Introduction

Globally, there are more studies and results on mycobiota of wheat kernels than on rachides. This is not in accordance with the importance of a rachis pathogen from the aspect of both resistance of small grains to Fusarium species (Jansen et al. 2005) and the biosynthesis of some fusariotoxins (Voigt et al. 2007). According to Mesterházy (2002), seven components (as stated by the author) or types (the term previously used) of resistance can be identified according to: pathogen invasion, pathogen distribution, grain infection, plant tolerance, toxin (deoxynivalenol) accumulation, late infection and death of a spike segment above the infection point. The component of the first type of resistance (I) is the infection distribution from the spikelet into the rachis, while the component of the second type of resistance (II) is the infection distribution from the rachis into other spikelets (Bai and Shaner 1996).

Genera of Fusarium and Alternaria are the dominant constituents of mycobiota isolated from wheat kernels worldwide (Trigo-Stockli et al. 1995; Krysinska-Traczyk et al. 2001; Bakutis et al. 2006; González et al. 2008). The other fungal species belonging to genera Absidia, Aspergillus, Bipolaris/Drechslera, Cladosporium, Cephalosporium, Chaetomium, Epicoccum, Oidiodendron, Penicillium, Rhizoctonia, Rhizopus, Stemphyllium and Trichothecium have not been always determined, and even if they have occurred they used to have a different status, because sometimes they have been dominant and sometimes they have occurred occasionally (Trigo-Stockli et al. 1995; Krysinska-Traczyk et al. 2001; Bakutis et al. 2006; Fakhrunnisa et al. 2006).

Many species of the genus Fusarium have been isolated worldwide from wheat kernels (Muthomi et al. 2008; González et al. 2008; Oerkea et al. 2010; Spanic et al. 2010). F. graminearum (Trigo-Stockli et al. 1995) or the combination of this species with F. poae (Ioos et al. 2004; Xu et al. 2005; González et al. 2008; Oerkea et al. 2010; Spanic et al. 2010) have been most often identified. Ioos et al. (2004) have clearly identified the increasing importance of F. graminearum and F. poae in the FEB complex in Europe.

In Serbia, the most important pathogenic species associated with wheat, barley and maize is F. graminearum. Furthermore, F. poae also is the most important pathogenic species for wheat and barely, while species of the section Liseola (F. verticillioides, F. subglutinans and F. proliferatum) are more important for maize and sorghum (Lević et al. 2009). The precipitation in Serbia during the wheat flowering time in recent decades has been of a local character and has had a significant effect on a variation of the intensity of the spike infestation with fusarioses and Fusarium head blight (FHB). In such a way, the grain yield reduction varied from 3.5 to 38.3 %, depending on agroecological conditions of the cultivation and resistance of wheat varieties (Lević et al. 2008a).

The aim of this study was to examine mycobiota, especially species of the genus Fusarium, and to determine whether there was interdependence in their occurrence on rachides and kernels of wheat.

Materials and methods

Field sampling

Thirty one field sites in 19 growing locations where 14 bread wheat varieties were grown were chosen mostly in the northern part of Serbia. Mature heads were hand collected from four sites each of 0.25 m2 from each field. The samples were packed in paper bags and immediately sent to the laboratory. The heads were manually threshed. Samples contained, on the average, 630 rachides and 725.1 g kernels. The kernel moisture ranged from 11.1 to 13.2 %. The samples were stored at 5 ± 1°C until analysed. Field sampling was done in cooperation with the Extension Services across Serbia.

Analysis of field samples

The rachis and kernel samples were rinsed under tap water for an hour and then were surface-sterilised by incubating for 1 min in 1 % (v/v) sodium hypochlorite solution and rinsed three times with distilled water. They were then dried, and one rachis or eight kernels were transferred onto 2 % water agar (WA) and incubated under ambient conditions, 25 ± 2°C and daylight. Six to seven days after incubation, samples were examined under a low magnification stereomicroscope. In order to determine species such as Chaetomium spp., Sordaria spp. and Thielaviopsis spp., incubated samples were once again examined after 5–7 days.

