#rsmy | Explore Tumblr posts and blogs

sockpansy · 6 years ago

Text

Gaining a crew

Welcome to part two (2) of 'lost at sea' sanders sides mermaid/pirste au!

A thanks to the lovely @thelazyblueshipper who drew the drawings that inspired thus lovely work!

Part one: https://sockpansy.tumblr.com/post/179000941959/lost-at-sea

Warning: kidnap mentioning, blood, mentions of drinking, let me know if there sre any others

______

"We need more crew members."

Logan had been the one to say it, but the other three had been thinking it as well.

"Logan's right. You guys got lucky with me, but not everyone will fall for the ghost ship, and we can't fight like them." Virgil pipped in.

Roman nods and was getting excited." A crew of mermaids being pirates! Of daring adventurers who face danger in order to save others! But who would join..."

"I could ask emile!" Patton bounced up and down happily. Emile Picani was Patton's older brother, and they got along greatly.

"He likes to study shipwrecks a lot, so im sure he could help us with fixing the ship up better, oh i mean 'The Siren.' "He giggled at the name they gave their ship.

That lead to now. Patton was swimming down to an old shipwreck. 'The duchmen' or something like that. He swam down and into a rotten hole in the side. "Emile?" He called out.

A pink head much like his own blue one popped out smiling."do you how do?" He swam over.

Patton gently lowered himself onto an old bench before asking, 'how would you like to join a pirate crew?"

Emile gasped, his eyes widening. "Patton, you know what pirates do to mermaids, joining a crew is dangerous!"

Patton wiggled hia tail playfully. 'Not if the entire crew is mermaids. Roman, Logan and I fixed up a boat to save Virgil, and it worked! Now we have decided to save more who are captured! You love boats a lot, and yeah our boat floats but we don't know as much as you do about them...and it is safer to have more people in our crew..."

Emile had started to wiggle his tail excitedly, the idea of actually working on a ship that was above water? It excited him.

"Oh it sounds like so much fun! I would love to join your crew!"

And with that, patton lead Emile to their ship, that perked on the island Roman found it on. Emile got to work with improving the boat, checking over jobs they did, and making suggestions.

"What if we had a hole in the bottom?"

"Then it would sink...Emile no offence but i thought you were smarter than that." Logan said gently.

Emile gave him a smirk."well yes it would, but i mean a hole that doesnt let water in, but that we can use to go in and out easier. It could add more to the 'ghost' idea as Well. Disappear inside the boat to appear on theirs."

Roman jumped in."perfect idea! Lets do it!"

Logan rolled his eyes. "Roman, it sounds good in theory, but to make it work will be a lot of trouble and toil, mostly trail and error. Plus, it wouls need to keep the water out when it is storming as well."

Virgil pat logan on the back, giving him a smile. "And with brains like you're I'm sure that we will have almost no errors. You are smart and think things out, plus Emile knows boats better than any of us."

Logan seemed in thought and glanced over to Emile, who was trying on glasses until he found some he could see out of.

"Well then....lets give it a try."

______

"So i said, at least buy me a drink before you try me for treason!" The bar of pirates laughed as Remy sipped his drink, finishing his story.

Remy went from town to town, staying as long as he wants, and leaving when he pleases, with nothing holding him back. He existed everywhere but yet no one had a record of him. No one knew how he traveled to so many places so quickly when he was never seen on a ship, yet could cross oceans and reach places before ships that leave a week before he does.

Honestly, it is a plus side to being a secret mermaid.

But remy knows that if he is caught, the only swimming he will do will be with the fishes.

So Remy takes a human form a lot, he goes to bars, tells stories (some are made up, some aren't, you can't prove anything) and drinks without ever actually paying for himself. He hides that he is a mermaid, but that doesnt mean he won't have fun!

Remy laughs at something when a pirate runs into the bar.

"I have a story to tell all of ye!"

Remy grinned as he sipped his drink. This was always fun. The stories never made since, and it gived Remy material to work with for stories later.

"Now I be helpin' to sail a ship through a storm, when we sees a small ship barely afloat! Nothin' out of the blue, but then, I be hearing calls for help. I help this wee lad out of the water and onto the ship, claims to be crew, and I believed him. Now i know i shouldn't have. So this boat is passing by, and I be thinkin' there is no one there! Then i see them, two pirates, a captain and what i assume be his first mate, they stare at us, as if challengin' us in the middle of a storm. We be strugglin' to keep the boat steady and watch this ship, and they hair stare. They only be two but they act like they not even tryin' to sail in this here storm! We pass them and get out of the storm, when i be going to check the cargo, and find a mermaid missin' then I can't find the pirate while fell overboard, we be thinkin' we csme across a ghost ship."

Remy sipped his drink as he watched the man walk around and explain his story. He began to put the pieces together and felt a smile growing on his face.

"Say, where did you see this ship?"

______

"Oh my god, Dee!" Remy ran along the beach to a short guy with an eye patch on. He looked up at the taller unamused and holding a sea urchin.

Remy slid over in the sand and grinned. "I think that some mermaids decided to become pirates,"

Dee stared at Remy before asking, "are you drunk agian? I,girl you, stick to teas or something."

Remy rolled his eyes, "no, i am not. Look, someone saw a ship with two people on it. They helped a pirate onto the boat and by the time they soft the storm, the pirate and a mermaid was missing! Lets just check it out, please Dee?" Remy asked.

"Whats in it for me?" Dee grinned as he started to pull spines out of the sea urchin.

"First off, stop being cruel to the thing-"

"No"

"Fair enough, second, I am sure they have races....wouldn't it be fun...beating an entire ship of mermaids in a race?" Remy wiggled his eyebrows.

Dee pulled out another spine. "Flattery gets you everywhere, lets go."

______

Dee made a trill noise and sighed, "Remy, this way!"

Remy has...horrible direction. If he tried to find the ship alone, he would fail. So dee was leading the way...kinda.

The pirate had told Remy which direction the ship had been in, but that was three weeks ago, so it could honestly be anywhere. Dee called agian as he swam through the water, "i think this is it!"

Remy swam over to dee and looked up at the boat above them. It was old alright, but in better condition that the old pirate had said. Remy swam around the bottom as he observed it. There was a hole near the back. He motioned Dee over to it.

The hole was flat, and it appeared as if no water got inside the bottom of then ship because of it. Why would a pirate ship float with a hole that didnt let water in? Why would a pirate ship need a way into the ocean?

For mermaids, of course.

Remy swam up and popped his head into the hole, looking about the bottom deck of the ship. Nothing much was down there, a bunch of old ropes and wood, and a pile of clothes. He motioned Dee to follow him as he climbed up and transformed into his human form.

Dee followed him up and looked around. "Looks like a shipwreck in here."he started to look through the pile of clothes. "Oooh, an eye patch!" Dee had lost his on the way over passing s coral reef, and was in need of s new one. Humans dont react well to different colored eyes, especially with the eyes glow from time to time. Dee tied the eyepatch on as remy threw clothing after clothing over his shoulder.

"Ew, gross, no, no way, oh maybe."

By the time they were done raiding the clothing pile, Remy had at least 10 pieces of jewelry on.

Now for the next part of the plan. Introduction.

______

Patton and Emile were making matching bracelets for everyone as logan and Virgil worked on the plan for the next boat raid and roman figured out how to steer a ship better.

Remy walked onto the deck like he owned it. "Wow, you guys need to really get some better clothes. Took me forever to pick an outfit!"

