Plant Disease • 2019 • 103:1156-1165 • https://doi.org/10.

1094/PDIS-06-18-1036-RE

Phylogeny, Distribution, and Pathogenicity of Lasiodiplodia Species Associated

With Cankers and Dieback Symptoms of Persian Lime in Mexico
M. A. Bautista-Cruz,1 G. Almaguer-Vargas,1 S. G. Leyva-Mir,2 M. T. Colinas-León,1 K. C. Correia,3
M. Camacho-Tapia,4 L. Robles-Yerena,1 S. J. Michereff,3 and J. M. Tovar-Pedraza5,†
1
Posgrado en Horticultura, Departamento de Fitotecnia, Universidad Autónoma Chapingo, Texcoco, 56230 Estado de México, Mexico;
2
Departamento de Parasitologı́a Agrı́cola, Universidad Autónoma Chapingo, Texcoco, 56230 Estado de México, Mexico; 3Centro
de Ciências Agrárias e da Biodiversidade, Universidade Federal do Cariri, Crato, 63.133-610 Ceará, Brazil; 4Laboratorio Nacional
de Investigación y Servicio Agroalimentario y Forestal, Universidad Autónoma Chapingo, Texcoco, 56230 Estado de México,
Mexico; and 5Laboratorio de Fitopatologı́a, Coordinación Culiacán, Centro de Investigación en Alimentación y Desarrollo,
Culiacán, 80110 Sinaloa, Mexico

Abstract
Persian lime (Citrus latifolia Tan.) is an important and widely cultivated identified as Lasiodiplodia pseudotheobromae, Lasiodiplodia theobro-
fruit crop in several regions of Mexico. In recent years, severe symptoms mae, Lasiodiplodia brasiliense, Lasiodiplodia subglobosa, Lasiodiplo-
of gummosis, stem cankers, and dieback were detected in the Persian dia citricola, and Lasiodiplodia iraniensis. All Lasiodiplodia species
lime-producing region in the states of Veracruz and Puebla, Mexico. of this study are reported for the first time in association with Persian lime
The aims of this study were to identify the species of Lasiodiplodia asso- in Mexico and worldwide. L. pseudotheobromae (46.9% of isolates) was
ciated with these symptoms, determine the distribution of these species, the most frequently isolated species followed by L. theobromae (28.1%)
and test their pathogenicity and virulence on Persian lime plants. In 2015, and L. brasiliense (12.5%). Pathogenicity on Persian lime young plants
symptomatic samples were collected from 12 commercial Persian lime using a mycelial plug inoculation method showed that all identified
orchards, and 60 Lasiodiplodia isolates were obtained. Fungal identifica- Lasiodiplodia species were able to cause necrotic lesions and gummosis,
tion of 32 representative isolates was performed using a phylogenetic but L. subglobosa, L. iraniensis, and L. pseudotheobromae were the most
analysis based on DNA sequence data of the internal transcribed spacer virulent.
region and part of the translation elongation factor 1-a and b-tubulin
genes. Sequence analyses were carried out using the Maximum Likeli- Keywords: Botryosphaeriaceae, Citrus latifolia, fungal diversity,
hood and Bayesian Inference methods. Six Lasiodiplodia species were pathogenicity, phylogenetic analysis

Mexico occupies the second place in world production of limes Al-Sadi et al. 2013; Alves et al. 2008; Coutinho et al. 2017; Davis
and lemons with a total of 2.4 million tonnes (Food and Agriculture et al. 1987; Ferrari et al. 1996; Guajardo et al. 2018; Linaldeddu
Organization [FAO] 2016). Currently, this country is considered et al. 2015).
the main producer and exporter of Persian lime (Citrus latifolia The main features that distinguish the genus Lasiodiplodia from
Tan.) worldwide, with a cultivated area of 82,900 ha and a total other closely related genera are the presence of pycnidial paraphy-
production of 1.01 million tonnes (SIAP 2013). The main produc- ses and longitudinal striations on mature conidia (Abdollahzadeh
tion area for Persian lime in Mexico is concentrated in the central et al. 2010). Currently, the identification of Lasiodiplodia species
coastal region of the Gulf of Mexico, and 46% of production is by morphology is impossible, and it is necessary to use DNA se-
exported to the U.S.A., Europe, Canada, and Japan. Fruits are used quence data, preferably combining sequences from multiple loci,
fresh, and they are also used for the extraction of pectin and essen- such as internal transcribed spacer (ITS), b-tubulin (BT2), and
tial oils for foreign markets (Mendoza-Tornez et al. 2016). Dieback translation elongation factor 1-a (TEF1) genes, for accurate identi-
of Persian lime trees is a disease that decreases the production and fication (Chen et al. 2014a, 2014b; Coutinho et al. 2017; Machado
longevity of orchards in the lime-producing region of the states et al. 2014; Phillips et al. 2013; Rosado et al. 2016; Slippers et al.
of Puebla and Veracruz, Mexico, but the causative agents are 2014; Zhou et al. 2015).
unknown. Previous studies have associated Lasiodiplodia spp. with symp-
The Botryosphaeriaceae family (Dothideomycetes, Botryosphaer- toms of dieback, gummosis, and canker in a wide diversity of
iales) includes 23 genera and 187 species, with cosmopolitan distri- Citrus species, including sweet orange (Citrus sinensis Osbeck)
bution on a broad range of woody hosts occurring as endophytes, in the U.S.A., Venezuela, Oman, Algeria, and Brazil (Al-Sadi
saprobes, or destructive plant pathogens (Dissanayake et al. 2016; et al. 2013; Coutinho et al. 2017; Davis et al. 1987; Ferrari
Phillips et al. 2013; Slippers and Wingfield 2007; Slippers et al. et al. 1996; Linaldeddu et al. 2015); acid lime (Citrus aurantifo-
2013). Several Botryosphaeriaceae species have been previously as- lia S.), sweet lime (Citrus limettioides Tan.), and mandarin
sociated with citrus diseases, including Diplodia spp., Dothiorella (Citrus reticulata Blanco) in Oman (Al-Sadi et al. 2013); sour
spp., Neofusicoccum spp. (Adesemoye et al. 2014), Neoscytalidium orange (Citrus aurantium L.) in the U.S.A. and Suriname (Alves
spp. (Adesemoye et al. 2014; Mayorquin et al. 2016), and Lasiodi- et al. 2008; Davis et al. 1987); grapefruit (Citrus paradisi Mac-
plodia spp. (Abdollahzadeh et al. 2010; Adesemoye et al. 2014; fad) in the U.S.A. and Venezuela (Davis et al. 1987; Ferrari et al.
1996); lemon (Citrus limon L. Osbeck) in Venezuela and Chile
(Ferrari et al. 1996; Guajardo et al. 2018); and Citrus species
†
Corresponding author: J. M. Tovar-Pedraza; juan.tovar@ciad.mx in Iran and the U.S.A. (Abdollahzadeh et al. 2010; Adesemoye
et al. 2014). However, there are no studies of Lasiodiplodia
The author(s) declare no conflict of interest.
species affecting Persian lime trees worldwide.
Accepted for publication 2 November 2018. The aims of this study were to identify the species of Lasiodiplodia
associated with these symptoms by phylogenetic inference, deter-
mine the distribution of these species, and test their pathogenicity
© 2019 The American Phytopathological Society and virulence on young Persian lime plants.

