Biological Characteristics of the Mycelium and Optimization of the Culture Medium for Phallus dongsun

Abstract

This study aimed to elucidate the influence of various culture medium components, including carbon sources, nitrogen sources, inorganic salts, suspension agents, and temperature, on the mycelial growth characteristics of Phallus dongsun. Employing single-factor experiments and response surface methodology within glass Petri dishes, the research identified that carrot powder, soybean powder, and ZnSO₄ notably enhanced the proliferation of aerial mycelium, significantly augmenting the growth rate of P. dongsun mycelium. The resultant mycelium was observed to be dense, robust, and fluffy in texture. In particular, ZnSO₄ markedly accelerated the mycelium growth rate. Furthermore, xanthan gum was found to effectively modulate the medium’s viscosity, ensuring a stable suspension and facilitating nutrient equilibrium. The optimal cultivation temperature was determined to be 25°C, with mycelial growth ceasing below 5°C and mycelium perishing at temperatures exceeding 35°C. The optimal medium composition was established as follows: wheat starch 5 g/l, carrot powder 5 g/l, soybean powder 7.50 g/l, glucose 10 g/l, ZnSO₄ 0.71 g/l, NH₄Cl 0.68 g/l, xanthan gum 0.5 g/l, and agar 20 g/l. Under these optimized conditions, the mycelium of P. dongsun exhibited a rapid growth rate (1.04 ± 0.14 mm/day), characterized by a thick, dense, and well-developed structure. This investigation provides a theoretical foundation for the conservation, strain selection, and breeding of P. dongsun.

Introduction

“Dongsun” denotes a folk term for an edible Phallus species found in Guizhou Province and southwest China (Li et al. 2015; Li et al. 2020). Its nomenclature derives from its growth period during the cooler seasons of autumn and winter. Additionally, it is classified within the same family as Dictyophora. Typically, the “Dongsun” fruiting body, comprising a cap, stalk, and receptacle, is classified under Basidiomycota, Phallo-mycetidae, Phallales, Phallaceae, and Phallus (Li et al. 2020). This species is widely distributed (Melanda et al. 2021; Li et al. 2023). As a culinary ingredient, “Dongsun” is flavorful, crunchy, nutrient-rich, and highly nutritious (Kang et al. 2020). It ranks among the most significant traditional edible mushrooms in China, including P. impudicus, P. fragrans, P. flavocostatus, and P. dongsun, among others (Li et al. 2020). Research indicates that “Dongsun” yields numerous bioactive compounds, such as crude polysaccharides, proteins, amino acids, flavonoids, phenols, fibrin, ergosterol, and mineral elements (Kikuchi et al. 1984; Wu et al. 2013; Li and Bao 2020; Shou et al. 2020). In traditional Chinese medicine, it is attributed with pharmaco logical properties including stimulating blood circulation, alleviating pain, dispersing wind, and eliminating dampness (Dai et al. 2009; Wu et al. 2019; Li and Bao 2020). Furthermore, it exhibits immunomodulatory (Gupta and Shinde 2016; Buko et al. 2019), anticoagulant, anti-edema, antiseptic, anti-tumor, anti-oxidant, anti-cholinesterase, anti-inflammatory, and antibacterial properties (Kuznecov et al. 2007; Kuznecova et al. 2007; Yoon and Lee 2019; Shou et al. 2020; Zheng et al. 2022; Qian et al. 2023). Currently, the functionalities of Phallus species are garnering increasing attention.

“Dongsun” has been artificially cultivated in Guizhou, China, and represents one of the edible mushroom species domesticated in China over the last decade (Li et al. 2023). It is currently cultivated similarly to the Dictyophora species, with its production and scale undergoing expansion (Yu et al. 2020). This mushroom has now been recognized as a product with a National Product of Geographical Indication in China. However, the widely cultivated “Dongsun” had been misidentified for a long time as the European material of P. impudicus. Fortunately, recent studies have confirmed that it constitutes a new species, distinct from any other Phallus taxon, proposed and described as P. dongsun (Li et al. 2020; Yang 2020). The results of this study contribute a new species to the roster of cultivated edible mushrooms within the genus Phallus.

As production and its scale expand, issues such as slow mycelium growth, low production efficiency, and a high contamination rate have become increasingly apparent in the strain production process (Zhang et al. 2021). In previous studies, glucose, fructose, and brown sugar have been identified as favorable carbon sources for P. impudicus or P. dongsun mycelium growth. Additionally, mycelium balls tend to form quickly in liquid culture (Zhang et al. 2021; Huang et al. 2022; Meng et al. 2022). Nitrogen sources are critical for mycelial growth; peptone, yeast powder, yeast extract, and beef extract have been recognized as favorable nitrogen sources for mycelial ball formation in liquid culture (Meng et al. 2022; Huang et al. 2022). Findings indicate that K₂PO₄, KCl, CaCl₂, VB₆, and 6-BA significantly enhance mycelial growth rate and density, whereas MgSO₄ · H₂O and NaCl deter mycelial growth (Zhang et al. 2021). Potato Dextrose Agar (PDA) is a commonly utilized medium for fungal isolation and purification. In the PDA medium, the P. impudicus colony appears white with rhizomorphs. The hyphae are achromatic and transparent, featuring branches, septa, and conspicuous clamps (Lu et al. 2010). However, “Dongsun” tissue blocks easily germinate but fail to germinate or exhibit slow growth in fresh PDA upon transfer to culture. Furthermore, research on the “Dongsun” mother seed medium remains scant. Most practitioners utilize various wood chips as substrates for the “Dongsun” mother seed culture medium, a model characterized by a lengthy production cycle and a high contamination rate, significantly hindering the positive development of the “Dongsun” industry.

