台灣入侵植物南美豬屎豆及其根瘤菌之共生關係

Symbiotic relationship between an invasive legume, Crotalaria zanzibarica, and its root-nodulating rhizobia in Taiwan

黃承泰(Cheng-Tai Huang)

指導教授 ： 高文媛

共同指導教授 ： 劉啟德(Chi-Te Liu)

國立臺灣大學/生命科學院/生態學與演化生物學研究所/博士(2018年)

https://doi.org/10.6342/NTU201800782

摘要

南美豬屎豆 (Crotalaria zanzibarica Benth.) 是台灣歸化豆科植物中分布最廣的一種，為多年生的木本植物，常見於道路旁、河岸及廢耕地。野外觀察發現南美豬屎豆普遍具有根瘤，因此推測與固氮菌(根瘤菌)共生有助其在貧瘠棲地建立族群，是讓此植物能夠在台灣廣泛分布的原因之一。然而，南美豬屎豆在台灣的共生根瘤菌未曾被研究。本論文探討南美豬屎豆與根瘤菌之間的共生關係，以了解此外來植物在台灣的共生根瘤菌之多樣性及可能來源，並檢驗不同根瘤菌株對植物的共生表現。
從種植在台大溫室的南美豬屎豆根瘤中分離出具多形態的菌株CzR2，分析此菌株之6個管家基因 (atpD、dnaK、glnII、gyrB、recA 和 rpoB) 序列，結果顯示CzR2屬於Bradyrhizobium arachidis。CzR2在游離狀態時受到甘露醇或果糖誘導會產生多形態細胞，此現象首次在根瘤菌中被發現。CzR2在與南美豬屎豆共生時也產生多形態的類菌體，然而有些類菌體其染色體具多倍體，有別於游離細胞的單倍體及二倍體。此結果顯示CzR2與南美豬屎豆共生時其染色體有內複製的現象。
調查新店溪沿岸的南美豬屎豆及常見的六種共域豆科植物，這些植物具有不同的根瘤(有限及無限)和類菌體(膨大及非膨大)形態。由分離菌株的16S rRNA序列分析，得知南美豬屎豆、蠅翼草、異葉山螞蝗和鍊夾豆皆與Bradyrhizobium建立共生，與田菁共生的根瘤菌為Neorhizobium和Rhizobium，白花菽草則為Rhizobium，含羞草的共生根瘤菌為Cupriavidus和Paraburkholderia。雖然這些植物具有不同的共生特徵，其葉片都具有類似的穩定性氮同位素比值(δ15N)，其值約為 -1 ‰；且這些植物根瘤的δ15N皆為正值(3.7-7.3 ‰)，其中無限根瘤比有限根瘤普遍具有較高的δ15N數值。
以多基因序列 (dnaK-glnII-recA-rpoB) 分析從台灣北、中、南三河岸南美豬屎豆族群所分離出之59株根瘤菌株，以及其他共域豆科植物族群所分離出之54株根瘤菌株，結果顯示這些菌株皆屬於Bradyrhizobium且可區分成21個支序群。同時比較分析其他共域豆科的根瘤菌群時，發現某些菌群似乎對南美豬屎豆有專一性，然而某些菌群則廣泛出現在多種植物的根瘤中。多數南美豬屎豆根瘤菌具有代表美洲起源的nodA共生基因，其次為亞洲起源及世界廣布型，顯示此物種在台灣能夠與來自不同地理區的的根瘤菌建立共生。
藉由溫室實驗比較接種不同菌株對南美豬屎豆的影響，結果發現南美豬屎豆與不同菌株共生時產生不同的根瘤且類菌體形態亦有所不同，顯示根瘤及類菌體形態亦受到共生菌株的影響，不全受到宿主決定；南美豬屎豆植株生物量及植株總氮量在不同菌株接種處裡下有顯著差異，然而，南美豬屎豆植株生物量分配、氮含量、穩定性同位素比值和共生效率(總生物量變化/總根瘤生物量變化)受不同接種菌株的影響並不顯著。
綜合野外調查和溫室實驗結果，證實南美豬屎豆能夠與多樣Bradyrhizobium菌種建立有效共生，顯示其為廣適性宿主，此特徵有助於該物種擴散到不同地點時能找到相容根瘤菌。另一方面，來自南美洲的外來根瘤菌對於南美豬屎豆在台灣與根瘤菌建立共生中扮演重要角色。

