what experiment is 606

Experiments 601, 606, and 303

Hey there everyone. This’ll be my second blog where three experiments will be the focus instead of one. Like the first time, the reason for this is that these experiments don’t really have much to go by, even in their own episodes. Because of this, there just wouldn’t be enough to write about when only focusing on one. With that in mind, I hope you all still enjoy.

I will be talking about experiments 601, 606, and 303. The names Lilo has given them are Kixx, Holio, and Amnesio respectively. While they’re all different from there appearance, abilities, and personalities, they do have two things in common. Sadly, one is that they are easily forgettable. While looking them up, the only one I had some remembrance of was Kixx and even then I don’t know how that is. It’s not that they are bad experiments. This is where they have the second thing in common. The plot for their episodes don’t do much for them. Let me explain.

First lets take a look at Kixx, experiment 601. He was created with the purpose of combat in mind. He has knowledge of kickboxing, super strength, and two sets of arms to fully use them. Kixx would have been a great experiment until you realize that his personality is that of a bully. He’s the muscle guy who beats people up and laughs at their misery. Heck, that’s what he does in the beginning of his episode.

It only keeps going downhill for 601 when knowing that his episode is for Stitch to overcome something. In the series, there are some episodes where Stitch becomes too cocky with himself or is somehow weakened. From this, it’s made that he loses against the first encounter with the experiment for the episode. This is what happened in Kixx’s episode and it was on the second option. For some reason, and this is what happens in the episodes, eating macadamia nuts and a tennis shoe together causes Stitch to somewhat forget how to fight. I wish I was making this up, but that’s what they give us. So Stitch has to learn things and he wins his next encounter with Kixx… that’s about it.

Next is experiment 606, Holio. He has the ability to turn into a blackhole and then revert when he pleases. He is also one of the more calmer experiment. Unlike Kixx who enjoys the suffering of others, Holio showed that he felt bad when he had done something wrong… and… that’s it… I’m not joking that’s all there is to talk about this one. Watch his episode and see for yourself.

Last is Amnesio, experiment 303. Okay, let me just say this. Out of the three, Amnesio is the only one that no memory of it existing came up for me. I said that Kixx was the only one that I could remember. With Holio, as I watched the episode, there was a bit of a feeling. With Amnesio, nothing. Which is fitting seeing that he’s able to erase his victims memories. This would be a cool experiment to talk about… if his ability didn’t make everything else more enjoyable them him.

Think about it, following characters that have no idea who they are. Instead of knowing what they’re going to do because that’s them, they do things you weren’t expecting. It’s more fun to watch them over the experiment that made them that way. As for 303’s personality, there just isn’t any. I think this is the first experiment that was like this.

I wanted to speak about each of them before saying what they one true place of belonging is. For Kixx, it’s at a gym, where he is a personal trainer and even has a few work out videos. Okay, that seems about right. For Holio, it’s cleaning up debris from construction sights… okay. For Amnesio… he doesn't get one. The bad guys get him… and the episode ends in a way that suppose to be funny.

Again, they are not bad experiments. I just feel like they were placed in situations that made things rough for them to be popular. I placed them together because I wanted to mostly see what you guys thought. Be honest with me here. If you watched the show, and based off the other experiments I’ve already spoken on, do you remember these three? Amnesio has to be real because I don’t remember him. Unless they were just going by what he can do. If that being the case, they did a good job.

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Lilo & Stitch: The Series #106

Lilo & Stitch: The Series » Lilo & Stitch: The Series #106 - Holio: Experiment 606 released by Disney on October 12, 2003.

Summary Short summary describing this episode.

Holio: Experiment 606 last edited by cloudguy on 01/28/22 02:49AM View full history

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Lilo & Stitch: The Series (season 1)

Pilot film | Seasons: 1 2 | Finale film | Main

• 1 Richter (Experiment 513) [1.01]
• 2 Phantasmo (Experiment 375) [1.02]
• 3 Clip (Experiment 177) [1.03]
• 4 Mr. Stenchy (Experiment 254) [1.04]
• 5 Holio (Experiment 606) [1.05]
• 6 Spooky (Experiment 300) [1.06: Halloween Special]
• 7 Cannonball (Experiment 520) [1.07]
• 8 Yapper (Experiment 007 a.k.a. Gigi) [1.08]
• 9 Yin-Yang (Experiments 501 & 502 a.k.a. Yin and Yang) [1.09]
• 10 Kixx (Experiment 601) [1.10]
• 11 Splodyhead (Experiment 619) [1.11]
• 12 Amnesio (Experiment 303) [1.12]
• 13 Swirly (Experiment 383) [1.13]
• 14 Fibber (Experiment 032) [1.14]
• 15 Tank (Experiment 586) [1.15]
• 16 Sprout (Experiment 509) [1.16]
• 17 Elastico (Experiment 345) [1.17]
• 18 Yaarp (Experiment 613) [1.18]
• 19 Experiment 627 [1.19: Thanksgiving Special]
• 20 The Asteroid [1.20]
• 21 Topper (Experiment 025) [1.21: Christmas Special]
• 22 Melty (Experiment 228) [1.22]
• 23 Houdini (Experiment 604) [1.23]
• 24 Sinker (Experiment 602) [1.24]
• 25 Nosy (Experiment 199) [1.25]
• 26 Finder (Experiment 158) [1.26]
• 27 Slushy (Experiment 523) [1.27]
• 28 Dupe (Experiment 344) [1.28]
• 29 Shortstuff (Experiment 297) [1.29]
• 30 Angel (Experiment 624) [1.30]
• 31 Felix (Experiment 010, also called "Oscar") [1.31]
• 32 Poxy (Experiment 222) [1.32]
• 33 Hunkahunka (Experiment 323) [1.33: Valentine's Day Special]
• 34 Sample (Experiment 258) [1.34]
• 35 Babyfier (Experiment 151) [1.35]
• 36 Bonnie & Clyde (Experiments 149 & 150) [1.36]
• 37 Slugger (Experiment 608) [1.37]
• 38 Bad Stitch [1.38]
• 39 Drowsy (Experiment 360) [1.39]
• 40 External links

Richter (Experiment 513) [1.01]

Phantasmo (experiment 375) [1.02], clip (experiment 177) [1.03], mr. stenchy (experiment 254) [1.04], holio (experiment 606) [1.05], spooky (experiment 300) [1.06: halloween special], cannonball (experiment 520) [1.07], yapper (experiment 007 a.k.a. gigi) [1.08], yin-yang (experiments 501 & 502 a.k.a. yin and yang) [1.09], kixx (experiment 601) [1.10], splodyhead (experiment 619) [1.11], amnesio (experiment 303) [1.12], swirly (experiment 383) [1.13], fibber (experiment 032) [1.14], tank (experiment 586) [1.15], sprout (experiment 509) [1.16], elastico (experiment 345) [1.17], yaarp (experiment 613) [1.18], experiment 627 [1.19: thanksgiving special], the asteroid [1.20], topper (experiment 025) [1.21: christmas special], melty (experiment 228) [1.22], houdini (experiment 604) [1.23], sinker (experiment 602) [1.24], nosy (experiment 199) [1.25], finder (experiment 158) [1.26], slushy (experiment 523) [1.27], dupe (experiment 344) [1.28], shortstuff (experiment 297) [1.29], angel (experiment 624) [1.30], felix (experiment 010, also called "oscar") [1.31], poxy (experiment 222) [1.32], hunkahunka (experiment 323) [1.33: valentine's day special], sample (experiment 258) [1.34], babyfier (experiment 151) [1.35], bonnie & clyde (experiments 149 & 150) [1.36], slugger (experiment 608) [1.37], bad stitch [1.38], drowsy (experiment 360) [1.39], external links.

• Lilo & Stitch: The Series seasons and films

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• Cast & crew
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Episode list

Lilo & stitch: the series.

S1.E1 ∙ Richter: Experiment 513

S1.E2 ∙ Phantasmo: Experiment 375

S1.E3 ∙ Clip: Experiment 177

S1.E4 ∙ Mr. Stenchy: Experiment 254

S1.E5 ∙ Spooky: Experiment 300

S1.E6 ∙ Holio: Experiment 606

S1.E7 ∙ Cannonball: Experiment 520

S1.E8 ∙ Yapper: Experiment 007

S1.E9 ∙ Yin-Yang: Experments 501 & 502

S1.E10 ∙ Kixx: Experiment 601

S1.E11 ∙ Splodyhead: Experiment 619

S1.E12 ∙ Amnesio: Experiment 303

S1.E13 ∙ Swirly: Experiment 383

S1.E14 ∙ Fibber: Experiment 032

S1.E15 ∙ Tank: Experiment 586

S1.E16 ∙ Sprout: Experiment 509

S1.E17 ∙ Elastico: Experiment 345

S1.E18 ∙ Yaarp: Experiment 613

S1.E19 ∙ 627: Experiment 627

S1.E20 ∙ The Asteroid

S1.E21 ∙ Topper: Experiment 025

S1.E22 ∙ Melty: Experiment 228

S1.E23 ∙ Houdini: Experiment 604

S1.E24 ∙ Sinker: Experiment 602

S1.E25 ∙ Nosy: Experiment 199

S1.E26 ∙ Finder: Experiment 158

S1.E27 ∙ Slushy: Experiment 523

S1.E28 ∙ Dupe: Experiment 344

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• Reformed Villains

List of Experiments

Many of Stitch's "cousins" are featured in this group photo taken at the end of Leroy & Stitch.

Experiments , also referred to as Stitch's cousins , are fictional genetically-engineered alien creatures in Disney's Lilo & Stitch franchise. In the franchise's fictional universe, they are creations of Kweltikwan biogeneticist (and self-proclaimed "ice spice genius") Dr. Jumba Jookiba , who made them with the shady funding of his former partner-in-crime (and one of the franchise's characters) Dr. Jacques von Hämsterviel . The most famous of these experiments is Experiment 626, better known as the franchise's half-namesake Stitch .

The idea of the experiments were partly conceived by Lilo & Stitch director and writer Chris Sanders during the development of the film's story. Initially, Stitch was just an alien creature who crash-landed in a forest on Earth. In later developments, he was meant to be the leader of an intergalactic criminal gang and Jumba was one of his former cronies sent by the Intergalactic Council to capture him. However, test audience responses to early versions of the film resulted in the change of Stitch and Jumba's relationship to that of creation and creator, respectively.

The genetic experiments other than Stitch were initially shown in prequel comics released in Disney Adventures magazine, which despite being made non-canonical after the 2005 release of Lilo & Stitch 2: Stitch Has a Glitch , are notable for introducing an early version of later major franchise character Experiment 625, also known as Reuben. Another early instance of a pre-626 experiment appearing came in the PlayStation 2 prequel video game, Disney's Stitch: Experiment 626 , released two days before Lilo & Stitch 's theatrical release. This game featured Experiment 621 (also known as "Chopsuey" after Leroy & Stitch ), a skinny green experiment who was featured as an antagonist. The video game was also later retconned by Lilo & Stitch 2 .

The experiments in general made their formal debut in the 2003 direct-to-video sequel Stitch! The Movie , which serves as the pilot to the Disney Channel and ABC animated series, Lilo & Stitch: The Series , in which they are featured as the main plot devices. In the series, Stitch and his best friend Lilo Pelekai must find the experiments who were scattered around Kauai as dehydrated pods that activate when the come in contact with water (as seen in Stitch! The Movie ). The duo capture, name, and rehabilitate the other experiments so that they use their powers to become productive members of society instead of the destructive creatures that Jumba initially intended them to be. Thanks to Lilo's influence, Stitch, inspired by Hawaiian terminology, calls almost every one of the experiments his "cousins" (with the notable exception of Experiment 624/Angel, who becomes his girlfriend instead) and sees them all as part of his ʻohana (family). By the end of the final Lilo & Stitch film Leroy & Stitch , Lilo and Stitch would successfully reform all 625 experiments, up to and including Stitch's immediate predecessor Experiment 625/Reuben, who served as a reluctant sidekick to secondary antagonist Gantu throughout the show. During the series and Leroy & Stitch , Jumba also creates three more experiments after Stitch: Experiment 627 (also known as "Evile" to the Lilo & Stitch fanbase), Experiment 628, and Leroy (eventually designated Experiment 629 in 2020 via spin-off manga Stitch & the Samurai ). However, none of these experiments were rehabilitated; 627 could not be turned good and was dehydrated back into pod form, 628 was never activated, and Leroy and his clones were shut down thanks to a fail-safe and were sent to prison.

• 1.1 Biology
• 1.2 Creation
• 2 Experiment series
• 3.1 0-Series: Jumba's test batch, including many household helpers
• 3.2 1-Series: Civic disturbances
• 3.3 2-Series: Technological and scientific
• 3.4 3-Series: Psychological
• 3.5 4-Series: Military
• 3.6 5-Series: Elemental manipulators
• 3.7 6-Series: Battlefield or doomsday experiments with galactic implications and world-ending properties, Jumba's strongest line
• 3.8 Other experiments
• 4.1 Continuity errors
• 5 References

Background [ ]

Biology [ ].

Due to different experiments' original purposes, the majority of experiments each have a unique appearance, powers, and weaknesses. Though all experiments have unique capabilities and functions, most of them have some abilities in common due to Jumba reusing DNA from one experiment as a prototype for a different experiment, like the similarities of body shape, appearance, and even for powers like in the case of Jumba reusing some of Twang (021)'s DNA in the creation of Bragg  (145), granting the latter musical ability. Most seem capable of scaling walls like a gecko (as Stitch frequently does); many are able to grow and retract an extra pair of arms. Since they are genetic experiments each created with multiple kinds of DNA that Jumba sampled throughout the galaxy, they all age quite differently than creatures on Earth as seen with Lilo and the rest of humanity when time is lapsed twenty years in "Skip".

It is also shown that some experiments' powers (such as Mr. Stenchy (254) 's cuteness, and Checkers (029) 's hypnotic effect) do not affect other experiments, possibly so that these powers do not prevent other experiments from carrying out their primary functions. If affected by other experiments' powers, it might be temporary as Stitch managed to recover from Drowsy (360)'s sleep-inducing program without being exposed to water. Though Angel (624) primarily reverts other experiments (before her time) to evil, Amnesio (303) and Retro (210) can do so as well.

Additionally, it is shown that when an experiment is dehydrated, they will deactivate and turn into a small orb called an experiment pod. If an experiment pod gets wet, the experiment will be reactivated and released. According to Jumba, a home food dehydrator is the only known method of deactivating an experiment a second time.

On numerous occasions, it has been shown that most, if not all, of the experiments have an intentional imperfection added to their design by Jumba: a "fail-safe" of some sort. These imperfections were presumably added in by Jumba in the event his experiments ran out of control, in order to provide him with a way of stopping them in their tracks. Even his most powerful experiments, like 627 and Leroy , both had their own intentional imperfection, with an overreactive sense of humor and the song " Aloha ʻOe " being their respective imperfections.

Creation [ ]

Upon each experiment's creation, it is important that their molecules be charged. If an experiment's molecules are not fully charged, about a year after their creation, they will suffer glitches, during which they will experience seizures and temporarily revert to their original programming. If the molecular charging process is not completed, these glitches will eventually burn out the experiment's circuits and kill them.

Experiment series [ ]

The first digit of the experiment numbers reflect what series of experiment they belong to. The official series of experiments, as stated by Jess Winfield, one of the executive producers, are as follows:

• 0-Series: Jumba's test batch, including many household helpers.
• 1-Series: Civic disturbances.
• 2-Series: Technological and scientific.
• 3-Series: Psychological.
• 4-Series: Military.
• 5-Series: Elemental manipulators.
• 6-Series: Battlefield or doomsday experiments with galactic implications and world-ending properties, Jumba's strongest line.

For the most part, the colors of the experiment pods correspond to the series numbers; however, some of the pods are colored incorrectly.

List of experiments [ ]

0-series: jumba's test batch, including many household helpers [ ].

1-Series: Civic disturbances [ ]

2-Series: Technological and scientific [ ]

3-Series: Psychological [ ]

4-Series: Military [ ]

5-Series: Elemental manipulators [ ]

6-Series: Battlefield or doomsday experiments with galactic implications and world-ending properties, Jumba's strongest line [ ]

Other experiments [ ]

• According to Jess Winfield , during the development of Lilo & Stitch: The Series , the idea of it focusing on "the other 625 experiments" was birthed from an idea by artist and director Steve Lyons who "suggested that an evil villain clone Stitch into a bunch of different creatures that Lilo and Stitch would chase." Also, the phrase "the other 625 experiments" was coined either by Winfield himself, Bobs Gannaway , or possibly Barry Blumberg, although Winfield doesn't remember who was first to say it. [1]
• Disney's Stitch: Experiment 626 featured an experiment series numbered as 700, which were mass-produced by Jumba and served as enemies in the game.
• In Stitch! , eleven previously unseen experiments created by Jumba were introduced in addition to two experiments created by someone other than Jumba. The first, called Skunkuna , was created by Hämsterviel , and the second was Dark End , created by  Delia . Since neither were created by Jumba, they do not fall under his numbering scheme.
• In Stitch & Ai , Jumba creates new experiments based on ancient scrolls he is given. Most are based on Chinese mythical creatures, while others appear to be derived from some creatures. It is unknown whether or not Jumba considers these experiments a part of his existing numbering scheme.
• According to the series episode "Skip" (as seen on a monitor) and the Magic Kingdom attraction Stitch's Great Escape! (as seen on wanted posters in the exit halls of the attraction), the official shorthand prefix for "Experiment" is "X-" (X with a hyphen). In addition, Jumba referred to Spooky as "X-300" (ex three hundred) and Kixx as "X-601" (ex six-oh-one) in their respective eponymous episodes, further supporting this.

Continuity errors [ ]

• Several experiments have been given multiple numbers, such as Bonnie and Clyde , who are called 349 and 350 in their episode, but called 149 and 150 in the end credits of Leroy & Stitch . The same goes for Finder and Ploot , who are called 458 and 515 in their respective episodes, but called 158 and 505 in the end credits of Leroy & Stitch .
• Many of the experiments that appear in the background during the Aloha Stadium battle in Leroy & Stitch are duplicates or recolors and slight edits of existing experiments. They were put in as screen fillers to make it appear as if all 626 experiments were actually there. For example, Houdini appears five times in the frame, while Cannonball appears eight times; twice recolored, twice re-edited and four times copied. Phoon is also seen twice in the scene, both in normal and mutated form.
• Experiment 272 was listed as "Mamf" in the end credits of Leroy & Stitch . However, in Stitch!, Wormhole is called 272 while 275 is given to Tickle-Tummy , who was mistakenly left off the list in the movie. When asked about the numbers of these three, Jess Winfield stated: "The best I can tell you about Tickle-Tummy/Wormhole is that Jumba's genius does not extend to keeping a tidy database. There is some confusion in his records regarding Tickle-Tummy, Wormhole, and a third experiment named Mamf, with different lists giving different numbers for them. Perhaps it will be sorted out someday."

References [ ]

• ↑ L&S Says Mahalo TV Tome (December 13, 2004). Archived from the original on March 19, 2016. Retrieved on May 27, 2018.
• 1 List of Experiments
• 2 Marvin “628”

Character » appears in 1 games

Holio, also known as Experiment 606, is one of Stitch's experiment "cousins" in Disney's "Lilo & Stitch" franchise. He is designed to transform into a black hole.

Summary short summary describing this character..

