Here is a great bit of advice for how to continue to prepare for your dental school interviews. Pretend that you are a member of the dentistry school admissions staff at one of the top dental schools. Carefully read over your own application as you simultaneously pretend that you do not know anything about yourself except what is written on your application that you have submitted to schools of dentistry. If you were on the dental admissions committee, what more would you want to know about yourself. For example, you may have listed a volunteer experience in an orthodontist's office or in a dental clinic for the poor. You may be asked to elaborate on what you did. For example, did you help make molds for the teeth or instruct children how to brush and floss with braces ? What new emotions did you experience when you interacted with poverty-stricken patients who did not come into the clinic until they had advanced dental disease?
Or, perhaps there are other aspects of your dental school application that you may be asked about. Why were your grades so low during sophomore year? Why was your DAT score in biology lower than your DAT scores on other sections? Why did you take organic chemistry at a community college instead of at your undergraduate university? These are important questions to ponder to prepare for your dental school interviews.
Take time to think about each part of your application and how you would give more details during your dental school interviews. Never put blame on someone else for one of your deficiencies. For example, don't blame a bad grade on a bad professor (even if it's true). If you do this, the dental school admission committee will think: "This person will blame us if he/she doesn't succeed here." Instead say something such as, "My study skills were not up to par when I took chemistry but since then, I've worked hard on learning how to focus my studying by practicing problems from old exams. My other grades in other science courses reflect my improvement".
TAKE HOME LESSON: Prepare for your school of dentistry interviews by role playing as the admissions officer. Think about how to elaborate on the information you provided on your dental school application. Never place blame for your any weakness on your dental school application on anyone else.


Cleft lip and cleft palate are openings or splits in the upper lip, the roof of the mouth (palate) or both. Cleft lip and cleft palate result when facial structures that are developing in an unborn baby don't close completely.
How common is cleft lip and cleft palate?
Cleft lip, with or without cleft palate, affects one in 700 babies annually, and is the fourth most common birth defect in the U.S. Clefts occur more often in children of Asian, Latino, or Native American descent. Compared with girls, twice as many boys have a cleft lip, both with and without a cleft palate. However, compared with boys, twice as many girls have cleft palate without a cleft lip. Please discuss with your doctor for further information.
What are the symptoms of cleft lip and cleft palate?
A cleft lip and cleft palate are usually noticed at birth, and your doctor may start coordinating care at that time. If your baby has signs and symptoms of a submucous cleft palate, make an appointment with your child's doctor.
What increases my risk for cleft lip and cleft palate?
In the United States, cleft lip and palate are reportedly most common in Native Americans and least common in African-Americans.
Males are twice as likely to have a cleft lip with or without cleft palate. Cleft palate without cleft lip is more common in females.
Exposure to certain substances during pregnancy. Cleft lip and cleft palate may be more likely to occur in pregnant women who smoke cigarettes, drink alcohol or take certain medications.
Having diabetes. There is some evidence that women diagnosed with diabetes before pregnancy may have an increased risk of having a baby with a cleft lip with or without a cleft palate.
What are some lifestyle changes or home remedies that can help me manage cleft lip and cleft palate?
Early dental care. Like other children, children born with cleft lip and cleft palate require proper cleaning, good nutrition, and fluoride treatment in order to have healthy teeth. Appropriate cleaning with a small, soft-bristled toothbrush should begin as soon as teeth erupt. If a soft children's toothbrush will not adequately clean the teeth because of the modified shape of the mouth and teeth, a toothette may be recommended by your dentist. A toothette is a soft, mouthwash-containing sponge on a handle that's used to swab teeth. Many dentists recommend that the first dental visit be scheduled at about 1 year of age or even earlier if there are special dental problems. Routine dental care can begin around 1 year of age.
Orthodontic care. A first orthodontic appointment may be scheduled before the child has any teeth. The purpose of this appointment is to assess facial growth, especially jaw development. After teeth erupt, an orthodontist can further assess a child's short and long-term dental needs. After the permanent teeth erupt, orthodontic treatment can be applied to align the teeth.
Prosthodontic care. A prosthodontist is a member of the cleft palate team. He or she may make a dental bridge to replace missing teeth or make special appliances called "speech bulbs" or "palatal lifts" to help close the nose from the mouth so that speech sounds more normal. The prosthodontist coordinates treatment with the oral or plastic surgeon and with the speech pathologist.
Cleft Lip and Cleft Palate. http://www.webmd.com/oral-health/cleft-lip-cleft-palate#2-3. Accessed August 1, 2017.
Cleft lip and cleft palate. http://www.mayoclinic.org/diseases-conditions/cleft-palate/basics/definition/con-20024619. Accessed August 1, 2017.


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Robert Sypniewski Supports Future Programming by Naming WTTW and WFMT Beneficiaries of His Estate
Robert Sypniewski gave WTTW and WFMT's Kathy Kielar a tour of the Brookfield Zoo, including a chance to feed a rhinoceros, after announcing his plans to name the two stations and the Zoo as the co-equal beneficiaries of his estate.
As a lifelong educator Robert Sypniewski deeply appreciated the quality programming available on WTTW and WFMT, which is why he specified in his trust that each station would receive one-third of his estate through the Robert and Patricia Sypniewski Charitable Trust.
Robert and his late wife Patricia were teachers who lived on one salary while investing the other. In retirement, they converted a box truck into an RV and traveled the country for years before Patricia developed Alzheimer's disease. Robert would spend the next two decades caring for his wife at home before her death in 2007.
The couple loved WTTW and WFMT programming, especially travel and cooking shows, which is why Robert announced in 2013 his plan to name the stations as beneficiaries of their trust. To make his intentions clear to everyone involved, he called a meeting of his estate-planning attorney, his financial advisor, and WTTW and WFMT's Vice President of Development Kathy Kielar to finalize his plans.
"Robert enjoyed on-air recognition in honor of him and his wife for many years before he passed away in 2021," says Kathy Kielar, Vice President of Development at WTTW and WFMT. "We will continue to honor Robert's memory with on-air recognition for the next few years. With his deep passion for our mission and careful financial planning, he really has made a difference at WTTW and WFMT."

redpajama_set_name: RedPajamaCommonCrawl


Oil India Limited is calling out for interested and eligible candidates to apply for the post of Senior Accounts Officer. The selection process will comprise a written test, a group discussion and a round of viva voce. Selected candidates have the scope of earning up to INR 180000. The application deadline is on June 6, 2018.
Step 1: Log on to the Oil India Limited official website.
Step 3: Select Current Openings.
Step 4: The list of openings will be displayed on the screen.
Step 5: Select the application link for Senior Accounts Officer that will be active between May 31, 2018 and June 6, 2018. Follow the subsequent pages to complete the registration process.


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Television Collision
CC2K
The Nexus of Pop-Culture Fandom
Album Review :: Make Do and Mend :: Don't Be Long
Written by: Andrea Janov, CC2K Music Editor
Make Do and Mend :: Don't Be Long :: Stay Close / Rise Records
Since the first note that I heard many years ago, I was a Make Do and Mend fan. They perfectly meld heavy hitting music, with meaningful lyrics, and hooks that will grab you. Don't Be Long follows the great style they have been perfecting through the years. I have listened to this album once a day for the last three weeks and I am not sure exactly what to tell you about it. It is a solid punk rock record. It is heavy, it is catchy, it is accessible, it has substance.
Like everyone else, I get in moods for different music. Some days are reserved for the classic rock I grew up on, some days are for grunge/alt rock that made me the weird kid in middle school, and some days are for punk. The first days of summer are always overtaken by Screeching Weasel, NOFX, and ALL. But no matter what mood I am in, some gruff punk with a hook will get me every time. And Don't Be Long was one of those records.
It starts off with the title track, Don't Be Long, that sets the stage for the album, it is gruff, slow paced, hardcore punk track, with great moments of melody and great moments of unrestrained passion. The simple line, "Every time I see you, I still hear my favorite song" pulled me into this song so much deeper. I identified with that sentiment completely, from boyfriends, to best friends, to first dates, to break ups, I instantly accessed all those songs and all those emotions and put that into my backstory while listening to this track.
Ever Since, is the catchiest track on the album. It is almost rock and roll, until it reaches is break down. It will have the entire pit singing, the opening lines "Measure the moments /by the almost and the came close/ by the 'what the fuck's and 'I don't know's" drawing on their own experiences and with the conviction as if those words were written just for them. The track progresses sonically and lyrically until we reach the conclusion, "I guess we had it to lose/ but that just makes it harder now that it's through/ you chose the other side of a mountain that I can't move".
Bluff is a call to arms and a calling out of punk bands. It is aggressive, with raw strained vocals, putting bands and scene kids on the spot, "play me a song from the legion halls / back of the van, and truck stop walls / show me you've got something to say". Old Circles will have you singing along and Each of Us is a hardcore breakup song, "Was it the 'I love you's, and the 'I'll be home soons / or the 'Baby please don't wait up's / that start to sound like rain drops / a skipping record, a ticking clock".
