Cultivable mushroom growth ‐ promoting bacteria and their impact on Agaricus blazei productivity

The objective of this work was to identify growth‐promoting bacteria isolated from Agaricus blazei and to evaluate their effect on mushroom mycelial growth and productivity. A total of 56 A. blazei‐associated bacterial isolates were obtained from casing soil and identified by 16S rRNA gene sequencing. Bacteria were evaluated as to phosphate‐solubilization ability, nitrogen‐fixation capability, and secretion of cellulase. Superior isolates were tested for their to effect on A. blazei productivity, micelial growth, and on the contents of the polysaccharide‐protein complex and of N, P, K, Ca, and Mg. Bacterial isolates were identified as actinobacteria (60%), firmicutes (20%), and proteobacteria (20%). Among them, ten isolates had phosphate‐solubilization ability, eight showed nitrogen‐fixation capability, and 12 isolates promoted A. blazei mycelium growth. Bacterial inoculation reduces time till harvest in up to 26 days, increases fresh mushroom yield up to 215%, and increases total polysaccharide‐protein complex content twofold when compared to the non‐inoculated control. The actinobacteria group is the predominant A. blazei‐associated phylum.


Introduction
Plant root exudates influence the diversity and activity of rhizobacteria, and may play important roles in nutrient mobilization and plant growth (Kumar et al., 2012).Growth-promoting rhizobacteria (PGPR) in the vicinity of plant root tissues have been reported to stimulate plant growth (Laslo et al., 2012;Sessitsch et al., 2012;Young et al., 2013).However, the screening of mushroom growth-promoting bacteria (MGPB) for mushroom culturing is still limited.Zarenejad et al. (2012) screened the casing layer of 14 edible mushroom farms and identified 23 strains that were potent MGPB.Further fieldwork indicated that Pseudomonas putida was the best suited growth-promoting inoculant for increasing Agaricus bisporus mushroom yield in production farms.In another study, bacteria were isolated from the mycelial surface of Pleurotus ostreatus and assessed as to their roles in the induction of fruiting body (Cho et al., 2003).The authors found that the inoculation of pure culture of the mycelium with strains of fluorescent Pseudomonas spp., isolated from the mycelial plane of commercially produced mushrooms, promoted the formation of primordia and enhanced the development of the basidiome of P. ostreatus.These studies strongly suggest that screening of specific bacteria may have beneficial applications for mushroom production and serve as a basis for future research.
Edible mushrooms with high amounts of protein, minerals, and vitamins are important components of many ethnic diets (Gbolagade et al., 2006).Agaricus blazei contains active organic compounds associated with the maintenance of human health and the healing of diseases.Pharmacological studies have shown that bioactive substances, such as polysaccharide-protein complexes (PSPC) from A. blazei, function as antioxidants, antimutagenics, antitumorigenics, and anticancer agents (Kimura et al., 2004;Firenzuoli et al., 2008).However, the harvesting time of A. blazei is relatively long (approximately 60 days) when compared to other related Agaricus sp., including A. bisporus (approximately 30 days) (Pardo-Gimenez et al., 2010;Chu et al., 2012).Attempts to improve A. blazei productivity have mainly focused on the application of different combinations of culture media and casing soils (Pardo-Gimenez et al., 2010;Chu et al., 2012).Young et al. (2012) studied six A. blazei-associated soil bacteria and successfully identified several beneficial microbes that could significantly improve A. blazei productivity and PSPC content.Therefore, it would be beneficial to characterize mushroom-associated microorganisms and to identify biological agents that can be used to decrease the time and energy required to culture A. blazei.Although metagenomic approaches are widely used in the characterization of microbial communities associated with various crop or soil systems, specific functions of those native microbes and their practical implications remain largely unexplored.
The objective of this work was to identify growth-promoting bacteria isolated from A. blazei casing soil and to evaluate their effect on mushroom mycelial growth and productivity.

