Cell-wall-degrading enzymes produced in vitro and in vivo by Rhizoctonia solani, the causative fungus of peanut sheath blight

Fungal isolate

R. solani isolate CLL-01 used in this study was collected from naturally infected peanut tissue in a field in Wangjia Town, Liaoyang City, Liaoning Province, China. The isolate was cultured on potato dextrose agar (PDA) at 25 °C for 36 h and identified as AG1-IA by molecular evidence (NCBI Accession number: MH395844, with 99% identity to R. solani AG1-IA isolated from Salvia miltiorrhiza; also available in Supplemental Dataset S1) (Yi et al., 2016).

Enzyme production and growth in liquid culture

A modified Richard’s liquid medium (Zhang, Bruton & Biles, 2014) containing KNO₃ (10 g l⁻¹), KH₂PO₄ (5 g l⁻¹), MgSO₄·7H₂O (2.5 g l⁻¹) and FeCl₃·6H₂O (0.02 g l⁻¹) was used. Carbon sources used in the liquid medium were: 1% (w/v) orange pectin, 0.5% (w/v) orange pectin + 0.5% (w/v) carboxymethyl cellulose (CMC), or 1% (w/v) CMC. The initial pH of autoclaved liquid medium containing pectin, pectin plus CMC, or CMC was measured before inoculation. Liquid medium (100 ml) was dispensed into 250 ml flasks, which were inoculated with two 0.5 cm. PDA plugs obtained from the periphery of 36-h-old cultures of the isolate growing at 25 °C. The flasks were placed on an orbital shaker (100 rpm) at 25 °C. Every second day, from day 2 until day 16, the contents were filtered through Miracloth™ (Calbiochem, EMD Millipore Corp., Billerica, MA, USA). The filtrates were centrifuged at 16,000×g for 20 min and stored at −20 °C until use for detection of enzyme activity. The pH of the filtrates was determined using a pH electrode (PB-10; Sartorius, Gottingen, Germany) soon after harvest. Fungal mycelia were removed using the Miracloth and then both the mycelia and Miracloth were dried to constant weight at 80 °C and weighed. The Miracloth was pre-dried and weighed for fungal mass calculation.

Plant material and inoculation

Two peanut cultivars, Baisha (Bs) and Silihong (Slh), were used for the following examination. The peanut seeds were surface-sterilized with 70% ethanol for 10 s followed by 1.4% NaOCl for 20 min, then extensively washed with sterile water. The seeds were germinated in sterile conditions at 25 °C. Three germinant peanut seeds were placed in each pot (10 cm diameter, seven cm depth) containing a sterile potting mix of soil and peat moss (4:1). The peanut plants were grown in a greenhouse at 27 ± 1 °C, humidity 60∼99%, day 14∼15 h and night 9∼10 h, at light intensity of 8.6∼61.0 klx.

Inoculum was produced for in vivo experiments in peanut using the following procedures. Isolate CLL-01 was grown on PDA at 25 °C for 36 h. Two 0.5 cm diameter mycelial plugs obtained from the periphery of the culture were added to a 250 ml flask containing 100 g sterile corn niblets which had been boiled for 30 min and dipped in water overnight before autoclaving. The corn kernel inocula was incubated at 25 °C for 5 days. The flasks were shaken once per day to ensure the grains were evenly and fully incubated. Five cultured kernels were placed approximately one cm deep in the soil around the base of each 5-week-old peanut plant. Each pot of three plants was placed in a clear plastic bag as needed to maintain high humidity. The infected stalk and leaf tissues were collected 10 days post-inoculation (dpi) and stored at −20 °C until enzyme extraction. The controls were inoculated with sterile corn kernels. The pH values of exudate of the control and infected peanut samples were examined using precise pH indicator paper.

Extraction of enzymes from infected tissue

The extraction of PG, PMG, Cx and β-glucosidase from decayed or healthy peanut plant tissue was conducted according to procedures described previously (Zhang et al., 1999) with some modifications. Decayed tissue (one g) was placed in five ml of 0.1M Na-acetate buffer (pH 5.0) containing 1M NaCl and one mM EDTA in a precooled mortar, on ice. The mixture was ground with silica sand for 2 min. The slurry was filtered through Miracloth, followed by centrifugation of filtrates at 16,000×g for 20 min, and then the supernatants were collected and stored at −20 °C until further processing.

