Species of Lasiodiplodia associated with mango in Brazil - Springer Link

Fungal Diversity (2013) 61:181–193 DOI 10.1007/s13225-013-0231-z

Species of Lasiodiplodia associated with mango in Brazil Marília W. Marques & Nelson B. Lima & Marcos Antônio de Morais Jr & Maria Angélica G. Barbosa & Breno O. Souza & Sami J. Michereff & Alan J. L. Phillips & Marcos P. S. Câmara

Received: 23 January 2013 / Accepted: 27 March 2013 / Published online: 7 April 2013 # Mushroom Research Foundation 2013

Abstract Mango (Mangifera indica) is a major tropical fruit species cultivated in Brazil. The objective of this study was to identify species of Lasiodiplodia associated with dieback and stem-end rot of mango in the semi-arid region of Northeastern Brazil, and compare the species in relation to mycelial growth, pathogenicity and virulence. A total of 120 isolates of Lasiodiplodia were used and identifications were made using a combination of morphology and phylogenetic analysis based on partial translation elongation factor 1-α sequence (EF1-α) and internal transcribed spacers (ITS). The following species were identified: Lasiodiplodia crassispora, L. egyptiacae, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. theobromae and Lasiodiplodia sp.. Lasiodiplodia theobromae was the most frequently isolated species, which represented 41 % of all the isolates. Only this species had been previously reported on mango in Brazil, while the other species represent the first report associated with mango tree diseases in this country. Lasiodiplodia crassispora is reported for the first time associated with mango diseases worldwide. There were significant differences in mycelial growth rates among the Lasiodiplodia species and M. W. Marques : N. B. Lima : B. O. Souza : S. J. Michereff : M. P. S. Câmara (*) Departamento de Agronomia, Universidade Federal Rural de Pernambuco, 52171-900 Recife, Brazil e-mail: [email protected] M. A. de Morais Jr Departamento de Genética, Universidade Federal de Pernambuco, 50732-970 Recife, Brazil M. A. G. Barbosa Embrapa Semi-Árido, 56302-970 Petrolina, Brazil A. J. L. Phillips Centro de Recursos Microbiológicos, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal

also in the optimum temperature for growth. All species of Lasiodiplodia were pathogenic on mango fruit. There were significant differences in virulence among the species, wherein L. hormozganensis and Lasiodiplodia sp.were the most virulent, while the least virulent were L. iraniensis, L. pseudotheobromae, L. crassispora and L. egyptiacae. Keywords Botryosphaeriaceae . Mangifera indica . ITS . EF1-α . Phylogeny . Virulence

Introduction Mango tree (Mangifera indica L.) is a major tropical fruit species cultivated in Brazil. In 2010, Brazilian production reached 1,197,694 t of fruit in an area of approximately 75,416 ha, generating about US$ 334 million. Considering both income and exported volume, mango occupied the third place in the segment of fresh fruit, with revenues of US$ 119,929 million and 124,694 t of fruit, respectively (Agrianual 2012). All mango exported is produced in the semiarid area of the Northeast region, mainly in the São Francisco Valley and Assú Valley (Costa et al. 2010). This crop also plays an important social role, generating over 25,000 direct and 75,000 indirect jobs in the São Franscico Valley alone (Souza et al. 2002). Among the wide range of diseases that impact on mango production in Brazil, dieback and stem-end rot have become increasingly important (Costa et al. 2010). The first report of mango dieback in Brazil was in 1947 (Batista 1947) and since then the intensity of the disease has increased leading, in some cases, to the complete loss of production and elimination of entire orchards (Tavares et al. 1991; Tavares 2002). Mango dieback and stem-end rot are caused by a complex of fungi, but members of the Botryosphaeriaceae are considered to be the most important (Johnson 1992; Al

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Adawi et al. 2003; Slippers et al. 2005; Javier-Alva et al. 2009; Costa et al. 2010; Ismail et al. 2012). The Botryosphaeriaceae is a genus-rich family in the Dothideomycetes, comprising numerous species with a cosmopolitan distribution (Crous et al. 2006; Phillips et al. 2008; Liu et al. 2012). Species of the Botryosphaeriaceae are associated with several host plants, and can act as primary or secondary pathogens, or as endophytes that under stress conditions of the host plant can become pathogenic (Denman et al. 2000; Crous et al. 2006). Lasiodiplodia theobromae (Pat.) Griff. & Maubl, a member of the Botryosphaeriaceae, occurs mainly in tropical and subtropical regions, causing severe damage in almost 500 host plants (Punithalingam 1980; Burgess et al. 2006). The fungus has been reported as a mango pathogen worldwide associated with several disease symptoms including dieback, stem-end rot, decline, gummosis and canker (Ploetz et al. 1996; Jacobs 2002; Khanzada et al. 2004; Abdollahzadeh et al. 2010; Costa et al. 2010; Sakalidis et al. 2011a; Ismail et al. 2012). Besides mango, several other crops of economic importance are affected by L. theobromae in Brazil, especially avocado (Persea americana Mill.), banana (Musa spp.), barbados cherry (Malpighia glabra L.), cashew (Anacardium occidentale L.), citrus (Citrus spp.), coconut palm (Cocos nucifera L.), custard apple (Annona squamosa L.), grapevine (Vitis sp.), guava (Psidium guajava L.), muskmelon (Cucumis melo L.), papaya (Carica papaya L.), passion fruit (Passiflora edulis Sims), soursop (Annona muricata L.) and watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) (Tavares 2002; Freire et al. 2003). According to Sutton (1980) and Phillips et al. (2008) the main features that distinguish Lasiodiplodia from other closely related genera are the presence of pycnidial paraphyses and longitudinal striations on mature conidia (Sutton 1980). Recent studies based on ITS and EF-1α sequence data have led to the identification of cryptic species within the L. theobromae species complex (Pavlic et al. 2004; Burgess et al. 2006; Damm et al. 2007; Alves et al. 2008; Pavlic et al. 2008; Abdollahzadeh et al. 2010; Begoude et al. 2010; Úrbez-Torres et al. 2012; Ismail et al. 2012). Presently, 17 species are recognized in Lasiodiplodia. Several species of Lasiodiplodia have been associated with mango diseases worldwide. Lasiodiplodia theobromae was recorded in Australia (Slippers et al. 2005), Brazil (Costa et al. 2010), Egypt (Ismail et al. 2012), Iran (Abdollahzadeh et al. 2010), South Africa (Jacobs 2002) and USA (Ploetz et al. 1996). Recently, four new species of Lasiodiplodia (L. citricola Abdollahzadeh, Zare & A.J.L. Phillips, L. gilanensis Abdollahzadeh, Javadi & A.J.L. Phillips, L. hormozganensis Abdollahzadeh, Zare & A.J.L. Phillips and L. iraniensis Abdollahzadeh, Zare & A.J.L. Phillips) and L. pseudotheobromae A.J.L. Phillips, A. Alves & Crous were associated with this host in Iran (Abdollahzadeh et al. 2010). In Australia, L. iraniensis

