Abstract
Neonectria ditissima is the causal agent of apple canker. Here, we present the draft genome sequences of two isolates of N. ditissima that differ in virulence. Comparative genomics will enable pathogenicity determinants to be identified in this plant-pathogenic fungus.
GENOME ANNOUNCEMENT
Neonectria ditissima (Tul. & C. Tul.) Samuels & Rossman (synonym Neonectria galligena) is a fungal pathogen causing canker disease in many broad-leaved trees, including iconic Northern Hemisphere forest and specimen trees and the horticultural genera Malus (apple) and Pyrus (European pear [1, 2]). In these fruit trees, the disease in apple (European canker) is especially severe and is of importance to growers across both the Northern and Southern Hemispheres, although it is particularly damaging in mild, wet climates (3, 4). Apple canker is currently controlled by pruning and fungicides. Different apple varieties show different susceptibilities to canker, hinting at the possibility of breeding a highly tolerant or resistant cultivar (5). Variation is also seen in the pathogen population. At the level of host range determination, formae speciales probably exist within N. ditissima sensu lato (1), and variations in virulence (6) and conidial morphology (7) are seen within the populations of N. ditissima able to infect apple. Comparative genomics of isolates varying in virulence against apple may provide details on which pathogenicity determinants are essential for virulence and pathogen fitness.
Thus, two single-ascospore isolates of N. ditissima from New Zealand were selected for whole-genome sequencing: RS305p, isolated from Malus x domestica cv. ‘Brookfield,’ a naturally occurring isolate with very low virulence; and RS324p, isolated from M. x domestica cv. ‘Golden Delicious,’ a virulent isolate (6). For each of the isolates, genomic DNA was extracted from axenic in vitro-grown fungal material. Two paired-end libraries (insert sizes, 180 bp and 400 bp) and two mate-pair libraries (5 kb and 10 kb) were constructed for each isolate and sequenced on the Illumina HiSeq 2000 platform. The genomes were estimated to be 45 Mb in size, based on the average size of related Fusarium genomes (8), giving a sequence depth >200× for both isolates. The de novo assembly was carried out using Platanus-1.2.1 (9) and scaffolded with SSPACE version 2.0 (10). Gap filling was performed with GapCloser version 1.12 in the SOAP Tools package (http://soap.genomics.org.cn/soapdenovo.html). For isolate RS305p, the assembled draft genome was 43.56 Mb in size, consisting of 189 scaffolds, with an N50 metric of 1.17 Mb and a longest scaffold of 5.5 Mb, while the genome of isolate RS324p was 44.95 Mb in size, consisting of 172 scaffolds, with an N50 metric of 1.81 Mb and a longest scaffold of 4.05 Mb. To check the completeness of the genome assemblies, CEGMA version 2.4.010312 (11) was run, and both assemblies were >95.5 percent complete. All of the N. ditissima (n = 107) and N. galligena (n = 83) sequences available from NCBI were correctly assembled in both genomes. Using AUGUSTUS version 3.0.3 (12) with the Fusarium model, 12,561 protein-coding genes were predicted for both isolates, and 471 and 461 tRNAs were detected using tRNAscan-SE in the RS305p and RS324p genomes, respectively (11). Among the predicted proteins, a search of the Pathogen-Host Interactions database (PHI-base [13]) identified those with similarity to two effectors (avirulence determinants) and six proteins with a mutant phenotype of hypervirulence in both isolates.
Nucleotide sequence accession numbers.
This whole-genome shotgun project of N. ditissima has been deposited at DDBJ/EMBL/GenBank under BioProject PRJNA285413 with the accession no. LDPK00000000 for isolate RS305p and LDPL00000000 for RS324p. The version described in this paper is version 1.0.
ACKNOWLEDGMENTS
This work was supported by The New Zealand Institute for Plant & Food Research Limited.
Sequencing was carried out by Macrogen, South Korea. We thank Erik Rikkerink and Mark Andersen for critically reviewing the manuscript.
Footnotes
Citation Deng CH, Scheper RWA, Thrimawithana AH, Bowen JK. 2015. Draft genome sequences of two isolates of the plant-pathogenic fungus Neonectria ditissima that differ in virulence. Genome Announc 3(6):e01348-15. doi:10.1128/genomeA.01348-15.
