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The influence of photosynthetic bacteria treatments on the crop yield, dry matter content, and protein content of the mushroom Agaricus bisporus
Scientia Horticulturae 82 (1999) 171±178
Short communication
The influence of photosynthetic bacteria treatments on the crop yield, dry matter content, and protein content of the mushroom Agaricus bisporus Jianrong Han* Department of Life Science, Shanxi University, Taiyuan 030006, People's Republic of China Accepted 10 February 1999
Abstract Experiments were performed to determine the effect of spraying suspensions of photosynthetic bacteria (PSB) onto casing soil on the yield, dry matter content, and protein content of the fruit bodies of Agaricus bisporus. The results showed that spraying PSB suspension at casing and between flushes could significantly increase the mushroom yield, and the increased rate of yield had a positive correlation with the PSB density of the suspension. When spraying 5000 ml of PSB suspension which contained 3.3 109 viable PSB cells/ml onto each tray (0.54 m2), increases in mushroom yield of 39.53% were obtained. No significant differences were found in either the dry matter contents or the protein contents of fruit bodies among all treatments. The mechanisms of mushroom yield increase resulting from spraying PSB suspension were discussed. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Agaricus bisporus; Photosynthetic bacteria; Yield; Dry matter content; Protein content
1. Introduction In China, cultivation of the mushroom Agaricus bisporus has developed rapidly in recent decades (Chang, 1994). However, the unit yield is lower than in many advanced countries such as France, Netherlands and the USA, in which many * E-mail address: [email protected] (J. Han) 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 4 3 - 6
172
J. Han / Scientia Horticulturae 82 (1999) 171±178
advanced methods such as the use of delayed release nutrient supplementation at spawning or at casing (Carroll and Schisler, 1976), have been extensively applied to the mushroom production to increase yield while decreasing production costs. Therefore, many Chinese research workers have made attempts to find the ways to enhance the yield of A. bisporus (Liu and Liu, 1994; Jiang et al., 1998). Photosynthetic bacteria (PSB) have been noted for their extensive applied value (Zhu et al., 1991). For example, the PSB cells may be used as a potential source of livestock and fish feed in addition to being used as a fertilizer for fruit and grain crops (Tanaka et al., 1992). Kobayashi (1978) determined the influence of photosynthetic bacteria (PSB) on the yield of rice (Oryza sativa) when applied as a nutritional liquid manure of active PSB, believed that active substances excreted by PSB play a role in increasing rice yield. Furthermore, the effects of nutritional liquid manure of active PSB on grape (Vitis vinifera) were investigated by Shi et al. (1995). Less is known, however, about whether PSB might influence mushroom production. This study was undertaken to determine the influence of spraying PSB suspensions containing large amounts of viable cells onto casing soil on crop yield, dry matter content, and protein content of the fruit bodies of A. bisporus. 2. Material and methods 2.1. Preparation of PSB suspension The purple non-sulfur bacterium Rhodopseudomonas palustris, obtained from the PSB Research Institute, Shanxi University, was used in all experiments. The R. palustris was cultured in the modified Ormerod et al. (1961) medium which contained the following per liter of distilled water: malate, 1.64 g; (NH4)2SO4, 1.32 g; KH2PO4, 0.6 g; K2HPO4, 0.9 g; MgSO47H2O, 0.2 g; CaCl22H2O, 0.075 g; FeSO47H2O, 0.27 g; EDTA, 0.05 g; biotin, 20 mg; -aminobenzoic acid, 20 mg; thiamine, 20 mg; nicotinic acid, 20 mg; mineral solution, 0.1 ml. Minerals (per liter of distilled water) consisted of MnSO4H2O, 18 g; ZnSO47H2O, 2.4 g; H3BO3, 28 g; CuSO45H2O, 0.79 g; Na2MoO42H2O, 12.6 g. The pH was adjusted to 6.8±7.2. After sterilization, the medium was dispensed aseptically into 500 ml screw capped bottles, leaving a small air bubble in each bottle. The bottles were inoculated with 2 ml of 48 h culture of R. palustris and incubated at 308C for 20 days under illumination of 8000 lx provided with incandescent lighting for photosynthetic growth. Then, the cultures were counted by a method of determination of the most probable number (MPN) (Zhou, 1993) of viable PSB cells, and were diluted into four different suspensions with water. The MPN values of viable PSB cells of four different suspensions
