Pathogenicity of nine Fusarium species causing head blight and crown rot in Iraqi wheat cropping system, Basra province

This experiment was conducted to determine pathogenicity of 32 isolates of nine Fusarium spp. (F. chlamydosorum, F. equiseti, F. graminearum, F. pseudograminearum, F. solani, F. avenaceum, F. culmorum, F. cerealis and F. nygamai) on the heads, stems and seeds of soft winter wheat cultivar, Abu Ghraib 3 (AG 3). For pathogenicity test on head bleaching, the effects of disease severity and percent Fusarium head blight (FHB) were significant (P ≤ 0.05) for each of Fusarium species and isolates as well as time were analyzed individually. Analysis by Fusarium species displayed that F. culmorum, F. cerealis and F. graminearum caused the highest percent FHB at 21 days after inoculation, while F. chlamydosporum and F. nygamai were the least pathogenic. F. Pseudograminearum had the greatest effect on seed germination (40-48%), followed by F. culmorum (53-60%). The effects of most examined Fusarium species and isolates were significant (P ≤ 0.05) for discoloration rating of basal stem as indications of Fusarium crown rot (FCR). F. pseudograminearum had the highest average percent disease severity of FCR (60.58%), followed by F. cerealis (51.13%), F. culmorum (49.58%), F. avenaceum (48.61%) and F. graminearum (43.92%). Infected heads and seeds might be resulted in basal stem infections, consequently serving Fusarium inoculum to be survived over time. Infected ground and underground plant parts could then become an inoculum sources for head infection in the next seasons.


INTRODUCTION
According to (Leslie and Summerell, 2008), the genus Fusarium encompasses more than 70 worldwide species, arising in natural conditions in various areas of the world. They are saprophytically widespread in soil. Similarly, they can develop on plant debris and other organic material. Under favorable conditions, a number of them are facultative parasites. In such conditions, Fusarium spp. might give rise to plant diseases of both upper and under organs of plants. Thereupon, species for the genus Fusarium are believed to be a number of the most risky pathogens of cereals and other varieties of plants, producing noteworthy economic losses. The majority of Fusarium spp. are polyphagous, contaminating diverse plant species. In either case, any growth stages of plants can be subjected by Fusarium spp., leading to Fusarium head blight (FHB), Fusarium crown rot (FCR), Fusarium root rot (FRR), seedling blight, and leaf necrosis in cereals (Champeil et al., 2004;Łukanowski and Sadowski, 2002). Specifically, in wheat agricultural fields, the highest substantial problems are FCR and FHB diseases. Reduction of the occurrence of those diseases is tremendously difficult, because of the massive share of cereals cultivation globally. Correspondingly, weather conditions are another factors effecting the presence of those diseases that influence both growth of plants and the infection occurrence (Doohan et al., 2003;Narkiewicz-Jodko et al., 2005). A number of Fusarium species that give rise to FHB, can likewise be pathogens to FCR (Mitter et al., 2006). The best example of these species are F. graminearum, F. culmorum, F. aveaceum and F. acuminatum. Furthermore, many researches have reported that the biology and epidemiology of FCR and FHB pathogens are related. As stated by (Dill-Macky, 2003), F. graminearum has a great prominence in its ability for variation. This ability is due to the differences in the destructive infections of diverse isolates to small grain cereals as wheat, barley and maize. Differentiations in pathogenicity among these small grain cereals are renowned, however explanation of the relationships between pathogen species or isolates and type of crops is still unclear. The presence of diverse pathogenic isolates seasonally may be highly related to the fluctuations in environmental conditions. In view of that, observation of such different isolates on crowns and stem bases of wheat crops may result in extreme head blight, on the other hand crown rot is generated only by isolates from diseased stem bases or crowns. Previously, the authors of this study have isolated and identified morphologically and molecularly nine Fusarium species (F. pseudograminearum, F. graminearum, F. culmorum, F. chlamydosprum, F. equiseti, F. solani, F. avenaceum, F. cerealis, and F. nygamai) from Iraqi wheat crops as causal agents of FHB and FCR (Minati and Mohammed-Ameen, 2019d). Also, they have studied the interaction between FHB and FCR disease incidences and cultural practices on wheat in the south of Iraq (Minati and Mohammed-Ameen, 2019b). Furthermore, they have studied the interaction between FHB and FCR disease incidences and environmental factors and soil physiochemical analysis on wheat in Basra Province (Minati and Mohammed-Ameen, 2019c). Moreover, they investigated the co-occurrence of FHB and FCR on several wheat cultivars in the south of Iraq (Minati and Mohammed-Ameen, 2020). Additionally, they have conducted an important study to detect and quantify three mycotoxins Dioxynivalenol, Nivalenol and Fumonisin B2 in seeds of seven wheat cultivars contaminated with those nine Fusarium species in 17 wheat fields distributed in the south of Iraq (Minati and Mohammed-Ameen, 2019a). For comprehensive and complementary study, the objective of our study was conducted to determine the pathogenicity of 32 isolates of those nine Fusarium species on heads, stems and seeds of a susceptible winter wheat cultivar.