1156 Plant Disease / Vol. 103 No. 6

Materials and Methods Ið%Þ = ðni=NÞ × 100;  where  I = incidence  of  disease;   ni = total  number  of 
symptomatic  trees;   and  N = total  number  of  evaluated  trees:
Field survey. In September 2015, a survey was conducted in 12
commercial Persian lime orchards distributed in the states of Puebla Isolates. Fragments of branches (4 to 5 mm in diameter) were col-
and Veracruz in the central coastal region of the Gulf of Mexico. lected from the margin between necrotic and healthy tissues, surface
Symptomatic plant tissues (4 to 14 years old) showing stem cankers, sterilized with 1% sodium hypochlorite for 1 min, rinsed three times
gummosis, and branches dieback were collected (Fig. 1). Disease in- with sterile distilled water, and dried on sterile paper. Fragments were
cidence was calculated using the following formula: placed in petri plates containing 2% potato dextrose agar (PDA;

Fig. 1. Symptoms of dieback and canker of Persian lime (Citrus latifolia) associated with Lasiodiplodia spp. in Mexico. A, Tree showing dieback of branches. B, Tree exhibiting
severe defoliation. C and D, Sunken cankers on branches. E, Branch with longitudinal cracking of the bark. F, Branch with canker and internal tissue necrosis. G, Diseased trunk
with severe wood necrosis.

Plant Disease / June 2019 1157

Difco, Detroit, MI) supplemented with 0.5 g/L streptomycin sulfate DNA extraction, polymerase chain reaction amplification,
(Sigma-Aldrich Co, St. Louis, MO) and incubated at 25°C in the and sequencing. Aerial mycelium from 6-day-old culture was
dark. Pure cultures were obtained by transferring hyphal tips from scraped directly from the medium using a sterile spatula and placed
the colony margin onto fresh PDA and incubated at 25°C in the dark. in 2-ml microtubes. Total genomic DNA was extracted using a
The isolates used in this study were deposited in the Culture Collec- DNeasy Plant Mini Kit (Qiagen, Valencia, CA) following the manu-
tion of Phytopathogenic Fungi of the Department of Agricultural Par- facturer’s recommendation. DNA concentrations were quantified us-
asitology at the Chapingo Autonomous University. Stock cultures ing a NanoDrop Lite Spectrophotometer (Thermo Fisher Scientific,
were stored in 2.0-ml cryovials in 15% glycerol at −80°C and sterile Madison, WI), and the samples were diluted to 100 ng/ml for poly-
water at 4°C. merase chain reaction (PCR).

