Epitypification, characterization and phylogenetic ...

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Received: 13 July 2017 Accepted: 14 March 2018 DOI: 10.1111/efp.12434

ORIGINAL ARTICLE

Epitypification, characterization and phylogenetic positioning of Pseudobeltrania cedrelae, the causal agent of pseudobeltrania spot on Cedrela fissilis C. A. Milagres | D. M. Q. Azevedo | O. L. Pereira

Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil Correspondence Gleiber Furtado, Departamento de Fitopatologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil. Email: [email protected] Funding information Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES-Proex); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG) Editor: J. Stenlid

| G. Q. Furtado

Summary The fungus Pseudobeltrania cedrelae, the type species of the genus, is the causal agent of an important leaf spot in seedlings and adult plant of cedar (Cedrela fissilis). Due to the contradictory phylogenetic position of the genus Pseudobeltrania, epitypification of P. cedrelae was carried out based on a culture obtained from the same locality and host of the original type. Samples were collected, and 10 isolates of P. cedrelae associated with lesions on cedar leaflets were obtained. For morphological characterization, conidia, conidiophores, conidiogeneous cells, conidiogeneous loci and basal cells were taken both from the fungus obtained from leaf lesions and from that obtained in slide culture. Mycelial growth rates and sporulation were evaluated in six different culture media. For molecular phylogeny, maximum parsimony analyses were performed from the ITS and 28S sequences of the isolates. Both the morphological characteristics of the fungal structures obtained from symptomatic leaves and the slide culture technique presented variations. In foliar lesions, isolates presented the same morphological characteristics as the type material. Mycelial growth rate and sporulation of P. cedrelae were greatest on malt extract agar and V8 juice agar. Pseudobeltrania cedrelae was pathogenic when inoculated into healthy cedar plants. According to the phylogenetic tree, isolates grouped in the same clade, but in a distinct clade of Pseudobeltrania ocoteae. The results suggested that P. ocoteae belongs to the genus Hemibeltrania. This paper presents new information on P. cedrelae that contributes to clarifying the phylogeny of the Beltraniaceae.

The genera in the Beltraniaceae—Beltrania, Beltraniella,

1 | INTRODUCTION

The genus Pseudobeltrania, identified for the first time associated with leaf lesions on Cedrela fissilis Vell. (Meliaceae) in São Paulo, SP, Brazil, was proposed based on the type species Pseudobeltrania cedrelae by Gusmão & Grandi (1996), Hennings (1902). This genus, in the Ascomycota, Beltraniaceae, is characterized by the presence of conidiophores with lobed basal cells, conidia with a hyaline transversal band and the absence of setae and separating cells (Rajeshkumar, Crous, Groenewald, & Seifert, 2016; Zucconi, 1991).

Forest Pathology. 2018;e12434. https://doi.org/10.1111/efp.12434

Beltraniopsis, Pseudobeltrania, Hemibeltrania and Rhombostilbella— were reviewed taxonomically and the family considered an innate group (Pirozynski, 1963). In 1980, Kendrick studying generic concepts of the hyphomycetes proposed that members of the Beltrania complex should possess at least three of five common characteristics: dark setae, setae or conidiophores with lobed basal cells, separating cells, biconic conidia and conidia with a hyaline medium band. Thus, the genera Hemibeltrania and Rhombostilbella were

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excluded from the Beltrania complex because they had only one of

characteristics of the type. For plant pathogenic fungi, the epi-

these characteristics (Gusmão & Grandi, 1996; Rajeshkumar et al.,

type must trigger the same symptoms in the reported host and

2016).

be deposited in a collection in the country of origin. However, the

The genera Parapleurotheciopsis and Subramaniomyces were

objectives of this study were to epitypify, characterize and gener-

considered members of the Beltraniaceae by Maharachchikumbura

ate information about the phylogenetic positioning of P. cedrelae,

et al. (2016). However, Rajeshkumar et al. (2016), based on a phy-

based on the revised guidelines (Ariyawansa et al., 2014; Hyde &

logenetic analysis (maximum parsimony) of the 28S region, showed

Zhang, 2008).

