Introduction

Fusarium head blight (FHB) is a devastating disease of wheat. FHB has received more attention because of the impact that infection may cause yield losses of up to 50–70% and quality reduction due to the accumulation of several mycotoxins [especially deoxynivalenol (DON)] in the grain that poses a significant risk to food and feed safety (Parry et al. 1995; Dahl and Wilson 2018). The disease complex is associated with at least seventeen Fusarium species and two Microdochium causal agents like F. graminearum, F. culmorum and F. avenaceum are the most pathogenic and widely distributed species in most infected areas worldwide. F. equiseti, F. poae and F. cerealis are less frequent, and F. proliferatum, F. solani and F. verticillioides are the least frequent species of FHB (Saharan and Sharma 2009).

Assessment of quantitative components related to pathogenicity is a fundamental aspect in the FHB-wheat pathosystem (Wu et al. 2005). The expression of aggressiveness is not only controlled by the broad range of Fusarium species associated and the host, but also by the environment and their interactions (Singh and Aujla 1994; Dweba et al. 2017). Aggressiveness describing the quantitative pathogenicity may vary among FHB species and even among different isolates of a given species (Xue et al. 2004; Xu et al. 2008; Saharan and Sharma 2009; Xu and Nicholson 2009). There are very limited data on the differences in aggressiveness of the less frequent FHB species compared to the most commonly distributed (Zhang et al. 2012). No complete resistance to FHB was detected and limited number of resistant wheat genotypes are available (Kharbikar et al. 2019), and two primary types of polygenic resistance in wheat to disease infection were recorded (Mesterhazy et al. 1999): type 1 (resistance to initial penetration of the pathogen) and Type II (resistance to spreading within a spike).

Traditionally, pathogenic reaction of wheat to FHB pathogen to quantify aggressiveness is commonly evaluated under controlled and field conditions at mid-anthesis stage in adult plants by determining the visual scoring of head symptoms. It has the disadvantage of influenced by environment and leads to experimental error (Wu et al. 2005; Pakdaman et al. 2006; Imathiu et al. 2014). Thus, the testing of highly aggressive isolates without the risk of escaping into wheat production areas is the major constraint in traditional method. During the last two decades, less costly in vitro assays permitting for simple, accurate and reliable prediction of the head blight reaction in whole plant have been studied.

Germination rate reduction, lesion length, coleoptile length reduction and area under disease progress curve (AUDPC) have been utilized effectively to examine aggressiveness of different FHB isolates and several fungal species (Brennan et al. 2003; Wu et al. 2005; Imathiu et al. 2009; Opoku et al. 2011; Purahong et al. 2012; Khaledi et al. 2017; Sakr 2017, 2018a, c, d). In addition, several reports have showed that disease reactions revealed in diverse in vitro assays on juvenile plants strongly correlated with pathogenicity determined on mature plants (Wu et al. 2005; Purahong et al. 2012; Khaledi et al. 2017; Sakr 2017, 2018a, c, d, 2019b, 2020). Sakr (2019c) reported that AUDPC and latent period differentiated FHB isolates on barley plants. Furthermore, these criteria were indicators of aggressiveness occurring in the adult plant during FHB infection (2019c). The novel method of choice to analyze responses to individual FHB species should account for their aggressiveness on individual tissues or parts and their capacity to induce significant disease ratings at specific growth stages under favorable experimental conditions. Incubation period and LP of detached leaf assay and percentage infection of seedling assay are components of quantitative wheat resistance to FHB infection (Browne and Cooke 2004; Browne 2007; Shin et al. 2014; Sakr 2019a). Also, these criteria are measured as indicators of aggressiveness in several pathosystems (Lannou 2012). Till recently, disease reaction as measured by more distinct in vitro methodologies, i.e., detached leaf and seedling assays, to assess aggressiveness in several FHB species on durum and bread cultivars has not been combined in one experiment.