J. Han / Scientia Horticulturae 82 (1999) 171±178
173
were 1.650 109, 1.980 109, 2.745 109 and 3.300 109 mlÿ1, respectively, of PSB suspension. 2.2. The mushroom culture Starting material for the production of mushroom compost (consisting of a mixture of horse manure, straw, chicken manure, gypsum and water) were obtained from a grower in the suburbs of Taiyuan. The composting of substrates was processed using the method of Yang (1986). The total outdoor composting process (Phase I) took 28 days (including prewetting period). The second treatment (Phase II) was processed indoor for 7 days. The nitrogen contents following Phase II was approximately 2.15%. Forty kg of wet compost (was equal to 12 kg of dry compost) were filled into a tray (90 60 20 cm) with 400 g of grain spawn, Strain Fujian 2796. The running was in growing room under controlled conditions. The relative humidity was kept at 85±90% and the compost temperature to 23±258C. After 20 days of running, a 5 cm layer of pasteurized top soil was covered over the compost. Ten days after casing, the room temperature was lowered to 16±188C, and the relative humidity was kept at 95±99%. The diluted PSB suspensions were sprayed onto the casing soil at casing and between flushes as outlined in Table 1. Crop harvesting began 16 days after casing and lasted 40 days (a flush of 10 days). All mushrooms were picked before the veil was broken. The fresh weight and number of harvested mushrooms were recorded daily for each tray. For each treatment and each flush, 3±6 representative fruit bodies were dried at 1008C to constant weight to determine their dry matter Table 1 Experimental design of spraying photosynthetic bacteria (PSB) suspension onto casing soil during fruit bodies growth Treatment No.
1 2 3 4 5 6 a
Times of sprayingb
PSB suspension MPNa/ml
Amount of per tray (ml)
1.650 109 1.980 109 2.475 109 3.300 109 0 0
5000 5000 5000 5000 0c 0d
4 4 4 4 4 4
Most probable number of viable PSB cells. The PSB suspension were sprayed at casing and between flushes (four flushes) respectively, each times 1250 ml. c Spraying 5000 ml of diluent of medium diluted at 1 : 15 with water. d Spraying 5000 ml of water. b
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J. Han / Scientia Horticulturae 82 (1999) 171±178
content. The protein content of fruit bodies was calculated from the nitrogen content (N 6.25) as determined by micro-Kjeldahl method. 2.3. Experimental design The experiment included six treatments, Treatments 1±4 of spraying PSB suspensions of different densities and Treatments 5 and 6 of spraying water or medium served as two controls (Table 1). Each treatment was replicated in five trays, each having a surface area of 0.54 m2. The trays were arranged in five tiers from top to bottom in the cropping room, each tier having six trays. Six different treatments were assigned at random to six trays of each tier. Duncan's multiple range test (Ray, 1985) was used on the isolation treatment means to test for significant differences at the 1% level of confidence. 3. Results The mushroom yields during a harvesting period of 40 days are shown in Table 2. Means represent the data obtained from five replicates for each treatment. The differences in yield among Treatments 1±6 were statistically significant (p < 0.01). The mushroom yields of treatments of spraying PSB suspension were apparently higher than that of two controls, spraying water or medium, respectively (Treatments 5 and 6). This indicates that spraying PSB suspension in casing soils layer can enhance significantly the mushroom yield. Fig. 1 showed that the increased rate of yield of four treatments of spraying PSB suspension compared with the water control (Treatment 6) had a positive correlation with the PSB densities of the suspensions (r 0.9783). The higher the MPN value of PSB suspension was, the higher the mushroom yield. The MPN Table 2 The yield (fresh weight), size (fresh weight) and quantity of the harvested mushroom during a harvesting period of 40 days (means of five trays per treatment) Treatment No.
