Influence of Wheat Cultivars, Infection Level, and Climate after Anthesis on Efficacy of Fungicide for Control of Fusarium Head Blight in the Huang-Huai-Hai Plain of China

Abstract

Fusarium head blight (FHB), caused by the Fusarium graminearum species complex, causes significant losses in grain yield and quality of wheat (Triticum aestivum) by inducing floret sterility. Grains become contaminated with mycotoxins, especially deoxynivalenol (DON), making them unsuitable for consumption. To clarify the impact of wheat cultivar resistance, infection level, and climate after anthesis on the efficacy of a fungicide for the control of FHB, we treated two moderately susceptible cultivars and 11 susceptible cultivars with fungicide (48% phenamacril + tebuconazole) at anthesis over two years. FHB incidence (INC), disease severity index (DSI), Fusarium-damaged kernels, DON contamination, thousand-kernel weight, and yield were evaluated under artificially inoculated and naturally infected field trials in 2018 and 2021. The results of multi-factor variance analysis show that the control efficacy with respect to INC and DSI is affected by cultivar, fungicide, infection level, and climatic conditions including the average daily temperature, average daily relative humidity, and total rainfall from anthesis to 21 days after anthesis (p < 0.01). Notably, cultivar resistance (deviance = 13.34, 9.55, and 11.22) is more important than fungicide (deviance = 5.77, 6.66, and 6.69) to control the efficacy of INC, DSI, and DON. The results also suggest that infection level appears to be more important than cultivars and fungicide to control the efficacy of INC, and more important than fungicide to control the efficacy of DSI. Total rainfall is more important than other climatic factors. Our results reveal that fungicide is more effective in moderately susceptible cultivars (‘Zhengmai 9023’ and ‘Xinong 979’, 89.5%~98.9%) and some susceptible cultivars than in other susceptible cultivars (‘Zhengmai 7698’ and ‘Zhoumai 27’, 51.9%~67.2%). Thus, integrating cultivar resistance with fungicide application can be an effective strategy for the management of FHB and DON in winter wheat in the Huang-huai-hai Plain of China.

1. Introduction

Fusarium head blight (FHB), also known as scab, is a fungal disease caused by the Fusarium graminearum species complex and other related species. It causes a destructive disease in wheat (Triticum aestivum) and other small-grained cereals [1]. Since the early 1990s, FHB has occurred regularly in the winter wheat region in the middle and lower reaches of the Yangtze River, and in the spring wheat region of Heilongjiang, with occasional occurrence in the winter wheat region of the Huang-huai-hai Plain (HHP) [2,3]. Since 2010, wheat FHB has spread to considerably more places, becoming more frequent in the HHP; however, its incidence has remained relatively constant in the middle and lower reaches of the Yangtze River, where it has long infected crops. FHB occurrence in the HHP of China was a serious problem in 2010, 2012, 2014, 2015, 2016, and 2018. In 2012 alone, the area affected by FHB occurrence exceeded 9.949 million ha [4,5,6]. FHB not only causes yield losses, but also lowers the commercial value of the grain when the percentage of diseased kernels exceeds 4% or the content of the mycotoxin deoxynivalenol (DON) produced by FHB in the grain exceeds 1 mg/kg [7].

Several strategies have been employed to manage FHB infestation and DON contamination, including deploying resistant cultivars, practicing crop rotation, applying fungicides, and biological control [8,9,10,11,12,13,14,15]. Of these methods, using resistant cultivars is one of the most effective ways of controlling FHB [1]. Most of the Yangmai and Ningmai varieties grown in the middle and lower reaches of the Yangtze River are moderately resistant cultivars. Yao et al. [16] demonstrated that moderately resistant cultivars show little or no accumulation of mycotoxins compared with the low-to-moderate accumulation seen in susceptible cultivars. However, these varieties are adapted to the climate and growth conditions of southern China and are not suitable for planting in the HHP. Of the various commercially available pesticides for fighting FHB—mainly benzimidazole, phenamacril, prochloraz, and triazole fungicides—phenamacril stands out because of its good efficacy, resulting in relatively low toxin levels in grains and improvement in wheat quality and output [17]. Phenamacril is a cyanoacrylate fungicide developed by Jiangsu Pesticide Research Institute in 1998 [18]. Mixed formulations of phenamacril and tebuconazole (so-called Jingxing fungicide) are among the most popular fungicides among Chinese farmers and are highly effective against FHB.

Several studies in China and elsewhere have shown that, in years with heavy FHB outbreaks, individual management strategies are not sufficient for decreasing the percentage of Fusarium-damaged kernels (FDK) or the DON content to levels below those required by the grain industry [19,20]. Effective control of FHB using a single strategy is, therefore, not possible [4]. Since the 2012 FHB outbreak in the HHP, it has been difficult to achieve control through the use of single chemical treatments or the deployment of resistant cultivars: both disease incidence and DON levels have often not met national wheat acquisition standards. The integration of resistance cultivar and fungicide applications was more effective than management strategies alone for controlling the efficacy of FHB.

