Symptoms and pathogens diversity of Corn Fusarium sheath rot in Sichuan Province, China

To elucidate the symptoms and pathogens diversity of corn Fusarium sheath rot (CFSR), diseased samples were collected from 21 county-level regions in 12 prefecture-level districts of Sichuan Province from 2015 to 2018 in the present study. In the field, two symptom types appeared including small black spots with a linear distribution and wet blotches with a tawny or brown color. One hundred thirty-seven Fusarium isolates were identified based on morphological characteristics and phylogenetic analysis (EF1-α), and Koch’s postulates were also assessed. The results identified the isolates as 8 species in the Fusarium genus, including F. verticillioides, F. proliferatum, F. fujikuroi, F. asiaticum, F. equiseti, F. meridionale, F. graminearum and F. oxysporum, with isolation frequencies of 30.00, 22.67, 15.33, 7.33, 6.00, 5.33, 3.33 and 1.33%, respectively. Fusarium verticillioides and F. proliferatum were the dominant and subdominant species, respectively. Two or more Fusarium species such as F. verticillioides and F. proliferatum were simultaneously identified at a mixed infection rate of 14.67% in the present study. The pathogenicity test results showed that F. proliferatum and F. fujikuroi exhibited the highest virulence, with average disease indices of 30.28 ± 2.87 and 28.06 ± 1.96, followed by F. equiseti and F. verticillioides, with disease indices of 21.48 ± 2.14 and 16.21 ± 1.84, respectively. Fusarium asiaticum, F. graminearum and F. meridonale showed lower virulence, with disease indices of 13.80 ± 2.07, 11.57 ± 2.40 and 13.89 ± 2.49, respectively. Finally, F. orysporum presented the lowest virulence in CFSR, with a disease index of 10.14 ± 1.20. To the best of our knowledge, this is the first report of F. fujikuroi, F. meridionale and F. asiaticum as CFSR pathogens in China.

Maize (Zea mays L.) is one of the largest food staples worldwide and is one of the most economically important crops in China 1 . Maize yields as high as 260.77 billion kilograms have been attained in China, greatly contributing to ensuring food safety and increasing farmer income [2][3][4] . In China, the maize-sowing area reached approximately 44.96 million hm 2 in 2015, after which it decreased to 42.39 million hm 2 in 2017 5 . Chinese maize production of 259 million tons has been reported, accounting for 39% of Chinese cereal crop production and 22.8% of the global maize output 6 . However, several diseases caused by fungi, bacteria and viruses are a major factor limiting maize production. Among these diseases, Fusarium spp. can cause ear rot, stalk rot, seedling blight and root rot 7 .
Corn Fusarium sheath rot (CFSR) is one of the most serious crop diseases in China 8,9 . The results of previous studies suggest that Fusarium proliferatum can not only affect maize production but also produces the toxin fumonisins, posing great risks to human and livestock 10 . F. graminearum, F. verticillioides and F. equiseti have also been identified as causal agents of CFSR 8,11 . In fields, these pathogens primarily infect the sheath from the late growth period to the grain-forming stage 11 . Initial symptoms appear as irregular circular brown necrotic spots, after which the entire sheath gradually appears water-soaked and finally dies 12,13 . A severely infected sheath can eventually reduce lodging resistance and yield. According to statistical data, lodging results in major economic losses of approximately 15-25% and can even result in total crop failure 14 . A positive correlation has been demonstrated between yield loss and disease severity 15 . Additionally, wounds caused by aphid feeding can exacerbate the sheath rot severity of maize 16 .
Studies have shown that CFSR has occurred in more than 12 provinces in China 17 . However, the disease has not been reported in Sichuan Province, a major maize-producing region in China. Therefore, the goal of our present study was to characterize the disease severity, symptoms and Fusarium spp. pathogens of CFSR, which will provide an important basis for effective integrated control of this disease.

