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CHEMOTYPING OF THE FUSARIUM GRAMINEARUM ISOLATES AND VARIATION IN AGGRESSIVENESS AGAINST WHEAT HEADS.

Byline: F. Mert-TA1/4rk R. Gencer F. and Kahriman

: Abstract

Fusarium head blight (FHB) caused mainly by Fusarium graminearum is a devastating disease of wheat and other small grain cereals. FHB lowers grain yield and quality and contaminates grain with mycotoxins predominantly trichotecenes i.e nivalenol (NIV) deoxynivalenol (DON). A survey conducted at three Provinces in Turkey for FHB and 17 isolates were identified as F. graminearum using morphological and molecular markers. A PCR assay was carried out to identify the chemotypes of the isolates. Using Tri13 gene cluster all 17 isolates that were identified to 15-AcDON type of DON chemotype. None of the isolates displayed 3-ADON or NIV chemotypes. In order to assess variation in aggressiveness among isolates all isolates were inoculated to a susceptible wheat spikes at field conditions and disease severity and a thousand kernel weight were measured.

Aggressiveness (measured as FHB severity or TKW) differed significantly among 17 F. graminearum isolates inoculated onto wheat spikes of FHB susceptible cultivar GAlnen (P=005). Means of FHB severity ranged from 39.75 to 86.33% averaging 63.29% in total. Reduction in TKW was also reduced significantly by different isolates. Differences in aggressiveness among isolates may due to genetic recombination mutation or selection in the surveyed area.

Key words: Fusarium graminearum mycotoxins chemotypes DON NIV pathogenicity aggressiveness.

INTRODUCTION

The fungal pathogen Fusarium graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch] is the most common causal agent of Fusarium head blight (FHB) in many parts of the world. This destructive disease so-called scab affects wheat barley and other small grains both in temperate and in semitropical areas and has the capacity to destroy crop within a few weeks of harvest (McMullen et al. 1997). Primary inoculum comes from infected plant debris on which the fungus overwinters as saprophytic mycelia. In spring warm moist weather conditions are favourable for the development and maturation of conidia and perithecia that produce ascospores concurrently with the flowering of cereal crops (Markell and Francl 2003). The principal mode of fungal spread in wheat from floret to floret inside a spikelet and from spikelet to spikelet is through the vascular bundles in the rachis and rachilla (Ribichich et al. 2000).

Infection of wheat heads by F. graminearum reduces grain yield by degrading starch granules in the kernels (Jackowiak et al. 2005). It also reduces the quality of the grain by contaminating it with harmful mycotoxins such as the trichothecenes rendering it unsafe for human and livestock consumption. Isolates of Fusarium graminearum can be categorised depending on the type B trichothecene they produce. Some isolates produce deoxynivalenol (DON chemotypes) while others produce nivalenol (NIV chemotypes). DON chemotypes can be further divided depending on where DON is acetylated with 3-acetyl DON (3-AcDON) producers and 15-acetyl DON (15-AcDON) (Miller et al. 1991; Jennings et al. 2004).

Several studies reported variation in aggressiveness among F. graminearum isolates sampled from various parts of the world within a country and even within populations from individual fields (Bai and Shaner 1996; Miedaner et al. 1996; Miedaner et al. 2010). Aggressiveness of F. graminearum is not geographically structured since isolates with low medium and high levels of aggressiveness make up the population in a single location. Most studies revealed a high level of genetic diversity in F. graminearum within individual field populations or populations sampled across a definite geographical scale. Knowledge of isolate characteristics in terms of aggressiveness in any location is necessary for predicting the pathogenic potential of FHB pathogens and for the deployment of resistance in a given location.

Lee at al. (2002) and Brown et al (2002) reported that Tri13 from the Fusarium trichothecene biosynthetic cluster is responsible from converting DON to NIV. Differences in the functional and unfunctional Tri13 genes can be ascertained by PCR assays. The objective of this research is to characterize the chemotypes of F. graminearum collected from the blighted ears in North-West of Turkey and to compare aggressiveness of the isolates at field conditions.

MATERIALS AND METHODS

Collection of the Fusarium graminearum isolates: Wheat crops were surveyed FHB from May until the beginning of June in the 2009-2010 wheat growing season. Three provinces Canakkale Tekirdag and Balikesir were surveyed covering 4500 ha area. The geographic origins of the isolates collected are given in Table 1.