Virgil and Roman had their swords drawn, patton and Emile dropped their half finished bracelets, and Logan was observing the intruder.

"How did you get here." Virgil demanded.

"Oh, how you would." Remy grinned. "I swam."

Silence fell over the crew. Roman ran down from the wheel grinning. "Your a mermaid too!" He exclaimed.

Remy grinned."better believe it! Heard about a Ghost ship and a missing mermaid...thought you might need some help..."

Patton smiled. "Yes, more crew members are also good! Im Patton, this is my brother Emile, the captain there is Roman, the guy in purple who looks ready to kill you is Virgil, and the guy with glasses in front of him is Logan!"

Remy hummed as he looked around. "Cute bunch. Names Remy, the kid behind me-"

"-i am not a kid-"

"Is named Dee. He is a half siren. And short." Remy stretched as Dee walked out into the sunlight.

"So what do you guys even do?" Dee asked.

Logan said."we rescue captured mermaids...oe well, that is the plan atleast."

Emile smiled."I'm new to the crew, but im the one in charge of making sure the boat floats. Like the hidden hatch i added? It makes it easier for us to go in and out of the bait without worrying about the clothes or being caught by pirates."

Rsmy nods and leaned on the railing as Dee explored the ship. "Yeah, it was pretty cool, the bait does still need some work..what does everyone else do?"

Roman took over."well i am the captain! I am the one in charge here." He beemed with pride, "Logan is my first mate, he makes sure the plans will work ideally. Patton is our savor. He gets on the ships and frees the mermaids. Virgil is the one we saved first, but his job will be to sneak onto the boats and help Patton, or steal gold or other things from the pirates." He smiled." So really, the question is...what can you offer us?"

Remy laughed."I like your flare cap! And what can i offer?" He smirked, leaning forward. "A spy. I go into bars,and chat up pirates all the time! I can find out who has mermaids and where they plan to go and report back to you easily!"

Dee laughed."maybe if you had a good sense of direction!"

Remy nods."yeah but i can get people to trust me, if you were to go, you would get into a fight." He teased.

"So you are a spy while he is your escort?" Logan asked.

Dee scoffed."uh escuse you, i am more than that. Im the fastest swimmer around! Nothing says a pirate ship will just end up in a storm all the time, you need something for them to follow." Dee put his hands on his hips confidently. " i am part siren, luring pirates to their deaths is kinda my thing. I just gotta get them to chase me into a storm and you guys take care of the rest."

Virgil was silent. "What if you get caught?"

Dee greeted his silence with one of his own. "I was in a barrel before, i can handle being in one agian until you guys save me. " he took a breath and grinned getting cocky. "Besides, who says ill be caught?"

And so Remy and Dee joined the crew.

____

For a few months, eveeything worked out great. They all had matching bracelets thanks to the sunshine brothers, Remy and Dee would also find out information easily. After a while, Patton would board ships sooner. He would board at the docks, or as a 'ship wrecked sailor' trying to get to land. It got the pirates to trust him quicker and got him on the ship sooner. Emile painted the name of the ship onto it and ' The Siren. ' officially was set sail.

Virgil had gathered gold and jewels. He would knock barrels of drinking water off, and would take the food reserves from time to time. Once he stabbed someone who had grabbed Patton when he tried to jump over the railing, so he had a new job added to it. Dee was fast, like he promised, and could easily tempt ships to,follow him.

But then Remy heard about Thomas.

Remy ran onto the deck, with Dee close behind him (he swims fast, not run)

"They have a multi colored!" He announced out of breath.

Multi colored meant that the tail of the mermaid was more than one color. They were extremely rare. Having just two colors together was surprising and often times, these types of,mermaids where higher value to pirates.

"They won't keep them with the others." Virgil said."pat, you will have to-"

"Let me finish!" Remy called over Virgil. He took a breath.

"They are a rainbow tail."

The silence was deafening. Having two colors was rare and surprising, but six different colors? That would be like finding a four leaf clover at 11:11 on the luckest day of the year, on your birthday. Nearly impossible.

And extremely valuable.

"They wont leave them by themselves...and,no where near any kind of water..." Emile stated.

"Then we sink the ship."

All eyes turned to Logan. They never sunk a ship before, only boarded.

"Think for a second, we still have a small crew, on a small ship, and we have barely any kind of actual training with swords. A rainbow tailed mermaid will be guarded, and inside, and even if we could get to them, they will be locked up...if we sink the ship, we could get them free easier..."

"But how do we sink a pirate ship?" Virgil asked in disbelief. "They have ships so much bigger than ours! We don't even know if our canons even work, and if they did, they wont be enough to sink it."

"They we take it apart from under it." Emile,spoke up. This time, all eyes fell to emile surprised.

He smiled."I know boats, i can figure out which boards to remove to sink it...logan can help me with removing them. He is smart, and I dont know what tools to use to remove boards, just to put them on."

And with a few more ideas and making sure the plan cant go,wrong, they were ready.

_____

Dee had to get this ship to follow him. Rumors had began to spread about their ship, and about missing mermaids. Whether or not they believe in those rumors didn't matter, no one would risk loosing something like a multi colored. So Dee had to pull out all the stops this time.

He swam up quickly towards the surface, break through the water and into the air.

He flew up a little ways from his speed, and grabbed hold of the bowsprit. He pulled himself up and gained the attention of some pirates. He sat on it, ready to jump in and swim.

"Captain, a mermaid be sittin' on our ship!" A pirate called.

Dee gasped."mermaid?,no way! I am part siren!" He had his yellow eye glow.

The captain came out of his quarters. "Half siren eh? Sirens be tricky to catch, and worth a lot of treasure...a half siren be easier to nab...grab 'em!" He ordered.

Dee jumped into the water below and began to swim to a storm a ways away. The boat turned to,follow him. He swam quickly, staying at the surface and keep the attention on him.

The ship followed him, althought he did have to get their attention by letting them get close at times. The storm was heading to the ship, and even if they changed direction now, they would be caught. Dee swam down deep and to the ship to get ready to ghost.

_____

"Ok, everyone ready?" Roman stated, going over the rolls agian. "Emile and Logan will work to break the underside of the boat, Patton will distract the crew while Virgil boards to see if he can find the rainbow. Remy, dee and i stay here and play the ghosts, keeping the ship distracted." The others nodded.

Roman looked at them all and said."be safe ok? And if something happens, get to safety first Alright?" He gave them all a look of worry, this was his crew. He didnt knwo what he would do if something happened to any of them.

They all gave a nod

_____

Roman stood at the end of the bowspirt with his sword drawn at his side, just....staring at the ship in the storm. A few pirates were watching him confused, some looked scared even, good.

Off to the back, roman could see Patton freeing a few normal mermaids into the ocean, some looked like they decided to help logan and Emile under the ship.

That was taking some time. Of course it would, ships were made to last and not fell apart easily. That didnt mean roman wasnt worried. The longer they were down there, the longer they had to be caught.

Movement of something purple caught roman's eye as he saw Virgil climbing the back of the ship and crawl through a window into tge captain's quarters. He hoped Virgil would be ok.

Virgil on the other hand, was focused. He slid down behind a bookshelf (pirates read? Wait no, it was covered in treasure) and watched as the captain yelled at a crew member.

"I aint be leavin' this here rainbow tail! It be worth a lot on dry land, now ye be going out to keep yar ship strudy! Go! Off with ya!" He shouted.

