Australasian Plant Pathol. DOI 10.1007/s13313-014-0275-x
Pseudocercospora lonicerigena a leaf spot fungus on the invasive weed Lonicera japonica in Brazil Bruno E. C. de Miranda & Bruno Wesley Ferreira & Janaína L. Alves & Davi M. de Macedo & Robert W. Barreto
Received: 31 October 2013 / Accepted: 22 January 2014 # Australasian Plant Pathology Society Inc. 2014
Abstract Lonicera japonica (Japanese honeysuckle), a plant of Asian origin introduced as an ornamental into many countries, has become an invasive weed in some exotic situations, such as in southern Brazil. Classical biological control (CBC) with natural enemies collected in the centre of origin is considered to be the best alternative for sustainable management of L. japonica. In the context of this strategy, previous knowledge of the natural enemies already present in the target area is of great importance in order to avoid un-necessary introductions of CBC agents from the weed’s native range. Recently, examination of populations of L. japonica in localities in the Brazilian states of Rio de Janeiro and Paraná revealed the presence of leaf spots consistently associated with a cercosporoid hyphomycete. A molecular and morphological study of this fungus led to its recognition as Pseudocercospora lonicerigena. There were few differences when the Brazilian isolate was compared with a strain found in North America. This is the first report of this fungus in Brazil. The fungus was isolated in pure culture and inoculation of healthy leaves of L. japonica eventually led to leaf yellowing and necrosis, indicating that P. lonicerigena is not highly pathogenic, as evidenced by the continuing advance of this weed in southern Brazil. Therefore, the introduction of additional co-evolved natural enemies collected in Asia, perhaps piggy-backing on an ongoing project already underway in New Zealand, will be necessary to control this invasive alien weed in Brazil. Keywords Classical biological control . Cercosporoid fungi . Japanese honeysuckle . Molecular phylogeny . Ornamental
B. E. C. de Miranda : B. W. Ferreira : J. L. Alves : D. M. de Macedo : R. W. Barreto (*) Departamento de Fitopatologia, Universidade Federal de Viçosa, 36570-000 Viçosa, MG, Brazil e-mail:
[email protected]
Introduction Lonicera japonica Thunb. (Japanese honeysuckle), common name in Brazil madressilva, is a plant of Asian origin belonging to the family Caprifoliaceae. It is a semi-woody vine species that is very vigorous, and produces highly fragrant white or yellow inflorescences which attract hummingbirds and butterflies. It is widely used as an ornamental species in Brazil, mainly as a fence cover (Lorenzi and Souza 1995). In some Asian countries it is used as a medicinal plant and is important in traditional Chinese medicine (Kumar et al. 2005, Shang et al. 2011). However, in the USA (including Hawaii), Australia and New Zealand, it has escaped cultivation to become a serious weed in natural ecosystems (Cronk and Fuller 1995). Recognition of L. japonica as a weed problem is recent in Brazil but invasions in southern Brazil, especially in Rio Grande do Sul (Schneider 2007), have been recorded. The plant was recorded (R. W. Barreto, pers. obs.) infesting a significant part of an area of critical importance for the preservation of the rare animal species Peripatus acacioi Marcus and Marcus, 1955, (Onycophora, Peripatidae) in the Reserva Biológica de Tripuí (Ouro Preto, state of Minas Gerais, Brazil), together with another exotic weed, Hedychium coronarium J. Koenig - wild ginger (Fig. 1). The latter is already recognized as a major threat to the survival of this iconic species (an evolutionary link between annelids and arthropods) as there are indications that environmental conditions imposed by such infestations are deleterious for the animal populations (Soares and Barreto 2008). Biological control with natural enemies from the Asian centre of origin is considered to be the sole sustainable method of control for L. japonica. Investigations are underway in New Zealand aimed at finding potential biocontrol agents for this weed species (Waipara et al. 2007). Recently, an insect natural enemy of Japanese honeysuckle – the admiral butterfly (Limenitis glorifica Fruhstorfer, 1909, from Japan) - was
B.E.C. de Miranda et al.