The mycelia of Fusarium species growing out of the rachides or kernels were transferred to potato dextrose agar (PDA) and carnation leaf agar (CLA) under sterile conditions to obtain pure cultures for the morphological identification of species according to Burgess et al. (1994). Subcultures on PDA were incubated in the dark at 25°C, while those on CLA were incubated at 12 h light (combined fluorescent and NUV light)/dark regime. All mentioned media were prepared according to procedures described by Burgess et al. (1994). Forty rachides and 128 kernels of each sample were analysed in four replications.

Statistical analysis

The prevalence of fungi was expressed as the percentage of infected samples (isolation frequency) and the infection levels as the percentage of infected kernels (isolation incidence).

The isolation frequency (F) and the incidence (I) of fungus were estimated as follows (Ghiasian et al. 2004): F (%) = [Number of samples in which a species occurred/Total number of samples] x 100; and I (%) = [Number of rachides/kernels in which a species occurred/Total number of rachides or kernels] x 100. The distribution of the infection levels in five frequency classes was performed according to Ioos et al. (2004).

The interrelation of the fungal species on rachides and kernels were determined by the Pearson Product–moment Correlation. The median value of the incidence of F. graminearum and Alternaria spp. in rachides and kernels of different wheat varieties in various environments in Serbia was calculated using Microsoft Office Excel 2007.

Results

Fungal enumeration

Out of 31 surface-disinfected wheat samples, 30 and nine fungal genera were isolated and identified from rachides and from kernels, respectively (Table 1). Phoma spp. was the only species that was not isolated from the rachides, but was isolated from kernels.

Table 1 Frequency and incidence of fungi on wheat rachides and kernels
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Species of the genera Fusarium, Alternaria, Chaetomium and Bipolaris were determined in 100 %, 96.8 %, 90.3 % and 61.3 % rachis samples, respectively, with the incidence of 39.0 (±21.7), 87.0 (±8.8), 18.1 (±18.3) and 6.7 (±6.8) percentages, respectively (Table 1). The most often identified species of the genus Bipolaris was B. sorokiniana, and much less B. tetramera and others. The distribution of the infection levels of other fungal species was within the frequency range of >5 to ≤10 (Epicoccum spp., Aspergillus spp., Paecylomices spp., Sordaria spp., Sporotrix spp., Thielaviopsis spp., Microdochium spp., Mucor spp. and Papulospora spp.), and of >10 to ≤25 (Acremoniella atra, Gonatobotrys spp., Nigrospora oryzae, Rhizopus spp., Trichoderama spp., Cladosporium spp., Cephalosporium spp. and Penicillium spp.).

Out of fungi identified on wheat kernels, species of the genera Fusarium and Alternaria were the most frequent (100 %) with the incidence of 22.0 (±18.2) and 48.5 (±21.5) percentages, respectively (Table 1). One species was in the class within a frequency range of >5 % to ≤10 % (Phoma spp.), five species within the range of >10 % to ≤25 % (Cephalosporium spp., Bipolaris spp., Cladosporium spp., Epicoccum spp. and Nigrospora oryzae), while the frequency of Chaetomium spp. was over 25 %.

Twelve and 11 species of the genus Fusarium were identified on rachides and kernels, respectively (Table 2). On the average, Fusarium species were more frequent on rachides (mean 24.0 %) than on kernels (mean 15.2 %). F. chlamydosporum, F. oxysporum and F. solani were detected only on rachides, while F. arthrosporioides and F. avenaceum were determined only on kernels. The remaining Fusarium species were recorded on both, rachides and kernels.

Table 2 Frequency and incidence of the Fusarium species on rachides and kernels of wheat
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F. graminearum was detected in 30 out of 31 samples (in 96.8 % cases) on both, rachides and kernels. The incidence of this species on rachides and kernels were almost the same (24.0 % vs. 22.0 %).

F. poae was the second most prevalent Fusarium species on both, rachides (93.5 %) and kernels (51.6 %). However, the difference in the incidence of this species on rachides and kernels was significant: 11.6 (±9.1) and 1.8 (± 1.4) percentages, respectively (Table 2). Furthermore, F. sporotrichiodes was more frequent on rachides (16.1 %) than on kernels (6.5 %).