Previous studies have reported that the use of moderately resistant cultivars, combined with fungicide applications, can effectively control FHB and DON levels in grains. For example, Wegulo et al. [21] showed that the disease index and DON content of treated grains from moderately resistant cultivars were significantly lower than those from susceptible cultivars. Mesterházy et al. [22] also found that chemical control of DON levels in grains was most effective in moderately resistant cultivars, followed by moderately susceptible and then susceptible cultivars. Koch et al. [23] also reported that the content of DON in grains from moderately resistant cultivars subjected to chemical control was significantly lower than that in similarly treated susceptible cultivars. However, Horsley et al. [24] also noticed that integration of moderately resistant cultivars and fungicide (tebuconazole) treatment in barley (Hordeum vulgare) did not result in lower DON content in grains. Whereas the effectiveness of FHB control is clearly affected by cultivar resistance and fungicide use, it may also be influenced by other meteorological factors after anthesis.

Resistant cultivars are one of the most effective ways of controlling FHB and DON [1,16]. However, the fungicide efficacy varies among environments and cultivars with different levels of resistance, and it may also vary with infection level. In the present study, the combined effect of fungicide application and cultivars in the HHP was valuable for determining whether and how the efficacy of fungicide applications is influenced by cultivar resistance, environments, and infection level. In this region, more than 90% of the main wheat cultivars are susceptible to Fusarium graminearum infestation, with a few moderately resistant or moderately susceptible cultivars. There is a particularly strong need to study the effects of fungicide use coupled with cultivar resistance in this region, as well as for wider application of these results. The objectives of this study are to (i) evaluate the effects of integrating cultivar resistance and fungicide application on mitigating FHB and DON levels in winter wheat in the HHP, China, and (ii) quantify the contribution of factors (cultivar, fungicide application, climatic data after anthesis, and infection level) that affect the effectiveness of FHB prevention and control.

2. Materials and Methods

2.1. FHB Integrated Management Trials

Integrated management experiments 1 to 4 were conducted during the 2017–2018 and 2020–2021 winter wheat growing seasons on the Tongzhaipu Farm and Chengjiao Farm, respectively, in Tanghe County, China (longitude: 112.7753, latitude: 32.7802, elevation: 124.1 m). Thirteen cultivars, comprising the moderately susceptible cultivars ‘Zhengmai 9023’ and ‘Xinong 979’ and the susceptible cultivars ‘Hengguan 35’, ‘Zhongmai 895’, ‘Yumai 49-198’, ‘Zhoumai 18’, ‘Aikang 58’, ‘Zhengmai 379’, ‘Zhoumai 27’, ‘Zhengmai 366’, ‘Zhengmai 103’, ‘Zhengmai 7698’, and ‘Bainong 207’, were planted in early November 2017 and 2020 in fields previously planted with maize (

Table 1. Summary of analysis of variance from experiments conducted in 2018 and 2021.

		INC		DSI			FDK		DON		Yield
Source of Variation	df	MS y	P > F	MS	P > F	df	MS	P > F	MS	P > F	MS	P > F
Experiment 1 x, 2018
Block	1	48.0	0.2970	5.0	0.5480	1	0.0	0.9860	0.8	0.7640	571,097.0	0.2276
Cultivar (C)	12	2519.0	<0.0001	1147.5	<0.0001	12	655.2	<0.0001	91.2	<0.0001	2,236,838.0	<0.0001
FungicideI(F)	1	56,234.0	<0.0001	27,773.9	<0.0001	1	3957.0	<0.0001	1955.9	<0.0001	90,727,734.0	<0.0001
C × F	12	774.0	<0.0001	289.4	<0.0001	12	349.1	<0.0001	26.4	0.0028	1,459,042.0	0.0004
Error	77	44.0		13.0		51			8.7		382,970.0
Experiment 2, 2018
Block	1	4.0	0.6680	1.1	0.5930	1	6.2	0.5650	3.8	0.3205	1,024,246.0	0.1263
Cultivar (C)	12	2992.8	<0.0001	393.2	<0.0001	12	490.0	<0.0001	8.1	0.0323	2,089,157.0	<0.0001
FungicideI (F)	1	8767.4	<0.0001	2322.8	<0.0001	1	631.6	<0.0001	128.2	<0.0001	38,155,627.0	<0.0001
C × F	12	571.2	<0.0001	93.6	<0.0001	12	91.2	<0.0001	5.9	0.1372	959,416.0	0.0218
Error	77	20.6		4.0		51	18.5		3.8		424,060.0
Experiment 3, 2021
Block	1	37.8	0.2370	7.8	0.2250	1	51.0	0.1722	18.5	0.4675	162,624.0	0.4160
Cultivar (C)	12	2484.8	<0.0001	302.0	<0.0001	12	1175.4	<0.0001	1563.8	<0.0001	9,396,753.0	<0.0001
FungicideI (F)	1	1842.3	<0.0001	417.0	<0.0001	1	1717.4	<0.0001	772.9	<0.0001	4,697,643.0	<0.0001
C × F	12	199.3	<0.0001	45.2	<0.0001	12	108.9	0.0002	89.8	0.0082	1,083,043.0	<0.0001
Error	77	26.6		5.2		51	26.6		34.6		241,930.0
Experiment 4, 2021
Block	1	7.1	0.5630	0.8	0.5600	1	21.	0.2700	6.1	0.6541	361.0	0.9751
Cultivar (C)	12	1926.4	<0.0001	153.7	<0.0001	12	1299.5	<0.0001	1437.1	<0.0001	10,348,843.0	<0.0001
FungicideI (F)	1	1552.6	<0.0001	155.5	<0.0001	1	1101.6	<0.0001	1012.4	<0.0001	10,521,162.0	<0.0001
C × F	12	293.0	<0.0001	29.8	<0.0001	12	100.4	<0.0001	85.3	0.004	618,408.0	0.0963
Error	77	21.1		2.3		51	16.9		30.0		365,434.0