Fig. 1 Pseudocercospora lonicerigena on Lonicera japonica. a Healthy leaves and flowers of honeysuckle in invasive situation; b Diseased leaves; c Conidiophores of P. lonicerigena; d Conidia of P.lonicerigena (Scale bars: 10 μm)
proven to be host-specific enough for release in New Zealand. Lonicera japonica is also a likely future candidate to be a target for classical biological control in Brazil. In this context, previous knowledge of natural enemies already established in Brazil is of relevance to avoid unnecessary introductions of biocontrol agents from the weed’s native range that are already present in Brazil but which have gone unnoticed (Barreto 2009). Recently, separate populations of L. japonica were found at several localities in the municipality of Nova Friburgo (state of Rio de Janeiro, Brazil), and also in the state of Paraná, bearing leaves with chlorotic areas that become necrotic with age. A Cercospora–like fungus was regularly found in association with the necrotic tissues. There is only one record of a fungus attacking Lonicera sp. in Brazil, in the main database of fungi on plants in Brazil. This is an old record of Helminthosporium lonicerae Viégas (Viégas 1946) from Poços de Caldas (state of Minas Gerais). Nevertheless, upon examination of Viegas’ description it is likely that the fungus he described now belongs to another genus segregated from Helminthosporium (possibly Drechslera or Bipolaris) (Alcorn 1988). Moreover, there are no records or images of the fungus from Poços de Caldas. Most fungi listed in association with L. japonica have been recorded
from the Far East (USDA Fungal Database, http://nt.ars-grin. gov/fungaldatabasews), which is the centre of origin of L. japonica, and some belong to the broad group traditionally called “cercosporoids”. The aim of this study was to confirm the identity of the fungus involved with leaf spots of L. japonica in Brazil and to verify its pathogenicity.
Materials and methods Morphological characterization Samples of diseased leaves of L. japonica were collected in the field, dried in a plant press and brought to the laboratory for further examination. Representative specimens of the fungus were deposited in the herbarium at the Universidade Federal de Viçosa (VIC). Pure cultures were obtained by transferring conidia, using a sterile fine-pointed needle, from lesions onto plates containing VBA (vegetable broth-agar), as described in Pereira et al. (2003). Cultures were deposited in the culture collection at the Universidade Federal de Viçosa – Coleção Oswaldo Almeida Drummond (COAD). Slides containing fungal structures were mounted in lactophenol or lactofuchsin. Sections were prepared with the
Pseudocercospora lonicerigena on Lonicera japonica in Brazil
help of a freezing microtome (Microm HM 520). Observations of fungal structures and measurements, as well as preparation of photographs, were performed with an Olympus BX 51 light microscope fitted with an Olympus E330 camera and a drawing tube. Colony description was based on observation of 6 day-old colonies formed on potato dextrose agar (PDA) and potato carrot agar (PCA) (Crous et al. 2009), on plates incubated at 25 °C either in the dark or under a daily light regime of 12 h (light provided by 40 W fluorescent daylight lamps placed 30 cm above the plates). Colony colours (surface and reverse) were recorded following the terminology of Rayner (1970). DNA sequence comparisons and phylogenetic analyses For molecular studies of the sample, a pure culture was grown on potato dextrose broth at 25 °C for 7 days. Total genomic DNA isolation was extracted from mycelium using Wizard ® Genomic DNA Purification Kit (Promega corporation, WI, USA) according