การพัฒนาเห็ดตับเต่าดำสำหรับเป็นเห็ดเศรษฐกิจอย่างยั่งยืน

Abstract

This study surveyed black bolete (Phlebopus portentosus) in northern Thailand and investigated the suitable growth conditions, plant growth promoting abilities, sporocarp production without a host plant and the status of this mushroom. Moreover, the preservative methods of P. portentosus sporocarp for the commercial sale were evaluated. A total of 18 samples were collected from Chiang Mai, Chiang Rai and Lumphun Provinces in northern Thailand. The identification based on morphological and molecular characteristics indicated that all samples were agreed well with the taxonomic of P. portentosus. Pure cultures of P. portentosus were isolated from each sporocarp. The results indicated that mycelia of P. portentosus grew well at 30°C and the optimum pH was 4.0. Among 15 culture media tested, modified Murashige & Skoog and fungal-host media were the best for mycelial growth. Glucose and ammonium salts produced the largest radial growth. Moreover, the in vitro abilities of selected fungi such as Astraeus odoratus, P. portentosus, Pisolithus albus and Scleroderma sinnamariense, to produce indole-3-acetic acid (IAA) and siderophore, to solubilize different toxic metal (Co, Cd, Cu, Pb, Zn)-containing isoluble minerals, and metal tolerance were investigated. The result indicated that all fungi were able to produce IAA in liquid medium and fungal IAA could simulate coleoptile elongation, and increase seed germination and root length of tested plant. The highest IAA yield (65.29±1.17 μg ml-1) was obtained from P. portentosus isolate CMU51-210-2 after 40 days of cultivation in liqiud medium supplemented with 4 mg ml-1of L-tryptophan. All selected fungi produced both catecholate- and hydroxamate-type siderophores and P. portentosus isolate CMU51-210-2 showed the highest yield of catecholate- and hydroxamate-type siderophores 236.43±2.34 and 530.06±3.16 mg l-1, respectively. All selected fungi could solubilize insoluble metal containing minerals which varied for different minerals and fungal species. The solubilization and tolerance indexes decreased when the concentration of metal minerals increased. Astraeus odoratus showed the lowest tolerance to metals. The sporocarp production ability in the absence of a host plant condition of five isolates of P. portentosus such as CMU51-110-1, CMU51-210-2, CMU51-241-1, CMU51-261-1 and CMU51-320-2 were examined. The result indicated that the fungal mycelia grew fastest on sorghum grains supplemented with fungal-host solution. The primordia were formed under low temperature, high humidity and a 12 h photoperiod. Fungal isolate CMU51-320-2 produced primordia at higher number than other isolates. The primordia were developed into young sporocarps and matured later within 815 days in the in vitro culture, pot culture and bag culture experiments. Cultivation experiment indicated that both the highest number of primordial formation and yield of sporocarp were obtained on 1:1 (v/v) of mixed sorghum grains supplemented with fungal host solution and sawdust. As a supplemental solution, fungal host solution was found to be better comparing with Murashige & Skoog and vitamin solutions. The identification of mature cultivated sporocarps was confirmed by both morphological and molecular methods. ECM status of P. portentosus was evaluated by a host range under greenhouse experiment and a stable carbon (δ13C) and nitrogen (δ15N) isotopic evidences analyses. A peat:vermiculite mixed with fungal-host solution was the best solid substrate for mycelial inoculum production. Six plant seedling species such as Castanopsis tribuloides, Dimocarpus longan, Elaeocarpus hygrophilus, Mangifera indica, Pinus kesiya and Syzygium cumini were tested. The results indicated that P. portentosus isolate CMU51-320-2 able to form ECM association with P. kesiya and E. hygrophilus after one and three years after inoculation. The 57 samples of ECM fungi and 25 samples of saprotophic fungi were collected in northern Thailand for the isotopic comparison experiment. New spices of ECM fungus, Scleroderma suthepense was found and Morganella purpurascens was a new report in Thailand. The stable carbon and nitrogen comparisons separated fungal samples into three groups such as ECM fungi, saprotrophic fungi and cultivated P. portentosus in the absence of a host plant conditions. The collected P. portentosus samples in nature were fall in an ECM group. This study indicated that P. portentosus was a facultative ECM fungus by a host range, the stable isotopic evidence and the ability of sporocarp formation without host plant analyses. Three processes for the preservation of P. portentosus sporocarps such as brining, drying and freezing were investigated and chemical composition of preserved product was analyzed. The results indicated that the preserved products are rich in protein (17.089.23% dry weight) and carbohydrates (60.6263.10% dry weight) contents, and low fat (0.891.09% dry weight) content. The brining processes decreased protein and fat contents but increased carbohydrate content. Moreover, sensory test of black bolete spicy curry and tomato-mushroom sauce made from the preserved products was evaluated. The results showed that favor, taste, texture and color were changed in different preservative methods. Brining product had the highest overall acceptability scores than other persevered products.

Keywords: Ectomycorrhizal fungi, black bolete, cultivation, plant growth promoting substance, status, preservation