Identification of Eight High Yielding Strains via Morpho-Molecular Characterization of Thirty-Three Wild Strains of Calocybe indica.

Calocybe indica, generally referred as milky mushroom, is one of the edible mushroom species suitable for cultivation in the tropical and sub-tropical regions of the world. However, lack of potential high yielding strains has limited its wider adaptability. To overcome this limitation, in this study, the germplasms of C. indica from different geographical regions of India were characterized based on their morphological, molecular and agronomical attributes. Internal transcribed spacers (ITS1 and ITS4)-based PCR amplification, sequencing and nucleotide analysis confirmed the identity of all the studied strains as C. indica. Further, evaluation of these strains for morphological and yield parameters led to the identification of eight high yielding strains in comparison to the control (DMRO-302). Moreover, genetic diversity analysis of these thirty-three strains was performed using ten sequence-related amplified polymorphism (SRAP) markers/combinations. The Unweighted Pair-group Method with Arithmetic Averages (UPGMA)-based phylogenetic analysis categorized the thirty-three strains along with the control into three clusters. Cluster I possesses the maximum number of strains. Among the high yielding strains, high antioxidant activity and phenol content was recorded in DMRO-54, while maximum protein content was observed in DMRO-202 and DMRO-299 as compared with the control strain. The outcome of this study will help the mushroom breeders and growers in commercializing C. indica.

Identification and morpho-molecular characterization of low spore strain in oyster mushroom.

BACKGROUND: Sporocarps of oyster mushroom liberate enormous spores and cause allergic reactions to workers involved in its cultivation. These spore-related allergies include stiffness or pain in the forearms, limbs, itchy throat, grogginess, and respiratory problems and are major problems during oyster mushroom cultivation. METHODS AND RESULTS: In this study, we have generated seven hybrids using single-spore isolates (SSIs) of Pleurotus ostreatus var. florida (DMRP-49) and P. ostreatus (DMRP-30). Chimera was observed during cultivation trial of these hybrids and led to the development of low spore-producing/sporeless strain (DMRP-395) as evident from spore print and microscopic analysis. Further, the cultivation trial of this sporeless strain revealed a bunchy fruiting pattern and required 20-24 °C temperature for fruiting. At par yield was observed in sporeless strain. Notably, a prominent infundibuliform-shaped pileus along with central attachment of stipe was observed in the sporeless strain. Moreover, genetic diversity and principal component biplot analysis revealed resemblance of sporeless strain with one of the parental strain, i.e., P. ostreatus var. florida (DMRP-49). CONCLUSIONS: The developed sporeless strain (DMRP-395) contains high protein and at par yield as compared with the control (DMRP-136). This sporeless strain will be helpful to reduce spore-related allergic responses in mushroom growers.

Identification of Key Regulatory Pathways of Basidiocarp Formation in Pleurotus spp. Using Modeling, Simulation and System Biology Studies.

Pleurotus (Oyster mushroom) is an important cultivated edible mushroom across the world. It has several therapeutic effects as it contains various useful bio-molecules. The cultivation and crop management of these basidiomycete fungi depends on many extrinsic and intrinsic factors such as substrate composition, growing environment, enzymatic properties, and the genetic makeup, etc. Moreover, for efficient crop production, a comprehensive understanding of the fundamental properties viz. intrinsic-extrinsic factors and genotype-environment interaction analysis is required. The present study explores the basidiocarp formation biology in Pleurotus mushroom using an in silico response to the environmental factors and involvement of the major regulatory genes. The predictive model developed in this study indicates involvement of the key regulatory pathways in the pinhead to fruit body development process. Notably, the major regulatory pathways involved in the conversion of mycelium aggregation to pinhead formation and White Collar protein (PoWC1) binding flavin-chromophore (FAD) to activate respiratory enzymes. Overall, cell differentiation and higher expression of respiratory enzymes are the two important steps for basidiocarp formation. PoWC1 and pofst genes were participate in the structural changes process. Besides this, the PoWC1 gene is also involved in the respiratory requirement, while the OLYA6 gene is the triggering point of fruiting. The findings of the present study could be utilized to understand the detailed mechanism associated with the basidiocarp formation and to cultivate mushrooms at a sustainable level.

