Population and Pathogenicity of Fusarium Species Associated with Fusarium Head Blight in a Wheat–Maize Cropping System in Sichuan Province, China

doi:10.21203/rs.3.rs-5307730/v1

Fusarium head blight (FHB) is a serious disease worldwide that leads to substantial economic losses. Wheat‒maize cropping is the dominant system in Sichuan Province, China. However, FHB has become increasingly severe in this system, such that Fusarium rot disease is also becoming a severe threat to maize. To understand the composition and pathogenicity of the Fusarium species associated with FHB, samples of typical symptomatic wheat spikes were collected from wheat‒maize cropping fields in 16 administrative districts of Sichuan Province, and Fusarium perithecia were obtained from both wheat straw and maize stubble. On the basis of morphological and molecular identification, 175 isolates from symptomatic wheat spikes were identified as five Fusarium species: F. asiaticum, F. avenaceum, F. graminearum, F. meridionale and F. proliferatum. Among them, F. asiaticum and F. graminearum were the dominant pathogenic species, with isolation frequencies of 75.43% and 20.57%, respectively. Additionally, 136 single-ascospore isolates from wheat straw or maize stubble were identified as F. asiaticum, F. equiseti, F. graminearum, F. meridionale, F. proliferatum and F. temperatum. Pathogenicity assays revealed that the Fusarium strains from all sources were capable of successfully infecting both wheat and maize. F. graminearum was highly pathogenic to both crops, whereas F. asiaticum appeared to be more strongly pathogenic to wheat than maize. This work will be helpful for understanding the cyclic infection caused by Fusarium species in wheat‒maize cropping systems and provide valuable data for the effective control of Fusarium rot disease in both wheat and maize.

Biological sciences/Microbiology/Fungi

Biological sciences/Microbiology/Pathogens

Fusarium head blight

Wheat‒maize cropping system

Wheat straw

Maize stubble

Population composition

Pathogenicity assays

Fusarium head blight (FHB), also known as wheat scab, is a destructive disease of wheat worldwide. Fusarium species can not only infect kernels, thus reducing the yield of wheat and other small-grain cereals but also produce mycotoxins that are harmful to both humans and animals^1,2. FHB was first reported in 1936 in China, and the Yangtze River valleys were known as the most severe FHB epidemic areas for a long period of time^3–5. The disease has spread northward since the last decade and now in the Huanghuai region, which is currently the main wheat production area of China⁶. Over the past decade, the annual average area affected by Fusarium head blight has exceeded approximately 4 million hectares, especially in 2012, when there was a severe outbreak^7,8.

The causal agents of FHB are multiple species of Fusarium, such as F. acuminatum, F. asiaticum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. verticillioides, and F. tricinctum^9–11. F. graminearum is the primary causal agent of FHB in North America and Australia^12,13. However, except for F. graminearum, F. meridionale and F. poae are also significant causative agents of FHB in southern Brazil^14,15. In Europe, the F. graminearum species complex (FGSC) has become increasingly prevalent in wheat^16–18. F. asiaticum and F. graminearum are the predominant pathogens of FHB in Asian^19–21. In China, at least 15 Fusarium species have been reported to cause FHB, among which F. graminearum and F. asiaticum are dominant^1,5,7,19,22. Usually, F. graminearum is the most prevalent species in the cooler region of northeastern China, whereas F. asiaticum is widely distributed in the warmer area of southern China. Therefore, climate and geographical location are likely pivotal factors in the prevalence of FHB epidemics²³.

Crop rotation is a common agricultural practice, effective crop rotation practices improve soil fertility in low-productivity soils²⁴. Moreover, strategic crop rotation reduces nitrogen fertilizer use and environmental losses, boosts grain yield, and supports sustainable agriculture and environmental health^25,26. Wheat‒maize rotation system can reduce weed density and biomass, thereby reducing the dosage of herbicides and other chemical pesticides while also increasing grain yield^27,28. During the last decade, the leftover straw from crop rotation has typically been treated by returning it to fields. As an important measure for improving soil structure and increasing soil fertility, this practice has been widely promoted and adopted in China^24,29. However, this has led to a significant increase in the number of pathogen sources in the field, which increases the risk of disease prevalence and disaster³⁰. Specifically, in wheat‒maize cropping rotation systems, the incidence of Fusarium maize stalk rot (MSR) and ear rot as well as FHB in wheat has increased over time^31,32. Many studies indicate that continuous wheat cropping or a cropping system of wheat following maize significantly increases the incidence and severity of FHB^12,33–35. Besides, crop residues (e.g., wheat straw, maize stubble and other host plants) can provide natural hotbeds for Fusarium species, increasing FHB risk. These residues may play an important role in genetic exchange between cereal and noncereal populations^31,36,37.

Wheat‒rice and wheat‒maize cropping systems have long coexisted in Sichuan Province, China. However, in the past decade, wheat‒maize cropping systems have gradually become more popular because of the promotion of straw return and no-tillage practices. Currently, research on the host selection of Fusarium in cyclic infection between wheat and maize is rather limited. In this planting system, crop residues carrying Fusarium propagules are considered inoculum sources for both FHB and MSR. However, the relations between FHB and crop residues remain unknown, and it is unclear whether all Fusarium species are pathogenic to subsequent crops. Therefore, the objectives of this study were (1) to define the population composition of Fusarium species in wheat‒maize cropping system and (2) to determine the aggressiveness of Fusarium isolates on both wheat straw and maize stubble by cross-infection.

