蝴蝶蘭與朵麗蝶蘭耐寒性快速檢測與蘭共生菌接種源 之生產及保存

Abstract

蝴蝶蘭與朵麗蝶蘭為台灣重要之外銷花卉，冬季低溫逆境會影響蝴蝶蘭與朵麗蝶蘭之生理。接種絲核菌屬(Rhizoctonia spp.)蘭共生菌對蘭科植物具多方面之益處，本研究目的為評估蝴蝶蘭耐寒性之科學方法，並探討施用植物生長物質提高蝴蝶蘭耐寒性之可行性。另外，為提高蘭菌之應用價值，探討其接種源之生產及保存方法。 四個蝴蝶蘭品種KC1111(Phalaenopsis Taisuco Snow × Dtps. White Wonder)、KC1938(Phal. Taisuco Kaaladian)、KC2902(Phal. Mount. Lip × Dtps. (Mt. Beauty × Happy Valentine ‘Hamakite Beauty’))、KHM469 (Phal. Brother Girly)與朵麗蝶蘭KHM192 (Doritaenopsis Sinica Ruby ‘SCL16 #1’)，給予7 ℃低溫逆境十天後再回溫一星期，並利用PSII光化學效率值(PSII photochemical efficiency, Fv/Fm)、測量電解質滲漏(electrolyte leakage)與葉綠素含量(SPAD)，作為評估低溫傷害之生理指標參數，並比較這些檢測方法與各品種蘭花之實際耐寒性之相關性。結果顯示各品種之耐寒性以KC2902最高，其次為KHM469，KC1111的抗寒性較差，而對低溫最敏感的品種為KC1938與KHM192。其中以葉綠素螢光分析儀測量Fv/Fm值之技術最為準確，建議未來可應用於蝴蝶蘭耐寒性之檢測。另以蝴蝶蘭KC1111與KC1938作為試驗材料，噴施水楊酸0.143 g/L、Weather King 200 X及過氧化氫0.025 M後，置於生長箱進行低溫7 ℃處理十天後，測定回溫後一周之PSII光化學效率值。實驗結果證實，於低溫逆境前噴施過氧化氫可增加KC1111之耐寒性，但KC1938則無有效之藥劑，施用濃度或施用次數的增加能否再提高抗性，需要進一步探討。 本實驗室自蘭科植物根部分離出絲核菌屬(Rhizoctonia spp.)蘭菌，其對蘭科植物具多方面之益處，為提高蘭菌之應用價值，必須研究其菌種接種母源之生產及保存方法。以冷凍乾燥建立蘭菌之接種母源，蘭共生菌R01冷凍乾燥適用之保護劑，以5%(w/v)脫脂奶粉配合5%麥芽糊精存活率最高，另外，除5%(w/v)脫脂奶粉配合乳糖或水溶性澱粉無法保持活力外，配合其他糖類，如果糖、葡萄糖、乳糖、麥芽糊精、甘露糖、半乳糖、蔗糖、海藻糖、木糖皆能成功保持圓球體菌絲團活力。蘭共生菌R02適用之保護劑種類最多，上述九種外加糖類皆可，但以含5%脫脂奶粉與5%麥芽糊精為保護劑的存活率最高，達50%。蘭共生菌R15推薦之保護劑配方為5%脫脂奶粉配合5%麥芽糊精配合5%蔗糖或果糖或木糖，存活率可至32%。R19最適合之保護劑配方為10%脫脂奶粉配合10%麥芽糊精與10%蔗糖，且外加5%之含氮化合物，如酵母抽出物、大豆萃取物、牛肉萃取物或胰化蛋白酮，或是谷胺酸，復水後圓球體菌絲團存活率介於10至32%。蘭共生菌R04目前適用之保護劑配方為5%脫脂奶粉配合5%麥芽糊精與5%蔗糖與5%谷胺酸，存活率為40%。 利用固態發酵，生產菌種接種母源及接種源，並探討其保存方法。麥粒接種源其耐儲藏之能力，高於本實驗室沿用已久之水苔接種源，水苔接種源則有長期儲存下容易遭受其他真菌汙染之問題。與25 ℃之結果相較，接種源推薦儲存於4 ℃，各菌種之推薦儲存接種源如下：蘭共生菌R01之吸米與燕麥接種源可儲存至十六個月無汙染。R02之燕麥、小米與吸米接種源可儲存至十六個月無汙染。R04之蛭石與珍珠石接種源可儲藏至六個月無汙染，活力維持於100%至90%之間。R15之燕麥、小米、蛭石與珍珠石接種源可儲存至六個月無汙染；R19之蛭石與珍珠石接種源可儲存至六個月無汙染，燕麥接種源則可儲存至七個月無汙染。根據上述結果，固態發酵為建立蘭菌之接種母源與接種源良好方法。