that Parapleurotheciopsis did not group with other members of the Beltraniaceae, and proposed that the positioning of this genus is uncertain, possibly varying with the sequences and the phylogenetic methods utilized in analyses. Currently, based partly on molecular analyses, the Beltraniaceae comprises eight genera, including the five mentioned above, along Hemibeltrania, Porobeltraniella and Subsessila (Lin et al., 2017; Rajeshkumar et al., 2016). As there are no molecular data at present for Rhombostilbella and Beltraniomyces, the position of these genera requires further clarification (Rajeshkumar et al., 2016). There have been few molecular analyses of Beltraniaceae, but recently a study carried out by Rajeshkumar et al. (2016) yielded the phylogenetic positioning of a number of genera in the family. However, Pseudobeltrania, which according to Mycobank (2017) has 11 valid species (P. angamosensis, P. cedrelae, P. chumrungensis, P. cristaspora, P. guerensis, P. havanensis, P. macrospora, P. ocoteae, P. penzigii, P. selenoides and P. summa), was represented by P. ocoteae alone, as it is the only species for which DNA sequences are available in GenBank. The fungus P. cedrelae was reported in Spain on leaves dead on Ruscus aculeatus L. and in Brazil on living and decomposing leaves of

2 | M ATE R I A L S A N D M E TH O DS 2.1 | Collection, isolation and preservation of the cultures Isolates were obtained from leaves of C. fissilis, collected in Viçosa county, state of Minas Gerais, Brazil, from the SIF/UFV nursery (20.774°S, 42.876°W), in the community of Sapé (20.767°S, 42.835°W) and Rua Nova Street – Highway MG-280 (20.779°S, 42.964°W). To obtain cultures, conidia were transferred, under aseptic conditions, to Petri dishes containing potato dextrose agar (PDA, Sigma-Aldrich®). Symptomatic leaves (Figure 1a,b) were collected and dried in a botanical press. Single conidia cultures were obtained on malt extract agar (MEA) and preserved in sterilized distilled water, glycerol (15%) and silica gel (Zauza, Alfenas, & Mafia, 2007). The epitype was deposited in the Herbarium of the Universidade Federal de Viçosa (VIC). The ex-epitype was deposited in the Octavio de Almeida Drumond Collection (COAD) at the Universidade Federal de Viçosa.

C. fissilis and also on leaves of Cedrela odorata L. (Batista, Matta, & Bezerra, 1964; Cvetkovié, Llimona, & Hoyo, 1996; Gusmão & Grandi, 1996; Hanada, Gasparotto, & Ferreira, 2005; Hennings, 1902). The disease symptoms described on C. odorata include light brown spots, circular to angular, surrounded by a chlorotic halo, which can coalesce and cause extensive necrotic areas, leading to intense defoliation (Hanada et al., 2005). Pseudobeltrania cedrelae was first reported in Brazil over 100 years ago. The type material (SP 32718) was deposited in the Maria Eneyda Pacheco Kauffmann Fidalgo Herbarium, the Botanical Institute of São Paulo, Brazil, and is duplicated (B 14155) in the Herbarium Berolinense, Berlin, Germany. However, epitypification of P. cedrelae is required as there are no cultures available in culture collections (ex-type) or genome sequences available in GenBank. In addition, when analysing the type material deposited in Brazil (SP 32718), it was observed that the host plant leaflets were in an advanced state of deterioration and no fungal structures could be observed.

2.2 | Morphological characterization Fungal structures from lesions on C. fissilis leaflets and from cultures (slide culture) were placed on glass slides in a drop of lactic acid. To obtain slide cultures, a small fragment of MEA culture medium was placed on a sterile glass slide. The slide was suspended on glass rods, in a Petri dish containing moistened filter paper. A fragment of the mycelium of the isolate was transferred to the edge of the culture medium and covered with a coverslip. Petri dishes containing slides were incubated at 25°C with a 12-hr photoperiod (32 μE m−2 s−1). After 10 days, the coverslip was removed and placed on a glass slide. Fungal structures (conidia, conidiophores, conidiogeneous cells and conidiogeneous loci) were measured, with 30 replicates for each structure, under an Olympus BX 53 compound microscope equipped with an Olympus Q-COLOR5 digital camera.

Epitypification is recommended in cases where there is an erroneous interpretation of the representative taxon of the genus and there is no way to acquire the genome sequence of the type species. However, it was pointed out that the epitype must be

2.3 | Mycelial growth rate and sporulation of P. cedrelae

morphologically identical to the type itself (Ariyawansa et al.,

A mycelial disc of approximately 1 cm in diameter was transferred

2014; Hyde & Zhang, 2008). Ariyawansa et al. (2014) proposed a

to six different culture media: V8 juice agar, MEA, oat agar, potato

set of guidelines to better designate an epitype. These include col-

dextrose agar, carrot agar and corn meal agar (Zauza et al., 2007).

lections from the same host, when possible from the same place

Cultures were incubated in a growth chamber at a temperature not

where the type was originally obtained, and observation of all the

exceeding 25°C, with a 12-hr photoperiod (32 μE m−2 s−1).