Table 1. GenBank accession numbers of DNA sequences of Lasiodiplodia spp. included in the phylogenetic studya
GenBank accession number
Species Isolate code Host Location ITS TEF1 BT2
Lasiodiplodia avicenniae CMW41467 Avicennia marina South Africa KP860835 KP860680 KP860758
Lasiodiplodia avicenniae LAS199 (DNA) Avicennia marina South Africa KU587957 KU587947 KU587868
Lasiodiplodia brasiliense CMM4015 Mangifera indica Brazil JX464063 JX464049 N/A
Lasiodiplodia brasiliense CMW35884 Adansonia madagascariensis Madagascar KU887094 KU886972 KU887466
Lasiodiplodia brasiliense UACH257 Citrus latifolia Mexico MH277910 MH286537 MH279903
Lasiodiplodia brasiliense UACH266 Citrus latifolia Mexico MH277911 MH286535 MH279905
Lasiodiplodia brasiliense UACH267 Citrus latifolia Mexico MH277912 MH286536 MH279906
Lasiodiplodia brasiliense UACH269 Citrus latifolia Mexico MH277913 MH286538 MH279904
Lasiodiplodia brugulerae CMW41470 Bruguiera gymnorrhiza South Africa KP860833 KP860678 KP860756
Lasiodiplodia brugulerae CMW42480 Bruguiera gymnorrhiza South Africa KP860832 KP860677 KP860755
Lasiodiplodia caatinguensis IBL271 Anacardium occidentale Brazil KT154756 KT154750 KT154763
Lasiodiplodia caatinguensis CMM1325 Citrus sinensis Brazil KT154760 KT008006 KT154767
Lasiodiplodia chinensis CGMCC3.18061 Unknown China KX499889 KX499927 KX500002
Lasiodiplodia chinensis CGMCC3.18044 Vaccinium uliginosum China KX499875 KX499913 KX499988
Lasiodiplodia citricola UACH262 Citrus latifolia Mexico MH277948 MH286541 MH279934
Lasiodiplodia citricola CBS124707 Citrus sp. Iran GU945354 GU945340 KU887505
Lasiodiplodia citricola IRAN1521C Citrus sp. Iran GU945353 GU945339 KU887504
Lasiodiplodia crassispora CBS118741 Santalum album Australia DQ103550 EU673303 KU887506
Lasiodiplodia crassispora CMW13488 Eucalyptus urophylla Venezuela DQ103552 DQ103559 KU887507
Lasiodiplodia euphorbicola CMM3609 Jatropha curcas Brazil KF234543 KF226689 KF254926
Lasiodiplodia euphorbicola CMM3651 Jatropha curcas Brazil KF234553 KF226711 KF254937
Lasiodiplodia exigua BL184 Retama raetam Tunisia KJ638318 KJ638337 N/A
Lasiodiplodia exigua CBS137785 Retama raetam Tunisia KJ638317 KJ638336 KU887509
Lasiodiplodia gilanensis IRAN1523C Unknown Iran GU945351 GU945342 KU887511
Lasiodiplodia gilanensis IRAN1501C Unknown Iran GU945352 GU945341 KU887511
Lasiodiplodia gonubiensis CBS115812 Syzigium cordatum South Africa AY639595 DQ103566 DQ458860
Lasiodiplodia gonubiensis CMW14078 Syzigium cordatum South Africa AY639594 DQ103567 EU673126
Lasiodiplodia gravistriata CMM4564 Anacardium humile Brazil KT250949 KT250950 N/A
Lasiodiplodia gravistriata CMM4570 Anacardium humile Brazil KT250948 KT266814 N/A
Lasiodiplodia hyalina CGMCC3.17975 Acacia confusa China KX499879 KX499917 KX499992
Lasiodiplodia hyalina CGMCC3.18383 Unknown China KY767661 KY751302 KY751299
Lasiodiplodia hormozganensis IRAN1500C Olea sp. Iran GU945355 GU945343 KU887515
Lasiodiplodia hormozganensis IRAN1498C Mangifera indica Iran GU945356 GU945344 KU887514
Lasiodiplodia iraniensis IRAN1502C Juglans sp. Iran GU945347 GU945335 KU887517
Lasiodiplodia iraniensis IRAN1520C Salvadora persica Iran GU945348 GU945336 KU887516
Lasiodiplodia iraniensis UACH275 Citrus latifolia Mexico MH271621 MH286542 MH279933
Lasiodiplodia iraniensis CMM3610 Jatropha curcas Brazil KF234544 KF226690 KF254927
Lasiodiplodia iraniensis CMW36237 Adansonia digitata Mozambique KU887121 KU886998 KU887499
Lasiodiplodia laeliocattleyae BOT 29 Mangifera indica Egypt JN814401 JN814428 N/A
Lasiodiplodia laeliocattleyae CBS130992 Mangifera indica Egypt JN814397 JN814424 KU887508
Lasiodiplodia lignicola CBS134112 Deadwood Thailand JX646797 KU887003 JX646845
Lasiodiplodia macrospora CMM3833 Jatropha curcas Brazil KF234557 KF226718 KF254941
Lasiodiplodia mahajangana CMW27818 Terminalia catappa Madagascar FJ900596 FJ900642 FJ900631
Lasiodiplodia mahajangana CMW27801 Terminalia catappa Madagascar FJ900595 FJ900641 FJ900630
Lasiodiplodia margaritaceae CBS122519 Adansonia gibbosa Australia EU144050 EU144065 KU887520
Lasiodiplodia margaritaceae CBS122065 Adansonia gibbosa Australia EU144051 EU144066 N/A
(Continued on next page)
a Newly deposited sequences are shown in bold. BL, personal number of B. T. Linaldeddu; BOT, A. M. Ismail, Plant Pathology Research Institute, Giza, Egypt;
BT2, b-tubulin; CBS, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands; CGMCC, China General Microbiological Culture Collection, Beijing,
China; CMM, Culture Collection of Phytopathogenic Fungi “Professora Maria Menezes,” Universidade Federal Rural de Pernambuco, Recife, Brazil; CMW,
Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa; CPC, working collection of P. W. Crous housed at the CBS;
N/A, sequence not available in GenBank; IBL, personal culture collection of I. B. L. Coutinho; IRAN, Culture Collection of the Iranian Research Institute of Plant
Protection, Tehran, Iran; ITS, internal transcribed spacer; STE-U, Culture Collection of the Department of Plant Pathology, University of Stellenbosch, Stellenbosch,
South Africa; TEF1, translation elongation factor 1-a; UACH, Culture Collection of Phytopathogenic Fungi of Department of Agricultural Parasitology, Chapingo
Autonomous University, Texcoco, Mexico; UCD, Phaff Yeast Culture Collection, Department of Food Science and Technology, University of California, Davis,
U.S.A.; WAC, Department of Agriculture Western Australia Plant Pathogen Collection, Perth, Australia.