^x Experiments 1 and 2 were conducted under inoculated and natural conditions in Tanghe County, Nanyang city, in 2018, respectively; experiments 3 and 4 were conducted under inoculated and natural conditions in Tanghe County, Nanyang city, in 2021, respectively. ANOVA was performed to determine the combined effect of cultivar resistance and fungicide application on Fusarium head blight (FHB) incidence (INC), disease severity index (DSI), Fusarium-damaged kernels (FDK), deoxynivalenol (DON) concentration (mg/kg), and yield (kg/ha) in winter wheat grown in 2018 and 2021. ^y Mean square.

and

Table 2. Control efficacy of fungicide treatment among cultivars ^a.

Cultivar	Experiments 1 and 2 in 2018			Experiments 3 and 4 in 2021
	CE.INC b	CE.DSI	CE.DON	CE.INC	CE.DSI	CE.DON
Zhengmai 9023	92.1 a	96.6 a	80.6 ab	92.7 a	89.5 ab	69.3 ab
Xinong 979	77.8 ab	90.3 ab	90.3 a	98.6 a	98.9 a	78.4 a
Hengguan 35	84.0 ab	89.1 ab	79.9 ab	66.5 bc	72.5 cd	41.7 cd
Zhongmai 895	81.0 ab	88.6 abc	75.3 ab	95.1 a	96.3 a	78.8 a
Yumai 49-198	80.4 ab	77.4 abc	60.4 ab	96.6 a	97.2 a	81.9 a
Zhoumai 18	74.7 abc	86.5 abc	77.9 ab	96.4 a	97.1 a	32.5 d
Aikang 58	84.8 ab	89.2 ab	87.4 a	81.6 ab	77.5 bcd	63.7 abc
Zhengmai 379	50.2 cde	71.9 bcd	70.8 ab	95.8 a	96.7 a	65.9 abc
Zhoumai 27	44.0 de	67.2 cd	67.1 ab	51.3 cd	62.2 de	−7.3 e
Zhengmai 366	59.5 bcd	69.2 bcd	69.2 ab	92.4 a	93.4 a	70.5 ab
Zhengmai 103	58.1 bcd	77.5 abc	68.6 ab	70.3 b	77.8 bc	28.4 d
Bainong 207	−3.4 f	28.1 e	43.5 b	89.1 a	91.8 ab	47.4 bcd
Zhengmai 7698	23.6 e	51.9 d	71.5 ab	45.4 d	56.6 e	33.7 d

^a Fungicide control efficacy (as percentage) was calculated for Fusarium head blight (FHB) incidence (CE.INC), disease severity index (CE.DSI), and deoxynivalenol (CE.DON) concentration from experiments 1–2 and experiments 3–4 to determine the combined effect of cultivar resistance and fungicide application on winter wheat during the growing seasons of 2018 and 2021. ^b Means followed by the same letter within a column are not different according to Fisher’s least significant difference test at p = 0.05.

). The seeding rate was 187.5 kg seeds/ha. The plot size was 5.5 m × 2 m. Each cultivar was associated with control and fungicide treatments. The fungicide treatments were under either artificial inoculation or natural conditions with four replicates (field conditions with no inoculation). Experiments 1 and 2 were established for artificial inoculation and natural conditions, respectively, at Tongzhaipu Farm in 2018. Experiments 3 and 4 were established for artificial inoculation and natural conditions, respectively, at Chengjiao Farm in 2021. In two growing seasons, the Jingxing fungicide (36% [w/v] phenamacril + 12% [w/v] tebuconazole; Jiangsu Pesticide Research Institute Co., Ltd, Nanjing, China.) was used on thirteen cultivars. There were 52 treatments (fungicides and control × thirteen cultivars × two conditions) in each season (Tables S1 and S2). The experimental design used a randomized block with four replications, giving a total of 104 plots. FHB susceptibility or resistance of the 13 cultivars was determined from information provided by the National Crop Accreditation Committee and Henan Crop Accreditation Committee.

2.2. Inoculum Preparation

To obtain Fusarium-graminearum-colonized wheat inoculum, wheat grains were soaked for 12 h in tap water and placed in a sterile polyethylene plastic bag. Each bag was filled with 50 g of soaked grains and sterilized by autoclaving at 120 °C for 1 h. After 2 days at room temperature in the dark, bags were autoclaved again for 1 h and allowed to cool down for later use. Five F. graminearum isolates (14LY9-2-4, 14AY1-2, 14YY1-3, 14KF3-8, and 14ZK1-4 of the 15-acetyldeoxynivalenol chemotype from Luoyang, Anyang, Yuanyang, Kaifeng, and Zhoukou cities of Henan Province in China) were first grown on potato dextrose agar (PDA) plates (Figure S3). These strains were stored at the China General Microbiological Culture Collection Center. Ten hyphae blocks, each 5 mm in diameter, were collected using a sterilized loop punch and introduced into the polyethylene plastic bag containing wheat grains using a sterilized toothpick. The bag was sealed and rubbed once every 3 days to ensure uniform growth. After 3 weeks, wheat grains were completely covered in fungus and were placed at 4 °C until use.