In this study, we investigated the effects of carbon sources, nitrogen sources, inorganic salts, suspending agents, and incubation temperature on the biological characteristics of P. dongson in Petri dishes utilizing single-factor experiments and response surface methodology. The aim of this research is to develop an optimal mother culture medium for the growth of P. dongson, thereby providing scientific support for its conservation, strain selection, and large-scale production.

Experimental

Materials and Methods

Test strain

P. dongsun GZCC0351 was isolated and purified from fresh fruiting bodies. The sample was collected from Liulong Town, Dafang County, Bijie City, Guizhou Province, China, and is preserved in the Species Preservation Room at the Guizhou Institute of Biology. Slants were incubated at 25°C on PDA for 7–10 days in glass petri dishes (90 mm in diameter).

Source of materials

All experimental materials were sourced from a consistent supplier and were newly acquired for each use to guarantee substrate stability and uniformity. Brown sugar, wheat bran, carrot powder, potato starch, corn starch, wheat starch, and soybean powder, identified as natural substrates, were procured from Xinghua Lüshuai Food Co., Ltd., (China). Wood powder, a natural substrate, was sourced from Caoxian Luyi Wood Industry Co., Ltd. (China). The following reagents, all of the analytical grade, were acquired from Tianjin Kemiou Chemical Reagent Co., Ltd. (China): KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, MgCl₂ · 6H₂O, ZnSO₄, CaCl₂, CaSO₄ · 2H₂O, Fe₂(SO₄)₃, CaCO₃, NH₄Cl, (NH₄)₂SO₄, KNO₃, NaNO₃, carbamide, glucose, fructose, sucrose, maltose, lactose, cellulose, xylogen, chitosan, dimethyl sulfoxide, and Tween 80. Additionally, all the analytical grade, sodium carboxymethyl cellulose, sodium alginate, gum arabic, and xanthan gum were purchased from Tianjin Yongda Chemical Reagent Co., Ltd. (China). Furthermore, biochemical reagents such as peptone, yeast powder, yeast extract, beef extract, and soybean powder were procured from Shanghai Bowei Biotechnology Co., Ltd. (China).

Base medium (PDA)

Potatoes (200 g) were peeled, sliced thinly, boiled in water until they reached a soft yet firm consistency, and subsequently strained to extract the juice. The extracted juice, along with dextrose (20 g) and agar (20 g), was mixed, and the volume was adjusted to 1000 ml, ensuring the maintenance of the natural pH.

Mycelium culture method

After preparing the medium, it was sterilized at 121°C for 30 minutes, then cooled and aliquoted into 90 mm petri dishes, with a volume of 20 ml/dish. Following solidification, the medium was inoculated with a P. dongsun inoculum block (approximately 5 mm in diameter) and incubated at 25 ± 2°C. Cultivation proceeded in darkness for 28 days in a constant-temperature incubator.

Screening of single factors for nutrient content

Natural substrates

This study aimed to investigate the effects of various natural substrates on the biological properties of P. dongsun mycelium. Substitutes for the base medium (PDA) included 200 g/l peeled potato,wheat bran, and 10 g/l of carrot powder, potato starch, corn starch, wheat starch, and wood powder, respectively. A control group was established by using the base medium without any natural substrate.

Suspension agent

Following prior research outcomes, this study examined the impact of various suspension agents on the medium’s physical state and the biological properties of P. dongsun mycelium. 5 g/l of sodium carboxymethyl cellulose, sodium alginate, gum arabic, xanthan gum, and 5 ml/l of dimethyl sulfoxide, Tween 80 were added to the medium. A control group was employed utilizing the medium without any suspension agent.

Carbon source

In light of prior experimental outcomes, various carbon sources were evaluated for their effects on the biological properties of P. dongsun mycelium. Each medium was supplemented with 20 g/l of glucose, fructose, sucrose, maltose, lactose, brown sugar, cellulose, xylogen, and chitosan. A control group was established using a medium devoid of any carbon source.

Nitrogen source

Drawing upon the findings from previous experiments, diverse nitrogen sources were investigated for their influence on the biological properties of P. dongsun mycelium. The medium was respectively augmented with 10 g/l of peptone, yeast powder, yeast extract, beef extract, soybean powder, and 1 g/l of carbamide, KNO₃, NH₄Cl, (NH4)₂SO₄, NaNO₃. A control group was established with the medium lacking any nitrogen source.

Inorganic salt

Following the outcomes of preceding experiments, various inorganic salts were investigated for their influence on the biological characteristics of P. dongsun mycelium. Each medium was supplemented with 1 g/l of KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · · 7H₂O, MgCl₂ · 6H₂O, ZnSO₄, CaCl₂, CaSO₄ · 2H₂O, Fe₂(SO₄)₃, CaCO₃, NH₄Cl, KNO₃, respectively. A control group was constituted without the addition of any inorganic salt.

Plackett-Burman design

The Plackett-Burman design was employed to identify components that had significant influence on medium optimization. Two natural substrates, one suspension agent, one carbon source, one nitrogen source, and two inorganic salts were investigated for their impact on colony diameter. Utilizing the Plackett-Burman design, a 12-run experiment was conducted to evaluate nine factors, including two virtual variables. Each factor was set at two levels: -1 representing the low level and +1 representing the high level, with the high level being 1.5 times the magnitude of the low level (refer to Table I).

Table I.

Range of factors investigated using Plackett-Burman design.

