Cisgenics as a next generation GMO crops. This concept is new and alternative to transgenic crops...can avoid fear of transgenics w.r.t health and environment problems.
Cisgenics as a next generation GMO crops. This concept is new and alternative to transgenic crops...can avoid fear of transgenics w.r.t health and environment problems.
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
Marker assisted breeding of biotic stress resistance in Rice Senthil Natesan
A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation
Elucidation of cow tick Rhipicephalus microplus (formerly Boophilus microplus...Innspub Net
Ticks comprise one of the most significant groups of arthropods in terms of effects on animal health. They incapacitate the host by feeding on it. The cattle tick, economically impact cattle industry in tropical and subtropical regions of the world, is a cautiously serious external parasite affecting, primarily, cattle. These ticks are adapted to the advantages of specialising to feed on cattle and with all the feeding stages occurring on one individual host in a rapid sequence of reproduction. Cattle tick’s reproduction and life cycle occurs on body of only one host. This stage takes approximately 21 days, during which the tick changes from a minute larva to a nymph and finally an adult. With the use of a thin-tipped tweezers or forceps with a steady even pressure, ticks were removed straight upward from different body parts of cattle. Ticks were identified to the species level based on their morphologic features under a dissecting microscope and their genus and species were identified under the stereo microscope in the laboratory. Several parameters were taken as to with its life cycle. As observed, the period of tick’s life cycle varies due to some factors. This study aims to elucidate the reproduction process and life cycle of cattle ticks to serve as a guide in controlling and managing these parasitic creatures. Get more articles at: http://www.innspub.net/volume-6-number-4-april-2015-jbes/
B4FA 2012 Tanzania: Plant breeding and GM technology - Chris Leaverb4fa
Presentation at the November 2012 dialogue workshop of the Biosciences for Farming in Africa media fellowship programme in Arusha, Tanzania.
Please see www.b4fa.org for more information
It is a presentation prepared to tell people more about male sterility in brief. I have also included one case study to explain and help you. Hope you like it. Thanks!
brief presentation about the environmental and health issues associated with transgenic crops
or
impact of transgenic crops or GMO crops on environment and health
Inheritance of soybean resistance to soybean rust in Uganda’s soybean germplasmInnspub Net
Understanding the genetic mechanisms of soybean rust resistance is important for effective selection and breeding procedures. This study was hence conducted to determine the combining ability and gene action controlling soybean rust using a 10×10 half diallel mating design. The F2 segregating populations along with their parents were evaluated for rust severity and sporulation level at two reproductive stages (R4 and R6) in screen house and field conditions during the second season of 2016 and first season of 2017 at MUARIK using an alpha lattice design replicated thrice. Significant differences were observed among the parents and F2 generations for both disease severity and sporulation level. General and specific combining abilities were highly significant. The GCA/SCA ratio (1.50-2.30) and the Baker’s ratio (0.75-0.82) showed the predominance of additive gene action in the inheritance of soybean rust resistance. The broad-sense (0.94-0.99) and narrow-sense (0.73-0.82) heritability estimates indicated the possibility of improving resistance to soybean rust through selection in the early generations. UG 5, Maksoy 3N, Maksoy 4N and Maksoy 5N had negative GCA effects. The F2 populations derived from these parents crossed with Wonder soya and Nam 2 had also negative SCA effects. The use of these parents and F2 populations can, therefore, increase the response to selection for improving resistance to soybean rust.
International Winter Wheat Improvement Program: breeding strategies and meth...ICARDA
A. Morgunov (CIMMYT-Turkey)
B. Akin (CIMMYT-Turkey),
Y. Kaya (B. Dagdas International Agric. Research Institute, Turkey)
M. Keser (ICARDA-Turkey)
K. Nazari (ICARDA-Syria)
Z. Mert (Central Field Crop Research Institute, Turkey),
R. Sharma (ICARDA-Uzbekistan)
T. Wuletaw (ICARDA-Syria)
Development of biotic stress resistance technologiesMamtaChoudhary75
This ppt is regarding the topic 'Development of biotic stress resistance technologies'. in it, I've discussed biotic stress, effects on crop production, disease and insect resistance mechanisms, case studies, etc.
Introgression of Plantain Fruit Parthenocarpy (pfp) Genes in Land Races of Pl...Premier Publishers
Trials were conducted at the Teaching and Research Farm of Rivers State University of Science and Technology, Port Harcourt, to investigate the transfer and effects of parthenocarpy gene on ploidy and bunch traits of plantain and banana hybrids using five different banana and plantain clones with each serving differently as a male and female parent that gave a total of 10 crosses. The ten crosses were laid out in Randomized complete block design (RCBD) and replicated three times. The Experimental materials consisted of three plantain cultivarsS: Agbagba, ObinaiEwai and UST Px/02/01; and two banana clones: Calcutta 4 and Yangambi (km5). The potential for indirect marker- assisted election through the utilization of predictive multiple regression equations, heritability (h2) and repeatability (R) in the selection of parthenocarpy progenies was tested. In all viable crosses between ObinaiEwai and Calcutta 4, one of these pfp genes segregates in euploid hybrid progenies resulting in the production of parthenocarpy and non-parthenocarpy progenies. Linear correlation and regression analysis showed that bunch weight, fruit size, bunch hand and fruit number were positively influenced by each ploidy increases and change of recessive to dominant in pfp genes alleles. Although ploidy increase and pfp allele substitution accounted for more than 50 per cent of the Heritability(h) value for the afore-mentioned traits, other genetic factors (pleiotropic) also affect the manifestation of these traits. This implies that ploidy and the effect of allele substitution did not explain all the genetic variation for bunch and fruit traits. This work has proved the use of indirect marker assisted selection which is cheap and reliable for the selection of the F1 2n and 4n parthenocarpy progenies which would be used for the production of superior secondary 3x progenies.
The use of the term cisgenesis is an attempt to distinguish GM plants or other organisms produced in this way from transgenics that is GM plants that contain DNA from unrelated organisms. Schouten et al. (2006) introduced the term cisgenesis and defined cisgenesis as the modification in the genetic background of a recipient plant by a naturally derived gene from a cross compatible species including its introns and its native promoter and terminator flanked in the normal sense orientation. Since cisgenes shared a common gene pool available for traditional breeding the final cisgenic plant should be devoid of any kind of foreign DNA viz., selection markers and vector- backbone sequences. Sometimes the word cisgenesis is also referred to as Agrobacterium-mediated gene transfer from a sexually compatible plant where only the T-DNA borders may be present in the recipient organism after transformation (EFSA, 2012). The cisgenesis precludes linkage drag, and hence, prevents hazards from unidentified hitch hiking genes (Schouten, and Jacobsen, 2008). Compared to transgenesis, one of the disadvantages shared by cisgenesis is that characters outside the sexually compatible gene pool cannot be introduced. Furthermore, development of cisgenic crops involves extraordinary proficiency and time compared to transgenic crops. Therefore, the required genes or fragments of genes may not be readily accessible but have to be isolated from the sexually compatible gene pool (Holme et al., 2013).
On 16 February 2012, European Food Safety Authority (EFSA, 2012) reported the detail study concerning the safety aspects of cisgenic plants and validated that cisgenic plants are secure to be used in terms of environment, food and feed, similar to the traditionally bred plants. However, the present GMO regulation keeps the cisgenic micro-organisms out from its supervision. The first scientific statement of bringing forth a true plant obtained by cisgenic approach was reported in apple through the insertion of the internal scab resistance gene HcrVf2 influenced by their own regulatory genes into the cultivar Gala, a scab susceptible cultivar (Vanblaere et al., 2011). Barley with improved phytase activity was produced successfully by Holme et al. 2011, through cisgenic approach. Late blight resistant potatoes have developed by cisgene stacking of R- gene (jo et al., 2014).
