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\begin{document}

\title{Tar Spot of Maize: Current knowledge of genetic interactions and future
research prospects to improve disease resistance}



\author[1]{Matthew Helm}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 22, 2024}


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Matthew Helm,\textsuperscript{1*} Raksha
Singh,\textsuperscript{1}Stephen B. Goodwin,\textsuperscript{1} Denise
Caldwell,\textsuperscript{2} and Anjali S.
Iyer-Pascuzzi\textsuperscript{2}

\textsuperscript{1} Crop Production and Pest Control Research Unit, U.S.
Department of Agriculture-Agricultural Research Service (USDA-ARS), West
Lafayette, IN 47907-2054, U.S.A.

\textsuperscript{2} Department of Botany and Plant Pathology, Purdue
University, West Lafayette, IN 47907-2054, U.S.A.

\textbf{* Corresponding author} : M. Helm; Email: Matthew.Helm@usda.gov

Funding: This work was supported by the United States Department of
Agriculture, Agriculture Research Service (USDA-ARS) research project
5020-21220-019-00D.

\par\null

\emph{Abstract}

The emergence and spread of crop plant pathogens pose a significant risk
to global food and nutritional security. Of particular concern are those
fungal pathogens that can significantly reduce yields of staple crop
plants such as maize (\emph{Zea mays} subsp. \emph{mays} ). One such
plant pathogen is \emph{Phyllachora maydis} Maubl (\emph{P. maydis} ),
which causes tar spot disease on maize. Though it was only recently
identified within the United States, \emph{P. maydis} is now considered
one of the most economically important foliar diseases of maize and has
since spread to most maize production areas within the United States and
Canada, resulting in substantial yield losses. In this mini-review, we
aim to summarize recent advancements in our understanding of the genetic
interactions between maize and \emph{P. maydis} whilst also highlighting
future research prospects for developing genetic-based resistances.

Keywords: Maize, \emph{Phyllachora maydis} , tar spot, disease
resistance

\par\null

\emph{Introduction}

Maize (\emph{Zea mays} subsp. \emph{mays} ) is one of the most widely
cultivated cereal grains and serves as a significant source of calories
for human and animal consumption. Though there are a multitude of
threats to global maize production, substantial yield losses caused by
diseases significantly challenge our ability to achieve global food and
nutritional security sustainably and impose significant financial
constraints to growers (Savary et al., 2019). Of particular concern are
fungal pathogens capable of causing global pandemics and significantly
reducing maize yields (Savary et al., 2019). One such fungal pathogen is
the ascomycete \emph{Phyllachora maydis} Maubl (\emph{P. maydis} ), a
foliar pathogen of maize that causes tar spot disease (Ruhl et al.,
2016; Figure 1A-B). First documented in Mexico in 1904, \emph{P.
maydis}is endemic to Central and South America where it has the
potential to significantly reduce grain yields, especially under
favorable environmental conditions (Maublanc 1904; Valle-Torres et al.,
2020). Though \emph{P. maydis} is often associated with tropical maize
production areas, it emerged in Illinois and Indiana during 2015 and has
since spread to several other maize-growing areas within the United
States and Canada (Rocco da Silva et al., 2021; Ruhl et al., 2016;
Valle-Torres et al., 2020). Such rapid emergence and spread of \emph{P.
maydis} across varying geographical climates strongly suggests
commercial maize hybrids in the United States are susceptible to this
fungal pathogen. To this end, Telenko et al. (2019) evaluated a subset
of commercially grown maize hybrids for their responses to \emph{P.
maydis} and showed that all hybrids tested were susceptible. Notably, a
severe tar spot epidemic within the United States in 2018 resulted in an
estimated \textasciitilde{}\$840 million USD economic loss,
demonstrating \emph{P. maydis} is capable of imposing severe financial
constraints to growers (Crop Protection Network, 2021). The current lack
of durable, genetic-based resistance coupled with its potential to
significantly reduce maize yields makes \emph{P. maydis} one of the most
economically important foliar diseases of maize (Mueller et al., 2020).
Hence, future research should, in part, investigate the genetic
mechanisms underlying maize--\emph{P. maydis} interactions to improve
tar spot resistance.

