**5.3 Characterization of bacterial strains isolated from SMS**

Four bacteria species were isolated from spent mushroom substrate used in this study. They were coded isolate B1, B2, B3 and B4. These microorganisms were identified and characterized as *Bacillus, Clostridium, Pseudomonas,* and *Escherichia coli*.

The isolation of these bacteria from composting agricultural substrates suggests that a form of fermentation had taken place during the composting process. Addition of straw in the soil caused an increase in the number of total bacteria, actinomycetes and fungi of the rhizosphere [49].

Different substrate harbor different kind and number of microorganisms and the variation in microbial population in different substrates is due to nutritional or chemical composition of the substrate.

Similar isolation of *Bacillus* and *Clostridium* species were reported from fermenting cocoa beans [50, 51]. The presence of *Pseudomonas* sp. (Isolate B3) in the fermenting SMS may be related to its ability to survive in vast number of habitats. From the results obtained, it can be concluded that various bacteria genera were involved in the decomposition of further microbial SMS. The pure cultures of these bacteria could be incorporated into agricultural wastes in a controlled fermentation unit.

Effect of rice straw compost on soil microbial population reported that, compost application resulted in marked increase of organic matter content in the soil in relation to initial value of plain sandy soil which affirmatively exaggerated the bacterial and fungal populations and that microbial population increased with the increase in dosage of compost [52].

Similar results were reported in maize by using *Agaricus bisporus* spent mushroom compost [53] using recomposted button mushroom spent substrate with wheat crop and with *Pleurotus florida* spent substrate on tomato crop.

#### **5.4 Anti-fungal activity of bacterial strains isolated from SMS extracts**

Spent mushroom compost from *Pleurotus* sp., used in this study harbored bacterial population including, *Bacillus, Clostridium, Pseudomonas* and *E. coli*. This is in support of the findings on the microbial composition of spent mushroom compost of *Pleurotus* sp. [53]. SMS used for soil amendment has been found to be more efficient than commercial fungicides and nematicide in controlling soil borne pathogens like *Meloidogyne* sp. in tomato, *Venturia inaequalis* in apple [53]. Some of these microorganisms have been reported to possess antagonistic property in several

**261**

*Screening and Potential Uses of Contaminated Spent Mushroom (*Pleurotus *spp.)*

studies [54] and this was confirmed in this study from the result obtained from the dual culture assay involving the SMS microbial isolates and plant pathogenic fungi. Fungal pathogens such as *Fusarium oxysporum* and *Phytophthora* cause severe plant diseases, limiting plant yields as well as the quality of the products. Moreover, they have wide host spectra, causing diseases in economically important agricultural crops worldwide [55]. These fungal phytopathogens are difficult to control not only because of their wide host spectra, but also because of their soil borne nature [56]. *Aspergillus flavus* is the most deleterious fungus in stored rice grains, and it receives particular attention because of its ability to produce potent carcinogenic

*Alternaria solani*, *Alternaria alternata*, *Fusarium solani*, *Phytophthora megasperma* and *Verticillium dahlia* are ubiquitous and cosmopolitan phytopathogens causing

The findings on antifungal activity of four bacterial strains *Bacillus* sp., *Clostridium* sp., *Pseudomonas* sp. and *E. coli* isolated from SMS are shown in **Table 7** and **Figure 1**. Using antibiotic producing bacteria to control plant fungal diseases is a popular topic and has extensively been studied [60]. Compared with chemical biocides, many antibiotics produced by antagonistic strains have the advantage of being easily decomposed in nature, leaving no harmful residues behind. The results, of the present study on the **in vitro** sensitivity of phytopathogenic fungi to antagonistic bacteria revealed that the isolates of *B. subtilis* were suppressive, though with different degrees, to the tested isolates of phytopathogenic fungi, are consistent with

*Bacillus* and *Pseudomonas* sp. show antifungal effect against soil borne plant

**Bacillus subtilis** showed strong ability against many common plant fungal pathogens **in vitro** [61]. The investigation of bio control activity of the strains in this study revealed that they could produce extracellular secondary metabolites with antifungal activity against the tested fungi. These strains inhibited the mycelial growth of the fungus in dual-culture assays. The Pseudomonas isolates obtained from soil were shown to reduce growth of *Aspergillus niger, Fusarium sp., Alternaria* 

