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Lasiodiplodia hormozganensis causing basal stem rot on Ricinus communis in Brazil

  • Fábio Alex Custódio
  • Alexandre Reis Machado
  • Dartanhã José Soares
  • Olinto Liparini PereiraEmail author
Article
  • 628 Downloads

Abstract

Ricinus communis plants showing symptoms of root and stem rot were observed in the states of Bahia and Paraíba, Brazil. Based on the morphology and phylogenetic analyses of ITS and TEF-1α combined, the causal agents of the observed symptoms were identified as Lasiodiplodia hormozganensis and L. theobromae, pathogenicity was confirmed by fulfilling Koch’s postulates. To our knowledge, this is the first report from any part of the world of L. hormozganensis causing root and stem rot in R. communis.

Keywords

Botryosphaeriales Biofuel Phylogeny Ricinus communis Stem rot 

Castor (Ricinus communis) is a non-edible oilseed crop belonging to the family Euphorbiaceae. This crop is of great importance to the chemical industry as a raw material used in a variety of products such as lubricants, pharmaceuticals, cosmetics, paints, and plastics, since it is the only commercial source of a hydroxylated fatty acid (Severino et al. 2012). In Brazil, castor is commonly cultivated in the semiarid areas of the Northeast region, where few diseases have been reported (Severino et al. 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 observed 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 relocated to the Plant Pathology Laboratory of Embrapa Algodão 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 had been reported causing stem rot and also dieback of castor plants in Brazil. The dieback 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 et 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 between 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 50 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 with M. phaseolina. All six isolates obtained were deposited in the Coleção de Culturas de Microrganismos Fitopatogênicos of the Embrapa Algodão (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 I Himedia®) and incubated at 25 °C for 12 days. Subsequently, the growing tips of hyphae of colonies were cut out and transferred to Petri dishes with PDA. The isolates were grown on Petri dishes containing 2% WA overlaid with triple-sterilized corn straw and incubated at 25 °C with a photoperiod of 12 h to induce the formation of pycnidia and spores. Observations, measurements and photographs were taken on an Olympus BX 53 microscope equipped with a digital camera Q-Color 5 Olympus.

All isolates had similar morphology: conidiomata stromatic, pycnidial, produced on corn 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, turning to dark brown, striate didymospores when mature (Fig. 1), typical of the genus Lasiodiplodia.
Fig. 1

Morphological characteristics of L. hormozganensis isolates, a colony of L. hormozganensis on PDA grown in the dark, b pycnidia with conidia exuded as a cirrus on corn straw, c Pycnidia releasing conidia immature hyaline, becoming pigmented with age, d conidia pigmented, septate and striate. Bars: c = 20 μm, d = 20 μm

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 microcentrifuge tube containing 600 μL 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 described by Pinho et al. (2012).

Target sequences of the Internal Transcribed Spacer regions 1 and 2 including the 5.8S rRNA gene (ITS) and Translation Elongation Factor 1-α (TEF1-α) were amplified using primers ITS1 and ITS4 for ITS (White et al. 1990); EF1-728F (Carbone and Kohn 1999) and EF2R (Jacobs et al. 2004) or EF1-688F (Alves et al. 2008) and EF1-986R (Carbone and Kohn 1999) for partial TEF1-α. The PCR conditions 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 first identification. The ITS and TEF1-α sequences of additional species were retrieved from GenBank (Table 1) and aligned with sequences generated in this study using the multiple sequence alignment MUSCLE® program (Edgar 2004), an application of the MEGA v. 6 software program (Tamura et al. 2013). Alignments were checked, and manual adjustments were made where necessary.
Table 1

Isolate number, location, host/substrate and genbank accession numbers of DNA sequences of Lasiodiplodia spp. used in phylogenetic analyses

Species

Isolates

Location

Host/Substrate

Reference

Genbank acession number

ITS

TEF1-α

Lasiodiplodia brasiliense

CMM4015

Brazil

Mangifera indica

Netto et al. 2014

JX464063

JX464049

L. brasiliense

CMM2313

Brazil

Carica papaya

Netto et al. 2014

KC484793

KC481524

L. caatinguensis

IBL 40

Brazil

Spondias purpurea

Coutinho et al. 2016

KT154762

KT154755

L. caatinguensis

CMM 1325

Brazil

Citrus sinensis

Coutinho et al. 2016

KT154760

KT008006

L. citricola

IRAN1521C

Iran

Citrus sp.

Abdollahzadeh et al. 2010

GU945353

GU945339

L. citricola

IRAN1522C

Iran

Citrus sp.