"But captain, the ship outside has-"

"I know what ye think it has! Let me yell yar this now, I dont be caring if there be a ghost ship or nah, we be sailin' to shore!"

Virgil looked around the room as the captain continued. He saw a crate with a lock on it. That must be the rainbow tailed. But it was across the room and, as mentioned before, locked.

Virgil watched things roll around and shift as the boat was thrown by the waves, then he noticed that everything started to mostly roll one way. He assumed that mean-

"Captain! We be sinking!" The door flew open as a pirate ran in in a panic.

"What?! what do ye mean we be sinking?" And the captain stormed out of the room as the ship was tilting more. The door slammed closed and virgil was alone in the room.

He grabbed a random trinket from the book shelf and went over to the crate with the rainbow tailed in it. He raised the trinket, which appeared to be a golden statue of some kind, and hit the lock. He hit it over and over agian until it finally came off.

Virgil opened the crate too only be splashed by water right away. He looked down at the sight before him.

There was a mermaid with a rainbow tail. Honestly it was an honor to even see one. The mermaid looked at Virgil with pleading eyes, ropes tied around him and his mouth gagged. Virgil knew the look in his eyes, he had the look himself back when he had been captured.

"Ever hear of ' the siren ' before?" Virgil asked as he removed the gag from his mouth and got to work untying him.

He shook his head no.

Virgil nods as he got one knot undone and worked on another, "figured you hadn't, we arent too popular yet. Ill explain it like this for you. There is another ship in this storm, it isn't sinking, go to it, it will be safe there," he smirked as he saw the confusing on the rainbow's face.

"Lets just say we dont have our sea legs." And with a wink, Virgil finished the last knot and helped the rainbow out. The ship was sinking faster now, which was no problem for them.

The problem was the door slamming open to a furious captain, who drew his sword.

"You best be puttin' that rainbow tailed back!"

Virgil held the mermaid princess style snd looked at the captain scared, what was he suppose to do? The back window was too high up now for virgil to go out that way, making the door the only place left, and the captain was blocking the way out. And he had a sword that was very pointy.

"Uh I.,i can't..do that...you see, im a ghost." Virgil nods, trying to Roman his way out of this mess. "Totally a ghost. From the ghost ship over there. So uh.,you can't kill me."

Virgil turns out to be very bad at Roman-ing a situation.

The captain laughed. "Ye be a ghost? There be no such thing! And if ye be a ghost, then ye won't bleed now, will ye?"

Virgil will bleed. Virgil does not want to bleed. Blood belongs on the inside and he would like to keep it there.

The ship was taking water in faster, and nearly vertical now, giving Virgil an idea.

Time to Roman this another way.

By being dramatic.

"Well if i wasn't a ghost..could I...do this?" And Virgil jumped up. The captain was holsing into the door to stay at the room and not fall down to the waters that had taken over his boat.

Because the boat was almost veritcal, when Virgil jumped, instead of landing on the wood of the boat, he fell towards the door the captain was at, feet first.

Virgil hit the captain with his feet, making him loose his grip on the door and fall into the raging waters. Virgil was still holding the rainbow when he landed in the water.

Virgil transformed, grabbing his clothes and quickly swimming towards the ship.

He grabbed the rainbow's hand as he swam.

______

By the time Virgil got the rainbow on the ship, the entire crew had ran down to meet them.

"You did it!"

"We saw the captain go in-"

"Wow a real rainbow tail."

"Did you get hurt?"

Everything meshed together as Virgil smiled at everyone. Patton and Emile were helping the rainbow tail get adjusted, logan and Roman were helping virgil up, Remy was picking an outfit for the rainbow, and dee was stating st Virgil silently, which was weird.

"We did it." Virgil grinned. Everything was fine. They saved the rainbow and probably down other mermaids on the sinking ship.

"Your bleeding."

Everyone froze and they looked at Dee, who was pointing at virgil's leg.

As Virgil slowly looked down, he saw a gash in his leg going from the side of his foot up to his hip.

He remembered when he kicked the captain, and how he had his sword drawn when he did.

He remembered it cutting his leg.

And now he felt it.

Virgil collapsed as the pain finally hit him. He could hear yelling and feel arms and hands on him, he thinks he heard someone say 'its okay', but he wasn't sure.

And he passed out.

____

When virgil work up, his leg was wrapped and Patton was leaning over him. He turned and yelled,

"He's awake!"

Virgil sat up and stretched as the crew ran over.

Emile took over first. "Nothing is broken, but until it is healed, i would suggest staying in human form for a while ok? It will heal faster since this is the body you were injured with."

Virgil nods a bit, slowly making sense of everything.

He saved the rainbow, got cut in the elf, swam to the bait, got on, found out he was bleeding, passed out from shock of blood and suddenly knowing of pain, work up. Yep, sounds about right.

"Where is the rainbow?" Virgil looked around, until a familar face smiled at him.

"Hi, name is Thomas," the rainbow smiled and held out his hand. Virgil noticed he had a rainbow friendship bracelet on, which had ended up beconing the Mark of the crew. Virgil couldn't help but smiked as he took his hand.

"Virgil, and welcome aboard."

_____

Every tale has two sides, and so does every legend.

The legend on land was known by pirates. Of a ghost ship that only appears in storms and a crew that will take your mermaids. The legend is a warning.

The legend of the see was known by the mermaids themselves, of a ship filled with pirates. Pirates who were mermaids, who sink ships and free those who were caught, promising them safety above the water, the legend is a promise, a promise of protection.

And every legend holds truth.

_________

And done! Hope you all wnjoy and wilk stick sround for the next part!

#Sanders sides #mermaid au #pirate au #lost at sea au #that is what im calling it

8 notes · View notes

juniperpublishersmicrobiology · 5 years ago

Text

Molar Fraction of Carbon, Nitrogen and Mineral Sources Regulate Antibiotic Biosynthesis Pattern in Pseudomonas Fluorescens CHA0

Authored by Archana G,

Abstract

Fluorescent Pseudomonas species inhibit fungal phytopathogens under laboratory and greenhouse conditions, but in field trials the inconsistent performance is observed, which is thought to be due to variations in abiotic and biotic factors in/around the rhizosphere. Combination of nutrients and mineral components relevant to root zone and its effect on the antibiotic biosynthesis by fluorescent Pseudomonas species important for the understanding of biocontrol activity in the rhizosphere. Using Stat ease 8, and fractional factorial design sixteen combinations of four macronutrient (glucose, citrate, nitrate, phosphate) and three essential elements (Fe, Zn and Mo) obtained, and studied for the main effects and higher order interactions, on the culture growth, antifungal activity toward Rhizoctonia bataticola, and on the biosynthesis of pyrrolnitrin (PRN), 2, 4-diacetyphloroglucinol (DAPG) and pyoluteorin (PLT) in the model biocontrol strain Pseudomonas fluorescens CHA0 (Pf CHA0).

Citrate, glucose, Fe2+, Zn2+ individually and citrate with glucose, found to be supportive while triple combination of citrate, glucose and nitrate is highly inhibitory to Pf CHA0 growth, antifungal activity as well as antibiotic biosynthesis. Principal Component Analysis revealed that 2 day antifungal is positively correlation with PLT maximally followed by OD, PRN and DAPG. Fifth day antifungal activity showed positive correlation maximally with PRN followed by OD, and PLT. Bio-control supporting nutrient combinations showed effective protection of Vigna radiata from R. bataticola. Present study will be helpful for improving the reliability and application potential to the various soil types and different plant rhizosphere types.

