Optimization of cultural conditions for pectinase production by Diaporthe isolate Z1-1N and its pathogenicity on kiwifruit

Plant material and pathogenic fungi

Kiwifruit (A. chinensis cv. Hongyang) of similar size and consistent maturity was purchased from the local market in Liupanshui, Guizhou Province, China. The pathogenic fungi Diaporthe spp. Z1-1N, isolated in our previous studies from rotten ‘Hongyang’ kiwifruit during cold storage (Ling et al., 2023; Ling et al., 2024b), was cultured on potato dextrose agar (PDA) medium at 25 °C in the dark for 6 days for the enzyme assays and pathogenicity trials.

Optimization of pectinase production in liquid culture

In this study, we employed a modified Marcus liquid medium, as described in our previous research, to investigate the production of two pectinases: PG and PMG (Ling et al., 2024a). Five millimeter mycelial discs from a 6-day-old PDA culture were inoculated into 100 ml of liquid medium contained in 250 ml flasks, which were then incubated on an orbital shaker. Optimization of pectinase production was conducted through a series of single factor experiments, assessing variables such as incubation periods (1, 2, 3, 4, 5, 6, and 7 days), temperature (18, 23, 28, 33, and 38 °C), and the initial pH of the medium (ranging from 4 to 9 in 0.5 intervals). The optimal incubation time was determined based on the experimental design. When investigating the other two factors, cultures were harvested on the third day. The contents were filtered using a vacuum to remove the fungal mycelia. The filtrates were subsequently centrifuged at 10,000 g for 15 min at 4 °C, and the supernatant was utilized to assess the activitiesof PMG and PG using methods described in our previous study (Ling et al., 2024a).

Based on the results of the single-factor experiment, a three-factor, three-level standard orthogonal table, L₉ (3³), was selected to optimize the production conditions for two pectinases (PG and PMG) as shown in Table 1. The incubation temperature (A) was set at 25 °C, 28 °C, and 31 °C; the initial pH of the medium (B) was set at 6.5, 7.0, and 7.5; and the incubation time was set at 2, 3, and 4 days. These factors and their corresponding levels were incorporated into the L₉ (3³) orthogonal table, resulting in a total of nine experimental groups with various combinations of the three parameters in this study.

Measurement of pectinase activity

The activities of PMG and PG were determined using the 3,5-dinitrosalicylic acid (DNS) colorimetric method, following the detailed procedures outlined by our previous study (Ling et al., 2024a). Three biological trials were conducted.

Purification of crude enzyme extracts and incubation on kiwifruit fruit

Pectinase was induced for production from the liquid culture of the Diaporthe isolate Z1-1N using orange pectin (Shanghai Aladdin Industries, Shanghai, China) as the carbon source. The purification process adhered to the method described (Ling et al., 2024a). The pathogenicity of various treatments, including pectinase extract and mycelial plugs from the Diaporthe isolate Z1-1N, was assessed by incubating them at 28 °C in darkness. Sterile water and sterile plugs served as controls, respectively. Ten kiwifruit fruits were used in each treatment. The lesions are classified based on their diameters (Li et al., 2019): grade 0 (0 cm); grade 1 (0 cm−1.5cm); grade 3 (1.5–3 cm); grade 5 (3–4.5 cm); grade 7 (4.5–6 cm); grade 9 (more than six cm). The Disease Index (DI) is calculated as follows: ${\displaystyle \text{Disease Index (DI)}=\frac{\sum \left(\text{The rotten fruits of each grade}\times \text{the corresponding grade}\right)}{\text{Total number of tested fruits}\times \text{the topmost grade}}\times 100\text{\%}.}$

Data and statistical analysis

Each experiment of enzymic assay was conducted in triplicate, and lesion size measurements were taken from ten kiwifruit fruits per treatment group. From the gathered data, we calculated the mean and standard error to provide a comprehensive analysis. To discern significant differences among the means, we employed one-way analysis of variance (ANOVA) using SPSS version 19.0 for Windows. Following the ANOVA, we applied Duncan’s multiple range test to separate the means, establishing statistical significance at the P < 0.05 threshold. Additionally, we leveraged the Orthogonal Designing Assistant II V3.1 software for the rigorous evaluation of our statistical experimental design, ensuring the precision of our experimental outcomes.

Effect of initial pH of medium on two pectinase production

The initial pH of the medium is a crucial factor in the production of pectinases, as it influences both the type and quantity of enzymes produced by fungi. In this study, we found that the activities of pectin methylgalacturonase (PMG) and polygalacturonase (PG) produced by Diaporthe Z1-1N showed the similar trends but also exhibited some differences (Fig. 1). For example, significant differences in enzyme activities were only observed at pH levels above 7.5, specifically in the range of pH 7.5 to 8.0. Within this range (7.5–9.0), PG activity significantly decreased compared to that of PMG (Fig. 1). In addition, PMG activity remained relatively stable across different pH levels, with a significant peak at pH 7.5 (10.14 U/ml). Similarly, PG activity reached its highest level at pH 7.5 (9.70 U/ml) but it was lower than that of PMG. Therefore, these results indicate that the optimal production of both PMG and PG by Diaporthe Z1-1N occurs at pH 7.5, with PMG exhibiting a higher activity than PG.