Identification of Fusarium species in a wheat‒maize cropping system

A total of 311 single-spore isolates identified morphologically as Fusarium spp. were obtained (Fig. 1). The details of all single-spore isolates are listed in supplementary Table S1.

Using the species-specific primer pair Fg16F/Fg16R, 92 isolates were identified as F. asiaticum, accounting for 74.2%, and 32 isolates were F. graminearum, accounting for 25.8%. A maximum parsimony tree containing the remaining isolates and partial isolates subjected to specific PCR amplification was constructed on the basis of these TEF1α sequences (TL = 564, CI = 0.832, RI = 0.955, and RCI = 0.794), as shown in Fig. 2. In the phylogenetic tree, 234 Fusarium isolates were clustered into four clades with their corresponding species from GenBank. Those in the first clade, including all F. asiaticum, F. graminearum and F. meridionale isolates, belong to the FGSC. F. equiseti grouped into the second clade with a 99% bootstrap value. F. temperatum and F. proliferatum were grouped into the third clade. Four isolates associated with FHB symptoms clustered in the F. avenaceum clade.

Fusarium collection from wheat

On the basis of morphological and molecular analyses, 175 Fusarium isolates were identified as F. asiaticum (132 strains, 75.43%), F. graminearum (36 strains, 20.57%), F. avenaceum (4 strains, 2.29%), F. meridionale (2 strains, 1.14%) and F. proliferatum (1 strain, 0.57%) (Table 1). F. asiaticum and F. graminearum were the dominant species. F. asiaticum was found at each sampling location, but F. avenaceum was found only in Guang’an. F. meridionale and F. proliferatum were distributed only in Ziyang and Mianyang, respectively.

Saprophytic Fusarium collection

Thirty-two strains were collected from maize stubble, and 104 strains were collected from wheat straw. Six Fusarium species were identified from maize stubble: F. asiaticum (10 strains, 31.25%), F. graminearum (10 strains, 31.25%), F. meridionale (5 strains, 15.63%), F. temperatum (3 strains, 9.38%), F. equiseti (2 strains, 6.25%) and F. proliferatum (2 strains, 6.25%). The isolates collected from wheat straw included five species: F. asiaticum (46 strains, 44.23%), F. graminearum (41 strains, 39.42%), F. meridionale (13 strains, 12.50%), F. proliferatum (3 strains, 2.88%) and F. equiseti (1 strain, 0.96%) (Table 1). Thus, the Fusarium composition of maize stubble and wheat straw differed at the same sampling site. F. asiaticum and F. meridionale were isolated from wheat straw, whereas F. graminearum was only isolated from maize stubble at the sampling site in Ya’an. F. proliferatum was isolated from wheat straw but not from maize stubble in Chengdu.

Sources	FHB	Wheat straw	Maize stubble	Total
F. asiaticum	132	46	10	188
F. graminearum	36	41	10	87
F. meridionale	2	13	5	20
F. proliferatum	1	3	2	6
F. avenaceum	4	0	0	4
F. equiseti	0	1	2	3
F. temperatum	0	0	3	3
Total	175	104	32	311

Table 1. The population composition of Fusarium isolates from different sources in a wheat‒maize cropping system.

Pathogenicity assays

Pathogenicity of Fusarium spp. from wheat spikes

All tested strains from wheat spikes caused apparent FHB symptoms, and the organisms were successfully reisolated from symptomatic tissues. No symptoms were observed on the control plants. The disease index varied among the different Fusarium species (Fig. 3). F. asiaticum had the greatest disease index (47.13), followed by F. graminearum (42.12). F. asiaticum and F. graminearum are considered highly pathogenic. The remaining three species with disease indices less than 21 were weakly pathogenic.

Aggressiveness of the strains between F. asiaticum and F. graminearum

All the Fusarium strains tested for pathogenicity in the six wheat cultivars were able to produce FHB symptoms but showed variation in aggressiveness. Averaged across the six cultivars, the mean disease index of the F. asiaticum strains was greater than 45, indicating that it was highly aggressive, and no significant differences were found among the 5 strains (P > 0.05). The F. graminearum strains, with a mean disease index of less than 45, were less aggressive. Furthermore, there was a significant difference among the F. graminearum strains (P < 0.05) (Table 2). The cultivars differed significantly in terms of the main effect on the FHB disease index (P = 0.001). However, there was no significant two-way interaction effect between the cultivar and the Fusarium strains tested (P = 0.065) (Table 3).

*Fusarium* spp.	Isolate	Location	Disease index
*Fusarium* spp.	Isolate	Location	Chuanyu 20	Mianyang 31	Chuanmai 104	Shumai 969	Sumai 3	Wangshuibai	Mean
F. asiaticum	4a	Shifang, Deyang	67.33	53.89	45.56	40	26.67	17.78	41.87 ± 7.35 ab
	8a	Jiangyou, Mianyang	54.22	57.78	48.33	53.33	31.67	26.11	45.24 ± 5.36 a
	20a	Shuangliu, Chengdu	55.2	63.89	60	50	27.78	24.44	46.89 ± 6.85 a
	39a	Wenjiang, Chengdu	63	61.11	52.67	47.78	29.44	21.67	45.95 ± 6.91 a
	69a	Jiangyou, Mianyang	68.89	75.56	58.89	51.11	33.89	25.56	52.32 ± 7.99 a
F. graminearum	1a	Yucheng, Ya’an	45.33	47.22	38.89	33.89	24.44	20.67	35.07 ± 4.43 abc
	5a	Yucheng, Ya’an	42	35	22.78	20	21.11	15.56	26.08 ± 4.15 bc
	10a	Renshou, Meishan	24.25	29.33	25	13.89	7.78	6.67	17.82 ± 3.94 c
	16a	Wenjiang, Chengdu	33.67	34	27.22	10.56	8.89	4.44	19.80±5.44 c

