Fusarium Species Associated with Maize Leaf Blight in Heilongjiang Province, China

Fusarium spp. are among the most important plant pathogens in the world. A survey on maize leaf blight was carried out in Heilongjiang province from 2019 to 2021. Based on morphological characteristics and a phylogenetic analysis on translation elongation factor (tef1) and second-largest subunit of RNA polymerase II (rpb2) genes, 146 Fusarium isolates were obtained and grouped into 14 Fusarium species, including F. ipomoeae (20.5%), F. compactum (17.1%), F. sporotrichioides (9.59%), F. graminearum (9.59%), F. citri (8.9%), F. asiaticum (6.85%), F. verticillioides (6.85%), F. acuminatum (5.48%), F. glycines (5.48%), F. temperatum (2.74%), F. armeniacum (2.74%), Fusarium sp. (2.05%), F. flagelliforme (1.4%), and F. annulatum (0.68%). The Fusarium incarnatum-equiseti species complex (FIESC, including F. ipomoeae, F. compactum, F. citri, and F. flagelliforme) was the most prevalent, indicating an evolving occurrence of the Fusarium species causing maize leaf blight. The typical symptoms observed on the maize leaves were oval to long strip lesions, with a gray to dark gray or brownish red coloration in the center and a chlorotic area at the edges. Based on the tef1 gene, seven haplotypes of FIESC were identified in Heilongjiang province, suggesting a population expansion. This is the first report of F. ipomoeae, F. compactum, F. flagelliforme, F. citri, F. sporotrichioides, F. graminearum, F. asiaticum, F. acuminatum, F. glycines, F. temperatum, F. armeniacum, Fusarium sp., and F. annulatum causing maize leaf blight in Heilongjiang province, China. The current research is informative for managing disease, exploring the phylogenetic relationship among Fusarium species, and clarifying the diversity of Fusarium species associated with maize leaf blight.


Introduction
Fusarium spp. can cause several diseases in maize, such as Fusarium ear rot [1][2][3], Fusarium stalk rot and root rot [2,4], seedling blight [5], and maize leaf blight [6]. Regarding maize leaf blight, Fusarium verticillioides was the first pathogen, reported in 1968 [6], to cause the disease, and the only reported one up to now. However, the pathogenicity and diversity of Fusarium spp. causing maize leaf blight are still unclarified. Maize leaf blight is characterized by symptoms of irregular or spindle lesions, with gray to reddish brown coloration in the lesions' center surrounded by a chlorotic halo. Sometimes, this disease is misjudged as northern corn leaf spot due to the similar symptoms in the field. Thus, the identification of the pathogens based only on disease symptoms in the field is difficult.
To our knowledge, the genus Fusarium includes more than 300 phylogenetic species [7] and is one of the most important plant pathogens in the world [8]. Most species within the genus can produce a diverse range of mycotoxins, causing varying degrees of acute or chronic toxic effects [1]. Therefore, the accurate identification of these mycotoxin producers is a considerable endeavor [9]. For the identification of fungi and the investigation of molecular ecology, the internal transcribed spacer (ITS) is the most sequenced DNA region [10].
However, the ITS region cannot distinguish the species complex of Fusarium due to its conservation [11]. By contrast, the tef1 gene can be used to discriminate Fusarium species at the species or subspecies level [11,12], and the rpb2 gene is also more informative and frequently employed, so it has been recommended that they are sequenced for Fusarium species identification. However, although the partial beta-tubulin gene has been used to identify several Fusarium species, it was not universally informative within Fusarium [13].
The members of Fusarium incarnatum-equiseti species complex (FIESC) are considered important plant pathogens. FIESC is rarely considered the major pathogen of disease epidemics, but it has been identified as a co-occurring fungal pathogen during an infection [14]. Thirty phylogenetic species within the FIESC (FIESC 1 through FIESC 30) were recognized through Multi-locus Sequence Typing (MLST) [15,16], and the species containing multiple haplotypes are designated by the addition of a lowercase letter to the phylogenetic species designation [9].
Phylogenetic and genetic diversity analyses based on multiple sequences can reveal evolutionary relationships associated with geographical regions [9]. High genetic diversity indicates greater adaptability to changing environmental conditions. In some complex evolutionary scenarios, appropriate and sufficient information may not be obtained from phylogenetic trees [17,18]. By comparison, haplotype networks can be employed to analyze the intraspecific diversity of populations, genetic processes, and the biogeography and history of populations [18,19].
To date, there has been little research on pathogenicity, genetic diversity, and the haplotype groups of pathogenic Fusarium species isolated from symptomatic maize leaves in China. Hence, the purposes of the present study were to: (i) describe the morphological characterization and phylogenetic relationships based on tef1 and rpb2 genes of Fusarium species responsible for maize leaf blight in Heilongjiang province, (ii) evaluate the pathogenicity of different Fusarium species, and (iii) determine the haplotype diversity of FIESC based on tef1 associated with maize leaf blight.