Results
Occurrence of corn Fusarium sheath rot (CFSR) in the field. In the present study, we investigated the occurrence of CFSR from 2015 to 2018. CFSR is characterized by two primary types of disease symptoms in the field (Fig. 1), including small black spots with a linear distribution (Fig. 1A,B) and wet blotches with a tawny or brown color (Fig. 1C,D). Disease incidence primarily ranged from 55 to 70% ( Fig. 2A), and the disease index primarily ranged from 14.00 to 22.00 for CFSR (Fig. 2B). Among 84 investigated spots, the disease incidence ranged from 49.59-84.16%, with an average of 66.83 ± 0.89%, while the disease index ranged from 7.32 to 37.62, with an average of 19.42 ± 0.75 (Table 1).   www.nature.com/scientificreports/ Identification of Fusarium species associated with CFSR. One hundred thirty-seven Fusarium isolates from 150 maize sheath samples were divided into six types according to the color and shape of their colonies and the morphology of their conidia (Fig. 3). The features of the macroconidia are described in Table 2

Pathogenicity test of Fusarium species.
To assess the pathogenicity of the Fusarium species identified from the maize sheaths in Sichuan Province, symptoms of CFSR were observed at 25 days after inoculation with twenty-three representative Fusarium isolates from eight Fusarium species (Table 2), and the disease index was calculated according to disease severity caused by Fusarium species. Four maize cultivars were tested here.
The symptoms of small black spots with a homogeneous distribution (caused by F. proliferatum, F. fujikuroi, F. equiseti, F. verticillioides, F. meridonale, F. asiaticum and F. graminearum) and wet blotches with a tawny color (caused by F. orysporum) were observed after inoculation, while the control plants showed no significant symptoms (Fig. 6).
All Fusarium isolates were pathogenic and caused CFSR, with disease indices ranging from 10.14-30.28. Among the assayed isolates, F. proliferatum and F. fujikuroi showed significantly higher virulence than the other Fusarium species (P < 0.05), with average disease indices of 30.28 ± 2.87 and 28.06 ± 1.96 (Table 3), followed by F. equiseti and F. verticillioides, which had similar disease indices of 21.48 ± 2.14 and 16.21 ± 1.84, respectively. F. asiaticum, F. graminearum and F. meridonale, members of the FGSC, also caused CFSR following inoculation but

Discussion
Many studies have demonstrated that various Fusarium species, such as pathogens isolated in Henan, Hebei, Shandong and Gansu are associated with CFSR, which has significantly affected the quality and quantity of maize since it was first reported in northeast China in 2008 11,18 . In the present survey, the occurrence of CFSR was commonly observed, with two primary types of disease symptoms detected in the field (Fig. 1), including small black spots with a linear distribution and wet blotches with a tawny or brown color, similar to that described by Zhai 11 . Based on a survey conducted from 2015 to 2018, the disease incidence was very high, at 49.  21,22 . In the present study, F. verticillioides and F. proliferatum were consistently observed in mixed infections in CFSR on maize, with a mixed infection rate of 14.67%.
Fusarium proliferatum is a ubiquitous, polyphagous, highly adaptable fungal pathogen of different plant species that attacks plants both in the field and during postharvest storage, causing blights, rots, and wilts on maize, garlic, soybean, tomato and Aloe vera 7,19,[23][24][25][26] . Interestingly, several studies have shown that F. proliferatum is also the predominant pathogen of some commercial crops, such as Polygonatum cyrtonema, date palm and Cymbidium [27][28][29] . F. proliferatum was observed as the dominant fungus in infected garlic bulbs, with a high disease incidence of 35.40%, and it was confirmed as the causal agent of dry rot in garlic postharvest 30 . F. proliferatum was also highly pathogenic, and significant symptoms were also observed 2 weeks after being inoculated on onion 31 . On soybean, F. proliferatum easily infected seeds, with an observed disease severity index of 43.33-49.16% 32 . In www.nature.com/scientificreports/ our present study, F. proliferatum exhibited the highest virulence among the evaluated species, with a disease index of 30.28 in four different maize varieties, which is consistent with results of previous studies 8,16 . Additionally, F. proliferatum was also widely distributed, with an isolation frequency of 24.82%. F. equiseti, F. verticillioides and F. graminearum were successively reported as pathogens in CFSR 11,33 . Interestingly, F. fujikuroi, F. meridionale and F. asiaticum species were recorded as causing CFSR for the first time in China in the present study (Fig. 4). Several studies have shown that F. fujikuroi causes rice bakanae disease and ear and stalk rot in maize 34,35 . In our present study, F. fujikuroi was the primary pathogen of CFSR, and F. verticillioides and F. proliferatum also exhibited strong pathogenicity, with an isolation frequency of 16.79% and a disease index of 28.06. F. graminearum is perhaps the best-known pathogen for causing head blight in wheat and ear and stalk rot in maize 36,37 . F. graminearum was detected in ≥ 80% of all Fusarium head blight (FHB) samples, sometimes even 100% 38 . In addition, the frequency of F. graminearum isolated from ears ranged from 30 to 71% with an average of 57%, and from stalks ranged from 43 to 81%, with the average of 65% 39 . Although the isolation frequency of F. graminearum from CFSR was only 3.65% in our present study, the potential risk of mycotoxins produced by F. graminearum to human health cannot be ignored 40 .
