction with their hosts. On other hand, harpin causes a hypersensitive reaction (HR) when it is infiltrated into the intercellular space of non-host species (1,24). The foliar application of harpin does not cause a visible HR, but it activates plant defense responses, inducing systemic resistance in Arabidopsis thaliana against P. syringae pv. tomato (6), in cucumber plants against Gliocladium sp., in tomato plants against Ralstonia solanacearum (23), and in bell pepper plants against Beet curly top virus (12). These results stimulated the company EDEN Bioscience to develop a commercial product named Messenger®, a resistance inducer having 3% of harpin as the active ingredient. In the case of elicitors of different natures, some commercial products were already developed like Agromos®, based on mannan-oligosaccharides isolated from the S. cerevisiae cell wall, and Elexa®, where chitosan is the active ingredient.
Regarding L. edodes, peak V contains 34% of the proteins present in the precipitate p40-80 (Table 2), and approximately 15% of the proteins present in the crude aqueous extract from basidiocarps. Peroxidase activity in cucumber was similar in seedlings treated with peak V or precipitate p40-80 (Table 3), showing a progress in the purification of the elicitors present in L. edodes basidiocarps. However, although peak III also induced the same level of peroxidases as peak V (Table 3), it did not reduce anthracnose severity in the same way. This indicates that peroxidases are components of the defense responses of plants, and can even be considered as a marker for resistance (17), but do no act alone. There should be other defenses being activated by peak V, since defense is a multifunctional component in plants (7).
New studies involving the application of the fractions of interest in plants could indicate the viability of obtaining commercial products from the shiitake mushroom, in an attempt to control plant diseases in a healthy and ecological-safe way.
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Submitted: July 01, 2004; Returned to authors for corrections: March 22, 2005; Approved: March 15, 2006
* Corresponding Author. Mailing address: Departamento de Entomologia, Fitopatologia e Zoologia Agrícola, ESALQ, USP. 13418-900, Piracicaba, SP, Brasil. Tel.: (+5519) 3429-4124, Fax: (+5519) 3434-4839. E-mail: email@example.com