‘Austalasian Plant Disease Notes (2018) 13:25. Innps//dirg/10.1007/13314-018-0308-3 Ou Lasiodiplodia hormozganensis causing basal stem rot on Ricinus communis in Brazil Fabio Alex Custédio" - Alexandre Reis Machado? « Dartanha José Soares? - Olinto Liparini Pereira Received: 7 May 2018 Aceptet: 13 July 2018 Published online: 29 July 2018 © Australasian Plant Pathelogy Society I. 2018, Abstract, Ricinus communis plants showing symptoms of root and stem rot were observed in the states of Bahia and Paraiba, Brazil. Based ‘on the morphology and phylogenetic analyses of ITS and TEF-1o combined, the causal agents of the observed symptoms were identified as Lasiodiplodia hormozganensis and L. theobromae, pathoge ‘our knowledge, tis isthe first report from any part ofthe world of. orm icity was confirmed by fulfilling Koch’s postulates. To =ganensis causing root and stem rot in R. communis Keywords Borryasphacriales - Biofuel - Phylogeny - Ricinus communis - Stem rot Castor (Ricinus communis) is a non-edible oilseed crop belonging to the family Euphorbiaceae. This erop is of ‘great importance to the chemical industry as a raw mate- rial used in a variety of produets such as lubricants, phar- maceuticals, cosmetics, paints, and plasties, since it is the ‘only commercial source of a hydroxylated fatty acid (Severino et al, 2012), In Brazil, castor is commonly cul tivated in the semiarid areas of the Northeast region, where few diseases have been reported (Severino et all 2012). During a survey of castor diseases in the states of Bahia and Paraiba, castor plants were found showing symptoms of stem, collar, and root rot, eventually resulting in plant demise. Symptoms were usually ob- served on adult plants during capsule maturing stages, despite soil type, but usually more frequent under water- deficit stress. Symptomatic plants were collected in the field, and both roots and basal portion of the stem were 152 Olin Lipari Porica olparini@utcbe Departamento de Fitopatologia, Universidade Federal de Vigoss, Vigosa 3650-000, Brazil * Departamento de Micologia, Universidad Federal de Pernambuco, Recife 50740.600, Bz Bnpres Brasileira de Pesquisa Agropeuiia, Embrapa Algodio, Campina Grande 5828-095, Bra relocated to the Plant Pathology Laboratory of Embrapa Algodio for examination, Black pycnidia with two-celled spores, initially hyaline but turning brown on maturity, ‘were observed suggesting that a Lasiodiplodia sp. was associated with the symptoms. Up to now, only L. ‘theobromae bad been reported causing stem rot and also dieback of eastor plants in Brazil. The dicback is usually a consequence of injury caused during the harvesting of racemes in biannual varieties (Batista et al. 1996; Lima et al. 1997). On the other hand, basal stem and root rot of castor plants in Brazil are usually associated with Macrophomina phaseolina (Severino ct al. 2012; Claudino and Soares 2014). Thus, the aim of the present ‘work was to elucidate the etiology of the Lasiodiplodia sp. associated with basal stem and root rot of castor plants in Brazil Isolation of Lasiodiplodia sp. was secured by direct transfer of the spore mass to Petri dishes filled with Potato Dextrose Agar (PDA), as well as by transferring small tissue fragments obtained from transition areas be- ‘ween healthy and symptomatic tissues. These fragments ‘were disinfected in 70% ethanol for 1 min followed by 1% sodium hypochlorite for 3 min and washed in sterile distilled water. Approximately $0 samples showing stem and root rot symptoms were collected during above- mentioned survey. From those, only six samples resulted in colonies with morphology compatible with Lasiodiplodia sp., while the majority were compatible D Springer25 Page2016 Australasian lant Os, Notes (2018) 13:25. With M. phaseotina. All six isolates obtained were depos- ited in the Colegio de Culturas de Microrganismos, Fitopatogénicos of the Embrapa Algodio (CCMF- CNPA) with