Materials and Methods
The casing soil associated with A. blazei stipe was collected from an indoor farm located in Nantou, Taiwan (N23°57'26", E120°58'29").The physicochemical properties of the culture medium and loamy casing soil were previously described in Young et al. (2012) and are summarized in Table 1.The casing soil was soaked in sterile water at a ratio of 1:1 (w/w) and shook for 0.5 hour at 25 o C. Aliquots (100 ml) of the soil solution were plated on nutrient agar (HiMedia Laboratories, Mumbai, India) by the serial dilution method, and incubated at 25 o C for three days.Pure cultures of morphologically different bacterial colonies were isolated and individually cultured on nutrient broth for DNA extraction with a Microbial DNA Isolation Kit Ultraclean, (MO BIO Laboratories, Inc., Carlsbad, CA, USA).The 16S rRNA gene was amplified using 1F (5'-GAGTTTGATCATGGCTCAG-3') and 9R (5'-AAGGAGGTGATCCAACCGCA-3') universal primers (Edwards et al., 1989), with a PCR Master Mix reaction kit (GeneMark Technology Co., Ltd., Tainan, Taiwan) and a Prime Thermal Cycler (Techne Flexigene, Cambridge, UK).Amplification was set at the following conditions: 95 o C for 5 min, followed by 35 cycles of 95 o C for 1 min, 60 o C for 1 min, 72 o C for 2 min, and final extension at 72 o C for 7 min.DNA fragments of approximately 1,500 bp were purified using a QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and were sequenced as described in Young et al. (2012).The cycle sequencing primers used were: 3F: 5'-CCTACGGGAGGCAGCAG-3', 4R: 5'-TTA CCGCGGCTGCTGGCAC-3', and 5F: 5'-AAACTC AAATGAATTGACGGGG-3' (Edwards et al., 1989).The 16S rRNA gene sequences (approximately 1,500 continuous nucleotides) were analyzed using EzBioCloud (2013).Only the identity and accession number that showed the highest level of similarity in the 16S rRNA gene sequence was considered (Table 2).
Cellulase activity of bacterial isolates was determined by incubating in medium containing 0.5% carboxymethyl cellulose for 24 hours and subsequent staining with 0.1% Congo red in triplicates (Sakthivel et al., 2010).Phosphate-solubilization activity was performed using Pikovskaya's medium supplemented with Ca 3 (PO 4 ) 2 , AlPO 4 or FePO 4 , and the presence of clearing zone formation was observed in triplicates (Husen, 2003).Nitrogen-fixation capabilities were assessed using the acetylene reduction assay (Koch   & Evans, 1966).Briefly, bacteria were inoculated in 10 mL nitrogen-free bromothymol blue (Nfb) medium using a 30 mL screw cap test tube for 24 hours.Afterwards, 3 mL of enclosed air were replaced with 3 mL of acetylene.The reaction took place at room temperature for 24 hours.A total of 0.5 mL of air from the test tube was extracted with a syringe and injected in a gas chromatograph, model 163 (Hitachi, Ltd., Tokyo, Japan) equipped with a flame ionization detector in order to analyze ethylene quantity.The separation tube was loaded with 80-100 mesh Porapark R, (Hitachi, Ltd., Tokyo, Japan); the injector was set at 100 o C; the chamber was set at 60 o C; H 2 and N 2 gas flow was set at 40 mL min -1 ; and air flow was set at 0.4 mL min -1 .
Duncan's multiple range test was used as the statistical method for measuring the three replicates.
The effect of the 56 individual isolates on A. blazei mycelium growth was tested as described below.A 0.5-cm diameter block of A. blazei mycelium was spotted on a Difco potato dextrose agar (Voigt Global Distribution Inc., Lawrence, KS, USA) plate along with an individual bacterial strain, which was drawn in a line approximately 2 cm away from the spotted A. blazei mycelium block with a sterilized inoculation loop.After seven days at 28 o C, with a 12 hour/12 hour light/dark cycle, the average mycelium diameter of A. blazei was measured in triplicates and compared to the control without microbial inoculation (Young et al., 2012).
Fresh A. blazei was cultured on sterilized wheat to produce A. blazei grain spawn (Chu et al., 2012).Sterilized sawdust medium (Chu et al., 2012) 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 A. blazei 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 lx, measured with the TES-1335 Digital Light Meter (Tes Electrical Electronic Corp., Taipei, Taiwan).
