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Neonectria castaneicola and Neo. rugulosa in Japan

     Department of International Agricultural Development, Tokyo University of Agriculture. Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Differences between Neonectria castaneicola, which causes stem and perennial canker of trees, and Neo. rugulosa have not been clearly shown in previous studies. In this study these two species were compared in detail using 17 Japanese isolates consisting of 10 strains of Neo. castaneicola and seven of Neo. rugulosa. Fourspored asci were constantly found in Neo. castaneicola and this species produced larger ascospores and macroconidia than Neo. rugulosa which produced eight-spored asci. The mating system of Neo. castaneicola was homothallic while Neo. rugulosa was heterothallic. Characters in each species, such as the number of ascospores in an ascus and mating system, were constantly transferred to the 3rd generation. Molecular analysis revealed that the 10 isolates of Neo. castaneicola and seven of Neo. rugulosa were differentiated using rDNA sequence data from the nuclear rDNA ITS region. Moreover, Neo. castaneicola and Neo. rugulosa were separated into different clades. From these results, it was concluded that Neo. castaneicola should be maintained as an independent species, separate from Neo. rugulosa. The isolates of Neo. rugulosa used in this study were the first reported in Japan and found on Castanea crenata, Castanopsis sp., Myrica rubra and Quercus acutissima.

Key words: Cylindrocarpon-anamorph, heterothallic, homothallic, Hypocreales

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The genus Nectria Fries (Fries) has long been known as a member of the Nectriaceae (Hypocreales) containing more than 700 species (Schroers 2001). It consists of plant pathogens, hyperparasites and mycotoxin-producing species. Recently, fungi belonging to Hypocreales were restudied intensively and reclassified into three to six families (Rogerson 1970, Rossman 1996, Rossman et al 1999, Samuels et al 1990, Samuels and Brayford 1994). The genus Nectria also was restudied and divided into several genera based on the reaction of their perithecia to potassium hydroxide (KOH) or lactic acid, and on their anamorphs. In recent classifications the species of Nectria, which have a Cylindrocarpon anamorph and perithecia color change to dark red in KOH or yellow in lactic acid, were transferred to the genus Neonectria (Aoki 2001, Rossman et al 1999). In 1959 Booth placed these fungi in two groups, Coccinea and Mammoidea. At present, nectrioid species with Cylindrocarpon anamorphs are divided into five groups; the Nectria coccinea/galligena, N. mammoidea, N. rugulosa, N. radicicola and N. veuillotiana groups (Rossman et al 1999). These groups are identified by the surface structure of the ascospores, structure of the perithecial wall and micro- or macro-conidia formation.

Previously the causal fungus of perennial cankers of Abies veitchii Lindl. and Acer crataegifolium Sieb. et Zucc. was identified as Neonectria castaneicola (Yamamoto and Oyasu 1958) Tak. Kobay. et Hirooka (Hirooka et al 2003; Kobayashi et al 2002, 2005), instead of the morphologically similar species Neonectria rugulosa (Pat. & Gaill.) Mantiri & Samuels (Mantiri et al 2001). Neo. castaneicola, which has a Cylindrocarpon castaneicola Tak. Kobay anamorph, was first described in 1958 in Japan as Nectria castaneicola W. Yamam. et Oyasu (Yamamoto 1958, 1962; Yamamoto and Oyasu 1958; Yamamoto et al 1957). It was reported that Neo. rugulosa causes bark death, often in association with cankers and the rapid decline of Macadamia trees in the United States (Ko and Kunimoto 1991, Samuels and Brayford 1994). The differences between Neo. castaneicola and Neo. rugulosa are shown in the number of ascospores in the ascus, four and eight, respectively, and by the production of macroconidia, which are septated more than 8x in Neo. castaneicola, but not at all in Neo. rugulosa.