Keywords: Nutrients; Antibiotics biosynthesis; Pf CHA0

Go to

Introduction

Many species of root-associated beneficial bacteria have the ability to reduce the severity of diseases caused by soil borne fungal phytopathogens and to increase plant yields under laboratory and greenhouse conditions and are considered important biocontrol inoculants for sustainable agricultural practices [1-3.], Whipps et al. (2001). However, in field trials the inconsistent performance of bacterial biocontrol agents has been observed from site to site and from year to year [4.,5.] which is a major concern for their large-scale application [6]. A thorough understanding the sources of variability in field performance, which could be due to variations in environmental conditions, abiotic and biotic, that the bacterial inoculants confront in the rhizosphere is an important aspect of study. Many factors are found to affect the performance of biocontrol agents, for example, there is a clear link between soil pH and mineral concentrations and the variability in biocontrol activity of both fungal and bacterial inoculants against root diseases [7-9].

Biosynthesis of antimicrobial compounds by bio-control bacteria is modulated by the total concentration and type of carbon (C) source, nitrogen (N) source, amino acids, metal ions such as Fe2+, Mo2+ and Zn2+ and other compounds found abundantly in plant root exudates but lacking in bulk soil [10-13]. C- Sources contribute to the variability of bio-control in different soils and on host crops that differ in root exudates composition Latour et al. (1996). Comparison of root exudates composition of cucumber, tomato, sweet pepper and grass revealed that it contained more organic acids than sugars and citric, malic, and succinic acid were major organic acids and fructose, glucose and to a minor extent, xylose, were major sugars Kuiper et al. (2002). Plant specificity of bio-control bacteria has been demonstrated at both the species and cultivar level which is generally been attributed to differential utilization of the various carbon and nitrogen compounds found in exudates and its effects on growth [14].

Fluorescent pseudomonads are one of the most well- studied group of biocontrol and plant growth promoting rhizobacteria (PGPR) that have the ability to produce diverse antifungal metabolites of different efficacy, mode of action and stability and thereby protect the crop from attack by different fungal pathogens Raaijmakers et al. (2002) [6,15]. Important antifungal metabolites which contribute to biocontrol by fluorescent Pseudomonas include 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), pyrrolnitrin (PRN), phenazines (PHZ), lipopeptides and hydrogen cyanide Nielsen et al. (2002), Raaijmakers et al. (2002), Haas & Keel (2003) [16,17]. Certain biocontrol strains of Pseudomonas produce multiple antibiotics with overlapping or different degrees of activity against specific pathogens. For instance, the biocontrol agents Pf CHA0 and Pf-5 produce the phenolic compounds DAPG, PLT and PRN Loper et al. (1997), Raaijmakers et al. (2002) [18].

Regulatory pathways responding to these nutritional and environmental factors are generally integrated into to respond to these signals on various additional regulatory elements, global environmental and physiological regulators such as GacS-GacA, RpoS, and CRP [19,20] and there is evidence that rhizospheric microbes such as Pseudomonas sense the availability of diverse nutritional factors as exogenous signal, activate small RNAs (sRNAs) that bind RsmA/ CsrA proteins are typically produced under the positive control of a two-component system termed GacS/GacA for global activation of antibiotic biosynthesis [21], Babitzke & Romeo et al. (2007).

Pf CHA0 respond to exogenous regulatory signal(s) by activating Gac/ Rsm a membrane-bound sensor kinase Laville et al. (1992) [22] the biosynthesis of bacterial auto inducers, rpoD and the cellular levels of mRNAs, stable RNAs (i,e. rRNAs and tRNAs), small RNAs (RsmX, RsmY and RsmZ) when cells reach high population densities [23]; Valverde et al. (2003); Kay et al. (2005). When bacterial cells are growing in batch culture, the GacS-GacA-dependent phenotypes are expressed mostly when the culture is in the transition from exponential to stationary phase Heeb & Haas (2001). An additional level of control may be provided by the relative concentrations of the housekeeping a factor RpoD and the stress and stationary phase a factor RpoS in the bacterial cell. In Pf CHA0, over expression of the housekeeping a factor RpoD enhances DAPG and PLT biosynthesis in vitro and in the rhizosphere, resulting in improved bio control depending on the plant species Maurhofer et al. (1992) [24,25].

Likewise, an rpoS mutant of the related strain Pf-5 shows strongly enhanced PLT and DAPG biosynthesis [26] whereas rpoS over expression in strain Pf CHA0 was found to shut off PLT biosynthesis Haas & Keel, et al. (2003). Two-component regulatory cascade composed of the sensor kinase GacS and the response regulator GacA that positively controls antibiotic biosynthesis upon activation by a yet unidentified quorum sensing-signal Haas & Keel, et al. (2003); Valverde et al. (2003) [22]. Numerous biotic and abiotic signals may also influence the production of these antifungal compounds, including different mineral and carbon sources as well as metabolites released by micro-organisms and plants [27-29]. Quantitative and/ or qualitative differences in the sugar, nitrogen, phosphate and mineral component of root exudates could determine the optimal environment for the effective bio-control mechanism in given crop-pathogen systems [24,30].

Maria Pechy-Tarr et al. [31] reported that rpoN is a key regulator of bio control activity in plant-beneficial pseudomonad and recently, Kumar and Shimizu, 2010 reported that as C/N ratio get increased, and transcript level of rpoN (which encodes σ54) increases in E. coli.So the role of rpoN in bio control activity by fluorescent pseudomonads under varied C/N ratio could not be ignored. The importance of organic acids as carbon sources for growth of Pseudomonas spp in the rhizosphere was shown previously by De Weert et al. [32] who demonstrated that mutants of the good colonizer P. fluorescens WCS365 with mutations in genes encoding malate/quinone oxidoreductase or cisaconitate hydratase, enzymes of the tricarboxylic acid cycle, are poor competitive colonizers of the tomato root compared with the parental strain. Our results add another point to plant specificity i.e. the influence of combination of root exudates components on the biosynthesis of antimicrobial metabolites by fluorescent Pseudomonas and increase its competence and survival efficiency in rhizosphere.

In the present study, we have studied the well-characterized biocontrol agent Pf CHA0 [2,18] to get insight into the pattern of biosynthesis of the antifungal compounds DAPG, PLT and PRN that are major determinants of the biocontrol activity in this strain under the different combinations of nutritional factors. Along with glucose, citrate is also studied as an alternate C source, since citrate is preferred C source Pseudomonas and it is the one of major constituent of root exudates. The results of this study provide

Insight into the possible biosynthetic regulation of antibiotics by complex of organic and inorganic component present in the rhizosphere,

Limited number of factors and their combinations for intensive study in situ,

The factors that can be manipulated to improve bacterial inoculants and

The application prospect of the biocontrol strains based on the rhizosphere and soil type for better bio-control potential.

