Open Life Sci. 2017; 12: 76–81

Research Article Open Access

Shuling He, Lingfa Ma, Kentian Zhao*, Jingjun Yang, Yuwei Chang Survey analysis of soil physicochemical factors that influence the distribution of Cordyceps in the Xiahe Region of Province

DOI 10.1515/biol-2017-0008 Received February 16, 2016; accepted September 13, 2016 1 Introduction

Abstract: In this paper, we studied the influence of soil Cordyceps is a fungus form the Clavicipitaceae family. It physicochemical factors on the distribution of Cordyceps. comprises a stroma and sclerotia component resulting We found that in the soil layers at different depths, the from the endoparasitic infection of swift moth larvae [1]. differences in pH values, total N (TN), total P (TP), available Dried fungi-larva sporocarp is highly valued in traditional P (AP) and available K (AK) were not significant, but the Chinese medicine. Sources report that its ingestion has differences in soil water content (WC), soil organic matter positive influence on human immune system. Cordyceps (OM), hydrolysable nitrogen (HN) and AK were significant. is primarily used for treating chronic cough, post-illness In the 5-10 cm layer, WC, pH and TP were significant factors weakness, insomnia, emaciation, impotence, among that influence the distribution of Cordyceps - the number of other conditions [2-4]. The fungi life cycle is extremely Cordyceps was the largest and the characterization of the sophisticated. It undergoes a relatively independent Cordyceps was also the best. TP was an important factor for conidia stage as well as anascospore stage. This combined the first principle component of the soil physicochemical with host swift moth 5-6-year life cycle, 85% of which is properties that influenced the population distribution of underground, result in its extremely peculiar habitat Cordyceps. pH value was an important factor for the the requirements [5]. Increased market demand for Cordyceps second principal component of the soil physicochemical makes its wild environment severely endangered [6]. properties that influenced population distribution, and WC Accordingly, recent research has focused on protecting was an important factor for the third principal component, the wild environment of this fungus. the soil physicochemical properties. This demonstrates that Cordyceps has very strict requirements for ideal the requirement of Cordyceps for specific ranges of WC, soil environmental conditions. It grows primarily on and acidity-alkalinity and AP in soil layers is very high. around the -Tibet Plateau, including Tibet, Qinghai, , Gansu, Yunnan and other provinces. Keywords: Cordyceps, soil environment, soil layer, Normally it can be found on the alpine shrub meadows physicochemical factor, habitat and alpine meadows at 3600-4500 m a.s.l. The suitable soil for its development is the fertile and loose alpine meadow soil with thick soil layer and moderate moisture. The occurrence of Cordyceps is characterized by a very clear zonal and vertical distribution pattern, which is consistent with its host distribution [7-10], which relies *Corresponding author: Kentian Zhao, College of Agricultural and heavily on soil habitat. Rcent research has primarily Animal Husbandry, Tibet University, Linzhi 860000, , E-mail: focused on associated plants, altitude limits, eating [email protected] patterns of swift moth larva and the relationship between Shuling He, Yuwei Chang, College of Agricultural and Animal Hus- soil microorganisms and the formation of Cordyceps [8-15]. bandry, Tibet University, Linzhi 860000, China Shuling He, Lingfa Ma, Jingjun Yang, Institute of High and Cold So far, there are no reports on the soil microenvironment of Ecology, Gansu Normal University for Nationalities, 747000, Cordyceps within its distribution area in the Xiahe Region China of Gansu Province. This paper presents new information Lingfa Ma, Cold and Arid Regions Environmental and Engineering about the relationship between Cordyceps population Research Institute, Chinese Academy of Sciences, 730000, distribution and soil nutrients in this area. We hope the China

© 2017 Shuling He et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Survey analysis of soil physicochemical factors that influence the distribution 77 results will encouragethe environmentally-sensitive use sealed using the diagonal method. After being recorded of of Cordyceps’ natural habitat and the sustainable use ofits the type, they were carried to the laboratory in freshness semi-artificial cultivation. protection packages for physicochemical property detection. We also recoded surrounding environment information, including soil type, altitude, slope gradient, 2 Materials and methods grassland type and major vegetation.