Fusarium Isolates Collection
From 2019 to 2021, a total of 132 symptomatic maize leaves were collected from 10 different maize-growing counties or cities in Heilongjiang province. The symptomatic maize leaves were cut with a sterilized scalpel, superficially disinfected with a 2% solution of sodium hypochlorite for 1 min and 75% ethanol for 30 s, rinsed thrice with sterile distilled water, and air-dried on sterile filter papers under aseptic conditions. Pure cultures were obtained by single-spore isolation and maintained on PDA (potato dextrose agar) at 25 • C for 7 days. Fusarium isolates were obtained and preserved on PDA slants at 4 • C and 20% glycerol at −80 • C for temporary storage and long-term storage, respectively.

Morphological Characterization
All Fusarium isolates were incubated on PDA plate in the dark at 25 • C for 7 days. Colony color and colony texture were observed for each isolate. To determine the size of well-developed macroconidia (n = 30) and the number of septa, these Fusarium isolates were incubated on PDA plates at 25 • C for 7 days with light/dark cycle of 8/16 h. The macroconidia were observed under light microscopy (Zeiss Axiolab5 equipped with an Axiocam 208 color industrial digital camera).

DNA Extraction and Sequence Analysis
Fresh mycelia were harvested from cultures grown on PDA supplemented with streptomycin (50 mg/L) and tetracycline (50 mg/L) for 7 days at 28 • C. The extraction of fungal genomic DNA was performed as Ramdial et al. described [9]. The sequences of the translation elongation factor 1-alpha (tef1) gene, second-largest subunit of RNA polymerase II gene (rpb2), and partial beta-tubulin gene were amplified by the primers EF-1/EF-2, RPB2-5f2/RPB2-7cr, and Bt2a/Bt2b [13,20], respectively. The PCR products were sent to Jilin Comate Bioscience Co. Ltd. for purification and sequencing. Sequences of 146 Fusarium isolates were searched against GenBank and FUSARIOID-ID database (www.fusarium.org, accessed date: 1 September 2022) [21] by Basic Local Alignment Search Tool (BLAST) analysis and then deposited into the NCBI GenBank (Table 1).
HQ728164 -Bold accession numbers were generated from other studies.

Phylogenetic Relationships among Fusarium Isolates
The rpb2 (794-896 bp), tef1(546-686 bp), and β-tubulin (332-356 bp) gene sequences of Fusarium isolates were also compared to the sequences available in the FUSARIOID-ID database (www.fusarium.org, accessed date: 1 September 2022) to collect related sequences for inclusion in phylogenetic analysis. Multiple sequence alignments were correspondingly inferred in Molecular Evolutionary Genetics Analysis (MEGA) 7 software [22] using the MUSCLE (multiple sequence comparison by log-expectation) program [23] and refined manually if necessary. To generate concatenated datasets, single gene sequences (tef1 and rpb2) were manually combined utilizing BioEdit [24]. Phylogenetic tree based on the concatenated sequences of tef1 and rpb2 genes was built using the maximum likelihood (ML) method in MEGA 7, respectively. ML tree was generated from bootstrapping 1000 replicates. Bootstrap values ≥ 70% were shown in phylogenetic trees. The sequences from the Fusarium spp. type strains, initially identified as closely related to the sequences herein, were finally included by the preliminary BLAST searches.

