Mycotoxin DON Accumulation in Wheat Grains Caused by Fusarium Head Blight Are Significantly Subjected to Inoculation Methods

The disease severity and mycotoxin DON content in grains caused by fusarium head blight (FHB) have been two prioritized economical traits in wheat. Reliable phenotyping is a prerequisite for genetically improving wheat resistances to these two traits. In this study, three inoculation methods: upper bilateral floret injection (UBFI), basal bilateral floret injection (BBFI), and basal rachis internode injection (BRII), were applied in a panel of 22 near-isogenic lines (NILs) contrasting in Fhb1 alleles. The results showed that inoculation methods had significant influence on both disease severity and mycotoxin accumulation in grains, and the relationship between them. UBFI method caused chronic FHB symptom characterized as slow progress of the pathogen downward from the inoculation site, which minimized the difference in disease severity of the NILs, but, unexpectedly, maximized the difference in DON content between them. The BBFI method usually caused an acute FHB symptom in susceptible lines characterized as premature spike death (PSD), which maximized the difference in disease severity, but minimized the difference in DON content in grains between resistant and susceptible lines. The BRII method occasionally caused acute FHB symptoms for susceptible lines and had relatively balanced characteristics of disease severity and DON content in grains. Therefore, two or more inoculation methods are recommended for precise and reliable evaluation of the overall resistance to FHB, including resistances to both disease spread within a spike and DON accumulation in grains.


Introduction
Fusarium head blight (FHB) is a worldwide fungal disease mainly caused by Fusarium graminearum species complex and has been reported in America [1,2], Asia [3,4], Europe [5], Australia [6], and some other countries. When favorable conditions, such as optimal temperature and humidity, occur during the flowering period of wheat, the ascospores of fusarium in soil, stubble, and other carriers will spread with rain and wind, and then This study focused on type II and III resistance using three relatively novel inoculation methods including BBFI [30], BRII [25], and UBFI. BBFI and UBFI were modified from SFI method. BBFI was performed by injecting inoculum into the bilateral florets of the 5th spikelet from the bottom of a spike [30]; and UBFI was performed by injecting inoculum into the bilateral florets of the 5th spikelet in the upper part of a spike. The effect of Fhb1 on reducing severity has been well acknowledged, whereas its effect on toxin accumulation disagreed among reports [31,32] and no convincing explanation has been acknowledged. BBFI and BRII are able to cause premature spike death (PSD) in susceptible wheat genotypes. Different from the BBFI and BRII methods, the UBFI method allows pathogens to spread downward from the inoculation point and the spikelets unreached by pathogen keep normal growth and development, thus, the PSD symptom is avoided. In this study, a panel of 22 near-isogenic lines contrasting in Fhb1 alleles with relatively simple genetic background and relatively stable resistance level was used to compare the consequences of the three methods in evaluating disease severity and DON content in grains, advantages and disadvantages of each method, and also to re-explore the relationship between disease severity and DON content in grains. We hope the outcome of this study would be useful for further improving the accuracy and reliability of FHB phenotyping, and helpful for untangling the uncertain relationship between FHB severity and DON accumulation.

Phenotypic Variation of PSS under the Three Methods
The basic statistics and distributions of PSS under the three methods were summarized in Table S1 and Figure S1, respectively. For bilateral floret inoculation, PSS of NILs showed a bias distribution in 2020 (UBFI, 0.2-0.4; BBFI, 0.8-1.0), whereas they were mainly between 0.5 and 0.9 in 2021; under the BRII method, more lines were distributed between 0.0 and 0.2 in 2021, and even with a lower mean PSS in 2020 (Table S1 and Figure S1A,B). Correlations of PSS between the two years were significant (p < 0.05) under UBFI method and highly significant (p < 0.01) across the BBFI and BRII methods.
Fhb1 + group and Fhb1 − groups significantly differed (p < 0.01) in PSS across the three methods in each season (Figures 1 and 2). Smaller variation in PSS among the NILs and narrower PSS gap between the two groups were observed under the UBFI method ( Figure 2). In comparison, larger variation of PSS among the lines and wider difference in PSS between the two groups were observed under BBFI (Figure 2), and PSS values of the lines without Fhb1 were close to or equal to 1.0 (Table 1). Compared with the two bilateral floret inoculation methods, the variation of PSS was smaller under the BRII method, ranging from 0.0 to 0.5 ( Figure 2). The rachis of the inoculated spikes of the lines carrying Fhb1 had an obvious bleaching phenomenon, but visible symptomatic spikelets were not/seldom observed, with PSS being close to or equal to 0.0 (Table 1 and Figure S2), significantly differing from those of the lines without Fhb1 (Figure 2), suggesting that the pathogen was easier to spread from rachis into spikelet in susceptible lines than in the resistant lines. Highly significant correlations in PSS were observed among the three methods (Figure 3), and the BLUP values over the two seasons for each method showed the order of BBFI > BRII > UBFI in PSS gap between Fhb1 − and Fhb1 + groups (Table 1), suggesting the BBFI and BRII methods were advantageous over UBFI in measuring FHB severity.

