Prospecting Russula senecis: a delicacy among the tribes of West Bengal

DNA extraction, polymerase chain reaction and sequencing

Genomic DNA was extracted from dried herbarium specimens (10–50 mg) using the ‘Fungal gDNA Mini Kit’ (Xcelris Genomics, Ahmedabad, India). ITS region 1 and 2, and the 5.8S rDNA, were amplified using universal primers pair ITS1 (5′ TCC GTA GGT GAA CCT GCG G 3′) and ITS4 (5′ TCC TCC GCT TAT TGA TAT GC 3′) (White et al., 1990). The DNA fragments were amplified on an Applied Biosystems^® 2,720 automated thermal cycler (Applied Biosystems, Carlsbad, California, USA) following the protocol as described by Abd-Elsalam et al. (2003) with little modifications. A hot start of 4 min at 94 °C was followed by 35 cycles consisting of 1 min at 94 °C, 1 min at 56 °C, 1 min at 72 °C, and a final elongation step of 7 min at 72 °C. PCR products were checked on 2% agarose gel stained with ethidium bromide. PCR products were purified using QIAquick^® Gel Extraction Kit (QIAGEN, Hilden, Germany) and was subjected to automated DNA sequencing based on Sanger dideoxy sequencing technique, on ABI3730xl DNA Analyzer (Applied Biosystems, Carlsbad, California, USA) using primers identical with amplification for ITS rDNA region. The newly generated sequences were then deposited in GenBank (www.ncbi.nlm.nih.gov) with the accession numbers KJ768982 and KP142981.

Taxon sampling

Twenty eight Internal Transcribed Spacer (ITS) nrDNA sequences representing nineteen species were used in the analyses, of which two sequences of Russula senecis S. Imai were generated as part of this study. The sequences represent sixteen species of Russula distributed over five subgenus, namely Compacta (Fr.) Bon (Russula delica Fr.), Heterophyllidia Romagn. (Russula cyanoxantha (Schaeff.) Fr. and Russula virescens (Schaeff.) Fr.), Amoenula Sarnari (Russula amoenicolor Romagn.), Ingratula Romagn. (Russula cf. laurocerasi, Russula cf. subfoetens, Russula fellea (Fr.) Fr., Russula foetens Pers., Russula insignis Quél., Russula grata Britzelm. (in the present study represented as Russula laurocerasi Melzer), Russula ochroleuca Fr., Russula pulverulenta Peck and Russula senecis S. Imai), Russula emend. Sarnari (Russula emetica (Schaeff.) Pers.) and Incrustatula Romagn. emend. (Russula rosea Pers.). Stereum hirsutum (Willd.) Pers., Amylostereum laevigatum (Fr.) Boidin, and Bondarzewia mesenterica (Schaeff.) Kreisel (here represented as Bondarzewia Montana (Quél.) Singer) were selected as outgroup taxa for rooting purpose following Buyck et al. (2008). The accession numbers of newly generated two ITS sequences of R. senecis and those pulled from GenBank for the purpose of conducting phylogenetic analysis for this study are cited in Fig. 3.

Phylogenetic analysis

Sequences were edited with the CodonCode Aligner software (CodonCode Corporation, Dedham, Massachusetts, USA). The newly generated two ITS1-5.8S-ITS2 sequences of R. senecis and those retrieved from GenBank were aligned with the help of ClustalX (Thompson et al., 1997) using the default setting. A final set of 28 sequences were aligned. The appropriate substitution model was determined using Bayesian information criterion (BIC) in MEGA6 (Tamura et al., 2013). The K2 + G model (with lowest BIC scores of 4931.469) was selected as the best-fit model.

Phylogenetic analyses was performed in MEGA6 (Tamura et al., 2013) using the Maximum Likelihood (ML) method based on the Kimura 2-parameter model. Initial tree(s) for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.5337)).

Beside ML method, phylogenetic analyses were also carried out using Neighbor-Joining (NJ) method (Saitou & Nei, 1987) to determine whether different methods (Maximum Likelihood versus Neighbor-Joining) alter the resulting phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980) and are in the units of the number of base substitutions per site. The sum of branch length of the optimal tree was 1.12069257. In both the cases, all positions containing gaps and missing data were eliminated and a bootstrap test of 1,000 replicates was performed to obtain the percentage of replicate trees for clustering the associated taxa.

Test bacteria

Listeria monocytogenes MTCC Code 657, Salmonella typhimurium MTCC Code 98, Bacillus subtilis MTCC Code 736, Escherichia coli MTCC Code 68, Pseudomonas aeruginosa MTCC Code 8158 and Staphylococcus aureus MTCC Code 96 were obtained from the culture collection of the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, India. They were incubated for 24 h by inoculation into nutrient broth.

Disk diffusion method

The determination of the inhibitory effect of RusePre on test bacteria was carried out by the agar-disc diffusion method (Bauer et al., 1966). Nutrient agar was poured into each sterilized petri dish (90 mm diameter) after injecting cultures (100 µl) of bacteria and medium was distributed homogeneously. Paper discs (5 mm) were loaded with 20 µl of 20 mg/ml concentrated RusePre. The impregnated discs were air dried before being place in on the petri dishes with the test microorganisms. Plates were incubated as per the bacterial requirements. Studies were performed in triplicate and the inhibition zones were compared with those of blank discs.

