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|標題:||The spore production of Entrophospora kentinensis in aeroponic system
叢枝菌根菌Entrophospora kentinensis 於氣霧環境產孢之研究
|關鍵字:||AMF;叢枝菌根菌;P;areoponical culture;spore production;Entrophospora kentinensis;Glomus;磷;氣霧栽培;產孢;Entrophospora kentinensis;繡球菌屬||出版社:||土壤環境科學系||摘要:||
It seems to be common that most researchers of arbuscular mycorrhizal fungi (AMF) either used mixed inoculum or ignored what species they used. They are more interested in the inoculation effect of AMF on the crop growth. Recently, some scientists switched their attention to the species specificity, because different AMF species may be suitable for different hosts or environmental conditions. The key step to study the relationship between them is to produce pure inoculum of AMF. Without pure culture of spores, could not run any further experiment. Up to dates, there are three major inoculum production techniques of AMF, i.e., pot culture, aeroponical culture and root organ culture.In this study, an aeroponic system was set up to assay the effect of different phosphorus concentrations on the spore production of Entrophospora kentinensis, originally described from Taiwan. A correspondent pot culture of bahia(Paspalum notatum)was made to reconfirm the nutrient effect on the spore production. The P concentration of nutrient solution may express certain inhibition on the spore production and colonization of Entrophospora kentinensis, and this effect seems to be dominated by the P requirement of host plant. The P requirement of plant is normally more at high light intensity than at low light intensity, because of active plant growth. Therefore, the strength of P in nutrient solution was relative to the P requirement of plant. Between two P concentrations (0.18 and 0.09 mg L-1) of nutrient solution, the higher P solution could be depressive to the spore production and colonization, if host plant grew at low light intensity and temperature. However, the same P concentration may show opposite effect when host plants grow under high light intensity and temperature.Similar inhibition effect on the spore production and colonization of Entrophospora kentinensis was also observed in pot cultures at higher P fertilization (16 mg L-1), if compared with the other treatment of P (8 mg L-1). In this experiment, the correlation between spore production and root colonization was not concluded.After two consecutive experiments, more than one species of spores was produced in aeroponic systems, even though pure spores of E. kentinensis were carefully isolated as starter inoculum. There were four more species identified, namely Glomus microaggregatum, G. intraradices, G. mosseae and G. pansihalos. The hyphal system of Entrophospora kentinensis is different from four other species. The diameter of external hyphe of Entrophospora kentinensis is smaller than that of internal hyphae; nevertheless, the hyphal diameter of four Glomus species are quite the reverse. Furthermore, the intracellular hyphal growth (such as hyphal coils) seems more in Entrophospora kentinensis than that in the four Glomus species.Besides, the most distinctive feature for external hyphae of Entrophospora kentinensis is that they very often form a hyphal net, which bridge the gaps across roots. However, most external hyphae of Glomus species extend along the root surface. If compared with other four species of Glomus, the colonization rate of Entrophospora kentinensis along root axis is relatively slow, and this phenomenon was referred to the difference in hyphal morphology and the type of hyphal growth. To build up a hyphal net interconnecting roots is more difficult and time-consuming than to extend a single hypha concordantly with the root growth. Because the root growth in aeroponic culture is faster than hyphal colonization of Entrophospora kentinensis , the colonization rate started to decline at mid—term of experiment. If the growth rate of plant root could be adjusted somehow, it may be useful in maintaining or even promoting colonization of Entrophospora kentinensis. The decline of colonization of E. kentinensis, however, was not observed in sandy pot cultures. Slower root growth rate and medium in pot culture (such as sand or pearlite), that may serve as infrastructure for further hyphal growth, are assumed two possible explanations.Competition between E. kentinensis and G. intraradices may occur, when both infect the same host plant. When both species colonize the same host plant, the spore production of G. intraradices is more than that of single species in aeroponic culture. pH in the nutrient solution of aeroponic culture seems to place certain selection pressure on the fungal species, when more than one species mixed with the inoculant, E. kentinensis. Glomus mosseae and Glomus pansihalos only appeared in experiment Ⅱ when the nutrient solution was slightly alkaline. Glomus intraradices seemed to fit in wider spectrum of pH range, so it appeared in two experiments.In order to pursuit the truth, pure inoculum was thought necessary to run the experiments, but it is very difficult to handle especially in an open environment. Even the spores of target species were carefully picked up, it is still hard to detect the hyphal sections of other species on the spores at microscope. If the activity of the hyphal fragment of other species was stronger than the target species, the contaminated species may overgrow and become the dominant species. After this study, we suggest researchers to check the purity of inoculum and spore production before and after the experiments.Contamination seems not easy to be avoided either in pot culture or in aeroponic systems, because these two systems are operated in open condition. In the future, we expect to see an innovative combination between root organ culture and aeroponic systems, which may guarantee the inoculum quality and provide a better model to study the mechanism of spore production in different nutrient condition.
