Keywords

Fusarium head blight; FHB; Fusariumspp; Pathogenicity; Bread wheat

Introduction

The necrotrophic Fusarium head blight (FHB) of wheat is a major head disease with an overwhelming impact on yield, and grain quality mainly during wet seasons that favor FHB disease development, and higher mycotoxin accumulation in grains [1,2]. Generally, up to 19 species in the genus Fusarium were reported in causing FHB disease of wheat [3]. In addition to wheat, FHB disease infects several crops including barley, oats, rye, corn, canary seed, forage grasses, sugarcane, and rice, but wheat, barley, and maize are the most affected crops [4-7].

Kernel infection by FHB pathogens can cause poor seed germination, kernel shriveling, reduction in the number of kernels per spike, low protein content, and low baking quality that contribute to a significant loss both in yield and quality. Besides, the pathogen produces toxic metabolites that have health problems both to humans and animals when consumed [8-11].

Globally, due emphasize has given for FHB because of its impact on grain yield as seedling blight, shriveled kernels, and infertility of spikes [1], grain quality such as low protein content, and low baking quality [12], and mycotoxin contaminations in grains, and in straws which had health problem when feed by humans and animals [8,9].

However, there is a limited research effort done on FHB disease of wheat in Ethiopia. One of the efforts was, yield loss of 60% and more were recorded on wheat under natural infection at Holeta [13]. The other was, identification of 17 and 13 Fusarium species from stored wheat grains, and blighted wheat spikes across Arsi, Bale, Gojam, Gonder, Shoa, and Wollo areas [14], but their pathogenicity tests were not verified. Additionally, a new novel species F. aethiopicum has phylogenetically identified from 31 Fusarium c F. graminearum isolates originated from Amhara and Oromia regions of Ethiopia [15]. This may indicate the existence of species diversity in the country. So far, all the past studies of FHB do not enclose Jimma, Buno-Bedele, and West-Wellega zones where wheat is grown as one of the staple food crops. Therefore, this study had aimed to identify, characterize, and test the pathogenicity of Fusarium spp. responsible for causing FHB disease of wheat in southwestern Ethiopia.

Materials and Methods

Description of study areas

Blighted spikes of wheat were sampled at Dedo, and Seka- Chekorssa districts of Jimma zone, Bedele and Gechi districts of the Buno-Bedele zone, and Begi district of West-Wollega zone of Oromia region (Table 1).

Table 1: Coordinates, elevations, annual rainfall and mean temperatures of the study area by districts, 2017

a. Data obtained from National Meteorology Agency of Ethiopia, Jimma Meteorology Center, 2017

b. Data obtained from National Meteorology Agency of Ethiopia, Assosa Meteorology Center, 2017. Coordinate and altitude ranges were obtained from the respective district agriculture and natural resource development office.

c. Data obtained from the respective district agriculture and natural resource development office.

Sample collection

Four wheat spikes with a typical FHB symptom were sampled per field and placed inside paper bags. The paper bags were labeled with sample code, date of sampling, Altitude, Latitude, Longitude, and collectors' names. The samples were placed in the icebox and also ventilated overnight to avoid excess water. Finally, the samples were taken to the Plant Pathology Laboratory of Jimma University College of Agriculture and Veterinary Medicine (JUCAVM) for isolation and identification of the causal agents.

Growth media

Malachite-Green Agar (MGA) and Potato Dextrose Agar (PDA) were used to isolating Fusarium species from the samples. Spezieller Nahrstoffarmer Agar (SNA) media with two sterile filter paper pieces had used to enhance the sporulation of isolates. Water agar (3% WA) media had used for single conidial purification. Besides, PDA and potato sucrose agar (PSA) had used for studying the colony characteristics (such as pigmentation and mycelial growth). All media used in this study had prepared according to 'The Fusarium Laboratory Manual' and 'Fusarium species: an illustrated manual for identification' [16,17]. Also, all the media were amended by 250 mg of Chloramphenicol per liter of media to inhibit bacterial contaminants.

Isolation of Fusarium spp.