A very high frequency of species of the section Liseola was determined on rachides. This was particularly attributed to F. verticillioides (64.5 %). F. proliferatum and F. subglutinans were detected on rachides in 12.9 % and 9.8 % of samples, respectively (Table 2). The most often isolated species of this section from grain was F. verticillioides (19.4 %), while F. proliferatum and F. subglutinans were detected in 9.7 % of samples. The incidence of these fungi on kernels amounted to 1.7 (±1.1), 3.1 (±4.0) and 1.4 (±0.7) percentages, respectively.

The distribution of the infection levels of Fusarium species on rachis samples was in the classes within a frequency range of >5 to ≤10 (F. chlamydosporum, F. equiseti, F. semitectum, F. solani, F. subglutinans and F. tricinctum), and >10 to ≤25 (F. oxysporum and F. sporotrichiodes), while on kernels they were distributed only in one frequency class (>5 to ≤10).

Fungal relationships

The positive correlation was determined for the frequency and incidence of fungi in both, rachides (r = 0.6191**) and kernels (r = 0.8524**). Furthermore, there was also a positive correlation (r = 0.8074**) between the incidences of fungi on rachides and kernels, as well as, between the incidences of fungi in all samples (r = 0.9584**).

The isolation incidence of F. graminearum and Alternaria spp. in rachides and kernels of different wheat varieties in diverse environments in Serbia is presented in Table 3. The positive correlation was established between frequencies of F. graminearum on rachides and kernels (r = 0.5356**), while this correlation, although positive, was not statistically significant for Alternaria spp. (r = 0.1729). The correlation between frequencies of F. graminearum and Alternaria spp. was negative on both, rachides (r = −0.3783*) and kernels (r = −0.4863**).

Table 3 Incidence of isolated F. graminearum and Alternaria spp. in rachides and kernels of different wheat varieties in diverse environments in Serbia
Full size table

Discussion

Generally, greater abundance of different fungal species was determined in rachis samples than in kernel samples of the 14 wheat cultivars grown under diverse agroecological conditions (19 locations). However, differences between rachis and kernel mycobiota related to the frequency and incidence of certain species of the genus Fusarium are much smaller.

The positive correlation established between infections of rachides and kernels caused by F. graminearum, has a scientific explanation. During the plant infection, F. graminearum spreads by systemic growth through the rachis from one spike to another (Ribichich et al. 2000; Wanjiru et al. 2002). Histopathological studies showed that the colonisation of wheat heads by F. graminearum occurred in two ways, either by a horizontal or by a vertical path (Ribichich et al. 2000). In the horizontal path, the fungus invaded anthers and bracts of contiguous florets in the first spikelet invaded, then moved through the rachis and the rachilla to the contiguous spikelet. In the vertical path, the fungus moved through vascular bundles and parenchyma to invade spikelets above and below the originally infected spikelet. At later stages of infection, the pathogen switched from predominately vertical to lateral growth and accumulated below the surface of the rachis (Brown et al. 2010). Delayed hyphal colonisation of the vascular bundles in the rachis was observed in the type II resistant genotypes (Foroud and Eudes 2009). Transmission electron microscope studies showed that at 5 days after inoculation, the hyphae had reached the rachis from the infected ovary, lemma and palea (Wanjiru et al. 2002). The fungus extended inter- and intracellularly in the cortical tissue and vascular bundles of the rachis.

Jansen et al. (2005) found that hyphae of F. graminearum reaching the rachis proceeded to apically located developing kernels. In the absence of trichothecenes, the fungus is blocked by the development of heavy cell wall thickenings in the rachis node of wheat, a defence inhibited by the mycotoxin. In barley, hyphae of both wild-type and trichothecene knockout mutant are inhibited at the rachis node and rachilla, limiting infection of adjacent florets through the phloem and along the surface of the rachis.

