American Journal of Biochemistry and Biotechnology

Original Research Paper

Characterization of Fungal Communities in Limed and Unlimed Lands Contaminated with Metals: Phospholipid Fatty Acid (PLFA) Analysis and Soil Respiration

1Kassandre Goupil, 1,2 Kabwe K. Nkongolo and 1Sabah Nasserulla

1Department of Biology, 2Biomolecular Sciences Program, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada

Article history Abstract: Northern Ontario (Canada) especially the Greater Sudbury Received: 05-04-2015 region is highly known for its nickel, copper and other metal deposits. The Revised: 28-04-2015 mining, roasting and smelting of these elements have caused disastrous Accepted: 13-05-2015 effects on the vegetation and overall environment. Dolomitic lime which

contains calcium and magnesium carbonate was applied to soils from 1980 Corresponding Author: Kabwe K. Nkongolo to 1995 at different locations across Northern Ontario. The objective of Department of Biology, the present study is to determine fungi diversity and abundance in Laurentian University, selected limed and unlimed areas contaminated with metals in the Region. Sudbury, Ontario, P3E 2C6, Soil respiration, fungi cultures and Phospholipid Fatty Acid (PLFA) were Canada analyzed. The liming did maintain an increase in soil pH from extremely Email: [email protected] acid to slightly acid, even 25 to 30 years after liming applications. A total of 52 fungi species belonging to 34 genera were identified on growth media. The majority of fungi (up to 70%) in all the sites belong to the phylum. Some species were specific to one or two sites, while others were present in the majority of the sites. Fungal diversity and abundance were higher in limed soils compared to unlimed samples based on SDA medium growth. The rates of soil respiration in limed sites were also higher compared to unlimed areas. Phospholipid Fatty Acid (PLFA) analysis revealed a significantly higher total microbial biomass in samples from limed areas compared to unlimed samples. Total and arbuscular mycorrhizal fungi abundance based on this analysis followed the same trend. Surprisingly, there were 7 to 10 fold more bacteria than fungi in all the sites. Moreover, there was twice more Gram (-) bacteria than Gram (+) indicating that the sites are still severely stressed. Soil pH appears to be the most important factor for microbial abundance, diversity and activities than total metal content.

Keywords: Soil Liming, Fungi Diversity and Abundance, Phospholipid Fatty Acid, Soil Respiration, Northern Ontario

Introduction also reported a decrease of the levels of colonization of roots of oxeye daisy ( Chrysanthemum leucanthemum ) Mining and smelting processes and activities have and devil’s paintbrush ( Hieracium aurantiacum ) by caused a variety of negative effects to the Northern vesicular arbuscular mycorrhizal fungi in the Sudbury Ontario environment. SO 2 fumigation and the barrens (Balsillie et al ., 1978). accumulation of numerous metal particulates have A decrease in industrial emissions and remediation damaged soil quality and ultimately, the vegetation and projects were initiated to restore Northern Ontario fungi populations inhabiting the region (Winterhalder, ecosystems (Winterhalder, 1996; Nkongolo et al ., 2013; 1996). Balsillie et al . (1978) have documented a Theriault et al ., 2013). Remediation consisted of decrease in soil fungi and bacteria near the location of neutralizing soil pH with the addition of lime as well as smelter sites within the Greater Sudbury Region. They adding fertilizer, seed distribution and tree planting

© 2015 Kassandre Goupil, Kabwe K. Nkongolo and Sabah Nasserulla. This open access article is distributed under a Creative Commons Attribution (CC-BY) 3.0 license. Kassandre Goupil et al . / American Journal of Biochemistry and Biotechnology 2015, 11 (2): 45.56 DOI: 10.3844/ajbbsp.2015.45.56