to the manufacturer’s instructions. The primers ITS4 and ITS5 (White et al. 1990) were used to amplify the ITS region as well as the 5.8S rRNA gene. Part of the translation elongation factor (TEF) gene was amplified using the primers EF728F and EF–2 (Carbone and Kohn 1999). The primers LSU forward and LR5 were use to amplify part of the large subunit ribosomal. The polymerase chain reaction conditions were the same for all regions and the reaction mixture had a total volume of 12.5 μl: 1 μl of diluted gDNA, 1× PCR buffer, 48 μM each of the dNTPs, 2.5 pmol of each primer, 1.5 mM of MgCl2 and 0.7 units Taq polymerase (Bioline GmbH, Luckenwalde, Germany). The amplification reactions were done on a GeneAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT). The initial denaturation step was done at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C (30 s), annealing at 52 °C (30 s), and elongation at 72 °C (30 s). A final elongation step at 72 °C (7 min) was included in the run. The PCR products were separated by electrophoresis at 80 V for 40 min on a 0.8 % (wt/vol) agarose gel containing GelRed Nucleic acid stain (Biotium) at 0.1 μg/ml in 1× Tris-acetate EDTA buffer (0.4 M Tris, 0.05 M NaAc, and 0.01 M EDTA, pH 7.85) and visualized under UV light. The amplicons were sequenced in both directions using the PCR primers and a DYEnamic ET Terminator Cycle Sequencing kit (Amersham Biosciences, Roosendal, The Netherlands) according to the manufacturer’s recommendations. The products were analyzed on an ABI Prism 3700 DNA Sequencer (Perkin-Elmer, Foster City, CA). A consensus sequence was generated using the staden package (Staden 1996). The sequences generated from DNA of the cercosporoid fungus on L. japonica were compared through a BLAST (blast.ncbi.nlm.nih.gov/) search with existing
sequences in GenBank and a more complete phylogenetic study was performed with sequences from these isolates (Table 1). Sequences were assembled and added to the outgroup using MEGA 5.0 software package and were manually adjusted where necessary. The best-fit evolutionary model was determined for each data set by comparing different evolutionary models via the Akaike information criterion using PAUP (version 4.0b10) and MrModeltest 2.2 (Nylander 2004). The Bayesian analysis was performed using Markov chain Monte Carlo sampling and were run with two chains 1,000,000 generations by which time the average standard deviation of split frequencies was close to or less than 0.01. The program Tracer v1.5 (Rambaut and Drummond 2003) was used to ensure the convergence of the chains and then the consensus tree was calculated using the software MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001) where the first 25 % of the sampled trees were discarded as “burnin”. Phylogenetic trees were visualized with the program FigTree v1.3.1 (Rambaut 2009).
Pathogenicity screening A pathogenicity test was conducted to verify the pathogenic status of the fungus. As the fungus on L. japonica did not sporulate in culture, mycelial discs were used as inoculum. These were obtained from actively-growing colonies formed on VBA which were placed onto healthy leaves of six potted L. japonica individuals. The plants were kept in a moist chamber produced by covering individual potted plants with plastic bags together with water-soaked cotton wool placed at the base of the stems. VBA culture medium disks (without fungal colonies) were deposited on control plants. After 48 h, the plastic bags were removed and the plants were transferred to a greenhouse bench. Plants were observed regularly for the emergence of disease symptoms and the appearance of fungal structures on diseased tissues.