Eight kernels had separated from each blighted wheat spike samples and surface-sterilized in 4% (v/v) sodium hypochlorite solution for a minute, followed by thrice rinsing in sterilized distilled water. The kernels were well drained under laminar flow. Then, four kernels had placed on PDA plate and the other four kernels had placed on MGA plate. All plates were labeled, sealed with parafilm and incubated at 25°C. After 4 to 5 days of incubation, all Fusarium resembling colonies were cut alone with the help of sterile needle and transferred onto SNA plates. Both side of the fungal agar block sterile filter paper pieces were placed to enhance conidia formation. The needle used was dipped in ethanol and burned off between each colony transfer. After the colony purification, all the Petri dishes were labeled, sealed with parafilm and incubated at 25°C for 7 to 17 days until sporulation.

Single conidium isolate development

For single conidial isolation, a small fungal plug was taken from sporulated SNA cultures and transferred to 3% WA and a drop of autoclaved distilled water was added onto the fungal plug and the conidia were dislodged by sterile glass road. The dislodged conidia were spread over the WA by sterile glass road spreader and the plates were incubated at 25°C for 24 hours. Then after, a hyphal tip derived from a single conidium was cut and transferred to SNA with two sterile filter paper pieces [17]. The Petri dishes were then labeled, sealed with parafilm and incubated at 25°C for 7 to 17 days until sporulation. These isolates were used for examination of microscopic and macroscopic features.

Identification of Fusarium spp.

Isolates of Fusarium recovered from blighted wheat spikes sampled across southwestern Ethiopia was identified in to species level based on cultural and morphological characteristics as described by [16-18].

Pathogenicity test

Experimental design and kernel disinfection: Pathogenicity experiment was conducted from February 2018 to June 2018 on Danda'a (a susceptible) bread wheat variety at JUCAVM, Jimma, Ethiopia. RCBD design with three replications had used. Fusarium species were used as test treatments, while sterile distilled water was used as a control. The experimental units were plastic pots (having a size of 15 cm × 11 cm × 15 cm), which had filled with an autoclaved potting mix (1:3:1 v/v sand/peat/ compost). Before sowing, the wheat kernels had washed under running tap water for five minutes. Then, disinfected in 75% ethanol for 30 seconds and 0.5% NaOCl (sodium hypochlorite) solution for a minute. Finally, the kernels were rinsed twice in sterile distilled water and allowed to dry under laminar flow [19]. The four well-dried kernels had seeded at a depth of 2 cm in each pot. Each pot was fertilized with 5 g urea before emergence, 5 g NSP at tillering, and 5 g urea at booting and also watered twice daily.

Preparation of inocula: The nine identified Fusarium spp. were recovered on SNA with sterile filter paper and incubated for 7-17 days at 25°C until sporulation. Then, 10 ml of sterilized distilled water was poured onto each sporulated plate and the conidia were dislodged by using sterile glass road cell spreader. The suspension was filtered through two layers of sterilized cheesecloth [19] and the final concentration was adjusted to 5 × 105 conidia ml-1 with the help of hemocytometer. From the adjusted inoculum, 200μl of each Fusarium species was kept in a 5 ml Falcon tube at 4ºC pending for inoculation [20,21].

Inoculation: A single centrally positioned floret of two spikes per pot were injected [22] at Zadok’s growth stage 65 [23] by the already prepared 10 μl inoculum of each Fusarium species. Control (check) spikes were inoculated in the same way by 10 μl of sterile distilled water. Simultaneously, the spikes were tagged and covered with polythene bags for 48 hours to maintain high humidity that can facilitate infection process [24-26].

Collected data

Fusarium morphology data: Primary and secondary morphology data were collected for identification Fusarium isolates in to species level according to the description of species described by [16-18]. The primary characters such as a) Macroconidia characteristics like phialides, shape, size, number of septa, shape of the apical and basal cells was noted; b) Microconidia characteristics including presence or absence of microconidia, if present their shape, size and the manner in which they are formed (phialides) were noted; and c) Chlamydospores presence or absence, if present their form (chain or single). Secondary characters; a) Colony morphology features that includes color on PDA, pigmentation and hyphal colony growth on PDA and PSA.