Symbol	Variables	Experimental value
		Low (-1)	High (+1)
X1	Glucose (g/l)	10	15
X2	Wheat starch (g/l)	5	7.5
X3	Virtual 1	-1	+1
X4	Carrot powder (g/l)	5	7.5
X5	Soybean powder (g/l)	10	15
X6	Virtual 2	-1	+1
X7	ZnSO4 (g/l)	0.5	0.75
X8	NH4Cl (g/l)	0.5	0.75
X9	Virtual 3	-1	+1

Steepest ascent path

Based on the results of the Plackett-Burman design, the steepest ascent method was applied to ZnSO₄ and NH₄Cl to determine the centroid of the response surface design. The main effects, significance, step size, and direction of change of the factors were analyzed through the regression equation of the Plackett-Burman design model.

Response surface optimization experiment

Based on the single-factor design, Plackett-Burman design, and steepest ascent path design, a response surface methodology (central composite design, CCD) encompassing 20 runs was employed. The variables tested (soybean powder, ZnSO₄, NH₄Cl) were designated as A, B, and C, respectively, and were evaluated at five distinct levels, incorporating factorial points (-1, +1), axial points (-1.6818, +1.6818), and a central point (0), as depicted in Table II.

Table II.

Factors and levels of response surface central composite design.

Symbol	Variables	Code level
		-1.6828	–1	0	1	1.6818
A	Soybean powder (g/l)	5.80	7.50	10	12.50	14.20
B	ZnSO4 (g/l)	0.45	0.63	0.88	1.13	1.30
C	NH4Cl (g/l)	0.47	0.57	0.72	0.86	0.98

Testing the temperature gradient

Building upon the outcomes of preceding experiments, the impact of varying temperatures on the biological characteristics of P. dongsun mycelium was investigated. Subsequently, medium plates were inoculated and incubated within a climate chamber at temperatures of 5 ± 2°C, 10 ± 2°C, 15 ± 2°C, 20 ± 2°C, 25 ± 2°C, 30 ± 2°C, and 35 ± 2°C, respectively.

Statistical Analysis

Each experiment was conducted with five replicates. Upon completion of the cultivation period, the colony diameter resulting from mycelial elongation was measured using the criss-cross method. For colonies exhibiting incomplete diameters, measurements were recorded at their widest points (Zhang et al. 2021). Furthermore, the mycelial growth rate was calculated employing the subsequent mathematical equation (1).

$$\begin{array}{l}Mycelialgrowthrate\frac{(mm)}{d}=\hfill \\ =\frac{Colonydiameter(mm)-Inoculumblockdiameter(mm)}{2\times culturetime(d)}\hfill \end{array}$$ 1

Colony diameter and mycelial growth rate were presented as mean values ± standard deviation (SD). An analysis of variance (ANOVA), followed by Tukey’s post hoc test, was utilized to conduct multiple comparisons for significant differences at p < 0.05. The DesignExpert® software, version 13.0.1.0 (StatEase Inc., USA, www.statease.com), was employed to design experiments, regression, and graphical analysis of the data obtained. Bar and line graphs were generated using the OriginPro® 2021 software package, version 9.8.0.200 (OriginLab Corporation, USA).

Results

Effect of natural substrates on the biological properties

The effect of natural substrates on the biological properties was investigated. Adding wood powder to the medium resulted in the fastest mycelial growth and largest colony diameter (0.85 ± 0.13 mm/d), followed by wheat bran, wheat starch, and carrot powder. However, the differences among these substrates were not statistically significant. Upon adding carrot powder, mycelial growth was observed to be both dense and vigorous, in contrast to the other test groups, which exhibited either dense or moderately dense growth. Adding carrot powder, wood powder, and wheat bran to the medium facilitated the most vigorous mycelial growth. The poorest mycelial growth was observed with the addition of potato powder. Considering colony diameter, growth density, and overall mycelial development, the incorporation of wheat bran, wood powder, wheat starch, and carrot powder into the medium proved to be beneficial for the growth of P. dongsun mycelium (Table III, Fig. 1). The study aimed to investigate the effects of composite natural substrates on the biological characteristics of P. dongsun mycelium, with wheat starch, carrot powder, and wood powder being added individually or in combination to the culture media (Table IV). Results indicated that the FA4 treatment exhibited the fastest mycelial growth rate and the largest colony diameter (67.07 ± 4.79 mm). FA4 and FA6 treatments demonstrated the most vigorous mycelial growth, albeit with sparse colony density. The FA5 treatment resulted in the densest mycelial density and superior growth. In conclusion, the FA5 treatment was identified as the optimal natural substrate combination, comprising 10 g/l wheat starch and 10 g/l carrot powder (Table IV, Fig. 2).

Table III.

Effects of natural substances on the growth of Phallum dongsun.

Natural substance	Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
Control group	29.64 ± 7.07de	0.44 ± 0.13de	#	++
Peeled potato	20.75 ± 0.79e	0.28 ± 0.01e	##	+
Carrot powder	42.17 ± 3.23bcd	0.66 ± 0.06bcd	###	+++
Wood powder	54.75 ± 5.1a	0.88 ± 0.09a	##	+++
Wheat bran	52.71 ± 7.22ab	0.85 ± 0.13ab	##	++++
Potato starch	37.63 ± 3.63bcd	0.58 ± 0.06bcd	#	+
Corn starch	37.58 ± 5.35bcd	0.58 ± 0.10bcd	#	+
Wheat starch	48.12 ± 9.48abc	0.77 ± 0.17abc	##	++

¹Different small letters in the same column indicate significance at the 5% level

¹#– sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 1.

Effects of natural substances on the growth of Phallus dongsun.

A – control group, B – peeled potato, C – carrot powder, D – wood powder, E – wheat bran, F – potato powder, G – corn starch, H – wheat starch

Table IV.

Effects of composite natural substances on the growth of Phallus dongsun.