(Winterhalder, 1996). In fact, dolomitic lime which Materials and Methods contains calcium and magnesium carbonate was applied to soils from 1980 to 1995 at different locations across Soil Sampling and Processing Northern Ontario. This resulted in an increase of organic Soil sampling was completed as described in Goupil matter that requires adequate microbial biomass for and Nkongolo (2014) at seven sites that include mineralization’s processes. The intricacies of soil-fungal Wahnapitae Hydro-Dam, Daisy Lake, Kingsway, Kelly interactions are crucial in determining fungi diversity Lake, St.Charles (Hagar), Onaping Falls and Capreol (Fig. and abundance for a particular region. Thus, soil 1). Each sample was dried at room temperature (25°C) for chemistry, metal binding capacity and metal speciation 48 h. It was then sifted with a 2 mm sieve to remove all within the soil are key concepts that need to be forms of debris. Three soil samples from each of the adequately assessed and properly understood in order to limed, unlimed or reference sites were mixed to produce a sufficiently identify metal-fungal dynamics for a ‘master’ limed, unlimed or reference soil sample to be specific region. The examination of fungal diversity used later for fungal analysis. These “master mix” for a particular area is critical as it is an essential samples were used for serial dilutions. Only the top 5 cm factor in the determination of their sustainability (LFH layer) of organic soil was analyzed because it was (Nielsen and Winding, 2002). Additionally, fungal hypothesized that the majority of fungi colonies would abundance is another prime indicator of their be present within this soil horizon. population health (Nielsen and Winding, 2002). Since fungi play such a crucial role in soil formation, organic Fungal Isolation Method matter decomposition as well as in the transformations All materials, except the soil samples, were of toxic metabolites found in the soil (Ritz and Young, autoclaved before use. Serial dilutions were 2004), soil amendment strategies adopted in damaged subsequently prepared. For that purpose, 10.0 g of soil ecosystems, such as liming, require extreme diligence was mixed with 90.0 mL of sterile H 2O resulting in a to ensure that all fungal nutrient, metal, substrate dilution of 1/10. A sterile stopper was then firmly placed requirements are addressed and achieved. on the Erlenmeyer flask containing the 1/10 dilution. The objective of the present study is to determine The solution was shaken vigorously for 1 h. Four fungi diversity and abundance in selected limed and successive dilutions by a factor of 10 were then unlimed areas contaminated with metals in the Northern prepared. They include 1/100, 1/1000, 1/10000, Ontario Region. Soil respiration, fungi cultures and 1/100000 and 1/1000000. Phospholipid Fatty Acid (PLFA) were analyzed.

Fig. 1. Locations of sampling areas from the Greater Sudbury Region. Site 1: Wahnapitae Hydro-Dam; Site 2: Kingsway; Site 3: Kelly Lake; Site 4: St.Charles (Hagar); Site 5: Capreol and Site 6: Onaping Falls. Sites 4, 5 and 6 were used as reference sites. St.Charles (Hagar) is outside the map as a reference site and is about 50-60 km from Sudbury. Source: Edited from Google Map 2014

46 Kassandre Goupil et al . / American Journal of Biochemistry and Biotechnology 2015, 11 (2): 45.56 DOI: 10.3844/ajbbsp.2015.45.56

