臺灣兩種固有塊菌之親緣關係及溫度對青剛櫟接種塊菌在菌根形成與苗木生長之影響

Abstract

塊菌為珍貴的共生性食用真菌之一，普遍引起世界各地研究學者的興趣。在台灣，目前僅有台灣塊菌（Tuber formosanum）與屑塊菌（Tuber furfuraceum）被發現報導，雖然這兩種塊菌的形態特徵已被描述，然缺乏分子鑑定的證據可確立二者與其他塊菌間的關係。本研究的目的有二，一在利用5.8S-ITS2與&szlig;-微管蛋白質(&szlig;-tubulin)基因序列，建立台灣塊菌及屑塊菌與世界其他塊菌屬種類的親緣關係，並以分子鐘（molecular clock）模式建立台灣與大陸塊菌間之分化時間（divergence time）；二為利用台灣塊菌及夏塊菌（Tuber aestivum）為材料，探討塊菌接種青剛櫟後，溫度對於菌根形成、形態、菌根感染率及塊菌接種對青剛櫟苗木生長之影響。利用5.8S-ITS2序列建立的親緣演化樹顯示，所分析塊菌屬種類可分為五個主要的支序群(clade)，台灣塊菌位於第五支序群(clade V)，並與黑孢塊菌、喜馬拉雅塊菌、印度塊菌、中國塊菌形成第二次支序群(subclade V-2)；屑塊菌則與會東塊菌、T. ferrugineum、棕紅塊菌、T. candidum、T. quercicola形成第三支序群(clade III)。而利用&szlig;-微管蛋白質基因所建立的親緣演化樹，則得到三個主要的支序群(clade)，屑塊菌與棕紅塊菌位於第一支序群；第三支序群亦可分成四個次支序群，台灣塊菌與印度塊菌B群形成第四次支序群(subclade III-4)，subclade III-1包括偽凹孔塊菌，subclade III-2包括黑孢塊菌，subclade III-3包括印度塊菌A群。親緣分析結果，與各塊菌間的形態特徵大致相符，而台灣塊菌與印度塊菌B群最為親近，屑塊菌則是與會東塊菌最親近。根據分子鐘(molecular clock)推算，台灣塊菌與印度塊菌B群的分化時間在4.1百萬年(Ma)前，屑塊菌與會東塊菌的分化時間則在10.2百萬年前。基於本研究結果，可以推測台灣的兩種塊菌，應是與大陸相近的塊菌種類有著共同的祖先，然因為較長的分化時間及地理隔離效應後，進而演化為台灣特有塊菌種類。 在研究不同溫度對青剛櫟接種台灣塊菌與夏塊菌兩種塊菌在菌根形成與苗木生長之影響方面，由試驗結果得知，在不同溫度下生長的台灣塊菌及夏塊菌之菌根，以立體解剖顯微鏡觀察，外部形態並無明顯差異，台灣塊菌菌根呈單根或不規則羽狀，淡黃色至深褐色，具有或無明顯之剛毛；夏塊菌菌根主要為單根狀，淡黃色至深褐色，具有羊毛狀之菌絲。進一步以掃描電子顯微鏡觀察菌根，則可發現在較高溫度的組合（35/30℃、30/25℃）中生長的菌根，其菌毯厚度較薄，結構較為鬆散，且其皮層細胞內，澱粉粒累積出現之頻率亦較高。台灣塊菌之菌毯厚15~25 μm，哈替氏網延伸菌絲可侵入至第二層皮層細胞間；夏塊菌之菌毯厚18~30 μm，哈替氏網延伸菌絲亦侵入至第二層皮層細胞間。菌根感染率以25/20℃為最高，且與其他溫度組呈顯著差異，20/15℃與30/25℃次之，15/12℃再次之，35/30℃為最低；兩種菌根感染率在接種處理間，則以台灣塊菌較夏塊菌高，但兩者間之差異不顯著。苗高方面，各處理均以35/30℃為最高，與其他溫度組呈顯著差異；不同接種試驗方面，苗高以接種夏塊菌者最高，台灣塊菌次之，未接種者最低，三者間呈顯著差異。苗木根頸方面，則是以25/20℃最高，35/30℃、30/25℃與20/15℃等次之，但四者間之差異呈不顯著，而15/12℃則最低；根頸以接種台灣塊菌者最高，夏塊菌次之，兩者差異不顯著，未接種者最差，與有接種者互呈顯著差異。葉部養分元素方面，在碳、氮、鉀、鈣、鎂、鈉、磷等元素方面，都是35/30℃者最高，且與其他溫度組合相互呈顯著差異，顯示高溫組合對苗木之生長與養分元素吸收有利，但對菌根形成是不利的。溫度與菌根感染率呈負相關（r＝-0.57），溫度與苗高、根頸、及葉部之碳、氮、鉀、鈣與鈉濃度（r＝0.52、0.54、0.69、0.43、0.51、0.47、0.55），均呈顯著正相關（P<0.05）；菌根感染率與苗高、根頸、及葉部之鉀、磷濃度（r＝0.49、0.47、0.42、0.67），呈顯著正相關（P<0.05）；苗高與根頸、葉部之氮、鉀、磷濃度（r＝0.51、0.71、0.46、0.42），呈顯著正相關（P<0.05）；根頸同樣與葉部之氮、鉀、磷濃度（r＝0.62、0.44、0.47）呈顯著正相關（P<0.05）；葉部之鉀、鈣與鎂濃度三者之間，則是互呈負相關（r＝-0.48、-0.54、-0.43）。