Test group	Natural substance (g/l)				Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
	Peeled potato	Wheat starch	Carrot powder	Wood powder
FA0	200	0	0	0	26.78 ± 4.76c	0.38 ± 0.09c	###	++
FA1	0	20	0	0	56.19 ± 6.44b	0.91 ± 0.12b	##	+++
FA2	0	0	20	0	51.57 ± 7.10b	0.83 ± 0.13b	##	+++
FA3	0	0	0	20	34.34 ± 4.57c	0.52 ± 0.08c	#	++
FA4	0	10	0	10	67.07 ± 4.79a	1.10 ± 0.09a	#	+++
FA5	0	10	10	0	52.35 ± 5.21b	0.84 ± 0.09b	###	++++
FA6	0	0	10	10	56.19 ± 8.14b	0.91 ± 0.15b	#	++++
FA7	0	10	10	10	50.66 ± 9.46b	0.81 ± 0.17b	##	+++

¹Different small letters in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 2.

Effects of composite natural substances on the growth of Phallus dongsun.

A – FA0, B – FA1, C – FA2, D – FA3, E – FA4, F – FA5, G – FA6, H – FA7

Effect of suspension agent on the biological properties

When suspension agents were added to the medium, P. dongsu mycelium grew adherent to the surface of the medium with less aerial mycelium, resulting in faster mycelial growth, denser colonies, and vigorous growth. However, there were differences in the rate of mycelial growth and overall growth (Table V, Fig. 3). A medium suspension can be formed when sodium carboxymethyl cellulose, sodium alginate, gum arabic, and xanthan gum are added. However, only with the addition of xanthan gum do the medium become more viscous and the suspension stabilize. Adding dimethyl sulfoxide and Tween 80 to the medium resulted in no suspension formation and led to medium precipitation. The results indicated that xanthan gum is the best-suspending agent. A higher concentration of xanthan gum significantly affected the viscosity of the medium. Varying concentrations of xanthan gum had negligible effects on the growth of P. dongsu mycelium, which all exhibited average, vigorous growth and high colony density (Table VI, Fig. 4). However, considering the physical state of the medium, increased concentrations of xanthan gum made it more difficult to disperse and increased the viscosity, adversely affecting the medium’s partitioning. In summary, an optimal concentration of 0.5 g/l xanthan gum was determined for the culture medium.

Table V.

Effects of suspension agents on the physical state of the medium and the growth of Phallus dongsun.

Suspension agent	Medium physical state	Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
Control group	Sediment	44.45 ± 6.Γ’	0.70 ± 0.11**	##	+++
Sodium carboxylmethyl cellulose (g/l)	Unstable suspension	45.05 ± 8.48*	0.72 ± 0.15*	##	++++
Sodium alginate (g/l)	Unstable suspension	44.10 ± 8.64*	0.70 ± 0.15*	###	++++
Dimethyl sulfoxide (ml/l)	Sediment	33.11 ± 8.35c	0.50 ± 0.15c	##	++++
Tween 80 (ml/l)	Sediment	38.95 ± 6.89bc	0.61 ± 0.12bc	#	++++
Gum arabic (g/l)	Unstable suspension	50.73 ± 5.4a	0.82 ± 0.1a	##	++++
Xanthan gum (g/l)	Stable suspension, high viscosity	48.03 ± 6.06*	0.77 ± 0.11*	###	++++

¹Different small letters in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 3.

Effects of suspension agents on the physical state of the medium and the growth of Phallus dongsun.

A – control group, B – sodium carboxylmethyl cellulose, C – sodium alginate, D – dimethyl sulfoxide, E – Tween 80, F – gum arabic, G – xanthan gum

Table VI.

Effects of xanthan gum concentration on the physical state of the medium and the growth of Phallus dongsun.

Xanthan gum (g/l)	Medium physical state	Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
Control group	Sediment	60.41 ± 7.95a	0.98 ± 0.14a	##	+++
0.5	Stable suspension, Suitable viscosity	65.34 ± 6.52a	1.08 ± 0.12a	##	+++
1.0	Stable suspension, Suitable viscosity	66.04 ± 5.46a	1.09 ± 0.10a	##	+++
1.5	Stable suspension, Higher viscosity	62.63 ± 7.24a	1.03 ± 0.13a	##	+++
2.0	Stable suspension, Higher viscosity	61.06 ± 5.46a	1.00 ± 0.10a	##	+++
2.5	Stable suspension, high viscosity	56.23 ± 6.40a	0.91 ± 0.11a	##	+++
3.0	Stable suspension, high viscosity	60.68 ± 4.23a	0.99 ± 0.08a	#	+++

¹Small letter in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 4.

Effects of xanthan gum concentration on the physical state of the medium and the growth of Phallus dongsun.

A – Control group, B – 0.5 g/l, C – 1.0 g/l, D – 1.5 g/l, E – 2.0 g/l, F – 2.5 g/l, G – 3.0 g/l

Effect of carbon source on the biological properties

When glucose, maltose, and cellulose were utilized as carbon sources in the culture, P. dongsun mycelium exhibited rapid growth, high colony density, and an optimal growth rate, notably with glucose, which demonstrated a mycelial growth rate of 0.77 ± 0.06 mm/d. When fructose, sucrose, lactose, and brown sugar served as carbon sources, the mycelium grew and maintained a high density; however, its performance was comparable to the control group, showing no significant difference. Utilizing chitosan as a carbon source resulted in suboptimal and slower mycelium growth, which was inferior to the control; conversely, when lignin served as the carbon source, the mycelium failed to germinate and grow (Table VII, Fig. 5). In conclusion, glucose was determined to be the optimal carbon source.

Table VII.

Effects of carbon sources on the growth of Phallus dongsun.