Phalaenopsis spp. and Doritaenopsis spp. are important export orchids in Taiwan. Low temperature during winter is harmful on physiological and morphological growth of Phal. spp. and Dtps. spp, The orchid mycorrhizal fungi (OMF) are beneficial to the orchids. The objectives of first study were to compare three detection methods of chilling tolerance of Phal. spp., inculding PSII photochemical efficiency(Fv/Fm), electrolyte leakage and chlorophyll meter reading(SPAD), and to study the possibilities of increasing chilling tolerance of phal. spp. by using plant growth substance. The second study were to research that using submerged fermentation (SmF), freeze-drying(FD) or lyophilization and solid state fermentation (SSF) to establish OMF’s mother inocula. In the experiment of setting up the quick monitoring methods of chilling tolerance for four species of Phal. spp. and one Dtps. spp. were treated 7 ℃ cold temperature for 10 days and re-warned for 7 days. Four species of Phal. spp. included KC1111(Phalaenopsis Taisuco Snow × Dtps. White Wonder), KC1938(Phal. Taisuco Kaaladian), KC2902(Phal. Mount. Lip × Dtps.(Mt. Beauty × Happy Valentine ‘Hamakite Beauty’), KHM469 (Phal. Brother Girly) and KHM192 (Dtps. Sinica Ruby ‘SCL16 #1’). PSII photochemical efficiency (Fv/Fm), electrolyte leakage and chlorophyll meter reading (SPAD) values were used to evaluate the indexes of chilling tolerance. The result showed that Fv/Fm were reliable index to predict the chilling tolerance of orchids. In the another experiment of increase chilling tolerance of Phalaenopsis spp. plants by using plant growth substances, pre-application of H2O2 (0.025 M) could increase chilling tolerance of KC1111, however, there had no suitable plant growth substance to increase chilling tolerance of KC1938. Overall, there need further experiment to test the concentrations, treatment munber of plant growth substances. The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the Rhizoctonia spp. were studied. Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polymers and nitrogen compounds were tested either alone or in combination with skim milk. Using 5%(w/v) skim milk combined with 5% maltodextrin as cryoprotectants to lyophilize, the survival rates of rehydrated OMF R01 and R02 pellets were 40% and 50%. Skim milk combined with maltodextrin and sucrose or fructose or xylose at 5% were the best protective agents tested alone but the viability of freeze-dried R15 pellets was always <35%. Survival of freeze-dried R19 pellets was maintained from 10% to 32% by using appropriate protective media containing combinations of skim milk, sugars and other protectants such as 5% glycine or 5% tryptone or 5% nutrient broth or 5% soytone or 5% yeast extract. However, the best cryoprotectant was 10% skim milk combined with 10% maltodextrin and 10% sucrose.The highest survival rate of rehydrated OMF R04 pellets was 40% by using 5% skim milk combined with 5% maltodextrin and 5% sucrose and 5% glycine as cryoprotectants to lyophilize, and use 5% soytone to replace the glycine, may obtain the similar result. Using solid state fermentation(SSF) to establish OMF’s mother inocula and inocula. Compare with 25 ℃ results, 4 ℃ was the suggestion storage temperature and the recommendation storage inocula to be as follows: The broomcorn millet and oat inocula of OMF R01 can store for sixteen months with no contamination. The broomcorn millet, millet and oat inocula of OMF R02 can store for sixteen months with no contamination. The millet, oat, vermiculite and perlite inocula of OMF R15 can store for six months with no contamination. The vermiculite and perlite inocula of OMF R04 and R19 can store at 4 ℃ for six months with no contamination.