Yield (kgmÿ2)
1 2 3 4 5 6
12.92 13.39 13.87 14.33 11.74 10.27
Da C B A E F
Size (g mushroomÿ1)
Quantity (mushrooms mÿ2)
11.20 10.91 10.71 10.57 10.93 10.79
1154 1277 1295 1356 1074 952
A A A A A A
D C B A E F
a Means in the same column followed by the same letter are not significantly different at the P < 0.01 level according to Duncan's multiple range test.
J. Han / Scientia Horticulturae 82 (1999) 171±178
175
Fig. 1. MPN values of viable PSB cells versus mushroom yield increased rate of four treatments of spraying PSB suspension.
value of Treatment 4 was the highest among the four treatments, its mushroom yield was the highest with an increased rate of 39.53% as compared with water control (Treatment 6). The spraying of PSB suspension also influenced significantly the number of harvested mushrooms (Table 2). Similarly, the differences in number of mushrooms among treatments were related with the PSB densities of the suspensions. A correlation coefficient (r) of 0.9707 was calculated for the number of mushrooms and the MPN values of PSB suspensions of Treatments 1±4. This means that, within the density range from 1.65 109 to 3.3 109 cells per ml of PSB suspension, the higher the MPN value of PSB suspension sprayed was, the more the number of harvested mushrooms. There were no apparent differences in size of mushrooms (i.e. the average weight of sporophores) among treatments (Table 2). This indicates that spraying PSB suspension can stimulated the formation of more mushroom primordia, which results in the increase of mushroom yield. Table 3 The dry matter content, protein content of the harvested mushrooms during a harvesting period of 40 days (means of five trays per treatment) Treatment No.
Dry matter content (%)
Protein content (%)
1 2 3 4 5 6
8.24 8.47 8.65 8.93 9.14 7.99
34.82 34.30 33.50 34.60 34.08 32.92
A A A A A A
A A A A A A
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J. Han / Scientia Horticulturae 82 (1999) 171±178
The effect of spraying PSB suspension on the dry matter content and protein content of the fruit bodies is shown in Table 3. No significant differences were found in either the dry matter contents or the protein contents of fruit bodies among all treatments. Due to the increase of mushroom yield by spraying PSB suspension, it is likely that spraying a PSB suspension onto the casing soil may increase the total dry weight of all harvested fruit bodies from the crop cycle. 4. Discussion The results from the experiments mentioned above indicate that spraying PSB suspension may result in a 39.53% increase in yield and, hence, has profound commercial significance. The question may be raised, `what is the mechanism of PSB making mushroom yield increase by a big margin?' At this point a satisfactory explanation cannot be offered. Generally, the casing soil should be of low nutritional content (Masaphy et al., 1989) to benefit the formation of sporophore. However, in our experiment that was not the case. For example, only spraying the diluent of medium onto casing soil (Treatment 5) also resulted in a 14.3% increase in yield as compared with water control (Treatment 6). This indicates that the nutrients in the medium used for R. palustris favor the mushroom growth. Nevertheless, this cannot account for the reason why spraying PSB suspension within the density ranges from 1.65 109 to 3.3 109 mlÿ1 can result in a higher increased rate of yield than that of only spraying diluent of medium. It is likely that the PSB suspensions contain one or more kinds of growth-stimulating substances, which can stimulate the formation of fruit bodies. Many research workers (Tanaka et al., 1991; Sasaki et al., 1993) reported that the purple non-sulfur bacterium could accumulate large amounts of 5-aminolevulinic acid (ALA) in cell, and the ALA could be excreted extracellularly. Recently, Tanaka et