The foliar fungal pathogen Bipolaris maydis (Nisikado et Miyake) Shoem (teleomorph, Cochliobolus heterostrophus (Drechsler) Drechsler), the causative microorganism of southern corn leaf blight (SCLB), is one of the most important biotic restrictive factors, and contributes to substantial yield losses worldwide on corn (Zea mays L.), which is a crucial source of human food, industrial ingredient, and animal feed (Zwonitzer et al., 2009; Wang et al., 2014a). The SCLB symptoms initially appear on the bottom corn leaves, whereafter diffuse to the mid leaves. In susceptible corn cultivars, the necrotic lesions expand and cause significant harm to the functional leaves, thereby dramatically impairing the photosynthetic ability of corn plants (Chen and Xu, 2007). Despite the occurrence of SCLB outbreaks worldwide resulting in more than 58% yield losses in susceptible corn cultivars, these epidemics incline to be most continual in warm and moist regions (Carson et al., 2004).

There are several control strategies for SCLB in the field, such as the application of fungicides, use of resistant cultivars, and crop rotation (Wang et al., 2006). Nevertheless, despite the development of several available measures to reduce SCLB severity under natural conditions, this destructive disease still causes massive grain output losses ranging from 10% to 50% being reported in Fujian Province, China (Dai et al., 2020a). The warm and moist conditions conducive to the epidemic of SCLB are remarkably representative of Fujian, wherein the disease has developed as a crucial constraint affecting corn production (Dai et al., 2020a). Further constraints that have been responsible for enhancing the epidemics of SCLB in Fujian are the resistance development to demethylation inhibitor fungicides (Gan et al., 2016), susceptibility of sweet corn cultivars (Dai et al., 2020a), few genes that afford complete immunity to the disease (Kump et al., 2011; Li et al., 2018), and an increase in continuous corn cropping, which have facilitated massive accumulation of initial infections in the field (Dai et al., 2020a).

Information on the plant pathogens’ genetic structure is crucial to comprehend the influencing factors that contribute to their variability (McDonald, 1997; Ahmadpour et al., 2018). Additionally, this knowledge could provide suggestions for formulating available disease management strategies and guidance for further resistance breeding efforts. Molecular marker techniques have been broadly applied to analyzing genetic diversity and population structure of plant pathogens, and several molecular marker techniques have been successfully applied to evaluating the genetic diversity of B. maydis populations from diverse geographical origins in different countries (Nicholson et al., 1993; Karimishahri and Sharma, 2016; Wang et al., 2017; Chang et al., 2020). Among these molecular markers, inter-simple sequence repeat (ISSR) is a powerful tool to detect genotypes in heterotic groups at DNA level with little influence of environmental conditions (Kantety et al., 1995). Furthermore, ISSR can synchronously analyze plentiful fingerprint profiles and has a remarkable capacity to sensitive and repetitive identify high level of polymorphisms in large scale genome regions (Kantety et al., 1995). Hence, it is suitable for simple and fast polymorphism identify. In two B. maydis populations from the Hainan Province and Huang-Huai-Hai regions of China, high DNA polymorphism was measured in each sampling region using ISSR markers, with evident association between isolate groups according to their sampling locations as concluded using a clustering analysis (Chang et al., 2020). In another previous investigation, 60 race O B. maydis isolates with additional control race O, T, and C isolates collected from 15 corn growing locations in South China revealed high diversity and limited correlation among phylogenetic clustering, geographic location, and the virulence matrix as determined by amplified fragment length polymorphism markers (Wang et al., 2017). Genetic variation inferred through hierarchical clustering analysis of DNA fingerprinting patterns was reported in additional studies of B. maydis isolates from India (Gogoi et al., 2014; Karimishahri and Sharma, 2016; Manzar et al., 2022), Pakistan (Nadeem et al., 2024), and other regions (Condon et al., 2013). However, most B. maydis populations in these investigations originated from a very limited number of corn cultivars. There are few comparative studies on the role of the corn cultivars in B. maydis population structure.