\textbf{Population screens and linkage mapping have contributed to our
understanding of the maize--\emph{P. maydis} pathosystem}

Our general knowledge regarding \emph{P. maydis} resistance in
temperate-derived maize remains limited given its recent emergence
within the continental United States and Canada. However, sources of
resistance to \emph{P. maydis} have been identified in diverse maize
germplasm collections. Mahuku et al. (2016) and Cao et al. (2017)
performed Genome-Wide Association Studies (GWAS) on diverse mapping
populations and identified a large-effect Quantitative Trait Locus (QTL)
on chromosome 8 (bin 8.03), referred to as \emph{qRtsc8-1} .
Fine-mapping of this locus revealed two candidate genes GRMZM2G063511
and GRMZM2G073884, encoding an integral membrane protein and a putative
leucine-rich repeat receptor-like protein, respectively, as likely
having a functional role in \emph{P. maydis} recognition (Ren et al.,
2022). It is worth noting that integral membrane proteins and
leucine-rich repeat receptor-like proteins are known to have a
functional role in Surface Receptor-Mediated Immunity (SRMI) against
several plant pathogens (Ding et al., 2021; Yadeta et al., 2013). It
remains unclear, however, what pathogen-derived proteins are recognized
by these host proteins and how their activation induces resistance
to\emph{P. maydis} .

To identify new allelic diversity against \emph{P. maydis} , Lipps et
al. (2022) screened several maize accessions developed by the Germplasm
Enhancement of Maize (GEM) project. Though no line showed complete
resistance, two accessions, GEMS-0066 and GEMS-0226, consistently
displayed partial resistance to \emph{P. maydis} across multiple
geographical locations. These lines may thus serve as important sources
of new resistance specificities (Lipps et al., 2022). Furthermore, 200
GEM accessions and a subset of 600 inbred lines from the Wisconsin
Diversity Panel-942 (WiDiv-942) were evaluated from their responses
to\emph{P. maydis} (A. Thompson, \emph{personal communication} ). GWAS
analysis revealed more than 100 Single Nucleotide Polymorphisms (SNPs)
were associated with \emph{P. maydis} tolerance (A.
Thompson,\emph{personal communication} ). Intriguingly, several of the
identified SNPs were linked to defense-related genes and may thus be
important for\emph{P. maydis} resistance (A. Thompson, \emph{personal
communication} ).

The maize Nested Association Mapping (NAM) recombinant inbred lines
(RILs) representing global maize diversity were derived from 26 inbred
founder lines and are a valuable genetic resource that may contain
additional allelic diversity against \emph{P. maydis} (Gage et al.,
2020). Indeed, the 26 inbred NAM founders were recently evaluated for
their responses to \emph{P. maydis} with the goal of identifying inbreds
that are either resistant or susceptible to this fungal pathogen (R.
Singh, \emph{personal communication} ). Though all inbred lines
evaluated were susceptible, there were significant variations in their
responses (Figure 1C). Nine inbred lines were scored as tolerant without
complete resistance, six as moderately tolerant, two moderately
susceptible, and the remaining eight were susceptible (R. Singh,
\emph{personal communication} ). Intriguingly, most of the
CIMMYT-derived maize lines (CMLs) were evaluated as tolerant or
moderately tolerant. In contrast, the North American-derived lines,
including B73, displayed varying levels of susceptibility but most
inbreds were susceptible (R. Singh,\emph{personal communication} ).
Variations in susceptibility of the 26 founder lines to \emph{P. maydis}
could help guide the selection of appropriate NAM mapping populations
for future genetic studies. Future research efforts should thus focus on
fine mapping the genetic loci responsible for \emph{P. maydis}
resistance via GWAS and/or SNP linkage map analysis. Collectively, these
studies demonstrate that diverse maize germplasm collections harbor
genetic-based disease resistances to\emph{P. maydis} and have been
instrumental in our initial understanding of the genetic architecture
underlying resistance to \emph{P. maydis} .

\emph{Research prospects to improve disease resistance to P. maydis}

The data thus far suggest \emph{P. maydis} can cause significant yield
losses in maize and should thus be considered a serious concern for
maize growers. We, therefore, would like to propose high-priority
research avenues likely to advance our fundamental understanding of the
maize--\emph{P. maydis} pathosystem.

\textbf{Screen additional maize germplasm to identify new sources of
resistance to \emph{P. maydis~}}

Immediate efforts should focus, in part, on evaluating additional maize
germplasm collections for resistance to \emph{P. maydis} . In addition
to screening the aforementioned natural germplasm collections,
artificially generated germplasms also may serve as a useful resource
for identifying new sources of resistance to \emph{P. maydis} (Figure
2A). Ethyl methanesulfonate (EMS)-mediated mutagenesis of pollen grains
is often used to generate mutagenic populations of maize that can then
be subsequently used to screen for specific phenotypes (Candela and
Hake, 2008). For example, Marla et al. (2018) used an EMS-mutagenized
population of maize to generate and functionally characterize novel
mutant alleles of the \emph{Hm1} resistance gene. It is thus likely a
similar chemical mutagenesis-based strategy could be deployed to
identify new sources of genetic resistance against multiple \emph{P.
maydis} isolates. One advantage of screening an EMS-mutagenized
population is the likelihood of identifying susceptibility genes, which
are host proteins required by the pathogen to facilitate infection
(Garcia-Ruiz et al., 2021; Langner et al., 2018). For this reason,
inactivation of host susceptibility genes often compromises pathogen
infection, thereby conferring resistance (Garcia-Ruiz et al., 2021;
Langner et al., 2018). Therefore, screening an EMS-mutagenized
population of maize for loss of susceptibility to \emph{P. maydis} may
be an attractive approach for identifying new resistance loci.

