**6. Morphological changes after electrical stimulation**

were 60, 111, 90 and 89 g in the control, 50, 100 and 125 kV stimulation groups, respectively. Compared with the control group, the total yield increased when applying a voltage of 50 and 100 kV. The harvested weight for 15 days after the first crop (day 18) was approximately 50% of the total in the control group. However, the crop weight during this period increased to 86% of the total when applying voltages of 50 and 100 kV. This result indicates that the mushrooms can be harvested in fewer days by applying high voltage as electrical stimulation. **Figure 15** shows the crop weight of *L. decaste* stimulated with three different voltage amplitudes: 50, 90 and 130 kV. The yield of the fruiting body at the first flash in substrate cultivation

**Figure 14.** Time-history of the total amount of harvested fruit bodies for various stimulation voltages [3].

108 Physical Methods for Stimulation of Plant and Mushroom Development

**Figure 15.** Yield of *Lyophyllum decastes* fruit bodies for various stimulation conditions. Vertical bars indicate the standard

errors of the mean (number of samples; *n* = 20). Asterisks indicate the significant differences at *p* < 0.05 (\*) [3].

It is very difficult to reveal how electric stimulation affects fruiting body induction in mushroom species. Because molecular mechanisms for fruiting body induction in mushroom species have not still been well understood yet. Therefore, we focused on morphological changes after electrical stimulation.

**Figure 17(a)** and **(b)** shows images of *L. edodes* hyphae before (a, red) and after (b, blue) application of electric pulses. **Figure 17(c)** shows a superimposed image of (a) and (b) with purple (red + blue) indicating that hyphae retained the same position before and after applying the pulsed electric fields. Red and blue colored hyphae in **Figure 17(c)** show displaced hyphae. Displacement can be explained by the slightly negative charge of mushroom hyphae. When an electrical field *E* is applied, hyphae will thus be subjected to a Coulomb force *f* (*f* = *qE*; *q* means total charge of the hypha) from the electrical field. As a result, the hyphae are accelerated towards the positive electrode according to the equation *f* = *ma*, where m and a mean mass of the hypha and acceleration of the hypha, respectively. The application of electric pulses, resulting in hyphal displacement and sometimes damage, can be considered as a form of physical stress. Other physical stresses such as scrapping of surface hyphae (Kinkaki) have been known to induce fruiting body formation in several mushrooms, suggesting that electric pulses that induce fruiting body formation act through a similar mechanism. **Figure 17(d, e)** shows scanning electron microscope (SEM) images of hyphae before and after applying an electrical pulse of 10 kV between wire electrodes with a gap length of 9 cm. It was observed in the SEM image that after

some hyphae were broken by the electric pulse (**Figure 17(e)** arrow). This suggests that the electric pulse will be a similar stimulation as scratching mycelia on the surface of the sawdust media for mushroom production. Furthermore, it would be possible that new hyphae will be generated after electric pulse stimulation and Kinkaki. Hydrophobin, which is involved in hyphal structure and architecture in fungi [15, 16], would be involved in new

High-Voltage Methods for Mushroom Fruit-Body Developments

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**Figure 18** shows typical photographs 10 days after cultivation at various amplitudes of the applied voltage. The pulsed voltage was applied after 5 days of cultivation of *L. edodes* hyphae. The tip positions of the hyphae after 5 days of cultivation were marked by the inner dotted circles. The hyphae grew from the inner to the outer circle positions after 5 days cultivation from the pulse voltage stimulation. From the microscopic observation, the growth direction of the hyphae changed perpendicular to the surface of the agar medium between the inner and

High-voltage electrical stimulation on fruiting body formation in cultivating mushrooms was described. The compact high-voltage pulsed power supplies were developed for the electrical stimulation to promote fruiting body formation on cultivation bed-logs and sawdust substrate (bed-block). The promotion effects of high-voltage stimulation of sawdust-based substrate of *L. decastes* and natural logs hosting *L. edodes*, *P. microspora* and *H. lateritium* were confirmed through the evaluation using a developed compact pulsed power generator. The fruiting body formation of mushrooms increases 1.3–2.0 times in terms of the total weight. The accumulated yield of *L. edodes* for four cultivation seasons was improved from 160 to 320 g by applying voltages of 50 or 100 kV. However, the yield was decreased from 320 to 240 g upon increasing the applied voltage from 100 to 130 kV. The yield of the other types of mushrooms show tendencies similar to those of *L. edodes* when voltage was applied. An optimal voltage was confirmed for efficient fruiting

Securing profitability of the electrical stimulation is important for the widespread to the mushroom famers. The pulse voltage stimulation systems for improvement of mushroom yield have been developed and sold by some companies. Typical price of the stimulation system is around 5000 USD. The increment of *L. edodes* yield is around 155 g/(1-log, 2-year) at 50 kV. The price of the *L. edodes* is around 20 USD/1-kg at natural-log cultivation in Japan. If the mushroom farmer uses 1612 logs, the initial cost of 5000 USD can be recovered with

The authors of this chapter confirm that they have received permission to reuse all the tables

hyphae generation after pulse stimulation.

**7. Conclusions**

body induction.

increment of the mushroom yield.

and figures in their current work.

**Acknowledgements**

the outer dotted circles as the result of applying a high voltage.

**Figure 17.** Microscopic images of *Lentinula edodes* hypha (a) before and (b) after applying 5 kV/cm pulse electric field with pulse width of 100 ns and 500 times of repetition. (c) Superimposed image of two images (a) and (b). (d) and (e): SEM images of *L. edodes* hyphae before (d) and after (e) applying 10 kV pulse voltages. White bar indicates 100 μm in (a), (b), (c) and 10 μm in (d) and (e).

**Figure 18.** Influence of the pulsed voltage stimulation on hypha growth in agar medium cultivation. The diameter of the petri dishes is 10 cm in the all cases. The inner and outer dotted circles indicate growth positions of hyphae at 5- and 10-days cultivation, respectively [3].

some hyphae were broken by the electric pulse (**Figure 17(e)** arrow). This suggests that the electric pulse will be a similar stimulation as scratching mycelia on the surface of the sawdust media for mushroom production. Furthermore, it would be possible that new hyphae will be generated after electric pulse stimulation and Kinkaki. Hydrophobin, which is involved in hyphal structure and architecture in fungi [15, 16], would be involved in new hyphae generation after pulse stimulation.

**Figure 18** shows typical photographs 10 days after cultivation at various amplitudes of the applied voltage. The pulsed voltage was applied after 5 days of cultivation of *L. edodes* hyphae. The tip positions of the hyphae after 5 days of cultivation were marked by the inner dotted circles. The hyphae grew from the inner to the outer circle positions after 5 days cultivation from the pulse voltage stimulation. From the microscopic observation, the growth direction of the hyphae changed perpendicular to the surface of the agar medium between the inner and the outer dotted circles as the result of applying a high voltage.
