Impaginato 199 Adv. Hort. Sci., 2017 31(3): 199-203 DOI: 10.13128/ahs-20585 Aggressiveness of four Fusarium head blight species on wheat cultivars N. Sakr Department of Agriculture, Atomic Energy Commission of Syria, P.O. Box 6091, Damascus, Syria. Key words: disease development, diseased-head severity, Fusarium species, soft dough stage, Syrian wheat cultivars. Abstract: Aggressiveness of four Fusarium head blight species (F. culmorum, F. solani, F. verticillioides and F. equiesti) was studied on six Syrian wheat culti- vars under controlled conditions. Two aggressiveness criteria: diseased-head severity (DHS, Fusarium infection) and disease development (DD, Fusarium spread) were visually estimated as percentage of heads showing Fusarium symptoms in wheat cultivars at the soft dough stage. Results showed signifi- cant differences among fungal isolates and wheat cultivars for the two tested criteria. The mean values of DHS evaluations ranged from 33.27 to 45.49% among fungal isolates, and from 29.62 to 42.22% among tested cultivars. The mean DD rating varied from 25.58 to 35.43% among fungal isolates, and from 25.33 to 34.01% among tested cultivars. Results in the current research high- lighted that the level of resistance in Syrian cultivars to Fusarium species is characterized with low to moderate DHS and DD evaluations (%). Also, the results were comparable with those previously obtained using the same fungal isolates and wheat cultivars in vitro. The current study confirmed the suitability of in vitro method to be used as fast and reliable test to analyze aggressiveness in Fusarium species. 1. Introduction Fusarium head blight is one of the most destructive global diseases of wheat. In infected plants, it leads to kill the developing seed (prematurely bleached spikes) within moist conditions and moderate temperatures prevail during flowering. Since it was identified in 1884, severe epidemic outbreaks caused quantitative losses in yield of up to 50-75% (Parry et al., 1995; McMullen et al., 2012). It also reduces grain quality due to contami- nation of harvest with large amount of mycotoxins that cause toxicities to human and livestock (Maresca, 2013). At least seventeen Fusarium species with several habitats and types of mycotoxins produced have been associated with Fusarium species (Parry et al., 1995). Fusarium graminearum is the main causal agent of this disease and has been subdi- vided into at least 11 cryptic species (O’Donnell et al., 2004). Other species can cause Fusarium disease on a lesser scale such as F. ave- naceum, F. culmorum, F. solani, F. equiseti, F. verticillioides and F. poae (*) Corresponding author: ascientific@aec.org.sy Citation: SAkr N., 2017 - Aggressiveness of four Fusarium head blight species on wheat cultivars. - Adv. Hort. Sci., 31(3): 199-203. Copyright: © 2017 Sakr N. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. received for publication 24 April 2017 Accepted for publication 6 June 2017 AHS Advances in Horticultural Science Adv. Hort. Sci., 2017 31(3): 199-203 200 (Xu et al., 2008). Understanding the interaction between wheat plants and Fusarium populations requires more detailed knowledge about the variation of aggres- siveness (Wu et al., 2005). Van der Plank (1968) defined aggressiveness as a quantitative ability of an isolate to cause disease on a susceptible host plant in a non-race-specific pathosystem. Aggressiveness is an important factor determining the potential ability of Fusarium isolates to cause Fusarium epidemics. Variability of quantitative component of pathogenici- ty in F. graminearum has been the subject of several studies (Parry et al., 1995; Leonard and Bushnell, 2003; Wu et al., 2005; McMullen et al., 2012). However, other Fusarium species have attracted less pathogenic analyses (Xu et al., 2008; Bakri et al., 2012; Sakr, 2017). Fusarium resistance in wheat plants is conferred by quantitative trait loci (QTL) detected on all chromosomes (Loffler et al., 2009). It is necessary to combine type I (resistance to initial infection) and type II (resistance to spreading) to get Fusarium resistant wheat plants (Loffler et al., 2009). In Syria, about 1.7 million hectares were sown to wheat, with an annual production of 3.9 million tons in 2011. Host-pathogen interactions were evaluated for several local wheat cultivars inoculated with fun- gal isolates of different species associated with Fusarium species, and differential reactions on culti- vars were detected (Alazem, 2007; Talas et al., 2011; Bakri et al., 2012). recently, Sakr (2017) analyzed aggressiveness of four Fusarium species in vitro, and significant differences were detected between pathogen isolates and wheat genotype. In order to underline pathogenic variation for Fusarium species collected from Ghab Plain, one of the principal Syrian wheat production areas, the objectives of the current study were to (1) evaluate aggressiveness of four iso- lates [F2 (F. culmorum), F27 (F. verticillioides), F35 (F. solani), and F43 (F. equiesti)] on six wheat Syrian cul- tivars under controlled conditions, and (2) compare results previously obtained by Sakr (2017) in vitro with the current data from floret inoculation under controlled conditions. 