Response of wheat (Triticum spp.) and barley (Hordeum vulgare) to Fusarium poae
Respuesta del trigo (Triticum spp.) y la cebada (Hordeum vulgare) a Fusarium poae
Sebastián A. Stenglein*, M. Inés Dinolfo, Fabricio Bongiorno, M. Virginia Moreno
Facultad de Agronomía de Azul, UNCPBA, República de Italia No. 780, Azul (7300), Argentina. Author for correspondence. (stenglein@faa.unicen.edu.ar).
]]> Recibido: October, 2011.
Aprobado: February, 2012.
Abstract
Fusarium head blight is an important disease attacking wheat (Triticum spp.), barley (Hordeum vulgare) and other grains worldwide. Among the Fusarium species causing this disease, Fusarium poae is less often implicated, but is a fungus of increasingly recognized importance and it is associated with human and animal toxicoses. The aim of this study was to examine the responses of wheat and barley varieties to inoculation by different F. poae isolates, in order to observe contamination by this fungus in the grains. The analyses were performed during 2008, 2009, and 2010 under natural conditions at the Facultad de Agronomía de Azul-UNCPBA, province of Buenos Aires, Argentina. Statistical analyses were carried out and the identities of re-isolated isolates were tested by a primer-specific PCR reaction and by comparing DNA-ISSR amplifications. Differences among varieties in fungal symptoms were significant (p≤0.05) only in 2008. Although the number of re-isolated isolates in wheat was greater than the number of samples with observable symptoms, no significant correlations were found. However, there were correlations in barley and the linear regression analyses allow suggesting that for each grain with visual symptoms, two barley grains could contain the fungus. Thus it can be concluded that the real number of grains contaminated with F. poae is significantly higher than the number with observable disease symptoms, and therefore the real extent of contamination with F. poae is currently underestimated and should be considered for food risk analysis in the near future.
Key words: disease symptoms, Fusarium poae, grain contamination.
Resumen
La fusariosis de la espiga es una enfermedad importante que ataca al trigo (Triticum spp.), la cebada (Hordeum vulgare) y otros granos en el mundo. Entre las especies de Fusarium que causan esta enfermedad, Fusarium poae es una de las menos frecuentes, pero es un hongo cuya importancia es cada vez más reconocida y se le asocia con la toxicosis en humanos y animales. El objetivo de este estudio fue examinar las respuestas de las variedades de trigo y cebada a la inoculación de diferentes aislamientos de F. poae, a fin de observar la contaminación producida por este hongo en los granos. Los análisis se realizaron durante 2008, 2009 y 2010 en condiciones naturales, en la Facultad de Agronomía de Azul-UNCPBA, provincia de Buenos Aires, Argentina. Se hicieron análisis estadísticos, y las identidades de los aislamientos re-aislados se probaron con reacciones de iniciadores específicos PCR y por comparación de amplificaciones de ADN-ISSR. Las diferencias entre variedades en los síntomas del hongo fueron significativas (p≤0.05) sólo en 2008. Aunque el número de aislamientos re-aislados en trigo fue mayor que el número de muestras con síntomas observables, las correlaciones no fueron significativas. Sin embargo, hubo correlaciones significativas en la cebada y los análisis de regresión lineal sugiriendo que por cada grano con síntomas visibles, dos granos de cebada podrían contener el hongo. Asi puede concluirse que el número de granos contaminados con F. poae es significativamente mayor que el de aquellos con síntomas observables de la enfermedad y, por tanto, el alcance real de la contaminación con F. poae es subestimado y se debiera considerar para el análisis de riesgos alimentarios en un futuro próximo.
]]> Palabras clave: síntomas de la enfermedad, Fusarium poae, contaminación del grano.