Mycochemical analyses

The content of total phenolic compounds in RusePre was estimated using Folin-ciocalteu reagent and gallic acid as standard (Singleton & Rossi, 1965). The results were expressed as µg of gallic acid equivalents per mg of dry extract. Total flavonoid content was determined using aluminium nitrate and potassium acetate. Quercetin (5–20 µg/ml) was used to calculate the standard curve (Park et al., 1997). The results were expressed as µg of quercetin equivalents per mg of dry extract. β-carotene and lycopene were estimated by measuring absorbance at 453, 505 and 663 nm (Nagata & Yamashita, 1992). Ascorbic acid was determined by titration against 2, 6-dichlorophenol indophenol dye (Rekha et al., 2012).

Determination of phenolic profile by HPLC

For quantitative analysis of phenolic compounds, a 3-level calibration curve was obtained by injection of known concentrations (10–50 µg/ml) of eleven standard compounds: gallic acid (y = 34.773x − 9.2238; R² = 0.9991), chlorogenic acid (y = 13.776x–2.9025; R² = 0.9993), vanillic acid (y = 19.225x + 0.2588; R² = 0.9994), p-coumaric acid (y = 49.773x − 10.541; R² = 0.9994), ferulic acid (y = 30.425x − 2.8188; R² = 0.9995), myricetin (y = 5.0676x − 6.0375; R² = 0.9937), salicylic acid (y = 4.4974x − 0.4763; R² = 0.9994), quercetin (y = 5.2478x − 5.9763; R² = 0.9954), cinnamic acid (y = 108.07x − 111.55; R² = 0.9979), pyrogallol (y = 10.8x + 0.3333; R² = 0.9999) and kaempferol (y = 18.667x − 80.875; R² = 0.9997). The results were expressed as µg/mg of dry extract.

A 0.5 mg amount of RusePre was dissolved in 1 ml of methanol and water (1:1 v/v) and filtered through 0.2 µm filter paper. 20 µl filtrate was loaded on the HPLC system (Agilent, Santa Clara, California, USA). Separation was achieved on an Agilent Eclipse Plus C18 column (100 mm × 4.6 mm, 3.5 µm) using a flow rate of 0.8 ml/min at 25 °C. The mobile phase consisted of eluent A (acetonitrile) and eluent B (aqueous phosphoric acid solution, 0.1% v/v). A gradient program was used for elution: 0–2 min, 5% A; 2–5 min, 15% A; 5–10 min, 40% A; 10–15 min, 60% A; 15–18 min, 90% A. The absorbance of standard and sample solution was measured at 280 nm. Sample compounds were identified on the basis of retention times and absorption spectra of standard materials. Components were quantified by comparing their peak areas with those of standard curves.

Statistical analysis

All the assays were carried out in triplicate. Data were recorded as mean values and standard deviation (SD). The results were analyzed by Student’s t Test, using Microsoft^® Office Excel (Microsoft^®, Redmond, Washington, USA), where values of p < 0.05 were considered as statistically significant.

Russula senecis S. Imai

Pileus 5.5–7(–13) cm broad, convex when young, becoming plano-convex to applanate at old, usually with broad central depression, glabrous, slightly viscid when wet, hygrophanous, bay, pale ochraceous buff to ochraceous-tawny towards centre, pallid to ochraceous buff towards margin, surface turns translucent rust to rusty-tawny with KOH; margin decurved, tuberculate striate; cuticle not easily separable from the context, cracking up into patches near margin; context up to 3.5 mm thick, creamy buff, unchanging color when exposed (Figs. 1A–1B). Lamellae 4.5–6 mm broad, adnexed, regular, bifurcate near the attachment of stipe, rarely one tiered, creamy buff, entire, even, edge discolorous, with fine brown to sienna buff margin. Stipe 5.5–7.5(–14) × 1.1–1.3(–2.4) cm towards top × 1.2–2.5 cm towards base, tapered towards the base, central to slightly eccentric, fleshy, slightly curved, cylindrical, becoming compressed, multi chambered at maturity; surface smooth, moist, slight shiny, creamy buff to dull yellow, often with fine dark brown warts, becoming clay buff on bruising, turns rusty-tawny to bay with KOH (Fig. 2A). Odor strong. Taste very acrid. Spore print creamy white.