1158 Plant Disease / Vol. 103 No. 6

 The ITS region of ribosomal DNA was amplified using the primers conducted in a Bio-Rad C1000 thermocycler (Bio-Rad Laboratories,
ITS5/ITS4 (White et al. 1990). Parts of TEF1 and BT2 genes were Hercules, CA). The PCR products were separated by electrophoresis
amplified using the primers EF1-688F/EF1-1251R (Alves et al. in 1% agarose gel stained with ethidium bromide and viewed under ul-
2008) and Bt2A/Bt2B (Glass and Donaldson 1995), respectively. traviolet light. The amplified PCR products were purified using the
The amplification conditions were as follows: an initial denaturation QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and se-
step at 94°C for 2 min followed by 35 cycles of denaturation at 94°C quenced by Macrogen (Macrogen Inc., Seoul, Korea) in both direc-
for 30 s; annealing at 55°C for the ITS region, 54°C for the TEF1 gene, tions with the same primers that were used for the PCR reactions.
and 58°C for BT2 gene for 30 s; extension at 72°C for 1 min; and a Phylogenetic analyses. Forward and reverse sequences were as-
final extension step at 72°C for 10 min. The PCR assays were sembled using the Staden Package (Staden et al. 1998). Sequences

Table 1. (Continued from previous page)

GenBank accession number

Species Isolate code Host Location ITS TEF1 BT2
Lasiodiplodia mediterranea CBS137784 Vitis vinifera Italy KJ638311 KJ638330 KU887522
Lasiodiplodia mediterranea CBS137783 Quercus ilex Italy KJ638312 KJ638331 KU887521
Lasiodiplodia missouriana UCD2193MO Vitis sp. U.S.A. HQ288225 HQ288267 HQ288304
Lasiodiplodia missouriana UCD2199MO Vitis sp. U.S.A. HQ288226 HQ288268 HQ288305
Lasiodiplodia parva CBS494.78 Cassava field soil Colombia EF622084 EF622064 EU673114
Lasiodiplodia parva CBS456.78 Cassava field soil Colombia EF622083 EF622063 KU887523
Lasiodiplodia plurivora STE-U5803 Prunus salicina South Africa EF445362 EF445395 KU887524
Lasiodiplodia plurivora STE-U4583 Vitis vinifera South Africa AY343482 EF445396 KU887525
Lasiodiplodia pontae IBL12 Spondias purpurea Brazil KT151794 KT151791 KT151797
Lasiodiplodia pseudotheobromae CMM3887 Jatropha curcas Brazil KF234559 KF226722 KF254943
Lasiodiplodia pseudotheobromae CBS116459 Gmelina arborea Costa Rica EF622077 EF622057 EU673111
Lasiodiplodia pseudotheobromae UACH258 Citrus latifolia Mexico MH277917 MH286514 MH279929
Lasiodiplodia pseudotheobromae UACH259 Citrus latifolia Mexico MH277926 MH286522 MH279919
Lasiodiplodia pseudotheobromae UACH260 Citrus latifolia Mexico MH277921 MH286518 MH279924
Lasiodiplodia pseudotheobromae UACH261 Citrus latifolia Mexico MH277919 MH286516 MH279926
Lasiodiplodia pseudotheobromae UACH265 Citrus latifolia Mexico MH277916 MH286513 MH279930
Lasiodiplodia pseudotheobromae UACH271 Citrus latifolia Mexico MH277920 MH286517 MH279925
Lasiodiplodia pseudotheobromae UACH274 Citrus latifolia Mexico MH277923 MH286520 MH279922
Lasiodiplodia pseudotheobromae UACH276 Citrus latifolia Mexico MH277915 MH286512 MH279931
Lasiodiplodia pseudotheobromae UACH277 Citrus latifolia Mexico MH277918 MH286515 MH279927
Lasiodiplodia pseudotheobromae UACH278 Citrus latifolia Mexico MH277927 MH286524 MH279918
Lasiodiplodia pseudotheobromae UACH279 Citrus latifolia Mexico MH277922 MH286519 MH279923
Lasiodiplodia pseudotheobromae UACH281 Citrus latifolia Mexico MH277924 MH286521 MH279921
Lasiodiplodia pseudotheobromae UACH283 Citrus latifolia Mexico MH277914 MH286511 MH279932
Lasiodiplodia pseudotheobromae UACH286 Citrus latifolia Mexico MH277925 MH286523 MH279920
Lasiodiplodia pseudotheobromae UACH268 Citrus latifolia Mexico MH277928 MH286525 MH279928
Lasiodiplodia pyriformis CBS121771 Acacia mellifera Namibia EU101308 EU101353 KU887528
Lasiodiplodia pyriformis CBS121770 Acacia mellifera Namibia EU101307 EU101352 KU887527
Lasiodiplodia rubropurpurea CMW15207 Eucalyptus grandis Australia DQ103554 DQ103572 KU887530
Lasiodiplodia rubropurpurea CBS118740 Eucalyptus grandis Australia DQ103553 DQ103571 EU673136
Lasiodiplodia thailandica CPC22795 Mangifera indica Thailand KJ193637 KJ193681 N/A
Lasiodiplodia thailandica CPC22755 Phyllanthus acidus Thailand KJ193637 KJ193681 N/A
Lasiodiplodia theobromae CBS111530 Unknown Unknown EF622074 EF622054 KU887531
Lasiodiplodia theobromae CBS164.96 Fruit along coral reef coast New Guinea AY640255 AY640258 KU887532
Lasiodiplodia theobromae UACH263 Citrus latifolia Mexico MH277691 MH286528 MH279908
Lasiodiplodia theobromae UACH264 Citrus latifolia Mexico MH277692 MH286534 MH279909
Lasiodiplodia theobromae UACH272 Citrus latifolia Mexico MH277693 MH286533 MH279915
Lasiodiplodia theobromae UACH273 Citrus latifolia Mexico MH277694 MH286526 MH279907
Lasiodiplodia theobromae UACH280 Citrus latifolia Mexico MH277695 MH286527 MH279910
Lasiodiplodia theobromae UACH284 Citrus latifolia Mexico MH277696 MH286532 MH279914
Lasiodiplodia theobromae UACH285 Citrus latifolia Mexico MH277697 MH286531 MH279913
Lasiodiplodia theobromae UACH287 Citrus latifolia Mexico MH277698 MH286530 MH279912
Lasiodiplodia theobromae UACH288 Citrus latifolia Mexico MH277699 MH286529 MH279911
Lasiodiplodia sterculiae CBS342.78 Sterculia oblonga Germany KX464140 KX464634 KX464908
Lasiodiplodia subglobosa CMM4046 Jatropha curcas Brazil KF234560 KF226723 KF254944
Lasiodiplodia subglobosa CMM3872 Jatropha curcas Brazil KF234558 KF226721 KF254942
Lasiodiplodia subglobosa UACH270 Citrus latifolia Mexico MH271619 MH286539 MH279916
Lasiodiplodia subglobosa UACH282 Citrus latifolia Mexico MH271620 MH286540 MH279917
Lasiodiplodia venezualensis CBS118739 Acacia mangium Venezuela DQ103547 DQ103568 KU887533
Lasiodiplodia venezuelensis WAC12540 Acacia mangium Venezuela DQ103548 DQ103569 KU887534
Lasiodiplodia viticola UCD2604MO Vitis sp. U.S.A. HQ288228 HQ288270 HQ288307
Lasiodiplodia viticola UCD2553AR Vitis sp. U.S.A. HQ288227 HQ288269 HQ288306
Lasiodiplodia vitis CBS124060 Vitis vinifera Italy KX464148 KX464642 KX464917
Neoscytalidium dimidiatum CBS145.78 Homo sapiens United Kingdom KF531816 KF531795 KF531796