Holio last edited by Veilor on 09/28/18 03:50PM View full history

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Lilo & Stitch – Experiments: 6-Series » Characters

Lilo & Stitch franchise character index Lilo Pelekai and Stitch (Experiment 626) | Major Characters | Minor Characters | Stitch! anime | Stitch & Ai Experiments : 0-Series | 1-Series | 2-Series | 3-Series | 4-Series | 5-Series | 6-Series

List of 6-Series and unnumbered experiments

• X-600: Woops — A purple, skinny, Stitch-like experiment with huge yellow buckteeth, a football -shaped head with three short white-tipped antennae and a short white-tipped tail. He is designed to be indestructible and able to do anything. He has all the powers of Stitch, but is a total klutz who accidentally bumps into everything. The only word he says is "Woops!" His pod is blue. His one true place is being a valued member of Pleakley's bowling team, since the one thing he can actually do is knock things over, including bowling pins. He is the first prototype of Stitch and is considered a failure due to his extreme clumsiness. He is voiced by Rob Paulsen . ( Appearances: "Woops", Leroy & Stitch )
• X-601: Kixx — See folder below
• X-602: Sinker — See folder below
• X-603: Zap — A bright yellow living laser beam-like experiment with lightning-shaped antennae, dark blue eyes, and a small limbless vaguely dog-like body. He was caught by Gantu with the help of Experiment 627 in "627", but was later rescued in "Snafu". ( Appearances: "627", "Snafu", Leroy & Stitch , Stitch! , Disney Sorcerer's Arena )
• X-604: Houdini — See folder below
• X-605: Warpstron
• X-606: Holio — See folder below
• X-607: Launch — A tan naked mole-rat-like experiment with four spines on its back. It is designed to warp the fabric of time and space, causing the entire universe to collapse upon itself. This experiment itself never actually appeared in The Series , but it was the blue pod on the sandcastle in "627". Rufus the naked mole-rat was mistaken for this experiment in the Kim Possible crossover episode named after him until Stitch identified him as not being one of the "cousins". This experiment may have briefly appeared in the Japanese intro sequence (and international end credits) of the Stitch! anime's second season, ~The Mischievous Alien's Great Adventure~ , when Stitch accidentally brings down the walls of the enclosed venue he was performing a rock concert in after fighting 627; a pink creature with a reddish-pink nose and large front teeth that stick out much like a naked mole rat's teeth can be partially seen behind Thresher, looking up at the collapsing walls.
• X-608: Slugger — A small, yellow pterosaur-like experiment who is designed to deflect projectiles with his tail shaped like a baseball bat . His pod is blue. His one true place is as a Little League Baseball coaching assistant. ( Appearances: "Slugger", "Glitch", Leroy & Stitch , Stitch! , Lilo & Stitch 2: Hämsterviel Havoc , Disney Stitch Jam note  Non-playable character )
• X-609: Heat — A small, red-orange vaguely dog-like experiment with small eyes, a big mouth, small drooping ears, and a large shiny black oval embedded in his forehead. He is designed to fire rays of flaming heat from the blowtorch-like feature on his forehead. He was found (in pod form), activated, named, and trained by Gantu. This experiment was sent to Dr. Hämsterviel, but was rescued by Lilo and Stitch in "Snafu". ( Appearances: "Dupe", "Remmy", "Snafu", Leroy & Stitch , Stitch! )
• X-610: Witch — A small, purple bat/ghost-like experiment. The back of her head is shaped like a cone and curved at the bottom to resemble a witch hat, and her ears resemble small bat wings. Her eyes are blue and she has eyelashes. Her stomach is light purple and her chest has a yellow heart. Her witch-like hat head has a pattern of purple and then light purple stripes. She was designed to have many supernatural abilities. These include the abilities of a ghost and being able to possess others in order to turn them into evil witches too. Once possessed, the victim is under Witch's complete control and also has her variety of abilities such as animation, spontaneous generation, levitation, demonic transformation, and other forms of reality-bending abilities. Her pod is green. She is voiced by Susanne Blakeslee . ( Appearance: Stitch! )
• X-611: El Fin (a.k.a. Doomsday) — An experiment designed to make the universe implode when Jumba speaks the password. Fortunately, Jumba forgot the password. Called the "ultimate super weapon", Gantu thought that he had X-611's blue pod in "Houdini" but he was actually reading X-119's pod upside down. His original name was Doomsday, but this was changed due to a copyright issue.
• X-612: Blandzilla
• X-613: Yaarp — See folder below
• X-614: Gunner — This experiment's blue pod was seen in the experiment profiler special feature on the Lilo & Stitch 2: Stitch Has a Glitch DVD. Its function is unknown.
• X-615: Yogee
• X-616: Joey
• X-617: Plasmoid — A large green scorpion-like experiment with a pincer-less tail. He is designed to shoot explosive balls of plasma from his tail . Plasmoid was found (in pod form), activated, named, and trained by Gantu. This experiment was sent to Dr. Hämsterviel, but was rescued in "Snafu". ( Appearances: "Dupe", "Snafu", Leroy & Stitch , Stitch! , Disney Tsum Tsum note  Was originally a non-playable event character and later cameos in Gantu's ability )
• X-618: Crystallene
• X-619: Splodyhead — See folder below
• X-620: Page
• X-621: Chopsuey — See folder below
• X-622: Jumby
• X-623: Trainer
• X-624: Angel — See folder below
• X-625: Reuben — See folder below
• X-626: Stitch — See the franchise's title characters' page
• X-627 — See folder below
• X-628 — Created around the same time as 627, it was seen only as a blue pod at the end of "627" as Jumba locked it away in a vault, saying, "So much for Experiment 627. Perhaps I will have better luck next time." This experiment never physically appeared in the franchise, nor was it mentioned ever again after "627".
• X-629: Leroy — See folder below
• Skunkuna — A black and white skunk-like experiment with a beak, three antennae on his head and two antennae on his tail. According to the English dub of the Stitch! anime, he was made by Dr. Hämsterviel instead of Jumba, and thus is not part of Jumba's numbering system. He is able to shoot explosive pellets that release an unbearable stench upon impact. He falls in love with Mr. Stenchy, who has a similar stench-releasing ability. When the stenches of Skunkuna and Mr. Stenchy combine, they form a perfect aroma. Many fans assumed that the experiment is actually Experiment 126/Stank and the English dub completely changed his identity, but Jess Winfield has stated that the Japanese original did name the experiment "Skunkuna" as it was written on the translated scripts the English dubbing team received for the episode's "re-versioning". ( Appearance: Stitch! )

Notable 6-Series experiments

• Adaptational Villainy : He's one of the experiments that Hämsterviel has control over in the anime, although his cameo in Stitch and the Planet of Sand implies that he's become good again.
• The Bully : Before he was reformed, he loved to pick fights. This trope is what he was designed to do, really.
• Expy : See the above description.
• Hair-Trigger Temper : Before he’s reformed, it doesn't take much to get him violent.
• Multi-Armed and Dangerous : He has four muscular arms, perfect for beating up others. However, unlike his cousins, they are not retractable as far as we know.
• No-Neck Chump : He doesn’t have a neck.
• Purple Is Powerful : He’s a purple experiment with super-strength and hand-to-hand combat skills.
• Spectacular Spinning : Like his inspiration, he can spin like a tornado.
• Purple Is Powerful : He is a strong purple experiment who is powerful enough to challenge Stitch in a fight.
• Would Hit a Girl : He was willing to attack Lilo.
• Would Hurt a Child : Again, he tried to attack Lilo.
• Wrestler in All of Us : Some of his fighting styles employ professional wrestling moves.

• Adaptational Villainy : He's one of the experiments that Hämsterviel has control over in the anime, although he reforms by the end of "Stitch Ahoy!" .
• Aliens Speaking English : He verbally speaks in the anime.
• Flipping Helpless : In the anime , Sinker (602) has a blind spot on his large fin that makes him immobile on land if he falls over to his side.
• Height Angst : He scowls when Stitch laughs at him for being small.
• Mundane Utility : He gets a job at a sushi restaurant cutting food. Also, at the end of the anime episode "Stitch Ahoy!", he uses his fin to cut Slushy's ice balls into shave ice.
• Suddenly Voiced : He was silent in The Series , but is able to speak in the anime, possibly due to Hämsterviel's transmutation.
• Super Not-Drowning Skills : He can't actually breathe underwater, but he can stay under there for a long time.
• Threatening Shark : Resembles a shark. He has a big shark-like dorsal fin that can cut things in half.
• Weaksauce Weakness : In the anime, it's shown that if he's forced into a horizontal position, he'll become immobile due to his large dorsal fin.

• Bunnies for Cuteness : He looks like a rabbit, has big cute eyes, and is very timid.
• Invisibility : He can turn things invisible by blinking, including himself.
• Lovable Coward : He is a cute bunny-like experiment who cowers and trembles at everything.
• Named After Someone Famous : Named after Harry Houdini , a famous magician.
• Power Incontinence : If his eyes are irritated by flashing lights, he'll blink rapidly and turn many things invisible by accident.
• Shrinking Violet : He is very shy and turns invisible whenever he is scared.

• Black-Hole Belly : He sucks things into his mouth but he can give some of it back like Mertle's dolls.
• Gonk : He has a wide yet flat-looking head compared to the other experiments.
• Gravity Sucks : He can turn into a black hole, sucking up everything in its vicinity.
• Early Installment Character-Design Difference : In his debut episode, he had no tail. In "Checkers", he had the typical stumpy tail seen on several experiments.
• Extreme Omnivore : He can eat anything using his black hole.
• Mundane Utility : Now he uses his ability to clean up litter and debris.
• Sequel Non-Entity : He does not appear in the Stitch! anime.
• Unrealistic Black Hole : He can summon black holes at any time even when he's not in space. Also, they're not quite as powerful as actual black holes, and anything that goes into his black holes does not undergo spaghettification and can be retrieved.
• Vacuum Mouth : His main function is to suck things into his mouth, even after redemption where he cleans up debris.
• Volumetric Mouth : He can turn his mouth into a black hole and inhale anything like a vacuum cleaner.

• Bizarre Alien Biology : His horn is of the sound-producing definition and is able to curl up when not in use.
• Bizarre Alien Locomotion : Cuter than most examples of this trope, he goes around by bouncing on his tail like Tigger .
• Blue Is Heroic : He is turquoise and his one true place is a warning alarm at Lilo's halau.
• Cute, but Cacophonic : He's small, blue, and fuzzy, but makes extremely loud noises from the horn on his head.

• Gale-Force Sound : He can make sounds so loud that they knock people off their feet.
• Multi-Armed and Dangerous : Subverted ; he has four seemingly non-retractable arms but they're not what makes him dangerous.
• Super-Scream : His power is to emit deafeningly loud noises through the horn-like appendage on his head.
• Series Continuity Error : He accidentally appeared in the episode before his debut, already reformed. He also appeared in the E.A.R.W.A.X. fight in "Spike" despite not being part of the group, as he's only meant to appear in the trivia competition in the same episode.

• Bioweapon Beast : Like most of the 6-series, but particularly noticeable in his case due to being based on a specific weapon (a Plasma Cannon ).
• The Cameo : In Big Hero 6 , his image appears on a pillow in Fred's bedroom, making him the first of Stitch's cousins to appear in the Disney Animated Canon in some form.
• Elemental Rivalry : Had one with Slushy (523) , Splody's opposite in elemental power, where they had an epic fight against one another in Slushy's episode. They also started the fight during Pleakley's E.A.R.W.A.X. session in "Spike" when Slushy threw a snowball at Splody, who returned fire only for Slushy to bat it away and break out Spike in the process. They eventually resolved their issues with one another after seeing Pleakley successfully use hugging therapy on Spike, leading the elemental opposites to hug each other.
• Evil Smells Bad : In the anime, his ability was modified by Hämsterviel to fire purple balls of a terrible odor . Said modified ability would be reversed so he would expel a more pleasant smell.
• Inconsistent Coloring : Splody's pod is blue in his debut episode, but red in the anime.
• Plasma Cannon : He is a living one, although the plasma he fires look like fireballs, not the green blobs we see throughout the franchise.
• Playing with Fire : His main ability, although it's actually hot plasma that he fires.
• Series Continuity Error : Jumba mistakenly referred to Splodyhead as Experiment 515 in "Slushy"; 515 is the number of Deforestator.
• Your Size May Vary : His size has been somewhat inconsistent in his appearances, most notably in "Spike" when in one scene, he appeared a lot bigger due to a layering error.

• Aliens Speaking English : He speaks fluent English with a very nasal voice like Stitch.
• Evil Laugh : He randomly bursts into maniacal laughter.
• Green and Mean : He has green fur, he's one of the main antagonists and bosses of the Stitch: Experiment 626 video game.
• Green-Eyed Monster : He was very jealous of 626 and tried to prove himself to be the superior experiment over the younger experiment. It didn't work.
• Flawed Prototype : Has Stitch's powers and abilities, but he's not as capable as him.
• Multi-Armed and Dangerous : Has four arms, but they don't seem to be retractable.
• No Name Given : In his only appearance as it was a prequel to the events of the original film. He was named by the time of Leroy & Stitch , however.
• One-Shot Character : Only appeared in a Spin-Off prequel video game that was removed from canon after a sequel film came out.
• One-Winged Angel : After his mutation, getting a stretched-out face with bulging bugged-out eyes, a wide chest with an even skinnier waist, longer arms with narrow upper arms and very thick forearms, longer legs with skinny thighs and thick lower legs, and longer spines (doubled from three to six), claws, and toes.
• "Well Done, Son" Guy : He seeks his creator's approval despite Jumba being disappointed with his results.

• Action Girl : She isn't as limited in capabilities as most of her predecessors. She knows how to fight, as seen when she busted herself, Stitch, and others out in "Snafu", and when she quickly took down four Leroy clones to protect Stitch in Leroy & Stitch .
• Adaptational Jerkass : She became more short-tempered in the anime, getting angry at Stitch for petty reasons.
• Affectionate Nickname : She and Stitch refer to each other as their "boojiboo", which is a term of endearment meaning "beloved" or "loved one" in their native Tantalog language . Reuben, who was more attracted by her looks, called her "Hotcakes".
• Alien Hair : Her antennae vaguely resemble long human hair. She can use them like tendrils in lieu of a second pair of arms, but the only time they do something was in her debut episode where she uses one to push an out-of-reach button .
• Aliens Speaking English : Not in The Series , as she spoke just two non-Tantalog words in "Snafu", but by the time of the anime she's become a pretty proficient English speaker, though she still speaks Tantalog most of the time. Justified considering that she probably needed to learn English in order to have her singing appeal to humans. This is also averted in Disney Speedstorm where Tara Strong reprises her role.
• Ascended Extra : As explained below, she went from intended One-Shot Monster of the Week to becoming one of the most prominent characters of the franchise.
• Beauty, Brains, and Brawn : The Beauty of the main trio of experiments (which includes Angel, Reuben, and Stitch). She is the most attractive, the best singer, note  Reuben can carry a tune and his original voice actor does sing, though Tara Strong and Kate Higgins are professional singers. And does anyone really want to hear the extremely nasal Stitch (who is voiced by a filmmaker and illustrator with no real singing ability) sing? and the only female of the three. That said, she has shown some of the other two parts of this trope in The Series and Leroy & Stitch .
• Becoming the Mask : First pretended to love Stitch and be his new girlfriend, but his sheer kindness and siren song immunity caused her to actually fall in love with him and become his girlfriend.
• Breakout Character : She was in only two episodes of The Series , note  three if you count her appearance in a dream in "Remmy", one of them being the show's last regular episode , and in Leroy & Stitch she was only in the final battle against the Leroys and in the big family photo at the end. She was also not meant to appear ever again after her debut episode . Despite this, she is the most popular experiment besides Stitch himself that the franchise ever introduced. The anime featured her much more often, she has made several appearances in Disney's crossover video games including Disney Magic Kingdoms and Disney Speedstorm , and she nowadays gets more merchandise than Lilo.
• The Cameo : And to also continue from the above trope, she even appears as a plush toy in the third episode of Monsters at Work .
• Cute Monster Girl : Not that the other experiments aren't cute themselves , but her Stitch-like body has a few humanlike traits and we see very few females.
• Character Development : She originally acted sweet to Stitch simply to sell him out to Hämsterviel, but later does have true affection for him.
• Damsel out of Distress : She breaks out of her own containment capsule after Stitch gets himself captured and he confesses his love to her.
• Defecting for Love : Thanks to Stitch being the first experiment to treat her kindly (helped by the fact that he's immune to her siren song), she ended up feeling guilty for her manipulation of him. As a result, she turned on Gantu and Hämsterviel by reverting the experiments back to good just before the cousins could beat up a weakened and reluctant-to-fight-back Stitch. Unfortunately, this decision did lead to her capture by Gantu at the end of her episode.
• Distaff Counterpart : To Stitch. The only real differences between them are her color, her long antennae, and her markings, especially the white V-shaped chest marking that (fittingly) resembles angel wings and the back marking that ( normally ) looks like an upside-down heart.
• Dude Magnet : She utilizes this to attract reformed experiments so she can revert them with her singing.
• Evil Diva : Her song itself has evil repercussions. Also, yes, she is a diva; the anime shows quite well how her international and intergalactic fame has affected her.
• Face of an Angel, Mind of a Demon : At first as she pretended to be sweet to take advantage of Stitch, but she drops the "demon" side later. This is also the basis for her name since Lilo named her in sarcasm (see Shout-Out below).
• Flanderization : Angel actually did have a small bit of a diva personality in The Series , but the anime made it a defining aspect of her character.
• Flat Character : The Series ' s version of Angel didn't have much of a defined personality other than being a pretty girl version of Stitch with a siren song who was initially antagonistic. Stitch! gave her a job that fits her abilities and the character flaws to go along with it.
• Hair Substitute Feature : She has two long antennae that resemble human hair.
• Heel–Face Revolving Door : While not exhibited by her herself, this is the basis of her primary function, able to invoke a Face–Heel Turn in any experiment made before her (and Jumba) with a song, and can invoke a Heel–Face Turn in the same by singing it backward .
• Heel–Face Turn : Used to work for Hämsterviel, but changed sides after realizing her feelings for Stitch.
• Heroic Sacrifice : In her debut, although technically Gantu captured her moments after the experiments were freed and he has to blast Stitch in a net while their guard was down. She was later rescued.
• Kissing Cousins : Although she's not actually directly related to Stitch beyond their similar appearances and sharing the same creator, since they are artificial lifeforms with their own unique DNA mixtures, and the "cousins" thing with the experiments are based on Hawaiian slang where it's used to refer to friends , not necessarily relatives. Still, they are in love, though tellingly, Stitch never calls or considers Angel a "cousin" to get around this problem.
• Iconic Sequel Character : The most famous of Stitch's predecessors, but it took a long while to achieve this status. She didn't appear at all in the 2002 original film, was only hinted at through a mini-poster and a bonus feature included with 2003's Stitch! The Movie , officially debuted in the thirtieth aired episode of Lilo & Stitch: The Series in January 2004, did not make her only other major appearance in the show until the penultimate aired episode in June 2006, finally became a recurring regular in the Stitch! anime that debuted in 2008 (whose English dub first aired in 2009), and eventually gained a steady stream of merchandise sold worldwide (including the United States, where the anime that she appears most frequently in had only a brief run) from The New '10s onward. She even makes video game appearances years after her debut.
• Her fur was a deeper shade of pink in her debut episode compared to her official artwork and all her later appearances.
• Her appearances in Gameloft 's Disney games, including Disney Magic Kingdoms and Disney Speedstorm , color both sides of the tips of her ears purple rather than only the insides of the ear tips.
• Ironic Nickname : At first since Lilo gave 624 her name in sarcasm, although Stitch believed her name to be completely unironic before she actually reformed.
• Jerk with a Heart of Gold : She may be a bit selfish and snobbish, but she cares for Stitch greatly.
• Love at First Sight : Subverted at first; she caused this to Stitch but didn't truly have feelings for him until a day later.
• Love Redeems : How she was reformed instead of Defeat Means Friendship . She realizes that she does have feelings for the dedicated, sweet-hearted Stitch, and decides to defy Gantu and Hämsterviel to save him.
• Magic Music : Not quite magic, since this comes from an artificial alien lifeform and is known to work on only similar artificial alien lifeforms made before her (as well as her creator), but hearing a song like hers normally won't affect one's morality so suddenly.
• Manipulative Editing : Due to her being reformed, Reuben had to trick her into singing to him (he and Stitch are the only experiments unaffected by her singing) so that he could record her voice and play it backward, turning the experiments tamed by Lilo evil again. Experiment 120 (Snafu) would ruin these plans sometime later.
• Mascot : The de facto one of the experiments other than Stitch, considering she's usually the only one of the first 625 that Disney is willing to promote.
• Messy Male, Fancy Female : The Fancy Female to Stitch's Messy Male. Stitch has spiked tufts of fur on his head and chest, while Angel's fur is smooth, with a white V-shaped marking on her chest. In addition, Stitch has notches in his slightly pointed ears and his retractable antennae are short with budded tips, while Angel's slightly rounder ears lack notches and her non-retractable antennae are long, curved, and smooth with rounded tips.
• Morality Pet : She quickly establishes herself as the second one of these for Stitch (Lilo's the first, obviously) in her debut. He behaves a lot more politely whenever she's around him.
• In her eponymous debut episode when Stitch is captured by the experiments she turned to evil.
• Also in her first anime episode ("Angel's Flight") when she accidentally made Stitch evil again, eventually leading to him breaking the wind chime Yuna made for her grandmother, causing the human girl to snap at Angel for all the problems she caused during her visit.
• No-Sell : Her siren song can turn any experiment evil, but if that experiment is created after her, they will be completely unaffected by it, though they may still compliment her beautiful singing nonetheless. However, the anime shows that it's (apparently) actually possible to convert experiments made after her to evil if it's performed with a different voice; when she got a head cold in the episode, her singing voice became croaky as she was singing her song repeatedly to entertain herself and Stitch on their date. The worse-performed but still-in-tune song affected Stitch and turned him to evil again.
• Now Do It Again, Backwards : Singing or playing her siren song in reverse around her affected victims turns them good again.
• Our Sirens Are Different : An alien genetic experiment siren who seduces other alien experiments made before her, singing her siren song to turn them to evil . Acoota chi-meeto igatta no mootah, nagga to nala itume tidooka Translation Heed me, you are in my power. Henceforth, nothing shall be too cruel or too abominable for you. You cannot escape.
• Pink Girl, Blue Boy : With Stitch given their fur colors.
• Pink Means Feminine : She has pink fur and is girly, especially in Stitch! .
• Pint-Sized Powerhouse : Well, not to the extent of her boyfriend as far as we know, but she is strong enough to break open containment capsules with only a headbutt . She's also strong enough to knock Stitch out with a headbutt.
• Retractable Appendages : Does not seem to have these (her antennae are always exposed and she is too thin at the waist to have the extra arms), but she does have retractable claws. (Pay close attention to her first scene in her debut when she threatens Lilo.)
• Satellite Love Interest : To an extent in that the main reason she was introduced was to give Stitch a love interest. The anime sanded down the "Satellite" part by giving her more of a personality.
• Sequel Non-Entity : Despite that her popularity allowed her to gain more appearances in the anime, this did not follow through with Stitch & Ai .
• Shout-Out : Her name is most likely a reference to the subject of Elvis Presley 's "(You're the) Devil in Disguise", especially since Lilo named her "Angel" as sarcasm.
• Single Woman Seeks Good Man : Although she did not intend to fall in love with him, Stitch's kindness is what won her over, although he is immune to her siren song. Compare to how she primarily acts around Reuben who behaves more like a sleazeball around her.
• Suddenly Speaking : In the anime. In the original series, she only speaks Stitch's alien language, but she speaks English in the anime, albeit not as much as more fluent Stitch in that show.
• Tertiary Sexual Characteristics : She looks like a version of Stitch with a very high-pitched voice, pink fur, long eyelashes, an hourglass figure, a heart-shaped marking on her back, and long antennae that look like hair.
• Took a Level in Jerkass : In the anime where she gains the personality of a vain, spoiled celebrity (due to becoming a popular singer in the years since Leroy & Stitch ) and gets charmed by someone else at least three times (even though she always came back to him).
• The Unintelligible : In the original series she only speaks in Tantalog and never speaks English once.
• Use Your Head : Angel has a surprisingly strong headbutt for someone who was only made to be a siren. In the The Series episode "Snafu", she headbutts her capsule to shatter it open, then headbutts Stitch's capsule to free him, and then both do so to free Lilo and the other trapped experiments from their capsules . In the Stitch! anime episode "Angel's Flight", she headbutts Stitch after she accidentally made him evil again when she, coming down with an awful head cold, sang her song beside him with a croaky voice to knock him out cold and end his sudden rampage .