An important challenge is to analyze more effective and accurate in vitro disease evaluation methods for detecting pathogen differences and investigating if aggressiveness observed in individual plant organs and earlier growth stages may relate to disease expression in the whole plant (Purahong et al. 2012; Sakr 2017, 2018a, c, d, 2019b, 2020). In this context, we report the evaluation of quantitative components related to pathogenicity in FHB species complex using in vitro detached leaf and seedling assays on six durum and bread wheat cultivars. We have also examined the relationships between the present data and the findings previously and currently generated using in vitro Petri-dish test and artificial inoculations under controlled and field conditions.

Materials and methods

Fungal isolates

Sixteen fungal isolates representing the four Fusarium species (F. culmorum (F1, F2, F3, F28 and F30), F. solani (F7, F20, F26, F29, F31 and F35), F. verticillioides (synonym F. moniliforme) (F15, F16, F21 and F27) and F. equiseti (F43)) were recovered from symptomatic wheat spikes originating from Ghab Plain, one of the principal Syrian wheat production regions, during the 2015 growing season. On potato-dextrose agar, single-spore derived cultures were identified to species by microscopic observations of sporulation shape and type (spores and phialides morphology), along with optical observations of the cultural characteristics, such as colony shape and morphology, pigmentation and density of aerial and medial parts (Leslie and Summerell 2006). The 16 fungal isolates were verified to be belonged to four FHB species (F. culmorum, F. verticillioides, F. solani and F. equiseti) based on molecular identification (Sakr unpublished data). For long term storage, fungal cultures were stored in sterile distilled water at 4 °C and freezing at − 16 °C (Sakr 2018b). Conidia were dislodged and harvested by flooding the cultures with 10 ml of sterile distilled water and the suspensions of the 16 FHB isolates were filtered through two layers of sterile cheesecloth to remove mycelia. Concentrations of resulting spore suspensions were determined using a haemocytometer and adjusted to a desirable concentration.

Wheat cultivars

Six Syrian durum (Acsad65 released in 1984, Cham7 in 2004 and Cham9 in 2010) and bread (Cham4 released in 1986, Douma4 in 2007 and Bohoth10 in 2014) wheat cultivars were used for evaluating of the quantitative components. These cultivars with highest agronomic characteristics and resistance to biotic and abiotic stresses were selected because they are currently the most important wheat cultivars in Syria. Furthermore, pathogenic reactions in these cultivars were determined using in vitro component (AUDPC) of Petri-dish inoculation methodology (Sakr 2018a, c, d, 2020) and head and floret artificial inoculations under controlled conditions (Sakr 2019b). Therefore, we were able examine the relationships between the current findings with the previous results of in vitro AUDPC, spraying and point inoculations under controlled conditions.

Quantitative component tests in vitro

The capacity of 16 FHB isolates of four Fusarium species to confer disease on young plant organs was evaluated using a detached leaf assay according to Browne and Cooke (2004) and a seedling assay described by Shin et al. (2014) (Fig. 1). These methodologies were used by Sakr (2019a, c) to assess quantitative components of resistance in wheat to FHB infection and quantify aggressiveness of FHB agents on barley plants. Three aggressiveness components were assessed in the detached leaf assay: incubation period (days from inoculation to first appearance on the leaf surface, of a dull gray-green water-soaked lesion), latent period (days from inoculation to sporulation), and lesion length (measured after 7 days as a visible chlorotic area). Three replicates of each isolate based on observations on 120 detached leaves were set up, and the experimentation was conducted twice. Regarding seedling inoculation, the percentage of infected seedlings was assessed using pin-point and foliar-spraying methods. Lesion length was assessed utilizing a clip-dipping method. For these three experiments, three replications of each isolate were set up, and the experiment was conducted twice.