Plant Disease / June 2019 1159

of the type and reference strains of Lasiodiplodia species of each 2). The first clade with nine isolates (Culture Collection of Phyto-
locus analyzed were downloaded from GenBank and combined pathogenic Fungi of Department of Agricultural Parasitology,
with the newly generated sequences (Table 1). Multiple sequence Chapingo Autonomous University, Texcoco, Mexico [UACH]
alignments for each locus independently were performed using 263, UACH264, UACH272, UACH273, UACH280, UACH284,
the MEGA 7.0.14 (Kumar et al. 2016), and manual adjustments UACH285, UACH287, and UACH288) clustered with Lasiodi-
were done where necessary. Alignment of each locus was loaded plodia theobromae (Centraalbureau voor Schimmelcultures,
in SequenceMatrix v.1.8 (Vaidya et al. 2011) to build the concat- Utrecht, The Netherlands [CBS] 111530 and CBS164.96), with
enated matrix. 83% bootstrap support. The second clade with four isolates
The phylogeny for each locus (ITS, TEF1, and BT2) and the (UACH257, UACH266, UACH267, and UACH269) clustered
concatenated matrix were inferred under the Maximum Likeli- with Lasiodiplodia brasiliense (Culture Collection of Phytopath-
hood (ML) and Bayesian Inference (BI) criterion. The ML analy- ogenic Fungi “Professora Maria Menezes,” Universidade Federal
ses were done in RAxML-HPC2 (Stamatakis 2014), and Bayesian Rural de Pernambuco, Recife, Brazil [CMM] 4015 and Forestry
phylogenetic estimates were done in MrBayes 3.2.6 (Ronquist and Agricultural Biotechnology Institute, University of Pretoria,
et al. 2012) implemented on the CIPRES Science Gateway portal Pretoria, South Africa [CMW] 35884), with 86% bootstrap sup-
(https://www.phylo.org/portal2/home.action). The ML tree searches port. In the third clade, only one isolate (UACH275) clustered
were performed under the GTRGAMMA model with 1,000 pseudo- with Lasiodiplodia iraniensis (CMM3610 and CMW36237), with
replicates. The BI trees were constructed using the GTR + I + G 100% bootstrap support. The fourth clade with two isolates
(ITS), HKY + G (TEF1), and GTR + G (BT2) nucleotide substitution (UACH270 and UACH282) clustered with Lasiodiplodia subglo-
models, with four runs and four MCMC chains per run; Markov bosa (CMM3872 and CMM4046), with 97% bootstrap support.
chains were run over 100,000,000 generations, and trees and param- The fifth clade had the highest number of isolates (UACH258,
eter values were sampled every 10,000 generations. Sequences gen- UACH259, UACH260, UACH261, UACH265, UACH268,
erated in this study were deposited in GenBank (Table 1), and the UACH271, UACH274, UACH276, UACH277, UACH278,
alignments and tree are in TreeBASE (http://purl.org/phylo/tree- UACH279, UACH281, UACH283, and UACH286), which clus-
base/phylows/study/TB2:S23111). tered with Lasiodiplodia pseudotheobromae (CBS116459 and
Pathogenicity and virulence tests. Pathogenicity of 32 repre- CMM3887), with 85% bootstrap support. The sixth clade has only
sentative isolates was tested on healthy 18-month-old Persian one isolate (UACH262) clustered with Lasiodiplodia citricola
lime plants obtained from a commercial nursery. For inoculation, (CBS124707 and Culture Collection of the Iranian Research Insti-
branches of Persian lime plants were wounded using a sterile scal- tute of Plant Protection, Tehran, Iran 1521C), with 100% boot-
pel, and a colonized PDA plug (5-mm diameter) from a 6-day-old strap support (Fig. 2).
culture was placed on a wounded area. The inoculation site was Distribution of Lasiodiplodia species. L. pseudotheobromae was
sealed with parafilm to prevent desiccation. Four branches (two the most frequently isolated species (46.9%) from Persian lime
per plant) were inoculated with each isolate. Ten control branches trees followed by L. theobromae (28.1%), L. brasiliense (12.5%),
were inoculated with noncolonized PDA plugs. Immediately after L. subglobosa (6.3%), L. iraniensis (3.1%), and L. citricola
inoculation, each plant was covered with a plastic bag for 72 h to (3.1%). The distribution of Lasiodiplodia species varied among the
maintain humidity. All plants were maintained in a glasshouse un- five populations distributed in the states of Puebla and Veracruz,
der natural light conditions, and the temperature was regulated at Mexico (Fig. 3). L. pseudotheobromae was found in all populations.
30 °C ± 5°C by air conditioning. Virulence of isolates was assessed L. theobromae and L. subglobosa were found in at least one popula-
30 days after inoculation by removing the bark and measuring the le- tion of each state. L. brasiliense was recorded only in the two popu-