The natural persistence of the foliar fungus B. maydis is a heterothallic ascomycete that has a specific mating type (MAT) gene locus, which has two alternative allelic forms, named MAT1-1 and MAT1-2 (Turgeon et al., 1993). Despite the sexual structures of B. maydis (e.g., perithecium or ascospore) having not been discovered under natural conditions, they can be easily formed using nutritive substances with suitable inductive materials and proper environmental conditions in a laboratory (Dai et al., 2020a, b). Random mating interactions among individuals influence population structure and genetic diversity of the phytopathogens through restructuring of alleles in multilocus individuals, resulting in random linking between unassociated alleles (Milgroom, 1996). Sexual recombination of new genotypes could increase multilocus haplotypic diversity and benefit plant pathogens by obtaining more adaptive characteristics, such as overcoming resistance to hosts or developing resistance to fungicides (Bi et al., 2014; Ahmadpour et al., 2018). However, there are few comprehensive studies that explore the reproductive strategies of natural B. maydis populations.

Due to the high frequency of SCLB outbreaks and the high proportion of epidemic individuals of B. maydis with highly pathogenicity to local sweet corn cultivars in Fujian Province (Dai et al., 2020a), this study used 13 ISSR markers to compare the population structure and genetic diversity of natural B. maydis isolates from single and multiple sweet corn cultivars with various genetic backgrounds, which afforded a comprehensive assessment of the role that geographical origins and corn cultivars play in B. maydis genetic differentiation. In addition, we explored the potential mode of reproduction of the natural B. maydis population in Fujian Province. This study will enhance the information on the role that geographical origins and corn cultivars play in the population structure of B. maydis as well as the reproductive strategies in B. maydis in Fujian Province.

In the present study, population genetic analyses were used to assess the major role of geographical origins and corn cultivars in B. maydis population differentiation in Fujian Province. The results indicated that significant differences in genetic diversity were observed in the B. maydis populations from different geographical locations, with high deviations in genetic diversity being observed in those from single and multiple sweet corn cultivars, implying that Fujian B. maydis isolates showed a greater genetic association with geographic origin and corn cultivars. A moderate to high genetic differentiation (Φ_PT > 0.1, P<0.001) was detected between the B. maydis isolates from the three sampling locations, which was higher than that when isolates were grouped by corn cultivar (Φ_PT <0.04, P<0.001). In addition, the genetic variation among populations was higher when isolates were grouped based on location (13.0%), compared to that when isolates were grouped according to corn cultivar (4.0%). The results obtained in the present study show that low genetic similarity and higher genetic differentiation being detected among B. maydis isolates from different sampling locations but not different corn cultivars implies that the geographical factor played a more important role in the population differentiation of B. maydis isolates than corn cultivars. The main reason for lack of genetic differentiation in B. maydis populations from single and multiple sweet corn cultivars may be due to high level of gene exchange (Nm > 12) within population. The findings in this study were consistent with analogical investigations revealing genetic differentiation of B. maydis on corn from north and south China and Villosiclava virens on rice in the Hubei Province of China, where both geographical origins and cultivars had an impact on the differentiation of B. maydis and V. virens populations, but the geographical origins played a more important role in the selection of B. maydis and V. virens isolates when compared with that of cultivars (Wang et al., 2014b; Chang et al., 2020). However, the AMOVA results from our study were in contrast with previously similar studies exhibiting host differences of Exserohilum turcicum and Monolinia fructicola on different cereal crops (corn and sorghum) and fruits (apricot, cherry, peach, and plum), respectively, where among-population differentiation was higher when isolates were grouped based on host rather than geographical location (21% and 2%, P < 0.022; 24% and 7%, P < 0.001, respectively) (Papavasileiou et al., 2015; Nieuwoudt et al., 2018).