Pathogenicity test data:Blighted spikelets per spikes due to the infection of inoculated Fusarium spp. was carefully inspected at weekly basis. The spikelet bleaching severity caused by each Fusarium spp. was recorded as a percentage of blighted spikelets over the total number of spikelets per spike [28] at 7, 14, 21 and 28 days after inoculation [29]. Finally, each inoculated spike was separately taken to laboratory and re-isolation was performed to confirm the identity of the test pathogen.

Data analysis

From the pathogenicity test experiment, the area under disease progress curve (AUDPC) for the nine Fusarium spp. was determined as described by [30].

Where; AUDPC is the area under disease progress curve, n is total number of observation days at the ith observation, yi is spikelet bleaching severity at the ith observation, t is time at the ith observation.

Analysis of variance for spikelet bleaching severity and AUDPC data was performed using the general linear model procedure of SAS version 9.3 statistical software [31]. The means were separated by LSD test at a probability level of 0.05. The spikelet infection rate of each inoculated species was determined by Minitab 17 software. The RCBD model used for analyzing AUDPC and spikelet bleaching severity is described as follows:

Where; Yij is the response (AUDPC or spikelet bleaching severity) for treatment i observed in block j; μ is the overall mean, α is the effect of the ith treatment, β is the effect of the jth block, εij is the error term for the ith treatment in the jth block.

Finally, aggressiveness of Fusarium spp. used in pathogenicity test on Danda’a wheat variety was determined from spikelet infection severity and AUDPC [32,33].

Results and Discussion

Fusarium spp. associated with blighted wheat spikes

A total of 269 single conidial purified Fusarium isolates had recovered from blighted wheat spikes collected during the 2017 main cropping season in Jimma, Buno-Bedele and West-Wollega zones of Oromia, southwestern Ethiopia. Based on their cultural and microscopic characteristics as described by [16-18], all isolates were grouped into nine Fusarium species (Figure 1) with varied isolation frequency across the study area (Table 2). The variation may be due to factors such as field location, climatic conditions, soil management, crop rotation and cultivation methods [34].

Figure 1: F. graminearum; A: conidiophore, and B: and amp; C: conidia

Based on this provisional identification, F. culmorum and F. ussurianum were isolated from blighted wheat spikes which were not reported by the previous study conducted in Ethiopia, though this needs further confirmation.

On the other hand, F. graminearum, F. avenaceum, F. poae, F. semitectum, F. sambucinum, F. heterosporum, and F. lateritium had recovered from stored wheat grains and blighted wheat spikes sampled from Arsi, Bale, Gojam, Gonder, Shoa, and Wollo areas [14].

Among the nine species, F. graminearum and F. culmorum were the two most frequently isolated species comprised of 29.0% and 26.4% of the total number of Fusarium isolates, respectively. Whereas, F. avenaceum, F. poae, F. ussurianum, F. semitectum, F. sambucinum, and F. lateritium had made up of 10.4%, 7.4%, 6.7%, 6.3%, 6.0%, and 6.0%, respectively.

On the other hand, the least isolated species was F. heterosporum which had only 1.9% of the total isolates (Table 2). These revealed that F. graminearum and F. culmorum were the two most predominately isolated species followed by F. avenaceum from blighted wheat spikes in southwestern Ethiopia.

Table 2: Isolation frequency (%) of identified Fusarium spp. from wheat blighted heads in SWE, 2017 main cropping season.

Previously in Ethiopia, F. graminearum and F. avenaceum were reported among the predominant species isolated from stored wheat grains, and blighted wheat spikes sampled from Arsi, Bale, Gojam, Gonder, Shoa, and Wollo areas [14].

Further, in neighboring country Kenya, these two species (F. graminearum and F. avenaceum) were predominately isolated from wheat spikes in Narok County, and kernels in Nakuru County [35].