Infected grain samples were surface sterilized with 1% sodium hypochlorite solution and rinsed three times with sterile distilled water. After drying in a fume hood to eliminate excess moisture they were placed in potato dextrose agar (PDA) medium containing 100 g/mL streptomycin sulfate and incubated at 250C for 5-7 days. Monoconidial isolates were obtained by streaking spore suspensions onto water agar plates and single conidia were transferred onto new plates.

Identification of the Fusarium graminearum isolates: The isolates were examined after 10 days and selected based on pigmentation on PDA. The selected isolates were grown on carnation leaf agar (CLA) and identified as F. graminearum by conidial morphology and colony characteristic as described by Leslie and Summerell 2006.

Fungal DNA Extraction and PCR: The DNA extraction technique was modified from Saitoh et al (2006) adding some further cleaning steps. A mycelial mass was picked with a pipette tip and put in a 2 ml Eppendorf tube. The mycelial mass was then homogenised with a micro pestle for a few seconds. One ml of lysis buffer (200 mM Tris- HCl 50 mM ethylenediaminetetra acetic acid 200 mM NaCl 1% n-lauroylsarcosine sodium salt pH 8.0) was added immediately to the mycelia. The mycelial mass was dispersed in the buffer by vortexing for 10 seconds. Two l of RNAse A (Fermentas Lithuania) and 4 l of proteinase K (Fermentas Lithuania) was added and incubated for about 15 min at 370C. The mixtures were centrifuged at 13000 rpm for 5 min. The supernatant was removed to a clean tube containing 1 ml of chloroform. The tubes were inverted briefly before centrifugation for a further 10 min.

The supernatant (about 800 l) was removed into a new tube containing 80 l sodium acetate (5 M) and 550 l ice cold isopropanol then inverted gently. The mixure was centrifuged at 13000 rpm for 10 min following incubation on ice for several minutes. The pellet was washed twice with 70% ice cold ethanol. After drying at room temperature for 30 min the DNA was dissolved in 100 l of TE buffer.

PCR amplification was carried out in a 50 l of reaction mix containing 25 l of 2X PCR master mix (0.05 units/ l TaqDNA polymerase in reaction buffer 0.4 mM of each dNTPs Fermentas Lithuania) 8 l (25-50 ng) fungal DNA 2.5 l (300 nM) each of primer and 12 l nuclease free water. A thermal cycler (Bio-RAD USA) was used for amplification of the specific fragment of DNA.

PCR conditions used for F. graminearum detection were: 95C for 5 min followed by 35 cycles of denaturation at 95C for 30 s annealing at 62C for 30 s and extension at 72C for 40 s followed by a final extension of 72C for 5 min. The same conditions were applied for Tri13DON and Tri13NIV with an exception that denaturation temperature was 94C. The PCR conditions for the generic primer Tri13P was: 94C for 4 min followed by 35 cycles of 94C for 1 min 60C for 40 s 72C for 40 s then a final extension of 72C for 6 min. PCR products were separated by electrophoresis on 2 % agarose gels stained with ethidium bromide and photographed under UV light in a Bio-Imaging system (Bio-RAD USA). All assays were repeated at least twice. The primers used to identify F. graminearum and its chemotypes are listed in Table 2.

Inoculation of Wheat Head and Disease Assessment: In order to assess variation in aggressiveness among the isolates a field trial was conducted. The experimental design was a completely randomized block design comprising four replications for each isolate. The size of each plot was 1 m X 5 m and the spacing between rows was 12.5 cm.

Pathogenicity testing was performed using GAlnen wheat variety one of the most common varieties in the Region. Twenty five-30 individual heads per plot were inoculated with the fungus at mid-anthesis stage. Approximately 10 l of spore suspension containing 5X104 macroconidia was injected in both sides of the florets at the middle of heads with a hypodermic needle of syringe. Plastic bags were covered over inoculated heads for 2 days for high level of humidity. The percentage of blighted kernels was recorded 14 days after inoculation. The mean percentage of infected kernels per infected head and the percentage of infected heads per plot were calculated for parameters of disease severity. Severity of FHB=blighted kernels% X infected heads% /100 (Snijders and Perkowski 1990). Relative a thousand kernel weights (TKW) were calculated by dividing a TKW from blighted plot to TKW from respective control plots (Miedaner et al. 2003).

