Rice (Oryza sativa L.) is one of the most important crops with a worldwide production of 509 million metric tons annually (FAOSTAT, 2019). Rice provides a major source of energy for more than half of the world population (FAOSTAT, 2019). In 2019, the US rice production was estimated to be 10 million metric tons. Arkansas, California, Louisiana, Mississippi, Missouri, and Texas are the major rice producers in the US (USDA, 2020). Rice kernel smut, caused by Tilletia horrida Tak., causes partial or full bunt in rice grains, resulting in a direct reduction in grain yield and quality (Whitney and Cartwright, 2018). Rice kernel smut was first reported in 1896 in Japan (Takahashi, 1896); currently, rice kernel smut is widespread across rice-growing countries and its distribution is expected to be wider than recently reported (Carris et al., 2006).

Average losses from rice kernel smut have been reported around 15%; however, losses as high as 87 and 100% in Pakistan and China have been reported (Biswas, 2003). Major losses from kernel smut are due to the depletion in grain quality with countries restricting the maximum permissible limit for the smutted grains. Milled rice in the US has a restriction of 3% smutted rice (USDA, 2020). Similarly, certified rice seeds in India have a restriction of 0.5% smutted rice grains (Chahal, 2001). Historically kernel smut was considered a minor disease; however, persistent occurrence and frequent outbreaks of the disease in recent years have made kernel smut as one of the most economically important diseases in rice in many countries (Elshafey, 2018; Wang et al., 2019b; Allen et al., 2020; Zhou et al., 2020). In the US, kernel smut occurrence and severity have been on the rise for the past decade and pose a serious threat to the US rice production (Espino, 2019, 2020; Allen et al., 2020; Way and Zhou, 2020). In 2021, severe outbreaks of kernel smut occurred widely across the Texas rice areas and southwest Louisiana, with the percentage of affected panicles ranged up to 50% and the infected kernels ranged up to 20 percent (Zhou et al., 2021a). In states such as Arkansas and California where rice industry is valued as billion-dollar industry, potential economic losses are even higher (USDA, 2020). With continual increase in acreage compounded with the use of susceptible cultivars, kernel smut has also threatened organic rice production in California and Texas, the two leading states in the US organic rice production (Zhou et al., 2021b).

Rice kernel smut is caused by a basidiomycota fungus, belonging to Tilletia genus and Tilletiaceae family. More than 80 genera and 4,200 species of smut fungi have been reported as the pathogens to many plant species (Vánky, 1987). Phylogenetically Tilletia species have been considered to separate their lines from those of other smut fungi, Ustilago and Sporisorium (Roux et al., 1998). Tilletia horrida forms thick walled dark teliospores which can be present widely on the soil, plant debris, and rice seeds (Carris et al., 2006; Whitney and Cartwright, 2018). Kernel smut taxonomy has been turbulent through the years of many studies. Tilletia horrida Tak., was first described by Takahashi in 1896; however, over the years, various authors reclassified the fungus to different genus and species: T. barclayana (Bref.) Sacc. & Syd., Neovossia barclayana (Bref.), and Neovossia horrida (Tak.; Tullis and Johnson, 1952). Through the years, T. barclayana and T. horrida have been interchangeably used to describe kernel smut of rice. However, a distinction between T. horrida and T. barclayana has been demonstrated by various molecular and phylogenetic studies (Levy et al., 2001; Carris et al., 2006). Currently, T. horrida has been more commonly used to describe kernel smut of rice in the literature (Wang et al., 2015, 2019a,b; Allen et al., 2020).

