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Dr. Ferre is a Utah native who completed his undergraduate work at the University of Utah. In 2003, he graduated from the Medical College of Wisconsin. He completed his residency in emergency medicine at Maine Medical Center, where he received the award for best resident teacher and the MMCRI best annual research presentation. Following residency, Dr. Ferre served 3 years on active duty for the United States Air Force at Wilford Hall Medical Center where he was the Emergency Ultrasound Director for the SAUSHEC Emergency Medicine Residency program. During his 3 years of active duty service he completed 2 tours in Iraq with the 332 EMDG in support of Operation Iraqi Freedom.
Following his active duty commitment to the Air Force, Dr. Ferre completed an Emergency Ultrasound Fellowship at Palmetto Health Richland in Columbia South Carolina where he received the SAEM award for best fellow research presentation.
Dr. Ferre spent 9 years at Vanderbilt University Medical Center where he founded the Emergency Ultrasound Fellowship and the Emergency Ultrasound Division. He moved to Indiana in 2019, where he currently serves as the Chief of the Point of Care Ultrasound Division of the Department of Emergency Medicine. He also directs the IUSM Point of Care Ultrasound Initiative where we has the privilege of working with talented and dedicated educators.
An avid teacher of emergency ultrasound, Dr. Ferre has trained thousands of physicians of all different specialties and medical students throughout the United States, Guyana, Nepal and Iraq. When not working, Dr. Ferre enjoys spending time with his wife and 4 children doing just about anything, but especially kayaking, fishing and hiking.
Titles & Appointments
- Associate Professor of Clinical Emergency Medicine
- Chief, Point of Care Ultrasound Division
- Director, IUSM Point of Care Ultrasound Initiative
Professional Organizations
Board Certifications


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JACKSONVILLE – It's loud outside, louder than it has been in three years.
But when it comes to Blake Bortles and his teammates' belief in him, Allen Hurns said outside means nothing. Noise means nothing. Criticism, analysis, Twitter? Nothing. Nothing. Nothing.
What means something is what happens inside. Actually, as Hurns sees it, what happens inside means everything, which is why the third-year receiver made sure the Jaguars' starting quarterback – his starting quarterback – knew what was going inside the Jaguars' locker room this week.
"We're all in this together," Hurns said as the Jaguars (0-3) prepared to play the Indianapolis Colts (1-2) at Wembley Stadium in London, England, Sunday at 9:30 a.m.
Yes, outside there is noise surrounding Bortles. He is enduring more outside criticism than at any other time in his 32 games as an NFL starter. That's the nature of the NFL quarterback position, particularly when the player playing that position has committed six turnovers in two games.
That noise is real.
But Hurns and other Jaguars players said what's going in inside is realer – and that the belief in Bortles remains strong. Hurns said he has told Bortles as much this week in the wake of a 19-17 loss to the Ravens in which two of Bortles' three interceptions came in the fourth quarter.
"That's the guy," Hurns said of Bortles. "We can relate. It's unique because we all came in at the same time. No matter what happens throughout our career, we're always going to relate to each other. I know he was disappointed [after Baltimore] and you should be disappointed. We were all disappointed after that loss. It was very frustrating. But I was just letting him know that we're here with him.
"No matter what happens and what people talk about, we are what matters. We're all we've got."
Hurns said in no way is Bortles' solely responsible for the Jaguars' early offensive struggles.
"Fans are frustrated, but sometimes people don't understand things that are going on on the field," Hurns said. "There are tipped passes, or it could be an interception where people say it's a silly throw, but maybe I didn't get my depth on the route. There are situations like that.
"He already knows that no matter what I have his back. I'm going to ride for him."
Hurns was far from alone this week. Throughout the locker room, teammates expressed support for Bortles.
"We all have total trust in him," left guard Luke Joeckel said.
Wide receiver Allen Robinson said there's a reason teammates believe so strongly in their quarterback – and that reason goes beyond the common bond he, Hurns, receiver Marqise Lee and Bortles share from entering the league in the same year, 2014.
Robinson said Bortles has earned the belief not only on the field but in the locker room, in meeting rooms, in his daily approach.
"He doesn't focus on the outside noise too much," Robinson said. "He knows he has a job that he has to go out there and do. He has to go out there and lead and at the end of the day that's what he's trying to do. He's not about what people say. That's why he's so good mentally. He knows his task at hand."
And the criticism? How does Bortles respond to that?
"He doesn't too flustered or let it influence what he does," Robinson said. "He doesn't let it impact things he does. You see guys across the league start to point blame on other people. He takes responsibility for his action and actions of others."
Robinson said that's how Bortles has been since arriving with the Jaguars, and since Day One Bortles indeed has handled the quarterback profession with a maturity that believes years and experience.
That remained true this week. With the preseason storyline of "Bortles as Rising Star" suddenly – and prematurely – transformed into "What's Wrong with Blake," Bortles handled his Wednesday media availability as he always does. With candidness, class and ease. He said indeed he and the offense have been in a funk. He answered questions about body language and mechanics for the umpteenth and umpteenth-plus-one time, respectively, and said the issues that have plagued him and the offense so far this season are absolutely correctable.
What matters, of course, isn't what Bortles says. It's whether or not Bortles plays better on Sunday than in the first three games of the season. The Jaguars can't win if Bortles plays as he has the last two weeks, and if he continues in this vein, all sorts of questions will continue about whether or not he's the franchise quarterback the Jaguars oh-so-need him to be.
That's a legitimate question right now, one that only will be answered in time. But there's no question within the Jaguars' locker room.
Because whatever the outside noise, inside Jaguars players are riding with the guy who has earned that trust on the field and off.

subset: commoncrawl


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Congratulations to Nicola Challis of That Lovely Stuff who has been awarded Plastic Free Champion Status!
Nicola stocks a wide range of beautiful ethical and hand made products. Most products are either not wrapped in plastic or are wrapped in plant-based alternatives, for example, all hampers and candles. Nicola explained that the locally sourced hand made products she sells tend to be environmentally conscious in terms of the packaging materials used compared to mass-market products. House of Bluebell is an example of a supplier which is very environmentally conscious in terms of packaging, and many others are also moving to or already using environmentally friendly options.
Nicola is in communication with suppliers encouraging them to take the issue of single-use plastic seriously and recently refused to stock Christmas wrapping paper that was unrecyclable. She also buys Christmas Crackers in bulk and sells them individually, reducing the need for packaging. She offers paper, not plastic bags, although most customers now bring their own reusable bags. Nicola is bringing out a range of candles that will be in a glass jar with a wooden top - these beautiful items will need no wrapping. On top of this, she has either persuaded her other candle suppliers to stop using single-use plastic wrapping or is stopping stocking those items.
Well done Nicola!


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Upsolver components
Upsolver architecture and its components.
API Server
The API Server is responsible for user interaction. Every time a user triggers an action that requires a response, the request interacts with the API Server.
Upsolver fully managed architecture does not require a dedicated API Servers. It utilizes the Global API Server to interact with Upsolver.
Private VPC deployment architecture requires access to the data in order to display information in the UI. The API Server is required to be deployed in the user's private VPC. The users will need access to the API Server to access the website. The API server will typically have the HTTPS port open for the users to access through a component such as VPN.
Compute Cluster
Upsolver Compute Cluster is a group of EC2 instances that are responsible for data processing. These servers are the compute power for transforming, aggregating, and enriching the data in Upsolver. They don't interact with outside processes. The instances pull work and process the data, then write the data to S3.
Query Cluster
Query cluster is for users that want to populate Lookup Tables for dashboards or applications. It allows Lookup Tables to be loaded into memory and waits for user requests. The Query Cluster is an optional component. It is not a requirement for Upsolver. If the Lookup Tables are used for joins between data streams, a Query Cluster is not required because Upsolver manages the Lookup Tables automatically. A Query Cluster is only required when a user wants to manually interact with Lookup Tables.
Kinesis Stream
Upsolver utilizes the Kinesis Stream to allow servers to communicate with each other. Rather than communicating with each other directly, all servers report information to Kinesis, each server will pull data from Kinesis to discover information about other servers.
All servers will send and pull information to the Kinesis Stream to communicate state and synchronize the work between the servers that are running in the account.
Upsolver fully managed architecture deployed in Upsolver's environment
Private VPC deployment architecture deployed in the user's AWS account
Metadata store
The Metadata Store is a global component for Upsolver fully managed deployment model and Private VPC deployment model. It's a centralized space to store the configurations. If a user creates an object in Upsolver, the definitions will always be stored in the global Metadata Store. The Metadata Store is a Key-Value store that the clients communicate using the API Server. Clients don't interact with the Metadata Store directly. Outgoing traffic will go through the API server, the purpose of it is to store and request information from the KV store. The same entities are reflected on S3 in the user's account to provide durability. The production servers will only pull data from S3. In the unlikely event of the global environment being unavailable, it will not impact any data being processed.
The servers report to several Kinesis streams including status of the tasks and operational metrics such as CPU and memory. It keeps track of servers' health and the information is used to replace servers if necessary. Servers report some of the metrics directly to the user's CloudWatch environment, which is used for scaling and auto-healing of the cluster. The metrics are also used for spinning up and down servers. Servers report additional metrics to Upsolver managed Kinesis streams such as billing information and telemetry data. The data being reported directly to CloudWatch is operational data about the servers.
Logs and environment
By default, logs are sent to an Upsolver bucket. Application logs are sent to a centralized location for easy debugging. Optionally, the users can choose to send the logs to their own dedicated bucket if direct access to the logs is desired.
The Upsolver environment pulls data from various locations. Environment configurations including geo IP map, user agent map files are pulled from global configurations as well as static initialization files. They're pulled from various buckets on Amazon S3. During the initialization phase, the servers also install components such as Java and Docker containers pulled from Upsolver's dockerhub repository. Servers also report to the monitoring infrastructure which is InfluxDB.
Upsolver's web interface is hosted on a CDN out of an Amazon S3 bucket. The web interface accesses the private API directly to populate the entities.
Last updated