RESULTS

Collection and Identification: A total of 17 isolates were described as F. graminearum using morphological identification techniques. All isolates were confirmed as F. graminearum by species-specific PCR analysis with Fg16 and Fg16N primers developed by Nicholson et al. (2004). All samples produced a common band of 280 bp with Fg16N and 450 bp with Fg16 (Figure 1). These data and the morphological examination confirmed that all studied isolates were belonged to F. graminearum clade (Table 2).

Determination of Chemotypes: Chemotyping of collected isolates was done by PCR assays using a set of specific primer of Tri13 gene sequence including: Tri13NIV Tri13DON and Tri13P (Table 1). The results obtained from PCR reaction with Tri13NIV showed that none of the isolates were NIV chemotypes; however all the isolates gave specific 282 bp fragment with Tri13DON assay indicating all population was DON- producer (Figure 2).

The generic PCR detection of 3-AcDON- and 15-AcDON- chemotypes based on Tri13P assay detected only two of acetylated DON chemotypes in a single reaction. All isolates produced a fragment of approximately 583 bp indicating 15-AcDON-chemotypes at the population. The results indicated that although NIV-chemotypes is absent in the surveyed area only 15- AcDON-chemotype is present among two acetylated DON-producers.

Differences in Aggressiveness: FHB was assessed at 14 days after inoculation. All F. graminearum isolates caused visible head blight symptoms in field experiments. No symptoms of disease occurred in the control inoculations. Seventeen isolates of F. graminearum tested showed broad genetic variation for aggressiveness as measured head blight ratings (%). Means of FHB severity ranged from 39.75 % to 86.33 % averaging 63.29 % (Table 3). The results show that although there are some isolates associated with the low level of disease symptoms 14 days after inoculation (T608) some have a high level of aggressiveness such as B114.

TKW was also assessed after harvest and values obtained from each isolate differed greatly (Table 3). Relative TKW from inoculated heads ranged from 0.22 to 0.83 proving high level of variation in yield. The data showed that infection by T608 influenced TKW the least among the isolates whereas C402 B018 and T118 reduced TKW dramatically.

Table 1. Code origin of isolates amplification through PCR using F. graminearum specific primers (Fg16 and Fg16N)

###and Tri13DON Tri13NIV and Tri13P assays for NIV and DON and acetylated forms of the isolates.

###Tri13P1

###Code###Province###Fg16###Fg16 N###Tri13NIV Tri13DON 3-AcDON 15-AcDON###NIV

###C-601###Canakkale###+###+###-###+###-###+###-

###C-402###Canakkale###+###+###-###+###-###+###-

###C-302a###Canakkale###+###+###-###+###-###+###-

###C-601a###Canakkale###+###+###-###+###-###+###-

###C-606b###Canakkale###+###+###-###+###-###+###-

###B-006###Balikes ir###+###+###-###+###-###+###-

###B-018###Balikes ir###+###+###-###+###-###+###-

###B-114###Balikes ir###+###+###-###+###-###+###-

###B-440###Balikes ir###+###+###-###+###-###+###-

###T-604###Tekirda###+###+###-###+###-###+###-

###T-709###Tekirda###+###+###-###+###-###+###-

###T-113###Tekirda###+###+###-###+###-###+###-

###T-507###Tekirda###+###+###-###+###-###+###-

###T-608###Tekirda###+###+###-###+###-###+###-

###T-919###Tekirda###+###+###-###+###-###+###-

###T-118###Tekirda###+###+###-###+###-###+###-

###T-119###Tekirda###+###+###-###+###-###+###-

Table 2. Oligonucleotide primer sequences and sizes of the PCR products.

Primer name###Specificity###Fragment size (bp)

Fg161###F. graminearum###450

Fg16N1###F. graminearum###280

Tri13DON1###DON chemotypes###282

Tri13NIV1###NIV chemotypes###312

Tri13P2###Generic (15-###583 644 859

###AcDON###respectively

3-AcDON and NIV)

Table 3. Mean and significance of isolate variation for percent infected kernels and a thousand seed weight in the GAlnen wheat genotype.

