Hi Daudi, we are doing a research on plant-microbe interactions,...

Hi Daudi,
Thanks so much for your reply. It's quite a relief to have some professional guidance.
Just three more questions:
(1) if it's still hard to fill the whole leaf with one-time infiltration after the spray, should we just leave it be and use disks near the infiltration site for emumeration, or infiltrate twice to fill the whole leaf?

(2) Since Pst cfu of the disks from near the infiltration site is significantly higher than that of disks from the other half, we wonder where these disks for enumeration are usually excised from. Is it necessary to excise from both halves of the leaves, or just about 0.5cm2 from near the infiltrate site would be fine?

(3) We notice a "n=6~8" rule in a lot of pathology protocols. Does it mean 6~8 leaves or 6~8 plants? We've been following the rule of "6-8 plants per treatment(i.e. 12-16 per genotype), 2-3 similar leaves per plant, disks from 2 plants (i.e 4-6 leaves) pooled together for a tissue sample and 3 samples for a enumeration data point", and we found it very time-consuming when a experiment involves more than 5 genotypes. Is there any alternative designs for such a experiment?

Just one infiltration per leaf should be fine if you control the experiment, so if you have an adequate number of individual leaves that you test, perhaps 3 plants and 3 leaves each or something designed along those lines. Keeping mocks a little distance away like at least 1 foot is probably a good idea too to be safe but we did not find this essential, other than making sure they're not in physical contact with one another - but yes perhaps for volatiles it is safe to keep away

Thanks for your question! What we found was if you sprayed the abaxial surface of the leaf with water (just a regular gentle water sprayer) it assisted in the update of liquid during syringe infiltration.