Go to

Methods

Bacterial maintenance and growth conditions

Pf CHA0 (kindly provided by Dr. Fabio Rezzonico, Agroscope Changins-Wadenswil Zurich, Switzerland, was routinely maintained on King's B medium plates (Hi-media, India) at 28 °C and stored for long term in 0.8% nutrient broth with 0.5% yeast extract (NBY) broth with 40% glycerol at -80 °C. Starter cultures were grown in 10 ml dilute (1/5-strength) NBY broth in 20 ml test tubes for 12 h at 28 °C at 150 rpm, yielding approximately 109 CFU/ml. For further growth, 20 ml of dilute (1/5-strength) NBY broth in 100-ml Erlenmeyer flasks was inoculated with 100 |il of starter bacterial. Autoclaved medium was amended with different combinations of following seven components: filter-sterilized solutions of KNO3 (10mM), KH2PO4 (Pi,10mM), FeSO4.7H2O (Fe2+,0.5mM), Mo7(NH4)6O24.4H2O (Mo2+,0.5mM),ZnS04.7H20 (Zn2+,0.35m) and with autoclaved stock solutions of glucose and citrate to give final concentration of 100mM. Rhizoctonia bataticola causal organism of dry root rot of Vigna radiata, was maintained on potato dextrose agar slants (PDA, Hi-media, India).

Design of experiment for the combinations of nutritional factors

The study of the effect of combinations of nutritional factors for the enhancement of bio-control traits by Pf CHA0 was carried out by using Stat ease 8. For seven factors, a total of 16 combinations were used (Table 1). Each column has equal number of high and low values of given nutrient factor. 50 ml of all the 16 media combination in 250 ml conical flask was inoculated with 100ul of overnight grown bacterial and kept at 26oC in shaking condition at 150 rpm in darkness. After 2 day and 5 days, 20 ml of bacterial was transferred in sterile 50 ml centrifuge tubes for metabolite extraction. Absorbance of the culture was noted at 600nm to monitor the growth of the organism in the medium. pH of all media was in the range of 6.6 to 6.8 at time of inoculation and between 7.5 to 7.8 after 2 day and 5 day of growth.

Extraction, identification and quantification of antifungal metabolites

Antibiotics were extracted from bacterial supernatants and quantified with high-performance liquid chromatography (HPLC, Shimadzu 10) as described by Duffy and Defago, 1997. Metabolites were identified and by comparison with the pure DAPG, PRN and PLT. Metabolite quantity was estimated from standard curves of reference compounds and normalized for the bacterial absorbance at 600nm prior to extraction. Briefly, liquid culture of 20 ml were acidified to pH 2 with 400 to 700 µl of 1 N HCl and extracted with 20 ml of ethyl acetate for 60 min with vigorous shaking at 200 rpm. Phase separation was accelerated by 15 min of centrifugation at 5000 rpm. The organic phase was transferred to a round-bottomed glass flask flash evaporated, and the residue was dissolved in 1 ml of HPLC-grade methanol and quantified by established HPLC procedures Keel et al. (1992]; Maurhofer et al. (1992)[24].

In vitro tests of fungal antagonism

The plate test screening for in vitro antagonism against the plant-pathogenic fungus Rhizoctonia bataticola was performed by placing an agar plug with in the centre of a Potato Dextrose Agar (Hi-media, Mumbai, India] plate and by adding 100^l ethyl acetate extract in the well bored at four places equidistance from centre at plate periphery. The plates were incubated at 30°C for fungal growth and checked for zones of mycelia growth inhibition after approximately 2 and 5 days, when the fungal mycelium had reached the edge of the plate. Percentage of fungal inhibition was calculated as: radial growth of fungus from centre -radial growth of fungus in presence of extract)/radial growths of fungus from centre X 100. All tests were performed 4 times, with new extract used each time.

Principal Component Analysis

PCA was used to establish combinations of variables e.g. OD600 nm, PRN, DAPG and PLT biosynthesis to describe the principal tendencies. PCA study provided the corresponding Eigen values, which were extracted by each factor, and the variance percentages (accounted for and accumulative] corresponding to the principal components by formula Fn=A1 X1+A2 X2+...+An Xn. Given Xobservations on n variables, PCA reduce the dimensionality of the data matrix by finding r new variables, where r is less than n. PCA Analysis revealed that data can be summarizing with just five variables and it contains all 5 principal components and their corresponding Eigen values for 2 day and 5 day.

Seed sterilization and plant inoculation study

Equal sized seeds Vigna radiata were thoroughly washed 3-4 times by sterile distilled water and treated with 1% HgCl2 for 2 minute followed by 2 minute treatment of 70% ethanol with vigorously shaking. Then the seeds were given two or three washes of sterile distilled water and were transferred to sterile petriplates containing wet filter paper. The seeds were incubated in dark. Sterile distil water was again added on the second day to maintain humidity for proper germination. Seeds were germinated up to the time when radical size reached to 1 cm. Overnight grown bacterial culture were used to inoculate germinated sterile seedlings of Vigna radiata.The germinated seedlings were incubated in saline washed bacterial suspension for 30 minutes and then three seedlings were planted in 75 ml Murashige and Skoog medium with in 500 ml glass tube (Hi- Media Ltd, India] and the various supplementations according to different combination types containing 0.7 % agar. The seedlings were allowed to grow for 7 days at 30±2 o C in alternate dark and light period.

Plant protection assay

For the in vitro antifungal assay MS medium was supplemented with biocontrol supportive combinations. The germinated seedlings were incubated in saline washed bacterial suspension for 30 minutes. After bacterial treatment seedling were treated with fungal broth aliquot from same stock broth of grown fungus in potato dextrose broth. Three seedlings were planted in 75ml Murashige and Skoog medium with in 500 ml glass tube (Hi-Media Ltd, India] and the various supplementations according to different combination types containing 0.7 % agar. The seedlings were allowed to grow for 7 days at 30±2 oC in alternate dark and light period.

Go to

Results and Discussion

Effect of nutrient combinations on growth and antifungal activity

Numerous biotic and abiotic signals influence the production of antifungal compounds, including different mineral and carbon sources as well as metabolites released by micro-organisms and plants [27-29]. Quantitative and/or qualitative differences in the sugar, nitrogen, phosphate and mineral component of root exudates could determine the optimal environment for the effective bio-control mechanism in given crop-pathogen systems [24,30]. Our results add another point to plant specificity i.e. the influence of combination of root exudates components and edaphic factors on the growth, biocontrol traits and biosynthesis of antimicrobial metabolites by fluorescent Pseudomonas which will be helpful to increase its competence and survival efficiency in rhizosphere.

Use of 1/5-strength nutrient broth yeast extract broth only and further amendment with different combinations of glucose, citrate, KNO3, KH2PO4, FeSO4, ZnSO4 , and (NH4)6Mo7O24 resulted into the variations in Pf CHA0 response for the growth ,antifungal activity and antibiotics productions. Effect of nutrient combinations on 2 day growth (p=0.0026) and 5 day growth (p=0.011) and antifungal activity by the 2 day (p=0.0082) and 5 day (p=0.0007) ethyl acetate extract for the antibiotics from different nutrient combinations have shown significant variations (Table 1]. The inhibition of R.bataticola by the ethyl acetate extract of different nutrient combinations is shown in (Figure 1]. Combination amended with KNO3, (NH4]6Mo7O24 and ZnSO4 did not supported the Pf CHA0 growth. Combination amended with citrate, FeSO4, glucose and KH2PO4 combination amended with citrate ,FeSO",KNO" and ZnSO" and the combination amended with citrate ,glucose, FeSO4 , KH2PO4, KNO3, (NH4]6Mo7O24 and ZnSO4 have shown highest antifungal activity of 70% on 5 day and high growth( OD 600nm = 3±0.5]. Combination amended with citrate, FeSO4, glucose and KH2PO4 and combination amended with glucose, KH2PO4, KNO3 and ZnSO4 has supported growth on both the days of sampling with OD 600nm value of 4±0.5 but earlier combination supported more to antifungal activity on 5 day

Combination amended only with all inorganic components viz., FeSO4, KH2PO4, (NH4]6Mo7O2, and ZnSO4 showed green fluorescent pigment biosynthesis and antifungal activity up to 40% on 5 day. Combination with Zn, Mo and nitrate only did not support growth because of very low C/N ratio (C/N<1] and the stress effect of heavy metal Zn2+ and Mo2+ and it was not included in the further data analysis and graphs. Combination supplemented with glucose, citrate, Zn2+ and Mo2+ supported well to growth and showed declined growth on 5 day due to low nitrogen content but good antifungal activity on 2 day and 5 day due to very high C/N ratio. The amount of available nitrogen seems to be the crucial factor in deciding the metabolic response especially under N- limitation. Most the pathways used for N assimilation under N- limitation utilize high amount of ATP.