Our results on the antifungal activity of the microbes isolated from SMS (**Table 7** and **Figure 1**) which showed that the culture filtrates of bacterial strains, *Bacillus megaterium* KU143*, Microbacterium testaceum* KU313, and *Pseudomonas* 

The SMS isolate of *Pseudomonas* sp. was tested positive for antifungal activity against *Phytophthora* sp. (**Table 7** and **Figure 1**). Similarly this bacterium was found efficient in inhibiting the mycelial growth and the antifungal compounds extracted were found inhibitory to the growth of *Rhizoctonia* sp.*, Phytophthora parasitica,* 

The inhibitory properties of spent mushroom substrate remained unaffected even after autoclaving and filter sterilization of extract [54]. Unsterilized spent mushroom compost had a better inhibition potential than sterilized compost, this suggested that the pathogen inhibitory properties of spent mushroom compost could be more due to the biotic components than the abiotic components, i.e., more due to the activities of the inherent microorganisms rather than the chemical

The results of the present study confirm that spent mushroom substrate contains a large number of indigenous beneficial microbes capable of suppressing soil-borne pathogens. This character of the SMS can be utilized as an alternate substrate for the in mass production of biocontrol agents for field application which may lead to

properties or the organic matter content. Similar views were reported [65].

suppression of diseases leading to increased crop productivity.

pathogenic fungi *c, Fusarium solani* and *Fusarium oxysporum* [63].

*protegens* significantly inhibited the growth of *A. flavus*.

*DOI: http://dx.doi.org/10.5772/intechopen.93863*

severe diseases in wide range of crops [58, 59].

those obtained by others [61, 62].

*solani, Drechslera oryzae* etc., [64].

*P. palmivora* and *Fusarium solani*.

aflatoxins [57].

#### *Screening and Potential Uses of Contaminated Spent Mushroom (*Pleurotus *spp.) DOI: http://dx.doi.org/10.5772/intechopen.93863*

studies [54] and this was confirmed in this study from the result obtained from the dual culture assay involving the SMS microbial isolates and plant pathogenic fungi.

Fungal pathogens such as *Fusarium oxysporum* and *Phytophthora* cause severe plant diseases, limiting plant yields as well as the quality of the products. Moreover, they have wide host spectra, causing diseases in economically important agricultural crops worldwide [55]. These fungal phytopathogens are difficult to control not only because of their wide host spectra, but also because of their soil borne nature [56]. *Aspergillus flavus* is the most deleterious fungus in stored rice grains, and it receives particular attention because of its ability to produce potent carcinogenic aflatoxins [57].

*Alternaria solani*, *Alternaria alternata*, *Fusarium solani*, *Phytophthora megasperma* and *Verticillium dahlia* are ubiquitous and cosmopolitan phytopathogens causing severe diseases in wide range of crops [58, 59].

The findings on antifungal activity of four bacterial strains *Bacillus* sp., *Clostridium* sp., *Pseudomonas* sp. and *E. coli* isolated from SMS are shown in **Table 7** and **Figure 1**. Using antibiotic producing bacteria to control plant fungal diseases is a popular topic and has extensively been studied [60]. Compared with chemical biocides, many antibiotics produced by antagonistic strains have the advantage of being easily decomposed in nature, leaving no harmful residues behind. The results, of the present study on the **in vitro** sensitivity of phytopathogenic fungi to antagonistic bacteria revealed that the isolates of *B. subtilis* were suppressive, though with different degrees, to the tested isolates of phytopathogenic fungi, are consistent with those obtained by others [61, 62].

*Bacillus* and *Pseudomonas* sp. show antifungal effect against soil borne plant pathogenic fungi *c, Fusarium solani* and *Fusarium oxysporum* [63].

**Bacillus subtilis** showed strong ability against many common plant fungal pathogens **in vitro** [61]. The investigation of bio control activity of the strains in this study revealed that they could produce extracellular secondary metabolites with antifungal activity against the tested fungi. These strains inhibited the mycelial growth of the fungus in dual-culture assays. The Pseudomonas isolates obtained from soil were shown to reduce growth of *Aspergillus niger, Fusarium sp., Alternaria solani, Drechslera oryzae* etc., [64].

Our results on the antifungal activity of the microbes isolated from SMS (**Table 7** and **Figure 1**) which showed that the culture filtrates of bacterial strains, *Bacillus megaterium* KU143*, Microbacterium testaceum* KU313, and *Pseudomonas protegens* significantly inhibited the growth of *A. flavus*.