Fungal Diversity (2013) 61:181–193

and L. pseudotheobromae were isolated from cankers and dieback of mango (Sakalidis et al. 2011a). In 2012, L. pseudotheobromae and a new species (L. egyptiacae A.M. Ismail, L. Lombard & Crous) were recorded in Egypt causing dieback of mango (Ismail et al. 2012). In Brazil, for a long time, dieback and stem-end rot of mango were attributed exclusively to L. theobromae, but recent studies based on molecular methods revealed the presence of other Botryosphaeriaceae species Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips, Fusicoccum aesculi (Corda) Crous, Slippers & A.J.L. Phillips and Pseudofusicoccum stromaticum (Mohali, Slippers & M.J. Wingf.) Mohali, Slippers & M.J. Wingf. associated with these diseases (Costa et al. 2010; Marques et al. 2012). The increasing economic importance of plant diseases caused by Lasiodiplodia and the recent discovery of several new species of fungus associated with tropical plants led us to speculate that more than one species of Lasiodiplodia may be associated with mango diseases in Northeastern Brazil. The disease etiology is crucial for epidemiological studies and for a better understanding of the distribution and importance of individual species, as well as finding effective management strategies to each pathogen. Therefore, the objective of this study was to identify species of Lasiodiplodia from a large number of isolates associated with dieback and stem-end rot of mango in the semi-arid region of Northeastern Brazil.

Materials and methods Sampling and fungal isolation From January to February 2010, stems and fruits showing dieback and stem-end rot symptoms were collected from 14 mango orchards (25 samples per orchard) located in São Francisco Valley and Assú Valley, Northeastern Brazil. Samples were recovered from the cultivars Tommy Atkins, Keitt, Haden and Palmer. Plant tissues were surface disinfested in 70 % ethanol for 30 s and 1 % NaOCl for 1 min. Samples were then rinsed in sterile distilled water for 30 s and dried before small pieces (4–5 mm) of tissue were taken from the margin between necrotic and apparently healthy tissue and plated onto potato dextrose agar (PDA) (Acumedia, Lansing, USA) amended with 0.5 g l−1 streptomycin sulfate (PDAS). Plates were incubated at 25 °C in the dark for 3 to 4 days. Fungal colonies emerging from plant tissue pieces that were morphologically similar to species of Botryosphaeriaceae (Sutton 1980; Phillips 2006) were transferred to PDA plates and incubated at 25 °C in the dark, with observation after 3, 5 and 15 day. To obtain single-spore isolates, pycnidia were obtained on 2 % water agar (WA) with autoclaved pine needles as a substrate after 3-week


incubation at 25 °C under a 12 h daily photoperiod with near-ultraviolet light (Slippers et al. 2004). A single pycnidium was cut from each isolate under a stereo microscope (Zeiss Stemi DV4; Carl Zeiss, Berlin, Germany) and placed in 250 μl of sterile water to produce a conidial suspension. A 20 μl aliquot was spread on PDAS and incubated at 28 °C in the dark for 24 h. A single-pycnidiospore isolate was recovered for an individual sample and transferred to a fresh PDA plate. All isolates were morphologically identified as Lasiodiplodia when characteristics typical of genus were present, namely conidiomatal paraphyses, conidia that were initially hyaline and aseptate, but in time developed a single median septum, the wall became dark brown and melanin granules deposited longitudinally on the inner surface of the wall gave the conidia a striate appearance (Sutton 1980; Alves et al. 2008). Stock cultures were stored in PDA slants at 5 °C in the dark. DNA isolation, PCR amplification and sequencing A portion of the translation elongation factor 1α (EF1-α) gene was sequenced for all the Lasiodiplodia isolates collected from mango orchards. The internal transcribed spacer (ITS) region of rDNA was sequenced to confirm the identity of representative isolates within each EF1-α identified species. Using a sterile 10 μl pipette tip, a small amount of aerial mycelium was scraped from the surface of a 5 day old culture on PDA at 25 °C and genomic DNA was extracted using the AxyPrep™ Multisource Genomic DNA Miniprep Kit (Axygen Scientific Inc., Union City, USA) following the manufacturer’s instructions. The ITS region was amplified using the primers ITS1 and ITS4 (White et al. 1990) as described by Slippers et al (2004) and EF1-α gene was amplified using the primers EF1-688F and EF1-1251R (Alves et al. 2008) as described by Phillips et al. (2005). Each 50 μl polymerase chain reaction (PCR) mixture included 21 μl of PCR-grade water, 1 μl of DNA template, 1.5 μM of each primer, and 1 μl of PCR Master Mix (2X) (0.05 u μl−1 de Taq DNA polimerase, reaction buffer, 4 mM MgCl2, 0.4 mM of each dNTP; Thermo Scientific, Waltham, USA). PCR reactions were carried out in a thermal cycler (Biocycler MJ 96; Applied Biosystems, Foster City, USA). The PCR amplification products were separated by electrophoresis in 1.5 % agarose gels in 1.0× Tris-acetate acid EDTA (TAE) buffer and were photographed under UV light after staining with with ethidium bromide (0.5 μg ml−1) for 1 min. PCR products were purified using the AxyPrep™ PCR Cleanup Kit (Axygen) following the manufacturer’s instructions. ITS and EF1-α regions were sequenced in both directions using a ABI PRISM® 3100-Avant Genetic Analyzer (Applied Biosystems) at the Sequencing Platform LABCEN/CCB in the Universidade Federal de Pernambuco (Recife, Brazil).