Premature Spike Death Could Be an Alternative Method for Measuring Disease Severity
The BBFI method easily caused premature death of the inoculated wheat spikes in the lines without Fhb1 ( Figure 1B), and the BRII method was next to BBFI in PPSD. PSD phenomenon was not observed under the UBFI method since the pathogen progressed chronically downward from the point of inoculation ( Figure 1A and Table S1). The extremum, mean, and distributions of PPSD for NILs under BBFI and BRII are shown in Table

UBFI Method Maximized DON Accumulation in Grains
Among the three methods, the maximum, mean and range of DON content under UBFI method were consistently the largest across the two years (Table S1 and Figure S1D,E), and the mean DON content exceeded 1000 µg·kg −1 (Table S1). In contrast, mean DON content of the NILs under BRII method was lower than those of the other two methods in each year (Table S1). A smaller variation of DON content was observed in 2021 than that in 2020 ( Figure S1D,E), but the mean value in 2021 was higher (Table S1). Correlations of DON content in grains between the two years were significant (p < 0.05) only under UBFI method, but not significant under the other two methods (p > 0.05).
The average DON accumulation varied from the inoculation methods with a trend of UBFI > BBFI > BRII (Table S1). DON content of most lines without Fhb1 exceeded 1000 µg·kg −1 under the UBFI method ( Figure 2), with the highest reaching 4224.38 µg·kg −1 (Table S1). Highly significant differences (p < 0.01) in DON content between the groups contrasting in Fhb1 alleles under the UBFI method and significant differences (p < 0.05) under the BRII method were observed, respectively, but there was no significant difference (p > 0.05) under the BBFI method ( Figure 2). The gap of DON content between Fhb1 − and Fhb1 + groups under the three methods followed the order of UBFI > BRII > BBFI, with intergroup gap of UBFI being about 10 times more than that of BRII (Table 1). A significant correlation in DON content (p < 0.05) was also observed between the UBFI and BRII methods, but neither of the two methods correlated with BBFI in DON content ( Figure 3). All these results suggested that UBFI was advantageous over the BBFI and BRII methods in assaying DON accumulation potential.

Premature Spike Death Could Be an Alternative Method for Measuring Disease Severity
The BBFI method easily caused premature death of the inoculated wheat spikes in the lines without Fhb1 (Figure 1B), and the BRII method was next to BBFI in PPSD. PSD phenomenon was not observed under the UBFI method since the pathogen progressed chronically downward from the point of inoculation ( Figure 1A and Table S1). The extremum, mean, and distributions of PPSD for NILs under BBFI and BRII are shown in Table S1 and Figure S3. PPSD under the BBFI method was mainly distributed between 0.5 and 1 over two years; PPSD under the BRII method ranged from 0 to 0.5 in 2020, and even less PSD occurred in 2021 ( Figure S3 and Table S1). The correlation coefficient of PPSD between the two years was significant (p < 0.01) only under the BBFI method.
One-way ANOVA analysis of PPSD showed that there was a highly significant difference (p < 0.01) between the two groups with contrasting Fhb1 alleles across the two years under the BBFI method, and the lines without Fhb1 were close to or equal to 1.0 ( Figure 4 and Table 1). PSD also occurred in some lines with Fhb1, but significantly less than those lines without Fhb1. PPSD under the BRII method was significant at p < 0.01 between the two groups in 2020, but not significant in 2021 ( Figure 4). PPSD of the lines carrying Fhb1 were close to 0.0 under the BRII method ( Figure 4 and Table 1), indicating that PSD under the BRII method tended to segregate in a qualitative manner. The BLUP values of PPSD over the two years showed a significant positive correlation between the BBFI and BRII methods (p < 0.05) (Figure 3), and BBFI caused more prematurely dead spikes than BRII did with a differential BLUP value up to 0.49 between the Fhb1 − and Fhb1 + groups (Table 1). These results indicated that PPSD under BBFI could be an alternative index for phenotypic assessment of FHB severity and an optimal index to assay the genetic effect of Fhb1.
that PSD under the BRII method tended to segregate in a qualitative manner. The BLUP values of PPSD over the two years showed a significant positive correlation between the BBFI and BRII methods (p < 0.05) (Figure 3), and BBFI caused more prematurely dead spikes than BRII did with a differential BLUP value up to 0.49 between the Fhb1 -and Fhb1 + groups (Table 1). These results indicated that PPSD under BBFI could be an alternative index for phenotypic assessment of FHB severity and an optimal index to assay the genetic effect of Fhb1.