I Don't Wonder At All is an acoustic track that somehow does not sound out of place at all. Sanctimony has a cool a capella opening that builds into a rock and roll song full of fury and urgency before in devolves into a chanting section. Begging for the Sun To Go Down is the final song on the album, it is slow, deep, and brooding, it is the perfect punctuation to this album.
Don't Be Long is an album that takes you on a journey, it ebbs and flows as it progresses. The raw vocals draw you in and the hints of melody carry you along. And if, I've said it once, I've said it a million times, lyrics matter. Yes, a song can be great with nonsense lyrics, but those song that I really love, the ones that changed my life, are the ones with lyrics that I was able to connect with, lyrics that were crafted, lyrics that mean something. Don't Be Long is full of those lyrics.
Author: Andrea Janov, CC2K Music Editor
A little magic goes a long way: A personal childhood journey with Matilda
By Bianca Garner, CC2K Staff Writer
A strong cast cannot save the bizarrely bad thriller 'Nomis'
By Fiona Underhill, CC2K Staff Writer
"The Secret Life of Pets 2" ~ our favourite pets are back for a worthy sequel
By Peggy Marie, Staff Writer for CC2K
"Dark Phoenix" ~ Ending X-Men saga doesn't rise to the occasion
"Trial by Fire" – a decent feel-bad movie with a serious message
Hulu's Shrill is a cathartic balm for everyone who doesn't fit the mold
Tony Lazlo's Thoughts on The New She-Ra
By Tony Lazlo, CC2K Staff Writer
AFI Fest Review: 'I am the Night' preview is a devilish, intriguing tribute to the LA noir
By Adriana Gomez-Weston, CC2K Staff Writer
South Park ruined a generation of young people, but not for the reasons you think
By Robert Hamer, CC2K Staff Writer
The End of Days: Has American Horror Reached its Final Chapter?
Comic Preview: 'The Sequels' questions whether our nostalgia is endangering our future
By Laura Hong-Tuason, CC2K Comics Editor
Todd's back. Oh, joy.
Learning to love comics again
The ordinary life of 'American Splendor' 15 years later
By Jessica Pena, CC2K Staff Writer
'The Death of Superman' is a dumb, boring story based on a lie (and it's okay to finally admit that)
The Six Seasons of Twin Peaks – Robert J. Peterson, Author on The Six Seasons of Twin Peaks
Tony Lazlo's Thoughts on The New She-Ra – Robert J. Peterson, Author on Tony Lazlo's Thoughts on The New She-Ra
Vic on Sex Week: The Allure of the Sex Quiz
Allan Beamer on Disney's 'Christopher Robin' is a silly old bore
Terry Hanks on Review: 'Greta' Is A Terrifying Twisted Delight
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Backstreet Boys' 'DNA' may very well be their best album ever
By Phoebe Raven, CC2K Staff Writer
In Music, Music Current Reviews
August New Music Roundup: 6 Songs you should be listening to
By Ally Johnson, CC2K Music Editor
July New Music Roundup: 7 Songs you should be listening to
In Music, Music RoundUp
Album Review :: Pkew Pkew Pkew :: Self Titled
By Andrea Janov, CC2K Music Editor
In Music Current Reviews
Album Review :: Burst One's Side :: Tight
From Novel to Game: Adapting a Story into a Playable Format
In Books, Books Meta Critiques, Games, Games Essays Etc
TellTale Games and the video game industry's labor crisis
In Games
Review: PlayerUnknown's Battleground: PUBG Global Invitational 2018
In Games, Games Essays Etc
Rebuild 3: Gangs of Deadville
By Adam "ManKorn" Korenman, CC2K Video Games Editor
In Games Current Reviews
The Hype Stuff: How Game Developers Can Learn from Bethesda and Fallout 4
In Games Essays Etc
Q&A: Mark Falkin, author of 'The Late Bloomer'
The Big Picture: The Fight for the Future of Movies
Review: Painless by Jamie Mayer
Review: Slade House by David Mitchell
About CC2K
Founded in 2004, CC2K offers geeks of all stripe an array of portals into pop-culture fandom. Aficionados of movies, TV, books, music, comics and video games all have a home at CC2K, which publishes reviews and in-depth essays about all kinds of media. We attract more than 5 million page views annually.
Email: info@cc2konline.com
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THE M/V STELLA AUSTRALIS & THE M/V VENTUS AUSTRALIS take you on an amazing journey to the "uttermost end of the earth" during a four-night voyage along the winding passages of the fjords of Tierra del Fuego. The natural splendor of Patagonia is on full display all along this route between Ushuaia (Argentina) and Punta Arenas (Chile) as you travel on one of the industry's most comfortable expedition cruise ships, designed to make your trip as pleasant and remarkable as possible through the Strait of Magellan and Beagle Channel. This remarkable journey, traveling through Patagonia along one of the world's most iconic sea routes, runs a broad gamut from glaciers and iceberg-filled fjords to wildlife colonies and historic places where long-ago humans left their imprint on the landscape. You can also enjoy the rare opportunity to walk on legendary Cape Horn (weather permitting) which few others have had.
Full day by day itinerary available on request.
Prices are per person based on two persons sharing a stateroom. Cruise itinerary is subject to change. During the months of May thru August these itineraries are not available due to the winter season. Single rates are available in all types of cabins and the rate will be the 150% of the category rate. Rate does not include port tax, migration fee, and national park fee. The cost per person is $60 for the 4-night cruises and $70 for the 8-night cruises. Children Policy: Infants between 0 and 3 years old have a 100% discount, sharing the bed with their parents. Children between 4 and 12 years old have a 50% discount, sharing a cabin with an adult.


Flintlocks & Fancy, LLC makes Live Action Games.
If you are interested in our Live Action Roleplaying Game (LARP) writing and editing services, please contact us for details. Some more information on our LARP world is provided on the Renaissance World system page.
Contact us by writing a Comment on this site or by emailing us. Our email address is eponymous with the site's website address at gmail.
If you are an artist who has a portfolio of Victorian, Steampunk, Renaissance, Baroque, or Medieval work and are willing to create new works for compensation, please let us know as we have future projects for which your work may be welcomed.
What is a LARP? A LARP is an acronym for "Live Action Roleplaying Game." Essentially the games we present are interactive Murder Mystery Theatre where you play a role, much as an actor plays a role in a play. Our "no contact" games are an excellent introduction for people who love acting but who may have never previously played a Roleplaying Game. If you enjoy acting, you can LARP. A bevy of characters are available for both experienced and not-so experienced actors. Our helpful GMs will be glad to assist you either before or during the game.


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Inhibitory Abilities of Bacillus Isolates and Their Culture Filtrates against the Gray Mold Caused by Botrytis cinerea on Postharvest Fruit
Xiaomeng Chen, Yajie Wang, Yu Gao, Tongguo Gao, Dongdong Zhang
DOI : https://doi.org/10.5423/PPJ.OA.03.2019.0064
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Plant Pathol J > Volume 35(5); 2019 > Article
Chen, Wang, Gao, Gao, and Zhang: Inhibitory Abilities of Bacillus Isolates and Their Culture Filtrates against the Gray Mold Caused by Botrytis cinerea on Postharvest Fruit
The Plant Pathology Journal 2019;35(5):425-436.
Published online: October 01, 2019
DOI: https://doi.org/10.5423/PPJ.OA.03.2019.0064
Xiaomeng Chen†, Yajie Wang†, Yu Gao, Tongguo Gao, Dongdong Zhang*
College of Life Science, Hebei Agricultural University, Baoding 071001, China
*Corresponding author.: Phone) +86-3127528273, FAX) +86-3127528273, E-mail) zhangdongcumt@163.com
Handling Editor : Kim, Hun
(Received on March 25, 2019; Revised on July 10, 2019; Accepted on July 18, 2019)
© The Korean Society of Plant Pathology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Botrytis cinerea, a major phytopathogenic fungus, has been reported to infect more than 200 crop species worldwide, and it causes massive losses in yield. The aim of this study was to evaluate the inhibitory abilities and effects of Bacillus amyloliquefaciens RS-25, Bacillus licheniformis MG-4, Bacillus subtilis Z-14, and Bacillus subtilis Pnf-4 and their culture filtrates and extracts against the gray mold caused by B. cinerea on postharvest tomato, strawberry, and grapefruit. The results revealed that the cells of Z-14, culture filtrate of RS-25, and cells of Z-14 showed the strongest biocontrol activity against the gray mold on the strawberry, grape, and tomato fruit, respectively. All the strains produced volatile organic compounds (VOCs), and the VOCs of Pnf-4 displayed the highest inhibition values. Based on headspace solid-phase microextraction in combination with gas chromatography-mass spectrometry, esters accounted for the largest percentage of the VOCs produced by RS-25, MG-4, Z-14, and Pnf-4 (36.80%, 29.58%, 30.78%, and 36.26%, respectively). All the strains showed potent cellulase and protease activities, but no chitinase activity. RS-25, Z-14, and MG-4, but not Pnf-4, grew on chrome azurol S agar, and an orange halo was formed around the colonies. All the strains showed biofilm formation, fruit colonization, and lipopeptide production, which may be the main modes of action of the antagonists against B. cinerea on the fruit. This study provides the basis for developing natural biocontrol agents against the gray mold caused by B. cinerea on postharvest fruit.
