Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis .

HomePlant DiseaseVol. 107, No. 2Whole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalis PreviousNext RESOURCE ANNOUNCEMENT OPENOpen Access licenseWhole-Genome Resource of Lasiodiplodia pseudotheobromae BaA, the Causative Agent of Black Root Rot Morinda officinalisXiaoyi Li, Mei Luo, Handa Song, and Zhangyong DongXiaoyi LiInnovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, 510225 Guangzhou, Guangdong, ChinaSearch for more papers by this author, Mei Luo†Corresponding authors: M. Luo; E-mail Address: [email protected], and Z. Y. Dong; E-mail Address: [email protected]https://orcid.org/0000-0002-1950-4204Innovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, 510225 Guangzhou, Guangdong, ChinaKey Laboratory of Fruit and Vegetable Green Prevention and Control in South China, Ministry of Agriculture and Rural Affairs, 510225 Guangzhou, Guangdong, ChinaSearch for more papers by this author, Handa SongInnovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, 510225 Guangzhou, Guangdong, ChinaKey Laboratory of Fruit and Vegetable Green Prevention and Control in South China, Ministry of Agriculture and Rural Affairs, 510225 Guangzhou, Guangdong, ChinaSearch for more papers by this author, and Zhangyong Dong†Corresponding authors: M. Luo; E-mail Address: [email protected], and Z. Y. Dong; E-mail Address: [email protected]https://orcid.org/0000-0001-7524-0226Innovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, 510225 Guangzhou, Guangdong, ChinaKey Laboratory of Fruit and Vegetable Green Prevention and Control in South China, Ministry of Agriculture and Rural Affairs, 510225 Guangzhou, Guangdong, ChinaDeqing Zhongkai Agricultural Technical Innovation Research Co. Ltd., 526600 Zhaoqing, Guangdong, ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Xiaoyi Li1 Mei Luo1 2 † Handa Song1 2 Zhangyong Dong1 2 3 † 1Innovative Institute for Plant Health, Zhongkai University of Agriculture and Engineering, 510225 Guangzhou, Guangdong, China 2Key Laboratory of Fruit and Vegetable Green Prevention and Control in South China, Ministry of Agriculture and Rural Affairs, 510225 Guangzhou, Guangdong, China 3Deqing Zhongkai Agricultural Technical Innovation Research Co. Ltd., 526600 Zhaoqing, Guangdong, China Published Online:31 Dec 2022https://doi.org/10.1094/PDIS-06-22-1507-AAboutSectionsView articlePDFSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat View articleGenome AnnouncementMorinda officinalis F. C. How. (Rutaceae, MO) is widely cultivated in subtropical and tropical areas, including China and Northeast Asia (Zhang et al. 2018). In China, this species is cultivated as a traditional medicinal plant in Guangdong, Guangxi, Fujian, and Hainan provinces. Its roots have been used as a tonic to nourish the kidneys, strengthen the bones, and enhance immune function in the treatment of impotence, osteoporosis, depression, and inflammatory diseases such as rheumatoid arthritis and dermatitis (Cai et al. 2021; Zhang et al. 2018). Since 2018, a serious black root rot disease caused by Lasiodiplodia pseudotheobromae has resulted in significant economic losses by affecting the medicinal quality of M. officinalis (Dong et al. 2019).Lasiodiplodia spp., members of the Botryosphaeriaceae family, is widely found in tropical and subtropical regions (Phillips et al. 2013). L. pseudotheobromae has many hosts globally, with 229 hosts recorded in fungal databases as of 12 May 2022 (Farr and Rossman 2022). It causes many plant diseases, including rot, canker, leaf necrosis, leaf spot, and shoot dieback (Alam et al. 2021; Bao et al. 2021; Chen et al. 2021; Dong et al. 2019).In this study, we sequenced the genome of L. pseudotheobromae BaA strain, which we previously isolated as black root rot pathogens from M. officinalis in Guangdong Province (23°08′36′′N, 111°46′33′′E, Dong et al. 2019). The culture of L. pseudotheobromae BaA strain (ZHKUCC 22-0124) was preserved in the culture collection of Zhongkai University of Agriculture and Engineering, Guangzhou City, Guangdong, China (ZHKUCC). The BaA strain was cultured on PDA medium for 7 days at 28°C. Total DNA was extracted from fresh mycelia using the cetyltrimethylammonium bromide method (Kim et al. 2010). Genome sequencing was performed using a HiSeq 2000 Sequencer (Illumina, San Diego, CA, U.S.A.) operating in 150-bp paired-end mode, with a library insert size of 350 bp. In total, 6.56 million raw reads were generated, from which low-quality data were filtered using fastqc (version 0.11.9) and Trimmomatic tools (version 0.39). After quality filtering, high-quality reads were assembled de novo using Abyss (version 2.0.2), Velvet (version 1.2.10), and SOAPdenovo2 software with various K-mers (Shu et al. 2021). The assembly consisted of 8,846 scaffolds with an N50 value of 1,647,118 bp. The overall G+C content of the strain assembly was 54.58% and the genome coverage was 225×. The completeness of the assembly about gene set was verified with Benchmarking Universal Single-Copy Orthologs (BUSCO, version 5.3.2) (Simão et al. 2015) software, which is based on fungi_odb10, ascomycota_odb10, and dothideomycetes_odb10 database. About 99.2% (752/758), 98.6% (1,683/1,706), and 95.0% (3,596/3,786) of the BUSCOs in full length genes were found in the assembly, respectively, indicating the near-completeness of the assembly. De novo gene prediction was performed from the assembly results using AUGUSTUS version 3.3.3 (Stanke et al. 2006), trained with L. theobromae gene models. We predicted 12,742 protein-encoding genes in the L. pseudotheobromae strain BaA genome (Table 1).Table 1. Genome statistics of Lasiodiplodia pseudotheobromae fungiFeatureStatisticsGenBank assembly accessionJAMJPI000000000GCA_023087545.1GCA_009829805.1StarinBaAKET9CBS 116459HostMorinda officinalisPrunus persicaAcacia mangiumTotal length of sequence (bp)43,699,98645,888,43243,008,851Number of scaffolds8,8463,456403Scaffolds N50 (bp)1,647,118283,597236,232G+C content (%)54.5853.7054.70BUSCO completeness (%)a99.2099.1099.10Number of genes12,74212,84912,869Genome225×151×80×ReferencesThis studyYu et al. 2022Castro-Medina et al. 2014aBUSCO here is based on a set of 758 common fungal genes.Table 1. Genome statistics of Lasiodiplodia pseudotheobromae fungiView as image HTML The results of functional annotation indicated that 12,597 (99.00%), 8,373 (65.80%), 8,985 (70.61%), 8,660 (68.06%), 7,606 (59.78%), 2,513 (19.75%), 773 (6.07%), and 4,944 (38.86%) of the genes were annotated in Nr, Swiss-Prot, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Eukaryotic Orthologous Groups, Cytochrome P450, Carbohydrate-Active Enzymes (CAZy), and PHI-base databases, respectively. To analyze the secreted proteome, we used a series of analysis software tools, including SignalP (version 5.0, Armenteros et al. 2019), TargetP (version 2.0, https://services.healthtech.dtu.dk/service.php?TargetP-2.0), TMHMM (version 2.0, https://services.healthtech.dtu.dk/service.php?TMHMM-2.0), WoLF PSORT (https://wolfpsort.hgc.jp/), ProtComp (version 9.0, http://linux1.softberry.com/berry.phtml?topic=protcomppl&group=programs&subgroup=proloc), and PredGPI predictor (http://gpcr.biocomp.unibo.it/predgpi/pred.htm). A total of 785 proteins were considered components of the secretome, and 294 of these were annotated in CAZy. Of the 785 secreted proteins, 337 were annotated in PHI-base. Of these, 139 were associated with reduced virulence, 19 were effectors, one was related to loss of pathogenicity, 133 were not related to pathogenicity, eight were predicted to increase virulence, and the remaining 37 were annotated as mixed outcomes (Supplementary Table S1).Many studies have shown that secondary metabolites of fungi play important roles in fungi, other microorganisms, and insects (Rohlfs 2015; Zeilinger et al. 2016). We identified 58 key biosynthesis genes that may be associated with secondary metabolites in the genome of the BaA strain, including 29 genes encoding nonribosomal peptide synthase, 19 genes encoding polyketone synthase, nine genes encoding terpene synthase, and one gene encoding betalactone.We compared the genome of BaA with 22 known genomes of other members of the Botryosphaeriaceae family in the National Center for Biotechnology Information (NCBI) database to determine the differences between L. pseudotheobromae and other species (Table 2). Two known L. pseudotheobromae genomes of strain CBS 116459 (GCA_009829805.1, Castro-Medina et al. 