Marker assisted breeding of biotic stress resistance in Rice Senthil Natesan
A marker is a DNA sequence which serves as a signpost/flag post
linked to the trait/gene of interest and is co-inherited along with
the trait
Presence of specific allele of marker = Presence of specific allele of target gene based on the concept the MAS practiced -R.M. Sundaram
Directorate Rice of Research, Hydrabad , July 3rd 2009, CPMB&B, TNAU presentation
Elucidation of cow tick Rhipicephalus microplus (formerly Boophilus microplus...Innspub Net
Ticks comprise one of the most significant groups of arthropods in terms of effects on animal health. They incapacitate the host by feeding on it. The cattle tick, economically impact cattle industry in tropical and subtropical regions of the world, is a cautiously serious external parasite affecting, primarily, cattle. These ticks are adapted to the advantages of specialising to feed on cattle and with all the feeding stages occurring on one individual host in a rapid sequence of reproduction. Cattle tick’s reproduction and life cycle occurs on body of only one host. This stage takes approximately 21 days, during which the tick changes from a minute larva to a nymph and finally an adult. With the use of a thin-tipped tweezers or forceps with a steady even pressure, ticks were removed straight upward from different body parts of cattle. Ticks were identified to the species level based on their morphologic features under a dissecting microscope and their genus and species were identified under the stereo microscope in the laboratory. Several parameters were taken as to with its life cycle. As observed, the period of tick’s life cycle varies due to some factors. This study aims to elucidate the reproduction process and life cycle of cattle ticks to serve as a guide in controlling and managing these parasitic creatures. Get more articles at: http://www.innspub.net/volume-6-number-4-april-2015-jbes/
B4FA 2012 Tanzania: Plant breeding and GM technology - Chris Leaverb4fa
Presentation at the November 2012 dialogue workshop of the Biosciences for Farming in Africa media fellowship programme in Arusha, Tanzania.
Please see www.b4fa.org for more information
It is a presentation prepared to tell people more about male sterility in brief. I have also included one case study to explain and help you. Hope you like it. Thanks!
brief presentation about the environmental and health issues associated with transgenic crops
or
impact of transgenic crops or GMO crops on environment and health
Inheritance of soybean resistance to soybean rust in Uganda’s soybean germplasmInnspub Net
Understanding the genetic mechanisms of soybean rust resistance is important for effective selection and breeding procedures. This study was hence conducted to determine the combining ability and gene action controlling soybean rust using a 10×10 half diallel mating design. The F2 segregating populations along with their parents were evaluated for rust severity and sporulation level at two reproductive stages (R4 and R6) in screen house and field conditions during the second season of 2016 and first season of 2017 at MUARIK using an alpha lattice design replicated thrice. Significant differences were observed among the parents and F2 generations for both disease severity and sporulation level. General and specific combining abilities were highly significant. The GCA/SCA ratio (1.50-2.30) and the Baker’s ratio (0.75-0.82) showed the predominance of additive gene action in the inheritance of soybean rust resistance. The broad-sense (0.94-0.99) and narrow-sense (0.73-0.82) heritability estimates indicated the possibility of improving resistance to soybean rust through selection in the early generations. UG 5, Maksoy 3N, Maksoy 4N and Maksoy 5N had negative GCA effects. The F2 populations derived from these parents crossed with Wonder soya and Nam 2 had also negative SCA effects. The use of these parents and F2 populations can, therefore, increase the response to selection for improving resistance to soybean rust.
International Winter Wheat Improvement Program: breeding strategies and meth...ICARDA
A. Morgunov (CIMMYT-Turkey)
B. Akin (CIMMYT-Turkey),
Y. Kaya (B. Dagdas International Agric. Research Institute, Turkey)
M. Keser (ICARDA-Turkey)
K. Nazari (ICARDA-Syria)
Z. Mert (Central Field Crop Research Institute, Turkey),
R. Sharma (ICARDA-Uzbekistan)
T. Wuletaw (ICARDA-Syria)
Development of biotic stress resistance technologiesMamtaChoudhary75
This ppt is regarding the topic 'Development of biotic stress resistance technologies'. in it, I've discussed biotic stress, effects on crop production, disease and insect resistance mechanisms, case studies, etc.
Introgression of Plantain Fruit Parthenocarpy (pfp) Genes in Land Races of Pl...Premier Publishers
Trials were conducted at the Teaching and Research Farm of Rivers State University of Science and Technology, Port Harcourt, to investigate the transfer and effects of parthenocarpy gene on ploidy and bunch traits of plantain and banana hybrids using five different banana and plantain clones with each serving differently as a male and female parent that gave a total of 10 crosses. The ten crosses were laid out in Randomized complete block design (RCBD) and replicated three times. The Experimental materials consisted of three plantain cultivarsS: Agbagba, ObinaiEwai and UST Px/02/01; and two banana clones: Calcutta 4 and Yangambi (km5). The potential for indirect marker- assisted election through the utilization of predictive multiple regression equations, heritability (h2) and repeatability (R) in the selection of parthenocarpy progenies was tested. In all viable crosses between ObinaiEwai and Calcutta 4, one of these pfp genes segregates in euploid hybrid progenies resulting in the production of parthenocarpy and non-parthenocarpy progenies. Linear correlation and regression analysis showed that bunch weight, fruit size, bunch hand and fruit number were positively influenced by each ploidy increases and change of recessive to dominant in pfp genes alleles. Although ploidy increase and pfp allele substitution accounted for more than 50 per cent of the Heritability(h) value for the afore-mentioned traits, other genetic factors (pleiotropic) also affect the manifestation of these traits. This implies that ploidy and the effect of allele substitution did not explain all the genetic variation for bunch and fruit traits. This work has proved the use of indirect marker assisted selection which is cheap and reliable for the selection of the F1 2n and 4n parthenocarpy progenies which would be used for the production of superior secondary 3x progenies.
Breeding for Development of Climate Resilient Chickpea.pptxKanshouwaModunshim
The breeding for the development of Climate Resilient Chickpea is a critical initiative aimed at enhancing the productivity and adaptability of chickpea genotypes under challenging environmental conditions. Chickpea, a vital pulse crop globally, faces yield limitations due to the combined impact of heat, cold, drought, and salinity stresses. The average yields, currently far below the potential, necessitate the development of highly productive and resilient chickpea cultivars. Traditional breeding methods and modern genomic resources, including molecular markers, genetic maps, and QTL identification, have been instrumental in enhancing grain yields and stress adaptation. Marker-assisted backcrossing has successfully produced cultivars like Pusa Manav, demonstrating the effectiveness of genomic technologies. Additionally, the adoption of gene-editing technologies, such as CRISPR-Cas9, holds promise in accelerating genetic gain for stress-related traits.
