Isaria farinosa is a pathogen of alpine Thitarodes larvae that are hosts for the Chinese medicinal fungus, Ophiocordyceps sinensis. A matrix analysis indicated that the optimal culture conditions for the mycelial growth of I. farinosa are a 50-mL liquid broth in a 250-mL flask at more than 100-rpm rotation and 15–25 °C. Illumination does not affect the mycelial growth. The optimal nutrition requirements are D-(+)-galactose and D-(−)-fructose as carbon resources and D-cysteine as well as yeast powder, peptone, and beef extract as nitrogen resources at a carbon-to-nitrogen ratio of 1:1 to 1:7. The mineral component and vitamins also significantly increase the mycelial growth of I. farinosa. Based on the optimal culture conditions and nutrition requirements for the mycelial growth of I. farinosa, the effects of altitude on mycelial growth and its metabolome were evaluated using quadrupole-time-of-flight/mass spectrometry, principal component analysis and partial least squares discriminant analysis. The altitude did not affect the mycelial production but significantly regulated its metabolome. The study presents a new approach to better select a method for producing more useful metabolites and highlights the necessity of establishing standards for culturing methods related to altitude to preserve fungal quality; additionally, the results indicate that the use of a fermenter may meet the demands of large-scale mycelial production.
Isaria farinosa (Holmsk.) Fr. 1832 [formerly Paecilomyces farinosus (Holmsk.) A.H.S. Br. & G. Sm. 1957] has been an important fungus in agriculture and pharmacy, and many studies have been conducted to identify the biological characteristics of the fungus; these studies have included collecting high-quality samples, finding the suitable temperature and moisture for its mycelial and spore growth, determining the range of its host, and additional topics1. Once the disease in Thitarodes (formerly Hepialus) larvae (Lepidoptera: Hepialidae), the host of Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones & Spatafora 2007, was found to be caused by I. farinosa2, the study of I. farinosa intensified, including not only its virulence characteristics3,4, fungicide5,6, biochemical characterisation7, biotransformation activity8 and molecular biology9,10 but also its secondary metabolites and pharmacological activity11.
The secondary metabolites extracted from I. farinosa have included alkaloids12, polysaccharides13, ketones14, quinones15 and more than 20 compounds4. Some of them have shown many pharmacological activities. For example, the paecilosetin extracted from I. farinosa can inhibit cellular proliferation of leukaemia16; Militarinones B isolated from I. farinosa was active against Staphylococcus aureus Rosenbach 1884, Candida albicans (C.P. Robin) Berkhout 1923 and Streptococcus pneumoniae (Klein) Chester 188417; Jiang et al.18 reported that the intra- and extracellular polysaccharides from I. farinosa had the same antioxidant efficiency. In addition to the above, some chemical compositions from I. farinosa have shown hypoglycaemic action19 and antineoplastic activity14,18,20. Therefore, I. farinosa has the potential to be developed for medicinal purposes on a large scale.
There have been some studies on the factors that affect the mycelial growth of I. farinosa, such as the carbon and nitrogen sources and the optimal medium and temperature6,21,22,23. However, most previous studies were either aimed at biological control or were partial studies, and there have been no studies on systematically screening the suitable nutrition and culture conditions for mycelial growth from the perspective of preserving medicinal quality. Additionally, because the disease in Thitarodes larvae caused by I. farinosa develops differently at different altitudes during the practical production of artificially culturing Chinese Cordyceps, and because the strain used in the present study was obtained from Thitarodes larvae from the Tibetan Plateau, it was inferred that mycelial growth and its metabolome might be affected by altitude. Although the strains used in the studies6,24 were obtained from high altitude, the purpose of their works were biological control or increasing the survival rate of the larvae, and there have been no experiments about the effect of altitude on the mycelia and metabolome.
Therefore, based on these unexplored research questions and the previous studies, the effect of altitude on the mycelial growth and metabolome of I. farinosa were investigated after optimising the nutritional and cultured conditions used for mycelial growth. We hope that the results of this study provide parameters for more effectively utilizing the fungus.
The results showed that the mycelial weight was the highest in the treatment with the 50-mL capacity in the 250-mL Erlenmeyer flask (EF), and the weight was significantly different from the weights recorded in the other treatments (all P values were 0.00). The mycelial weight was the lowest in the treatment with a 200-mL capacity in the 250-mL EF, and the weight was significantly different from the weights recorded in the other treatments (all P values were 0.00). In the 50–200 mL capacity range for the 250-mL EFs, higher capacities led to lower mycelial weights. There was no significant difference between the mycelial weights in the treatment with 100-mL capacity in the 500-mL EF and the treatment with 100-mL capacity in the 250-mL EF (P = 0.87) (Table 1).
The results indicated that higher inoculum concentrations led to higher mycelial weights (Table 2). The mycelial weight was the highest in the treatment with the 20% inoculum concentration and lowest in the treatment with the 2% inoculum concentration. The one-way analysis of variance (ANOVA) results indicated that there was no significant difference among mycelial weights for the treatments with inoculum concentrations of 2, 5, 10 and 15% (P2–5% = 0.49; P2–10% = 0.09; P2–15% = 0.17; P5–10% = 0.28; P5–15% = 0.48; P10–15% = 0.70). The difference in the mycelial weight from the inoculum concentration of 20% was not significantly different from the treatments with the inoculum concentrations of 15% (P = 0.08) and 10% (P = 0.15).