Results Molecular phylogeny Sequences obtained for the representative isolate obtained from L. japonica were aligned with sequences of Pseudocercospora downloaded from GenBank (Table 1). Comparison of sequences of the large subunit ribosomal (LSU) and small subunit ribosomal (ITS) of fungi in Pseudocercospora are not regarded as sufficient for species separation (Crous et al. 2012). Results obtained for another region regarded as more reliable for species distinction in this genus, namely EF–1α, indicated with certainty that the isolate on L. japonica collected in Brazil does not cluster within any
B.E.C. de Miranda et al. Table 1 List of isolates of Pseudocercospora, including the outgroup sequence (Passalora eucalypti), in the phylogeny study, with their country of origin and hosts Species
Strain
Host
Origin
GenBank number (EF-1α)
Passalora eucalypti Pseudocercospora corylopsidis Pseudocercospora dodonaeae Pseudocercospora exosporioides
CBS111318 MUCC874 CBS114647 MUCC893
Eucalyptus saligna Hamamelis japonica Dodonaea viscosa Sequoia sempervirens
Brazil Japan New Zealand Japan
GU384558 GU384437 GU384413 GU384423
Pseudocercospora flavomarginata Mycosphaerella fori (Pseudocercospora fori) Mycosphaerella gracilis (Pseudocercospora gracilis) Pseudocercospora guianensis Pseudocercospora kaki Pseudocercospora lonicericola Pseudocercospora lonicerigena Pseudocercospora lythracearum Pseudocercospora natalensis Pseudocercospora paederiae Pseudocercospora pancratii Pseudocercospora prunicola Pseudocercospora pyracanthae Pseudocercospora ranjita Pseudocercospora stephanandrae Pseudocercospora subsessilis Pseudocercospora tereticornis Pseucercospora timorensis
CPC13492 CMW9095 CBS243.94 MUCC855 MUCC900 MUCC889 COAD 1272 CPC10707 CBS111069 CPC10007 CBS137.94 CPC14511 MUCC892 CPC11141 MUCC914 CBS136.94 CBS124996
Eucalyptus camaldulensis Eucalyptus grandis Eucalyptus urophylla Lantana camara Diospyros kaki Lonicera gracilipesvar. glabra Lonicera japonica Lagerstroemia indica Eucalyptus nitens Paederia foetida **** Prunus x yedoensis Pyracantha angustifolia Gmelina sp. Stephanandra incisa **** Eucalyptus nitens
Thailand South Africa Indonesia Japan Japan Japan Brazil South Korea South Africa South Korea Cuba South Korea Japan Indonesia Japan Cuba Australia
GU384512 DQ211664 DQ211666 GU384435 GU384442 JQ324999 KF623108 GU384452 JQ325000 GU384468 GU384470 GU384393 GU384479 GU384500 GU384526 GU384527 GU384377
MUCC819
Ipomoea indica
Japan
GU384536
Pseudocercospora variicolor Pseudocercospora viticicola Pseudocercospora vitis Pseudocercospora weigelae
MUCC746 MUCC777 CPC11595 MUCC899
Paeonia lactiflora var. trichocarpa Vitex trifolia Vitis vinifera Weigela coraeensis
Japan Japan South Korea Japan
GU384538 GU384540 GU384541 GU384543
clade from other species of Pseudocercospora and is not conspecific with P. lonicericola (Fig. 2). Morphology Morphology of conidiophores and conidia of the newly collected fungus from Brazil on L. japonica was recognized as typical of members of Pseudocercospora. A comparison of the specimen VIC 31823 with another three species of Pseudocercospora reported on Lonicera spp. indicated that the fungus is morphologically similar to P. lonicerigena. Unfortunately, no cultures or sequences of this species are available in culture collections or in public databases for comparison. Pseudocercospora lonicerigena U. Braun and Crous, Fungal Diversity, 26: 62, 2007 (Fig. 1) Lesions on living leaves, amphigenous, 1.5−8×1−5.5 mm, irregular, yellow becoming light brown to black with age,
coalescing as leaf ages covering large areas of the leaf and leading to blight and defoliation. Sexual morph unknown Internal mycelium intra and intercelular, 2−3 μm, branched, septate, dark brown. External mycelium brown, septate, branched, 2−5 μm. Stromata semi-immersed to immersed, globose, abaxial, 13−42×19−38 μm, composed of dark brown textura angularis. Conidiophores either fasciculate (primary) or separate and emerging from external mycelium (secondary) – primary conidiophores subcylindrical, 19–45 × 3−4 μm, 1–2 septate, unbranched, brown, smooth – secondary conidiophores cylindrical somewhat sinuose, 12−33 × 3−4 μm, septate, branched, brown, smooth. Conidiogenous cells terminal, integrated, subcylindrical, 9−23×3 μm, light brown. Conidiogenous loci 1.5−2 μm not darkened, unthickened. Conidia single, subcylindrical, straight to curved or somewhat sigmoid, subhyaline, 54−78×2−4 μm, rounded at apex, truncate at base, 2–6 septate, hilum unthickened, not darkened, sub-hyaline, smooth.