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Homepage Development for DMC & incoming Agencies
Today a homepage is an essential tool to be successful in business.
Forward Destinations would be delighted to develop your company's homepage for Germany, Austria and Switzerland. Your homepage would not only deliver high qualified sales leads but does also support your online performance in your home country and its markets.
But Forward Destinations goes one step further. Being our partner, we will develop a "Micro-Site" within our homepage and we can create a social media platform in our language for the digital promotion of your products and services. This will engage consumers & travel agents in communication and generate relevant sales leads and messages.
Top rankings in the major search engines is a "must have" for us.
Online Marketing
Once your singular homepage for the German spaking markets has been established, our goal is to generate maximum traffic.
Forward Destinations will use all available online tools to position your dedicated German speaking homepage on top of the relevant search engines.
If you want to promote your product or your destination online, we`re able to arrange a Webinar for you.
In addition, Forward Destinations is able to offer a dedicated web-based platform to promote your products and generate incremental revenue with our sales & marketing tool within the travel trade, "Tours2go".


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Advent of digital payment and increasing popularity of buy-now-pay-later (BNPL) model in India has stimulated series of initiatives to introduce relevant services and strengthen portfolio.
Payments and merchant solutions platform, Mosambee has reportedly announced 100% strategic acquisition of Benow, a EMI technology-based payments platform owned by Cashless Technologies under a cash and stock deal.
Under the terms of acquisition, Mosambee will aid Benow in enhancing its product portfolio and help focus on robust growth.
In exchange, the platform will use Benow's solid partner network encompassing more than 35 brands and 15 leaders to solidify its presence throughout the country under the leadership of the current CEO, Soorraj VS.
For last 2-3 years, the company has been focused on growth of its digital-first as a part of transaction lending solutions, offering facilitated via brand-sponsored EMI transactions.
In the past year, Benow has successfully developed a solid ecosystem of brand partnerships and has emerged as the second biggest and fastest growing offline BNPL service provider in the country.
Speaking on the recent takeover, Sameer Chugh, Co-founder and Director of Mosambee, mentioned that Benow's product offering blends seamlessly with Mosambee's vision of solutions catering to the needs of small retails to support them compete with the online giants.
Mr. Chugh appreciated Benow's drive for BNPL ecosystem and claims to have build a habitable environment for the EMI platform to work as a startup within a startup.
Soorraj VS, Managing Director and CEO of Benow, commented that the platform has been committed to the development of MSMEs by leveraging an innovative digital solution and believes the acquisition will allow merchants with the apt tools to enhance business performance.
In a span of eight years, Mosambee has scarcely raised some $2.0 million and generated a revenue of more than $53.5 million, reflecting profits for last 5 years.
Last fiscal year, the company reported 25% growth and aims for additional 50% growth this fiscal year.
Source Credit: https://www.thehindubusinessline.com/companies/mosambee-acquires-bnpl-focussed-start-up-benow/article65070988.ece


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$ 75.00
The Sleek Dash Lariat has become a must have in my line.
Made with super high polished silver chain. The dash style links are so shiny, they almost look like little mirrors!
It's straight lines of this piece make any look instantly a bit more sexy and edgy.
Wear it alone or stack with other pieces form the collection and create your own look.
All handmade and original designs by me.
Made to sit at the base of a 13 inch neck but can be customized to fit you perfectly if needed.
If the custom option is chosen, please state the size of your neck in inches in the notes section at checkout.
HRH logo tag not included.


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The best removals to Amsterdam services offer customs clearance
Because the UK's break up from your European Union in January 2020, shifting treatments are becoming more difficult. Earlier, prior to Brexit, any citizen of the participant nations could move freely without the need of going through very complicated customs procedures. Now, this all is passed on. Shifting from London to Madrid entails far more forms than simply the most educated can handle.
While not all is shed, removals on the Netherlands, like other places, have set up in their transferring programs and packages the digesting of your required permits for your shift in their merchandise from England or any other Commonwealth country to European territory.
These types of services include the correct wrapping according to the degree of fragility of your respective belongings, and also insurance policy that will safeguard you and also cover any harm or crash that takes place throughout the relocate. Though removals to Amsterdam businesses method this particular records, you must not wait a few days before your relocate to think about doing the formalities essental to customs. For this reason it is most a good idea that, if you already possess a date set up to get started on your moving method, make contact with the corporation of your liking and allow the pros take care of the issue. Give all of the wanted information, and you will definitely surely not have any inconvenience throughout your vacation.
removals to Amsterdam organizations provide you with the most comprehensive services available on the market. All of your offers contains the loading and preparing of your own belongings, which must be done in ways that can guarantee the integrity of your respective products. Usually do not take too lightly the assistance of specialists in this connection. Any blunder could cause some regrettable damage.
The assistance will even let you keep an eye on your valuables during the entire whole trip. Each one of the transfer cars comes with a GPS that allows knowing its location and option anytime. The device songs your items round the clock, provided that needed until you get to your location. The removals to Netherlands support also permit you to shop your items inside a risk-free location if possible. Whenever you retain the services of the company's providers, you can also purchase insurance in your stuff, which safeguards from any damage, burglary, or other scenario that triggers damage to your house.