Two medium types were used for this study, namely between limed, unlimed and reference soil samples for Sabouraud Dextrose Agar (SDA) and Malt Extract Agar fungal diversity and abundance. Individual differences were (MEA). SDA is a multipurpose growing medium for determined using Tukey’s multiple range test. numerous fungal species whereas, MEA is an ideal medium for the cultivation of and . For each Results medium (SDA and MEA), six plates including a control and different diluted solutions were prepared and Soil pH Analysis duplicated. The labelled plates were inoculated with 0.1 mL of each solution respectively. The inocula were evenly The pH in unlimed sites was consistent with that spread over the surface of the agar plate using a sterile documented for soils on coarser textured soils with glass rod. The plates were then incubated at 26°C until coniferous vegetation on the Canadian Shield of at <4, colony masses were easily observed. The plates were classified as extreme acid (15, 16). The pH in limed examined every day starting at 3-4 days after incubation. sites was significantly higher ranging from 4.12 to 6.75 in the top organic layer compared to unlimed sites Slide Preparation (Table 1). The liming did maintain an increase in soil pH from extremely acid to slightly acid, even 20 to 35 All standard microscope slides were cleaned with years after liming applications. 95% ethanol. A few drops of lactophenol cotton blue were then placed on the slides. With the aid of a pin Fungi Diversity and Abundance probe, parts of a fungal colony were placed onto the slide. A cover slip was then placed onto the mount. Slides were Figure 2 shows the long term effect of liming on plant properly labeled and air dried before storage. Fungi vegetation and population complexity. All fungal species identification was performed using classification guides identified in limed and unlimed sites as well as in the and was based on microscopic morphology and colony reference sites are described in Fig. 3-5. Venn diagrams appearance on the media. The reference guides included (Fig. 3-5) also show different fungi species and their detection and isolation of soil fungi (Davet and Rouxel, shared or specific site location. Mean number of fungi and 2000); Compendium of soil fungi (Domsch et al ., mean level of fungi abundance for limed, unlimed and 2007); Identification of fungi: An illustrated reference sites are presented in Table 2 and 3. The most introduction with keys, glossary and guide to literature abundant fungi species are described in Table 4. (Dugan, 2006); A laboratory guide to common Penicillium species (Pitt and Hocking, 1985); Pictorial Table 1. Mean pH for limed, unlimed and reference sites in atlas of soil and seed fungi: Morphologies of cultured Northern Ontario fungi and key to species (Watanabe, 2002); and the Site pH H 2O pH CaCl 2 (Zycha and Siepmann, 1970). Kelly Lake limed 6.41 6.13 Kelly Lake unlimed 3.75 3.37 Phospholipid Fatty Acid (PLFA) Analysis and Soil Kingsway limed 4.67 4.35 Respiration Kingsway unlimed 3.87 2.35 Phospholipid Analysis (PLFA) was performed at Wahnapitae Hydro-Dam limed 6.75 6.34 FAME Lab, Microbial ID. Inc, Newark, Delaware Wahnapitae Hydro-Dam unlimed 3.82 3.56 Capreol (reference site) 3.92 3.43 (USA) as described in Buyer and Saaser (2012). Soil St.Charles (Hagar) (reference site) 3.50 3.23 respiration was assessed using the Solvita Soil Test as Onaping Falls (reference site) 4.84 3.92 described in Goupil and Nkongolo (2014). Soil samples were dried in an incubator at 45°C for 24 h prior to the test. Dried soil samples were weighed into a capillary Table 2. Number of different fungi species within medium for limed, unlimed and reference sites cup after placing a fiber filter in the bottom of the cup. Each beaker, containing 40 g of dried soil, was placed Number of species (Mean ± SE)* into a glass jar with the use of forceps. Following this, ------Treatment SDA MEA 20 mL of distilled water was placed into the glass jar. A Limed sites 7.25 a 5.13 a CO 2 probe was then inserted into the glass jar using ±0.53 ±0.95 forceps. Jars were opened 24 h later to remove the Unlimed sites 5.13 b 5.63 a probes. Probe color was determined using a Digital ±0.44 ±0.42 Color Reader (DCR) as per the manufacturer Reference sites 5.83 ab 7.50 a instructions. Interpretations of the DCR provided data ±0.31 ±0.85 were based on Solvita’s overall guidelines. Limed and unlimed sites include: Wahnapitae Hydro-Dam, Statistical Analysis Daisy Lake, Kingsway and Kelly Lake. Reference sites include: St.Charles (Hagar), Onaping Falls and Capreol Fungal diversity and abundance in unlimed and limed *Means in columns with a common letter are not significantly soil samples were compared using Student t-tests. One-Way different based on Tukey multiple comparison test (p≥0.05). Analysis of Variance (ANOVA) tests were also conducted SE = Standard error

47 Kassandre Goupil et al . / American Journal of Biochemistry and Biotechnology 2015, 11 (2): 45.56 DOI: 10.3844/ajbbsp.2015.45.56

(a) (b)