Pathogenicity Tests
All Fusarium isolates were used to evaluate their pathogenicity based on the method described by Xu et al. [25]. To fulfill Koch's postulates, 10 healthy, surface-sterilized, and four to five leaf-stage maize seedlings (var. Demeiya 3) for each Fusarium isolate were inoculated with Fusarium spore suspension (1 × 10 6 spores/mL). Twenty maize seedlings sprayed with sterile distilled water served as controls. All seedlings sealed with plastic bags were maintained in a greenhouse at 25 • C with 90% relative humidity and a light/dark cycle of 12/12 h.

DNA Polymorphism
DNA Sequence Polymorphism software version 6 was used to individually determine the DNA polymorphism relative degree of the tef1 gene sequences [27]. Furthermore, Tajima's D, Fu and Li's D, and Fu and Li's F were used to determine neutrality test statistics. Significant values of these tests indicate the presence of population changes [28,29]. DNA polymorphism analyses were only performed on FIESC and not on other Fusarium species on account of the limited number of isolates from those species obtained in the current study.

Haplotype Analysis
Haplotype networks were individually generated based on the tef1 gene sequences of 70 FIESC isolates (including 30 F. ipomoeae isolates, 25 F. compactum isolates, 13 F. citri isolates, and 2 F. flagelliforme isolates in the present study) using PopART v. 1.7 (Allan Wilson Centre Imaging Evolution Initiative) to evaluate genealogy pattens of the haplotypes [19]. The aligned haplotype sequences were used to construct a TCS network [30,31].
Seventy isolates were identified as the members of FIESC and produced white to light yellow aerial mycelia. The bottom of the plate turned white to pale brown with time. The macroconidia were slightly curved at the apex with three to five septa and ranged from 39.6 to 83.5 × 3.9 to 5.2 µm (n = 30, Figures 1a-d and 2a-d) in size.   Fourteen F. sporotrichioides isolates produced dense, pinkish white to carmine red aerial mycelia, whose macroconidia were moderately curved to straight with three to five septa, but mostly three-septate, and measured 20.5 to 47.3μm × 2.8 to 4.2 μm (n = 30, Figures 1n and 2f).
The colonies of four F. armeniacum isolates were white to light pink. The macroconidia were prominently curved with three to five septa and had sizes ranging from 35.6 to 59.3 μm × 4 to 4.6 μm (n = 30, Figures 1g and 2g).  Ten isolates producing pink to fluffy dark red aerial mycelia, and red to aubergine pigmentation with age, were classified under F. asiaticum. Their macroconidia were falcate with three to five septa and measured 25.2 to 61.5 × 3.9 to 4.7 μm (n = 30, Figures 1h and  2h).
Fourteen F. graminearum isolates produced white-pink aerial mycelia and had dark red pigmentation. Their macroconidia were straight or slightly curved with five to seven septa and measured 25.4 to 97.7 × 3.4 to 5.8 μm (n = 30, Figures 1k and 2i).
Three Fusarium sp. isolates produced white to yellow colonies and red pigmentation. Their macroconidia were curved with three to five septa and measured 34.0 to 71.6 × 3.2 to 4.7 μm (n = 30, Figures 1j and 2j).
The colonies of eight F. acuminatum isolates were whitish-pink or carmine to rose red. Their macroconidia were slender with a distinct curve of the apical cell, mostly three-to five-septate, and measured 31.3 to 65.3 × 4.0 to 6.5 μm (n = 30, Figures 1f and 2k).
The colonies of eight F. glycines isolates produced fluffy, white aerial hyphae and a dark red pigment. Their macroconidia were three-to seven-septate, slightly curved, and ranged from 53.3 to 117.9 μm × 3.3 to 4.5 μm (n = 30, Figures 1l and 2l) in size.
The aerial mycelia of the F. annulatum isolates were white to cream-colored and turned violet with age, and their macroconidia were straight or slightly curved and contained three to five septa, with sizes of 21.5 to 58.3 × 2.1 to 3.6 μm (n = 30, Figures 1i and  2m). Fourteen F. sporotrichioides isolates produced dense, pinkish white to carmine red aerial mycelia, whose macroconidia were moderately curved to straight with three to five septa, but mostly three-septate, and measured 20.5 to 47.3µm × 2.8 to 4.2 µm (n = 30, Figures 1n and 2f).
The colonies of four F. armeniacum isolates were white to light pink. The macroconidia were prominently curved with three to five septa and had sizes ranging from 35.6 to 59.3 µm × 4 to 4.6 µm (n = 30, Figures 1g and 2g).
Ten isolates producing pink to fluffy dark red aerial mycelia, and red to aubergine pigmentation with age, were classified under F. asiaticum. Their macroconidia were falcate with three to five septa and measured 25.2 to 61.5 × 3.9 to 4.7 µm (n = 30, Figures 1h and 2h).
Fourteen F. graminearum isolates produced white-pink aerial mycelia and had dark red pigmentation. Their macroconidia were straight or slightly curved with five to seven septa and measured 25.4 to 97.7 × 3.4 to 5.8 µm (n = 30, Figures 1k and 2i).
Three Fusarium sp. isolates produced white to yellow colonies and red pigmentation. Their macroconidia were curved with three to five septa and measured 34.0 to 71.6 × 3.2 to 4.7 µm (n = 30, Figures 1j and 2j).
The colonies of eight F. acuminatum isolates were whitish-pink or carmine to rose red. Their macroconidia were slender with a distinct curve of the apical cell, mostly three-to five-septate, and measured 31.3 to 65.3 × 4.0 to 6.5 µm (n = 30, Figures 1f and 2k).
The colonies of eight F. glycines isolates produced fluffy, white aerial hyphae and a dark red pigment. Their macroconidia were three-to seven-septate, slightly curved, and ranged from 53.3 to 117.9 µm × 3.3 to 4.5 µm (n = 30, Figures 1l and 2l) in size.
The aerial mycelia of the F. annulatum isolates were white to cream-colored and turned violet with age, and their macroconidia were straight or slightly curved and contained three to five septa, with sizes of 21.5 to 58.3 × 2.1 to 3.6 µm (n = 30, Figures 1i and 2m).
Ten F. verticillioides isolates formed cottony white to greyish-purple colonies with a dark yellow to purple-gray underside. Their microconidia were abundant and mainly showed clavate shapes measuring 4.2 to 7.5 × 2.1 to 3.8 µm (n = 30, Figures 1m and 2e). However, there were no macroconidia of the F. verticillioides isolates observed in this study.
Ten F. verticillioides isolates formed cottony white to greyish-purple colonies with a dark yellow to purple-gray underside. Their microconidia were abundant and mainly showed clavate shapes measuring 4.2 to 7.5 × 2.1 to 3.8 μm (n = 30, Figures 1m and 2e). However, there were no macroconidia of the F. verticillioides isolates observed in this study.

