Beneficial bacterial strains on Agaricus blazei cultivation

The objective of this work was to evaluate the effects of six bacterial strains isolated from Agaricus blazei (ABM) on its cultivation. The six strains were characterized as to their effects on the productivity, polysaccharide-protein complex (PSPC), and polysaccharide contents of ABM cultured on sterilized casing soils. Three isolates enhanced ABM mycelium growth. Inoculation of Arthrobacter sp. or Exiguobacterium sp. on sterile peat casing soil resulted in 64% increase in ABM mushroom total fresh matter yield compared to the uninoculated control. Inoculation of Exiguobacterium sp., Microbacterium esteraromaticum or Pseudomonas resinovorans on sterilized loamy casing soil resulted in 62, 95, and 59% increase in ABM mushroom total fresh matter yield, respectively. The PSPC content in ABM increased 7 to 10% in casing soil inoculated with five of the six isolates compared to the uninoculated control. Exiguobacterium sp. inoculated on casing soil resulted in a mushroom‐polysaccharide content 15% higher than the control. Moreover, inoculation of five of the six isolates on the casing soil reduced the harvesting time from 10 to 27 days. The evaluated beneficial microbes improve the yield, PSPC, and polysaccharide contents, besides reducing the harvesting time in ABM culture.


Introduction
Agaricus blazei Murrill is a medicinal mushroom that contains bioactive polysaccharides and polysaccharide-protein complexes (PSPC) which have been shown to function as potent antioxidants, antitumorigenic, and anticancer agents (Endo et al., 2010;Ishii et al., 2011). Due to its pharmacological activities, ABM has drawn the attention of food scientists, and biotechnologists, as well as industrial growers.
Conventional culturing of ABM requires the initial spawning of the mycelium on a solid culture medium (15 to 21 days), and subsequent covering with suitable casing soil to stimulate fruiting-body formation, which normally takes around 45 to 60 days (Colauto et al., 2010;Chu et al., 2012). The time required to culture ABM from spawning to a harvestable mushroom size is long, compared to related Agaricus sp., such as A. bisporus (25 to 35 days, Pardo-Gimenez et al., 2010). Furthermore, the biological efficiency (BE) -defined as the ratio of the productivity of an organism to its Pesq. agropec. bras., Brasília, v.47, n.6, p.815-821, jun. 2012 energy supply -for ABM cultures are often in the range of 40-70% (Mendonça et al., 2005;Donini et al., 2006;Andrade et al., 2007;Pokhrel & Ohga, 2007;Colauto et al., 2010;Chu et al., 2012). As such, it would be beneficial to identify techniques to decrease the time and energy required to culture ABM.
A previous report showed that a loamy soil can substitute peat soil (PS) as a casing material and significantly improve the total fresh yield, production time and BE of ABM (Chu et al., 2012). In addition, chemical constituents as Cu and Zn, in the casing soil, have shown significant negative correlation with ABM total yield (Chu et al., 2012).
Biological approaches to improve crop growth have been practiced worldwide. In animal systems, beneficial microbes (BM) or probiotics are essential for maintaining the health of an individual (Malo et al., 2010). Similarly, endophytes and BM that dwell in the rhizosphere contribute to plant growth and development (Chen et al., 2006;Young & Shen, 2007;Hardoim et al., 2008). Reports on the application of BM for improving the productivity of edible mushrooms are scarce. The thermophilic fungus Scytalidium thermophilum, identified in the early 1990s, was shown to enhance mycelial growth and improve the productivity of A. bisporus (Straatsma et al., 1994). The bacterium Pseudomonas sp. P7014 was shown to enhance the mycelial growth and reduce the production time of the edible mushroom Pleurotus eryngii (Kim et al., 2008). In addition, the effects of Trichoderma sp. or Chaetomium olivacearum co-cultures with spawned ABM mycelium on the productivity of ABM was assessed, and the results showed co-culturing of these two fungi did not affect ABM productivity (Andrade et al., 2007). Further reports on the effects of BM in ABM cultivation are limited.
Several novel bacterial species were recently identified from the base of ABM stipes, including Agaricicola taiwanensis, Microbacterium agarici sp. nov., Microbacterium humi sp. nov., and Microbacterium pseudoresistens sp. nov. , and the evaluation of the possible beneficial effects of these and other species on ABM productivity are still needed.
The objective of this work was to evaluate the effects of six bacterial strains isolated from ABM on its cultivation, specifically their effects on ABM productivity, harvesting time, and polysaccharide and PSPC contents.

