Genome Genome and transcriptome analysis of the latent pathogen Lasiodiplodia theobromae, an emerging threat to the cacao industry Journal: Genome Manuscript ID gen-2019-0112.R1 Manuscript Type: Article Date Submitted by the 05-Sep-2019 Author: Complete List of Authors: Ali, Shahin; Sustainable Perennial Crops Laboratory, United States Department of Agriculture Asman, Asman; Hasanuddin University, Department of Viticulture & Enology Draft Shao, Jonathan; USDA-ARS Northeast Area Balidion, Johnny; University of the Philippines Los Banos Strem, Mary; Sustainable Perennial Crops Laboratory, United States Department of Agriculture Puig, Alina; USDA/ARS Miami, Subtropical Horticultural Research Station Meinhardt, Lyndel; Sustainable Perennial Crops Laboratory, United States Department of Agriculture Bailey, Bryan; Sustainable Perennial Crops Laboratory, United States Department of Agriculture Keyword: Cocoa, Lasiodiplodia, genome, transcriptome, effectors Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 46 Genome 1 Genome and transcriptome analysis of the latent pathogen Lasiodiplodia 2 theobromae, an emerging threat to the cacao industry 3 4 Shahin S. Ali1,2, Asman Asman3, Jonathan Shao4, Johnny F. Balidion5, Mary D. Strem1, Alina S. 5 Puig6, Lyndel W. Meinhardt1 and Bryan A. Bailey1* 6 7 1Sustainable Perennial Crops Laboratory, USDA/ARS, Beltsville Agricultural Research Center-West, 8 Beltsville, MD 20705, USA. 9 2Department of Viticulture & Enology, University of California, Davis, CA 95616 10 3Department of Plant Pests and Diseases, Hasanuddin University, South Sulawesi, Indonesia. 11 4USDA/ARS, Northeast Area, Beltsville, MDDraft 20705, USA. 12 5 Institute of Weed Science, Entomology and Plant Pathology, University of the Philippines, Los Banos, 13 Laguna 4031, Philippines. 14 6Subtropical Horticultural Research Station, USDA/ARS, Miami, FL 33158, USA 15 16 17 18 *Corresponding author: 19 Phone: 1-301-504-7985; Fax: 1-301-504-1998 20 E-mail: [email protected] 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 46 21 22 Abstract 23 Lasiodiplodia theobromae (Ltheo), a member of the Botryosphaeriaceae family, is becoming a 24 significant threat to crops and woody plants in many parts of the world, including the major 25 cacao growing areas. While attempting to recover Ceratobasidium theobromae, causal agent of 26 vascular streak dieback (VSD), from symptomatic cacao stems, 74% of recovered fungi were 27 Lasiodiplodia spp. Sequence-based identification of 52 putative Lasiodiplodia isolates indicates 28 that diverse Lasiodiplodia species are associated with cacao in the studied areas, and the isolates 29 showed variation in aggressiveness when assayed using cacao leaf discs. The current study 30 reports on the 43.75 Mb de novo assembledDraft genome of a Ltheo isolate from cacao. Ab initio gene 31 prediction has generated 13,061 protein-coding genes, of which 2,862 are unique to Ltheo, when 32 compared to other closely related Botryosphaeriaceae fungi. Transcriptome analysis revealed 33 that 11,860 predicted genes were transcriptionally active and 1,255 were more highly expressed 34 in planta compare to cultured mycelia. The predicted genes differentially expressed during 35 infection were mainly those involved in carbohydrate, pectin and lignin catabolism, cytochrome 36 P450s, necrosis-inducing proteins and putative effectors. These findings significantly expand our 37 knowledge of the Ltheo genome and Ltheo genes involved in virulence and pathogenicity. 38 Keywords: Cocoa, Lasiodiplodia, genome, transcriptome, effectors 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 46 Genome 39 40 Introduction 41 Lasiodiplodia theobromae (Pat.) Griffon & Maubl., (Ltheo), a member of the 42 Botryosphaeriaceae family, is often considered a latent plant pathogen attacking more than 500 43 plant species in the tropics and subtropics (Burgess et al. 2006; Slippers and Wingfield 2007). 44 The significance of disease caused by Ltheo appears to be increasing in many parts of the world, 45 perhaps in association with global climate change. Environmental factors like temperature and 46 drought are known to influence the interactions between Ltheo and their plant hosts (Paolinelli- 47 Alfonso et al. 2016; Yan et al. 2017; Songy et al. 2019). The effects of climate change on cacao, and 48 the tropics in general, are of increasing Draftconcern (Medina and Laliberte 2017). Theobroma cacao 49 L. (cacao), the source of chocolate, is the major source of income for six million farmers located 50 around the world in tropical climates (World Cocoa Foundation 2014). Most cacao farmers have 51 small plots of land and many suffer major income losses due to destructive diseases (Ploetz 52 2016). Though Ltheo was first reported to cause pod rot and dieback in cacao in 1923 (Nowell 53 1923), it was never considered as a major pathogen of cacao. However, Ltheo has been 54 suggested as a significant constraint for cacao production in some locations, and isolates from 55 symptomatic tissues can cause stem cankers and diebacks when artificially inoculated onto cacao 56 tissues (Mbenoun et al. 2008; Alvindia and Gallema 2017; del Castillo et al. 2016). Typical 57 symptoms on cacao caused by Ltheo can resemble those of other diseases and there is 58 speculation concerning associations of Ltheo with other cacao pathogens, such as canker caused 59 by Phytophthora species (Jaiyeola et al. 2014) and vascular streak dieback (VSD) caused by 60 Ceratobasidium theobromae (Alvindia and Gallema 2017; McMahon and Purwantara 2016). 