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Phylogenetic analyses Sequences were edited with Chromas v. 2.32 (Technelysium Pty Lda, Brisbane, Australia). Sequences of both DNA regions of additional isolates were retrieved from GenBank. Sequences were aligned with ClustalX v. 1.83 (Thompson et al. 1997) and manually adjusted when necessary. Phylogenetic information contained in indels (gaps) was incorporated into the phylogenetic analyses using simple indel coding as implemented by GapCoder (Young and Healey 2003). A partition homogeneity test determined the possibility of combining the ITS and EF1-α datasets (Farris et al. 1995; Huelsenbeck et al. 1996). Sequences of Lasiodiplodia species obtained from GenBank were included in the analyses (Table 1). Diplodia seriata De Not. (CBS 112555) and D. mutila Fr. (CBS CBS 112553) were used as outgroup. Phylogenetic analyses were performed using PAUP v. 4.0b10 (Swofford 2003) for maximum-parsimony and MrBayes v. 3.0b4 (Ronquist and Huelsenbeck 2003) for Bayesian analyses. Maximum-parsimony analyses were performed using the heuristic search option with 1,000 random taxa addition and tree bisection and reconnection (TBR) as the branch-swapping algorithm. All characters were unordered and of equal weight and gaps were treated as missing data. Branches of zero length were collapsed and all multiple, equally parsimonious trees were saved. The robustness of the most parsimonious trees was evaluated from 1,000 bootstrap replications (Hillis and Bull 1993). Other measures used were consistency index (CI), retention index (RI) and homoplasy index (HI). Bayesian analyses employing a Markov Chain Monte Carlo method were performed. The general time-reversible model of evolution (Rodriguez et al. 1990), including estimation of invariable sites and assuming a discrete gamma distribution with six rate categories (GTR+Γ+G) was used. Four MCMC chains were run simultaneously, starting from random trees for 1,000,000 generations. Trees were sampled every 100th generation for a total of 10,000 trees. The first 1,000 trees were discarded as the burn-in phase of each analysis. Posterior probabilities (Rannala and Yang 1996) were determined from a majority-rule consensus tree generated with the remaining 9,000 trees. This analysis was repeated three times starting from different random trees to ensure trees from the same tree space were sampled during each analysis. Phylogenetic trees were viewed with Treeview (Page 1996). Sequences generated in this study were deposited in GenBank and the alignment in TreeBASE (S13281). Representative isolates of different Lasiodiplodia species obtained in this study were deposited in the Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes” (CMM) at the Universidade Federal Rural de Pernambuco (Recife, Brazil).

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Table 1 Isolates of Lasiodiplodia species used in this study Taxon

Culture Accession No. a

Host

Location

Diplodia mutila D. seriata Lasiodiplodia citricola

CBS 112553 CBS 12555 CBS 124707

Vitis vinifera Vitis vinifera Citrus sp.

Portugal Portugal Iran

L. L. L. L. L. L. L. L. L.

IRAN 1521C CMW 13488 CMW 14691 CMM 3982 CBS 130992 BOT 29 CMM 3981 CMM 1485 IRAN 1501C

Citrus sp. Eucalyptus urophylla Santalum album Mangifera indica M. indica M. indica M. indica M. indica Unknown

Iran Venezuela Australia Brazil Egypt Egypt Brazil Brazil Iran

L. gilaniensis

CBS 124704

Unknown

Iran

L. gonubiensis L. gonubiensis L. hormozganensis

CMW 14078 CBS 115812 CBS 124709

Syzigium cordatum S. cordatum Olea sp.

South Africa South Africa Iran

L. hormozganensis

IRAN 1498C

M. indica

Iran

L. L. L. L. L. L. L.

hormozganensis hormozganensis hormozganensis hormozganensis hormozganensis hormozganensis iraniensis

CMM 1546 CMM 3983 CMM 3984 CMM 3985 CMM 3986 CMM 3987 CBS 124710

M. indica M. indica M. indica M. indica M. indica M. indica Salvadora persica

Brazil Brazil Brazil Brazil Brazil Brazil Iran

L. L. L. L. L. L. L. L. L. L. L. L. L.

iraniensis iraniensis iraniensis iraniensis iraniensis iraniensis iraniensis iraniensis mahajangana mahajangana margaritaceae margaritaceae missouriana

IRAN 1502C CMM 3990 CMM 1483 CMM 3993 CMM 3995 CMM 3996 CMM 3998 CMM 4051 CBS 124927 CMW 27801 CBS 122065 CBS 122519 UCD 2199MO

Juglans sp. M. indica M. indica M. indica M. indica M. indica M. indica M. indica Terminalia catappa T. catappa Adansonia gibbosa A. gibbosa Vitis vinifera

Iran Brazil Brazil Brazil Brazil Brazil Brazil Brazil Madagascar Madagascar Western Australia Western Australia Missouri, USA

L. missouriana

CBS 128311

V. vinifera

Missouri, USA

L. L. L. L. L. L.

CBS 494.78 CBS 456.78 CBS 120832 STE-U4583 CBS 116459 IRAN 1518C

Cassava-field soil Cassava-field soil Prunus salicina V. vinifera Gmelina arborea Citrus sp.

