Erasing the Past: A New Identity for the Damoclean Pathogen Causing South American Leaf Blight of Rubber

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Background: South American leaf blight (SALB) of rubber has been the main constraint to production in its neotropical centre of origin since commercial plantations were first established. The fungal causal agent was identified and described more than
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  Erasing the Past: A New Identity for the DamocleanPathogen Causing South American Leaf Blight of Rubber Braz Tavares da Hora Ju´ nior 1 , Davi Mesquita de Macedo 1 , Robert Weingart Barreto 1 , Harry C. Evans 1,2 ,Carlos Raimundo Reis Mattos 3 , Luiz Antonio Maffia 1 , Eduardo S. G. Mizubuti 1 * 1 Departamento de Fitopatologia, Universidade Federal de Vic¸osa, Vic¸osa, Minas Gerais, Brazil,  2 CAB International, E-UK Centre, Egham, Surrey, United Kingdom, 3 Plantac¸o˜es Michelin da Bahia, Itubera´, Bahia, Brazil Abstract Background:   South American leaf blight (SALB) of rubber has been the main constraint to production in its neotropicalcentre of srcin since commercial plantations were first established. The fungal causal agent was identified and describedmore than a century ago but its precise placement within the Ascomycota still remains uncertain. Indeed, such is theambiguity surrounding the pathogen that each of the spore morphs would, according to their present classification, beplaced in different ascomycete families: the  Microcyclus  sexual morph in the Planistromellaceae and the two purportedasexual morphs -  Fusicladium  and  Aposphaeria  – in the Venturiaceae and Lophiostomataceae, respectively. Given thehistorical importance of the fungus and the ever-menacing threat that it poses to rubber production in the Palaeotropics –and, thus to the rubber industry and to the global economy – its phylogeny, as well as its biology, should be resolved as amatter of urgency. Methods and Results:   Here, six genomic regions (LSU rRNA, mtSSU, MCM7, EF-1 a , Act and ITS) were used for reconstructingthe molecular phylogeny of the SALB fungus based on material collected throughout Brazil. The analyses support theclassification of the fungus in the family Mycosphaerellaceae s. str. (Capnodiales, Dothideomycetes) and place it firmlywithin the clade  Pseudocercospora  s. str., now accepted as one of the distinct genera within Mycosphaerellaceae. The newcombination  Pseudocercospora ulei   is proposed and the life cycle of the fungus is confirmed, based on both experimentaland phylogenetic evidence, with the  Aposphaeria  morph shown to have a spermatial rather than an infective-dispersalfunction. Conclusions:   Because the phylogeny of the SALB fungus has now been clarified, new insights of its epidemiology andgenomics can be gained following comparison with closely-related, better-researched crop pathogens. Citation:  Hora Ju´nior BTd, de Macedo DM, Barreto RW, Evans HC, Mattos CRR, et al. (2014) Erasing the Past: A New Identity for the Damoclean Pathogen CausingSouth American Leaf Blight of Rubber. PLoS ONE 9(8): e104750. doi:10.1371/journal.pone.0104750 Editor:  Daniel Ballhorn, Portland State University, United States of America Received  April 19, 2014;  Accepted  July 17, 2014;  Published  August 15, 2014 Copyright:    2014 Hora Ju´nior et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the srcinal author and source are credited. Data Availability:  The authors confirm that all data underlying the findings are fully available without restriction. The data set has been deposited to Genbank and are available under the accession numbers: KC800717-KC800782. Funding:  The work was funded by Plantac¸o˜es Michelin da Bahia. The funders had no role in study design, data collection and analysis, decision to publish, orpreparation of the manuscript. Competing Interests:  The authors take this opportunity to declare that the affiliation of Carlos R. R. Mattos with Plantac¸o˜es Michelin da Bahia does not alter anyadherence to PLOS ONE policies on sharing data and materials.