2.1 Research area 2.3 Calculation of the amount of Cordyceps The research region included seven townships in of Gannan Tibetan : We investigated and analyzed every quadrat, including Ganjia Township, Damei Township, Zhayou Township, the Cordyceps number, vegetation component, plant Tanggaang Township, Bola Township, Sangke Township, coverage, density and height, distribution status, growth and Kecai Township. Xiahe County is located in the west status, soil type, and soil physicochemical properties. We edge of the southern part of Gansu Province. The county selected 1 small quadrat from each major plot and dug the is surrounded by Zhuoni County in the east, small quadrat to the depth of 50 cm. Then we inspected the in the south, Linxia Prefecture in the north and Qinghai number of host insects, weighed their biomass, measured Province in the west. The altitude of this region is between the body length of the insect and the length of stroma and 3000 m and 3800 m. The climate there is cold and humid calculated its resource mass. For some major plots, due with clear high plateau climate characteristics. The average the severe damages caused medicine farmers digging, the temperature is 2.6°C, average rainfall is 516 mm, average number of Cordyceps could not be accurately calculated. frost-free period lasts 56 days and annual sunshine time In such cases, we could only estimate it using the number is 2,296 h. The primary vegetation in the region includes of holes left after digging. Gentianaceae, Scrophulariaceae, Compositae, Rosaceae, Polygonaceae, and Gramineae, with a coverage rate of 78.5%. The soil thickness is between 32 and 42 cm and the 2.4 Analysis of the soil physiochemical soil types are primarily alpine meadow soil and dark felty properties soil. (1) The soil organic matter (OM) was tested using the electric sand bath and potassium dichromate titrimetric 2.2 Sample plot selection and soil sample method. (2) The total nitrogen (TN) was tested using collection the Semimicro Macro Kjeldahl method. (3) The total phosphorus in soil (TP) was tested using the NaOH Under the guidance of local medicine farmers, forestry melting method —Mo-Sb anticolorimetric method. (4) The bureaus, Tibetan hospitals and Cordyceps markets, we total potassium (TK) was tested using the NaOH melting chose sampling sites and established plots and visited method- flame photometer method. (5) The hydrolysable them from early May to late June for four consecutive years nitrogen (HN) was tested using the alkaline hydrolysis 2013-2016. Plots were established in regions that reflected diffusion method. (6) The rapidly-available phosphorus Cordyceps distribution characteristics and random (AP) was tested using the sodium bicarbonate method. (7) sampling method was applied. Every year, we selected The rapidly-available potassium (AT) was tested using the 10 new typical 10 m × 10 m plots (major plots). In each ammonium acetate—flame photometer method (1 mol/L major plot, we selected 5 small 1 m × 1 m quadrats through neutral NH4OAc extraction). (8) The water content (WC) the five-point method. 50 small sampling quadrats were was tested using the oven drying method. (9) The soil pH created in this way. A deep hole (30-40 cm) was dug in value was measured using the potentiometry method. teach quadrat. Soil samples at the layers of 0-5, 5-10, 10-15 and 15-20 cm were collected and 3 random samplings were conducted. The soil type was recorded. In the same 2.5 Data analysis sample site, soil samples in 5 quadrates at the layers of 0-5, 5-10, 10-15 and 15-20 cm were mixed, which was then We used DPS 7.5 to analyze, process and conduct principle used as the soil sample of corresponding quadrates. 20 soil component analysis of the data. After we obtained the samples were collected in this way. The samples were then principle component, we conducted a hierarchical cluster 78 S. He, et al. analysis on it to confirm the principle component of the in soil layer 20-25 cm was significantly 85.67% less than soil nutrient selection of the Cordyceps. We then analyzed that in the soil layer 5-10 cm. This indicates that Cordyceps the influence of water content, pH value, organic matter, primarily lives in soil layer 5-10 cm in the Xiahe Region in total nitrogen, hydrolysable nitrogen, total phosphorus, Gannan Tibetan Autonomous Prefecture and that the soil AP and AT on the population distribution of Cordyceps. layer over 20 cm is not suitable for Cordyceps to grow, as suggest the sources [10-13]. 3 Results 3.2 Environmental characteristics of 3.1 Influence of soil depth on the Cordyceps main distribution areas characteristics and number of Cordyceps Table 2 suggests that in Zhayou Township,Tanggaang We found that the numbers of larva differed greatly in Township, Bola Township, Sangke Township and Kecai different soil depths. From Table 1, we can see that as Township and Nawu Township which are located at the soil depth increased, the number of Cordyceps first an upper slope, over 3000 m in altitude and having an increased and then decreased and so did the diameter annual precipitation of 340 mm, the number of Cordyceps and size of Cordyceps. The number of Cordyceps was the is more,In contrast, the number of Cordyceps in Ganjia largest in the soil layer 5-10 cm and was the smallest in Township and Damei Township was less. Vegetations soil layer 20-25 cm. In addition, the number of Cordyceps in the habitat primarily include Gentianaceae,

Table 1. Influence of soil depth on the characteristics and number of Cordyceps.