• Adaptation Dye-Job : He's teal-colored in Comic Zone: Lilo & Stitch but was changed to golden-colored for the films and TV series to better differentiate him from Stitch.
• Affably Evil : For an experiment designed to cause massive destruction (and one of Hämsterviel's employees), he's surprisingly laid-back and easygoing.
• Aliens Speaking English : Unlike his cousin Stitch, he's perfectly fluent in English, speaking it with a Brooklyn accent . This is one of the very few advantages he has over his younger cousin, although the anime's English dub puts them on near-equal ground in English fluency. He is also implied to know several other Earth-based languages. It's justified in that he has "advanced language programming", though it's never explained why he has such programming and Stitch doesn't .
• Appropriated Appellation : After a few attempts he wasn't proud of, he takes to Lilo's last name suggestion, Reuben.
• Beauty, Brains, and Brawn : The Brains of the main trio of experiments (which includes Angel, Reuben, and Stitch). Although he has the exact same strength as Stitch, his laziness negates the Brawn aspect, and he isn't as "cute" or attractive as the more popular Stitch or Angel (not to say that he isn't cute, though). But he's easily the Brains considering he's more fluent in English than Stitch and Angel (both of whom do not speak English as fluently), makes tons of snarky quips, knows a lot about the experiments before his time, and is able to use Awesomeness by Analysis the most effectively. note  Stitch has the same supercomputer levels of thinking, but he is quicker to make brash decisions and is more temperamental.
• Because You Were Nice to Me : Lilo wins him over by naming him and actually befriending him.
• Big Eater : Mostly of sandwiches, and he's not picky about what ingredients he puts in them.
• Bioweapon Beast : Like Stitch, he's a genetic experiment meant to be an ultimate superweapon. Unlike Stitch, he's too lazy to use his powers properly .
• Bookends : He is one of the first experiments to be reactivated since Stitch's creation (which happens in Stitch! The Movie ), but the very last one to be rehabilitated and named (which happens in Leroy & Stitch ).
• Brilliant, but Lazy : He has all of Stitch's abilities, including super-intelligence, but not the motivation. Some episodes do show how clever he really is, such as figuring out Experiment 627's Achilles' Heel just by noticing his heavy laughter .
• Bullying a Dragon : Often insults and otherwise antagonizes Gantu, who dwarfs him in size and is armed. Since 625 lacks the motivation to use his Stitch-like strength, he is thus often fried by a blaster or stomped on.
• Canon Immigrant : He originally appeared in a series of Disney Adventures Comic Zone Prequels to the first movie.
• Card-Carrying Villain : Unlike Gantu, who sees himself as the hero, Reuben calls himself and Gantu "the bad guys" in "Angel".
• Reuben stars in his own game, 625 Sandwich Stacker .
• He's also the deuteragonist of Leroy & Stitch , playing an important role in helping Lilo reunite with Stitch, Jumba, and Pleakley.
• Stitch! gives him an third-season episode where he actually plays an active (for once) role of being a villain with "Reuben 2.0".
• Deadpan Snarker : One of his main functions. Gantu (injured from his fight with X-601/Kixx) : I will not be made a fool of! X-625/Reuben (immediately without even facing Gantu) : Too late.
• Deuteragonist : In Leroy & Stitch , where he gets his Heel–Face Turn thanks to Gantu abandoning him and Lilo giving him a name. He helps her out in rescuing Stitch, Jumba, and Pleakley after they find out that the experiment they were talking to was not Stitch at all.
• Flawed Prototype : To Stitch. He has all the same powers and abilities as Stitch, but he's also extremely lazy and an incredible coward.
• Freudian Excuse : He claims in the Grand Finale that the only reason he worked against Lilo and Stitch is because Lilo never bothered to try to help or even name him. There is some justification here if you've read the prequel comics; Jumba considered 625 a failure and frequently disparaged him... except for his sandwiches.
• Friendly Enemy : Arguably towards Lilo and Stitch when he was one of their enemies in The Series , and had helped them out on occasions, although it's likely due to not caring to actually fight them.
• Heel–Face Turn : In Leroy & Stitch after Gantu sets out on a small, two-man ship to break Hämsterviel out of prison (the second spot being for Hämsterviel), and leaves Reuben stranded on Earth. Unfortunately, this does not stick in the anime, in which he's back working for Hämsterviel and is slightly more proactive than before.
• Heterosexual Life-Partners : With Gantu; even at the end of Leroy & Stitch , he reconciles with Gantu and finds his one true place as Gantu's first mate and galley cook.
• Iconic Sequel Character : Although dwarfed in popularity by his immediate successor and predecessor, he is one of the major characters of the franchise and the most often seen experiment in the canon besides Stitch himself.
• The Imp : He is just as powerful as Stitch, but he is never a real threat. He just spends most of his time making wisecracks (and sandwiches) and eating.
• Inconsistent Coloring : Reuben's pod is blue in Stitch! The Movie , although it appeared as purple in some frames of that film. In the anime episode "Stitch's Birthday, Part One", when Reuben was dehydrated, his pod is white.
• Informed Ability : A justified version: it has been stated that he has all the same powers as Stitch, but he is never seen using them because, from the start, Jumba explains that he's also a lazy coward. Finally ends in Leroy & Stitch , when Lilo finally convinces him to get off his lazy patookie and do something for himself.
• Irony : He was in his one true place all along: by Gantu's side.
• Late-Arrival Spoiler : His name, which he didn't get until the last film. The Stitch! anime and other official Disney media released since usually use "Reuben" instead of his experiment number, note  Oddly, however, a Lilo & Stitch: The Series -themed event in Disney Tsum Tsum did not refer to him as "Reuben", but rather as "Experiment 625" like in the show, despite Leroy appearing in the same event. and his costumed character also signs his autographs as "Reuben". note  The signature shown on Character Central (a Disney fan site about costumed characters) shows that Reuben's performer at the catered event where the photographer got the signature misspelled his name as "Rueben".
• Lazy Bum : He'll get around to helping Gantu in the field once he's done eating sandwiches and watching TV.
• The Load : He's only useful when it comes to sandwiches. Everything else? Yeah, no.
• Lovable Coward : He's absolutely useless as a sidekick, but he's still an adorable-looking experiment.
• Meaningful Name : He's named after a type of sandwich, the very food he loves. Also, since Reuben is behaviorally the most human-like experiment, his name is a more natural-sounding human given name, which he prefers over the sillier names Lilo gives the other experiments; 625 even rejected a few Punny Name suggestions from her before settling on "Reuben".
• Minion with an F in Evil : Or really, "Minion Who Can't Be Bothered to Do Any Evil". Although Sloth—which he excels at being— is one of the Seven Deadly Sins , he can't be bothered to help out Gantu or Hämsterviel unless he's either forced to or gets some benefit for doing so.
• Multi-Armed and Dangerous : Subverted ; he has the retractable lower arms like Stitch does but is no real threat to anyone because he just doesn't care to be threatening.
• Mundane Utility : He has extendable claws, but the first time we see them, he's using them like butter knives to spread some grape jelly on a sandwich.
• No Name Given : He had this status (regarding names other than his experiment number) until he was named "Reuben" in Leroy & Stitch , although he was called "Sandwich Boy" by Lilo once in the episode "627".
• Redemption Promotion : Once he decides to stop being lazy and help Lilo, he actually proves himself to be quite useful much like his successor. He even manages to repair Gantu's ship, which Gantu himself was incapable of doing.
• Retractable Appendages : Revealed in Leroy & Stitch that he does have a lower pair of arms much like Stitch. Before that, an episode of The Series also reveals that he does have retractable claws.
• Satiating Sandwich : Loves making these and is incredibly good at doing so, to the point that he was named after one.
• Sequel Non-Entity : Despite his regular appearances throughout the first two TV shows, he did not show up or was even mentioned in Stitch & Ai .
• Supreme Chef : Reuben loves making sandwiches and they prove to be very delicious. The problem is that he is obsessed with making them due to his programming from Jumba.
• Took a Level in Badass : In Leroy & Stitch after Lilo encourages him to use his powers to save the galaxy.
• Trademark Favorite Food : Sandwiches; he loves anything that can be placed between two slices of bread.
• Vitriolic Best Buds : With Gantu.
• Vocal Evolution : A minor case; in Stitch! The Movie , Rob Paulsen gave Reuben a sort of low, husky voice. In The Series , however, Paulsen pitched his Reuben voice up a little and made it a slight bit more nasal.
• You Are Number 6 : He remained number 625 through The Series up until Leroy & Stitch when he was given the name "Reuben" by Lilo.

    Stitch (Experiment 626)  Voiced by : Chris Sanders , Ben Diskin ( Stitch! , Stitch & Ai ) Voiced in Japanese by : Kōichi Yamadera Voiced in Mandarin Chinese for Stitch & Ai by : Li Zhengxiang Pod color : N/A; Stitch was never dehydrated into pod form.

A blue koala-like experiment who was designed to "destroy everything he touches", but ended up defying his programmed function after being adopted by Lilo Pelekai as her pet "dog". He is The Protagonist of the franchise.

See the characters sheet for the title characters for tropes and more information.

• Always Someone Better : He is the evolution of Stitch; greater strength, higher intelligence, fewer weaknesses, and more powers than ever before.
• Big Little Brother : Well, "cousin", but still 627 is taller and younger than Stitch.
• Black Eyes of Evil : Oh, yes. His eyes are black and can't become good in the slightest.
• The Bully : Really what his personality is in general. He could kill anything he pleases but doesn't because he finds torturing others more fun.
• Combo Platter Powers : He has the abilities of many other experiments, not just having improved versions of Stitch's powers.
• Evil Counterpart : To Stitch, being specifically made by Jumba to stay evil and not ever be reformed.
• Evil Laugh : Has a lower-pitched one in comparison to Stitch's.
• Ear Notch : One on each ear like Stitch, but they are symmetrical to one another near the tip.
• Evil Redhead : He has red fur and he is one of the few experiments who never become good.
• Evil Sounds Deep : Has a deeper, gravely sounding voice compared to Stitch. Ben Diskin further exaggerates this in the anime, except in 627's duke disguise.
• Forehead of Doom : He is designed to be even more powerful than Stitch and has a large cone-shaped head.
• For the Evulz : He loves demoralizing others, to the point that he will even ruin bad moments just because he can.
• Giggling Villain : He is rather easily amused for an evil experiment; Jumba even says that he has an extra sense of humor.
• Gone Horribly Right : Meant to have extra everything Stitch had. This includes an extra sense of humor, causing his laughter to inconvenience him .
• The Juggernaut : Good grief, when he gave Stitch —who's designed to be this very trope—a No-Holds-Barred Beatdown ...
• Heel–Face Turn : Jumba designed 627 to defy this trope , making it impossible to rehabilitate him. Doesn't stop him from sort of doing so in the anime.
• The Hyena : Laughs a lot with an extra sense of humor. Deconstructed as his laughter is also his Achilles' Heel ; he goes into cataplexy and will collapse if he laughs uncontrollably .
• Multiple Head Case : He has a retractable second head.
• My Brain Is Big : He has a large, cone-shaped head ( two, in fact ) to match with his higher intelligence.
• Nested Mouths : He can extend his jaws (with the tongue inside) out from his mouth.
• Nigh-Invulnerable : Most likely more so than Stitch, though his laughter undermines this.
• No Name Given : Since he could not be rehabilitated, Lilo never named him.
• Olympus Mons : The most powerful experiment that Jumba ever created.
• One-Shot Character : The only other appearances this experiment made in The Series was a cameo in Lilo's dream in "Remmy" (which is also the only time he is seen as "good") and the reuse of footage from his original appearance in "Ace" . He would appear again in the anime, but it too became a one-time appearance within that show, although he also appears in " Stitch Meets High School Musical " as well (again, seen as "good").
• One-Word Vocabulary : The only word he could say was "evil". He would gain a greatly expanded vocabulary in the anime.
• The Only One Allowed to Defeat You : In the anime, he tells Stitch this after saving his and Angel's lives .
• Retractable Appendages : Has more of these than Stitch; he can gain four extra arms instead of just two, four spines (compared to Stitch's three) and another head , with each head having its own retractable antennae.
• The Rival : To Stitch, at least in 627's only Western appearance, as the egotistical Stitch suddenly finds direct competition with this experiment in experiment hunting, only to find himself on the losing end. It took an unusual strategy for Stitch to exploit 627's weakness and defeat him.
• Smug Super : In his original appearance, he tortured Stitch with a No-Holds-Barred Beatdown rather than just killing him, just to prove that he is better in every way.
• Suddenly Speaking : In the anime, as Hämsterviel reprogrammed him to be able to speak. Quite eloquently, too.
• Superior Successor : Designed to be a better (albeit worse) Stitch, having improved versions of his powers, none of his weaknesses, and some of the other experiments' abilities.
• Superpower Lottery : Not only does he have improved versions of Stitch's powers, but he has a lot of powers that some other experiments have as well. And then some.
• Villain Respect : To Gantu in The Series , judging by how easily he tends to obey him. It could be that 627 was intrigued by Gantu's ambitions.
• Weaksauce Weakness : Laughter. He has a sense of humor so immature that when Stitch makes a fool of himself on purpose, he laughs so hard that he faints .
• Wicked Cultured : In his anime appearance, where he even gains a greater vocabulary.
• You Are Number 6 : He is the only activated experiment who retains his experiment number as a name. (Note that Experiment 628 was never activated.)