Fig. 2. Wahnapitae Hydro-Dam site (a) Limed area and (b) Unlimed area

Fig. 3. Venn diagram illustrating common fungi species among limed areas based on SDA and MEA medium analysis

A total of 52 fungi species belonging to 34 genera while 17 species were observed in samples from were identified. Some species were specific to one or unlimed sites. A total of 18 fungi species were found two sites, while others were present in the majority of in the reference sites. These results indicate that the sites including the reference sites. A total of 23 fungal diversity is higher in limed soils compared to fungi species were identified in samples from limed unlimed soils based on SDA medium growth areas based on fungi growth on SDA medium plates, (Table 2).

48 Kassandre Goupil et al . / American Journal of Biochemistry and Biotechnology 2015, 11 (2): 45.56 DOI: 10.3844/ajbbsp.2015.45.56

Fig. 4. Venn diagram illustrating common fungi species among unlimed areas based on SDA and MEA medium analysis

Fig. 5. Venn diagram illustrating common fungi species among reference sites based on SDA and MEA medium analysis

49 Kassandre Goupil et al . / American Journal of Biochemistry and Biotechnology 2015, 11 (2): 45.56 DOI: 10.3844/ajbbsp.2015.45.56

No significant difference was observed for fungi belong to the Ascomycota phylum, 12% , diversity based on MEA medium growth. Overall, fungi 9% , 6% Deuteromycota and 3% Oomycota. growth on the MEA medium revealed the presence of 21 At the species level, and species in samples from limed areas and 19 species from Rhizopus stolonifer were found in all the areas. Five fungi unlimed areas. A total of 20 species were found in the species were specific to Wahnapitae Hydro-Dam, three three reference sites based on MEA growth analysis. different species specific to Kingsway, five to Daisy Lake and ten to Kelly Lake sites (Fig. 3). Fungi Abundance Based on Medium For unlimed sites, 65% of the fungi species belong to Optimal fungal growth was observed on SDA medium the Ascomycota phylum, 8% Basidiomycota, 11% at dilution factors of 10-1 to 10-4 and at 10-1 to 10-3 Zygomycota, 8% Deuteromycota and 8% Oomycota. dilution factors for the MEA medium. In both media, the mentagrophytes was a common species to majority of the fungal growth was observed within the first all the areas, while three species were specific to 7 to 10 days post inoculation. Table 4 described for each Wahnapitae Hydro-Dam, two to Kingsway, four to site the species that was the most abundant based on SDA Daisy Lake and five to Kelly Lake sites. Other species and MEA growth. The most prevalent fungi species on the were present in some sites and absent in others (Fig. 4).

SDA medium include Cryptococcus neoformans, Table 3. Mean fungi abundance in soil samples from limed, Acremonium strictum, , Cunninghamella unlimed and reference sites based on growth on SDA bertholletiae, Penicillium fellutanum, Sporotrichum and MEA media schenckii , Trichophyton mentagrophytes and Penicillium Abundance (Mean c.f.u./g. ± SE)* citrinum . For the MEA medium, Cryptococcus neoformans , ------Streptomyces scabies , Sprorotrichum schenckii, Treatment SDA MEA Acremonium strictum, Mycoderma aceti, Penicillium Limed sites 1.82 ×10 8 a 1.54 ×10 8 a montanense, Trchosporon mucoides, Sporotrichum ±5.17 ×10 7 ±7.84 ×10 7 schenckii and Trichophyton tonsurans were the most Unlimed sites 8.11 ×10 7 b 1.61 ×10 8 a prevalent species. In general, 41 species were identified on Reference sites ±1.73 ×10 7 ±4.44 ×10 7 8 9 SDA medium and 36 species on MEA medium. 4.0 ×10 a 1.36 ×10 b ±1.22 ×10 8 ±8.43 ×10 8 Fungi Species Distribution in Limed and Unlimed Limed and unlimed sites include: Wahnapitae Hydro-Dam, Areas Daisy Lake, Kingsway and Kelly Lake. Reference sites Distribution Among Sites include: St.Charles (Hagar), Onaping Falls and Capreol *Means in columns with a common letter are not significantly Fungi species distribution was based on growth on SDA different based on Tukey multiple comparison test (p≥0.05). and MEA media. For limed sites, 70% of the fungi species SE = Standard Error