Keywords: Bacillus isolate, Botrytis cinerea, gray mold, inhibitory ability, postharvest fruit
Botrytis cinerea is a major phytopathogenic and necrotrophic fungus that causes the gray mold disease (Fekete et al., 2012). The fungus has been reported to infect more than 200 crop species worldwide, such as grapes, stone fruit, berries, and vegetables, and the losses in yield before and after harvest are extensive (Williamson et al., 2007). B. cinerea is not host-specific, and its virulence varies in different plant hosts (Derckel et al., 1999; Mirzaei et al., 2009). Because it has a broad host spectrum and causes significant economic losses, B. cinerea has been considered as the second most important fungal pathogen worldwide (Dean et al., 2012).
In particular, postharvest losses due to fungal infections are more important for highly perishable fresh fruit than for field crops. Generally, postharvest losses are attributed to pathological decomposition due to fungal and bacterial infections; pathophysiological damage during storage due to excessive cooling or lighting or abnormalities in the atmospheric gaseous components; and physical damage, such as mechanical damage (Schnaubelt, 2005).
Synthetic fungicides are commonly used to protect crops, especially fruit, in the postharvest period. However, effective and safe non-fungicides are urgently required to control postharvest pathogens because synthetic fungicide residues are toxic to humans and the environment (Conway et al., 2005; Droby, 2006; Soylu et al., 2010). Therefore, researchers worldwide are trying to develop safe, natural, and biodegradable alternatives to replace synthetic fungicides (Grzegorczyk et al., 2017; Nalinia and Parthasarathi, 2014).
Previous studies have confirmed the efficacy of biocontrol agents against several postharvest phytopathogenic fungi, such as species that belong to the genera Botrytis, Penicillium, and Monilinia (Bautista-Rosales et al., 2013; Mari et al., 2012; Parafati et al., 2015; Platania et al., 2012). Species that belong to the genus Bacillus can form endospores, which ensures the feasibility of commercial products with low storage requirements but a long shelf life (Posada et al., 2016). Thus, Bacillus strains can colonize the surface or wounds of postharvest fruit and produce broad-spectrum antibiotics that suppress a variety of plant pathogens (Stein, 2005). Bacillus subtilis V26 displays high antifungal activity against several fungi, including B. cinerea, and has powerful chitosanase activity (Kilani-Feki et al., 2016). Bacillus atrophaeus CAB-1 displays high inhibitory activity against various fungal pathogens and suppresses cucumber powdery mildew and tomato gray mold. All the compounds extracted from CAB-1 (e.g., C15-C17 fengycin A, a protein, and volatile compounds) also effectively prevented the occurrence of the cucumber powdery mildew caused by Sphaerotheca fuliginea under greenhouse conditions (Zhang et al., 2013). Bacillus cereus S42 screened from Nicotiana glauca plants native to the Tunisian Centre-East demonstrated a strong ability to suppress tomato Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici and showed proteolytic and chitinolytic activities (Abdallah et al., 2016).
In this study, we have reported the efficacy of four antagonists or their culture filtrates in controlling the gray mold caused by B. cinerea on postharvest fruit and discussed their ability to colonize the wounds on the fruit and synthesize antifungal metabolites.
Growth of bacterial isolates and production of culture filtrates
Bacterial isolate RS-25 from jujube fruit, MG-4 from strawberry fruit, Z-14 from wheat rhizosphere soil and Pnf-4 from wheat plant significantly inhibited the growth of various phytopathogenic fungi and were maintained at 4°C on streak-inoculating nutritive agar (NA) slants (Zhang et al., 2017a, 2017b). The strains were cultured overnight at 37°C on NA slants, transferred to Erlenmeyer flasks containing 50 ml of the seed culture medium, and shaken at 220 rpm for 48 h at 37°C. The vegetative cells and supernatant were obtained after centrifugation at 10,000 ×g for 15 min. The cells were suspended in sterile distilled water (SDW) and adjusted to the appropriate concentration (1 × 108 cells/ml), and the supernatant was filtered through a 0.22-μm syringe to obtain the culture filtrate.
Antifungal activities of the Bacillus isolates and their culture filtrates against B. cinerea
Using the dual-culture in vitro assay described by Kilani-Feki et al. (2016), the antifungal activities of the four isolates against B. cinerea were evaluated. A single 5-mm-diameter mycelial disc of the test fungus was placed at the center of potato dextrose agar (PDA) plates. The samples were treated with the bacterial isolates at four symmetrical sites, a distance of 3 cm from the center of the plate, and then allowed to grow for 5 days at 27°C in darkness. The antifungal activity of the culture filtrate was measured using a diffusion plate assay modified from that described by Pretorius et al. (2015). Four evenly spaced wells (7 mm in diameter) were made 2.5 cm from the center of PDA plates containing 40 μg/ml of streptomycin sulfate. A plug of B. cinerea was placed at the center of the plate, and an aliquot (50 μl) of the filtrate or sterile water (control) was added to each well. The plates were incubated at 27°C, and the inhibition zones were measured after clear halos became visible.
Extraction of lipopeptides produced by the antagonists
HCl (6 M) was used to lower the pH of the culture filtrates of the four antagonistic bacteria to 2. The filtrates were maintained overnight at 4°C and centrifuged to isolate the precipitate, which was then washed twice using diluted HCl (pH 2) and extracted twice using methanol (Zhang et al., 2017b). The extract was dried using a rotary vacuum evaporator and resuspended in an equivalent volume of sterile water to detect the biocontrol effects of the extract in controlling the gray mold caused by B. cinerea on postharvest fruit.
Efficacy of the antagonists and their culture filtrate and extracts in controlling the gray mold caused by B. cinerea on postharvest fruit
To evaluate the efficiency of the four antagonist bacteria, RS-25, MG-4, Z-14, and Pnf-4, and their culture filtrates and extracts in controlling the gray mold on the postharvest fruit of tomato, strawberry, and grape, the in vivo assay reported by Panebianco et al. (2014) and Parafati et al. (2015) was used with slight modifications. Tomato (Lycopersicon esculentum Mill. 'Qianxi'), strawberry (Fragaria × ananassa Duch. 'Shuangliu'), and grape (Vitis vinifera L. 'Red Globe') fruit were obtained from commercial orchards in Baoding, China. In this study, the tomato, strawberry, and grapefruit did not receive any preharvest fungicide treatment, and only healthy and homogenous fruit were selected. Before treatment and inoculation, the fruits were washed with tap water, superficially disinfected for 1 min in 0.1% (v/v) sodium hypochlorite, rinsed with SDW, and air-dried at room temperature.
A sterilized needle was used to make artificial wounds (0.3 cm in depth × 0.5 cm in width) at equatorial areas of the fruit (2 wounds on each fruit). A drop (10 μl; 1 × 106 conidia/ml) of B. cinerea was inoculated in each wound and air-dried for 2 h; then, another drop (10 μl; 1 × 108 cells/ml) of each bacterium (culture filtrate or extract) was added to each wound. The fruits were placed in plastic packaging cartons (5 fruit/carton) and incubated at 25°C and 95% relative humidity for 5 days. Fruits treated with SDW and infected with the pathogen were used as the negative control. Three replicates of 5 fruit each were used per treatment (15 fruit/treatment), and the experiment was repeated thrice. The disease index (DI) and disease reduction (DR) of the gray mold were calculated according to the formulas published by Gong et al. (2017). The degree of disease severity due to inoculation with B. cinerea was calculated as follows: 0 = no lesion; 1 = a few scattered lesions covering < 2% of the fruit surface; 2 = extensive lesions covering > 2% but < 5% of the fruit surface; 3 = extensive lesions covering > 5% but < 25% of the fruit surface, and 4 = extensive lesions covering > 25% of the fruit surface. The DI was calculated using the following formula:
DI=(∑i=04ni×i)/(4×∑i=04ni)×100%
Where i is the disease severity (0-4), and ni is the number of fruit with a severity of i. The DR was calculated using the following formula: DR (%) = 100 × (DI-Bc − DI-test)/DI-Bc. Where Bc is B. cinerea.