2. Materials and Methods Fungal isolates and inoculum production The fungal isolates of four Fusarium species [F. culmorum (F2), F. verticillioides (F27), F. solani (F35), and F. equiesti (F43)] were collected in 2015 from naturally wheat spikes exhibiting Fusarium symptoms from different locations of Ghab Plain in 2015. Isolates were identified morphologically according to Nelson et al., (1983). The cultures were maintained in sterile distilled water at 4°C and freezing at -16°C until needed. For inoculum preparation, four to six agar plugs out of the stored isolates were put over the surface of PDA in 9-cm Petri dishes and incubated for 10 days, at 22°C in the dark to allow mycelial growth and sporulation. Ten ml of sterile distilled water were added to each dish, and the resulting spore suspen- sions were adjusted to 5 × 104 spores/ml for inocula- tion following a count in a hemacytometer (Bakri et al., 2012). Wheat cultivars and growth chamber conditions In the current study, aggressiveness for the four Fusarium head blight isolates was measured on six wheat cultivars previously analyzed in vitro (‘Cham1’, ‘Cham7’, ‘Acsad65’, ‘Cham4’, ‘Cham6’ and ‘Douma4’, most cultivated in different Syrian areas) under con- trolled conditions. Wheat seeds were surface-sterilized with 5% sodi- um hypochlorite solution for 8 min and then washed six times in sterile distilled water (Purahong et al., 2012). They were sown into plastic pots (15-cm) filled with 2 kg of sterilized soil (ten seeds per pot), and arranged in a complete randomized design with three replicates. Three plots per replicate were left non-inoculated as control treatment. Pots were placed in a growth chamber operated at 20°C during day and night with an 16-h photoperiod. Following emergence, plants were thinned to three per pot and nitrogen fertilizer was applied twice at two dates: emergence and tillering. Aggressiveness tests At 10-14 days after heading, spore suspensions of the four Fusarium head blight isolates or sterile dis- tilled water (control) were sprayed one time into flowering spikes. Six flowering spikes were randomly selected within each replicate of the six cultivars. After the inoculum dried for 30 min, inoculated spikes were then kept covered for 48 h using poly- thene bags to ensure 100% rH. Head blight symptoms were evaluated as percent- age of spikes showing Fusarium symptoms after 7, 14, and 21 days, when plants were at the soft dough stage. Fusarium disease severity was visually estimat- ed in situ for each inoculated spike using the Xue’s et al., (2004) scale. This scale includes nine levels of incidence expressed in percentage of bleached spike area on a head: 0 (no visible Fusarium symptoms) to Sakr - Aggressiveness of four Fusarium head blight species on wheat cultivars 201 9 (severely diseased, spike dead). Each head was assessed separately in all experiments. The values of diseased-head severity as percent- age of infected spikes measured 21 days after inocu- lation (DAI) were considered, for each cultivar, a parameter to determine initial infection. The values of disease development calculated by the means of each evaluation; 7, 14 and 21 DAI over the estimation time were considered, for each cultivar, a parameter to determine pathogen spreading. Statistical analyses Statistical analyses of aggressiveness data were performed using StatView, 4.57® Abacus Concepts, Berkley, Canada. Before statistical analysis, the per- centages were transformed using the Arcsines func- tion. A complete randomized design with two factors (Fusarium isolate and wheat genotype) and 3 replica- tions was used for aggressiveness analysis. Fisher’s LSD test was used to compare the means at P <0.05. 3. Results and Discussion Understanding the interaction between Fusarium head blight species and wheat plants requires knowl- edge of the variation of quantitative component of pathogenicity (Wu et al., 2005). With this in mind, aggressiveness variability for four local Fusarium species was analyzed by using a floret inoculation in a growth chamber on six wheat cultivars most cultivat- ed in different Syrian areas. Differences in aggressiveness of four Fusarium species (F. culmorum, F. verticillioides, F. solani, and F. equiesti) are indicated when isolates vary in the amount of damage that they cause in wheat plants. The results demonstrated that none of the six tested cultivars was immune from disease. However, typical Fusarium symptoms induced by the four isolates (F2, F27, F35 and F43) were clear and easy to score in the inoculated spikes, while no symptoms were present in the control (Fig. 1). The mean values of diseased- head severity (DHS) ranged from 33.27% to 45.49% as compared with 0% for the control treatment (Table 1). There were significant differences among four isolates (F isolates=4.376; Probability=0.0084). The mean values of disease development (DD) varied from 25.58% to 35.43% (Table 1). Significant differ- e n c e s a m o n g f o u r i s o l a t e s ( F i s o l a t e s = 4 . 