Pathogenicity of Asymptomatically Residing Fusarium Species in Non-Gramineous Plants and Weeds to Spring Wheat under Greenhouse Conditions

Despite significant efforts in recent decades to combat Fusarium head blight (FHB), this disease remains one of the most important and widely studied diseases of wheat and other cereal plants. To date, studies have focused on small grain cereals as hostplants for these pathogens, but it was recently discovered that asymptomatic non-gramineous plants and weeds can serve as alternative sources of fungi associated with FHB. The aim of this study was to evaluate the pathogenicity of Fusarium avenaceum, F. culmorum, F. graminearum and F. sporotrichioides isolated from non-gramineous plants and weed species to spring wheat under greenhouse conditions. A total of 91 Fusarium isolates, including 45 from weeds and 46 from non-gramineous plants were floret inoculated at mid anthesis. The FHB incidence and severity (%) of inoculated heads and the area under the disease progress curve (AUDPC) were calculated. To determine yield losses, the weight of 1000 grains (TGW) was evaluated. Results of the research showed that FHB severity (%) values in Fusarium spp.-inoculated heads from non-gramineous plants varied from 9.3% to 69.6% and AUDPC values ranged from 161.5% to 1044.6%. TGW was most significantly reduced by the F. culmorum isolates BN26r and BN39fl from Brassica napus and isolates BV15.1l and BV142.1pe from Beta vulgaris (37%, 30%, 28.8% and 31.8% respectively, compared to the water control). In Fusarium-inoculated heads from weeds, FHB severity values ranged from 6.2% to 81.0% and AUDPC values varied from 134.2% to 1206.6%. TGW was most significantly decreased by CBP1401r isolate from Capsella bursa-pastoris (52%). The study results suggest that the pathogenicity of Fusarium species isolated from different hosts to wheat more strongly depends on the Fusarium species and strain than the hostplant. Under greenhouse conditions, F. culmorum strain groups obtained from weeds, non-gramineous plants and Triticum were more pathogenic to wheat than the water control and other Fusarium species.


Introduction
Fusarium head blight (FHB), which is primarily caused by several Fusarium species and their complexes, is a disease that affects wheat and other small-grain cereals [1][2][3][4][5]. Although FHB has been heavily combated in recent years, it continues to be one of the most globally significant and extensively researched diseases of wheat and other cereal plants [6]. This disease is a major concern worldwide, as it can not only reduce grain quality, but also cause losses in grain yields [7][8][9][10]. In the past, FHB and the resulting yield losses posed a minimal threat to farmers in Lithuania, though the disease has continued to be a severe issue in Lithuanian fields since its first outbreaks were discovered in 2012 [4,11]. Though it is reported that, globally, F. graminearum and F. culmorum are the main causal agents of The results indicated that the most frequently isolated and important species in terms of FHB incidence in Croatia is F. graminearum. Recently conducted studies in Lithuania demonstrated that oilseed rape, potatoes, sugar beet, peas and 56 weeds species (all detected in the field) were asymptomatically colonized by nine Fusarium species: F. avenaceum, F. culmorum, F. graminearum, F. equiseti, F. tricinctum, F. sporotrichioides, F. poae, F. oxysporum and F. redolens [38][39][40]. In pathogenicity tests, all tested F. graminearum (91 isolates) were able to cause FHB symptoms in spring wheat and the disease severity values were comparable to those isolated from the primary hostplants of wheat and barley [40]. The results of a study conducted in 2019 showed that F. culmorum isolates from asymptomatic weeds and non-gramineous plants had similar effects to F. graminearum on spring wheat. However, the roles of other Fusarium spp. in the epidemiology of the FHB remained unclear [41]. Previously, similar findings were published by Pereyra and Dill-Macky, who indicated that F. graminearum isolated from Digitaria sanguinalis residues caused FHB in wheats [29].