INTRODUCTION
In cereal grain production, Fusarium head blight (FHB) is one of the most important and insidious diseases. It has a severe impact through yield reductions and the mycotoxin contamination that it causes. Increasing worldwide concern about food safety has enhanced interest in Fusarium infections and mycotoxin production in food products. Fusarium graminearum is the predominant FHB mycotoxin-producing agent worldwide, although F.culmorum, F. avenaceum and F. poae are commonly isolated from cereal grains. In recent years, some changes were found in the predominance of the various Fusarium species. For example, F. poae prevails in Argentina, Hungary, Ireland, Italy and the UK, in cereal (Xu et al., 2005; González et al., 2008). However, the predominance and distribution, or both, of Fusarium species depend on climatic factors. Thus, F. graminearum is associated with warmer/humid conditions, F. poae with relatively drier and warmer conditions, and both F. culmorum and F. avenaceum with niches of cooler/wet/humid conditions (Xu et al., 2008).
Among the mycotoxins produced by Fusarium species, trichothecenes are considered the most important, since they are potent inhibitors of eukaryotic protein synthesis (Bennet and Klich, 2003), and can cause a wide range of adverse effects in animals and humans through ingestion of food and feed prepared from contaminated cereal grains (D'Mello et al., 1999). Fusarium poae is a relatively weak pathogen compared with F. graminearum and F. culmorum, but it can produce a large number of mycotoxins, such as trichothecenes of type A (T-2, HT-2, diacetoxyscirpenol, monoacetoxyscirpenol, scirpentriol) and B (nivalenol, fusarenone-X), beauvericin and enniatins (Vogelgsang et al., 2008; Stenglein, 2009).
The strategies used to prevent Fusarium colonization and mycotoxin contamination in cereal grains caused by Fusarium species include crop rotation, optimized tillage and straw management, and the use of varieties with low susceptibility (Edwards, 2004). The aim of this study was to examine the responses of wheat and barley varieties to inoculation by different F. poae isolates, in order to observe the contamination of grains infected by the fungus.
MATERIALS AND METHODS
The study was carried out at the Facultad de Agronomía de Azul-UNCPBA (36° 41' S, 59° 48' W; 132 m altitude), province of Buenos Aires, Argentina.
Four monosporic F. poae isolates obtained from wheat (TSS1a, TSa1a, T-MICA-01 and T-MICA-08) and one obtained from barley grains (Hsu1a) were used individually (Dinolfo etal., 2010). Fungal inoculum was produced by placing individual agar plugs with mycelium and conidia onto potato dextrose agar (PDA, Britania®) in petri dishes (90×20 mm) and incubating for 7 d at 25±2 °C under 12 h light/dark. Conidial harvested were taken by flooding the plates with 5 mL of sterilized distilled water (SDW) and dislodging the conidia with a bent glass rod. The resulting suspension was filtered through cheesecloth and the conidial suspension was adjusted to 1×105 conidia mL-1 using a haemacytometer (Neubauer) and a binocular microscope. Tween® 20 (0.05 %) was added as surfactant.
]]> Four wheat cultivar were used: Apogee [(with susceptibility to FHB and F. poae, Mackintosh et al. (2006); Vogelgsang et al. (2008)]; Klein Chajá (bread wheat); Buck Biguá (bread wheat); Chagual (durum wheat), and two barley varieties, Stander (6-row and susceptible to FHB, Urrea et al., 2005); Quilmes Scarlett (2-row). Seeds were surface-sterilized by immersing them 3 min in 50 % ethanol, 3 min in sodium hypochlorite (commercial 55 g Cl L-1), and by washing them three times with SDW. Three seeds were sown in pots (3 L) filled with soil (mixture of farm soil, organic soil and sand, 2:1:1) and after emergence, seedlings were selected based on size uniformity, leaving one seedling per pot. Eighteen replicates (pots) of each variety were used in a completely randomized design.Plants were grown in the absence of any nutritional, pest or water stress, except that no fungicides were applied. The experiment was performed during each of three consecutive years (2008, 2009, and 2010) under natural conditions. The sowing dates for the different varieties and species were adjusted according to growth progress observed in plants grown in 2007, to ensure that all varieties were at similar growth stages at the same time.