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1 in 5 Americans have tried or are currently using mHealth apps and fitness trackers. At least 5% of adults in the US regularly use a health tracking application to track their medication and illness. These statistics are phenomenally significant.
The astronomical growth of mHealth apps can be attributed to the increase in smartphone usage, internet and social media availability, need for remote monitoring of health, rising demand for better healthcare solutions, and technological advancements in the global healthcare system.
So, what difference does a health tracking application make?
Eventually, it can vastly reduce healthcare costs for patients and providers since we are focusing on preventive methods to stay healthy.
Even with a large market, there were hardly any apps that offered women-centric services. With half the world's population as the target market, the FemTech segment has finally started to grow.
According to Vision Research Reports, the women's health app market size is predicted to reach $4.9 billion by 2028 globally.
FemTech is using digital technology to develop software, applications, products or services that cater to women's health. Although a large chunk of the health apps in the market right now cater to period or pregnancy tracking, FemTech covers many more subjects in women's health.
Why is it important to develop women's Health Tracking Apps?
The women's health market has never been looked at as a niche market that needs catering to. With the 'she-economy' growing expansively, the idea of having a specific health tracking application catering to the needs of women has gained attention.
It is important for women to have greater control over their health. These apps will help women track their periods, understand their body health, predict ovulation, anticipate the need for birth control pills and more.
It will help women understand their overall health and bodies better, reduce unwanted pregnancies and help deal with medical issues.
These apps also provide a scientific understanding of health conditions, timely diagnosis and treatment. For instance, if women track their periods regularly, they would detect any abnormality in their cycles. You can use this timely information to seek immediate medical attention.
One of the significant advantages of FemTech apps is that it ensures health tracking is available to women living in remote locations. Besides, some places consider women's health issues as taboo topics. Women –from remote areas – can also seek medical help and advice on topics such as unwanted pregnancies, infertility, birth control, menstrual health, and sexual health.
Read more : 8 Key Advantages of a Home Workout App
Developing Women's Health Tracking Apps
Before you give the go-ahead for health tracking app development, understand the target market. Design an app to satisfy a specific need, and understand which need-gap your app will fill. Then, identify your resources, functions of the app and data compliance. Pay special attention to security, as the information gathered is sensitive.
Lets' look at some key features a good women health tracking app should have:
1. Onboarding or Inboarding
If you want women to start using your app, you should make it convenient and practical. To get users onboard or sign up for your app, try the time-tested email notification and password approach method. These days, fitness tracking application development companies are also using social media integration for onboarding. Inboarding is when users discover the app while actually using it.
2. Complete Health Profile
If you can get users to sign up and register, then it's great. Still, if you don't want to scare your new users with a lengthy registration process, allow them to skip it. Either way, the next step in the process should be gathering important specific information.
It is crucial to pay attention here – gather only specific information that will allow you to offer personalized content. Since users divulge highly private information, any hint that you might be asking additional questions will surely scare them away.
3. Functionality
Choose a functionality that has a broader need and market. You don't have to be confined to designing only a basic fitness tracking application. You should create an app that tracks women's health during the cycle to evaluate the changes and offer constructive analysis.
Provide current-day in the cycle information, ovulation period, approximate date for next period, fertility and more.
Also, make sure your app is intuitive in nature and that it doesn't overburden users with constant questions.
4. Symptoms Tracking
Include an additional feature of symptom tracking in the app where users can monitor their symptoms, detect patterns and identify irregularities to provide accurate analysis.
Many health tracking app development companies are using AI technology and machine learning to collate large amounts of shared user data to generate customized reports for each user.
Besides this, adding a wearable feature in the app helps collect additional data – blood pressure, sleep cycle and heart rate, among others.
5. Notification
Having pop-up notifications is a great way of reminding users to log in entries, consume medications on time and get intimation of the next cycle. Notifications also help you to stay engaged with users.
You should maintain a fine balance in having the correct number of notifications/reminders on the app. One too many and users can find the app intruding.
It would help if you tried to build in algorithms that constantly use the information to analyze, evaluate and identify health concerns.
6. Information Sharing
Information sharing is a two-pronged feature. Many apps allow users to share information about their health with their partners and doctors. This makes it easier for them to start conversations about topics they are uncomfortable with.
It is important (from a mental health perspective) to have a social network to connect and share personal health issues. Considering this, health tracking app development companies try to have in-built social groups where users can share their health concerns and knowledge.
7. Consultation
To make the fitness tracking application more meaningful and reliable, it is important to provide a digital consultation facility. While it is great to get instant information via an app, sometimes women find it easier to talk to a real specialist.
Apps that provide online consultations, medical advice from specialists, and help with medical prescriptions through calls or messages can have a winning edge over other healthcare apps.
Wrapping Up:
FemTech is no longer just a growing trend. It has become a necessity to have applications that specifically cater to women's health. Certain health conditions can be prevented with early intervention and screening. The advancement of mHealth apps designed to meet women's health needs goes a long way in ensuring fewer hospital visits and a better understanding of personal health.