Colombia Colombia South Africa South Africa Costa Rica Iran

citricola crassispora crassispora crassispora egyptiacae egyptiacae egyptiacae egyptiacae gilaniensis

parva parva plurivora plurivora pseudotheobromae pseudotheobromae

Collector

A.J.L. Phillips A.J.L. Phillips J. Abdollahzadeh & A. Javadi A. Shekari S. Mohali T.I. Burgess & G. Pegg M.W. Marques A.M. Ismail A.M. Ismail M.W. Marques V.S.O. Costa J. Abdollahzadeh & A. Javadi J. Abdollahzadeh & A. Javadi D. Pavlic D. Pavlic J. Abdollahzadeh & A. Javadi J. Abdollahzadeh & A. Javadi V.S.O. Costa M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques J. Abdollahzadeh & A. Javadi A. Javadi M.W. Marques V.S.O. Costa M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques J. Roux J. Roux T.I. Burgess T.I. Burgess K. Striegler & G.M. Leavitt K. Striegler & G.M. Leavitt O. Rangel O. Rangel U. Damm F. Halleen J. Carranza-Velásquez J. Abdollahzadeh/ A. Javadi

GenBank Accession No.b ITS

EF-1α

AY259093 AY259094 GU945354

AY573219 AY573220 GU945340

GU945353 DQ103552 DQ103550 JX464058 JN814397 JN814401 JX464072 JX464076 GU945352

GU945339 DQ103559 DQ103557 JX464015 JN814424 JN814428 JX464035 JX464044 GU945341

GU945351

GU945342

AY639594 DQ458892 GU945355

DQ103567 DQ458860 GU945340

GU945356

GU945344

JX464077 JX464069 JX464080 JX464084 JX464085 JX464094 GU945348

JX464053 JX464033 JX464052 JX464101 JX464022 JX464034 GU945336

GU945347 JX464067 JX464073 JX464068 JX464097 JX464100 JX464059 JX464071 FJ900597 FJ900595 EU144051 EU144050 HQ288226

GU945335 JX464028 JX464023 JX464029 JX464046 JX464019 JX464045 JX464030 FJ900643 FJ900641 EU144066 EU144065 HQ288268

HQ288225

HQ288267

EF622084 EF622083 EF445362 AY343482 EF622077 GU973874

EF622064 EF622063 EF445395 EF445396 EF622057 GU973866


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Table 1 (continued) Taxon

Culture Accession No. a

L. L. L. L. L. L. L. L. L. L.

pseudotheobromae pseudotheobromae pseudotheobromae pseudotheobromae pseudotheobromae gonubiensis gonubiensis rubropurpurea rubropurpurea theobromae

CMM 3999 CBS 116459 CMM 4001 CMM 4002 CMM 4008 CMW 14078 CBS 115812 WAC 12535 WAC 12536 CBS 164.96

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae theobromae venezuelensis venezuelensis viticola

L. viticola Lasiodiplodia Lasiodiplodia Lasiodiplodia Lasiodiplodia Lasiodiplodia Lasiodiplodia Lasiodiplodia

sp. sp. sp. sp. sp. sp. sp.

Host

Location

Collector

GenBank Accession No.b ITS

EF-1α

Brazil Costa Rica Brazil Brazil Brazil South Africa South Africa Queensland Queensland New Guinea

M.W. Marques J. Carranza-Velásquez M.W. Marques M.W. Marques M.W. Marques D. Pavlic D. Pavlic T.I. Burgess & G. Pegg T.I. Burgess & G. Pegg A. Aptroot

JX464075 EF622077 JX464092 JX464086 JX464090 AY639594 DQ458892 DQ103553 DQ103554 AY640255

JX464018 EF622057 JX464039 JX464020 JX464016 DQ103567 DQ458877 DQ103571 DQ103572 AY640258

CBS 111530 CMM 1476 CMM 1481 CMM 1517 CMM 4019 CMM 4033 CMM 4039 CMM 4041 CMM 4042 CMM 4043 CMM 4046 CMM 4047 CMM 4048 CMM 4050 CMM 4052 CMM 4053 CMM 4054 CMM 4021 WAC 12539 WAC 12540 UCD 2604MO

M. indica G. arborea M. indica M. indica M. indica S. cordatum S. cordatum E. grandis E. grandis Fruit on coral reef coast Unknown M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica Acacia mangium A. mangium V. vinifera

Unknown Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Venezuela Venezuela USA

AY622074 JX464083 JX464095 JX464060 JX464096 JX464081 JX464065 KC184891 JX464070 JX464087 JX464091 JX464082 JX464093 JX464062 JX464088 JX464089 JX464099 JX464064 DQ103547 DQ103548 HQ288228

AY622054 JX464057 JX464021 JX464054 JX464026 JX464032 JX464041 JX464042 JX464017 JX464056 JX464027 JX464025 JX464048 JX464024 JX464050 JX464051 JX464038 JX464047 DQ103568 DQ103569 HQ288270

CBS 128313

V. vinifera

USA

HQ288227

HQ288269

CMM CMM CMM CMM CMM CMM CMM

M. M. M. M. M. M. M.

Brazil Brazil Brazil Brazil Brazil Brazil Brazil

Unknown M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques S. Mohali S. Mohali K. Striegler & G.M. Leavitt R.D. Cartwright & W.D. Gubler V.S.O. Costa V.S.O. Costa M.W. Marques M.W. Marques M.W. Marques M.W. Marques M.W. Marques