2.3. Disease Evaluation and Yield

Fusarium-graminearum-colonized wheat inoculum (prepared using the five isolates collected from Henan Province, China) was applied to the soil surface in the wheat plots at full flag leaf emergence (Feeke’s GS 9) on 1 April 2018 and 2021 at a rate of 60 kg/ha. Plots were not irrigated. Natural rainfall rehydrated the wheat kernel inocula, initiating fungal development. Each subplot was divided into two sub-subplots: one was treated with the Jingxing fungicide (36% [w/v] phenamacril + 12% [w/v] tebuconazole; Jiangsu Pesticide Research Institute Co., Ltd., No. 31-1, Hengjing Road, Nanjing Economic and Technological Development Zone, Nanjing, China) at a rate of 750 mL/ha, as recommended in the summary of the product’s characteristics; the other sub-subplot was left untreated. Fungicide application was performed when approximately 50% of the main tillers had reached anthesis (Feeke’s GS 10.5.1), using a backpack sprayer calibrated to deliver approximately 400 L/ha. Application date varied depending on cultivar maturity. In experiments 1 and 2, cultivars ‘Hengguan 35’ and ‘Zhengmai 9023’ were treated on 17 April 2018; the other cultivars were treated on 19 April 2018. In experiments 3 and 4, cultivars ‘Zhengmai 366’ and ‘Zhengmai 9023’ were treated on 18 April 2021, ‘Zhoumai 18’, ‘Bainong 207’, and ‘Yumai 49-198’ were treated on 20 April 2021, and the other cultivars were treated on 19 April 2021. Disease severity and incidence (INC, percentage of diseased spikes) was assessed on 400 spikes per plot at the soft dough growth stage (Feeke’s GS 11.2), 21 days after anthesis. The severity of the disease symptoms on each spike was scored using a 0–4 rating scale, where 0 = no diseased spikelets, 1 = ≤ 25% diseased spikelets per spike, 2 = 25.1–50% diseased spikelets per spike, 3 = 50.1–75% diseased spikelets per spike, and 4 = ≥ 75% diseased spikelets per spike [25,26]. The disease severity index (DSI) was calculated according to the following formula [27]:

DSI = 100 × [(0 × P0 + 1 × P1 + 2 × P2 + 3 × P3 + 4 × P4)]/(100 × 4),

(1)

where P0, P1, P2, P3, and P4 represent the number of spikes scored at the rating scale of 0, 1, 2, 3, and 4, respectively, and the values 100 and 4 represent the total number of spikes scored and the highest possible disease rating value, respectively.

Grains were harvested from three small 1 m² plots per main plot, using a plot thresher (QKT-320, Weihui Agricultural Machinery Factory of Henan Province, Weihui, China) with a fan speed of 1240 rpm. After drying, the harvested grains were packed in mesh bags and weighed separately. The yield per hectare (kg) and thousand-kernel weight (TKW, g) were estimated from the yield of a small plot (1 m²). Fusarium-damaged kernels (FDK) were counted to ascertain the number of diseased kernels, using 1000 kernels from each small plot. A 10 g grain sample from each small plot was then ground to flour using grinders (ZM200, Wuhan Branch of Retsch China Co., Ltd., Wuhan, China); the resulting powder was submitted to the Institute of Quality Standards and Testing Technology for Agro-products (Henan Academy of Agricultural Sciences, Zhengzhou, China) for DON content determination (mg/kg) using high-performance liquid chromatography (HPLC)/electrospray-ionization–tandem-mass-spectrometry.

2.4. Climatic Data

Climatic data were obtained from the Tanghe County weather station nearest to each sampling field (< 30 km); these stations were managed by the China Meteorological Data Network (http://www.cma.gov.cn/, accessed on 1 October 2023.). The average daily temperature (°C, TEM), average daily relative humidity (%, RH), and total rainfall (mm, PRE) from anthesis to 21 days after anthesis were estimated from daily weather data.

2.5. Data Analysis

A one-way factorial analysis of variance (ANOVA) in R (v.4.0.0; R Foundation for Statistical Computing, Vienna, Austria) was used to determine the differences in INC, DSI, FDK, DON, TKW, and yield among treatments. Data from experiments 1 to 4 were analyzed separately to determine differences between treated and untreated cultivars. Prior to analysis, those response variables quantified on a percentage scale (INC and FDK) were arcsine square root transformed (arcINC and arcFDK, respectively). An ANOVA was conducted for each experiment to determine the effect of combining cultivar resistance and fungicide application on INC, DSI, yield, FDK, and DON in winter wheat for the 2018 and 2021 growing seasons. The study was conducted by following a randomized complete block design. F values for cultivar and fungicide treatment effects and their interaction were considered significant at p < 0.05.