Isolates###FHB (%)###TKW

C601###48.06 fg###0.65 de

C402###76.85 bc###0.22 j

C302a###58.84 d###0.62 ef

C601a###46.41 fg###0.78 b

C606b###71.59 c###0.45 h

B006###49.60 ef###0.60 f

B018###77.84 bac###0.23 j

B114###86.33 a###0.44 h

B440###43.18 fg###0.71 c

T604###71.43 c###0.50 g

T709###83.92 ba###0.30 i

T113###41.27 fg###0.67 cd

T507###82.31 ba###0.44 h

T608###39.75 g###0.83 a

T919###60.26 d###0.61 ef

T118###81.42 ba###0.25 j

T119###56.93 de###0.61 ef

Average###6329###052

Tukey MSD 86.767###0.0493

Rep###769.689###0.00046329

Isolates###1098.61347###0.14687040

Error###1.130.447###0.00036550

DISCUSSION

Wheat fields in three provinces in North-West of Turkey were surveyed for FHB symptoms and F. graminearum isolates were selected from other Fusarium species collected. F. graminearum was first diagnosed morphologically and then by PCR using two sets of species-specific primers. The ability of the isolates to produce trichothecenes was evaluated using chemotype- specific PCR markers (Table 1). Only one chemotype DON was identified and all displayed the 15-AcDON chemotype (Table 1 Figure 2).

It has been suggested that knowledge of Fusarium chemotypes and their distribution could be crucial in forecasting schemes for disease development and mycotoxin contamination within a wheat growing area (Jennings et al. 2004). Using a molecular approach in current work the trichothecene chemotype distribution analysis among the wheat cultivation of these three provinces revealed only one dominant chemotype (DON) furthermore 15-AcDON type was the only sub- chemotype throughout the sampling area.

Carter et al. (2002) reported that DON chemotype was dominant comparing NIV in Europe and North America. Previous observations from Luxembourg showed the presence of two populations (Pasquali et al. 2009). Strains of F.graminearum complex from maize of northwest Argentina were DON and NIV producers (Sampietro et al. 2012). Employing 11 F. graminearum isolates the previous report from Turkey by YAlrA1/4k and Albayrak (2012) showed that although there was a single NIV-producing isolate in the population the DON- producers were dominant.

In different USA areas Gale et al. (2007) identified 15-ADON as the prevailing chemotype of F. graminearum sensu stricto followed by 3-ADON (5.1% of total) and one to NIV chemotype. In southern Russia 90% of the isolates was 15-ADON (Yli-Mattila et al. 2008) Jennings et al. (2004) found DON (75%) and NIV (25% ) with the predominant 15-ADON chemotype (95%) in England and Wales the 15-ADON chemotype was the major population (94.3%) the 3-ADON chemotype was not detected and the NIV chemotype was detected sporadically (5.8%) in Luxembourg (Pasquali et al.2010). YAlrA1/4k and Albayrak (2012) from Turkey reported all DON chemotypes were 15-AcDON- producers.

We previously reported NIV 3-AcDON and 15- AcDON chemotypes of F. culmorum in the same surveyed area (Mert-TA1/4rk and Gencer 2013). However employing 17 F. graminearaum isolates all were described as 15-AcDON-producers in the current study.

In order to analyse the differences in aggressiveness among the isolates we conducted a field trial. We assessed disease progress as well as a thousand kernel weight. FHB severity was rated visually as the percentage of infected spikelets per plot (0 to 100%). Mean FHB ratings and TKW in wheat showed a wide and significant (P = 0.05) range of variation within population (Table 3). Some isolates produced on average 86% disease severity on the wheat cultivar tested whereas others were much less virulent on average 22% (Table 3). TKW was also affected variously when inoculated by different isolates.

Differences in aggressiveness among isolates may due to genetic recombination mutation or selection.

In F. graminearum there is large genetic variation within a given population even in samples collected from a small area within a field (McMullen et al. 1997). Miedaner et al. (210) analyzed the aggressiveness on young winter rye plants of populations of F. graminearum and F. culmorum collected from natural field epidemics of FHB and rye foot rot. They found high genotypic variance in the two species and a high degree of diversity of aggressiveness within single field populations of either species.

This study demonstrated that F. graminearum isolates differ in aggressiveness in planta and that the same chemotype- producers could show different degree of virulence in field conditions. The existence of variability in aggressiveness among isolates suggests that wheat breeders should use mixtures of isolates to screen for FHB resistance in wheat lines. In addition the information can be used by small grain producers to make decisions pertaining to deployment of FHB management strategies.

Acknowledgements: This research was partly supported by TUBITAK Turkey (Project code: TOVAG- 109O830).