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- Soil Microbiology Lab, Department of Soil Science, "Luiz de Queiroz" College of Agriculture, University of São Paulo, Piracicaba, Brazil
The rhizosphere harbors one of the most complex, diverse, and active plant-associated microbial communities. This community can be recruited by the plant host to either supply it with nutrients or to help in the survival under stressful conditions. Although selection for the rhizosphere community is evident, the specific bacterial traits that make them able to colonize this environment are still poorly understood. Thus, here we used a combination of community level physiological profile (CLPP) analysis and 16S rRNA gene quantification and sequencing (coupled with in silico analysis and metagenome prediction), to get insights on bacterial features and processes involved in rhizosphere colonization of sugarcane. CLPP revealed a higher metabolic activity in the rhizosphere compared to bulk soil, and suggested that D-galacturonic acid plays a role in bacterial selection by the plant roots (supported by results of metagenome prediction). Quantification of the 16S rRNA gene confirmed the higher abundance of bacteria in the rhizosphere. Sequence analysis showed that of the 252 classified families sampled, 24 were significantly more abundant in the bulk soil and 29 were more abundant in the rhizosphere. Furthermore, metagenomes predicted from the 16S rRNA gene sequences revealed a significant higher abundance of predicted genes associated with biofilm formation and with horizontal gene transfer (HGT) processes. In sum, this study identified major bacterial groups and their potential abilities to occupy the sugarcane rhizosphere, and indicated that polygalacturonase activity and HGT events may be important features for rhizosphere colonization.
Of the various niches that comprise the plant microbiome – i.e., phyllosphere, spermosphere, rhizosphere, internal tissues (Turner et al., 2013; Andreote et al., 2014)- the most complex, diverse, and active microbial community is located in the rhizosphere, the soil layer immediately influenced by the rhizodeposition (Prashar et al., 2014). In the rhizosphere, there is a selection effect imposed by the physicochemical changes created in the roots' surroundings that shape the microbial composition. This selection can happen both indirectly, favoring the growth of opportunistic microbes adapted to specific chemical conditions, or by the active recruitment of microbes that will support plant development, support plant growth/nutrition, and/or enhancing the plant's ability to resist biotic and abiotic stressors (Bulgarelli et al., 2013; Philippot et al., 2013; Mendes R. et al., 2014). These microbial partners are understood to receive labile forms of carbon and a continuing supply of nutrients from the plants in exchange to their services (Bais et al., 2006).
Bulk soil, i.e., the zone of soil not under the influence of rhizodeposition, houses a huge microbial diversity and is considered the ultimate reservoir of microbes available for plant colonization, from which microbes are selected to the rhizosphere microbiome (Uroz et al., 2010; Peiffer et al., 2013; Mendes L.W. et al., 2014). However, the set of features required for bacteria to efficiently colonize the rhizosphere is yet to be properly described. For instance, studies have shown the importance of motility (de Weger et al., 1987) and lipopolysaccharide (LPS) production (de Weger et al., 1989) for the colonization of potato roots by a Pseudomonas fluorescens strain. The capacity to form biofilm was shown to be related to the rhizosphere colonization in Bacillus amyloliquefaciens and rhizobia species (Rinaudi and Giordano, 2010; Tan et al., 2013), but not in P. fluorescens (Barahona et al., 2010). Fast growth rate was suggested to be important for rhizosphere colonization in Pseudomonas spp. and B. amyloliquefaciens (Simons et al., 1996; Tan et al., 2013). In addition, the chemotaxis exerted by some organic acids was significant for the colonization of tomato roots by B. amyloliquefaciens (Tan et al., 2013). Another less explored ecological aspect possibly involved in the rhizosphere colonization by soil bacteria is the suitability of bacteria for horizontal gene transfer (HGT) processes, which could hypothetically support the rapid adaptation of bacteria in the face of environmental shifts. There are increasing indications that the rhizosphere could be a hot spot of HGT events, for example, by the increase of the transference of conjugative plasmids between rhizosphere inhabitants (Lilley et al., 1994; Pukall et al., 1996; Van Elsas et al., 1998). Although some studies have identified traits related to the colonization of specific bacterial species, there is still a gap in the knowledge of key features involved in rhizosphere colonization by the soil microbiome.
Thus, the aim of the present study was to identify key bacterial traits for rhizosphere colonization using sugarcane as a model system. We studied the bacterial communities and assessed shifts in the taxonomic and functional profiles in bulk soil in comparison to the rhizosphere. Analyzes were performed using a combination of bacterial quantification, metabolic capacity to degrade carbon sources, high-throughput sequencing and metagenome prediction. Using this approach we gained a better understanding about the taxonomic and ecological relationships that microorganisms establish in the sugarcane rhizosphere, as well as of some of the characteristics needed for bacterial communities to colonize this soil habitat, including polygalacturonase activity and the possible importance of HGT in the rhizosphere.
Materials and Methods
Bulk Soil and Rhizosphere Sampling
Bulk soil and rhizosphere were sampled in a sugarcane cultivation field (cultivar SP-3250) located at ESALQ/USP (Piracicaba, Brazil). The sugarcane crop is being used in this field for 10 years under a green harvest management. Plants sampled in this study were at 9-months of cultivation (average height of 2.0 m) and did not show evidence for pest attack, disease, or nutritional deficiency. Bulk soil samples were made of soils free of roots, collected in the interline area of planting, at the layer of 0–10 cm. Rhizosphere samples were obtained by separating soil from plants roots (similar soil depth), focusing on a soil layer not thicker than 2 mm from the roots surface. Six biological replicates were used, each comprising of a single plant from which bulk soil and rhizosphere samples were collected, generating a total of 12 samples.
DNA Extraction
Total DNA was extracted from each sample using a MoBio Power Soil DNA Isolation Kit (Mobio, USA) according to the manufacturer's instructions. The resulting DNA was checked for integrity by electrophoresis in a 1% agarose gel, stained with ethidium bromide and visualized under UV light, and stored at -20°C.
Quantification of Bacterial Community
The 16S rRNA gene copy numbers in bulk soil and rhizosphere were assessed to investigate whether these two habitats harbor significantly different bacterial abundances. Real-time PCR amplification was performed using P1/P2 primers (Muyzer et al., 1993) in a reaction with 12.5 μL of Sybr Green (1X), 10 mM of each primer, and 1 μL of template DNA for a total volume of 25 μL. The amplification was conducted using a StepOne Real-Time System (Applied Biosystems) under the following conditions: 1 cycle of 95°C for 3 min, 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 30 s, followed by a melting curve analysis. For the standard curve, soil-derived amplicons of the 16S rRNA gene were diluted from 10-1 to 10-8 and quantified. These dilutions were submitted to amplifications under the same conditions described above. In addition, the efficiency of the reaction was calculated and was 101.5%, between the acceptable values of 90 and 110%, indicating the absence of PCR inhibitors.
Sequencing of the 16S rRNA Gene
We performed a sequence-based analysis using the Illumina MiSeq platform and the Nextera XT index kit for library preparation (Illumina, USA), targeting the V3-V4 region of the 16S rRNA gene. A nested protocol was performed. In the first reaction the whole gene was amplified using the universal primers 27F (Lane, 1991) and 1387R (Marchesi et al., 1998). This first reaction contained 1 μL of template, 2 μL of dNTPs (2.5 mM), 3.75 μL of MgCl2 (25 mM), 2.5 μL of Taq buffer (10X), 0.1 μL of each primer (100 mM) and 0.3 μL of Taq polymerase (5U/μL) for a total volume of 25 μL. The thermal cycles consisted of 1 cycle of 94°C for 4 min, 25 cycles of 94°C (30 s), 63°C (1 min) and 72°C (1 min), ending with 1 cycle of 72°C for 10 min. In the second reaction, primers S-D-Bact-0341-b-S-17/S-D-Bact-0785-a-A-21 (Klindworth et al., 2013) were used (coupled with Illumina adapters), which cover the hypervariable regions V3-V4 of the 16S rRNA gene. This reaction was conducted using 1 μL of the amplicons produced in the first reaction, 2 μL of dNTPs (2.5 mM), 3 μL of MgCl2 (25 mM), 3 μL of Taq Buffer (10X), 0.1 μL of each primer and 0.3 μL of Taq DNA polymerase for a total volume of 25 μL. The amplification was conducted following the thermal cycles: an initial denaturation cycle of 95° for 3 min, 30 cycles of 95°C for 45 s (denaturation), 57°C for 1 min and 45 s (annealing) and 72°C for 1 min (extension), followed by a final extension cycle of 72°C for 4 min. The primers sequences are found in Supplementary Table S1. Nextera indices and sequencing adapters were ligated to amplicons from the second reaction, and submitted for paired-end sequencing on the Illumina MiSeq platform. The reads were deposited at the SRA database (NCBI), BioProject PRJNA319762, and the BioSample accession numbers are: SAMN04904299 (B1); SAMN04904300 (B2); SAMN04904301 (B3); SAMN04904302 (B4); SAMN04904303 (B5); SAMN04904304 (B6); SAMN04904305 (R1); SAMN04904306 (R2); SAMN04904307 (R3); SAMN04904308 (R4); SAMN04904309 (R5); and SAMN04904310 (R6) (R = Rhizosphere and B = Bulk Soil samples).
Next Generation Sequence Analysis
The sequenced paired-end reads were separated by sample and analyzed using the QIIME software pipeline (Caporaso et al., 2010). Quality control analyses were performed to eliminate low quality reads, short reads, chimeric sequences, and to trim the low quality 3′ region of individual reads in order to achieve a minimum quality of Q28 (Phred scale). The UCLUST algorithm was used to cluster the reads in operational taxonomic units (OTUs) with a 97% cutoff, and to assign the taxonomy using the Greengenes database (version gg_13_8_otus) with a minimum fraction of 0.51. The reads were then aligned using the PyNAST algorithm and filtered. An OTU table was generated, the singletons were excluded and the OTU table was rarified (170,000 sequences) to avoid bias related to different number of reads in the samples. Richness and diversity indices were calculated, and the OTU table was exported to STAMP (Parks et al., 2014) for statistical analyzes.
In addition to the amplicon-based analysis, we also used a metagenome prediction approach to infer probable functions performed by the bacterial communities, which is more informative than a purely taxonomic community structure approach. For this, we used the PICRUSt software package, which uses 16S rRNA libraries to make a predicted reconstruction of the metagenome (Langille et al., 2013). The PICRUSt software uses an evolutionary approach to handle the OTUs that match with unavailable genomes by using their sequenced relatives as a reference for the prediction. This means that there is some uncertainty in the genome prediction, as all microbiome sequencing produces some OTUs that match with unavailable genomes and different strains of the same species/OTUs have some distinct gene content. The nearest sequenced taxon index (NSTI) value show the level of uncertainty of the metagenome prediction, increasing the accuracy of the prediction the smaller are its values. First, a new OTU table was created using a closed-reference picking OTU protocol against the Greengenes database (version gg_13_5_otus) at 97% identity, which was then normalized by dividing the abundances of each OTU by known or predicted 16S rRNA gene copy number abundances. The normalized data was then submitted to metagenome prediction and categorized by function using KEGG level 2 Gene Ontology (GO) terms for classification. The NSTI was used to quantify the availability of nearby genome representatives in the samples. The prediction tables (the raw gene prediction table and the classified table with the GO terms) were then exported to STAMP software (Parks et al., 2014) for statistical analysis.
Community Level Physiological Profile (CLPP) Analysis
To better characterize the functional profile of the microbial communities in our samples, we performed a CLPP analysis using BIOLOG Ecoplates (Biolog Inc., USA), which contains 31 different carbon sources. For this analysis, 10 g of either soil or rhizosphere samples were weighed, and a soil suspension was obtained by shaking the samples in 90 mL of saline solution (0.1 M NaCl) for 30 min. This suspension was then centrifuged for 30 min at 250 rpm, the supernatant was diluted to 10-3 and 150 μl was used to inoculate the Ecoplates. Plates were incubated at 25°C and read at the 590 nM wavelength in 24 h intervals for a total of 168 h. Absorbance readings were corrected using blank samples as controls.
Statistical Analysis
Results of qPCR were analyzed using the PAST software (Hammer et al., 2001), where ANOVA and Tukey pairwise comparison tests were performed to assess differences in rhizosphere and bulk soil samples.
The OTU table of the 16S rRNA gene sequencing was exported and analyzed in PRIMER-6 software (Clarke and Gorley, 2006) where a Non-metric Multidimensional Scaling (NMDS) as well as an Analysis of Similarity (ANOSIM) were performed using the Bray–Curtis dissimilarity matrix with the aim of detecting differences in bacterial community structure between the 2 habitats studied (rhizosphere × bulk soil). These identified differences were further explored using the STAMP software (Parks et al., 2014), where bacterial groups with significant differences were identified using the Welch's t-test (Parks et al., 2014). The same approach was used to identify significant differences in the predicted metagenomes of rhizosphere and bulk soil samples. The Benjamini–Hochberg P-value correction was utilized to avoid type 1 and 2 errors. In addition, a Bonferroni P-value correction was applied to the raw predicted genes table to account for potential bias related to PICRUSt predictions and statistical analysis, as this correction is even more conservative in detecting significant differences.
The PAST software was used to perform an ANOVA and Tukey test for each variable (C-sources) of the CLPP results. Alternatively, the results were exported to the CANOCO 4.5 software, where a biplot Principal Component Analysis (PCA) was performed to check the differences in the metabolic profiles of the microbial communities of rhizosphere and bulk soil, as well as to detect any correlation with the C-sources (ter Braak and Smilauer, 2002). An ANOSIM was also performed to validate the significance of these differences using the PRIMER-6 software package (Clarke and Gorley, 2006). The niche of a given community, bulk soil or rhizosphere, was calculated according to Salles et al. (2009). This analysis is based on the performance of the total community in each of the carbon sources, calculated as the sum of the best performances on each source present on the environment where that community is functioning.
Distinctions of Bacterial Abundance and Community Structure between Rhizosphere and Bulk Soil
Quantification of total bacterial communities in bulk soil and rhizosphere samples revealed significant differences in bacterial abundance (P < 0.05). The average bacterial abundance in rhizosphere samples was 4.96 × 109 rRNA gene copies per gram of soil, while bulk soil samples contained an average of 1.67 × 109 (Figure 1A). This distinction was complemented by the differential structuring of bacterial communities in each niche, as indicated by the results from partial sequencing of the 16S rRNA gene. After trimming sequences of low quality and rarification of sequences per sample, a total of 2,040,000 sequences – 1,020,000 from each rhizosphere and bulk soil samples – were used in the analysis.
FIGURE 1. (A) Quantification of 16S rRNA gene in samples of bulk soil and rhizosphere. Different letters means significant differences according to the pairwise comparison test of Tukey (P < 0.05). (B) Non-Metric Multidiomensional Scaling (NMDS) comparing the structure of bacterial community in bulk soil and rhizosphere on the basis of the OTU table.
Non-metric Multidimensional Scaling results indicated the distinct structure of bacterial communities in rhizosphere and bulk soil samples – confirmed by ANOSIM (P = 0.008/R = 0.82). The two groups of samples were separated in the first axis, and replicates grouped together, clearly indicating the differential selection exerted by each of these niches upon the bacterial community (Figure 1B). Although there was a clear difference in β-diversity, no differences concerning the α-diversity estimators (P < 0.05) were observed between the niches (Table 1).
TABLE 1. The output of sequences analysis after quality filtering per sample, before and after the OTU table rarefaction.
Sequences were classified into 24 known and 31 candidate bacterial phyla, with prevalence of sequences affiliated to Acidobacteria and Proteobacteria in both communities (Figure 2A). Divergences in the relative abundance of phyla Actinobacteria, Bacteroidetes, Cyanobacteria and Gemmatimonadetes were observed, which were more abundant in the bulk soil (P < 0.01). On the other hand, Verrucomicrobia, Nitrospirae and Tenericutes were more abundant in the rhizosphere (P < 0.01) (Figure 2B). Higher divergences were observed at family level (Figure 3), from which 24 were significantly more abundant in the bulk soil, and 29 were more abundant in the rhizosphere (P < 0.05).
FIGURE 2. (A) Taxonomic classification of OTUs (at level of phyla) based on Greengenes database using the QIIME software. (B) Statistical comparison (Welch's t-test) between the phyla abundance on rhizosphere and bulk soil using the Benjamini–Hochberg P-value correction (P < 0.05).
FIGURE 3. Statistical comparison (Welch's t-test) between the families abundance on rhizosphere and bulk soil using the Benjamini-Hochberg P-value correction (P < 0.05).
Metabolic Profiles of Rhizosphere and Bulk Soil Microbial Communities
CLPP analysis indicated differential metabolic patterns of the microbial communities found in bulk soil and rhizosphere. A broad view of such patterns was obtained with a PCA, which revealed clear distinctions between the carbon degradation profiles of the communities from each niche, supported by ANOSIM (P = 0.002; r2 = 0.51) (Figure 4). From the 31 C-sources, 11 were significantly more utilized by the rhizosphere community while 4 were more utilized by the bulk soil community (P < 0.05). The performances of the communities on individual carbon sources were used to calculate the community niche of bulk soil and rhizosphere samples, and were significantly higher (P < 0.05) in the rhizosphere (29.03 ± 2.90) compared to bulk soil (23.57 ± 0.30). The most catabolized C-sources by the microbial community from rhizosphere (P < 0.05) were D-galacturonic acid, D-galactonic acid, D-glucosaminic acid, L-asparagine, 4-hydroxybenzoic acid, D,L,a-glycerolphosphate, L-phenylalanine, L-threonine, L-serine, tween-40 and putrescine. On the other hand, A,D-lactose, D-xylose, i-erythritol and y-hydroxybutyric acid were the most consumed C-sources by the microbial community from the bulk soil (P < 0.05) (Figure 4).
FIGURE 4. Principal component analysis (PCA) showing the ordination of rhizosphere and bulk soil samples according to the oxidation of C-sources in the CLPP analysis. Variables (C-sources) colored in red means significantly different between rhizosphere and bulk soil in the Tukey test (P < 0.05). Samples represented by green squares are from rhizosphere and samples represented by brown circles are from bulk soil (PC1 = 32.5%, PC2 = 21.3%).
Metagenome Prediction of Bulk Soil and Rhizosphere Bacterial Communities
Metagenomes were predicted from the 16S rRNA gene sequences with an accuracy based on a NSTI average value of 0.23 ± 0.02, which is typical for soil samples analyzed in other studies (Zarraonaindia et al., 2015; Chen et al., 2016). Despite such a high NSTI value, Zarraonaindia et al. (2015) found a significant similarity between the sequenced and predicted metagenome. Metagenome prediction resulted in more than 6,900 protein-coding genes, of which 153 were differentially predicted in accordance to their frequencies in bulk soil or rhizosphere (P < 0.05); 80 were significantly more abundant in the rhizosphere and 73 were significantly higher in bulk soil (Supplementary Figure S1). The predicted protein-coding genes were categorized by function using the KEGG level 2 GO, revealing the prevalence (∼50%) of genes related to amino acid metabolism, carbohydrate metabolism, membrane transport, replication and repair and energy metabolism, in both bulk soil and rhizosphere samples (Figure 5A). The other half of the predicted functions were affiliated with more than 25 other functional categories. A Welch's t-test indicated that rhizosphere and bulk soil were enriched in 6 and 7 functional categories (KEGG level 2), respectively (P < 0.05). More specifically, predicted genes more abundant in the rhizosphere were mainly associated with Cellular Processes and Signaling, Cell Growth and Death, Carbohydrate Metabolism, Metabolism, Glycan Biosynthesis and Metabolism and Transcription (Figure 5B). On the other hand, genes related to Metabolism of Amino Acids, Lipids, Nucleotide, Terpenoids and Polyketides, and Cell Communication and Environmental Adaptation were more abundant in the bulk soil (Figure 5B).
FIGURE 5. (A) Metagenome predicted functions classified using KEGG level 2 database in PICRUSt software, showing the most abundant functions throughout the 12 samples. (B) Statistical comparison (Welch's t-test) between the predicted functions abundance on rhizosphere and bulk soil using the Benjamini–Hochberg P-value correction (P < 0.05).
We observed a significantly higher abundance of predicted genes associated with HGT in the rhizosphere (Figure 6B) than in bulk soil. We found a higher prevalence of genes associated with conjugation, such as the pilus assembly protein CpaE and type IV pilus assembly protein PilV (Cabezón et al., 2015); as well as genes involved in transformation/conjugation, such as the type IV secretion system proteins VirB4, VirB5, VirB6, and VirB9 (Figure 6B) (Christie et al., 2005; Cabezón et al., 2015; Juhas, 2015). We also observed a greater number of predicted genes indirectly related to transduction, like DNA polymerase bacteriophage-type. Another interesting predicted gene, also more abundant in the rhizosphere, was related to colanic acid biosynthesis protein WcaH, which is associated with biofilm formation. To validate the results from the PICRUSt analysis, we looked for predicted genes coding for enzymes involved in the metabolism of the carbon sources present in the Biolog plate assays. We found a predicted gene associated with polygalacturonase, which was more abundant in the rhizosphere (Figure 6A). Likewise, the rhizosphere samples had higher degradation levels of D-galacturonic acid in the Biolog assays (Figure 4).
FIGURE 6. Predicted genes on PICRUSt related to (A) CLPP analysis and (B) HGT, which showed significantly higher abundance in the rhizosphere (Welch's t-test, P < 0.05) using the Bonferroni P-value correction.
This study accessed the bacterial communities present in the sugarcane rhizosphere and bulk soil using real-time PCR and high-throughput sequencing in order to identify differences in bacterial abundance, diversity and taxonomic structure. Furthermore, the metabolic profile of the microbial communities was also assessed using CLPP and metagenome prediction with the aim of identifying potential traits important for the rhizosphere colonization.
Results of bacterial quantification (based on the quantity of 16S rRNA gene copies) provided the first indication of growth stimulus in the roots surroundings in our study, corroborating other studies (Wessén et al., 2010; Rachid et al., 2013). Differences in the bacterial community structure and composition between rhizosphere and bulk soil support the hypothesis that the rhizosphere selects a bacterial community from a pool in the bulk soil, which changes the abundance of specific groups according to their ability to adapt to the rhizosphere conditions. In this line, the similar values of richness and α-diversity found in these two environments provide supporting evidences that the rhizosphere shapes the bacterial community by modulating its composition (Mendes L.W. et al., 2014). Such distinction is better observed in the community structure based on the analysis of OTUs, where samples from each niche are separately plotted in the NMDS indicating a high β-diversity between rhizosphere and bulk soil.
Despite the fact that the bacterial communities were very different considering the analysis of OTUs, small changes were observed at the phyla level, where the taxonomic profile is quite similar (Figure 1A), suggesting that the shifts in abundances of specific groups are not phylogenetically dramatic. Three phyla, Acidobacteria, Proteobacteria and Actinobacteria were the most abundant in our study, similarly to those observed by Rachid et al. (2013), who analyzed bacterial communities of sugarcane fields under two different managements. In our study, from these 3 phyla only Actinobacteria was present at relatively higher abundance in bulk soil. Members of this phylum are known for their resistance to stress conditions (particularly drought stress) and/or ability to degrade complex carbon sources, explaining their higher abundance in the oligotrophic bulk soil environment (DeBruyn et al., 2011; Roes-Hill et al., 2011; Thomas et al., 2011; Kavamura et al., 2013). On the other hand, less abundant phyla, such as Nitrospirae, Verrucomicrobia, and Tenericutes, were enriched in the rhizosphere. Nunes da Rocha et al. (2013) showed that groups affiliated to Verrucomicrobia might be important to the rhizosphere of several plants, such as grasses, leek, and potatoes. Similarly, Verrucomicrobia and Nitrospirae were also detected with significant higher abundance in the rhizosphere of oats (DeAngelis et al., 2009) and soybean (Mendes L.W. et al., 2014). This evidence suggests that although understudied, these phyla can be commonly selected by the roots of several different plants and might play a critical role for their development. Proteobacteria is another bacterial phylum known for colonizing the rhizosphere (Uroz et al., 2010; Peiffer et al., 2013). Here we did not find differences when compared the abundance of members of this phylum in each niche. However, we found relatively higher abundances of families such as Burkholderiaceae, Pseudomonadaceae and Rhizobiaceae, which belong to Proteobacteria and are widely described as plant growth promoters (García-Fraile et al., 2012; Suárez-Moreno et al., 2012; Redondo-Nieto et al., 2013), in the rhizophere (Figure 3). These observations indicate the need to compare these communities in more detailed taxonomy levels than phyla, where they seem more similar.
Community level physiological profile results provide the first evidence of how the plant roots select microbes for its rhizosphere microbiome. PCA based on C-sources oxidation showed that the rhizosphere samples were closer to each other, while the bulk soil samples were more dispersed in the chart, suggesting that the microbial communities from the rhizosphere share a very similar functional community structure (Figure 4). This suggests that the rhizosphere selection effect can be possibly more effective for functional attributes than for taxonomy – as observed in the ordination of samples based on OTUs (Figure 1B).
We could conclude from both the number of carbon sources consumed in each habitat and the community niche analysis that the rhizosphere microbial community is more metabolically active. It might be that cells in an active state preferentially colonize the rhizosphere microbiome, as the roots are constantly supplying nutrients to this niche. In counterpart, the bulk soil community is more prone to undergo long periods without labile carbon, which might decrease the metabolic rate of most microbes. Our results also indicate that the labile C-sources released in the rhizosphere might be the main plant compounds acting in the microbiome selection. This is supported by the preferential use of the D-galacturonic acid, and its reduced form D-galactonic acid, in the rhizosphere.D-galacturonic acid is the most abundant component of pectin (Zhang et al., 2011), a major constituent of plant cell walls, which is consequently released in the rhizosphere. D-galacturonic acid has also been found as a component of root exudates (Tawaraya et al., 2015). It is important to note that CLPP is culture dependent, meaning that the measurements of C-sources oxidation reflect the activity of only part of the community in the sample, and is therefore limited in its ability to provide the real functional capacity of a microbial community in vivo.
The only common plant-produced C-source that was more utilized by the bulk soil bacterial community was D-xylose, a component of hemicellulose. Nevertheless, hemicellulose is one of the polysaccharide constituents of lignocellulose, the most recalcitrant component of the plant cell wall, which is only degraded by specialized enzymes (Mansour et al., 2016). In sugarcane field soils, the presence of hemicellulose is even higher, as leaves are constantly falling and accumulating on the soil surface (Chandel et al., 2012).
Although evidence for selection in the rhizosphere is clear, the bacterial abilities that enable rhizosphere colonization are still poorly described. To gain more insights on these abilities, PICRUSt was used to predict the metagenome of each of the samples. PICRUSt is dependent on available sequenced genomes and some bias can be produced if references for the species inhabiting the sampled environment are missing. However, it is a useful tool to provide insights about the community functional potential in the absence of shotgun metagenomic data. The combination of metagenome prediction and CLPP provided insights into community functional structure and reduced the bias related to each approach. For both communities, rhizosphere and bulk soil, the most prevalent predicted functions were Amino Acid Metabolism and Carbohydrate Metabolism, corroborating a previous study of sugarcane rhizosphere communities by metaproteomics (Lin et al., 2013). Some significant differences between bulk soil and rhizosphere were found, but the overall functional profiles classified by KEGG (level 2) were very similar, meaning that all higher functions are probably executed in both environments. Functions related to basic metabolism were found in higher abundance in the rhizosphere compared to the bulk soil (Cell Growth and Death, Metabolism, Carbohydrate Metabolism, Glycan Biosynthesis and Transcription), what can be linked to the higher metabolic activity in the rhizosphere communities detected on the CLPP analysis. These convergent results also suggest that the fast growth rate observed in previous studies of single species can be a widespread feature for rhizosphere colonization (Simons et al., 1996; Tan et al., 2013). In contrast, the bulk soil community showed a higher ratio of functions related to secondary metabolism, including the degradation of complex compounds and environmental resistance (Metabolism of Terpenoids and Polyketides, and Environmental Adaptation).
A deeper investigation of the metagenome prediction showed a greater abundance of genes related to Colanic Acid Biosynthesis Protein WcaH in the rhizosphere. Colanic acid is associated with biofilm formation in Escherichia coli (Danese et al., 2000), which is in agreement with previous data that showed the importance of biofilm formation for colonization of the roots surface (Ramey et al., 2004; Rinaudi and Giordano, 2010; Tan et al., 2013). Another interesting result obtained from the metagenome prediction was the higher abundance of genes related to HGT in the rhizosphere. We found genes related to bacterial transformation and conjugation, such as those associated with the Type IV Secretion System, enriched in the rhizosphere (Christie et al., 2005; Cabezón et al., 2015; Juhas, 2015). We also found a higher abundance of genes related to conjugation, as indicated by the occurrence of genes codifying the pilus assembly protein CpaE and type IV pilus assembly protein PilV (Cabezón et al., 2015); and genes possibly related to transduction, such as genes related to bacteriophages. These results suggest that HGT could be an important mechanism for bacterial adaptation in the rhizosphere. Given that the accumulation of beneficial mutations is a slow process to generate genetic variability in the short time scale (Gogarten et al., 2002; Jain et al., 2003), the horizontal transfer of entire genes may be a more efficient way for bacteria to rapidly adapt to the rhizosphere niche. This process is widely understood as a powerful tool for the rapid evolution of antibiotic resistance in bacteria inhabiting hospital environments (Palmer and Kishony, 2013), and may happen in an analogous way for rhizosphere colonization.
Although there is some bias in using 16S rRNA gene libraries to predict metagenomes, other studies based on real metagenome sequencing found results consistent with and supporting our data. Mendes L.W. et al. (2014) found an enrichment of Type IV Secretion System genes in the rhizosphere of soybean. Similarly, Alzubaidy et al. (2016) found a significant higher number of genes linked to Phages, Prophages, Transposable Elements, and Plasmids in the rhizosphere of the mangrove plant Avicennia marina. Together with our results, this evidence suggests that HGT may be a widespread mechanism that facilitates rhizosphere colonization in plants.
Moreover, to support the results of the functional analyzes, we checked for matches between the C-sources degradation in the CLPP analysis and the enzyme-coding genes predicted by PICRUSt. Even considering the bias in each approach, a match was observed for genes related to polygalacturonase, more abundant in the rhizosphere samples, and the preferential degradation of D-galacturonic acid by the rhizospheric microbial community. This meaningful finding indicates that the capacity to metabolize the galacturonic acid may be an important trait for rhizosphere colonization. Other genes related to the degradation of specific CLPP C-sources were found in the metagenome prediction, but no significant differences were observed between rhizosphere and bulk soil. The lack of other matches might be related to the fact that CLPP measures differences in gene content as well as differential expression of genes, while metagenome prediction is based exclusively on the prediction of genotypes. It might also be that the low frequency of groups hosting these genes limited its prediction in the PICRUSt approach, or even that this approach may not have the accuracy to properly detect small changes in gene abundance of functional groups. The high NSTI values found in our samples and the lack of other approach to confirm the prediction also tell that the finding of HGT-related genes higher in the rhizosphere is an interesting suggestion that this activity can be enriched in this environment, but we highlight the need to experimentally confirm this issue in next studies.
Taken together, our results show important findings concerning the taxonomic and functional selection exerted by the sugarcane rhizosphere on the bacterial community, as well as identified potential traits that allow bacteria to colonize the rhizosphere environment. We firstly highlight the ability to degrade the D-galacturonic acid, a compound potentially used by the plant in a biochemical selection; and secondly the potential ability to perform HGT, which can spread important genes related to rhizosphere colonization, providing the bacteria an evolutionary and ecological advantage.
Author Contributions
LL performed the molecular, bioinformatics and statistical analyzes, designed the experiment, interpreted the data and wrote the manuscript; MdS performed the Biolog and bioinformatics analyzes, designed the experiment, interpreted the data and wrote the manuscript; FA contributed to the conception of the work and design of the experiment, interpreted the data and wrote the manuscript.
We acknowledge CAPES for the scholarship support provided to LL and MdS, and FAPESP-NWO cod 2013/50353-7 for the financial support in the publication of the article.
Supplementary Material
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.01341
FIGURE S1 | Statistical comparison (Welch's t-test) between the predicted genes abundance on rhizosphere and bulk soil samples using the Bonferroni P-value correction (P < 0.05).
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Peiffer, J. A., Spor, A., Koren, O., Jin, Z., Tringe, S. G., Dangl, J. L., et al. (2013). Diversity and heritability of the maize rhizosphere microbiome under field conditions. Proc. Natl. Acad. Sci. U.S.A. 110, 6548–6553. doi: 10.1073/pnas.1302837110
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Keywords: microbiome, sugarcane, metagenome prediction, plant selection, horizontal gene transfer
Citation: Lopes LD, Pereira e Silva MC and Andreote FD (2016) Bacterial Abilities and Adaptation Toward the Rhizosphere Colonization. Front. Microbiol. 7:1341. doi: 10.3389/fmicb.2016.01341
Received: 13 April 2016; Accepted: 15 August 2016;
Published: 25 August 2016.
Edited by:
Paul Bodelier, Koninklijke Nederlandse Akademie van Wetenschappen, NetherlandsReviewed by:
Marc Gregory Dumont, University of Southampton, UKChristopher Blackwood, Kent State University, USA
Copyright © 2016 Lopes, Pereira e Silva and Andreote. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Fernando D. Andreote, email@example.com