Plant pathogen populations with few private loci are usually characterized by a lack of population subdivision and continual gene drift among isolates from different regions (Slatkin, 1995). In the present study, a limited number of private loci were observed among the B. maydis populations from single and diverse sweet corn cultivars at three sampling locations in the Fujian Province, which was consistent with the findings reported by Onaga et al. (2015), who detected few private loci in populations of another important pathogen, Magnaporthe oryzae, from East Africa with high gene flow between subpopulations. Nevertheless, our results are in contrast with those from a similar population genetic study of E. turcicum in South Africa, where numerous private alleles were observed in E. turcicum populations from corn and sorghum with limited gene flow between the two hosts (Nieuwoudt et al., 2018). The limited number of private loci and absence of shared multilocus haplotypes between isolates from the single and multiple corn cultivars imply that high levels of gene flow resulted in no genetic differentiation between the B. maydis isolates from single and multiple sweet corn cultivars in Fujian Province. This inference was supported by the results from our population structure and genetic differentiation analyses, in which a high level of gene flow (Nm > 12) was detected between the populations from the single and multiple corn cultivars in Fujian Province. In addition, high levels of haplotypic diversity (H_S =1.0) were detected from B. maydis populations in all situations, suggesting that multiple infection sources occur on individual sampling fields. Meanwhile, no shared multilocus haplotypes were detected from the single and multiple corn cultivars at the three locations in Fujian Province, indicating that large scale migration of B. maydis isolates did not occur in Fujian Province. Furthermore, disease severities in the three sampling fields growing multiple sweet corn cultivars were lower than that growing single sweet corn cultivar ( Table 1 ), suggesting rational distribution of resistant and susceptible corn cultivars being used for effective control SCLB.

Heterothallic plant pathogens undergoing frequent sexual reproduction are commonly characterized by high haplotypic diversity, equal mating type distribution, and linkage equilibrium (Milgroom, 1996). In the present study, we used these three approaches to evaluate the potential reproductive strategies of B. maydis populations from single and multiple sweet corn cultivars at three locations in Fujian Province. The detected haplotypic diversity values were relatively high in the B. maydis populations (1.00) from single and multiple sweet corn cultivars in Fujian Province. These high levels of haplotypic diversity were not related to a strictly clonal population, which afford proof for sexual reproduction. The haplotypic diversity values of the B. maydis isolates were consistent with those of high-haplotypic diversity populations of Cercospora sojina (0.41–0.69) (Kim et al., 2013), Mycosphaerella musicola (0.55–0.89) (Hayden et al., 2005), Bipolaris oryzae (0.51–1.00) (Castell-Miller and Samac, 2012), Setosphaeria turcica (0.81–0.91) (Ferguson and Carson, 2007), and Dothistroma septosporum (0.94–1.00) (Dale et al., 2011) which were reported in similar population genetic studies characterized by frequent sexual reproduction. The values obtained for the two mating type ratios of the B. maydis isolates from all three datasets showed no significant difference from the expected 1:1 ratio using the full ISSR datasets, confirming the hypothesis of frequency-dependent selection (Milgroom, 1996; May et al., 1999) and providing evidence of random mating interactions. However, significant differences in linkage disequilibrium (I_A and r̄_d ) (P < 0.01) were detected between populations in all datasets; therefore, we gained no forceful proof in favor of random mating in the Fujian B. maydis populations. Previous studies suggested that many factors (e.g. gene drift and flow, selection) could result in linkage disequilibrium analysis deviation from random mating, despite linkage disequilibrium is a crucial measurement for random mating (Milgroom, 1996; Mullett et al., 2015). As the gene drift between some sampling locations is high, especially the locations cultivated the same crop, gene flow may well impact on linkage disequilibrium at these locations. In addition, the gene exchanges between sampling locations and between STRUCTURE population groups was detected to be high, hence high the effect of gene flow on linkage disequilibrium in these populations (Milgroom, 1996). This is likely a contributing factor in this study.