Abdollahzadeh et al. 2010

GU945354

GU945340

L. crassispora

CBS110492

Australia

Unknown

Burgess et al. 2006

EF622086

EF622066

L. crassispora

CMW22653

Australia

Pterocarpus angolensis

Burgess et al. 2006

FJ888465

FJ888452

L. egyptiacae

CBS130992

Egypt

Mangifera indica

Ismail et al. 2012

JN814397

JN814424

L. egyptiacae

BOT-29

Egypt

Mangifera indica

Ismail et al. 2012

JN814401

JN814428

L. euphorbiicola

CMM3651

Brazil

Jatropha curcas

Machado et al. 2014

KF234553

KF226711

L. euphorbiicola

CMM3652

Brazil

Jatropha curcas

Machado et al. 2014

KF234554

KF226715

L. euphorbicola

CMM3609

Brazil

Jatropha curcas

Machado et al. 2014

KF234543

KF226689

L. exigua

CBS 137785

Tunisia

Retama raetam

Linaldeddu et al. 2014

KJ638317

KJ638336

L. exigua

BL 184

Tunisia

Retama raetam

Linaldeddu et al. 2014

KJ638318

KJ638337

L. gilanensis

IRAN1523C

Iran

Unknown

Abdollahzadeh et al. 2010

GU945351

GU945342

L. gilanensis

IRAN1501C

Iran

Unknown

Abdollahzadeh et al. 2010

GU945352

GU945341

L. gonubiensis

CBS115812

South Africa

Syzygium cordatum

Pavlic et al. 2004

DQ458892

DQ458877

L. gravistriata

CMM 4564

Brazil

Anacardium humile

Netto et al. 2016

KT250949

KT250950

L. gravistriata

CMM 4565

Brazil

Anacardium humile

Netto et al. 2016

KT250947

KT266812

L. hormozganensis

IRAN1500C

Iran

Olea sp.

Abdollahzadeh et al. 2010

GU945355

GU945343

L. hormozganensis

IRAN1498C

Iran

Mangifera indica

Abdollahzadeh et al. 2010

GU945356

GU945344

L. hormozganensis

CCMF-CNPA 0553

Brazil

Ricinus communis

This study

MG787090

MG806911

L. hormozganensis

CCMF-CNPA 0555

Brazil

Ricinus communis

This study

MG787091

MG806912

L. hormozganensis

CCMF-CNPA 0603

Brazil

Ricinus communis

This study

MG787092

MG806913

L. hormozganensis

CCMF-CNPA 0556

Brazil

Ricinus communis

This study

MG787093

MG806914

L. iraniensis

IRAN1517C

Iran

Citrus sp.