* Email: Introduction South American leaf blight (SALB) of the rubber tree  Heveabrasiliensis  (Willd. ex A. L. Juss.) Muell.-Arg., caused by  Microcyclus ulei  (Henn.) Arx (Ascomycota), is recognized as themost serious threat to the natural rubber industry worldwide [1– 4]. Epidemics of SALB led to the failure of rubber plantations intropical America in the early 20 th century, as epitomized by thedemise of Fordlandia in the Lower Amazon region of Brazildespite enormous investment in research and development [5,6].Currently, the world supply of natural rubber is highly dependenton the plantations established in Southeast Asia [3]. Themagnitude of the threat represented by the SALB fungus ishighlighted by Money [7] who described the relevance of naturalrubber as an irreplaceable prime matter for the world’s industry ina plethora of applications besides tires, machinery belts andcondoms and as an industry itself providing the livelihood of 30million people, concluding that ‘‘Nothing else (but  M. ulei  ) has thepower to terminate the global flow of latex.’’ Because of thepotential serious economic consequences, there are strict quaran-tine measures in place to prevent SALB from establishing in therubber tree production areas in the Palaeotropics, especially inSoutheast Asia, a SALB-free zone [3,8]. The fungus infects young leaves, stems and fruits of   Hevea brasiliensis , as well as  H.benthamiana  Muell.-Arg.,  H. spruceana  (Benth.) Muell.-Arg.,  H. guianensis  Aublet and  H. camporum  Ducke [2], resulting indefoliation and, potentially, after repeated outbreaks in tree death.The fungus was first observed and collected by E. Ule in 1900 inthe Upper Amazon region of Peru and Brazil and was laterdescribed by Hennings [9]. Initially, two spore morphs wererecognised: the sexual morph,  Dothidella ulei ; and a supposedasexual pycnidial morph,  Aposphaeria ulei . The hyphomycete PLOS ONE | 1 August 2014 | Volume 9 | Issue 8 | e104750  asexual morph was described soon after by J. Kuyper in Surinamin 1911 as  Fusicladium macrosporum . In 1917, G. Stahel observedthe connection of hyphae from different fungal structures withinthe leaf tissue and linked the sexual and asexual morphs of thefungus and renamed the former as  Melanopsammopsis ulei  [1].Much later, von Arx (in [10]) transferred this to the genus  Microcyclus  and suggested a close relationship with the genus  Mycosphaerella , based on the morphology of the hyphomycete  Passalora -type morph. Subsequently, he suggested that  Fusicla- dium  should only be used for asexual morphs belonging to thefamily Venturiaceae rather than to the Mycosphaerellaceae [11].  Microcyclus  is characterized by erumpent ascostromata on living leaves having a foot-like hypostroma, similar to some  Myco- sphaerella  pathogens of pine trees [12].Each of the spore morphs in  M. ulei ’s life cycle would,according to the present classification for the genera where theyare placed, belong to a different ascomycete family, namely:Planistromellaceae/incertae sedis (   Microcyclus  sexual morph) [13– 15], Venturiaceae (   Fusicladium  asexual morph) [16] and Lophios-tomataceae (   Aposphaeria  asexual morph) [17,18]. This is clearlyinadequate and requires an explanation. Although there would begrounds for speculating that SALB is a disease complex involving three unrelated fungal species, perhaps with the involvement of amycoparasite, previous authors that have dealt with the SALBdisease and its etiology have not reached such conclusionNevertheless, Langford [19] and more recently, Guyot and Doare´[20] have inoculated conidia and ascospores on rubber plants andwere able to reproduce the symptoms of SALB, demonstrating that the  Microcyclus  and  Fusicladium  morphs are part of the cycleof a single fungus. Ascospores were shown to play an