Table 4. Fungi abundance Site Most abundant species Abundance (C.F.U./g) SDA medium Daisy Lake Limed Sporotrichum schenckii 3.38 ×10 6 Daisy Lake Unlimed Acremonium strictum 5.26 ×10 7 Kingsway Limed Candida albicans 1.12 ×10 8 Kingsway Unlimed Cunninghamella bertholletiae 3.42 ×10 7 Kelly Lake Limed Cryptococcus neoformans 2.66 ×10 8 Kelly Lake Unlimed Cryptococcus neoformans 1.06 ×10 8 Wahnapitae Hydro-Dam Limed Penicillium fellutanum 7.77 ×10 7 Wahnapitae Hydro-Dam Unlimed Trichophyton mentagrophytes 1.22 ×107 Reference sites St.Charles (Hagar) Trichophyton mentagrophytes 1.01 ×10 8 Onaping Falls Penicillium citrinum 2.79 ×10 8 Capreol Trichophyton mentagrophytes 1.05 ×10 8 MEA medium Daisy Lake Limed Sporotrichum schenckii 6.18 ×10 6 Daisy Lake Unlimed Acremonium strictum 1.37 ×10 8 Kingsway Limed Mycoderma aceti 6.60 ×10 7 Kingsway Unlimed Cryptococcus neoformans 1.66 ×10 8 Kelly Lake Limed Streptomyces scabies 4.89 ×10 8 Kelly Lake Unlimed Penicillium montanense 3.86 ×10 8 Reference sites St.Charles (Hagar) Trichophyton tonsurans 1.59 ×10 8 Onaping Falls mucoides 1.23 ×10 8 Capreol Sporotrichum schenckii 1.61 ×10 8

50 Kassandre Goupil et al . / American Journal of Biochemistry and Biotechnology 2015, 11 (2): 45.56 DOI: 10.3844/ajbbsp.2015.45.56

For reference sites, 69% of the fungi species belong to and Verticillium dahlia were the most frequent in the Ascomycota phylum, 10% Basidiomycota, 14% unlimed site . Zygomycota, 3% Deuteromycota and 4% Oomycota. For Kingsway , Mycoderma aceti and Trichophyton At the species level, and Trichophyton mentagrophytes were the most common while tonsurans were common to all the sites. Six species Cryptococcus neoformans , Saprolegnia parasitica , where specific to Capreol reference site, seven to Sporotrichum schenckii and Trichophyton Onaping Falls and four to St.Charles (Hagar) (Fig. 5). mentagrophytes were most frequent in unlimed site . Distribution within Sites For Kelly Lake, Penicillium lividum and Streptomyces scabies were the most abundant in limed SDA Medium area while Acremonium strictum , Actinomyces bovis and Alternaria alternata were the most prevalent in For Wahnapitae Hydro-Dam site, Penicillium fellutanum , Rhodotorula mucilaginosa , Trichoderma unlimed site. harzianum and Trichophyton mentagrophytes were the For reference sites, Penicillium crustaceum, most commonly found in limed areas while Rhizopus stolonifer, Streptomyces scabies, Eupenicillium shearii , Penicillium fellutanum , Trichophyton concentricum, Trichophyton Sporotrichum schenckii and Trichophyton mentgrophytes mentagrophytes and Trichophyton tonsurans were the were the most frequent in unlimed sites . most common in St.Charles (Hagar) ; Blastomyces For Daisy Lake , Mortierella isabellina , Penicillium dematitidis , immitis , Mucor indicus , chrysogenum and Sporotrichum schenckii were most Penicillium citreonigrum , Penicillium citrinum , common species in limed site while Acremonium Penicillium montanense , Pythium debaryanum , strictum , Penicillium lividum and Rhizo