It was clear that the mycelial weight was the lowest in the treatment with a rotation speed of 0 rpm; in contrast, the weight was the highest in the treatment with a rotation speed of 120 rpm (Table 3). The ANOVA results indicated that the difference in the mycelial weight in the treatment with a rotation speed of 0 rpm was significantly different compared with the mycelial weights in the treatments with other rotation speeds (all P values were 0.00). However, there were no significant differences among the mycelial weights in the treatments with any rotation speed above 0 in the present experiment (P100–120 rpm = 0.50; P100–150 rpm = 1.00; P100–180 rpm = 0.69; P120–150 rpm = 0.50; P120–180 rpm = 0.30; P150–180 rpm = 0.70).
The research indicated that the mycelial weight was the highest in the treatment with 24 h of darkness, and the weight was the lowest in the treatment with 24 h of light. The ANOVA results indicated there were no significant differences among the mycelial weights of the three treatments (P12 h of light/12 h of darkness−24 h of light = 0.54; P12 h of light/12 h of darkness−24 h of darkness = 0.90; P24 h of darkness−24 h of light = 0.46) (Table 4).
The growth rate of the mycelia at 20 °C was the largest after 5 d, followed by 25 °C, and the mycelia did not grow at 0 and 35 °C (Table 5). The differences in growth rates among the 15, 20 and 25 °C conditions were not significant (P15–20 °C = 0.08; P15–25 °C = 0.25; P20–25 °C = 0.54), and all them were significantly different from the growth rates of the mycelia at 5, 10 and 30 °C (Except P15–10 °C = 0.10, P15–30 °C = 0.08 and P15–5 °C = 0.02, P25–10 °C = 0.01, P25–30 °C = 0.01, the other P values were 0.00). The result of verification showed that the differences in mycelial yield in liquid fermentation were not significant (P20–15 °C = 0.66, P20–25 °C = 0.40 and P25–15 °C = 0.68); however, the mycelial dry weight at 20 °C was the highest, consistent with the results on agar medium (Table 5).
Among the 16 tested carbon sources, the organic acid did not benefit the growth of the mycelia. The highest mycelial yield was obtained with D-(+)-galactose, followed by D-(−)-fructose. Very weak growth was observed in the medium containing citric acid and in the control medium (Table 6). However, the ANOVA results showed that the mycelial yield in the D-(+)-galactose medium was not significantly different from that in the media containing D-(−)-fructose (P = 0.97), soluble starch (P = 0.45), D-(+)-trehalose (P = 0.34), D-(+)-cellobiose (P = 0.25), sucrose (P = 0.18) and D-(+)-glucose (P = 0.05). Very weak growth was observed in the media with glucitol or citric acid as the carbon sources, and both mycelial yields treated with these carbon sources were significantly low (Pcitric acid-glucitol = 0.75; Pcitric acid-control = 0.97; Pglucitol-control = 0.79).
Among the 19 nitrogen sources examined in this study, yeast powder was the most effective for increasing mycelial growth, followed by peptone. The mycelial yield was the lowest in the medium that lacked a carbon source or a nitrogen source (Table 7). The ANOVA results showed there were no significant differences among the mycelial yields in the treatments with yeast powder, peptone and beef extract (Pyeast powder-peptone = 0.82; Pbeef extract-peptone = 0.30; Pbeef extract-yeast powder = 0.20). The media supplemented with yeast powder, peptone or beef extract resulted in significantly higher mycelial yields than the yields that resulted from any other nitrogen sources (all P values were 0.00). Among all tested amino acids, L-cystine was the best nitrogen source for mycelial growth, followed by DL-glutamic acid. There were no significant differences among the mycelial yields for L-cystine, DL-glutamic acid, L-arginine, asparagine and DL-serine (PL-cystine-DL-glutamic acid = 0.52; PL-cystine-L-arginine = 0.39; PL-cystine-asparagine = 0.14; PL-cystine-DL-serine = 0.12; PDL-glutamic acid-L-arginine = 0.82; PDL-glutamic acid-asparagine = 0.39; PDL-glutamic acid-DL-serine = 0.36; PL-arginine-asparagine = 0.53; PL-arginine-DL-serine = 0.49; Pasparagine-DL-serine = 0.96). The mycelial yields of glycine, L-histidine and L-aspartic acid were not significantly different from the yields of Control 1 and Control 2 (Pglycine-Control 1 = 0.94; Pglycine-Control 2 = 0.33; PL-histidine-Control 1 = 0.83; PL-histidine-Control 2 = 0.40; PL-aspartic acid-Control 1 = 0.82; PL-aspartic acid-Control 2 = 0.40).
Among the eight C/N ratios, the mycelial yield for the treatment with a 1:1 ratio was the highest, followed by the treatment with the 7:1 ratio (Table 8). The ANOVA results showed there was no significant difference between the treatment with 1:1 and 7:1 ratio (P = 0.07), and both treatments were significantly different from the other treatments (all P values were 0.00). As the C/N ratio increased, the mycelial yield decreased, except for the observed increase in the treatment with the 56:1 ratio.
Among all macro-element treatments, the mycelial yield was the highest in the complete medium without sodium, followed by the complete medium (Table 9). There were no significant differences among the yields of the treatments with Control 1, the complete medium without sodium, the complete medium without potassium and the complete medium without calcium (PControl 1-the complete medium without sodium = 0.69; PControl 1-the complete medium without potassium = 0.79; PControl 1-the complete medium without calcium = 0.15; Pthe complete medium without sodium-the complete medium without potassium = 0.50; Pthe complete medium without sodium-the complete medium without calcium = 0.07; Pthe complete medium without potassium-the complete medium without calcium = 0.23). The mycelial yield of Control 2 was the lowest, and it was significantly different from the yield of any other treatment (all P values were 0.00). Except for Control 2, the mycelial yield of the complete medium without magnesium was the lowest, but it was not significantly different from the complete medium without calcium (P = 0.08). It seems that magnesium is more indispensable than the other tested macro-elements.