PG and PMG activity assays

Polygalacturonase activity was assayed by the 3,5-dinitrosalicylic acid (DNS) method, as described by Cao, Jiang & Zhao (2007). The substrate was 1% polygalacturonic acid in 50 mM Na-acetate buffer (pH 5.5). The reaction mixture consisted of 0.5 ml sample, 1.0 ml Na-acetate buffer (50 mM, pH 5.5) and 0.5 ml substrate, and it was incubated for 60 min at 37 °C. The reaction was terminated by adding 1.5 ml DNS and boiling for 5 min, and the absorbance was measured at 540 nm using a Spectrophotometer (Multiskan GO; ThermoFisher Scientific, Boston, USA). Boiled enzyme was used as a control. Galacturonic acid at various concentrations was used to establish a standard curve. Activity of PMG was measured using the same procedure as that used for assaying PG activity, except that the substrate was 1% pectin in 50 mM Na-acetate buffer (pH 5.5).

Cx and β-glucosidase activity assays

Cellulase activity was assayed by the DNS method as described by Cao, Jiang & Zhao (2007). The substrate was 1% CMC in 50 mM citric acid-sodium citrate buffer (pH 5.0). The reaction mixture consisted of 0.5 ml sample and 1.5 ml substrate, and it was incubated for 60 min at 37 °C. The reaction was terminated by adding 1.5 ml DNS and boiling for 5 min, and the absorbance was measured at 540 nm using a Spectrophotometer. Boiled enzyme was used as a control. Glucose at various concentrations was used to establish a standard curve. Activity of β-glucosidase was measured using the same procedure as that used for assaying Cx activity, except that the substrate was 1% salicin in 50 mM citric acid-sodium citrate buffer (pH 4.5).

In this study, one unit of enzyme activity was defined as one μmol of reducing groups released by the enzyme per min at 37 °C. All enzyme activities of culture extracts are expressed in units ml⁻¹, while those in infected tissue extracts are expressed in units mg⁻¹ protein.

Protein assay

Protein concentrations were estimated by the method of Bradford (Cao, Jiang & Zhao, 2007). Bovine serum albumin was used to establish a standard curve.

Preparation of crude enzyme extracts

The culture filtrate from medium containing 0.5% (w/v) orange pectin plus 0.5% (w/v) CMC, collected 10 dpi as described above, was used for the preparation of crude enzymes as this material showed the greatest PG enzyme activity. Ammonium sulfate was added to the culture filtrate to 90% saturation and incubated at 4 °C for 24 h, followed by centrifugation at 16,000×g for 30 min at 4 °C. The precipitate was dissolved in distilled water and extensively dialyzed (MWCO 10,000 Da) over 24 h at 4 °C against distilled water (replaced every 8 h), and then freeze-dried. The obtained crude enzyme extracts were stored at −20 °C until inoculation and use in ultrathin layer polyacrylamide isoelectric focusing (IEF) electrophoresis.

Effects of crude enzyme extracts on peanut plant tissue

We examined the effect of the above crude enzymes extracted from culture filtrate on peanut plant tissue. The crude enzyme preparation from 10 dpi shaken culture with pectin plus CMC as the carbon source was inoculated onto peanut leaf (Bs cultivar). Two five μl drops of crude enzyme were placed on one side of the midrib of leaves and controls including heated crude enzyme extracts and distilled water were placed symmetrically on the other side. Incubation was in moist chambers at 27 °C, while the petiole was moistened with cotton wool and the bottom of the box contained water. Observations were made every 24 h for 7 days. To compare symptoms induced by crude enzyme extract and pathogens, a mycelial plug (five mm in diameter) obtained from 36-h-old cultures of the R. solani isolate CLL-01 growing on PDA at 25 °C was placed on the adaxial surface of the leaf at the main vein center with the mycelial side facing the leaf and incubated in a moist chamber in the same conditions. Sterile PDA plugs were used as controls.

Isoelectric focusing electrophoresis of PG

Extract from 10 dpi peanut plant tissue (two g) was performed in 10 ml 0.1M Na-acetate buffer (pH 5.0) containing 1M NaCl and one mM EDTA, and then dialyzed and freeze-dried in the same manner as extracts from medium described in the section “crude enzyme extraction” but without ammonium sulfate precipitation. The crude enzymes from culture filtrate and infected tissue extract were subjected to thin-layer polyacrylamide gel IEF (Liuyi, DYCP-37B, Beijing, China) and evaluated for PG isoenzymes. The IEF used 0.4 mm thick polyacrylamide gels containing 4% (v/v) ampholytes (Bio-Lyte 3/10, 40%; BIO-RAD, Hercules, CA, USA) covering the pH range 3.5–10.0. The electrode strips were soaked with 1M phosphoric acid (anode) and 1M sodium hydroxide (cathode) and placed on opposing sides of the gel. The pre-run was carried out at 100–150 V for 30 min, followed the application of 20 μl protein samples to application strips placed directly on the gel. Heat-treated crude enzyme solution was used as a negative control. IEF protein standards (Isoelectric Focusing Calibration Kit, Broad pH 3–10; GE Healthcare, Amersham, UK) were diluted according to the manufacturer’s instructions before loading. After electrophoresis at 100–200 V for 1–1.5 h, the application strips were removed. Thereafter, electrophoresis was conducted at constant 580 V for about 3.5 h at the same temperature. Ultrathin overlay gels (0.9 mm) for PG isoenzyme detection were prepared as described by Ried & Collmer (1985) with the following modifications: the 10 g l⁻¹ agarose gel contained one g l⁻¹ polygalacturonic acid and 10 mM EDTA buffered at pH 5.0 with 50 mM Na-acetate. The polyacrylamide IEF gels overlaid with ultrathin agarose gels for PG detection were incubated at 100% humidity and 37 °C for 30–60 min. To visualize activity bands, the agarose overlay was stained for 10 min in 0.5 g l⁻¹ ruthenium red solution. PG appeared as a white band visualized using a light box. The pI values of the PG isoenzymes were estimated from a regression equation of standard proteins vs. the distance migrated.