Wheat heads were inoculated at mid-anthesis (Vogelgsang et al., 2008) and barley when >50 % of the plants had reached anthesis (Buerstmayr et al., 2004). Conidial suspensions of each fungal isolate were applied to separate plants, until run off, using a gravity spray gun. Five heads of each plant variety randomly selected per isolate were inoculated. Sterilized distilled water with Tween® 20 was used to inoculate two heads per plant variety to serve as control treatment. Visual disease assessment was conducted at 20 d post inoculation by counting the number of symptomatic grains (lesions or bleaching of grains or glumes with a dark margin) from each inoculated head.
Temperature and RH data (from inoculation to the time of visual disease assessment) were obtained from the Boletín Agrometeorológico del Centro-Sur de la Provincia de Buenos Aires (2008, 2009, 2010), located 50 m from the experimental site.
The five heads of each variety per inoculated isolate and the controls were individually threshed and grains were collected. All grains (with and without symptoms) were transferred to petri dishes containing PDA with 250 mg chloramphenicol L-1 and incubated 7d at 25±2 °C under 12 h light/dark. Fungal isolates were then taken from the incubated grains.
To be sure that the re-isolated isolates obtained were the same as the ones originally inoculated, the DNA of all isolates was extracted using a cetyltrimethylammonium bromide (CTAB) method according to Stenglein and Balatti (2006), amplified by a species specific-F. poae PCR reaction using primers Fp82F 5'-ACGACGAAGGTGGTTATG-3' and Fp82R 5'-GAAGAGCCTGTTTGCTTG-3' (Parry and Nicholson 1996), and compared in five ISSR primer amplifications, CTC(GT)8, (GAG)5CAG, CAC5, CT(GA)8, and (GCC)5, with the DNA of the original isolates used for inoculations (Dinolfo et al., 2010). Each ISSR reaction was performed at least twice, running eight DNA samples plus the DNA ladder and a negative control in one gel, simultaneously. Products from PCR reactions were examined by electrophoresis in 1.5 % (w v-1) agarose gels containing GelRedTM (Biotium, Hayward, USA). Fragments were visualised under UV light. The size of the DNA fragments were estimated by comparing the DNA bands with a 1 kb and 100 bp DNA ladder (Genbiotech S.R.L., Buenos Aires, Argentina).
A factorial experiment with 30 treatments (five different isolates X six different plant varieties) in a completely random design was used. An analysis of variance of the number of infected (visible symptoms) grains/total grains per head, was performed for each year in order to determine differences between varieties and isolates, and their interaction. Percentage data were arcsin (square root) transformed.
A Chi-square test was carried out to test independence between plant varieties and F. poae isolates re-isolated from grains. A correlation and linear regression analyses were performed to test the relationship between the number of grains with visible symptoms (x) and the number of grains from which isolates could be re-isolated (y) with and without symptoms. All statistical analyses were performed using INFOSTAT version 2006 (Infostat group, FCA, Universidad Nacional de Córdoba, Argentina).
RESULTS AND DISCUSSION
]]> No symptoms were observed on control head cultivars. The five isolates differed in the rate of F. poae symptoms developments on the six plant genotypes (Table 1). Apogee was the most susceptible wheat variety to all F. poae isolates tested, whereas the barley variety Stander was more susceptible to all isolates, except to MICA-T-08, than Scarlett, for the three years (Table 1). In general, all varieties inoculated with the different F. poae isolates showed a low incidence of disease symptoms, ranging from 0.0 to 7.94 % (Table 1). These results agree with reports that F. poae is a weak pathogen for wheat and barley (Xue et al., 2006; Vogelgsang et al., 2008). However, data analyses showed in 2008 differences (p=0.009) between varieties in disease symptoms development. In that year, greater differences in relativel humidity (average maximum RH, 81.9 %; average minimum RH, 33.2 %) and especially in temperature (average maximum T, 28.4 °C; average minimum T, 13.4 °C) were registered, as compared to 2009 (average maximum RH, 80.1 %; average minimum RH, 34.7 %; average maximum T, 21.8 °C; average minimum T, 6.6 °C), and 2010 (average maximum RH, 76.9 %; average minimum RH, 46.8 %; average maximum T, 22.5 °C; average minimum T, 8.6 °C).Fusariumpoae was associated with relatively warmer and drier conditions than other Fusarium species (Turner and Jennings, 1997; Xu et al., 2008). Average RH was not different (p>0.05) between years; however, in 2008 there were higher temperatures which could explain the statistical significances for 2008 only, a result in agreement with reports from Turner and Jennings (1997) and Xu et al. (2008).