sion length in the wood with a digital caliper. The experiment was con- lations from the state of Puebla. L. iraniensis was identified only in
ducted twice. Differences in virulence caused by Lasiodiplodia species the Papantla population. L. citricola was only isolated from an or-
were determined by one-way analysis of variance, and means were chard located in Acateno, Puebla.
compared by LSD test at a = 0.05 using SAS (version 9.1; SAS Insti- Pathogenicity and virulence on branches. Thirty days after inoc-
tute, Cary, NC). ulation, all isolates belonging to the six species of Lasiodiplodia
identified in this study were pathogenic on Persian lime young plants
Results using a mycelial plug inoculation method. The symptoms produced
Field survey. Symptoms of gummosis, stem cankers, and dieback on the wood were necrotic lesions that extended upward and down-
were detected in all surveyed orchards of Persian lime distributed in ward from the inoculation site (Fig. 4). All isolates of Lasiodiplodia
the states of Puebla and Veracruz. The highest incidence of disease induced the formation of gum exudations on the branches (Fig. 4),
was observed in the municipality of Hueytamalco with 97% followed whereas no disease symptoms were observed on the control plants.
by Martinez de la Torre with 96.5% and Acateno with 94.3%. The Lasiodiplodia isolates were consistently recovered from the diseased
lowest incidence rates were detected in Tlapacoyan and Papantla branches, whereas no Lasiodiplodia spp. were isolated from healthy
with 89 and 86%, respectively. control plants, thus fulfilling Koch’s postulates. The two experiments
Collection of samples and isolation. In this study, a total of 60 did not differ significantly (P = 0.05); thus, the data from both exper-
Lasiodiplodia isolates were obtained from Persian lime diseased tis- iments were combined for statistical analyses. The mean lesion
sues collected in 12 commercial orchards (5 isolates per orchard) dis- lengths caused by species of Lasiodiplodia for the combination of
tributed in the municipalities of Martinez de la Torre, Tlapacoyan, two experiments are shown in Fig. 5.
and Papantla in the state of Veracruz as well as in Acateno and There were significant differences (P = 0.001) in internal necro-
Hueytamalco in the state of Puebla, Mexico. Based on colony mor- sis length produced by the different Lasiodiplodia species. The lon-
phology, a subsample of 32 representative isolates (at least two per gest mean lesions (>19 mm) were produced by L. subglobosa,
orchard) was used for the phylogenetic analyses and pathogenicity L. iraniensis, and L. pseudotheobromae, which were the most
tests. virulent species. In addition, these species caused longitudinal
Phylogenetic analyses. The isolates of Lasiodiplodia species cracks on the bark of the branches. However, the shorter mean
were identified based on ML and BI phylogenetic analyses of lesions (<14 mm) were induced by L. theobromae, L. brasi-
the ITS region and parts of TEF1 and BT2 genes. The combined liense, and L. citricola, which were considered the least virulent
ITS, TEF1, and BT2 datasets consisted of 100 taxa, including 1 species.
outgroup taxon. The alignment contained 1,309 characters, of
which 348 corresponded with the TEF1 gene, 430 corresponded Discussion
with the BT2 gene, and 531 corresponded with the ITS region. This study showed the first findings about Lasiodiplodia species
The combined datasets resulted in six well-supported clades cor- infecting Persian lime trees in Mexico. Six Lasiodiplodia species
responding to previously described Lasiodiplodia species (Fig. were isolated from stem cankers and dieback of branches, and they
1160 Plant Disease / Vol. 103 No. 6
were identified based on phylogenetic analyses of combined ITS, Lasiodiplodia isolates was not performed, because the identification
BT2, and TEF1 sequence datasets. These species included L. pseudo- of Botryosphaeriaceae species using morphology is virtually impos-
theobromae, L. theobromae, L. brasiliense, L. subglobosa, L. citri- sible, and it should be only used to distinguish between genera and
cola, and L. iraniensis. All Lasiodiplodia species of this study are complement the DNA phylogeny for the description of new species
reported for the first time in association with Persian lime in Mexico (Ismail et al. 2012; Osorio et al. 2017; Phillips et al. 2013; Slippers
and worldwide. A detailed morphological characterization of et al. 2014). The most appropriate way to differentiate species within