Control efficacy with respect to INC, DSI, DON concentration for each treatment was calculated as:

[(C − T)/C] × 100

where C is the check treatment value and T is the fungicide treatment or other cultivar treatment value. Control efficacy with respect to yield under each treatment was calculated as:

[(T − C)/C] × 100

The check treatment represents the variety with the highest DSI (‘Zhengmai 366’, ‘Zhengmai 379’, ‘Hengguan 35’, and ‘Zhengmai 7698’ check treatment in experiments 1 to 4, respectively) in each experiment. When analyzing the fungicide efficacy among cultivars with respect to INC, DSI, and DON to determine the effect of combining cultivar resistance and fungicide application on winter wheat, experiments 1 and 2 in 2018 and experiments 3 and 4 in 2021 were combined prior to analysis, respectively.

Pearson’s correlations among weather variables (TEM, RH, PRE) and the control efficacy among cultivars with respect to INC, DSI, FDK, DON, and yield were calculated using the psych package [28] in R. The level of the epidemic was determined by calculating the INC mean of all cultivars for each year. In this study, the 2018 epidemic was considered to be high (INC mean > 40) and the epidemic in 2021 was considered to be low (INC mean < 20) for FHB intensity. A generalized linear model (GLM) was used to assess the relationships of cultivar, fungicide, epidemic, and weather variables with the control efficacy among cultivars with respect to INC, DSI, FDK, DON, and yield, in which residual errors were assumed to follow a quasibinomial distribution. During GLM analysis, negative values were converted to 0. Each explanatory variable (cultivar, fungicide, epidemic, TEM, RH, and PRE) was included separately into the model to estimate its contribution (as the deviance which was accounted for) to the observed variability. All statistical analyses were performed in R (v.4.0.0; R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. Experiments 1 and 2 in 2018

In Experiments 1 and 2 from 2018 (a year with high FHB intensity, with the mean INC of all varieties > 40%), the mean FHB incidence (INC, defined as the percentage of diseased spikes among the total number of spikes evaluated) on the moderately susceptible varieties ‘Zhengmai 9023’ and ‘Xinong 979’ was 53.3% and 70.3% vs. 6.6% and 29.6% for artificially inoculated and natural conditions, respectively (Table S1, Figure 1). For the susceptible varieties, INC ranged from 71.7% to 99.7% without fungicide treatment and from 19.5% to 68.0% with fungicide treatment (Table S1, Figure 1). Thus, the treatment with the Jingxing fungicide significantly decreased the INC and DSI for all cultivars relative to their untreated controls (p < 0.05), except for ‘Zhengmai 9023’ in experiment 2 (Table S1, Figure 2).

Under the artificial inoculation (Experiment 1) condition in 2018, the Jingxing fungicide did not affect FDK values for cultivars ‘Zhengmai 9023’, ‘Xinong 979’, ‘Hengguan 35’, ‘Zhongmai 895’, or ‘Zhoumai 27’. However, the fungicide treatment did result in lower DON content in all cultivars, except for ‘Zhengmai 9023’. Under natural inoculation (Experiment 2) conditions in 2018, the application of the Jingxing fungicide had no effect on FDK or DON in ‘Xinong 979’, ‘Zhongmai 895’, ‘Zhoumai 18’, ‘Zhengmai 379’, ‘Bainong 207’, or ‘Zhengmai 7698’, on FDK in ‘Yumai 49-198’ or ‘Zhoumai 27’, or on DON in ‘Zhengmai 9023’, ‘Hengguan 35’, or ‘Zhengmai 103’ (Table S1). Under artificial inoculation (Experiment 1) conditions in 2018, the fungicide treatment did not affect the TKW of ‘Xinong 979’, ‘Hengguan 35’, ‘Zhengmai 366’, or ‘Zhengmai 7698’. The overall yield of all cultivars was significantly higher under the fungicide treatment compared with that of untreated controls, except for the case of ‘Zhengmai 7698’ (Table S1). Under natural inoculation (Experiment 2) conditions in 2018, there was no effect of the fungicide treatment on the TKW of ‘Zhengmai 9023’, ‘Xinong 979’, ‘Zhoumai 18’, ‘Zhengmai 103’, ‘Zhongmai 895’, ‘Hengguan 35’, ‘Zhengmai 379’, ‘Zhengmai 7698’, or ‘Bainong 207’. The yield of all cultivars under the fungicide treatment was significantly higher than that of untreated controls, except for ‘Xinong 979’, ‘Aikang 58’, ‘Zhoumai 18’, ‘Hengguan 35’, and ‘Zhengmai 366’.

3.2. Experiments 3 and 4 in 2021

In Experiments 3 and 4 in 2021 (a year with low FHB intensity, the mean INC of all varieties < 20%), the mean INC of the moderately susceptible varieties ‘Zhengmai 9023’ and ‘Xinong 979’ was 14.4% and 7.0% vs. 6.7% and 1.9% for artificially inoculated and natural conditions, respectively (Table S2). The range of INC values for susceptible varieties was 2.4% to 62.7% (artificial inoculation, experiment 3) and 3.0% to 73.4% (natural inoculation, experiment 4) (Table S2). The Jingxing fungicide did not decrease the INC, DSI, FDK, or DON, and it did not increase TKW or the yield compared with the respective untreated controls (p < 0.05), except for the INC, DSI, FDK, and DON of ‘Hengguan 35’ in experiments 3 and 4, INC of ‘Yumai 49-198’, DSI of ‘Aikang 58’, DON and TKW of ‘Zhengmai 103’ in experiment 3, DON and TKW of ‘Zhengmai 103’, and INC, DSI, FDK, and DON of ‘Zhengmai 7698’ in experiment 4 (Table S2).