CHARACTERIZATION OF STREPTOMYCES SCABIES ISOLATESijabjournal
Potato, (Solanum tuberosum L,) have various biotic constraints in its production due to pest attack. Among these, common scab caused by streptomyces scabies in an important disease in potato which causes economic loss with respect to plant yield and quality of tubers. The present study was conducted to determine the pathogenicity, pathogenic variation, characterization of morphological, physiological and
biochemical aspects of Streptomyces specie associated with potato tubers grown in Rawalpindi district.Severity data and pathogenic variation of disease was studied by using different isolation and characterization techniques. Isolation and characterization of Streptomyces spp. From potato tubers will
guide the researchers about the causative strains of common scab of potato present in the particular area.
The development of more efficient and sustainable technologies in the production of materials in ‘in vitro’ cassava (Manihot esculenta Crantz), favor the improvement of seed quality and sanitation of the plant material. The purpose of the research is to evaluate the effectiveness of Pectimorf® (mixed oligo-galacturonide), it’s safe and natural availability in Cuba. It is used as a possible complement or substitute for growth regulators traditionally used in the culture medium for the propagation of this crop in vitro. In this study, the results obtained indicate that, at least, under the experimental conditions, the Pectimorf®, altered patterns of development and distribution of stomata in the leaves of cassava plants, where the effect was most evident when the product is added to the culture medium. The new results contribute to the elucidation of the mechanisms of action of this substance.
Identification of Ralstonia Solanacearum in Kyrgyzstan’s Potato Fields and th...Agriculture Journal IJOEAR
Abstract— In this study, we have used well-known, efficient methods and bioassay for systematic screening of R. solanacearum for identification of its phenotype and biochemical profile, as well as for pathogenicity and virulence. As a result, an aggressive race — Biovar 3 — was most isolated from the potato fields of the Issyk-Kul region, especially in fields where the Picasso variety was grown. The isolated indigenous strains of Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3 has the potential to be used as a biocontrol agent for the management of the bacterial wilt of potatoes, as indicated by the reduced percentage wilt incidence. Root zone and soil application of Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3 at a dose of 108 cell/ml significantly reduced disease incidence and increased the growth of potato plants. The disease’s progress was reduced by 60% and 56% in plants inoculated with Streptomyces diastatochromogenesstrain sk-6 and Streptomyces bambergiensis strain k1-3, respectively.
Genome wide association studies (GWAS) analysis of karnal bunt resistance in ...Innspub Net
Karnal bunt (KB) disease is one of the most important challenges posed on of wheat (Triticum aestivum L.) industry of Pakistan because of itsinclusionin quarantine list around the globe. This disease is caused by the fungus Tilletia indica M. (Neovossia indica). It affects the grain quality of wheat and hampers its movement in international market resulting in economic losses. Presence of >3% infected grains in wheat lot makes it unsuitable for human consumption. Eradication of this disease is very difficult as no resistant cultivar has been found against KB in Pakistan so far. Genome wide association study (GWAS) was conducted on a set of 199 wheat germplasm collected from Pakistan. In this study 31,000 single nucleotide polymorphism markers were developed by 90K SNP array technology. A linear mixed model in GWAS, accounting for population structure, was fitted to identify significant genomic regions [-log(P) ≥ 4.0] on 6 different chromosomes i.e. 1A, 1D, 2D, 3B, 4A, 5A with novel loci. Candidate genes, through wheat genome assembly, were identified as putative genes related to KB resistance including kinase like protein family. The results of this study can be useful in wheat breeding through marker assisted selection for KB resistant varieties.
Ethical and bio-safety issues related to GM cropsMahammed Faizan
a seminar presentation on ethical and bio-safety issues related GM crops.
impact of gm crops on human, animal and environmental health.
safety measure related transgenic crops.
international governmental bodies
CNR: Sustainable Soil Management to reduce agricultural inputs: What is the r...ExternalEvents
This presentation was presented during Day 3 of the Global Soil Partnership Plenary Assembly – 5th Session that took place at FAO Hq in Rome, Italy, from 20 to 22 June 2017. The presentation was made by Raffaella M. Balestrini
Genetic Variability, Heritability and Genetic Advance of Kabuli Chickpea (Cic...Premier Publishers
The present study was carried out to assess the extent of genetic variability among yield and yield related traits in selected kabuli chickpea genotypes. Forty-nine kabuli chickpea genotypes were studied for thirteen traits at Debre Zeit and Akaki using 7x7 simple lattice design in 2018 cropping season. Combined analysis of variance revealed that there was a significant difference among genotypes for all traits studied, indicating the presence of considerable amount of variability among genotypes. High Phenotypic coefficients of variation and moderate genotypic coefficients of variation value were shown for number of pods per plant and number of seeds per plant, respectively, indicating the possibility of genetic improvement in selection of these traits. High broad sense heritability coupled with high genetic advance were obtained for hundred-seed weight (91.88 and 23.81), number of pods per plant (68.07 and 28.13), number of secondary branches (80.92 and 27.80), number of seeds per plant (67.86 and 31.840), grain yield (62.33 and 24.42) and harvest index (75.70 and 28.17), respectively. This indicates that these characters could be improved easily through selection.
Explore the innovative world of trenchless pipe repair with our comprehensive guide, "The Benefits and Techniques of Trenchless Pipe Repair." This document delves into the modern methods of repairing underground pipes without the need for extensive excavation, highlighting the numerous advantages and the latest techniques used in the industry.
Learn about the cost savings, reduced environmental impact, and minimal disruption associated with trenchless technology. Discover detailed explanations of popular techniques such as pipe bursting, cured-in-place pipe (CIPP) lining, and directional drilling. Understand how these methods can be applied to various types of infrastructure, from residential plumbing to large-scale municipal systems.
Ideal for homeowners, contractors, engineers, and anyone interested in modern plumbing solutions, this guide provides valuable insights into why trenchless pipe repair is becoming the preferred choice for pipe rehabilitation. Stay informed about the latest advancements and best practices in the field.
CFD Simulation of By-pass Flow in a HRSG module by R&R Consult.pptxR&R Consult
CFD analysis is incredibly effective at solving mysteries and improving the performance of complex systems!
Here's a great example: At a large natural gas-fired power plant, where they use waste heat to generate steam and energy, they were puzzled that their boiler wasn't producing as much steam as expected.
R&R and Tetra Engineering Group Inc. were asked to solve the issue with reduced steam production.
An inspection had shown that a significant amount of hot flue gas was bypassing the boiler tubes, where the heat was supposed to be transferred.
R&R Consult conducted a CFD analysis, which revealed that 6.3% of the flue gas was bypassing the boiler tubes without transferring heat. The analysis also showed that the flue gas was instead being directed along the sides of the boiler and between the modules that were supposed to capture the heat. This was the cause of the reduced performance.
Based on our results, Tetra Engineering installed covering plates to reduce the bypass flow. This improved the boiler's performance and increased electricity production.
It is always satisfying when we can help solve complex challenges like this. Do your systems also need a check-up or optimization? Give us a call!
Work done in cooperation with James Malloy and David Moelling from Tetra Engineering.
More examples of our work https://www.r-r-consult.dk/en/cases-en/
Final project report on grocery store management system..pdfKamal Acharya
In today’s fast-changing business environment, it’s extremely important to be able to respond to client needs in the most effective and timely manner. If your customers wish to see your business online and have instant access to your products or services.
Online Grocery Store is an e-commerce website, which retails various grocery products. This project allows viewing various products available enables registered users to purchase desired products instantly using Paytm, UPI payment processor (Instant Pay) and also can place order by using Cash on Delivery (Pay Later) option. This project provides an easy access to Administrators and Managers to view orders placed using Pay Later and Instant Pay options.