Fig. 2. Phylogenetic tree of Lasiodiplodia species inferred from a concatenated alignment of an internal transcribed spacer, b-tubulin, and translation elongation factor 1-a
sequence alignments. Bootstrap values by the Maximum Likelihood method and probabilities by the Bayesian Inference analyses are shown on the respective branches.
Bootstrap values below 80% and posterior probabilities below 0.90 are not shown. The tree is rooted to Neoscytalidium dimidiatum CBS 145.78. Epi- and ex-type strains are
indicated in bold. The scale bar indicates the average number of substitutions per site.

Plant Disease / June 2019 1161

Fig. 3. Collection sites of six Lasiodiplodia spp. associated with Persian lime (Citrus latifolia) dieback in five populations distributed in the states of Puebla (PUE) and Veracruz
(VER), Mexico. Circles represent association frequency of each species with Persian lime trees showing symptoms of dieback in each population sampled, v is the number of
commercial orchards sampled in each population, and n is the number of isolates analyzed in each population. L. brasiliense, Lasiodiplodia brasiliense; L. citricola, Lasiodiplodia
citricola; L. iraniensis, Lasiodiplodia iraniensis; L. pseudotheobromae, Lasiodiplodia pseudotheobromae; L. subglobosa, Lasiodiplodia subglobosa; L. theobromae, Lasiodiplodia
theobromae.

Fig. 4. Symptoms developed in the branches of young Persian lime (Citrus latifolia) plants artificially inoculated with Lasiodiplodia spp. 30 days after inoculation. A and B, Branches
producing gum exudations. C, Symptomatic branch with necrosis and gummosis. D, Branch with necrosis around inoculation site.