Relationships among PSS, DON Content in Grains and PPSD
The relationships among PSS, DON content, and PPSD across two years are shown in Figure 3. PSS under the three methods showed significantly positive correlations with DON content under the UBFI method (p < 0.05), but had no, or weak, correlations with DON content under the BBFI and BRII methods. There was an expectedly significant correlation between PPSD and PSS under the BBFI method (p < 0.05). No significant correlations were found between PPSD and DON content under the BBFI and BRII methods, but PPSD of BBFI and BRII had significantly positive correlations with DON content under the UBFI method (p < 0.05). The BLUP results also suggested that correlations among PSS, DON content in grains and PPSD depended on inoculation methods ( Figure 3C).

Discussion
Limitations on precise phenotyping have been perceived as a constraint to both genetic study and breeding efforts [33]. A reliable and suitable phenotyping method is a prerequisite for understanding the function and the mechanism of a gene, evaluation of germplasm, breeding cultivars, and for genetic study of economically important traits including wheat FHB resistances to disease spread (type II) and DON accumulation (type III). In this study, three different inoculation methods (UBFI, BBFI, and BRII) were applied to understand their relative advantages and limitations in assessment of disease severity,

Relationships among PSS, DON Content in Grains and PPSD
The relationships among PSS, DON content, and PPSD across two years are shown in Figure 3. PSS under the three methods showed significantly positive correlations with DON content under the UBFI method (p < 0.05), but had no, or weak, correlations with DON content under the BBFI and BRII methods. There was an expectedly significant correlation between PPSD and PSS under the BBFI method (p < 0.05). No significant correlations were found between PPSD and DON content under the BBFI and BRII methods, but PPSD of BBFI and BRII had significantly positive correlations with DON content under the UBFI method (p < 0.05). The BLUP results also suggested that correlations among PSS, DON content in grains and PPSD depended on inoculation methods ( Figure 3C).

Discussion
Limitations on precise phenotyping have been perceived as a constraint to both genetic study and breeding efforts [33]. A reliable and suitable phenotyping method is a prerequisite for understanding the function and the mechanism of a gene, evaluation of germplasm, breeding cultivars, and for genetic study of economically important traits including wheat FHB resistances to disease spread (type II) and DON accumulation (type III). In this study, three different inoculation methods (UBFI, BBFI, and BRII) were applied to understand their relative advantages and limitations in assessment of disease severity, DON accumulation potential in grains, and their relationships. We found that no method was versatile for both traits.

