Identification and trichothecene genotypes of Fusarium graminearum species complex from wheat in Taiwan

Fungal isolates collected in this study were from areas of northern and central Taiwan located from North 23°50? to North 25°02? which represented one of the wheat production areas at the lowest latitude in the world. FHB pathogens were not detected from samples collected from southern Taiwan near or lower than the Tropic of Cancer. It would be interesting to dissect the reasons of no detection in southern Taiwan, including the presence or absence of pathogens, pathogen survival ability in high temperature, alternative hosts and environmental factors for disease development. In this study, all pathogenic isolates were considered to be members of FGSC by PCR amplification with primers Fg16F and Fg16R and morphological characteristics of asexual and sexual stages. Because species of FGSC were established based on the molecular characteristics and cannot be identified through the morphological characteristics (O’Donnell et al. 2004; Sarver et al. 2011), we used molecular methods to determine the species of each isolate. As mentioned earlier, F. asiaticum and F. graminearum s. str. have been reported as predominate species of FGSC in China and Japan (Shen et al. 2012; Zhang et al. 2007; Suga et al. 2008; Zhang et al. 2012). A PCR–RFLP method has been reliable to diagnose 867 isolates of F. graminearum s. str. and F. asiaticum in Japan, China and US (Shen et al. 2012; Gale et al. 2011; Suga et al. 2008). We, therefore, applied the PCR–RFLP diagnosis method to detect F. asiaticum and F. graminearum s. str. from collected isolates. A phylogenetic analysis based on RED and TEF–1? sequences complied with the diagnosed results, and further identified a F. meridionale isolate in our collection. As shown in studies, RED and TEF–1? sequences were able to separate most species of FGSC and used at the initial stage to discover a novel species (O’Donnell et al. 2004; Starkey et al. 2007). These identifications of F. asiaticum, F. graminearum s. str. and F. meridionale were also supported by the fixed nucleotides presenting in the 2 gene sequences and by the morphology of the sexual and conidial stages. Nevertheless, there was still one isolate in query. More informative genes such as genes in the mating-type locus are required to resolve the relationship between the unknown isolate and other species of FGSC (O’Donnell et al. 2004).

Fusarium asiaticum was identified as a predominate species with 98% of the population in this study. Fusarium asiaticum and F. graminearum s. str. were the major species in many Asian countries (van der Lee et al. 2015; Wang et al. 2011). Factors like temperatures and hosts have been ascribed to species distribution. In China, F. asiaticum was mainly distributed in the southern provinces, whereas F. graminearum s. str. was mostly in the northern provinces (Qu et al. 2008b; Shen et al. 2012). The same trend was observed in an analysis of Japanese isolates (Suga et al. 2008). From a study of FHB of wheat, Qu et al. (2008b) concluded that F. asiaticum was mainly obtained from warmer regions where the annual average temperature were above 15 °C, and the majority of F. graminearum s. str. was isolated from cooler regions with the annual average temperature at 15 °C or lower. A BIOCLIM analysis suggested that areas where the warmest quarter with the mean temperature above 22 °C and the precipitation over 320 nm favored the occurrence of F. asiaticum (Backhouse 2014). In addition, the host preference was implicated by a study of rice population in South Korea in which F. asiaticum (FGSC lineage 6) dominated over F. graminearum s. str. (FGSC lineage 7) and F. boothii (FGSC lineage 3) (Lee et al. 2009). The study showed that F. asiaticum produced more perithecia on rice straw than the other two species in an experiment of mixed-species inoculum. Zhang et al. (2012) found a strong association between the predominate crops and the occurrence of F. asiaticum and F. graminearum s. str. They showed that F. asiaticum was more frequently obtained from the middle and low valleys of Yangtze River (southern China) where rice acreages were higher than maize acreages. Fusarium graminearum s. str. was prevalent in northern China with higher maize acreages (Zhang et al. 2012). In our study, the average annual temperatures of sampling areas in Taiwan were 22 °C and higher, and the precipitation of the warmest quarter were from 322 to 608 nm. All areas where samples were collected were operated under the rice–wheat rotation system. The result of predominate F. asiaticum in Taiwan was consistent with the notion of those previous studies.