al. (1992) reported primitive effects of ALA on the growth and photosynthesis of crops and vegetables such as rice, barley (Hordeum spp. ), potato (Solanum tuberosum) and garlic (Allium sativum) by applying small amounts of ALA on the leaves and roots of the plants. Yields of crops were also enhanced by the application of ALA during the vegetation growth stage in the life cycle of the plants. In our research, no attempt was made to determine the ALA of the PSB suspension. Certainly, the possibility that the PSB suspension contains other unknown growth hormones which were excreted by PSB cannot be ruled out. The purple non-sulfur bacterium R. palustris was selected for this experiment rather than other PSB, such as purple sulfur bacteria and green sulfur bacteria. Two reasons were considered for this selection. First, the purple non-sulfur bacteria are facultative anaerobes and phototrophs. They readily undergo interchange between light anaerobic and dark aerobic modes of life depending
J. Han / Scientia Horticulturae 82 (1999) 171±178
177
on the conditions of growth (He, 1981). Therefore, under dark condition in which the mushroom is grown, the purple non-sulfur bacteria may remain viable and active theoretically in casing soil layer. Second, the purple non-sulfur bacteria can utilize extensive organic acids as carbon source for their phototrophic growth (Zhu et al., 1991), so they are easier to be cultivated than other PSB. In order to indicate whether the PSB can remain alive or not in the casing soil layer, we tried to isolate PSB again from the casing soils at the completion of this experiment; however, our attempt was unsuccessful due to infestation by the bacteria Bacillus spp. coming from mushroom bed. The importance of further studies on whether the PSB can remain alive or not in the casing soil layer can readily be seen. It was reported that the quality of fruit was improved after spraying nutritional liquid manure of active PSB on grape leaves (Shi et al., 1995). Whether spraying PSB suspension could also affect flavor, quality, and shelf life of the mushroom A. Bisporus remains a question. So, a further research on this question should be conducted. As for occurrence of mushroom disease during the course of this investigation, in the visual assessment, the occurrence of mushroom disease was low, there were no obvious differences among the treatments. There is an obvious advantage of spraying PSB suspension onto casing soil over supplementation by release nutrients, that is, the operation is simple and convenient; therefore, it would be benefit both of conventional bed and tray mushroom-growing operations. In this research, it had not been considered as to how much production cost would be increased or decreased if this technique was used under commercial cultivation conditions. However, it is certain that the PSB suspension can be produced cheaply by utilizing agricultural wastes or by-products to culture the PSB by means of advanced fermentative techniques (Shipman et al., 1977). Acknowledgements The author would like to thank Dr. F. Zhang for his advice on experimental design and Dr. Z.M. Zhang for his practical assistance during these experiments. References Carroll, A.D., Schisler, L.C., 1976. Delayed release nutrient supplement for mushroom culture. Appl. Environ. Microbiol. 31, 499±503. Chang, S.T., 1994. Mushroom biology: the impact on mushroom production and mushroom products, Edible Fungi of China 13 (2) 3±5 (in Chinese). He, Y.Q., 1981. Regulation of nitrogenase synthesis in resting cells of Rhodopseudomonas capsulata: effect of light. Acta Phytophysiologia Sinica 7(2), 193±201. Jiang, Z.H., Zhu, D., Yang, P.Y., 1998. Comparison of the effect by adding 864 culture solution into the compost and by using double fermentation in mushroom cultivation, Edible Fungi of China 17 (3) 38±39 (in Chinese).