essential rolein the perpetuation of the disease outside the host’s growthperiods, in the resumption of epidemics, and in long-distancedispersal and the conidia contributed primarily to the stepwise andshort-distance spread of the disease [21]. A conclusive Koch’spostulates have never been performed with the pycnidial morph of the fungus. This might play a different role in the life cycle of thefungus or even be a mycoparasite of   M. ulei . Conversely, thispuzzling situation may just result from the lack of properunderstanding of the life cycle and classification of the fungusbehind SALB.The general lack of DNA sequence data for all three purportedmorphs (   Microcyclus ,  Fusicladium  and  Aposphaeria  ) contributes tothe confusion surrounding the taxonomy of the causal agent of SALB. Until relatively recently, the genus  Microcyclus  wasclassified in the Mycosphaerellaceae (order Capnodiales), as astromatic counterpart of the family [22,23], but has since been re-classified in the Planistromellaceae (Dothideales), initially toaccommodate genera with ascostromatal locules that openschizogenously by a periphysate ostiole [13,14]. More recently, aphylogenetic analysis showed that the core Planistromellaceaebelong in the Botryosphaeriales, from which  Microcyclus  – represented only by ITS sequences of   M. ulei  – was excludedbased on BLAST searches of GenBank, and its familial positionwas considered to be uncertain [15]. After a taxonomic review of the hyphomycete conidial morph, this asexual morph was retainedin  Fusicladium  s. lat. [16]; some species of which have now beenassigned to the newly recognised family Sympoventuriaceae in thenew order Venturiales [24]. However, in the absence of typematerial, the species was neotypified and the name changed to  F. heveae  since it was adjudged that the srcinal epithet could beconfused with  F. macrosporium  Bonord. 1864 [16,25]. The latterauthors added the rider that: ‘‘  Fusicladium heveae  is an unusualspecies, since its teleomorph,  Microcyclus ulei , is placed in theMycosphaerellaceae and not in the Venturiaceae’’. The coelomy-cete genus  Aposphaeria  is recognized as a member of the familyLophiostomataceae (order Pleosporales), as a well-supported group[17,18]. Thus, questionable issues regarding the classification of both the purported asexual morphs, as well as the sexual morph, atthe genus, family and order levels of the causal agent of SALBneed to be addressed. ‘‘Clearly, a re-examination of its taxonomicposition would be justified’’ [26] and a single unifying genericname should be adopted in accordance with the new nomencla-tural rules of one fungus one name system and the promotion of progressive plant pathology [27]. Additionally, knowledge about the evolutionary history of   M. ulei  and of related species is scarce and molecular studies couldhelp to resolve the true affinity of this fungus [15,25,28]. Thus, theobjectives of the present study were: i) To obtain molecularevidence of the connection of the three spore morphs of   M. ulei ; ii)to elucidate the phylogenetic relationships of   M. ulei  using molecular approaches; iii) to determine the adequate nomencla-tural treatment for the fungus causing SALB; iiii) to obtainexperimental evidence on the function of the intermediatepycnidial morph; iv) to prepare an updated model of life-cycle of the SALB fungus. Conceivably, this should also lead to a betterunderstanding of the biology and ecology of one of the mostthreatening plant pathogens to mankind’s welfare. Material and Methods Ethics statement No specific permits were required for the described field studies.No endangered or protected species were involved in the studies. Sampling, isolation and DNA extraction Leaves with lesions of South American leaf blight were sampledin commercial fields of rubber in Brazil. Sampling was aimed atareas with records of high incidence of SALB in the Brazilianstates of Acre, Rondoˆnia, Mato