Overhauling the Effect of Surface Sterilization on Analysis of Endophytes in Tea Plants

Abstract

Increasing evidence shows that plant Endophytes play a crucial role in the fitness and productivity of hosts. Surface sterilization is an indispensable process before high-throughput sequencing (HTS) and tissue separation of plant endophytes, but its potential impact on the composition and diversity of endophytes has rarely been investigated. In the present work, the influence of sodium hypochlorite (NaClO) on the diversity of endophytic bacteria and fungi in leaves and stems of tea plants was investigated. We found that the diversity of bacterial endophytes was significantly affected by the concentration of NaClO as well as the pretreatment time. Pretreatment with 0.5% NaClO for 8 min and 2.0% NaClO for 3 min were suitable for the tea plant leaves and stems, respectively, but the effects of NaClO on the diversity of fungal endophytes were limited according to the results from HTS. Regardless of NaClO sterilization, most of the endophytes in tissues, such as the dominant taxa, could not be Isolated by using the regular culture-dependent approaches. Collectively, our results demonstrated that the pretreatment with NaClO should be modified to precisely understand the diversity of endophytes from different tissues of tea plants and also indicate that more attention should be paid to establish specific culture-dependent protocols for the isolation of plant endophytes.

In Planta Detection of Beauveria bassiana (Ascomycota: Hypocreales) Strains as Endophytes in Bean (Phaseolus vulgaris L.)

“…Our strain-nested PCR technique also complemented the traditional technique of plating tissue, since the frequent use of sodium hypochlorite to sterilize tissue surfaces may affect the diversity of fungal endophytes [28] or result in non-viable applied endophytes, as observed in our results. Subsequent evaluations will be essential to authenticate the efficacy of this method in the field-based monitoring of single or multiple strains in beans or other plants.…”

“…Techniques to study natural or artificially applied endophytes include direct observation, and culture-dependent and culture-independent methods, in which plant surface microorganisms are first removed [ 27 ]. However, this process may affect the diversity [ 28 ] and viability of endophytes of interest [ 29 ] in the plant. Therefore, it is essential to use culture-independent methods for the accurate and fast detection of endophytes, even if the microorganism is not viable.…”

Why Are There So Few Basidiomycota and Basal Fungi as Endophytes? A Review

“…Successful methods for endophyte isolation have been reviewed [67,73]. Yu et al [74] studied the effect of different concentrations of sodium hypochlorite employed and exposure times on the efficacy of surface sterilization on endophyte diversity from tea plants (Camellia sinensis). They found that stem and leaf tissues need different conditions, with mature stem tissue requiring a higher concentration and longer exposure time to achieve complete surface sterilization.…”

Anti-Fungal Properties of Plant Extracts in vitro against Alternaria solani, the Cause of Early Blight in Potato

“…The excess Collection, Isolation, Culture and Maintenance of A. solani Early blight was characterized by the visualization of leaf lesions and leaf samples with typical symptoms of A. solani were collected from Potato Crop Development Centre (PCDC) Nigale, Sindhupalchowk. Samples were surface sterilized with 1% Sodium hypochlorite for 5 minutes (Yu et al, 2022) and washed three times with distilled water. They were then dried and incubated under a 5 mm thick potato tuber slice on top of a moistened filter paper in petri plates (9 mm Ø) at 24 ± 2 After 24 hrs, fresh sporulation (whitish cottony fungal growth) was visible on abaxial surface of leaves.…”