Phylogenetic Analysis
The sequences of the tef1, rpb2, and beta-tubulin genes of all the Fusarium isolates obtained in this study were searched against the FUSARIOID-ID database (www.fusarium.org, accessed date: 1 September 2022) using a BLAST analysis (Table S1). For further molecular verification, a multilocus phylogenetic analysis (MLSA) was further performed based on the concatenated sequences (tef1 and rpb2 genes) of all the Fusarium isolates ( Figure 3). These results indicated that all the Fusarium isolates could be grouped into 14

Pathogenicity Tests
Two weeks after inoculation, the pathogenicity test revealed that all the Fusarium species could cause similar maize leaf blight symptoms (Figure 4). Small oval to fusiform or long striped spots initially appeared on the maize leaves three days post-inoculation, in which the lesions' centers were gray to reddish brown and surrounded by a chlorotic area. The lesions gradually enlarged with time and merged into each other. In a severe case, the infected leaves were withered. The symptoms observed under greenhouse conditions were similar to the symptoms of maize leaf blight in the field (Figure 4a). No symptoms were observed in the control group. In addition, all the Fusarium species were consistently reisolated and confirmed based on morphological and molecular methods, while no Fusarium isolates were obtained from the control group, thus fulfilling Koch's postulates. The average disease incidence and average disease index caused by the Fusarium species ranged from 23 to 74% and from 52 to 85, respectively (Figures 5 and 6; Table S2). Moreover, all the Fusarium isolates were pathogenic towards maize leaves (var. Demeiya 3) and caused maize leaf blight in the inoculation study. In addition, F. graminearum showed the highest virulence, followed by Fusarium sp., F. glycines, F. acuminatum, F. compactum, F. temperatum, F. asiaticum, F. citri, F. verticillioides, F. armeniacum, F. ipomoeae, F. annulatum, F. sporotrichioides, and F. flagelliforme.

