to ranks below species and subtypes, respec-
tively (data S10). Strains of G1, G4, G7 (three
species-level groups), and G8 (three species-
level groups) of BV1 as well as BV2 were di-
vided into 124 unique genotypes. Type I–III
Ti plasmids were divided into 40 distinct plas-
mid clusters (data S11 to S19).
Multiple patterns underscore aspects of ag-
ricultural ecosystems that promote the diver-
sification and spread of pathogens (Fig. 6 and
fig. S25). Nonpathogenic, plasmid-lacking strains
were isolated from healthy and diseased in-
dividuals (data S12, S15, S16, and S17). For
example, three nonpathogenic strains were
cultured from symptomatic plants at facility
C7_N18, a location that also had pathogenic
strains, differing by ~130,000 to ~290,000 SNPs
(Fig. 6A and data S17). As potential recipients
of virulence plasmids, nonpathogenic strains,
which are common in soils and in association
with plants, represent a standing pool of ge-
netic diversity for the evolution of new path-
ogen genotypes. Strain-plasmid combinations
can persist over the long term within agricul-
tural ecosystems. Strains LMG 267 (type II
Ti) and B140/95 (type II Ti) were collected
60 years apart yet are members of the same
genotype and plasmid cluster, with 7 and 0 SNP
differences, respectively (Fig. 6B and fig. S25B).
Pathogens can be transmitted between agri-
cultural and unmanaged ecosystems. Three
members of a BV2 genotype (type I.a Ti) linked
a natural ecosystem, an agricultural ecosystem,
and an undocumented location in a different
country (Fig. 6C and fig. S25C). The three
strains and their Ti plasmids have≤5and
0 SNP differences, respectively, among them.
S6_N30 and S6_N25 are almost 240 km apart,
but two other plant production facilities are
located within 1.5 km of S6_N30 and could
have bridged the link to S6_N25.
Agricultural ecosystems can have recur-
rent infections. Locations in which different
genotype-plasmid combinations were detected
had likely experienced independent infections.
Facility S2_N7 had eight strains representing
seven different genotype-plasmid combina-
tions (Fig. 6D and fig. S25, I to M). Over a 12-year
span, S8_N32 was infected by G1 (type III Ti)
strains that have >50,000 and >350 SNP differ-
ences among strains and plasmids, respectively
(fig. S25, N to P). An agricultural ecosystem
can also have disease reservoirs; this idea is
supported by the presence of multiple strains
of the same genotype-plasmid combination on
different individuals. Location C7_N18 had
27 strains of the same G7 genotype (type III
Ti) on four individuals of three different host
species (Fig. 6A and fig. S25Q).
There were at least seven cases in which
global distribution of plants is hypothesized to
have contributed to the transmission of a strain-
plasmid combination. These are patterns in
which genotype and plasmid nodes are con-
nected by multiple edges. Patterns by themselves
are insufficient for differentiating between di-
rect and indirect transmission routes. How-
ever, one case is highlighted that includes a
facility that produces plants for wholesalers
and could be a common source. Strains belong-
ing to a G1 genotype (type III Ti) were identi-
fied from facility C4_N13 (Fig. 6E and fig. S25E).
Strains of the same genotype-plasmid combina-
tion were later identified in two other facilities.
The strains and plasmids from these three loca-tions have≤10 and 0 SNP differences, respec-
tively, among them. The dataset has other
instances in which sets of closely related strains
marginally exceeding the >15 SNP difference
threshold have plasmids that belong to the
same cluster. It is thus possible that there
were more strain-plasmid transmissions than
reported.
Horizontal transmission of plasmids greatly
diversifies and amplifies the spread of patho-
gens. Fifteen networks have a plasmid clusterWeisberget al.,Science 368 , eaba5256 (2020) 5 June 2020 6of8
Fig. 5. Model of the evolution of oncogenic Ti and Ri plasmids.Genes in boxes were acquired from
unknown sources. The cluster oftzs,vir[mas] genes is predicted to have been acquired once (dotted arrows)
and then transferred from one plasmid backbone to the other (solid arrows). Genes in purple were acquired
horizontally from the indicated sources. Purple arrows represent major horizontal acquisition events.
Circle with“X”depicts an undefined plasmid hypothesized to be the donor of the prominent T-DNAs that
swept through the type I–IV Ti plasmids.Fig. 6. Spatiotemporal transmission of strains and plasmids.(AtoH) Patterns revealed in undirected
networks combining strain genotypes and Ti plasmid clusters (fig. S25). Key: gray square, agricultural ecosystem;
large circle, species-level group; small circle, type or subtype of Ti plasmid. The location is labeled with a
coded identifier (top); strain name(s) (left or right side); date isolated (bottom; unk, unknown). Double-
headed arrows link locations and show approximate distance (d, kilometers) and/or time (t, years). The large
circle without a small circle in (A) represents a nonpathogenic strain that lacks an oncogenic plasmid.RESEARCH | RESEARCH ARTICLE