N: Number of Isolates; PDA: % of Isolates on Potato Dextrose Agar; MGA: % of Isolates on Malchet-Green Agar; TN: Total Isolation Frequency (%); TIF: Total Isolation Frequency (%)

Distribution of Fusarium spp. in southwestern Ethiopia

F. graminearum, F. culmorum, F. lateritium, F. avenaceum, F. poae, and F. heterosporum were isolated from samples collected from the five assessed districts in southwestern Ethiopia (Table 3). However, F. sambucinum, F. ussurianum, and F. semitectum were isolated from four assessed districts.

The most dominant F. graminearum was mainly isolated from samples of the Buno-Bedele zone (44.9%) and West-Wollega zone (34.6%). In particular, Begi, Bedele, and Gechi districts were attributed 24.4%, 23.1%, and 21.8% of F. graminearum isolation, respectively (Table 3).

Table 3: Distribution (%) of Fusarium spp. by zones and districts in southwestern, 2017.

Whereas, the second predominant F. culmorum was frequently isolated from samples of the Buno-Bedele zone (59.2%) and Jimma zone (25.4%). It was mainly recovered from samples of Gechi district (39.4%), Bedele district (19.7%), and Seka-Chekorssa (19.7%).

The third predominant F. avenaceum was mainly isolated from samples of Jimma zone (53.6%) particularly in Seka- Chekorssa that attributed 38.5% isolation frequency (Figure 2).

Figure 2: F. culmorum; A: conidiophore, and B: conidia

Values in parenthesis is percent frequency; -shows the specie does not recovered from the samples.

The occurrence and distribution of Fusarium species can vary with the changing climate, crop rotation, cultivar resistance and interactions among different species [34,36]. For instance, in some parts of Europe, the predominant species were varied among F. graminearum, F. poae, F. avenaceum and F. culmorum [36], however, F. graminearumwas also reported in displacing the F. culmorum [37]. Moreover, a four-year study in Belgium revealed that the most frequent causal agent of FHB in wheat was F. graminearummainly in areas where corn was cultivated and F. culmorum, mainly in areas where small grains were grown (Figure 3) [38]. This clearly revealed the effect of cultural practices on Fusarium species abundance.

Figure 3: F. avenaceum; A: conidiophore, and B: conidia

Pathogenicity test

The pathogenicity of all Fusarium spp. identified in this study was assessed using point (single spikelet) injection method (Figure 4) [22]. The results indicated that all the tested Fusarium spp. caused FHB symptoms on spikes of Danda’a variety (Figure 5). However, no FHB symptoms were observed on spikes inoculated with sterile distilled water (control). Re-isolation from the kernels of inoculated spikes agrees with descriptions of the inoculated species, which confirms their pathogenicity under Lath-house condition (Figure 6).

Figure 4: F. lateritium; A: conidiophore, and B: conidia

Figure 5: F. poae; A: conidiophore, and B: conidia

Figure 6: F. semitectum A: conidiophore, and B: conidia

Mean values in a column with different letters are significant at p<0.05; AUDPC: Area Under Disease Progress Curve; DAI: Days After Inoculation; LSD: Least Significant Difference; r: Rate of Spikelet Bleaching

Fusarium spp. had shown significantly varied spikelet bleaching severity and AUDPC on Danda'a wheat variety (Table 4).F. avenaceum was the most aggressive species that caused the highest spikelet bleaching severity of 100% at 28 days after inoculation (DAI) and AUDPC of 1067.2 on Danda'a variety.Statistically comparable spikelet bleaching severities had produced by F. culmorum, F. graminearum, F. lateritium, F. sambucinum, F. poae, and F. heterosporum. Likewise, F. poae, F. sambucinum, F. lateritium, and F. culmorum were generated statistically similar AUDPC as compared to that of F. avenaceum. However, F. ussurianum and F. semitectum had produced the lower AUDPC of 175.2 and 116.2, respectively (Figure 7).