Pathogenicity Tests
Two weeks after inoculation, the pathogenicity test revealed that all the Fusarium species could cause similar maize leaf blight symptoms (Figure 4). Small oval to fusiform or long striped spots initially appeared on the maize leaves three days post-inoculation, in which the lesions' centers were gray to reddish brown and surrounded by a chlorotic area. The lesions gradually enlarged with time and merged into each other. In a severe case, the infected leaves were withered. The symptoms observed under greenhouse conditions were similar to the symptoms of maize leaf blight in the field (Figure 4a). No symptoms were observed in the control group. In addition, all the Fusarium species were consistently re-isolated and confirmed based on morphological and molecular methods, while no Fusarium isolates were obtained from the control group, thus fulfilling Koch's postulates. The average disease incidence and average disease index caused by the Fusarium species ranged from 23 to 74% and from 52 to 85, respectively (Figures 5 and 6; Table S2). Moreover, all the Fusarium isolates were pathogenic towards maize leaves (var. Demeiya 3) and caused maize leaf blight in the inoculation study. In addition, F. graminearum showed the highest virulence, followed by Fusarium sp., F. glycines, F. acuminatum, F. compactum, F. temperatum, F. asiaticum, F. citri, F. verticillioides, F. armeniacum, F. ipomoeae, F. annulatum, F. sporotrichioides, and F. flagelliforme.

Pathogenicity Tests
Two weeks after inoculation, the pathogenicity test revealed that all the Fusarium species could cause similar maize leaf blight symptoms (Figure 4). Small oval to fusiform or long striped spots initially appeared on the maize leaves three days post-inoculation, in which the lesions' centers were gray to reddish brown and surrounded by a chlorotic area. The lesions gradually enlarged with time and merged into each other. In a severe case, the infected leaves were withered. The symptoms observed under greenhouse conditions were similar to the symptoms of maize leaf blight in the field (Figure 4a). No symptoms were observed in the control group. In addition, all the Fusarium species were consistently re-isolated and confirmed based on morphological and molecular methods, while no Fusarium isolates were obtained from the control group, thus fulfilling Koch's postulates. The average disease incidence and average disease index caused by the Fusarium species ranged from 23 to 74% and from 52 to 85, respectively (Figures 5 and 6; Table S2). Moreover, all the Fusarium isolates were pathogenic towards maize leaves (var. Demeiya 3) and caused maize leaf blight in the inoculation study. In addition, F. graminearum showed the highest virulence, followed by Fusarium sp., F. glycines, F. acuminatum, F. compactum, F. temperatum, F. asiaticum, F. citri, F. verticillioides, F. armeniacum, F. ipomoeae, F. annulatum, F. sporotrichioides, and F. flagelliforme.

Haplotype Analyses and DNA Polymorphism
The haplotype networks based on the tef1 gene sequences of 70 FIESC isolates (including 30 F. ipomoeae isolates, 25 F. compactum isolates, 2 F. flagelliforme isolates, and 13 F. citri isolates) obtained in this study were used to determine evolutionary relationships among the haplotypes. Most haplotypes within one species were closely related and separated by one to three mutations.
A total of seven haplotypes were identified: the F. ipomoeae isolates were assigned to Hap 1 and 4; F. compactum isolates were assigned to Hap 2, 5, and 6; F. flagelliforme isolates were assigned to Hap 3; and F. citri isolates were assigned to Hap 7 ( Figure 7).
Meanwhile, Hap 1, 2, 4, 5, and 7 were shared haplotypes ( Figure 7). Hap 1 was the most predominant haplotype, and presented in six locations (Harbin city, Wuchang city, Daqing city, Suihua city, Jixi city, and Qiqihar city). Hap 2 was found in Harbin city and Jixi city. Hap 4 was found in Harbin city and Wuchang city. Hap 5 was distributed in Harbin city and Shuangyashan city. Hap 7 was detected in Harbin city and Qitaihe city. Furthermore, two private haplotypes (Hap 3 and 6) were present in Harbin city and Jixi city, respectively. However, there was no obvious center between these predominant haplotypes. In addition, A low degree of nucleotide diversity (0.02706) and a high degree of haplotype diversity (Hd) (0.778) were found. Tajima's D, Fu and Li's D, and Fu and Li's F tests were negative with no significance (p > 0.10, Table S3).