Statistical evaluation

Statistical evaluation of the results obtained was performed using SPSS 20.0. Correlation analysis was performed between pH and mycelial dry weight, pH and enzyme production, and mycelial dry weight and enzyme production, respectively, for shaken-flask cultures.

Production of cell-wall-degrading enzymes in infected tissue

The pH of the exudate from healthy tissue including stalks and leaves of both Bs and Slh cultivars was 5.4, whereas from rotted tissue this increased to 6.7. The extracts of infected leaf and stalk of the two peanut cultivars showed notable PG activities, while healthy tissue showed only trace activity (Fig. 1A). The PG activities of the extract of infected leaf and stalk of Bs cultivar were, respectively, 56.7- and 51.0-fold those in healthy tissue; for Slh the activities were 6.6- and 13.4-fold those in healthy tissue, respectively. These results indicate that peanut tissue was alkalified in the infection process and high PG activity was induced by fungal infection.

The extracts of infected tissue showed high PMG activity, while healthy tissue showed very low level activity (Fig. 1B). The PMG activities of the extracts of infected leaf and stalk of Bs were 15.0- and 73.9-fold those in healthy tissue, respectively, and for Slh the activities were 24.2- and 70.4-fold those in healthy tissue. These results indicate that high PMG activity was induced by fungal infection.

Only traces of Cx activity were detected in the extract of healthy tissue, and no activity was detectable in extracts of leaf from the Bs cultivar. However, high Cx activity was detected in extracts of infected tissue, including leaf and stalk of both cultivars (Fig. 1C). The Cx activity of extract from infected Bs stalk was 188.6-fold that in healthy tissue, and the activities in infected Slh leaf and stalk were 302.6- and 145.7-fold those in healthy tissue, respectively. These results indicate that relatively high Cx activity was induced by fungal infection.

Very low β-glucosidase activity was detected in the extract of healthy tissue from the two cultivars. The β-glucosidase activity of the extract of infected tissue was higher (Fig. 1D): the β-glucosidase activities of the extract of infected leaf and stalk of Bs were, respectively, 50.7- and 22.9-fold those in healthy tissue, and for Slh the relative increases in activity were 24.6- and 23.2-fold. These results indicate that relatively high β-glucosidase activity was induced by fungal infection.

Production of cell-wall-degrading enzymes in liquid media (in vitro culture)

Polygalacturonase activity was first detected two dpi in the medium containing pectin as the sole carbon source, and four dpi in the medium containing CMC alone or pectin plus CMC as the carbon source(s) (Fig. 2A). In the three types of medium, the PG activity continued to increase to a peak on day 10, and then it tended to decrease until the end of the experiment on day 16. The highest amount of PG was produced in the medium containing pectin plus CMC, a little more than in medium with pectin alone, while the PG activity was low throughout the cultivation period in the CMC medium (Fig. 2A). On day 10 (the peak activity), PG activity in the medium containing pectin and pectin plus CMC was 4.90- and 5.38-fold that in the CMC-containing medium, respectively. The results indicated that PG activity was induced in the three media; the highest amount of PG activity was produced in the medium containing pectin plus CMC, followed by medium with pectin as the sole carbon source, and low activity was produced in CMC-containing medium.

Polymethyl-galacturonase activity was detected two dpi in all three types of medium (Fig. 2B). The activity in the medium containing pectin plus CMC reached its maximum on day 8 and remained high thereafter. On day 8, the activity was 3.5- and 4.5-fold that in the medium containing pectin or CMC as the carbon source, respectively. The activity in medium containing pectin as the sole carbon source peaked on day 12 and was 2.7-fold the activity in the medium containing CMC alone on that day, but was much lower than that in the medium containing pectin plus CMC. The activity in the medium containing CMC as the sole carbon source peaked on day 6 and then began to decrease; this medium resulted in the lowest PMG activity (Fig. 2B). The results indicated that PMG activity was induced in the three media. The highest activity was produced in medium containing pectin plus CMC, followed by medium with pectin as the sole carbon source, and then CMC-containing medium.