Table 2. Disease indices of six wheat cultivars inoculated with 5 strains of Fusarium asiaticum and 4 strains of F. graminearum from FHB. Data are presented as mean ± standard deviation. Different lowercase letters in the same column indicate that there is a significant difference among the means, as determined by Fisher's LSD test at P = 0.05.

Sources of variation	Degrees of freedom	Mean square	F value	P value
Isolates	1	6313.543	126.045	0.001
Cultivars	5	1590.684	31.757	0.001
Isolates × Cultivars	5	113.522	2.266	0.065
Error	42	50.089

Table 3. Analysis of variance for the disease indices of six wheat cultivars inoculated with 5 strains of Fusarium asiaticum and 4 isolates of F. graminearum.

Pathogenicity of saprophytic Fusarium species

All the Fusarium strains from either the maize stubble or the wheat straw were able to cause FHB and MSR to different extents. Among the five Fusarium species isolated from wheat straw, F. graminearum was the most aggressive species for causing FHB, with a disease index of 55.67, followed by F. equiseti (40.97) and F. asiaticum (36.60). F. meridionale and F. proliferatum were relatively weakly aggressive in wheat, with disease indices of 16.91 and 16.37, respectively (Fig. 4a). Among the six Fusarium species isolated from maize stubble, F. graminearum was the most aggressive at causing FHB, with a disease index of 54.21, which was significantly greater than that of the other species. Moreover, the F. temperatum, F. asiaticum, F. meridionale and F. equiseti strains were highly aggressive, with disease indices greater than 36.00. F. proliferatum showed relatively weak aggressiveness, with a disease index of 31.07 (Fig. 4b).

Among the five species isolated from wheat straw, F. graminearum displayed the greatest ability to cause MSR, with a disease index of 46.07, followed by F. proliferatum (33.33), F. equiseti (32.92) and F. meridionale (26.16). F. asiaticum, with a disease index of 13.41, exhibited the weakest aggressiveness in maize (Fig. 4c). With disease indices ranging from 39.74 to 47.29, F. graminearum, F. equiseti, F. meridionale and F. proliferatum from maize stubble displayed high pathogenicity in maize. F. asiaticum was also the weakest pathogenic species, with a disease index of 12.27 (Fig. 4d).

Among the five Fusarium species from MSR, F. graminearum, with a disease index of 63.73, was the most aggressive species in wheat, followed by F. verticillioides (58.86). F. equiseti and F. proliferatum were less pathogenic, with disease indices of 26.94 and 23.77, respectively. With a disease index of 10.51, F. kyushuense was the species with the weakest effect on wheat (Fig. 5a). Among the five Fusarium species isolated from FHB, F. graminearum was the most aggressive species for infecting maize stems, with a disease index of 51.75, followed by F. proliferatum (34.20) and F. asiaticum (23.11). F. meridionale and F. avenaceum were relatively weakly pathogenic, with disease indices of 18.32 and 11.85, respectively (Fig. 5b).

FHB is the most serious fungal disease in wheat production. Fusarium species can not only infect a range of cereal crops during the growing season but also survive saprophytically on crop residues, where many ascospores released from perithecia serve as the primary inoculum for subsequent crops^11,29,30. FHB affects a large area and has a negative effect on the yield and quality of wheat in China^5,6,8. In this study, we confirmed that F. asiaticum was the dominant species of FHB in the wheat‒maize rotation system in Sichuan Province. F. graminearum was also frequently found, with an isolation frequency of 20.57% in wheat spikes. Previous studies have suggested that wheat‒maize rotation selects for F. graminearum, whereas wheat‒rice rotation trends toward F. asiaticum selection^6,31,38. However, the dominant species varied by climate and region-specific conditions. F. asiaticum is the dominant Fusarium species responsible for FHB^7,38,39, which is in agreement with our results. However, our study revealed that the prevalence of F. graminearum was significantly greater than that in other provinces along the Yangtze River. The wheat‒maize cropping system is more prevalent in Sichuan than in other provinces in this region. Therefore, an important reason may be the pressure by maize in rotation. In addition, F. graminearum was more aggressive than F. asiaticum toward maize in our study, which confirms that a wheat‒maize rotation may contribute to the selection of F. graminearum.

Fusarium meridionale has previously been reported on wheat and barley but typically occurs at low frequencies^7,40. In Nepal, Brazil and northern Argentina, F. meridionale is the predominant species on maize^41–43, and small populations of F. meridionale have also been reported in China and Korea^21,44. In the present study, the isolation frequency of F. meridionale was low, although it was greater in the saprophytic stage than in the wheat spikes. There was also a trend for F. meridionale strains to be more aggressive in maize than in wheat. Thus, F. meridionale appears to prefer maize. F. temperatum, which is closely related to F. subglutinans, has previously been reported as a new biological species causing maize ear rot in Jilin, Hubei, and Guizhou in China⁴⁵. Nonetheless, this study is the first report of F. temperatum on maize stubble in Sichuan. F. temperatum was highly pathogenic in the pathogenicity assays,indicating that it is a potential threat to maize and wheat.