Abdollahzadeh et al. 2010

GU945349

GU945337

L. iraniensis

IRAN1519C

Iran

Mangifera indica

Abdollahzadeh et al. 2010

GU945350

GU945338

L. jatrophicola

CMM3610

Brazil

Jatropha curcas

Machado et al. 2014

KF234544

KF226690

L. jatrophicola

CMM 4472

Brazil

Anacardium occidentale

Netto et al. 2014

KT325572

KT325585

L. laeliocattleyae

CBS 167.28

Italy

laeliocattleyae

Rodríguez-Gálvez et al. 2016

KU507462

KU507429

L. laeliocattleyae

LAREP1

Peru

Mangifera indica

Rodríguez-Gálvez et al. 2016

KU507484

KU507451

L. margaritacea

CBS122519

Australia

Adansonia gibbosa

Pavlic et al. 2008

EU144050

EU144065

L. macrospora

CMM3833

Brazil

Jatropha curcas

Machado et al. 2014

KF234557

KF226718

L. mahajangana

CMW27801

Madagascar

Terminalia catappa

Begoude et al. 2010

FJ900595

FJ900641

L. mahajangana

CMW27820

Madagascar

Terminalia catappa

Begoude et al. 2010

FJ900597

FJ900643

L. mediterranea

CBS 137783

Italy

Quercus ilex

Linaldeddu et al. 2014

KJ638312

KJ638331

L. mediterranea

CBS 137784

Italy

Vitis vinifera

Linaldeddu et al. 2014

KJ638311

KJ638330

L. missouriana

UCD2193MO

USA

Vitis vinifera

Úrbez-Torres et al. 2011

HQ288225

HQ288267

L. missouriana

UCD2199MO

USA

Vitis vinifera

Úrbez-Torres et al. 2011

HQ288226

HQ288268

L. parva

CBS456.78

Colombia

Cassava-field soil

Alves et al. 2008

EF622083

EF622063

L. parva

CBS495.78

Colombia

Cassava-field soil

Alves et al. 2008

EF622085

EF622065

L. plurivora

STE-U5803

South Africa

Vitis vinifera

Damm et al. 2007

EF445362

EF445395

L. pontae

CMM1277

Brazil

Spondias purpurea

Coutinho et al. 2016

KT151794

KT151791

L. pseudotheobromae

CBS116459

Costa Rica

Gmelina arborea

Alves et al. 2008

EF622077

EF622057

L. pseudotheobromae

CMM3887

Brazil

Jatropha curcas

Machado et al. 2014

KF234559

KF226722

L. pyriformis

CBS 121770

Namibia

Acacia mellifera

Slippers et al. 2014

EU101307

EU101352

L. pyriformis

CBS 121771

Namibia

Acacia mellifera

Slippers et al. 2014

EU101308

EU101353

L. rubropurpurea

WAC12536

Australia

Eucalyptus grandis

Burgess et al. 2006

DQ103554

DQ103572

L. subglobosa

CMM3872

Brazil

Jatropha curcas

Machado et al. 2014

KF234558

KF226721

L. subglobosa

CMM4046

Brazil

Jatropha curcas

Machado et al. 2014

KF234560

KF226723

L. thailandica

CPC 22755

Thailand

Phyllanthus acidus

Trakunyingcharoen et al. 2014

KM006433

KM006464

L. thailandica

CPC22795

Thailand

Mangifera indica

Trakunyingcharoen et al. 2014

KJ193637

KJ193681

L. theobromae

CBS164.96

Papua New Guinea

Fruit along coral reef coast

Phillips et al. 2005

AY640255

AY640258

L. theobromae

CBS124.13

USA

Unknown

Alves et al. 2006

DQ458890

DQ458875

L. theobromae

CBS111530

Unknown

Unknown

Alves et al. 2008

EF622074

EF622054

L. theobromae

CCMF-CNPA 0554

Brazil

Ricinus communis

This study

MH485394

MH491477

L. theobromae

CCMF-CNPA 0585

Brazil

Ricinus communis

This study

MH485395

MH491478

L. venezuelensis

CMW13513

Venezuela

Acacia mangium

Burgess et al. 2006

DQ103549

DQ103570

L. venezuelensis

WAC12539

Venezuela

Acacia mangium

Burgess et al. 2006

DQ103547

DQ103568

L. viticola

UCD2553AR

USA

Vitis vinifera

Úrbez-Torres et al. 2011

HQ288227

HQ288269

L. viticola

UCD2604MO

USA

Vitis vinifera

Úrbez-Torres et al. 2011

HQ288228

HQ288270

Diplodia mutila

CBS 136015

Portugal

Populus alba

Alves et al. 2014

KJ361838

KJ361830

ITS internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal rRNA gene, TEF1-α translation elongation factor 1-α

Bayesian Inference (IB) analyses of the concatenated gene regions were performed as described by Machado et al. (2014). The K80 + I evolution models for the ITS region were used in the analyses while HKY + I + G was selected for TEF1-α. 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-1α gene regions, four isolates were identified as Lasiodiplodia hormozganensis, since they grouped with the type isolate of L. hormozganensis (IRAN1500C), and two isolates were identified as L. theobromae, the tree was rooted to Diplodia mutila (CBS 136015) (Fig. 2).
Fig. 2

Multilocus phylogenetic tree of Lasiodiplodia species inferred from Bayesian analysis based on the combined sequences of the ITS and TEF-1α. Bayesian posterior probabilities are indicated above the nodes and the tree was rooted to Diplodia mutila (CBS 136015). The bar represents the number of changes in the nucleotide sequence of each 100 bp. The species in this study are highlighted in bold

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 sterilized 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 each disk and then covered with plastic film for the first 48 h after inoculation to maintain moisture. The control treatment consisted of PDA plugs, with no fungus grown, deposited on the injured stalks as described above. The plants were then kept in a greenhouse for 30 days. All inoculated plants showed a necrosis 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.
Fig. 3

Pathogenicity test on R. communis, a control, b plant inoculated with Lasiodiplodia hormozganensis

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 described in Iran associated with Olea sp. and Mangifera indica (Abdollahzadeh et al. 2010). In Brazil, this species has been reported to cause dieback in mango and grape, and stem-end rot in mango and papaya in the Northeast region of the country (Marques et al. 2013; Netto et al. 2014; Correia et al. 2015). Thus, the results of this work suggest this is the first report of L. hormozganensis causing basal stem and root rot on R. communis.

Notes

Acknowledgments

The authors wish to thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico and Fundação de Amparo à Pesquisa do Estado de Minas Gerais for financial support.

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Copyright information

© Australasian Plant Pathology Society Inc. 2018

Authors and Affiliations

  1. 1.Departamento de FitopatologiaUniversidade Federal de ViçosaViçosaBrazil
  2. 2.Departamento de MicologiaUniversidade Federal de PernambucoRecifeBrazil
  3. 3.Empresa Brasileira de Pesquisa Agropecuária, Embrapa AlgodãoCampina GrandeBrazil

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