花生根瘤菌之多基因譜系分析及其與不同栽培型花生之共生親合性研究

Abstract

根瘤菌與豆科植物共生，透過生物固氮作用可以將空氣中的氮轉化為植物可利用的氮源。施用根瘤菌等微生物肥料有助於減少化學肥料的投入，因此被視為一種對環境友善的施肥方式。花生 (Arachis hypogara L.) 屬豆科蝶型花亞科，因其宿主專一性感染機制較不嚴謹，在田間施用花生根瘤菌時 （註：慢生型根瘤菌），若是土壤中已存在有更高宿主親和性之原生共生菌，則花生會優先與其結瘤，因而降低了微生物肥料的接種效率。豆科植物與根瘤菌之間的共生親和性高低主要取決於宿主與共生菌雙方的基因型。本研究利用不同栽培型的花生品種（包括瓦倫西亞型的黑金剛、西班牙型的台南選9號、中國遠雜9102以及中國商花5號）從臺大試驗田土壤中捕集花生根瘤菌。根據PCR檢驗結果，試驗田土壤中至少存在著帶有Clade III.3與Clade VIII這兩類nodA基因型的根瘤菌。從花生根瘤所分離出的24株共生菌經多基因序列以及結瘤基因分析鑑定，皆為帶有Clade VIII 的nodA基因之慢生型根瘤菌。本研究進一步評估接種單一根瘤菌株之共生固氮能力，結果發現將帶有Clade VIII結瘤基因的NTU-T-2菌株接種在台南選9號花生可展現最佳的共生親和性，而該菌株與其他兩種栽培型花生亦可表現較優良的結瘤能力。在共接種競爭結瘤試驗中，我們也證實了帶有Clade VIII nodA之花生根瘤菌顯然比帶有Clade III.3 nodA的花生根瘤菌具有結瘤能力上的優勢。花生雖然是被視為共生關係較不專一的豆科植物，然而本研究發現花生宿主對於帶有Clade VIII 這種nodA基因型的根瘤菌有較高的共生親和性，可作為篩選具有競爭優勢的花生根瘤菌之依據，將有助於提高花生根瘤菌的接種效率。

Rhizobia are bacterial symbionts of legumes that can convert atmospheric nitrogen into ammonia via biological nitrogen-fixation within root-nodules. Successful application of rhizobial inoculant can reduce the usage of chemical fertilizer without a decrease in crop yield, and it has been regarded as an environmental friendly approach. Peanut (Arachis hypogaea L.) is an annual herbaceous plant in the Fabaceae. It is usually nodulated by Bradyrhizobium spp., and is regarded as a promiscuous (or less-selective) legume due to its infection is via a less stringent pathway, called crack entry. However, if the indigenous rhizobia with high affinity presented in soil, peanut host will form nodules with them rather than the rhizobial inoculant. This feature causes a severe defect of rhizobial inoculant application in peanut cultivation. The symbiotic fitness between legumes and rhizobia is affected by the genotype of either host or symbiont. In this study, four peanut cultivated varieties (including the Valencia type: Taiwanese cv. BK, and some Spanish type cultivars: Taiwanese cv. TNS 9, Chinese cv. Yuanza9102, and Chinese Shanghua 5) were applied to trap peanut rhizobia inhabited the soil of NTU experimental field. According to the PCR results, there were Bradyrhizobium spp. with Clade III.3 and VIII nodA genotypes in the soil sample. Total 24 strains were isolated from the peanut root-nodules. All of them belonged to Bradyrhizobium genus carrying Clade VIII nodA genotype by multilocus sequence analysis (MLSA) and nodA sequence analysis. Furthermore, comparing the N-fixation ability between Clade III.3 and VIII strains by single strain inoculation test showed that the symbiont with Clade VIII nodA (i.e strain NTU-T-2) performed better symbiotic fitness on cv. TNS 9, and also showed good nodulation ability on the other two cultivars (i.e Valencia type cultivar: Taiwanese cv. TN 16, and Virginia type cultivar: Taiwanese cv. TC 2). Furthermore, in the co-inoculation experiment on cv. TNS 9, the Clade VIII nodA strain (i.e NTU-T-2) also showed better nodulation efficiency than that of type III.3 strains. These results indicated that although peanut is considered as a less-selective legume host, it still showed advantaged nodulation fitness with the Clade VIII nodA strains. This finding not only gives new insights into symbiotic fitness, but can be used as a screening platform for high nodulation competitiveness inoculants to increase peanut rhizobial inoculation efficiency.