accession numbers CCMF-CNPA 0553, CCMF-CNPA 554, CCMF-CNPA. 0555, CCMF-CNPA (0556, CCMF-CNPA 585, and CCMF-CNPA 0603. The isolates of Lasiodiplodia sp. obtained were grown in Petri dishes with 2% Water Agar (WA ~ Agar Agar, type | Himedia®) and incubated at 25 °C for 12 days. Subsequently the growing tips of hyphae of colonies were cet out and transfered to Pet dishes with PDA. The isolates ‘were grovn on Petr dishes containing 2% WA overlaid with triple-strilized com straw and incubated at 25 °C with a pho- toperiod of 12 h to induce the formation of pyenidia and spores. Observations, measurements and photographs were taken on an Olympus BX $3 microscope equipped with a digital camera Q-Color $ Olympus, All isolates had similar morphology: conidiomata stromatic, pyenidial, produced on com straw on WA, superficial, dark, with an apical ostiole, stromatic wall composed of several layers of dark brown, thick-walled cells; conidia elipsoidal to cylindrical, one-celled and hyaline when immature, tuning to dark brown, strate idymospores when mature (Fig. 1), typical of the ge- nus Lasiodiplodia. To extract genomic DNA, the isolates were grown on PDA at 25 °C for 1 week, Approximately 40 mg of mycelia were collected and placed in a 2 mL. Fig. 1 Morphological characteris off. Ihormogamonsi isolates, a colony of hormorganensis on PDA grown inthe dak, b pycnidia with conidia exuded asa ‘ius oncom stra, ¢ Pye leasing conidia immature Iyalinc, becoming pigmented swith age, d conidia pigmented, ‘sept and striate, Bas: 20 um, d= 20 um D springer microcentrifuge tube containing 600 ul of Nuclei Lysis Solution of the Wizard® Genomic DNA Purification Kit (Promega Corporation, WI, U.S.A.), 100 mg of Polyvinylpyrrolidone (PVP; Sigma-Aldrich Co.) and four steel beads. Next, the samples were mixed and crushed in the L-Beader 3 (Loccus Biotecnologia). ‘After maceration, the extraction was continued as de- seribed by Pinho et al. (2012). Target sequences ofthe Internal Transeribed Spacer regions. 1 and 2 including the 5.85 rRNA gene (ITS) and Translation Elongation Factor 1-« (TEF1-a) were amplified using primers ITSI and ITS4 for ITS (White et al. 1990); EFI- 728F (Carbone and Kohn 1999) and EF2R Jacobs et al 2004) or EF1-688F (Alves et al. 2008) and EFI-986R (Carbone and Kohn 1999) for partial TEF I-c. The PCR con- ditions and reagents were the same as those described by Machado et al. (2014). ‘Consensus sequences were compared against GenBank’s database using their Mega BLAST program for a firs identi- fication. The ITS and TEF1-« sequences of additional species were retrieved from GenBank (Table 1) and aligned with se- {quences generated in this study using the multiple sequence alignment MUSCLE® program (Falgar 2004), an application Of the MEGA v. 6 software program (Tamura et al. 2013). Alignments were checked, and manual adjustments were male where necessary. Bayesian Inference (IB) analyses of the concatenated ‘gene regions were performed as described by Machado-Astalasian Pant Dis Notes (2018) 13:25, Page3of6_25 Table 1 Isolate number, lesion, hostaubstate and genbank accession numbers of DNA sequences of Lasidpldia spp used in phylogenetic analyses Species Isolates Location HosvSubsrate Reference Genbank aesson number MS TEFF-« “Lasioipladia trastense MNOS Baw! Mangere inliea Neto etal 2014 IXMGHDG—_IXA6AO49 E beasiione cx Bruit Carica papava Neto etal 2014 KCAHTI | KCHRIS2S E. catinguensis ‘L40 Bruit Spondis pupuca (Coutn etal. 2016 KTIS¢762 | KTISATSS| E.caatinguensis exon 1325 Bail Cir sinensis (Couto etal. 2016 Kris760 TO Lctricola IRANIS2IC fan Carus sp ANbdoshndeh etl. 2010 GUMS3S3_GUIIS339 Le ciricola RANIS22¢ inn Carus 9. ‘Nbdolsndeh etal. 2010 GUMSIS¢ GU9NS340 1 enaspora cstiows2 Asean Unknown Burges ea 2006 FROGS ER622066 1 erassipora CMW265)—Aualin Preocars angolensis args etal, 2006 FIBRES FIBRES? E egpiacae CBSI3092 ype ‘Manger nea Ismail etal 2012 INSI4397 INSI4#24 © axpiacae BOT29 Et Manger nde Ismail etal 2012 INsI4IOL INS1428 L aphorbicols cxn3651 Baal arp cures Machado etal 2018 KF2H553._KF226711 L aphorbicola cexin3652 Baal “ropa cures Machado etal 2018 KA2H554KF226715 L.ephorbicla cxn3609 Brat “ropa cures Machado etal 2018 KAZHS43 _KF226609 1 exiua CBS 137785 Tania Retama ractom inaldedd ea. 2018 KGS KJG3K336 1 exiua BL st Tunis Retama ration inal et a. 2018 KGS KJGIK337 planers IRANIS23C an Unksowe Alboltzadeh etal. 2010 GUMS3S1_GU9KS342 L planensis IRANISOIC an Unksowe Abdolsaadeh etal 2010 GUMS3S? _GUSKS341 L. gomubensis CBSIISKI2 South Afica Sint condanum ——_—Pavicctal. 20 | DDQESHKH2 _DOSSKATT 1 grvisrana Can 45654 Brat ‘Anacandion mile Neto. 2016 7250049 KT250050 1 gravis Can 5655 Bruit ‘anacandion haile Neos a. 2016 1250047 | KT265612, TL hormorganenss——IRANISOOC Ian (Ola sp. Abdoldvadeh etal. 2010 GUMS3SS _GUMS343 Lhormorganowss—IRANL@SC Ian Mangere ili ANbdolzadeh etal. 2010 GUSS3S6GUSMS36 E hormerganents ——CCME-CNPA 0$$3. Brut icin commas ‘This say MG87050. MG806911 LE hormocgancns ——CCME-CNPA 0555. Brut ‘Reus commun ‘This say MGTS7091 MG306012 L hormocganens —— CCMECNPA 0403. Brut ‘Reins communis ‘This suds MGTS7002 -MGE06913 LL hormorganomis——CCMP-ENPA.0556 Brat ‘Ricinus communis ‘Ths sy MG7s7083 MGs0eO14 LL inanionic IRANISITC Inn Cir 5p Abolseadeh eta. 2010 GUSMSM9_GU9S337 L irtonts IRANISIC Inn Manger indica Abdolradeh etal. 2010 GUMS3S0_GUBMS338 E jatrplecole canc6t0 Brat dcropha crt Machado tl 2014 KFDASE4KF226690 L jarophicola cunt 4472 Baal Anacardiam occidentale Neto eal. 2014 KT25572_KTIOSS85| Ltaelociteyve BS 16728 ly Tcloateyae Rodrigue Galvez etal 2016 KUSOT462 KUSOTA29 1 taetocateyne LAREPL Peru ‘Manger nda Roviigvez-Gilvezetal 2016 KUSOTARS KUSOTAS1 1. marganiacea cusiz2s9, Attain ‘Adansonia gibbosa—_—_‘Pavicet al. 2008 u1440so ura 1. macrospora| Cains Brat aropha cars| Machado etl 2018 KF23ASS7._KF226718 1. mahajiangana CMW27801———Madugncar_ Terminal cata ——_—Bagoute etal. 2010, F900595 FISHO6SI 1. mahajiangana CMW27K20 Madagascar ——Terminaiacaappa———_Begoute ea. 2010 FI900397 — FIsOO6E E medteranea CAS I3783 aly ues ee Linaldedd etal. 2014 KU63312 — KIG38331 E mediterranea cBs7784 aly Mis aera inal eta. 2014 RUGS KUGGRI30 E miscuriana UCD2I93MO «USA Vii tea Ube Tomes etal 2011 HQD8K225 | HODRS267 1. miscuriana UCD2I99MO USA Wiis wnt Urbee Tomes etal 2011 HQD8K226 HQDRK768 L parwe cssis678 Colombia Cassin fl si Alves ea 2008 ERODES EFS22063 Cpe casins.78 Colorbia Caswell Alves et al 2008 FROGS — ERS2206S plate STE-US8OR South Afica Wis wines Dasa ta 2007 PRUs ERUSI9S pene cMMi277 Baa Spondissprpurea Cato etal, 2016 KTISI?N | KTISITL Lpeudotheobromae — CBSIIG4S9 Cost Rica Gretna arborea Alves et al. 2008 ERGOT EFG2I0S7 LL peudotheobromae — CMMBES7 Baal arpa carcas Machado eta, 2014 KF2HS59. | KF226722 L pmiformis: CBS 1217 Namibia ‘acacta mellfo Slippers tal 2014 EUIOISO7 EUIOISS2, L pmiformis: Bs 21771 Namibia ‘Acacia mellfra Slippers etal 2014 EUIOIS08 EUIONSSS| Lrubropurpurea—— WACI25%6, ‘Avwiaie Eucalp grandis ‘Barges ta 2006 DQWUSSSs DO1OIST2 1 subgiobosa cuss Brat Latropha cares, Machado ta 2018 KF234558 KF226721 L subglobosa CaN06 Baal “arp cars Machado et 2014 KFDH4S6OKE226723 Ethan cre 22755 Thad Pilantus acidus ——Tranyingceroen ea. 2014 KMOO6433KMOV6A64 haan crenns Thailand ‘Manger nea ‘Trakunyingcharoen tal 2014 KII93637— KIIO3681 . theobromae ‘cHS16496 Papua New Guinea Fait along core oust Pils al. 2008 AVGHRSS AYGHDSS 1. theobromae csi28.13 USA. Unknown Alves ea 2006 DOssHHHO_DQESEETS 1 theobromae cusitiso Unknown Unknown ‘Aves al. 2008 ERRDIUTS FG2I084 L beobranae CCME-CNPA.0SS4 Reval icin commas ‘This soy Mrlags304 MHG91477 1 theabromae CCMECNPA 0585. Bri icin canis ‘This sy MHia8S395. M4147 E venecttosis CMWI3SIS Veneta ‘Acacia mangium Burges a. 2006, DQ!Ess:9 _DO103s70 E venesulosis wacirs3» Venemela ‘Acaca mangium Burges tal 2006 DQLGRS!T _DO103S6s D Springer28 Page dot Australasian