2 5 7 ; Probability=0.0096) were detected. results in the current study showed that there was no interaction between Fusarium isolates and host plant for the two parameters. This indicates to non-race-specific inter- action described for this pathosystem (Loffler et al., 2009). High diseased-head severity and disease develop- ment values represent high aggressiveness (Parry et al., 1995; Wu et al., 2005; Xu et al., 2008). results shown in Table 1 indicate that the isolate F35 of F. solani was the most aggressive with a mean value of DHS of 45.49% and DD of 35.43%; followed by other tested isolates. These results are in accordance with previous analysis on the aggressiveness of theses iso- lates in vitro; Sakr (2017) observed that the isolate F35 was the most aggressive one, followed by other analyzed isolates. The current study confirmed the suitability of in vitro modified Petri-dish method to be used as fast and reliable test to analyze aggres- siveness in Fusarium species. results in the current study are comparable with those found by Alazem (2007) and Bakri et al., (2012) for F. culmorum, F. solani, F. verticillioides and F. equiesti in which signifi- cant differences were detected for aggressiveness among fungal isolates in each Fusarium head blight species in a growth chamber. The mean value of DHS and DD rating for six Fig. 1 - Fusarium head blight symptoms on spike of Syrian wheat cultivar Cham4 inoculated with isolate F35 (Fusarium solani) compare with control (water). Adv. Hort. Sci., 2017 31(3): 199-203 202 wheat cultivars (Table 1) reflects the ability of the same isolate of the pathogen (F2, F27, F35 and F43) t o d i s t i n g u i s h d i f f e r e n t l e v e l s o f r e s i s t a n c e a s observed for the same pathosystem (Alazem, 2007; Talas et al., 2011). Also, the resistance of a given wheat cultivar is not related to a certain Fusarium species (Table 1). Significant differences were under- lined for DHS (F cultivars=2.907; Probability=0.0226) and DD (F cultivars=1.870; Probability=0.1171) crite- ria among wheat cultivars (Table 1). The mean values of DHS evaluations ranged from 29.62 to 42.22% among tested cultivars. The mean DD rating varied f r o m 2 5 . 3 3 t o 3 4 . 0 1 % a m o n g t e s t e d c u l t i v a r s . Quantitative resistant wheat cultivars are identified by low DHS and DD values of the fungus compared with the susceptible one (Parry et al., 1995). results in the current research highlighted that the level of resistance in Syrian cultivars to Fusarium species is characterized with low to moderate DHS and DD evaluations (%). These results are in accordance with previous analysis on the comportment of local wheat cultivars in which differential reactions on cultivars were detected (Alazem, 2007; Bakri et al., 2012; Talas et al., 2011). However, Fusarium resistance scores ranged one fold and half between resistant and sus- ceptible cultivars for the two tested parameters (Table 1). Thus our observation suggests that in resis- t a n t w h e a t c u l t i v a r s , t h e d e v e l o p m e n t o f t h e pathogen was slowed, and may be due to resistance mechanisms expressed by accumulation of QTL in host cultivars (Alazem, 2007; Talas et al., 2011). results in the current study showed that the level of quantitative resistance in the six wheat cultivars made it possible to detect significant differences between isolates of four Fusarium species. These results are in accordance with our previous analysis on the behavior of theses cultivars in vitro (Sakr, 2017). The variability of resistance for the Syrian cul- tivars is interesting and promising for ecological framing/breeding and also for improving resistance of wheat cultivars. For F. graminearum, Purahong et al., (2012) vali- dated the modified Petri-dish method (used by Sakr, 2017) by highly significant correlation with the data from floret inoculation in adult plants in a growth chamber. results indicted that the Petri-dish aggres- siveness test conducted on other Fusarium species (F. culmorum, F. verticillioides, F. solani, and F. equiesti) is repeatable and stable with the six wheat cultivars in a growth chamber (Table 1). It will be nec- essary to analyze the pathogenic variation in a large number of Fusarium isolates on several wheat culti- vars under controlled and field conditions to screen Fusarium resistance in Syrian wheat cultivars. Acknowledgements The author would like to thank Director General of AECS, and the Head of the Agriculture Department Fusarium head blight incidence scores were evaluated as percentage of spikes showing Fusarium species symptoms using the Xue’s et al., (2004) scale. F tests (P<0.05), NS= not significant. Table 1 - Diseased-head severity and disease development scores in % among isolates of four Fusarium head blight species measured on six Syrian wheat cultivars Isolate ‘Cham1’ Cham7' Acsad65' Cham4' Cham6' Douma4' Mean Diseased-head severity scores (%) F2 25.92 25.92 29.62 37.03 44.44 44.44 35.24 F27 29.62 29.62 37.03 33.33 37.04 36.66 33.27 F35 33.33 33.33 40.74 58.88 47.77 47.77 45.49 F43 29.62 29.62 44.44 40.00 37.04 40 37.40 Mean 29.62 29.62 37.96 42.31 41.57 42.22 F isolates=4.376; Probability=0.0084 F cultivars=2.907; Probability=0.0226 F interactions=0.601 NS; Probability=0.8594 Diseased development scores (%) F2 23.56 24.69 21.16 30.86 40.4 29.62 28.38 F27 24.69 23.56 28.49 25.64 24.69 26.45 25.58 F35 30.30 31.74 29.10 45.58 32.09 43.77 35.43 F43 22.79 25.64 37.03 33.95 26.45 29.10 29.16 Mean 25.33 26.41 28.94 34.01 30.91 32.23 F isolates=4.257; Probability=0.0096 F cultivars=1.870; Probability=0.1171 F interactions=1.199 NS; Probability=0.3051 Sakr - Aggressiveness of four Fusarium head blight species on wheat cultivars 203 for their support. 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