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