MATERIAL AND METHODS Isolate sources of Fusarium species
Isolates of the F. chlamydosorum, F. equiseti, F. pseudograminearum, F. solani, F. avenaceum and F. culmorum were isolated from wheat roots, stems and heads. F. graminearum isolates were isolated from wheat roots, stems and rhizospere. While, isolates of F. cerealis and F. nygamai were isolated only from wheat stems and roots respectively. Based on field assessments of the disease incidence experiment (Minati and Mohammed-Ameen, 2020), Abu Ghraib 3 (AG 3) was the most FCR and FHB-susceptible cultivar.

Planting
Seeds of the AG 3 cultivar were planted in 10-cm diameter pots. The soil mixture contained of 50% clay loam soil and 50% Turkish sphagnum peat moss. The peat moss was sterilized by autoclaving at 15 lbs.' pressure (121°C) for 20 minutes.

Inoculum preparation
For pathogenicity evaluation of seed germination and seedling survival as well as occurrence of stem lesions (FCR symptoms), one square centimetre of mycelial plug of PDA agar from energetically growing edges of the each Fusarium isolate was placed in a depth of 5 cm in the soil mixture for each pot. Pots then watered lightly and covered with a transparent plastic bag for 48 hours following inoculation. Seeds of the AG 3 cultivar, surface-sterilized by moistening with 70% ethanol for two minutes, soaking in 2% sodium hypochlorite for one minute, rinsing in sterile distilled water several times and then air dried on a filter paper. Sterilized seeds were planted on the 30 th November 2018 at rate of 10 seed / pot after 60 days of vernalisation at 4ºC. Pots with no fungal agar plug on them were used for the control treatment. Pots were kept under semi-controlled conditions (25°C day/18°C night ±3). The pots were located on a greenhouse worktop and fertilized weekly. European fertilizer contained of 20:20:20 NPK added weekly at a rate of tow tablespoon per 10 litres throughout regular watering. For pathogenicity evaluation of head bleaching, seeds were planted on the 15 th of November 2018 at rate of three seed / pot (10-cm diameter pots) after 60 days of vernalisation at 4ºC. The soil mixture was not inoculated with mycelial plugs. The pots were also located on a greenhouse worktop and fertilized weekly. European fertilizer contained of 20:20:20 NPK added weekly at a rate of tow tablespoon per 10 litres throughout regular watering. Plants were kept until ripening stage under the same conditions used prior to inoculation. At ripening stage, three spikes / pot were sprayed (using hand-held sprayer) to overflow with a spore suspension (1 × 10 -6 conidia / ml) of each isolate and concealed with a transparent plastic bag for 72 h. Spore suspension was facilitated by adding one drop of Tween 20 (polyoxyethylene sorbitan monolaurate) to each 250 ml. Fusarium isolates were grown on PDA for 7-10 days in 9-cm-diameter Petri dishes in a low temperature incubator set at 25ºC ±2. Control plants were sprayed in the same way with sterile distilled water. Each spike was rated in a visual manner for percent surface area with FHB symptoms at one, two, and three weeks after inoculation. A design of these experiments was chosen to be a split plot randomized complete block with three replications for each isolates (each replicate containing of a pot with three plants).