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Colony diameters in two perpendicular directions were mea-

(BSA, Sigma-A ldrich®, St. Louis, MO, USA), 2 μL genomic DNA

sured daily until the P. cedrelae mycelium on at least one of the plates

(25 ng/μL) and 2.5 μL nuclease-free water to a total volume of

reached the edge of the dish and the daily mycelial growth rate cal-

25 μL.

culated (Oliveira, 1991). Sporulation was evaluated by counting the

The regions rDNA ITS1–5.8S–ITS2 (ITS) and 28S rDNA (28S)

conidia in a Neubauer chamber after 15 days of incubation of the

of the isolates were amplified and sequenced using the primers

isolate, by washing the culture surface with 10 mL of distilled water.

ITS-1 and ITS-4 (White, Bruns, Lee, & Taylor, 1990), LROR and

To determine the production of conidia in each culture media, the

LR5 (Vilgalys & Hester, 1990), respectively. PCR was carried out in

number of conidia obtained was divided by the colony area on each

C1000™ Thermal Cycler (BIO-R AD) with the following program: ini-

medium.

tial denaturation at 95°C for 5 min, followed by 35 cycles of dena-

The experiment was carried out in a completely random-

turation at 94°C for 1 min, annealing at 52°C (ITS) for 1 min or 53°C

ized design with four replicates, each one comprising a Petri dish

for 45 s (28S) and an extension at 72°C for 1 min. The final extension

(Ø = 90 mm). Tukey’s test was utilized to compare the means at the

included an additional annealing at 72°C for 5 min. PCR products

5% probability level, using the R Studio© software program v3.1.1 (R

were analysed by electrophoresis on 2% agarose gels, stained with

Core Team, 2016).

GelRed™ (Biotium Inc., Hayward, CA, EUA) in buffer TAE (1×) and visualized under UV light to verify the size and quality of the amplifications. Amplicons were purified and sequenced by Macrogen Inc.,

2.4 | Molecular characterization

South Korea (http://www.macrogen.com).

For DNA extraction, monosporic cultures were cultured on moist

For phylogenetic analyses, the nucleotide sequences were

and sterile cellophane overlayed on PDA, at 25°C for 7 days.

assembled into contigs and edited using the DNADragon soft-

Mycelium was scraped off the cellophane with a sterilized wooden

ware (http://www.dna-dragon.com). All sequences were manually

toothpick and transferred to a 1.5-mL microcentrifuge tube. The

checked and nucleotides with ambiguous positions corrected by an-

extraction was performed by mechanical disruption of cells using

alysing the sequences obtained with both primers.

microspheres. Total DNA was extracted using the Wizard® Genomic

Sequences of the Beltraniaceae isolates used in the phylo-

DNA Purification Kit (Promega Corporation, WI, EUA) following the

genetic analyses were obtained from Rajeshkumar et al. (2016)

protocol described by Pinho, Firmino, Ferreira-Junior, and Pereira

and from Megablast of NCBI GenBank (Table 1). These sequences were obtained in FASTA format and aligned with the sequences

(2013). The following reagents were used for each PCR: 12.5 μL Dream

of the isolates from this study using the multiple sequence align-

Taq TM PCR Master Mix 2X (MBI Fermentas, Vilnius, Lithuania),

ment Muscle ® program (Edgar, 2004), implemented in MEGA 6

1 μL each primer (forward and reverse) synthesized by Invitrogen

software (Tamura, Stecher, Peterson, Filipski, & Kumar, 2013).

(Carlsbad, U.S.A), 1 μL dimethyl sulfoxide (DMSO, Sigma-A ldrich,

Alignments were manually checked and readjustments made

St. Louis, MO, USA), 5 μL 100× (10 mg/mL) Bovine Serum Albumin

when necessary.