JX464061 JX464079 JX464078 JX464074 JX464066 JX464098 JX464063

JX464040 JX464036 JX464043 JX464037 JX464055 JX464031 JX464049

1472 1491 4010 4011 4013 4014 4015

indica indica indica indica indica indica indica

a

CBS Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; CMW Forestry and Agricultural Biotechnology Institute, University of Pretoria, South Africa; WAC Department of Agriculture Western Australia Plant Pathogen Collection, University of Western Australia, Perth, Australia; CMM Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes”, Universidade Federal Rural de Pernambuco, Recife, Brazil; STE-U Culture Collection of the Department of Plant Pathology, University of Stellenbosch, Stellenbosch, South Africa; UCD Phaff Yeast Culture Collection, Department of Food Science and Technology, University of California, Davis, USA; BOT A. M. Ismail, Plant Pathology Research Institute, Giza, Egypt; IRAN Culture Collection of the Iranian Research Institute of Plant Protection, Tehran, Iran. Isolate numbers in bold represents ex-type specimens

b

Sequence numbers in bold were obtained in the present study

186

Fungal Diversity (2013) 61:181–193 CMM 4039 61/-- CMM 4047

CMM 4042 CMM 4050 CMM 4043 CMM 4033 59/0.55 CMM 1481 CMM1476 L. theobromae CBS 164.96 58/0.93 CMM 4019 CMM 4021 CMM 1517 67/-- CMM 4046 CMM 4053 CMM 4054 CMM 4052 CMM 4048 L. theobromae CBS 111530 CMM 4041 CMM 4011 CMM 1472 CMM 4013 CMM 1491 CMM 4010 CMM 4014 CMM 4015 64/1.00 L. viticola CBS 128313 99/1.00 L. viticola UCD 2604MO L. iraniensis CBS 124710 CMM 3993 CMM 3996 CMM 3990 CMM 3998 99/1.00 CMM 1483 CMM 3995 97/0.65 CMM 4051 68/0.90 L. iraniensis IRAN1502C L. gilanensis CBS 124704 L. gilanensis IRAN 1501C L. missouriana UCD 2199MO L. missouriana CBS 128311 L. plurivora STE-U 4583 L. plurivora CBS 120832 99/0.99 97/0.99 L. mahajangana CBS 124927 100/1.00 L. mahajangana CMW 27801 CMM 3986 94/1.00 L. hormozganensis IRAN 1498C 80/0.72 CMM 3987 CMM 3983 CMM 3985 CMM 1546 L. hormozganensis CBS 124709 CMM 3984 99/1.00 CMM 4008 L. pseudotheobromae CBS 116459 L. pseudotheobromae IRAN 1518C CMM 3999 CMM 4002 CMM 4001 74/0.90 L. egyptiacae BOT-29 CMM 3981 L. egyptiacae CBS130992 CMM1485 L. citricola IRAN 1521C L. citricola CBS 124707 L. parva CBS 456.78 97/1.00 L. parva CBS 494.78 L. rubropurpurea WAC 12535 65/0.95 100/1.00 L. rubropurpurea WAC 12536 L. venezuelensis WAC 12539 99/1.00 L. venezuelensis WAC 12540 L. crassispora CMW 13488 CMM 3982 100/1.00 L. crassispora CMW 14691 L. margaritacea CBS 122519 99/1.00 L. margaritacea CBS 122065 L. gonubiensis CBS 115812 100/1.00 L. gonubiensis CMW 14078 D. seriata CBS 112555 D. mutila CBS 112553

100/1.00

10


Fig. 1

One of 4 most parsimonious trees (length = 327; CI = 0.758; RI = 0.918; HI = 0.242) obtained from combined ITS and EF1-α sequence data. Maximum parsimony bootstrap support values from 1,000 replications and Bayesian posterior probability scores are shown at the nodes. Ex-type isolates are in bold

Morphological characterization The isolates that were identified in the phylogenetic analysis were used to study colony morphology and conidial characteristics. The color and aerial hyphal growth from isolates were recorded during 15 days of growth on PDA 2 % at 25 °C in the dark. Conidial characteristics were observed after placing cultures on 2 % WA containing autoclaved pine needles and incubation under near-ultraviolet light, as previously described. Conidia and other structures were mounted in 100 % lactic acid and digital images recorded with a Leica DFC320 camera on a Leica DMR HC microscope fitted with Nomarski differential interference contrast optics (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). The length and width of 50 conidia per isolate were measured with the Leica IM500 measurement module. Mean and standard errors of the conidial measurements, including mean length to width ratio (L/W) of the conidial measurements were calculated. Isolates were also used to determine the effect of temperature on colony growth of different species. A 3-mm-diameter mycelial plug from the growing margin of a 3-day-old colony was placed in the center of a 90-mm-diameter 2 % PDA plate, and four replicates of each isolate were incubated at temperatures ranging from 5 °C to 35 °C in 5 °C intervals in the dark. After a 2-days incubation period, the colony diameter (mm) was measured in two perpendicular directions. The experiment was done twice. Colony diameters were plotted against temperature and a curve was fitted by a cubic polynomial regression (y=a+bx+cx2 +dx3). Optimal temperature was estimated from the regression equation and numeric summary with TableCurve™ 2D v. 5.01 (SYSTAT Software Inc., Chicago, USA). Optimum temperature was defined as the temperature that produced the maximum mycelial growth rate. The colony diameter data at 30 °C were used to calculate the mycelial growth rate (mm/day). One-way analyses of variance (ANOVA) were conducted with data obtained from optimum temperature and mycelial growth rate experiments, and means were compared by Fisher’s least significant difference (LSD) test at the 5 % significance level using STATISTIX v. 9.0 (Analytical Software, Tallahassee, USA). Pathogenicity and virulence in fruits The isolates used in the morphological characterization were selected for this test. Mango fruits (cv. Tommy Atkins) at stage three of maturation (Assis 2004), which were not treated with fungicides, were washed in running water,

