TABLE 1.

Overview of several known mechanisms by which phyllosphere microbes can inhibit pathogen growth.

In vitro screening	In silico screening	Compound	Mechanism/specific activity	Identified in	References	BC	A
1.1 Antibiotic metabolites
Binary inhibitory interactions, purification and identification of compounds in supernatant	Screening for biosynthetic gene clusters using the antiSMASH tool	Antimicrobial secondary metabolites	Various	Various species	Helfrich et al., 2018	X
	srfAC, srfAD	Lipopeptide, surfactin	Triggers biofilm formation	Bacillus spp.	Chen et al., 2007; Ongena and Jacques, 2008; Dunlap et al., 2013; Kim et al., 2015		X
	fenF, mycABC	Lipopeptide, iturin	Interferes with lipid layers	Bacillus spp.	Chen et al., 2007; Ongena and Jacques, 2008; Dunlap et al., 2013; Kim et al., 2015	X
	ppsABCDE	Lipopeptide, fengycin	Interferes with lipid layers	Bacillus spp.	Chen et al., 2007; Ongena and Jacques, 2008; Dunlap et al., 2013; Kim et al., 2015	X
	phz gene cluster, ehp gene cluster	Phenazine	Interferes with histone acetylation and biofilm formation	Pseudomonas spp., Pantoea spp.	Giddens et al., 2002; Chin-A-Woeng et al., 2003	X	X
	ddaA-I	Herbicolin I		Pantoea vagans C9-1	Kamber et al., 2012	X
1.2 Hydrolytic enzymes
Zymogram, or specific colorimetric assays	chiA, chiB, chiC or other genes encoding for glycosyl hydrolases from family 18 or 19 in the CAZy database	Hydrolytic enzymes: e.g., chitinase	Hydrolyses fungal cell wall	Bacillus subtilis	Essghaier et al., 2012	X
	msp1 (p75)	Bifunctional peptidoglycan hydrolase/chitinase	Inhibits hyphae formation	Lactobacillus casei group species	Allonsius et al., 2019	X
1.3 Quorum quenching and sensing
	nis gene cluster (nisin), spa gene cluster (subtilin), luxI and luxR (AHLs)	Signalling molecules. Some gr- bacteria use bacteriocins (nisin and subtilin) that also have a signalling function	Quorum sensing	Nisin in lactococcus lactis and subtilin in Bacillus subtilis	Kleerebezem, 2004		X
Screening of bacteria interfering with the transcription of a reporter gene, induced by the signalling molecule of interest	carAB (degradation signaling molecule of Xylella fastidiosa), aiiA (AHL lactonase)	Enzymes involved in degradation signalling factors	Quorum quenching	Bacillus, Paenibacillus, Microbacterium, Staphylococcus, and Pseudomonas	Newman et al., 2008; Morohoshi et al., 2009; Alymanesh et al., 2016	X
1.4 Competition for nutrients and space
Carbon source profiling and calculation of NOI	Genes related in carbohydrate metabolism (e.g., glycosyl hydrolases), or transport (e.g., Tonb receptors), using the CAZy database	Enzymes ensuring flexible carbohydrate metabolism, e.g., high diversity of TonB receptors	Increased competitiveness in a carbon limited environment	Sphingomonas spp.	Delmotte et al., 2009	X	X
Selective media with methanol as sole carbon source	mxaF	Conserved enzyme responsible for methanol dehydrogenase	Methylotrophy, increased adaptability in a carbon limited environment	Methylobacterium spp.	Mcdonald and Murrell, 1997		X
1.5 Siderophores
Plate assay with indicator for detection of siderophores (Chrome azurol S assay)	Screening for siderophore gene clusters, using antiSMASH	Siderophores	Primary function is iron chelation. Siderophores can also have antibacterial properties trough the production of ROS and play a role in motility on the phyllosphere	Pseudomonas protegens CS1	Burbank et al., 2015; Santos Kron et al., 2020	X	X
2.1 Modulation plant hormone levels
Colorimetric assays	iac gene cluster	Enzymes responsible for the degradation of indole-3-acetic acid (IAA)	IAA is used as an energy source and modulation of IAA levels induces physiological changes in the plant	Pseudomonas putida 1290	Leveau and Gerards, 2008	X?	X
HPLC analysis of extracts of the supernatant	ipdC/aldH or dcc/aldH or iaaM/iaaH or nthA	Enzymes involved in the production of IAA, several pathways possible, described in text	Modulation of IAA levels can enhance plant growth, enhanced protection against pathogens has not been demonstrated so far	P. agglomerans	Brandl et al., 2001; Duca et al., 2014; de Souza et al., 2019	X?	X
Cultivation with 1-aminocyclopropane-1 carboxylate as nitrogen source and by measuring production α-ketobutyrate (end-product) spectrophotometrically	acdS or accD	Enzymes responsible for lowering ethylene levels	1-aminocyclopropane-1-carboxylate deaminase, modulation of ethylene levels induces physiological changes in the plant. Enhanced protection against pathogens has not been demonstrated so far	Methylobacterium spp., R. fascians	Chinnadurai et al., 2009; Francis et al., 2016	X?	X
HPLC analysis of extracts of the supernatant	fas4 or IPT	Enzymes responsible for production cytokinins	Isopentenyl transferase, modulation of cytokinins levels induces physiological changes in the plant. Enhanced protection against pathogens has not been demonstrated so far	Methylobacterium spp., R. fascians	Madhaiyan et al., 2006; Francis et al., 2016; Jorge et al., 2019	X?	X
2.2 Induced systemic response
Transcriptomics of the host plant	Creation of a MAMP database, compare between beneficial and pathogenic microbes	MAMPs that trigger an immune response, that increases protection against pathogens	Detection results in immune response	Sphingomonas melonis fr1	Ryffel et al., 2016; Vogel et al., 2016	X	X
Transcriptomics of the host plant	Creation of an effector database, screening for type III secretion system gene clusters	effectors that trigger an immune response, that increases protection against pathogens	Detection results in immune response	Pseudomonas spp., Parabulkholderia sp.	Stringlis et al., 2019; Herpell et al., 2020	X	X

The table includes (i) information on in vitro assays to test for the presence of these mechanisms, (ii) known genes involved in these mechanisms (in silico screening), (iii) the compound and (iv) the mechanism resulting in antipathogenic activity, (v) microbes in which the mechanism has been identified, (vi) references and the last two columns indicate whether the mechanism is (vii) a biocontrol factor (BC) and/or (viii) an adaptation factor (A). The screening methods, strains and references are not exhaustive but rather examples, which are also discussed in the text. The table follows the same structure as the review.