Numerous previous studies showed the extensive adaptations of Fusarium species in colonizing the internal tissues of various non-gramineous agroecosystem plants and weeds. However, it is still challenging to fully comprehend the true role of F. graminearum and other Fusarium species in FHB epidemiology, as morphologically and genetically distinct Fusarium species may have high phenotypic diversity. It is, therefore, necessary to investigate the ability of asymptomatically existing Fusarium species (especially other than F. graminearum) in alternative hosts to cause FHB symptoms in cereals and to determine whether these species are more aggressive to spring wheats. The aim of this study was to evaluate the pathogenicity of Fusarium avenaceum, F. culmorum, F. graminearum and F. sporotrichioides isolated from non-gramineous plants and weed species to spring wheat under greenhouse conditions.

Isolation of Fusarium Fungi from Plant Material
This research was conducted in 2021 at the Institute of Agriculture, Lithuanian Research Centre for Agriculture and Forestry (55 • 23 50" N; 23 • 51 40" E). The presence of F. avenaceum, F. culmorum, F. graminearum and F. sporotrichioides was assessed in non-gramineous plants (Brassica napus, Pisum sativum and Beta vulgaris) and in weeds (Tripleurospermum inodorum, Viola arvensis, Fallopia convolvulus, Capsella bursa-pastoris and Poa annua). The aforementioned plants were collected from cropping system fields, taken to the laboratory, identified and processed for further experiments. Selected visually asymptomatic plants were thoroughly washed, dried, numbered and identified by growth stage and species. Fusarium fungi were isolated from all morphological parts of the plant, including roots, crowns, stems, leaves, florets, pods, petioles and fruits. Isolation of Fusarium spp. was performed according to Suproniene et al. [39]. Several segments of different parts of the plant (1 cm in size) were sterilized for 3 min in 1% sodium hypochlorite (NaClO) solution and then rinsed 3 times in sterile distilled water (SDW) and dried on sterile filter paper in a laminar. Three segments of different parts of the plant were placed on potato dextrose agar (PDA, Merck) medium and the plates were incubated at 22 ± 2 • C in the dark for 2-4 days. Fusarium fungi that appeared were purified via PDA and grown on a Spezieller Nährstoffarmer Agar medium (SNA) for spore mass formation [42].

Identification of Fusarium Fungi
Monosporic cultures were grown on PDA and SNA at 25 ± 2 • C for 10-30 days until the formation of macroconidia. Species were identified based on Nelson et al. [43] and Leslie et al. [44] as descriptors based on visual colonies and microscopic morphological features of conidia typical for each species. During identification a microscope at 10×, 20× and 40× magnification was used to evaluate all possible signs. Once Fusarium species were identified, fungal cultures were purified on water agar (WA) media [44]. Three suspension dilutions (10 −1 , 10 −2 and 10 −3 ) were prepared for each identified Fusarium isolate. Then, 10 µL of the suspension (10 −3 ) was spread in plates with WA and dispersed with a sterile L-shaped spreader. Inverted plates with Fusarium cultures on WA were grown in an incubator at 25 ± 2 • C for 2-3 days until the first mono-sporous colonies appeared. Using a microscope, in laminar under aseptic conditions, we determined whether the colony was formed from one conidia or did not come into contact with other conidia. After the assessment, the germinated conidia/monosporic colony was transferred to a PDA medium and incubated at 25 ± 2 • C for 3-7 days until further colony growth. After that, a colony fragment~5 mm Ø from the peripheral zone was transferred onto an SNA medium and incubated at the same temperature for 10-30 days until the formation of macroconidia.

Preparation of Spore Suspensions
Spore suspensions were prepared according to Purahong et al. [46] and Suproniene et al. [39,40]. For suspension preparation, Fusarium isolates were grown on an SNA medium at a temperature of 22 ± 2 • C for 14-30 days (until they formed a spore mass of macroconidia in sporodochia). For the preparation of spore suspensions, 10 mL of SDW was added to the plate and the spores formed in the micelle and on the surface of the medium were separated by sweeping the surface of the medium with circular movements using an L-shaped spreader. The suspension was filtered through a sterile cotton strainer. The concentration of spores in the suspension was counted using a Neubauer cell counting chamber. A suspension of 1 × 10 5 spores/mL concentration was used for the wheat inoculation.