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Kobayashi, T., 1978. The ecological role in nature and practical uses as well as interrelation with higher plant of photosynthetic bacteria, Fermentation and Industry 36, 574±583 (in Japanese). Liu, H.Z., Liu, Q.Y., 1994. Application of the acid proteinase inhibitor to the production of mushrooms, Edible Fungi of China 13(3), 31±33 (in Chinese). Masaphy, S., Levanon, D., Kanai, O., Henis, Y., 1989. Nutritional supplementation to the casing soil: Ecological aspects and mushroom production Part I. Mushroom Sci. 12, 417±426. Ormerod, T.G., Ormerod, K.S., Gest, H., 1961. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch. Biochem. Biophys. 94, 449±463. Ray, A.A., 1985. SAS Users Guide: Statistics, 1982 edn., SAS Institute Statistical Analysis System, Cary, NC, 584 p. Sasaki, K., Tanaka, T., Nishio, N., Nagai, S., 1993. Effect of culture pH on the extracellular production of ALA by Rhodobacter sphaeroides from volatile fatty acids. Biotechnol. Lett. 15, 859±864. Shipman, R.H., Kao, I.C., Fan, L.T., 1977. Single-cell protein production by photosynthetic bacteria. Adv. Appl. Microb. 21, 161±183. Shi, Q.L., Yang, S.P., Ma, Y.Z., Zhang, Z.M., 1995. The effect of nutritional liquid manure of active PSB on grape. J. Shanxi Univ. 18(3) 329±331 (in Chinese, with English abstract). Tanaka, T., Watanabe, K., Hotta, Y., Lin, D., Sasaki, K., 1991. Formation of 5-aminolevulinic acid under aerobic/dark condition by a mutant of Rhodobacter Sphaeroides. Biotechnol. Lett. 13, 589±594. Tanaka, T., Hotta, T., Takeuchi, Y., Konnai, M., 1992. Proceedings of the Nineteenth Annual Meeting on Plant Growth Regulator Society of America. San Francisco, pp. 237±241. Yang, X.M., 1986. The Cultivation of Edible Mushroom in China. The Agriculture Press. Beijing, pp. 259±270. Zhou, D.Q., 1993. Microbiology Course. Higher Education Press. Beijing, pp. 184±185. Zhu, Z.Y., Yu, J.A., Ling, Z.X., 1991. Research on the photosynthetic bacteria and their applications. Shanghai Jiaotong University Press, Shanghai, pp. 250±288.
Short communication
The influence of photosynthetic bacteria treatments on the crop yield, dry matter content, and protein content of the mushroom Agaricus bisporus Jianrong Han* Department of Life Science, Shanxi University, Taiyuan 030006, People's Republic of China Accepted 10 February 1999
Abstract Experiments were performed to determine the effect of spraying suspensions of photosynthetic bacteria (PSB) onto casing soil on the yield, dry matter content, and protein content of the fruit bodies of Agaricus bisporus. The results showed that spraying PSB suspension at casing and between flushes could significantly increase the mushroom yield, and the increased rate of yield had a positive correlation with the PSB density of the suspension. When spraying 5000 ml of PSB suspension which contained 3.3 109 viable PSB cells/ml onto each tray (0.54 m2), increases in mushroom yield of 39.53% were obtained. No significant differences were found in either the dry matter contents or the protein contents of fruit bodies among all treatments. The mechanisms of mushroom yield increase resulting from spraying PSB suspension were discussed. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Agaricus bisporus; Photosynthetic bacteria; Yield; Dry matter content; Protein content
1. Introduction In China, cultivation of the mushroom Agaricus bisporus has developed rapidly in recent decades (Chang, 1994). However, the unit yield is lower than in many advanced countries such as France, Netherlands and the USA, in which many * E-mail address: [email protected] (J. Han) 0304-4238/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 0 4 3 - 6
172
J. Han / Scientia Horticulturae 82 (1999) 171±178