BBFI Was Suitable for Assessing FHB Severity Rather Than for DON Assay
Single-floret inoculation (SFI) has been widely used for resistance evaluation of FHB [20]. SFI has high labor cost and low inoculation throughput, and it is relatively stable and becomes a popular method for evaluating type II resistance [21]. Based on SFI, a BBFI method has been developed to further improve the successful infection rate in field conditions without moistening facility [30]. BRII is a relatively novel method that we have recently reported, and is used in evaluating type II resistance [25]. One of the prominent merits of BRII method lies in exemption of moisture-maintaining system when compared with other existing methods [25]. UBFI method was developed here to avoid PSD that frequently occurred in susceptible FHB-genotypes by SFI and BBFI inoculation methods ( Figure 1 and Table 1).
PSD is a common phenomenon in the outbreak of FHB, which affects the normal grain filling process of the whole spike [34]. In this study, we defined the ratio of the number of prematurely dead spikes to the total inoculated wheat spikes under a certain inoculation method as proportion of premature spike death (PPSD). UBFI, BBFI, and BRII had different effects on PPSD. Under the UBFI method, the pathogen mainly spread downward and had little influence on the growth and development of the pathogen-unreached spikelets below the inoculation point, so the bleaching of spikes went progressively downward instead of causing acute premature death, i.e., PSD. Additionally, the UBFI method resulted in a negligible difference in disease severity between the two contrasting groups of Fhb1 vs. non-Fhb1 within 14 days after infection, and the difference in disease severity between the contrasting groups of Fhb1 vs. non-Fhb1 became remarkable at a later stage of infection (Figure 2A-C). In a word, the UBFI method seemed to reflect the nature of resistance to disease spread within the spike without causing acute PSD. In contrast, BBFI frequently caused acute death of the whole wheat spike in susceptible genotypes ( Figure S3 and Table S1). In this case, the pathogen entering the rachis damaged the vascular tissue of the spike, and the spikelets above the infection point eventually starved to death due to the lack of nutrient and water supply. The probability of PSD in the lines without Fhb1 under BBFI was close to 100% ( Figure 4 and Table 1), so BBFI maximized the phenotypic differences between resistant and susceptible genotypes (Figure 2A-C). In this case, attributed to the hemi-biotrophic lifestyle of Fusarium graminearum [35], it is suspicious to define the acute spikelets death as type II resistance since the pathogen may not spread within the dead spike due to the lack of humidity and nutrients required for pathogen growth.
For the BRII method, the pathogen inoculum was injected into the basal rachis internode, and the spikelets could be infected only when the pathogen passed through the rachis node, which demonstrated that the rachis node was crucial for hindering the spread of pathogen from one spikelet to another. The majority of lines carrying Fhb1 had no visible diseased spikelets at 21 DAI (Table 1 and Figure S2), which confirmed our previous observation [25]. The lines without Fhb1 also had a high PPSD but lower than that of BBFI ( Figure 4 and Table 1). PPSD under the BBFI and BRII methods were significantly different between the NILs with contrasting Fhb1 alleles (p < 0.01) (Figure 4), indicating that PPSD, instead of PSS, could be an optimal and practical index of FHB severity and could be used to reflect the genetic effect of Fhb1 on disease severity.
The robustness and accuracy of phenotypic evaluation is the prerequisite for fine mapping, positional cloning, functional characterization, and mechanism understanding. The mixture of PSS with PPSD brought to mind the controversial issues of the candidate genes, conflicting functions, and mysterious mechanism of Fhb1 [36][37][38]. In addition to phenotypic issue, other factors including the huge and complex wheat genome, the complexity of pathogen-wheat interaction and sensitivity to environmental cues might also contribute to the difficulty of unveiling the mysterious of Fhb1.

