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First report of Seimatosporium hypericinum causing leaf spots on the ornamental shrub Hypericum × inodorum and in Brazil

  • A. Colmán
  • L. Freitas
  • R. W. Barreto
Article
  • 468 Downloads

Abstract

Hypericum ×inodorum is an ornamental plant that has become popular for the cut flower industry in Brazil, in recent years. No pathogen has been reported on this plant in Brazil. A coelomycete asexual morph was found associated with leaf spot and severe foliage blight on this host and is reported here for the first time. A combination of morphological and molecular features allowed it to be identified as Seimatosporium hypericinum. Koch’s postulates were performed and demonstrated, for the first time, that S. hypericinum is pathogenic to H. × inodorum.

Keywords

Coelomycete asexual morph Cut flower Etiology Foliage blight Ornamental plant Taxonomy 

There is little information published in the scientific literature about the pathogens that affect many of the ornamentals grown in Brazil, particularly for “minor” or novelty ornamental crops. A case in point is sweet amber or tutsan (hipérico in Brazil) Hypericum × inodorum (Hypericicaceae), a hybrid of H. androsaemum and H. hircinum, two perennial shurbs from Eurasia. It is grown for its stems which bear yellow flowers and red and pink coloured fruits (de Assis 2015) (Fig. 1a), and has been growing in popularity in the cut flower market in Brazil (Lorenzi and Souza 2008).

In May 2015, H. inodorum plants cultivated in an experimental plot measuring the effect of light regimes on flowering, on the campus of the Universidade Federal de Viçosa (Viçosa, state of Minas Gerais, Brazil), had severe foliar necrosis (Fig. 1b). Samples of leaves with necrotic symptoms were collected, dried in a plant press, and deposited in the herbarium at the Universidade Federal de Viçosa (Acc. No VIC 43000). All leaves were examined under a dissecting microscope and fungal structures were consistently associated with the necrotic tissues. Sections of infected leaves bearing fungal structures were prepared and slides were mounted in lactophenol or lactofuchsin. Observations of the fungal morphology were made under an Olympus BX 51 light microscope equipped with DIC and an Olympus E 330 camera.

The fungus had the following morphology: Lesions beginning as numerous, small irregular or angular blackened areas on leaves 1 ̶ 6 mm wide later surrounded by reddish brown poorly defined necrotic areas developing a greyish center, coalescing and leading to entire necrosis of leaves and defoliation of plants; Conidiomata acervular, sub-epidermal, 26–106 μm diam., dark brown; Conidiogenous cells cylindrical, holoblastic, phialidic, 4–12 × 2–3 μm, hyaline, smooth; Conidia fusiform, straight or curved, 12–24 × 3–5 μm, 3 − septate, two median cells, medium brown, apical cells hyaline, each terminal cell bearing two unbranched, whip-like, hyaline appendages, 8–23 μm long (Fig. 1c-e). This morphology is equivalent to that of Seimatosporium hypericinum, as described in Sutton (1980) and Nag Raj (1993).
Fig. 1

a. Hypericum× inodorum flowering and fruiting; b. Symptoms of leaf blight caused by Seimatosporium hypericinum on H. × inodorum c. Section of an acervulus with conidia; d. Conidia and conidiogenous cells; e. Mature conidia with pigmented median cells and hyaline apical cells ending in whip-like hyaline appendages; f. Colony on PCA after 15 days at 24 °C. Scale bars = 20 μm

Isolation in pure culture was performed by direct transfer of spores or other fungal structures onto plates containing potato dextrose-agar (PDA), with a sterile finely pointed needle. Pure cultures were preserved in 10% glycerol and silica-gel. One sporulating single-spore culture was deposited in the culture collection of the Universidade Federal de Viçosa (Acc. No COAD 2362b). Culture morphology was described based on observation of the colonies formed after 10 days in plates containing PDA and potato carrot-agar (PCA) and incubated at 25 °C with a 12 h daily light regime. Colour terminology followed Rayner (1970). Colonies were slow growing (reaching 3–3.5 cm diam. after 8 days), low convex to umbonate, margin entire, aerial mycelium wooly white to saffron; reverse centrally salmon or cinnamon, white to salmon at periphery; sporulation on PCA after 14 days, concentrated in black and globular masses (Fig. 1f).

Additionally, in order to better clarify the identity of the pathogen, a molecular characterisation of the isolate was performed. COAD 2362b was grown in plates containing potato dextrose and incubated at 23 ± 2 C for four days. DNA was extracted from the fungus mycelium with the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. The internal transcribed spacer (ITS) region was amplified with primers ITS1 and ITS4 (White et al. 1990). PCR amplification was performed as described by Tanaka et al. (2011). Sequences were generated by MACROGEN, Inc. (South Korea). Bayesian inference analyses were performed with the CIPRES web portal using MrBayes v. 3.2.6. (Miller et al. 2015). The jmodeltest (Darriba et al. 2012) was used for estimation of sequence evolution. Phylogenetic analysis was performed with COAD 2362b and selected sequences from additional taxa withdrawn from GenBank (Table 1). Phylogenetic trees were visualised in FigTree (Rambaut 2009).
Table 1

GenBank Accession numbers of the sequences used in phylogenetic analysis

Fungal Species

Strain Number

GenBank Acc. No.