advanced methods such as the use of delayed release nutrient supplementation at spawning or at casing (Carroll and Schisler, 1976), have been extensively applied to the mushroom production to increase yield while decreasing production costs. Therefore, many Chinese research workers have made attempts to find the ways to enhance the yield of A. bisporus (Liu and Liu, 1994; Jiang et al., 1998). Photosynthetic bacteria (PSB) have been noted for their extensive applied value (Zhu et al., 1991). For example, the PSB cells may be used as a potential source of livestock and fish feed in addition to being used as a fertilizer for fruit and grain crops (Tanaka et al., 1992). Kobayashi (1978) determined the influence of photosynthetic bacteria (PSB) on the yield of rice (Oryza sativa) when applied as a nutritional liquid manure of active PSB, believed that active substances excreted by PSB play a role in increasing rice yield. Furthermore, the effects of nutritional liquid manure of active PSB on grape (Vitis vinifera) were investigated by Shi et al. (1995). Less is known, however, about whether PSB might influence mushroom production. This study was undertaken to determine the influence of spraying PSB suspensions containing large amounts of viable cells onto casing soil on crop yield, dry matter content, and protein content of the fruit bodies of A. bisporus. 2. Material and methods 2.1. Preparation of PSB suspension The purple non-sulfur bacterium Rhodopseudomonas palustris, obtained from the PSB Research Institute, Shanxi University, was used in all experiments. The R. palustris was cultured in the modified Ormerod et al. (1961) medium which contained the following per liter of distilled water: malate, 1.64 g; (NH4)2SO4, 1.32 g; KH2PO4, 0.6 g; K2HPO4, 0.9 g; MgSO47H2O, 0.2 g; CaCl22H2O, 0.075 g; FeSO47H2O, 0.27 g; EDTA, 0.05 g; biotin, 20 mg; -aminobenzoic acid, 20 mg; thiamine, 20 mg; nicotinic acid, 20 mg; mineral solution, 0.1 ml. Minerals (per liter of distilled water) consisted of MnSO4H2O, 18 g; ZnSO47H2O, 2.4 g; H3BO3, 28 g; CuSO45H2O, 0.79 g; Na2MoO42H2O, 12.6 g. The pH was adjusted to 6.8±7.2. After sterilization, the medium was dispensed aseptically into 500 ml screw capped bottles, leaving a small air bubble in each bottle. The bottles were inoculated with 2 ml of 48 h culture of R. palustris and incubated at 308C for 20 days under illumination of 8000 lx provided with incandescent lighting for photosynthetic growth. Then, the cultures were counted by a method of determination of the most probable number (MPN) (Zhou, 1993) of viable PSB cells, and were diluted into four different suspensions with water. The MPN values of viable PSB cells of four different suspensions
J. Han / Scientia Horticulturae 82 (1999) 171±178
173
were 1.650 109, 1.980 109, 2.745 109 and 3.300 109 mlÿ1, respectively, of PSB suspension. 2.2. The mushroom culture Starting material for the production of mushroom compost (consisting of a mixture of horse manure, straw, chicken manure, gypsum and water) were obtained from a grower in the suburbs of Taiyuan. The composting of substrates was processed using the method of Yang (1986). The total outdoor composting process (Phase I) took 28 days (including prewetting period). The second treatment (Phase II) was processed indoor for 7 days. The nitrogen contents following Phase II was approximately 2.15%. Forty kg of wet compost (was equal to 12 kg of dry compost) were filled into a tray (90 60 20 cm) with 400 g of grain spawn, Strain Fujian 2796. The running was in growing room under controlled conditions. The relative humidity was kept at 85±90% and the compost temperature to 23±258C. After 20 days of running, a 5 cm layer of pasteurized top soil was covered over the compost. Ten days after casing, the room temperature was lowered to 16±188C, and the relative humidity was kept at 95±99%. The diluted PSB suspensions were sprayed onto the casing soil at casing and between flushes as outlined in Table 1. Crop harvesting began 16 days after casing and lasted 40 days (a flush of 10 days). All mushrooms were picked before the veil was broken. The fresh weight and number of harvested mushrooms were recorded daily for each tray. For each treatment and each flush, 3±6 representative fruit bodies were dried at 1008C to constant weight to determine their dry matter Table 1 Experimental design of spraying photosynthetic bacteria (PSB) suspension onto casing soil during fruit bodies growth Treatment No.