In vitro production of volatile organic compounds
A dual-culture method was used to assess the efficacy of volatile organic compounds (VOCs) produced by the antagonists in decreasing the growth of B. cinerea (Gao et al., 2017; Grzegorczyk et al., 2017). Twenty-microliter aliquots of the bacterial suspensions (108 cells/ml) were seeded on NA plates and incubated at 37°C for 24 h. Then, discs (5-mm circular plugs) of actively growing fungal mycelia of B. cinerea were placed at the center of Petri dishes with PDA. Each pathogen plate was covered with a plate containing a bacterial culture incubated for 24 h. Three plates were used for each antagonist, and unseeded PDA plates were used as the negative control. The two plates were superimposed with Parafilm around the edges to prevent the leakage of air and incubated at 25°C. The mycelial diameter was measured after 5 days, and the rate of inhibition of radial mycelial growth was calculated using the equation published by Gao et al. (2018). Three replicates were used for each treatment, and the experiment was repeated thrice. VOCs from the four antagonists were analyzed using solid-phase micro extraction-gas chromatography-mass spectrometry (SPME-GC-MS). For collecting the VOCs, the antagonists were inoculated in NA medium with 25 ml of headspace for 7 days at 28°C. The NA medium without the antagonistic bacteria was used as the control. DVB/CAR/PDMS fiber (Supelco, Inc., Bellafonte, PA, USA) was applied for headspace SPME of the VOCs at 55°C for 40 min. The SPME fiber was immediately inserted into the injection port of the gas chromatography-mass spectrometry (GC-MS; Agilent 7820A-5977E, Santa Clara, CA, USA) for thermal desorption at 250°C for 5 min with a DB-5MS column (30 m × 0.25 mm i.d. × 0.25 μm film thickness; Agilent J&W Scientific, Folsom, CA, USA). The initial oven temperature was 60°C for 2 min, and it was increased to 100°C at a rate of 10°C/min, then increased to 180°C at a rate of 5°C/min, and further increased to 240°C at a rate of 10°C/min and held for 5 min. The mass spectrometer was operated in the electron ionization mode (70 eV). The ion source, quadruple, and GC-MS interface temperatures were set at 230°C, 150°C, and 250°C, respectively. Mass spectrometry (MS) was performed in the full-scan mode with the m/z ranging from 50 to 400 for the analysis of VOCs. The mass spectra data of the volatile compounds were compared with those in the NIST/EPA/NIH Mass Spectrometry Library and Mainlib and/or Replib databases (Gao et al., 2017).
Detection of the in vitro activities of protease, chitinase, and cellulase of the antagonists
The protease activity of the antagonists, indicated by casein degradation, was measured by inoculation on skimmed milk agar with the cross-line method, and the width of each halo was measured as an indicator of the level of protease activity (Yang et al., 2008). The cellulase activity of the antagonists was determined by observing the widths of the clear zones on carboxymethyl cellulose (CMC) agar plates (Ghose, 1987). The chitinase activity of the antagonists was determined by measuring the widths of the clear zones on chitin agar (CA) plates (Roberts and Selitrennikoff, 1988). To measure the activity of the metabolites secreted by the antagonists, four evenly spaced wells (7 mm in diameter) were formed 2.2 cm from the center of the plate by excision, and a 30-μl aliquot of the culture filtrate was added to each well.
Qualitative estimation of siderophore production
The chrome azurol S (CAS) assay was performed to evaluate the effectiveness of the Bacillus antagonists in siderophore production (Kurabachew and Wydra, 2013; Schwyn and Neilands, 1987). After 5 days at 28°C, the siderophore-producing isolates generated orange zones, and the diameter of the clear zone of these colonies was visually rated. Siderophore production in the culture filtrate was measured as described above.
Biofilm formation by the antagonists
The antagonists were cultured in nutritive broth and shaken to midexponential growth; then, 10 μl of the cultured medium was transferred to 1 ml of minimal salts glycerol glutamate (MSgg) medium (Branda et al., 2001) in 24-well polyvinylchloride microtiter plates (Thermo Fisher Scientific, Waltham, MA, USA) and incubated under stationary conditions at 37°C for 4 days (Dietel et al., 2013).
Colonization of the wound sites by the antagonists
The ability of the antagonists to colonize the wounds on the grape, strawberry, and tomato fruit was tested using previously described and partially modified procedures (Cirvilleri et al., 2005; Parafati et al., 2015). A sterile needle was used to make artificial wounds (4 wounds/fruit) on superficially sanitized fruit, and 10 μl aliquots of the bacterial suspensions (1 × 106 cells/ml) were individually inoculated into the wounds; the fruit were incubated for 5 days under the conditions mentioned previously (6 fruit/box). Tissue samples containing the whole wound were cut using a sterile knife after inoculation for 0, 24, 48, 72, 96, and 120 h. The samples were weighed, crushed with a sterilized mortar and pestle, serially diluted, and plated on NA. The bacterial colonies were counted after overnight culture at 37°C to calculate the mean colony forming units (log10 cfu) on the basis of fresh weight. Three replicates of each fruit type were used per treatment (6 fruit per treatment), and the experiment was repeated twice.
Identification of lipopeptides produced by the antagonists
The methanol extracts of the culture filtrates of the four antagonistic bacteria were analyzed using the 5800 matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometer (AB SCIEX, Redwood, CA, USA) operated in the positive reflectron mode; α-cyano-4-hydroxycinnamic acid was used as the matrix (prepared by dissolving 5 mg in 1 ml of 50:50 acetonitrile: water containing 0.1% trifluoroacetic acid) (Velho et al., 2011).
Identification of the antagonist isolates
Genomes extracted from the antagonists isolates were analyzed for the 16S rRNA gene sequence by using universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1495R (5′-CTACGGCTACCTTGTTACGA-3′), that amplified a 1,400-bp region of the 16S rRNA gene. Phylogenetic analysis was conducted using maximum likelihood in MEGA 5.10 (Tamura et al., 2011). The topology of the phylogenetic tree was evaluated by 1,000 resamplings.
The data from the replicates were expressed as mean ± SD values. SPSS version 17.0 software package (SPSS Inc., Chicago, IL, USA) was used to perform the calculations and comparisons of the treatment means for each experiment. Duncan's test (P ≤ 0.05) and one-way analysis of variance were used to determine whether the means differed significantly.
Activity of the antagonists and their culture filtrates and extracts in controlling the gray mold on the postharvest fruit
The four isolates, RS-25, MG-4, Z-14, and Pnf-4, and their culture filtrates all demonstrated significant antifungal activities against B. cinerea. The culture filtrate of RS-25 demonstrated the strongest antifungal activity, whereas that of Pnf-4 was the weakest (Fig. 1). The efficacies of RS-25, MG-4, Z-14, and Pnf-4 and their culture filtrates and extracts against the gray mold caused by B. cinerea on the postharvest tomato, strawberry, and grapefruit are reported in Table 1 and Fig. 2. Generally, Z-14, MG-4, and their culture filtrates and extracts were more active against B. cinerea. Pnf-4, RS-25, and their culture filtrates and extracts were relatively ineffective in reducing the DI of the strawberry fruit. The DR of B. cinerea was observed to be the highest (82.14) when the strawberry fruits were treated with the cells of Z-14, whereas the culture filtrate of RS-25 was the most ineffective and yielded the lowest DR value (55.08). The DR values of the tomato fruit treated with Z-14, RS-25, and the culture filtrate and extract of Z-14 were higher (82.62%, 80.46%, 79.25%, and 75.23%, respectively) than those of the tomato fruit treated with Pnf-4, MG-4, and the culture filtrate of RS-25 (68.65%, 67.65%, and 67.29%, respectively). The DR values of the tomato fruit treated with the culture filtrates of Pnf-4 and MG-4 were the lowest (55.27% and 54.03%, respectively). The DR values of the grapefruit treated with MG-4 and RS-25 and culture filtrates of Pnf-4, MG-4, and RS-25 were the highest (84.69%, 86.66%, 83.24%, 86.28%, and 86.81%, respectively), whereas the culture filtrate of Z-14 showed the lowest efficiency in controlling the gray mold (12.20%) on grapefruit.
Inhibitory effects of VOCs produced by the antagonists
The variable inhibition efficiency of the VOCs produced by RS-25, MG-4, Z-14, and Pnf-4 against the mycelial growth of B. cinerea is shown in Fig. 3. The highest inhibition values were detected for Pnf-4 against B. cinerea (70.71 ± 3.94%), followed by RS-25 (62.57 ± 3.26%) and MG-4 (53.44 ± 2.71%). In contrast, the VOCs of Z-14 were the most ineffective in reducing the mycelial growth of B. cinerea (24.21 ± 1.85%). Based on the headspace SPME-GC-MS, 109 potential VOCs produced by RS-25, 93 VOCs by MG-4, 92 by Z-14, and 91 by Pnf-4 were identified. The main components of the VOCs were ketone, alcohol, aldehyde, olefin, ester, alkane, and phenol, and esters accounted for the highest percentage in the VOCs produced by RS-25, MG-4, Z-14, and Pnf-4 (36.80%, 29.58%, 30.78%, and 36.26%, respectively) (Table 2).