No isolates of F.poae were obtained from control grains. All re-isolated isolates correspond to F. poae (no other Fusarium species were isolated from inoculated grains) and we used the ISSR method to check that the re-isolated isolates corresponded to each F. poae isolate used to inoculate each plant genotype in all the experiments. These reisolation tests are essential to show the absence of cross-infection with other Fusarium species or other F. poae isolates, or both, used in our study that could interfere with the results and conclusions.
All F. poae isolates were re-isolated from each inoculated plant varieties, except for the barley Scarlett variety inoculated with MICA-T-01 (Table 2). Surprisingly, the number of grains from which we recovered isolates was always higher than the number of grains with visible symptoms in the three years (Tables 1 and 2). For example, the wheat variety Apogee presented 7.94 % of grains with symptoms and we re-isolated isolates from 23.98 % grains (Tables 1 and 2), and the wheat variety Chagual showed ±10 % differences between symptomatic and infected grains (Table 1 and 2). Another interesting result was that, although in 2008 the conditions favored F. poae infections and was the only year for which we observed statistical significances, the numbers of re-isolated isolates were similar during each of the three years analyzed (data not shown).
The Chisquare test to prove independence between plant varieties and F. poae isolates re-isolated showed that only in 2008 we can reject the null hypothesis; thus, these variables were dependent (p=0.01). This allows confirming that the degree of success for F. poae infection depends on the environmental conditions, a result in agreement with reports by Turner and Jennings (1997) and Xu et al. (2008).
The number of grains from which isolates could be re-isolated was higher than the number of grains with observable symptoms in wheat varieties, but no correlations were found. However, in barley there were positive correlations, and therefore linear regressions analyses were performed. The 2008 analysis showed a correlation of 0.82 and the linear regression equation was: y=2.3049x + 0.2073 (r2=0.67). For 2009 and 2010 the correlation coefficients were 0.84 and 0.63 and the regression equations were: y=3.0198x – 0.4455 (r2=0.71), and y = 2.3043x + 1.2609 (r2=0.40). These results allow suggesting that for each grain with visual symptoms two barley grains could actually contain the fungus. Stenglein (2009) highlights the increasingly recognized importance of F. poae in different countries and summarizes the potential mycotoxin production, with particular regard to possible human and animal health issues. Niessen (2008) indicate that the level of contamination with certain mycotoxins may be related to the amount of pathogen biomass present in plant material. Correlations between DNA level and enniatins and nivalenol in barley grains samples were found using TaqMan assays (Yli-Mattila et al., 2008). However, in asymptomatic wheat samples contaminated with F. poae and using a TaqMan assay, no positive correlation was found between F. poae DNA and the quantity of enniatins B+B1 (Kulik and Jestoi, 2009).
CONCLUSIONS
The results confirm the importance of Fusariumpoae. Thus, the environmental conditions during anthesis are important for the development of the symptoms but they do not seem to be determinant for fungus colonization of the grains. Moreover, the actual number of grains contaminated with F. poae is significantly higher than the number with observable disease symptoms. Therefore, the actual degree of grain contamination with F. poae is currently underestimated and should be considered for a food risk analysis.
]]> ACKNOWLEDGEMENTS
This work was supported by FONCYT-SECYT PICT-PRH 2008/110 and PIP 167 CONICET.
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