Carbon source (g/l)	Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
Control group	36.84 ± 1.29bcd	0.56 ± 0.02bcd	##	++
Glucose	48.32 ± 3.63a	0.77 ± 0.06a	###	+++
Fructose	37.92 ± 6.61bcd	0.58 ± 0.12bcd	##	++
Sucrose	32.43 ± 3.27d	0.49 ± 0.06d	##	+++
Maltose	48.18 ± 2.97ab	0.77 ± 0.05ab	###	+++
Lactose	37.13 ± 6.36bcd	0.57 ± 0.11bcd	###	++
Brown sugar	36.57 ± 4.02cd	0.56 ± 0.07cd	##	+++
Cellulose	47.89 ± 5.17abc	0.76 ± 0.09abc	##	+++
Xylogen	0.00 ± 0.00f	0.00 ± 0.00f	n.d.	n.d.
Chitosan	18.07 ± 6.54e	0.23 ± 0.12e	##	+

¹Different small letters in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth, n.d. – no data for the experimental group

Fig. 5.

Effects of carbon sources on the growth of Phallus dongsun.

A – control group, B – glucose, C – fructose, D – sucrose, E – maltose, F – lactose, G – brown sugar, H – cellulose, I – xylogen, J – chitosan

Effect of nitrogen source on the biological properties

Nitrogen source screening experiments demonstrated that the NaNO₃ exhibited the fastest mycelial growth rate (0.74 ± 0.09 mm/d), with vigorous growth, yet the colony density was notably sparse. In contrast, the soybean powder demonstrated a faster mycelial growth rate (0.69 ± 0.09 mm/d), experiencing vigorous growth and dense colonies; however, the difference from the NaNO₃ was minimal. Adding peptone, yeast powder, yeast paste, and carbamide to the culture medium significantly inhibited the growth of P. dongsun mycelium, resulting in a slow growth rate and suboptimal growth (Table VIII, Fig. 6). In conclusion, soybean powder was determined to be the best nitrogen source.

Table VIII.

Effects of nitrogen sources on the growth of Phallus dongsun.

Nitrogen source (g/l)	Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
Control group	36.01 ± 5.30c	0.55 ± 0.09c	##	+++
Peptone	19.16 ± 4.26cd	0.25 ± 0.08cd	##	+
Yeast powder	9.16 ± 2.35cd	0.07 ± 0.04cd	##	+
Yeast extract	5.72 ± 0.63d	0.01 ± 0.01d	#	+
Beef extract	39.86 ± 5.18*	0.62 ± 0.09*	##	++
Soybean powder	44.18 ± 5.17*	0.69 ± 0.09*	###	+++
KNO3	40.62 ± 4.33*	0.63 ± 0.08*	##	+++
Carbamide	5.70 ± 0.16d	0.01 ± 0.00d	#	+
NH4Cl	37.73 ± 5.98*	0.58 ± 0.11*	##	++
(NH4)2SO4	35.66 ± 3.32b	0.54 ± 0.06b	##	++
NaNO3	46.49 ± 5.31a	0.74 ± 0.09a	#	+++

¹Different small letters in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 6.

Effects of nitrogen sources on the growth of Phallus dongsun.

A – control group, B – peptone, C – yeast powder, D – yeast extract, E – beef extract, F – soybean powder, G – KNO₃, G – carbamide, I – NH₄Cl, J – (NH4)2SO₄, K – NaNO₃

Effect of inorganic salt on the biological properties

Inorganic salt screening experiments indicated that the ZnSO₄ exhibited the fastest mycelial growth rate (0.80 ± 0.16 mm/d), characterized by vigorous growth, and was followed by NH₄Cl (0.76 ± 0.13 mm/d) and Fe2(SO₄)₃ (0.65 ± 0.18 mm/d). Incorporating MgSO₄ · 7H₂O and CaCO₃ into the culture medium significantly inhibited the growth of P. dongsun mycelium (Table IX, Fig. 7). To explore the effects of composite inorganic salts on the biological characteristics of P. dongsun mycelium, ZnSO₄, NH₄Cl, and Fe₂(SO₄)₃ were each added either individually or in combination to the culture media (Table X). The findings revealed that FM-F demonstrated the fastest mycelial growth rate (1.00 ± 0.13 mm/d). In conclusion, FM-F emerged as the optimal inorganic salt combination, comprising ZnSO₄ 0.5 g/l and NH₄Cl 0.5 g/l (Table X, Fig. 8).

Table IX.

Effects of inorganic salts on the growth of Phallus dongsun.

Inorganic salt (g/l)	Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
Control group	40.00 ± 7.53abc	0.62 ± 0.13abc	##	+++
KH2PO4	33.66 ± 8.64bcd	0.51 ± 0.15bcd	##	+++
K2HPO4·3H2O	36.14 ± 5.64bcd	0.55 ± 0.10bcd	###	++
MgSO4·7H2O	29.51 ± 4.16cd	0.43 ± 0.07cd	##	++
MgCl2·6H2O	39.37 ± 4.56abc	0.61 ± 0.08abc	##	++
ZnSO4	50.14 ± 9.16a	0.80 ± 0.16a	###	++++
CaCl2	37.11 ± 8.19abc	0.57 ± 0.15abc	###	+++
CaSO4·2H2O	37.09 ± 4.72abcd	0.57 ± 0.08*cd	#	++
Fe2(SO4)3	41.85 ± 9.97abc	0.65 ± 0.18abc	###	++++
CaCO3	20.79 ± 2.32d	0.28 ± 0.04d	#	+
NH4Cl	48.00 ± 7.14ab	0.76 ± 0.13*	###	+++
KNO3	40.40 ± 6.27abc	0.63 ± 0.11**	###	++

¹Different small letters in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 7.