Description of Greenhouse Experiment
Experiments included evaluation of the pathogenicity of F. avenaceum, F. culmorum, F. graminearum and F. sporotrichioides strains isolated from different alternative hostplants under greenhouse conditions. The pathogenicity of Fusarium strains isolated from nongramineous hostplants to spring wheat was determined according to Purahong et al. [46]. The experiment was divided into two parts: the pathogenicity of Fusarium spp. isolates from non-gramineous plants (experiment I) and the pathogenicity of isolates from weeds (experiment II). In experiment I, a total 46 of Fusarium isolates, including 35 from non-gramineous plants and 11 from spring wheat, were used. In experiment II, a total of 45 Fusarium isolates, including 34 from weeds and 11 from spring wheat, were used. Isolates of Fusarium spp. were obtained from the collection of the Microbiology Laboratory, Lithuanian Research Centre for Agriculture and Forestry and were used for floret inoculation. Pots (LxWxH: 13.0 × 8.8 × 11.5 cm) were filled with a commercial pH-adjusted (5.5-6.5) substrate and four spring wheat seeds were planted per pot. The FHB-susceptible spring wheat breeding line "DS-1403-3-DH" was used for pathogenicity tests. The greenhouse was maintained at ±25 • C during the day and ±19 • C at night with a 14-h light and 10-h dark mode. Wheat plants with mineral fertilizer complex (NPK, 11-11-21) were fertilized one week after planting (3 g of fertilizer per pot) and watered 2 times a week. The spikes were inoculated during mid anthesis. Twenty microliters (10 µL/floret) of each Fusarium isolate suspension (spore concentration of 1 × 10 5 spores mL −1 ) and sterile water as a negative control were injected into two adjacent florets at the center of the head (without wounding). The inoculated heads were covered with polyethylene bags for 72 h to ensure the required moisture. The suspension of each isolate was used for the inoculations of 15 heads (3 heads × 5 replicates).

Analysis of FHB Parameters of Inoculated Spring Wheat
FHB incidence and severity (%) were assessed after the 7th (BBCH 69-71), 14th (BBCH 73) and 21st (BBCH 73-75) days post inoculation (DPI). The area under the disease progress curve (AUDPC) was calculated after the 28th (BBCH 75-77) DPI. All inoculated plants were evaluated. The incidence of disease showed the number of heads affected by the disease, expressed as a percentage. For the assessment of disease severity, we used the visual evaluation scale for the disease developed by Engle et al. [47]. When the grain was fully ripe (BBCH 89), each bundle of 5 heads (replication of each treatment) was cut and packed in a paper bag upon which the date, the treatment of the study and the replication were indicated. The grain was threshed using a laboratory single-ear thresher (Precision Machine model WHTA010002, Co. Inc. Lincoln, NE, USA), cleaned, packed in small paper bags with all the above information and stored in a dry room for no longer than two weeks until the grain analyses. The total number and weight of grains of five heads, biological yield (grain weight per head) and weight of 1000 grains were calculated (TGW).

Statistical Analysis
The statistical analyses were carried out using SAS software package, version 9.3 (SAS Institute Inc., USA) (p ≤ 0.05), to identify the significance of pathogenicity between Fusarium species obtained from different hostplants. Research data were processed via Tukey's HSD (honestly significant difference) test (p = 0.05). The mean ± SE (standard error of the mean) was used to describe the variability of measurements. The area under the FHB disease progress curve (AUDPC), FHB severity and 1000-grain weight (TGW) were statistically evaluated, and calculations were performed in Microsoft Office Excel 2007. This program was also used to present the data graphically.