1 2 3 4 5 6 a
Times of sprayingb
PSB suspension MPNa/ml
Amount of per tray (ml)
1.650 109 1.980 109 2.475 109 3.300 109 0 0
5000 5000 5000 5000 0c 0d
4 4 4 4 4 4
Most probable number of viable PSB cells. The PSB suspension were sprayed at casing and between flushes (four flushes) respectively, each times 1250 ml. c Spraying 5000 ml of diluent of medium diluted at 1 : 15 with water. d Spraying 5000 ml of water. b
174
J. Han / Scientia Horticulturae 82 (1999) 171±178
content. The protein content of fruit bodies was calculated from the nitrogen content (N 6.25) as determined by micro-Kjeldahl method. 2.3. Experimental design The experiment included six treatments, Treatments 1±4 of spraying PSB suspensions of different densities and Treatments 5 and 6 of spraying water or medium served as two controls (Table 1). Each treatment was replicated in five trays, each having a surface area of 0.54 m2. The trays were arranged in five tiers from top to bottom in the cropping room, each tier having six trays. Six different treatments were assigned at random to six trays of each tier. Duncan's multiple range test (Ray, 1985) was used on the isolation treatment means to test for significant differences at the 1% level of confidence. 3. Results The mushroom yields during a harvesting period of 40 days are shown in Table 2. Means represent the data obtained from five replicates for each treatment. The differences in yield among Treatments 1±6 were statistically significant (p < 0.01). The mushroom yields of treatments of spraying PSB suspension were apparently higher than that of two controls, spraying water or medium, respectively (Treatments 5 and 6). This indicates that spraying PSB suspension in casing soils layer can enhance significantly the mushroom yield. Fig. 1 showed that the increased rate of yield of four treatments of spraying PSB suspension compared with the water control (Treatment 6) had a positive correlation with the PSB densities of the suspensions (r 0.9783). The higher the MPN value of PSB suspension was, the higher the mushroom yield. The MPN Table 2 The yield (fresh weight), size (fresh weight) and quantity of the harvested mushroom during a harvesting period of 40 days (means of five trays per treatment) Treatment No.
Yield (kgmÿ2)
1 2 3 4 5 6
12.92 13.39 13.87 14.33 11.74 10.27
Da C B A E F
Size (g mushroomÿ1)
Quantity (mushrooms mÿ2)
11.20 10.91 10.71 10.57 10.93 10.79
1154 1277 1295 1356 1074 952
A A A A A A
D C B A E F
a Means in the same column followed by the same letter are not significantly different at the P < 0.01 level according to Duncan's multiple range test.
J. Han / Scientia Horticulturae 82 (1999) 171±178
175
Fig. 1. MPN values of viable PSB cells versus mushroom yield increased rate of four treatments of spraying PSB suspension.
value of Treatment 4 was the highest among the four treatments, its mushroom yield was the highest with an increased rate of 39.53% as compared with water control (Treatment 6). The spraying of PSB suspension also influenced significantly the number of harvested mushrooms (Table 2). Similarly, the differences in number of mushrooms among treatments were related with the PSB densities of the suspensions. A correlation coefficient (r) of 0.9707 was calculated for the number of mushrooms and the MPN values of PSB suspensions of Treatments 1±4. This means that, within the density range from 1.65 109 to 3.3 109 cells per ml of PSB suspension, the higher the MPN value of PSB suspension sprayed was, the more the number of harvested mushrooms. There were no apparent differences in size of mushrooms (i.e. the average weight of sporophores) among treatments (Table 2). This indicates that spraying PSB suspension can stimulated the formation of more mushroom primordia, which results in the increase of mushroom yield. Table 3 The dry matter content, protein content of the harvested mushrooms during a harvesting period of 40 days (means of five trays per treatment) Treatment No.
Dry matter content (%)
Protein content (%)
1 2 3 4 5 6
8.24 8.47 8.65 8.93 9.14 7.99
34.82 34.30 33.50 34.60 34.08 32.92
A A A A A A
A A A A A A
176
J. Han / Scientia Horticulturae 82 (1999) 171±178
The effect of spraying PSB suspension on the dry matter content and protein content of the fruit bodies is shown in Table 3. No significant differences were found in either the dry matter contents or the protein contents of fruit bodies among all treatments. Due to the increase of mushroom yield by spraying PSB suspension, it is likely that spraying a PSB suspension onto the casing soil may increase the total dry weight of all harvested fruit bodies from the crop cycle. 