61 Alvindia and Gallema (2017) reproduced many of the symptoms commonly associated with 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 46 62 VSD on cacao seedlings by inoculating the young leaves with Ltheo. Symptoms included leaf 63 chlorosis and necrotic blotches and leaf scar and stem vascular discoloration. 64 How Ltheo causes disease on such a wide range of host is a question of considerable 65 interest. To establish an infection, Ltheo and related members of the family Botryosphaeriaceae 66 must overcome both preformed and inducible host defenses (Yan et al. 2017), which can vary 67 significantly among hosts. Recently published draft genomes of woody plant pathogens in the 68 Botryosphaeriaceae have provided information about a range of potential virulence factors such 69 as effectors and cell wall modifying enzymes (Blanco-Ulate et al. 2013; Morales-Cruz et al. 70 2015; Paolinelli-Alfonso et al. 2016; van der Nest et al. 2014; Yan et al. 2017). These studies 71 reported the presence of expanded gene families associated with cell wall degradation, 72 membrane transport, nutrient uptake andDraft secondary metabolism, which contribute to adaptations 73 for degrading grapevine tissue (Yan et al. 2017). Although the genome and transcriptome 74 analysis of Ltheo strains pathogenic to grapevine, along with other, mostly grapevine-associated, 75 Botryosphaeriaceae species, has provided a better understanding of Ltheo biology, the study of a 76 strain pathogenic on cacao would help our understanding of how widespread some basic aspects 77 of Ltheo biology are. 78 In this study, we describe the genome and transcriptome of a Ltheo isolate AM2A which 79 was isolated from a cacao stem showing symptoms of vascular streak dieback. The genome of 80 Ltheo isolate AM2As is compared to the genomes of closely related Botryosphaeriaceae 81 pathogens. Expressed genes within the Ltheo genome are characterized through RNA-Seq 82 analysis using infected cacao leaves, and potential effectors expressed during the infection 83 process are identified. These insights add to our understanding of the Ltheo genome and 84 transcriptome. 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 46 Genome 85 Material and method: 86 Isolation and maintenance of fungi 87 Cacao stems showing symptoms of vascular streak dieback (VSD) were collected from Wotu 88 District of South Sulawesi Province, Indonesia (Coordinate point S: 02°33'33.30" E: 89 120°47'51.74", Elevation 39 m) and Davao Region of Philippines between 2014 and 2016. 90 Samples were shipped to USDA-APHIS-PPQ facility at Beltsville, USA and transferred to 91 USDA-ARS Sustainable Perennial Crops Laboratory in Beltsville after inspection. Bark was 92 removed and remaining stem material was cut into one cm long segments. Stem segments were 93 surface sterilized by submerging in 6% (v/v) bleach solution (Clorox, USA) for three minutes 94 followed by three rinses in sterile water.Draft Stem segments were placed on 1.5% water agar (Difco 95 Laboratories, USA) in 100 mm diameter plastic Petri plates and incubated at 25oC under dark. 96 After 2-3 days, stem segments were examined for hyphal growth from the stem segment in 97 contact with the agar. Fungal mycelia were transferred from water agar to Corticium Culture 98 Media (Samuels et al. 2012) containing 100 µg/ml ampicillin. Note the procedure to this point is 99 typically used when attempting to isolate C. theobromae, causal agent of vascular streak dieback 100 (VSD), from cacao tissues and other organisms are commonly isolated from tissues showing 101 symptoms of VSD. Subsequently, fungal isolates were transferred to 20% clarified V8 (CV8)- 102 agar plates and maintained at room temperature. 103 Molecular identification of Lasiodiplodia spp. 104 DNA Extraction 105 For DNA extraction, 2-3 agar plugs (0.25 cm2) from 5 day old cultures of each isolate were 106 transferred to 50 ml falcon tubes containing 20 ml liquid CV8 and grown at room temperature 5 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 46 107 for 5 days, while shaking at 100 rpm. Mycelia were harvested and DNA was extracted as 108 previously described by Ali et al. (2016). 109 PCR amplification and DNA sequencing of ITS region and EF1α gene 110 For molecular identification and phylogenetic analysis, PCR amplification of the ITS region and 111 elongation factor 1-alpha (EF1α) of the template DNA from 52 Lasiodiplodia isolates was 112 performed using the primers ITS4 and ITS5 described by White et al.