Evaluation of FHB Severity, AUDPC and TGW under Wheat Inoculation with Fusarium Fungi Isolated from the Non-Gramineous Plants
Overall, 43 out of 46 of Fusarium spp. isolates obtained from non-gramineous plants present in the crop rotation fields were confirmed to be pathogenic to spring wheat according to the ability to cause FHB symptoms during the floret inoculation test in the greenhouse. After 21 DPI, in Fusarium spp.-inoculated heads, the FHB severity ranged from 9.3% to 69.6% and was significantly higher (p < 0.01) compared to the water control (4.3%) ( Figure 1). In F. avenaceum-inoculated heads, the FHB severity values varied from 9.3% to 19.0% (on average, 12.6%). The isolate BN19c from B. napus showed the highest FHB severity (19%), while the isolates BV33.3s from B. vulgaris and PS10fl from P. sativum were the least pathogenic (7.8% and 8.0%) and did not differ from the water control. In F. culmorum-inoculated heads, FHB severity values ranged from 9.5% to 45.7% (on average, 31.8%), with the highest value (45.7%) observed in isolate BV15.1l from B. vulgaris. In F. graminearum-inoculated heads, the FHB severity values varied from 11.7% to 69.6% (on average, 26.8%). The isolate 5PS3p3-1 from P. sativum showed the highest FHB severity value (69.6%). In F. sporotrichioides-inoculated heads, the FHB severity values ranged from 9.7% to 16.7% (on average, 11.9%). The highest FHB severity (16.7%) showed isolate 9SWSP17 from spring wheat. Isolate PS37s from P. sativum was the least pathogenic (8.2%) among the F. sporotrichioides isolates and did not differ from the water control.
Fusarium species obtained from different hostplants. Research data were processed via Tukey's HSD (honestly significant difference) test (p = 0.05). The mean ±SE (standard error of the mean) was used to describe the variability of measurements. The area under the FHB disease progress curve (AUDPC), FHB severity and 1000-grain weight (TGW) were statistically evaluated, and calculations were performed in Microsoft Office Excel 2007. This program was also used to present the data graphically.

Evaluation of FHB Severity, AUDPC and TGW under Wheat Inoculation with Fusarium Fungi Isolated from the Non-Gramineous Plants
Overall, 43 out of 46 of Fusarium spp. isolates obtained from non-gramineous plants present in the crop rotation fields were confirmed to be pathogenic to spring wheat according to the ability to cause FHB symptoms during the floret inoculation test in the greenhouse. After 21 DPI, in Fusarium spp.-inoculated heads, the FHB severity ranged from 9.3% to 69.6% and was significantly higher (p <0.01) compared to the water control (4.3%) ( Figure 1). In F. avenaceum-inoculated heads, the FHB severity values varied from 9.3% to 19.0% (on average, 12.6%). The isolate BN19c from B. napus showed the highest FHB severity (19%), while the isolates BV33.3s from B. vulgaris and PS10fl from P. sativum were the least pathogenic (7.8% and 8.0%) and did not differ from the water control. In F. culmorum-inoculated heads, FHB severity values ranged from 9.5% to 45.7% (on average, 31.8%), with the highest value (45.7%) observed in isolate BV15.1l from B. vulgaris. In F. graminearum-inoculated heads, the FHB severity values varied from 11.7% to 69.6% (on average, 26.8%). The isolate 5PS3p3-1 from P. sativum showed the highest FHB severity value (69.6%). In F. sporotrichioides-inoculated heads, the FHB severity values ranged from 9.7% to 16.7% (on average, 11.9%). The highest FHB severity (16.7%) showed isolate 9SWSP17 from spring wheat. Isolate PS37s from P. sativum was the least pathogenic (8.2%) among the F. sporotrichioides isolates and did not differ from the water control. After 28 DPI, in Fusarium-inoculated heads, the AUDPC varied from 161.5% to 1044.6% and was significantly higher (p < 0.01) compared to the water control (70.9%) (Figure 2). In F. avenaceum-inoculated heads, the AUDPC values ranged from 161.5% to 331.6% (on average, 254.8%). The isolate BN19c from B. napus showed the highest AUDPC value (331.6%). In F. culmorum-inoculated heads, the AUDPC values ranged from 332.5% to 683.7% (on average, 480.9%), with the highest value (683.7%) observed in isolate 8SW5SP2 from spring wheat. In F. graminearum-inoculated heads, the AUDPC values ranged from 191.5% to 1044.6% (on average, 438.2), with the highest value (1044.6%) observed in isolate 5PS3p3-1 from P. sativum. In F. sporotrichioides-inoculated heads, the AUDPC values ranged from 189.0% to 291.8% (on average, 220.7%), with the highest value (291.8%) observed in isolate 9VKV17 from spring wheat.