ITS

Discostroma fuscellum

NBRC 32625

AB594794

 

NBRC 32680

AB594806

 

418-BB

GU244511

Discostroma stoneae

NBRC 32690

AB594797

Discostroma tostum

NBRC 32626

AB594795

Pseudopestalotiopsis theae

MFLUCC 12–0055

JQ683727

Sarcostroma bisetulatum

CBS 122695

EU552155

Sarcostroma restionis

CBS 118153

DQ278923

 

CBS 118154

DQ278922

Seimatosporium biseptatum

CPC 13584

JN871199

Seimatosporium botan

H4619

AB594799

Seimatosporium botan

H4619

AB594779

Seimatosporium discosioides

H4621

AB594800

 

KACC 42490

EF600969

Seimatosporium elegans

NBRC 32674

AB594801

Seimatosporium elegans

NBRC 32674

AB594801

Seimatosporium foliicola

NBRC 32676

AB594802

Seimatosporium glandigenum

NBRC 32677

AB594803

Seimatosporium grevilleae

ICMP 10981

AF405304

Seimatosporium hakeae

NBRC 32678

AB594804

Seimatosporium hypericinum

NBRC 32647

AB594805

Seimatosporium lichenicola

MFLUCC 14–0623

KT198724

Seimatosporium mariae

NBRC 32681

AB594807

Seimatosporium obtusum

CPC 12935

JN871206

Seimatosporium parasiticum

NBRC 32682

AB594808

Seimatosporium physocarpi

MFLUCC 14–0625

KT198722

Seimatosporium pistaciae

CPC 24455 / CBS 138865

KP004463

 

CPC 24457

KP004464

Seimatosporium rosae

MFLUCC 14–0621

KT198726

Seimatosporium walkeri

CPC 17644

JN871207

Seimatosporium hypericinum

RWB 2362b

MG385139

Discosia pini

MAFF10149

AB594776

A BLAST search of GenBank accessions resulted in 100% homology between COAD 2071b and a previously deposited sequence of S. hypericinum (isolate NBRC32647 -Accession No. AB594805). The sequence of COAD 2071b was deposited in GenBank under the Accession No, MG385139. The combination of morphological features and phylogenetic analyses (Fig. 2) placed the fungus unequivocally in S. hypericinum.
Fig. 2

Phylogenetic tree inferred from Bayesian analysis based on combined ITS sequences. Isolate from Brazil in bold face. Bayesian posterior probabilities are indicated at the nodes. The tree was rooted with Discosia pini (isolate MAFF 410149). Note that the name Discostroma has been generally used for the sexual morph of Seimatosporium

Conidia were produced on PDA and a suspension (1.0× 106 conidia/ml) was used to inoculate four healthy plants. Two plants were sprayed with sterile water and served as controls. The plants were covered with internally humidified plastic bags, containing a cotton plug soaked in water, and maintained in a greenhouse with a temperature at 22 and 26 °C for 48 h. After that period, the plastic bags were removed and the plants were observed daily for symptom development. All inoculated plants showed typical leaf spot symptoms 5 days after inoculation and the pathogen was re-isolated from diseased tissues. Controls remained healthy.

Seimatosporium was regarded by Tanaka et al. (2011) as a monophyletic genus of saprobic or plant pathogenic coelomycetous species of so-called ‘pestalotioid fungi’. Norphanphoun et al. (2015) stated that “Seimatosporium species are common as saprobes and pathogens on plants, but are not considered as destructive foliar pathogens”. However, from observations of this fungus on H. perforatum, Zimowska (2004) stated that it is frequently isolated from plants showing symptoms of necrosis and cracks in the bark of stems as well as from leaves showing red-amber necrotic spots. Our observations of S. hypericinum on H. × inodorum showed it is a damaging pathogen to tutsan.

Seimatosporium hypericinum has been reported on H. perforatum in Italy (Nag Raj 1993; Sutton 1980) and Poland (Adamska 2007) on H. quadrangulum in Sweden (Sutton 1975) and on an undetermined species of Hypericum in Australia (Cunnington 2003). Additionally there is a record of this fungus on Lonicer astandishii (Caprifoliaceae) in Ukraine. This record, however, requires confirmation as it is the only existing record of this fungus on a non-Hypericum host and on a genus belonging to a plant family different from Hypericicaceae. It appears that S. hypericinum is a pathogenic fungus specialised on members of Hypericum. Hypericum × inodorum represents a new host for this fungus, which is newly reported from Brazil.

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Copyright information

© Australasian Plant Pathology Society Inc. 2018

Authors and Affiliations

  1. 1.Departamento FitopatologiaUniversidade Federal de ViçosaViçosaBrazil

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