Therefore, it appears critical for the cell to shut down activities of such pathways under certain circumstances to save ATP which causes the cell concentration decreases [33]. Combination without any amendments showed low growth but high antifungal activity on 5 day than on 2 day which suggest that in diluted nutrient broth even growth is low and not much difference in 2 and 5 day growth but antifungal trait get induced on 5 day which indicates that as bacterial ages antifungal traits get intensified and such trend should be followed in every combinations and this combination should be considered as negative control for the amendments. Kumar and Shimizu 2010 reported that, as C/N ratio increases the transcript level of rpoN get increased, which encodes σ54. So the induction of rpoN was thought to be possible reason for the induced antifungal traits.

Effect of nutritional factors and their contribution to growth of Pf CHA0 at 2 day and 5 day is depicted in (Figure 2]. Citrate, glucose, Zn2+, citrate and glucose and triple combination of citrate, glucose and nitrate have shown positive effect on growth. Citrate and Pi, citrate and zinc, Mo2+, citrate and Mo2+, triple combination of citrate, glucose and KNO3 have shown negative effects on growth (Figure 2]. Citrate and Pi individually have shown positive effect and contribution of 9% and 5% respectively on 2 day and 10 % and 1 % respectively on 5 day however the combination of citrate and Pi showed negative effect on growth and contributed to 18% on 2 day and 19 % on 5 day. Contribution order of the factors acting positively on the 2 day growth is, Glucose > Zn2+>citrate +glucose>citrate>Pi>KNO3 while for the 5 day growth the order is Zn2+> citrate+ glucose > citrate > glucose >Pi=KNO3. In our result, citrate and glucose individually and in combination, have shown strong positive effect on Pf CHA0 growth on 2 day and 5 day which is in accordance to previous finding that an organic acid or a tricarboxylic acid cycle intermediate, not glucose, is usually the preferred carbon source in Pseudomonas species [34]. Of the minerals, zinc was found to be supportive much to Pf CHA0 growth on 2 day and 5 days. Sodeberg et al. (1990] explained that the zinc exhibits different bacterial affinity with gram-positive and gram-negative bacteria, it may be ascribed to the difference in the protein constituents of their cell walls zinc and other minerals are essential for growth, they influence cell membrane integrity, and they are key components and/or catalysts of over 300 enzymes and other proteins [35].

However the contribution order of negatively acting factors on 2 day growth is, citrate +Pi = Citrate +glucose + KNO3 > Mo2+ and for 5 day growth order is, citrate +Pi >citrate +glucose + KNO3 >citrate+ Zn2+. Ammonium molybdate has been reported to be a strong inhibitor of acid phosphatase activity [36] and the process of phosphorylation / dephosphorylation plays a crucial role in many metabolic processes [37].

Effect of nutritional factors and their contribution to 2 day and 5 day antifungal activity shown in Figure 2. Citrate has strong positive effect on antifungal activity on 5 day and contribution 29% and 2 day with contribution 14%. However glucose showed more positive effect on 2 day than 5 day with contribution 33% on 2 day and only 3% on 5 day. Citrate with glucose has positive effect and shown 16% contribution on 2 day and 12% on 5 day (Figure 2]. Although carbon sources differentially influence medium acidification during growth [38], which may then indirectly affect antibiotic biosynthesis [39] and bio control activity [9] but in our result we did not observe any significant change medium pH after 2 day and 5 day of growth. On 2 day, Pi, Fe2+, Citrate with Pi, citrate with Fe2+ and citrate with Mo2+ have shown negative effect on antifungal activity. On 5 day only glucose with nitrate and citrate with glucose and nitrate have shown negative effect on antifungal activity. Glucose with nitrate contributed 14% on 2 day and only 1% on 5 day. Citrate with nitrate contributed 6% on 2 day to 15% on 5 day. Contribution of triple combination of citrate, glucose and nitrate was 25% on 5 day but no contribution on 2 day. Citrate with nitrate showed 6% contribution on 2 day and 15% on 5 day.

Contribution order of factors acting positively to antifungal activity on 2 day is, Glucose>citrate>citrate +glucose while on 5 day antifungal activity the order is, citrate> citrate +glucose>Fe2+>glucose>citrate +Pi=citrate+Fe2+. Combination with Fe2+ and citrate have shown increased antifungal activity on 5 day and was thought to be mediated by induction of antifungal traits specifically. It is in support of previous observation that iron stimulates bicontrol activity [13] and cyanide production [2]. However the antifungal activity on 2 day is negatively regulated in following order, Citrate +Pi > citrate +glucose + KNO3>> citrate+Fe2+ and on 5 day in the order citrate + glucose +KNO3> citrate+KNO3> glucose + KNO3>Pi=KNO3. Glucose and citrate individually and in combinations, supported well to growth and antifungal activity. Combination with mineral supplementation of Fe2+, Mo2+, Zn2+ and Pi showed pigmentation after 5 day of growth and 40% antifungal activity and thought to positive effect of Pi on growth and positive effect of Fe2+ on antifungal traits. Combination with glucose, citrate and nitrate and did not have any amended mineral nutrient and have shown more growth (2 day OD600nm > 4.0) and antifungal activity on 2 day and less growth (OD600nm 5 day>3) and antifungal activity on 5 day which could be possible due to high C/N ratio which will induce rpoN and also antifungal traits and have not any interference of heavy metals.

Combination with Fe2+, nitrate and phosphate showed weak growth and antifungal activity which could be possible due to very low C/N ratio but only low growth because of supplementation of Pi and Fe2+. Effect of factors on antifungal Table 2: Biosynthesis of PRN,DAPG and PLT under different nutrient activity showed many variations in effect (+/-] and contribution compare to its effect on growth. Possible reason could be that the growth is result of basic metabolism and cumulative effect of many supportive pathways but antifungal activity is contributed by limited number of pathways so the variation in effect was observed. Citrate and Pi individually have positive effect on the growth but their combination have shown negative effect which show a kind of the shift of bacterial physiology/behaviour to nutrients present individually and in combinations.'