Evaluation of FHB Severity, AUDPC and TGW under Wheat Inoculation with Fusarium Fungi Isolated from Weeds
After 21 DPI, in Fusarium-inoculated heads, the FHB severity ranged from 6.2% to 81.0% and was significantly higher (p < 0.01) compared to the water control (1.9%) (Figure 4). In F. avenaceum-inoculated heads, the FHB severity varied from 6.2% to 19

Comparison of the Pathogenicity of Fusarium Fungi Isolated from Different Hostplants
In this study, the aggressiveness of different Fusarium species isolated from different hostplant groups was compared (weeds, non-gramineous plants and Triticum). The results showed that FHB severity was highest (46%) when spring wheat was inoculated with a F. culmorum species group isolated from weeds; similar results were obtained for the species isolated from wheat (42%). Meanwhile, F. graminearum and F. culmorum isolates from non-gramineous plants caused FHB severity in wheat of 27% and 29%, respectively. These data indicate that Fusarium isolates from various hostplants can produce different disease severities. Although F. graminearum is noted to be the most pathogenic among Fusarium species for wheat, in our case, the conditions were more favorable for the development of F. culmorum. F. avenaceum and F. sporotrichioides isolates, which caused disease with similar severity (8-12%) in all hostplant groups (Figure 7). Figure 5. AUDPC values (%) in spring wheat inoculated with Fusarium isolates isolated from weeds at 28 DPI. Spring wheat florets were inoculated with the spore suspension (1 × 10 5 ) in the middle of anthesis. AUDPC values are the means of 5 replicates ± standard error (SE). Different letters above the bars indicate significant differences according to Tukey's HSD (honestly significant difference) test (confidence level = 0.95) compared to the water control (p < 0.01). TI-Tripleurospermum inodorum, VA-Viola arvensis, CBP-Capsella bursa-pastoris, PA-Poa annua, FC-Fallopia convolvulus, SW-spring wheat, G-glyphosate soil, c-crown, f-fruit, fl-flower, l-leave, r-root, s-stem, sp-head.
At the full ripening stage (BBCH 89), F. culmorum isolates CBP1147c and CBP1401r from C. bursa-pastoris (27.5 and 19.0 g), PA1129c and PA1129f from P. annua (24.7 and 27.7 g) and FC1088r from F. convolvulus (29.2 g) showed significantly (p < 0.01) lower TGW values compared to the water control (39.7 g) ( Figure 6). The F. graminearum isolates PA1130c from P. annua (30.0 g) and 6SW5SP1 from spring wheat (32.2 g) showed a significantly lower (p < 0.01) 1000 grain weight compared to the water control. Figure 6. The 1000 grain weight (g) (TGW) in spring wheat inoculated with Fusarium isolates isolated from weeds at the full ripening stage. Spring wheat floret values are the means of 5 replicates Figure 6. The 1000 grain weight (g) (TGW) in spring wheat inoculated with Fusarium isolates isolated from weeds at the full ripening stage. Spring wheat floret values are the means of 5 replicates ± standard error (SE). Different letters above the bars indicate significant differences according to Tukey's HSD (honestly significant difference) test (confidence level = 0.95) compared to the water control (p < 0.01). TI-Tripleurospermum inodorum, VA-Viola arvensis, CBP-Capsella bursa-pastoris, PA-Poa annua, FC-Fallopia convolvulus, SW-spring wheat, G-glyphosate soil, c-crown, f-fruit, fl-flower, l-leave, r-root, s-stem, sp-head.