• Conservation of Ninjutsu : The original Leroy can defeat Stitch in a battle, but the army of clones are unable to defeat the rest of the experiments, despite outnumbering them 80 to 1.
• The Dragon : For Hämsterviel in Leroy & Stitch , usurping the role from Gantu.
• Evil Counterpart : To Stitch.
• Evil Doppelgänger : Not only does he looks like a red Stitch, but he can also shapeshift somewhat to perfectly resemble him.
• Evil Redhead : He has red fur and like 627 he is one of the few experiments who never become good.
• Evil Twin : Disney Tsum Tsum and some official promotional descriptions for his film describe him as this towards Stitch, since they have identical powers and Leroy was made (from the same 626 template, no less) so that Hämsterviel would have his own version of Stitch. Thus, it could be that Leroy is Stitch's only actual brother.
• Fake Ultimate Mook : The clones despite looking intimidating, are extremely weak to the point of relying on a numerical superiority of 80 to 1 in order to defeat the other experiments.
• Final Boss : The last experiment and opponent Stitch faces in the original Western Animation era of the franchise.
• Flat Character : Being Stitch's Evil Counterpart is all there is to Leroy, as despite the fact that he is one of the main antagonists of Leroy & Stitch , he does not get any character development. You could almost take him out of the movie and replace him with Stitch clones since he doesn't contribute much despite sharing the title with Stitch.
• Flight, Strength, Heart : All of Stitch's powers, plus he can instantly regrow his fur and change colors.
• Good Colors, Evil Colors : He's a red, unreformed Stitch. Hämsterviel deliberately wanted Leroy colored red, because one, he hates the color blue, and two, he wanted an experiment that matches his red cape.
• Identical Twin ID Tag : After Hamsterviel creates a clone army of him, the original is told apart from them by the captain's uniform he took from Stitch.
• Ironic Name : Leroy's name means "King" or "The King" in French. His one weakness is "Aloha ʻOe", a song covered by Elvis Presley and programmed into him specifically through one of his records.
• Me's a Crowd : Mass-produced by Hämsterviel soon after his creation.
• Mooks : Leroy is a mass-produced version of Stitch.
• No Name Given : invoked This was Inverted until June 2020; he was named by Hämsterviel, but not numbered. Jumba tried to number him 627, but Gantu reminded him that he already made Experiment 627. Most fans gave Leroy the number 629, while some Japanese and Disneyland Paris merchandise give him the number 628, except that there was already an Experiment 628 made, too, but no one in the franchise knows that besides Jumba, who's likely also forgotten about 628. In 2018/2019, Disney Tsum Tsum reaffirmed the lack of a number in both the Japanese and Global versions for a Lilo & Stitch: The Series -based event. This flip-flopping of Leroy's experiment number in official Disney media finally ended in June 2020, when a side story of the Feudal Japan -set manga Stitch & the Samurai was released exclusively through the Japanese version of the mobile game and gave Leroy the title of Experiment 629, though Disney, for whatever reason, did not translate his part of the side story into English . It took until April 2021 for Lilo & Stitch fans in English-speaking regions (including the U.S.) to get their confirmation of Leroy being 629 through a licensed sticker book of all things.
• Our Clones Are Different : Leroy is only related to his blue doppelgänger Stitch in that he's a new experiment who was created based on Stitch's template. That being said, Hämsterviel uses a machine which creates an instant clone of the subject in a flash of lightning at the press of a button to clone Leroy. Many times.
• Paper-Thin Disguise : Played with. He looks exactly like Stitch when he turns blue, but can't really pull off the act.
• Tom the Dark Lord : Is christened the name "Leroy" by Hämsterviel, something almost everyone scoffs at.
• Theme Music Power-Up : Inverted; as a fail-safe added by Jumba, if Leroy hears "Aloha ʻOe", he will shut down. This also applies to his clones.
• The Unintelligible : He mainly speaks in Tantalog, the same gibberish-sounding language Stitch and the experiments speak.

Video Example(s):

Kixx (Experiment 601) is an experiment with four arms, making him four times as strong.

Example of: Multi-Armed and Dangerous

Stitch's Sudden...

Experiment 621'...

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Experiment 607: Launch

Stats/strengths  [ ].

"For the last time, I'm not Rufus!"

Pod Color:  Blue

Gender:  Male

Special Abilities:

Oh dear, this is  very  terrifying experiment, even for Jumba! Sole purpose is to be prototype doomsday device, one never fully realized until 611, but regrettably, we shall get to him soon enough.

607 uses his large front teeth to literally tear a hole in the fabric of space and time and if he was to create a large enough hole it would cause the universe to collapse in on itself. Experiment 607's small tears in the fabric of reality repair themselves after a few seconds.

Thanks to fellow fabric of reality benders like 606 , 607 was reprogrammed to tear holes in reality to get rid of nasty chemicals in power plants, not to destroy whole universe. Sometimes, Little Girl's resourcefulness surprises even Evil Genius self. As or the name, it is highly possible it was short for being launch-ed into oblivion by 607's powers, but this hasn't been fully proven (and for good reason).

True place: At first, I confused this experiment with a Mole Rat named Rufus. But then, when Little Girl and 626 freed the experiments that Gantu had caught, they discovered his capsule. Maybe this capsule that 627 stole from the boy was 607! Oh, if I had known that, I would not have made such a mistake in front of Kim Possible's friend! Well, what's done is done. Eventually, this experiment found his true place by safely disposing of nuclear waste.

• 1 Experiment 607: Launch
• 2 Experiment 628
• 3 Experiment 623: Trainer

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Article Contents

Introduction, materials and methods, acknowledgements, author contributions, conflicts of interest, data availability.

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Colonization compatibility with Bacillus altitudinis confers soybean seed rot resistance

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Ping-Hu Wu, Hao-Xun Chang, Colonization compatibility with Bacillus altitudinis confers soybean seed rot resistance, The ISME Journal , Volume 18, Issue 1, January 2024, wrae142, https://doi.org/10.1093/ismejo/wrae142

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The plant microbiome and plant-associated bacteria are known to support plant health, but there are limited studies on seed and seedling microbiome to reveal how seed-associated bacteria may confer disease resistance. In this study, the application of antibiotics on soybean seedlings indicated that seed-associated bacteria were involved in the seed rot resistance against a soil-borne pathogen Calonectria ilicicola , but this resistance cannot be carried to withstand root rot. Using PacBio 16S rRNA gene full-length sequencing and microbiome analyses, 14 amplicon sequence variants (ASVs) including 2 ASVs matching to Bacillus altitudinis were found to be more abundant in the four most resistant varieties versus the four most susceptible varieties. Culture-dependent isolation obtained two B. altitudinis isolates that both exhibit antagonistic capability against six fungal pathogens. Application of B. altitudinis on the most resistant and susceptible soybean varieties revealed different colonization compatibility, and the seed rot resistance was restored in the five varieties showing higher bacterial colonization. Moreover, quantitative PCR confirmed the persistence of B. altitudinis on apical shoots till 21 days post-inoculation (dpi), but 9 dpi on roots of the resistant variety TN5. As for the susceptible variety HC, the persistence of B. altitudinis was only detected before 6 dpi on both shoots and roots. The short-term colonization of B. altitudinis on roots may explain the absence of root rot resistance. Collectively, this study advances the insight of B. altitudinis conferring soybean seed rot resistance and highlights the importance of considering bacterial compatibility with plant varieties and colonization persistence on plant tissues.

The plant microbiome constitutes a vast and complex community of microbes, playing a crucial role in supporting plant health, productivity, and resilience [ 1 ]. These microbes engage in various beneficial interactions with their hosts, such as nutrient acquisition, growth promotion [ 2 , 3 ], and improving resistance to biotic and abiotic stresses [ 4–6 ]. As a result, the plant microbiome has received significant research attention due to its potential to sustain agriculture and the environment [ 7 ]. Although research over the past decade has predominantly concentrated on the phyllosphere and rhizosphere microbiomes, seed and seedling microbiome have been relatively overlooked despite its fundamental importance in plant growth and agricultural production.

The microbiome composition is shaped by factors such as plant species, genotypes, and environmental conditions. For example, seed endophytes are suggested to co-evolve with Zea species throughout domestication and geographical expansion [ 8 ]. In addition, domestication has been shown to decrease microbial diversity in wheat seeds, whereas domesticated rice exhibits a greater microbial diversity than their wild ancestors [ 9 , 10 ]. Moreover, investigations indicate that the seed endophytic microbiome of rapeseed, pumpkin, and tomato can be influenced by host cultivar/genotype and environmental factors [ 11–13 ], whereas the seed epiphytic microbiome is primarily affected by environmental factors such as location [ 14 ]. Methodological variations, including the surface disinfestation procedure, can impact the detected species richness in different studies [ 15–17 ]. Nevertheless, there is a consensus across studies that seeds harbor fewer microbial species compared with other plant tissues [ 17 ]. This reduction in diversity raises interests to study how plants select the seed-associated microbes and their roles on plant health.

One of the initial perspectives on the seed-associated microbes stemmed from the recognition of seed-borne pathogens, which can cause diseases and significantly impact seedling health [ 18 ]. This understanding led to the widespread adoption of physical and chemical seed treatments, such as hot water soaking and seed coating, to eliminate seed-associated microbes in conventional farming. However, recent research is reshaping this perspective, unveiling a diverse range of beneficial bacteria and fungi in seeds. These seed-associated microbes are now recognized for playing crucial roles in seed germination, seedling development, and seedling protection from pathogens [ 19 ]. For instance, studies have demonstrated that rice and millet seeds treated with antibiotics, resulting in the absence of bacteria, exhibited slower germination processes [ 20 , 21 ]. Similarly, maize and pearl millet seedlings treated with antibiotics showed reduced growth [ 22 , 23 ]. Furthermore, endophytic bacteria such as Bacillus , Pseudomonas , and Sphingomonas have been identified as contributors to protect young seedling through the production of antimicrobial substances [ 5 , 24 ] or the stimulation of plant defense responses [ 25 , 26 ]. Some of these microbes can be vertically transmitted from parents to offspring plants, ensuring the continuity of a beneficial holobiont across generations [ 11 , 17 , 27 , 28 ]. Accordingly, these findings suggest that seeds are the initial microbial reservoir, supporting the establishment of primary seedling holobiont. This concept not only reshapes the traditional approach of seed sterilization for disease management [ 29 ], but also underscores the potential of harnessing seed-associated microbes to develop sustainable disease management.

Soybean ( Glycine max ) holds significant agricultural importance worldwide. One of the primary challenges in soybean cultivation is the prevalence of seed-borne and soil-borne diseases, which can hinder seed germination through seed rot, root rot, and damping-off [ 30 ]. In the USA, these diseases together known as seedling diseases, accounting for 76% of soybean yield loss related to diseases from 2018 to 2020 [ 31 ]. Various fungal pathogens like Athelia rolfsii , Fusarium oxysporum , Macrophomina phaseolina , Rhizoctonia solani , and Sclerotinia sclerotiorum have become persistent endemic problems [ 32 ], and a recently emerging disease, red crown rot (RCR) caused by Calonectria ilicicola , has gained attention globally in recent years [ 33 , 34 ]. Currently, seed coating with fungicides is commonly suggested to manage both endemic and emerging diseases [ 32 , 35 ]. Alternatively, strategies such as plant resistance and beneficial microbes may offer more sustainable options for disease management. However, studies have pointed out that limited resistance in the global soybean germplasms to RCR, therefore, regional varieties should be evaluated to extend the search of resistant source [ 36 , 37 ]. Regarding seed-associated bacteria of soybean, ~30 bacterial genera such as Bacillus , Pantoea , and Sphingomonas were identified from soybean seeds [ 38 ], and some of these seed-associated bacteria exhibited in vitro antagonistic activity against various soybean pathogens [ 39 ].

In this study, we found seed rot resistance but not root rot resistance against C. ilicicola among 16 local soybean varieties in Taiwan. We discovered and hypothesized the seed rot resistance may be attributed to seed-associated bacteria, rather than plant innate immunity. To verify the hypothesis, we employed antibiotic treatment to confirm the contribution of seed-associated bacteria, and utilized PacBio 16S rRNA gene full-length sequencing, which is a better technology than the 16S rRNA gene V3-V4 short read sequencing to uncover species-level resolution [ 40 , 41 ] and explore the differential abundance of soybean seedling microbiome. We identified and characterized the colonization of Bacillus altitudinis on compatible soybean varieties is crucial for preventing seed rot, and the impersistent colonization of B. altitudinis on roots is the cause that the seed rot resistance cannot be carried to root rot resistance. Collectively, this study presents the instance of seed-associated B. altitudinis , exhibiting antagonistic capability depending on the bacterial density and persistence on soybean varieties and tissues.

Preparation of fungal and plant materials

Fungal materials including A. rolfsii isolate a31, C. ilicicola isolate F018, F. oxysporum isolate R1031, M. phaseolina isolate 1-4-03, R. solani AG-7 isolate WDG070, S. sclerotiorum isolate 1980 were routinely cultured on potato dextrose agar (PDA) plates (HIMEDIA, India), and maintained on filter papers at −20°C or preserved in 20% glycerol at −80°C for long-term storage.

Upon experimental setup, soybean seeds were triply washed with tap water and then immersed in 1% NaOCl for 10 min, followed by repeatedly rinsing with sterile distilled H 2 O for five times. The completeness of surface disinfestation was assessed by spreading 100 μL aliquot of the last rinse on nutrient agar (NA) plates (HIMEDIA), and then incubated at 28°C for 5 days. Seeds were considered surface-disinfested and would be used in subsequent experiments if no microbial colony was formed on the NA plates. There are 16 varieties of soybean seeds used in this study ( Table S1 ).

Phenotyping soybean disease resistance to C. ilicicola

The seed rot assay was conducted according to Broders et al. [ 42 ] with slight modifications. In brief, conidia were collected from a 10-day-old C. ilicicola colony on ½-strength PDA (½PDA) and diluted to 2 conidia per microliter. A total of 100 μL of the diluted conidia (200 conidia) were spread on 1.5% water agar (WA) plates and incubated at 25°C in the dark for 2 days. Subsequently, eight surface-disinfested soybean seeds were placed on each WA plate and cultured at 25°C in the dark for 5 days. Surface-disinfested soybean seeds of each variety on the WA plates without conidia were included as the controls. Soybean seeds were considered dead for situations including no germination, the radicle length was <1 cm, or the cotyledon and radicle were fully colonized by mycelia. The seed mortality rate was represented by the number of dead seeds divided by the eight seeds in each WA plate. Each plate was counted as one biological replicate, and each experiment was constituted of three biological replicates. The experiment was repeated three times independently.

For the cotyledon rot assay, the severity of cotyledon rot was scored on a six-grade scale based on the lesion size and severity on the cotyledon: 0 = no lesion; 1 = lesion <25%; 2 = lesion less ranging from 26 to 50%; 3 = lesion less ranging from 51 to 75%; 4 = lesion over 75%; 5 = seeds with no germination or all cotyledon and radicle were colonized by mycelia. The scores of eight seedlings were averaged to represent one biological replicate; in other words, each plate was considered as one biological replicate. Each experiment was constituted of three biological replicates. The experiment was repeated three times independently. Finally, the seed rot severity was calculated by standardizing the seed mortality and the cotyledon rot to range from 0 to 1 and averaging these two indices.

For the root rot assay, the fungal inoculum was freshly prepared according to the previous methods [ 35 ], and 15 mL of the fungal inoculum or control inoculum was mixed with commercial potting soils (T033, Garden Castle Ltd, Taiwan) in 500 mL pots. Each pot contained four soybean seeds, and these pots were placed in a greenhouse at 25°C in a 16 h–8 h light–dark cycle with daily irrigation. After 21 days, soybean seedlings were collected to separate roots from soils by gently rinsing with tap water. The disease severity was visually scored using a six-grade scale: 0 = no symptom; 1 = small brown necrotic lesions on the primary root; 2 = brown necrotic lesions extending over the primary root and some lateral roots; 3 = over half root lost and rotted with brown necrosis on the subterranean stem; 4 = almost all roots lost and rotted; and 5 = dead seedlings [ 36 ]. The scores of four seedlings were averaged to represent one biological replicate; in other words, each pot was considered as one biological replicate. The experiment included 8 varieties × 2 treatments (inoculation or not) in a complete randomized design, and each factorial combination included 3 biological replicates (pots). The experiment was repeated three times independently.

Elimination of seed-associated bacteria by antibiotics

To assess the role of seed-associated bacteria in the seed rot resistance, soybean seeds were firstly surface-disinfested as abovementioned method, and then soaked in a solution containing ampicillin (100 μg/mL), rifampicin (50 μg/mL), and streptomycin (100 μg/mL) for 16 h ( Fig. S1 ). The control group was soaked in sterile distilled H 2 O or DMSO solution for the same period. Following treatment, the seeds were rinsed seven times with sterile distilled H 2 O to remove antibiotics residue. To ensure the completeness of eliminating seed-associated bacteria, the seeds were ground using a mortar and pestle in 2-fold volume (v/w) of phosphate-buffered saline (PBS) buffer, and 100 μL of the grinding aliquot was spread onto tryptic soy agar (TSA) plates (STBIO MEDIA). After incubating the TSA plates at 28°C for 5 days, the elimination of seed-associated bacteria was considered successful if no colony was observed.

PacBio 16S rRNA gene full-length sequencing and microbiome analyses

DNA was extracted from 5 day-post-germination soybean seeds using the CTAB method. Each biological replicate was a pool of eight seeds within a Petri plate, and five biological replicates were included from each variety. Based on the seed rot resistance, the 4 most resistant and the 4 most susceptible soybean varieties were included, therefore, a total of 40 DNA samples were subjected to PacBio 16S rRNA gene full-length sequencing. The bacterial 16S rRNA gene was amplified by the universal primers 27f and 1492r ( Table S2 ), sourced from the PacBio library preparation kit. To minimize the amplification of host DNA, the PNA blocker targeting soybean chloroplast DNA [ 43 ] was incorporated at a final concentration of 2.5 pmole, and the LNA blockers were added to target soybean mitochondria DNA [ 44 ] at a final concentration of 5 pmole (2.5 pmole for each direction). The sequencing libraries were prepared according to the workflow of the PacBio SMRTbell kit, and sequencing was performed on the PacBio Sequel IIe platform with 10 h movie collection time.

The raw sequencing files were filtered, trimmed, and de-replicated using the PacBio single-molecule real-time link software to generate circular consensus sequencing reads. The R package “DADA2” v1.26 was utilized to denoise and construct amplicon sequence variants (ASVs) following the adjusted parameters [ 45 ]. The chimeras were removed before classifying the ASVs using the Bayesian classifier in DADA2 and the SILVA v138.1 database at 99% similarity [ 46 ]. The non-prokaryotic and unclassified ASVs were removed before finalizing the ASV table. To assign the taxonomy to each ASV, the sequences were aligned using BLAST+ to NCBI 16S rRNA gene database with an E-value at 10 −5 . The classification of each ASV was determined by the lowest E-value, followed by the highest Bit score, and then the highest identity. The BLAST results were processed by the R package “taxize” v0.9.1, resulting in a final taxonomy table. The ASV sequences were aligned using MAFFT [ 47 ], and a maximum likelihood phylogenetic tree was constructed with IQ-TREE2 with 1000 bootstrap replicates [ 48 ].

The ASV table, taxonomy table, and phylogenetic results were imported into the R package “phyloseq” v1.42, and four samples with fewer than 2000 reads were filtered [ 49 ]. ASVs with a mean of relative abundance below 0.01% across 36 samples or with occupancy below 5% (lower than 2 samples) were excluded from the subsequent analyses. To assess α-diversity indices, all samples were rarefied to the sample with the lowest sequencing depth using the R package “vegan” v2.64-4 and “picante” v1.8.2. To assess the β-diversity, ASVs were normalized by median sequencing depth before subjected to non-metric multidimensional scaling (NMDS) based on the Bray–Curtis distance. PERMANOVA was conducted using the “adonis2” function in “vegan” package with 999 permutations. The differential abundance of ASVs between the resistant and susceptible varieties was analyzed using the R package “DESeq2” v1.38.3 [ 50 ], and the significance was determined at the BH-adjusted P values at 0.05.

Isolation of seed-associated bacteria and in vitro antagonistic assay against fungal pathogens

Surface-disinfested soybean seeds were germinated on 1.5% WA plates for 5 days before grinding using a mortar and a pestle. The ground aliquots were serial diluted till 10 −5 folds using PBS buffer, and the 100 μL diluted aliquot was spread on soymilk agar (SA) plates, NA plates, Luria-Bertani agar (LA) plates, and 0.1% TSA plates. These plates were incubated at 28°C for 7 days, with three plates for each dilution fold. After incubation, colonies were differentiated and selected based on their morphological features. The selected colonies were subsequently single colony purified using the streak plate technique. Finally, the purified colonies were preserved in 25% glycerol at −80°C for a long-term storage.