In order to develop an e-commerce website, a number of Technologies must be studied and understood. These include multi-tiered architecture, server and client-side scripting techniques, implementation technologies, programming language (such as PHP, HTML, CSS, JavaScript) and MySQL relational databases. This is a project with the objective to develop a basic website where a consumer is provided with a shopping cart website and also to know about the technologies used to develop such a website.
This document will discuss each of the underlying technologies to create and implement an e- commerce website.
Hierarchical Digital Twin of a Naval Power SystemKerry Sado
A hierarchical digital twin of a Naval DC power system has been developed and experimentally verified. Similar to other state-of-the-art digital twins, this technology creates a digital replica of the physical system executed in real-time or faster, which can modify hardware controls. However, its advantage stems from distributing computational efforts by utilizing a hierarchical structure composed of lower-level digital twin blocks and a higher-level system digital twin. Each digital twin block is associated with a physical subsystem of the hardware and communicates with a singular system digital twin, which creates a system-level response. By extracting information from each level of the hierarchy, power system controls of the hardware were reconfigured autonomously. This hierarchical digital twin development offers several advantages over other digital twins, particularly in the field of naval power systems. The hierarchical structure allows for greater computational efficiency and scalability while the ability to autonomously reconfigure hardware controls offers increased flexibility and responsiveness. The hierarchical decomposition and models utilized were well aligned with the physical twin, as indicated by the maximum deviations between the developed digital twin hierarchy and the hardware.
Cosmetic shop management system project report.pdfKamal Acharya
Buying new cosmetic products is difficult. It can even be scary for those who have sensitive skin and are prone to skin trouble. The information needed to alleviate this problem is on the back of each product, but it's thought to interpret those ingredient lists unless you have a background in chemistry.
Instead of buying and hoping for the best, we can use data science to help us predict which products may be good fits for us. It includes various function programs to do the above mentioned tasks.
Data file handling has been effectively used in the program.
The automated cosmetic shop management system should deal with the automation of general workflow and administration process of the shop. The main processes of the system focus on customer's request where the system is able to search the most appropriate products and deliver it to the customers. It should help the employees to quickly identify the list of cosmetic product that have reached the minimum quantity and also keep a track of expired date for each cosmetic product. It should help the employees to find the rack number in which the product is placed.It is also Faster and more efficient way.
1. 1Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
www.nature.com/scientificreports
Transgenic banana plants
expressing Xanthomonas wilt
resistance genes revealed a stable
non-target bacterial colonization
structure
Jean Nimusiima1,2,*
, Martina Köberl3,*,†
, John Baptist Tumuhairwe2
, Jerome Kubiriba1
,
Charles Staver4
&Gabriele Berg3
Africa is among the continents where the battle over genetically modified crops is currently being
played out.The impact ofGM inAfrica could potentially be very positive. In Uganda, researchers
have developed transgenic banana lines resistant to banana Xanthomonas wilt.The transgenic lines
expressing hrap and pflp can provide a timely solution to the pandemic. However, the impact of
the transgenes expression on non-target microorganisms has not yet been investigated.To study
this effect, transgenic and control lines were grown under field conditions and their associated
microbiome was investigated by 16S rRNA gene profiling combining amplicon sequencing and
molecular fingerprinting.Three years after sucker planting, no statistically significant differences
between transgenic lines and their non-modified predecessors were detected for their associated
bacterial communities.The overall gammaproteobacterial rhizosphere microbiome was highly
dominated by Xanthomonadales, while Pseudomonadales and Enterobacteriales were accumulated
in the pseudostem. Shannon indices revealed much higher diversity in the rhizosphere than in the
pseudostem endosphere. However, the expression of the transgenes did not result in changes in the
diversity of Gammaproteobacteria, the closest relatives of the target pathogen. In this field experiment,
the expression of the resistance genes appears to have no consequences for non-target rhizobacteria
and endophytes.
Banana Xanthomonas wilt (BXW) triggered by the plant pathogen known as Xanthomonas campestris pathovar
musacearum is a highly devastating disease in banana production, ranked first in the Great Lakes region of East
and Central Africa1–4
. The economic impact of the banana wilt has been disastrous, because it affects almost all
commonly grown banana cultivars, leading to yield collapse as it continues to spread. Currently, there are no com-
mercial pesticides, biological control agents or resistant banana cultivars available to bring the wilting disease under
control, although rigorous cultural practices have been shown to minimize disease damage5,6
. Due to carryover
of soil-borne inoculum, infested fields cannot be replanted with bananas for at least half a year2
. To address this
problem, Tripathi et al.7
and Namukwaya et al.8
from the International Institute of Tropical Agriculture (IITA) and
the National Agricultural Research Organisation (NARO) in Uganda have developed transgenic banana lines with
resistance to BXW mediated by the constitutive expression of the resistance genes hrap (hypersensitive response
assisting protein) and pflp (plant ferredoxin-like protein), both originating from sweet pepper Capsicum annuum.
These genetically modified banana lines have already proven their enhanced resistance against X. campestris pv.
musacearum under greenhouse7,8
and field conditions6
. Both transgenes are associated with the harpin-elicited
1
National Agricultural Research Organisation, National Agricultural Research Laboratories, Kampala, Uganda.
2
Makerere University, College ofAgricultural and Environmental Sciences, Department ofAgricultural Production,
Kampala, Uganda. 3
Graz University ofTechnology, Institute of Environmental Biotechnology, Austria. 4
Bioversity
International, Montpellier, France. *
These authors contributed equally to this work.†
Present address: Pacific Northwest
National Laboratory, Biological Sciences Division, Richland,WA, USA. Correspondence and requests for materials
should be addressed to M.K. (email: martina.koeberl@tugraz.at)
received: 30 June 2015
accepted: 03 November 2015
Published: 10 December 2015
OPEN
2. www.nature.com/scientificreports/
2Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
hypersensitive response (HR) in plants challenged with Gram-negative pathogens. Hrap intensifies the HR acti-
vation by dissociating harpin multimers into dimers and monomers which triggers a stronger hypersensitive cell
death (HCD) necrosis leading to a systemic acquired resistance (SAR) of the plant9,10
. Overexpression of Pflp leads
to increased production of reactive oxygen species (ROS) in harpin-activated cells and consequently to HCD and
SAR11
. The lack of natural resistance to BXW in any banana cultivar and the difficulties in conventional breeding
with this highly sterile crop favor an effective transgenic approach. However, nothing is known about non-target
effects, especially on Gammaproteobacteria, the closest relatives of the target pathogen.
Globally, transgenic or genetically modified (GM) crops are considered regulated products subject to reg-
ulatory oversight during testing and environmental release. The main concern about GM crops centers on the
lack of studies on the side effects, in terms of adverse impacts on the environment and human health, although
substantial research was performed in Europe12–15
. The controversies surrounding GM crops highlight the need
to establish regulatory frameworks and data of these technologies in Africa16–18
. Despite clear benefits to countries
and farmers who grow GM crops, there is concern about suspected potential risks associated with GMOs19,20
. To
move beyond the 30 years debates on existing GM crops, where they are often discussed in “black and white”, we
need a bigger picture which is “nuanced, equivocal and undeniably messy”, a data-driven debate as well as risk
assessment studies for GM crops also for Africa17
.