In this wheat‒maize cropping system, F. asiaticum, F. graminearum, F. meridionale and F. proliferatum are common species isolated from all sources, including wheat spikes, wheat straw and maize stubble. However, F. avenaceum was isolated only from spikes. F. equiseti and F. temperatum were present only at the saprophytic stage. Our data indicate that the Fusarium population of saprophytic sources was more diverse than that of infected kernels. Both F. asiaticum and F. graminearum from saprophytic sources were dominant, with almost equal isolation frequencies. F. asiaticum may be the dominantpathogen of FHB, whereas F. graminearum is the dominantpathogen of MSR. In the whole wheat‒maize rotation system, the Fusarium population is forced to undergo two selection cycles (wheat or maize) every year, which ultimately leads to significant competition between F. asiaticum and F. graminearum during both the infection stage and the saprophytic stage.

Preceding crop residues are both important diversity reservoirs and inoculums of pathogenic Fusarium to subsequent crops^30,46,47. In this study, Fusarium strains from the saprophyticstage were pathogenic to both wheat and maize, indicating that crop residues might act as a primary source of inoculums for FHB and MSR.

In the genus Fusarium, some species are pathogenic to a wide range of economically important crops, usually causing seedling blight, root rot, stem rot, and ear rot^15,18,48. Fusarium species may interact among different hosts under certain environmental conditions, such as the rotation of wheat with maize or soybean intercropped with maize combined with the use of no or minimum tillage. Our pathogenicity assays verified that strains from different sources have no specialization and are similarly aggressive when causing FHB and MSR. Crop rotation systems are very common and play a role in reducing the use of herbicides and other chemical pesticides, as well as increasing grain production^25–27. However, the aggressiveness of Fusarium strains might increase due to crop rotation⁴⁹. Hence, further research is needed to better understand the influence of a rotation system with different crops on the ecology and epidemiology of Fusarium populations. Wide variation among Fusarium strains in wheat has been reported previously^37,50,51. In this study, variations in the aggressiveness of Fusarium strains were also observed in wheat and maize. The aggressiveness of F. asiaticum and F. graminearum strains significantly differed (P < 0.05) within species. This finding suggests that screening crop genotypes for resistance requires a mixture of several different highly aggressive strains that are prescreened on target hosts.

In conclusion, F. asiaticum, F. graminearum, F. avenaceum, F. meridionale, F. proliferatum, F. temperatum and F. equiseti were successfully identified from wheat‒maize cropping fields in Sichuan Province. These Fusarium species can maintain cyclic infection to cause Fusarium rot in this cropping system under certain environmental conditions, and crop residues are the most crucial link. In this wheat‒maize rotation system, F. graminearum was distributed among all sources with a high isolation frequency and was highly pathogenic to both wheat and maize. However, no obvious host selectivity was observed. F. asiaticum was found ata very high frequency in the wheat spike samples, but itappeared to favorwheat and be more strongly pathogenic to wheat than to maize. Thus, the findings of the current study may be helpful for understanding the temporal and spatial dynamics of Fusarium populations in local rotation systems. Thus, long-term monitoring of the composition of Fusarium species in this system is imperative.

The wheat‒maize cropping system is a prevalent cultivation pattern in Sichuan Province, China. In this system, FHB has become increasingly severe, such that Fusarium rot disease is also becoming a severe threat to maize. Here, we present the first report of the population composition and pathogenicity of Fusarium species present in naturally infected wheat spikelets and crop residues in a wheat‒maize cropping system in Sichuan Province. We confirmed that F. graminearum is a highly pathogenic species in both wheat and maize, whereas F. asiaticum appears to favor wheat and to be more strongly pathogenic in wheat than in maize. This work will be helpful for understanding the cyclic infection of Fusarium species in wheat‒maize cropping systems and provide valuable references for the effective control of Fusarium rot disease. This information will be helpful for predicting and preventing Fusarium-related diseases and will suggest possibilities for disease management through agricultural practices.

Sample collection and isolation

Samples of typical wheat spikes characterized by clear symptoms of FHB were collected during the wheat growing season from 16 different administrative districts throughout the wheat‒maize cropping regions in Sichuan Province, China. Furthermore, samples of wheat straw exhibiting black-colored Fusarium perithecia were collected during the maize jointing stage, and samples of maize stubbles exhibiting black-colored Fusarium perithecia were collected during the wheat elongation stage from the same sampling sites distributed in five wheat‒maize cropping regions: Chengdu, Ya’an, Deyang, Mianyang, and Meishan in Sichuan Province. All the sampling sites were distributed in different villages or fields. The samples from each site were investigated via a five-point sampling method. For each sample, at least ten affected wheat spikes, straws or stubble from each sample were collected. All the samples were air dried at room temperature (25 to 30°C) for 24 h and then stored at –20°C until processing.