Biochemical Profiling and Cultivation of Medicinal Fungus Isaria cicadae (Ascomycetes) from India.

Isaria cicadae is an entomopathogenic fungus possessing several therapeutic properties and has a potential role in traditional Chinese medicine. The present study was designed to describe the taxonomic details of a new isolate of I. cicadae collected from the Northern Himalayas of India and to study its vegetative and reproductive growth responses under in vitro conditions. Proximate composition, biochemical profiling, and radical scavenging activities were studied to establish the bioactivity of the isolate. Micromorphological characteristics of conidia and conidiophore formation were studied using scanning electron microscopy. The optimum temperature and pH for mycelial growth was 25°C and 7.0, respectively. Pinhead initiation was observed at day 10 after inoculation, but the fully developed, branched, and coral to club-shaped fruiting bodies could be observed after 30 days of inoculation. Proximate analysis indicated that carbohydrates are the major constituents (50.2%) of the fruit bodies, along with a lower quantity of protein (4.46%), crude fat (6.4%), and crude fiber (1.55%). Vitamin D content of I. cicadae was 3,605.84 IU/g. Radical scavenging activity based on the DPPT (1,1-diphenyl-2-picrylhydrazyl) assay was 21.2%. ABTS [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid] and potassium ferricyanide reducing activity were quite high, at around 93% and 99.3%, respectively. The findings of this study provide insight into the biochemical constituents of I. cicadae and its cultivation practices for further exploitation of this mushroom at a larger scale.

Nutritional and Biochemical Characterization of Panus lecomtei Mushroom (Agaricomycetes) from India and Its Cultivation.

Panus lecomtei is emerging as an edible mushroom found worldwide and particularly in the Northern Hemisphere. The mushroom contains a substantial amount of useful nutritional and medicinal compounds. In the present study, we have examined a specimen of P. lecomtei submitted to the ICAR-Directorate of Mushroom Research gene bank. The specimen was examined for taxonomical characters using classical and molecular tools. Attempts were made for cultivation of this mushroom under controlled conditions using sawdust-based substrate. The specimen was characterized by its purplish fruiting body having coarse, rigid, dense hairs on the cap, pubescent stipe, and abundant metuloids. Molecular identification through conserved ITS region was done and the sequence was deposited in NCBI GenBank under accession number MN332200. Nutritional profiling and biochemical analysis showed that the mushroom contained high carbohydrate but low fat contents. The mushroom showed the presence of phenolics, ß-carotene, and lycopene. The analysis also showed substantial antioxidant properties in the mushroom. The findings presented herein point out that P. lecomtei can be used as a potential edible mushroom for diversification of mushroom production in India.

Substrate sterilization with thiophanate-methyl and its biodegradation to carbendazim in oyster mushroom (Pleurotus ostreatus var. florida).

Residue analysis to detect thiophanate-methyl and its primary metabolite (carbendazim) during oyster mushroom (Pleurotus ostreatus var. florida) cultivation was done for two consecutive years 2017 and 2018. Wheat straw substrate was chemically treated with different treatments of thiophate-methyl, viz, thiophanate-methyl 30 ppm + formalin 500 ppm (T1), thiophanate-methyl 40 ppm + formalin 500 ppm (T2), thiophanate-methyl 50 ppm + formalin 500 ppm (T3), thiophanate-methyl 60 ppm + formalin 500 ppm (T4), and formalin 500 ppm (T5 as control and recommended concentration), and utilized for cultivation of oyster mushroom. Treatments T3 and T4 exhibited significant difference in pH levels during both the trials. Minimum spawn run, pinhead formation, and fruit body formation time were recorded in treatments T3 and T4. Significantly higher biological efficiency (%) was recorded in treatments T3 and T4 as compared with all other treatments. No incidence of competitor molds was recorded in T3 and T4. Pesticide residue analysis for detection of thiophanate-methyl and its metabolite (carbendazim) was done in the fruit body produced in T3 and T4 treatments using liquid chromatography with tandem mass spectrometry method. No residue of thiophanate-methyl and carbendazim was detected at 50 ppm concentration of thiophanate-methyl during both the trials. However, in trial II, residue of carbendazim (5.39 µg/kg) was detected at 60 ppm. Based on the findings of the trials I and II, T3 (thiophanate-methyl 50 ppm + formalin 500 ppm) may be utilized for substrate sterilization for oyster mushroom cultivation and Pleurotus ostreatus var. florida could be recognized as microorganism which could play a role in degradation of thiophanate-methyl.