Figure 7: F. ussurianum; A: conidiophore, and B and C: conidia

Table 4: Blighted spikelet severity and AUDPC of Fusarium spp. under lath-house, 2018

All nine Fusarium species had shown the different rates of FHB disease development on Danda'a wheat variety (Table 4). Seven of the tested species had caused FHB symptoms at 7 DAI, while the others at 14 DAI and 28 DAI. This finding almost agrees with the comparative aggressiveness study conducted in Canada that reported F. graminearum, F. avenaceum, F. culmorum, and F. poae had produced visible spikelet bleaching at 21 and 28 DAI on wheat spikes [39]. On the other hand, delayed symptom development was observed by F. ussurianum and F. semitectum after seven and 21 DAI (Figure 8).

Figure 8: F. sambucinum; A: conidiophore and B: conidia

Based on spikelet bleaching severity and AUDPC results, F. avenaceum, F. poae, F. sambucinum, F. lateritium, F. culmorum, F. heterosporum, and F. graminearum were more aggressive on Danda'a wheat variety. These seven species had caused spikelet bleaching severity and AUDPC beyond or equal to 57.8% and 546.8, respectively. Whereas, F. semitectum and F. ussurianum were showed less aggressiveness on Danda'a variety with spikelet bleaching severity of 33.19% and 29.78%, and AUDPC of 116.2 and 175.2, respectively (Figure 9). These findings concurred with the aggressiveness study that reported F. graminearum and F. culmorum as an aggressive species causing more than 35% of spikelet bleaching severity of wheat in Canada [39].

Figure 9: F. heterosporum; A: conidiophore, and B: conidia

In addition to causing blighted wheat spikes, F. culmorum, F. graminearum, and F. avenaceum had responsible for crown rot of bread wheat and durum wheat inTurkey [40], and root rot of corn, soybean, and wheat in Nebraska [41]. Likewise, F. culmorumhad reported in causing higher seedling blight, while F. graminearum had responsible for causing severe crown rot of wheat [42-44].

Conclusion

A total of 269 single conidial purified isolates had recovered from blighted wheat spikes sampled across Jimma, Buno-Bedele, and West-Wellega zones of Oromia, southwestern Ethiopia. Based on their cultural and microscopical characteristics, all isolates had classified into nine Fusarium species. Among the nine specie, F. graminearum and F. culmorum were the most predominant ones, followed by F. avenaceumin southwestern Ethiopia. Besides, all the nine species had pathogenic, and F. avenaceum, F. poae, F. sambucinum, F. lateritium, F. culmorum, F. heterosporum, and F. graminearum were shown more aggressiveness on Danda'a wheat variety.

Acknowledgements

The authors would like to thank Ethiopian Institute of Agricultural Research and Assosa Agricultural Research Center for the financial supports. We also extend a sincere thanks to Jimma University College of Agriculture and Veterinary Medicine (JUCAVM) Department of Horticulture and Plant Science for allowing us to use their plant pathology laboratory and Lathhouse facilities for successful execution of this work. Also, our gratitude goes to Kulumssa Agricultural Research Center for the provision of the susceptible test crop; Danda'a wheat variety, for pathogenicity test.

Conflict of Interest

The authors declare that there is no conflict of interest.

Ethical Statement

This study did not involve any human or animal testing.

