Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris

Abstract

The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1-2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly influenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations.

Keywords: Cordyceps militaris; degeneration; fruiting body formation; heterokaryotic strains; heterothallic fungus; mating-type loci; sexual reproduction; spore ratios; successive culturing generations.

Figure 1

Fruiting body formation in different C. militaris strains. (A) The mating-type loci (MAT1-1, MAT1-2) in five tested C. militaris strains (G12, H8, M5, B12, and HQ1) were examined with PCR using the primer pairs specific for the MAT1-1-1 and MAT1-2-1 genes. PCR products were analyzed on 0.7% agarose gels. All five C. militaris strains were artificially cultivated to evaluate the fruiting body formation ability. (B) The C. militaris strains were cultured on the PDA medium for five successive generations (first to fifth) using the spore isolation method. All five generations were inoculated to the BR medium with the culture conditions of 22 °C, a humidity of 85–90%, and a lighting cycle of 12 h light/12 h dark for 45 days to promote fruiting body formation.

Figure 2

Examining opposite mating-type ascospores from the heterokaryotic strains for fruiting body formation. (A) Isolation of ascospores from three heterokaryotic strains, G12, B12, and HQ1. Fungal colonies derived from ascospores were confirmed for the mating-type loci (MAT1-1 and MAT1-2) by PCR. (B) Monokaryotic isolates from a representative heterokaryotic strain (G12) were cultured as separated or combined to evaluate fruiting body formation. Fruiting body formation assays were conducted on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark.

Figure 3

Effects of different mating-type spore ratios in combinations on fruiting body formation in three heterokaryotic C. militaris strains. (A) Combinations of monokaryotic isolates G4 (MAT1-1) and G2 (MAT1-2) derived from strain G12. (B) Combinations of monokaryotic isolates B5 (MAT1-1) and B6 (MAT1-2) derived from strain B12. (C) Combinations of monokaryotic isolates H3 (MAT1-1) and H2 (MAT1-2) derived from HQ1. Spore mixtures were inoculated on the BR medium for 45 days at 22 °C, with a humidity of 85–90% and a lighting cycle of 12 h light/12 h dark to promote the formation of fruiting bodies.

Figure 4

Maintaining the fruiting body formation ability of the heterokaryotic strains. (A) Monokaryotic isolates of the MAT1-1 mating type and the MAT1-2 mating type were preserved separately. These isolates were successively cultured for five generations. The opposite mating-type spores from each generation were mixed and cultivated on the BR medium for fruiting body formation. (B) Fruiting body formation in three heterokaryotic strains (G12, B12, and HQ1) after five successive culturing generations of the monokaryotic isolates and respective spore combinations by mating at a 1:1 ratio. (C) Quantification of fruiting bodies from the combinations. Three parameters, namely number of fruiting bodies (FBs), height of FBs, and weight of FBs, were documented. Experiments were conducted in triplicate, and data are presented as means ± standard deviations. Error bars represent the standard deviations, and different lowercase letters indicate significant differences (p < 0.05).