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Sounds like a slight error caused by the upscaling algorithm.  Upscaling is done by the program making a guess to fill in the new pixels created by the larger size, and sometimes the guess isn't great.

There's a few different things you can try to get around it, but I'm not sure which one would be better for your particular problem.  You can try a different program, a different upscaling algorithm, upscaling by only one tile at a time, taking out the error and filling in the gap by copying a pixel from the side or another upscaling attempt, or just drawing it in with your own best guess.

I'm not able to make an upscale of my own right now, but I'll try to remember to make one in a few weeks when I will hopefully be free.

Thank you for your reply, I tried a different program and its still doing it. I've noticed it does it with all the trees on A2 most specifically. Is there any way I can show you a screenshot of it to make sure that I'm explaining the problem correctly? I appreciate your time, Id really like to get this fixed as this is perfect for the game i'm working on and is very unique.

Thanks again

Got it to work!, changed the upsacaling to from cubic or bicubic to none and worked, thank you very much for your time!

That makes sense.  "None" would preserve the transparency.  I don't remember the layout, but I suspect that it was taking information from either over the edge of the document (which on some programs equals black), or there was bleed over from the neighboring tiles.

The only problem with "none" is that it can make things look pixelated, since it's adding in the exact same color blocks instead of guessing at in-betweens, but the original art is a touch on the blurry side, so it's probably fine.  I'm glad you got it working!