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surface disinfested in 70 % ethanol for 1 min and 1 % NaOCl for 5 min, then rinsed in sterile distilled water. After drying, the fruits were placed on plastic trays, on the base of each were four layers of paper towels wetted with distilled water to increase humidity. Each fruit was put on a sterilized Petri plate to avoid direct contact with water. Since non-wounded treatment caused no lesions of Lasiodiplodia (non published data), each fruit was wounded at the medium region by pushing the tip of four sterile pins through the surface of the skin to a depth of 3 mm. A mycelial plug (5 mm in diameter) removed from the margin of a 5-day-old PDA culture grown at 28 °C in the dark of each isolate was immediately placed on the wound. A non-colonized agar plug was used for the control. The trays were enclosed in plastic bags and incubated at 25 °C in the dark. The plastic bags and paper towels were removed after 48 h and the fruits were kept at the same temperature. Isolates were considered pathogenic when the lesioned area advanced beyond the 5-mm diameter initial injury. The virulence of the isolates was evaluated by measurement of the lesion length at 72 h after inoculation in two perpendicular directions on each fruit. The experiment was arranged in a completely randomized design with six replicates per treatment (isolate) and one fruit per replicate. The experiment was conducted twice. Differences in virulence caused by Lasiodiplodia species were determined by one-way ANOVA and means were compared by LSD test at the 5 % significance level using STATISTIX.

Results DNA sequencing and phylogenetic analyses A total of 120 isolates of Lasiodiplodia spp. were obtained from mango stems and fruits. From this total, seven species of Lasiodiplodia were identified based on phylogenetic analysis of the partial translation elongation factor 1α (EF1-α) gene: L. crassispora Burgess & Barber, L. egyptiacae, L. hormozganensis, L. iraniensis, L. pseudotheobromae, L. theobromae and Lasiodiplodia sp.. To confirm the identity of the isolates, the internal transcribed spacer (ITS) sequence was obtained for 44 isolates representing each putative species. PCR fragments for the ITS were approximately 580 bp in size, while those for EF1-α was 450 bp. A partition homogeneity test showed no significant difference between the data from the different gene regions, indicating that they could be combined in a single dataset (P=0.08). The combined data set consisted of 78 taxa and 2 outgroup species. The alignment contained 738 characters including coded alignment gaps. Of these characters 548 were constant, 43 were variable and parsimony uninformative and 147 were parsimony-informative. Heuristic search generated four equally parsimonious trees with (TL = 327; CI = 0.758; RI = 0.918; HI = 0.242). The phylogenetic

188


analyses of Maximum-parsimony and Bayesian methods produced nearly identical topologies (Bayesian tree not shown). Sequences of ex-type isolates of various Lasiodiplodia species from GenBank were included in the analysis together with isolates obtained in this study (Table 1). The combined dataset resulted in 17 well supported clades. Each clade corresponds to previously described species. The isolates from this study grouped in seven clades. The majority of isolates (17 isolates) clustered together in a large clade containing the species L. theobromae (CBS 164.96; CBS111530). These isolates subdivided into five sub-clades, with low support. The second group with seven isolates formed a sub-clade that clustered together with L. viticola, with low bootstrap support (64 %) but high Bayesian support (100 %). Seven isolates that clustered with L. iraniensis presented a well-supported clade (MP / MB: 99/1.00), subdivided into two other sub-clades. Six isolates clustered together with L. hormozganensis (MP/MB: 80/0.72). Four isolates showed sequences nearly identical to the ex-type isolate of L. pseudotheobromae strongly supported by bootstrap values (MP/MB: 99/1.00). Two Isolates grouped with a species recently described from mango, L. egyptiacae (MP/MB: 74/0.90) and one isolate resided in a clade with the ex-type isolate of L. crassipora strongly supported by a bootstrap value of 100 % (Fig. 1). Lasiodiplodia theobromae was the predominant species isolated (44 %) followed by Lasiodiplodia sp. (18 %), L. iraniensis (17 %), L. pseudotheobromae (13 %), L. hormozganensis (5 %), L. egyptiacae (2 %) and L. crassispora (1 %). The distribution of Lasiodiplodia species differed between Assú Valley and São FranciscoValley. Lasiodiplodia theobromae was the predominant species in the two regions. L. pseudotheobromae, L. iraniensis, L. hormozganensis and Lasiodiplodia sp. were also found in both regions, while L. crassipora and L. egyptiacae were found only in São Francisco Valley (Fig. 2).

species obtained in this study were similar to conidial dimensions of Lasiodiplodia species previously described in the literature, although in all cases they were slightly larger (Table 2). There were significant differences (P≤0.05) in growth rate among the Lasiodiplodia species and differences in the optimum temperature for mycelial growth. The optimum temperature for growth of L. iraniensis (28.3 °C) was significantly lower than that of L. hormozganensis (31.1 °C), Lasiodiplodia sp. (29.9 °C) and L. theobromae (29.9 °C). The other species (L. crassispora, L. egyptiacae and L. pseudotheobromae) presented intermediate values of optimum temperature for growth, without differing of observed extremes. The mycelial growth rates of L. hormozganensis and Lasiodiplodia sp. (49.1 mm/day) were significantly higher than L. crassispora, L. iraniensis, L. pseudotheobromae and L. theobromae, which varied from 35.9 to 41.4 mm/day. Only L. egyptiacae (41.9 mm/day) did not differ significantly from these extremes (Table 3).