3.3. Cultivar, Fungicide, and Their Interaction Effects

Both cultivar and fungicide treatment had a significant effect on INC, DSI, FDK, yield, and DON (p < 0.0001). However, the F values for the cultivar × fungicide interaction were significant for INC, DSI, FDK, yield, and DON (p < 0.01) in all four experiments (Table 1), except for DON in experiment 2 from 2018 (p = 0.1372) and yield in experiment 4 from 2021 (p = 0.0963).

3.4. Control Efficacy of Fungicide Treatment among Cultivars

Differences among cultivars with respect to fungicide efficacy (calculated as percentage decrease in INC, DSI, FDK, and DON, and percentage increase in yield) varied across different experiments (Table 2 and Tables S3–S6). There was no significant difference in fungicide efficacy with respect to INC, DSI, or DON between moderately susceptible cultivars (‘Zhengmai 9023’ and ‘Xinong 979’) and susceptible cultivars (‘Hengguan 35’, ‘Zhongmai 895’, ‘Yumai 49-198’, ‘Zhoumai 18’, ‘Aikang 58’, ‘Zhengmai 379’, ‘Zhengmai 366’, and ‘Zhengmai 103’) in 2018 (Figure S1). However, fungicide efficacy was lower in terms of INC, DSI, and DON in ‘Zhoumai 27’, ‘Bainong 207’, and ‘Zhengmai 7698’ than in moderately susceptible cultivars (‘Zhengmai 9023’ and ‘Xinong 979’) under the high FHB intensity of 2018. Fungicide efficacy was lower with respect to INC, DSI, and DON in ‘Zhoumai 27’, ‘Hengguan 35’, and ‘Zhengmai 7698’ than in moderately susceptible cultivars (‘Zhengmai 9023’ and ‘Xinong 979’) under the low FHB intensity of 2021. Fungicide efficacy showed a similar effect on INC, DSI, and DON in 2018 and 2021, except for ‘Hengguan 35’ and ‘Bainong 207’ (Table 2; Figures S1 and S2).

3.5. Effect of Cultivar, Fungicide, Infection Level, and Climate Data after Anthesis on Control Efficacy of FHB

There was a strong and positive correlation between control efficacy with respect to DSI and INC (r = 0.934, p < 0.0001) and DSI and DON (r = 0.619, p < 0.0001). The control efficacy relative to DSI, INC, and FDK was negatively correlated with TEM (r = −0.433, p < 0.0001; r = −0.524, p < 0.0001; r = −0.353, p < 0.0001) and positively correlated with RH (r = 0.422, p < 0.0001; r = 0.510, p < 0.0001; r = 0.351, p < 0.0001) (

Table 3. Pairwise Pearson’s correlation coefficients for control efficacy (%) among FHB-related indices for all cultivars ^a.

	CE.DSI	CE.Yield	CE.FDK	CE.DON	TEM c	RH	PRE
CE.INC	0.934 ***	−0.082	0.093	0.481 *** b	−0.524 ***	0.510 ***	0.117
CE.DSI	…	0.127	0.192	0.619 ***	−0.433 ***	0.422 ***	0.147
CE.Yield	…	…	0.287 **	0.462 ***	0.427 ***	−0.435 ***	−0.001
CE.FDK	…	…	…	0.232 *	−0.353 ***	0.351 ***	−0.019
CE.DON	…	…	…	…	0.027	−0.054	−0.045
TEM	…	…	…	…	…	−0.994	0.083
RH	…	…	…	…	…	…	−0.050

^a Correlation coefficients were calculated between values for Fusarium head blight (FHB) incidence (CE.INC), disease severity index (CE.DSI), Fusarium-damaged kernels (CE.FDK), deoxynivalenol (CE.DON) concentration, yield (CE.Yield), and the three climatic variables in experiments 1–4 in the growing seasons of 2018 and 2021. ^b Asterisks indicate correlation coefficient significant at p = 0.05 (*), p = 0.01 (**), and p = 0.001 (***). ^c TEM, RH, PRE = Average daily temperature (°C), average daily relative humidity (%), and total rainfall (mm) from anthesis to until 21 days after anthesis.

). The control efficacy relative to yield was positively correlated with TEM (r = 0.427, p < 0.0001) and negatively correlated with RH (r = 0.435, p < 0.0001) (Table 3). A GLM was used to assess the relationships of cultivar, fungicide, epidemic, and weather variables with the control efficacy (%) of DSI, INC, FDK, DON, and yield. Of all the variables studied, the deviances of the control efficacy of DSI from a perfect model due to cultivar, fungicide, epidemic, TEM, RH, and PRE were 9.55, 6.66, 7.13, 0.87, 0.83, and 3.13, respectively, all of which were statistically significant (

Table 4. Deviance in control efficacy with respect to FHB incidence (INC), disease severity index (DSI), Fusarium-damaged kernels (FDK), deoxynivalenol (DON) concentration, and yield among cultivars.

