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

Abstract

Background: Tea, 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. Results:A 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. Conclusions:This 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.

The necrotroph Botrytis cinerea promotes disease development in Panax ginseng by manipulating plant defense signals and antifungal metabolites degradation

“…Real-Time quantitative PCR was performed as previously described [ 29 ]. Primer sequences are listed in Table S7 .…”

“…The compounds were mixed with sterile melting PDA medium to obtain final concentrations. The incubation of fungi, the measurement of mycelial growth, and the relative inhibition ratio were performed as previously [ 29 , 32 ].…”

Infection timing affects Fusarium poae colonization of bread wheat spikes and mycotoxin accumulation in the grain

“…Fungal development in various plant tissues is often measured through the use of highly sensitive DNA-based methods such as qPCR. In particular, a procedure that normalizes pathogenic DNA in relation to plant DNA provides a precise measurement of pathogen growth in various host plants and has been widely used 61,62 being DNA a realiable approximation of fungal biomass. 63 In the present study, F. poae DNA related to wheat DNA, quantified by qPCR, was used as an estimation of fungal biomass in grain and chaff tissues.…”

The Necrotroph Botrytis cinerea BcSpd1 Plays a Key Role in Modulating Both Fungal Pathogenic Factors and Plant Disease Development

“…Real-time quantitative PCR was performed as previously described ( He et al, 2020 ). BcActin was used for normalization.…”