Truffles are one of the most valuable edible fungi that have drawn extensive research interests worldwide. In Taiwan, two species of truffles, Tuber formosanum and T. furfuraceum, have been identified and reported. Although morphological features of these two truffles have been described, lack of molecular identification can firmly establish their relatedness to other truffles. The goals of this study are to: 1) utilize the ITS and β-tubulin gene sequences to generate the phylogenetic relationship and divergence time of T. formosanum and T. furfuraceum with other taxonomic relatives; 2) study the effects of temperature on the mycorrhizae formation and growth of Cyclobalanopsis glauca seedlings inoculated with T. formosanun and T. aestivum. Our phylogenetic analyses revealed five major phylogenetic clades according to the 5.8S-ITS2 gene sequences and corroborated with their morphological characterization. In the 5.8S-ITS2 phylogenetic tree, T. formosanum was included in Clade V and T. furfuraceum was located in Clade III. Clade V was divided into two subclades. Subclade V-1 was formed by two species, T. brumale and T. pseudoexcavatum, and Subclade V-2 was formed by five species, T. melanosporum, T. himalayense, T. indicum, T. sinense and T. formosanum. Clade III consisted of six species, T. ferrugineum, T. rufum, T. candidum, T. quercicola, T. furfuraceum and T. huidongense. Based on the β-tubulin gene sequences, three major phylogenetic clades were revealed. Clade I contained two species, T. rufum and T. furfuraceum. Clade III was divided into four subclades. Subclade III-1 and subclade III-2 comprised of T. pseudoexcavatum and T. melanosporum respectively. Subclade III-3 contained T. indicum A complex. T. formosanum and T. indicum B complex were located in subclade III-4. In conclusion, T. formosanum highly resembles to T. indicum B complex, while T. furfuraceum is most similar to T. huidongense. Based on molecular clock, we estimated that T. furfuraceum and T. formosanum would have diverged from the close relatives in mainland China between 10.2 and 4.1 Ma, respectively. Taken together, we propose that these two Tuber species found in Taiwan might originate from the common ancestors with some truffle species in China. However, with long divergence time and geographical separation, they have evolved into indigenous species of Taiwan. In the second part of this study, we set to determine the effects of temperature on the mycorrhizae formation and growth of Cyclobalanopsis glauca seedlings inoculated with T. formosanun and T. aestivum. No obvious morphological difference was seen in the ectomycorrhizae formed by T. formosanum and T. aestivum with different temperature treatment under stereomicroscope. The ectomycorrhizae formed by T. formosanum were monopodial or pinnate, and the color was light yellow to dark brown with or without bristles. The ectomycorrhizae formed by T. aestivum were monopodial, and the color was light yellow to dark brown with woolly hyphae. However, futher examination by scanning electron microscope revealed different structures. In the 35/30℃ and 30/25℃ treatment, fungal mantle was thinner and mycorrhizal structure was looser. In addition, the starch grain appeared more frequently in the cortical cells. The fungal mantle of T. formosanum was 15~25 μm thick, and the Hartig nets extended to the second layer of the cortex cells. The fungal mantle of T. aestivum was 18~30 μm thick, and the Hartig nets also extended to the second layer of the cortex cells. The mycorrhizal infection rate was highest in the 25/20℃ treatment, which was significantly different (P<0.05) with other treatments. Treatments with 20/15℃ and 30/25℃ were second and their mycorrhizal infection rates showed no significantly different (P>0.05). Treatments with 15/12℃ and 35/30℃ showed the lowest infection rate. The mycorrhizal infection rate was higher in seedlings inoculated with T. formosanum; however, the rate was not significantly different (P>0.05) from that inoculated with T. aestivum. The temperature effect on the height of seedlings was also examined. Treatment with 35/30℃ showed highest seedlings, which was significantly different (P<0.05) from other treatments. Seedlings inoculated mycorrhizae exhibited higher growth than noninfected plants; whereas T. formosanum displayed better growth effects than T. aestivum. Treatment with 25/20℃ showed best growth in the root collar diameter of seedlings and treatments with 35/30℃, 20/15℃ and 30/25℃ were second. However, no significant difference (P>0.05) was seen among these four treatments. Treatment with 15/12℃ showed the worst growth of root collar diameter. Seedlings inoculated mycorrhizae also exhibited better root collar growth than noninfected plants; whereas T. formosanum also enhanced better root collar growth than T. aestivum, but both treatments showed no significant difference (P>0.05). The concentrations of all macro-nutrient elements in the leaves exhibited the highest level in the 35/30℃ sample, which was significantly different (P<0.05) from other treatments. Overall, these results indicate that plants grow faster with higher nutrient contents in the high temperature; however, high temperature displayed negative effects on mycorrhizal development. Temperature exhibited negative relationship with mycorrhizal infection rate (r= -0.57), but showed positive relationships with the height and root collar diameter of seedlings, and the contents of C, N, K, Ca and Na (r= 0.52, 0.54, 0.69, 0.43, 0.51, 0.47, 0.55) in the leaves. As for the mycorrhizal infection rate, it showed positive relationships with the height and root collar diameter of seedlings, and the contents of K and P (r= 0.49, 0.47, 0.42, 0.67) in the leaves. The height of seedlings showed positive relationships with root collar diameter of seedlings, and the contents of N, K and P (r= 0.51, 0.71, 0.46, 0.42) in the leaves and the root collar diameter of seedlings showed positive relationship with the contents of N, K and P (r= 0.62, 0.44, 0.47) in the leaves as well. Finally, K, Ca and Mg showed negative relationships with each other (r= -0.48, -0.54, -0.43).