Effects of inorganic salts on the growth of Phallus dongsun.

A – control group, B – KH₂PO₄, C – K₂HPO₄ · 3H₂O, D – MgSO₄ · 7H₂O, E – MgCl₂ · 6H₂O, F – ZnSO₄, G – CaCl₂, H – CaSO₄ · 2H₂O, I – Fe₂(SO₄)₃, J – CaCO₃, K – NH₄Cl, L – KNO₃

Table X.

Effects of composite inorganic salt on the growth of Phallus dongsun.

Test group	Inorganic salt (g/l)			Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
	ZnSO4	Fe2(SO4)3	NH4Cl
FM-A	0	0	0	42.23 ± 8.15b	0.66 ± 0.15b	##	++
FM-B	1	0	0	50.04 ± 4.62ab	0.80 ± 0.08ab	###	+++
FM-C	0	1	0	39.01 ± 2.45b	0.61 ± 0.04b	##	++
FM-D	0	0	1	39.07 ± 0.24ab	0.61 ± 0.00ab	##	++
FM-E	0.5	0.5	0	49.51 ± 6.03ab	0.79 ± 0.11ab	###	++
FM-F	0.5	0	0.5	61.10 ± 7.26a	1.00 ± 0.13a	###	++++
FM-G	0	0.5	0.5	50.05 ± 8.99ab	0.80 ± 0.16ab	###	++
FM-H	0.5	0.5	0.5	44.52 ± 5.92b	0.71 ± 0.11b	###	+++

¹Different small letters in the same column indicate significance at the 5% level

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth

Fig. 8.

Effects of composite inorganic salts on the growth of Phallus dongsun.

A – FM-A, B – FM-B, C – FM-C, D – FM-D, E – FM-E, F – FM-F, G – FM-G, H – FM-H

Screening of important variables using Plackett-Burman design

The data indicated that process optimization plays a crucial role in enhancing the efficiency of colony diameter (Table XI). Analysis of the regression coefficients for 9 factors (Table XII) revealed that X₁, X₇, and X₈ positively affected colony diameter. X2, X4, and X₅ demonstrated negative impacts. Variables affecting a confidence level above 95% are deemed significant factors. Based on these results (Table XII), two factors, X₇ (ZnSO₄) and X₈ (NH₄Cl), were identified as significant for the colony diameter of P. dongsun mycelium.

Table XI.

Plackett-Burman design and response values.

Run	Variables								Y Colony diameter (mm)
	X1	X2	X3	X4	X5	X6	X7	X8
1	1	1	-1	1	1	1	-1	-1	49.92 ± 3.47
2	-1	1	1	-1	1	1	1	-1	59.97 ± 6.54
3	1	-1	1	1	-1	1	1	1	74.45 ± 3.25
4	-1	1	-1	1	1	-1	1	1	60.35 ± 2.28
5	-1	-1	1	-1	1	1	-1	1	59.05 ± 4.68
6	-1	-1	-1	1	-1	1	1	-1	61.95 ± 5.93
7	1	-1	-1	-1	1	-1	1	1	63.52 ± 4.59
8	1	1	-1	-1	-1	1	-1	1	58.35 ± 4.82
9	1	1	1	-1	-1	-1	1	-1	62.39 ± 4.49
10	-1	1	1	1	-1	-1	-1	1	53.93 ± 6.15
11	1	-1	1	1	1	-1	-1	-1	50.62 ± 4.74
12	-1	-1	-1	-1	-1	-1	-1	-1	48.70 ± 2.69

¹X₁ –X₈ code values are shown in Table I

Table XII.

Results of the regression analysis of the Plackett–Burman design.

Source	Coefficient estimate	df	Sum of squares	Means quares	F–value	p –value (Prob > F)
Model	58.60	6	486.42	81.07	5.14	0.0464*
X1	1.28	1	19.53	19.53	1.24	0.3162
X2	-1.11	1	14.90	14.90	0.9453	0.3756
X4	-0.0640	1	0.0491	0.0491	0.0031	0.9576
X5	-1.36	1	22.24	22.24	1.41	0.2882
X7	5.17	1	321.04	321.04	20.37	0.0063**
X8	3.01	1	108.66	108.66	6.90	0.0468*
Residual		5	78.79
Cor Total		11	565.20

¹* – significant at 0.05 level, ** – significant at 0.01 level

Table XIII.

Experimental design and results of the steepest ascent path.

Factor level	Inorganic salt (g/l)		Colony diameter (mm)	Mycelium growth rate (mm/d)	Colony density	Hyphal growth
	ZnSO4	NH4Cl
Step size (△)	0.125	0.073	n.d.	n.d.	n.d.	n.d.
Origin (O)	0.5	0.5	48.13 ± 3.00	0.77 ± 0.05	###	+++
O + 1 △	0.625	0.573	52.99 ± 5.85	0.85 ± 0.10	###	+++
O + 2 △	0.750	0.646	54.20 ± 7.01	0.87 ± 0.13	###	+++
O + 3 △	0.875	0.719	61.07 ± 2.20	1.00 ± 0.04	###	++++
O + 4△	1.000	0.792	60.43 ± 10.18	0.99 ± 0.18	###	++++
O + 5△	1.125	0.865	59.56 ± 4.15	0.88 ± 0.30	###	++++
O + 6△	1.250	0.938	48.82 ± 4.68	078 ± 0.08	###	++++
O + 7△	1.375	1.011	51.52 ± 9.49	0.83 ± 0.17	###	+++
O + 8 △	1.500	1.084	32.24 ± 6.24	0.48 ± 0.11	###	++
O + 9△	1.625	1.157	35.39 ± 12.32	0.54 ± 0.22	###	+++
O + 10△	1.75	1.230	29.47 ± 15.78	0.43 ± 0.28	##	+
O + 11△	1.875	1.303	26.49 ± 8.02	0.38 ± 0.14	##	+
O + 12△	2.00	1.376	23.38 ± 7.40	0.32 ± 0.13	##	+