Fusarium meridonale is a member of the FGSC that is well-known to cause FHB in wheat and barley worldwide 41 . In addition, F. meridionale was also recently reported as pathogen in root rot on soybean under monoculture and ear rot on maize 42,43 . Moreover, F. asiaticum has been reported as a major and dominant causal agent of FHB on wheat and barley in China [44][45][46] and was also detected as a pathogen causing Gibberella ear rot of maize and seedborne diseases of soybean 47,48 . Although F. meridonale and F. asiaticum were not predominant species in the present study, with low isolation frequencies, they were reported in China for the first time. On the other hand, F. meridonale and F. asiaticum also exhibited typical pathogenicity, with disease indices of 13.89 and 13.80, respectively.
In summary, in the present study, eight species of Fusarium were recovered from maize fields in Sichuan Province, China. Interestingly, three species, F. fujikuroi, F. meridionale and F. asiaticum, were reported to cause CFSR in China for the first time. All isolates could infect the maize sheath and had disease indices ranging from 10.14 to 30.28. F. proliferatum and F. fujikuroi were the primary pathogens of CFSR, exhibiting high isolation frequencies and disease indices. The results of the present study provides the theoretical basis for integrated control of CFSR in Sichuan Province of China.

Methods
Survey and sampling. Twenty-one county-level regions of 12 municipal administrations were surveyed for sampling between 2015 and 2018 in Sichuan, China. The disease percentage and severity of CFSR under natural conditions were investigated as described by Huang et al. 49 . Four fields that were at least 1 km apart were contained in each area, and five sites were evaluated in each field, with 100 ear leaf sheaths sampled per site. In addition, 150 diseased maize sheaths with dark brown spots or bronzing and tawny blotch were collected in valve bags and stored in a large ice box before cultural isolation.
Isolated and morphological observations of Fusarium spp.. The pathogens were isolated from maize sheaths by tissue and single spore isolation 50,51 . Maize sheaths were cut into 0.5 cm × 0.5 cm pieces at the junction between disease and healthy tissue prior to isolation. The sheaths were surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed extensively with sterilized water, soaked in 75% ethanol for 30 s, and then rinsed extensively with sterilized water again. Subsequently, all sample pieces were placed on potato dextrose agar (PDA; 200 g·L −1 potato, 10 g·L −1 glucose anhydrous, and 15 g·L −1 agar) plates supplemented with 0.2 g chloramphenicol for fungal isolation for 3 days at approximately 25 °C under 12-h lighting.
Additionally, all strains were transferred to liquid carboxymethyl cellulose medium (CMC; 7.5 g·L −1 carboxymethyl cellulose sodium, 0.5 g·L −1 yeast extract, 2.5 g·L −1 K 2 HPO 4 , and 0.25 g·L −1 MgSO 4 ·7H 2 O) 52 . Then, the inoculated cultures were incubated for 5 days in a shaking incubator at 27 °C, 120 rpm. The culture characteristics and conidia of Fusarium species were morphologically assessed under a light microscope (Axio Imager Z2, ZEISS, Germany). One hundred thirty-seven isolates from 6 groups were identified as Fusarium spp. 53,54 . PCR amplification of rDNA-ITS and EF-1α sequences. All 137 obtained isolates were sub-cultured on PDA for 10 days at 25 °C with 12-h lighting. Approximately 20 mg of mycelia of each isolate was then scraped from the PDA plates with a sterilized ladle, and mycelia were ground to a powder in liquid nitrogen with a mortar. DNA was extracted using the cetyl-trimethylammonium bromide (CTAB) method 55 , and DNA concentration and quality were estimated using a Thermo Scientific NanoDrop 2000 Spectrophotometer (Massachusetts, USA) with the default setting for DNA assays.