Morphology and cultural characteristics

Discussion

The isolates that were identified in the phylogenetic analysis using the combined data set were used to study colony morphology and conidial characteristics. Anamorphic structures formed on the pine needles on WA within 2–4 week. No sexual (teleomorph) structures were observed during this study. All species showed morphological features typical of the genus, namely slowly maturing conidia with thick walls and longitudinal striations resulting from melanin deposition on the inner surface of the wall (Punithalingam 1976, 1980). All isolates on PDA grew rapidly, covering the entire surface of the Petri dishes within 4 days. The aerial mycelium was initially white, turning dark greenish-grey or greyish after 4–5 days at 25 °C in the dark. There were differences in conidial dimensions between the Lasiodiplodia spp. (Table 2). The conidia produced by

Seven species of Lasiodiplodia associated with dieback and stem-end rot of mango in Brazilian Northeast were identified in the present study: L. crassispora, L. egyptiacae, L. hormozganensis, L. iraniensis, L. pseudotheobromae, Lasiodiplodia sp. and L. theobromae. Currently, several Lasiodiplodia species have been reported in mango worldwide (Jacobs 2002; Slippers et al. 2005; Abdollahzadeh et al. 2010; Costa et al. 2010; Sakalidis et al. 2011a; Ismail et al. 2012). In this study, L. theobromae was the most frequently isolated species with 48 % and 42 % of all the isolates in the São Francisco Valley and Assú Valley, respectively, indicating it is the most widespread Lasiodiplodia species in Northeastern of the Brazil. This species is known for its cosmopolitan distribution and its wide range of hosts

Pathogenicity and virulence in fruits All isolates of Lasiodiplodia were pathogenic to mango fruits. Observed symptoms on the fruit surface were dark brown necrotic lesions with roughly circular shape around the inoculation sites. There were significant (P≤0.05) differences in virulence among the species, wherein Lasiodiplodia sp. and L. hormozganensis were most virulent, causing the largest lesions (33.8 mm and 33.6 mm, respectively). The less virulent species were L. iraniensis, L. pseudotheobromae, L. crassispora and L. egyptiacae, with lesions varying from 22.5 to 17.2 mm. The lesion length induced by L. theobromae differs from these extremes, and represented intermediate virulence (Fig. 3).


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Fig. 2 Frequency (%) of Lasiodiplodia species associated with dieback and stem-end rot of mango in Assú Valley and São Francisco Valley, Northeastern Brazil

Assú Valley (n = 72 ) L. hormozganensis 3% L. iraniensis 22%

L. theobromae 42% L. pseudotheobromae 14%

Lasiodiplodia sp. 19%

São Francisco Valley (n = 48) L. egyptiacae 4% L. hormozganensis 8%

L. crassispora 2%

L. iraniensis 8% L. theobromae 48%

L. pseudotheobromae 13%

Lasiodiplodia sp. 17%

Table 2 Comparison of conidial dimensions of Lasiodiplodia species examined in this study and previous studies

Species

Conidial size (μm)

L/W ratio

References

Lasiodiplodia crassispora

26.61–28.61×16.34–18.84 27–30×14–17 20.69–22.96×10.86–13.14 20–24×11–13 20.62–22.89×11.8–13.09 19.6–23.4×11.7–13.3 22.51–26.09×12.75–14.97 18.7–23.0×12.1–13.9 25.07–28.23×13.4–15.6 25.5–30.5×14.8–17.2 24.49–27.49×13.3–14.79 23.6–28.8×13–15.4 25.1–27.3×14.1–15.0

1.6 1.8 1.8 1.8 1.8 1.7 1.7 1.6 1.8 1.7 1.8 1.9 1.8

Present study Burgess et al. 2006 Present study Ismail et al. 2012 Present study Abdollahzadeh et al. 2010 Present study Abdollahzadeh et al. 2010 Present study Alves et al. 2008 Present study Alves et al. 2008 Present study

L. egyptiacae L. hormozganensis L. iraniensis L. pseudotheobromae L. theobromae Lasiodiplodia sp.

190 Table 3 Optimum temperature for mycelial growth and mycelial growth rate at 30 °C of Lasiodiplodia species associated with dieback and stem-end rot of mango in Northeastern Brazil

Mean ± standard error. Values within columns followed by the same letter do not differ significantly according to Fisher’s LSD test (P≤0.05)


Species

n

Optimum temperature (°C) ± SE

Mycelial growth rate (mm/day) ± SE

Lasiodiplodia crassispora L. egyptiacae L. hormozganensis L. iraniensis L. pseudotheobromae L. theobromae

1

29.0±1.27 ab

35.9±4.20 b

1 5 7 4 20

29.3±1.27 31.1±0.57 28.2±0.48 29.8±0.64 29.9±0.28

41.9±4.20 49.1±1.88 41.4±1.67 36.4±2.30 41.3±0.96

Lasiodiplodia sp.

6

29.9±0.52 a

ab a b b b

49.0±1.71 a

Lasiodiplodia sp. was the second most prevalent species in the São Francisco Valley with 17 % and the third in the Assú Valley with 19 % of all isolate. Considering the phylogenetic data, the mango isolates that grouped together with L. viticola formed a subgroup, strongly supported in the Bayesian analysis (1.00) but with only moderate support in MP (64 %). Since these isolates have sequences in the EF-1α that were identical to L. viticola additional analysis is needed to clarify this subgroup. Such a study is currently in progress and will be addressed in a future paper. The species L. hormozganensis and L. iraniensis were recently described in Iran associated with several hosts, including mango (Abdollahzadeh et al. 2010). In this work, L. iraniensis was the third most prevalent species, and L. hormozganensis together with Lasiodiplodia sp. were the most virulent species in mango fruit. A similar result was found by Sakalidis et al. (2011a), where L. hormozganensis isolates produced the largest lesions in mango branches. In our study L. crassispora was the least abundant species with only one isolate. This species has notably thicker cell walls in the immature spores and the striations appear to be wider and the cytoplasm wart-like in appearance, which

