Optimization of Fermentation Conditions of Lentinula edodes (Berk.) Pegler (Shiitake Mushroom) Mycelia as a Potential Biopesticide

The shiitake mushroom [Lentinula edodes (Berk.) Pegler], is a new source of biopesticide against bacterial spot of tomato having similar efficacy as that of traditional antibiotics in vitro. The culture-filtrates of fifteen commercially available L. edodes strains were screened for antibacterial activity against Erwinia amylovora (Ea) and Xanthomonas campestris pv. vesicatoria (Xcv), based on fermentation time and carbon source for minimum oxalic acid production. Two different carbon sources, glucose and sucrose for fifteen and 30 days were used for fermentation. The detection and quantification of oxalic acid in culture filtrates were performed by using HPLC. Most of the L. edodes strains inhibited the growth of Ea (13 out of 15) and Xcv (14 out of 15) with similar efficacy as that of the control treatment of 100 μg mL streptomycin sulfate. Two of the L. edodes strains, ATCC 38164 and ATCC 28760 released the least amount of oxalic acid in both carbon sources, but did not differ from each other significantly. The concentration of oxalic acid in glucose medium was generally lower than that in sucrose medium, while strains ATCC 20635 and ATCC 38167 released the highest and similar concentrations in both carbon sources. This study provides preliminary evidence that L. edodes strains may be potential alternatives to streptomycin and copper compounds as a source of metabolites against bacterial spot of tomato and fire blight of apple [Malus × domestica (Borkh.)] and merits further investigation.


Introduction
Sustainable agricultural systems particularly in plant protection of organic production systems face numerous challenges due to lack of robust organic pesticides as summarized by Crowder and Harwood (2014). The management of bacterial plant diseases, such as, bacterial spot of tomato (Solanum lycopersicum L.) and fire blight of apple [Malus × domestica (Borkh.)] and pear (Pyrus communis L.) caused by Xanthomonas campestris pv. vesicatoria (Xcv) and Erwinia amylovora (Ea), respectively is even more challenging (McManus & Stockwell, 2000;Potnis et al. 2015). Numerous authors (McManus & Stockwell, 2000;McManus, 2014;Potnis et al., 2015;Griffin et al., 2017) have addressed the issue that, farmers can no longer rely on the traditional control measures involving the use of copper compounds and streptomycin for these plant diseases. In the United States, streptomycin had been permitted to manage fire blight of apple and pear as a standard control measure in conventional and organic orchards (McManus & Stockwell, 2000;Anonymous, 2012). But residual streptomycin detected in apple fruits (Mayerhofer et al., 2009) and streptomycin resistant genes, discovered in E. amylovora isolates from shoots, blossom and rootstock in apple orchards (Tancos et al., 2016), indicate the possibibily of horizontal gene transfer to the non-target micro-organisms. While there is no direct evidence that the use of streptomycin in agriculture introduces antibiotic resistance to non-taget microorganisms or human pathogens through the food chain (McManus, 2014), the overall concerns about antibiotic resistance need to be addressed (Mayerhofer et al., 2009;Shade, 2013;McManus, 2014;Singer & Williams-Nguyen, 2014). Therefore, research focusing on developing alternatives to streptomycin for use in organic apple and pear production is of high priority (Anonymous, 2012).
Research efforts focused on streptomycin alternatives involve the use of non-pathogenic bacteria, plant or compost extracts, essential oils, antibiotics, synthetic chemical compounds, bacteriophages, and nano-materials (Griffin et al., 2017). Of the many products, one of the most effective against bacterial spot and fire blight is acibenzolar-S-methyl (ASM) (Johnson et al., 2016;Griffin et al., 2017). However, ASM is not as effective as standard control measures using copper compounds and streptomycin sulfate against the bacterial spot of tomato and fire blight of apple, respectively (Johnson et al., 2016;Griffin et al., 2017).
In our previous study (Kaur et al., 2016), findings indicated that L. edodes culture-filtrate suppressed bacterial spot symptoms in tomato with the same efficacy as that of 100 µg mL -1 streptomycin sulfate in vitro, however, oxalic acid phytotoxicity symptoms were observed under natural conditions. According to Heleno et al. (2015), the most common secondary metabolites in L. edodes fruiting bodies are sugars (fructose, mannitol and trehalose), fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid and α-linolenic acid), phenolic compounds (protocatechuic acid, p-hydroxybenzoic acid and cinnamic acid), and organic acids (fumaric acid, citric acid, malic acid, quinic acid, and oxalic acid). Oxalic acid, a toxic organic acid is one of the most common fungal virulence factors which causes phytotoxicity in plants (Cessna et al., 2000;Williams et al., 2011;Kaur et al., 2016).
The concentration of fungal metabolites such as oxalic acid in the fermentation medium varied with the type of carbon source and time (Ishikawa et al., 2001;Hassegawa et al., 2005;Mandal & Banerjee, 2005). In this study, we report the antibacterial activity of the culture-filtrate from various strains of L.edodes against Xcv, Ea, and optimize fermentation conditions with an appropriate carbon source for the least oxalic acid production.