In this respect, we analyzed the plant-associated microbiome of transgenic banana lines expressing sweet
pepper hrap and pflp genes in comparison to their not genetically modified predecessors grown under natural soil
conditions in a confined field trial in Uganda. The experiment was a randomized complete block design (RCBD)
with two different breeding lines of the East African Sukari Ndizi (AAB genome). Each breeding line expressing
each of the transgenes was replicated four times and had four non-transgenic control plants (Fig. S1). We mon-
itored the effect of the transgenes on the composition and diversity of the banana-associated microbiome in the
rhizosphere and the pseudostem endosphere, with special focus on the gammaproteobacterial community which
comprises the causal agent of BXW.
Results
Molecular fingerprinting of total bacterial communities. Molecular fingerprinting of the total bac-
terial community associated with genetic-modified banana plants and their non-modified predecessors using
SSCP analysis showed significant differences in their endophytic colonization of the pseudostem between the two
investigated breeding lines (p < 0.001, permutation test) (Fig. 1). High variability was found in the rhizospheric
soil communities within the breeding lines. No significant effect from genetic modification on the banana-associ-
ated bacterial microbiome was found in either the rhizosphere or the endosphere. The rhizosphere soil exhibited
a highly diverse bacterial community composition, while the pseudostem endosphere was characterized by a
much lower abundance of species. Interestingly, different species of the genus Methylobacterium (closest database
matches M. mesophilicum, M. phyllosphaerae, and M. adhaesivum) were found as dominant endophytic banana
colonizers in all investigated pseudostem samples. Furthermore, Bacillus (closest database match B. flexus, 100%
similarity to GenBank accession number NR_024691) and Paenibacillus (closest database match P. barcinonensis,
100% similarity to GenBank accession number NR_042272) were identified as dominant banana endophytes.
Amplicon sequencing-based 16S rRNA gene profiling of the gammaproteobacterial com-
munity. The 796,445 quality sequences with a read length ≥ 200 nucleotides (between 1,594 and 43,012
quality reads per sample), generated through deep sequencing-based analysis, provided detailed insights into
the gammaproteobacterial community composition and diversity. Rarefaction analyses of the normalized
Gammaproteobacteria-specific sequencing data at three different cut-off levels (3%, 5%, and 10% genetic dissimi-
larity), corresponding to the taxonomic levels of species, genera and families (Fig. S2) showed higher numbers of
operational taxonomic units (OTUs) in the rhizosphere than in the endophytic pseudostem tissue. Comparisons
of observed OTUs with their estimated richness by the Chao1 index revealed relatively high coverages for the
individual samples between 58.4 and 100% (S: 58.4–77.1%; P: 72.8–100%) at order level (Table S1). The sequencing
efforts at genus and species level reached 42.5–84.7% (S: 42.5–60.9%; P: 45.1–84.7%) and 30.4–66.1 (S: 34.3–56.7;
P: 30.4–66.1%), respectively. The rhizosphere soil exhibited a significantly higher diversity within the gammaprote-
obacterial community in comparison to the pseudostem of the banana plants (p < 0.001, t-test). Shannon diversity
indices (H’) for the rhizosphere samples ranged from 5.12 to 6.86 at a dissimilarity level of 3%, while values for
endosphere samples were in the range of 1.53 to 3.14 (Table S1). The expression of the transgenes hrap and pflp did
not result in changes in gammaproteobacterial diversity (S: p = 0.525; P: p = 0.979, Tukey post hoc test).
While all quality sequences from the pseudostem could be assigned at least to a gammaproteobacterial fam-
ily, in the rhizosphere soil, on average, 3.4% per sample could not be taxonomically assigned accurately below
the class level, and 5.4% not below the order level (Fig. 2). At genus level, the pseudostem reads which could be
unambiguously affiliated to a gammaproteobacterial genus were much greater than for rhizospheric reads, 64.2%
versus 21.1%. Highest abundances in the rhizosphere soil were found for Xanthomonadales (average 62.3% per
sample), Legionellales (16.3%), Pseudomonadales (6.8%), and Enterobacteriales (3.2%) 16S rRNA gene sequences.
The overall accumulation of Pseudomonadales (54.8%) and Enterobacteriales (44.7%) in the pseudostem endosphere
of the banana plants was notable.
At lower taxonomic levels, Xanthomonadales could be assigned to Sinobacteraceae (genera Steroidobacter,
and Nevskia), and Xanthomonadaceae (genera Dokdonella, Stenotrophomonas, Luteimonas, Arenimonas,
Pseudoxanthomonas, and Rhodanobacter). Pseudomonadales reads could be classified in Moraxellaceae
(Acinetobacter) and Pseudomonadaceae (Pseudomonas). The enterobacterial fraction was dominated by Erwinia,
and the order Legionellales could be divided into the families Legionellaceae (Legionella) and Coxiellaceae
(Rickettsiella). Further genera identified for taxonomic groups with a relative abundance over 1% in any sample
belonged to the Alteromonadales (Cellvibrio, and Marinobacter), exclusively found in some rhizosphere samples.
3. www.nature.com/scientificreports/
3Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
Principal coordinate analysis based on weighted UniFrac distances visualized a clear separation of the two
investigated microenvironments, rhizosphere and endosphere, resulting from the remarkable differences in their
hosted Gammaproteobacteria communities (Fig. 3). The statistical significance was additionally confirmed by
adonis test (p = 0.001). In contrast to the highly similar gammaproteobacterial community profiles found in
pseudostem samples, the rhizosphere samples showed a much broader scattering. Within microenvironments,
no statistically significant differences could be observed between the two individual breeding lines (S: p = 0.249;
P: p = 0.643, adonis test) nor between plants with genetic modifications and their non-modified predecessors
(p > 0.05, adonis test; Table S2).
Figure 1. Comparative analyses of PCR-SSCP profiles of the total bacterial communities in rhizosphere
soil (A) and pseudostem (B) of transgenic and non-transgenic banana plants. Left: Unweighted pair group
method with arithmetic mean (UPGMA) trees. The dendrograms were generated with GelCompar II using
Pearson correlation. Samples were encoded using abbreviations indicating (1) breeding line (1, 2), (2) genetic
modification, if any (1 = hrap, 2 = pflp), and (3) independent replicate sample (1–4). Right: Multidimensional
scaling (MDS) ordination plots based on Pearson similarity matrices. Colors indicate genetic modification and
correspond to squares in the tree, and samples of the two investigated breeding lines are grouped together.
4. www.nature.com/scientificreports/
4Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
Figure 2. Taxonomic composition of the gammaproteobacterial communities inhabiting rhizosphere soil
(S) and pseudostem (P) of banana plants with and without genetic modifications. Sequences obtained by
Illumina MiSeq sequencing were classified at order, familiy and genus level. From each genetic modification,
four independent replicate samples were investigated in comparison to non-modified control plants. Sample
abbreviations indicate: (1) microenvironment (S = rhizosphere soil, P = pseudostem), (2) breeding line (1, 2),
(3) genetic modification, if any (1 = hrap, 2 = pflp), and (4) independent replicate sample (1–4).
5. www.nature.com/scientificreports/
5Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
Discussion
In our risk assessment study for transgenic banana lines resistant to banana Xanthomonas wilt (BXW), we inves-
tigated two microenvironments and found statistically significant differences for the composition and diversity of
rhizosphere and endosphere bacterial communities. This shows that the applied sampling design and the methods
were appropriate to detect statistical differences. The importance of rare taxa for bacterial diversity, shown recently
in the rhizosphere of Bt- and conventional maize varieties15
, was established. Three years after sucker planting, we
found no differences between transgenic lines and their non-modified predecessors, indicating that in our field
experiment the insertion of the BXW resistance genes in the banana genome appears to have no consequences
for non-target rhizobacteria and endophytes of healthy banana plants. Longer-term studies are needed to track
further changes.