Enzyme activity and siderophore production assays
All the tested Bacillus strains and their culture filtrates were able to hydrolyze CMC, and they produced obvious clear zones in the CMC agar plates. The culture filtrate of Z-14 exhibited the strongest cellulase activity; the diameter of the clear zone was 3.4 cm, whereas those of RS-25, Pnf-4, and MG-4 were 2.4 cm, 2.3 cm, and 2.8 cm, respectively (Fig. 4A and B). The four antagonists and their culture filtrates all exhibited potent protease activity, and the hydrolysis zones of RS-25, Z-14, and MG-4 were relatively bigger than that of Pnf-4; this is similar to the protease activity results of the culture filtrates of the antagonists (Fig. 4C and D). The four antagonists and their culture filtrates did not produce clear zones in CA plates, which demonstrated they did not synthesize chitinase. These results are consistent with the findings of Ren et al. (2013), who suggested that Bacillus pumilus JK-SX001 does not secrete chitinase but instead secretes protease and cellulase.
Biofilm formation and colonization of fruit
All four antagonists exhibited the ability to form biofilms on the inert surface of liquid MSgg medium and evolved into whitish aerial structures that appeared to be dry and had a cotton-like texture (Fig. 4G). The population dynamics of RS-25, MG-4, Z-14, and Pnf-4 strains on artificially wounded tomato, strawberry, and grapefruit are shown in Fig. 5. The population trends of the antagonists in the wound tissue of tomato fruit, starting from a similar concentration (log10 cfu/g of tissue = 3.50), remained relatively stable for a period of 24 h; increased for MG-4, Z-14, and Pnf-4 and a few drops of RS-25 up to 72 h; and were similar (log10 cfu/g of tissue = 4.30) after 120 h of incubation (Fig. 5A). The population trend of the antagonists in the wound tissue of strawberry fruit showed fluctuations to a certain extent during 120 h after inoculation; RS-25 showed the highest concentration (log10 cfu/g of tissue = 3.76), whereas MG-4 showed the lowest concentration (log10 cfu/g of tissue = 3.06) (Fig. 5B). The population trend of the antagonists in the wound tissue of grapefruit, starting from similar concentrations, ranged from 3.11 (log10 cfu/g of tissue) for MG-4 to 3.37 (log10 cfu/g of tissue) for Pnf-4; growth appeared to be quite stationary up to 24 h, but it showed variations after that period. The population densities of Z-14 and Pnf-4 increased significantly (log10 cfu/g of tissue = 4.07 and 4.05, respectively), whereas the population density of MG-4 remained relatively stable (log10 cfu/g of tissue = 3.10) and that of RS-25 showed a notable decline (log10 cfu/g of tissue = 2.41) (Fig. 5C). The results prove that the population densities of the four antagonists in the three types of fruit showed different colonization capacities. The colonization abilities of the antagonists were similar in the wounds of the tomato fruit but different in those of the strawberry and grapefruit. The colonization ability of RS-25 was the strongest in the wounds of the strawberry fruit, but weakest in those of the grapefruit.
MALDI-TOF mass spectrometry was used to analyze the lipopeptide antibiotics produced by the antagonists. The mass spectra of the crude extract from RS-25 revealed two major clusters with peaks at m/z 1,016.69-1,074.70 Da, which correspond to the isoforms of surfactin B, and peaks at m/z 1,433.88-1,529.88 Da, which correspond to the isoforms of fengycin (Fig. 6A). The mass spectra of the crude extract from Pnf-4 revealed three major clusters with peaks at m/z 1,030.70-1,074.72 Da, which correspond to surfactin B, peaks at m/z 1,079.62-1,125.57 Da, which correspond to iturin A, and peaks at m/z 1,435.87-1,543.88 Da, which correspond to fengycin (Fig. 6B). Surfactin B, iturin A, and fengycin were detected from Z-14 (Fig. 6C), and iturin A and fengycin, from MG-4 (Fig. 6D).
Identification of the antagonists
When compared with the GenBank database, the 16S rRNA sequences of the strains were matched to those of Bacillus spp. The constructed unrooted phylogenetic tree revealed that antagonist Pnf-12 was most similar to B. subtilis, according to the alignment results. Antagonist RS-25 was identified as Bacillus amyloliquefaciens; MG-4, Bacillus licheniformis; and Z-14, B. subtilis (Fig. 7).
Previous studies have shown that biological control, such as the application of antagonist microorganisms or their secondary metabolites, is a promising method for decreasing the decay of harvested fruit (Li et al., 2016; Wu et al., 2017). Postharvest treatments with antagonist microorganisms such as Bacillus (Gao et al., 2017; Zhang et al., 2013), Clonostachys rosea (Gong et al., 2017), and yeast strains (Parafati et al., 2015) have been reported to suppress the development of B. cinerea and decay of harvested fruit.
In this study, the main biocontrol modes of action of the four Bacillus antagonists against the in vitro growth of B. cinerea, such as biofilm formation; competition for iron; and production of VOCs, cell-wall-degrading enzymes, and lipopeptide antibiotics, were evaluated on the basis of the mechanisms that play a significant role in the biocontrol activities of antagonistic bacteria. Secondary metabolites play key roles in the antifungal activities of antagonistic bacteria; however, the production of these antagonistic metabolites is greatly affected by the environment (Zhang et al., 2017a). The direct use of antimicrobial substances synthesized by microorganisms as the basis of microbial pesticides can overcome such limitations (Kilani-Feki et al., 2016). Antifungal substances readily decompose and do not pollute the environment (Gu et al., 2017). No significant differences in biocontrol activities were observed between the antagonists and their culture filtrates against the gray mold on the strawberry fruit; however, the crude extract of Z-14 showed lower efficiency than the cells and culture filtrate. The biocontrol efficiency of Z-14, including that of its culture filtrate and extract, was the lowest of the four antagonists on the grapefruit, but the extract of Z-14 showed higher efficiency than the vegetable cells. The vegetable cells of the antagonists demonstrated higher biocontrol activities against the gray mold on the tomato fruit than their culture filtrates and extracts (Table 1, Fig. 2). The different biocontrol activities of the antagonists in the three fruits showed that the effects of the antagonists were influenced by the host fruit.
There is limited information on the antifungal activities of VOCs from microorganisms. Naturally occurring VOCs of endophytic bacteria are regarded as sources of new antifungal agents that are safe for humans and the environment and do not have a harmful influence on the host plants (Gao et al., 2017). Microbial VOCs inhibit the growth of pathogenic fungi; improve plant growth (Park et al., 2015); mediate relationships, interactions, and communications between organisms (Cernava et al., 2015); identify bacterial species; and induce systemic resistance in plants (Zamioudis et al., 2015). GC-MS has revealed that 29 unique VOCs produced by Bacillus velezensis ZSY-1 isolated from Chinese catalpa exhibit significant antifungal activity against plant pathogenic fungi (Gao et al., 2017). VOCs may act in both direct and indirect modes during the interaction between bacterial agents and fungal pathogens (Zhang et al., 2013). The antifungal activity of the VOCs of Z-14 was the weakest of the four antagonists; however, Z-14 displayed the strongest biocontrol effect on the strawberry and tomato fruit. This may indicate that the production of VOCs is not the main mechanism exhibited by Bacillus strains against plant pathogenic fungi.
Aureobasidium pullulans (three strains) and Wickerhamomyces anomalus (four strains) have been tested for extracellular lytic enzyme activity, and all seven strains showed β-1,3-glucanase and protease activities but no chitinase activity (Parafati et al., 2015). Physical damage of the hypha prevents its penetration into the host cell wall. Loss of turgidity and tearing of the pathogen hyphae may both be attributable to cellulase and protease activities. These metabolites may be involved in the control of phytopathogenic fungi through mycoparasitism, which is characterized by colonization of pathogen hyphae (Grzegorczyk et al., 2017). RS-25, Z-14, and MG-4 grew on CAS agar; an orange halo was formed around the colonies, except those of Pnf-4; this is consistent with the results of the culture filtrates of these antagonists (Fig. 3E and F). Generally, siderophores are produced by microorganisms to bind Fe3+ from the environment, transport it to the microbial cell, and make it available for growth (Leong, 1996). Therefore, competition for iron may also be a possible mechanism for controlling phytopathogens (Xue et al., 2013).
Generally, biofilm formation is regarded as a major mechanism of antagonists against plant pathogenic fungi, as biofilms act as physical and chemical barriers (Díaz Herrera et al., 2016; Dietel et al., 2013). Haggag and Timmusk (2008) showed that Paenibacillus polymyxa strains produced biofilms to control the crown root rot disease, which is caused by Aspergillus niger. A previous study has shown that W. anomalus and Metschnikowia pulcherrima strains were able to adhere to polystyrene plates and maintain a high film-forming capacity after 48 h and 72 h of incubation, even after repeated washes (Parafati et al., 2015).