Basidiospores (7.5–)8.2–8.6–8.9(–9.7) × 7.8–8.3–8.6 µm, Q= 0.95–1.04–1.18, globose to subglobose, ornamentation amyloid, up to 2.1–3.2 µm high, composed of large wings and isolated warts, never forming reticulum (Figs. 1C and 1D; 2B). Basidium 32–38 × 10–10.7 µm, clavate, 4–spored (Fig. 2C). Hymenial cystidia (61–)64–68(–82) × 8.6–9.7(–10.7) µm, lanceolate to fusoid or elongated fusoid, with mucronate to moniliform apex, thin-walled, mostly with heteromorphous contents (Fig. 2D). Lamellar trama ca. 143–150 µm broad towards middle, 96 µm broad towards edge, mainly composed of sphaerocytes. Subhymenium pseudoparenchymatous. Pileipellis orthochromatic in cresyl blue, sharply delimited from underlying sphaerocytes of the context, distinctly divided into a dense, gelatinized, ca. 143–157(–161) µm deep subpellis composed of horizontally oriented hyphae, 3.2–3.6(–4.3) µm wide, mostly scattered with oleiferous fragments, (5.7–)6.4–7.2(–8.6) µm wide, and a less gelatinized, 36–72(–89) µm deep suprapellis of erect or repent hyphal ends. Incrustations absent. Pileocystidia up to 4.3–7.2 µm broad, mostly lanceolate, apex cylindrical to often with a minute rounded capitulum, thin-walled, recognizable by their distinct heteromorphous contents (Fig. 2E). Underlying sphaerocytes globose to sub-globose, ca. 12.5–13.9(–14.3) × 13.6–14.3 µm, hyaline. Stipitipellis up to 107–143 µm thick, composed of 3.6–3.9 µm broad hyphae, frequently with interspersed oleiferous hyphae, measuring 5.7–8.9 µm broad. Caulocystidia absent. Stipe trama composed of nested subglobose sphaerocytes, measuring 21–36(–44) µm diam.

Habit and habitat: common, ectomycorrhizal with Shorea robusta C.F.Gaertn. and Castanopsis sp.

Specimen examined: INDIA: West Bengal, Burdwan district, Malandighi, 11 July 2008, Prakash Pradhan, CUH AM103; Burdwan district, Malandighi, 25 August 2008, Prakash Pradhan, CUH AM104; Bankura district, Bishnupur, 10 August 2009, Prakash Pradhan, CUH AM105; Bankura district, Manjhulia, 15 July 2010, Prakash Pradhan, CUH AM106; Birbhum district, Gonpur, 08 July 2011, Arun Kumar Dutta and Prakash Pradhan, CUH AM107; East Midnapur district, Ramnagar, Kasaphaltalya, 24 July 2011, Arun Kumar Dutta and Prakash Pradhan, CUH AM108; Darjeeling district, Jawbari, 28 June 2012, Prakash Pradhan, CUH AM102; Darjeeling district, 7th mile Jungle, near Gurdum, 1 July 2012, Prakash Pradhan, CUH AM081.

Notes: Russula senecis was originally described as being from Japan (Imai, 1938), and reported to frequently grow in association with Vateria indica plants among the dipterocarp forests of Western Ghats (Natarajan et al., 2005), and in mixed forests under Lithocarpus and Castenopsis plant from Sikkim Himalaya, India (Das, 2009; Das, Van de Putte & Buyck, 2010). This well-known, widely distributed species can be easily recognized by the combination of an ochraceous-tawny pileus which turns rust to rusty-tawny with KOH, ochraceous buff tuberculate striate margin; creamy buff lamellae which often bifurcate near the attachment of stipe, discolorous lamellae with fine brown to sienna buff edges; creamy buff to dull yellow coloured, multi chambered stipe; acrid taste; strong odor; cream spore print; globose to sub-globose basidiospores (7.5–9.7 × 7.8–8.6 µm) with large wings and isolated warts, often with ridges (up to 2.1–3.2 µm high), but never form reticulum, absence of amyloid suprahilar spot; lanceolate to fusoid or elongated fusoid hymenial cystidia with mostly mucronate to moniliform apex; and lanceolate pileocystidia. The presence of these morphological features categorize Russula senecis within the subgen. Ingratula Romagn., series Ingratae (Quél.) Maire and subsect. Foerentinae (Melzer & Zvára) Singer (Sarnari, 1998).

Being a member of series Ingratae (of subgenus Ingratula), R. senecis closely resembles Russula laurocerasi and Russula foetens. However, R. laurocerasi differs from the present species by a light yellow to brilliant yellow or orange yellow coloured pileus with viscid to sticky surface, yellowish white lamellae, presence of lamellulae, pale yellow coloured spore-print, and up to 5 µm broad pileocystidia; and R. foetens differs by having characters like brilliant to dark or deep orange yellow or soft yellowish brown pileus, yellowish white coloured lamellae with lamellulae of two series, a stipe with veined surface, pale yellow spore-print, partially amyloid and mostly conic to acute tipped isolated warts basidiospores, and fusoid shaped hymenial and pileocystidia. A recently described species from India, Russula dubdiana K. Das, Atri & Buyck, differs from R. senecis by having a white coloured lamellae which turns sienna after bruising, white stipe when young, becoming faintly greying in places at maturity or hazel which turns fulvous to cinnamon towards base on bruising, smaller (5.2–7 × 4.2–5.5 µm) broadly ellipsoid to ellipsoid basidiospores with mostly of cylindrical warts and very few ridges and fertile lamellae edge (Das, Atri & Buyck, 2013).