3. DiscussionOur study revealed for

3. Discussion

Our study revealed for the first time the possibility of Illumina

sequencing protocol to evaluate microbiota present in plant tissues–

bacterial endophytes. The sequencing can be improved with good

choice of primer pair to amplify a longer stretch of the 16S rRNA gene.

Our empirical results highlight the utility of this platform for precise

and high resolution microbiota profiling (N90% at species level) of endo-
phytic communities, or perhaps extended to other resources/samples.

The improvement to the various analyses tools was equally important

to minimise the biasness introduced by the host DNA (chloroplast)

and chimaera which was removed without affecting the overall quality

of the reads. Mothur pipeline relatively provides us a good opportunity

to effectively process the read sequence on a single platform. This

minimised the probable loss of quality of reads. The use of novel

shotgun 16S rRNA gene by NGS has also revealed the overall richness

and diversity of microbiota communities in plant tissues to encompass

both the culturable and unculturable endophytic bacteria. The

α-diversity analysis indicated the richness and inverse Simpson diversi-
ty index of the bacterial endophyte communities for the leaf, root and

stem tissues to be 2.221, 6.603 and 1.491 respectively. It further eluci-
dates the microbial colonisation of plant tissues as revealed by the

Venn diagram which illustrates the distribution of the bacterial commu-
nities across the tissues and the total shared richness (Fig. 2).

The colonisation pattern as illustrated by the Venn diagram of the

OTU distribution indicated that 41% of microbes found in the root

tissues were also present in all the three tissues, such as Pseudomonas,

Bacilli, Klebsiella and unclassified families of Pseudomonadaceae, Entero-
bacteriaceae and Bacillaceae (refer supplementary Table S2). It was

interesting to note that the Klebsiella genus was not captured in the stem and leaf tissues from culturable isolation method (unpublished),

strengthening the use of NGS in this study. It was also noted that 9% of

the microbes were present only in both root and leaf tissues, and 5% of

microbes identified were present only in both root and stem tissues.

Nevertheless, the stem tissues accommodated some genera (6%) such

as Gluconacetobacter and Anoxybacillus which were not detected in

both the root and leaf tissues. This further collaborated the facts that

there are many routes by which these microbes enter into the plant

tissues [28,29]. Conclusively, four prominent phyla were identified to

colonise A. vera plant tissues; Proteobacteria, Firmicutes, Actinobacteria

and Bacteriodetes, which have been shown to produce beneficial bioac-
tive compounds (unpublished).

Kết quả (Việt) 1:

3. thảo luậnNghiên cứu của chúng tôi tiết lộ lần đầu tiên khả năng Illuminaxác định trình tự các giao thức để đánh giá microbiota hiện diện trong mô cây-vi khuẩn endophytes. Trình tự có thể được cải thiện với tốtsự lựa chọn của mồi cặp để khuyếch đại một đoạn dài hơn 16S rRNA gene.Kết quả thực nghiệm của chúng tôi làm nổi bật các tiện ích của nền tảng này cho chính xácvà độ phân giải cao microbiota profiling (N90% loài cấp) endo-cộng đồng phytic, hoặc có lẽ có thể mở rộng tới các mẫu/tài nguyên khác.Cải tiến các công cụ phân tích khác nhau là bằng nhau quan trọngđể giảm thiểu nguy cơ biasness được giới thiệu bởi máy chủ DNA (Lạp lục)và khảm đã được gỡ bỏ mà không ảnh hưởng đến chất lượng tổng thểlần đọc. Mothur đường ống tương đối cung cấp cho chúng tôi một cơ hội tốtđể có hiệu quả xử lý các trình tự đọc trên một nền tảng duy nhất. Điều nàygiảm thiểu mất chất lượng đọc, có thể xảy ra. Việc sử dụng của cuốn tiểu thuyếtshotgun 16S rRNA gene bởi Spincam cũng tiết lộ tổng thể phong phúvà sự đa dạng trong cộng đồng các mô thực vật bao gồm microbiotacả hai dễ và unculturable còn vi khuẩn. CácPhân tích đa dạng α chỉ ra sự phong phú và nghịch đảo Simpson diversi-ty index của cộng đồng vi khuẩn endophyte cho lá gốc vàthân cây mô là 2.221, 6.603 và 1.491 tương ứng. Nó tiếp tục eluci-từ thuộc địa vi khuẩn của các mô thực vật như tiết lộ bởi cácSơ đồ Venn mà minh hoạ việc phân phối các vi khuẩn commu-nities across the tissues and the total shared richness (Fig. 2).The colonisation pattern as illustrated by the Venn diagram of theOTU distribution indicated that 41% of microbes found in the roottissues were also present in all the three tissues, such as Pseudomonas,Bacilli, Klebsiella and unclassified families of Pseudomonadaceae, Entero-bacteriaceae and Bacillaceae (refer supplementary Table S2). It wasinteresting to note that the Klebsiella genus was not captured in the stem and leaf tissues from culturable isolation method (unpublished),strengthening the use of NGS in this study. It was also noted that 9% ofthe microbes were present only in both root and leaf tissues, and 5% ofmicrobes identified were present only in both root and stem tissues.Nevertheless, the stem tissues accommodated some genera (6%) suchas Gluconacetobacter and Anoxybacillus which were not detected inboth the root and leaf tissues. This further collaborated the facts thatthere are many routes by which these microbes enter into the planttissues [28,29]. Conclusively, four prominent phyla were identified tocolonise A. vera plant tissues; Proteobacteria, Firmicutes, Actinobacteriaand Bacteriodetes, which have been shown to produce beneficial bioac-tive compounds (unpublished).