Significant differences were observed between groups of Fusarium strains in the AU-DPC values. F. culmorum strains isolated from weeds showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control under greenhouse conditions (Figure 8). In the non-gramineous hostplant group, F. culmorum and F. graminearum strains showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control. In the Triticum group, F. culmorum strains were also the most aggressive and showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control.
The thousand grain weight (TGW) values between wheat inoculated with Fusarium isolates obtained from different hostplants (weeds, non-gramineous plants and Triticum) were found to be very similar (Figure 9). A statistically significant decrease in TGW was observed when wheats were inoculated with F. culmorum isolated from weeds, non-gramineous plants and Triticum (on average, decreases of 25.4%, 23.3% and 21.8% respectively), compared to the water control. F. avenaceum, F. graminearum and F. sporotrichioides reduced TGW compared to the control, but the results were statistically insignificant. species isolated from wheat (42%). Meanwhile, F. graminearum and F. culmorum isolates from non-gramineous plants caused FHB severity in wheat of 27% and 29%, respectively. These data indicate that Fusarium isolates from various hostplants can produce different disease severities. Although F. graminearum is noted to be the most pathogenic among Fusarium species for wheat, in our case, the conditions were more favorable for the development of F. culmorum. F. avenaceum and F. sporotrichioides isolates, which caused disease with similar severity (8-12%) in all hostplant groups (Figure 7). Significant differences were observed between groups of Fusarium strains in the AUDPC values. F. culmorum strains isolated from weeds showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control under greenhouse conditions (Figure 8). In the non-gramineous hostplant group, F. culmorum and F. graminearum strains showed significantly higher AUDPC values (p < 0.01) com- Figure 7. FHB severity (%) in spring wheat inoculated with Fusarium species isolated from different hostplants. FHB severity values are the means of all isolates of one Fusarium species ± standard error (SE). Different letters above the bars indicate significant differences according to Tukey's HSD (honestly significant difference) test (confidence level = 0.95).
Significant differences were observed between groups of Fusarium strains in the AUDPC values. F. culmorum strains isolated from weeds showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control under greenhouse conditions (Figure 8). In the non-gramineous hostplant group, F. culmorum and F. graminearum strains showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control. In the Triticum group, F. culmorum strains were also the most aggressive and showed significantly higher AUDPC values (p < 0.01) compared to other Fusarium species and the water control. The thousand grain weight (TGW) values between wheat inoculated with Fusarium isolates obtained from different hostplants (weeds, non-gramineous plants and Triticum) were found to be very similar (Figure 9). A statistically significant decrease in TGW was observed when wheats were inoculated with F. culmorum isolated from weeds, non-gramineous plants and Triticum (on average, decreases of 25.4%, 23.3% and 21.8% respectively), compared to the water control. F. avenaceum, F. graminearum and F. sporotrichioides reduced TGW compared to the control, but the results were statistically insignificant.

