New Methods to Remove Rhizoplane Bacterial DNA of Banana

The aim of this study was to evaluate the effects of different surface sterilization protocols on retained rhizoplane bacterial DNA of banana. Viable rhizoplane bacteria and bacterial DNA copies of banana roots were treated with four sterilization agents: 75% ethanol and sodium hypochlorite solution (5% available chlorine), chlorine dioxide (0.2%), peroxyacetic acid (0.4%), and formaldehyde (36%) with different incubation times. Retained viable bacteria and bacterial DNA of banana roots treated were compared based on viable count, and qPCR and MPN-PCR methods. Root treatments with peroxyacetic acid (0.4%) and formaldehyde (36%) for 5 min could remove most (99.9999%) of viable rhizoplane bacteria. Chlorine dioxide (0.2%), peroxyacetic acid (0.4%), and formaldehyde (36%) could remove 99.9% of bacterial DNA segments of 16S rRNA gene, whereas, formaldehyde (36%) could remove 99.99% of whole 16S rRNA genes of rhizoplane bacteria. The surface sterilization protocol that included incubation with formaldehyde (36%) for 9 min and further treatment with 0.1 mol l-1 NaOH for 10 min might be an effective sterilization method to remove rhizoplane bacterial rRNA genes in the study of endophytic bacterial communities of banana roots. Research Article New Methods to Remove Rhizoplane Bacterial DNA of Banana Miaomiao Yin, Mingyue Wang, Hongming Tan and Lixiang Cao* School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China Dates: Received: 24 January, 2017; Accepted: 15 February, 2017; Published: 16 February, 2017 *Corresponding author: Lixiang Cao, School of Life Sciences, Sun Yat-sen University, Guangzhou, China; Tel: 8620-84110238; Fax: 8620-84036215; E-mail:


Introduction
Endophytes are conventionally defi ned as bacteria or fungi that reside internally in plant tissues, can be isolated from the plant after surface disinfection and cause no negative effects on plant growth (i.e., they are either benefi cial or commensal) [1]. Endophytes are considered as a subset of the root microbiota. The composition of the root microbiota can affect important plant traits, such as stress tolerance, productivity, and fi tness [2]. Although the importance of the rhizosphere microbiome for plant growth has been widely recognized, the vast majority of rhizosphere microorganisms (including endosphere microbiota) are still poorly understood [3].
Until recently, most studies on endophytes involved fi rst isolating organisms into pure culture to identify them, the species that do not grow or grow very slowly in culture media are overlooked [4]. By the culture-dependent methods, surface sterilization was the critical step to isolate endophytes from plant tissues [5,6]. Molecular-based methods overcome the culturability problem of many microbes by analyzing the PCRamplifi ed DNA products from total DNA extracted from plant tissues [7]. However, rhizosphere bacterial cells tightly attach to plant cells, so the bacterial DNA extracted from plant tissues may be contaminated by surface bacteria [8]. In order to avoid the isolation of surface bacterial DNA, potato stems and tubers were peeled asceptically [9]; however, it was not possible to peel tiny roots, mechanical removal of rhizoplane bacterial populations using vigorous shaking with glass beads had been demonstrated previously, however, the removal effi ciency of rhizosphere bacterial DNA was not demonstrated [8]. Reliable surface sterilization methods are needed to be developed for analysis of endosphere microbiota.
Hypochlorite is known to be a very effective killer of bacteria, which is partly attributable to lethal DNA damage [6]. Hypochlorites have been widely used to surface sterilize plant samples to remove viable bacteria [7,[10][11][12][13][14][15][16]. Nevertheless, the removal effi ciency of rhizosphere bacterial DNA by surface sterilization protocols was not demonstrated. After surface sterilization, it is possible that some residual DNAs are still remained on the root surfaces and might be misconsidered as endophytic bacteria by molecular-based methods.
To evaluate the effi ciency of surface sterilization methods to remove rhizoplane bacterial DNA, different surface sterilization methods (hypochlorite, chlorine dioxide, peroxyacetic acid, formaldehyde) were proposed to sterilize banana roots and the residual DNAs were quantifi ed by real-time PCR and MPN-PCR in the study.

Sample Collection
Healthy roots of banana plants (Musa sp., AAA, Cavendish subgroup, cv. Williams) were collected from a banana plantation in the suburbs of Guangzhou, China. Three banana plantlets were dug out carefully to ensure that maximal amount of root materials was collected. The samples were placed in plastic bags and taken to the laboratory and processed within 4 h of collection.