The SMS isolate of *Pseudomonas* sp. was tested positive for antifungal activity against *Phytophthora* sp. (**Table 7** and **Figure 1**). Similarly this bacterium was found efficient in inhibiting the mycelial growth and the antifungal compounds extracted were found inhibitory to the growth of *Rhizoctonia* sp.*, Phytophthora parasitica, P. palmivora* and *Fusarium solani*.

The inhibitory properties of spent mushroom substrate remained unaffected even after autoclaving and filter sterilization of extract [54]. Unsterilized spent mushroom compost had a better inhibition potential than sterilized compost, this suggested that the pathogen inhibitory properties of spent mushroom compost could be more due to the biotic components than the abiotic components, i.e., more due to the activities of the inherent microorganisms rather than the chemical properties or the organic matter content. Similar views were reported [65].

The results of the present study confirm that spent mushroom substrate contains a large number of indigenous beneficial microbes capable of suppressing soil-borne pathogens. This character of the SMS can be utilized as an alternate substrate for the in mass production of biocontrol agents for field application which may lead to suppression of diseases leading to increased crop productivity.

*Emerging Contaminants*

shoot and root weight.

crop requirement.

and fungi of the rhizosphere [49].

dosage of compost [52].

chemical composition of the substrate.

compost (SMC) of *Pleurotus ostreatus* improved the agronomic characters and yield (pod no, fresh weight and dry weight) when it was added as soil conditioner to

Reason for poor root and shoot growth of the seedlings of the selected plants in 100% SMS may be that the paddy straw based. SMS may only be used as an amendment and not as a basic growth medium. While growth on straw, *Pleurotus* releases humic acids like fractions which when added to soil would increase its fertility. In addition, humic substances may affect the plant biochemical process [48]. Present findings confirm the efficacy of SMS in growth promotion in terms of seedling

SMS from *Agaricus bisporus, Hericium erinaceus* and *Pleurotus ostreatus* are effective to the growth promotions of pea, pepper and tomato plants respectively [29, 36]. The results of the present study revealed that the compost has a good impact in promoting better growth and yield. Further in order to promote growth and yield, it becomes imperative to optimize the usage of organic manure according to the

Four bacteria species were isolated from spent mushroom substrate used in this study. They were coded isolate B1, B2, B3 and B4. These microorganisms were identified and characterized as *Bacillus, Clostridium, Pseudomonas,* and *Escherichia coli*. The isolation of these bacteria from composting agricultural substrates suggests that a form of fermentation had taken place during the composting process. Addition of straw in the soil caused an increase in the number of total bacteria, actinomycetes

Different substrate harbor different kind and number of microorganisms and the variation in microbial population in different substrates is due to nutritional or

Similar isolation of *Bacillus* and *Clostridium* species were reported from fermenting cocoa beans [50, 51]. The presence of *Pseudomonas* sp. (Isolate B3) in the fermenting SMS may be related to its ability to survive in vast number of habitats. From the results obtained, it can be concluded that various bacteria genera were involved in the decomposition of further microbial SMS. The pure cultures of these bacteria could be

Effect of rice straw compost on soil microbial population reported that, compost application resulted in marked increase of organic matter content in the soil in relation to initial value of plain sandy soil which affirmatively exaggerated the bacterial and fungal populations and that microbial population increased with the increase in

Similar results were reported in maize by using *Agaricus bisporus* spent mushroom compost [53] using recomposted button mushroom spent substrate with

incorporated into agricultural wastes in a controlled fermentation unit.

wheat crop and with *Pleurotus florida* spent substrate on tomato crop.

**5.4 Anti-fungal activity of bacterial strains isolated from SMS extracts**

Spent mushroom compost from *Pleurotus* sp., used in this study harbored bacterial population including, *Bacillus, Clostridium, Pseudomonas* and *E. coli*. This is in support of the findings on the microbial composition of spent mushroom compost of *Pleurotus* sp. [53]. SMS used for soil amendment has been found to be more efficient than commercial fungicides and nematicide in controlling soil borne pathogens like *Meloidogyne* sp. in tomato, *Venturia inaequalis* in apple [53]. Some of these microorganisms have been reported to possess antagonistic property in several

soybean at different levels of its concentrations [14].

**5.3 Characterization of bacterial strains isolated from SMS**

**260**

*Emerging Contaminants*