Haplotype Analyses and DNA Polymorphism
The haplotype networks based on the tef1 gene sequences of 70 FIESC isolates (including 30 F. ipomoeae isolates, 25 F. compactum isolates, 2 F. flagelliforme isolates, and 13 F. citri isolates) obtained in this study were used to determine evolutionary relationships among the haplotypes. Most haplotypes within one species were closely related and separated by one to three mutations.
A total of seven haplotypes were identified: the F. ipomoeae isolates were assigned to Hap 1 and 4; F. compactum isolates were assigned to Hap 2, 5, and 6; F. flagelliforme isolates were assigned to Hap 3; and F. citri isolates were assigned to Hap 7 ( Figure 7).
Meanwhile, Hap 1, 2, 4, 5, and 7 were shared haplotypes ( Figure 7). Hap 1 was the most predominant haplotype, and presented in six locations (Harbin city, Wuchang city, Daqing city, Suihua city, Jixi city, and Qiqihar city). Hap 2 was found in Harbin city and Jixi city. Hap 4 was found in Harbin city and Wuchang city. Hap 5 was distributed in Harbin city and Shuangyashan city. Hap 7 was detected in Harbin city and Qitaihe city. Furthermore, two private haplotypes (Hap 3 and 6) were present in Harbin city and Jixi city, respectively. However, there was no obvious center between these predominant haplotypes. In addition, A low degree of nucleotide diversity (0.02706) and a high degree of haplotype diversity (Hd) (0.778) were found. Tajima's D, Fu and Li's D, and Fu and Li's F tests were negative with no significance (p > 0.10, Table S3).

Figure 7.
TCS analyses and the haplotype distribution based on the tef1 gene sequences of 70 FIESC isolates obtained in this study. Each haplotype is represented by a circle, the size of which is proportional to the haplotype frequency.

Discussion
As far as we know, this is the first systematic study of the Fusarium species associated with maize leaf blight. In this study, 146 Fusarium isolates delimited to 14 Fusarium species were obtained from symptomatic maize leaves in Heilongjiang province. To analyze the genetic relationship between these Fusarium isolates obtained in the current study, phylogenetic trees were constructed only based on the concatenated sequences of tef1 and rpb2 genes because these two genes were more informative and frequently employed, while the beta-tubulin gene was not universally informative in Fusarium [13]. A total of 14 Fusarium species were identified, including F. ipomoeae, F. compactum, F. sporotrichioides, F. citri, F. graminearum, F. asiaticum, F. verticillioides, F. acuminatum, F. glycines, F. temperatum, F. armeniacum, Fusarium sp., F. flagelliforme, and F. annulatum. Except for F. verticillioides, which was the only reported pathogen inciting maize leaf blight [6], the remaining Fusarium species were all first reported in Heilongjiang province, China, suggesting that the composition of Fusarium species causing maize leaf blight may have changed.
Furthermore, considerable pathogenicity differences were found among the different Fusarium species. F. graminearum showed significantly greater average disease incidence and average disease indices than those of other Fusarium species, followed by Fusarium Figure 7. TCS analyses and the haplotype distribution based on the tef1 gene sequences of 70 FIESC isolates obtained in this study. Each haplotype is represented by a circle, the size of which is proportional to the haplotype frequency.