Discussion
Although numerous studies have already investigated the epidemiology of Fusarium species, there is still a lack of information about how alternative cropping system plants and weeds contribute to the spread of FHB. The present study reports the results of the pathogenicity of Fusarium spp. isolates from asymptomatic non-gramineous plants and weeds to spring wheat under greenhouse conditions. As previously reported, weeds and non-gramineous plants can serve as alternative hosts for Fusarium species [9,18,32,33,37,39,40], leading cereals to become contaminated by pathogenic fungi. Our first experiment with non-gramineous cropping system plants confirmed that species such as B. napus, P. sativum and B. vulgaris can harbor FHB-associated Fusarium fungi such as F. avenaceum, F. culmorum, F. graminearum and F. sporotrichioides. These findings agree with the previous study of Rasiukeviciute et al. [38], which presented non-cereal plants as alternative hostplants. Isolates from the above-mentioned plants produced FHB symptoms in the tested spring wheat inoculated with the spore suspension (1 × 10 5 ) and some were more pathogenic compared to those isolated from primary hostplants. F. avenaceum isolate BN19c from B. napus, F. culmorum isolate BV15.1l from B. vulgaris and F. graminearum isolate 5PS3p3-1 from Pisum sativum showed the highest FHB severity (%). Our results indicated that FHB severity differs between isolates and between species. Additionally, Fusarium species also play a key role in the infection of FHB, as F. graminearum from non-gramineous plants was most pathogenic. These findings are consistent with other study results [33,48,49]. A study conducted under field conditions by Matelionienė et al. [48] showed that F. graminearum isolates from both wheat and weeds cause severe FHB disease, whereas F. avenaceum species did not show heavy disease symptoms. Notably, other crucial factor for pathogenicity to wheat include the production of different mycotoxins by Fusarium species. A previous study by Janaviciene et al. [50] investigated mycotoxin concentrations in spring wheat inoculated with F. graminearum strains isolated from weeds, including P. annua, T. inodorum, V. arvensis, F. convolvulus, B. napus and T. aestivum. The authors observed that the levels of mycotoxin production depended not only on the trichothecene genotype but also (and mostly) on the strain and environmental conditions. In addition to other studies showing that F. graminearum from non-cereal plants is pathogen to cereals under field conditions [38], our findings also illustrate that F. graminearum (5PS3p3-1) from P. sativum can cause severe FHB symptoms. The aforementioned isolate showed the highest FHB severity and highest AUDPC value (%) under greenhouse conditions. It is also evident, that F. culmorum isolates from non-gramineous plants were as pathogenic as F. graminearum isolates, since isolate BV15.1 l from B. vulgaris showed higher FHB severity (%) than isolates from wheat. In addition, our results indicate that F. culmorum isolates BN26r and BN39fl from B. napus, isolates BV15.1l and BV142.1pe from B. vulgaris decreased the 1000 grain weight by 37%, 30%, 28.8% and 31.8%, respectively, compared to the water control. These findings agree with those of Brennan et al. [12], who showed that F. culmorum and F. graminearum strains caused a losses of 54.3% and 46.9%, respectively, to 1000 grain weight in wheat cultivars. Our results also confirm that although F. avenaceum is reported to cause FHB symptoms in cereals, it is not as pathogenic as F. culmorum and F. graminearum. There is, however, very little information about the pathogenicity of F. sporotrichioides to spring wheat. Our study showed that F. sporotrichioides isolates from non-gramineous plants were able to cause FHB-associated symptoms, although the pathogenicity of the isolates was less strong than that of other Fusarium strains.
