Molecular Identification of Pathogenic Bacteria in Kantong Semar Plants (Nepenthes Gracillis) Based on Mitochondrial 16S rRNA Gene

Rahmad Agus Prasetio,Isnawati Isnawati,Dwi A Rahayu,I.H.A Wahab,R Mardian,A.P Bayuseno

doi:10.1051/e3sconf/202132808005

Rahmad Agus Prasetio, Isnawati Isnawati + Show 4 more

Open Access

https://doi.org/10.1051/e3sconf/202132808005

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Abstract

Nepenthes is a protected and unique carnivorous plant because it has a fluid-filled pouch at the end of the leaf that serves as an insect trap. The existence of Nepenthes gracillis in ecosystems is endangered, one of the reasons is caused by the attack of pathogenic bacteria, so it is necessary to identify pathogenic bacteria that attack N. gracillis as an effort to handle. The purpose of this study was to identify pathogenic bacteria in N. gracillis using 16S rRNA gene sequencing results. This study aims to identify the 16S rRNA sequence and phylogenetic of bacteria attached in N. Gracillis leaves, and the identification was confirmed using molecular data. The construction of phylogenetic topology was made based on ML and NJ method using Kimura-2 parameter model. Subsequently, molecular characterization and identification was conducted based on 720 bp of 16S rRNA gene similarity, sequence variation, genetic distance, and phylogenetic topology. According to the results, the bacteria species attached in N. Gracillis identified as Lelliottia nimipressuralis, and closely related with Erwinia aphidicola. Genetic distance between sample and related species was 0,0173, with 98,26% similarities, then strongly 100 bootstrap analyses. Thus, this study provides more information for Lelliottia nimipressuralis phylogenetic studies using different marker.

Highlights

Nepenthes is a tropical carnivorous plant that has evolved modified leaves as external digestive organs via the leaf epiascidiation process in order to acquire nutrients from prey trapped within pitcher cups [1]
The results showed that the cause of Nepenthes disease caused by pathogenic bacteria from the genus Erwinia causing wet brown spots on the leaves of N. mirabilis
The materials used in this study included Nutrient Agar (NA) and Nutrient Both (NB) media for bacterial growth, 5.3% sodium hypochlorite, sterile distilled water, 70% alcohol, 96% alcohol, crystal violet, lugol, and safranin, DNA isolation kit, GT1 buffer, GT2 buffer, W2 buffer, elution buffer, agarose gel, 1x TAE buffer, loading dye, ethidium bromide (EtBr), proteinase K, universal 16S primer

Summary

Introduction

Nepenthes (locally known as monkey cup) is a tropical carnivorous plant that has evolved modified leaves as external digestive organs via the leaf epiascidiation process in order to acquire nutrients from prey trapped within pitcher cups [1]. Nepenthes is a monotypic family Nepenthaceae, known as tropical pitcher plants or monkey cups. Nepenthes is the only genus in the Nepenthaceae tribe that is classified as a carnivorous plant, as it survives in low-nitrogen environments by eating insects and small animals [2]. Nepenthes can thrive in soils with high acidity and very low levels of essential nutrients such as nitrogen, phosphorus, and potassium, in which other plants struggle [3]. Nepenthes has a symbiotic relationship with chitinolytic bacteria found in Nepenthes cotton fluid, and these bacteria can be used as potential and environmentally friendly biological controllers to deal with pests and pathogens, insects, in cultivated plants [4]

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