# Addition of fallen wood from Pinus massoniana improved the growth of Plagiomnium acutum in a substrate culture

### Experimental site

The experiment was carried out from March to July 2021 at the West Campus of Guizhou University located in Guiyang, Guizhou Province, China (106°65′ E, 27°22′ N), which has a subtropical climate, typical humid and mild, with plateau and monsoon characteristics. The average annual precipitation, average annual temperature, average annual relative humidity and average annual sunshine hours in Guiyang were 1129.5mm, 15.3℃, 77% and 1148.3h respectively. Temperature and humidity changes were recorded with a thermohygrometer at 8:00-9:00 a.m., 12:00-1:00 p.m., and 5:00-6:00 p.m. each day during the experiment (Fig. 8). P.acutum preferred a shaded one and the proper light range for this is 70% to 90% shade30, so the cultivation area was treated with three layers of shading net to ensure that the shading rate was over 70%. Spray and moisturize daily according to the actual situation.

### Growing medium and plant material

The soil substrate includes granular soil, general purpose nutrient soil, charcoal soil and garden soil. P. massoniana fallen wood was collected from the West Campus of Guizhou University and divided into different degrees of decomposition (Table 2) with respect to the classification and criteria for determining the decomposition degrees of forest coarse wood residues by studies previous13,31,32. The mosses were harvested from Guanshan Lake Park in Guiyang City, Guizhou Province.

### Experimental design

The soils were disinfected by exposure to the sun for 7 days and the bark, sapwood and heartwood of fallen wood were removed and then ground and mixed separately before planting. Two groups of treatments were implemented in the cultivation trials (Table 3), namely the mixed equal-volume soil-soil homogeneous mixed (SS) treatment and the mixed equal-volume mixed soil-wood homogeneous treatment (SW). . After laying the non-woven fabric in the planting pots, put the same volume of mixed substrate in the pots and water thoroughly before transplanting. The cultivation of moss was done by culturing gametophytes. The cultivation method borrowed from the mixed sowing method (area method + weight method) of Sun33that is, the foam crushed by a crusher was weighed, and then it was evenly laid flat on the substrate with an area of ​​4 cm × 4 cm, and each treatment was repeated three times.

### Indicator measurement method

#### Determination of the index of the growing medium

Indices of physical and chemical properties of the substrate include bulk density, water-holding capacity, total porosity, aeration porosity, water-holding porosity, and void ratio, which were measured before culture, relying on the determination methods of Li et al.34 and Zhao et al.35. Indicators of nutrient properties include total nitrogen, total phosphorus and total potassium. The solution to be measured was prepared by H2SO4-HCLO4 decoction method, the total nitrogen content was determined by the CleverChem automatic interrupted chemical analyzer, the phosphorus content was determined by the colorimetric molybdenum yellow method, and the total potassium content was determined by the photometric method of flame.

#### Determination of growth and physiological indices of P.acutum

Length measurement: After 30 days of cultivation, ten mosses were randomly selected for each treatment every 10 days and their length was measured (accuracy 0.1 cm).

Cover measurement: After 30 days of cultivation, take a photo every 10 days, then measure the moss area with AutoCAD 2016 plotting, and the moss cover calculation formula was as follows.

$${text{C}} = left( {{text{A}}/{text{B}}} right) times 100% .$$

Formula: C-moss blanket; A-zone foam; Planting area B.

#### Determination of physiological indicators

Determination of chlorophyll content in P.acutum refers to the determination method of Bao et al.36, with slight modifications. After 120 days of growth in different growing mediums, the moss was removed, washed and drained for later use, 0.2 g of new branches were weighed and placed in a 10 ml centrifuge tube. Then 8 ml of 95% ethanol was added to the centrifuge tube. After 24 h of extraction in the dark, the liquid in the centrifuge tube and the foam residue were filtered several times and fixed in a 25 ml brown volumetric flask, then the extract was poured into a colorimetric dish of 1 cm optical diameter. Finally, absorbance at wavelengths 665 nm and 649 nm was measured with a spectrophotometer with 95% ethanol as a blank control, then chlorophyll a content, chlorophyll b content, chlorophyll a/ b and the total chlorophyll content were calculated according to the Arnon method. The soluble sugar content was determined by the anthrone colorimetric method, and the soluble protein content was determined by Komas Brilliant Blue G-250 (Bradford method).

#### A comprehensive assessment of the growth and development status of mosses

The fuzzy affiliation function assessment method was used to comprehensively assess the growth and development status of P.acutum. The calculation equation is as follows.

$${text{F}}_{1} left( {{text{X}}_{{text{i}}} } right) = left( {{text{X}} _{{text{i}}} – {text{X}}_{min } } right)/left( {{text{X}}_{max } – {text{X }}_{min } } right),$$

$${text{F}}_{2} left( {{text{X}}_{{text{i}}} } right) = left( {{text{X}} _{{text{i}}} – {text{X}}_{min } } right)/left( {{text{X}}_{max } – {text{X }}_{min } } right).$$

Formula: X denotes the measured value of the ith index, Xmin denotes the measured minimum value of the ith index, and Xmaximum denotes the maximum measured value of the ith index. A Pearson correlation analysis was performed on the growth and physiological indices of the P.acutum. If the indices were opposed to the growth indices of the P.acutumthe value of its affiliate function was calculated using the inverse affiliate function formula F2. The values ​​of the subordinate functions of the indices of growth and development of P.acutum under different growing mediums were accumulated and averaged to obtain the complete plant morphology evaluation index. The larger the index value, the better the plant growth under the substrate cultivation treatment37.38.

### statistical analyzes

Data were analyzed by one-way ANOVA with Excel 2010 and SPSS19.0 software. Multiple comparisons were performed by Duncan (P

### Plant Harvest Permit

The collection of plant material complies with relevant institutional, national and international guidelines and legislation.