Grosso, Minas Gerais, Espı´ritoSanto and Bahia between 2008 and 2010 (Table 1). Single conidiawere transferred from fungal structures formed on lesions toculture media, using a sterilized fine-needle under a dissecting microscope. Monosporic cultures of   F. heveae  were grown on M4culture medium [29] in the dark for 2 months at 24  6  1  u C.Pycnidial stromata of   A. ulei  and ascostromata of   M. ulei  wereexcised from a single lesion of an infected leaf with a sterilizedrazor blade. Each lesion was examined under the microscope tocheck for possible contamination by mycoparasites and selectedstromata (approximately 10 structures) were transferred to amicrotube (1.5 mL). The procedure was repeated from anotherlesion on the same leaf. To break up the melanised cell walls, themicrotubes containing fungal material (mycelium, pycnidia orascostromata) were placed in liquid nitrogen and macerated using a micropestle. DNA extraction was carried out following standardcetyltrimethyl ammonium bromide extraction procedures [30]. DNA phylogeny  All phylogenetic analyses were performed using DNA sequenceof six loci as the first 900 bp at the 5 9  end of the 28S rRNA gene(LSU), the first and second internal transcribed spacer (ITS), themitochondrial region of the mtSSU-rDNA and partial sequencesof nuclear genes such as the mini-chromosome maintenanceprotein (MCM7), translation elongation factor 1-alpha (EF-1 a  )and actin (ACT). Specific primers utilized were LR0R [31] andLR5 [32], ITS1 and ITS4 [33], NMS1 and NMS2 [34], Mcm7-709for and Mcm7-1384rev [35], EF1-728F and EF1-986R and ACT-512F and ACT-783R [36], respectively. Phylogeny of   Microcyclus ulei  PLOS ONE | 2 August 2014 | Volume 9 | Issue 8 | e104750  The polymerase chain reaction (PCR) was done with a mixturecontaining 20  g g of DNA, 0.2  m M of each primer and 1 6 of DreamTaq DNA Polymerase Master mix as described by themanufacturer (Thermo Fisher Scientific). PCR cycles were carriedout in a PTC100 thermal cycler (MJ Research, Incline Village,NV) and consisted of a 5 min denaturation step at 94  u C, followedby 35 cycles of 30 s at 94  u C, 30 s at 60  u C for LSU, mtSSU, EF-1 a , ACT and ITS primers or 57  u C for MCM7 primers and 1 minat 72  u C with a final extension of 10 min at 72  u C. PCR productswere visualized by ultraviolet fluorescence following 1% agarosegel electrophoresis in 1 6  TBE buffer and GelRed (Biotium)staining. Single-band products were purified using the E.Z.N.Acycle-pure kit (OMEGA Bio-tek). DNA concentration wasmeasured by NanoDrop 2000 Spectrophotometer (Thermo FisherScientific). The same primers used for PCR amplification wereused for the sequencing reactions using the DYEnamic ETTerminator Cycle Sequencing Kit (GE Healthcare) according tothe manufacturer’s recommendations. The purified PCR productswere sequenced using a MegaBACE 1000 DNA Sequencing System (GE Healthcare). A consensus sequence was generatedafter manually editing with The Staden Package, v. 1.6.0 [37].Genbank accession numbers are provided in Table 1. Additionalsequences used in the analyses were obtained from GenBank andthe Fungal Genomics Portal of the Joint Genome Institute[38](Table S1). Sequences were aligned with the Muscle v. 3.6software [39] implemented in the MEGA 5.0 program [40].Statistics resulting from sequence alignment such as variable,parsimony-informative and uninformative sites were estimated inMEGA.Bayesian analysis was conducted with MrBayes v. 3.1.2 [41] todetermine generic relationships based on the LSU, mtSSU andMCM7. Aligned datasets were inspected with MrModeltest v.2.2[42] to select the suitable nucleotide substitution model and alltrees were rooted with  Aspergillus niger . Additionally, anotherdataset at species level was constructed and Bayesian phylogenywas derived from the concatenated ITS, EF-1 a  and ACTalignments with  Pseudocercospora  s. str. sequences.  