Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Development of RAPD-SCAR markers for Lonicera japonica (Caprifolicaceae) variety authentication by improved RAPD and DNA cloning / Desarrollo de marcadores RAPD-SCAR para Lonicera japonica Thunb. (Caprifolicaceae) para la autenticación de variedades mediante RAPD mejorados y clonación de ADN

Genetic diversity within a species is a common feature, which plays a vital role in its survival and adaptability, and is important for the identification and authentication of a species. Lonicera japonica is a traditionally used medicinal plant, which have been recently genetically characterized by an improved ran- dom amplified polymorphic DNA (RAPD) analysis. In this study, the molecular markers on the basis of these RAPD fragments have been developed to identify specific L. japonica variety. The DNAs were extracted from fresh young leaves of different samples of L. japonica collected from Shenzhen, Yichang, Leshan, Emei and Loudi, China. The DNA materials were amplified using improved RAPD PCR. Different RAPD bands were excised, cloned and developed for stable sequence-characterized amplified region (SCAR) markers with differ- ent species. Two SCAR markers, JYH3-3 and JYH4-3, have been successfully cloned from improved RAPD fragments. The SCAR marker JYH3-3 was found specific for all of the L. japonica samples collected from the different regions, and another marker JYH 4-3 was strictly specific to the Shenzhen sample from Guangdong province, which is geographically distant from Hubei, Sichuan and Hunan Provinces (source of other L. japonica samples). The marker JYH3-3 was found as specific molecular marker for the identification of L. japonica, while JYH4-3 was found as molecular marker strictly specific for the Shenzhen sample. The developed SCAR mark- ers might serve as more specific molecular markers for L. japonica variety authentication. The combination of improved RAPD analysis and SCAR marker development have resulted useful tools to study the genetic variety of any organism, which we have successfully applied here in L. japonica.

La diversidad genética dentro de una especie es una característica común, que juega un papel vital en su supervivencia y adaptabilidad, y es importante para la identificación y la autenticación de una especie. Lonicera japonica es una planta medicinal utilizada tradicionalmente, que han sido recientemente caracterizada genéticamente por amplificación aleatoria mejorada de ADN polimórfico (RAPD). En este estudio, los marcadores moleculares basados en estos fragmentos de RAPD se han desarrollado para identificar una variedad específica de L. japonica. Los ADN se extrajeron de las hojas jóvenes frescas de diferentes muestras de L. japonica recogidas de Shenzhen, Yichang, Leshan, Emei y Loudi, China. Los materiales de ADN fueron amplificados utilizando el RAPD PCR mejorado. Diferentes bandas RAPD fueron extraídas, clonadas y desarrolladas para las regiones amplificadas de secuencia conocida (SCAR) con marcado- res de diferentes especies. Dos marcadores SCAR, JYH3-3 y JYH4-3, se clonaron con éxito de los RAPD mejorados. El marcador SCAR JYH3-3 se encontró específico para todas las muestras de L. japonica recolectadas en las diferentes regiones, mientras que el otro marcador JYH4-3 era estrictamente específico para la muestra de Shenzhen de la provincia de Guangdong, que está geográficamente distante de Hubei, Sichuan y Provincias Hunan (fuente de otras muestras de L. japonica). Se encontró que JYH3-3 es un marcador molecular específico para la identificación de L. japonica, mientras que JYH4-3 se encontró como marcador molecular estrictamente específico para la muestra de Shenzhen. Los marcadores SCAR desarrollados podrían servir como marcadores moleculares más específicos para la autenticación de la variedad L. japonica. La combi- nación de RAPD mejorado y el desarrollo del marcador SCAR han dado como resultado herramientas útiles para el estudio de la variedad genética de cualquier organismo, que hemos aplicado con éxito en L. japonica.