Soil layer Larva length Larva diameter Stroma length Stroma diameter Larva number [cm] [cm] [mm] [cm] [mm]

0-5 2.31 ± 0.12bB 3.67 ± 0.12aA 2.55 ± 0.09bB 2.46 ± 0.47bB 2.31 ± 0.12bB 5-10 2.67 ± 0.13bB 4.35 ± 0.17aA 3.27 ± 0.43bB 2.78 ± 0.44bB 3.84 ± 0.38cB 15-20 3.84 ± 0.38cB 4.97 ± 0.34bB 4.53 ± 0.23bcB 6.33 ± 0.44aA 2.67 ± 0.13bB 20-25 0.55 ± 0.12abAB 0.65 ± 0.13aA 0.46 ± 0.13bcAB 0.36 ± 0.09cB 0.55 ± 0.12abAB

Table 2. Environmental characteristics of the main distribution areas of Cordyceps.

Environmental Ganjia Damei Township Zhayou Tanggaang Bola Sangke Town- Kecai characteristics Township Township Township Township ship Township

Altitude 2950 2780 3020 3210 3012 3100 3000 [m a.s.l.] Annual average 415 432 332 510 630 450 340 precipitation [mm] Annual average 3.4 4.6 0.3 2.8 1.9 1.2 1.6 temperature [°C] Cordyceps number 1-2 0-2 2-5 2-6 2-5 1-5 2-5 [item/m2] Soil altitudinal Dark brown soil Dark brown soil Brown felty Dark brown soil Brown soil Brown soil Brown soil zone soil Vegetation type Gentianaceae Compositae Papaveraceae Gramineae Primulaceae Gramineae Gentianaceae Scrophulariaceae Papaveraceae Gramineae Rosaceae Gramineae Compositae Gramineae Compositae Leguminosae Rosaceae Equisetaceae Rosaceae Rosaceae Rosaceae Equisetaceae Polygonacee Compositae Compositae Polygonaceae Polygonaceae Compositae Rosaceae Polygonacae Soil type Wet and dark felty Dark felty Dark felty Thin felty cold and brown Cold calcic Cold and dark calcic calcic Slope position Upper Upper Upper Upper Middle Middle Upper Survey analysis of soil physicochemical factors that influence the distribution 79

Scrophulariaceae, Compositae, Equisetaceae, Rosaceae, 3.4 The eigen values and variance contri- Polygonaceae and Gramineae. Soil types primarily bution rates of relevant coefficients in the include wet and dark felty soils, dark felty soils, felty principal component analysis of soil physico- soils, thin felty soils, cold and brown calcic soils, cold chemical properties calcic soils, and cold and dark calcic soil. The primary plants were Kobresia myosuroides, Potentilla anserine, From the principal component analysis (Table 4), we can Equisetum arvense, Bistorta macrophylla, Polygonum see that that the eigen values of the first two principle viviparum, Potentilla fruticosa. The vegetation coverage components were K1 = 6.4908 > 1.0000 and K2 = 1.5868 rate was 75% and the soil thickness was 30-40 cm. > 1.0000 and the contribution rate was the highest; the accumulative contribution rate was 200% > 85%, indicating that the first three principal components 3.3 Physicochemical properties of soils at basically included 100% of the information of the nine different layers parameters (WC, OM, pH, TN, HN, TP, AP, TK and AK) in soil layers 0-5, 5-10, 10-15 and 15-20 cm. We conducted single-factor variance analysis on the physicochemical properties of the soil from 50 quadrates, the results suggests (Table 3) that the differences in pH 3.5 Factor rotation of soil physicochemical value, TN, TP , AP and AK of the three soil layers were not significant (P > 0.05) but that the differences in WC, properties OM, HN and TK were extremely significant (P < 0.01). On the basis of principal component analysis, we selected The content of OM, HN and AK in the soil in the major the first three principal components from the factor distribution area of Cordyceps was high. The pH value matrix after varimax orthogonal rotation and used them gradually decreased as the soil layer depth increased. to calculate the eigenvector of variable. Table 5 suggested Other soil nutrient indicators also exhibited this inverse that AP had the highest correlation coefficient in the first relationship.

Table 3. Investigation on the soil physicochemical properties of different soil layers.

Variables Soil layer [cm]