The seed-associated bacteria were tested for their antifungal activity against fungal pathogens in the dual culture assay on TSA plates. A mycelial plug (5 mm in diameter) from the actively growing edge of each fungal species was placed on the center of a medium plate. The bacteria were cultured in tryptic soy broth (TSB) for 16 h and adjusted to OD 600 value of 1. Subsequently, 2 μL of the bacterial aliquot was placed 3 cm from the mycelial plug on both sides of a TSA plate. This method tested fungal pathogens under varying conditions of temperature and time. Specifically, A. rolfsii and C. ilicicola were measured at 7 days post-inoculation (dpi), M. phaseolina and S. sclerotiorum at 5 dpi, and R. solani at 2 dpi. All fungi were cultured at 28°C in the dark, except for S. sclerotiorum that was cultured at 25°C. The inhibition rate was calculated by:

For identifying the species of bacterial isolate TN5S8 and TN3S3, the genomic DNA were extracted using the Presto gDNA Bacteria Advanced Kit (Geneaid Biotech Ltd, Taiwan), and subjected to PCR using the 27F/1492R for 16S rRNA gene and UP1/2r for gyrB gene [ 51 ] ( Table S2 ). The amplicons were submitted for Sanger sequencing (Genomics, Taiwan), and sequencing results were subjected to BLAST search in the NCBI database the alignment with ASVs, and phylogenetic analysis.

Colonization of B. altitudinis TN5S8 on different soybean varieties

Antibiotics-treated soybean seeds were soaked for 16 h in the freshly prepared aliquot of B. altitudinis TN5S8 (hereafter abbreviated as TN5S8). Meanwhile, the control group was soaked in PBS buffer. The inoculant was prepared from a 24-hour-old bacterial culture in TSB, pelletized by centrifugation at 14 000 g for 3 min, before being resuspended and adjusted to a concentration of 10 7  CFU/mL using PBS buffer. The treated seeds were air-dried in a laminar flow hood. Seed rot resistance was evaluated using the plate assay method abovementioned. The colonization efficiency of TN5S8 on each soybean variety was assessed by re-isolating TN5S8 from seeds using the method aforementioned. The colony numbers per gram of seeds were assessed by:

To study the impact of TN5S8 on seed germination, two different treatments were tested, including the surface-disinfested seeds + TN5S8, and the surface-disinfested seeds + cell-free culture filtrate of TN5S8. The cell-free culture filtrate was prepared from a 3-day-old bacterial culture in TSB, centrifuging at 14 000 g for 3 min and filtering the supernatant through a 0.2 μm Millex filter (Merck KGaA, Germany). The impact of TN5S8 on seed germination was assessed by germination rate at 5 dpi.

Quantitative PCR detection of B. altitudinis TN5S8

Soybean seeds of the resistant variety TN5 and the susceptible variety HC were surface-disinfested and inoculated with TN5S8, and then planted in pots containing a mixture of peat and perlite at a ratio of 4:1 in the greenhouse using the methods mentioned above. Plant tissue samples were collected at 5 timepoints. The cotyledons and epicotyls were collected for samples at 3 dpi, and the apical shoot and the first node were sampled for samples from 6 dpi onwards. The root samples were washed with sterile water to remove soil, and the taproots were collected for DNA extraction. Each biological replicate consisted of tissues from four plants in a pot; in other words, each pot was considered as the biological replicate, and there were five biological replicates obtained for each time point. The experiment was repeated twice independently.

Approximately 350 mg of plant tissues were homogenized in liquid nitrogen with a mortar and postal, and DNA was extracted using the CTAB method. Specific primers were designed to amplify a 106-bp fragment of the gyrB gene of B. altitudinis ( Table S2 ). The quantitative PCR (qPCR) was performed on the CFX Connect Real-Time System (Bio-Rad Hercules, CA, USA) using genomic DNA, iQ SYBR green supermix kit (Bio-Rad), and 0.4 μM of each primer under the following thermocycling conditions: 95°C for 3 min; 40 cycles of 95°C for 10 s and 57°C for 30 s, with a melting curve processing from 60°C to 95°C for quality control. Genomic DNA of TN5S8 and soybean were serially diluted 10-fold to build standard curves for the mean Ct values against the DNA concentrations. Soybean actin gene Glyma.15G050200 was used as an internal control. Each biological replicate was technically repeated twice.

Sequencing, assembly, and analyses of B. altitudinis TN5S8 genome

The genomic DNA extracted by Presto gDNA Bacteria Advanced Kit (Geneaid) was sent for the Oxford Nanopore Technologies (ONT) whole genome sequencing (BIOTOOLS Co, Ltd, Taiwan). DNA concentration, purity, and integrity were checked by the Qubit 4.0 fluorometer (Thermo Scientific) and the Qsep 100 system (Bioptic Inc, Taiwan). To construct ONT sequencing library, DNA fragments smaller than 10 kb were removed by Short Read Eliminator XS (PacBio). Subsequently, 1 µg of high molecular weight genomic DNA underwent end-repairing and dA-tailing using the KAPA End Repair and A-Tailing reagent (Roche), followed by the barcode and adapter ligation using the ONT Native Barcoding Kit 24 V14. The resulting DNA libraries were cleaned up to enrich fragments larger than 1 kb before being sequenced on the PromethION 24 device using the FLO-PRO114M flow cell (R10.4.1). In addition, the NEBNext DNA Library Prep Kit (New England Biolabs) was used to construct the sequencing library for the NovaSeq 6000 (Illumina) paired end 150-bp platform. Long-read sequences generated by PromethION were processed using Guppy’s Super-accurate basecalling 400 bps model. Reads with an average quality score above Q10 were assembled using Flye [ 52 ]. The Flye contigs were further polished with Medaka ( https://github.com/nanoporetech/medaka ), and a final sequence polishing was conducted using Homopolish [ 53 ]. Additionally, short-read sequences from the NovaSeq 6000 system were quality controlled using FastQC and Cutadapt. Filtered reads were then mapped to the contigs using BWA [ 54 ], and corrections were made with Pilon [ 55 ]. The corrected contigs were evaluated using QUAST [ 56 ] and BUSCO [ 57 ] to assess the genome quality. Gene locations were predicted using Prokka [ 58 ]. Annotation of the protein-coding sequence was conducted using the BLAST against the clusters of orthologous groups (COG) database. The final annotated chromosome was plotted using CIRCOS to show the gene locations, GC content, and COG annotation. Secondary metabolite biosynthesis-related gene clusters (BGCs) were predicted by the antiSMASH 7.0 [ 59 ], and 47 complete B. altitudinis genomes were obtained from NCBI database for a comparative analysis on the BGCs.

Statistical analysis

All statistical analyses were conducted using the R environment 4.2.3. For the data analyses using the t -test, ANOVA, and Tukey’s HSD test, the normality was checked by the Shapiro test and Q-Q plot, and homoscedasticity was checked by the Levene’s test. For the data not fitting parametric assumptions, the Kruskal–Wallis test and Dunn’s test was applied. The P values of the Tukey’s HSD test and Dunn’s test were adjusted by the BH method for multiple comparisons. The significance of the statistical analysis was determined by α at 0.05.

In assessing soybean resistance to C. ilicicol a, significant differences were observed for both seed mortality ( P  < 0.001) and cotyledon rot ( P  < 0.001) across 16 local varieties of Taiwan. Soybean variety SS exhibited the highest levels of seed mortality rate and cotyledon rot score, whereas TN5 displayed the lowest seed mortality rate and TN11 displayed the lowest cotyledon rot score ( Table S3 ). By averaging the seed mortality rate and the cotyledon rot score to obtain the seed rot severity, the results showed that TN11, HBS, TN3, and TN5 were the four most resistant varieties, whereas SS, KS9, KS7, and HC were identified as the four most susceptible varieties ( Fig. 1 ).

Seed rot resistance and root rot resistance of 16 soybean varieties to C. ilicicola . Seed rot severity was calculated by averaging the seed mortality rate and the cotyledon rot indices. Root rot severity was determined by the pot assay. The colors indicate the color of soybean seed coat. The Kruskal–Wallis and the Dunn’s test were used to determine significant difference at α = 0.05. There were three biological replicates (Petri plates or pots) for each variety and the experiment was repeated three times ( n  = 9).

In contrast, no significant difference ( P  = 0.293) was observed for root rot resistance across the same 16 varieties ( Fig. 1 ). These findings suggested that the soybean resistance to only seed rot, but not root rot, may not be solely determined by soybean innate immunity. It is possible that seed-associated bacteria play a role in seed rot resistance to C. ilicicola . Using antibiotics-treated seeds of the four most resistant and the four most susceptible soybean varieties, the susceptibility of the four susceptible varieties remained unchanged ( Fig. 2A ), but the four resistant varieties became susceptible ( Fig. 2B, C ). As the antibiotic treatment did not affect seed germination in the control groups and did not impact the growth of C. ilicicola , the increased seed rot severity in these four resistant varieties (TN11, HBS, TN3, and TN5) may be attributed to the elimination of seed-associated bacteria.

Seed rot assay using the antibiotics-treated seeds reveals that seed-associated bacteria confer the seed rot resistance . The antibiotics included ampicillin, rifampicin, and streptomycin. The control was treated with ddH 2 O. (A) The four most susceptible soybean varieties showed no difference between the control and antibiotic treatment. (B) The four most resistant soybean varieties showed significant difference in seed rot between the control and antibiotic treatment. Both seed mortality rate and cotyledon rot score were increased for the antibiotics-treated seeds, and the four initially resistant varieties became susceptible. (C) Seed rot severity. There were three biological replicates (Petri plates) for each factorial combination of variety and treatment, and the experiment was repeated three times ( n  = 9). The asterisks indicate significance based on the Tukey’s HSD test ( * : P  < 0.01, * * : P  < 0.001).

A total of 803 220 PacBio 16S rRNA gene full-length raw reads were acquired. Following quality controls and the exclusion of chloroplast, mitochondrial, and non-characterized sequences, 588 419 reads were retained ( Table S4 ). Despite the inclusion of PCR blockers, two varieties (KS7 and KS9) still exhibited interference from Plant DNA ( Fig. S2A ). Consequently, two samples with fewer than 2000 reads from each of the KS7 and KS9 varieties were excluded from subsequent analyses. For the remaining 36 samples, rarefaction curves indicated satisfactory sampling depth, as all curves reached saturation status ( Fig. S2B ). After quality control, 145 ASVs were identified in the 36 samples, and the BLAST results showed an identity range of 85.33 to 100% for these ASVs according to the NCBI reference taxa ( Table S5 ). Among them, 114 ASVs displayed identities greater than 99% to the reference taxa ( Fig. S2C ). Therefore, the microbiota obtained from PacBio 16S rRNA gene full-length sequences yielded a high-quality taxonomic profile at the species level for downstream analyses.

The taxonomic profile unveiled four bacterial phyla associated with soybean, with the predominant taxa being Bacillota (56.4%), Pseudomonadota (41.0%), Bacteroidota (1.9%), and Actinomycetota (0.7%). At the family level, Bacillaceae emerged as the most abundant family (55.7%), followed by Moraxellaceae (15.0%) and Rhizobiaceae (11.7%) ( Fig. 3A , Table S5 ). At the species level, the 145 ASVs were attributed to 44 bacterial species. Two bacterial species, Priestia aryabhattai and Priestia megaterium , were identified in seven varieties except for KS7. Seven bacterial species were present in six varieties ( Fig. 3B , Fig. S3A ). Additionally, 29 bacterial species were found in fewer than four varieties. The results indicated a considerable variability of the bacterial composition across the eight soybean varieties.

PacBio 16S rRNA gene full-length analyses to identify the seed-associated bacteria of the eight soybean varieties. (A) Maximum likelihood phylogenetic tree of the 145 ASVs the presence in each soybean variety. (B) Heatmap of log 10 (relative abundance) for the bacterial species in each soybean variety. There were eight seeds per plate and five Petri plates for each variety ( n  = 5).

α-Diversity, β-diversity, and differential abundance analyses

α-Diversity indices such as richness, Shannon diversity, and Pielou’s evenness, as well as β-diversity analysis using the NMDS based on the Bray–Curtis distance did not identify a clear separation between the four most resistant and the four most susceptible varieties ( Fig. S3B-D ). Indeed, PERMANOVA disclosed that the seed source contributed to 39.7% ( P  = 0.001) and soybean variety contributed to 6.8% ( P  = 0.003) of the total variance in microbial composition ( Table S6 ). However, the seed rot resistance still accounted for 7.1% of the total variance ( P  = 0.002), suggesting microbial differences between the resistant and susceptible varieties.

Differential abundance analysis identified 14 ASVs being more abundant in the four most resistant varieties versus the four most susceptible varieties ( Fig. 4 ). These 14 ASVs were assigned to seven bacterial species, including Acinetobacter johnsonii ASV4, ASV11 and ASV12, Acinetobacter oryzae ASV13 and ASV14, Agrobacterium cavarae ASV31 and ASV34, Agrobacterium larrymoorei ASV2 and ASV5, B. altitudinis ASV20 and ASV48, P. aryabhattai ASV29 and ASV49, Pseudomonas oryzihabitans ASV38. ( Fig. 4 ).

Differential abundance analysis of the seed-associated bacteria between the resistant and susceptible varieties by DEseq2. The Manhattan plots showing the ASVs, which are represented by circles or triangles. Whereas the circles are non-significant ASVs in the differential abundance analysis, the triangles are ASVs significantly enriched (filled) or depleted (empty) in the resistant varieties. The triangle size indicates the log 2 fold change of the ASV. The y-axis indicates -log 10 (adjusted P value) and the x-axis represent the categorization of bacteria genus.

In pairwise comparison of these resistant and susceptible soybean varieties, A. cavarae , A. larrymoorei , P. aryabhattai were found as the significant species in the resistant variety HBS versus the other four susceptible varieties. Priestia aryabhattai was found in the resistant variety TN3 versus the other four susceptible varieties. As for the resistant variety TN5, B. altitudinis and P. aryabhattai were found as the significant species. Lastly, A. johnsonii , A. larrymoorei , and P. oryzihabitans were found in the resistant variety TN11 ( Fig. S4 ). Collectively, the pairwise differential abundance analyses suggested the likelihood that different seed-associated bacteria may be involved to confer the seed rot resistance to C. ilicicola .

Identification of B. altitudinis to inhibit fungal pathogens

A total of 300 bacterial isolates were obtained from the seedlings of eight soybean varieties, and 93 isolates with distinct colony morphology were selected for the in vitro antagonistic assay against C. ilicicola . The results identified 29 bacterial isolates that could inhibit at least 20% of the mycelial growth of C. ilicicola ( Table S7 ). Among these isolates, one from TN3 and one from TN5 displayed a clear inhibition zone. Molecular identification and phylogenetic analysis of 16S rRNA and gyrB gene sequences revealed that both isolates belonged to B. altitudinis ( Fig. S5A-B ). The 16S rRNA gene sequences of these two bacterial isolates (TN3S3 and TN5S8) exactly matched B. altitudinis ASV20 and exhibited a single nucleotide difference with ASV48 ( Fig. S5C ).

These two bacterial isolates (TN3S3 and TN5S8) exhibited antagonistic activity against other soil-borne pathogens, inhibiting the mycelial growth over 30% for A. rolfsii , M. phaseolina , R. solani , and S. sclerotiorum , and over 20% for F. oxysporum ( Fig. 5 ). Using TN5S8 in the subsequent experiments, the results demonstrated that re-inoculating TN5S8 to the antibiotics-treated soybean seeds significantly mitigated seed rot caused by C. ilicicola across the four most resistant varieties and the susceptible variety KS9 ( Fig. 6 ). These findings strongly suggested that B. altitudinis TN5S8 played a pivotal role in conferring the seed rot resistance against C. ilicicola .

In vitro assay of B. altitudinis against six soil-borne fungal pathogens. (A) Representative image of the dual culture assay using TN5S8 to antagonize six fungal pathogens. (B) The inhibition rate, which contains three biological replicates (Petri plates) and experiment was repeated three times ( n  = 9). Ar: Athelia rolfsii , Ci: Calonectria ilicicola , Fo: Fusarium oxysporum , Mp: Macrophomina phaseolina , Rs: Rhizoctonia solani , Ss: Sclerotinia sclerotiorum.

Seed rot assay using the antibiotics-treated seeds inoculated with or without B. altitudinis TN5S8. (A) The four most susceptible soybean varieties with three showed no difference with or without the inoculation of TN5S8. KS9 is the only variety being rescued by the inoculation of TN5S8. (B) The four most resistant soybean varieties showed significant reduction in the seed rot severity after the inoculation of TN5S8. (C) Seed rot severity. There were three biological replicates (Petri plates) for each factorial combination of variety and treatment, and the experiment was repeated three times ( n  = 9). The asterisks indicate significance based on the Tukey’s HSD test ( * : P  < 0.01, * * : P  < 0.001).

The whole genome of B. altitudinis TN5S8 was sequenced to uncover the potential antifungal mechanisms. The genome of B. altitudinis TN5S8 comprises a 3747 068 bp circular chromosome with a GC content of 41.4% and 3771 coding sequences ( Table S8 , Fig. S6A ). There were 10 secondary metabolite biosynthetic gene clusters identified in the TN5S8 genome ( Fig. S6B ), including two non-ribosomal peptide synthetase (NRPS) clusters with 85% similarity to the lichenysin gene cluster and 53% similarity to the fengycin gene cluster. Both lichenysin and fengycin were cyclic lipopeptides with antifungal properties [ 60 , 61 ]. Additionally, a gene cluster encoding a siderophore was 60% similar to the schizokinen gene cluster, which may also contribute to the antifungal activity through nutrient competition [ 62 ]. Comparative analysis of secondary metabolite biosynthetic gene clusters across 48 B. altitudinis strains revealed that these three gene clusters were highly conserved within the B. altitudinis species ( Fig. S7 ), suggesting the possible mechanism of antibiosis and nutrient competition for B. altitudinis to antagonize C. ilicicola .

Colonization compatibility and persistence of B. altitudinis TN5S8 is a prerequisite to gain the seed rot resistance

The introduction of TN5S8 did not provide seed rot resistance for three susceptible varieties—HC, KS7, and SS. We postulated that the colonization compatibility between TN5S8 and soybean varieties might be a pivotal factor in gaining the seed rot resistance. Therefore, we re-isolated and quantified the TN5S8 population on the eight soybean varieties inoculated with TN5S8, and we observed a significantly higher density of TN5S8 in the four resistant varieties and the susceptible variety KS9, and a lower density for HC, KS7, and SS ( Fig. 7A ). The highest recovery of TN5S8 was observed from its original variety TN5. In addition, TN5S8 significantly impeded the seed germination of soybean varieties HC, KS7, and SS ( Fig. 7B ), and the non-germinated seeds of HC, KS7, and SS exhibited dark hue and soft rot. Furthermore, the germination reduction and diseased symptoms were not induced by the cell-free culture filtrate of TN5S8. These findings underscored the importance of colonization compatibility between TN5S8 and soybean varieties to confer the seed rot resistance.