Effect on nutritional factors on antibiotic biosynthesis

PRN biosynthesis under different nutrient combinations is depicted in Table 2. Significant variation in biosynthesis level of PRN was observed across all combinations on 2 day (p=0.0016) and 5 day (p=0.0045). Nutrient combination amended with FeSO4, KH2PO4 and KNO3 , combination amended with,KH2PO4, (NH4)6Mo7O2, FeSO4 and ZnSO4, combination amended with FeSO4, Glucose, KNO3 and (NH4)6Mo7O24, and combination amended with citrate, FeSO4 , glucose, KH2PO4, KNO3, (NH4]6Mo7O24 and ZnSO4 supported PRN biosynthesis on 2 day.Nutrient combination amended with citrate, FeSO4, glucose and KH2PO4, combination amended with citrate,FeSO4, KNO3 and ZnSO4 , combination amended with citrate, FeSO4 ,glucose, KH2PO4, KNO3, (NH4)6Mo7O24 and ZnSO4, combination amended with citrate,glucose,(NH4]6Mo7O24 and ZnSO4 and combination amended with citrate , KH2PO4, KNO3 and (NH4]6Mo7O24 supported PRN biosynthesis on 5 day. It supports the previous observation by Duffy and Defago, 1999 that the production of pyrrolnitrin by Pf CHA0 was stimulated by Mo2+.

It suggests that direct improvement in the bio control effectiveness by NH4-Mo could be indirectly mediated by altered enzymatic activity. Effect of nutritional factors and their contributions to PRN biosynthesis is depicted in Figure 3. Fe2+, Pi, citrate with glucose and triple combination of nitrate, glucose with nitrate showed positive effect on PRN biosynthesis on 2 day. On 5 day, PRN biosynthesis is primarily influenced by citrate. Fe2+ showed strong positive effect on PRN biosynthesis with contribution 29% on 2 day and 13 % on 5 day PRN biosynthesis. It is in support of previous observation that iron stimulates biosynthesis of a variety of antifungal metabolites (e.g., zwittermycin A [11], kanosamine [12], phenazine [13].

Pi showed positive effect with 8% contribution on 2 day but negative effect with 1% contribution on 5 day PRN biosynthesis which is not significant effect and supports the previous finding that Pyrrolnitrin production by Pf CHA0 was not affected by 200 mM phosphate [16]. Contribution order of factors acting positively to PRN biosynthesis on 2 day is, Fe2+ > citrate +glucose >glucose+KNO3 > Pi> KNO3>citrate +Pi while on 5 day PRN biosynthesis the order is, Citrate>Fe2+>citrate+KNO3> citrate+Fe2+ > Zn2+> KNO3=citrate +Pi. The negative effect of factors on PRN biosynthesis on 2 day is in the order, Citrate +glucose + KNO3 >Citrate +Fe2+ > citrate >Zn2+ and on 5 day in the order, glucose + KNO3> citrate+glucose +KNO3>Pi>Citrate +glucose.

The DAP G biosynthesis levels under different nutrient combinations are shown in Table 2. DAPG biosynthesis varies significantly in all 15 combinations on the 2 day (p=0.009] but non significantly on 5 day (p=0.10) Glucose, citrate with Fe2+, citrate with Zn2+ and triple combination of citrate, glucose and nitrate have shown significantly positive effect on DAPG biosynthesis at 2 day (p<0.05].The effect of nutrients on the DAPG biosynthesis on 2 day and 5 day depicted in Figure 3. Positive contribution of factors acting on DAPG biosynthesis on 2 day follows the order, glucose>citrate+ Zn2+.

DAPG biosynthesis on 2 day is stimulated by glucose followed by combination of citrate and Zn2+ confirming the previous observation that increased DAPG biosynthesis by glucose [16]. However the negatively acting factors on DAPG biosynthesis on 2 day follow the order, citrate + Fe2+ > citrate+ glucose +KNO3. On 5 day DAPG is repressed by Pi which supports previous observation that DAPG production by Pf CHA0 was almost abolished by 10 mM phosphate. Growth was increased 5- to 10fold by 100 mM phosphate amendment [16].

The biosynthesis level of PLT under different nutrient combinations shown in Table 2. PLT biosynthesis level varies significantly in all 15 combinations on the 2 day (p=0.020) and 5 day (p=0.0098]. PLT biosynthesis level was high in combination in FeSO4, KH2PO4, (NH4]6Mo7O2, and ZnSO4, combination amended with citrate ,glucose, (NH4]6Mo7O24 and ZnSO4, combination amended with citrate ,glucose, and KNO3 and combination amended with citrate, FeSO4 ,glucose, KH2PO4, KNO3, (NH4]6Mo7O24 and ZnSO4 on 2 day. High PLT biosynthesis on 5 day was observed in the combination amended with citrate, FeSO4, glucose and KH2PO4 , combination amended with ZnSO4, KH2PO4, (NH4]6Mo7O24, FeSO4 and combination amended with citrate, FeSO4 ,glucose, KH2PO4, KNO3, (NH4)6Mo7O24 and ZnSO4 (Table 2). Table 2 shows the significant effect of nutritional factors and their combinations on PLT biosynthesis (p<0.05). Zinc increased PLT biosynthesis confirming the previous finding of Duffy and Defago, 1999 that zinc increased PLT biosynthesis in all fluorescent Pseudomonas strains able to produce this antibiotic, but the level of stimulation varied.

Citrate, glucose and combination of citrate with glucose showed positive effect on 2 day PLT biosynthesis and combination of citrate with glucose and citrate with nitrate and glucose with nitrate showed positive effect on 5 day PLT biosynthesis. Citrate with glucose contributed 37%, citrate with Pi contributed to 18 %, citrate with Fe2+ contributed to 18% and citrate, glucose individually contributed to only 7% on 2 day PLT biosynthesis. For the 5 day, citrate with glucose, citrate with nitrate and glucose with nitrate has shown strong positive effect on PLT biosynthesis. Positive contribution of factors on PLT biosynthesis on 2 day showed following order, citrate+ glucose> glucose=citrate> glucose+ KNO3> citrate+KNO3 while on 5 day PLT biosynthesis the order is, citrate +glucose> citrate+KNO3 > glucose+KNO3.

The negatively acting factors on PLT biosynthesis on 2 day showed the order, citrate +Fe2+ =Citrate +Pi >>citrate+glucose +KNO3 and on 5 day in the order citrate+ glucose +KNO3> KNO3>> glucose>citrate. PLT biosynthesis is maximally repressed by combination of citrate and Fe2+, citrate and phosphate on 2 day confirming the previous observation, that production of PLT by Pf CHA0 was completely inhibited by 100 mM but only slightly reduced by 10 mM phosphate. However on 5 day PLT biosynthesis is maximum repressed by triple combination of citrate, glucose and nitrate followed by nitrate confirming the previous finding that PLT is repressed by glucose [16].

Combination of citrate and glucose has shown positive effect on 2 day and 5 day antifungal activity and well correlated by its positive effect on PRN and PLT production. Glucose showed the positive effect on 2 day antifungal activity and was correlated with its positive effect on DAPG production. Combination of citrate and Fe2+ has shown negative effect on 2 day antifungal activity and it was supported well by negative effect of this combination on 2 day PRN, DAPG and PLT production. Citrate has shown positive effect on 5 day antifungal activity and correlated with its positive effect on 5 day PRN production. Triple combination of citrate, glucose and nitrate has negative effect on antifungal activity and correlated also with its negative effect on PRN and PLT production. The importance of nutrient status to pyoluteorin production is corroborated by the observation that pyrrolquinoline quinone, a cofactor required by glucose and alcohol dehydrogenases, represses pyoluteorin production confirm that pyoluteorin production is linked to the physiological status of the cell [40].