The presence of a high number of antagonists in the wounded tissues would protect the plants or fruit from pathogens because tissue colonization by biocontrol agents is critical for the effective control of phytopathogens (Zeriouh et al., 2014). The antagonists can successfully compete with the phytopathogens for the niches and nutrients, such as organic compounds, that are essential for the reactivation of propagules and/or subsequent proliferation and colonization of tissues (Budiharjo et al., 2014). The colonization capacities of antagonists in tissues may have a key role in disease suppression, even if competition or antibiosis occurs between the antagonists and pathogens (Huang et al., 2013). The four antagonists showed similar colonization abilities but different biocontrol effects against the gray mold in the tomato fruit, which indicates that secondary metabolites may play a major role in the control activity (Table 1, Figs. 2 and 5A). The colonization abilities and control activities of the antagonists did not have a direct correlation in the strawberry and grapefruit. RS-25 displayed the strongest colonization ability and lowest biocontrol effect in the strawberry fruit (Table 1, Figs. 2 and 5B), but the strongest biocontrol effect and lowest colonization ability in the grapefruit (Table 1, Figs. 2 and 5C).
Lipopeptide antibiotics, such as iturins, fengycins, and surfactins, are an important class of antifungal substances synthesized by Bacillus (Moyne et al., 2004). Lipopeptide antibiotics have direct or indirect inhibitory effects on fungal plant pathogens, but they are not toxic to the plant itself (Ongena and Jacques, 2008; Zhang et al., 2017b). MALDI-TOF mass spectrometry can be easily and conveniently used for component identification (Sandrin et al., 2013; Xie et al., 2012). This study showed that MALDI-TOF mass spectrometry could become a new, sensitive, and rapid technique for detecting metabolites. MALDI-TOF mass spectrometry has been used to show that B. amyloliquefaciens FZB42 promotes plant growth, suppresses plant pathogenic organisms in the rhizosphere of plants, and synthetizes and excretes bacillomycin D, fengycin, and surfactin (Koumoutsi et al., 2004). Three types of lipopeptides, surfactin A, iturin A, and fengycin, produced by B. subtilis CMB32 isolated from the soil by Kim et al. (2010) have been reported for their potential to control the anthracnose disease caused by Colletotrichum gloeosporioides. Lipopeptide antibiotics are relatively stable in nature and resistant to acids and bases, which ensure that the lipopeptide antibiotics can exert antagonistic effects over a long period (Fan et al., 2017; Guo et al., 2014). Iturins and fengycins exhibit a wide range of strong inhibitory activities against fungal plant pathogens. Romero et al. (2007) concluded that the iturin and fengycin families of lipopeptides have a major role in the antagonism of B. subtilis towards Podosphaera fusca, which is the main causal agent of cucurbit powdery mildew in Spain. Fengycin-type lipopeptides produced by B. subtilis NCD-2 were the main antifungal compounds that restricted the population of Rhizoctonia solani in the cotton rhizosphere and suppressed cotton damping-off disease (Guo et al., 2014).
In summary, the cells of Z-14, culture filtrate of RS-25, and cells of Z-14 showed the strongest biocontrol activity against the gray mold on the postharvest strawberry, grape, and tomato fruit, respectively. Biofilm formation; strong colonization ability in fruit tissues; and subsequent production of VOCs, cellulase and protease, siderophores, and lipopeptide antibiotics may be the main modes of action of the antagonist bacteria against B. cinerea on the postharvest fruit. This study provides theoretical support and material basis for developing agents against the gray mold caused by B. cinerea on postharvest fruit.
We would like to thank the native English-speaking scientists of Elixigen Company (Huntington Beach, California) for editing our manuscript. This work was supported by the Hebei Provincial Natural Science Foundation of China (grant number C2014204027).
Antifungal activities of the antagonists (A) and their cultural filtrates (B) against Botrytis cinerea.
Efficacy of the vegetable cells, culture filtrates, and crude extracts of the antagonists in controlling the gray mold caused by Botrytis cinerea on postharvest fruit (strawberry, tomato, and grape). VC, vegetable cells; CF, culture filtrate; CE, crude extract.
Evaluation of the efficacy of volatile organic compounds produced by the antagonists in reducing the growth of Botrytis cinerea.
Detection of the in vitro activities of cellulase, protease, and chitinase; siderophore production; and biofilm formation by the antagonists and their culture filtrates. (A, C) The in vitro activities of cellulase and protease of the antagonists. (B, D) The in vitro activities of cellulase and protease of the culture filtrate. (E, G) Siderophore production and biofilm formation by the antagonists, respectively. (F) Siderophore production by the culture filtrate; 1-4 represent antagonists RS-25, Pnf-4, Z-14, and MG-4, respectively.
Population dynamics of the antagonists RS-25, Pnf-4, Z-14, and MG-4 in the wound tissue of tomato fruit (A), strawberry fruit (B), and grapefruit (C). Vertical bars indicate the standard error of the mean. cfu, colony forming unit.
Fig. 6 (A-D)
Mass spectroscopy analysis of the lipopeptides produced by the antagonists.
Unrooted phylogenetic tree based on the 16S rRNA sequences of the antagonists.
Efficacy of the vegetable cells, culture filtrates, and crude extracts of the four antagonists in controlling the gray mold caused by Botrytis cinerea on strawberry, grape, and tomato fruit in a greenhouse
DR (%)
Pathogen control 67.03 ± 4.75 a - 70.18 ± 5.77 a - 62.13 ± 4.61 a -
Vegetable cell of Z-14 11.97 ± 1.47 d 82.14 40.40 ± 6.70 d 42.43 10.80 ± 2.57 f 82.62
Culture filtrate of Z-14 14.03 ± 1.43 d 79.07 61.62 ± 4.85 b 12.20 12.89 ± 3.35 ef 79.25
Crude extract of Z-14 19.47 ± 1.90 c 70.95 52.31 ± 4.43 c 25.46 15.39 ± 3.01 def 75.23
Vegetable cell of Pnf-4 25.19 ± 2.92 b 62.42 21.09 ± 5.52 e 69.95 19.48 ± 3.08 cde 68.65
Culture filtrate of Pnf-4 26.71 ± 2.53 b 60.15 11.76 ± 3.13 f 83.24 27.79 ± 4.47 b 55.27
Crude extract of Pnf-4 27.75 ± 1.73 b 58.60 19.69 ± 3.81 e 71.94 25.25 ± 2.58 bc 59.36
Vegetable cell of MG-4 15.42 ± 3.13 cd 77.00 10.75 ± 3.11 f 84.69 20.10 ± 4.36 cd 67.65
Culture filtrate of MG-4 13.75 ± 2.61 d 79.49 9.63 ± 2.93 f 86.28 28.56 ± 4.32 b 54.03
Crude extract of MG-4 19.42 ± 1.95 c 71.03 14.58 ± 2.94 ef 79.22 25.94 ± 4.04 bc 58.25
Vegetable cell of RS-25 26.41 ± 1.61 b 60.60 9.36 ± 2.87 f 86.66 12.14 ± 3.58 f 80.46
Culture filtrate of RS-25 30.11 ± 3.46 b 55.08 9.26 ± 2.38 f 86.81 20.32 ± 4.94 cd 67.29
Crude extract of RS-25 29.15 ± 3.55 b 56.51 17.06 ± 2.08 ef 75.69 24.78 ± 4.39 bc 60.12
Values are mean ± SD of three replicates per treatment (5 fruit each). As determined by Fisher's protected least significant difference test, the means within a column followed by different letters are significantly different from each other at P = 0.05.
DI, disease index; DR, disease reduction.
Volatile organic compounds (VOCs) produced by the antagonists
Possible VOCs
MG-4
Pnf-4
Relative peak area (%)
Ketone 35 15.08 21 8.75 12 3.23 19 5.27
Alcohol 5 5.97 11 2.88 18 8.33 5 0.95
Aldehyde 5 7.02 8 1.12 6 5.37 1 0.94
Olefin 5 1.41 3 0.50 4 0.44 3 0.09
Ester 18 36.80 22 29.58 19 30.78 29 36.26
Alkane 30 29.71 17 4.32 16 8.86 30 16.24
Phenol 2 4.41 3 5.57 1 3.20 1 0.26
Others 9 14.22 8 2.47 16 4.94 3 0.08
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Johns Hopkins University Press, 9780801890635, 300pp.
Battling bad guys. High-tech hideouts. The gratitude of the masses. Who at some point in their life hasn't dreamed of being a superhero? Impossible, right? Or is it?
Zehr discusses the physical training necessary to maintain bad-guy-fighting readiness while relating the science underlying this process, from strength conditioning to the cognitive changes a person would endure in undertaking such a regimen. In probing what a real-life Batman could achieve, Zehr considers the level of punishment a consummately fit and trained person could handle, how hard and fast such a person could punch and kick, and the number of adversaries that individual could dispatch. He also tells us what it would be like to fight while wearing a batsuit and the amount of food we'd need to consume each day to maintain vigilance as Gotham City's guardian.