Colonization compatibility and persistence of B. altitudinis TN5S8 on soybean varieties. (A) The recovery of TN5S8 population on the antibiotic-treated seeds at 5 dpi. There were three biological replicates (Petri plates) for each variety, and the experiment was repeated three times ( n  = 9). ANOVA and the Tukey’s HSD test were used to determine the significance at α = 0.05. (B) The germination rates of the eight soybean varieties. The white bars indicate the inoculation of TN5S8 on the surfaced-disinfested seeds. The yellow bars indicate the application of TN5S8 culture filtrate on the surfaced-disinfested seeds. There were three biological replicates (Petri plates) for each variety, and the experiment was repeated three times ( n  = 9). The Kruskal–Wallis and the Dunn’s test were used to determine significant difference at α = 0.05. (C) qPCR to quantify TN5S8 on the apical shoots and roots of the resistant soybean variety TN5 at different timepoints. (D) qPCR to quantify TN5S8 on the apical shoots and roots of the susceptible soybean variety HC at different timepoints. The y axis indicates the transformed values of the absolute gyrB gene amount of B. altitudinis in soybean tissues represented by the absolute soybean actin gene amount. There were five biological replicates for each timepoint in each experiment. The experiment was repeat twice ( n  = 10).

Additionally, using qPCR to detect the presence of TN5S8 on the resistant variety TN5 and the susceptible variety HC, TN5S8 was found to persist on the apical shoot of soybean seedlings until 21 dpi, but it was not detected on the roots after 9 dpi on the resistant variety TN5 ( Fig. 7C ). In contrast, TN5S8 was rarely found on the apical shoots or roots of the susceptible variety HC after 6 dpi ( Fig. 7D ). Accordingly, the colonization compatibility of TN5S8 on seeds and the impersistent colonization on roots of the resistant TN5 provide an explanation for the seed rot resistance which cannot be carried to root rot.

Plant innate immunity has been recognized as the major underlying source of plant disease resistance, not only for soybean [ 63 ] but also for most important crops [ 64 ]. However, some studies have observed that fungal infection on different tissues such as seed, root, node, or leaf of the same plant genotype could result in different levels of resistance [ 65 , 66 ]. For example, a study of soybean resistance to Pythium revealed the phenotypic correlation between seed rot and root rot was ranged from 0.1 to 0.17 [ 65 ]. Another study on the pea resistance to S. sclerotiorum reported the phenotypic correlation of nodal resistance and leaf resistance was only 0.19. Recently, it has been known that disease resistance can also be provided by the plant-associated microbes [ 67–69 ], therefore, the importance of considering the plant holobiont (including the plant host and the plant-associated microbes) as an entity has been increasingly recognized to uncover the mechanism of plant health [ 70 ].

Among the plant-associated microbes inhabiting on different tissues such as fruit, leaf, or roots, the seed-associated bacteria are the front line group in fighting against soil-borne diseases that mostly damage plants at the seedling stage [ 71 ]. For example, distinct bacterial compositions in the seeds of different oilseed rape cultivars were correlated with varying resistance levels to Verticillium wilt and Plasmodiophora brassicae [ 13 , 72 ]. In another study, a seed endophytic bacterium Sphingomonas melonis , which can be vertically transmitted to the next generation of seeds, confers rice seedlings resistance against Burkholderia plantarii [ 5 ]. Similarly, Bacillus velezensis isolated from maize seeds and Bacillus subtilis found in millet seeds were shown to protect seedlings from Fusarium infection [ 22 , 23 ]. In assessing 16 local soybean varieties, this study identified a discrete disease resistance, which is present only for seed rot, but not for root rot. The source of this discrete resistance may be something other than plant innate immunity, leading us to the hypothesis that seed-associated bacteria confer the seed rot resistance, which cannot be carried to the roots.

Based on the experiments using the antibiotics-treated seeds, the results confirmed that seed-associated bacteria were involved in the seed rot resistance of soybean. As previously reported that the α-diversity or co-occurrence network properties between the resistant and susceptible plants were different [ 72 , 73 ] and may protect the resistant plants from pathogen [ 74 ], this study applied 16S rRNA gene full-length sequencing and microbiome analyses to compare the seedling microbiome between the resistant and susceptible soybean varieties. Nevertheless, there was no significant differences in the α-diversity or β-diversity. Instead, the differential abundance analysis discovered 14 ASVs that were significantly enriched in the four most resistant varieties, suggesting that a certain group of seed-associated bacteria may contribute to the seed rot resistance.

These 14 ASVs belong to the bacteria species such as A. johnsonii , A. oryzae , A. larrymoorei , A. cavarae , B. altitudinis, and P. oryzihabitans . Acinetobacter johnsonii has been previously isolated from soybeans [ 75 ], exhibiting antagonistic capabilities against soil-borne pathogens. In addition, A. larrymoorei and P. oryzihabitans have been reported as soybean endophytic bacteria, showing potential in the nitrogen fixation and phosphate solubilization [ 76 , 77 ]. Moreover, P. oryzihabitans strains have antagonistic capability against pathogens such as Acidovorax citrulli in cucurbits and Pythium in cotton [ 78 , 79 ]. Although literature suggested that these bacteria may play roles in the seed rot resistance, our culture-dependent isolation obtained two isolates, TN5S8 and TN3S3, which matched to another enriched ASVs identified as B. altitudinis . In our experiments, loss-of-function evidence through the antibiotic treatment and the gain-of-function evidence through the re-inoculation of B. altitudinis to soybean seeds confirmed the contribution of B. altitudinis in the seed rot resistance.

Bacillus altitudinis was first isolated from extreme UV-stressed air samples collected in the stratosphere [ 80 ]. It has been identified as an endophyte in various plants, including soybean [ 81 ] and others [ 82–89 ]. Several strains of B. altitudinis have shown biocontrol capabilities, such as cotton Verticillium wilt [ 82 ], grape downy mildew [ 85 ], kiwi fruit root-knot nematodes [ 89 ], soybean Phytophthora damping-off [ 81 ], and sweet potato black rot [ 87 ]. It has been shown that B. altitudinis can inhibit plant pathogens by producing antimicrobial lipopeptides lichenysin [ 60 , 85 ] and inducing plant defense responses [ 81 , 89 ]. Moreover, genome analysis also identified gene clusters similar to the fengycin and schizokinen gene clusters, and these compounds may be associated with the antagonistic ability [ 61 , 62 ]. Our comparative genomics analyses have found that these secondary metabolite biosynthesis gene clusters are highly conserved in different strains of B. altitudinis , and more recently, B. altitudinis has been suggested to have an open pangenome with 42.7% genes characterized as accessory genes. These results indicated that B. altitudinis may tend to acquire new genes to enhance its antagonistic capability and ecological competitiveness [ 85 ].

We further observed this seed rot resistance depends on the colonization compatibility of TN5S8 on soybean varieties. The relationship between bacterial population and disease suppression echoes previous findings on the biocontrol efficacy of Pseudomonas fluorescens was proportional to their density [ 90 ], and the effective threshold ranges from 10 5 to 10 6 bacteria per gram of root against wheat take-all decline disease [ 90 ]. Similarly, the suppression of other Pythium root diseases in sugar beets also depended on the population density of the Pseudomonas [ 91 , 92 ]. In rice, the abundance of Sphingomonas was observed to be lower in plants susceptible to seedling blight disease [ 5 ]. Specifically for the cases within the Bacillus genus, colonization and formation of biofilm on the phyllosphere or root surface is critical for the success of biocontrol [ 93 ]. On tomato, Bacillus strains with less colonization ability on the phyllosphere showed a reduced biocontrol ability against Botrytis cinerea [ 94 ]. Application of plant extracts such as pectin can enhance the Bacillus amyloliquefaciens population on tobacco roots and increase the biocontrol efficacy to tobacco bacterial wilt [ 95 ]. Mutation of B. amyloliquefaciens abrB gene, which is a negative transcription regulator of chemotaxis and biofilm formation, increased colonization and biocontrol capability against the cucumber Fusarium wilt [ 96 ]. In addition, the colonization of B. subtilis surfactin deletion mutant reduced 4 to 10-fold on the melon roots and leaves, which ended up losing the biocontrol efficacy [ 97 ]. However, it has not be reported whether the colonization of Bacillus can affect seed resistance, and our finding provided the evidence that the colonization compatibility of B. altitudinis on soybean seeds is an important factor to confer the seed rot resistance.

The prevalence of B. altitudinis was not uniform in all PacBio sequencing samples, meaning the ASVs assigned to B. altitudinis could be detected in some but not all samples of the four most resistant varieties ( Fig. S3A ). One possibility is that other seed-associated bacteria provide the seed rot resistance in samples where B. altitudinis was absent. Indeed, the differential abundance pointed out additional bacteria that may confer the seed rot resistance, with B. altitudinis was one of these bacteria. In other words, the seed rot resistance observed in other varieties where B. altitudinis was absent may be provided by other seed-associated bacteria that were not recovered from our culture-dependent isolation. Another possible cause may have been the nature of the seed bacterial community which was highly variable and stochastic according to the seed source and planting location ( Fig. S8 ). The PERMANOVA results suggested a great proportion of microbiome variance was explained by the seed source. Moreover, because the seedling microbiome is assembled from the seed bacterial community, the process becomes a selection bottleneck to increase the variability of seedling microbiome.

Only a small fraction of seed taxa is transmitted to the seedlings [ 98 , 99 ]. A recent study on oak showed that 63% of fungal taxa and 45% of bacterial taxa on the seeds can be transmitted to the seedlings [ 15 ]. Another study on tomato demonstrated that some seed-associated microbes such as P. aryabhattai , Bacillus nakamurai , Ralstonia pickettii , and Stenotrophomonas maltophilia could persist from seeds to seedlings for at least two generations [ 11 ]. However, even though the seed-associated bacteria can be transmitted to seedlings, their colonization on the shoots or roots may be different. A study on soybeans in an axenic environment demonstrated that the seed-transmitted bacterial ASVs dominant in the shoots can be rare or absent in the roots [ 100 ]. This report aligns with our observation on TN5S8, which was detected on the apical shoots for at least 21 dpi, but it could not be detected after 9 dpi on the root of the compatible and resistant variety TN5. As for the incompatible and susceptible variety HC, TN5S8 was rarely detected after 6 dpi, and the absence of TN5S8 on roots may result in no protection in the root rot assays.

In summary, this study identified that the seed-associated bacterium B. altitudinis could provide antagonistic capability to fungal pathogens, and B. altitudinis confers only the seed rot resistance in certain soybean varieties based on its colonization compatibility and persistence. The results highlight the future application of seed-associated bacteria in disease management to consider not only the antagonistic capability, but also the colonization compatibility and persistence on the plant varieties and tissues.

We thank Dr Kuo-Lung Chou and Dr Min-Nan Tseng at the Kaohsiung District Agricultural Research and Extension Station, Ministry of Agriculture, Taiwan, for providing soybean seeds.

Ping-Hu Wu and Hao-Xun Chang conceptualized the project. Ping-Hu Wu completed the assays for disease resistance, microbiome analyses, bacterial isolation and re-inoculation assays, antagonistic experiments, quantification of bacterial colonization as well as the qPCR for bacterial persistence on plant tissues. Ping-Hu Wu and Hao-Xun Chang prepared the figures, tables, and wrote the manuscript. All authors proofread and agreed on the manuscript and results. Hao-Xun Chang supervised the project.

The authors declare no conflict of interest.

This project was mainly supported by the Ministry of Education, Taiwan (The Yushan Young Scholar Program: MOE-113-YSFAG-0003-003-P2) to Dr H.X.C., and partially supported by the Ministry of Agriculture (111AS-1.3.2-ST-aQ) to Dr H.X.C.

The raw microbiome sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject IDs PRJNA1099878. The whole genome sequence of B. altitudinis TN5S8 is available in GenBank with the accession number CP155530.1 . Code to analyze microbiome data and generate figures and tables are located on GitHub at: https://github.com/wuphw/Soybean_seed_microbiome_resistance .

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• Talking experiments
• Video game characters
• Ultimate Monster Experiment
• Recurring Experiments
• Transmutated Experiments
• Titular protagonists

Stitch (626)

Stitch , also known as Experiment 626 , is an illegal genetic experiment created by Jumba Jookiba , one of the titular protagonists of the Lilo & Stitch franchise alongside Lilo , and the most prominent lead character of the franchise overall, appearing in all major media. His original primary function is to cause chaos across the galaxy by destroying everything he touches. He is designed to be abnormally strong, virtually indestructible, super-intelligent, and very mischievous. His one true place is with Lilo and her ʻohana .

• 1.1 Lilo & Stitch
• 1.2 Lilo & Stitch 2: Stitch Has a Glitch
• 1.3 Stitch! The Movie
• 1.4 Lilo & Stitch: The Series
• 1.5 Leroy & Stitch
• 1.6 Stitch! anime
• 1.7 Stitch & Ai
• 1.8 Stitch & the Samurai
• 2 Personality
• 3.1 Physical appearance
• 3.2 Special abilities
• 3.3 Weaknesses
• 5 References

Background and appearances

Experiment 626 was the 626th genetic experiment created by Jumba , and the first to be made without Hämsterviel 's funding. He was designed to be virtually indestructible, fireproof, bulletproof, shockproof, think faster than a supercomputer, have super sight and hearing, and lift objects 3,000 times his own weight (but not an ounce more). He was created by Jumba from six or more of the galaxy's deadliest predators , though instead of him appearing vicious, he appeared to be cute and fluffy. Shortly after his creation, the intergalactic police arrived to arrest 626 and Jumba, preventing 626's molecules from being fully charged in the process.

Lilo & Stitch

Stitch shortly after arriving on Earth

Jumba and 626 were taken to the planet Turo to stand trial. When 626 replied to the Grand Councilwoman 's attempts to reason with him with an obscene phrase, he and Jumba were declared guilty, and 626 was escorted by Captain Gantu to be exiled on a desert asteroid.

However, 626 was able to escape his detention cell (thanks to the incompetence of the lieutenant left to guard him) and steal a federal police cruiser . 626's cruiser was soon fired upon by a fleet of police cruisers , and, in an attempt to escape, 626 activated his cruiser's hyperdrive, but the damage inflicted on the police cruiser by the fleet caused it to exit hyperspace over Earth and crash-land on the island of Kauai .

Stitch providing shade to sea turtles

Shortly after landing, 626 was run over by a convoy of trucks , knocking him out, and taken to the local dog shelter . When 626 awoke, he attempted to escape the shelter but was forced to retreat back inside by an attack from his own creator. Soon after, 626 saw Lilo looking for a pet dog. Seeing the little girl as an unaware hostage, 626 altered his appearance to look less otherworldly and pose as a dog in order for her to adopt him. The plan worked and Lilo adopted 626 and named him Stitch.

Using Lilo as an unsuspecting hostage and Pleakley 's concern for humans, Jumba was unable to try and capture Stitch whenever he was near Lilo. At first, Lilo only mattered to Stitch as a hostage, but over time, Lilo taught Stitch how to love others as his ʻohana . As a result, Stitch eventually left to find his own ʻohana when he saw the damage he was causing Lilo's. Instead, Stitch discovered the evil behind his origins from Jumba.

Stitch discovers his origins

When Jumba confronted Stitch, the latter engaged Jumba in a standoff to protect Lilo, which ended in a tie when Jumba's plasma cannon exploded. Stitch then revealed his alien origins to Lilo and later helped rescue her using a tanker truck when she was mistakenly captured by Gantu. Afterwards, Stitch was captured and arrested by the intergalactic police, but then allowed to stay on Earth as exile due to his change of heart and Lilo's legal ownership of him.

Lilo & Stitch 2: Stitch Has a Glitch

Jumba noticing Stitch's glitch

About a year after the events of the previous film, Stitch was fearing that he would revert to his destructive programming.

Though all seemed well for Stitch at first, he began to suffer from glitches (caused by his molecules not being fully charged during his creation before his capture), during which he would experience seizures and temporarily revert to his original, destructive programming.

The damage Stitch's glitches caused began to drive a wedge between him and Lilo and was ruining the latter's chances of winning the hula competition. Because Lilo was so concerned with winning the hula competition, she failed to notice Stitch's glitches or that he had no control over it. Thus, Lilo blamed Stitch for the destruction his glitches were causing and neglected to help him.

Eventually, the two devised a hula based on the legend of Hiʻiaka, but as Stitch's glitches ruined their practice sessions, Lilo became increasingly mad with him.

Just before the competition, Stitch attempted to make up with Lilo, but he suffered another glitch during which he accidentally scratched Lilo. Visibly upset by this, Stitch stole Jumba's ship to banish himself to an uninhabited planet where his glitches could not cause any harm. However, before he could activate the hyperdrive to send him to one, he suffered a massive glitch attack, which caused him to crash the ship into the Hawaiian mountains.

Lilo then arrived and dragged a near-death Stitch from the wreckage and into Jumba's fusion chamber , praying that he would be okay as she pulled the switch. Unfortunately, she was too late and Stitch temporarily died. However, Lilo's love for Stitch successfully revived him and fully charged his molecules.

Stitch! The Movie

Stitch dressed in a hula costume

At the start of Stitch! The Movie , Stitch is shown having a difficult time connecting with the island locals during a beach party, feeling that he doesn't fit in and is too much of an outcast. Though Lilo tries to comfort him, he seeks Jumba in hopes of learning about having others like he and the evil scientist. However, Jumba explains that, in the vast universe, he and Stitch are alone and lack, what Lilo calls, "cousins".

Later on, Stitch discovers Jumba's first 625 genetic experiments locked in a crate, in the form of dehydrated pods . In order to rescue a kidnapped Jumba, a trade between Dr. Jacques von Hämsterviel and Jumba for the experiments is organized, but it instead results in the freedom of the experiments and the capture of Lilo and Stitch. Hämsterviel then tries to clone Stitch, but he was rescued by Experiment 221 ( Sparky ). Stitch then rescued Lilo and landed Hämsterviel's spacecraft back in Kauai , where the Grand Councilwoman waited to arrest the evil rodent. She then puts Stitch and Lilo in charge of capturing and taming the other illegal genetic experiments scattered on the island.

Lilo & Stitch: The Series

Stitch behaving like a dog

In Lilo & Stitch: The Series , Stitch and Lilo have to capture Jumba 's experiments , turn them from bad to good, and find them each a home before Gantu , 625 , and Hämsterviel capture them and use them for evil. Due to being the most physically capable, Stitch is often the one who defeats Gantu and subdues the experiment, leaving it up to Lilo to rehabilitate them. Along the way, Stitch meets his match 627 (who is tougher than him), and he eventually defeats him.

He is also affected by fourteen experiments' powers: Amnesio wipes out his memory; Babyfier turns him into a baby (along with Nani , Jumba, and Pleakley ); Spike makes him goofy; Bugby turns him into an insect; Spooky turns into water and nearly drowns him (thus traumatizing him in the process until he overcomes it); Lax makes him lazy; Dupe clones him into four, weakening his strength; Frenchfry serves him unhealthy meals (along with Lilo and Pleakley) and fattens him up into a giant bowling ball; Swapper switches Stitch and Lilo's minds; Yaarp blasts his megaphone, temporarily deafening Stitch; Snooty sucks out his mucus, temporarily putting him out of commission; Swirly hypnotizes him; Drowsy (in the episode " PJ ") puts him to sleep; and Houdini makes him invisible. He also meets Experiment 624 ( Angel ), who is introduced as a love interest for Stitch.

Over the course of the series, he and Lilo have also had adventures with Kim Possible , Jake Long , Penny Proud , and the Re ce ss ga ng .

Leroy & Stitch

Stitch as captain of the Galactic Armada

After all of the first 624 experiments were captured and rehabilitated, Stitch was assigned to replace Captain Gantu by the Grand Councilwoman as a reward, but found himself missing Lilo and his ʻohana .