(a)

(c)

(e)

(b)

(d)

(f) F I G U R E 1 Pseudobeltrania cedrelae on leaflets of Cedrela fissilis (a) Symptoms of pseudobeltrania spot in the field; (b) Signs of the pathogen in the abaxial leaf face; (c) Branched conidiophore originating from lobate basal cells; (d) Detail of conidiogeneous loci denticulated; (e) Biconic conidia; (f) Detail of the conidium with a hyaline band in the median region and a thick conidial scar. Bars = 20 μm

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TA B L E 1 Sequences of Beltraniaceae isolates used in phylogenetic analysis, including isolates of Pseudobeltrania cedrelae GenBank accession number Species

Isolates

ITS

28 S

Host

Location

Collector/References

Beltraniella portoricensis

CX1

KU212349

–

Eugenia uruguayensis (Myrtaceae)

Canelones, Uruguay

García-L aviña, Bettucci, and Tiscornia (2016)


BCRC 34590

GU905993

–

Unknown

Taiwan

–


NFCCI 3993

KX519516

KX519522

Mangifera indica fallen leaves

Kerala, India

Rajeshkumar et al. (2016)


NBRC 9079

907901

907901

Unknown

Unknown

–

Beltraniella endiandrae

CPC 22193

KJ869128

KJ869185

Endiandra introrsa

Australia: New South Wales

Crous et al. (2014)

Beltraniella botryospora

TMQa1A18

–

AB496426

Quercus acuta fallen leaves

Japan: Tokyo, Meiji Jingu Shrine

Shirouzu, Hirose, Tokumasu, To-Anun, and Maekawa (2010)

Beltraniella carolinensis

NBRC 9502

950202

–

Persea borbonia leaf

Unknown

–


9502IFO

–

DQ810233

Persea borbonia leaf

Southeastern Forest Exp. Sta., USDA

–

Beltraniella japonica

NBRC 30443

3044301

3044301

Quercus phillyraeoides litter

Kagoshima, Japan

–

Beltraniella odinae

NBRC 6774

677401

677401

Unknown

Unknown

–

Beltraniella sp.

37.1.1

KP133183

–

Persea pseudofasciculata

Ecuador: Reserva Los Cedros, Imbabura

Thomas, Vandegrift, Ludden, Carroll, and Roy (2016)

Beltraniella sp.

37.2.1

KP133182

–



Thomas et al. (2016)

Beltraniella sp.

37.2.2

KP133176

–




Beltraniella sp.

37.3.1

KP133175

–




Beltraniella sp.

37.3.2

KP133178

–




Beltraniella sp.

37.4.1

KP133180

–





111.1.2

KP133177

–

Nectandra lineatifolia




111.3.3

KP133179

–





111.4.1

KP133173

–




Beltrania sp.

RV-2015 isolate 114.2.1

KP133187

–

Myrcia fallax



(Continues)

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TA B L E 1 (Continued) GenBank accession number Species

Isolates

ITS

28 S

Host

Location


Beltrania sp.

RV-2015 isolate 114.1.1

KP133186

–

Myrcia fallax



Beltrania sp.

RV-2015 isolate 114.3.1

KP133189

–

Myrcia fallax



Beltrania sp.

RV-2015 isolate 38.1.1

KP133188

–

Myrcia fallax



Beltrania sp.


KP133185

–

Myrcia fallax



Beltrania sp.


KP133184

–




Beltrania pseudorhombica

CPC 23656

KJ869158

KJ869215

Pinus tabulaeformis

China: Beijing

Crous et al. (2014)

Beltrania pseudorhombica

JSP 01-10 A 1.2

KR093912

–

Atta capiguara

Brazil: Botucatu, Sao Paulo

Pereira et al. (2016)

Beltrania querna

ICMP 15825

EF029240

–

Quercus ilex

New Zealand

Cooper (2005)

Beltrania querna

BCRC 34620

GU905994

–

Unknown

Taiwan

–

Beltrania querna

NBRC 32637

3263701

3263701

Quercus dead leaf

Spain

–

Beltrania querna

NBRC 6884

688401

688401

Unknown

Unknown

–

Beltrania querna

NBRC 7543

754301

754301

Unknown

Unknown

–

Beltrania rhombica

DWM34

KM357317

–

Plant

Yunnan,China

–

Beltrania rhombica

10353

–

AB496423

Quercus myrsinaefolia fallen leaves

Japan

Shirouzu et al. (2010)

Beltrania rhombica

CPC 27482

KX519515

KX519521

Acacia crassipes leaves

Malaysia


Beltrania rhombica

NBRC 8857

885701

885701

Unknown

Unknown

–

Beltrania rhombica

NBRC 100226

10022601

10022601

Litter

Chiba, Japan

–

Beltrania rhombica

NBRC 100558

11055001

11055001

Abies firma needles

Ishikawa, Japan

–

Beltraniopsis sp.