0-5 5-10 10-15 15-20

Water content (WC) 35.08 ± 0.07cC 33.76 ± 0.06bB 32.32 ± 0.04aA 28.14 ± 0.05dD Organic matter (OM) 40.01 ± 0.05bB 45.28 ± 0.08aA 36.32 ± 0.07cC 30.35 ± 0.10dD pH 6.85.00 ± 0.08aA 6.88 ± 0.12aA 6.89 ± 0.05aA 6.90 ± 0.07aA Total nitrogen (TN) 4.21 ± 0.05aA 4.29 ± 0.07aA 4.18 ± 0.028aA 4.16 ± 0.11aA Hydrolysable nitrogen (HN) 38.72 ± 0.05bB 42.05 ± 0.08aA 35.21 ± 0.07cC 32.20 ± 0.09dD Total phosphorus (TP) 1.51 ± 0.07aA 1.53 ± 0.06aA 1.52 ± 0.06aA 1.48 ± 0.09aA Avidly-available phosphorus (AP) 18.39 ± 0.05aA 18.47 ± 0.05aA 18.34 ± 0.05aA 18.34 ± 0.06aA Total potassium (TK) 4.70 ± 0.07bB 4.89 ± 0.05aA 4.62 ± 0.06cC 4.47 ± 0.06dD Available potassium (AK) 142.01 ± 0.06aA 143.65 ± 0.10aA 142.79 ± 0.05aA 144.82 ± 0.09aA

Note: Lowercase letters indicate significance at P<0.05 Uppercase letters indicatesignificance at P<0.01.

Table 4. The eigen values and variance contribution rates of soil physicochemical properties.

Principal component Eigenvalue Contribution rate /% Accumulative contribution rate /%

1 6.491 72.120 72.120 2 1.587 17.632 89.752 3 0.922 10.248 100 80 S. He, et al. principal component, indicating that an important factor the first four factors that influence the distribution of influencing the soil physiocochemical properties was Cordyceps in this layer were respectively AP > TK > pH AP [7-10]. In the second principal component, pH had a > TN. In soil layer 5-10 cm, PH and AP had an extremely relatively high correlation coefficient, which reflected the significant influence on the distribution of Cordyceps, strict requirement of Cordyceps on the acidity-alkalinity. In WC and OM had a significant influence and the first four the third principal component, WC had the relatively high factors that influence the distribution of Cordyceps in this correlation coefficient, suggesting that WC was an important layer were respectively AP > pH > WC > TK. In soil layers factor that influenced the distribution of Cordyceps in soil. 15-20 cm and 20-25 cm also the first four factors that influence the distribution of Cordyceps in these layers were AP > pH > WC > TK (Table 6). 3.6 The influence of soil physicochemical properties on the distribution of Cordyceps 4 Discussion

After standardizing the original data using the clustering The research results suggested that the water content analysis method, we conducted a statistical analysis of in soil layer 0-5 cm was the highest, which was likely the influence of the physicochemical properties factors of associated with the precipitation in that region. In the three layers of soil on the distribution of Cordyceps. contrast with Maqu and Luqu, the Xiahe Region was The results showed that in soil layer 0-5 cm, AP and TK rather dry. The water content in soil layer 15-20 cm was the had an extremely significant influence on the distribution lowest. The larva of swift moth preferred high humidity of Cordyceps, pH and TN had a significant influence and (soil humidity 40%-46%) [6]. Through the analysis of

Table 5. Transposed matrix of the variable eigenvector of soil habitat of Cordyceps.

Variable The first eigenvector The second eigenvector The third eigenvector

Water content (WC) 0.2567 -0.1985 0.9459 Organic matter (OM) 0.8609 -0.3044 0.4077 pH -0.3113 0.9501 -0.0197 Total nitrogen (TN) 0.9743 -0.0456 0.2207 Hydrolysable nitrogen (HN) 0.8942 -0.3303 0.3023 Total phosphorus (TP) 0.5890 -0.1665 0.7908 Avidly-available phosphorus (AP) 0.9951 -0.0695 0.0709 Total potassium (TK) 0.8972 -0.1992 0.3941 Available potassium (AK) -0.0035 0.8505 -0.5259

Table 6. Influence of the soil physicochemical properties on the distribution of Cordyceps.

Soil layer/cm Variable 0-5 5-10 15-20 20-25

Water content (WC) 0.68bB 0.79bA 0.67cC 0.62cC Organic matter (OM) 0.87aA 0.89aA 0.81bA 0.79bA pH 0.72bA 0.74bB 0.74bB 0.76bA Total nitrogen (TN) 0.74bA 0.89aA 0.72bB 0.71bB Hydrolysable nitrogen (HN) 0.95aA 0.93aA 0.92aA 0.90aA Total phosphorus (TP) 0.92aA 0.95aA 0.91aA 0.88aA Avidly-available phosphorus (AP) 0.95aA 0.99aA 0.95aA 0.95aA Total potassium (TK) 0.63bB 0.72bB 0.82bA 0.82bA Available potassium (AK) 0.72aA 0.97aA 0.95aA 0.93aA Survey analysis of soil physicochemical factors that influence the distribution 81 the physicochemical properties of the soil, we found sustainable use of wild resources and provide theoretical that the soil in which Cordyceps were growing in were references for protecting the grasslands and maintain primarily wet and dark felty soil and wet felty soil, whose ecological balance in the Gannan Plateau. humidity is compatible with biological needs. 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