Materials and Methods
Farm-grown ABM was collected from Nantou, Taiwan (23°57'26" N, 120°58'29" E). The base of the mushroom stipe was excised, minced, soaked and incubated in distilled water by shaking for 30 min at 25 o C. Aliquots of the stipe-soil-solution were plated on nutrient agar (Himedia, Mumbai, India) by serial dilution, and incubated at 25 o C for 3 days. Bacterial colonies were subcultured into single colonies, which were individually cultured on nutrient broth for DNA extraction, using MO BIO Ultraclean Microbial DNA Isolation Kit (MO BIO, Carlsbad, CA, USA).
Amongst the identified bacteria, six isolates (Table 1) were selected for this study, due to their similar fast-growing characteristics. All six microbes belong to the risk group 1 microbia, based on the German Collection of Microorganisms and Cell Cultures (2011) and, thus, are potential BM for improving ABM production. Individual microbes were co-cultured with ABM on potato dextrose agar, in order to determine their effects on the mycelium growth.
The ABM strain BCRC36814 was purchased from The Food Industry Research and Development Institute, Hsin-Chu, Taiwan. To assess the effect of these six bacteria on ABM mycelium growth, a 0.5 cm diameter block of ABM mycelium was spotted on a potato dextrose agar (Difco, Detroit, MT, USA) plate along with individual microbium. The microbium was drawn in a line approximately 2 cm away from the spotted ABM mycelium block with a sterilized inoculation loop. After 7 days at 28 o C, the ABM mycelium diameter was measured and compared to the control without microbial inoculation. The above ABM mycelium growth tests for each bacteria treatment were performed with three replicates.
Sawdust medium, purchased from Q-YO (Bio Technology Farm, Ta-Chun, Chang-Hua, Taiwan), was used as the culture medium for the spawning of ABM mycelium (Chu et al., 2012). In addition to the commonly used peat soil (PS), local loamy soil was also used as the casing soil for culturing ABM (Pokhrel & Ohga, 2007;Colauto et al., 2010;Chu et al., 2012). The physicochemical properties of the culture medium and casing soils are summarized in Table 2. The contents of soil ash, organic matter, and organic carbon were analyzed according to Nelson & Sommers (1982), and total N was analyzed using the Kjeldahl method (Bremner & Mulvaney, 1982). The culture medium and casing soils were soaked separately in water at a 1:5 ratio (w/v), and the pH and electrical conductivity were determined using a pH-electroconductivity meter. Bulk density (BD) was determined by measuring the ratio (DW/TV) between soil dry matter weight (DW) and its fixed volume (TV) (Blake & Hartge, 1986). The particle density (PD) of the soil samples was averaged from two measurements (Blake & Hartge, 1986). Porosity (f) was calculated by the equation: f = [(1-BD/ PD) x 100%]. Water-holding capacity (WHC) was calculated using the weight of water-saturated casing soil divided by the weight of oven-dried casing soil.
The ABM stock was activated and subcultured on potato dextrose agar medium to produce fresh ABM and, subsequently, cultured on sterilized wheat to produce ABM grain spawn (Chu et al., 2012). Sterilized sawdust medium was evenly packed in bag logs in a 250 mL pot with a total weight of 100 g. The moisture content of the sawdust medium was 57%. Two grams of ABM grain spawn were inoculated on sterilized sawdust medium and placed in an incubator at 28ºC for spawning. The light intensity in the growth chamber was 20±2 lux, measured at 45±5 cm (Digital Light Meter, TES-1335, Taiwan). After complete spawning (1 month), two separate casing treatments of imported PS -peat soil (Saprists, Histosols; FAO, 1988) -or loamy soil (loamy, mixed, nonacid, hyperthermic, Typic Udorthent;FAO, 1988) were covered on top of the medium with 2 to 2.5-cm thickness (Chu et al., 2012). The casing soils and sawdust medium were sterilized before use (121 o C, 1.033 kg cm -2 , 1 hour for 3 times), in order to avoid possible microbial contaminants. We also included an uninoculated nonsterile casing soil as control.
To assess the effects of the bacterial isolates on the productivity of ABM, individual bacterial strains were inoculated on sterilized casing soil instead of the culture medium because the bacteria strains were isolated from the mushroom stipe along with the casing   soil. Five milliliters of nutrient broth-cultured microbe (10 9 cfu mL -1 ; calculated using the serial dilution method) were evenly applied directly on top of the casing soil weekly until harvesting. The same volume of bacterial-free nutrient broth was applied on the uninoculated control. Each experiment was conducted in a randomized block design with three replicates.
Harvesting of the fruiting body takes place when the mushrooms reach their highest biomass, which occurs during the immature stage, when the veil membrane is enclosed and the gills are intact (Mendonça et al., 2005;Pokhrel & Ohga, 2007;Chu et al., 2012). Total fresh matter yield (FW, g per pot) was measured, and the BE was determined after harvesting by the equation BE=FW/DWC (Andrade et al., 2007), where DWC is the dry weight of the culture medium.
Polysaccharide-protein complex content was determined according to Sarangi et al. (2006). Lyophilized PSPC was used to determine the polysaccharide content, using the phenol-sulfuric acid method (Sarangi et al., 2006). All standards were purchased from Sigma Chemical Co., St. Louis, MO, USA.
Statistical analyses were performed using the CoStat's statistical procedures (CoHort Software, 2002), and means were compared by Duncan's test, at 5% probability.