During a survey of Japanese nectrioid fungi, 10 specimens of Neo. castaneicola and seven of Neo. rugulosa were collected. The latter species, collected for the first time (TABLE I), tentatively identified by their eight-spored asci. In this study, several stable characters differentiating Neo. castaneicola and Neo. rugulosa were identified using these 17 specimens and monoascospore isolates.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Morphological observation.— – Perithecia from fresh materials were tested directly for their color reaction to 3% KOH solution and 100% lactic acid, respectively. Perithecia from old, dried collections were also tested for their color reaction to KOH and lactic acid after rehydration with a drop of Shear’s solution (Udagawa et al 1978). Perithecia were hand-sectioned and crushed for observation of morphological characteristics by light microscopy. Anamorphs of the Neo. castaneicola and Neo. rugulosa were grown at 25 C on PDA in 9 cm plastic petri dishes in the dark to evaluate their colony color and odor. Colonies on synthetic low-nutrient agar (SNA) or carnation leaf agar (CLA) grown for 2 wk at 25 C in complete darkness were used to examine the production and morphology of conidia. The morphological characteristics of these fungi were examined in detail by light and scanning electron microscopy. The size of conidia was measured by evaluating 100 spores selected randomly from individual isolates incubated under each culture condition. The colors of the colony reverse were determined according to Kornerup and Wanscher (1978).

Colony growth.— – Discs 4 mm in diam taken from the edge of young colonies were placed in the center of PDA plates and incubated at temperatures from 5 to 40 C at 5 C intervals. After 16 d in complete darkness, the diam of colonies in five plates at each of the eight temperatures were measured.

DNA extraction and sequencing.— – Mycelia grown on PDA at 25 C were harvested after 1 to 2 wk. Genomic DNA was extracted from lyophilized hyphae based on the method of O’Donnell et al (1997) with some modifications or with a DNeasy Plant Mini Kit (QIAGEN®). The nuclear ribosomal internal transcribed spacer (ITS) region was amplified with primer pairs ITS1 and ITS4 (White et al 1990). Polymerase chain reaction (PCR) amplification of ribosomal DNA (rDNA) was performed by TaKaRa ExTaq (TaKaRa Biomedical, Japan) with 30 cycles of incubation for 1 min at 96 C, 1 min at 52 C and 2 min at 72 C. Sequencing was conducted with the ABI-PRISM 377 DNA sequencing system (Applied Biosystems, Foster City, California) and DNA sequencing kit (Perkin-Elmer, USA) following the ABI protocol.

Phylogenetic analysis.— – Sequence alignment and homology analysis were carried out using AssemblyLIGNTM 1.0.9c (Accelrys, USA) and CLUSTAL W package with Mac Vector 6.5.3 (Accelrys, USA) (Thompson et al 1994). The aligned sequences were analyzed by the neighbor-joining method (Saitou and Nei 1987), using PAUP 4.0b. The distance matrix was calculated using DNADIST with the Kimura’s two-parameter method, and the topology was tested with 1000 bootstrap trials.

Examination of holomorphism and mating systems.— – To examine holomorphism, each of the four or eight ascospores in the asci were individually transferred onto PDA using a micromanipulator. Holomorphism was confirmed by the production of mature perithecia originating from monoascospores on sterilized twigs of poplar (Populus sp.) on PDA after 1 to 2 mo. Each dish was sealed with surgical tape (Micropore^TM) to allow natural air circulation. The dishes were incubated for 3 to 4 wk under natural irradiation after having been maintained in dark conditions for 2 wk. Mature perithecia in 4 (ascospores in one ascus) x 10 (specimens in TABLE I) isolates of Neo. castaneicola and in 8 (ascospores in one ascus) x 7 (specimens in TABLE I) isolates of Neo. rugulosa were observed.

Stability of ascospores, number in different generations.— – Stability of the number of ascospores in the asci was examined to determine whether this character can be used as a criterion for classification. Stability of the number of ascospores in an ascus was examined in perithecia obtained by cultivation of Neo. castaneicola and Neo. rugulosa. The number of ascospores in each ascus was examined from 100 perithecia obtained independently from each monoascospore isolate of Neo. castaneicola and Neo. rugulosa.

	RESULTS

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Morphological characteristics.— – Since the morphological characteristics of Neo. castaneicola were described previously (Hirooka et al 2003, Kobayashi et al 2002, 2005), only those of Neo. rugulosa are described herein based upon the seven newly recorded Japanese isolates in this study. It was thought that these fungi could be either Neo. castaneicola or Neo. rugulosa from the morphological characterizations. The definitions of Neo. castaneicola and Neo. rugulosa are briefly summarized as follows.