Our study also agrees with the findings of other scientists who reported that nonsymptomatic weeds can harbor FHB-associated Fusarium fungi [18,39,40]. In our second experiment, F. avenaceum, F. culmorum, F. graminearum and F. sporotrichioides were observed in weeds such as F. convolvulus. C. bursa pastoris, P. annua, T. inodorum and V. arvensis without any visible symptoms. Suproniene et al. [39,40] reported that F. graminearum is one the most colonizing and pathogenic Fusarium species among weeds along with F. culmorum. Our study also indicated that these two Fusarium species are the most pathogenic. Specifically, spring wheat inoculated with isolates from these species showed the highest levels of FHB severity, while the AUDPC and 1000 grain weight were the lowest. Among all Fusarium spp. isolates from weeds, the F. culmorum isolate CBP1401r from C. bursa-pastoris was the most pathogenic and showed the highest FHB severity and AUDPC value. This isolate decreased the 1000 grain weight by 52% compared to the water control. These findings agree with previous study by Jenkinson and Parry [49], who reported that C. bursa-pastoris was among the weed hostplants that harbor F. avenaceum and caused FHB symptoms in winter wheat within the UK. The F. avenaceum isolate FC1178fl from F. convolvulus, F. culmorum isolate CBP1401r from C. bursa-pastoris and F. graminearum isolate PA1130c from P. annua showed highest FHB severity and were more pathogenic to spring wheat compared to isolates from the primary hostplant-wheat. These results show that not only primary hostplants, but also non-symptomatic weeds can harbor pathogenic Fusarium fungi and produce FHB symptoms in spring wheat. Moreover, isolates from F. avenaceum and F. sporotrichioides did not have any significant difference in 1000 grain weight and were less pathogenic than the other two Fusarium species. These findings are similar to those reported in the study by M. Gerling et al. [51], which demonstrated that weeds were 15 times more strongly infected with Fusarium fungi than herbaceous plants. The difference in the hostplant colonization of fungi is explained by the greater genetic diversity of weeds than cultivated plants. Due to this diversity, weeds are less susceptible to diseases themselves and do not show any signs of disease associated with FHB. Linde et al. [52] also confirmed that pathogens from genetically diverse hosts, such as weeds, may be more virulent than pathogens from monocultural hosts.
In our research, we also evaluated the aggressiveness of different Fusarium species isolated from various hostplants. We determined that, under greenhouse conditions, F. culmorum strains from weeds and primary hostplant Triticum were more pathogenic than the other three investigated Fusarium species. Additionally, in non-gramineous plants, F. culmorum was the most pathogenic alongside F. graminearum, which is already known to be one of the most aggressive Fusarium strains. Therefore, our findings relating to F. culmorum are very important as they show not only that Fusarium strains from F. graminearum complex can cause FHB-associated symptoms, but also that F. culmorum should be considered one of the most aggressive Fusarium strains against spring wheat. Despite this factor, it remains necessary to further investigate other Fusarium species isolated from alternative hostplants and their ability to cause FHB. We should also obtain more information on which species contributes most to the spread of pathogenic disease.

Conclusions
In conclusion, our results indicate that asymptomatic non-gramineous crop rotation plants such as Brassica napus, Pisum sativum and Beta vulgaris and weed species such as Tripleurospermum inodorum, Fallopia convolvulus, Poa annua, Capsella bursa-pastoris and Viola arvensis, which are found in cereal-based crop rotations, can serve as reservoirs of pathogenic Fusarium species. In the present study, we showed that isolates from these plants can cause FHB symptoms in spring wheat under greenhouse conditions. We determined that F. culmorum strains isolated from weeds and Triticum aestivum were more pathogenic to spring wheat, whereas in non-gramineous plant groups F. culmorum and F. graminearum were the most pathogenic to spring wheat. A significant thousand grain weight reduction was caused only by F. culmorum strains isolated from all hostplant groups. Despite these results, further research is necessary to investigate the significance and role of other Fusarium species from alternative plants as such species play a key role in FHB epidemiology.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pathogens11121467/s1, Table S1: Treatments in Experiment I for assessing the pathogenicity of Fusarium spp. isolated from non-gramineous plants to spring wheat under greenhouse conditions; Table S2: Treatments in Experiment II for assessing the pathogenicity of Fusarium spp. isolated from weeds to spring wheat under greenhouse conditions. Author Contributions: Conceptualization, S.S. and G.K.; methodology, E.Z. and A.G.; validation, E.Z., S.S. and N.M.; formal analysis, E.Z.; investigation, E.Z., S.S. and G.K.; data curation, E.Z.; writing-original draft preparation, E.Z.; writing-review and editing, N.M. and S.S.; visualization, E.Z. and A.G.; supervision, S.S. All authors have read and agreed to the published version of the manuscript.