He was soon assigned to recapturing Hämsterviel , but got in a fight with the newly-created Leroy , his red evil twin. Though Stitch had the upper hand for most of the battle, Pleakley 's untimely arrival caused Stitch to drop his guard, enabling Leroy to strike the final blow and get Stitch locked in a capsule.

Hämsterviel attempted to dispose of Stitch, Jumba and Pleakley by sending them into a black hole, but Stitch broke free and was able to use a rock to cause the black hole to instead transport them to Turo in time to save Lilo, Gantu and Reuben from a group of Leroys.

They then returned to Earth in time to save the other experiments from Hämsterviel and the Leroys, and Stitch led the experiments in a battle against the Leroy army.

Stitch, Lilo and Reuben eventually defeated the winning Leroy army with the song " Aloha ʻOe ", and Stitch willingly resigned and returned to Earth to his ʻohana .

Stitch! anime

Stitch and Yuna doing a pinky swear

In the anime spin-off, set years after the events of Leroy & Stitch , Lilo left for college and Stitch felt enough of a void in his life that he chose to leave Kauai and go back into space.

He started reverting to his destructive programming and escaped while Jumba and Pleakley tried to locate and rescue him, but years later, he would land back on Earth on Izayoi Island , off the coast of Okinawa . Soon joined by Jumba and Pleakley, they make a new life with a young girl named Yuna , and Stitch seeks the magic powers of the Spiritual Stone , an object that can grant any wish he wants, in this case, wanting power. But the magic only works by doing good deeds, and Stitch now faces the return of Hämsterviel and a once-again disgraced Captain Gantu , along with various experiment threats.

Eventually, Stitch reunites with Lilo again when she, now fully grown, visits Okinawa with her identical looking daughter, Ani , though things get tricky when Gantu and Hämsterviel use Morpholomew to take advantage of the situation. Stitch decides to stay with Yuna in her hometown instead for the time being.

Also, it is revealed that a special power cell was hidden inside Stitch during his creation called the "Neo-PowerChip". This immense power is latent, but it can be used in times of dire situation. For instance, increased love and support from his friends activated this boost of power inside of Stitch, which he then used to defeat Dark End .

Stitch & Ai

Stitch's newly-introduced "destructive form" as depicted in this promotional poster

This Chinese spin-off series is set chronologically after the events of Leroy & Stitch but on a separate timeline from the anime, since it was produced without any consideration towards the latter.

In this series, it is revealed that Stitch has additional programming which causes him to under go a metamorphosis when activated. This transforms him into a large monster capable of destroying not only cities, but star systems as well. The information about Stitch's programming is hacked and spreads throughout the galaxy. This causes Stitch to be kidnapped by the Jaboodies , who had failed in their own attempts at re-creating Jumba's Experiment 626 research and wish to use Stitch to end their space war against the Woolagongs. When their ship is raided by the Woolagongs , Stitch uses the confusion to escape and falls back to Earth. After surviving re-entry, he ends up landing in the Huangshan Mountains in China and befriends a young girl named Wang Ai Ling . In order to help contain Stitch and stop the Jaboodies and Woolagongs, the Galactic Federation sends Jumba and Pleakley to monitor Stitch in China.

Stitch & the Samurai

In this manga, Stitch escapes the United Galactic Federation in a stolen police cruiser spaceship by using its hyperdrive to make a blind jump that ends up on Earth. However, instead of traveling through just space, he also travels through time; he crash-lands in Japan during the Sengoku period, in which he meets battle-weary samurai warlord Meison Yamato . After a brief struggle, Yamato is not only impressed by Stitch's strength, but the sight of the little blue "tanuki" causes him to have a thought that he has never had before: "cute". Thus, the warlord takes in the alien, not only making Stitch part of his army, but also developing an unlikely bond.

Personality

Stitch snuggling his little stuffed turtle after a nightmare

Before Stitch was rehabilitated, he was a vicious, hyperactive, selfish, and extremely aggressive and mischievous creature. After he was rehabilitated by Lilo, although his mischievous, destructive, and aggressive tendencies remained, he possessed a heart of gold.

He also seems to like turtles, being there is a picture of Stitch shading two sea turtles in the credits of the original Lilo & Stitch movie and snuggling a stuffed turtle after a nightmare in Lilo & Stitch 2: Stitch Has a Glitch . As well as having a soft spot for turtles, he also seems to like frogs. He gently moves frogs out of harm's way constantly and seems to talk to them as if they are his friends. These likes may be a sign that Stitch may be growing fond of Earth creatures.

Starting off bad when he was programmed by Jumba Jookiba, Stitch was originally designed to create pandemonium and chaos wherever he was. He was drawn to large cities where he would back up sewers, reverse street signs, and steal individuals' left shoe, but Lilo changed his ways a while after adopting him at an animal shelter .

Stitch is still prone to having temper tantrums when things don't go his way, or he sees that he's been squirted by a water bottle for something that he thought was good. So he isn't good all of the time. Or more accurately, he isn't well-behaved all of the time. He also gets easily angered by inanimate objects, especially the toaster. The toaster has popped toast in his face once, and since then, he has beaten up the toaster and juiced it at least twice. He is a complex character. According to his creator Jumba, Stitch is a destructive machine who was not given a greater purpose in life. Since Stitch is trapped on an island in Hawaii, where there are relatively few things to destroy, he has many occasions to reflect on occupations other than evil. It is implied that Stitch is only destructive because that is what he was programmed to do; but since he has a personality of his own (a fact that only Lilo, and Jumba truly understand), he also has the choice, and even the will, not to be destructive.

Despite having superhuman strength and a high level of computer intelligence, Stitch is emotionally fragile and has a childlike personality beneath his monsteresque exterior. Lilo believes that one reason why he originally pursued his destructiveness was for reasons similar to her own naughtiness—that it was because he was emotionally scarred (for having been made a monster by Jumba), but received little or no understanding or sympathy from others. Stitch becomes much more well-meaning after his betterment but is frequently unable to control all of his destructive impulses. Fortunately for him, Lilo (and later, Jumba, Reuben and Angel) is always there to support him and he returns her support willingly.

Stitch has a huge appetite and gets very irritable if he doesn't get what he wants to eat. Also, despite his temper tantrums, Stitch can sometimes have a little common sense and be more mature than Lilo. For example, when Lilo used the other experiments for her own personal gain (most notably, Sprout , Slugger , Checkers , Morpholomew , and PJ ), Stitch thought she went too far involving such experiments in her schemes.

Physical appearance

Stitch is a blue-furred, koala-like experiment with aqua countershading around his eyes and running from under his chin down to his stomach. The upper rims of the countershading around the eye sockets have black eyebrows. He has two dark blue markings; one on his occiput (the back of his head), and the other on his back. His head features large rabbit-like ears with pink insides and dark blue tips on the back, a wide mouth with a pink inside, white teeth, a navy round nose, navy claws and toes, two nose wrinkles, one chin wrinkle, and large black, tear-shaped, pupil-less eyes. He also has a small, short, stumpy tail, stubby legs, three tufts of sharp fur on top of his head and on his chest, as well as two extra, retractable arms, sharp navy retractable claws on his front and back paws (hands and feet), three thick, long, sharp, retractable porcupine-like spines tipped with white and a very dark navy that run down his back, and two long dark blue-tipped retractable bug-like antennae on his head. His blood is neon pink in color, as shown in the first film when his blood is drawn as a DNA sample for the prison turrets to lock onto.

In some promotional art with his disguised "dog form", he wears a red flea collar with a gold name tag. However, he almost never wears a collar in the actual film or the rest of the animated canon.

In his true alien form, he formerly wore a red spacesuit with an upside-down triangle consisting of a red border and yellow center on his chest, with each of his sleeves and patches consisting of a dark red border and orange center on each of his legs, black belt with a dark red square buckle, both black collar, cuffs, and anklets. His spacesuit was shredded to bits when he was run over by three trucks shortly after crash-landing on Earth.

Covered in blue fur and having exceptionally large ears for his body, which measures around a third of his height, Stitch is an unusual thing to see wandering the streets. Usually, his smile scares people, as his gums are lined with nothing but sharp teeth.

Stitch's ears both have a little notch missing, though both in different places (although in Lilo & Stitch 2: Stitch Has a Glitch , it was shown that he initially had pierced ears); his lower left ear and upper right ear have a triangular piece of flesh that is missing from them. He has got large black eyes that turn green when using his night vision, red when using infrared vision, bright green when using x-ray vision, purple when using ultraviolet vision, and white when using binocular vision.

His arms differ from his hind legs, as his front paws look more like little hands and his back feet have flat pads. Each of his paws have four digits (fingers and toes) that are tipped with sharp claws that help Stitch to attack his enemies, although the toe claws are shorter and more blunt.

There have been a few instances in the franchise in which several creatures' DNA from which Stitch's genetic template was created are discussed. In the Disney Adventures comic titled: "Experiment 626!", we see four fearsome creatures whose DNA Stitch possesses in addition to a domesticated dog from Earth, although the comics were later rendered non-canon. In The Origin of Stitch , we learn that Stitch has had some of his DNA sampled from the following creatures:

• Fearsome Manglyoid of Upmoridian Four
• Goo-Gobbling Booger-Beast
• People-Eating Puss Monkey
• Deadly Disemboweler
• Boiling Tongue-Boid
• Bottom Feeding Scum Sucker

Special abilities

While explaining his creation to the Galactic Council early on in Lilo & Stitch , Dr. Jumba gives the following rundown of Stitch's powers: "He is bulletproof, fireproof, shockproof, and can think faster than [a] supercomputer. He can see in the dark and can move objects 3,000 times his size. His only instinct: to destroy everything he touches!"

• Feral mind (temporarily) : In the original Lilo & Stitch film, Jumba claimed that Stitch's "destructive tendency was taking effect" and that he would be irresistibly drawn to large cities to "back up sewers, reverse street signs, and steal everybody's left shoe."
• Superhuman durability/Dense body tissue : Even though we rarely see Stitch shot at with a bullet, he does survive the crash of his spaceship without even a scratch, is only briefly stunned by a fall of several thousand feet, and has to be run over by three tractor trailers in succession to be knocked out.
• Fireproof skin : He drives a tanker truck of gasoline into a volcano and ends up only a little singed from the resulting explosion.
• Bulletproof skin : Rounds fired from plasma guns seem to cause him some discomfort, although he can hold the material in his hands long enough to throw the plasma back at his enemies.
• Shockproof : He has some resistance to electricity since he was able to take a direct electrical attack from Sparky , quickly recovering from it. However, he can still feel pain from these types of shocks despite his immunity to it.
• Hyper-cognition : Thinking faster than a supercomputer is harder to quantify, but he does escape from captivity fairly ingeniously; builds a model of San Francisco after only glancing at a postcard; grabs a crossword puzzle from the table and finishes it in about seven seconds; builds a bomb out of a plasma bolt, a doll , and a roller skate; creates a "bucking bronco" out of a toaster, vacuum cleaner, and a lamp; reads aloud a description of a local museum's display, showing he is able to understand written language at a remarkable speed; manipulates his moral compass by using a human being as a shield in Jumba and Pleakley's presence; and generally picks up quickly on what is happening around him.
• Lingual intuition : Stitch can converse fluently in over twenty different languages, but prefers Tantalog (his native language).
• Parallel computing : He is an information repository, containing an internal dictionary, thesaurus, encyclopedia, and experiment catalog, and records every moment of his life in a reserved portion of his brain that can theoretically store up to 300 PB.
• Instrument player : He is also skilled at playing musical instruments, most notably the ukulele, thanks to his quick learning.
• Vehicle intuition : Stitch can drive any sort of vehicle from something as simple as a tricycle to complex machinery like intergalactic spaceships .
• Superhuman strength : His ability to lift objects 3,000 times his own size and weight is seen several times throughout the franchise, including incidents where he picks up a descending blasted door, hits Dr. Jumba with a thrown Volkswagen Beetle (shouting gleefully, "Blue punch buggy! No punch back!"), and stops an 18-wheeled tanker of gasoline dead in its tracks. The reason why Stitch can lift 3,000 times his own weight, even though he has small arms, is because his muscles contain excessively compressed amounts of myofibril within one muscle cell. This muscular arrangement makes Stitch's muscles contract 1,000 times faster than a human's, making him much, much more powerful. The ability is sometimes joked about in the later series; for example, in Stitch! The Movie , when Hämsterviel has Stitch physically restrained for a cloning experiment, Stitch's restraints counter his strength at, as Hämsterviel loudly declares, to "three thousand and one!" times Stitch's weight, though he later admits it was intended as a joke. This was further validated in the Lilo & Stitch: The Series episode " Swirly ", when Gantu correctly guessed that Stitch cannot lift even an ounce more than 3,000 times his size when he adds a small ticket to the given load.
• Quadrupedalism : Stitch can run and walk on all fours in a quadrupedal manner, similar to a dog.
• Wall crawling : While Stitch's claws are very strong, the pads on his hands and feet can secrete a sticky substance, allowing him to scale and adhere to almost any surface, such as buildings and walls. He can also stand on walls and ceilings.
• Spherical form : His skeletal system is very flexible, allowing him to put his feet into his mouth and roll like a ball. This allows him to squeeze through tight spaces.
• Superhuman speed : Stitch can move and run faster as shown when moving past Lilo and Pleakley while carrying Jumba, during a bet made with Lilo to catch Experiments 501 ( Yin ) and 502 ( Yang ), and hurrying from Gantu's ship back to the fundraiser while carrying Lilo. His speed is also usually used when he's in his spherical form. When Stitch runs, his speed doesn't automatically increase and can only achieve this by putting effort into his muscles.
• Superhuman jump : Stitch's legs are small but, thanks to the muscle advancements mentioned above, are powerful enough to enable him to jump several feet into the air.
• Superhuman agility : Stitch has extraordinarily greater agility, dexterity, balance, and body coordination, capable of doing feats far beyond an acrobat, such as leaping far distances, jumping off from wall to wall, swinging from vines, and standing on branches, wires, and narrow platforms. He is also capable of standing and walking on his paws. The reason is Stitch has a very flexible skeletal system that also allows him to squeeze through tight spaces.
• Superhuman reflexes : Stitch is able to react and dodge plasma bolts from the plasma guns from Jumba and Gantu. When Jumba tried to shoot him, Stitch caught the plasma bolts in his hands. He was also able to catch some tennis balls with his four arms and even threw them back in the pitching machine.
• Superhuman stamina : Stitch's advanced musculature produces fewer fatigue toxins, allowing him to exert himself for extended periods of time. Stitch can still fight, run, and even eat without tiring out; even when hunting for other experiments, he would hunt them for an entire day to when night comes.
• Night vision : When activated, Stitch's eyes turn green, permitting him to see clearly at night or in dark places.
• Infrared vision : When activated, Stitch's eyes turn red, permitting him to locate targets by their body heat. He most notably uses this in " Houdini " to track the titular experiment who can turn things, including himself, invisible.
• X-ray vision : When activated, Stitch's eyes turn bright green, permitting him to look through walls and other obstacles.
• Audio amplifier : He can act as an audio amplifier, radio, and/or microphone, illustrated when he uses his claw as the needle on a record player and plays music through his open mouth like a sound horn.
• Enhanced hearing and smell : He has an acute sense of smell and hearing. Concept designs detailing Stitch's anatomy describe the sensory filaments of his nose being so dense and extensive that, if unraveled, could blanket Earth. While listening for Jumba and Pleakley, who were hiding on a hill outside the dog shelter , he was able to detect Pleakley speaking at a whisper.
• Skilled hand-to-hand combat : He is dexterously skilled in hand-to-hand combat, whether using all four arms or just two.
• Marksmanship : He is also dexterously skilled in marksmanship, using plasma blasters (even loaded with net to capture experiments).
• Venomous spines : In anatomy drawings of Stitch from the film's production, his spines are described as being poisonous and connected to venom sacs in his spinal cord.
• Shapeshifting : In his alien form, he has three spines on his back, two antennae on his head, and an extra pair of arms. However, he is able to retract all of these features to look more like a dog while on Earth and used this to become Lilo's "pet".
• Semi-immortality : Stitch cannot age. When Skip flashed ten years ahead, he did not age whatsoever, but, for some reason, Babyfier 's powder regressed him into an infant. This could simply mean he stops aging once he is physically mature.
• Vacuum adaptation : Along with the rest of his "cousins", Stitch can survive in the vacuum of space due to being a genetic alien.
• Power immunity : Stitch has some resistance but not total immunity to the other experiments' powers. He is shown to be immune to some of the experiments' powers, such as Angel 's siren song and Checkers ' hypnosis, since Stitch was created after them. While Stitch was put to sleep by Drowsy , he was able to wake up on his own without the need to be sprayed with water.

Both of the two East Asian animated spin-offs add additional abilities to Stitch not shown in the original Western continuity.