Discussion
As far as we know, this is the first systematic study of the Fusarium species associated with maize leaf blight. In this study, 146 Fusarium isolates delimited to 14 Fusarium species were obtained from symptomatic maize leaves in Heilongjiang province. To analyze the genetic relationship between these Fusarium isolates obtained in the current study, phylogenetic trees were constructed only based on the concatenated sequences of tef1 and rpb2 genes because these two genes were more informative and frequently employed, while the beta-tubulin gene was not universally informative in Fusarium [13]. A total of 14 Fusarium species were identified, including F. ipomoeae, F. compactum, F. sporotrichioides, F. citri, F. graminearum, F. asiaticum, F. verticillioides, F. acuminatum, F. glycines, F. temperatum, F. armeniacum, Fusarium sp., F. flagelliforme, and F. annulatum. Except for F. verticillioides, which was the only reported pathogen inciting maize leaf blight [6], the remaining Fusarium species were all first reported in Heilongjiang province, China, suggesting that the composition of Fusarium species causing maize leaf blight may have changed.
Furthermore, considerable pathogenicity differences were found among the different Fusarium species. F. graminearum showed significantly greater average disease incidence and average disease indices than those of other Fusarium species, followed by Fusarium sp., F. glycines, F. acuminatum, F. compactum, F. temperatum, F. asiaticum, F. citri, F. verticillioides, F. armeniacum, F. ipomoeae, F. annulatum, F. sporotrichioides, and F. flagelliforme. Members of FIESC are generally considered co-occurring pathogens [32,33], and the moderate aggressiveness of FIESC in this study seems to confirm the previous conclusion. FIESC was the most predominant in this study. Members of FIESC have been frequently isolated from maize, soybean, rice, barley, wheat, and so on [34][35][36][37][38][39] and have also been reported to cause leaf blight in peanut plants [40] and Cyperus iria [41].
The haplotype groups of FIESC associated with maize leaf blight were first identified in this work. The predominant haplotype (Hap 1) represented multiple locations (Harbin city, Wuchang city, Daqing city, Suihua city, Jixi city, and Qiqihar city). It is well-known that older haplotypes may have a wider geographic distribution, which suggests that Hap 1 has lasted in the population for a long time [42]. The rest of the haplotypes may represent recently evolved lineages [4]. Furthermore, haplotypes 2, 5, and 6 belonged to the F. compactum clade; haplotypes 1 and 4 belonged to the F. ipomoeae clade; haplotype 3 belonged to the F. flagelliforme clade; and haplotype 7 belonged to the F. citri clade. These FIESC isolates were distributed in different clades in the haplotype network, which suggests that the haplotype network could effectively differentiate the Fusarium species complex and further confirmed our identification results. Moreover, the F. flagelliforme haplotype (Hap 3) and F. citri haplotype (Hap 7) were observed in external parts of the haplotype network and showed more mutation events from their nearest haplotypes, which indicated that these two species have an older evolutionary relationship. In addition, the high haplotype diversity and low nucleotide diversity indicated a population expansion [43].
In conclusion, the current study focused on the pathogenicity and genetic diversity of Fusarium species causing maize leaf blight in Heilongjiang province, China, and is the first to report F. ipomoeae, F. compactum, F. flagelliforme, F. citri, F. sporotrichioides, F. graminearum, F. asiaticum, F. verticillioides, F. acuminatum, F. glycines, F. temperatum, F. armeniacum, Fusarium sp., and F. annulatum as the causal agents. Fusarium can cause various maize diseases; therefore, clarifying the population composition of Fusarium spp. on maize leaves will provide information for the overall control of maize diseases.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/jof8111170/s1, Table S1. Tef1 gene sequences similarity to reference strain; Table S2. Disease index and disease incidence on maize leaves inoculated with different Fusarium isolates; Table S3. DNA polymorphism data for FIESC isolates based on tef1 gene sequences.
Author Contributions: X.X., L.Z., X.Y., G.S. and S.W. performed the experiments. H.T. and C.Y. prepared the figures and tables. X.X. and X.L. analyzed the data. X.W., W.X. and J.Z. designed the experiments and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Data Availability Statement: Sequences have been deposited in GenBank. The data presented in this study are openly available in NCBI. Publicly available datasets were analyzed in this study. These data can be found here: https://www.ncbi.nlm.nih.gov/, accessed on 3 September 2022.

Conflicts of Interest:
The authors declare that there are no conflict of interest.