Surface Sterilization
The root samples were washed with tap water to remove soil particles and sterilized by the following protocols: (1) sequential immersion in 75% (v/v) ethanol for 5 min, and sodium hypochlorite solution (5% available chlorine) for 5 min; (2) immersion in 0.2% chlorine dioxide solution for 5 and

Measurements of Rhizosphere Bacteria and Bacterial DNA
Surface sterilization of the banana roots was checked:

Results
Sterilization with 75% ethyl alcohol did not reduce the number of viable bacteria on the rhizoplanes, further immersion in sodium hypochlorite solution (5% available chlorine) for 5 min removed 99% of rhizoplane bacteria. Chlorine dioxide (0.2%) removed 99.9% of rhizoplane bacteria after immersed for 5 min, extending treatment time to 7 min did not remove more bacteria [ Table 1]. However, the chlorine dioxide only retarded the bacterial growth.
The number of bacterial colony was 33 ~ 83 cfu ml-1 when the plates were incubated for 48 hours, and more bacterial colonies (137 ~ 330 cfu ml-1) appeared when the plates were incubated more than 72 hours. Peroxyacetic acid (0.4%) and formaldehyde (36%) removed most (99.9999%) of rhizoplane bacteria when the roots were treated for 5 min. The 7 min treatment did not enhance the sterilization effi ciency. The Table 1: The number of viable bacteria in aliquots (10 ml) of the sterile water used in the fi nal rinse (cfu/ml) from different sterilization agent treatments (sodium hypochlorite solution (5%), chlorine dioxide (0.2%), peroxyacetic acid (0.4%), and formaldehyde (36%) with different time).

Treatments
Roots collected a Roots soaked in bacterial suspension b Without  Table 2].
However, more viable bacteria on roots were found from the procedure. Therefore, the procedure was a sensitive method for validating surface sterilization. The two methods validated the effectiveness of sterilization with peroxyacetic acid (0.4%) and formaldehyde (36%), thus the rhizoplane bacterial DNA was further quantifi ed.
The number of bacterial DNA copies in sterile water used in the fi nal rinse determined by real-time PCR is one order of magnitude higher than that determined by plate counting [ rRNA genes were removed [ Table 4].
Although formaldehyde (36%) could remove 99.99% of bacterial whole 16S rRNA genes, the remained bacterial DNA could still be amplifi ed by PCR. NaOH, NaHCO3 and DNase was further used to remove rhizoplane bacterial DNA [ Table 5].
Compared with negative controls, the Ct value of retained bacterial DNA after sterilization with formaldehyde (36%) for 9 min and further treatment with 0.1 mol l-1 NaOH solution for 10 min is similar to negative control. The protocols could remove the disturbance from surface bacterial DNA of banana roots and could be used to study the plant endosphere microbiome.   a The roots were collected from banana plantation. b The roots collected from banana plantation were further soaked with Pseudomonas putida (2.0×109 cfu ml-l).
The total DNA was extracted from banana roots sterilized with formaldehyde (36%) for 9 min and 0.1 mol l-1 NaOH solution for 10 min and amplifi ed for bacterial whole 16S rRNA genes. The whole 16S rRNA genes (1.5 kb) could be amplifi ed from two banana root samples [ Figure 1]. So the DNA extracted from surface sterilized banana roots could be used to analyze bacterial community by high-throughput sequencing technologies based on PCR (such as pyrosequencing and Illmina-based analysis).

Discussion
The microbiota colonizing the rhizosphere and the endophytic compartment contribute to plant growth, productivity, carbon sequestration, and phytoremediation. bacteria should be excluded. However, the effects of dead rhizoplane bacterial DNA on the endophytic communities have not been demonstrated. Although the rhizoplane bacteria were killed by sterlization agents, the bacterial DNA could be amplifi ed by PCR with primers towards part of or whole 16S rRNA genes. Some rhizoplane bacteria might be misconsidered as endophytic bacteria in previous reports [7,[10][11][12][13][14][15][16]. The endophytic bacterial communities in previous reports might contain some rhizoplane bacteria.
In the study, although peroxyacetic acid (0.4%) and Compared with peroxyacetic acid (0.4%), formaldehyde (36%) is stable and low cost, so formaldehyde (36%) was suggested to sterilze the banana roots. DNase was exluded for retained DNase might degrade total DNA extracted from surface sterilized banana roots. NaOH was used to remove rhizoplane bacterial DNA due to its low price and stability.

Conclusions
In general, the killed rhizoplane bacterial DNA could still be amplifi ed by PCR with primers towards part of or whole 16S rRNA genes. Some rhizoplane bacteria might be misconsidered as endophytic bacteria by PCR-based sequence analysis.   However, genetic principles governing the derivation of hostspecifi c endophyte communities from soil communities are poorly understood [19]. The use of DNA-based metagenomic sequencing for analyzing endophytic bacterial communities has revealed that the excess bacterial cells mostly represent uncultured bacteria [20]. So the errors derived from rhizoplane