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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!