Statistical analysis
Based on the percent symptoms of FCR and FHB, discoloration ratings, seed germination and seedling survival for each plant, average values were considered for each replicate (pot). The effects of Fusarium species, and isolates at different three times on percent FHB in the head bleaching test, and the effects of Fusarium species, and isolates on seed germination, seedling survival and discoloration ratings of basal stem in the seed and seedling tests, were evaluated using the statistical program (SPSS ® ver. 21) software (SPSS, 2012). Percentage data of FHB and FCR tests were arcsine-transformed before analysis to stabilize variance. When F values were significant (P ≤ 0.05), least significant differences were calculated. Interaction between species and time, isolates and time, species and isolate source as well as isolates and source were done to make comparisons among percent FHB in the head test, and among seed germination, seedling survival and discoloration ratings of basal stem in the seed and seedling tests.

Pathogenicity test on head bleaching
The interactions of Fusarium species × time and Fusarium isolates × time and species × source were not significant (P > 0.05) for most of the measurements employed. The only one-way interaction that was significant was Fusarium isolates × isolate sources (P ≤ 0.01). However, when Fusarium species and isolates as well as time were analyzed individually, the effects of disease severity and percent FHB were significant (P ≤ 0.05 to P ≤ 0.01) for each of the measurements taken. The majority of examined nine Fusarium species with their 32 isolates were shown significant percent FHB (P ≤ 0.05 to P ≤ 0.01) at 7, 14 and 21 days after inoculation, except F. nygamai, F. chlamydosporum and some isolates of other Fusarium species their effects were less than 15%, which is not significant. There was no significant differences between (7 and 14) days after inoculation, whereas, the difference between (21 and 7) days was highly significant (P ≤ 0.01), and between (21 and 14)  Isolates of F. graminearum, F. culmorum (originated from stem) and F. cerealis (originated from root) seemed to be more pathogenic to heads compared to other isolates of the same species. Whereas, only one isolate of F. solani (originated from head) had significant effect on examined wheat heads as shown in (Figure 1).

Pathogenicity test on seeds
Analysis of the seed test of AG 3 cultivar indicated that the interaction of Fusarium species × seed germination and Fusarium species × seedling survival was not significant (P > 0.05). However, when isolate sources were analyzed, the source effects of Fusarium species and isolates were significant (P ≤ 0.05 to P ≤ 0.01) for most measurements taken, except F. chlamydosporum had the least effect on seed germination and development of surviving plants, which were not significantly different (P ˃ 0.05) from the control.  Table 4). Few seedlings emerged from seeds inoculated with isolates of these pathogens, or were still healthy at the end of experiment. Development of the surviving plants was stunted most by F. pseudograminearum (from 0 -7%), followed by culmorum (7-28%) and F. cerealis (14-21%), ( Table 4).