REFERENCES

Bai G.H. and G. Shaner (1996). Variation in Fusarium graminearum and cultivar resistance to wheat scab. Plant Dis. 80(9): 975-979.

Carter J. H. Rezanoor D. Holden A.E. Desjardins R.D. Plattner and P. Nicholson (2002). Variation in pathogenicity associated with the genetic diversity of Fusarium graminearum. Eur. J. Plant Pathol. 108: 573-583.

Gale L.R. T.J. Ward V. Balmas and H.C. Kistler (2007).

Population subdivision of Fusarium graminearum sensu stricto in the upper Midwestern United States. Phytopathology. 97: 1434-1439.

Jackowiak H. D. Packa M. Wiwart and J. Perkowski (2005). Scanning electron microscopy of Fusarium damaged kernels of spring wheat.

International J. Food Microbiol. 98: 113-123.

Jennings P. M.E. Coates J.A. Turner E.A. Chandler and P.Nicholson (2004). Determination of deoxynivalenol and nivalenol chemotypes of Fusarium culmorum isolates from England and Wales by PCR assay. Plant Pathol. 53: 182190.

Markell S.G. and L.J. Francl (2003). Fusarium head blight inoculum: species prevalence and Gibberella zeae spore type. Plant Dis. 87: 814820.

McMullen M. R. Jones and D. Gallenberg (1997). Scab of wheat and barley: a re-emerging disease of devastating impact. Plant Dis. 81: 13401348.

Mert-TA1/4rk F. and R. Gencer (2013). Distribution of the 3- AcDON 15-AcDON and NIV Chemotypes of

Fusarium culmorum in the North-West of Turkey. Plant Protect. Sci. 49(1): 5764.

Miedaner T. G. Gang and H.Geiger (1996). Quantitative- genetic basis of aggressiveness of 42 isolates of Fusarium culmorum for winter rye head blight. Plant Dis. 80(5): 500-504.

Miedaner T. A. Schilling and H. Geiger (2001).

Molecular genetic diversity and variation for aggressiveness in populations of Fusarium graminearum and Fusarium culmorum sampled from wheat fields in different countries. J. Phytopathol. 149(11): 641-648.

Miedaner T. M. Moldovan and M. Ittu (2003).

Comparison of Spray and Point Inoculation To Assess Resistance to Fusarium Head Blight in a Multienvironment Wheat Trial. Phytopathology. 93(9): 1068-1072.

Miedaner T. C. Bolduan and A.Melchinger (2010).

Aggressiveness and mycotoxin production of eight isolates each of Fusarium graminearum and Fusarium verticillioides for ear rot on susceptible and resistant early maize inbred lines. Eur. J. Plant Pathol. 127(1): 113-123.

Miller J.D. R. Greenhalgh Y.Z. Wang and M. Lu (1991). Trichothecene chemotypes of three Fusarium species. Mycologia. 83: 121-130.

Pasquali M. F. Giraud C. Brochot E. Cocco L. Hoffmann and T. Bohn (2010). Genetic Fusarium chemotyping as a useful tool for predicting nivalenol contamination in winter wheat. Int. J. Food Microbiol. 137: 246-253.

Ribichich K.F. S.E. Lopez and A.C. Vegetti (2000).

Histopathological spikelet changes produced by Fusarium graminearum in susceptible and resistant wheat cultivars. Plant Dis. 84: 794802.

Sampietro D.A. M.E. Ficoseco C.M. Jimenez M.A. Vattuone and C.A. Catalan (2012). Trichothecene genotypes and chemotypes in Fusarium graminearum complex strains isolated from maize fields of northwest Argentina. Int. J. Food Microbiol. 153(1-2): 229-33

Snijders C.H.A. and J. Perkowski (1990). Effects of Head Blight Caused by Fusarium culmorum on Toxin Content and Weight of Wheat Kernels. Phytopathology. 80: 566-570.

Yli-Mattila T. K. O'Donnell T. Ward and T. Gagkaeva (2008). Trichotecene chemotype composition of Fusarium graminearum and related species in Finland and Russia. J. Plant Pathol. 90(3): 60-64.

YAlrA1/4k E. and G. Albayrak (2012). Chemotyping of Fusarium graminearum and F. culmorum isolates from Turkey by PCR Assay. Mycopathologia. 173(1): 53-61.
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