Perithecia of the Neo. castaneicola tested are reddish to orange, warted, globose to subglobose, low papillate, solitary to caespitose in groups of five to 70, 250 – 470 µm in height and 350 – 430 µm in diam, not collapsing when dry, stained uniformly dark red in KOH and yellow in lactic acid. They grow superficially on erumpent basal stroma. Cells at the surface of the perithecial wall and of warts are almost circular in outline, and 15–25 µm in diameter. The perithecial wall is 40–87 µm thick and composed of two layers. The outer layer is 22–48 µm thick, composed of circular cells, 12–24 µm in diam, and pigmented; the inner layer is ca. 13 µm thick, composed of flat cells, 10–15 µm long, lumina 2-wide, and walls 2–3 µm thick. The asci are unitunicate, clavate, without a differentiated apical structure, and 50–89 x 9–13 µm. Ascospores are narrowly ellipsoid to fusiform, equally two-celled, not constricted at the septa, finely striate, often appearing smooth, colorless or yellowish brown, and 18–28 x 7.5–11 µm. Mycelium is not visible on or around the stroma. On PDA, Neo. castaneicola produces white to beige colony with a yellow center on the surface and reverse sides. Odor is a little acidic. The colony surface is cottony to powdery with aerial mycelia. Microconidia and macroconidia are abundant on CLA, PDA, and SNA, but conidia are not produced on old colonies. Cylindrical microconidia with rounded ends are produced in false heads on short conidiophores. Conidiogenous cells are 25– 45 x 2– 4.5 µm broad at their base, tapering to 2– 4 µm, with a distinct collarette. Macroconidia are formed on sparse, scattered, buff or orange sporodochia on the agar surface after 14–20 d or sometimes absent in degenerated cultures. Conidiogenous cells of sporodochia are cylindrical, 15–20 x 2.5–4 µm, and borne terminally on dense, irregularly branching conidiophores. Macroconidia are fusiform, usually broadest in their upper third, curved, 5–9-septate, 5–septate: 60–82 x 5–10 µm, 6-septate: 67–85 x 5–10 µm, 7-septate: 77–87 x 5–10 µm, 8-septate: 77–87 x 5–10 µm, 9-septate: 80–95 x 5–10 µm, with flattened basal and tapering apical cells. Phialides are simple with a collar and a single apical pore. Monophialidic conidiophores are cylindrical, often proliferated, branched or unbranched. Chlamydospores were rarely observed.

Perithecia of the Neo. rugulosa tested are reddish to orange, warted, globose to subglobose, low papillate, solitary to caespitose in groups of 5–40, 345–470 µm in height and 350–410 µm in diam, not collapsing when dry, stained uniformly dark red in KOH and yellow in lactic acid. They grow superficially on erumpent basal stroma. Cells at the surface of the perithecial wall and of warts are almost circular in outline, and 10–22 µm in diam. The perithecial wall is 58–94 µm thick and composed of two layers. The outer layer is 27–50 µm thick, composed of circular cells, 10–20 µm in diam, and pigmented; the inner layer is ca. 15 µm thick, composed of flat cells, 10–15 µm long, lumina 1–3 µm wide, and walls 2–3 µm thick. The asci are unitunicate, clavate, without differentiated apical structure, and 64–89 x 10–15 µm. Ascospores are narrowly ellipsoid to fusiform, equally 2-celled, not constricted at the septa, finely striate, often appearing smooth, colorless or yellowish brown, and 18–24 x 6–7.5 µm. Mycelium is not visible on or around the stroma. On PDA, Neo. rugulosa produces white to beige colony with a yellow center on the surface and reverse sides. Odor is a little acidic. The colony surface is cottony to powdery with aerial mycelia. Microconidia and macroconidia are abundant on CLA, PDA and SNA, but conidia are not produced on old colonies. Cylindrical microconidia with rounded ends are produced in false heads on short conidiophores. Conidiogenous cells are 21– 43 x 3–4.5 µm broad at base, tapering to 2– 4 µm, with a distinct collarette. Macroconidia are formed on sparse, scattered, buff or orange sporodochia on the agar surface after 14–20 d or sometimes absent in degenerated cultures. Conidiogenous cells of sporodochia are cylindrical, 15–20 x 2.5–3 µm, and borne terminally on dense, irregularly branching conidiophores. Macroconidia are fusiform, usually broadest in their upper third, curved, 5–7-septate, 5-septate: 47–75 x 5–7.5 µm, 6-septate: 55–77 x 5–7.5 µm, 7-septate: 65–87 x 5–7.5 µm, with flattened basal and tapering apical cells. Phialides are simple with a collar and a single apical pore. Monophialidic conidiophores are cylindrical, often proliferated, branched or unbranched. Chlamydospores were rarely observed.

