Fusarium Head Blight and Deoxynivalenol Accumulation of Wheat in Marmara Region and Reactions of Wheat Cultivars and Lines to F. graminearum and F. culmorum

MATERIALS AND METHODS

Samples collection: During June of 2000 and 2001 spikes of winter and spring wheat with symptoms of scab were collected from farm fields and Agricultural Research Institutes experimental plots in Sakarya. Collections were at 34 sites in four provinces in June of 2000 at 20 sites in one province in June of 2001 at maturity.

Kernels from all collected wheat spikes were surface sterilized with 1% sodium hypochlorite for 3 mi rinsed sterile distilled water. Then seed were cultured on acidified half-strength potato dextrose agar (AHSPDA) (Salas et al., 1999) with Streptomycin sulfate (100 mg L^-1) and Ampicilin (100 mg L^-1). Fungal isolates were incubated at 24±1°C for 7 days. Fusarium isolates were transferred Synthetic Nutrient Agar (SNA).Culture plates were incubated at 24±1°C with 12 h photoperiod provided by cool white fluorescent and black lights tubes. After 7 to 10 days Fusarium species were identified based on descriptions given by (Booth, 1977; Burgess et al., 1994; Gerlach and Nirenberg, 1982; Nelson et al., 1983; Toussoun and Nelson, 1995). A small scrape of macro conidia from a sporodochium was preferred to obtain 1-10 conidia in a drop under a stereomicroscope. Water Agar (WA) plates were seeded by pouring the conidial suspension over the surface. The plates were incubated described by Burgess et al. (1994). Plates were examined using a stereo microscope. Fusarium species and their isolates were transferred to fresh potato dextrose agar (1/2PDA) (10 g of dextrose, 100 g of potato and 20 g agar per liter of water) test tubes. Slants were incubated for 5 to 7 days and stored in a refrigerator at 4°C until needed. Confirmation of some species was made by L.W. Burgess, Department of Crop sciences, Fusarium Research Laboratory Sydney, Australia.

Deoxynivalenol analysis: Samples from collection sites and from 59 wheat cultivars and lines artificially inoculated with F. graminearum and F. culmorum were assayed for DON concentration by HPLC. The samples finely ground and mixed well. A one-gram sample was taken from the ground material. To prepare extracts, 5 mL of methanol/water were added to 1 g samples in glass tubes, which were then subjected to end over end mixing for 1 h then centrifuged for 5 min at 2000rpm. The supernatant solution passed through a filter. The filtrate was used for clean-up procedures. A 2 mL extract washed with Ethyl acetate. The solution was evaporated to dryness vacuum and the residues were treated with dichloromethane. A prepared Pasteur pipette was pre-washed with toluene/acetone then washed with dichloromethane. The sample solution was added to a test tube and toluene/acetone mixture. This solution was discharged after that dichloromethane/methanol was treated and collected into tube and mixed. This solution was evaporated to dryness and used for analysis in HPLC. The residue dissolved into 0.5 mL methanol: water transferred to vials of HPLC (Sinha and Savard, 1996).

Artificial inoculation tests for reactions of cultivars and lines: Experiments with commercial cultivars and breeding lines of winter and spring wheat were conducted in the summer of 2002 and 2004. Thirty nine lines and commercial cultivars were used for experiments in 2002. Experiments were also conducted with twenty CIMMYT’s breeding lines and Chinese cultivars in 2004. F. graminearum and F. culmorum single spore isolates used to study head blight resistance of winter wheat commercial cultivars and breeding lines. Testing was done at the mid-flowering stage. Thirty Individual flower heads were inoculated with F. graminearum and F. culmorum at mid-anthesis (Zadoks scale 65) growth stage. Approximately 50 μL of spore suspension was injected in to the left and right floret of the central spikelet on both sides of each head with a hypodermic needle of syringe (Rudd, 1996). A spore suspension containing 5x104 F. graminearum and 105 F. culmorum macroconidia per milliliter of H₂O was used for each inoculation. One drop of Tween 20 was used per liter of macro conidial suspension. Plastic bags were misted with top water and paced over inoculated heads for 2 days. This period provided 90 to 100% relative humidity for infection and prevented movement of isolates to other plants. The percentage of blighted spikelets was recorded after 20 days.

Disease assessment: Thirty heads were collected at harvest from inoculated and noninoculated (control) plots. The heads were visually assessed for FHB infection, visually assessment of FHB was based on soft, lightweight, shriveled or normal-sized kernels having pale white or carmine red discoloration. The mean percentage of infected florets per infected head and the percentage of infected heads per plot were calculated for parameters of disease severity. Severity of FHB = blighted spikelets% X infected heads%/100 (Snijders and Perkowski, 1990). Number of kernels and kernel weight (1000 kernel weight) was measured for 30 heads according to the methods described by (Chelkowski, 1994).