Fg16F and Fg16R were designed as specific primers for FGSC isolates (Nicholson et al. 1998). The various amplicons from Fg16F/R PCR were used for the SCAR analysis of FGSC isolates to reveal the genetic variation (Carter et al. 2000, 2002; Qu et al. 2008a, b; Desjardins et al. 2004). Later studies showed that SCAR type 1 and type 5 were fully congruent with F. graminearum s. str. and F. asiaticum, respectively, and were used to screen these 2 phylogenetic species (Chandler et al. 2003; Qiu et al. 2014; Zhang et al. 2007). SCAR type 2 was congruent with F. meridionale, but contained the same PCR band size as SCAR type 5 (Chandler et al. 2003). In this study, isolates of SCAR type 1 and most SCAR type 5 were confirmed as F. graminearum s. str. and F. asiaticum, respectively, by PCR–RFLP and phylogenetic analysis. Some F. asiaticum isolates were of SCAR type 3 and SCAR type 4, which has been reported elsewhere (Chandler et al. 2003). Notably, the one F. meridionale isolate (Fanyuan1-11) was determined to be SCAR type 5, rather than SCAR type 2. The unknown species isolate (Daya272-3) was of SCAR type 2, but was phylogenetically closest to F. cortaderiae rather than F. meridionale. These results suggested that additional caution was needed to be taken to infer FGSC species by SACR types.

It was surprising to find that F. asiaticum isolates were either 15-ADON or NIV genotype in this Taiwanese population. The 3-ADON genotype isolate was not detected in current collection. The 15-ADON genotype isolates were more prevalent than NIV genotype isolates in all geographic areas, especially in northern Taiwan where 15-ADON genotype isolates accounted for 99% of the population. In Japanese and Chinese populations of F. asiaticum, all 3 trichothecene genotypes (3-ADON, 15-ADON and NIV genotypes) were detected. Both 3-ADON and NIV genotype isolates usually dominated over 15-ADON genotype isolates (Qiu et al. 2014; Suga et al. 2008). Outside of Asia, NIV genotype isolates of F. asiaticum were prevalent in southern Louisiana, United States and southern Brazil (Gale et al. 2011; Gomes et al. 2015). The 15-ADON genotype isolates of F. asiaticum were a typical minority and may be absent in culture collections (Karugia et al. 2009; Shen et al. 2012). In China, the 3-ADON and NIV genotype isolates were prevalent in the warmer southern regions whereas the 15-ADON genotype was mainly presented in the cooler northern regions. The geographic difference of trichothecene genotypes were explained by the unevenly distribution of phylogenetic species. F. asiaticum, the warmer-region inhabitant, mainly belonged to 3-ADON and NIV genotypes based on 1903 isolates while F. graminearum s. str., the cooler-region inhabitant, contained more 15-ADON genotype isolates based on 383 isolates, according to several studies in China and Japan (Karugia et al. 2009; Qiu et al. 2014; Shen et al. 2012; Suga et al. 2008; Zhang et al. 2007). Regarding the geographic locations of 15-ADON genotype isolates which were mostly in the north of East Asia, this Taiwanese F. asiaticum 15-ADON isolates represented a unique population locating in the south of East Asia. This result suggested that 3-ADON genotype isolates were possibly not existed in Taiwanese populations or several factors may favor the 15-ADON genotype isolates to establish in Taiwan. It is notable that the wheat cultivar Taichung Sel. 2 derived from the wheat line Au-Maya74“S” was the only wheat cultivar used for wheat production in Taiwan currently. All FGSC isolates were isolated from diseased plants of the cultivar. The pathogenicity assay, however, indicated that the host cultivar was equally susceptible to 15-ADON and NIV genotype isolates. It needed further evaluations if the Taichung Sel. 2 cultivar favors F. asiaticum 15-ADON genotype isolates for growth, survival, sporulation, or other biological traits that would facilitate these 15-ADON genotype isolates to outcompete other genotype isolates.