Fig. 1
figure 1

in vitro inoculation techniques used to assess Fusarium head blight aggressiveness on plant materials of Syrian durum wheat cultivar Acsad65 infected with isolate F29 (F. solani), a foliar-spraying assay, c clip-dipping assay, d pin-point assay and e detached-leaf assay

Full size image

Quantitative component tests under pot conditions

The 16 FHB isolates were separately inoculated on Acsad65, Cham7, Cham9, Cham4, Douma4 and Bohoth10 to measure disease incidence (DI) for aggressiveness as indicator of quantitative pathogenicity of the fungal isolates. The six wheat cultivars were cultivated at the Deir Al-Hajar Agricultural Experiment Station, located south east of Damascus, Syria (33°20′ N, 36°26′ E) at 617 m above sea level over the growing season 2018/19.

Wheat plants were grown in plastic 15-cm pots containing soil pasteurized at 5 k Gray of Gamma Ray (GR) with 60Co source using a gamma irradiator (ROBO, Russia). The soil used was a clay soil (57% clay, 39% loam and 2% sand) collected from Sojji research station (located east of Damascus, Syria, 3330′54.4″N 36°07′33.2″E) with the following characteristics: pH = 7.8; P = 13.4 ppm; K, Na, Ca, Mg = 1.81, 2.99, 33.1, 14 mg/100 g soil respectively, and organic matter = 1.25%. Each plastic pot was filled with 2 kg of air-dried, sieved (2 mm) soil. The experimental layout was a randomized complete design with three replicates (pots). Three pots per replicate were left non-inoculated as control treatment. Following emergence, plants were thinned and nitrogen fertilizer was applied twice at two dates: thinning and tillering. The plants were watered when needed. At mid-anthesis stage, plants of a pot were sprayed with a spore suspension at 5 × 104 spores/ml of 16 FHB isolates or sterile distilled water (control). Wheat heads were covered with clear plastic bags for 2 days to provide constant high humidity to promote primary infection. The experiment was repeated twice on Acsad65, Cham7, Cham9, Cham4, Douma4 and Bohoth10. Disease incidence (% symptomatic spike) for aggressiveness was estimated as the percentage of spikes showing pathogenic symptoms.

Statistical analyses

Data were subjected to analysis of variances (ANOVA) using StatView, 4.57® Abacus Concepts, Berkley, Canada. Prior statistical analysis, the percentages of infected seedlings were converted using the angular transformation to stabilize variances. Fisher’s LSD test was used to compare the means at p = 0.05. The sample correlation coefficients (Pearson r) were calculated using overall values per isolates at p = 0.05.

Results and discussion

It has been adopted that under natural conditions, FHB occurs unpredictably and the disease in not equally spread across fields, hence artificial infection is the most suitable for FHB bioassay assessment (Dweba et al. 2017). Quantitative components in wheat cultivars to FHB complex depends on percentage of diseased spike in growth chamber and field, however, this visual scoring of head symptoms incorporate several constraints (Imathiu et al. 2014). Such in vitro tests making it possible to predict aggressiveness at earlier plant stages relative to disease scoring in whole plant have been developed (Wu et al. 2005; Purahong et al. 2012; Khaledi et al. 2017; Sakr 2017, 2018a, c, d, 2019b, 2020). For the first time, we showed that the latent period (LP) of detached-leaf in vitro assay is a reliable and rapid method to discriminate aggressiveness in FHB species in wheat in the laboratory in a short period of time. In addition, we investigated the potential use of LP in predicting FHB data generated under several experimental conditions.

During our experiment, differences in inoculated treatment on young plant organs relative to water controls were found (Table 1), indicating that the FHB species used in this research were found to be suitable for the differential expression of all tested quantitative components. Detached leaves and seedlings of six wheat cultivars growing in the presence of 16 Fusarium isolates showed typical in vitro FHB symptoms according to two tested inoculations, suggesting a strong effect of the four Fusarium species on the growth of these cultivars whereas the control plants did not show any head blight symptoms. This also refers that the two different tested in vitro detached leaf and seedling assays (varying in incoculum concentration, infection methods, growth conditions and target young plant parts) conducted on Microdochium nivale, F. graminearum and the four tested FHB species to screen quantitative wheat resistance (Browne and Cooke 2004; Shin et al. 2014; Sakr 2019a) can be used to assess pathogenicity components of FHB species: F. culmorum, F. verticillioides, F. solani and F. equiseti on several bread and durum wheat plants.