Passaloraeucalypti  was used as the outgroup. For this analysis, the alignmentgaps were treated as a fifth character state and the MrModeltest v.2.2 selected the best nucleotide substitution model for eachpartition. The Markov Chain Monte Carlo (MCMC) analysis usedfour chains that started with a heating parameter of 0.2 from arandom tree topology and lasted 50 million generations. Treeswere saved each 1000 generations, resulting in 50,000 saved trees.Burn-in was set at 5,000,000 generations after which the likelihood values were stationary, leaving 35,000 trees from which the 50%majority rule consensus trees and posterior probabilities werecalculated. Quality of mixing and convergence to the stationarydistribution were assessed from three independent runs using Tracer v. 1.5 [43]. The resulting phylogenetic trees were preparedusing FigTree v. 1.4 ( ). All alignments and resulting trees were deposited into TreeBASE(14357), and the nomenclatural novelty in MycoBank [44]. Taxonomy Based on newly obtained information and information availablein the literature and on the re-examination of newly collectedmaterial a model was prepared. Observations of the morphologyof fungal structures belonging to each morph in the life cycle weremade based on the examination of microscope slides containing sections of such structures mounted in lactophenol or lactofuchsinand observed under a light microscope (Olympus BX 51)equipped with a drawing tube. At least 30 measurements weremade of each fungal structure. Nomenclature The electronic version of this article in Portable DocumentFormat (PDF) in a work with an ISSN or ISBN will represent apublished work according to the International Code of Nomen-clature for algae, fungi, and plants, and hence the new namescontained in the electronic publication of a PLOS ONE article areeffectively published under that Code from the electronic editionalone, so there is no longer any need to provide printed copies.In addition, the new combination introduced in this work hasbeen submitted to MycoBank from where they will be madeavailable to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewedthrough any standard web browser by appending the MycoBank  Table 1.  Origin of the  Microcyclus ulei   isolates used in the phylogenetic study. Isolate Location 1 Coordinates in decimals(Lat/Lon)GenBank accession number (ITS, ACT, EF-1 a , LSU,MCM7, mtSSU) Fusicladium heveae  UFVMu01RO Buritis-RO -10.211944/-63.828889 KC800717, KC800725, KC800733, KC800741, KC800755,KC800768 Fusicladium heveae  UFVMu05MT Itiquira-MT -17.208889/-54.150000 KC800718, KC800726, KC800734, KC800742, KC800756,KC800769 Fusicladium heveae  UFVMu01ES Sooretama-ES -19.220087/-40.121414 KC800719, KC800727, KC800735, KC800743, KC800757,KC800770 Fusicladium heveae  UFVMu77BA Porto Seguro-BA -16.378001/-39.366433 KC800720, KC800728, KC800736, KC800744, KC800758,KC800771 Microcyclus ulei   AC Xapuri-AC -10.651944/-68.503889 KC800721, KC800729, KC800737, KC800745, KC800759,KC800772 Microcyclus ulei   MG Orato´rios-MG -20.415833/-42.908889 KC800722, KC800730, KC800738, KC800746, KC800760,KC800773  Aposphaeria ulei   RO Ariquemes-RO -9.913333/-63.040833 KC800723, KC800731, KC800739, KC800747, KC800761,KC800774  Aposphaeria ulei   ES Cachoeiro do Itapemirim-ES -20.752609/-41.290358 KC800724, KC800732, KC800740, KC800748, KC800762,KC800775 1 Brazilian states: Acre (AC), Bahia (BA), Espı´rito Santo (ES), Mato Grosso (MT), Minas Gerais (MG) and Rondoˆnia (RO).doi:10.1371/journal.pone.0104750.t001 Phylogeny of   Microcyclus ulei  PLOS ONE | 3 August 2014 | Volume 9 | Issue 8 | e104750  number contained in this publication to the prefix The online version of this work is archivedand available from the following digital repositories: [PubMedCentral, LOCKSS]. Assessments of the pleomorphic development of  Microcyclus ulei   under natural conditions The development of the pathogen in the rubber leaf wasmonitored under environmental conditions favorable to the Figure 1. Bayesian