Variability assessment of fodder cowpea (vigna unguiculata (L.) walp) for mosaic resistance using SSR markers

Abstract

The study entitled “Cultivation of Trametes spp. and assessment for laccase activity and cytotoxicity” was conducted at Department of Plant Pathology, College of Agriculture, Vellayani during 2023-2025 with the objectives of standardization of cultivation of Trametes spp. for laccase activity, polysaccharide production and assessment of its cytotoxicity. Survey was conducted at different locations of Thiruvananthapuram, Kollam and Pathanamthitta districts (AEU-1 and 2) for the collection of native Trametes sp. and five mushrooms were obtained viz, M1. M2, M3, M4 and M5. These native mushrooms were isolated and made into pure culture. Cultural characterization of all the isolates (I1. I2, I3, I4 and I5) was carried out using Potato Dextrose Agar (PDA) and observations on mycelial characters, growth rate, and time taken for complete growth in Petri dish (9 cm) were made. Among the isolates I4 was found to be best in terms of the mycelial characters and growth. The molecular characterization was conducted using LSU primers and confirmed the identity of I4 as Trametes sp. (PVO12560). This isolate was selected and compared with Trametes versicolor (DMRO-211). The physiological studies to identify the best media was carried out with I4 and T. versicolor on five different media viz., Potato dextrose agar (PDA), Malt extract agar (MEA), Peptone potato dextrose agar (PPDA), Potato malt agar (PMA) and Czapek Dox agar (CDA). MEA medium recorded the highest mycelial growth rate (1.80 cm day-1) and minimum of 5 days for complete growth in Petri dish for both the isolates. This was followed by PPDA and PMA in I4 and T. versicolor respectively. The optimum temperature for the growth of both the isolates were evaluated at range of 15°C to 35°C in MEA medium. Both the isolates showed optimal growth at 30°C. Higher and lower temperatures (35ºC and 15ºC) were inhibitory for the mycelial growth of I4 while growth was observed in T. versicolor at 15 and 35º C. The cultures of both isolates exhibited rapid growth when incubated at 30ºC. The optimal pH was assessed using MEA medium adjusted to a pH range of 4.5 to 8.0. Isolate I4 demonstrated the highest growth within the pH range of 5.5 to 6.5, whereas T. versicolor showed optimal growth between pH 5.5 and 6.0. Both isolates exhibited growth reduction at pH levels below 5 and above 7. 93

Four different substrates viz., paddy grains, wheat grains, sorghum grains and sawdust (supplemented with 10% rice husk) were evaluated as spawn substrates for the growth and colonization of I4 and T. versicolor. Sorghum grains were observed to be the most suitable substrate, with short spawn run period of 10 days for I4 and 9.75 days for T. versicolor. Substrates suited for sporocarp production of I4 and T. versicolor was evaluated using different media viz., paddy straw, sawdust (amended with 10% rice bran and wheat bran 10%), sawdust + paddy straw (2:3), and spent mushroom substrate. Among these, sawdust amended with 10% rice bran was found to be the efficient substrate for both species. I4 completed spawn run in 38 days and produced 42.7 g of sporocarps, while T. versicolor took 39.5 days for spawn run and yielded 29.5 g. Sawdust supplemented with wheat bran resulted in significantly lower yield. The substrates paddy straw and spent mushroom substrate failed to produce any sporocarp attributed to heavy contamination and poor mycelial colonization. Polysaccharide content was estimated from dried fruiting bodies, where I4 yielded 62.1 % and T. versicolor yielded 56.9 %. GC-MS analysis of mushroom extracts revealed diverse bioactive compounds in both species, including polysaccharides, fatty acids, phenols, and organic compounds. Of these several compound beta D glucopyranose and 1,3,5, cyclohexanetetrol has been reported to possess anticancerous, antioxidant and antimicrobial properties. The isolate I4 exhibited the highest laccase activity during the mycelial stage (64.197 U ml⁻¹), followed by pin head formation stage (52.957 U ml⁻¹) and young fruiting body stage (45.980 U ml⁻¹). T. versicolor exhibited the highest laccase activity at the pin head formation stage (55.863 U ml⁻¹) followed by young fruiting body stage (46.420 U ml⁻¹,) and mycelial stage (41.720 U ml⁻¹). The cytotoxic activity of both isolates was determined using the MTT assay against human colon cancer (HCT-116) cell lines. The ethanolic extract of I4 demonstrated an IC50 value of 371.85 μg mL⁻¹, while T. versicolor showed slightly higher cytotoxicity (308.44 μg mL⁻¹). The present study concluded occurrence