A fun foray of escapism grounded in sound science, Becoming Batman provides the background for attaining the realizable--though extreme--level of human performance that would allow you to be a superhero.
E. Paul Zehr is a professor of neuroscience and kinesiology at the University of Victoria, British Columbia, where he is also a biomedical research scholar. He holds black belts in both empty hand and armed martial arts. For more information about finding your inner superhero, visit www.becomingbatman.com.


text:
Spartans edge Stanford
PASADENA, Calif. – Connor Cook passed for a career-high 332 yards and hit Tony Lippett with a tiebreaking 25-yard touchdown pass early in the fourth quarter, leading No. 4 Michigan State to a 24-20 victory over No. 5 Stanford on Wednesday night in the 100th Rose Bowl.
Cook also threw a TD pass to Trevon Pendleton, and Jeremy Langford rushed for 84 yards and a score in the first Rose Bowl victory in 26 years for the Big Ten champion Spartans (13-1), who finished the season with 10 straight wins.
Michigan State's defense capped its dominant season with one more old-school, smash-mouth performance during the centennial celebration of the Granddaddy of Them All.
"It's a special time for all Spartans, and we came here in force," coach Mark Dantonio said. "I'm very happy for our football team, the resilience we showed all season long."
The nation's best defense ended it by stopping the Pac-12 champion Cardinal (11-3) on fourth-and-1 with 1:46 to play, utterly stuffing a run play up the middle. Kyler Elsworth, who started in place of suspended senior leader Max Bullough, hurdled the pile to deliver an electrifying, head-on hit to fullback Ryan Hewitt.
"When I saw their offensive linemen's stance, I knew the way to make a play was to go over the top," said Elsworth, selected the game's defensive MVP.
Tyler Gaffney ran for 91 yards and an early TD for Stanford, and linebacker Kevin Anderson returned an interception 40 yards for a score late in the first half. But the Cardinal couldn't follow up last season's victory in Pasadena with back-to-back Rose Bowl wins, managing just three points from their offense after the first quarter.
The Spartans have long labored in the shadow of Michigan and Ohio State, but coach Dantonio's seven-year rebuilding project in East Lansing has put them on top of the Midwest this season with an unbeaten run through conference play.
After knocking off the unbeaten Buckeyes in the league title game, Michigan State earned only the Big Ten's second Rose Bowl win since 2000, even rallying from its first double-digit deficit of the entire season to do it.
Cook led the way in his own inimitable fashion, making incredible plays and huge mistakes along the way. Along with his costly interception to Anderson, he also threw two passes that went through the hands of Cardinal defenders, and an interception in the third quarter was wiped out by a defensive holding call.
But when the Spartans needed big plays in the second half, Cook repeatedly delivered, finishing 22 for 36. Stanford's Kevin Hogan beat Wisconsin in last year's Rose Bowl, but couldn't match Cook, going 10 for 18 for 143 yards.


On Sunday we said goodbye to our youngest son at the entrance to Hellenic College, a college that has shaped him through academia,service, friendship, and most importantly – faith.
We said goodbye in early evening, when the sun still had a long while before it set, reflecting golden rays off of Jamaica Pond.
We said goodbye to the many years of college that come with five children. We said goodbye to the joy we had in watching a child grow to be a man. We said goodbye to those who came into our lives through him.
A short while after we said goodbye, he boarded a plane to Albania; from there his plans include travel and study for the next year. We raised our children on travel and the uncertainty that comes with frequent moves, so there is a deep satisfaction knowing that he is choosing to grow through travel.
Letting go of our children is a series of stages that begins early in their lives. We proudly, but fearfully, watch as they make their way onto buses or across playgrounds, their first venture into a world we can no longer control. Each stage and step gives them a bit more independence until we face the reality that we are ancillary to their adult lives. When we began the journey of parenthood, we created their world, we were their world. But through the years we gradually step aside and let them shine, apart from us.
And our son – he shines, and it is the work of God.
The gratefulness I feel is complicated by post-surgery exhaustion and the tears from saying goodbye. It comes in waves, and I try not to overthink, over analyze, instead allowing myself to just be, to feel what I'm feeling without defending or accusing.
…the best thing I do as I pack him off and say goodbye is place him where I have placed him countless times before — in the arms of the Father. The Father who does not walk, but pulls up his robe and runs to greet his beloved children.
While the journey of parenthood continues until the day we die, there are pivotal turning points within that journey – and this is one of them. So I say goodbye with open arms, a glad heart, and tear-filled eyes. Somehow, all of those emotions belong to this moment.


text:
Doctorate in Oceanography
Doctorado en Oceanografía (Doctorate in Oceanography )
PhD (ISCED 2011 level 8)
Entry level PhD
Cost Five thousand euros
The Doctoral Program in Oceanography trains specialists in the integrated study of the ocean with a solid background in the four fundamental areas of oceanography: biological, physical, chemical and geological, with specialization in one of these basic areas through the thesis . This training aims to provide the student with the necessary tools to develop independent research and participate in the training of undergraduate and graduate students in academic settings. Scientific training will also allow them to lead technical assistance projects that involve the integrated study of the marine environment. The Program is focused on the training of doctors in all areas of oceanography. Biological oceanography is the most developed and includes research lines in pelagic and benthic ecology, fishing oceanography, population biology, marine organisms genetics, and aquaculture. Research areas in physical oceanography are, among others, internal waves, remote sensors, marine instrumentation and fluid mechanics. In chemical oceanography, the main research areas are marine organic geochemistry, biogeochemical cycles in the water and sediment column, radiochemistry, natural products chemistry, organic matter flows to the water-sediment interface. Geological oceanography research is oriented towards sediment transport and paleoceanography.
The graduate of the Program will have the competences to apply concepts and methodologies relevant to oceanography, analyze and solve scientific problems associated with the area of specialization, formulate research projects and integrate into multidisciplinary work teams, and communicate scientific knowledge in their area of specialization in academic and non-academic contexts. Graduates of the Doctorate in Oceanography will be prepared to:
- Integrate scientific concepts and methodologies for the generation of new knowledge in Oceanography.
- Organize and communicate scientific information about the discipline in academic and non-academic contexts.
- Formulate autonomously research projects in oceanography.
- Develop oceanographic research autonomously or by integrating multidisciplinary work teams.
- Critically evaluate information relevant to the discipline.
- Have a Bachelor's degree in Marine Biology or related careers, or a Master's degree in disciplines related to natural sciences (i.e. Sciences with a mention in Fisheries, Economics of Natural Resources and Environment, Exact or engineering sciences).
- A verifiable academic trajectory: pre- and post-graduate assistantships, participation in publications and congresses as co-author and as principal author, short-term academic stays abroad, participation in seasonal courses or any other verifiable relevant academic activity.
- Present two letters of recommendation from researchers of recognized trajectory, one of which must be from a teacher of the House of Studies (Alma Mater) where the applicant was trained.
- Undergo a face-to-face personal interview for Chilean applicants from the VIII Region and adjacent Regions, and by video conference for foreign and Chilean applicants from other Regions.
- In the application, the line of research to be carried out must be indicated according to the lines of research of the Program.
- Instrumental English evaluable in the admission interview, or by means of an English level certificate from an accredited institution.
Physical and chemical oceanography
Facultad de Ciencias Naturales y Oceanográficas
4030000 Concepción
Universidad de Concepción (University of Concepción)


text:
Ironside's Notes
Ironside's Notes on Selected Books
The Failed Testimony
It pleased the God of heaven, in bringing about an answer to His servant's petition to attract the attention of the Persian ruler to the grief-stricken face of Nehemiah. Kindly the monarch inquires after the cause of this change of countenance, for the son of Hachaliah had been wont to exhibit a cheerful mien, as became one whose confidence was in the Lord. "Why is thy countenance sad," asks the king, "seeing thou art not sick? This is nothing else but sorrow of heart." Fearful of his sovereign's displeasure, his cup-bearer replies, "Let the king live forever: why should not my countenance be sad, when the city, the place of my fathers' sepulchres, lieth waste, and the gates thereof are consumed with fire?" (ver. 23). Nehemiah could not be indifferent to a matter of this kind. He was no misanthropical pessimist-rather indeed the very opposite-but he could not be unmoved by the terrible break-down on the part of his loved people and the desolate condition of that city that should have been the glory of the whole earth.
But, observe, he did not stand aside and write pamphlets on the failure of his brethren; nor wash his hands of the whole matter and conclude that because the failure had indeed come in, he was justified in giving up all concern about the testimony committed to Judah. Not at all. His was a grief deep and genuine; but it was one that led to exercise before God, and an earnest desire to be an instrument in the hand of the Lord for the establishment of the truth and the recovery and encouragement of the feeble few who had broken clown so sadly in the very place where Jehovah had set His name.
