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When escorting Princess Peach to Little Fungitown, she is attacked by Trunkle, and the bros must save her. In battle, Trunkle has the ability to throw boulders at the brothers, which have to be countered with Hammer strikes. Trunkle will use his right arm to target Mario and left arm to target Luigi if the boulders simply roll straight. If the boulders also spin sideways while rolling, then they will target the opposite brother. Trunkle also has the ability to vacuum objects into his mouth that will pass the brothers along the way; these include Gritty Goombas, poisonous mushrooms, and standard Mushrooms. Any of these objects can be dodged by jumping, though touching the Mushrooms will heal the brother that comes into contact with one. After finishing this attack, Trunkle will heal approximately 10 HP. When Trunkle's HP is reduced by half, he splits into four tiny Trunkles. One of them has a berry in its mouth, and Mario and Luigi have to keep an eye on that one while all four shuffle around. A Mini-Trunkle can attack by simply rolling into one of the brothers, or burrowing underground and popping up in front of him, attempting to strike him afterwards. The former attack can be countered by jumping, whereas the latter can be countered by hammering. When the other Mini-Trunkles are attacked, they will simply crumble, although the one with the berry will continue to lose HP until it is finally defeated, to the brothers' victory. However, should the brothers fail to do this in a timely manner, the Mini-Trunkles will merge into Trunkle again, with full HP and the whole process restarts. Alternatively, if Mario and Luigi attack Trunkle's "tree" on top of his head, they can defeat him without him splitting into four tiny Trunkles. His "tree" has much lower HP and higher defense. It is also extremely resistant to jump attacks and will only take more damage from regular Chopper Bros. attacks and Knockback Bros. attacks. Defeating Trunkle by using the former method is more challenging and rewarding; defeating him by using the latter method is easier, but less rewarding.
If the brothers follow the path to Little Fungitown before Princess Peach comes to the Beanbean Kingdom, Trunkle is still blocking the path, but is sleeping and cannot be woken.
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