¹# – sparse colony density, ## – dense colony density, ### – densest colony density, + – weak mycelial growth, ++ – moderate mycelial growth, +++ – favorable mycelial growth, ++++ – strong mycelial growth, n.d. – no data for the experimental group

Steepest ascent path

Based on the coefficients from the colony diameter regression equation derived from the Plackett-Burman design model, the steepest ascent path for optimizing colony diameter involved primarily adjusting the levels of ZnSO₄ and NH₄Cl. The magnitude and direction of changes were dictated by the regression equation from the Plackett-Burman design model, as illustrated in Table XIII. Colony diameter reached its peak at the “ O + 3Δ” factor level before decreasing. This “O + 3Δ” factor level, marking the maximum response, was subsequently chosen as the central point for the central composite design.

Response surface optimization experiment

To assess the impact of medium components on colony diameter, an evaluation involving soybean powder, ZnSO₄, and NH₄Cl was conducted using a response surface optimization experiment. Detailed experimental designs and results are presented in Table XIV. A regression analysis was conducted to align the response function with the experimental data. Based on the variables presented in Table XV, the model, denoted as equation (2), articulates the colony diameter (Y) in relation to the concentrations of soybean powder (A), ZnSO₄ (B), and NH₄Cl (C):

$$\begin{array}{c}Y=62.04-3.24\times A-1.15\times B-0.23\times C+0.16\times AB+1.14\times \\ \times AC-0.81\times BC-1.40\times A^2-0.32\times {\text{B}}^2-0.96\times C^2\end{array}$$ 2

Table XIV.

Response surface central composite design and matching results.

Run	Variables			Y Colony diameter (mm)
	A	B	C
1	-1	-1	-1	64.56 ± 5.88
2	1	-1	-1	54.69 ± 4.98
3	-1	1	-1	62.58 ± 7.79
4	1	1	-1	54.35 ± 7.41
5	-1	-1	1	63.78 ± 5.37
6	1	-1	1	59.50 ± 4.29
7	-1	1	1	59.59 ± 9.21
8	1	1	1	54.91 ± 3.34
9	-1.6818	0	0	63.33 ± 3.93
10	1.6818	0	0	53.13 ± 4.86
11	0	-1.6818	0	62.65 ± 4.13
12	0	1.6818	0	59.95 ± 4.14
13	0	0	-1.6818	60.88 ± 1.57
14	0	0	1.6818	58.10 ± 4.80
15	0	0	0	62.62 ± 2.36
16	0	0	0	61.43 ± 1.60
17	0	0	0	61.72 ± 7.81
18	0	0	0	64.65 ± 4.31
19	0	0	0	59.99 ± 2.18
20	0	0	0	61.76 ± 1.83

¹A, B and C code values are shown in Table II

Table XV.

ANOVA for response surface quadratic polynomial model.

Source	df	Sum of squares	Means quares	F-value	p-value (Prob > F)
Modle	215.88	9	23.99	13.54	0.0002**
A – Soybean powder	143.14	1	143.14	80.81	< 0.0001**
B – ZnSO4	17.91	1	17.91	10.11	0.0098**
C – NH4Cl	0.6925	1	0.6925	0.3910	0.5458
AB	0.1922	1	0.1922	0.1085	0.7487
AC	10.44	1	10.44	5.89	0.0356*
BC	5.22	1	5.22	2.94	0.1169
A 2	28.37	1	28.37	16.02	0.0025**
B 2	1.46	1	1.46	0.8214	0.3861
C 2	13.22	1	13.22	7.46	0.0211*
Residual	17.71	10	1.77
Lack of Fit	5.81	5	1.16	0.4882	0.7750
Pure Error	11.90	5	2.38
Cor Total	233.60	19
R 2	0.9242
Adjusted R2	0.8559

¹* – significant at 0.05 level, ** – significant at 0.01 level

The F -test analysis of variance (ANOVA) results demonstrated that the regression achieved statistical significance at the 99% confidence level (Table XV), with a high goodness of fit value (R² = 0.9242), signifying the model’s accurate representation of the relationship between the investigated factors and response values. The “ F- value” for “Lack of Fit” was found to be insignificant, indicating the model’s retention of fit. Furthermore, the linear terms of A and B, the interaction term of AC, and the quadratic terms of A² and C² were identified as significant model terms for colony diam-eter. The findings reveal that the maximum values for colony diameter are attained through the regression equation and two-dimensional contour plots (Fig. 9). The model forecasted a maximum colony diameter of 64.75 mm at the optimized medium component levels of soybean powder 7.50 g/l, ZnSO₄ 0.71 g/l, and NH₄Cl 0.68 g/l. Validation testing demonstrated the rapid growth of P. dongsun mycelium, with the colony diameter and mycelial growth rate achieving 63.00 ± 3.89 mm and 1.04 ± 0.14 mm/d at 28 days, respectively. These outcomes were closely aligned with the model’s predicted values and can facilitate the evaluation of medium components and mycelial growth in P. dongsun.

Fig. 9.