KaTaQsal10LF3

–

AB496424

Quercus salicina fallen leaves

Japan

Shirouzu et al. (2010)

Beltraniopsis neolitseae

CPC 22168

KJ869126

KJ869183

Neolitsea australiensis

Australia: New South Wales

Crous et al. (2014)

Beltraniopsis sp.


KP133171

–

Myrcia fallax



Beltraniopsis sp.


KP133172

–




Pseudobeltrania ocoteae

CPC 26219

KT950856

KT950870

Ocotea obtusata leaves

France: La Reunion

Crous et al. (2015)

Subramaniomyces fusisaprophyticus

CBS 418.95

EU040241

EU040241

Leaf litter

Cuba

Crous, Braun, Schubert, and Groenewald (2007) (Continues)

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TA B L E 1 (Continued) GenBank accession number Species

Isolates

ITS

28 S

Host

Location


Hemibeltrania sp.

CL12WA

JQ621881

–

Cinnamomum iners

Malaysia

Refaei and Santhanam unpubl.

Hemibeltrania cinnamomi

NFCCI 3695

KT119564

KT119565

Cinnamomum malabathrum

Kerala, India


Hemibeltrania cinnamomi

NFCCI 3997

KX519517

KX519523

Cinnamomum malabatrum leaves

Kerala, India


Porobeltraniella porosa

NFCCI 3994

KX519518

KX519524

Gnetum ula leaf litter

Mulshi, Maharashtra, India



NFCCI 3995

KX519519

KX519525





NFCCI 3996

KX519520

KX519526




Pseudobeltrania cedrelae

PF1

X

X

Cedrela fissilis

Paraíso, Brazil – Viçosa-MG

This paper


PF2

X

X

Cedrela fissilis


This paper


PF3

X

X

Cedrela fissilis


This paper


PF4

X

X

Cedrela fissilis


This paper


PF5

X

X

Cedrela fissilis

SIF, Brazil – Viçosa-MG

This paper


COAD 2098 a

X

X

Cedrela fissilis


This paper


PF6

X

X

Cedrela fissilis


This paper


PF7

X

X

Cedrela fissilis

Comunidades do Sapé, Brazil – Viçosa-MG

This paper


PF8

X

X

Cedrela fissilis


This paper


PF9

X

X

Cedrela fissilis


This paper

ITS = internal transcribed spacer regions 1 and 2, including the 5.8S ribosomal RNA gene; and 28S = large-subunit ribosomal RNA gene. Isolates obtained in this study are highlighted in bold. COAD = Coleção Octávio Almeida Drummond at the Universidade Federal de Viçosa. a Ex- epitype.

Maximum parsimony (MP) analyses were performed for each gene/locus (ITS and 28S) using MEGA 6. MP trees were inferred based on the Tree Bisection Reconnection (TBR) method. The ro-

2.5 | Pathogenicity test Petri dishes containing P. cedrelae incubated for 15 days were

bustness of the most parsimonious trees was evaluated by 1,000

washed with sterile distilled water (SDW) and the conidial suspen-

bootstrap replicates resulting from MP analysis, each with 10 repe-

sion transferred to a beaker containing Tween 80 (0.05% v/v). After

titions of random addition (Felsenstein, 1985). Statistics were calcu-

stirring for one minute, the suspension was filtered through a dou-

lated for tree length (TL), consistency index (CI), retention index (RI)

ble layer of gauze and spore density adjusted to 4 × 105 conidia/mL,

and rescaled consistency index (RC). The trees were visualized using

following replicate Neubauer chamber counts. Five C. fissilis plants

the FigTree software program (Rambaut, 2009) and edited in the

were inoculated by spraying with the conidia suspension; two con-

CorelDRAW X7 (64-Bit). The tree was rooted with Subramaniomyces

trol plants were sprayed with SDW only. After inoculation, plants

fusisaprophyticus (CBS 418.95).

were conditioned in a humid chamber and kept in the dark for 48 h.