Cellulase activity was first detected two dpi in the medium containing CMC or pectin plus CMC, and six dpi in the medium containing pectin as the sole carbon source (Fig. 2C). In the medium containing pectin plus CMC, the activity increased rapidly to reach its maximum at day 6, when it was 21.4- and 1.7-fold the activity in the medium containing pectin or CMC as the carbon source, respectively; thereafter the activity remained high until the end of the experiment (day 16). In CMC-containing medium, the activity reached its maximum on day 8; the level of the activity was slightly lower than that in the medium containing pectin plus CMC through most of the cultivation process (Fig. 2C). In the medium containing pectin alone, only trace Cx activity was observed throughout the cultivation. The results indicated that Cx activity was induced in the three media; the highest activity was produced in the medium containing pectin plus CMC, followed by medium with CMC as the sole carbon source, and trace Cx activity was produced in pectin-containing medium.

β-glucosidase activity was low in all three types of medium. Activity was first detected at four dpi in the medium containing CMC alone; it peaked on day 8, followed by a decrease (Fig. 2D). β-glucosidase activity was first detected on day 6 in the other two media. The activity in the medium containing pectin plus CMC, or pectin alone, varied throughout the remainder of the experimental period. These results indicate that only trace β-glucosidase activity was induced in the three media.

Fungal growth and culture filtrate pH in vitro

The mycelial dry weight and pH in the three media were determined after filtrates were obtained. The growth of mycelium was different in the three media. In medium containing pectin as the sole carbon source, the amount of mycelium remained low and roughly constant to 10 dpi, and then increased (Fig. 3A). In medium containing pectin and CMC, mycelium grew slowly between day 0 and 4, and rapidly from day 4 to 6, after which the growth rate levelled off. The overall growth rate was much higher than in the other two media (Fig. 3B). In the medium containing CMC as the sole carbon source, the mycelium increased a little between day 0 and 4 and then remained roughly constant throughout the experimental period (Fig. 3C). The results indicated that mycelium grew rapidly in medium containing pectin and CMC, but slowly in the other two media.

During growth, the pH of the culture medium increased (from different starting values) coincident with the increasing mycelial dry weight. The pH of the medium containing pectin plus CMC increased sharply after 4 days, and that of the other two media slightly accumulated through the experiment (Fig. 3). The correlations between pH value and mycelial dry weight were found to be significant in shaken cultures containing pectin (r = 0.837, p = 0.005), pectin plus CMC (r = 0.953, p = 0.000), and CMC (r = 0.803, p = 0.009). Moreover, we found that the production of PG, PMG and Cx appeared to have a similar pattern to the mycelial growth and pH in the culture containing pectin plus CMC (Figs. 2A–2C and 3B). Hence, the correlations between enzyme activity and pH of the culture, and enzyme activity and mycelial growth, were respectively analyzed. PG, PMG and Cx activity were found to correlate extremely significantly with pH and fungal growth in pectin plus CMC shaken cultures (Table 1), showing strong association between the activity of these enzymes and the culture pH, and between activity of the enzymes and fungal growth. PG and PMG activity in shaken cultures in medium containing pectin or CMC as the sole carbon source, Cx activity in CMC medium, and β-glucosidase in pectin medium showed lower correlations with pH and fungal growth that significantly positively correlated with pH and fungal growth (Table 1).

Effect of crude enzyme solution on tissue

Crude enzyme extract obtained from a 10 dpi culture containing pectin plus CMC was tested by inoculation onto peanut leaves. The crude enzymatic extract was able to induce symptoms of necrotic lesion, whereas the control samples such as heated enzyme and distilled water did not produce any symptoms of necrotic lesion (Fig. 4A), indicating the possible involvement of CWDEs in lesion development. The results also showed that the leaves treated with crude enzyme extract developed symptoms similar to those caused by infection with R. solani mycelium (Figs. 4A and 4B), while control leaves inoculated with sterile PDA plugs showed no lesions (Fig. 4C).

Isoelectric focusing of polygalacturonase activity

The dialyzed extracts collected 10 dpi from culture filtrates and from infected tissues including stems and leaves of Bs and Slh peanut cultivars were subjected to thin-layer polyacrylamide gel IEF and evaluated for the presence of PG isoenzymes. One PG band, with pI ∼9.2, was observed in all the samples (Fig. 5). R. solani produced the same single PG isoform in vitro (i.e., in shaken flask cultures) and during host–pathogen interaction. The control (heat-treated extracts) showed no PG band (Fig. 5). Experiments indicated that the same, single PG isoenzyme was induced in infected leaf tissue and in in vitro fungal cultivation.