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A love for boating, undying dedication to the Tragically Hip, and a completely blank canvas were the inspiration behind a recent basement renovation from the creative minds of dynamic design duo Denise Hulaj and Jason Bellaire of StyleHaus Interiors. The creation was of epic proportions, and gave them an appreciation for a detailed, interesting, outside the box environment.
Having previously worked with clients Brian Turner and Jennifer George on a living room, family room, and kitchen space in their single-family Kanata Lakes home, the StyleHaus Interiors pair was brought back for a complete overhaul of the 900 square foot unfinished basement. Entrepreneurship runs deep throughout this family of four. Brian owns a microchip company while his wife, Jennifer, is a consultant within the field of human resources. With a Sea Ray Sport Yacht named Four Turtles, both parents and their teenage daughters spend plenty of time on the water and, like most homegrown Canadians, they carry a love for the iconic Kingston rock band The Tragically Hip—both boat and band were big influences in the design.
Explains Denise, "Our approach is very individualized for each project and we approach them with fresh eyes each time. We don't have a look that we repeat for each client. We like our designs to reflect the homeowners—things that they love—so at the end of the day it feels like their home. Our style isn't imposed on them. It's just us interpreting their style and making it special."
Although the overall style was inspired by, and took many elements from, a nautical design theme, Jason insists that they did not want to go overboard (no pun intended). Instead, details reminiscent of a summer on Cape Cod are sprinkled throughout. The cabinetry is navy blue and soft grey, and the custom closets wrap around the bathroom door, hiding its entrance, much like you might see on board the Four Turtles. Walking into the bathroom you'll spot some very interestingly patterned tiles, invoking memories of a steering wheel at the helm of a ship. Surrounding the living area is a wall of horizontal whitewashed tongue and groove shiplap which brings with it a coastal vibe, while a bar with a unique cross design on the front of the millwork boasts two taps for whichever local brews Brian is in favor of at the time (right now, he's drinking a cold one courtesy of Calabogie Brewing). Another impressive detail that Jason emphasized is the cold wall system, which offers an alternative to a traditional fireplace, redirecting heat buildup from within the enclosure back into the room, allowing material to be brought right up to the base of the glass, without fear of heat damage. And because this family knows how to enjoy some friendly competition on game nights, an underutilized alcove was the perfect spot to create a custom banquette, wrapped around three walls. Adding in a large roundtable with an ornate chandelier and that coastal shiplap, it's reminiscent of the built-in dining aboard a ship. Jason explains, "There are little references like that [so when] you're in the space you're not obviously saying 'oh this is very nautical', but it has an homage to all things boating."
Inarguably, the pièce de résistance is the custom mural, executed by Doug Jackson, who owns local Ottawa company ManMade Art. A bright yellow canvas that showcases an ancient nautical map is the backdrop from which The Tragically Hip appear to emerge and scrawled throughout are a collection of the Hip's lyrics that hold special meaning to the family. Denise gushes, "it's a very nice piece." "We were engaged with the project right around the time that Gord Downie died so it was sort of memorializing him in a way. It was an homage to their favourite band, and to Canada in general," adds Jason.
From an unfinished storage area springs a comfortable and cool coastal space where the family can do it all: work, eat and play. Denise is confident that the basement will be well used, "I think they'll probably spend more time there than they will upstairs to be honest."