\textbf{Investigate the infection strategy of \emph{P. maydis} using
high-resolution microscopy}

In parallel to screening natural and artificial maize populations for
new resistance alleles, additional studies should also aim to
investigate the infection strategy used by \emph{P. maydis} at the
individual plant scale (Figure 2B). Currently, \emph{P. maydis} is
presumed an obligate biotrophic fungal pathogen as it likely requires
photosynthetically active tissue for survival and is unable to be
cultured in the laboratory (Rocco da Silva et al., 2021; Telenko et al.,
2020). However, \emph{P. maydis} has also been shown to overwinter on
maize debris in the United States (Kleczewski et al., 2019b; Groves et
al., 2020), suggesting it can survive as a saprophyte and may exhibit a
bi-phasic lifestyle. Hence, \emph{P. maydis} may be considered a
hemibiotroph. Furthermore, our current understanding regarding the
mechanisms underlying initial host infection and systemic spread is
limited but is urgently needed to understand host immune mechanisms.
High-resolution microscopy, in which the host and associated microbe are
clearly observed at cell-type resolution (Caldwell et al., 2019), offers
the potential to help elucidate the pathogen lifestyle and infection
strategy of \emph{P. maydis} within the host plant. For example,
confocal microscopy revealed that \emph{Fusarium oxysporum} f.
sp.\emph{lycopersici} first infects tomato root hairs and colonizes
junctions between root epidermal cells (Lagopodi et al. 2002). Confocal
microscopy also revealed differences in the root infection strategy
between highly and weakly virulent strains of \emph{Fusarium
oxysporum}f. sp. \emph{phaseoli} in susceptible common bean
(Nino-Sanchez et al. 2015). The highly virulent strain reached the root
xylem faster than the weakly virulent strain, which predominantly
colonized the root cortex (Nino-Sanchez et al. 2015). Microscopy has
also revealed differences in\emph{Fusarium} colonization between
resistant and susceptible cultivars in several species, including banana
(Chen et al. 2019) and chickpea (Jimenez-Fernandez et al. 2013). Using
such microscopy methods in plants with differing responses to \emph{P.
maydis} will help us elucidate whether this fungal pathogen is
biotrophic or hemibiotrophic as well as shed light on the underlying
resistance mechanisms in maize.

\emph{P. maydis} \textbf{genomic surveillance and isolate profiling
using a field pathogenomics strategy}

A detailed understanding of the current genetic diversity and population
structure of \emph{P. maydis} populations is limited. Such information
is critical for monitoring \emph{P. maydis} outbreaks and for the
emergence of new \emph{P. maydis} populations with potential resistance
to fungicides. To this end, Broders et al. (2021) assessed the sequence
diversity of \emph{P. maydis} collected from 186 single stroma derived
from 16 hosts in an effort to initially understand the genetic diversity
within \emph{P. maydis} populations. Intriguingly, sequencing of the
internal transcribed spacer (ITS) region from these samples revealed
that there are likely three genetically distinct groups of \emph{P.
maydis} in the United States capable of infecting maize (Broders et al.,
2021). However, the phylogenetic analysis performed was based only on
sequencing of the ITS region and historical representatives from
herbaria and may not be representative of current populations.
Alternatively, whole transcriptome sequencing of \emph{P. maydis}
-infected tissue collected directly from the field offers a more
thorough and detailed understanding of host-pathogen interactions
(Figure 2C). This approach, termed field pathogenomics, presents a
detailed analysis of the population structure and genetic diversity
of\emph{P. maydis} as well as informative gene expression data for both
the host and pathogen. Field pathogenomics has been used to identify new
lineages of wheat yellow rust populations in the United Kingdom (Hubbard
et al., 2015) and the origins of wheat blast in Bangladesh (Islam et
al., 2016; Kamoun et al., 2019). For these reasons, the field
pathogenomics strategy may be an attractive approach to actively
monitor\emph{P. maydis} populations for the emergence of novel isolates.