Molecular phylogeny through multi-locus sequence typing (MLST) offers an excellent means to parse bacterial population structure with the use of housekeeping gene sequences and hence found a rightful reliable place in disease epidemiology (Maiden et al., 1998). MLST characterizes bacterial strain by their unique allelic profiles by measuring the variations in housekeeping genes. MLST provides a discriminatory power to differentiate different bacterial strains. Although use of MLST is less prevalent in mycology, it has also become a useful tool for studying to understand the fungal populations (Taylor and Fisher, 2003). The method represents an important tool to determine the population of fungi that are pathogenic to humans (Bougnoux et al., 2003; Bain et al., 2007) and plants (Kellner et al., 2011; Choi et al., 2013; Sun et al., 2013; Gurjar et al., 2021). MLSA has also been used to study the genetic diversity of various smut fungi (Kellner et al., 2011; Sun et al., 2013; Gurjar et al., 2021; Sedaghatjoo, 2021). Previous phylogenetic studies of T. horrida populations have been rare. One phylogenetic study conducted with T. horrida isolates collected from seven different provinces in China did not find any genetic variation (Wang et al., 2018). In the current study, we used the MLSA approach to understand the genetic diversity of the T. horrida populations in the US. Understanding the genetic diversity will help in designing and improving rice breeding programs to develop new cultivars with improved kernel smut resistance and in developing effective chemical management strategies for control of kernel smut. The results of our multi-gene phylogeny analyses showed, for the first time, the presence of genetic diversity in the rice kernel smut populations in the US, with all the T. horrida isolates clustering into four different genetic groups.

Kernel smut of rice has emerged as one of the most important diseases, threatening the US rice production. Economic impact of kernel smut is more significant from the loss of quality than from the loss of yield. In recent years, an increase in the severity and incidence of kernel smut and in cases of rejection of rice at selling point has been reported across the US, especially in Texas. In this study, we investigated the genetic diversity of the T. horrida populations in the US. Genomic DNA was extracted from the 63 isolates of T. horrida collected from Arkansas, California, Louisiana, Missouri, Mississippi, and Texas and subjected to multi-locus sequence analysis (MLSA). The results of our study showed the presence of genetically diverse T. horrida populations in the US. To our knowledge, this is the first study to analyze multi-locus region of the T. horrida populations in rice.

Our research reveals that there were five groups of the T. horrida populations in the US. Tilletia horrida clusters did not correspond with the geographical origin of the isolates collected. Only the isolates collected from Arkansas were clustered together in only one clade (clade I) with the reference isolate T. horrida strain QB1. Along with the Arkansas population, the isolates from California were the least diverse, whereas the population in Louisiana and Mississippi were most diverse as they grouped together in 3 out of 5 clades. Low diversity within the California population found in the current study can be attributed to quarantine practice that has been enforced in the state to prevent the introduction of disease, insect, and weed pests in rice seed into California from the southern region of the US and from foreign countries for many years (Oscar, 2006). The results of our research here are in contrast with those of Ustilaginoidea virens, the causal agent of false smut of rice, where a higher level of genetic differentiation among the U. virens populations found in the study with the isolates collected from geographically distant rice-growing areas of China (Sun et al., 2013). Unlike another bunt pathogen T. indica (Singh and Gogoi, 2011), the lack of correlation between genetic diversity and geographical specificity among the T. horrida populations in the current study may be attributed to unrestricted trading of rice seeds and lack of quarantine restriction in the southern US. In addition, the current study indicates that there are some levels of genetic differentiation in the T. horrida populations. These results have a direct implementation on the development of new rice cultivars with improved resistance to kernel smut. Future efforts toward breeding for resistant cultivars against kernel smut should consider selecting representative isolates from each of genetically diverse groups in the process of kernel smut resistance screening.