Table 1 Mean aggressiveness components determined using in vitro detached leaf and seedling assays among 16 fungal isolates of four Fusarium head blight species measured on six wheat cultivars
Full size table

The four tested species were found to provide distinct and observable symptoms on detached wheat leaves (Fig. 1). All FHB isolates caused obvious lesions on detached leaves of six wheat cultivars by the sixth day after inoculation. Lesions, almost oval in shape, appeared as dark-brownish water-soaked patches and characterized later by more chlorosis and less necrosis on lesions developed on wheat leaves (Fig. 1). This observation is of great importance in FHB research since the Microdochium genus has been used to study quantitative components compared with other FHB complex species, it provides more obvious and observable symptoms on leaf segments (Browne and Cooke 2004). The type of chlorosis formation in our investigation was comparable to its observed type in the detached leaf assay conducted on the methodology described by Browne and Cooke (2005); lesions being accompanied by chlorosis of leaf tissues and later by less necrosis on lesions developed on wheat leaves. This may imply that delay of death of cells and living tissue occurs in wheat leaves irrespective of the 16 FHB isolates involved, as necrosis occurs as a result of cell death (Imathiu et al. 2009). Also, wheat seedlings inoculated with FHB conidia showed visible fungal head blight symptoms within a few days after inoculation (Fig. 1). Brown lesions started from the infected portions in sections in 3-day-old seedling coleoptiles and stems. Seven-day-old visually necrotic area exhibited varying degrees of necrosis as a consequence of the pathogen severity. Thus, the symptoms observed on wheat seedling in the present research were similar to its noted pattern caused by F. graminearum (Shin et al. 2014).

The two in vitro assays: detached leaf and seedling provide favorable conditions for FHB species to actively grow, thus ensure that their aggressiveness is stable and/or correctly distinguished or assessed. All the fungal isolates treated with the two in vitro assays fulfilled the requirement to induce FHB disease, thus they are pathogenic. The four FHB species used in this experiment are known for mycotoxin production (Xu and Nicholson 2009). Thus, the capability of 16 fungal isolates to cause chlorosis and necrosis in varying amounts on wheat leaves and seedlings (Fig. 1) might be mainly the result of phytotoxic action of these metabolites. Furthermore, it is important to measure directly DON or other toxins characteristics to the given fungal species in the research relevance to pathogeneicity and disease development.

The significant reductions observed with incubation period (IP) and LP, long lesion length (LL) and high percentage infection are indications of aggressiveness (Lannou 2012). The five aggressiveness components: IP (P = 0.652) and LL (P = 0.936) of detached leaf assay, percentage of infected seedlings (PIS) (P = 0.941 and P = 0.927, respectively) (of foliar-spraying and pin-point inoculations) and lesion length (P = 0.150) of clip-dipping inoculation did not distinguish isolates within and among species (Table 1). Mean IP and LL ranged from 2.0 to 2.3 days and 7.2 to 8.1 mm, respectively. The PIS (of spraying inoculation) varied from 41.5 to 45.7%. The PIS (of pin-point inoculation) varied from 39.8 to 43.8%. The LL (of clip-dipping inoculation) varied from 1.9 to 2.2 cm. Our data are not in accordance with those found by Imathiu et al. (2009), Opoku et al. (2011) and Khaledi et al. (2017), they observed highly significant differences in the aggressiveness of F. graminearum and F. langsethiae as measured by LL of detached-leaf assay.