The rhizosphere microbiome is mainly a result of microbe attraction by root secretions and other rhizodeposits
released by the plant. These chemical stimulants are highly controlled by the plant genotype21–23
. The presence and
expression of hrap and pflp genes in transgenic banana plants exhibited no impact in this study on composition
and diversity within the bacterial rhizosphere microbiome. This suggests that inserting these genes in banana
lines has not significantly affected chemical functioning of root exudates or that rhizobacteria compensate these
changes. Xanthomonadales was the predominant gammaproteobacterial order in the rhizosphere of transgenic and
non-transgenic banana plants. In contrast in the rhizosphere of dessert bananas investigated in Central America,
where the BXW disease is not present2,3
, Pseudomonadales and Legionellales were observed as the most dominant
gammaproteobacterial orders24
. Several members of Xanthomonadales are known as phytopathogens that cause a
variety of serious diseases in a number of crops, including banana which is threatened by BXW6,25
. In most rhiz-
osphere samples for both the transgenic and non-transgenic lines, the Xanthomonadales family Sinobacteraceae
dominated over Xanthomonadaceae; the genus Xanthomonas was not found in any sample. Instead, Dokdonella
was the most identified genus within the Xanthomonadaceae family, and Steroidobacter within the Sinobacteraceae,
which are both common soil bacteria and not associated with plant pathogenicity. However, only a relatively small
proportion of sequences could be classified down to the genus level, and samples originated only from healthy
banana plants without disease symptoms. Evidence that the community composition of the rhizosphere micro-
biome was not influenced by constitutive expression of transgenic BXW resistance genes indicates that the useful
key contributions of banana root exudates to the rhizosphere ecology were not affected.
In the inner plant tissue, microorganisms are protected against the competitive and sometimes hostile rhizos-
phere environment. In turn, endophytes have often close and advantageous interactions with their host plants26
.
The endosphere of the succulent banana pseudostem has been found to be an extraordinary microenvironment
due to its generally dense bacterial colonization and the presence of a remarkably high number (9.4%) and broad
spectrum of antagonistic strains27
. The dominant genera among the banana endophytes, Methylobacterium,
Bacillus, and Paenibacillus, revealed through molecular fingerprinting have all been previously detected as endo-
phytes of a variety of plants, primarily associated with beneficial plant-microbe interactions. A broad diversity of
Methylobacterium spp. was, for instance, observed for the endosphere of citrus plants, where they were described
as main players in interactions with the phytopathogen Xylella fastidiosa, also belonging to the Xanthomonadaceae
family28,29
. Endophytic Bacillus and Paenibacillus isolates of medicinal plants were identified as being amongst
the most efficient broad-spectrum antagonists against soil-borne plant diseases of Egypt30
. The apparent lack
Figure 3. Principal coordinate analysis (PCoA) plot of the gammaproteobacterial microbiome inhabiting
rhizosphere soil and pseudostem of two different banana breeding lines (1 and 2) expressing different
transgenes (hrap and pflp). PCoA biplots are based on weighted UniFrac distances of gammaproteobacterial
16S rRNA gene amplicon sequencing data jackknife-supported by ten replicates. Statistical comparisons based
on the underlying distance matrices are shown in the supplementary information (Tables S2).
6. www.nature.com/scientificreports/
6Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
of interference of the transgenic banana lines expressing hrap and pflp genes on endophytic interactions is an
important achievement which should be taken into account broadly in breeding programs. Just as with the rhiz-
osphere, the endosphere community remained stable, irrespective of the foreign genes inserted into the banana
genome. While the gammaproteobacterial rhizosphere colonization was highly dominated by Xanthomonadales,
the banana endosphere was almost exclusively inhabited by Pseudomonadales and Enterobacteriales. The clas-
sifiable pseudostem endophytes were assigned to the genera Acinetobacter, Pseudomonas, and Erwinia. All of
them are well-known plant colonizers. However, while Acinetobacter and Pseudomonas are often accountable for
disease-suppressive antagonism, plant growth promotion and stress reduction31,32
, Erwinia has so far mainly been
recognized as a phytopathogen, causing, for instance, the devastating fireblight disease in Rosaceae plants33,34
and
pseudostem wet rot in plantain35
. The high presence in the pseudostem of fecal enterobacteria could be explained by
the manure applications used in the experiment twice a year. Manure applications, widely used in Uganda’s banana
production, affect the native soil and plant-associated microbiome, and a masking of potential transgene effects
cannot be completely excluded under these circumstances. The endophytic gammaproteobacterial colonization
patterns are well in accordance with those found for the Gros Michel banana investigated in Central America24
and
the East African Highland banana of Uganda27
. However, the difference in the rhizosphere, which in this field trial
was so highly inhabited by Xanthomonadales, and the rigorous selection process of enterics and pseudomonads
was even more notable in the present study compared to previous studies.
It is crucial that the release of GM crops does not bring new risks with irretrievable consequences for envi-
ronmental and human health. In our short-term study, we found no detectable impact on the inhabiting bacterial
communities resulting from any genetic modification, by the expression either of the hrap or of the pflp transgene.
Additional studies should address the microbiome stability over a longer time frame, confirm the stability in other
soil types and under different management practices, and investigate the effects in the presence of the disease.
This encouraging result, however, also serves to remind us that the agricultural challenges facing the developing
world are very broad and diverse16
. Investment in soil management to improve soil fertility and resilience through
alternative approaches already established in Africa like agroforestry, intercropping and crop-livestock integra-
tion need to be considered, including their effects on the soil and plant microbiome. Moreover, in the push for
greater productivity often achieved through specialization, we have the opportunity to avoid the loss in diversity of
human gut microbiota found in the developed world compared to the higher diversity found in African people36,37
,
acknowledged to be under the influence of diets and lifestyles.
Methods
Experimental design and sampling. The study was carried out on an on-going confined field trial of
transgenic bananas at the National Agricultural Research Laboratories (NARL) located about 13 km north of
Kampala at an altitude of 1,190 m above sea level. Average annual rainfall is 1,250 mm distributed bimodally, and
the annual temperature is 27.3/15.3 °C (mean maximum/mean minimum) with only 1–2 degrees difference between
coolest and warmest months. The clay soil where the transgenic banana trial was established was slightly acidic
(pH 5.2) with low nutrient content. The cultivar under study was the East African Sukari Ndizi (AAB genome) with
two different breeding lines originating from tissue culture, each expressing vector-inserted hrap (hypersensitive
response assisting protein) and pflp (plant ferredoxin-like protein) genes7,8
. Tissue culture plants were planted at
a spacing of (3 × 3) m. The experiment was a randomized complete block design (RCBD) organized in four block
repetitions with six genotypically different plants per block (Fig. S1). Each breeding line expressing each of the
genes was replicated four times and had four non-transgenic control plants. Guard row plants were planted around
the experimental plot. Cow dung manure at a rate of 10,000 kg ha−1
was applied at planting. Both cow manure and
mulch were applied subsequently twice a year at the same rates as at planting. Banana plant and field management,
de-trashing, de-suckering, de-budding and weeding, were done monthly. The plantation was three years old at
sampling time. From each plant, samples were collected from both the rhizosphere and the pseudostem endosphere
and stored under cooled conditions until workup in the laboratory.