The spikelets were cut into pieces 3–4 mm in length, surface sterilized in 75% ethanol (V/V) for 3 min, followed by 0.5% NaOCl (W/V) for 2 min, and then rinsed three times with sterilized distilled water. After drying on sterile filter paper, the spikelets were placed onto potato dextrose agar (PDA) and incubated for 4 days at 25°C. Mycelia were subsequently inoculated on PDA or carboxymethyl cellulose (CMC) media to produce spores. Single-spore isolates from wheat spikes were finally acquired via the method described by Qu et al.¹⁹ Maize stubbles and wheat straws exhibiting perithecia were cut into small segments. The surfaces were disinfected in 75% ethanol (V/V) for 2 min and 0.5% NaOCl (W/V) for 30 s, rinsed three times with sterilized distilled water, and blotted dry on sterile filter paper. Under a microscope, a single perithecium was removed from the segment and crushed for single-ascospore isolation. Single ascospore was purified via the method of Pereyra⁴⁶ and Dill-Macky³⁶. One or two isolates were derived from each sampling location. The colony characteristics and spore morphology of these single-spore isolates on PDA or CMC were observed and recorded according to the methods described by Leslie and Summerell⁵².

DNA extraction

Each isolate was cultured on PDA at 27°C for 3‒5 d, and genomic DNA was extracted from mycelia via the cetyltrimethylammonium bromide (CTAB) method⁵³. The DNA concentration and quality were estimated via a NanoDrop^TM 2000 spectrophotometer (Thermo Fisher Scientific, Delaware, USA). The DNA was diluted to 50 ng/μL and stored at -20 °C.

PCR amplification and sequencing

PCR amplification was carried out in a final volume of 20 μL containing 1 μL of genomic DNA (approximately 50 ng), 0.5 μL of each primer (10 μM), 10 μL of Taq PCR Master Mix (Sangon Biotech, Shanghai, China), and 8 μL of ddH2O. The primer pairs Fg16F (5’-CTCCGGATATGTTGCGTCAA-3’) and Fg16R (5’-GGTAGGTATCCGACATGGCAA-3’) were used for the detection of F. asiaticum and F. graminearum described by Nicholson et al.⁵⁴. The cycling parameters were 95 °C for 5 min, followed by 35 cycles of 45 s at 95 °C, 30 s at 55 °C, and 1 min at 72 °C and 10 min at 72 °C⁵⁵. The F. graminearum isolate amplicon is 410 bp, and the F. asiaticum amplicon is 497 bp. The translation elongation factor 1α (TEF1α) sequence was amplified via the primer pair EF-1 (5’-ATGGGTAAGGAGGACAAGAC-3’) and EF-2 (5’-GGAAGTACCAGTGATCATG-3’)⁵⁶. The PCR conditions were as follows: initial denaturation for 2 min at 96 °C; 35 cycles of 30 s at 94 °C, 30 s at 53 °C, and 30 s at 72 °C; and a final extension for 10 min at 72 °C. The PCR products were assessed via 1.5% agarose gel electrophoresis. TEF1α sequences were obtained from Sangon Biotech Co., Ltd. (Shanghai, China). All TEF1α sequences of the Fusarium isolates obtained in this study were deposited in GenBank.

Phylogenetic analysis

The TEF1α sequences from the Fusarium isolates were compared to sequences in the National Center for Biotechnology Information (NCBI) database via BLASTn (BLAST network services) and then aligned together with reference sequences obtained from GenBank via Clustal X v2.0, with characters weighted equally. Phylogenetic trees were constructed in MEGA 6.0 via the maximum parsimony approach based on the Kimura 2-parameter model, rooted with Bipolaris oryzae (KJ939510) as the outgroup. The bootstrap values provided on the phylogenetic dendrogram were generated with 1000 replicates, and alignment gaps were excluded. In addition, descriptive tree statistics, such as parsimony (Tree Length [TL], Consistency Index [CI], Retention Index [RI], and Related Consistency Index [RCI]), were calculated.

Plant materials

Six winter wheat cultivars with different levels of resistance to FHB and one maize cultivar were used. “Mianyang 31” (susceptible), “Chuanyu 20” (moderately susceptible), “Chuanmai 104” (moderately susceptible) and “Shumai 969” (moderately resistant) are the main cultivars in Sichuan, and the resistance of these cultivars to FHB has been evaluated in our laboratory for several years. “Sumai 3” (resistant) and “Wangshuibai” (resistant) are the most widely used sources of FHB resistance worldwide³. One maize cultivar, “Chuandan 428” (susceptible) was used, which is a primary maize cultivar in Sichuan. Each wheat cultivar was planted in three blocks (2 m × 4 m per block) according to standard agronomic practices at Wenjiang Farm (wheat‒maize continuously planted for a minimum of 6 years) in Chengdu (the experimental farm of Sichuan Agricultural University). After the wheat was harvested, maize was planted in three blocks (4 m × 6 m per block) according to standard agronomic practices. The experiment involved a randomized complete block design with three replicates.

Preparation of Fusarium inoculums

A conidial suspension was prepared by scraping mycelia into Czapek-Dox media and shaking at 120 rpm for 6 d at 27 °C. The suspension was filtered with layers of cheesecloth and adjusted under a microscope to a concentration of 1 × 10⁵ conidia in 1 mL of sterile water containing 0.01% Tween 20.

