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|標題:||產氣內生真菌Nodulisporium sp. PDL-005之特性與培養條件暨防治貯藏病害評估|
Characteristics and culture conditions of the volatile-producing endophytic fungus Nodulisporium sp. PDL-005 and evaluation for controlling postharvest diseases
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|摘要:||植物內生真菌（endophytic fungi）為棲息於健康植物組織中的一群真菌，可產生和寄主植物產物相似的活性物質，其中可產生有機揮發性化合物（volatile organic compounds, VOCs）者可稱為產氣內生真菌（volatile-producing endophytic fungi）。本研究自石禪草（Peperomia dindygulensis）葉片組織中分離出7株具產氣活性之內生真菌，以Nodulisporium sp. PDL-005最佳。培養於potato sucrose agar（PSA）平板三天之PDL-005菌株所產生之活性揮發物質，能抑制多種貯藏病原真菌之菌絲生長，對草莓灰黴病菌（Botrytis cinerea）等病原真菌之抑制率可達100.0%，而對柳橙綠黴病菌（Penicillium digitatum）之抑制率介於88.2-99.4%，雖具防治潛力但抑制效果變異度較高，為篩選可穩定表現PDL-005菌株拮抗能力之基質，以察氏培養基（Czapek's medium）添加不同碳氮素源進行測試。結果得知，添加單一供試碳氮素源之察氏培養基，無法促進PDL-005菌株產生拮抗綠黴病菌的氣體，然將PDL-005菌株培養於potato dextrose agar（PDA）、malt extract agar（MEA）或oat meal dextrose agar（OMDA）等培養基時，可提高該菌株之拮抗能力，其中以培養在MEA培養基時之抑菌效果最佳，抑制率可達100%並具有致死效果。進一步測試PDL-005菌株培養於芝麻粕、苦茶粕、大豆粕、小麥粒、小麥麩、燕麥、米糠、粗糠及蔗渣等9種基質的生長情形，並評估PDL-005菌株抑制綠黴菌生長的效果，結果得知，培養於蔗渣之PDL-005菌株生物量雖低，但抑制效果最好（90.6-100.0%）且具致死效果，而培養於小麥麩上之PDL-005菌株生物量與最高之芝麻粕組別無顯著差異，且對柳橙綠黴病菌的抑制率次高（約86.4%）。為了提高培養於蔗渣之PDL-005的生物量，將小麥麩以1：3、1：6及1：9比例（w / w）混合蔗渣，測試對PDL-005菌株生長與活性影響。顯示隨著小麥麩比例越高，PDL-005菌株生物量增加越多，然抑制率卻逐漸下滑，並失去對柳橙綠黴病菌之致死作用。續將PDL-005菌株培養於蔗渣且經吹乾磨碎後製成PDL-005粉粒，並將該粉粒以2：3比例（w / w）與蔗渣混合後放入中藥袋製成PDL-005製劑，於加水活化1天後，實際測試對貯藏病害之防治效果。得知6 L之容器內，20 g之PDL-005製劑對草莓灰黴病之防治率可達100%；而30 g之PDL-005製劑對柳橙綠黴病之防治率亦可達100%。利用GC-MS分析PDL-005菌株所產生之主要氣體成份，得知隨培養基質與培養天數不同，PDL-005菌株所產生之揮發性物質種類與含量亦有所變化，其中主要活性物質為2-ethyl-2-hexenal與2,4-dimethyl-1,3-cyclopentanedione。
Endophytic fungi inhabiting healthy plants can produce some phytochemicals with the same characteristic of their hosts. Some endophytic fungi that can produce volatile organic compounds（VOCs）are called volatile-producing endophytic fungi. In this study, seven volatile-producing endophytic fungi were obtained from the leaf of Peperomia dindygulensis, among all endophytic fungi, the bioactivity of Nodulisporium sp. PDL-005 was best. VOCs produced by 3-day-old PDL-005 isolate cultured on PSA could inhibit several postharvest fungal pathogens mycelial growth, for example, it can completely inhibit several fungal pathogens such as Botrytis cinerea, while to Penicillium digitatum, the ratio of inhibition is from 88.2% to 99.4%. The efficacy of controlling P. digitatum was obvious but varied a lot. For screening the nutrients to stabilize antifungal ability, the PDL-005 isolate was cultured on the Czapek's medium which different carbon or nitrogen sources were added to. The results indicated that the single carbon or nitrogen source could not enhance the ability against tested postharvest pathogens. However, the activity of PDL-005 isolate could be enhanced while it was cultured on potato dextrose agar（PDA）、malt extract agar（MEA）and oat meal dextrose agar（OMDA）.Among all medium, the inhibition effect of PDL-005 isolate cultured on MEA was the best（100%）with a killing effect. The nine substrates, including sesame meal, tea seed pomace, soybean meal, wheat grain, wheat bran, oatmeal, rice bran, rice hulls and bagasse were tested in efficiency of enhancing mycelial growth of PDL-005 and the inhibition of P. digitatum. The result showed that the biomass of PDL-005 cultured on bagasse was low, while the inhibition effect against P. digitatum was the best（90.6-100.0%）and had a killing effect. Furthermore, the biomass of PDL-005 cultured on wheat bran was no difference against the highest biomass group-sesame meal and the inhibition rate of PDL-005 isolate against P. digitatum was the second highest（about 86.4%）among all substances. In order to enhance the biomass of PDL-005 cultured on bagasse, the effect was examed that bagasse was mixed with wheat bran in the ratio of 1:3, 1:6 and 1:9 on biomass and inhibition ability of PDL-005 against P. digitatum was examed. The result showed that the biomass of PDL-005 increased more in substrate containing higher proportion of wheat bran, but the inhibition rate of PDL-005 isolate against P. digitatum gradually decreased and lost the killing effect. Next, the PDL-005 grain was made from PDL-005 isolate cultured on bagasse under the process of dried-and-grinded. Furthermore, the PDL-005 grain was mixed with bagasse in the ratio of 2:3 and put into a medicine bag as the PDL-005 product. After rehydration of PDL-005 product for one day, the control value of PDL-005 product against postharvest diseases was examed. The result showed that the control value of 20 g PDL-005 product against gray mold of strawberry, the control value of 30 g PDL-005 product against green mold of orange were 100% in 6L food storage box. The major compound of VOCs produced by PDL-005 isolate was analyzed by GC-MS. The type and content of VOCs produced by PDL-005 isloate changed with culture day and different medium or substances, and the major active compound produced by PDL-005 isolate were 2-ethyl-2-hexenal and 2,4-dimethyl-1,3-cyclopentanedione. The PDL-005 product developed in this study showed good potential on controlling postharvest diseases. In the future, the formulation and usage of the PDL-005 product can be further optimized. Furthermore, the risk of using PDL-005 should be assessed and the control effect of PDL-005 against other pest should be further examed, or the major active compound produced by PDL-005 could be artificially generated as a biological source fumigant, instead.
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