And so when the king inquired, "For what dost thou make request?" he did not answer till he had "prayed to the God of heaven." What an atmosphere of prayer surrounds this man! It is his constant resource throughout all his varied experiences. He walked with God because he talked with God. Now, assured of the Lord's mind, he made request for permission to visit the land of Judah and the city of Jerusalem, that he might "build it." This was morally very lovely. He desired to build, to edify. Any one with a small measure of discernment can stand off and either bewail or criticize the failures of others, but one must needs be in touch, with God to be a true builder. Such an one was Paul, "a wise master-builder," and he, by the Spirit, directs that "all things be done unto edifying." "Knowledge," he tells us, "puffeth up, but love edifieth" (or, buildeth up). This is an all-important truth.
Many there are who entered on the path of separation with high hopes and fond expectations; eagerly they drank in the precious truths the Holy Spirit of God was making known in the place where He had liberty to work as He would. But today, alas, alas! many of these have turned away disheartened, and that because of breakdown on the part of brethren whom these others deem less clear of sight, less devoted and less intelligent than themselves. So they stand off and bewail the divided condition, the worldliness, the cold-heartedness that has come in. But to what end? Such a course profits neither those who so judge, nor those judged. Better, a thousand times better, to rise up in the spirit of Nehemiah, and throw oneself in the breach as a builder. The heart may be grieved and the countenance sad, but there will be a deep-toned joy in seeking thus to enlighten, instruct, and edify weaker brethren; endeavoring in the fear of God to close up the breaches sin has made, and occupy saints with the blessed Gatherer Himself instead of the failure of those gathered.
Yes, as the days darken and the dispensation fast hastens to its close, it is men of the Nehemiah stamp who will be of real value to the people of God, and who shall thus save themselves and those who hear them.
In the presence of his consort, Artaxerxes gave the desired permission, stipulating a defined leave of absence, in which Nehemiah would be free to carry out the desire of his heart, and go to his brethren as a true prophet to speak words of exhortation, edification and encouragement (ver. 6). All that may be needed for the work of building is granted by the king, even as the King of kings, who is also head of His body, the Church, delights to supply His walling workers with all things that pertain to the ministry committed to them. And here we note that Ezra and Nehemiah were men of like mind in tracing every blessing to the good hand of God (ver. 8).
The intervening journey soon completed (for a burning love urged him on), Nehemiah crosses the river and presents the king's letters to the governors of the mixed Samaritan people, who had been settled in the land of the ten tribes since the days of Esar-haddon. At once we read of two men who are grieved and displeased; they were Sanballat the Horonite and Tobiah the Ammonite, called contemptuously, Tobiah the servant. When they heard of his arrival, "it grieved them exceedingly that there was come a man to seek the welfare of the children of Israel" (ver. 10). As these men caused Nehemiah much trouble and concern later on, it will be well to inquire here as to who or what they might represent, and to ask if any such adversaries are likely to be encountered to-day in connection with the defense of the "present truth?"
Sanballat is called a Horonite, generally sup- posed to mean a native of Horonaim, a city of Moab. Of Tobiah's ancestry we are left in no doubt. We have therefore in these two foes representatives of those hostile races of whom it was written, "The Moabite and the Ammonite should not come into the congregation of God forever," as we are reminded later in chapter 13:1. The prohibition in Deuteronomy 23:3-6 gives the reason for this: "An Ammonite or Moabite shall not enter into the congregation of the Lord; even to their tenth generation shall they not enter into the congregation of the Lord forever: because they met you not with bread and water in the way, when ye came forth out of Egypt; and because they hired against thee Balaam the son of Beor of Pethor of Mesopatamia, to curse thee. Nevertheless … the Lord thy God turned the curse into a blessing unto thee, because the Lord thy God loved thee. Thou shalt not seek their peace nor their prosperity all thy days forever."
Reading such a command, we naturally ask why such a doom upon Moab and Ammon for refusing aid to Israel when about to enter the land of promise? Why should it have been expected of them and not of others? The answer is very simple. There were ties of blood that gave Israel to expect their assistance, but these ties were utterly repudiated. Moab and Ammon were the natural sons of Lot, but by his own daughters! They were really then "bastards, and not sons" (Hebrews 12:8). They surely speak to us of those professing to be children of God, but not born of the Spirit. And so they ever, as born only of the flesh, persecuted the spiritual seed. They are the representatives of fleshly religion, of carnal profession, and as such they detest reality, and hate the truth that "Except a man be born again he cannot see the kingdom of God." They feel they have as much right to the ordinances of God, and as much liberty to participate in His service and worship as any; but they are only natural men with a veneer of religiousness, and such have ever been the bitterest opponents of what really honors Christ and glorifies God. They abound to-day as they have abounded all down the centuries, and their object is still, as ever, to corrupt if they can, and to destroy if they cannot corrupt.
Leaving Sanballat and Tobiah for the present, gnashing their teeth in their rage and vexation, we follow Nehemiah to the city of God. Reaching Jerusalem, he rested three days. Then, conferring not with flesh and blood, but taking a few men with him, though telling none what God had put in his heart, he arose in the night and went out to view in silence the ruin that had come in. This night journey around the walls of the city is deeply pathetic. Who that has any real care for the people of God has not known something of it? The nobles and rulers and all the people are wrapt in slumber, but this lonely man, whose heart God has touched, keeps his midnight vigil, and goes from gate to gate and tower to tower, noting with deepest sorrow and concern the breaches sin has made. "I went out by night," he says, "by the gate of the valley, even before the dragon well, and to the dung port, and viewed the walls of Jerusalem, which were broken down, and the gates thereof were consumed with fire. Then I went on to the gate of the fountain, and to the king's pool: but there was no place for the beast that was under me to pass. Then went I up in the night by the brook, and viewed the wall, and turned back and entered by the gate of the valley, and so returned" (vers. 13-15). It was no carping critic viewing with indifferent feelings the defenselessness of his brethren; but a man of purpose and prayer, beholding what stirred his soul to its depths, with the desire to build up what carnal ease and self-seeking had permitted to fall into ruin.
It was not till after this night view that he called the people, with their rulers and the priests of the Lord together, to give them cognizance of his mission. He does so most delicately. There are no reproaches, no Pharisaic and odious comparisons or contrasts, but he identifies himself fully with them and says: "Ye see the distress that we are in; how Jerusalem lieth waste, and the gates thereof are burned with fire: come, and let us build up the wall of Jerusalem, that we be no more a reproach" (ver. 17). Such an one is a God-sent and Spirit-qualified leader. He does not say, "You are in distress;" but "We are." He does not command, "Go, and build," but he entreats, "Let us build." He does not say, "You are a reproach," but he pleads, "Let us be no more a reproach." And then he tells of the good hand of his God upon him, and of the king's commission.
The people are aroused and encouraged, and cry at once, "Let us rise up and build;" and so they join hands with God's dear servant for the work he has planned. No doubt there was not the exercise of soul in all that conditions called for; but the work must be done nevertheless, and there will be more exercise as they go on.
And now we hear of Sanballat and Tobiah again; and with them a third adversary, Geshem the Arabian. This man is either an Edomite or an Ishmaelite, more probably the latter; but in either case he speaks of the flesh warring against the Spirit. Both Ishmael and Esau were types of the natural man-hence of the flesh-and were opposed to Isaac and Jacob, the seed of promise. Geshem is elsewhere in this book called Gashmu. When this unworthy trio hear of the work contemplated at the place of the Name, they indulge in sarcastic merriment. Nehemiah noted that "They laughed ns to scorn, and despised us, and said, What is this thing that ye do? will ye rebel against the king?" (ver. 19).
Heretofore the line of demarkation between the outwardly separated Israelites and these mixed nations had been almost obliterated; hence there was peace and quietness. But now a man has come who contemplates rearing <!-- TRANSIT - HYPERLINK --><!-- .http://afres.hr. -->afresh the wall of exclusion, and this is bitterly resented, though at first they attempt but to laugh down the determination of the remnant. To all their sneers Nehemiah calmly replies: "The God of heaven, He will prosper us; and therefore we His servants will arise and build: but ye have no portion, nor right, nor memorial, in Jerusalem!" (ver. 20). He has thrown down the gauntlet and declares his uncompromising attitude in a manner not to be misunderstood. Henceforth he will be hated as only those can hate who resent having their false religious claims made nothing of!
The out-and-out worldling does not hate what is truly of God so bitterly as the Christless professor who has a name that he lives but is dead. Such cannot bear spiritual realities; for when confronted with them the hollowness of his profession is exposed, like Dagon when the ark of Jehovah was set down before it. This explains the bitterness with which these adversaries opposed the work of God going on at Jerusalem.
Ironside, H. A. "Commentary on Nehemiah 2". Ironside's Notes on Selected Books. https://www.studylight.org/commentaries/eng/isn/nehemiah-2.html. 1914.