In our study, LP varied from one fungal isolate to another irrespective of origin in wheat cultivars (P = 0.001). F35 (F. solani), the least aggressive isolate, showed maximum LP (6.4 days), however, F29 (F. solani) (4.3 days) was significantly more aggressive (Table 1). Until now, LP applies more easily to pathogens with no specific biological features, i.e., fungi like Fusarium species and also for pathogens with specific biological features (Lannou 2012). Recently, inter and intraspecific differences in aggressiveness of the four tested FHB species were observed towards barley plants as measured by LP (Sakr 2019c). Regarding in vitro standardized area under disease progress curve (AUDPC) component determined using a Petri-dish assay, variability in aggressiveness of the same isolates was also detected on the same wheat cultivars (Sakr 2018a, c, d, 2020). Mutation, genetic recombination or selection may play a basic role in pathogenesis (Imathiu et al. 2009). The five mentioned above aggressiveness components showed similar results for the various FHB isolates; while the individual aggressiveness component, LP, differentially influence aspects of FHB disease development in wheat plants at early stages.

The mean disease incidence (% symptomatic spike) (DI) values generated under field conditions of FHB fungi varied from 35.0% for the least aggressive isolate F27 (F. verticillioides) to 57.0% for the most aggressive isolate F20 (F. solani) on wheat cultivars as compared with 0% for the non-inoculated controls (P = 0.001) (Table 2). Inoculation of heads carried out to determine Type I FHB resistance showed substantial differences in the resistance of wheat cultivars. The mean portion of plants exhibiting FHB symptoms ranged from 38.7 to 53.4% (P = 0.001) (Table 2). Cham4 and Bohoth10 showed the lowest infection levels, with DI values of 38.7 and 40.5% respectively, whereas Acsad65 was the most affected cultivar, with DI value of 53.4%. Based on Type I resistance, Bohoth10 and Cham4 were moderately resistant cultivars, Douma4 was moderately susceptible, the two durums, Cham7 and Cham9, were susceptible to moderately susceptible, and Acsad65 was susceptible.

Table 2 Disease incidence (% symptomatic spike) (DI) determined using a head artificial inoculation under field conditions for six wheat cultivars infected with 16 fungal isolates of four Fusarium head blight species
Full size table

Although large differences among isolates of individual species for aggressiveness were detected, analysis of our results showed that the four FHB species did not vary in their comparative aggressiveness measured by LP on bread and durum wheat plants (Fig. 2) because of the relative homogeneity in pathogenic level among the 16 fungal isolates. Fernandez and Chen (2005) noted an obvious lack of a difference in aggressiveness between F. graminearum and F. culmorum on wheat. Similarly, Sakr (2018a, c, d, 2020) did not cluster the same fungal species on wheat cultivars using AUDPC. Our results did not support previous reports showing that FHB species varied in their aggressiveness (Bottalico and Perrone 2002; Xue et al. 2004; Saharan and Sharma 2009). The differences in these data may be due to the contrasting fungal isolates and host wheat cultivars used in this study and previous work. Origin of the 16 FHB cultures may contribute to this relative pathogenic similarity (Sakr 2018a).

Fig. 2
figure 2

Mean latent period (days) of four Fusarium head blight species on six bread and durum wheat cultivars detected in an in vitro detached leaf assay. Bars represent the standard errors of means

Full size image

Variation in aggressiveness in FHB populations can lead to break down of host resistance (Xu and Nicholson 2009). Correlation values of LP component among the six wheat cultivars showed that all possible comparisons were not significantly correlated, suggesting that aggressiveness mechanisms and genes probably different to head blight disease caused by individual FHB species. So, results shown in this research indicated that a complex genotype interaction exists among bread and durum cultivars and pathogens for LP component. Our results agree with pervious in vitro AUDPC data showing the presence of a cultivar-specific pathogenicity in the same fungal isolates infected wheat cultivars, i.e., Acsad65, Cham7, Cham4, Douma4 (Sakr (2018a, c, d, 2020)). This type of specific interaction has previously been reported by Foroud et al. (2012), who noted that F. graminearum pathogenicity is host-dependent in wheat.