UBFI Was Suitable for Assessing Mycotoxin Accumulation Potential
Mycotoxins, such as DON, NIV, etc., are virulence factors in the process of pathogenhost interaction, which are not only one of the factors that aggravate the severity of FHB, but are also harmful to the health of consumers [7]. Therefore, it is necessary to assess the degree of toxin contamination in wheat grains and to develop varieties with low mycotoxin accumulation potential. Until now, the genetic research on type III resistance has lagged behind type II resistance due to a lack of appropriate inoculation methods, high cost of assay, large within-genotype variations, virulence of pathogen, etc., which obviously requires more effort and time. Most QTL for type III coincide with those for type II [17]. The wheat spikes without Fhb1 were more seriously contaminated by DON than the spikes with Fhb1, but several lines carrying Fhb1 have also been reported to have extremely high DON content [30]. A transcript mapping study showed that the DON-responsive transcripts were associated with, but not exclusively located within, Fhb1 [31]. In practice, the visual rating finishes 3-4 weeks after inoculation, while mycotoxin analysis is carried out after wheat harvest [23], and various uncertain factors during this additional time have a high probability to affect the evaluation of type III resistance.
A suitable inoculation method that significantly distinguishes different wheat genotypes in mycotoxin content is essential for research on type III resistance. At present, SFI and spray/grain-spawn inoculation, as common inoculation methods for assessing FHB severity, have been used directly in mycotoxin analysis [30,39,40], whereas little was known about the effects of different inoculation methods on mycotoxin accumulation. Unexpectedly, the BBFI method did not distinguish DON content between the NILs differing in Fhb1 in this study ( Figure 2D-F). The current data clearly demonstrated that DON content was highly significantly different (p < 0.01) between resistant and susceptible genotypes under the UBFI method, and significantly different (p < 0.05) under the BRII method ( Figure 2D-F), which indicated that the effect of Fhb1 on mycotoxin accumulation depended on inoculation methods, and also might explain why DON content had been highly variable among plants within a genotype/different replications under spray/spawn methods due to random infection points.
The mechanism by which the infection point of pathogenic fungi affects the mycotoxin accumulation in grains is not easy to explain. Mycotoxins are water-soluble and, theoretically, can be transported up and down the vascular tissue of spike and stem. UBFI and BBFI were performed by injecting inoculum into spikelets at the top and bottom of the spike, respectively. One possible explanation for the phenomenon of high DON content under UBFI method is that no PSD occurs and the pathogen progresses chronically downward; thus, the pathogen-unreached spikelets stay alive and sustain the supply of nutrients that induce the pathogen to produce DON. Another possible explanation may be that DON tends to be transported downwards, so DON may have a greater probability of remaining in the healthy grains. Our previous studies in a population of recombinant lines derived from Ning7840 and Clark showed that the effects of Fhb1 on DON content between the BBFI and BRII methods were similar [30]. The discrepancy between the previous and current experiments may be attributed to the genetic backgrounds. DON content and disease severity are quantitative traits. In current study, the genetic backgrounds of the NILs were of recurrent parent Clark, and the foreground was determined mainly by Fhb1 locus.

The Relationship between PSS and DON Content Was Subjected to Inoculation Methods
The relationship between FHB severity and toxin accumulation has been somewhat complex and conflicting. Varying degrees of relationship between FHB severity and DON content have been reported, including high positive correlations, low positive correlations, and no correlations [28,41,42]. A meta-analysis across 163 studies supported the high correlation between FHB severity and DON content (p < 0.001) [27]. As discussed above, we thought that BBFI and UBFI were beneficial for assessment of FHB severity and DON accumulation, respectively, and, in this case, the positive and significant correlation (p < 0.05) between the two traits validated most of the previous studies ( Figure 3). In addition, no correlation (p > 0.05) between FHB severity and DON content in grains was found under the BBFI method, whereas the correlation was weakly significant under the BRII method (p < 0.05, r ≤ 0.4) (Figure 3). Therefore, the degree of relationship between the two traits was greatly affected by inoculation methods or infection points. The visual FHB rating has become a useful parameter for DON prediction [28], but large variations in DON levels would be expected in the case of random infection points or the occurrence of PSD. If the infection point occurs in the upper part of a spike, the wheat grains may be contaminated by more mycotoxins, while the whole spike with severe premature death may accumulate less toxin.

Conclusions
In this study, the near-isogenic lines contrasting in Fhb1 alleles were used as materials to analyze the advantages, disadvantages, and applicability of the three phenotyping methods for disease severity and DON accumulation in grains. We concluded no inoculation method was versatile, and infection point (or inoculation method) was one of the important factors contributing to the complicated and uncertain relationship between disease severity and mycotoxin accumulation. UBFI narrowed the difference in PSS between resistant and susceptible lines, which might not be competent for evaluating disease severity for breeders. However, UBFI had prominent advantages over other methods for DON assay. BBFI was an optimized method from SFI, which maximized phenotypic differences between resistant and susceptible genotypes of FHB, and was suitable for evaluating the disease severity of a genotype, but would not be recommended for evaluation of mycotoxin accumulation potential. BRII was complementary to BBFI and UBFI. PPSD can be a key index to evaluate wheat resistance to FHB. Therefore, it is critical to select an appropriate phenotyping method or a combination of two or more methods to develop wheat elite cultivars with overall resistance to FHB.