Pathogenicity test on seedlings
The interaction of Fusarium species × isolate sources and Fusarium isolates × isolate sources was not significant (P > 0.05) for all of the percent disease severity. However, when both measurements were analyzed together, the effects of most examined Fusarium species and isolates were significant (P ≤ 0.05 to P ≤ 0.01) for discoloration rating of basal stem. F. pseudograminearum had the highest average percent disease severity of FCR (60.58%), followed by F. cerealis (51.13%), F. culmorum (49.58%), F. avenaceum (48.61%) and F. graminearum (43.92%). Only inoculation with F. nygamai and F. chlamydosporum caused a lower average percent disease severity (11.36 and 19) % respectively compared to the other species. In turn, F. equiseti caused almost similar discoloration of basal stem as F. solani ( Table 3). In the seedling test, the discoloration of basal stem detected at harvest time elongated up to the basal stem of plants on an average of (60%, 51% and 49%) of the seedlings inoculated with F. pseudograminearum, F. cerealis and F. culmorum respectively and 48% and 43% of the seedlings inoculated with F. avenaceum and F. graminearum respectively, and the discoloration frequently touched and sometime passed the first node (Figure 2). The obvious spread of the discoloration for the rest of examined Fusarium species on the basal stem ranged between 19-30%, except F. nygamai, which caused less discoloration rate (11.36%).  In general, all Fusarium isolates caused discoloration of the basal stems. The isolates of F. pseudograminearum (ranged between 45 to 70.83%), F. cerealis (ranged between 47.72 to 54.54%), F. culmorum (ranged between 41 to 57.14%) and F. graminearum (ranged between 41.07 to 54.61%) caused the greatest discoloration ratings. Furthermore, even though there were some differences in pathogenicity among isolates within some of the Fusarium species, but on the whole, there were not significant differences among isolates of the rest of examined species, except that isolate of F. nygamai that caused the least discoloration (11.36%), which was not different (P > 0.05) from the control treatments (Table 4). In combination of all Fusarium isolate treatments, a positive correlation was detected between seed germination and plant survival in the seed test (r = 0.93, P ≤ 0.05). Also, simple correlations was executed among all measurements performed in both tests showed that the survival of inoculated plants in the seed test was correlated with that in the seedling test for Fusarium 10 species (r = 0.95, P ≤ 0.05) and Fusarium isolates (r = 0.71, P ≤ 0.01). In contrast, there was a great negative correlation between percent disease severity of FHB at 21 days and seed germination and seedling survival in the seed test (r = -0.89 to -0.93, P ≤ 0.01 for Fusarium species; r = -0.87 to -0.95, P ≤ 0.01 for Fusarium isolates), and between percent disease severity of FHB at 21 days and plant survival (r = -0.89, P ≤ 0.05 for Fusarium species; r = 0.71, P < 0.05 for Fusarium isolates) in the seedling test.