• Battery storage : The third season of the Stitch! anime shows that he also has a natural battery in his body that can supercharge his powers.
• Transformation : In Stitch & Ai , it shows that Stitch has a metamorphosis code in his DNA to assist him in carrying out his primary function. When the metamorphosis programming is activated, he transforms into a gigantic and unstoppable monstrosity with four tentacle-like blasters capable of destroying not just cities, but planets and potentially entire star systems.
• Quill retraction : In " Tell the World ", he is also seen sprouting quills around his neck.
• Gliding : In " The Phoenix ", Stitch sprouts a patagium—a parachute-like membrane found in bats, birds, and other animals that assists in gliding or flight—to glide through the air, mainly for the sake of having some fun while he, Ai , and her cousin Bao head to a shrine that the titular phoenix heads to. (The trio were using a hang glider during their journey, and Stitch deliberately jumps off it so he can glide through the air without hanging on to the glider itself.) Unlike the metamorphosis program, neither the quills nor the patagium have any major factor into the episodes' plots or the metamorphosis program itself.
• Water : Stitch's greatest weakness is his inability to float, let alone swim, in water due to his molecular structure being much denser than the average human or animal. This causes him to instantly sink like a stone. Stitch originally had an instinctive aquaphobia (which was even used against him by Spooky ), but he was able to overcome it thanks to Lilo's influence.
• Glitch (formerly) : In Stitch Has a Glitch , it is revealed that Stitch's molecules were not fully charged after his creation, which is proven to be very deadly once his molecules run out of power. As a result, Stitch frequently experiences periodic glitches that have him revert to his original destructive programming; basically, suffering from a seizure disorder and having nightmares of destroying everything and everyone in his path, including Lilo. However, his glitches were permanently resolved after Lilo's love for him caused him to be fully charged at last.
• Memory erase from Crezonyte : It is shown in " Kixx " that if Stitch consumes two different elements which form to create a particular chemical compound called Crezonyte, all of his training skills will be wiped. Eating the rubber of a tennis shoe and palmitoleic acid (found in macadamia nut oil) together will result in this.
• Mucus drain : Stitch is 62.7% mucus, which is similar in chemical composition to Snootonium. If this is drained from his body, he will become temporarily exhausted from dehydration (evidenced when Snooty siphoned Stitch's mucus).
• Sonic blasts/Sensory overload : Another weakness is the fact that his super-sensitive hearing can lead to temporary deafness when exposed to sonic blasts, such as Yaarp 's destructive sound blast.
• Strength limit : As stated above, he cannot lift even an ounce past the "3,000 times his weight" limit.
• Experiments' powers : While Stitch is immune to Angel and Checkers ' powers since he was created after them, there are some experiments whose powers he is vulnerable to, such as Amnesio , Babyfier , Spike , Bugby , Lax , Dupe , Frenchfry , Swapper , Swirly , and Houdini . Stitch can also be put to sleep by Drowsy , but he was able to wake up on his own without the need to be sprayed with water.
• Blades/Durability limit : While Stitch can withstand plasma bolts, being beaten, and even the most hazardous environments, it is implied that Stitch isn't resistant to bladed objects. This is shown when Stitch was pierced by a Sample Extractor and able to take out his blood.
• Trash ingestion : If Stitch consumes vast amounts of trash, he will become incredibly sick as shown in " Ploot ". He first gets sick to his stomach and sneezes, then his fur turns yellow, his tongue becomes orange, and his nose turns red. Another symptom is, with each sneeze, an arm grows from random areas of his body. Stitch's garbage-eating illness was theorized as a rare space disease by Jumba. Stitch was temporarily cured when he drank a barrel of orange juice and took medicine from an eyedropper, though it was only for a few seconds. When he sneezed again, he developed yellow stripes and grew legs instead. The only permanent cure is inhaling steam from a vaporizer.
• Sauerkraut : According to Lilo, Stitch is apparently allergic to sauerkraut. However, it was never shown what kind of allergic reaction he has to sauerkraut or if he even actually has an allergy to it.
• Pollen : It is shown in Stitch & Ai that if Stitch smells flowers, he will sneeze.
• According to Chris Sanders ' initial designs, Stitch was originally going to have green fur and smaller eyes. His ears flared at their bases instead of close to their tips, and his nose was below the eye line instead of above. He also had white or silver claws instead of dark blue claws, and wore a yellow-orange spacesuit with burnt orange pants instead of the more uniform red-orange spacesuit that he wears in the original film 's first act.
• Stitch is the only Lilo & Stitch character to have his name in every title of the franchise .
• Despite not appearing in the show itself (as the show ran for about one year before the movie came out, although the film came out during its run on television), Stitch was featured with Lilo in some online games for the television series House of Mouse .
• Also, the original version of the scene where Jumba attacks Stitch which results in the Pelekai residence accidentally being burned down was actually going to have more violent scenes like Stitch using a chainsaw to attack Jumba, as well as Stitch setting the house on fire by biting apart a gas pipe and tricking Jumba into shooting it with his blaster . The final version of the same scene has everything violent edited out and replaced in favor of making the scene more slapstick-oriented (Jumba knocking the ceiling down with dishes instead of laser blasts, Jumba's "shuriken" being made of hairbrushes and a tube of toothpaste instead of knives and a pizza cutter, and the entire chainsaw scene shortened), having more comedic scenes added in (exploding Scrump , Stitch catching Jumba's laser, Stitch yelling, "Blue Punch Buggy! No punch back!" when hitting Jumba with Nani's VW, the Elvis Presley song " Hound Dog " playing in the background, and Lilo saying, "Oh, good. My dog found the chainsaw."), and the house being destroyed by having Stitch stop up Jumba's blaster with a carrot and then having them both play " hot potato " with it until the blaster overloads and explodes.
• A similar treatment was done to the climax , where Jumba, Pleakley, Stitch, and Nani fly through the sky to pursue Gantu after he accidentally captures Lilo as a result of the terrorist attacks on September 11, 2001.
• When Jumba attacks Stitch on the ceiling using plates like shurikens in the original film, he claims that he wants to reprogram Stitch to be "taller" and "not so fluffy", hence highlighting Stitch's failure as Jumba's "ultimate monster" experiment, and foreshadowing the creations of both Experiment 627 and 629/Leroy in Lilo & Stitch: The Series and Leroy & Stitch , respectively; coincidentally, both of them are improved versions of Stitch that are more evil and destructive and less fluffy than Stitch himself.
• Stitch is considered to be one of the first successful prototypes for Jumba's "ultimate monster" experiment, though he is technically a failure because he wanted to belong and find a family. Other notable attempts for this prototype are 000 , 262 , 600 , and 625 , and each failed for its own reason: 000 is uncontrollable, 262 is good, 600 is clumsy, and 625 is lazy. 627 and Leroy are additional attempts Jumba made after Stitch, but both of them also failed: 627 is weak to laughter while Leroy is weak to a song . However, Leroy can also be considered a success, since Jumba deliberately programmed the weakness into him.
• In said parody, when Stitch and the other experiments are looking up at the ceiling, Stitch is colored red and 627 is colored blue due to a coloring error.
• In terms of the actual production, his limited intelligible speech is due to Chris Sanders not wanting the character to speak anymore than what's absolutely necessary and keeping his dialogue to a bare minimum.
• Lilo & Stitch 2: Stitch Has a Glitch continues from the first film by having Stitch become more proficient in English, though with occasional moments of his more familiar childish English, and he almost never speaks in Tantalog. However, after he has his final glitch at the end of the film, which causes him to crash-land Jumba's ship , he only says a few words in brief sentence fragments from then on out.
• Lilo & Stitch: The Series and its films has Stitch speaking mostly in Tantalog, which was developed by the show's writing staff based on Chris Sanders' alien gibberish in the original film. When Stitch does speak English, it's usually as brief, broken sentence fragments with some brief pauses between words at times and a few possible instances of echolalia .
• The Stitch! anime series gives Stitch more dialogue than in any other work in the franchise, making him more talkative compared to his Western and Chinese portrayals. In the Japanese original, he maintains his broken and simplistic speech patterns, including his trademark illeism, and more clearly has echolalia, showing a tendency to repeat that last thing someone else says. However, in the English dub, his English improves, he talks in more complete sentences, and he develops a considerable wit. This is likely to reflect the long passage of time since the events of Leroy & Stitch , as Stitch may have spent enough time on Earth actually get a grasp on the English language. (It should also be noted that English is heavily taught as a second language in Japan .)
• Stitch & Ai has Stitch's speech revert back to his broken, childish English, and he mainly speaks Tantalog again, much like in Lilo & Stitch: The Series . Interestingly, despite having the same English voice actor as his anime counterpart ( Ben Diskin ), he speaks with a lower-pitched and somewhat less energetic voice than that used in the anime.
• Interestingly, he only does this twice in the original Lilo & Stitch film, after he was apprehended by the Grand Councilwoman . He says "Stitch" instead of "I" when he asks her if he has to go into her ship , and if he can say goodbye to Lilo and Nani. All other times he refers to himself in the original film have been in the correct first-person. It is likely that, since this trait of Stitch's is more prominent in the sequels and spin-offs, his illeism (along with his broken English in a franchise where aliens can speak English fluently) ended up being a case of Flanderization for his character.
• In Stitch Has a Glitch , he is shown to do this less often up until the climax.
• In the English dub of the Stitch! anime, he does it even less often than before, but over the course of the series, he seems to have grown out of this habit: he talks in complete sentences, his English has improved, and he has more dialogue.
• Adding on to this, Chris Sanders published a TikTok video in June 2022 for the film's 20th anniversary in which he unofficially (as it was not made nor authorized by Disney) voices Stitch again, albeit in narration. The video has Stitch speaking in fluent English throughout, with only his famous illeism sticking around. This suggests that Sanders sees Stitch as eventually becoming naturally fluent or near-fluent in English over time.
• However, in Stitch & Ai , when he reunites with Jumba and Pleakley, he tells Ai that they're "cousins", despite never having called either of them "cousins" before.
• The plot revolving around the book mirrors Stitch questioning his purpose, as it revolves around his loneliness and desire to find a family, to which he did in the end.
• Stitch also developed a fondness for ducks as he showed some ducklings the storybook, as seen in the credits of Lilo & Stitch .
• Stitch apparently has a fondness for frogs as seen in Lilo & Stitch , where Stitch saves one from being blasted by a plasma gun of Gantu's ship .
• In the end credits of Lilo & Stitch , Stitch is also seen covering a turtle and its baby with a leaf.
• Stitch likes to dress up as Elvis Presley and is very skilled at playing the ukulele.
• Stitch also enjoys soda, as he steals a can from the Pelekais' fridge in the original film, angrily drinks the rest of the soda from a pitcher at Macki Macaw's restaurant in " Phantasmo " when Pleakley doesn't pour him some after Lilo had Stitch wait his turn, and enjoys a can after stuffing Mr. Stenchy into the Pelekais' pantry in " Mr. Stenchy " before being interrupted by Gantu.
• However, in " Shortstuff ", Stitch, as a giant, drank some lemonade from the cart he accidentally smashed without expressing any disgust, and in " Swapper ", he drank a whole bowl of lemonade (albeit modified by Jumba to cause those who drink it to temporarily change colors), again without expressing any disgust, which would contradict this supposed dislike of lemons. It could be that either Stitch doesn't like organically-grown foods (as his heightened senses might have the unintended side effect of him tasting even the tiniest amounts of manure residue), he doesn't like the taste of unsweetened lemons or lemon juice, or him spitting out the organic lemon was nothing more than a simple joke.
• In the Stitch! anime, he also loves Yuna 's blue crab sushi.
• Due to a continuity error, Stitch's ear notches have a tendency to disappear or change position in some works and merchandise. This has also caused countless fan art and even some official artwork to reflect these and other errors, such as reducing his four toes on each foot to three or changing his paw pads or body markings from dark blue or indigo to the same aqua color as his countershading.
• Additionally, following after Stitch Has a Glitch , Stitch's fur color has changed from dark blue to light blue.
• Since then, Stitch has developed trypanophobia (a fear of needles) as evidenced in " Sample ", when he showed fear of going to the vet to get his shots.
• Ironically, in the original film, Stitch's eyes were shown green after he crash-landed on Earth, but he displayed no glitch whatsoever.
• In Stitch Has a Glitch , the animation was reused in the beginning.
• Usually when Stitch rolls into a ball, he puts his feet into his mouth and covers his face with his butt. In " Dupe " and " Snafu ", his ball form resembles Cannonball 's ball form in Leroy & Stitch .
• This may also provide additional context when he interrupts the Grand Councilwoman to clarify his name in the denouement of Lilo & Stitch , as he may also imply that it would no longer be considered proper to refer to him as "Experiment 626" in any further formal or legal matters. Since he is referred to as "Captain Stitch" when he joins the Galactic Armada in Leroy & Stitch , it could also be that the United Galactic Federation does formally acknowledge "Stitch" as his proper legal name; if they didn't, then he would have been "Captain 626" instead.
• Notably, Stitch appeared on more covers for issues of Disney Adventures than any other character, appearing on the cover of twelve issues.
• In the Lilo & Stitch: The Series episode " Shortstuff ", Stitch uses Jumba's Protoplasmic Growth Ray to make himself bigger, but he is considerably clumsier in this larger size since he was not designed to be large. The episode's titular experiment also becomes bigger and defeats the enlarged Stitch at the carnival. Stitch would later be returned to his normal size in order to fight back and win.
• In the Stitch! anime episode " Experiment-a-palooza ", Shrink zaps Stitch with an energy ray after the latter falls into a swimming pool, causing him to grow into a giant. Due to Retro reverting Stitch to his original destructive programming, Stitch goes on a rampage in this giant form until Yuna reaches out to him and reminds him of his promise. Shrink would be used again to revert Stitch to his original size.
• However, a deleted version of the opening of the original film contradicts this. In this opening, the Galactic Council shows video footage of Experiment 626 – in his familiar small size – causing chaos and destruction in an alien city, including tipping over a skyscraper by lifting it from its base. The existence of this scene strongly suggests that Chris Sanders, Stitch's creator and original voice actor, never intended or at least considered giving Stitch this metamorphosis ability, thus likely making the monstrous "destruction form" in Stitch & Ai a retcon or a " revision " of the character. (Sanders did not work on either Stitch & Ai or the Stitch! anime series in any capacity.)
• Sanders left Disney to join DreamWorks Animation (a rival animation company to Disney) in 2007 due to creative differences over Disney rejecting his original Bolt script and designs. Despite his departure, though, Sanders still continues to voice Stitch in video games, such as Kingdom Hearts II , Birth by Sleep , Disney Infinity 2.0 , and in many other appearances of Stitch, having established a contract with Disney Character Voices. The notable exceptions are the Japanese Stitch! anime and the Chinese Stitch & Ai series, which had their English voice acting done with Studiopolis instead of in-house at Disney.
• In Big Hero 6 , Stitch is referenced twice: the first as a costume worn by Mochi in a painting, and the second as one of the pillows on Fred 's bed (alongside a Splodyhead pillow).
• The number 626 is the area code for San Gabriel Valley in Southern California, the place where one of the writers of the film used to live, albeit unconfirmed. [1]
• ↑ Disney Hidden Secrets

• Characters

Main characters • • • • • • • • • • main continuity characters • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • characters • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • characters • • • • • • • • • • • • • • • • Video game characters characters • • Others ( • ) • • • • Guest characters • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ( ) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • ( ) Other • • ( ) • 1 Angel (624) 2 Reuben (625) 437 COMMENTS Holio (606) Holio, also known as Experiment 606, is an illegal genetic experiment created by Jumba Jookiba and a character in the Lilo & Stitch franchise. He is designed to transform into a matter-sucking black hole that can destroy entire galaxies. His one true place is sucking up construction debris and litter. Experiment 606 was the 606th genetic experiment created by Jumba with Hämsterviel's funding ... Holio Holio, also known as Experiment 606, is an illegal genetic experiment created by Jumba Jookiba and a character in the Lilo & Stitch franchise. He is designed to transform into a matter-sucking black hole that can destroy entire galaxies. His one true place is sucking up construction debris and litter. Despite his destructive and chaotic function, Holio has an eerily calm nature when he is not ... Experiment 606: Holio Aha! Now, we come to one of the top three most diabolical experiments of all: Number 606! Pod Color: Blue Gender: Male Special Abilities: Designed to turn itself into a black hole that sucks in entire planets! Only a certain discharging bomb can disrupt this, reverting him to normal. Ironically, I got the idea from wanting a new type of garbage disposal, and wasn't interested in trying to ... "Lilo & Stitch: The Series" Holio: Experiment 606 (TV Episode 2003 Holio: Experiment 606: Directed by Mike Girard, Don MacKinnon. With Daveigh Chase, David Ogden Stiers, Chris Sanders, Jillian Henry. Mertle gets a bracelet for her birthday with an experiment pod on it. Lilo and Stitch set out to retrieve it before the experiment can be activated. Lilo & Stitch: The Series S1 E6 "Holio" Recap Experiments who appeared or were mentioned in this episode:. Holio (X-606): This episode's Monster of the Week; a small, red chipmunk-like experiment with a wide and large mouth, two thin antennae, and three thin back spines who is designed to create a matter-sucking black hole when he opens his mouth. Richter (X-513): He makes a brief cameo at the end of the episode, helping Nani to ... Experiments 606 Blue Holio: A small, red chipmunk-like experiment with a wide, large mouth, two thin antennae, and three thin back spines. Designed to create a matter-sucking black hole when he opens his mouth. His one true place is working in construction sites, clearing up waste by sucking in the unneeded matter. He is voiced by Frank Welker. 116, Leroy ... Experiments 601, 606, and 303 With that in mind, I hope you all still enjoy. I will be talking about experiments 601, 606, and 303. The names Lilo has given them are Kixx, Holio, and Amnesio respectively. While they're all different from there appearance, abilities, and personalities, they do have two things in common. Sadly, one is that they are easily forgettable. Holio (606)/Gallery Holio (606)/Gallery < Holio (606) View source History Talk (0) Images of Holio (606). Contents. 1 Lilo & Stitch: The Series. 1.1 "Holio" 1.2 "Checkers" 1.3 "Snafu" 2 ... Misidentified as Experiment 130 (Bonez) Miscellaneous [] Holio in Stitch!Now. v • e Image galleries; Works "Stitch Meets High School Musical" • McDonald's: Lilo & Stitch: The Series season 1 Holio: Experiment 606 Picks up where STITCH: THE MOVIE left off, with the experiment pods scattered all over the Hawaiian islands! The series followed Lilo and Stitch, as they rounded up Stitch's cousins (the other experiments) and repurposed those that they caught to do what they do best. ===== The series only had 2 seasons. The items listed in Season 3 of this guide are movies or other stuffAnother straight to ... Holio Lilo notices the experiment pod and realizes she must get that bracelet from Mertle. Soon she and Stitch are practically stalking Mertle, but are constantly being foiled by a local police officer. Stitch finally is able to discern the number on the pod: Experiment 606, a black hole experiment that will suck in the whole world! Lilo and Stitch Experiment 606 Holio In this episode we're going through the life of Experiment 606 HolioTwitter: https://twitter.com/WiredAnomaly#liloandstitch #stitch #lilo #Holio Lilo & Stitch: The Series #106 Lilo & Stitch: The Series » Lilo & Stitch: The Series #106 - Holio: Experiment 606 released by Disney on October 12, 2003. Holio: Experiment 606 last edited by cloudguy on 01/28/22 02:49AM View ... "Lilo & Stitch: The Series" Holio: Experiment 606 (TV Episode 2003 Summaries. Mertle gets a bracelet for her birthday with an experiment pod on it. Lilo and Stitch set out to retrieve it before the experiment can be activated. Lilo & Stitch: The Series (season 1) Lilo: [opens up Jumba's computer and searches up Experiment 606; horrified] Oh, no. [on walkie-talkie] Stitch. Come in, Stitch! Experiment 606 is a black hole that will suck in the whole world! Lilo: [shouting out to Mertle in warning about not getting the experiment pod wet as Nani drags her away] Don't get it wet! It'll destroy everything! Lilo & Stitch: The Series (TV Series 2003-2006) Fri, Nov 14, 2003. While at the post office mailing a letter to her pen-pal, Lilo finds experiment 509 pod. Jumba reveals it is an extraterritorial weed that grows out of control, bent on destruction. He deems too dangerous and locks away in a draw. Later, Lilo bets Myrtle that she can win the orchid competition. List of Experiments 606 Blue Holio: A small, red, chipmunk-like experiment with a large mouth, two thin antennae, and three thin back spines. Designed to create a matter-sucking black hole when he opens his mouth. His one true place is in construction, clearing up waste. He is voiced by Frank Welker. Holio (Character) Holio. Character » appears in 1 games. Holio, also known as Experiment 606, is one of Stitch's experiment "cousins" in Disney's "Lilo & Stitch" franchise. He is designed to transform into a black hole. Holio last edited by Veilor on 09/28/18 03:50PM View full history. Characters in Lilo & Stitch This experiment was sent to Dr. Hämsterviel, but was rescued by Lilo and Stitch in "Snafu". X-610: Witch — A small, purple bat/ghost-like experiment. The back of her head is shaped like a cone and curved at the bottom to resemble a witch hat, and her ears resemble small bat wings. Her eyes are blue and she has eyelashes. Experiment 607: Launch Experiment 607's small tears in the fabric of reality repair themselves after a few seconds. Thanks to fellow fabric of reality benders like 606, 607 was reprogrammed to tear holes in reality to get rid of nasty chemicals in power plants, not to destroy whole universe. Sometimes, Little Girl's resourcefulness surprises even Evil Genius self. Colonization compatibility with Bacillus altitudinis confers soybean The experiment was repeated three times independently. Elimination of seed-associated bacteria by antibiotics To assess the role of seed-associated bacteria in the seed rot resistance, soybean seeds were firstly surface-disinfested as abovementioned method, and then soaked in a solution containing ampicillin (100 μg/mL), rifampicin (50 μg/mL ... Stitch (626) Stitch, also known as Experiment 626, is an illegal genetic experiment created by Jumba Jookiba, one of the titular protagonists of the Lilo & Stitch franchise alongside Lilo, and the most prominent lead character of the franchise overall, appearing in all major media. His original primary function is to cause chaos across the galaxy by destroying everything he touches. He is designed to be ... essay homework paper phd project resume review speech thesis