摘要
蛇足石杉是一种重要的药用蕨类植物,目前还没有大面积人工栽培的报道。蛇足石杉生活史与内生真菌的关系密切是其一个重要的生物学特征。本文对蛇足石杉内生真菌的分离技术、内生真菌的形态、及其对蛇足石杉扦插生根、生长发育的影响等内容进行了研究,以期加深对这种低等蕨类植物生物学、生理学上的了解,为人工栽培技术的建立提供理论参考。
本文主要内容和结论如下
(1)蛇足石杉内生真菌的分离方法。比较适合蛇足石杉茎表面灭菌的方法是75%乙醇灭菌2min后用1%NaClO灭菌4min;叶的最佳灭菌方法是75%乙醇灭菌0.5min后用1%NaClO灭菌3min;植物样品剪碎法处理,以1/2PDA(马铃薯葡萄糖培养基)作分离培养基。根部内生真菌的分离可采用无菌水洗涤法。
(2)蛇足石杉内生真菌分离结果。从江西宜黄县、安徽金寨县、安徽宣城市三个产地的蛇足石杉中共分离到内生真菌59株,其中从江西宜黄县蛇足石杉中分离到内生真菌17株,安徽金寨县蛇足石杉中分离到内生真菌20株,安徽宣城市蛇足石杉中分离到内生真菌22株。从内生真菌的分布部位来看,根部的内生真菌种类多于茎、叶。
(3)蛇足石杉的菌根结构。通过对蛇足石杉根部显微观察发现蛇足石杉根部生有大量根毛,部分根毛中生有菌丝,属于内生菌根菌。
(4)蛇足石杉内生真菌的形态学研究。蛇足石杉内生真菌从形态上看种类比较丰富。部分蛇足石杉内生真菌可在PDA培养基上产生孢子,产孢真菌占56%,鉴定分别属于木霉属、单梗曲霉属、青霉属、根霉属、曲霉属、聚孢霉属、粘帚霉属、瓶梗青霉属等。青霉属9株,占15%;聚孢霉属8株,占14%;木霉属5株,占8%。
(5)建立实验室蛇足石杉扦插方法。进行了人工气候箱内的扦插试验,模拟蛇足石杉原生境6月份的条件,设为湿度90%,光照33%(经测定800-12001x),白昼温度22℃,14h,夜间14℃,10h。春季插条在人工气候箱中扦插后的44天左右长出不定根,生根率为77%。
(6)内生真菌对蛇足石杉扦插生根的影响及机理。在上述(5)人工气候箱条件下,蛇足石杉分别在原生境土壤(土壤I)、接种了内生真菌的灭菌土壤(土壤Ⅱ)、灭菌土壤(土壤Ⅲ)上进行扦插,调查蛇足石杉扦插生根率及生根过程中内源物质的变化,结果表明与土壤Ⅲ上的扦插蛇足石杉相比,原生境土壤和施入内生真菌土壤上植株生根率,分别提高了10%和16%,内生真菌影响蛇足石杉体内酚类物质和ⅠAA(吲哚乙酸)的含量,以及POD(多酚氧化酶)和PPO(过氧化物酶)的活性,并提高其扦插生根率,增加生根量。内生真菌促进蛇足石杉插条切口处的愈伤组织形成、分化,从而提高插条切口处生根率。证明内生真菌与蛇足石杉的生长有密切关系。
(7)内生真菌对蛇足石杉生理生化指标的影响。在上述(5)人工气候箱条件下,蛇足石杉分别在原生境土壤(土壤Ⅰ)、接种了内生真菌的灭菌土壤(土壤Ⅱ)、灭菌土壤(土壤Ⅲ)上进行扦插,扦插120天后测定可溶性蛋白含量、可溶性糖含量、PPO、POD活性、黄酮类化合物和石杉碱甲含量。与土壤Ⅲ上的扦插蛇足石杉相比,土壤Ⅱ上扦插植株茎中的可溶性蛋白含量显著提高(P<0.05),土壤Ⅰ上扦插植株叶片和茎中的可溶性糖含量显著提高(P＜0.05),土壤Ⅰ和土壤Ⅱ上的扦插植株的黄酮类化合物含量显著提高。原生境土壤微生物、内生真菌可以提高蛇足石杉体内的代谢水平。
(8)内生真菌发酵产物的植物活性物质研究。根据形态学初步鉴定结果,选取同时出现在两个产地的蛇足石杉中内生真菌,这种真菌与蛇足石杉共生专一性较强,共选取7株进行研究,xj1,xj2,xj3,xj4,xj5,dg5,dg14。发现2株内生真菌能够分泌ⅠAA,3株真菌发酵液具有PPO活性,3株真菌发酵液具有POD活性。
(9)对蛇足石杉内生真菌发酵产物的AchE(乙酰胆碱酯酶)抑制活性进行了研究。发现有9株内生真菌发酵可产生生物碱,其中4株内生真菌发酵产生的生物碱具有Ache抑制活性,其中抑制活性最强的是xj14菌株发酵产生的生物碱,它对Ache的ⅠC50为134.8280μg.mL~(-1)。
(10)内生真菌的分子鉴定。具有较强Ache抑制活性的菌株xj14属于木霉属。与蛇足石杉共生专一性较强的7株内生真菌分别属于接合菌门和子囊菌门,有3株属于木霉菌属,2株被孢霉属,1株小克银汉属,1株根毛霉属。
Huperzia serrata is a kind of important Chinese medicinal herb,and at present the large-scale cultivation has not been reported.The symbiosis of H.serrata with endophtic fungi is its important biological characteristic.In order to understand plant biology and physiology,provide the theory reference for cultivation,in this paper,the isolation technology of endophytic fungi,its morphology and the effects on cuttings rooting,growth and development of H.serrata were studied.The main contents and conclusions are as follow:
(1) Isolation method of endophytic fungal.The optimal method for isolation of endophytic fungal:Stem surface-sterilization with 75%alcohol(2min),1%NaClO(4min) and leaf surface-sterilization with 75%alcohol(0.5min),l%NaClO(3min).The sample treatment with dissection method and used 1/2PDA as isolation medium.Aseptic water method was used to isolate endophytic fungi from the root.