Parry et al. (1995) showed no strong evidence for specific aggressiveness interactions among fungal species implicated in the FHB complex and wheat plants. It seems that a minor gene–for–minor gene interaction may exist between six wheat cultivars and 16 fungal isolates, suggesting that the isolate-specific effectiveness may lead to erosion of wheat quantitative resistance to FHB invasion. However, further investigation is required in order to draw any final conclusions. The six aggressiveness components involved in detached leaf and seedling assays were found to be not correlated, indicating that these components are genetically different, and also reflecting into complex polygenic nature of aggressiveness in the interaction in the FHP-wheat system, which are not fully understood (Castiblanco et al. 2018).

Correlations were obtained between the data of LP, AUDPC, disease incidence and disease severity previously generated in vitro and under controlled and field conditions for the six tested wheat cultivars (Table 3). The repeatability and the stability of LP test over several experimental conditions were proved using different bread and durum cultivars by yielding correlations with the data from in vitro, head and floret inoculations. Current durum wheat cultivars are highly susceptible to FHB infection than bread cultivars (Mesterhazy et al. 1999), thus in our study we chose six wheat cultivars with different resistance levels under controlled and field conditions (Sakr 2019b, Table 2) (two moderately resistant, one moderately susceptible, two susceptible to moderately susceptible and one susceptible) that would be sufficient to investigate the stability and repeatability of LP test in vitro and under controlled and field conditions. In accordance with our data, a weak and negative correlation (r = − 0.47) of wheat seed germination value caused by M. majus and FHB rating obtained by head inoculation of F. graminearum in the field was observed by Browne (2007). In parallel, Purahong et al. (2012) reported positive relationships of AUDPC estimations and FHB evaluations obtained by spray inoculation of F. graminearum across four durum wheat cultivars in the growth chamber and field. They found highly correlations between these two parameters (Purahong et al., 2012), which is similar to our results.

Table 3 Correlation coefficients between aggressiveness components generated under several experimental conditions on six wheat cultivars infected with 16 fungal isolates of four Fusarium head blight species determined by Pearson correlation coefficient
Full size table

Thus, our findings showed that the LP test is repeatable and stable with a large diversity depending on the six used wheat cultivars. The situation in the detached leaf assay was similar to artificial head and floret inoculations because FHB species need to overcome the morphology of the head and spikelet and they could directly penetrate and infect leaves. Thus, disease development is manifested through appearance of symptoms such as discoloured, malformed, necrotic or chlorotic areas on the affected plant part (Browne and Cooke 2004). Therefore, the in vitro component, LP, predicts aggressiveness occurring at the earliest and latest wheat development stages during FHB infection. In parallel, Sakr (2019c) revealed the potential use of in vitro indicator, LP, in predicting FHB data generated under controlled and field conditions on barley. The biological clarification for an association between the in vitro and in planta responses to FHB infection remains largely speculative, but it can be hypothesized that similar genetic pathways become stimulated at both developmental stages (Sakr 2019a, c).

Screening for disease reaction of different FHB species on several wheat cultivars in the growth chamber and field has been found to be time-consuming and is influenced by climatic conditions under field conditions making interpretation of data difficult. LP assay can be applied for screening the most aggressive isolates of different FHB species for FHB resistance breeding of wheat as LP has proved to be very useful tool in identifying quantitative resistance in wheat to FHB infection (Browne and Cooke 2004; Sakr 2019a). LP test has a high potential to simplify the advance of research into the wheat-FHB pathosystem, especially in Europe where genetically modified FHB species are not allowed to be sprayed in the field. On the other hand, LP test required only 21 days from seed sowing to obtaining results and the data correlates significantly with inoculations conducted under several experimental conditions.