Results and Discussion
JN12, JN16 and JN17 bacterial isolates were able to positively affect ABM mycelium growth, when compared to the control without bacteria co-culturing. However, JN03, JN05 and JN10 isolates have not affected ABM mycelium growth. Several reports have indicated that co-cultivation with certain microbial strains results in positive effects on mushroom mycelial growth and subsequent mushroom production (Straatsma et al., 1994;Kim et al., 2008). Naturally occurring thermophilic fungi such as S. thermophilum have been reported to be important for the preparation of matured compost for mushroom culture. Furthermore, inoculation of sterilized culture medium with S. thermophilum increased the productivity of A. bisporus (Straatsma et al., 1994).
After 90 days in lab conditions, nonsterilized uninoculated PS casing (BK) produced many primordia, but very few of them developed into harvestable mushrooms. This resulted in a very low total fresh matter yield and BE (Table 3). On the contrary, sterilized PS casing without microbe inoculation (CK) resulted in higher total fresh matter yield and BE. Compared to the CK treatment, inoculation of both Exiguobacterium sp. (JN03) and Arthrobacter sp. (JN12) on the sterile PS casing resulted in a significant 64% increase in ABM total fresh matter yield. The increase in the total fresh matter yield of the fruiting body also reflected a significant increase in the BE. However, inoculations with B. psychrodurans (JN05) and P. resinovorans (JN16) on the sterile PS casing did not show significant effects on ABM total fresh matter yield and BE, in comparison to CK treatment. Furthermore, inoculation of M. esteraromaticum (JN10) and P. alcaliphila (JN17) resulted in a significant reduction in the total fresh matter yield and BE, in comparison to CK treatment. It is interestingly to note that the enhancement of ABM mycelial growth by individual isolates (JN12, JN16, JN17) does not correlate with the total fresh matter yield. Similar observations were reported for A. bisporus (Wiegant et al., 1992). Results warrant further development of some of the microbes identified here as BM for application in commercial ABM production.
Previous reports have shown that loamy soil can substitute PS as a casing soil for the cultivation of ABM (Pokhrel & Ohga, 2007;Colauto et al., 2010;Chu et al., 2012). Individual isolates were inoculated on loamy casing soil to assess their effects on ABM production. Inoculation of all isolates, except for B. psychrodurans (JN05), on the sterilized loamy casing soil, during the cultivation period, resulted in a 10 to 27-day reduction of the harvesting time (Table 4). Exiguobacterium sp. (JN03) and and P. alcaliphila (JN17), which showed the best fruiting-body promotion, are alkaliphilic bacteria (Yumoto et al., 2001;Vishnivetskaya et al., 2009). Studies on the relationship between the alkaliphilic properties of these bacteria and ABM fruiting-body production will be a topic of future research. Inoculation of all individual isolates, except for B. psychrodurans (JN05), on the loamy casing soil increased the total fresh matter yield from 29 to 95% of the control without inoculation (Table 4). In particular, M. esteraromaticum (JN10), Exiguobacterium sp. (JN03) and P. resinovorans (JN16) inoculation resulted in total fresh yield increases of 95, 62 and 59% over the CK, respectively. Interestingly, the effect of M. esteraromaticum (JN10) and P. alcaliphila (JN17) in PS was opposite to their effect in local loamy casing soil. The most extreme difference was for M. esteraromaticum (JN10) inoculation; 78.8 vs 195% of the control (100%) in PS and loamy soil casing, respectively. This disparity may be due to differences in microbe count after application to the soils, despite weekly application of individual microbes. More likely, the difference in the physicochemical characteristics between the two casing soils -for instance, the relatively acidic pH of PS -may have affected the alkaliphilic microbium M. esteraromaticum (Habe et al., 2004). In any case, further characterization of microbial viability and microbial exudation in the two different environments could be insightful.
Pharmacologically active substances, including PSPC and polysaccharides, were measured in the dried fruiting bodies harvested from flush 1. None of the microbe inoculation treatments showed significant differences in PSPC content from the uninoculated control. Only Exiguobacterium sp. (JN03) inoculation resulted in a significant increase in polysaccharide content (Table 4).
Beneficial microbes are important components of plant ecosystems, where they function to improve plant growth, increase yield, and enhance resistance to diseases (Young & Shen, 2007). Cultivation of ABM to a harvestable size takes a long time, often with low BE. Thus, approaches that could improve ABM productivity are of crucial importance, considering its commercial value.

Conclusions
1. Beneficial microbes can be identified and applied in Agaricus blazei (ABM) cultivation.
2. Application of Exiguobacterium sp. on sterilized casing soil can increase biomass, shorten harvesting time and increase polysaccharide production in ABM. (1) Means followed by equal letters do not significantly differ by Duncan's multiple range test, at 5% probability.
(2) PSPC and polysaccharide contents were analyzed using mushroom harvested from first flush. (3) Increase or reduction of variables in relation to uninoculated control (CK).