(2) Results of the isolation of endophytic fungi:There were 59 strains endophytic fungi isolated from H.serrata,17 strains isolated from H.serrata of Jiangxi Yihuang;20 strains isolated from H.serrata of Anhui Jinxian and 22 strains isolated from H.serrata of Anhui Xuancheng.The endophytic fungal species from root were more than that from stem and leaf.
(3) The mycorrhiza structure of H.serrata:A large number of root hairs was found around the roots,fungi hypha was observed in root hairs.The results suggested that mycorrhiza of H.serrata belonged to endotrophic mycorrhiza.
(4) Morphological studying on endophytic fungal.The identification result showed the biodiversity of endophytic fungi in H.serrata.There were 56%endophytic fungi can produce spore on the PDA medium,they were identified as Trichoderma, Aspergillus,Penicillium,Rhizopus etc.There were 9 strains that belong to Penicillium, account for 15%,8 strains that belong to Glomerularia,account for 14%,5 strains that belong to Trichoderma,account for 8%in totals isolaled endophytic fungi。
(5) The method of cutting was established in the laboratory.The cutting test simulated June climate of the habitat in artificial climate box.Humidity was setted at 90%,illumination was setted at 33%(800-1200Lx),temperature was setted at the day 22℃in day(14h),and 14℃(10h)in night.Cutting was found rooting after 44 days.The rooting rate of spring cutting is 77%.
(6) The effect and mechanism of endopythe on cutting rootings.The cuttings of H.serrata were planted on soilⅠof primary habitat,on soilⅡwhich had been inoculated with endophytic fungi,on soilⅢwhich had been autoclaved.The rooting rate was examined,the change of endogenous substances contents during rooting were determined too.Results showed that the rooting rate of H.serrata planted on SoilⅢincreased by 10%and 16%compared with SoilⅠand SoilⅡ.The effect of endophy fungi on the content of Phenols,IAA and the activity of POD and PPO was observed. The endopythe could increase the rooting rate and the number of roots.The endophytic fungi promoted callus formation and cell differentiation then could increase rooting rate.The close relation between endopythe and H.serrata can be demonstrated.
(7) Effects of endophytic fungi on physiological and biochemical indexes of H.serrata.The cuttings of H.serrata were planted on soilⅠof primary habitat,on the soilⅡhad been inoculated endophytic fungi,on the soilⅢhad been autoclaved.Soluble protein and soluble sugar content,activities of PPO and POD,flavonoids and huperzine A contents were determined after cutting 120d.Results:SoilⅠincreased the soluble protein content of stem of H.serrata(P＜0.05),soilⅠincreased the soluble sugar contents of leaves and stem of H.serrata(P＜0.05),soilⅠand soilⅡincreased the flavonoids contents of H.serrata(P＜0.05).Soil microbes from primary habitat, endophytic fungi promote rooting,they also increase plant metabolism level of H.serrata.
(8) The study on plant-active substance in fermentation products by endopytic fungi.7 strains of endopythe(xj1,xj2,xj3,xj4,xjS,dg5,dg14)which have specifical symbiosis with H.serrata were researched.Results showed that two of the 7 strains were able to produce IAA,three of them strains have activity of PPO,three of them have POD activity.
(9) The AchE inhibitory effect of the fermentation products by endopythe was studied.Results showed that 9 strains endopythe could produce alkaloid,and 4 strains of them have the inhibitory effect on AchE.The strongest inhibitory effect was the alkaloid produced by xj14,its IC50 is 134.8280μg.mL~(-1).
(10) Molecular identification of endophytic fungi.The strain xj14 was suggested belonging to Trichoderma 7 strains endopythe that have specifical symbiosis with H.serrata belonged to Zygomycetes and ascomycetes.3 strains belong to Trichoderma,2 strains belong to Mortierella,1strain belongs to Rhizomucor.
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