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A student strapped for cash was approached for feet pics in exchange for draining her sugar daddies credit card.
Amy Williams, a student from Glasgow moaned (as you do) on twitter one day as she was skint. She was then approached by Richard who offered to take away your financial worries in exchange for feet pics. The 21-year-old thought this was the solution to her problems even if she had to send a few feet pics.
The law student explained that Richard would physically beg Amy to spend his money in exchange for the pics and Amy did just that…
Amy said: "I've had the arrangement since May. I tweeted saying I was skint and needed a sugar daddy and he direct messaged me straight away offering money in return for feet pictures.
I thought he was talking rubbish, as there's loads of fake people like that on Twitter, so I said money first, and he sent me one of his credit cards and told me to buy myself something so I did and it worked.
As time went on, he started sending me money literally just for pictures of my feet, which I'm sure anyone would do."
I'm sure there is a lot of students up and down the country who wouldn't pass up the opportunity. Richard also encourages Amy to spend his money on her friends too, which she happily does.
Amy makes the point that although people may judge her, people have to remember it is only feet, everyone has them!
She says "Just recently he's sent me credit cards with big amounts on them like £2,500 and told me to 'drain' the lot of them and treat me and my friends.
There was so much stuff, I had to label it all up in my room with everyone's name on it so I didn't get confused."
After Amy did some shopping on MissGuided for her and 15 friends, the story went viral on twitter when her friend tweeted. Now MissGuided are offering a girls chat the entire wish list! All you have to do is comment on their post why they should pick you! We don't need a sugar daddy after all