PCR amplification of rDNA-ITS 56 was performed with PCR primers ITS1 and ITS4 using the amplification conditions described by Schoch et al. 57 . Amplification of the Fusarium translation elongation factor 1α (EF1-α) gene was performed with the primer pair EF1 and EF2 using the amplification conditions described by O'Donnell et al. 58 . Molecular identification of Fusarium species was confirmed by PCR amplification using the primers ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (GCT GCG TTC TTC ATC GAT GC) for the partial rDNA-ITS gene and the primers EF1-728F (CAT CGA GAA GTT CGA GAA GG) and EF4-986R (TAC TTG AAG GAA CCC TTA CC) for the partial EF1-α gene. PCR amplification was performed in a final volume of 25 μL containing 12.5 μL of 2 × PCR Master Mix (Vazyme, Nanjing, China), 0.5 μM of each primer and 10 ng of genomic DNA. The thermocycling conditions used for PCR amplification were as follows: 4 min at 94 °C followed by 35  www.nature.com/scientificreports/ Phylogenetic analyses. Sequence analysis of the rDNA-ITS region was first performed using BLAST at the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). Then, the EF-1α sequences from Fusarium species were compared to those in the NCBI database using the DNA BLAST program and the FUSARIUM-ID database (http://isola te.fusar iumdb .org/guide .php). The sequences were aligned using the software ClustalX 2 59 . Phylogenetic analyses were performed using MEGA5 software package with the default parameters 60 . The alignments were manually edited to delete trimmed regions and discard incomplete sequences. Phylogenetic trees for each genomic region and their tandem sequences were constructed using the neighbor-joining (NJ) approach 61 with 1000 bootstrap repeats and the pairwise deletion option 62 . Bipolaris oryzae was used as an outgroup (GenBank accession no. KJ 939510).
Pathogenicity tests. Twenty-three isolates from eight identified species (3 representative isolates from each species, except for F. oxysporum with only 2 isolates) were assessed for pathogenicity on maize during the flowering period in 2016-2017. Subsequently, they were cultured on PDA medium at 25 °C under 12 h of ambient room lighting per day for 5 days. Thereafter, five mycelial plugs of each strain (5 mm in diameter) were transferred to 100 mL of CMC liquid medium and cultured for 3 days in a shaker incubator (25 °C, 160 rpm). The conidial suspensions were then filtered and adjusted to a final concentration of 1 × 10 5 spores/mL. Four maize cultivars (Chuandan 455, Chuandan 428, Zhenghong 6 and Ruiyu 16) were sown in field plots, with each field plot containing 23 × 4 lines and 40 plants per line. Then, a 2-mL aliquot of a conidial suspension for each strain was injected into the first sheath above the ear leaf in 20 plants of each maize cultivar, and 20 control plants treated following the same procedure but were inoculated with sterile water 63 . Disease symptoms were assessed 25 days after inoculation, and disease severity was scored based on the average severity in 30 plants as described by Huang et al. 49 . Then, the disease index was calculated based on the average for each species. To confirm Koch's postulates, isolation from maize sheaths infected with one representative isolate of each Fusarium species was attempted. Subsequently, symptomatic maize sheath tissue was sectioned, and 0.5-cm pieces were placed on PDA for reisolation under the same culture conditions. Data analysis. Differences in the field survey, growth rate, conidial length and width and pathogenicity were analyzed using Statistical Package for Social Sciences (SPSS) (version 22.0 for Windows). Analysis of variance was performed using the general linear model, and means were compared using Duncan's New Multiple Range test with SPSS, with differences considered significant at P ≤ 0.05.