(Punithalingam 1980; Burgess et al. 2006), and is reported commonly as an important pathogen associated with disease of mango trees in tropical and subtropical regions (Ploetz et al. 1996; Jacobs 2002; Khanzada et al. 2004; Abdollahzadeh et al. 2010; Costa et al. 2010; Sakalidis et al. 2011a; Ismail et al. 2012). The phylogenetic analysis of the L. theobromae clade reveals low support in the analyses of Maximumparsimony and Bayesian method, indicating a great intraspecific diversity. This intraspecific variation was observed also in other studies with this species (Pavlic et al. 2004; Begoude et al. 2010; Ismail et al. 2012). During the past 150 years this fungus has had many names and has been treated as many different species. This trend ended with the monograph of Punithalingam (1976) which reduced most species to synonymy with L. theobromae. However, lately several species have been described in the L. theobromae complex, mostly because of the increase in the application of DNA sequence data, but also because of the increased sampling of relatively unexplored areas, including Venezuela (Burgess et al. 2006), Australia (Pavlic et al. 2008), Iran (Abdollahzadeh et al. 2010), Egypt (Ismail et al. 2012) and Brazil (this study). Fig. 3 Mean lesion lengths (mm) caused by Lasiodiplodia species associated with dieback and stem-end rot of mango in Northeastern Brazil, 72 h after inoculation with mycelium colonized agar plugs onto wounded fruits of Tommy Atkins cultivar. Bars above columns are the standard error of the mean. Columns with same letter do not differ significantly according to Fisher’s LSD test (P≤0.05)

ab a b ab a

40

a

a b

Lesion length (mm)

30

c c 20

c

c

10

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lod

La

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La

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p egy

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cae

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tia

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a ozg

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L

plo

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Species

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is different from all other species (Burgess et al. 2006). At present the host range is considered to be limited and this species has been reported only on Santalum album L., Eucalyptus urophylla S. T. Blake., V. vinifera, Corymbia sp. Hook., and Syzygium spp. Gaertn. (Sakalidis et al. 2011b; Perez et al. 2010; Burgess et al. 2006; ÚrbezTorres et al. 2010). Information about pathogenicity of this species is scarce. Lasiodiplodia crassispora was isolated as a stem endophyte from Adansonia gregorii F. Muell. and the pathogenicity trials demonstrated that it is pathogenic (Sakalidis et al. 2011b). It is likely that this fungus also occurs as an endophyte on mango but with the ability to cause disease. This study represents the first report of this species associated with mango in the world, with proven pathogenicity but low virulence compared to other species found in Northeastern Brazil. Lasiodplodia pseudotheobromae differs from L. theobromae in its bigger conidia that are more ellipsoid and do not taper as strongly towards the base. This species was described from Acacia, Citrus, Coffea, Gmelina and Rosa species (Alves et al. 2008). Worldwide, L. pseudotheobromae has been reported on numerous hosts, but in Brazil it has been reported only on Vitis spp. (Correia et al. 2012). The present work represents the first report on mango in Brazil. Regarding the pathogenicity, Sakalidis et al. (2011a) reports L. pseudotheobromae as the most virulent species in mango fruits in Australia. In another work, a pathogenicity test on mango tree saplings revealed that some isolates of L. pseudotheobromae were more virulent than L. theobromae (Ismail et al. 2012). In Terminalia catappa L., this species was also considered to be the most aggressive (Begoude et al. 2010). However, in the present work, compared with other species, L. pseudotheobromae did not show much virulence in mango fruit. Pathogenicity data in many crops is still scarce, as for a long time this species was identified as L. theobromae. However, recent works suggest that, like L. theobromae, L. pseudotheobromae also has worldwide distribution and a wide range of hosts (Abdollahzadeh et al. 2010; Begoude et al. 2010; Sakalidis et al. 2011a; Ismail et al 2012). Another species found in this study was L. egyptiacae, with only two isolates. This species was recently described on mango in Egypt. This is the first report of L. egyptiacae outside Egypt. Pathogenicity tests done by Ismail et al. (2012) showed that this species is less virulent on mango fruit than other Lasiodiplodia species. In this study, L. egyptiacae showed low levels of virulence on mango fruit. More sampling is necessary to understand the host range, distribution and variability of this species. Regarding cultural characteristics, the optimum temperature for mycelial growth for the Lasiodiplodia species varied between 28 and 31 °C. In addition, all the species in this study grew at a temperature of 10 °C. This growth at low

191

temperature corroborates the work of Abdollahzadeh et al. (2010) and is in contrast to other studies that show only L. pseudotheobromae as capable of growing at this temperature (Alves et al. 2008; Ismail et al. 2012). As can be observed, cultural characteristics vary greatly amongst isolates of the same species and are therefore, of limited value in the determination of species. All the species found in Northeastern Brazil have potential to cause disease to mango, but L. hormozganensis and Lasiodiplodia sp. were the most virulent species. Information about these species is scarce because of its recent descriptions. Studies are needed on the epidemiology and impact on mango production together with information referring to ecology, distribution and host range of all species of Lasiodiplodia found in this study. The correct identification of plant pathogenic fungi is of utmost importance for quarantine and control measures, once the control measures are specific for each pathogen. This study found seven species of Lasiodiplodia associated with dieback and stem end rot in mango tree. Future research on the distribution and epidemiology are important and could aid in finding effective management strategies of these pathogens that represent a threat to the mango crop in Brazil. Acknowledgments We are grateful to Sequencing Platform LABCEN/CCB in the Universidade Federal de Pernambuco for use of its facilities. This work was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 141275/2009-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES/BEX 0245/12-7). M. P. S. Câmara, Marcos A. Morais Junior and S. J. Michereff also acknowledge the CNPq research fellowship. A. J.L. Phillips thanks Fundação para a Ciência e a Tecnologia (Portugal) for financial support through grant PEst-OE/BIA/UI0457/2011

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