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Quality work that our Customers can trust
So many of our customers return to us time and time again, because they are going to get honest answers and high quality work. We value the relationships built with our clients and strive to provide, not only the best products and services, but also friendly competent customer service. Spending time and money on home repairs can be stressful, and its our goal to make the process as simple and easy as possible. Our customers truly appreciate the care taken and have had many kind words to say over the years.
"My family and I are grateful for your patience and perseverance through the process of our purchasing and your installing a Generac generator for our home in Sherman. The process took longer than usual and was complicated by our family not being on the property. But you and your colleagues, in your office and on your technical staff, were always courteous, quick to respond to our many questions and concerns, and got the job done. When we realized that we needed a new generator - and would not work with our former contractor because of our unsatisfactory experience — I contacted five friends living in the area to ask for their recommendations, and 5 out of 5 recommended Deak. Therefore, our expectations were high, and Deak Electric exceeded them. We look forward to our ongoing relationship."
Richard, Alice, Alyson and Greg
"I just wanted to thank you all for so much help and great work. Kevin is amazing! The Runtal install yesterday was a little tricky - and he solved all of the problems to perfection. I thought I would need a handyman for touch ups -- but there is not a scratch. The units you ordered look great and they are the perfect scale. We are very appreciative. Hope you have nice holiday plans ahead. Many thanks."
Julia in Washington
"We want to tell you that Kevin and Dylan, who worked at our home today, are very, very nice people, plus being professional and knowledgeable. Thank you for employing these kind of people. It says a lot about your company, who you are and what you stand for :) We appreciate you. "
Sue and Charlie
"I wanted to let you know how impressed I was with Friday's installation. The team was efficient, sensitive to my suggestions, creative (specifically around the crawl space water pipe bonding) and willing to explain the whole setup in detail. I was very impressed with your son and I'm sure you are very proud of him and his work ethic. He is a terrific representative for your business."
Leo, Brookfield, CT
"Alex deserves more than 5 Stars!! He is fabulous, kind, very easy to talk to and helped me to work out the best option for the deck lighting. I look forward to working with him and Deak Electric in the future. "
Patti B from Danbury
"Deak installed Generac Generator. Their staff was very careful and respectful of our safety during this Covid pandemic. I don't think any other company would have pleased us more than they did. Thank you Vinny and crew for our peace of mind if and when lights go out again. "
Richard, Bethel, CT
"Deak Electric is the most Professional & Family run business I have come across. From Bill Jr to Mrs Deak, I couldn't have had a better experience. Price was extremely competitive & they explained the process in detail & answered all questions. The crew had the Generac generator & transfer switch up & running in 1 day. Very responsive, would use them again in a second. Very highly recommended as they treat you like family! "
Frank, Brewster, NY
"We just had Robert at our home to get our generator back online…he did an excellent job and said he wasn't leaving til it was perfect… he was professional,and he was very proud of the fact that Deak was top dealer in northeast. Nice to know your employees have such high regard for your company…always impressed with everyone we have to deal with at Deak!"
Susan M
"I wanted to say thank you for the great service. Thank you for the integrity you exhibited in not changing your price as many other companies were doing throughout this pandemic. You have a great company with wonderful employees. I will certainly recommend your company whenever the opportunity presents itself. "
John W
"Deak Electric installed a generator that I purchased through them. I was more than pleased with their service from Bill the owner to Helena the office manager and every crew member that worked on my job. I felt I was working with family and would not hesitate to recommend them. "
Ann, Brookfield, CT
"Thank you for your service today and Steven was exceptional. Glad to utilize your business and professional tech. His knowledge and customer interaction were the best I have seen. Will always recommend your company to others. "
Ken, Ridgefield, CT
"We used DEAK Electric to design and install our whole house generator. We had many quotes, but Bill Deak was very transparent with what we needed and the best way to set the system up in our home. Helena was super responsive to our needs, as the permit process was very, very long for our town. I highly recommend them for a generator install, or any other electrical work needed for your home. "
Randall, South Salem, NY
"I had a notification of a recall for my generator, I called Deak and they replaced it. I will tell you they know their business, very professional, trustworthy, and helpful. I will plan on doing business with them from now for everything electric related. The tech Vinny even spoke with my gas company to alert them of a high pressure natural gas problem. I'd give them ten stars if I could. "
Louis, Danbury, CT
"Wanted to let you know how great the installers were! Double checked with me regarding power shut off time. Made sure no mess went on any carpets I had put out for them to stand on. Everything looks extremely neat, tidy, and organized. Very much appreciate the high degrees of professionalism. "
Paul, Brookfield, CT
"We wanted to take a few minutes to personally forward a "Thank You" for your employee, Steve. He came into our facility twice and both times was very professional and mindful of our patients. Thank you! "
Childcare Associates, Danbury, CT
"I wanted to take the time out to let you know how great the technicians were who hooked up/installed my generator. All of the technicians were great. Vinny in particular was awesome and really took the time to explain everything to me and answer the hundreds of questions I had. He spent a lot of time going over my concerns and making sure that I was confident with the whole process before he left. He and they were all very professional and worked very hard all day at my home in order to connect my generator. I wanted to take time out from my day and mention this to you to pass along because I think it is important to know when people go above and beyond and do a great job. "
Josh, Sandy Hook, CT
"The city inspector was here yesterday and of course the install was passed with flying colors! Thanks for a great install of the generator. Your employees were a pleasure to deal with and very good at explaining the operation to me."
Al, Danbury, CT
"The job turned out perfect and your son is sharp and was very neat with the install. The other guys were polite as well. A great experience all the way through. I will be giving many references in your behalf!"
George, Sherman, CT
"We always recommend Deak Electric to all of our clients. Your guys are always professional, on time and leave the job site cleaner than they found it."
Tom, General Contractor, Brookfield, CT
"Great job as usual. Many Thanks. He is a good guy and always cares about the quality of his work."
Mitch, New Milford, CT
"Thanks so much! I truly appreciate your prompt response."
Oslene, Patterson, NY
"I wanted to let you know that your son Billy, Jr. and your team terrific! I will highly recommend you to everyone."
Frank, New Milford, CT
"Billy and Vinny were very helpful. Thanks again for all your work."
Patricia, Sherman, CT
"Final inspection on our generator installation went well and we received a passing report. Once the inspector saw Deak Electric had performed the work, they said it would be a positive inspection. It is clear that Deak Electric has built alot of credibility. Thank you for your professionalism with this service."
Jeff, Brookfield, CT
"Just wanted to give a shout out to Deak Electric. During the last wind storm our power went out and unfortunately our portable generator malfunctioned. Deak Electric came out after regular business hours, took our generator and gave us a loaner. They saved our groceries and our sanity!. We were without power for over three days. They also did a great job with the repair. Absolutely went above and beyond for us."
Pamela, New Milford, CT