DISCUSSION
Neither the difference in pathogenicity of the nine Fusarium species nor the pathogenicity of F. pseudograminearum, F. graminearum and F. equiseti on wheat has been previously studied. The high pathogenicity appeared on wheat heads caused by F. culmorum and F. graminearum compared to the other Fusarium species examined in this study agree with a number of studies, even though the use of a spore suspension in which was varied such as injection of the spore suspension into, or dropping it onto a part or an entire spike (Specht and Rush, 1988;Walker et al., 2001;Wilcoxson et al., 1988;Wong et al., 1995). Moreover, we found that the pathogenicity on heads caused by F. culmorum was greater than F. graminearum, which is also in concurrent with (Balmas et al., 1995;Mihuta-Grimm and Forster, 1989;Wong et al., 1995), however it does not agree with (Fernandez and Chen, 2005), who reported no real differences between the two species in pathogenicity on wheat heads. The pathogenicity of F. cerealis to wheat heads in this study was less than F. culmorum and higher than F. graminearum. This finding is in accordance with (Xue et al., 2006), who stated that F. crealis is highly pathogenic to heads but not higher than F. culmorum. (Sugiura et al., 1994) and (Xue et al., 2004) also confirmed that Fusarium cerealis is severe pathogen causing FHB symptoms on wheat heads in Canada. Furthermore, we also found that F. avenaceum and F. equiseti were relatively moderate pathogenic species to wheat heads, while (Fernandez and Chen, 2005;Wilcoxson et al., 1988;Wong et al., 1995) found them least pathogenic species. This disagreement can be at least relatively, attributed to the diverse inoculation techniques applied. However, in contrast to our findings, (Golinski et al., 2002) found that F. avenaceum had a greater effect on wheat heads than F. culmorum when a spore suspension was sprayed onto heads in the field trial. In general, the pathogenicity results of seed and seedling tests in this study also go along with other researches on Fusarium pathogenicity to basal stems of wheat, even though comparative differences among the examined Fusarium species have in some circumstances varied. Similar to our results, F. pseudograminearum was described as more pathogenic to wheat seedling than other Fusarium species (Kazan and Gardiner, 2018), lead to seedling death before or after development, particularly when planted seeds were collected from previously infected fields by different Fusarium species (Simpfendorfer, 2013), and it may cause wide browning basal stem directly after infection and during plant growth (Kazan and Gardiner, 2018). We also found that F. cerealis was more pathogenic than F. culmorum and F. graminearumon on wheat in causing FCR. This result is completely in agreement with (Liddell, 1985) who stated that Fusarium cerealis is severe pathogenic on wheat, causing FCR and FRR higher than F. culmorum and F. graminearum. What's more, our result agree with many studies that reported that the pathogenicity of F. culmorum to wheat seedling was a greater than F. avenaceum (Arseniuk et al., 1993;Colhoun et al., 1968), to wheat seed was a greater than F. graminearum and F. avenaceum (Uoti, 1976), and cause broad crown rot to wheat seedling compared to F. graminearum (Corazza et al., 2001). Besides, according to (Arseniuk et al., 1993), the pathogenicity of F. culmorum, F.  11 graminearum, and F. avenaceum to winter wheat seedling was highly a greater than F. equiseti, which is also similar to our result. However, as opposed to our results, the pathogenicity of F. avenaceum to winter wheat seedling was a greater that F. culmorum and F. graminearum (Arseniuk et al., 1993) and (Jenkinson and Parry, 1994), while (Kane and Smiley, 1987) and (Specht and Rush, 1988) have reported that F. avenaceum was a weak pathogen to winter wheat. This research has generally revealed parallel proportional susceptibility of wheat heads and basal stems to the most of Fusarium species examined, except F. nygamai and F. chlamydosporum that were less pathogenic species in both tests. However, the rest of Fusarium species were unevenly pathogenic to wheat heads and basal stems, except F. cerealis and F. avenaceum were in the second and fourth level of effects respectively for both tests, but with different percent disease severities. This study also shown that F. cerealis is extremely pathogenic and can possibly be a main causative agent of FHB and FCR on wheat in regions where it is present. F. Pseudograminearum was affected basal stems to a greater extent than F. culmorum, F. avenaceum and F. graminearum, whereas F. culmorum and F. graminearum were affected wheat heads to a greater extent than others. The higher pathogenicity of F. graminearum and F. culmorum on wheat heads compared to basal stems might be associated with stimulatory compounds existent at ripening stage in anthers. Similar explanation was given by (Stack and McMullen, 1985) when F. graminearum was more pathogenic to wheat heads than ground and underground wheat parts. Relative to the other Fusarium species examined, F. solani and F. equiseti were moderate pathogenic to heads and stems in both tests. The observation that the examined Fusarium species were differed in pathogenicity to heads than to basal stems or vice versa may be clarified, at least partially, by dissimilarities in the maturity of plant tissue between seed germination and infected plant parts (seedling, basal stems and heads) (Fernandez and Chen, 2005), might be due to the different sources of Fusarium species and their isolates resulted in a different adaptation of these isolates to the different infected wheat parts, and also may possibly affected by weather conditions, as they influence both disease incidences and development of plants (Doohan et al., 2003).

CONCLUSION
Overall, although the number of isolates for each Fusarium species was varied in this study, the high number of isolates did not indicate a real adaptation of such isolates to specific plant parts. For example, differences in pathogenicity among the nine isolates of F. culmorum did not seem correlated to the source of these isolates for both tests. This explanation, and the results of Fusarium species obtained from different sources had various pathogenicity on wheat heads, basal stems, seed germination and seedling survival, suggest that infected heads and seeds might be resulted in basal stem infections, consequently serving Fusarium inoculum to be survived over time. Infected ground and underground plant parts could then become an inoculum sources for head infection in the next seasons. Further research is needed to investigate the adaptation of these different isolates of each Fusarium species to another wheat cultivars and their developments on these cultivars in other Iraqi provinces, which have various weather conditions.