The influence of nutritional and physical stress on sporulation, conidial germination and vegetative biomass of Ophiocordyceps sinensis, one of the most important medicinal fungi in China and now globally, was evaluated using a two-stage culture method. All the treatments, except nutrient deprivation, enhanced conidial production and vegetative biomass to some extent. However, conidia produced under stress showed decreased germination in comparison with those continuously cultured on the enriched potato dextrose agar (PDA; as the control). Among 10 treatments tested, the physical stress of frozen-shock produced the largest number of conidia, 7.5 times higher than that of the control, followed by heat-shock treatment. These results demonstrate that the fungus has strong physiological adaptations to environmental stress that may have evolved because it is endemic to the Tibetan Plateau. This report will be relevant to the study of the pathogenicity and artificial cultivation of this endangered fungus.
Ophiocordyceps sinensis (previously Cordyceps sinensis), an entomogenous fungus on moth larvae, is one of the most valued traditional Chinese medicines. It has been used as a tonic in China for hundreds of years and has been officially classified as a drug in the Chinese Pharmacopeia (Committee of Pharmacopeia, 2010). In recent studies, O. sinensis has also been shown to have multiple pharmacological effects, including immunomodulatory (Wu et al., 2006), hypocholesterolemic (Koh et al., 2003), hypoglycemic (Zhang et al., 2006), anti-tumor (Wu et al., 2005), anti-oxidation (Dong & Yao, 2008) and anti-aging (Ji et al., 2009) activities.
The fungus is endemic to the Tibetan Plateau and may be found in alpine meadow and shrub habitats from above 3000 m in altitude up to the snow line (Li et al., 2011). Plentiful sunshine, strong UV radiation, hypoxia, cold environment and great difference in temperature during day and night are the most important ecological factors, which have profoundly affected organism survival on the plateau. As a native species of the plateau, constantly challenged by the harsh environmental stress during their long evolutional history, O. sinensis has adapted to the severe conditions at high altitude. However, natural production of the fungus is limited and annual yield has been declining continually over recent decades owing to its strict host-specificity (Wang & Yao, 2011), confined geographic distribution (Li et al., 2011) and over exploitation by humans (Yao, 2004). It is therefore currently listed as an endangered species under the second class of state protection (State Forestry Administration & Ministry of Agriculture, 1999).
Entomopathogenic fungi usually produce asexual reproductive spores for dispersal, transmission and infection of host organisms. The conidia of several fungi are some of the most efficient infection-initiating propagules (Zeng et al., 2012). Furthermore, conidia are often able to survive unfavorable environmental conditions and are generally more stable in the environment than somatic cells or mycelia (Wraight et al., 2007). In addition, conidia are useful for scientific research on pathogenic mechanisms (Andersen et al., 2006; Song & Feng, 2011) and other aspects of biology (Kim et al., 2010). Therefore, it is important to obtain optimal production of conidia under artificial conditions.
Conidia are produced mainly by solid fermentation, and conidial production is affected by media, culture conditions and other methods employed during artificial growth. The effects of nutritional and environmental conditions on the sporulation and conidial germination of some fungi have been previously investigated, including carbon concentration and the carbon to nitrogen ratio for several biocontrol fungi (Gao et al., 2007), the pH, moisture content and nitrogen concentration of the growth substrate for Metarhizium anisopliae (Prakash et al., 2008), oxygen concentrations for Beauveria bassiana (Garza-Lopez et al., 2012), etc. These studies have demonstrated that nutritional and environmental conditions play an important role in the sporulation and conidial germination of fungi.
In addition to nutritional and environmental conditions, culture methods are important factors in sporulation and conidial germination. A two-stage fermentative protocol has been reported to produce good spore yields (Glare, 2004). Xu et al. (2009) recently proposed a method involving the use of hydrogen peroxide (H2O2) treatment and a two-stage culture that significantly promoted conidial production in Pochonia chlamydosporia. According to Rangel et al. (2008a), the germination, adhesion and virulence of M. anisopliae conidia can be strongly influenced by stress encountered during growth. Information on the sporulation phase of fungi in response to nutritional and environmental stress conditions will be a useful tool to improve conidial production.
The objective of this study was to develop an easy, rapid and simple method to evaluate the vegetative growth, conidial production and germination using two-stage culture of O. sinensis in conjunction with both nutritional and physical treatments.
