Effects of Different Proportions of Spent Pleurotus ostreatus Substrate on Seed Germination and Seedling Growth of Mung Beans

Ling WANG Shuang YANG Yihong HU Chenzhong JIN Zhi ZENG

Abstract [Objectives]This study was conducted to investigate the effects of different proportions of spent Pleurotus ostreatus substrate on the germination and seedling growth of mung beans.[Methods]The cellulosedegrading bacteria HB8 and HF1 were mixed with a commercially available microbial composting agent， respectively， for the composting of spent P. ostreatus substrate. Mung beans were cultivated with different proportions of spent mushroom substrate compost and soil. The seed germination rate， plant height， fresh weight and chlorophyll content of mung bean were used as indicators to investigate the effects of the organic fertilizer from spent P. ostreatus substrate on the growth of mung bean seedlings.[Results]The addition of cellulosedegrading bacteria can significantly improve the composting effect of the spent mushroom substrate. After 8 d of cultivation of mung beans with different ratios of the mushroom substrate organic fertilizer， 50% of the organic fertilizer can make the plant height， fresh weight and leaf chlorophyll content of mung bean seedlings reach the highest value and was suitable for mung bean breeding and cultivation.[Conclusions]This study provides scientific basis and technical indicators for the rapid and harmless treatment of spent mushroom substrate and its application in crop cultivation and nursery.

Key words Cellulosedegrading bacteria; Organic fertilizer from spent Pleurotus ostreatus substrate; Mung bean; Seed germination rate

Mushroom dregs are solid substrates discarded after harvesting fruit bodies of edible fungi. They are mainly composed of residual mycelia， wood chips， cottonseed hulls， corn cobs and wheat bran， and belong to agricultural solid waste. In general， about 5 kg of spent mushroom substrate will be produced for every 1 kg of edible fungus produced[1]. China is a big producer of edible fungi. According to the statistics of China Edible Fungi Association， the total output of edible fungi in China reached 35.97 million tons in 2016， and the production of mushroom dregs exceeded 150 million tons. A large amount of spent mushroom substrate is randomly discarded or landfilled without effective treatment， causing serious environmental pollution and huge waste of agricultural organic resources. There are still a large amount of organic matter， carbon and nitrogen nutrients， various mineral elements and mycelial active ingredients that can be directly absorbed and utilized by the crop in mushroom dregs， and basically no pathogenic bacteria， parasitic eggs and toxic and harmful substances are contained. Therefore， the application as fertilizer is the most effective way to utilize mushroom dregs at current[2]. Appropriate application of organic fertilizer manufactured from mushroom dregs in the soil can inhibit some soilborne diseases， significantly reduce the occurrence of crop diseases and insect pests， and promote plant growth[3].

Mushroom dregs rot slowly under natural conditions （generally 2-3 years）， which affects the rooting and survival of plants， and also causes inconvenience in production management. The use of specific microorganisms to decompose mushroom dregs can significantly improve the quality of mushroom dreg composting[4]. If EMs （effective microorganisms） act as a promoter， they can effectively promote the composting of mushroom dregs[5]. Studies have shown that fermented mushroom dreg has the advantages of loose structure and rich organic matter， and is capable of improving the organic matter content of the culture substrate， improving the rhizosphere environment of plants， increasing vegetable yield and improving quality[6]. Adding a proper amount of decomposed mushroom substrate to the cultivation substrate can effectively improve the yield and quality of fruits and vegetables such as rape， pepper， tomato， okra and grape， and decomposed mushroom substrate can be used as a new fertilizer for fruit and vegetable cultivation[7-11].

The cellulose component in mushroom dregs is one of the important factors restricting the degradation and reuse of mushroom dregs， and the addition of cellulosedegrading bacteria to promote compost maturity is an economical and effective method[12]. At present， there are many researches on microbial composting， but there are few reports on the compounding of commercial microbial composting agents and cellulosedegrading bacteria and its synergistic effect. Moreover， in practical application， mushroom dregs still have some disadvantages such as large pH value， high EC value， small bulk density and high N， P and K contents and should not be used directly as a cultivation substrate alone. Therefore， in this study， the commercially available microbial composting agent was compounded with the cellulosedegrading microbes for the composting treatment of the fresh spent substrate of Pleurotus ostreatus as the main raw material， the compost from which was mixed with soil at different proportions and used to study its effect on the germination of mung bean seeds and seedling growth. This study provides a scientific basis and technical indicators for the rapid and harmless treatment of mushroom dregs and their application in crop cultivation and seedling cultivation.