References

1. Mcmullen M, Bergstrom G, De Wolf E, Dill-Macky R, Hershman D, et al. (2012) A unified effort to fight an enemy of wheat and barley: Fusarium head blight. Plant Dis 96: 1712-1728.
2. Kelly AC, Clear RM, O’donnell K, Mccormick S, Turkington TK, et al. (2015) 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.
3. Liddell CM (2003) Systematics of Fusarium species and allies associated with Fusarium head blight. Fusarium head blight of wheat and barley 35-43.
4. Clear R, Patrick S (2003) Fusarium Head Blight In Western Canada.
5. Hsuan H, Zakaria L, Salleh B (2010) Characterization of Fusarium isolates from rice, sugarcane and maize using RFLP-IGS. J plant protecresear 50: 409-415.
6. Bashyal B, Aggarwal R, Sharma S, Gupta S, Rawat K, et al. (2016) Occurrence, identification and pathogenicity of Fusarium species associated with bakanae disease of basmati rice in India. Eur J Plant Pathol. 144: 457-466.
7. Ferre FS (2016) Worldwide occurrence of mycotoxins in rice. Food Control 62: 291-298.
8. Grabowski A, Siuda R, Lenc L, Jaroszuk-Ściseł J (2012) Effect of the degree of fusariosis on the physical characteristics of individual wheat kernels. Int J Food Sci Techno 47: 1122-1129.
9. Darwish WS, Ikenaka Y, Nakayama SM, Ishizuka M (2014) An overview on mycotoxin contamination of foods in Africa. J Vet Med Sci 76: 789-797.
10. Martinez-Espinoza A, Campus G, Ethredge R, County S, Youmans J, et al. (2014) Identification and Control of Fusarium Head Blight (Scab) of Wheat in Georgia. pp:1-8.
11. Nielsen LK, Cook DJ, Edwards SG, RayRV (2014) The prevalence and impact of Fusarium head blight pathogens and mycotoxins on malting barley quality in UK. Int J Food Microbiol 179: 38-49.
12. Gärtner BH, Munich M, Kleijer G, Mascher F (2008) Characterisation of kernel resistance against Fusarium infection in spring wheat by baking quality and mycotoxin assessments. Eur J Plant Pathol 120: 61-68.
13. Snijders C (1989) Current status of breeding wheat for Fusarium head blight resistance and the mycotoxin problem in the Netherlanas.  Workshop on Fusarium Head Blight and Related Mycotoxins, 1989. 17.
14. Bekele E (1990) Identification of Fusarium spp. and Mycotoxins Associated with Head Blight of Wheat in Ethiopia: A dissertation presented to the faculty of the graduate school.
15. O’donnell K, Ward TJ, Aberra D, Kistler HC, Aoki T, et al. (2008) Multilocus genotyping and molecular phylogenetics resolve a novel head blight pathogen within the Fusarium graminearum species complex from Ethiopia. Fungal Genet Biol 45: 1514-1522.
16. Summerell BA, Salleh B, Leslie JF (2003) A utilitarian approach to Fusarium identification. Plant disease 87: 117-128.
17. Leslie JF, Summerell BA, Bullock S (2006) The Fusarium laboratory manual, Wiley Online Library.
18. Refai M, Hassan A, Hamed M (2015) Monograph on the Genus Fusarium. Cairo University, Giza.
19. Gargouri-Kammoun L, Gargouri S, Rezgui S, Trifi M, Bahri N, et al. (2009) Pathogenicity and aggressiveness of Fusarium and Microdochium on wheat seedlings under controlled conditions. Tunisian Journal of Plant Protection, 4, 2009135.
20. Malbrán I, Mourelos CA, Girotti JR, Aulicino MB, Balatti PA, et al. (2012) Aggressiveness variation of Fusarium graminearum isolates from Argentina following point inoculation of field grown wheat spikes. Crop Prot 42: 234-243.
21. He X, Singh PK, Duveiller E, Dreisigacker S, Singh RP (2013a) Development and characterization of International Maize and Wheat Improvement Center (CIMMYT) germplasm for Fusarium head blight resistance. Fusarium Head Blight in Latin America. Springer 241-262.
22. Dill-Macky R, Leonard KJ, Bushnell W (2003) Inoculation methods and evaluation of Fusarium head blight resistance in wheat. Fusarium Head Blight Of Wheat And Barley. 184-210.