The main morphological differences between Neo. castaneicola and Neo. rugulosa (TABLE II) were: (i) the number of ascospores in the ascus, 4 in Neo. castaneicola and 8 in Neo. rugulosa (FIG. 1); (ii) the width of the ascospores in Neo. castaneicola compared to Neo. rugulosa, which showed a l/b ratio of 2.67 and 2.91, respectively, and; (iii) larger macroconidia with the production of 8- and 9-septated macroconidia in Neo. castaneicola, but only seven or fewer septate conidia in Neo. rugulosa.

View larger version (92K): [in this window] [in a new window]   FIG. 1. A: Neonectria castaneicola (No. 1) 4-spored ascus: B: Neo. rugulosa (No. 97) 8-spored ascus. Scale bar: 25 µm.

View larger version (104K): [in this window] [in a new window]   FIG. 2. Morphological characteristics of both Neo. castaneicola and Neo. rugulosa has same points. A–F: Neo. castaneicola and anamorph, Cy. castaneicola. A: Perithecia. B: Median section of perithecia. C: Close-up of perithecial wall. D: Ascospores, E: Conidia, F: Chlamydospore. G–L: Neo. rugulosa and anamorph, Cy. rugulosum. G: Perithecia. H: Median section of perithecia. I: Close-up of perithecial wall. J: Ascospores, K: Conidia, L: Chlamydospore. (Scale bar: A, G = 500 µm; B, H = 100 µm; C, I = 100 µm; D–F, J–L = 20 µm).

Comparison of DNA sequences and Molecular phylogenetic relationship.— – Nucleotide sequences of the amplified the rDNA ITS region were determined for 27 cultures of genus Neonectria and Gibberella. Number of nucleotide of the rDNA ITS region ranged in length from 461 to 486-bp in these cultures. Based on these aligned sequences, we constructed a rooted molecular phylogenetic tree of 10 taxa of Neonectria and Gibberella species after 1000 bootstrap replications (FIG. 3). In phylogenetic studies, Neo. castaneicola and Neo. rugulosa were examined with Gibberella xylarioides as outgroup. The phylogenetic tree for Neo. castaneicola and Neo. rugulosa were separated into two groups. The topologies of the two groups were well supported by 100% bootstrap values (FIG. 3). Aligned nucleotide sequences of the nuclear ribosomal ITS region of 10 isolates of Neo. castaneicola and seven of Neo. rugulosa isolates were 461 bp long. Neo. castaneicola and Neo. rugulosa were differentiated by 12 nucleotide-fixed differences within the ITS region.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Phylogenetic tree constructed by neighbor-joining method for 27 collections of Neonectria and Fusarium species based on nucleotide sequences of ITS and 5.8s regions. The values at the nodes are the confidence levels from 1000 replicate bootstrap samplings. *Database accession No. AB233175 [GenBank] **Database accession No. AB233176

Holomorphism and mating systems.— – Four monoascospore isolates dissected from an ascus of Neo. castaneicola formed mature perithecia. A total of 40 monoascospore isolates from 10 perithecia of Neo. castaneicola were judged homothallic. Conversely, 56 monoascospore isolates from the 8-ascospore containing asci of one of the seven Neo. rugulosa isolates did not produce any mature perithecia. However, in the seven isolates of Neo. rugulosa, each of the four ascospores in the upper part of one ascus crossed with each of the four ascospores in the lower part of the same ascus and formed mature perithecia. These results show that Neo. rugulosa is heterothallic. Thus, Neo. castaneicola and Neo. rugulosa have different waiting systems (FIG. 4).