Materials and Methods

Experimental materials

The spent substrate of P. ostreatus was provided by Hunan Zhongshi Mushroom Industry Co.， Ltd. and was pulverized into granules with a diameter less than 1 cm after drying. The FJZ microbial composting agent was purchased from the market and consists of bacillus， actinomycetes， yeasts and lactic acid bacteria; HB8 bacteria and HF1 molds having strong ability to degrade cellulose were preserved by the Microbiology Laboratory of Hunan University of Humanities， Science and Technology. The soil was ordinary soil in the greenhouse of the Training Center of College of Agriculture and Biotechnology， Hunan University of Humanities， Science and Technology. The soil had not been planted with crops， and it was dried， pulverized and sieved for later use. Mung beans were purchased in the market.

Reagents

Acetone （analytical pure）; calcium carbonate powder; quartz sand.

Instruments

FA1004 analytical electronic balance （Shanghai Liangping Instrument Co.， Ltd.）; 9240A constant temperature blast drying oven （Shanghai Jinghong Experimental Equipment Co.， Ltd.）; GZX type light incubator （Beijing Zhongxing Weiye Instrument Co.， Ltd.）; SP722 visible spectrophotometer （Shanghai Spectrum Instruments Co.， Ltd.）; vernier caliper （Shanghai Shoufeng Precision Instrument Co.， Ltd.）.

Determination indexes and methods

Fermentation and compounding of the spent substrate

A total of four treatment groups were set， and each group treated 20 kg of spent substrate. Among them， treatment group I was spent substrate as the control check （CK）; treatment group II was spent substrate+FJZ microbial agent （1%）; treatment group III was spent substrate+FJZ （1%）+HB8 （1%）; and treatment group IV was spent substrate+FJZ microbial agent （1%）+HF1 microbial agent （1%）. After mixing evenly according to the ratio， the water content was adjusted to about 60%， that is， the water is oozing out of the spent substrate when held in hand but does not drop. The material was placed in a 1.2 m≠0.6 m≠0.6 m plastic barrel， and three thermometers were inserted into each barrel to the middle of the material at the measuring point of temperature. Each barrel was covered with plastic film which was then perforated. The center temperature of each treatment group was measured at noon every day， followed by taking the average. The experiment was carried out in the outdoor corridor of the experimental building of College of Agriculture and Biotechnology， Hunan University of Humanities， Science and Technology from March to April 2018， which lasted for 60 d. The material was not turned over at ordinary times， but was turned over to ventilate properly in the high temperature period with the addition of clear water in a timely manner to keep the spent substrate moist. Soil was mixed with the fermented spent substrate from treatment IV （volume ratio）， and the material ratios are shown in Table 1.

Mung bean culture and index determination

The same volume of compound media were filled in culture dishes， and each treatment was set with five replicates. In each dish， 10 full mung bean seeds were sown， and the media were watered to saturation. The dishes were placed in a light incubator and observed. Watering was performed timely to prevent drought， and the amount of water was the same for each dish. The germination rate was counted on the 4th d， and the plant height， fresh weight and chlorophyll content of the seedlings were measured on the 8th d.

Ten seedlings were randomly selected for each treatment. The length from the highest point of the leaf to the hypocotyl was measured with a vernier caliper， and the average value was taken as the plant height. The whole plant weight was weighed， and the average value served as the seedling fresh weight. The chlorophyll content of the leaves was determined by the acetone method[13]. In a dark room， 0.2 g of seedling leaves were accurately weighed， cut into pieces and placed in a mortar. A small amount of 80% acetone， calcium carbonate and quartz sand were added to fully grind the material into a homogenate. An appropriate amount of 80% acetone was then added， followed by grinding until the tissue turned white. The homogenate was filtered into a brown volumetric flask， washed several times with 80% acetone and made up to 50 ml. The absorbance was measured at wavelengths of 663 and 645 nm using a 80% acetone solution as a control. The chlorophyll a and chlorophyll b contents and total chlorophyll content in the sample was calculated according to the following formula： CT=Ca+Cb， Ca=（12.7A663-2.59A645）*VT/m， Cb=（12.7A645-2.59A663）*VT/m. Wherein Ca is the chlorophyll a content （mg/g）; Cb is the chlorophyll b content （mg/g）; CT is the total chlorophyll content （mg/g）; V is the extract volume （L）; T is the dilution factor; and m is the fresh weight of sample （g）.