Lentinula edodes Strains and Fermentation
The shiitake mushroom strains of L. edodes, American Type Culture Collection (ATCC) # 20546, 20635, 24462, 28760, 36558, 38164, 38165, 38166, 38167, 38169, 38172, 38173, 44744, 48858 and 56004 were purchased from ATCC (ATCC, Rockville, MD, USA). Lentinula edodes mycelia were grown and fermented using the same procedure (except time and carbon source) as in our previous study (Kaur et al., 2016). Lentinula. edodes mycelia-cultures, from American Type Culture Collection (ATCC) # 38169 used by Kaur et al. (2016), were fermented for 15 days in glucose and sucrose carbon sources. Since oxalic acid concentrations in glucose and sucrose were similar, culture-filtrates fermented for 30 days in sucrose carbon source from fourteen more L. edodes strains were screened for their antibacterial activity against Xcv and Ea cells in vitro. A new carbon source as glucose and fermentation time of 15 days were included in the fermentation for this study. All fermentations were carried out by using three biological replicates of each L. edodes strain. After fermentation, culture-filtrates were processed (filtered through sterile Whatman filter sheets followed by 0.45 µm sterile syringe filtration, stored at -80 o C).

Bacterial Phytopathogens
Xanthomonas campestris pv. vesicatoria (Xcv) and Erwinia amylovora (Ea) cells were locally isolated and maintained in pure cultures on agar plates as described in a previous study (Kaur et al., 2016). The plant pathogenicity assays for Xcv and Ea were performed on tomato plants and pear detached leaves, respectively as per Koch's postulates (isolation of an organism from diseased plant tissue, infection of a healthy plant and re-isolation from infected plant tissue).

Antibacterial Assays
The antibacterial assays were conducted in three biological replicates of freshly prepared inocula of Xcv and Ea. All L .edodes culture filtrates were tested for their antibacterial activity on a bacterial concentration of 10 8 colony forming units (cfu). Microbial cell population for the assays was determined using optical density (OD 625 ) and spread plating (serial dilutions) methods. The bactericidal activity of culture-filtrates was compared to 100 µg mL -1 streptomycin sulfate which is the concentration used in commercial and organic pome fruit orchards (Johnson & Temple, 2013) as a positive control and bacterial cell suspension as a negative control. Please refer to our previous study (Kaur et al., 2016) for detailed methodology.

Oxalic Acid Detection and Quantification
The oxalic acid content analysis was carried out by modifying the method developed by Tormo and Izco (2004). The oxalic acid separation was achieved by Varian Prostar HPLC system equipped with a 335 Ultraviolet-Visible (UV-VIS) Photodiode detector, Prostar 410 autosampler, reverse phase c 18 column (Pecosil 5 cm length, 5 mm internal diameter), and a Prostar 240 quaternary pump. The mobile phase was 1% (v/v) acetonitrile (Thermo Fisher Scientific, Waltham, MA, USA) in 20 mM of the sodium phosphate buffer adjusted to 2.2 pH with phosphoric acid (prepared in de-ionized double-distilled water filtered through a 0.45 µm nylon membrane). The solvent was re-filtered after preparation. A standard oxalic acid stock solution of 2000 mg mL -1 was prepared by dissolving pure oxalic acid (Sigma Chemical Company, St. Louis, MO, USA), in double-deionized distilled-water and filtered through a 0.45 µm nylon membrane. The oxalic acid calibration curve ( Figure A1) was developed by plotting the chromatogram area from six concentrations (100-600 µg mL -1 ) in triplicates with duplicate injections. The mobile phase flow rate was 1.5 mL min -1 . The injection volume of 100 µL was run for 10 min with two min equilibration time. An ambient column temperature was maintained through the analysis. Oxalic acid was detected at 210 nm wavelength. The data were acquired and analyzed using the software LC Workstation (version 6.41 HPLC). In culture-filtrates, oxalic acid was detected and quantified (three BRs of each strain, duplicate injections) by comparing the retention time and peak areas with the standard curve.

