Abstract:
The genus Phyllachora contains numerous obligate fungal parasites
that produce raised, melanized structures called stromata on their plant
hosts. Most members of this genus are not of significant economic
concern, with the exception of P. maydis , the causal agent of tar
spot of maize (Zea mays ). Tar spot of maize has emerged as a
major threat to maize production throughout the Americas and continues
to spread throughout North America. To date, species designations forPhyllachora have been based on host associations and morphology,
and the origin and diversity of the pathogen that causes tar spot is
unknown. We assessed the sequence diversity of 186 single stroma
isolates collected from 16 hosts representing 15 countries by
amplification of the ITS and LSU gene regions. Samples included both
herbarium and contemporary strains that covered a temporal range from
1905-2019. These 186 isolates were grouped into 5 distinct species with
strong bootstrap support. We found three closely related, but
genetically distinct groups of Phyllachora are capable of
infecting maize in the United States, we refer to these as the P.
maydis species complex. Based on herbarium species, we hypothesize that
these three groups in the P. maydis species complex originated
from Central America, Mexico and the Caribbean. Although two of these
groups were only found on maize, the third and largest group contained
contemporary strains found on maize and other grass hosts, as well as
herbarium specimens from maize and other grasses that include 10 species
of Phyllachora . The herbarium specimens were identified based on
morphology and host association, but our data indicates there may be
significant synonymy in the Phyllachora genus and additional work
on species delineation and host specificity should be considered.
Introduction
Phyllachorales is a monophyletic order of biotrophic fungi comprised of
approximately 1,226 recognized species (Maharachchikumbura et al. 2016,
Mardones et al. 2017), but global estimates of species within this order
approach 160,000 (Cannon 1997). The Phyllachorales largely contain plant
parasitic fungi and are commonly associated with monocotyledonous plants
across a range of habitats. These fungi are often referred to as “tar
spot” fungi due to the production of stromata on plant hosts that
resemble black flecks of tar (Fig 1) (Mardones et al. 2017).
Tar spot of maize (Figure 1 A-C), caused by the fungus Phyllachora
maydis , emerged in the United States (U.S.) in 2015, with the disease
expanding each year since the initial report and continuing to have a
significant economic impact on maize across many production regions in
the U.S. (Kleczewski et al. 2020a, Valle-Torres et al. 2020). Since
first being identified in North America in 2015, P. maydis has
spread rapidly throughout the U.S. and Canada (Kleczewski and Bowman
2020, Kleczewski et al. 2020), and resulted in yield losses exceeding
$US 658 million in 2018 (Mueller et al. 2020). Phyllachora
maydis caused significant losses again in 2021, appearing to be on par
or greater than 2018 levels (authors personal observations). Although
tar spot symptoms caused by members of the genus Phyllachora have
been commonly observed on a number of grasses (Figure 1 D-F) and shrub
species throughout North, Central and South America, historically the
fungus has rarely been known to cause significant plant damage. However,
tar spot has been occasionally reported to cause severe damage to maize
in Mexico, Central America and several Caribbean Islands (Valle-Torres
et al. 2020).
The origin of P. maydis within the U.S. is not currently known,
although the presence of two distinct epicenters of maize tar spot in
the Midwest and Southeast indicates at least two separate emergence
events. While tar spot is a new disease on maize in the U.S. and Canada,
it has been present in Mexico, several Caribbean islands including
Puerto Rico, Cuba and the Dominican Republic as well as Central American
Countries, such as Guatemala, Honduras, Nicaragua and Costa Rica for the
last century but only caused limited damage. In addition, tar spot signs
and symptoms caused by Phyllachora species are common on several
native and weedy grass species in North America (Figure 1 D-F) (Orton
1944). The monographic work by Orton (1944) was completed solely by
morphological identification and host affinity. Given our understanding
of phenotypic plasticity of many fungi and the ability of biotrophic
pathogens to infect multiple hosts (Morris and Moury 2019), it is
possible that cryptic species or species complexes may be present.
Species definitions within the Phyllachorales have historically been
based largely on morphological characteristics and assumption of high
host specificity, due to their presumed biotrophic nature. However,
there are examples in the genus where this assumption of host
specificity does not hold true (Cannon 1991, Cannon 1997). Furthermore,
species designations based on host specificity are highly dependent on
accurate identification of the host species, which may be difficult or
impossible in some instances. For example, P. graminis (Pers.)
Fuckel is considered a “dustbin” species where many specimens of
isolates infecting grasses are deposited with the host not often
identified to species (Parbery 1967). Furthermore, factors such as
nutrients available to the fungus, temperature, light quality, light
cycles, substrate type, host, and epigenetic factors may also result in
alterations in fungal morphology that may result in inaccurate species
designations (Slepecky and Starmer 2009, Stockinger et al. 2009, Money
2013, Francisco et al. 2019). Thus, our current understanding of the
genetic diversity, host range and species delimitation within the genusPhyllachora is relatively limited and requires reevaluation.
The recent emergence of P. maydis in the U.S. and Canada may also
be associated with the ability of the fungus to better persist and
spread than previously thought. Once established, the fungus can survive
at least one winter at subzero temperatures on corn residue as
ascospores within stromata, which are believed to be the main inoculum
source the following season (Kleczewski et al. 2019, Groves et al.
2020). Under periods of moderate temperatures and wet weather it is
believed that ascospores are dispersed by wind and rain-splash where
they land on the foliage, stalks, and husks of corn. After spore
germination and infection of the host, the fungus remains dormant for at
least 2 weeks after which stromata, and associated spermatia and
ascospores, are produced. Data from Central America indicated a
relatively steep dispersal curve of P. maydis ascospores from a
source (Hock et al. 1995). However, the rapid spread of this fungus
throughout the Midwest, coupled with observations of “top down”
infestations in fields with no history of disease and observations of
infestations of isolated plots located 1,200 m from potential inoculum
sources, indicate that the pathogen can travel much further across
local/regional topographies than estimated previously (Kleczewski et al.
2020).
Based on this information, the emergence of P. maydis on corn in
the U.S. and Canada could have been the result of many factors including
the introduction of the fungus on infected plant material, natural
northern dispersal through wind, establishment in the U.S. favored by
climate change, changes in hybrid genetics, a host-jump from a grass
species, or a combination of any of these four. In this study we use DNA
sequence data to understand the genetic diversity of P. maydispopulations in contemporary maize production regions in the U.S., and
compare this to historical specimens of P. maydis from herbarium
samples from Mexico, Central and South America, the Caribbean, and
contemporary and herbarium species of Phyllachora species
associated with grass hosts in the U.S. The goal of this study is to
understand the genetic diversity and population dynamics within theP. maydis population infecting corn in North America. Differences
in genetic backgrounds may imply dissimilar biology, and potentially
interactions with the host. Furthermore, understanding the overall
phylogenetic diversity and the potential host and geographic range ofPhyllachora populations associated with maize and other grasses
in the Americas will help to infer the potential evolutionary origins
and speciation patterns in this genus.
MATERIAL AND METHODS