Two-dimensional contour plots of response surface on mycelial growth of Phallus dongsun.

a – soybean powder vs. ZnSO₄, b – soybean powder vs. NH₄Cl, c – ZnSO₄ vs. NH₄Cl

The impact of various temperatures on the biological characteristics

The impact of various temperatures on the biological characteristics of P. dongsun mycelium was significant, as illustrated in Fig. 10 and 11. The findings indicated that under culture conditions at 20 and 25°C, the colonies were dense, vigorous, and robust, significantly surpassing those of other treatments. Mycelium growth was slow and weak under culture conditions at 10, 15, and 30°C. Furthermore, at 35°C, the mycelium failed to germinate, and the mycelium on the inoculum block subsided, indicating death. In conclusion, the optimal temperature range for the growth of P. dongsun mycelium is 20–25°C, with growth ceasing below 5°C and death occurring above 35°C.

Fig. 10.

Effects of different temperature on colony diameter and mycelial growth rate of Phallus dongsun.

Fig. 11.

Effects of different temperature on mycelial growth of Phallus dongsun.

A – 5°C, B – 10°C, C – 15°C, D – 20°C, E – 25°C, F – 30°C, G – 35°C

Discussion

This investigation focused on how medium nutrients, the physical state of the medium, and temperature gradients influence the biological characteristics of P. dongsun mycelium. In the carbon source screening phase, adding natural substrates (wheat starch and carrot powder) and a single carbon source (glucose) to the culture medium resulted in the dense, thick, and rapid growth of P. dongsun mycelium. This suggests that P. dongsun can utilize monosaccharides, disaccharides, and polysaccharides derived from natural ingredients. This outcome slightly diverges from prior research, which identified brown sugar as the optimal carbon source due to its content of vitamins, amino acids, trace elements, and other nutrients conducive to mycelial growth (Zhang et al. 2021). The study concluded that P. dongsun mycelium extensively utilizes various carbon sources. Incorporating wheat starch, carrot powder, and glucose into the medium, which collectively encompasses mono-, di-, and polysaccharide forms, ensures a continuous and efficient carbon supply, significantly enhancing the growth of P. dongsun mycelium.

According to previous studies, organic nitrogen sources, particularly yeast paste, and peptone, foster denser and more rapidly growing mycelium than inorganic nitrogen sources (Zhang et al. 2021; Huang et al. 2022). This research found NaNO₃ to accelerate mycelial growth, while soya flour contributed to denser and thicker mycelium. Conversely, peptone, yeast powder, yeast paste, and urea were found to inhibit mycelial growth. Inorganic salt screening indicated that ZnSO₄, Fe₂(SO₄)₃, and NH₄Cl enhanced P. dongsun mycelial growth, aligning with previous findings (Zhang et al. 2021), whereas magnesium sulfate was inhibitory. This nuanced understanding of how different nitrogen sources and inorganic salts affect mycelial growth can guide future cultivation strategies for P. dongsun. It was also observed that NaNO₃, KNO₃, NH₄Cl, (NH4)₂SO₄,ZnSO₄, and Fe₂(SO₄)₃ enhanced mycelial growth, with the compositions containing NO₃-, NH4+, and SO₄2-either individually or in combination. Nitrogen is a critical factor in plant growth and development, with NO₃-and NH4+ serving as plants’ primary sources of inorganic nitrogen. NH4+ can be directly utilized in nitrogen assimilation following its uptake by plants. NO₃-must undergo an energy-intensive reduction process to be assimilated by plant cells (Xiao et al. 2023). Elemental sulfur, a vital component of cells, exists in diverse forms within living organisms and the environment, playing crucial roles in signaling, redox balance, gene expression promotion, and the maintenance of fundamental metabolic processes (Wasmund et al. 2017). Microorganisms play pivotal roles in all significant sulfur meta bolic pathways and are key drivers of the sulfur biocycle (Wang et al. 2022). Consequently, NO₃^-, NH₄⁺, and SO₄^2- are involved in nitrogen and sulfur metabolism during P. dongsun growth and their underlying biological mechanisms warrant further investigation

Upon incorporating sodium carboxymethyl cellulose and sodium alginate into the liquid medium, Morchella sextelata mycelium balls exhibited homogeneity and high biomass (Zheng et al. 2020). This study revealed that adding carrot, wheat starch, and soya flour to the medium led to precipitation and nutrient imbalance. Including xanthan gum in the medium resulted in a low concentration, stable suspension, reduced propensity for precipitation, and favorable mycelium growth. Suspension aids, or thickeners, can significantly alter the viscosity of liquids and also modify the morphology of fungal hyphae (Dahlstrom et al. 2000).

In the one-factor temperature test, mycelial growth rate and momentum initially increased and decreased as the temperature rose. At the optimal temperature of 25°C, the mycelium exhibited its fastest growth; at temperatures below 5°C, growth ceased, and at temperatures above 35°C, the mycelium perished, aligning with the findings of Luo and Chen (2016). The study demonstrated that temperature stress altered the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in macrofungal mycelium. This imbalance in intracellular reactive oxygen species (ROS) metabolism and subsequent accumulation of free radicals inhibited mycelial growth, as Liu et al. (2010) observed.

This study evaluated the composition, physical state, and temperature gradient of the P. dongsun medium using single-factor experiments, Plackett-Burman design, and response surface methodology. The results indicated that an optimized medium composition comprising 5 g/l wheat starch, 5 g/l carrot powder, 7.50 g/l soybean powder, 10 g/l glucose, 0.71 g/l ZnSO₄, 0.68 g/l NH₄Cl, 0.5 g/l xanthan gum, 20 g/l agar, and an incubation temperature of 25°C resulted in vigorous, uniform, thick, and dense growth of P. dongsun mycelium, with a growth rate of 1.04 ± 0.14 mm/d. These findings offer a theoretical foundation for the conservation, selection, and breeding of P. dongsun strains, and scientific support for its large-scale and industrial production.