After complete spawning (one month), loamy soilloamy, mixed, nonacid, hyperthermic, Typic Udorthent (Food and Agriculture Organization of the United Nations, 1988) -was covered on top of the medium to a depth of 2-2.5 cm (Chu et al., 2012).The casing soil was sterilized before use (121 o C, 1.033 kg cm -2 , 1 hour for three times) to avoid possible microbial contaminants and was subsequently inoculated with individual bacterial strains.Seven bacterial isolates were chosen as to their effects on A. blazei productivity.Five milliliters of nutrient broth-cultured bacteria (10 9 cfu mL -1 , determined using the serial dilution method) were evenly applied directly on top of the casing soil, weekly until harvest.The same volume of bacteria-free nutrient broth was applied on the casing soil of the non-inoculated control.Each experiment was conducted with three replicates, in a randomized block design.
Harvesting time of the fruiting body took place when the mushroom reached its 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 mushroom fresh yield (g per pot) and PSPC content were measured from the first mushroom flush.PSPC content was determined according to Sarangi et al. (2006) and the ones of N, P, K, Ca, and Mg were measured using double acid (HClO 4 :HNO 3 = 1:4, v/v) analysis (Jones Junior, 2001).All standards were purchased from Sigma-Aldrich Co. (St.Louis, MO, USA).Data were analyzed statistically using CoStat's statistical procedures (CoHort Software, Pacific Grove, CA, USA), and means were compared by Duncan's multiple range test, at 5% probability.
Actinobacteria isolates belonged to 14 genera, making it the most phylogenetically diverse group.Among them, Microbacterium sp.(29%) and Streptomyces sp.(21%) were dominant, which have been reported to be active indole-3-acetic acid (IAA) phytohormone producers and biological control agents, respectively (Saharan & Nehra, 2011).Notably, Arthrobacter sp. and Mycobacterium sp. have been described as important non-symbiotic nitrogen-fixers and as being able to exert beneficial effects on plant growth (Tilak et al., 2005).Within the identified firmicute phylum, 82% belong to the genus Bacillus, which is the most abundant genus in the rhizosphere.The growth-promoting activity and Pesq. agropec. bras., Brasília, v.48, n.6, p.636-644, jun. 2013 DOI: 10.1590/S0100-204X2013000600009 the plant disease suppressing capabilities of some of these strains have resulted in a broad knowledge of the mechanisms involved (Probanza et al., 2002).The phylum proteobacteria accommodates Enterobacter, Pseudomonas, and Serratia genera, which are known to be associated with the plant rhizosphere and are able to exert growth-promoting effects (Tilak et al., 2005;Zarenejad et al., 2012).In fact, Pseudomonas sp. are ubiquitous bacteria in agricultural soils and have many traits that make them well suited as growth-promoting bacteria (Saharan & Nehra, 2011).Therefore, the A. blazei-associated bacteria identified here could prove useful for the development of biotechnological products for the improvement of A. blazei production.
Functional characterization revealed that 29% of the bacterial isolates were able to secrete cellulase (Figure 1; Table 3).In addition, other growth promotion capabilities were tested for further potential development into MGPB.Approximately 18% of the isolates confer phosphate-solubilization and 14% had nitrogen-fixation ability.Among the A. blazei-associated bacteria, 21% of the isolates promote A. blazei mycelium growth, whereas 14% inhibit its growth.Although there are no known reports on the usage of Gordonia hydrophobica (SBCK202) in agriculture, it was able to solubilize all three forms of insoluble P (Fe-P, Ca-P, and Al-P) and acted as a nitrogen fixer in the present study.This observation merits further investigation for its conversion into beneficial microorganisms in agricultural practices.Strains such as A. taiwanensis (SBABM117), M. agarici (SBCK209), and M. soli (16107) were considered to be novel nitrogen fixers as well.Moreover, A. taiwanensis confers phosphate-solubilization and cellulase secreting abilities.Conventional culturing of A. blazei utilizes plant residues, such as sawdust or rice straw, as a medium for initial spawning of the mycelium (Chu et al., 2012).Since A. blazei is a secondary saprophyte, bacterial isolates that could degrade cellulose may be beneficial to mycelial growth.Promotion of mycelium growth by inoculants has been reported to enhance mushroom productivity (Kim et al., 2008).Overall, the above data provided benchmarks to select and shortlist seven isolates from the total of 56 to test their effects on A. blazei productivity.