Pseudocercospora lonicerigena on Lonicera japonica in Brazil Fig. 2 Phylogenetic relationships of Pseudocercospora isolates based on the consensus tree from a Bayesian analysis of EF–1α sequences. The Bayesian posterior probabilities are given at the nodes. The tree was rooted with Passalora eucalypti (Genbank access: GU384558)
Culture characteristics Slow growing (up to 23 mm diam after 15 days), flat (raised centrally) lanose or felty, with entire edges, olivaceous gray to olivaceous at periphery, pale olivaceous gray to smoke gray centrally, with diurnal zonation, olivaceous or dark mouse gray reverse, sporulation absent.
The fungus was re-isolated from diseased tissues and the colonies obtained were identical to those originally utilized in the inoculation of the plant, therefore fulfilling Koch’s postulates.
Specimens examined Brazil, state of Rio de Janeiro, Nova Friburgo, on living leaves of Lonicera japonica, 10 Nov 2012, R.W. Barreto (VIC 31823), living culture COAD 1272.
Discussion
Additional specimens Brazil, state of Paraná, Araucaria, on living leaves of Lonicera japonica, 8 Jan 2013, R.W. Barreto (VIC 39071). (GenBank accession number for VIC 31823: EF -1α=KF623108). A table is provided (Table 2) for comparison of the morphology of the fungus collected in Brazil with other species of Pseudocercospora recorded on L. japonica. Pathogenicity screening Symptoms identical to those observed in the field appeared 30 days after inoculation. These started as faint ill-delimited yellow areas and led to necrosis and sporulation 40 days postinoculation. No disease symptoms appeared on control plants.
Three species of Pseudocercospora have been described on members of the genus Lonicera: Pseudocercospora lonicerigena U. Braun and Crous, P. lonicerae Guo (Guo and Hsieh 1995) and P. lonicericola (W. Yamam.) Deighton (Deighton 1976). Unfortunately, there are no sequences available in GenBank or other public databases for P. lonicerigena and P. lonicerae and comparison with these species was based only on morphology. A preliminary search on GenBank, using the tool Blast, for sequences similar to those obtained for our fungus for ITS, LSU and EF–1α indicated a considerable similarity with other species belonging to the genus Pseudocercospora. These results were further confirmed after a more comprehensive study through Bayesian analyses. The alignment of sequences of taxa listed in Table 1 consisted of 29 ITS sequences including an outgroup and containing 520 characters of which 66 sites were variable and 27 were parsimony informative. For
B.E.C. de Miranda et al. Table 2 Morphology of selected Pseudocercospora spp on Lonicera spp. Structures
P. lonicericola
P. lonicerigena
P. lonicerae
P. lonicerigena (Brazilian isolate)
External mycelium
Light olivaceous, branched, septate, 1.5−4 μm Lacking
Indistinct
Not mentioned
Present Up to 40 5−25×1.5−3.5 Pale olivaceous to olivaceous brown Acicular to narrowly obclavate Apex subacutate Base truncate to obconally truncate 20−100×2−4 3−9-septate Subhyaline to very pale olivaceous Lonicera japonica Braun and Crous (2007)
Present 65−120 15−96×3.2−5.6 Pale olivaceous brown
Brown, septate, branched, 2−5 μm Present 13−42 19−45×3−4 Very pale to olivaceous brown Subcylindrical Apex rounded Truncate
Stromata Diam. (μm) Conidiophores (μm)
Conidia (μm)
Host Reference
10−65×2−4 Pale to olicaceous brown Obclavate-cylindrical Apex subobtuse Base subtruncate to obconally truncate 30−120×2−4 Indistinctly 3-to-11- multiseptate Subhyaline to very pale olivaceous Lonicera japonica Deighton (1976)