Variables	Deviance a
	CE.INC b	CE.DSI	CE.FDK	CE.DON	CE.Yield
Cultivar	13.3375 ***	9.5478 ***	10.5844	11.2158 ***	0.9202
Fungicide	5.7684 ***	6.6590 ***	1.5846 *	6.6905 ***	1.2200 ***
Epidemic c	14.6220 ***	7.1310 ***	2.9653	0.1943 *	3.0231
TEM d	0.5646 ***	0.8652 ***	0.4606	0.0250 *	0.0124
RH	0.7009 **	0.8272 ***	1.5071	0.8935	0.1290
PRE	2.9386 ***	3.1298 ***	0.0508	1.0155 *	0.0416
Total	51.5380	36.494	60.1230	36.9490	12.7063

^a The deviance in control efficacy was calculated for Fusarium head blight (FHB) incidence (CE.INC), disease severity index (CE.DSI), Fusarium-damaged kernels (CE.FDK), deoxynivalenol (CE.DON) concentration, and yield (CE.Yield) accounting for cultivar, fungicide, and the three climatic variables. ^b Asterisks indicate whether the correlation coefficient is significant at p = 0.05 (*), p = 0.01 (**), or p = 0.001 (***). ^c Epidemic indicates INC in each year. In 2018, with a high INC, the INC mean of all treatments was above 40. In 2021, with low INC, the INC mean of all treatments was below 20. ^d TEM, RH, PRE = Average daily temperature (°C), average daily relative humidity (%) and total rainfall (mm) from anthesis to until 21 days after anthesis.

). The deviances of the control efficacy with respect to INC from a perfect model due to cultivar, fungicide, epidemic, TEM, RH, and PRE were 13.34, 5.77, 14.62, 0.56, 0.70, and 2.94, respectively, all of which were statistically significant (Table 4). The control efficacy with respect to DON was mainly affected by cultivar (deviance = 11.22), fungicide (deviance = 6.69), epidemic (deviance = 0.19), TEM (deviance = 0.03), and PRE (deviance = 1.02), but not by RH (Table 4). However, the control efficacy with respect to FDK and yield was only affected by fungicide application, and not by weather variables from the time of anthesis to until 21 days after anthesis.

4. Discussion

In this study, the efficacy of the control of FHB INC, DSI, and DON was not only affected by cultivar and fungicide application, but also by the severity of the FHB epidemic and post-anthesis weather (TEM, RH, and PRE). Utilizing resistant cultivars has been shown to be one of the most efficacious strategies for managing FHB in previous study [1]. Cultivar resistance was not only more important than fungicide treatment for the efficacy of the control (%) of FHB, INC, and DSI in the HHP in this study, but also for DON. Multiple factors have been analyzed to assess the impact of varieties and fungicide application on the efficacy of the control of FHB and DON. However, the results also indicated that the infection level was more significant than both the cultivars and the fungicide regarding the efficacy of the control of INC. The infection level was more important than fungicide in terms of the efficacy of the control of DSI. To control DON, cultivar was the most important factor in this study. The chemical control of DON levels in grains has been found to be most effective in moderately resistant cultivars, followed by moderately susceptible and then susceptible ones in previous studies [22,23]. In addition, post-anthesis weather can greatly influence the success of prevention and control measures with respect to FHB, with PRE being the most important factor in this study.

Cultivation of resistant varieties is usually seen as key to the prevention and control of FHB. The disease index and DON content from moderately resistant cultivars were significantly lower than those from susceptible cultivars [21]. In the heavy FHB outbreak year of 2018 (Tables S3 and S4; Figure S1), the disease appeared to be controlled more effectively by fungicide application on moderately susceptible cultivars and some susceptible cultivars (for example: ‘Zhongmai 895’ and ‘Zhoumai 18’) rather than on some susceptible cultivars (for example: ‘Zhengmai 7698’ and ‘Zhoumai 27’). In a year with low FHB intensity such as 2021 (Tables S5 and S6; Figure S2), it may not be necessary to apply fungicide to moderately susceptible cultivars or certain susceptible cultivars, as use of these cultivars alone may be effective in preventing economic loss. Therefore, we propose that adequate control can be achieved by planting moderately susceptible cultivars and some susceptible cultivars (for example: ‘Zhongmai 895’ and ‘Zhoumai 18’) in areas where and/or in years when the incidence of disease is forecast to be low to obtain high yields and high-quality grains while limiting the use of fungicides, especially in the absence of moderately resistant and moderately susceptible cultivars in the HHP, China.