Results and Analysis

Change of the compost pile

Temperature is an important parameter of composting， reflecting the growth and reproduction of microorganisms during composting. The temperature change curve of each treatment group in the experiment had a temperature rising stage， a high temperature stage， a cooling stage and a steady stage， and followed the change rule of general composting. It can be seen from the composting temperature curves （Fig. 1） that at the initial temperature of the piles at 21， the temperature rising stage of treatment group III and treatment group IV was short， and the temperatures rose rapidly 3 d after stacking to the maximum temperatures of 41 and 44.2 respectively. However， treatment group I and treatment group II entered the high temperature stage at the 9th d， and had their temperatures rose to 36.8 and 38.1， respectively， and the temperatures began to decrease slowly after the high temperature stage was maintained only for 4 d. With the prolongation of composting time， various factors were gradually unsuitable for the proliferation of microorganisms， and the temperature decreased. Every time the piles were turned over， the temperature increased and then dropped again. After composting for 36 d， treatment group IV entered the steady stage. After composting for 46 d， treatment group III entered the steady stage， while treatment group I and treatment group II entered the steady stage after composting for 54 d， which meant that the composting process was basically completed.

In general， the results of treatment group I and treatment group II were basically the same， and the addition of microbial composting agent in spent substrate did not show significant synergy， which is basically the same as that of Wan et al.[14]. Treatment group III and treatment group IV with the addition of the cellulosedegrading microbes were maintained at 40 at the high temperature stage for more than 11 and 16 d， respectively， while treatment group I and treatment group II continued to have a temperature of 40 or more at the high temperature stage for only 2 and 7 d， respectively. The cellulosedegrading microbes had a synergistic effect on the fermentation with the composting agent， under which the time required for composing was short， the high temperature lasted for a long time， and the temperature can be maintained at a higher level during the steady stage.

Morphological appearance of decomposed spent substrate

Observation and comparison showed that the spent substrate particles after decomposing were hard and soft， odorless， and the color changed from yellow to dark brown， in completely rotted state. The spent substrates of the treatment group III and the treatment group IV were sticky and soft， and might be degraded by the cellulosedegrading microbes. Because the spent substrate was well degraded， it produced active substances such as extracellular polysaccharides which caused the sticking to hands.

Effect of the content of the spent substrate in the culture medium on the germination rate of mung beans

The effect of the spent substrate content in the culture medium on the germination of mung bean seeds had a range （Fig. 2）. The culture medium with the content of 0%-20% of the spent substrate was likely to be hardened due to water loss， resulting in poor water permeability and rapid evaporation. The lack of sufficient moisture was not conducive to soil breaking of mung beans， resulting in less seed germination. The culture medium with the content of 70%-90% of the spent mushroom substrate was loose and not tight， and the water that was poured was mostly absorbed by the spent substrate， resulting in insufficient water around the mung beans. After the mung beans broke through the seed coat， they can absorb the water retained in the spent mushroom substrate. The results showed that the culture medium with the content of spent substrate in the range of 30%- 60% had the least effect on seed germination.

Effects of the content of the spent substrate in the culture medium on the height and fresh weight of mung beans sprouts

On the 4th to 6th d  after sowing of mung beans， the growth rate of mung bean seedlings in the culture medium with 0%-30% of spent substrate was higher， but after the 6th d， the growth rate was significantly lower than other treatment groups， and the growth was not obvious. The reason might be that the growth of mung beans was mainly to absorb water and to decompose the nutrition stored in the cotyledons to support their own growth in the early growth period， and to rely on the nutrients in the culture medium to supply their own needs in the later period， so the growth rate in the later period should be significantly lower than the media with a higher content of spent mushroom substrate. The growth rate of mung bean seedlings in the culture medium with 40%-70% of spent substrate was lower at the beginning， but after 6 d， the growth rate was significantly higher and the growth was the best. It can be seen from Fig. 3 that the plant height of the treatment group with the spent substrate content of 40%-70% was significantly higher than that of the control group. It can be seen from Fig. 4 that the fresh weight of mung bean seedlings increased with the increase of the proportion of the spent substrate， and reached the highest value at the spent substrate content of 50% and then decreased， and at this time， the average yield of mung bean sprouts was the highest.