23. Zadoks JC, Chang TT, Konzak CF (1974) A decimal code for the growth stages of cereals. Weed Research 14: 415-421.
24. Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal crops. Mol Plant Pathol 5: 515-525.
25. Matarese F, Sarrocco S, Gruber S, Seidl-Seiboth V, Vannacci G (2012) Biocontrol of Fusarium head blight: interactions between Trichoderma and mycotoxigenic Fusarium. Microbiology 158: 98-106.
26. He X, Singh PK, Duveiller E, Schlang N, Dreisigacker S, Singh RP (2013b) Identification and characterization of international Fusarium head blight screening nurseries of wheat at CIMMYT, Mexico. Eur J Plant Pathol 136: 123-134.
27. Summerell BA, Salleh B, Leslie JF (2003) A utilitarian approach to Fusarium identification. Plant Disease 87: 117-128.
28. Stack RW, Mcmullen MP (2011) A visual scale to estimate severity of Fusarium head blight in wheat. NDSU. Extension Service, North Dakota State University.
29. ŠÍP V, Chrpova J (2008) Screening inoculum sources for tests of resistance to Fusarium head blight in wheat. Cereal Research Communications 36: 531-534.
30. Madden LV, Hughes G, Bosch F (2007) The study of plant disease epidemics, American Phytopathological Society (APS Press).
31. SAS, 2010. SAS System for Windows, Version 9.3. Cary, NC: SAS Institute.
32. Mardi M, Buerstmayr H, Ghareyazie B, Lemmens M, Moshrefzadeh N, et al. (2004) Combining ability analysis of resistance to head blight caused by Fusarium graminearum in spring wheat. Euphytica 139: 45-50.
33. Contreras-Medina LM, Torres-Pacheco I, Guevara-González R, Romero-Troncoso R, Terol-Villalobos I, et al. (2009) Mathematical modeling tendencies in plant pathology. Afr J Biotechnol. 8: 7399-7408.
34. Scala V, Aureli G, Cesarano G, Incerti G, Fanelli C, et al. (2016) Climate, Soil Management, and Cultivar Affect Fusarium Head Blight Incidence and Deoxynivalenol Accumulation in Durum Wheat of Southern Italy. Front Microbiol 7: 1014.
35. Wagacha JM, Njeru NK, Okumu OO, Muthomi JW, Mutegi CK (2016) Occurrence of Fusarium head blight of wheat and associated mycotoxins in Narok and Nakuru Counties, Kenya. World Journal of Agricultural Research 4: 119-127.
36. Xu X.-M, Parry D, Nicholson P, Thomsett M, Simpson D, et al. (2005) Predominance and association of pathogenic fungi causing Fusarium ear blightin wheat in four European countries. Eur J Plant Pathol 112: 143-154.
37. Waalwijk C, Kastelein P, De Vries I, Kerényi Z, Van Der Lee T, et al. (2003) Major changes in Fusarium spp. in wheat in the Netherlands. Eur J Plant Pathol 109: 743-754.
38. Isebaert S, De Saeger S, Devreese R, Verhoeven R, Maene P, Heremans B, Haesaert G (2009) Mycotoxin‐producing Fusarium species occurring in winter wheat in Belgium (Flanders) during 2002–2005. J Phytopatho 157: 108-116.
39. Xue A, Armstrong K, Voldeng H, Fedak G, Babcock C (2004) Comparative aggressiveness of isolates of Fusarium spp. causing head blight on wheat in Canada. Canad J Plant Patho 26: 81-88.
40. Gebremariam ES, Sharma-Poudyal D, Paulitz T, Erginbas-Orakci G, Karakaya A, et al. (2018) Identity and pathogenicity of Fusarium species associated with crown rot on wheat (Triticum spp.) in Turkey. Eur J Plant Pathol 150: 387-399.
41. Parikh L, Kodati S, Eskelson MJ, Adesemoye AO (2018) Identification and pathogenicity of Fusarium spp. in row crops in Nebraska. Crop Prot 108: 120-127.
42. Dyer AT, Johnston RH, Hogg AC, Johnston JA (2009) Comparison of pathogenicity of the Fusarium crown rot (FCR) complex (F. culmorum, F. pseudograminearum and F. graminearum) on hard red spring and durum wheat. Eur J Plant Pathol 125: 387-395.
43. Kazan K, Gardiner DM (2018) Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: recent progress and future prospects. Mol Plant Pathol 19: 1547-1562.
44. Ch'en LF, BAI GH, DESJARDINS AE (2000) Recent advances in wheat head scab research in China.