Statistical analysis: The original data for FHB severity, DON concentration and TKW (Thousand inoculated Kernel Weight), TCKW (Thousand Control Kernel Weight), using analysis of variance, correlation and regression analysis. Means of cultivars and lines were compared with the Least Significant Difference test (LSD) at the 0.01 level.

RESULTS

Fifteen Fusarium species were isolated from spikes of winter and spring wheat with symptoms of scab during 2000 and 2001 from Marmara region (Northwest part of Anatolia). During two years of the study 256 isolates of Fusarium species were identified (Table 1). Fusarium species constituted 15.9% of all fungi isolates. Fusarium species were tested for DON concentrations in natural infection conditions (Table 1). The highest DON concentrations were found in F. graminearum infected wheat spikes. The overall mean percentage in Fusarium species were 20.7% F. graminearum, 10.1% F. culmorum, 2.7% F. poae, 2.3% F. sporotrichoides and 1.6% F. avenaceum in 2001.

Table 1:	Percentage of Fusarium isolates in different species from scabby wheat and DON concentration in nine counties of Marmara Region in 2000 and 2001

Table 2:	Means of FHB% severity, thousand kernel weight (TKW) and thousand control kernel weight (THKW) and deoxynivalenol (DON) concentration (ppm g-1) of 39 Turkish and east European and 20 CIMMYT cultivars and lines in artificial inoculation tests with F. graminearum and F.culmorum at Sakarya
**: Significant at 0.01 level

Table 3:	Correlation coefficient between FHB%; thousand kernel weight, thousand control kernel weight and DON content in 39 Turkish and east European and 20 CIMMYT cultivars and lines after inoculation with F. graminearum and F. culmorum in 2002 and 2004
**: Significant at 0.01 level

Other Fusarium species isolated from wheat kernels in survey included F. oxysporum, F. chlamydosporum, F. heterosporum, F. equiseti, F. compactum, F. concolor, F. crookwellense and F. verticilloides. The highest mean percentage of kernels infected with F. oxysporum, 26.1%.

The FHB%, TKW, THKW and DON concentrations for 59 wheat cultivars and lines from F. graminearum and F. culmorum inoculated plots in 2002 and 2004 are shown in Table 2. FHB severity ranged from 21.33 to 96.20%. Significant differences were found between FHB reactions of wheat cultivars and lines (significant at = 0.01). Catbird (2279), Pamukova 97, Pehlivan, Marmara 86, Martar, BVD-7, SHA3/CBRD(38) and SABUF/3/BCN/CETA.AE.SQUAROSSA (895).(253) were found have a low resistance to the disease, where as, Ceyhan 99, Momtchil, Seri-82, Basribey, QT 4118(0), Saraybosna and MAYOOR/TKSN108/AE: SQUARROSA (222) were appeared as susceptible genotypes (Table 2). In inoculated kernels, the highest accumulation of DON was observed in Turkish lines and cultivars except BVD-7 (0.20%), Ceyhan 99 (0.26 ppm g^-1) and Acar1.The highest DON accumulated cultivar contained 22.34 ppm g^-1 DON was Turkish cultivar Adana 99. The east Eurepean cultivar Martar had the lowest accumulation of DON (0.10 ppm g^-1). CIMMYT lines DOY1/AESquarrossa (0), SABUF/3/BCN/CETA.AE. SQUAROSSA (895)-(253) and QT.4118 (0) also had low level of DON accumulation. Mean accumulation of DON in all CIMMYT lines and Chinese cultivars were lower than Turkish cultivars. The correlation between FHB and DON concentration (ppm g^-1) was significant in both years. Thousand kernel weight in all inoculated cultivars and lines was negatively correlated with Fusarium head blight percentage at p = 0.01 level (Table 3). The greatest reduction in TKW of cultivars and lines was observed in cultivars of Timgalen, Yüreğir 89 and Kaþif Bey. The lower reduction in TKW of cultivars and lines was observed in cultivars and line of Basribey, Saraybosna, Cumhuriyet and BVD-7.

DISCUSSION

F. graminearum was the primary pathogen responsible for FHB epidemics in Marmara region that has been generally identified as the principal pathogen by authors (Clear and Patrick, 2000; Fernando et al., 2000; Miedenar et al., 2003; Choo et al., 2004; Mc Callum et al., 2004). Marmara region has high humidity and warmer climatic conditions. F. culmorum has been proved most aggressive to wheat plants and was a secondary pathogen in Marmara Region. Survey studies were conducted in 1994/95 and 2000 to 2004 find more than 10 species of Fusarium isolated from crown and sub-crown tissue with F. culmorum being the most commonly isolated Fusarium in Central Anatolia Plateau (dryland environment) of Turkey (Aktas et al., 1999). Parry et al. (1995) suggested that the geographical distribution of species is related to temperature requirements. F. graminearum is the most important species in hotter regions with F. culmorum predominating in the cooler regions where F. poae and Microdochium nivale (=Syn. F. nivale) also assume a greater importance. F. avenaceum is isolated over a range of climatic zones.