以往研究叢枝菌根菌的試驗大多採用混合菌種的接種劑或不重視為何種菌種，而主要研究叢枝菌根菌對於作物的接種效應，但近來的學者開始注意到不同的菌種適合不同的宿主與環境。但要研究不同菌種與不同宿主或環境間的關係，首先必須先繁殖純的接種劑。繁殖菌種的方式目前共有三種：盆栽繁殖、氣霧式繁殖與根器官繁殖技術。本試驗主要是針對叢枝菌根菌的繁殖技術之一的氣霧式繁殖法(aeroponic culture)，研究在不同磷濃度下對於1995年於台灣發現的新菌種Entrophospora kentinensis的產孢影響。並同時觀察比較在同一試驗期間使用高磷與低磷營養液在砂耕繁殖方式下，對E. kentinensis產孢的影響。試驗結果發現在氣霧繁殖系統中，營養液的磷濃度是否對E. kentinensis造成抑制效應取決於植物對磷的需求。當植物對磷的需求量大時，原本可能對E. kentinensis造成抑制效應的磷濃度（0.18 mg L-1），在較長日照情況與氣溫較高適合植物生長時不產生抑制效應。以砂耕繁殖E. kentinensis，發現當營養液磷濃度為16 mg L-1時，植株的感染率與產孢量均遜於營養液磷濃度為8 mg L-1。因此當磷濃度為16 mg L-1時，即對E. kentinensis造成抑制效應。分析E. kentinensis在植株上的拓殖率與產孢量間的相關性，發現兩者間並沒有絕對的相關性，這方面的資料仍需要較多的數據與研究來加以佐證。在試驗期間發現不只繁殖出E. kentinensis，同時也另外繁殖出Glomus屬的四種菌種（G. microaggregatum、G. intraradices、G. mosseae與G. pansihalos）。Entrophospora kentinensis的菌絲型態不同於四種Glomus屬的菌種。Entrophospora kentinensis的根內菌絲直徑大於根外菌絲，但上述之四種Glomus屬的菌種，根內菌絲的直徑均小於根外菌絲。Entrophospora kentinensis細胞內菌絲捲曲的情況較Glomus屬的菌種多。Entrophospora kentinensis的根外菌絲會在植株根系間形成綿密的菌絲網。Glomus屬的菌種（如Glomus intraradices）的根外菌絲則會緊貼根系向下延伸拓殖。這些型態上的差異，可能就是造成E. kentinensis在氣霧環境下拓殖緩慢的原因，同時也因為如此E. kentinensis在氣霧繁殖系統下的競爭力弱於上述四種Glomus屬菌種。由於在氣霧環境下植物根系的生長速度較E. kentinensis的拓殖速度快，因此造成感染率在試驗中期後便開始下降。若能適當調整根系的生長速度，或可利於E. kentinensis的拓殖。Entrophospora kentinensis在砂耕環境下則由於有介質做為媒介，因此能夠順利拓殖，不影響產孢。氣霧環境下Entrophospora kentinensis與Glomus intraradices同時感染植株時，似乎存在有彼此競爭的作用。這種現象使得Glomus intraradices的產孢量，較其單獨感染植株的產孢量大。當所繁殖的菌根菌種原出現一種以上時，氣霧環境中的營養液pH會影響感染植株的菌種。其中Glomus mosseae與Glomus pansihalos較喜好偏弱鹼性的營養液，因此出現在試驗Ⅱ。Glomus intraradices則是較常出現在營養液pH呈微弱酸性至中性的試驗Ⅰ中。在使用菌種作為接種劑時，必須注意該接種劑是否真的為單一菌種，而這種情形往往很難掌握。因為在解剖顯微鏡下，即使所挑取的孢子是單一菌種，但卻很難察覺孢子上所可能附著的其它菌種的菌絲片段。這些菌絲的活力有可能超越原先的孢子，而在接種後造成鵲巢鳩佔的情形。例如本試驗所用菌種一開始判定為純菌。但接下來兩次的繁殖結果都意外繁殖出Glomus屬的菌種。因此在試驗結束後，對於所繁殖出的孢子是否為原先接種的菌種，應再予以確認。以氣霧繁殖菌種的方法仍無法避免其它腐生性雜菌的污染，因為不論是氣霧箱或是砂耕均為開放性系統，只要是開放性系統就無法上述的現象發生。未來希望結合根器官與氣霧繁殖菌種的方式，在封閉系統內大量繁殖；既可避免雜菌的污染，又可大量繁殖純淨的種原。
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