Abstract
ABSTRACTCulture-dependent and culture-independent methods were compared and evaluated in the study of the endophytic diversity of Dendrobium officinale. Culture-independent methods consisted of polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) and metagenome methods. According to the results, differences were found between the three methods. Three phyla, namely Firmicutes, Proteobacteria, and Actinobacteria, were detected using the culture-dependent method, and two phyla, Firmicutes and Proteobacteria, were detected by the DGGE method. Using the metagenome method, four major phyla were determined, including Proteobacteria (76.54%), Actinobacteria (18.56%), Firmicutes (2.27%), and Bacteroidetes (1.56%). A distinct trend was obtained at the genus level in terms of the method and the corresponding number of genera determined. There were 449 genera and 16 genera obtained from the metagenome and DGGE methods, respectively, and only 7 genera were obtained through the culture-dependent method. By comparison, all the genera from the culture-dependent and DGGE methods were contained in the members determined using the metagenome method. Overall, culture-dependent methods are limited to ‘finding’ endophytic bacteria in plants. DGGE is an alternative to investigating primary diversity patterns; however, the metagenome method is still the best choice for determining the endophytic profile in plants. It is essential to use multiphasic approaches to study cultured and uncultured microbes.
Highlights
Dendrobium officinale Kimura et Migo, of the family Orchidaceae, which is mainly distributed in South-east and South Asian countries, is a traditional Chinese medicinal plant associated with immunostimulating [1] and anti-tumour [2] functions
PCR products were analysed by computer sequencing of segments reading a length of 100 bp, and the results showed that the sample sequencing error rate was low, which proved that the D. officinale sample DNA bases were of good quality, ensuring that the sequencing results were of high accuracy
Diversity of endophytic bacteria based on the culture-dependent method
Summary
Dendrobium officinale Kimura et Migo, of the family Orchidaceae, which is mainly distributed in South-east and South Asian countries, is a traditional Chinese medicinal plant associated with immunostimulating [1] and anti-tumour [2] functions. D. officinale usually grows on nutrient-poor cliffs on mountains at altitudes between 900 m and 1500 m, mainly in the Yunnan, Sichuan, Guizhou, Guangxi, and Zhejiang provinces of China [3]. In the wild, it grows slowly, and microbes may provide vital nutrition for it as they do for other orchids. Fungi were reported to play a critical role in the growth of D. officinale [4,5,6]. Prokaryotes are directly related to the function of plants, and varied endophytes were found in D. officinale. Clarification of the diversity and function of the endophytes may more effectively help to elucidate their roles in their hosts
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