Metagenomic DNA isolation. To isolate total community DNA, 2 g of each rhizosphere soil sample and
15 ml of sterile 0.85% NaCl were mixed for 10 sec on the vortex. For the isolation from the banana endosphere,
5 g of pseudostem were washed with sterile distilled water, transferred to Whirl-Pak bags (Nasco, Fort Atkinson,
WI, USA), and after 10 ml 0.85% NaCl were added, homogenized using mortar and pestle. From the liquid parts,
4 ml were centrifuged at high speed (16,000 × g, 4 °C) for 20 min and resulting pellets were stored at − 70 °C. Total
community DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, Solon, OH, USA) accord-
ing to the manufacturer’s protocol. Metagenomic DNA samples were encoded using abbreviations indicating: (1)
microenvironment (S = rhizosphere soil, P = pseudostem), (2) breeding line (1, 2), (3) genetic modification, if any
(1 = expressing hrap gene [hypersensitive response assisting protein], 2 = expressing pflp gene [plant ferredoxin-like
protein]), and (4) independent replicate sample (1–4).
Fingerprinting of the total bacterial community by single-stranded conformational polymor-
phism analysis of the 16S rRNA genes (PCR-SSCP). Fingerprinting by SSCP analysis was carried out
as described by Schwieger & Tebbe38
. Bacterial 16S rRNA gene sequences were PCR-amplified using the eubac-
terial primer pair Unibac-II-515f and Unibac-II-927rP
. Separation and analysis were performed according to
Köberl et al.39
. Comparisons of generated bacterial community profiles were performed using GelCompar II 5.1
(Applied Maths, Kortrijk, Belgium). Cluster analyses were performed with the following settings: dendrogram
type: unweighted pair group method with arithmetic mean (UPGMA); similarity coefficient: curve based: Pearson
correlation; position tolerances: optimisation: 0.2%, position tolerance: 1%. Multidimensional scaling (MDS)
ordination plots were constructed based on the Pearson similarity matrices. These matrices were additionally
subjected to significance tests of pair-wise similarities by applying permutation analyses (p ≤ 0.01) using the
7. www.nature.com/scientificreports/
7Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
permtest package of R statistics 3.2.0 (The R Foundation for Statistical Computing, Vienna, Austria) with 105
random permutations of sample elements40,41
. Excised and re-amplified DNA fragments were sequenced at LGC
Genomics (Berlin, Germany).
Gammaproteobacterial 16S rRNA gene profiling by Illumina MiSeq Sequencing. In a
deep-sequencing approach, we focused on the colonization by Gammaproteobacteria. The hypervariable V4 region
of the 16S rRNA gene was amplified in a nested PCR approach with the Gammaproteobacteria specific primer pair
Gamma395f/Gamma871r42
and the universal primer pair 515F/806R43
according to Köberl et al.24
. PCR products
of three independent reactions were pooled in equal volumes and purified by employing the Wizard SV Gel and
PCR Clean-Up System (Promega, Madison, WI, USA). Amplicon libraries were generated and sequenced by a
paired-end approach using the Illumina MiSeq platform (LGC Genomics, Berlin, Germany). The nucleotide
sequences are available in the European Nucleotide Archive (www.ebi.ac.uk/ena) under the BioProject accession
number PRJEB9422.
Data analysis was performed by employing the software package QIIME 1.7 and 1.844
. Joined paired-end
reads with more than three consecutive low quality base calls (Phred quality score ≤ 20) were truncated at the
position where their quality began to drop, and only reads with > 75% consecutive high quality base calls, with-
out any ambiguous characters, and longer than 200 nucleotides in length were retained for further analyses. All
quality sequences were adjusted in the same orientation and clustered into operational taxonomic units (OTUs)
with uclust45
, using 3%, 5%, and 10% dissimilarity thresholds. From each OTU the most abundant sequence was
selected as the representative one, and the taxonomy of the representative set was assigned with the uclust-based
consensus taxonomy assigner using an 80% confidence threshold. The representative sequence set was aligned
with PyNAST46
. Chimera check was performed with ChimeraSlayer and potentially chimeric sequences were
discarded. OTU tables at the different dissimilarity levels were constructed, and OTUs not assigned to the class of
Gammaproteobacteria as well as singletons were removed from the dataset. For alpha and beta diversity analyses,
OTU tables were rarefied at 1,590 reads. Diversity indices Shannon47
, Chao148
and observed species were deter-
mined based on the normalized clustering data. Significant differences were calculated with PASW Statistics 18
(SPSS Inc., Chicago, IL, USA) using the independent samples t-test and the Tukey post hoc test. Beta diversity was
analyzed based on weighted UniFrac distances49
and ten jackknife replicates of the total rarefied datasets. Statistical
analyses were performed using the adonis test (p ≤ 0.05) with 999 permutations.
References
1. Aritua, V. et al. Characterization of the Xanthomonas sp. causing wilt of enset and banana and its proposed reclassification as a strain
of X. vasicola. Plant Pathol. 57, 170–177 (2008).
2. Tripathi, L. et al. A threat to banana production in east and central Africa. Plant Dis. 93, 440–451 (2009).
3. Wasukira, A. et al. Genome-wide sequencing reveals two major sub-lineages in the genetically monomorphic pathogen Xanthomonas
campestris pathovar musacearum. Genes 3, 361–377 (2012).
4. Pemsl, D. E. et al. Results of a global online expert survey: major constraints, opportunities and trends for banana production and
marketing and priorities for future RTB banana research. Lima (Peru). CGIAR Research Program on Roots, Tubers and Bananas
(RTB). RTB Working Paper 2014‐2. (2014) Available at: www.rtb.cgiar.org (Accessed: 2nd
June 2015).
5. Biruma, M. et al. Banana Xanthomonas wilt: a review of the disease, management strategies and future research directions. Afr. J.
Biotechnol. 6, 953–962 (2007).
6. Tripathi, L. et al. Field trial of Xanthomonas wilt disease-resistant bananas in East Africa. Nat. Biotechnol. 32, 868–870 (2014).
7. Tripathi, L., Mwaka, H., Tripathi, J. N. & Tushemereirwe, W. K. Expression of sweet pepper Hrap gene in banana enhances resistance
to Xanthomonas campestris pv. musacearum. Mol. Plant Pathol. 11, 721–731 (2010).
8. Namukwaya, B. et al. Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease. Transgenic
Res. 21, 855–865 (2012).
9. Ger, M. J. et al. Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial
pathogens by induction of a hypersensitive response. Mol. Plant Microbe Interact. 15, 764–773 (2002).
10. Pandey, A. K. et al. Expression of the hypersensitive response-assisting protein in Arabidopsis results in harpin-dependent
hypersensitive cell death in response to Erwinia carotovora. Plant Mol. Biol. 59, 771–780 (2002).
11. Huang, H. E. et al. A hypersensitive response was induced by virulent bacteria in transgenic tobacco plants overexpressing a plant
ferredoxin-like protein (PFLP). Physiol. Mol. Plant Pathol. 64, 103–110 (2004).
12. Lottmann, J., Heuer, H., Smalla, K. & Berg, G. Influence of transgenic T4-lysozyme-producing potato plants on potentially beneficial
plant-associated bacteria. FEMS Microbiol. Ecol. 29, 365–377 (1999).
13. Heuer, H., Kroppenstedt, R. M., Lottmann, J., Berg, G. & Smalla, K. Effects of T4 lysozyme release from transgenic potato roots on
bacterial rhizosphere communities are negligible relative to natural factors. Appl. Environ. Microbiol. 68, 1325–1335 (2002).
