In recent years, greenhouse-grown tomato (
Solanum lycopersicum
) plants showing vascular wilt and yellowing symptoms have been observed between 2015 and 2018 in North Carolina (NC) and considered as an emerging threat to profitability. In total, 38 putative isolates were collected from symptomatic tomatoes in 12 grower greenhouses and characterized to infer pathogenic and genomic diversity, and mating-type (
MAT
) idiomorphs distribution. Morphology and polymerase chain reaction (PCR) markers confirmed that all isolates were
Fusarium oxysporum
f. sp.
lycopersici
(FOL) and most of them were race 3. Virulence analysis on four different tomato cultivars revealed that virulence among isolates, resistance in tomato cultivars, and the interaction between the isolates and cultivars differed significantly (
P
< 0.001). Cultivar ‘Happy Root’ (
I-1, I-2
, and
I-3
genes for resistance) was highly resistant to FOL isolates tested. We sequenced and examined for the presence of 15 pathogenicity genes from different classes (
Fmk1
,
Fow1
,
Ftf1
,
Orx1
,
Pda1
,
PelA
,
PelD, Pep1
,
Pep2
,
eIF-3
,
Rho1
,
Scd1
,
Snf1
,
Ste12
, and
Sge1
), and 14
S
ecreted
I
n
X
ylem
(
SIX
) genes to use as genetic markers to identify and differentiate pathogenic isolates of FOL. Sequence data analysis showed that five pathogenicity genes,
Fmk1, PelA
,
Rho1
,
Sge1
, and
Ste12
were present in all isolates while
Fow1
,
Ftf1
,
Orx1
,
Peda1
,
Pep1
,
eIF-3
,
Scd1
, and
Snf1
genes were dispersed among isolates. Two genes,
Pep2
and
PelD
, were absent in all isolates. Of the 14
SIX
genes assessed,
SIX1, SIX3, SIX5, SIX6, SIX7, SIX8, SIX12
, and
SIX14
were identified in most isolates while the remaining
SIX
genes varied among isolates. All isolates harbored one of the two mating-type (
MAT-1
or
MAT-2
) idiomorphs, but not both. The
SIX4
gene was present only in race 1 isolates. Diversity assessments based on sequences of the effector SIX3
-
and the translation elongati...