Effect of incubation temperature on two pectinase production

Temperature is directly related to the metabolic activities of microorganisms and significantly influences both growth and product formation. Figure 2 illustrates the effects of various temperatures on the production of two types of pectinase. The activity of PMG was consistently higher than that of PG across different temperatures, with the exception of 33 °C, where PG exhibited greater activity than PMG. Additionally, both PMG and PG displayed a similar trend, characterized by an initial increase in activity followed by a decline. Our results indicate that maximum pectinase production occurred at 28 °C, yielding 10.14 U/ml for PMG and 9.70 U/ml for PG. Subsequently, the second most favorable temperature for PMG production was 23 °C (8.87 U/ml), while for PG, it was 33 °C (8.92 U/ml). Conversely, the lowest production levels for both pectinases were observed at 38 °C, with values of 7.32 U/ml for PMG and 6.39 U/ml for PG. Therefore, these findings suggest that the optimal temperature for the growth and production of PMG and PG by Diaporthe Z1-1N is 28 °C.

Effect of incubation periods on two pectinase production

The incubation period is crucial for maximizing enzyme yield. Over the course of 1 to 7 days, we monitored the activities of PMG and PG, as illustrated in Fig. 3. Throughout this period, PMG generally exhibited a higher activity than PG, with the exception of the second and fifth days. Our data further indicated that both pectinases displayed significant fluctuations in activity and showed a similar trend with a slight variation. For PG, activity gradually increased from the start, peaking on the third day at a concentration of 10.52 U/ml, before gradually declining by the seventh day. PMG activity began to rise on the second day, dipped slightly on the third day, and then reached its maximum on the fourth day at 10.43 U/ml. Subsequently, PMG activity decreased, reaching its lowest point at the 7-day mark with a yield of 7.42 U/ml, as depicted in Fig. 3. These findings suggest that the optimal times for PMG and PG production are the third and fourth days of incubation, respectively.

Optimization of cultural conditions for pectinase productivity

Culture conditions, such as pH levels, incubation temperatures, incubation duration, and the sources of carbon, nitrogen, and mineral salts, are pivotal in dictating the synthesis and secretion of extracellular enzymes by microorganisms (Jia et al., 2010; Koirala et al., 2014; Patidar et al., 2018). Hence, meticulous adjustment of these parameters can obtain the highest activity the major extracellular enzymes secreted by pathogenic fungi. An orthogonal experiment demonstrated that the highest activity of PMG, at 11.228 U/mL, was attained at a temperature of 28 °C, an initial pH of 7.5, and an incubation period of 2 days (Table 2). Under these same conditions, PG achieved its peak activity of 10.056 U/mL (Table 2). This finding demonstrated that under the optimal conditions identified through an orthogonal experimental design, PMG exhibited a higher activity than PG.

In this study, we examined the impact of three key factors—incubation temperature (A), initial medium pH (B), and incubation duration (C)—on PMG production by Diaporthe Z1-1N. A higher extreme R value for a factor indicates that it has a more significant effect on the outcome. By analyzing the magnitude of the extreme R values, we ranked the influence of these factors on PMG production activity as follows: A >C >B (Table 3). This ranking indicates that incubation temperature had the greatest impact, followed by incubation duration, and then initial pH value. As previously mentioned, the optimal conditions were determined to be an incubation time of 2 days, an incubation temperature of 28 °C, and an initial pH of 7.0. To confirm the effectiveness of the optimization approach based on the orthogonal experiment’s results, we conducted a verification experiment using the refined parameter combination. The findings revealed that PMG activity increased significantly to 11.50 U/mL (Table 3), surpassing the activity observed in all previous orthogonal experiments. This outcome validates the efficacy of the optimization strategy employed.

Pathogenicity of the pectinase extract on kiwifruit

In this study, we investigated the effects of a purified pectinase extract on kiwifruit. The pectinase was prepared from a three-day shaken lipid culture using orange pectin as the sole carbon source, based on the optimal conditions for PMG production. Our results indicated that the pectinase extract induced significant necrotic lesions, with the DI value reaching 47.62% after seven days of treatment (Table 4). Additionally, we observed that the symptoms caused by the pectinase extract were similar to those resulting from infection with Diaporthe Z1-1N mycelium. However, the necrotic lesions induced by the pectinase extract were considerably less severe than those caused by the mycelium plug, which exhibited a DI of 94.81%. In contrast, the control fruits inoculated with sterile water or PDA plugs showed no lesions. Therefore, these results suggest that pectinase plays a role in lesion development associated with Diaporthe Z1-1N.