Materials and methods
A strain of O. sinensis, no. 1621, was selected from a screen of 135 strains of the species based on conidial production and used in this study. This strain was originally isolated from a field collection of the fungus from Guoluo (Qinhai Province, China) by our laboratory. It was maintained on slants of potato dextrose agar (PDA) supplemented with 5% (w/v) wheat bran at 4 °C.
To ensure the correctness of the strain used, the internal transcribed spacer (ITS1–5.8S–ITS2) of nuclear ribosomal DNA (nrDNA) was amplified from the culture and DNA sequencing identified 570 bp of this fragment. The identity of the strain was confirmed by comparing the sequence with a data set generated in our laboratory containing ITS sequences from dried specimens and living strains of O. sinensis obtained from various regions of the Tibetan Plateau, in addition to the morphological characters observed in culture.
Inoculum preparation and two-stage culture conditions
For inoculation, the strain was first incubated at 18 °C for 90 days in a Petri dish with PDA supplemented with 5% wheat bran, 0.5% fish peptone and 0.1% yeast extract as a basic medium. For the first preculture, 40 mL of the basic medium without agar (natural pH) was prepared in a 250-mL flask. The first preculture was inoculated with a 10-mL mycelial suspension (10 mL of sterile water washed across the surface of the colony from the Petri dish culture) and incubated for 14 days at 18 °C on a rotary shaker (100 r.p.m.). For the second preculture, 45 mL of the same medium was prepared in a 250-mL flask, inoculated with 5 mL of the first preculture, and then incubated for 14 days at 18 °C on a rotary shaker (100 r.p.m.). A solid medium (as the enriched PDA) was prepared by supplementing the basic medium with 2.5% dried silkworm-pupa meal. The enriched PDA was inoculated with 0.5 mL of the second preculture in the center of a cellophane sheet (6.0 × 7.0 cm) on the surface of plates. The second preculture broths were spread evenly on the cellophane sheets with a bent glass rod. The cultures were incubated in the dark at 15 °C (determined by preliminary experiments to be the optimal sporulation temperature). Unless otherwise stated, these conditions were maintained throughout all experiments.
After 20 days, vegetative colonies on the enriched PDA were transferred to PDA or water agar plates for a further 14 days of culture, which applied gradient descent nutritionally to the fungus for sporulation. For comparison, vegetative colonies continuously cultured on the enriched PDA for a further 14 days served as the control (CK).
Colonies on the enriched PDA were transferred 20 days after inoculation to PDA plates and subjected to heat-shock, frozen-shock, osmotic or oxidative stresses as follows: (1) transferred without any further physical treatment as the PDA nutritional stress; (2) transferred and exposed to heat-shock at 28 °C for 1 h; (3) transferred and exposed to frozen-shock at −20 °C for 1 day; (4) transferred and placed with a temperature cycle of 15/4 °C for 24/24 h (15 and 4 °C day−1); (5) transferred and placed with a 16 h day−1 photoperiod at 4800 lux; (6) transferred and placed with a cycle of 15/4 °C for 16/8 h (15 °C for 16 h and 4 °C for 8 h) with 16 h day−1 photoperiod at 4800 lux; (7) transferred and treated with a 20 mM hydrogen peroxide solution for 5 min; and (8) transferred to new PDA medium amended with 0.1 M potassium chloride. All of the above treatments, except (4), (5) and (6), were incubated in an artificial climate chamber (PRX-350A, Saife Instruments Co., Ltd, Ningbo, China), in the dark at 15 °C for a further 14 days after stress. The above-mentioned parameters were defined by preliminary experiments (data not shown).
To measure conidial production under different stresses, colonies on cellophane were collected with a sterile pincer and transferred to a 100-mL flask containing 50 mL sterile 0.1% (v/v) Tween 80 solution with 10 sterile beads (5 mm diameter). A rotary shaker was used to vigorously shake the flasks at 200 r.p.m. for 1 h to dislodge the spores, and the process of washing the colonies was repeated three times to obtain a homogenous conidial suspension. The solutions were combined and harvested in order through three sieves with mesh sizes of 150 (100 μm), 300 (50 μm) and 500 (30 μm) to remove hyphal bodies. The solutions were centrifuged at 12 401 g for 15 min and the supernatant discarded. Harvested conidia were re-suspended in 5 mL sterile 0.1% Tween 80 solution. Conidia were counted with a hemocytometer and a light microscope (Zeiss Axiophot) at ×400 magnification. To obtain sufficient conidia for the bioassay, five plates were required for each replicate. The experiment was repeated three times.
