Development of a DNA-based real-time PCR assay for the quantification of Colletotrichum camelliae growth in tea (Camellia sinensis)

Shengnan He,Yi Wei,Tai An,Shouan Liu,Huchen Chen

doi:10.1186/s13007-020-00564-x

Abstract

BackgroundTea, which is produced from new shoots of existing tea plants (Camellia sinensis), is one of the most popular, non-alcoholic, healthy beverages worldwide. Colletotrichum camelliae is one of the dominant fungal pathogens of tea. The interaction of C. camelliae with tea could be a useful pathosystem to elucidate various aspects of woody, medicinal plant-fungal interactions. Currently, many studies characterizing resistance or virulence and aggressiveness use lesion size at the infection sites on the leaves to quantify the growth of the pathogen. However, this method does not offer the sensitivity needed for the robust quantification of small changes in aggressiveness or the accurate quantification of pathogen growth at the early stages of infection.ResultsA quantitative real-time polymerase chain reaction (qRT-PCR) assay was developed for the quantification of C. camelliae growth on tea plant. This method was based on the comparison of fungal DNA in relation to plant biomass. This assay was used to investigate the phenotypes of tea plant cultivars in response to C. camelliae infection. Two cultivars, Zhongcha 108 (ZC108) and Longjing 43 (LJ43), were tested with this method. ZC108 was previously reported as an anthracnose-resistant cultivar against C. camelliae, while LJ43 was susceptible. The traditional lesion measurement method showed that both cultivars were susceptible to a virulent strain of C. camelliae, while the qRT-PCR approach indicated that very little fungal growth occurred in the anthracnose-resistant cultivar ZC108. The observed results in this study were consistent with previously published research. In addition, the DNA-based real-time PCR method was applied for analysis of pathogenic differences in general C. camelliae isolates and among several Colletotrichum spp that infect tea.ConclusionsThis study showed that the DNA-based qRT-PCR technique is rapid, highly sensitive and easily applicable for routine experiments and could be used in screening for resistant tea plant cultivars or to identify differences in pathogen aggressiveness within and among Colletotrichum species.

Highlights

Tea (Camellia sinensis) is one of the most economically important crops in the world
The internal transcribed spacer (ITS) primers designed in this study did not amplify the DNA from P. camelliae-sinensis, Neopestalotiopsis sp. or M. oryzae. These results indicated that the ITS primer could quantify the Colletotrichum spp. including C. camelliae, C. fructicola, C. siamense and C. fioriniae
A procedure for the quantification of C. camelliae using quantitative real-time polymerase chain reaction (qRT-PCR) was studied as a new method for assessing the growth of this fungus on tea

Summary

Introduction

Tea (Camellia sinensis) is one of the most economically important crops in the world. Tea is widely grown in Asian, African and South American countries to produce He et al Plant Methods (2020) 16:17. Pathogens of tea include Colletotrichum spp., a large genus of Ascomycete fungi causing anthracnose disease on a wide range of host genera [13–19]. Colletotrichum camelliae, C. fructicola, C. siamense and C. fioriniae have been isolated in the main tea growing region of China and caused anthracnose on Ca. sinensis [20–23]. Colletotrichum camelliae and C. fructicola were the species most often isolated and were proposed as dominant pathogens of tea [20–23]. The interaction of tea and C. camelliae would be a useful pathosystem to elucidate various aspects of woody medicinal plant-fungi interactions. The interaction of C. camelliae with tea could be a useful pathosystem to elucidate various aspects of woody, medicinal plant-fungal interactions. This method does not offer the sensitivity needed for the robust quantification of small changes in aggressiveness or the accurate quantification of pathogen growth at the early stages of infection

Methods

Results

Discussion

Conclusion