F. oxysporum, F. chlamydosporum, F. solani, F. heterosporum, F. equiseti and F. poae, occurred at least in 4 counties of 9 wheat growing counties in our study. No DON detected in Fusarium species other than F. graminearum and F.culmorum. But, Salas et al. (1999) reported that, DON was detected in barley spikes with at least one isolates of F. poae, F. avenaceum and F. sporotrichoides.

Wilcoxon et al. (1988); Stack and McMullen (1985) reported that inoculated wheat spikelets with bore many of the species tested in their study and also found that only F. graminearum and F.culmorum spread from the inoculated spikelets. Each of their other Fusarium species caused necrosis only in the inoculated spikelets.

The survey studies was showed that disease incidence in fields were fewer than 2% in Marmara Region in 2001. We think that ecological conditions, conventional tillage and inoculum’s density may have prevented severe disease ratio in the Region. FHB is currently not a severe problem in Turkey and the disease only locates in northwest part of Anatolia. The farmers produce maize intensively in this region and this might be the reason for the disease occurrence crop rotation between maize and wheat for many years. Teich and Nelson (1984) reported that, the average incidence of Fusarium head blight on wheat following maize was six to seven times greater than on wheat following other cereals and in crop rotation. It may be a major reason of FHB incidence in this region of Turkey. However, after warm winter period, infection was occurred at high humidity and warm winter temperatures in spring time in 1997 and 1998 in Marmara Region. After these two years, the disease ratio is low as usual.

The correlation between FHB and DON concentration (ppm) were significant for both years (2002, 2004). DON levels were from 0.1 to 22.34 ppm after two years experiments (Table 2). Rabenstein et al. (2004) found that a significant correlation FHB disease severity and DON content in artificially infected cereals in Böhnshausen (Germany). In contrast Wisniewska et al. (2004) found no correlation between DON content and other parameters. (Arsenuik et al., 1999) found that, susceptible genotypes often accumulated more DON in kernel, but this is not general rule and on an intra specific basis, the associations were not very strong. It was reported that, the ability of some genotypes have the ability to degrade mycotoxins in plant tissue (Miller and Arnison, 1986). Plants that resist mycotoxin accumulation in their kernels by blocking DON translocation from the chaff may also explain the lack of a strong association between FHB and DON level (Snijders and Kreching, 1992). The mechanisms of plant resistance to scab are very complex (Chelkkowski et al., 1987; Foremska et al., 1994; Snijders, 1990). The lack of correlation between FHB/DON and %FDKL/DON in Polish cultivars and spring accession of CIMMYT suggests that there exist at least three different resistant composition to pathogen spread, to kernel colonization and to toxin (DON) accumulation (Wisniewska et al., 2004). Snijders and Perkowski (1990) also found a lack of correlation between FHB/DON ratio and FHB%, DON and reduction of yield and they concluded that this was due to different mechanisms determining disease severity and mycotoxin accumulation.

In this study, Thousand Kernel Weight (TKW) in all inoculated cultivars and lines was negatively correlated with Fusarium head blight percentage (FHB%). Yield lost was calculated from 2.0 to 84.0% ranged at total fifty nine breeding lines and cultivars in 2002 and 2004 year’s experiment plots. DON content was not significantly correlated with TKW.

Wisniewska et al. (2004), found that, the percentage of Fusarium-damaged kernels (%FDK) was positively correlated with disease score (Fi) and negatively correlated with Kernel Weight per Head (KWH). DON content was not significantly correlated with any parameter.

FHB % severity ranged from 21.0 to 94.53. Significant differences were found between FHB reactions of wheat cultivars and lines (significant at ≤0.01). Mean levels of FHB% and DON in samples of eight wheat cultivars ranged from 21.0 to 49.2% and from 0.1 to 1.50 ppm, respectively, in contrast mean levels of FHB% and DON have another 13 wheat cultivars ranged from 69.0 to 96.2% and from 6.22 to 22.34 ppm, respectively (Table 2).

On the basis of the results obtained, we can conclude that inoculation with F. graminearum and F. culmorum reduced kernel weight of each cultivar. Cultivars from China which were known as resistant cultivars that had low kernel weight and high FHB% incidence but in contrast their DON accumulation from 2.22 to 1.08 in inoculated plots. It seems that mixtures isolates highly pathogenic isolates of F. culmorum and F. graminearum, cause more severe disease than individual isolates. It was conclusion that none of the wheat cultivars and lines was immune or highly resistant to FHB. However, some of these cultivars and lines may have useful levels of FHB resistance against F. graminearum and F. culmorum. They can use in breeding programs if they could have suitable agronomical parameters and well adapting to the areas. DON accumulation also is an important parameter should take into breeding programs with the other parameters those are FHB severity, isolation frequency of Fusarium species, TKW or yield losses and climatic conditions.

ACKNOWLEDGMENTS

We thank General Directorate of Agricultural Research for financial supports and Marc Savard and his staff for teaching DON analysis, from Agri-Food, Ottawa, Canada. Lester Burgess for identified some Fusarium species.