4. Discussion The results from the experiments mentioned above indicate that spraying PSB suspension may result in a 39.53% increase in yield and, hence, has profound commercial significance. The question may be raised, `what is the mechanism of PSB making mushroom yield increase by a big margin?' At this point a satisfactory explanation cannot be offered. Generally, the casing soil should be of low nutritional content (Masaphy et al., 1989) to benefit the formation of sporophore. However, in our experiment that was not the case. For example, only spraying the diluent of medium onto casing soil (Treatment 5) also resulted in a 14.3% increase in yield as compared with water control (Treatment 6). This indicates that the nutrients in the medium used for R. palustris favor the mushroom growth. Nevertheless, this cannot account for the reason why spraying PSB suspension within the density ranges from 1.65 109 to 3.3 109 mlÿ1 can result in a higher increased rate of yield than that of only spraying diluent of medium. It is likely that the PSB suspensions contain one or more kinds of growth-stimulating substances, which can stimulate the formation of fruit bodies. Many research workers (Tanaka et al., 1991; Sasaki et al., 1993) reported that the purple non-sulfur bacterium could accumulate large amounts of 5-aminolevulinic acid (ALA) in cell, and the ALA could be excreted extracellularly. Recently, Tanaka et al. (1992) reported primitive effects of ALA on the growth and photosynthesis of crops and vegetables such as rice, barley (Hordeum spp. ), potato (Solanum tuberosum) and garlic (Allium sativum) by applying small amounts of ALA on the leaves and roots of the plants. Yields of crops were also enhanced by the application of ALA during the vegetation growth stage in the life cycle of the plants. In our research, no attempt was made to determine the ALA of the PSB suspension. Certainly, the possibility that the PSB suspension contains other unknown growth hormones which were excreted by PSB cannot be ruled out. The purple non-sulfur bacterium R. palustris was selected for this experiment rather than other PSB, such as purple sulfur bacteria and green sulfur bacteria. Two reasons were considered for this selection. First, the purple non-sulfur bacteria are facultative anaerobes and phototrophs. They readily undergo interchange between light anaerobic and dark aerobic modes of life depending
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on the conditions of growth (He, 1981). Therefore, under dark condition in which the mushroom is grown, the purple non-sulfur bacteria may remain viable and active theoretically in casing soil layer. Second, the purple non-sulfur bacteria can utilize extensive organic acids as carbon source for their phototrophic growth (Zhu et al., 1991), so they are easier to be cultivated than other PSB. In order to indicate whether the PSB can remain alive or not in the casing soil layer, we tried to isolate PSB again from the casing soils at the completion of this experiment; however, our attempt was unsuccessful due to infestation by the bacteria Bacillus spp. coming from mushroom bed. The importance of further studies on whether the PSB can remain alive or not in the casing soil layer can readily be seen. It was reported that the quality of fruit was improved after spraying nutritional liquid manure of active PSB on grape leaves (Shi et al., 1995). Whether spraying PSB suspension could also affect flavor, quality, and shelf life of the mushroom A. Bisporus remains a question. So, a further research on this question should be conducted. As for occurrence of mushroom disease during the course of this investigation, in the visual assessment, the occurrence of mushroom disease was low, there were no obvious differences among the treatments. There is an obvious advantage of spraying PSB suspension onto casing soil over supplementation by release nutrients, that is, the operation is simple and convenient; therefore, it would be benefit both of conventional bed and tray mushroom-growing operations. In this research, it had not been considered as to how much production cost would be increased or decreased if this technique was used under commercial cultivation conditions. However, it is certain that the PSB suspension can be produced cheaply by utilizing agricultural wastes or by-products to culture the PSB by means of advanced fermentative techniques (Shipman et al., 1977). Acknowledgements The author would like to thank Dr. F. Zhang for his advice on experimental design and Dr. Z.M. Zhang for his practical assistance during these experiments. References Carroll, A.D., Schisler, L.C., 1976. Delayed release nutrient supplement for mushroom culture. Appl. Environ. Microbiol. 31, 499±503. Chang, S.T., 1994. Mushroom biology: the impact on mushroom production and mushroom products, Edible Fungi of China 13 (2) 3±5 (in Chinese). He, Y.Q., 1981. Regulation of nitrogenase synthesis in resting cells of Rhodopseudomonas capsulata: effect of light. Acta Phytophysiologia Sinica 7(2), 193±201. Jiang, Z.H., Zhu, D., Yang, P.Y., 1998. Comparison of the effect by adding 864 culture solution into the compost and by using double fermentation in mushroom cultivation, Edible Fungi of China 17 (3) 38±39 (in Chinese).
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