REFERENCES
- 1.Flack NJ, Swinburne TR. 1977. Host range of Nectria galligena Bres. and the pathogenicity of some Northern Ireland isolates. Trans Br Mycol Soc 68:185–192. doi: 10.1016/S0007-1536(77)80007-7. [DOI] [Google Scholar]
- 2.Farr DF, Bills GF, Chamuris GP, Rossman AY. 1989. Fungi on plants and plant products of the United States. The American Phytopathological Society, St. Paul, MN. [Google Scholar]
- 3.Beresford RM, Kim KS. 2011. Identification of regional climatic conditions favorable for development of European canker of apple. Phytopathology 101:135–146. doi: 10.1094/PHYTO-05-10-0137. [DOI] [PubMed] [Google Scholar]
- 4.Grove GG. 1990. Nectria canker, p 34–45. In Jones AL, Aldwinckle HS (ed), Compendium of apple and pear diseases. The American Phytopathological Society, St. Paul, MN. [Google Scholar]
- 5.Weber RWS. 2014. Biology and control of the apple canker fungus Neonectria ditissima (syn. N. galligena) from a Northwestern European perspective. Erwerbs Obstbau 56:95–107. doi: 10.1007/s10341-014-0210-x. [DOI] [Google Scholar]
- 6.Scheper RWA, Frijters L, Fisher BM, Hedderley D. 2015. Effect of freezing of Neonectria ditissima inoculum on its pathogenicity. N Z Plant Protect 68:257–263. [Google Scholar]
- 7.Scheper RWA, Fisher BM, Amponsah NT, Walter M. 2014. Effect of culture medium, light and air circulation on sporulation of Neonectria ditissima. N Z Plant Protect 67:123–132. [Google Scholar]
- 8.Ma L-J, van der Does HC, Borkovich KA, Coleman JJ, Daboussi M-J, Di Pietro A, Dufresne M, Freitag M, Grabherr M, Henrissat B, Houterman PM, Kang S, Shim W-B, Woloshuk C, Xie X, Xu J-R, Antoniw J, Baker SE, Bluhm BH, Breakspear A, Brown DW, Butchko RAE, Chapman S, Coulson R, Coutinho PM, Danchin EGJ, Diener A, Gale LR, Gardiner DM, Goff S, Hammond-Kosack KE, Hilburn K, Hua-Van A, Jonkers W, Kazan K, Kodira CD, Koehrsen M, Kumar L, Lee Y-H, Li L, Manners JM, Miranda-Saavedra D, Mukherjee M, Park G, Park J, Park S-Y, Proctor RH, Regev A, Ruiz-Roldan MC, Sain D, Sakthikumar S, Sykes S, Schwartz DC, Turgeon BG, Wapinski I, Yoder O, Young S, Zeng Q, Zhou S, Galagan J, Cuomo CA, Kistler HC, Rep M. 2010. Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 464:367–373. doi: 10.1038/nature08850. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kajitani R, Toshimoto K, Noguchi H, Toyoda A, Ogura Y, Okuno M, Yabana M, Harada M, Nagayasu E, Maruyama H, Kohara Y, Fujiyama A, Hayashi T, Itoh T. 2014. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res 24:1384–1395. doi: 10.1101/gr.170720.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Boetzer M, Henkel CV, Jansen HJ, Butler D, Pirovano W. 2011. Scaffolding pre-assembled contigs using SSPACE. Bioinformatics 27:578–579. doi: 10.1093/bioinformatics/btq683. [DOI] [PubMed] [Google Scholar]
- 11.Parra G, Bradnam K, Korf I. 2007. CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes. Bioinformatics 23:1061–1067. doi: 10.1093/bioinformatics/btm071. [DOI] [PubMed] [Google Scholar]
- 12.Stanke M, Schöffmann O, Morgenstern B, Waack S. 2006. Gene prediction in eukaryotes with a generalized hidden Markov model that uses hints from external sources. BMC Bioinformatics 7:62. doi: 10.1186/1471-2105-7-62. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Urban M, Pant R, Raghunath A, Irvine AG, Pedro H, Hammond-Kosack K. 2015. The Pathogen-Host Interactions database (PHI-base): additions and future developments. Nucleic Acids Research 43:D645–D655. doi: 10.1093/nar/gku1165. [DOI] [PMC free article] [PubMed] [Google Scholar]