Pathogenicity assays

Four experiments were carried out to evaluate the aggressiveness of these Fusarium strains: (i) 30 strains of F. asiaticum, 15 strains of F. graminearum and all F. avenaceum (4 strains), F. meridionale (2 strains), and F. proliferatum (1 strain) from spikes were used to test the pathogenicity to wheat plants (variety Chuanyu 20). (ii) 5 representative strains of F. asiaticum and 4 strains of F. graminearum from wheat were selected to evaluate the aggressiveness to six wheat cultivars (variety Chuanyu 20, Mianyang 31, Chuanmai 104, Shumai 969, Sumai 3 and Wangshuibai). (iii) 27 strains from wheat straw and 32 strains from maize stubbles were selected to test the pathogenicity in wheat (variety Chuanyu 20) and maize (variety Chuandan 428), respectively. And (iv) 52 strains from wheat and 64 strains from maize (previously identified) were selected to cross-infect on maize (variety Chuandan 428) and wheat (variety Chuanyu 20).

During the inoculation process of wheat, a precise single-floret injection technique was employed to assess the pathogenicity of the samples in wheat³⁸. Twenty spikes at the 50% anthesis stage were randomly selected from each block for treatment with each representative strain. Twenty microliters of conidial suspensions were injected into the central spikelet. In addition, twenty spikes from each block were inoculated with 20 µL of sterile water as controls. After inoculation, each wheat spike was covered with a plastic bag for 72 h to maintain humidity. Pathogenicity was assessed as the percentage of infected spikelets at 21 days after inoculation. Disease severity was estimated visually on a 0-4 scale as described in Zhang et al.⁴⁸ Disease index =(∑(A×B))/(∑B×4) ×100, where A represents the disease scale (0, 1, 2, 3, or 4) and where B represents the number of plants at each disease level.

During the inoculation of maize, a channel inoculation method was used to inoculate the maize stems. The penultimate stem internode section was pricked with an obtuse needle, and 1 mL of conidial suspension was injected into the wounded tissue at the tasseling stage via a sterile syringe. Maize stems inoculated with sterile water were used as negative controls. Fifteen maize plants were inoculated with each strain in each block in a randomized design. Disease levels were assessed on day 20 after inoculation. Each inoculated maize stem was divided, and the MSR severity was rated on a scale of 1-9, as described in Zhang et al.⁴⁸ Disease index = (∑(A×B))/(∑B×9) ×100, where A represents the disease scale (1, 3, 5, 7, or 9), and B represents the number of plants at each disease level.

Statistical analyses

The aggressiveness of Fusarium strains on wheat and maize was analyzed via one-way analysis of variance (ANOVA). The data were tested for a normal distribution and equal variance prior to ANOVA. When ANOVA indicated a significant difference between treatments (P < 0.05), the treatment data were compared via Fisher's least significant difference (LSD) test. The data of the nine F. asiaticum and F. graminearum strains tested in the six cultivars were assessed for homogeneity of variance via Levene’s test. The variability in the observations in the experiments for the disease index was comparable, and an ANOVA could be validly carried out. Fisher's LSD was calculated at the 5% level to determine the significance differences in the treatment means. All analyses were performed with IBM SPSS Statistics v21.0 (Armonk, New York, USA).

Author Contribution

Xiaofang Sun: Writing–original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization, Software, Validation, Visualization. Rui Yang: Writing–review & editing, Formal analysis, Validation. Huimin Tang: Resources, formal analysis. Miaomiao Ma: Writing–review & editing. Huabao Chen: Data curation. Xiaoli Chang: Writing–review & editing, Project administration. Min Zhang: Methodology, Project administration, Conceptualization. Guoshu Gong: Methodology, Writing–review & editing, Visualization, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

Acknowledgement

We are grateful to Shan He, Qinjun Tao, and Nian Li for their help in sample collection and field trials. We appreciate the support provided by the Plant Pathology Laboratory of the Department of Plant Protection at Sichuan Agricultural University.

Data Availability

The data generated during this study are available upon reasonable request to the corresponding author.

Funding

This research was supported by the Sichuan Innovational Team of Industry Technology System of Modern Agriculture (SCCXTD-2024-2).

Competing interests

The authors declare no competing interests.

Additional information

The following supporting information can be downloaded from Table S1: Information on Fusarium species isolated from wheat spikes, wheat straw and maize stubble collected from different wheat‒maize cropping regions in Sichuan, China.

Correspondence and requests for materials should be addressed to G. Gong.

Reprints and permissions information is available at www.nature.com/reprints.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