Determination of biomass
Biomass was determined based on dry mycelial weight. As in the ‘conidial production’ section, biomasses were dried at 60 °C for 24 h to determine the dry weights. Mean values for three independent repetitions were calculated, and five plates were used for each replicate.
Conidial germination assay
The germination rate of conidia produced after the different treatments was assessed by incubation in 5 mL water suspension (with ca. 106 conidia) in a 30-mL centrifugal tube. Tubes were incubated in the dark at 15 °C, and germinated conidia, determined based on the germ tube being greater than or equal to the width of the conidia, were counted at ×400 magnification under the microscope at 3, 6, 9 and 12 days. A total of 300 conidia were scored for each treatment.
Fluorescence microscopy assay
Fluorescence microscopy with Calcofluor White (Sigma), a vital stain that binds to β-glucans, was used to determine if different treatments affected the carbohydrate composition at the surface of the conidial cell wall following the method described by Ibrahim et al. (2002). Briefly, sporulating cultures grown under each of the treatments for 34 days were used to prepare separate conidial suspensions (105 conidia mL−1). For each conidial suspension, 5 mL of culture was centrifuged for 15 min at 12 401 g, and the resulting pellet was washed three times in 5 mL 0.05% Tween 80 and re-suspended in 5 mL of 0.01% aqueous fluorescent brightener 28 (Calcofluor White M2R; Sigma). The suspension was then incubated overnight at 15 °C in the dark. Following incubation, stained conidia were washed three times in sterile distilled water and re-suspended in 5 mL of 0.05% Tween 80 before examination under a fluorescence microscope (Zeiss Axio).
All experiments were performed in triplicate. The statistical analysis of the data for mycelial growth, sporulation and germination was performed using one-way anova, and significant differences were determined by Duncan's multiple-range tests at P =0.05 using spss 20.0 (SPSS Inc.).
Results and discussion
Effects of nutritional stress on sporulation and mycelial growth of O. sinensis
The two-stage fermentative protocol involves liquid–solid and solid–solid fermentative processes. The former promotes the mycelial growth in liquid medium to reduce the time required for the initiation of sporulation, and the latter, conducted on two different solid media, stimulates conidial production. In the present study, O. sinensis was cultured on the enriched PDA to grow vegetatively for 20 days (the fungus began to sporulate at later time points, as determined in pilot studies; data not shown) and was then transferred to a new nutritionally decreased medium (PDA or Water), where the fungus sporulated for 14 days. Conidial production and mycelial growth under the different nutritional treatments are shown in Fig. 1, and both were significantly affected compared with CK (P <0.05). In general, the effects of nutritional treatments on both sporulation and mycelial growth were similar. The best conidial and biomass production were found when colonies were transferred to PDA, reaching 2 and 0.5 times greater levels compared with those of CK, respectively (Fig. 1). However, the water agar condition resulted in the poorest production of conidia and biomass and was significantly decreased compared with CK. The difference between the two nutritional treatments was nearly 6 and 3.6 times for conidial production and biomass, respectively (Fig. 1).
Generally, fungi produce more spores under low nutrient conditions but need high nutritional requirements for vegetative growth (Elson et al., 1998). Unlike other species with increased sporulation by transfer to water agar (Xu et al., 2009), O. sinensis did not perform well after the transfer as observed in this study. It appeared that water agar could not meet the nutritional requirements of conidial and biomass production of O. sinensis. By contrast, O. sinensis could produce more conidia and biomass by transfer to a lower nutritional condition, indicating it required definite nutritional support for conidial production.
Effects of physical stress on sporulation and mycelial growth of O. sinensis
Evaluation of the effect of supplemental physical stress on sporulation and mycelial growth was carried out based on nutritional treatments. As shown in Fig. 2, both sporulation and mycelial growth were significantly affected (P <0.05) by physical stress. For biomass, optimal growth (0.88 g dried weight per five plates) was obtained on the PDA nutritional stress (assigned as PDA), resulting in a 5–35% increase compared with the other physical treatments, but no significant differences were observed among PDA, heat-shock (28 °C), frozen-shock (−20 °C), osmotic (0.1 M) and oxidative (H2O2) treatments. The temperature-photoperiod treatment (15/4 °C for 16/8 h) produced the poorest mycelial growth in physical treatments, possibly due to the length of time used for the low temperature and light intensity. However, no significant differences were observed with the treatments of temperature (15/4 °C for 24/24 h) or photoperiod (16/8 h) alone. In terms of conidial production, the stimulation of physical stress was more significant on sporulation than on mycelial growth. The highest number of conidia (3.6 × 107 per five plates) was observed on frozen-shock treatment and was 1.6 times and 7.5 times higher than the production level achieved on PDA (1.4 × 107 per five plates) and on CK, the continuous culture on the enriched PDA (4.2 × 106 per five plates), respectively. However, there was no significant difference between the frozen-shock and heat-shock treatments.