Neutrality tests suggest there was no significant departure from the mutation drift equilibrium, indicating the T. horrida population does not deviate from natural expectation in Tajima’s D and Fu and Li’s D* and F* tests (Tajima, 1989; Fu and Li, 1993). Individual phylogenetic analyses of all four regions showed higher nucleotide substitution in RPB1 and ITS as compared to the EF1⋅ and LSU regions. Along with higher nucleotide substitution per site, RPB1 also had highest nucleotide diversity (ND), Watterson’s theta (θ_w), and segregating sites. Similarly, based on Tajima’s D test, only RPB1 deviates from the mutation equilibrium with significant and positive Tajima’s D. Variation statistics showed 50 recombination events among the concatenated sequence. This might be due to the possible sexual recombination between different isolates. Tilleita horrida is a hemi-biotrophic fungus, which probably facilitates such recombination through mating of compatible types. However, no clear evidence of compatible sexual mating is still unknown in Tilletia horrida (Carris et al., 2006) or in similar non-systematic bunt Tilletia indica (Goates, 1988; Gupta et al., 2015).

Along with multi-locus analysis of the T. horrida populations in the US, we also analyzed 33 other Tilletia spp., including T. horrida and T. barclayana reported from eight different countries. Due to the lack of global information on multi-locus regions on the T. horrida isolates, we analyzed ITS regions-only reported in the NCBI database. Our analysis revealed that there are three groups of T. horrida isolates distributed in the world. The majority (76%) of the T. horrida isolates from this study, along with six other isolates reported previously from the US (Levy et al., 2001), was clustered together along with 15 other smut isolates from eight different countries. Similarly, four T. horrida isolates from this study were clustered together with seven T. horrida isolates reported from China (Zhou et al., 2006; Wang et al., 2015). Kernel smut is seedborne and rice is one of the most traded crops around the world (USDA, 2020). Our ITS regions-only analysis suggests potential multiple entries of the kernel smut pathogen from foreign countries into the US.

In the current study, clade III also clustered with T. australiensis isolated from wild rice (O. rufipogon) in Australia (McTaggart and Shivas, 2018) in addition to T. horrida isolates isolated from rice. ITS region sequence showed 99.75% similarity between T. australiensis and T. horrida strain 54,899 isolated from rice in Australia. Such high percentage of similarity between the kernel smut isolates from wild and cultivated rice indicates that the kernel smut fungus can infect multiple hosts and that wild rice potentially serves as an alternative host to the pathogen. The results of previously unverified reports indicate that T. horrida may infect Digitaria Haller, Leeria Sw., and Panicum L. (Tracy and Earle, 1896) and Pennisetum L. C. (Tullis and Johnson, 1952). However, until this date, there is no clear evidence that the T. horrida fungus can infect multiple hosts other than rice under natural conditions (Carris et al., 2006). Out of six T. barclayana isolates only two strains, DAOM 236425 and DAOM 238028, clustered together in clade III. Two T. barclayana strains clustered in clade III were the only T. barclayana isolated from rice (Liu, 2014), whereas other T. barclayana strains were isolated either from Paspalum spp. or Pennisetum orientale. Other strains formed a separate group from T. horrida isolates. Kernel smut isolated from other grasses clearly form different branches in the phylogenetic tree. Difference in the lineage of T. horrida and T. barclayana isolates have also been demonstrated in previous studies (Levy et al., 2001; Castlebury et al., 2005).

In conclusion, we can parse finger genetic diversity in the kernel smut fungus, T. horrida, using multi-locus sequence analysis. This is the first study analyzing the genetic diversity among the T. horrida populations in the US. Our study demonstrates the presence of genetically diverse of T. horrida isolates in different rice-growing states. Higher than 99% similarity in ITS region sequences between T. horrida isolates and between different countries may be attributed to the wide distribution of smutted rice along with global trade. The understanding of the genetic diversity of the T. horrida populations from the current study will help researchers develop effective host resistance and chemical management strategies, especially cultivar resistance, for the control of kernel smut of rice.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

SK, SA-B, SG, and X-GZ conceived and designed the experiments. SK and SG performed the isolations of the fungus and genomic DNA extractions. SK performed all sequencing experiments and wrote the manuscript. SK and SA-B analyzed the data. All authors have read and approved the manuscript.

This work was supported, in part, by USDA NIFA OREI (2015-51300-24286) and Texas Rice Research Foundation (TRRF 2018–2021).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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