To our knowledge, this work is the first detailed study aimed at understanding the effect of Jingxing fungicide application on moderately susceptible and susceptible cultivars for the efficient control of FHB and DON accumulation in winter wheat planted in the HHP. Previous studies focusing on the integration of cultivar resistance and fungicide application have mainly compared moderately resistant cultivars with susceptible cultivars [21,29,30,31,32,33]. In those studies, the INC and DSI of moderately resistant cultivars in the presence of fungicide have been found to be significantly lower than those of susceptible cultivars subjected to fungicide treatment [21,29,30,31]. Few comparisons have looked only at susceptible varieties, which are more suitable for growing in certain regions. It is, therefore, necessary to compare the control effects of fungicide application on different susceptible cultivars. Fungicide application had a similar beneficial effect on the key susceptible cultivars ‘Hengguan 35’, ‘Zhongmai 895’, ‘Aikang 58’, and ‘Zhoumai 18’, achieving a similar effect to that seen in moderately susceptible cultivars treated with fungicide. However, we recommend avoiding some susceptible wheat cultivars (such as ‘Zhengmai 7698’ and ‘Zhoumai 27’) when high levels of FHB are forecast in the HHP, as the disease pressure will remain high even with fungicide treatment.

In this study, cultivar × fungicide interactions were consistently significant, except for DON in experiment 2 in 2018 (p = 0.1372). Although we mostly tested susceptible cultivars, the effect of fungicide application on the variables measured varied among cultivars, suggesting that fungicide efficacy for controlling FHB and DON might be greater in some cultivars. A previous study also reported cultivar × fungicide interactions for all measured variables (DSI, DON, FDK, and yield) [22]. By contrast, Wegulo et al. [21] tested fungicides on winter wheat cultivars differing in their levels of resistance to FHB and did not always observe significant cultivar × fungicide interactions, possibly owing to the small (six) number of cultivars tested [21]. Several fungicides (such as tebuconazole) did not reduce FHB and DON [32], whereas Menniti et al. [33] found that the fungicides effectively controlled FHB and DON in durum wheat. The levels of DON accumulation in the grains of spring wheat cultivars with moderate resistance and moderate susceptibility has been shown to remain unaffected by the application of fungicides [34]. The variability in the response of FHB and DON to fungicides can be attributed not only to the timing and coverage of fungicide application and the species of pathogens involved, but also to the level of infection and post-anthesis weather.

The efficacy of fungicides for controlling FHB is highly variable and often unsatisfactory [13,22,24]. Sources of variability in previous studies have included the timing of fungicide application, the severity of the infection, the species and virulence of the pathogen, the level of resistance of the cultivars planted, and the level of FHB epidemic [35,36,37]. In this study, although fungicide treatments significantly decreased INC and DSI values in most cases, they did not have a significant control effect on FDK or DON accumulation in grains in the moderately susceptible cultivar ‘Zhengmai 9023’ under high disease outbreak conditions. Consistent with our results, other studies have shown that chemical control is effective for limiting FDK and DON accumulation in susceptible varieties when compared with untreated controls [17,23,38,39,40], but it does not reduce DON accumulation in grains of moderately susceptible and moderately resistant spring wheat cultivars [23,41]. The efficacy of fungicide application also depends on the severity of the disease. When disease severity was low, there was no significant difference in the INC, DSI, FDK, or DON values between treated and untreated cultivars in most cases. The inconsistent performance of wheat cultivars under high and low disease pressure has been reported in previous studies [21,31].

The efficacy of fungicides for controlling FHB in terms of TKW and yield is also highly uncertain [42]. For most cultivars in this study, plants treated with the Jingxing fungicide showed significantly higher TKW and yield relative to untreated controls, especially when the disease infestation was severe. When disease severity was low, there was no significant effect on the yield of ‘Xinong 979’, ‘Aikang 58’, ‘Zhoumai 18’, ‘Hengguan 35’, or ‘Zhengmai 366’. Among these cultivars, when disease severity was low, there was also no significant difference in the FDK, DON, TKW, or yield values of ‘Xinong 979’ or ‘Zhoumai 18’ treated with or without fungicide. These two cultivars, therefore, exhibited better disease resistance, as measured by FDK, DON, TKW, and yield, under low disease pressure than under high disease pressure.

The inexpensive and effective benzimidazole fungicide MBC has been extensively applied to control FHB and DON for more than four decades in China. Multiple investigations have demonstrated that the efficacy of MBC in managing FHB has been compromised by the advent of resistant pathogen populations in the field [43]. A novel cyanoacrylate fungicide, phenamacril, has been widely employed for the management of FHB and DON [44,45]. The widely utilized formulation for managing FHB and DON contamination in wheat growing areas in the middle and lower reaches of the Yangtze River is the Jingxing fungicide, comprising phenamacril (36%, w/v) and tebuconazole (12%, w/v), manufactured by Jiangsu Pesticide Research Institute Co., Ltd, No. 31-1, Hengjing Road, Nanjing Economic and Technological Development Zone, Nanjing, China. This investigation represents a preliminary assessment of the efficacy of the fungicide Jingxing applied to moderately susceptible and susceptible cultivars across various disease severities in the HHP region of China.

Importantly, the efficacy of the Jingxing fungicide in some susceptible cultivars can achieve the same effects as those of a fungicide treatment applied to moderately susceptible cultivars. There are no moderately resistant varieties, and only few moderately susceptible varieties, grown in the HHP. To control FHB more efficiently in this region, we suggest combining moderately susceptible or some susceptible cultivars with the application of the Jingxing fungicide for the control of FHB. We also recommend not planting highly susceptible wheat cultivars (such as ‘Zhengmai 7698’ and ‘Zhoumai 27’) when high levels of FHB are forecast in the HHP.