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F I G U R E 2 Features of Psedobeltrania cedrelae in slide culture, colony and symptoms on Cedrela fissilis leaves (a) Branched hyphae; (b) Branched conidiophore with 6–13 septa; (c) Conidiogeneous cells with sympodial proliferation. Arrows indicating conidiogeneous loci; (d) Biconic conidia with a hyaline transverse band in the chain; (e) Conidia forming on conidiophore; (f) Conidia with germ tube and appressorium; (g) Culture in malt extract agar after 9 days of growth; (h) Leaf spot symptoms 10 days after inoculation. Bars = 20 μm

(a)

(b)

(c)

(d)

(e)

(f)

(g)

Subsequently, photoperiod (225 μE m−2 s−1) was adjusted to 12 hr and

(h)

dark brown. External mycelium absent. Erect conidiophores, 1–2

the plants maintained in a humid chamber until symptoms appeared.

septa, branched, 35–48 × 3–5 μm, light brown and originating from

Evaluations were carried out daily until the onset of symptoms and

lobed basal cells (Figure 1c); conidiogeneous cells terminal, holoblastic,

signs. The pathogen was reisolated from the sporulating lesions, and

mono-or polyblastic, sympodial proliferation, light brown, cylindri-

the cultures obtained were compared with those used in inoculations.

cal, 19–28 × 3–7 μm. Conspicuous conidiogeneous loci, denticulated, 1–9 per cell, 1.0–2.5 × 0.8–2.5 μm (Figure 1d). Separating cells absent.

3 | R E S U LT S 3.1 | Isolation of Pseudobeltrania cedrelae Ten isolates of P. cedrelae (Table 1) were obtained from the abaxial face of C. fissilis foliage. On PDA, colonies were light brown in colour with mycelium predominantly immersed in agar.

Biconic conidia, holoblastic, 19–29 × 8–11 μm, smooth, solitary, light brown, with a hyaline band in the median region, thick conidial scar with a diameter varying from 0.83 to 2.08 μm (Figure 1e,f).

3.2.2 | Material examined In Viçosa, Minas Gerais, Brazil, on leaves of Cedrela fissilis (VIC 44134, epitype), 15 April 2015, COAD2098. Collectors: Furtado GQ

3.2 | Morphological characterization

and Milagres CA. In the Public Gardens, São Paulo, state of São Paulo, Brazil,

The epitype presented, branched erect conidiophores, lobed basal

leaves of Cedrela fissilis, April, 1901, P. Hennings. N. SP32718 and

cells, biconic conidia with a hyaline transversal band and lacked of

B14155 (holotype).

separating cells or setae, indicating that the isolate in question was a species of Pseudobeltrania.

On MEA, the colony reached 84 mm in diameter in 9 days. The mycelium was initially white in appearance, turning greyish after 15 days incubation, when abundant sporulation was observed

3.2.1 | Pseudobeltrania cedrelae P. Hennings, Hedwigia 41:310, 1902 (Figure 1).

(Figure 2g). In slide culture, branched hyphae were visible (Figure 2a) and a number of differences in relation to those observed in leaf lesions. Lobed basal cells were not observed. Erect conidiophores,

Circular, brown lesions were apparent along the leaf lamina, with

with 6–13 septa, branched, 95.5–229.5 × 3.0–5.0 μm, light brown

diameters between 7 and 14 mm; branched internal mycelium, septate,

(Figure 2b). Conidiogeneous cells terminal, holoblastic, sympodial

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TA B L E 2 Mycelial growth rate (mm/day) and sporulation (conidia/cm2 of fungal colony) of Pseudobeltrania cedrelae in different culture media

trees were obtained, one of which is shown in Figure 4 (TL = 78, CI = 0.667, RI = 0.894 e RCI = 0.596). Based on this region, the isolates were placed in a separate and well-supported clade indicating

Culture

Mycelial growth rate

Sporulation

Malt extract agar

8.54 a

4.13 × 10 4 a

V8 juice agar

7.04 ab

0.51 × 10 4 ab

Corn meal agar

6.55 bc

0.36 × 10 4 ab

Potato dextrose agar

6.09 bcd

2.66 × 10 4 ab 4

Oat agar

5.03 cd

3.35 × 10 ab

Carrot agar

4.67 d

0b

CV(%)

24

122

that they belonged to a novel genus in the Beltraniaceae. The species P. ocoteae was also positioned in a clade separate from the isolates of P. cedrelae.

