Identification And Genetic Study Of Canker-inducing Pathogens (Fusarium Spp.) Of Prickly Ash

Prickly ash(Zanthoxylum bungeanum)is an economically important tree species across north to southwest China.Its pericarp is not only a common spice in Asian cuisine and also a Chinese traditional medicine(Zhou et al.2016;Tang et al.2014).Canker disease has caused damage to the prickly ash trees in several regions of northern China and induced dieback and even tree mortality,seriously declined the production of Chinese prickly ash.To clarify the species and genetics of Fusarium causing stem rot of Zanthoxylum bungeanum,isolates from the main production areas of prickly ash in Shaanxi,Gansu,Shanxi,Hebei and Shandong Province have been identified by phylogenetic,morphological and biological study.Research has also conducted on their sexual reproduction and mitochondrial inheritance.The main results were as follows: 1.Identification of the dominant canker-inducing pathogens of prickly ashTo identify the main pathogenic Fusarium,38 isolates were recovered from cankers of prickly ash trees in Shaanxi,Gansu,Shandong,etc.five provinces in China.These isolates were identified by BLASTn queries as well as phylogenetic analyses with molecular markers of the nuclear ribosomal internal transcribed spacer region(ITS-rDNA),partial translation elongation factor 1-α gene(TEF1),RNA polymerase II largest subunit gene(RPB1)and RNA polymerase II second largest subunit gene(RPB2).Results indicated that 30/38 isolates represent two novel phylogenetic species: Fusarium zanthoxyli X.Zhou,T.Aoki,K.O’Donnell & Z.M.Cao and Fusarium continuum X.Zhou,T.Aoki,K.O’Donnell & Z.M.Cao,which are closely related to Fusarium torreyae,the pathogen of the Florida torreya(Torreya taxifolia)in America.These three tree canker-inducing pathogens constitute a novel clade within Fusarium,which was designated as Fusarium torreyae species complex(FTOSC).In addition,distinctions of morphological characters and optimal temperaturses were found between these two prickly ash pathogens via studying their cultural characters,microstructure and growth rate.Investigations indicated that F.zanthoxyli was distributed across Shaanxi,Gansu and Shanxi Province;F.continuum was mainly distributed in Shandong Province.Pathogenicity test revealed that F.zanthoxyli,F.continuum and FSSC 6 were able to induce cankers on 1–2 year old branches of Z.bungeanum cultivar Doujiao and cutivar Fengjiao.The virulence of F.zanthoxyli and F.continuum on resistant cultivar Fengjiao were significant higher than FSSC 6.2.Sexual reproduction of dominant canker-inducing pathogens of prickly ashMating loci of 11 representative isolates within F.torreyae,F.lateritium,F.buharicum species complex have been mined from whole-genome sequence data via BLASTn algorithm in CLC Genomics Workbench.Search revealed that 6 isolates are MAT1-1 and the other 5 isolates are MAT1-2.Separate synteny comparison of two mating type isolates within 3 Fusarium species complex revealed that MAT1-1-1 and MAT1-2-1 are the most conserved genes of each mating type.Specific PCR primers were designed according to these two conserved genes to screen the mating type.PCR assay indicated that 5 F.torreyae isolates,30 F.zanthoxyli isolates and 5 F.continuum isolates were either MAT1-1 or MAT1-2,which suggested that these three Fusarium species are heterothallic.Further lab crosses between MAT1-1 and MAT1-2 isolates of each species were conducted,all 30 isolates of F.zanthoxyli and 3/5 isolates of F.continuum tested were female fertile,producing abundant mature perithecia of Gibberella.By comparison,only one pairing of the five tested isolates of F.torreyae have the ability to produce perithecia with the majority of the asci aborted.Among the three tested temperatures(22 C,25 C,27 C),the optimal temperature of perithecium production was 22–25 C for F.zanthoxyli,25 C for both F.continuum and F.torreyae.Ascospore isolated from three crosses of F.zanthoxyli,three crosses of F.continuum and one cross of F.torreyae were genotyped,76%–90% progeny of F.zanthoxyli and 81%–85% of F.continuum were recombinant.However,only 36% of the progeny from the F.torreyae cross were recombinant.The success of lab crosses confirmed that F.zanthoxyli,F.continuum and F.zanthoxyli are different biological species.3.Mitochondrial inheritance of dominant canker inducing pathogens of prickly ashPolymorphic regions contained SNPs and InDels were identified by comparative analyses of parental mitogenomes of both F.zanthoxyli and F.continuum.PCR primers were designed flanking the polymorphic regions to test the progeny mitochondrial genotype.Progeny genotype tested from a reciprocal cross of F.zanthoxyli NRRL 66714(MAT1-1)× NRRL 66285(MAT1-2),and single cross of F.continuum ♀ NRRL 66286(MAT1-2)× ♂ NRRL 66218(MAT1-1)revealed maternal uniparental mitochondrial inheritance.In addition,the reciprocal cross of F.zanthoxyli suggested mating type was not involved in mitochondrial inheritance.Research showed that the mitochondrial genomes of both F.zanthoxyli and F.continuum contains 14 polypeptide-encoding genes associated with oxidative respiration,1 intronic ribosomal protein gene(rps3),2 rRNA genes and 26–28 tRNA genes.The gene order in the cicular mitogenomes are identical to other members of Hypocreales.Parental mitogenomes of F.zanthoxyli are 80.9 kb and 98.7 kb in length.In contrast parental mitogenomes of F.continuum are 63.4 kb,which are relatively shorter than F.zanthoxyli.The size difference in these two species are mainly due to variable numbers of introns and ORFs.