Actinomycetales bacterium N12, Advenella incenata, A. taiwanensis, Curtobacterium citreum, G. hydrophobica, M. humi, and Streptomyces violaceorubidus were selected to be tested.Sterilized soil casing without bacterial inoculation (control) resulted in a harvest time of 67 days after casing (Table 4).Inoculation of Actinomycetales bacterium N12 or S. violaceorubidus in the casing soil increased the time till harvest, whereas the inoculation of the other five isolates resulted in reductions ranging from 21 to 26 days, compared to control.Among the seven tested isolates, A. taiwanensis and M. humi were tested as to their effects on mycelium growth in a co-culture plate assay, which resulted in shorter harvesting time.The isolates A. incenata, C. citreum, and G. hydrophobica were neutral to the mycelium growth of A. blazei but also reduced the time till harvest.These results suggest that the promotion of mycelium growth does not necessarily reflect faster fruiting body development.Other studies are necessary to identify unknown biotic factors (e.g.microbial exudates) that may contribute more significantly to mushroom development.
Regarding mushroom yield in the first flush, inoculation of all seven isolates individually exhibited significant increase in total fresh yield, ranging from 170 to 215% of the non-inoculated control.Again, this result does not correlate with promotion of mycelium growth, as reported in other edible mushrooms (Kim et al., 2008).It is interesting to note that P. putida was the best suited growth-promoting inoculant for increasing A. bisporus mushroom yield (Zarenejad et al., 2012).However, in a previous report, inoculation of P. resinovorans (103% of the non-inoculated control) and P. alcaliphila (74% of the non-inoculated control) did not improve A. blazei productivity (Young et al., 2012).Future works on possible microbial inducing substances of fruiting body and on different combinations of bacterial isolates should be conducted in order to determine the relation of MGPB with mushroom development.Inoculation of G. hydrophobica in the casing soil significantly increased PSPC content when compared to that of the non-inoculated control (Table 4).
Inoculations of the other six isolates had no effect or showed slight decrease in PSPC content in comparison to that of the control.However, the increase in fresh yield from the first mushroom flush in most treatments can boost the total extractable PSPC content to almost twofold of the control.

Figure 1 .
Figure 1.Frequency and affiliation of isolates from Agaricus blazei casing soil conferring various metabolic and growth promoting activities.The column of total isolates shows the phylogenetic affiliation of each population for comparison.

Table 1 .
Physicochemical properties of loamy casing soil and sawdust medium used to culture Agaricus blazei.

Table 2 .
Identification of bacterial strains isolated from Agaricus blazei casing soil based on most similar 16S rRNA gene sequence.

Table 5 .
Effect of microbe inoculation in loamy casing soil on inorganic element concentrations in fruiting bodies from first flush of Agaricus blazei(1).
Means followed by equal letters do not differ by Duncan's multiple range test, at 5% probability.(2)Control,sterile soil without inoculation.All microbe tests were inoculated on sterile soil.Sawdust medium was used for culturing of A. blazei in all tests.

Table 4 .
Effect of bacterial inoculation in loamy casing soil on harvesting time, fresh yield, and polysaccharide protein complex (PSPC) content of Agaricus blazei(1).