Parry, D. W., Jenkinson, P. & Mcleod, L. Fusarium ear blight (scab) in small grain cereals—a review. Plant. Pathol. 44, 207–238 (1995).
Shah, L. et al. Integrated control of Fusarium head blight and deoxynivalenol mycotoxin in wheat. Plant. Pathol. 67, 532–548 (2018).
Bal, G. Scab of wheat: prospects for control. Plant. Dis. 78, 760 (1994).
Li, W., Hu, Y. C., Chen, Y., Zhang, A. X. & Chen, H. G. Phylogenetic analysis, chemotype diversity, and pathogenicity of the Fusarium graminearum clade in the Yangtze basin. Mycosystema. 29, 51–58 (2010).
Chen, H. et al. Carbendazim-resistance of Gibberella zeae associated with Fusarium head blight and its management in Jiangsu Province, China. Crop Prot. 124, 104866 (2019).
Hao, J. J. et al. Analysis of Fusarium graminearum species complex from wheat–maize rotation regions in Henan (China). Plant. Dis. 101, 720–725 (2017).
Zhang, H. et al. Population analysis of the Fusarium graminearum species complex from wheat in China show a shift to more aggressive isolates. PLoS ONE. 7, e31722 (2012).
Huang, C., Jiang, Y. Y., Wu, J. W., Qiu, K. & Yang, J. J. Occurrence characteristics and reason analysis of wheat head blight in 2018 in China. Plant. Prot. 45, 160–163 (2019).
Starkey, D. E. et al. Global molecular surveillance reveals novel Fusarium head blight species and trichothecene toxin diversity. Fungal Genet. Biol. 44, 1191–1204 (2007).
Van Der Lee, T., Zhang, H., Van Diepeningen, A. & Waalwijk, C. Biogeography of Fusarium graminearum species complex and chemotypes: a review. Food Addit. Contam. Part. A. 32, 453–460 (2015).
Karlsson, I., Persson, P. & Friberg, H. Fusarium head blight from a microbiome perspective. Front. Microbiol. 12, 628373 (2021).
Ward, T. J. et al. An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America. Fungal Genet. Biol. 45, 473–484 (2008).
Burlakoti, R. R., Tamburic-Ilincic, L., Limay‐Rios, V. & Burlakoti, P. Comparative population structure and trichothecene mycotoxin profiling of Fusarium graminearum from corn and wheat in Ontario, central Canada. Plant. Pathol. 66, 14–27 (2017).
Scoz, L. B. et al. Trichothecene mycotoxin genotypes of Fusarium graminearum sensu stricto and Fusarium meridionale in wheat from southern Brazil. Plant. Pathol. 58, 344–351 (2009).
Pereira, C. B. et al. Five-year survey uncovers extensive diversity and temporal fluctuations among Fusarium head blight pathogens of wheat and barley in Brazil. Plant. Pathol. 70, 426–435 (2021).
Lindblad, M. et al. Deoxynivalenol and other selected Fusarium toxins in Swedish wheat — Occurrence and correlation to specific Fusarium species. Int. J. Food Microbiol. 167, 284–291 (2013).
Aamot, H. U. et al. Genetic and phenotypic diversity within the Fusarium graminearum species complex in Norway. Eur. J. Plant. Pathol. 142, 501–519 (2015).
Beccari, G. et al. Causal agents of Fusarium head blight of durum wheat (Triticum durum Desf.) in central Italy and their in vitro biosynthesis of secondary metabolites. Food Microbiol. 70, 17–27 (2018).
Qu, B. et al. Geographic distribution and genetic diversity of Fusarium graminearum and F. asiaticum on wheat spikes throughout China. Plant. Pathol. 57, 15–24 (2008).
Suga, H. et al. Molecular characterization of the Fusarium graminearum species complex in Japan. Phytopathology. 98, 159–166 (2008).
Lee, J. et al. Population structure of and mycotoxin production by Fusarium graminearum from maize in south Korea. Appl. Environ. Microbiol. 78, 2161–2167 (2012).
Shen, C. et al. Geographic distribution of trichothecene chemotypes of the Fusarium graminearum species complex in major winter wheat production areas of China. Plant. Dis. 96, 1172–1178 (2012).
Yan, Z. et al. Evaluation of Fusarium head blight resistance in 410 Chinese wheat cultivars selected for their climate conditions and ecological niche using natural infection across three distinct experimental sites. Front. Plant. Sci. 13, 916282 (2022).
Ye, X. et al. The influence of a year-round tillage and residue management model on soil N fractions in a wheat-maize cropping system in central China. Sci. Rep. 9, 4767 (2019).
Shu, X. et al. Response of soil N₂O emission and nitrogen utilization to organic matter in the wheat and maize rotation system. Sci. Rep. 11, 4396 (2021).
Zhang, L. et al. Long term effects of crop rotation and fertilization on crop yield stability in southeast China. Sci. Rep. 12, 14234 (2022).
Brankov, M., Simić, M. & Dragičević, V. The influence of maize – winter wheat rotation and pre-emergence herbicides on weeds and maize productivity. Crop Prot. 143, 105558 (2021).
Simić, M. et al. Integrated effects of crop rotation and different herbicide rates in maize (Zea mays L.) production in central Serbia. Crop Prot. 187, 106913 (2025).
Chen, Y., Wang, J. Q., Yang, R. M. & Ma, Z. H. Current situation and managements trategies of Fusarium head blight in China. Plant. Prot. 43, 11–17 (2017).
Ma, Z. H., Chen, Y. & Yin, Y. N. Epidemiological analysis and management strategies of Fusarium head blight of wheat. China Acad. J. Electron. Publ House. 34, 464–469 (2020).
Zhang, H. et al. Crops are a main driver for species diversity and the toxigenic potential of Fusarium isolates in maize ears in China. World Mycotoxin J. 9, 701–716 (2016).
Xi, K. et al. Species diversity and chemotypes of Fusarium species associated with maize stalk rot in Yunnan province of southwest China. Front. Microbiol. 12, 652062 (2021).