The distribution of O. sinensis at high altitude emphasizes the cool-loving or psychrophilic nature of the fungus. Accordingly, mechanisms of protection and adaptation of this fungus under low temperature may have been formed during their long evolutional history. Nevertheless, strains of the species can withstand higher temperature for certain periods, e.g. 20 days at 25 °C and 10 days at 28 °C (Dong & Yao, 2011). The results obtained here also revealed that the best condition for sporulation was not the same as that for the best mycelial growth, but the biomass under the best sporulation condition (frozen-shock) had no significantly difference from that under the best mycelial growth condition (PDA).
In general, conidial production was enhanced to some extent under all of the physical stresses tested in this study, while a converse result was reported by Rangel et al. (2008b) that conidial yield of M. anisopliae was reduced, in some cases severely, by nutritional and osmotic stresses and that oxidative and heat-shock stresses did not alter levels of spore production. The disagreement between these results may be due to the different species involved.
The present study showed that O. sinensis could survive under challenging environmental conditions such as nutritional, temperature, osmotic and oxidative stresses, indicating that the fungus has a strong physiological tolerance to environmental stress that may have evolved because the fungus is endemic to the Tibetan Plateau.
Similar to the work of Garza-Lopez et al. (2012) on B. bassiana, conidia of O. sinensis produced under stress germinated less than those from CK that had the highest germination rate (Fig. 3). In general, stress conditions had a negative effect on conidial germination. After 6 days incubation, germination rates of conidia were similar among the different treatments (P <0.05), except for under the photoperiod (16/8 h), water agar nutritional (Water), and temperature-photoperiod (15/4 °C for 16/8 h) treatments, where low germination rates were found (Fig. 3). The highest number of conidia was produced under the heat-shock (28 °C) and frozen-shock (−20 °C) treatments (Fig. 2), but the germination rates were 46.3% and 52.1% after 12 days incubation, which is less 35.3% and 27.2% than that of conidia produced on CK (71.6%), respectively (Fig. 3). Garza-Lopez et al. (2012) reported that low oxygen (16%) promoted conidial production and that the germination rate was lowered by almost 27% compared with those produced in normal atmosphere. Taken together, these results suggest that stress conditions generally reduce the capacity for conidial germination. In general, the frozen-shock (−20 °C) treatment is the best method to maximally produce high-quality conidia, followed by the heat-shock (28 °C) treatment. The former would produce around 6.2-fold more healthy conidia than the control (calculated based on 8.5 folds × 52.1%/71.6%).
Fluorescence microscopy assay
As an insect parasite, good conidial adhesion, which has been considered an attribute of virulent strains of entomogenous fungi (Al-Aidroos & Roberts, 1978), of O. sinensis would help the fungus to establish the infection on its host. The fluorescence of conidia produced under stress after calcofluor staining in this study was greater than those under CK (Fig. 4), indicating that stress conditions may affect the carbohydrate composition at the surface of conidial walls. The surface properties of conidia produced under stress, as indicated by increased calcofluor-stain fluorescence, may also influence the binding of spores to insect cuticles by hydrophobic forces to some extent (Ibrahim et al., 2002; Song & Feng, 2011). Further research is needed to determine the characteristics of conidia and their tolerance to stress, persistence, adhesion to the host cuticle and pathogenicity.
In this study, the physical stress of frozen-shock, among 10 treatments tested, produced the greatest number of conidia, reaching 7.5 times higher than the control. As a result, an easy, rapid and simple method for mass production of conidia was developed for O. sinensis. This method may become useful for further studies of the species and possibly other less sporulating fungi.
This work was supported by the National Natural Science Foundation of China (31170017), the Chinese Academy of Sciences (KSCX2-YW-G-076) and the Ministry of Science and Technology of China (2012CB126308, 2012FY111600, 2013BAD16B01).