Contradicting results were obtained from the analyses of haplotypic diversity, ratios of the two mating types, and linkage disequilibrium. The high levels of haplotypic diversity and equal ratios of mating type supported that sexual reproduction is considerable in the B. maydis lifecycle in Fujian Province despite evident proof of linkage disequilibrium. In similar previous investigations of other Cercospora, Dothistroma, and Exserohilum fungi, although linkage disequilibrium analysis did not support sexual reproduction in all datasets tested, high haplotypic diversities and near equal mating type ratios suggested that these populations experience both sexual and frequent clonal reproduction (Dale et al., 2011; Kim et al., 2013; Human et al., 2016; Nieuwoudt et al., 2018). Thus, the conflicting results between the haplotypic diversity, mating type distribution, and linkage disequilibrium analyses obtained in the present study imply a mixed reproductive strategy within the B. maydis populations from single and multiple sweet corn cultivars in Fujian Province. This reproductive strategy was beneficial to B. maydis adaptability. Because wide new genotypes generating in sexual reproduction could help the pathogen to obtain more adaptive characteristics, such as overcoming host resistance or developing fungicides resistance (McDonald and Linde, 2002; Bi et al., 2014). The perithecium produced in sexual reproduction is a good protective structure to help B. maydis survival in the adverse environment. In addition, asexual reproduction is beneficial to rapid growth and expansion. This challenge warrants the utilization of management strategies to prolong the service life period of these control methods (Liu and Mundt, 2020). Furthermore, the results in this study also showed that neither geographical origins nor corn cultivars have an impact on reproductive strategy of B. maydis populations. The main reason may be that the reproductive strategy of B. maydis is not only influenced by environmental factors, but by other factors, such as mating type distribution, alternative hosts.

Collectively, the results of STRUCTURE analysis showed that two genetic clusters occur in the B. maydis population from single and multiple sweet corn cultivars in Fujian Province. Intriguingly, B. maydis populations from single and multiple sweet corn cultivars at FQ exhibited obvious cultivar-specific differences, although the isolates of B. maydis were collected from the two neighboring fields. A reasonable interpretation of this situation is that there are two different proveniences of the pathogen that undergo mixing through sexual reproduction. A mixed reproduction strategy might explain the consolidation of two genetic groups at a comparatively slow speed (Liu and Mundt, 2020). The mechanisms of this inherent differentiation should be further considered, and continued genetic study of additional more samples, since such study is necessary, and will be beneficial to clarifying this controversy.

In conclusion, we comparatively studied the influence of geographical origin and corn cultivars on the differentiation of B. maydis populations in Fujian Province, given the more important role of the geographical origin in this pathogen differentiation. Our results showed high haplotypic diversity, an equal ratio of mating type, and remarkable linkage disequilibrium, and therefore are indicative of a mixed reproductive strategy among the B. maydis isolates from single and multiple sweet corn cultivars in Fujian Province. Sexual reproduction mostly took place on senescent phytodetritus at the later stage of crops or alternative hosts. Therefore, the existence of perithecia or ascospores on corn debris or alternative hosts needs to be further investigated to clarify the full lifecycle of B. maydis. The wide variation of gene recombination in sexual reproduction can increase the adaptability of offspring to natural selection and also promote the spread of advantaged mutations in the population. However, asexual reproduction can enable offspring to maintain the parental excellent characteristics, which can accelerate growth and achieve rapid population expansion. Hence, further evaluation of B. maydis is necessary to carefully assess the relative importance of both sexual and asexual stages in the lifecycle of B. maydis. In addition, a further temporal investigation of B. maydis isolates collected from the same site over continuous years will be beneficial to clarifying the influence of sampling time on genetic diversity and population structure.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

YD, XY, and WL: design the experiment. YD, LG, and CL: methodology and technology. YD, LG, CL, XL, and XY: samples and isolates collection. YD, LG, and CL: investigation and data analysis. YD: writing the draft manuscript. YD and WL: further review and editing. WL and XY: project supporting. All authors contributed to the article and approved the submitted version.