\textbf{Characterize \emph{P. maydis} virulence (effector) proteins and
their host targets in maize}

Virulence proteins translocated by fungal pathogens often have a
functional role in promoting plant disease either by suppressing or
activating host immune responses and, therefore, can serve as important
resources for introducing novel host resistance specificities (Figueroa
et al., 2021). Though \emph{P. maydis} is predicted to secrete effector
proteins (Telenko et al., 2020), our general understanding of
how\emph{P. maydis} utilizes its effector repertoire for virulence as
well as their host targets in maize remain unknown. Such studies are
complicated by the observation that \emph{P. maydis} is genetically
intractable and culturing of this pathogen has not been successful
(Rocco da Silva et al., 2021; Telenko et al., 2020). Hence, genetic
manipulation of the fungal pathogen is not yet possible. Furthermore,
controlled inoculation protocols using \emph{P. maydis} have yet to be
developed, thus greenhouse-based disease assays aimed at evaluating host
responses to \emph{P. maydis} cannot be performed. Despite these
limitations, screening the effector repertoire of \emph{P. maydis} can
be performed through the use of heterologous expression systems in
non-host plants such as \emph{Arabidopsis thaliana} and \emph{Nicotiana
benthamiana} (Figueroa et al., 2021; Lorrain et al., 2018). This
strategy has been effective at revealing the specific plant cell
compartments targeted by effectors from other obligate biotrophic fungi
as well as their host protein interactors (Figueroa et al., 2021;
Lorrain et al., 2018). Future work should thus aim to exploit the use of
heterologous plant systems to investigate the subcellular compartments
targeted by \emph{P. maydis} effector proteins as well as identify their
host targets in maize (Figure 2D). Such information is likely to advance
our understanding of the molecular interactions between \emph{P.
maydis}and host as well as provide initial insight into the
pathogenicity mechanisms used by \emph{P. maydis} to promote disease.

\emph{Concluding remarks}

The rapid emergence and spread of \emph{P. maydis} within the United
States poses a significant threat to maize production. Most commercial
U.S. maize hybrids are susceptible to \emph{P. maydis} , and naturally
occurring resistance genes have yet to be identified and functionally
characterized. For these reasons, we aspire to synergize collaborations
among multidisciplinary research experts in an attempt to: 1) evaluate
additional germplasm collections for \emph{P. maydis} resistance; 2)
determine the lifestyle and host infection strategy of this fungal
pathogen; 3) assess its population~diversity; and 4) characterize
the\emph{P. maydis} effector repertoire and identify their host targets
in maize (Figure 2). Such multidisciplinary research collaborations will
likely accelerate the development and deployment of maize hybrids with
genetic-based disease resistance to \emph{P. maydis} .

\textbf{Author Contributions}

We sincerely apologize to colleagues whose works we were not able to
cite due to space limitations. MH conceived this minireview. MH produced
the first draft, and all authors critically revised the manuscript. All
authors approved the final version of the manuscript.

\textbf{Funding}

This research was funded, in part, by the United States Department of
Agriculture, Agriculture Research Service (USDA-ARS) research project
5020-21220-019-00D. The funding body had no role in designing the
experiments, collecting the data, or writing the manuscript. Mention of
trade names or commercial products in this publication is solely for the
purpose of providing specific information and does not imply
recommendation or endorsements by the United States Department of
Agriculture. USDA is an equal opportunity provider and employer.

\textbf{Acknowledgements}

We thank Dr. Morgan E. Carter (University of Arizona) for critical
reading of this manuscript and helpful comments. The authors also thank
Dr. Gurmukh Johal (Purdue University) for providing photographs
of\emph{P. maydis} -infected leaf tissue.

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\textbf{Figure Legends}

\textbf{Figure 1. \emph{Phyllachora maydis} disease symptoms on
maize.A,} Tar spot symptoms on foliage and husk leaves. Severe\emph{P.
maydis} infection often leads to premature senescence and blighting.
\textbf{B,} Cross section of \emph{P. maydis} -infected leaf tissue
depicting a single perithecium with ascospores.
\textbf{C,}Representative leaves from four maize accessions phenotyped
as tolerant and susceptible to \emph{P. maydis} .

\textbf{Figure 2. Proposed research strategies to improve disease
resistance to \emph{P. maydis}.} \textbf{A,} Screening of additional
maize germplasms may lead to the identification of new sources of
resistance. \textbf{B,} High-resolution microscopy will help shed light
on the pathogen lifestyle and infection strategy.
\textbf{C,}Whole-transcriptome sequencing of \emph{P. maydis} -infected
tissue may be an effective approach to monitor pathogen populations for
the emergence of novel isolates. \textbf{D,} Functionally characterizing
the\emph{P. maydis} effector repertoire and identifying their host
targets in maize will help elucidate the virulence mechanisms that
promote disease. Figure created with\emph{www.BioRender.com}.

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