2014) and KET9 (GCA_023087545.1, Yu et al. 2022) have been submitted to NCBI. All three L. pseudotheobromae strains are pathogens isolated from symptomatic plants with different hosts. Compared with these three strains’ genomes, strain BaA had a higher scaffold number, longer N50 length, and deeper genome coverage, while having the fewest predictive genes (Table 1). In addition, five BUSCO databases were used to assess the assembly quality of the genome, and the BaA’s assembly was better than the other two (Castro-Medina et al. 2014; Yu et al. 2022; Supplementary Table S2). The BaA genome was compared with known genomes in the NCBI database using the screening criteria of e-value < 1e−20 and identity >99%. The strain BaA of L. pseudotheobromae genome had 10,622 genes in common with the other two L. pseudotheobromae genomes. The remaining 2,120 genes were the differential genes (Supplementary Table S3). Thirty of them were predicted as disease-related effectors (marked in red font in Supplementary Table S3), which may be related to colonization and infection of pathogens on different hosts. For example, BaA_g7118.t1, a secreted aspartic peptidase (SAP2), encodes the protein of Candidapepsin-2, which is a group of 10 acidic hydrolases considered as key virulence factors. These enzymes supply the fungus with nutrient amino acids and are able to degrade the selected host’s proteins involved in the immune defense (Bocheńska et al. 2013). This may be the reason for different host and geography. It was then compared with 20 other genomes from the same family. In total, 657 unique genes were identified in L. pseudotheobromae. Twenty-six of these 657 unique genes were found to be secreted. Of note, a putative hypersensitive response-inducing protein elicitor (BaA_g5863.t1) was identified. Chen et al. (2012) found that the expression of some pathogenesis-related genes and genes involved in signal transduction are induced by an elicitor from Magnaporthe oryzae. Eighty of the 657 unique proteins were annotated in PHI-base. Of these, 29 showed reduced virulence, one was an effector, two were related to loss of pathogenicity, three were related to lethality, and eight were annotated as mixed outcomes. Moreover, 35 proteins were not related to pathogenicity and two showed increased virulence (Supplementary Table S4).Table 2. Details of fungal species used for phylogenetic and genomic comparative analysisSpeciesStrainAssemblyTotal seq. lengthBotryosphaeria agavesFJII-L1-SW-P2GCA_022813555.125,639,012B. dothideasdau11-99GCA_011503125.251,758,482B. kuwatsukaiPG2GCA_004016305.147,871,327Diplodia corticolaCBS 112549GCA_001883845.134,986,079D. intermediaM45-28GCA_021495925.138,134,010D. mutilaCBS 112553GCA_022560015.144,660,741D. sapineaCMW190GCA_000671355.136,053,350D. scrobiculataCMW30223GCA_001455585.134,931,051D. seriataDS831GCA_001006355.137,120,944Lasiodiplodia gonubiensisCBS 115812GCA_009829795.141,142,063L. pseudotheobromaeCBS 116459GCA_009829805.143,008,851L. pseudotheobromaeKET9GCA_023087545.145,888,432L. theobromaeAM2AsGCA_012971845.143,694,574Macrophomina phaseolinaCBS 205.47GCA_022204945.147,085,788M. pseudophaseolinaWAC 2767GCA_022204955.146,619,302Neofusicoccum cordaticolaCBS 123638GCA_009830905.143,555,352N. kwambonambienseCBS 123642GCA_009829855.144,213,875N. laricinumMAFF 410183GCA_022609205.138,802,059N. parvumUCRNP2GCA_000385595.142,592,847N. ribisCBS 121.26GCA_009829435.143,124,176N. umdonicolaCBS 123644GCA_009829365.142,293,999N. dimidiatumUM 880GCA_900092665.142,687,746Table 2. Details of fungal species used for phylogenetic and genomic comparative analysisView as image HTML Comparisons with the 21 genomes of the Botryosphaeriaceae family showed that the number of secondary metabolites within members of the same genus was relatively similar (Supplementary Fig. S1), indicating that the genome had a certain degree of conservation. Similarly, we found a similar number of carbohydrate-active enzymes in members of the same genus (Supplementary Fig. S2), and the distribution of carbohydrate-active enzymes in Macrophomina and Neoscytalidium were highly similar, which is consistent with the phylogenetic tree constructed by Coutinho et al. 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