Statistical Analysis
Data generated by each experiment were analyzed for statistical significance using the Statistical Analysis System (SAS®9.2 SAS Institute Inc., Cary, NC, USA) software. Analysis of Variance (ANOVA) and General Linear Model (GLM) procedures of SAS were utilized, followed by treatment mean separation by LSD test at 5% level of probability.

Xanthomonas campestris pv. vesicatoria (Xcv) Inhibition
The culture filtrates from 14 L. edodes strains collected after 30 d fermentation in sucrose carbon source, inhibited the growth of Xcv cells with the same level of efficacy (P < 0.0001) as that of 100 µg mL -1 streptomycin sulfate ( Figure 1A).

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Discussion
Of the abundant research strategies tested to search for new and effective streptomycin alternatives to combat antimicrobial resistance in the Xcv population, ASM (acibenzolar-S-methyl) has emerged as one of the most effective products. However, its use is limited by the low efficacy as a stand-alone compound, and yield reductions in the host plants (Johnson et al., 2016;Griffin et al., 2017). It has been reported that foliar application of L. edodes culture-filtrates suppresses bacterial leaf spot symptoms in tomato in vitro, without adversely impacting plant height and flowering in vivo; however, oxalic acid phytotoxicity was observed on tomato foliage when tested in growth chambers (Kaur et al., 2016).
Oxalic acid in culture-filtrates of different strains of L edodes fermented for different times in sucrose or glucose media varied between 222.8-709.1 µg mL -1 . In general, all L. edodes strains released lower oxalic acid in glucose compared to sucrose carbon source. There are no previous reports on the oxalic acid concentration of L. edodes culture-filtrates after fermenting in different carbon sources. However, immobilized particles of Aspergillus niger have been reported to release a higher amount of oxalic acid in glucose compared to sucrose and lactose carbon sources during 7 d fermentation at 30 o C, and 215 rpm (Mandal & Banerjee, 2005). The difference in extracellular oxalic acid concentration released by different L. edodes strains in the same medium during fermentation may be due to genetic variation among strains. Despite the variability in oxalic acid concentration, almost all of the L. edodes strains exhibited similar antibacterial activity against the Xcv cells.
Mycelial-culture-filtrate of several strains of L. edodes, tested in this study inhibited the in vitro growth of E. amylovora cells with the same efficacy as that of streptomycin. Pacumbaba et al., (1999) also reported similar findings of in vitro inhibition of Ea cells by L. edodes filtrates. Moreover, the antibacterial activity of culture-filtrates remained unaffected during and after storing for 120 d at -80 o C.
Although culture-filtrates were not tested on pear or apple leaves in this study, oxalic acid has been known to be a phytotoxin in model plants including tomato (Cessna et al., 2000;Williams et al., 2011;Kaur et al., 2016). In future, tomato, apple or pear plant assays it would be important to use L. edodes strains which release the least oxalic acid in the media during fermentation.
In this study, L. edodes 28760 exuded the lowest amount of oxalic acid in both sugars and exhibited Xcv and Ea growth inhibition similar to streptomycin during the initial screening of the fifteen strains. Lentinula edodes culture-filtrate may also become a potential alternative to streptomycin against fire blight of apple and pear, and other toxic chemicals in agriculture. Lentinula edodes strains, 28760, and 38164 are good candidates for further assessment as a potential biopesticide against the bacterial spot of tomato. Studies involving genomics for the genetic identification of these strains would yield beneficial results.