Effect of the content of the spent substrate on the chlorophyll content of mung bean leaves

The chlorophyll content is directly proportional to the photosynthetic rate of the crop， which directly affects its growth ability， physiological characteristics and accumulation of dry matter， and ultimately affects its yield and quality[15]. It can be seen from Fig. 5 that the chlorophyll content of mung bean seedlings cultured with the organic fertilizer from spent mushroom substrate was higher than that of the control group， and the highest value was reached at the spent substrate content of 50%， indicating that adding appropriate amount of spent mushroom substrate organic fertilizer can promote the growth of mung bean seedlings and achieved the effect of increasing yield to a certain extent.

Effect of the optimum treatment on the growth of mung bean seedlings

In summary， after 8 d of cultivation of mung beans with different ratios of P. ostreatus substrate organic fertilizer， compared with the control group， the culture medium containing 50% of the decomposed mushroom substrate can make the mung bean seed germination rate， plant height， fresh weight and leaf chlorophyll content reach the highest value， and can promote the growth of mung bean seedlings. Compared with the pure soil control group， the 1≥1 mixture of the decomposed mushroom substrate and soil can significantly increase the growth potential of mung bean seedlings， indicating that applying appropriate amount of the spent mushroom substrate organic fertilizer in the soil can improve crop yield and enhance disease resistance （Fig. 6）.

Conclusions and Discussion

Studies have shown that the inoculation of exogenous flora can promote compost to reach maximum temperature， improve the maximum temperature of compost fermentation and prolong the duration of the high temperature[16]. In this study， the temperature of the compost pile during the temperature rising stage rose rapidly， but a higher fermentation temperature did not appear in the high temperature period， which is basically the same as that of Wu et al.[17]. In general， treatment IV after adding the cellulosedegrading microbes and microbial composting agent reached the temperature above 40， which was kept for 15 d at the high temperature stage. Although the pile temperature was not too high， excessive nitrogen loss caused by a toohigh temperature of the pile can be lowered.

Under appropriate initial carbonnitrogen ratio （C/N） conditions， direct composting with pure spent mushroom substrate can still achieve a better rapid composting effect. In the rapid composting process， the optimal initial carbonnitrogen ratio （C/N） is generally 25≥1-30≥1， and if the C/N ratio and the compost pile temperature continue to decrease until a stable condition during the composting process， it indicates that the compost is close to maturity[18]. In this study， organic fertilizer was prepared from the spent substrate of P. ostreatus containing wood chips and cottonseed hulls， which was determined to have an initial C/N ratio of about 36≥1. Excessive C/N ratio would cause nitrogen deficiency， thus affecting microbial growth and the metabolism velocity of organic matter. The general commercially available microbial composting starters are mainly composed of EM microbial agents， mainly containing bacillus， photosynthetic bacteria， lactic acid bacteria， yeast and actinomycetes， and are suitable for a pile having a relatively low initial C/N. With the addition of cellulosedegrading microbes in this study， it can well adapt to the high C/N ratio pile， and can obviously speed up the fermentation of the spent substrate and shorten the composting time compared with the control group. In addition， due to the small mass and volume of the pile material during the experiment， the temperature of the pile body was easily affected by the environment， resulting in the pile body being unable to maintain a higher temperature. Therefore， in the rapid composting with spent mushroom dregs， an appropriate amount of nitrogen source such as urea can be added to lower the initial C/N ratio， and at the same time， the quality and volume of the compost materials can be increased to reduce the environmental impact.

The decomposed mushroom substrate is an excellent horticultural substrate material suitable for the cultivation and breeding of horticultural crops[19]. However， in view of the particularity of the components of spent mushroom substrate， it does not completely replace the soil as a cultivation medium， and excessive application may have an inhibitory effect. At present， people lack indepth study on the physical and chemical properties and fertility factors of edible fungus substrate organic fertilizer， and the application effect and influence of different spent mushroom substrate organic fertilizers on different crops are still to be further studied. Subsequently， a variety of cellulosedegrading microbes and fungi can be mixed for fermentation， and the effect of the synergistic effect on the pure spent mushroom substrate composting can be studied. The ratio of the organic fertilizer from spent mushroom substrate to the compound fertilizer or other substrates can be further studied， so that the novel substrate can be more widely used in seedling raising and cultivation of crops and organic agricultural products in facility agriculture.

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