1162 Plant Disease / Vol. 103 No. 6

Botryosphaeriaceae is through of DNA sequence data, preferably This finding contrasts with that found by Marques et al. (2013),
combining sequences from multiple loci (Dissanayake et al. 2016; who determined that L. theobromae was more virulent than L. pseu-
Phillips et al. 2013; Rodrı́guez-Gálvez et al. 2017; Slippers et al. dotheobromae in inoculated mango fruits. In the case of citrus, Al-
2014). Sadi et al. (2014) found that L. theobromae was more virulent than
L. pseudotheobromae was the most frequently isolated species Lasiodiplodia hormozganensis on inoculated twigs of acid lime
from Persian lime plants with dieback symptoms, and it occurred and sweet lime, because L. hormozganensis induced symptoms of
in the five populations analyzed in this study. This fungal species gummosis and dieback, similar to those induced by L. theobromae
has been previously associated with diseases on some citrus spe- observed in our study.
cies, including sour orange in Suriname (Alves et al. 2008), Citrus L. brasiliense was the third most common species of all of the iso-
species in Iran (Abdollahzadeh et al. 2010), and lemon in Turkey lates examined in our study. This species was previously reported to
(Awan et al. 2016). Similarly, this fungus has been commonly iso- be a pathogen of mango (Coutinho et al. 2017; Netto et al. 2014;
lated from mango (Mangifera indica L.) tissues with symptoms of Rodrı́guez-Gálvez et al. 2017), papaya (Netto et al. 2014), coconut
dieback and/or stem end rot worldwide (Ismail et al. 2012; Marques palm (Rosado et al. 2016), table grapes (Correia et al. 2016), sapo-
et al. 2013; Munirah et al. 2017; Rodrı́guez-Gálvez et al. 2017; dilla (Manilkara zapota L.), hog plum (Coutinho et al. 2017), cashew
Sakalidis et al. 2011; Trakunyingcharoen et al. 2014; 2015). In ad- (Netto et al. 2017), custard apple (Annona squamosa L.) (Cardoso
dition, it has been recorded in Brazil on fruit trees, including et al. 2017), and apple (Martins et al. 2018) in Brazil. In addition, this
cashew (Anacardium occidentale L.) (Coutinho et al. 2017; Netto fungus was recorded on Tectona grandis in Thailand (Doilom et al.
et al. 2017), hog plum (Spondias purpurea L.), and Tamarindus 2015) and Eucalyptus hybrid (Li et al. 2018) and mango (Zhang et al.
indica L. (Coutinho et al. 2017). Results of pathogenicity tests 2018) in China. Thus, this study represents the first report of this fun-
revealed that isolates of L. pseudotheobromae were more virulent in gus on a citrus worldwide. In 2017, Cruywagen et al. (2017) consid-
Persian lime branches than L. theobromae isolates. These results agree ered L. brasiliense as a hybrid that could have arisen from
with those reported by Begoude et al. (2010, 2011) and Chen et al. hybridization between L. theobromae and another currently un-
(2011), who found that L. pseudotheobromae was more virulent than known species; however, additional studies are needed to confirm
L. theobromae when inoculated on stems of tropical almond (Termi- this hybrid status. In our study, L. brasiliense and L. theobromae
nalia catappa), Eucalyptus spp., and Terminalia spp. In contrast to exhibited low levels of virulence, whereas L. pseudotheobromae
our results, Correia et al. (2016), Marques et al. (2013), and Netto was highly virulent in Persian lime young plants. A recent study
et al. (2017) determined that L. theobromae isolates were more viru- by Netto et al. (2014) showed that L. brasiliense, L. theobromae,
lent than L. pseudotheobromae isolates when inoculated on mango and L. pseudotheobromae did not differ with respect to their viru-
fruits, detached green shoots of grapes, and detached green shoots lence in papaya fruits. In contrast to our results, Correia et al.
of cashew. (2016) found that L. brasiliense and L. theobromae were highly vir-
L. theobromae was the second most abundant species isolated ulent species, and L. pseudotheobromae exhibited an intermediate
from Persian lime orchards in Mexico. This species has a cosmopol- virulence in detached green shoots of grapes. Rosado et al. (2016) ob-
itan distribution, particularly in tropical and subtropical regions, served that L. theobromae was highly virulent in coconut fruits com-
where it causes a variety of diseases on a wide range of host plants pared with L. brasiliense and L. pseudotheobromae. Subsequently,
(Punithalingam 1980). Studies based on the combination of morpho- Netto et al. (2017) noted that L. brasiliense and L. theobromae
logical characters and DNA sequence data confirmed that this fungus exhibited intermediate virulence, and L. pseudotheobromae pre-
is a common pathogen in fruit crops of economic importance, such as sented a low level of virulence in detached green shoots of cashew.
mango (Abdollahzadeh et al. 2010; Al-Jabri et al. 2017; Al-Sadi et al. All of the studies mentioned above show a wide range of virulence
2013; de Oliveira Costa et al. 2010; Ismail et al. 2012; Marques et al. of L. brasiliense isolates in different tissues of fruit crop hosts.
2013; Munirah et al. 2017; Ni et al. 2012; Rodrı́guez-Gálvez et al. L. subglobosa was described from physic nut collected in Brazil
2017; Sakalidis et al. 2011; Trakunyingcharoen et al. 2014), papaya (Machado et al. 2014), and later, it was reported by Poletto et al.
(Carica papaya L.) (Netto et al. 2014), coconut palm (Cocos nucifera (2016) to be causing stem canker of pecan (Carya illinoinensis
L.) (Rosado et al. 2016), kiwifruit (Actinidia chinensis Planch.) [Wangenh.] Koch). Thus, this study represents the first report of
(Zhou et al. 2015), Vitis spp. (Correia et al. 2016; Rodrı́guez-Gálvez
et al. 2015), cashew (Coutinho et al. 2017; Netto et al. 2017), hog
plum, and Talisia esculenta (Coutinho et al. 2017). Similarly, L. the-
obromae has been recorded as the second most frequently isolated
species after L. pseudotheobromae in symptomatic tissues of the or-
namental tree known as tropical almond in Madagascar and South
Africa (Begoude et al. 2010). Regarding citrus species, Davis et al.
(1987) recorded a trunk disease of young citrus trees caused by
two isolates of L. theobromae in Texas, U.S.A. and also showed that
some isolates were apparently avirulent. This pathogen also has been
associated with dieback and gummosis symptoms on acid lime,
sweet orange, and sweet lime in Oman (Al-Sadi et al. 2013) and
lemon in Venezuela (Ferrari et al. 1996) and Chile (Guajardo et al.
2018). In Mexico, L. theobromae was previously associated as the
causal agent of bot canker disease of grapevines based on morpho-
logical and cultural characters as well as analyses of ITS, BT2, and
TEF1 sequences (Úrbez-Torres et al. 2008). Subsequently, other
studies have mentioned the occurrence of L. theobromae as a com-
mon pathogen on fruit crops in Mexico, including Pouteria sapota
(Vásquez-López et al. 2009), mango (Sandoval-Sánchez et al. Fig. 5. Virulence of six Lasiodiplodia species associated with dieback of Persian lime
2013), and physic nut (Uc-Várguez et al. 2017). However, all of these (Citrus latifolia) as measured by mean internal lesion lengths (millimeters). Data are
isolates were identified based on morphological characteristics and lesion sizes measured 30 days after inoculation with mycelium-colonized agar
analysis of the ITS region, and therefore, it is necessary to verify plugs inserted into wounded branches of young lime trees. Bars above columns are
the standard errors of the means. Columns with the same letter do not differ
and update the inventory of Botryosphaeriaceae species associated significantly according to Fisher’s LSD test (P # 0.05). L. brasiliense, Lasiodiplodia
with the main fruit crops in Mexico by analysis of sequences of at brasiliense; L. citricola, Lasiodiplodia citricola; L. iraniensis, Lasiodiplodia iraniensis;
least three genes. In this study, L. theobromae was less virulent than L. pseudotheobromae, Lasiodiplodia pseudotheobromae; L. subglobosa, Lasiodiplodia
L. pseudotheobromae in inoculated young plants of Persian lime. subglobosa; L. theobromae, Lasiodiplodia theobromae.

Plant Disease / June 2019 1163

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