14. Germaine, K. et al. Colonisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiol. Ecol. 48, 109–118 (2004).
15. Dohrmann, A. B. et al. Importance of rare taxa for bacterial diversity in the rhizosphere of Bt- and conventional maize varieties. ISME
J. 7, 37–49 (2013).
16. Okeno, J. A., Wolt, J. D., Misra, M. K. & Rodriguez, L. Africa’s inevitable walk to genetically modified (GM) crops: opportunities and
challenges for commercialization. N. Biotechnol. 30, 124–130 (2013).
17. Whitty, C. J., Jones, M., Tollervey, A. & Wheeler, T. Biotechnology: Africa and Asia need a rational debate on GM crops. Nature 497,
31–33 (2013).
18. Nang’ayo, F., Simiyu-Wafukho, S. & Oikeh, S. O. Regulatory challenges for GM crops in developing economies: the African experience.
Transgenic Res. 23, 1049–1055 (2014).
19. Adenle, A. A. Response to issues on GM agriculture in Africa: are transgenic crops safe? BMC Res. Notes 4, 388 (2011).
20. Kikulwe, E. M., Wesseler, J. & Falck-Zepeda, J. Attitudes, perceptions, and trust. Insights from a consumer survey regarding genetically
modified banana in Uganda. Appetite 57, 401–413 (2011).
21. Walker, T. S., Bais, H. P., Grotewold, E. & Vivanco, J. M. Root exudation and rhizosphere biology. Plant Physiol. 132, 44–51 (2003).
22. Bais, H. P., Weir, T. L., Perry, L. G., Gilroy, S. & Vivanco, J. M. The role of root exudates in rhizosphere interactions with plants and
other organisms. Annu. Rev. Plant Biol. 57, 233–266 (2006).
23. Berg, G. & Smalla, K. Plant species and soil type cooperatively shape the structure and function of microbial communities in the
rhizosphere. FEMS Microbiol. Ecol. 68, 1–13 (2009).
24. Köberl, M., Dita, M., Martinuz, A., Staver, C. & Berg, G. Agroforestry leads to shifts within the gammaproteobacterial microbiome
of banana plants cultivated in Central America. Front. Microbiol. 6, 91 (2015).
25. Tushemereirwe, W. K. et al. Status of banana bacterial wilt in Uganda. Afr. Crop Sci. J. 14, 73–82 (2006).
8. www.nature.com/scientificreports/
8Scientific Reports | 5:18078 | DOI: 10.1038/srep18078
26. Ryan, R. P., Germaine, K., Franks, A., Ryan, D. J. & Dowling, D. N. Bacterial endophytes: recent developments and applications. FEMS
Microbiol. Lett. 278, 1–9 (2008).
27. Rossmann, B. et al. Banana-associated microbial communities in Uganda are highly diverse but dominated by Enterobacteriaceae.
Appl. Environ. Microbiol. 78, 4933–4941 (2012).
28. Araújo, W. L. et al. Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants. Appl.
Environ. Microbiol. 68, 4906–4914 (2002).
29. Lacava, P. T., Araújo, W. L., Marcon, J., Maccheroni, W. Jr. & Azevedo, J. L. Interaction between endophytic bacteria from citrus plants
and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis. Lett. Appl. Microbiol. 39, 55–59 (2004).
30. Köberl, M. et al. Bacillus and Streptomyces were selected as broad-spectrum antagonists against soilborne pathogens from arid areas
in Egypt. FEMS Microbiol. Lett. 342, 168–178 (2013).
31. Weller, D. M. Pseudomonas biocontrol agents of soilborne pathogens: looking back over 30 years. Phytopathology 97, 250–256 (2007).
32. Sarode, P. D., Rane, M. R., Chaudhari, B. L. & Chincholkar, S. B. Siderophoregenic Acinetobacter calcoaceticus isolated from wheat
rhizosphere with strong PGPR activity. Malays. J. Microbiol. 5, 6–12 (2009).
33. Miller, P. W. (1928) A preliminary report on studies of fireblight of apple. Science 68, 386–388.
34. Eastgate, J. A. Erwinia amylovora: the molecular basis of fireblight disease. Mol. Plant Pathol. 1, 325–329 (2000).
35. CABI, CAB Thesaurus 2014. (2014) Available at: http://www.cabi.org/cabthesaurus/mtwdk.exe?k= default&l
= 60&w= 11023&n= 1&s= 5&t= 2 (Accessed: 2nd
June 2015).
36. Schnorr, S. L. et al. Gut microbiome of the Hadza hunter-gatherers. Nat. Commun. 5, 3654 (2014).
37. O’Keefe, S. J. et al. Fat, fibre and cancer risk in African Americans and rural Africans. Nat. Commun. 6, 6342 (2015).
38. Schwieger, F. & Tebbe, C. C. A new approach to utilize PCR-single-strand-conformation polymorphism for 16S rRNA gene-based
microbial community analysis. Appl. Environ. Microbiol. 64, 4870–4876 (1998).
39. Köberl, M., Müller, H., Ramadan, E. M. & Berg, G. Desert farming benefits from microbial potential in arid soils and promotes
diversity and plant health. PLoS One 6, e24452 (2011).
40. Kropf, S., Heuer, H., Grüning, M. & Smalla, K. Significance test for comparing complex microbial community fingerprints using
pairwise similarity measures. J. Microbiol. Methods 57, 187–195 (2004).
41. R Development Core Team, R: a language and environment for statistical computing. R Foundation for Statistical Computing. (2011)
Available at: www.r-project.org (Accessed: 2nd
June 2015).
42. Mühling, M., Woolven-Allen, J., Murrell, J. C. & Joint, I. Improved group-specific PCR primers for denaturing gradient gel
electrophoresis analysis of the genetic diversity of complex microbial communities. ISME J. 2, 379–392 (2008).
43. Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. USA
108 Suppl 1, 4516–4522 (2011).
44. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).
45. Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).
46. Caporaso, J. G. et al. PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26, 266–267 (2010).
47. Shannon, C. E. The mathematical theory of communication. 1963. MD Comput. 14, 306–317 (1997).
48. Chao, A. & Bunge, J. Estimating the number of species in a stochastic abundance model. Biometrics 58, 531–539 (2002).
49. Lozupone, C. A., Hamady, M., Kelley, S. T. & Knight, R. Quantitative and qualitative beta diversity measures lead to different insights
into factors that structure microbial communities. Appl. Environ. Microbiol. 73, 1576–1585 (2007).
Acknowledgements
We thank the National Banana Research Program and the International Institute of Tropical Agriculture (Uganda)
for conducting the confined field trial. This study was supported by the Federal Ministry for Europe, Integration
and Foreign Affairs (BMEIA) of the Republic of Austria through the Austrian Development Agency (ADA).
AuthorContributions
Conceived and designed the experiments: G.B., C.S., J.K. and J.B.T. Performed the experiments: J.N. and M.K.
Analyzed the data: J.N., M.K. and G.B. Contributed reagents/materials/analysis tools: C.S. and G.B. Wrote the
paper: J.N., M.K. and G.B.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Nimusiima, J. et al. Transgenic banana plants expressing Xanthomonas wilt resistance
genes revealed a stable non-target bacterial colonization structure. Sci. Rep. 5, 18078; doi: 10.1038/srep18078
(2015).
This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/