analysis showing the phylogenetic relationships of   Microcyclus ulei   based on the LSU sequence alignment. Bayesian posterior probabilities are given at the nodes and coded according to the colored scale bar. The black line scale bar shows 0.2 expectedchanges per site. The tree was rooted with  Aspergillus niger  .doi:10.1371/journal.pone.0104750.g001Phylogeny of   Microcyclus ulei  PLOS ONE | 4 August 2014 | Volume 9 | Issue 8 | e104750  development of SALB. At the Michelin Plantation of Bahia(Brazil), 90 leaves at the B2 developmental stage [45] of eightrubber trees of the RO38 clone were tagged with a label andobservations of the disease were made until maturity (stage D),from December 15, 2011 to February 24, 2012 (Experiment 1)and September 19 to December 03, 2012 (Experiment 2). All treeswere pruned 45 days before each experiment started. Scoring of sporulation in lesions naturally infected was performed at everyfour days using a 1–6 scale for sporulation intensity of the asexualmorph (conidia) adapted from Junqueira et al. [46], where 1 =necrotic non-sporulating lesions, 2 = chlorotic non-sporulating lesions, 3 = slight sporulation on lower side of the leaflets, 4 =moderate sporulation on lower side of the leaflets, 5 = highsporulation on lower side of the leaflets, and 6 = high sporulationon both sides of the leaflets. Pycnidial and ascostromata densitywas assessed at the same time interval using a 0–4 scale where 0 =no stroma, 1 = 1–5 stromata per leaflet, 2 = 6–15 stromata perleaflet, 3 = 16–50 stromata per leaflet, and 4 = more than 50stromata per leaflet. The weighted average was computed in eachobservation from total of leaves in each phenological stage andscore of conidial sporulation intensity and spermogonia andascostromata density. Test of infectivity and germination of pycniospores of  Microcyclus ulei   under controlled conditions Suspension of pycniospores was obtained from pycnidia formedin near mature leaves (C/D stage) of the RO38 rubber clone.There were no conidia or ascospores. Suspension of hyphomyceteasexual morph was used as positive control. Both inoculumsuspensions were adjusted to 2 6 10 5 spores/mL in a Tween 80 at0.05% solution. The lower surface of three young leaves from theFx 3864 rubber tree clone were spray-inoculated until runoff withan inoculum suspension of pycniospores or conidia separatelyusing a HS Airbrush Complete set (Paasche Airbrush company) inan inoculation chamber at 24 u C, relative humidity greater than85%, artificial daylight of 2000 lux and 12 h photoperiod. The0.05% Tween 80 solution was used as a negative control.Sporulation was scored after 12 days on all inoculated leaves.The suspensions of pycniospores and conidia were incubated inthe dark at 25  u C on both water agar and M4 culture media.Germination assessments were conducted at 6, 12, 24 and 120 hof incubation at 24 6 1 u C. The experiment was conducted twice. Results Phylogeny: LSU, mtSSU and MCM7 datasets Strongly supported clades provide molecular evidence of asexual-sexual morph connection between the three morphs of the SALB fungus and thus the holomorph belongs to the familyMycosphaerellaceae s. str., order Capnodiales (Figures 1–3). Forthis study, two specimens each of   M. ulei  and  A. ulei  and four of   F. heveae  collected in Brazil were analyzed. The generic relationshipswere determined with datasets for LSU, mtSSU and MCM7 thatincluded 89, 55 and 36 taxa, respectively (available in TreeBASE)and the same nucleotide substitution model, GTR + I + G was usedin all analyses.The alignment of the partial sequence of the LSU region had838 sites including alignment gaps, of which 243 sites wereparsimony-informative, 57 were variable and parsimony-uninfor-mative, and 534 were constant. The LSU phylogeny (Figure 1)resulted in  Aposphaeria populina  and  A. corallinolutea  (membersof the Lophiostomataceae: Pleosporales), species of the genus  Fusicladium  (members of the Sympoventuriaceae: Venturiales),species of   Kellermania  (members of Planistromellaceae: Botryo-sphaeriales), as well as members of the Dothideales forming well-supported monophyletic groups. Representatives of the Capno-diales grouped within well-established families as Cladosporiaceae,Capnodiaceae, Teratosphaeriaceae, Schizothyriaceae, Dissoconia-ceae and Mycosphaerellaceae. In the Mycosphaerellaceae, severalwell-supported clades were formed with  Mycosphaerella  s. str.