In case of Pf-5 as bacterial ages, the stationary phase and stress response sigma factor RpoS (aS) and Lon protease, are implicated in regulation of antibiotic production. Relative levels of aS and the housekeeping sigma factor RpoD (aD) influence pyoluteorin, 2, 4-diacetylphloroglucinol, and pyrrolnitrin production. Abundant aS is required for the production of pyrrolnitrin but is repressive to pyoluteorin and 2,4-diacetylphloroglucinol production [25,26]. So the role of relative levels of aS and the housekeeping sigma factor RpoD (aD) in the increased antifungal trait on 5 day in low/moderate growth supporting combinations could not be ignored.

Contribution of PRN,DAPG and PLT for antifungal activity

Based on antifungal activity under all combinations and the biosynthesis level of antifungal metabolites, the percentage contribution of PRN,DAPG and PLT to 2 day and 5 day antifungal activity by Pf CHA0 depicted in Figure 4. For the 2 day antifungal activity, PLT has contributed 59%,PRN has contributed 26 % and remaining 15% by DAPG. For the 5 day antifungal activity PRN has contributed 59%,PLT 37% and DAPG only 4%. Regulation of DAPG and PLT production in Pf CHA0 involves a molecular balance in which each antibiotic induces the expression of its own biosynthetic genes while repressing the expression of the biosynthetic genes of the other antibiotic [41,42]. This could be possible reason of not obtaining significant variations in DAPG production under different nutrient combinations on day 5 and day 2. Another possible explanation for these deviations is that other, yet unknown effectors could interfere with the fine-tuned regulation of the DAPG-PLT balance. Similar deviation in DAPG production was obtained by Baehler et al. [41] as they have used GFP-based reporters to study the antibiotic gene expression at the transcriptional level. The possibility of involvement of some post-transcriptional regulatory mechanisms such as those controlled by the GacS/GacA two-component system Haas & Keel (2003) may be involved in the modulation of the observed effects as well.

2 day antifungal found to have maximum positive correlation with PLT followed by PRN however both PLT and PRN itself had shown strong positive correlation with OD600nm. Up to 2 day DAPG is found to be not dependent much on OD600nm and PLT 5 day antifungal found to have maximum positive correlation with PRN followed by PLT however both PLT and PRN itself had shown a positive correlation with OD6OOnm. S day PLT have shown but negative correlation with DAPG production. S day DAPG have shown negative correlation with OD600nm, PRN and PLT which proves the earlier observation by Baehler et al. [41].

Principal Component Aanalysis for 2 day and 5 day antifungal activity

For the 2 day as biplot graph (Figure S) as shown in that 2 day antifungal, OD and PLT biosynthesis showed strong correlation and fall in same zone (-x, +y).While 2 day DAPG and PRN fall in other zone (+x, +y). Based on PCA analysis for the five variables with sixteen different combinations, Pearson correlation between different variables was obtained (Figure S) which shows the degree to which the variables are related with each other. Only the variables with the Pearson value (n) > 0.3S have been considered with significant relatedness between them. 2 day antifungal is positively correlation with PLT maximally (n=0.8l8) followed by OD (n=0.S44), PRN and DAPG.2 day PRN has shown positively correlation with 2 day OD (n=0.44), antifungal activity (n=0.38). DAPG is nearly independent of OD and PLT (n<0.1).2 day PLT has shown high positive correlation with OD6OOnm (n=0.694) and antifungal activity.S day antifungal activity have shown positive correlation with OD (n=0.S22), PRN (n=0.l68) and PLT (n=0.4lS). S day PLT have shown positive correlation with OD (n=0.33) and antifungal activity (n=0.4lS] but negative correlation with DAPG production (n= -0.16). DAPG have shown negative correlation with OD (n= -0.187), PRN (n=- 0. 131) and PLT.

For 2 day the results showed that of the first three components, the first component accounted for about 54.52%, the second component about 22.36% and the third component about 13.88% of the total variance in the data set These three components together accounted for about 90.71% of the total variance and the rest of the components only accounted for about 9.29%.For 5 day biplot graph (Figure 5). shows that PLT and OD shows correlation and falls in same zone (+x, +y] while 5 day antifungal and PRN falls in other zone (+x,-y].For the 5 day PCA analysis first three components, the first component accounted for about 45.72%, the second component about 22.72% and the third component about 19.44% of the total variance in the data set. These three components together accounted for about 87.89 % of the total variance and the rest of the components only accounted for about 12.11% (Figure 5).

Plant protection assay

The plant growth promoting effect of Pf CHA0 on Vigna radiata under biocontrol trait supporting combinations has been shown in Figure 6. Combination with glucose, FeSO4, KNO3 and ZnSO4 supplemented to MS medium and combination with glucose,citrate, nitrate,Pi, ZnSO4, FeSO4 and Mo2+ have shown inhibition of plant growth in compare to non supplemented combination while inhibitory effect was minimized in inoculated with Pf CHA0.Plant inoculation study for the antifungal activity of Pf CHA0 under antifungal traits supportive combinations has been shown in Figure 6. Non supplemented combination supported to plant growth in uninoculated control and growth suppression in R.bataticola treated set. Pf CHA0 have shown highest plant growth promotion out of all combination and MS medium control. Pf CHA0 showed an effective inhibition of R.bataticola and plant protection in combination supplemented with glucose, FeSO4, KNO3 and ZnSO4 supplemented to MS medium and combination with glucose, citrate, nitrate, Pi, ZnSO4, FeSO4 and Mo2+ in compare to un-inoculated control.

In biocontrol supportive combination with Mo and Zn it could be deleterious effect of metals on plant growth which could be the possible reason of less growth in compare to non supplemented combination .Combination 16 have three metals so it has retorted the plant growth effectively in compare to combination 9 which have Fe2+ and Zn2+. Combination 9 retardation effect was minimized by PfCHAO inoculation compare to uninoculated. In bio control supportive combination the effective suppression of R.bataticola by Pf CHA0 was observed. Non Supplemented combination have supported to plant growth in uninoculated control and bio control support against R.bataticola. Pf CHA0 have shown highest plant growth promotion out of all combination and MS medium control. Possible reason could be that this combination does not have any metal which is inhibitory to plant growth and it is also supportive to Pf CHA0 growth in compare to MS medium. Pf CHA0 showed effective bio-control against R.bataticola in bio control supporting combinations.?

Go to

Conclusion

Present work establish the connection between multiple combinations of nutrients and edapic factors relevant to root zone and its effect on the growth, antifungal activity and biosynthetic of pyrrolnitrin, 2, 4-DAPG and pyoluteorin by Pf CHA0. Result showed that citrate, glucose, combination of citrate and glucose, ferrous iron and zinc have positive effect on growth, antifungal activity and antibiotic biosynthesis. Triple combination of citrate, glucose and nitrate inhibited the growth, antifungal activity and antibiotic biosynthesis. Antifungal activity is maximum contributed by pyoluteorin initially but by pyrrolnitrin later stage of growth. Bio-control supporting nutrient combinations showed effective protection of Vigna radiata from R.bataticola infection in plant inoculation assay.

Go to

Acknowledgement

Authors express a gratefully acknowledgement to University Grant Commission-Research Fellowship for Meritorious Student (UGC-RFMS) and Gujarat State Biotech Mission (GSBTM), India, for the financial support.

For more open access journals in juniper publishers please click https://juniperpublishers.com/

For more articles on Advances in Biotechnology & Microbiology Please click on https://juniperpublishers.com/aibm/

Advances in Biotechnology & Microbiology in Full text in Juniper Publishers

#Juniper publishers Juniper publishers in USA Juniper Publishers Reviews Juniper Publishers Group

1 note · View note