3.5 | Pathogenicity test Pseudobeltrania cedrelae was pathogenic when inoculated into healthy C. fissilis plants. The first symptoms were observed 6 days after inoculation (dai) with the appearance of chlorotic areas and

Means followed by the same letter in each column do not differ by Tukey test at 5% probability.

brown spots diffused over the leaf blade (Figure 2h); affected foli-

proliferation, light brown, cylindrical, 9.64–32.28 × 2.96–4.87 μm

and the characteristics of pure culture and conidia were identical to

(Figure 2c).

those of the inoculated isolate.

age abscised prematurely. Abundant sporulation occurred at 10 dai. Control plants remained asymptomatic. The fungus was reisolated,

Denticulated

conidiogeneous

loci,

1.0–3.5 × 0.7–

2.0 μm. Separating cells absent. Biconic conidia, holoblastic, 15.5– 26.0 × 6.5–10.0 μm, smooth, light brown, with a hyaline band in the median region, thick conidial scar with a diameter ranging from 0.8

4 | D I S CU S S I O N

to 1.9 μm, either isolated or in chains (Figure 2d). Conidiophore formation from conidia and appressoria formation were also observed

Morphological characterization along with phylogenetic analysis and

(Figure 2e,f).

pathogenicity tests confirmed the identity of P. cedrelae obtained

3.3 | Mycelial growth rate and sporulation of Pseudobeltrania cedrelae

this work suggested that the isolates of P. cedrelae were not closely related to P. ocoteae, as described by Crous et al. (2015). The mor-

The evaluation of mycelial growth continued until the ninth day

idiophores, with lobed basal cells, absence of setae and separating

of incubation and the measurement of sporulation on day 15. The

cells, and biconic to piriform conidia with a hyaline transversal band

from C. fissilis in São Paulo city. The phylogenetic trees generated in

phological characteristics of P. ocoteae included non-branched con-

highest growth rates were on MEA (8.54 mm/day) and V8 juice agar

indistinct in vivo and absent in vitro (Crous et al., 2015). In the iso-

(7.04 mm/day). All media tested supported sporulation except carrot

lates examined in this study, the band was observed both in vivo and

agar (Table 2).

in vitro. This feature is one of the important characteristics which separates the genus Pseudobeltrania from other genera in the same

3.4 | Phylogenetic analysis

family, the most commonly used being form and colour of the conidia for species differentiation (Zucconi, 1991).

Phylogenetic analysis using the ITS region was based on 57 se-

In addition, the characteristics reported by Crous et al. (2015)

quences, including the outgroup sequence (Figure 3). The alignment

were also observed in the genus Hemibeltrania. Hemibeltrania urban-

comprised 601 characters, of which 75 were parsimony-informative,

odendrii, for example, has a non-branched conidiophore originating

119 variable, 44 un-informative and 477 constant. A more parsimo-

from basal cells with isolated oval, obovoid, bicononic to limoni-

nious tree is shown in Figure 3 (TL = 169, CI = 0.568, RI = 0.859 e

form conidia lacking setae and separating cells (Fernandes, Lustosa,

RCI = 0.489). The species Pseudobeltrania ocoteae grouped with the

Barreto, & Bezerra, 2007). Therefore, based on the morphological

genus Hemibeltrania in a separate clade with 77% support, along

characteristics and phylogenetic analyses in the present work, the

with Porobeltraniella. The ten isolates obtained in this study formed

species P. ocoteae may possibly be more correctly placed in the

a separated group within the Beltrania clade with a well-supported

genus Hemibeltrania.

clade (99%). The 28S region analysis used 35 sequences, including those

In addition to P. ocoteae, the taxonomic placements of the species P. chumrungensis B. Sutton (1970), P. selenoides Hoog

of the outgroup (Figure 4). The alignment comprised 553 charac-

(1977) and P. cristaspora (Matsush.) de Hoog (1977) should be re-

ters of which 31 were parsimony-informative, 23 variable and un-

viewed because they exhibit characteristics that diverge from the

informative and 492 characters constant. Two more parsimonious

genus Pseudobeltrania. According to Heredia, Arias, Reyes, and

F I G U R E 3 Maximum parsimony tree resulting from ITS sequence analysis for species of the Beltraniaceae. Bootstrap values are indicated next to nodes. The length of the branches is indicated in the scale below the tree. Species under study are in bold type. The tree was rooted with Subramaniomyces fusisaprophyticus CBS 418.95

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F I G U R E 4 Maximum parsimony tree resulting from 28S sequence analysis for species of the Beltraniaceae. Bootstrap values are indicated next to nodes. The length of the branches is indicated in the scale below the tree. Species under study are in bold type. The tree was rooted with Subramaniomyces fusisaprophyticus CBS 418.95 Castañeda-Ruíz (2002), these species lack the hyaline transversal

in the genus Hemibeltrania; the name of the two species was not

band in the conidia and the conidia of P. selenoides are selenoids

changed as materials were not available for review. Moreover, the

with acute ends. Furthermore, the morphological characteristics of

authors proposed an identification key for Pseudobeltrania spp.,

P. chumrungensis and P. cristaspora bear greater similarity to species

whereby P. cedrelae was considered to have a simple conidiophore.