Champeil, A., Doré, T. & Fourbet, J. F. Fusarium head blight: epidemiological origin of the effects of cultural practices on head blight attacks and the production of mycotoxins by Fusarium in wheat grains. Plant. Sci. 166, 1389–1415 (2004).
Kelly, A. C. et al. Diversity of Fusarium head blight populations and trichothecene toxin types reveals regional differences in pathogen composition and temporal dynamics. Fungal Genet. Biol. 82, 22–31 (2015).
Tillmann, M., Von Tiedemann, A. & Winter, M. Crop rotation effects on incidence and diversity of Fusarium species colonizing stem bases and grains of winter wheat. J. Plant. Dis. Prot. 124, 121–130 (2017).
Dill-Macky, R. & Jones, R. K. The effect of previous crop residues and tillage on Fusarium head blight of wheat. Plant. Dis. 84, 71–76 (2000).
Kadziene, G. et al. Tillage and cover crop influence on weed pressure and Fusarium infection in spring cereals. Crop Prot. 127, 104966 (2020).
Yang, M. et al. Host and cropping system shape the Fusarium population: 3ADON-producers are ubiquitous in wheat whereas NIV-producers are more prevalent in rice. Toxins. 10, 115 (2018).
Shi, W. Q. et al. Analysis on the population structure of Fusarium pathogenic spp. and its mycotoxin chemotypes in Fusarium head blight epidemic region. Acta Phytopathol. Sin. 41, 486–494 (2011).
Boutigny, A. L. et al. Analysis of the Fusarium graminearum species complex from wheat, barley and maize in South Africa provides evidence of species-specific differences in host preference. Fungal Genet. Biol. 48, 914–920 (2011).
Desjardins, A. E. & Proctor, R. H. Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from maize in Nepal and identification of a putative new lineage. Fungal Biol. 115, 38–48 (2011).
Sampietro, D. A., Ficoseco, M. E. A., Jimenez, C. M., Vattuone, M. A. & Catalán, C. A. Trichothecene genotypes and chemotypes in Fusarium graminearum complex strains isolated from maize fields of northwest Argentina. Int. J. Food Microbiol. 153, 229–233 (2012).
Kuhnem, P. R. et al. Composition and toxigenic potential of the Fusarium graminearum species complex from maize ears, stalks and stubble in Brazil. Plant. Pathol. 65, 1185–1191 (2016).
Duan, C. et al. Identification of pathogenic Fusarium spp. causing maize ear rot and potential mycotoxin production in China. Toxins. 8, 186 (2016).
Wang, J. et al. Molecular identification, mycotoxin production and comparative pathogenicity of Fusarium temperatum isolated from maize in China. J. Phytopathol. 162, 147–157 (2014).
Pereyra, S. A. & Dill-Macky, R. Colonization of the residues of diverse plant species by Gibberella zeae and their contribution to Fusarium head blight inoculum. Plant. Dis. 92, 800–807 (2008).
Lu, J. et al. The impact of different rotation regime on the soil bacterial and fungal communities in an intensively managed agricultural region. Arch. Microbiol. 204, 142 (2022).
Zhang, Z. B., Gao, Z. G., Zhang, X. F., Zhuang, J. H. & Sui, H. Pathogenicity of Fusarium graminearum causing wheat head scab and maize stalk rot. Liaoning Agric. Sci. 1–4 (2010).
Sella, L., Gazzetti, K., Castiglioni, C., Schäfer, W. & Favaron, F. Fusarium graminearum possesses virulence factors common to Fusarium head blight of wheat and seedling rot of soybean but differing in their impact on disease severity. Phytopathology. 104, 1201–1207 (2014).
Xue, A. G., Armstrong, K. C., Voldeng, H. D., Fedak, G. & Babcock, C. Comparative aggressiveness of isolates of Fusarium spp. causing head blight on wheat in Canada. Can. J. Plant. Pathol. 26, 81–88 (2004).
Kuhnem, P. R., Ponte, D., Dong, E. M. & Bergstrom, G. C. Y. Fusarium graminearum isolates from wheat and maize in New York show similar range of aggressiveness and toxigenicity in cross-species pathogenicity tests. Phytopathology 105, 441–448 (2015).
Leslie, J. F. & Summerell, B. A. Techniques for growing and maintaining Fusarium. Fusarium Lab. Man. 21–30. 10.1002/9780470278376.ch4 (2006).
Nicholson, P. & Parry, D. W. Development and use of a PCR assay to detect Rhizoctonia cerealis, the cause of sharp eyespot in wheat. Plant. Pathol. 45, 872–883 (1996).
Nicholson, P. et al. Detection and quantification of Fusarium culmorum and Fusarium graminearumin cereals using PCR assays. Physiol. Mol. Plant. Pathol. 53, 17–37 (1998).
Carter, J. P., Rezanoor, H. N., Desjardins, A. E. & Nicholson, P. Variation in Fusarium graminearum isolates from Nepal associated with their host of origin. Plant. Pathol. 49, 452–460 (2000).
O’Donnell, K., Kistler, H. C., Cigelnik, E. & Ploetz, R. C. Multiple evolutionary origins of the fungus causing Panama disease of banana: concordant evidence from nuclear and mitochondrial gene genealogies. Proc. Natl. Acad. Sci. 95, 2044–2049 (1998).

No competing interests reported.

TableS1.pdf

Population and Pathogenicity of Fusarium Species Associated with Fusarium Head Blight in a Wheat–Maize Cropping System in Sichuan Province, China

Status:

Version 1

Abstract

Figures

Introduction

Results

Identification of Fusarium species in a wheat‒maize cropping system

Fusarium collection from wheat

Saprophytic Fusarium collection

Pathogenicity assays

Pathogenicity of Fusarium spp. from wheat spikes

Aggressiveness of the strains between F. asiaticum and F. graminearum

Pathogenicity of saprophytic Fusarium species

Discussion

Conclusion

Materials and methods

Sample collection and isolation

DNA extraction

PCR amplification and sequencing

Phylogenetic analysis

Plant materials

Preparation of Fusarium inoculums

Pathogenicity assays

Statistical analyses

Declarations

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1