The research began in the field. Specimens were collected from Gool in District Ramban, at altitudes of roughly 2,500 to 3,500 meters above sea level, during the monsoon months between June and August. The club-shaped, orange stromata the researchers found were closely associated with insect larvae and pupae, whose bodies the fungus had colonized and mummified with its mycelium. Back at the Plant Pathology, Mycology and Microbiology Laboratory in Srinagar, the team documented the fungus’s macroscopic anatomy in detail, noting stromata measuring 3.8 to 6.2 centimeters in length, semi-immersed ovoid perithecia, and the darker fertile heads that signal maturity.

Identification did not rest on appearance alone. Microscopic examination revealed subcylindrical conidiophores, slender flask-shaped phialides, and variably shaped conidia, along with eight-spored cylindrical asci containing filiform ascospores. The researchers then turned to molecular tools, extracting DNA by the CTAB method and amplifying the internal transcribed spacer region of the ribosomal DNA with the universal primers ITS-1F and ITS-4R. Sequencing showed 99.78 percent identity with a reference strain of C. militaris, and the sequence was deposited in GenBank under accession number PQ810010. A maximum-likelihood phylogenetic analysis of 18 ITS sequences placed the Kashmir isolate firmly within the C. militaris species complex, supported by a bootstrap value of 100 percent. The specimen itself was preserved as voucher number 9337-KASH in the herbarium at the University of Kashmir.

With identity confirmed, the researchers set out to solve a practical problem: the fungus grows slowly. On ordinary Sabouraud’s dextrose agar, C. militaris needed roughly 27 to 28 days to cover a plate, and the full cultivation cycle to mature stromata in prior work stretched to two or three months. For a fungus whose value lies in compounds such as cordycepin, adenosine, gamma-aminobutyric acid, ergothioneine, lovastatin, carotenoids and a suite of minerals, time is money. The team compared four solid media, Sabouraud’s dextrose agar, potato dextrose agar, corn meal agar and Richard’s synthetic agar, measuring mycelial diameter every three days over three weeks of incubation at 25 degrees Celsius.

Sabouraud’s dextrose agar emerged as the clear winner, producing the maximum mycelial growth of 54.0 millimeters after 21 days, ahead of potato dextrose agar, corn meal agar and Richard’s synthetic agar. But the researchers did not stop there. Knowing that the carbon-to-nitrogen ratio strongly influences mycelial growth in this species, they supplemented the best medium with malt extract, a rich carbon source, and yeast extract, a rich nitrogen source. Malt extract at 6 grams per liter pushed the colony diameter to 70.30 millimeters, but adding more than that brought no further gain. The decisive step came with yeast extract: the optimal combination of 6 grams per liter malt extract and 4 grams per liter yeast extract produced the maximum diameter of 84.60 millimeters after just 17 days of incubation, roughly ten days faster than unsupplemented Sabouraud’s medium.

Faster mycelium is only half the battle; producing the fruiting bodies, or stromata, that contain the medically interesting compounds requires moving the fungus onto solid substrates. Here the team prepared liquid inoculum in two nutrient broths of differing sugar and nitrogen composition, agitating flasks on a shaker at 170 revolutions per minute for up to eight days. Broth I, based on dextrose with peptone and yeast extract plus magnesium sulfate, potassium phosphate and vitamin B1, produced visible growth after seven days, five days sooner than broth II. The liquid mycelial culture also colonized grain substrates faster than mycelium grown on solid media, confirming that liquid spawn is the more efficient route to fruiting.

For the cultivation trials, the researchers chose four locally available and inexpensive grains: brown rice, white rice, corn kernels and wheat. Each 20-gram portion of grain received 45 milliliters of one of the two nutritional broths before sterilization and inoculation. Jars were incubated in darkness at 20 degrees Celsius and 65 to 70 percent relative humidity for the spawn run, then exposed to fluorescent light at 800 to 1,000 lux for twelve hours daily at 85 to 90 percent humidity to trigger primordia formation and stroma development. The logic of using local grains was deliberate: they are cheap, produced by regional farmers, and sustainable, reducing production costs for would-be growers in Jammu and Kashmir.

The results were unambiguous. Brown rice combined with nutritional broth I delivered the fastest performance on every measure: a spawn run of only 14 days, primordia after 11.5 days, and mature stromata after about 25.75 days, for a total cultivation cycle of 51.25 days from inoculation to harvest, the shortest of any combination tested. This substrate pairing also produced the longest stromata, at 78.0 millimeters on average, and the greatest number of stromata per jar, roughly 63 compared with a low of about 28 on wheat grain with broth II. Most strikingly, brown rice with broth I achieved a biological efficiency, the ratio of fresh yield to dry substrate weight, of 72.75 percent, far outperforming corn, white rice and wheat. Wheat grains did produce the thickest stromata, at just over 4 millimeters in diameter, but nowhere near the overall yield of brown rice.

The findings align with a growing international literature. Previous studies have found brown rice superior for C. militaris fruiting, reported maximum stroma yields on whole rice grains at slightly acidic pH, and shown that grain type and vitamin supplementation significantly shape mycelial growth and antioxidant capacity. Other work has optimized liquid culture conditions, identifying ideal glucose, yeast extract and mineral concentrations for biomass production. What distinguishes the Kashmir study is its focus on a wild, locally isolated strain rather than a commercial or imported one. Local strains may carry physiological adaptations to regional climate and substrates, and the authors argue that strain-specific optimization of this kind offers new insight into how cultivation protocols should be tailored to geographic origin rather than assumed universal.

The practical implications reach beyond mycology. C. militaris was approved by China’s Ministry of Public Health in 2009 as the first novel food of its kind, and its bioactive profile is considered comparable to that of the rare and costly caterpillar fungus Ophiocordyceps sinensis, making it a viable substitute in both traditional and modern therapeutic applications. Cordycepin and adenosine, its flagship compounds, show antibacterial, antioxidant, anti-inflammatory and anticancer activities in laboratory studies. By shortening the cultivation cycle, specifying cheap local substrates, and demonstrating conditions, 20 degrees Celsius, moderate humidity and a simple light regimen, that small facilities can maintain, the Kashmir team has laid a foundation for sustainable commercial cultivation that could benefit mushroom growers and entrepreneurs across the Indian Himalayas while easing pressure on wild populations.

Subject of Research: Optimization of nutrient media and grain substrates for the commercial cultivation of the wild medicinal fungus Cordyceps militaris from Jammu and Kashmir.

Article Title: Nutrient media and substrate optimization for commercial cultivation of Cordyceps militaris (L.) Fr., a novel medicinal mushroom from Jammu and Kashmir

Article References: Shrikhandia, P., Lone, S. A., Wani, A. H., & Bhat, M. Y. (2026). Nutrient media and substrate optimization for commercial cultivation of Cordyceps militaris (L.) Fr., a novel medicinal mushroom from Jammu and Kashmir. Discover Biotechnology, 3(1), Article 3. https://doi.org/10.1007/s44340-026-00048-z

Image Credits: AI Generated

DOI: 10.1007/s44340-026-00048-z

Keywords: Cordyceps militaris, medicinal mushroom, cultivation, substrate optimization, brown rice, cordycepin, mycelial growth, biological efficiency, Jammu and Kashmir, entomopathogenic fungus, Sabouraud dextrose agar, stroma formation