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MILAGRES et al.

However, according to the characteristics observed here, P. cedrelae

period in relation to that found in this work, with differences of 2

has branched conidiophores.

and 6 days, respectively. These differences in the latent period be-

The different dimensions of conidiophores found in leaf lesions

tween the two studies may result from the use of different host spe-

and slide culture may be attributed to the differences in nutrient

cies and the different conditions in which the inoculated plants were

composition found in these substrates. The phenotypic plasticity

incubated.

observed in some fungi is a response to environmental stimuli.

Based on the results presented in this paper, the morphological

Aureobasidium pullulans, for example, forms different types of col-

characteristics of the genus Pseudobeltrania remain confused and

onies depending on the temperature and the substrate in which

there is a need to populate database with further DNA sequences

it is growing (Slepecky & Starmer, 2009). Another example is

to generate more robust phylogenetic trees for this group of fungi.

Arthrobotrys oligospora which produces different structures to infect and parasitize nematodes or fungi in response to environmental signals (Nordbring-Hertz, 2004). In relation to the culture media, the highest mycelial growth rates

AC K N OW L E D G M E N T S The authors would like to acknowledge the Coordenação de

for P. cedrelae occurred on MEA and V8 juice agar. Bogo, Maffioletti,

Aperfeiçoamento Pessoal de Nível Superior (CAPES) for finan-

Valdebenito-Sanhueza, and Casa (2008) also reported more rapid

cial support, Conselho Nacional de Desenvolvimento Científico e

colony development in these two culture media for Cryptosporiopsis

Tecnológico (CNPq) and Fundação de Amparo a Pesquisa do Estado

perennans. The composition and nutrient concentration in the sub-

de Minas Gerais (FAPEMIG).

strate, the nutritional requirements and the physiological variability of the isolate, in addition to environmental conditions, all influence the growth of fungi (Cruz, Prestes, & Maciel, 2009). Phylogenetic analysis of the 28S and ITS regions presented a number of divergences. Based on the 28S region, which is better conserved than the ITS region, the isolates examined here grouped

ORCID O. L. Pereira

http://orcid.org/0000-0002-0274-4623

G. Q. Furtado

http://orcid.org/0000-0001-5842-3389

in the same well-supported clade and were different from the other genera in a Beltrania sibling group. When comparing sequences from the ITS region, the isolates were not placed in a separate clade, but formed a group with a well-supported node within the Beltrania complex. The species Beltrania rhombica and Beltrania querna were interrelated, as also observed by Rajeshkumar et al. (2016), who proposed that studies using sequences obtained from type material should be initiated to determine whether they represent a complex with numerous cryptic species or only one species with significant sequence variation. In relation to the genus Beltraniella, species of B. portoricensis should also be sequenced to better designate the species, as this was placed in different positions in the phylogenetic tree, as also observed by Rajeshkumar et al. (2016). The symptoms observed on C. fissilis seedlings inoculated with P. cedrelae consisted of a single and extensive brown lesion along the leaf blade, while in leaves naturally infected with the pathogen a greater number of circular lesions distributed across the entire leaf area were observed. Furthermore, all symptomatic leaves collected to obtain isolates had black spots in the centre of the lesions. According to the literature, these spots correspond to another leaf pathogen of C. fissilis, Phyllachora balansae, which forms punctiform ascomata that are distributed circularly in the leaflets (Ferreira, 1989). Hanada et al. (2005), describing the symptoms of P. cedrelae leaf lesion on C. odorata, also mentioned the presence of black spots in the lesions. Further work is needed to elucidate the role of P. balansae in the development of the pseudobeltrania spot on cedar leaves. In the study by Hanada et al. (2005), plants of C. odorata inoculated with P. cedrelae presented a longer incubation and latency

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How to cite this article: Milagres CA, Azevedo DMQ, Pereira OL, Furtado GQ. Epitypification, characterization and phylogenetic positioning of Pseudobeltrania cedrelae, the causal agent of pseudobeltrania spot on Cedrela fissilis. For. Path. 2018;e12434. https://doi.org/10.1111/efp.12434