Genetic improvement in Pleurotus (oyster mushroom): a review.

Pleurotus is an important genus comprising several edible species of great commercial significance. These species are grown all across the world. The production areas of Pleurotus mainly belong to the Asian part and are gaining wide popularity across the globe owing to their promising nutritional gains. The demand for improved strains with high productivity has also been rising. The genetic improvement in Pleurotus started with a simple selection technique, which later utilized hybridization (intraspecific, interspecific and intergeneric) and mutation breeding. The traits such as productivity, sporelessness and quality improvement are important objectives on which most of the works have been done so far. However, new generation approaches such as molecular breeding, genetic transformation and genome editing techniques also added pace to the present improvement process. Hitherto, seven species of Pleurotus have been sequenced and a sizable data has been generated that can be used in further breeding programs. This paper discusses and summarizes various research findings on genetic improvement of Pleurotus and gives an outlook for future breeding programs.

Root endophyte Piriformospora indica DSM 11827 alters plant morphology, enhances biomass and antioxidant activity of medicinal plant Bacopa monniera.

Unorganized collections and over exploitation of naturally occurring medicinal plant Bacopa monniera is leading to rapid depletion of germplasm and is posing a great threat to its survival in natural habitats. The species has already been listed in the list of highly threatened plants of India. This calls for micropropagation based multiplication of potential accessions and understanding of their mycorrhizal associations for obtaining plants with enhanced secondary metabolite contents. The co-cultivation of B. monniera with axenically cultivated root endophyte Piriformospora indica resulted in growth promotion, increase in bacoside content, antioxidant activity and nuclear hypertrophy of this medicinal plant.

The root endophyte fungus Piriformospora indica leads to early flowering, higher biomass and altered secondary metabolites of the medicinal plant, Coleus forskohlii.

This study was undertaken to investigate the influence of plant probiotic fungus Piriformospora indica on the medicinal plant C. forskohlii. Interaction of the C. forskohlii with the root endophyte P. indica under field conditions, results in an overall increase in aerial biomass, chlorophyll contents and phosphorus acquisition. The fungus also promoted inflorescence development, consequently the amount of p-cymene in the inflorescence increased. Growth of the root thickness was reduced in P. indica treated plants as they became fibrous, but developed more lateral roots. Because of the smaller root biomass, the content of forskolin was decreased. The symbiotic interaction of C. forskohlii with P. indica under field conditions promoted biomass production of the aerial parts of the plant including flower development. The plant aerial parts are important source of metabolites for medicinal application. Therefore we suggest that the use of the root endophyte fungus P. indica in sustainable agriculture will enhance the medicinally important chemical production.

Mushroom cryopreservation and its effect on survival, yield and genetic stability.

Mycelial stock cultures of Agaricus bisporus, A. Bitorquis, Pleurotus flabellatus, P. Sajor-caju, P. Ostreatus, P. Sapidus, Auricularia polytricha, Lentinula edodes, Morchella esculenta and Volvariella volvacea were maintained by frequent subculturing at an interval of two months and separately as wheat grain spawn in liquid nitrogen with 15 percent glycerol. Preservation of mushroom stock cultures as wheat grain spawn under liquid nitrogen proved to be the better method of maintenance. The percent recoveries of stored samples were unchanged from the first recovery after six months to the last recovery after 42 months in nine out of 11 stock cultures preserved under liquid nitrogen. However, a marginal decline in survival of 10 % was recorded in Auricularia polytricha and Volvariella volvacea. Yields before preservation of mushroom stock cultures and after 30 months of preservation exhibited static biological efficiency and fruitbody weight. The comparison of Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacers (ITS) PCR amplified products did not exhibit DNA fragment variation in banding patterns at the intraspecific level during preservation of stock cultures by either method. The modified cryopreservation protocol and experimental demonstration of genetic stability of stock cultures reported here validate the use of mushroom cryopreservation techniques and supports studies on genetic stability of preserved biological materials.