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Ritchie Bros. launches new PriorityBid feature for live auctions
September 17, 2019 By Ritchie Bros.
Sept. 17, 2019, Vancouver, B.C. – Ritchie Bros. wants to make buying equipment easier than ever. With its new PriorityBid feature, buyers will be able to make proxy bids online for items in its live auctions up to a week before the sale. These PriorityBids put them first in line to win the item, provided no one outbids them.
"Launched in mid-August, PriorityBid has quickly become a valuable feature for our live auction customers – allowing them to set a max bid on an item, go back to work, and let the system bid for them," said Ravi Saligram, Ritchie Bros CEO. "PriorityBid also provides us increased insight into demand ahead of the auction, which is extremely valuable information for us and our sellers as it allows us to be more nimble in optimizing our marketing efforts right up to the day of the auction. We believe this will take online bidding in our live auctions to the next level."
Since it was launched in mid-August, more than 25,000 PriorityBids have been made, with approximately 45 per cent of those bids coming through Ritchie Bros.' mobile application.
"PriorityBid was initially introduced on IronPlanet; its recent implementation with our live auctions now provides customers a more consistent buying experience across platforms," said Matt Ackley, chief marketing officer, Ritchie Bros. "As we continue to leverage our technology capabilities, capitalizing on online buying trends, we will become more flexible, allowing us to meet the diverse needs of our customer base. For example, the PriorityBid experience in our mobile app is the ultimate example of convenience for our buyers – they can place bids while they're in the yard inspecting the equipment at our sites. It's that simple."
How it works
Submit a maximum bid and the PriorityBid system will bid the lowest amount required for you to win, up to your max bid. Bids are confidential and the system will only raise your bid if someone outbids you. If you're outbid, you'll receive an email notification. In the case of a tie, the first bid placed wins. To learn more, visit rbauction.com/PriorityBid.
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The most obvious time to arrive at West Mersea is on the afternoon high water. Visiting yachts should contact the launch man as they approach the Nass Beacon on Ch 37 and will be allocated a mooring on arrival. The launch service will take you ashore.
Saturday- High water Burnham 0514 Low water Burnham 1125 High water West Mersea 1712
West Mersea Yacht Club – Pre-dinner drinks from 1900 followed by Dinner at 2000 – Be prepared to leave the Yacht Club soon after 2200 as the launch service finishes at 2300.
Shallow draft fleet, most motor boats and some larger sailing cruisers have the option of crossing from the Crouch to the Blackwater via the Raysand channel. Larger yachts usually head further seawards and cross the Swin Spitway.
Opportunities for cruising in company on the Friday or Saturday and returning on the Sunday or Monday will evolve as the forecast becomes more reliable. I will advise details of the options as I see them later this week by email and via the WhatsApp group. If you are not on the Cruiser Fleet email or WhatsApp Groups and wish to be in the loop please contact me asap.
Unfortunately for crews of smaller boats, being Bank Holiday Weekend, there is very little suitable accommodation ashore so my advice is to only make the trip if you can overnight onboard your boat.
I have made provisional bookings to secure moorings and dinner so please let me know if you would like to join us for this event. It is not a firm commitment at this stage, numbers will have to be confirmed later in the week.
Steve Barham – Cruiser Fleet Captain
21st May 2019
07792 788401


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Missing London woman found in camp for ISIS brides with son
A MISSING Brit who left London aged 18 has been found six years later in a camp for ISIS brides in Syria.
Nasra Abukar, who now has a three-year-old son, prompted a widespread search when she disappeared from her family home in Lewisham in 2014.
She was found at the Kurdish run al-Hol detention and refugee camp in northeastern Syria.
The camp holds around 70,000 people, most of them women and children displaced by conflict in Syria, but around 3,000 thought to be the wives of ISIS fighters.
After leaving London, Abukar made her way to Syria and married an ISIS fighter from Cardiff, the Times reports.
She had two sons, Faris and Talha, but Faris was later killed in a coalition airstrike, while Talha lives at al-Hol with his mother.
Abukar and husband Aseel Muthana are believed to have remained with ISIS until its last piece of territory - the town of Bargouz in eastern Syria - was recaptured in March 2019.
Speaking to the Mirror last month, Muthana said he had last seen Abukar when the town fell.
"We were separated at that point and she was taken to a camp," he said.
"I wrote to her and I believe the letter was passed on but I have never received a reply."
Abukar's social media posts from when ISIS still controlled large portions of Iraq and Syria reportedly advertise life under the group and celebrate its attacks abroad.
Following the attacks in Paris in November 2015, which claimed a total of 130 victims, Abukar posted the word "Paris" followed by a "crying with laughter" emoji.
Abukar's mother, Kaha, has previously said she was not particularly religious before being recruited and leaving for Syria.
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