View larger version (26K): [in this window] [in a new window]   FIG. 4. Holomorphism and mating types in ascospores of Neo. castaneicola and Neo. rugulosa.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Stability of the number of spores in asci of Neo. castaneicola and Neo. rugulosa over 3 generations. + Fertile cross, perithecia formed; – Infertile cross, no perithecia formed.

	DISCUSSION

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Since 2002, eight specimens of Neo. castaneicola, which have 4-spored asci, and seven new specimens of Neo. rugulosa, which have 8-spored asci, previously unrecorded in Japan, were collected (TABLE I and FIG. 1). In this study, biological, molecular and morphological differences between Neo. castaneicola and Neo. rugulosa were examined. In the teleomorph, the number of ascospores in an ascus was constantly different between the two species. At the same time, Neo. castaneicola consistently produced longer ascospores than those of Neo. rugulosa. In the anamorph stage, which was observed on CLA, the macroconidia with more than 5-septa in Neo. castaneicola were longer than those of Neo. rugulosa. Moreover, Neo. castaneicola sometimes formed 8 and 9-septated macroconidia, but those of Neo. rugulosa never contained more than seven septa (TABLE II). Molecular analysis allowed comparisons of the aligned 461-bp nucleotide sequences of the ITS rDNA region from 10 isolates of Neo. castaneicola and seven of Neo. rugulosa. In the molecular phylogenetic relationship, Neo. castaneicola and Neo. rugulosa were separated into two groups (FIG. 3). In the ITS region, Neo. castaneicola and Neo. rugulosa were also shown as closely related species with 97% homology, but they were clearly differentiated at 12-bp nucleotide differences within the ITS region. Nectria rugulosa-group (Neo. castaneicola and Neo. rugulosa) and Neo. veuillotiana-group were distinguished by Rossman et al (1999) based upon the morphology of the surface of ascospore and presence of microconidia even though their structure of perithecial wall was same. Especially, these fungi produced similar perithecial walls with thick-walled cells. Moreover, structure of perithecial wall is an important characteristic in recent taxa (Brayford et al 2004). Our molecular results support that structure of the perithecial wall was significant in the classification of genus Neonectria. From mating experiments, Neo. castaneicola was shown to be homothallic because it produced perithecia from every single ascospore tested. On the other hand, cultures from monoascospore of Neo. rugulosa never formed mature perithecia. However, crosses between monoascospores obtained from the upper half of one ascus and those from the lower half of the same ascus formed mature perithecia (FIG. 4). Based on these results, Neo. rugulosa was confirmed as being heterothallic and the number of ascospores in the asci was shown to be stable at least through the third generation. Because of these differences, Neo. castaneicola and Neo. rugulosa should be classified as separate species (FIG. 5).

In some Nectriaceous fungi such as Albonectria rigidiuscula (Anamorph: Fusarium decemcellulare) (Rossman et al 1999), Neonectria austroradicicola (Anamorph: Cylindrocarpon austrodestructans) (Samuels and Brayford 1990) and Xenonectriella ornamentata (Anamorph: unknown) (Rossman et al 1999) 4-spored and 8-spored asci are produced in two same species. Because this is the first study to demonstrate the stability of ascospore number in the Nectriaceae, and to use this feature for classification, re-examination of 4-spored and 8-spored strains in other species based on similar analysis should be conducted.

	ACKNOWLEDGMENTS

 
We thank Norikazu Kameyama, University of Ryukyu for his support in sample collection in Okinawa Prefecture, and Yasunori Ono, Lead Discovery Research Laboratories, Sankyo Co., Ltd., for his technical advice.

	FOOTNOTES

 
Accepted for publication August 29, 2005.

¹ Corresponding author. E-mail: keiko{at}nodai.ac.jp

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This Article Abstract Full Text (PDF) An erratum has been published Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Hirooka, Y. Articles by Natsuaki, K. T. Search for Related Content PubMed Articles by Hirooka, Y. Articles by Natsuaki, K. T. Agricola Articles by Hirooka, Y. Articles by Natsuaki, K. T.