關鍵字

入侵植物 ； 南美豬屎豆 ； 豆科與根瘤菌共生 ； 慢生型根瘤菌 ； 共生特徵

並列摘要

Crotalaria zanzibarica Benth., a perennial shrub native to Africa, is the most widely-distributed naturalized legume in Taiwan. The plant, commonly distributed along roadsides and riverbanks, and in abandoned fields, established symbiosis with rhizobia forming root nodules. Root nodules are capable of fixing nitrogen. Accordingly, the symbiosis with nitrogen-fixing rhizobia might help C. zanzibarica colonizing nutrient-poor habitats. In this dissertation, I studied the symbiotic relationship between rhizobia and this legume, aiming to understand the diversity and possible origins of the symbiotic rhizobia and the beneficial effects of the symbiosis to C. zanzibarica.
A rhizobial strain, designed as CzR2, was isolated from the nodules of C. zanzibarica grown in a greenhouse. This strain displayed pleomorphism, cell size ranging from 2 to 10μm, in free-living state when cultivated in standard YEM medium which significantly differs from any known rhizobia. Based on the analysis of atpD-dnaK-glnII-gyrB-recA-rpoB gene set, CzR2 belongs to Bradyrhizobium arachidis. Results of further experiments revealed that pleomorphism in this strain in its free-living state could be induced by mannitol, or fructose, but not by glucose. Accordingly, the pleomorpism is substrate-dependent. CzR2 in its free-living state contained haploid and diploid cells, while that in symbiosis with C. zanzibarica was elongated with polyploidy, suggesting the occurrence of genomic endo-reduplication.
Legume-rhizobia symbioses of C. zanzibarica and six common legume species growing sympatrically along Xindian riverbank were investigated in Chapter 2. I found that these legumes form either determinate or indeterminate types of root nodules and harbored swollen or non-swollen bacteroids. Based on the 16S rRNA sequences, the symbionts of these legumes were classified as Bradyrhizobium, Neorhizobium, Rhizobium, Cupriavidus and Paraburkholderia. Irrespective of their possessing of diverse symbiotic traits and nodule symbionts, the seven legume species had similar and consistently negative leaf δ15N values (mean of -1.2 ‰), and showed 15N enrichment (varying from 3.7 to 7.3 ‰) in their nodules. In addition, variations in the values of leaf δ13C (varying from -29 to -34‰) among the seven legumes were measured, indicating their photosynthetic water use efficiencies were different. The results also suggested that C. zanzibarica could be nodulated by diverse rhizobia.
To compare the symbionts of C. zanzibarica and sympatric legumes growing along three distant riverbanks in Taiwan, I collected 59 isolates from this plant and 54 isolates from coexisting legumes. Based on the multilocus sequence analysis of concatenated dnaK-glnII-recA-rpoB gene sequences, the C. zanzibarica isolates were highly diverse, belonging to 14 clades and varied among sampling sites, which can be either phylogenetically similar to or distinct from the isolates of coexisting legumes. The majority of C. zanzibarica isolates had nodA genes of American origin, following by Asian origin, while others might be cosmopolitan.
To confirm the field isolates are able to nodulate C. zanzibarica and to compare the effects of symbionts on growth of this plant, I conducted single-strain inoculation experiment and investigated growth response, nodulation response, symbiotic efficiency and nitrogen relationship of C. zanzibarica inoculated with six rhizobial strains. The greenhouse inoculation experiment revealed that nodule and bacteroid morphologies in C. zanzibarica were rhizobial strain-dependent. Furthermore, C. zanzibarica plants showed significant variation in total plant biomass and nitrogen accumulation among the strains inoculated, while there was very little variation in biomass allocation, nitrogen content, δ15N value and symbiotic efficiency among these tested plants.
Results of the greenhouse experiments and field investigations indicated that C. zanzibarica was capable of forming effective symbiosis with diverse rhizobia, which might confer the plant the ability of colonizing various habitats and contribute to its widely distribution in Taiwan.

並列關鍵字

invasive plant ； Crotalaria zanzibarica ； legume-rhizobia symbiosis ； Bradyrhizobium ； symbiotic traits

參考文獻

Adams MA, Turnbull TL, Sprent JI, Buchmann N (2016) Legumes are different: Leaf nitrogen, photosynthesis, and water use efficiency. Proc. Natl. Acad. Sci. 113:4098-4103

Appunu C, N’Zoue A, Moulin L, Depret G, Laguerre G (2009) Vigna mungo, V. radiata and V. unguiculata plants sampled in different agronomical–ecological–climatic regions of India are nodulated by Bradyrhizobium yuanmingense. Syst. Appl. Microbiol.32:460-470

Andam CP, Parker MA (2008) Origins of Bradyrhizobium nodule symbionts from two legume trees in the Philippines. J. Biogeogr. 35:1030-1039

Aserse AA, Räsänen LA, Aseffa F, Hailemariam A, Lindström K (2012) Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia. Mol. Phylogenet. Evol. 65:595-609

Azevedo H, Lopes FM, Silla PR, Hungria M (2015) A database for the taxonomic and phylogenetic identification of the genus Bradyrhizobium using multilocus sequence analysis. BMC Genomics 16(Suppl 5):S10