Plant Materials
Twenty-two wheat near-isogenic lines (NILs) contrasting in Fhb1 alleles were used in this study. The NILs for Fhb1 were generated from the heterozygotes at Fhb1 locus derived from the cross of R75 and S98, two advanced backcrossed lines contrasting in Fhb1 alleles, which were previously developed from the cross of Ning7840 and Clark by Dr. Guihua

Fusarium graminearum Strain and Inoculum Preparation
A Fusarium graminearum strain Fg0865 (15-ADON chemotype) with strong pathogenicity and high sporulation was used for inoculation [25]. The strain was provided by Professor Huaigu Chen in Jiangsu Academy of Agricultural Sciences. The strain was activated by culturing on potato dextrose agar (PDA) medium at 25 • C for 4-5 days. The blocks of activated strain were then taken with a puncher with a diameter of seven millimeters (mm) and cultured in the mung bean soup with a volume of 10 milliliters (mL) per block at 25 • C and 150 r/min for 3-5 days to induce sporulation. The spore density was determined by a hemocytometer under the microscope. The inoculum reached at least 10 5 spores per microliter (µL) for UBFI and BBFI methods, and 10 6 spores/µL for BRII inoculation method.

FHB Inoculation and Phenotypic Evaluation
At the early flowering stage of wheat, three inoculation methods of UBFI, BBFI, and BRII were used for FHB inoculation. UFBI and BBFI methods were performed by injecting about 10 µL inoculum into the bilateral florets of a spikelet positioned at the fifth spikelet in the upper part of a spike and at the fifth spikelet from the bottom of a spike, respectively. The BRII inoculation was performed by injecting about 1 µL concentrated inoculum into the basal rachis internode of a spike. At least 15 spikes were inoculated for each line per replicate under each method.
At the seventh day after inoculation (DAI), the wheat spikes without visible symptoms at the inoculated site were removed due to inoculation failure. The proportion of symptomatic spikelets (PSS) per spike was counted at 21 DAI, which was calculated as "number of symptomatic spikelets/total number of spikelets of a spike". The proportion of premature spike death (PPSD) under BBFI and BRII methods was calculated as "number of prematurely dead spikes/total number of inoculated spikes".

Extraction and Quantification of DON in Grains
At maturity, all the inoculated spikes of each line were harvested and threshed manually to retain FHB-infected grains, and then ground into powder. DON in a gram of powder sample was extracted with 4 mL mixed solution of 49.5% acetonitrile,1% formic acid, and 49.5% distilled water. The extract was filtered sequentially through pore diameters of 0.45 µm and 0.22 µm needle-type organic phase filters into the autosampler insert (8 mm, 200 µL) of the autosampler vial (8 mm, 2 mL). All prepared samples were stored at −20 • C for subsequent determination. DON content in grains was determined following the protocol as described by Mao et al. [43], using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS, TSQ-Vantage, Thermo Fisher SCIENTIFIC, Waltham, MA, USA).

Data Analysis
Statistical analysis was performed using Excel 2016 (Microsoft Office Inc., Redmond, DC, USA) and IBM SPSS Statistics 21.0 (IBM, Armonk, NY, USA). Moreover, best linear unbiased prediction (BLUP) was also performed to remove environmental effects in an R package called lme4 operated by RStudio 1.4 software (Rstudio, Boston, MA, USA). The effects of Fhb1 alleles and interannual variation on PSS, DON content and PPSD under the three inoculation methods were determined by one-way ANOVA and t test, respectively, and the linear correlations among PSS, DON content, and PPSD under the three methods were measured using correlation analysis set from the simple Pearson correlation coefficient, using significance levels of 0.05 and 0.01. OriginPro 2021 (OriginLab, Northampton, MA, USA) and Adobe Photoshop CC 2019 (Adobe System Incorporated, Mountain View, CA, USA) were used for graphic processing.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/toxins14060409/s1, Table S1: Variation of proportion of PSS, DON content and PPSD in the Fhb1 near-isogenic lines. Figure S1: Distribution of proportion of symptomatic spikelets (PSS) and deoxynivalenol (DON) content in the Fhb1 near-isogenic lines under the three inoculation methods. Figure S2: The rachis and spikelets of the wheat spikes inoculated by basal rachis internode injection (BRII). Figure