(asexual morph  Ramularia  ) and mycosphaerella-like with theasexual morphs  Cercospora ,  Pallidocercospora ,  Pseudocercospora ,pseudocercospora-like,  Ramulispora ,  Septoria , and  Zymoseptoria ,amongst others.  Microcyclus ulei  and its morphs  A. ulei  and  F. heveae  were identical and grouped in the well-defined  Pseudo-cercospora  s. str. clade of the Mycosphaerellaceae, distinct from  Mycosphaerella  s. str. (   M. punctiformis , represented by  Ramulariaendophylla  ), showing clearly that the holomorph of the SALBfungus is a species of   Pseudocercospora  in the Mycosphaerellaceae.The phylogeny reconstructed with the partial sequence of themtSSU sequences (Figure 2) had 724 characters (248 parsimony-informative and 99 singletons), while the dataset of the partialsequence of the MCM7 region (Figure 3) was based on a datasetwith 466 characters (254 variables sites of which 221 wereparsimony-informative). The OTUs from the Venturiales, Pleos-porales, Dothideales and Capnodiales (Capnodiaceae, Cladospor-iaceae, Dissoconiaceae and Mycosphaerellaceae) for mtSSUregion, and those from the Pleosporales (Lophiostomataceae andPleosporaceae), Venturiales and Capnodiales (Teratosphaeriaceaeand Mycosphaerellaceae) for MCM7 formed well-supportedclades. In both analyses, OTUs of the genus  Pseudocercospora  inMycosphaerellaceae were the nearest relatives of the holomorph of the SALB pathogen. Phylogeny: Concatenated ITS, EF-1 a  and ACT datasets  After the analyses at the genus level, phylogeny at species levelwas conducted with some OTUs of   Pseudocercospora  s. str. using sequences of ITS, EF-1 a  and ACT regions combined (Figure 4).The nucleotide substitution models, GTR + I + G, GTR + G andSYM + I + G, were used for each partition, respectively. For thisdataset, 1126 characters were used, 517 were constant, 367 wereparsimony-informative and 144 were singletons. Two well-definedclades were observed, both with posterior probability of 0.96, andthe holomorph was closely related to  Pseudocercospora angolensis . Pleomorphic development and function of intermediatepycnidial morph in the life cycle of   Microcyclus ulei  The SALB symptoms were assessed in two consecutiveexperiments from trees after pruning. In the first period,December 15, 2011 to February 24, 2012, conidial lesions startedin leaves in the B2 stage on December 19 and were observed up tothe D stage leaves, which corresponded to 26 days of monitoring (Figure 5A).  A. ulei  first emerged from the upper side of infectedleaves in the C/D stage on December 29. Ascostromata arose after32 days (January 17) and were found only in the upper side of Dstage leaves. In the second period, September 19 to December 3,2012, conidial lesions were found on September, 28 in B2 stageleaves and in D leaves within a 28 day-period (October 17)(Figure 5B).  A. ulei  appeared in the C/D stage on October 9 (20days) and ascostromata arose after 36 days of monitoring and werefound only in stage D leaves. Both stages occurred in the adaxialside of leaves.The main weather descriptors during the course of theexperiment 1 (72 days) and experiment 2 (75 days) were,respectively: average maximum temperature 29 and 27.7  u C;average minimum temperature 22 and 20.2  u C; average relativehumidity 83% and 83.9%. Total (cumulative) rainfall was 267 and267.8 mm. Phylogeny of   Microcyclus ulei  PLOS ONE | 5 August 2014 | Volume 9 | Issue 8 | e104750
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