LSU region, the alignment consisted of 30 LSU sequences including an outgroup and containing 1301 characters of which 58 sites were variable and 21 were parsimony informative. As for part of the EF–1α, the alignment consisted of 26 EF–1α sequences including an outgroup and containing 475 characters of which 209 sites were variable and 123 were parsimony informative. Both ITS and LSU regions were confirmed to be inadequate to allow a clarification of the true affinities of our isolate. There were not enough phylogenetic signals to separate our isolate from others used in the analyses. As for EF–1α, the distinction of species was possible (Fig. 2), and in fact, the fungus collected on L. japonica represents a distinct taxon, with a probability a posteriori equal to 1, and clustered in a distinct clade; having as a sister clade, a grouping of two other species (P. natalensis and Mycosphaerella fori – sexual morph of Pseudocercospora fori G.C. Hunter, Crous and M.J. Wingf.). Our specimen (VIC 31823) is both a morphologically distinct species and phylogenetically separated from other taxa included in the study with a high statistical value supporting it for the EF–1α. The views of Crous et al. (2012), about the usefulness of EF–1α for species separation in Pseudocercospora, are confirmed here. In this study, the Pseudocercospora on L. japonica from Brazil was demonstrated to be phylogenecically distinct from P. lonicericola. As for the two other species of Pseudocercospora described in association with Lonicera spp. - P. lonicerigena (originally described from North America) and P. lonicerae (known only from China) - a comparison of morphological features with the fungus from Brazil showed that P. lonicerae differs by having larger stromata (65−120 μm diam), much longer conidiophores
Acicular to cylindrical Obtuse at the apex Truncate at the base 40−120×3.2−6.0 3−10-septate Pale olivaceous to olivaceous Lonicera sp. Guo and Hsieh (1995)
54−78×2−4 2−6-septate Subhyaline Lonicera japonica This paper
(up to 96 μm), and wider conidia (3−6 μm) (Guo and Hsieh, 1995). In addition to the clear distinction indicated by EF1α-based phylogeny, the Brazilian fungus differs from P. lonicericola in having well-defined stromata. The morphology of our fungus is similar to that described for P. lonicerigena, based on the North American type material, but, in contrast to the Brazilian specimens, no external mycelium was observed on the type of P. lonicerigena (Braun and Crous 2007). It is assumed here that the differences observed between the Brazilian and the North American specimens represent geographic (ecotypic) variation within the same species. Until sequences of an epitype of P. lonicerigena become available for a DNA-based comparison, we prefer to place the fungus on L. japonica from Brazil in P. lonicerigena. This is the first report of this species from Brazil. Although P. lonicerigena, as with other species of Pseudocercospora, is likely to be highly host-specific, the disease caused by this fungus was observed to be slowdeveloping - both in controlled conditions and in the field and, although leading to some defoliation, the impact on the host is insufficient to mitigate invasion by L. japonica. A similar conclusion was also reached by Waipara et al. (2007) concerning Pseudocercospora lonicerae which already occurs in New Zealand but seemingly without causing significant deleterious effects on invasive stands of L. japonica. This work represents the first contribution towards the knowledge of the existing mycobiota of L. japonica in Brazil. There are 55 records of fungi associated with L. japonica (USDA Fu nga l D ata bas e, h t t p : / / n t . a r s - g r i n . g o v / fungaldatabasews) worldwide, several of which are restricted
Pseudocercospora lonicerigena on Lonicera japonica in Brazil
to the plant’s native range and, therefore, potentially useful for classical biocontrol. In the future, cooperation with scientists in New Zealand, Japan and other countries in the west of Asia should be sought in order to survey and screen classical biocontrol agents for introduction into Brazil.
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