lant Os, Notes (2018) 13:25. ‘Table (continved) Species Isolates Location HostSubstrate Reference ‘Gentnk aession umber MS TEFI-« 1 ico UUCDISSSAR USA Vai ira Grbee-Tomes etal 2011 HQD8K227HODNKZOD 1. wicola ucDas0MO USA Wei infra Hqpsx228 1HQ288070 Dipl mata CRS 36015 Portugal Pops eas KIKI KI361830 {TS interval transcribed spacer regions | and 2, including the $8 ribosomal RNA gene, TEFI-ctaslation elongation ctor I-e €t al, (2014). The K80+I evolution models for the ITS region were used in the analyses while HKY +1+G was selected for TEFI-a. The resulting trees were visualized in the Figtree v.1.3.1 program (Rambaut 2009) and later exported to graphic programs. Based on the results of combined phylogenetic analyses of ITS and TEF-l« gene regions, four isolates were identified as Lasiodiplodia hormozganensis, since they grouped with the type isolate of L. hormozganensis (IRAN1SOOC), ‘and two isolates were identified as L. theobromae, the tree was rooted to Diplodia mutila (CBS 136015) (Fig. 2), For the pathogenicity test (Fig. 3), castor plants of BRS Energia cultivar, approximately 3 months old ‘grown in a greenhouse, were inoculated with isolates obtained during the course of this study, using five plants per isolate. For inoculation, the isolates were ‘grown in petri dishes with PDA at 25 °C for 7 days. Healthy plants had their stalks wounded with a steri- ized scalpel and a 6-mm-diameter PDA disk containing mycelium of each isolate was deposited on the wound. A portion of cotton moistened with autoclaved distilled ‘water was placed on cach disk and then covered with plastic film for the first 48 h after inoculation to. main- tain moisture. The control treatment consisted of PDA plugs, with no fungus grown, deposited on the injured stalks as deseribed above. The plants were then kept in 4 greenhouse for 30 days. All inoculated plants showed ‘a neerosis and constriction on inoculation area and a severe wilt symptom. The fungus was re-isolated from the all inoculated plants and its identity was confirmed as previously described thereby satisfying Koch's Postulates. No fungus was re-isolated from control plants. ‘As above-mentioned, L. theobromae was already known to be associated to stem rot of castor plants in Brazil. On the other hand, L. hormozganensis. was de- scribed in Iran associated with Olea sp. and Mangifera indica (Abdollahzadeh et al. 2010). In Brazil, this spe- cies has been reported to cause dieback in mango and ‘grape, and stem-end rot in mango and papaya in the D springer Fig. 2 Mullocus phylogenetic wee of Lasiolplodia species infeed from Bayesian analysis based on the combined sequences of the ITS and TEF-1a, Bayesian posterior probable are indicated above the odes and the mee was roted to Diplodia mula (CBS 136015). The ‘ar represents the number of changes in the nuclootde sequence of ‘ach 100 bp, The species in his stay ae highligh in bold-Astalasian Pant Dis Notes (2018) 13:25, PageSof6_25 Fig. 3 Pathogenicity text om ‘communis, a conto, b plant inoculated with Lasodipodia ormosganensi 2013; Thus, the results Northeast region of the country (Marques et al Netto et al. 2014; Correia et al. 2015). of this work suggest this is the first report of L. hormozganensis causing basal stem and root rot on R. ‘Acknowledgments The authors wish to thank Coordenagao de Apereigaaments de Pesca de Nivel Superior, Conselho Nacional de Desenvolvimento Cienlificoe Teenoligico and Fundagio de Amparo & Pesquisa do Estado de Minas Gerais for Financial support References Alsollahradch vad A, Mohansmad-Goltpch E, Zar R, Philips AIL (2010) Phylogeny and morphology of four new species of Lasiipla fom rn, Persona 25:1-10 Alves A, Correia A, Philips AJL (2006) Muli-gene genealogies and ‘morphological data suppoetDipladia cypress sp. nov recognize a D. pine sp. cress 8 & distinc species, Fungal Divers 23:1-15 [Alves A. 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