According to the literature more studies have been conducted on kernel mycobiota than on rachis mycobiota. Brandfass and Karlovsky (2006) have stated that the only Fusarium species found apart from F. graminearum and F. culmorum was F. poae, occurring just in three instances in rachides, which also contained F. graminearum or F. culmorum, and in four instances in rachides where none of the other two Fusarium spp. were detected. Other fungal species occasionally recovered from the rachides were Alternaria spp., Rhizoctonia cerealis and Epicoccum spp. In comparison with these results, our results show that a significantly higher number of fungi (22 fungal genera) were determined on wheat rachides and that the genera Fusarium, Alternaria, Chaetomium and Bipolaris were dominant. Out of 11 species of the genus Fusarium, F. graminearum (96.8 %) and F. poae (93.5 %) had the highest incidence, while F. culmorum was not detected.

It should be mentioned that the frequency of the Fusarium species of the section Liseola (F. verticillioides, F. proliferatum and F. subglutinans) was relatively high on rachides and kernels of wheat. Recent literature data has been indicating the importance of these species in the aetiology of diseases of wheat kernels and contamination by mycotoxins. Desjardins et al. (2006) characterised nine F. proliferatum strains from wheat from Nepal for ability to cause wheat kernel black point under greenhouse conditions, and for fumonisin contamination of infected kernels. According to Stanković et al. (2012) a large number of stored wheat kernel samples (53.1–64.0 %) contained FB1 at levels higher than 1,000 mg kg−1.

Our studies confirmed that in Serbia, as well as, in Europe (Ioos et al. 2004), F. graminearum and F. poae were the most frequent species in the FEB complex. Our previous studies show that under agroecological conditions of Serbia, F. graminearum mainly developed along the rachides (Lević et al. 2008a). These points out that cultivated varieties of wheat are not the type II resistant or resistant to spreading of pathogens on spikes. On the other hand, F. poae was the most often developed on the rachis tips, and although it was very frequent it did not express pathogenic properties against wheat seedlings (Lević et al. 2008b).

Unlike F. graminearum and F. poae, other species of the genus Fusarium did not spread beyond kernels (F. arthrosporioides and F. avenaceum) or rachides (F. chlamydosporum, F. oxysporum and F. solani). These results point out to smaller effects of these Fusarium species on the development of FHB.

Different incidences of F. graminearum on wheat kernels were established in dependence on the location in Serbia in which the varieties were grown. For example, the incidence of this species in kernels of the variety cv-10 varied from 0 % to 71.9 % over seven locations. These differences can be explained by different impacts of agrometeorological conditions on the development of spike infection, as well as, by the application of fungicides. We have previously determined that precipitation during the stage of wheat flowering varied over locations and significantly affected the variation of the incidence of Fusarium head blight over the environments in Serbia (Lević et al. 2008a). For the particular years, the climatic conditions reported by Ioos et al. (2004) were quite favourable for FHB development with high levels of moisture at the cereal flowering stage.

Our results are in accordance with results obtained by other researchers who have studied FHB and determined a great variation in the species composition and in dominant species in the fungal complex that caused FHB. This variation related not only to their year-to-year variation (Ioos et al. 2004) or variations in previous decade (Tomczak et al. 2002), but also to region-to-region variation (Xu et al. 2005) or zone-to-zone variation (Muthomi et al. 2008), and also to their field-to-field variation or even within-field variation (Birzele et al. 2002; Oerkea et al. 2010). Results obtained in eastern Croatia, located eastwards from the northern part of Serbia, differed in frequencies of certain Fusarium species in comparison with our results, although the spatial distance between the two countries is small.

The results of Kosaik et al. (2004) indicated a negative interaction between F. graminearum and Alternaria spp. in Norwegian grains, as well as, between F. graminearum and other Fusarium spp. Our results, related to a negative interaction between F. graminearum and Alternaria spp. (r = −0.4863**), are in accordance with results gained by these authors. Moreover, we have also determined a negative interaction between these two species in rachides (r = −0.3783*).

In conclusion, the majority of rachis samples contained more notable quantities of fungal species than kernel samples of wheat collected from 19 locations in Serbia characterised with different agroecological conditions. But, the number of Fusarium species was approximately the same on rachides and kernels. The high frequency and incidence of F. graminearum, F. poae and Alternaria species in wheat is a matter of concern for wheat production.